Oxidative Stress and Antioxidant Defense

Complimentary Contributor Copy

NEW DEVELOPMENTS IN MEDICAL RESEARCH
OXIDATIVE STRESS AND

ANTIOXIDANT DEFENSE
BIOMEDICAL VALUE IN HEALTH AND DISEASES
No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or
by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no

expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No
liability is assumed for incidental or consequential damages in connection with or arising out of information
contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in
rendering legal, medical or any other professional services.
Additional books and e-books in this series can be found

on Nova’s website under the Series tab.

OXIDATIVE STRESS AND

ANTIOXIDANT DEFENSE
BIOMEDICAL VALUE IN HEALTH AND DISEASES
MD. SAHAB UDDIN

AND
AMAN B. UPAGANLAWAR
EDITORS

Copyright © 2019 by Nova Science Publishers, Inc.
All rights reserved. No part of this book may be reproduced, stored in a retrieval system or
transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical
photocopying, recording or otherwise without the written permission of the Publisher.
We have partnered with Copyright Clearance Center to make it easy for you to obtain permissions
to reuse content from this publication. Simply navigate to this publication’s page on Nova’s website
and locate the “Get Permission” button below the title description. This button is linked directly to
the title’s permission page on copyright.com. Alternatively, you can visit copyright.com and search
by title, ISBN, or ISSN.
For further questions about using the service on copyright.com, please contact:
Copyright Clearance Center
Phone: +1-(978) 750-8400 Fax: +1-(978) 750-4470 E-mail: info@copyright.com.
NOTICE TO THE READER

The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or
implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability
is assumed for incidental or consequential damages in connection with or arising out of information
contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary
damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any
parts of this book based on government reports are so indicated and copyright is claimed for those
parts to the extent applicable to compilations of such works.
Independent verification should be sought for any data, advice or recommendations contained in
this book. In addition, no responsibility is assumed by the Publisher for any injury and/or damage
to persons or property arising from any methods, products, instructions, ideas or otherwise contained
in this publication.
This publication is designed to provide accurate and authoritative information with regard to the
subject matter covered herein. It is sold with the clear understanding that the Publisher is not
engaged in rendering legal or any other professional services. If legal or any other expert assistance
is required, the services of a competent person should be sought. FROM A DECLARATION OF
PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR
ASSOCIATION AND A COMMITTEE OF PUBLISHERS.
Additional color graphics may be available in the e-book version of this book.
Library of Congress Cataloging-in-Publication Data
ISBN: HERRN

Library of Congress Control Number:2019943053
Published by Nova Science Publishers, Inc. † New York

Md. Sahab Uddin would like to dedicate this book to his aunt, Shraboni Azad and
uncle, AK Azad Sarker.
Aman B. Upaganlawar would like to dedicate this book to his father,

Late Shri Babanrao Upaganlawar.

CONTENTS
Preface xi
Acknowledgments xiii
Section I Antioxidant Defense System in Health and Diseases 1

Chapter 1 Aging, Oxidative Stress and Antioxidants 3
Shalini Mani and Aarushi Singh
Chapter 2 Free Radicals and Antioxidants in Human Health and Diseases 23
Meenakshi Kaira, Abhilasha Ahlawat, Vaibhav Walia
and Munish Garg
Section II Antioxidant Signaling in Lifestyle Choices 55

Chapter 3 Antioxidant Status and Cigarette Smoking 57
Tanuj Joshi and Archana Negi Sah
Chapter 4 Oxidants, Antioxidants in Physical Exercise 73
Vaishali R. Undale
Section III Antioxidative Defense Mechanism in Neurodegeneration 93

Chapter 5 Antioxidants Signaling in Neurodegenerative Disorders 95
Abhilasha Ahlawat, Vaibhav Walia, Meenakshi Kaira
and Munish Garg
Chapter 6 Antioxidants against Stress-Induced Memory Dysfunction 131
S. B. Chandrasekar, Anil T. Pawar, M. S. Lokesh Prasad
and G. Hemavathi
Chapter 7 Neuroprotective Strategies for Oxidative Stress-Mediated
Alzheimer’s Disease 167
Manoj Govindarajulu, Sindhu Ramesh, Ayaka Fujihashi,
Mansour Alturki, Tyler Lynd, Gayatri Devi Donepudi,
Vishnu Suppiramaniam, Timothy Moore
and Muralikrishnan Dhanasekaran

viii Contents
Chapter 8 Oxidant/Antioxidant Imbalances in Huntington’s Disease 203

Arup Kumar Misra, Surjit Singh, Pramod Kumar Sharma,
Sneha Ambwani and Sushil Kumar Varma
Section IV Antioxidant Activity in Chronic Degenerative Diseases 229

Chapter 9 Oxidative Damage and Antioxidative Defense Mechanisms
in Inflammation 231
Newman Osafo, Joseph Owusu-Sarfo and David Darko Obiri
Chapter 10 Free Radicals and Antioxidant Defense in Asthma 257
Rashmi Pandey, Priyanka Tiwari, Ved Prakash,
Vivek Kumar Gupta and Bechan Sharma
Chapter 11 Antioxidant Activity and Cancer 275
Newman Osafo, Joseph Owusu-Sarfo,
Eyitayo Adeyemi Oyindamola,
Sophia Naa Ayikailey Ankrah and David Darko Obiri
Chapter 12 Oxidants and Antioxidant Defense in HIV/AIDS 297
Hrushikesh E. Velis
Section V Antioxidants in Cardiovascular Diseases 323

Chapter 13 Antioxidants and Antihypertensive Potency 325
Abdulla Sherikar, Aman Upaganlawar, Sameer Goyal
and Chandrashekhar Upasani
Chapter 14 Radical Approaches to Stroke 345
Vipin Dhote, Avinash Singh Mandloi, Durgesh Nandan Shukla,
Priyanshi Kapoor and Aditya Ganeshpurkar
Section VI Antioxidant Status in Metabolic Disorders 391

Chapter 15 Free Radicals, Antioxidants and Diabetes 393
Seetha Harilal and K. Rajesh
Chapter 16 Antioxidant Defense in Diabetic Nephropathy 427
Manojkumar S. Mahajan, Vishal S. Gulecha, Aman Upaganlawar
and Chandrashekhar D. Upasani
Chapter 17 Antioxidants in Glycation Related Diseases 465
Ahmad Ali, Additiya Paramanya, Prairna,
Koula Doukani and Sadaf Zehra

Contents ix
Section VII Antioxidant Defense in Sexual Health and Pregnancy 489

Chapter 18 Oxidative Stress and Antioxidant Therapy in Erectile Dysfunction 491
Yogesh Chand Yadav
Chapter 19 Oxidative Stress and Antioxidant Status in Pregnancy 507
Parag A. Pathade and Vinod A. Bairagi
About the Editors 523
List of Contributors 525
Index 531
Related Nova Publications 541

PREFACE
Oxidative stress is a state in which oxidative forces exceed the antioxidant systems that
lead to structure and functional deterioration of biomolecules. In the pathogenesis of copious
diseases and degenerative processes, oxidative stress plays a crucial role by direct damage to
macromolecules or liberation of toxic byproducts. However, oxidative stress also has a
beneficial role in physiologic adaptation and in the regulation of intracellular signal
transduction. Understanding the impact of oxidation in typical cellular function and disease
pathogenesis, as well as the molecular target of antioxidants, remains the primary focus of many
scientists for better preventive and therapeutic intervention. Oxidative Stress and Antioxidant
Defense: Biomedical Value in Health and Diseases represent current findings on the impact of
oxidative stress in the pathogenesis of diseases and underlying mechanisms of antioxidants
influencing health and disease processes. This book is divided into 7 sections consist of 19
chapters, describes how antioxidants defend oxidative stress-mediated diseases as well as
recent developments, future opportunities, and challenges. Sections 1 analyzes the role of
oxidative stress in aging and associated diseases as well as the use of antioxidants in health
maintenance, preventing and repairing injuries caused by oxidative stress. Section 2 represents
the status of various antioxidants in cigarette smoking and antioxidant defense against exercise-
induced oxidative stress. Section 3 focuses on the effect of oxidative stress in the pathogenesis
of neurodegeneration and the existing status of antioxidant therapy. Section 4 covers the impact
of oxidative stress at different levels of chronic degenerative diseases, as well as treatment with
antioxidants to revert and diminish the cellular injury. Section 5 offers the importance of
antioxidants in abating the pathological processes involved in hypertension and stroke. Section
6 presents the complexity associated with oxidative stress and metabolic disorders as well as
the potential of antioxidants used in amelioration of related pathologies. Section 7 discusses the
antioxidant defense against oxidative stress-mediated erectile dysfunctions and the significance
of antioxidants in pregnancy.
This book represents the copious set of specific research updates. All over the world
numerous erudite, experienced and eminent academicians, researchers and scientists had
participated to write the texts of this book to give a precise and diaphanous understanding of
oxidative stress-mediated cellular damages and role of antioxidants in disease processes at a
more advanced level with excellent presentation.
This book is suitable for professionals, academicians, students, researchers, scientists and
industrialists around the world. Biomedical, health, and life science departments can use this
book as a crucial textbook. Researchers and scientist from research institutes can use this book

xii Md. Sahab Uddin and Aman B. Upaganlawar
as efficient research info. Nutritionists, pharmacists, physicians, and other healthcare

professionals can use this book as a complete reference book. Furthermore, for interested
readers, this book is a storehouse of knowledge to comprehend the mechanisms of
oxidant/antioxidant altering factors accountable for cell injury. The organizations of this book
provided a profound knowledge and also maintain the reader's interest.
It is expected that readers shall find this book very informative and enormously useful.
Since science is constantly changing readers are strongly recommended to check the recent
update. The editors are ebulliently ready to accept any comment, suggestion, advice or critique.
Md. Sahab Uddin

Department of Pharmacy, Southeast University, Dhaka, Bangladesh
Aman B. Upaganlawar
Department of Pharmacology, SNJBs SSDJ College of Pharmacy, Maharashtra, India

ACKNOWLEDGMENTS
Editing is a complex process, the editors would like to thank the people involved in this
project for their quality time, expertise, and countless efforts. The editors are extremely
indebted to the following:
The authors for the countless time and expertise that they have put into their texts. Those
authors who submitted works to contribute to this project but unfortunately rejected. The
reviewers for their constructive reviews in improving the quality and presentation of the
contents. The teachers, colleagues, friends, and supporters for their endless inspiration and
assists.
Especially the Nova Science Publishers for giving us a great opportunity to edit this book
and their aid in the preparation of this edition
Last but not least, the editors would like to express the heartiest gratitude to almighty Allah
and their parents.
Md. Sahab Uddin

Aman B. Upaganlawar

SECTION I: ANTIOXIDANT DEFENSE SYSTEM IN
HEALTH AND DISEASES

In: Oxidative Stress and Antioxidant Defense ISBN: 978-1-53615-687-4
Editors: Md. Sahab Uddin and A. B. Upaganlawar © 2019 Nova Science Publishers, Inc.
Chapter 1
AGING, OXIDATIVE STRESS AND ANTIOXIDANTS
Shalini Mani and Aarushi Singh

Jaypee Institute of Information Technology, Noida, India
ABSTRACT
Intrinsic, extrinsic or even the environmental factors have been the source of the
progressive and time-dependent deterioration of a living cell. The pivotal mechanism of
this aging process is the genesis of free radicals. Not only the genes but also the
environmental circumstances and the dietary habits cause variations in average lifespan of
humans. The human body has a natural antioxidant pool which protects it from such cause
of aging, but they are compromised due to overwhelming oxidative stresses. Dietary
antioxidants are found to be a great influencer in the life history commutations.
Antioxidants having the power to inhibit this oxidative stress are the focus of numerous
studies. Homeostasis is maintained by the balance between the assembly and scavenging
of reactive oxygen species (ROS) and thus, shifting of this balance leads to the creation of
free radicals. Researchers are now keen to know more about the antioxidants as these can
neutralize the free radicals and can be a source to slow down the inevitable and irreversible
process of aging. This chapter focuses on the role of ROS in aging and function of
antioxidants in reducing the aging process.
1. INTRODUCTION
In the present modernistic society, neurodegenerative diseases allied to aging, are cause for
great concern. The concept of “healthy lifespan” has been promoted by the World Health
Organization (WHO) which focuses to elevate the proportion of healthy individuals to the total
lifespan of one (Peng et al., 2014). In 1532, Mohammad conducted the first study on aging in
his book “Ainul Hayat” and now with the passing of nearly five centuries the exact mechanism
of aging still remains unclear (Peng et al., 2014).

Corresponding Author’s Email: mani.shalini@gmail.com.

4 Shalini Mani and Aarushi Singh
Oxygen in concentrations greater than that of normal air can become toxic to aerobic
organisms. Toxic properties of oxygen remained unclear till the free radical theory of oxygen
toxicity made it through and was published in 1954 by Gershman, stating that the intracellular
reduction of oxygen results in the formation of highly reactive chemical species, known as the
free radicals (Gershman et al., 1954; Wickens, 2001). In the same year, Commoner observed
electron paramagnetic resonance (EPR) signals in lyophilized biological materials which were
attributed to the free radicals (Commoner et al., 1954). This idea of free radicals related to aging
is always credited to Denham Herman, who in 1956 observed that these free radicals reduced
the lifespan of living beings and produced changes that resembled aging (Wickens, 2001).
Most of the physiological functions depend on the equilibrium between reactive oxygen
species and antioxidant defense systems (Catoni et al., 2008; Halliwell and Gutteridge, 2006).
There are trade-offs involved in the metabolism of the human body. Normal metabolism leads
to the production of oxidant by-products (reactive species) which are crucial for energy supply,
detoxification and chemical signaling (Halliwell and Gutteridge, 1985). But an excessive
exposure to these reactive species can cause damage to the cells and become a serious cause of
extensive impair to the deoxyribonucleic acid (DNA), lipids, and proteins (Ames et al., 1993).
Elevated levels of oxidative stress have a negative result on life history traits such as immunity
and growth (Catoni et al., 2008). It is a leading factor in premature aging followed by age-
related disorders and degenerative diseases such as brain dysfunctions that continues to
deteriorate over time (Ames et al., 1993; Finkel and Holbrook, 2000; Halliwell and Gutteridge,
2006).
Therefore, there is a substantiating need to widen our perceptions regarding the
mechanisms associated with oxidative stress, antioxidants, and aging-related issues. This
chapter emphasizes all of such mechanisms, the biological hypothesis of aging, ROS, function
of antioxidants in reducing the aging process and the progress so far in knowing the age-related
disorders.
2. AGING THEORIES
In order to diminish the incidence of diseases related to aging, it is necessary to understand
the molecular mechanism associated with aging. Ample studies are being conducted to
recognize the underlying relationship between diet and health. Some of the biological theories
related to aging are listed below.
2.1. Free Radical Theory of Aging
Harman first propounded the free radical theory of aging (FRTA) affirming that free
radicals cause oxidative damages to the tissues (Harman, 1956). The free radicals have free
electrons that destructively react with the healthy molecules in body (Guttridge and Halliwell,
2000; Peng et al., 2014). These free radicals are produced in large amounts from exposure to
oxygen and radiation and even from the environmental toxins like pesticides and herbicides
(Sroka and Madeja, 2009). Reactive oxygen species (ROS) are the most copious free radicals
present in cells. These ROS species have a major role in cell differentiation, multiplication and

Aging, Oxidative Stress and Antioxidants 5
cell defense response (Douglas et al., 2003). ROS species cause lipid oxidation in cells, as the
cell membranes consist of polyunsaturated fatty acids that has multiple double bonds with
active hydrogen molecules and thus making them more vulnerable to free radical attack
specially hydroxyl radicals which further leads to increased permeability of cell membrane and
cellular dysfunction (Douglas et al., 2003; Krystonet al., 2011; Liu et al., 2002). ROS also lead
to DNA damage and oxidation of proteins. Two antioxidant systems work on scavenging these
radicals, namely enzymatic and non-enzymatic ones. The antioxidant system comprises of
some enzymes such as catalase (CAT), glutathione reductase (GR), superoxide dismutase
(SOD) and glutathione peroxidase (GPx). This is the foremost defense system opposing the
ROS species and is illustrated in Figure 1 (Matés and Sánchez-Jiménez, 1999). ROS species
and other mechanisms will be discussed in detail further in the chapter.
Figure 1. Scavenging of free radicals by the enzymatic antioxidant defense system. ROS, Reactive
oxygen species; GSH, Glutathione; SOD, Superoxide dismutase; CAT, Catalase; GPx, Glutathione
peroxidase; GR, Glutathione reductase; GSSG, Oxidised glutathione.
2.2. Mitochondrial Decline Theory of Aging
Mitochondrial respiration capacity decreases with aging. Cytochrome c oxidase (CcO), the
oxidoreductase present at the extremes of mitochondrial electron transport chain (ETC)
persistently dwindle in the aged invertebrates and vertebrates (Beckman and Ames, 1998;
Harman, 1972). It has been observed that deficiency of CcO decreases the overall activity of
electron transport chain, due to the enhanced assembly of superoxide anion radicals or
hydrogen peroxide. Consequently, mitochondrial decline theory of aging (MDTA) and FRTA
are supposed to be linked together (Ren et al., 2010). Intake of antioxidants will not only reduce
the formation of free radicals but will also protect the mitochondria from functionally declining
(Peng et al., 2014; Ren et al., 2010).

2.3. Decline Theory of Ubiquitin Proteasome System
Inappropriate folding of proteins and their aggregations are the genesis of aging processes
and many times it leads to degenerative diseases like Parkinson’s and Alzheimer’s (Lee et al.,
2009). They can be cleared mainly by ubiquitin-proteasome system (UPS). Lower activity of
26S proteasome is reported to be analogous to the aging aspects (Savitt et al., 2006). The 26S
proteasome is made up of a core of 20S and caps which are of 19S on either side. The 20S core
itself can’t degrade the multi-ubiquitinated proteins as the catalytic chamber consists of pores
to the catalytic chamber and they remain closed (Thomas and Beal, 2007; Verma and
McDonald, 2001). 19S caps help in penetrating into these pores. Rpn11 is one of the main
components of the 19S. Reduce in the activity of Rpn11 can lead to accumulation of
ubiquitinated proteins with age (Tonoki et al., 2009). This knocking down of Rpn11 reduces
lifespan and if there is an overexpression of this then extended lifespan can be expected. Thus,
for longevity the maintenance of this 26S proteasome is essential and this can be achieved by
the intake of antioxidants (Thrower et al., 2000; Tonoki et al., 2009).
2.4. Genetic Theory of Aging
Single gene mutations can be used to study aging mechanisms at the molecular level
(Murakami and Johnson, 1996; Hekimi et al., 2001). Many of the studies based on
Caenorhabditis elegans and Drosophila led to the discovery of such mutations which extended
their lifespan (Cooley et al., 1988; Spradling et al., 1995). Some of the longevity determined
genes found in Drosophila are mentioned in Table 1.
Table 1. Longevity determined genes recognized in fruit flies
Longevity Molecular Mechanism References

Determined
Genes
Indy Longevity with the insertion of P-element at Indy Rogina and Helfand,
2013
Sirtuins Enhanced lifespan by upregulation of genes of Merksamer et al., 2013
sirtuins
Mth 35% expansion in lifespan by a P-element insertion Lin et al., 1998
at Mth
Chico Heterozygous for chico shows an increase in median Clancy et al., 2001
lifespan
dTOR Hindrance of the target of rapamycin (TOR) pathway Kapahi et al., 2004
leads to 24–26% lifespan extension
P element insertion mutations are found to increase the lifespan of Drosophila by 35%
(Cooley et al., 1988). Other than this Mth mutant flies are found to be resistant to high
temperatures and stresses. Mth proteins belong to a class of G protein-coupled receptors
(GPCRs) (Kikkou et al., 2007; Lin et al., 1998). It was demonstrated that flies expressing Mth
antagonist peptide live significantly longer (Harmar, 2001). APG1 gene in humans is
homologous with Mth and that makes it a gene to be focused upon for development of anti-

aging drugs (Rogina and Helfand, 2013; Ja et al., 2007). Many more evidences have shown that
a fat body is present in Drosophila that acts as a nutrient sensor and uses TOR signaling to
generate a humoral signal for modulating insulin signaling and this modulation activity is
considered to increase the lifespan (Clancy et al., 2001). Many other genes are also involved in
this process of aging (Kapahi et al., 2004).
3. REACTIVE OXYGEN SPECIES AND OXIDATIVE STRESS

Normal cellular metabolism leads to the fabrication of ROS, as well as reactive nitrogen
species (RNS) (Valko et al., 2006). At lower concentrations, ROS plays its role in defense
against infections and also in the induction of mitogenic responses. When ROS reaches to such
concentrations, it starts causing probable biological damages entitled as the oxidative stress
(Kovacic and Jacintho, 2001; Ridnour et al., 2005; Valko et al., 2001). Overproduction of ROS
leads to the incidence of oxidative stress and simultaneously a reduction in antioxidant
formation also occurs. The flowchart in Figure 2, depicts the detailed pathway of ROS
formation and also lists the part of GSH and the antioxidants in directing the oxidative stress.
3.1. Endogenous Reactive Oxygen Species Production
Endogenous production of ROS is more extensive as it is a part of the overall lifespan of a

cell. Among these endogenous sources of ROS, mitochondria, endoplasmic reticulum and
peroxisomes are the main sources (Mani, 2015).
3.1.1. Reactive Oxygen Species Production in Mitochondria

Several major oxygen derivatives are produced with the formation of ROS, where
superoxide and hydrogen peroxide (H2O2) is produced in substantial quantities. The
mitochondria consist of a multicomponent system which is ETC and it involves a series of
oxidation-reduction reactions (Lodish et al., 2000). Transfer of electrons and protons in NADH,
FAD and also in cytochromes occur all through these oxidation-reduction reactions.
Cytochrome oxidase, a terminal enzyme in ETC uses O2 and never releases oxygen radicals
freely. Nevertheless, while the electron transport is going on in earlier sections of ETC, a few
electrons do leak out directly on to O2, resulting in the origination of •O2– (Lodish et al., 2000;
Nohl and Hegner, 1978; Turrens and Boveris, 1980). Complex I and complex III are considered
to be the major sites of ROS formation and they are the respiratory complexes involved in ETC.
3.1.2. Reactive Oxygen Species Production in Endoplasmic Reticulum

Endoplasmic reticulum (ER) produces ROS predominantly by two mechanisms and that
too at stressful conditions (Bhandary et al., 2003). Firstly, when the transfer of electrons from
protein thiol to molecular oxygen takes place then ROS are created as a by-product. This
transfer occurs by endoplasmic reticulum oxidoreductin-1 (ERO-1) and protein disulfide-
isomerase (PDI) (Bhandary et al., 2003). Alternatively, depletion of GSH leads to protein
misfolding and then occurrence of ROS (Santos et al., 2009; Tu et al., 2002), soon after GSH
gets devoured, thiols are overhauled enabling them to interact with ERO-1/PDI and their re-

oxidation takes place (Bhandary et al., 2003). These lead to the initiation of consecutive cycles
of disulfide bond formation and breakage, with formation of ROS by-products in every cycle
(Hega and Chevet, 2012). The build-up of unfolded proteins in the ER prompts Ca2+ leakage
into the cytosol, thus enhancing ROS generation in the mitochondria (Malhotra and Kaufman,
2007).
Figure 2. Detailed pathway of ROS formation, role of GSH and antioxidants managing oxidative stress.
ROS, Reactive oxygen species; ETC, Electron transport chain.
3.1.3. Reactive Oxygen Species Production in Peroxisomes

Peroxisomes participate in fatty acid oxidation and contain peroxide-producing enzymes.
Peroxisomes are the prime origin of total cellular H2O2 production (Angermüller et al., 1987).
Peroxisomes consist of several enzymes that generate H2O2 as part of their normal catalytic
cycle. These enzymes, which are essentially flavoproteins, include acyl-CoA oxidases, urate
oxidase, D-amino acid oxidase, D-aspartate oxidase, L-pipecolic acid oxidase, L-α-hydroxy
acid oxidase, polyamine oxidase, and xanthine oxidase (Fransen et al., 2012). By using all these
different enzymes different types of ROS such as hydrogen peroxide, superoxide, nitric oxide
radicals, hydroxyl radical, and peroxynitrites are produced. Peroxisomes produce H2O2, under
various physiologic constraints (Valko et al., 2004). These are the vital areas of oxygen
utilization in the cell and are the ones engaged in several metabolic outcomes in the body

requiring oxygen. Variety of molecules requires H2O2 for oxidation which they get from these
peroxisomes (Valko et al., 2007). The organelle also contains catalase which does not allow
the accumulation of hydrogen peroxide which can be toxic in higher concentrations. Thus, the
peroxisome perpetuates exquisite stability in the production of ROS (Valko et al., 2004). This
maintained stability is still unclear. Destruction of peroxisomes leads to down-regulation of
their H2O2 consuming enzymes which further leads to release of H2O2 into the cytosol and these
toxic surroundings notably contribute to oxidative stress (Valko et al., 2004; Valko et al., 2007).
3.2. Oxidative Damage to DNA, Lipids and Proteins
Higher concentrations of hydroxyl radicals are a damaging factor for the body. Destruction
of the purine and pyrimidine bases and the deoxyribose backbone occurs when these hydroxyl
radicals react with the DNA molecule (Halliwell and Gutteridge, 1999). Such damages cause
perpetual moderation of genetic material resulting in carcinogenesis, mutagenesis, and aging
(Siems et al., 1995; Valko et al., 2006). Re-arrangement of peroxyl radicals (ROO•) occurs
according to some cyclisation reactions at their formation and they get converted to
endoperoxides which are the forerunners of malondialdehyde (Fedtke et al., 1990; Fink et al.,
1997; Mao et al., 1999; Marnett, 1999; Wang et al., 1996). Lipid peroxidation also gives an
extensive aldehyde product which is 4-hydroxy-2-nonenal (HNE). All amino acid residues of
proteins peculiarly cysteine and methionine have side chains making them more vulnerable to
oxidation by ROS/RNS (Stadtman, 2004). Oxidation of these residues leads to the reversible
formation of mixed disulfides between protein thiol groups (–SH) and low molecular weight
thiols, like GSH (S-glutathionylation). Protein oxidation mediated by ROS can be estimated by
the concentration of carbonyl groups contemplating all the mechanisms involved in their
production (Dalle-Donne et al., 2003; Dalle-Donne et al., 2005). Aging is a cumulative effect
of oxidative stress on cells and tissues. Several things are evident and supportive of this
hypothesis:
 The disparity in species lifespan is associated with metabolic rate and defensive
antioxidant activity;
 Increase in longevity can be seen by the enhanced expression of antioxidative
enzymes;
 Cellular levels of free radical destruction get elevated with age; and
 Diminished intake of calorie and proper diet leads to a dwindling in the fabrication of
reactive oxygen species and growth in lifespan (Wickens, 2001).
Effect of ROS onto age-related diseases and complications is understood through this
concept of oxidative stress as elaborated by Sies (Sies, 1986). The balance between ROS and
the antioxidant defense gets shifted in favor of pro-oxidants resulting in oxidative stress with
aging. This disbalance is proposed to link in the etiology of various human diseases and age-
related disorders. The free radical activity also oxidizes and cross-links proteins and even
enzymes and this damage is supposed to be increased in the body as a function of age
(Stadtman, 1995). Some of the ROS species are listed in Table 2.

Table 2. Features of reactive oxygen species
ROS Symbol Half-Life Remarks References

(second)
Superoxide O2• - 10-6 Generated in mitochondria Halliwell and
Guttridge, 1999
Peroxyl ROO• 1 Reactive and formed from lipids, Harman, 1972
Radical proteins, DNA, sugars etc.during
oxidative damage
Hydroxyl •OH 10-9 Highly reactive, Generated during Halliwell and
Radical iron overload in the body Guttridge, 1999
Hydrogen H2O2 Stable Numerous reactions in body yield this Halliwell and
Peroxide especially ones linked to peroxisomes Guttridge, 1999
Nitric NO• 1 Neurotransmitter and blood pressure Harman, 1972
Oxide regulator
The repair of oxidatively impaired nucleic acids is done by specific enzymes. Oxidized
proteins are eliminated by proteolytic systems, and oxidized lipids are restored by
phospholipases, peroxidases, and acyltransferases (Cheeseman and Slater, 1993). Failures of
these repair systems are considered to contribute more to aging then the disbalance between
antioxidants and free radicals (Gems and Doonan, 2009; Jang and Remmen, 2009; Pérezet al.,
2009).
4. MITOCHONDRIAL BIOGENESIS IN AGING

Everyday functioning of cells essentially requires mitochondrial biogenesis. Mitochondria
are the target and originators of radicals in cells, therefore, are essential organelles to
understand aging (Vina et al., 2009). Mitochondrial synthesis is triggered by the PGC-1α–
NRF1–TFAM pathway. First the PGC-1α spark the mitochondrial biogenesis. NRF1, a
transcription factor is an intermediate stimulator in this biogenesis and cause the synthesis of
the final effector called mitochondrial transcription factor A (TFAM), which activates the
duplication of mitochondrial DNA molecule. This pathway is impeded in aging (Vina et al.,
2009).
4.1. Mitochondriogenic Pathway
The regular removal of mitochondria persistently takes place from the body as these
mitochondria play a vital role in the functioning of the cell. Thus, the mechanisms associated
with the production of mitochondria should be essentially understood. PGC-1α is the chief
controller of the antioxidant defense system and is a co-activator of PPARγ (Puigserver et al.,
1998). PGC-1α has the capacity to administer the responses to oxidative stress at the cellular
level (St-Pierre, 2006). Similarly, it initiates the formation of nuclear respiratory factors such
as NRF-1 and NRF-2 which take part in mitochondrial genesis. The expression of TFAM is
dominantly stimulated by NRF-1 and NRF-2 and this TFAM is the chief stimulator in

duplication of mitochondrial DNA (Puigserver et al., 1998). When mitochondrial genesis is

required PGC-1α gets activated which further activates NRF-1 and TFAM. Enhanced copies of
DNA in cell thus lead to duplication of mitochondria (Puigserver et al., 1998; Vina et al., 2009).
This pathway is depicted in Figure 3.
Figure 3. Mitochondriogenic pathway. TFAM, Mitochondrial transcriptional factor A; mtDNA,

Mitochondrial DNA; PGC-1α, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha;
CREB, cAMP response element binding protein; NRF, Nuclear respiratory factor; TF, Transcription
factor; FKHR, Forkhead protein.
4.2. Impaired Mitochondriogenesis in Aging
Miquel's mitochondrial free radical theory of aging was based on the studies which
depicted reduced production of NRF-1 and decreased expression of PGC-1α in the aged
animals (Gadaleta et al., 1998; Puigserver et al., 1998). Figure 4 shows age-related
mitochondrial dysfunctions.
5. ANTIOXIDANTS
Antioxidants neutralize the effect of toxic oxygen and free radicals. There are generally
three categories of antioxidants (Salavkar et al., 2011):
1. Antioxidant enzymes – These enzymes catalyze the reactions required in the

transformation of free radicals leading to the production of oxygen and water. Catalase,
glutathione peroxidase, and superoxide dismutase are some of the enzymes involved
in this antioxidant activity (Shindo et al., 1993; Stone and Smith, 2004).
2. Chain breaking – Such antioxidants avert the propagation of oxidative chain reactions
by aborting the free radicals. These include tocopherols, retinol, flavonoids, ascorbic
acid (Shindo et al., 1993; Stone and Smith, 2004).

3. Preventive – Metal binding proteins like these seize the free iron or copper hindering
the free radicals from producing other hydroxyl radicals. These include ferritin,
transferrin, lactoferrin (Shindo et al., 1993; Stone and Smith, 2004).
Exposure to free radicals has led organisms to evolve several defense mechanisms against
oxidative stress (Cadenas, 1997). These include preventative mechanisms, restorative
mechanisms, physical defenses, and antioxidant defenses.
Figure 4. Mitochondrial dysfunctions related to aging. mtDNA, Mitochondrial DNA; ROS, Reactive
oxygen species; ETC, Electron transport chain.
5.1. Endogenous Antioxidants
Glutathione is abundantly produced in the cytosol of the cell, nuclei, and mitochondria. It
is a soluble antioxidant in the cell (Masella et al., 2005). In the nucleus, GSH takes care of DNA
repair by maintaining the redox state of vital protein sulfhydryls. Accumulation of oxidized
glutathione (GSSG) inside the cells predicts oxidative stress and the ratio of GSH/GSSG
becomes a good measure of oxidative stress in any organism (Jones et al., 2000; Nogueira, et
al., 2004). High concentrations of GSSG have a destructive effect on many enzymes.
Glutathione demonstrates several effects against oxidative stress (Masella et al., 2005):
 Releases assorted detoxifying enzymes in opposition to the oxidative stress like

glutathione peroxidase (GPx), glutathione transferase which requires glutathione as a
cofactor;
 GSH engages in amino acid delivery through the plasma membrane;

 Glutathione peroxidase has a catalytic action on the scavenging activity of GSH in

which it rummages onto the hydroxyl radical and detoxifies H2O2;
 Regeneration of most of the antioxidants and vitamins like vitamin C and E (Pastore
et al., 2003).
Further antioxidants like SOD, carotenoids, vitamin C and E also are effective in
deteriorating the oxidative stress (Burton and Ingold, 1984; Cameron and Pauling, 1976; Miller
et al., 2005; Schrauzer, 2006).
5.2. Dietary Antioxidants
Secondary metabolites produced by plants to safeguard themselves against oxidative stress

are the dietary antioxidants. These come under four different classes: vitamin C; vitamin E;
carotenoids and polyphenolic antioxidants (Halliwell and Gutteridge, 2006). This grouping is
based on their chemistry. There are two broad classes of antioxidants which include the
lipophilic antioxidants present in the cell membrane and the hydrophilic antioxidants present
in the cytoplasm (Suraj et al., 2000). Vitamin E and other carotenoids lie in the first category
leaving vitamin C and polyphenols in the other. Liver, adipose tissue and skin have higher
concentrations of vitamin E and carotenoids. Vitamin C and polyphenolic antioxidants are more
concentrated in the lungs, brain, and testes (Kojo, 2004; Suraj, 2002; Talavera et al., 2005).
5.2.1. Mechanism of Action

Dietary antioxidants scavenge the free radicals by three mechanisms:
1. Hydrogen ion is donated by the tocopherols and polyphenols;

2. Quenching of oxygen singlets is done by the carotenoids like a- and b- carotenes;
3. Transferring of electrons done by ascorbate (Halliwell and Gutteridge, 2006; Vertuani
et al., 2004).
The end results of all these reactions are the neutralization of the free radicals. The
productivity of antioxidants is called antioxidants potency. After reducing the free radicals the
antioxidants get oxidized and these are then catabolized and excreted (Packer et al., 1979;
Vertuani et al., 2004).
5.3. Levels of Antioxidant Action
Neutralization of free radicals takes place at different stages. They act at the levels of
precaution, interception, and restoration. Antioxidants which are preventive such as SOD and
catalase venture to hinder the formation of ROS. Dismutation of superoxide to H2O2 is
catalyzed by SOD and it is broken down to water by catalase (Cadenas and Packer, 1996; Sies,
1996). Radical scavenging leads to the interception of free radicals. Various antioxidants act as
effectors. Repair enzymes are involved in repair and reconstitution level (Cadenas and Packer,
1996; Halliwell and Aruoma, 1993; Sies, 1996).

6. ANTIAGING NUTRACEUTICALS AND FUNCTIONAL FOODS

According to FRTA, any substance with great antioxidant properties can be used to delay
the aging process. Thus, most of the nutraceuticals, herbal plants and functional foods are an
eminent supplier of antioxidants.
Black tea extracts have catechins. Green tea and black tea intake cause a notable elevation
in antioxidant potential of the plasma (Leenen et al., 2000). Catechins from tea also deduct
DNA oxidation and lipid peroxidation. Tea catechins showed longevity in the mean lifespan of
Drosophila by 10-16% depicting their anti-aging properties (Meng et al., 2001; Rietveld, 2003).
Endogenous antioxidant enzymes like SOD also get greatly expressed due to green catechins
(Li et al., 2007; Li et al, 2008; Peng et al., 2009).
Apple consists of numerous amounts of phytochemicals, specifically polyphenols which
have higher antioxidant properties which include chlorogenic acid, phloretin and
proanthocyanidin B2. However, the antiaging activity of apple and the repressed mechanisms
are still evasive (Feskanich et al., 2000; Marchand et al., 2000). Black rice anthocyanins have
several health benefits such as it is an anti-cancerous, anti-inflammatory, anti-diabetic and anti-
inflammatory disease (Peng et al., 2009). Many such nutraceuticals with antiaging properties
are mentioned in Table 3 with their mechanisms of action.
Table 3. Anti-aging properties of nutraceuticals/functional foods and their mechanisms
Nutraceuticals/ Molecular Mechanism References

Functional Foods
Apple polyphenols Upregulation of SODs, catalase, and Rpn11 genes Peng at al., 2011
of peroxisomes. Downregulation
of MTH gene
Green tea catechin CuZnSOD, MnSOD, and catalase genes get Li et al., 2007; Li
upregulated et al., 2008
Black tea theaflavins Enhanced CAT activitySOD1 and catalase genes Peng et al., 2009
upregulated
Black rice anthocyanin Upregulation SOD1, SOD2, CAT, and Rpn11 Peng et al., 2012
Downregulation of MTH gene
Curcumin Reduced age-related genes expression, including Lee et al., 2000
TOR, InR, Hep, sun, and mth
Soyabean isoflavones Upregulation of endogenous antioxidants SOD1, Borrás et al., 2006
SOD2 and CAT at mRNA and protein level
7. ANTIOXIDANTS: A CURE FOR AGE-RELATED DISEASES

Natural compounds obtained from dietary sources are a great source of antioxidants. These
include the tea catechins discussed above and many other medicinal plants also exist.
Consumption of tea not only has anti-aging properties but also can reduce the risks of aging-
related diseases like cardiovascular diseases. Moreover, it can reduce the risk of cancer as well
as kidney stones.
Indian medicinal plants also provide antioxidants. Aeglemarmelos(Bengal quince), Allium
cepa(Onion), Allium sativum (Garlic), Aloe vera (Ghritkumari), Amomumsu bulatum (Greater

cardamom), Asparagus racemosus (Shatavari), Azadirachtaindica (Neem), Bacopamonnieri

(Brahmi), Camellia sinensis(Green tea), Cinnamomumverum (Cinnamon), Cinnamomum
tamala (Tejpat), Curcuma longa (Turmeric), Emblica officinalis (Amlaki), Glycyrrhizaglabra
(Yashtimadhu), Momordica charantia (Bitter gourd), Nigella sativa (Black cumin), Ocimum
sanctum (Tulsi) are some of the Indian medicinal plants which are a great source of
antioxidants. A great number of many ayurvedic formulations of these medicinal plants also
exist which showed pronounced antioxidant properties (Devasagayam et al., 2004; Tilak et al.,
2001). Indian medicinal plants with higher levels of essential vitamins and nutrients (eg.
vitamin E, lycopene, vitamin C, bioflavonoids etc.) contribute to a rich source of antioxidants
(Devasagayam et al., 2004). Such sources of antioxidants are not only providing anti-aging
benefits but also a preventive measure to various aging-related problems like
neurodegenerative diseases, diabetes etc.
Apart from these natural medicinal plants, dietary supplements are also a source of
antioxidants. These supplements include vitamin A (retinoids, carotenes), vitamins C and E
(tocopherols), ubiquinone, glutathione, polyphenols (flavonoids), resveratrol, and N
acetylcysteine (Glaser, 1999; Raskin et al., 2002). The use of multivitamin/mineral
supplements has raised in the past decades. The equilibrium between ROS and antioxidants is
more important as the extent of oxidative stress and “antioxidative stress” both are menacing
for the health (Dundar and Aslan, 2000). Therefore, it is necessary to know about the individual
oxidative stresses in order to provide proper subscriptions of supplementary antioxidants
(Poljsak and Milisav, 2000).
Aging-associated diseases such as neurodegenerative diseases are caused by the alterations
in antioxidant enzymes such as catalase, Mn-SOD. Increased stress and protein oxidation take
place in patients suffering from these diseases. Antioxidant supply balances this homeostatic
imbalance. Natural compounds are more effective for this antioxidant supply as antioxidative
stress issues hardly occur through them (Yoshikawa et al., 2000).
8. RELATIONSHIP BETWEEN ANTIOXIDANT

PROTECTION AND LIFESPAN
“There is good scientific evidence that eating a diet with lots of vegetables and fruits is
healthful and lowers risks of certain diseases,” said lead author Annlia Paganini-Hill of the
Clinic for Aging Research and Education at the University of California, Irvine (Doyle, 2015).
Longer-lived species were supposed to have more active levels of antioxidant enzymes and
defense systems. Tolmasoff was the first one to study this activity in 14 types of mammals. He
measured superoxide dismutase (SOD) activity in brain, liver, and heart and he found a
significant relationship between SOD activity and lifespan (Tolmasoff et al., 1980). Longer-
lived species were found to have a slow rate of metabolism and high SOD protection. However,
few studies have also shown no correlation (Sohal et al., 1992) amongst aging and SOD
protection. It is possible that this variation in species in SOD could have arisen epigenetically
all throughout the development or impacts on gene regulation (Sohal et al., 1990). Despite all
of this it is possible to identify the lifespan of flies by measuring the antioxidant activity and
free radical damage at the mid-point of their life. The rate of oxidant generation can be better
correlated to changes in aging than the level of antioxidant defenses (Sohal, 1986).

9. RECENT DEVELOPMENTS, FUTURE OPPORTUNITIES

AND CHALLENGES
Numerous behavioral and neuronal changes occur in aging even when the degenerative
disorders are not present. Various researches state the importance of foods with a high
concentration of dietary antioxidants in reducing neurodegenerative disorders.
Lately, a new class of SOD mimetic drugs is developed which reduce tissue damage by
inflammation and these have the ability to increase antitumor effects. Building up of genetically
engineered plants to achieve plants with elevated levels of antioxidants are some of the new
approaches to provide a richer source of dietary antioxidants (Lachnicht et al., 2002). Some of
these genetically engineered plants include tomatoes with 3 times the lycopene concentration,
orange cauliflower which is a great source of antioxidants (Devasagayam et al., 2004;
Lachnicht et al., 2002).
After several years of research on oxidants and antioxidants, it is time to focus on the fields
to improve and increase the antioxidant contents of fruits and vegetables. Presently, it is
strenuous to assess the oxidative stress of the organism because the dissimilar criteria of
oxidative stress are non-segregated with one another. As there is no ubiquitous “scale” of
oxidative stress, determination of total antioxidants and oxidative stress levels in different body
fluids (urine, saliva, blood, and cytosol) still remains a future challenge (Yu, 1996).
CONCLUSION
Antioxidants have now given a solution for aging and age-related issues. Antioxidants are
hope, if not to stop aging but at least to slow it down and even to prevent neurodegenerative
diseases caused due to aging. Nutraceuticals, functional foods, and medicinal plants are active
oxygen scavengers and studies are being conducted to improve them for better antioxidant
content. Novel drugs with great antioxidant activities are coming into the limelight. For
longevity, antioxidants intake is essential and higher dietary antioxidants will provide a greater
resistance to oxidative stress. ROS materialize to be vital regulatory factors in molecular
pathways associated with tumor development and tumor dissemination, which offer
prospective therapeutic invention points. New technologies such as genetic engineering should
now be the main focus of researchers for new and improved sources of antioxidants.
REFERENCES
Ames, B. N., Shigenaga, M. K., Hagen, T. M. Oxidants, antioxidants, and the degenerative
diseases of aging. Proc. Natl. Acad. Sci. USA. 1993;90:7915-7922.
Angermüller, S., Bruder, G., Völkl, A et al., Localization of xanthine oxidase in crystalline
cores of peroxisomes. A cytochemical and biochemical study. Eur J Cell Biol.
1987;45:137-144.
Beckman, K. B., Ames, B. N. Mitochondrial aging: open questions. Annals of the New York
Academy of Sciences. 1998;85:4118-127.
Bhandary, B., Marahatta, A., Kim, H. R. et al., An involvement of oxidative stress in
endoplasmic reticulum stress and its associated disease. Int J Mol Sci. 2003;14:434-456.

Borrás C., Gambini, J., Gómez-Cabrera, M. C. Genistein, a soy isoflavone, up-regulates

expression of antioxidant genes: involvement of estrogen receptors, ERK1/2, and
NFkappaB. The FASEB Journal. 2006;20:2136-2138.
Burton, G. W., Ingold, K. U. Beta-carotene—An unusual type of lipid antioxidant. Science.
1984;224:569-573.
Cadenas, E, Packer, L. (eds) Hand Book of Antioxidants. Plenum Publishers, New York; 1996.
Cadenas, E. Basic mechanisms of antioxidant activity. Biofactors. 1997;6:391-397.
Cameron, E., Pauling, L. Supplemental ascorbate in supportive treatment of cancer –
prolongation of survival times in terminal human cancer. Part 1. Proc. Natl. Acad. Sci.
USA. 1976;73:3685-3689.
Catoni, C., Peters, A., Schaefer, H. M. Life history trade-offs are influenced by the diversity,
availability and interactions of dietary antioxidants. Animal Behaviour. 2008;76:1107-
1119.
Cheeseman, K. H., Slater, T. F. An introduction to free radical biochemistry. British Medical
Bulletin. 1993;49:481-493.
Clancy, D. J., Gems, D., Harshman, L. G. et al., Extension of lifespan by loss of CHICO, a
Drosophila insulin receptor substrate protein. Science. 2001;292:104-106.
Commoner, B., Townsend, J., Pake, G. E. Free radicals inbiological materials. Nature.
1954;174:689-691.
Cooley, L., Berg C., Spradling, A. Controlling P element insertional mutagenesis. Trends
inGenetics. 1988;4:254-258.
Dalle-Donne, I., Giustarini, D., Colombo, R., Rossi, R., Milzani, A. Protein carbonylation in
human diseases. Trends Mol. Med. 2003;9:169-176.
Dalle-Donne, I., Scaloni, A., Giustarini, D., Cavarra, E., Tell, G., Lungarella, G. Proteins as
biomarkers of oxidative/nitrosative stress in diseases: The contribution of redox
proteomics. Mass Spectrom. Rev. 2005;24:55-99.
Devasagayam, T. P. A., Tilak, J. C., Boloor, K. K., Sane, K. S., Ghaskadbi, S. S., Lele, R. D.
Free Radicals and Antioxidants in Human Health: Current Status and Future Prospects.
The Journal ofthe Association of Physicians of India. 2004;52:794-804.
Douglas, J. G., Bakris, G. L., Epstein, M. Management of high blood pressure in African
Americans: consensus statement of the hypertension in African Americans Working Group
of the International Society on Hypertension in blacks. Archives of InternalMedicine. 2003;
163:525-541.
Doyle, K. Extra antioxidants may make little difference in lifespan. Reuters Health News. 2015.
Dundar, Y., Aslan, R. Antioxidative stress. Eastern Journal of Medicine. 2000; 5:45-47.
Fedtke, N., Boucheron, J. A., Walker, V. E., Swenberg, J. A. Vinyl chloride-induced DNA
adducts. 2. Formation and persistenceof 7-2_-oxoethylguanine and n 2,3-ethenoguanine in
rat-tissue DNA. Carcinogenesis. 1990;11:1287-1292.
Feskanich, D., Ziegler, R. G., Michaud, D. S. Prospective study of fruit and vegetable
consumption and risk of lung cancer among men and women. Journal of the National
Cancer Institute. 2000;92:1812-1823.
Fink, S. P., Reddy, G. R., Marnett, L. J. Mutagenicity in Escherichia coli of the major DNA
adduct derived from the endogenous mutagen malondialdehyde. Proc. Natl. Acad. Sci.
USA. 1997; 94:8652-8657.
Finkel, T., Holbrook, N. J. Oxidants, oxidative stress and the biology of ageing. Nature.
2000;408:239-247.

Fransen, M., Nordgren, M., Wang, B et al., Role of peroxisomes in ROS/RNS- metabolism:
implications for human disease. Biochim Biophys Acta. 2012;1822:1363-1373.
Gadaleta, M. N., Cormio A, Pesce V, Lezza AM, Cantatore P. Aging and mitochondria.
Biochimie. 1998;80:863-870.
Gems, D., Doonan, R. Antioxidant defense and aging in C. elegans: is the oxidative damage
theory of aging wrong. Cell Cycle. 2009;8:1681-1687.
Gerschman, R., Gilbert, D.L., Nye SW, Dwyer P, Fenn WO. Oxygen poisoning and x-
irradiation—A mechanism in common. Science. 1954;119:623-626.
Glaser, V. Billion-dollar market blossoms as botanicals take root. Nature Biotechnology.
1999;17:17-18.
Gutteridge, J. M., Halliwell, B. Free radicals and antioxidants in the year 2000. A historical
look to the future. Annals of the New York Academy of Sciences. 2000;899:136-147.
Halliwell, B., Aruoma OI (eds) DNA and Free Radicals, Boca Raton Press; 1993.
Halliwell, B., Gutteridge, J. M. C. Free Radicals in Biology and Medicine. Oxford: Oxford
University Press; 2006.
Halliwell, B, Gutteridge, J. M. C. Free radicals in biology and medicine. 3rd ed.
OxfordUniversity Press; 1999.
Halliwell, B., Gutteridge, J. M. C. Oxygen radicals and the nervous-system. Trends in
Neurosciences. 1985;8:22-26.
Harman D. Aging: a theory based on free radical and radiation chemistry. Journal of
Gerontology. 1956;11:298-300.
Harman, D. Free radical theory of aging: dietary implications. American Journal of Clinical
Nutrition. 1972;25:839-843.
Harmar, A. J. Family-B G-protein-coupled receptors. Genome Biology. 2001; 2: 1-10.
Heart mitochondria. Biochem J. 1980;191:421-427.
Hekimi, S., Burgess, J., Bussière, F., Meng, Y., Bénard, C. Genetics of lifespan in C. elegans:
molecular diversity, physiological complexity, mechanistic simplicity. Trends in Genetics.
2001;17:712-718.
Higa, A., Chevet, E. Redox signalling loops in the unfolded protein response. Cell Signal.
2012;24:1548-1555.
Ja, W. W., West Jr., AP, Delker, S. L., Bjorkman, P. J., Benzer, S., Roberts, R. W. Extension
of Drosophila melanogaster life span with a GPCR peptide inhibitor. Nature Chemical
Biology. 2007;3:415-419.
Jang, Y. C., Remmen., H. V. The mitochondrial theory of aging: insight from transgenic and
knockout mouse models. Experimental Gerontology. 2009;44:256–260.
Jones, D. P., Carlson, J. L., Mody, V. C., Cai, J. Y., Lynn M. J., Sternberg, P. Redox state of
glutathione in human plasma. Free Radic. Biol. Med. 2000;28:625-635.
Kapahi, P., Zid, B. M., Harper T., Koslover, D., Sapin, V., Benzer, S. Regulation of lifespan in
Drosophila by modulation of genes in the TOR signaling pathway. Current Biology.
2004;14:885-890.
Kikkou, T., Matsumoto, O., Ohkubo, T., Kobayashi, Y., Tsujimoto, G. NMR structure of a
human homologous methuselah gene receptor peptide. Biochemical and Biophysical
Research Communications. 2007; 352:17-20.
Kojo, S. Vitamin C: basic metabolism and its function as an index of oxidative stress. Current
Medicinal Chemistry. 2004;11:1041-1064.

Kovacic, P, Jacintho, J. D. Mechanisms of carcinogenesis: Focus on oxidative stress and

electron transfer. Curr. Med. Chem. 2001;8:773-796.
Kryston, T. B., Georgiev, A. B., Pissis, P., Georgakilas, A. G. Role of oxidative stress and DNA
damage in human carcinogenesis. Mutation Research-Fundamental and Molecular
Mechanisms of Mutagenesis. 2011;711:193-201.
Lachnicht, D., Brevard, P. B., Wagner T. L., DeMars, C. E. Dietary oxygen radical absorbance
capacity as a predictor of bone mineral density. Nutr Res. 2002;22:1389-1399.
Lee, F. K., Wong, A. K., Lee, Y. W., Wan, O. W., Chan, H. Y. E., Chung, K. K. K. The role of
ubiquitin linkages on 𝛼-synuclein induced-toxicity in a Drosophila model of Parkinson’s
Disease. Journal of Neurochemistry. 2009;110:208-219.
Lee, K. S., Lee, B. S., Semnani, S. Curcumin extends lifespan, improves health span, and
modulates the expression of age-associated aging genes in Drosophila melanogaster.
Rejuvenation Research. 2010;13:561-570.
Leenen, R., Roodenburg, A. J. C., Tijburg, L. B. M., Wiseman, S. A. A single dose of tea with
or without milk increases plasma antioxidant activity in humans. European Journal of
Clinical Nutrition. 2000;54:87-92.
Li, Y. M., Chan, H. Y. E., Huang, Y., Chen, Z. Y. Green tea catechins upregulate Superoxide
dismutase and catalase in fruitflies. Molecular Nutrition and Food Research. 2007;51; 546-
554.
Li, Y. M., Chan, H. Y. E., Yao, X. Q., Huang, Y., Chen, Z. Y. Green tea catechins and broccoli
reduce fat-induced mortality in Drosophila melanogaster. Journal of Nutritional
Biochemistry. 2008;19:376-383.
Lin, Y. J., Seroude, L., Benzer, S. Extended life-span and stress resistance in the Drosophila
mutant methuselah. Science. 1998;282:943-946.
Liu, J., Head, E., Gharib, A. M. Memory loss in old rats is associated with brain mitochondrial
decay and RNA/DNA oxidation: partial reversal by feeding acetyl-L-carnitine and /or R-
alpha lipoic acid. Proceedings of the National Academy of Sciences of the United States of
America. 2002; 99:1876-1881.
Lodish, H., Berk, A., Zipursky, S. L. et al., Electron transport and oxidative phosphorylation.
W. H. Freeman, New York, Section 16.2. 4th ed. Molecular cell biology; 2000.
Malhotra, J. D., Kaufman, R. J. Endoplasmic reticulum stress and oxidative stress: a vicious
cycle or a double edged sword? Antioxid Redox Signal. 2007;9:2277-2293.
Mani, S. Production of Reactive Oxygen Species and Its Implication in Human Diseases. In:
Rani V, Yadav UCS, editors. Free Radicals in Human Health and Disease. Springer India;
2015.
Mao, H., Schnetz-Boutaud, N. C., Weisenseel, J. P, Marnett, L. J., Stone, M. P. Duplex DNA
catalyzes the chemical rearrangement of a malondialdehyde deoxyguanosine adduct.
Proc.Natl.Acad. Sci. USA. 1999;96:6615-6620.
Marchand, L. L., Murphy, S. P., Hankin, J. H., Wilkens, L. R., Kolonel, L. N. Intake of
flavonoids and lung cancer. Journal of the National Cancer Institute. 2000;92:154-160.
Marnett, L. J. Lipid peroxidation—DNA damage by malondialdehyde. Mut. Res-Fund. Mol.
Mech Mutagen. 1999;424:83-95.
Masella, R., Di Benedetto, R., Vari, R., Filesi, C., Giovannini, C. Novel mechanisms of natural
antioxidant compounds in biological systems: Involvement of glutathione and glutathione
related enzymes. J. Nutr. Biochem. 2005;16:577-586.

Matés, J. M., Sánchez-Jiménez, F. Antioxidant enzymes and their implications in

pathophysiologic processes. Frontiers in Bioscience. 1999;4:339-345.
Meng, J., Ren, B., Xu, Y., Kamendulis, L. M., Dum, N., Klaunig, J. E. Reduction of oxidative
DNA damage (comet assay) in white blood cells by black tea consumption in smokers and
non-smokers. Toxicological Sciences. 2001;60:411-412.
Merksamer, P. I., Liu, Y., He, W., Hirschey, M. D., Chen, D., Verdin, E. The sirtuins, oxidative
stress and aging: an emerging link. Ageing. 2013;5:144-150.
Miller, E. R., Pastor-Barriuso, R., Dalal, D., Riemersma, R. A., Appel, L. J., Guallar, E. Meta-
analysis: High-dosage Vitamin E supplementation may increase all-cause mortality. Ann.
Intern.Med. 2005;142:37-46.
Murakami, S., Johnson, T. E. A genetic pathway conferring life extension and resistance to UV
stress in Caenorhabditis elegans. Genetics. 1996;143:1207-1218.
Nogueira, C. W., Zeni, G., Rocha, J. B. T. Organo selenium and organo tellurium compounds:
Toxicology and pharmacology. Chem. Rev. 2004;104:6255-6285.
Nohl, H., Hegner, D. Do mitochondria produce oxygen radicals in vivo? Eur J Biochem.
1978;82:563-567.
Pérez, V. I., Bokov, A., Remmen, H. V. et al., Is the oxidative stress theory of aging dead?
Biochimicaet Biophysica Acta. 2009;1790:1005-1014.
Packer, J. E., Slater T. F, Willson R. L. Direct observation of a free-radical interaction between
vitamin-E and vitamin-C. Nature. 1979;278:737-738.
Pastore, A., Federici, G, Bertini, E, Piemonte, F. Analysis of glutathione: Implication in redox
and detoxification. Clin. Chim. Acta. 2003;333:19-39.
Peng C, Chan H. Y., Li Y. M., Huang, Y., Chen Z. Y. Black tea theaflavins extend the lifespan
of fruit flies. Experimental Gerontology. 2009;44:773-783.
Peng C., Chan H. Y.E., Huang Y., Yu H., Chen Z. Apple polyphenols extend the mean lifespan
of Drosophila melanogaster. Journal of Agricultural and Food Chemistry. 2011;59:2097-
2106.
Peng, C., Wang, X., Chen, J., Jiao, R., Wang, L, Li, Y., Zuo Y., Liu Y., Lei L., Ying Ma K,
Huang Y, Chen Z. Biology of Ageing and Role of Dietary Antioxidants. Bio Med Research
International. 2014;2014:1-13.
Peng, C, Zuo, Y., Kwan K. M. Blueberry extract prolongs lifespan of Drosophila melanogaster.
Experimental Gerontology. 2012;47:170-178.
Poljsak, B., Milisav, I. The neglected significance of ‘Antioxidative Stress’. Oxidative
Medicine and Cellular Longevity. 2012;95:1-12.
Puigserver, P., Wu, Z., Park, C. W., Graves R., Wright M., Spiegelman B. M. A cold inducible
coactivator of nuclear receptors linked to adaptive thermogenesis. Cell. 1998;92:829-839.
Raskin, I., Ribnicky, D. M., Komarnytsky, S. Plants and human health in the twenty-first
century. Trends in Biotechnology. 2002;20:522-531.
Ren, J. C., Rebrin, I, Klichko, V., Orr, W. C., Sohal, R. S. Cytochrome c oxidase loses catalytic
activity and structural integrity during the aging process in Drosophila melanogaster.
Biochemical and Biophysical Research Communications. 2010;401:64-68.
Ridnour, L. A., Isenberg, J. S., Espey, M. G., Thomas, D. D., Roberts, D. D., Wink, D. A. Nitric
oxide regulates angiogenesis through a functional switch involving thrombo spondin-1.
Proc. Natl. Acad. Sci. USA. 2005;102:13147-13152.
Rietveld, A., Wiseman, S. Antioxidant effects of tea: evidence from human clinical trials.
Journal of Nutrition. 2003;133:3285S-3292S.

Rogina, B., Helfand, S. L. Indy mutations and Drosophila longevity. Frontiers in Genetics.
2013;4:1-8.
Salavkar, S. M., Tamanekar, R. A., Athawale, R. B. Antioxidants in skin ageing – Future of
dermatology. International Journal of Green Pharmacy. 2011;5:161-168.
Santos, C. X., Tanaka, L. Y., Wosniak, J et al., Mechanisms and implication of reactive oxygen
species generation during the unfolded protein response: roles of endoplasmic reticulum
oxidoreductase, mitochondrial electron transport, and NADPH oxidase. Antioxid Redox
Signal. 2009;11:2409-2427.
Savitt, J. M., Dawson, V. L., Dawson, T. M. Diagnosis and treatment of Parkinson disease:
molecules to medicine. Journal of Clinical Investigation. 2006;116:1744-1754.
Schrauzer, G. N. Interactive effects of selenium and chromium on mammary tumor
development and growth in MMTV infected female mice and their relevance to human
cancer. Biol. Trace Element Res. 2006;109:281-292.
Shindo, Y., Witt, E., Packer, L. Antioxidant defence mechanisms in murine epidermis and
dermis and their response to ultraviolet light. J Invest Dermatol. 1993;100:260-265.
Siems, W. G., Grune, T., Esterbauer H.4-Hydroxynonenal formation during ischemia and
reperfusion of rat small-intestine. Life Sci. 1995;57:785-789.
Sies, H. (ed.) Antioxidants in Disease, Mechanisms and Therapy, Academic Press, New York;
1996.
Sies, H. Biochemistry of Oxidative Stress. Angew Chem Internat Ed Eng. 1986;25:1058-71.
Sohal, R. S., Orr, W. C. Relationship between antioxidants, pro oxidants and the aging process.
Ann. NY Acad. Sci. 1992; 663:74-86.
Sohal, R. S., Sohal, B. H., Brunk, U. T. Relationship between antioxidant defences and
longevity in different mammalian species. Mech. Aging Dev. 1990;53:217-227.
Sohal, R. S. The rate of living theory: a contemporary interpretation. In: Collatz, K.G.,Sohal,
R.S, editors. Insect Aging. Springer, Berlin. 1986;23-44.
Spradling, A. C., Stern, D. M., Kiss, I., Roote, J., Laverty, T., Rubin, G. M. Gene disruptions
using P transposable elements: an integral component of the Drosophila genome project.
Proceedings of the National Academy of Sciences of the United States of America.
1995;92:10824-10830.
Sroka, J., Madeja, Z. Reactive oxygen species in regulation of cell migration. The role of
thioredoxin reductase. Postepy Biochemii. 2009;55:145-152.
Stadtman, E. R. Role of oxidant species in aging. Curr. Med. Chem. 2004;11:1105-1112.
Stadtman, E. R. The status of oxidatively modified proteins as a marker of aging. In: Esser K,
Martin GM, editors. Molecular Aspects of Aging. Wiley, Chichester. 1995;129–144.
Stone, W. L., Smith, M. Therapeutic uses of antioxidant liposomes. Mol Biotech. 2004;27:217-
230.
St-Pierre, J., Drori, S., Uldry, M., Silvaggi, J. M., Rhee, J., Jager, S., Handschin, C., Zheng, K.,
Lin, J., Yang, W., Simon, D. K., Bachoo, R., Spiegelman, B. M. Suppression of reactive
oxygen species and neurodegeneration by the PGC-1 transcriptional coactivators. Cell.
2006;127:397-408.
Surai P. F. Vitamin E. In: Natural Antioxidants in Avian Nutrition and Reproduction. Surai PF
(Ed). Nottingham: Nottingham University Press. 2002;27-128.
Surai, P.F., Royle, N. J., Sparks, N. H. C. Fatty acid, carotenoid and vitamin A composition of
tissues of free living gulls. Comparative Biochemistry and Physiology A.Molecular and
Integrative Physiology. 2000;126:387-396.

Talavera, S., Felgines, C, Texier, O., Besson, C., Gil-Izquierdo, A., Lamaison, J. L., Remesy,
C. Anthocyanins metabolism in rats and their distribution to digestive area, kidney and
brain. Journal of Agricultural and Food Chemistry. 2005;53:3902-3908.
Thomas, B., Beal, M. F. Parkinson’s disease. Human Molecular Genetics. 2007;16:R183-
R194.
Thrower, J. S., Hoffman, L., Rechsteiner, M., Pickart, C. M. Recognition of the polyubiquitin
proteolytic signal. EMBO Journal. 2000;19:94-102.
Tilak, J. C., Devasagayam, T. P. A., Lele, R. D. Antioxidant activities from Indian medicinal
plants: A review - Current status and future prospects. International Conference on Natural
Antioxidants and Free Radicals in Human Health and Radiation Biology (NFHR). July 21-
24, 2001. Mumbai, India. (Abstract book, page no. 5).
Tolmasoff, J. M., Ono, T., Cutler, R. G. Superoxide dismutase: correlation with life span and
specific metabolic rate in primate species. Proc. Natl. Acad. Sci. 1980;77:2777-2781.
Tonoki, A., Kuranaga, E., Tomioka, T. Genetic evidence linking age-dependent attenuation of
the 26S proteasome with the aging process. Molecular and Cellular Biology.
2009;29:1095-1106.
Tu, B. P., Weismann, J. S. The FAD and O (2) dependent reaction cycle of Erol-mediated
oxidative protein folding in the endoplasmic reticulum. Mol Cell. 2002;10:983-994.
Turrens, J. F., Boveris, A. Generation of superoxide anion by the NADH dehydrogenase of
bovine.
Valko, M, Izakovic, M., Mazur, M., Rhodes, C. J., Telser, J. Role of oxygen radicals in DNA
damage and cancer incidence.Mol. Cell. Biochem. 2004;266:37-56.
Valko, M., Morris, H., Mazur, M., Rapta, P., Bilton, R. F. Oxygen free radical generating
mechanisms in the colon: Do the semi quinones of Vitamin K play a role in the aetiology
of colon cancer? Biochim. Biophys. Acta. 2001;1527:161-166.
Valko, M., Rhodes, C. J., Moncol, J., Izakovic, M., Mazur, M. Free radicals, metals and
antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact. 2006;160:1-40.
Valko, M. Leibfritz, D., Moncola, J., Cronin, M. T. D., Mazura, M., Telser, J. Free radicals and
antioxidants in normal physiological functions and human disease. The International
Journal of Biochemistry & Cell Biology. 2007;39:44-84.
Verma, R., McDonald, H., Yates III, J. R., Deshaies, R. J. Selective degradation of
ubiquitinated Sic1 by purified 26Sproteasome yields active S phase cyclin-Cdk. Molecular
Cell. 2001;8:439-448.
Vertuani, S., Angusti, A., Manfredini, S. The antioxidants and pro-antioxidants network: an
overview. Current Pharmaceutical Design. 2004;10:1677-1694.
Vina J., Gomez-Cabrera, M. C., Borras, C., Froio, T., Sanchis-Gomar, F., Martinez-Bello, V.
E., Pallardo, F. V. Mitochondrial biogenesis in exercise and in ageing. Advanced Drug
Delivery Reviews. 2009;11913:1-6.
Wickens, A. P. Ageing and the free radical theory. Respiration Physiology. 2001;128:379-391.
Yoshikawa, T., Toyokuni, S., Yamamoto, Y., Naito, Y. Free Radicals in Chemistry Biology
and Medicine, OICA International, London; 2000.
Yu, B. P. Aging and oxidative stress: modulation by dietary restriction. Free Radical Biology
&Medicine. 1996;21:651- 668.

Editors: Md. Sahab Uddin and A. B. Upaganlawar © 2019 Nova Science Publishers, Inc.
Chapter 2
FREE RADICALS AND ANTIOXIDANTS IN HUMAN

HEALTH AND DISEASES
Meenakshi Kaira, Abhilasha Ahlawat,

Vaibhav Walia and Munish Garg*
Maharshi Dayanand University, Rohtak, India
ABSTRACT
Oxidative stress (OS) is characterized by the increased production of the reactive
oxygen species (ROS), which further results in the oxidative damages to the cell and its
components. ROS produced in the body in the normal conditions due to the leakage of the
electrons from the electron transport chain. ROS plays a key role in the various normal
processes that occur inside the human body, but the uncontrolled production of ROS give
rise to the various pathological conditions. Of all the body parts, the human brain is very
much susceptible to the oxidative damage because it consumes approximately 20 percent
of the oxygen. ROS mediated oxidative damage to the neurons in the brain region is thus
responsible for the neuronal dysfunction in the various neuropsychiatric and
neurodegenerative disorders. Thus the attenuation of the ROS and their downstream
signaling events might exert the beneficial role in these pathologies. In this chapter, the
authors describe the role of oxidative stress in health and diseases and the current use of
antioxidants as therapeutics.
1. INTRODUCTION
OS represents an imbalance between ROS production and the cellular antioxidant defense
system. In stress conditions, ROS level increases and, because of their high reactivity,
participates in a variety of chemical reactions (De Marchi et al., 2013). ROS plays both
beneficial as well as a destructive role in the body (De Marchi et al., 2013). ROS are also
produced under normal conditions in the body (Wei et al., 2001) but the uncontrolled and the
*
Corresponding Author’s Email: mgarg2006@gmail.com.

24 Meenakshi Kaira, Abhilasha Ahlawat, Vaibhav Walia et al.
excessive ROS production is responsible for pathological changes in the body which increase
with age of an individual (Harman, 1972). ROS is considered as the by-product of oxygen
metabolism, environmental factors such as pollutants, heavy metals, UV radiations, ionizing
radiations, and drugs also contribute to increased ROS production (Pizzino et al., 2017).
Mitochondria are the main site of the production of ROS (Inoue et al., 2003). However,
besides mitochondria, ROS in the body can be produced enzymatically by the action of
enzymes such as xanthine oxidase, etc. (Valko et al., 2004). Peroxisomes are the organelles that
are also responsible for degradation of fatty acids and various other molecules and peroxisomal
β-oxidation of fatty acids provides another source of production of oxygen radicals by
generating H2O2 as a by-product (Beckman and Ames, 1998). Peroxisomes possess higher
levels of catalase, so whether H2O2 leakage from peroxisomes contributes significantly to
cytosolic OS under normal circumstances is yet to be known. Phagocytic cells, another key
source of oxidants, protect the central nervous system against invading pathological
microorganisms and remove debris from the damaged cells by oxidative burst of NO, H2O2 and
O2−. Ultimately, cytochrome P450 enzymes are known to be the first defense against natural
toxins from plants.
Further, the oxidation of the various biomolecules such as unsaturated fatty acids,
hemoglobin, and myoglobin also produce ROS (Stamler et al., 1992). ROS play several
beneficial roles for the organism. For example, they are needed to synthesize some cellular
structures and to be used by the host defense system to fight pathogens. The pivotal role of
ROS for the immune system is well exemplified by patients with the granulomatous disease.
These individuals are unable to produce O2•− because of a defective NADPH oxidase system,
so they are prone to multiple and in most of the cases persistent infections (Valko et al., 2007;
Droge, 2002). Probably, the most well-known free radical acting as a signaling molecule is
nitric oxide (NO). It is an important cell-to-cell messenger required for a proper blood flow
modulation, involved in thrombosis, and is crucial for the normal neural activity (Pacher et al.,
2007). The exact mechanism by which ROS damages the body cells is not known completely
but ROS mediated inflammation and vascular changes that might be responsible for the damage
(Gu et al., 2011). Inflammation occurs as a consequence of OS has been found to be responsible
for the pathological changes mediated by ROS (Pashkow, 2011). Central nervous system (CNS)
consumes approximately 20 percent of the oxygen (O2) making itself very much susceptible to
the oxidative damage (OD).
The brain is a vital organ in our body found to consume a large amount of O2 and therefore
is very much prone to OS (Inoue et al., 2003). OS is thus linked with increase ROS level which
plays a lead role in the pathogenesis of various disorders (Seet, 2010; Smith, 2000). Thus, the
attenuation of ROS/OD/OS might be a beneficial treatment in the related disorders. In the
present chapter, the role of OS and antioxidants is described in details in various disorders.
2. MITOCHONDRIA, ELECTRON TRANSPORT

CHAIN AND PRODUCTION OF ROS
Mitochondria are the powerhouse of the cell and implicated in the production of cellular
ATP. Structurally, the outer membrane of mitochondria is porous in nature and having porins,
allow the small and uncharged molecule to pass through it and maintain membrane potential

Free Radicals and Antioxidants in Human Health and Diseases 25
(Bayrhuber et al., 2008). The ionic rearrangement through the transporter, present in the inner
membrane of mitochondria, the membrane potential of about 180 mV developed and used for
the synthesis of ATP. There are three major complexes of mitochondrial electron transport
chain (ETC) named as complex I (NADH dehydrogenase), III (cytochrome c reductase) and IV
(cytochrome c oxidase). In ETC (shown in Figure 1), electron (e-) is transferred from NADH
to quinol by complex I which is utilized to pump four e- to intermembrane space.
Then, complex III pump one e- into the intercellular membrane by transferring e- from
reduced quinol to cytochrome c (cyt c). Finally, cyt c transfers the e- from cyt c to molecular
O2 and converts it into the water by combining each molecule of O2 with four protons. It is
suggested that complex II (succinate dehydrogenase) transfer e- directly to quinol from
succinate but does not contribute to developing potential gradient (Kuhlbrandt, 2015). Complex
V is a rotor ring, having Fo and F1 complex (Watt et al., 2010) involved in the generation of the
ATP from ADP and phosphate (Zhang et al., 2012).
Approximately 1-5% of the molecular O2 gets converted into ROS at physiological
conditions (Wei et al., 2001). ROS production majorly occurs in mitochondria but also may
occur outside mitochondria (Murphy, 2009) (shown in Figure 2 and Figure 3). In mitochondria,
there are two major sites of superoxide radicals (O2-) production which are complex I and
complex III (Selivanov et al., 2011). In normal conditions, complex III produce more ROS as
compared to complex I (Finkel and Holbrook, 2000). It is suggested that impairment of
mitochondrial ETC lead to the production of ROS (Balaban 2005). When free radicals are
produced chemically, firstly they require activation of the molecular O2 (Smith et al., 2007).
Figure 1. Mitochondrial electron transport chain and its components. Cyt-c, cytochrome-c; e-, Electron;
ATP, Adenosine triphosphate; NADH, Nicotinamide adenine dinucleotide; O2, Oxygen.
In detail mechanism inside, the superoxide, highly reactive and produced by the complex
I and II of ETC, cross the inner membrane and get reduced to form H2O2 (Muller et al., 2004).
H2O2 undergo Fenton’s reaction to produce highly reactive hydroxyl radicals (OH-), most
harmful ROS in the presence of ferrous ion (Fe2+) (reduced metal) can cause damage to
mitochondrial macromolecules (Van Houten et al., 2006). Long term exposure to ROS results
in the damage of various cellular macromolecule. Further ROS exposure also inhibits the iron-
sulfur (Fe-S) complex I, III, and IV and TCA cycle of mitochondria (Ghezzi and Zeviani, 2012).
ROS also induce apoptosis and neurodegeneration in various studies (Perez-Pinzon, 2005).
Mitochondrial production of free radical contributes to the age-related damage (Gruber et al.,

2008; Ames et al., 1995), further suggesting the link between mitochondrial dysfunction and
aging (Sohal et al., 1994; Sohal and Sohal, 1991). OS mediated by the ROS thus determines
the cell fate and cell death (Kyriakis and Avruch, 2001).
Figure 2. Mitochondrial electron transport chain components involved in the production of reactive
oxygen species. cyt-c, Cytochrome-c; e-, Electron; ATP, Adenosine triphosphate; NADH,
Nicotinamide adenine dinucleotide; O2, Oxygen; ROS, Reactive oxygen species.
Figure 3. Mitochondrial and extramitochondrial sources of reactive oxygen species. SOD, Superoxide
dismutase; CAT, Catalase; OH−, Hydroxide ion; H2O2, Hydrogen peroxide; O2−, Superoxide anion;
ROS, Reactive oxygen species.

3. OXIDATIVE STRESS AND MENTAL DISORDERS

OS is known to be the primary reason in the pathogenesis of various neurological and
psychiatric diseases (Sorce and Krause, 2009). Accumulation of ROS in brain tissues increase
the susceptibility towards the damage in brain tissues and triggering numerous molecular
cascades further altering the brain morphology, neuroinflammation, neuronal death and shifting
the balance towards the abnormal functionality of brain (Gu et al., 2011). Both ROS and
glucocorticoids receptors have been shown to modulate the activity of each other in the various
previous studies, further suggesting the link between them (Makino et al., 1996). ROS mediated
OS further upregulate the activity of NMDA receptor resulting in the glutamate excitotoxicity
(Nguyen et al., 2011). Figure 4 represents the role of oxidative stress in anxiety and depression.
3.1. Anxiety
Anxiety is described as a defensive mechanism exhibited by an organism in response to

novelty. OS also plays an important role in the pathogenesis of anxiety-related behavior (Salim
et al., 2010a, b). Further OS status of the CNS and emergence of the anxiety has been explained
previously (Berry et al., 2007). Several reports have mentioned that anxiety-related disorders
increases with the increase in age especially older individuals are more prone to anxiety attacks
and a decline in cognitive behavior (Brenes et al., 2008; Palmer et al., 2007).
Glyoxalase (GLO)-1 and glutathione reductase (GSR)-1 are the antioxidant enzymes
involved in anxiety-like behavior and OS pathway. It is suggested that the treatment of the
rodents with the pro-oxidant buthionine sulfoximine (BSO). PDE2 inhibitor and apocynin
antagonize anxiety-like behaviors induced by BSO (Massod et al., 2008). Also, the reduction
in the level of GLO1 and GSR1 increase the anxiety related behavior responsible for anxiety-
like behavior in rodents. Thus GLO1 might be the effective target for the treatment of anxiety
and the related disorders (Hovattay et al., 2005).
3.2. Depression
Depression is a heterogeneous disorder described by the behavioral despair and the

suicidality. It is seen that OS plays a key role in the pathogenesis of depression (Ng et al., 2008).
Several evidence indicate the possible link between the OS and major depression (MDD) (Maes
et al., 2011a, b). Depression is accompanied by the increased levels of ROS and NO (Dhir and
Kulkarni, 2011) also by the reduced levels of the antioxidant defenses, such as reduced
glutathione (GSH)(Gawryluk et al., 2011). Antidepressant treatments reduce the OS and
augmented the antioxidant defenses to exert the neuronal protection (Behr et al., 2012).

Figure 4. Reactive oxygen species and reactive nitrogen species-mediated anxiety and depression.
NMDA, N-methyl-D-aspartate; 5-HT, 5-Hydroxytryptamine; NO, Nitric oxide; GABA, Gamma-
Aminobutyric Acid; O2−, Superoxide anion.
4. OXIDATIVE STRESS AND NEURODEGENERATIVE DISORDERS

Degeneration of neurons in many regions of the brain leads to neurodegenerative disorders.
Neurodegeneration has been thought to occur due to the interaction of various factors such as
environmental, genetic factors and damage to redox metal. ROS production contributes to
neuroinflammation and neurodegeneration (Balaban et al., 2005). The present section describes
the role of ROS in the neuronal degeneration in the AD and the PD pathology.
4.1. Alzheimer’s Disease
Alzheimer’s disease (AD) which is characterized by accumulation of amyloid beta (Aβ)

and neurofibrillary tangles (NFTs) (Christen, 2000) characterized by the accelerate production
of ROS which binds to mitochondrial membranes leads to mitochondrial dysfunctions by
altering energy metabolism resulting in neurotoxicity (Garcia-Matas et al., 2010; Shah et al.,
2009). Membrane lipid peroxidation induced by Aβ causes neuronal dysfunction (Morris et al.,
2005). N-methyl-D-aspartate (NMDA) receptors activation results in the production of ROS in
a person suffering from AD leads to neuronal excitotoxicity and synaptic dysfunction (Rai et
al., 2013). AD resulting in the production of toxic Aβ aggregates which are known to confer

the OS and cytotoxicity. Aβ then causes phosphorylation of tau proteins via redox metals leads
to their release from microtubules and form NFTs (Boom et al., 2009).
Phosphorylated tau proteins affect the activity of complex I of electron transport chain and
contribute mitochondrial dysfunction and ROS production (shown in Figure 5) (Mondragon-
Rodriguez et al., 2013). This could explain why the accumulation of both Aβ and tau may be
required to initiate neurodegeneration in AD patients. In senile and classical plaques, ROS plays
a role in the generation of advanced glycation end products (AGEs) (Munch et al., 2000) via
glycation process.
Figure 5. Amyloid beta-mediated oxidative stress in Alzheimer’s pathology. Aβ, Amyloid beta; ETC,
Electron transport chain; ROS, Reactive oxygen species; NMDA, N-methyl-D-aspartate.
Role of AGEs in association with Aβ has been demonstrated in AD (Smith et al., 1995).
Aβ interacts with biometals such as copper, iron, and zinc (Kozlowski et al., 2012) and forms
ROS. Aβ form complexes with OH ions which further crosslinked with Aβ peptides and form
toxic oligomer (Valko et al., 2005) which suggested that the interaction of heavy metals with
ROS leads to neuronal toxicity (Adlard et al., 2008). Thus, treatment with the metal chelators
reduces the burden of Aβ plaques thus restores the normal physiology in AD patients (Cristovao
et al., 2016).
4.2. Parkinson’s Disease
Parkinson’s disease (PD), which is first observed by James Parkinson in the mid-region of
the brain, is characterized by (Dauer and Przedborski, 2003) dopaminergic neuronal damage

particularly in substantia nigra (SN) region of the midbrain. SN region of the brain having a
large population of dopaminergic neurons and damage of these neurons leads to a deficiency
of dopaminergic neurons (Forno, 1996; Hornykiewicz and Kish, 1987). The actual cause of
neurodegeneration in SN region is still not known but it is suggested that the metabolism of
dopamine results in the production of a large amount of ROS making the SN neurons
susceptible to OS mediated damage (Bender et al., 2006).
Metabolism of the dopamine further produces dopamine quinone, which is capable of
covalently modifying cellular nucleophiles, including low molecular weight sulfhydryls such
as GSH and protein cysteinyl residues. Dopamine quinone also modifies the α-synuclein
monomer to produce cytotoxic protofibril (Conway et al., 2001) responsible for the
dopaminergic neuronal damage SN region of the brain (Figure 6).
Further, dopamine, when comes in contact with α-synuclein, exerts its neurotoxic effect.
α-Synuclein also causes mitochondrial complex I impairment and the accumulation of α-
synuclein into mitochondria leading to energy deficit (Parihar et al., 2008, 2009) mitochondrial
dysfunctioning and neurodegeneration in PD. OS in PD promotes the neurotoxicity by causing
mitochondrial dysfunction (Ved et al., 2005), mitochondrial membrane disarrangement
(Conway et al., 2001) and suppression of lysosomal protein breakdown (Martinez-Vicente et
al., 2008).
5. OXIDATIVE STRESS AND DIABETES MELLITUS

Pancreatic beta cells are more prone to ROS mediated damage because beta cells express
less antioxidant defensive enzymes (Pi et al., 2007). Thus, ROS might play a key role in the
development and the pathogenesis of both the types of diabetes mellitus (DM) (Li et al., 2009)
characterized by endothelial dysfunction. In case of diabetes, endothelium fails to produce a
sufficient amount of nitric oxide (NO) which is accountable for the relaxation of blood vessels
(Ceriello et al., 2008). Elevated ROS levels, results in the reaction of NO with the superoxide
radicals to form peroxynitrite in diabetic individuals (Ceriello et al., 2001) and further results
in the reduced availability of NO to the endothelium responsible for the endothelial dysfunction
and damage (Tannous et al., 1999). The rise in blood glucose level in diabetes is responsible
for the production of the ROS viz. activation of diacylglycerol (DAG)-protein kinase C (PKC)
pathway (Ahmad et al., 2005; Brownlee, 2001) involving an increased formation of the
glycolytic intermediate like dihydroxyacetone phosphate and through the ligation of AGE
receptors (Ahmad et al., 2005).
PKC and nuclear factor kappa light chain enhancer of activated B cells (NF-κB) are the
potent activators of NADPH oxidase (Venugopal et al., 2002) and responsible for the excessive
production of ROS (Brownlee, 2001). The elevated levels of the blood glucose further increase
the activity of the enzyme aldose reductase responsible for the conversion of glucose into
sorbitol which is then utilized by the enzyme sorbitol dehydrogenase accompanied by the
conversion of the NAD+ to NADH (Ahmad et al., 2005). Elevated NADH:NAD+ results in the
activation of enzyme PKC responsible for the production of ROS (Brownlee, 2001).
Resveratrol, a nonflavonoid polyphenolic compound, is also known to mitigate the harmful
effects of DM (Szkudelski and Szkudelska, 2015). The administration of resveratrol have been
reported to promote the pancreatic β cell population (Fiori et al., 2013); increase insulin

secretion (Chang et al., 2014); induce glycogenesis (Su et al., 2006) and increase the uptake of
the glucose via the translocation of GLUT-4 (Penumathsa et al., 2008). Further the
administration of resveratrol has been shown to ameliorate the disturbances in lipid and protein
metabolism (Hussein and El-Maksoud, 2013; Chen et al., 2011); improve glucose tolerance
(Lagouge et al., 2006); downregulate the expression of RAGE (Khazaei et al., 2016); enhance
the expression of antioxidant enzymes (Hamadi et al., 2012) and inhibits the OD in the diabetic
models (Schmatz et al., 2012).
Figure 6. Dopamine-mediated oxidative stress in Parkinson’s disease. DA, Dopamine; E2ub, E2

Ubiquitin; NADPH, Nicotinamide adenine dinucleotide phosphate; GSH, Glutathione; SH, Sulfhydryl;
O2−, Superoxide anion; OS, Oxidative stress; SN, Substantia nigra.
6. OXIDATIVE STRESS AND SKIN DISORDER

Aging and associated degenerative diseases are consequences of imbalance between free
radical and endogenous antioxidant defense system which ultimately damage the cell. Initially,
this theory stated that molecular oxygen is involved in various reactions which further leads to
the generation of free radicals by oxidative enzymes in cells (Harman, 1956). Later the theory
was modified and stated that mitochondria are mainly responsible for the physiological process
of aging (Harman, 1972). Free radicals are highly reactive as they consist of an unpaired e-.
ROS are even more reactive than O2. In aerobic organism, ROS deteriorates biological
macromolecules (such as amino acid, lipids, etc.) as it forms continuously by pairing its
unpaired e- with O2.
ROS leads to functional and structural loss of cells when interacts with normal cells (Rossi
et al., 2008). According to current studies, ROS may be involved in intracellular signaling

pathways (e.g., response to growth factor stimulation or generation of the inflammatory

response to bacterial defense) by acting as physiological regulators or cellular messengers
(Finkel, 2011). This indicates the need for developing an efficient antioxidants defense system
to protect cellular constituents from OD as free radicals are not only harmful but also necessary
for human beings. However, OD will be increased for sure when there is an increase in ROS
generation and a decrease in antioxidants. All other factors such as lipid peroxidation (Schafer
and Buettner, 2000), protein oxidation (Stadtman, 2004) and DNA damage (Kil et al., 2006)
ultimately leads to cell death by modulating normal cells into abnormal ones which are
incompatible for the proper functioning of the cell. This damage leads to the age-related
degenerative diseases and conditions (Goswami et al., 2006). By concluding the information,
it can be stated that there is a need to build an equilibrium between ROS and antioxidants to
protect cell damage or death (Sastre et al., 2000; Kregel and Zhang, 2007).
Skin aging includes two clinically and biologically different and independent processes
i.e., intrinsic and extrinsic skin aging (Tzellos et al., 2009). The former one is also known as
chronological aging as it affects all internal organs and skin in the same way (Makrantonaki
and Zouboulis, 2007). The later one as its name indicates is a result of extrinsic factors which
includes environmental factors such as exposure to UV radiations or sunlight, pollution and
some individual related factors such as smoking, insomnia, imbalanced nutritional diet, etc.
The best way to prevent skin aging in response to extrinsic factors is to adapt a well-organized
lifestyle which includes regular exercise, low caloric intake, balanced nutritional diet, etc. and
avoid stressful conditions. Many studies have been done on skin aging and related diseases.
Most of them concluded that compounds having antioxidants (carotenoids, tocopherols and
flavonoids, as well as vitamins A, C, D and E) as their chemical constituents are found to be
beneficial in protecting against skin aging and can be used for enhancing skin beauty and health
(Ristow and Schmeisser, 2011; Ristow et al., 2009).
Recently many studies have been done to find a proper balance between the increased level
of ROS and its anti-aging effects. ROS production in the response of exercise has been found
to act as an internal signal which further protects the cell and aging. ROS induce the expression
of antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT), etc. when
translocates into nucleus from cytoplasm after getting activated via stimulation of STE-like 20
protein kinase 1 (MST1) and Jun N-terminus kinase (JNK) specific phosphorylations of
forkhead box class O transcription factor (FoxO transcription factors). Increase in nuclear FoxO
level decreases cell proliferation and cell death. The expression and upregulation of the cell’s
own intrinsic antioxidative enzyme systems ultimately fight against increased ROS production
and protect the cells from the harmful effects of ROS (Huang and Tindall, 2007).
7. OXIDATIVE STRESS AND CANCER

In cancer, the exact mechanism of free radicals is still not known but various studies have
shown that some of the antioxidants are related to lowering the incidence of some specific types
of cancer. For example in some of the experiments, vitamin E has not just shown the most
significant reduction in the incidence of prostate cancer but also shown a reduction in breast,
lung, and colon cancers. In a study, supplementation with α-tocopherol has been found to show

preventive measures against the incidence and mortality of prostate cancer in subjects that
include all male smokers (Albanes et al., 1995).
Many studies have given evidence for the beneficial effects of antioxidants
supplementation on the incidence of cancer only in healthy individuals (Hercberg et al., 2006).
However, some studies reveal that high dose of antioxidant supplementation may prove harmful
in an individual that already has started carcinogenesis and also may be ineffective in a healthy
individual with adequate antioxidant level (Hercberg et al. 2006). In colorectal cancer,
supplementation with multivitamin has shown minor lowering risk in the incidence of cancer
(Jacobs et al., 2003). In spite of many favorable studies, no study has shown antioxidants
supplementation effective against gastrointestinal (GI) cancers (Bjelakovic et al. 2004).
8. ANTIOXIDANTS
Antioxidants are the substances which reduce the damaging effects of the ROS produced
in the body and thus confer the protection to the various cellular molecules. Various
antioxidants which are produced inside the body or which are produced inside the body or
which are supplied from the external source provide protection from the damaging effect of the
various physicochemical and environmental factors which predispose the body to the damaging
effect of the ROS (Lobo et al., 2010). Antioxidants work either by reducing the levels of the
ROS by inhibiting their production or by increasing their scavenging or decomposition (Young
and Woodside, 2001).
Antioxidants itself gets oxidized to neutralize the harmful effects of ROS and protects the
cells thus prevents the disease. Antioxidant function either by breaking the chain or steals an
electron. There are many pieces of evidence for the beneficial effects of antioxidants but several
factors hinder the systematic use of supplements including the lack of perspective and
controlled studies, the long-term effects and the dosages necessary for each type of diseases.
Antioxidant may interact with other medication and may show adverse effects thus consulting
professional healthcare is necessary before taking antioxidant as a supplement with any other
drug therapy (Pham-Huyet al., 2008).
8.1. Endogenous Antioxidant System
Excessive formation of ROS cause OD of cellular constituents (Halliwell, 2006). The

antioxidant enzyme system (SOD, CAT, glyoxalase, glutathione reductase, glutathione
peroxidase [GPx]) (Griendling et al., 2000) and the low-molecular-weight antioxidants
(glutathione, uric acid, vitamin C, and melatonin) have a protective mechanism against ROS in
the brain (Kohen et al., 2000). Superoxide dismutases (SODs) act by the spontaneous
dismutation of O2- to generate H2O2, which is further removed by enzymes like glutathione
peroxidase and CAT (Saso and Firuzi, 2014).
8.1.1. Catalase
Catalase reduces H2O2 into H2O and O2. Firstly, catalase heme Fe+3 reduces one molecule
of H2O2 to H2O, generating a covalent oxyferryl (Fe+4=O) species and a porphyrin cation radical

(Compound I). Then, compound I further oxidize another H2O2 forming molecular O2 and H2O.
Catalase over-expression has been found to enhance cognition and reduce the measures of
anxiety (Heck et al., 2010).
8.1.2. Reduced Glutathione

Reduced glutathione (GSH) consists of three amino acids cysteine, glutamic acid, and
glycine and decrease the toxicity of endogenous metabolic products. GSH exists in 2 forms,
oxidized disulfide form (GSSG) and reduced (GSH) state and enzymes like GPx, glutathione-
S-transferase (GST) and γ-glutamyltransferase (GGT) maintain their ratio which indicates
redox status of cells (Allen and Bradley, 2011). Toxicity of xenobiotic and endogenous
compounds is decreased by glutathione and therefore, the low levels of glutathione and/or
transferase activity are associated with various disorders of the CNS. Glutathione directly
scavenges diverse oxidants such as O2-, OH- and NO radicals. Glutathione catalytically
detoxifies hydroperoxides, peroxynitrites and lipid peroxides (Pizzorno, 2014). Since the
glutathione transferase enzyme plays an important role in the detoxification and thus the
deletion of the genes i.e., GSTM1 and GSTT1 represent a risk factor for anxiety and other mood
disorders (Odebrecht Vargas Nunes, 2014).
8.1.3. Glyoxalase System

Glyoxalase system consists glutathione as a cofactor and two enzymes named as
glyoxalase-I and glyoxalase-II which plays a vital role in the detoxification of the aldehydes
such as methylglyoxal (MG) and alpha-oxo-aldehydes which give rise to the AGES (Wu et al.,
2002). Aging is characterized by the reduced activity of enzyme glyoxalase 1 and by the
increased accumulation of glycated products leading to tissue damage (Thornalley, 2003) and
neuronal dysfunction in the body of a living organism (Peculis et al., 2013).
9. ANTIOXIDANT PROCESS
An antioxidant maintains the balance between free radicals and oxidants by oxidizing itself
this is why there is a constant need to restore antioxidant resources as once an antioxidant is
utilized in one system it cannot be used in another system. In some cases, it acts as pro-oxidants
and may produce toxic ROS/reactive nitrogen species (RNS).
The antioxidant process can work either of the two ways i.e., chain- breaking or prevention.
In the former one, a series of radical release and next radical formation occurs until the free
radical is converted into a harmless product or gets stabilized by a chain-breaking antioxidant
such as vitamin C, E, etc. for example, lipid peroxidation. In the later one, oxidation is
prevented by reducing the rate of chain initiation either by scavenging initiating free radicals
or by stabilizing transition metal radicals such as copper and iron via an antioxidant enzyme
like superoxide dismutase, catalase and glutathione peroxidase (Young and Woodside, 2001).

9.1. Antioxidant: Advantages and Inconveniences
Antioxidant supplements can be extracted from natural foods or synthesized chemically

but their composition definitely varies from natural antioxidants present in foods which arose
a question that whether the antioxidant supplements have same beneficial effects as of
antioxidants naturally present in food (Fusco et al., 2007). The systematic use of supplements
is hindered by several factors such as lack of perspective and controlled studies, the long-term
effects and the dosages necessary for each type of diseases, although it has been suggested
through various studies that antioxidants play a vital role in many chronic diseases.
If possible one should consume the antioxidants from their diet rather than through
supplements as it may show drug interaction or side effect as other conventional medicines do.
When antioxidant supplements are consumed at significantly higher levels than the
recommended dietary intakes (RDI), they act as pro-oxidants and may induce OS. However, in
some situations, for example, poor people who are unable to afford to consume balanced diet
on daily purpose, dietary supplements can provide an alternative to natural antioxidants that
can be extracted from diet and may be helpful to maintain good health of an individual. But
yes, one should always consult a healthcare professional before combining a dietary supplement
with conventional medical treatment as it may be proven to be harmful if taken in high doses.
9.2. Antioxidants Used in Clinical Studies
Various antioxidants exert their action by inhibiting the production of ROS or by the ROS
neutralization (shown in Table 1). Antioxidants that have been used in the various clinical
studies include:
9.2.1. α-Lipoic Acid

α-Lipoic acid (ALA) is a natural compound having chemical name 1,2-dithiolane-3-
pentanoic acid synthesized from octanoic acid by the help of enzymes. ALA serves as a cofactor
for α-ketoacid dehydrogenases and plays a vital role in energy metabolism in mitochondria.
Role of ALA has been explored as a potent antioxidant molecule in various disorders such as
diabetes, cardiovascular disease, neuromuscular disorders (Smith et al., 2004; Suh et al., 2004).
LA and dihydrolipoic acid (DHLA) inhibit the heavy metal-induced OD (Suh et al., 2004),
inhibits the development of diabetes (Haugaard and Haugaard, 1970; Singh and Bowman,
1970), increased the glucose uptake by stimulation of GLUT4 translocation to cell (Yaworsky
et al., 2000), mimics the action of insulin and promotes the insulin-stimulated glucose disposal
and evoke beneficial effects in diabetic patients (Coppey et al., 2001).
9.2.2. Curcumin
Curcumin, derived from the rhizome of Curcuma longa (Bhullar et al., 2013). It is
metabolized into tetrahydrocurcumin, an active metabolite by a reductase enzyme which is
present in intestine on oral ingestion (Sadowska-Bartosz and Bartosz, 2014). Curcumin acts as
an antioxidant, antimutagenic, antiprotozoal, anticancer and antibacterial agent (Bhullar et al.,
2013; Brondino et al., 2014). Recently curcumin has been explored as a neuroprotector against
hemin (oxidized heme) which damage the cerebellar neurons. Curcumin also stimulates an

inflammatory and antioxidant response against hemin-induced neuronal death by translocating

nuclear factor related factor-2 (Nrf2) into the nucleus (Gonzalez-Reyes et al., 2013) and exerts
neuroprotection.
9.2.3. Rutin
Rutin is a flavonol (Harborne, 1986) that exhibits various pharmacological activities (Javed
et al., 2012). Rutin treatment has been shown to decrease the oxaliplatin-induced peroxidative
changes and lipid peroxidation (Azevedo et al., 2013) and exerts protection against the
glutamate toxicity and OS (Pu et al., 2007). It has been reported that the administration of rutin
have reduced the blood glucose levels and increased the levels of insulin in a study (Kappel et
al., 2013) and through the increased the translocation of GLUT4 responsible for the increased
glucose uptake and thus exerts beneficial effects in diabetes mellitus (Hsu et al., 2014).
9.2.4. Ferulic Acid

Ferulic acid (FA) is a metabolic product of phenylamine and tyrosine-derived from plants.
FA having electron donating groups, 4-hydroxy and 3-methoxy, on benzene ring which
contribute to the termination of the free radical chain reaction. In addition, FA contains a
carboxylic acid group and this group act as a platform for attacking free radicals. Due to which
free radical don’t attack membrane and prevent lipid peroxidation (Kanaski et al., 2002).
Resonance stabilized phenoxy radical in FA, formed due to the presence of a phenolic nucleus
and an unsaturated side chain, provide antioxidant property to FA.
FA can easily abstract hydrogen atom by colliding with any reactive radical to form
phenoxy radical having an unpaired electron which can be delocalized not only to O2 but to the
entire molecule which makes it highly resonance stabilized. Unsaturated side chain having an
extended conjugation also provide additional stabilization to phenoxy radical. Moreover, an
additional hydroxyl group (by providing more resonance stabilization as well as o-quinone
formation) improve antioxidant and radical scavenging activity (Graf, 2000). Free radical in
brain generates ROS and RNS to induce a cascade of events viz., protein damage,
neurodegeneration, DNA damage and lipid peroxidation (Joshi et al., 2006) and thus the
treatment with FA confers protection in these conditions (Joshi et al., 2006).
9.2.5. Vitamin E
Vitamin E is a very important antioxidant and exists in 4 isoforms such as α tocopherol, β
tocopherol, γ tocopherol and δ tocopherol (Bartlett and Eperjesi, 2008). Vitamin E participates
in various processes including apoptosis of tumoral cells; inhibition of platelet aggregation;
modulation of immune response; gene expression and stabilization of the cellular membrane
(Colombo, 2010; Kataja-Tuomola et al., 2011). Supplementation with vitamin E exerts the
beneficial effect on blood pressure, glucose and antioxidant levels (Rafighi et al., 2013;
Goldenstein et al., 2013), reduced the risk of onset of diabetes and diabetic complications
(Ebesunun and Obajobi, 2012; Illison et al., 2011) maintain neuronal structure and function
(Muller and Goss-Sampson, 1990) and exerts neuroprotective effect (Nelson et al., 1981).
9.2.6. Vitamin C
Vitamin C (ascorbic acid) is a water-soluble vitamin (Wannamethee et al., 2006). It exists
in two isoforms including reduced form and oxidized form. The reduced form of ascorbic acid,

that is present in anion form (Ascorbate) at physiological pH, while the oxidized form is
dehydroascorbic acid (DHA) (Du et al., 2012). Vitamin C plays a vital role in scavenging of
ROS (Calder et al., 2009; Bartlett and Eperjesi, 2008) and protects the body against the OD
mediated by ROS (Calder et al., 2009; Young et al., 1995). It helps in generation of the vitamin
E and glutathione (Calder et al., 2009; Aguirre and May 2008).
Vitamin C also acts as a co-factor in the synthesis of neurotransmitters especially
catecholamines i.e., dopamine and norepinephrine (Figueroa-Mendez et al., 2015) and
modulates the release and action of neurotransmitters for their receptors (Rebec and Pierce,
1994; Serra et al., 2000). Ascorbic acid also modulates the activity of glutamate and GABA
receptors (Kara et al., 2014) and prevent glutamate-mediated excitotoxicity (Sandstrom and
Rebec, 2007; Harrison and May 2009). The greatest limitation is that vitamin C has not been
adequately investigated in clinical trials for aging-related diseases with other molecules rather
than used alone (Salonen et al., 2003).
9.2.7. Naringin
Naringin is a flavanone glycoside and is a strong antioxidant (Renugadevi and Prabu, 2009;
Jung et al., 2004). Naringin acts as a scavenger of ROS (Cavia-Saiz et al., 2010) and inhibits
the enzyme xanthine oxidase whose expression is linked with the production of the O2- (Russo
et al., 2000). Naringin supplementation improved the levels and the action of various
antioxidants (Mamdouh et al., 2004) and prevents ROS mediated OD and lipid peroxidation
(Jung et al., 2004).
9.2.8. Quercetin
Quercetin has been found as the important constituent of the various herbal drugs and it
has been reported that the administration of quercetin results in the reduction in blood glucose
levels as compared to the control (Ahmad et al., 2017). Further, quercetin treatment inhibits the
enzyme aldose reductase and prevent the polyol accumulation (Chaudhry et al., 1983) and
ameliorate retinopathy and neuropathy (Valensi et al., 2005).
9.2.9. Resveratrol
Resveratrol belongs to stilbene class, is a phytoalexin accumulated in the glycosylated form
in many plants, such as peanuts, blueberries, pine nuts and grapes (Harikumar and Aggarwal,
2008). It blocks LDL oxidation, a biological phenomenon associated with the risk of coronary
heart disease and myocardial infarction and decreases cardiovascular risk factor (Khurana et
al., 2013). It also acts as an antioxidant and anti-inflammatory by mediating various molecular
mechanisms (Szkudelski and Szkudelska, 2015). Further, the administration of resveratrol has
been shown to ameliorate the disturbances in lipid and protein metabolism (Hussein and El-
Maksoud, 2013; Chen et al., 2011); improve glucose tolerance (Lagouge et al., 2006);
downregulate the expression of RAGE (Khazaei et al., 2016); enhance the expression of
antioxidant enzymes and inhibits the OD (Schmatz et al., 2012). It also has neuroprotective
action as it suppresses NF-κB activity induced by beta-amyloid in PC12 neuron cell lines, (Jang
and Surh, 2003) suggesting an important role in neurodegenerative disorders, such as PD and
AD pathology (Albani et al., 2009).

9.2.10. Hydroxytyrosol
Hydroxytyrosol is an ortho-diphenol (a catechol), a metal chelator, abundant in olive, fruits
and extra virgin olive oil (Waterman and Lockwood, 2007). It acts as an antioxidant and
scavenges ROS, RNS and peroxyl radicals due to its catechol structure. It also inhibits low-
density lipoprotein (LDL) oxidation, platelet aggregation and endothelial cell activation and
protects DNA from oxidative damage (Notomista et al., 2011; Bulotta et al., 2014). The
scavenging activity of hydroxytyrosol was found to be comparable with hypochlorous acid
(HOCl) which is a potent oxidant produced in vivo at the site of inflammation (Visioli et al.,
1998).
Table 1. Outline of the mechanism implicated in the

protection mediated by the antioxidants
Antioxidants Mechanism of Action

α-Lipoic acid It reacts with reactive oxygen species (ROS) and prevents the damage
caused by them
Curcumin It neutralizes ROS
Rutin It neutralizes ROS
Ferulic acid It has e- donating groups which terminate free radical chain reaction
Vitamin E It acts as a potent chain-breaking antioxidant and inhibits ROS
production
Vitamin C It is an electron donor and scavenges ROS
Naringin It acts as ROS scavenger and inhibits the enzyme xanthine oxidase
Quercetin It has ROS scavenging property especially for hydroxyl ion, superoxide
anion
Resveratrol It has ROS scavenging property and inhibits lipid peroxidation
Hydroxytyrosol It has ROS, reactive nitrogen species (RNS) and peroxyl radicals
scavenging property
10. PRACTICAL ASPECTS ON THE USE OF ANTIOXIDANTS

Antioxidants are used for neutralizing the imbalance between ROS and antioxidants level.
In the treatment of dermatoses, antioxidants can be used as orally or topically to reduce or
prevent the structural damage of the cells. These can be used in line with treatments where re-
establishment of the redox balance is the main objective in any situation (Guaratini et al., 2007).
Antioxidant effects vary with concentrations but the concentrations close to the
physiological ones should be preferred as they can be adjusted easily, also it reduces the risk of
toxicity or drug interaction (Sadowska-Bartosz and Bartosz, 2014). Antioxidants should only
be taken when prescribed by a physician for a purpose.
However, it is necessary to mention that supplementation of an antioxidant is not a
replacement for the normal diet with fruit or vegetable consumption as it does not leads to any
risk (Rizwan et al., 2011; Martinez et al., 2012). Association of antioxidants with
complementary mechanisms allows a broader neutralizing action, with adequate safety of use
during the period of OS (Matsui et al., 2009).

11. THE PARADOX OF ANTIOXIDANTS

It is suggested that a balance between ROS and antioxidants is needed for proper
functioning of the cell. Some of the studies on chronological aging have shown that some
stressful situations like physical activity, increase mitochondrial activity which further
increases the ROS generation, in the response of which an adaption is provoked i.e., an
improved defense mechanism and stress resistance (Ristow and Schmeisser, 2011). This
indicates the use of antioxidants in a situation where there are both the mechanisms of
neutralization i.e., by the ROS excess and by the decline of the endogenous system gets failed,
that generally occurs in most of the diseases.
The combination therapy of antioxidants has been used as the safest alternative for its use.
For example, supplementation with vitamin E (an antioxidant) to vitamin C synergies the
regeneration of product of alpha-tocopherol oxidation (Keller and Fenske, 1998).
Also, supplementation with beta-carotene to lycopene and a probiotic increases the
polymorphic light eruption (Marini et al., 2014). These studies suggest the use of combination
therapy in low doses over monotherapy of antioxidants in large doses not only because it
amplifies the effect of antioxidant but also due to its synergistic effect (Addor et al., 2013).

AND CHALLENGES
Antioxidants have shown the potential in the pharmacotherapy in various preclinical and
the clinical studies, but despite this several studies shows that the majority of antioxidants
available for human use has found to be ineffective (Gelain et al., 2012). Therefore, it is
important to explore these beneficial moieties for the treatment of the disorders. The research
should aim to explore the newer, safer and the effective antioxidants. The majority of the drugs
or the antioxidants which are used till date have been discovered in the 1950s or 60s. So, if one
thinks that the same drug which was discovered in the 1950s will work in 2018 or today at same
efficacy does not make sense actually. The reason is the change in human and the change in the
environment. So, we cannot rely on these moieties of the past and have to explore the newer
one.
Antioxidants are used mainly as the adjunct therapy and therefore, there are very fewer
studies which report their true therapeutic values. Thus, the preclinical and clinical studies
should focus on the exploration of the full-scale therapeutic potential of these drugs. Further,
one of the approaches is to combine the antioxidant with the drug, so the patients do not need
to take two drugs/therapies. Thus, the approach is to combine the antioxidant with the drug
molecules. Selected antioxidants combined with available drugs to design codrugs to avoid
many problems. These codrugs have been designed by combining a particular group of
antioxidant with available drugs so that it can resolve various issues of permeation, oral
absorption, stability, lipophilicity, solubility of the drug and drug resistance.
However, there are only a few candidates approved for clinical use (Cacciatore et al., 2012).
Further, the safety, quality and purity of antioxidants will always remain in the questions. Also,
which antioxidant is suitable for which pathology it is clearly unknown until now. Further,

which antioxidant is suitable to which age group patients, this notion again requires the
exploration studies in various preclinical and the clinical settings.
CONCLUSION
It is concluded that OS plays a key role in the pathogenesis of the various disorders of the
human being. Therefore, the mitigation of the OS by the use of the antioxidants may be
beneficial in the treatment of these disorders. Antioxidants work either by reducing the levels
of ROS by inhibiting their production or by increasing their scavenging or decomposition. But
the main problem is the incomplete information or the knowledge regarding the use of the
antioxidants. Further, it has also not yet known which antioxidant is required for which
pathology. Also, the mechanism of action of various antioxidants are generally similar or they
act in the same way in the body. It is also not known for how much duration the person should
be on the antioxidant therapy. Thus, future work is required to explore the beneficial and
therapeutic effect of the antioxidants to confirm their therapeutic role in different pathologies.
REFERENCES
Addor, F. A.; Camarano, P.; Agelune, C. P. Increase in the minimum erythema dose level based
on the intake of a vitamin supplement containing antioxidants. Surg Cosmet Dermatol.,
2013,5(3),2125.
Adlard, P. A., Cherny, R. A., Finkelstein D. I., Gautier E., Robb E., Cortes M., Volitakis I., Liu
X., Smith J. P., Perez K., Laughton K. Rapid restoration of cognition in Alzheimer’s
transgenic mice with 8-hydroxy quinoline analogs is associated with decreased interstitial
Aβ. Neuron., 2008, Jul 10,59(1),43-55.
Aguirre, R., May, J. M. Inflammation in the vascular bed: importance of vitamin C.
Pharmacology & therapeutics., 2008, Jul 1,119(1),96-103.
Ahmad, F. K., He, Z., King, G. L. Molecular targets of diabetic cardiovascular complications.
Current drug targets., 2005, Jun 1,6(4),487-94.
Ahmad, M., Sultana, M., Raina, R., Pankaj, N. K., Verma, P. K., Prawez, S. Hypoglycemic,
hypolipidemic, and wound healing potential of quercetin in streptozotocin-induced diabetic
rats. Pharmacognosy magazine., 2017 Oct,13(3),S633.
Albanes, D., Heinonen, O. P., Huttunen, J. K., Taylor, P. R., Virtamo, J. Edwards, B. K.,
Haapakoski, J., Rautalahti, M., Hartman, A. M., Palmgren, J. Effects of alpha-tocopherol
and beta-carotene supplements on cancer incidence in the Alpha-Tocopherol Beta-
Carotene Cancer Prevention Study. The American journal of clinical nutrition., 1995, Dec
1,62(6),1427S-30S.
Albani, D Polito, L Batelli, S De Mauro, S Fracasso, C Martelli, G; Colombo, L Manzoni, C
Salmona, M; Caccia, S Negro, A. The SIRT1 activator resveratrol protects SK‐N‐BE cells
from oxidative stress and against toxicity caused by α-synuclein or amyloid‐β (1‐42)
peptide. Journal of neurochemistry., 2009 Sep,110(5),1445-56.

Allen, J; Bradley, RD. Effects of oral glutathione supplementation on systemic oxidative stress
biomarkers in human volunteers. The Journal of Alternative and Complementary
Medicine., 2011, Sep 1,17(9),827-33.
Ames, BN; Shigenaga, MK; Hagen, TM. Mitochondrial decay in aging. Biochimica et
Biophysica Acta (BBA)-Molecular Basis of Disease., 1995, May 24,1271(1),165-70.
Azevedo, MI; Pereira, AF; Nogueira, RB; Rolim, FE; Brito, GA; Wong, DV; Lima-Júnior, RC;
de Albuquerque Ribeiro, R; Vale, ML. The antioxidant effects of the flavonoids rutin and
quercetin inhibit oxaliplatin-induced chronic painful peripheral neuropathy. Molecular
Pain., 2013 Dec,9(1),53.
Balaban, RS; Nemoto, S; Finkel, T. Mitochondria, oxidants, and aging. cell., 2005 Feb,
25,120(4),483-95.
Bartlett, HE; Eperjesi, F. Nutritional supplementation for type 2 diabetes: a systematic review.
Ophthalmic and Physiological Optics., 2008 Nov,28(6),503-23.
Bayrhuber, M; Meins, T; Habeck, M; Becker, S; Giller, K; Villinger, S; Vonrhein, C;
Griesinger, C; Zweckstetter, M; Zeth, K. Structure of the human voltage-dependent anion
channel. Proceedings of the National Academy of Sciences., 2008, Oct 7,105(40),
15370-5.
Beckman, KB; Ames, BN. The free radical theory of aging matures. Physiol Rev., 1998, 78,547.
Behr, GA; Moreira, JC; Frey, BN. Preclinical and clinical evidence of antioxidant effects of
antidepressant agents: implications for the pathophysiology of major depressive disorder.
Oxidative Medicine and Cellular Longevity., 2012,2012,609421.
Bender, A; Krishnan, KJ; Morris, CM; Taylor, GA; Reeve, AK; Perry, RH; Jaros, E;
Hersheson, JS; Betts, J; Klopstock, T; Taylor, RW. High levels of mitochondrial DNA
deletions in substantia nigra neurons in aging and Parkinson disease. Nat Genet., 2006
May,38(5),515.
Berry, A; Capone, F; Giorgio, M; Pelicci, PG; De Kloet, ER; Alleva, E; Minghetti, L; Cirulli,
F. Deletion of the life span determinant p66 Shc prevents age-dependent increases in
emotionality and pain sensitivity in mice. Experimental gerontology., 2007, Feb
28,42(1),37-45.
Bhullar, KS; Jha, A; Youssef, D; Rupasinghe, HP. Curcumin and its carbocyclic analogs:
structure-activity in relation to antioxidant and selected biological properties. Molecules.,
2013, May 10,18(5),5389-404.
Bjelakovic, G; Nikolova, D; Simonetti, RG; et al. Antioxidant supplements for prevention of
gastrointestinal cancers: a systematic review and meta-analysis. Lancet., 2004,364,1219–
28.
Boom, A; Authelet, M; Dedecker, R; Frédérick, C; Van Heurck, R; Daubie, V; Leroy, K;
Pochet R; Brion, JP. Bimodal modulation of tau protein phosphorylation and conformation
by extracellular Zn2+ in human-tau transfected cells. Biochimica et Biophysica Acta
(BBA)-Molecular Cell Research., 2009, Jun 1,1793(6),1058-67.
Brenes, GA; Penninx, BW; Judd, PH; Rockwell, E; Sewell, DD; Wetherell, JL. Anxiety,
depression and disability across the lifespan. Aging and Mental Health., 2008, Jan
1,12(1),158-63.
Brondino, N; Re, S; Boldrini, A; Cuccomarino, A; Lanati, N; Barale, F; Politi, P. Curcumin as
a therapeutic agent in dementia: a mini systematic review of human studies. The Scientific
World Journal., 2014,2014.

Brown, DI; Griendling, KK. Regulation of signal transduction by reactive oxygen species in
the cardiovascular system. Circulation research., 2015, Jan 30,116(3),531-49.
Brownlee, M. Biochemistry and molecular cell biology of diabetic complications. Nature.,
2001, Dec 13,414(6865),813.
Bulotta, S; Celano, M; Lepore, SM; Montalcini, T; Pujia, A; Russo, D. Beneficial effects of the
olive oil phenolic components oleuropein and hydroxytyrosol: focus on protection against
cardiovascular and metabolic diseases. Journal of translational medicine., 2014
Dec,12(1),219.
Cacciatore, I; Baldassarre, L; Fornasari, E; Mollica, A; Pinnen, F. Recent advances in the
treatment of neurodegenerative diseases based on GSH delivery systems. Oxid Med Cell
Longev., 2012, Jun 3,2012.
Calder, P. C; Albers, R; Antoine, J. M; Blum, S.; Bourdet-Sicard, R.; Ferns, G. A; Folkerts, G;
Friedmann, PS; Frost, GS; Guarner, F; Løvik, M. Inflammatory disease processes and
interactions with nutrition. British Journal of Nutrition., 2009 May,101(1),1-45.
Cavia‐Saiz, M; Busto, M. D; Pilar‐Izquierdo, M. C.; Ortega, N; Perez‐Mateos, M; Muñiz, P.
Antioxidant properties; radical scavenging activity and biomolecule protection capacity of
flavonoid naringenin and its glycoside naringin: a comparative study. Journal of the
Science of Food and Agriculture., 2010 May,90(7),1238-44.
Ceriello, A; Esposito, K; Piconi, L; Ihnat, MA; Thorpe, J. E; Testa, R; Boemi, M; Giugliano,
D. Oscillating glucose is more deleterious to endothelial function and oxidative stress than
mean glucose in normal and type 2 diabetic patients. Diabetes., 2008, May 1,57(5),1349-
54.
Ceriello, A; Mercuri, F; Quagliaro, L; Assaloni, R; Motz, E; Tonutti, L; Taboga, C. Detection
of nitrotyrosine in the diabetic plasma: evidence of oxidative stress. Diabetologia., 2001,
Jul 1,44(7),834-8.
Chang, C. C.; Yang, M. H; Tung, H. C; Chang, C. Y.; Tsai, Y. L; Huang, J. P; Yen, T. H; Hung,
L.M. Resveratrol exhibits differential protective effects on fast-and slow-twitch muscles in
streptozotocin-induced diabetic rats. Journal of diabetes., 2014 Jan,6(1),60-7.
Chaudhry, PS; Cabrera, J; Juliani, HR; Varma, SD. Inhibition of human lens aldose reductase
by flavonoids, sulindac and indomethacin. Biochemical pharmacology., 1983, Jul
1,32(13),1995-8.
Chen, K. H; Hung, C. C; Hsu, H. H; Jing, Y. H; Yang, C. W; Chen, J. K. Resveratrol ameliorates
early diabetic nephropathy associated with suppression of augmented TGF-β/smad and
ERK1/2 signaling in streptozotocin-induced diabetic rats. Chemico-biological
interactions., 2011, Mar 15,190(1),45-53.
Christen, Y. Oxidative stress and Alzheimer disease. Am J Clin Nutr., 2000, Feb 1,71(2),621S-
9S.
Colombo, M. L. An update on vitamin E, tocopherol and tocotrienol—perspectives. Molecules.,
2010,15(4),2103-13.
Conway, K. A; Rochet, J. C; Bieganski, R. M; Lansbury, P. T. Kinetic stabilization of the α-
synuclein protofibril by a dopamine-α-synuclein adduct. Science., 2001, Nov
9,294(5545),1346-9.
Coppey, L. J; Gellett, J S; Davidson, E. P; Dunlap, J. A; Lund, D. D; Yorek, M. A. Effect of
antioxidant treatment of streptozotocin-induced diabetic rats on endoneurial blood flow,
motor nerve conduction velocity, and vascular reactivity of epineurial arterioles of the
sciatic nerve. Diabetes., 2001, Aug 1,50(8),1927-37.

Cristóvão, J. S; Santos, R; Gomes, C. M. Metals and neuronal metal binding proteins implicated
in Alzheimer’s disease. Oxidative medicine and cellular longevity., 2016,2016.
Dauer, W; Przedborski, S. Parkinson’s disease: mechanisms and models. Neuron., 2003, Sep
11,39(6),889-909.
De Marchi, E; Baldassari, F; Bononi, A; Wieckowski, M. R; Pinton, P. Oxidative stress in
cardiovascular diseases and obesity: role of p66Shc and protein kinase C. Oxidative
Medicine and Cellular Longevity., 2013,2013,564961.
De Marchi, E; Baldassari, F; Bononi, A; Wieckowski, M. R; Pinton, P. Oxidative stress in
cardiovascular diseases and obesity: role of p66Shc and protein kinase C. Oxidative
medicine and cellular longevity., 2013,2013,564961.
Dhir, A; Kulkarni, S. K. Nitric oxide and major depression. Nitric Oxide., 2011,24(3),125–31.
Dikalov, S. Cross talk between mitochondria and NADPH oxidases. Free Radical Biology and
Medicine., 2011, Oct 1,51(7),1289-301.
Droge, W. Free radicals in the physiological control of cell function. Physiological Reviews.,
2002,82,47–95.
Du, J; Cullen, J. J; Buettner, G R. Ascorbic acid: chemistry, biology and the treatment of cancer.
Biochimica et Biophysica Acta (BBA)-Reviews on Cancer., 2012, Dec 1,1826(2),443-57.
Ebesunun, M. O; Obajobi, E. O. Elevated plasma homocysteine in type 2 diabetes mellitus: a
risk factor for cardiovascular diseases. Pan African Medical Journal., 2012,12(1).
Figueroa-Méndez, R; Rivas-Arancibia, S. Vitamin C in health and disease: its role in the
metabolism of cells and redox state in the brain. Frontiers in physiology., 2015, Dec
23,6,397.
Finkel, T; Holbrook, N. J. Oxidants, oxidative stress and the biology of ageing. Nature., 2000,
Nov 9,408(6809),239.
Finkel, T. Signal transduction by reactive oxygen species. The Journal of cell biology., 2011,
Jul 11,194(1),7-15.
Fiori, J. L; Shin, Y. K; Kim, W; Krzysik-Walker, S. M; González-Mariscal, I; Carlson, OD;
Sanghvi, M; Moaddel, R; Farhang, K; Gadkaree, SK; Doyle, ME. Resveratrol prevents β-
cell dedifferentiation in non-human primates given a high fat/high sugar diet. Diabetes.,
2013, Jul 18,DB_130266.
Forno, L. S. Neuropathology of Parkinson’s disease. Journal of Neuropathology and
Experimental Neurology., 1996,55(3),259-272.
Fusco, D; Colloca, G; Monaco, M. R; Cesari, M. Effects of antioxidant supplementation on the
aging process. Clinical interventions in aging., 2007 Sep,2(3),377.
Garcia-Matas, S; de Vera, N; Aznar, A. O; Marimon, J. M; Adell, A; Planas, A. M; Cristòfol,
R; Sanfeliu, C. In vitro and in vivo activation of astrocytes by amyloid-β is potentiated by
pro-oxidant agents. Journal of Alzheimer’s Disease., 2010, Jan 1,20(1),229-45.
Gawryluk, J. W; Wang, J. F; Andreazza, A. C; Shao, L; Young, L. T. Decreased levels of
glutathione, the major brain antioxidant, in post-mortem prefrontal cortex from patients
with psychiatric disorders. International Journal of Neuropsychopharmacology., 2011,
Feb 1,14(1),123-30.
Gelain, D. P; Antonio Behr, G; Birnfeld de Oliveira, R; Trujillo, M. Antioxidant therapies for
neurodegenerative diseases: mechanisms, current trends, and perspectives. Oxidative
medicine and cellular longevity., 2012, Dec 5,2012.

Ghezzi, D; Zeviani, M. Assembly factors of human mitochondrial respiratory chain complexes:

physiology and pathophysiology. In Mitochondrial Oxidative Phosphorylation, 2012,65-
106.
Giordano, E; Davalos, A; Nicod, N; Visioli, F. Hydroxytyrosol attenuates tunicamycin‐induced
endoplasmic reticulum stress in human hepatocarcinoma cells. Molecular nutrition & food
research., 2014 May,58(5),954-62.
Gonzalez-Reyes, S; Guzmán-Beltrán, S; Medina-Campos, O. N; Pedraza-Chaverri, J.
Curcumin pretreatment induces Nrf2 and an antioxidant response and prevents hemin-
induced toxicity in primary cultures of cerebellar granule neurons of rats. Oxidative
medicine and cellular longevity., 2013,2013.
Goswami, A; Dikshit, P; Mishra, A; Mulherkar, S; Nukina, N; Jana, N. R. Oxidative stress
promotes mutant huntingtin aggregation and mutant huntingtin-dependent cell death by
mimicking proteasomal malfunction. Biochemical and biophysical research
communications., 2006, Mar 31,342(1),184-90.
Graf, E. Antioxidant potential of ferulic acid. Free radical biology and medicine., 1992, Oct
1,13(4),435-48.
Griendling, K. K; Sorescu, D; Lassegue, B; Ushio-Fukai, M. Modulation of protein kinase
activity and gene expression by reactive oxygen species and their role in vascular
physiology and pathophysiology. Arteriosclerosis, thrombosis, and vascular biology.,
2000 Oct,20(10),2175-83.
Gruber, J; Schaffer, S; Halliwell, B. The mitochondrial free radical theory of ageing—where
do we stand. Front Biosci., 2008, May 1,13,6554-79.
Gu, Y; Dee, C. M; Shen, J. Interaction of free radicals, matrix metalloproteinases and caveolin-
1 impacts blood-brain barrier permeability. Front Biosci (Schol Ed)., 2011, Jun
1,3(3),1216-31.
Guaratini, T; Medeiros, M. H; Colepicolo, P. Antioxidantes na manutenção do equilíbrio redox
cutâneo: uso e avaliação de sua eficácia [Antioxidants in maintaining redox cutaneous
balance: use and evaluation of its effectiveness]. Química Nova., 2007,30(1),206-13.
Halliwell, B. Oxidative stress and neurodegeneration: where are we now?. Journal of
neurochemistry., 2006 Jun,97(6),1634-58.
Harborne, J B. Nature, distribution and function of plant flavonoids. Progress in clinical and
biological research., 1986,213,15-24.
Harikumar, K. B; Aggarwal, B. B. Resveratrol: a multitargeted agent for age-associated chronic
diseases. Cell cycle., 2008, Apr 15,7(8),1020-35.
Harman, D. Aging: a theory based on free radical and radiation chemistry. J Gerontol.,
1957,2,298–300.
Harman, D. The biologic clock: the mitochondria?. Journal of the American Geriatrics Society.,
1972, Apr 1,20(4),145-7.
Harrison, F. E; May, J. M. Vitamin C function in the brain: vital role of the ascorbate transporter
SVCT2. Free Radical Biology and Medicine., 2009, Mar 15,46(6),719-30.
Haugaard, N; Haugaard, E. S. Stimulation of glucose utilization by thioctic acid in rat
diaphragm incubated in vitro. Biochimica et Biophysica Acta (BBA)-General Subjects.,
1970, Dec 29,222(3),583-6.
Heck, D. E; Shakarjian, M; Kim, H. D; Laskin, J. D; Vetrano, A. M. Mechanisms of oxidant
generation by catalase. Annals of the New York Academy of Sciences., 2010
Aug,1203(1),120-5.

Hercberg, S; Czernichow, S; Galan, P. Antioxidant vitamins and minerals in prevention of

cancers: lessons from the SU. VI. MAX study. British Journal of Nutrition., 2006
Aug,96(1),28-30.
Hornykiewicz, O. Biochemical pathophysiology of Parkinson’s disease. Parkinson’s
disease.Advances in Neurology., 1986,45,19-34.
Hovatta, I; Tennant, R. S; Helton, R; Marr, R. A; Singer, O; Redwine, JM; Ellison, JA; Schadt,
EE; Verma, IM; Lockhart, DJ; Barlow, C. Glyoxalase 1 and glutathione reductase 1
regulate anxiety in mice. Nature., 2005 Dec,438(7068),662-6.
Hsu, C. Y; Shih, H. Y; Chia, Y. C; Lee, C. H; Ashida, H; Lai, Y. K; Weng, CF. Rutin potentiates
insulin receptor kinase to enhance insulin‐dependent glucose transporter 4 translocation.
Molecular nutrition and food research., 2014 Jun,58(6),1168-76.
Huang, H; Tindall, D. J. Dynamic FoxO transcription factors. J Cell Sci., 2007, Aug
1,120(15),2479-87.
Hussein, M. A; El-Maksoud, H. A. Biochemical effects of Resveratrol and curcumin
combination on obese diabetic rats. Molecular and Clinical Pharmacology., 2013,4(1),1-
0.
Illison, V. K; Rondo, P. H; de Oliveira, A. M; D’Abronzo, F. H; Campos, K. F. The relationship
between plasma α-tocopherol concentration and vitamin E intake in patients with Type 2
diabetes mellitus. International Journal for Vitamin and Nutrition Research., 2011, Jan
1,81(1),12.
Inoue, M; Sato, E. F; Nishikawa, M; Park, A. M; Kira, Y; Imada, I; Utsumi, K. Mitochondrial
generation of reactive oxygen species and its role in aerobic life. Current medicinal
chemistry., 2003, Dec 1,10(23),2495-505.
Jacobs, E. J; Connell, C. J; Chao, A; et al. Multivitamin use and colorectal cancer incidence in
a US cohort: does timing matter? Am J Epidemiol., 2003,158(7),621–8.
Jang, J. H; Surh, Y. J. Protective effect of resveratrol on β-amyloid-induced oxidative PC12
cell death. Free Radical Biology and Medicine., 2003, Apr 15,34(8),1100-10.
Javed, H; Khan, M. M; Ahmad, A; Vaibhav, K; Ahmad, M. E; Khan, A; Ashafaq, M; Islam, F;
Siddiqui, MS; Safhi, MM. Rutin prevents cognitive impairments by ameliorating oxidative
stress and neuroinflammation in rat model of sporadic dementia of Alzheimer type.
Neuroscience., 2012, May 17,210,340-52.
Joshi, G; Perluigi, M; Sultana, R; Agrippino, R; Calabrese, V; Butterfield, D. A. In vivo
protection of synaptosomes by ferulic acid ethyl ester (FAEE) from oxidative stress
mediated by 2, 2-azobis (2-amidino-propane) dihydrochloride (AAPH) or Fe2+/H2O2:
Insight into mechanisms of neuroprotection and relevance to oxidative stress-related
neurodegenerative disorders. Neurochemistry international., 2006, Mar 1,48(4),318-27.
Jung, UJ; Lee, MK; Jeong, KS; Choi, MS. The hypoglycemic effects of hesperidin and naringin
are partly mediated by hepatic glucose-regulating enzymes in C57BL/KsJ-db/db mice. The
Journal of nutrition., 2004, Oct 1,134(10),2499-503.
Kanski, J; Aksenova, M; Stoyanova, A; Butterfield, DA. Ferulic acid antioxidant protection
against hydroxyl and peroxyl radical oxidation in synaptosomal and neuronal cell culture
systems in vitro, structure-activity studies. The Journal of nutritional biochemistry., 2002,
May 1,13(5),273-81.
Kappel, V. D; Zanatta, L; Postal, B. G; Silva, FR. Rutin potentiates calcium uptake via voltage-
dependent calcium channel associated with stimulation of glucose uptake in skeletal
muscle. Archives of biochemistry and biophysics., 2013, Apr 15,532(2),55-60.

Kara, Y; Doguc, D. K; Kulac, E; Gultekin, F. Acetylsalicylic acid and ascorbic acid

combination improves cognition: Via antioxidant effect or increased expression of
NMDARs and nAChRs?. Environmental toxicology and pharmacology., 2014, May
1,37(3),916-27.
Kataja-Tuomola, M. K; Kontto, J. P; Männistö, S; Albanes, D; Virtamo, J. Intake of
antioxidants and risk of type 2 diabetes in a cohort of male smokers. European journal of
clinical nutrition., 2011 May,65(5),590.
Keller, K. L; Fenske, N. A. Uses of vitamins A, C, and E and related compounds in
dermatology: a review. Journal of the American Academy of Dermatology., 1998, Oct
1,39(4),611-25.
Khazaei, M; Karimi, J; Sheikh, N; Goodarzi, M. T; Saidijam, M; Khodadadi, I; Moridi, H.
Effects of resveratrol on receptor for advanced glycation end products (RAGE) expression
and oxidative stress in the liver of rats with type 2 diabetes. Phytotherapy research., 2016
Jan,30(1),66-71.
Khurana, S; Venkataraman, K; Hollingsworth, A; Piche, M; Tai, TC. Polyphenols: benefits to
the cardiovascular system in health and in aging. Nutrients., 2013, Sep 26,5(10),3779-827.
Kil, I. S; Huh, T. L; Lee, YS; Lee, Y. M; Park, J. W. Regulation of replicative senescence by
NADP+-dependent isocitrate dehydrogenase. Free Radical Biology and Medicine., 2006,
Jan 1,40(1),110-9.
Kohen, R; Vellaichamy, E; Hrbac, J; Gati, I; Tirosh, O. Quantification of the overall reactive
oxygen species scavenging capacity of biological fluids and tissues. Free Radical Biology
and Medicine., 2000, Mar 15,28(6),871-9.
Kozlowski, H; Luczkowski, M; Remelli, M; Valensin, D. Copper, zinc and iron in
neurodegenerative diseases (Alzheimer’s, Parkinson’s and prion diseases). Coordination
Chemistry Reviews., 2012, Oct 1,256(19-20),2129-41.
Kregel, K. C; Zhang, H. J. An integrated view of oxidative stress in aging: basic mechanisms,
functional effects, and pathological considerations. American Journal of Physiology-
Regulatory, Integrative and Comparative Physiology., 2007 Jan,292(1),R18-36.
Kuhlbrandt, W. Structure and function of mitochondrial membrane protein complexes. BMC
biology., 2015 Dec,13(1),89.
Kyriakis, J. M; Avruch, J. Mammalian mitogen-activated protein kinase signal transduction
pathways activated by stress and inflammation. Physiological reviews., 2001, Apr
1,81(2),807-69.
Lagouge, M; Argmann, C; Gerhart-Hines, Z; Meziane, H; Lerin, C; Daussin, F; Messadeq, N;
Milne, J; Lambert, P; Elliott, P; Geny, B. Resveratrol improves mitochondrial function and
protects against metabolic disease by activating SIRT1 and PGC-1α. Cell., 2006, Dec
15,127(6),1109-22.
Li, N; Brun, T; Chop, M; Cunha, D. A; Eizirik, D. L; Maechler, P. Transient exposure of β-
cells to oxidative stress interrupts the transduction of signals normally coupling glucose
metabolism to insulin secretion. J. Biol. Chem., 2009,284,23602-12.
Lobo, V; Patil, A; Phatak, A; Chandra, N. Free radicals, antioxidants and functional foods:
Impact on human health. Pharmacognosy Reviews., 2010,4(8),118-26.
Maes, M; Galecki, P; Chang, Y. S; Berk, M. A review on the oxidative and nitrosative stress
(O&NS) pathways in major depression and their possible contribution to the (neuro)
degenerative processes in that illness. Progress in Neuro-Psychopharmacology and
Biological Psychiatry., 2011, Apr 29,35(3),676-92.

Maes, M. An intriguing and hitherto unexplained co-occurrence: depression and chronic fatigue
syndrome are manifestations of shared inflammatory, oxidative and nitrosative (IO&NS)
pathways. Progress in Neuro-Psychopharmacology and Biological Psychiatry., 2011, Apr
29,35(3),784-94.
Makino, Y; Tanaka, H; Dahlman-Wright, K; Makino, I. Modulation of glucocorticoid-
inducible gene expression by metal ions. Molecular pharmacology., 1996, Apr
1,49(4),612-20.
Makrantonaki, E; Zouboulis, C. C. The skin as a mirror of the aging process in the human
organism–state of the art and results of the aging research in the German National Genome
Research Network 2 (NGFN-2). Experimental gerontology., 2007, Sep 1,42(9),879-86.
Mamdouh, M. A; Monira, A. A. The influence of naringin on the oxidative state of rats with
streptozotocin-induced acute hyperglycaemia. Zeitschrift für Naturforschung C., 2004, Oct
1,59(9-10),726-33.
Marini, A; Jaenicke, T; Grether‐Beck, S; Le Floc’h, C; Cheniti, A; Piccardi, N; Krutmann, J.
Prevention of polymorphic light eruption by oral administration of a nutritional supplement
containing lycopene, β‐carotene, and L actobacillus johnsonii: results from a randomized,
placebo‐controlled, double‐blinded study. Photodermatology, photoimmunology &
photomedicine., 2014 Aug,30(4),189-94.
Martínez, M. E; Jacobs, E. T; Baron, J. A; Marshall, J. R; Byers, T. Dietary supplements and
cancer prevention: balancing potential benefits against proven harms. Journal of the
National Cancer Institute., 2012, Apr 25,104(10),732-9.
Martinez-Vicente, M; Talloczy, Z; Kaushik, S; Massey, A. C; Mazzulli, J; Mosharov, E. V;
Hodara, R; Fredenburg, R; Wu, D. C; Follenzi, A; Dauer, W. Dopamine-modified α-
synuclein blocks chaperone-mediated autophagy. The Journal of clinical investigation.,
2008, Feb 1,118(2),777-88.
Masood, A; Nadeem, A; Mustafa, S. J; O’Donnell, J. M. Reversal of oxidative stress-induced
anxiety by inhibition of phosphodiesterase-2 in mice. Journal of Pharmacology and
Experimental Therapeutics., 2008, Aug 1,326(2),369-79.
Matsui, M. S; Hsia, A; Miller, J. D; Hanneman, K; Scull, H; Cooper, KD; et al. Non-sunscreen
photoprotection: antioxidants add value to a sunscreen. J Investig Dermatol Symp Proc.,
2009,14,56–59.
Mikhed, Y; Daiber, A; Steven, S. Mitochondrial Oxidative Stress, Mitochondrial DNA Damage
and Their Role in Age-Related Vascular Dysfunction. International Journal of Molecular
Science., 2015,16(7),15918-53.
Mondragon-Rodriguez, S; Perry, G; Zhu, X; Moreira, P. I; Acevedo-Aquino, M. C; Williams,
S. Phosphorylation of tau protein as the link between oxidative stress, mitochondrial
dysfunction, and connectivity failure: implications for Alzheimer’s disease. Oxidative
medicine and cellular longevity., 2013, Jul 10,2013.
Morris, MC; Evans, D. A; Tangney, C C; Bienias, J. L; Wilson, R. S; Aggarwal, N. T; Scherr,
P. A. Relation of the tocopherol forms to incident Alzheimer disease and to cognitive
change–. The American journal of clinical nutrition., 2005, Feb 1,81(2),508-14.
Muller, DP; Goss-Sampson, MA. Neurochemical, neurophysiological, and neuropathological
studies in vitamin E deficiency. Critical reviews in neurobiology., 1990,5(3),239-63.
Muller, FL; Liu, Y; Van Remmen, H. Complex III releases superoxide to both sides of the inner
mitochondrial membrane. Journal of Biological Chemistry., 2004, Aug 17.

Münch, G; Lüth, HJ; Wong, A; Arendt, T; Hirsch, E; Ravid, RA; Riederer, P. Crosslinking of
α-synuclein by advanced glycation end products—an early pathophysiological step in
Lewy body formation? Journal of chemical neuroanatomy., 2000, Dec 1,20(3-4),253-7.
Murphy, M. P. How mitochondria produce reactive oxygen species. Biochemical journal.,
2009, Jan 1,417(1),1-3.
Nelson, J. S; Fitch, C. D; Fischer, V. W; Broun, Jr. GO; Chou, A. C. Progressive
neuropathologic lesions in vitamin E-deficient rhesus monkeys. Journal of
Neuropathology and Experimental Neurology., 1981, Mar 1,40(2),166-86.
Ng, F; Berk, M; Dean, O; Bush, A. I. Oxidative stress in psychiatric disorders: evidence
base and therapeutic implications. International Journal of Neuropsycho-pharmacology.,
2008,11(6),851–876.
Nguyen, D; Alavi, M V; Kim, K Y; Kang, T; Scott, R. T; Noh, Y. H; Lindsey, J. D; Wissinger,
B; Ellisman, M. H; Weinreb, R. N; Perkins, G. A. A new vicious cycle involving glutamate
excitotoxicity, oxidative stress and mitochondrial dynamics. Cell death and disease., 2011,
Dec 8,2(12),e240.
Notomista, E; Scognamiglio, R; Troncone, L; Donadio, G; Pezzella, A; Di Donato, A; Izzo, V.
Tuning the specificity of the recombinant multicomponent toluene o-xylene
monooxygenase from Pseudomonas sp. strain OX1 for the biosynthesis of tyrosol from 2-
phenylethanol. Applied and environmental microbiology., 2011, Aug 1,77(15),5428-37.
Núñez, M. T; Urrutia, P; Mena, N; Aguirre, P; Tapia, V; Salazar, J. Iron toxicity in
neurodegeneration. Biometals., 2012, Aug 1,25(4),761-76.
Odebrecht Vargas Nunes, S; Pizzo de Castro, M. R; Ehara Watanabe, M. A; Losi
Guembarovski, R; Odebrecht Vargas, H; Vissoci Reiche, EM; Berk, M. Genetic
polymorphisms in glutathione-S-transferases are associated with anxiety and mood
disorders in nicotine dependence. Psychiatric Genetics., 2014,24(3),87–93.
Pacher, P; Beckman, J. S; Liaudet, L. Nitric oxide and peroxynitrite in health and disease.
Physiological Reviews., 2007,87,315–424.
Palmer, K; Berger, AK; Monastero, R; Winblad, B; Bäckman, L; Fratiglioni, L. Predictors of
progression from mild cognitive impairment to Alzheimer disease. Neurology., 2007, May
8,68(19),1596-602.
Parihar, M. S; Parihar, A; Fujita, M; Hashimoto, M; Ghafourifar, P. Alpha-synuclein
overexpression and aggregation exacerbates impairment of mitochondrial functions by
augmenting oxidative stress in human neuroblastoma cells. The international journal of
biochemistry and cell biology., 2009, Oct 1,41(10),2015-24.
Pashkow, F. J. Oxidative Stress and Inflammation in Heart Disease: Do Antioxidants Have a
Role in Treatment and/or Prevention? International journal of inflammation.,
2011,2011,514623.
Peculis, R; Konrade, I; Skapare, E; Fridmanis, D; Nikitina-Zake, L; Lejnieks, A; Pirags, V;
Dambrova, M; Klovins, J. Identification of glyoxalase 1 polymorphisms associated with
enzyme activity. Gene., 2013, Feb 15,515(1),140-3.
Penumathsa, S. V; Thirunavukkarasu, M; Zhan, L; Maulik, G; Menon, V. P; Bagchi, D; Maulik,
N. Resveratrol enhances GLUT‐4 translocation to the caveolar lipid raft fractions through
AMPK/Akt/eNOS signalling pathway in diabetic myocardium. Journal of cellular and
molecular medicine., 2008, Dec 1,12 (6a),2350-61.

Perez-Pinzon, M. A; Dave, K. R; Raval, A. P. Role of reactive oxygen species and protein

kinase C in ischemic tolerance in the brain. Antioxidants and redox signaling., 2005, Sep
1,7(9-10),1150-7.
Pham-Huy, L. A; He, H; Pham-Huy, C. Free radicals, antioxidants in disease and health.
International journal of biomedical science: IJBS., 2008 Jun,4(2),89.
Pi, J; Bai, Y; Zhang, Q; Wong, V; Floering, L. M; Daniel, K; Reece, J. M; Deeney, J T;
Andersen, M. E; Corkey, B. E; Collins, S. Reactive oxygen species as a signal in glucose-
stimulated insulin secretion. Diabetes., 2007, Jul 1,56(7),1783-91.
Pizzino, G; Irrera, N; Cucinotta, M; Pallio, G; Mannino, F; Arcoraci, V; Squadrito, F; Altavilla,
D; Bitto, A. Oxidative stress: harms and benefits for human health. Oxidative medicine and
cellular longevity., 2017,2017.
Pizzorno, J. Glutathione! Integrative Medicine: A Clinician’s Journal., 2014,13(1),8–12.
Pu, F; Mishima, K; Irie, K; Motohashi, K; Tanaka, Y; Orito, K; Egawa, T; Kitamura, Y;
Egashira, N; Iwasaki, K; Fujiwara, M. Neuroprotective effects of quercetin and rutin on
spatial memory impairment in an 8-arm radial maze task and neuronal death induced by
repeated cerebral ischemia in rats. Journal of pharmacological sciences., 2007,104(4),329-
34.
Rafighi, Z; Shiva, A; Arab, S; Yusuf, R. M. Association of dietary vitamin C and E intake and
antioxidant enzymes in type 2 diabetes mellitus patients. Global journal of health science.,
2013 May,5(3),183.
Rai, S; Kamat, P. K; Nath, C; Shukla, R. A study on neuroinflammation and NMDA receptor
function in STZ (ICV) induced memory impaired rats. Journal of neuroimmunology.,
2013, Jan 15,254(1-2),1-9.
Rebec, G. V; Pierce, R. C. A vitamin as neuromodulator: ascorbate release into the extracellular
fluid of the brain regulates dopaminergic and glutamatergic transmission. Progress in
neurobiology., 1994, Aug 1,43(6),537-65.
Reid, M. E; Duffield-Lillico, A. J; Garland, L; Turnbull, B. W; Clark, L. C; Marshall, J. R.
Selenium supplementation and lung cancer incidence: an update of the nutritional
prevention of cancer trial. Cancer Epidemiology and Prevention Biomarkers., 2002, Nov
1,11(11),1285-91.
Renugadevi, J; Prabu, S. M. Naringenin protects against cadmium-induced oxidative renal
dysfunction in rats. Toxicology., 2009, Feb 4,256(1-2),128-34.
Ristow, M; Schmeisser, S. Extending life span by increasing oxidative stress. Free radical
biology and medicine., 2011, Jul 15,51(2),327-36.
Ristow, M; Zarse, K; Oberbach, A; Klöting, N; Birringer, M; Kiehntopf, M; Stumvoll, M;
Kahn, CR; Blüher, M. Antioxidants prevent health-promoting effects of physical exercise
in humans. Proceedings of the National Academy of Sciences., 2009, May
26,106(21),8665-70.
Rizwan, M; Rodriguez‐Blanco, I; Harbottle, A; Birch‐Machin, M. A; Watson, R. E; Rhodes,
L. E. Tomato paste rich in lycopene protects against cutaneous photodamage in humans in
vivo, a randomized controlled trial. British Journal of Dermatology., 2011 Jan,164(1),154-
62.
Rossi, P; Marzani, B; Giardina, S; Negro, M; Marzatico, F. Human skeletal muscle aging and
the oxidative system: cellular events. Current aging science., 2008, Dec 1,1(3),182-91.

Russo, A; Acquaviva, R; Campisi, A; Sorrenti, V; Di Giacomo, C; Virgata, G; Barcellona, ML;

Vanella, A. Bioflavonoids as antiradicals, antioxidants and DNA cleavage protectors. Cell
biology and toxicology., 2000, Apr 1,16(2),91.
Sadowska-Bartosz, I; Bartosz, G. Effect of antioxidants supplementation on aging and
longevity. BioMed Research International., 2014,2014.
Salim, S; Asghar, M; Chugh, G; Taneja, M; Xia, Z; Saha, K. Oxidative stress: a potential recipe
for anxiety, hypertension and insulin resistance. Brain research., 2010, Nov 4,1359,178-
85.
Salim, S; Sarraj, N; Taneja, M; Saha, K; Tejada-Simon, MV; Chugh, G. Moderate treadmill
exercise prevents oxidative stress-induced anxiety-like behavior in rats. Behavioural brain
research., 2010, Apr 2,208(2),545-52.
Salonen, R. M; Nyyssönen, K; Kaikkonen, J; Porkkala-Sarataho, E; Voutilainen, S; Rissanen,
TH; Tuomainen, TP; Valkonen, VP; Ristonmaa, U; Lakka, HM; Vanharanta, M. Six-year
effect of combined vitamin C and E supplementation on atherosclerotic progression: the
Antioxidant Supplementation in Atherosclerosis Prevention (ASAP) Study. Circulation.,
2003, Feb 25,107(7),947-53.
Sandstrom, M. I; Rebec, GV. Extracellular ascorbate modulates glutamate dynamics: role of
behavioral activation. BMC neuroscience., 2007 Dec,8 (1),32.
Saso, L; Firuzi, O. Pharmacological applications of antioxidants: lights and shadows. Current
Drug Targets., 2014, Dec 1,15(13),1177-99.
Sastre, J; Pallardo, F. V; García de la Asuncion, J; Vina, J. Mitochondria, oxidative stress and
aging. Free radical research., 2000, Jan 1,32(3),189-98.
Schafer, F. Q; Buettner, G. R. Acidic pH amplifies iron-mediated lipid peroxidation in cells.
Free Radical Biology and Medicine., 2000, Apr 15,28(8),1175-81.
Schmatz, R; Perreira, L. B; Stefanello, N; Mazzanti, C; Spanevello, R; Gutierres, J; Bagatini,
M; Martins, C. C; Abdalla, FH; da Silva Serres, J. D; Zanini, D. Effects of resveratrol on
biomarkers of oxidative stress and on the activity of delta aminolevulinic acid dehydratase
in liver and kidney of streptozotocin-induced diabetic rats. Biochimie., 2012, Feb
1,94(2),374-83.
Seet, R. C; Lee, C. Y; Lim, E. C; Tan, J. J; Quek, A. M; Chong, W. L; Looi, W. F; Huang, S.
H; Wang, H; Chan, Y. H; Halliwell, B. Oxidative damage in Parkinson disease:
measurement using accurate biomarkers. Free Radical Biology and Medicine., 2010, Feb
15,48(4),560-6.
Selivanov, V. A; Votyakova, T. V; Pivtoraiko, V. N; Zeak, J; Sukhomlin, T; Trucco, M; Roca,
J; Cascante, M. Reactive oxygen species production by forward and reverse electron fluxes
in the mitochondrial respiratory chain. PLoS computational biology., 2011, Mar
31,7(3),e1001115.
Serra, P. A; Esposito, G; Delogu, MR; Migheli, R; Rocchitta, G; Grella, G; Miele, E; Miele,
M; Desole, MS. Analysis of 3‐morpholinosydnonimine and sodium nitroprusside effects
on dopamine release in the striatum of freely moving rats: role of nitric oxide, iron and
ascorbic acid. British journal of pharmacology., 2000 Oct,131(4),836-42.
Shah, S. B; Nolan, R; Davis, E; Stokin, GB; Niesman, I; Canto, I; Glabe, C; Goldstein,
LS.Examination of potential mechanisms of amyloid-induced defects in neuronal
transport.Neurobiology of disease., 2009, Oct 1,36(1),11-25.

Singh, H. P; Bowman, R. H. Effect of DL-α-lipoic acid on the citrate concentration and

phosphofructokinase activity of perfused hearts from normal and diabetic rats. Biochemical
and biophysical research communications., 1970, Nov 9,41(3),555-61.
Smith, A. R; Shenvi, S. V; Widlansky, M; Suh, J. H; Hagen, T. M. Lipoic acid as a potential
therapy for chronic diseases associated with oxidative stress. Current medicinal chemistry.,
2004, May 1, 11 (9),1135-46.
Smith, D. G; Cappai, R; Barnham, K. J. The redox chemistry of the Alzheimer’s disease
amyloid β peptide. Biochimica et Biophysica Acta (BBA)-Biomembranes., 2007, Aug
1,1768(8),1976-90.
Smith, M. A; Rottkamp, C. A; Nunomura, A; Raina, A. K; Perry, G. Oxidative stress in
Alzheimer’s disease. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease.,
2000, Jul 26,1502(1),139-44.
Smith, M. A; Sayre, LM; Monnier, VM; Perry, G. Radical Ageing in Alzheimer’s disease.
Trends in neurosciences., 1995, Apr 1,18(4),172-6.
Sohal, RS; Ku, HH; Agarwal, S; Forster, MJ; Lal, H. Oxidative damage, mitochondrial oxidant
generation and antioxidant defenses during aging and in response to food restriction in the
mouse. Mechanisms of ageing and development., 1994, May 1,74(1-2),121-33.
Sohal, RS; Sohal, B. H. Hydrogen peroxide release by mitochondria increases during aging.
Mechanisms of ageing and development., 1991, Feb 1,57(2),187-202.
Sorce, S; Krause, K. H. NOX enzymes in the central nervous system: from signaling to disease.
Antioxidants and redox signaling., 2009, Oct 1,11(10),2481-504.
Stadtman, E. R. Role of oxidant species in aging. Current medicinal chemistry., 2004, May
1,11(9),1105-12.
Stamler, J. S; Simon, D. I; Osborne, J. A; Mullins, M .E; Jaraki, O; Michel, T; Singel, DJ;
Loscalzo, J. S-nitrosylation of proteins with nitric oxide: synthesis and characterization of
biologically active compounds. Proceedings of the National Academy of Sciences., 1992,
Jan 1, 89 (1),444-8.
Su, H. C; Hung, L. M; Chen, J K. Resveratrol, a red wine antioxidant, possesses an insulin-like
effect in streptozotocin-induced diabetic rats. American Journal of Physiology-
Endocrinology and Metabolism., 2006 Jun,290(6),E1339-46.
Suh, J. H; Shenvi, S. V; Dixon, B. M; Liu, H; Jaiswal, A. K; Liu, R. M; Hagen, T. M. Decline
in transcriptional activity of Nrf2 causes age-related loss of glutathione synthesis, which is
reversible with lipoic acid. Proceedings of the National Academy of Sciences., 2004, Mar
9,101(10),3381-6.
Szkudelski, T; Szkudelska, K. Resveratrol and diabetes: from animal to human studies.
Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease., 2015, Jun 1, 1852(6),
1145-54.
Tannous, M; Rabini, R. A; Vignini, A; Moretti, N; Fumelli, P; Zielinski, B; Mazzanti, L; Mutus,
B. Evidence for iNOS-dependent peroxynitrite production in diabetic platelets.
Diabetologia., 1999, Apr 1, 42(5), 539-44.
Thornalley, P. J. Glyoxalase I–structure, function and a critical role in the enzymatic defence
against glycation. Biochem Soc Trans., 2003, Dec 1, 31(6), 1343-48.
Tzellos, T. G; Klagas, I; Vahtsevanos, K; Triaridis, S; Printza, A; Kyrgidis, A; Karakiulakis,
G; Zouboulis, CC; Papakonstantinou, E. Extrinsic ageing in the human skin is associated
with alterations in the expression of hyaluronic acid and its metabolizing enzymes.
Experimental dermatology., 2009 Dec, 18(12), 1028-35.

Valensi, P; Le Devehat, C; Richard, J. L; Farez, C; Khodabandehlou, T; Rosenbloom, R. A;

LeFante, C. A multicenter, double-blind, safety study of QR-333 for the treatment of
symptomatic diabetic peripheral neuropathy: a preliminary report. Journal of Diabetes and
Its Complications., 2005, Sep 1, 19(5), 247-53.
Valko, M; Izakovic, M; Mazur, M; Rhodes, C. J; Telser, J. Role of oxygen radicals in DNA
damage and cancer incidence. Molecular and cellular biochemistry., 2004, Nov 1, 266(1-
2), 37-56.
Valko, M; Leibfritz, D; Moncola, J; Cronin, M. D; Mazur, M; Telser, J. Free radicals and
antioxidants in normal physiological functions and human disease. The International
Journal of Biochemistry & Cell Biology., 2007, 39, 44–84.
Valko, MM; Morris, H; Cronin, M. T. Metals, toxicity and oxidative stress. Curr Med Chem.,
2005, May 1, 12 (10), 1161-208.
Van Houten, B; Woshner, V; Santos, J. H. Role of mitochondrial DNA in toxic responses to
oxidative stress. DNA repair., 2006, Feb 3, 5(2), 145-52.
Ved, R; Saha, S; Westlund, B; Perier, C; Burnam, L; Sluder, A; Hoener, M; Rodrigues, CM;
Alfonso, A; Steer, C; Liu, L. Similar patterns of mitochondrial vulnerability and rescue
induced by genetic modification of α-synuclein, parkin, and DJ-1 in Caenorhabditis
elegans. J Biol Chem., 2005, Dec 30, 280(52), 42655-68.
Venugopal, S. K; Devaraj, S; Yang, T; Jialal, I. α-Tocopherol decreases superoxide anion
release in human monocytes under hyperglycemic conditions via inhibition of protein
kinase C-α. Diabetes., 2002, Oct 1, 51(10), 3049-54.
Visioli, F; Bellomo, G; Galli, C. Free radical-scavenging properties of olive oil polyphenols.
Biochemical and biophysical research communications., 1998, Jun 9, 247(1), 60-4.
Wannamethee, S. G; Lowe, G. D; Rumley, A; Bruckdorfer, K. R; Whincup, P. H. Associations
of vitamin C status, fruit and vegetable intakes, and markers of inflammation and
hemostasis–. The American journal of clinical nutrition., 2006, Mar 1, 83(3), 567-74.
Waterman, E; Lockwood, B. Active components and clinical applications of olive oil.
Alternative Medicine Review., 2007, Dec 1, 12(4).
Watt, I. N; Montgomery, M. G; Runswick, M. J; Leslie, A. G; Walker, J E. Bioenergetic cost
of making an adenosine triphosphate molecule in animal mitochondria. Proceedings of the
National Academy of Sciences., 2010, Sep 28, 107(39), 16823-7.
Wei, M. C; Zong, W. X; Cheng, E. H; Lindsten, T; Panoutsakopoulou, V; Ross, A. J; Roth, K.
A; MacGregor, G. R; Thompson, C. B; Korsmeyer, S. J. Proapoptotic BAX and BAK: a
requisite gateway to mitochondrial dysfunction and death. Science., 2001, Apr 27,
292(5517), 727-30.
Wu, L; Davies, G. F; Roesler, W. J; Juurlink, B. H. Regulation of the glyoxalase pathway in
human brain microvascular endothelium: effects of troglitazone and tertiary
butylhydroperoxide. Endothelium., 2002, Jan 1, 9(4), 273-8.
Yaworsky, K; Somwar, R; Ramlal, T; Tritschler, H. J; Klip, A. Engagement of the insulin-
sensitive pathway in the stimulation of glucose transport by α-lipoic acid in 3T3-L1
adipocytes. Diabetologia., 2000, Mar 1, 43(3), 294-303.
Young, IS; Tate, S; Lightbody, J. H; McMaster, D; Trimble, E. R. The effects of
desferrioxamine and ascorbate on oxidative stress in the streptozotocin diabetic rat. Free
Radical Biology and Medicine., 1995, May 1, 18(5), 833-40.
Young, I. S; Woodside, J. V. Antioxidants in health and disease. Journal of clinical pathology.,
2001, Mar 1, 54(3), 176-86.

Zhang, Y; Wang, M; Li, H; Zhang, H; Shi, Y; Wei, F; Liu, D; Liu, K; Chen, D. Accumulation
of nuclear and mitochondrial DNA damage in the frontal cortex cells of patients with HIV-
associated neurocognitive disorders. Brain research., 2012, Jun 6, 1458, 1-1.

SECTION II: ANTIOXIDANT SIGNALING IN
LIFESTYLE CHOICES

Editors: Md. Sahab Uddin et al. © 2019 Nova Science Publishers, Inc.
Chapter 3
ANTIOXIDANT STATUS AND CIGARETTE SMOKING
Tanuj Joshi and Archana Negi Sah*

Kumaun University, Nainital, India
ABSTRACT
Normal aerobic metabolism leads to the emergence of oxidative species. Oxidative
species play a protective as well as a destructive role in the body. Nitric oxide is a mediator
of vasodilation and plays a role in inflammation in normal circumstances but when there
is an excess of nitric oxide it plays a destructive role in the body and is associated with
various pathological conditions. This is the case with any disease in which oxidative
species are involved. Scientists are now studying in detail the link of oxygen and nitrogen
species in the progression of various diseases. Various biomarker studies point towards an
enhancement in oxidative stress due to cigarette smoking and this increased oxidative stress
results in dangerous consequences of smoking cigarette. Thus, scavengers of reactive
oxidative and reactive nitrosative species can counteract the dangerous consequences of
smoking cigarette. The current chapter highlights the status of various antioxidants in
cigarette smoking.
1. INTRODUCTION
In normal circumstances, oxidants are produced in the human body and sufficiently
scavenged by antioxidants present in our body. This balance between the oxidants and
antioxidants is disturbed if the level of oxidants increase to such an extent that they cannot be
scavenged adequately by the antioxidants present in the body (Kelly, 2002a; Kelly, 2002b;
Kelly, 2003). There is an increased accumulation of oxidative species in the bodies of cigarette
smokers which can lead to harmful consequences like deoxyribonucleic acid (DNA) damage,
lipid peroxidation, cancers, cardiovascular diseases, etc. Scientific evidence suggested that
there is a depletion in the level of antioxidants like vitamin C in cigarette smokers (Kelly, 2003).
The low levels of antioxidant in cigarette smokers might be due to the low dietary intake in
*
Corresponding Author’s Email: archanansah@gmail.com.

58 Tanuj Joshi and Archana Negi Sah
them, but in many cases it is seen that the dietary intake of antioxidants like ascorbic acid is the
same in smokers as well as non-smokers, still cigarette smokers have low levels of antioxidants
(Kelly, 2002a; Kelly, 2002b; Kelly, 2003). This might be due to the fact that the constituents
of cigarette smoke like nicotine alter the metabolism, absorption, and elimination of
antioxidants like ascorbic acid and result in their depletion. Thus, supplementation of
antioxidants either in the form of fruits, vegetables, edible plants or individual antioxidants like
vitamin C, reduces the harmful consequences of smoking to a certain extent. Various biomarker
studies have been carried out to study the consequences of antioxidant preparations (Kelly,
2003). Effects of antioxidants on biomarkers associated with DNA damage, nicotine
metabolism, oxidative stress, and endothelial function were studied in various experiments
(Kelly, 2002a; Kelly, 2002b; Kelly, 2003). Different effects on these biomarkers studies were
produced by antioxidants, like ascorbic acid dose-dependently depressed the cytochrome P450
metabolism of nicotine, but endothelial dysfunctions in cigarette smokers could not be
normalized by ascorbic acid. Also in studies, ascorbic acid showed a beneficial effect of
protecting cigarette smokers against atherosclerosis (Kelly, 2002a; Kelly, 2002b; Kelly, 2003).
Thus, antioxidant supplementation reduces oxidative damage in cigarette smokers to a certain
extent, yet there is no full beneficial effect of antioxidants in reducing cancers and
cardiovascular complications in humans. Also in some studies combination of antioxidants
have shown a better effect in reducing harmful effects of cigarette smoking than individual
antioxidants used alone, whereas in other studies the individual antioxidant showed a much
better result than the combination of antioxidants (Kelly, 2002a; Kelly, 2002b; Kelly, 2003).
Lipid peroxidation is a result of smoking cigarette (Pryor et al., 1983; Wiencke et al., 1995).
The attachment of free radicals with unsaturated fatty acids, which are present in cellular
membranes lead to chain reactions and the formation of lipid hydroperoxides (LOOHs).
LOOHs decompose to produce malondialdehyde (MDA). LOOHs as well as malondialdehyde
are both genotoxic and are involved in the formation of adducts of DNA. Generation of these
adducts is involved in cancers and cardiovascular conditions caused by cigarette smoking
(Blair, 2001; Nair et al., 2007a; Yanbaeva et al., 2007). Various biomarkers are evaluated for
assessing the level of damage to the body by smoking cigarette.
Changes in the level of these biomarkers determine the status of oxidative stress or
involvement of antioxidants in decreasing dangerous consequences of smoking cigarette.
Examples of this are like identifying LOOH and MDA-DNA adducts. Also, determination of
DNA adducts in peripheral blood lymphocytes (PBL) is another example of a biomarker
(Nesnow et al., 1993). Some studies have used biomarkers like the metabolism of nicotine,
sperm quality, endothelial function, monocyte adhesion, DNA damage, etc. to evaluate the
benefits of ascorbic acid on cigarette smoking (Kelly, 2003). Thiobarbituric acid reactive
substances (TBARS), which serve to assess MDA are also used as markers of oxidative damage
due to cigarette smoking. Formation of TBARS and the number of cigarettes consumed per day
are related to each other, also smoking of cigarette involve higher MDA levels (Brude et al.,
1997). This chapter represents the linkage of oxidants, antioxidants, and biomarkers associated
with smoking.

Antioxidant Status and Cigarette Smoking 59
2. CIGARETTE SMOKING,
OXIDATIVE STRESS AND ASSOCIATED DISEASES
Numerous harmful molecules are present in cigarette smoke (Figure 1). This gives an idea
about the enhanced rate at which smoking cigarette leads to oxidative stress in humans (Wang
et al., 2013). Different markers have been evaluated to study the oxidative stress produced by
cigarette smoking. These studies have given different results. It was seen that 8-isoprostane,
formed as a result of lipid peroxidation elevated fifteen minutes after acute cigarette smoking.
Also, hydrogen peroxide levels elevated thirty minutes after acute cigarette smoking. At one
and ten minutes the levels of nitric oxide increased after smoking. But in another study nitric
oxide decreased five minutes after acute cigarette smoking (van der Vaart et al., 2004).
The markers associated with oxidative stress were evaluated in bronchoalveolar lavage
fluid (BALF) after cigarette smoking. It was seen that from the BALF leukocytes, the release
of superoxide increased (van der Vaart et al., 2004). There was no difference in intracellular
oxidized glutathione or reduced glutathione levels in leukocytes. Also, the levels of
thiobarbituric acid reactive substances showed no changes in BALF and epithelial fluid lining.
Smoking also had different effects on inflammatory mediators. It was found that there was an
enhancement of activity and recruitment of macrophages and neutrophils. One hour after
cigarette smoking there was an increase in elastase activity in BALF and there was a decrease
in the release of leukotriene (LTB4) from alveolar macrophages. Twenty minutes after acute
cigarette smoking the levels of leukotrienes B4, D4, E4 increased in peripheral blood (van der
Vaart et al., 2004).
Figure 1. Some chemicals present in cigarette smoke. DDT, Dichlorodiphenyltrichloroethane.
As a consequence of smoking six cigarettes, the level of LTE4 increased by two folds in
the urine. After acute cigarette smoking, neutrophil elastase increased immediately and one

hour after smoking (van der Vaart et al., 2004). The outcome of cigarette smoking on the above-
mentioned markers of oxidative stress is variable in terms of species and whether the data is
obtained from animal studies or human research. Cigarette smoke comprises various oxidants.
Oxidants like superoxide anion and nitric oxide that occur in cigarette smoke immediately
interact with one another to generate highly reactive peroxynitrite. The tar-phase of cigarette
smoke contains long-lived semiquinone radicals that can react with superoxide anion to
generate hydrogen peroxide and hydrogen radical. Tar phase of cigarette smoke has the
efficient metal chelating ability, due to this ability it can bind iron and can lead to the formation
of tar-Fe2+ and tar semiquinone. These products are highly devastating as these can
continuously produce hydrogen peroxide. The presence of metal ions especially irons in
cigarette smoke and lung-linning fluid can lead to Fenton reaction with hydrogen peroxide.
This further generates hydroxyl radicals and reactive oxygen species (Faux et al., 2009).
Free radicals released from neutrophils and epithelial cells in lungs can further enhance the
oxidative destruction by cigarette smoking. The imbalance between levels of prooxidants and
oxidants, in favor of oxidants, leads to oxidative stress and damage to airspace epithelial cells
(Isik et al., 2007). Smoking besides increasing oxidants in the body also weakens antioxidant
defenses of our body. Various studies have indicated that the activity of paraoxonase is
inhibited by cigarette smoke extract. Paraoxonase hydrolyzes paraoxon. Paraoxon acts as a
potent inhibitor of cholinesterases. According to scientists, paraoxonase inhibits oxidative
modification of low-density lipoproteins and acts as an antioxidant. Smoking thus weakens this
beneficial effect of paraoxonase by reducing its level (Isik et al., 2007). Thus cigarette smoke
is involved in impairing our antioxidant system. Glutathione is an important antioxidant in the
human body (Faux et al., 2009). It leads to detoxification of electrophilic compounds,
metabolism and signal transduction. Epidermal growth receptor is linked to cancers like lung
cancer. Hydrogen peroxide is an abundant component of the gas phase of smoke. Epidermal
growth factor receptor undergoes aberrant phosphorylation by hydrogen peroxide. This
oxidative stress produced by hydrogen peroxide leads to activation and stabilization of
epidermal growth factor receptor (Faux et al., 2009). Epidermal growth factor receptor’s
enhanced activation in human airway epithelial cells (HAE) leads to activation of protein kinase
B and extracellular signal-regulated kinase. Stabilization of epidermal growth factor receptor
occurs by loss of c-Cbl binding and receptor ubiquitination. The complexity of cigarette smoke
can be the reason behind the toxic consequences of the smoke of cigarette (Faux et al., 2009).
There are numerous disorders that can occur as a consequence of cigarette smoking.
Pulmonary disorders like chronic obstructive pulmonary disease (COPD) are a result of
smoking cigarette. Also, cigarette smoking has an influence on the immune system (Zuo et al.,
2014). In COPD there is a decrease in both forced expiratory volume in 1 second (FEV 1) and
maximum expiratory flow. It is observed that there is a decrease in FEV 1 along with oxidative
stress in patients having cigarette smoke-induced COPD. It is observed that nonvolatile
components contained within lyophilized cigarette smoke extract as well as components like
acetaldehyde and acrolein can cause inhibitory effects on human airway epithelial cell
chemotaxis (Zuo et al., 2014). Thus by the above-mentioned facts, it can be said that cigarette
smoking leads to pathophysiological complications in airways, that is observed in COPD
patients. Numerous experiments point that due to reactive oxygen species (ROS) present in the
smoke of a cigarette, the smoker is exposed to oxidative stress. Both acute and chronic smokers
experience elevated reactive oxygen species by leukocytes in airways. If the production of ROS

becomes excessive, the antioxidants in the body are not able to scavenge it and it results in
damage within the body (Zuo et al., 2014).
Smoking of cigarette produces oxidative stress on the alveolar type II and bronchiolar
epithelial cells. There is a higher expression of superoxide dismutase in central bronchial and
alveolar epithelium of smokers with COPD as compared to nonsmokers (Zuo et al., 2014). This
indicates the increased role of antioxidants during oxidative stress. Involvement of oxidative
stress produced as a consequence of smoking cigarette, in the pathogenesis of COPD has been
explained by certain scientists. According to one of them, ROS activate NF-κBand p38
mitogen-activated protein kinase (MAPK) and this activates proinflammatory/ inflammatory
cytokine and chemokine genes. Increased elastolysis and accelerated damage to the lungs is a
consequence of an imbalance of endogenous proteases/antiproteases by ROS (Zuo et al., 2014).
Excessive lipid peroxidation, DNA damage and protein carbonylation of airway endothelial
and alveolar cells by ROS enhances the symptoms of COPD (Zuo et al., 2014). Cigarette
smoking has harmful consequences on skeletal muscles. Lower peripheral muscle fatigue
resistance has been demonstrated in smokers when compared to non-smokers (Zuo et al., 2014).
Oxidative modifications change muscle proteins in a way that they become more susceptible to
break down and would produce loss of muscle and dysfunction in smokers suffering from
COPD. On exposure of animals to cigarette smoke, it was found that three months after
exposure to cigarette smoke oxidative stress was produced in their muscles (Zuo et al., 2014).
The above-mentioned event occurred before the characteristic bronchiolar and parenchymal
changes induced by cigarette smoking in the lungs. This study demonstrates that cigarette
smoking on skeletal muscle proteins produces a direct toxic effect (Barreiro et al., 2010).
Antigen-antibody complexes can be formed due to antigenic chemicals in cigarettes and these
complexes can induce pulmonary and peripheral injuries. There is involvement of cigarette
smoke in restriction of anti-inflammatory mediators, activation of inflammatory cells and
release of proinflammatory cytokines. Due to cigarette smoking, there is an enhancement of
circulation of inflammatory molecules like acute-phase proteins and proinflammatory
cytokines (Zuo et al., 2014).
The levels of interleukins (IL-1, IL-6, IL-8), TNFα, TNFα receptor, and granulocyte-
macrophage-colony-stimulating factor are elevated by cigarette smoking. The increase in the
levels of cytokines and/or chemokines after cigarette smoking acts as a chemoattractant. This,
in turn, enhances the recruitment of dendritic cells, macrophages, and neutrophils and
ultimately enhances the inflammatory cascade (Zuo et al., 2014). The inflammatory process
continues with the recruitment of new inflammatory cells through secretion of cytokines,
phagocytosis, and initiation of both reactive oxygen species and surface antigen formation to
mediate adaptive and innate immune responses. Smoking of cigarette enhances the number of
polymorphonuclear neutrophils but decreases their phagocytic and antimicrobial activity (Zuo
et al., 2014). Above mentioned facts lead to the conclusion that the immune system of cigarette
smokers has decreased the ability to fight bacterial infections as compared to persons who do
not smoke and bacterial infections enhance the symptoms of cigarette smoking. Various
components of the innate immune system like natural killer cells, neutrophils, dendritic cells,
etc. play a role in the initiation and progression of COPD (Zuo et al., 2014).
T cells play an important role in COPD. Mediators derived from CD8+ T cells in the later
stages of COPD lead to alveolar wall destruction, inflammation and small airway fibrosis in
later stages of COPD. Neutrophils and lymphocytes level is increased in smokers and these

results are consistent with the findings mentioned in the previous lines. Enhancement of CD8+
T cells and a subsequent reduction of CD4+/CD8+ T cell ratio has been seen in airway
submucosa of smokers. Elevated levels of CD8+ T cells are seen in smokers suffering from
COPD in comparison with smokers without COPD (Zuo et al., 2014).
Smoking cessation is mainly encouraged at a younger age, and much attention is not paid
to elderly smokers. Age-related decrease in antioxidant activity in alveolar macrophages occurs
due to smoking (Donohue, 2006). As compared to younger adults very few attempts have been
performed in elderly to reduce oxidative stress associated with smoking by antioxidant
supplementation and by increasing antioxidant stores within the body. Also, older smokers are
less likely to be advised to stop smoking than their younger counterparts. But the cessation of
smoking at any age leads to a decreased decline rate in FEV 1, a decrease in risks of myocardial
infarction and stroke. Thus smoking cessation might be beneficial at any age (Donohue, 2006).
Smoking of cigarette also results in age-related macular degeneration (AMD). Blindness
in the elderly is frequently associated with AMD. Probably the development of AMD is related
to oxidative stress produced by smoking (Cano et al., 2010). Smoking also leads to various
cardiovascular diseases. Cardiovascular complications like myocardial infarction and sudden
death are associated with smoking. Endothelial dysfunction and cardiovascular events arise due
to an imbalance between the activity of antioxidant defenses and oxidants (Glantz and Parmley,
1996; Celermajer et al., 1993; Raij et al., 2001; Morrow et al., 1995; Heitzer et al., 2001;
Tanriverdi et al., 2006).
Superoxide radical and other reactive oxygen species can inactivate nitric oxide and cause
destructive effects on the cardiovascular system. Peroxynitrite is formed by the reaction of
superoxide with nitric oxide. It is a potent oxidant found in atherosclerotic lesions and is
identified as nitrotyrosine. In the arterial wall superoxide dismutase (SOD) plays an important
role of an antioxidant enzyme. The strategic location of this enzyme is between endothelium
and vascular smooth muscle cells. SOD is insufficient amount in the arterial wall to counter the
harmful effects of superoxide, such as formation of highly destructive peroxynitrite with nitric
oxide (Glantz and Parmley, 1996; Celermajer et al., 1993; Raij et al., 2001; Morrow et al., 1995;
Heitzer et al., 2001; Tanriverdi et al., 2006). Another important enzyme that counters the
harmful effects of oxidants is reduced glutathione. Smoking leads to elevation of oxidants and
reduction of antioxidants (Glantz and Parmley, 1996; Celermajer et al., 1993; Raij et al., 2001;
Morrow et al., 1995; Heitzer et al., 2001; Tanriverdi et al., 2006). Thus cardiovascular diseases
are associated with smoking and smoking plays the role of a major risk factor for the production
of atherosclerosis. Impairment of endothelium-dependent vasodilation occurs in microvascular
beds as well as macrovascular beds. Increased production of isoprostanes is a consequence of
cigarette smoking, and these are products of lipid peroxidation (Glantz and Parmley, 1996;
Celermajer et al., 1993; Raij et al., 2001; Morrow et al., 1995; Heitzer et al., 2001; Tanriverdi
et al., 2006). The endothelial damage in smokers might also be due to enhancement in levels of
endothelial angiotensin II, which decreases the activity of nitric oxide. Reduced serum
plasminogen levels and enhanced platelet and serum fibrinogen levels, results in impaired
endothelial function in smokers. MDA levels are also increased in smokers (Glantz and
Parmley, 1996; Celermajer et al., 1993; Raij et al., 2001; Morrow et al., 1995; Heitzer et al.,
2001; Tanriverdi et al., 2006).
Antioxidants have a significant role in the body by guarding the body against the dangerous
consequences of free radicals particularly in conditions like ischemia. In certain studies, it has
been found that smokers have a high level of MDA and SOD. SOD levels might have increased

in smokers as a compensatory measure to counteract oxidative damage. Smokers also had low
glutathione compared to control subjects (Tanriverdi et al., 2006). Various studies have shown
active or passive smoking results in vasomotor dysfunction, atherogenesis, and thrombosis in
multiple vascular beds. Oxidative stress due to free radicals is associated with the formation of
atherothrombotic diseases in cigarette smoking. Oxidative stress due to cigarette smoking leads
to the development of endothelial dysfunction and in individuals with angiographically normal
coronary arteries produces slow flow in coronary arteries (Tanriverdi et al., 2006). A number
of carcinogens have been detected in cigarette smoke and tar. There are around 60 known
mutagens and carcinogens found in cigarette smoke.
3. STATUS OF ANTIOXIDANTS IN CIGARETTE SMOKING

3.1. Ascorbic Acid
In the bodies of smokers, low levels of ascorbic acid are found. This might be due to a lack
of ascorbic acid in the diet of smokers or its altered metabolism in smokers (Kelly, 2003). Like
in smokers there can be decreased absorption of ascorbic acid, increased metabolic demands,
increased elimination of ascorbic acid from the body. The points mentioned above alone or
together can lead to a decrease in the level of ascorbic acid in smokers (Raitakari et al., 2000;
Lykkesfeldt et al., 2000; Smith and Hodges, 1987; Kelly, 2003).
A study comparing the serum level of ascorbic acid was performed by scientists in those
who smoked and those who did not smoke and it was found that though smokers took almost
the same diet as nonsmokers, there was a relative vitamin C deficiency in smokers compared
to nonsmokers (Smith and Hodges, 1987). Thus the scientists involved in the study, Smith, and
Hodges made the observation that due to relative ascorbic acid deficiency smokers should
consume 59 mg of ascorbic acid additionally (Smith and Hodges, 1987). Also, scientists like
Schectman made observations that the serum of smokers contained low levels of ascorbic acid
as compared to nonsmokers. It was noted that despite providing sufficient ascorbic acid to
smokers, the serum levels of ascorbic acid was found to be low in smokers in comparison to
nonsmokers. This could be due to changes in the metabolism of ascorbic acid in smokers due
to cigarette smoke (Schetman, 1993).
The dietary and supplemental intake of ascorbic acid was also found to be low in people
smoking a large number of cigarettes (Schetman, 1993). Thus, by the above studies, one thing
becomes clear that there is both dietary deficiency of ascorbic acid in smokers as well as the
altered metabolism of ascorbic acid in smokers. The increased amount of ascorbic acid can thus
be recommended in smokers. Various biomarker experiments were performed to determine the
effect of ascorbic acid supplementation on modification of biomarkers associated with cigarette
smoking (Kelly, 2003). It was found that there was a modification in some of these biomarkers
in a positive manner by ascorbic acid, whereas in other cases no effect was observed on these
biomarkers. Nicotine is metabolized by cytochrome P450 to cotinine metabolites, and these
cotinine metabolites are more toxic than nicotine. Ascorbic acid dose-dependently exerts a
beneficial effect by depressing the cytochrome P450 metabolism of nicotine (Kelly, 2003).
Ascorbic acid did not have a very positive impact on reducing oxidative stress as observed
in some studies. Positive modification of in vitro low-density lipoprotein (LDL) oxidation

kinetics, or in vivo lipid peroxidation measured through TBARS was not produced by ascorbic
acid (Kelly, 2003). Endothelial function was not positively modified by ascorbic acid
supplementation but ascorbic acid did have a favorable effect on monocyte adhesion to
endothelial cells when it was given as a supplement for a short period of ten days. The above-
mentioned actions associated with ascorbic acid can provide a favorable effect on the
prevention of atherosclerosis. Dose at which ascorbic acid should be used in smokers varies
with respect to the number of cigarettes smoked daily by the individual, genetics, dietary habits
and lifestyle of the individual (Kelly, 2003). This same dose of ascorbic acid cannot be given
to all individuals. Various doses of ascorbic acid had various effects on biomarkers of oxidative
stress, due to cigarette smoking. At a dose of 515 mg given daily for two months and 2 g given
daily for five days, 2 g daily dose of ascorbic acid showed improvement in monocyte adhesion.
Also, ascorbic acid in doses of 200 mg daily and 1 g daily was used in a study to improve sperm
quality in cigarette smokers (Kelly, 2003). It was found that with the low dose of ascorbic acid
no significant effect was observed in the improvement of sperm quality, but at a higher dose, a
reduction in the percent of total sperm agglutination and abnormalities in morphology was
observed. Thus the above-mentioned observations suggest that a daily dose of ascorbic acid
ranging from 500-2000 mg given for short periods sufficiently produced positive modification
of some aspects of disturbed functions in smokers (Kelly, 2003).
Certain studies also point that with respect to TBARS formation ascorbic acid acted as a
prooxidant. This fact was supported by a study in which ascorbic acid was given at a daily dose
of 500 mg for two months in cigarette smokers. In this study, ascorbic acid increased the
byproducts of lipid peroxidation (Kelly, 2003). Thus the safety of ascorbic acid when used at
high doses and for long periods should also be checked, when ascorbic acid is being used to
decrease the harmful effects of cigarette smoking. Also in certain studies, ascorbic acid was
used in combination with other antioxidants to counter the harmful effects of cigarette smoking
(Kelly, 2003). It was observed in these studies that in some situations ascorbic acid exerted a
beneficial effect in combination with other antioxidants, whereas in other situations ascorbic
acid was better than the combination. For example, ascorbic acid in combination with alpha-
tocopherol was useful in preventing the decrease in the levels of carotenes (Kelly, 2003). Also,
this combination was effective in positively modifying aspects of in vitro LDL-lipid
peroxidation kinetics. But this combination was not useful in preventing damage caused to
DNA. In a study, a combination of antioxidants which consisted of ascorbic acid, lipoic acid,
and tocopherols was less efficient in reducing the levels of F2-isopentanes than ascorbic acid
used alone (Kelly, 2003). Combined preparation of α-tocopherols, β-carotene, and ascorbic
acid, added to a tomato juice base had a positive impact on breath pentane output. Breath
pentane output (BPO) is evaluated by collecting exhaled air and determining the number of
pentanes (Kelly, 2003). The formation of pentanes in the body occurs as a result of peroxidation
of polyunsaturated fatty acids (PUFAs). A fraction of these pentanes is volatile and its
elimination occurs in respiration. Therefore BPO provides a functional surrogate measure of
oxidative stress to lipids. It has been suggested that BPO is enhanced as oxidative stress
increases. Thus this combination was effective in reducing lipid peroxidation (Kelly, 2003).
It is observed in smokers that the rate of disappearance of vitamin E from their bodies is
enhanced due to oxidative stress caused by smoking (Bruno et al., 2006). An experiment found
that ascorbic acid supplementation for two weeks was beneficial in reducing the disappearance
of α- and β-tocopherols in cigarette smokers. The reason for the decrease in the disappearance
of vitamin E could be due to the fact that ascorbic acid protects α-tocopherol from oxidation

and regenerates a reduced α- tocopherol from an α-tocopheroxyl radical (Bruno et al., 2006).
The reduction caused in the disappearance rates of α-tocopherol and β-tocopherol produced by
ascorbic acid may be due to the property of ascorbic acid to decrease an oxidized tocopherol
moiety rather than prevent lipid peroxidation. Thus, according to this study oxidative stress
caused due to cigarette smoking leads to enhanced disappearance of α-tocopherol and β-
tocopherol in smokers. Supplementation with ascorbic acid reduced the disappearance rates of
these tocopherols (Bruno et al., 2006).
3.2. Carotenoids, Vegetables and Fruits
It has been suggested epidemiologically that chronic cigarette smokers consume fewer
vegetables and fruits (Kelly, 2002a). Thus, they take less amount of phytonutrient-rich food.
They make up for the calorie deficit by taking more fats in the diets, especially saturated fats.
Trials carried out in 282 patients of lung cancers and an equal number of controls suggested a
protective effect of vegetable and fruit intake on lung cancer (Kelly, 2002a). With the increase
in the frequency of vegetable and fruit intake, the protective role of cigarette smoking also
increases. The value of different fruits and vegetables differ in their protective effects on
cigarette smokers. Thus individual vegetables and fruits should be evaluated for their beneficial
effect on cigarette smoking (Kelly, 2002a). It has been observed that carrots and broccoli have
a special place in protecting cigarette smokers from the harmful effects of cigarette smoking.
Data from nurses’ health study indicate that alpha-carotene is the primary carotenoid effective
in lung cancer. Various studies have found that supplementation with certain antioxidants in
cigarette smokers is not always beneficial to reduce the harmful effects of cigarette smoking.
The antioxidant supplementation might also be harmful (Kelly, 2002a). An example of this
finding is that of beta-carotene. Studies have suggested that beta-carotene shows variable
responses in smokers; also a combination of other nutrients and beta-carotene did not produce
any benefit to smokers of cigarette. Also, biomarker studies carried out in smokers, have not
suggested any positive role of beta-carotene in smokers (Kelly, 2002a). Thus beta-carotene
supplementation is not justified in smokers and it should even be avoided. Thus, by the above-
mentioned findings, it can be suggested that eating of whole vegetables and fruits with
antioxidant activities might be beneficial in reducing the harmful effects of cigarette smoking,
but this fact also is not very well proven and a large number of extensive studies are needed to
support this fact (Kelly, 2002a).
3.3. α-Tocopherol
α-Tocopherol was also analyzed in many studies to ascertain its beneficial effect on
cigarette smokers. In these studies, it was found that α-tocopherol supplementation increased
the levels of α-tocopherol in serum and tissues (Kelly, 2002b). On the other hand, α-tocopherol
might have undesired effects on the level of other nutrients like ascorbic acid, β-carotene,
lycopene, γ- and β-tocopherols. Additional pharmacokinetic interaction experiments are
necessary to evaluate whether administration of α-tocopherol influences the level of other
antioxidants or not (Kelly, 2002b). According to the different biomarker studies, the
administration of α-tocopherol might be useful, have no influence or may even be dangerous
in cigarette smokers.

Alpha-tocopherol has a positive effect on certain markers associated with in vitro lipid
peroxidation but not much beneficial effect was exerted on these markers in vivo (Kelly,
2002b). There was some decrease in BPO levels over a course of two weeks by supplementation
with α-tocopherol, but this was not sufficient as the decrease in BPO levels was nowhere near
that found in nonsmokers. Alpha-tocopherol also did not have a positive impact on F2-
isopentane levels and did not alter DNA damage. Also, α-tocopherol did not produce any
positive modification in endothelial function, with the exception of patients with
hypercholesterolemia. Thus, until very strong evidence is obtained about the efficacy of α-
tocopherol in preventing the harmful effects of cigarette smoking, it should be used with caution
(Kelly, 2002b).
3.4. Natural Products and Herbs
There are also many natural products and herbs which due to their antioxidant activity
might be very useful in decreasing dangerous consequences associated with cigarette smoking.
In a study, the antioxidant property of honey was studied to reduce oxidative stress in smokers
(Ghazali et al., 2015). Supplementation of tualang honey was studied for a period of 12 weeks,
on the levels of superoxide dismutase (SOD), F2 isoprostanes, catalase (CAT), glutathione
peroxidase (GPx) and total antioxidant status (TAS) among chronic smokers (Ghazali et al.,
2015).
Around 64 chronic smokers and 34 nonsmokers were taken in the experiment (Ghazali et
al., 2015). The smokers were randomly distributed into two groups. Every group contained 32
individuals. One group received no supplementation with honey, another group received
supplementation with honey in a dose of 20 g/day for a total period of 12 weeks (Ghazali et al.,
2015). Blood samples were taken from nonsmokers only once. Blood was taken from smokers
twice, before and after supplementation with honey. The blood samples were analyzed for the
oxidative stress status of the individuals in different groups. It was found that in smokers before
receiving supplementation with honey the activity/level of catalase, F2-isoprostanes, total
antioxidant status of the body was significantly higher, whereas the levels of superoxide
dismutase and glutathione peroxidase were found to be lower than nonsmokers (Ghazali et al.,
2015). After supplementation with honey, the activity/level of F2-isoprostanes was decreased
and total antioxidant status of the body, glutathione peroxide, catalase activity/level were
increased in smokers as compared to the smokers which did not receive supplementation with
honey (Ghazali et al., 2015).
No significant difference was found in smokers receiving supplementation with honey
regarding SOD pre and posttest levels. In smokers which were not given honey
supplementation, there were no significant differences for plasma F2-isoprostanes, total
antioxidant status, as well as the activities of glutathione peroxidase and catalase, but there was
a significant decrease in activity of erythrocyte superoxide dismutase after 12 weeks (Ghazali
et al., 2015). F2-isoprostane in smokers served as an indicator for lipid peroxidation in the
experiment. It was found that the levels of F2-isoprostanes were lowered in smokers that
received supplementation with honey. The results also indicate that levels of SOD remained
unchanged in smokers that receive honey supplementation. This indicates that a lower
formation of free radicals occurred due to the free radical scavenging effect of honey (Ghazali
et al., 2015). In nonsmokers there was a reduction in the level of SOD, this could be due to

inactivation of SOD by reactive oxygen species. Before supplementation, the levels of GPx
was found to be lower, but after giving supplementation with honey the levels of GPx were
increased and this, in turn, decreased the oxidative stress in smokers (Ghazali et al., 2015).
Before supplementation smokers demonstrated a higher level of CAT, this increase could be
due to the capability of this antioxidant to overcome the enhancement in oxidative stress by
continuously converting hydrogen peroxide into water and oxygen (Ghazali et al., 2015).
Also, the enhanced levels of CAT after treating smokers with honey indicated the increased
bioavailability of CAT to scavenge hydrogen peroxide by honey supplementation (Ghazali et
al., 2015). The levels of TAS were found to be higher in smokers as compared to nonsmokers,
this probably reflects the compensatory measures taken by the body of the smoker to fight the
increase in oxidative stress. The significant rise in TAS after giving honey supplementation to
smokers might be due to the capability of honey to enhance plasma antioxidant capacity to
reduce oxidative stress (Ghazali et al., 2015). This fact is supported by the increase in levels of
CAT and GPx found after supplementing smokers with honey. Tulang honey, found in regions
like Malaysia, contains a number of antioxidants like phenolic compounds such as trans-
cinnamic acids, gallic acid, syringic acid, and benzoic acid. Also, a number of flavonoids like
kaempferol and catechin are present in it. All these chemicals have strong free radical
scavenging activity (Ghazali et al., 2015). Thus, supplementation of Tulang honey in smokers
produced a beneficial effect in reducing oxidative stress due to its antioxidant ability. Thus
honey supplementation can be a very good remedy to counteract the harmful effects of smoking
in active as well as passive smokers (Ghazali et al., 2015).
Another study was conducted which involved the use of medicinal herbal tea in reducing
smoking withdrawal symptoms and helping in smoking cessation. In this study, around twenty-
one, medicinal herbs were studied for nicotine degradation (ND) activity and antioxidant
activity in vitro (Lee and Lee, 2005). Among these twenty-one herbs, eleven herbs that showed
the highest antioxidant activity and nicotine degradation activity in vitro were chosen for the
preparation of medicinal herbal tea (MHT) (Lee and Lee, 2005). Evaluation of this tea for the
cessation of smoking and decreasing withdrawal symptoms were studied in 100 smokers (Lee
and Lee, 2005). The eleven plants chosen for the preparation of medicinal herbal tea showing
highest antioxidant and ND activity were as follows: Glycyrrhiza uralensis Fisch, Ligusticum
tenuissimum Nakai, Raphani seed, Platycodon grandiflorum (Jacq.) A. DC., Crataegus
pinnatifida, Aloe arborescens, Acathopanax sessiliflorus (Ruprecht et Maximowicz) Seemann,
Anthricus sylvestris Hoffmann, Eugenia aromaticum, Citrus reticulate, Polygonum
multiflorum (Lee and Lee, 2005).
Around 100 male volunteers were used in the study who smoked cigarettes (Lee and Lee,
2005). They were taken from schools, government organizations, and companies. Eighty
individuals were given MHT and twenty individuals were given placebo tea. MHT and placebo
tea was administered three times a day at a dose of 3 gm with 200 ml of warm water (Lee and
Lee, 2005). The IC50 produced by MHT was 50.6 µg/ml and ND activity value shown was 1.24.
Before the start of the experiments, subjects were asked about smoking habits through the use
of Fagerstrom questionnaire sheet. The individuals were evaluated on smoking withdrawal
symptoms, the basal level of craving and number of cigarettes that were smoked in a day (Lee
and Lee, 2005). Interviews and urinary analysis for cotinine levels, which is a metabolite of
nicotine, was done on 0, 1st, 2nd, 3rd and 4th weeks. Whether the individuals stopped cigarette
smoking or not was evaluated with the help of survey data and cotinine content in urine (Lee
and Lee, 2005).

The daily consumption of cigarettes was determined by measuring cotinine content in

urine. Linkert scale 1 (very low) to 5 (very high) was used to evaluate cigarette taste and craving
(Lee and Lee, 2005). The value obtained from the first week was subtracted from the value
obtained after the fourth week. Negative and positive scores indicated a decrease or increase in
the effects respectively, in terms of the taste of cigarette and craving. Total withdrawal scale
(TWS) was used to evaluate smoking withdrawal symptoms. Five important parameters were
included in this scale like increased appetite, insomnia, depressed mood, difficulty in
concentration and restlessness scored on a Linkert scale 1 (very low) to 5 (very high) (Lee and
Lee, 2005). Total scores sum ranged from 5 to 25. Severe smoking withdrawal symptoms were
shown by high score. Total withdrawal scale’s testing was done at 0, 2nd and 4th week. It was
found in the study that in the MHT treated group there was an increase in cotinine levels in the
first two weeks, followed by a decline in the cotinine level from the second to the fourth week.
The smoking withdrawal symptoms in the MHT treated group was decreased when compared
with non-MHT taking group (Lee and Lee, 2005). Around 38% of people taking MHT were
successful in the cessation of smoking as compared to the non-MHT group in which only 12%
people stopped smoking (Lee and Lee, 2005). Also, it was observed that the MHT treated group
showed a reduction in the taste and craving for cigarettes as compared to the placebo group.
Thus MHT successfully helped in reducing the cigarette taste, craving, withdrawal symptoms
in cigarette smokers and even helped them to stop cigarette smoking. The long-term benefits
of this study still need to be explored. Also, there might be some contribution of the synergistic
antioxidant activity of the eleven selected herbs in the beneficial effects of MHT on cigarette
smoking (Lee and Lee, 2005).
Tahitian noni juice (Morinda citrifolia) was used in another study to evaluate its effect in
reducing cancer risk in cigarette smokers (Wang et al., 2009). The parameter evaluated in this
study was the measurement of aromatic DNA adducts since a determination of these adducts
in peripheral blood lymphocytes acts as a useful surrogate biomarker in assessing carcinogen
exposure and or chemopreventive intervention. The trial was carried for a period of one month
(Wang et al., 2009). Smokers were divided into groups, one of which received 1 oz of noni
juice and the other group received 4 oz of noni juice. An equal number of healthy male and
female smokers was used in these groups (Wang et al., 2009). A control group, consisting of
nonsmokers was also used in this study so as to obtain a baseline value for aromatic DNA
adduct, that can be used for comparison between smokers and nonsmokers. Nuclease P1
procedure of 32P-post-labelling assay was the method used for determining DNA adducts
(Wang et al., 2009). It was found in the study that by drinking 1-4 oz of noni juice significant
reduction in aromatic DNA adducts by around 44.9% occurred in all the smokers who
participated in the study (Wang et al., 2009). Dose dependently there was a reduction of 49.7%
in aromatic DNA adducts level in the participants who received 1 oz of noni juice and there
was a reduction of 37.6% in the level of aromatic DNA adducts in participants who received 4
oz of noni juice (Wang et al., 2009).
No significant difference was noted on the basis of gender differences on the effect of noni
juice in the reduction of aromatic DNA adduct levels in the group that received 4 oz of noni
juice, whereas a significant difference on the basis of gender was observed in case of the group
that received 1 oz of noni juice (Wang et al., 2009). The percentage decrease in the level of
aromatic DNA adducts shown by males in the 1 oz noni juice group was 43.1% and in females,
the reduction was of 56.1%. The findings of these study point that by consuming 1-4 oz of

Tahitian noni juice the chances of cancer in individuals who smoke a large number of cigarettes
can be reduced (Wang et al., 2009).
Experiments were done on the ability of noni juice to reduce DNA adducts and cancer in
smokers of cigarette. A double-blind, placebo-controlled and the randomized clinical trial were
performed on 245 heavy cigarette smokers (Wang et al., 2013). The effect of the juice of noni
was studied on lipid hydroperoxides (LOOH) and MDA-DNA adducts in peripheral blood
lymphocytes. Volunteers were divided into groups such that some received 118 ml placebo
juice, some 29.5 ml noni juice and some 118 ml noni every day for one month. Both male and
females were recruited in equal proportions (Wang et al., 2013). The levels of LOOH and
MDA-DNA adducts were determined because both these parameters point towards the
oxidative damage caused by cigarette smoking. Noni treatment groups received Tahitian noni
juice whereas the placebo group received a combination of blueberry juice and grape juice. The
cheese flavor was also incorporated in this combination to resemble the taste of Tahitian noni
juice (Wang et al., 2013).
The placebo juice was also considered as a control in which iridoid glycosides, which are
important phytochemicals of noni juice were not present. LOOH-DNA and MDA-DNA adduct
levels were analyzed before and after treatment with noni juice. Evaluation of the adducts was
done by 32P-post-labelling analysis (Wang et al., 2013). It was found in the study that
consuming 29.5 ml to 118 ml of the juice of noni significantly decreased the levels of LOOH-
DNA adducts as well as MDA-DNA adducts by 44.6 to 57.4% in cigarette smokers (Wang et
al., 2013). Whereas no significant reduction in the above-stated parameters was observed with
smokers treated with placebo juice. Phytochemical analysis of the placebo and noni juice
revealed that chlorogenic acid, rutin, total polyphenols were found in both the placebo and noni
juices, but iridoid glycosides were found only in noni juice (Wang et al., 2013). The iridoid
glycosides like deacetylasperulosidic acid and asperulosidic acid contributed significantly
towards the protective effect of the juice of noni on cigarette smoking, whereas the placebo
juice which was devoid of the iridoids had no significant reducing effect on the levels of LOOH
and MDA-DNA adducts. The beneficial effects of noni juice in cigarette smokers may be due
to its iridoid content since iridoids have antioxidants activities (Wang et al., 2013).

AND CHALLENGES
The present chapter gives an idea about the role of cigarette smoke in producing oxidative
stress and the diseases produced subsequent to the smoking of cigarettes. The chapter throws
light on the status of antioxidants in countering the harmful effects of smoking cigarettes. As
mentioned in the current chapter studies have shown beneficial effects also as well as harmful
effects also of antioxidants in relation to smoking of cigarettes. Thus, the present studies are
not sufficient to establish the status of antioxidants in smoking.
Antioxidants in the future should be evaluated by long-term studies at genetic and
molecular levels. Only by going to the level of genes and molecules we will be able to
understand newer mechanisms by which cigarette smoking produces harmful consequences in
the human body and then only the status of antioxidants in cigarette smoking will become clear.
The challenges associated with these studies will be that these studies will be long, laborious

and very expensive, but will be necessary to establish the beneficial effects of antioxidants in
cigarette smoking. The use of gene mapping and pharmacogenomics should establish in future
which antioxidants should be used in a particular individual and which antioxidants should be
avoided to counter the harmful consequences of smoking cigarette. Thus, these studies can
dictate the future role of antioxidants in smokers of cigarettes (Saha et al., 2007).
CONCLUSION
Antioxidants like ascorbic acid counteract the harmful effects of cigarette smoking whereas
antioxidants like beta-carotene can actually be harmful in cigarette smoking. Combination of
antioxidants was found to be better than individual antioxidant in some studies whereas in other
studies individual antioxidant showed better effect than the combination. Also, some
researchers suggested that eating whole vegetables and fruits might be useful in cigarette
smoking, whereas isolated antioxidants can cause harmful effects in the body. Thus, in
conclusion, it can be said that extensive experiments are required for evaluation of the impact
of antioxidants on cigarette smokers.
REFERENCES
Barreiro E, Peinado V. I, Galdiz J. B., Ferrer E, Marin-Corral J, Sa’nchez F et al. Cigarette
smoke–induced oxidative stress: A role in chronic obstructive pulmonary disease skeletal
muscle dysfunction. Am J Respir Crit Care Med. 2010; 182(4):477-488.
Blair I. A. Lipid hydroperoxide-mediated DNA damage. Exp Gerontol. 2001; 36(9):1473-1481.
Brude I. R, Drevon C. A, Hjermann I, Seljeflot I, Lund-Katz S, Saarem K et al. Peroxidation
of LDL from combined-hyperlipidemic male smokers supplied with omega-3 fatty acids
and antioxidants. Arterioscler Thromb Vasc Biol. 1997; 17(11):2576-2588.
Bruno R. S, Leonard S. W, Atkinson J, Montine TJ, Ramakrishnan R, Bray TM et al. Faster
plasma vitamin E disappearance in smokers is normalized by vitamin C supplementation.
Free Radic Biol Med. 2006; 40(4):689-697.
Burlakova E. B, Zhizhina G. P, Gurevich S. M, Fatkullina L. D, Kozachenko A. I, Nagler L. G
et al. Biomarkers of oxidative stress and smoking in cancer patients. J Cancer Res Ther.
2010; 6(1):47-53.
Cano M, Thimmalappula R, Fujihara M, Nagai N, Sporn M, Wang AI et al. Cigarette smoking,
oxidative stress, the anti-oxidant response through Nrf2 signaling, and age-related macular
degeneration. Vision Res. 2010; 50(7):652-664.
Celermajer D. S, Sorensen K. E, Georgakopoulos D, Bull C, Thomas O, Robinson J et al.
Cigarette smoking is associated with doserelated and potentially reversible impairment of
endothelium-dependent dilation in healthy young adults. Circulation. 1993; 88:2149–
2155.
Donohue J.F. Ageing, smoking and oxidative stress. Thorax. 2006; 61(6):461-462.
Faux S. P, Tai T, Thorne D, Xu Y, Breheny D, Gaca M. The role of oxidative stress in the
biological responses of lung epithelial cells to cigarette smoke. Biomarkers. 2009;
14(S1):90-96.

Ghazali W. S. W, Mohamed M, Sulaiman SA, Aziz A. A, Yusoff H. M. Tualang honey

supplementation improves oxidative stress status among chronic smokers. Toxicol Environ
Chem. 2015; 97(8):1017-1024.
Glantz S. A, Parmley W. W. Passive and active smoking: A problem for adults. Circulation.
1996; 94(4):596-598.
Heitzer T, Schlinzig T, Krohn K, Meinertz T, Munzel T. Endothelial dysfunction, oxidative
stress and risk of cardiovascular events in patients with coronary artery disease.
Circulation. 2001; 104(22):2673–2678.
Isik B, Ceylan A, Isik R. Oxidative Stress in smokers and non-smokers. Inhal Toxicol. 2007;
19(9):767-769.
Kelly G. The interaction of cigarette smoking and antioxidants. Part II: alpha-tocopherol. Altern
Med Rev. 2002b; 7(6):500-511.
Kelly G. The interaction of cigarette smoking and antioxidants. Part I: diet and carotenoids.
Altern Med Rev. 2002a; 7(5):370-388.
Kelly, G. The interaction of cigarette smoking and antioxidants. Part III: Ascorbic Acid. Altern
Med Rev. 2003; 8(1):43-54.
Lee H. J., Lee J. H. Effects of medicinal herb tea on the smoking cessation and reducing
smoking withdrawal symptoms. Am J Chin Med. 2005; 33(1):127-138.
Lykkesfeldt J, Christen S, Wallock L. M., Chang H. H., Jacob R. A., Ames, B. N. Ascorbate is
depleted by smoking and repleted by moderate supplementation: a study in male smokers
and nonsmokers with matched dietary antioxidant intakes. Am J Clin Nutr. 2000;
71(2):530-536.
Morrow J.D., Frei B, Longmire A.W., Gaziano J.M., Lynch S.M., Shyr Y et al. Increase in
circulating products of lipid peroxidation (F2-isoprostanes) in smokers: Smoking as a cause
of oxidative damage. N Engl J Med. 1995; 332 (18):1198– 1203.
Nair J, De Flora S, Izzotti A, Bartsch H. Lipid peroxidation-derived etheno-DNA adducts in
human atherosclerotic lesions. Mutat Res. 2007; 621(1-2):95-105.
Nesnow S, Ross J, Nelson G, Holden K, Erexson G, KligermanA et al. Quantitative and
temporal relationships between DNA adduct formation in target surrogate tissues:
implications for biomonitoring. Environ Health Perspect. 1993; 101(S3):37-42.
Pryor W. A, Prier D. G., Church D. F. An electro-spin resonance study of mainstream and side
stream cigarette smoke: nature of the free radicals in gas-phase smoke and in cigarette tar.
Environ Health Perspect. 1983; 47:345-355.
Raij L, DeMaster E. G., Jaimes E. A. Cigarette smoke-induced endothelium dysfunction: Role
of superoxide anion. J Hypertens. 2001; 19(5):891 – 897.
Raitakari O. T, Adams M. R, McCredie R. J, et al. Oral vitamin C and endothelial function in
smokers: short-term improvement, but no sustained beneficial effect. J Am Coll Cardiol.
2000; 35(6):1616-1621.
Saha S. P., Bhalla D. K., Whayne Jr T. F., Gairola C. G. Cigarette smoke and adverse health
effects: An overview of research trends and future needs. Int J Angiol. 2007; 16(3):77-83.
Schectman G. Estimating ascorbic acid requirements for cigarette smokers. Ann N Y Acad Sci.
1993; 686:335-345.
Smith J. L., Hodges R. E., Serum levels of vitamin C in relation to dietary and supplemental
intake of vitamin C in smokers and nonsmokers. Ann N Y Acad Sci. 1987; 498:144-152.

Tanriverdi H., Evrengul H., Kuru O., Tanriverdi S., Seleci D., Enli Y et al. Cigarette Smoking
induced oxidative Stress may impair endothelial function and coronary blood flow in
angiographically normal coronary arteries. Circ J. 2006; 70(5): 593 -599.
Van der Vaart H, Postma D. S., Timens W, ten Hacken N.H. Acute effects of cigarette smoke
on inflammation and oxidative stress: a review. Thorax. 2004; 59(8):713-721.
Wang M-Y, Peng L, Jensen C. J., Deng S, West B.J., Noni juice reduces lipid peroxidation–
derived DNA adducts in heavy smokers. Food Sci Nutr. 2013; 1(2):141-149.
Wang M-Y, Peng L, Lutfiyya M. N., Henley E, Weidenbacher-Hoper V, Anderson G.
MorindaCitrifolia (Noni) reduces cancer risk in current smokers by decreasing aromatic
DNA adducts. Nutr Cancer. 61(5): 634-639.
Wiencke J. K., Kelsey K. T., Varkonyi A, Semey K, Wain J. C., Mark E et al. Correlation of
DNA adducts in blood mononulear cells with tobacco carcinogen-induced damage in
human lung. Cancer Res. 1995; 55(21):4910-4914.
Yanbaeva D. G, Dentener M. A., Creutzberg E. C., Wesseling G, Wouters E. F. Systemic
effects of smoking. Chest. 2007; 131(6):1557-1566.
Zuo L., He F., Sergakis G. G., Koozehchian M. S., Stimpfl J. N., Rong Y et al. Interrelated role
of cigarette smoking, oxidative stress, and immune response in COPD and corresponding
treatments. Am J Physiol Lung Cell Mol Physiol. 2014; 307(3):L205-L218.

Chapter 4
OXIDANTS, ANTIOXIDANTS IN PHYSICAL EXERCISE
Vaishali R. Undale*
Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, India
ABSTRACT
Physical exercise means carrying out some physical activity regularly, to attain and
maintain overall health. Exercises are broadly classified as flexibility, aerobic and
anaerobic exercises. Acute aerobic and anaerobic exercises increase the oxidative free
radicals or reactive oxygen species (ROS) in the biological system inducing the oxidative
stress. An inherent cellular antioxidant system consisting of antioxidant enzymes, vitamins,
proteins, and non-protein thiol, etc. protects the biological systems from the deleterious
effects of oxidants to a certain extent. ROS generated during physical exercise challenge
these antioxidants. A profound session of exercise stimulates the inherent antioxidant
system providing a defense to the cell under oxidative stress while prolonged exercise may
lead to its momentary reduction leading to its deficiency. Antioxidant nutrients such as
vitamin C, vitamin E, etc. supports innate antioxidant defense system to combat the
exercise-induced oxidative stress. Deficiency of these nutrients may worsen the oxidative
damage, while nutritional supplementation rich in antioxidant substances may help to
reduce oxidative deterioration of the tissues. The chapter elaborates on sources, mechanism
of ROS generation, antioxidant defense system with the support from nutritional
supplementation during exercise-mediated oxidative stress.
1. INTRODUCTION
Physical exercise can be defined as carrying out some sort of body activity with an
objective to regain sustained fitness and overall health. Persistent physical activity plays a
pivotal role in enhancing athletic capacity, as well as prevention and management of chronic
diseases, such as the cardiovascular diseases, diabetes, and obesity (Powers et al., 2011).
Depending upon the integral physiological changes produced on the human body, exercises are
of three types – flexibility, aerobic, and anaerobic. Flexibility exercises involve movements of
*
Corresponding Author’s Email: vaishaliundale@gmail.com.

74 Vaishali R. Undale
muscles and joints, such as extension and flexion. These exercises improve stretchability of the
skeletal muscles and overall flexibility of the body. Aerobic exercises include brisk walking
and running, focused on augmenting cardiovascular tolerance. The demand for energy and
oxygen is found to be increased in aerobic exercises. Anaerobic exercises include weight
bearing, fast running for a while, and the exercises which improve muscle power. These
exercises involve cellular respiration in the absence of oxygen and energy utilization.
Consistent and regular physical activity is thought to play a vital role in overall health,
through multiple actions, such as maintaining the health of overall body tissues such as bones,
muscles, and joints; enhancing physiological functions of organs, thereby leading to overall
well-being; decreasing the risk of surgeries and boosting of the immune system. In a heavy
physical exercise, especially the skeletal muscles and whole-body cells take up and utilize most
of the oxygen for metabolism and energy production i.e., adenosine triphosphate (ATP)
generation (Banerjee et al., 2003). Mitochondria generate ATP through electron transport chain
in which the last electron is accepted by oxygen and the water molecule is formed. However,
during this chain, the 4 - 5% of oxygen molecules is not reduced completely and form molecules
consisting of one or more unpaired electrons in its outer orbit. Such molecules having unpaired
electrons are known as free radicals, which are highly reactive and unstable. They seek stability
by donating their unpaired electron. Thus, the chain of free radical generation continues still
stable molecule is formed. After every twenty-five oxygen molecules utilized, one free radical
is generated. Free radicals are reported to produce deleterious effects on proteins, lipids, and
nucleic acid biomolecules. Numerous studies have proposed that, the physical exercise
promotes oxygen utilization and subsequently increasing the free radical formation (Chance et
al., 1979; Clarkson and Thompson, 2000).
During exercise, about 10-15 times rise in the oxygen consumption, while a hundred times
increase in oxygen movement in physically active muscles may be observed (Sen, 1995). A
part of oxygen may get transformed univalently into the number of intermediates (i.e., O2-•,
H2O2, OH•) that subsequently escape the electron transport chain (Chance et al., 1979).
Superoxide radical (O2-•) and hydroxyl radical (OH•) and hydrogen peroxide (H2O2) are
collectively known as reactive oxygen species (ROS). Only O2-• and OH• radicals are identified
as free radicals due to the presence of an unpaired electron in their atomic structure (Chakraborti
et al., 1998).
To protect the body cells and tissues from the damaging effects of oxidative free radicals,
an innate antioxidant defense system, comprising of enzymes, such as glutathione, catalase,
superoxide dismutase, glutathione peroxidase, etc. exists in human beings. This system relies
on dietary intake of antioxidant nutrients such as vitamin C and E, beta-carotene, and minerals
(Machlin and Bendich, 1987). Exercisers, athletes, fitness leaders and health professionals
always doubt the need for an antioxidant dietary supplementation (Clarkson and Thompson,
2000). This chapter briefly focuses on the types of free radicals, exercise and training-induced
changes in antioxidant markers and the effects of antioxidants supplementation in exercise-
prompted oxidative stress.

Oxidants, Antioxidants in Physical Exercise 75
2. FREE RADICALS
2.1. Reactive Oxygen and Nitrogen Species
A molecule consisting of an unpaired electron in its outer orbit, which can be present
independently, is said to be a free radical (Halliwell and Gutteridge, 1989). Generally reactive
oxygen and nitrogen species (RONS) are considered as free radicals. Most biological molecules
are non-radicals, which have only paired electrons. The three mechanisms reported for
unpairing and the free radical formation are homolysis of covalent bond, addition of single
electron to a neutral atom, and loss of a single electron from a neutral atom. Free radicals are
considered highly reactive as they rapidly search for another electron donor molecule. The
donation of an electron by the donor further leads to the formation of additional free radicals.
Thus, induces a chain reaction of a free radical generation that lasts until a chain termination
reaction takes place (Bloomer, 2008). The common reactive oxygen and nitrogen species are
shown in Table 1.
Table 1. Common reactive oxygen and nitrogen species
Reactive Oxygen Species Reactive Nitrogen Species

Superoxide O2- Nitric oxide NO-
Hydroxyl HO Nitrogen dioxide NO2
Peroxyl ROO Nitrous acid HNO2
Perhydroxyl radical HO2 Peroxynitrite ONOO-
Alkoxy RP Alkyl peroxynitrite ROONO
Hydrogen peroxide H2O2
Singlet oxygen 1
O2
2.2. Mechanisms of Free Radicals Generation in Exercise
Various mechanisms involved in the free radical generation during exercise are as
following:
2.2.1. Sympathetic System Activation

It is a well-known fact that, sympathetic activity is increased during exercise due to which,
the biogenic amines like adrenaline, noradrenaline, and dopamine rises in the biological tissues.
Respective enzymes metabolize some of these biogenic amines, however, some proportion is
oxidized to aminochrome, which further leads to the formation of oxidative radicals by the
action of cytochrome P450 reductase enzyme (Ji and Leichtweis, 1997).
2.2.2. Lactic Acid Activation

Lactic acid is formed during exercise, as a result of anaerobic respiration. Increased
formation of lactic acid can induce the transformation of superoxides into hydroxyl radicals,
which are known for causing more tissue damage than superoxides (Demopoulos et al., 1986).

2.2.3. Inflammatory Responses

Overexertion of the muscular tissue during exercise may result in an inflammatory
response through the activation of arachidonic acid derivatives. These derivatives also induce
free radical formation and produce secondary muscles responses such as the muscular injury
(Aruoma, 1994; Ji, 1995; Tiidus and Houston, 1995; Clarkson, 1995; Maxwell, 1995; Sen,
1995; Dekkers et al., 1996; Alessio, 1993; Packer, 1997; Ji et al., 1998; Ashton et al., 1998;
Kanter, 1998).
2.2.4. Tissue Hypoxia

During physical exercise, although increased oxygen consumption is involved, a sudden
decrease in tissue oxygen (i.e., hypoxia), may occur. One of the reasons for hypoxia is increased
ROS formation. In a state of hypoxia, hydrogen ions are found to be increased, which react
with the superoxide ions inducing the formation of more ROS (Jenkins, 1993). During tissue
hypoxia, transition metals such as iron and copper also become free from their transporters.
These free metals ions also accelerate the formation of free radicals (Chakraborti et al., 1999).
2.3. Sources and Generation of Free Radicals in Exercise
During acute exercise, free radicals can be formed from various sources. When free radicals
are generated as a direct response to the presented situation, it is known as a primary source,
however, free radicals generated as a secondary response to the damage induced through other
mechanisms, it is known as a secondary source (Bloomer, 2008).
2.3.1. Primary Source

Mitochondrial utilization of oxygen through a respiratory chain transport for ATP
generation is a prime source for free radical generation. Under normal physiological situation,
enzyme cytochrome oxidase completely reduces oxygen to water. Nevertheless, during this
process, some minor portion of total oxygen (1 - 5%) may lead to the production of superoxide
anions, further processing to the generation of hydrogen peroxide and hydroxyl free radicals
(Nohl, 1994). Another strong oxidant peroxynitrite (ONOO−) is also formed in mitochondria,
through nitric oxide generation. (Giulivi, 1998). The mitochondria possess inherent antioxidant
enzyme, superoxide dismutase, to protect against superoxide-initiated damage, however,
during long-lasting exercise, it is not sufficient to prevent the impairment of mitochondria. It is
also postulated that, momentous variations in temperature during exercise and reduced
mitochondrial PO2 (PmO2), could be a key mechanism of free radical production (Di Meo and
Venditti, 2001; Bailey et al., 2000).
Apart from a mitochondrial mechanism, some other mechanisms are also suggested behind
free radical generation these includes:
2.3.1.1. Prostanoid Metabolism

Prostaglandins, inflammatory mediators are liberated in muscles during acute stress, such
as excessive contractile activity, which further initiates RONS production. A predecessor of
prostaglandin, arachidonic acid is also acted by lipoxygenase and transformed into an active
metabolite leading to radical species (Jackson, 2000).

2.3.1.2. Catecholamines Metabolism

Catecholamines substantially rise during exercise. They exhibit a process of autooxidation,
as well as metal ion, catalyzed oxidation leading to increased RONS synthesis during exercise
(Jackson, 2000).
2.3.1.3. Xanthine Oxidase-Catalyzed Reaction

Usually, during a high-intensity short duration strenuous exercise, where the muscle is
metabolically imperiled and damaged, xanthine oxidase mediates RONS production. During
strenuous exercise diminution of ATP, vis-à-vis, the intracellular rise in ADP promotes
conversion of xanthine dehydrogenase to xanthine oxidase, a generator of superoxide radicals
(Knight, 1999).
2.3.1.4. NADPH Oxidase-Catalyzed Reaction

NADPH oxidase present in neutrophils and other cells also acts as a site for generation of
RONS (Bloomer, 2008). The primary sources of free radicals are shown in Figure 1.
Figure 1. Primary sources of free radicals.
2.3.2. Secondary Source

Exercise-induced tissue injury is followed by the generation of free radicals. The chains of
events followed by tissue injury are considered as secondary sources:
 The first invasion of phagocytic cells (neutrophils, macrophages, etc.) into injured
tissue can produce a substantial amount of RONS, including superoxide and hydrogen
peroxide (Bloomer, 2008).

 The process of erythrolysis and myoglobin disruption during heavy exercises might
increase free iron that catalyzes radical reactions. Furthermore, anaerobic exercises
induce acidosis and superfluous lactate accumulation leading to release of iron from
transferrin (Demopoulos et al., 1986). Enhanced oxidation of iron-containing proteins,
hemoglobin, and myoglobin during forceful exercise can cause RONS formation.
Hemoglobin autooxidation leads to methemoglobin and superoxide (Wallace et al.,
1982) while myoglobin autooxidation produces hydrogen peroxide (Brantley et al.,
1993).
 Imbalance in calcium homeostasis is also linked with the free radical generation.
Extreme intracellular calcium initiates RONS production through the activation of
phospholipase and proteolytic enzymes (Bloomer, 2008). Secondary sources of free
radicals are indicated in Figure 2.
The exercise stimulates RONS generation through above mentioned ways, as a

consequence of increased oxygen consumption to provide energy for muscular work or via
primary and secondary sources. As numerous RONS generation pathways are induced, increase
in oxidative stress is observed, ensuing aerobic and anaerobic exercise sessions. It is still
unpredictable that, in exercising humans, whether the increased RONS generation is a direct
exercise stimulus or is secondary stimulus to muscular injury. It is likely that, RONS production
involves several pathways, all of which collectively lead to the presence of oxidized molecules
in biological sample analysis (Bloomer, 2008).
Figure 2. Secondary sources of free radicals.

2.4. Free Radicals Actions on Biomolecules
Increased in free radicals may start oxidation of a variety of biomolecules and impair their
physiological function potentially leading to weakened health and physical performance. Some
of the commonly affected biomolecules by free radicals are:
2.4.1. Lipids
Lipids are the most abundant biomolecules present in the human body. Lipid bilayer
composed of phospholipids which constitute about 75% of the biological membranes.
Polyunsaturated fatty acids, triglycerides, cholesterol are other significant bio-lipids present in
biosystems (Waugh and Grant, 2014). Biomembranes plays a characteristic role in maintaining
selective permeability of the cells and homeostasis between an internal and external
environment of the cell. During an acute bout of exercise, increased oxygen uptake is related
to increased mitochondrial respiration thus, inducing imbalance in electron transport chain and
lipid peroxidation. Lipid peroxidation involves a deleterious effect on polyunsaturated fatty
acids and phospholipids implicated by free radicals produced in this chain reaction (Alessio,
2000). A variety of oxidation products are formed due to lipid peroxidation. The primary
product is lipid hydroperoxides, while different aldehydes such as malondialdehyde (MDA),
propanal, hexanal and 4-hydroxynonenal (4-HNE) are the other products formed. With extreme
lipid peroxidation, normal physiological functions such as membrane fluidity and permeability
may be diminished. The normal membrane fluidity and permeability maintains intracellular
and extracellular proteins and their physiological enzymatic functional integrity. Cell death
results in extreme loss of these characteristic properties of the membrane, thereby implicating
the loss of physiologically important proteins and enzymes (Bloomer, 2008).
The process of lipid peroxidation is also associated with a rise in age-related pigmentation.
Accumulation of lipid peroxidation end products, such as hydrogen gases e.g., pentane, ethane,
and aldehydes facilitate the process of aging (Knight, 1999). To evaluate the degree of lipid
peroxidation, lipid hydroperoxides (LOOH), thiobarbituric acid reactive substances (TBARS)
and MDA formed in biological systems are measured with an appropriate analytical method.
2.4.2. Proteins
Similar to lipid peroxidation, RONS also mediate protein oxidation, however much
attention has not been focused on it, as the proteins constantly turnover and RONS affected
damaged proteins do not accumulate in the body. Nowadays, studies on the effects of exercise-
induced oxidative stress on the protein are attracting more attention. Oxidative radicals either
directly react with proteins or they generate secondary radicals which react with lipids or sugars
before reacting with proteins. Aromatic and sulfhydryl residues on the protein side chain or the
peptide bond are prone to the oxidative process by oxidant intermediates especially, when
transition metal ions are present (Griyths, 2000). The exposure of proteins to free radical
induces tyrosine cross-linking and/or nitrosylation of thiol and other proteins group. The
oxidized proteins may lead to loss of catalytic, contractile or structural function of the affected
proteins thus, increasing their vulnerability to proteolytic degeneration (Levine and Stadtman,
2001). Protein carbonyl content, dityrosine, and 3-nitrotyrosine are the markers of protein
oxidation (Bloomer, 2008).

2.4.3. DNA
Deoxyribonucleic acid (DNA) is composed of nucleotide bases adenosine, guanine,
cytosine and thymine bound with phosphate bridges. RONS damages to single bases of DNA.
The oxidative radicals may affect both the nuclear as well as mitochondrial DNA. Diverse
modifications are observed in affected DNA, depending on the RONS interacting with DNA
(Halliwell and Gutteridge, 1989). 8-hydroxy-2’-deoxyguanosine (8-OHdG) is a biomarker to
assess DNA destruction (Lodovici et al., 2000).
3. IMPLICATIONS OF OXIDATIVE STRESS IN EXERCISE

The situation when the formation of ROS becomes disproportionate with the innate
antioxidant protective mechanism in the body, the physiological system becomes
overburdened, and is defined as the oxidative stress (Ji, 2002). Numerous physiologically
normal as well as abnormal situations induce the formation of free radicals or impair the innate
antioxidant defense system. In the year 1978, the concept of exercise-induced oxidative stress
was put forth, for the first time (Powers and Jackson, 2008). Although, regular physical activity
and intensive exercise provides benefits of health promotion and reduces the risk of chronic
diseases, it is thought to be an inducer of the oxidative burden. Excess of RONS such as
superoxide anion (O2-), hydroxyl (OH-), peroxyl radical (RO2) and peroxynitrite are formed in
exercise (Powers et al., 2011). Though, multiple ideas in the concept of exercise-induced
oxidative stress are postulated, the debate still continues. In response to the stimulus of exercise,
the mitochondria produce ROS to remodel muscles. The ROS formation during exercise is
programmed in the cell and is necessary as a prime signaling pathway (Powers et al., 2011,
Powers et al., 2011; Jackson, 2008). Role of exercise-mediated free radical production in the
prevention of type-2 diabetes has also been proposed by some studies. It has been suggested
that, the free radical promoted insulin sensitivity aid to prevent type-2 diabetes (Goldstein et
al., 2005; Ristow et al., 2009). ROS also stimulates few redox-dependent signaling pathways
and initiates the action of the inherent antioxidant protection system (Ji, 2002; Ji et al., 2006).
4. BIOMARKERS OF OXIDATIVE STRESS IN PHYSICAL EXERCISE

Biomarker or biological marker is any molecule, structure or a phenomenon that can be
quantitatively measured in the biological tissues (WHO, 2001). To be a clinically useful
biomarker, the following criteria must be accomplished (Frijhoff et al., 2015):
 It should be distinct and selective for a certain physiological situation and/or an ailment
(diagnostic).
 It should help in the prediction of the progress of disease or medical treatment.
 It should be consistent with the disease process.
 It should be stable in biological samples collected and during the evaluation procedure.
 It should be present in easily approachable tissue.
 Its evaluation should be economical.

During the evaluation of biomarkers to estimate exercise-mediated oxidative stress, the

time of sample collection, the number of samples collected and the correlation of the biomarker
assessed should be considered. During exercise, the RONS generation and biomolecular
oxidation may occur at a particular time. The possibility of missing the exact time remains, if
the samples are collected too early or too late after a bout of exercise. It is suggested that, the
collection of repeated biological samples at a specific time, over a period of 24 hours, or days
following an exercise will be the best strategy to measure oxidative stress (Bloomer, 2008).
Common biomarkers of exercise-induced oxidative stress are shown in Table 2.
Table 2. Common biomarkers of exercise-induced oxidative stress (Bloomer, 2008)
Molecule Biomarkers Method for Assessment

Lipids Isoprostanes Gas chromatography-Mass spectroscopy (GC-
MS), Enzyme-linked immunosorbent assay
(ELISA)
Lipid hydroperoxides Spectrophotometric, ELISA
Malonaldehyde (MDA) High-performance liquid chromatography
(HPLC), Spectrophotometric
Thiobarbituric acid reactive HPLC, Spectrophotometric, Fluorometric
substances
Conjugated dienes Spectrophotometric
Oxidized low-density lipoproteins ELISA
Proteins Protein carbonyls Spectrophotometric, ELISA, Western Blot
Individual oxidized amino acids GC-MS, Western blot,
Nitrotyrosine ELISA
DNA 8-Hydroxydeoxyguanosine HPLC, ELISA
Oxidized DNA bases HPLC
Strand breaks Comet assay
5. ANTIOXIDANT SYSTEM IN EXERCISE-INDUCED STRESS

All living organisms have an inherent antioxidant system that protects cells from oxidative
stress generated through physiological or non-physiological mechanisms. The antioxidant
system comprises of certain enzymes such as superoxide dismutase, catalase, GSH peroxidase,
vitamins, trace elements, glutathiones, and thiols, etc. The deficiency of the antioxidant system
has proven to worsen oxidation induced tissue damage, while inconsistent results are reported
with the supplementation of various antioxidants. To relate the effect of existing antioxidant
system in a biological tissue with an exercise-induced oxidative damage, various studies have
been conducted in the past (Banerjee et al., 2003).
5.1. Antioxidant Enzymes
A drastic session of exercise has proven to elevate the action of antioxidant enzymes in
skeletal muscles, heart, and liver (Chakraborti et al., 1998). The verge and extent of activation
differ among enzymes and tissues. The exact mechanism of how a short period of exercise

induces antioxidant system is unknown. It is proposed that, exercise may be activating some
allosteric or covalent alterations in the enzyme molecule, thereby activating them. It is also
been considered that, the partial binding of the substrates might increase their catalytic activity
(Chakraborti et al., 1998; Ghosh et al., 1996; Roychoudhury et al., 1996).
5.2. Antioxidant Vitamins
Vitamin E, is a significant lipid soluble vitamin, reported to be a free antioxidant. It is

amphipathic in nature, due to the presence of both hydrophobic tocols and tocotrienols as well
as hydrophilic hydroxyl substitutions. Various isomers of vitamin E exists, of which alpha
tocopherols are biologically important isomer. Vitamin E is a key soluble antioxidant
constituent of biomembranes (Clarkson and Thompson, 2000). It partitions into lipoproteins of
membranes. Vitamin E is found in varying amounts in different body tissues e.g., approximate
30 – 50 nmol/g is found in voluntary muscles while 60 – 70 mmol/g in liver and heart. It protects
the polyunsaturated lipids present in the membranes from oxidative attack by free radicals
generated. It also acts as a membrane stabilizer. It conjugates with by-products of membrane
lipid peroxidation, phospholipids, and free fatty acids and prevents deleterious effects on
biological membranes (Wang and Quinn, 2000).
Many studies have previously reported the effects of vitamin E in exercise-induced
oxidative damage. After physical training reduction in vitamin E content within vital tissues
has been reported in a study conducted in rats (Packer et al., 1989). Davies reported detrimental
effects of vitamin E deficiency on skeletal muscles and liver, increased lipid peroxidation and
malfunctioning of the mitochondria in exhaustively exercised rats. (Davies et al., 1982).
Depletion in vitamin E store has been observed, after a session of endurance training, in an
individual consuming a normal diet. An increase in daily dietary vitamin E is suggested among
individuals following a physically active lifestyle (Packer et al., 1989).
Vitamin C is well-known and widely used antioxidant. However, the action of vitamin C
in relieving oxidative stress produced due to exercise is not yet fully understood. Reactivation
of vitamin E, by converting its radical form to vitamin form, is postulated to be through vitamin
C (Kanter, 1998). Vitamin E and vitamin C as supplements are further described in this chapter.
5.3. Glutathione and Thiols
A tripeptide, glutathione is formed from the conjugation of glutamic acid, cysteine, and
glycine. Thiols, the sulfhydryl-containing organic compounds, are found in biological cells at
a concentration of about 5 mM/g. Some plants, animals, fungi, and bacteria also consist of
thiols. As a co-enzyme, it is involved in numerous oxidation-reduction reactions within cells.
In the living system, thiols are present in two forms: a reduced form GSH, and an oxidized form
GSSG (glutathione disulfide). Glutathione gets oxidized to glutathione disulfide by oxidation,
while by receiving an electron from NADPH, it is reduced back to glutathione reductase (Couto
et al., 2013). Its ratio of reduced to oxidized form is used to assess oxidative stress in biological
cells. The exercise-induced oxidative damage is prevented by reduced glutathione by multiple
actions:

 It reduces hydrogen and organic peroxides via glutathione peroxidase enzymes.

 It scavenges singlet oxygen and hydroxyl radicals
 It reduces alpha-tocopherol by direct or indirect action
 It prevents lipid peroxidation.
Various experiments conducted on rats have shown that, the exercise increases skeletal
muscle’s GSH and GSSG levels by importing it from the plasma (Bloomer, 2008). The rise
was found to be dependent on the extent of exercise performed. Moderate decrease in the ratio
of GSH: GSSG has been reported after a session of forceful exercise. The rise in myocardial
GSH has also been found after swim training in rats. On the contrary, the reduction in GSH
content in rat soleus muscles after exercise training has also been reported. In the situation of
oxidative stress, tissues not only import GSH but also export GSSG thus, a net reduction in
GSH may be seen when the rate of GSH consumption increases than that of import. During
acute and chronic exercise, thiol content in the body reduces as it is utilized for the oxidation
process, and its deficiency can lead to oxidative damage. Supplementation with thiol during
exercise may prove helpful for the prevention of oxidative impairment. To provide thiol as
supplement N-acetylcysteine (NAC), free GSH, and GSH acetyl monoesters are thought of as
excellent composites (Ji, 1995).
5.4. Trace Elements and Antioxidant Enzymes
The metal elements like copper, zinc, manganese, iron, selenium, also known as trace
elements, are required for the activity of antioxidant enzymes. Flavin adenine dinucleotide
(FAD) is essential for the activity of glutathione reductase enzyme, as a prosthetic factor. The
trace elements play indirect role in the balancing of antioxidant enzymes. Thus, in acute and
chronic physical workout induced stress, apart from the extent of stress produced, the
accessibility of trace elements may also determine the activity of antioxidant enzymes (Kanter,
1998; Ji, 1995).
Experiments conducted on rats have reported that, the selenium deficiency has decreased
glutathione peroxidase activity by 80% in exercise-induced oxidative stress, while other
parameters such as GSSG, Vitamin E were not affected. In the case of short as well as long
duration exercise, appropriate amount of trace elements should be present to carry out normal
antioxidant activity in oxidatively stressed cells (Clarkson and Thompson, 2000).
5.5. Effects of Antioxidant Supplements
5.5.1. Vitamin C
Vitamin C (ascorbic acid) is proven as highly antioxidant. Its discovery was reported in the
year 1923 by Hungarian Nobel Laureate Szent-Gyorgyi, while Howarth and Hirst synthesized
it, the first time in year 1933 (Haworth and Hirst, 1933). Many vegetables and fruits e.g.,
Cauliflower, Spinach, Amla, Citrus fruits, etc. are a rich source of vitamin C. It exists in an
interchangeable dual forms, dehydroascorbic acid and ascorbate as the oxidized and the reduced
form, respectively. Vitamin C acts directly by donating the electrons to free peroxy radical or

indirectly by restoring the antioxidant property of vitamin E, another potent antioxidant

molecule. Thus, it protects the biological tissues from multiple harmful actions of free radicals
and other toxicants. Vitamin C oxidizes rapidly, and decomposes by oxygen, alkali and high
temperature (Askari et al., 2012).
Numerous studies on the actions of vitamin C in exercise-induced stress have been
reported. A study conducted by Maxwell et al., to evaluate the effects of addition of vitamin C
rich supplements in the exercise, reported rise in blood vitamin C concentration, unchanged
MDA level and overall increase in antioxidant capacity in supplement treated group than
control group suggesting importance of vitamin C supplementation to prevent oxidative
damage due to free radicals generated during exercise (Maxwell et al., 1993). Similarly,
reduced damage to muscles in the vitamin C supplemented group has been suggested in a study
conducted by Jakeman and Maxwell (Jakeman and Maxwell, 1993).
In spite of ambiguity about the results related to vitamin C supplementation, no
advantageous effect on either stamina or strength has been reported in several well-controlled
studies (Bucci, 1993; Clarkson, 1991; Keith, 1989; Gey et al., 1970; Keren and Epstein, 1980;
Keith and Driskell, 1982; Keith and Merrill, 1983, Van der Beek et al., 1990). Although,
addition of vitamin C supplements has not shown enhanced exercise performance, it might
reduce exercise-induced muscle damage.
5.5.2. Vitamin E
Group of fat dissolving tocopherols and tocotrienols compounds is known as vitamin E. It
scavenges free radicals by donating a hydrogen atom to free radicals. Each molecule of alpha-
tocopherol inhibits two lipid free radicals terminating the chain reaction of free radicals. It also
participates in muscle growth at large, via regulating the activity and an enzyme protein kinase
C and its gene expression. It is incorporated in the cell membranes and protects the cell
membranes from oxidative damage (Clarkson and Thompson, 2000).
Numerous experiments conducted to evaluate the function of vitamin E in controlling
exercise-induced oxidative damage, concluded that vitamin E effectively reduces lipid
peroxidation. Alpha-tocopherol administered in different doses and duration has proven the
protective role of the vitamin E (Dillard et al., 1978; Sumida et al., 1989). It is proven that the
repetition of exercise-induced muscle damage initiates a rapid adaptation of tissues in response.
As a result of adaptation, fewer changes in indicative marker are reported, after a bout of the
same exercise done the second time. For example, in the second turn of exercise, amount of
muscle enzymes going out into blood is reduced as compared to the first act of exercise
(Clarkson and Tremblay, 1988; Clarkson et al., 1992). The lower MDA level after recurrent
physical acts may also be due to adaptation but is not yet proved.
The lesser rise in serum MDA and creatinine kinase has been reported in a study involving
a long-term supplementation of vitamin E (Rokitzki et al., 1994). However, several studies
reported no change in oxidative or performance parameters such as the endurance or aerobic
capacity and muscle soreness after the administration of vitamin E (Helgheim et al., 1979;
Francis and Hoobler, 1986; Tiidus and Houston, 1995; Gerster, 1991; Sharman et al., 1971;
Talbot and Jamieson, 1977; Sharman et al., 1976; Lawrence et al., 1975; Lawrence et al., 1975;
Shephard et al., 1974; Watt et al., 1974; Shephard, 1983; Williams, 1989). In summary, vitamin
E supplementation does not augment physical performance but may safeguard exercise-induced
muscle injury, although the scientific reports are indecisive.

5.5.3. Antioxidant Mixtures

Various antioxidants as an individual supplement are reported to be useful in combating
oxidative stress induced due to short- and long-term exercise. The combination of these
numerous antioxidants is also reported to be helpful in oxidative stress induced due to exercise
in many of studies conducted.
An antioxidant mixture of alpha-tocopherol, ascorbic acid, beta-carotene was studied for
an effect of exercise on lipid peroxidation. The results obtained recommends that the
antioxidant blend blunts lipid peroxidation in treated individuals (Kanter et al., 1993). The
decrease in MDA level by the combination of supplements containing selenium, vitamin E,
glutathione, and cysteine, as compared to the placebo-treated group, has been reported in
trained cyclists when administered for three weeks (Dragan et al., 1991). Similarly, the
combination of vitamin C and glutathione has been found to decrease the GSSG level in a
treated group suggesting a decrease in oxidative stress (Sastre et al., 1992). Minor variation in
blood GSSG level is reported in people supplemented with N-acetylcysteine as an antioxidant.
Similarly, the combination of vitamin E and vitamin C has proven to be protective in oxidative
stress, in treated individuals as compared to placebo-treated individuals (Rokitzki et al., 1994).
5.5.4. Other Antioxidant Supplements

The superoxide dismutase containing extract of pollens was studied in the exercising group.
The MDA level in pollens extract treated group was found to be decreased as compared to
placebo-treated (Krotkiewski et al., 1994). The decline in MDA during exercise has been
reported on the supplementation of Selenium (100 mg/day) for two weeks (Dragan et al., 1990).
Rise in muscle glutathione peroxidase activity, in response to forceful exercise in the selenium-
supplemented group, in contrast to the null effect in resting muscle, has also reported previously
(Tessier et al., 1995).
5.6. Oxygen Radical Absorbance Capacity
Oxygen radical absorbance capacity (ORAC) is a parameter used to quantify the

antioxidant ability of biomolecules in vitro, while the total antioxidant capacity (TAC)
describes the antioxidant capacity of nutritional supplementation. Both the terms are
synonymously used in describing the antioxidant capacity of antioxidant supplements (Cao et
al., 1993)
United States Department of Agriculture (USDA) database for the ORAC of certain food
was published in 2010, but in 2012 USDA’s Nutrient Data Laboratory removed the database
due to increasing evidence that, the antioxidant capacity has no relevance with the biological
effects. A variety of nutritional supplements are available in the market focusing on the ORAC
as one of the hit words to attract the exercising consumers.


AND CHALLENGES
The lifestyle modernization, the use of advanced technologies and pesticides in the
agriculture, the rise in environmental pollutants is enhancing the formation of oxidative radicals
in human beings, consequently, increasing the oxidative stress. An appropriate set of exercise
is reported to be associated with the activation of an innate antioxidant system and the balance
between oxidant and antioxidant systems. As the exercise is thought to be useful in combating
the oxidative stress, the awareness of exercise and inclination towards antioxidant nutritional
supplementation is constantly on the rise.
At present, world is focusing on a set of well-planned variable sets of exercise such as
running, gym training, cardio, and aerobics, etc. and supplementation of antioxidants in the
form of nutrition or diet. A vast range of antioxidant system boosting products are freely
available in the market. Plants being a source of numerous macro and micro antioxidant
nutrients, they are explored widely for antioxidant potential. Exploring a range of unexplored
flora and fauna for antioxidant potential is the need of present time. Providing new antioxidant
combinations and products for exercising individuals with proven and additional antioxidant
potential will be a future challenge for the nutrition industry.
CONCLUSION
Physical exercise promotes an increase in free radical generation. The chronic exercise may
induce oxidative damage due to the imbalance between oxidant generation and antioxidant
defense mechanism. Despite studies have been conducted focusing on the exercise-induced free
radical generation, oxidative stress and antioxidants, investigators have failed to identify exact
phenomenon and the role of antioxidant supplementation in protection from oxidative damage.
Nowadays, advanced and sophisticated instruments are available for further investigating the
molecular mechanisms underlying oxidative process. Numbers of issues regarding the
antioxidant requirements in the physically active individuals are yet to be elucidated. In future
utilizing newer sophisticated methodologies, advanced tools of molecular research and
increasing availability of phytopharmaceuticals may provide answers to many of these
questions.
REFERENCES
Alessio, H. M. (1993). Exercise-induced oxidative stress. Med Sci Sports Exerc, 25:218–224.
Alessio, H. M. (2000). Lipid peroxidation in healthy and diseased models: Influence of
divergent types of exercise. In: Sen CK, Packer L, Hanninen O, editors. Handbook of
Oxidants and Antioxidants in Exercise. Amsterdam: Elsevier Science: 115-127.
Aruoma, O. I. (1994). Free radicals and antioxidant strategies in sport. J Nutr Biochem, 5: 370–
381.

Ashton, T; Rowlands, C. C; Jones, E; et al. (1998). Electron spin resonance spectroscopic

detection of oxygen-centered radicals in human serum following exhaustive exercise. Eur
J Appl Physiol, 77: 498–502.
Askari, G; Ghiasvand, R; Karimian, J; Feizi, A; Paknahad, Z; Sharifirad, G. (2012).Does
quercetin and vitamin C improve exercise performance, muscle damage, and body
composition in male athletes. J Res Med Sci., 17(4): 328–331.
Bailey, D. M.; Davies, B; Davison, G. W.; Young, I. S. (2000).Free radical damage at high-
altitude, isolating the source and implications for the pathophysiology of acute mountain
sickness. News Int Soc Mount Med., 10: 3–13.
Banerjee, A. K.; Mandal, A; Chanda, D; Chakraborti, S. (2003).Oxidant, antioxidant and
physical exercise. Molecular and Cellular Biochemistry., 253: 307–312.
Bloomer, R. J. (2008). Exercise and oxidative stress. In: Makowski GS, editor. Advances in
clinical chemistry., Elsevier Ltd.Vol. 46: 1-50.
Brantley, R. E; Smerdon, S. J; Wilkinson, A. J; Singleton, E. W; Olson, JS. (1993). The
mechanism of autooxidation of myoglobin. J Biol Chem., 268(10): 6995–7010.
Bucci, L. (1993). Nutrients as ergogenic aids for sports and exercise. Boca Raton, FL: CRC
Press, 32–39: 42–45.
Cao, G; Alessio, H. M; Cutler, R. G. (1993). Oxygen-radical absorbance capacity assay for
antioxidants Free Radic. Biol. Med, 14 (3): 303–311.
Chakraborti, T; Das, S; Mondal, M; Roychoudhury, S; Chakraborti, S. (1999). Oxidant,
mitochondria, and calcium: An overview. Cell Signal., 2: 77–85.
Chakraborti, T; Ghosh, S. K; Michael, JR; Batabyal, S. K; Chakraborti, S. (1998). Targets of
oxidative stress in the cardiovascular system. Mol Cell Biochem., 187: 1–10.
Chance, B; Sies, H; Boveris, A. (1979). Hydroperoxide metabolism in mammalian organs.
Physiol Rev., 59: 527–605.
Clarkson, P. M.; Nosaka, K; Braun, B. (1992). Muscle function after exercise-induced muscle
damage and rapid adaptation. Med Sci Sports Exerc., 24: 512–520.
Clarkson, P. M.; Thompson, H. S. (2000). Am J Clin Nutr., 72(suppl) : 637S–46S.
Clarkson, P. M.; Tremblay, I. (1988). Rapid adaptation to exercise-induced muscle damage. J
Appl Physiol., 65: 1–6.
Clarkson, P. M. (1995). Antioxidants and physical performance. Clin Rev Food Sci Nutr, 35:
131–141.
Clarkson, P. M. (1991). Vitamins and trace minerals. In: Lamb DR, Williams MH, eds.
Perspectives in exercise science and sports medicine., Vol 4. Carmel, IN Benchmark
Press.: 123–176.
Couto, N; Malys, N; Gaskell, S. J.; Barber, J. (2013). Partition and turnover of glutathione
reductase from Saccharomyces cerevisiae: a proteomic approach. Journal of Proteome
Research., 12 (6): 2885–2894.
Davies, K. J.; Quintanilha, A. T.; Brooks, G. A.; Packer, L. (1982). Free radicals and tissue
damage produced by exercise. Biochem Biophys Res Commun., 107: 1198–1205.
Dekkers, J. C.; Van Doornen, L. J. P; Kemper, H. C. G. (1996). The role of antioxidant vitamins
and enzymes in the prevention of exercise-induced muscle damage. Sports Med, 21: 213–
238.
Demopoulos, H. B.; Santomier, L. M.; Seligman, Hogan P. I.; Pietronigro, D. D. (1986). Free
radical pathology: Rationale and toxicology of antioxidants and other supplements in sports

medicine and exercise science. In: Katch FJ, editors. Sport, Health and Nutrition.
Champaign, IL: Human Kinetics. : 189.
Di Meo, S; Venditti, P. (2001). Mitochondria in exercise-induced oxidative stress. Biol Signals
Recept., 10(1–2) : 125–140.
Dillard, C. J.; Litov, R. E.; Savin, WM; Dumelin, E. E.; Tappel, AL. (1978). Effects of exercise,
vitamin E, and ozone on pulmonary function and lipid peroxidation. J Appl Physiol., 45:
927–932.
Dragan, I; Dinu, V; Cristea, E; Mohora, M; Ploesteanu, E; Stroescu, V. (1991). Studies
regarding the effects of an antioxidant compound in top athletes. Rev Roum Physiol, 28:
105–108.
Dragan, I; Dinu, V; Mohora, M; Cristea, E; Ploesteanu, E; Stroescu, V. (1990). Studies
regarding the antioxidant effect of selenium on top swimmers. Rev Roum Physiol, 27: 15–
20.
Francis, K. T.; Hoobler, T. (1986). Failure of vitamin E and delayed muscle soreness. J Med
Assoc Alabama, 55:15–18.
Frijhoff, J; Winyard, P. G.; Zarkovic, N; Davies, S. S.; Stocker, R; Cheng, D. (2015). Clinical
Relevance of Biomarkers of Oxidative Stress. Antioxidants & redox signaling., 23(14) :
1144-1170.
Gerster, H. (1991). Function of vitamin E in physical exercise: a review. Z Ernahrungswiss.,
30: 89–97.
Gey, GO; Cooper, K. H.; Bottenberg, R. A. (1970). Effect of ascorbic acid on endurance
performance and athletic injury. JAMA, 211: 105.
Ghosh, S. K.; Chakraborti, T; Banerjee, A.B., Roychoudhury, S; Chakraborti, S. (1996). Role
of hydroxyl radical in superoxide induced microsomal lipid peroxidation: Protective effect
of anion channel blocker. J Biosci., 21: 35–43.
Giulivi, C. (1998). Functional implications of nitric oxide produced by mitochondria in
mitochondrial metabolism. Biochem J., 332(Pt 3) : 673–679.
Goldstein, B. J.; Mahadev, K; Wu, X. (2005). Redox paradox: insulin action is facilitated by
insulin-stimulated reactive oxygen species with multiple potential signaling targets.
Diabetes., 54(2) :311–321.
GriYths, H. R. (2000). Antioxidants and protein oxidation. Free Radic Res, 33(Suppl).
Halliwell, B; Gutteridge, JMC. (1999). Free radicals in Biology and Medicine, 3rd, New
York:Oxford University Press.: 1-25
Haworth, W. N.; Hirst, E. L. (1933). Synthesis of ascorbic acid. J Soc Chem Ind., 52: 645–647.
Helgheim, I; Hetland, Q; Nilsson, S; Ingjer, F; Stromme, S. B. (1979). The effects of vitamin
E on serum enzyme levels following heavy exercise. Eur J Appl Physiol, 40: 283–289.
Jackson, M. J. (2000).Exercise and oxygen radical production by muscle. In: Sen CK, Packer
L, Hanninen O, editors. Handbook of Oxidants and Antioxidants in Exercise. Amsterdam:
Elsevier Science: 57-68.
Jackson, M. J. (2008). Free radicals generated by contracting muscle: by-products of
metabolism or key regulators of muscle function? Free Radic Biol Med., 44(2) : 132–141.
Jakeman, P; Maxwell, S. R. J. (1993). Effect of antioxidant vitamin supplementation on muscle
function after eccentric exercise. Eur J Appl Physiol., 67: 426–430.
Jenkins, R. R. (1988). Free radical chemistry. Relationship to exercise. Sports Med., 5: 156–
170.

Jenkins, R. R. (1993). Exercise, oxidative stress, and antioxidants: A review. Int J Sports Nutr.,
3: 356–375.
Ji, L. L. (1995).Oxidative stress during exercise: Implication of antioxidant nutrients. Free
Radical Biol Med., 18: 1079–1086.
Ji, L. L. (2002). Exercise-induced modulation of antioxidant defense. Ann NY Acad Sci, 959:
82-92.
Ji, L. L; Gomez-Cabrera, MC; Vina, J. (2006). Exercise and hormesis: activation of the cellular
antioxidant signaling pathway. Ann N Y Acad Sci., 1067: 425–435.
Ji, L. L; Leeuwenburgh, C; Leichtweis, S; et al. (1998). Oxidative stress and aging. Role of
exercise and its influences on antioxidant systems. Ann N Y Acad Sci, 854: 102–107.
Ji, L. L; Leichtweis, S. (1997). Exercise and oxidative stress: sources of free radicals and their
impact on antioxidant systems. Age, 20: 91-106.
Ji, L. L. (1995). Oxidative stress during exercise: implication of antioxidant nutrients. Free
Radic Biol Med, 18: 1079–1086.
Kanter, M. (1998). Free radicals, exercise and antioxidant supplementation. Proc Nutr Soc, 57:
9–13.
Kanter, M. M.; Nolte, L. A; Holloszy, J. O. (1993). Effects of an antioxidant vitamin mixture
on lipid peroxidation at rest and post exercise. J Appl Physiol., 74: 965–969.
Keith, R. E.; Driskell, J. A. (1982). Lung function and treadmill performance of smoking and
nonsmoking males receiving ascorbic acid supplements. Am J Clin Nutr, 36: 840–845.
Keith, R.E.; Merrill, E. (1983). The effects of vitamin C on maximal grip strength and muscular
endurance. J Sports Med, 23: 253–256.
Keith, R. E. (1989). Vitamins in sport and exercise. In: Hickson JE, Wolinsky I, eds. Nutrition
in exercise and sport. Boca Raton, FL: CRC Press: 233–253.
Keren, G; Epstein, Y. (1980). The effect of high dosage vitamin C intake on aerobic and
anaerobic capacity. J Sports Med, 20: 145–148.
Knight, J. A. (1999). Free Radicals, Antioxidants, Aging, and Disease. Washington, DC:
American Association for Clinical Chemistry Press: 2319-2325.
Krotkiewski, M; Brzezinska, Z; Liu, B; Grimby, G; Palm, S. (1994). Prevention of muscle
soreness by pretreatment with antioxidants. Scand J Med Sci Sports, 4: 191–199.
Lawrence, J. D.; Bower, R. C; Riehl, W. P; Smith, J. L. (1975). Effects of alpha-tocopherol
acetate on the swimming endurance of trained swimmers. Am J Clin Nutr, 28: 205–208.
Lawrence, J. D; Smith, J. L; Bower, R. C; Riehl, W. P. (1975). The effect of alpha-tocopherol
(vitamin E) and pyridoxine HCL (vitamin B6) on the swimming endurance of trained
swimmers. J Am Coll Health Assoc, 23: 219–222.
Levine, R. L; Stadtman, E. R. (2001). Oxidative modification of proteins during aging. Exper
Geront., 36: 1495–1502.
Lodovici, M; Casalini, C; Cariaggi, R; Michelucci, L; Dolara, P. (2000). Levels of 8-
hydroxydeoxyguanosine as a marker of DNA damage in human leukocytes. Free Radic
Biol. Med., 28(1) : 13–17.
Machlin, L.J.; Bendich, A. (1987). Free radical tissue damage: protective role of antioxidant
nutrients. FASEB J., 1: 441–445.
Maxwell, S. R. J.; Jakeman, P; Thomason, H; et al. (1993).Changes in plasma antioxidant status
during eccentric exercise and the effect of vitamin supplementation. Free Radic Res
Commun., 19: 191–202.
Maxwell, S. R. J. (1995). Prospects for the use of antioxidant therapies. Drugs, 49: 345–361.

Nohl, H. (1994). Generation of superoxide radicals as byproducts of cellular respiration. Ann

Biol Clin., 52:199–204.
Packer, L; Almada, AL; Rothfuss, LM; Wilson, DS. (1989). Modulation of tissue vitamin E
levels by physical exercise. Ann NY Acad Sci., 570:311– 321.
Packer, L. (1997).Oxidants, antioxidant nutrients, and the athlete. J Sports Sci, 15: 353–63.
Powers, S. K.; DeRuisseau, K. C.; Quindry, J; Hamilton, K. L. (2004).Dietary antioxidants and
exercise. J Sports Sci., 22 (1): 81–94.
Powers, S. K.; Jackson, MJ. (2008). Exercise-induced oxidative stress: cellular mechanisms
and impact on muscle force production. Physiol Rev., 88(4): 1243–1276.
Powers, S. K.; Nelson, W. B; Hudson, M. B. (2011). Exercise-induced oxidative stress in
humans: Causes and consequences. Free Radic Biol Med., 51(5): 942–950.
Powers, S.K; Talbert, E. E; Adhihetty, P. J. (2011). Reactive oxygen and nitrogen species as
intracellular signals in skeletal muscle. J Physiol., 589(Pt 9):2129–2138.
Ristow, M; Zarse, K; Oberbach, A; Kloting, N; Birringer, M; Kiehntopf, M; et al. (2009).
Antioxidants prevent health-promoting effects of physical exercise in humans. Proc Natl
Acad Sci U S A., 106(21):8665–8670.
Rokitzki, L; Logemann, E; Huber, G; Keck, E; Keul, J. (1994). Alpha-Tocopherol
supplementation in racing cyclists during extreme endurance training. Int J Sports Nutr.,
4:253–264.
Rokitzki, L; Logemann, E; Sagredos, A. N; Murphy, M; Wetzel-Roth, W; Keul, J. (1994). Lipid
peroxidation and antioxidative vitamins under extreme endurance stress. Acta Physiol
Scand, 151: 149–158.
Roychoudhury, S; Ghosh, S. K; Chakraborti, T; Chakraborti, S. (1996). Role of hydroxyl
radical in the oxidant H2O2-mediated Ca2+ release from pulmonary smooth muscle
mitochondria. Mol Cell Biochem., 159:95–103.
Sastre, J; Aseni, M; Gasco, E; et al. (1992). Exhaustive physical exercise causes oxidation of
glutathione status in blood: prevention by antioxidant administration. Am J Physiol., 263:
992–995.
Sen, C.K; Rankinen, T; Vaisanen, S; Rauramaa, R. (1994). Oxidative stress after human
exercise: effect of N-acetylcysteine supplementation. J Appl Physiol., 76:2570–2577.
Sen, C.K. Oxidants and antioxidants in exercise. (1995). J Appl Physiol., 79: 675– 686.
Sharman, I. M; Down, M. G; Norgan, N. G. (1976).The effects of vitamin E on physiological
function and athletic performance of trained swimmers. J Sports Med, 16: 215–225.
Sharman, I. M.; Down, M. G.; Sen, R. N. (1971).The effects of vitamin E and training on
physiological function and athletic performance in adolescent swimmers. Br J Nutr,
26:265–276.
Shephard, R. J.; Campbell, R; Pimm, P; Stuart, D; Wright, G. R. (1974).Vitamin E, exercise,
and recovery from physical activity. Eur J Appl Physiol., 33:119–126.
Shephard, R. J. (1983).Vitamin E and athletic performance. J Sports Med, 23: 461–470.
Sumida, S; Tanaka, K; Kitao, H; Nakadomo, F. (1989). Exercise-induced lipid peroxidation
and leakage of enzymes before and after vitamin E supplementation. Int J Biochem.,
21:835–838.
Talbot, D; Jamieson, J. (1977). An examination of the effect of vitamin E on the performance
of highly trained swimmers. Can J Appl Sports Sci, 2: 67–69.
Tessier, F; Hida, H; Favier, A; Marconnet, P. (1995). Muscle GSH-Px activity after prolonged
exercise training and selenium supplementation. Biol Trace Element Res, 47: 279–285.

Tessier, F; Margaritis, I; Richard, M; Moynot, C; Marconnet, P. (1995). Selenium and training

effects of the glutathione system and aerobic performance. Med Sci Sports Exerc, 27:390–
396.
Tiidus, P. M; Houston, M. E. Vitamin E status and response to exercise training. (1995). Sports
Med., 20:12–23.
Van der Beek, E. J.; Van Dokkum, W; Schrijver, J; Wesstra, A; Kistemaker, C; Hermus, R. J.
J. (1990). Controlled vitamin C restriction and physical performance in volunteers. J Am
Coll Nutr., 9:332–329.
Wallace, W. J; Houtchens, R. A; Maxwell, J. C; Caughey, W. S. (1982). Mechanism of
autooxidation for hemoglobins and myoglobins. Promotion of superoxide production by
protons and anions. J Biol Chem., 257(9): 4966–4977.
Wang, X. 1; Quinn, P. J. (2000). The location and function of vitamin e in membranes. Mol
Membr Biol., 17(3) :143-156.
Watt, T; Romet, T. T; McFarlane, I; McGuey, D; Allen, C; Goode, R. C. (1974). Vitamin E
and oxygen consumption. Lancet, 2:354–355.
Waugh, A; Grant, A (2014). Anatomy and physiology in health and illness. 10th, Elsevier Ltd:
30-31.
WHO. (2001). Biomarkers in Risk assessment: Validity and Validation. Geneva: WHO: 12.
Williams, M. H. (1989). Vitamin supplementation and athletic performance: an overview. Int
J Vitam Nutr Res,30: 161-191.

SECTION III: ANTIOXIDATIVE DEFENSE
MECHANISM IN NEURODEGENERATION

Chapter 5
ANTIOXIDANTS SIGNALING IN
NEURODEGENERATIVE DISORDERS
Abhilasha Ahlawat, Vaibhav Walia, Meenakshi Kaira

and Munish Garg
ABSTRACT
Neurodegenerative disorders involve neuronal damage in one or more areas of the
brain. Overproduction of reactive oxygen species (ROS) and the diminished amount of the
antioxidants amount to the development and propagation of neurodegenerative disorders.
The reduction in the levels of the antioxidants leads to increased neuronal insults/damage
which has been noticed in conditions including Parkinson’s disease (PD), Alzheimer’s
disease (AD) and Huntington’s disease (HD). Further, the oxidative damage plays a
prominent role in the neuronal damage in the aforementioned pathologies. Antioxidants
are either exogenous or endogenous compounds that remove free radicals, scavenge ROS,
inhibit production of ROS and bind to metal ions that catalyze ROS production. This
chapter describes the process of the neuronal degeneration in AD, PD, and HD, the role of
the oxidative stress in these pathologies and the role of antioxidant signaling in these
disorders.
1. INTRODUCTION
Neurodegeneration occurs due to the interaction of various factors such as environmental,
genetic and damages due to redox metal. ROS is the main culprit of neurodegeneration (Uttara
et al., 2009) in various pathologies which are characterized by the increased levels, production
or action of the ROS. For example, degeneration of dopaminergic neurons in the substantia
nigra (SN) region of the brain (Forno, 1996; Hornykiewicz and Kish, 1987) due to a large
amount of ROS making neurons in the SN (Bender et al., 2006; Kraytsberg et al., 2006). OS in

Corresponding Author’s Email: mgarg2006@gmail.com.

96 Abhilasha Ahlawat, Vaibhav Walia, Meenakshi Kaira et al.
PD promotes neurotoxicity by causing mitochondrial dysfunction (Ved et al., 2005) and by

stimulating the misfolding and aggregation of α-synuclein (Lotharius and Brundin, 2002).
Further, the various toxins which produce PD like symptoms in rodents also work by evoking
the oxidative stress (OS) in the targeted neurons (Tatton and Kish, 1997; Jackson-Lewis et al.,
1995; Mazzio et al., 2004).
Similarly, AD is characterized by the deposition of amyloid beta (Aβ) plaques in various
regions of the brain and is found to be dependant on the increased levels of ROS responsible
for tau phosphorylation mediated neuronal damage (Tamagno et al., 2003). Aβ mediated
accelerated production of ROS is also responsible for altered mitochondrial dynamics,
abnormality in energy metabolism and subsequent synaptic dysfunction (Christen, 2000; Beal,
2005; Baloyannis et al., 2004). Moreover, OS also plays a key role in the pathogenesis of other
neurodegenerative disorders i.e., HD (Sorolla et al., 2008; Perluigi et al., 2005).
Various molecular phenomenons such as dysregulation of transcription, mitochondrial
dysfunction, and protein aggregation have been found to be responsible for the HD (Kumar et
al., 2014; Luthi-Carter et al., 2000; Achour et al., 2015; Iannicola et al., 2000). It is suggested
that mHTT gene in HD enhances the ROS levels (Wyttenbach et al., 2002; Hands et al., 2011)
contributing to mitochondrial dysfunction (Sorolla et al., 2008; Sorolla et al., 2010).
Furthermore, HD is characterized by reduced levels of the various antioxidant enzymes (Sorolla
et al., 2008).
In the light of these findings, it is suggested that the ROS mediated oxidative damage is
the underlying cause of neurodegeneration in the mentioned pathologies such as AD, PD, and
HD. Further, there is an emerging role of ROS in age-associated disorders, there is debate
emerging in this topic (Gandhi and Abramov, 2012). Therefore, the objective of the present
chapter is to highlight the mechanism of neurodegeneration in these pathologies, role of ROS
in the process of neurodegeneration and how the antioxidant treatments counteract the
destructive effects of these ROS in neurodegenerative pathologies.
2. OXIDATIVE STRESS AND NEURODEGENERATIVE DISORDERS

Common neurodegenerative disorders compromising of motor or cognitive dysfunction
and damage to neuronal cells include AD, PD, and HD (Figure 1) and spinocerebellar ataxia
(SCA) (Alzheimer’s Association, 2016). ROS is the major consequence of aerobic cellular
metabolism. Free radicals have minimum one unpaired electron in their outermost shell which
makes them highly reactive. These free radicals utilizing oxygen are considered as ROS (Aiken
et al., 2011). Endogenous antioxidants play an important role in counteracting any kind of loss
caused by the overproduction of ROS. OS occur due to the imbalance between ROS production
and the body’s capacity to detoxify these highly reactive free radicles (Ienco et al., 2011). OS
due to the attack of free radicals on neuronal cells causes neurodegeneration and thus ROS
toxicity leads to protein misfolding, glial cell activation, mitochondrial dysfunction and further
cellular apoptosis (Fulda et al., 2010). Generally, ROS is not considered as the major triggering
factor for these disorders, but it is known to exacerbate the disease progression by causing
oxidative damage and interacting with mitochondria (Dias and Junn, 2013).

Antioxidants Signaling in Neurodegenerative Disorders 97
Figure 1. The role of oxidative stress in neurodegeneration.
AD is a neurodegenerative disorder identified by the presence of cognitive decline,

functional deterioration, psychiatric impairment, etc. Patients suffering from AD often exhibit
symptoms including memory deficits, language impairment, mental confusion, poor judgment,
hallucination, sleep disturbances, mood swings, etc. (Waldemar et al., 2007). AD corresponds
to nearly 60-80% of all types of cases of dementia which largely influence individuals above
65 years (Plassman et al., 2007). Approximately 1-6% of AD cases have younger-onset (<60-
65 years) while nearly 60% of early-onset AD patient (EOAD) are inherited, and 13% cases
are heritable in nature (Campion et al., 1999). The difference between the familial AD with
EOAD (beginning at the age of <60-65 years) and familial AD with late-onset (LOAD)
(beginning at the age of >60-65 years) is somewhat irrational (Brickell et al., 2006). EOAD
patients when compared to LOAD patients show fewer memory deficits but higher neurological
symptoms (Gerritsen et al., 2016), decreased overall morbidities (Gerritsen et al., 2016) more
neocortical atrophy in the parietal cortex and less atrophy in the mesial temporal lobe (MTL)
(Cho et al., 2013) and prominent pattern of damage in the posterior areas (Daianu et al., 2016).
Furthermore, hippocampal atrophy has been seen in typical amnestic EOAD patients with
language problems due to more left parietal atrophy in comparison to variant phenotypes of
EOAD with visuospatial presentations which have more right parietal-occipital atrophic
changes (Caso et al., 2015; Cerami et al., 2014).

AD is one of the familial causes of primary dementia distinguished by the accumulation

and deposition of insoluble plaques of Aβ and is the neuropathological hallmark of AD (Mirra
et al., 1991). Aβ is synthesized by amyloid precursor protein (APP) that plays an important role
in the transport of synaptic vesicles by acting as a receptor for them (Kamal
et al., 2001). APP is metabolized by the enzymes β-secretase and γ-secretase into Aβ peptide
(Cole and Vassar, 2007; Burdick et al., 1992; Gustaw et al., 2008) and the increased
accumulation of Aβ is responsible for the neuronal cell death into the affected regions of the
brain (Selkoe 1998; Shankar and Walsh, 2009). Aβ peptide in its monomeric form is cleared
easily and does not exert toxicity (Wegiel et al., 2007). But when aggregation of Aβ occurs,
this peptide develops toxic oligomers (Hartley et al., 1999; Roher et al., 1996) which exert
synaptic (Selkoe, 2008) and cognitive dysfunction (Tomic et al., 2009). Aggregation of Aβ
further results in the phosphorylation of tau proteins (Zheng et al., 2002) responsible for the
neuronal death via disruption of the cytoskeletal network (Iqbal et al., 1998; Brion et al., 2001;
Billingsley and Kincaid, 1997). Also, it has been observed that Aβ treatment increases the levels
of ROS responsible for the activation of enzymes resulting in the tau phosphorylation which
further exert neurotoxicity (Tamagno et al., 2003). Abnormal gathering of Aβ usually arises in
the early stages of AD (Sun et al., 2002; Phiel et al., 2003; Li et al., 2003; Su et al., 2004).
Disruption of axonal transport mechanism by abnormal phosphorylation of tau may, therefore,
lead to disturbances in the synaptic transmission and neuronal death (Rajmohan and Reddy,
2017). Phosphorylated tau exerts neuronal damage by interacting with Fyn which then
sensitizes the NMDA receptors, rendering the neurons more vulnerable to Aβ (Rajmohan and
Reddy, 2017). However, it has been reported that the substantial deposition of neocortical Aβ
may occur in the deprivation of prominent impairment of cognition or loss of neurons
(Giannakopoulos et al., 2003). Acetylcholine plays a crucial role in the maintenance of
cognition and therefore the cholinergic system is targeted in the context of AD and AD-related
dementia (Wilcock et al., 1982). Further, the cholinergic deficits influence cognition, behavior,
memory (Bartus, 2000) and therefore the disruption of cholinergic transmission and damage to
the cholinergic neurons affects the process mediated by these neurons in the brain (Muir et al.,
1992). Aβ also modulates the functions of cholinergic system (Blusztajn and Berse, 2000;
Hellstrom-Lindahl, 2000; Auld et al., 2002; Dolezal and Kasparova, 2003) and impose damage
to the cholinergic neurons in the forebrain region of AD brain (Francis et al., 1999; Blusztajn
and Berse, 2000). Aβ peptides presence and loss of neurons, or change in presynaptic
cholinergic markers, altered cholinergic transmission and degeneration within determined brain
areas further forms the basis of AD (Giovannelli et al., 1995; Harkany et al., 1995; Itoh et al.,
1996; Boncristiano et al., 2002; Bronfman et al., 2000; Wong et al., 1999). Aβ oligomer toxicity
at the cholinergic synapses (Wong et al., 1999; Bell et al., 2006) results in the cognitive
impairment (Terry et al., 1991; Selkoe, 2002) and memory loss in AD patients (Wilcock et al.,
1982).
2.1.1. Oxidative Stress and Neuronal Degeneration in Alzheimer’s Disease

OS is considered as an altered balance between prooxidant and antioxidant level (Jones,
2006) and is characterized by the enhanced level of ROS including superoxide radical anions
(O2–), hydrogen peroxide (H2O2), hydroxyl radicals (OH•), nitric oxide (NO) and peroxynitrite
(ONOO–) production. Mitochondria are the largest contributor to ROS (Ray et al., 2012;
Holmstrom and Finkel, 2014). ROS has been found to play a role in the glycation process
(Mattson et al., 1995) giving rise to the generation of advanced glycation end products (AGEs),

observed in the senile and classical plaques (Munch et al., 2000). AGEs are found to be in
association with Aβ (Vitek et al., 1994) demonstrating the role of AGEs in AD pathogenesis
(Colaco and Harrington, 1994; Smith et al., 1995).
Aβ deposition in AD results (Table 1) in the accelerated production of ROS by binding
directly to the mitochondrial membranes, responsible for altered mitochondrial dynamics,
function abnormality in energy metabolism and subsequent synaptic dysfunction (Christen,
2000; Beal, 2005; Baloyannis et al., 2004). Aβ mediated oxidative damage (Szabados et al.,
2004; Ichimura et al., 2003) and neurotoxicity (Garcia-Matas et al., 2010) has been observed
in AD patients (Shah et al., 2009). Aβ not only increases the levels of ROS but also depletes
the levels of antioxidant (Crouch et al., 2008). Aβ further causes membrane lipoperoxidation
and also affects the neuronal functions (Morris et al., 2016). Aβ oligomers stimulate the
formation of ROS by the activation of N-methyl-D-Aspartate (NMDA) receptors (De Felice et
al., 2007; Ferreira et al., 2007) resulting in the increased calcium influx and neuronal
excitotoxicity (Mattson and Chan, 2003) and synaptic dysfunction (De Felice et al., 2007; Rai
et al., 2013).
Table 1. Aβ promotes oxidative damage responsible for the neurodegeneration

in Alzheimer’s disease
Alzheimer’s Promote Consequences

Hallmark
Aβ Aggregation Increased generation of ROS
Aggregation to form the Altered mitochondrial dynamics and energy
protofibrils metabolism
Aβ itself Diminished antioxidant levels
Promotes membrane Altered neuronal function
lipoperoxidation
Activate NMDA receptors Increased Ca++ influx, excitotoxicity and
synaptic dysfunction
Interacts with heavy metal Increased ROS production due to the
formation of toxic oligomers
Phosphorylation of tau Formation of NFTs, increased ROS, disruption
of complex I of ETC
Aβ also increases the production of ROS through the interaction with biometals such as
copper, iron, and zinc (Kozlowski et al., 2012). For example, Fenton reaction, where metals
participate in the conversion of O2– and H2O2 into HO– species responsible for the process of
lipid peroxidation (Stohs and Bagchi, 1995). Aβ binds to copper via histidine bridge resulting
in the conversion of Cu2+ to Cu+ and generation of H2O2 (Huang et al., 1999). Further, APP and
Aβ possess the binding sites for heavy metals such as copper and zinc on N-terminal metal-
binding domains (Barnham et al., 2003). Aβ also forms complexes with the more toxic OH-
ions to form dityrosine crosslinked with Aβ peptides to form toxic oligomer (Valko et al., 2005;
Atwood et al., 2004; Barnham et al., 2004). Therefore, it is suggested that the neuronal toxicity
of Aβ is also mediated through the interaction between heavy metals and ROS. Treatment with
metal chelators thus restores the normal physiology in AD patients (Adlard et al., 2008; Cherny
et al., 2001; Opazo et al., 2003) and further reduces the burden of Aβ plaques in AD patients
(Cristovao et al., 2016). Besides the production of Aβ aggregates, redox metals also promote

phosphorylation of tau proteins to form neurofibrillary tangles (NFTs) resulting in the further
production of ROS and disruption of complexes (complex-1 mainly) of electron transport chain
(ETC) (Sayre et al., 2000; Boom et al., 2009; Mondragon-Rodriguez et al., 2013). This may
explain the requirement of the aggregation of both Aβ and pTau to begin the neurodegeneration
process in AD patients.
2.2. Parkinson’s Disease
James Parkinson first introduced the PD, arising due to the gradual degeneration of
dopaminergic neurons in the SN region of the midbrain (Dauer and Przedborski, 2003). In PD,
neuronal cell death in SN area induces the deficiency of dopamine (Forno, 1996; Hornykiewicz
and Kish, 1987) responsible for PD symptom which usually develops above 65 years, whereas
few individuals with PD in childhood have also been observed (Olanow andTatton, 1999;
Tanner, 2003). Dopaminergic neuronal death in PD results in the motor symptoms of PD such
as resting tremor, bradykinesia (slower movements and reflexes), stiffness and posture
imbalance (Goldenberg et al, 2008; Clarke, 2002). Rest tremor is generally a pill-rolling type
and is minimal damaging in nature (DeMaagd and Philip, 2015). Apart from rest tremors, PD
individuals frequently show the presence of several other forms of tremor. About 90% of cases
of PD exhibit resting tremor, 88-92% cases exhibit postural tremor and 17% cases exhibit head
tremor (Thenganatt and Louis, 2012). Extremely slower movements (Bradykinesia) is a critical
motor symptom of PD that may develop at the first or may appear after tremors (DeMaagd and
Philip, 2015) and is one of the common debilitating condition due to which patients are not able
to do the tasks (Goldenberg, 2008). Rigidity is defined as the enhanced resistance to the
movements (Baradaran et al, 2013) and is well expressed by the phrase/word cogwheel rigidity
in PD patients (DeMaagd and Philip, 2015). Rigidity usually occurs when the balance between
muscles get impaired and generally lead to masked face expression (DeMaagd and Philip,
2015). Instability in posture often progresses in the later stages of PD (Santamato et al, 2015)
responsible for the fall of the patients or increases the risk and incidence of fall in PD patients
(DeMaagd and Philip, 2015; Goldenberg, 2008) further predisposing the patients to injuries
and fracture (Santamato et al, 2015). The postural instability in PD denotes the presence of
damage in nondopaminergic areas of the brain (Santamato et al, 2015).
2.2.1. Oxidative Stress and Neuronal Degeneration in Parkinson’s Disease

Neurons of SN region are pigmented, have pacemaking activity, generate intensified
oxidative species caused by the dopamine (DA) metabolism and this becomes more particularly
susceptible to OS (Bender et al., 2006; Kraytsberg et al., 2006). Dopamine and its metabolites
contain two hydroxyl residues that are known to exert cytotoxicity in dopaminergic neuronal
cells due to the production of highly reactive DA and quinone (Asanuma et al., 2003).
Generally, DA quinone species have the potential of altering cellular nucleophiles covalently
which includes lower molecular weight sulfhydryl groups and protein cysteinyl residues (Table
2). DA quinone generally modifies various proteins for e.g., α-synuclein, DJ-1, UCH-L1, and
parkin, involved in PD pathology. DA quinone modifies and enhances the conversion of α-
synuclein to cytotoxic protofibril form (Conway et al., 2001). DA also covalently modifies
Parkin and it becomes insoluble leading to deactivation of activity of E2 ubiquitin ligase
(LaVoie et al., 2005). DA quinone also causes deactivation of dopamine transporter and

tyrosine hydroxylase (Kuhn et al., 1999) further resulting in dopamine deficiency. DA quinone
undergoes cyclization to convert into highly reactive compound aminochrome responsible for
the superoxide radical production and cellular NADPH exhaustion (Zecca et al., 2008).
Dopaminergic neuronal loss is also found to be linked with the presence of neuromelanin
(Perfeito et al., 2012). Neuromelanin is an intracellular store for iron and interacts with metals
(Bohic et al., 2008) resulting in iron overload in PD patients leading to the oxidative damage
in PD patients (Double et al., 2003; Faucheux et al., 2003; Jellinger et al.,1993). Ultimately,
neuromelanin leaking from the neurons also contributes to the neurodegeneration through the
activation of microglia and by precipitating inflammation in neurons (Zecca et al., 2008;
Karlsson and Lindquist, 2013).
Table 2. Dopamine itself promotes oxidative damage in Parkinson’s disease
Parkinson’s Pathology Promote Consequences

DA converts to Deactivation of DA deficiency
DA quinone tyrosine hydroxylase
Enhance conversion Cytotoxic protofibril form
of α-synuclein
Modifies Parkin Deactivation of E2 ubiquitin ligase activity
Conversion to Formation of superoxide radicals and NADPH
aminochrome exhaustion
OS in PD promotes the neurotoxicity by causing mitochondrial dysfunction (Ved et al.,

2005), mitochondrial membrane disarrangement (Conway et al., 2001) and suppression of
lysosomal protein breakdown (Martinez-Vicente et al., 2008). Alpha-Synuclein is found to be
over-expressed in particular melanized neurons where its aggregates switch to neuromelanin in
the SN early in PD (Grundemann et al., 2011; Li et al., 2012; Xuan et al., 2011). OS further
stimulates the misfolding and aggregation of α-synuclein resulting in neuronal toxicity
(Lotharius and Brundin, 2002). Accumulation of α-synuclein and its conformational changes
is also brought by the exposure to OS and heavy metal presence (Bendor et al., 2013; Breydo
et al., 2012; Fujiwara et al., 2002; Giasson et al., 2000). α-synuclein upon activation get
transferred in mitochondria in an energy-based process (Devi et al., 2008) and impaired the
activity of complex I of ETC resulting in reduced ΔΨm (mitochondrial membrane potential
gradient) responsible for the increased generation of ROS (Devi et al., 2008; Parihar et al.,
2008, 2009).
Levels of iron, increased by OS through various pathways inclusive of increased release of
iron from ferritin with the help of superoxide anion radical obtained from heme proteins (Kaur
and Andersen, 2004; Berg et al., 2001). Iron further produces ROS as ferric (Fe3+) and ferrous
form of iron (Fe2+) that may show a reaction with superoxide ion and H2O2, respectively, to
form most reactive OH- responsible for the oxidation of dopamine (Nunez et al., 2012).
However, no remarkable enhancement in iron levels is observed in the initial stages of PD, as
compared to symptomatic PD patients. Increased iron depositions further fasten and aggravate
the neuronal atrophy through OS-mediated pathways (Dexter et al., 1994).
Mitochondrial dysfunction mediated by toxins also aggravates the PD pathology by
promoting damage to the dopaminergic neurons (Greenamyre and Hastings, 2004). For
example, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is a widely used toxin for the

induction of PD and has the capability to generate progressive PD in human also (Davis et al.,
1979; Langston et al., 1983). MPTP is a lipophilic toxin that, usually after subcutaneous (SC)
or intraperitoneal (IP) injection, cross the BBB quickly (Riachi et al., 1989) and get transformed
by monoamine oxidase B (MAO-B) into the intermediate product, 1-methyl-4-phenyl-
2,3,dihydropyridinium (MPDP+) ion and then oxidized to the toxic moiety spontaneously, 1-
methyl-4-phenylpyridinium (MPP+) inside the brain (Chiba et al., 1984), which is taken up into
the dopaminergic neurons via DAT where it accelerates the production of ROS contributing to
neurotoxicity (Javitch et al., 1985). MPP+ also accumulate in the mitochondria and causes
impairment of complex-1 of ETC responsible for the inhibition of the mitochondrial respiration
(Nicklas et al., 1987), further resulting in the decreased production of ATP and increased
generation of the ROS. The integrated effect of decreased cellular ATP and enhanced
production of ROS is certainly responsible for the neuronal cell death, which finally leads to
cell death (Tatton and Kish, 1997; Jackson-Lewis et al., 1995). Another toxin, 6-OHDA, have
the ability to induce dopaminergic neurons degeneration in the nigrostriatal tract (Ungerstedt,
1968). It is taken up into the dopaminergic neurons via DAT (dopamine transporter) after its
injection in the brain where it commences degeneration via OS pathway. 6-OHDA oxidizes
freely to form H2O2 (Mazzio et al., 2004), decrease the levels of antioxidants such as GSH (total
glutathione) or SOD (superoxide dismutase) in striatum (Perumal et al., 1992; Kunikowska and
Jenner, 2001), increase the iron content (Oestreicher et al., 1994) and further inhibit the
mitochondrial respiration (Glinka et al., 1997). Another toxin, paraquat or 1,1′-dimethyl-4,4′-
bipyridinium and fungicide, maneb or manganese ethylene-bis-dithiocarbamate are found to be
linked with the increased occurrence of PD (Ascherio et al., 2006; Costello et al., 2009).
Paraquat is taken inside the neurons (Shimizu et al., 2001) where it is responsible for the
inhibition of mitochondrial complex I of the ETC (at higher doses) (Miller, 2007). Further
maneb also inhibit complex III of ETC responsible for the inhibition of mitochondrial
respiration (Zhang et al., 2003). Another toxin, rotenone, readily crosses the BBB and diffuses
into the neurons in the same way as MPTP and aggregates in mitochondria and inhibits complex
I of ETC. Rotenone, through the generation of ROS, depletes glutathione (Sherer et al., 2003a)
and induces the protein carbonyl formation observed in PD patients (Sherer et al., 2003a; Alam
et al., 1997). Huge activation of microglia has been observed in the various regions of the brain
after rotenone injection (Sherer et al., 2003b).
2.3. Huntington’s Disease
HD is a degenerative disorder of central nervous system (CNS), prominently affects the

striatum, cerebral cortex and other subcortical regions of the brain. Symptoms include
dementia, chorea (brief, abnormal, nonrhythmic movements that flows from one muscle to the
other), athetosis (slow, writhing movements), dysregulated coordination, abnormality in
speech, and difficulties in swallowing and neuropsychiatric symptoms including depression
(Blackstone, 2014). The typical motor features are involuntary and unwanted movements.
These movements mostly affect the distal extremities including fingers, toes during onset but
also affect small facial muscles in the later stages. Unstable walking may represent a person in
slightly drunk state. These undesirable movements usually affect other muscles also from distal
to axial and proximal part (Roos, 2010). Choreatic movements occur usually the entire time
when an individual is awake. Choreatic movements of the face may cause continual movements

in facial muscles such as eyebrow gets raised, an eye gets closed, head often bent or turned with
protruded tongue along with the pouting lips (Roos, 2010). The most recognized movements
are the extension movement of the long back muscles. In some patients, swallowing and talking
becomes more troublesome which gradually lead to choking at any time and can make patient
even mute in later stages. Dysarthria (speech disorder) and dysphagia (difficulty in swallowing)
are commonly seen. Nearly, all HD patients develop symptoms related to slower speed such as
hypokinesia, akinesia, and dystonia, slow movements along with an increased muscle tone
leading to an abnormality in postures, such as torticollis and trunk or limbs rotation (Roos,
2010). Other rare undesirable movements comprise of tics. Cerebellar signs may be visible
regularly along with the presence of both hypo- and hypermetria. Walking is frequently related
to ‘drunk’ or ‘cerebellar ataxia’-like. It is very difficult to distinguish between choreatic and
ataxic walking. Pyramidal signs or Babinski sign appears coincidentally. The effect of motor
disturbance on daily life activities increases slowly with time. Presence of hypo- and
hyperkinesia causes difficulty in standing, walking and often leads to an ataxic gait and frequent
falls. Moreover, routine tasks also become burdensome (Roos, 2010).
Psychiatric manifestations are frequently present in the initial phases of the disease even
before motor manifestations appears and varies between 33% and 76% of patients with HD
(van Duijn et al., 2007). As psychiatric symptoms have a considerable impact on routine life,
these signs and symptoms generally have a high adverse influence on routine functioning and
on the family (Wheelock et al., 2003), with depression, being most often arising manifestation.
Irritability is often the very first sign observed and occurs commonly during every stage of
disease (van Duijn et al., 2007). Expression of irritability differs remarkably from serious
disputes to physical aggressiveness. Apathy syndrome contributes to losing interest and
elevated passive behavior. It may be difficult to differentiate apathy from depression (Roos,
2010). The diagnosis of HD is usually troublesome due to apathy, loss of weight and lethargy
occurring in HD along with anxiety, feeling of no confidence and guilt. Instead of anxiety and
depression, apathy is considered to lead to disease stage. Suicide is more frequent in early
symptomatic patients and in premanifest genetic carriers. Anxiety also appears commonly (34-
61%). Obsessions and compulsions also lead to irritability and aggression and can adversely
affect the life of the patient (Roos, 2010). Psychosis appears primarily in the late phases of the
disease. In most of the cases, psychosis combines with cognitive impairment. These clinical
symptoms are similar to schizophrenia along with paranoid and acoustic hallucinations. In the
early stages, hyper-sexuality may pose serious issues in a relationship followed by hypo-
sexuality in the later stages (Roos, 2010).
Cognitive impairment/decline is another prominent symptom of HD and shows its presence
even before the first motor sign emerges, however, it may be minimal in later phases of the
disease. These cognitive alterations are especially related to the executive functions.
Individuals with HD lose the ability to differentiate between important and unimportant things.
These individuals can no longer organize or plan things which were quite simple in the past
(Bates et al., 2002). They can not make proper judgments. Impaired judgments often lead to
complex conditions where individuals can not recall what they did in the past or do not react in
a particular manner that everyone presumes. Language is comparatively spared. Memory gets
impaired to a certain extent, but semantic memory is spared certainly. Psychomotor processes
are critically hampered (Bates et al., 2002). Weight loss has also been seen. Hypothalamic
neuronal loss, slower functioning, reduced hunger, trouble in handling food and swallowing
also contribute to weight loss (Kremer et al., 1991; Aziz et al., 2007). Recognition has been

only gained for sleep- and circadian rhythm disorders in patients of HD (Arnulf et al., 2008).
Autonomic disruptions may result in profuse sweating.
Cleavage of htt protein by proteolysis is the main step in the neurotoxicity pathway. The
HTT gene codes for a particular protein made up of 3,144 amino acids along with the glutamine
tract initiating at codon 18. HD is known to be caused by a repeated expansion of ≥40 CAG
repeats of DNA trinucleotide (triplet) within the huntingtin gene (HTT, OMIM 613004).
Repeated lengths varying in the range of 27 to 35 are not found to be associated with the
occurrence of HD. Expansion upon transmission is more likely to occur at higher repeat lengths
and is more common during male transmission (Zühlke et al., 1993). In HD patients, amount
of CAG repeats extending from 16-20 repeats to more than 35 repeats resulting in extended
polyglutamine tract at the amino terminal of the transformed huntingtin protein are related to
protein aggregation and a gain-of-function toxicity (Munoz-Sanjuan and Bates, 2011; Warby
et al., 2011; Williams and Paulson, 2008). Mutant huntingtin (mHTT) gene undergoing higher
accumulation and forming cytoplasmic accumulates and nuclear inclusion in the brain is the
foremost salient hallmark feature of HD (Davies et al., 1997; DiFiglia et al., 1997). HTT gene
acts as a medium for proteolytic separation done by caspases and calpains (Gafni, 2002;
Goldberg et al., 1996; Wellington et al., 2002). The augmentation of an amino-terminal
truncation product in HD and in other polyQ diseases has led to the “toxic fragment hypothesis”
(Ross, 2002). As per this hypothesis, polyQ disease protein cleaves into a polyQ-containing
peptide representing the important toxic class at the molecular level (Slow et al., 2005).
Various evidence considers the short oligomeric organization of mutant HTT gene as the
major toxic species and suggests that the generation of huge inclusions may illustrate another
coping scheme in which mHTT is divided into a lesser widespread structure (Miller et al., 2011).
Aggregate formation of mHTT involves a complex multi-step process where mHTT monomers
gather to form an intermediary oligomeric species range before inclusions generated (Jeong et
al., 2009; Gu et al., 2009; Tam et al., 2009; Thakur et al., 2009; Kar et al., 2011). mHTT forms
a complex challenging pattern of oligomeric conformations making it difficult to understand
the pathological role of individual mHTT monomers, oligomers, and large inclusions that may
co-exist and disturb various cellular pathways and affect disease progression (Ren et al., 2009).
2.3.1. Oxidative Stress and Neuronal Degeneration in Huntington’s Disease

OS is the key event in the neuropathology of HD (Browne et al., 1999; Polidori et al., 1999;
Stack et al., 2008; Sorolla et al., 2008; Perluigi et al., 2005). Various molecular phenomenon
such as dysregulation of transcription, mitochondrial dysfunction, and protein aggregation is
related to pathology of HD, but it is not yet known that oxidative stress is caused by HD, or it
is an outcome of other key events (Kumar et al., 2014; Luthi-Carter et al., 2000; Achour et al.,
2015; Iannicolaet al., 2000). Metal dyshomeostasis may contribute to HD pathogenesis (Muller
and Leavitt, 2014). Transitional metals possess the powerful characteristic of depriving or
obtaining electrons simply because of the occurrence of unpaired electrons in their outermost
orbital shell. This kind of chemical property permits these metals (Fe, Zn, Cu) to be involved
in various physiological reactions (Brazier et al., 2014). Particularly, iron (Fe) and copper (Cu)
has shown to be involved as mediators of pathology as they are found to be accumulated in HD
(Bartzokiset al., 2007; Rosas et al., 2012; Fox et al., 2007). Raised iron aggregation is observed
in particular brain region (basal ganglia and cortex) of HD individuals which establishes its
connection between the CAG repeats number and seriousness of disease pathologic process
(Rosas et al., 2012).

Mutant huntingtin gene has shown to raise the double strand breaks by causing impairment
in DNA repair mechanism (Table 3) by interfering with non-homologous end joining repair
protein ku70 (Enokidoet al., 2010). Restoration of damaged DNA leads to the enlargement and
instability of CAG repetition in mHTT gene. It aids the notion that the level of DNA destruction
in specified tissue determines the extent of trinucleotide repeat instability. Although, Goula et
al., has highlighted that FEN1, a base excision repair (BER) enzyme and HMGB1, a BER co-
factor, are possible elements involved in regulating the variation in repeat instability (Goula et
al., 2009).
mHTT gene has also manifested to enhance the ROS levels (Wyttenbachet al., 2002; Hands
et al., 2011). In HD pathogenesis, mitochondrial dysfunction is considered as the key defect.
Creatine kinase and other mitochondrial proteins such as cytochrome b-c1 complex subunit 2
proteins and proteins of alpha subunit of ATPase and Krebs cycle, involved in the oxidative
phosphorylation, have recently shown to exert remarkable enhancement in carbonylation
(protein oxidation reaction catalyzed by ROS) in the striatum brain region (Sorolla et al., 2008;
Sorolla et al., 2010). Furthermore, the protein antioxidant enzymes (peroxiredoxin 1, 2 and 6,
glutathione peroxidases 1 and 6, and the activities of mitochondrial superoxide dismutase and
catalase) levels have shown to be markedly increased in autopsied brain region specifically
striatum and cortex of HD individuals (Sorolla et al., 2008). In various trials, complexes II, III,
and IV activities were also found to be significantly decreased in the striatum of HD patients
(Browne et al., 1997; Brennan et al., 1985; Stahl and Swanson, 1974; Gu et al., 1996). In
another study involving HD patients, various oxidative markers levels, such as leukocyte 8-
OHDG and plasma malondialdehyde (MDA) levels, were found to be elevated, but the
antioxidant activities, for e.g., Cu/Zn-SOD and GPx1 in erythrocytes were diminished in HD
patients when differentiated with healthy individuals (Chen et al., 2007).
Table 3. mHTT mediated neuronal damage in Huntington’s disease
Mutant Huntingtin Consequences

mHTT Impair DNA repair mechanism
Decreased activity of complex II, III, IV of ETC
Increased ROS
Increased levels of oxidative markers
Decreased Cu/Zn-SOD and GPx1 levels
Increased accumulation of Fe and Cu
3. ANTIOXIDANTS IN THE TREATMENT

OF NEURONAL DEGENERATION
Antioxidants are either exogenous or endogenous compounds that remove free radicals,
scavenge ROS, inhibit production of ROS and bind to metal ions that are frequently needed for
catalyzing ROS production. Natural antioxidants are further divided into enzymatic and non-
enzymatic type. Enzymatic antioxidants commonly include catalase, GPx, SOD. Non-
enzymatic antioxidants are either directly acting antioxidants such as ascorbic acid, lipoic acid,
polyphenols, flavonoids, carotenoids, generally obtained from dietary sources or indirectly

acting antioxidants such as chelating agents (Gilgun-Sherki et al., 2001). Figure 2 shows the
neuroprotection mediated by the antioxidants.
Figure 2. Neuroprotective effects of antioxidants to reduce neurodegeneration.
3.1. Alpha-Lipoic Acid
Alpha-lipoic acid (LA) is a dithiol compound known to boost cardiovascular, cognitive and
neuromuscular insufficiency (Smith et al., 2004; Scott et al., 1994; Devasagayam et al., 1993;
Liu et al., 2002; Suh et al., 2004; Lodge et al., 1998; Anuradha and Varalakshmi, 1999; Han et
al., 1997). Dithiolane ring of LA is mainly responsible for its action. Both oxidized and reduced
forms i.e., LA and dihydrolipoic acid (DHLA) are considered the most potent naturally derived
antioxidants (Searls and Sanadi, 1960). LA and DHLA terminates singlet oxygen (Scott et al.,
1994; Devasagayam et al., 1993; Suzuki et al., 1991; Kaiser et al., 1989; Devasagayam et al.,
1991; Haenen and Bast, 1991) but do not show activity against H2O2 (Scott et al., 1994; Haenen
and Bast, 1991). DHLA is also known to regenerate various other endogenous antioxidants for
e.g., vitamin C and E (Bast and Haenen, 2003). LA significantly enhances the intracellular
levels of glutathione (GSH) in various cells and tissues (Bast and Haene, 1998; Busse et al.,
1992).

3.2. Curcumin
Curcumin is a powerful antioxidant known to alleviate the symptoms of AD caused by

oxidation and inflammation (Frautschy and Hu, 2001). Curcumin significantly provides
protection against the Aβ insult (Kim et al., 2001) and also reduce the amount of Aβ (Yang et
al., 2005). Curcumin also inhibits the metabolism of APP (Narlawar et al., 2008) and decreases
the existing senile plaques (Garcia-Alloza et al., 2007) by increasing the phagocytosis of Aβ
(Fiala et al., 2007). Curcumin suppresses inflammatory damage by inhibiting the induction of
NF-Kβ (Baum and Ng, 2004; Shukla et al., 2003) and further inhibit the activation of TNF-α
and IL-1β (Park and Kim, 2002; Kim et al., 2005).
3.3. Ferulic Acid
Ferulic acid (FA) possesses various structural motifs that confer it a strong antioxidant
(Srinivasan et al., 2007). FA possesses the scavenging activity against the ROS and provides
protection against lipid peroxidation (Kanski et al., 2002). FA possesses the anti-amyloidogenic
activity by inhibiting the self-assembling activity and blocks the expansion of protofibrils and
increase the fragmentation of the mature fibrils (Picone et al., 2009; Bondi et al., 2009). FA
also directly alters the Aβ fibril formation (Hamaguchi et al., 2010) and provides protection
against learning and memory impairments caused by the administration of Aβ (Yan et al., 2001;
Yan et al., 2001).
3.4. Quercetin
Quercetin is a common flavonol known to exert the neuroprotective effects (Ossola et al.,
2009). Quercetin is a strong scavenging agent for ROS (O2∙ -) and RNS (reactive nitrogen
species such as nitric oxide [NO] and peroxynitrite [ONOO]) (Boots et al., 2008). It has been
reported that the administration of quercetin (0.5–50 mg/kg) protects from the oxidative injury
and neurotoxicity (Ishisaka et al., 2011; Das et al., 2008). Quercetin has been shown to
ameliorate the AD pathology and related cognitive disorders in an animal model of AD
(Sabogal-Guáqueta et al., 2015).
3.5. Vitamin E
Vitamin E is considered as an important antioxidant, and its antioxidant property is found

to be due to the presence of hydroxyl group of the aromatic ring of tocochromanols, which
further donates hydrogen to neutralize the free radicals, such as ROS. The antioxidant activities
of α, β, and γ isoforms of tocopherol and tocotrienol are the same whereas the δ isoform has
weaker activity (Brigelius-Flhoe, 2009; Muller et al., 2010). It is suggested that AD patients
have reduced plasma levels of vitamin E (Grundman, 2000). Vitamin E decreases the lipid
peroxidation in AD patients in comparison to normal control (Morris et al., 2005). Vitamin E
application in rat neuronal cultures arrests Aβ-associated ROS production, the oxidative stress

marker (Yatin et al., 2000). Also, the findings from the human studies suggested that vitamin
E supplementation improve the cognitive skills in AD patients (Sano et al., 1997).
3.6. Ascorbic Acid
Ascorbic acid (AA) possesses various effects on redox oxidative and mitochondrial
pathways (Padayatty et al., 2003; Naidu, 2003; Grosso et al., 2013; Duarte and Lunec, 2005).
Vitamin C plays an essential role in the catecholamines biosynthesis, peptide amination, myelin
formation, synaptic function, and exerts neuroprotection against glutamate toxicity (Nualart et
al., 2014; Harrison et al., 2014). AA homeostasis imbalance has been implicated in
degeneration of neurons (Covarrubias-Pinto et al., 2015). AA exerts various favorable outcome
in AD pathogenesis (Feng and Wang, 2012; Yao et al., 2004). AA neutralizes ROS reactivity
and provides neuroprotection by scavenging ROS, suppressing neuroinflammation and
fibrillation of Aβ (Choudhry et al., 2012) and provides protection against the glutamate
excitotoxicity in the brain (Qiu et al., 2007; Ballaz et al., 2013).
3.7. Rutin
Rutin or quercetin-3-O-rutinoside, a versatile naturally occurring flavonoid glycoside,

possess various beneficial properties such as antioxidant, anti-inflammatory, neuroprotective,
antidiabetic, cardiovascular and anticancer property (Habtemariam, 2016; Al-Dhabi et al.,
2015; Perk et al., 2014). Rutin is generally a glycone of quercetin, having flavonol structure,
responsible for alterations in cognitive and various other behavioral signs, as rutin and its
metabolites may cross the blood-brain barrier, thus, regulating numerous cellular functions
under pathological milieu (Arjumand et al., 2011; Habtemariam, 2016). Rutin also acts as a
scavenger for ROS (Hanasaki et al., 1994). Rutin also enhances the generation of glutathione
(GSH) and raises the levels of numerous antioxidant enzymes (Al-Enazi, 2014; Ahmed and
Zaki, 2009). Rutin inhibits the enzyme xanthine oxidase responsible for the generation of ROS
(Kostic et al., 2015). Rutin also inhibits nitric oxide synthase enzyme responsible for the
generation of NO (Teles et al., 2018). Rutin decreases and alters Aβ fibril formation, thus
overcome its toxicity to neurons (Jiménez-Aliaga et al., 2011). Rutin also inhibits Aβ
aggregation (Wang et al., 2012).
3.8. Naringin
Naringin, a flavanone glycoside commonly found in citrus fruits and grapefruits (Singh et
al., 2003; Choi et al., 2010; Golechha et al., 2011; Rong et al., 2012; Xianchu et al., 2016)
shows potential protective action due to its antioxidant and neuroprotective activities (Choi et
al., 2010; Golechha et al., 2011; Leem et al., 2014; Kim et al., 2016). Naringin regulates the
oxidative stress and induces neurotrophic factors and stimulates anti-apoptotic pathways (Rong
et al., 2012; Leem et al., 2014; Kim et al., 2016). Naringin protects the nigrostriatal DA
projections from neurotoxicity in PD (Leem et al., 2014; Kim et al., 2016). Naringin also
activates mTORC1 in adult DA neurons in vivo (Leem et al., 2014; Kim et al., 2016) and

provide protection to DA neurons (Leem et al., 2014; Kim et al., 2016). Naringin also attenuates
microglial activation responsible for the production of anti-inflammatory actions including
reduced cytokines (Leem et al., 2014; Kim et al., 2016).
3.9. Hesperidin
Hesperidin is a flavanone glycoside commonly found in citrus fruits having

neuroprotective and free radical scavenging properties. Hesperidin stimulates and promotes
neuronal survival (Nones et al., 2012). Hesperidin reduces APP expression, inhibits amyloid
beta accumulation and reduces microglial activation (Li et al., 2014). Hesperidin acts on glial
cells, microglia, and astrocytes by stimulating a decrease in neuroinflammation and oxidative
stress. Hesperidin also promotes synapse formation in various brain areas involved in memory
and decision-making (Teles et al., 2018) and thus might be beneficial in the treatment of AD
and related pathway.
3.10. Silibinin
Silibinin is a flavonoid, derived from plant milk thistle belonging to species Silybum
marianum, is a potential therapeutic agent for neurodegenerative disorders having
neuroprotective properties (Teles et al., 2018; Song et al., 2017). Silibinin has shown to reverse
memory damage and spatial learning and improves the ability to recognize new objects and
memory flexibility. Silibinin may suppress the expression of phosphorylated p53, responsible
for cell death (Song et al., 2017). Silibinin administration in aging animals decreases dopamine
depletion in the striatum region and improves motor behavior. Silibinin decreases the MDA
levels remarkedly and increases the GSH levels (Chen et al., 2015). Silibinin further protects
the dopaminergic neurons (Lee et al., 2015).

AND CHALLENGES
Recently, the effectiveness of natural antioxidants in the prevention and treatment of

various disorders has gained more attention. Various studies have been conducted to explore
their bioactive constituents which will provide greater practical significance for the exploitation
and utilization of natural antioxidants in various disorders (Li et al., 2014).
Modifications in brain GSH metabolism has shown to be involved in oxidative stress and
neurodegenerative disorders. So, reduced GSH levels in these disorders attract various
researchers to explore new potential approaches for retaining GSH levels. Due to poor stability
and low bioavailability, GSH delivery to the CNS is limited. Various approaches including
administration utilizing nanocarriers and codrug may be used in neurodegenerative disorders
related to deficiency of GSH. GSH and cysteinyl codrugs do not alter the pharmacodynamic
and physiochemical properties of the drug. These codrugs have been designed by combining a
particular group of antioxidant with available drugs so that it can resolve various issues of

permeation and oral absorption, stability, and solubility of the drug, drug resistance, and brain
delivery. However, there are only a few candidates approved for clinical use (Cacciatore et al.,
2012). This approach could be adopted in order to selectively deliver GSH to tissues in
sufficient concentrations to reduce the oxidative damage, but few data are available about the
clinical studies. Although the potential use of these strategies needs further exhaustive studies
as it offers a promising therapeutic alternative for reducing the GSH functional loss related to
human diseases such as PD and AD.
Despite the presence of several studies showing the effectiveness of antioxidants, the
majority of antioxidants available for human use has found to be ineffective (Gelain et al.,
2012). One such example includes antioxidant vitamin A, which may act as potent pro-oxidant
(Gelain et al., 2012). A clinical study has shown that there is a higher risk for lung cancer on
the oral intake of beta-carotene and retinol in humans but this study was discontinued due to
increased incidence of lung cancer, cardiovascular disease and increased death rate (Gelain et
al., 2012).
However, some authors consider that even pro-oxidants that earlier needed to be avoided
may play an important role in maintaining healthy status as less exposure to the free radicals
may regulate an effective antioxidant defense system through the physiological process, also
known as “oxidative stress preconditioning.” So, it is clear that antioxidants and antioxidant-
based therapies must be carefully addressed by researchers in the field of redox biology (Gelain
et al., 2012).
CONCLUSION
ROS accumulation results in the various damages, such as the cellular macromolecule
damage, neuronal dysfunction, synaptic dysfunction, altered release of the neurotransmitters
and the neuronal cell death. OS is common in the process of the neuronal degeneration. Since
the oxidative damage plays a key role in the neuronal damage, therefore the treatment with
antioxidants in these disorders might be effective as compared to the standard available therapy.
Further, various studies have described the benefits of antioxidants in the treatment of
neurodegenerative disorders. However, they are only used as an adjunct therapy, which hides
their full-scale potential in treatment in the clinical settings. So, the clinical studies should focus
on the use of these antioxidants for the treatment of these pathologies to explore their full-scale
potential in this regard.
REFERENCES
Achour M, Le Gras S, Keime C, Parmentier F, Lejeune F.X., Boutillier A.L., et al., Neuronal
identity genes regulated by super-enhancers are preferentially down-regulated in the
striatum of Huntington’s disease mice. Hum Mol Genet. 2015;24(12):3481-3496.
Adlard P.A., Cherny R. A., Finkelstein D. I., Gautier E, Robb E, Cortes M, Volitakis I, Liu X,
Smith J. P., Perez K, Laughton K. Rapid restoration of cognition in Alzheimer’s transgenic
mice with 8-hydroxy quinoline analogs is associated with decreased interstitial Aβ.
Neuron. 2008 Jul 10;59(1):43-55.

Ahmed M. M., Zaki N. I. Assessment the ameliorative effect of pomegranate and rutin on
chlorpyrifos-ethyl-induced oxidative stress in rats. Nature and Science. 2009;7(10):49-61.
Aiken C.T., Kaake R.M., Wang X, Huang L. Oxidative stress-mediated regulation of
proteasome complexes. Mol Cell Proteomics. 2011 May; 10(5):R110.006924.
Alam Z.I., Daniel S.E., Lees A.J., Marsden D.C., Jenner P, Halliwell B. A generalised increase
in protein carbonyls in the brain in Parkinson’s but not incidental Lewy body disease. J
Neurochem. 1997 Sep;69(3):1326-9.
Al-Dhabi N. A., Arasu M. V. , Park C. H., Park S. U. An up-to-date review of rutin and its
biological and pharmacological activities. EXCLI Journal. 2015;14:59.
Al-Enazi M. M. Protective effects of combined therapy of rutin with silymarin on
experimentally-induced diabetic neuropathy in rats. Pharmacology and Pharmacy.
2014;5(9):876–889.
Alzheimer’s Association. 2016 Alzheimer’s disease facts and figures. Alzheimers Dement.
2016 Apr; 12(4):459-509.
Anuradha B., Varalakshmi P. Protective role ofdl-α-lipoic acid against mercury-induced neural
lipid peroxidation. Pharmacol Res. 1999 Jan 1;39(1):67-80.
Arjumand W, Seth A, Sultana S. Rutin attenuates cisplatin induced renal inflammation and
apoptosis by reducing NFκB, TNF-α and caspase-3 expression in wistar rats. Food Chem
Toxicol. 2011 Sep; 49(9):2013-21.
Arnulf I., Nielsen J., Lohmann E., Schieffer J., Wild E., Jennum P., Konofal E., Walker M.,
Oudiette D., Tabrizi S., Durr A. Rapid eye movement sleep disturbances in Huntington
disease. Arch Neurol. 2008 Apr 1;65(4):482-8.
Asanuma M, Miyazaki I, Ogawa N. Dopamine-or L-DOPA-induced neurotoxicity: the role of
dopamine quinone formation and tyrosinase in a model of Parkinson’s disease.
Neurotoxicity research. 2003 Jan 1;5(3):165-76.
Ascherio A, Chen H, Weisskopf M. G., O’Reilly E., McCullough M. L., Calle E. E.,
Schwarzschild M. A., Thun M.J. Pesticide exposure and risk for Parkinson’s disease.
Annals of Neurology: Official Journal of the American Neurological Association and the
Child Neurology Society. 2006 Aug;60(2):197-203.
Atwood CS, Perry G, Zeng H, Kato Y, Jones WD, Ling KQ, Huang X, Moir RD, Wang D,
Sayre L.M., Smith MA. Copper mediates dityrosine cross-linking of Alzheimer’s amyloid-
β. Biochemistry. 2004 Jan 20;43(2):560-8.
Auld, D. S., Kornecook, T. J., Bastianetto, S. and Quirion, R., 2002. Alzheimer’s disease and
the basal forebrain cholinergic system: relations to β-amyloid peptides, cognition, and
treatment strategies. Prog Neurobiol, 68(3), pp. 209-245.
Aziz NA. Hypothalamic dysfunction and neuroendocrine and metabolic alterations in
Huntington disease: clinical consequences and therapeutic implications. Rev Neuroscience.
2007;18(3-4):223-52.
Ballaz S, Morales I, Rodríguez M, Obeso J.A.. Ascorbate prevents cell death from prolonged
exposure to glutamate in an in vitro model of human dopaminergic neurons. J Neurosci
Res. 2013 Dec;91(12):1609-17.
Baloyannis S. J., Costa V, Michmizos D. Mitochondrial alterations Alzheimer’s disease. Am J
Alzheimers Dis Other Demen. 2004 Mar;19(2):89-93.
Baradaran N, Tan S. N., Liu A, Ashoori A, Palmer S. J., Wang Z. J., Oishi M. M., McKeown
M. J., Parkinson’s disease rigidity: relation to brain connectivity and motor performance.
Front Neurol. 2013 Jun 5;4:67.

Barnham K. J., Haeffner F, Ciccotosto G. D., Curtain C. C., Tew D, Mavros C, Beyreuther K,
Carrington D, Masters C. L., Cherny R. A., Cappai R. Tyrosine gated electron transfer is
key to the toxic mechanism of Alzheimer’s disease β-amyloid. The FASEB J. 2004
Sep;18(12):1427-9.
Barnham KJ, McKinstry W. J., Multhaup G, Galatis D, Morton C. J., Curtain C. C., Williamson
N. A., White A. R., Hinds M. G, Norton R. S., Beyreuther K. Structure of the Alzheimer’s
disease amyloid precursor protein copper binding domain A regulator of neuronal copper
homeostasis. J Biol Chem. 2003 May 9;278(19):17401-7.
Bartus R.T. On neurodegenerative diseases, models, and treatment strategies: lessons learned
and lessons forgotten a generation following the cholinergic hypothesis. Exp Neurol. 2000
Jun 1;163(2):495-529.
Bartzokis G, Lu P. H., Geschwind D. H., Tingus K, Huang D, Mendez M. F., Edwards N, Mintz
J. Apolipoprotein E affects both myelin breakdown and cognition: implications for age-
related trajectories of decline into dementia. Biol Psychiatry. 2007 Dec 15;62(12):1380-7.
Bast A, Haenen GR. Interplay between lipoic acid and glutathione in the protection against
microsomal lipid peroxidation. Biochimica et Biophysica Acta (BBA)-Lipids and Lipid
Metabolism. 1988 Dec 16;963(3):558-61.
Bast A, Haenen GR. Lipoic acid: a multifunctional antioxidant. Biofactors. 2003;17(1‐4):207-
13.
Bates G, Harper P, Jones L. Huntington’s disease. 2002. Oxford, Oxford University press. 3.
Bates G, Harper P, Jones L. Huntington’s disease. 3. Oxford, Oxford University press; 2002.
Baum L, Ng A. Curcumin interaction with copper and iron suggests one possible mechanism
of action in Alzheimer’s disease animal models. J Alzheimer’s Dis. 2004 Jan 1;6(4):367-
77.
Beal M. F. Mitochondria take center stage in aging and neurodegeneration. Annals of
Neurology: Official Journal of the American Neurological Association and the Child
Neurology Society. 2005 Oct;58(4):495-505.
Bell K. F., Ducatenzeiler A, Ribeiro-da-Silva A, Duff K, Bennett D.A., Cuello A.C. The
amyloid pathology progresses in a neurotransmitter-specific manner. Neurobiol Aging.
2006 Nov 1;27(11):1644-57.
Bender A, Krishnan K. J., Morris C. M., Taylor G. A., Reeve A. K., Perry R. H., Jaros E,
Hersheson J. S., Betts J, Klopstock T, Taylor R. W. High levels of mitochondrial DNA
deletions in substantia nigra neurons in aging and Parkinson disease. NatGenet. 2006
May;38(5):515.
Bendor J. T, Logan T. P., Edwards R. H. The function of α-synuclein. Neuron. 2013 Sep
18;79(6):1044-66.
Berg D, Gerlach M, Youdim M.B., Double K.L., Zecca L, Riederer P, Becker G. Brain iron
pathways and their relevance to Parkinson’s disease. J Neurochem. 2001 Oct 15;79(2):225-
36.
Billingsley M. L., Kincaid R. L. Regulated phosphorylation and dephosphorylation of tau
protein: effects on microtubule interaction, intracellular trafficking and neurodegeneration.
Biochemical Journal. 1997 May 1;323(3):577-91.
Blackstone C. Huntington’s disease: from disease mechanisms to therapies. Drug Discov
Today. 2014 Jul;19(7):949–950.
Blusztajn J. K., Berse B. The cholinergic neuronal phenotype in alzheimer′ s disease. Metab
Brain Dis. 2000 Mar 1;15(1):45-64.

Bohic S, Murphy K, Paulus W, Cloetens, Salomé M, Susini J, Double K. Intracellular chemical

imaging of the developmental phases of human neuromelanin using synchrotron X-ray
microspectroscopy. AnalChem. 2008;80(24):9557-9566.
Boncristiano S, Calhoun M. E., Kelly P. H., Pfeifer M, Bondolfi L, Stalder M, Phinney A. L.,
Abramowski D, Sturchler-Pierrat C, Enz A, Sommer B. Cholinergic changes in the APP23
transgenic mouse model of cerebral amyloidosis. J Neuroscience. 2002 Apr 15;22(8):3234-
43.
Bondi ML, Montana G, Craparo E. F., Picone P, Capuano G, Carlo MD, Giammona G. Ferulic
acid-loaded lipid nanostructures as drug delivery systems for Alzheimer’s disease:
Preparation, characterization and cytotoxicity studies. Curr Nanosci. 2009 Feb 1;5(1):26-
32.
Boom A, Authelet M, Dedecker R, Frédérick C, Van Heurck R, Daubie V, Leroy K, Pochet R,
Brion J. P. Bimodal modulation of tau protein phosphorylation and conformation by
extracellular Zn2+ in human-tau transfected cells. Biochimica et Biophysica Acta (BBA)-
Molecular Cell Research. 2009 Jun 1;1793(6):1058-67.
Brazier M, Wedd A, Collins S. Antioxidant and metal chelation-based therapies in the treatment
of prion disease. Antioxidants. 2014 Apr 21;3(2):288-308.
Brennan Jr., W. A., Bird ED, Aprille J. R. Regional mitochondrial respiratory activity in
Huntington’s disease brain. J Neurochem. 1985 Jun;44(6):1948-50.
Breydo L, Wu J. W., Uversky VN. α-Synuclein misfolding and Parkinson’s disease. Biochimica
et Biophysica Acta (BBA)-Molecular Basis of Disease. 2012 Feb 1;1822(2):261-85.
Brickell K. L., Steinbart E. J, Rumbaugh M, Payami H, Schellenberg G. D., Van Deerlin V,
Yuan W, Bird T. D. Early-onset Alzheimer disease in families with late-onset Alzheimer
disease: a potential important subtype of familial Alzheimer disease. ArchNeurol. 2006
Sep 1;63(9):1307-11.
Brigelius-Flohé R. Vitamin E: the shrew waiting to be tamed. Free Radic Biol Med. 2009 Mar
1;46(5):543-54.
Brion JP, Anderton B. H., Authelet M., Dayanandan R., Leroy K., Lovestone S, Octave JN,
Pradier L, Touchet N, Tremp G. Neurofibrillary tangles and tau phosphorylation.
InBiochemical Society Symposia 2001 Feb 1 (Vol. 67, pp. 81-88). London; Portland on
behalf of The Biochemical Society; 1999.
Bronfman FC, Moechars D, Van Leuven F. Acetylcholinesterase-positive fiber deafferentation
and cell shrinkage in the septohippocampal pathway of aged amyloid precursor protein
london mutant transgenic mice. NeurobiolDis. 2000 Jun 30;7(3):152-68.
Browne S. E, Bowling A. C, Macgarvey U, Baik M. J, Berger S. C, Muquit M. M, Bird E. D,
Beal M. F. Oxidative damage and metabolic dysfunction in Huntington/s disease: selective
vulnerability of the basal ganglia. Annals of Neurology: Official Journal of the American
Neurological Association and the Child Neurology Society. 1997 May;41(5):646-53.
Browne S. E, Ferrante R. J, Beal M. F. Oxidative stress in Huntington/s disease. Brain Pathol.
1999 Jan;9(1):147-63.
Burdick D, Soreghan B, Kwon M, Kosmoski J, Knauer M, Henschen A, Yates J, Cotman C,
Glabe C. Assembly and aggregation properties of synthetic Alzheimer/s A4/beta amyloid
peptide analogs. J Biol Chem. 1992 Jan 5;267(1):546-54.
Busse E, Zimmer G, Schopohl B, Kornhuber B. Influence of alpha-lipoic acid on intracellular
glutathione in vitro and in vivo. Arzneimittel-Forschung. 1992 Jun;42(6):829-31.

Cacciatore I, Baldassarre L, Fornasari E, Mollica A, Pinnen F. Recent advances in the treatment

of neurodegenerative diseases based on GSH delivery systems. Oxid Med Cell Longev.
2012 Jun 3;2012.
Campion D, Dumanchin C, Hannequin D, Dubois B, Belliard S, Puel M, Thomas-Anterion C,
Michon A, Martin C, Charbonnier F, Raux G. Early-onset autosomal dominant Alzheimer
disease: prevalence, genetic heterogeneity, and mutation spectrum. Am J Hum Genet. 1999
Sep 1;65(3):664-70.
Caso F, Agosta F, Mattavelli D, Migliaccio R, Canu E, Magnani G, Marcone A, Copetti M,
Falautano M, Comi G, Falini A. White matter degeneration in atypical Alzheimer disease.
Radiol. 2015 May 27;277(1):162-72.
Cerami C, Crespi C, Della Rosa PA, Dodich A, Marcone A, Magnani G, Coppi E, Falini A,
Cappa S. F., Perani D. Brain changes within the visuo-spatial attentional network in
posterior cortical atrophy. J Alzheimer/s Dis. 2015 Jan 1;43(2):385-95.
Chen C. M., Wu Y. R, Cheng M. L., Liu J. L., Lee YM, Lee P. W., Soong B. W., Chiu D. T.
Increased oxidative damage and mitochondrial abnormalities in the peripheral blood of
Huntington’s disease patients. Biochem Biophys Res Commun. 2007 Jul 27;359(2):335-40.
Chen H, Wang X, Wang M, Yang L, Yan Z, Zhang Y, Liu Z. Behavioral and neurochemical
deficits in aging rats with increased neonatal iron intake: silibinin’s neuroprotection by
maintaining redox balance. Front Aging Neurosci. 2015 Oct 28;7:206.
Chen M, Gu H, Ye Y, Lin B, Sun L, Deng W, Zhang J, Liu J. Protective effects of hesperidin
against oxidative stress of tert-butyl hydroperoxide in human hepatocytes. Food Chem
Toxicol. 2010 Oct 1;48(10):2980-7.
Cherny R. A., Atwood C. S, Xilinas M. E, Gray D. N, Jones W. D., McLean C. A., Barnham
K. J., Volitakis I, Fraser F. W., Kim Y. S., Huang X. Treatment with a copper-zinc chelator
markedly and rapidly inhibits β-amyloid accumulation in Alzheimer/s disease transgenic
mice. Neuron. 2001 May 1;30(3):665-76.
Chiba K, Trevor A, Castagnoli Jr., N. Metabolism of the neurotoxic tertiary amine, MPTP, by
brain monoamine oxidase. Biochem Biophys Res Commun. 1984 Apr 30;120(2):574-8.
Cho H, Seo S. W., Kim J. H, Kim C, Ye BS, Kim G. H., Noh Y, Kim H. J, Yoon C. W., Seong
J. K., Kim C. H. Changes in subcortical structures in early-versus late-onset Alzheimer/s
disease. NeurobiolAging. 2013 Jul 1;34(7):1740-7.
Choi B. S., Sapkota K, Kim S, Lee H. J., Choi HS, Kim S. J. Antioxidant activity and protective
effects of Tripterygium regelii extract on hydrogen peroxide-induced injury in human
dopaminergic cells, SH-SY5Y. Neurochem Res. 2010;35:1269–1280.
Choudhry F, Howlett D. R, Richardson J. C., Francis P. T., Williams R. J. Pro-oxidant diet
enhances β/γ secretase-mediated APP processing in APP/PS1 transgenic mice. Neurobiol
Aging. 2012 May 1;33(5):960-8.
Christen Y. Oxidative stress and Alzheimer disease. Am JClin Nutr. 2000 Feb 1;71(2):621S-
9S.
Ciancarelli I, De Amicis D, Di Massimo C, Di Scanno C, Pistarini C, D’Orazio N, Tozzi
Ciancarelli MG. Peripheral biomarkers of oxidative stress and their limited potential in
evaluation of clinical features of Huntington’s patients. Biomarkers. 2014 Sep 1;19(6):452-
6.
Clarke, C. E. Medical management of Parkinson’s disease. J Neurol Neurosurg Psychiatry.
2002 Mar 1;72(suppl 1):i22-7.

Colaco, C. A., Harrington CR. Glycation: a pathological modification in neuropathies?: a

hypothesis. Neuroreport. 1994 Apr;5(8):859-61.
Cole, S. L., Vassar R. The Alzheimer’s disease β-secretase enzyme, BACE1. Mol
Neurodegener. 2007 Dec;2(1):22.
Conway, K. A., Rochet J. C., Bieganski R. M., Lansbury P. T., Jr Kinetic stabilization of the
alpha-synuclein protofibril by a dopamine-alpha-synuclein adduct. Science.
2001;294:1346–49.
Costello, S, Cockburn M, Bronstein J, Zhang X, Ritz B. Parkinson’s disease and residential
exposure to maneb and paraquat from agricultural applications in the central valley of
California. Am JEpidemiol. 2009 Mar 6;169(8):919-26.
Covarrubias-Pinto A, Acuña A. I., Beltrán F. A., Torres-Díaz L, Castro M. A,. Old things new
view: ascorbic acid protects the brain in neurodegenerative disorders. Int JMol Sci. 2015
Nov 27;16(12):28194-217.
Cristóvão, J. S., Santos R, Gomes C. M. Metals and neuronal metal binding proteins implicated
in Alzheimer’s disease. Oxidative Med Cell Longev. 2016;2016:9812178.
Crouch, P. J, Harding SM, White AR, Camakaris J, Bush AI, Masters CL. Mechanisms of Aβ
mediated neurodegeneration in Alzheimer’s disease. Int JBiochem Cell Biol. 2008 Jan
1;40(2):181-98.
Daianu, M, Mezher A, Mendez MF, Jahanshad N, Jimenez EE, Thompson PM. Disrupted rich
club network in behavioral variant frontotemporal dementia and early‐onset Alzheimer’s
disease. HumBrain Mapp. 2016 Mar;37(3):868-83.
Das, S, Mandal AK, Ghosh A, Panda S, Das N, Sarkar S. Nanoparticulated quercetin in
combating age related cerebral oxidative injury. Curr Aging Sci. 2008 Dec 1;1(3):169-74.
Dauer, W, Przedborski S. Parkinson’s disease: mechanisms and models. Neuron. 2003 Sep
11;39(6):889-909.
Davies, S. W., Turmaine M, Cozens B. A., DiFiglia M, Sharp A. H., Ross C. A., Scherzinger
E, Wanker E. E., Mangiarini L, Bates G. P. Formation of neuronal intranuclear inclusions
underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell. 1997
Aug 8;90(3):537-48.
Davis, G. C., Williams A. C., Markey S. P., Ebert M. H., Caine E. D., Reichert C. M., Kopin I.
J,. Chronic parkinsonism secondary to intravenous injection of meperidine analogues.
Psychiatry Res. 1979 Dec 1;1(3):249-54.
De Andrade Teles RB, Diniz T. C., Pinto C, Coimbra T, de Oliveira Júnior R. G., Gama e Silva
M, de Lavor ÉM, Fernandes A. W., de Oliveira A. P., de Almeida Ribeiro F. P., da Silva
A. A. Flavonoids as Therapeutic Agents in Alzheimer’s and Parkinson’s Diseases: A
Systematic Review of Preclinical Evidences. Oxid Med Cell Longev. 2018;2018.
De Felice F. G., Velasco P. T., Lambert M. P., Viola K, Fernandez S. J., Ferreira S. T., Klein
W. L. Aβ oligomers induce neuronal oxidative stress through an N-methyl-D-aspartate
receptor-dependent mechanism that is blocked by the Alzheimer drug memantine. J Biol
Chem. 2007 Apr 13;282(15):11590-601.
DeMaagd G, Philip A. Parkinson’s disease and its management: part 1: disease entity, risk
factors, pathophysiology, clinical presentation, and diagnosis. Pharm Ther. 2015
Aug;40(8):504-32.
Devasagayam, T. P., Di Mascio P, Kaiser S, Sies H. Singlet oxygen induced single-strand
breaks in plasmid pBR322 DNA: the enhancing effect of thiols. Biochimica et Biophysica
Acta (BBA)-Gene Structure and Expression. 1991 Mar 26;1088(3):409-12.

Devasagayam T. P., Subramanian M, Pradhan D. S., Sies H. Prevention of singlet oxygen-

induced DNA damage by lipoate. Chemico-biological interactions. 1993 Jan 1;86(1):79-
92.
Devi L, Raghavendran V, Prabhu B. M., Avadhani N. G., Anandatheerthavarada H. K.
Mitochondrial import and accumulation of α-synuclein impair complex I in human
dopaminergic neuronal cultures and Parkinson disease brain. J Biol Chem. 2008 Apr
4;283(14):9089-100.
Dexter DT, Sian J, Rose S, Hindmarsh J. G., Mann V. M., Cooper J. M., Wells F. R., Daniel S.
E., Lees A. J., Schapira A. H., Jenner P. Indices of oxidative stress and mitochondrial
function in individuals with incidental Lewy body disease. Annals of Neurology: Official
Journal of the American Neurological Association and the Child Neurology Society. 1994
Jan;35(1):38-44.
Dias V, Junn E, Mouradian M. M. The role of oxidative stress in Parkinson’s disease.
DiFiglia M, Sapp E, Chase K. O., Davies S. W., Bates G. P., Vonsattel J. P., Aronin N.
Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in
brain. Science. 1997 Sep 26;277(5334):1990-3.
Doležal V, Kašparová J. β-amyloid and cholinergic neurons. Neurochem Res. 2003 Apr 1;28(3-
4):499-506.
Double K. L., Gerlach M, Schünemann V, Trautwein A. X., Zecca L, Gallorini M, Youdim M.
B., Riederer P, Ben-Shachar D. Iron-binding characteristics of neuromelanin of the human
substantia nigra. BiochemPharmacol. 2003 Aug 1;66(3):489-94.
Duarte T. L., Lunec J. When is an antioxidant not an antioxidant? A review of novel actions
and reactions of vitamin C. Free Radic Res. 2005 Jul;39(7):671-86.
Enokido Y, Tamura T, Ito H, Arumughan A, Komuro A, Shiwaku H, Sone M, Foulle R, Sawada
H, Ishiguro H, Ono T. Mutant huntingtin impairs Ku70-mediated DNA repair. J Cell Biol.
2010 May 3;189(3):425-43.
Entezam A, Lokanga AR, Le W, Hoffman G, Usdin K. Potassium bromate, a potent DNA
oxidizing agent, exacerbates germline repeat expansion in a fragile X premutation mouse
model. Human Mutation. 2010 May;31(5):611-6.
Faucheux B. A , Martin M. E , Beaumont C, Hauw J. J., Agid Y, Hirsch E. C. Neuromelanin
associated redox‐active iron is increased in the substantia nigra of patients with Parkinson’s
disease. J Neurochem. 2003 Sep;86(5):1142-8.
Feigin A, Kieburtz K, Como P, Hickey C, Abwender D, Zimmerman C, Steinberg K, Shoulson
I. Assessment of coenzyme Q10 tolerability in Huntington’s disease. Movement disorders:
official journal of the Movement Disorder Society. 1996 May;11(3):321-3.
Feng Y, Wang X. Antioxidant therapies for Alzheimer’s disease. OxidMed Cell Longev. 2012
Jul 25;2012.
Ferreira ST, Vieira MN, De Felice FG. Soluble protein oligomers as emerging toxins in
Alzheimer’s and other amyloid diseases. IUBMB Life. 2007;59(4‐5):332-45.
Fiala M, Liu PT, Espinosa-Jeffrey A, Rosenthal MJ, Bernard G, Ringman JM, Sayre J, Zhang
L, Zaghi J, Dejbakhsh S, Chiang B. Innate immunity and transcription of MGAT-III and
Toll-like receptors in Alzheimer’s disease patients are improved by
bisdemethoxycurcumin. Proceedings of the National Academy of Sciences. 2007 Jul
31;104(31):12849-54.
Forno LS. Neuropathology of Parkinson’s disease. Journal of Neuropathology and
Experimental Neurology. 1996;55(3):259-272.

Fox J. H., Kama J. A., Lieberman G, Chopra R, Dorsey K, Chopra V, Volitakis I, Cherny RA,
Bush AI, Hersch S. Mechanisms of copper ion mediated Huntington’s disease progression.
PloS one. 2007 Mar 28;2(3):e334.
Francis PT, Palmer AM, Snape M, Wilcock GK. The cholinergic hypothesis of Alzheimer’s
disease: a review of progress. Journal of Neurology, Neurosurgery and Psychiatry. 1999
Feb 1;66(2):137-47.
Frautschy SA, Hu W, Kim P, Miller SA, Chu T, Harris-White ME, Cole GM. Phenolic anti-
inflammatory antioxidant reversal of Aβ-induced cognitive deficits and neuropathology.
Neurobiology of aging. 2001 Nov 1;22(6):993-1005.
Fujiwara H, Hasegawa M, Dohmae N, Kawashima A, Masliah E, Goldberg MS, Shen J, Takio
K, Iwatsubo T. α-Synuclein is phosphorylated in synucleinopathy lesions. Nature cell
biology. 2002 Feb;4(2):160.
Fulda S, Gorman AM, Hori O, Samali A. Cellular stress responses: cell survival and cell death.
Int J Cell Biol. 2010; 2010():214074.
Gafni J, Ellerby LM. Calpain activation in Huntington’s disease. Journal of Neuroscience. 2002
Jun 15;22(12):4842-9.
Gandhi S, Abramov AY. Mechanism of oxidative stress in neurodegeneration. Oxidative
medicine and cellular longevity. 2012 May 16;2012.
Garcia‐Alloza M, Borrelli LA, Rozkalne A, Hyman BT, Bacskai BJ. Curcumin labels amyloid
pathology in vivo, disrupts existing plaques, and partially restores distorted neurites in an
Alzheimer mouse model. Journal of neurochemistry. 2007 Aug;102(4):1095-104.
García-Matas S, de Vera N, Aznar AO, Marimon JM, Adell A, Planas AM, Cristòfol R,
Sanfeliu C. In vitro and in vivo activation of astrocytes by amyloid-β is potentiated by pro-
oxidant agents. Journal of Alzheimer’s Disease. 2010 Jan 1;20(1):229-45.
Gaur V, Kumar A. Hesperidin pre-treatment attenuates NO-mediated cerebral ischemic
reperfusion injury and memory dysfunction. Pharmacological Reports. 2010 Jul
1;62(4):635-48.
Gelain DP, Antonio Behr G, Birnfeld de Oliveira R, Trujillo M. Antioxidant therapies for
neurodegenerative diseases: mechanisms, current trends, and perspectives. Oxidative
medicine and cellular longevity. 2012 Dec 5;2012.
Gerritsen AA, Bakker C, Verhey FR, de Vugt ME, Melis RJ, Koopmans RT, Oosterveld SM,
Kessels RP, Rikkert MO, Hamel R, Ramakers IH. Prevalence of comorbidity in patients
with young-onset Alzheimer disease compared with late-onset: a comparative cohort study.
Journal of the American Medical Directors Association. 2016 Apr 1;17(4):318-23.
Giannakopoulos P, Herrmann FR, Bussière T, Bouras C, Kövari E, Perl DP, Morrison JH, Gold
G, Hof PR. Tangle and neuron numbers, but not amyloid load, predict cognitive status in
Alzheimer’s disease. Neurology. 2003 May 13;60(9):1495-500.
Giasson BI, Duda JE, Murray IV, Chen Q, Souza JM, Hurtig HI, Ischiropoulos H, Trojanowski
JQ, Lee VM. Oxidative damage linked to neurodegeneration by selective α-synuclein
nitration in synucleinopathy lesions. Science. 2000 Nov 3;290(5493):985-9.
Gilgun-Sherki Y, Melamed E, Offen D. Oxidative stress induced-neurodegenerative diseases:
the need for antioxidants that penetrate the blood brain barrier. Neuropharmacology. 2001
Jun; 40(8):959-75.
Giovannelli L, Casamenti F, Scali C, Bartolini L, Pepeu G. Differential effects of amyloid
peptides β-(1–40) and β-(25–35) injections into the rat nucleus basalis. Neuroscience. 1995
Jun 1;66(4):781-92.

Glinka Y, Gassen M, Youdim MB. Mechanism of 6-hydroxydopamine neurotoxicity.

InAdvances in Research on Neurodegeneration 1997 (pp. 55-66). Springer, Vienna.
Goldberg YP, Nicholson D, Rasper DM, Kalchman MA, Koide HB, Graham RK, Bromm M,
Kazemi-Esfarjani P, Thornberry NA, Vaillancourt JP, Hayden MR. Cleavage of huntingtin
by apopain, a proapoptotic cysteine protease, is modulated by the polyglutamine tract.
Nature genetics. 1996 Aug;13(4):442–49.
Goldenberg MM. Medical Management of Parkinson’s Disease. Pharmacy and Therapeutics,
2008;33(10):590-606.
Golechha M, Chaudhry U, Bhatia J, Saluja D, Arya DS. Naringin protects against kainic acid-
induced status epilepticus in rats: evidence for an antioxidant, anti-inflammatory and
neuroprotective intervention. Biol Pharm Bull. 2011;34:360–365.
Goula AV, Berquist BR, Wilson III DM, Wheeler VC, Trottier Y, Merienne K. Stoichiometry
of base excision repair proteins correlates with increased somatic CAG instability in
striatum over cerebellum in Huntington’s disease transgenic mice. PLoS genetics. 2009
Dec 4;5(12):e1000749.
Greenamyre JT, Hastings TG.Parkinson’s--divergent causes, convergent mechanisms. Science.
2004 May 21;304(5674):1120-2.
Grosso G, Bei R, Mistretta A, Marventano S, Calabrese G, Masuelli L, Giganti MG, Modesti
A, Galvano F, Gazzolo D. Effects of vitamin C on health: a review of evidence. Front
Biosci (Landmark Ed). 2013 Jun 1;18:1017-29.
Gründemann J, Schlaudraff F, Liss B. UV-laser microdissection and mRNA expression
analysis of individual neurons from postmortem Parkinson’s disease brains. InLaser
Capture Microdissection 2011 (pp. 363-374). Humana Press.
Grundman M. Vitamin E and Alzheimer disease: the basis for additional clinical trials–. The
American journal of clinical nutrition. 2000 Feb 1;71(2):630S-6S.
Gu M, Gash MT, Mann VM, Javoy‐Agid F, Cooper JM, Schapira AH. Mitochondrial defect in
Huntington’s disease caudate nucleus. Annals of Neurology: Official Journal of the
American Neurological Association and the Child Neurology Society. 1996
Mar;39(3):385-9.
Gu X, Greiner ER, Mishra R, Kodali R, Osmand A, Finkbeiner S, Steffan JS, Thompson LM,
Wetzel R, Yang XW. Serines 13 and 16 are critical determinants of full-length human
mutant huntingtin induced disease pathogenesis in HD mice. Neuron. 2009 Dec
24;64(6):828-40.
Gustaw KA, Garrett MR, Lee HG, Castellani RJ, Zagorski MG, Prakasam A, Siedlak SL, Zhu
X, Perry G, Petersen RB, Friedland RP. Antigen–antibody dissociation in Alzheimer
disease: a novel approach to diagnosis. Journal of neurochemistry. 2008 Aug;106(3):1350-
6.
Habtemariam S. Rutin as a natural therapy for Alzheimer’s disease: Insights into its
mechanisms of action. Current medicinal chemistry. 2016 Mar 1;23(9):860-73.
Haenen GR, Bast A. Scavenging of hypochlorous acid by lipoic acid. Biochemical
pharmacology. 1991 Nov 6;42(11):2244-6.
Hamaguchi T, Ono K, Yamada M. Curcumin and Alzheimer’s disease. CNS Neuroscience and
Therapeutics. 2010;16(5):285-297.
Han D, Sen CK, Roy S, Kobayashi MS, Tritschler HJ, Packer L. Protection against glutamate-
induced cytotoxicity in C6 glial cells by thiol antioxidants. American Journal of

Physiology-Regulatory, Integrative and Comparative Physiology. 1997 Nov 1;273(5):

R1771-8.
Hanasaki Y, Ogawa S, Fukui S. The correlation between active oxygens scavenging and
antioxidative effects of flavonoids. Free Radic Biol Med. 1994 Jun; 16(6):845-50.
Hands S, Sajjad MU, Newton MJ, Wyttenbach A. In vitro and in vivo aggregation of a fragment
of huntingtin protein directly causes free radical production. Journal of Biological
Chemistry. 2011 Dec 30;286(52):44512-20.
Harkany T, Lengyel Z, Soós K, Penke B, Luiten PG, Gulya K. Cholinotoxic effects of β-
amyloid (1–42) peptide on cortical projections of the rat nucleus basalis magnocellularis.
Brain research. 1995 Oct 9;695(1):71-5.
Harrison F, Bowman G, Polidori M. Ascorbic acid and the brain: rationale for the use against
cognitive decline. Nutrients. 2014 Apr 24;6(4):1752-81.
Hartley DM, Walsh DM, Chianping PY, Diehl T, Vasquez S, Vassilev PM, Teplow DB, Selkoe
DJ. Protofibrillar intermediates of amyloid β-protein induce acute electrophysiological
changes and progressive neurotoxicity in cortical neurons. Journal of Neuroscience. 1999
Oct 15;19(20):8876-84.
Hellström‐Lindahl E, Moore H, Nordberg A. Increased levels of tau protein in SH‐SY5Y cells
after treatment with cholinesterase inhibitors and nicotinic agonists. Journal of
neurochemistry. 2000 Feb 1;74(2):777-84.
Hersch SM, Gevorkian S, Marder K, Moskowitz C, Feigin A, Cox M, Como P, Zimmerman C,
Lin M, Zhang L, Ulug AM. Creatine in Huntington disease is safe, tolerable, bioavailable
in brain and reduces serum 8OH2′ dG. Neurology. 2006 Jan 24;66(2):250-2.
Holmström KM, Finkel T. Cellular mechanisms and physiological consequences of redox-
dependent signalling. Nature reviews Molecular cell biology. 2014 Jun;15(6):411–421.
Hornykiewicz O. Biochemical pathophysiology of Parkinson’s disease. Parkinson’s disease.
Advances in Neurology. 1986;45:19-34.
Huang SM, Tsai SY, Lin JA, Wu CH, Yen GC. Cytoprotective effects of hesperetin and
hesperidin against amyloid β‐induced impairment of glucose transport through
downregulation of neuronal autophagy. Molecular nutrition and food research. 2012
Apr;56(4):601-9.
Huang X, Atwood CS, Hartshorn MA, Multhaup G, Goldstein LE, Scarpa RC, Cuajungco MP,
Gray DN, Lim J, Moir RD, Tanzi RE. The Aβ peptide of Alzheimer’s disease directly
produces hydrogen peroxide through metal ion reduction. Biochemistry. 1999 Jun
15;38(24):7609-16.
Huntington Study Group. A randomized, placebo-controlled trial of coenzyme Q10 and
remacemide in Huntington’s disease. Neurology. 2001 Aug 14;57(3):397-404.
Huntington Study Group. Safety and tolerability of the free-radical scavenger OPC-14117 in
Huntington’s disease. Neurology. 1998 May 1;50(5):1366-73.
Iannicola C, Moreno S, Oliverio S, Nardacci R, Ciofi‐Luzzatto A, Piacentini M. Early
alterations in gene expression and cell morphology in a mouse model of Huntington’s
disease. Journal of neurochemistry. 2000 Aug;75(2):830-9.
Ichimura H, Parthasarathi K, Quadri S, Issekutz AC, Bhattacharya J. Mechano-oxidative
coupling by mitochondria induces proinflammatory responses in lung venular capillaries.
The Journal of clinical investigation. 2003 Mar 1;111(5):691-9.

Ienco EC, LoGerfo A, Carlesi C, Orsucci D, Ricci G, Mancuso M, et al., Oxidative stress
treatment for clinical trials in neurodegenerative diseases. J Alzheimers Dis. 2011; 24 Suppl
2():111-26.
Iqbal K, Alonso AD, Gong CX, Khatoon S, Pei JJ, Wang JZ, Grundke-Iqbal I. Mechanisms of
neurofibrillary degeneration and the formation of neurofibrillary tangles. InAgeing and
Dementia 1998 (pp. 169-180). Springer, Vienna.
Ishisaka A, Ichikawa S, Sakakibara H, Piskula MK, Nakamura T, Kato Y, Ito M, Miyamoto
KI, Tsuji A, Kawai Y, Terao J. Accumulation of orally administered quercetin in brain
tissue and its antioxidative effects in rats. Free Radical Biology and Medicine. 2011 Oct
1;51(7):1329-36.
Itoh A, Nitta A, Nadai M, Nishimura K, Hirose M, Hasegawa T, Nabeshima T. Dysfunction of
cholinergic and dopaminergic neuronal systems in β‐amyloid protein‐infused rats. Journal
of neurochemistry. 1996 Mar;66(3):1113-7.
J Parkinsons Dis. 2013; 3(4):461-91.
Jackson-Lewis V, Jakowec M, Burke RE, Przedborski S. Time course and morphology of
dopaminergic neuronal death caused by the neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-
tetrahydropyridine. Neurodegeneration. 1995 Sep 1;4(3):257-69.
Jarem DA, Wilson NR, Delaney S. Structure-dependent DNA damage and repair in a
trinucleotide repeat sequence. Biochemistry. 2009 Jun 24;48(28):6655-63.
Javitch JA, D’Amato RJ, Strittmatter SM, Snyder SH. Parkinsonism-inducing neurotoxin, N-
methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine: uptake of the metabolite N-methyl-4-
phenylpyridine by dopamine neurons explains selective toxicity. Proceedings of the
National Academy of Sciences. 1985 Apr 1;82(7):2173-7.
Jellinger KA, Kienzl E, Rumpelmaier G, Paulus W, Riederer P, Stachelberger H, Youdim MB,
Ben-Shachar D. Iron and ferritin in substantia nigra in Parkinson’s disease. Advances in
neurology. 1993;60:267.
Jeong H, Then F, Melia TJ, Mazzulli JR, Cui L, Savas JN, Voisine C, Paganetti P, Tanese N,
Hart AC, Yamamoto A. Acetylation targets mutant huntingtin to autophagosomes for
degradation. Cell. 2009 Apr 3;137(1):60-72.
Jiménez-Aliaga K, Bermejo-Bescós P, Benedí J, Martín-Aragón S. Quercetin and rutin exhibit
antiamyloidogenic and fibril-disaggregating effects in vitro and potent antioxidant activity
in APPswe cells. Life sciences. 2011 Dec 19;89(25-26):939-45.
Jones DP. Redefining oxidative stress. Antioxidants and redox signaling. 2006 Sep 1;8(9-
10):1865-79.
Kaiser S, di Mascio P, Sies H. Lipoat und Singulettsauerstoff. In: Borbe HO, Ulrich H, editors.
Thioctseure.pmi Verlag GmbH; Frankfurt.1989;69–76.
Kamal A, Almenar-Queralt A, LeBlanc JF, Roberts EA, Goldstein LS. Kinesin-mediated
axonal transport of a membrane compartment containing β-secretase and presenilin-1
requires APP. Nature. 2001 Dec;414(6864):643.
Kanski J, Aksenova M, Stoyanova A, Butterfield DA. Ferulic acid antioxidant protection
against hydroxyl and peroxyl radical oxidation in synaptosomal and neuronal cell culture
systems in vitro: structure-activity studies. The Journal of nutritional biochemistry. 2002
May 1;13(5):273-81.
Kar K, Jayaraman M, Sahoo B, Kodali R, Wetzel R. Critical nucleus size for disease-related
polyglutamine aggregation is repeat-length dependent. Nature Structural and Molecular
Biology. 2011 Mar;18(3):328–336.

Karlsson O, Lindquist NG. Melanin affinity and its possible role in neurodegeneration. Journal
of neural transmission. 2013 Dec 1;120(12):1623-30.
Kaur D, Andersen J. Does cellular iron dysregulation play a causative role in Parkinson’s
disease?. Ageing research reviews. 2004 Jul 1;3(3):327-43.
Kim DS, Park SY, Kim JY. Curcuminoids from Curcuma longa L.(Zingiberaceae) that protect
PC12 rat pheochromocytoma and normal human umbilical vein endothelial cells from βA
(1–42) insult. Neuroscience letters. 2001 Apr 27;303(1):57-61.
Kim GY, Kim KH, Lee SH, Yoon MS, Lee HJ, Moon DO, Lee CM, Ahn SC, Park YC, Park
YM. Curcumin inhibits immunostimulatory function of dendritic cells: MAPKs and
translocation of NF-κB as potential targets. The Journal of Immunology. 2005 Jun
15;174(12):8116-24.
Kim HD, Jeong KH, Jung UJ, Kim SR. Naringin treatment induces neuroprotective effects in
a mouse model of Parkinson’s disease in vivo, but not enough to restore the lesioned
dopaminergic system. J Nutr Biochem. 2016;28:140–146.
Kim HJ, Song JY, Park HJ, Park HK, Yun DH, Chung JH. Naringin Protects against Rotenone-
induced Apoptosis in Human Neuroblastoma SH-SY5Y Cells. Korean J Physiol
Pharmacol. 2009;13:281–285.
Kostić DA, Dimitrijević DS, Stojanović GS, Palić IR, Đorđević AS, Ickovski JD. Xanthine
oxidase: isolation, assays of activity, and inhibition. Journal of Chemistry. 2015;2015:8.
Kovtun IV, Liu Y, Bjoras M, Klungland A, Wilson SH, McMurray CT. OGG1 initiates age-
dependent CAG trinucleotide expansion in somatic cells. Nature. 2007
May;447(7143):447.
Kozlowski H, Luczkowski M, Remelli M, Valensin D. Copper, zinc and iron in
neurodegenerative diseases (Alzheimer’s, Parkinson’s and prion diseases). Coordination
Chemistry Reviews. 2012 Oct 1;256(19-20):2129-41.
Kremer HP, Roos RA, Dingjan GM, Bots GT, Bruyn GW, Hofman MA. The hypothalamic
lateral tuberal nucleus and the characteristics of neuronal loss in Huntington’s disease.
Neuroscience letters. 1991 Oct 28;132(1):101-4.
Kuhn DM, Arthur Jr RE, Thomas DM, Elferink LA. Tyrosine hydroxylase is inactivated by
catechol‐quinones and converted to a redox‐cycling quinoprotein: possible relevance to
Parkinson’s disease. Journal of neurochemistry. 1999 Sep 1;73(3):1309-17.
Kumar A, Vaish M, Ratan RR. Transcriptional dysregulation in Huntington’s disease: a failure
of adaptive transcriptional homeostasis. Drug discovery today. 2014 Jul 1;19(7):956-62.
Kunikowska G, Jenner P. 6-Hydroxydopamine-lesioning of the nigrostriatal pathway in rats
alters basal ganglia mRNA for copper, zinc-and manganese-superoxide dismutase, but not
glutathione peroxidase. Brain research. 2001 Dec 13;922(1):51-64.
Langston JW, Ballard P, Tetrud JW, Irwin I. Chronic Parkinsonism in humans due to a product
of meperidine-analog synthesis. Science. 1983 Feb 25;219(4587):979-80.
LaVoie MJ, Ostaszewski BL, Weihofen A, Schlossmacher MG, Selkoe DJ. Dopamine
covalently modifies and functionally inactivates parkin. Nature medicine. 2005
Nov;11(11):1214-1221.
Lee Y, Park HR, Chun HJ, Lee J. Silibinin prevents dopaminergic neuronal loss in a mouse
model of Parkinson’s disease via mitochondrial stabilization. Journal of neuroscience
research. 2015 May;93(5):755-65.

Leem E, Nam JH, Jeon MT, Shin WH, Won SY, Park SJ, Choi MS, Jin BK, Jung UJ, Kim SR.
Naringin protects the nigrostriatal dopaminergic projection through induction of GDNF in
a neurotoxin model of Parkinson’s disease. J Nutr Biochem. 2014;25:801–806.
Li B, Ryder J, Su Y, Zhou Y, Liu F, Ni B. FRAT1 peptide decreases Aβ production in
swAPP751 cells. FEBS letters. 2003 Oct 23;553(3):347-50.
Li C, Zug C, Qu H, Schluesener H, Zhang Z. Hesperidin ameliorates behavioral impairments
and neuropathology of transgenic APP/PS1 mice. Behavioural brain research. 2015 Mar
15;281:32-42.
Li J, Yang J, Zhao P, Li S, Zhang R, Zhang X, Liu D, Zhang B. Neuromelanin enhances the
toxicity of α-synuclein in SK-N-SH cells. Journal of Neural Transmission. 2012 Jun
1;119(6):685-91.
Li S, Chen G, Zhang C, Wu M, Wu S, Liu Q. Research progress of natural antioxidants in foods
for the treatment of diseases. Food Science and Human Wellness. 2014 Sep 1;3(3-4):110-
6.
Liu J, Head E, Gharib AM, Yuan W, Ingersoll RT, Hagen TM, Cotman CW, Ames BN.
Memory loss in old rats is associated with brain mitochondrial decay and RNA/DNA
oxidation: partial reversal by feeding acetyl-L-carnitine and/or R-α-lipoic acid.
Proceedings of the National Academy of Sciences. 2002 Feb 19;99(4):2356-61.
Lodge JK, Traber MG, Packer L. Thiol chelation of Cu2 by dihydrolipoic acid prevents human
low density lipoprotein peroxidation. Free Radical Biology and Medicine. 1998 Aug
1;25(3):287-97.
Luthi-Carter R, Strand A, Peters NL, Solano SM, Hollingsworth ZR, Menon AS, Frey AS,
Spektor BS, Penney EB, Schilling G, Ross CA. Decreased expression of striatal signaling
genes in a mouse model of Huntington’s disease. Hum Mol Genet. 2000 May 22;9(9):1259-
71.
Martinez-Vicente M, Talloczy Z, Kaushik S, Massey AC, Mazzulli J, Mosharov EV, Hodara
R, Fredenburg R, Wu DC, Follenzi A, Dauer W. Dopamine-modified α-synuclein blocks
chaperone-mediated autophagy. The Journal of clinical investigation. 2008 Feb
1;118(2):777-88.
Mattson MP, Carney JW, Butterfield DA. A tombstone in Alzheimer’s?. Nature. 1995
Feb;373(6514):481.
Mattson MP, Chan SL. Neuronal and glial calcium signaling in Alzheimer’s disease. Cell
calcium. 2003 Oct 1;34(4-5):385-97.
Mazzio EA, Reams RR, Soliman KF. The role of oxidative stress, impaired glycolysis and
mitochondrial respiratory redox failure in the cytotoxic effects of 6-hydroxydopamine in
vitro. Brain research. 2004 Apr 9;1004(1-2):29-44.
Menze ET, Tadros MG, Abdel-Tawab AM, Khalifa AE. Potential neuroprotective effects of
hesperidin on 3-nitropropionic acid-induced neurotoxicity in rats. Neurotoxicology. 2012
Oct 1;33(5):1265-75.
Miller GW. Paraquat: the red herring of Parkinson’s disease research. Toxicological Science.
2007;100,:1–2.
Miller J, Arrasate M, Brooks E, Libeu CP, Legleiter J, Hatters D, Curtis J, Cheung K, Krishnan
P, Mitra S, Widjaja K. Identifying polyglutamine protein species in situ that best predict
neurodegeneration. Nature chemical biology. 2011 Dec;7(12):925–34.
Mirra SS, Heyman A, McKeel D, Sumi SM, Crain BJ, Brownlee LM, Vogel FS, Hughes JP,
Van Belle G, Berg L. The Consortium to Establish a Registry for Alzheimer’s Disease

(CERAD) Part II. Standardization of the neuropathologic assessment of Alzheimer’s

disease. Neurology. 1991 Apr 1;41(4):479-86.
Møllersen L, Rowe AD, Illuzzi JL, Hildrestrand GA, Gerhold KJ, Tveterås L, Bjølgerud A,
Wilson III DM, Bjørås M, Klungland A. Neil1 is a genetic modifier of somatic and
germline CAG trinucleotide repeat instability in R6/1 mice. Hum Mol Genet. 2012 Aug
21;21(22):4939-47.
Mondragón-Rodríguez S, Perry G, Zhu X, Moreira PI, Acevedo-Aquino MC, Williams S.
Phosphorylation of tau protein as the link between oxidative stress, mitochondrial
dysfunction, and connectivity failure: implications for Alzheimer’s disease. Oxidative
medicine and cellular longevity. 2013 Jul 10;2013.
Morris MC, Evans DA, Tangney CC, Bienias JL, Wilson RS, Aggarwal NT, Scherr PA.
Relation of the tocopherol forms to incident Alzheimer disease and to cognitive change–.
The American journal of clinical nutrition. 2005 Feb 1;81(2):508-14.
Muir JL, Dunnett SB, Robbins TW, Everitt BJ. Attentional functions of the forebrain
cholinergic systems: effects of intraventricular hemicholinium, physostigmine, basal
forebrain lesions and intracortical grafts on a multiple-choice serial reaction time task.
Experimental Brain Research. 1992 Jun 1;89(3):611-22.
Müller L, Theile K, Böhm V. In vitro antioxidant activity of tocopherols and tocotrienols and
comparison of vitamin E concentration and lipophilic antioxidant capacity in human
plasma. Molecular nutrition and food research. 2010 May;54(5):731-42.
Muller M, Leavitt BR. Iron dysregulation in Hunting-ton’s disease. Journal of Neurochemistry.
2014;130(3):328–350.
Münch G, Lüth HJ, Wong A, Arendt T, Hirsch E, Ravid RA, Riederer P. Crosslinking of α-
synuclein by advanced glycation endproducts—an early pathophysiological step in Lewy
body formation?. Journal of chemical neuroanatomy. 2000 Dec 1;20(3-4):253-7.
Munoz-Sanjuan I, Bates GP. The importance of integrating basic and clinical research toward
the development of new therapies for Huntington disease. The Journal of clinical
investigation. 2011 Feb 1;121(2):476-83.
Naidu KA. Vitamin C in human health and disease is still a mystery? An overview. Nutrition
journal. 2003 Dec;2(1):7.
Narlawar R, Pickhardt M, Leuchtenberger S, Baumann K, Krause S, Dyrks T, Weggen S,
Mandelkow E, Schmidt B. Curcumin‐Derived Pyrazoles and Isoxazoles: Swiss Army
Knives or Blunt Tools for Alzheimer’s Disease?. ChemMedChem: Chemistry Enabling
Drug Discovery. 2008 Jan 11;3(1):165-72.
Nicklas WJ, Youngster SK, Kindt MV, Heikkila RE. IV. MPTP, MPP+ and mitochondrial
function. Life sciences. 1987 Feb 23;40(8):721-9.
Nones J, Costa AP, Leal RB, Gomes FC, Trentin AG. The flavonoids hesperidin and rutin
promote neural crest cell survival. Cell and tissue research. 2012 Nov 1;350(2):305-15.
Nualart F, Mack L, García A, Cisternas P, Bongarzone ER, Heitzer M, Jara N, Martínez F,
Ferrada L, Espinoza F, Baeza V. Vitamin C transporters, recycling and the bystander effect
in the nervous system: SVCT2 versus gluts. Journal of stem cell research and therapy.
2014 May 19;4(5):209.
Núñez MT, Urrutia P, Mena N, Aguirre P, Tapia V, Salazar J. Iron toxicity in
neurodegeneration. Biometals. 2012 Aug 1;25(4):761-76.

Oestreicher E, Sengstock GJ, Riederer P, Olanow CW, Dunn AJ, Arendash GW. Degeneration
of nigrostriatal dopaminergic neurons increases iron within the substantia nigra: a
histochemical and neurochemical study. Brain research. 1994 Oct 10;660(1):8-18.
Olanow CW, Tatton WG. Etiology and pathogenesis of Parkinson’s disease. Annual review of
neuroscience. 1999 Mar;22(1):123-44.
Opazo C, Barría MI, Ruiz FH, Inestrosa NC. Copper reduction by copper binding proteins and
its relation to neurodegenerative diseases. Biometals. 2003 Mar 1;16(1):91-8.
Ossola B, Kääriäinen TM, Männistö PT. The multiple faces of quercetin in neuroprotection.
Expert opinion on drug safety. 2009 Jul 1;8(4):397-409.
Padayatty SJ, Katz A, Wang Y, Eck P, Kwon O, Lee JH, Chen S, Corpe C, Dutta A, Dutta SK,
Levine M. Vitamin C as an antioxidant: evaluation of its role in disease prevention. Journal
of the American college of Nutrition. 2003 Feb 1;22(1):18-35.
Parihar MS, Parihar A, Fujita M, Hashimoto M, Ghafourifar P. Alpha-synuclein overexpression
and aggregation exacerbates impairment of mitochondrial functions by augmenting
oxidative stress in human neuroblastoma cells. The international journal of biochemistry
and cell biology. 2009 Oct 1;41(10):2015-24.
Parihar MS, Parihar A, Fujita M, Hashimoto M, Ghafourifar P. Mitochondrial association of
alpha-synuclein causes oxidative stress. Cellular and molecular life sciences. 2008 Apr
1;65(7-8):1272-84.
Park SY, Kim DS. Discovery of natural products from Curcuma l onga that protect cells from
beta-amyloid insult: A drug discovery effort against Alzheimer’s disease. Journal of
natural products. 2002 Sep 27;65(9):1227-31.
Peña-Sánchez M, Riverón-Forment G, Zaldívar-Vaillant T, Soto-Lavastida A, Borrero-
Sánchez J, Lara-Fernández G, Esteban-Hernández EM, Hernández-Díaz Z, González-
Quevedo A, Fernández-Almirall I, Pérez-López C. Association of status redox with
demographic, clinical and imaging parameters in patients with Huntington’s disease.
Clinical biochemistry. 2015 Dec 1;48(18):1258-63.
Perfeito R, Cunha-Oliveira T, Rego AC. Revisiting oxidative stress and mitochondrial
dysfunction in the pathogenesis of Parkinson disease--resemblance to the effect of
amphetamine drugs of abuse. Free Radic Biol Med. 2012 Nov 1; 53(9):1791-806.
Perk AA, Shatynska-Mytsyk I, Gerçek YC, Boztaş K, Yazgan M, Fayyaz S, Farooqi AA. Rutin
mediated targeting of signaling machinery in cancer cells. Cancer Cell Int. 2014;
14(1):124.
Perluigi M, Poon HF, Maragos W, Pierce WM, Klein JB, Calabrese V, Cini C, De Marco C,
Butterfield DA. Proteomic Analysis of Protein Expression and Oxidative Modification in
R6/2 Transgenic Mice A Model of Huntington Disease. Molecular and Cellular
Proteomics. 2005 Dec 1;4(12):1849-61.
Perumal AS, Gopal VB, Tordzro WK, Cooper TB, Cadet JL. Vitamin E attenuates the toxic
effects of 6-hydroxydopamine on free radical scavenging systems in rat brain. Brain
research bulletin. 1992 Nov 1;29(5):699-701.
Phiel CJ, Wilson CA, Lee VM, Klein PS. GSK-3α regulates production of Alzheimer’s disease
amyloid-β peptides. Nature. 2003 May;423(6938):435–439.
Picone P, Bondi ML, Picone P, Bondi ML, Montana G, Bruno A, Pitarresi G, Giammona G,
Di Carlo M. Ferulic acid inhibits oxidative stress and cell death induced by Ab oligomers:
improved delivery by solid lipid nanoparticles. Free radical research. 2009 Jan
1;43(11):1133-45.

Plassman BL, Langa KM, Fisher GG, Heeringa SG, Weir DR, Ofstedal MB, Burke JR, Hurd
MD, Potter GG, Rodgers WL, Steffens DC. Prevalence of dementia in the United States:
the aging, demographics, and memory study. Neuroepidemiology. 2007;29(1-2):125-32.
Polidori MC, Mecocci P, Browne SE, Senin U, Beal MF. Oxidative damage to mitochondrial
DNA in Huntington’s disease parietal cortex. Neuroscience letters. 1999 Sep 3;272(1):53-
6.
Polyzos A, Holt A, Brown C, Cosme C, Wipf P, Gomez-Marin A, Castro MR, Ayala-Peña S,
McMurray CT. Mitochondrial targeting of XJB-5-131 attenuates or improves
pathophysiology in HdhQ150 animals with well-developed disease phenotypes. Hum Mol
Genet. 2016 Feb 21;25(9):1792-802.
Qiu S, Li L, Weeber EJ, May JM. Ascorbate transport by primary cultured neurons and its role
in neuronal function and protection against excitotoxicity. Journal of neuroscience
research. 2007 Apr;85(5):1046-56.
Rai S, Kamat PK, Nath C, Shukla R. A study on neuroinflammation and NMDA receptor
function in STZ (ICV) induced memory impaired rats. Journal of neuroimmunology. 2013
Jan 15;254(1-2):1-9.
Rajmohan R, Reddy PH. Amyloid-beta and phosphorylated tau accumulations cause
abnormalities at synapses of Alzheimer’s disease neurons. Journal of Alzheimer’s Disease.
2017 Jan 1;57(4):975-99.
Ray PD, Huang BW, Tsuji Y. Reactive oxygen species (ROS) homeostasis and redox
regulation in cellular signaling. Cellular signalling. 2012 May 1;24(5):981-90.
Raza SS, Khan MM, Ahmad A, Ashafaq M, Khuwaja G, Tabassum R, Javed H, Siddiqui MS,
Safhi MM, Islam F. Hesperidin ameliorates functional and histological outcome and
reduces neuroinflammation in experimental stroke. Brain research. 2011 Oct 28;1420:93-
105.
Ren PH, Lauckner JE, Kachirskaia I, Heuser JE, Melki R, Kopito RR. Cytoplasmic
penetration and persistent infection of mammalian cells by polyglutamine aggregates.
Nature cell biology. 2009 Feb;11(2):219.
Riachi NJ, LaMANNA JC, Harik SI. Entry of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine
into the rat brain. Journal of Pharmacology and Experimental Therapeutics. 1989 Jun
1;249(3):744-8.
Rimbach G, Minihane AM, Majewicz J, Fischer A, Pallauf J, Virgli F, Weinberg PD.
Regulation of cell signalling by vitamin E. Proceedings of the Nutrition Society. 2002
Nov;61(4):415-25.
Roher AE, Chaney MO, Kuo YM, Webster SD, Stine WB, Haverkamp LJ, Woods AS, Cotter
RJ, Tuohy JM, Krafft GA, Bonnell BS. Morphology and toxicity of Aβ-(1-42) dimer
derived from neuritic and vascular amyloid deposits of Alzheimer’s disease. Journal of
Biological Chemistry. 1996 Aug 23;271(34):20631-5.
Rong W, Wang J, Liu X, Jiang L, Wei F, Hu X, Han X, Liu Z. Naringin treatment improves
functional recovery by increasing BDNF and VEGF expression, inhibiting neuronal
apoptosis after spinal cord injury. Neurochem Res. 2012;37:1615–1623.
Roos RA. Huntington’s disease: a clinical review. Orphanet journal of rare diseases. 2010
Dec;5(1):40.
Rosas HD, Chen YI, Doros G, Salat DH, Chen NK, Kwong KK, Bush A, Fox J, Hersch SM.
Alterations in brain transition metals in Huntington disease: an evolving and intricate story.
Archives of neurology. 2012 Jul 1;69(7):887-93.

Ross CA. Polyglutamine pathogenesis: emergence of unifying mechanisms for Huntington’s

disease and related disorders. Neuron. 2002 Aug 29;35(5):819-22.
Sabogal-Guáqueta AM, Munoz-Manco JI, Ramírez-Pineda JR, Lamprea-Rodriguez M, Osorio
E, Cardona-Gómez GP. The flavonoid quercetin ameliorates Alzheimer’s disease
pathology and protects cognitive and emotional function in aged triple transgenic
Alzheimer’s disease model mice. Neuropharmacology. 2015 Jun 1;93:134-45.
Sano M, Ernesto C, Thomas RG, Klauber MR, Schafer K, Grundman M, Woodbury P,
Growdon J, Cotman CW, Pfeiffer E, Schneider LS. A controlled trial of selegiline, alpha-
tocopherol, or both as treatment for Alzheimer’s disease. New England Journal of
Medicine. 1997 Apr 24;336(17):1216-22.
Santamato A, Ranieri M, Cinone N, Stuppiello LA, Valeno G, De Sanctis JL, Fortunato F,
Solfrizzi V, Greco A, Seripa D, Panza F. Postural and balance disorders in patients with
parkinson’s disease: A Prospective open-label feasibility study with two months of action
observation treatment. Parkinson’s Disease. 2015;2015.
Sayre LM, Perry G, Harris PL, Liu Y, Schubert KA, Smith MA. In situ oxidative catalysis by
neurofibrillary tangles and senile plaques in Alzheimer’s disease: a central role for bound
transition metals. Journal of neurochemistry. 2000 Jan 1;74(1):270-9.
Scott BC, Aruoma OI, Evans PJ, O’neill C, Van Der Vliet A, Cross CE, Tritschler H, Halliwell
B. Lipoic and dihydrolipoic acids as antioxidants. A critical evaluation. Free radical
research. 1994 Jan 1;20(2):119-33.
Searls RL, Sanadi DR. α-Ketoglutaric Dehydrogenase VIII. Isolation and some properties of a
flavoprotein component. Journal of Biological Chemistry. 1960 Aug 1;235(8):2485-91.
Selkoe DJ. Alzheimer’s disease is a synaptic failure. Science. 2002 Oct 25;298(5594):789-91.
Selkoe DJ. Soluble Oligomers of the Amyloid β-Protein: Impair Synaptic Plasticity and
Behavior. InSynaptic Plasticity and the Mechanism of Alzheimer’s Disease 2008 (pp. 89-
102). Springer, Berlin, Heidelberg.
Selkoe DJ. The cell biology of β-amyloid precursor protein and presenilin in Alzheimer’s
disease. Trends in cell biology. 1998 Nov 1;8(11):447-53.
Shah SB, Nolan R, Davis E, Stokin GB, Niesman I, Canto I, Glabe C, Goldstein LS.
Examination of potential mechanisms of amyloid-induced defects in neuronal transport.
Neurobiology of disease. 2009 Oct 1;36(1):11-25.
Shankar GM, Walsh DM. Alzheimer’s disease: synaptic dysfunction and Aβ. Molecular
neurodegeneration. 2009 Dec;4(1):48.
Sherer TB, Betarbet R, Kim JH, Greenamyre JT. Selective microglial activation in the rat
rotenone model of Parkinson’s disease. Neuroscience letters. 2003 May 1;341(2):87-90.
Sherer TB, Betarbet R, Testa CM, Seo BB, Richardson JR, Kim JH, Miller GW, Yagi T,
Matsuno-Yagi A, Greenamyre JT. Mechanism of toxicity in rotenone models of
Parkinson’s disease. Journal of Neuroscience. 2003 Nov 26;23(34):10756-64.
Shimizu K, Ohtaki K, Matsubara K, Aoyama K, Uezono T, Saito O, Suno M, Ogawa K, Hayase
N, Kimura K, Shiono H. Carrier-mediated processes in blood–brain barrier penetration and
neural uptake of paraquat. Brain research. 2001 Jul 6;906(1-2):135-42.
Shukla PK, Khanna VK, Khan MY, Srimal R C. Protective effect of curcumin against lead
neurotoxicity in rat. Human and experimental toxicology. 2003 Dec;22(12):653-8.
Singh D, Chander V, Chopra K. Protective effect of naringin, a bioflavonoid on ferric
nitrilotriacetate-induced oxidative renal damage in rat kidney. Toxicology. 2003;201:1–8.

Slow EJ, Graham RK, Osmand AP, Devon RS, Lu G, Deng Y, Pearson J, Vaid K, Bissada N,
Wetzel R, Leavitt BR. Absence of behavioral abnormalities and neurodegeneration in vivo
despite widespread neuronal huntingtin inclusions. Proceedings of the National Academy
of Sciences. 2005 Aug 9;102(32):11402-7.
Smith AR, Shenvi SV, Widlansky M, Suh JH, Hagen TM. Lipoic acid as a potential therapy
for chronic diseases associated with oxidative stress. Current medicinal chemistry. 2004
May 1;11(9):1135-46.
Smith MA, Sayre LM, Monnier VM, Perry G. Radical AGEing in Alzheimer’s disease. Trends
in neurosciences. 1995 Apr 1;18(4):172-6.
Song X, Zhou B, Cui L, Lei D, Zhang P, Yao G, Xia M, Hayashi T, Hattori S, Ushiki-Kaku Y,
Tashiro SI. Silibinin ameliorates Aβ 25-35-induced memory deficits in rats by modulating
autophagy and attenuating neuroinflammation as well as oxidative stress. Neurochemical
research. 2017 Apr 1;42(4):1073-83.
Sorolla MA, Reverter-Branchat G, Tamarit J, Ferrer I, Ros J, Cabiscol E. Proteomic and
oxidative stress analysis in human brain samples of Huntington disease. Free Radical
Biology and Medicine. 2008 Sep 1;45(5):667-78.
Sorolla MA, Rodríguez-Colman MJ, Tamarit J, Ortega Z, Lucas JJ, Ferrer I, Ros J, Cabiscol E.
Protein oxidation in Huntington disease affects energy production and vitamin B6
metabolism. Free Radical Biology and Medicine. 2010 Aug 15;49(4):612-21.
Srinivasan M, Sudheer AR, Menon VP. Ferulic acid: therapeutic potential through its
antioxidant property. Journal of clinical biochemistry and nutrition. 2007;40(2):92-100.
Stack EC, Matson WR, Ferrante RJ. Evidence of oxidant damage in Huntington’s disease:
translational strategies using antioxidants. Annals of the New York Academy of Sciences.
2008 Dec;1147(1):79-92.
Stahl WL, Swanson PD. Biochemical abnormalities in Huntington’s chorea brains. Neurology.
1974 Sep 1;24(9):813-819.
Stohs SJ, Bagchi D. Oxidative mechanisms in the toxicity of metal ions. Free Radical Biology
and Medicine. 1995;18(2):321-336.
Su Y, Ryder J, Li B, Wu X, Fox N, Solenberg P, Brune K, Paul S, Zhou Y, Liu F, Ni B. Lithium,
a common drug for bipolar disorder treatment, regulates amyloid-β precursor protein
processing. Biochemistry. 2004 Jun 8;43(22):6899-908.
Suh JH, Shenvi SV, Dixon BM, Liu H, Jaiswal AK, Liu RM, Hagen TM. Decline in
transcriptional activity of Nrf2 causes age-related loss of glutathione synthesis, which is
reversible with lipoic acid. Proceedings of the National Academy of Sciences. 2004 Mar
9;101(10):3381-6.
Sun X, Sato S, Murayama O, Murayama M, Park JM, Yamaguchi H, Takashima A. Lithium
inhibits amyloid secretion in COS7 cells transfected with amyloid precursor protein C100.
Neuroscience letters. 2002 Mar 15;321(1-2):61-4.
Suzuki YJ, Tsuchiya M, Packer L. Thioctic acid and dihydrolipoic acid are novel antioxidants
which interact with reactive oxygen species. Free radical research communications. 1991
Jan 1;15(5):255-63.
Szabados T, Dul C, Majtényi K, Hargitai J, Pénzes Z, Urbanics R. A chronic Alzheimer’s model
evoked by mitochondrial poison sodium azide for pharmacological investigations.
Behavioural brain research. 2004 Sep 23;154(1):31-40.

Tam S, Spiess C, Auyeung W, Joachimiak L, Chen B, Poirier MA, Frydman J. The chaperonin
TRiC blocks a huntingtin sequence element that promotes the conformational switch to
aggregation. NatStruct Mol Biol. 2009 Dec;16(12):1279-1285.
Tamagno E, Parola M, Guglielmotto M, Santoro G, Bardini P, Marra L, Tabaton M, Danni O.
Multiple signaling events in amyloid β-induced, oxidative stress-dependent neuronal
apoptosis. Free Radic Biol Med. 2003 Jul 1;35(1):45-58.
Tanner CM. Is the cause of Parkinson’s disease environmental or hereditary? Evidence from
twin studies. AdvNeurol. 2003;91:133.
Tatton NA, Kish SJ. In situ detection of apoptotic nuclei in the substantia nigra compacta of 1-
methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-treated mice using terminal
deoxynucleotidyl transferase labelling and acridine orange staining. Neuroscience. 1997
Feb 14;77(4):1037-48.
Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, Hill R, Hansen LA, Katzman R.
Physical basis of cognitive alterations in Alzheimer’s disease: synapse loss is the major
correlate of cognitive impairment. Annals of Neurology: Official Journal of the American
Neurological Association and the Child Neurology Society. 1991 Oct;30(4):572-80.
Thakur AK, Jayaraman M, Mishra R, Thakur M, Chellgren VM, Byeon IJ, Anjum DH, Kodali
R, Creamer TP, Conway JF, Gronenborn AM. Polyglutamine disruption of the huntingtin
exon 1 N terminus triggers a complex aggregation mechanism. Nat Struct Mol Biol. 2009
Apr;16(4):380–389.
Thenganatt MA, Louis ED. Distinguishing essential tremor from Parkinson’s disease: bedside
tests and laboratory evaluations. Expert Rev Neurother. 2012 Jun 1;12(6):687-96.
Tomic JL, Pensalfini A, Head E, Glabe CG. Soluble fibrillar oligomer levels are elevated in
Alzheimer’s disease brain and correlate with cognitive dysfunction. NeurobiolDis. 2009
Sep 1;35(3):352-8.
Ungerstedt U. 6-Hydroxy-dopamine induced degeneration of central monoamine neurons. EurJ
Pharmacol. 1968 Dec 1;5(1):107-10.
Uttara B, Singh AV, Zamboni P, Mahajan RT. Oxidative stress and neurodegenerative diseases:
a review of upstream and downstream antioxidant therapeutic options. Curr
Neuropharmacol. 2009 Mar 1;7(1):65-74.
Valko MM, Morris H, Cronin MT. Metals, toxicity and oxidative stress. CurrMed Chem. 2005
May 1;12(10):1161-208.
van Duijn E, Kingma EM, van der Mast RC. Psychopathology in verified Huntington’s disease
gene carriers. J Neuropsychiatry Clin Neurosci. 2007;19:441–8.
Ved R, Saha S, Westlund B, Perier C, Burnam L, Sluder A, Hoener M, Rodrigues CM, Alfonso
A, Steer C, Liu L. Similar patterns of mitochondrial vulnerability and rescue induced by
genetic modification of α-synuclein, parkin, and DJ-1 in Caenorhabditis elegans. J Biol
Chem. 2005 Dec 30;280(52):42655-68.
Vitek MP, Bhattacharya K, Glendening JM, Stopa E, Vlassara H, Bucala R, Manogue K,
Cerami A. Advanced glycation end products contribute to amyloidosis in Alzheimer
disease. Proceedings of the National Academy of Sciences. 1994 May 24;91(11):4766-70.
Waldemar G, Dubois B, Emre M, Georges J, McKeith IG, Rossor M, Scheltens P, Tariska P,
Winblad B. Recommendations for the diagnosis and management of Alzheimer’s disease
and other disorders associated with dementia: EFNS guideline. Eur J Neurol. 2007
Jan;14(1):e1-26.

Wang SW, Wang YJ, Su YJ, Zhou WW, Yang SG, Zhang R, Zhao M, Li YN, Zhang ZP, Zhan
DW, Liu RT. Rutin inhibits β-amyloid aggregation and cytotoxicity, attenuates oxidative
stress, and decreases the production of nitric oxide and proinflammatory cytokines.
Neurotoxicol. 2012 Jun 1;33(3):482-90.
Wang XF, Li S, Chou AP, Bronstein JM. Inhibitory effects of pesticides on proteasome activity:
implication in Parkinson’s disease. Neurobiol Dis. 2006 Jul 1;23(1):198-205.
Warby SC, Visscher H, Collins JA, Doty CN, Carter C, Butland SL, Hayden AR, Kanazawa I,
Ross CJ, Hayden MR. HTT haplotypes contribute to differences in Huntington disease
prevalence between Europe and East Asia. Eur J Hum Genet. 2011 May;19(5):561-66.
Wegiel J, Kuchna I, Nowicki K, Frackowiak J, Mazur-Kolecka B, Imaki H, Wegiel J, Mehta
PD, Silverman WP, Reisberg B, Wisniewski T. Intraneuronal Aβ immunoreactivity is not
a predictor of brain amyloidosis-β or neurofibrillary degeneration. Acta Neuropathol. 2007
Apr 1;113(4):389-402.
Wellington CL, Ellerby LM, Gutekunst CA, Rogers D, Warby S, Graham RK, Loubser O, van
Raamsdonk J, Singaraja R, Yang YZ, Gafni J. Caspase cleavage of mutant huntingtin
precedes neurodegeneration in Huntington’s disease. J Neuroscience. 2002 Sep
15;22(18):7862-72.
Wheelock VL, Tempkin T, Marder K, Nance M, Myers RH, Zhao H, Kayson E, Orme C,
Shoulson I. Huntington Study Group. Predictors of nursing home placement in Huntington
disease. Neurol. 2003;60:998–1001.
Wilcock GK, Esiri MM, Bowen DM, Smith CC. Alzheimer’s disease: correlation of cortical
choline acetyltransferase activity with the severity of dementia and histological
abnormalities. JNeurol Sci. 1982 Dec 1;57(2-3):407-17.
Williams AJ, Paulson HL. Polyglutamine neurodegeneration: protein misfolding revisited.
Trends Neurosci. 2008 Oct 1;31(10):521-8.
Wong TP, Debeir T, Duff K, Cuello AC. Reorganization of cholinergic terminals in the cerebral
cortex and hippocampus in transgenic mice carrying mutated presenilin-1 and amyloid
precursor protein transgenes. J Neuroscience. 1999 Apr 1;19(7):2706-16.
Wyttenbach A, Sauvageot O, Carmichael J, Diaz-Latoud C, Arrigo AP, Rubinsztein DC. Heat
shock protein 27 prevents cellular polyglutamine toxicity and suppresses the increase of
reactive oxygen species caused by huntingtin. Hum Mol Genet. 2002 May 1;11(9):1137-
51.
Xianchu L, Lan PZ, Qiufang L, Yi L, Xiangcheng R, Wenqi H, Yang D. Naringin protects
against lipopolysaccharide-induced cardiac injury in mice. Environ Toxicol Pharmacol.
2016;48:1–6.
Xuan Q, Xu SL, Lu DH, Yu S, Zhou M, Uéda K, Cui YQ, Zhang BY, Chan P. Increase
expression of α-synuclein in aged human brain associated with neuromelanin
accumulation. JNeural Transmission. 2011 Nov 1;118(11):1575-83.
Yan JJ, Cho JY, Kim HS, Kim KL, Jung JS, Huh SO, Suh HW, Kim YH, Song DK. Protection
against β‐amyloid peptide toxicity in vivo with long‐term administration of ferulic acid.
British J Pharmacol. 2001 May 1;133(1):89-96.
Yang F, Lim GP, Begum AN, Ubeda OJ, Simmons MR, Ambegaokar SS, Chen PP, Kayed R,
Glabe CG, Frautschy SA, Cole GM. Curcumin inhibits formation of amyloid β oligomers
and fibrils, binds plaques, and reduces amyloid in vivo. J Biol Chem. 2005 Feb
18;280(7):5892-901.

Yao Y, Chinnici C, Tang H, Trojanowski JQ, Lee VM, Praticò D. Brain inflammation and
oxidative stress in a transgenic mouse model of Alzheimer-like brain amyloidosis. J
Neuroinflammation. 2004 Dec;1(1):21.
Yatin SM, Varadarajan S, Butterfield DA. Vitamin E prevents Alzheimer’s amyloid ß-Peptide
(1-42)-induced neuronal protein oxidation and reactive oxygen species production. J
Alzheimer’s Dis. 2000 Jan 1;2(2):123-31.
Zecca L, Wilms H, Geick S, Claasen JH, Brandenburg LO, Holzknecht C, Panizza ML, Zucca
FA, Deuschl G, Sievers J, Lucius R. Human neuromelanin induces neuroinflammation and
neurodegeneration in the rat substantia nigra: implications for Parkinson’s disease. Acta
Neuropathol. 2008 Jul 1;116(1):47-55.
Zhang J, Fitsanakis VA, Gu G, Jing D, Ao M, Amarnath V, Montine TJ. Manganese ethylene‐
bis‐dithiocarbamate and selective dopaminergic neurodegeneration in rat: a link through
mitochondrial dysfunction. J Neurochem. 2003 Jan;84(2):336-46.
Zühlke C, Riess O, Bockel B, Lange H, Thies U. Mitotic stability and meiotic variability of the
(CAG)n repeat in the Huntington disease gene. HumMol Genet. 1993 Dec;2(12):2063-7.

Chapter 6
ANTIOXIDANTS AGAINST STRESS-INDUCED

MEMORY DYSFUNCTION
S. B. Chandrasekar1, Anil T. Pawar2,,

M. S. Lokesh Prasad1 and G. Hemavathi1
1
Hi-Tech Lab, Drug Testing Laboratory, Drug Control Department,
Bangalore, India
2
MAEER’s Maharashtra Institute of Pharmacy, Pune, India
ABSTRACT
Brain’s biochemical integrity is very important for the physiological working of the
central nervous system (CNS). Oxidative stress stands as one of the major factors causing
cerebral dysfunction. When the antioxidant response mechanism fails to neutralize the
oxidative stress conditions, the consequence leads to the generation of free radicals in
excess. Since the brain constitutes lipid matter in abundance and also consumes oxygen in
large amounts, it is vulnerable to oxidative stress. Thus, damage caused by oxidative stress
to the brain results in a harmful influence on the normal physiology of CNS. Though the
association of neurodegenerative illnesses (Alzheimer’s disease, Parkinson’s disease, and
Huntington’s disease) with oxidative stress has been studied and also been reported, its
participation in neuropsychiatric disorders like depression and anxiety are yet to be
determined. The present chapter covers the discussion of cerebral oxidative stress, its
consequence in impairing memory and the role of antioxidants in treating the memory
impairment.
1. INTRODUCTION
The term “learning” encompasses acquiring of data (information) as well as expertise.
Ensuring storage of the attained information in the course of the learning process is called

Corresponding Author’s Email: anil_pawar31@yahoo.co.in.

132 S. B. Chandrasekar, Anil T. Pawar, M. S. Lokesh Prasad et al.
memory (Gisquet-Verrier and Riccio, 2012). The memory system is the way for the brain to
process information that will be available for use at a later time. The brain utilizes the preserved
information through the organization of “memory system” whose availability may be useful at
later point of time. Memory dysfunction is one of the most impairing neurological ailments
together with neuro-degenerative disorders, hypoxia stroke, tumor, depression, cardiac surgery,
head trauma, attention deficit disorder (ADD), anxiety, adverse effects of medication,
malnutrition and lastly aging (Budson and Prince, 2005). The memory loss consequently alters
the patient’s day-to-day conducts along with impacting their families.
The brain is extremely susceptible to oxidative injury. This might be accounted to the
factors such as high percentage of body’s overall oxygen utilization (approximately 20%), a
large part of polyunsaturated fatty acids (PUFA) content and relatively reduced levels of
endogenous antioxidant performance to remaining tissues (Head, 2009). Dysfunction in the
process of learning as well in memory contributes as the manifestation of Alzheimer’s disease
(AD). AD affects certain parts of the brain due to amyloid-β protein (Aβ) deposition (Hwang
et al., 2011). Studies have been reported that Aβ protein as well the Aβ protein linked reactive
oxidative species have a major part in the progress of AD (Pappolla et al., 1998). Other
important causes that might be the reasons for the progression of AD include intake of diet that
is devoid or scanty of molecules possessing antioxidant properties and lessened endogenous
enzyme performance (Hwang et al., 2011). The chief enzymes include catalase, glutathione
peroxidase (GPx) and superoxide dismutase (SOD). Memory dysfunction related to age is
correlated with increased oxidative stress along with a decline in brain and plasma antioxidants
defense mechanism (Haider et al., 2014). All these facts represent how oxidative stress along
with shortfall of the antioxidant system are involved in the pathological progression of memory
dysfunction.
The outcome of human reaction (in the form of physical as well as mental) towards the
proceedings, alterations, and circumstances around him/her are understood by means of stress.
In the course of acute stress, huge changes occur in organ systems (endocrine, cardiovascular,
nervous and immune systems). Response to stress is generally adaptive in nature, however, the
response to a severe and long-lasting one result in tissue damage consequently leading to a state
of disease which includes memory loss (Schneiderman et al., 2005). A stressor works by
causing oxidative stress and thus promoting cellular injury and tissue damage. In order to
neutralize the harmful results of oxidative stress, the body has evolved itself with some
mechanism including antioxidant defense system. Several types of antioxidant agents are
available and are primarily chosen agents to treat the oxidative damage (Lobo et al., 2010).
Endogenously available antioxidant mechanisms comprise of the enzyme as well as non-
enzyme molecules. These molecules play an essential role in supporting life through
maintenance of reduction-oxidation equilibrium within the cells along with reducing the
unwanted cell injury triggered by reactive oxygen species (ROS).
ROS are generated abundantly as a result of the response to stress (Rahul et al., 2014).
Natural food-derived compounds such as flavonoids possess free radical scavenging activities
and therefore prevent oxidative injury and cell death. Certain primary studies conducted
clinically have revealed that medicines that are of plant origin proved to make the process of
learning as well as memory better in AD victims (mild-to-moderate) (Jivad and Rabiei, 2014).
This chapter discusses the association between the oxidative stress, the complications which
the CNS undergoes during oxidative stress (mainly memory) and the role of antioxidants and
related factors combating the consequences of oxidative stress.

Antioxidants against Stress-Induced Memory Dysfunction 133
2. MEMORY
Memory has been considered as one of the most complicated activities of the brain. This
activity has attracted the attention of several intellectuals and research scholars from quite a
long time. Memory is presently perceived as a process that comprises of mind involvement
which utilizes numerous buffers of varying capacity and period of time meant for storage of
information (Well, 2010). Neurotransmitters play a vital role in learning and memory (Table
1). Memory is composed of numerous systems that function individually and thus it should not
be considered as a single unit mechanism which depends on a single neuro-anatomical track.
A memory system revolves between the declarative memory (explicit memory) and non-
declarative memory (implicit memory) (Squire and Dede, 2015).
The memory system which is organized deliberately/purposefully and submissively is
termed as the declarative or explicit memory. This system utilizes determination and purpose.
To achieve this, certain memory approaches for instance mnemonics are employed so as to
recollect the information. The mechanism is facilitated through the hippocampus region as well
as the frontal lobes of the brain. Any injury caused to these critical regions of the brain would
lead to the complete loss of explicit memory (Mancia, 2006). Explicit memory tests (e.g., recall
or recognition) are employed to measure explicit memory. In this case, the subject is conscious
of the test he/she is undergoing. The test measures are seen to reduce generally with increased
age. Subsystems of declarative memory include working memory, episodic memory and
semantic memory (Voss and Paller, 2008). Working memory is described as a short period
system where the restricted amount of information is preserved and processed for instantaneous
use. Working memory persists for a very short period (2 to 18 seconds). This type of memory
system is witnessed in the course of calculations performed mentally. Episodic memory is a
relatively long duration memory which allows the storage of particular proceedings or incidents
associated with that individual’s life and this memory system is helpful in remembering former
happenings. The semantic memory system is again a long-term memory meant for storing
common information (Goshen‐Gottstein, 2003).
Table 1. Neurotransmitters in learning and memory
Neurotransmitters/Modulators Receptor System

Glutamate N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid (AMPA) receptors
Acetylcholine Muscarinic, nicotinic receptors
Dopamine D1 (dopamine 1), D2 receptors
Serotonin 5HT3 (serotonin 3), 5HTIA receptors
Noradrenaline Alpha and beta receptors
Neuropeptides G-protein coupled receptors
Gamma Amino Butyric Acid GABAA/BZD (benzodiazepines) receptor
(GABA)
Neurosteroids NMDA/GABAA receptors
The type of memory system which has an impact on an individual’s existing view and
activities in absence of one’s consciousness, purpose or knowledge is termed as nondeclarative
memory. This memory system is of unintentional use and does not require any exertion. Similar

to declarative memory, even implicit memory is evaluated through different implicit memory
tests. However, during these tests, the individual undergoing the test is unacquainted about the
process (Schacter, 1987). The circuit of the nondeclarative memory system involves the basal
ganglia, the cerebellum as well as the cortical areas of the brain. Implicit memory continues to
stay unwavering with the typical aging process. Some of the instances to relate nondeclarative
memory may include bicycle riding, swimming, speaking similar words repeatedly, etc.
Priming, procedural memory, and classical conditioning are the subsystems of the implicit
memory system (De Renzi, 1989; Squire and Clark, 2001; Squire and Dede, 2015). A memory
process that is involuntary or the one involving total unawareness but has the ability to augment
the swiftness and the exactness of a reaction due to the previous familiarity is termed as
priming. Priming is one of the methods used for assessing implicit memory. Here basically the
person under the test is unaware of the fact that his/her memory is being measured. Priming
makes the remembering process more efficacious by activating series of related thoughts.
Procedural memory is concerned with the finishing of work where the work is learned well
(skilled) and is now involuntary. Classical conditioning is often referred to as the Pavlovian or
respondent conditioning. In this type of memory system, a biologically effective stimulus is
connected with a formerly neutral stimulus (Clark, 2004).
The types of information processed and the principle lying behind the memory operation
is the base for the categorization of diverse memory systems. In implicit memory, the capability
of extracting the frequent elements within the sequence of different events is the basis, while,
in explicit memory the capacity of noticing and determining the distinctive in a particular event
occurring at a particular place and time (Well J, 2010).
3. STRESS
Stress is a psychological as well as physical response shown by individuals to the
modifications, proceedings, and circumstances occurring in their lives. Stress is encountered
by individuals in several ways and for various causes. The term "homeostasis" is explained as
balancing the body’s internal environment steadily with regard to the outer changing
environment. The term “stress” was coined to symbolize those effects which critically hamper
the system of homeostasis (Selye, 1998). The “stressor” is the one responsible (also witnessed)
for causing a real hazard to the homeostasis. The stress response is the reaction generated due
to the stressor. In most of the cases stress responses progress as adaptation, however, a critical
or long-term stress responses progress to tissue injury and ultimately lead to disease
(Schneiderman et al., 2005).
Stress is classified into eustress and distress. Eustress is the positive or good stress that
happens during pleasant situations such as the exhilaration experienced during receipt of a
promotion or hike in salary. Unlike eustress, distress is the negative or bad stress that occurs
when stress is perceived as dangerous, difficult, unfair or painful such as sleeplessness, legal
issues, etc. (Well J, 2010).
Stress is categorized based on the extent and commencement into acute stress and chronic
stress. Acute stress is generally short-lived and can be either eustress or distress. An example
includes performing in front of a group (Well, 2010). In contrast, chronic stress prevails for
long-term and is a result of important events. An example includes long-term sickness. As such,

chronic stress is considered to be the most health-hazardous since it is liable to cause negative
effects (Well J, 2010).
The condition that put in danger, or alleged to endanger the individual for continued
existence, is known to be a stressor (Van de Kar and Blair, 1999). The stressors are grouped
into the following categories:
1. Psychosomatic stressors are dependent on a reaction to the risk of an approaching

adverse situation such as fright or nervousness, exposure to new surroundings.
2. Stressors incorporated with physical stimuli and associated with a powerful emotional
constituent, for instance, pain, immobilization, etc.
3. Stressors which hinder cardio-vascular homeostasis. Examples of such settings include
hemorrhage, orthostatic stress or upright tilt, exercise, heat exposure.
The circuit through which the stress response is intervened is located in the CNS as well
as the peripheral nervous network. The chief effector cells are confined to the regions namely
the paraventricular nucleus (PVN) of the hypothalamus, pituitary gland (the anterior lobe) as
well as the adrenal gland. The above mentioned confined region is collectively named as
hypothalamic-pituitary-adrenal (HPA) axis (Smith and Vale, 2006). Extending the HPA axis,
quite a few other constitutions also take part in the maintenance of adaptive response to stress.
The constitutional network includes brain stem noradrenergic neurons, sympathetic
adrenomedullary circuits and parasympathetic systems (Habib et al., 2001; Whitnall, 1993).
4. STRESS AND HYPOTHALAMIC-PITUITARY-ADRENAL AXIS

The primary controller of the HPA axis is the corticotropin-releasing factor (CRF). This
factor is produced by the hypophysiotropic neurons situated in the medial parvo-cellular section
of the PVN (Vale et al., 1981; Rivier and Vale, 1983a). The CRF is allowed to flow into the
region of hypophysial portal vessels in response to stress (Figure 1). The hypophysial portal
vessels have the means of entering the anterior pituitary gland. The CRF then binds to its
corresponding receptors present on the pituitary corticotropes (basophilic cells in the anterior
pituitary). This association leads to the adrenocorticotropic hormone (ACTH) release into the
systemic circulation. The released ACTH in the circulatory system reaches its chief effector
i.e., the adrenal cortex. Upon reaching the adrenal cortex, ACTH causes the synthesis and
secretion of glucocorticoids from the zona fasciculate region of adrenal cortex. Glucocorticoids
are hormones which act as HPA axis downstream effectors. The physiological activities of
glucocorticoids are controlled by means of widely distributed intracellular receptors (Munck et
al., 1984; Bamberger et al., 1996). In normal physiology, the glucocorticoids produce biological
effects that are adaptive in nature. Any imbalance in the glucocorticoids levels (insufficient or
too much) caused due to abnormal activation of HPA axis, lead to pathological state (Munck et
al., 1984; McEwen and Stellar, 1993). CRF is also involved in the sympathetic nervous system
activation (Penalva et al., 2002). The PVN of the hypothalamus comprises of CRF neurons that
extend outwards to noradrenergic cell bodies present in the locus coeruleus (the principal site
of norepinephrine [noradrenaline] in the brain. The norepinephrine regulates the
sympathoadrenal system’s stress-induced stimulation (Koob, 1999; Valentino et al., 1983). In

addition, CRF neurons are also involved in the production of enhanced norepinephrine levels
in terminal regions, for instance, frontal cortex (Curtis et al., 1997; Lavicky and Dunn, 1993).
Serotonin (5-hydroxytryptamine or 5-HT) is involved in the stimulation of noradrenergic
neurons as well as CRF. In contrast, they are inhibited by ACTH, glucocorticoids as well as
gamma-aminobutyric acid (GABA), (Aghajanian and Vandermaelen, 1982; Calogero et al.,
1988; Stratakis and Chrousos, 1995).
Figure 1. Effect of stress on hypothalamic-pituitary-adrenal axis. PVN, Paraventricular nucleus; GABA,

Gamma-aminobutyric acid; +, Activation; −, Inhibition.
4.1. Corticotropin-Releasing Factor
CRF was firstly extracted from ovine hypothalamic preparation in the year 1981. CRF is
basically a polypeptide consisting of 41 amino acids (Vale et al., 1981). After the original
recognition of CRF, it has been experimented and proven to be involved in various vital
activities. The list includes control of the autonomic nervous system (ANS) (Valentino et al.,
1983; Croiset et al., 2000), chief controller of ACTH secretion from pituitary gland (anterior
pituitary corticotropes) (Rivier and Vale, 1983a), learning as well as memory (Croiset et al.,
2000), reproduction (Chatterton, 1990). CRF is extensively distributed covering the CNS along
with several peripheral tissues. CRF is densely found in the medial parvocellular subdivision
of the PVN in the brain. It is also confined to a preoptic area, bed nucleus of the stria terminalis
(BNST), olfactory bulb, central nucleus of the amygdala and medial preoptic area (Sawchenko

et al., 1993). Peripheral distribution of CRF includes skin, gastrointestinal tract and placenta
(Bale and Vale, 2004), adrenal gland (Bruhn et al., 1987), testis (Audhya et al., 1989) and
adrenal gland (Bruhn et al., 1987).
The CRF activities are facilitated by means of G-protein coupled receptors. Two subtypes
are identified, namely CRF1 and CRF2 (Carrasco and Van de Kar, 2003). Only one functional
variant i.e., α, besides numerous splice variants that are non-functional are encoded through the
CRF type 1 receptor (CRFR1) gene observed in rodents as well as humans. In case of CRF type
2 receptor (CRFR2), two functional splice variants (α and β) are available in rodents while in
humans three functional splice variants (α, β, and γ) are available (Smith and Vale, 2006).
The CRFR1 expression is densely populated in areas of the brain such as the hippocampus,
olfactory bulb, anterior pituitary, cerebellum, and cerebral cortex. CRFR1 is also expressed in
peripheral tissues in scarce amounts. These regions include testis, ovary and adrenal gland
(Potter et al., 1994; Van Pett et al., 2000). The CRFR2’s expression is exactly opposite to
CRFR1’s expression. CRFR2’s expression is extreme in peripheral regions but confined in a
restricted number of nuclei in the brain (Kishimoto et al., 1995). The CRF type 2β is mainly
distributed in the gastrointestinal tract (GIT), skeletal muscle, heart, and skin, thereby
expressing in the periphery (Perrin and Vale, 1999; Dautzenberg et al., 2001). The CRF’s
neuroendocrine activities are facilitated by means of CRFR1 receptors present in the anterior
pituitary. When the CRF binds to CRF1 receptors, the adenylate cyclase enzyme is activated.
This stimulation leads to activation of the cAMP pathway resulting ultimately in the ACTH
release from pituitary corticotropes (Perrin and Vale, 1999; Dautzenberg et al., 2001;
Bilezikjian and Vale, 1983).
Ever since the CRF’s recognition (in 1981), a hypothesis has been existing, that the CRF
is involved in the process of combining several constituents of the stress response (Vale et al.,
1981). Besides controlling the corresponding activities in the hypothalamus, pituitary as well
as the adrenal region, CRF has also been known to stimulate effects similar to stress. The
examples include suppression of eating, ANS stimulation, behavior similar to anxiety,
immunity suppression behavior (Bale et al., 2000; Brown and Fisher, 1985; Dunn and Berridge,
1990, Owens and Nemeroff, 1991, Spina et al., 2000; Sutton et al., 1982). These activities of
CRF which are centrally regulated, appropriately ease the fight or flight responses (Korte et al.,
1993).
The pathology of AD involves deposition of amyloid β protein, oxidative stress and
ultimately neuronal apoptosis. In one of the studies, the primary neurons along with the clonal
cells were employed to establish the neuroprotection by CRF in CRFR1 expressing the neurons
to prevent cell death due to oxidation. Also, CRF causes NF-kB (the redox-sensitive
transcription factor) suppression mediated by CRH-R1 which portrays CRF as endogenous
neuroprotective against oxidative cell death. These functions display the defensive activity of
CRF and offer a connection between the CRFR1 system and the oxidative stress-related events
(Lezoualc’h et al., 2000).
4.2. Adrenocorticotropic Hormone
Proopiomelanocortin (POMC) is a precursor hormone (prohormone). This hormone is

expressed mainly in the hypothalamic as well as the pituitary region of the brain. The
processing of POMC results in the generation of several peptides that are biologically active,

namely β-lipotropic hormone, melanocortins, β-endorphin, β-lipotropic hormone and ACTH

(Chang et al., 1980; Lacaze-Masmonteil et al., 1987; Sanson et al., 2003). ACTH gets released
into the systemic circulation from pituitary corticotropes, in response to CRF. This ACTH then
binds to the precise and corresponding receptor present in the adrenal cortex. The parenchyma
cells of the adrenocortical zona fasciculata consist of the receptor namely melanocortin type 2
receptor (MC2-R). These are the specific receptors to which the ACTH binds. Upon binding of
ACTH to the receptor, activation of the receptor occurs which in turn triggers the cAMP
pathway. The cAMP pathway gives rise to a course in which cholesterol is transformed to
steroidal hormones (steroidogenesis) such as androgenic steroids, mineralocorticoids, and
glucocorticoids that are endogenously active (Mountjoy et al., 1992). ACTH plays a vital role
by stimulating the synthesis of Δ5-pregnenolone from cholesterol in the course of
glucocorticoid biogenesis biosynthesis (Sanson et al., 2003; Simpson and Waterman, 1988).
Increased secretion of ACTH, corticosterone, and corticotropin-releasing hormone (CRH)
are observed during the upsurge of HPA activity which is prompted by oxidative stress. Aged
adults display a modified HPA reclamation to pharmacological as well as psychosomatic
encounters (Wilkinson et al., 1997; Otte et al., 2003). The older adults proved to have reduced
sensitivity to HPA’s negative feedback block, which was verified by dampened plasma ACTH
suppression in response to ACTH challenge (Boscaro et al., 1998). Inadequate response to
cortisol feedback inhibition is related to memory dysfunctions (O’Brien et al., 1994).
4.3. Vasopressin
Antidiuretic hormone (ADH), also known as argipressin or vasopressin or arginine

vasopressin (AVP), is a nonpeptide. ADH is expressed greatly in various segments of
hypothalamus such as the suprachiasmatic nucleus, PVN and supraoptic nucleus (SON)
(Swanson and Sawchenko, 1983; Brownstein, 1983). The endocrine cells present in the
hypothalamus, namely the magnocellular neurons (MCN) of the PVN and SON are considered
as the large units residing in the brain responsible for the genesis of oxytocin (OT) as well as
ADH. The released ADH reaches the systemic circulation and functions as an osmotic
homeostasis controller (Brownstein et al., 1980; Verbalis, 1993). Apart from MCN, the parvo-
cellular neurons arising from PVN participate significantly in the process of production as well
as the discharge of ADH into the circulation associated with liver and GIT (Portal circulation).
The released ADH, in turn, stimulates the CRF so as to release ACTH from the anterior pituitary
(Whitnall, 1993; Rivier and Vale, 1983; Antoni, 1993). The AVP’s additive effects on the
release of ACTH are facilitated by means of vasopressin V1b receptor (V1BR) on pituitary
corticotropes (Hernando et al., 2001). AVP binds to the V1BR and subsequently couples to Gq
proteins resulting in activation of phospholipase C (PLC). The PLC is involved in the
stimulation of protein kinase C (PKC). This eventually leads to an increased effect of ACTH
release (Birnbaumer, 2000). Investigational reports by several researchers have revealed that in
case of the chronic stress, the AVP’s (residing in parvocellular neurons of the PVN) gene
expression is increased considerably along with an increase in V1b receptor concentration
confined in pituitary corticotropes (Sawchenko, 1987; Kovacs and Sawchenko, 1996). The
study reports thus indicate and uphold the theory that ADH has a significant part in response to
stress through regulating the reaction of ACTH towards new stressors in the course of chronic
stress. During the chronic stress conditions, the AVP gene expression at parvo-cellular neurons

in the PVN of the hypothalamus is raised. Also eventually increased secretion of ADH is
observed in the pituitary portal circulation. In due course, the HPA axis is stimulated
(Goncharova, 2013). In addition, the V1BR are controlled by stress thereby potentiating the
vasopressin’s ACTH releasing activity (Rabadan-Diehl et al., 1995). Thus, vasopressin appears
to play significantly in several tasks that are performed by the brain. For instance, learning,
memorizing, raising emotions, etc (Ebner et al., 2000).
A preclinical study performed using vascular dementia (VD) rats demonstrated that
vasopressin expression in their hippocampi was very much lesser in immunohistochemistry.
Also, the infusion of AVP normalized the upsurge of malondialdehyde (MDA) concentration
and the reduced superoxide dismutase (SOD) activity at the hippocampus. This suggested an
ancillary association between AVP transmission, oxidative status and spatial memory (Chun-
Ying et al., 2017).
4.4. Serotonin
Serotonin also called 5-hydroxytryptamine (5-HT), is a monoamine commonly circulated

in the brain. It is involved in mood and impulse control (Fink et al., 1998; Young, 1996).
Impairment of serotonergic neurotransmission has been allied with numerous mood disorders
like panic disorder, anxiety, depression, obsessive-compulsive disorder and eating disorders
(Graeff et al., 1997; Levy and Van de Kar, 1992; Mora et al., 1997). Serotonergic neurons are
situated in the midbrain raphe innervate of the hypothalamus. Several of these neurons also
transmit collaterals to the amygdala (Petrov et al., 1994) and probably to limbic forebrain
regions also. Thus, changes in the serotonergic contribution to several limbic forebrain regions
can reflect these changes in the serotonergic input into the hypothalamus.
Except for 5-HT3 receptors, six families of serotonin receptors (5-HT1 to 5-HT7) are have
been cloned (Hoyer et al., 2002). 5-HT3 receptors are ligand-gated ion channels. The cloned
receptors are G-protein transmembrane peptides. The 5-HT1 receptors have been divided into
five subtypes (5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT1F) which are coupled to Gi/o/z
proteins. 5-HT2 receptors have been divided into three subtypes (5-HT2A, 5-HT2B, 5-HT2C)
which are coupled to Gq/11 proteins (Albert and Tiberi, 2001; Hoyer et al., 2002).
In stress condition, extracellular levels of serotonin vary in the brain areas like
hypothalamus, amygdala, frontal cortex and raphe nuclei (Adell et al., 1997; Fujino et al.,
2002). Tryptophan hydroxylase enzyme activity is elevated by glucocorticoid receptors in the
sound stress condition which is reverted back to normal by adrenalectomy (Singh et al., 1990).
Even foot shock-induced stress which increased level of serotonin synthesis/metabolism was
considered as an adaptive response to counterbalance neuronal depletion of serotonin (Dunn
and Berridge, 1990; Saphier and Welch, 1995; Chaouloff et al., 1999). In the limbic regions,
synthesis/metabolism of serotonin elevated by immobilization or restraint stress (Dunn and
Berridge, 1990; Shimizu et al., 1989). ACTH, oxytocin, and corticosterone are the hormones
released by 5-HT1A receptors stimulation (Serres et al., 2000; Van de Kar, 2000). Serotonin is
a monoamine neurotransmitter which plays an important role in the stress response. In humans,
decreased level of brain serotonin leads to elevation of anxiety. The treatment of generalized
anxiety and panic disorders such as selective serotonin uptake inhibitors elevate the serotonin
level and lead to anxiolytic effects (Leonard, 1993). In neuroendocrine brain regions, stress

hormones (e.g., ACTH, corticosterone, oxytocin, prolactin and renin) are secreted and
regulated by CRF (Parsons et al., 1996).
The endogenous N-acetylserotonin (NAS) is a precursor of melatonin and is known to
possess antioxidant activity (Pevet et al., 1980). However, the antioxidant activity of NAS on
neuronal cells is still not known. Some of the studies suggest that NAS causes activation of
antioxidant enzymes thereby inducing neuroprotection against oxidative stress-induced
cytotoxicity. Also, N-palmitoyl serotonin is well known to hold powerful antioxidant activity.
Conversely, it has to be clinically experimented yet so as to find its clinical application (Jin
et al., 2014).
4.5. Norepinephrine
Norepinephrine (NE) is a catecholamine synthesized from L-tyrosine. It is one of the chief

neurotransmitters of the peripheral sympathetic nervous system, although its presence is
established in certain areas of the brain too (Tripathi, 2013). The sub-types of nor-adrenergic
receptors comprises of alpha (α) adrenergic receptors and beta (β) adrenergic receptors. The ɑ
adrenergic receptors are comprised of two subclasses namely: α1, and α2. Similarly, β adrenergic
receptors are comprised of three subclasses namely β1, β2, and β3 (Barar, 2007).
Postsynaptically, the G proteins of Gs type are stimulated by β adrenergic receptors which lead
to adenylyl cyclase (AC) activation. The activation of AC is responsible for a series of
physiological responses (Tripathi, 2013). In contrast to β adrenergic receptors, the α2 adrenergic
receptors are involved in the diminution of AC enzyme function. They are also known to
stimulate certain potassium channels mediated by the G protein-coupled receptor-Gi/Go
(Nestler eta l., 1999). The α2 adrenergic receptors are located presynaptically as well as
postsynaptically (Ruffalo et al., 1981; Boehm, 1999). The phospholipase C enzyme is activated
by the α1 adrenergic receptor by coupling with Gx. α1 adrenergic receptors are found in the
postsynaptic terminals (Wu et al., 1992). However, β2 and α2 receptors are present on
presynaptic terminals as well as postsynaptic terminals (Tripathi, 2013). NE is involved in the
activation of α as well as β receptors which are located in the CNS and peripheral regions
(McCorry, 2007). This is based on the equilibrium existing among both receptors presence and
their stimulation (Ramos and Arnsten, 2006).
The activities of NE are facilitated and regulated through molecules or peptides that
participate in the activities of the brain (neurochemicals). Several of these neurochemicals are
known to affect the behavioral mechanism and any modification observed in any of the
neurotransmitters, undoubtedly hinder the actions of other related neurochemicals which
ultimately result in behavioral disorders (Choudhury et al., 2018).
The neurochemicals known to act together with noradrenergic system includes
acetylcholine (Beani et al., 1978; Vizi, 1979; Bianchi et al., 1979; Vizi and Pasztor, 1981),
corticotropin-releasing hormone (Melia and Duman, 1991; Veith et al, 1993), serotonin (Jaim-
Etcheverry, 1982; Murphy et al., 1998; Gillespie et al., 1988), dopamine (Soderpalm et al.,
1987; Yamamoto and Novotney, 1988; Gresch et al, 1995), gamma-aminobutyric acid (Ferraro
et al., 1993), neuropeptide Y (Kunik et al., 1994) and somatostatin (Gerner and Yamada, 1982).
The above-listed neurotransmitters and neurochemicals interaction have been known to
cause various types of behaviors which include depression, aggression, and anxiety in non-
sufferers of dementia (de Wied and van Ree, 1989).

NE has been established in treating AD by offering short period defense against the
characteristic neurotoxicity induced by the β amyloid. Treatment with NE has shown to reduce
intracellular ROS considerably thereby signifying its antioxidant characteristics. Depending
upon this factor NE is also projected to offer long-lasting neuroprotection, possibly by
combating the oxidative stress (Alvarez-Diduk et al., 2015).
4.6. Glucocorticoids
Glucocorticoids (GCs) are often the first-line therapy for autoimmune diseases including
many neurological conditions. Hydrocortisone and betamethasone are the weakest and
strongest GCs, respectively (Charmandari et al., 2005; Sapolsky et al., 2000). The HPA axis is
known to be affected by GC. This is evident by its role in homeostasis regulation as well as in
the response perceived due to stress (Habib et al., 2001). The activities of GC are facilitated by
means of the glucocorticoid receptor (GR). GRs are of two types, namely mineralocorticoid
receptor and glucocorticoid receptor. The mineralocorticoid receptor offers greater
corticosterone binding capacity (Kdc1 nM) while the glucocorticoid receptor offers lower
corticosterone binding capacity (Kdc5 nM). The GRs are mediated through transcription factors
(ligand-dependent). The glucocorticoid receptors reside in areas of the brain which include the
hypothalamic PVN and the frontal cortex, while the mineralocorticoid receptors reside mainly
in the hippocampal region of the brain (Meijer and Dekloet, 1998). GC is mainly associated
with vital body mechanisms such as growth, immunity and metabolism. GC also has
intervention in the functioning of the brain such as learning, memory which is fundamentally
dependent on adapted behaviour (Stratakis and Chrousos, 1995; Gesing et al., 2001). The
regulation of neuroendocrine activities on the HPA axis is also performed by GC. By exercising
the negative feedback mechanism at the regions of pituitary, hypothalamus and certain areas of
supra hypothalamic region, the stress response is ended (Gesing et al., 2001; De Kloet et al.,
1986). The functions of HPA axis is inhibited when the hippocampal mineralocorticoid
receptors are activated (Gesing et al., 2001).
Extreme activity of glucocorticoids reduces the activity of mitochondria. This leads to
oxidative stress. Hence the oxidative stress may be linked to the excessive response in the field
of neuronal dysfunctions (Tang, 2012). ROS is induced by augmented corticosterone levels in
serum. This is brought about by serum protein glycation and more detrimental oxidative effect
caused to the hippocampus. Enhanced production of glucocorticoid is succeeded after the HPA
has been made dysfunctional by oxidative stress. The ultimate result of the overall process is
the impairment of cognitive functions (Kobayashi et al., 2009).
4.7. Vasoactive Intestinal Polypeptide
Vasoactive intestinal polypeptide (VIP) is basically a peptide comprising of 28 amino acid

monomers. It functions as a neuropeptide and found in CNS as well as the peripheral nervous
system (PNS) (Said and Rosenberg, 1976). VIP is structurally analogous to various hormones
and associated neuropeptides such as a gastric inhibitory peptide, glucagon, growth hormone
releasing factor (GRF), glucagon-like peptide (GLP-1 and GLP-2) helodermin and secretin
(Delgado and Ganea, 2011). Peripheral location of VIP includes certain motor nerves which

include autonomic nerves supplied to exocrine glands, sympathetic ganglia, smooth muscle
(vascular and nonvascular), sciatic nerve (Henning and Sawmiller, 2001). CNS locations of
VIP include the suprachiasmatic nucleus of the hypothalamus, bed nucleus of stria terminalis,
cerebral cortex, striatum, hippocampus, sacral spinal cord, central gray of the midbrain
(periaqueductal gray), medial preoptic nucleus, anterior olfactory nuclei, amygdala and nucleus
accumbens (Said, 2013). In the PVN of the hypothalamus, VIP is found existing along with
CRF (Ceccatelli et al., 1989). In the pituitary, VIP is found the zona medularis and in the adrenal
gland it is found in the glomerulosa as well as in the chromaffin cells (Arnaout et al., 1986;
Cunningham and Holzwarth, 1988; Murase et al., 1993). In summary, VIP is found extensively
dispersed including all the regions of HPA axis. Several physiological activities of VIP are
mediated through particular G-protein coupled receptors (GPCR). The receptors include
VPAC1, VPAC2 and PAC1. The physiological functions including bone metabolism, gastric
motility, hormonal regulation, bronchodilation, smooth muscle relaxation, neurotrophic effects,
learning and behavior, increased cardiac output, systemic vasodilatation, hyperglycemia and
analgesia (Morell et al., 2012). The response of VIP due to stress was initially verified by an
upsurge in pituitary secretion of ACTH (Nicosia et al., 1983; Westendorf and Schonbrunn,
1985). Additionally, reports suggested that CRF and VIP act in a synergistic manner cause
ACTH release from pituitary cells (Leonard et al., 1988). Also, VIP is found to be involved
actively in the release of corticosterone and aldosterone (Cunningham and Holzwarth, 1988).
Stearyl-Nle17-VIP (SNV) is an analog of VIP. It is known to be 100 times more powerful
compared to the native peptide and thereby provides noteworthy protection of neurons in case
of oxidative stress by enhancing the cellular resistance. Thus, further research of VIP
derivatives may offer more advantages in therapeutics against the detrimental effects of
neurodegenerative diseases like AD (Offen et al., 2000).
4.8. Neuropeptide Y
Neuropeptide Y (NPY) is a neurotransmitter and neurohormone consisting of 36 amino

acid monomers that is secreted in the pancreas (Holzer et al., 2012). NPY is found widely
distributed in CNS as well as PNS. Centrally, it is found in the regions of hindbrain,
hypothalamus, cortex, and hippocampus (Chronwall et al., 1985). The brain activities in
conjunction with the physiological functions of NPY include cognition, neuroprotection,
homeostasis, circadian rhythm, food intake, neurogenesis and response to stress (Catalani
et al., 2017). Peripherally NPY I associate with the sympathetic nervous system (SNS). Here it
is involved in the control of functions related to blood vessels and heart by interacting with
ATP and NE (Brothers and Wahlestedt, 2010).
The functions of NPY are facilitated by means of GPCRs namely Y1, Y2, Y4, Y5 and Y6
(subtypes of NPY receptors) (Li et al., 2015). NPY represents itself as a stress molecule
(Zukowska-Grojec and Vaz, 1988) and its levels in plasma are seen to escalate as a response to
stress (Enman et al., 2014). Also, studies on NPY have confirmed that it facilitates the recovery
from stress through its defensive neuro-chemical activity (Reichmann and Holzer, 2015).
The effect of a single intracerebroventricular (ICV) administration of neuropeptide Y was
studied in order to study the neurochemical and behavioral parameters connected to oxidative
stress. Spatial memory impairments and depression-like responses induced by Aβ1-40 were
found to be prevented with NPY pre-treatment. Aβ1-40 infusion displayed an enhanced lipid

peroxidation in prefrontal cortex and hippocampus that were initially diminished by the
administration of NPY. The outcome of the study thus suggested that NPY administered
centrally prevents depressive behavior and Aβ1-40-induced spatial memory shortfalls. This
response is possibly mediated by Y2 receptors activation and inhibition of oxidative stress (dos
Santos et al., 2013).
4.9. Cholecystokinin
Cholecystokinin (CCK) is a peptide which is found in high density in the regions of

cerebral cortex, amygdala, and hippocampus of the mammalian brain (Bourin et al., 1991).
Furthermore, it co-localizes with several key neurotransmitter and neuromodulator systems.
CCK is involved in a diversity of normal behaviors such as learning and memory, feeding,
nociception, satiety, and it is also strongly linked to several CNS disorders including anxiety,
panic attacks, deficits in fear extinction, schizophrenia and epilepsy (Lee and Soltesz, 2011). It
binds to 2 receptors, CCK-A, and CCK-B that are found within the central nervous system
(Rotzinger and Vaccarino, 2003). These receptors are found in brain areas involved with
cognitive or emotional aspects of behavior, such as prefrontal cortex, cingulate, hippocampus,
amygdala, and the locus coeruleus (Hill and Woodruff, 1990, Gulpinar and Yegen, 2004).
Stressful condition activates HPA axis with the participation of these central CCK receptors
(Gulpinar and Yegen, 2004).
A huge number of studies has been performed to evaluate the participation of CCK system
in panic, anxiety, and stress associated behavior. Significant reduction in extracellular levels of
CCK was observed during stress, proposing that CCK is actively involved in the stress-induced
memory dysfunction (Dauge et al., 2003).
4.10. Substance P
Substance P (SP) is a neuropeptide consisting of 10 amino acid monomers and belongs to

the family of tachykinins (Steinhoff et al., 2014). The tachykinins function as neuromodulator
and neurotransmitters. They are widely found in both CNS as well as PNS (Steinhoff et al.,
2014). Quite a large number of physiological activities are performed by SP. It plays a vital
part in regulating processes involving cardiovascular smooth muscle, respiratory system and
GIT. SP also mediates glandular secretions, nociception, allergic reactions and inflammation
(Xu et al., 2013). The activities involving vascular permeability, blood flow, intestinal
secretions and GI motility are regulated by tachykinins in the periphery. In CNS, the SP
modulates the sensory activities such as hearing, sense of smell and vision as well as pain
perception (Strand, 1999). SP is shown to be connected in a wide variety of ailments
(neurological and psychiatric) including migraine, pain, emesis, movement disorders,
inflammatory bowel disease (IBD), psoriasis, psychosis, anxiety, asthma and stroke (Turner
and Vink, 2013). All the effects caused by SP are intervened through its binding to tachykinin
NK receptors (Rosso et al., 2012). Tachykinin NK receptors are family 1 (rhodopsin-like) of
GPCRs (Garcia-Recio and Gascon, 2015). The subtypes of tachykinin NK receptors include
NK1, NK2, and NK3 (Maggi, 1995). NK1 receptor has high affinity towards SP (Schwarz and
Ackenheil, 2002). SP is generally transmitted when stress or anxious conditions are triggered

(Ebner and Singewald, 2006). Tachykinins regulate the functions of the HPA axis as they reside
in their secretory cells as well as nerve fibers (Nussdorfer and Malendowicz, 1998).
Interactions of NK-1R with SP are known to be linked with CNS disorders such as AD.
Reduced SP concentrations have been reported in animal models pertaining to CNS motor
disorders as well as in the brain tissues obtained from the postmortem of Parkinson’s disease
patients (Chen et al., 2004). Similarly, in AD, reduced SP levels are observed in the cortical
regions of post-mortem patient’s cerebrospinal fluid and in brain tissues (Quigley and Kowall,
1991).
4.11. Oestrogen and Stress
Studies in humans have reported valuable positive effects of estrogen in response to stress.
However, the mode of action and the sites responsible for the effect is still under ambiguity
(Luine, 2014). Dementia linked with a deficiency of estrogen, when treated with estrogen
therapy resulted in reversing the memory impairment in several studies thus indicating estrogen
as a promising treatment to reduce the possibility of emerging dementia.
One of the clinical studies involving postmenopausal women evaluated the link between
the alterations in blood oxidative stress activity and memory. Oestrogen progestin therapy
(EPT) for a period of sixteen weeks resulted in a significant development in memory recovery
among the patients. However, the mechanism is still under ambiguity whether the improvement
in memory is due to the inherent antioxidant activity of estrogen or due to enhanced catalase
(CAT) and glutathione peroxidase (GPx) activity in plasma. The ratio of reduced glutathione
to oxidized glutathione (GSH:GSSG) and superoxide dismutase levels was confidently
associated with the performance of immediate memory in the study (Shafin et al., 2013).
4.12. Dopamine and Stress
Dopamine occupies greater than half of CNS catecholamine content. Most of the part is
present in some portions of the mesolimbic system as well as corpus striatum which is a portion
of the extrapyramidal motor system (Haber, 2014; Bissonette and Roesch, 2015). The
mechanism of how dopamine acts in the course of stress is still a debatable subject. Results
obtained from non-clinical studies have revealed variable responses of dopamine towards
various stressful inducements (Arnsten et al., 2014; Juarez Olguin et al., 2016). Increased
dopamine outflow was observed in the ventral striatum when a severe, regulated/avoidable
physical stress was applied, while the outflow of dopamine was diminished when a long-lasting,
unregulated/unavoidable stress was applied (Kumar et al., 2013).
Inclusion bodies are also known as Lewy bodies are seen in Parkinson’s disease (PD).
These are the depositions of α-synuclein found in the substantia nigra. The mutation of α-
synuclein has been observed and reported in AD (Gasser, 2001). Although dopamine (DA) is
well known as a neurotransmitter, it plays a crucial role to produce noxious free radicals due to
its metal chelating as well as electron donor activity. DA has a high affinity towards Fe3+ and
Cu2+ thereby initiating Fenton’s reaction leading the generation of H2O2 (Gerard et al., 1994).

4.13. Ascorbic Acid
Ascorbic acid (AA) is synthesized in the liver and is readily absorbed from GIT. Following
absorption, the ascorbic acid flows in the plasma and gets accumulated in the tissues of glands.
A part of accumulated AA undergoes metabolism and subsequently gets excreted in the form
of oxalate through urine. The other leftover part of Ascorbic acid gets excreted as such.
Enormous quantities of ascorbic acid are concentrated in the regions of adrenal cortex as well
as adrenal medulla (Patak et al., 2004). The oxidation of amino acids, namely, phenylalanine
(essential amino acid) as well as tyrosine (non-essential amino acid) occurs by the involvement
of AA. AA is consumed in great quantities in case of stress which is accountable to the
enhanced synthesis of steroids (Shohami et al., 1999). In addition, one of the pre-clinical studies
conducted in stress-induced rats reported that AA yielded defensive activity against immunity
related or hormonal or biological or chemical modifications (Kowalski and Jakubowski, 2000).
AA renders defense against damage induced by oxidative stress due to its antioxidant
property. The mechanism includes neutralizing of lipid hydroperoxyl (LHP) radicals and
thereby avoiding the alkylation of proteins mediated by electrophilic lipid peroxidation (Traber
and Stevens, 2011). Oxygen free radicals and other products of oxidative metabolism have been
shown to be neurotoxic. Restraint stress may cause generation of free radicals and result in
oxidative injury to the brain. Studies have shown that dehydroascorbic acid, the oxidized form
of ascorbic acid, enters the brain by means of facilitated transport. The oxygen free radicals, as
well as by-products of oxidative metabolism, are known to cause neurotoxicity. Free radicals
may be generated by restraint stress which leads to oxidative damage to the brain. Several
studies have established that the oxidized product of AA, namely dehydroascorbic acid gains
entry into the brain mediated by facilitated transport when ascorbic acid is orally administered
during restraint stress conditions. This probably could be the probable cause of neuroprotection
against the harmful effects of free radicals. Therefore, AA may be useful in the effective
treatment of stress-induced neurological disorders associated with cognitive impairment (Agus
et al., 1997).
5. PSYCHOLOGICAL, BEHAVIORAL AND BIOLOGICAL

DETERMINANTS OF STRESS
An immense flow of modifications occurs in vital organ systems such as the immune
system, nervous system, endocrine system and cardiovascular system (CVS), during an event
of acute stress. Generally, response to stress is adaption in nature, whereas, long-term stress has
shown to damage tissues and also causes disease (Selye, 1998). In case the acute stress is of
recurrent or incessant type, the response received for the same would be a non-adaptive or
poorly adjustable one. For instance, when the sympathetic nervous system (SNS) related to the
CVS is activated chronically, induced by stress, results in enlargement of blood vessels along
with an upsurge in blood pressure (Henry et al., 1975). Less intensity and steady natured stress
cause the overall body’s functional structures such as muscles, lungs, vessels, and brain to
progress severely either as hyper-stimulated or hypo-stimulated. This response ultimately
results in physical as well as mental impairment over a period of time.

A sequence of synchronized reactions is observed when in contact with stress. The

responses are such that they are systemized to improve the survival possibility. These structured
reactions include behavioral shifts, functional modification in autonomic system, production
and discharge of several hormones such as adrenal catecholamines, prolactin, oxytocin,
adrenocorticotropic hormone (ACTH), renin and corticosterone (Van de Kar and Blair, 1999).
Certain functional alterations that are linked with stress response are listed below:
 Energy utilization which controls the physiology of muscles and brain

 Intense as well as concentrated devotion on the observed risk
 Increased blood supply to cerebral regions and glucose consumption in confined
cerebral areas
 Upsurge in respiration and cardiac output, an imbalance in energy supply and substrate
utilization in muscles as well as in brain and reorganization of blood circulation
 Physiological inflection of immunity
 Diminishing of reproductive growth including sexual performance
 Reduced craving for food and food intake
 Weakening of short-term memory
6. OXIDATIVE STRESS
The oxygen gas exhibits dual nature due to its two contrasting effects (beneficial as well
as deteriorating) observed biologically (Burton and Jauniaux, 2011). The link existing amid the
oxidative stress and illness occurring due to the way of living has recently been well
established. The condition in which the degree of oxidation surpasses the antioxidant
organization present in the body is termed as oxidative stress and occurs chiefly when there is
an imbalance among them (Yoshikawa and Naito, 2002). When the movement in equilibrium
between the oxidant and antioxidants are considered and if the movement is more towards the
oxidant, then the condition is referred to as oxidative stress (Birben et al., 2012). Oxidative
stress has been revealed to be the main culprit in causing several mortal diseases. The list
includes atherosclerosis, hypertension, ischemia/perfusion (Holliwell and Gutteridge, 1990;
Devasagayam et al., 2003; Devasagayam et al., 2004), neurological disorders (Burton and
Jauniaux, 2011; Rahman et al., 2012; Hermes-Lima, 2004), cancer (Valko et al., 2006), asthma
(Birben et al., 2012) and diabetes, acute respiratory distress syndrome, idiopathic pulmonary
fibrosis, chronic obstructive pulmonary disease (Stadtman, 1992). Oxidative stress has also
shown beneficial effects in certain cases such as empowering of biological resistance system
through oxidative stress taking place during work-out or ischemia, initiation of apoptosis for
preparing the birth canal during the delivery (Yoshikawa and Naito, 2002), etc. Hence,
oxidative stress can thus be defined as a state where oxidation exceeds the antioxidant systems
because the balance between them has been lost (Yoshikawa and Naito, 2002).

6.1. Free Radicals
Free radicals are extremely reactive species of atoms or molecules. These are usually
composed of a lone pair of electrons. Generally, free radicals may evolve from several sources;
however, the biological generation of free radicals chiefly encompasses the two vital elements;
nitrogen and oxygen (Burton and Jauniaux, 2011). The elimination or addition of an electron
carried out by means of a non-radical species, not necessarily give rise to free radicals (Hermes-
Lima, 2004; Jauniaux, 2011).
The functions performed by free radicals are as follows:
 Production of adenosine triphosphate (ATP) from adenosine diphosphate by means of

oxidative phosphorylation in mitochondria
 Removal of toxins from xenobiotics (medicines) through cytochrome P450 (CYP-450)
enzymes
 Death of faulty cells
 Removal of cells which are neoplastic in nature along with microorganisms by means
of immune mechanisms involving cells such as cytotoxic lymphocytes, macrophages,
etc.
 Production of enzymes (cyclo-oxygenases (COX)) involved in the generation of
prostaglandins (PG) and leukotrienes (LT) (Devasagayam et al., 2004)
 Free radicals also function as signal molecules accountable for the “on” and “off” of
genes
 Cells associated with the immune system produce free radicals (superoxide and nitric
oxide) in plenty which are toxic to microorganisms
 Free radicals also eradicate tumor cells. This information has been the basis for the
development of certain drugs for treating cancer (Sarma et al., 2010).
6.2. Reactive Oxygen Species
Free radicals along with its intermediates which are non-radical together compose the
reactive species (RS) (Burton and Jauniaux, 2011). Free radicals comprise of a lone pair of
electrons (one or more) and thus render the molecule extremely reactive. Nonradicals are
formed when the unpaired electrons of two free radicals are split in between them. Reactive
intermediates namely, Reactive nitrogen intermediate (RNI) and reactive oxygen intermediate
(ROI) are produced biologically in a continuous process (Rahman et al., 2012). The reactive
oxygen species (ROS) comprise species such as hydroxyl radical (OH.), superoxide anion (O2.-
.) and hydrogen peroxide (H O ) whereas; reactive nitrogen species (RNS) comprise species
2 2
such as peroxynitrite (ONOO-), s-nitrosothiol (HNOS) and nitric oxide (NO). These species are
biologically important (Birben et al., 2012; Rahman et al, 2012). The reaction of biomolecules
(carbohydrates, lipids, and proteins) with RNS and ROS occur due to an imbalance in
homeostasis (both extracellular and intracellular) ultimately result in possible apoptosis
followed by regeneration (Rahman et al., 2012).
ROS is generated through the endogenous and exogenous source. ROS formed in the
period of cellular digestion as a result of processes involving enzymes, constitutes the

endogenous source. The enzymes involved in the endogenous source include cyclooxygenase
(COX), lipoxygenases (LOX), xanthine oxidases (XO), mitochondrial oxidases and nitric oxide
synthases (NOS). Unlike endogenous source, the exogenous source comprises of reaction
between organic compounds and oxygen as well as those initiated by ionizing radiations, which
are not enzymatic reactions (Pham-Huy et al., 2008). In some instances, free radicals are
generated by means of radiations which cause ionization. Apart from biological sources
external sources also contribute to the free radicals generation examples include solvents used
in industries, ozone deformation, hazardous radiations, certain toxic drugs, pollutants,
ultraviolet light, pesticides and cigarette smoke (Phaniendra et al., 2014).
6.3. Molecular Damage Induced by Free Radicals
The reaction of free radicals is very hazardous and hence if they are not stopped, entire
biological macromolecules (nucleic acids, proteins, carbohydrates, and lipids) will be
rampaged. The situation can be well understood by an instance of the destruction caused by
free radicals on proteins. The chemical reactivity of free radicals on proteins causes cataract.
Similarly, DNA damage by free radicals leads to cancer (Kumar, 2011).
6.3.1. Lipids and Lipid Peroxidation

Free radicals react with membrane lipids residing in the organelles within the cell and cause
lipid peroxidation (LP). LP constitutes a series of events which are highly destructive in nature.
The by-products of LP include 4-hydroxynonenal (4-HNE), malondialdehyde (MDA) and
various 2-alkenals. These products are significantly toxic. The process of arachidonic acid-lipid
peroxidation leads to the generation of Isoprostanes. The LP causes damage in such a way that
it hinders the cell physiology to the highest extent (Devasagayam et al., 2003).
6.3.2. Oxidative Damages to Carbohydrates

Hydroxyl radical (OH•) is a free radical. The reaction between the carbohydrates and the
hydroxyl radical lead to the formation of a radical that is carbon-centered. The process involves
the extraction of a hydrogen atom from the carbohydrate’s carbon atoms. This results in the
breakdown of hyaluronic acid. Hyaluronic acid is a significant component of synovial fluid.
The synovial fluid is found adjacent to the joints performing the lubrication of joints. During
inflammation, neutrophils are stimulated and get accumulated in the synovial fluid. These
neutrophils generate oxyradicals in considerable quantities and are well known to be associated
with one of the most hazardous immune disorder rheumatoid arthritis (Devasagayam et al.,
2004).
6.3.3. Oxidative Damages to DNA

The reactive species ROS or RNS are prone to interact with DNA and cause oxidation of
DNA. Free radical OH•, the chief class of active oxygen is the main malefactor that damages
the structural units of DNA molecule (bases and deoxyribose residues) as well as free
nucleotides. Oxidation of protein or lipid molecules is also caused by ROS. This leads to
intermediates generation that eventually forms adducts by interacting with DNA. Several repair
pathways repair DNA damage (Halliwell and Aruoma, 1993). 8-OHdG (8-Oxo-2'-

deoxyguanosine), an oxidized derivative of DNA has been associated in causing cancer and is
utilized as a trustworthy marker for oxidative DNA damage (Devasagayam et al., 2004;
Halliwell and Aruoma, 1993).
6.3.4. Oxidative Damage to Proteins

Several stable and unstable products are produced when the protein is oxidized by RNS or
ROS. Protein hydroperoxides are one among the products which are capable of producing some
more radicals when they undergo interaction with transition elements. The oxidized products
of proteins which are not active functionally will be excluded from the body in most cases.
However, few of the derivatives may slowly gather in course of long periods and become the
causative factor for various disease conditions including aging. Lipofuscin is one such example
which is composed of products formed as a result of LP and protein oxidation and seen
accumulated in lysosomal parts of older cells as well as neurons of AD patients (Stadtman,
1992).
6.4. Antioxidants
Antioxidants are molecules that are involved in acting against the cell damage triggered by
free radicals. Antioxidants function through two categories namely primary antioxidants or
secondary antioxidants (Buettner, 1993; Vertuani et al., 2004). The primary antioxidants
(chain-breaking antioxidants) cause interruption of chain reaction which is involved in the
production of reactive radicals. This interruption gives rise to radicals that are relatively less
reactive. The secondary antioxidants (preventive antioxidants) produce non-radicals that are
non-reactive. The secondary antioxidants function by means of various processes such as
chelating, scavenging highly reactive and highly toxic singlet oxygen or by ROS removal
(Chaiyasit et al., 2007).
The mechanism of antioxidant might be either through enzyme or non-enzyme. The ROS
is attacked (directly or indirectly) by the antioxidants enzyme mechanism. The enzymes
contributing to the antioxidant enzyme system includes thioredoxin (Trx), glutathione
peroxidase (GPx), superoxide dismutase (SOD), glutathione reductase (GR), catalase (CAT)
(Valko et al., 2006). The non-enzymatic antioxidant system chiefly comprises of vitamin C,
metal binding proteins (MBPs), vitamin E, uric acid (UA), melatonin (MEL), glutathione
(GSH), bilirubin (BIL) and polyamines (PAs) (Mironczuk-Chodakowska et al., 2018).
6.4.1. Superoxide Dismutase

The SOD enzyme is considered as the utmost efficient antioxidant found within the cells.
This enzyme functions as a catalyst for the partitioning process of superoxide radical to an
oxygen molecule (O2•− to O2) or hydrogen peroxide (O2•− to H2O2) molecule. SOD of three
types is known in humans, viz., extracellular SOD (EC-SOD), cytosolic Cu, Zn-SOD and
mitochondrial Mn-SOD. The action of SOD is to abolish superoxide by means of oxidation
followed by reduction of transition metal ion in a consecutive manner (Landis and Tower,
2005).

6.4.2. Catalase
Catalase (CAT) enzyme resides intracellularly in the peroxisome. It is involved mainly in
the catalytic transformation of hydrogen peroxide to molecular oxygen and water (Heck et al.,
2010). Catalase has one of the highest turnover rates of all enzymes with the capacity to
transform millions of molecules of hydrogen peroxide to water and oxygen (Ibrahim et al.,
2015).
6.4.3. Glutathione Peroxidase

Glutathione peroxidase (GPx) belongs to the body’s antioxidant system which is enzymatic
in nature. Two distinct types of glutathione peroxidase are known based on its association with
selenium, they are, selenium-dependent and selenium-independent (GPx and glutathione-S-
transferase, GST) (Mates et al., 1999). The variation between the enzymes is accountable to
type of bond formed between the selenium and the active center of the enzyme, the number of
subunits and the mode of catalysis. Four types of glutathione peroxidases (Se-dependent) are
recognized in humans (Mates et al., 1999). The process of adding two electrons to form
selenoles (Se-OH) resulting in peroxide reduction is a common mechanism to every type of
GPx. The reduced peroxides act as substrates and undergo Fenton reaction and subsequently
get eliminated. GPx performs in association with glutathione (GSH) which is a tripeptide,
present intracellularly in high amounts. The peroxides are reduced to water/alcohol by GPx and
consequently, GSH is oxidized (Kurutas, 2016).
6.4.4. Vitamin C
Ascorbic acid, also known as vitamin C is one among the most essential as well as potent
antioxidants. This antioxidant is found to function in watery surroundings within the body.
Carotenoids (tetraterpenoids) along with vitamin E (alpha-tocopherol) constitute as the chief
associates of vitamin C. The α-tocopherol is restored from the radicals of α-tocopherol. The
process is mediated by the association of vitamin E with ascorbic acid and takes place in
lipoproteins and membranes (Kojo, 2004). Ascorbic acid is basically a water solvable vitamin.
Its chief function involves intra- and extra-cellular antioxidant activity which encompasses the
rummaging of oxygen free radicals. Another vital role of ascorbic acid comprises of the back
conversion of free radicals of vitamin E to vitamin E. However, the increasing age is shown to
reduce the ascorbic levels in plasma (Lee et al., 1995; Barceloux, 1999).
6.4.5. Glutathione
Glutathione (GSH) is one of the members of the body’s antioxidant system which is non-
enzyme. It is present in every compartment of every cell. GSH stands apart from other
antioxidants for being the foremost solvable antioxidant. The antioxidant activity of GSH is
observed through various mechanisms (Birben et al., 2012). GSH removes the toxicity effects
of lipid peroxides and hydrogen peroxide through the GPx. Thus, the GPx enzyme is considered
to be a vital protector of cell membranes against peroxidation of lipids. In fact, the cofactor for
various toxin removing enzymes (e.g., GPx and transferase) is the GSH. The mechanism of
GSH for protection of lipids and membranes against the hydrogen peroxide (H2O2) involves
the donation of an electron from GSH to H2O2 to form reduced glutathione. The reduced form
further involves in the protonation of lipids and cell membranes and thereby prevents and
guards them against damaging effects of oxidant (Beckman and Ames, 1997).

6.4.6. Carotenoids
Carotenoids are generally the coloring part (pigments) of the plants. Carotenoids are also
involved in the antioxidant activity. They are revealed to be the most proficient singlet oxygen
(1O2) as well as ROS quenchers. Hence the carotenoids play a vital role in curbing the disorders
that are mediated through the ROS. Among the carotenoids, β-carotene is known to interact
with hydroxyl radical (OH•), peroxyl radical (ROO•) as well as superoxide radical (O2•-)
(Dizdarogluet al., 2002).
6.4.7. Flavonoids
Polyphenols comprise the larger part and abundantly occurring vegetal metabolites. These
compounds signify an essential portion of human nutrition. Polyphenolic compounds have
attained great attention currently (especially flavonoids) due to their antioxidant efficiency
implicating direct positive benefits (Panche et al., 2016). The major chronic disorders such as
cardiovascular disease (CVD), malignancy and others are treated effectively by polyphenols
(Rice-Evans et al., 1997). The destructive effects of lipid peroxidation are prevented by the
polyphenol compounds. The polyphenols perform as chelating agents for metal ions which are
capable of redox reactions. These metal ions are involved in catalysis of lipid peroxidation.
Chelation of metal ions ultimately leads to the ending of free radical reactions as well as lipid
peroxidation (Schroeter et al., 2002).
6.5. Herbal Antioxidants for Management of Stress
The nutrients that are required in large amounts and are acquired by the food consumed,
are termed as macronutrients. Examples of macronutrients include ascorbic acid, tocopherol,
beta-carotene, etc. Herbal antioxidants commonly used in the management of stress is
summarised in Table 2. Furthermore, there are some medicinal plants which are reported to
possess potent memory enhancing potential (Table 3).
Table 2. Herbal antioxidants for management of stress
Plants Proposed Mechanism Therapeutic Effects

of Action
Panax ginseng Stimulate ACTH and Assists in increasing the effect of response due to
(Head and Kelly, subsequently cortisol acute stress, thereby creating an enhanced level
2009). production of adaption. This permits the physiological
function of the body to respond effectively to the
stress.
Withania Stimulate ACTH and The endogenous alterations such as
somnifera subsequently cortisol modifications in normal adrenal weight or blood
(Head and Kelly, production sugar levels or cortisol levels etc. occurring due
2009) to the high-stress conditions are neutralized.
Rhodiola rosea Acts primarily upon Effects chiefly in the hypothalamic region and
(Hamidpour hypothalamus and regulates HPA balance.
et al., 2015) maintains equilibrium with
HPA

Table 3. Medicinal plants with memory-enhancing potential
Plants Useful Parts Active Constituents

Bocopa monnieri (Singh and Dhawan, 1982) Whole plant Bacosides A and bacosides B
Celastrus paniculatus (Gattu et al., 1995) Seeds Celapagine and celapanigine
Withania somnifera (Schliebs et al., 1997) Roots Withanolides
Ricinus communis (Ferraz et al., 1999) Beans Ricinine
Salvia officinalis (Dhingra et al., 2003) Leaves Monoferpenoid
Ginkgo biloba (Taylor, 1986) Leaves Ginkgolides
Huperzia serrata (Cheng et al, 1996) Moss Huperzine
Hypericum perforatum (Khalifa, 2001) Roots -
Physostigma venenosum (Dhingra et al., 2003) Beans Physostigmine
Acorus calamus (Vohora et al., 1990) Rhizomes Asarone and methyl isoeugenol
Curcuma longa (Yu et al., 2002) Rhizomes Curcumin
Terminalia chebula (Dhingraet al., 2003) Rhizomes Chebulic acid
Centella asiatica (Nalini et al., 1992) Leaves Madicasoside, asiaticoside and
brahmoside
7. RECENT DEVELOPMENT, FUTURE OPPORTUNITIES

AND CHALLENGES
New approaches to safeguard neurons from being injured or deteriorated are gaining more
interest from the research community worldwide. The common principle for developing drugs
in this regard includes prevention of neuronal apoptosis and therefore the drugs are rightly
termed as antiapoptotic drugs (Rutland-Brown et al., 2006). These antiapoptotic drugs are being
developed as antioxidants by employing novel techniques like genetic engineering and
molecular biology.
The inference of oxidative stress in the cause of numerous chronic as well as deteriorating
diseases recommends treatment with antioxidants. An approach in therapeutics to enhance the
cell’s antioxidant activity to reinforce the long-term treatment could be a strategy in the future.
But even for the present scenario, the use of antioxidant supplements in combating the disease
conditions caused due to oxidative stress is still debatable. Advanced investigation is required
before any supplements are officially suggested as adjuvant therapy. Meanwhile, sources of
oxidative stress such as smoking and alcohol intake should be avoided completely and replaced
with a healthy, nutritious diet that is filled with antioxidants.
CONCLUSION
The pathogenesis of memory dysfunction involves increased oxidative stress and a decline
in brain and plasma antioxidants defense system. The free radicals are generated as a response
to the stress applied. The endogenous antioxidants (enzymatic and non-enzymatic), are the first
line of choice to prevent or minimize unwanted impairment of the cell triggered through the
ROS generated in excess by stress responses. Natural plant-derived compounds such as
flavonoids possess free radical scavenging activities and therefore prevent oxidative injury and

cell death. These plant-derived compounds are, therefore, useful for the management of stress-
induced memory dysfunction.
REFERENCES
Adell A, Casanovas J. M, Artigas F. Comparative study in the rat of the actions of different
types of stress on the release of 5-HT in raphe nuclei and forebrain areas. Neuropharmacol.
1997; 36(4-5): 735-41.
Aghajanian G. K, VanderMaelen C. P. Intracellular identification of central noradrenergic and
serotonergic neurons by a new double labeling procedure. J Neurosci. 1982; 2(12): 1786-
92.
Agus D. B, Gambhir SS, Pardridge W. M, Spielholz C, Baselga J, Vera J. C, et al., Vitamin C
crosses the blood-brain barrier in the oxidized form through the glucose transporters. J Clin
Invest. 1997; 100(11): 2842-8.
Albert P. R, Tiberi M. Receptor signaling and structure: insights from serotonin-1 receptors.
Trends Endocrinol Metab. 2001; 12(10): 453-60.
Alvarez-Diduk R, Galano A. Adrenaline and noradrenaline: protectors against oxidative stress
or molecular targets? J Phys Chem B. 2015; 119(8): 3479-91.
Antoni F. A. Vasopressinergic control of pituitary adrenocorticotropin secretion comes of age.
Front Neuroendocrinol. 1993; 14(2): 76-122.
Arnaout M. A, Garthwaite T. L, Martinson D. R, Hagen T. C. Vasoactive intestinal polypeptide
is synthesized in anterior pituitary tissue. Endocrinol. 1986; 119(5): 2052-7.
Arnsten A. F, Raskind M. A, Taylor F. B, Connor D. F. The Effects of Stress Exposure on
Prefrontal Cortex: Translating Basic Research into Successful Treatments for Post-
Traumatic Stress Disorder. Neurobiol Stress. 2014; 1: 89-99.
Audhya T, Hollander C. S, Schlesinger D. H, Hutchinson B. Structural characterization and
localization of corticotropin-releasing factor in testis. Biochim Biophys Acta. 1989; 995(1):
10-6.
Bale T. L, Contarino A, Smith G. W, Chan R, Gold L. H, Sawchenko P. E, et al., Mice deficient
for corticotropin- releasing hormone receptor-2 display anxiety-like behavior and are
hypersensitive to stress. Nat Genet. 2000; 24(4): 410-4.
Bale T. L, Vale W. W. CRF and CRF receptors: role in stress responsivity and other behaviors.
Annu Rev Pharmacol Toxicol. 2004; 44: 525-57.
Bamberger C. M, Schulte H M, Chrousos G. P. Molecular determinants of glucocorticoid
receptor function and tissue sensitivity to glucocorticoids. Endocr Rev. 1996; 17(3): 245-
61.
Barar, F. S. K. Essentials of Pharmacotherapeutics, 4th ed.; S. Chand & Company Ltd.: New
Delhi, 2007.
Barceloux D. G. Nickel. J Toxicol ClinToxicol. 1999; 37(2): 239-58.
Beani L, Bianchi C, Giacomelli A, Tamberi F. Noradrenaline inhibition of acetylcholine release
from guinea-pig brain. Eur J Pharmacol. 1978; 48(2): 179-93.
Beckman K. B, Ames B. N, Oxidative decay of DNA. J Biol Chem. 1997; 272(32):19633-6.

Bianchi C, Spidalieri G, Guandalini P, Tanganelli S, Beani L. Inhibition of acetylcholine

outflow from guinea-pig cerebral cortex following locus coeruleus stimulation. Neurosci
Lett. 1979; 14(1): 97-100.
Bilezikjian L. M, Vale WW. Glucocorticoids inhibit corticotropin-releasing factor-induced
production of adenosine 3’,5’-monophosphate in cultured anterior pituitary cells.
Endocrinology. 1983; 113(2): 657-62.
Birben E, Sahiner U. M, Sackesen C, Erzurum S, Kalayci O. Oxidative stress and antioxidant
defense. World Allergy Organ J. 2012; 5(1): 9-19.
Birnbaumer M. Vasopressin receptors. Trends Endocrinol Metab. 2000; 11(10): 406-10.
Bissonette G. B, Roesch M. R. Development and function of the midbrain dopamine system:
what we know and what we need to. Genes Brain Behav. 2015; 15(1): 62-73.
Boehm S. Presynaptic alpha2-adrenoceptors control excitatory, but not inhibitory, transmission
at rat hippocampal synapses. J Physiol. 1999; 519 Pt 2(Pt 2): 439-49.
Boscaro M, Paoletta A, Scarpa E, Barzon L, Fusaro P, Fallo F, et al. Age-related changes in
glucocorticoid fast feedback inhibition of adrenocorticotropin in man. J Clin Endocr
Metab. 1998; 83(4): 1380-3.
Bourin M, Bradwejn J, Koszycki D. Is cholecystokinin a biological support in panic attacks?
Encephale. 1991; 17(5): 475-9.
Brothers S. P, Wahlestedt C. Therapeutic potential of neuropeptide Y (NPY) receptor ligands.
EMBO Molecular Medicine. 2010; 2(11): 429-439.
Brown M. R, Fisher L. A, Corticotropin-releasing factor: effects on the autonomic nervous
system and visceral systems. Fed Proc. 1985; 44(1 Pt 2): 243-8.
Brownstein M. J, Russell J. T, Gainer H. Synthesis, transport, and release of posterior pituitary
hormones. Science. 1980; 207(4429): 373-8.
Brownstein M. J. Biosynthesis of vasopressin and oxytocin. Annu Rev Physiol. 1983;45: 129-
35.
Bruhn T. O, Engeland W. C, Anthony E. L, Gann D. S, Jackson I. M. Corticotropin-releasing
factor in the adrenal medulla. Ann N Y Acad Sci. 1987; 512: 115-28.
Budson AE, Prince BH. Memory dysfunction in clinical practice. Discove Med. 2005; 5(26):
135-41.
Buettner G. R. The pecking order of free radicals and antioxidants: lipid peroxidation, alpha-
tocopherol, and ascorbate. Arch Biochem Biophys. 1993; 300(2): 535-43.
Burton G. J, Jauniaux E. Oxidative stress. Best Pract Res Clin Obstet Gynaecol. 2011; 25(3):
287-99.
Calogero A. E, Gallucci W. T, Gold P. W, Chrousos G. P. Multiple feedback regulatory loops
upon rat hypothalamic corticotropin-releasing hormone secretion: potential clinical
implications. J Clin Invest. 1988; 82(3): 767-74.
Carrasco G. A, Van de Kar L. D. Neuroendocrine pharmacology of stress. Eur J Pharmacol.
2003; 463(1-3): 235-72.
Catalani E, De Palma C, Perrotta C, Cervia D. Current Evidence for a Role of Neuropeptides
in the Regulation of Autophagy. Biomed Res Int. 2017; 2017: 1-10.
Ceccatelli S, Eriksson M, Hokfelt T. Distribution and coexistence of corticotropin-releasing
factor-, neurotensin-, enkephalin-, cholecystokinin-galanin-and vasoactive intestinal
polypeptide/peptide histidine isoleucine-like peptides in the parvocellular part of the
paraventricular nucleus. Neuroendocrinology. 1989; 49(3): 309-23.

Chaiyasit W, Elias R. J, McClements D. J, Decker E. A. Role of physical structures in bulk oils

on lipid oxidation. Crit Rev Food Sci Nutr. 2007; 47(3): 299-317.
Chang A. C, Cochet M, Cohen S. N. Structural organization of human genomic DNA encoding
the pro-opiomelanocortin peptide. Proc Natl Acad Sci U S A. 1980; 77(8): 4890-4.
Chaouloff F, Berton O, Mormede P. Serotonin and stress. Neuropsychopharmacol. 1999; 21(2):
28S-32S.s
Charmandari E, Tsigos C, Chrousos G. Endocrinology of the stress response. Annu Rev Physiol.
2005; 67: 259-84.
Chatterton R. T. The role of stress in female reproduction: animal and human considerations.
Int J Fertil. 1990; 35(1): 8-13.
Chen L. W, Yung K. K, Chan Y. S. Neurokinin peptides and neurokinin receptors as potential
therapeutic intervention targets of basal ganglia in the prevention and treatment of
Parkinson's disease. Curr Drug Targets. 2004; 5(2): 197-206.
Cheng D. H, Ren H, Tang X. C. Huperzine A, a novel promising acetylcholinesterase inhibitor.
Neuroreport. 1996; 8(1): 97-101.
Choudhury A, Sahu T, Ramanujam PL, Banerjee AK, Chakraborty I, Arun Kumar R, et al.,
Neurochemicals, behaviours and psychiatric perspectives of neurological diseases.
Neuropsychiatry (London). 2018; 8(1): 395-424.
Chronwall B. M, DiMaggio D. A, Massari V. J, Pickel V. M, Ruggiero D. A, O’Donohue T. L.
The anatomy of neuropeptide-Y-containing neurons in rat brain. Neuroscience. 1985;
15(4): 1159-81.
Chun-Ying L, Lei Z, Jing L, Chun-Li Q, Dong-Ying L, Xu L, et al., Effect of endogenous
arginine-vasopressin arising from the paraventricular nucleus on learning and memory
functions in vascular dementia model rats. BioMed Res Int. 2017: 3214918.
Clark RE. The classical origins of Pavlov’s conditioning. Integr Physiol Behav Sci. 2004; 39(4):
279-94.
Croiset G, Nijsen M. J, Kamphuis P. J. Role of corticotropin-releasing factor, vasopressin and
the autonomic nervous system in learning and memory. Eur J Pharmacol. 2000; 405(1-3):
225-324.
Cunningham L. A, Holzwarth M. A. Vasoactive intestinal peptide stimulates adrenal
aldosterone and corticosterone secretion. Endocrinology. 1988; 122(5): 2090-7.
Cunningham LA, Holzwarth M. A. Vasoactive intestinal peptide stimulates adrenal aldosterone
and corticosterone secretion. Endocrinology. 1988; 122(5): 2090-7.
Curtis G. C, Abelson J. L, Gold P. W. Adrenocorticotropic hormone and cortisol responses to
corticotropin-releasing hormone: changes in panic disorder and effects of alprazolam
treatment. Biol Psychiatry. 1997; 41(1): 76-85.
Dauge V, Pophillat M, Crete D, Melik-Parsadaniantz S, Roques BP. Involvement of brain
endogenous cholecystokinin in stress-induced impairment of spatial recognition memory.
Neuroscience. 2003; 118(1): 19-23.
Dautzenberg F. M, Kilpatrick G. J, Hauger R. L, Moreau J. Molecular biology of the CRH
receptors-in the mood. Peptides. 2001; 22(5): 753-60.
De Kloet E. R, Sybesma H, Reul H. M. Selective control by corticosterone of serotonin1
receptor capacity in raphe-hippocampal system. Neuroendocrinology. 1986; 42(6): 513-
21.
De Renzi E. Apraxia. In: Boller F, Grafman J, editors. Handbook of Neuropsychology. New
York: Elsevier Science; 1989.

de Wied D, van Ree JM. Neuropeptides: animal behaviour and human psychopathology. Eur
Arch Psychiatry Neurol Sci. 1989; 238(5-6): 323-31.
Delgado M, Ganea D. Vasoactive intestinal peptide: a neuropeptide with pleiotropic immune
functions. Amino Acids. 2011; 45(1): 25-39.
Devasagayam T. P, Boloor K. K, Ramsarma T. Methods for estimating lipid peroxidation:
Analysis of merits and demerits (minireview). Indian J Bioche Biophys. 2003; 40(5): 300-
8.
Devasagayam T. P, Tilak J. C, Boloor K. K, Sane K. S, Ghaskadbi S. S, Lele R. D. Free radicals
and antioxidants in human health: current status and future prospects. J Assoc Physicians
India. 2004; 52: 794-804.
Dhingra D, Parle M, Kulkarni S. K. Medicinal plants and memory. Indian Drugs. 2003; 40(6):
313-19.
Dizdaroglu M, Jaruga P, Birincioglu M, Rodriguez H. Free radical-induced damage to DNA:
mechanisms and measurement. Free Rad Biol Med. 2002; 32(11): 1102-15.
dos Santos V. V, Santos DB, Lach G, Rodrigues A. L, Farina M, De Lima T. C, et al.,
Neuropeptide Y (NPY) prevents depressive-like behavior, spatial memory deficits and
oxidative stress following amyloid-β (Aβ(1-40)) administration in mice. Behav Brain Res.
2013; 244: 107-15.
Dunn A. J., Berridge C. W. Physiological and behavioral responses to corticotropin-releasing
factor administration: is CRF a mediator of anxiety or stress responses? Brain Res Rev.
1990; 15(2): 71-100.
Ebner K, Wotjak C. T, Landgraf R, Engelmann M. A single social defeat experience selectively
stimulates the release of oxytocin, but not vasopressin, within the septal brain area of male
rats. Brain Res. 2000; 872(1-2): 87-92.
Ebner K, Singewald N. The role of substance P in stress and anxiety responses. Amino Acids.
2006; 31(3): 251-72.
Enman N. M, Sabban EL, McGonigle P, Van Bockstaele E. J. Targeting the Neuropeptide Y
System in Stress-related Psychiatric Disorders. Neurobiol Stress. 2014; 1: 33-43.
Ferraro L, Tanganelli S, Calo G, Antonelli T, Fabrizi A, Acciarri N, et al., Noradrenergic
modulation of gamma-aminobutyric acid outflow from the human cerebral cortex. Brain
Res. 1993; 629(1): 103-8.
Ferraz A. C, Angilucci M. E, Da Costa M. L, Batista I. L, Deoleveira B. H, DaCunha C.
Pharmacological evaluation of ricinine, a CNS stimulant isolated from the Ricinus
communis. Pharmacol Biochem Behav. 1999; 63(3): 367-75.
Fink G, Sumner B. E, McQueen J. K, Wilson H, Rosie R. Sex steroid control of mood, mental
state and memory. Clin Exp Pharmacol Physiol. 1998; 25(10): 764-75.
Fujino K, Yoshitake T, Inoue O, Ibii N, Kehr J, Ishida J, et al., Increased serotonin release in
mice frontal cortex and hippocampus induced by acute physiological stressors. Neurosci
Lett. 2002; 320(1-2): 91-5.
Garcia-Recio S. Gascon P. Biological and pharmacological aspects of the NK1-receptor.
Biomed Res Int. 2015; 2015: 495704.
Gasser T. Genetics of Parkinson’s disease. J Neurol. 2001; 248(10): 833-40.
Gattu M, Boss K. L, Terry AV, Buccafusco J. L. Reversal of scopolamine induced deficits in
navigational memory performance by the seed oil of Celastrus paniculatis. Pharmacol
Biochem Behav. 1995; 57(4): 793-9.

Gerard C, Chehal H, Hugel R. P. Complexes of iron (III) with ligands of biological interest:
dopamine and 8-hydroxyquinoline-5-sulfonic acid. Polyhedron. 1994; 13(4): 591-97.
Gerner R. H, Yamada T. Altered neuropeptide concentrations in cerebrospinal fluid of
psychiatric patients. Brain Res. 1982; 238(1): 298-302.
Gesing A, Bilang-Bleuel A, Droste S. K, Linthorst AC, Holsboer F, Reul J. M. Psychological
stress increases hippocampal mineralocorticoid receptor levels: involvement of
corticotropin-releasing hormone. J Neurosci. 2001; 21(13): 4822-9.
Gillespie D. D, Manier D. H, Sanders-Bush E, Sulser F. The serotonin/norepinephrine-link in
brain, II: role of serotonin in the regulation of beta adrenoceptors in the low agonist affinity
conformation. J Pharmacol Exp Ther. 1988; 244(1): 154-9.
Gisquet-Verrier P, Riccio D. C. Memory reactivation effects independent of reconsolidation.
Learn Mem. 2012; 19(9): 401-9.
Goncharova N. D. Stress responsiveness of the hypothalamic-pituitary-adrenal axis: age-
related features of the vasopressinergic regulation. Front Endocrinol (Lausanne). 2013; 4:
26.
Goshen‐Gottstein Y. Learning and memory. In: Encyclopedia of life sciences. New York: John
Wiley & Sons. 2003.
Graeff FG, Viana MB, Mora PO, Dual role of 5-HT in defense and anxiety. Neurosci Biobehav
Rev. 1997; 21(6): 791-9.
Gresch PJ, Sved A. F, Zigmond M. J, Finlay J. M. Local influence of endogenous
norepinephrine on extracellular dopamine in rat medial prefrontal cortex. J Neurochem.
1995; 65(1): 111-6.
Gulpinar M. A, Yegen B. C. The physiology of learning and memory: role of peptides and
stress. Curr Protein Pept Sci. 2004; 5(6): 457-73.
Haber SN. The place of dopamine in the cortico-basal ganglia circuit. Neuroscience. 2014; 282:
248-57.
Habib K. E, Gold P. W, Chrousos G. P. Neuroendocrinology of stress. Endocrinol Metab Clin
North Am. 2001; 30(3): 695-728.
Haider S, Saleem S, Perveen T, Tabassum S, Batool Z, Sadir S, et al., Age-related learning and
memory deficits in rats: role of altered neurotransmitters, acetylcholinesterase activity and
changes in antioxidant defense system. Age (Dordr). 2014; 36(3): 9653.
Halliwell B, Aruoma OI, editors. DNA and Free Radicals. Boca Raton, Florida: CRC Press;
1993.
Hamidpour R, Hamidpour S, Hamidpour M, Shahlari M,Sohraby M, Shahlari N, et al.,
Rhodiola rosea from the selection of traditional applications to the novel phytotherapy for
the prevention and treatment of serious diseases. Global Journal of Medical Research.
2015; 15(3): 1-10.
Head E. Oxidative damage and cognitive dysfunction: antioxidant treatment to promote healthy
brain aging. Neurochem Res. 2009; 34(4): 670-8.
Head KA, Kelly GS. Nutrients and botanicals for treatment of stress: adrenal fatigue,
neurotransmitter imbalance, anxiety, and restless sleep. Altern Med Rev. 2009; 14(2): 114-
40.
Heck D. E, Shakarjian M, Kim H. D, Laskin J. D, Vetrano A. M. Mechanisms of oxidant
generation by catalase. Ann N Y Acad Sci. 2010; 1203: 120-5.
Henning R. J, Sawmiller D. R. Vasoactive intestinal peptide: cardiovascular effects.
Cardiovasc Res. 2001; 49(1): 27-37.

Henry J. P, Stephens P. M, Santisteban G. A. A model of psychosocial hypertension showing

reversibility and progression of cardiovascular complications. Circ Res. 1975; 36(1): 156-
64.
Hermes-Lima M. Oxygen in biology and biochemistry: role of free radicals. In: Storey KB,
editors. Functional metabolism: regulation and adaptation. New York: John Wiley & Sons;
2004.
Hernando F, Schoots O, Lolait SJ, Burbach JP. Immunohistochemical localization of the
vasopressin V1b receptor in the rat brain and pituitary gland: anatomical support for its
involvement in the central effects of vasopressin. Endocrinology. 2001; 142(4): 1659-68.
Hill DR, Woodruff GN. Differentiation of central cholecystokin reception binding sites using
the non peptive antagonist MK-329 and L-365, 260. Brain Res. 1990; 526(2): 276-83.
Holliwell B, Gutteridge J. M. The antioxidants of human extracellular fluids. Arch Biochem
Biophys. 1990: 280(1): 1-8.
Holzer P, Reichmann F, Farzi A. Neuropeptide Y, peptide Y. Y and pancreatic polypeptide in
the gut-brain axis. Neuropeptides. 2012; 46(6): 261-74.
Hoyer D, Hannon JP, Martin GR. Molecular, pharmacological and functional diversity of 5-
HT receptors. Pharmacol Biochem Behav. 2002; 71(4): 533-54.
Hwang J. H, Lee I. T, Jeng K. C, Wang M F, Hou R. C, Wu S. M, et al., Spirulina prevents
memory dysfunction, reduced oxidative stress damage and augmentys antioxidant activity
in senescence-accelerated mice. J Nutr Sci Vitaminol (Tokyo). 2011; 57(2): 186-91.
Ibrahim M, A., Ghazy A. M, Masoud H. M. M. Catalase from larvae of the camel tick
Hyalomma dromedarii. Biochem Biophys Rep. 2015; 4: 411-6.
Jaim-Etcheverry G, Zieher LM. Coexistence of monoamines in peripheral adrenergic neurones.
In: Co-Transmission. Editors, Cuello AC. London: Macmillan, 1982.
Jin M. C, Yoo J. M, Sok D. E, Kim M. R. Neuroprotective effect of N-acyl 5-
hydroxytryptamines on glutamate-induced cytotoxicity in HT-22 cells. Neurochem Res.
2014; 39(12): 2440-51.
Jivad N, Rabiei Z. A review study on medicinal plants used in the treatment of learning and
memory impairments. Asian Journal of Tropical Biomedicine. 2014; 4(10): 780-789.
Juarez Olguin H, Calderon Guzman D, Hernandez Garcia E, Barragan Mejia G. The Role of
dopamine and its dysfunction as a Consequence of Oxidative Stress. Oxid Med Cell
Longev. 2016;2016:9730467.
Khalifa AE. Hypericum perforatum as a nootropic drug: enhancement of retrieval memory of
a passive avoidance conditioning paradigm in mice. J Ethnopharmacol. 2001; 76(1): 49-
57.
Kishimoto T, Pearse R. V, Lin C. R, Rosenfeld M. G. A sauvagine/corticotropin- releasing
factor receptor expressed in heart and skeletal muscle. Proc Natl Acad Sci U S A. 1995;
92(4): 1108-12.
Kobayashi N, Machida T, Takahashi T, Takatsu H, Shinkai T, Abe K, et al., Elevation by
oxidative stress and aging of hypothalamic-pituitary-adrenal activity in rats and its
prevention by vitamin E. J Clin Biochem Nutr. 2009; 45(2): 207-13.
Kojo S. Vitamin C: basic metabolism and its function as an index of oxidative stress. Curr Med
Chem. 2004; 11(8): 1041-64.
Koob G. F. Corticotropin-releasing factor, norepinephrine, and stress. Biol Psychiatry. 1999;
46(9): 1167-80.

Korte S. M, Bouws G. A, Bohus B. Central actions of corticotropin- releasing hormone (CRH)

on behavioral, neuroendocrine, and cardiovascular regulation: brain corticoid receptor
involvement. Horm Behav 1993; 27(2):167-83.
Kovacs K. J, Sawchenko P. E. Regulation of stress-induced transcriptional changes in the
hypothalamic neurosecretory neurons. J Mol Neurosci. 1996; 7(2): 125-33.
Kowalski A, Jawibowski K. Effect of different doses of vitamin C on selected hormonal,
biochemical and immunological indices in rats subjected to acute stress. NatSci. 2000; 4:
273-81.
Kumar A, Rinwa P, Kaur G, Machawal L. Stress: Neurobiology, consequences and
management. J Pharm Bioallied Sci. 2013; 5(2): 91-7.
Kumar S. Free Radicals and Antioxidants: Human and Food System. Advances in Applied
Science Research. 2011; 2(1): 129-35.
Kunik M. E, Yudofsky SC, Silver JM, Hales RE. Pharmacologic approach to management of
agitation associated with dementia. J Clin Psychiatry. 1994; 55 (Suppl): 13-7.
Kurutas E. B. The importance of antioxidants which play the role in cellular response against
oxidative/nitrosative stress: current state. Nutr J. 2016;15(1): 71.
Lacaze-Masmonteil T, de Keyzer Y, Luton J. P, Kahn A, Bertagna X. Characterization of
proopiomelanocortin transcripts in human nonpituitary tissues. Proc Natl Acad Sci U S A.
1987; 84(20): 7261-5.
Landis GN, Tower J. Superoxide dismutase evolution and life span regulation. Mech Ageing
Dev. 2005; 126(3): 365-79.
Lavicky J, Dunn A. J. Corticotropin-releasing factor stimulates catecholamine release in
hypothalamus and prefrontal cortex in freely moving rats as assessed by microdialysis. J
Neurochem. 1993; 60(2): 602-12.
Lee S. Y, Soltesz I. Cholecystokinin: a multi-functional molecular switch of neuronal circuits.
Developmental neurobiology 2011; 71(1): 83-91.
Lee Y. W, Klein C. B, Kargacin B, Salnikow K, Kitahara J, Dowjat K, et al., Carcinogenic
nickel silences gene-expression by chromatin condensation and DNA methylation: a new
model for epigenetic carcinogens. Mol Cell Biol. 1995; 15(5): 2547-57.
Leonard BE. The comparative pharmacology of new antidepressants. J Clin Psychiatry. 1993;
54(8 Suppl): 3-15.
Leonard J. F, Bluet-Pajot M. T, Oliver C, Kordon C. Interaction of vasoactive intestinal peptide
(VIP) and growth hormone releasing factor (GRF) with corticotropin releasing factor
(CRF) on corticotropin secretion in vitro. Neuropeptides. 1988; 12(3): 131-3.
Levy A. D, Van de Kar L. D. Endocrine and receptor pharmacology of serotonergic anxiolytics,
antipsychotics and antidepressants. Life Sci. 1992; 51(2): 83-94.
Lezoualc’h F, Engert S, Berning B, Behl C. Corticotropin-releasing hormone mediated
neuroprotection against oxidative stress is associated with the increased release of non-
amyloidogenic amyloid beta precursor protein and with the suppression of nuclear Factor-
kappaB. Mol Endocrinol. 2000; 14(1): 147-59.
Li J, Tian Y, Wu A. Neuropeptide Y receptors: a promising target for cancer imaging and
therapy. Regen Biomater. 2015; 2(3):215-9.
Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and functional foods: Impact
on human health. Pharmacogn Rev. 2010; 4(8): 118-26.
Luine V. N. Estradiol and cognitive function: past, present and future. Horm Behav.
2014;66(4):602-18.

Maggi C. A. The mammalian tachykinin receptors. Gen Pharmacol. 1995; 26(5): 911-44.
Mancia M. Implicit memory and early unrepressed unconscious: their role in the therapeutic
process (how the neurosciences can contribute to psychoanalysis). Int J Psychoanal. 2006;
87(Pt 1): 83-103.
Mates J. M, Perez-Gomez C, Nunez de Castro I. Antioxidant enzymes and human diseases.
Clin Biochem. 1999; 32(8): 595-603.
McCorry L. K. Physiology of the autonomic nervous system. Am J Pharm Educ. 2007;71(4):78.
McEwen BS, Stellar E. Stress and the individual mechanisms leading to disease. Arch Intern
Med. 1993; 153(18): 2093-101.
Meijer O. C, Dekloet E. R. Corticosterone and serotonergic neurotransmission in the
hippocampus-functional implications of central corticosteroid receptor diversity. Crit Rev
Neurobiol. 1998; 12(1-2): 1-20.
Melia K. R, Duman R. S. Involvement of corticotropin-releasing factor in chronic stress
regulation of the brain noradrenergic system. Proc Natl Acad Sci U S A. 1991; 88(19):
8382–6.
Mironczuk-Chodakowska I, Witkowska A. M, Zujko M .E. Endogenous non-enzymatic
antioxidants in the human body. Adv Med Sci. 2018; 63(1): 68-78.
Mora PO, Netto CF, Graeff FG. Role of 5-HT2A and 5-HT2A receptor subtypes in the two
types of fear generated by the elevated T-maze. Pharmacol Biochem Behav. 1997; 58(4):
1051-7.
Morell M, Souza-Moreira L, Gonzalez-Rey E. VIP in neurological diseases: more than a
neuropeptide. Endocr Metab Immune Disord Drug Targets. 2012;12(4):323-32.
Mountjoy K. G, Robbins L. S, Mortrud M. T., Cone R. D. The cloning of a family of genes that
encode the melanocortin receptors. Science. 1992; 257(5074): 1248-51.
Munck A, Guyre P. M, Holbrook N. J. Physiological functions of glucocorticoids in stress and
their relation to pharmacological actions. Endocr Rev. 1984; 5(1): 25-44.
Murase T, Kondo K, Otake K, Oiso Y. Pituitary adenylate cyclase-activating polypeptide
stimulates arginine vasopressin release in conscious rats. Neuroendocrinology. 1993;
57(6): 1092-6.
Murphy D. L, Andrews A. M, Wichems C. H, Li Q, Tohda M, Greenberg B. Brain serotonin
neurotransmission: an overview and update with an emphasis on serotonin subsystem
heterogeneity, multiple receptors, interactions with other neurotransmitter systems, and
consequent implications for understanding the actions of serotonergic drugs. J Clin
Psychiatry. 1998; 59(suppl 15): 4-12.
Nalini K, Aroor A. R, Karanth K. S, Rao A. Centella asiatica fresh leaf aqueous extract on
learning and memory and biogenic amine turnover in albino rats. Fitoterapia. 1992; 63(3):
232-7.
Nestler E. J, Alreja M, Aghajanian GK. Molecular control of locus coeruleus
neurotransmission. Biol Psychiatry. 1999; 46(9): 1131-9.
Nicosia S, Oliva D, Giannattasio G, Spada A. The role of vasoactive intestinal polypeptide
(VIP) as a hypothalamic neurohormone. J Endocrinol Invest. 1983; 6(3): 235-42.
Nussdorfer G. G, Malendowicz L. K. Role of tachykinins in the regulation of the hypothalamo-
pituitary-adrenal axis. Peptides. 1998; 19(5): 949-68.
O’Brien J. T, Schweitzer I, Ames D, Tuckwell V, Mastwyk M. Cortisol suppression by
dexamethasone in the healthy elderly: effects of age, dexamethasone levels, and cognitive
function. Biol Psychiatry. 1994; 36(6): 389-94.

Offen D, Sherki Y, Melamed E, Fridkin M, Brenneman DE, Gozes I. Vasoactive intestinal

peptide (VIP) prevents neurotoxicity in neuronal cultures: relevance to neuroprotection in
Parkinson's disease. Brain Res. 2000; 854(1-2): 257-62.
Otte C, Yassouridis A, Jahn H, Maass P, Stober N, Wiedemann K, et al. Mineralocorticoid
receptor-mediated inhibition of the hypothalamic-pituitary-adrenal axis in aged humans. J
Gerontol A Biol Sci Med Sci. 2003; 58(10): B900-5.
Owens M. J, Nemeroff CB. Physiology and pharmacology of corticotropin-releasing factor.
Pharmacol Rev. 1991; 43(4): 425-73.
Panche A. N, Diwan AD, Chandra S. R. Flavonoids: an overview. J Nutr Sci. 2016; 5: e47.
Pappolla M. A, Chyan Y. J, Omar R. A, Hsiao K, Perry G, Smith MA, et al., Evidence of
oxidative stress and in vivo neurotoxicity of β-amyloid in a transgenic mouse model of
Alzheimer’s disease. Am J Pathol. 1998; 152(4): 871-7.
Parsons LH, Koob GF, Weiss F. Extracellular serotonin is decreased in the nucleus accumbens
during withdrawal from cocaine self-administration. Behav Brain Res. 1996; 73(1-2): 225-
8.
Patak P, Willenberg H. S, Bornstein S. R. Vitamin C is an important cofactor for both adrenal
cortex and adrenal medulla. Endocr Res. 2004; 30(4): 871-5.
Penalva R. G, Flachskamm C, Zimmermann S, Wurst W, Holsboer F, Reul J. M, et al.,
Corticotropin-releasing hormone receptor type 1-deficiency enhances hippocampal
serotonergic neurotransmission: an in vitro microdialysis study in mutant mice.
Neuroscience. 2002; 109(2): 253-66.
Perrin MH, Vale W. W. Corticotropin releasing factor receptors and their ligand family. Ann N
Y Acad Sci. 1999; 885: 312-28.
Petrov T, Krukoff T. L, Jhamandas J. H. Chemically defined collateral projections from the
pons to the central nucleus of the amygdale and hypothalamic paraventricular nucleus in
the rat. Cell Tissue Res. 1994; 277(2): 289-95.
Pevet P, Balemans M. G, Legerstee W. C, Vivien-Roels B. Circadian rhythmicity of the activity
of hydroxyindole-O-methyl transferase (HIOMT) in the formation of melatonin and 5-
methoxytryptophol in the pineal, retina, and harderian gland of the golden hamster. J
Neural Transm. 1980; 49(4): 229-45.
Pham-Huy L. A, He H, Pham-Huy C. Free radicals, antioxidants in disease and health. Int J
Biomed Sci. 2008;4(2):89-96.
Phaniendra A, Jestadi DB, Periyasamy L. Free radicals: properties, sources, targets, and their
implication in various diseases. Indian J Clin Biochem. 2014; 30(1): 11-26.
Potter E, Sutton S, Donaldson C. Chen R, Perrin M, Lewis K, et al., Distribution of
corticotropin releasing factor receptor mRNA expression in the rat brain and pituitary. Proc
Natl Acad Sci U S A. 1994; 91(19): 8777-81.
Quigley B. J, Kowall N. W. Substance P-like immunoreactive neurons are depleted in
Alzheimer's disease cerebral cortex. Neuroscience. 1991; 41(1): 41-60.
Rabadan-Diehl C, Lolait SJ, Aguilera G. Regulation of pituitary vasopressin V1b receptor
mRNA during stress in the rat. J Neuroendocrinol. 1995; 7(12): 903-10.
Rahman T, Hosen I, Towhidul Islam M. M, Shekhar H. U. Oxidative stress and human health.
Advances in Biosciences and Biotechnology. 2012; 3(7): 997-1019.
Rahul A, Kumar A, Singh V, Yadav B, Tiwari R, Chakraborty S, et al., Oxidative stress,
prooxidants and antioxidants: the interplay. Biomed Res Int. 2014; 2014: 761264.

Ramos B. P, Arnsten A. F. Adrenergic pharmacology and cognition: focus on the prefrontal

cortex. Pharmacol Ther. 2006; 113(3): 523-36.
Reichmann F, Holzer P. Neuropeptide Y: A stressful review. Neuropeptides. 2015; 55: 99-109.
Rice-Evans C, Miller NJ, Paganga G. Antioxidant properties of phenolic compounds. Trends
Plant Sci. 1997; 2(4): 152-9.
Rivier C, Vale W. Interaction of corticotropin-releasing factor and arginine vasopressin on
adrenocorticotropin secretion in vivo. Endocrinology. 1983; 113(3): 939-42.
Rivier C, Vale W. Modulation of stress-induced ACTH release by corticotropin- releasing
factor, catecholamines and vasopressin. Nature. 1983a; 305: 325-7.
Rosso M, Munoz M, Berger M. The role of neurokinin-1 receptor in the microenvironment of
inflammation and cancer. Scientific World Journal. 2012; 2012: 747912.
Rotzinger S, Vaccarino FJ. Cholecystokinin receptor subtypes: role in the modulation of
anxiety-related and reward-related behaviours in animal models. J Psychiatry Neurosci.
2003; 28(3):171-81.
Ruffalo R, Waddell J, Yaden E. Postsynaptic alpha adrenergic receptor subtypes differentiated
by yohimbine in tissues from the rat: existence of alpha-2 adrenergic receptors in rat aorta.
J Pharmacol Exp Ther. 1981;217(2):235-40.
Rutland-Brown W, Langlois J. A, Thomas K. E, Xi Y. L. Incidence of traumatic brain injury
in the United States, 2003. J Head Trauma Rehabil. 2006; 21(6): 544-8.
Said S. I, Rosenberg R. N. Vasoactive intestinal polypeptide: abundant immunoreactivity in
neural cell lines and normal nervous tissue. Science. 1976; 192(4242): 907-8.
Said S. I. Vasoactive intestinal peptide. In: Mutt V, editors. Gastrointestinal hormones:
Advances in metabolic disorders. New York: Academic Press, 2013.
Sanson ML, de Keyzer Y, Bertagna X. Proopiomelanocortin, a polypeptide precursor with
multiple functions: from physiology to pathological conditions. Eur J Endocrinol. 2003;
149(2): 79-90.
Saphier D, Welch J. E. Effects of the serotonin1A agonist, 8-hydroxy-2-(di-n-propylamino)
tetralin on neurochemical responses to stress. J Neurochem. 1995; 64(2):767-76.
Sapolsky R. M, Romero L. M, Munck A. U. How do glucocorticoids influence stress
responses? Integrating permissive, suppressive, stimulatory, and preparative actions.
Endocr Rev. 2000; 21(1): 55-89.
Sarma A. D, Mallick A. R, Ghosh AK. Free radical and their role in different clinical
conditions: an overview. Intern. J Pharma Sciences and Res. 2010; 1(3): 185-92.
Sawchenko PE, Imaki T, Potter E, Kovacs K, Imaki J, Vale W. The functional neuroanatomy
of corticotropin-releasing factor. Ciba Found Symp. 1993; 172: 5-21.
Sawchenko P. E. Adrenalectomy-induced enhancement of CRF and vasopressin
immunoreactivity in parvocellular neurosecretory neurons: anatomic, peptide, and steroid
specificity. J Neurosci. 1987; 7(4): 1093-106.
Schacter D. L. Implicit memory: History and current status. Journal of Experimental
Psychology: Learning, Memory, and Cognition. 1987; 13: 501-18.
Schliebs R, Liebman A, Bhattacharya S. K, Kumar A, Ghosal A, Bigl V. Systemic
administration of defined extracts of Withania somnifera (Indian Ginseng) and Shilajit
differentially affects cholinergic but not glutametergic and GABArgic markers in rat brain.
Neurochem Int. 1997; 30(2): 181-90.
Schneiderman N, Ironson G, Siegel S. D. Stress and health: psychological, behavioral, and
biological determinants. Annu Rev Clin Psychol. 2005; 1: 607-8.

Schroeter H, Boyd C, Spencer J. P, Williams R. J, Cadenas E, Rice-Evans C. MAPK signaling

in neurodegeneration: influences of flavonoids and of nitric oxide. Neurobiol Aging. 2002;
23(5): 861-80.
Schwarz M. J, Ackenheil M. The role of substance P in depression: therapeutic implications.
Dialogues Clin Neurosci. 2002; 4(1): 21-9.
Selye H. Syndrome produced by diverse nocuous agents-1936. J Neuropsychaitry Clin
Neurosci. 1998; 10(2): 230-1.
Serres F, Li Q, Garcia F, Raap DK, Battaglia G, Muma NA, et al., Evidence that Gz proteins
couple to hypothalamic 5-HT1A receptors in vitro. J Neurosci. 2000; 20(9): 3095-103.
Shafin N, Zakaria R, Hussain NH, Othman Z. Association of oxidative stress and memory
performance in postmenopausal women receiving estrogen-progestin therapy. Menopause.
2013; 20(6): 661-6.
Shimizu N, Oomura Y, Kai Y. Stress-induced anorexia in rats mediated by serotonergic
mechanisms in the hypothalamus. Physiol Behav. 1989; 46(5): 835-41.
Shohami E, Gati I, Beit-Yannai E, Trembovler V, Kohen R. Closed head injury in the rat
induces whole body oxidative stress: overall reducing antioxidant profile. J Neurotrauma.
1999; 16(5): 365-76.
Simpson ER, Waterman MR. Regulation of the synthesis of steroidogenic enzymes in adrenal
cortical cells by ACTH. Annu Rev Physiol. 1988; 50: 427-40.
Singh H. K, Dhawan BN. Effect of Bacopa monnieri Linn. (Brahmi) on avoidance response in
rat. J Ethnopharmacol. 1982; 5(2): 205-14.
Singh V. B, Onaivi ES, Phan TH, Boadle-Biber MC. The increases in rat cortical and midbrain
tryptophan hydroxylase activity in response to acute or repeated sound stress are blocked
by bilateral lesions to the central nucleus of the amygdala. Brain Res. 1990; 530(1): 49-53.
Smith S. M, Vale WW. The role of the hypothalamic-pituitary-adrenal axis in neuroendocrine
responses to stress. Dialogues Clin Neurosci. 2006; 8(4): 383-95.
Soderpalm B, Andersson L, Carlsson M, Modigh K, Eriksson E. Serotonergic influence on the
growth hormone response to clonidine in rat. J Neural Transm. 1987; 69(1-2): 105-14.
Spina MG, Basso AM, Zorrilla EP, Heyser CJ, Rivier J, Vale W, et al., Behavioral effects of
central administration of the novel CRF antagonist astress in in rats.
Neuropsychopharmacology. 2000; 22(3): 230-9.
Squire L. R, Dede A. J. Conscious and unconscious memory systems. Cold Spring Harb
Perspect Biol. 2015; 7(3): a021667.
Squire L. R, Clark RE, Knowlton BJ. Retrograde amnesia. Hippocampus. 2001; 11(1): 50-5.
Squire L. R, Dede A. J. Conscious and unconscious memory systems. Cold Spring Harb
Perspect Biol. 2015; 7(3): a021667.
Stadtman E. R. Protein oxidation and aging. Science. 1992; 257(5074): 1220-5.
Steinhoff M. S, von Mentzer B, Geppetti P, Pothoulakis C, Bunnett N. W. Tachykinins and
their receptors: contributions to physiological control and the mechanisms of disease.
Physiol Rev. 2014, 94(1), 265-301.
Strand F. L. Tachykinins: substance P, neurokinin A, neurokinin B. In: Stevens C. F, editors.
Neuropeptides: regulators of physiological processes. Cambridge, MA: The MIT Press:
1999.
Stratakis CA, Chrousos GP. Neuroendocrinology and pathophysiology of the stress system.
Ann N Y Acad Sci. 1995; 771:1-18.

Sutton R. E, Koob G. F, Le Moal M, Rivier J, Vale W. Corticotropin releasing factor produces

behavioural activation in rats. Nature. 1982; 297(5864): 331-3.
Swanson L. W, Sawchenko P. E. Hypothalamic integration: organization of the paraventricular
and supraoptic nuclei. Annu Rev Neurosci. 1983; 6: 269-324.
Tang V. M. Glucocorticoid effects on oxidative stress and mitochondrial dysfunction. The
University of British Columbia. 2012.
Taylor J. E. Neuromediator binding to receptors in the rat brain-the effect of chronic
administration of Ginkgo biloba extract. Presse Med. 1986; 15(31): 491-3.
Traber M. G, Stevens J. F. Vitamins C and E: beneficial effects from a mechanistic perspective.
Free Radic Biol Med. 2011; 51(5): 1000-13.
Tripathi, K. D. Essentials of Medical Pharmacology, New Delhi: Jaypee Brothers Medical
Publishers (P) Ltd.; 2013.
Turner RJ, Vink R. The Role of Substance P in Ischaemic Brain Injury. Brain Sci. 2013; 3(1):
123-42.
Vale W, Spie J, Rivier C, Rivier J. Characterization of a 41-residue ovine hypothalamic peptide
that stimulates secretion of corticotropin and beta-endorphin. Science. 1981; 213(4514):
1394-7.
Valentino R. J, Foote SL, Aston-Jones G. Corticotropin-releasing factor activates noradrenergic
neurons of the locus coeruleus. Brain Res. 1983; 270(2): 363-7.
Valko M, Rhodes C. J, Moncola J, Izakovic M, Mazura M. Free radicals, metals and
antioxidants in oxidative stress-induced cancer. Chem Biol Interact. 2006; 160(1): 1-40.
Van de Kar L. D, Blair M. L. Forebrain pathways mediating stress induced hormone secretion.
Front Neuroendocrinol. 1999; 20(1): 1-48.
Van de Kar L. D. 5-HT receptors involved in the regulation of hormone secretion. In:
Baumgarten HG, Gothert M, editors. Serotoninergic neurons and 5-HT receptors in the
CNS. Handbook of experimental pharmacology. Berlin: Springer; 2000.
Van Pett K, Viau V, Bittencourt JC, Chan RK, Li HY, Arias C, et al., Distribution of mRNAs
encoding CRF receptors in brain and pituitary of rat and mouse. J Comp Neurol. 2000;
428(2): 191-212.
Veith RC, Lewis N, Langohr JI, Murburg MM, Ashleigh EA, Castillo S, et al., Effect of
desipramine on cerebrospinal fluid concentrations of corticotropin-releasing factor in
human subjects. Psychiatry Res. 1993; 46(1): 1-8.
Verbalis J. G. Osmotic inhibition of neurohypophysial secretion. Ann N Y Acad Sci. 1993; 689:
146-60.
Vertuani S, Angusti A, Manfredini S. The antioxidants and proantioxidants network: an
overview. Curr Pharm Des. 2004; 10(14): 1677-94.
Vizi E. S, Pasztor E. Release of acetylcholine from isolated human cortical slices: inhibitory
effect of norepinephrine and phenytoin. Exp Neurol. 1981; 73(1): 144-53.
Vizi E. S. Presynaptic modulation of neurochemical transmission. Prog Neurobiol. 1979; 12(3-
4): 181-290.
Vohora SB, Shah SA, Dandiya PC. Central nervous system studies on an ethanol extract of
Acorus calamus rhizomes. J Ethnopharmacol. 1990; 28(1): 53-62.
Voss J. L, Paller K. A. Brain substrates of implicit and explicit memory: the importance of
concurrently acquired neural signals of both memory types. Neuropsychologia. 2008;
46(13): 3021-9.
Well J. Balancing Stress for Healthy Living. Understanding Stress. 2010; 1-10.

Westendorf J. M, Schonbrunn A. Peptide specificity for stimulation of corticotropin secretion:

activation of overlapping pathways by the vasoactive intestinal peptide family and
corticotropin-releasing factor. Endocrinology. 1985; 116(6): 2528-35.
Whitnall M. H. Regulation of the hypothalamic corticotropin-releasing hormone
neurosecretory system. Prog Neurobiol. 1993; 40(5): 573-629.
Wilkinson CW, Peskind E. R, Raskind M. A. Decreased hypothalamic-pituitary-adrenal axis
sensitivity to cortisol feedback inhibition in human aging. Neuroendocrinology. 1997;
65(1): 79-90.
Wu D, Katz A, Lee C. H, Simon M. I. Activation of phospholipase C by alpha 1-adrenergic
receptors is mediated by the alpha subunits of Gq family. J Biol Chem.
1992;267(36):25798-802.
Xu D, Xie X, Li S, Zhang J, Hou L. Function of substance P and substance P receptor in
exercise. International Journal of Applied Physics and Mathematics 2013; 3(1): 78-81.
Yamamoto B. K, Novotney S. Regulation of extracellular dopamine by the norepinephrine
transporter. J Neurochem. 1998; 71(1): 274-80.
Yoshikawa T, Naito Y. What is Oxidative Stress? JMAJ. 2002; 45(7): 271-6.
Young E. A. Sex differences in response to exogenous corticosterone: a rat model of
hypercortisolemia. Mol Psychiatry. 1996; 1(4): 313-9.
Yu Z. F, Kong L. D, Chen Y. Antidepressant activity of aqueous extract of Curcuma longa in
mice. J Ethnopharmacol. 2002; 83(1-2): 161-5.
Zukowska-Grojec Z, Vaz A. C. Role of neuropeptide Y (NPY) in cardiovascular responses to
stress. Synapse. 1988; 2(3):293-8.

Chapter 7
NEUROPROTECTIVE STRATEGIES FOR OXIDATIVE

STRESS-MEDIATED ALZHEIMER’S DISEASE
Manoj Govindarajulu1, Sindhu Ramesh1, Ayaka Fujihashi1,

Mansour Alturki1,3, Tyler Lynd1, Gayatri Devi Donepudi2,
Vishnu Suppiramaniam1, Timothy Moore1
and Muralikrishnan Dhanasekaran1,*
Auburn University, Auburn, Alabama, US
1
J. J. M. Medical College, Davanagere, India

2
3
Abdulrahman Bin Faisal University, Damman, Saudi Arabia
ABSTRACT
Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases that
cause dementia in the elderly. Growing evidence and research in AD have recently
demonstrated convincing evidence on the role of oxidative processes in the pathogenesis
of the disease. The complex sequences of molecular changes demonstrate that oxidative
stress precedes the appearance of hallmark pathologies of the disease, namely
neurofibrillary tangles and senile plaques. Mitochondrial dysfunction and/or an abnormal
buildup of transition metals generate reactive oxygen species (ROS), yet the mechanisms
that produce free radicals and lead to redox imbalances remain elusive. No definite initial
source of oxidative stress has been identified in AD; thus, further investigation into the role
that oxidative stress mechanisms seem to play in the pathogenesis of AD may lead to novel
clinical interventions. The development of drugs such as antioxidants, which breaks the
vicious cycles of oxidative stress and neurodegeneration, offer new opportunities and may
be a possible therapeutic value in AD. This chapter discusses the role of mitochondrial
dysfunction and oxidative stress in the pathogenesis of AD and the role of antioxidants to
lessen oxidative stress.
*
Corresponding Author’s Email: dhanamu@auburn.edu.

168 Manoj Govindarajulu, Sindhu Ramesh, Ayaka Fujihashi et al.
1. INTRODUCTION
AD is one of the most prevalent dementia-inducing neurodegenerative diseases in the
elderly population. Dementia is characterized by a progressive and gradual decline in cognitive
functions and occupational ability, often resulting in death within a few years after diagnosis
(Querfurth and LaFerla, 2010). AD predominantly occurs sporadically in the elderly population
and has a complex etiology due to varying environmental conditions and genetic features of
individuals (Kalaria et al., 2008). The pathological hallmarks of AD brains include the presence
of extracellular senile Aβ plaques, neurofibrillary tangles (NFTs), and neuronal death. Beta-
amyloid peptides (Aβ) make up senile plaques, whereas NFTs are composed of aggregates of
paired helical filaments (PHFs) structures and are characterized by an insoluble and
phosphorylated microtubule-associated protein tau (Goedert and Spillantini, 2006). Mounting
evidence suggests that oxidative stress and mitochondrial dysfunction are involved in the
pathogenesis of AD in addition to the conventional pathologies of Aβ and NFT (Praticò, 2008).
Several reports indicate that the products of protein oxidation (protein carbonyls and 3-
nitrotyrosine) (Butterfield and Kanski, 2001), markers of oxidative damage to DNA and RNA
(8-hydroxydeoxyguanosine [8-OHdG] and 8-hydroxyguanosine) (Gabbita et al., 1998;
Nunomura et al., 1999), and products of lipid peroxidation (malondialdehyde [MDA], 4-
hydroxynonenal, and F2-isoprostanes) are elevated in AD brains (Praticò and Sung, 2004). In
addition, reduced activity or expression of antioxidant enzymes such as SOD and CAT have
also been observed in both central and peripheral nervous system tissues of AD patients
(Marcus et al., 1998; Padurariu et al., 2010). Patients with AD or mild cognitive impairment
(MCI)—conditions more localized to synapses—exhibited increased oxidative damage to
lipids and proteins and reduced glutathione activity. Additionally, antioxidant enzyme activities
correlated with the disease severity, signifying an involvement of oxidative stress in AD-related
synaptic loss (Ansari and Scheff, 2010). Importantly, previous studies have mentioned that
oxidative stress damage precedes the onset of AD, and elevated levels of oxidative stress may
be an intermediate state between normal aging and dementia present in MCI. These results
suggest that oxidative stress may be one of the most crucial and earliest changes that occur
during the initiation, development, and progression of AD (Huang et al., 2016).
Interestingly, the imbalances in redox states induce oxidative stress, leading to an excess
generation of ROS. Therefore, excess production of ROS as a result of oxidative stress can be
an important mediator of damage to cell components, structures, and, consequently, of various
disease states and aging. Excess ROS levels thus may have a lethal effect. This chapter
elaborates on the various markers of oxidative stress, mitochondrial dysfunction, the
mechanisms by which oxidative stress promotes Aβ and tau-mediated neurotoxicity, and how
each one contributes to the pathophysiology of AD. Furthermore, the roles of endogenous and
exogenous antioxidants in mitigating the effects of oxidative stress are discussed with a brief
note on why some of the commercially available antioxidants have failed to achieve therapeutic
efficacy in clinical trials.

Neuroprotective Strategies for Oxidative Stress-Mediated Alzheimer’s Disease 169
2. REACTIVE OXYGEN SPECIES

AND REACTIVE NITROGEN SPECIES
Free radicals, or species with one or more unpaired electrons in their outermost (valence)
shell, are natural byproducts of cellular metabolism. Due to the odd number of electrons in the
outer orbit of the atom or molecule, they are incredibly unstable and reactive. In order to achieve
stability, free radicals will obtain electrons from nearby compounds. The compound that has
lost its electron will then act as a free radical to obtain an electron to attack another molecule,
creating a chain reaction that eventually damages the cell (Halliwell, 1993). Two subtypes of
free radicals include ROS and reactive nitrogen species (RNS) (Pham-Huy et al., 2008). ROS
and RNS are a “double-edged sword,” per se, for they are beneficial to organisms in low to
moderate levels, playing crucial roles in certain physiological mechanisms as immunity, cell
signaling, cell cycle, and redox regulation (Nordberg and Arnér, 2001; Valko et al., 2007).
However, decreased activities of natural enzymatic and non-enzymatic antioxidants, in
conjunction with excess levels of ROS/RNS, can result in oxidative stress and nitrosative stress,
respectively. Both oxidative and nitrosative stress can potentially damage important
biomolecules such as lipids, proteins, and DNA and are implicated in cataracts as well as a
variety of neurodegenerative diseases, cardiovascular diseases, respiratory diseases, and
autoimmune diseases (diabetes mellitus and rheumatoid arthritis).
ROS occur in many forms, including the oxygen molecule, which consists of two unpaired
electrons and is hence referred to as biradical, superoxide, oxygen radical, hydroxyl, alkoxy
radical, peroxyl radical, nitric oxide, and nitrogen dioxide (Halliwell, 2015). These compounds
all contain an unpaired electron that will donate the lone electron or obtain another to achieve
stability, making the compounds highly reactive. The non-radical species include hydrogen
peroxide (H2O2), hypochlorous acid (HOCl), hypobromous acid (HOBr), ozone (O3), singlet
oxygen (1O2), nitrous acid (HNO2), nitrosyl cation (NO+), nitroxyl anion (NO-), dinitrogen
trioxide (N2O3), dinitrogen tetraoxide (N2O4), nitronium (nitryl) cation (NO2+), organic
peroxides (ROOH), aldehydes (HCOR) and peroxynitrite (ONOOH)(Kohen and Nyska, 2002).
These non-radical species are not free radicals but can easily lead to free radical reactions in
living organisms (Genestra, 2007).
The ROS/RNS can be produced from either endogenous or exogenous sources. Cellular
organelles depicted in Figure 1, are endogenous sources of ROS. Exogenous sources of free
radicals include radiation exposure (UV light, X-rays etc.), tobacco smoking, industrial
solvents, pesticides, inorganic particles, transition metals (Cd, Hg, Pb, As), heavy metals (Fe,
Cu, Co, Cr), and drugs like halothane, anticancer drugs, carbon tetrachloride etc. An imbalance
between ROS/RNS levels and the antioxidant defense system, wherein the former are produced
in excess concentrations, predispose the body to oxidative/nitrosative stress. These ROS/RNS
can further damage key biological components including nucleic acids, lipids, and proteins
(Dröge, 2002) and lead to several cellular and molecular alterations as illustrated in Figure 2.
The impact of ROS/RNS in the development of pathologies of AD is described in the following
sections.

Figure 1. Endogenous sources of free radicals. NOS, Nitric oxide synthases; cyp450, Cytochrome
P450; ROS, Reactive oxygen species.
3. OXIDATIVE STRESS IN ALZHEIMER’S DISEASE
3.1. Oxidative Stress and Amyloid Beta
Abnormal synthesis of Aβ occurs through sequential cleavage of amyloid precursor protein

(APP) by two membrane-bound proteases: β-secretase (β-site APP cleaving enzyme-BACE1)
and γ-secretase (a multiprotein complex consisting of presenilin [PS], nicastrin [NCT], anterior
pharynx-defective one [APH-1], and presenilin foil macromolecule a pair of [PEN-2]).
Contrarily, cleavage of APP by α-secretase inhibits Aβ generation (Esler and Wolfe, 2001).
Increase in Aβ synthesis or decrease in its clearance leads to Aβ accumulation, which further
activates cell signaling pathways that result in neurodegeneration and cognitive impairment
(Hsiao et al., 1996; Yankner and Lu, 2009). Several lines of evidence indicate that oxidative
stress promotes Aβ production. For instance, dysregulation of the antioxidant defense system
leads to oxidative stress and increased Aβ accumulation in transgenic mice overexpressing APP
mutant (Li et al., 2004). This was substantiated by another study which showed that in
transgenic mice overexpressing APP mutant, overexpression of manganese superoxide
dismutase (MnSOD) decreased protein oxidation and reduced Aβ levels in the brain while
increasing antioxidant defense capability (Dumont et al., 2009).
Various studies investigating the mechanisms by which oxidative stress enhances Aβ
production have revealed that oxidative stress promotes the expression and activity of β- and
γ- secretases while suppressing the activity of α-secretase (Quiroz-Baez et al., 2009; Tamagno
et al., 2002). Activation of the c-Jun N-terminal kinase (JNK) pathway has been found to play
a role in increasing β- and γ-secretase induced by oxidative stress (Shen et al., 2008).
Additionally, the promoter and untranslated 5’ region of the BACE gene contains binding sites

for redox-sensitive activator protein (AP1) and nuclear factor (NF)-kappa B. Activation of these
transcription factors by oxidative stress may consequently increase expression of BACE
(Sambamurti et al., 2004). Activation of the JNK pathway and increased BACE1 and PS1
expression/activity have been found in AD brains, indicating that increased oxidative stress
initiates a cascade of events including JNK, which increases BACE1 and PS1 expression,
eventually leading to Aβ production.
Figure 2. Effects of reactive oxygen and nitrosative species on key biological components.
On the other hand, several researchers have hypothesized that Aβ may have protective
effects against oxidative stress (Nunomura et al., 2006). For instance, picomolar or nanomolar
levels of Aβ has been shown to be neurotrophic (Luo et al., 1996; Plant et al., 2003). Inhibition
of lipoprotein oxidation in CSF and plasma (Kontush et al., 2001) and increased hippocampal
long-term potentiation were noted at physiological concentrations of Aβ, whereas high
concentrations of Aβ caused toxic effects (Puzzo et al., 2008). In addition to concentration,
aggregation states of Aβ and Aβ size-form specific also contributed to the dualistic effects of
Aβ (Plant et al., 2003; Zou et al., 2002). Amyloid beta, in turn, promotes oxidative stress, as

demonstrated from in vitro studies that revealed increased H2O2 and lipid peroxide levels as a
result of Aβ treatment (Behl et al., 1994; Mattson, 1997). In vivo studies utilizing transgenic
mice models of AD have shown increased H2O2, NO, and oxidative changes of proteins and
lipids that correlated with Aβ deposition, indicating that Aβ promotes oxidative stress
(Matsuoka et al., 2001; Smith et al., 1998). A vicious cycle continues, eventually leading to
neurodegeneration. Further elucidation of the pathological and physiological roles of Aβ may
lead to more effective strategies for AD interventions.
3.2. Oxidative Stress and Tau Pathology
Hyperphosphorylated tau (pTau) protein, another hallmark of AD, results in aggregation

of NFTs, leading to neurodegeneration and cognitive decline (Rodríguez-Martín et al., 2013).
Hyperphosphorylation impairs tau protein from binding with tubulin and inhibits its capacity
to promote microtubule assembly, causing f filaments to form as a result of self-aggregation
(Iqbal et al., 2009). The mechanisms by which abnormal phosphorylation of tau occurs include
several protein kinases and protein phosphatases, such as glycogen synthase kinase-3 beta
(GSK-3β), cyclin-dependent kinase 5 (CDK-5), mitogen-activated protein kinase (MAPK)
(Goedert et al., 1997; Zhu et al., 2000), calcium calmodulin kinase, and protein kinase C
(Billingsley and Kincaid, 1997), as depicted in Figure 3. It has been reported that Aβ
accumulation occurs before the tau pathology responsible for a cascade of molecular events
leading to tau hyperphosphorylation (Gotz et al., 2001; Zheng et al., 2002).
Figure 3. Mechanisms of abnormal phosphorylation of tau. GSK-3β, Glycogen synthase kinase 3β; p38
MAPK; p38 mitogen-activated protein kinase.
Cells overexpressing tau show increased susceptibility to oxidative stress— perhaps due to
depletion of peroxisomes—supporting the link between oxidative stress and pTau (Stamer et
al., 2002). Similarly, reduction of the genes thioredoxin reductase (TrxR) or mitochondrial
SOD2 enhanced tau-induced neurodegenerative histological abnormalities and neuronal
apoptosis in the Drosophila model of human tauopathy. Treatment with vitamin E attenuated
tau-induced neuronal cell death (Dias-Santagata et al., 2007).
P301L tau transgenic mice (carrying human tau gene with P301S or P301L mutations)
exhibited mitochondrial dysfunction with reduced NADH-ubiquinone oxidoreductase activity.

This was associated with increased ROS production and impaired mitochondrial respiration
and ATP synthesis (David et al., 2005). Furthermore, oxidative stress has been suggested as
playing a significant role in tau pathology as evidenced by increased protein carbonyl levels in
cortex mitochondria and alterations in the activity and content of mitochondrial enzymes
involved in ROS formation and energy metabolism (Dumont et al., 2011). Consistently,
administration of coenzyme Q10 to P301S mice significantly increased complex I activity and
reduced lipid peroxidation (Elipenahli et al., 2012). Proteomic analysis of triple transgenic mice
brain samples showed dysregulated mitochondrial proteins mainly associated with complexes
I and IV (Rhein et al., 2009). The effects of Aβ and tau on mitochondrial function were found
to be synergistic and age-associated, resulting in a decrease of mitochondrial respiratory
capacity and reduction of ATP synthesis, synaptic loss, and neuronal death. Oxidation of fatty
acids has been found to play a role in the polymerization of tau. Because fatty acid oxidation is
elevated in the brains of AD patients, this may be a possible link between oxidative stress and
neurofibrillary tangles characteristic in AD (Gamblin et al., 2000). In Tg2576 AD transgenic
mice, ROS has been suggested as playing a critical role in the hyperphosphorylation of tau as
evidenced by a deficiency in mitochondrial SOD2 (Melov et al., 2007) or reduction of
cytoplasmic SOD1-induced tau phosphorylation. p38 MAPK, which can be activated by
oxidative stress, is capable of phosphorylating tau protein in vitro (Goedert et al., 1997). In
hippocampal and cortical brain regions of AD patients, activated p38 is found exclusively
localized to NFT and coimmunoprecipitated with PHF-tau, suggesting that it might be involved
in the phosphorylation of tau in vivo (Zhu et al., 2000). Thus, p38 may be a candidate for the
link between the phosphorylation of tau with increased oxidative stress in AD.
3.3. Mitochondrial Dysfunction and Oxidative Stress in Alzheimer’s Disease
Mitochondria are unique organelles with essential cell functions such as ATP synthesis,
calcium homeostasis, and cell survival and death. Mitochondria are majors source of ROS
production in the cell, and thus, is vulnerable to oxidative stress (Tan et al., 1998).
Morphometric analysis of biopsies from AD brains exhibited reduced energy metabolism;
however, in the cytoplasm and vacuoles associated with lipofuscin—a lysosome suggested as
the site of mitochondrial autophagy—mitochondrial DNA and protein were increased (Hirai et
al., 2001; Zhu et al., 2006). These mitochondrial abnormalities were accompanied by oxidative
damage discernable by 8-hydroxyguanosine and nitrotyrosine, demonstrating damage to the
mitochondria during the progression of AD (Hirai et al., 2001). Correspondingly, the cortex of
AD brains exhibited decrease in mitochondrial cytochrome oxidase (complex IV) activity
(Mutisya et al., 1994).
Interestingly, in brains of AD patients, transgenic mice, and neuroblastoma cells expressing
human mutant APP, Aβ was found to be localized to the mitochondria (Caspersen et al., 2005;
Manczak et al., 2006). This Aβ in mitochondria correlated with impaired mitochondrial
metabolism and increased mitochondrial ROS production, as well as disrupted lipid polarity
and protein mobility and inhibition of key enzymes of the mitochondria respiratory chain,
leading to increased mitochondrial membrane permeability, cytochrome c release, caspase
activation, and apoptosis (Casley et al., 2002; Rodrigues et al., 2001). In double homozygous
knock-in mice models expressing APP and PS-1 mutants, MnSOD, a chief antioxidant that
protects the mitochondria from superoxide, was found to be nitrated and inactivated

(Anantharaman et al., 2006). Carrier proteins on the inner mitochondrial membrane are known
as uncoupling proteins (UCPs) (Rousset et al., 2004). UCP2 and UCP3, when stimulated by
ROS or products of lipid peroxidation, can diminish proton motive force and reduce
mitochondrial membrane potential and ATP production, causing mitochondria uncoupling and
a decrease in ROS generation (ECHTAY, 2007).
Therefore, the expression and stimulation of UCPs is a defense mechanism in response to
oxidative stress. The expression of UCP2, 4, and 5 is significantly decreased in AD brains
because this mechanism appears dysfunctional (de la Monte and Wands, 2006; Wu et al., 2009).
Moreover, in cells overexpressing APP, superoxide treatment was found to diminish the UCP2-
and UCP4-dependent upregulation of mitochondrial free calcium, demonstrating that Aβ
accumulation may be associated with dysfunctional mitochondria and increased cell sensitivity
to the loss of calcium homeostasis (Wu et al., 2009). Due to the presence of mitochondria-
associated Aβ, increase in H2O2, and decrease in cytochrome oxidase activity prior to the
appearance of Aβ plaques, mitochondrial dysfunction may be an early feature of AD (Wang et
al., 2009). Thus, early interventions focused on the mitochondria may delay the onset of
progression of AD (Caspersen et al., 2005; Manczak et al., 2006).
3.4. Compromised Oxidative Stress Defense Mechanisms in

Alzheimer’s Brain
The antioxidant defense system is important in combatting the various forms of oxidative
stress. Some of the major antioxidant defense systems include glutathione, heat shock
pathways, heme oxygenase, superoxide peroxidases, and thioredoxin (Thx) systems.
Glutathione (γ-l-glutamyl-l-cysteinyl-glycine) is an important low molecular weight
antioxidant synthesized in cells. It exists in a dynamic pool of thiol-reduced (GSH) and
disulfide-oxidized states. GSH can react with free radicals either independently or in the
reaction catalyzed by glutathione peroxidase (GPx) to form glutathione disulfide (GSSG),
which can be converted back to the reduced state by glutathione reductase (GR). Alternatively,
glutathione S-transferases (GST) can catalyze a reaction between GSH and nucleophilic
compounds that react with thiols in proteins. The GSH/GSSG ratio is considered to be an
indicator of cellular redox potential and oxidative stress and has been also analyzed to assess
levels of oxidative stress in AD (Lu, 2013).
Brain samples from AD patients have revealed a decrease in GSH levels with an associated
increase in GSSG levels. This decreased GSH/GSSH ratio has been demonstrated in post-
mitochondrial supernatant (PMS) as well as mitochondrial and synaptosomal fractions obtained
from frontal cortices of MCI and AD patients when compared to controls (Karelson et al.,
2001). Additionally, in vivo GSH analysis utilizing proton magnetic resonance spectroscopy
have shown reduced GSH levels in the frontal cortex and hippocampus of AD patients, which
correlated with the extent of cognitive impairments when assessed with Mini-Mental State
Examination (MMSE) and clinical dementia rate (CDR) scores (Mandal et al., 2015).
Decreased activity of GR, GPx, and GST enzymes in mitochondrial and synaptosomal fractions
was noted in AD patients when compared to control individuals. Interestingly, the magnitude
of the changes in the glutathione redox cycle seems to correspond with disease severity (Mandal
et al., 2015).

SOD and CAT are important antioxidants that catalyze toxic superoxide radicals to oxygen
and H2O2, which is further converted into water and oxygen. Patients with MCI and those
having severe AD both showed reduced activity of these enzymes, indicating compromised
antioxidant defense mechanisms (Ansari and Scheff, 2010). Reduced mRNA expression and
activity of methionine sulfoxide reductase (MsrA), an enzyme that converts methionine
sulfoxide to methionine, has been detected in AD-affected brain regions (Gabbita et al., 2002).
In addition, reduced levels of Thx, a potent ROS scavenger, have been observed in the
amygdala, hippocampus, and superior and middle temporal gyri of AD brains (Lovell et al.,
2000). Reduced transcription of detoxifying genes and diminished expression of antioxidant
enzymes are in line with decreased levels of nuclear factor-like 2 (Nrf2), a transcription factor
activated upon exposure to oxidative stress (Lee and Johnson, 2004), and in the nucleus of
hippocampal neurons in human AD brains (Ramsey et al., 2007).
In contrast, increased protein levels and/or activity of enzymes, which are induced as
protective mechanisms against oxidative stress, have been reported. Elevated thioredoxin
reductase activity and increased HO-1 protein expression have been reported in AD patients
when compared to controls (Lovell et al., 2000). Chronic upregulation of HO-1 may exacerbate
the degenerative process by promoting pathological iron deposition and oxidative
mitochondrial damage (Schipper and Song, 2015). Interestingly, detailed immunofluorescence
analysis of the HO-1 localization has revealed that the expression of this enzyme is elevated
both in neurons and glial fibrillary acidic protein- (GFAP-) positive astrocytes and that senile
plaques and neurofibrillary tangles are immunopositive for HO-1 (Schipper et al., 1995).
Importantly, the percentage of double GFAP and HO-1-positive astrocytes is much higher in
the temporal cortex and hippocampus of MCI and AD patients compared to controls, shows a
negative correlation with global cognitive performance, and exhibits a positive correlation with
AD pathology, with the latter apparent only in the temporal cortex (Schipper et al., 2006). The
induction of the expression of the protective enzymes has been proposed to be a result of
chronic exposure to high levels of oxidative stress in AD. Overall, the described studies indicate
that AD is associated with unsuccessful cellular attempts to induce oxidative stress defense
mechanisms and to compensate for the oxidative damage.
4. STRATEGIES TO COMBAT OXIDATIVE STRESS

Endogenous defense mechanisms include both enzymatic and non-enzymatic antioxidant

systems that protect against free radical-induced cellular damage. The three primary
endogenous enzymes that directly eliminate active oxygen species (superoxide radical and
H2O2) include SOD, CAT, and GPx. Secondary enzymes, or enzymes that decrease peroxide
levels and maintain the concentrations of metabolic intermediates like glutathione (GSH) and
NADPH, include glutathione reductase, glucose-6-phosphate dehydrogenase, and cytosolic
GST. The secondary enzymes aid in optimum functioning of the primary antioxidant enzymes
(Singh and Saini, 2003; Vendemiale et al., 1999).

Table 1. Endogenous antioxidants and their mechanisms
Antioxidant Major Mechanisms and Features References

Catalase (CAT)  Porphyrin-containing enzyme located mainly in Casado et al., 1998;
-Enzymatic peroxisomes Amir Aslani and
Ghobadi, 2016
Glutathione  Two forms: selenium-dependent and selenium independent Dickinson and
peroxidase (GPx)  Catalyzes reduction of H2O2 or organic peroxide to water Forman, 2002;
-Enzymatic or alcohol (in presence of GSH) Forsberg et al.,
 Four isoforms: cytosolic and mitochondrial (GPx1), 2001; Jan et al.,
cytosolic (GPx2), extracellular (GPx3) and phospholipid 2015
peroxide (GPx4)
Superoxide  Convert reactive superoxide radicals through dismutation Noor et al., 2002;
dismutase (SOD) reaction into H2O2 and O2 Weisiger and
-Enzymatic  Four isoenzymes SOD1, SOD2, SOD3, and SOD4. SOD1 Fridovich, 1973
is the master regulator of oxidative stress
Bilirubin  Potent in vitro scavengers of free radicals Scavenge NO Stocker et al., 1987;
-Non-enzymatic and NO‐related species Dore and Snyder,
 HO‐1 is a crucial inducible antioxidant system engaged to 1999; Kaur et al.,
assist against oxidative injury and other forms of cellular 2003
stress
Coenzyme Q  Directly involved in energy transduction and ATP Gille and Nohl,
-Non-enzymatic production 2000; Rohr-Udilova
 Functions as redox carrier, antioxidant, activator of et al., 2008
uncoupling proteins and influences the permeability
transition pore in the inner mitochondrial membrane
Ferritin  Iron-binding protein Nunomura et al.,
-Non-enzymatic  Limits Fe (II) available to participate in the generation of 2007; Balla et al.,
ROS 1992
Glutathione  Reduced form (GSH) is biologically active Lillig et al., 2005;
(GSH)  Antioxidant defense against ROS/RNS Ardite et al., 2000;
-Non-enzymatic  Part of detoxication enzymes like GSH peroxidases and Liu and Ames,
GSH-S-transferases 2005
 Also, increase the cofactors of mitochondrial enzymes
α-Lipoic acid  Modulates redox potential between different subcellular Liu, 2008; Gorąca
(LA) compartments and extracellularly et al., 2011; Packer
-Non-enzymatic  Acts through neutralizing ROS and by chelating transition et al., 1997;
metal ions Arivazhagan et al.,
 Counteracts NF-kB activation 2001
Uric acid  Major antioxidant in human plasma (24% of total Miller and Rice-
-Non-enzymatic antioxidant activity). Antioxidant mainly in a hydrophilic Evans, 1996; Ames
environment. et al., 1981
 Scavenge oxygen radicals and protect erythrocyte
membrane from lipid oxidation
 High levels of uric acid protect cardiac, vascular and
neuronal cells from oxidative injury.
 Acute elevations provide antioxidant protection, but
chronic elevations are associated with cardiovascular (CV)
risk

The endogenous non-enzymatic antioxidants in the body are glutathione, NADPH, uric
acid, bilirubin, the iron-binding proteins transferrin and ferritin, α-lipoic acid, melatonin, and
reduced CoQ10, all of which are involved in protecting the body from ROS and their respective
byproducts that are produced during normal cellular metabolism. GSH is one of the most
important components that play a major role in xenobiotic metabolism by preventing oxidative
damage caused by ROS (lipid peroxides like hydroxynonenal). GSH can become depleted
during xenobiotic neutralization, resulting in reduced availability to serve as an antioxidant.
Additionally, GSH is important in maintaining ascorbate (vitamin C) and α-tocopherol (vitamin
E) in their reduced form to prevent damage by ROS and function as antioxidants (Anderson et
al., 1996; Meister, 1994; Sies and Stahl, 1995). Both enzymatic and non-enzymatic antioxidant
systems and their major mechanisms are summarized in Table 1.
4.2. Exogenous Antioxidants
Exogenous antioxidants can be obtained through the diet. For instance, vitamin E, vitamin
C, and β-carotene are dietary chain-breaking antioxidants, which decrease neuronal cell damage
induced by free radicals and help inhibit the development of dementia.
4.2.1. Vitamin E
Vitamin E is a fat-soluble compound and an essential micronutrient for humans. Natural
vitamin E can be divided into two main groups—tocotrienols (TCTs) and tocopherols (TCPs)—
both of which are fat-soluble and each with four analogs such as α, β, γ, and δ. Vitamin E
maintains the integrity of cell membranes and is a major lipophilic antioxidant in the brain
(Chang et al., 2018), thereby protecting lipids from oxidative stress. Vitamin E is important for
maintaining neuronal homeostasis, and vitamin E deficiency is known to cause degeneration of
neurons. Studies have shown decreased levels of vitamin E in CSF (Jiménez-Jiménez et al.,
1997) and blood of AD patients compared to controls (Mullan et al., 2017; Sinclair et al., 1998;
ZAMAN et al., 1992). Oxidative stress induced by Aβ has been shown to be attenuated by
vitamin E in several in vitro models (Nourooz-Zadeh et al., 1999; Praticò et al., 1998). Some
of the effects include decreases in 4-hydroxynonenal (HNE), H2O2, lipid peroxidation, and
thiobarbituric acid reactive substances (TBARS) production. Additionally, prevention of
apoptosis by decreasing caspase-3 activation, cytochrome c release, and cleavage of poly-ADP
ribose polymerase (PARP) has been demonstrated. Several animal models of AD have shown
decreases in oxidative stress with improvements in behavioral and cognitive deficits. Vitamin
E administered orally in Aβ1–42-treated mice showed improvement in memory deficits and
decreased oxidative stress markers in the hippocampus and cerebral cortex. More recently,
tocotrienols—another member of the vitamin E family—have been shown to exhibit stronger
antioxidant and anti-inflammatory activities compared to α-tocopherol, thereby serving as a
potent neuroprotective agent (Chin et al., 2016). However, further studies are required to
validate its utility in humans.

4.2.2. Vitamin C
Vitamin C (ascorbic acid) is one of the most potent antioxidants that mediates numerous
beneficial effects on redox oxidative pathways, mitochondrial pathways, and on lipoprotein
metabolism (Naidu, 2003; Padayatty et al., 2003). L-ascorbic acid has been shown to attenuate
several specific factors linked to AD pathogenesis (Yao et al., 2004). Some of the
neuroprotective mechanisms include ROS scavenging activity, neuromodulation, Aβ fibril
formation suppression, and metal chelation (iron, copper, and zinc) (Choudhry et al., 2012).
4.2.3. Vitamin A
Vitamin A offers anti-oxidant and cytoprotective effects in several in vitro and in vivo
models of AD. Vitamin A deficiency is associated with cognitive impairment in the elderly.
Additionally, vitamin A deficiency promotes BACE1-mediated Aβ production and plaque
formation in mice models of AD, and its effects are reversed by treatment with therapeutic
doses of vitamin A (Zeng et al., 2017). Furthermore, vitamin A treatment reduced tau
hyperphosphorylation, neuroinflammation, and improved spatial learning and memory (Ding
et al., 2008).
4.2.4. Carotenoids
Carotenoids scavenge free radicals and oxidation interference, thereby protecting lipid
membranes (Paiva and Russell, 1999). In a randomized double-blind placebo-controlled study,
healthy subjects with age-related dementia and treated with astaxanthin, a xanthophyll
carotenoid with high potency and anti-peroxidative activity, showed improved cognitive
functioning (Katagiri et al., 2012; Satoh et al., 2009). However, in another double-blind
randomized clinical trial (Chew et al., 2015), oral supplementation with lutein/zeaxanthin had
no statistical significance on cognitive functioning in AD patients. Likewise, in an N-methyl-
4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of Parkinson’s Disease
(PD), treatment with β-carotene established neuroprotection (Yong et al., 1986).
Epidemiological studies have confirmed that individuals who consume carotenoids and β-
carotene as part of their diet have a significantly reduced risk of PD as compared to controls
(Miyake et al., 2011). Further studies and more investigation are needed for beneficial risk
reduction of vitamin A/carotenoids, as well as their role in treating neurodegenerative diseases.
4.2.5. Vitamin B12

Vitamin B12 has also been suggested in the treatment of AD. Most studies have found
significantly lower serum levels of vitamin B12 in AD patients as compared to controls, which
may contribute to neurodegeneration (Regland et al., 1991). Several studies indicated that
vitamin B12 supplementation increased choline acetyltransferase activity in cholinergic
neurons in cats (Nadeau and Roberge, 1988) and improved cognitive functions in AD patients
(Ikeda et al., 1992). Therefore, vitamin B12 may be an effective antioxidant therapy. Some of
the other dietary antioxidants, which have shown therapeutic benefit in AD, are described
in Table 2.

Table 2. Dietary antioxidants and their role in Alzheimer’s disease
Dietary Biological Functions References

Antioxidants
α-Tocopherol Most effective (lipid-soluble) antioxidant within the cell La Fata et al., 2017;
membrane, where it protects membrane fatty acids from Gugliandolo et al.,
lipid peroxidation. Decreases free-radical-mediated damage 2017
in neuronal cells and helps to inhibit AD pathologies.
β-Carotene Antioxidant effect to lipid-rich tissues like a brain. Also, Obulesu et al.,
inhibit the formation of Aβ. 2011;
Li et al., 2012;
Ono et al., 2006
Caffeine Potent antioxidant attenuates glutathione depletion, inhibits Kolahdouzan and
Aβ production and tau hyperphosphorylation both in vitro Hamadeh, 2017;
and in vivo. Carman et al., 2014;
Panza et al., 2015
Curcumin Antioxidant, anti-inflammatory, and anti-amyloid Chen et al., 2018;
pathology activity. Inhibits enzymes lipoxygenase and Serafini et al.,2017;
cyclooxygenase 2 that are responsible for the synthesis of Tang and
the pro-inflammatory leukotrienes, prostaglandins, and Taghibiglou, 2017
thromboxanes.
Quercetin Direct antioxidant; stimulate cellular defense against Costa et al., 2016;
oxidative stress by induction of Nrf-2 and antioxidant/anti- Sabogal-Guáqueta
inflammatory enzyme paraoxonase 2 (PON2), activation of et al., 2015
SIRT1.
Tocotrienol Vitamin E family with strong antioxidant and anti- Chin and Tay, 2018;
inflammatory effects. More effective against proxyl- Durani et al., 2018
radical-induced loss of cell viability.
Vitamin B12 An antioxidant that increases choline acetyltransferase de Wilde et al.,
activity in cholinergic neurons and improves cognitive 2017; Zhang et al.,
functions in AD patients. 2017
Vitamin C Water-soluble antioxidant and an inhibitor of lipid Rosales-Corral et
peroxidation. Powerful antioxidant in extracellular fluids. al., 2003; Sil et al.,
Neutralizes ROS in the acquis phase before lipid 2016
peroxidation is initiated. Decreases oxidative stress and
neuroinflammation mediated by AB fibrils.
5. PHARMACOTHERAPY TO COMBAT OXIDATIVE STRESS

IN ALZHEIMER’S DISEASE
Currently, there is no drug that can cure or halt the progression of AD. Drugs approved by
the FDA offer only symptomatic relief and are associated with various adverse effects
(Yiannopoulou and Papageorgiou, 2013). A conceptual approach is to prevent the disease onset
during the pre-AD stage, thereby reducing the pathologies of AD. This eventually leads to a
delay in the onset of disease, slows down its progression, or even repairs neurodegeneration.
Some of the novel strategies or therapies with this aim include antioxidants, nonsteroidal anti-
inflammatory drugs (NSAIDs), neurotrophins, and anti-diabetic drugs like pioglitazone, Aβ
vaccines, secretase inhibitors, and immunotherapy. All agents either show modest efficacy or
its utility is limited by serious adverse effects. Therefore, prevention and intervention strategies

for the eradication of AD require further investigation. Research to understand the

pathophysiology of AD has increased over the past decade to explore novel molecular targets
and develop appropriate therapeutics. Current evidence suggests that antioxidant therapy might
offer promising therapeutic benefits for AD. Several antioxidants have been investigated to
alter the pathophysiology of AD. The subsequent sections describe the conventional and novel
antioxidants, which have been investigated in AD.
5.1. Traditional Herbal Antioxidants
Several traditional herbal antioxidants offer intervention options for AD. There has been a
recent surge in the scientific and commercial significance of drugs based on medicinal plants
in health-related fields. These herbal medications have been standardized and their applications
specified.
5.1.1. Ginkgo biloba

Standardized extracts of Ginkgo biloba, a natural plant, have compounds such as
flavonoids and terpenoids that have the ability to scavenge free radicals and thus provide
neuroprotection. The beneficial effects are due to direct attenuation of ROS (Luo and Smith,
2004), as well as stabilization of cellular redox states by upregulating protein levels and activity
of antioxidant enzymes (Ahlemeyer and Krieglstein, 2003). The flavonoid fraction is the
primary antioxidant, for it directly scavenges ROS, chelates prooxidant transitional metal ions,
and increases antioxidant proteins such as SOD and GSH (Maitra et al., 1995; Oken et al., 1998;
Smith and Luo, 2003). There have been several serious side effects such as coma, bleeding, and
seizures that have been reported with use of commercially available ginkgo. Thus, there is little
evidence to support the use of Ginkgo biloba for AD treatment (Galluzzi et al., 2000; Tuppo
and Forman, 2001).
5.1.2. Bacopa monnieri

Bacopa monnieri (Brahmi) is a traditional plant commonly used in Ayurvedic medicine to
treat several neurological diseases. The principal constituents of Bacopa monnieri are saponins
and triterpenoid bacosaponins that include bacopasides III to V, bacosides A and B, and
bacosaponins A, B, and C. These constituents offer antioxidant activity by reducing divalent
metals, scavenging ROS, decreasing the formation of lipid peroxides, and inhibiting
lipoxygenase activity (Dhanasekaran et al., 2007). In AD, it improves memory and cognitive
function by inhibiting cholinergic degeneration (Stough et al., 2008; Uabundit et al., 2010).
5.1.3. Curcuma longa

Curcumin is the principal curcuminoid derived from turmeric Curcuma longa and is
predominantly used as a spice and coloring agent in Asia. In southeastern Asian countries where
turmeric is well incorporated into their diets, epidemiologic studies show a 4.4-fold lower
incidence of AD (Ganguli et al., 2000). Other studies indicate that turmeric has a non-steroidal
anti-inflammatory property, which is associated with a reduced risk of AD (Breitner et al.,
1995). Curcumin has been shown to inhibit lipid peroxidation and neutralize ROS in vitro with
a potency several times that of vitamin E (Butterfield et al., 2002).

5.1.4. Coptidis rhizoma

Several other traditional herbs exhibit antioxidant effects and are found to be of therapeutic
value in treating AD. Coptidis rhizoma, known as “Huang Lian” in China, is the dried rhizome
of medicinal plants from the family Ranunculaceae. Its major alkaloids, groenlandicine,
berberine, and palmatine, act through cholinesterase and Aβ pathways and inhibit ROS and
RNS, respectively (Jung et al., 2009).
5.1.5. Silybum marianum

Herb milk thistle, Silybum marianum contains silibinin (silybin), a flavonoid with
antioxidative properties. In AD mice, silibinin prevented memory impairment and oxidative
damage induced by Aβ. Thus silibinin may be a potential therapeutic agent for Alzheimer’s
disease (Lu et al., 2009).
5.2. Mitochondria-Targeted Antioxidants
Mitochondrial dysfunction and oxidative stress have been reported in several studies as
occurring early in the pathogenesis of AD (Moreira et al., 2010). Aβ accumulation reduces the
activity of mitochondrial respiratory chain complexes, decreases the activity of several
mitochondrial metabolic enzymes, and induces ROS production (de la Monte and Tong, 2014;
Swomley et al., 2014). The metabolic and oxidative stress renders neurons susceptible to
excitotoxicity and apoptosis. Hence, mitochondrial-targeted antioxidants may be a novel
therapy for modifying AD pathogenesis by protecting the mitochondria and reducing
mitochondrial oxidative damage. To date, the most effective molecules in counteracting
mitochondrial oxidative stress have been CoQ10, MitoQ, lipoic acid (LA), and SS peptides.
5.2.1. CoQ10
CoQ10 (ubiquinone) is an important cofactor of the electron transport chain where it
accepts electrons from complex I and II. CoQ10 is also a potent antioxidant (Beal, 2004), for,
during oxidative stress, ubiquinone preserves the mitochondrial membrane potential and
protects neuronal cells by attenuating Aβ overproduction and intracellular Aβ plaque deposits
(Moreira et al., 2010). Several in vivo studies utilizing CoQ10 have shown beneficial effects in
attenuating the pathologies of AD (Yang et al., 2008). However, a synthetic analog of CoQ10-
idebenone failed to show therapeutic benefits in AD patients (Su et al., 2010).
5.2.2. MitoQ
MitoQ is a novel antioxidant that targets the mitochondria and is produced by conjugating
a lipophilic triphenylphosphonium (TPP+) cation to coenzyme Q. Due to the large
mitochondrial membrane potential, coenzyme Q concentrates several 100-fold in the
mitochondria and reduces free radical-induced toxicity in the mitochondria (Murphy and Smith,
2007; Sheu et al., 2006). Coenzyme Q penetrates the membrane core with the help of lipophilic
TPP+, and in the mitochondrial matrix, is reduced to ubiquinol— its active form—by complex
II, which decreases lipid peroxidation and reduces oxidative damage (James et al., 2007).
Additionally, it exerts its cytoprotective effects by reducing free radicals, decreasing oxidative
damage, and maintaining mitochondrial functions (Dhanasekaran et al., 2004). AD mice treated

with MitoQ showed improvements in oxidative stress, Aβ accumulation, astrogliosis, and

synaptic loss (McManus et al., 2011). Chronic supplementation with mitoQ improved vascular
function in healthy older adults by reducing mitochondrial ROS and oxidizing LDL (a marker
of oxidative stress), indicating its potential for utilization in AD treatment.
5.2.3. SS Peptides
SS peptides—SS02, SS19, SS20, and SS31—are another class of mitochondria-targeted
antioxidants. These peptides concentrate in the inner mitochondrial membrane, scavenge H2O2
and ONOO-, and inhibit lipid peroxidation, thereby decreasing mitochondrial toxicity (Szeto,
2008, 2006). SS31 is the most effective among the SS peptides in terms of anti-oxidative effects
due to its extensive cellular uptake and localization in the mitochondria (Thomas et al., 2007).
Intracellular concentrations of [3H] SS-31 were 6-fold higher than extracellular concentrations.
[3H] SS31 was found at a concentration 6-fold higher intracellularly as compared to
extracellular conditions (Scheltens et al., 2012). Several in vivo studies have demonstrated SS-
31 reduces Aβ induced mitochondrial dysfunction and oxidative stress, thereby improving
axonal transport and synaptic plasticity (Calkins et al., 2011).

α-Lipoic acid (LA) is a cofactor for at least five enzyme systems. Among the enzyme
systems, pyruvate dehydrogenase complex and α-ketoglutarate dehydrogenase complex are the
most important. LA has potent antioxidant and iron chelator properties (Persson et al., 2003).
It also helps in recycling other antioxidants, such as vitamin C, vitamin E, and glutathione
(Bonda et al., 2010). Several in vitro and in vivo studies have demonstrated LA’s potent
antioxidant (Rochette et al., 2013), anti-inflammatory (Maczurek et al., 2008), and anti-
amyloidogenic properties (Ono et al., 2006b). Despite LA not being an efficient free radical
scavenger, the disulfide bond and five-membered cyclic structure of LA offer powerful
antioxidant and iron-chelation activity (Moreira et al., 2007; Persson et al., 2003). Thus, LA is
a promising therapeutic agent for AD, but the exact cellular and molecular mechanisms remain
unknown and mandate further investigation.
5.3. Other Drugs
5.3.1. N-Acetyl-L-Cysteine
N-acetyl-L-cysteine (NAC) is an acetylated precursor of cysteine, which plays an
important role in de novo synthesis of the endogenous antioxidant glutathione (GSH). NAC is
metabolized to cysteine in the liver, which is subsequently converted to GSH. A portion of
cysteine enters the systemic circulation and crosses the blood-brain barrier (BBB).
Additionally, NAC is de-aminated at the BBB to form cysteine. The cysteine thus formed
reaches the brain parenchyma via amino acid transporters and gets converted to GSH
(Cotgreave, 1997). Thus, NAC participates in antioxidant activities by contributing additional
cysteine for GSH production, which subsequently scavenges toxic by-products, reduce
oxidative and nitrosative stress, and reduces neuronal damage (Atkuri et al., 2007; Zafarullah
et al., 2003).

In vivo studies utilizing rodents have shown that NAC treatment increases GSH levels and
provides protection from pro-oxidant species (Farr et al., 2003; Koppal et al., 1999). NAC
reduced oxidative markers for protein oxidation/nitration (protein carbonyls and 3-nitro-
tyrosine) and protein-bound 4-hydroxy-2-nonenal (HNE) in brains of AD mice. In addition,
NAC increased the activity and expression of GPx, resulting in increased hydrogen peroxide
clearance (Huang et al., 2010). Proteomic studies on AD mice treated with NAC showed
beneficial effects on various targets altered by disease pathology. These targets included several
proteins involved in energy-related pathways, cellular defense, excitotoxicity, and synaptic
abnormalities (Robinson et al., 2011). Supplementation of 50 mg/kg/day of NAC has been
demonstrated to be safe, well tolerated, and potentially beneficial for humans.
NAC (600 mg/day) was included in two nutraceutical preparations tested on a multisite
Phase II clinical trial on AD and MCI individuals to evaluate effects on cognitive performance
with the hope that NAC will provide a precursor for GSH synthesis. One study reported
improvements in cognitive performance and behavioral and psychological symptoms of
dementia (BPSD) in the NAC treated group compared to the placebo treatment (Remington et
al., 2016, 2015). Other studies have been completed but data is not yet available.
5.3.2. Selenium
Selenium is an essential trace element important for normal brain function, and decreased
selenium levels have been associated with a significantly increased risk of AD (Akbaraly et al.,
2007; Loef et al., 2011). To substantiate, plasma selenium levels were lower in AD patients
when compared to healthy individuals (Vural et al., 2010). Selenium is incorporated with the
amino acid cysteine to form selenocysteine, which offers protection from free radical-induced
damage to the cells. Selenoprotein P (SelP), is a multifunctional protein with both enzymatic
and Se transport functions. SelP is critical for transporting selenium to the brain for the
synthesis of essential selenoproteins, such as GPx and thioredoxin reductase (TrxR).
Additionally, it contains a redox motif and exhibits antioxidant properties. SelP enhances
neuronal survival and prevents apoptotic cell death caused by Aβ induced oxidative stress
(Takemoto et al., 2010).
In vitro studies utilizing human neuroblastoma cells (SH-SY5Y expressing Swedish APP
mutant) treated with Se significantly reduced Aβ levels by reducing β- and γ-secretase
activities. This was also associated with a reduction in HNE induced β-secretase transcription
by selenium (Gwon et al., 2010).
A study by Ishrat et al. showed that selenium treatment in rats injected with
intracerebroventricular streptozocin improved learning and memory deficits and exhibited
increased levels of GSH, GPx, and glutathione reductase (GR) in the hippocampus and cerebral
cortex (Ishrat et al., 2009). Similarly, Se-enriched diets administered to APP/PS1 mice showed
lower Aβ deposition, decreased DNA and RNA oxidative damage, and significant increases in
GPx activity (Lovell et al., 2009).
Several randomized controlled trials in mild to moderate AD patients treated with selenium
(Se) have shown conflicting results (Scheltens et al., 2012, 2010; Shah et al., 2013). This
necessitates measuring selenium status, for there is no reason for giving supplemental Se to
population groups where Se status is already adequate. This was the case in a large randomized
controlled trial (PREADVise) conducted to investigate the effect of Se supplementation on AD
risk in elderly males and, unsurprisingly, found no effect (Kryscio et al., 2017). Symptoms of
Se toxicity (alopecia and dermatitis) were in fact shown in SELECT, the parent study of

PREADViSE, suggesting that the dose given had adverse effects in the population that already
had quite a high Se status (Lippman et al., 2009). Any further RCTs, if carried out in
populations of appropriately low Se status, may lead to novel strategies for the treatment of
AD.
5.3.3. Melatonin
Melatonin (N-Acetyl-5-methoxytryptamine) is a lipid and water-soluble hormone
synthesized mainly in the pineal gland. In 1993, it was reported to be an efficient endogenous
antioxidant (Tan et al., 1993). Melatonin stimulates the expression and activity of GPx, SOD,
and NO synthetase, allowing it to scavenge for oxygen and nitrogen free radicals in the
mitochondria (Kurutas, 2015). Melatonin levels decrease during aging, and AD patients have
been reported to have reduced CSF melatonin levels (Zhou et al., 2003).
Melatonin has been shown to attenuate Aβ induced increased intracellular Ca2+ and lipid
peroxidation (Pappolla et al., 1997). In mouse microglial BV2 cells, rat astroglioma C6 cells,
and PC12 cells, melatonin has attenuated Aβ-induced apoptosis (Feng and Zhang, 2004; Jang
et al., 2005; Pappolla et al., 1997). Furthermore, melatonin significantly attenuates ROS
production in Aβ treated mice by increasing the levels of the scavenging enzymes SOD, CAT,
and GSH when compared with the untreated group (Gunasingh et al., 2008). Though melatonin
has exhibited positive effects both in vitro and in vivo, melatonin has not shown any benefit
when administered to AD subjects. In fact, clinical trials have demonstrated that melatonin
lacks efficiency, which may be attributed to the true treatment effect or the high physiological
dose of melatonin used (Gehrman et al., 2009).
5.3.4. Resveratrol
Resveratrol, a polyphenol predominantly found in grapes, is known to possess diverse
biological activities including antioxidant, anti-inflammatory, cardioprotective, and
neuroprotective effects (Baur and Sinclair, 2006). Some of its effects are attributed to SIRT1
activity. The beneficial effects of resveratrol were validated through an epidemiological
research wherein moderate red wine consumption lead to lower incidences of cardiovascular
diseases, hence termed “French Paradox” (Ferriè, 2004). Mitochondrial ROS formation is
limited by resveratrol through (a) its scavenging activity on hydroxyl, superoxide, and metal-
induced free radicals; and (b) activation of SIRT1-PGC1α pathway, which enhances
mitochondrial function and biogenesis (Desquiret-Dumas et al., 2013; Khan et al., 2012;
Leonard et al., 2003). Additionally, resveratrol reduces iNOS and COX-2 levels with associated
increases in the heme-oxygenase-1 enzyme to attenuate oxidative stress. Downregulation of the
ROS producing enzyme Nox4 and increased expression of ROS-inactivating enzymes, SOD1
and GPx1, exhibit the additional anti-oxidative properties of resveratrol. Resveratrol has
several neuroprotective functions in AD, including its effects on Aβ, tau, neuroinflammation,
and apoptosis (Ma et al., 2014). However, to date, its utility in humans is limited by several
factors, including poor bioavailability and biotransformation, side effects, and complex
pathophysiology of AD (Braidy et al., 2016). Hence, resveratrol alone may not be an effective
therapy, thus requiring additional compounds to have effective therapy with multiple targets.

5.3.5. Honokiol
Honokiol belongs to a class of neolignans and is distributed naturally in several magnolia
species (Magnolia officinalis, Magnolia obovata, Magnolia dealbata, and Magnolia
grandiflora). It has been used as traditional medicine in Asian countries for their multiple
therapeutic properties, including antioxidant (Amorati et al., 2015), anti-inflammatory,
antimicrobial (Li et al., 2016), anticancer (Fried and Arbiser, 2009), antithrombotic (Lee et al.,
2017), antidepressant (Sulakhiya et al., 2015), and neuroprotective activities (Liou et al., 2003).
It finds its therapeutic application in several neurological disorders due to its BBB permeability
(Lin et al., 2012).
Honokiol possesses specific substituents that activate the intracellular signal cascade,
giving honokiol antioxidant capabilities. The antioxidant activity of the biphenolic moiety of
Honokiol has been attributed to its intramolecular hydrogen bonding between alcohol groups
with the aromatic and allyl π-systems (Amorati et al., 2015). Unlike other phenols without allyl
groups, such as α-tocopherol, that act as a free radical chain-breaking antioxidant (Ogata et al.,
1997), honokiol directly scavenges oxygen-derived free radicals, giving honokiol significantly
stronger antioxidant capabilities.
In unstressed conditions, the Nrf2-pathway is restricted from inducing antioxidant and
cytoprotective genes by Keap1-mediated ubiquitination and additional negative regulators
present in the nucleus; however, honokiol has the ability to upregulate the Nrf2-pathway,
possibly by disrupting Keap1 destabilization of Nrf2. This allows Nrf2 to be translocated to the
nucleus where antioxidant cellular response genes are activated, and, consequently, cellular
antioxidant defense mechanisms are also activated (Rajgopal et al., 2016). Honokiol has been
shown to attenuate oxidative stress and inflammation in neuronal and microglial cells. A study
done by Chuang et al. showed that Honokiol significantly inhibits superoxide formation
induced by NMDA but had no effects on neuronal superoxide production. Similarly, Honokiol
attenuated interferon-γ induced ROS production in microglial cells (Chuang et al., 2013).
Honokiol decreased ROS and lipid peroxidation, increased antioxidant enzymes, and improved
mitochondrial bioenergetics in Chinese Hamster Ovarian (CHO) cells transfected with amyloid
precursor protein-APP and Presenilin PS1 mutation (Ramesh et al., 2018). In rodents,
lipopolysaccharide treatment significantly increased both oxidative stress parameters
(malondialdehyde, glutathione) and nitrosative stress parameters (nitrite) which were
ameliorated by pretreatment with Honokiol (Sulakhiya et al., 2014). Similarly, Honokiol
attenuated oxidative stress and lipid peroxidation and restored glutathione levels in brains of
mice exposed to NMDA (Cui et al., 2007). These studies indicate that Honokiol might be an
effective therapeutic candidate for AD; however, clinical trials need to be evaluated for its
application in humans.

AND CHALLENGES
Antioxidant therapies have been studied for years as a promising therapeutic approach for
AD (Wojsiat et al., 2018). Nevertheless, the use of antioxidants as treatment remains
inconclusive and controversial in clinical settings. The failure of these clinical trials might be
attributed to the administration of incorrect doses or low BBB permeability to most of the

antioxidants currently used. Nanoparticles possess potential as a novel system for delivering
drugs into the central nervous system (CNS). The sensitive and elusive balance between
antioxidants and free radical production is also of importance to consider. Still, many other
factors must be examined, such as the use of a single molecule, appropriate dosing, and the
antioxidant status of each subject undergoing treatment.
Measuring levels of certain biomarkers that give insight into the antioxidant status of
subjects supplemented with the use of multiple antioxidants may yield positive outcomes.
Vascular factors, often neglected in clinical trials of antioxidant supplements, may be a strong
candidate for future AD research, and direct comparison of nutritional, micronutrient, and
antioxidant strategies to single-target strategies such as cholesterol or blood pressure, may be
one such aspect deserving research. In fact, in some trials, the presence of vascular risk factors
constituted an exclusion criterion for the study. Of course, several other factors that limit the
study of antioxidants in AD research must also be considered, such as the study design itself,
including the type of antioxidant and time window versus natural nutrition; lack of previously
well-established efficacy on oxidative stress biomarkers; lack of nutritional information; lack
of well-established neuropsychological measures and lifestyle endpoints; and differing disease
severity.
Hence, most antioxidants show overall success in animal models but are less successful in
human trials. Finally, the unsatisfactory results of the clinical trials might be correlated to the
inappropriate recruitment of patients, administration of drugs at the advanced AD stages, and
too short follow-ups to detect clinical benefits. The selection of patients and the monitoring of
the clinical trials can be improved by the design of novel noninvasive biomarker-based tools
allowing the detection of the disease at the pre-symptomatic stage and sensitive long-term
monitoring of therapeutic efficacies. Clinical trials for the use of antioxidants to decrease the
risk or slow the progression of AD remain in its infancy. Thus, further research is essential for
determining the efficacy of antioxidants in the prevention and treatment of AD.
CONCLUSION
Although multiple pathophysiologic mechanisms are associated with AD, oxidative stress
appears to be a major contributor to this process. The subtle balance of oxidant/antioxidant
defenses and ROS production may be disrupted by deficient antioxidant defenses, inhibited
electron flow, and/or mitochondrial dysfunction, leading to neuronal loss. Understanding the
molecular basis of dementia, particularly those involving oxidative stress, may lead to
elucidation of the etiopathogenesis of AD that will help to develop novel neuroprotective
treatment strategies. However, multicenter trials support the capacity of antioxidant treatment
effects to retard the progression of AD, suggesting the complexity of AD.
REFERENCES
Ahlemeyer B, Krieglstein J. Neuroprotective effects of Ginkgo biloba extract. Cell Mol Life Sci
2003;60:1779–92. doi:10.1007/s00018-003-3080-1.

Akbaraly N. T, Hininger-Favier I, Carri??re I, Arnaud J, Gourlet V, Roussel A-M, et al. Plasma

Selenium Over Time and Cognitive Decline in the Elderly. Epidemiology 2007;18:52–8.
doi:10.1097/01.ede.0000248202.83695.4e.
Ames B. N, Cathcart R, Schwiers E, Hochstein P. Uric acid provides an antioxidant defense in
humans against oxidant- and radical-caused aging and cancer: a hypothesis. Proc Natl Acad
Sci U S A 1981;78:6858–62.
Amir Aslani B, Ghobadi S. Studies on oxidants and antioxidants with a brief glance at their
relevance to the immune system. Life Sci 2016;146:163–73. doi:10.1016/j.lfs.2016.
01.014.
Amorati R, Zotova J, Baschieri A, Valgimigli L. Antioxidant Activity of Magnolol and
Honokiol: Kinetic and Mechanistic Investigations of Their Reaction with Peroxyl Radicals.
J Org Chem 2015;80:10651–9. doi:10.1021/acs.joc.5b01772.
Anantharaman M, Tangpong J, Keller J. N, Murphy M. P, Markesbery W. R, Kiningham K. K,
et al. Beta-amyloid mediated nitration of manganese superoxide dismutase: implication for
oxidative stress in a APPNLH/NLH X PS-1P264L/P264L double knock-in mouse model
of Alzheimer’s disease. Am J Pathol 2006;168:1608–18. doi:10.2353/AJPATH.
2006.051223.
Anderson R, Ramafi G, Theron A. J. Membrane stabilizing, anti-oxidative interactions of
propranolol and dexpropranolol with neutrophils. Biochem Pharmacol 1996;52:341–9.
Ansari MA, Scheff SW. Oxidative Stress in the Progression of Alzheimer Disease in the Frontal
Cortex. J Neuropathol Exp Neurol 2010;69:155–67. doi:10.1097/NEN.
0b013e3181cb5af4.
Ardite E, Sans M, Panés J, Romero F. J, Piqué JM, Fernández-Checa J. C. Replenishment of
glutathione levels improves mucosal function in experimental acute colitis. Lab Invest
2000;80:735–44.
Arivazhagan P, Ramanathan K, Panneerselvam C. Effect of DL-alpha-lipoic acid on
mitochondrial enzymes in aged rats. Chem Biol Interact 2001;138:189–98.
Atkuri KR, Mantovani JJ, Herzenberg LA, Herzenberg LA. N-Acetylcysteine—a safe antidote
for cysteine/glutathione deficiency. Curr Opin Pharmacol 2007;7:355–9.
doi:10.1016/j.coph.2007.04.005.
Balla G, Jacob H. S, Balla J, Rosenberg M, Nath K, Apple F, et al. Ferritin: a cytoprotective
antioxidant strategem of endothelium. J Biol Chem 1992;267:18148–53.
Baur J. A, Sinclair D. A. Therapeutic potential of resveratrol: the in vivo evidence. Nat Rev
Drug Discov 2006;5:493–506. doi:10.1038/nrd2060.
Beal M. F. Mitochondrial Dysfunction and Oxidative Damage in Alzheimer’s and Parkinson’s
Diseases and Coenzyme Q10 as a Potential Treatment. J Bioenerg Biomembr 2004;36:381–
6. doi:10.1023/B:JOBB.0000041772.74810.92.
Behl C, Davis J. B, Lesley R, Schubert D. Hydrogen peroxide mediates amyloid beta protein
toxicity. Cell 1994;77:817–27.
Billingsley M. L, Kincaid R. L. Regulated phosphorylation and dephosphorylation of tau
protein: effects on microtubule interaction, intracellular trafficking and neurodegene-
ration. Biochem J 1997;323 (Pt 3):577–91.
Bonda D. J, Wang X, Perry G, Nunomura A, Tabaton M, Zhu X, et al. Oxidative stress in
Alzheimer disease: A possibility for prevention. Neuropharmacology 2010;59:290–4.
doi:10.1016/j.neuropharm.2010.04.005.

Braidy N, Jugder B-E, Poljak A, Jayasena T, Mansour H, Nabavi S. M, et al. Resveratrol as a

Potential Therapeutic Candidate for the Treatment and Management of Alzheimer’s
Disease. Curr Top Med Chem 2016;16:1951–60.
Breitner J. C, Welsh K. A, Helms M. J, Gaskell P. C, Gau B. A, Roses A. D, et al. Delayed
onset of Alzheimer’s disease with nonsteroidal anti-inflammatory and histamine H2
blocking drugs. Neurobiol Aging n.d.;16:523–30.
Butterfield D, Castegna A, Pocernich C, Drake J, Scapagnini G, Calabrese V. Nutritional
approaches to combat oxidative stress in Alzheimer’s disease. J Nutr Biochem
2002;13:444.
Butterfield D. A, Kanski J. Brain protein oxidation in age-related neurodegenerative disorders
that are associated with aggregated proteins. Mech Ageing Dev 2001;122:945–62.
Calkins MJ, Manczak M, Mao P, Shirendeb U, Reddy PH. Impaired mitochondrial biogenesis,
defective axonal transport of mitochondria, abnormal mitochondrial dynamics and synaptic
degeneration in a mouse model of Alzheimer’s disease. Hum Mol Genet 2011;20:4515–29.
doi:10.1093/hmg/ddr381.
Carman A. J, Dacks P. A, Lane RF, Shineman DW, Fillit HM. Current evidence for the use of
coffee and caffeine to prevent age-related cognitive decline and Alzheimer’s disease. J
Nutr Health Aging 2014;18:383–92. doi:10.1007/s12603-014-0021-7.
Casado A, de la Torre R, López-Fernández E, Carrascosa D, Venarucci D. [Superoxide
dismutase and catalase levels in diseases of the aged]. Gac Med Mex n.d.;134:539–44.
Casley CS, Canevari L, Land J. M, Clark J. B, Sharpe M. A. Beta-amyloid inhibits integrated
mitochondrial respiration and key enzyme activities. J Neurochem 2002;80:91–100.
Caspersen C, Wang N, Yao J, Sosunov A, Chen X, Lustbader JW, et al. Mitochondrial Aβ: a
potential focal point for neuronal metabolic dysfunction in Alzheimer’s disease. FASEB J
2005;19:2040–1. doi:10.1096/fj.05-3735fje.
Chang K-H., Cheng M-L, Chiang M-C, Chen C-M. Lipophilic antioxidants in
neurodegenerative diseases. Clin Chim Acta 2018;485:79–87. doi:10.1016/J.CCA.2018.
06.031.
Chen M, Du Z-Y, Zheng X, Li D-L, Zhou R-P, Zhang K. Use of curcumin in diagnosis,
prevention, and treatment of Alzheimer’s disease. Neural Regen Res 2018;13:742.
doi:10.4103/1673-5374.230303.
Chew E. Y, Clemons T. E, Agrón E., Launer L. J., Grodstein F, Bernstein P. S. Effect of
Omega-3 Fatty Acids, Lutein/Zeaxanthin, or Other Nutrient Supplementation on Cognitive
Function. JAMA 2015;314:791. doi:10.1001/jama.2015.9677.
Chin K-Y, Pang K-L, Soelaiman I-N. Tocotrienol and Its Role in Chronic Diseases. Adv. Exp.
Med. Biol., vol. 928, 2016, p. 97–130. doi:10.1007/978-3-319-41334-1_5.
Chin K-Y, Tay S. A Review on the Relationship between Tocotrienol and Alzheimer Disease.
Nutrients 2018;10:881. doi:10.3390/nu10070881.
Choudhry F, Howlett D. R, Richardson J. C, Francis P. T, Williams R. J. Pro-oxidant diet
enhances β/γ secretase-mediated APP processing in APP/PS1 transgenic mice. Neurobiol
Aging 2012;33:960–8. doi:10.1016/j.neurobiolaging.2010.07.008.
Chuang D. Y, Chan M-H., Zong Y, Sheng W, He Y, Jiang J. H, et al. Magnolia polyphenols
attenuate oxidative and inflammatory responses in neurons and microglial cells. J
Neuroinflammation 2013;10:786. doi:10.1186/1742-2094-10-15.

Costa L. G, Garrick J. M, Roquè P. J, Pellacani C. Mechanisms of Neuroprotection by

Quercetin: Counteracting Oxidative Stress and More. Oxid Med Cell Longev 2016;2016:1–
10. doi:10.1155/2016/2986796.
Cotgreave I. A. N-acetylcysteine: pharmacological considerations and experimental and
clinical applications. Adv Pharmacol 1997;38:205–27.
Cui H. S, Huang L. S, Sok D-E, Shin J, Kwon B-M, Youn U. J, et al. Protective action of
honokiol, administered orally, against oxidative stress in brain of mice challenged with
NMDA. Phytomedicine 2007;14:696–700. doi:10.1016/j.phymed.2007.03.005.
David D. C, Hauptmann S, Scherping I, Schuessel K, Keil U, Rizzu P, et al. Proteomic and
Functional Analyses Reveal a Mitochondrial Dysfunction in P301L Tau Transgenic Mice.
J Biol Chem 2005;280:23802–14. doi:10.1074/jbc.M500356200.
de la Monte S. M, Tong M. Brain metabolic dysfunction at the core of Alzheimer’s disease.
Biochem Pharmacol 2014;88:548–59. doi:10.1016/j.bcp.2013.12.012.
de la Monte S. M, Wands J. R. Molecular indices of oxidative stress and mitochondrial
dysfunction occur early and often progress with severity of Alzheimer’s disease. J
Alzheimers Dis 2006;9:167–81.
de Wilde M. C, Vellas B, Girault E, Yavuz AC, Sijben J. W. Lower brain and blood nutrient
status in Alzheimer’s disease: Results from meta-analyses. Alzheimer’s Dement Transl Res
Clin Interv 2017;3:416–31. doi:10.1016/j.trci.2017.06.002.
Desquiret-Dumas V, Gueguen N, Leman G, Baron S, Nivet-Antoine V, Chupin S, et al.
Resveratrol Induces a Mitochondrial Complex I-dependent Increase in NADH Oxidation
Responsible for Sirtuin Activation in Liver Cells. J Biol Chem 2013;288:36662–75.
doi:10.1074/jbc.M113.466490.
Dhanasekaran A, Kotamraju S, Kalivendi S V., Matsunaga T, Shang T, Keszler A, et al.
Supplementation of Endothelial Cells with Mitochondria-targeted Antioxidants Inhibit
Peroxide-induced Mitochondrial Iron Uptake, Oxidative Damage, and Apoptosis. J Biol
Chem 2004;279:37575–87. doi:10.1074/jbc.M404003200.
Dhanasekaran M, Tharakan B, Holcomb L. A, Hitt A. R, Young K. A, Manyam B V.
Neuroprotective mechanisms of ayurvedic antidementia botanicalBacopa monniera.
Phyther Res 2007;21:965–9. doi:10.1002/ptr.2195.
Dias-Santagata D, Fulga TA, Duttaroy A, Feany MB. Oxidative stress mediates tau-induced
neurodegeneration in Drosophila. J Clin Invest 2007;117:236–45. doi:10.1172/JCI28769.
Dickinson DA, Forman HJ. Cellular glutathione and thiols metabolism. Biochem Pharmacol
2002;64:1019–26.
Ding Y, Qiao A, Wang Z, Goodwin JS, Lee E-S, Block M. L, et al. Retinoic Acid Attenuates -
Amyloid Deposition and Rescues Memory Deficits in an Alzheimer’s Disease Transgenic
Mouse Model. J Neurosci 2008;28:11622–34. doi:10.1523/JNEUROSCI.3153-08.2008.
Dore S, Snyder S. H. Neuroprotective Action of Bilirubin against Oxidative Stress in Primary
Hippocampal Cultures. Ann N Y Acad Sci 1999;890:167–72. doi:10.1111/j.1749-
6632.1999.tb07991.x.
Dröge W. Free Radicals in the Physiological Control of Cell Function. Physiol Rev 2002;82:47–
95. doi:10.1152/physrev.00018.2001.
Dumont M, Stack C, Elipenahli C, Jainuddin S, Gerges M, Starkova N. N, et al. Behavioral
deficit, oxidative stress, and mitochondrial dysfunction precede tau pathology in P301S
transgenic mice. FASEB J 2011;25:4063–72. doi:10.1096/fj.11-186650.

Dumont M, Wille E, Stack C, Calingasan N. Y, Beal M. F, Lin M. T. Reduction of oxidative

stress, amyloid deposition, and memory deficit by manganese superoxide dismutase
overexpression in a transgenic mouse model of Alzheimer’s disease. FASEB J
2009;23:2459–66. doi:10.1096/fj.09-132928.
Durani L. W, Hamezah H. S, Ibrahim N. F, Yanagisawa D, Nasaruddin M. L, Mori M, et al.
Tocotrienol-Rich Fraction of Palm Oil Improves Behavioral Impairments and Regulates
Metabolic Pathways in AβPP/PS1 Mice. J Alzheimer’s Dis 2018;64:249–67.
doi:10.3233/JAD-170880.
Echtay K. Mitochondrial uncoupling proteins—What is their physiological role? Free Radic
Biol Med 2007;43:1351–71. doi:10.1016/j.freeradbiomed.2007.08.011.
Elipenahli C, Stack C, Jainuddin S, Gerges M, Yang L, Starkov A, et al. Behavioral
Improvement after Chronic Administration of Coenzyme Q10 in P301S Transgenic Mice.
J Alzheimer’s Dis 2012;28:173–82. doi:10.3233/JAD-2011-111190.
Esler W. P, Wolfe M. S. A Portrait of Alzheimer Secretases--New Features and Familiar Faces.
Science (80- ) 2001;293:1449–54. doi:10.1126/science.1064638.
Farr S. A, Poon H. F, Dogrukol-Ak D, Drake J, Banks W. A, Eyerman E, et al. The antioxidants
alpha-lipoic acid and N-acetylcysteine reverse memory impairment and brain oxidative
stress in aged SAMP8 mice. J Neurochem 2003;84:1173–83.
Feng Z, Zhang J. Protective effect of melatonin on β-amyloid-induced apoptosis in rat
astroglioma c6 cells and its mechanism. Free Radic Biol Med 2004;37:1790–801.
doi:10.1016/j.freeradbiomed.2004.08.023.
Ferriè J. Coronary disease The French Paradox: Lessons for Other Countries. vol. 90. 2004.
Forsberg L, de Faire U, Morgenstern R. Oxidative Stress, Human Genetic Variation, and
Disease. Arch Biochem Biophys 2001;389:84–93. doi:10.1006/abbi.2001.2295.
Fried L. E, Arbiser J. L. Honokiol, a multifunctional antiangiogenic and antitumor agent.
Antioxid Redox Signal 2009;11:1139–48. doi:10.1089/ars.2009.2440.
Gabbita P, Lovell M, Markesbery iamR. Lippincoti-Raven Publishers. Philadelphia Increased
Nuclear DNA Oxidation in the Brain in Alzheimer’ s Disease. vol. 71. 1998.
Gabbita SP, Aksenov MY, Lovell MA, Markesbery WR. Decrease in Peptide Methionine
Sulfoxide Reductase in Alzheimer’s Disease Brain. J Neurochem 2002;73:1660–6.
doi:10.1046/j.1471-4159.1999.0731660.x.
Galluzzi S, Zanetti O, Binetti G, Trabucchi M, Frisoni GB. Coma in a patient with Alzheimer’s
disease taking low dose trazodone and gingko biloba. J Neurol Neurosurg Psychiatry
2000;68:679–80. doi:10.1136/JNNP.68.5.679A.
Gamblin T. C, King M. E, Kuret J, Berry R. W, Binder L. I. Oxidative regulation of fatty acid-
induced tau polymerization. Biochemistry 2000;39:14203–10.
Ganguli M, Chandra V, Kamboh M. I, Johnston J. M, Dodge H. H, Thelma B. K, et al.
Apolipoprotein E polymorphism and Alzheimer disease: The Indo-US Cross-National
Dementia Study. Arch Neurol 2000;57:824–30.
Gehrman P. R, Connor D .J, Martin J. L, Shochat T, Corey-Bloom J, Ancoli-Israel S. Melatonin
Fails to Improve Sleep or Agitation in Double-Blind Randomized Placebo-Controlled Trial
of Institutionalized Patients With Alzheimer Disease. Am J Geriatr Psychiatry
2009;17:166–9. doi:10.1097/JGP.0b013e318187de18.
Genestra M. Oxyl radicals, redox-sensitive signalling cascades and antioxidants. Cell Signal
2007;19:1807–19. doi:10.1016/j.cellsig.2007.04.009.

Gille L, Nohl H. The Existence of a Lysosomal Redox Chain and the Role of Ubiquinone. Arch
Biochem Biophys 2000;375:347–54. doi:10.1006/abbi.1999.1649.
Goedert M, Hasegawa M, Jakes R, Lawler S, Cuenda A, Cohen P. Phosphorylation of
microtubule-associated protein tau by stress-activated protein kinases. FEBS Lett
1997;409:57–62.
Goedert M, Spillantini M. G. A Century of Alzheimer’s Disease. Science (80- ) 2006;314:777–
81. doi:10.1126/science.1132814.
Gorąca A, Huk-Kolega H, Piechota A, Kleniewska P, Ciejka E, Skibska B. Lipoic acid -
biological activity and therapeutic potential. Pharmacol Rep 2011;63:849–58.
Gotz J, Chen F, van Dorpe J, Nitsch R. M. Formation of Neurofibrillary Tangles in P301L Tau
Transgenic Mice Induced by Abeta 42 Fibrils. Science (80- ) 2001;293:1491–5.
doi:10.1126/science.1062097.
Gugliandolo A, Bramanti P, Mazzon E. Role of Vitamin E in the Treatment of Alzheimer’s
Disease: Evidence from Animal Models. Int J Mol Sci 2017;18. doi:10.3390/
ijms18122504.
Gunasingh M. J, Philip JE, Ashok BS, Kirubagaran R, Jebaraj WCE, Davis GDJ, et al.
Melatonin prevents amyloid protofibrillar induced oxidative imbalance and biogenic amine
catabolism. Life Sci 2008;83:96–102. doi:10.1016/j.lfs.2008.05.011.
Gwon A-R, Park J-S, Park J-H, Baik S-H, Jeong H-Y, Hyun D-H, et al. Selenium attenuates
Aβ production and Aβ-induced neuronal death. Neurosci Lett 2010;469:391–5.
doi:10.1016/j.neulet.2009.12.035.
Halliwell B. The Chemistry of Free Radicals. Toxicol Ind Health 1993;9:1–21.
doi:10.1177/0748233793009001-203.
Halliwell B. Free Radicals and other reactive species in Disease. n.d.
Hirai K, Aliev G, Nunomura A, Fujioka H, Russell R. L, Atwood C. S, et al. Mitochondrial
abnormalities in Alzheimer’s disease. J Neurosci 2001;21:3017–23.
Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, et al. Correlative memory
deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science 1996;274:99–
102.
Huang Q, Aluise C. D, Joshi G, Sultana R, St. Clair D. K, Markesbery W. R, et al. Potential in
vivo amelioration by N-acetyl-L-cysteine of oxidative stress in brain in human double
mutant APP/PS-1 knock-in mice: Toward therapeutic modulation of mild cognitive
impairment. J Neurosci Res 2010;88:n/a-n/a. doi:10.1002/jnr.22422.
Huang W-J, Zhang X, Chen W-W. Role of oxidative stress in Alzheimer’s disease. Biomed
Reports 2016;4:519–22. doi:10.3892/br.2016.630.
Ikeda T, Yamamoto K, Takahashi K, Kaku Y, Uchiyama M, Sugiyama K, et al. Treatment of
Alzheimer-type dementia with intravenous mecobalamin. Clin Ther n.d.;14:426–37.
Iqbal K, Liu F, Gong C-X, Alonso A del C, Grundke-Iqbal I. Mechanisms of tau-induced
neurodegeneration. Acta Neuropathol 2009;118:53–69. doi:10.1007/s00401-009-0486-3.
Ishrat T, Parveen K, Khan M. M, Khuwaja G, Khan M. B, Yousuf S, et al. Selenium prevents
cognitive decline and oxidative damage in rat model of streptozotocin-induced
experimental dementia of Alzheimer’s type. Brain Res 2009;1281:117–27.
doi:10.1016/j.brainres.2009.04.010.
James A. M, Sharpley M. S, Manas A-R. B, Frerman F. E, Hirst J, Smith R. A. J, et al.
Interaction of the Mitochondria-targeted Antioxidant MitoQ with Phospholipid Bilayers

and Ubiquinone Oxidoreductases. J Biol Chem 2007;282:14708–18. doi:10.1074/jbc.

M611463200.
Jan A, Azam M, Siddiqui K, Ali A, Choi I, Haq Q. Heavy Metals and Human Health:
Mechanistic Insight into Toxicity and Counter Defense System of Antioxidants. Int J Mol
Sci 2015;16:29592–630. doi:10.3390/ijms161226183.
Jang M-H, Jung S-B, Lee M-H, Kim C-J, Oh Y-T, Kang I, et al. Melatonin attenuates amyloid
beta25–35-induced apoptosis in mouse microglial BV2 cells. Neurosci Lett 2005;380:26–
31. doi:10.1016/j.neulet.2005.01.003.
Jiménez-Jiménez F. J, de Bustos F, Molina J. A., Benito-León J, Tallón-Barranco A, Gasalla
T, et al. Cerebrospinal fluid levels of alpha-tocopherol (vitamin E) in Alzheimer’s disease.
J Neural Transm 1997;104:703–10. doi:10.1007/BF01291887.
Jung HA, Min B-S, Yokozawa T, Lee J-H, Kim Y. S, Choi J. S. Anti-Alzheimer and antioxidant
activities of Coptidis Rhizoma alkaloids. Biol Pharm Bull 2009;32:1433–8.
Kalaria R. N, Maestre G. E, Arizaga R, Friedland R. P, Galasko D, Hall K, et al. Alzheimer’s
disease and vascular dementia in developing countries: prevalence, management, and risk
factors. Lancet Neurol 2008;7:812–26. doi:10.1016/S1474-4422(08)70169-8.
Karelson E, Bogdanovic N, Garlind A, Winblad B, Zilmer K, Kullisaar T, et al. The
Cerebrocortical Areas in Normal Brain Aging and in Alzheimer’s Disease: Noticeable
Differences in the Lipid Peroxidation Level and in Antioxidant Defense. Neurochem Res
2001;26:353–61. doi:10.1023/A:1010942929678.
Katagiri M, Satoh A, Tsuji S, Shirasawa T. Effects of astaxanthin-rich Haematococcus pluvialis
extract on cognitive function: a randomised, double-blind, placebo-controlled study. J Clin
Biochem Nutr 2012;51:102–7. doi:10.3164/jcbn.D-11-00017.
Kaur H, Hughes M. N, Green CJ, Naughton P, Foresti R, Motterlini R. Interaction of bilirubin
and biliverdin with reactive nitrogen species. FEBS Lett 2003;543:113–9.
Khan R. S, Fonseca-Kelly Z, Callinan C, Zuo L, Sachdeva M. M, Shindler K. S. SIRT1
activating compounds reduce oxidative stress and prevent cell death in neuronal cells.
Front Cell Neurosci 2012;6:63. doi:10.3389/fncel.2012.00063.
Kohen R, Nyska A. Invited Review: Oxidation of Biological Systems: Oxidative Stress
Phenomena, Antioxidants, Redox Reactions, and Methods for Their Quantification.
Toxicol Pathol 2002;30:620–50. doi:10.1080/01926230290166724.
Kolahdouzan M, Hamadeh M. J. The neuroprotective effects of caffeine in neurodegenerative
diseases. CNS Neurosci Ther 2017;23:272–90. doi:10.1111/cns.12684.
Kontush A, Berndt C, Weber W, Akopyan V, Arlt S, Schippling S, et al. Amyloid-beta is an
antioxidant for lipoproteins in cerebrospinal fluid and plasma. Free Radic Biol Med
2001;30:119–28. doi:10.1016/S0891-5849(00)00458-5.
Koppal T, Drake J, Butterfield D. A. In vivo modulation of rodent glutathione and its role in
peroxynitrite-induced neocortical synaptosomal membrane protein damage. Biochim
Biophys Acta 1999;1453:407–11.
Kryscio R. J., Abner E. L, Caban-Holt A, Lovell M, Goodman P, Darke A. K, et al. Association
of Antioxidant Supplement Use and Dementia in the Prevention of Alzheimer’s Disease
by Vitamin E and Selenium Trial (PREADViSE). JAMA Neurol 2017;74:567.
doi:10.1001/jamaneurol.2016.5778.
Kurutas E. B. The importance of antioxidants which play the role in cellular response against
oxidative/nitrosative stress: current state. Nutr J 2015;15:71. doi:10.1186/s12937-016-
0186-5.

La Fata G, van Vliet N, Barnhoorn S, Brandt R. M. C, Etheve S, Chenal E, et al. Vitamin E

Supplementation Reduces Cellular Loss in the Brain of a Premature Aging Mouse Model.
J Prev Alzheimer’s Dis 2017;4:226–35. doi:10.14283/jpad.2017.30.
Lee J-M, Johnson J. A. An Important Role of Nrf2-ARE Pathway in the Cellular Defense
Mechanism. vol. 37. 2004.
Lee T-Y, Chang C-C, Lu W-J, Yen T-L, Lin K-H, Geraldine P, et al. Honokiol as a specific
collagen receptor glycoprotein VI antagonist on human platelets: Functional ex vivo and
in vivo studies. Sci Rep 2017;7:40002. doi:10.1038/srep40002.
Leonard S. S, Xia C, Jiang B-H, Stinefelt B, Klandorf H, Harris GK, et al. Resveratrol
scavenges reactive oxygen species and effects radical-induced cellular responses. Biochem
Biophys Res Commun 2003;309:1017–26. doi:10.1016/j.bbrc.2003.08.105.
Li F-J, Shen L, Ji H-F. Dietary Intakes of Vitamin E, Vitamin C, and β-Carotene and Risk of
Alzheimer’s Disease: A Meta-Analysis. J Alzheimer’s Dis 2012;31:253–8.
doi:10.3233/JAD-2012-120349.
Li F, Calingasan N. Y, Yu F, Mauck W. M, Toidze M, Almeida C. G, et al. Increased plaque
burden in brains of APP mutant MnSOD heterozygous knockout mice. J Neurochem
2004;89:1308–12. doi:10.1111/j.1471-4159.2004.02455.x.
Li W-L, Zhao X-C, Zhao Z-W, Huang Y-J, Zhu X-Z, Meng R-Z, et al. In vitro antimicrobial
activity of honokiol against Staphylococcus aureus in biofilm mode. J Asian Nat Prod Res
2016;18:1178–85. doi:10.1080/10286020.2016.1194829.
Lillig C. H, Berndt C, Vergnolle O, Lönn M. E, Hudemann C, Bill E, et al. Characterization of
human glutaredoxin 2 as iron-sulfur protein: a possible role as redox sensor. Proc Natl
Acad Sci U S A 2005;102:8168–73. doi:10.1073/pnas.0500735102.
Lin J-W, Chen J-T, Hong C-Y, Lin Y-L, Wang K-T, Yao C-J, et al. Honokiol traverses the
blood-brain barrier and induces apoptosis of neuroblastoma cells via an intrinsic bax-
mitochondrion-cytochrome c-caspase protease pathway. Neuro Oncol 2012;14:302–14.
doi:10.1093/neuonc/nor217.
Liou K-T, Lin S-M, Huang S-S, Chih C-L, Tsai S-K. Honokiol Ameliorates Cerebral Infarction
from Ischemia-Reperfusion Injury in Rats. Planta Med 2003;69:130–4. doi:10.1055/s-
2003-37707.
Lippman S. M, Klein E. A, Goodman P. J, Lucia M. S, Thompson I. M, Ford L. G, et al. Effect
of Selenium and Vitamin E on Risk of Prostate Cancer and Other Cancers. JAMA
2009;301:39. doi:10.1001/jama.2008.864.
Liu J. The Effects and Mechanisms of Mitochondrial Nutrient α-Lipoic Acid on Improving
Age-Associated Mitochondrial and Cognitive Dysfunction: An Overview. Neurochem Res
2008;33:194–203. doi:10.1007/s11064-007-9403-0.
Liu J, Ames B. N. Reducing mitochondrial decay with mitochondrial nutrients to delay and
treat cognitive dysfunction, Alzheimer’s disease, and Parkinson’s disease. Nutr Neurosci
2005;8:67–89. doi:10.1080/10284150500047161.
Loef M, Schrauzer G. N, Walach H. Selenium and Alzheimer’s Disease: A Systematic Review.
Lovell MA, Xie C, Gabbita SP, Markesbery WR. Original Contribution. Decreased
Thioredoxin and Increased Thioredoxin Reductase Levels in Alzheimer’s Disease Brain.
2000.

Lovell M. A, Xiong S, Lyubartseva G, Markesbery W. R. Organoselenium (Sel-Plex diet)

decreases amyloid burden and RNA and DNA oxidative damage in APP/PS1 mice. Free
Radic Biol Med 2009;46:1527–33. doi:10.1016/j.freeradbiomed.2009.03.008.
Lu P, Mamiya T, Lu L, Mouri A, Zou L, Nagai T, et al. Silibinin prevents amyloid β peptide-
induced memory impairment and oxidative stress in mice. Br J Pharmacol 2009;157:1270–
7. doi:10.1111/j.1476-5381.2009.00295.x.
Lu S. C. Glutathione synthesis. Biochim Biophys Acta 2013;1830:3143–53. doi:10.1016/j.
bbagen.2012.09.008.
Luo Y, Smith J. V. Studies on molecular mechanisms of Ginkgo biloba extract. Appl Microbiol
Biotechnol 2004;64:465–72. doi:10.1007/s00253-003-1527-9.
Luo Y, Sunderland T, Roth G. S, Wolozin B. Physiological levels of β-amyloid peptide promote
PC12 cell proliferation. Neurosci Lett 1996;217:125–8. doi:10.1016/0304-3940(96)13087-
1.
Ma T, Tan M-S, Yu J-T, Tan L. Resveratrol as a therapeutic agent for Alzheimer’s disease.
Biomed Res Int 2014;2014:350516. doi:10.1155/2014/350516.
Maczurek A, Hager K, Kenklies M, Sharman M, Martins R, Engel J, et al. Lipoic acid as an
anti-inflammatory and neuroprotective treatment for Alzheimer’s disease. Adv Drug Deliv
Rev 2008;60:1463–70. doi:10.1016/j.addr.2008.04.015.
Maitra I, Marcocci L, Droy-Lefaix M. T, Packer L. Peroxyl radical scavenging activity of
Ginkgo biloba extract EGb 761. Biochem Pharmacol 1995;49:1649–55. doi:10.1016/0006-
2952(95)00089-I.
Manczak M, Anekonda T. S, Henson E, Park B. S, Quinn J, Reddy P. H. Mitochondria are a
direct site of Aβ accumulation in Alzheimer’s disease neurons: implications for free radical
generation and oxidative damage in disease progression. Hum Mol Genet 2006;15:1437–
49. doi:10.1093/hmg/ddl066.
Mandal P. K, Saharan S, Tripathi M, Murari G. Brain Glutathione Levels – A Novel Biomarker
for Mild Cognitive Impairment and Alzheimer’s Disease. Biol Psychiatry 2015;78:702–
10. doi:10.1016/j.biopsych.2015.04.005.
Marcus D. L, Thomas C, Rodriguez C, Simberkoff K, Tsai J. S, Strafaci J. A, et al. Increased
Peroxidation and Reduced Antioxidant Enzyme Activity in Alzheimer’s Disease. Exp
Neurol 1998;150:40–4. doi:10.1006/EXNR.1997.6750.
Matsuoka Y, Picciano M, La Francois J, Duff K. Fibrillar beta-amyloid evokes oxidative
damage in a transgenic mouse model of Alzheimer’s disease. Neuroscience 2001;104:609–
13. doi:10.1016/S0306-4522(01)00115-4.
Mattson M. P. Cellular actions of beta-amyloid precursor protein and its soluble and
fibrillogenic derivatives. Physiol Rev 1997;77:1081–132. doi:10.1152/physrev.
1997.77.4.1081.
McManus M. J., Murphy M. P, Franklin J. L. The Mitochondria-Targeted Antioxidant MitoQ
Prevents Loss of Spatial Memory Retention and Early Neuropathology in a Transgenic
Mouse Model of Alzheimer’s Disease. J Neurosci 2011;31:15703–15. doi:10.1523/
JNEUROSCI.0552-11.2011.
Meister A. Glutathione, ascorbate, and cellular protection. Cancer Res 1994;54:1969s–1975s.
Melov S, Adlard PA, Morten K, Johnson F, Golden T. R, Hinerfeld D, et al. Mitochondrial
Oxidative Stress Causes Hyperphosphorylation of Tau. PLoS One 2007;2:e536.
doi:10.1371/journal.pone.0000536.

Miller N. J, Rice-Evans C. A. Spectrophotometric determination of antioxidant activity. Redox

Rep 1996;2:161–71. doi:10.1080/13510002.1996.11747044.
Miyake Y, Fukushima W, Tanaka K, Sasaki S, Kiyohara C, Tsuboi Y, et al. Dietary intake of
antioxidant vitamins and risk of Parkinson’s disease: a case-control study in Japan. Eur J
Neurol 2011;18:106–13. doi:10.1111/j.1468-1331.2010.03088.x.
Moreira PI, Harris P. L. R, Zhu X, Santos MS, Oliveira CR, Smith MA, et al. Lipoic acid and
N-acetyl cysteine decrease mitochondrial-related oxidative stress in Alzheimer disease
patient fibroblasts. J Alzheimers Dis 2007;12:195–206.
Moreira P. I, Zhu X, Wang X, Lee H, Nunomura A, Petersen RB, et al. Mitochondria: A
therapeutic target in neurodegeneration. Biochim Biophys Acta - Mol Basis Dis
2010;1802:212–20. doi:10.1016/j.bbadis.2009.10.007.
Mullan K, Williams MA, Cardwell CR, McGuinness B, Passmore P, Silvestri G, et al. Serum
concentrations of vitamin E and carotenoids are altered in Alzheimer’s disease: A case-
control study. Alzheimer’s Dement Transl Res Clin Interv 2017;3:432–9. doi:10.1016/J.
TRCI.2017.06.006.
Murphy M. P, Smith R. A. J. Targeting Antioxidants to Mitochondria by Conjugation to
Lipophilic Cations. Annu Rev Pharmacol Toxicol 2007;47:629–56. doi:10.1146/annurev.
pharmtox.47.120505.105110.
Mutisya E. M, Bowling A. C, Beal M. F. Cortical cytochrome oxidase activity is reduced in
Alzheimer’s disease. J Neurochem 1994;63:2179–84.
Nadeau A, Roberge A. G. Effects of vitamin B12 supplementation on choline acetyltransferase
activity in cat brain. Int J Vitam Nutr Res 1988;58:402–6.
Naidu K. A. Vitamin C in human health and disease is still a mystery? An overview. Nutr J
2003;2:7. doi:10.1186/1475-2891-2-7.
Noor R, Mittal S, Iqbal J. Superoxide dismutase--applications and relevance to human diseases.
Med Sci Monit 2002;8:RA210-5.
Nordberg J., Arnér E. S. Reactive oxygen species, antioxidants, and the mammalian thioredoxin
system. Free Radic Biol Med 2001;31:1287–312.
Nourooz-Zadeh J., Liu E. H. C, Yhlen B, Änggåard E. E, Halliwell B. F4 - Isoprostanes as
Specific Marker of Docosahexaenoic Acid Peroxidation in Alzheimer’s Disease. J
Neurochem 1999;72:734–40. doi:10.1046/j.1471-4159.1999.0720734.x.
Nunomura A, Castellani R. J, Zhu X, Moreira P. I, Perry G, Smith M. A. Involvement of
Oxidative Stress in Alzheimer Disease. J Neuropathol Exp Neurol 2006;65:631–41.
doi:10.1097/01.jnen.0000228136.58062.bf.
Nunomura A, Moreira P. I, Takeda A, Smith M. A, Perry G. Oxidative RNA damage and
neurodegeneration. Curr Med Chem 2007;14:2968–75.
Nunomura A, Perry G, Pappolla MA, Wade R, Hirai K, Chiba S, et al. RNA oxidation is a
prominent feature of vulnerable neurons in Alzheimer’s disease. J Neurosci 1999;19:1959–
64. doi:10.1523/JNEUROSCI.19-06-01959.1999.
Obulesu M, Dowlathabad MR, Bramhachari PV. Carotenoids and Alzheimer’s Disease: An
insight into therapeutic role of retinoids in animal models. Neurochem Int 2011;59:535–
41. doi:10.1016/j.neuint.2011.04.004.
Ogata M, Hoshi M, Shimotohno K, Urano S, Endo T. Antioxidant activity of Magnolol,
honokiol, and related phenolic compounds. J Am Oil Chem Soc 1997;74:557–62.
doi:10.1007/s11746-997-0180-3.

Oken BS, Storzbach D M, Kaye J. A. The efficacy of Ginkgo biloba on cognitive function in
Alzheimer disease. Arch Neurol 1998;55:1409–15.
Ono K, Hamaguchi T, Naiki H, Yamada M. Anti-amyloidogenic effects of antioxidants:
Implications for the prevention and therapeutics of Alzheimer’s disease. Biochim Biophys
Acta - Mol Basis Dis 2006a;1762:575–86. doi:10.1016/J.BBADIS.2006.03.002.
Ono K, Hirohata M, Yamada M. α-Lipoic acid exhibits anti-amyloidogenicity for β-amyloid
fibrils in vitro. Biochem Biophys Res Commun 2006b;341:1046–52. doi:10.1016/j.bbrc.
2006.01.063.
Packer L, Tritschler H. J, Wessel K. Neuroprotection by the metabolic antioxidant alpha-lipoic
acid. Free Radic Biol Med 1997;22:359–78.
Padayatty SJ, Katz A, Wang Y, Eck P, Kwon O, Lee J-H, et al. Vitamin C as an antioxidant:
evaluation of its role in disease prevention. J Am Coll Nutr 2003;22:18–35.
Padurariu M, Ciobica A, Hritcu L, Stoica B, Bild W, Stefanescu C. Changes of some oxidative
stress markers in the serum of patients with mild cognitive impairment and Alzheimer’s
disease. Neurosci Lett 2010;469:6–10. doi:10.1016/j.neulet.2009.11.033.
Paiva S. A. R, Russell R. M. β-Carotene and Other Carotenoids as Antioxidants. J Am Coll
Nutr 1999;18:426–33. doi:10.1080/07315724.1999.10718880.
Panza F, Solfrizzi V, Barulli M. R, Bonfiglio C, Guerra V, Osella A, et al. Coffee, tea, and
caffeine consumption and prevention of late-life cognitive decline and dementia: A
systematic review. J Nutr Health Aging 2015;19:313–28. doi:10.1007/s12603-014-
0563-8.
Pappolla M. A, Sos M, Omar R. A, Bick R. J, Hickson-Bick D. L, Reiter R. J, et al. Melatonin
prevents death of neuroblastoma cells exposed to the Alzheimer amyloid peptide. J
Neurosci 1997;17:1683–90.
Persson H. L, Yu Z, Tirosh O, Eaton J. W, Brunk U. T. Prevention of oxidant-induced cell
death by lysosomotropic iron chelators. Free Radic Biol Med 2003;34:1295–305.
Pham-Huy L. A, He H, Pham-Huy C. Free radicals, antioxidants in disease and health. Int J
Biomed Sci 2008;4:89–96.
Plant L. D, Boyle J. P, Smith I. F, Peers C, Pearson H. A. The production of amyloid beta
peptide is a critical requirement for the viability of central neurons. J Neurosci
2003;23:5531–5.
Praticò D. Oxidative stress hypothesis in Alzheimer’s disease: a reappraisal. Trends Pharmacol
Sci 2008;29:609–15. doi:10.1016/j.tips.2008.09.001.
Praticò D, M. Y Lee V, Trojanowski J. Q, Rokach J, Fitzgerald G. A. Increased F2-isoprostanes
in Alzheimer’s disease: evidence for enhanced lipid peroxidation in vivo. FASEB J
1998;12:1777–83.
Praticò D, Sung S. Lipid Peroxidation and Oxidative imbalance: Early functional events in
Alzheimer’s disease. J Alzheimer’s Dis 2004;6:171–5. doi:10.3233/JAD-2004-6209.
Puzzo D, Privitera L, Leznik E, Fà M, Staniszewski A, Palmeri A, et al. Picomolar amyloid-
beta positively modulates synaptic plasticity and memory in hippocampus. J Neurosci
2008;28:14537–45. doi:10.1523/JNEUROSCI.2692-08.2008.
Querfurth H. W, LaFerla F. M. Alzheimer’s Disease. N Engl J Med 2010;362:329–44.
doi:10.1056/NEJMra0909142.
Quiroz-Baez R, Rojas E, Arias C. Oxidative stress promotes JNK-dependent amyloidogenic
processing of normally expressed human APP by differential modification of α-, β- and γ-

secretase expression. Neurochem Int 2009;55:662–70. doi:10.1016/j.neuint.2009.

06.012.
Rajgopal A, Missler S. R, Scholten J. D. Magnolia officinalis (Hou Po) bark extract stimulates
the Nrf2-pathway in hepatocytes and protects against oxidative stress. J Ethnopharmacol
2016;193:657–62. doi:10.1016/j.jep.2016.10.016.
Ramesh S, Govindarajulu M, Lynd T, Briggs G, Adamek D, Jones E, et al. SIRT3 activator
Honokiol attenuates β-Amyloid by modulating amyloidogenic pathway. PLoS One
2018;13:e0190350. doi:10.1371/journal.pone.0190350.
Ramsey C. P, Glass C. A, Montgomery M. B, Lindl K. A, Ritson G. P, Chia L. A, et al.
Expression of Nrf2 in Neurodegenerative Diseases. J Neuropathol Exp Neurol
2007;66:75–85. doi:10.1097/nen.0b013e31802d6da9.
Regland B, Gottfries C-G, Oreland L. Vitamin B12-induced reduction of platelet monoamine
oxidase activity in patients with dementia and pernicious anaemia. Eur Arch Psychiatry
Clin Neurosci 1991;240:288–91. doi:10.1007/BF02189542.
Remington R, Bechtel C, Larsen D, Samar A, Doshanjh L, Fishman P, et al. A Phase II
Randomized Clinical Trial of a Nutritional Formulation for Cognition and Mood in
Alzheimer’s Disease. J Alzheimer’s Dis 2015;45:395–405. doi:10.3233/JAD-142499.
Remington R, Bechtel C, Larsen D, Samar A, Page R, Morrell C, et al. Maintenance of
Cognitive Performance and Mood for Individuals with Alzheimer’s Disease Following
Consumption of a Nutraceutical Formulation: A One-Year, Open-Label Study. J
Alzheimer’s Dis 2016;51:991–5. doi:10.3233/JAD-151098.
Rhein V, Song X, Wiesner A, Ittner LM, Baysang G, Meier F, et al. Amyloid- and tau
synergistically impair the oxidative phosphorylation system in triple transgenic
Alzheimer’s disease mice. Proc Natl Acad Sci 2009;106:20057–62. doi:10.1073/pnas.
0905529106.
Robinson R. A. S, Joshi G, Huang Q, Sultana R, Baker A. S, Cai J, et al. Proteomic analysis of
brain proteins in APP/PS-1 human double mutant knock-in mice with increasing amyloid
β-peptide deposition: Insights into the effects of in vivo treatment with N-acetylcysteine as
a potential therapeutic intervention in mild cognitive. Proteomics 2011;11:4243–56.
doi:10.1002/pmic.201000523.
Rochette L, Ghibu S, Richard C, Zeller M, Cottin Y, Vergely C. Direct and indirect antioxidant
properties of α-lipoic acid and therapeutic potential. Mol Nutr Food Res 2013;57:114–25.
doi:10.1002/mnfr.201200608.
Rodrigues C. M. P, Solá S, Brito M. A, Brondino C. D, Brites D, Moura J. J. G. Amyloid β-
Peptide Disrupts Mitochondrial Membrane Lipid and Protein Structure: Protective Role of
Tauroursodeoxycholate. Biochem Biophys Res Commun 2001;281:468–74. doi:10.1006/
bbrc.2001.4370.
Rodríguez-Martín T, Cuchillo-Ibáñez I, Noble W, Nyenya F, Anderton B. H, Hanger D. P. Tau
phosphorylation affects its axonal transport and degradation. Neurobiol Aging
2013;34:2146–57. doi:10.1016/j.neurobiolaging.2013.03.015.
Rohr-Udilova N V., Stolze K, Sagmeister S, Nohl H, Schulte-Hermann R, Grasl-Kraupp B.
Lipid hydroperoxides from processed dietary oils enhance growth of hepatocarcinoma
cells. Mol Nutr Food Res 2008;52:352–9. doi:10.1002/mnfr.200700149.
Rosales-Corral S, Tan D-X, Reiter R. J, Valdivia-Velázquez M, Martínez-Barboza G, Acosta-
Martínez J. P, et al. Orally administered melatonin reduces oxidative stress and

proinflammatory cytokines induced by amyloid-beta peptide in rat brain: a comparative, in

vivo study versus vitamin C and E. J Pineal Res 2003;35:80–4.
Rousset S, Alves-Guerra M-C, Mozo J, Miroux B, Cassard-Doulcier A-M, Bouillaud F, et al.
The biology of mitochondrial uncoupling proteins. Diabetes 2004;53 Suppl 1:S130-5.
Sabogal-Guáqueta A. M, Muñoz-Manco J. I, Ramírez-Pineda J. R, Lamprea-Rodriguez M,
Osorio E, Cardona-Gómez G. P. The flavonoid quercetin ameliorates Alzheimer’s disease
pathology and protects cognitive and emotional function in aged triple transgenic
Alzheimer’s disease model mice. Neuropharmacology 2015;93:134–45. doi:10.1016/
j.neuropharm.2015.01.027.
Sambamurti K, Kinsey R, Maloney B, Ge Y-W, Lahiri D. K. Gene structure and organization
of the human beta-secretase (BACE) promoter. FASEB J 2004;18:1034–6.
doi:10.1096/fj.03-1378fje.
Satoh A, Tsuji S, Okada Y, Murakami N, Urami M, Nakagawa K, et al. Preliminary Clinical
Evaluation of Toxicity and Efficacy of A New Astaxanthin-rich Haematococcus pluvialis
Extract. J Clin Biochem Nutr 2009;44:280–4. doi:10.3164/jcbn.08-238.
Scheltens P, Kamphuis P. J. G. H, Verhey F. R. J, Olde Rikkert M. G. M, Wurtman RJ,
Wilkinson D, et al. Efficacy of a medical food in mild Alzheimer’s disease: A randomized,
controlled trial. Alzheimer’s Dement 2010;6:1–10.e1. doi:10.1016/j.jalz.2009.10.003.
Scheltens P, Twisk J. W. R, Blesa R, Scarpini E, von Arnim C. A. F, Bongers A, et al. Efficacy
of Souvenaid in Mild Alzheimer’s Disease: Results from a Randomized, Controlled Trial.
Schipper H, Bennett D, Liberman A, Bienias J, Schneider J, Kelly J, et al. Glial heme
oxygenase-1 expression in Alzheimer disease and mild cognitive impairment. Neurobiol
Aging 2006;27:252–61. doi:10.1016/j.neurobiolaging.2005.01.016.
Schipper H, Song W. A Heme Oxygenase-1 Transducer Model of Degenerative and
Developmental Brain Disorders. Int J Mol Sci 2015;16:5400–19. doi:10.3390/
ijms16035400.
Schipper H. M, Cissé S, Stopa E. G. Expression of heme oxygenase-1 in the senescent and
alzheimer-diseased brain. Ann Neurol 1995;37:758–68. doi:10.1002/ana.410370609.
Serafini M. M, Catanzaro M, Rosini M, Racchi M, Lanni C. Curcumin in Alzheimer’s disease:
Can we think to new strategies and perspectives for this molecule? Pharmacol Res
2017;124:146–55. doi:10.1016/j.phrs.2017.08.004.
Shah R. C, Kamphuis P. J, Leurgans S, Swinkels S. H, Sadowsky C. H, Bongers A, et al. The
S-Connect study: results from a randomized, controlled trial of Souvenaid in mild-to-
moderate Alzheimer’s disease. Alzheimers Res Ther 2013;5:59. doi:10.1186/alzrt224.
Shen C, Chen Y, Liu H, Zhang K, Zhang T, Lin A, et al. Hydrogen peroxide promotes Abeta
production through JNK-dependent activation of gamma-secretase. J Biol Chem
2008;283:17721–30. doi:10.1074/jbc.M800013200.
Sheu S-S, Nauduri D, Anders M. W. Targeting antioxidants to mitochondria: A new therapeutic
direction. Biochim Biophys Acta - Mol Basis Dis 2006;1762:256–65.
doi:10.1016/j.bbadis.2005.10.007.
Sies H, Stahl W. Vitamins E and C, beta-carotene, and other carotenoids as antioxidants. Am J
Clin Nutr 1995;62:1315S–1321S. doi:10.1093/ajcn/62.6.1315S.
Sil S, Ghosh T, Gupta P, Ghosh R, Kabir S. N, Roy A. Dual Role of Vitamin C on the
Neuroinflammation Mediated Neurodegeneration and Memory Impairments in Colchicine

Induced Rat Model of Alzheimer Disease. J Mol Neurosci 2016;60:421–35.

doi:10.1007/s12031-016-0817-5.
Sinclair A. J, Bayer A. J, Johnston J, Warner C, Maxwell S. R. J. Altered plasma antioxidant
status in subjects with Alzheimer’s disease and vascular dementia. Int J Geriatr Psychiatry
1998;13:840–5. doi:10.1002/(SICI)1099-1166(1998120)13:12<840::AID-GPS877>3.0.C
O;2-R.
Singh M, Saini H. K. Resident Cardiac Mast Cells and Ischemia-Reperfusion Injury. J
Cardiovasc Pharmacol Ther 2003;8:135–48. doi:10.1177/107424840300800207.
Smith JV, Luo Y. Elevation of oxidative free radicals in Alzheimer’s disease models can be
attenuated by Ginkgo biloba extract EGb 761. J Alzheimers Dis 2003;5:287–300.
Smith MA, Hirai K, Hsiao K, Pappolla MA, Harris PL, Siedlak SL, et al. Amyloid-beta
deposition in Alzheimer transgenic mice is associated with oxidative stress. J Neurochem
1998;70:2212–5.
Stamer K, Vogel R, Thies E, Mandelkow E, Mandelkow E-M. Tau blocks traffic of organelles,
neurofilaments, and APP vesicles in neurons and enhances oxidative stress. J Cell Biol
2002;156:1051–63. doi:10.1083/jcb.200108057.
Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN. Bilirubin is an antioxidant of
possible physiological importance. Science 1987;235:1043–6.
Stough C, Downey L. A, Lloyd J, Silber B, Redman S, Hutchison C, et al. Examining the
nootropic effects of a special extract of Bacopa monniera on human cognitive functioning:
90 day double-blind placebo-controlled randomized trial. Phyther Res 2008;22:1629–34.
doi:10.1002/ptr.2537.
Su B, Wang X, Bonda D, Perry G, Smith M, Zhu X. Abnormal Mitochondrial Dynamics—A
Novel Therapeutic Target for Alzheimer’s Disease? Mol Neurobiol 2010;41:87–96.
doi:10.1007/s12035-009-8095-7.
Sulakhiya K, Kumar P, Gurjar S. S, Barua C. C, Hazarika N. K. Beneficial effect of honokiol
on lipopolysaccharide induced anxiety-like behavior and liver damage in mice. Pharmacol
Biochem Behav 2015;132:79–87. doi:10.1016/j.pbb.2015.02.015.
Sulakhiya K, Kumar P, Jangra A, Dwivedi S, Hazarika N. K, Baruah CC, et al. Honokiol
abrogates lipopolysaccharide-induced depressive like behavior by impeding
neuroinflammation and oxido-nitrosative stress in mice. Eur J Pharmacol 2014;744:124–
31. doi:10.1016/j.ejphar.2014.09.049.
Swomley A. M, Förster S, Keeney J. T, Triplett J, Zhang Z, Sultana R, et al. Abeta, oxidative
stress in Alzheimer disease: Evidence based on proteomics studies. Biochim Biophys Acta
- Mol Basis Dis 2014;1842:1248–57. doi:10.1016/j.bbadis.2013.09.015.
Szeto H. H. Development of Mitochondria-targeted Aromatic-cationic Peptides for
Neurodegenerative Diseases. Ann N Y Acad Sci 2008;1147:112–21. doi:10.1196/
annals.1427.013.
Szeto H. H. Mitochondria-targeted peptide antioxidants: Novel neuroprotective agents. AAPS
J 2006;8:E521–31. doi:10.1208/aapsj080362.
Takemoto A. S, Berry M. J, Bellinger F. P. Role of selenoprotein P in Alzheimer’s disease.
Ethn Dis 2010;20:S1-92–5.
Tamagno E, Bardini P, Obbili A, Vitali A, Borghi R, Zaccheo D, et al. Oxidative stress
increases expression and activity of BACE in NT2 neurons. Neurobiol Dis 2002;10:279–
88.

Tan D, Chen L, Poeggeler B, Manchester L, Reiter R, Reiter R, et al. Melatonin a potent

endogenous hydroxyl radical scavenger 1993.
Tan S, Sagara Y, Liu Y, Maher P, Schubert D. The regulation of reactive oxygen species
production during programmed cell death. J Cell Biol 1998;141:1423–32.
Tang M, Taghibiglou C. The Mechanisms of Action of Curcumin in Alzheimer’s Disease. J
Alzheimer’s Dis 2017;58:1003–16. doi:10.3233/JAD-170188.
Thomas D. A, Stauffer C, Zhao K, Yang H, Sharma V. K, Szeto H. H, et al. Mitochondrial
Targeting with Antioxidant Peptide SS-31 Prevents Mitochondrial Depolarization,
Reduces Islet Cell Apoptosis, Increases Islet Cell Yield, and Improves Posttransplantation
Function. J Am Soc Nephrol 2007;18:213–22. doi:10.1681/ASN. 2006080825.
Tuppo E. E, Forman L. J. Free radical oxidative damage and Alzheimer’s disease. J Am
Osteopath Assoc 2001;101:S11-5.
Uabundit N, Wattanathorn J, Mucimapura S, Ingkaninan K. Cognitive enhancement and
neuroprotective effects of Bacopa monnieri in Alzheimer’s disease model. J
Ethnopharmacol 2010;127:26–31. doi:10.1016/j.jep.2009.09.056.
Valko M, Leibfritz D, Moncol J, Cronin M. T. D, Mazur M, Telser J. Free radicals and
antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol
2007;39:44–84. doi:10.1016/j.biocel.2006.07.001.
Vendemiale G, Grattagliano I, Altomare E. An update on the role of free radicals and
antioxidant defense in human disease. Int J Clin Lab Res 1999;29:49–55.
Vural H, Demirin H, Kara Y, Eren I, Delibas N. Alterations of plasma magnesium, copper,
zinc, iron and selenium concentrations and some related erythrocyte antioxidant enzyme
activities in patients with Alzheimer’s disease. J Trace Elem Med Biol 2010;24:169–73.
doi:10.1016/j.jtemb.2010.02.002.
Wang X, Su B, Zheng L, Perry G, Smith MA, Zhu X. The role of abnormal mitochondrial
dynamics in the pathogenesis of Alzheimer’s disease. J Neurochem 2009;109 Suppl 1:153–
9. doi:10.1111/j.1471-4159.2009.05867.x.
Weisiger RA, Fridovich I. Superoxide dismutase. Organelle specificity. J Biol Chem
1973;248:3582–92.
Wojsiat J, Zoltowska K. M, Laskowska-Kaszub K, Wojda U. Oxidant/Antioxidant Imbalance
in Alzheimer’s Disease: Therapeutic and Diagnostic Prospects. Oxid Med Cell Longev
2018;2018:1–16. doi:10.1155/2018/6435861.
Wu Z, Zhang J, Zhao B. Superoxide Anion Regulates the Mitochondrial Free Ca 2+ Through
Uncoupling Proteins. Antioxid Redox Signal 2009;11:1805–18. doi:10.1089/ars.
2009.2427.
Yang X, Yang Y, Li G, Wang J, Yang E. S. Coenzyme Q10 Attenuates β-Amyloid Pathology
in the Aged Transgenic Mice with Alzheimer Presenilin 1 Mutation. J Mol Neurosci
2008;34:165–71. doi:10.1007/s12031-007-9033-7.
Yankner B. A, Lu T. Amyloid beta-protein toxicity and the pathogenesis of Alzheimer disease.
J Biol Chem 2009;284:4755–9. doi:10.1074/jbc.R800018200.
Yao Y, Chinnici C, Tang H, Trojanowski J. Q, Lee V. M, Praticò D. Brain inflammation and
oxidative stress in a transgenic mouse model of Alzheimer-like brain amyloidosis. J
Neuroinflammation 2004;1:21. doi:10.1186/1742-2094-1-21.
Yiannopoulou K. G, Papageorgiou S. G. Current and future treatments for Alzheimer’s disease.
Ther Adv Neurol Disord 2013;6:19–33. doi:10.1177/1756285612461679.

Yong V. W, Perry T. L, Krisman A. A. Depletion of glutathione in brainstem of mice caused

by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine is prevented by antioxidant
pretreatment. Neurosci Lett 1986;63:56–60. doi:10.1016/0304-3940(86)90012-1.
Zafarullah M, Li W. Q, Sylvester J, Ahmad M. Molecular mechanisms of N-acetylcysteine
actions. Cell Mol Life Sci 2003;60:6–20.
Zaman Z, Roche S, Fielden P, Frost PG, Niriella DC, Cayley ACD. Plasma Concentrations of
Vitamins A and E and Carotenoids in Alzheimer’s Disease. Age Ageing 1992;21:91–4.
doi:10.1093/ageing/21.2.91.
Zeng J, Chen L, Wang Z, Chen Q, Fan Z, Jiang H, et al. Marginal vitamin A deficiency
facilitates Alzheimer’s pathogenesis. Acta Neuropathol 2017;133:967–82. doi:10.1007/
s00401-017-1669-y.
Zhang D-M, Ye J-X, Mu J-S, Cui X-P. Efficacy of Vitamin B Supplementation on Cognition
in Elderly Patients With Cognitive-Related Diseases. J Geriatr Psychiatry Neurol
2017;30:50–9. doi:10.1177/0891988716673466.
Zheng W-H, Bastianetto S, Mennicken F, Ma W, Kar S. Amyloid beta peptide induces tau
phosphorylation and loss of cholinergic neurons in rat primary septal cultures.
Neuroscience 2002;115:201–11.
Zhou J-N, Liu R-Y, Kamphorst W, Hofman MA, Swaab DF. Early neuropathological
Alzheimer’s changes in aged individuals are accompanied by decreased cerebrospinal fluid
melatonin levels. J Pineal Res 2003;35:125–30.
Zhu X, Perry G, Moreira P. I, Aliev G, Cash A. D, Hirai K, et al. Mitochondrial abnormalities
and oxidative imbalance in Alzheimer disease. J Alzheimers Dis 2006;9:147–53.
Zhu X, Rottkamp C. A, Boux H, Takeda A, Perry G, Smith M. A. Activation of p38 kinase
links tau phosphorylation, oxidative stress, and cell cycle-related events in Alzheimer
disease. J Neuropathol Exp Neurol 2000;59:880–8.
Zou K, Gong J-S, Yanagisawa K, Michikawa M. A novel function of monomeric amyloid beta-
protein serving as an antioxidant molecule against metal-induced oxidative damage. J
Neurosci 2002;22:4833–41.

Chapter 8
OXIDANT/ANTIOXIDANT IMBALANCES
IN HUNTINGTON’S DISEASE
Arup Kumar Misra1,, Surjit Singh1, Pramod Kumar Sharma1,

Sneha Ambwani1 and Sushil Kumar Varma2
All India Institute of Medical Sciences, Jodhpur, India
1
2
Mahatma Gandhi Institute of Medical Sciences, Sevagram, India
ABSTRACT
Cytosine-adenine-guanine (CAG) expansion mutation of the huntingtin (HTT) gene
leads to a neurodegenerative disorder known as Huntington’s disease (HD). The
polyglutamine neurodegeneration leads to neuronal dysfunction that may eventually lead
to progressive psychiatric and cognitive impairment due to neuronal cell death. Oxidative
stress results in mutation of HTT gene that leads to mitochondrial dysfunction and building
up of reactive oxygen species (ROS). The oxidative damage to the structural components
of cells like proteins; lipids and DNA that account for the neuronal death. Antioxidants
have the therapeutic potential against free radical toxicity in many neurodegenerative
disorders and in the management of aging. Further, it has demonstrated promise in slowing
down the progression of HD in various models of transgenic mouse and in clinical trials.
They offer protection in HD by promoting the neurogenesis and neuroprotection of the
brain against the oxidative stress thus hold great promise to support and reduce the severity
of HD. It has also been proposed that transcribed antioxidant enzymes expression is the
better option for better management of HD in the future. The aim of the chapter is to
describe the pathogenesis of the HD and the potential role of antioxidants, which may slow
down the disease progression and improve the quality of life.

Corresponding Author’s Email: arup2003m@gmail.com.

204 Arup Kumar Misra, Surjit Singh, Pramod Kumar Sharma et al.
1. INTRODUCTION
Huntington’s disease (HD) is a polyglutamine nervous system disorder that cripples the
patient’s life by causing myriad of psychiatric symptoms and affecting insight. The prevalence
of HD in western countries is about 3 to 10 individuals per lakhs (Vonsattel and DiFiglia, 1998).
The pathogenesis of HD is due to an unstable elaboration of cytosine-adenine-guanine (CAG)
repeats of huntingtin protein coded within the HTT gene. This unstable elaboration of
huntingtin protein leads to stretching of glutamine residues which is present in the NH2-
terminal of huntingtin (The Huntington’s Disease Collaborative Research Group, 1993). This
uncommon stretching of the glutamine residues leads to the formation and accumulation of
intranuclear inclusions in neuron (DiFiglia et al., 1997). Cell deterioration happens mainly in
the striatum and some part of the cortex of the brain even though the abnormal intranuclear
inclusions of protein are ubiquitously expressed (Vonsattel and DiFiglia, 1998; Gil and Rego,
2008).
The incidence of HD is high in adults especially between 35 to 50 years of age. The
pathogenesis of HD has a progressive pattern. After the development of first mild symptoms,
it progresses slowly and becomes lethal after a gap of a decade or two. The classical symptoms
of HD are motor incoordination, especially the loss of voluntary movement which is followed
by bradykinesia and rigidity in the ensuing and fatal stage of the disorder. The first sign of
cognitive impairment in the patient of HD is the slowing down of intellectual processing (Gil
and Rego, 2008). The progressive nature of the disease reveals initial cognitive impairment
followed by the motor symptoms that are more prominent after a decade or two. There is a
progressive worsening of cognition and some patients may show dementia (Folstein et al.,
1983). Manic-depressive behaviour (Folstein et al., 1983) and psyche changes are often part of
the psychiatric syndrome seen in the patients of HD (Dewhurst et al., 1970; Jensen et al., 1993).
Muscle wasting due to incoordination and energy expenditure consequent to abnormal
metabolic processes are some of the clinical features of this disorder. Severe disability and
ultimately death supervenes usually after 15 to 20 years of the first symptoms.
In modern era of genetics, the detection of the genetic make-up of HD makes it possible
for the early intervention and management of the disease. This advancement prioritizes the need
for the introduction of disease-modifying therapies (Tabrizi et al., 2011). In 1993, the gene for
HD was discovered (The Huntington’s Disease Collaborative Research Group, 1993). A
number of potential drug targets, new drug discovery and delivery system, and therapeutic
approaches have been developed (Brocardo et al., 2012; Brocardo et al., 2013; Gil et al., 2013;
Gil-Mohapel, 2012). Despite the growing efforts of the international HD research community,
the goal for cure or satisfactory treatment of HD is farsighted.
The worldwide prevalence of HD varies due to the genetic diversity and geographical
distribution (Walker, 2007; Warby et al., 2011). The highest incidence of this disorder is
observed in Caucasians as compared to its Asian counterpart. The prevalence of HD is high in
Western Europe, Australia, and North America. India has the largest number of total HD cases
in Asia (Rawlins et al., 2016; Moily et al., 2014).
Novel and non-invasive therapeutic strategies with minimal side effects hold the promise
for the successful management of HD. In addition, physical activities, supplement enriched
diets, and palliative care are the other viable options that can be tried in conjunction with drug

Oxidant/Antioxidant Imbalances in Huntington's Disease 205
treatment. The chapter reviews the genetic variation of this dreaded disorder and how proper
use of antioxidants may retard the disease progression.
2. DESCRIPTIVE CHANGES IN HUNTINGTON’S DISEASE

2.1. Structural Changes in Huntington’s Disease
The onset of disorder at a particular age is due to the abnormal expression of CAG repeats
in the HTT gene. The remaining variation in the gene also correlates strongly with inheritance.
The variation in CAG repeats lengths corresponds with the period of the disorder. The length
of CAG repeats longer than 60 have been related to juvenile-onset HD (Vonsattel
et al., 1998). Genetic testing is the best methods to diagnose this disorder (Wexler et al., 2004).
As known, CAG repeat length correlates with predicting age of onset, but provides less
information about the progressive nature of the disease. The factors associated with the
progressive nature of the disease will help to detect and design rational therapeutic intervention
(Rosenblatt et al., 2006).
The factors involved in the pathogenesis in HD are responsible for lesions in the striatum
manifested as involuntary movements like chorea (uncontrollable dance-like movements). On
autopsy of brains of HD, loss of the medium-sized spiny neurons (MSNs) involved in
GABAergic transmission in the striatal tissue; damage to thalamus, hippocampus, amygdala
and atrophy of cortex have been seen (Hayden et al., 2001). The cortical atrophy in the motor-
sensory cortex progress to involve the occipital, parietal and limbic cortices (Rosas et al., 2008).
Before the onset of motor and cognitive dysfunctions, the pathophysiological changes in the
brain may precede by years. In HD, MSNs are the most vulnerable tissue yet some neurons
survive (Reiner et al., 1988; Albin et al., 1992). NADPH diaphorase/NO synthetase are the
enzymes that are spared in the pathogenesis of HD. The cholinergic interneurons and calretinin
interneurons are spared partly in the progression of the disease (Ferrante et al., 1985; Ferrante
et al., 1987). The loss of the GABA/enkephalin neurons projecting to the lateral globus pallidus
(indirect pathway) leads to the development of chorea in HD. Later, the motor incoordination
manifested as increase in muscle tone, abnormal eye movements, and dystonia are due to the
damage of GABA/substance P/dynorphin cells projecting to the substantia nigra pars reticulata
and the medial globus pallidus (direct pathway) (Albin et al., 1992; Albin et al., 1989).
2.2. Role of Genes in Huntington’s Disease
Degeneration of striatum correlates well with the mutation in the huntingtin’s protein. The
huntingtin’s protein consists of about 3,300 amino acids. The cytoplasm of the cell has the
protein and is found in every tissue with highest level in the brain and testes. The physiological
activities of the protein are not elucidated in the context but were shown to have a role in
neurogenesis and essential for the cell survival (White et al., 1997; Dragatsis et al., 2000; Reiner
et al., 2003). Wild-type of htt protein has been proposed to involve in many physiological roles
like for intracellular signalling endocytosis, RNA biogenesis, vesicle trafficking, mitosis, and
apoptosis. It has the inclination for binding with synaptic vesicles, microtubules and spindle

poles (Duyao et al., 1995; Rigamonti et al., 2001; DiFiglia et al., 1995; Harjeset al., 2003; Velier
et al., 1998; Cavistonet al., 2009; Godin et al., 2010). In HD, the mutant httproteins level that
disrupt the biological activities of the neurons and leads to toxicity. The toxicity manifests as
transcriptional modulation, aggregation of protein and excitotoxicity. On the other hand,
disruption of the function of wild-type htt may progress to diminish the elaboration of BDNF
gene and vesicle trafficking. The protein, mhtt should be target for intervention as it is
ubiquitously expressed in homozygous mice and in wild-type htt, with no particular selectivity.
These findings lead to the conclusion that there may be other characteristics of mhtt, or
abnormal neurons within these regions that makes the cells susceptible to mhtt toxicity (Duyao
et al., 1995).
Approximately 5% of the HTT in the cell is found in the nucleus suggesting that it transport
to and fro between the cytoplasm and nucleus (Sawa et al., 2005). The N-terminal 17-amino
acid chain of htt proposed to be binding to the nuclear exporter translocated promoter region
(Tpr) for the nuclear export signal. Augmentation of the repeat of polyglutamine (polyQ) is
proposed to interfere in the nuclear export signal leading to assembly of mhtt in the nucleus
(Imarisio et al., 2008). The inhibition of shuttling of the mhtt leads to its increase in nuclear
localization that enhances cytotoxicity (Baeet al., 2006). The aggregation of htt in the brain’s
neurons is a pathological hallmark which suggests that the proteolytic cleavage of htt in the
brain of the patients as well as in various preclinical models of rodent plays an important part
in the pathologic process of HD (Wheeler et al., 2000; DiFiglia et al., 1997). Protein truncation
in vitro and animal studies models exacerbated polyQ induced toxicity. N-terminal mhtt
fragments play a major role in inducing clinical syndromes related to HD in various animal
models (Palfi et al., 2007). It is proposed that truncated mhtt also induces apoptosis (Martindale
et al., 1998). Substance that inhibits the cleavage of mutant htt reduces toxicity, thus indicating
that defective htt proteolysis is key in pathophysiology of HD (Miller et al., 2010; Johri and
Beal, 2010).
Based on evidences, it is confirmed that specks of htt are found mostly in the cortical
neurons. The assembly of specks of mhtt in cortical neurons progresses to corticostriatal
dysfunction an important event in HD pathogenesis (Fusco et al., 1999). The fragmented htt
also binds to various transcription factors like the caspases, calmodulins, and transglutaminases
(Browne and Beal, 2006). These sequences of events may trigger the cascades of damage in
addition to compensatory molecular processing and genetic programming due to mhtt and its
fragments. These events may culminate into neuronal dysfunctional which make the neurons
prone to disordered neurophysiology, expression of potentiating inflammatory and pro-
apoptotic signals that activate caspase 8, increased transglutaminase activity, malfunctioning
proteolysis, oxidative injury, excitotoxic stress, and energy depletion.
The correlation between impaired function of mitochondria and huntingtin protein
mutation leads to abnormalities of dynamics and metabolic function of mitochondria.
Subsequent to mitochondrial dysfunction there occurs destruction and degeneration of neurons
in affected regions of the brain as have been demonstrated in many studies(Browne and Beal,
2004; Kim et al., 2010; Costa et al., 2010; Song et al., 2011; Johri et al., 2011; Shirendeb et al.,
2011). Binding of mhtt to several transcriptional regulators leads to aberrant transcription and
dysfunction of mitochondrial activity in animal models of HD. Impairment of mitochondria is
also proposed to be caused by the decrease elaboration of peroxisome proliferator-activated
receptor gamma coactivator-1α (PGC-1α), an important co-regulator for mitochondrial
metabolism and production of antioxidant enzymes (Cui et al., 2006; Weydt et al., 2006; McGill

and Beal, 2006; Chaturvedi et al., 2009; Chaturvedi et al., 2010; Johri et al., 2011). PGC-1α
reductions correlate well with the diminishing numbers of striatal mitochondria in the
pathologic process of HD (Kim et al., 2010). The important metabolic events like the
mitochondrial fission-fusion events and mitochondrial trafficking along axons and dendrites
are regulated by the interaction between mhtt proteins with mitochondria or with various
protein complexes (Reddy et al., 2009). The increased elaboration of mhtt may disrupt the
structural and biochemical functions of mitochondria like ultrastructure abnormality, impaired
buffering of calcium, defects of bioenergetics and deletions of mitochondrial DNA which will
lead to the failure of mitochondrial fission and fusion reactions (Costa et al., 2010; Song et al.,
2011). In patients of HD, the above abnormality plays a significant role in increased expression
of mitochondrial fission proteins (Drp1 and Fis1), mitochondrial matrix protein CypD;
decreased levels of mitochondrial fusion proteins (Mfn1, Mfn2, Opa1 and Tomm40), and
increase levels of CypD (Kim et al., 2010; Shirendeb et al., 2011). In patients of HD, there are
alarming levels of oligomers of mhtt with evidence of oxidative DNA damage and loss of
mitochondrial function in the cerebral cortex. Medium spiny neurons due to its long projections
make them more susceptible to increased mitochondrial fission (Kim et al., 2010; Shirendeb et
al., 2011). Decreased levels of PGC-1α reflect reduced numbers of mitochondria of the HD
medium spiny neurons (Kim et al., 2010). Presence of mhtt oligomers in the nuclei and
mitochondria of neurons have been documented by immunolabelling of brain samples of
patients with HD.
3. OXIDATIVE STRESS
Oxidative stress is defined by the disruption of the balance between reactive oxygen species
(ROS) production and scavenger action. The prolongation of oxidative stress makes the cells,
tissues, and organs prone to oxidative damage (Sies, 1991). Normal cellular metabolism in
mitochondria produces ROS as its by-product (Halliwell and Gutteridge, 1990). Various
metabolic pathways like xanthine oxidase and reduced nicotinamide adenosine dinucleotide
phosphate (NADPH) oxidase (NOX) pathways also produce ROS in the cytoplasm (Gao et al.,
2003; Infanger et al., 2006). ROS can also be produced by exposure to various inducing
oxidative agents like alcohol, ultraviolet radiation, chemical reagents, cigarette smoke, etc.
(Zadak et al., 2009).
Cellular and immune function get boast by the low to moderate concentrations of ROS and
RNS (Reactive Nitrogen Species) but their higher concentrations will cause oxidative stress,
and later damage to the structural components of cells (Halliwell, 2007). The most susceptible
organ to overproduction of ROS is brain as its physiological need of oxygen is high but its
vulnerability increases to the high amount of redox-active metals (such and copper and iron)
and oxidizable polyunsaturated fatty acids (Gerlach et al., 1994; Halliwell, 1992; Sokoloff,
1999).
As the age increases, the antioxidant defense system has an inverse relationship with the
oxidative stress (due either to overproduction of ROS or RNS) (Valko et al., 2007). This
mechanism will lead to neuronal damage, reducing neuroprotection and ultimately leading to
many neurodegenerative diseases, including HD.

Superoxide, hydroxyl, peroxyl free radicals and nitrogen intermediates (NO and
peroxynitrite) belongs to ROS. Mitochondria are the main source of superoxide production in
the cell (Cadenas and Sies, 1998). The addition of one electron to dioxygen forms superoxide
anion radical (O2·−) (Miller et al, 1990). In electron transport chain of the mammalian cell,
mitochondria is the major source of ATP which is the final source of energy to sustain life.
Superoxide is generated during the energy transduction process when a minimal number of
electrons prematurely add up to the oxygen molecule, in place of reducing oxygen molecule to
form water (Kovacic et al., 2005). Complexes I and III of the electron transport chain are
responsible for the formation of superoxide in the cells and the strong anionic charge of the ion
make it possible to transverse the inner membrane of mitochondria. Complex I-dependent
superoxide has been demonstrated to exclusively release into the matrix (Muller et al., 2004).
It is only under the conditions of oxidative stress that free iron from iron-containing molecules
is released by the excess superoxide which leads to the formation of highly reactive hydroxyl
radical,-OH (Fenton reaction). Peroxyl radicals (ROO-) and HOO-, a protonated form of
superoxide (O2−) known as hydroperoxyl radical or perhydroxyl radical are the other reactive
radicals formed in living systems from oxygen. Fatty acid peroxidation occurs in the cells by
the hydroperoxyl radical (Aikens et al., 1991). Peroxynitrite (ONOO−) is formed in the
extracellular space by the reaction between superoxide and NO; the product formed has the
ability to traverse the cell membranes and damage intracellular elements (Beckman et al.,
1996).
Reactive oxygen species are constantly assembled by the multiple metabolic chains in the
aerobic cells. ROS maintain homeostasis of the cells by serving as a marker of the cell
irrespective of its conditions and it plays an essential role to sustain the cellular function. It has
the ability to damage the cellular components of the cells by the oxidative damage when there
is an imbalance with the antioxidant systems (Barja, 2004; Boveris, 1998; Lenaz, 2001). The
oxidative damage (Figure 1) commences by ROS may lead to a wide span of phenotypic
responses that can be from gene expression activation to proliferation to growth arrest and
ultimately to senescence or cell death (Droge, 2002; Hensley et al., 2000; Finkel, 2001).
Figure 1. Sequence of events leading to oxidative stress in Huntington’s disease.

3.1. Oxidative Stress in Neurodegeneration
Proteins, polysaccharides, lipids or nucleic acids are the major macromolecules which are
affected by the ROS. In the brain, the neurons with its intrinsic properties are more susceptible
to oxidative stress. The intrinsic factors make the neurons prone to peroxidation; forms reactive
hydroxyl radicals and have low levels of antioxidants which make it prone to oxidative stress.
Mitochondria supply the required number of ATP for the intense energy demands of neurons.
Excess production of ROS in mitochondria makes it a potential target (Figure 2). In the
cultured cerebellar granule neurons, it demonstrated that oxidative stress stimulates
mitochondrial fission and mitochondrial fragmentation due to the addition of hydrogen
peroxide (Jahani-Asl et al., 2007). It was also shown in the stroke model of mouse that nitric
oxide induces mitochondrial fission in neurons which triggers the neuronal loss (Barsoum et
al., 2006). Minor expressions, dominant negative Drp1 were seen to be protective against
oxidative damage in cultured neurons (Jahani-Asl et al., 2007; Barsoum et al., 2006). Damaged
and compromised mitochondria in the cells are the potential source of increased generation of
ROS. The disruption of the mitochondrial energetics may lead to the permeability of
mitochondria and uncoupled oxidative phosphorylation which will cause mitochondrial
damage. High concentration of ROS in acute conditions can disrupt the mitochondrial function,
in the presence of calcium which further propagates the production of ROS.
Figure 2. Oxidative stress in Huntington’s disease leads to various cellular and metabolic dysfunction.
The progression of the disease can be assessed by the markers for nucleic acid oxidation
like 8-hydroxy-2-deoxyguanosine (8-OHDG) in DNA and 8-hydroxyguanosine in RNA of
neurons. The generation of hydroxyl radical causes damage to DNA by breaking strands, cross-
linking DNA-protein and base-modifications. ROS generation damages DNA and other cellular
components, which make them prone to oxidation (Siems et al., 1995). Malondialdehyde
(MDA), a product of peroxidation formed by the chemical reactions and cyclisation reaction of
endoperoxides and peroxyl radicals (ROO-), respectively. 4-Hydroxy-2-nonenal (HNE) is the

major aldehyde product of lipid peroxidation apart from malondialdehyde. Specifically, ROS
and RNS oxidized cysteine and methionine residues of proteins (Stadtman, 2004).
This specific oxidation will result in the formation of mixed disulfides between protein thiol
groups (–SH) and glutathione (GSH, S-glutathionylation), low molecular weight thiols in
particular. Carbonyl groups concentration in the cells reflects the ROS-mediated protein
oxidation. Increased concentration of isoprostanes in cerebrospinal fluid (CSF) of HD patients
reflects the lipid peroxidation in the neurons (Montine et al., 1999).
Increases in the brain age make the neurons prone to oxidative damage and decrease the
defensive ability of the cells. These neurodegenerative changes in the neuron are due to protein
damage induced by the ROS in the brain leading to HD.
3.2. Evidence for Oxidative Damage in Huntington’s Disease
Experimental models and clinical studies in HD demonstrated that oxidative stress has a
role in the dysfunction of mitochondria and thus induce degeneration of neurons in the brain.
Various studies in the past have reviewed the oxidative damage mechanisms (Browne et al.,
1999; Beal and Ferrante, 2004; Stack et al., 2008; Browne and Beal, 2006). The important
evidence are summarized below:
3.2.1. Corroboration from Post-mortem Studies of Huntington’s Patients

Oxidative damage is well corroborated from the post-mortem samples of HD patients like
the plasma, brain tissue, cerebrospinal fluid and lymphoblasts (Browne et al., 1999; Stack et
al., 2008; Browne and Beal, 2006; Klepac et al., 2007; Sorolla et al., 2008; Tasset et al., 2009).
The commonly used markers of oxidative stress as discussed above were found to be higher in
striatum and cortex of HD patients. The markers most commonly found in the post-mortem
samples are heme oxygenase, 3-nitrotyrosine, and malondialdehyde (Browne et al., 1999).
These markers correlate well with the extent and intensity of damage to the neuron at
neostriatum and reflect the degree of immunoreactivity in the dorsal striatum to the ventral
striatum which is less affected. Malondialdehyde and HNE show 8-fold greater increases in the
brain of HD patients which is considered to be highly significant (Stoy et al., 2005).
A positive association was demonstrated between the severity of symptoms in HD patients
and products of lipid peroxidation in plasma (Chen et al., 2007). It was also proposed that lipid
peroxidation could be a prospective biomarker for assessing the adequacy of the management
(Chen et al., 2007). Tunez et al., (2011) showed a correlation between the alarming global
oxidative stress (GOS), depleted antioxidant systems, and the phase of the disease. CAG repeat
length in HD patients also correlate well with the increased in cytoplasmic lipofuscin (a product
of unsaturated fatty acid peroxidation) and increased DNA fragmentation (Browne et al., 1999;
Braak and Braak, 1992; Tellez-Nagel et al., 1974; Butterworth et al., 1998; Portera-Cailliau et
al., 1995). In HD patients, molecular targets like aldolase C, tubulin, γ-enolase, and creatine
kinase B are the potential targets for oxidative modification in the brain (Sorolla et al., 2008).
More recently, pathogenesis of HD observed a link between bioenergetics deficits and
decreased catalytic activity due to the oxidation of mitochondrial enzymes in the striatum
samples of the patients (Sorolla et al., 2010). Pyridoxal kinase and antiquitin 1 oxidation may
result in decreased production of pyridoxal 5-phosphate, a precursor for the synthesis of
glutathione, GABA and dopamine. The decreased synthesis of these neurotransmitters plays a

significant part in the pathologic process of HD. Studies of Lim et al., (Lim et al., 2008) and
Tabrizi et al., (1999) also provided the extra indirect shreds of evidence in the pathologic
process. Lim et al., (Lim et al., 2008) investigated post-mortem tissues of the brain of HD
patients and found dysfunction of Ca2+ homeostasis in mitochondria of striatum as the mutant
striatal neurons are not able to handle large Ca2+ loads. It is proposed that increased reactivity
of Ca2+ to the porous transition pore leads to the release of accumulated Ca2+. Increase in mutant
cells and defective mitochondria especially those induced by complex II inhibitors produce
more harmful ROS. This will endanger the mitochondria of the mutant cells to the damage
induced by calcium ions and reactive oxygen species. It has been perceived that by decreasing
Ca2+ level in the cells, could prevent Ca2+ stress that is responsible for inducing irreversible
damage to mitochondria eventually causing neuronal death. Furthermore, aconitase, a
mitochondrial tricarboxylic acid (TCA)-cycle enzyme impairment may be attributed to Fe-S
clusters within the protein, which may jeopardize neuronal cells to the oxidative damage by the
free radicals (Tabrizi et al., 1999; Patel et al., 1996). There is documentation for a direct
correlation between excitotoxicity of glutamate/NMDA neurotransmitters and aconitase
inhibition via NO and mitochondrial free radical generation.
4. ANTIOXIDANT THERAPIES IN HUNTINGTON’S DISEASE

Free radical scavengers attenuate production of reactive oxygen species and consequent
damage by oxidative stress. Enzymatic and non-enzymatic antioxidants are the two types of
free radical scavengers. The first one is the principal line of protection against free radicals like
superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT). The major
non-enzymatic antioxidants are ascorbic acid (vitamin C), α-tocopherol (vitamin E),
glutathione (GSH), carotenoids, retinoic acid, and flavonoids. In Table 1, the potential
therapeutic molecules trialed in vitro are shown. In Table 2 and Table 3, potent antioxidant
agents tested in chemically induced rodent and murine models and potent antioxidant agents
tested in transgenic mouse models of HD are summarized, respectively.
Table 1.Potential antioxidant therapies tested in vitro in Huntington’s disease

(Adapted from Gil-Mohapel J et al., 2014)
Drug Targets Pharmacological Actions

Metalloporphyrins Reactive oxygen species Reduce cell death
Ascorbic acid (vitamin C) Reactive oxygen species Decrease neurodegeneration
α-Tocopherol (vitamin E) Glutamate-induced neuronal death Increase neuroprotection
Grape seed phenolic GSPE inhibit aggregation of mhtt and Attenuate spatial memory
extract (GSPE) reduced the carbonyl levels impairment

4.1. In Vitro Studies
4.1.1. Metalloporphyrins
It has transpired as a significant member of antioxidants. It has metal-containing catalytic
antioxidants which act as scavengers on the reactive oxygen species. In vitro model, manganese
porphyrin has been shown to significantly reduce cell death (Petersen et al., 2000).
Table 2. Potential antioxidant agents tested in chemically induced rodent and murine
models of Huntington’s disease (Adapted from Gil-Mohapel et al., 2014)

Melatonin Reduces -OH, CO32−and reactive Protects DNA and increase neuronal
nitrogen species cells survival
Selenium Reduced lipid peroxidation Increases GABA concentrations in
the striatum and improves behaviour
significantly
Idebenone Reduces glutamic acid Restoration of GAD
decarboxylase (GAD) immunoreactivity
Tauroursodeoxycholic Prevented striatal degeneration Improves locomotor and cognitive
acid (TUDCA) deficits
N-Acetylcysteine Neuroprotection against 3-NP Reduced striatal lesion volumes
(NAC)
Creatine Stabilizes the concentration of Reduces striatal lesion volumes
calcium in cell, ameliorate
mitochondrial transition pore
activation and maintain
intracellular energy
CoQ10 Antioxidant action against Neuroprotection and improvement in
malonate and 3-NP striatal volume
CoQ10 and creatine Blocks 3-NP, preserve Additive neuroprotective
glutathione levels and maintain
glutathione/glutathione disulfide
ratio in the striatum
FK-506 Reduces MDA and nitrite; Neuroprotection
maintain level
Lycopene Reduces oxidative stress Improves behavior
markers.
CDDO-MA It induces NAD(P)H Preserve glutathione/glutathione
dehydrogenase (quinone) 1 disulfide ratio, maintain glutathione
(NQO1) and suppresses iNOS level in striatum and maintain
reduced MDA level. Maintain
neuronal nuclei in striatum.

Table 3. Potential antioxidant agents tested in transgenic mouse models of Huntington’s

disease (Adapted from Gil-Mohapel et al., 2014)

Lipoic acid Co-factor for alpha-ketoglutarate Extension of survival of a transgenic
dehydrogenase and pyruvate mouse
dehydrogenase
Pyruvate Carbohydrate metabolism Neuroprotection
BN82451 Inhibits lipid peroxidation Improves survival, motor function
and increases the volume of striatal
areas
Tauroursodeoxycholic Reduces ROS, mitigates Neuroprotection
acid (TUDCA) mitochondrial insufficiency and
apoptosis
Creatine Phosphorylation of adenosine Improves motor performance,
diphosphate to adenosine survival extension maintains body
triphosphate weight and maintains brain weight
CoQ10 It involves oxidative Improves brain weight, improves
phosphorylation for electron motor performance and maintain
transfer and ATP production survival
L-Carnitine Free-radical It decreases aggregation mhtt in the
scavenger/antioxidants, nucleus, improves motor performance
peroxisomal oxidation of fatty and maintains survival
acids is blocked and maintain
acyl-coenzyme A/coenzyme A
ratio in mitochondria
Grape seed phenolic Exact target not known Extended lifespan and reduce the
extract motor deficit
Synthetic triterpenoids Activate Nrf2/ ARE Has antioxidant, anti-inflammatory
transcriptional pathways and neuroprotective action
Kynurenine-3- Inhibits receptors of excitatory Block neurodegeneration
monooxygenase amino acid in the brain
inhibitors
4.1.2. Ascorbic Acid

Ascorbic acid oxidizes to dehydroascorbic acid which has the potential to acts as an
antioxidant and acts as a scavenger for free radicals. The metabolite is formed in the presence
of ROS. Glutamate has the ability for significant neurodegeneration of neurons in the cortex of
rodent which was completely rescued with ascorbic acid (Majewska and Bell, 1990).
4.1.3.α-Tocopherol
α-Tocopherol has the potential scavenger action on reactive oxygen species. It significantly
attenuated neuronal death caused by glutamate in a neuronal cell-based assay (Miyamoto et al.,
1989). It is also found that in the same in vitro model, idebenone in a dose-dependent manner
has the ability to restore complete neuroprotection (Miyamoto et al., 1989).

4.1.4. Grape Seed Phenolic Extract

Wang et al., (Wang et al., 2010) demonstrated that grape seed phenolic extract (GSPE)
inhibited mhtt aggregation in PC-12 cells and thus also reduces the carbonyl levels caused by
the increased expression of mhtt protein. It has the property of metal chelator and antioxidant
effect in vivo and thus significantly reduces reactive oxygen species. In the mouse model of
AD, GSPE has the capability to diminished attenuate spatial memory impairment
(Wang et al., 2008).
4.2. Chemically Induced Rodent and Murine Models of Huntington’s Disease
4.2.1. Melatonin
Melatonin has a magnificent neuroprotective effect due to its scavenging action on free
radicals and organic ions. In the neurodegenerative rodent model induced by kainic acid,
melatonin demonstrated significant neuroprotection (Uz et al., 1996). Melatonin protects DNA
from oxidative damage and improves the longevity of the neurons in the brain. Melatonin has
been shown to significantly reduce superoxide dismutase activity, peroxidation of lipid and
protein carbonyl in the striatum of the 3-NP model of HD (Tunez et al., 2004).
4.2.2. Selenium
Selenium, a co-factor for glutathione peroxidase has an excellent scavenging action by
forming the active enzyme. In the rat models, quinolinic acid (N-methyl-D-aspartate
antagonist) induced striatal neurodegeneration which shown to be ameliorated by treating with
selenium in a dose-dependently manner. It improves the neuronal morphology of the striatum
of rats and it reduces lipid peroxidation (Santamaria et al., 2003). It has the property of
improving GABA concentrations in the striatum and has a positive influence on behaviour in
these animals evident by significant reduction in ipsilateral turning as compared with untreated
controls.
Pyruvate in a dose-dependent manner can be used in striatal lesion model of HD induced
by quinolinic acid (Ryu et al., 2003). At lower doses, it did not provide the neuroprotection,
although higher doses reduced striatal lesion and offered significant neuroprotection.
4.2.3. Idebenone
It has the potential of an antioxidant. It also has the property of penetrating the brain. In
the rat models of HD, intrastriatal injection of kainic acid in the striatum markedly reduced
glutamic acid decarboxylase (GAD), a presynaptic striatal marker. Idebenone treatment in the
affected rats had shown the significant result of revamping the GAD immunoreactivity
(Miyamoto et al., 1990). On the contrary, idebenone treatment had no effect on striatal lesions
induced by quinolinic acid and on GAD immunoreactivity.
4.2.4. Tauroursodeoxycholic Acid

It is a hydrophilic bile acid with effective scavenging action on free radicals. In the 3-NP
rat model, it prevented degeneration of striatum and improved the locomotor and cognitive
functions (Keene et al., 2001).

4.2.5. N-Acetylcysteine
N-Acetylcysteine (glutathione precursor) has the ability for neuroprotection against
oxidative stress induced by 3-NP. Electron paramagnetic resonance (EPR) and western blot
analysis were used to measure its antioxidant action by measuring protein carbonyl (Fontaine
et al., 2000). The striatal lesion volume was significantly reduced by N-acetylcysteine treatment
before administrating 3-NP. It is a useful therapeutic intervention for an imperative lesion of
striatum formed by 3-NP (Fontaine et al., 2000).
4.2.6. Creatine
Creatine has antioxidant action (Lawler et al., 2002) and its supplementation was found to
improve the striatal volumes in the lesions induced by the malonate and neurotoxins 3-NP
(Matthews et al., 1998; Shear et al., 2000). It acts as co-factors in many biochemical reactions
for neuroprotection and thus helps in the survival of neuronal cells. It stabilizes intracellular
calcium, buffers intracellular energy reserves and inhibits activation of the mitochondrial
transition pore (O’Gorman et al., 1997).
4.2.7. CoQ10
Antioxidant compound CoQ10 is a lipid-soluble benzoquinone that has the potential of
antioxidant agents. In murine models of HD, it has shown therapeutic efficacy. CoQ10,
ubiquinone is reduced to ubiquinol which enhances its antioxidant potential. CoQ10 has
additive action with vitamin E as it enhances its antioxidant efficacy. CoQ10 provides dose-
dependent neuroprotection and improves the striatal lesion volume as it detoxifies unwanted
stimuli to neurons (Beal et al., 1994; Matthews et al., 1998).
4.2.8. Combination of CoQ10 and Creatine

The combination treatment with CoQ10 and creatine has supplementary effects on cells
biology. The combined treatment of CoQ10 with creatine was demonstrated in the 3-NP model
of HD (Yang et al., 2009). The combination has significant protective effects on the striatal
neurons as the combination reduced lesions induced by 3-NP, maintained GSH levels in the
striatum and preserved GSH/GSSG ratio. It also reduced malondialdehyde and 8-hydroxy-2-
deoxyguanosine levels in the striatal lesion induced by 3-NP in rats (Yang et al., 2009).
4.2.9. FK-506 (Tacrolimus or Fujimycin)

FK-506, an immunosuppressant approved for the management of rejection of the allografts.
In the 3-NP model of rodent, FK-506 was demonstrated to have neuroprotective effects (Kumar
et al., 2010). Management of HD with FK-506 has shown reduced behavioral deficits in the
animal model. FK-506 maintains SOD and catalase levels; and also reduces malondialdehyde
and nitrite level. Dopamine and norepinephrine levels are maintained by FK-506 in the neurons
of the brain.
4.2.10. Lycopene
Lycopene is a pigment, which is naturally found in vegetables and fruits. In the 3-NP model
of HD in rodents, it has demonstrated the potential to improve behaviour and reduced markers
of oxidative stress (Kumar et al., 2009).

4.2.11. CDDO-Methyl Amide

CDDO-methyl amide (2-cyano-N-methyl-3,12-dioxooleana-1,9(11)-dien-28 amide or
CDDO-MA), a synthetic triterpenoid has the probable role in inactivating the Nrf-2/ARE
pathway which forms NAD(P)H dehydrogenase [quinone] 1 (NQO-1) and suppresses the
inflammatory marker, inducible nitric oxide synthase (iNOS). Intervention with CDDO-MA
maintain the integrity of striatal neurons from 3-NP, preserved the level of GSH in the striatum,
maintain GSH/GSSG ratio and reduce malondialdehyde levels. The induction of the Nrf2/ARE
pathway reduces the damage induced by the oxidative stress and may be a potential therapeutic
agent for HD in the future (Yang et al., 2009).
4.3. Transgenic Mouse Models of Huntington’s Disease
4.3.1. Lipoic Acid

Lipoic acid is found in the cells, which act as a catalyst for many enzymatic reactions. It
acts as a cofactor for the synthesis of alpha-ketoglutarate dehydrogenase and pyruvate
dehydrogenase in mitochondria. It has efficacy to treat disorders related to disrupted energy
metabolism in the cells in addition to its effective antioxidant action (Packer et al., 1997). Nrf-
2/ARE pathways are activated by lipoic acid (Shay et al., 2009). Dietary supplementation with
lipoic acid had shown an excellent result in improving weight and prolonging survival of N171-
82Q and R6/2 transgenic mice (Andreassen et al., 2001).
4.3.2. Pyruvate
Pyruvate is an essential element component in carbohydrate metabolism and has potent
antioxidant action. Pyruvate has been found to improve neuroprotection when given in addition
to dichloroacetate as the latter stimulates pyruvate dehydrogenase in genetically modified mice
like R6/2 and N171-82Q and due to its antioxidant capacity (Andreassen et al., 2001).
4.3.3. BN82451
Pyruvate is a compound which plays an important neuroprotective role and inhibits ROS
generation by inhibiting lipid peroxidation. In R6/2 mice, administration of BN82451
significantly improved survival, motor function and the morphology of neurons in the striatum.
Pyruvate significantly decreased ubiquitin positivity in R6/2 mice compared to control mice
(Klivenyi et al., 2003).
4.3.4. Tauroursodeoxycholic Acid

Tauroursodeoxycholic acid (TUDCA) has a potential antioxidant action as it prevents ROS
production, improves mitochondrial functions. It inhibits Bax translocation to mitochondria
from the cytosol and partly induces apoptosis. Keene et al., (Keene et al., 2002) showed that
TUDCA administered systemically has notably reduced lesions and apoptosis of striatal cells;
also decrease htt inclusions in the nucleus of ubiquitinated neurons. TUDCA improves
locomotor and sensorimotor deficits in R6/2 transgenic HD mouse.

4.3.5. Creatine
Free creatine and phosphocreatine (PCr) constitute the total creatine pool in the body.
Phosphocreatine acts as a buffer in the phosphorylation of adenosine diphosphate to adenosine
triphosphate in the tissues, which need high energy. Creatine kinase (CK) enzyme catalyzes the
transfer of this phosphoro group (Tarnopolsky and Beal, 2001). In transgenic mouse models,
supplementation of creatine improved motor performance, survival extension, maintained body
weight, reduced neuronal atrophy and thus maintained brain weight (Ferrante et al., 2000;
Andreassen et al., 2001).
4.3.6. CoQ10
CoQ10 is present in the inner mitochondrial membrane of the cell. It plays a pivotal role in
complex I and II electron transfer during oxidative phosphorylation and in ATP production.
CoQ10 administration significantly improves mitochondrial CoQ10 levels in the central
nervous system (Matthews et al., 1998). Its administration in transgenic mice (R6/2P) improved
motor performance and prolonged survival by improving brain weight; ameliorated brain
atrophy, ventricular enlargement and striatal neuron atrophy (Ferrante et al., 2002).
CoQ10 and creatine co-administration have additive effects in improving neuroprotection
by improving motor performance, preventing striatal atrophy and prolonging survival in
transgenic mouse model of HD (R6/2) (Yang et al., 2009).
4.3.7. L-Carnitine
L-Carnitine plays a major role in cell metabolic functions. It has pivotal role in maintaining
the ratio of acyl-coenzyme A/coenzyme A in the mitochondria; prevention of oxidation of fatty
acids and has a scavenging role for free radicals. It reduces reactive oxygen species and thus
prevents damage to the cell membrane. In N171-82Q transgenic mice, administration of a high
dose of L-carnitine was demonstrated to boost motor performance, decrease aggregation of
intranuclear mhtt and prolonged survival of neurons (Vamos et al., 2010).
4.3.8. Grape Seed Phenolic Extract

Grape seed phenolic extract (GSPE) administration prolonged lifespan in fly and R6/2
mouse models, and lessen motor deficits especially in the R6/2 mice models. In preclinical
models of HD, GSPE has demonstrated effectiveness (Wang et al., 2010).
4.3.9. Synthetic Triterpenoids

Synthetic triterpenoids, an analog of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid
(CDDO) is an antioxidant and anti-inflammatory agent. Triterpenoids administered to N171-
82Q transgenic mouse model of HD activated the Nrf2/ARE transcriptional pathways and
demonstrated to have a neuroprotective role in the transgenic mice (Tabrizi et al., 1999). The
neuroprotection was due to its ability to preserve the medium spiny neurons from degeneration
and maintaining its striatal volumes. It’s improved the behavioral functions, prolonged the
survival time and decreased the pathogenesis process in the periphery. Triterpenoids also
decreased the immunoreactivity of 8- OHdG, MDA and 3-nitrotyrosine in the striatum (Tabrizi
et al., 1999). CDDO-EA and CDDO-TFEA were found to cross the blood-brain barrier better
and therefore hold great promise for therapeutic application in neuronal disorders.

4.3.10. Kynurenine-3-Monooxygenase Inhibitors

Kynurenic acid is a metabolite of the kynurenine pathway. It has an inhibitory action on
excitatory amino acid receptors. In post-mortem analysis of brain tissue, marked decrease in
level of kynurenic acid was revealed (Beal et al., 1992). The glutamate-mediated excitotoxicity
and free radical formation are responsible for the decrease concentration of kynurenic acid in
the brain. Drosophila model of HD showed decrease neurodegeneration with the administration
of kynurenic acid (Campesan et al., 2011). A preclinical model of R6/2 transgenic mouse
demonstrated increase kynurenic acid concentration and reduce extracellular glutamate by the
administration of JM6, an inhibitor of kynurenine-3-monooxygenase (Zwilling et al., 2011). Its
chronic administration was found to prolong lifespan, avert the loss of synapses and decreased
activation of microglial (Zwilling et al., 2011).
5. CURRENT TREATMENT STRATEGY IN HUNTINGTON’S DISEASE

The treatment strategy of replacing the missing neurotransmitter has proven to be clinically
not effective in HD. Counseling and rehabilitative modalities do have effects to refine the
quality of life (QOL). GABA receptor agonist or inhibitors of GABA catabolism have also not
been found to be clinically effective in HD patients. However, maintaining the balance of
DA/ACh by antagonizing the DA over-activity has been effective. Antipsychotic drugs like
chlorpromazine, haloperidol, and olanzapine have been found to be effective. Tetrabenazine
(dopamine depleter) has been found to be useful in HD. One of the disadvantages of these drugs
is that they are not useful in advanced HD (Sharma and Sharma, 2017).
Minocycline, a second-generation tetracycline has an anti-apoptotic activity which has
been used for rheumatoid arthritis (RA), acne, rosacea, and is the treatment of various
infections. Minocycline has been found to have an inhibitory action on the caspase in mice,
which in turn will reduce proteolysis of mutant huntingtin and reduce death. In a recent trial in
HD patients, minocycline improved motor and neuropsychological functions and prevented
psychiatric symptoms.

AND CHALLENGES
There is an association between oxidative stress and mitochondrial dysfunction, thus the
improvement in one dysfunction lead to the betterment of another in HD. There are many
compounds with the therapeutic potential to block the sequential steps in the pathogenesis of
HD. Mitochondrial medicine is a new initiative where the therapeutic agents are directed to
manage mitochondrial dysfunction. This new approach has the capacity to ameliorate oxidative
stress and maintains the viability of the cell. Vitamins or other cofactors involved in the
electrons transport chain and other intermediates biochemical pathways have the capacity to
enhance the function of mitochondria or block dysfunction of mitochondria. The potential
antioxidants agents that specifically target mitochondria if used as an adjunct may enhance the
therapeutic benefits of the existing molecules (Johri and Beal, 2012).

The next therapeutic approach, which might have a beneficial role in reducing pathogenesis
of HD, is to enhance PGC-1α as it acts as co-regulator in significant metabolism of cells. The
beneficial effects of PGC-1α are it regulates uncoupling of proteins in mitochondria and
enhance antioxidant enzymes production in the cells. Superoxide dismutase (SOD1 and SOD2)
and glutathione peroxidase (GPx-1) are the antioxidant enzymes regulated by PGC-1α (St-
Pierre et al., 2006). In HD, decrease PGC-1α levels have a direct correlation with the pathology
of the disease (Cui et al., 2006; Weydt et al., 2006; McGill and Beal, 2006; Chaturvedi et al.,
2009; Chaturvedi et al., 2010; Johri et al., 2011). It is demonstrated that mice which are PGC-
1α deficit have shown clinical features of HD like, dysfunction of mitochondria, dysfunctional
bioenergetics, movement disorder and degeneration of striatum (Leone et al., 2005; Lin et al.,
2004). Deficiency of PGC-1α in mutated mice will have striatal volume shrinkage due to its
neuron degeneration and makes the neuronal cells vulnerable to the mitochondrial toxin 3-NP
(Cui et al., 2006). The impaired function and decreased expression of this molecule may lead
to the failure of the antioxidant systems. Thus targeting this important molecule could help in
improving the function of mitochondria and the propagation of cell metabolism. It will also
reduce the damage caused by oxidative stress and enhance the level of antioxidant enzymes.
Bezafibrate (pan-PPAR agonist) has the ability to increases the expression of PGC-1α. This
molecule has shown the beneficial benefit in the transgenic mouse model by ameliorating the
damage caused by the oxidative stress, and by improving the performance deficits, prolongation
of survival, and reduction in atrophy of striatum (Johri and Beal, unpublished observations).
Pioglitazone, a PPAR-γ agonist, and thiazolidinedione are the other agents, which also have a
role in improving the neurological dysfunctions in HD (Kalonia et al., 2010; Chiang et al.,
2010). Molecules, which have the capacity to trigger the Nrf-2/ARE pathway, will increase the
antioxidant enzymes, and chaperone proteins levels in HD and have a neuroprotective role by
reducing oxidants in HD (Stack et al., 2010). The multitude of approaches has the advantage to
modulate neuroprotective pathways endogenously and have a regenerative approach on the
catalytic processes of the cells and ongoing antioxidant effects. Transcriptional modulation of
antioxidant pathways and the newer, mitochondrial medicines are the future therapeutic
interventions for HD.
In modern world, sophistication and technology may be reflected in the human’s way of
life. With the increase in the standard of living, many problems become part and parcel of life.
It is a challenge for the physicians for managing patient for the long-term. The best management
for HD should be the combination of non-pharmacological and pharmacological approaches.
With the improvement in target characterization in disease and targeted drug delivery
technologies, we see immense opportunities in the field of management of this particular
disorder where limited therapeutic options are available until date.
CONCLUSION
Although the involvement of oxidative stress in HD is undisputable, it is still not clear
whether cellular metabolism and imbalance in the intracellular components, a result, or simply
a by-product of the neurodegenerative process. The availability of HD transgenic mouse models
has improved the methods to test the potential antioxidant agents in the preclinical phase. The
guidelines should include clinical setting, and clinical trials that consider potential placebo

effects, use of standardized and clinically relevant rating scales and include a large patient
sample while minimizing variables within the sampled population. Finally, the design of multi-
drug cocktails targeting oxidative stress as well as other intracellular pathways are strongly
encouraged as it will revolutionize the ways the potential drugs for HD are evaluated in
preclinical and clinical studies. The multifactorial approach is likely to result in better outcomes
for patients afflicted with this devastating neurodegenerative disorder.
REFERENCES
Aikens J, Dix TA. Perhydroxyl radical (HOO.) initiated lipid peroxidation. The role of fatty
acid hydroperoxides. J Biol Chem. 1991;266:15091–98.
Albin R. L, Reiner A, Anderson K. D, Dure L. S. T., Handelin B, Balfour R, et al., Preferential
loss of striato-external pallidal projection neurons in presymptomatic Huntington’s disease.
Ann Neurol. 1992;31:425–30.
Albin R. L, Young A B, Penney J. B. The functional anatomy of basal ganglia disorders.
TrendsNeurosci. 1989;12:366–75.
Andreassen O. A, Dedeoglu A, Ferrante R. J, Jenkins B. G, Ferrante K. L, Thomas M, et al.,
Creatine increase survival and delays motor symptoms in a transgenic animal model of
Huntington’s disease. Neurobiol Dis. 2001;8:479–91.
Andreassen O. A, Ferrante R. J, Dedeoglu A, Beal M. F. Lipoic acid improves survival in
transgenic mouse models of Huntington’s disease. Neuroreport. 2001;12:3371–3.
Andreassen O. A, Ferrante R. J, Huang H. M, Dedeoglu A, Park L, Ferrante K. L, et al.,
Dichloroacetate exerts therapeutic effects in transgenic mouse models of Huntington’s
disease. Ann Neurol. 2001;50:112–7.
Bae B. I, Hara M. R, Cascio M. B, Wellington C. L, Hayden M. R, Ross C. A, et al., Mutant
huntingtin: nuclear translocation and cytotoxicity mediated by GAPDH. Proc Natl Acad
Sci USA. 2006;103:3405–9.
Barja G. Free radicals and aging. Trends Neurosci.2004; 27:595–600.
Barsoum M. J, Yuan H, Gerencser A. A, Liot G, Kushnareva Y, Graber S, et al., Nitric oxide-
induced mitochondrial fission is regulated by dynaminrelated GTPases in neurons. EMBO
J. 2006;25:3900–11.
Beal M. F, Ferrante RJ. Experimental therapeutics in transgenic mouse models of Huntington’s
disease. Nat Rev Neurosci. 2004;5:373–84.
Beal MF, Henshaw D. R, Jenkins B. G, Rosen B. R, Schulz J B. Coenzyme Q10 and
nicotinamide block striatal lesions produced by the mitochondrial toxin malonate. Ann
Neurol. 1994;36:882–8.
Beal M. F, Matson WR, Storey E, Milbury P, Ryan EA, Ogawa T, et al., Kynurenic acid
concentrations are reduced in Huntington’s disease cerebral cortex. J Neurol Sci.
1992;108:80–7.
Beckman J. S, Koppenol W. H. Nitric oxide, superoxide, and peroxynitrite: the good, the bad,
and ugly. Am J Physiol. 1996; 271:C1424–37.
Boveris A. Biochemistry of free radicals: from electrons to tissues. Medicina (B Aires).
1998;58:350–6.

Braak H, Braak E. Allocortical involvement in Huntington’s disease. Neuropathol Appl

Neurobiol. 1992;18:539–47.
Brocardo P. S, Gil-Mohapel J. From preclinical to clinical trials: an update on potential
therapies for Huntington’s disease. Current Psychopharmacology. 2013;2:113-31.
Brocardo P. S, Gil-Mohapel J. Therapeutic strategies for Huntington’s disease: from the bench
to the clinic. Current Psychopharmacology. 2012;1:137-54.
Browne S. E, Beal M. F. Oxidative damage in Huntington’s disease pathogenesis.
AntioxidRedox Signal. 2006;8:2061–73.
Browne S. E, Beal M. F. The energetics of Huntington’s disease. Neurochem Res.
2004;29:531–46.
Browne S. E, Ferrante R. J, Beal M. F. Oxidative stress in Huntington’s disease.Brain Pathol.
1999;9:147–63.
Butterworth N. J, Williams L, Bullock J. Y., Love D. R, Faull R. L, Dragunow M. Trinucleotide
(CAG) repeat length is positively correlated with the degree of DNA fragmentation in
Huntington’s disease striatum. Neuroscience. 1998;87:49–53.
Cadenas E, Sies H. The lag phase. Free Radic Res. 1998;28:601–9.
Campesan S, Green E. W, Breda C, Sathyasaikumar K. V, Muchowski P. J, Schwarcz R, et al.,
The kynurenine pathway modulates neurodegeneration in a Drosophila model of
Huntington’s disease. Curr Biol. 2011;21:961–6.
Caviston J. P, Holzbaur E. L. Huntingtin as an essential integrator of intracellular vesicular
trafficking. Trends Cell Biol. 2009;19:147–55.
Chaturvedi R. K, Adhihetty P, Shukla S, Hennessy T, Calingasan N, Yang L, et al., Impaired
PGC-1alpha function in muscle in Huntington’s disease. Hum Mol Genet. 2009;18:3048–
65.
Chaturvedi R. K, Calingasan N. Y, Yang L, Hennessey T, Johri A, Beal M. F. Impairment of
PGC-1alpha expression, neuropathology and hepatic steatosis in a transgenic mouse model
of Huntington’s disease following chronic energy deprivation. Hum Mol Genet.
2010;19:3190–205.
Chen C. M, Wu Y. R, Cheng M. L, Liu J. L, Lee Y. M, Lee P. W, et al., Increased oxidative
damage and mitochondrial abnormalities in the peripheral blood of Huntington’s disease
patients. Biochem Biophys Res Commun. 2007;359:335–40.
Chiang M. C, Chen C. M, Lee M. R, Chen H. W, Chen H. M, Wu Y. S, et al., Modulation of
energy deficiency in Huntington’s disease via activation of the peroxisome proliferator-
activated receptor gamma. Hum Mol Genet. 2010;19:4043–58.
Costa V, Giacomello M, Hudec R, Lopreiato R, Ermak G, Lim D, et al., Mitochondrial fission
and cristae disruption increase the response of cell models of Huntington’s disease to
apoptotic stimuli. EMBO Mol Med. 2010;2:490–503.
Cui L, Jeong H, Borovecki F, Parkhurst C. N, Tanese N, Krainc D. Transcriptional repression
of PGC-1alpha by mutant huntingtin leads to mitochondrial dysfunction and
neurodegeneration. Cell. 2006;127:59–69.
Dewhurst K, Oliver J. E, McKnight A. L. Socio-psychiatric consequences of Huntington’s
disease. Br J Psychiatry. 1970;116:255-8.
DiFiglia M, Sapp E, Chase K, Schwarz C, Meloni A, Young C, et al., Huntingtin is a
cytoplasmic protein associated with vesicles in human and rat brain neurons. Neuron.
1995;14:1075–81.

DiFiglia M, Sapp E, Chase KO, Davies S. W, Bates G. P, Vonsattel J. P, et al., Aggregation of

huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science.
1997;277:1990–3.
DiFiglia M, Sapp E, Chase K. O. Aggregation of huntingtin in neuronal intranuclear inclusions
and dystrophic neurites in brain. Science 1997;277:1990-3.
Dragatsis I, Levine M. S, Zeitlin S. Inactivation of Hdh in the brain and testis results in
progressive neurodegeneration and sterility in mice. Nat Genet. 2000; 26:300–6.
Droge W. Free radicals in the physiological control of cell function. Physiol Rev. 2002;82:47–
95.
Duyao M. P, Auerbach A. B, Ryan A, Persichetti F, Barnes G. T., McNeil SM, et al.,
Inactivation of the mouse Huntington’s disease gene homolog Hdh. Science.
1995;269:407–10.
Ferrante RJ, Andreassen OA, Dedeoglu A, Ferrante KL, Jenkins BG, Hersch SM, et al.,
Therapeutic effects of coenzyme Q10 and remacemide in transgenic mouse models of
Huntington’s disease.J Neurosci. 2002;22:1592–9.
Ferrante R. J, Andreassen O. A, Jenkins B. G, Dedeoglu A, Kuemmerle S, Kubilus J. K, et al.,
Neuroprotective effects of creatine in a transgenic mouse model of Huntington’s disease.
J Neurosci. 2000;20:4389–97.
Ferrante R. J, Beal M. F, Kowall N. W, Richardson E. P Jr, Martin J. B. Sparing of
acetylcholinesterasecontaiing striatal neurons in Huntington’s disease. Brain Res. 1987;
411:162–6.
Ferrante R. J, Kowall N. W, Beal M. F, Richardson E. P Jr, Bird E. D, Martin J. B. Selective
sparing of a class of striatal neurons in Huntington’s disease. Science. 1985;230:561–63.
Finkel T. Reactive oxygen species and signal transduction. IUBMB Life. 2001;52:3–6.
Folstein S, Abbott M. H, Chase G. A, Jensen B. A, Folstein M. F. The association of affective
disorder with Huntington’s disease in a case series and in families. Psychol Med. 1983;13:
537-42.
Fontaine M. A, Geddes J. W, Banks A, Butterfield D. A. Effect of exogenous and endogenous
antioxidants on 3-nitropionic acid-induced in vivo oxidative stress and striatal lesions:
insights into Huntington’s disease. J Neurochem. 2000;75:1709–15.
Fusco F. R, Chen Q, Lamoreaux W. J, Figueredo-Cardenas G, Jiao Y, Coffman J. A, et al.,
Cellular localization of huntingtin in striatal and cortical neurons in rats: lack of correlation
with neuronal vulnerability in Huntington’s disease. J Neurosci. 1999;19:1189–202.
Gao H. M, Liu B, Zhang W, Hong J. S. Critical role of microglial NADPH oxidase-derived
free radicals in the in vitro MPTP model of Parkinson’s disease. FASEB J. 2003;17:1954-
6.
Gerlach M, Ben-Shachar D, Riederer P, Youdim M. B. Altered brain metabolism of iron as a
cause of neurodegenerative diseases? J Neurochem. 1994;63:793-807.
Gil J. M, Rego A. C. Mechanisms of neurodegeneration in Huntington’s disease. Eur J
Neurosci. 2008;27:2803-20.
Gil J. M, Rego A. C. The R6 lines of transgenic mice: A model for screening new therapies for
Huntington’s disease. Brain Res Rev. 2009;59:410-31.
Gil-Mohapel J, Brocardo 1PS, Christie B. R. The Role of Oxidative Stress in Huntington’s
Disease: Are Antioxidants Good Therapeutic Candidates? Current DrugTargets.
2014;15:1-16.

Gil-Mohapel J. M. Screening of Therapeutic Strategies for Huntington’s Disease in YAC128

Transgenic Mice. CNS Neurosci Ther. 2012;18:77-86.
Godin J. D, Colombo K, Molina-Calavita M, Keryer G, Zala D, Charrin B. C, et al., Huntingtin
is required for mitotic spindle orientation and mammalian neurogenesis. Neuron.2010;
67:392–406.
Halliwell B, Gutteridge J. M. Role of free radicals and catalytic metal ions in human disease:
an overview. Methods Enzymol. 1990;186:1-85.
Halliwell B. Biochemistry of oxidative stress. Biochem Soc Trans. 2007;35:1147-50.
Halliwell B. Reactive oxygen species and the central nervous system. J Neurochem.
1992;59:1609-23.
Harjes P, Wanker E. E. The hunt for huntingtin function: interaction partners tell many different
stories. Trends Biochem Sci. 2003;28:425–33.
Hayden MR, Kremer B. The metabolic and Molecular Basis of Inherited Disease. 8th Editions;
McGraw-Hill: New York; 2001.
Hensley K, Robinson K. A, Gabbita S. P, Salsman S, Floyd R. A. Reactive oxygen species, cell
signaling, and cell injury. Free Radic Biol Med. 2000;28:1456–62.
Imarisio S, Carmichael J, Korolchuk V, Chen C. W, Saiki S, Rose C, et al., Huntington’s
disease: from pathology and genetics to potential therapies. Biochem J. 2008;412:191–209.
Infanger D. W, Sharma R. V, Davisson R. L. NADPH oxidases of the brain: distribution,
regulation, and function. Antioxid Redox Signal. 2006;8:1583-96.
Jahani-Asl A, Cheung E. C, Neuspiel M, MacLaurin J. G, Fortin A, Park D. S, et al., Mitofusin
2 protects cerebellar granule neurons against injury-induced cell death. J Biol Chem.
2007;282:23788–98.
Jensen P, Sorensen S. A, Fenger K, Bolwig T. G. A study of psychiatric morbidity in patients
with Huntington’s disease, their relatives, and controls. Admissions to psychiatric hospitals
in Denmark from 1969 to 1991.Br J Psychiatry. 1993;163:790-7.
Johri A, Beal M. F. Antioxidants in Huntington’s disease. Biochim Biophys Acta. 2012;1822:
664–74.
Johri A, Beal M. F. Hunting-ton for new proteases: MMPs as the new target? Neuron.
2010;67:171–3.
Johri A, Chaturvedi R. K, Beal M. F. Hugging tight in Huntington’s. Nat Med. 2011;17:245–
6.
Johri A, Starkov A. A, Chandra A, Hennessey T, Sharma A, Orobello S, et al., Truncated
peroxisome proliferator-activated receptor-gamma coactivator 1alpha splice variant is
severely altered in Huntington’s disease. Neurodegener Dis. 2011;8:496–503.
Kalonia H, Kumar P, Kumar A. Pioglitazone ameliorates behavioral, biochemical and cellular
alterations in quinolinic acid induced neurotoxicity: possible role of peroxisome
proliferator activated receptor-Upsilon (PPARUpsilon) in Huntington’s disease.
Pharmacol Biochem Behav. 2010;96:115–24.
Keene C. D, Rodrigues C. M, Eich T, Chhabra M. S, Steer C. J, Low W. C.
Tauroursodeoxycholic acid, a bile acid, is neuroprotective in a transgenic animal model of
Huntington’s disease. Proc Natl Acad Sci USA. 2002;99:10671–6.
Keene C. D, Rodrigues C. M, Eich T, Linehan-Stieers C, Abt A, Kren B. T, et al., A bile acid
protects against motor and cognitive deficits and reduces striatal degeneration in the 3-
nitropropionic acid model of Huntington’s disease. Exp Neurol. 2001;171:351–60.

Kim J, Moody J. P, Edgerly C. K, Bordiuk O. L, Cormier K, Smith K, et al., Mitochondrial

loss, dysfunction and altered dynamics in Huntington’s disease. Hum Mol Genet.
2010;19:3919–35.
Klepac N, Relja M, Klepac R, Hecimovic S, Babic T, Trkulja V. Oxidative stress parameters
in plasma of Huntington’s disease patients, asymptomatic Huntington’s disease gene
carriers and healthy subjects: a cross-sectional study. J Neurol. 2007;254:1676–83.
Klivenyi P, Ferrante R. J, Gardian G, Browne S, Chabrier P. E, Beal M. F. Increased survival
and neuroprotective effects of BN82451 in a transgenic mouse model of Huntington’s
disease. J Neurochem. 2003;86:267–72.
Kovacic P, Pozos R. S, Somanathan R, Shangari N, O’Brien P. J. Mechanism of mitochondrial
uncouplers, inhibitors, and toxins: focus on electron transfer, free radicals, and structure-
activity relationships. Curr Med Chem. 2005;12:2601–23.
Kumar P, Kalonia H, Kumar A. Possible nitric oxide modulation in protective effect of FK-506
against 3-nitropropionic acid-induced behavioral, oxidative, neurochemical, and
mitochondrial alterations in rat brain. Drug Chem Toxicol. 2010;33:377–92.
Kumar P, Kumar A. Effect of lycopene and epigallocatechin-3-gallate against 3-nitropropionic
acid induced cognitive dysfunction and glutathione depletion in rat: a novel nitric oxide
mechanism. Food Chem Toxicol. 2009;47:2522–30.
Lawler J. M, Barnes W. S, Wu G, Song W, Demaree S. Direct antioxidant properties of creatine.
Biochem Biophys Res Commun. 2002;290:47–52.
Lenaz G. The mitochondrial production of reactive oxygen species: mechanisms and
implications in human pathology. IUBMB Life. 2001;52:159–64.
Leone T. C, Lehman J. J, Finck B. N, Schaeffer P. J, Wende A. R, Boudina S, et al., PGC-
1alpha deficiency causes multi-system energy metabolic derangements: muscle
dysfunction, abnormal weight control and hepatic steatosis. PLoS Biol. 2005;3:e101.
Lim D, Fedrizzi L, Tartari M, Zuccato C, Cattaneo E, Brini M, et al., Calcium homeostasis and
mitochondrial dysfunction in striatal neurons of Huntington disease. J Biol Chem.
2008;283:5780–9.
Lin J, Wu P. H, Tarr P. T, Lindenberg K. S, St-Pierre J, Zhang C. Y, et al., Defects in adaptive
energy metabolism with CNS-linked hyperactivity in PGC-1alpha null mice. Cell.
2004;119:121–35.
Majewska M. D, Bell J. A. Ascorbic acid protects neurons from injury induced by glutamate
and NMDA. Neuroreport. 1990;1:194–6.
Martindale D, Hackam A, Wieczorek A, Ellerby L, Wellington C, McCutcheon K, et al.,
Length of huntingtin and its polyglutamine tract influences localization and frequency of
intracellular aggregates. Nat Genet. 1998;18:150–4.
Matthews R. T., Yang L, Browne S, Baik M, Beal M. F. Coenzyme Q10 administration
increases brain mitochondrial concentrations and exerts neuroprotective effects. Proc
NatlAcad Sci USA. 1998;95:8892–7.
Matthews R. T, Yang L, Jenkins B. G, Ferrante R. J, Rosen B. R, Kaddurah-Daouk R, et al.,
Neuroprotective effects of creatine and cyclocreatine in animal models of Huntington’s
disease. J Neurosci. 1998;18:156–63.
McGill J. K, Beal M. F. PGC-1alpha, a new therapeutic target in Huntington’s disease? Cell.
2006;127:465–8.
Miller D. M, Buettner G. R, Aust S. D. Transition metals as catalysts of “autoxidation”
reactions. Free Radic Biol Med. 1990;8:95–108.

Miller J. P, Holcomb J, Al-Ramahi I, de Haro M, Gafni J, Zhang N, et al., Matrix

metalloproteinases are modifiers of huntingtin proteolysis and toxicity in Huntington’s
disease. Neuron. 2010;67:199–212.
Miyamoto M, Coyle J T. Idebenone attenuates neuronal degeneration induced by intrastriatal
injection of excitotoxins. Exp Neurol. 1990;108:38–45.
Miyamoto M, Murphy T. H, Schnaar R. L, Coyle J. T. Antioxidants protect against glutamate-
induced cytotoxicity in a neuronal cell line. J Pharmacol Exp Ther. 1989;250:1132–40.
Moily N. S, Kota L. N, Anjanappa R. M. Trinucleotide repeats and haplotypes at the huntingtin
locus in an Indian sample overlaps with European haplogroup A. PLoS Currents
Huntington Disease. 2014;6.
Montine T. J, Beal M. F, Robertson D, Cudkowicz M. E, Biaggioni I, O’Donnell H, et al.,
Cerebrospinal fluid F2-isoprostanes are elevated in Huntington’s disease. Neurology.
1999;52:1104–5.
Muller F. L, Liu Y, Van Remmen H. Complex III releases superoxide to both sides of the inner
mitochondrial membrane. J Biol Chem. 2004; 279:49064–73.
O’Gorman E, Beutner G, Dolder M, Koretsky AP, Brdiczka D, Wallimann T. The role of
creatine kinase in inhibition of mitochondrial permeability transition. FEBS Lett.
1997;414:253–7.
Packer L, Tritschler H. J, Wessel K. Neuroprotection by the metabolic antioxidant alpha-lipoic
acid. Free Radic Biol Med. 1997;22:359–78.
Palfi S, Brouillet E, Jarraya B, Bloch J, Jan C, Shin M, et al., Expression of mutated huntingtin
fragment in the putamen is sufficient to produce abnormal movement in non-human
primates. Mol Ther. 2007;15:1444–51.
Patel M, Day B. J, Crapo J. D, Fridovich I, McNamara J. O. Requirement for superoxide in
excitotoxic cell death. Neuron. 1996;16:345–55.
Petersen A, Castilho R. F, Hansson O, Wieloch T, Brundin P. Oxidative stress, mitochondrial
permeability transition and activation of caspases in calcium ionophore A23187-induced
death of cultured striatal neurons. Brain Res. 2000;857:20–9.
Portera-Cailliau C, Hedreen J. C, Price D. L, Koliatsos V. E. Evidence for apoptotic cell death
in Huntington disease and excitotoxic animal models. J Neurosci. 1995;15:3775–87.
Rawlins M. D, Wexler N. S., Wexler A. R. Theprevalence of Huntington’s disease.
Neuroepidemiology. 2016;46(2):144–53.
Reddy P. H, Mao P, Manczak M. Mitochondrial structural and functional dynamics in
Huntington’s disease. Brain Res Rev. 2009;61:33–48.
Reiner A, Albin R. L, Anderson K. D, D’Amato C. J, Penney J. B, Young A. B. Differential
loss of striatal projection neurons in Huntington disease. Proc Natl Acad Sci USA.
1988;85:5733–37.
Reiner A, Dragatsis I, Zeitlin S, Goldowitz D. Wild-type huntingtin plays a role in brain
development and neuronal survival. Mol Neurobiol. 2003;28:259–76.
Rigamonti D, Sipione S, Goffredo D, Zuccato C, Fossale E, Cattaneo E. Huntingtin’s
neuroprotective activity occurs via inhibition of procaspase-9 processing. J Biol Chem.
2001;276:14545–8.
Rosas H. D, Salat D. H, Lee S. Y, Zaleta A. K, Pappu V, Fischl B, et al., Cerebral cortex and
the clinical expression of Huntington’s disease: complexity and heterogeneity. Brain.
2008;131:1057–68.

Rosenblatt A, Liang K. Y, Zhou H, Abbott M. H, Gourley L. M, Margolis R. L, et al., The

association of CAG repeat length with clinical progression in Huntington disease.
Neurology. 2006;66:1016–20.
Ryu J. K, Kim S. U, McLarnon J G. Neuroprotective effects of pyruvate in the quinolinic acid
rat model of Huntington’s disease. Exp Neurol. 2003;183:700–4.
Santamaria A, Salvatierra-Sanchez R, Vazquez-Roman B, Santiago-Lopez D, Villeda-
Hernandez J, Galvan-Arzate S, et al., Protective effects of the antioxidant selenium on
quinolinic acid-induced neurotoxicity in rats: in vitro and in vivo studies. J Neurochem.
2003;86:479–88.
Sawa A, Nagata E, Sutcliffe S, Dulloor P, Cascio MB, Ozeki Y, et al., Huntingtin is cleaved by
caspases in the cytoplasm and translocated to the nucleus via perinuclear sites in
Huntington’s disease patient lymphoblasts. Neurobiol Dis. 2005;20:267–74.
Sharma, H. L.; Sharma, K. K. Anxiolytic and Hypnotics. Principles of Pharmacology, 2nd
Edition.; Paras Medical Publisher: New Delhi; 2017.
Shay K. P, Moreau R. F, Smith E. J, Smith A. R, Hagen T. M. Alpha-lipoic acid as a dietary
supplement: molecular mechanisms and therapeutic potential. Biochim Biophys Acta.
2009;1790:1149–60.
Shear D. A, Haik K. L, Dunbar G. L. Creatine reduces 3-nitropropionic-acid-induced cognitive
and motor abnormalities in rats. Neuroreport. 2000;11:1833–7.
Shirendeb U, Reddy A. P, Manczak M, Calkins M. J, Mao P, Tagle D. A, et al., Abnormal
mitochondrial dynamics, mitochondrial loss and mutant huntingtin oligomers in
Huntington’s disease: implications for selective neuronal damage. Hum Mol Genet.
2011;20:1438–55.
Siems W. G, Grune T, Esterbauer H. 4-Hydroxynonenal formation during ischemia and
reperfusion of rat small intestine. Life Sci. 1995;57:785–9.
Sies H. Oxidative stress: from basic research to clinical application. Am J Med. 1991;91:31S-
8S.
Sokoloff L. Energetics of functional activation in neural tissues. Neurochem Res. 1999;24:321-
9.
Song W, Chen J, Petrilli A, Liot G, Klinglmayr E, Zhou Y, et al., Mutant huntingtin binds the
mitochondrial fission GTPase dynamin-related protein-1 and increases its enzymatic
activity. Nat Med. 2011;17:377–82.
Sorolla M. A, Reverter-Branchat G, Tamarit J, Ferrer I, Ros J, Cabiscol E. Proteomic and
oxidative stress analysis in human brain samples of Huntington disease. Free Radic Biol
Med. 2008;45:667–78.
Sorolla M. A, Rodriguez-Colman M. J, Tamarit J, Ortega Z, Lucas J. J, Ferrer I, Ros J, et al.,
Protein oxidation in Huntington disease affects energy production and vitamin B6
metabolism. Free Radic Biol Med. 2010;49:612–21.
Stack C, Ho D, Wille E, Calingasan N. Y, Williams C, Liby K, et al., Triterpenoids CDDO-
ethyl amide and CDDO-trifluoroethyl amide improve the behavioural phenotype and brain
pathology in a transgenic mouse model of Huntington’s disease. Free Radic Biol Med.
2010;49:147–58.
Stack E. C, Matson W. R, Ferrante R. J. Evidence of oxidant damage in Huntington’s disease:
translational strategies using antioxidants. Ann N Y Acad Sci. 2008;1147:79–92.
Stadtman E. R. Role of oxidant species in aging. Curr Med Chem. 2004;1:1105–12.

Stoy N, Mackay G. M, Forrest C. M, Christofides J, Egerton M, Stone T. W, et al., Tryptophan

metabolism and oxidative stress in patients with Huntington’s disease. J Neurochem.
2005;93:611–23.
St-Pierre J, Drori S, Uldry M, Silvaggi J. M, Rhee J, Jager S, et al., Suppression of reactive
oxygen species and neurodegeneration by the PGC-1 transcriptional coactivators. Cell.
2006;127:397–408.
Tabrizi S. J, Cleeter M. W., Xuereb J, Taanman J. W, Cooper J. M, Schapira A. H. Biochemical
abnormalities and excitotoxicity in Huntington’s disease brain. Ann Neurol. 1999;45:25–
32.
Tabrizi S. J, Scahill R. I, Durr A. Biological and clinical changes in premanifest and early stage
Huntington’s disease in the TRACK-HD study: the 12-month longitudinal analysis. Lancet
Neurol. 2011;10:31-42.
Tarnopolsky M. A, Beal M. F. Potential for creatine and other therapies targeting cellular
energy dysfunction in neurological disorders. Ann Neurol. 2001;49:561–74.
Tasset I, Sanchez F, Tunez I. [The molecular bases of Huntington’s disease: the role played by
oxidative stress]. Rev Neurol. 2009;49:424–9.
Tellez-Nagel I, Johnson A. B, Terry R. D. Studies on brain biopsies of patients with
Huntington’s chorea. J Neuropathol Exp Neurol. 1974;33:308–32.
The Huntington’s Disease Collaborative Research Group. A novel gene containing a
trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes.
Cell. 1993;72:971-83.
Tunez I, Montilla P, Del Carmen Munoz M, Feijoo M, Salcedo M. Protective effect of
melatonin on 3-nitropropionic acid-induced oxidative stress in synaptosomes in an animal
model of Huntington’s disease. J Pineal Res. 2004;37:252–6.
Tunez I, Sanchez-Lopez F, Aguera E, Fernandez-Bolanos R, Sanchez FM, Tasset-Cuevas I.
Important Role of Oxidative Stress Biomarkers in Huntington’s Disease. J Med Chem.
2011;54:5602–6.
Uz T, Giusti P, Franceschini D, Kharlamov A, Manev H. Protective effect of melatonin against
hippocampal DNA damage induced by intraperitoneal administration of kainate to rats.
Neuroscience. 1996;73:631–6.
Valko M, Leibfritz D, Moncol J, Cronin M. T, Mazur M, Telser J. Free radicals and antioxidants
in normal physiological functions and human disease. Int J Biochem Cell Biol. 2007;39:
44-84.
Vamos E, Voros K, Vecsei L, Klivenyi P. Neuroprotective effects of L-carnitine in a transgenic
animal model of Huntington’s disease. Biomed Pharmacother. 2010;64:282–6.
Velier J, Kim M, Schwarz C, Kim TW, Sapp E, Chase K, et al., Wild-type and mutant
huntingtins function in vesicle trafficking in the secretory and endocytic pathways. Exp
Neurol. 1998;152:34–40.
Vonsattel J. P., DiFiglia M. Huntington disease. J Neuropathol Exp Neurol. 1998;57:369-84.
Walker F. O.Huntington’s disease.The Lancet. 2007;369 (9557):218–28.
Wang J, Ho L, Zhao W, Ono K, Rosensweig C, Chen L, et al., Grape-derived polyphenolics
prevent Abeta oligomerization and attenuate cognitive deterioration in a mouse model of
Alzheimer’s disease. J Neurosci. 2008;28:6388–92.
Wang J, Pfleger C. M, Friedman L, Vittorino R, Zhao W, Qian X, et al., Potential Application
of Grape Derived Polyphenols in Huntington’s Disease. Transl Neurosci. 2010;1:95–100.

Warby S. C., Visscher H, Collins J. A. HTT haplotypes contribute to differences in Huntington

disease prevalence between Europe and East Asia. European Journal of Human Genetics.
2011;19(5):561–6.
Wexler N. S, Lorimer J, Porter J, Gomez F, Moskowitz C, Shackell E. Venezuelan kindreds
reveal that genetic and environmental factors modulate Huntington’s disease age of onset.
Proc Natl Acad Sci USA. 2004;101:3498–503.
Weydt P, Pineda V. V, Torrence AE, Libby RT, Satterfield TF, Lazarowski ER, et al.,
Thermoregulatory and metabolic defects in Huntington’s disease transgenic mice implicate
PGC-1alpha in Huntington’s disease neurodegeneration. Cell Metab. 2006;4:349–62.
Wheeler V. C, White J. K, Gutekunst C. A, Vrbanac V, Weaver M, Li X. J, et al., Long
glutamine tracts cause nuclear localization of a novel form of huntingtin in medium spiny
striatal neurons in HdhQ92 and HdhQ111 knock-in mice. Hum Mol Genet. 2000;9:503–
13.
White J. K, Auerbach W, Duyao M. P, Vonsattel J. P, Gusella J. F, Joyner A. L, et al.,
Huntingtin is required for neurogenesis and is not impaired by the Huntington’s disease
CAG expansion. Nat Genet. 1997;17:404–10.
Yang L, Calingasan N Y, Thomas B, Chaturvedi RK, Kiaei M, Wille E J, et al., Neuroprotective
effects of the triterpenoid, CDDO methyl amide, a potent inducer of Nrf2-mediated
transcription. PLoS One. 2009;4:e5757.
Yang L, Calingasan N. Y, Wille E. J, Cormier K, Smith K, Ferrante R. J, et al., Combination
therapy with coenzyme Q10 and creatine produces additive neuroprotective effects in
models of Parkinson’s and Huntington’s diseases. J Neurochem. 2009;109:1427–39.
Zadak Z, Hyspler R, Ticha A. Antioxidants and vitamins in clinical conditions. Physiol Res.
2009;58:S13-S17.
Zwilling D, Huang S. Y, Sathyasaikumar K. V, Notarangelo F. M, Guidetti P, Wu H. Q, et al.,
Kynurenine 3-monooxygenase inhibition in blood ameliorates neurodegeneration. Cell.
2011;145:863–74.

SECTION IV: ANTIOXIDANT ACTIVITY IN
CHRONIC DEGENERATIVE DISEASES

Chapter 9
OXIDATIVE DAMAGE AND ANTIOXIDATIVE DEFENSE

MECHANISMS IN INFLAMMATION
Newman Osafo*, Joseph Owusu-Sarfo and David Darko Obiri

Kwame Nkrumah University of Science and Technology, Kumasi, Ghana
ABSTRACT
The immune system plays a role in the production of substances such as cytokines and
oxidant molecules which include free radicals. These cell products are meant to destroy
invading organisms that do cause tissue damage which contributes to recovery. This does
not rule out the fact that oxidants can have a deleterious effect on healthy tissues.
Henceforth, excessive production of oxidants plays a major role in mortality due to
infection, trauma and inflammatory diseases. Oxidants enhance cellular production of pro-
inflammatory markers like TNF-α IL-1 and IL-8 via activation of nuclear transcription
factor NF-κB. Antioxidant defense, therefore, has a direct and indirect protective function
on tissues by inhibiting damaging effects of cytokines and oxidants. Also, antioxidants
reduce the activation of the pro-inflammatory transcription factor, NF-κB hence regulating
cytokine production. This chapter discusses the in-depth effect of antioxidants on the
inflammatory pathways and the importance they play in inflammatory therapy.
1. INTRODUCTION
In the area of medicine, much attention has been given to free radicals, otherwise called
pro-oxidants. Their role is implicated in the etiology of many chronic diseases that affect
humans, such as cardiovascular diseases and atherosclerosis; cancer; a myriad of
neurodegenerative diseases; aging and inflammatory lesions (Phaniendra et al., 2014; Rahal et
al., 2014). Pro-oxidants refer to substances (which may be endogenous or exogenous), that have
the ability to oxidize target molecules. The oxidation may occur directly or indirectly. The
*
Corresponding Author’s Email: nosafo.pharm@knust.edu.gh.

232 Newman Osafo, Joseph Owusu-Sarfo and David Darko Obiri
direct mechanism is mediated via abstraction of electrons while the indirect mechanism is via
the production of intermediates that are highly reactive, e.g., free radicals (Rahal et al., 2014).
A free radical refers to a molecule or an atom that has at least one unpaired electron and is
able to independently exist (Poyton et al., 2009). Free radicals are very reactive, unstable and
relatively short-lived. This feature is as a result of the fact that these chemical entities have an
odd number of electrons. In order to attain stability, a free radical can quickly react with other
chemical entities to steal the required electron. When a free radical attacks a molecule, it steals
an electron from the attacked molecule. When this happens, the attacked molecule, in turn,
becomes a free radical. This is the beginning of a series of chemical processes that consequently
lead to cellular damage (Reuter et al., 2010).
Reactive oxygen species are chemical compounds that contain an oxygen atom, obviously.
One peculiarity that differentiates them from other oxygen-containing chemical compounds is
the fact that these compounds are very reactive, or they are rapidly converted to these oxygen
radicals in the cells. ROS include ozone (O3); singlet oxygen (1O2); molecular oxygen (O2);
superoxide anion (O2-); hydrogen peroxide (H2O2); hydroxyl radical (OH•); peroxyl radical
(RCOO•) and hypochlorous acid (HOCl). Singlet oxygen is oxygen with antiparallel spins. It
is not considered a free radical but it is very reactive. However, it is not considered an important
source of toxicity in vivo due to its rapid decomposition. Superoxide anion is in the same way
extremely reactive but its solubility in a lipid environment is woefully poor, so it cannot diffuse
far. Through the Haber-Weiss process, superoxide anion upon reaction with hydrogen peroxide
yields hydroxyl radical, a very powerful radical.
Another group of free radicals – the reactive nitrogen oxide species (RNOS) and reactive
nitrogen species (RNS) also exist (Nimse and Pal, 2015). They include nitric oxide (NO) and
peroxynitrite (ONOO-). RNS/RNOS are obtained majorly from nitric oxide, a free radical,
which is in turn produced by endogenous means via the activity of nitric oxidase synthase
(NOS) (Pham-Huy et al., 2008). RNOS/RNS are involved in the etiology of several disease
states including Parkinson’s disease, as well as chronic inflammatory disorders such as
atherosclerosis (Pennathur et al., 2004).
The Medical Dictionary for Dental Profession defines inflammation as a fundamental
pathological process consisting of a dynamic complex of histologically apparent cytologic
changes, cellular infiltration, and mediator release that occurs in the affected blood vessels and
adjacent tissues in response to an injury or abnormal stimulation caused by a physical, chemical
or biological agent (Farlex Partner Medical Dictionary, 2012). The inflammatory response is
an immune reaction and thus, protective, as it has the tendency to ameliorate the injurious
effects of any infection, eradicating foreign agents and damaged tissue debris, allowing
restoration of the tissues towards normality. However, persistent or chronic inflammation can
result in the development of several pathologies.
As part of the inflammatory response, leukocytes and mast cells consume a large amount
of oxygen and as such produce and release copious amounts of reactive oxygen species at the
damaged site (Coussens and Werb, 2002; Hussain et al., 2003). Nonetheless, the inflammatory
cells produce cytokines, arachidonic acid metabolites such as the prostaglandins, and
chemokines which act via active inflammatory cells in the infected site to release even
additional reactive species. These vital mediators can cause stimulation of signal transduction
pathways, together with alterations in transcription factors like nuclear Factor-kappa B (NF-
κB). This inflammatory cum oxidative environment initiates an unhealthy vicious cycle which

Oxidative Damage and Antioxidative Defense Mechanisms in Inflammation 233
can cause damage to healthy cells - the underlying mechanism of several chronic pathologies.
Henceforth, the chapter represents the role of antioxidants on the inflammatory paths.
2. INFLAMMATION
Inflammation is one area that has received much attention with regards to research. The
inflammation cascade is a series of long-chain chemical interactions and cellular activity. The
goal of inflammation is to repair tissue damage (Freire et al., 2013). The acknowledgment of
the inflammatory response takes us back to long ago. In the first century AD, the people of old
noted that in response to tissue injury, the characteristics of a tumor (i.e., swelling); rubor (i.e.,
redness); dolor (i.e., pain) and calor (i.e., heat) were observed (Punchard et al., 2004). The
redness is due to hyperemia; a tumor or swelling is due to enhanced microvasculature
permeability and protein leakage into the interstitium. The increased flow of blood and
enhanced metabolism is the cause of the heat while the pain that is recognized is the
consequence of alterations in the perivasculature and nerve endings. These were reported to be
the cardinal signs of inflammation (Chandrasoma and Taylor, 2005). In the 1850s, through the
publications of Rudolf Virchow, organ dysfunction became the fifth cardinal sign of
inflammation (Punchard et al., 2004). The inflammatory process gives a unifying mechanism
that underlies several diseases most especially chronic conditions like rheumatoid arthritis,
osteoporosis, osteoarthritis, cardiovascular disorders and diabetes (Hunter, 2012). It is therefore
not very surprising that inflammation has become one of the most serious, if not the most
serious study areas among biomedical scientists.
Research in the 21st-century era has provided a much more elaborate knowledge of how
the cardinal signs of inflammation observed by the ancient people occur (Hansson et al., 2002).
The host defense mechanism presents two very districts but interlinked pathways – innate and
adaptive immunity.
Rapid response to tissue is mounted by the innate arm of immunity. The innate immunity
is able to identify and recognize a wide array of patterns that are present on disease-causing
organism, however, alien to the host, called pathogen-associated molecular patterns (PAMPs).
The innate aspect of immunity does not have the fine specificity of recognition unlike the
adaptive aspect of immunity (Janeway and Medzhitov, 2002; Medzhitov and Janeway, 2000).
There is an expression of a set of recognition receptors such as the scavenger receptors and
Toll-like receptors (TLRs) (Krieger, 1997). Ligands of these receptors include the PAMPs -
aldehydes derivatized proteins, lipopolysaccharides (LPS), surface phosphatidylserine and
modified forms of low-density lipoproteins (LDL). Interaction of the ligands with the scavenger
receptors can result in the endocytosis of the infectious and consequent degradation in the
lysosome (Krieger et al., 1993; Pearson, 1996). On the other hand, interaction with TLRs
activates NF-κB and mitogen-activated protein kinase (MAPK) pathways (Faure et al., 2000;
Muzio et al., 1998). The secretion of cytokines, autacoids, and mediators that enhance the local
inflammatory reaction also occur when an interaction with the Toll-like receptors occurs (Guha
and Mackman, 2001; Wright, 1999; Libby, 2002; Takeda and Akira, 2005).
The other arm of the host defense mechanism, i.e., the adaptive immunity is much more
specific. It is a slow response and the recognition of certain molecular structures is required. It
is also reliant on the generation of abundant quantities of antigen receptors (i.e., T-cell receptors

and immunoglobulins) (Hansson et al., 2002). T-cells recognize foreign antigens that are
presented to them and the latter initiate specific response that aims at that particular antigen.
The response includes a direct attack on the cells that bear the antigen. This bout is mounted
by cytotoxic T-cells. The manufacture of antibodies by B-lymphocytes is also stimulated. T-
cells differentiate into subtypes, notable among them are T helper (Th) 1 and 2. The Th-1 cells
elaborate several cytokines including interferon gamma (IFN-γ). IFN-γ stimulates macrophages
to enhance their production of mediators such as ROS, lipid molecules, autacoids, and other
cytokines, all contributing to the inflammatory response (Robertson and Hansson, 2006;
Frostegard et al., 1999). Th2 cells, on the other hand, elaborate several cytokines that augment
the development of B-cells into plasma cells that produce antibodies. The cytokines cause the
promotion of B-cells class switching to elevate immunoglobulin E (IgE) production.
Furthermore, Th2 cells also help recruit and galvanize mast cells, potent effectors of allergic
response.
3. CLASSIFICATION OF INFLAMMATION
3.1. Acute Inflammation
As the name suggests, acute inflammation is a short process and it is ephemeral, its time
course ranging from few minutes to a few days. Key characteristics of acute inflammation
include seepage of plasma proteins and/or the exodus of WBCs into an extravascular domain
(Markiewski and Lambris, 2007). Chemical factors released from the plasma mediate these
reactions. They are also responsible for swelling, redness, pain, heat, and organ dysfunction. It
is reported that three major steps are characteristic of acute inflammation (Schmid-Schonbein,
2006; Markiewski and Lambris, 2007; Kobayashi et al., 2014). These steps are increased
perfusion to the inflammatory site; which is followed by an increased diameter of the blood
vessel and vascular permeability with plasma fluid leaking from the microcirculation. The last
step is the migration of phagocytes to the surrounding tissues.
The initial changes that are observed in the course of inflammation are alterations in the
vascular flow and changes in the nature of the smaller blood vessels. Angiogenesis leading to
the generation of newer capillaries and bigger arterioles increase the flow of blood to the
inflammatory site. Enhanced changes in the blood vessels endothelium increase vascular
permeability, which eventually leads to an escape of plasma fluid into the extravascular areas
(Markiewski and Lambris, 2007). With a reduction in the fluid volume in the lumen of the
blood vessel, the viscosity of the blood increases and flow rate dwindles. Following these
alterations in the circulation, leukocyte adhesion to the endothelium commences. Eventually,
the leukocytes transmigrate into the interstitium. This is the stage that the critical aim of acute
inflammation (i.e., the supply of WBCs and plasma mediators to the inflammatory site) is duly
accomplished (Markiewski and Lambris, 2007; Kobayashi et al., 2014).
When there is extensive tissue damage, immature neutrophils are released from their place
of synthesis – the bone marrow. The movement of neutrophils to the inflammatory site is
facilitated by chemical substances called chemotactic factors that diffuse from the damaged
area, creating a concentration gradient which the neutrophils follow. This one-way movement
of the neutrophils along the concentration gradient is referred to as chemotaxis (Froese, 1980).

Great numbers of neutrophils get to the inflammatory site usually about an hour after infection.
About 24-28 hours after initiation of inflammation, another group of WBCs, the monocytes
which eventually differentiate into macrophages are recruited.
Consequentially, the immune cells that are recruited identify the invading pathogens and
destroy them. Macrophages become more prevalent at the injury site only after a long period
of time i.e., several days to weeks, representing the cellular hallmark of chronic inflammation
(Male et al., 2006). Plasma components like antibodies assist the inflammatory process. The
complement system also plays a very important role to augment the inflammatory response.
The inflammatory mediators are vehemently powerful and do not show discrimination
between the host and microbial targets. Therefore, injury to host cells is inexorable (Nathan,
2002; Medzhitov, 2010). An effective acute inflammatory action is that which leads to the
eradication of infectious agents, after which tissue repair and resolution mediated via tissue-
resident macrophages occurs (Serhan and Savill, 2005). One vital aspect of the inflammation
to the resolution transition is the shift of prostaglandins which are pro-inflammatory substances
to lipoxins. Lipoxins possess anti-inflammatory effects, in that they reduce the activity of
neutrophils and upregulate that of monocytes. The monocytes are involved in the removal of
dead cells and the initiation of remodeling of tissues. Other factors that are crucial in the
inflammatory resolution include protectins, resolvins, TGF-β and other growth factors from
macrophages (Serhan and Savill, 2005; Serhan, 2007).
3.2. Chronic Inflammation
Unlike acute inflammation, chronic inflammation occurs without the presence of a definite
provocation. Usually, it results from infections that were not resolved completely (Eaves-Pyles
et al., 2008)). Also, chronic inflammation may come about due to physical or chemical
substances which were not completely broken down. Genetic susceptibility may also play a
vital role (Ferguson, 2010).
The process of chronic inflammation varies and it is reliant on the kind of cells involved
and the organ. For instance, it is reported that in the western countries, chronic liver disease is
chiefly associated with chronic hepatitis B infection; hepatitis C infection, alcohol
consumption, autoimmune causes, and drug/toxin-induced liver injury (Arulselvan et al.,
2016).
3.2.1. Chronic Inflammation and Associated Diseases

In patients with infections, environmental diseases as well as chronic diseases like diabetes
and gout, inflammation exists, and it seems to be an important factor in the etiology of these
ailments. Recent evidence has also revealed that a wide range of diseases has a link with
inflammation. These include chronic blood vessel diseases (Engler et al., 1983; Schmid-
Schonbein et al., 2001; Schmid-Schonbein and Hugli, 2005), myocardial ischemia (Entman et
al., 1991; Anselmi et al., 2004), Alzheimer’s disease, cancer and arterial hypertension
(Suematsu et al., 2002). In patients with depression, indicators of inflammation have been
revealed. Furthermore, research has been able to link obesity to chronic inflammation (Nicklas
et al., 2004; Calabro et al., 2005). Thus, chronic inflammation is central to several pathologies.

4. MEDIATORS OF INFLAMMATION
4.1. Histamine
Histamine is a biogenic amine and it is a chief mediator of gastric acid secretion,

anaphylaxis and inflammation (Goodman et al., 2011). Histamine is biosynthesized from L-
histidine, via the activity of the enzyme, histidine decarboxylase (Best et al., 2017). It is a
hydrophilic compound and it consists of an imidazole ring and two methylene groups
connecting an amino group to the imidazole ring. The mast cell is the dominant storage site for
histamine in most tissues, thus tissues like the skin, bronchial and intestinal mucosa that express
a lot of mast cells will have a particularly large amount of histamine. In the blood, the chief
storage site of histamine is the basophil (Goodman et al., 2011). Histamine is made in the cells,
stockpiled in granules and are released when mast cells are activated. In its storage form,
histamine is bound. It is positively charged at physiological pH of about 5.5 and forms an ion
complex with negatively charged groups like heparin and proteases (Goodman et al., 2011).
The release of histamine occurs following an interaction of an antigen with IgE antibodies on
the surface of mast cells. Some drugs like succinylcholine and d-tubocurarine may also cause
mast cells to release histamine. Histamine has several physiological roles (Goodman et al.,
2011) but for the purpose of this chapter, only the role histamine plays in the inflammatory
response will be particularly considered.
The primary response to an antigen results in the generation of IgE antibodies that go to
reside on the surface of mast cells and basophils via specific high-affinity Fc receptors,
specifically the FcεR1 receptors (Goodman et al., 2011). Upon secondary exposure, antigen
bridges the IgE antibodies and via the FcεR1 receptors, there is an activation of signaling
pathways which involves kinase and the subsequent phosphorylation of several protein
components. Protein kinases involved include Lyn and Syk. Important proteins that are
phosphorylated are the subunits of FcεR1, i.e., the β and γ subunits and phospholipase C
(PLC)γ1 and PLCγ2. This results in the generation of inositol triphosphate (IP3) with
subsequent immobilization of calcium intracellularly, leading to the exocytosis of secretory
granules. Other mediators apart from histamine are released, thus, the release of histamine does
not fully explain the effects that occur, during an inflammatory response (Goodman et al.,
2011).
4.1.1. Role of Histamine in Inflammation

A lot of the features of hypersensitivity and inflammation are mediated by the biogenic
amine, histamine. Some of these effects are the vasodilation, swelling/edema, enhanced
vascular penetrability and smooth muscle contraction (Rang et al., 1995). Fluid escape from
capillaries when there is increased permeability (Sompayrac, 1999). Histamine receptors are
G-protein coupled. The H1 receptors couple to Gq/11, activating the PLC-IP3-Ca2+ pathways. H2
receptors, on the other hand, couple to Gs, activating the adenylyl cyclase cyclic AMP-PKA
pathway (Figure 1).
During the initial stages of tissue injury, there is a transient vasoconstriction which is
followed by a pronounced vasodilation mediated by histamine H1 and H2 receptors.
Vasodilation mediated by H1 receptors has a quick commencement and it is ephemeral unlike
that mediated by H2 receptors which have a slow onset but last longer.

Figure 1. The effect of histamine in inflammation.
Histamine effect on the capillaries via H1 receptors causes efflux of protein and fluid into
the extravascular space, increasing the flow of lymph, leading to the edema. The exudate
comprises clotting factors, which are able to reduce the spread of infectious mediators (Benly,
2015). Some of the proteins are antibodies that destroy the attacking agents. With the
extravasation of fluids of plasma proteins, blood flow declines, falling out of the axial stream
in the center of the lumen to flow nearer the wall of the vessel. The role of H2 receptors in this
response is quite unclear. Afterward, WBCs adhere to the vessel wall (Sompayrac, 1999),
beginning the process of transmigration, a process known as diapedesis. Diapedesis is followed
by cellular events which consequentially destroys the invading agent.
4.2. Kinin System
During allergic reactions, infections and other inflammatory events, a sequence of reactions
are activated to generate bradykinin and kallidin in circulation (Goodman et al., 2011). Kinin
receptors are B1 and B2, not to be confused with the β receptors of the adrenergic system. The
B1 receptor expression is induced by inflammation and tissue injury. Kinin metabolites are
peptides that are released by carboxypeptidases. These peptides, as autacoids, contribute to the
inflammatory response to produce the pain, increased vascular permeability, and vasodilation
that occurs in the inflammatory response. A number of their activities is mediated by
stimulation of prostaglandins, nitric oxide or endothelium-derived hyperpolarising factor
(EDHF).

Bradykinin is produced by the action of plasma kallikrein on high molecular weight

kininogen while kallidin is also produced by the hydrolysis of low molecular weight kininogen.
However, aminopeptidase can also convert kallidin to bradykinin via cleavage of the lysine-
arginine peptide bond of kallidin.
The B2 receptor is the classical bradykinin receptor and it is constitutively expressed in
normal tissues. Here, it binds selectively to bradykinin and kallidins. Des-Arg metabolites of
bradykinin and kallidins that results from the activity of carboxypeptidases N and M activate
the B1 receptor. The B1 receptor expression is prompted by tissue damage and inflammation as
well as by growth factors, cytokines, and endotoxins (Bhoola et al., 1992; Leeb-Lundberg et
al., 2005).
The kinins partake in a wide range of inflammatory conditions. Permeability in the
microcirculation is enhanced by plasma kinins. This stems from the fact that, the kinins are able
to act on tiny venules to disrupt the enter-endothelial tight junctions. This effect coupled with
an increased hydrostatic pressure gradient leads to edema (Goodman et al., 2011).
4.3. Lipid-Derived Autacoids and Inflammation
Lipid autacoids derived from arachidonic acid include the prostaglandins (PGs). The
prostaglandins mediate several physiological mechanisms including inflammation. The
synthesis of prostaglandins is greatly increased in the inflamed tissues and as such, contributes
to the principal signs of inflammation and also, there has been the establishment of the pro-
inflammatory effects concerning the individual prostaglandins, however, their part in
inflammation resolution is a matter of controversy (Ricciotti et al., 2011).
Prostanoid is a collective name given to prostaglandins and thromboxane A2 (TXA2). The
prostanoids are biosynthesized from arachidonic acids that are liberated from the cell
membranes by the activity of phospholipases and further processed by successive activities of
prostaglandin synthase G/H (also called cyclooxygenase) and corresponding synthases.
In vivo, four major prostaglandins are made; PGE2, PGI2 (prostacyclin), PGD2 and PGF2α.
Prostaglandin production elevates sharply in acute inflammation preceding leukocyte
recruitment and infiltration of immune cells. The production of prostaglandin is dependent on
the activity of G/H synthases, also known as cyclooxygenase (COX), which exists as two
isoenzymes, i.e., COX-1 and COX-2. Unlike COX-1 that is constitutively expressed in most
tissues, COX-2 activity is prompted via inflammatory stimuli and growth factors.
PGE2 is of special interest in inflammation. This is due to the fact it is associated with every
process that results in the cardinal signs of inflammation (Funk, 2001). The swelling and
redness come about of enhanced blood flow in the inflammatory site due to PGE2-mediated
arterial dilation and enhanced microvascular permeability (Funk, 2001). The effect of PGE2 on
peripheral sensory neurons and sites in the central nervous system results in the pain.
PGI2 is immediately synthesized during inflammation or tissue injury, making it an
essential mediator of the pain and edema that results from acute inflammation. Its
concentrations also rise rapidly in inflammatory milieus (Bombardieri et al., 1981).

5. REACTIVE OXYGEN SPECIES AND THEIR METABOLISM

The capacity of WBCs, precisely speaking, neutrophils to fight off invading pathogens
requires the production of free radicals, most specifically reactive oxygen species (ROS). The
cytotoxicity of the ROS results in the death of the invading organism. The role of ROS in the
protection of the body from bacterial infections thus cannot be overemphasized. Also, it is
becoming a lot more evidence that some types of tissue injury due to inflammation are mediated
by reactive species and/or their metabolites (Conner and Grisham, 1996). The demonstration
that the administration of certain antioxidants like catalase and superoxide dismutase (SOD)
has the capacity to reduce tissue injury resulting from inflammation is a typical example. This
demonstration was shown in experimental models of myocardial ischemia and reperfusion;
arthritis; and chronic gut inflammation (Granger et al., 1981; Skaleric et al., 1991; Clark et al.,
1988; Klebanoff, 1992). Data from these indicate that directly or indirectly, ROS may facilitate
the inflammation and eventual dysfunction of tissue accompanying these pathologies.
Prominent sources of ROS are macrophages, neutrophils, monocytes, as well as eosinophils
that infiltrate the tissues. When pro-inflammatory agents or infectious products interact with
the receptors on the plasma membrane of white blood cells, the cells are activated. Activation
of the leukocytes causes the assembly of NADPH oxidase, a multicomponent enzyme. Upon
assembly, the enzyme system catalyzes the generation of the enormous amount of superoxide
radicals from molecular oxygen (Nimse and Pal, 2015).
Conversion of superoxide radical to hydrogen peroxide and molecular oxygen occurs

rapidly. It is important to note that the superoxide anion is less reactive towards a lot of
biological substrates (Nimse and Pal, 2015).
Unlike the superoxide anion, hydrogen peroxide is a more potent oxidant; however, it is
less reactive towards most substrates except its levels occur in unphysiologically high
concentrations (Surh, 2005). Through the Fenton’s reaction, hydrogen peroxide yields hydroxyl
radical via interaction with iron (Knight, 1999; Sharma et al., 2012; Halliwell and Gutteridge,
2015).
Hydroxyl radical is known to be the most highly reactive oxidant (Sharma et al., 2012;
Halliwell and Gutteridge, 2015; Phaniendra et al., 2014).

It is reported that the demonstration of NO production in humans has been difficult.

However, great amounts of NO are known to be produced by rodent macrophages and
neutrophils. This production is facilitated by iNOS, the inducible isoenzyme of nitric oxide
synthase (Weinberg et al., 1995; Miles et al., 1995). Nitric oxide has also been shown to act as
an antioxidant. This is made possible by its ability to react with a superoxide radical to promote
the breakdown of both radicals yielding NO3- (Assreuy et al., 1994; Wink et al., 1993;
Harbrecht et al., 1992; Florquin et al., 1994; Kurose et al., 1993). Hydroxyl radical is a very
reactive species. It is known to interact with virtually all known biological substrates. Its half-
life is very short and it reacts where it is generated. Lipids are known to be peroxidized by
hydroxyl radical. It has also been demonstrated to oxidize proteins and cause scission of DNA
strands (Grisham, 1992).
As part of the respiratory burst, activated leukocytes secrete myeloperoxidase, a heme
protein enzyme into the extracellular environment. There, the enzyme catalyzes the oxidation
of chloride anion by hydrogen peroxidase. The result of this reaction is the generation of
hypochlorous acid (HOCl) (Klebanoff, 1992).
The toxicity of HOCl exceeds that of superoxide radical or hydrogen peroxide by 100-1000
fold. HOCl is quite non-specific, interacting with a wide variety of biomolecules – DNA,
polyunsaturated fatty acids (PUFAs), amino acids, sulfhydryls, pyridine nucleotides in addition
to other nitrogen-containing compounds (Grisham, 1992). Unlike hydroxyl radical,
hypochlorous acid does not cause peroxidation of PUFA to generate aldehydes (such as
malondialdehyde). It does, however, yield chlorohydrin derivatives (PUFA, containing
chlorine atoms that are covalently bonded to the backbone of carbon) (Winterboum et al.,
1992). The reactivity of hypochlorous acid is also very high with primary amines, producing
derivatives that contain a bond of nitrogen and chlorine (RNHCl; N-chloramines).
The toxicity of RNHCl2 depends on their membrane permeability. Those that are lipophilic
have better membrane penetrating ability and thus, are more toxic (Thomas and Grisham, 1986;
Grisham et al., 1984).
The reactive species produced via this cascade can also obliquely enhance tissue injury
inflammation by shifting the protease/antiprotease balance. Typically, hypochlorous acid
causes inactivation of protease inhibitors like α1-protease inhibitors and α2-macroglobulin. The
activation of the protease inhibitors will result in excessive and uncontrolled proteolysis by
proteolytic enzymes such as elastase. Weiss et al. (1989) demonstrated that the
myeloperoxidase (HOCl) system is able to cause the activation of silent gelatinase and
collagenase that are released by neutrophils. Putting these data together, it is suggestive that
inactivation of essential protease inhibitors through oxidation, coupled with the oxidant-
induced activation of dormant proteases provides a conducive medium to allow the proteases,
namely elastase, gelatinase and collagenase to cause degradation of the interstitial matrix. The
perturbation of the protease/antiprotease balance is widely known for its detriment and it is

central to the pathogenesis of inflammatory conditions like chronic obstructive pulmonary

disease (COPD) and emphysema.
6. FREE RADICALS AND INFLAMMATION

Over the last few years, extensive studies have established the roles free radicals and
oxidants play in the cell to cell communication. The activation of certain immunologically
relevant transcription factors by free radicals as well as the contribution of free radicals in the
modulation of some signal transduction pathways have been fully established (Brigelius-Flohe
and Maiorino, 2013; Ray et al., 2012).
Proper functioning of the immune system is dependent largely on cell-to-cell signaling.
This process of communication involves cytokine release from the tissues in response to some
physiological insult; such as drugs, disease, antigens, and radicals. Drӧge et al., 1994 reported
that the optimal activation of certain genes and the function of lymphocytes depend to a
significant extent on cellular thiols. Generally speaking, high levels of free radicals pose a
serious threat to health due to the negative impact on the immune system. On the other hand,
antioxidants seem to augment certain aspects of the immune function (Knight, 2000).
Molecular mechanism accounting for these effects is not quite clear but may be due to a balance
between the roles radicals play as toxicants of the immune system and their roles in cell-to-cell
communication.
Free radicals have been reported to actually initiate and cause aggravation of existing tissue
injury (Kehrer and Klotz, 2015). This is significant in chronic inflammatory disorders like
atherosclerosis and rheumatoid arthritis. However, concerning the roles that radicals play in
inflammatory disorders, there is time to time and case to case variation as the process of
inflammation evolves (Kehrer and Klotz, 2015).
Inflammation is a usual reaction of tissue to injury, and therefore not pathologic, at least at
the onset (Freire et al., 2013) (Figure 2). The production of free radicals in the immune reactions
(except autoimmune) is controlled and targeted as possible as it can be (Phaniendra et al., 2014).
Nonetheless, this control is not absolute, thus, aggravation of the existing damage can occur.
When uncontrolled for a long period of time or initiated by an abnormal trigger, inflammation
which is an immune response can actually become a chronic disease process. This appears to
be the underlying mechanism of diseases like atherosclerosis and rheumatoid arthritis (Libby,
2012).
Vascular endothelial cells in their normal state show great resistance to contact with WBCs.
Upon exposure to a stimulus, such as microbial agents and cytokines, there is an expression of
a palette of E-selectin, P-selectin and vascular cell adhesion molecule-1 (VCAM-1) (Cybulsky
et al., 1991; Geng et al., 1990; Larson et al., 1989; Bevilacqua et al., 1989; Lasky et al., 1989).
The VCAM-1interacts with monocytes and T-lymphocytes (Cybulsky et al., 1991) while P-
selectin, on the other hand, interacts with neutrophils (Bevilacqua et al., 1989). Via the activity
of monocyte chemoattractant protein -1 (MCP-1), there is the transmigration of monocytes into
the arterial intima. The monocytes then undergo differentiation and proliferation upon exposure
to factors like the macrophage colony stimulating factor, a very powerful co-mitogenic
mediator. The macrophages then engulf modified lipoproteins via endocytosis (Brown and
Goldstein, 1983). The modification of low-density lipoproteins (LDLs) may occur via the

oxidation by ROS, making ROS and for that matter, free radicals crucial to the pathogenesis of
several chronic pathologies like atherosclerosis. The final stage involves the accumulation of
cholesteryl ester in the cytoplasmic droplets of the macrophages, creating foam cells which are
reported to be a hallmark of nascent atherosclerotic plaque (Brown and Goldstein, 1983). Foam
cells are required for the formation of fatty streak. From this stage to atherosclerosis, more
macrophages are recruited. The generation of ROS and RNOS, with the release of degradative
enzymes, cytokines, and growth factors, alongside the direct toxicity of oxidized LDL to
endothelial cells (Cathcart et al., 1985; Hessler et al., 1983) cause great destruction to the
endothelium resulting in the formation of the atherosclerotic lesion.
Figure 2. Mechanism of tissue injury leading to inflammation.
With rheumatoid arthritis, the stimulus that leads to the production of the autoantibodies
that attack the tissue of the joint has not been fully established. However, there is no doubt that
these autoantibodies are able to bind to several proteins in the joint leading to the accumulation
of activated neutrophils (Yoo et al., 2016). These activated cells produce large amounts of ROS
and other pro-inflammatory mediators. This step is thought to make a significant contribution
to the damage that occurs in the rheumatoid joint. More neutrophils are subsequently recruited.
This cascade results in chronic inflammation and the gradual joint degradation that is
characteristic of the rheumatoid arthritis disease (Gelderman et al., 2007).
A lot of investigations have made available some evidence concerning the contribution of
ROS in rheumatoid arthritis disease. In their study, Wruck et al. (2001), using the NrP2-
knockout mouse reported that ROS are actually germane factors in the breakdown of collagen
on experimental arthritis. Another study by Stamp et al. (2012), has also supported a role for
myeloperoxidase in the pathogenesis of rheumatoid arthritis. Even though correlation does not
always mean causation, it is still likely that ROS are culprits in at least some of the etiologies
of rheumatoid arthritis.

The important role played by ROS in rheumatoid arthritis does not relegate the roles played
by other numerous inflammatory mediators. The involvement of pro-inflammatory substances
and proteases secreted by neutrophils are significant and probably, the major contributors to
this disease process.
6.1. Amplification of Inflammatory Response by Reactive Oxygen Species
Aside from direct toxicity caused by reactive oxygen species, it has also become quite
apparent that these substances may initiate and/or even augment the inflammatory cascade.
This may occur through the upregulation of several important factors that are associated with
the inflammatory response. The upregulation of these genes takes place by activation of
transcription factor; important among them is NF-κB, a ubiquitous factor and a pleiotropic
regulator of several genes that are involved in the inflammatory and immune responses
(Sienbelist et al., 1994). Other transcription factors include NF-E2 related factor, Activator
Protein-1 (AP-1), signal transducer and activator of transcription 3 (STAT3), Hypoxia-
inducible factor-1α (HIF1-α) and nuclear factor of activated T-cells. Important cellular stress
reactions are mediated by these transcription factors.
It has also been shown that the expression of pro-inflammatory cytokines and chemokines,
as well as the inducibility of nitric oxide synthase, and the initiation of cyclooxygenase-2
(COX-2) contribute greatly to the oxidative stress-induced inflammation.
When an inflammation trigger is not present, NF-κB is associated with its inhibitor, IκB in
the cytosol. IκB prevents the movement of NF-κB into the cell nucleus so that it binding to
domains on the DNA molecules to activate and upregulate genes does not occur. Activation of
NF-κB occurs via several mediators such as lipid mediators, bacterial products, proteins from
viruses, cytokines and ultimately, oxidants. All these mediators are thought to augment the
metabolism of ROS endogenously, providing a subtle connection between the inflammatory
process and oxidative stress. Production of these substances triggers the activation of redox-
sensitive kinases, resulting in the phosphorylation of IκB selectively. Ubiquitination is followed
by selective degradation via the 26S proteasome complex. The destruction of the inhibitor
paves way for NF-κB to be translocated in the nucleus. When NF-κB is activated, there is a
surge in inflammatory response through the upregulation of several pro-inflammatory
mediators and enzymes including IL-2 (Hoyos et al., 1989), TNF-α (Shakhov et al., 1990), IL-
6 (Liberman and Baltimore, 1980), and iNOS (Xie et al., 1994). Some evidence also shows that
NF-κB plays a regulatory role in the transcription of the genes that are necessary for leukocyte
adhesion, typically, VCAM-1, ICAM-1, and E-selectin (Collins et al., 1995). These adhesion
molecules are very crucial for WBC adhesion to the endothelium, and subsequent migration
through the endothelium; a process called transendothelial cell migration. Diverse antioxidants
have been reported to cause inhibition of NF-κB activation mediated via various cytokines.
They are lipoic acid (Suzuki et al., 1992), N-acetylcysteine (Shibanuma et al., 1994), tocopherol
derivatives (Suzuki and Packer, 1993), among several others.

6.2. Antioxidants Inhibit Cell Injury and Gene Transcription
The body has natural antioxidants to guard the cells and tissues against excessive
production of ROS. The antioxidants also serve to neutralize these oxidants once they are
produced. The defense mechanism comprises both enzymatic and non-enzymatic antioxidants.
Catalase, SOD, and glutathione peroxidase are enzymatic antioxidants. They offer the first line
of protection in the case of oxidative stress. Non-enzymatic antioxidants also represent another
group of antioxidants. Non-enzymatic antioxidant effects in the body are provided by
glutathione, L-arginine, coenzyme Q10, melatonin and lipoic acid ((Drӧge, 2002; Willcox et
al., 2004).). Metal chelating proteins such as ceruloplasmin, transferrin, albumin, hemopexin,
and haptoglobin do not have direct interaction with ROS. However, they are still added to the
list of antioxidants for the reason that they are able to bind to active metals and ameliorate the
generation of metal-catalyzed free radicals (Conner and Grisham, 1996).
The vitamins, E and C are essential nutrients that are very potent protectors against ROS-
mediated cellular damage. It is reported that vitamin C reduces oxidized Vitamin E, helping to
regenerate this essential antioxidant (Chan, 1993; Halliwell and Gutteridge, 1990). Vitamin E
is a sequence breaking antioxidant, preventing oxidation of LDL, inhibiting the peroxidation.
It is also reported to attenuate the toxicity that occurs with glutathione depleting agents (Conner
and Grisham, 1996; Esterbauer et al., 1993; Comporti et al., 1991).
7. ANTIOXIDANTS IN INFLAMMATION
The morbidity and mortality accompanying a wide range of diseases have been connected
to the excessive generation of pro-inflammatory cytokines and oxidants (Halliwell and
Gutteridge, 2015). It is reported that directly or indirectly, nutrient intake influences antioxidant
defense. Recent studies have provided further evidence that via an interaction with the biology
cytokines and ROS, these nutrients antioxidants elaborate some anti-inflammatory effects
which may be of clinical relevance (Shigematsu et al., 2003; Middleton, 1999; Falchetti et al.,
2001; Zhao et al., 1998; Tsimikas et al., 1999; Wang et al.., 1999; Martinez and Moreno, 2000).
7.1. Vitamin C
Also referred to as ascorbic acid, vitamin C was first isolated from adrenal glands,
cabbages, and oranges. Ascorbic acid is regarded as an essential water-soluble vitamin. It is
essential, in that it cannot be produced by the human body due to the lack of gluconolactone
oxidase enzyme, an essential enzyme in its biosynthetic process.
Vitamin C has an immense reducing capacity in some vital redox reactions in the body. It
seems to be the initial line of protection in the control of oxidative stress. Vitamin C spares
other antioxidants from being used up. This, it does by serving as a reductant in physiologically
important redox reactions like the reduction of transition metal ions.

In vivo, ascorbate also scavenges free radicals like hydroxyl radical, nitric oxide and ozone.
It is also a potent neutralizer of peroxynitrous acid, hypochlorous acid, and singlet oxygen.
Ascorbic acid is also able to stabilize catecholamines. This prevents the formation of ROS from
catecholamines. The stabilization of catecholamines by ascorbate is due to its ability to
maintain the proper functioning of dopamine-β-hydroxylase (Halliwell and Gutteridge, 2015;
Hwang et al., 2000).
Very recently, ascorbic acid has been demonstrated to offer protection against LDL
oxidation. This is made possible via covalent modification of the lipoprotein (Alul et al., 2003).
Concerning the ascorbic acid status on the immune system function, extensive studies have
been done. Diseases like rheumatoid arthritis have been postulated to have inflammatory as
well as oxidative stress mechanisms underlying their pathogenesis. It is reported that vitamin
C tends to scavenge the ROS/RNS that results from the activated phagocytes present in the
disease site (Halliwell and Gutteridge, 2015).
Previous investigations have revealed that increased doses of vitamin C showed just little
effects on the function of the immune system during colds. Nonetheless, a significant decline
in the symptoms severity has been shown (Chalmers, 1975). It was later revealed that
movement of vitamin C into WBCs occurred at the same time with the increase in plasma
concentration after subjects received vitamin C (2000 mg) with aspirin (600 mg). Results from
this study indicated that the anti-inflammatory effects observed with aspirin during colds may
be related to its capacity to elevate the levels of vitamin C in the tissues and the direct effect on
vitamin C in the cells of the inflamed tissues.
7.2. Vitamin E
Vitamin E also referred to as tocopherol, is an essential lipid soluble vitamin. Eight isomers
of vitamin E exists, however, the most effective among these is alpha-tocopherol (Halliwell
and Gutteridge, 1990; Bhavnani et al., 2001).
Vitamin E neutralizes peroxyl radicals and it is reported to be the most vital inhibitor of
the sequence reaction of lipid peroxidation. In addition to this, alpha-tocopherol is able to both
neutralize and react with superoxide, singlet oxygen and hydroxyl radical. It also prevents nitric
oxide from undergoing a reaction with superoxide radical (Halliwell and Gutteridge, 1990;
Bhavnani et al., 2001; Uysal et al., 1998).
Important investigations have assessed the anti-inflammatory properties of vitamin E and
its clinical correlations. Some of these studies demonstrated that tocopherol may prevent and/or
slow down the rate of development of atherosclerosis. However, this effect does not seem to be
related to the inhibition of LDL oxidation (Stemme, 1994). Still, on atherosclerosis, vitamin E
has shown other effects that seem to frustrate the development and progression of the condition.
These include; the inhibition of isoprostane generation (Willcox et al., 2003); inhibition of
COX-2 (Abate et al., 2000); the enhancement of NO bioactivity and the downregulation of
scavenger receptor activity resulting in the limited production of inflammatory mediators by
macrophages (Beharka et al., 2000).
The inhibition of phospholipase A2 is of therapeutic relevance in the inflammatory process
owing to its function as the rate-limiting enzyme in the production of eicosanoids. Some
investigations have demonstrated that vitamin E retards the progression of arachidonic acid
metabolism due to inhibition of PLA2 and COX activities (Esposito et al., 2002). The vitamin

has been reported to limit pro-inflammatory events in the microglia due to its inhibition of PGE2
synthesis, secretion of migration inhibitory factor (MIF) and monocyte cell adhesion (Li et al.,
2001).
Non-steroidal anti-inflammatory drugs have the limitation of causing ulceration of the
gastrointestinal tract (GIT) mucosa. Recent investigations have revealed that the GIT ulcerative
effects of aspirin may be attenuated by co-administration of tocopherol, which increases the
sensitivity of aspirin to COX-2. This synergy was demonstrated in macrophage cell lines. The
researchers, therefore, suggested that the anti-inflammatory therapy may be successful with
smaller doses of aspirin when it is co-administered with tocopherol, hence, reducing the GIT
side effects associated the high dose of aspirin (Jaarin et al., 2002).
7.3. Carotenoids
Carotenoids were first isolated from carrots but they are well distributed in several
vegetables and fruits. Beta-carotene and lycopene are the major carotenoids (Halliwell and
Gutteridge, 2015; Furhman and Aviram, 2001). Transportation of carotenoids in plasma is via
complexation with LDL. Carotenoids are considered to be pro-vitamins in that they can be
biotransformed to active vitamin A. Pham-Huy et al. (2008) reported beta-carotene to be the
best neutralizer of singlet oxygen. Carotenoids have been shown to exhibit very high
antioxidant potency. As an antioxidant, beta-carotene is reported to inhibit peroxidation of
simple lipid at a reduced oxygen-oxygen concentration (Halliwell and Gutteridge, 2015;
Furhman and Aviram, 2001). Lycopene, on the other hand, interacts with peroxynitrite and
offer protection of LDL from peroxynitrite-induced oxidation.
These compounds have been shown to exhibit positive effects on the function of the
immune system; however, it has been well established that their most important role is to act
as precursor to retinol, which is very key for proper vision, and cell growth and differentiation
(Halliwell and Gutteridge, 2015). Furthermore, studies have shown that carotenoids such as
beta-carotene, bixin, canthaxanthin, and lutein suppress macrophage respiratory burst via
superoxide anion scavenging (Zhao et al., 1998).
7.4. Polyphenols
The polyphenols are plant phytochemicals with low molecular weight. It is reported that
over 4000 polyphenols have been identified and classified. They occur in seeds, nut, herbs,
fruits, vegetables, flowers, red wine, etc. They are classified into anthocyanidins, flavanols,
flavanones, flavonols, flavones and phenylpropanoids (Table 1) (Halliwell and Gutteridge,
2015).
The flavonoids appear to be the largest and most investigated group with flavanols being
the most widely distributed (Halliwell and Gutteridge, 2015; Wilson, 1978).
Phenols contain a hydroxyl group connected to a benzene ring. A polyphenol is made up
of a 3-ring system, i.e., an aromatic ring which condensed to a heterocyclic ring, which is also
connected to another aromatic ring. The particular hydroxylation pattern of ring C enhances the
activity of the flavonol (Middleton et al., 2000). This effect is particularly observed in the
inhibition of mast cell degranulation. Because polyphenols have both hydrophilic and

hydrophobic components, they are able to scavenge ROS that is generated in both aqueous and
lipid media.
Table 1. Classes of polyphenols and their main sources.
Compound Sources
Anthocyanidines: Red grapes, red wine
Malvidin Cherry, raspberry, strawberry, grapes
Cyanidin Colored fruit and peels
Apigenidin
Flavanones: Peel of citrus fruits
Taxifolin Citrus fruits
Maringin
Flavonols: Endive, leek, broccoli, radish, grapefruit, black tea
Kaempferol Onion, lettuce, apple skin, berries, olive, red wine
Quercetin Cranberry, grapes, red wine
Myricetin
Flavones: Fruit skin
Chrysin Celery, parsley
Apigenin
Phenylpropanoids: Apples, peaches, apricots, blueberries, tomatoes
Chlorogenic acid White grapes, olives, olive oil, asparagus
Caffeic acid White grapes, white wine, cabbage, asparagus
p-Coumaric acid
Polyphenols have shown a strong antioxidant effect in terms of guarding against LDL
oxidation (Halliwell and Gutteridge, 2015; Furham and Aviram, 2001a; Bhavnani et al., 2001;
Esposito et al., 2002; Fuhrman and Aviram, 2001b). From the studies of Esposito et al. (2002),
and Fuhrman and Aviram (2001b), it has been demonstrated that polyphenols are able to
scavenge peroxyl radical, hydroxyl radical, hypochlorous acid and perynitrous acid. The studies
also showed that polyphenols spare LDL associated antioxidants such as tocopherol and
carotenoids. Enhancement of hydrolyzation of oxidized LDL mediated via the preservation of
serum paraoxonase activity was also observed in these two studies. In the case of cardiovascular
disease, polyphenols have shown remarkable protective effects (Halliwell and Gutteridge,
2015; Furham and Aviram, 2001a; Fuhrman and Aviram, 2001b; Keli et al., 1996; Sesso et al.,
1999).
Resveratrol is a polyphenolic compound with a lot of biological effects both in vitro and in
vivo. This compound has been demonstrated to reduce hydrogen peroxide and superoxide
radical generation, and LDL oxidation (Halliwell and Gutteridge, 2015; Furham and Aviram,
2001a; Bhavnani et al., 2001; Frankel et al., 1993). Also, reduced arachidonic acid release from
macrophages (Martinez and Moreno, 2000) and inhibition of platelet aggregation (Pignatelli et
al., 2000; Bertelli et al., 1995) have been reported.
Anti-inflammatory effects of polyphenols have been evaluated in several studies. Their
inhibitory effects on mast cells and subsequent histamine release have been established (Sagin
and Sozmen, 2004). Flavonoids may also inhibit the generation of ROS by neutrophils and
other phagocytes. The inhibition of ROS generation by these immune cells are possibly

mediated via interference with myeloperoxidase and NADPH oxidase and in neutrophils (Sagin
and Sozmen, 2004).
The particular hydroxylation patterns of ring C enhance the activity of these agents,
improving their inhibition of mediators like histamine, IL-8, and IL-6, all of which are
associated with inflammation (Middleton et al., 2000). Matsuoka et al. (2002) reported that the
green tea polyphenol was observed to exhibit profound antioxidant and anti-inflammatory
effects. This occurred as a reduction in the production of cytokines as shown in human lung
alveolar epithelial cells (A549 cell line). The reduction in cytokine production was linked to
inactivation of protein kinases like the MAPK, by name Jun N-terminal kinase as well as p38
which mediated the production of IL-8.
Flavonoids have also been shown to inhibit the activity of lipoxygenase and COX, in
addition to their inhibition of PLA2 (Esposito et al., 2002; Shigematsu et al., 2003). With
blockade of the COX pathway, there is reduced formation of TXA2, a very potent platelet
aggregator and vasoconstrictor. The polyphenols have been shown to attenuate the reduction in
blood flow resulting from endothelial damage and the eventual adhesion of inflammatory cells
and platelets to the damaged site (Shigematsu et al., 2003; Shimada et al., 1999).
Data from recent works have revealed that polyphenols may also exhibit anti-inflammatory
effects by reducing the expression of the cytokines, IFN-γ, TNF-α, and IL-1. The reduced
expression of these cytokines goes a long way to downregulate the activation of NF-κB by
TNF-α.
Several other immune functions have been reported of the flavonoids. These include
modulation of antigen presentation and mononuclear cell adhesion, lymphocyte proliferation
and neutrophil respiratory burst (Middleton, 1998).

AND CHALLENGES
Antioxidants have shown a lot of potential biological properties. The polyphenols, for
instance, have been gaining much attention and interest as a result of their wide utilization in
various pathological situations (Arulselvan et al., 2016). A number of investigations have
established a link between oxidative stress and inflammation. Even though several studies have
shown that dietary intake of antioxidants is beneficial for the prevention and treatment of such
conditions, only a few clinical studies have been conducted to establish the relevance of the
animal and cell-based experiments for extrapolation in humans (Hussain et al., 2016).
Even further, a number of the well-known antioxidant vitamins and even the recently
developed agents like the nitrones have not been able to pass clinical trials for prevention and
treatment of various diseases (Firuz et al., 2011).
One novel and future approach in the area of antioxidant therapy includes gene therapy.
The purpose of this is to produce more antioxidants in the body. Genetic engineering can also
be employed to obtain plant products that have a higher level of antioxidants. Synthetic
antioxidant enzymes such as SOD mimetic agents, novel biomolecules and the utilization of
functional foods that are enriched with antioxidants also represent a new approach in the area
of antioxidant therapy (Devasagayam et al., 2004).

CONCLUSION
Dietary antioxidants offer a lot of protection against oxidative stress and certain related
disorders. The effects of antioxidants also have implications in inflammation. Antioxidant
intake via diet influences the progression of inflammation by attenuating the production of
RNOS/ROS by phagocytes. The reducing ability of dietary antioxidants downregulates NF-κB
activation, reducing the upregulation of iNOS, COX-2 and several mediators that participate in
the recruitment of phagocytes. Current knowledge suggests that the use of nutritional
modulation is an effective and safer method of preventing disorders in which oxidative and
inflammatory mechanisms are involved. With a lot of evidence backing the effects of dietary
antioxidants in the inflammatory response, more attention should be given to research
evaluating the impacts of these nutrients on various facets of inflammation.
REFERENCES
Abate A, Yang G, Dennery P. A, Oberle S, Schröder H. Free Rad. Biol. Med., 2000, 29 (11):
1135.
Alul R. H, Wood M, Longo J, Marcotte A. L, Campione A. L, Moore M. K, Lynch S. M. Free
Rad. Biol. Med., 2003, 34 (7), 881.
Anselmi A, Abbate A, Girola F, Nasso G, Biondi-Zoccai G. G, Possati G, et al. Myocardial
ischemia, stunning, inflammation, and apoptosis during cardiac surgery: a review of
evidence. European Journal of Cardio-Thoracic Surgery. 2004 Mar 1, 25(3):304-11.
Arulselvan P, Fard M. T, Tan WS, Gothai S, Fakurazi S, Norhaizan M. E, et al. Role of
antioxidants and natural products in inflammation. Oxid Med Cell Longev. 2016, 2016.
Assreuy J, Cunha F. Q, Epperlein M, Noronha‐Dutra A, O’Donnell C. A, Liew F. Y, et al.
Production of nitric oxide and superoxide by activated macrophages and killing of
Leishmania major. Eur. J. Immunol. 1994 Mar, 24(3):672-6.
Beckmen J. S, Beckman T. W, Chen J, Marshall P. A, Freeman B. A. Apparent hydroxyl radical
production by peroxynitrite: implication for endothelial injury from nitric oxide and
superoxide. Proc Natl Acad Sci USA. 1990, 87:1620-4.
Beharka A. A, Han S. N, Adolfsson M. S, Wu D, Smith D, Lipman R, Cao G, Meydani M,
Meydani S. H. Nutr. Res., 2000, 20 (2), 281.
Benito M. J, Veale D. J, FitzGerald O, van den Berg W. B, and Bresnihan B, 2005. Synovial
tissue inflammation in early and late osteoarthritis, Ann Rheum Dis, vol. 64, no. 9, pp.
1263–1267, 2005.
Benly. P. Role of Histamine in Acute Inflammation. 2015. J. Pharm. Sci. Res 7(6):373-376.
Bertelli A. A, Giovannini L, Giannesi D, Migliori M, Bernini W, Fregoni M, Bertelli A. Int. J.
Tissue React., 1995, 17, 1.
Best J, Nijhout H. F, Samaranayake S, Hashemi P, Reed M. A mathematical model for
histamine synthesis, release, and control in varicosities. Theor Biol Med Model. 2017,
14(1):24. Published 2017 Dec 12. doi:10.1186/s12976-017-0070-9.
Bevilacqua M. P, Stengelin S, Gimbrone M. A, Jr, Seed B. Endothelial leukocyte adhesion
molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins
and lectins. Science. 1989, 243:1160–1165.

Bhavnani B. R, Cecutti A, Gerulaht A, Woolever A C, Berco M. J, North. Am. Men. Soc., 2001,
8 (6), 408.
Bhoola K. D, Figueroa C. D, Worthy K. Bioregulation of kinins: Kallikreins, kininogens, and
kininases. Pharmacol Rev, 1992, 44:1–80.
Bombardieri S, Cattani P, Ciabattoni G, Di Munno O, Pasero G, Patrono C et al. The synovial
prostaglandin system in chronic inflammatory arthritis: differential effects of steroidal and
nonsteroidal anti-inflammatory drugs. Br J Pharmacol. 1981, 73:893–901.
[PubMed:6895043].
Brigelius-Flohe R, Maiorino M. Glutathione peroxidases. Biochim Biophys Acta
2013,1830:3289–3303.
Brown M. S, Goldstein J. L. Lipoprotein metabolism in the macrophage: implications for
cholesterol deposition in atherosclerosis. Annu Rev Biochem. 1983, 52:223–261.
Calabro P, Chang D. W, Willerson J. T, and Yeh E. T. H, “Release of C-reactive protein in
response to inflammatory cytokines by human adipocytes: linking obesity to vascular
inflammation,” J Am Coll Cardiol, vol. 46, no. 6, pp. 1112–1113, 2005.
Cathcart M. K, Morel D. W, Chisolm G. M. Monocytes and neutrophils oxidize low density
lipoprotein making it cytotoxic. J Leukoc Biol 1985,38:341–350.
Chalmers T C. Effects of ascorbic acid on the common cold: an evaluation of the evidence. Am
J Med. 1975 Apr 1,58(4):532-6.
Chan A. C. Partners in defense, vitamin E and vitamin C. Can J Phvsiol Pharmacol
1993,71:725.
Chandrasoma P, Taylor C. R. Part A. General Pathology, Section II. The Host Response to
Injury, Chapter 3. The Acute Inflammatory Response, sub-section Cardinal Clinical Signs.
Concise Pathology. 2005.
Clark D. A, Fomabaio D. M, McNeil1 H, Mullane K. M, Caravella S. J, Miller M. J. S.
Contribution of oxygen-derived free radicals to experimental necrotizing enterocolitis. Am
J Path 01 1988, 130:537.
Cohen MS, Britigan BE, Hassett DJ, Rosen GM. Do humans neutrophils form hydroxyl
radical? Evaluation of an unresolved controversy. Free Rad Biol Med. 1988 Jan 1,5(2):81-
8.
Collins T, Read MA, Neish AS, Whitley MZ, Thanos D, Maniatis T. Transcriptional regulation
of endothelial cell adhesion molecules: NF-κB and cytokine-inducible enhancers. FASEB
J 1995,9:899.
Comporti M, Maellaro E, Del-Bello B, Casini A. F. Glutathione depletion: Its effects on other
antioxidant systems and hepatocellular damage. Xenobiotica 1991,21: 1067.
Conner E. M, Grisham M. B, Inflammation, Free Radicals, and Antioxidants, Nutrition Vol.
12, No. 4, 1996.
Coussens L. M and Werb Z, “Inflammation and cancer,” Nature, vol. 420, no. 6917, pp. 860–
867, 2002.
Cybulsky M. I, Gimbrone M. A, Jr. Endothelial expression of a mononuclear leukocyte
adhesion molecule during atherogenesis. Science. 1991,251:788–791.
Devasagayam T. P, Tilak J. C, Boloor K. K, Sane K. S, Ghaskadbi S. S, Lele R. D. Free radicals
and antioxidants in human health: current status and future prospects. Japi. 2004
Oct,52(794804):4.
Dizdaroglu M, Jaruga P, Birincioglu M, Rodriguez H. Free radical-induced damage to DNA:
mechanisms and measurement1, 2. Free Rad Biol Med. 2002 Jun 1,32(11):1102-15.

Drӧge W, Schulze-Osthoff K, Mihm S, Galter D, Schenk H, Eck HP et al. Functions of

glutathione and glutathione disulfide in immunology and immunopathology. FASEB J
1994,8:1131–1138.
Drӧge W. Free radicals in the physiological control of cell function. Review. Physiol. Rev.
2002, 82: 47-95.
Eaves-Pyles T, Allen C. A, Taormina J, Swidsinski A, Tutt C. B, Jezek G. E, et al. Escherichia
coli isolated from a Crohn’s disease patient adheres, invades, and induces inflammatory
responses in polarized intestinal epithelial cells. Int J Med Microbiol. 2008 Jul 1,298(5-
6):397-409.
Engler R, Schmid-Schonbein G, and Pavelec R, “Leukocyte ¨ capillary plugging in myocardial
ischemia and reperfusion in the dog,” Am J Pathol, vol. 111, no. 1, pp. 98–111, 1983.
Entman M. L, Michael L, Rossen R. D, Dreyer W. J, Anderson D. C, Taylor A. A et al.
Inflammation in the course of early myocardial ischemia. The FASEB journal. 1991
Aug,5(11):2529-37.
Esposito E, Rotilio D, Di-Matteo V, Di-Giulio C, Cacchio M, Algeri S. Neurobiol. Aging.,
2002, 23, 719.
Esterbauer H, Wag G, Puhl H. Lipid peroxidation and its role in atherosclerosis. Br Med Bull
1993:49:566.
Falchetti R, Fuggetta M. P, Lanzilli G, Tricarico M, Ravagnan G. Effects of resveratrol on
human immune cell function. Life sciences. 2001 Nov 1,70(1):81-96.
Faure E, Equils O, Sieling P. A, Thomas L, Zhang F. X, Kirschning C. J, et al. Bacterial
lipopolysaccharide activates NF-κB through toll-like receptor 4 (TLR-4) in cultured human
dermal endothelial cells differential expression of TLR-4 and TLR-2 in endothelial cells. J
Biologic Chem. 2000 Apr 14,275(15):11058-63.
Ferguson L. R, “Chronic inflammation and mutagenesis,” Mutation Research—Fundamental
and Molecular Mechanisms of Mutagenesis, vol. 690, no. 1-2, pp. 3–11, 2010.
Firuzi O, Miri R, Tavakkoli M, Saso L. Antioxidant therapy: current status and future prospects.
Curr. Med. Chem. 2011 Sep 1,18(25):3871-88.
Florquin S, Amraoui 2, Dubois C, Decuyper J, Goldman M. The protective role of
endogenously synthesized nitric oxide in staphylococcal enterotoxin B-induced shock in
mice. J Exp Med 1994,180:1153.
Frankel E. N, Waterhouse A. L, Kinsella J. E. Lancet, 1993, 341, 1103.
Freire M. O, Van Dyke T. E. Natural resolution of inflammation. Periodontol 2000. 2013 Oct
1,63(1):149-64.
Froese A. 1980. Structure and function of the receptor for IgE. Crit. Rev. Immunol 1(2): 79-
132.
Frostegård J, Ulfgren A. K, Nyberg P, Hedin U, Swedenborg J, Andersson U, et al. Cytokine
expression in advanced human atherosclerotic plaques: dominance of pro-inflammatory
(Th1) and macrophage-stimulating cytokines. Atherosclerosis. 1999 Jul 1,145(1):33-43.
Fuhrman B, Aviram M. Handbook of Antioxidants, Second Edition (Ed: Enrique-Cadenas,
Lester-Packer), Marcel-Dekker, Inc: New York, 2001a, pp. 303-336.
Fuhrman B, Aviram M. Flavonoids protect LDL from oxidation and attenuate atherosclerosis.
Curr Opin Lipidol. 2001b Feb 1,12(1):41-8.
Funk C. D. Prostaglandins and leukotrienes: advances in eicosanoid biology. Science. 2001,
294:1871–1875. [PubMed: 11729303].

Gelderman K A, Hultqvist M, Olsson L. M, Bauer K, Pizzolla A, Olofsson P et al. Rheumatoid

arthritis: the role of reactive oxygen species in disease development and therapeutic
strategies. Antioxid Redox Signal. 2007 Oct 1,9(10):1541-68.
Geng J. G, Bevilacquat M. P, Moore K. L, Mclntyre T. M, Prescott S. M, Kim J. M et al. Rapid
neutrophil adhesion to activated endothelium mediated by GMP-140. Nature. 1990
Feb,343(6260):757.
Goodman L. S, Brunton L. L, Chabner B, Knollman B. C. 2011, Goodman and Gilman’s
Pharmacological Basis of Therapeutics. New York: McGraw-Hill.
Granger D N, Rutili G, McCord J. M. Superoxide radicals in feline intestinal ischemia.
Gastroenterology 1981,81:22.
Grisham M. B, Jefferson M. M, Thomas E. L. Chlorination of endogenous amines by isolated
neutrophils. J Biol Chem 1984,259:10,404.
Grisham M. B. Reactive oxygen metabolism. In: Grisham MB, ed. Reactive metabolites of
oxygen and nitrogen in biology and medicine. Austin: RG Landers Company, 1992:39.
Guha M, Mackman N. LPS induction of gene expression in human monocytes. Cell Signal.
2001,13:85–94.
Halliwell B, Gutteridge J. M. C. Free radicals in biology and medicine. Oxford University
Press, USA, 2015.
Halliwell B. Gutteridge J. M. C. The antioxidants of human extracellular fluids. Arch Bioihem
Biophys 1990,280:1.
Hansson G. K, Libby P, Schonbeck U, Yan Z. Q. Innate and adaptive immunity in the
pathogenesis of atherosclerosis. Circ Res. 2002,91:281–291.
Harbrecht B. G, Billiar T. R, Stadler J. O, Demetris A. J, Ochoa J B, Curran R. D et al. Nitric
oxide synthesis serves to reduce hepatic damage during acute murine endotoxemia. Crit.
Care Med. 1992 Nov,20(11):1568-74.
Hessler J. R, Morel D. W, Lewis L. J, Chisolm G. M. Lipoprotein oxidation and lipoprotein-
induced cytotoxicity. Arteriosclerosis 1983,3:215–222.
Hoyos B, Ballard D. W, Bohnlein E, Siekevity M, Greene W. C. Kappa-B specific DNA
binding proteins: Role in the regulation of human interleukin-2 gene expression. Science
1989,244:457.
Hunter P. The inflammation theory of disease: The growing realization that chronic
inflammation is crucial in many diseases opens new avenues for treatment. EMBO reports.
2012 Nov 1,13(11):968-70.
Hussain S. P, Hofseth L. J, and Harris C. C, “Radical causes of cancer,” Nature Reviews
Cancer, vol. 3, no. 4, pp. 276–285, 2003.
Hussain T, Tan B, Yin Y, Blachier F, Tossou M. C, Rahu N. Oxidative stress and inflammation:
what polyphenols can do for us?. Oxid Med Cell Longev. 2016,2016.
Hwang J, Peterson H, Hodis H. N, Choi B, Sevanian A. Atheroscl., 2000, 150, 275.
Inflammation. (n.d) Farlex Partner Medical Dictionary. (2012). Accessed 14 August 2018.
Available: https://medical-dictionary.thefreedictionary.com/inflammation.
Jaarin K, Gapor M. T, Nafeeza M. I, Fauzee A. M. Effect of various doses of palm vitamin E
and tocopherol on aspirin-induced gastric lesions in rats. Int J Exp Pathol. 2002, 83(6):295-
302.
Janeway C. A, Jr., Medzhitov R. Innate immune recognition. Annu Rev Immunol. 2002,20:197–
216.

Johnson K. J, Ward P. A. Role of oxygen metabolites in immune complex injury of lung. J

Immunol 1981, 126:2365.
Keli S. O, Hertog M. G, Feskens E. J, Kromhout D. Dietary flavonoids, antioxidant vitamins,
and incidence of stroke: the Zutphen study. Arch Intern Med. 1996 Mar 25,156(6):637-42.
Knight J. A. Free radicals, antioxidants, aging and disease. Amer Assn for Clinical Chemistry,
1999.
Knight J. A. Free radicals, antioxidants, and the immune system. Ann Clin Lab Sci
2000,30:145–158.
Kobayashi H, Higashiura Y, Shigetomi H, and Kajihara H, “Pathogenesis of endometriosis: the
role of initial infection and subsequent sterile inflammation (Review),” Mol Med Rep, vol.
9, no. 1, pp. 9–15, 2014.
Krieger M, Acton S, Ashkenas J, Pearson A, Penman M, Resnick D. Molecular flypaper, host
defense, and atherosclerosis. Structure, binding properties, and functions of macrophage
scavenger receptors. J Biol Chem. 1993,268:4569–4572.
Krieger M. The other side of scavenger receptors: pattern recognition for host defense. Curr
Opin Lipidol. 1997,8:275–280.
Kurose I, Kubes P, Wolf R, Anderson D. C, Paulson J, Miyasaka M et al. Inhibition of nitric
oxide production. Mechanisms of vascular albumin leakage. Circ. Res. 1993 Jul
1,73(1):164-71.
Larsen E, Celi A, Gilbert G. E, et al. PADGEM protein: a receptor that mediates the interaction
of activated platelets with neutrophils and monocytes. Cell. 1989,59:305–312.
Lasky L. A, Singer M. S, Yednock TA, Dowbenko D, Fennie C, Rodriguez H, et al. Cloning
of a lymphocyte homing receptor reveals a lectin domain. Cell. 1989 Mar 24,56(6):1045-
55.
Leeb-Lundberg L. F, Marceau F, Müller-Esterl W, Pettibone D. J, Zuraw B. L. International
union of pharmacology. XLV. Classification of the kinin receptor family: from molecular
mechanisms to pathophysiological consequences. Pharmacol Rev. 2005 Mar 1,57(1):27-
77.
Li Y, Liu L, Barger S. W, Mrak R. E, Griffin W. S. Vitamin E suppression of microglial
activation is neuroprotective. J. Neurosci. Res. 2001 Oct 15,66(2):163-70.
Libby P. Inflammation in atherosclerosis. Arterioscler Thromb Vasc Biol. 2012 Sep
1,32(9):2045-51.
Libby P. Inflammation in atherosclerosis. Nature. 2002, 420:868–874.
Liberman TA, Baltimore D. Activation of interleukin-6 gene expression through the NF-κB
transcription factor. Mol Cell Biol 1990, lo:2327.
MacGlashan D. Histamine: a mediator of inflammation. J. Allergy Clin. Immunol. 2003 Oct
1,112(4):S53-9.
Male D, Brostoff J, Roitt B. D (2006) Immunology, 7th edition. Canada: Mosby Elsevier.
Markiewski M. M and Lambris J. D, “The role of complement in inflammatory diseases from
behind the scenes into the spotlight,” Am J Pathol, vol. 171, no. 3, pp. 715–727, 2007.
Martinez J, Moreno J. J. Effect of resveratrol, a natural polyphenolic compound, on reactive
oxygen species and prostaglandin production. Biochem Pharmacol. 2000 Apr 1,59(7):865-
70.
Matsuoka K, Isowa N, Yoshimura T, Liu M, Wada H. Green tea polyphenol blocks H2O2-
induced interleukin-8 production from human alveolar epithelial cells. Cytokine. 2002 Jun
1,18(5):266-73.

Medzhitov R, “Origin and physiological roles of inflammation,” Nature, vol. 454, no. 7203,
pp. 428–435, 2008.
Medzhitov R, Janeway C, Jr. Innate immunity. N Engl J Med. 2000,343:338–344.
Middleton E, Kandaswami C, Theoharides T. C. The effects of plant flavonoids on mammalian
cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev. 2000 Dec
1,52(4):673-751.
Middleton EJ. Flavonoids in the Living System. Plenum Press: New York, 1998.
Miles A. M, Owens M. W, Milligan S, Johnson G. G, Fields J. Z, Ing T. S et al. Nitric oxide
synthase in circulating vs. extravasated polymorphonuclear leukocytes. J. Leukoc. Biol..
1995 Nov,58(5):616-22.
Muzio M, Natoli G, Saccani S, Levrero M, Mantovani A. The human toll signaling pathway:
divergence of nuclear factor kappaB and JNK/SAPK activation upstream of tumor necrosis
factor receptor-associated factor 6 (TRAF6). J Exp Med. 1998,187:2097–2101.
Nathan C, “Points of control in inflammation,” Nature, vol. 420, no. 6917, pp. 846–852, 2002.
Nicklas B. J, Ambrosius W, Messier S. P, Miller G. D, Penninx B. W, Loeser R. F et al. Diet-
induced weight loss, exercise, and chronic inflammation in older, obese adults: a
randomized controlled clinical trial. Am J Clin Nutr. 2004 Apr 1,79(4):544-51.
Nimse S. B, Pal D. Free radicals, natural antioxidants, and their reaction mechanisms. Rsc
Advances. 2015,5(35):27986-8006.
Padh H. Vitamin C: Newer insights into its biochemical functions. Nutr Rev 1991,49:65.
Pearson AM. Scavenger receptors in innate immunity. Curr Opin Immunol. 1996,8:20–28.
Pennathur S, Bergt C, Shao B, Byun J, Kassim S. Y, Singh P, et al. Human atherosclerotic
intima and blood of patients with established coronary artery disease contain high density
lipoprotein damaged by reactive nitrogen species. J Biol Chem. 2004 Oct 8,279(41):42977-
83.
Pham-Huy L. A, He H, Pham-Huy C (2008). Free Radical, Antioxidants in Disease and Health.
International Journal of Biomedical Science: IJBS, 4(2), 89-96.
Phaniendra A, Jestadi D. B, Periyasamy L. Free radicals: properties, sources, targets, and their
implication in various diseases. Indian J Clin Biochem. 2014,30(1):11-26.
Pignatelli P, Pulcinelli F. M, Celestini A, Lenti L, Ghiselli A, Gazzaniga P. P, Violi F. Am. J.
Clin. Nutr., 2000, 72, 1150.
Poyton R. O, Ball K. A, and Castello P. R, “Mitochondrial generation of free radicals and
hypoxic signaling,” Trends Endocrinol Metab, vol. 20, no. 7, pp. 332–340, 2009.
Punchard N. A, Whelan C. J, Adcock I. The Journal of Inflammation. J Inflamm (Lond).
2004,1(1):1. Published 2004 Sep 27. doi:10.1186/1476-9255-1-1.
Rahal A, Kumar A, Singh V, Yadav B, Tiwari R, Chakraborty S, et al. Oxidative stress,
prooxidants, and antioxidants: the interplay. BioMed research international. 2014,2014.
Rang H, Dale M, Ritter J, Gardner P. 1995. Pharmacol. New York, NY: Churchill Livingstone.
p. 226-229.
Ray PD, Huang B-W, Tsuji Y. Reactive oxygen species (ROS) homeostasis and redox
regulation in cellular signaling. Cell Signal 2012,24:981–990.

Reuter S, Gupta S. C, Chaturvedi M. M, and Aggarwal B. B, “Oxidative stress, inflammation,

and cancer: how are they linked?” Free Rad Biol Med, vol. 49, no. 11, pp. 1603–1616,
2010.
Ricciotti E, FitzGerald G. A. Prostaglandins and inflammation. Arterioscler Thromb Vasc Biol.
2011,31(5):986-1000.
Robertson A. K, Hansson G. K. T cells in atherogenesis: for better or for worse? Arterioscler
Thromb Vasc Biol. 2006,26:2421–2432.
Sagin F. G, Sozmen E. Y. Anti-inflammatory Effects of Dietary Antioxidants. Curr. Med.
Chem. – Anti-Inflammatory & Anti-Allergy Agents, 2004, 3, 19-30.
Schmid-Schonbein G. W and Hugli T. E, “A new hypothesis for ¨ microvascular inflammation
in shock and multiorgan failure: self-digestion by pancreatic enzymes,” Microcirculation,
vol. 12, no. 1, pp. 71–82, 2005.
Schmid-Schonbein GW, “Analysis of inflammation,” Annu Rev Biomed Eng, vol. 8, pp. 93–
151, 2006.
Schmid-Schonbein G. W, Takas S, and Bergan J. J, “New ¨ advances in the understanding of
the pathophysiology of chronic venous insufciency,” Angiology, vol. 52, no. 1, supplement,
pp. S27–S34, 2001.
Serhan CN and Savill J, “Resolution of inflammation: the beginning programs the end,” Nature
Immunol, vol. 6, no. 12, pp. 1191–1197, 2005.
Serhan C. N, “Resolution phase of inflammation: novel endogenous anti-inflammatory and
proresolving lipid mediators and pathways,” Ann Rev Immunol, vol. 25, pp. 101–137, 2007.
Sesso H. D, Gaziano J. M, Buring J. E, Hennekens C. H. Am. J. Epidemiol., 1999, 149, 162.
Shakhov A. N, Collart M. A, Vassilli P, Nedospasov S. A, Jongeneel C. V. kB-type enhancers
are involved in lipopolysaccharide-mediated transcriptional activation of the tumor
necrosis factor-α gene in primary macrophages. J Exp Med 1990, 171:35.
Sharma P, Jha A. B, Dubey R. S, Pessarakli M. Reactive oxygen species, oxidative damage,
and antioxidative defense mechanism in plants under stressful conditions. J. Bot.
2012,2012.
Shibanuma M, Kuroki T, Nose K. Inhibition by N-acetyl-L-cysteine of interleukin-6 mRNA
induction and activation of NF-κB by tumor necrosis factor a! in a mouse fibroblastic cell
line, Balb13T3. FEBS Lett 1994,353:62.
Shigematsu S, Ishida S, Hara M, Takahashi N, Yoshimatsu H, Sakata T, Korthuis R. J. Free
Rad. Biol. Med., 2003, 34 (7), 810.
Siebenlist U, Franzoso G, Brown K. Structure, regulation and function of NF-κB. Ann Rev Cell
Biol 1994: lo:405.
Skaleric U, Allen J. B, Smith P. D. Mergenhagen S. E, Wahl S. M. Inhibitors of reactive oxygen
intermediates suppress bacterial cell wall-induced arthritis. J Immunol 1991, 147:2559.
Sompayrac L. 1999. How the Immune System Works. Malden, MA: Blackwell Science, Ltd. p.
37-38, 88.
Stamp L. K, Khalilova I, Tarr J. M, Senthilmohan R, Turner R, Haigh R. C, Winyard P. G,
Kettle A. J. Myeloperoxidase and oxidative stress in rheumatoid arthritis. Rheumatology
2012,51:1796–1803.
Stemme S, Hansson G. K. Immune mechanisms in atherosclerosis. Coronary artery disease.
1994 Mar 1,5(3):216-22.

Suematsu M, Suzuki H, Delano F. A, and SchmidSchonbein G. W, “The inflammatory aspect

of the microcirculation “in hypertension: oxidative stress, leukocytes/endothelial
interaction, apoptosis,” Microcirculation, vol. 9, no. 4, pp. 259–276,2002.
Surh Y. J. Oxidative stress, inflammation, and health. CRC press, 2005 May 24.
Suzuki YJ, Packer L. Inhibition of NF-κB by vitamin E derivatives. Biochem Biophys Res
Commun 1993, 193:277.
Suzuki Y. J, Aggarwal BB, Packer L. a-Lipoic acid is a potent inhibitor of NF-κB activation in
human T cells. Biochem Biophys Res Commun 1992, 189:1709.
Takeda K, Akira S. Toll-like receptors in innate immunity. Int Immunol. 2005, 17:1–14.
Thomas E. L, Grisham M. B. Cytotoxicity of chloramines. Meth Enzymol 1986, 132585.
Tsimikas S, Philis-Tsimikas A, Alexopoulos S, Sigari F, Lee C, Reaven PD. Arterioscler.
Thromb. Vasc. Biol., 1999, 19, 122.
Uysal F, Girgin F. K, Tüzün S, Aldemir S, Sözmen E. Y. Biochem. Mol. Bio. Int., 1998, 44 (6),
1255.
Wang H, Nair M. G, Strasburg GM, Chang Y. C, Booren A. M, Gray J. I, DeWitt D. L. J. Nat.
Products, 1999, 62 (2), 294.
Weber A, Boege Y, Reisinger F, and Heikenwalder M, “Chronic liver inflammation and
hepatocellular carcinoma: persistence matters,” Swiss MedicalWeekly, vol. 141, no. 4.
Weinberg J. B, Misukonis M. A, Shami P J, Mason S. N, Sauls D. L, Dittman W. A, et al.
Human mononuclear phagocyte inducible nitric oxide synthase (iNOS): analysis of iNOS
mRNA, iNOS protein, biopterin, and nitric oxide production by blood monocytes and
peritoneal macrophages. Blood. 1995 Aug 1,86(3):1184-95.
Weiss S. J. Tissue destruction by neutrophils. N Engl J Med 1989,320:365.
Willcox J. K, Ash S. L, Catignani G. L. Antioxidants and prevention of chronic disease.
Review. Crit. Rev. Food. Sci. Nutr. 2004, 44: 275-295.
Willcox, J. K, Catignani, G. L, Roberst L. J. Free Rad. Biol. Med., 2003, 34 (7), 795.
Wilson, C. W. M, Grene, M. J. Clin. Pharmacol., 1978, 18 (1), 21.
Wink D. A, Hanbauer I, Krishna M. C, DeGraff W, Gamson J, Mitchell J. B. Nitric oxide
protects against cellular damage and cytotoxicity from reactive oxygen species. Proc Nat1
Acad Sci USA 1993,90:9813.
Winterboum C. C, van den Berg J J. M, Roitman E, Kuypers F. A. Chlorohydrin formation
from unsaturated fatty acids reacted with hypochlorous acid. Arch Biochem Biophys
1992,296:547.
Wright S. D. Toll, a new piece in the puzzle of innate immunity. J Exp Med. 1999,189:605–
609.
Wruck C. J, Fragoulis A, Gurzynski A, Brandenburg L-O, Kan Y. W, Chan K, Hassenpflug J,
et al. Role of oxidative stress in rheumatoid arthritis: insights from the Nrf2-knockout
mouse. Ann Rheum Dis 2011,70:844–850.
Xie Q, Kashiwabara Y, Nathan C. Role of transcription factor NF-κB/Rel in induction of nitric
oxide synthase. J Biol Chem 1994,269:4705.
Yoo S. J, Go E, Kim Y. E, Lee S, Kwon J. Roles of reactive oxygen species in rheumatoid
arthritis pathogenesis. J Rheum Dis. 2016 Dec 1,23(6):340-7.
Zhao, W., Han, Y., Zhao, B., Hirota, S., Hou, J., Xin, W. Biochim. Biophys. Acta, 1998, 1381
(1), 77.

Chapter 10
FREE RADICALS AND ANTIOXIDANT

DEFENSE IN ASTHMA
Rashmi Pandey1, Priyanka Tiwari2, Ved Prakash1,

Vivek Kumar Gupta2 and Bechan Sharma2,
1
King George Medical University, Lucknow, India
2
University of Allahabad, Allahabad, India
ABSTRACT
Asthma is one of the allergic diseases caused by multiple factors such as host genetic
factors, allergens, pollutants in the air, obesity, increase in eosinophils, and infections in
the respiratory tract. The antiasthmatics include a wide range of drugs including steroids
based compounds, leukotriene modifiers, long-acting β2-adrenoreceptor agonists and
several others. The long-term applications of antiasthmatics induce excessive production
of free radical oxygen and free radical nitrogen species causing an imbalance between the
oxidative and antioxidative systems resulting in lipid peroxidation induced damage at the
cellular and molecular levels. In order to alleviate oxidative stress-mediated toxicity, some
plant-based principles have been explored for their antioxidant and antiasthmatic
properties. Keeping in view the excessive generation of free radical-mediated toxicity
during asthma and also due to the inability of these drugs to manage severe asthma, there
is a need to develop safe, potential and cost-effective antiasthmatics. This chapter
illustrates a current account of asthma, symptoms, clinical diagnosis, treatment, the toxicity
of drugs, generation of oxidative stress and its proper management using antioxidants/plant
products. The information contained in the chapter may be of great use to the clinicians,
researchers and to those interested in the management of asthma.
1. INTRODUCTION
Asthma is known as a common inflammatory disease of the respiratory tract which affects
both the males and females at any age. If not treated in time, it can severely attack the

Corresponding Author’s Email: sharmabi@yahoo.com.

258 Rashmi Pandey, Priyanka Tiwari, Ved Prakash et al.
respiratory system and may prove to be fatal. The incidences of asthma and its burden are ever
on increase due to a variety of reasons worldwide. Currently, over 300 million people are
suffering from asthma (Braman, 2006). Children are found to be more affected (3-38%) than
the adults (2-12%). Recently Indian study on epidemiology of asthma, respiratory symptoms
and chronic bronchitis (INSEARCH) has published its report after studying the incidence of
asthma in 85,105 men and 84,470 women. The investigators had recruited these people from
12 urban and 11 rural sites in India. They found an occurrence of asthma in India to be 2.05%
among the people of age group of over 15 years. INSEARCH has estimated national burden of
asthma to be about 18 million (Koul and Dhar, 2018).
Based on specific characteristics and extent of inflammation, asthma has been broadly
classified into two major groups such as phenotypes and endotypes. The phenotypes primarily
include asthma induced by allergens or even non-allergic factors, exercise, infections, aspirin,
and asthma, with clinical presentations such as transient wheezing, non-atopic wheezing in
toddlers, and exacerbation-prone asthma (Stein and Martinez. 2004). The endotypes of asthma
relates the pathophysiological mechanisms associated with both the inflammation development
and its persistence in the respiratory tract (Lotvall et al., 2011). This is mainly accompanied by
the overproduction of reactive oxygen species (ROS) and nitrogen (RNS), which are believed
to significantly contribute towards the airway inflammation (Babusikova et al., 2012).
The sources of ROS may be categorized into two groups such as the endogenous and
exogenous. The endogenous sources of ROS comprise the following three sub-cellular
components including mitochondria, endoplasmic reticulum, and peroxisomes; enzymes such
as nitric oxide synthase, cytochrome P450, xanthine and NADPH oxidases; cells involved in
eliciting immune responses or even the non-immune cells such as monocytes and macrophages,
endothelium, activated eosinophils and neutrophils, phagocytes, airway epithelial and smooth
muscle cells; and other factors such as proteins of heme and metal ions reactions (Phanjendra
et al., 2015; Meo et al., 2016; Jesenak et al., 2017). The exogenous sources of ROS include the
use of tobacco, cigarette smoke, ultraviolet (UV) and ionizing radiations, pollutants, ozone,
organic solvents, metals, and some chemotherapeutics. These two sources of ROS are
considered to be responsible for pathogenesis and complicating the inflammatory conditions
through oxidative damage of certain biomolecules involved in the antioxidative defense of the
living systems (Erzurum, 2016; Jesenak et al., 2017; Iyer et al., 2017).
A person suffering from asthma is reported to suffer from different clinical complications
such as interference in breathing and sleep, recreations, permanent narrowing of the bronchial
tubes (airway remodeling), and toxicity due to long-term use of some medications. The current
challenges in the treatment of asthmatics include adequate diagnosis of asthma as sometimes
the symptoms stay latent due to treatment of the disease, as well as the serious side effects of
long-term and high dose use of inhaled corticosteroids for example fluticasone alone in children
or in combination with β-2 agonists (salmeterol/formoterol) (Becker, 2002; Cates, 2007; Souza
et al., 2013; Athari et al., 2017; Szefler and Chipps, 2018)). Keeping in view the growing
environmental pollution and increasing risk of onset of asthma throughout the world, it is
envisaged to take this chapter with an objective to present here a comprehensive and an updated
account of asthma, production of excessive free radicals due to different environmental factors,
causes and the symptoms of the disease and the use of antiasthmatics by patients, challenges in
treatment and possible remediation by improving therapeutic potential of drugs with increased
safety to the patients.

Free Radicals and Antioxidant Defense in Asthma 259
2. CAUSES OF ASTHMA
The exposure of a person to various irritants and substances that trigger allergies (allergens)
have been found to trigger the signs and symptoms of asthma, though its onset differs from one
individual to the other. The factors responsible to induce asthma include the pollens and fungal
spores, smoke, dust particles, wastes from different insects, low environmental temperature,
physical exercise, use of certain medications such as aspirin, ibuprofen, beta blockers, and
naproxen, preservatives like sulfites used in the food (shrimp, dried fruit, processed potatoes)
and drinks (beverages, beer and wine), as well as strong stress and emotional trauma etc.
In addition to these, the exposure of an individual to different pollutants and chemicals
used in farming, hairdressing and manufacturing, the overweight and the genetic factors have
also been reported to be involved as the risk factors causing asthma (Janssens and Ritz, 2013).
3. ASTHMATIC SYMPTOMS
Asthma symptoms may vary from one person to another. The asthmatic symptoms may be
characterized as including short breaths and restlessness in sleeping, congestion, and pain in
the chest, excessive coughing, and a whistling sound while exhaling mainly in children.
The symptoms in some people become more severe when an asthmatic person doing brisk
exercise, getting exposed to chemical fumes, gases, dust, particles of skin or dried saliva of pets
(Brussino et al., 2018).
4. ASTHMA AND ITS CLINICAL DIAGNOSIS

Asthma’s clinical diagnosis may be involving physical or chemical tests. The physical
examination includes the tests to measure lung function by spirometry (which measures the
exhale of volume and speed of air) and peak flow (a device to measure the quantum of force of
breath out) as well as imaging tests (especially for lungs and nose cavities (sinuses) using X-
ray or scanning employing high-resolution computerized tomography).
The additional tests to diagnose asthma include the methacholine challenge (Methacholine,
when inhaled, may cause mild constriction of airways. Any reactivity of the patient to
methacholine shows likelihood of asthma), the nitric oxide (NO) test (quantitative measurement
of NO exhaled; compared to the normal, higher NO levels exhaled denotes asthma), test for
allergy involving skin or blood in order to determine allergy to various factors such as mold,
pets, and pollen, test to measure the eosinophils count present in the sputum (determining the
number of eosinophils by staining with eosin in saliva or mucus or sputum released during
coughing) and the provocative testing by exposing the person to the vigorous exercise or
inhaling the cold air. Based on the severity of asthma, the treatment is imparted to the patients
(Esden and Pesta-Walsh, 2018; Kowalski, 2019).

5. RISK FACTORS ASSOCIATED TO ASTHMA

The factors contributing towards risk for causing asthma include:
 Infections by viruses (Rouse sarcoma viruses, coronavirus), mycoplasma, and

chlamydia
 Drugs
 Environmental pollutants
 Malfunction of lungs
 Genetic factors
 Allergens
 Exercise
 Anti-inflammatory non-steroidal drugs
 Stress or psychological factors
All these factors and their mechanisms in inducing asthma have been explained in detail
elsewhere (Duffy et al., 1997; Lemanske Jr. and Busse, 2011; Saraya et al., 2017).
6. CELLULAR AND MOLECULAR EVENTS INVOLVED IN ASTHMA

The cascade of cellular and molecular events which take place during asthma have been
found to be occurring in a pathophysiologically different manner in the people of different age
groups. For example, in the asthmatic children, there is several fold increase in the number of
blood cells (macrophages, eosinophils, and monocytes) in comparison to the healthy individual.
In addition, the levels of prostaglandins E2, cysteinyl leukotrienes (Parameswaran et al., 2002),
exhaled NO, and oxidative markers were also found to be significantly enhanced (Krawiec et
al., 2011).
Instead, in adults, the molecular expression of cellular indices is complex and variable. The
severity of airway inflammation in asthmatic adults depends on the different phases of the
disease. Overall, it involves eosinophils, mast cells (Fischer et al., 1994), Th2 lymphocytes,
cytokines (IL-3, IL-4, IL-5, IL-9 and IL-13) (Barnes, 2008), IgE (Bousquet et al., 2000), and
growth factor (VEGF) (Amrani et al., 2009). A clear and accurate pathological information
about this disease based on these molecular indices may help treat the patients specifically to
control the disease (reviewed by Lemanske Jr. and Busse, 2011).
7. ASTHMA AND OXIDATIVE STRESS

Asthma has been shown to be associated with a serious imbalance between the reducing
and non-reducing or oxidizing induce shifting it towards the production of the higher amount
of oxidative chemical species in asthma. The reactive oxygen species (ROS) include superoxide
anions, hydroxyl radicals, hypohalite radicals, and hydrogen peroxide, whereas the reactive

nitrogen species (RNS) include NO, peroxynitrite, and nitrite. All of these chemical species are
reported to rise during asthma causing airway inflammation and making asthma highly severe.
On the other hand, asthma also causes a drastic imbalance in the cellular redox potential in
the affected individuals. The endogenous non-enzymatic (glutathione) and the enzymatic
antioxidants such as catalase, glutathione peroxidase, superoxide dismutase, and glutathione-
S-transferase, NAD(P)H, quinine oxidoreductase (NQO1), and glucuronosyltransferase-1a6
(UGT-1a6) (Cho et al., 2002; Sahiner et al., 2011; Mishra et al., 2018) are known to be
responsible for neutralizing the free radicals so as to convert them in harmless factors. The
administration of antiasthmatics is known to further enhance this imbalance and generate
oxidative stress in the patients of asthma.
8. OXIDATIVE STRESS AND INFLAMMATION IN ASTHMATICS

The existing information indicates that generation of oxidative stress because of increased
levels of exogenous or endogenous RNS and ROS in asthmatics may complicate the
interactions between different cells of lungs and mediators causing airway inflammation
(Comhair et al., 2001; Sahiner et al., 2011). The mechanisms involved in the oxidative damage
include excessive generation of oxidative chemical species (ROS and RNS) and their
involvement in chronic inflammation (Ayer et al., 2017), deficiency of glutathione (intrinsic
factor) or vitamins and natural antioxidants present in food items (extrinsic factors) (Jiang et
al., 2014), modulation of functions of antioxidant enzymes (Babusikova et al., 2013) and
enhanced activity of pro-oxidative enzymes (Li and Li, 2016). The ROS severely damage
various biomolecules such as proteins, lipids, nucleic acids, and carbohydrates and their
conjugates with drastic alterations in their functional and structural properties.
The increased amount of RNS and ROS, however, may trigger cascades of intracellular
signaling such as mitogen-activated protein kinase (MAPK) and nuclear factor-kB (NF- kB),
(Hart et al., 1998; Pantano et al., 2008) which are proinflammatory in nature and have potential
to induce expression of chemokines, cytokines, and adhesion, the inflammatory molecules.
These cellular phenomena lead to the mitochondrial cytotoxic response, which is crucial for
cellular necrosis or apoptosis (Sahiner et al., 2011).
9. ENDOGENOUS FREE RADICAL SPECIES AND ASTHMA

The free radical species in the biological systems have been shown to be mainly produced
by the activated eosinophils, neutrophils, monocytes, and macrophages (inflammatory cells)
and the epithelial and muscular cells (resident cells) (Henricks and Nijkamp, 2001, Sahiner et
al., 2011). The pathways which consume the major amount of oxygen in the developed
organisms include nicotinamide adenine dinucleotide phosphate (NADP) oxidase-dependent
complex, the respiratory chain in mitochondria, as well as the cytosolic xanthine oxidase
system, and. hydrogen peroxide (H2O2), a potential prooxidant, produced due to dismutation of
O2. Further interaction of O2 and H2O2 in association with iron and some other metal ions in
the living systems lead to the production of OH- (Mac et al., 2006). The inflammatory cells as
mentioned above contain peroxidases which are shown to catalyze the reaction between H2O2

and halides. As a result of it, the hypohalides (HOCl) are formed. Further, the reaction involving
NO and the superoxide produces a very potential ROS i.e., peroxynitrite (ONOO-) (Mak and
Chan-Yeung, 2006). In the normal steady state condition, the concentration of H2O2 within the
cell has been found to be in the range of 0.002-0.2 mM. This level of H2O2 is increased to 0.5-
0.7 mM during oxidative stress condition or when the intracellular signaling is generated (Cho
and Moon, 2010).
The excess production of ROS due to asthma causes oxidation of proteins and convert them
into carbonyls which are shown to react with nitrogen oxide species mainly NO and tyrosine
to produce nitro derivatives of tyrosine (Iijima et al., 2001). The NO after autoxidation forms
nitrite which is utilized as a substrate by peroxidases (eosinophil peroxidize and
myeloperoxidase) to form superoxides. The NOs further react with these superoxides and form
highly reactive ONOO- which is able to modify the tyrosine residues of many structural proteins
and the enzymes leading to the inactivation of these important cellular constituents (Abu-Soud
and Hazen, 2000). Rise in the level of NO during severe asthma has been reported (Sanders,
1999). The lipid peroxidation at the membrane level is another important event caused by many
ROS. As a result, 8-isoprostane and ethane are produced, which can be detected in the breaths
of asthmatics (Pardi et al., 2000; Dworski et al., 2001). Wedes et al., (2009) have reported the
presence of increased contents of bromotyrosine and F2-isoprostanes in the urine of the
asthmatics. Further, the elevated concentration of malondialdehyde (a marker of oxidative
species) and the decreased concentration of glutathione (a marker of antioxidative agents) have
been reported into asthmatics, especially in children (Ercan et al., 2006) as compared to the
controls.
10. ASSOCIATION OF ENVIRONMENTAL FACTORS

WITH OXIDATIVE STRESS DURING ASTHMA
The environmental factors such as smoke arising from cigarettes and tobacco (Church and
Pryor, 1985; Gilmour et al., 2006), several air pollutants which include ozone and oxides of
sulphur, nitrogen, lead, cadmium and automobile exhausts, which are known causative agents
to produce excessive free radicals and hence the oxidative stress, have been shown to be related
with generation of asthma as they adversely influence the respiratory tract and lung cells,
though it depends on the chemical nature and dose of the toxicants, age, and sensitivity of the
person receiving the exposure as well as the sensitivity of an individual (Li et al., 2003; Liu et
al., 2009). The different phases of smoke, their major chemical constituents, and abilities to
generate a variety of ROS in humans and impacts on the respiratory system have been
summarized by Sahiner et al., (2011).
11. MODULATION OF INNATE ANTIOXIDANTS

BY OXIDATIVE STRESS DURING ASTHMA
Since oxidative stress develops due to increased levels of the oxidative species and a
significant reduction in the antioxidative indices in an individual; the concentration of the
former becomes higher than the later. Oxidative stress causes depletion of the levels of

protective agents such as the non-enzymatic (α-tocopherol, albumin, glutathione, β-carotene,

and uric acid) as well as enzymatic (CAT, GST, GPx, SOD, glutaredoxins, heme oxygenase-1,
peroxiredoxins, and thioredoxins) antioxidants in the lungs. The adequate intake of several
natural antioxidants like carotenes and vitamin C under this condition has been found to
significantly decrease the risk of tissue damage in the airways due to oxidative stress in the
asthmatics (Heffner and Repine 1989; Sackesen et al., 2008; Zhu et al., 2017).
12. ASSOCIATION OF POLYMORPHISMS OF ANTIOXIDATIVE GENES

WITH THE ONSET, PATHOGENESIS AND SEVERITY OF ASTHMA
The data obtained from a study conducted by Sahiner et al., (2011) in children have
indicated that there exists an association of polymorphisms of antioxidative genes with the
onset, pathogenesis, and severity of asthma. This group has demonstrated that children who
received exposure to ozone displayed an elevated level of respiratory symptoms and contained
GSTM1 null or GSTP1 val/val genotypes.GST-P1 and GST-M1 are subclasses of GST. The
genotype, GSTP1 val/val alone was found to be responsible for the severity of asthma in the
early stage of life (Sahiner et al., 2011).
Other workers have also reported similar observations (Garte et al., 2001; Repapi et al.,
2010). The genetic polymorphism in the genes encoding for SOD was found to be associated
with the pathogenesis or severity of asthma has not been concluded as yet, though some workers
have shown a link between the EC-SOD R213G polymorphism and the chronic obstructive
pulmonary disease (COPD) (Kinnula et al., 2004; Juul et al., 2006).
13. CLINICAL MARKERS OF OXIDATIVE STRESS

FOR DIAGNOSIS OF ASTHMA
The oxidative stress markers for clinical diagnosis of asthma include presence of molecules
reflecting peroxidation of cell-membrane lipids usually monitored in terms of alterations in the
levels of malondialdehyde (MDA), thiobarbituric acid-reactive substances (TBARS), 4-
hydroxyhexanal, 4-hydroxynonenal, 4-hydroxyhexanal, 4-hydroxynonenal acrolein, hexanal;
DNA damage such as formation of 8-hydroxy-2-deoxyguanosine; protein oxidative damage
such as appearance of reduced content of free thiol-groups, general total antioxidant capacity
of plasma; glutathione disulfide, nitrosothiols; and nitrotyrosine in the lavage fluid of
bronchoalveolar and tissues of airway, urine, plasma, fluid of epithelial lining, sputum, and
saliva of asthmatics. The higher concentrations of these molecules indicate the severity of the
disease (Mak et al., 2004; Beier et al., 2004; Bentur et al., 2006; Antus, 2016; Bishopp et al.,
2017).
However, direct measurement of other biomarkers such as free radicals or NO and H2O2
responsible for inflammation in the airways and present in the exhaled air has also been
suggested to act as indicators of oxidative stress in asthma but they are highly unstable and
appear for a short time only. In addition, the oxidation byproducts such as isopentane and ethane
may also serve as key indicators of asthmatic oxidative stress. The attributes of oxidative stress
are summarized in Figure 1.

Figure 1. Attributes of oxidative stress in asthma. The exogenous and endogenous sources of asthma
are shown which are responsible for generating excessive free radicals, which modulate the structures
and functions of different biomolecules. The accumulation of free radical species, production oxidative
stress, depletion of antioxidative indices and emergence of asthma have been demonstrated. ROS,
Reactive oxygen species; RNS, Reactive nitrogen species.
14. APPLICATION OF ANTIOXIDANTS

AS PROSPECTIVE ANTIASTHMATICS
Application of antioxidants is thought to play a key role in the amelioration of oxidative

stress-mediated risk in asthma patients. These antioxidants include thiol antioxidants (N-acetyl
cysteine, NAC and bucillamine), vitamin A, vitamin C and vitamin E, and nutrients (quercetin:
a flavonoid, genistein: a soy isoflavone, α-lipoic acid: a food product and epigallocatechin 3
gallate: a polyphenol found in green tea, resveratrol: a phytoalexin present in grape seeds). The
thiol antioxidants maintain the pool of reduced glutathione in the cells and thus help suppress
the hyperactivity or inflammation in the airway of humans (Kurtoglu et al., 2009; Aliyali et al.,
2010) as well as the animal experimental models (Whitekus et al., 2002; Blesa et al., 2002;
2003). The mechanisms of actions of nutritional antioxidants and the thiols have been updated
and discussed by some workers (Sahiner et al., 2011; Zhu et al., 2017; Pandey and Sharma,
2018).
15. PHARMACOLOGICAL AND THERAPEUTIC TARGETS IN ASTHMA

The information available about the therapeutics being used against asthma and their
specific targets do not indicate any authentic cellular site for their actions. Asthma therapy
follows strategies devised decades ago even prior to the advancement of knowledge derived
from cellular and molecular biology. It is therefore urgently felt to design and develop new
specific drugs against this disease which may target any specific cellular and/or molecular sites

with an enhanced level of safety (Sharma and Halayko, 2009). Since asthma is considered to
be a multifactorial and inflammatory chronic disease, it is thought that immune cell signaling
pathways or an array of the complex immunological network including the receptors involved
in causing inflammation may be considered as new therapeutic targets to develop new
molecules as effective antiasthmatics against respiratory diseases (Vyas and Vohora, 2016).
Barnes (2016) has suggested that both asthma and the COPD involve chronic inflammation
mediated by multiple kinases. These kinases are responsible for the activation of the signaling
pathways. As a result, the contraction of smooth muscles of the respiratory tract and the release
of inflammatory factors such as growth factors, chemokines, and cytokines take place. These
biochemical steps could be exploited for developing kinase inhibitors to arrest inflammation
during asthma. The development of such kinase inhibitors is not so easy as these molecules
may not be safe and effective if administered orally. However, the inhaled formulations of some
kinase inhibitor drugs are being developed to target Syk-kinases, isoenzyme-selective PI3-
kinases, Rho-kinase (Taki et al., 2007; Prado et al., 2014), p38 MAP kinases (Adcock et al.,
2008) and Janus-activated kinases. It is believed that such drugs may be highly useful in the
therapeutic management of asthma as well as COPD (Barnes 2016). Alves et al., (2018) have
proposed that interleukins could be exploited as a suitable target to treat asthma as they are
expected to modulate the functions of interleukins-13 (Adcock et al., 2008) as well as their
receptors. Some molecules targeting interleukins have been developed and grouped into two
categories:
1. Molecules of small size such as SP600125 and ligustrazine and

2. Molecules of large size such as dupilumab, lebrikizumab, and benralizumab
The targets for a few other drugs include proteinases (Campo and Henry, 2005), arginase,
nicotinic receptors and cholinergic systems (Prado et al., 2014).
16. DRUG-INDUCED TOXICITY IN ASTHMATICS AND MITIGATION

BY PHYTOCHEMICALS
Earlier some studies on the clinical aspects of asthma have suggested that the controlled
treatment of asthmatics with the drugs such as inhaled corticosteroids or long-acting β2 agonists
could induce oxidative stress in terms of rising in the LPO levels in the patients as compared
to the healthy individuals (Alzoghaibi and Bahammam, 2007; Zdanowicz, 2007). The
antiasthmatics in clinical practice and their toxic effects are summarized and reviewed by
Pandey and Sharma (2018). In order to alleviate oxidative stress-mediated drug toxicity in
asthmatics, some plant-based principles have been explored for their antioxidant and
antiasthmatic properties as alternative strategies. The herbal drugs being the part of
complementary and alternative medicines have been exploited to treat many diseases primarily
in India (Agrawal and Sharma, 2012; Singh and Sharma, 2013) and China (Zhu et al., 2018)
since long. There are reports to suggest that these plant-based compounds have the potential to
help patients suffering from many chronic diseases. In the Indian traditional medicine system,
about 45000 plant species have been mentioned which contain varied medicinal properties.
Some of these plants have been demonstrated to possess antiasthmatic, antihistaminic,
anticholinergic and antiallergic activities (Taur and Patil, 2011; Pandey and Sharma, 2018).

The saponins, a class of compounds derived from different plant species such as
Clerodendron serratum, Gardenia turgida, Albizia lebbeck, and Solanum xanthocarpum have
been shown to exhibit antiasthmatic potential. Similarly, several phytochemicals isolated from
many other plants have been found to offer respite from allergic reactions including
inflammation in asthma (Zhu et al., 2017; Pandey and Sharma, 2018). The toxic effects of some
corticosteroids and other drugs commonly used as antiasthmatics are summarised in Table 1.
Table 1. Toxic effects of some drugs used as antiasthmatics
Antiasthmatics Toxicity References

Used through inhalation Zdanowicz, 2007
Cromolyn, nedocromil Mouth and throat irritation Houglum, 2000
Beclomethasone Cough Zdanowicz, 2007
Budesonide Dysphonia Zdanowicz, 2007
Fluticasone Hoarseness Zdanowicz, 2007
Flunisolide Oral candidiasis Zdanowicz, 2007
Triamcinolone Toxicity at high doses Zdanowicz, 2007
Isoproterenol hydrochloride (> 5mg) Mouth and throat irritation, Light et al., 1979
and albuterol (10 mg) vomiting
Through systemic application Zdanowicz, 2007
Methylprednisone Growth suppression Zdanowicz, 2007
Prednisolone Adrenal suppression Zdanowicz, 2007
Prednisone Osteoporosis, peptic ulcers, Zdanowicz, 2007
myopathy, electrolyte Pandey and Sharma, 2018
imbalance, hypertension,
Cushing’s syndrome
Theophylline (20 ug/ml) Seizure, apnea, bradycardia Dasgupta, 2012
Aminophylline (>24 mg/L serum) Lethal Eason and Markowe, 1989
Caffeine (>50 ug/ml) Tachycardia, gastrointestinal Dasgupta, 2012
intolerance
Short-acting beta-agonists (albuterol) Hepatotoxicity van der Wouden et al., 2008
and long-acting beta-agonists
(salmeterol)

AND CHALLENGES
In addition to the challenges in the treatment of asthma as stated above, there are several
other factors associated with effective eradication of the disease. Regarding the prevention,
diagnosis, treatment, and control, main factors were deeply associated; one of them being the
patient, his family members or the attendants and the second concerns the health care providers
as summarised in Table 2. Apart from the noncooperation from the patients and his family
members, no adequate diagnosis of asthma (Pavord et al., 2015), not-adherence to drugs during
treatment, and no feedback to physicians about the level of recovery due to treatment poses
serious impediment in the management of the disease (Cabana and Le, 2005; Cates, 2007;
Taylor et al., 2008; Onyedum et al., 2013; Song and Wong, 2017; Athari et al., 2017).

Table 2. Challenges in the prevention, diagnosis, treatment and the control of asthma
(Anarella et al., 2017)
Parameters Associated Factors Challenges

Challenges Patients, family,  Database missing through proper screening about the
in the and attendants number of children, adolescents, and adults (both males
prevention and females) or by conducting proper surveillance to
of asthma identify those living with symptoms of allergy and
respiratory disorders in rural and urban areas.
 Lack of awareness programmes to educate people about
their food habits and health care especially concerning the
respiratory system.
 Lack of efforts to connect people seeking their
cooperation in asthma management.
Health care  Increasing environmental contamination and pollution
providers especially air pollution in developing countries including
India.
 Lack of monitoring of quality of food items and edibles.
Challenges Patients, family,  Lack of cooperation from the patients, family members or
in the and attendants their attendants.
diagnosis of  Lack of record maintenance about proper diagnosis of
asthma patients.
Health care  Poor infrastructure facilities and resource-limited setting
providers towards a proper clinical diagnosis of the disease
especially in the developing countries including India.
Lack of access to such specialized physicians in rural
areas.
Challenges Patients, family,  Lack of cooperation from patients towards taking regular
in the and attendants treatment, lack of adherence to the drugs, lack of
treatment of feedback about the level of recovery from patients.
asthma Health care  Lack of proper treatment centers for the efficient
providers management of the disease, treatment of patients in
particularly the developing countries in the resource-
limited setting, unavailability of low-cost diagnosis and
drugs, lack of access of specialized physicians to the
people of rural areas.
Challenges Patients, family,  Lack of maintenance of proper records concerning the
in control of and attendants disease profile of patients suffering from respiratory
asthma disorders, lack of trained and efficient human resource for
effective monitoring of the patients, and lack of
emotional and psychological support to the patients
offered by their family members and others.
Health care  Lack of proper coordination among different units of the
providers health departments of government agencies, lack of
financial resources with patients to afford the cost of
travel, diagnosis and treatment in developing countries,
lack of proper food and nutrition, tendency to ignore the
disease and consulting a physician only when the disease
reaches the next alarming level.

However, the easy access of specialized physicians, availability of pathology laboratories

for low-cost diagnosis, treatment centers to provide safe drugs with minimum price and proper
monitoring of such patients by establishing a database may improve the present scenario in
developing world towards eradication of this disease. The participation of NGOs may also be
encouraged to work in perfect coordination with those of the government agencies.
CONCLUSION
Oxidative stress plays a significant role in causing pathogenesis in the airway of asthmatics
via eliciting a pathway of intracellular signaling events resulting in drastic reduction in immune
tolerance and induction of allergic inflammation. Since therapeutic application of antioxidants
in the prevention of asthma is not proven by any clinical study, investigations are continued to
initiate antioxidant therapy of specific asthmatics as the application of several antioxidants as
drug supplements are expected to show promise to reduce free radicals generation and to
improve the health of asthmatics from further deterioration. Therefore, extensive research is
required to develop cost-effective, safe and clinically potential antiasthmatics for treatment and
management of asthma. The plant-based principles may offer a potential therapeutic solution
to this challenging health issue.
REFERENCES
Abu-Soud, H. M., and Hazen, S. L., (2000). Nitric oxide is a physiological substrate for
mammalian peroxidases. J Biol Chem, 275: 37524-32.
Adcock, I. M., Caramori, G., and Chung, K. F., (2008). New targets for drug development in
asthma. Lancet, 372(9643): 1073-87.
Agrawal, A., and Sharma, B., (2012). Natural products and their antioxidant potential. Nat
Products, 8 (2): 72-87.
Aliyali, M., Poorhasan, A. A., Sharifpoor, A., and Zalli, F., (2010). Effects of N-acetylcysteine
on asthma exacerbation. Iran J Allergy Asthma Immunol, 9:103–9.
Alves, M. F., da Fonseca, D. V., de Melo, S. A. L., Scotti, M. T., Scotti, L., Dos Santos, S. G.,
and de Fatima Formiga Melo Diniz, M., (2018). New therapeutic targets and drugs for the
treatment of asthma. Mini Rev Med Chem, 18(8): 684-696.
Alzoghaibi, M. A., and Bahammam, A. S., (2007). Lipid peroxides in stable asthmatics
receiving inhaled steroids and long-acting beta 2-agonists. Respirology. 12: 439–42.
Amrani, Y., Tliba, O., and Panettieri, R. A. Jr., (2009). Biology of Airway Smooth Muscle
Cells. In: Adkinson, N. F. Jr., Bochner, B. S., Busse, W. W., Holgate, S. T., Lemanske, R.
F. Jr., Simons, F. E. R., editors. Middleton’s Allergy Principles & Practice. 7. Philadelphia:
Elsevier; 399–411.
Anarella, J. P., Wagner, V. L., McCauley, S. G., Mane, J. B., and Waniewski, P. A., (2017).
Eliminating disparities in asthma care: identifying broad challenges in quality
improvement. Am J Med Qual, 32(6): 598-604.
Antus, B., (2016). Oxidative stress markers in sputum. Oxid Med Cell Longev, 2016:2930434.

Athari, S. M., Afshari, F., Eftekhari, A., and Athari, S. S., (2017). The surveillance system,
diagnosis and treatment challenges of asthma and health policy orientation of main
challenges. J Pain Manage Ther, 1(2): 1-5.
Babusikova, E., Jesenak, M., Evinova, A., Banovcin, P., and Dobrota, D., (2013). Frequency
of polymorphism -262 C/T in catalase gene and oxidative damage in Slovak children with
bronchial asthma. Arch Bronconeumol, 49: 507-12.
Babusikova, E., Jurecekova, J., Evinova, A., Jesenak, M., and Dobrota, D., (2012). Oxidative
damage and bronchial asthma. In: Ghanei M, editor. Respiratory Diseases. Rijeka: InTech,
151–76.
Braman, S. S., (2006). The global burden of asthma. Chest, 130(1): 4S-12S.
Barnes, P. J., (2008). Immunology of asthma and chronic obstructive pulmonary disease. Nat
Rev Immunol, 8(3): 183–92.
Barnes, P. J., (2016). Kinases as novel therapeutic targets in asthma and chronic obstrutive
pulmonary disease. Pharmacol Rev, 68(3): 788-815.
Becker, A. B., (2002). Challenges to treatment goals and outcomes in pediatric asthma. The J
Aller Clin Immunol, 109(6): S533-S538.
Beier, J., Beech, K. M., Semmler, D., Beike, N., and Buhl, R., (2004). Increased concentrations
of glutathione in induced sputum of patients with mild or moderate allergic asthma. Ann
Allergy Asthma Immunol, 92: 459–63.
Bentur, L., Mansour, Y., Brik, R., Eizenberg, Y., and Nagler, R. M., (2006). Salivary oxidative
stress in children during acute asthmatic attack and during remission. Respir Med, 100:
1195–201.
Bishopp, A., Sathyamurthy, R., Manney, S., Webbster, C., Krishna, M. T., and Mansur, A. H.,
(2017). Biomarkers of oxidative stress and antioxidants in severe asthma: a prospective
case-control study. Ann Allergy Asthma Immunol, 118: 445–51.
Blesa, S., Cortijo, J., Mata, M., Serrano, A., Closa, D., Santangelo, F., Estrela, J. M.,
Suchankova, J., and Morcillo, E. J., (2003). Oral N-acetylcysteine attenuates the rat
pulmonary inflammatory response to antigen. Eur Respir J, 21: 394–400.
Blesa, S., Cortijo, J., Martinez-Losa, M., Mata, M., Seda, E., Santangelo, F., and Morcillo, E.
J., (2002). Effectiveness of oral N-acetylcysteine in a rat experimental model of asthma.
Pharmacol Res.; 45:135–40.
Bousquet, J., Jeffery, P. K., Busse, W. W., Johnson, M., and Vignola, A. M., (2000). Asthma
from bronchoconstriction to airways inflammation and remodeling. Am J Respir Crit Care
Med, 161(5): 1720–45.
Brussino, L., Heffer, E., Bucca, C., Nicola, S., and Rolla, G., (2018). Eosinophils Target
Therapy for Severe Asthma: Critical Points. Biomed Res Int, 2018: 7582057.
Cabana, M. D., and Le, T. T., (2005). Challenges in asthma patient education. The J Allergic
Clin Immunol, 115(6): 1225-27.
Cates, C. J., (2007). Current challenges in Asthma. Br J Gen Practice, 57(536): 179-80.
Cho, H. Y., Jedlicka, A. E., Reddy, S. P., Kensler, T. W., Yamamoto, M., Zhang, L. Y., and
Kleeberger, S. R., (2002). Role of NRF2 in protection against hyperoxic lung injury in
mice. Am J Respir Cell Mol Biol, 26: 175.
Cho, Y. S., and Moon, H. B., (2010). The role of oxidative stress in the pathogenesis of asthma.
Allergy Asthma Immunol Res, 2(3): 183-87.
Church, D. F., and Pryor, W. A., (1985). Free-radical chemistry of cigarette smoke and
toxicological implications. Environ Health Perspect, 64: 111–26.

Comhair, S. A., Bhathena, P. R., Farver, C., Thunnissen, F. B., and Erzurum, S. C., (2001).
Extracellular glutathione peroxidase induction in asthmatic lungs: evidence for redox
regulation of expression in human airway epithelial cells. FASEB J, 15: 70–78.
Dasgupta, A., (2012). Introduction to therapeutic drug monitoring. In: Therapeutic Drug
Monitoring. (https://www.sciencedirect.com/topics/neuroscience/antiasthmatic-drugs)
David, L. D., (1997). Genetic epidemiology of asthma. Epidemiol Rev, 19(1): 129–43.
Campo, B. A., and Henry, P. J., (2005). Stimulation of protease-activated receptor-2 inhibits
airway eosinophilia, hyperresponsiveness and bronchoconstriction in a murine model of
allergic inflammation. Br J Pharmacol, 144: 1100-1108.
Dworski, R., Roberts, L. J. II., Murray, J. J., Morrow, J. D., Hartert, T. V., and Sheller, J. R.,
(2001). Assessment of oxidant stress in allergic asthma by measurement of the major
urinary metabolite of F2-isoprostane, 15-F2t-IsoP (8-iso-PGF2 alpha). Clin Exp Allergy,
31: 387–90.
Eason, J., and Markowe, H. L. J., (1989). Aminophylline toxicity how many hospital asthma
deaths does it cause? Respiratory Med, 83: 219-26.
Erzurum, S. C., (2016). New insights in oxidant biology in asthma. Ann Am Thorac Soc,
13(Suppl 1): S35–9.
Esden. J., and Pesta-Walsh, N., (2018). Diagnosis and Treatment of Asthma in Nonpregnant
Women. J Midwifery Womens Health, 64(1): 18-27.
Fischer, A. R., Rosenberg, M. A., Lilly, C. M., Callery, J. C., Rubin, P., Cohn, J., White, M.
V., Igarashi, Y., Kaliner, M. A., and Drazen, J. M., et al., (1994). Direct evidence for role
of the mast cell in the nasal response to aspirin in aspirin-sensitive asthma. J Allergy Clin
Immunol. 94: 1046–56.
Ercan, H., Birben, E., Dizdar, E. A., Keskin, O., Karaaslan, C., Soyer, O. U., Dut, R., Sackesen,
C., Besler, T., and Kalayci, O., (2006). Oxidative stress and genetic and epidemiologic
determinants of oxidant injury in childhood asthma. J Allergy Clin Immunol, 118: 1097–
104.
Garte, S., Gaspari, L., Alexandrie, A. K., Ambrosone, C., Autrup, H., Autrup, J. L., Baranova,
H., Bathum, L., Benhamou, S., Boffetta, P., Bouchardy, C., Breskvar, K., Brockmoller, J.,
Cascorbi, I., Clapper, M. L., Coutelle, C., Daly, A., Dell'Omo, M., Dolzan, V., Dresler, C.
M., Fryer, A., Haugen, A., Hein, D. W., Hildesheim, A., Hirvonen, A., Hsieh, L. L.,
Ingelman-Sundberg, M., Kalina, I., Kang, D., Kihara, M., Kiyohara, C., Kremers, P.,
Lazarus, P., Le Marchand, L., Lechner, M. C., van Lieshout, E. M., London, S., Manni, J.
J., Maugard, C. M., Morita, S., Nazar-Stewart, V., Noda, K., Oda, Y., Parl, F. F., Pastorelli,
R., Persson, I., Peters, W. H., Rannug, A., Rebbeck, T., Risch, A., Roelandt, L., Romkes,
M., Ryberg, D., Salagovic, J., Schoket, B., Seidegard, J., Shields, P. G., Sim, E., Sinnet,
D., Strange, R. C., Stücker, I., Sugimura, H., To-Figueras, J., Vineis, P., Yu, M. C., and
Taioli, E., (2001). Metabolic gene polymorphism frequencies in control populations.
Cancer Epidemiol Biomarkers Prev, 10: 1239–48.
Gilmour, M. I., Jaakkola, M. S., London, S. J., Nel, A. E., and Rogers, C. A., (2006). How
exposure to environmental tobacco smoke, outdoor air pollutants, and increased pollen
burdens influences the incidence of asthma. Environ Health Perspect, 114: 627–33.
Hart, L. A., Krishnan, V. L., Adcock, I. M., Barnes, P. J., and Chung, K. F., (1998). Activation
and localization of transcription factor, nuclear factor-kappa B, in asthma. Am J Respir Crit
Care Med, 158: 1585-92.

Heffner, J. E., and Repine, J. E., (1989). Pulmonary strategies of antioxidant defense. Am Rev
Respir Dis, 140: 531–54.
Henricks, P. A., and Nijkamp, F. P., (2001). Reactive oxygen species as mediators in asthma.
Pulm Pharmacol Ther, 14: 409–20.
Houglum, J. E., (2000). Asthma Medications: Basic pharmacology and use in the athlete. J
Athletic Training, 35(2): 179-87.
Iijima, H., Duguet, A., Eum, S. Y., Hamid, Q., and Eidelman, D. H., (2001). Nitric oxide and
protein nitration are eosinophil dependent in allergen-challenged mice. Am J RespirCrit
Care Med, 163: 1233–40.
Iyer, D., Mishra, N., and Agrawal, A., (2017). Mitochondrial function in allergic disease. Curr
Allergy Asthma Rep, 17: 29.
Janssens, T., and Ritz, T., (2013). perceived triggers of asthma: key to symptom perception and
management. Clin Exp Allergy, 43(9): 1000-08.
Jesenak, M., Zelieskova, M., and Babusikova, E., (2017). Oxidative Stress and Bronchial
Asthma in Children—Causes or Consequences? Front. Pediatr, 5: 162.
Jiang, L., Diaz, P. T., Best, T. M., Stimpfl, J. N., He, F., and Zuo, L., (2014). Molecular
characterization of redox mechanisms in allergic asthma. Ann Allergy Asthma Immunol,
113: 137–42.
Juul, K., Tybjaerg-Hansen, A., Marklund, S., Lange, P., and Nordestgaard, B. G., (2006).
Genetically increased antioxidative protection and decreased chronic obstructive
pulmonary disease. Am J Respi rCrit Care Med, 173: 858–64.
Kinnula, V. L., Lehtonen, S., Koistinen, P., Kakko, S., Savolainen, M., Kere, J., Ollikainen, V.,
and Laitinen, T., (2004). Two functional variants of the superoxide dismutase genes in
Finnish families with asthma. Thorax, 59: 116–19.
Kowalski, M. L., (2019). Heterogeneity of NSAID-Exacerbated Respiratory Disease: has the
time come for sub-phenotyping? Curr Opin Pulm Med, 25(1): 64-70.
Koul, P. A., and Dhar, R., Economic burden of asthma in India. Lung India. 2018; 35:281-83.
Krawiec, M. E., Westcott, J. Y., Chu, H. W., Balzar, S., Trudeau, J. B., Schwartz, L. B., Wenzel,
S. E., (2001). Persistent wheezing in very young children is associated with lower
respiratory inflammation. Am J Respir Crit Care Med, 163(6): 1338–43.
Kurtoglu, Y. E., Navath, R. S., Wang, B., Kannan, S., Romero, R., and Kannan, R. M., (2009).
Poly(amidoamine) dendrimer-drug conjugates with disulfide linkages for intracellular drug
delivery. Biomaterials, 30: 2112–21.
Lemanske, Jr. R. F., and Busse, W. W., (2010). Asthma: clinical expression and molecular
mechanisms. J Allergy Clin Immunol, 125(2 Suppl 2): S95–102.
Li, Y., and Li, G.P., (2016). Oxidative stress in asthma: a distinct clinical and pathological
feature? J Biol Regul Homeost Agents, 30: 1053–57.
Li, N., Sioutas, C., Cho, A., Schmitz, D., Misra, C., Sempf, J., Wang, M., Oberley, T., Froines,
J., and Nel, A., (2003). Ultrafine particulate pollutants induce oxidative stress and
mitochondrial damage. Environ Health Perspect, 111: 455–60.
Light, R. W., Taylor, R. W., and George, R. B., (1979). Albuterol and isoproterenol in bronchial
asthma efficacy and toxicity of drugs administered via intermittent positive pressure
breathing. Arch Intern Med, 139(6): 639-643.
Liu, L., Poon, R., Chen, L., Frescura, A. M., Montuschi, P., Ciabattoni, G., Wheeler, A., and
Dales, R., (2009). Acute effects of air pollution on pulmonary function, airway

inflammation, and oxidative stress in asthmatic children. Enviro Health Perspect, 117:
668–74.
Lotvall, J., Akdis, C. A., Bacharier, L. B., Bjermer, L., and Casale, T. B., Custovic, A.,
Lemanske, R. F. Jr., Wardlaw, A. J., Wenzel, S. E., and Greenberger, P. A., (2011). Asthma
endotypes: a new approach to classification of disease entities within asthma syndrome. J
Allergy Clin Immunol, 127: 355–60.
Mak, J. C., and Chan-Yeung, M. M., (2006). Reactive oxidant species in asthma. Curr Opin
Pulm Med, 12: 7–11.
Mak, J. C., and Chan-Yeung, M. M., (2006). Reactive oxidant species in asthma. Curr Opin
Pulm Med, 12: 7–11.
Mak, J. C., Leung, H. C., Ho, S. P., Law, B. K., Lam, W. K., Tsang, K. W., Ip, M. S., and Chan-
Yeung, M., (2004). Systemic oxidative and antioxidative status in Chinese patients with
asthma. J Allergy Clin Immunol, 114: 260–64.
Meo, S. D., Reed, T. T., Venditti, P., and Victor, V. M., (2016). Role of ROS and RNS sources
in physiological and pathological conditions. Oxid Med Cell Longev, 2016: 1245049
Mishra, V., Banga, J., and Silveyra, P., (2018). Oxidative stress and cellular pathways of asthma
and inflammation: therapeutic strategies and pharmacological targets. Pharmacol
Theraputics, 181: 169-82.
Onyedum, C. C., Ukwaja, K. N., Desalu, O. O., and Ezeudo, C., (2013). Challenges in the
Management of Bronchial Asthma Among Adults in Nigeria: A Systematic Review. Ann
Med Health Sci Res, 3(3): 324–29.
Pandey, R., and Sharma, B., (2018). Therapeutic Applications of Antiasthmatics,
Consequences and Remedies. EC Pharmacol Toxicol, 6(7): 580-589.
Pantano, C., Ather, J. L., Alcorn, J. F., Poynter, M. E., Brown, A. L., Guala, A. S., Beuschel,
S. L., Allen, G. B., Whittaker, L. A., Bevelander, M., Irvin, C. G., Janssen-Heininger, Y.
M. (2008). Nuclear factor-kappa B activation in airway epithelium induces inflammation
and hyperresponsiveness. Am J Respir Crit Care Med, 177: 959-69.
Parameswaran, K., Cox, G., Radford, K., Janssen, L. J., Sehmi, R., and O'Byrne, P. M., (2002).
Cysteinyl leukotrienes promote human airway smooth muscle migration. Am J Respir Crit
Care Med, 166: 738-42.
Pardi, P., Kharitonov, S. A., and Barnes, P. J., (2000). Elevation of exhaled ethane
concentration in asthma. Am J Respir Crit Care Med, 162: 1450–54.
Pavord, I. D., Bush, A., and Holgate, S., (2015). Asthma diagnosis: addressing the challenges.
The Lancet Resp Med, 3(5): 339-41.
Phanjendra, A., Jestadi, D. B., and Periyasamy, L., (2015). Free radicals: properties, sources,
targets, and their implication in various diseases. Indian J Clin Biochem, 30(1): 11–26.
Prado, C. M., Righetti, R. F., da Silva Pigati, P. A., Possa, S. S., dos Santos, A. S. A., Pinheiro,
N. M., de Toledo, A. C., Leick, E. A., Martins, M. A., and Tibério, I. F. L. C., (2014). New
pharmacological targets for asthma drug development. J Allergy Ther, 5: 170-83.
Repapi, E., Sayers, I., Wain, L. V., Burton, P. R., Johnson, T., Obeidat, M., Zhao, J. H.,
Ramasamy, A., Zhai, G., Vitart, V., Huffman, J. E., Igl, W., Albrecht, E., Deloukas, P.,
Henderson. J., Granell, R., McArdle, W. L., Rudnicka, A. R.,; Wellcome Trust Case
Control Consortium, Barroso, I., Loos, R. J., Wareham, N. J., Mustelin, L., Rantanen, T.,
Surakka, I., Imboden, M., Wichmann, H. E., Grkovic, I., Jankovic, S., Zgaga, L.,
Hartikainen, A. L., Peltonen, L., Gyllensten, U., Johansson, A., Zaboli, G., Campbell, H.,
Wild, S. H., Wilson, J. F., Gläser, S., Homuth, G., Völzke, H., Mangino, M., Soranzo, N.,

Spector, T. D., Polasek, O., Rudan, I., Wright, A. F., Heliövaara, M., Ripatti, S., Pouta, A.,
Naluai, A. T., Olin, A. C., Torén, K., Cooper, M. N., James, A. L., Palmer, L. J., Hingorani,
A. D., Wannamethee, S. G., Whincup, P. H., Smith, G. D., Ebrahim, S., McKeever, T. M.,
Pavord, I. D., MacLeod, A. K., Morris, A. D., Porteous, D. J., Cooper, C., Dennison, E.,
Shaheen, S., Karrasch, S., Schnabel, E., Schulz, H., Grallert, H., Bouatia-Naji, N.,
Delplanque, J., Froguel, P., Blakey, J. D.,; NSHD Respiratory Study Team, Britton, J. R.,
Morris, R. W., Holloway, J. W., Lawlor, D. A., Hui, J., Nyberg, F., Jarvelin, M. R.,
Jackson, C., Kähönen, M., Kaprio, J., Probst-Hensch, N. M., Koch, B., Hayward, C.,
Evans, D. M., Elliott, P., Strachan, D. P., Hall, I. P., and Tobin, M. D., (2010). Genome
wide association study identifies five loci associated with lung function. Nat Genet, 42:
36–44.
Sackesen, C., Ercan, H., Dizdar, E., Soyer, O., Gumus, P., Tosun, B. N., Büyüktuncer, Z.,
Karabulut, E., Besler, T., and Kalayci, O., (2008). A comprehensive evaluation of the
enzymatic and nonenzymatic antioxidant systems in childhood asthma. J Allergy
ClinImmunol, 122: 78–85.
Sahiner, U. M., Birben, E., Erzurum, S., Sackesen, C., and Kalayci, O., (2011). Oxidative Stress
in Asthma. WAO Journal, 4: 151–58.
Sanders, S. P., (1999). Nitric oxide in asthma. Pathogenic, therapeutic, or diagnostic? Am J
Respir Cell Mol Biol, 21: 147–49.
Saraya, T., Kimura, H., Kurai, D., Ishii, H., and Takizawa, H., (2017). The molecular
epidemiology of respiratory viruses associated with asthma attacks: A single-center
observational study in Japan. Medicine (Baltimore), 96(42): e8204.
Sharma, P., and Halayko, A. J., (2009). Emerging molecular targets for the treatment of asthma.
Indian J Biochem Biophys, 46: 447-60.
Singh, R. K., and Sharma, B., (2013). Certain Traditional Indian Plants and Their Therapeutic
Applications: A Review. VRI Phytomed, 1(1): 1-11.
Song, W-J., and Wong, G. W. K., (2017). Changing trends and challenges in the management
of asthma in Asia. J Allergy Clin Immunol, 140: 1272-4.
Souza, F. C., Gobbato, N. B., Maciel, R. G., Prado. C. M., Martins, M. A., Leick, E. A., and
Tibério, I. F., (2013). Effects of corticosteroid, montelukast and iNOS inhibition on distal
lung with chronic inflammation. Respir Physiol Neurobiol, 185: 435-45.
Stein, R. T., and Martinez, F. D., (2004). Asthma phenotypes in childhood: lessons from an
epidemiological approach. Paediatr Respir Rev, 5: 155–61.
Szefler, S. J., and Chipps, B., (2018). Challenges in the treatment of asthma in children and
adolescents. Annals of Allergy, Asthma and Immunology, 120(4): 382-88.
Taki, F., Kume, H., Kobayashi, T., Ohta, H., Aratake, H., and Shimokata, K., (2007). Effects
of Rho-kinase inactivation on eosinophilia and hyper-reactivity in murine airways by
allergen challenges. Clin Exp Allergy, 37: 599-607.
Taylor, D. R., Bateman, E. D., Boulet, L-P., Boushey, H. A., Busse, W. W., Casale, B., Chanez, P.,
Enright, P. L., Gibson, P. G., de Jongste, J. C., Kerstjens, H. A., Lazarus, S. C., Levy, M.
L., O'Byrne, P. M., Partridge, M. R., Pavord, I. D., Sears, M. R., Sterk, P. J., Stoloff, S. W.,
Szefler, S. J., Sullivan, S. D., Thomas, M. D., Wenzel, S. E., and Reddel, H. K., (2008). A
new perspective on concepts of asthma severity and control. Eur Resp J, 32: 545-54.
van der Wouden, J. C., Uijen, J. H., Bernsen, R. M., Tasche, M. J., de Jongste, J. C., and
Ducharme, F., (2008). Inhaled sodium cromoglycate for asthma in children. Cochrane
Database Syst Rev, 4: CD002173.

Vyas, P., and Vohora, D., (2016). Innovative targets for asthma and COPD: exploring the
existing and screening the new! Infectious Disorders - Drug Targets, 16(3): 162-71.
Whitekus, M. J., Li, N., Zhang, M., Wang, M., Horwitz, M. A., Nelson, S. K., Horwitz, L. D.,
Brechun, N., Diaz-Sanchez, D., and Nel, A. E., (2002). Thiolntioxidants inhibit the
adjuvant effects of aerosolized diesel exhaust particles in a murine model for ovalbumin
sensitization. J Immunol, 168: 2560–67.
Wedes, S. H., Khatri, S. B., Zhang, R., Wu, W., Comhair, S. A., Wenzel, S., Teague, W. G.,
Israel, E., Erzurum, S. C., and Hazen, S. L., (2009). Noninvasive markers of airway
inflammation in asthma. Clin Transl Sci, 2: 112–17.
Zdanowicz, M. M., (2007). Pharmacotherapy of asthma. Am J Pharm Educ, 71(5): 98-110.
Zhu, L. Y., Ni, Z. H., Luo, X. M., and Wang, X. B., (2017). Advance of antioxidants in asthma
treatment. World J Respirol, 7(1): 17-28.

Chapter 11
ANTIOXIDANT ACTIVITY AND CANCER
Newman Osafo1,*, Joseph Owusu-Sarfo1,

Eyitayo Adeyemi Oyindamola1, Sophia Naa Ayikailey Ankrah2
and David Darko Obiri1
1
2
Komfo Anokye Teaching Hospital, Kumasi, Ghana
ABSTRACT
Antioxidants are relevant in the maintenance of cellular architecture and are
henceforth important in maintaining the homeostasis of the host immune system. It is
prudent to maintain a healthy balance in the levels of pro- and antioxidants which protects
the genomic integrity by the maintenance of the redox status of the cell. When there is
perturbation of this balance, the rippling effect is seen as an alteration in the host immunity
that inadvertently affects the normal cellular signaling pathways. Some of these pathways
end up in unregulated cellular proliferation which could result in the promotion of
carcinogenesis. Currently, there is a conflicting report on the therapeutic and/or
prophylactic benefits of antioxidants with human cancers. No consistent results are
available concerning the impact of antioxidants during cancer treatment. Therefore, until a
lot is known concerning the impact of antioxidant supplements in cancer treatment, patients
must take supplements with caution. This chapter focuses on updates on the mechanistic
role of antioxidants in carcinogenesis or otherwise and the role they might play in cancer
therapy via their immunomodulatory function.
1. INTRODUCTION
The pathogenesis of cancer is reported to have a free radical link. Free radicals, otherwise
known as pro-oxidants are defined as extremely reactive chemicals possessing the tendency to
cause injury to living cells (Pham-Huy et al., 2008). The generation of these substances takes
place when there is the gain or loss of an electron by an atom or molecule. The human body
*
Corresponding Author’s Email: nosafo.pharm@knust.edu.gh.

276 Newman Osafo, Joseph Owusu-Sarfo, Eyitayo Adeyemi Oyindamola et al.
naturally produces free radicals, making free radicals play a very important role in normal cell
physiology (Halliwell and Gutterdge, 2007; Bahorun et al., 2006). For instance, the ability of
phagocytes– macrophages and neutrophils- to fight off invading pathogens requires the
formation of free radicals. The free radical generated by the phagocyte by a process called
respiratory burst is hypochlorous acid (HOCl) produced from chloride ion and hydrogen
peroxide (H2O2), requiring heme as a cofactor. The cytotoxicity of this free radical causes death
of the invading pathogen. The significance of free radicals to the human body, thus, cannot be
overemphasized.
At elevated concentrations, however, these free radicals become detrimental to the body
causing harm to major constituents of the cell such as DNA, cell membranes, proteins. The
damage free radicals cause to DNA may contribute to the pathogenesis of cancer as well as
other disorders such as atherosclerosis (Halliwell and Gutterdge, 2007; Bahorun et al., 2006).
Despite endogenous production of free radicals, they may also be produced at abnormally
high levels when the individual is exposed to ionizing radiation and certain toxins in the
environment. When the radiation encounters a molecule or atom in a living cell, there could be
a loss of an electron, resulting in the formation of reactive species. Environmental toxins such
as cigarette smoke and some metals may contain a lot of free radicals or may themselves have
the ability to stimulate the cells of the body to produce free radicals. Free radicals containing
oxygen appear to be the most common forms of free radicals generated by a cell (Halliwell and
Gutterdge, 2007). They are called reactive oxygen species (ROS).
The damage caused by free radicals is curtailed by certain defense mechanisms in the body.
Oxidative stress occurs when the scale tilts in favor of free radical formation and injury. In
other words, when the endogenous antioxidant defense system is not able to counterbalance the
free radical-mediated cellular damage, oxidative stress arises (Fuchs-Tarlovsky, 2013). When
there is perturbation of this balance, the rippling effect is seen as an alteration in the host
immunity that inadvertently impacts the usual cellular signaling pathways.
Chronic oxidative stress causes significant changes to many cellular components like
lipids, proteins, and DNA. Free radicals are thought to act as promoting agents in the
development of cancer. The damage caused by free radicals and for that matter ROS to DNA
and the subsequent development of cancer has necessitated the argument on the role of
antioxidants in carcinogenesis. This chapter gives an in-depth discussion of the mechanistic
involvement of antioxidants in carcinogenesis as well as their potential role in cancer therapy.
2. CLASSIFICATION OF ANTIOXIDANTS
The endogenous chemical substances may be categorized as enzymatic antioxidants and
non-enzymatic antioxidants (Pham-Huy et al., 2008). This section will consider the two classes
briefly.
The neutralization of free radicals, mainly reactive nitrogen species and reactive oxygen
species are done by some enzymes in the body. Major enzymes directly involved include
glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione reductase (GRx) and
catalase (CAT) (Willcox et al., 2004; Pacher et al., 2007; Genestra, 2007; Halliwell, 2007;
Young and Woodside, 2001; Valko et al., 2006; Valko et al., 2005).

Antioxidant Activity and Cancer 277
The first line of protection against free radical injury is SOD. This enzyme converts
superoxide anion radical (O2) to hydrogen peroxide. There is a further transformation of
hydrogen peroxide into water and oxygen by GPx or CAT. GPx is a selenoprotein enzyme and
upon conversion of H2O2 using reduced glutathione (GSH), the latter is oxidized to GSSH. To
continue the process of free scavenging, GSSH must be reduced back to GSH. This is where
GRx comes in, using NADPH as a source of reducing power (Bahorun et al., 2006).
The other aspect of free radical scavenging is carried out by non-enzymatic means. Pham-
Huy et al. (2008) classify this group into metabolic and nutrients antioxidants. Those in the
metabolic class are of endogenous origin and they are produced by metabolic processes in the
body. These antioxidants include glutathione, L-arginine, co-enzyme Q10, metal chelating
proteins, melatonin and lipoic acid (Droge, 2002; Willcox et al., 2004).
Nutrient antioxidants, on the other hand, are of exogenous origin since they cannot be
produced by the body. It is necessary that the individual gets these substances from the diet or
supplements. They include omega-3-fatty acids, omega-6-fatty acids, and trace metals like
selenium; carotenoids; vitamin E and vitamin C.
3. NUTRIENT ANTIOXIDANTS
Antioxidant supplementation from the diet plays a very crucial role in complementing the
endogenous ones to balance the oxidative stress scale. Deficiencies in these have been
implicated in several pathologies. Careful consideration is made concerning the most important
nutrient antioxidants as below:
3.1. Vitamin E
Also known as tocopherol, Vitamin E is a fat-soluble vitamin with antioxidant activity.

There are eight stereoisomers of tocopherol: α, β, γ, δ-tocopherol and α, β, γ, δ-tocotrienol
(Figure 1). Αlpha tocopherol is reported to be the most active stereoisomer in humans (Nguyen
et al., 2006). Due to its fat solubility, α-tocopherol protects the cell membrane from free radical
harm. Protection from lipid peroxidation is the main mechanism of its antioxidant activity.
Sources of vitamin E include eggs, poultry, meat, nuts, whole grains and cereals (Willcox et
al., 2004).
3.2. Vitamin C
Unlike vitamin E, vitamin C, commonly called ascorbic acid (Figure 2) is very water
soluble. Its benefits go beyond its antioxidant effects as it is essential for the synthesis of
collagen as well as neurotransmitters (Li and Schellhorn, 2007). Ascorbic acid has also been
demonstrated to be anti-atherogenic, anti-carcinogenic and it also acts as an efficient
immunomodulator. Ascorbic acid also works in synergy with vitamin E to counterbalance
oxidative stress. Ascorbic acid also helps to regenerate the reduced form of vitamin E. Vitamin
C is obtained from green vegetables and fruits (Naidu, 2003).

3.3. Beta-Carotene
Beta-carotene belongs to the carotenoid group (Figure 3). This group is considered to be
pro-vitamins in that they can be transformed into active retinol (Vitamin A). This compound in
vivo is converted to vitamin A, a substance that is important for good vision. Beta-carotene is
reported to be the best neutralizer of singlet oxygen (Pham-Huy et al., 2008). Sources include
fruits, grains, and vegetables (Willcox et al., 2004).
Figure 1. Chemical structures of the stereoisomers of vitamin E.
Figure 2. Chemical structure of vitamin C.
Figure 3. Chemical structure of beta-carotene.
3.4. Lycopene
Lycopene is a carotenoid (Figure 4). It has antioxidant and anti-proliferative properties in

animals. In vitro studies have also confirmed antioxidant and antiproliferative effects on breast,
lung, and prostate cell lines. There is still a lot of controversy concerning its anticancer
properties in humans (Willcox et al., 2004; Seren et al., 2008; Dahan et al., 2008). Dahan et al.
(2008) reported lycopene to be a very protective agent in the case of cancer of the prostate.

Results from some prospective cohort studies revealed some causalities between increased
consumption of lycopene and decreased occurrence of cancer of the prostate, however, several
of these studies have produced inconsistent results. Lycopene is available in tomatoes. Tomato
appears to be the chief source of this compound. It is also reported that the lycopene in cooked
tomatoes and tomato sauce is more bioavailable than the lycopene in raw tomato (Donaldson,
2004).
Figure 4. Chemical structure of lycopene.
3.5. Selenium
As a trace mineral, it is available in garlic, onion, grains, human liver (Willcox et al., 2004).
It is an integral constituent of the active site of many antioxidant enzymes of which glutathione
peroxidase is a part. Pham-Huy et al. (2001) reported selenium to be antioxidant, anti-
carcinogenic and immunomodulatory at low doses. The contribution of selenium in cancer
prevention is currently a matter of research and debate. Some clinical and cohort studies have
been done on the impact of selenium in the prevention of prostate cancer, lung cancer, and
colorectal cancer. Unfortunately, results from these are mixed and inconclusive (Young and
Woodside, 2001; Higdon et al., 2007).
3.6. Flavonoids
Flavonoids are polyphenolic in terms of their structure and they exist as phytochemicals in
many plants. It is reported that more than 4000 flavonoids have been identified as far as
chemical structure is concerned. They are categorized into catechins, flavonols, flavanones,
isoflavones, flavones, anthocyanins and proanthocyanidins (Pham-Huy et al., 2008).
Flavonoids are potent antioxidants (Miller, 1996) and have been shown to either avert or retard
the incidence of protracted ailments such as cancer, aging, inflammation, memory loss, and
stroke. The major sources of flavonoids are berries, grapefruit, red wine, olive, and celery.
3.7. Omega-3 and Omega-6 Fatty Acids
As long-chain polyunsaturated fatty acids are concerned, these compounds cannot be made
by the human body and as such must be provided in the diet. The omega-3-fatty acids sources
include salmon, tuna, sardines, walnut and nut oils. Omega-6-fatty acids have their sources in
nuts, vegetable oils, cereals, eggs, and poultry. A balance of these two fatty acids is very
important since the two work hand in hand (Logan, 2004). The omega-3s have been reported
to reduce inflammation, memory loss, heart, and cancer while the omega-6s have also been

shown to improve osteoporosis, diabetic neuropathy and aid in the treatment of cancer (Nitta
et al., 2007).
It should, however, be recognized that the antioxidant list goes beyond those that are
mentioned in this chapter.
4. ANTIOXIDANT PROCESS
The process of free radical scavenging is such that upon the destruction of a free radical by
an antioxidant, the antioxidant is itself oxidized. Thus, the restoration of the antioxidant
resources is essential to maintain the balance. The antioxidant mechanism can occur in any of
the following ways: prevention or chain breaking (Lobo et al., 2010).
In the prevention mechanism, an antioxidant enzyme, such as any of the ones mentioned
above, prevents oxidation by decreasing the chain initiation rate either by scavenging the free
radical that initiates the cascade or by stabilizing transition metals e.g., iron and copper (Young
and Woodside, 2001).
The chain breaking process occurs in the following manner: when an electron is released
or stolen by a free radical, another radical is generated. The newly formed radical exerts a
similar effect on other molecules. This goes on until termination occurs. Termination can occur
either by the stabilization of the free radical generated by a chain break antioxidants such as
vitamin C and E or disintegration of the radical into a harmless product (Lobo et al., 2010).
5. FREE RADICALS AND ANTICANCER THERAPY

Nevertheless, cancer chemotherapy aims at destroying cancerous cells via the formation of
free radicals. With radiotherapy, ionizing radiation causes lesions in DNA which ultimately
lead to the death of cancer cells (Figure 5). Thus, free radicals generated from cancer treatment
tend to have the deleterious outcome on healthy non-cancerous body cells as well (Fuchs-
Tarlovsky, 2013).
Carcinogenesis follows a three-stage model – initiation, promotion, and progression.
Reactive oxygen species seem to play a role in every stage of cancer development. The damage
to DNA that results from reactive oxygen species and other free radicals can be direct or
indirect. Indirect damage occurs through a reaction with cellular components such as
phospholipids in the cell membrane to generate a number of reactive intermediates. These
intermediates can couple to DNA bases, leading to formation of DNA adducts (Marnette,
2000). DNA adduct formation is very crucial to cancer development, the reason being that upon
escape from cellular repair machinery, the adducts will lead to mutations (Wogan et al., 2004).

Figure 5. Pathway of oxidative damage to DNA.
6. CANCER AND ANTIOXIDANT STATUS

Several studies have evaluated cancer and antioxidant status. Results from these research
works indicate that low antioxidant status, as well as increased oxidative stress, are observed
in people with cancer. Sharma et al. (2009) reported that in patients who have carcinoma of the
tongue, the levels of conjugated dienes and lipid peroxide in plasma were considerably
increased prior to treatment in comparison to control individuals. Also, a significantly reduced
amount of GSH, GPx, vitamin C, vitamin E, and SOD were seen in cancer patients. The
conclusion was that high levels of oxidative stress markers, with decreased amounts of
antioxidants in carcinoma of the tongue, indicating that a significant contribution is made by
markers of oxidative stress in carcinogenesis.
Badajatia et al. (2010) in their research similarly showed that levels of ascorbic acid and
tocopherol in serum; expressions of glutathione peroxidase and superoxide dismutase in whole
blood and serum showed significantly reduced antioxidant activity in cancer patients in
comparison with the control subjects.
Results from a study done by Klarod et al. (2011) revealed that in individuals with lung
cancer, statistically low levels of serum lycopene and vitamin A were observed at the initial
stages of the disease while serum levels of tocopherol, selenium, beta-carotene, and zinc were
even much lower at the latter stages of cancer (Fuchs-Tarlovsky, 2013).

7. ANTICANCER THERAPY AND ANTIOXIDANT STATUS

In a systematic review by Ladas et al. (2004), the authors reported a direct relationship
between chemotherapy and reduced antioxidant status. However, their report was based on a
limited number of studies (Durken et al., 2000; Durken et al., 1995; Erhola et al., 1996). Also,
this review found no consistent pattern between changes in the antioxidants - vitamin E, vitamin
C, and selenium - with chemotherapy.
One plausible explanation for the inconsistencies in the alteration in antioxidant status
following chemotherapy may be attributed to the depletion of antioxidants preceding treatment.
This is because cancer cells are able to use antioxidants in a more efficient way than normal
healthy cells, leading to reduced levels of circulating antioxidants. Studies that have found
decreased levels of antioxidants in cancer patients as compared to control subjects before
treatment support this claim (Ray et al., 2000; Agus et al., 1999; Bratakos et al., 1990;
Broghamer et al., 1976; Broghamer et al., 1978; Ambrosome et al., 1999).
This finding indicates that neoplastic activity and its related poor health is linked to low
antioxidant status, thereby, giving a suggestion that antioxidants may be beneficial in cancer
patients (Ladas et al., 2004).
8. IMMODULATORY FUNCTIONS OF ANTIOXIDANTS IN

CANCER THERAPY
Elimination, equilibrium, and escape are the phases of immunoediting, a mechanism by
which the immune system identifies and eradicates cancer cells (Teng et al., 2005; Bhatia and
Kumar, 2016; Jones et al., 2016; Schreiber et al., 2011). In the elimination phase, through
surveillance, the immune cells of the host recognize and attempt to remove nascent tumors
(Schreiber et al., 2011). The equilibrium phase commences when some of the tumor cells mount
resistance to the mechanisms of immune surveillance. The equilibrium phase actually exists
when there is an equilibrium between immune cell-mediated apoptosis and proliferation of
tumor cells. When tumor cells escape destruction by the immune system, the escape phase
becomes established. Immunosuppression in the tumor microenvironment also favors tumor
cell proliferation. At this stage, tumors can be detected clinically (Schreiber et al., 2011;
Quintana et al., 2010).
Currently, no direct connection between the modulation of cancer immunoediting and
antioxidants has been well established. One chemical compound, curcumin, a yellowish
curcuminoid of turmeric of the family Zingiberaceae, known widely for its anti-inflammatory
as well as antioxidant effects (Sahu et al., 2016; Anuradha et al., 2015; Kumaravel et al., 2013).
It has been shown to cause a modulation in the process of immunoediting, including the
revamping of the mechanisms of immune surveillance with cancer cells. Mechanism of
curcumin is via:
 Suppression of T-cells apoptosis,

 Restoration of CD4+/CD8+ T cells and

 Inhibition of immunosuppression mediated via the attenuation of immunosuppressive

regulatory T-cells (Tregs) (Bose et a., 2015; Ranjan et al., 1998; Jagetia and Aggarwal,
2007; Pal et al., 2005; Bhattacharyya et al., 2010)
Immunosuppression, specifically systemic immunosuppression, is implicated in several

malignancies including glioblastoma and melanoma (Finlay-Jones and Hart, 1997; Schoof et
al., 2011; Gustafson et al., 2010; Youn et al., 2008). Tumor dependent immunosuppression is
reported to be a very significant event in promoting circumvention of cancer cells from
antitumor defense machinery. Myeloid-derived suppressor cells (MDSCs) and Tregs, which
are suppressor cells provide tumor escape routes (Youn et al., 2008; Nagaraj et al., 2009;
Nagaraj and Gabrilovich, 2010; Gabrilovich, 2001; Schlecker et al., 2012; Steitz et al., 2001;
Wang et al., 2009). As a matter of importance, inhibitors of immune checkpoint including
programmed cell death protein 1 (PD1) and cytotoxic T-lymphocyte associated protein 4
(CTLA-4) have been demonstrated to facilitate Tregs-induced immunosuppression, which will
eventually enhance the advancement of cancer. Essentially, when inhibitors of this immune
checkpoint are blocked by antibodies like anti-PD1 and anti-CTLA-4 antibodies, there is a
substantial attenuation in tumor growth and increased effectiveness of cancer therapy
(Flammiger et al., 2009; Ye et al., 2013; Zhou et al., 2009; Curiel, 2008; Son et al., 2015;
Rossowska et al., 2014; Hu et al., 2014). Thus, agents which target these immunosuppressive
cells are being studied in clinical and preclinical models alongside known anticancer drugs
(Curiel, 2008; Son et al., 2015; Rossowska et al., 2014; Hu et al., 2014).
Some antioxidants have been demonstrated to contribute to attenuating tumor growth
mediated by immunosuppressive cells. Resveratrol, a polyphenolic compound known to
possess antioxidant properties at reduced doses is reported to inhibit lung metastasis of breast
cancer tumor (Lee-Chang et al., 2013). This effect was mediated by inactivation of STAT3; the
consequence being inhibition of the function of a tumor evoked regulatory B cells (tBregs) and
their subsequent transformation to Tregs. Similarly, Wang et al. (2013) have reported that by
combining fish oil and selenium, there is synergy in the suppression of the lung tumor growth.
This was mediated by reducing the numbers of MDSCs and Tregs in the spleen, increasing the
host’s immunity against the tumor.
Several compounds possessing antioxidant effects have been reported to augment
radiotherapy by increasing cytotoxicity on cancer cells as well as decreasing the toxicity on
normal healthy cells. Soy isoflavones such as genistein, daidzein, and glycitein, which are
known antioxidants have been extensively investigated concerning their utilization in
anticancer therapy. Raffoul et al. (2007) in their study observed that these antioxidants could
be utilized as radio-sensitizers to augment the suppression of growth and metastasis mediated
by radiotherapy. Another study found out that genistein and soy are able to cause inhibition of
the proliferation of both in vitro human PC-3 prostate cancer cells as well as in vivo orthotropic
PC-3 tumors. This effect was even increased when either genistein or soy was used alongside
radiotherapy (Raffoul et al., 2007). The mechanism of soy isoflavones was mediated by
reducing the radiation-induced increase in the apurinic/apyrimidinic endonuclease 1/redox
factor-1 expression and activation of NF-κB and its DNA binding activity. This resulted in the
potentiation of radiotherapy mediated effects, thus their utilization as radio-sensitizers. Another
in vitro study has revealed that soy isoflavones augment the suppression of the growth of human
A549 non-small cell lung cancer cells that is mediated by radiation. This effect occurs via

inducing increased DNA damage, inhibition of the DNA repair mediated by APE 1/ref-1 as
well as a reduced expression of NF-κB and HIF-1α (Singh-Gupta et al., 2010).
9. IMPERIOUS ANTIOXIDANTS AND THEIR ROLE IN

ANTICANCER THERAPY
As mentioned earlier, there still is much controversy and debate concerning the impact of
antioxidant in cancer therapy. A number of investigations have been done in this area and
varying results have been reported. This section looks at current evidence available on the
utilization of certain antioxidants in anticancer therapy.
Vitamin E is reported to be the major fat-soluble antioxidant that protects lipids from
peroxidation (Burton et al., 1983). Due to its hydrophobic nature, vitamin E works only in a
lipid environment. Thus, antioxidant effects of vitamin E, specifically α-tocopherol is limited
to its direct impact in the lipoprotein and cell membranes. In the cell membrane, vitamin E
reacts with lipid peroxyl radicals to generate a more stable hydroperoxide, which is also a lipid.
The tocopheroxyl radical actually terminates the chain reaction, providing a defense against
lipid peroxidation (Burton et al., 1982; Burton et al., 1983). This vitamin has been proposed for
the prevention of colon, prostate and breast cancers (Pham-Huy et al., 2008).
Ascorbic acid has also been shown to be the chief antioxidant as far as the extracellular
fluid is concerned even though it performs several cellular antioxidant activities (Moser and
Benich, 1991). Research has shown that vitamin C is able to scavenge peroxide, hydroxyl
radical, hydrogen, singlet oxygen, superoxide and peroxyl radicals with high efficiency (Valko
et al., 2006; Rukmini et al., 2012; Bendich et al., 1986). Ascorbic acid has been shown to be
important in immune system stimulation. This is made possible via the attenuation of chronic
inflammatory responses, the persistence of which is associated with many ailments including
cancer. By enhancing the activity of vitamin E, vitamin C also helps to shield membranes from
lipid peroxidation (Figure 6). Vitamin C exerts positive effects by reducing the occurrence of
colorectal lung cancers (Pham-Huy et al., 2008). In a randomized clinical trial, which made an
evaluation of ascorbic acid treatment concurrently with chemotherapy, a non-significant benefit
was shown in an objective response (complete response + partial response), which was greater
in the ascorbic acid supplemented group than in the placebo group (Moser and Benich, 1991).
Also, the two groups were observed to have significant reductions in mean lump diameter prior
to and after treatment.
Carotenoids are lipophilic natural compounds. The most prominent among substances in
this category appears to be beta-carotene. A large number of carotenoids possess an extended
system of alternating single and double bonds. This conjugated system is reported to be
responsible for their antioxidant properties (Knasmüller et al., 2009). As mentioned earlier,
beta-carotene is the principal neutralizer of singlet oxygen. Additionally, this compound
decreases trichloromethyl peroxy radical very efficiently. Despite these, supplementation of
beta-carotene in doses of 20 mg daily for a period of 5-8 years has been shown to increase the
risk of prostate and lung cancers. Mortality is even higher in cigarette smokers. However, these
effects do not seem to be observed in individuals who consume a diet with a high concentration
of beta-carotene.

Figure 6. Reaction of vitamin E with peroxyl free radicals and regeneration of vitamin E radical.
Some studies have also evaluated the impact of flavonoids on cancer. Quercetin is an
aglycone form of flavonoids derived from plants. This has been used as a nutritional supplement
when managing certain ailments like cancer. A study on the impact of quercetin on nitric oxide
and ROS generation in the lipopolysaccharide-stimulated human acute monocytic leukemia
cell line (THP-1) and acute monocytic leukemia cells showed that quercetin decreased LPS-
induced ROS to almost normal levels. It also decreased LPS-induced NO production (Zhang et
al., 2011). Another research showed that there is transient inhibition of the activity of
nocodazole and taxol by the flavonoid quercetin. This was mediated via interference with the
cell cycle of the cancer cells. Based on these observations, the authors thus made a conclusion
that with prolonged exposure of cancer cells to quercetin, the flavonoid is able to prevent cell
survival and proliferation. Furthermore, the ability of quercetin to interfere with the cell cycle
reduces the efficacy of drugs that target microtubule to arrest cells at G2/M (Samuel et al.,
2010).
10. ALLEVIATION OF ADVERSE EFFECTS OF ANTICANCER THERAPY

BY ANTIOXIDANTS
The protection of healthy cells during anticancer therapy is very important. Anticancer
therapy generates a lot of free radicals that could damage healthy cells. Free radicals and ROS
generation is the cause of several adverse effects of cancer therapy including cardiotoxicity,
nausea, and nephrotoxicity. The amelioration of these side effects could be important in
improving the patients’ compliance and adherence.
Current evidence shows that about 25 - 84% of cancer patients take antioxidant
supplements, usually at doses higher than the recommended (VandeCreek et al., 1999; Burstein
et al., 1999; Kelly et al., 2010). The antioxidants are taken by the patients either with or after
conventional treatment. This is to reduce adverse effects and improve general wellbeing. Quite
unfortunately, evidence supporting the efficacy of antioxidants in cancer chemotherapy is
sparse and indirect.
It has been established that certain antioxidants may alleviate some toxicities resulting from
chemotherapy, but there is a concern and even fear that in doing this, the antioxidants may
interfere with the conventional treatment (Labriola and Livingston, 1999; Agus et al., 1999).

The rationale behind taking antioxidant supplements is to make up for the depletion that
results as a consequence of cancer or of the treatment itself. Using micronutrients in serum
and/or total antioxidant capacity, some studies have evaluated the effects that conventional
chemotherapy has on the antioxidant status of the cancer patient. Other studies have also
investigated the effects of taking certain antioxidants or a combination of antioxidants
alongside chemotherapy, but no consistent review of the results is currently available (Mokhtari
et al., 2017).
Nonetheless, several substances possessing antioxidant activity have proved useful in
anticancer therapy. Amifostine is a cytoprotective agent used as an adjuvant in cancer
chemotherapy. This agent works by detoxification of the reactive metabolites of alkylating
agents (Kouvaris et al., 2007). It is also a potent free radical scavenger. Amifostine is also
reported to be a radioprotectant drug. Acceleration of DNA repair, modification of enzyme
activity and the induction of cellular hypoxia have also been proposed as mechanisms through
which amifostine exerts its actions (Kouvaris et al., 2007). The protection of healthy cells
results in the reduction in the occurrence of neutropenia-related fever and the nephrotoxicity
caused by organoplatinum anticancer agents (Spencer et al., 2007) as well as xerostomia in
individuals who are undergoing radiotherapy for head and neck cancers (Brizel et al., 2000).
Recently, studies have shown that antioxidant therapy attenuates the cardiotoxicity
resulting from doxorubicin therapy. In the first study, tannic acid, an antioxidant was used
concurrently with doxorubicin in experimental animals. It was observed that there was the
amelioration of the cardiotoxicity induced by doxorubicin. Also, potentiation of the anticancer
effect of doxorubicin was observed in both in vitro and in vivo studies using 7,12-
dimethylbenz(a)anthracene (DMBA)-induced mammary tumor animal models (Tikko et al.,
2011). The second study employed the use of procyanidins. The procyanidins have been
demonstrated to be a very potent scavenger of free radicals and have a high capacity to offer
protection against anthracycline-induced cardiotoxicity. Upon treatment of the rats with
procyanidins prior to doxorubicin therapy, the authors observed that there was attenuation of
cytoplasm vacuolization with an increase in the left ventricular pressure coupled with a
significant improvement in the electrocardiogram. A decrease in lipid peroxidation resulting
from high antioxidant potency was believed to be responsible for the cardioprotective effects
in the experimental rat category. Also, an in vitro investigation revealed that the procyanidins
offered cardiomyocytes protection against doxorubicin-induced cardiotoxicity. This was
mediated via oxidative stress suppression (Li et al., 2009).
The debate concerning the use of antioxidants concurrently with chemotherapy is based on
agents that accomplish their effects by producing free radicals. These include radiation and
alkylating agents. From the theoretical point of view, the use of antioxidants may neutralize
free radicals, reducing the efficacy of these agents. In their study, Lenzhofer et al. (1983)
reported an alteration in the metabolism of doxorubicin by vitamin E. An interaction such as
this may not necessarily attenuate efficacy of treatment. For instance, some adjuvants like
mesna may neutralize free radicals in the exertion of their effects but do not seem to reduce the
efficacy of chemotherapy. Also, amifostine, a potent free radical scavenger is used to alleviate
adverse effects associated with some anticancer agents without interfering with efficacy.
Just as patients on methotrexate may require leucovorin rescue, patients whose antioxidant
levels have fallen due to anticancer therapy may also require antioxidant supplementation
(Ladas et al., 2004). Since antioxidant supplements could alleviate toxicities associated with

anticancer therapy, they may make it possible for the administration of higher doses of
chemotherapy.
Two studies have suggested that selenium supplements might decrease hematologic
toxicity as well as the nephrotoxicity resulting from cisplatin therapy (Seija, 2000; Hu et al.,
1997). Yet, follow-up was too short for assessment of effects on survival. Some prospective
studies also report that the supplementation of ascorbic acid before the diagnosis of breast
cancer can be of benefit to the patient (Holm et al., 1993; Ingram, 1994, Jain and Millwe, 1994;
Rohan et al., 1993). The 2002 prostate cancer prevention trials in the USA also established that
tocopherol and selenium may decrease the incidence of cancer of the prostate (Brawley and
Parnes, 2000).
11. ANTIOXIDANTS AND THE RISK OF CANCER

Certain studies, both in vivo and in vitro, reveal that some antioxidants selectively prevent
the growth of the tumor, may terminate cellular differentiation and may change the redox state
of the intracellular environments. All these contribute to the cytotoxicity of cancer treatment
(Conklin, 2000; Conklin, 2002; Lamson and Brignall, 1999; Prasad et al., 1999; Prasad et al.,
2001). Additionally, results from two lung cancer chemoprevention trials revealed an increase
beta-carotene intake and increased lung cancer (Omenn et al., 1996; Albanes et al., 1995). This
has raised several concerns regarding the promotion of antioxidants usage in cancer
chemotherapy.
The hypothesis that antioxidants’ ability to safeguard against cancer as a result of their
capacity to neutralize free radicals that damage DNA has gained a lot of popularity. The
availability of elevated amounts of exogenous antioxidants has been demonstrated to avert
damage by free radicals, which are associated with the development of cancer. This observation
has been shown in both animal and laboratory studies.
However, a lot of large, randomized, placebo controller prevention trials have not been
able to validate this thought. A number of these trials had to be terminated because the patients
who were taking antioxidant supplements had a higher incidence of cancer than those who were
not given antioxidant supplements. For instance, in the 1980s, researchers conducted two large
clinical trials to assess the protective effects of β-carotene, vitamin E, and vitamin A in smokers
and found more cases of lung cancer in volunteers taking β carotene, leading to termination of
the trials. A more recent trial evaluating the ability of vitamin E and selenium in the prevention
of prostate cancer was terminated when prostate cancer turned out to be more common in the
vitamin E group (Kaiser, 2014).
Additionally, some experimental studies reveal a negative correlation between antioxidant
supplementation and cancer. Results from two recent studies using mice as models have
revealed that antioxidant supplementation via diet may actually increase the growth and
metastasis of the tumor. Authors from these two studies hence suggest that people with cancer
and even those at risk of developing cancer should limit or even avoid the intake of antioxidant
supplements (Sayin et al., 2014; Cebula et al., 2015).
The first experimental study, research led by Martin Bergö, to study human lung cancer
using mouse models revealed that adding tocopherol or N-acetylcysteine (NAC) to the diet of
mice which had small lung cancer significantly increased the size, stage, and a number of

tumors. Further work also revealed that the addition of the aforementioned antioxidants
significantly lowered the levels of ROS and DNA damage in cancer cells. There was also an
elimination of the expression of the p53 gene. The p53 gene is a tumor suppressor gene that is
activated by DNA damage. The reduced expression of this gene will allow abnormal and
uncontrolled proliferation, increasing metastasis of the tumor (Le Gal et al., 2015)
In the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) study in Finland,
published in 1994, it was observed that giving antioxidants to male smokers increased the
occurrence of lung cancer as compared to the placebo category. Findings from Bergö’s led
study give a plausible explanation for what was observed in this study (Le Gal et al., 2015).
Bergö and his team further looked at the impact of NAC on melanoma. The high sensitivity
of melanoma cells to oxidative stress and the existence of a mouse model of melanoma made
this experiment possible. It was reported that even though supplementation of NAC did not
increase the number and size of the primary melanoma tumors, there was a twofold rise in the
number of lymph node metastasis. The researchers further observed that the ratio of reduced
glutathione to oxidized glutathione (GSH:GSSH) was only slightly increased in the primary
melanomas while it was greatly increased in the metastasized tumors. This observation is
suggestive that the antioxidant provided was counteracting the oxidative stress in the metastatic
cells rather than the healthy cells. Since oxidative stress is the mechanism by which cancer cells
die from cancer therapy, reduction of oxidative stress in the cancer cells specifically promoted
growth and enhanced metastasis. Using human melanoma cell lines, there was an observation
that the analog of vitamin E, trolox, increased the cells’ ability to migrate and invade (Le Gal
et al., 2015).
The other study was carried out by Morrison and his colleagues (Quintana et al., 2015).
Results from this study gave evidence concerning the promotion of metastasis by antioxidants.
Using mouse models of melanoma, the researchers observed that comparing cancer cells in
primary tumors to circulating cancer cells, the oxidative stress was higher in the latter than the
former. Upon treatment with antioxidants, it was realized that there was an increase in
metastasis. This indicates that antioxidants promoted the survival of the metastasizing cells,
giving them the ability to metastasize, even more, elevating the metastatic burden. These
findings buttress the point that the reduction in the levels of oxidative stress gives more benefit
to tumor cells than healthy cells. These results also support the popular idea that treatment of
patients with pro-oxidants could prevent metastasis. Methotrexate, which is a popular
anticancer drug is known to have pro-oxidant properties. As a potent inhibitor of DHFR,
methotrexate plays a significant role in pathways that yield GSH, as well as the metabolic
pathways that yield new bases for DNA synthesis. Available data thus, suggest strongly that
antioxidant supplementation may pose a serious danger in cancer such as lung cancer and
melanoma (Piskounova et al., 2015).
In their study, Sayin et al. (2014) demonstrated that the antioxidants (NAC and vitamin E)
supplementation of the diet decreases ROS levels and damage to DNA, and prevents the
activation of the p53 gene. Also, a marked increase in tumor cell proliferation and tumor growth
in mice were reported. The researchers observed that there was a faster proliferation of tumor
cells upon oxidative stress suppression, a reason which is consistent with some previous studies
(Singh et al., 2008; DeNicola et al., 2011; Bauer et al., 2011).
Moving from animal and laboratory studies, a large number of observational studies such
as cohort and case-control studies have been performed to evaluate the impact of dietary
antioxidants on cancer in humans. The studies have provided varied results. Nine randomized

controlled trials of supplementation of dietary antioxidants for the prevention of cancer have
been performed worldwide, a significant number of them done with the sponsorship of the
National Cancer Institute. Results from the nine studies failed to give evidence that antioxidant
supplementation via diet is of benefit in the prevention of primary cancer. Additionally, a
review of evidence concerning the utilization of mineral and vitamin supplements for
prevention of chronic diseases, of which cancer is a part, done by the United State Prevention
Service Task Force (USPSFT) gave no lucid evidence of the benefit in cancer prevention
(Fortmann et al., 2013).

AND CHALLENGES
The majority of patients undergoing therapy for cancer use antioxidants. However, not all
antioxidants may be beneficial. Also, the mechanism of action on the cellular systems and the
interaction of the antioxidants with anticancer drugs have not been largely explored
(Thyagarajan and Sahu, 2018). As such, a lot more clinical and preclinical investigations are
required to validate the clinical effects of the antioxidants’ doses and timing based on disease
stage and treatment regimen.
It is also essential to look at new targets and identify new agents following an in-depth
assessment of their efficacy, metabolism, safety, and bioavailability using the appropriate
models. Further clinical trials should be done using the appropriate patient population. Current
research looks at single agent antioxidants, however future research should also look at “whole
foods” due to the recent assertion that the flavonoids and other phytochemicals that are
available in vegetables and fruits have the potential to augment the bioavailability of the other
different nutrients, and can target several molecular pathways - the result being a better
response (Ahmad and Mukhtar, 2013).
CONCLUSION
The assessment of the impact of antioxidant supplementation in cancer is largely limited
by variations in dosing, timing, and duration in the studies done. Several studies make use of
the recommended dietary allowances (RDA). However, RDA may not be applicable since it
may not be enough to maintain levels of antioxidant in such individuals. Also, evidence that
supplementation of antioxidants reduces adverse reactions resulting from cancer therapy is
limited by sample size and quality. Additional studies are required to evaluate the impact of
antioxidants. These studies should make use of appropriate sample size and procedures so that
the results will have some clinical relevance.
REFERENCES
Agus, D. B; Vera, J. C; Golde, D. W. Stromal cell oxidation: A mechanism by which tumors
obtain vitamin C. Cancer Res., 1999, 59, 4555-8.

Ahmad, N; Mukhtar, H. Antioxidants meet molecular targets for cancer prevention and
therapeutics. Antioxid Redox Signal., 2013, 19(2), 85–88.
Albanes, D; Heinonen, OP; Huttunen, J. K; Taylor, P. R; Virtamo, J; Edwards, B. K; et al.
Effects of alpha-tocopherol and beta-carotene supplements on cancer incidence in the
Alpha-Tocopherol Beta-Carotene Cancer Prevention Study. Am J Clin Nutr., 1995, 62(6),
1427S-30S.
Alpha Tocopherol Beta-Carotene Cancer Prevention Study Group. The effect of vitamin E and
beta carotene on the incidence of lung cancer and other cancers in male smokers. N Engl J
Med., 1994, 330(15), 1029-35.
Ambrosone, C. B; Freudenheim, J. L; Thompson, P. A; Bowman, E; Vena, J. E; Marshall, J.
R; et al. Manganese superoxide dismutase (MnSOD) genetic polymorphisms, dietary
antioxidants, and risk of breast cancer. Cancer Res., 1999, 59(3), 602-6.
Anuradha, B. R; Bai, Y. D; Sailaja, S; Sudhakar, J; Priyanka, M; Deepika, V. Evaluation of
anti-inflammatory effects of curcumin gel as an adjunct to scaling and root planning: a
clinical study. J Int Oral Health., 2015, 7, 90-93.
Badajatia, N; Satyam, A; Singh, P; Seth, A; Sharma, A. Altered antioxidant status and lipid
peroxidation in Indian patients with urothelial bladder carcinoma. Urol Oncol., 2010, 28,
360–7.
Bahorun, T; Soobrattee, M. A; Luximon-Ramma, V; Aruoma, OI. Free radicals and
antioxidants in cardiovascular health and disease. Int J Med Update., 2006, 1, 1-17.
Bauer, A. K; Cho, H. Y; Miller-DeGraff, L; Walker, C; Helms, K; Fostel, J; et al. Targeted
deletion of Nrf2 reduces urethane-induced lung tumor development in mice. PloS One.,
2011, 21, 6(10), e26590.
Bendich, A; Machlin, L. J; Scandurra, O; Burton, G. W; Wayner, D. D. W. The antioxidant
role of vitamin C. Adv Free Radic Biol Med., 1986, 2, 419–44.
Bhatia, A; Kumar, Y. Cancer stem cells and tumour immunoediting: putting two and two
together. Expert Rev Clin Immunol., 2016, 12, 605-7.
Bhattacharyya, S; Hossain, D. M; Mohanty, S; Sen, G. S; Chattopadhyay, S; Banerjee, S; et al.
Curcumin reverses T cell-mediated adaptive immune dysfunctions in tumor-bearing hosts.
Cell Mol Immunol., 2010, 7(4), 306.
Bose, S; Panda, A. K; Mukherjee, S; Sa, G. Curcumin and tumor immune-editing: resurrecting
the immune system. Cell division., 2015, 10(1), 6.
Bratakos, M. S; Vouterakos, T. P; Loannou, P. V. Selenium status of cancer patients in Greece.
Sci Total Environ., 1990, 92, 207-22.
Brawley, O. W; Parnes, H. Prostate cancer prevention trials in the USA. Eur J Cancer., 2000,
36, 1312-5.
Brizel, D. M; Wasserman, T. H; Henke, M; Strnad, V; Rudat, V; Monnier, A; et al. Phase III
randomized trial of amifostine as a radioprotector in head and neck cancer. J Clin Oncol,
2000, 18, 3339–45.
Broghamer, Jr. W. L; McConnell, K. P; Grimaldi, M; Blotcky, A. J. Serum selenium and
reticuloendothelial tumors. Cancer., 1978, 41(4), 1462-6.
Broghamer, W. L; McConnell, K. P; Blotcky, A. L. Relationship between serum selenium
levels and patients with carcinoma. Cancer., 1976, 37, 1384-8.
Burstein, H. J; Gelber, S; Guadagnoli, E; Weeks, J. C. Use of alternative medicine by women
with early-stage breast cancer. N Engl J Med., 1999, 340(22), 1733-9.

Burton, G. W; Joyce, A; Ingold, K. U. First proof that vitamin E is the major lipidsoluble, chain
breaking antioxidant in human blood plasma. Lancet., 1982, 2, 327–8.
Burton, G. W; Joyce, A; Ingold, K. U. Is vitamin E the only lipid-soluble, chainbreaking
antioxidant in human blood plasma and erythrocyte membranes? Arch Biochem Biophys.,
1983, 221, 281–90
Cebula, M; Schmidt, E. E; Arnér, E. S. J. TrxR1 as a potent regulator of the Nrf2-Keap1
response system. Antioxid. Redox Signal., 2015, 23, 823–53.
Chen, S; Xiong, G; Wu, S; Mo, J. Downregulation of apurinic/apyrimidinic endonuclease
1/redox factor-1 enhances the sensitivity of human pancreatic cancer cells to radiotherapy
in vitro. Cancer Biother Radiopharm., 2013, 28(2), 169-76.
Conklin, K. A. Dietary antioxidants during cancer chemotherapy: Impact on chemotherapeutic
effectiveness and development of side effects. Nutr Cancer., 2000, 37, 1-18.
Conklin, K. A. Dietary polyunsaturated fatty acids: Impact on cancer chemotherapy and
radiation. Altern Med Rev., 2002, 7, 4-21.
Curiel, T. J. Regulatory T cells and treatment of cancer. Curr Opin Immunol., 2008, 20, 241-6.
Dahan, K; Fennal, M; Kumar, N. B. Lycopene in the prevention of prostate cancer. J Soc Integr
Oncol., 2008, 6, 29-36.
DeNicola, G. M; Karreth, F. A; Humpton, T. J; Gopinathan, A; Wei, C; Frese, K; et al.
Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis.
Nature., 2011, 475(7354), 106.
Donaldson, M. S. Nutrition and cancer: A review of the evidence for an anti-cancer diet. Nutr
J., 2004, 3, 19-25.
Droge, W. Free radicals in the physiological control of cell function. Physiol Rev., 2002, 82,
47-95.
Dürken, M; Agbenu, J; Finckh, B; Hübner, C; Pichlmeier, U; Zeller, W; et al. Deteriorating
free radical-trapping capacity and antioxidant status in plasma during bone marrow
transplantation. BMT., 1995, 15(5), 757-62.
Dürken, M; Herrnring, C; Finckh, B; Nagel, S; Nielsen, P; Fischer, R; et al. Impaired plasma
antioxidative defense and increased nontransferrin-bound iron during high-dose
chemotherapy and radiochemotherapy preceding bone marrow transplantation. Free Radic
Biol Med., 2000, 28(6), 887-94.
Erhola, M; Kellokumpu-Lehtinen, P; Metsä-Ketelä, T; Alanko, K; Nieminen, M. M. Effects of
anthracyclin-based chemotherapy on total plasma antioxidant capacity in small cell lung
cancer patients. Free Radic Biol Med., 1996, 21(3), 383-90.
Finlay-Jones, J. J; Hart, P. H. Ultraviolet irradiation, systemic immunosuppression and skin
cancer: role of urocanic acid. Australas J Dermatol., 1997, 38(suppl 1), S7-S12.
Flammiger, A; Weisbach, L; Huland, H; Tennstedt, P; Simon, R; Minner, S; et al. High tissue
density of FOXP3+ T cells is associated with clinical outcome in prostate cancer. Eur J
Cancer., 2013, 49(6), 1273-9.
Fortmann, S. P; Burda, B. U; Senger, CA; Lin, J. S; Whitlock, E. P. Vitamin and mineral
supplements in the primary prevention of cardiovascular disease and cancer: an updated
systematic evidence review for the US Preventive Services Task Force. Ann Intern Med.,
2013, 159(12), 824-34.
Fuchs-Tarlovsky, V. Role of antioxidants in cancer therapy. Nutrition., 2013, 29(1), 15-21.

Gabrilovich, D. I.; Velders, M. P; Sotomayor, E. M; Kast, W. M. Mechanism of immune

dysfunction in cancer mediated by immature Gr-1+ myeloid cells. J Immunol., 2001, 166,
5398-406.
Genestra, M. Oxyl radicals, redox-sensitive signalling cascades and antioxidants. Cell Signal.,
2007, 19, 1807-19.
Gustafson, M. P; Lin, Y; New, KC; Bulur, P. A; O’neill, B. P; Gastineau, DA; et al. Systemic
immune suppression in glioblastoma: the interplay between CD14+ HLA-DRlo/neg
monocytes, tumor factors, and dexamethasone. J Neurooncol., 2010, 12(7), 631-44.
Halliwell, B; Gutteridge, JMC. Free radicals in biology and medicine. 4th ed. Oxford, UK:
Clarendon Press., 2007.
Halliwell, B. Biochemistry of oxidative stress. Biochem Soc Trans., 2007, 35, 1147-50.
Higdon, J; Drake, V. J; Whanger, P. D. Selenium. Linus Pauling Institute. Oregon State
University. Micronutrient Information Center., 2007, Accessed 1, August 2018. Available:
http://lpi.oregonstate.edu/infocenter/minerals/selenium/.
Holm, L. E; Nordevang, E; Hjalmar, M. L; Lidbrink, E; Callmer, E; Nilsson, B. Treatment
failure and dietary habits in women with breast cancer. J Natl Cancer Inst., 1993, 85(1),
32-6.
Hu, M; Li, K; Maskey, N; Xu, Z; Peng, C; Wang, B; et al. Decreased intratumoral Foxp3 Tregs
and increased dendritic cell density by neoadjuvant chemotherapy associated with
favorable prognosis in advanced gastric cancer. Int J Clin Exp Pathol., 2014, 7(8), 4685.
Hu, Y. J; Chen, Y; Zhang, Y. Q; Zhou, M. Z; Song, X. M; Zhang, B. Z; et al. The protective
role of selenium on the toxicity of cisplatin-contained chemotherapy regimen in cancer
patients. Biol Trace Elem Res., 1997, 56(3), 331-41.
Ingram, D. Diet and subsequent survival in women with breast cancer. Br J Cancer., 1994, 69,
592- 5.
Jagetia, G. C; Aggarwal, B. B. “Spicing up” of the immune system by curcumin. J Clin
Immunol., 2007, 27, 19-35.
Jain, M; Miller, A. B; To, T. Premorbid diet and the prognosis of women with breast cancer. J
Natl Cancer Inst., 1994, 86, 1390-7.
Jones, L. M; Broz, M. L; Ranger, J. J; Ozcelik, J; Ahn, R; Zuo, D; et al. STAT3 establishes an
immunosuppressive microenvironment during the early stages of breast carcinogenesis to
promote tumor growth and metastasis. Cancer Res., 2016, 76(6), 1416-28.
Kaiser, J. Antioxidants could spur tumors by acting on cancer gene. Science., 2014, 343(6170),
477.
Kelly, K. M; Jacobson, J. S; Kennedy, D. D; et al. Use of unconventional therapies by children
with cancer at an urban medical center. J Pediatr Hematol Oncol., 2000, 22, 412-6.
Klarod, K; Hongsprabhas, P; Khampitak, T; Wirasorn, K; Kiertiburanakul, S;
Tangrassameeprasert, R; Daduang, J; Yongvanit, P; Boonsiri, P. Serum antioxidant levels
and nutritional status in early and advanced stage lung cancer patients. Nutrition., 2011,
27(11-12), 1156-60.
Knasmüller, S; De Marini, D. M; Johnson, I; Gerhäuser, C. Chemoprevention of cancer and
DNA damage by dietary factors. Chapter 23. Carotenoids and Vitamin A. Federal Republic
of Gernamy: Wiley-BlackWell, 2009.
Kumaravel, M; Sankar, P; Latha, P; Benson, C. S; Rukkumani, R. Antiproliferative effects of
an analog of curcumin in Hep-2 cells: a comparative study with curcumin. Nat Prod
Commun., 2013, 8, 183-6.

Labriola, D; Livingston, R. Possible interactions between dietary antioxidants and

chemotherapy. Oncology (Huntingt)., 1999, 13, 1003-8.
Lamson, D. W; Brignall, M. S. Antioxidants in cancer therapy; their actions and interactions
with oncologic therapies. Altern Med Rev., 1999, 4, 304- 29.
Le Gal, K; Ibrahim, M. X; Wiel, C; Sayin, V. I; Akula, M. K; Karlsson, C; et al. Antioxidants
can increase melanoma metastasis in mice. Sci Transl Med., 2015, 7(308), 308re8.
Lee-Chang, C; Bodogai, M; Martin-Montalvo, A; Wejksza, K; Sanghvi, M; Moaddel, R; et al.
Inhibition of breast cancer metastasis by resveratrol-mediated inactivation of tumor-
evoked regulatory B cells. J Immunol., 2013, 1300606.
Li, W; Xu, B; Xu, J; Wu, X. L. Procyanidins produce significant attenuation of doxorubicin-
induced cardiotoxicity via suppression of oxidative stress. Basic Clin Pharmacol Toxicol.,
2009, 104, 192–7.
Li, Y; Schellhorn, H. E. New developments and novel therapeutic perspectives for vitamin C.
J. Nutr., 2007, 137, 2171-84.
Lobo, V; Patil, A; Phatak, A; Chandra, N. Free radicals, antioxidants and functional foods:
Impact on human health. Pharmacogn Rev., 2010, 4(8), 118–26.
Logan, AC. Omega-3 fatty acids and major depression: A primer for the mental health
professional. Lipids Health Dis., 2004, 3, 25-33.
Marnette, L. J. Oxyradicals and DNA damage. Carcinogenesis., 2000, 21, 361–70.
Miller, A. L. Antioxidant Flavonoids: Structure, Function and Clinical Usage. Alt. Med Rev.,
1996, 1, 103-11.
Mokhtari, R. B; Homayouni, T. S; Baluch, N; Morgatskaya, E; Kumar, S; Das, B; Yeger, H.
Combination therapy in combating cancer. Oncotarget., 2017, 8(23), 38022–43.
Moser, U; Benich, A; Vitamin, C. In: Machlin LJ, editor. Handbook of Vitamins. 2nd ed. New
York: Marcel Dekker, 1991, p. 195–232.
Nagaraj, S; Collazo, M; Corzo, CA; Youn, J. I; Ortiz, M; Quiceno, D; Gabrilovich, D. I.
Regulatory myeloid suppressor cells in health and disease. Cancer Res., 2009, 69, 7503-6.
Nagaraj, S; Gabrilovich, D. I. Myeloid-derived suppressor cells in human cancer. Cancer J.,
2010, 16, 348-53.
Naidu, A. K. Vitamin C in human health and disease is still a mystery? An overview. Nutr J.,
2003, 2, 1-10.
Nguyen, L. A; He, H; Pham-Huy, C. Chiral drugs. An overview. Int J Biomed Sci., 2006, 2,
85-100.
Nitta, H; Kinoyama, M; Watanabe, A; Shirao, K; Kihara, H; Arai, M. Effects of nutritional
supplementation with antioxidant vitamins and minerals and fish oil on antioxidant status
and psychosocial stress in smokers: an open trial. Clin Exp Med., 2007, 7(4), 179-83.
Omenn, G. S; Goodman, G. E; Thornquist, M. D; Balmes, J; Cullen, M. R; Glass, A; et al.
Effects of a combination of beta carotene and vitamin A on lung cancer and cardiovascular
disease. N Engl J Med., 1996, 334(18), 1150-5.
Pacher, P; Beckman, J. S; Liaudet, L. Nitric oxide and peroxynitrite in health and disease.
Physiol Rev., 2007, 87, 315-424.
Pal, S; Bhattacharyya, S; Choudhuri, T; Datta, G. K; Das, T; Sa, G. Amelioration of immune
cell number depletion and potentiation of depressed detoxification system of tumor bearing
mice by curcumin. Cancer Detect Prev., 2005, 29, 470-8.

Pham-Huy, C; Nguyen, P; Marchand, V; Dumery, B; Warnet, JM; Thevenin, M; et al. Selenium

and tobacco smoke tars: In vitro effects on different immunocompetent cells. Toxicol.,
2001, 164(1-3), 111-2.
Pham-Huy, LA; He, H; Pham-Huy, C. Free Radical, Antioxidants in Disease and Health. J
Biomed Sci., 2008, 4(2), 89-96.
Piskounova, E; Agathocleous, M; Murphy, MM; Hu, Z; Huddlestun, SE; Zhao, Z; et al.
Oxidative stress inhibits distant metastasis by human melanoma cells. Nature., 2015,
527(7577), 186.
Prasad, K. N; Cole, W. C; Kumar, B; Prasad, K. C. Scientific rationale for using high-dose
multiple micronutrients as an adjunct to standard and experimental cancer therapies. J Am
Coll Nutr., 2001, 20(suppl 5), 450S-63S.
Prasad, K. N; Kumar, A; Kochupillai, V; Cole, W. C. High doses of multiple antioxidant
vitamins: essential ingredients in improving the efficacy of standard cancer therapy. J Am
Coll Nutr., 1999, 18(1), 13-25.
Quintana, E; Piskounova, E; Shackleton, M; Weinberg, D; Eskiocak, U; Fullen, DR; Johnson,
TM; Morrison, SJ. Human melanoma metastasis in NSG mice correlates with clinical
outcome in patients. Sci Transl Med., 2012, 4(159), 159ra149.
Quintana, E; Shackleton, M; Foster, H. R; Fullen, D. R; Sabel, M. S; Johnson, TM; et al.
Phenotypic heterogeneity among tumorigenic melanoma cells from patients that is
reversible and not hierarchically organized. Cancer cell., 2010, 18(5), 510-23.
Ranjan, D; Johnston, T. D; Wu, G; Elliott, L; Bondada, S; Nagabhushan, M. Curcumin blocks
cyclosporine A–resistant CD28 costimulatory pathway of human T-cell proliferation. J
Surg Res., 1998, 77, 174-8.
Ray, G; Batra, S; Shukla, N. K; Deo, S; Raina, V; Ashok, S; et al. Lipid peroxidation, free
radical production and antioxidant status in breast cancer. Breast cancer research and
treatment., 2000, 59(2), 163-70.
Rohan, T E; Hiller, J. E; McMichael, A. J. Dietary factors and survival from breast cancer. Nutr
Cancer., 1993, 20, 167-77.
Rossowska, J; Pajtasz-Piasecka, E; Anger, N; Wojas-Turek, J; Kicielinska, J; Piasecki, E; et al.
Cyclophosphamide and IL-12-transduced DCs Enhance the Antitumor Activity of Tumor
Antigen–stimulated DCs and Reduce Tregs and MDSCs Number. J Immunother., 2014,
37(9), 427-39.
Rukmini, A; Raharjo, S; Hastuti, P; Supriyadi, S. Antiphotooxidative effect of ascorbic acid
microemulsion in virgin coconut oil. 12th ASEAN Food conference. June, 2011
(Thailand). Consulted, 2012.
Sahu, P. K; Sahu, P. K; Sahu, PL; Agarwal, D. D. Structure activity relationship, cytotoxicity
and evaluation of antioxidant activity of curcumin derivatives. Bioorg Med Chem Lett.,
2016, 26, 1342-7.
Samuel, T; Fadalla, K; Yehualaeshet, T. E. The flavonoid quercetin transiently inhibits the
activity of taxol and nocodazole through interference with the cell cycle. Nutr Cancer,
2010, 62, 1025–35.
Sayin, V. I; Ibrahim, M. X; Larsson, E; Nilsson, J. A; Lindahl, P; Bergo, M. O. Antioxidants
accelerate lung cancer progression in mice. Sci Transl Med., 2014, 6(221), 221ra15.
Schlecker, E; Stojanovic, A; Eisen, C; Quack, C; Falk, C. S; Umansky, V; et al. Tumor-
infiltrating monocytic myeloid-derived suppressor cells mediate CCR5-dependent

recruitment of regulatory T cells favoring tumor growth. J Immunol., 2012, 189(12), 5602-
11.
Schoof, N; Iles, M. M; Bishop, D. T; Newton-Bishop, J. A; Barrett, J. H. Pathway-based
analysis of a melanoma genome-wide association study: analysis of genes related to
tumour-immunosuppression. PLoS One., 2011, 6(12), e29451.
Schreiber, R. D; Old, L. J; Smyth, M. J. Cancer immunoediting: integrating immunity’s roles
in cancer suppression and promotion. Science., 2011, 331, 1565-70.
Seren, S; Lieberman, R; Bayraktar, U. D; Heath, E; Sahin, K; Andic, F; et al. Lycopene in
cancer prevention and treatment. Am J Ther., 2008, 15(1), 66-81.
Sharma, M; Rajappa, M; Kumar, G; Sharma, A. Oxidant-antioxidant status in Indian patients
with carcinoma of posterior one-third of tongue. Cancer Biomak., 2009, 5, 253–60
Sieja, K. Protective role of selenium against the toxicity of multi-drug chemotherapy in patients
with ovarian cancer. Pharmazie., 2000, 55, 958- 9.
Singh, A; Boldin-Adamsky, S; Thimmulappa, R. K; Rath, S. K; Ashush, H; Coulter, J; et al.
RNAi-mediated silencing of nuclear factor erythroid-2–related factor 2 gene expression in
non–small cell lung cancer inhibits tumor growth and increases efficacy of chemotherapy.
Cancer Res., 2008, 68(19), 7975-84.
Singh-Gupta, V; Joiner, M. C; Runyan, L; Yunker, CK; Sarkar, F. H; Miller, S; et al. Soy
isoflavones augment radiation effect by inhibiting APE1/Ref-1 DNA repair activity in non-
small cell lung cancer. J Thorac Oncol., 2011, 6(4), 688-98.
Son, CH; Bae, J. H; Shin, D. Y; Lee, H. R; Jo, W. S; Yang, K; et al. Combination effect of
regulatory T-cell depletion and ionizing radiation in mouse models of lung and colon
cancer. Int J Radiat Oncol Biol Phys., 2015, 92(2), 390-8.
Spencer, A; Horvath, N; Gibson, J; Prince, H. M; Herrmann, R; Bashford, J; et al. Australasian
Leukemia and Lymphoma Group. Prospective randomized trial of amifostine
cytoprotection in myeloma patients undergoing high-dose melphalan conditioned
autologous stem cell transplantation. BMT., 2005, 35, 971–7.
Steitz, J; Brück, J; Lenz, J; Knop, J; Tüting, T. Depletion of CD25+ CD4+ T cells and treatment
with tyrosinase-related protein 2-transduced dendritic cells enhance the interferon α-
induced, CD8+ T-cell-dependent immune defense of B16 melanoma. Cancer Res., 2001,
61, 8643-6.
Teng, M. W; Galon, J; Fridman, W. H; Smyth, M. J. From mice to humans: developments in
cancer immunoediting. J Clin Invest., 2015, 125, 3338-46.
Thyagarajan, A; Sahu, R. P. Potential Contributions of Antioxidants to Cancer Therapy:
Immunomodulation and Radiosensitization. Integr Cancer Ther., 2018, 17(2), 210-6.
Tikko, K; Sane, M. S.; Gupta, C. Tannic acid ameliorates doxorubicin-induced cardiotoxicity
and potentiates its anticancer activity: potential role of tannins in cancer chemotherapy.
Toxicol Appl Pharmacol., 2011, 251, 191– 200.
Valko, M; Morris, H; Cronin, M. T. D. Metals, toxicity and oxidative stress. Curr Med Chem.,
2005, 12, 1161-208.
Valko, M; Rhodes, C. J; Monocol, J; Izakovic, M; Mazur, M. Free radicals, metals and
antioxidants in oxidative stress-induced cancer. Chem Biol Interact., 2006, 160, 1–40.
VandeCreek, L; Rogers, E; Lester, J. Use of alternative therapies among breast cancer
outpatients compared with the general population. Altern Ther Health Med., 1999, 5, 71-
6.

Wang, H; Chan, Y. L.; Li, T. L.; Bauer, B. A; Hsia, S; Wang, C. H et al. Reduction of splenic
immunosuppressive cells and enhancement of anti-tumor immunity by synergy of fish oil
and selenium yeast. PloS One., 2013, 8(1), e52912.
Wang, W; Lau, R; Yu, D; Zhu, W; Korman, A; Weber, J. PD1 blockade reverses the
suppression of melanoma antigen specific CTL by CD4+ CD25(Hi) regulatory T cells. Int
Immunol., 2009, 21, 1065-107.
Willcox, J. K; Ash, S. L; Catignani, G. L. Antioxidants and prevention of chronic disease. Crit
Rev Food Sci Nutr., 2004, 44, 275-95.
Wogan, G. N; Hecht, S. S; Felton, J. S; Conney, A. H; Loeb, L. A. Environmental and chemical
carcinogenesis. Semin Cancer Biol., 2004, 14, 437–86.
Ye, L; Li, X; Sun, S; Guan, S.; Wang, M; Guan, X; et al. A study of circulating anti-CD25
antibodies in non-small cell lung cancer. Clin Transl Oncol., 2013, 15(8), 633-7.
Youn, J. I; Nagaraj, S; Collazo, M; Gabrilovich, D. I. Subsets of myeloid-derived suppressor
cells in tumor-bearing mice. J Immunol., 2008, 181, 5791-802.
Young, I; Woodside, J. Antioxidants in health and disease. J Clin Pathol., 2001, 54, 176-86.
Zhang, M; Swarts, S. G; Yin, L; Liu, C; Tian, Y; Cao, Y; et al. Antioxidant properties of
quercetin. Adv Exp Med Biol., 2011, 915, 283–9.
Zhou, J; Ding, T; Pan, W; Zhu, L. Y; Li, L; Zheng, L. Increased intratumoral regulatory T cells
are related to intratumoral macrophages and poor prognosis in hepatocellular carcinoma
patients. Int J Cancer., 2009, 125, 1640-8.

Chapter 12
OXIDANTS AND ANTIOXIDANT DEFENSE

IN HIV/AIDS
Hrushikesh E. Velis*
Amrutvahini College of Pharmacy, Sangamner, India
ABSTRACT
Oxidative stress (OS) induced by human immunodeficiency virus (HIV), plays an
important role in the progression of acquired immunodeficiency syndrome (AIDS). HIV
activates cellular antioxidant enzymes to counter HIV induced OS. However, in the
absence of micronutrient support the cellular antioxidant stores are exhausted, which
increases the level of highly reactive oxygen sepsis (ROS), triggering apoptosis and death.
Hence, understanding the mechanism of HIV induced OS stress and its management
becomes important. The present chapter elaborates the mechanism of OS induced by HIV
and antiretroviral therapy (ART). The clinical studies discussed in this chapter clearly show
that the use of antioxidants significantly decreases OS and increase CD4 + cell count in
AIDS patients. Various supplements are available to manage the OS in AIDS such as,
vitamins, minerals, amino acids, and herbs. However, the irrational use of antioxidants for
AIDS can have a negative impact on the health of an AIDS patient. Therefore, there is a
need for the development of a standard treatment protocol and individualized dosage
regimen of antioxidants to ensure their safe and effective use against OS induced by HIV
and ART.
1. INTRODUCTION
Since the end of the 20th century, AIDS has proved to be a challenge for the existence of
the human race. Gottlieb et al., first noticed AIDS in 1981, when they found Pneumocystis
carinii pneumonia infection in five homosexual men and a report on these findings was
published by the US Center for Disease Control and Prevention (Oppenheimer and Bayer,
*
Corresponding Author’s Email: hrushikeshvelis@gmail.com.

298 Hrushikesh E. Velis
2011). Subsequently, in 1983 Luc Montagnier and Robert Gallo isolated a retrovirus, which
later came to be known as HIV, the causative microorganism of AIDS (Papadopulos-Eleopulos
et al., 2004). HIV is a retrovirus of the subgroup of retrovirus known as lentiviruses or slow
virus. Genetically, there are two types of HIV- HIV-1, and HIV-2. HIV-1 is endemic globally
whereas, HIV-2 is confined to western Africa. HIV-1 is found to be more virulent than HIV-2
(Klimas, and Koneru, 2008).
Once inside the body, HIV infects and kills vital cells of the immune system, like T-helper
cells (CD4+), macrophages and dendritic cells (Freed and Martin, 2007). The CD4+ cells are
destroyed by the HIV virus through mechanisms like apoptosis and cell membrane destruction,
and by continuous budding of viruses from the infected cells. Moreover, both infected and non-
infected CD4+ cells are destructed by syncytium formation (infected T cells induce membrane
fusion between adjacent cells to form a giant multinucleated cell called syncytium which
accelerates destruction) during advanced stage of disease (reviewed by Paranjape, 2005).
Progressive depletion of CD4+ cells leads to AIDS, with an increased likelihood of getting
opportunistic infections and other clinical problems associated with HIV. These include muscle
wasting and death. WHO, has recommended immunological criterion for diagnosis of advanced
HIV (including AIDS), which states that CD4+ count less than 350 cells per mm3 in adults and
child above 5 years is an indicator of AIDS. It also serves as a major determinant of initiating
ART for better virological outcomes (WHO, 2007). AIDS is a highly depleting disease with
catastrophic effects on the immune system, triggering various adverse events like opportunistic
infections (e.g., Pneumocystis carinii causing pneumonia), weight loss, neurological
deterioration (eg. AIDS dementia complex), cancers (e.g., Kaposi’s sarcoma) (Hughes, 2002).
Currently, AIDS has been effectively treated (though not cured) with highly active
antiretroviral therapy (HAART) leading to a 50% decline in the rate of AIDS-associated
mortalities, opportunistic infections and the incidences of HIV-associated cognitive
dysfunction (reviewed by Sharma, 2014).
Though HAART has proved to be effective in managing AIDS, there is still a long way to
go for treating AIDS, as there is currently no curative therapy available. The HAART therapy
itself presently has various limitations like pill burden, resistance, and induction of OS (Da-
Yong Lu et al., 2017). Therefore, there is a wide scope for exploring various other ways of
dealing with AIDS. One such area that will be discussed in the current chapter is an antioxidant
defense in AIDS. Once inside the host CD4+ cells, the HIV virus triggers OS by enhancing
reactive oxygen species (ROS) production, which severely elevates levels of oxidized nucleic
bases such as 8-oxoguanine (8-oxoG) and lipid peroxidation products, including monoaldehyde
(MDA) in plasma (reviewed by Ivanov et al., 2016). The mechanism by which HIV develops
OS and produces ROS is through its trans-activator of transcription (Tat) protein and envelope
glycoprotein (gp120) (Banerjee et al., 2010). Further, HIV also accumulates ROS by activating
macrophages via TNF-α release and activation of polymorphonuclear leukocytes (Robinson,
2009).
Consequently, there is an imbalance between OS and antioxidant defense within the cells
leading to a pro-oxidative state, which leads to cellular damage and death. Several in vitro
studies have also linked OS with increased HIV replication, functional and numerical
impairment of CD4+ cells, with altered immune response, and toxicity of antiretroviral drugs
(Masiá et al., 2016). HIV infection creates a situation of imbalance between pro-oxidants and
antioxidants due to depletion of micronutrients in CD4+ cells, which may be a major cause of
the progression of HIV to AIDS. The above facts clearly show that antioxidant defense is of

Oxidants and Antioxidant Defense in HIV/AIDS 299
paramount importance for the survival of CD4+ cells infected with HIV. Various antioxidants
and micronutrients have been tried in HIV/AIDS patients like, vitamin A, C, E, β-carotene,
selenium, etc. and some of these have shown promising results in preventing progression of
HIV to AIDS and improving overall health of the AIDS patient (Friis, 2005 and Bilbis et al.,
2010). Therefore, this chapter attempts to explore the preventive and therapeutic role of
antioxidant defense in HIV/AIDS.
2. OXIDATIVE STRESS IN HIV/AIDS

2.1. Mechanism of Oxidative Stress in HIV/AIDS
OS in any cell is a result of increase in ROS, like superoxide (O2• −), hydrogen peroxide
(H2O2), hypochlorous acid (HOCl), singlet oxygen (1O2), phenoxyl radicals (PhO•), lipid
peroxides (ROOH), hydroxyl radical (OH•), nitric oxide radical (NO•), and peroxynitrite
(ONOO−) (Couret and Chang, 2016). These ROS or free radicals have an unpaired electron,
making them highly reactive, causing oxidative damage to various biomolecules within cells
such as proteins, lipids, and nucleic acids. HIV induces OS by deregulating the OS pathway,
with an increase in ROS production by inducing mitochondrial dysfunction. In an HIV infected
cell, there is an increased concentration of ROS, due to viral envelope and functional proteins
like gp120, Tat, Nef, viral protein R (Vpr), and reverse transcriptase (RT) (reviewed in Ivanov
et al., 2016). The envelope protein gp120 enhances the intracellular level of ROS through the
upregulation of cytochrome P450 2E1 (CYP2E1), proline oxidase (POX), and activation of
NOX2 and NOX4 (Shah et al., 2013) (see Figure 1).
An experiment on immortalized endothelial cell lines from rat brain capillaries showed that
HIV proteins gp120 and Tat significantly decrease the level of various intracellular antioxidant
enzymes like glutathione (GSH), glutathione disulfide (GSSG), catalase (CAT), glutathione
peroxidase (GPx), and significantly reduce GSH/GSSG ratio. These observations clearly show
that HIV induces an OS in immortalized BBB endothelial cells, which can be involved in
developing dementia in AIDS patients. (Price et al., 2005). HIV-1 Tat freely enters the neuron
cell membrane and increases the level of lipid peroxidation by producing ROS, O2- and H2O2.
HIV Tat also activates inducible nitric acid synthase (iNOS) to generate nitric oxide (NO),
which binds with O2• − to yield a highly reactive ONOO-. This can damage neurons and lead to
cognitive dysfunction (reviewed in Kim et al., 2015). In another study, while elucidating the
stress pathway of neuropathogenesis, in patients with HIV-1 induced dementia, it was found
that HIV-1 protein Vpr, induces OS within microglia cells via accumulation of hypoxia-
inducible factor-1α (HIF-1α). The HIF-1α accumulation was due to a Vpr induced increase in
reactive oxygen species through increased production of H2O2, which also influences HIV-
1gene expression and deregulates multiple host cellular pathways (Deshmane et al., 2009) (see
Figure 1).
HIV-1 small protein Nef, which is expressed abundantly in astrocytes was studied by using
primary human fetal astrocytes (PHFAs), which utilizes an adenovirus. One of the findings of
the study shows that HIV-1 Nef released in extravascular vesicles (EVs), induces OS, as
indicated by a decrease in glutathione levels, which may contribute to the Nef-associated
neurotoxicity (Saribas et al., 2017) (see Figure 1). A study demonstrated for the first time that

the HIV-1 RT enzyme induces OS in human embryonic kidney cells (HEK293) (Figure 1).
Quantitative RT-PCR of RT in HEK293 showed a 10 to 15--fold increase in transcription of
the phase-2 detoxifying enzymes like human NADPH: quinone oxidoreductase (Nqo1) and
heme oxygenase-1 (HO-1), through the Nrf2/ARE pathway of OS. Further, ROS and Nrf2/ARE
modulate the ability of RT genes to induce Th1-type of the immune response in mice
(Isaguliants et al., 2013). OS stress triggers apoptosis of CD4+ cells, which increases the viral
replication by consuming the antioxidant enzymes like glutathione oxidase (Sandstrom et al.,
1998). Further, OS also increases HIV replication and amount of cytokine-like tumor necrosis
factor-alpha (TNF-α), via activation of nuclear factor-kappa binding (NF-κB) and by indirect
stimulation of genes, further promoting OS (Okechukwu, 2015). The mechanism can be
explained as follows (Figure 1): An inhibitory factor, I-κB, retains NF-κB in the cytoplasm,
ROS releases I-κB from NF-κB, the free NF-κB translocates into the nucleus, where it binds to
DNA and stimulates transcription of a large variety of genes, including cytokines, like TNF-α
and IL-1, cell adhesion molecules, hematopoietic growth factors, and promoter regulatory
regions of HIV-1 and HIV-2, thus increasing the viral replication in the infected cells (Allard,
2002).
2.2. Antiretroviral Therapy and Oxidative Stress
Antiretroviral therapy which is popularly known as HAART uses a combination of drugs,

like nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase
inhibitors (NNRTIs), protease inhibitors (PIs), integrase inhibitors (INIs), fusion inhibitors
(FIs) and chemokine co-receptor type-5 (CCR5). The decision to start HAART in an AIDS
patient depends on the WHO recommended guideline, which considers CD4+ cells count of less
than 350 cells/µl as a parameter for starting HAART. However, one of the major limitations of
HAART is the induction of OS in AIDS patients. The majority of oxidative reactions in a cell
are taking place in the mitochondria which contain DNA pool (mtDNA) maintained by DNA
polymerase-γ enzyme (Lee et al., 2003). The mtDNA encodes proteins responsible for electron
transport complexes of oxidative phosphorylation (OXPHOS). The phosphorylated-NRTI
inhibits a polymerase-γ enzyme that leads to depletion of mtDNA, altering OXPHOS protein
synthesis, energy deprivation, and tissue dysfunction, which contributes in ROS production
leading to OS (Valle et al., 2013) (see Figure 1).
A clinical investigation of 116 HIV-1 infected patients, which included 86 HAART treated
patients, 30 untreated patients and 46 patients as negative controls. The study clearly showed
that there was a significant increase in serum ROS and decrease in plasma total antioxidant
level. Further, it was also observed that patients who strictly adhered to the HAART therapy
displayed a higher level of OS compared to patients with intermittent HAART adherence
(Mandas et al., 2009). To understand the role of OS on progression of disease in HIV infected
patients receiving ART, a study was carried out on 35 HIV infected participants, eighteen on
d4T+3TC+EFV (stavudine + lamivudine + efavirenz), eight on AZT+3TC+EFV (zidovudine
+ lamivudine + efavirenz), nine subjects without ART, 20 HIV-negative and normal subjects
as controls. In the above 12-month study, it was observed that patients on HAART therapy
showed a significant increase in activities of glutathione peroxidase with a significant reduction
in glutathione reductase, which is a clear indication of increased OS in the patients on HAART
(Sundaram et al., 2008).

Figure 1. The mechanism of oxidative stress in AIDS and antioxidant defense. It explains various
mechanisms involved in human immunodeficiency virus (HIV) induced oxidative stress (OS) through
structural and functional proteins. This has been summarized as follows: The viral envelope
glycoprotein (gp120) enhances the intracellular level of reactive oxygen species (ROS) through
upregulation of cytochrome P4502E1 (CYP2E1), proline oxidase (POX), and activation of NADPH
oxidase (NOX2 and NOX4). Trans-activator of transcription (Tat) of HIV stimulates proapoptotic
mitochondrial proteins like Bcl-associated X protein (Bax), Bcl-2-like protein 11 (BIM), and B-cell
lymphoma-extra large (Bcl-xL) to release mitochondrial cytochrome-3 which in turn stimulates
procaspase-9 to trigger apoptosis. Tat is also involved in OS through lipid peroxidation and cognitive
dysfunction by activating inducible nitric acid synthase (iNOS) in neurons leading to the generation of
highly reactive ONOO-, which damages neurons. Viral protein R (Vpr), follows the same pathway as
that of Tat to induce apoptosis. Vpr also damages microglia by inducing OS through hypoxia-inducible
factor-1α (HIF-1α). HIF-1α accumulation stimulates HIV-1gene expression and deregulates multiple
host cellular pathways viz. pyruvate kinase muscle isozyme M2 (PKM2) and signal transducer and
activator of transcription (STAT) (Deshmane et al., 2009). HIV-1 negative regulatory factor (Nef)
released by astrocytes in extravascular vesicles (EVs) induces OS, which is indicated by a decrease in
glutathione levels, leading to the Nef-associated neurotoxicity (Saribas et al., 2017). HIV-1 reverse
transcriptase (RT) enzyme induces OS in human embryonic kidney cells (HEK293) (Banerjee et al.,
2010, Ivanov et al., 2016). Moreover, OS also stimulates viral replication via phosphorylation of IκB by
IκB kinase (IKK) resulting in activation of nuclear factor kappa B (NF-κB), which in turn increases
expression of cytokines like tumor necrosis factor (TNFα) and interleukin (IL) (Okechukwu, 2015).
Use of antiretroviral therapy (ART) also leads to OS, which is explained by examples like the
phosphorylation of nucleoside reverse transcriptase inhibitors (NRTIs) inhibits a polymerase-γ enzyme
that leads to depletion of mtDNA, altering oxidative phosphorylation, protein synthesis, energy
deprivation, and tissue dysfunction, which contributes in ROS production leading to OS (Valle et al.,
2013). Non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz (EFV) also induces OS via
mitochondrial BAX pathway. HIV induced OS activates antioxidant enzymes such as superoxide

dismutase (SOD), catalase (CAT), glutathione (GSH) and glutathione peroxidase-1(GPX-1), which
neutralize OS and as a result get exhausted (Okechukwu, 2015). Antioxidant supplements like vitamins,
minerals, amino acids, and herbs can replenish the antioxidant defense and counter the HIV induced OS
(Chan and Collins, 1997), which can increase chances of survival of cells (Some parts of the figure
have been adopted from www.googleimage.com, navalwiki.info, www.cronodon.com, and e-Enzyme-
HIV Virus Research).
A study was designed to link HIV associated neurocognitive disorder (HAND) with ART,
where the effects of NNRTI efavirenz (EFV) on neural stem cells (NSCs), and C57BL/6J mice
were tested (Jin et al., 2016). Both, in vitro and in vivo experiments showed that EFV, decreases
the proliferation of NSC by activation of OS pathway (reduced ATP storage and mitochondrial
membrane potential, MMP), leading to upregulation of Bcl-associated X protein (Bax) and
active caspase-3, with promotion of p38 mitogen-activated protein kinase (MAPK)
phosphorylation causing Bax induced apoptosis (Ghatan et al., 2000) (Figure 1). The above
study shows that EFV has the ability to cause oxidative stress, which can be directly linked to
Bax induced apoptosis, a possible mechanism for ART-induced HAND (Jin et al., 2016). HIV-
1 PIs are another group of drugs that can be used in combination with NRTIs and NNRTIs, but
these drugs also known to have many adverse effects called as metabolic syndrome, which
includes lipodystrophy, insulin resistance syndrome (IRS) and cardiovascular dysfunction. In
a study performed by Chandra et al., (2009), rat pancreatic beta cells (INS-1), were exposed to
PIs like, nelfinavir (5-10 µM), saquinavir (5-10 µM) and atazanavir (8-20 µM). All the PIs
decreased glucose-stimulated insulin release from INS-1, whereas nelfinavir significantly
increased ROS production and decreased the cytosolic, but not mitochondrial SOD. The above
study was able to establish an important link between PIs induced IRS and OS, which was
reversed by antioxidants thymoquinone and black seed oil. On the other hand, a comparative
study on INIs (darunavir, raltegravir and rilpivirine) and efavirenz, using human
hepatoblastoma Hep3B cells and cerebral cortex neurons from rat fetuses, showed that INIs
lacked the ability to alter the mitochondrial function, and cause cytotoxicity and oxidative and
endoplasmic reticulum stress (Blas-Garcia et al., 2014). Pharmacological profile of FI,
enfuvirtide (Hardy and Skolnik, 2004) and CCR5 inhibitor, maraviroc (Cooper et al., 2014)
revealed that these drugs have a good safety profile compared to other antiretroviral drugs.
3. ROLE OF ANTIOXIDANTS IN HIV/AIDS

Antioxidants are an integral part of the cellular defense mechanism. These are the
molecules that neutralize or quench free radicals and delay or decrease cellular damage (Nimse
and Pal, 2015).
As discussed so far, OS is not only induced by HIV, but the treatment of AIDS also induces
significant OS in a cell leading to various deleterious effects on the body like wasting,
opportunistic infections, neurocognitive disorders, HIV replication, cytotoxicity, etc (Valle et
al., 2013). Apart from conventional HAART therapy for management of AIDS, other non-
conventional approaches have been explored by many researchers and clinicians, for example
the use of antioxidants as an adjunct to HAART for effective treatment of AIDS and to improve
life expectancy of the infected patient. In context to HAND, antioxidants target upstream and
downstream pathways (Uttara et al., 2009). The upstream antioxidant therapy involves
preventive treatment by decreasing generation of free radicals with metals like iron (Fe) and

maintaining normal cellular metabolism, for example, vitamin E can prevent the neurotoxicity
induced by CSF of HIV patient with dementia. Whereas, the downstream antioxidant therapy
covers post oxidative stress events like inflammation, for example, non-steroidal anti-
inflammatory drugs (NSAIDS), were found to decrease the infiltration of macrophages and can
block the inflammatory cascade induced by oxidative stress (Louboutin and Strayer, 2014).
3.1. Role of Enzymatic Antioxidants in Oxidative Stress Induced

by HIV/AIDS

In humans, based on the co-factor they harbor, superoxide dismutase (SOD) is of three
types, cytosolic Cu, Zn-SOD, mitochondria (Mn-SOD) and extracellular SOD (ECSOD)
(Okechukwu, 2015). The antioxidant action of SOD is in a ping-pong manner, where there are
sequential oxidation and reduction of transition metal ion with simultaneous oxidation and
reduction of superoxide radical (Abreu and Cabelli, 2010). The main role of SOD is to
neutralize the O2• − produced in the cytoplasm, mitochondria and endoplasmic reticulum of the
cell. SOD acts by catalytically converting O2• − to O2 and hydrogen peroxide (H2O2) (Figure 1
and Table 1). However, SODs are not only antioxidants but can also be pro-oxidants as
dissociation of O2• − produces H2O2, which is toxic to cells. So, the presence of other antioxidant
enzymes like CAT and GPx becomes necessary (Carillon, et al, 2013).
HIV infection expresses Tat protein to suppress the expression of Mn-SOD in the cell and
to reduce the antioxidant defense against TNF (Okechukwu, 2015). As HIV depletes the stores
of antioxidants like SOD, micronutrients can be given to compensate for the cellular loss of
antioxidants. In a study, Lecithinized SOD (PC-SOD) was found to inhibit HIV-1 and 2 in MT-
4 cell types; PC-SOD does not inhibit viral enzymes like RTs, INs, and PIs (Table 2). Thus, the
study tries to convey that there may be another mechanism for the action of PC-SOD such as,
inhibition of the OS-induced depletion of sulfhydryls, which causes diminished antioxidant
defense in HIV/AIDS patients. (Premanathan et al., 1997).
Table 1. Classification of Antioxidants (Nimse and Pal, 2015)
Based on Their Activity Mechanism of Action Examples

Enzymatic Breaks down and removes free radicals. Superoxide dismutase (SOD),
Enzymes convert dangerous oxidative by- catalase (CAT), and glutathione
products to H2O2 and then to H2O. (GSH)
Non-enzymatic Interrupt free radical chain reactions Plant polyphenol, carotenoids, and
GSH, vitamin C and E
Based on Their Solubility Location Examples
Water soluble They are located in the cellular fluids vitamin C
such as cytoplasmic matrix or cytoplasm
Lipid soluble Located in cell membranes Carotenoids, and lipoic acid
vitamin E
Based on Their Size Mechanism of Action Examples
Small molecular size ROS is neutralized by the process called Carotenoids, and GSH vitamin C
antioxidants free radical scavenging and E
Large molecular size ROS are absorbed and prevent the attack SOD, CAT, and GSH
antioxidants on proteins

3.1.2. Catalase
Catalase (CAT) is an enzyme consisting of tetrameric heme with four identical
tetrahedrally arranged subunits of 60 kDa (Ścibior and Czeczot 2006). All cells contain CAT,
which acts by neutralizing the toxic H2O2, which is produced as a result of redox reaction by
SOD to counter the O2• −. H2O2 is detoxified in two steps: in the first step, molecule of H2O2
oxidizes the heme to an oxyferryl species. Then one oxidation equivalent is removed from iron
and one from the porphyrin ring to generate porphyrin cation. In the second step, hydrogen
peroxide molecule produces a molecule of oxygen and water by acting as a reducing agent to
regenerate the resting state enzyme (Figure 1) (Vašková et al., 2012). As HIV infection
progresses the catalase activity also increases with an increase in OS and subsequent
antioxidant defense. Catalase activity has also been related to HIV disease progression and
serves as a marker enzyme, where it increases the level of GSH, SOD and other antioxidants
(Table 2) (Pasupathi et al., 2008).
3.1.3. Glutathione System

Glutathione (GSH), a tripeptide composed of amino acids like L-cysteine, L-glutamic and
glycine, is a water-soluble and most abundant cellular thiol, providing antioxidant defense by
neutralizing toxic peroxides (Meschino, 2009). Evidence suggests that the depletion of GSH
below a critical level in the liver is an important sign of the conversion of HIV into serious
problems and complications of AIDS (Shriner and Goetz, 1992). A clinical study directly links
the lower survival rate of HIV patients (2-3 years) with low GSH levels in CD4+
(Table 2). The study also concludes that the NAC supplement is able to increase GSH levels in
subjects infected with HIV, which can be associated with longer survival rates of HIV patients
(Herzenberg et al., 1997). Advance HIV patients with systemic deficiency of GSH were given
a cysteine-rich supplement of whey proteins. A short-term oral administration of whey proteins
showed an increased plasma level of GSH (Micke et al., 2001).
3.1.4. Glutathione Peroxidase 1

Glutathione peroxidase 1 (GPx-1) is an enzyme containing selenium. Though CAT is a
major enzyme involved in H2O2 metabolism, GPx-1 also metabolizes a low level of H2O2 in
cells, while playing a major detoxifying role in RBCs. Further, GPx-1 not only metabolizes
H2O2 but also various types of organic peroxides, like long-chain fatty acid peroxides and
cholesterol (Arthur, 2000). A study was carried out on cell line 8E5, which is a chronically HIV
infected human T cell line with extreme susceptibility to apoptosis induced by H2O2, due to a
deficiency of catalase associated with HIV infection. The study showed that due to high H2O2
susceptibility, the HIV- infected cells are not able to convert 15-hydroperoxyeicosatetraenoic
acid (15-HPETE) to 1S-hydroxyeicosatetraenoic acid (15-HETE), as a result oxidized lipids
accumulate within the cells, leading to apoptosis and CD4+ cell depletion, which has been
directly correlated with GPx-1 deficiency (Table 2) (Sandstrom et al., 1994).
3.1.5. Glutathione S-Transferase

Glutathione S-transferase (GST) is a widely distributed enzyme and virtually present in
every living organism. The major role of GST in a cell is to detoxify xenobiotics and
endogenous electrophilic compounds by catalyzing the conjugation of electrophile with the
GSH, thus providing protection to the cell from environment and OS (Eklund, 2006) (Table

2). However, an oral supplement of GST to 42 healthy volunteers did not have any significant
effect on the biomarkers of OS (Allen and Bradley, 2011).
3.1.6. Glutathione Reductase

Glutathione reductase (GR) is a homodimeric enzyme belonging to the family of
flavoprotein disulfide oxidoreductases. It is a ubiquitous enzyme catalyzing the reduction of
oxidized disulfide (GSSG) to GSH (Carlberg and Mannervik 1985). A US patent application
claims, GR used for the preparation of medicament can also be used for therapy and prophylaxis
of AIDS. The major finding in the patent application shows that GR is able to inhibit the viral
cycle and interfere with HIV replication. GR can be used advantageously in combination with
antiretroviral drugs like NNRTIs, NRTIs, INIs and PIs (Ansovini, 2004) (Table 2).
Table 2. Enzymatic defense in HIV/AIDS
Enzyme Defense in HIV/AIDS References

Superoxide  HIV expressed Tat protein suppresses cellular Mn-SOD expression Okechukwu,
dismutase and reduces the antioxidant defense against TNF 2015
 Lecithinized SOD found to inhibit HIV-1 and 2 in MT-4 cell types Premanathan, et
may be due to inhibition of OS al., 1997
Catalase  Serves as a marker enzyme in HIV, increases glutathione, SOD and Pasupathi et al.,
other antioxidants 2008
 CAT level increases with HIV infection and resulting OS
Glutathione  Glutathione: The survival rate of HIV patients is low (2-3 years) Herzenberg et
system with low levels of GSH in CD4+ al., 1997
 Glutathione peroxidase: Study on cell line 8E5 shows that oxidized Sandstrom et
lipids get accumulated in the cell leading to apoptosis with CD4+ al., 1994
cell depletion, which is directly correlated with GPx-1 deficiency
 Glutathione transferases: Detoxifies xenobiotics and endogenous Eklund, 2006
electrophilic compounds by catalyzing the conjugation of
electrophile with the GSH, thus providing protection to the cell from
environment and OS
 Glutathione reductase: Inhibits the viral cycle and interferes with Ansovini, 2004
HIV replication, can be used advantageously in combination with
antiretroviral drugs
3.2. Nutraceuticals as Antioxidants for HIV/AIDS Therapy
In 1989, the term nutraceutical (functional food), was coined by Stephen Defelice from the
words ‘nutrition’ and ‘pharmaceutical.’ According to Defelice nutraceuticals are food or part
of food, which provide medical and health benefits, including the prevention and treatment of
diseases (Srivastava et al., 2015).
3.2.1. Nutrients as Antioxidants for HIV/AIDS Therapy

Nutritional deficiencies are commonly observed in HIV/AIDS patients. HIV infection
leads to heavy loss of micronutrients like vitamins A, C, E and B-complex, and elements such
as selenium and zinc (Zn). Deficiency of these micronutrients leads to OS, loss of immunity

and acceleration of HIV replication (Gedle, 2015). The micronutrients in a cell get exhausted
in countering the OS induced by HIV. Many studies have concluded that nutritional
supplementation can strengthen the immunity, replace lost micronutrients, counter OS, and
decrease the severity or impact of opportunistic infection in AIDS (Oguntibeju et al., 2009).
However, mega dosing of micronutrients like vitamin A and C and minerals Zn and selenium
can lead to adverse effects. Therefore, certain guidelines should be followed before initiating
micronutrient supplements such as, ESPEN Guidelines on Enteral Nutrition: wasting in HIV
and other chronic infectious diseases (Ockenga et al., 2006).
3.2.1.1. Vitamin E
Vitamin E, a fat-soluble vitamin is composed of several tocopherols and tocotrienols, with
α-tocopherol as the most active form. The primary dietary source of vitamin E is nuts,
sunflower seeds, wheat germ, vegetables and seed oils like soybean, safflower, and corn (Maria,
2010). As vitamin E is lipophilic in nature, it is found in the cellular membrane, where it binds
with free radicals to protect the membrane from oxidative damage. Several studies on
HIV/AIDS patients have shown a low plasma level of vitamin E, which has been correlated
with a decline in immune response with opportunistic infections because of increased levels of
ROS such as H2O2 (Kashou and Agarwal 2011). Further, vitamin E supplementation in a mice
model with a murine version of AIDS improved immune response by balancing cytokines like
IL2, interferon gamma and tumor necrosis factor (reviewed in Chan and Collins, 1997). While
analyzing serum levels of antioxidant vitamins and minerals in HIV positive subjects, living in
Sokoto, Nigeria, there was a progressive decline in vitamin E in the serum of HIV subjects,
when compared to control. The study also showed that the risk of progression of HIV to AIDS
was decreased by doubling the dose of vitamin E (Table 3) (Bilbis et al., 2010).
In contrast to the above studies on vitamin E, a study on 67 Kenyan women, showed that
vitamin E supplements may have an adverse effect on HIV patients, by accelerating HIV
disease progression and increasing the mortality rate. The pre-infection level of vitamin E has
been attributed to a decrease in antiviral chemokine RANTES, which leads to increased
synthesis of viral CCR5 on the surface of HIV infected CD4+ cells. This further facilitates HIV
entry and replication (Graham et al., 2007). However, the role of vitamin E as an antioxidant
in HIV/AIDS looks to be promising despite some contradictory studies raise some doubts on
the utilization of vitamin- E as an adjunct for AIDS therapy.
3.2.1.2. Vitamin A and Beta-Carotene

Vitamin A is a lipid-soluble vitamin stored in the liver and divided into two classes:
preformed vitamin and provitamin carotenoids. Vitamin A is found in liver, milk and animal
foods and absorbed in the form of retinol whereas, provitamin carotenoids are found in colorful
fruits and vegetables. They can be converted to vitamin A (Kashou and Agarwal, 2011).
However, vitamin A and carotenoids are antioxidants and their role in the management of
HIV/AIDS remains controversial. As per a report by WHO, supplementation with vitamin A in
malnourished HIV-infected individuals corrected the deficiency, but how much this contributes
to restoring immune function remains unclear (Annan, 2011).
Further, on the basis of various clinical studies, WHO has not recommended use of vitamin
A in pregnant women infected with HIV, as vitamin A has not been able to prevent mother to
child transmission (MTCT) of HIV, instead some studies on vitamin A have even shown to
increases MTCT of HIV (WHO, 2011). On the other hand, an earlier study on 52 HIV positive

patients showed that the supplementation of beta-carotene (60 mg/day), improved overall
oxidative status without affecting the CD4+ cell count (Constans et al., 1996). Interestingly, in
a clinical study on a placebo-controlled group of 21 volunteers with HIV; it was found that
beta-carotene supplementation significantly increased the total white cell and CD4+ count with
an increase in CD4/CD8 ratio (Coodley et al., 1993) (see Table 3).
Table 3. Role of nutraceuticals as antioxidants in HIV/AIDS
Nutraceuticals in HIV/AIDS References

Vitamins Bilbis et al., 2010
 Vitamin E: Risk of progression of HIV to AIDS can be decreased by a
double dose of vitamin E
 β-Carotene: β-carotene supplement (60 mg taken daily), improved overall Constans et al., 1996
oxidative status, with an increase in total leukocyte count, CD4+ count and Coodley et al., 1993
CD4/CD8 ratio
 Vitamin A: Used to correct a deficiency in malnourished HIV-infected Annan, 2011
individuals. On the other hand, vitamin A is not recommended by WHO in WHO, 2011
HIV infected pregnant women as it increases MTCT of HIV.
 Vitamin C: Vitamin C acts by enhancing O2• − anion and OH• radical Tang et al., 1993
scavenging, and boosting immunity. Kashou et al., 2011
 Higher intake of vitamin C may be linked with lower risk of HIV to AIDS
progression
 Vitamin D: Lower level of vitamin D has been observed in HIV infection Kivuyo, 2011
Minerals Batterham et al., 2005
 Selenium: Supplementation leads to significant improvement in oxidative Hurwitz et al., 2007
defense. A daily dose of selenium (200 μg) for 9 months elevates the serum
level of selenium and blocks the progression of HIV-1.
 Zinc: Immune boosting effect of zinc sulfate, which significantly improved Asikin et al., 2012
the CD4+ cell count. Zn + AZT therapy to stage 3 and 4 AIDS patients, Mocchegiani, 1995
maintains weight and increase CD4+ cell count.
 Amino acid Wu et al., 1989
 N-Acetylcysteine: It improves the ability of cells to produce T- cell
colonies in individuals with AIDS
Other Dave, 2011
 α-Lipoic acid: It can recycle GSH thus, improving the body’s own
antioxidant defense in HIV infected men and protecting nerves from
inflammation and oxidative damage induced by HIV
Herbs Winter et al., 2014
 Spirulina is an effective antioxidant in HIV
 Plectranthus barbatus, plant extract showed an in vitro and in vivo anti-HIV Kapewangolo et al.,
activity due to its antioxidant and anti-inflammatory action 2013
Phytochemicals Cohly et al., 2003
 Turmerin at a dose of 80 ng/ml + AZT inhibited HIV infection by 26% and
increased CD4+ count and has an anti-proliferative action on HIV infected
lymphocytes

3.2.1.3. Vitamin C
Vitamin C or ascorbic acid is a water-soluble antioxidant, important for normal growth of
body tissues and their repair. Vitamin C shows O2• − and OH• radical scavenging, and immune
boosting effects in the body (Kashou and Agarwal, 2011) (see Table 3). The effectiveness of
vitamin C in HIV/AIDS has been observed in the in vitro experiment, where it inhibits p24
antigen levels by 90%, and reverse transcriptase enzyme by more than 99%, but the same effect
has not been significantly reflected clinically.
In HIV the patients’ ascorbate level was depleted, though dietary intake was adequate,
which shows that vitamin C utilization in HIV infected cell increases to counter the oxidative
stress induced by the infection. Therefore, a higher intake of vitamin C can help to prevent
oxidative damage and maintain normal immunity (Stephensen et al., 2006). A study on
Canadian patients showed that vitamin C and E supplements decrease OS and severity of HIV
infection (Allard et al., 1998). Whereas, an observational study in US men demonstrated that a
higher intake of vitamin C can lower the risk of progression of HIV to AIDS (Tang et al., 1993)
(Table 3). The results from these interventional studies could not be completely credited to
antioxidative components of the supplement, but micronutrients can undress the antioxidant
deficiencies and diminish the severity of HIV infection (Stephensen et al., 2006). Further,
mega-dosage of vitamin C may cause kidney stones, diarrhea, acid/base imbalance, promote
the destruction of vitamin B12 and interfere with the action of vitamin E (Kashou and Agarwal,
2011).
3.2.1.4. Vitamin D
Vitamin D is a membrane antioxidant with active metabolites like 1, 25-
dihydroxycholecalciferol (1,25-(OH)2D), vitamin D2 (ergocalciferol) and 7-
dehydrocholesterol (pro-vitamin D3). All these metabolites of vitamin D inhibit iron-dependent
liposomal lipid peroxidation (Wiseman, 1993). Vitamin D deficiency augments the production
of advanced oxidation proteins, nitric oxide metabolites, oxidative stress index, and decrease
total radical-trapping capacity, which compromises the redox state of the patients (Preedy and
Watson, 2018).
Various clinical studies have tried to correlate 1, 25-(OH)2D, a deficiency with HIV
infection (Table 3). The studies suggested that the low level of 1, 25-(OH)2 D during HIV
infection can be due to increased utilization of 1,25-(OH)2 D for maturation and proliferation
of T lymphocytes. Advanced stage of HIV disease was associated with lower serum 1,25-(OH)2
D concentration, possibly due to a decrease in process of hydroxylation of 25-(OH)-D to
1,25(OH)2D in the macrophages (Kivuyo, 2011). Though other studies have shown that
hypovitaminosis D is not only due to HIV infection, there may be other reasons for the
deficiency as well, such as high intensity of solar radiation, the location of the study site, and
low latitude (Canuto et al., 2015).
3.2.1.5. Selenium
In 1817, Jons Jacob Berzelius discovered selenium (Se), a toxic metalloid and named it
after Selene, the Greek goddess of the moon (Schomburg, 2007). The best-known example of
selenoenzyme is GPx, which with other enzymes such as CAT, and SOD, neutralizes the
oxidative stress in the cells by breaking H2O2 and another ROS (Savitha, 2014). As discussed
earlier, HIV infection adversely affects the antioxidant mechanism of the infected CD4+ cell.

Researchers have found that the presence of selenium increases the level of
selenoproteins/enzymes like GPx, thioredoxin reductase, phospholipids, and hydroperoxide in
the normal T cell, but the same enzymes are depleted when T cell gets infected with HIV
(reviewed in Stone et al., 2010). Several preclinical and clinical studies on selenium have been
done to know its utility as an antioxidant in HIV/AIDS patients. In an in vitro study, the latently
infected T lymphocytes activated HIV replication through the actions of NFκB. Antioxidant
enzymes like, CAT and GPx, which require selenium decrease the NFκB, mediated activation
of HIV replication (Sappey et al., 1994).
Further, a clinical study using an antioxidant regimen (vitamin A as beta-carotene 5,450
IU, vitamin C 250 mg, vitamin E 100 IU, selenium 100 µg, and coenzyme Q10 50 mg), showed
a significant improvement in antioxidant measures like, selenium, GSH, GPx, and lipid
peroxides in HIV/AIDS infected men (Batterham et al., 2005). A comprehensive placebo-
controlled, randomized, double-blind trial was conducted in a community-based cohort study
in HIV-infected men and women to observe the effect of a daily dose of selenium on the CD4+
cell count. The result from the study showed that a daily dose of selenium (200 μg) for 9 months
elevated the serum selenium level and suppressed the HIV-1 progression and viral load (Table
3). The author suggested that the above effect is because of the HIV-1, which requires selenium
from host to produce its own selenoenzymes, thereby decreasing selenium resources leading to
OS in the host cell. The study supports the use of selenium as a simple, inexpensive, and safe
adjunct therapy for HIV/AIDS (Hurwitz et al., 2007). From the above studies, selenium seems
to be an attractive micronutrient for adjunct therapy of HIV/AIDS, but the use of selenium is
having various limitations (Shivakoti et al., 2014).
In Kenya, a randomized controlled trial was carried out by McClelland et al., (2004), where
400 participants received a supplement containing B-complex vitamins, vitamins C and E, and
200 μg of selenium over 6 weeks. The study showed that, HIV shedding was increased 2.5-fold
in infected cells and HIV-RNA in vaginal secretions was increased by 0.37 log10 units, both
these effects are the potential adverse reactions of the given micronutrient regimen. Moreover,
dose of selenium should not exceed 1mg/day, although some nutritionists have cautioned
against use of selenium above 250μg/day. Excess intake of selenium is immunosuppressive
with symptoms, which include vomiting, diarrhea, and skin lesions, loss of hair and nails, and
nervous system disorders (Chan and Collins, 1997). Hence, the use of selenium as a
micronutrient for HIV/AIDS needs to be properly weighed against risk: benefit ratio. Treatment
of HIV induced oxidative stress with a regimen containing selenium should follow standard
treatment protocol and depend upon the careful judgment of the physician and severity of the
infection.
3.2.1.6. Zinc
Zinc (Zn) is a trace element and a divalent metallic cation bound to proteins inside the cells
and plasma membranes. Though Zn itself is not an antioxidant, it plays an important catalytic,
structural and regulatory role for the functioning of near about 200 Zn metalloenzymes (Table
2) (Okechukwu, 2015). Zn is an important co-factor for enzymes that maintain the cellular
antioxidant defense system. In addition to this, Zn also protects the cell against oxidative
damage by stabilizing membranes and inhibiting a pro-oxidant enzyme nicotinamide adenine
dinucleotide phosphate oxidase (NADPH-Oxidase). Further, Zn also induces synthesis of
metallothionein, which is involved in the reduction of OH• radicals and quenching ROS under
conditions inducing OS (Marreiro et al., 2017).

Zn also acts as an antioxidant by another mechanism, viz. through the expression of

glutamate-cysteine ligase, which is the rate-limiting enzyme for glutathione de novo synthesis
(Eide, 2011). After recognizing that Zn deficiency can cause serious clinical problems like
growth retardation and male hypogonadism (Prasad, 2009); several studies have been
conducted to know the biological role of Zn in the human body. In context to HIV/AIDS, Zn
deficiency has been observed in HIV infected/AIDS patients, which can contribute to the
clinical syndrome of AIDS, such as immunodeficiency, gastrointestinal malfunction with
diarrhea, impaired taste and appetite, alopecia, epithelial lesions, hypogonadism and
hypospermia (Baum, et al, 2000). Oral supplementation of Zn as an adjunct to zidovudine
therapy to stage III and IV AIDS patients has shown to maintain weight, augment CD4+ cell
count and blood levels of Zn bound thymulin (Table 3). A Zn treated group showed marked
reductions in opportunistic infections, such as Candida and Pneumocystis carinii (Mocchegiani
et al., 1995).
A hospital-based study in ICUID of Dr. Soetomo Hospital in Surabaya, Indonesia,
demonstrated that zinc sulfate in HIV/AIDS patients has an immune-boosting effect (see Table
3), with significantly improved CD4+ cell count in the treatment group compared to control
(Asikin et al., 2012). Ironically, excessive Zn has been reported to improve HIV replication by
utilization of Zn as a co-factor by HIV enzymes, like reverse transcriptase, integrase, and
protease. A daily dose of 300 mg, which is 20 times the recommended dose can result in
immune impairment, interfere with copper and iron utilization and affect HDL cholesterol
concentration and monocyte function, all of which contribute to HIV-1 disease progression.
The study also shows that a high intake of Zn can lead to faster disease progression and death
in HIV-1 infected homo/bisexual men (Baum et al., 2000). From the above studies, it can be
concluded that Zn supplementation is only beneficial in Zn deficient HIV patients rather than
Zn sufficient patients. So, while initiating Zn supplementation or including Zn with other
micronutrients for adjunct therapy of HIV/AIDS, plasma Zn concentration should be taken into
account to know the Zn deficient or sufficient state of the patient.
3.2.1.7. N-Acetylcysteine
N-Acetylcysteine (NAC) a precursor of GSSH, is the acetylated variant of amino acid
cysteine, it is also a rich source of sulfhydryl (SH) groups. NAC is a GSH regenerator and can
be utilized in disease conditions, which are characterized by decreased GSH or OS, such as
cancer, heart diseases and HIV infection (Patrick, 2000). HIV infection depletes the cysteine
and GSH level inducing OS, consequently decreasing the number of CD4+ cells. A study in
1989, by Wu et al., showed that NAC improves the capability of cells to produce T-cell
colonies, in AIDS patients (Table 3).
Further, according to a study by Herzenberg et al., (1997) low levels of GSH predict poor
survival in otherwise indistinguishable HIV infected patients (Table 2). The study suggested
that since oral administration of NAC replenishes GSH stores in the subjects, it may be used as
a supplement to improve the survival of AIDS patients. In a placebo-controlled double-blind
clinical study, NAC supplementation in seropositive HIV individuals showed increased plasma
cysteine levels, and slowed the decrease in CD4+ count, when compared with placebo
(Akerlund et al., 1996). All these observations show that NAC can be a valuable supplement in
HIV patients with low GSH levels.

3.2.1.8. α-Lipoic Acid

α-Lipoic acid (ALA) is a powerful antioxidant, which is produced in tiny amounts by the
human body. ALA is a potent antioxidant and has an ability to neutralize free radicals like,
superoxide and peroxyl, which are produced during lipid peroxidation. Dihydrolipoic acid
(DHLA), a reduced form of ALA is a more powerful antioxidant than ALA. Both ALA and
DHLA are ROS scavengers and form chelates with Cu2+, Cd2+, and Fe2+. DHLA also
replenishes GSH, vitamin E, and C by removing the ROS from these antioxidants (Ambrosi et
al., 2018). When 450 mg of ALA was given to 11 AIDS diagnosed patients for 14 days, it was
found that ALA increased total plasma thiols, GSH, ascorbate, CD4 and CD4/CD8 ratios and
decreased lipid peroxide level (reviewed in Patrick, 2000). An in vitro study demonstrated that
at a concentration of 2 mM, ALA inhibits HIV replication by blocking activation of NF-κB
(Packer and Suzuki, 1993). A US patent (No. 5,728,735) of a formulation containing R-α-lipoic
acid or S-α-lipoic acid clearly states that the formulation significantly inhibits replication of
HIV-1 and HIV-2 virus.
Further, R-α-lipoic acid or S-α-lipoic acid can be effectively combined with antiretroviral
drugs, to improve survival rates of HIV infected patients (Ulrich et al., 1998). So, ALA has
proved to be a safe and effective micronutrient which can be used in combination with ART to
treat HIV infection effectively.
3.2.1.9. Herbs and Phytochemicals as Antioxidants for HIV/AIDS Therapy

Historically, herbs have been a treasure for treating many diseases, and HIV/AIDS is not
an exception. As antioxidant constituents are an integral part of any herb, many studies have
been done on the herbs to prove their utility as an adjuvant for the management of HIV/AIDS.
The following discussion is an attempt to summarise a few important studies done on the herbs
for treating HIV. Hoodia gordonii an herb found in South Africa exhibited significant
antioxidant activity in DPPH and reducing power assay. The ethyl acetate and ethanol extracts,
also showed significant inhibition of the HIV-1 RT enzyme in a colorimetric assay, having IC50
values 73.55 ± 0.04 and 69.81 ± 9.45μg/mL respectively, when compared with standard drug
doxorubicin (Kapewangolo et al., 2016). A clinical study involving 73 HIV-infected women
was carried out by using Arthrospira platensis, popularly known as Spirulina (cyanobacterium,
which is rich in proteins and micronutrients). The pre-HAART HIV-infected population of
adult females were administered supplementation of 5g/day of Arthrospira platensis for 3
months. Although Arthrospira platensis supplementation had no significant effect on viral load
and CD4+ count, the total antioxidant status in blood serum (TAOS), was significantly
improved (Table 3). The above study supports the role of Arthrospira platensis as an effective
antioxidant, which can be used as an adjunct for HIV/AIDS therapy (Winter et al., 2014).
Plectranthus barbatus, a widely used medicinal plant in African countries was investigated
by in-vitro assays for anti-HIV, antioxidant, and anti-inflammatory activity (Table 3). Results
from the study showed an in vitro, anti-HIV activity of the plant extract, which can be due to
direct action as well as through protective antioxidant and anti-inflammatory response.
(Kapewangolo et al., 2013). Owing to the excellent antioxidant property of turmeric (T) and its
derivatives like lipid-soluble curcumin (Cu) and water-soluble extract turmerin (Tm), studies
have been done to examine their possible role in the management of HIV. The study clearly
showed that Tm at a dose of 80 ng/ml with AZT inhibited HIV infection by 26% and increased
CD4+ count; and also that it has an anti-proliferative action on HIV infected lymphocytes (Table
3). However, the authors did not investigate the link between the anti-HIV activity and the

antioxidant property of turmerin (Cohly et al., 2003). Apart from these studies, there are many
other antioxidant herbs, which claim to benefit the HIV infected patient. However, these have
not been experimentally tested for HIV/AIDS patients. Few examples can be given like, a green
tea containing Chinese herb Astragalus propinquus claims to cure HIV/AIDS (Ravikumar,
2014), Herbal medicine IMOD containing Rosa canina, Urtica dioica, and Tanacetum vulgare
was found to enhance CD4+ lymphocyte count after a period of one to three months therapy
(reviewed in Saeidnia and Abdollahi, 2014). Although HIV infected patients often take herbal
remedies, their effectiveness against HIV is questionable as there are no dependable proofs to
show that a particular herb or herbal combination can cure the HIV infection. Moreover, herbal
remedies can also cause some adverse effects, for example, herbs like Astragalus, Echinacea,
and Siberian ginseng, which act as non-specific immune boosters, may enhance rather than
suppress the HIV replication. Using herbs as adjunct to the ART can also lead to many serious
drug interactions like, St John’s wort (Hypericum), an antidepressant herb reduces the level of
PI Indinavir in the body, while garlic and milk thistle (Silybum marianum) also reduce the
concentration of Saquinavir (Ladenheim et al., 2008).
4. ROLE OF ANTIOXIDANTS IN HIGHLY ACTIVE ANTIRETROVIRAL

THERAPY-INDUCED OXIDATIVE STRESS DURING
HIV/AIDS THERAPY
Once the patient moves from a stage of HIV to AIDS, a decision is taken to start highly
active antiretroviral therapy (HAART), based on CD4+ cell count. Cell count of fewer than 350
cells/µl is considered as a parameter to start HAART (WHO, 2007). HAART has been found
to be highly effective in decreasing mortalities due to AIDS and has completely transformed
the lives of HIV/AIDS patients. Although HAART is pivotal for HIV/AIDS therapy, a major
problem arising out of use of HAART is OS. To give a few examples which have been
discussed previously in the chapter, drugs used in HAART, such as NRTIs and PIs induced OS
by mitochondrial interface and activation of the P450 hepatic system, respectively (reviewed
in Mandas et al., 2009). To counter this problem associated with HAART, many studies have
been carried out on nutraceuticals to prove their utility as antioxidants to manage HAART
induced OS. A supplement containing micronutrients such as B-complex, vitamin E, C, and
NAC showed a significant improvement in the immunity of AIDS patients, on stable HAART
regimens (Patrick, 2000).
Thus, improvement in the CD 4+ count due to micronutrients may be attributed to their
antioxidant properties (Kaiser et al., 2006). OS induced by AZT damages DNA and triggers
the process of apoptosis, which is reversed by supplementation of vitamin C and E (Allard et
al., 1998). Other anti-HIV drug nelfinavir a PI, was found to induce OS by upregulating
mitochondrial uncoupling protein, affecting ATP levels in the cell. The OS induced by
nelfinavir was reversed by thymoquinone, an active ingredient of Nigella sativa oil (Chandra
et al., 2009). OS developed due to anti-retroviral drugs can be managed by use of different
antioxidants like vitamins A, E, C, along with cysteine glutathione, and whey proteins, which
are able to supply precursor for synthesis of glutathione, thus improving overall antioxidant
defense in people living with HIV/AIDS (reviewed in Sharma, 2014). Nevirapine-induced
spermiotoxicity in Sprague-Dawley rats was better managed by vitamin C compared to

kolaviron, an active component of Garcinia kola (antioxidant biflavonoid). The above effect
of vitamin C was mainly due to its antioxidant property, which was evident from the significant
increase of testicular SOD and GST activities. So, vitamin C can be used as an adjunct with
nevirapine therapy for HIV/AIDS (Ajayi et al., 2016). Supplementation of glutathione or
antioxidants like NAC can improve immunity and virological indices in HIV patients
(Sundaram et al., 2008).
5. LIMITATIONS OF ANTIOXIDANT THERAPY FOR HIV/AIDS

The patients living with HIV/AIDS and even the general public consider micronutrients
such as vitamins, minerals and other supplements as a popular complementary therapy
(Oguntibeju, 2009). Nutraceuticals or food supplements are mostly taken by AIDS patients
without any expert consultation, presuming that all that is natural is safe. On the contrary, the
micronutrients or nutraceuticals may show several adverse events in the AIDS patients and
instead of benefiting the patient, the condition of the patient can become worse. Several
examples can be given, such as Use of vitamin A by HIV positive pregnant women is not
recommended due to its teratogenic effect and increased danger of vertical i.e mother to child
transmission of HIV (WHO, 2011). Excessive doses of selenium can be immuno-suppressive,
with symptoms, which include vomiting and diarrhea, skin lesions, nervous system disorders
and loss of hair and nails. An excessive dose of Zn (300 mg/day) can result in an increased
progression of HIV-1 infection and mortality in adults with AIDS.
Currently, AIDS patients are not having any choice but to become victims of polypharmacy
leading to pill burden so, while prescribing or initiating micronutrient or any other adjunct
therapy attention should also be given to potential drug interactions, which can have deleterious
effects on the AIDS patient. For example, herbs like St John’s wort, milk thistle, and garlic
decrease the serum levels of antiretroviral drugs indinavir and saquinavir respectively. A
comprehensive survey in three specialist HIV outpatient clinics in central London was carried
out, where 293 patients received ART. The survey concluded that there is a potential health
risk to AIDS patients taking ART with complementary and alternative medicines (CAM)
(Ladenheim et al., 2008). A few examples can be cited from the study viz. Aloe vera increases
gastrointestinal transit reducing absorption leading to failure of ART (Fugh-Berman, 2000),
Ginkgo biloba (Yale and Glurich, 2005), Liquorice (Kent et al., 2002), and Cat’s claw
(Budzinski, 2000) interact with NNRTI and/or PI inhibit CYP3A4 enzyme leading to increased
risk of ART-related side effects. Use of Echinacea in HIV patient acts as a theoretical immune
stimulant resulting in increased viral load (Block and Mead, 2003).
6. RECENT DEVELOPMENTS, FUTURE OPPORTUNITIES,

AND CHALLENGES
In a quest for searching effective ways for treating HIV/AIDS and to counter the OS arising
out of infections, researchers are constantly engaged in exploring new therapies. These have
been summarized in the following discussion. A recent study on Sprague-Dawley rats showed
that steroidal glycoside ginsenoside Rb1 reversed the oxidative damage of BBB induced by a

combination of HIV1-Tat and methamphetamine. Moreover, ginsenoside Rb1 treated rats

showed an elevated level of SOD and GSH, which clearly demonstrates the antioxidant
potential of ginsenoside Rb1, which can be a therapeutic option for individuals with HIV
associated neurocognitive and neurodegenerative disorders (Li et al., 2018). However, in
another study, adult male Sprague-Dawley rats were administered HAART and alcohol which
led to severe degeneration of seminiferous epithelium, decreased semen quality and damage to
the basement membrane and interstitium. The above effect was reversed by a high dose of
virgin coconut oil (5 ml/kg/bw), with a simultaneous increase in parameters like sperm count,
hormone FSH and antioxidative level of GSH (Ogedengbe et al., 2018).
Further, randomized double-blind placebo-controlled crossover clinical study was
conducted on 92 patients, who were on 6 months ART with viral suppression. The patients
receiving 69 g of chocolate or 3 g of mate tea for 15 days showed significant improvement in
HDL-C concentrations, which may be attributed to the presence of stearic acid, polyphenols,
and theobromine in chocolate or mate tea (Souza et al., 2017). AIDS, a severe depleting
infection, due to profound immunosuppression, leads to opportunistic invasion by several
pathogens. Apart from problems like pill burden and opportunistic infections, AIDS patient
suffers from OS induced by HIV infection as well as ART, which contribute to disease
progression and death of the patient.
There are several challenges in the way of combating OS including adverse effects of
micronutrients, improper dosage schedule of the CAM, lack of individualized treatment
protocols for adjunct therapies in AIDS patient, proper optimization of the dosage forms of
CAMs, insufficient clinical data on therapeutic and safety implications of long-term use of
micronutrient supplements in AIDS patients, etc. Challenges themselves provide opportunities
and AIDS is not an exception to this fact. Antioxidants and micronutrients will be important
components of future AIDS treatment strategies. Future research in the area of antioxidant
therapy of AIDS will revolve around designing unique combinations of various macro and
micronutrients which will be individualized, optimized, and safe. Owing to ever-growing HIV
resistance against conventional HAART therapy, antioxidant adjuncts will play a pivotal role
in the mitigation of HIV/AIDS to improve longevity.
CONCLUSION
OS is a part and parcel of AIDS and has depleting effects on the patients, such as an
increase in HIV replication and disease progression leading to lower life expectancy. Hence,
antioxidant therapy becomes an obvious choice for physicians treating AIDS patients. There
are a number of CAMs available which claim to be effective antioxidants and can be used as
an adjunct with the conventional ART. However, antioxidant supplements should be used
judiciously as higher doses of supplements like selenium, Zn and vitamin A can cause serious
adverse effects and worsen the disease condition. There are also multiple factors that govern
the level of OS in HIV patients like initial micronutrient and antioxidant status, use of HAART,
the presence of opportunistic infections, etc. Thus, there is a need to device standard treatment
protocol with an individualized dosage regimen for use of micronutrients and nutraceuticals,
which can deal with HIV induced OS effectively and prevent the progression of AIDS.

REFERENCES
Abreu, I. A., Cabelli, D. E. Superoxide dismutases - A review of the metal-associated
mechanistic variations. Biochimica et Biophysica Acta. 2010; 1804: 263–274.
Ajayi, A. S., Azu, O. O., Chuturgoon A, Phulukdaree, A., Komolafe, A. O, Ofusori, A. D.,
Comparison of Antioxidant Effects of Kolaviron and Vitamin C Interventions on Testicular
Structures Following Nevirapine Administration in Sprague-Dawley Rats. Int. J. Morphol.
2016; 34(1): 102-109.
Akerlund, B., Jarstrand, C., Lindeke, B. Effect of N-acetylcysteine(NAC) treatment on HIV-1
infection: a double-blind placebo-controlled trial. Eur J Clin Pharmacol. 1996; 50: 457-
461.
Allard, J. P., Aghdassi, E., Chau, J., Tam, C., Kovacs, C. M., Salit, I. E., et al., Effects of vitamin
E and C supplementation on oxidative stress and viral load in HIV-infected subjects. AIDS.
1998; 12(13): 1653-1659.
Allen, J., Bradley, R. D. Effects of Oral Glutathione Supplementation on Systemic Oxidative
Stress Biomarkers in Human Volunteers. J Altern Complement Med. 2011; 9:17.
Ambrosi, N., Guerrieri, D., Caro, F., Sanchez, F., Haeublein, G., Casadei, D., et al., Alpha
Lipoic Acid: A Therapeutic Strategy that Tend to Limit the Action of Free Radicals in
Transplantation. Int. J. Mol. Sci. 2018; 19(102): 1-13.
Annan, R. A. Vitamin A supplementation and disease progression in HIV-infected adults.;
2011.
Ansovini R, inventor; Glutathione reductase for therapy and prophylaxis of AIDS. Perugia
patent 7045323. 2004.
Arthur, J. R. The glutathione peroxidases. CMLS, Cell. Mol. Life Sci. 2000; 57: 1825–1835.
Asikin, A, Bambang, W., Soeroso, J. Zinc Sulfate Increases Lymphocyte CD4 Count in HIV.
AIDS Patients at ICUID Dr. Soetomo Hospital Surabaya. Folia Medica Indonesiana. 2012;
48(1): 17-19.
Banerjee, A, Zhang, X., Reddy, K. M., William, B. A., Ercal, N. HIV proteins (gp120 and Tat)
and methamphetamine in oxidative. Free Radic Biol Med. 2010; 48(10): 1388-1398.
Batterham, M., Gold, J., Naidoo, D., Lux, O., Sadler, S., Bridle, S., et al., A preliminary open
label dose comparison using an antioxidant regimen to determine the effect on viral load
and oxidative stress in men with HIV/AIDS. Eur J Clin Nutr. 2005; 55(2): 107-114.
Baum, M. K., Posner, G. S., Campa, A. Zinc Status in Human Immunodeficiency Virus
Infection. J. Nutr. 2000; 130: 1421S-1423S.
Bilbis, L. S., Idowu, D. B., Saidu, Y., Lawal, M., Njoku, C. H. Serum levels of antioxidant
vitamins and mineral elements of human immunodeficiency virus positive subjects in
Sokoto, Nigeria. Annals of African Medicine. 2010; 9(4): 235-239.
Blas-Garcia, A., Polo M., Alegre, F., Funes, H. A., Martinez, E., Apostolova, N., et al., Lack
of mitochondrial toxicity of darunavir, raltegravir and rilpivirine in neurons and
hepatocytes: a comparison with efavirenz. J Antimicrob Chemother. 2014; 69: 2995–3000.
Block, K. I., Mead, M. N. Immune system effects of echinacea ginseng, and astragalus: a
review. Integr Cancer Ther. 2003; 2: 247–267.
Budzinski, J. W., Foster, B. C., Vandenhoek, S., Arnason, J. T. An in vitro evaluation of human
cytochrome P450 3A4 inhibition by selected commercial herbal extracts and tinctures.
Phytomedicine. 2000; 7: 273–282.

Canuto, J. M. P., Canuto, V. M. P., Alves de Lima MH, Silva de Omena ALC, Lima Morais
TMd, Paiva AM, et al., Risk factors associated with hypovitaminosis D in HIV/aids-
infected adults. Arch Endocrinol Metab. 2015; 59(1): 34-41.
Carillon, J., Rouanet, J. M., Cristol, J. P., Brion, R. Superoxide Dismutase Administration, A
Potential Therapy Against Oxidative Stress Related Diseases: Several Routes of
Supplementation and Proposal of an Original Mechanismof Action. Pharm Res. 2013;: 1-
11.
Carlberg, I., Mannervik, B. Glutathione reductase. Meth.Enzymol. 1985; 113: 485-490.
Chan, G, Collins, J. The Body.com. [Online].; 1997. Accessed 14 May 2018. Available from:
HYPERLINK “http://www.thebody.com/content/art13259.html?ts=pf.”
Chandra, S., Mondal, D., Agrawal, K. C. HIV-1 Protease Inhibitor Induced Oxidative Stress
Suppresses Glucose Stimulated InsulinRelease: Protection with Thymoquinone. Exp Biol
Med. 2009; 234(4): 442-453.
Chandra, S., Mondal, D., Agrawal, K. C. HIV-1 protease inhibitor induced oxidative stress
suppresses glucose stimulated insulin release: protection with thymoquinone. Exp Biol
Med. 2009; 234(4): 442-453.
Cohly, H. H. P., Asad S, Das, K. S., Angel, M. F., Rao, M. Effect of Antioxidant (Turmeric,
Turmerin and Curcumin) on Human Immunodeficiency Virus. Int. J. Mol. Sci. 2003; 4: 22-
33.
Constans, J., Delmas-Beauvieux, M. C., Sergeant, C. One-yearantioxidant supplementation
with beta-carotene or selenium forpatients infected with HIV: a pilot study. Clin. Infect.
Dis. 1996; 23: 654-656.
Coodley, G. O., Nelson, H. D., Loveless M. O., Folk, C. Beta-carotene in HIV infection. J
Acquir Immune Defic Syndr. 1993; 6(3): 272-276.
Cooper, D. A., Heera, J, Ive, P., Botes, M, Dejesus, E., Burnside, R., et al., Efficacy and safety
of maraviroc vs. efavirenz intreatment-naive patients with HIV-1:5-year findings. AIDS.
2014; 28:717–725.
Couret, J., Chang, T. L. Reactive Oxygen Species in HIV Infection. EC Microbiol. 2016; 3(6):
597–604.
Dave R. http://neuropathyandhiv.blogspot.com/. [Online].; 2011. Accessed 11 December 2018.
Available from: Hyperlink https://neuropathyandhiv.blogspot.com/search?q=Dave+2011
”\l.”XA_bhFUzbIV.
Da-Yong, L., Hong-Ying, W, Nagendra, S. Y., Bin, X., Jian, D., Ting-Ren, L. HAART in
HIV/AIDS Treatments, a Current Limitation. EC Orthopaedics. 2017; 6(4):140-146.
Deshmane, S. L., Mukerjee, R, Fan, S, Valle, L. D., Michiels, C., Sweet. T., et al., Activation
of the Oxidative Stress Pathway by HIV-1 Vpr Leads to Induction of Hypoxia-inducible
Factor1alpha Expression. J Biol Chem. 2009; 284(17):11364 –11373.
Eide DJ. The oxidative stress of zinc deficiency. Metallomics. 2011; 3(11): 1124-1129.
Eklund, B. I. Role of Multiple Glutathione Transferases in Bioactivation of Thiopurine
Prodrugs. Dissertation. Sweden:; 2006.
Friis, H. Micronutrients and HIV Infection. 1st ed. Boca Raton, Florida: CRC Press; 2002.
Friis, H. Micronutrients and HIV infection. Durban, South Africa:; 2005.
Fugh-Berman, H. Herb–drug interactions. Lancet. 2000; 355: 134–138.
Gedle, D. Effects of HIV/AIDS on Micronutrients. SM J Food Nutri Disord. 2015; 1(1): 1-2.

Ghatan, S., Larner, S., Kinoshita, Y., Hetman, M., Patel, L, Xia, Z., et al., p38 MAP kinase
mediates bax translocation in nitric oxide-induced apoptosis in neurons. J Cell Biol. 2000;
150(2): 335–47.
Graham, S. M., Baeten J. M., Richardson, B. A., Bankson, D. D., Lavreys, L., Ndinya-Achola,
J. O., et al., Higher pre-infection vitamin E levels are associated with higher mortality in
HIV-1-infected Kenyan women: a prospective study. BMC Infectious Diseases. 2007;
7(63): 1-5.
Hardy, H., Skolnik, P. R. Enfuvirtide, a new fusion inhibitor for therapy of human
immunodeficiency virus infection. Pharmacotherapy. 2004; 24(2):198-211.
Herzenberg, L. A., De Rosa, S. C., Dubs JG, al. e. Glutathione deficiency is associated with
impaired survival in HIV disease. Proc Natl Acad Sci USA. 1997; 94: 1967-1972.
Herzenberg, L. A., De Rosa, S. C. , Dubs, J. G., Roederer, M., Anderson, M. T., Ela, S. W., et
al., Glutathione deficiency is associated with impaired survival in HIV disease. Proc. Natl.
Acad. Sci. USA. 1997; 94:1967–1972.
Hughes, J. R. HIV: Structure, Life Cycle, and Pathogenecity. University of Tennessee, Honors
Thesis. Human immunodeficiency virus type 1 viral load with selenium supplementation:
a randomized controlled trial. Arch Intern Med. 2007; 167(2): 148-154.
Hurwitz, B. E., Klaus, J. R., Llabre, M. M., Gonzalez, A., Lawrence PJ, Maher KJ, et al.,
Suppression of human immunodeficiency virus type 1 viral load with selenium
supplementation: a randomized controlled trial. Arch Intern Med. 2007; 167(2): 148-154.
Isaguliants, M., Smirnova, O., Ivanov, A. V., Kilpelainen, A., Kuzmenko, Y., Petkov, S., et al.,
Oxidative stress induced by HIV-1 reverse transcriptase modulates the enzyme’s
performance in gene immunization. Hum Vaccin Immunother. 2013; 9(10): 2111–2119.
Ivanov, A. V., Valuev-Elliston, V. T., Ivanova, O. N., Kochetkov, S. N., Starodubova, E. S.,
Bartosch, B., et al., Oxidative Stress during HIV Infection: Mechanisms and. Oxidative
Medicine and Cellular Longevity. 2016;: 1-18.
Jin, J., Grimmig, B., Izzo, J., Brown, L. A. M., Hudson, C., Smith, A. J., et al., HIV Non-
Nucleoside Reverse Transcriptase Inhibitor Efavirenz Reduces Neural Stem Cell
Proliferation In Vitro and In Vivo. Cell Transplant. 2016; 25(11): 1967–1977.
Kaiser, J. D., Campa, A. M., Ondercin, J. P., Leoung, G. S., Pless, R. F, Baum, M. K.
Micronutrient Supplementation Increases CD4 Count in HIV-Infected Individuals on
Highly ActiveAntiretroviral Therapy: A Prospective:Double-Blinded, Placebo-Controlled
Trial. Acquir Immune Defic Syndr. 2006; 42(5): 523- 528.
Kapewangolo, P., Hussein, A. A,, Meyer, D. Inhibition of HIV-1 enzymes, antioxidant and
anti-inflammatory activities of Plectranthus barbatus. J Ethnopharmacol. 2013; 149(1):
184-190.
Kapewangolo, P., Knott, M, Shithigona, R. E. K., Uusiku, S. L., Schulz, M. K. In vitro anti-
HIV and antioxidant activity of Hoodia gordonii (Apocynaceae), a commercial plant
product. BMC Complementary and Alternative Medicine. 2016; 16:411.
Kashou, A. H., Agarwal, A. Oxidants and Antioxidants in the Pathogenesis of HIV/AIDS. The
Open Reproductive Science Journal. 2011; 3:154-161.
Kent, U. M., Aviram, M, Rosenblat, M, Hollenberg, P. F. The licorice root derived isoflavan
glabridin inhibits the activities of human cytochrome P450S 3A4, 2B6 and 2C9. Drug
Metab Dispos. 2002; 30: 709–715.

Kim, S. H., Smith, A. J., Tan, J., Shytle, R. D., Giunta, B. MSM ameliorates HIV-1 Tat induced
neuronal oxidative stress via rebalance of the glutathione cycle. Am J Transl Res. 2015;
7(2): 328-338.
Kivuyo, S. L. Vitamin D status among HIV infected and HIV/TB co-infected patients attending
Haydom Lutheran Hospital, Rural Tanzania. University of Oslo, Master Thesis Projects
Norway; June 2011.
Ladenheim, D., Horn, O., Werneke, U., Phillpot, M., Murungi, A., Theobald, N., et al.,
Potential health risks of complementary alternative medicines in HIV patients. HIV
Medicine. 2008; 9: 653–659.
Lee, H., Hanes, J., Johnson, K. A. Toxicity of Nucleoside Analogues Used to Treat AIDS and
the Selectivity of the Mitochondrial DNA Polymerase. Biochemistry. 2003; 42:14711-
14719.
Li, J., Zeng, B., Hu, X., Li, Z., Zhang, D., Yang, G., et al., Protective Effects of Ginsenoside
Rb1 against Blood-Brain Barrier Damage Induced by Human Immunodeficiency Virus-1
Tat Protein and Methamphetamine in Sprague-Dawley Rats. Am J Chin Med. 2018; 46(3):
551-566.
Louboutin, J. P., Strayer, D. Role of Oxidative Stress in HIV-1-Associated Neurocognitive
Disorder and Protection by Gene Delivery of Antioxidant Enzymes. Antioxidants. 2014; 3:
770-797.
Mandas, A, Iorio E. L., Congiu, M. G., Balestrieri C., Mereu, A., Cau, D., et al., Oxidative
Imbalance in HIV-1 Infected Patients Treated with Antiretroviral Therapy. J Biomed
Biotechnol. 2009;:1-7.
Maria LC. An Update on Vitamin E, Tocopherol and Tocotrienol-Perspectives. Molecules.
2010; 15: 2103-2113.
Marreiro DdN, Cruz, K. J. C, Morais J. B. S., Beserra J. B., Severo, J. S., Oliveira ARSd. Zinc
and Oxidative Stress: Current Mechanisms. Antioxidants. 2017; 6(24): 1-9.
Masiá, M., Padilla S, Fernández, M., Rodríguez, C., Moreno, A., Oteo, J. A., et al., Oxidative
Stress Predicts All-Cause Mortality in HIV-Infected Patients. PLOS ONE. 2016;:1-12.
McClelland, R. S., Baeten, J. M., Overbaugh, J., Richardson, B. A., Mandaliya, K., Emery, S.,
et al., Micronutrient Supplementation Increases Genital Tract Shedding of HIV-1 in
Women. J Acquir Immune Defic Syndr. 2004; 37:1657–1663.
Meschino, J. P. Glutathione: The Body’s Master Detoxifier and Antioxidant. Dynamic
Chiropractic. 2009 December 2:1-11.
Micke P, Beeh KM, Schlaak JF, Buhl R. Oral supplementation with whey proteins increases
plasma glutathione levels of HIV‐infected patients. Eur J Clin Invest. 2001; 31(2): 171-
178.
Mocchegiani, E., Veccia, S., Ancarani, F., Scalise, G., Fabris, N. Benefit of oral zinc
supplementation as an adjunct to zidovudine (AZT) therapy against opportunistic
infections in AIDS. Int J Immunopharmacol. 1995; 17(9): 719-727.
Nimse, S. B., Pal, D. Free radicals, natural antioxidants, and their reaction mechanisms. RSC
Adv. 2015; 5: 27986–28006.
Ockenga J, Grimble R, Jonkers-Schuitema C, Macallan D, Melchior JC, Sauerwein HP, et al.,
ESPEN Guidelines on Enteral Nutrition: Wasting in HIV and other chronic infectious
diseases. Clinical Nutrition. 2006; 25: 319–329.

Ogedengbe OO, Naidu E, Akang EN, Offor U, Onanuga IO, Peter AI, et al., Virgin coconut oil
extract mitigates testicular-induced toxicity of alcohol use in antiretroviral therapy.
Andrology. 2018; 6(4): 616-626.
Oguntibeju, O. O., Esterhuyse, A. J., Truter, E. J. Possible benefits of micronutrient
supplementation in the treatment and management of HIV infection and AIDS. Afr. J.
Pharm. Pharmacol. 2009; 3(9): 404-412.
Okechukwu, I. B. open access book. Oxidative Stress, Redox Regulation and Elite Controllers
of HIV Infection: Towards a Functional Cure. In Trends in Basic and Therapeutic Options
in HIV Infection - Towards a Functional Cure.: Intech Open; 2015.183-207.
http://dx.doi.org/ 10.5772/60806.
Oppenheimer, G. M., Bayer, R. An Epidemic of Unknown Proportion: The First Decade of
HIV/AIDS. In John CH, Brian JH, Clay JC editors . HIV/AIDS in the Post-HAART Era:
Manifestations, Treatment and Epidemiology. New York: PMPH-USA; 2011:1-17.
Packer, L., Suzuki, Y. J. Vitamin E and alphalipoate:role in antioxidant recycling and activation
of the NF-kappa B transcription factor. Mol Aspects Med. 1993; 14:229-239.
Paranjape, R. S. Immunopathogenesis of HIV infection. Indian J Med Res. 2005; 121: 240-
255.
Pasupathi, P., Bakthavathsalam, G., Saravanan G., Chinnaswamy, .P, Devaraj, A. Oxidative
Stress, CD4+ Cell Count and Antioxidant Status in HIV/AIDS Patients. J. Cell Tissue
Research. 2008; 8(2):1437-1442.
Patrick, L. Nutrients and HIV: Part Three-N-Acetylcysteine, Alpha-Lipoic Acid, L-Glutamine,
and L-Carnitine. Altern Med Rev. 2000; 5(4):290-305.
Patrick, L. Nutrients and HIV:Part 2–Vitamins A and E, Zinc, B-Vitamins, and Magnesium.
Altern Med Rev. 2000; 5(1):39-51.
Prasad, A. S. Impact of the Discovery of Human Zinc Deficiency on Health. J Am Coll Nutr.
2009; 28(3): 257–265.
Preedy, V. R, Watson RR. e-book. HIV/AIDS Oxidative Stress and Dietary Antioxidants
London: Academic Press; 2018.
Premanathan, M., Nakashima, H., Igarashi, R., Mizushima, Y., Yamada, K. Lecithinized
superoxide dismutase: an inhibitor of human immunodeficiency virus replication. AIDS
Res Hum Retroviruses. 1997; 13(4): 283-290.
Price, T. O., Ercal, N., Nakaoke, R., Banks, W. A. HIV-1 viral proteins gp120 and Tat induce
oxidative stress in brain endothelial cells. Brain Research. 2005; 1045: 57 – 63.
Projects. Knoxville; June, 2002. https://trace.tennessee.edu/utk_chanhonoproj/554.
Ravikumar, C. Review on Herbal Teas. J. Pharm. Sci. Res. 2014; 6(5): 236-238.
Robinson, J. M. Phagocytic leukocytes and reactive oxygen species.. Histochem Cell Biol.
2009; 131: 465–469 d.
Saeidnia, S., Abdollahi, M. Role of micronutrients and natural antioxidants in fighting against
HIV; aquick mini-review. RJP. 2014; 1(4): 49-55.
Sandstrom, A. P. A., Murray, J. B., Folks, A T. M., Diamond, B. A. M. Antioxidant Defenses
Influence HIV-1 Replication and Associated Cytopathic Effects. Free Radical Biology and
Medicine. 1998; 24(9): 1485-1491.
Sandstrom, P. A., Tebbe, P. W., Cleave, S. V., Buttke, T.M. Lipid Hydroperoxides Induce
Apoptosis in T Cells Displaying a HIV-associated GlutathionePeroxidase Deficiency. J
Biol Chem. 1994; 269(2): 798-801.

Sappey, C., Legrand-Poels, S., Best-Belpomme M., Favier A., Rentier, B., Piette, J. Stimulation
of glutathione peroxidase activity decreases HIV type 1 activation after oxidative stress.
AIDS Res Hum Retroviruses. 1994; 10(11):1451-1461.
Saribas, S. A., Cicalese, S., Ahooyi, T. M., Khalili, K., Amini, S., Sariyer I. K. HIV-1 Nef is
released in extracellular vesicles derived from astrocytes: evidence for Nef-mediated
neurotoxicity. Cell Death Dis. 2017; 8 (e2542): 1-11.
Savitha, P. Role of Selenium. J. Pharm. Sci. Res. 2014; 6(1): p. 56- 59.
Schomburg, L. Selene, the goddess of the moon: Does she shine on men only? European Heart
Journal. 2007; 28(16): 2043-2044.
Ścibior, D., Czeczot, H. Catalase: structure, properties, functions. Postepy Hig Med Dosw.
(online). 2006; 60: 170-180.
Shah, A., Kumar, S., Simon, S. D., Singh, D. P., Kumar, A. HIV gp120- and methamphetamine-
mediated oxidative stress induces astrocyte apoptosis via cytochrome P450 2E1. Cell
Death and Disease. 2013; 4(10): article e850.
Sharma, B. Oxidative Stress in HIV Patients Receiving Antiretroviral Therapy. Current HIV
Research. 2014; 12:13-21.
Shivakoti, R., Gupte, N., Yang, W. T., Mwelase, N, Kanyama, C, Alice, M. T., et al., Pre-
Antiretroviral Therapy Serum Selenium Concentrations Predict WHO Stages 3, 4 or Death
but not Virologic Failure Post-Antiretroviral Therapy. Nutrients. 2014; 6: 5061-5078.
Shriner, K., Goetz, M. B. Severe hepatotoxicity in a patient receiving both acetaminophen and
zidovudine. Am J Med. 1992; 93: 94-96.
Singh, J., Sinha, S. Classification, Regulatory ACTS and Applications of Nutraceuticals for
Health. Int J Pharm Bio Sci. 2012; 2(1): 177-187.
Souza, S. J., Petrilli, A. A., Teixeira, A. M., Pontilho, P. M., Carioca, A. A., Luzia., L. A., et
al., Effect of chocolate and mate tea on the lipid profile of individuals with HIV/AIDS on
antiretroviral therapy: A clinical trial. Nutrition. 2017; 43(44): 61-68.
Srivastava, S., Sharma, P. K., Guru, S. K. Nutraceuticals: A Review. Journal of Chronotherapy
and Drug Delivery. 2015; 6(1): 1-10.
Stephensen, C. B., Marquis, G. S., Jacob, R. A., Kruzich, L. A., Douglas, S. D., Wilson, C. M.
Vitamins C and E in adolescents and young adults with HIV infection. Am J Clin Nutr.
2006; 83: 870-879.
Stone, C. A., Kawai, K., Kupka, R., Fawzi, W. W. The Role of Selenium in HIV Infection.
Nutr Rev. 2010; 68(11): 671–681.
Sundaram, M., Saghayam, S., Priya, B., Venkatesh, K. K., Balakrishnan, P., Shankar E. M., et
al., Changes in antioxidant profile among HIV-infected individuals on generic highly
active antiretroviral therapy in southern India. Int J Infect Dis. 2008; 12: e61- e66.
Tang, A. M., Graham, N. M., Kirby, A. J., McCall, L. D., Willett, W. C., Saah, A. J. Dietary
micronutrient intake and risk of progression to acquired immunodeficiency syndrome
(AIDS) in human immunodeficiency virus type 1 (HIV-1)-infected homosexual men. Am
J Epidemiol. 1993; 138(11): 937-951.
Ulrich, H., Weischer, C. H., Engel, J., Hettche, H. Inventors; Pharmaceutical Composition
Containing R-alpha-Lipoic acid or S--alpha-Lipoic acid as Active Ingredient. Germany
patent 5,728,735. 1998 March 17.
Uttara, B., Singh, A. V., Zamboni, P., Mahajan, R. T. Oxidative stress and neurodegenerative
diseases: A review of upstream and downstream antioxidant therapeutic options. Curr.
Neuropharmacol. 2009; 7: 65–74.

Valle, LGd., Hernández, R. G., Ávila, J. P. Oxidative Stress Associated to Disease Progression
and Toxicity during Antiretroviral Therapy in Human Immunodeficiency Virus Infection.
Journal of Virology Microbiology. 2013; 2013:1-15.
Vašková, J., Vaško, L., Kron, I. Oxidative Processes and Antioxidative Metaloenzymes. In El-
Missiry MA. Antioxidant Enzymes. Rijeka, Croatia: InTech; 2012:11-12.
WHO. WHO case definitions of HIV for surveillance and revised clinical staging and
immunological classification of HIV-related disease in adults and children. Geneva:; 2007.
WHO. Guideline: Vitamin A supplementation in pregnancy for reducing the risk of mother-to-
child transmission of HIV. Geneva:; 2011.
Winter, F. S., Emakam, F., Kfutwah, A., Hermann, J., Kenfack, M. A., Krawinkel, M. B. The
Effect of Arthrospira platensis Capsules on CD4 T-Cells and Antioxidative Capacity in a
Randomized Pilot Study of Adult Women Infected with Human Immunodeficiency Virus
Notunder HAART in Yaoundé, Cameroon. Nutrients. 2014; 6: 2973-2986.
Wiseman, H. Vitamin D is a membrane antioxidant. Ability to inhibit iron-dependent lipid
peroxidation in liposomes compared to cholesterol, ergosterol and tamoxifen and relevance
to anticancer action. FEBS Lett. 1993; 326(1-3): 285-288.
Wu , J., Levy, M., Black, P. H. 2-Mercaptoethanol and n-acetylcysteine enhance T cell colony
formation in AIDS and ARC. Clin Exp Immunol. 1989; 77: 7-10.
Yale, S. H., Glurich, I. Analysis of the inhibitory potential of Ginkgo biloba, Echinacea
purpurea and Serenoa repens on themetabolic activity of cytochrome P450 3A4, 2D6, and
2C9. J Altern Complement Med. 2005; 11: 433–439.

SECTION V: ANTIOXIDANTS IN
CARDIOVASCULAR DISEASES

Chapter 13
ANTIOXIDANTS AND ANTIHYPERTENSIVE POTENCY
Abdulla Sherikar1,, Aman Upaganlawar2,

Sameer Goyal1 and Chandrashekhar Upasani2
1
SVKM’s Institute of Pharmacy, Maharashtra, India
2
SNJB’s Shriman Sureshdada Jain College of Pharmacy, Nashik, India
ABSTRACT
Cardiovascular diseases (CVDs) are a becoming global burden problem in modern
lifestyle of both developed and undeveloped countries. Today’s modern life, hypertension
is one of the major representatives of CVDs. An exaggerated generation of reactive oxygen
species (ROS) is regarded as one of the major causes for dysfunction of endothelium in
blood vessels as in hypertension. Any deviation in synthesis and detoxification process, the
oxygen free radicals causes oxidative stress. The numerous examples of ROS in the
biological system include superoxide (O2ˉ), hydrogen peroxide (H2O2), hydroxyl radical
(OH•), lipid peroxy radical (LOO), nitric oxide (NO), nitrogen dioxide radical (NO2),
nitrosonium cation (NO+) and peroxynitrite (ONOO−). The biological agents such as both
enzymatic and non-enzymatic antioxidants are familiar as free radical scavenger thereby
maintaining the concentration of free radicals or ROS within the acceptable level. The aim
of this chapter is to focus on the major role of oxidative free radicals and the role of both
enzymatic and non-enzymatic antioxidants in the management of hypertension.
1. INTRODUCTION
The cardiac system and blood vessel system plays an important role in the pathogenesis of
various CVDs. Universally the mortality rate due to CVDs each year is 17.3 million and
expenditure for treatment of these diseases is near about $316.1 billion each year (Benjamin,
2017). In the modern era of life, the changes in lifestyle and habits, directly and indirectly are
thought to link with risk pathogenesis of CVDs disorders (Ambrose, 2004; D’Agostino, 2008;
Bjorntorp, 1990; Ezzati, 2002; Hubert, 1983). An altered endothelial function and oxidative

Corresponding Author’s Email: abdullasherikar@rediffmail.com.

326 Abdulla Sherikar, Aman Upaganlawar, Sameer Goyal et al.
stress burden in blood vessels cause pathogenesis of CVDs (Fearon, 2009; Keaney, 2000). An
elevated reactive oxygen species (ROS) level due to an imbalance between genesis and
detoxification of free radical results in the dysfunction of endothelium in blood vessels. This is
an important link between the development of hypertension, atherosclerosis, ischemia, vascular
dysfunction, cardiac toxicity, cardiomyopathy and heart failure (Ahmad, 2017; Clempus, 2006;
Dhalla, 2000; Giordano, 2005; Heistad, 2006; Ji, 1999; Montezano, 2012).
Physiologically blood pressure is defined as resistance to the blood flow by the peripheral
blood vessels (Mayorov, 2016; Tsiropoulou, 2016). The tone of blood pressure is regulated by
the central and peripheral nervous system and the abnormalities in the function of the hormone,
renal disorders and disorders of the peripheral vascular framework can result in the
development of hypertension in human (Mayorov, 2016; Tsiropoulou, 2016). Hypertension is
nothing but a persistent elevation of blood pressure more than systolic/diastolic (140/90 mm
Hg) and it is regarded as mild, moderate and severe (Mayorov, 2016; Tsiropoulou, 2016).
Pathophysiologically, hypertension is classified as primary hypertension (essential and 90 to
95%) and renal hypertension (secondary and 05 to 10%) (Mayorov, 2016; Tsiropoulou, 2016).
At an elevated blood pressure (systolic ≥ 210 and/or diastolic ≥ 120 mmHg), hypertension is
associated with endothelial injury and intimal proliferation which develops renal failure,
hypertensive encephalopathy, congestive cardiac failure and pulmonary edema (Mayorov,
2016; Tsiropoulou, 2016).
The precipitating condition for hypertension involves renal diseases, endocrine diseases,
steroid diseases, excess of growth hormone, catecholamine excess, vascular causes, renal artery
stenosis and drugs (Goodman and Gillman, 2011). Oxidative stress causes an elevated level of
angiotensin II, aldosterone and hydrogen peroxide (H2O2) (Mayorov, 2016; Tsiropoulou, 2016).
The various biological processes occurring in the human body are under the great influence of
oxygen. On the darker side, the imbalance in the genesis of reactive oxygen species (ROS)
results in state of oxidative stress where oxygen interacts with certain molecules such as DNA,
RNA, proteins, enzymes and cell membrane in the body and produces free radicals (Brieger,
2012; D’Autréaux, 2007; Lobo, 2010; McCord,2000). The highlighted examples of ROS
include superoxide (O2ˉ), H2O2, hydroxyl radical (OH•) and lipid peroxy radical (LOO•). In
addition to this, there are numerous nitrogen species such as nitric oxide (NO), nitrogen dioxide
radical (NO2•), nitrosonium cation (NO+) and peroxynitrite (ONOO−) (Dedon, 2004; Lushchak,
2014; Pratico, 2000).
Usually, antioxidants are known for their best as a free radical scavenger by preventing
their oxidation, an important step involved in the free radical generation. The prime important
role of antioxidants is to keep the concentration of ROS within the acceptable level (Peuchant,
2004). There are two types of antioxidants such as enzymatic and non-enzymatic antioxidants.
The important examples of enzymatic antioxidants are glutathione peroxidase, superoxide
dismutase, glutathione reductase and catalase. On the other hand, the important examples of
non-enzymatic antioxidants are alpha-tocopherol, glutathione and ascorbic acid (Peuchant,
2004). The present chapter highlighted the potential beneficial role of both enzymatic and non-
enzymatic antioxidant in the management of hypertension.

Antioxidants and Antihypertensive Potency 327
2. CLASSIFICATION OF ANTIOXIDANTS
The classification of antioxidants is based on their solubility, a line of defense and
supplementation source as shown in Figure 1 and Table 1 (Birben, 2012; Sen, 2010).
Figure 1. The classification of antioxidants.
Table 1. Classification of antioxidants as an antihypertensive potency

(Birben, 2012; Sen, 2010)
Class Subclass Notes

Based on Hydrophilic or  E.g., ascorbic acid, glutathione, lipoic acid, uric acid, etc.
solubility water-loving (Ames, 1994; Halliwell, 1994; Jadhav, 2004; Sies, 1992).
Hydrophobic  E.g., vitamin E, ubiquinol, etc. (Ames, 1994; Halliwell, 1994;
or lipid loving Jadhav, 2004; Sies, 1992).
Based on the First line  Abolishes genesis of free radicals
line of defense defense  Very fast acting neutralizer of any free radical
(preventive  They act by disguising superoxide radical and conversion of
antioxidants) hydrogen peroxides and hydroperoxides to non-toxic
compounds such as water and oxygen
 E.g., superoxide dismutase (SOD), catalase (CAT), glutathione
peroxidase (GTX), glutathione reductase and some minerals
like selenium, copper, manganese, transferrin, caeruloplasmin,
etc. (Ames, 1994; Halliwell, 1994; Jadhav, 2004; Sies, 1992).
Second line  Scavenge or neutralize active free radicals by donating an
defense electron and thereby arresting initiation of chain and causes
(radical breakdown of chain propagation
scavenging  Mainly are hydrophilic (e.g., ascorbic acid, uric acid,
antioxidants) glutathione) and lipid soluble antioxidants (tocopherol i.e.,
vitamin E and ubiquinol)

Class Subclass Notes

 Detoxify free radicals and various lipid hydroperoxides along
with detoxifying ozone (Ames, 1994; Halliwell, 1994; Jadhav,
2004; Sies, 1992).
Third Line  Only act after free radical damage and rebuild the injured cell
defense (repair membrane
and de-novo  Responsible for repair of damaged DNA, protein, and lipids
enzymes)  Functions as “clean up duty”
 E.g., DNA repair enzyme such as polymerases, glycosylases
and nucleases and proteolytic enzymes such as proteinases,
proteases, and peptidases.
 Other example includes some beverages, food, and cosmetic
products (Ames, 1994; Halliwell,1994; Jadhav,2004;
Sies,1992)
Fourth line  Plays an important role adaptation mechanism i.e., both inhibit
defense the synthesis of free radical and their harmful effects in the
antioxidants cell (Niki, 1993).
Based on Minerals  Acts as a cofactor
supplementation  Important for carbohydrate metabolism
source  E.g., selenium, copper, iron, zinc, and manganese (Ames,
1994; Halliwell, 1994; Jadhav, 2004; Sies, 1992)
Vitamins  Essentials for body metabolic functions
 E.g., vitamin C, vitamin E and vitamin B (Ames, 1994;
Halliwell, 1994; Jadhav, 2004; Sies, 1992)
Phytochemicals  Are phenolic compounds
 Are neither vitamins nor minerals (Ames, 1994; Halliwell,
1994; Jadhav, 2004; Sies, 1992)
Flavonoids  Are phenolic compounds
 Presents in some vegetable fruits, grains, seeds leaves and
flowers (Ames, 1994; Halliwell, 1994; Jadhav, 2004; Sies,
1992)
Carotenoids  Are fat soluble
 Found in fruits and vegetables (Ames, 1994; Halliwell, 1994;
Jadhav, 2004; Sies, 1992)
3. SOURCES OF REACTIVE OXYGEN SPECIES IN HYPERTENSION

There are two types of sources of ROS which are involved in the pathogenesis of
cardiovascular system. The first source is called an endogenous source which includes factors
such as NADPH oxidase, mitochondria, xanthine oxidase and uncouples nitric oxide synthase.
The second source is called exogenous sources which involve factors such as environmental
toxins, chemical toxins, radiations, ultraviolet light, smoking and some pharmaceuticals agents
as shown in Table 2.

Table 2. Sources of reactive oxygen species in cardiovascular diseases

(Elahi, 2009; Srivastava, 2004; Sugamura, 2011; Zhang, 2007)
Name of Roles
Source
NADPH  Most important enzymatic source
oxidase  Membrane-bound proteins
 Responsible for intracellular and extracellular production of ROS such as a
cellular membrane, nucleus, endoplasmic reticulum and mitochondria
 Has two types of subunits such as catalytic (e.g., Nox1, Nox2, Nox3, Nox4,
Nox5, Duox1and Duox2) and accessory subunits (p22phox, p47phox, p67phox,
and Rac1-2)
 Generates superoxide (O2-) or hydrogen peroxide (H2O2) (Anilkumar, 2013;
Altenhofer, 2012; Bedard, 2007; Kuroda, 2010; Lassegue, 2010; Panieri, 2015)
Mitochondria  Significant mediator in the genesis of ROS
 Causes oxidative phosphorylation on the interior of the mitochondrial membrane
through electron transport chain machinery
 Causes reduction of molecular oxygen to water through four single-electron
transfer reactions
 Responsible for reduction of 95% of oxygen to water (H2O) and rest of 5% of
oxygen to superoxide radical (Becker, 2004; Chen, 2003; Murphy, 2009; Sena,
2012; Turrens, 2003)
Xanthine  Are member of xanthine oxidoreductase family
oxidase  Present in endothelial cells
 Donates the electron to molecular which initiates oxidation of hypoxanthine into
xanthine and then to uric acid (Dawson, 2006; George, 2009; Harrison, 2004;
Viel,2008)
Uncoupled  It is a transmitter produces by endothelial nitric oxide synthase (eNOS)
nitric oxide  Important role in vasodilatation of blood vessels
synthase  Responsible for endothelial dysfunction leading to hypertension (Forstermann,
2006; Gorren, 2002; Landmesser, 2003; Moncada, 1991; Szabo, 1997)
4. IDEAL PROPERTIES OF NATURAL ANTIOXIDANTS

The process of oxidation in the human body produces unstable chemicals which are called
as free radicals having tendency to damage cell membranes and other structures. Antioxidants
are compounds having ability to scavenge and neutralize free radicals. Ideally, the natural
antioxidants should have:
 Well oral bioavailability,

 Administered as once or twice daily dosing,
 Local optimum concentration in different tissues such as kidney, brain, and/or
vasculature,
 Block genesis of ROS,
 Maximum safety profile with minimum side effects,
 Useful in a wide variety of hypertension and

 Should not produce any clinical toxicity with other concomitant antihypertensive drug
therapy (Kizhakekuttu, 2010)
5. ANTIOXIDANTS
5.1. Herbal Antioxidants
5.1.1. Silymarin
Silymarin is an important annual polyphenolic herbal antioxidants belonging to family
Asteraceae (Karkanis, 2011). Silymarin is best known for its beneficial properties in the
management of various diseases such as liver, cardiovascular, CNS and cancer (Blumenthal,
2003; Vasanthi, 2012). The seeds of milk thistles are used as a source of extraction of silymarin
in the form of a mixture of three structural isomers such as silybin & isosilybin; silydianin &
isosilydianin and silychristin & isosilychristin along with minimum quantity of quercetin and
taxifolin (Ding, 2001; Kvasnicka, 2003). Silibinin is regarded as silybin isomer which has
potent antioxidant and anti-inflammatory property (Haddad, 2011). The silymarin is best
known for its cardioprotective mechanism of action (Benjamin, 2017). Silymarin possesses
beneficial properties such as capacity to scavenge the free radicals, ferrous and copper chelation
(Ezzati, 2002) and inhibition of NAPDH oxidase activity (Bjorntorp, 1990). Adding more to
this, silymarin also causes activation of antioxidant enzymes, prevention of lipid peroxidation
(Hubert, 1983) and control the permeability and stability of cell membrane. Besides these
properties, silymarin also plays an important role in the acceleration of ribosomal protein
synthesis through activation of ribonucleic acid (RNA) polymerase (Ambrose, 2004), directing
the signal mechanism by activating the Nrf2 and causes inhibition of NF-κB (D’Agostino,
2008). Being as an antagonist of angiotensin at AT1 receptor, silymarin causes the blockade of
angiotensin II and produces an increase in smooth muscle relaxation and accelerate the
elimination of water and salt. The beneficial antihypertensive effects of silymarin are thought
to parallel with classical antihypertensive agents such as candesartan (Bahem, 2015).
In rodents and humans, silymarin has low oral bioavailability property (Fraschini, 2002).
Therefore the solubilizing agents are added to its formulation to achieve optimum plasma
concentration (El-Samaligy, 2006; Li, 2010). Silymarin undergoes phase II metabolism process
in the liver as conjugates of sulfated and glucuronide. The elimination half-life of silymarin is
6 to 8 hours. The 80% administered dose of silymarin is cleared from the body via biliary route
(Fraschini, 2002). The commercially silymarin is given in the form of capsules and tablets at
doses of 120 mg, 160 mg, 250 mg, and 300 mg. The usual dose of silymarin is 280 to 800
mg/kg of body weight with 1-2 tablets twice a day after a meal (Lee, 2006). The principle use
of silymarin is directed against the treatment of a wide range of diseases such as oxidative
stress-induced hypertension, atherosclerosis and cardiac toxicity (Karimi, 2011; McCord, 2000;
Tamayo, 2007). Among the notable rare adverse reactions of silymarin involves mild
gastrointestinal disturbance, nausea and headache (Karimi, 2011; McCord, 2000; Tamayo,
2007).

5.1.2. Apocynin
Structurally apocynin is a 4'-hydroxy-3'-methoxyacetophenone. It is extracted from
medicinal herb named as Picrorhiza kurroa. Pharmacologically apocynin is considered as a
potent inhibitor of the Nox complex i.e., mitochondria and NAD(P)H oxidase (Stolk, 1994;
Touyz, 2008). The antioxidant mechanism of apocynin involves the blockade of Nox-derived
O2• − release through inhibition of migration of p47phox to the membrane. In order to show this
antioxidant property, neutrophil oxidative metabolism of apocynin is essential to form
diapocynin, the metabolically active compound of apocynin (Wang, 2008; Ximenes, 2007).
Angiotensin II along with cytokines and growth factors plays an important role in
controlling the activity of Nox. Angiotensin II not only involved in the acceleration and
stimulation of Nox but also causes elevated expression of Nox isoforms thereby generating
ROS. The angiotensin II derived ROS are principally found in various tissues such as vascular
smooth muscle cells, endothelial cells, adventitial fibroblasts, and intact arteries (Touyz, 2003).
The link between antihypertensive property of apocynin involves the attenuation of both
elevation of ROS synthesis and collagen deposition and reduction in arterial stiffness (Chen,
2013).
5.1.3. Curcumin
Since from ancient time, the useful therapeutic medicinal use of Curcuma longa has been
proved (Brouk, 1975). The chemical constituents of Curcuma spp. has been categorized into
three types such as a water-soluble peptide, essential oils and curcuminoids. The turmeric is a
water-soluble peptide and the examples of essential oils are turmerones, atlantones and
zingiberene. Lastly curcumin is a curcuminoids which is chemically [1,7-bis-(4-hydroxy-3-
methoxyphenyl)-1,6-heptadiene-3,5-dione]. The percentage of curcumin is 2 – 8% which is the
most biological constituent of turmeric (Milobedzka, 1910; Heath, 2004). The curcumin has
potential antioxidant property. The known mechanism of action of curcumin includes the
blockade of COX-2 isoenzyme and inducible nitric oxide synthase (iNOS). Moreover curcumin
also causes attenuation of synthesis of cytokines such as interferon-γ by slowing the Janus
kinase (JAK)–STAT signaling mechanism through exerting the effects on the Src homology 2
domain-containing protein tyrosine phosphatases (SHP)-2 (Bharti, 2003).
In addition to this curcumin has a tendency to increase the myocardial protein expression
of inducible nitric oxide synthase (iNOS) which is responsible for the production of NO thereby
decreases the appearance of NADPH oxidase subunit p47phox (Aggeli, 2013; Mito,2011).
After oral administration, the bioavailability of curcumin is low i.e., 60%. It is highly
concentrated in colorectal tissue and undergoes the first-pass metabolism. It is metabolized in
the intestinal tract as glucuronide and sulfate conjugates such as tetrahydrocurcumin and
hexahydrocurcumin. The curcumin also undergoes enterohepatic circulation and 50% of
administered dose is excreted as metabolites in bile and rest drug is excreted as unchanged drug
(Ravindranath, 1981). The curcumin has a wide range of activity including anti-inflammatory,
anticarcinogenic, antirheumatic activity and antioxidant. The antioxidant property of curcumin
is useful in the treatment of hypertension. The usual dose of curcumin is 1 to 3 gm/day/oral
(Sharma, 2004). The curcumin has fewer tendencies to cause adverse reactions but occasionally
it is associated with diarrhea, nausea and increased serum alkaline phosphatase and lactate
dehydrogenase levels (Sharma, 2004).

5.1.4. Berberine
Berberine is one of the main compounds found in various species of Berberis (Imanshahidi,
2008; Yang, 2007). The endothelial microparticles released from endothelium plays an
important role in the pathogenesis of cardiovascular diseases. In case of hypertension, this
endothelial microparticle causes increased activity of NADPH oxidase and adenosine
monophosphate-activated protein kinase (AMPK) by increasing the expression of
nonphagocytic NADPH oxidase (Nox) proteins 2 and 4.
Due to its antioxidant property, berberine blocks the expression of these Nox proteins and
inhibits the NADPH oxidase and reverse the pathogenesis of hypertension (Kim, 2008; Wang,
2010). Berberine is widely used in the management of various cardiovascular diseases
including cardiac failure, hypertension, stroke and thromboembolic syndrome (Bernal-
Mizrachi, 2003; Mohammadzadeh, 2017).
5.1.5. Catechin
The catechin is a polyphenolic antioxidant isolated from tea leaves of Camellia sinensis
(Conney, 2002; Feng, 2016; Zhang, 2005). Catechin is a flavan-3-ol, a type of natural phenolic
antioxidant. Chemically catechin contains four different chemical entities such as
epigallocatechin gallate, epicatechin gallate, epigallocatechin and epicatechin (Chen, 2005;
Rameshrad, 2017).
The catechin contains the optimum concentration of epigallocatechin gallate which has
potent antioxidant property. Epigallocatechin gallate acts as an inhibitor of angiotensin-
converting enzyme thereby preventing endothelial dysfunction and prevents the pathogenesis
of hypertension (Müller, 2016). In addition to this, it also causes a reduction in NADPH oxidase
activity thereby suppressing the synthesis of reactive oxygen species. Adding to this more,
epigallocatechin gallate is responsible for remodeling of endothelial dysfunction by causing
stimulation of eNOS, acceleration of NO release and reduction in the level of angiotensin II
(Abo, 1991; Ihm, 2009; Potenza, 2007). Catechin has poor oral bioavailability (Hu, 2012;
Tagashira, 2012; Zhang, 2004).
5.1.6. Resveratrol
The polyphenol such as resveratrol is a natural antioxidant and an anti-inflammatory agent.
It has a wide range of therapeutic potential in the management of multiple diseases such as
hypertension (Rueda-Clausen, 2012; Tain, 2017). It is obtained from various herbs such as
Vratrum grandiflorum, and Polygonum cuspidatum grapes and also from various berries (Baur,
2006). Prolonged treatment with resveratrol produces antihypertensive and atherosclerotic
effect which results in marked restoration of cardiovascular function. In addition to this, it also
causes a blockade of platelet aggregation and elevation of endogenous antioxidants (Toklu,
2010).
The utility of resveratrol possibly is dependent on its effects on SIRT1 and AMPK
signaling. The usefulness of resveratrol as an antihypertensive agent in adult offspring was
proven by using a different animal model of hypertension such as prenatal hypoxia and
postnatal high-fat diet rat model. Resveratrol, when given to pregnant women, causes
attenuation of hypertension in adult offspring (Bonnefont-Rousselot, 2016; Care, 2016; Li,
2012).

5.1.7. Melatonin
An endogenous hormone such as melatonin which is secreted from pineal gland has good
therapeutic potential against various diseases such as hypertension. Fundamentally because of
its antioxidant property, melatonin controls the various physiological processes such as
inflammatory process, redox homeostasis, epigenetic direction, biological clock cycle, blood
pressure regulation and regulation of development of fetus growth (Chen, 2013; Simko, 2007).
The therapeutic utility of melatonin as an antihypertensive agent was studied in a various
animal model of hypertension such as glucocorticoid exposure (Chang, 2016; Tain, 2004; Wu,
2014), high-fructose consumption (Tain, 2014) and high-fat diet (Tain, 2015).
5.1.8. Quercetin
Being a polyphenol, quercetin possesses good antioxidants property. Due to the generation
of superoxide anion by NADPH oxidase, decreased eNOS activity causes decreased liberation
of NO from endothelium. This underlying pathological mechanism results in endothelial
dysfunction which is reversed by administration of quercetin. This mechanistic approach with
quercetin administration suggests that quercetin has a coronary vasodilatory effect, anti-
aggregatory effect and results in the betterment of endothelial function (Duarte, 1993;
Gryglewski, 1987; Perez-Viizcaino, 2002).
5.1.9. Ginkgo biloba

The leaf of Ginkgo biloba was proven to be as an antihypertensive herb because it has the
antioxidant property that prevents vascular dysfunction and inhibits vasoconstriction (Xiong,
2014). The presence of flavone glycosides and terpenoids makes the best choice of Ginkgo
biloba as a powerful antioxidant agent in the treatment of hypertension (Gaby, 1996). The
Chinese Pharmacopoeia stated the other advantageous effects of Ginkgo biloba in the treatment
of congestive cardiac failure, angina pectoris, myocardial disorders and cerebral infarction
(National Pharmacopoeia Committee, 2010). The proposed antihypertensive mechanism of
Ginkgo biloba involves attenuation of vascular smooth muscle activity, inhibition of
angiotensin-converting enzyme and maintenance of both vascular endothelium-dependent and
independent effects of vasodilators (Kubota, 2006; Mansour, 2011).
5.1.10. Vitamin C
Vitamin C (ascorbic acid) is a well-known antioxidant agent having the antihypertensive
property. The administration of vitamin C has multiple biological effects and it is best known
as a scavenger of superoxide, an activator of smooth muscle cell guanylyl cyclase, a stimulator
of nitric oxide (NO) synthesis by NO synthetase and reducer of oxidation of low-density
lipoprotein (LDL) (May, 2000). In addition to this vitamin C ingestion also causes reduced
single-electron-free radical generation (Jackson, 1998) and inhibition of NADPH oxidase
activity (Chen, 2001; Ulker, 2003).
5.1.11. Vitamin E
Vitamin E (α-tocopherol) has excellent antioxidant property and also acts as an inhibitor
of LDL and prevents the oxidation of membrane phospholipid (Upston, 2001). Moreover,
vitamin E is a good inhibitor of NADPH oxidase, lipoxygenase and COX (Azzi, 2002). In

addition to this, vitamin E also causes a decrease in blood pressure response which is useful in
the management of hypertension (Galley, 1997).

Being as a good antioxidant, alpha-lipoic acid (LA) has attracted the attention of many
researchers. After absorption, LA is rapidly converted to its reduced form dihydrolipoic acid in
the tissues (Dincer, 2002). It is a dithiol derivative obtained from octanoic acid (Smith, 2004).
The LA causes recovery of coupling of eNOS and has anti-inflammatory property (Zhang,
2001) and decrease the blood pressure in an animal model of diabetes and hypertension
(Vasdev, 2000).
5.1.13. L-Arginine
Being as an important amino acid, L-arginine is required for the synthesis of nitric oxide
from eNOS (Tiefenbacher, 2001). The antihypertensive mechanism of L-arginine involves the
re-establishment of nitric oxide bioavailability thereby reduces the chances of development of
hypertension (Matsuoka, 1997). L-arginine also acts as a competitor of circulating
dimethylarginine for eNOS in case of hypertension (Matsuoka, H 1997). Moreover, L-arginine
acts as an inhibitor of angiotensin II and endothelin-1 and potentiator of insulin actions (Gokce,
2004).
5.1.14. Coenzyme Q10

Structurally coenzyme Q10 (CoQ) is a 2,3-dimethoxy-5-meth-6-decaprenyl benzoquinone
(Overvad, 1999). It is synthesized from mevalonic acid and phenylalanine (Overvad, 1999).
Coenzyme Q10 plays an important role in the electron transport chain (McCarty, 1999) and
attenuates the mitochondrial superoxide generation (Houston, 2005). The antioxidant property
of coenzyme Q10 has been proposed due to the reduction in lipid peroxidation (Houston, 2005).
It also acts as an antihypertensive agent (Langsjoen, 1994).

AND CHALLENGES
A lot of literature is available regarding the role of ROS in the development of hypertension
and the exploration of antioxidant therapy in the management of hypertension is also poor. Still,
there are several problems for effective antioxidant therapy in humans because of inadequate
dose and duration of the treatment period and influence of other stimuli which mask the useful
effects of an externally ingested antioxidant. Moreover, the preclinical dose of antioxidant
tested is more than a clinical dose and adding all the problems make a difficulty in achieving
the effective level of the antioxidant in the neutralization of ROS.
While performing the clinical trial of antioxidant, the recruitment of human volunteer is
not done according to the state of redox. So there is need and scope for the discovery of the
novel potential and beneficial antioxidant therapy in the management of hypertension. In order
to improve the antioxidant therapy, further extensive research has to be carried to specify the
sources and targets of ROS with their deleterious or beneficial effects along with the molecular
study in order to discover novel antioxidant treatment in the ideal clinical area.

CONCLUSION
Hypertension is regarded as prominent CVDs in the modern era of a human being.
Oxidative stress due to free radical production is one of the major causes involved in the
pathogenesis of hypertension. Along with the endogenous antioxidants, the non-endogenous
antioxidants such as polyphenol herbal constituents, some vitamins, and mitochondrial
antioxidants have useful antihypertensive potency and can be used as an antihypertensive
remedy due to their powerful antioxidant property. There are numerous synthetic drugs
available for the treatment of hypertension in the market. But these drugs have the potential to
cause adverse effects. So antioxidants such as polyphenol herbs, some vitamins, and
mitochondrial antioxidants can be used as antihypertensive agents. There is a lot of scopes to
study these antioxidants at the molecular, genetic and cellular level to understand their
pharmacology and to find out better one to treat hypertension.
REFERENCES
Abo, A., Pick, E., Hall, A., et al. Activation of the NADPH oxidase involves the small GTP-
binding protein p21rac1. Nature 1991; 353:668.
Aggeli, I. K., Koustas, E., Gaitanaki, C., Beis, I. Curcumin Acts as a Pro‐Oxidant Inducing
Apoptosis Via JNKs in the Isolated Perfused Ranaridibunda Heart. Journal of
Experimental Zoology Part A: Ecological Genetics and Physiology, 2013; 319:328-339.
Ahmad, K. A, et al. Antioxidant therapy for management of oxidative stress induced
hypertension, Free Radic. Res. 2017; 51 (4):428–438.
Altenhofers, et al. The NOX toolbox: validating the role of NADPH oxidases inphysiology and
disease, Cell. Mol. Life Sci. 2012;69 (14):2327–2343.
Ambrose, J. A., Barua, R. S. The pathophysiology of cigarette smoking and
cardiovasculardisease: an update, J. Am. Coll. Cardiol. 43 (10) (2004) 1731–1737.
Ambrose, J. A., and Barua, R. S. The pathophysiology of cigarette smoking and
cardiovasculardisease: an update, J. Am. Coll. Cardiol. 2004;43 (10):1731–1737.
Ames, B. Natural Antioxidants in Human Health and Disease. Academic Press. San Diego
1994. 14-25.
Anilkumar, N., et al. A 28-kDa splice variant of NADPH oxidase-4 is nuclear-localized and
involved in redox signaling in vascular cells significance, Arterioscler. Thromb. Vasc. Biol.
2013;33 (4):e104–e112.
Azzi, A., Ricciarelli, R., Zingg, J. M. Non-antioxidant molecular functions of alpha-tocopherol
(vitamin E). FEBS Lett 2002;519:8–10.
Bahem, R., et al. Modulation of calcium signaling of angiotensin AT1, endothelin ETA, and
ETB receptors by silibinin, quercetin, crocin, diallyl sulfides, and ginsenosideRb1, Planta
Medica 2015;81 (08):670–678.
Baur, J. A., and Sinclair, D. A. Therapeutic potential of resveratrol: the in vivo evidence. Nature
reviews Drug discovery 2006;5:493-506.
Becker, L. B. New concepts in reactive oxygen species and cardiovascular reperfusion
physiology, Cardiovasc. Res. 2004;61 (3):461–470.

Bedard, K. and Krause, K. H. The NOX family of ROS-generating NADPH oxidases:

physiology and pathophysiology, Physiol. Rev. 2007; 87 (1):245–313.
Benjamin, E. J., et al. Heart disease and stroke statistics—2017 update: a report from the
American Heart Association, Circulation 2017;135 (10):e146–e603.
Benjamin, E. J. Heart disease and stroke statistics—2017 update: a report from the American
Heart Association, Circulation 135 (10) (2017) e146–e603.
Bernal-Mizrachi, L., Jy, W., Jimenez, J. J., et al. High levels of circulating endothelial
microparticles in patients with acute coronary syndromes. American heart journal
2003;145:962-970.
Bharti, A. C., Donato, N. and Aggarwal, B. B. Curcumin (diferuloylmethane) inhibits
constitutive and IL-6-inducible STAT3 phosphorylation in human multiple myelomacells.
J Immunol 2003; 171: 3863–3871.
Birben E, et al. Oxidative stress and antioxidant defense, World Allergy Org. J. 2012;5(1):9.
Bjorntorp, P. " Portal" adipose tissue as a generator of risk factors for cardiovascular disease
and diabetes, Arterioscler. Thromb.Vasc. Biol. 1990;10 (4):493–496.
Björntorp, P. " Portal" adipose tissue as a generator of risk factors for cardiovascular disease
and diabetes, Arterioscler. Thromb.Vasc. Biol. 10 (4) (1990)493–496.
Blumenthal, M. The ABC Clinical Guide to Herbs, American Botanical Council, 2003.
Bonnefont-Rousselot, D. Resveratrol and cardiovascular diseases, Nutrients 2016;8:250.
Brieger, K et al. Reactive oxygen species: from health to disease, Swiss Med. Wkly. 2012; 142:
w13659.
Brouk, B. Plants Consumed by Man. New York: Academic Press, 1975, p. 331.
Care AS, Sung MM, Panahi S, et al. Perinatal resveratrol supplementation to spontaneously
hypertensive rat dams mitigates the development of hypertension in adult offspring,
Hypertension 2016;67:1038–1044.
Chang, H. Y. and Tain, Y. L. Postnatal dexamethasone-induced programmed hypertension is
related to the regulation of melatonin and its receptors, Steroids 2016;108:1–6.
Chen Q et al. Production of reactive oxygen species by mitochondria central roleof complex
III, J. Biol. Chem. 2003;278 (38):36027–36031.
Chen, C. H., Ho, M. L., Chang, J. K., et al. Green tea catechin enhances osteogenesis in a bone
marrow mesenchymal stem cell line. Osteoporosis International 2005;16:2039-2045.
Chen, Q. Z., Han, W.Q., Chen, J., et al. Anti-stiffness effect ofapocynin in deoxycorticosterone
acetate-salt hypertensive rats via inhibition of oxidative stress, Hypertens. Res.
2013;36:306–312.
Chen, X, Touyz, R. M., Park, J. B., et al. Antioxidant effects of vitamins C and E are associated
with altered activation of vascular NADPH oxidase and superoxide dismutase in stroke-
prone SHR. Hypertension 2001;38:606–611.
Chen, Y. C., Sheen JM, Tiao MM, et al. Roles of melatonin in fetal programming in
compromised pregnancies, Int. J. Mol. Sci. 2013;14:5380–5401.
Clempus, R. E. and Griendling, K. K. Reactive oxygen species signaling in vascular smooth
muscle cells, Cardiovasc. Res. 2006;71 (2):216–225.
Conney, A.H., Lu, Y.P., Lou, Y.R., et al. Inhibitory effects of tea and caffeine on UV-induced
carcinogenesis: relationship to enhanced apoptosis and decreased tissue fat. European
journal of cancer prevention: the official journal of the European Cancer Prevention
Organization (ECP) 2002;11:S28-36.

D’Agostino, R. B., et al., General cardiovascular risk profile for use in primary care,
Circulation 2008;117 (6):743–753.
D’Agostino, R. B. General cardiovascular risk profile for use in primary care, Circulation 117
(6) (2008) 743–753.
D’Autréaux, B. and Toledano, M. B. ROS as signaling molecules: mechanisms that generate
specificity in ROS homeostasis, Nat. Rev. Mol. Cell Biol. 2007;8 (10) :813.
Dawson, J. and Walters, M. Uric acid and xanthine oxidase: future therapeutic targetsin the
prevention of cardiovascular disease? Br. J. Clin. Pharmacol. 2006;62 (6):633–644.
Dedon, P. C. and Tannenbaum, S. R. Reactive nitrogen species in the chemical biologyof
inflammation, Arch. Biochem. Biophys. 2004;423 (1) :12–22.
Dhalla, N. S., Temsah, R. M. and Netticadan, T. Role of oxidative stress in cardiovascular
diseases, J. Hypertens. 2000; 18 (6) :655–673.
Dincer, Y, Telci, A, Kayali, R, et al. Effect of lipoic acid on lipid peroxidation and anti-oxidant
enzyme activities in diabetic rats. Clin Exp Pharmacol Physiol 2002; 29: 281-4.
Ding, T. M. et al., Determination of active component in silymarin By RP-LC and LC/MS, J.
Pharm. Biomed. Anal. 2001;26 (1):155–161.
Duarte, J., Vizcaíno, F. P, Utrilla P, et al. Vasodilatory effects of flavonoids in rat aortic smooth
muscle. Structure-activity relationships. General Pharmacology: The Vascular System
1993;24:857-862.
Elahi, M. M, Kong, Y. X. and Matata, B. M. Oxidative stress as a mediator of cardiovascular
disease, Oxid. Med. Cell. Longev. 2009;2 (5):259–269.
El-Samaligy M, Afifi N and Mahmoud E. Increasing bioavailability of silymarin using abuccal
liposomal delivery system: preparation and experimental design investigation, Int. J.
Pharm. 2006;308 (1-2):140–148.
Ezzati M, et al. Selected major risk factors and global and regional burden of disease, Lancet
2002; 360 (9343):1347–1360.
Ezzati M. Selected major risk factors and global and regional burden of disease, Lancet 360
(9343) (2002) 1347–1360.
Fearon, I. M. and Faux, S. P. Oxidative stress and cardiovascular disease: novel tools give (free)
radical insight, J. Mol. Cell. Cardiol. 47 (3) (2009) 372–381.
Feng, L, Chong, M. S, Lim, W. S, et al. TEA consumption reduces the incidence of
neurocognitive disorders: Findings from the Singapore Longitudinal Aging Study. The
journal of nutrition, health & aging 2016;20:1002-1009.
Forstermann, U and Münzel, T. Endothelial nitric oxide synthase in vascular disease,
Circulation 2006; 113 (13):1708–1714.
Foster, S. Milk thistle: Silybummarianum. Botanical series no. 305, EnCompositae,
Nomenclature, Milk Thistle, Food, Active_Principles, Medicines, History, Plant_Parts,
Review, Silybum_marianum, Hepatoprotective, Liver, Seeds, Clinical Trials (EBBD,
190001873), American Botanical Council, Austin, Texas,1991, p. 8.
Fraschini, F, Demartini, G and Esposti, D. Pharmacology of silymarin, Clin. DrugInvest.
2002;22 (1):51–65.
Gaby, A. R. Ginkgo biloba extract: a review. Altern. Med. Rev. 1996;1 (4): 236–242.
Galley, H. F., Thornton, J., Howdle, P. D., et al. Combination oral antioxidant supplementation
reduces blood pressure. ClinSci 1997;92:361–365.
George, J. and Struthers, A. D. Role of urate, xanthine oxidase and the effects of allopurinolin
vascular oxidative stress, Vasc. Health Risk Manage. 2009;5:265.

Giordano, F. J. Oxygen, oxidative stress, hypoxia, and heart failure, J. Clin. Invest. 2005;115
(3):500.
Gokce, N. L-arginine and hypertension. J Nutr 2004;134(Suppl 10):2807S–2811S.
Goodman and Gillman: Pharmacological Basis of Therapeutics, McGraw-Hill, Medical
Publishing Division New York, 12th edition, 2011.
Gorren, A. and Mayer, B. Tetrahydrobiopterin in nitric oxide synthesis: A novel biologicalrole
for pteridines. Curr. Drug Metab. 2002;3 (2):133–157.
Gryglewski, R. J., Korbut, R., Robak, J. On the mechanism of antithrombotic action of
flavonoids. Biochemical pharmacology 1987;36:317-322.
Haddad, Y., et al., Antioxidant and hepatoprotective effects of silibinin in a rat model of
nonalcoholic steatohepatitis, Evid. Based Compl. Altern. Med. 2011:nep164.
Hagen, T. M., Moreau R, Suh, J. H., et al. Mitochondrial decay in the aging rat heart: Evidence
for improvement by dietary supplementation with acetyl-L-carnitine and/or lipoic acid.
Ann NY Acad Sci USA 2002;959:491–507.
Halliwell, B. Free Radicals, Antioxidants, and Human Disease: Curiosity, Cause, or
Consequence. Lancet 1994; 344(8942):721-724.
Harrison, R. Physiological roles of xanthine oxidoreductase, Drug Metab. Rev.
2004;36(2):363–375.
Heath, D. D., Khwaja, F. and Rock, C. L. Curcumin content of turmeric and currypowders.
FASEB J 2004;18: A125.
Heistad, D. D. Oxidative stress and vascular disease, Arterioscl. Thromb. Vasc. Biol. 2006;26
(4):689–695.
Houston, M. C. Nutraceuticals, vitamins, antioxidants, and minerals in the prevention and
treatment of hypertension. Prog Cardiovasc Dis 2005;47:396–449.
Hu, B., Ting, Y., Zeng, X., et al. Cellular uptake and cytotoxicity of chitosan–case in
ophosphopeptides nanocomplexes loaded with epigallocatechin gallate. Carbohydrate
polymers 2012;89:362-370.
Hubert, H. B., et al. Obesity as an independent risk factor for cardiovascular disease:a 26-year
follow-up of participants in the Framingham heart study, Circulation 1983;67 (5):968–977.
Hubert, H. B. Obesity as an independent risk factor for cardiovascular disease:a 26-year follow-
up of participants in the Framingham heart study, Circulation 67 (5) (1983) 968–977.
Ihm, S. H., Lee, J. O., Kim, S. J., et al. Catechin prevents endothelial dysfunction in the
prediabetic stage of OLETF rats by reducing vascular NADPH oxidase activity and
expression. Atherosclerosis 2009;206:47-53.
Imanshahidi, M. and Hosseinzadeh, H. Pharmacological and therapeutic effects of Berberis
vulgaris and its active constituent, berberine. Phytotherapy research 2008; 22: 999-1012.
Jackson, T. S, Xu, A., Vita, J. A., et al. Ascorbate prevents the interaction of superoxide and
nitric oxide only at very high physiological concentrations. Circ Res 1998;83:916–922.
Jadhav, S. S., Salunkhe, V. R. and Magdum, C. S. Daily Consumption of Antioxidants:-
Prevention of Disease is better than Cure. Journal of Nutrition 2004; 3(1): 34-40.
Ji, L. L. Antioxidants and oxidative stress in exercise, Exp. Biol. Med. 1999; 222 (3):283–292.
Karimi, G., et al., “Silymarin,” a promising pharmacological agent for treatment of diseases,
Iran. J. Basic Med. Sci. 2011;14 (4):308.
Karkanis, A., Bilalis, D. and Efthimiadou, A. Cultivation of milk thistle (Silybummarianum L.
Gaertn.), a medicinal weed, Ind. Crops Prod. 2011;34 (1):825–830.

Keaney, J. F. Atherosclerosis: from lesion formation to plaque activation and endothelial-dys

function, Mol. Aspects Med. 21 (4) (2000) 99–166.
Kim, J. E., Kim, Y. W., Lee, I. K., et al. AMP-activated protein kinase activation by 5-
aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR)inhibits palmitate-induced
endothelial cell apoptosis through reactive oxygen species suppression. Journal of
pharmacological sciences 2008;106:394-403.
Kizhakekuttu, T. J. and Widlansky, M. E. Natural Antioxidants and Hypertension: Promise and
Challenges. Cardiovascular Therapeutics 2010;28:e20–e32.
Kubota Y, Tanaka N, Kagota S, et al. Effects of Ginkgo biloba extract on blood pressure and
vascular endothelial response by acetylcholine in spontaneously hypertensive rats. J.
Pharm. Pharmacol. 2006;58 (2):243–249.
Kuroda J, et al. NADPH oxidase 4 (Nox4) is a major source of oxidative stress in the failing
heart, Proceedings of the National Academy of Sciences 2010; 107 (35):15565–15570.
Kvasnicka, F., et al., Analysis of the active components of silymarin, J. Chromatogr. 2003;A
990 (1):239–245.
Landmesser U, et al., Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell
nitric oxide synthase in hypertension, J. Clin. Invest. 2003;111 (8):1201.
Langsjoen, P, Langsjoen, P, Willis, R, et al. Treatment of essential hypertension with coenzyme
Q10. Mol Aspects Med 1994;(Suppl 15):S265–S272.
Lassègue, B. and Griendling, K. K. NADPH oxidases: functions and pathologies in the
vasculature, Arterioscler. Thromb. Vasc. Biol. 2010;30 (4):653–661.
Lee, J. I., et al. Separation and characterization of silybin, isosilybin, silydianinandsilychristin
in milk thistle extract by liquid chromatography–electrospray tandemmass spectrometry,
J. Chromatogr. 2006;A1116 (1-2):57–68.
Li, H, Xia, N and Förstermann, U. Cardiovascular effects and molecular targets of resveratrol,
Nitric Oxide 2012;26:102–110.
Li, X, et al., Development of silymarin self-microemulsifying drug delivery system with
enhanced oral bioavailability, Aaps Pharmscitech. 2010;11 (2):672–678.
Lobo, V, et al. Free radicals, antioxidants and functional foods: impact on human health.
Pharmacognosy Reviews 2010; 4 (8) :118.
Lushchak, V. I. Free radicals, reactive oxygen species, oxidative stress and its classification,
Chem. Biol. Interact. 2014;224:164–175.
Mansour, S.M, Bahgat, A. K., El-Khatib, A. S. and Khayyal, M. T. Ginkgo biloba extract (EGb
761) normalizes hypertension in 2K, 1C hypertensive rats: role of antioxidant mechanisms,
ACE inhibiting activity and improvement of endothelial-dysfunction. Phytomedicine
2011;18(8): 641–647.
Matsuoka, H., Itoh, S., Kimoto, M., et al. Asymmetrical dimethylarginine, an endogenous nitric
oxide synthase inhibitor, in experimental hypertension. Hypertension 1997;29:242–247.
May, JM. How does ascorbic acid prevent endothelial dysfunction. Free radical Biol & med
2000;28:1421-1429.
Mayorov, V., et al. Scavenging of reactive isolevuglandins in mitochondria reduces vascular
oxidative stress and attenuates hypertension, Free Radic. Biol. Med. 2016;100:S66.
McCarty, M. F. Coenzyme Q versus hypertension: Does CoQ decrease endothelial superoxide
generation? Med Hypotheses 1999;53:300–304.
McCord, J. M. The evolution of free radicals and oxidative stress, Am. J. Med.
2000;108(8):652–659.

McCord, J. M. The evolution of free radicals and oxidative stress, Am. J. Med. 2000;108(8) :
652–659.
Milobedzka, J., Kostanecki, V. and Lampe, V. Structure. Chem Ber 1910; 43: 2163.
Mito, S., Thandavarayan, R. A., Ma, M., et al. Inhibition of cardiac oxidative and endoplasmic
reticulum stress-mediated apoptosis bycurcumin treatment contributes to protection against
acute myocarditis. Free radical research 2011; 45:1223-1231.
Mohammadzadeh, N., Mehri, S., and Hosseinzadeh, H. Berberis vulgaris and its constituent
berberine as antidotes and protective agents against natural or chemical toxicities. Iranian
journal of basic medical sciences 2017;20:538.
Moncada, S., Palmer, R., and Higgs, E. Nitric oxide: physiology, pathophysiology,
andpharmacology, Pharmacol. Rev. 1991;43 (2):109–142.
Montezano, A. C., and Touyz, R. M. Molecular mechanisms of hypertension—reactiveoxygen
species and antioxidants: a basic science update for the clinician, Can. J. Cardiol. 2012;28
(3):288–295.
Müller, L., Caris-Veyrat, C., Lowe, G., et al. Lycopene and its antioxidant role in the prevention
of cardiovascular diseases—a critical review. Critical reviews in food science and nutrition
2016;56:1868-1879.
Murphy, M. P. How mitochondria produce reactive oxygen species, Biochem. J. 2009;417 (1):
1–13.
National Pharmacopoeia Committee. Pharmacopoeia of People’s Republic of China. China
Medical Science Press, 2010; part 1.
Niki, E. Antioxidant defenses in eukaryotic cells. In: Poli G, Albano E, Dianzani MU, eds. Free
radicals: from basic science to medicine. Basel, Switzerland: BirkhauserVerlag; 1993:365–
373.
Overvad, K., Diamant, B., Holm, L., et al. Coenzyme Q10 in health and disease. Eur J Clin
Nutr 1999;53:764–770.
Panieri, E., and Santoro, M. M. ROS signaling and redox biology in endothelial cells, Cell.
Mol. Life Sci. 2015;72 (17):3281–3303.
Perez-Vizcano F., Ibarra M., Cogolludo A. L., et al. Endothelium-independent vasodilator
effects of the flavonoid quercetin and its methylated metabolites in rat conductance and
resistance arteries. Journal of Pharmacology and Experimental Therapeutics 2002;302:
66-72.
Peuchant E., Brun J., Rigalleau V., et al. Oxidative and antioxidative status in pregnant women
with either gestational or type 1 diabetes. Clin Biochem 2004;37:293–8.
Potenza M. A., Marasciulo F. L., Tarquinio M., et al. EGCG, a green tea polyphenol, improves
endothelial function and insulin sensitivity, reduces blood pressure, and protects against
myocardial I/R injury in SHR. American Journal of Physiology-Endocrinology and
Metabolism 2007;292:E1378-E1387.
Pratico, D. and Delanty, N. Oxidative injury in diseases of the central nervous system: focus
on Alzheimer's disease. Am J Med 2000; 109:577–85.
Rameshrad, M., Razavi, B. M., and Hosseinzadeh, H. Protective effects of green tea and its
main constituents against natural and chemical toxins: A comprehensive review. Food and
chemical toxicology 2017;100:115-137.
Ravindranath, V. and Chandrasekhara, N. Metabolism of curcumin: Studies with
[3H]curcumin. Toxicology, 1981; 22:337–344.

Rueda-Clausen, C. F., Morton, J. S. , Dolinsky, V. W., et al. Synergistic effects of prenatal

hypoxia and postnatal high-fat diet in the development of cardiovascular pathology in
young rats, Am. J. Physiol RegulIntegr Comp Physiol 2012;l303:R418–R426.
Ruggenenti, P., Cattaneo, D., Loriga, G., et al. Ameliorating hypertension and insulin resistance
in subjects at increased cardiovascular risk: Effects of acetyl-L-carnitine therapy.
Hypertension 2009;54:567–574.
Sen, S., et al. Free radicals, antioxidants, diseases and phytomedicines: current status and future
prospect, Int. J. Pharm. Sci. Rev. Res. 2010;3 (1):91–100.
Sena, L. A., and Chandel, N. S. Physiological roles of mitochondrial reactive oxygen species,
Mol. Cell 2012;48 (2):158–167.
Sharma, R. A., Euden S. A., Platton, S. L., et al. Phase I clinical trial of oral curcumin:
Biomarkers of systemic activity and compliance. Clin Cancer Res 2004;10:6847–6854.
Sies, H. Antioxidant Function of Vitamins. Analysis of the new Academy of Science. 1992;
669:7-20.
Simko, F., and Paulis L. Melatonin as a potential antihypertensive treatment, J. Pineal Res.
2007;42:319–322.
Smith, A. R., Shenvi S. V., Widlansky M, et al. Lipoic acid as a potential therapy for chronic
diseases associated with oxidative stress. Curr Med Chem 2004;11:1135–1146.
Srivastava, A. K., Pandey N. R, and Blanc A. Activation of mitogen-activated proteinkinases
and protein kinase B/Akt signaling by oxidative stress in vascular smoothmuscle cells:
Involvement in vascular pathophysiology, Pathophysiology of Cardiovascular Disease,
Springer, 2004, pp. 405–416.
Stolk, J., Hiltermann T. J, Dijkman J. H and Verhoeven A. J. Characteristics of the inhibitionof
NADPH oxidase activation in neutrophils by apocynin, amethoxy-substituted catechol, Am
J Respir Cell Mol Biol 1994;11:95–102.
Sugamura, K. and Keaney J. F. Reactive oxygen species in cardiovascular disease, Free Radic
Biol Med 2011;51 (5):978–992.
Szabo, C, et al., Endothelial dysfunction in a rat model of endotoxic shock. Importanceof the
activation of poly (ADP-ribose) synthetaseByperoxynitrite, J.Clin. Invest. 1997;100
(3):723.
Tagashira T., Choshi, T., Hibino, S., et al. Influence of Gallate and Pyroga o Moieties on the
Intestina A sorption of −)‐Epicatechin and −)‐Epicatechin Gallate. Journal of food science
2012;77:H208-H215.
Tain, Y. L., Chen, C. C., Sheen, J. M., et al. Melatonin attenuates prenatal dexamethasone-
induced blood pressure increase in a rat model, J. Am. Soc. Hypertens 2014;8:216–226.
Tain, Y. L., Leu, S., Wu, K. L., et al. Melatonin prevents maternal fructose intake-induced
programmed hypertension in the offspring: roles of nitric oxide and arachidonic acid
metabolites, J. Pineal Res 20014; 57:80–89.
Tain, Y. L., Lin, Y. J., Sheen, J. M., et.al Resveratrol prevents the combined maternal plus
postweaning high-fat-diets-induced hypertension in male offspring, J Nutr Biochem 2017;
48:120–127.
Tain, Y. L., Sheen, J. M., Yu, H. R., et al. Maternal melatonin therapy rescues prenatal
dexamethasone and postnatal high-fat diet induced programmed hypertension in male rat
offspring, Front. Physiol 2015;6:377.
Tamayo, C and Diamond, S. Review of clinical trials evaluating safety and efficacy of milk
thistle (Silybum marianum [L.] Gaertn.), Integr Cancer Ther 2007;6 (2):146–157.

Tiefenbacher, C. P. Tetrahydrobiopterin: A critical cofactor for eNOS and a strategy in the

treatment of endothelial dysfunction? Am J Physiol Heart Circ Physiol 2001;280:H2484–
H2488.
Toklu, H. Z., Sehirli O., Ersahin, M., et al. Resveratrol proves cardiovascular function and
reduces oxidative organ damage in the renal, cardiovascular and cerebral tissues of two‐
kidney, one‐clip hypertensive rats. Journal of Pharmacy and Pharmacology 2010;62:
1784-1793.
Touyz, R. M. Apocynin, NADPH oxidase, and vascular cells: a complex matter, Hypertension,
2008; 51:172–174.
Touyz, R. M. Reactive oxygen species in vascular biology: role in arterial hypertension, Expert.
Rev Cardiovasc Ther 2003;1:91–106.
Tsiropoulou, S., et al. Biomarkers of Oxidative Stress in Human Hypertension, Hypertension
and Cardiovascular Disease, Springer, 2016, pp. 151–170.
Turrens J. F. Mitochondrial formation of reactive oxygen species, J. Physiol. 2003;552(2): 335–
344.
Ulker, S., McKeown, P. P., Bayraktutan, U. Vitamins reverse endothelial dysfunction through
regulation of eNOS and NAD(P)H oxidase activities. Hypertension 2003;41:534–539.
Upston, J. M., Witting, P. K., Brown, A. J., et al. Effect of vitamin E on aortic lipid oxidation
and intimal proliferation after arterial injury in cholesterol-fed rabbits. Free RadicBiol Med
2001;31:1245–1253.
Vasanthi, R., ShriShriMal, H. N., and Das, D. K. Phytochemicals from plants to combat
cardiovascular disease, Curr. Med. Chem. 2012;19 (14):2242–2251.
Vasde, S., Ford, C. A., Parai, S, et al. Dietary alpha-lipoic acid supplementation lowers blood
pressure in spontaneously hypertensive rats. J Hypertens 2000;18:567–573.
Viel EC, et al. Xanthine oxidase and mitochondria contribute to vascular superoxideanion
generation in DOCA-salt hypertensive rats, Am J Physiol Heart Circ Physiol 2008;295
(1):H281–H288.
Wang, Q, Smith R. E., Luchtefeld, R., et al. Bioavailability of apocynin through its conversion
to glycoconjugate but not to diapocynin, Phytomedicine, 2008;15:496–503.
Wang S, Zhang M and Liang B. AMPKα2 deetion causes a errant expression and activation of
NAD P) H oxidase andconsequent endothelial dysfunction in vivo. Circulation research
2010;106:1117-1128.
Wu, T. H., Kuo, H. C., Lin I. C., et al. Melatonin prevents neonatal dexamethasone induced
programmed hypertension: histone deacetylase inhibition, J Steroid Biochem Mol Biol
2014; 144:253–259.
Ximenes, V. F., Kanegae, M. P, Rissato, S. R., and Galhiane, M. S. The oxidation of apocynin
catalyzed by myeloperoxidase: proposal for NADPH oxidase inhibition. Arch Biochem
Biophys 2007;457:134–141.
Xiong, X. J, Liu, W., Yang, X. C., et al. Ginkgo biloba extract for essential hypertension: A
systemic review. Phytomedicine 2014; 21(10):1131–1136.
Yang, J. and Lyn, J. Advance on study in anti-tumor mechanism of bererine (Ber).Zhongguo
Zhongyaozazhi= Zhongguozhongyaozazhi= China journal of Chinese material medica
2007;32:881-883, 934.
Zhang, D. X., and Gutterman, D. D. Mitochondrial reactive oxygen species-mediated signaling
in endothelial cells, Am. J. Physiol.-Heart Circ. Physiol. 2007;292 (5):H2023–H2031.

Zhang, L., Zheng, Y., Chow, M. S., et al. Investigation of intestinal absorption and disposition
of green tea catechins by Caco-2 monolayer model. International Journal of
Pharmaceutics 2004;287:1-12.
Zhang, W. J., and Frei, B. Alpha-lipoic acid inhibits TNF-alpha-induced NF-kappaB activation
and adhesion molecule expression in human aortic endothelial cells. FASEB J
2001;15:2423–2432.
Zhang, Z. M., Jiang, B., and Zheng, X. X. Effect of l-tetrahydropalmatine on expression of
adhesion molecules induced by lipopolysaccharides in human umbilical vein endothelium
cell. Zhongguo zhongyaozazhi= Zhongguo zhongyaozazhi= China journal of Chinese
material medica 2005;30:861-864.

Chapter 14
RADICAL APPROACHES TO STROKE
Vipin Dhote1,*, Avinash Singh Mandloi1, Durgesh Nandan Shukla1,

Priyanshi Kapoor1 and Aditya Ganeshpurkar2
VNS Group of Institutions, Bhopal, India
1
2
Shri Ram Institute of Technology-Pharmacy, Jabalpur, India
ABSTRACT
Stroke and ensuing reperfusion injury is the third major cause of morbidity and
mortality across the developed and developing nations. Neurological damage leads to
cognitive impairment, a major deficit in behavioral, visual, sensory movement and muscle
tone functions. In the absence of timely intervention, mortality seems to be the most
common outcome of stroke incidence; however, restoration of blood flow does not
guarantee complete recovery. Rehabilitation and post-injury management of stroke patient
is the key to revive and ensure the normal functioning of the patient after stroke and
reperfusion injury. Antioxidant supplements could enhance the chances of neurological
and cognitive recovery while preventing further generation of free radicals responsible for
cellular injury in massive stroke. Antioxidant actions are exhibited by agents obtained from
an array of sources. This chapter shed light on various deleterious mechanisms of free
radicals, its generation and available agents for restricting the cellular injury caused by the
formation of reactive oxygen species (ROS) in stroke.
1. INTRODUCTION
Ischemic stroke is induced by permanent or transient obstruction of blood flow to any
region of the brain which results in cellular and functional injury to cortex and hypothalamus.
The obstruction of blood flow, in turn, results in deprivation of oxygen and glucose supply
which instigate the cascade of major detrimental events responsible for cellular damage
(Kalogeris et al., 2012; Carmichael et al., 2005). Similarly, restriction or obstruction of blood
flow to any vital physiological organ lead to deprivation of oxygen and can result in severe
*
Corresponding Author’s Email: vvdhote@yahoo.com.

346 Vipin Dhote, Avinash Singh Mandloi, Durgesh Nandan Shukla et al.
ischemic condition. Restoration of the blood flow to this deprived region is the pre-requisite to
restrict and revive the cells at risk (Choi and Pile-Spellman, 2018). Paradoxically, this delayed
restoration of blood supply through occluded blood vessels lead to debilitating consequences
responsible for instigating severe metabolic energy deficiency, inflammation and leading to
permanent loss of functional cellular activity. The extensive cell injury due to this late
replenishment of the ischemic tissue or organ is termed as reperfusion injury (Aronowski et al.,
1997; Verma et al., 2002). The intensity of ischemic insult and the period of deprivation of
nutrients to cells determine the reversible or irreversible nature of stroke injury. This cell death
can be characterized microscopically by extensive swelling which may include detrimental
anatomical changes in affected tissue, deformed protein band and hypertrophic mitochondrial
structure (Piper et al., 2004).
Ischemic reperfusion injury can occur to any of the major organs in the body due to various
circumstances. However, brain and heart seem to be the most vulnerable organs to encounter
damage due to abnormal variations in blood supply. This blood flow restriction could be
transient which closely resembles clinical situations of vasospasm in a coronary artery,
resulting in angina, or cerebral hemorrhage resulting in mild to moderate cellular damage
(Kloner and Jennings, 2001; Kloner and Jennings, 2001). However, with prolong and severe
ischemia, extensive loss of functional ability with an array of pathological implications to
organ's normal physiology, collectively called reperfusion injury (Yellon et al., 2000). Stroke
or cerebral reperfusion injury occurs due to development of obstructions in a small vessel or
large artery resulting in neuronal cell death could be associated with vascular endothelial
damage or vasospasm, dysregulated homeostatic mechanism and abnormal hemodynamic
activity (Kalogeris et al., 2012).
In most of the developed and developing countries, cardiovascular diseases and stroke
account for nearly 2/3 of the total mortality and morbidity cases. Half of the estimated 32
million patients with acute vascular dysfunction are those having pre-established coronary heart
diseases (CHD) or major cerebrovascular diseases (CVD) (Saposnik et al., 2009; Hallenbeck
and Dutka, 1990). The risk of CHD and CVD recurrence is highest among pre-exposed patients
with cardiac and cerebral ischemic episodes. Stroke can lead to recurrence in survivors at about
7% per annum, while mortality is fivefold higher in patients with recurrent myocardial
infarction than those without any predisposition to CHD (Saposnik et al., 2009).
In the USA, stroke incidences are regarded as the third most relevant cause of death or
disability across all population groups. The economic and social burden entrusted by stroke
drains national coffers by the estimated annual expenditure of $ 23.2 billion. The alarming
mortality rate from stroke, more than 1,50,000 deaths per year, baffles researcher world over.
The enormity of stroke-related social and economic implications had ushered extensive
investigation of neuroprotective agents. The rehabilitation of the stroke patient is crucial for
complete recovery. One estimate highlight the plight of stroke survivors as 76% of them end
up being disabled for the rest of their life (Carmichael, 2005; Shi and Wardlaw, 2016). A large
chunk of $53.5 billion expenditure is incurred by the cost of supportive measures required for
long-term stroke survivors. Thus, stroke disables more than it kills. These facts and figures
gave an impetus to recent efforts to develop strategies for neural repair and rehabilitation after
stroke (Carmichael, 2005).
Oxidative stress generated by ROS regulates basic mechanisms of cellular dysfunction that
occur during ischemia/reperfusion injury (Granger et al., 2015; Kloner and Jennings, 2001).
Ischemia-reperfusion injury induced ROS can damage almost all vital cellular components,

Radical Approaches to Stroke 347
including cellular membranes to nucleic acids of the cells in contentions (Marczin et al., 2003).
The agents scavenging oxidative stress can be of exogenous and endogenous origin and these
compounds generally termed as antioxidants, have multifaceted activity including scavenging
of ROS, curtailing ROS generation by chelating various ions involved this process. Various
endogenous antioxidant supplementations found to be beneficial in restoring the antioxidant
enzymes during reperfusion injury (Pizzino et al., 2017; Hill et al., 2011). However, the
exogenous supplementation of lipoic acid (Dong et al., 2015; Cao and Philis, 1995; Shmonin
et al., 2012), coenzyme Q10 (Liang et al., 2017; Lu et al., 2017) was found to be protective in
the reperfusion injury induced animals.
Numerous reports highlighted the potential of antioxidants and their combinations with
other neuroprotective agents on the reperfusion injury of brain and heart. The studies
emphasizing the role of antioxidants in the management of stroke would further help to clarify
the controversies associated with the exact mechanism of antioxidants and its clinical
applicability in ischemia/reperfusion injury. Herein, the chapter explores the multifaceted
mechanisms involved in ischemia and reperfusion injury in the brain. The fundamental ROS
generating mechanisms, as well as the endogenous scavenging tools for limiting the oxidative
stress injury, are discussed in details. This section also provides an overview of the therapeutic
as well as experimental approaches available for the treatment of cerebral stroke and
reperfusion injury.
2. PATHOPHYSIOLOGICAL CONDITION RESPONSIBLE

FOR PROGRESSION OF STROKE AND CEREBRAL INJURY
Cerebral infarction may result from one or more pathophysiological mechanism, which
includes atherosclerosis, cardioembolism, reduced systemic pressure, hematological disorder,
and metabolic disorder. The diseases of large arteries and small vessels along with channel
disorders can also play a vital role in the initiation of cerebral damage.
2.1. Atherosclerosis
A pathological condition where lipids deposits, generally calcified on the innermost intimal
wall of the blood vessel which in progression hardens these arteries is termed as atherosclerosis.
Plaques develop especially at bifurcations of major arteries including carotid artery. The
biochemical processes ranging from fatty acid to formation of streaks leading to plaque
formation due to excessive proliferation of smooth muscle cells. Moreover, an already hardened
wall narrow down lumen and obstructs blood flow (Al Kasab et al., 2018). This obstructing of
blood flow leads to turbulence in blood and increases kinetic energy, which dislodges the
plaque in the bloodstream. Thus obstructed and narrower blood vessels restrict blood flow
sometimes resulting in thrombus formation. Of all stroke cases, almost 33% cases have an
association with atherosclerotic thrombosis (Kannel et al., 1971).

2.2. Cardioembolic Stroke
Cardiovascular dysfunction leads to ischemic stroke. The common disorders are familial
arterial myxomas, hereditary conduction defects and hypertrophic obstructive cardiomyopathy
(Hassan et al., 2000).
2.3. Reduced Systemic Pressure
Multiple mechanisms responsible for stroke and related complications arise from
obstruction of blood flow in cerebral arteries leading to ischemic damage in and around the
localized region (Alloubani et al., 2018). Turbulent blood flow weakens the walls of cerebral
and cardiac vessels. This would greatly affect normal hemodynamic functions and result in
altered systemic pressure among vital arteries adding to the factors responsible for the
development of ischemia (Hademenos and Massound, 1997).
2.4. Hematological Disorders
Several mechanisms are suggested for the increased risk of ischemic cerebral stroke in
patients with sickle cell disease; these include sickling of red blood cells during a crisis and
thrombotic infarction of both large and small arteries (Ilesanmi, 2010). Patients with sickle cell
anemia are more prone to develop stroke as compared to others.
2.5. Small Vessel Disease
In CNS, blood supply to deep white matter and basal ganglia is obstructed due to the
structural changes in the small penetrating end arterioles is the hallmark of small vessel disease.
Lacunar stroke syndrome is the most common clinical phenotype of this malformation. These
lacunar infarcts may lead to cognitive impairment and disorder of gait (Shi and Wardlaw,
2016).
2.6. Vasospasm
Spasticity of subarachnoid spaces due to sudden and excessive bleeding coupled with
muscular contractions is known as cerebral vasospasm. The vasospasm may aggravate focal
constriction leading to total occlusion (Wilkins, 1988). Any cerebral blood vessel encountering
vasospasm, for the varied time period, induces cerebral ischemia.
3. TREATMENT STRATEGIES FOR ACUTE ISCHEMIC CONDITIONS

Increased understanding of the biological implications of ischemic injury led to the
development of various treatment strategies to limit the extent of injury in the subject. The

primary objective of the treatment is to assure immediate survival of the patient and to restrict
the damage in critical ischemic condition. However, the major limitation of the current
development is lack of ability to prevent cell injury in the sudden ischemic milieu. Initiation of
necrotic and apoptotic cell death in core and penumbra is contributing to the neurological deficit
and impaired locomotors activity. The current drug discovery and development efforts need to
focus on recovery as well as management of the ischemic cells and related physiological
functions (Zheng et al., 2006).
3.1. Management of Acute Ischemic Stroke
Evident neurological deficit and a history of abrupt onset of symptoms in the absence of
trauma suggest the onset of a stroke. Restoration of blood flow to threatened vulnerable tissue
incidentally before it progress to infarction presents realistic opportunity to salvage penumbral
tissue (Kalogeris et al., 2012). The reperfusion therapies significantly restrict infarct area and
provide a window to limit cognitive deficit as well as bring a marked improvement in
neurological outcome. General treatment includes the administration of supplemental oxygen
tissue and isotonic intravenous fluid and anti-thrombotic/fibrinolytic therapy (Snow, 2016).
3.1.1. Intravenous Thrombolytics

Tenecteplase (TNK), a genetically modified form of tissue plasminogen activator (tPA) is
highly selective for fibrin and prevents any inhibition of plasminogen activator type 1. The long
lifetime of TNK allows it to administer in a single bolus dose and its specificity to fibrin ensures
complete clot lysis (Snow, 2016). Presently, desmoteplase, abundantly available in the saliva
of vampire bats, Desmodus rotundus, induces faster recanalization than tPA and is clinically
evaluated in phase 2 trials of ischemic stroke (Molina and Saver, 2005). A recombinant peptide
developed from human tPA consists of kringle 2 and protease domains of human tPA and works
as a potent recanalizing agent and this recombinant tPA id marketed as reteplase. Similarly,
antagonists of glycoprotein (GP) IIb/IIIa are shown to have the ability to improve flow by
dissolving platelet-rich clots in coronary and cerebral microcirculation (Molina and Saver,
2005; Snow, 2016).
Intra-arterial thrombolytic therapy can be performed with a microcatheter that is placed
into, beyond, and proximal to the arterial occlusion. Urokinase is conventionally used while
intraarterial tPA and prourokinase have been extensively studied in recent investigations
(Snow, 2016).
3.1.2. Combined Pharmacological Approach

The combination therapy is put forth to expand the fibrinolysis time beyond 3 h. The
combination therapies studied are anti-thrombotic (reteplase) and fibrinolytic (abciximab)
provided faster, consistent and sustained reperfusion (Broussalis et al., 2012). Treatment with
neuroprotective strategy (hypothermia) with thrombolytic (tPA) agent was promising.
Neuroprotective stabilizes threatened tissue and extends the time window for the subsequent
administration of reperfusion agent. The other neuroprotective strategies used in ischemia are
glutamate receptor antagonist, calcium channel inhibitors and antioxidants (Mestre et al., 2013;
Molina and Saver, 2005). Administering agents that are vasoprotective (matrix

metalloproteinase inhibitor batimastat) along with tPA may improve recanalization rates and
improve the permissible time window for reperfusion therapy by enhancing the benefit/risk
ratio in stroke as well as myocardial infarction incidences (Molina and Saver, 2005).
3.1.3. Sonothrombolysis
Although tPA is the most favored option in the management of ischemic stroke, its
transportation and delivery to the site of thrombus are crucial to achieving desirable outcomes.
Recent clinical evidence has demonstrated the ability of ultrasound technique to enhance
enzymatic thrombolysis (Teng et al., 2017; Chen et al., 2014). Timely application of optimum
frequency of ultrasound helps in transporting tPA into the thrombus and promoting the opening,
cleavage of the fibrin polymers (Marczin et al., 2003).
Most of the strategies discussed above are aimed at providing the prompt and complete
reperfusion of the severely ischemic tissue. Reperfusion injury is the paradox of recanalization
of obstructed arteries resulting in major cellular dysfunctions (Aronowski et al., 1997). The
recent research in stroke highlighted the implication of sudden reperfusion-induced cellular
damage and hence the emphasis, in drug development for stroke treatment, is on understanding
mechanisms of cell injury due to stroke and designing strategies to effectively counter
reperfusion injury (Cuzzocrea et al., 2001).
In the last two decades, the research on reperfusion injury is revolving around the concept
of oxidative stress management and the restoration of the metabolic balance of the cells prone
to injury (Marczin et al., 2003; Cuzzocrea et al., 2001). Various herbs (Thiyagarajan and
Sharma, 2004; Du and Ko, 2006), hormones (Zhai et al., 2000; Zhao et al., 2015) and chemical
agents (Dong et al., 2015) were experimentally evaluated for its effect on reperfusion injury.
Few of these agents were found to be effective but none of these could be taken to the next step
of developing an ideal therapy for the reperfusion injury.
4. CROSSTALK BETWEEN OXIDATIVE STRESS AND CELLULAR INJURY

The impact of reperfusion injury related to ischemic myocardial and cerebral conditions is
enormous and this enormity of the cerebrovascular and cardiovascular diseases has driven the
scientific world in the pursuit of the ideal therapy or strategy to restrict the inflicted damage
(Saposnik et al., 2009). Coronary heart diseases and coronary heart diseases take a heavy toll
on the population of lower and middle-income group countries. Almost 75% of the CVD burden
in the form of mortality and disability of the productive human resource is bored by this group
of countries (Alves et al., 2016).
The scarcity of data makes more difficult to analyze the actual extent of these diseases in
the developing countries. The major reason for the slow and agonizing progress towards
developing an intervention to treat these pathological complications is the absence of a strategy
for secondary prevention in CVD and CHD (Alves et al., 2016; Saposnik et al., 2009). The
intriguing mechanisms of stroke have always been a quagmire for researchers as well as
clinicians. Hence, the lack of therapeutic response to interventions targeting a single
mechanism has warranted a study design that analyzes the approach of a combination of various
pharmacologically active drugs. This approach gives us the liberty to study the effect of

multiple active drugs acting independently or synergistically in the combination to target more
than one underlying mechanism in stroke or reperfusion injury (D'Ascenzo, 2015).
To start with, the extensive review of the mechanisms of injury involved in cerebral
ischemia/reperfusion injury was carried out. Various factors assisting the ischemic damage and
ensuing disability are discussed in detail while diagrammatic representation provides a view of
ROS generation as shown in Figure 1.
Figure 1. Generation and metabolism of reactive oxygen species. Superoxide radical (O2.-) is produced
by ultraviolet radiations, stress, DNA damage, and atmospheric pollution. Superoxide is converted by
superoxide dismutase (SOD) to H2O2, which in turn is reduced to water by catalase and glutathione
peroxidases (GPx). In the presence of Fenton reaction (iron and hydrogen peroxide are capable of
oxidizing a wide range of substrates), H2O2 can undergo spontaneous conversion to hydroxyl radical
(OH•), which is extremely reactive. GPx neutralizes hydrogen peroxide by taking hydrogen from GSH
molecules resulting in two H2O (water) and one GSSG. GPx oxidizes GSH to GSSH with the help of
GR. SOD, Superoxide dismutase; CAT, Catalase; GPx, Glutathione peroxidase; H2O2, Hydrogen
peroxide; GSH, Glutathione; GR, Glutathione reductase; GSSH, Glutathione disulfide.
4.1. Oxidative Stress Induced by ROS Generation in Myocardial

Reperfusion Injury
The myocardial injury is accentuated by the generation of free radicals from different
sources during the process of reperfusion injury. ROS can be termed as any chemical species
that contains one or more unpaired electrons in outer most orbit making the molecule unstable

and highly reactive. Hence it quickly gets coupled with an adjacent molecule by sharing an
electron. This conjugation chemically modifies itself and also become excessively reactive
owing to the availability of an extra electron. Consequently, the adjacent molecule follows the
pattern of this oxidation and kick starts the chain reaction to generate ROS and in turn damage
cell structures (Jennings and Steenbergen, 1985). The free radicals are generated in three ways:
1. Homolytic cleavage of the covalent bond

2. Loss of electron from a stable molecule
3. Addition of an electron to a stable molecule
This generation of free radical depends on the stimuli exerted to the cell. The ROS and
others are generated by nitrogen termed as reactive nitrogen species (RNS) (Mimic-Oka et al.,
1999). ROS can be divided into radicals and nonradicals. Radicals are superoxide radical (O2.-
), hydroxyl radical (OH•), peroxyl radical (RO2•) and alkoxyl radical (RO•) come in this
category. The nonradicals are hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and
singlet oxygen (1O2). Nitric oxide (NO) and nitrogen dioxide (NO2) are two major nitrogen
species responsible for cell damage. The nonradicals in this category are nitrous oxide (HNO2),
peroxynitrite (ONOO-) and alkyl peroxynitrites (ROONO) (Cuzzocrea et al., 2001).
The initial surge in ROS generation during reperfusion has a unique mechanism and
different source of generation than ischemic milieu. However, the exact source of ROS
generation is not yet precisely identified but these cellular redox conditions are thought to be
responsible for regulation of vital cell pathways (energy metabolism, survival/stress, and
inflammatory reactivity). These are reports contesting conventional views about the exact role
of ROS in cerebral as well as myocardial reperfusion injury (Marczin et al., 2003).
Generation of free radicals occurs when oxygen gets reduced though univalent pathway,
however, the percentage of total oxygen reduction by univalent pathway is just 5%. Generally,
the reduction of 95% oxygen is carried out by mitochondrial electron transport using tetravalent
reduction pathway to H2O where no ROS is produced. Superoxide anion is formed when
oxygen accepts an electron and establishes equilibrium with protonated form HO2• which is
favored and more reactive especially during ischemic acidosis conditions (Jones et al., 2003).
Dismutation (SOD) of O2 to H2O2 is the key to prevent potential oxidative cellular damage by
RO2•. Moreover, it is acknowledged that H2O2 is only directly toxic only at high concentration
which is unlikely to occur under normal conditions. The cells have the endogenous mechanism
to neutralize this formed H2O2 by catalase and glutathione system. Superoxide formed as a
byproduct of the respiratory chain is safely neutralized to water. However, in
ischemia/reperfusion condition this delicate balance is destabilized (Lefer and Granger, 2000).
As the ischemia intensifies the defensive mechanism of the cells deplete and H2O2 become more
empowered to generate the destructive hydroxyl radical (OH•) (Sugamura and Keaney, 2011).
These hydroxyl radicals are highly reactive and are implicated in lipid peroxidation, destruction
of protein as well as sulfhydryl bonds of the cellular structures. Similarly, metal ions like iron
and copper are responsible for generating free radical. Especially the chelation of metals is
considered to be an approach to reduce oxidative stress owing to the vital role played by iron
and copper in oxidative stress-induced cellular damage (Jomova and Valko, 2011).
The Haber-Weiss reaction generates OH· from O2˙ˉ and H2O2, accelerates the non-
enzymatic oxidation of molecules such as epinephrine and glutathione (Jomova and Valko,
2011).

O2·- + H2O2 O2 + HO· + HO-
Oxidative stress induced by ROS generation during reperfusion injury is considered as a

defining factor for exacerbating the cell injury in ischemia region of any vital organ (Pizzino et
al., 2017). As the reintroduction of oxygen to ischemic cells produce oxygen free radicals after
sequential reduction of the oxygen molecule, the formation of superoxide anion, hydroxyl
radical, and peroxynitrite is envisaged in early first few minutes of reflow (Kalogeris et al.,
2016). Other major contributing components in the generation of ROS are enzymes, such as
xanthine oxidase, cytochrome oxidase, and cyclooxygenase, and the oxidation of
catecholamines.
In addition to free oxygen radicals, reperfusion also increases inflammatory reactivity
through activation of neutrophils (Vinten-Johansen, 2004), consequently, this infiltration of
neutrophil also serves as stimuli for generation of ROS. Lipid peroxides are a precursor for
lipid peroxidation which impairs functions of enzyme system which are bound to membranes
(Rubbo et al., 1994). Poly-unsaturated lipids serve as a primary source of lipid peroxides after
interaction with highly reactive free radicals. Endothelial dysfunction occurring in excessive
oxidative stress can be traced back to improper release as well as the sensitivity of vessels to
nitric oxides which may precipitate in microvascular dysfunction (Radi et al., 1991).
4.2. Key Mechanisms Responsible for Cellular Injury in Stroke
Few realms of disease are as devastating as neurological ones, because such damage can
destroy our very essence of life. Stroke and following reperfusion injury is a major source of
disability and thus much research is being placed on the treatment and prevention neurological
well being (Snow, 2016). Various neuroprotective strategies include free radical scavengers,
excitotoxicity inhibitors, and calcium channel blockers. Numerous new interventions are being
investigated for the prevention of the severe damage caused by the cerebral reperfusion injury
(Molina and Saver, 2005). To list, few of these are various hormones, tissue plasminogen
activators and antibodies to adhesion molecules.
Pathogenetically, stroke integrates a heterogeneous group of diseases. Maximum
incidences of stroke occur due to occlusion of vessels and account for 85% of all strokes, while
few cases represent excessive intracerebral bleeding. Herein, emboli formation in arterio-
arterial or cardiac origin is responsible for 75% of all cerebral vessel occlusions and resultant
ischemic injury (Soler and Ruiz, 2010). In rare cases, approximately 5% cases of stenoses of
cerebral arteries infarction may result in massive neurological deficit (Pongmoragot and
Saposnik, 2012; Kumral et al., 2012). The reperfusion of ischemic brain brought by various
interventions leads to cellular injury. There are numerous murine models for the induction of
focal cerebral ischemia which resembles clinical ischemic stroke (Pant, et al., 2012).
Cellular injury in the brain following stroke is a consequence of multifactorial events
occurring with a very short span of time. If the resourceful blood supply is not restored within
3 minutes of the recovery window the cells start experiencing energy deficit which generally
ends up in cells resorting to apoptosis or even experience sudden necrotic damage (Piper et al.,
2004; Kalogeris et al., 2012; Carmichael, 2005). Deficiency of oxygen and glucose to high
demand organ like brain potentially reduces its functional ability and accumulates toxic

metabolites which even induce structural damage to cells of the brain (Kalogeris et al., 2012;
Granger and Kyietvs, 2015).
The duration and extent of deficient blood flow determine the changes and sometimes the
reversibility/irreversibility of the damage to affected cells. Consequently, this situation
regulates the intensity variables responsible for future changes (Aronowski et al., 1997).
Alteration of electron transport and reduced ATP are two vital variables modulating the extent
of blood flow reduction while ROS generation is the third one partially dependent on flow rate
(Katsura et al., 1993). However, oxidative stress and ROS generation is a complex phenomenon
which also includes the role of electron transport and available oxygen in its activation (Dirnagl
et al., 1995). Hence it will be omnipresent even if blood flow is minimum, as slight as just
above zero. Incidences of ischemia induce specific and extensive changes which result in cell
death. The direct inhibition of oxidative phosphorylation is one of the most important activities
in this cascade of events which include decreased pH, reduced ATP availability, ROS
generation and increased accumulation of Na+ leading to loss of ATP substrate for the Na+-K+
pump. The significant change in ionic concentration initiates secondary changes which finally
result in damage of ischemic cells (Lipton, 1999).
Almost all focal ischemic models which closely resemble clinical settings involve
occlusion of one middle cerebral artery (MCAO) (Ginsberg and Busto, 1989). The partial
occlusion of blood flow with a nylon filament induces significant gradation of ischemia from
the core of the lesion to penumbra which is an area at risk of cell death lying on the outermost
boundary. The core and penumbra of the ischemic lesion exhibit different metabolic conditions
within the affected ischemic site. Modification of MCAO can be achieved by coating the
filament with poly-L-Lysine or silicon and inserting it into the carotid artery to be advanced at
the origin of a middle cerebral artery. This technique generally ensures minimum variation in
lesion size and blood flow restriction (Guan et al., 2012). With MCA occlusion and sudden
reperfusion of the affected region induces large infarct area of 250 mm3 at 24 h in rats (Guan
et al., 2012; Koizumi et al., 1986; Aspey et al., 1998). Transient obstruction of blood flow
increases the temperature in the core area up to 1.5oC due to reduced circulation and irreversible
damage to hypothalamus as observed in Wistar rats. In addition, ischemia damages the
endothelium of small arteries and also weakens muscles (Nishigaya et al., 1991). The core area
of ischemic lesion comprises of the lateral portion of the caudate putamen and somatosensory
cortex while the penumbra which can be revived consists of the neocortex and medial caudate-
putamen (Belayev et al., 1997). The ischemia and reperfusion injury can affect the brain
extensively and events involved in this injury revolve around the ionic balance and ROS
generation.
5. MAJOR BIOCHEMICAL CHANGES

AND CELLULAR MECHANISMS OF CEREBRAL INJURY
Detailed insight and molecular changes in ischemic cells can provide more information
about the extent of injury as well as regulates the chances of recovery from initial insult due to
severe ischemia in affected cells.

5.1. Ionic and Metabolic Changes in the Core and Penumbra
The biochemical and ionic changes triggered by stroke strikingly different in core and
penumbra. These differences in metabolism strongly suggest a different mechanism for cell
death in these two regions (Duverger et al., 1988). The infarct and edema in core and penumbra
is the result of massive ion homeostasis disruption and the influx of water into cells of affected
tissue (Back et al., 1995). The critical window of 1-3 minutes, experiencing rapid anoxic
depolarization with a concomitant rise in extracellular K+ to 70 mM and decrease Ca2+ at flow
levels entering in a cell that is present in the core are critical for the cellular death (Martin et
al., 1994). During the reperfusion period, after 2 h temporary focal ischemia, extracellular K+
returns to control levels for 6 h before rising slightly or the remainder of a 24-h recirculation
period (Gido et al., 1997). Despite this return to near normalcy, the insult produces severe
damage in the form of infarct encompassing entire core (Fiolet and Baartscheer, 2000) and
further development of tissue edema due to water influx in the cells. These changes account for
the osmotic lysis and the lytic cell death is also referred to as necrosis, which is as a hallmark
of the core of the infarct (Nagasawa and Kogure, 1989).
However, the limited damage to penumbra or risk of damage to the area receiving collateral
blood supply through smaller arteries is due to lack of permanent anoxic depolarization with
concomitant ion changes during the transient period (Back et al., 1995). Moreover, the ATP
deficit, which generally maintained in this area at 50–70% of normal, do not fall enough to
allow the anoxic depolarization. However, some transient depolarization (Ginsberg and
Pulsinelli, 1994; Mizoue et al., 2015) actually originates from glutamate release in the infarct
core but prompt reperfusion can salvage the area at risk of damage due to infarct.
5.2. Energy Metabolism in Stroke
The ATP levels in core fall to 25% of basal values and remain there between 5 min and 4
h of ischemia during transient ischemia (Folbergrova et al., 1992). The damage is severe even
after restoring the ATP level up to two-thirds of the normal levels with 4 h of ischemic period.
Whereas the ATP levels in penumbra remain around 50-70% of normal during this ischemic
period (Folbergova et al., 1995). The glucose utilization fluctuates during the early times and
3.5 h of the ischemia. During ischemia, the blood flows in penumbra declines but it is still much
higher than that of the core. Glucose utilization in the cells of contention is one of the reliable
indicators of the extent of the damage to core or penumbra (Belayev et al., 1997). Even after
complete reperfusion, the required levels of ATP cannot be replenished to their pre-ischemic
values (Piper et al., 2003; Kalogeris et al., 2012). In some cells of penumbra glucose utilization
is reduced to well below normal during early reperfusion, and if not restored the cells can later
become part of the infarction (Robbins and Swanson, 2014). Reperfusion cannot guarantee
prevention of damage despite the rapid recovery of ion homeostasis recovery by reoxygenation.
Any single intervention is unable to limit or recover the damage to cells when ischemia lasts
for 2-3 h. It will be advisable to administer drugs alone or in combination well before
reperfusion to garner the maximum benefits of these interventions in stroke.

5.3. Free Radical Generation in Cerebral Reperfusion Injury
In normal conditions, homeostasis is maintained between free radical generation and

elimination of it which is very well tolerated by the cells. However, ischemia-reperfusion injury
represents a peculiar sequence of events leading to excessive generation of ROS like
superoxide, hydrogen peroxides and hydroxyl radicals. Similarly, the scavenging activity of
these ROS is deficient at the massive extent which ultimately leads to dysfunction of the cells
in the event of increased oxidative stress (Hammer et al., 1993). Highly reactive peroxynitrite
radical, generated from the reaction of superoxide and nitric oxide, can react with virtually any
cellular component (carbohydrates amino acids, DNA, and phospholipids) and damage them
(Iadecola, 1997).
5.3.1. Xanthine/Hypoxanthine Oxidation

Initial stages of ischemia, first 10 minutes after onset, lead to the breakdown of adenine
nucleotides which may be metabolized by xanthine oxidase (XO) or xanthine dehydrogenase
(XDH). Out of these two, XDH converts adenine nucleotides to nontoxic metabolites but XO
generates free radicals which are higher in the brain and other vital organs like liver and
intestine (Kinuta et al., 1989). Generation of superoxide radical and hydrogen peroxide in
expressed in the given reaction:
Hypoxanthine + O2 O2·- + H2O2 + Urate
5.3.2. Nitric Oxide and Peroxynitrite Generation

Highly reactive peroxynitrite is formed as a result of the reaction between nitric oxide and
superoxide which both appears to rise during focal ischemia. Neuronal or endothelial NOS
generates NO by activation of Ca2+/calmodulin in most neurons and endothelial cells (Dawson
et al., 1994). This Ca2+/calmodulin movement directly activates the NOS enzyme and may also
remove a phosphorylation-induced block by activating calcineurin (Yan et al., 2004). During
ischemic episode upregulation of both neuronal and inducible NOS initiates reactions resulting
in the generation of peroxynitrite:
O2·- + NO· ONOO
5.3.3. Eicosanoid Accumulation in Ischemic Brain Regions

Generation of eicosanoids is one more contributing factor for oxidative stress-induced
damage. Accumulation of arachidonic acid, via cyclooxygenase (COX) or lipoxygenase (LOX)
pathway, serves as a substrate for oxidative metabolism and produces superoxides and hydroxyl
radicals (Katsuki and Okuda, 1995; Kontos, 1986; Ikeda and Long, 1990). Break down of
phospholipids into Arachidonic acid is increased in ischemia manifold due to the presence of
free fatty acid (FFA) which also results in an increased free radical generation (Au et al., 1985;
Nakano et al., 1990). During transient ischemia activation of NOS and COX is closely related
to ROS generation within 6-24 h period. Similarly, global ischemia also follows an identical
path for the free radical generation. The iNOS is present in endothelial cells and invading
neutrophils (Nakano et al., 1990), whereas the COX-2 regulated ROS generation in neurons. In
the reperfusion phase of injury, COX-2 (prostaglandin H synthase) produces superoxide

radicals and iNOS generated NO spearheads the destruction of affected cells. However different
cells do have a mode of activation of NOS and COX enzymes (Ahmad et al., 2009; Beckman,
1994).
5.3.4. Ischemia-Induced Alteration in Mitochondrial Function

Free radicals are generated at a brisk pace by the organelles in mitochondria. When electron
carried becomes highly reduced, as much as 2% of the total electrons undergo single-electron
reduction of oxygen molecule ultimately forming superoxide (Freeman and Crapo, 1982).
These reactions very prominently occur in focal ischemia. Accumulation of Ca2+ and opening
of mitochondrial permeability transition pore (MPT) at some stage of ischemia assist in the
generation of free radicals.
5.3.5. Role of Monoamine Accumulation

Monoamine oxidase metabolizes accumulated catecholamines to H2O2 within first 5
minutes of ischemia. Superoxide and NO together produce peroxynitrite which mediates much
of its toxicity (Birben et al., 2012). Ischemia also brings down the pH of the cell which favors
the generation of OH. (Birben et al., 2012; Dirnagl et al., 1999) and also by free iron (Sun et
al., 2018), as shown below in the reaction generally termed as Fenton reaction:
Fe2+ + H2O2 Fe3+ + OH- + OH
5.4. pH Changes during Stroke
Severe impairment of blood flow during cerebral ischemia leads to a decrease in

intracellular pH (pHi) by 0.5-1 unit. Interestingly pHi at core falls to 6.6, 6.2, and 6.1 when
blood flow is reduced to 25% at 10 min and to 10% by 3 and 4 h respectively. However,
penumbra at the same time (3h after ischemia) had no significant change in pHi even after a
40% reduction in blood flow (Meyer et al., 1986). Cerebral damage could be exacerbated by
this increased acidity due to change in the rate of ROS generation and hence the pH profile
during reperfusion is a vital parameter as well as a major contributing factor. Early dip in pHi
is associated with immediate blockade of oxidative phosphorylation and the glycolytic flux
(Lemasters et al., 1996). Deficient ATP supply also activates glycolysis stemming leading to
increased lactate concentration as blood glucose increased and fall in pHi (Banani et al., 2000).
These events during focal termed as lactate–acidosis-hypothesis is frequently cited in order to
explain the glucose paradox of cerebral ischemia and following cellular injury. The paradox is
that there is experimental evidence that high supply of glucose, the most important source of
energy of the brain, in cerebral ischemia surprisingly induces more damage instead of limiting
it as would have expected due to replenishment of ATP during reperfusion. However, this view
of paradox is contested by another school of thought (Payne et al., 2003).
5.5. Anoxic Depolarisation and Na+-K+ Pump in Cerebral Reperfusion Injury
Anoxic depolarization is the unique feature of anoxia/ischemia in vivo, this anoxic

depolarisation is observed especially in the core region and not in the penumbra (Chen et al.,

1993). The significant extracellular negative direct-current shift (10-30 mV) in cell bodies of
the ischemic tissue is often accompanied by the precipitous decrease in extracellular Na+, Cl-
and Ca2+ and considered as a characteristic feature of this phenomenon. Anoxic depolarization
needs ATP for its initiation and the condition like hypoglycemia where the rate of ATP
depletion is increased in hippocampal slices shortens the time to anoxic depolarization (Lipton
and Whittinghan, 1984). The average value of ATP is 40% at the time of anoxic depolarization
and the depolarization occurs within 5 sec of the time at which ATP suffered a precipitous fall
(60-90 Sec). This anoxic depolarization is due to Na+-K+ pump inhibition as it is essentially
independent of extracellular Ca+ but produces rapid changes in ion concentrations (Nishizawa,
2001). It is still to establish that the pump inhibition is indeed the basis for the anoxic
depolarization but the shift in K+ could be responsible for the majority of the depolarization
(Ferrazzano et al., 2011) and indicates that the neuronal damage is strongly correlated with
depolarization (Kaminogo et al., 1998).
5.6. Inflammation in Reperfusion Injury
As discussed earlier iNOS and COX-2 enzyme do regulate inflammatory reactivity in

cerebral ischemia but the role of cytokines generated from activated leukocytes play a crucial
role in adhesion and transmigration in brain parenchyma. Within a few hours of onset of
ischemia, the expression of adhesion molecules at the vascular endothelium and circulating
leukocytes induces stroke-induced brain inflammation (Ahmad et al., 2009). Neutrophil
granulocytes accumulation in the cerebral microvessels of the penumbra disrupts
microcirculation. Similarly, activated leukocytes (granulocytes, monocyte/macrophages,
lymphocytes) neurons and glial cells (astrocytes, microglia) produce cytokines and chemokine.
TNF-α, IL-1, and IL-6 are proinflammatory, play an important role as mediators of the
inflammatory phase (Del et al., 2000), while IL-1 β acts as a substrate for free radical generation
and inflammation. Anti-inflammatory cytokines like TGF-β1 and IL-10 downregulates
inflammation and have a protective effect in the context of cerebral ischemia (Bogdan et al.,
1992). The iNOS and especially COX-2, by means of the production of free oxygen radicals as
well as prostanoids, have a more detrimental effect on the penumbral tissue (Del et al., 2000).
5.7. Phospholipid Metabolism
Phospholipids serve as a major tool for the execution of membrane function and signaling
capabilities due to the presence of phosphoinositides (Zhang and Sun, 1995). During ischemic
episodes, the breakdown of phospholipids increases the levels of FFA and potentially enhances
the rate of ROS generation. The reperfusion, in turn, activates arachidonic acid and result in
greater free radical formation. This upregulated arachidonic acid and FFA concentrations and
observed in CA1 for more than 1 day after 5 min of ischemia (Abe et al. 1991). Total FFA was
increased 4- to 10-fold between the first 15 min and the end of 60 min of MCA occlusion
(Zhang and Sun, 1995). The concentration of FFA is dependent on the duration of the ischemic
period and the onset of reperfusion.
Metabolism of phospholipids is coupled with large Ca2+ influx and energy failures. In
addition to energy failure, activation COX and free radical generation during cerebral ischemia

present a conducive environment to initiate apoptosis (Rao et al., 1999). It also activates the
platelet activating factor which is also responsible for the explicit increase in Ca2+ in neurons.
The membrane damage induced by this phospholipids break down is of importance in an
organelle other than plasmalemma (e.g., mitochondria). Even the loss in phospholipids content
is small in the nonmitochondrial membrane may be small but the studies emphasize the
importance of membrane damage (Fukai et al., 2011).
5.8. Permeability of Blood-Brain Barrier in Stroke
The integrity of the blood-brain barrier (BBB) depends on the interaction of the cellular
matrix, which is composed of endothelial cells and astrocytes. Because of cerebral ischemia,
this cellular matrix and the intercellular signal exchange is damaged. Proteases, especially
matrix-metalloproteases (MMPs) play an important role in the disruption of the basic fabric of
BBB. Upregulation of MMP-9 in endothelial cells during the first 24 h of permanent ischemia,
is a result of cytokines production (Gottlieb, 2011) while extracellular edema development is
due to activation of Na+ transport across the BBB (Simard et al., 2007). Inflammatory reactivity
increases temperature and as BBB is highly sensitive to temperature variation and this
temperature sensitive nature of BBB causes extensive brain damage to endothelial tight
junctions. Once the permeability of BBB is increased the transfer of leukocyte recruitment
mediators across, creates havoc with respect to cellular damage during ischemia in a rat model
of stroke (Venkat et al., 2017).
5.9. Apoptosis
Although the majority of cell death stroke occurs due to necrosis but the role of apoptosis
cannot be ignored (Buja et al., 1993; James, 1994). Apoptosis is programmed cell death in the
event of deficient energy still aerobic state of metabolism. Programmed cell death proceeds in
a genetically programmed series of biochemical and morphological steps designed to avoid the
indiscriminate release of cytosolic contents and the ensuing inflammatory response (Martin et
al., 1994). The salient features of this type of cell death are chromatin condensation,
endonuclease fragmentation of internucleosomal DNA to multiples of 180 to 200 base pair
fragments, and cell fragmentation into small membrane-bound vesicles. On the other hand,
necrosis, a catastrophic breakdown of cellular homeostasis occurs if oxygen supply crosses the
minimum required level. It was however observed that apoptosis is an actively regulated
process of cellular self-destruction, which requires energy and de novo gene expression, and
which is directed by an inborn genetic program. The final result of this program is the
fragmentation of nuclear DNA into typical nucleosomal ladder (Majno and Joris, 1995; Yuan
et al., 2016).
Apoptosis, a programmed cell death takes place in a similar time frame as inflammation
and involves caspase enzymes resulting in the development of characteristic histological
features of apoptosis are membrane-blebbing, chromatin condensation, shrunken cytoplasm
with intact organelles, apoptotic bodies in destructed cells (Majno and Joris, 1995; He and
Simon, 2013).

Caspases are a unique class of aspartate-specific proteases (McIlwain et al., 2013), which
targets nuclear proteins; proteins involved in signal transduction, and cytoskeletal targets (Yuan
et al., 2016; Oliver and Vallette, 2005). Internucleosomal DNA fragmentation requires the prior
cleavage of a cytoplasmic inhibitor of the apoptosis-specific endonuclease (inhibitor of
caspase-activated DNAse, DNA fragmentation factor) (Ktazumi and Tsukahara, 2011) and
TNF-α mediated caspase activation (Teringova and Tousek, 2017; Kichev et al., 2014).
Multiple isoforms of caspase especially, caspase 3 has a central part in the apoptotic signaling
cascade, not only in cerebral ischemia (Friedlander, 2003).
DNA-repair enzymes like poly (ADP-ribose) polymerase (PARP) and DNA-dependent
protein kinases (DNA-PK) are the substrates that are metabolized by caspase-3 (Friedlander,
2003). PARP identifies single strand disruption and mediate DNA-repair through the
generation of ADP-ribose polymers. However, PARP reduces the energy pool of the cell
because it consumes ATP. Although partially controversial, the use of ATP by PARP is
considered cytotoxic (Yu et al., 2002). The cleavage of the inhibitor of caspase-activated
DNAse (ICAD) is of particular importance, as caspase-activated DNAse (CAD) is activated by
this mechanism. CAD leads to the characteristic DNA-laddering. Besides the destruction
genetic information, structural proteins (e.g., laminin, actin, gelsolin) that are essential for the
integrity of the nucleus and the cell are cleaved by caspases (Cao et al., 2001; Niizuma et al.,
2010).
Antioxidants reduce the extent and rate of apoptosis, hence oxidative stress is expected to
constitute an important intermediary step in signaling cascade of apoptosis (Ludwig et al.,
1996). Apoptosis can be prevented by proteins or survival factors like interleukin-3, nerve
growth factor (NGF), insulin-like growth factor-1 (IGF-1), or platelet-derived growth factor.
Many neuroprotective agents like erythropoietin activate the PI 3K/Akt-pathway, plays an
essential role in this neuroprotective pathway (Hernadez et al., 2017). Chromatin condensation
and margination that result in a half-moon or horseshoe appearance of the nucleus are typical
features of apoptotic cell death. The most commonly used techniques to detect apoptosis are
based on the fact that during apoptosis the genomic DNA is cleaved within internucleosomal
DNA segments by an endonuclease selectively activated during apoptosis (Lu et al., 2005).
6. MARKERS OF THE ISCHEMIA/REPERFUSION INJURY

Various mediators and their interrelated mechanisms accentuate the reperfusion injury. In
the last few decades, the need for early prognosis of the injury was considered as a vital
prerequisite for the treatment and the management of the reperfusion injury (Sinning et al.,
2017; Kumral et al., 2012). The research was focused on the development of new markers of
the injury for a proper clinical approach to avoid excessive damage and further complications.
Few of the bio-indicators of the reperfusion injury are discussed in this section.
6.1. Oxidative Stress/Antioxidant Biomarkers
Oxidative stress caused by the production of free radicals is postulated as one of the earliest
mechanisms for the damage to the tissue in the reperfusion injury (Robbins and Swanson,

2014). The generation of various ROS can be assessed by the measurement of endogenous
antioxidant enzymes and the level of reduced glutathione. Lipid peroxidation during the injury
is also of great importance to predict the damage caused by oxidative stress (Nakano et al.,
1990).

Oxygen, under normal conditions, is reduced to water (H2O) by mitochondrial electron
transport which reduces 95% of O2 by tetravalent reduction to H2O without any free radical
intermediates (Sinning et al., 2017) while remaining 5% of oxygen is reduced via the univalent
pathway ending up with free radicals production. Superoxide anion is generated after the
acceptance of an electron by oxygen. Ischemia-induced acidosis alters the equilibrium of
superoxide with its protonated form, HO2• and becomes more reactive. HO2• is capable of
oxidative injury to fatty acids and cell membranes, which is presented within the cell by
dismutation (primarily by superoxide dismutase) to hydrogen peroxide (H2O2). Under normal
conditions, H2O2 does not occur in cells, which at higher concentrations is directly toxic to the
cells. Cells do convert H2O2 to H2O via catalase or glutathione system ensuring safe metabolism
of superoxide, a byproduct of respiration to a humble water molecule (Dunning et al., 2013).
It is observed that a significant decrease in antioxidant enzymes especially superoxide
dismutase (SOD) due to ischemic injury leaves affected tissue extremely vulnerable to
oxidative stress-induced damage (Fukai and Ushio-Fukai, 2011). Superoxide is one of the most
reactive ROS leading to oxidative stress and is generated at various stages of reperfusion
following ischemia. SOD is an endogenous ROS scavenging enzyme which is subdivided based
on the location where it is highly expressed. In the cytosolic and lysosomal fractions as well as
in the mitochondrial intermembrane spaces has a maximum expression of CU/Zn-SOD
(cytosolic or SOD1) (Okado and Fridovich, 2001). Similarly, inflammatory cytokines like
TNF-α, interleukins (IL-1 and 6) and interferon-γ (INF- γ) induce expression of MnSOD
(mitochondrial or SOD2) in the mitochondrial matrix. However, overexpression of MnSOD
has a protective effect on tissue against reperfusion injury (Ighodaro and Akinloye, 2001). In
addition to these isoforms of SOD, brain exhibit extracellular SOD (EC-SOD also termed as
SOD3), a tetrameric protein secreted in extracellular compartment and is unregulated by iNOS
after activation of NFκB (Petersen et al., 2007; Qin et al., 2008).
Thus from all these data, it is clear that SOD enzyme has a critical pathophysiological role
in the oxidative stress-induced damage and the mimetic of the SOD may have the clinical
importance to restrict the injury.
6.1.2. Activity of Catalase

Hydrogen peroxide is produced by dismutation of superoxide by SOD. This hydrogen
peroxide is moderately reactive and has the ability to freely cross lipid bi-layer of the cell
(Lafon-Cazal et al., 1993). Hydrogen peroxide is converted to water, however, the intermediate
hydroxyl radical formed is more reactive than hydrogen peroxide which is metabolized through
the iron-catalyzed Haber–Weiss reaction (superoxide-driven Fenton chemistry) (Zweier and
Talukder, 2006). Elimination of hydrogen peroxide is therefore critical to the efficacy of SOD
in reducing oxidative stress. Catalase (CAT) and glutathione peroxidase (GPx) serve this
purpose (Day, 2009). CAT is mainly localized to peroxisomes.
Thus catalase metabolizes H2O2, prevents possible lipid peroxidation. NADPH has a vital
role in CAT activity during oxidative stress. NADPH assists CAT by preserving it from

excessive H2O2 inactivation and provides a source of GPx during stress (Ighodaro and
Akinloye, 2017).
6.1.3. Glutathione Depletion and Role of Glutathione Peroxidases

Glutathione is a tripeptide (g-L-glutamyl-L-cysteinyl-glycine) that is the reductant for
glutathione peroxidase. This glutathione (GSH) which is made up of cysteine sulfhydryl group
is vital for defense against oxidative injury. During ischemic insult, oxidation of the cysteine
sulfhydryl group joins two GSH molecules with a disulfide bridge to form glutathione disulfide
(GSSG). This oxidized metabolite is restricted as well as recovery of total GSH is catalyzed by
NADPH dependent glutathione reductase. The ratio of GSH/GSSG determines the status of
oxidative damage, higher the GSH/GSSG ratio smaller is the oxidative damage (Namba et al.,
2001). Vital organs like the brain and heart maintain a high ratio of GSH/GSSG which ensures
maximum antioxidant defenses in the event oxidative stress in the situations of ischemia and
ensuing reperfusion. Even though the GSH/GSSG ratio is key to prevention of damage to the
brain but severe depletion of GSH following ischemia/reperfusion may take up to 7 h to restore
pre-ischemic values of GSH/GSSG ratio (Warner et al., 2004; Ozer et al., 2005; Namba et al.,
2001; Dhalla et al., 2000). Principle consequence of GSH loss during oxidative stress in the
brain is the formation of protein-glutathione mixed disulfides (PrSSG) and loss of protein thiols
(Purucker et al., 1991). The loss of GSH and formation of PrSSG in the brain results in
dysfunction of various membrane functions in cells, such as inhibition of Na+K+-ATPase
activity and mitochondrial enzyme activities (Nakamura et al. 2000).
In this reaction reduced glutathione (GSH) is used as a co-substrate to metabolize H2O2,
resulting in H2O and oxidized glutathione (GSSG). Oxidized glutathione (GSSG) can be
reduced back to GSH by the enzyme GSH reductase (GR), a reaction requiring NADPH
regenerated by glucose 6-phosphate dehydrogenase (G6PDH). The capacity to recycle GSH
makes the GSH cycle a pivotal antioxidant defense mechanism for cells and prevent the
depletion of cellular thiols (Ighodaro and Akinolye, 2017; Day, 2009).
Glutathione reductase which removes GSSG recycles GSH and makes it available for reuse
to avoid the stress injury. The individuals with GSH deficiency may well experience excessive
oxidative injury as RBCs hemolysis and renders the endogenous defense system vulnerable to
damage. Glutathione peroxidase overexpression was found to reduce the necrotic and apoptotic
cell death in the cerebral injury. Various hydrophilic antioxidants (GSH and ascorbic acid) were
protective on the myocardial ischemia-reperfusion injury (Ighodaro et al., 2017).
6.1.4. Lipid Peroxidation as Bio-Indicator

The brain has the liability of more easily peroxidizable fatty acids, which consumes a large
quantum of total oxygen consumption. Except for a moderate expression of EC-SOD, brain
lacks an enriched defensive antioxidant system (Schonfeld et al., 2013). Arachidonic acid
concentration is at the highest level due to an increased flux of Ca2+ and ensuing energy failures
during cerebral reperfusion injury causing damage to cellular membrane (Hendrickson et al.,
1997). Inhibition of oxidative phosphorylation (Filmore et al., 2014), formation of
proinflammatory mediators (Wang et al., 2016; Filmore et al., 2014), free radical generation
and lipid peroxidation-mediated chain reactions (Sun et al., 2018), and cytotoxicity from lipid
peroxidation products are major undesirable effects associated with FFA metabolism. These
events may stimulate cell death in the vulnerable tissue, particularly in the penumbra region
(Ramana et al., 2013). The extent of lipid peroxidation by any noxious stimuli can be measured

by estimation of formed malondialdehyde (Grotto et al., 2009) which serves a reliable indicator
of lipid peroxidation are shown in Figure 2.
Figure 2. Mechanism of lipid peroxidation. During the metabolism of oxygen, superoxide anion (O2-) is
formed in presence of NADPH P450 reductase, further superoxide undergoes dismutation to hydrogen
peroxide (H2O2). Myeloperoxidase (MPO) converts H2O2 to hypochlorous acid (HOCl), which is a
strong oxidant that acts as a bactericidal agent. During Fenton reaction Fe ++ is oxidized to Fe+++ and
H2O2 is converted in the highly reactive hydroxyl radical (OH•), this radical result in lipid peroxidation.
6.2. Creatine Kinase
In search of the markers for ischemia and early predictors of infarct, creatine kinase (CK)
was found to be a reliable marker of ischemic injury. Numerous reports considered depletion
of CK in cardiac tissue and an index for infarction (Kitzenberg et al., 2016). CK is an important
cellular enzyme mediating energy transduction in muscle cells. CK is highly sensitive to
oxidation at the exposure of ROS due to the presence of SH groups in cardiac tissue. CK plays
a key role in energy production and utilization by ensuring the availability of high energy
phosphates throughout the cell (Kjekshus and Sobel, 1970; Saks et al., 1994). The rupture of
myocardial cells and destruction of cellular integrity results in leakage of intracellular enzymes
like CK from myofibrils after ischemic reperfusion injury. CK activity proved to be a pivotal
cardiac marker due to its rapid appearance and marked an increase in serum after acute ischemia
(Watts, 1973; Dreyfus et al., 1960). It appears that CK-MB is derived from the necrotic
myocardial cells and levels of free release of CK indicate cell death. It was observed that CK
release and its correlation with histology and ECG establishes a strong correlation to increased
damage to myocardial tissue and is now considered to be a specific diagnostic tool in
myocardial infarction (Al-Hadi et al., 2009).
Creatine kinase (MM and BB) is also found in brain tissue and may be elevated in the
diseased condition. Mlinarik et al., (1998) studied the rate constants of the CK reaction in the
brain after chronic ischemic condition. However, further research is required to establish CK
as a reliable marker of cerebral reperfusion injury.

6.3. Myeloperoxidase
Myeloperoxidase (MPO), a polymorphonuclear neutrophil-derived heme protein, is a

biomarker index for neutrophil infiltration and inflammatory reactivity. MPO activity is
determined by an accumulation of neutrophils which are enhanced during insults like
myocardial reperfusion injury is well established (Baldus et al., 2004; Stendahi et al., 1984;
Matsuo et al., 1994). Recruitment of polymorphonuclear neutrophils (PMN) attributed to
decreased bioavailability of nitric oxide (NO) is responsible for impaired microvascular
functions. Whereas MPO facilitates oxidative NO consumption and impairs vascular function
in animal models of acute inflammation. Upon activation, PMN not only generates superoxide
and H2O2 but they also secrete MPO. MPO in the presence of H2O2 is anatomically poised to
catalytically consume endothelial-derived NO; thereby impairing NO-dependent signaling in
smooth muscle cells and subsequent vascular relaxation thus contributes in the endothelial
dysfunction (Matsuo et al., 1994; Davies, 2011).
The molecular mechanism MPO mediated toxicity to the cell is halogenation and oxidation.
Halogenation involves the covalent binding of the halide co-factor to the target cell or molecule.
Among the possible products formed with protein acceptors and iodide as the cofactor are
iodotyrosines, iodohistidines and sulfenyl iodides (Baldus et al, 2004). The presence of these
chemical configurations near the active site could cause loss of biological activity of the cells.
The oxidation may be mediated by such peroxidase system intermediates as halogens,
hypohalous acid, chloramines, aldehydes, and singlet molecular oxygen. These MPO
components react with extracellular halogen and damage the tissue in the vicinity of the
inflammatory response as the cell-free peroxidase system destroys leukocyte, platelets and
other mammalian cells (Davies, 2011).
Increased brain MPO activity (in pericore region) after transient focal ischemia virtually
reflects the neutrophil infiltration and this neutrophil infiltration into the ischemic brain is
implicated in post-ischemic brain injury (which was evident from the histological observations)
(Matsuo et al., 1994). Thus MPO can be considered as a biomarker to indicate the inflammatory
response in the reperfusion injury of heart and brain.
6.4. Measurement of Plasma Fibrinogen
Ischemic injury due to obstruction of blood flow could be the result of inappropriate
thrombus formation and profound exploration of links of thrombosis and risks of stroke or
myocardial infarction suggested crosstalk between plasma fibrinogen and ischemia
(Wilhelmsen et al., 1984). A synergistic effect of fibrinogen levels and blood pressure on stroke
indicated high plasma fibrinogen is a risk factor for stroke and myocardial infarction (Stone
and Thorpe, 1985). Thus, estimation of plasma fibrinogen could be a better diagnostic tool for
exploring the thrombogenic potential of this protein in ischemic incidences and providing
impetus to develop new strategies for the treatment and management of stroke (Gawaz et al.,
2004).

7. PHARMACOLOGICAL STRATEGIES
TO PROTECT THE REPERFUSED TISSUE
Maintaining and recovering the reperfused tissue is the foremost goal of the
pharmacological interventions carried out during the reperfusion injury (Zheng et al., 2006).
The objective of achieving the protection of the ischemic tissue must be:
 Restoration of obstructed blood flow to the ischemic tissue

 Limiting injury due to sudden reperfusion using interventions
 Reduction in intensity of damage due to reperfusion and restoration of cellular
functions (Morel et al., 2012).
The goal mentioned to restore normal physiology of the ischemic and reperfused cells can
be achieved by various approaches which include:
 Use of agents restricting ischemic damage also considered being anti-ischemic agents
 Ensuring complete restoration using reperfusion interventions
 Exploration and utilization of innate (adaptive) responses (Margaill et al., 2005; Morel
et al., 2012).
7.1. Neuroprotection in Cerebral Reperfusion Injury
Injury scare to ischemic stroke is highest in prolonging obstruction of blood flow; however,
a narrow window of the first hour is available to achieve maximum neuroprotection. The
recanalization within the first hour of ischemic onset is the best approach to rescue cells at risk
of damage (Belayev et al., 1997). The most obvious target for achieving the goal of survival of
cells, prevention of post-stroke complications and recurrence of stroke incidences are molecular
mechanisms like glutamate accumulation, aberrant calcium fluxes, free radical formation and
lipid peroxidation (Kalogeris et al., 2016).
Recanalisation of the blood vessel can be achieved by anti-thrombolytic, fibrinolytic and
anti-platelet drugs which are already discussed in the stroke therapy section of the introduction
chapter. Here, the neuroprotective therapy and the new advances in the treatment of stroke and
reperfusion injury are mentioned.
7.2. Antioxidants and the Therapy Based on Antioxidant Markers
Oxidative stress which peaks after the reperfusion phase of ischemic tissue is considered
to be a major culprit for the instigation and aggravation of the post-ischemic injury. Reperfusion
is accompanied by the excessive release of glutamate and extensive infiltration of cells by
neutrophil responsible for the generation of ROS (Vinten-Johansen, 2004; Matsuo et al, 1994).
Generated ROS amplify the profound imbalance found in the neurons and astroglial cells
of the ischemic core and penumbra. Although tPA is approved for the treatment of stroke, very
few treatments are clinically suitable as of now (Snow, 2016). Hence, considering antioxidant

as the most promising therapeutic class available for the management as well as prevention of
further damage in the ischemic cells enhances the chances of achieving the set goal of the
treatment. Antioxidants have a longer therapeutic window than that of other strategies and can
be economically viable options for the treatment. Stroke is considered as an outcome associated
with a plethora of overlapping etiologies, strategies targeting multiple mechanisms with help
of the combination of a various antioxidant could add on to the benefits individual therapy
using right dose and correct timing for the administration of antioxidant agents. However, the
translation of these antioxidants strategies in clinics still remains a distant dream (Dhalla et al.,
2000)
7.2.1. Inhibition of Lipid Peroxidation

Inhibitors of lipid peroxidation are tested using tirilazad, a non-glucocorticoid steroid in
first phase II trial (study of tirilazad mesylate in patients with acute ischemic stroke), while
phase III studies were started as RANTTAS (randomized trial of tirilazad in acute stroke)
(Johnston et al., 2002). The patients from North America were enrolled whereas patients from
Europe, Israel, and New Zealand were tested for the parallel trial of the same agent (tirilazad
efficacy stroke study). However, the results from most of these trials are not significant in
reducing neuronal damage (Margaill et al., 2005).
7.2.2. Inhibition of Xanthine Oxidase

Allopurinol and oxypurinol were scrutinized for their antioxidant potential. Allopurinol is
oxidized by xanthine oxidase to oxypurinol, which in turn brings about marked inhibition of
xanthine oxidase by binding to the active site of xanthine oxidase inhibition (Nihei et al., 1989).
Thus, allopurinol, as well as oxypurinol, can be administered with the same net mechanistic
effect. The cerebral edema can be reduced by either of the agents as allopurinol decreases post-
ischemic cerebral uric acid while xanthine and conjugated diene concentrations (Marro et al.,
1994; Nihei et al., 1989), preserves ATP (Williams et al., 1992) preventing accumulation of
fluid in affected cells (Van et al., 1998). The experimental outcomes of allopurinol in rats are
mixed and a possible reason for this could be the inability of the agent to scavenge superoxide
and hydrogen peroxide generated by other routes.
Many herbal agents found to have significant protective activity in stroke. Curcuma longa
(Mohanty et al., 2004) and Ginkgo biloba (Clark et al., 2001), were studied for its effect on the
murine models for stroke and further research is going on these herbs to develop an ideal herbal
therapy for stroke.
7.2.3. SOD and Advance SOD Mimetics

The major endogenous SODs, which is studied widely for its effect on the ischemic brain
are CuZn SOD, MnSOD and ECSOD (extracellular). Abundant availability of Cu-Zn-SOD and
MnSOD in neural tissue had created major interest in the study of the modulation of these
enzymes for the prevention of cellular damage (Ighodaro and Akinloye, 2001). Cu-Zn-SOD
overexpression reduces ischemic damage resulting from ischemia/reperfusion (Yang et al.,
1994). However, neither Cu-Zn-SOD overexpression nor Cu-Zn-SOD targeted deletion alter
the outcome from permanent focal ischemia (Fujimura et al., 2001), indicating the requirement
of reperfusion for this enzyme to play a role. Some studies highlighted the role of MnSOD in
cellular activity after targeted deletion of this enzyme leading to worsening of the outcome

from both temporary and permanent middle cerebral artery occlusion (Kim et al., 2002;
Murakami et al., 1998). Similarly, over-expression of Cu-Zn-SOD contributed to inhibiting
post-ischemic damage. EC-SOD overexpressing mice have increased tolerance to both focal
and global cerebral ischemia, while EC-SOD knock-outs exhibit enhanced damage (Sheng et
al., 1999). Administration of M40401, a SOD mimetic exhibited beneficial effects in gerbils
(Mollace et al., 2003).
7.2.4. Catalase and Glutathione Peroxidase Activities

Catalase and glutathione peroxidase (GPx) are essential for the elimination of hydrogen
peroxide generated during reperfusion. Although both enzymes are present in the brain the
activity of GPx is sevenfold greater than that of catalase (Ighodaro and Akinloye, 2017).
Further, while glutathione peroxidase is present in the cytosol, catalase is localized mainly in
peroxisomes. Overexpression of GPx is implicated in significant reduction in necrotic and
apoptotic cell death, astrocytic/microglial activation and neutrophil infiltration. Ebselen is a
nonselective, synthetic mimetic of glutathione peroxidase (Muller et al., 1984) inhibits protein
kinase C, 5-lipooxygenase, cyclooxygenase and NADPH oxidase (Schewe, 1995) is being
studied in ongoing clinical trials (Saito et al., 1998; Yamaguchi et al., 1998). The Japanese
Ebselen Study Group performed studies on the efficacy of ebselen in ischemic stroke and got
approval for the treatment.
The other two antioxidants on the clinical trial at various phases are edaravone, a free
radical scavenger, which delays infarct and edema evolution and decreased the mortality in
acute stroke patients. However, improvement in functional recovery is not significant. NXY-
059 is another free radical scavenger, which is studied in different continents and the results
are encouraging. Its primary outcome is overall recovery and recovery of motor function (Green
et al., 2003).
7.2.5. Nitric Oxide Synthase Inhibitor

Nitric oxide is synthesized by more than one isoforms of NOS, inhibition of any of these
(eNOS, iNOS, and cNOS) affects the ability of NO to induce beneficial or detrimental effects.
Pharmacologic eNOS inhibition would be expected to worsen outcome, secondary to cerebral
vasoconstriction and reduced blood flow observed in eNOS-deficient mice (Lo et al., 1996)
that have worsened ischemic outcomes. In contrast, upregulation of eNOS activity by treatment
with 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors (e.g., simvastatin) caused
increased intra-ischemic blood flow and reduced infarct size (Amin-Hanjani et al., 2001).
Generation of peroxynitrite seems to be the major source of cellular damage and is correlated
with upregulation of iNOS (Suzuki et al., 2002) hence inhibition of iNOS exhibited beneficial
effects on ischemic injury in the brain.
7.3. Poly (ADP-ribose) Polymerase Inhibitors
Poly (ADP-ribose) polymerase (PARP) is an enzyme imparting a regenerating effect on

nucleic acids after degradation of some codons or entire strands (Enders et al., 1997). Poly
(ADP-ribose) is synthesized from NAD through a reaction catalyzed by PARP while its
degradation is mediated by poly(ADP-ribose) glycohydrolase (PARG). The beneficial effect

on that PARP knock-out on cerebral infarct in mice, when compared to wild-type counterparts,
highlighted the possible role of PARP in neuroprotection (Eliasson et al., 1997).
DNA damage is a basic trigger for the activation of PARP which serves as a repair
mechanism however it also results in NAD and ATP depletion which might potentially
exacerbate the ischemic injury. As discussed earlier, peroxynitrite generation and its activity is
a principal source of DNA damage (Giovannelli et al., 2002; Mandir et al., 2000). Interestingly,
Cu-Zn-SOD overexpressing mice do not exhibit post-ischemic PARP activation suggesting that
there is a close correlation between process involved in ROS generation and events leading to
disruption of DNA strands or amino acid sequences in each strand. (Effects of pharmacological
antioxidants on PARP activation have not been reported. It is warranted that any antioxidant
having a major effect on PARP activation needs to be systematically evaluated in ischemic cell
death or reperfusion injury. Antagonism of PARP or systemic administration of NAD could be
a useful tool to improve ischemic outcomes under sudden reperfusion milieu (Yang et al.,
2002).
7.4. Spin Traps Application
The spin trap is a method to capture ROS allowing their detection and quantification. Spin
trap approach is first studied in the ischemic brain using salicylate, which reacts with hydroxyl
radical to form a relatively stable adduct, 2,3-DHBA (Lapchak and Araujo, 2002). In order to
establish this approach or restrict reperfusion injury, nitroxide spin probes, nitrone spin traps
were developed to capture ROS, allowing detection by electron paramagnetic spectroscopy.
Recognizing the potential for nitrones to scavenge ROS, it was considered that these
compounds might present therapeutic potential (Marshall et al., 2003). Indeed, administration
of the spin trap a-phenyl-N-t-butyl-nitrone (PBN) is reported to be protective against both
global and focal ischemic insults. Moreover, the second generation spin trap, NXY-059
(disodium 4-[(tert-butylimino)-methyl]benzene-1,3-disulfonate N-oxide) has been found to
improve ischemic outcome in primates even after 10 weeks of permanent occlusion of the
middle cerebral artery, even when treatment was begun as late as 4 h after onset of ischemia
(Marshall et al., 2003). NXY-059 has been shown to maintain Akt activation and inhibit
cytochrome c release after ischemia (Yoshimoto et al., 2002).
7.5. Glutamate Antagonists
Glutamate-induced excitotoxicity is implicated in the reperfusion injury damage. Hence

the neuroprotective therapy can avail one more avenue of glutamate antagonists. A new
glycine-site NMDA receptor antagonist SM-31900 is a potent neuroprotective agent without
adverse effects and indicates that it is a promising therapeutic for stroke (Ohtani et al., 2003;
Hoque et al., 2016). SM-31900 exhibits nanomolar affinity for this receptor site and selectivity
relative to other neurotransmitter receptors and ion channels. In rat models of transient and
permanent focal cerebral ischemia, postischemic administration of SM-31900 significantly
decreased infarct volume, with potency and efficacy superior to gavestinel, another glycine-
site NMDA receptor antagonist. Unlike other NMDA receptor antagonists (dizocilpine,
selfotel, eliprodil), neuroprotective doses of SM-31900 were not associated with adverse

effects, such as psychotomimetic effects, neuronal vacuolization, renal toxicity or

cardiovascular effects. (Prous Science, 2006). YM872, an AMPA receptor antagonist, is a
neuroprotective compound that ameliorates the deterioration of the hemodynamic penumbra
after focal ischemia (Shimizu et al., 1998).
7.6. Inhibition of Interleukins
Interleukin 1 (IL-1) receptor antagonist, anakinra treatment groups showed

neuroprotection, particularly in the cortex, with a significant increase in the frequency of
surviving cortical neurons. A nonsignificant reduction in the area of focal infarction in the
striatum was also seen in the IL-1 receptor antagonist (IL-1ra)-treated groups (Emsley et al.,
2005).
7.7. Matrix Metalloproteinases Inhibitors
Evidence implicates the involvement of matrix metalloproteinases (MMPs) in the

disintegration of the vasculature and BBB disruption following cerebral ischemia (Misra et al.,
2018). Investigators at University of California at San Diego examined the effects of
pharmacological inhibition of MMPs with the relatively nonspecific inhibitor batimastat (BB-
94; British Biotech) in a rabbit model of tPA-induced hemorrhage after thromboembolic stroke.
This study reported that pretreatment with an MMP inhibitor may improve the safety of tPA in
the management of acute stroke, preventing intracerebral hemorrhage, although further studies
are necessary (Lopchack et al., 2000).
7.8. Heat Shock Proteins
Hypothalamus, especially CA1 and CA3 and dentate granule cells, exhibited strong
expression of HSP70 and HSP72. Penumbra which is considered to be the region of cells with
salvageable neurons due to collateral blood supply showed marked ability to produce HSP70
(Weinstein et al., 2004). It has been an intriguing correlation between the expression of HSP
and resistance to ischemia in focal ischemia. Activation of the transcription factor NF-κB,
responsible for inflammatory reactivity during ischemia is inhibited by HSP expression in cells
under ischemic insult. The 70-kDa HSP is proven to be highly protective and this effect could
be attributed to protection against apoptosis, by blocking the downstream actions of caspases
on activation of PLA2, the cell nucleus, and other processes (Li et al. 1993; Sharp et al., 2013).
As established in previous approaches use of single therapeutic agents may show potential
promise in animal models, its efficacy in the clinical setup is unconvincing. Consequently,
exploration of combined therapeutic approaches in stroke is a need of hour owing to
multifaceted etiology of stroke-related cell injury. Herein, it is worth to study various agents
and evaluated their effects on reperfusion injury alone or in combination with various
hormones. The selection of the agents is based on the antioxidant properties exhibited by the
drug may offer promising clues for the management of stroke and other ischemic injuries
(Margaill et al., 2005).

8. AGENTS WITH ESTABLISHED ANTIOXIDANT ACTIVITY

IN REPERFUSION INJURY
This section highlights some of the agents considered therapeutically viable and are
considered for experimental as well as clinical therapeutics.
8.1. Lipoic Acid
Lipoic acid (thioctic acid) is 1,2-dithiolane-3-pentanoic acid, is a molecule which contains

two thiol groups and 1,2-dithiolane ring, first isolated from liver samples in the early 1950s.
Dihydrolipoic acid and lipoic acid are the natural constituents of biological membranes, acting
as mitochondrial lipoamide co-factor of α-ketoacid dehydrogenase (Richard et al., 2011;
Panigrahi et al., 1996; Packer et al., 1995). Lipoic acid is highly protein bound by an amide
linkage to a lysine residue; this linkage is called as lipoyl group, which plays a lead role in
many multienzyme complexes (Packer et al., 1995).
A dual acting alpha-lipoic acid has the ability to act as an antioxidant in fat- and water-
soluble tissues in both its oxidized and reduced forms. Its major advantage seems to be rapid
absorption in GIT after oral administration and minimum undesirable changes in other
gastrointestinal activity makes lipoic acid a potentially effective therapeutic agent in clinical
conditions associated with free radical damage (Cardoso do Vale et al., 2003). Lipoic acid is
reported to be an excellent antioxidant in studies and radially taken up by the cells. The
exogenous α-lipoic acid is metabolized in the liver to where NADH and NADPH systems play
a major role in intracellular conversion. Dihydrolipoic acid, the reduced product of lipoic acid
produced in intracellular spaces acts as an antioxidant in extracellular space (Packer et al., 1995;
Cardoso do Vale et al., 2003).
Rosenberg and Culik in (1959) discovered the ability of lipoic acid as a potent antioxidant
when it exhibited protective effect in both scurvy symptoms in vitamin C-deficient guinea pigs
and vitamin E deficiency in rats fed a diet lacking α-tocopherol (Chidlow et al., 2002, Packer
et al., 1995; Sige et al., 1978).
Lipoic acid is studied for its antioxidant property in various models of ischemia and
reperfusion injury. It found to have an antioxidant effect in cerebral ischemia-reperfusion injury
owing to its ability to improve survival and protecting the rat brain against reperfusion injury
following cerebral ischemia (Panigrahi et al., 1996). The mitochondria are the main site of
antioxidant action of α-lipoic acid. The lipoic acid increases the intracellular glutathione levels;
it also has a significant influence on ion Fe2+ chelation, nitric oxide and other reactive oxidative
species scavenging. S-enantiomer of lipoic acid was more effective than the R-enantiomer
when administered 1 hour before ischemia (Cardoso do Vale et al., 2003; Panigrahi et al.,
1996). Lipoic acid administration attenuates liver ischemia-reperfusion injury (IRI) via the PI3-
kinase/AKT pathway which play a pivotal protective role in IRI. Lipoic acid pretreated organs
reduce postischemic activation of NF-kappa B and activating protein-1 (Panigrahi et al., 1996).
To addition of its protective antioxidant effects, Haramaki et al., 1993, proposed that lipoic acid
improve the cardiac recovery after ischemia, similarly, lipoic acid is reported for its effect on
glucose uptake activity (Jacob et al., 1996).

Alpha-lipoic acid is not without adverse effect if the dose and the route of administration
are not carefully regulated. Patients exposed to very high dose of lipoic acid suffer adverse
effects if thiamine deficiency is ignored. It is advisable to test the patient for thiamine
deficiency before administration of lipoic acid (Gal, 1965).
Intraperitoneal administration of high doses of alpha-lipoic acid intraperitoneally to rats
may cause fatal complications which can be averted with the prior administration of thiamine.
Researchers are in general agreement that alpha-lipoic acid is capable of scavenging hydroxyl
radicals, hypochlorous acid, and singlet oxygen, but not hydrogen peroxide, peroxyl, and
superoxide (Freisleben et al., 2000). It prevents DNA strand break up induced by singlet oxygen
(Passwater et al., 1995). Lipoic acid is rightly so regarded as a dual antioxidant owing to its
pro-oxidant effect at higher doses and very effective antioxidant action at low doses along with
its characteristic ability to act as an antioxidant in fat- and water-soluble tissues in both its
oxidized and reduced forms.
8.2. Coenzyme Q10
Coenzyme Q10 (CoQ) is also termed to be ubiquinone owing to its omnipresence in almost
all cells. A lipid-soluble benzoquinone, COQ, is an essential component for electron transport
in oxidative phosphorylation of mitochondria (mitochondrial respiratory chain) (Sunamori et
al., 1991). Principally it acts as an electron carrier between the NADH and succinate
dehydrogenases and the cytochrome system (Sunamori et al., 1991; Kagan et al., 1998). CoQ
functions as an intracellular antioxidant, presumably by preventing both the initiation and
propagation of lipid peroxidation (Ernster, 1993; Ernster, 1993).
When the heart experiences oxidative stress, the amount of CoQ decreases which triggers
a signal for increased CoQ synthesis (Das and Maulik, 1995 and Crestanello et al., 1996). As it
is synthesized by de novo synthesis CoQ can be endogenously regenerated and is capable of
acting as an antioxidant to reduce oxidative damage during reperfusion (Beyer, 1992; Sugawara
et al., 1990). CoQ has a potential antioxidant effect in the brain by reducing lactate
concentration, inhibiting lipid peroxidation and improves recovery of brain metabolism in
ischemic condition (Mattews et al., 1998; Liang et al., 2017).
Mitochondria are not the only site of action for antioxidant activity of CoQ, any cell
exhibiting CoQ is expected to have beneficial effects of its protective actions (Sohal and
Forster, 2007) making CoQ an ideal therapeutic agent to investigate in cerebral ischemia-
reperfusion injury (Garrido-Maraver et al., 2014).
8.3. Trimetazidine
Trimetazidine, 1-(2,3,4-trimethoxybenzyl)piperazine hydrochloride is a FDA approved an

anti-ischemic drug used in myocardial infarction and angina pectoris (Kutala et al., 2006).
Trimetazidine (TMZ) exerts its actions without any alteration of hemodynamics of the heart
(Lopaschuk et al., 2003). Principally it suppresses mitochondrial 3-ketoacyl-CoA thiolase (3-
KAT) thereby reducing the free fatty acid metabolism and balancing the cardiac metabolism.
TMZ positively modulates the energy homeostasis of the cells under ischemic stress or cells
deficient of the energy substrates (Kantor et al., 2000; Guarnieri et al., 1993). TMZ is reported

to preserve the cellular homeostasis by controlling intracellular acidosis and calcium overload
in the cells thereby conserving the valuable ATP stores to meet the energy requirements
(Mironova et al., 2001). The administration of TMZ is found to have beneficial effects on
antioxidant enzymes in the brain by limiting the depletion of GSH (Abdel-Salam et al., 2011).
The pathogenesis of cerebral ischemia and myocardial ischemia leading to an array of
critical cellular changes has similarities in a mechanism of cell death. These changes in cellular
and mitochondrial levels are responsive to TMZ (Harpey et al., 1989; Kay et al., 1995). Being
lipophilic, TMZ crosses the blood-brain barrier (BBB) ensures the availability at the target site
providing prominent neuroprotective agent in cerebral ischemia/reperfusion (I/R) injury. The
metabolic effects of TMZ during ischemia are attenuation of intracellular acidosis while in
reperfusion it was accompanied by an acceleration of the resynthesis of phosphocreatine and a
larger reconstitution of the ATP pool. Hence, TMZ has protected the ATP reserves by
streamlining the ATP-producing processes, probably at the mitochondrial level (Dezsi, 2016;
Dhote and Balaraman, 2008).

AND CHALLENGES
Table 1. Neuroprotective agents in phases of clinical development
Stage of Clinical
Neuroprotective Agents Mechanism of Action
Development
Desmoteplase Activates plasminogen conversion to the Phase 2, 3 and 4
(Broussalis et al., 2012) serine protease, plasmin ongoing
Tenecteplase Selectively converts thrombus-bound Phase 2
(Broussalis et al., 2012) plasminogen to plasmin
Ancrod Cleaves fibrinogen rather than fibrin Phase 3
(Broussalis et al., 2012)
Edaravone Increases eNOS and decreases iNOS Phase 4
(Broussalis et al., 2012)
Eptifibatide Reversibly binds to the platelet receptor Phase 1 and phase 2
(Mestre et al., 2013) glycoprotein (GP) IIb/IIIa of human
platelets
Ebselen Reduction of ROS Phase 3
(Azad and Tomar, 2014)
Minocycline Reduction of microglial activation Phase 2
(Veltkamp and Gill, 2016) reduced NO production, and inhibition
of apoptotic cell death
Triiodothyronine (T3) Non-genomic T3 activation of nitric _
nanoparticle oxide signaling and vasodilation.
(Mdzinarishvili, 2012) Crosses the BBB via a specific
transporter called monocarboxylate 8
(MCT8)

Table 2. List of various herbal antioxidants investigated using

the various model of ischemia/reperfusion injury
Biomarkers
Antioxidant Stroke Model Dose and Route
Evaluated
Canna indica roots BCCA; Male Wistar rats 400-800 SOD,CAT, and
(Talluria et al., 2018) mg/kg; PO MPO
Hexahydro-curcumin MCAO; Male Wistar rats 20-40 mg/kg; IP SOD, GSH, and
(Wicha et al., 2017) GPx
Grape seed Transient MCAO; male SD 50 mg/kg; IP GPx
Procyanidin rats
(Kong et al., 2017)
Kukoamine A Permanent MCAO; Male 5,10, 20 mg/kg; IV SOD
(Liu et al., 2016) SD rats,
Nobiletin Permanent MCAO; Male 25 mg/kg; IP HO1, SOD, and
(Zhanga et al., 2016) SD rats, GSH
Pentapetes phoenicea BCCA; Male Wistar rats 100 mg/kg; PO SOD, CAT, and
(Sravanthi et al., GPx
2016)
Arjunolic acid MCAO; Wistar rat 10 and 20 mg/kg; PO SOD, CAT,
(Yaidikar et al., 2015) GSH, and GPx
Pinocembrin BCCA; Male Wistar rats 10 mg/kg; PO LPO, GSH
(Saad et al., 2015)
Solasodine BCCA; 100 and 200 mg/kg; SOD, CAT,
(Sharma et al., 2014) Male Wistar rats PO GSH, and LPO
Sesamin MCAO; Swiss Albino mice 30 mg/kg; IP SOD, GSH, and
(Ahmada et al., 2014) LPO
Calycosin MCAO; Sprague Dawley 7.5,15,30 mg/kg; PO SOD, CAT and
(Guo et al., 2012) rat GPx
S-Nitrosoglutathione MCAO; Wistar rats 3 micro mol/kg; IP SIN-1,
(Khan et al., 2012) GSNO
Cymbopogon martini BCCA; Wistar rats 50-100 mg/kg; PO SOD, CAT,
(Buch et al., 2012) GSH, and GPx
Ginsenoside MCAO; Swiss albino mice 50 mg/kg; IP SOD, CAT,
(Ye et al., 2011) GSH, and GPx
Shikonin MCAO; male Kunming 50, 25, and 12.5mg/kg; SOD, CAT, and
(Wang et al., 2010) mice PO GPx
Bacopa monniera Transient ICA; Wistar rats 120 mg/kg; PO LPO
(Saraf et al., 2010)
BCCA, Bilateral carotid artery occlusion; MCAO, Middle cerebral artery occlusion; GSNO, S-
Nitrosoglutathione; SIN-1, 3-Morpholinosydnone imine HO-1, Heme oxygenase-1; ICA,
Intracarotid artery occlusion; PO, Per oral; IV, Intravenous; IP, Intraperitoneal
In the recent past, the renewed interest in stroke and reperfusion injury has driven numerous
investigations of neuroprotective agents using various murine models of stroke. Many of these
agents exhibited potent antioxidant activity indicated by marked changes in biomarkers of
oxidative stress. The pharmacological activity extends from scavenging of superoxide anions
to attenuating the oxidation of lipid peroxides. Many of these antioxidants do have a major
effect on inflammation as well as cell damage due to apoptosis and necrosis. A summarized

display of the various stages of pharmacological and clinical research has been given in the
tabular form (Table 1 and Table 2).
Having said that extensive research is driving the development of new antioxidant agents
which may have potent neuroprotective activity in preclinical stages, their translation in clinical
therapeutics is still debatable (Gilgun-Sherki et al., 2002; Flores et al., 2014). Looking at the
stringent criterion for clinical studies and slow pace of antioxidant-based neurotherapy
development, use of the innovative approach to ensure realistic translation of preclinical gains
in robust clinical strategies is the need for scientific research.
Very often reports of potent neuroprotection promised in preclinical studies by agents
having an excellent reduction in infarct size fails to in translating realistic improvement in
clinical trials. The reasons for this discrepancy are associated with disagreements in etiology,
age, co-morbidities, therapeutic time window, optimum doses to its appropriate route of
administration in preclinical and clinical milieu during the stroke (Gilgun-Sherki et al., 2002;
Flores et al., 2014). Classical example reinforcing these limitations is disodium 2,4-
disulfophenyl-N-tert-butylnitrone (NXY-059).
NXY-059 acts by trapping free radicals during reperfusion and ischemic episode which
attributed to its anti-ischemic activity (Bath et al., 2009; Day et al., 2009). NXY-059 exhibited
43% attenuation of infarct volume indicating potent neuroprotection in a stroke model.
Similarly, non-human primates (marmosets) model which closely resembles clinical etiology
of stroke also confirmed neuroprotection induced by NXY-059. It fulfilled all STAIR criteria
which revolves around post-stroke neurological recovery and hence promised a breakthrough
in the treatment of stroke. The initial clinical study, Stroke-Acute Ischemic NXY Treatment
(SAINT I), encouraged researches as it indicated a significant reduction in disability after 3
months of stroke when patients treated with NXY-059 in combination with thrombolysis (Lees
et al., 2006). More elaborate and statistically powerful SAINT II study could not endorse the
earlier potent outcomes and pooled data of two studies indicated a lack of functional
improvements and benefits after NXY-059 (Diener et al., 2008). Other examples in the list are
tirilazad (U74006F), a 21-aminosteroid which scavenges ROS, inhibits lipid peroxidation
(Umemura et al., 1994) and edaravone (3-methyl-1-phenyl-2-pyrazoline-5-one) (Feng et al.,
2011) (117 of SMI). However, edaravone is approved in Japan in-spite of limited clinical data
for acute ischemic stroke.
These examples emphasize the need for streamlined and aligning preclinical studies with
robust clinical study criteria. The existing discrepancies are lack of pharmacokinetic
considerations before treatment optimization, neglected co-morbidity status, inappropriate
therapeutic time window, missed primary endpoints on efficacy and homogeneity of animals
induced with stroke. These discrepancies seem to be just a broad overview of major limitations
of current preclinical models to match clinical etiology of stroke and criteria of clinical studies.
It has been suggested that preclinical studies can also be phased out on the lines of clinical trials
were phase I would aim to discover or investigate pathophysiological mechanisms of a
proposed treatment. While phase II pre-should evaluate the efficacy and safety of a drug and
phase III must be conducted in the international, multicentre preclinical setup is required to
confirm the efficacy of the proposed therapeutic agent. Similarly, the registration of preclinical
studies for primary and secondary endpoint and its definitions in the registry, which are
regularly followed in clinical trial registration protocol, must be made mandatory for all
preclinical studies.

CONCLUSION
There are numerous more promising approaches which are aimed toward neuroprotection
as well as cognitive recovery. Despite the pitfalls of current techniques, the translational
activities are being refined with continuous enrichment of current scientific know-how of stroke
etiology and glaring gaps in transition to clinics. Further innovative concepts and continuous
exploration of the novel hypothesis will definitely result in the development of the new drug
for the treatment of stroke in the near future.
REFERENCES
Abdel-Salam O. M. E, Mohammed N. A, Sleem A. A. The effects of trimetazidine on
lipopolysaccharide-induced oxidative stress in mice. EXCLI J. 2011; 10: 162–72.
Abe K, Yoshidomi M, Kogure K. Arachidonic acid metabolism in ischemic neuronal damage.
Ann NY Acad Sci. 1991; 559: 259-68.
Ahmad M, Zhang Y, Liu H, Rose M, Graham SH. Prolonged opportunity for neuroprotection
in experimental stroke with selective blockade of cyclooxygenase-2 activity. Brain Res.
2009; 1279: 168–73.
Ahmad S, Elsherbiny NM2, Haque R, Khan MB4, Ishrat T, Shah ZA, et al. Sesamin attenuates
neurotoxicity in mouse model of ischemic brain stroke. Neurotoxicology. 2014;45:100-10.
Al Kasab S, Derdeyn C P, Guerrero WR, Limaye K, Shaban A, Adams HP Jr. Intracranial
Large and Medium Artery Atherosclerotic Disease and Stroke. J Stroke Cerebrovasc Dis.
2018;27(7):1723-32.
Al-Hadi HA, Fox KA. Cardiac Markers in the Early Diagnosis and Management of Patients
with Acute Coronary Syndrome. Sultan Qaboos Univ Med J. 2009; 9(3): 231–46.
Alloubani A, Saleh A, Abdelhafiz I. Hypertension and diabetes mellitus as predictive risk
factors for stroke. Diabetes Metab Syndr. 2018;12(4):577-84.
Alves A. J, Viana J. L, Cavalcante Sl, Oliveira N. L, Duarte JA, Mota J,et al. Physical activity
in primary and secondary prevention of cardiovascular disease: Overview updated. World
J Cardiol. 2016; 8(10): 575–83.
Amin-Hanjani S, Stagliano N. E, Yamada M, Huang P. L, Liao J. K, Moskowitz M. A.
Mevastatin, an HMG-CoA reductase inhibitor, reduces stroke damage and upregulates
endothelial nitric oxide synthase in mice. Stroke. 2001;32(4):980-86.
Aronowski J, Strong R, Grotta J. C. Reperfusion injury: demonstration of brain damage
produced by reperfusion after transient focal ischemia in rats. J Cereb Blood Flow Metab.
1997;17(10):1048-56.
Aspey B. S, Cohen S, Patel Y, Terruli M, Harrison M. J. Middle cerebral artery occlusion in
the rat: consistent protocol for a model of stroke. Neuropathol Appl Neurobiol.
1998;24(6):487-97.
Au A. M, Chan P. H, Fishman R. A. Stimulation of phospholipase A2 activity by oxygen-
derived free radical in iso-lated brain capillaries. J Cell Biochem. 1985;27(4):449-53.
Azad GK, Tomar RS. Ebselen, a promising antioxidant drug: mechanisms of action and targets
of biological pathways. Mol Biol Rep. 2014;41(8):4865-79.

Back T, Zhao W, Ginsberg M. D. Three-dimensional image analysis of brain glucose

metabolism-blood flow uncoupling and its electrophysiological correlates in the acute
ischemic penumbra following middle cerebral artery occlusion. J. Cereb. Blood Flow
Metab. 1995;15(4): 566–77.
Baldus S, Heitzer T, Eiserich JP, Lau D, Mollnau H, Ortak M, et al. Myeloperoxidase enhances
nitric oxide catabolism during myocardial ischemia and reperfusion. Free Radl Biol &
Med. 2004;37(6):902-11.
Banani HE, Bernard M, Baetz D, Cabanes E, Cozzone P, Lucine A, et al. Changes in
intracellular sodium and pH during ischaemia–reperfusion are attenuated by trimetazidine
Comparison between low- and zero-flow ischemia. Cardiovascular Research. 2000; 47(4):
688–96.
Bath P. M. W, Gray L, Bath A, Buchan A, Miyata T, Green A. R. Effects of NXY-059 in
experimental stroke: An individual animal meta-analysis. Br J Pharmacol.
2009;157(7):1157-71.
Beckman J. S. Peroxynitrite versus hydroxyl radical: the role of nitric oxide in superoxide-
dependent cerebral injury. Ann NY Acad Sci. 1994;738:69-75.
Belayev L, Zhao W, Busto R, Ginsberg M. D. Ginsberg. Transient middle cerebral artery
occlusion by intraluminal suture. I. Three-dimensional autoradiographic image analysis of
local cerebral glucose metabolism-blood flow interrelationships during ischemia and early
recirculation. J. Cereb. Blood Flow Metab. 1997;17(12):1266-80.
Beyer R. An analysis of Coenzyme Q in free radical generation and as an antioxidant. Biochem
Cell Biol. 1992;70(6):390-403.
Birben E, Sahiner U. M, Sackesen C, Erzurum S, Kalayci O. Oxidative Stress and Antioxidant
Defense. World Allergy Organ J. 2012; 5(1): 9-19.
Bogdan C, Paik J, Vodovotz Y, Nathan C. Contrasting mechanisms for suppression of
macrophage cytokine release by transforming growth factor-beta and interleukin-10. J Biol
Chem. 1992;267(32):23301-08.
Broussalis, E, Trinka E, Killer M, Harrer A, McCoy M, Kraus J. Current therapies in ischemic
stroke. Part B. Future candidates in stroke therapy and experimental studies. Drug
Discovery Today. 2012; 17(13-14): 671-84.
Buch P, Patel V, Ranpariya V, Sheth N, Parmar S. Neuroprotective activity of Cymbopogon
martinii against cerebral ischemia/reperfusion-induced oxidative stress in rats. J
Ethnopharmacol. 2012;142(1):35-40.
Buja L. M, Eigenbrodt M. L, Eigenbrodt E. H. Apoptosis and necrosis: basic types and
mechanisms of cell death. Arch Pathol Lab Med. 1993;117(12):1208-14.
Cao G, Pei W, Lan J, Stetler RA, Luo Y, Nagayama T. Caspase-activated DNase/DNA
fragmentation factor 40 mediates apoptotic DNA fragmentation in transient cerebral
ischemia and in neuronal cultures. J Neurosci. 2001;21(13):4678-90.
Cao X, Philis J. W. The free radical scavenger alpha- lipoic acid protects against cerebral
ischemia-reperfusion injury in gerbils. Free Radic Res. 1995;23:365-70.
Carmichael S. T. Rodent Models of Focal Stroke: Size, Mechanism, and Purpose. NeuroRx.
2005;2(3): 396–409.
Chen Q, Chopp M, Bodzin G, Chen H. Temperature modulation of cerebral depolarization
during focal cerebral ischemia in rats: correlation with ischemic injury. J Cereb Blood Flow
Metab. 1993;13(3):389-94.

Chen X, Leeman J. E, Wang J, Pacella J. J, Villanueva F. S. New insights into mechanisms of

sonothrombolysis using ultra-high-speed imaging. Ultrasound Med Biol. 2014;40(1): 258-
62.
Chidlow G, Schmidt K. G, Wood J. P, Melena J, Osborne N. N. Alpha-lipoic acid protects the
retina against ischemia-reperfusion. Neuropharmacol. 2002;43(6):1015-25.
Clark W. M, Rinker L. G, Lessov N. S, Lowery S. L, Cipolla M. J. Efficacy of antioxidant
therapies in transient focal ischemia in mice. Stroke. 2001;32(4):1000-04.
Crestanello J. A, Kamelgard J, Lingle D, Mortensen S. A, Rhode M, Whitman G. J. Elucidation
of a tripartite mechanism underlying the improvement in cardiac tolerance to ischemia by
Coenzyme Q10 pretreatment. J Thorac Cardiovasc Surg. 1996;111(2):443-50.
Cuzzocrea S, Riely D. P, Caputi A. P, Salvemini D. Antioxidant therapy: A new
pharmacological approach in shock, inflammation, and ischemia/reperfusion injury.
Pharmacol. Rev. 2001;53(1):135-59.
Das D. K, Maulik N. Protection against free radical injury in the heart and cardiac performance.
In: Exercise and Oxygen Toxicity, edited by C. K. Sen, L. Packer, O. Ha¨ nninen.
Amsterdam, The Netherlands: Elsevier Science, 1995:359-88.
D'Ascenzo F, Taha S, Moretti C, Omedè P, Grossomarra W, Persson J et al. Meta-analysis of
randomized controlled trials and adjusted observational results of use of clopidogrel,
aspirin, and oral anticoagulants in patients undergoing percutaneous coronary intervention.
Am J Cardiol. 2015; 115(9):1185-93
Davies M. J. Myeloperoxidase-derived oxidation: mechanisms of biological damage and its
prevention. J Clin Biochem Nutr. 2011; 48(1): 8-19.
Dawson T. M, Dawson V. L, Snyder S. H. Molecular mechanisms of nitric oxide actions in the
brain. Ann. NY Acad Sci. 1994;738:76-85.
Day BJ. Catalase and glutathione peroxidase mimics. Biochem Pharmacol. 2009;77(3): 285-
96.
Del ZG, Ginis I, Hallenbeck JM, Iadecola C, Wang X, Feuerstein G. Z. Inflammationand
stroke: Putative role for cytokines, adhesion molecules and iNOS in brain response to
ischemia. Brain Pathol. 2000;10(1):95-112.
Dezsi C. A. Trimetazidine in Practice: Review of the Clinical and Experimental Evidence. Am
J Ther. 2016; 23(3): e871-79.
Dhalla N. S, Elmoselhi A. B, Hata T, Makino N. Status of myocardial antioxidants in ischemia–
reperfusion injury. Cardiovascular Research. 2000;47(3):446-56.
Dhote V, Balaraman R. Anti-oxidant activity mediated neuroprotective potential of
trimetazidine on focal cerebral ischaemia-reperfusion injury in rats. Clin Exp Pharmacol
Physiol. 2008;35:630-37.
Diener H. C, Lees K. R, Lyden P, Grotta J, Davalos A, Davis S. M, et al. NXY-059 for the
treatment of acute stroke: pooled analysis of the SAINT I and II Trials. Stroke.
2008;39(6):1751-58.
Dirnagl U, Iadecola C, Moskowitz M. A. Pathobiology of ischaemic stroke: An integrated view.
Trends Neurosci. 1999;22(9):391-97.
Dirnagl U, Lindauer U, Them A, Schreiber S, Pfister HW, Koedel U. et al. Global cerebral
ischemia in the rat: online monitoring of oxygen free radical production using
chemiluminescence in vivo. J. Cereb. Blood Flow Metab. 1995; 15(6): 929–40.
do Vale O. C, Fonteles D. S, Cabral F. R, Fonteles M. C. A dual action of alpha-lipoic acid in
the brain: an electrophysiological evaluation. Arq Neuropsiquiatr. 2003;61(3B):738-45.

Dong Y, Wang H, Chen Z. Alpha-Lipoic Acid Attenuates Cerebral Ischemia and Reperfusion
Injury via Insulin Receptor and PI3K/Akt-Dependent Inhibition of NADPH Oxidase. Int J
Endocrinol. 2015; 2015: 903186.
Dreyfus J. C, Schapira G, Rasnais J, Scebat L. La creatine-kinase sérique dans le diagnostic de
l'infarctus myocardique, Revue Française d'études Cliniques et Biologiques. 1960;5:386-
89.
Du Y, Ko K. M. Effect of pharmacological preconditoning by emodin/oleanolic acid treatment
and/or ischemic precondtioning on mitochondrial antioxidant components as well as the
susceptibility to ischemia-reperfusion injury in rat hearts. Mol Cell Biochem.
2006;288:135-42.
Dunning S, Rehman A, Tiebosch M. H, Hannivoort R. A, Haijer F. W Woudenber J, et al.
Glutathione and antioxidant enzymes serve complementary roles in protecting activated
hepatic stellate cells against hydrogen peroxide-induced cell death. Biochim Biophys Acta
Mol Basis Dis. 2013;1832:2027-34.
Duverger D, MacKenzie E. T. The quantification of cerebral infarction following focal
ischemia in the rat: influence of strain, arterial pressure, blood glucose concentration and
age. J. Cereb. Blood Flow Metab. 1988;8(4): 449-61.
Eliasson M. J., Sampei K, Mandir A. S, Hurn P. D, Traystman R. J, Bao J, et al. Poly(ADP-
ribose) polymerase gene disruption renders mice resistant to cerebral ischemia. Nature
Med. 1997;3:1089-95.
Emsley H, Smith C, Georgiou R, Vail A, Hopkins S, Rothwll N, et al. A randomised phase II
study of interleukin-1 receptor antagonist in acute stroke patients. J Neurol Neurosurg
Psychiatry. 2005; 76: 1366-72.
Endres M, Wang ZQ, Namura S, Waeber C, Moskowitz MA. Ischemic brain injury is mediated
by the activation of poly(ADP-ribose)polymerase. J Cereb Blood Flow Metab.
1997;17(11):1143-51.
Ernster L, Forsmark AP. Ubiquinol: an endogenous antioxidant in aerobic organisms. Clin
Invest. 1993;71:S60-65.
Ernster L. Ubiquinol as a biological antioxidant. In: Active Oxygens, Lipid Peroxides and
Antioxidants, edited by K. Yagi. Tokyo: Japan Sci Soc. 1993;1-38.
Feng S, Yang Q, Liu M, Li W, Yuan W, Zhang S Edaravone for acute ischaemic stroke.
Cochrane Database Syst Rev. 2011; available at doi: 10.1002/14651858.CD007230.pub2.
Ferrazzano P, Shi Y, Manhas N, Wang Y, Hutchinson B, Chen X, et al. Inhibiting the Na+/H+
exchanger reduces reperfusion injury: a small animal MRI study. Front Biosci (Elite Ed).
201; 3: 81-88.
Filmore N, Mori J, Lopaschuk G. D. Mitochondrial fatty acid oxidation alterations in heart
failure, ischaemic heart disease and diabetic cardiomyopathy. Br J Pharmacol. 2014; 171:
2080-90.
Fiolet J. W. T., Baartscheer A. Cellular calcium homeostasis during ischemia; a thermodynamic
approach. Cardiovascular Research. 2000;45:100-06.
Folbergrová J, Memezawa H, Smith M. L, Siesjö B. K. Focal and perifocal changes in tissue
energy state during middle cerebral artery occlusion in normo- and hyperglycemic rats. J
Cereb Blood Flow Metab. 1992;12:24-33.
Folbergrová J, Zhao Q, Katsura K, Siesjö B. K. N-tert-butyl-a-phenylnitrone improves recovery
of brain energy state in rats following transient focal ischemia. Proc. Natl. Acad. Proc Natl
Acad Sci U S A. 1995; 23;92(11):5057-61.

Freeman B. A, Crapo J. D. Biology of disease: free radicals and tissue injury. Lab Invest.
1982;47:412-26.
Freisleben H. J. Lipoic acid reduces ischemia-reperfusion injury in animal models. Toxicol.
2000;148:159-71.
Friedlander R. M. Apoptosis and caspases in neurodegenerative diseases. N Engl J Med.
2003;348:1365-75.
Fujimura M, Morita-Fujimura Y, Copin J, Yoshimoto T, Chan P. H. Reduction of copper,zinc-
superoxide dismutase in knockout mice does not affect edema or infarction volumes and
the early release of mitochondrial cytochrome c after permanent focal cerebral ischemia.
Brain Res. 2001;889:208-13.
Fukai T, Ushio-Fukai M. Superoxide Dismutases: Role in Redox Signaling, Vascular Function,
and Diseases. Antioxid Redox Signal. 2011; 15: 1583–1606.
Garrido-Maraver J, Cordero M. D, Oropesa-Avila M, Vega A. F, Mata M. D. L, PAvon AS.
Coenzyme Q10 Therapy. Mol Syndromol. 2014; 5: 187-97.
Gawaz M. Role of platelets in coronary thrombosis and reperfusion of ischemic myocardium.
Cardiovascular Research.2004;61: 498-511.
Gidö G, Kristián T, Siesjö BK. Extracellular potassium in a neocortical core area after transient
focal ischemia. Stroke. 1997;28:206-10.
Ginsberg M. D, Busto R. Rodent models of cerebral ischemia. Stroke. 1989;20:1627-42.
Ginsberg M. D, Pulsinelli WA. The ischemic penumbra, injury thresholds and the therapeutic
window for acute stroke. Ann Neurol. 1994;36:553-54.
Giovannelli L, Cozzi A, Guarnieri I, Dolara P, Moroni F. Comet assay as a novel approach for
studying DNA damage in focal cerebral ischemia: differential effects of NMDA receptor
antagonists and poly(ADP-ribose) polymerase inhibitors. J Cereb Blood Flow Metab.
2002;22:697-704.
Gottlieb R. A. Cell Death Pathways in Acute I/R Injury. J Cardiovasc Pharmacol Ther.
2011;16:233-38.
Granger D N, Kyietvs P. R. Reperfusion injury and reactive oxygen species: The evolution of
a concept. Redox Biol. 2015;6: 524-51.
Green A. R, Ashwood T, Odergren T, Jackson DM. Nitrones as neuroprotective agents in
cerebral ischemia, with particular reference to NXY-059. Pharmacol Ther. 2003;100:195-
214.
Grotto D, Maria L. S, Valentini J, Paniz C, Pomblum V. J, Garcia GSeSC, et al. Imporatnce of
lipid peroxidation biomarkers and methodological aspects for malondialdehyde
quantification. Quim Nova. 2009;32:169-74.
Guan Y, Wang Y, Yuan F, Lu H, Ren Y, Xiao T, et al. Effect of suture properties on stability
of middle cerebral artery occlusion evaluated by synchrotron radiation angiography.
Stroke. 2012;43(3):888-91.
Guarnieri C, Muscari C. Effect of trimetazidine on mitochondrial function and oxidative
damage during reperfusion of ischemic hypertrophied rat myocardium. Pharmacology.
1993;46:324-31.
Guo C, Tong L, Xi M, Yang H, Dong H, Wen A. Neuroprotective effect of calycosin on
cerebral ischemia and reperfusion injury in rats. J Ethnopharmacol. 2012;144:768-74.
Hademenos G. J, Massoud T. F. Biophysical mechanisms of stroke. Stroke. 1997;27: 2067-77.
Hammer B, Parker W. D. Jr, Bennett J P. Jr. NMDA receptors increase OH radicals in vivo
by using nitric oxide synthase and protein kinase C. Neuroreport. 1993;25;5(1):72-74.

Haramaki. N, Assadnazari. H, Zimmer. G, Schepkin, V, Packer, L. The influence of vitamin E

and dihydrolipoic acid on cardiac energy and glutathione status under hypoxia-
reoxygenation. Biochem Mol Biol Int. 1995;37:591-97.
Harpey C, Clausen P, Labrid C, Freyria J. L., Poirier, J. P. Trimetazidine, a cellular anti-
ischemic agent. Cardiovasc Drug Rev. 1989;6:292-312.
Hassan A., Markus H. S. Genetics and ischemic stroke. Brain. 2000;123:1784-812.
He Z, Simon H. U. A novel link between p53 and ROS. Cell Cycle. 2013;15:12(2):201-02.
Hendrickson, SC1, St Louis, J. D, Lowe, J. E, Abdel-aleem S. Free fatty acid metabolism during
myocardial ischemia and reperfusion. Mol Cell Biochem. 1997;166:85-94.
Hernandez, C. C., Burgos, C. F, Gajardo, A. H, Sliva-Grecchi T, Gavilan, J,Toledo, J. R., et al.
Neuroprotective effects of erythropoietin on neurodegenerative and ischemic brain
diseases: the role of erythropoietin receptor. Neural Regen Res. 2017;12:1381-89.
Hill, M. D, Martin, R. H, Palesch, Y. Y, Tamariz, D, Waldman, B. D, Ryckborst K. J, et al. The
Albumin in Acute Stroke Part 1 Trial: An exploratory efficacy analysis. Stroke.
2011;42:1621-25.
Hoque, A, Hossain, M. I., Ameen, S. S, Ang, C. S, Williamson N, Ng D. C., et al. A beacon of
hope in stroke therapy-Blockade of pathologically activated cellular events in excitotoxic
neuronal death as potential neuroprotective strategies. Pharmacol Ther. 2016;160:159-79.
Iadecola C. Bright and dark sides of nitric oxide in ischemic brain injury. Trends Neurosci.
1997;20:132-39.
Ighodaro O M, Akinloye OA. First line defence antioxidants-superoxide dismutase (SOD),
catalase (CAT) and glutathione peroxidase (GPX): Their fundamental role in the entire
antioxidant defence grid. Alex J Med. 2017; In press.
Ighodaro O. M, Akinolye O. A. First line defence antioxidants-superoxide dismutase (SOD),
catalase (CAT) and glutathione peroxidase (GPX): Their fundamental role in the entire
antioxidant defence grid. Alexandria Journal of Medicine. 2018; 54: 287-93.
Ikeda, Y, Long, D. M. The molecular basis of brain injury and brain edema: the role of oxygen
free radicals. Neurosurgery. 1990; 27:1-11.
Ilesanmi, O. O. Pathological basis of symptoms and crises in sickle cell disorder: implications
for counseling and psychotherapy. Hematol Rep. 2010; 2: e2.
Jacob, S, Streeper, R. S, Fogt, D. L, Hokama, J. Y, Henriksen, E. J, Dietze, G. J, et al. The
Antioxidant α-Lipoic Acid Enhances Insulin-Stimulated Glucose Metabolism in Insulin-
Resistant Rat Skeletal Muscle. Diabetes. 1996; 45: 1024-29.
James, T. N. Normal and abnormal consequences of apoptosis in the human heart: from
postnatal morphogenesis to paroxysmal arrhythmias. Circulation. 1994;90:556-73.
Jennings, R. B, Steenbergen C Jr. Nucleotide metabolism and cellular damage in myocardial
ischemia. Annu Rev Physiol. 1985;47:727-49.
Johnston, K. C, Wagner, D. P, Haley Jr, E. C, Connors Jr, A. F; RANTTAS Investigators.
Randomized Trial of Tirilazad Mesylate in Acute Stroke. Combined clinical and imaging
information as an early stroke outcome measure. Stroke. 2002;33(2):466-72.
Jomova, K, Valko, M. Importance of iron chelation in free radical-induced oxidative stress and
human disease. Curr Pharm Des. 2011;17: 3460-73.
Jones, S. P, Hoffmeyer, M. R, Sharp, B. R, Ho Y. S, Lefer, D. J. Role of intracellular antioxidant
enzymes after in vivo myocardial ischemia and reperfusion. Am J Physiol Heart Circ
Physiol. 2003;284(1):H277-82.

Kagan, V. E., Arroyo, A, Tyurin, V. A, Tyurina Y. Y., Villalba, J. M., Navas P. Plasma
membrane NADH-coenzymeQ10 reductase generates semiquinone radicals and recycles
vita-min E homologue in a superoxide-dependent reaction. FEBS Lett. 1998;428:43-46.
Kalogeris, T, Baines, C. P., Krenz, M., Korthuis, R. J. Cell biology of Ischemia/reperfusion
injury. Int Rev Cell Mol Biol. 2012; 298: 229-317.
Kalogeris T, Baines CP, Krenz M, Korthuis RJ. Cell Biology of Ischemia/Reperfusion Injury.
Int Rev Cell Mol Biol. 2012; 298: 229-317
Kalogeris, T., Baines, C. P, Krenz, M, Korthuis, R. J. Ischemia/Reperfusion. Compr Physiol;
2016; 7: 113-70.
Kaminogo M1, Suyama K, Ichikura A, Onizuka M, Shibata S. Anoxic depolarization
determines ischemic brain injury. Neurol Res. 1998;20:343-8.
Kannel W. B, Castelli W. P, Gordon T, McNamara P. M. Serum cholesterol, lipoproteins and
risk of coronary heart disease: the Framingham study. Ann Intern Med. 1971;74:1-12.
Kantor P. F, Lucien A, Kozak R, Lopaschuk G. D. The antianginal drug trimetazidine shifts
cardiac energy metabolism from fatty acid oxidation to glucose oxidation by inhibiting
mitochondrial long-chain 3-ketoacyl coenzyme A thiolase. Circ Res. 2000;86:580-88.
Katsuki H, Okuda S. Arachidonic acid as a neurotoxic and neurotrophic substance. Prog
Neurobiol. 1995;46:607-36.
Katsura K, Rodriguez de Turco E. B, Folbergrová J, Bazan N. G, Siesjö, B. K. Coupling among
energy failure, loss of ion homeostasis and phospholipase A2 and C activation during
ischemia. J. Neurochem. 1993;61: 1677-84.
Kay L, Finelli C, Aussedat J, Guarnieri C, Rossi A. Improvement of long term preservation of
the isolated arrested rat heart by Trimetazidine: Effects on the energy state and
mitochondrial function. Am J Cardiol. 1995;76:45B-9B.
Khan, M, Dhammu, T. S., Sakakima, H., Shunmugavel, A., Gilg, A. G., Singh, A. K, et al. The
inhibitory effect of S-nitrosoglutathione on blood-brain barrier disruption and peroxynitrite
formation in a rat model of experimental stroke. J Neurochem. 2012; 123 (Suppl 2): 86-97.
Kichev, A, Rousset, C. I., Baburamani, AA, Levison, S. W, Wood, T. L, Gressens P. et al.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling and cell death
in the immature central nervous system after hypoxia-ischemia and inflammation. J Biol
Chem. 2014;289:9430-39.
Kim G. W., Kondo T, Noshita N, Chan P. H. Manganese superoxide dismutase deficiency
exacerbates cerebral infarction after focal cerebral ischemia/reperfusion in mice:
implications for the production and role of superoxide radicals. Stroke. 2002;33:809-15.
Kinuta Y, Kimura M, Itokawa Y, Ishikawa M, Kikuchi H. Changes in xanthine oxidase in
ischemic brain. J Neurosurg. 1989;71(3):417-20.
Kitzenberg D, Colgan SP, Glover LE. Creatine kinase in ischemic and inflammatory disorders.
Clin Transl Med. 2016; 5: 31.
Kjekshus J. K., Sobel, B. E. Depressed myocardial creatine phosphokinase activity following
experimental myocardial infarction in rabbit. Circ Res. 1970;27:403-14.
Kloner R. A, Jennings R. B. Consequences of brief ischemia: stunning, preconditioning, and
their clinical implications: part I. Circulation. 2001;104:2981-89.
Kloner R. A., Jennings R. B. Consequences of brief ischemia: stunning, preconditioning, and
their clinical implications: part II. Circulation. 2001;104:3158-67.

Koizumi J, Yoshida Y, Nakazawa T, Ooneda G. Experimental studies of ischemic brain edema.

I. A new experi-mental model of cerebral embolism in rats in which recirculation can be
introduced in the ischemic area. Jpn J Stroke. 1986;8:1-8.
Kong X, Guan J, Gong S, Wang R. Neuroprotective Effects of Grape Seed Procyanidin Extract
on Ischemia-Reperfusion Brain Injury. Chin Med Sci J. 2017;32:92-99.
Kontos H. A. Oxygen radicals in cerebral vascular responses. In: Physiology of Oxygen
Radicals, edited by A. E. Taylor, S. Matalan, and P. A. Ward. Bethesda, MD: Am Physiol
Soc. 1986;207-16.
Ktazumi I, Tsukahara M. Regulation of DNA fragmentation: The role of caspases and
phosphorylation. FEBS J. 2011;278:427-41.
Kumral E1, Polat F, Uzunköprü C, Callı C, Kitiş Ö. The clinical spectrum of intracerebral
hematoma, hemorrhagic infarct, non-hemorrhagic infarct, and non-lesional venous stroke
in patients with cerebral sinus-venous thrombosis. Eur J Neurol. 2012;19:537-43.
Kutala V. K., Khan M, Mandal R, Ganesan L. P., Tridandapani S, Kalai T, et al. Attenuation
of Myocardial Ischemia-reperfusion Injury by Trimetazidine Derivatives Functionalized
with Antioxidant Properties. J Pharmacol Exp Ther. 2006;317(3):921-28.
Lafon-Cazal M, Culcasi M, Gaven F, Pietri S, Bockaert J. Nitric oxide, superoxide and
peroxynitrite: putative mediators of NMDA-induced cell death in cerebellar granule cells.
Neuropharmacology. 1993;32(11):1259-66.
Lapchak P. A., Araujo D. M. Spin Trap Agents: A New Approach to Stroke Therapy. Drug
News Perspect. 2002;15(4):220-25.
Lees K. R., Zivin J. A., Ashwood T, Davalos A, Davis S. M., Diener H. C., et al. NXY-059 for
acute ischemic stroke. N Engl J Med. 2006;354:588-600.
Lefer D. J., Granger D. N. Oxidative stress and cardiac disease. Am J Med. 2000;109(4):315-
23.
Lemasters J. J., Bond J. M., Chacon E, Harper I. S, Kaplan S. H, Ohata H, et al. The pH paradox
in ischemia-reperfusion injury to cardiac myocytes. EXS. 1996;76:99-114.
Li Y, Chopp M, Zhang Z. G, Zhang R. L, Garcia J. H. Neuronal survival is associated with 72-
kDa heat shock protein expression after transient middle cerebral artery occlusion in the
rat. J Neurol Sci. 1993;120:187-94.
Liang S, Ping Z, Ge J. Coenzyme Q10 Regulates Antioxidative Stress and Autophagy in Acute
Myocardial Ischemia-Reperfusion Injury. Oxidative Medicine and Cellular Longevity.
2017; available https://doi.org/10.1155/2017/9863181.
Lipton P, Whittingham T. S. Energy metabolism and brain slice function. In: Brain Slices,
edited by R. Dingledine. New York: Academic.1984;113-54.
Lipton P. Ischemic Cell Death in Brain Neurons. Physiol Rev. 1999;79(4):1431-68.
Liu J, Jiang X, Zhang Q, Lin S, Zhu J, Zhang Y,et al. Neuroprotective effects of Kukoamine A
against cerebral ischemia via antioxidant and inactivation of apoptosis pathway.
Neurochem Int. 2017;107:191-97.
Lo EH, Hara H, Rogowska J, Trocha M, Pierce AR, Huang PL, et al. Temporal correlation
mapping analysis of the hemodynamic penumbra in mutant mice deficient in endothelial
nitric oxide synthase gene expression. Stroke. 1996;27:1381-85.
Lopaschuk G. D, Barr R, Thomas P. D, Dyck J. R. Beneficial effects of trimetazidine in ex-
vivo working ischemic hearts are due to a stimulation of glucose oxidation secondary to
inhibition of long-chain 3-ketoacyl coenzyme a thiolase. Circ Res. 2003;93:e33-e37.

Lopchack P. A., et al., Novel MMP inhibitor, batimastat, on BBB of mice. Stoke. 2000;31:3034-
35.
Lu C. J, Guo Y. Z, Zhang Y, Yang L, Chang Y, Zhang J. W., et al. Coenzyme Q10 ameliorates
cerebral ischemia reperfusion injury in hyperglycemic rats. Pathol Res Pract;
2017:213:1191-99.
Lu Z, Zhang C, Zhai Z. Nucleoplasmin regulates chromatin condensation during apoptosis.
Proc Natl Acad Sci U S A. 2005;102: 2778-83.
Ludwig R. L., Bates S, Vousden KH. Differential activation of target cellular promoters by p53
mutants with impaired apoptotic function. Mol Cell Biol. 1996;16:4952-60.
Majno G, Joris I. Apoptosis, oncosis, and necrosis: an overview of cell death. Am J Pathol.
1995;146:3-15.
Mandir A. S., Poitras M. F., Berliner A. R., Herring W. J., Guastella D. B., Feldman A, et al.
NMDA but not non-NMDA excitotoxicity is mediated by Poly(ADP-ribose) polymerase.
J Neurosci. 2000;20:8005-11.
Marczin N, El-Habashi N, Hoare G. S, Bundy R. E, Yacouba M. Antioxidants in myocardial
ischemia–reperfusion injury: therapeutic potential and basic mechanisms. Arc of Biochem
Biophy. 2003;420:222-36.
Marczin N, El-Habashi N, Hoare G. S, Bundy R. E, Yacouba M. Antioxidants in myocardial
ischemia–reperfusion injury: therapeutic potential and basic mechanisms. Arc of Biochem
Biophy. 2003;420:222-36.
Margaill I, Plotkine M, Lerouet D. Antioxidant strategies in the treatment of stroke. Free Radic
Biol Med. 2005; 39:429-43.
Marro, P. J, McGowan J. E, Razdan B, Mishra O. P, Delivoria P. M. Effect of allopurinol on
uric acid levels and brain cell membrane Na +/K + -ATPase activity during hypoxia in
newborn piglets. Brain Res. 1994; 650: 9-15.
Marshall J. W. B., Duffin K. J., Green A. R., Ridley R. M. NXY-059, a free radical-trapping
agent, substantially lessens the functional disability resulting from cerebral ischemia in a
primate species. Stroke. 2003; 32: 190-98.
Martin R. L., Lloyd H. G., Cowan A. I. The early events of oxygen and glucose deprivation:
setting the scene for neuronal death. Trends Neurosci.1994; 17: 251-57.
Martin S. J., Green D. R., Cotter T. G. Dicing with death: dissecting the components of the
apoptosis machinery. Trends Biochem Sci. 1994; 19: 26-30.
Matsuo Y, Onodera H, Shiga Y, Nakamura M, Ninomiya M, Kihara T, et al. Correlation
between myeloperoxidase-quantified neutrophil accumulation and ischemic brain injury in
the rat. Effects of neutrophil depletion. Stroke. 1994; 25: 1469-75.
Matsuo Y, Onodera H, Shiga Y, Nakamura M, Ninomiya M, Kihara T, et al. Correlation
between myeloperoxidase-quantified neutrophil accumulation and ischemic brain injury in
the rat. Effects of neutrophil depletion. Stroke. 1994; 25: 1469-75.
Matthews R. T., Yang L, Browne S, Baik M, Beal M. F. Coenzyme Q10 administration
increases brain mitochondrial concentrations and exerts neuroprotective effects. Proc Natl
Acad Sci USA. 1998; 95: 8892-97.
McIlwain D. R. Berger T, Mak T. W. Caspase functions in cell death and disease. Cold Spring
Harb Perspect Biol. 2013; 5: available doi: 10.1101/cshperspect.a008656.
Mdzinarishvili A. Engineering triiodothyronine (T3) nanoparticle for use in ischemic brain
stroke. Drug Delivery and Translational Research. 2012;3: 309-31.

Mestre H, Cohen-Minian C, Zajaias-Fainsod D, Ibarra A. Pharmacological Treatment of Acute

Ischemic Stroke. In: Kishore U, editor. Neurodegenerative Disease. Intech Open. 2013;
Available at https://www.intechopen.com/books/neurodegenerative-diseases/pharma
cological-treatment-of-acute-ischemic-stroke.
Meyer F. B, Anderson R. E, Sundt Jr, T. M, Yaksh T. L. Intracellular brain pH, indicator tissue
perfusion and electroencephalography and histology in severe and moderate focal cortical
ischemia. J Cereb Blood Flow Metab. 1986;6:71-78.
Mimic-Oka J, Simic D. V, Simic V. Free Radicals In Cardiovascular Diseases. Med Biol.
1999;6(1):11-22.
Mironova O. P., Zarubina I. V., Krivoruchko B. I., Smirnov A. V. Antioxidant effects of
amtizol and trimetazidine in brain ischemia. Patol Fiziol Eksp Ter. 2001;7(3):13-16.
Misra S, Talwar P, Kumar A, Kumar P, Sagar R, Vibha D, et al. Association between matrix
metalloproteinase family gene polymorphisms and risk of ischemic stroke: A systematic
review and meta-analysis of 29 studies. Gene. 2018; 25;672:180-94.
Mizoue R, Takeda Y, Sato S, Takata K, Morimatsu H. Cerebral Blood Flow Threshold Is
Higher for Membrane Repolarization Than for Depolarization and Is Lowered by
Intraischemic Hypothermia in Rats. Crit Care Med. 2015;43:e350-55.
Mlynárik V, Kasparová S, Liptaj T, Dobrota D, Horecký J, Belan V. Creatine kinase reaction
rates in rat brain during chronic ischemia. Magn Reso Mat in Bio Phy and Med.
1998;7:162-65.
Mohanty I, Singh Arya D, Dinda A, Joshi S, Talwar K. K, Gupta S. K. Protective effects of
Curcuma longa on ischemia-reperfusion induced myocardial injuries and their
mechanisms. Life Sciences. 2004;75:1701-11.
Molina C. A, Saver J. L. Extending Reperfusion Therapy for Acute Ischemic Stroke: Emerging
Pharmacological, Mechanical, and Imaging Strategies. Stroke. 2005;36:2311-20.
Mollace V, Iannone M, Muscoli C, Palma E, Granato T, Modesti A, et al. The protective effect
of M40401,a superoxide dismutase mimetic, on post-ischemic brain damage in Mongolian
gerbils. BMC Pharmacology. 2003;3:1-9.
Morel O, Perret T, Delarche N, Jouve B, Elbax M, Piot C, et al. Pharmacological approaches
to reperfusion therapy. Cardiovascular research. 2012; 94:246-52.
Müller A, Cadenas E, Graf P, Sies H. A novel biologically active seleno-organic compound-I.
Glutathione peroxidase-like activity in vitro and antioxidant capacity of PZ 51 (Ebselen).
Biochem. Pharmacol. 1984;15:3235-39.
Murakami K, Kondo T, Kawase M, Li Y, Sato S, Chen S. F., et al. Mitochondrial susceptibility
to oxidative stress exacerbates cerebral infarction that follows permanent focal cerebral
ischemia in mutant mice with manganese superoxide dismutase deficiency. J Neurosci.
1998;18:205-13.
Nagasawa H, Kogure K. Correlation between cerebral blood flow and histological change in a
new rat model of cerebral artery occlusion. Stroke. 1989;20:1037-43.
Nakamura M, Thourani V. H. , Ronson R. S, Velez D. A., Ma X. L., Katzmark S, et al.
Glutathione reverses endothelial damage from peroxynitrite, the byproduct of nitric oxide
degradation, in cristaloid cardioplegia. Circulation. 2000; 102:III332-38.
Nakano S, Kogure K, Abe K, Yae T. Ischemia-induced alterations in lipid metabolism of the
gerbil cerebral cortex. I. Changes in free fatty acid liberation. J Neurochem. 1990;54:1911-
16.

Namba K, Takeda Y, Sunami K, Hirakawa M. Temporal profiles of the levels of endogenous

antioxidants after four-vessel occlusion in rats. J Neurosurg Anesthesiol. 2001;13:131-37.
Nihei H, Kanemitsu H, Tamura A, Oka H, Sano K. Cerebral uric acid, xanthine, and
hypoxanthine after ischemia: the effect of allopurinol. Neurosurgery. 1989;25:613-17.
Niizuma K, Yoshioka H, Chen H, Kim G. S., Jung J. E., Katsu M, et al. Mitochondrial and
apoptotic neuronal death signaling pathways in cerebral ischemia. Biochim Biophys Acta.
2010; 1802: 92-99.
Nishigaya K, Yoshida Y, Sasuga M, Nukui H, Ooneda G. Effect of recirculation on
exacerbation of ischemic vascular lesions in rat brain. Stroke. 1991;22:635-42.
Nishizawa Y. Glutamate release and neuronal damage in ischemia. Life science. 2001;69:369-
81.
Ohtani K, Tanaka H, Ohno Y. SM-31900, a novel NMDA receptor glycine-binding site
antagonist, reduces infarct volume induced by permanent middle cerebral artery occlusion
in spontaneously hypertensive rats. Neurochem Int. 2003;42(5):375-84.
Okado M. A, Fridovich I. Subcellular distribution of superoxide dismutases (SOD) in rat liver.
J Biol Chem. 2001;276:38388-93.
Oliver L, Vallette F. M. The role of caspases in cell death and differentiation. Drug Resistance
Updates. 2005;8:163-70.
Ozer M. K., Parlakpinar H, Cigremis Y, Ucar M, Vardi N, Acet A. Ischemia-reperfusion leads
to depletion of glutathione content and augmentation of malondialdehyde production in the
rat heart from overproduction of oxidants: can caffeic acid phenethyl ester (CAPE) protect
the heart? Mol Cell Biochem. 2005;273:169-75.
Packer L, Witt E. H, Tritschler H. J. Alpha-lipoic acid as a biological antioxidant. Free Rad
Biol Med. 1995;19:227-50.
Packer L, Witt E. H, Tritschler H. J. alpha-Lipoic acid as a biological antioxidant. Free Radic
Biol Med. 1995;19(2):227-50.
Panigrahi M, Sadguna Y, Shivakumar B. R, Kolluri S. V, Roy S, Packer L, et al. alpha-lipoic
acid protects against reperfusion injury following cerebral ischemia in rats. Brain Res.
1996;717:184-88.
Pant S, Deshmukh A, Neupane P. Middle cerebral artery preponderance in ischemic stroke: A
coincidence or fate? Medical Hypotheses. 2012; 79: 63-64.
Passwater R. A. Lipoic Acid: The Metabolic Antioxidant. New Canaan, CT: Keats Publishing
Inc.1995;1-47.
Payne RS1, Tseng M. T, Schurr A. The glucose paradox of cerebral ischemia: evidence for
corticosterone involvement. Brain Res. 2003;971:9-17.
Petersen S. V, Valnickova Z, Oury T. D, Crapo J. D, Nielsen N. C, Enghild J. J. The subunit
composition of human extracellular superoxide dismutase (EC-SOD) regulate enzymatic
activity. BMC Biochem. 2007; 8:19.
Piper H. M, Abdallah Y, Schafer C. The first minutes of reperfusion: a window of opportunity
for cardioprotection. Cardiovas Res. 2004;61:365-71.
Piper H. M, Meuter K, Schafer C. Cellular mechanism of ischemia-reperfusion injury. Ann
Thorac Surg. 2003;75:S644-48.
Pizzino G, Irrera N, Cucinotta M, Pallio G, Mannino F, Arcoraci V, et al. Oxidative Stress:
Harms and Benefits for Human Health. Oxid Med Cell Longev. 2017; 2017: available at
10.1155/2017/8416763.

Pongmoragot J, Saposnik G. Intracerebral Hemorrhage from cerebral venous thrombosis. Curr

Atherosclcr Rcp. 2012;14:382-89.
Purucker E, Egri L, Hamar H, Augustin A. J, Lutz J. Differences in glutathione status and lipid
peroxidation of red and white muscles: alterations following ischemia and reperfusion. Res
Exp Med (Berl). 1991;191:209-17.
Qin Z, Resxka K. J, Fukai T, Weintraub N. L. Extracellular superoxide dismutase (ecSOD) in
vascular biology: an update on exogenous gene transfer and endogenous regulators of
ecSOD. Transl Res. 2008; 151:68-78.
Radi R, Beckman J. S, Bush K. M, Freeman B. A. Peroxynitrite-induced membrane lipid
peroxidation: The cytotoxic potential of superoxide and nitric oxide. Arch Biochem
Biophys 1991;288:481-87.
Ramana K. V, Srivastava S, Singhal S. S. Lipid Peroxidation Products in Human Health and
Disease. Oxid Med Cell Longev. 2013; Available at http://dx.doi.org/10.1155/2013/
583438.
Rao A. M, Hatcher J. F, Kindy M. S, Dempsey R. J. Arachidonic acid and leukotriene C4: role
in transient cerebral ischemia of gerbils. Neurochem Res. 1999;24:1225-32.
Richard M. J, Connell B. J, Khan B. V, Saleh T. M. Cellular mechanisms by which lipoic acid
confers protection during the early stages of cerebral ischemia: a possible role for calcium.
Neurosci Res. 2011;69(4):299-307.
Robbins N. M, Swanson R. A. Opposing effects of glucose on stroke and reperfusion injury:
acidosis, oxidative stress, and energy metabolism. Stroke. 2014; 45: 1881-86.
Rubbo H, Radi R, Trujillo M, Telleri R, Kalyanaraman B, Barnes S, et al. Nitric oxide
regulation of superoxide and peroxynitritedependent lipid peroxidation. Formation of
novel nitrogen-containing oxidized lipid derivatives. J Biol Chem. 1994; 269:26066-75.
Saad M. A, Abdel Salam R. M, Kenawy S. A, Attia A. S. Pinocembrin attenuates hippocampal
inflammation, oxidative perturbations and apoptosis in a rat model of global cerebral
ischemia reperfusion. Pharmacol Rep. 2015;67:115-22.
Saito I, Asano T, Sano K, Takakura K, Abe H, Yoshimoto T, et al. Neuroprotective effect of
an antioxidant, ebselen, in patients with delayed neurological deficits after aneurysmal
subarachnoid hemorrhage. Neurosurgery. 1998;42:269-77.
Saks, V. A, Khuchua, Z. A, Vasilyeva, E. V, Belikova, O. Yu, Kuznetsov, A. V. Metabolic
compartmentation and substrate channeling in muscle cells: role of coupled creatine
kinases in in vivo regulation of cellular respiration: a synthesis. Mol Cell Biochem.
1994;133:155-92.
Saposnik G, Goodman S. G, Leiter L. A, Yan R. T, Fitchett D. H, Bayer N. H, et al. Applying
the evidence: do patients with stroke, coronary artery disease, or both achieve similar
treatment goals? Stroke. 2009;40:1417-24.
Saraf M. K, Prabhakar S, Anand A. Neuroprotective effect of Bacopa monniera on ischemia
induced brain injury. PharmacolBiochemBehav. 2010;97:192-97.
Schewe T. Molecular actions of ebselen--an antinflammatory antioxidant. Gen. Pharmacol.
1995;26:1153-69.
Schönfeld P, Reiser G. Why does brain metabolism not favor burning of fatty acids to provide
energy? - Reflections on disadvantages of the use of free fatty acids as fuel for brain. J
Cereb Blood Flow Metab. 2013; 33: 1493-99.

Sharma T, Airao V, Panara N, Vaishnav D, Ranpariya V, Sheth N, et al. Solasodine protects

rat brain against ischemia/reperfusion injury Through its antioxidant activity. Eur J
Pharmacol. 2014;725:40-46.
Sharp F. R, Zhan X, Liu D. Z. Heat shock proteins in the brain: role of Hsp70, Hsp 27, and HO-
1 (Hsp32) and their therapeutic potential. Transl Stroke Res. 2013;4(6):685-92.
Sheng H, Brady T. C, Pearlstein R. D, Crapo J. D, Warner D. S. Extracellular superoxide
dismutase deficient mice have decreased resistance to focal cerebral ischemia. Neurosci
Lett. 1999;267:13-17.
Shi Y, Wardlaw J. M. Update on cerebral small vessel disease: a dynamic whole-brain disease.
Stroke Vasc Neurol. 2016; 1: 83-92.
Shimizu S. M, Kano T, Rogowska J, Wolf G. L, Moskowitz M. A, Lo E. H. YM872, a highly
water-soluble ampa receptor antagonist, preserves the hemodynamic penumbra and
reduces brain injury after permanent focal ischemia in rats. Stroke. 1998;29:2141-48.
Shmonin A. A, Melnikova E. V, Churilova I. V, Vlasov T. D. Experimental Study of the
Neuroprotective Properties of Alpha-Lipoic Acid and Superoxide Dismutase in Cerebral
Ischemia in Rats. Neuroscience and Behavioral Physiology; 2012;42:429-33.
Sige H, Prijs B, McCormick D. B, Shih J. C. Stability of binary and ternary complexes of alpha-
lipoate and lipoate derivatives with Mn2+, Cu2+, and Zn2+ in solution. Arch Biochem
Biophys. 1978;187:208-14.
Simard J. M, Kent T. A, Chen M, Tarasov K. V, Gerzanich V. Brain oedema in focal ischaemia:
molecular pathophysiology and theoretical implications. Lancet Neurol. 2007; 6: 258-68.
Sinning C, Westermann D, Clemmensen P. Oxidative stress in ischemia and reperfusion:
current concepts, novel ideas and future perspectives. Biomark Med. 2017;11:11031-40.
Snow S. J. Stroke and t-PA -Triggering New Paradigms of Care. N Engl J Med. 2016;
374(9):809-11.
Sohal R. S., Forster M. J. Coenzyme Q, oxidative stress and aging. Mitochondrion. 2007;
7(Suppl): S103-11.
Soler E. P, Ruiz V. C. Epidemiology and Risk Factors of Cerebral Ischemia and Ischemic Heart
Diseases: Similarities and Differences. Current Cardiology Reviews, 2010; 6:138-49.
Sravanthi K. N, Rao N. R. Cerebroprotective activity of Pentapetesphoenicea on global cerebral
ischemia in rats. Indian J Pharmacol. 2016;48:694-700.
Stendahi, O, Coble B. I, Dahlgren C, Hed J, Molin L. Myeloperoxidase modulates the
phagocytic activity of polymorpho neutrophil leukocytes. Studies with cells from a
myeloperoxidase deficient patient. J Clin Invent. 1984;73:366-73.
Stone M. C, Thorpe J. M. Plasma fibrinogen a major coronary risk factor. J R Coll Gen Pract.
1985;35:565-69.
Sugamura K, Keaney J. F. Reactive Oxygen Species in Cardiovascular Disease. Free Radic
Biol Med. 2011; 51: 978-92.
Sugawara H, Yamamoto T, Shimizu S, Momose K. Inhibition of ubiquinone synthesis in
isolated rat heart under ischemic condition. Int J Biochem. 1990;22:477-80.
Sun M. S, Jin H, Sun X, Huang S, Zhang FL, Guo Z. N, et al. Free Radical Damage in Ischemia-
Reperfusion Injury: An Obstacle in Acute Ischemic Stroke after Revascularization
Therapy. Oxid Med Cell Longev. 2018; 2018: available 10.1155/2018/
3804979.
Sun M. S, Jin H, Sun X, Huang S, Zhang F. L, Guo A. N, et al. Free Radical Damage in
Ischemia-Reperfusion Injury: An Obstacle in Acute Ischemic Stroke after

Revascularization Therapy. Oxidative Medicine and Cellular Longevity. 2018: available at

https://doi.org/10.1155/2018/3804979.
Sunamori M, Tanaka H, Maruyama T, Sultan I, Sakamoto T, Suzuki A. Clinical experience of
coenzyme Q10 to enhance intraoperative myocardial protection in coronary artery
revascularization. Cardiovasc Drugs Ther. 1991;5:297-300.
Suzuki M, Tabuchi M, Ikeda M, Tomita T. Concurrent formation of peroxynitrite with the
expression of inducible nitric oxide synthase in the brain during middle cerebral artery
occlusion and reperfusion in rats. Brain Res. 2002;951:113-20.
Talluria M. R., Killari K. N., Manepalli V. M., Konduri P., Bandaru K. K. Protective effect of
Canna indica on cerebral ischemia-reperfusion 1 injury in rats. Agricul Nat Reso.
2017;51:470-77.
Teng Y, Jin H, Nan D, Li M, Fan C, Liu Y et al. In vivo evaluation of urokinase-loaded hollow
nanogels for sonothrombolysis on suture embolization-induced acute ischemic stroke rat
model. Bioact Mater. 2017; 24(3):102-09.
Teringova E, Tousek P. Apoptosis in ischemic heart disease. J Transl Med. 2017;15:87.
Thiyagarajan M, Sharma SS. Neuroprotective effect of curcumin in middle cerebral artery
occlusion induced focal cerebral ischemia in rats. Life Sciences. 2004;74:969-85.
Umemura K, Wada K, Uematsu T, Mizuno A, Nakashima M. ffect of 21-aminosteroid lipid
peroxidation inhibitor, U74006F, in the rat middle cerebral artery occlusion model. Eur J
Pharmacol. 1994;251:69-74.
Van BF, Shadid M, Moison RM, Dorrepaal CA, Fontijn J, Monteiro L, et al. Effect of
allopurinol on postasphyxial free radical formation, cerebral hemodynamics, and electrical
brain activity. Pediatrics. 1998;101:185-93.
Veltkamp R, Gill, D. Clinical Trials of Immunomodulation in Ischemic Stroke.
Neurotherapeutics. 2016; 13, 791-800.
Venkat P, Choppa M, Chen J. Blood–Brain Barrier Disruption, Vascular Impairment, and
Ischemia/Reperfusion Damage in Diabetic Stroke. J Am Heart Assoc. 2017; 6: available
10.1161/JAHA.117.005819.
Verma S, Fedak PWM, Weisel RD, Butany J, Rao V, Maitland A, et al. Fundamentals of
reperfusion injury for the clinical cardiologist. Circulation. 2002;105:2332-36.
Vinten-Johansen J. Involvement of neutrophils in the pathogenesis of lethal myocardial
reperfusion injury. Cardiovasc Res. 2004;61:481-97.
Wang X, Feng A, Zhu C. Cerebrospinal fluid levels of free fatty acid associated with ischemic
stroke recurrence and functional outcome. Neurol Sci. 2016;37:1525-29.
Wang Z, Liu T, Gan L, Wang T, Yuan X, Zhang B, et al. Shikonin protects mouse brain against
cerebral ischemia/reperfusion injury through its antioxidant activity. Eur JPharmacol.
2010;643:211-17.
Warner D. S, Sheng H, Batinić-Haberle I. Oxidants, antioxidants and the ischemic brain. The J
Exptal Biol. 2004;207:3221-31.
Watts D. C. Creatine kinase (adenosine 5'-triphosphate-creatine phosphotransferase). In: Boyer
PD, ed. The Enzyme. New York, NY: Academic Press. 1973;8:383-455.
Weinstein P. R, Hong S, Sharp F. R. Molecular identification of the ischemic penumbra. Stroke.
2004;35:2666-70.
Welln L, Svardsudd K, Wllhelmsen L, Larsson B, Tlbblln G. Analysis for risk factors of stroke
In a cohort of men bom In 1913. N Engl J Med. 1987;317:521-26.

Wicha P, Tocharus J, Janyou A, Jittiwat J, Changtam C, Suksamrarn A, et al.

Hexahydrocurcumin protects against cerebral ischemia/reperfusion injury, attenuates
inflammation, and improves antioxidant defenses in a rat stroke model. Plosone.
2017;12:e0189211.
Wilhelmsen L, Svärdsudd K, Korsan-Bengtsen K, Larsson B, Welin L, Tibblin G. Fibrinogen
as a risk factor for stroke and myocardial Infarction. N Engl J Med. 1984;311:501-05.
Wilkins R. H. Cerebral vasospasm. Contemp Neurosurgery. 1988;10:1-66.
Williams G. D, Palmer C, Heitjan D. F, Smith M. B. Allopurinol preserves cerebral energy
metabolism during perinatal hypoxia-ischemia:a 31P NMR study in unanesthetized
immature rats. Neurosci Lett. 1992;144:103-06.
Yaidikar L, Thakur S. Arjunolic acid, a pentacyclic triterpenoidal saponin of Terminalia arjuna
bark protects neurons from oxidative stress associated damage in focal cerebral ischemia
and reperfusion. Pharmacol Rep. 2015;67:890-95.
Yamaguchi T, Sano K, Takakura K, Saito I, Shinohara Y, Asano T, et al. Ebselen in acute
ischemic stroke: a placebo-controlled, double-blind clinical trial. Ebselen Study Group.
Stroke. 1998;29:12-17.
Yan X. B, Meng F. J, Song B, Zhang G. Y. Brain ischemia induces serine phosphorylation of
neuronal nitric oxide synthase by Ca(2+)/calmodulin-dependent protein kinase II in rat
hippocampus. Acta Pharmacol Sin. 2004;25:617-22.
Yang G, Chan P. H, Chen J, Carlson E, Chen S. F, Weinstein P, et al. Human copper-zinc
superoxide dismutase transgenic mice are highly resistant to reperfusion injury after focal
cerebral ischemia. Stroke. 1994;25:165-70.
Yang J, Klaidman L. K, Chang M L, Kem S, Sugawara T, Chan P, et al. Nicotinamide therapy
protects against both necrosis and apoptosis in a stroke model. Pharmacol Biochem Behav.
2002;73:901-10.
Ye R, Kong X, Yang Q, Zhang Y, Han J, Zhao G. Ginsenoside Rd attenuates redox imbalance
and improves stroke outcome after focal cerebral ischemia in aged mice.
Neuropharmacology. 2011;61:815-24.
Yellon D. M, Baxter G. F. Protecting the ischaemic and reperfused myocardium in acute
myocardial infarction: distant dream or near reality? Heart. 2000;83:381-87.
Yoshimoto T, Kanakaraj P, Ying Ma J, Cheng M, Kerr I, Malaiyandi L, et al. NYX-059
maintains Akt activation and inhibits release of cytochrome c after focal cerebral ischemia.
Brain Res. 2002;947:191-98.
Young J. N, Aitken P G, Somjen G. G. Calcium, magnesium, and long-term recovery from
hypoxia in hippocampal tissue slices. Brain Res. 1991;548:343-45.
Yu S. W, Wang H, Poitras M. F, Coombs C, Bowers W. J, Federoff H J, et al. Mediation of
poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor.
Science. 2002;297:259-63.
Yuan J, Najafov A, Py B. F. Roles of Caspases in Necrotic Cell Death. Cell. 2016; 167:1693–
1704.
Zhai P, Eureli T. E, Cotthaus R, Jeffery E. H, Bahr J. M, Gross D. R. Effect of estrogen on
global myocardial ischemia-reperfusion injury in female rats. Am J Physiol Heart Circ
Physiol. 2000;279:H2766-775.
Zhang J. P, Sun G. Y. Free fatty acids, neutral glycerides and phosphoglycerides in transient
focal ischemia. J Neurochem. 1995;64:1688-95.

Zhang L, Zhang X, Zhang C, Bai X, Zhang J, Zhao X,et al. Nobiletin promotes antioxidant and
anti-inflammatory responses an delicits protection against ischemic stroke in vivo. Brain
Res. 2016;1636:130-41.
Zhao Y. Z, Zhang M, Liu H. F, Wang J. P. Progesterone is neuroprotective by inhibiting
cerebral edema after ischemia. Neural Regen Res. 2015; 10: 1076-81.
Zheng C. J, Han L. Y, Yap CW, JI Z. L, Cao Z. W, Chen Y. Z. Therapeutic Targets: Progress
of Their Exploration and Investigation of Their Characteristics. Pharmacol. Rev. 2006;
58:259-79.
Zweier J. L, Talukder M. A. H. The role of oxidants and free radicals in reperfusion injury.
Cardiovas Res. 2006;70:181-90.

SECTION VI: ANTIOXIDANT STATUS IN
METABOLIC DISORDERS

Chapter 15
FREE RADICALS, ANTIOXIDANTS AND DIABETES
Seetha Harilal* and K. Rajesh

Kerala University of Health Sciences, Kerala, India
ABSTRACT
Diabetes is a carbohydrate metabolic disorder that affects the pancreatic beta cells
either at a much younger age as in type 1 diabetes or at a longer run as in type 2 diabetes;
special category as in gestational and other specific types of diabetes. Both type 1 and type
2 diabetes mellitus lead to microvascular as well as macrovascular complication as a result
of uncontrolled management of blood sugar level for a prolonged period of time. These
have specific pathways involved and science is advancing to the level of targeting specific
pathways to halt further progress of the complication. A number of genetic factors are
involved in the causation of diabetes mellitus which will be discussed in this chapter in
order to give an insight to the readers on complexity of the disorder and its treatment in
different populations. Oxidative stress plays a key role in all diseases including diabetes.
Targeting the reactive oxygen or nitrogen species can be an adjuvant to the currently
available antidiabetics. Hence the chapter also includes the signaling pathway and the role
of oxidative stress in disease formation and its advancement and the effect of various
antioxidants in diabetes and its complications.
1. INTRODUCTION
Diabetes refers to both diabetes mellitus and diabetes insipidus out of which diabetes
mellitus is the one generally referred to as diabetes. In spite of the similarity in the term, they
are unrelated. Diabetes mellitus is derived from the Greek word “diabainein” meaning “to pass/
go through” and Latin word “mellitus”, meaning “sweetened with honey”. In diabetes insipidus
the latin word "insipidus" means "lacking taste or flavor". Diabetes insipidus is a condition of
polyuria accompanied by polydipsia ((Ahmed, 2002; Farlex 2012). Diabetes mellitus is a
metabolic disorder of carbohydrate causing either a reduced production of insulin to neutralize
*
Corresponding Author’s Email: seethaharilal1989@gmail.com.

394 Seetha Harilal and K. Rajesh
the glucose or resistance of the produced insulin which can be a result of environmental and
hereditary factors. This is characterized by hyperglycaemic condition making the patients
experience symptoms such as polyuria, polydipsia, and polyphagia and affects a population at
various stages such as infancy, pregnancy, adulthood and oldness (Farlex 2012).
Type 1 diabetes mellitus (DM) also known as autoimmune diabetes can occur in the
presence or absence of autoimmune responses with a strong hereditary component and results
in pancreatic beta cell destruction. Type 2 DM is the dysfunction of pancreatic beta cells and is
developed due to factors such as obesity, smoking, and physical inactivity. There is an increased
occurrence of type 2 DM in men and women within the age band of 40- 49 (Chaturvedi et al.,
1998; Connolly et al., 2000). Gestational diabetes occurs in pregnant women and subsides after
delivery. This may cause growth disorders in the fetus and an increased risk of developing
diabetes mellitus. Later, women with gestational diabetes are at an increased risk of developing
type 2 DM due to the factors such as obesity contributing to insulin resistance as well as
autoimmune mediated type 1 DM (American Diabetes Association, 2004; Langer, 2011).
Another category of diabetes includes specific types of DM which have distinguished
features in comparison to other types of diabetes occurring secondary to specific disease
conditions (Kahn, 2007). Globally 382 million people are diabetic and this is expected to rise
to 592 million by the year 2035. Epidemiological studies reveal a 2-5% increase in the
incidence of type 1 DM occurrence (Shojaeian et al., 2018); 20% increase in developed
countries and 69% in developing countries by 2030 (Shaw et al., 2010); 1.2- 3.1% in European
and 1.9- 13.7% in pregnant women of Southeast Asia (Bellamy et al., 2009; Chen et al., 2016;
Zheng et al., 2018).
In the long run, diabetes further affects organs and organ systems such as nerves, eye,
kidney, cardiovascular system, cerebrovascular system, blood vessels, male genitalia causing
complications which are severe and less common, categorized as the microvascular and
macrovascular complications. These are a result of protein kinase C activation causing an
increased release of various inflammatory mediators and reactive nitrogen as well as oxygen
species thereby inducing oxidative stress and damage to various systems in the body
(Aghadavod et al., 2016; Baynes, 1991; Chou et al., 2002; Kahn, 2007; Lukovits et al., 1999).
This damage can be corrected by the use of antioxidants which scavenges the free radicals and
protects the body from oxidative stress-induced damage and can be of natural or synthetic
origin (Madsen et al., 1995). This chapter aims to highlight the various pathways involved in
diabetes mellitus causation and how antioxidants modulate oxidative stress induced diabetes
and improve the condition of the diabetics.
2. CLASSIFICATION OF DIABETES MELLITUS

According to WHO, diabetes mellitus can be classified based on etiological factors such as
pancreatic beta cell dysfunction or destruction, other genetic factors involved and disruption in
glucose homeostasis seen in conditions such as pregnancy as mentioned in Figure 1 (Alberti et
al., 1998; American Diabetes Association, 2014; Kahn, 2007). Classification of diabetes
mellitus is given in Table 1.

Free Radicals, Antioxidants and Diabetes 395
Table 1. Classification of diabetes mellitus with reference to its etiological

factors and characteristics
Types of Diabetes Characteristics/ Etiological Factors

1. Type 1 Pancreatic beta cell destruction
I. Type1 A: Presence of autoimmune responses
Autoimmune
II. Type 1 B: Idiopathic The absence of autoimmune responses
2. Type 2 Pancreatic beta cell dysfunction or a gradual pancreatic beta cell failure
3. Other specific types Distinctive features compared to other types of diabetes; Secondary to
etiologically varying diseases/ syndromes/ other specific conditions
I. Genetic defects of β- Associated with specific monogenic defects; impaired insulin secretion
cell function
MODY 1 Chromosome 20, HNF-4α
MODY 2 Chromosome 7, glucokinase
MODY 3 Chromosome 12, HNF-1α
MODY 4 Chromosome 13, IPF1
MODY 5 Chromosome 17, HNF-3β
Mitochondria DNA A-G mutation of Leu UUR gene at position 3243 resulting in diabetes
mellitus linked deafness
Others SUR1 mutation- causes diminished ability to secrete insulin and
congenital hyperinsulinemia and secretory capacity in adults
II. Genetic defects in
insulin action
Type A insulin Rare disorder with severe insulin resistance; in women develops primary
resistance amenorrhea or oligomenorrhea, ovarian cysts, hirsutism whereas in
males, develop hypoglycemia and, in some acanthosis, nigricans
Leprechaunism Genetic modification causing defects in insulin action
Rabson-Mendenhall A disorder that occurs rarely and characteristic feature is severe insulin
syndrome resistance; in women develops features such as muscle atrophy,
polycystic ovary, organ enlargements such as genitalia, heart, kidneys,
nipples, and lack of subcutaneous fat
Lipoatrophic diabetes Association with insulin-resistant diabetes and hypertriglyceridemia
III. Diseases of the Islet of Langerhans damage alters α, β and pancreatic polypeptide cells
exocrine pancreas hormonal secretion contributing to a lesser production of glucagon,
insulin and pancreatic polypeptide resulting in glucose level fluctuation
Fibrocalculous Malnutrition-related diabetes
pancreatopathy
Pancreatitis Inflammation of pancreas; caused due to activation of digestive enzymes
in the pancreas
Trauma/ Impaired metabolism of glucose and production of endocrine and
pancreatectomy exocrine hormones
Neoplasia Mechanism not completely elucidated; prevalence in a pancreatic or
pancreatogenic diabetes mellitus (T3cDM) due to chronic pancreatitis or
insulin treatment
Cystic fibrosis A characteristic feature is the presence of thick and sticky mucus which
develops scar on the pancreas thereby preventing the pancreas to secrete
insulin required normally
Hemochromatosis Occurs most commonly in European whites due to homozygosity for
p.C282Y of the HFE gene
Wolcott-Rallison Mutation of PERK, EIF2AK3; characterized by early-onset diabetes with
syndrome skeletal dysplasia

Table 1. (Continued)

IV. Endocrinopathies
Cushing’s syndrome Develops diabetes due to excess glucocorticoid which impairs insulin
secretion and various steps involved in insulin secretion within
pancreatic beta cells
Acromegaly Occurs as a result of excess GH; progresses to cardiovascular
complications, diabetes mellitus, osteoarthritis, goiter, polyps etc.
Pheochromocytoma These are rare tumors that are developed as a result of the production,
storage and metabolism of catecholamines (predominantly
norepinephrine) from extra- or intra-adrenal chromaffin cells.
Epinephrine and norepinephrine due to its affinity to β2 adrenoceptors
in the liver and pancreatic β-cells causes hyperglycemia.
Glucagonoma Occurs in those with a family history of MEN1
Hyperthyroidism Is associated with type 2 DM and cause insulin resistance; produces
effects directly (by the promotion of gluconeogenesis due to the
elevation in basal hepatic glucose output) as well as indirectly (by
hypothalamus mediated sympathetic activity stimulation)
Somatostatinoma Rarely occurring neuroendocrine tumor due to the presence of
somatostatin inhibits most of the gut wall secretions including
gastrointestinal hormones, exocrine secretory action of the pancreas
and pancreatic hormones (such as insulin, glucagon) release.
Abnormal elevation of somatostatin can result in reduced insulin
secretion and increased resistance.
V. Drug or chemical- Certain drugs and chemicals cause a reduction in the peripheral
induced (such as insulin sensitivity, elevate fatty acids, promote hepatic
glucocorticoids, interferon- gluconeogenesis thereby increase production of glucose, pancreatic
α, nicotinic acid, beta cell damage or dysfunction causing impairment in the release of
pentamidine, phenytoin, insulin.
pyriminil [vacor],
thiazides, thyroid hormone,
α-adrenergic agonists, β-
adrenergic agonists)
VI. Infections
Congenital rubella The probable mechanism can be rubella causes a reduction in the
pancreatic beta cells thereby inhibiting insulin production
Cytomegalovirus (CMV) Pancreatic beta cell damage can occur directly by CMV infection of
the beta cells accompanied by cytopathic effects. In addition to this
increased release of pro-inflammatory cytokines due to the buildup of
late-differentiated CD4+ as well as CD8+ T-cells increases cellular
immunogenicity. CMV affects pancreatic duct cells indirectly via IL-
1 β production causing the production of TNF -α leading to apoptosis.
Others
VII. Uncommon forms of
immune-mediated
Insulin autoimmune Characterized by the production of autoantibodies against endogenous
syndrome (IAS) insulin in people who haven’t been exposed previously to exogenous
insulin. The pathophysiology is unclear but drugs (such as captopril,
carbimazole, diltiazem, D-penicillamine, glutathione, gold
thioglucose, hydralazine, imipenem, interferon-α, isoniazid,
methimazole, penicillin G, procainamide, tiopronin, tolbutamide, α-
lipoic acid (ALA), α-mercaptopropionyl glycine) containing


sulfhydryl group contribute to autoantibodies against endogenous
insulin.
Anti-insulin receptor Autoantibodies compete with insulin against the insulin receptors by
antibodies desensitizing them causing severe insulin resistance and behaving
similarly to insulin in the activation of lipogenesis within the
adipose tissues.
Stiff-man syndrome Rarely occurring autoimmune or neurological disorder characterized
by a slow progression of muscle rigidity and spasms due to decreased
GABA levels as a result of GAD blockade.
4. Gestational Diabetes Increased insulin resistance and altered insulin sensitivity in
mellitus pregnancy
EIF2AK3, Eukaryotic translation initiation factor 2α kinase 3; GABA, Gamma-aminobutyric acid; GAD,
Glutamic acid decarboxylase; GH, Growth hormone; HFE, Human hemochromatosis protein; HNF,
Hepatic nuclear factor; IAS, Insulin autoimmune syndrome; IPF, Insulin promoting factor; Leu UUR
gene, Leucine UUR gene; MEN1, Multiple endocrine neoplasia type 1; MODY, Maturity-onset
diabetes of the young; PERK, PKR-like endoplasmic reticulum kinase; SUR, Sulfonylurea receptors;
T3Cdm, Type 3 diabetes mellitus.
Figure 1. Various factors affecting diabetes mellitus (Adapted from Kahn, 2007; Singh et al., 2009).
GAD, Glutamic acid decarboxylase.
2.1. Type 1 Diabetes Mellitus
Type 1 diabetes is a result of pancreatic beta cell destruction which can occur in the
presence or absence of autoimmune responses. In such a condition, the individual has normal
metabolism until the clinical manifestation of the disease and is dependant on insulin for
survival. Type 1 diabetes can further be classified into Type 1A and Type 1B diabetes (Gavin
et al., 1997). Type 1A diabetes also known as autoimmune diabetes is characterized by the
occurrence of autoimmune responses directing towards the destruction of the pancreatic beta

cell due to the presence of autoantibodies such as anti-insulin antibodies, anti-islet cell, and
anti-GAD. Studies reveal the strong association of human leukocyte antigen (HLA) mainly the
DQ- A and DQ-B loci with specific alleles in case of type 1A diabetes and the rate of destruction
of the pancreatic beta cell varies in individuals.
A rapid rate of destruction is observed in infants and children whereas slower in case of
adults (Greenbaurn et al., 2000; Kahn, 2007; Zimmet et al., 1994). In adults, the pancreatic beta
cell functioning remains preserved for a longer period and autoimmune diabetes mellitus
progresses slowly characterized by the presence of pancreatic autoantibodies such as glutamic
acid decarboxylase (GAD-65) and ICA. Such a condition is called as the latent autoimmune
diabetes in adults (LADA), also coined as latent autoimmune diabetes in young (LADY- like)
by Lohmann et al., (Unger, 2010, Lohmann et al., 2000).
It is differentiated from type 1 DM and it is insulin independent and develops dependency
at a later period of time on autoantibody development (Unger, 2010). Concomitant autoimmune
disorders are more likely to occur in type 1A DM patients (Kahn, 2007). Type 1B diabetes also
referred to as idiopathic diabetes is characterized by the absence of autoimmune antibodies and
development of ketoacidosis; C-peptide and insulin deficiency. Studies reveal this type of
diabetes has a strong hereditary component and has been reported in the non-whites, African,
Asian and Caucasian populations (Imagawa et al., 2000; Tiberti et al., 2000; Tanaka, 2000;
Kahn, 2007).
2.2. Type 2 Diabetes Mellitus
Type 2 is a result of pancreatic beta cell dysfunction or a gradual pancreatic beta cell failure
which can occur due to insulin resistance or decreased insulin secretion (Turner et al., 1999).
In such condition, insulin develops an increased uptake of glucose and glycogen synthesis
within the skeletal muscles due to decreased P13- kinase and tyrosine phosphorylation activity
associated with insulin receptor substrate-1 (IRS-1) and an elevation of free fatty acids (FFA)
level in the circulation by suppression of lipolysis in adipose tissue (Bjornholm et al., 1997;
Zierath et al., 1998; Groop et al., 1991). No reports of autoimmune responses are observed but
certain variants like Gly972Arg (Stumvoll et al., 2001; Withers et al., 1998), Arg409Gln
(Baynes et al., 2000) appears to take part in β cell dysfunction and resistance to insulin.
Insulin sensitivity is impacted by the polymorphism of genes such as glycogen synthase
(Thorburn et al., 1991). According to Hotamisligil et al., 1993, TNF-α genes may have a role
in the causation of Type 2 DM as well as insulin resistance (Hotamisligil et al., 1993). Many
studies suggest the linkage of type 2 DM to the chromosomal regions of Hepatic nuclear factor
1α (HNF1α) and hepatic nuclear factor 4α (HNF4α) [Mahtani et al., 1996; Ji et al., 1997;
Malecki et al., 1998]. According to Hansen et al., IPF1 genetic variability causes impairment
of insulin gene activation and an increased risk of type 2 DM development (Hansen et al.,
2000). Investigation in populations such as that of the UK showed a strong association of
uncoupling proteins (UCPs) to obesity and obesity-induced type 2 DM (Halsall et al., 2001;
Meirhaeghe et al., 2000). Genome screening has revealed the involvement of calpain 10 gene
and chromosome 2q37 in type 2 DM development (Hanis et al., 1996; Horikawa et al., 2000).

2.3. Other Specific Types of Diabetes Mellitus
These are categorized based on genetically defective functioning of the β-cells or activity
of insulin, exocrine pancreas related diseases, endocrinopathies, infections associated with the
use of certain drugs and rarely occurring diabetes as a result of compromising immune system
(Kahn, 2007; World Health Organization, 1999). Table 1 describes other specific types of DM
and the factors associated with it. Other specific types of DM are genetic defects of β-cell
function, genetic defects in insulin action, diseases of the exocrine pancreas, endocrinopathies,
drug or chemical-induced infections, uncommon forms of immune-mediated diabetes, other
genetic syndromes sometimes associated with diabetes (Ferner, 1992; Jaeckel et al., 2002;
Kahn, 2007; Stoss et al., 1982; World Health Organization, 1999).
2.4. Gestational Diabetes
This type of diabetes occurs in pregnant women due to an abnormality in glucose

homeostasis a result of inadequate insulin supply probably caused by increased insulin
resistance and altered insulin sensitivity which improves post-delivery (Gavin JR et al., 1997;
Kahn, 2007; World Health Organization, 1999). Progression in insulin resistance can be due to
a reduction in IRS-1 concentration which prevents activation of PT 3-kinase leading to PIP3
stimulation, hence preventing GLUT-4 transporter in aiding the entry of glucose within the cell.
According to Catalano et al., (2002) TNF- α is linked with increased insulin resistance which
might be probably due to its correlation with hyperinsulinemia and increased maternal
adiposity. Other factors attributing to insulin resistance particularly in obese pregnant woman
includes IL-6, circulating FFA and CRP (Catalano et al., 2002; Kahn, 2007; World Health
Organization, 1999).
3. DIABETIC COMPLICATIONS
Improper glucose maintenance over a long period of time can affect different body organs
severely and can be life-threatening if unchecked. These are referred to as diabetic
complications (Kahn, 2007; World Health Organization, 1999). Table 2 describes the various
types of complications associated with diabetes mellitus and their etiology.
3.1. Acute Complications
The major acute complications of diabetes are hypoglycemia, diabetes ketoacidosis, and
hyperosmolar state. Diabetes ketoacidosis occurs in type 1 diabetics and can be seen in
superimposed acute infectious conditions; serious medical stress might cause diabetes
ketoacidosis (DKA) in type 2 diabetics. This state occurs when the body is incapable of
producing insulin to aid glucose transfer intracellularly, a hyperglycemic condition leading to
dehydration and ketones builds up. (Kahn, 2007; Kitabchi et al., 1995; Hirsch et al., 2017).

Hyperglycemic hyperosmolar syndrome (HHS) is observed in type 2 diabetes mellitus

mainly in elderly patients. It is a state where insulin requirement when compared to insulin
deficiency, causes a hyperglycemic condition leading to dehydration (Kahn, 2007). This results
in a hyperosmolar state. The plasma sugar level will be in the range of 600 mg/dl or greater and
plasma osmolarity in the range of 320 mOsm/L and above with no state of ketosis (Pasquel et
al., 2014).
Table 2. Complications related to diabetes mellitus with reference to its etiological

factors and characteristics
Types of diabetic Characteristics/Etiological factors

complications
1. Acute
complication
I. Hypoglycemia Occurs as a result of oral hypoglycemic drugs or insulin administration
II. Diabetes Insulin deficiency occurs in IDDM and rarely occurs in NIDDM due to the
ketoacidosis contribution of stress induced by acute illness.
III. Hyperosmolar It is rarely occurring and is associated with NIDDM; Presence of insulin
state deficiency, hyperglycemia accompanied by dehydration, stupor in absence of
ketosis.
2. Chronic
complication
I. Microvascular
complications
Neuropathy Nerve damage due to a prolonged period of improper blood glucose and
triglyceride levels. Chronic hyperglycemia accelerates ROS production in
mitochondria followed by breakage of nuclear DNA strand thereby activating
poly ADP-ribose polymerase. This is associated with the activation of
pathways such as increased activity of aldose reductase and polyol pathway,
activation of DAG- PKC pathway, increased nonenzymatic glycosylation and
hexosamine pathway activation causing AgE generation and NF-κB
activation.
Retinopathy Retinal blood vessels damage due to a prolonged period of improper blood
glucose. Hyperglycemia leads to oxidative stress due to an alteration in a
number of pathways such as the DAG-PKC pathway (specifically by PKC β1
activation), glucose auto-oxidation, increased activity of aldose reductase and
advanced glycation end products (AGEs), giving rise to ROS production.
Nephropathy Occurs in both type 1 and 2 diabetics which can be a result of alteration in
many biochemical or physiological factors including nonenzymatic
glycosylation, polyol pathway activation, overexpression of GFAT in
glomerular cells via HBSP, metalloproteases activity reduction causing an
increased deposition in the matrix of kidney cells, hypertension, mononuclear
cells infiltration, fibrotic and proinflammatory cytokines overexpression due
to proteinuria
Diabetic foot Peripheral sensory neuropathy results in ischemia and trauma finally
resulting in diabetic foot ulceration. Neuropathy via motor, sensory and
autonomic pathway causes limited joint and motor mobility, callus, dry skin
and fissures contributing to high planar pressure resulting in foot ulceration.
III. Macrovascular
complications
Hypertension Blood pressure increases due to vasoconstriction caused by increased
endothelial and angiotensin II. In the case of type 1 DM, diabetic
nephropathy is usually expressed as hypertension whereas, in type 2 DM,

Types of diabetic Characteristics/Etiological factors

complications
clustering of hypertension with that of cardiometabolic syndrome
components is observed.
Cardiovascular Decreased nitric oxide along with release of inflammatory mediators lead to
disease atherosclerosis development. Cardiovascular diseases are interrelated to
diabetes and develop as a result of other disease conditions such as
hypertension, abnormality in cholesterol and triglyceride level, obesity, lack
of exercise and uncontrolled sugar level.
Cerebrovascular Diabetes affects the cerebrovascular system due to a number of mechanisms
disease such as endothelial dysfunction, insulin resistance, impairment of fibrinolysis
and increased oxidation and glycation, platelet aggregation
Peripheral vascular Plaque formation causes blockage of blood vessels in diabetics due to a
disease number of mechanisms such as dysfunction of endothelial, smooth muscles
of the blood vessels, production of inflammatory factors which can
contribute to ischemia
Erectile dysfunction In association with diabetes, such conditions are rarely reversible and can be
due to a number of mechanisms such as PKC activation, activation of
hexosamine pathway, increased AGEs accumulation and arginase
upregulation leading to limited acetylcholine synthesis; decreased Na+-K+-
ATPase activity; increased prosclerotic cytokine, TGF-β1 resulting in
decreased penile elasticity
ADP, Adenosine diphosphate; AGEs, Advanced glycation end products; DAG, Diacyl Glycerol; DM,
Diabetes mellitus; DNA, Deoxyribonucleic acid; GFAT, Glutamine fructose-6-phosphate
aminotransferase; HBSP, Hexosamine biosynthetic pathway; IDDM, Insulin-dependent diabetes
mellitus; Na+-K+-ATPase, Sodium-potassium adenosine triphosphatase; NF-κB, Nuclear factor
kappa-light-chain-enhancer of activated B cells; NIDDM, Non-insulin dependent diabetes meliitus;
PKC, Protein kinase C; ROS, Reactive oxygen species; TGF-β1, Transforming growth factor β
3.2. Chronic Complications
3.2.1. Microvascular Complications
3.2.1.1. Neuropathy
Diabetic neuropathy is a microvascular complication of diabetes that causes damage of
nerves due to a longer period of uncontrolled blood glucose as well as triglyceride levels. It can
be categorized as peripheral, autonomic, focal and proximal neuropathies. Peripheral
neuropathy occurs in about 50% of diabetic patients (Kahn, 2007; Pop-Busui et al., 2017). It is
involved in damage of autonomic as well as somatic nerves and can be observed in regions of
legs and feet and rarely in the area of arms and hands. If not taken proper care it can lead to
diabetic foot conditions. Autonomic neuropathy (in about 30% of diabetics) affects the nerves
regulating vital organs causing dysfunction of the digestive system, blood vessels, sex organs,
sweat glands, urinary system, and eyes (Pop-Busui et al., 2017).
Focal neuropathies are less common and damage single nerves located in the hand, leg,
torso or head. Most common of this type is the carpal tunnel syndrome (compression of a nerve
in the wrist) observed in about 25% diabetics. Proximal neuropathy is rare and affects a part of
the body disabling the regions of thigh, buttock or hip. This type causes weakness or severe
pain (Pop-Busui et al., 2017). Figure 2 illustrates the pathophysiology and signaling pathway
involved in diabetic neuropathy. Chronic hyperglycemia accelerates production of ROS in

mitochondria followed by breakage of nuclear DNA strand thereby activating poly ADP-ribose
polymerase. This is associated with the activation of different pathway such as increased
activity of aldose reductase and polyol pathway, activation of DAG- PKC pathway increased
nonenzymatic glycosylation and hexosamine pathway activation. Accumulation of glucose in
Schwann cells of the neurons initiates aldose reductase activity and polyol pathway activation
producing an increase in sorbitol accumulation accompanied by effects such as edema of the
nerve cells, nerve conduction impairment and apoptosis.
Figure 2. Pathophysiology and the signaling pathway involved in diabetic neuropathy (Adapted from Pop-
Busui et al., 2017). DNA, Deoxyribonucleic acid; PARP, Poly (ADP-ribose) polymerase; ADP, Adenosine
diphosphate; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; G-6-P, Glucose-6-phosphate; F-6-P,
Fructose-6-phosphate; G-3-P, Glyceraldehyde 3-phosphate; DHAP, Dihydroxyacetone phosphate; NAD+,
Nicotinamide adenine dinucleotide (oxidized form); NADH, Nicotinamide adenine dinucleotide (reduced
form); NADPH, Nicotinamide adenine dinucleotide phosphate (reduced form); NADP+: Nicotinamide
adenine dinucleotide phosphate; MAPK, Mitogen-activated protein kinases; GFAT, Glutamine fructose-6-
phosphate aminotransferase; UDP, Uridine diphosphate; ROS, Reactive oxygen species; NF-κB, Nuclear
factor kappa-light-chain-enhancer of activated B cells; AGE, Advanced glycation end products.
Sorbitol accumulation along with a reduction in the uptake of myo-inositol and sodium-
potassium adenosine triphosphatase (Na+K+ATPase) gives rise to retention of sodium ions,
edema, a disjunction of axoglial, swelling of myelin and degeneration of myelin. This causes
an increase in ROS (O2-, OH-, H2O2), RNS (NO, peroxynitrite, ONOO-), nicotinamide adenine
dinucleotide phosphate reduced form (NADPH) oxidase and xanthine reductase creating an
oxidative or nitrative stress condition which further activates mitogen-activated protein kinases
(MAPK) giving rise to nerve dysfunction which ultimately causes structural or functional
damage to the nerve cells such as axonopathy and demyelination.
Activation of diacylglycerol (DAG) causes Protein kinase-β2 (PKC-β2) activation which in
turn activates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) leading
to the dysfunction of endothelial cells (due to a reduction in PGI2, NO) resulting in nerve
dysfunction contributing to structural or functional damage to the nerve cells. Increased non-
enzymatic glycosylation produce AGE generation and NF-κB activation resulting in

dysfunction of neurons which ultimately causes structural or physiological nerve damage

(Vinik et al., 2000; Boulton, 2007; Tesfaye et al., 2005; Brownlee, 2005; Nishikawa et al.,
2000).
3.2.1.2. Retinopathy
Diabetic retinopathy includes in the category of microvascular complication of diabetes
where there is damage occurring in the retinal blood vessels over a period of time due to
improper blood glucose or blood pressure maintenance (Farlex 2012). This can be of non-
proliferative as well as proliferative type. Non-proliferative type of diabetic retinopathy occurs
in the early stages of disease where there is no angiogenesis in the eye leading to fluid and
blood leakage into the eye. Proliferative type of diabetic retinopathy is the advanced stage of
retinopathy initiating angiogenesis within the retina (Mayoclinic, 2018). In proliferative
retinopathy, both the pericytes as well as the capillary endothelium are subjected to mechanisms
such as PKC activation and oxidative stress. But a tremendous loss of pericytic cells is observed
compared to that of the endothelial (Chou et al., 2002). In this condition, modification of the
vascular morphology takes place causing impairment or loss of vision and these include
increased expression in VEGF and apoptosis in pericytes of the retinal areas.
The change in vascular morphology includes an accelerated thickening of inner capillary
basement membrane of the retina which consists of ganglionic and nerve layer, pericytic cells
reduction, and associated capillary microaneurysms, increase in the permeability of blood
vessels, along with decreased NO bioavailability causing a decrease in vasodilation which is
endothelium-dependent, new blood vessel formation, hemorrhage, occlusion of capillary,
detachment of retinal traction and formation of fibrotic tissue (Murata et al., 2002; Romeo et
al., 2002; Pomero et al., 2003; Yamagishi et al., 2002; Ho et al., 2000).
Hyperglycemia leads to oxidative stress due to an alteration in a number of pathways such
as the DAG-PKC pathway (specifically by PKC β1 activation), glucose auto-oxidation,
increased activity of aldose reductase and AGEs, giving rise to ROS production thereby
inflicting oxidative stress (Kahn, 2007). Figure 3 illustrates the pathophysiology and signaling
pathway involved in diabetic retinopathy. Nuclear factor erythroid-2-related factor 2 (Nrf2), a
neuroprotective transcription factor when sequestered within the cytoplasmic matrix in
association with Kelch-like ECH-associated protein 1 (Keap1), on ROS exposure causes
oxidation of Keap1 and further dissociation from Nrf2. This permits the migration of Nrf2 to
the nucleus and its binding to the antioxidant response element (ARE) region initiating target
gene transcription (Batliwala et al., 2017).
3.2.1.3. Nephropathy
Diabetic nephropathy is a category of microvascular complication whereby the kidney is
affected within 20 years after diabetes diagnosis and observed in both type 1 as well as type 2
diabetic patients (Cooper, 1998). This can be a result of alteration in number of biochemical or
physiological factors including nonenzymatic glycosylation, polyol pathway activation,
overexpression of glutamine fructose-6-phosphate aminotransferase (GFAT) in glomerular
cells via hexosamine biosynthetic pathway (HBSP), metalloproteases activity reduction
causing an increased deposition in the matrix of kidney cells, hypertension, mononuclear cells
infiltration, fibrotic and proinflammatory cytokines overexpression due to proteinuria
(Aghadavod et al., 2016). The schematic representation containing the signaling pathway
involved in diabetic nephropathy is given in Figure 4.

Figure 3. Signaling pathway involved in diabetic retinopathy (Adapted from Batliwala et al., 2017).
MAPK, Mitogen-activated protein kinases; ROS, Reactive oxygen species; AGE, Advanced glycation
end products; NF-κB, Nuclear factor kappa-light-chain-enhancer of activated B cells; DAG, Diacyl
Glycerol; PKC, Protein kinase C; TGF-β1, Transforming growth factor β.
Figure 4. Signaling pathway involved in diabetic nephropathy (Adapted from Aghadavod et al., 2016). G-6-
P, Glucose-6-phosphate; F-6-P, Fructose-6-phosphate; G-3-P, Glyceraldehyde 3-phosphate; DHAP,
Dihydroxyacetone phosphate; NAD+, Nicotinamide adenine dinucleotide (oxidized form); NADH,
Nicotinamide adenine dinucleotide (reduced form); NADPH, Nicotinamide adenine dinucleotide phosphate
(reduced form); NADP+, Nicotinamide adenine dinucleotide phosphate; MAPK, Mitogen-activated protein
kinases; GFAT, Glutamine fructose-6-phosphate aminotransferase; ROS, Reactive oxygen species; NF-κB,
Nuclear factor kappa-light-chain-enhancer of activated B cells; AGE, Advanced glycation end products;
TGF-β1, Transforming growth factor β1; TNF, Tumor necrosis factor; IL, Interleukin.

3.2.2. Macrovascular Complications

Macrovascular complications occur as a result of large blood vessel damage and are
associated with MDA and lipid hydroperoxides. The various complications include
hypertension, cardiovascular disease, cerebrovascular disease, peripheral vascular disease, and
erectile dysfunction (Huang et al., 2017).
3.2.2.1. Hypertension
According to Ferrannini et al., (1987) essential hypertension can be considered as a state
of insulin resistance (Ferrannini et al., 1987; Kahn, 2007). Diabetic nephropathy is usually
expressed as hypertension in case of type 1 DM and results in worsening of each other.
Clustering of hypertension with that of cardiometabolic syndrome components is observed in
type 2 DM. A decrease in nitric oxide accompanied by increased endothelial and angiotensin
II causes constriction of blood vessels which bring forth an increase in blood pressure (Kahn,
2007).
3.2.2.2. Cardiovascular Disease

Diabetes and cardiovascular diseases are inter-related and develop as a result of other
disease conditions such as hypertension, abnormality in cholesterol and triglyceride level,
obesity, lack of exercise and uncontrolled sugar level. A decrease in nitric oxide accompanied
by increased release of inflammatory mediators such as cytokines, chemokines and other
factors such as platelet activation, thrombosis, decreased fibrinolysis, hypercoagulation
altogether contributes to the development of atherosclerosis (Kahn, 2007; Rambhade et al.,
2011).
3.2.2.3. Cerebrovascular Disease

Diabetes is one of the risk factors causing ischemic stroke. The probable mechanisms are
endothelial dysfunction, insulin resistance, impairment of fibrinolysis, increased oxidation and
glycation, as well as platelet aggregation (Lukovits et al., 1999).
3.2.2.4. Peripheral Vascular Disease

Peripheral vascular disease (PVD) is a medical condition occurring in diabetic patients
where the blood vessels are blocked due to plaque formation. This can occur as a result of
dysfunction of endothelium, smooth muscle of the blood vessels, and production of
inflammatory factors which can contribute to ischemia (Huysman. et al., 2009).
3.2.2.5. Erectile Dysfunction

Erectile dysfunction (ED) in association with diabetes is reversible rarely and can be of
neurogenic, vasculogenic and endocrinologic types. Neurogenic ED occurs as a result of the
impairment of cholinergic neurons mediated nitric oxide (NO) synthesis which aids in relaxing
smooth muscles of the penis. In diabetics, there is a limited synthesis of acetylcholine and hence
the activation of endothelium-mediated smooth muscle relaxation is hindered probably due to
PKC activation, activation of hexosamine pathway, increased AGEs accumulation and arginase
upregulation (Blanco et al., 1990; Lue, 2000; Penson et al., 2004).
In vasculogenic ED, activation of DAG causes activation of PKC which decreases the
activity of Na+-K+-ATPase which increases the prosclerotic cytokine, transforming growth

factor β1 (TGF-β1) which in turn causes a change in collagen types and synthesis of collagen
increases resulting in decreased penile elasticity, compliance, and impairment in vein
occlusion. This prevents the penis from attaining required erection period and rigidity (Lue,
2000; Zhang et al., 2008). In endocrinologic ED, deficiency of androgen causes a decrease in
libido and nocturnal erections. Adding to hypogonadism other diseases such as
hyperprolactinemia, pituitary disorders, thyroid, and adrenal diseases may contribute to ED
associated diabetes (Lue, 2000).
4. OXIDATIVE STRESS AND DIABETES

4.1. Oxidative Stress
Oxidative stress is an imbalance in the production of reactive species and antioxidants to

defend the body against these reactive molecular species such as reactive nitrogen species
(RNS) or reactive oxygen species (ROS) (Rosen et al., 2001). In small quantity, RNS and ROS
are beneficial to the body but increased production causes nitrosative and oxidative stress
respectively thereby causing damage to cellular structures like protein, lipids, carbohydrates,
nucleic acids and alter their functions (Mukherji et al., 1986; Pham-Huy et al., 2008; Yla-
Herttuala, 1999; Stadtman et al., 2000; Marnett, 2000). This imbalance occurs when the body
fails to meet the antioxidant requirement to scavenge the free radicals formed due to a
deficiency in the production of enzymatic such as catalase (CAT), superoxide dismutase
(SOD), glutathione peroxidase (GPx) as well as non-enzymatic antioxidants such as
glutathione, and vitamins (A, C, E) (Finkel et al., 2000; Yla-Herttuala, 1999).
4.2. Reactive Oxygen Species and Reactive Nitrogen Species
Generally, oxidants or prooxidants are termed RNS or ROS among which ROS is of more
importance. The oxidants from exogenous sources include cigarette smoke air pollutants etc
which have many oxidants and free radicals such as nitric oxide, superoxide; hyperoxia, which
leads to greater level of reactive nitrogen and oxygen species; ionizing radiations which convert
superoxide, hydroxyl, and organic radicals to hydroperoxides and hydrogen peroxide in the
presence of oxygen and lead to oxidative stress; ozone exposure, which may produce lipid
peroxidation, release inflammatory mediators and lowers pulmonary function even in healthy
people; heavy metal ions such as copper, cadmium, nickel, mercury, iron, arsenic, and lead
which can damage cellular functions. ROS from endogenous sources includes free radicals
which have one or more electrons unpaired and non-radicals, in which two free radicals share
their unpaired electrons (Mukherji et al., 1986; Phaniendra et al., 2015).
The major endogenous oxidants are hydrogen peroxide, superoxide anion, hypochlorous
acid, hydroxyl radical, hydroperoxyl radical and peroxyl radicals. Superoxide anion is mainly
formed in the mitochondria (Kohen et al., 2002; Halliwell, 2001; Genestra, 2007). Nitric oxide
radical is a reactive radical that play an important role in signaling a wide variety of
physiological processes such as blood pressure regulation, neurotransmission etc. It has one
unpaired electron and is produced by specific nitric oxide synthases. Immune cells produce

nitric oxide and superoxide anion during inflammation, which further reacts to produce
peroxynitrite, a strong oxidizing agent which can cause lipid oxidation and DNA fragmentation
(Beckman et al., 1996; Douki et al., 1996; Ischiropoulos et al., 1995; Czapski et al., 1995). The
hydroxyl radical can damage the pyrimidine and purine bases, mainly guanine and the
deoxyribose backbone of the DNA. This alteration of genetic material is the first step towards
carcinogenesis and mutagenesis.
The metal generated ROS can also attack cell components having polyunsaturated fatty
acid residues (PUFA). The lipid peroxidation produces malondialdehyde (MDA) and 4-
hydroxy-2-nonenal (HNE) which are mutagenic and toxic products. ROS oxidizes proteins and
hydroxyl radicals or a mixture with superoxide radicals are formed. The methionine and
cysteine residues of proteins can undergo oxidation by reactive nitrogen species or reactive
oxygen species. Oxidation of cysteine forms mixed disulfides between protein thiol mainly
reduced glutathione (GSH). The carbonyl group concentration measure is a good indicator of
reactive oxygen species induced protein oxidation. Glycation (non-enzymatic glycosylation),
in which biomolecular amino groups react with free carbonyl group of carbohydrates then
produces advanced glycation end products (AGEs) which are very unstable (e.g., pentosidine,
carboxymethyl lysine) reducing carbohydrates generate reactive oxygen species via glycation
and both play a role in aging and related disorders (Phaniendra et al., 2015).
4.3. Oxidative Stress and Diabetes
Chronic hyperglycemia accelerates production of ROS in mitochondria followed by

breakage of nuclear DNA strand thereby activating poly ADP-ribose polymerase. This is
associated with the activation of different pathway such as increased activity of aldose
reductase and polyol pathway, activation of DAG- PKC pathway increased nonenzymatic
glycosylation and hexosamine pathway activation. In neuropathy, glucose accumulation within
the Schwann cells present in neurons initiates aldose reductase activity and polyol pathway
activation. This causes an increase in accumulation of sorbitol accompanied by neuronal
edema, impairment of nerve conduction and apoptosis. Sorbitol accumulation along with
reduced uptake of myo-inositol and Na+K+ATPase action give rise to retention of sodium ions,
edema, disjunction of axoglia, swelling of myelin and degeneration of myelin. This leads to an
increased level of NADPH oxidase, RNS, ROS and xanthine reductase creating a nitrative or
oxidative stress condition. This leads to activation of MAPK bringing about nerve dysfunction
which results in structural or functional neuronal damage such as axonopathy and
demyelination.
DAG activation causes activation of PKC-β2. This causes activation of NF-κB leading to
the dysfunction of endothelial cells (due to a reduction in PGI2, NO) resulting in nerve
dysfunction contributing to structural or functional damage to the nerve cells. Increased non-
enzymatic glycosylation bring forth ACE generation and NF-κB activation resulting in
neuronal dysfunction which ultimately causes structural or physiological damage to the nerves
(Vinik et al., 2000; Boulton, 2007; Tesfaye et al., 2005; Brownlee, 2005; Nishikawa et al.,
2000). In retinopathy and nephropathy, alteration in pathways such as DAG-PKC pathway (by
activating PKC β1), glucose auto-oxidation, increased aldose reductase, and AGEs activity,
lead to increased ROS production and oxidative stress (Kahn, 2007). Similar to microvascular
complications, macrovascular complications occur due to PKC activation, activation of

hexosamine pathway, increased AGEs accumulation and arginase upregulation causing a

hindrance to smooth muscle relaxation (Blanco et al., 1990; Lue, 2000; Penson et al., 2004).
5. ANTIOXIDANTS USEFUL IN DIFFERENT TYPES

OF DIABETES AND RELATED COMPLICATIONS
Oxidative stress plays a key role in diabetes mellitus and associated complications by
disrupting the insulin signaling and increasing insulin resistance thereby causing an increase in
glucose level in the serum. This damage can be minimized by antioxidants (Figure 5) which
can be of natural or synthetic origin. Natural antioxidants can be obtained from various dietary
sources to improve the body's defense mechanism and these include ascorbic acid, carotenoids,
flavonoids, tocopherols, vitamin E, and from few culinary herbs and spices.
Phenolic compounds such as butylated hydroxyl anisole (BHA), butylated hydroxyl
toluene (BHT), esters of gallic acid (propyl gallate), nordihydroguaiaretic acid (NDGA),
tertiary butyl hydroquinone (TBHQ) are synthetic antioxidants which act by retarding lipid
oxidation (Madsen et al., 1995). Several antioxidants such as ascorbic acid, carotenoids,
flavonoids, tocopherols, and vitamin E are known to protect the body against damage caused
by oxidative stress and complications associated with it as in diabetes mellitus. The most
commonly studied of all antioxidants is polyphenols.
Figure 5. Mechanism of antioxidants in diabetes (Adapted from Preedy, 2014). MAPK, Mitogen-
activated protein kinases; ROS, Reactive oxygen species; AGE, Advanced glycation end products; NF-
κB, Nuclear factor kappa-light-chain-enhancer of activated B cells; DAG, Diacylglycerol; PKC, Protein
kinase C; TGF-β1, Transforming growth factor β.

5.1. Antioxidants Effective Against Diabetes
5.1.1. Flavonoids
Flavonoids have been known to improve diabetes through various pathways. Quercetin
acts on human adipocytes by attenuation of inflammation mediated by TNFα and improve
insulin resistance (Chuang et al., 2010). Cyanidin 3-O-β-glucoside shows insulin mimetic
activity within the human adipocytes by activation of PPARγ (Scazzocchio et al., 2011).
5.1.2. Citrus Flavonoids

The citrus flavonoids such as hesperetin and naringenin block the FFA secretion in mouse
adipocytes stimulated by TNFα (Yoshida et al., 2010); as well as increased glucose uptake by
increased adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle cells
thereby causing an increase in glucose uptake (Zygmunt et al., 2010). Activation of PPARγ in
3T3-L1 adipocytes by luteolin increased insulin sensitivity (Ding et al., 2010). Caffeic acid
phenethyl ester and cinnamic acid via AMPK pathway stimulated glucose uptake into skeletal
muscle cells and insulin-resistant hepatocytes of mouse respectively (Eid et al., 2010; Huang
et al., 2009).
5.1.3. Culinary Herbs

Studies reveal that few culinary herbs belonging to the family Lamiaceae such as Salvia
officinalis (sage), oregano, peppermint, thyme (Thymus vulgaris), basil show antidiabetic
activity via α-amylase and α-glucosidase inhibition. Among these, sage was found to be less
effective (Cazzola et al., 2011; Kwon et al., 2006; Buyukbalci, 2008; Koga, 2006).
5.1.3.1. Salvia officinalis

Salvia officinalis (Sage) consists of antioxidants and other chemical constituents such as
β-carotene, β-sitosterol, γ-terpinene, ascorbic acid, apigenin, camphene, carnosol, carnosic
acid, cirsileol, cirsimaritin hispidulin, labiatic acid, nevadensin, oleanolic acid, salvigenin,
selenium, terpinen-4-ol, ursolic acid, and several studies demonstrates that inhibition of
glucagon-stimulated production of hepatic glucose, fasting gluconeogenesis reduction and an
increase in glucose consumption in hepatocytes cause a significant reduction in the fasting
plasma glucose level. The presence of rosmarinic acid in greek sage produce modulation of
SGLT-1 trafficking thereby contributes to its antidiabetic property (Azevedo et al., 2011; Suhaj,
2006).
5.1.3.2. Oregano
Two species oregano (Origanum vulgaris) consisting of camphene, carvacrol, gamma-
terpinene, thymol, terpinen-4-ol, myricene, linalyl-acetate and marjoram and Origanum
majorana consisting of ascorbic acid, beta-carotene, caffeic acid, rosmarinic acid, tannin,
eugenol, hydroquinone, myrcene, phenol, terpinen-4-ol, trans-anethole, ursolic acid, beta-
sitosterol, oleanolic acid were traditionally used in controlling diabetes (Suhaj, 2006).
5.1.3.3. Peppermint
Peppermint (Mentha x piperita) consists of chemical constituents such as ascorbic acid, β-
carotene, caffeic acid, diosmin, eriocitrin, eriodictyol, eriodictyol 7-O-β-glucoside, hesperidin,

isorhoifolin, lithospermic acid, luteolin 7-O-β-glucoside, luteolin 7-O-rutinoside, menthoside,

narirutin, piperitoside and rosmarinic acid (Fecka et al., 2007). Despite the presence of
appreciable phenolic content antidiabetic studies are scarce on this plant. According to
Barbalho et al., (2011) aqueous extracts of peppermint improved lipidemic and blood glucose
level in animal models.
5.1.3.4. Onion and Garlic

Onion (Allium cepa) consisting of allicin, cyanidin glucosides, kaempferol, peonidin
glucosides, quercetin, taxifolin and garlic (Allium sativum) consisting of allicin, caffeic, p-
coumaric acids, p-hydroxybenzoic and vanillic belonging to the family Liliaceae are widely
used in flavouring and are rich sources of sulfur-containing compounds such as S-alk(en)yl-l-
cysteine sulfoxides (ACSOs) converted to thiosulfates reported to have anti-inflammatory and
antioxidant property (Chan et al., 2013; Briggs et al., 2000; Butt et al., 2009; Ali et al., 2000;
Slimestad et al., 2007; Beato et al., 2011; Vagen, 2011). Both onion and garlic have been
reported of for its hypoglycemic property in various animal models (Srinivasan, 2005; Liu et
al., 2007; Kumar et al., 2013; Taj et al., 2010).
5.1.3.5. Bay Leaf

Bay leaf/ Laurel (Laurus nobilis) belonging to the family Lauraceae consisting of chemical
constituents 8-cineole, α-terpinyl acetate, ascorbic acid, β-carotene, catechin, cinnamtannin B1,
eugenol, eudesmol, kaempferol, kaempferol-3,7-dirhamnopyranoside, kaempferol-3-
rhamnopyranoside, methyl eugenol, terpinen-4-ol and tocopherols (Dall'Acqua et al., 2009;
Conforti et al., 2006) have been used in digestive ailments since ancient days. Being a rich
source of polyphenol ensures its antioxidant property in various extracts (Conforti et al., 2006).
Clinical studies on type 2 diabetics by Khan et al., (2009) revealed the promising antidiabetic
potential of this plant.
5.1.4. Spices
Apart from the culinary herbs certain spices also contribute to large extent antioxidant,
anti-inflammatory properties that outperform the antioxidants of synthetic origin and various
studies report anti-glycant and blood glucose lowering property proving its antidiabetic
potential. These include cumin, coriander, ginger, mustard and turmeric (Carlsen et al., 2010).
5.1.4.1. Cumin
Cumin (Cuminum cyminum) consisting of apigenin, β-pinene, cuminal, cuminaldehyde,
cumin alcohol, cuminic alcohol, γ-terpinene, luteolin, p-cymene and coriander (Coriandrum
sativum) consisting of beta-carotene, beta-sitosterol, camphene, caffeic acid, gamma-terpinene,
isoquercitrin, myristicin, myrcene, protocatechuic-acid, p-hydroxybenzoic-acid, quercetin,
rutin, rhamnetin, scopoletin, tannin, trans-anethole, terpinen-4-ol, vanillic-acid belonging to the
family Apiaceae are commonly used in oriental region for the purpose of flavouring and are
known to be rich in flavonoids and phenolic acids (El-Ghorab et al., 2010; de et al., 2005; Suhaj,
2006). In vitro and various animal models demonstrate antioxidant and antidiabetic activities
via hypoglycemic and anti-glycant activity (El-Ghorab et al., 2010; Skrovankova et al., 2012;
Srinivasan, 2005; Saraswat et al., 2009; Sowbhagya, 2013; Jagtap et al., 2010; Kumar et al.,
2009).

5.1.4.2. Ginger and Turmeric

Ginger (Zingiber officinalis) consisting of ascorbic acid, β-carotene, camphene, caffeic
acid, γ-terpinene, 6-Gingerol, p-coumaric-acid, 6-shogaol, terpinen-4-ol and turmeric
(Curcuma domestica) consisting of constituents, ascorbic acid, curcumin carotenes, caffeic acid
and p-coumaric-acid belonging to the family Zingiberaceae are rhizomes used widely in
traditional medicine and a good source of antioxidants mainly due to the presence of various
phenolic substances such as gingerols (in ginger) and polyphenolics (called as curcuminoids in
turmeric) (El-Ghorab et al., 2010; Gupta et al., 2013; Skrovankova et al., 2012; Suhaj, 2006).
Apart from the antioxidant property, studies reveal ginger possessing anti-glycant, anti-
hyperglycemic activity and protection against diabetic complications, metabolic and lipid
disorders associated with diabetes and improving insulin signaling (Dearlove et al., 2008; Li et
al., 2012; Saraswat et al., 2009). The anti-inflammatory property of turmeric makes it useful in
treating various diseases related to skin, liver, GIT, and wounds. Studies reveal that the
modulation of certain signaling molecules such as adhesion molecules, apoptotic proteins,
cyclooxygenases, lipoxygenases, transcription factors particularly NF-κB by curcumin proves
itself a potent immunomodulant and antioxidant (Gupta et al., 2013). Furthermore, the anti-
inflammatory property of turmeric in part contributes to its ability in improving insulin
sensitivity, hyperglycemia and various diabetic complications such as diabetic
microangiopathy and nephropathy (Gupta et al., 2013; El-Moselhy et al., 2011).
5.1.4.3. Mustards
Mustards (Brassica nigra, alba, juncea) belonging to the family Brassicaceae consists of
carotenes, and glucosinolates as constituents (Steinkellner et al., 2001). Glucosinolates are the
most investigated and the reason for its flavour. Its hydrolysis gives rise to a pungency due to
the formation of isothiocyanates. Both isothiocyanates and glucosinolates have multiple
properties such as anti-inflammatory, antioxidant and immunomodulatory activities
(Steinkellner et al., 2001). Few in vivo studies have shown antidiabetic effect (Srinivasan, 2005;
Thirumalai et al., 2011).
5.1.4.4. Black Pepper

Black pepper (Piper nigrum) belongs to the family Piperaceae and consists of constituent’s
ascorbic acid, β-carotene, γ-terpinene, camphen, carvacrol, eugenol, methyl eugenol, piperine
and ubiquinone (Suhaj, 2006). It is a good antioxidant due to the presence of an alkaloid,
piperine but no antidiabetic property is reported except for an invitro anti-glycant property
(Saraswat et al., 2009; Srinivasan, 2007). Studies demonstrate piperine has the ability to quench
ROS, lipid peroxidation and influence the cellular thiol condition in a positive manner. The
probable mechanism by which this spice might work is by potentiating the antioxidant and
antidiabetic activities of other spices (Srinivasan, 2007).
5.1.5. Resveratrol
Resveratrol (3,5,4′-trihydroxy-trans-stilbene), a stilbenoid occurs in grapevine and also in
berries, cocoa and peanuts as a result of injury or infection (Fremont, 2000; Takaoka, 1939).
Studies reveal its ability to prevent or treat diabetes mellitus via a number of mechanisms as
mentioned in Figure 5 due to its antioxidant activity (Frombaum et al., 2012). Resveratrol
causes an increase in insulin secretion similar to that of sulfonylureas when bound to

sulfonylurea receptors (SUR) as a result of adenosine triphosphate (ATP) sensitive K+ channel

closure present within the pancreas (Zhang et al., 2004); by uncoupling protein 2 (UCP2)
inhibition (Vetterli et al., 2011) and Forkhead box O (FOXO) deacetylation (Liu et al., 2008;
Schwer et al., 2008). FOXOA3 induction decreases the synthesis of adiponectin which in turn
causes a decrease in the oxidative stress-induced insulin resistance by reducing the
inflammatory cytokines and NADPH oxidase. IRS-1 phosphorylation improves insulin
signaling thereby reducing insulin resistance (Vinayagamoorthi et al., 2008). Insulin resistance
is also improved by peroxisome proliferator-activated receptor-γ coactivator (PGC-1a)
activation leading to the expression of endothelial nitric oxide synthase (Enos) gene in
endothelial cell by AMPK activation (Jonk et al., 2007).
5.1.6. α-Tocopherol
Tocopherols are compounds belonging to the class of fat-soluble vitamin E classified as α-
, β, γ- and δ-tocopherols with varying biological activity. Among these, α-tocopherol is known
to possess the highest biological activity. α-Tocopherol is a known potent antioxidant capable
of scavenging alkyl peroxy radicals, hindering lipid peroxidation, a probable mechanism
involved in protection against oxidative stress induced diabetes (Azzi et al., 2004; Preedy,
2014; Young et al., 2001) Studies suggest that vitamin E is also involved in inhibition of protein
kinase C activity, regulation of PPARγ thereby improving insulin resistance and glucose
tolerance in type 2 DM (Azzi et al., 2004; Preedy, 2014).
5.1.7. Vitamin D
It is a compound synthesized endogenously within the body on exposure to an ultraviolet
component present in sunlight. It is well known for its calcemic functions such as bone mass
maintenance, calcium and phosphate homeostasis (Adams et al., 2012; Adorini et al., 2008). It
also possesses non-calcemic functions such as antimicrobial and antioxidant properties,
cardiovascular health maintenance, immune modulation, muscle health maintenance,
regulation of cell growth and differentiation. According to Mitri et al., (2011) vitamin D has a
beneficial effect on blood glucose but is still under debate (Mitri et al., 2011; Preedy, 2014).
5.1.8. Puerarin
Puerarin, an estrogen analog was known to be isolated from Pueraria lobate. According to
Wang et al., puerarin demonstrated a significant reduction in glucose level in mice and rats with
type 1 DM and promoted the activity of glucose-6-phosphate dehydrogenase (G6PD) which
caused a reduction in oxidative stress and apoptosis in pancreatic β-cells (Wang et al., 2017).
5.2. Antioxidants Effective against Diabetes Retinopathy
5.2.1. Salvia miltiorrhiza

The dried roots of Salvia miltiorrhiza, danshen has been popularly used in cardiovascular
and related diseases for more than a century by the people residing in Korea, Japan, and China.
Danshen is mainly used in combination with different extracts out of which compound danshen
or fufang danshen is the most commonly used. Compound danshen dripping pills (CDDP), a
combination of hydrophilic extract of borneol, radix notoginseng, and S. miltiorrhiza has

demonstrated protection of endothelial cells of the retina in diabetics by increasing the GPx
activity and NO levels and reducing plasminogen activator activities (Zhou et al., 2002).
Consumption of vitamin E as a supplement over a period of 8 months demonstrated
normalization of blood flow in the retina in case of patients with less or no retinopathy (Bursell
et al., 1999).
5.3. Antioxidants Effective against Diabetes Nephropathy
5.3.1. Salvia miltiorrhiza

Studies demonstrated that Salvia miltiorrhiza combination, CDDP ameliorated the
functioning of kidney cells in the diabetic rats by preventing apoptosis and activation of NF-
κB (Chen et al., 2011; Lu et al., 2010).
5.4. Antioxidants Effective against Macrovascular Complications
5.4.1. Cardiovascular Disease
5.4.1.1. Ginkgo biloba

Flavonoid fraction from Ginkgo biloba leaves act by reduction of ROS, enhancement of
antioxidant metabolites level and antioxidant protein expression. Various studies showed that
consumption of extracts of Ginkgo biloba leaves up-regulated the enzymes responsible for
scavenging free radicals and reduced lipoprotein (a) (Lp(a)) and oxidized LDL levels thereby
producing an atherosclerotic effect (Libby et al., 2005; Rodriguez et al., 2007); prophylactic
activity prior to cardiac surgery by prevention of cardiac dysfunction and necrosis; reduced
ROS production post ischemic condition and antiplatelet effect by ginkgolide B;
cardioprotective activity by terpenoid and vasodilatory effect produced by EGb761 releasing
prostaglandins and nitric oxides (Auguet et al., 1982; Mahady, 2002).
5.4.1.2. Salvia miltiorrhiza

Salvia miltiorrhiza is widely used in regions such as Korea, China, and Japan for the
purpose of treating ischemic stroke, cardiovascular diseases and chronic hepatitis B,
demonstrated ability to ameliorate chronic heart disease in the diabetics (Qu et al., 2008; Cheng,
2007). It is mainly used in combination with other herbs and the most commonly used is
compound danshen in the form of tablets, injections, and dripping pills each with a combination
of different herbs/ ingredients (Preedy, 2014). The dripping pill administration demonstrated
cardioprotective activity probably through coronary microcirculation improvement,
antiplatelet, antioxidant and sedative properties (Ng et al., 2012).
5.4.2. Cerebrovascular Disease

A number of studies reveal that Ginkgo biloba extracts have many pharmacological effects
on the cerebrovascular system. Its ability to suppress RNS and ROS production in the

inflammatory cells demonstrate its anti-inflammatory property which can benefit conditions
such as acute ischemic stroke most probably via expression of heme oxygenase-1 mainly due
to the presence of flavonoids (Kressmann et al., 2002; Smith et al., 2003; Gohil et al., 2002;
Oken et al., 1998). Flavonoids such as isorhamnetin, kaempferol, and quercetin improve
contraction of blood vessels in response to catecholamine release and its antiplatelet activity
renders it useful in improving coronary blood flow (Auguet et al., 1982; Mahady, 2002). Apart
from this, Ginkgo biloba extracts have high potential against dementia or aging-associated
cognitive dysfunctions and other disorders such as depression and Alzheimer's as a result of its
antioxidant and neuroprotective properties most probably through the action of terpenoids and
phenolic compounds present in Ginkgo biloba (Kressmann et al., 2002).
5.4.3. Peripheral Vascular Disease

Meta-analysis and various studies reveal that Ginkgo biloba extracts have good therapeutic
potential in treating PVD. It showed potent antiplatelet, anti-thrombotic, anti-atherosclerotic
effects when co-administered with drugs such as cilostazol (Pittler et al., 2000; Ryu et al., 2009;
Ryu et al., 2009; Jung et al., 2012).
5.4.4. Erectile Dysfunction
5.4.4.1. Quercetin
Quercetin is the most commonly found flavonoid which is consumed frequently through
vegetables, nuts, fruits and has been reported to possess a number of health benefits including
anti-allergic, anticancer, anti-inflammatory, antiulcer, antiviral activity, cardiovascular
protection and cataract prevention. Additionally, quercetin is known to be involved against lipid
peroxidation, free radical scavenging and metal ion chelation thereby preventing oxidant injury
and death of cells. According to Zhang et al., 2011, quercetin ameliorated erectile dysfunction
by the inhibition of oxidative stress developed in cavernosum tissues.
5.4.4.2. Vitamin E
A study conducted by De et al. proved the synergistic effect of vitamin E with that of a
phosphodiesterase type 5 (PDE5) inhibitor in improving erectile function and impaired
cavernosal relaxation by improving the circulating NO level. Apart from these Vitamin E
demonstrates its antioxidant activity via a number of mechanisms such as enhancement of
endothelial adhesion, trapping of oxygen free radicals, reduction in glucose-induced vascular
dysfunction occurring in diabetes by reversing protein kinase C mechanism (De et al., 2004).
5.4.4.3. α- Lipoic Acid

α- lipoic acid is a potent antioxidant known to improve sensory and motor conduction
velocity, glutathione level and blood flow to nerves. According to Keegan et al. long-term use
of α- lipoic acid partially corrected corpus cavernosum smooth muscle relaxation mediated by
nitric oxide (Keegan et al., 1999).

5.4.4.4. Pomegranate Extract

Arteriogenic ED is a result of long-term exposure of the erectile tissue to nutrient
deficiency, hypoxic, ischemic conditions, lacunae in clearance of metabolic waste and
impairment of haemodynamic events. Studies showed that pomegranate extract possesses
prophylactic role in improving erectile activity, blood flow in intracavernosal region, relaxing
smooth muscles and protecting the structural integrity of endothelial, caveolae and
mitochondrial region probably by preventing upregulation of AR gene, SOD and reduction in
oxidative products (Zhang et al., 2011).

AND CHALLENGES
In spite of the advancement in science and development of pathway specific inhibitors such
as DAG inhibitors, PKC inhibitors, NADPH-oxidase inhibitors, there is no approach so far
available to avoid development of diabetic complications (Volpe et al., 2018). Targeting Nrf2
pathway can be promising in the treatment of diabetic retinopathy as it can increase the intrinsic
antioxidants and inhibit the inflammatory mediators thereby delaying the degeneration of
retina.
But limited information is available on the therapeutic approaches correcting Nrf2 pathway
during oxidative stress. A recently published study by Nakagami et al., (2016) describes RS9,
a novel Nrf2 activator delaying degeneration of retina (Batliwala et al., 2017; Nakagami et al.,
2016). Culinary herbs and spices are a good source of antioxidants and a number of them are
known to ameliorate diabetes and its complications by synergistic action along with antiglycant,
anti-inflammatory, and hypoglycemic effects. Available scientific information is limited and
there are a lot of spices, culinary herbs and other natural products that need to be further
investigated and mechanisms elucidated (Preedy, 2014).
CONCLUSION
The advancement in science and pathway-specific studies have developed a lot of novel
therapeutic strategies. One such approach is the study of various isomers of NADPH-oxidase
inhibitors (Nox) and development of Nox inhibitors as a strategy against vascular diseases.
Culinary herbs and spices are rich in antioxidant compounds which are effective against
diabetes and its complications. As oxidative stress plays a role in diabetes, the antioxidant as
well as antiglycant, anti-inflammatory, hypoglycemic properties contribute synergistically to
exert antidiabetic action. But further studies are the requisite of the hour as limited scientific
evidence is available. Daily intake of spices and culinary herbs add up to daily antioxidant
intake. Vitamins such as C, D, E shows good antioxidant properties among which vitamin E
specifically α-tocopherol is a potent antioxidant and is involved in inhibiting various pathways
involved in oxidative stress-induced diabetes, hence regular dietary intake of vitamins
specifically E will serve as a protective function against various oxidative stress-induced
diseases including diabetes.

REFERENCES
Adams J. S, Hewison M. Extrarenal expression of the 25-hydroxyvitamin D-1-hydroxylase.
Arch Biochem Biophys. 2012;523(1): 95–102.
Adorini L, Penna G. Control of autoimmune diseases by the vitamin D endocrine system. Nat
Clin Pract Rheumatol. 2008;4(8):404–12.
Aghadavod E, Khodadadi S, Baradaran A, Nasri P, Bahmani M, Rafieian-Kopaei M. Role of
oxidative stress and inflammatory factors in diabetic kidney disease. Iran J Kidney Dis.
2016;10(6):337-43.
Ahmed A. M. History of diabetes mellitus. Saudi medical journal. 2002;23(4):373-78.
Alberti K. G, Zimmet P. F. Definition diagnosis and classification of diabetes mellitus and its
complications. Part 1 Provisional report of a WHO consultation. Diabetic med.
1998;15(7):539-53.
Ali M, Thomson M, Afzal M. Garlic and onions their effect on eicosanoid metabolism and its
clinical relevance. Prostag Leukotr Ess. 2000; 62:55–73.
Anonymous. American Diabetes Association. Clinical Practice Gestational diabetes mellitus.
Diabetes care suppl Alexandria. 2004; 27:88- 90.
Anonymous. American Diabetes Association. Diagnosis and classification of diabetes mellitus.
Diabetes care. 2014;1(37 Suppl 1):81-90.
Anonymous. Definition, diagnosis, and classification of diabetes mellitus and its
complications: report of a WHO consultation. Diagnosis and classification of diabetes
mellitus. Geneva: World health organization; 1999.
Auguet M, DeFeudis F. V, Clostre F. Effects of Ginkgo biloba on arterial smooth muscle
responses to vasoactive stimuli. Gen Pharmacol. 1982;13(2):169–71.
Azevedo M. F, Lima C. F, Fernandes-Ferreira M, Almeida M. J, Wilson J. M, Pereira-Wilson
C. Rosmarinic acid a major phenolic constituent of Greek sage herbal tea modulates rat
intestinal SGLT1 levels with effects on blood glucose. Mol Nutr Food Res. 2011;55(1):15–
25.
Azzi A, Gysin R, Kempna P, Munteanu A, Villacorta L, Visarius T, et al. Regulation of gene
expression by α-tocopherol. Biological chemistry. 2004;385(7):585-91.
Barbalho S. M, Damasceno D. C, Spada A. P, da Silva V. S, Martuchi K. A. Metabolic profile
of offspring from diabetic Wistar rats treated with Mentha piperita Peppermint. Evid-
Based Compl Alt. 2011; 430237.
Batliwala S, Xavier C, Liu Y, Wu H, Pang I. Involvement of Nrf2 in Ocular Diseases. Oxid
Med Cell Longev. 2017.
Baynes J. W. Role of oxidative stress in development of complications in diabetes. Diabetes.
1991;40(4):405-12.
Baynes K. C, Beeton C. A, Panayotou G, Stein R, Soos M, Hansen T, et al. Natural variants of
human p85α phosphoinositide 3-kinase in severe insulin resistance a novel variant with
impaired insulin-stimulated lipid kinase activity. Diabetologia. 2000; 43:321-331.
Beato V. M, Orgaz F, Mansilla F, Montano A. Changes in phenolic compounds in garlic Allium
sativum L owing to the cultivar and location of growth. Plant Foods Hum Nutr. 2011;
66:218–23.

Beato V. M, Orgaz F, Mansilla F, Montano A. Changes in phenolic compounds in garlic Allium

sativum L owing to the cultivar and location of growth. Plant Foods Hum Nutr. 2011;
66:218–23.
Beckman J. S, Koppenol W. H. Nitric oxide superoxide and peroxynitrite the good the bad and
ugly. Am J Physiol. 1996; 271:1424–37.
Bellamy L, Casas J. P, Hingorani A. D, Williams D. Type 2 diabetes mellitus after gestational
diabetes a systematic review and meta-analysis. Lancet. 2009; 373:1773–79.
Bjornholm M, Kawano Y, Lehtihet M, Zierath J. R. Insulin receptor substrate-1
phosphorylation and phosphatidylinositol 3-kinase activity in skeletal muscle from
NIDDM subjects after in vivo insulin stimulation. Diabetes. 1997;46(3):524–27.
Blanco R, Saenz de Tejada I, Goldstein I. Dysfunctional penile cholinergic nerves in diabetic
impotent men. J Urol. 1990; 144:278–80.
Bluher M, Michael M. D, Peroni O. D, Ueki K, Carter N, Kahn B. B. Adipose tissue selective
insulin receptor knockout protects against obesity and obesity-related glucose intolerance.
Developmental cell. 2002;3(1):25-38.
Boulton A. J. Diabetic neuropathy classification measurement and treatment. Curr Opin
Endocrinol Diabetes Obes. 2007; 14:141–45.
Briggs W. H, Xiao H, Parkin K. L, Shen C, Goldman I. L. Differential inhibition of human
platelet aggregation by selected Allium thiosulfinates. J Agric Food Chem. 2000; 48:5731–
35.
Brownlee M. The pathobiology of diabetic complications a unifying mechanism. Diabetes.
2005; 54(6):1615–25.
Bursell SE, Clermont A. C, Aiello L. P, Aiello L. M, Schlossman D. K, Feener E. P, et al. High-
dose vitamin E supplementation normalizes retinal blood flow and creatinine clearance in
patients with type 1 diabetes. Diabetes Care. 1999; 22:1245–51.
Butt M. S, Sultan M. T, Iqbal J. Garlic nature's protection against physiological threats. Crit
Rev Food Sci Nutr. 2009; 49:538–51.
Buyukbalci A, El S. N. Determination of in vitro antidiabetic effects antioxidant activities and
phenol contents of some herbal teas. Plant Foods Hum Nutr. 2008; 63:27–33.
Carlsen M. H, Halvorsen B. L, Holte K, Bohn S. K, Dragland S, Sampson L, et al. The total
antioxidant content of more than 3100 foods beverages spices herbs and supplements used
worldwide. Nutr J. 2010.
Catalano P. M, Nizielski S. E, Shao J, Preston L, Qiao L, Friedman J. E. Downregulated IRS-
1 and PPARγ in obese women with gestational diabetes relationship to FFA during
pregnancy. Am J Physiol-Endoc M. 2002;282(3):522-33.
Cazzola R, Camerotto C, Cestaro B. Anti-oxidant anti-glycant and inhibitory activity against
alpha-amylase and alpha-glucosidase of selected spices and culinary herbs. Int J Food Sci
Nutr. 2011; 62:175–84.
Chan J. Y, Yuen A. C, Chan R. Y, Chan S. W. A review of the cardiovascular benefits and
antioxidant properties of allicin. Phytother Res. 2013; 27:637–46.
Chaturvedi N, Jarrett J, Shipley M. J, Fuller J. H. Socioeconomic gradient in morbidity and
mortality in people with diabetes cohort study findings from the Whitehall Study and the
WHO Multinational Study of Vascular Disease in Diabetes. BMJ. 1998;316(7125):100-5.
Chen L, Mayo R, Chatry A, Hu G. Gestational diabetes mellitus its epidemiology and
implication beyond pregnancy. Curr Epidemiol Rep. 2016; 3:1–11.

Chen P, Zhang Q. L, Chen W. Y, Wang Y. Q, Xu X. J. The effects of danshen dripping pills

on the expressions of HIF-1α and VEGF in renal tissues of type 2 diabetic rats Chinese.
Chin J Health Care Med. 2011;13(3):200–3.
Cheng T. O. Cardiovascular effects of danshen. Int J Cardiol. 2007;121(1):9–22.
Chou E, Suzuma I, Way K. J, Opland D, Clermont A. C, Naruse K, et al. Decreased cardiac
expression of vascular endothelial growth factor and its receptors in insulin-resistant and
diabetic states a possible explanation for impaired collateral formation in cardiac tissue.
Circulation. 2002; 105:373–379.
Chuang C. C, Martinez K, Xie G, Kennedy A, Bumrungpert A, Overman A, et al. Quercetin is
equally or more effective than resveratrol in attenuating tumor necrosis factor-α−mediated
inflammation and insulin resistance in primary human adipocytes. Am J Clin Nutr. 2010;
92:1511–21.
Conforti F, Statti G, Uzunov D, Menichini F. Comparative chemical composition and
antioxidant activities of wild and cultivated Laurus nobilis L leaves and Foeniculum
vulgare subsp piperitum Ucria coutinho seeds. Biol Pharm Bull. 2006; 29:2056–64.
Connolly V, Unwin N, Sherriff P, Bilous R, Kelly W. Diabetes prevalence and socioeconomic
status a population-based study showing increased prevalence of type 2 diabetes mellitus
in deprived areas. J Epidemiol Commun H. 2000;54(3):173-77.
Cooper ME. Pathogenesis prevention and treatment of diabetic nephropathy. Lancet. 1998;
352:213–219.
Czapski G, Goldstein S. The role of the reactions of NO with superoxide and oxygen in
biological systems a kinetic approach. Free Radic Biol Med. 1995;19(6):785–94.
Dall'Acqua S, Cervellati R, Speroni E, Costa S, Guerra M. C, Stella L, et al. Phytochemical
composition and antioxidant activity of Laurus nobilis L leaf infusion. J Med Food. 2009;
12:869–76.
Damm P, Handberg A, KÜhl C, Beck Nielsen H, Mølsted Pedersen L. Insulin receptor binding
and tyrosine kinase activity in skeletal muscle from normal pregnant women and women
with gestational diabetes. Obstet Gynecol. 1993;82(2):251-59.
De Young L, Yu D, Bateman R. M, Brock G. B. Oxidative stress and antioxidant therapy: Their
impact in diabetes-associated erectile dysfunction. J Androl. 2004; 25(5): 830-836.
De Almeida M. E, Mancini F. J, Barbosa G. N. Characterization of antioxidant compounds in
aqueous coriander extract Coriandrum sativum L. LWT Food Sci Technol. 2005;38:15–19.
Dearlove, R. P, Greenspan, P, Hartle, D. K, Swanson, R. B, Hargrove, J. L. Inhibition of protein
glycation by extracts of culinary herbs and spices. J Med Food. 2008; 11:275–81.
Ding, L, Jin, D, Chen, X. Luteolin enhances insulin sensitivity via activation of PPARg
transcriptional activity in adipocytes. J Nutr Biochem. 2010; 21:941–47.
Douki H, Cadet J. Peroxynitrite mediated oxidation of purine bases of nucleosides and isolated
DNA. Free Rad Res. 1996;24(5):369–80.
Eid, H. M., Vallerand D, Muhammad A, Durst T, Haddad, P. S, Martineau LC. Structural
constraints and the importance of lipophilicity for the mitochondrial uncoupling activity of
naturally occurring caffeic acid esters with potential for the treatment of insulin resistance.
Biochem Pharmacol. 2010; 79:444–54.
El-Ghorab A. H., Nauman M, Anjum F. M., Hussain S, Nadeem M. A comparative study on
chemical composition and antioxidant activity of ginger Zingiber officinale and cumin
Cuminum cyminum. J Agric Food Chem. 2010; 58:8231–7.

El-Moselhy M. A, Taye A, Sharkawi S. S, ElSisi S. F, Ahmed A. F. The antihyperglycemic

effect of curcumin in high fat diet fed rats Role of TNF-alpha and free fatty acids. Food
Chem Toxicol. 2011; 49:1129–40.
El-Remessy A. B., Behzadian M. A, AbouMohamed G, Franklin T, Caldwell R. W., Caldwell
R. B. Experimental diabetes causes breakdown of the blood-retina barrier by a mechanism
involving tyrosine nitration and increases in expression of vascular endothelial growth
factor and urokinase plasminogen activator receptor. Am J Pathol. 2003;162(6):1995-2004.
Engerman R. L., Kern T. S., Larson M. E. Nerve conduction and aldose reductase inhibition
during 5 years of diabetes or galactosaemia in dogs. Diabetologia. 1994; 37:141–144.
Farlex. Farlex Partner Medical Dictionary. 2012.
Fecka I, Turek S. Determination of water-soluble polyphenolic compounds in commercial
herbal teas from Lamiaceae peppermint melissa and sage. J Agric Food Chem.
2007;55(10):908–17.
Ferner R. E. Drug-induced diabetes. Baillieres Clin Endocrinol Metab. 1992;6:849–66.
Ferrannini E, Buzzigoli G, Bonadonna R, Giorico M. A., Oleggini M, Graziadei L, et al. Insulin
resistance in essential hypertension. New Engl J Med. 1987;317(6):350-57.
Finkel T, Holbrook N. J. Oxidants oxidative stress and the biology of aging. Nature.
2000;408(6809):239.
Fremont L. Biological effects of resveratrol. Life Sci. 2000;66(8):663-73.
Frombaum M, Le Clanche S, Bonnefont R. D., Borderie D. Antioxidant effects of resveratrol
and other stilbene derivatives on oxidative stress and NO bioavailability Potential benefits
to cardiovascular diseases. Biochimie. 2012;94(2):269–76.
Gavin J. R., Alberti, Davidson M. B., DeFronzo R. A., Drash A, Gabbe S. G., et al. Report of
the expert committee on the diagnosis and classification of diabetes mellitus. Diabetes
Care 1997;20(7):1183-97.
Genestra M. Oxyl radicals redox sensitive signaling cascades and antioxidants. Review Cell
Signal. 2007;19(9):1807–19.
Ghosh S, Watanabe R. M., Hauser E. R., Valle T, Magnuson V. L., Erdos M. R., et al. Type 2
diabetes evidence for linkage on chromosome 20 in 716 Finnish affected sib pairs. Proc
Natl Acad Sci. 1999; 96(5): 2198-2203.
Gohil K, Packer L. Global gene expression analysis identifies cell and tissue specific actions
of Ginkgo biloba extract EGb 761. Cell Mol Biol. 2002;48(6):625–31.
Greenbaum C. J., Schatz D. A., Cuthbertson D, Zeidler A, Eisenbarth G. S., Krischer J. P.
Diabetes Prevention Trial Type 1 Study Group Islet cell antibody-positive relatives with
human leukocyte antigen DQA1* 0102, DQB1* 0602: identification by the Diabetes
Prevention Trial type 1. J Clin Endocr Metab. 2000;85(3):1255-60.
Groop L. C., Saloranta C, Shank M, Bonadonna R. C., Ferrannini E, Defronzo R. A. The role
of free fatty acid metabolism in the pathogenesis of insulin resistance in obesity and
noninsulin dependent diabetes mellitus. J Clin Endocrinol Metab. 1991;72(1):96–107.
Gupta S. C., Patchva S, Aggarwal B. B. Therapeutic roles of curcumin lessons learned from
clinical trials. AAPS J. 2013; 15:195–218.
Halliwell B. Free Radicals and other reactive species in disease. Nature Encyclopedia of life
sciences. 2001; 1–7.
Halsall D. J., Luan J, Saker P, Huxtable S, Farooqi I. S., Keogh J, et al. Uncoupling protein 3
genetic variants in human obesity: the c-55t promoter polymorphism is negatively

correlated with body mass index in a UK Caucasian population. Int J Obes.

2001;25(4):472- 477.
Hanis C. L., Boerwinkle E, Chakraborty R, Ellsworth D. L., Concannon P, Stirling B, et al.
Effects of 50% ethanol extract from rosemary (Rosmarinus officinalis) on α-glucosidase
inhibitory activity and the elevation of plasma glucose level in rats and its active
compound. J Food Sci. 2006; 71:507–12.
Hansen L, Urioste S, Petersen H. V., Jensen J. N., Eiberg H, Barbetti F, et al. Missense
mutations in the human insulin promoter factor-1 gene and their relation to maturity-onset
diabetes of the young and late-onset type 2 diabetes mellitus in Caucasians. J Clin
Endocrinol Metab. 2000;85(3):1323-1326.
Hirsch I. B., Emmett M. Diabetic ketoacidosis and hyperosmolar hyperglycemic state in adults:
Clinical features, evaluation, and diagnosis.2017.
Ho, F. M., Liu, S. H., Liau, C. S., Huang, P. J., Lin-Shiau, S. Y. High glucose-induced apoptosis
in human endothelial cells is mediated by sequential activations of c-Jun NH(2)-terminal
kinase and caspase-3. Circulation. 2000; 101(22):2618–2624.
Horikawa, Y., Oda, N., Cox, N. J., Li, X., Orho-Melander, M., Hara, M., et al. Genetic variation
in the gene encoding calpain-10 is associated with type 2 diabetes mellitus. Nat Genet.
2000; 26(2):163-175.
Hotamisligil, G. S., Shargill, N. S., Spiegelman, B. M. Adipose expression of tumor necrosis
factor-alpha: direct role in obesity-linked insulin resistance. Science. 1993;259(5091):87-
91.
Huang, D., Refaat, M., Mohammedi, K., Jayyousi, A., Al Suwaidi, J., Abi Khalil, C.
Macrovascular complications in patients with diabetes and prediabetes. Biomed Res Int.
2017;1-9.
Huang D. W., Shen S. C, Wu J. S. B. Effects of caffeic acid and cinnamic acid on glucose
uptake in insulin-resistant mouse hepatocytes. J Agric Food Chem. 2009; 57:7687–92.
Huysman E., Mathieu C.. Diabetes and peripheral vascular disease. Acta Chir Belg.
2009;109(5):587-94.
Imagawa, A., Hanafusa, T., Miyagawa, J. I., Matsuzawa, Y. A novel subtype of type 1 diabetes
mellitus characterized by a rapid onset and an absence of diabetes-related antibodies. New
Engl J Med. 2000;342(5):301-7.
Ischiropoulos, H., Al-Mehdi, A. B. Peroxynitrite-mediated oxidative protein modifications.
FEBS Lett. 1995;364(3):279–82.
Jaeckel, E, Manns M., Von Herrath, M. Viruses and diabetes. Ann N Y Acad Sci. 2002; 958:7–
25.
Jagtap A. G, Patil P. B. Antihyperglycemic activity and inhibition of advanced glycation end
product formation by Cuminum cyminum in streptozotocin-induced diabetic rats. Food
Chem Toxicol. 2010; 48:2030–6.
Ji L, Malecki, M., Warram J. H., Yang Y, Rich S. S, Krolewski A. S. New susceptibility locus
for NIDDM is localized to human chromosome 20q. Diabetes. 1997;46(5):876-881.
Jonk, A. M., Houben, A. J., de Jongh, R. T., Serne, E. H., Schaper, N. C., Stehouwer, C. D.
Microvascular dysfunction in obesity: a potential mechanism in the pathogenesis of
obesity-associated insulin resistance and hypertension. Physiology Bethesda. 2007;
22:252–60.

Jung, I. H., Lee, Y. H., Yoo, J. Y., Jeong, S. J., Sonn, SK, Park, J. G., et al. Ginkgo biloba
extract (GbE) enhances the anti-atherogenic effect of cilostazol by inhibiting ROS
generation. Exp Mol Med. 2012;44(5):311–8.
Kahn C. R. Joslin's Diabetes Mellitus. 14th ed. Philadelphia: Lippincott Williams & Wilkins
Wolters Kluwer; 2007.
Keegan A, Cotter M. A., Cameron N. E. Effects of diabetes and treatment with the antioxidant
α-lipoic acid on endothelial and neurogenic responses of corpus cavernosum in rats.
Diabetologia.1999; 42(3), 343-350.
Khan, A, Zaman, G, Anderson, R. A. Bay leaves improve glucose and lipid profile of people
with type 2 diabetes. J Clin Biochem Nutr. 2009; 44:52–6.
Kitabchi, A. E., Wall, B. M. Diabetic ketoacidosis. Med Clin N Am. 1995;79(1):9-37.
Koga K, Shibata H, Yoshino K, Nomoto K. Effects of 50% ethanol extract from rosemary
(Rosmarinus officinalis) on α-glucosidase inhibitory activity and the elevation of plasma
glucose level in rats, and its active compound. J Food Sci. 2006; 71:507–12.
Kohen R, Nyska A. Invited review Oxidation of Biological Systems: Oxidative Stress
Phenomena, Antioxidants, Redox Reactions, and Methods for Their Quantification.
Toxicol Pathol. 2002;30(6):620–50.
Kowluru R. A. Effect of reinstitution of good glycemic control on retinal oxidative stress and
nitrative stress in diabetic rats. Diabetes. 2003;52(3):818-23.
Kressmann S, Muller W. E, Blume H. H. Pharmaceutical quality of different Ginkgo biloba
brands. J Pharm Pharmacol. 2002;54(5):661–9.
Kumar P. A., Reddy P. Y., Srinivas P. N., Reddy G. B. Delay of diabetic cataract in rats by the
antiglycating potential of cumin through modulation of alpha-crystallin chaperone activity.
J Nutr Biochem. 2009; 20:553–62.
Kumar R, Chhatwal S, Arora S, Sharma S, Singh J, Singh N, et al. Antihyperglycemic,
antihyperlipidemic, anti-inflammatory and adenosine deaminase- lowering effects of garlic
in patients with type 2 diabetes mellitus with obesity. Diabetes Metab Syndr Obes. 2013;
6:49–56.
Langer O, editor. Diabetes in Pregnancy Dilemma. 2nd ed. USA: People’s Medical Publishing
House; 2011.
Li Y., Tran V. H., Duke C. C., Roufogalis B. D. Preventive and protective properties of Zingiber
officinale (ginger) in diabetes mellitus, diabetic complications, and associated lipid and
other metabolic disorders: a brief review. Evid Based Complement Alternat Med. 2012;
2012:516-870.
Libby P, Theroux P. Pathophysiology of coronary artery disease. Circulation. 2005;
28;111(25):3481- 3488.
Liu C. T., Sheen L. Y., Lii C. K. Does garlic have a role as an antidiabetic agent. Mol Nutr
Food Res. 2007; 51:1353–64.
Liu L, Wang Y, Lam K. S., Xu A. Moderate wine consumption in the prevention of metabolic
syndrome and its related medical complications. Endocr Metab Immune Disord Drug
Targets. 2008;8(2):89-98.
Lohmann T, Nietzschmann U, Kiess W. Lady-like: is there a latent autoimmune diabetes in the
young. Diabetes Care.2000; 23:1707–1708.
Lu W. H., Shi B. Y., Li S. L., Wei D. G., Liu W. D. Effect of compound danshen dropping pills
on type 2 diabetes mellitus with microalbuminuria. J Shanxi Med Univ. 2010;41(4): 022.
Lue T. F. Erectile dysfunction. New Engl J Med. 2000;342(24):1802-13.

Lukovits T. G., Mazzone T. M., Gorelick T. M. Diabetes mellitus and cerebrovascular disease.
Neuroepidemiology. 1999;18(1):1-14.
Madsen H. L., Bertelsen G. Spices as antioxidants. Trends food sci tech trends in food science
& technology. 1995;6(8):271-7.
Mahady G. B. Ginkgo biloba for the prevention and treatment of cardiovascular disease: a
review of the literature. J Cardiovasc Nurs. 2002;16(4):21–32.
Mahtani M. M, Widen E, Lehto M, Thomas J, McCarthy M, Brayer J, et al. Mapping of a gene
for type 2 diabetes associated with an insulin secretion defect by a genome scan in Finnish
families. Nat Genet. 1996;14(1):90-96.
Malecki M. T, Antonellis A, Casey P, Ji L, Wantman M, Warram J. H, et al. Exclusion of the
hepatocyte nuclear factor 4-alpha as a candidate gene for late-onset NIDDM linked with
chromosome 20q. Diabetes. 1998;47(6):970-973.
Marnett L. J. Oxyradicals and DNA damage. Carcinogenesis. 2000;21(3):361–70.
Meirhaeghe A, Amouyel P, Helbecque N, Cottel D, Otabe S, Froguel P, et al. An uncoupling
protein 3 gene polymorphism associated with a lower risk of developing Type II diabetes
and with atherogenic lipid profile in a French cohort. Diabetologia. 2000;43(11):1424-
1428.
Mitri J, Muraru M. D, Pittas A. G. Vitamin D and type 2 diabetes: a systematic review. Eur J
Clin Nutr. 2011;65(9):1005–15.
Mukherji S. M, Singh S. P. Reaction mechanism in organic chemistry. Madras: Macmillan
India press; 1986.
Murata M, Ohta N, Fujisawa S, Tsai J. Y, Sato S, Akagi Y, et al. Selective pericyte degeneration
in the retinal capillaries of galactose-fed dogs results from apoptosis linked to aldose
reductase-catalyzed galactitol accumulation. J Diabetes Complications. 2002; 16(5):363–
370.
Ng C. S, Wang S. P, Cheong J. L, Wu Y. D, Jia Y. L, Leung S. W. Systematic review and meta-
analysis of randomized controlled trials comparing Chinese patent medicines Compound
danshen Dripping Pills and Di'ao Xinxuekang in treating angina pectoris. 2012;10(1):25–
34.
Nishikawa T, Edelstein D, Du X. L, Yamagishi S, Matsumura T, Kaneda Y, et al. Normalizing
mitochondrial superoxide production blocks three pathways of hyperglycemic damage.
Nature. 2000; 404:787–90.
Oken B. S, Storzbach D. M, Kaye J. A. The efficacy of Ginkgo biloba on cognitive function in
Alzheimer disease. Arch Neurol. 1998;55(11):1409–15.
Pasquel F. J, Umpierrez G. E. Hyperosmolar hyperglycemic state: a historic review of the
clinical presentation, diagnosis, and treatment. Diabetes care. 2014;37(11):3124-31.
Penson D. F, Wessells H. Erectile dysfunction in diabetic patients. Diabetes Spectrum.
2004;17(4):225-30.
Pham-Huy L. A, Hua, He, Pham-Huy, C. Free Radicals, Antioxidants in Disease and Health.
Int J Biomed Sci. 2008;4(2):89–96.
Phaniendra A, Jestadi DB, Periyasamy L. Free radicals: properties, sources, targets, and their
implication in various diseases. Indian J Clin Biochem. 2015;30(1):11-26.
Pittler M. H, Ernst E. Ginkgo biloba extract for the treatment of intermittent claudication: a
meta-analysis of randomized trials. Am J Med. 2000;108(4):276–81.

Pomero F, Allione A, Beltramo E, Buttiglieri S, Dalu F, Ponte E, et al. Effects of protein kinase
C inhibition and activation on proliferation and apoptosis of bovine retinal pericytes.
Diabetologia. 2003; 46 (3):416–419.
Pop-Busui R, Boulton AJ, Feldman E. L, Bril V, Freeman R, Malik R. A. Diabetic neuropathy:
a position statement by the American Diabetes Association. Diabetes care.
2017;40(1):136-54.
Preedy V. R. Diabetes: Oxidative stress and Dietary Antioxidants. Academic Press; 2014.
Qu F. N, Qi L. W, Wei Y. J, Wen X. D, Yi L, Luo H. W, et al. Multiple target cell extraction
and LC-MS analysis for predicting bioactive components in Radix Salviae miltiorrhizae.
Biol Pharm Bull. 2008;31(3):501–6.
Rambhade S, Singh S, Goswami R, Rambhade A. Occurrence, complications, and interventions
of diabetes: A new understanding of an old problem. Sys Rev Pharm. 2011;2(1):8.
Rodriguez M, Ringstad L, Schafer P, Just S, Hofer H. W, Malmsten M, et al. Reduction of
atherosclerotic nanoplaque formation and size by Ginkgo biloba (EGb 761) in
cardiovascular high-risk patients. Atherosclerosis. 2007; 192(2):438- 444.
Romeo G, Liu W. H, Asnaghi V, et al. Activation of nuclear factor-kappaB induced by diabetes
and high glucose regulates a proapoptotic program in retinal pericytes. Diabetes. 2002;
51:2241–3348.
Rosen P, Nawroth P. P, King G, Moller W, Tritschler H. J, Packer L. The role of oxidative
stress in the onset and progression of diabetes and its complications. Diabetes Metab Res
Rev. 2001; 17:189–212.
Ross S. R, Graves R. A, Greenstein A, Platt K. A, Shyu H. L, Mellovitz B et al. A fat-specific
enhancer is the primary determinant of gene expression for adipocyte P2 in vivo. Proc Natl
Acad Sci. 1990;87(24):9590-4.
Ryu K. H, Han H. Y, Lee S. Y, Jeon S. D, Im G. J, Lee B. Y, et al. Ginkgo biloba extract
enhances anti-platelet and antithrombotic effects of cilostazol without prolongation of
bleeding time. Thromb Res. 2009;124(3):328–34.
Saraswat M, Reddy P. Y., Muthenna P, Reddy G. B. Prevention of non-enzymic glycation of
proteins by dietary agents: prospects for alleviating diabetic complications. Br J Nutr.
2009; 101:1714–21.
Scazzocchio B, Var R, Filesi C, D’Archivio M, Santangelo C, Giovannini C, et al. Cyanidin-
3-O-β-glucoside and protocatechuic acid exert insulin-like effects by upregulating PPARγ
activity in human omental adipocytes. Diabetes. 2011; 60:2234–44.
Schwer B, Verdin E. Conserved metabolic regulatory functions of sirtuins. Cell Metab.
2008;7(2):104–12.
Shaw JE, Sicree R. A, Zimmet P. Z. Global estimates of the prevalence of diabetes for 2010
and 2030. Diabetes Res Clin Pract. 2010; 87:4–14.
Shojaeian A, Mehri-Ghahfarrokhi A. An overview of the Epidemiology of Type 1 Diabetes
Mellitus. Int J Metab Syndr. 2018;2(1):1-4.
Singh PP, Mahadi F, Roy A, Sharma P. Reactive oxygen species, reactive nitrogen species and
antioxidants in etiopathogenesis of diabetes mellitus type-2. Ind J Clin Biochem.
2009;24(4):324-42.
Skrovankova S, Misurcova L, Machu L. Antioxidant activity and protecting health effects of
common medicinal plants. Adv Food Nutr Res. 2012; 67:75–139.
Slimestad R, Fossen T, Vagen I. M. Onions: a source of unique dietary flavonoids. J Agric
Food Chem. 2007; 55:10067–80.

Smith JV, Luo Y. Elevation of oxidative free radicals in Alzheimer's disease models can be
attenuated by Ginkgo biloba extract EGb 761. J Alzheimers Dis. 2003;5(4):287–300.
Sowbhagya H. B. Chemistry, technology, and nutraceutical functions of cumin (Cuminum
cyminum L): an overview. Crit Rev Food Sci Nutr. 2013; 53:1–0.
Srinivasan K. Black pepper and its pungent principle-piperine: a review of diverse
physiological effects. Crit Rev Food Sci Nutr. 2007;47: 735–48.
Srinivasan K. Plant foods in the management of diabetes mellitus: Spices as beneficial
antidiabetic food adjuncts. Int J Food Sci Nutr. 2005; 56:399–414.
Stadtman E. R, Levine R. L. Protein oxidation. Ann N Y Acad Sci. 2000;899:191–208.
Steinkellner H, Rabot S, Freywald C, Nobis E, Scharf G Chabicovsky M, et al. Effects of
cruciferous vegetables and their constituents on drug metabolizing enzymes involved in
the bioactivation of DNA-reactive dietary carcinogens. Mutat Res. 2001;285–97.
Stoss H, Pesch H. J, Pontz B, et al. Wolcott-Rallison syndrome: diabetes mellitus and
spondyloepiphyseal dysplasia. Eur J Pediatr. 1982; 138:120–129.
Stumvoll M, Fritsche A, Volk A, Stefan N, Madaus A, Maerker E. The Gly972Arg
polymorphism in the insulin receptor substrate-1 gene contributes to the variation in insulin
secretion in normal glucose-tolerant humans. Diabetes. 2001;50(4):882-885.
Suhaj M. Spice antioxidants isolation and their antiradical activity: a review. J Food Composit
Anal. 2006; 19:531–7.
Taj Eldin IM, Ahmed EM, Elwahab HMA. A preliminary study of the clinical hypoglycemic
effects of Allium cepa (Red Onion) in Type 1 and Type 2 diabetic patients. Environ Health
Insights. 2010; 4:71–7.
Takaoka M. Resveratrol, a new phenolic compound, from Veratrum grandiflorum. Nippon
Kagaku Kaishi. 1939; 60:1090-100.
Tanaka S. A novel subtype of type 1 diabetes mellitus. N Engl J Med. 2000; 342:1835-7.
Tesfaye S, Chaturvedi N, Eaton S. E, Ward J. D, Manes C, IonescuTirgoviste C, et al. Vascular
risk factors and diabetic neuropathy. N Engl J Med. 2005; 352:341–50.
Thirumalai T, Therasa S. V, Elumalai E. K, David E. Hypoglycemic effect of Brassica juncea
(seeds) on streptozotocin-induced diabetic male albino rat. Asian Pacific J Tropical
Biomed. 2011; 1:323–5.
Thorburn A. W, Gumbiner B, Bulacan F, Brechtel G, Henry R. R. Multiple defects in muscle
glycogen synthase activity contribute to reduced glycogen synthesis in non-insulin
dependent diabetes mellitus. J Clin Invest. 1991;87(2):489- 495.
Tiberti C, Buzzetti R, Anastasi E, Dotta F, Vasta M, Petrone A, et al. Autoantibody negative
new onset Type 1 diabetic patient lacking high risk HLA alleles in a Caucasian population:
are these Type 1b diabetes cases? Diabetes/metabolism research and reviews.
2000;16(1):8-14.
Turner RC, Cull C. A, Frighi V, Holman R. R. Glycemic control with diet, sulfonylurea,
metformin, or insulin in patients with type 2 diabetes mellitus: progressive requirement for
multiple therapies (UKPDS 49). JAMA. 1999;281(21):2005–2012.
Unger J. Latent Autoimmune Diabetes in Adults. Am Fam Physician. 2010;81(7):843-847.
Vagen I. M. Onions: a source of unique dietary flavonoids. J Agric Food Chem. 2007;
55:10067–80.
Vetterli L, Brun T, Giovannoni L, Bosco D, Maechler P. Resveratrol potentiates glucose-
stimulated insulin secretion in INS-1E beta-cells and human islets through a SIRT1-
dependent mechanism. J Biol Chem. 2011;286(8):6049–60.

Vinik AI, Park T. S, Stansberry K, Pittenger G. L. Diabetic neuropathies. Diabetologia. 2000;

43:957–73.
Wang T., Liu Y., Huang C., Mansai H. A, Wei W., Zhang X., et al. Puerarin promotes MIN6
cell survival by reducing cellular reactive oxygen species. Mol Med Rep. 2018;17(5):7281-
7286.
Wapelhorst B., Spielman R. S, Gogolin-Ewens K. J, Shephard J. M. A genome wide search for
human non-insulin dependent (type 2) diabetes genes reveals a major susceptibility locus
on chromosome 2. Nat Genet. 1996;13(2):161-166.
Withers D. J, Gutierrez J. S, Towery H., Burks D. J, Ren J. M., Previs S. Disruption of IRS-2
causes type 2 diabetes in mice. Nature. 1998;391(6670):900-904.
Yamagishi S, Amano S, Inagaki Y, Okamoto T, Koga K, Sasaki N et al. Advanced glycation
end products induced apoptosis and overexpression of vascular endothelial growth factor
in bovine retinal pericytes. Biochem Biophys Res Commun. 2002; 290:973–978.
Yla-Herttuala S. Oxidized LDL and atherogenesis. Ann N Y Acad Sci. 1999; 874:134–7.
Young IS, Woodside JV. Antioxidants in health and disease. J Clin Pathol. 2001; 54:176–86.
Zhang L. W, Piao S, Choi M. J, Shin H. Y, Jin H. R, Kim W. J. Role of increased penile
expression of transforming growth factor‐β1 and activation of the Smad signaling pathway
in erectile dysfunction in streptozotocin‐induced diabetic rats. J Sex Med.
2008;5(10):2318-29.
Zhang Q, Radisavljevic Z. M, Siroky MB, Azadzoi KM. Dietary antioxidants improve
arteriogenic erectile dysfunction. Int J Androl. 2011; 34(3), 225-235.
Zhang W, Wang Y, Yang Z, Qiu J, Ma J, Zhao Z, Bao T. Antioxidant treatment with quercetin
ameliorates erectile dysfunction in streptozotocin-induced diabetic rats. J Biosci Bioeng.
2011; 112(3), 215-218.
Zhang Y, Jayaprakasam B, Seeram N. P, Olson L. K, DeWitt D, Nair M. G. Insulin secretion
and cyclooxygenase enzyme inhibition by cabernet sauvignon grape skin compounds. J
Agric Food Chem. 2004;52(2):228–33.
Zheng Y, Ley S. H., Hu F. B. Global aetiology and epidemiology of type 2 diabetes mellitus
and its complications. Nat Rev Endocrinol. 2018;14(2):88-98.
Zhou Y. P, Guo Z. Y, Tong X. L, Pan L, Zhao J. B. Effect of composite salvia pellets on diabetic
retinopathy in streptozotocin-induced diabetic rats. Chin J Integrated Tradit West Med.
2002;30(1):174–8.
Zierath J. R, Krook A, Wallberg-Henriksson H. Insulin action in skeletal muscle from patients
with NIDDM. Mol Cell Biochem. 1998; 182:153–160.
Zimmet P. Z, Tuomi T, Mackay I. R. Rowley M. J, Knowles W, Cohen M. Latent autoimmune
diabetes mellitus in adults (LADA) the role of antibodies to glutamic acid decarboxylase
in the diagnosis and prediction of insulin dependency. Diabet Med. 1994;11(3):299-303.

Chapter 16
ANTIOXIDANT DEFENSE IN DIABETIC NEPHROPATHY
Manojkumar S. Mahajan*, Vishal S. Gulecha,

Aman Upaganlawar and Chandrashekhar D. Upasani
ABSTRACT
Diabetes mellitus (DM) is the most common of the systemic diseases involving the
kidney. Renal function plays a major role in the homeostatic maintenance of body functions
which is challenged during DM. Thus, one of the chronic microvascular complications
resulting from DM due to chronic hyperglycemia is diabetic nephropathy (DN), the leading
causes of end-stage renal disease (ESRD) and increased mortality in the patients with DM.
Oxidative stress is at the center in the development of DN and underlying kidney damage.
In the diabetic state, oxidative stress induced due to a disturbance in the normal antioxidant
defense and increased free-radical formation triggers multiple downstream signaling
mechanisms that interfere with normal renal structural and functional features. Presently,
the therapies used in the treatment of DN have not been fully efficacious due to the diverse
etiologies in the development of DN. This results in difficulty in selecting the best possible
treatment strategy and a therapeutic agent. Therefore more effective and new therapeutic
approaches are needed in the treatment of DN than currently available. Many studies
reported that therapies with antioxidant have a beneficial effect on DN but the antioxidant
agents in current use lack target specificity. The chapter focuses on the beneficial effects
of antioxidants in the DN in addition to their targets proved clinically or identified during
the preclinical evaluation.
1. INTRODUCTION
DM is a group of heterogeneous disorders characterized by a common endpoint i.e.,
hyperglycemia which is due to either a decrease in circulating levels of insulin resulting in
insulin deficiency or decreased tissue sensitivity to it i.e., insulin resistance or combination of
*
Corresponding Author’s Email: mahajan.mscop@snjb.org.

428 Manojkumar S. Mahajan, Vishal S. Gulecha, Aman Upaganlawar et al.
both insulin deficiency and insulin resistance (Maitra, 2015; Masharani, 2008). DM has two
major forms such as type 1 DM (T1DM), type 2 DM (T2DM). Type 1 DM which occurs due
to autoimmune destruction of pancreatic β-cells or absolute deficiency of insulin is also known
as insulin dependent diabetes mellitus (IDDM). Type 2 DM, is due to insulin resistance with
relative insulin deficiency therefore known as non-insulin dependent diabetes mellitus
(NIDDM) (Cooke and Plotnick, 2008). Other types of DM include gestational DM, which
develops during pregnancy due to insulin resistance and maturity onset DM resulting from a
genetic defect in β-cell functions (Rubin and Strayer, 2012).
Both the forms of DM possess a strong genetic component in addition to the acquired
pathogenic causes. There is considerable evidence indicating that chronic hyperglycemia
causes many of the chronic complications of DM. There are several macrovascular
complications like coronary artery, cerebrovascular and peripheral vascular disease arising due
to damaged blood vessels (Ramachandran et al., 2012). The primary microvascular
manifestations of DM include diabetic nephropathy (DN), diabetic neuropathy and diabetic
retinopathy (Pal et al., 2014; Geraldes et al., 2009).
DN is a leading reason for chronic kidney disease (CKD) and end-stage renal disease
(ESRD). It is traditionally defined as a glomerular disease inclusive of five distinct stages such
as glomerular hyperfiltration, incipient nephropathy, microalbuminuria, overt proteinuria and
end-stage renal disease (Mogensen, 1983). It is a progressive and irreversible loss of renal
function characterized by initial hyperfiltration, albuminuria, glomerular mesangium expansion
due to the accumulation of extracellular matrix, interstitial fibrosis, thickening of basement
membranes, and renal cell damage. This means DN leads to structural changes and functional
abnormalities in the kidneys (Vora and Ibrahim, 2003).
Initially, there is an increase in glomerular filtration rate (GFR), called the stage of
hyperfiltration, leading to marked glomerular injury without affecting urinary albumin
excretion rate (UAER). Subsequently, there is the development of microalbuminuria leading,
structural and functional changes in glomerulus causing mesangial expansion and thickening
glomerular basement membrane (GBM), and altered permeability (Cooper and Gilbert, 2003).
This stage is known as incipient nephropathy. Persistent microalbuminuria in diabetic patients,
therefore, put them on at high risk of overt DN characterized by proteinuria, elevated blood
pressure (hypertension), and reduced renal function along with histopathological changes in
basement membrane that shows thickening, mesangial expansion causing interstitial fibrosis
(Cooper and Gilbert, 2003). Persistent proteinuria for several years may progress to ESRD
(Caramori and Mauer, 2001).
About 20-40% of total diabetic patients get affected by DN (Thorp, 2005; Rizvi et al.,
2014). DN is also a major contributor for cardiovascular disease (CVD), as most of the patients
with DN die due to either CVD or other related causes even before ESRD (Parving et al., 2000;
Foley et al., 1997). Therefore this chapter highlights the pathophysiological aspects of DN. The
emphasis is given to the generation oxidative stress due to excess free radicals in DM and
during chronic hyperglycemia and its role in the development of DN. The chapter is thus aimed
primarily to represents the clinically beneficial and experimentally proved renoprotective
antioxidants used in amelioration of DN.

Antioxidant Defense in Diabetic Nephropathy 429
2. DIFFERENT STAGES OF DIABETIC NEPHROPATHY

DM is resulting in a range of macro and microvascular complications as shown in Figure
1. As noted earlier, DN is one of the major complications of chronic hyperglycemia. For a better
understanding of the progression and development of DN, it is divided into the following
stages:
Figure 1. Complications of diabetes mellitus.
2.1. Stage 1
Glomerular hyperfiltration (Table 1), due to glomerular hypertrophy which is detected in

early diabetes. This stage is indicated by the increased demand for kidneys that leads to
increased or normal glomerular filtration rate (GFR) (Vora and Ibrahim, 2003).
2.2. Stage 2
Renal hyperfiltration (Table 1) is due to structural alterations including mesangial

expansion and thickening of GBM. This stage occurs with the progression of DM. The GFR is
increased or has returned to normal. There are signs of glomerular damage along with a small
increase in albumin excretion (normoalbuminuria, excretion of < 30 mg of albumin in the 24-
hour urine). This stage is therefore called incipient nephropathy (Lin et al., 2018).
2.3. Stage 3
This is the stage of microalbuminuria (Table 1) in which urinary albumin excretion rate
(UAER) is about 30 mg-300 mg or the ratio of urine albumin over urine creatinine ranging

from 30-300. There is a significant progression of glomerular damage. Hypertension typically

develops during stage 3 (Vora and Ibrahim, 2003).
2.4. Stage 4
This stage is of proteinuria and also referred to as overt nephropathy (Table 1). It develops
in the later stage of DM. Glomerular damage continues, with increased urinary albumin. Renal
function decline and indicated by elevated blood urea nitrogen (BUN) and creatinine (Cr) with
reduced GFR. Patients at this stage exhibit hypertension (Wolf, 2004).
2.5. Stage 5
It is the stage of ESRD with the development of fibrosis/sclerosis (Table 1). There is
progressive decline GFR continues and reaches to <10 mL/min. Renal replacement therapy like
hemodialysis, peritoneal dialysis or even kidney transplantation is highly required (Sharma and
Sharma, 2013, Lin et al., 2018).
Table 1. Different Stages of diabetic nephropathy
Stage of Diabetic Nephropathy Structural and Functional Renal Manifestations

Glomerular hyperfiltration Renal hyperfiltration indicated by increased GFR (GFR↑),
renal hypertrophy
Silent stage or Renal hyperfiltration (GFR↑), normal UAER
normoalbuminuria Early histologic changes: Non-specific, thickening of GBM,
increased mesangial matrix
Microalbuminuria Incipient nephropathy (UAER 30-300mg/24 h), GFR may
increase or decrease to normal
Histological alterations: Mesangial expansion, thickening of
GBM, arteriolar hyalinosis
Established or overt nephropathy Proteinuria, nephrotic syndrome, decreased GFR,
hypertension
Histological appearance: Interstitial fibrosis
End-stage renal disease ESRD, GFR declined to <10 mL/min, renal replacement
therapy, kidney transplantation
3. MOLECULAR MECHANISMS OF DIABETIC NEPHROPATHY

Several studies showed the involvement of many pathways in the development of DN
including hyperglycemia, oxidative stress, advanced glycation end products (AGEs), polyol
pathway hexosamine pathway, protein kinase C (PKC) activation, growth factors, and
cytokines (Figure 2).
Hyperglycemia is the key player in the development of DN. Recent clinical studies have
shown that persistent hyperglycemia in DM can induce oxidative stress by a number of
mechanisms that involve, glucose auto-oxidation, protein glycation through nonenzymatic

means, polyol pathway activation and acceleration, and reduced antioxidant defense system
(Schena and Gesualdo, 2005). Hyperglycemia may act through PKC activation, acceleration of
the polyol pathway, production of reactive oxygen species (ROS) and over-expression of
transforming growth factor-β (TGF- β). In addition to these factors, most of the recent studies
have reported roles of the immune system, inflammation and related processes in the
pathogenesis of DN (Navarro-González et al., 2011; Navarro-González and Mora-Fernández,
2008). There is significant involvement of cytokines like IL-18 and tumor necrotic factor-
α(TNF-α) in the pathogenesis of DN (Castro et al., 2014; Mahmoud et al.,2004). It is also found
that overexpression of cell adhesion molecules and chemokines inducing leukocyte infiltration
are recognized in DN (Usui et al., 2003). Taken together, immune system and inflammatory
processes may cause structural and functional changes to kidney leading to DN.
Figure 2. Contributors to the development of diabetic nephropathy. ROS, Reactive oxygen species;
PKC, Protein kinase C; AGEs, Advanced glycation end products.
3.1. Polyol Pathway
The polyol pathway is also known as sorbitol pathway that involves the two enzymes
namely aldose reductase (AR) and, sorbitol dehydrogenase (SDH). AR is responsible for the
formation of sorbitol from glucose by means of reduction in the presence of cofactor
nicotinamide adenine dinucleotide phosphate (NADPH) (Brownlee, 2005). Sorbitol thus
formed is converted to fructose by SDH with its co-factor, a reduced form of nicotinamide
adenine dinucleotide (NAD+). This leads to increased NADPH/NAD+ ratio considered an
important contributor to oxidative stress and PKC activation (Ramana, 2011). This is due to the
reason that the recycling of the powerful antioxidant glutathione reductase that reduces
oxidized glutathione into reduced glutathione depends on NADPH supplies. Utilization of
NADPH by AR results in the depletion in its concentration. In this way, an increase in the flow
of metabolites through the sorbitol pathway may shift the balance of oxidants/ antioxidants to
the oxidative side (Bernobich et al., 2004).

3.2. Advanced Glycation End Products Pathway
Advanced glycation end products (AGEs) are complex and heterogeneous compounds
having a significant role in DM-induced manifestations like nephropathy (Singh et al., 2001).
In this pathway, nonenzymatic interaction of glucose with amino groups in proteins (Maillard
reaction), nucleic acids and lipids in a takes place in a sequential manner results in the formation
of Schiff bases and Amadori products. In the subsequent stage Amadori products are degraded
through autocatalysis, dehydration, oxidation, cleavage or rearrangements to form a number of
secondary products, such as aldehydes and dicarbonyls those in the later stage undergo complex
polymerization reactions leading to the production of AGEs (Singh et al., 2001). Some AGEs
have been identified structurally e.g., carboxymethyl lysine (CML), pentosidine and pyralline
(Wolf, 2004; Ziyadeh et al., 1997). As these processes occur for a longer duration, long-lived
proteins get affected causing structural damage to tissue matrix especially GBM. One of the
examples of such damage is inhibition of the interactions required for collagen IV and laminin
due to glycation which further leads to oxidation of proteins undergone glycation. This process
of oxidation of glycated proteins is known as glycoxidation which is found to be increased
during oxidative stress (Charonis et al., 1992).
3.3. Protein Kinase C Pathway
Protein kinase C (PKC) is an intracellular serine/threonine kinase playing multiple roles in

signal transduction and internalization mechanisms. It is the family consisting of eleven
isoforms out of which nine PKCs are activated by diacylglycerol (DAG), a lipid-derived the
second messenger. The intracellular level of DAG gets increased in response to hyperglycemia
that in turn activates PKC (Koya and King, 1998). A preferential increase in PKC-β isoform
has been shown in experimentally induced diabetes (Ishii et al., 1996).
Activated PKC regulates vascular functions by modulating enzymes including intracellular
phospholipase A2(PLA2) and Na+/K+ATPase in addition to the control of gene expression of
extracellular matrix components and contractile proteins (Meier and King, 2000). Poor
regulation of PKC action may alter renal blood flow resulting from reduced nitric oxide (NO)
production, mesangial expansion, albuminuria, and increased GFR and vascular permeability.
It is also shown to cause increased synthesis of cytokines with increased production of
extracellular matrix due to increased cell proliferation and angiogenesis in vascular cells. All
these events may lead to vascular anomalies (Song et al., 2010; Gugliucci, 2000).
3.4. Hexosamine Pathway
This is an additional pathway involved in the progression and development of DN. During
hyperglycemia, excess glucose is moved into the hexosamine pathway. In the initial step of this
pathway, fructose-6-phosphate is converted to glucosamine-6-phosphate in the presence of
enzyme hexokinase. The rate-limiting enzyme involved in this of this pathway is glutamine:
fructose-6-phosphate aminotransferase (GFAT) which is activated by hyperglycemia and
circulating angiotensin II (Ang II) via activation of its promoter in mesangial cells (Singh et
al., 2008). Overexpression of GFAT in mesangial cells leads to increased expression of both

TGF- β and fibronectin (Zheng et al., 2008). Overactivation of the hexosamine pathway may
cause PKC activation, increased TGF-β expression and extracellular matrix production, all
these events are linked to the progression of DN (Hao et al., 2012).
The normal kidney due to its high metabolic activity is capable of generating considerable
oxidative stress that is balanced by an extensive antioxidant system. Accumulating research
showed that a significant contributor to diabetic complications is persistent hyperglycemia that
shifts this balance to a pro-oxidant state leading to tissue damage and vascular injury (Vasavada
and Agarwal, 2005). It is shown that almost all pathways contributing to the DN induce
oxidative stress by one or other mechanism.
Oxidative stress, a deleterious phenomenon defined as the increased production and/or
inadequate elimination of highly reactive molecules like reactive oxygen species (ROS) and
reactive nitrogen species (RNS) (Turko et al., 2001). ROS include free radicals such as
superoxide (O2•-), hydroxyl (OH•), peroxyl (RO2•), hydroperoxyl (HRO2•-), etc. It is also
capable of generating nonradical species including hydrogen peroxide (H2O2) and hydrochloric
acid (HOCl). RNS include free radicals like nitric oxide (NO•) and nitrogen dioxide (NO2•-),
along with nonradicals such as peroxynitrite (ONOO-), nitrous oxide (HNO2) and alkyl
peroxynitrates (RONOO). Among these reactive molecules, superoxide, nitric oxide, and
peroxynitrite are considered as an important player in the development the DM-induced
complications (Ceriello and Motz, 2001; Turko et al., 2001; Maritim et al., 2003; Evans et al.,
2002). These are electron deficient molecules which renders them highly reactive and are
capable of an oxidizing variety of cellular components including macromolecules resulting in
damage to cell organelles (Rashid and Sil, 2015; Manna et al., 2013).
In addition to this, several other pathways and mechanisms can if become faulty,
potentially induce oxidative stress in DM. These include mitochondrial dysfunction,
mitochondrial uncoupling, signal transduction and amplification through cytokine and growth
factor, the glutathione pathway, the xanthine oxidase pathway, nitric oxide synthase (NOS)
uncoupling, alteration in antioxidant and catalytic activity of iron and sequestration of nitric
oxide to peroxynitrite (Hakim and Pflueger, 2010; Forbes et al., 2008).
Therefore, at the molecular level, alterations in proteins by hyperglycemia like Amadori
products and AGEs are considered as important factors in the pathogenesis of DN. Recent
studies showed that persistent hyperglycemia induces ROS formation which is regarded as an
early event in the development of diabetic complications (Taylor, 2004).
4. OXIDATIVE STRESS AND KIDNEYS

4.1. Oxidative Balance in the Normal Kidney
Normal oxygen metabolism produces oxidant species that take part in many crucial
processes including cell signaling, aging, and degenerative disease. During normal
circumstances there exists a balance between oxidant species and antioxidants. Oxidant species
are considered as signaling molecules involved in normal physiological processes whereas

antioxidants prevent potential damaging effects caused by excess oxidant species accumulating
in tissues (Brownlee, 2001). Therefore disturbed equilibrium between the oxidant species and
antioxidant arising from situations where there is increased oxidant formation or weakened
antioxidant defense induces oxidative stress leading to subsequent pathological alterations and
tissue injury (Figure 3) (Brownlee, 2001; Remacle et al., 1995).
Figure 3. Role of free radicals and antioxidants in diabetic nephropathy.
As stated earlier, normal kidney produces a significant amount of ROS due to the addition
of electrons to oxygen in presence of NADPH oxidase. The ROS generated after the addition
of an electron include superoxide anion (O2●-) and further addition of electron generates
hydrogen peroxide (H2O2), along with formation of highly reactive hydroxyl ion (OH●-) due to
Haber-Weiss reaction a kind of redox reactions converting partial oxygen metabolites i.e.,
superoxide anion and hydrogen peroxide in the presence of iron. Also peroxynitrite, a potent
oxidizing agent is produced from the interaction between the superoxide anion and NO. This
peroxynitrite is involved in lipid and protein oxidation (Besarab et al., 1999; Onozato et al.,
2002; Leeuwenburgh et al., 1997).
Renox is a recently identified renal NADPH oxidase found to be highly expressed in
proximal convoluted tubule (PCT) epithelial cells in the renal cortex. Renox primarily involved

in the generation of ROS of the renal origin. It is also evident that highly reactive hypochlorous
acid is produced from H2O2 in the presence of myeloperoxidase (MPO) present in neutrophils
(Vissers and Winterbourn, 1986). Hypochlorous acid thus formed is thought to be involved in
the carbonylation of proteins known as carbonyl stress (Geiszt et al., 2000; Vasavada and
Agarwal, 2005).
4.2. Sources of Oxidative Stress in Kidneys
As discussed, the different key mechanisms which play an important role in

hyperglycemia-induced renovascular damage in both the forms of DM by means of diverse
mechanisms (Figure 2) include:
 Potentiation of polyol pathway

 Stimulation of AGE production
 PKC pathway activation
 Increased hexosamine pathway flux
 Activation inflammatory mediators like cytokines and growth factors like IL-18,
TNF- α, nuclear factor-κB(NF- κB), stimulation of Ang II synthesis
All of these pathways could reflect a common origin of overproduction of ROS induced by
hyperglycemia (Brownlee, 2001). Early structural and functional renal damage may arise due
to high glucose mediated oxidative stress. This vulnerability of kidney to oxidative stress is as
a result of a variety of factors that contribute either directly or indirectly in this event. Glucose
uptake mechanism in the mesangial cells and tubular cells do not require insulin. Therefore
these cells are unable to control glucose movement across them. Chronic hyperglycemia thus
stimulates the production of AGEs, potentiates the polyol pathway and activation of PKC, all
this ends as increased ROS turnover and oxidative stress in kidneys.
4.2.1. NADPH Oxidase Pathway

It is considered as the most important contributor to ROS overproduction in DM patients.
NADPH oxidase, a cytosolic enzyme is involved in the formation of highly reactive ROS like
O2●-and H2O2 by transferring the electron from NADPH to molecular oxygen (O2). It is actually
an enzyme complex, constituting several isoforms, known as the NOX family (Gorin et al.,
2005). NOX is said to be involved in carrying several important physiological functions
associated with organs like intestines, kidney, thyroid, testis and lymphoid tissues. The NADPH
oxidase is particularly associated with the phagocytic cells like it is found in the lysosomal
membrane of neutrophils. Apart from this, NADPH oxidase is located in the plasma membrane
of various kidney cells including mesangial and proximal tubular cells, vascular smooth muscle
cells, endothelial cells and fibroblasts (Thomas et al., 2008, Li and Shah, 2003).
In a healthy kidney, NADPH oxidase serves as a signaling molecule. However, under
influence of Ang II or TGF-β, increased PKC activates NADPH oxidase, leading to
overproduction of ROS, imposing harmful effects on renal cells. In experimentally induced of
diabetes, it is observed that NADPH oxidase is potentiated by hyperglycemia and elevated
circulating levels of free fatty acids. Ang II is the potent inducer of NADPH oxidase and thereby

involved in ROS formation in early DN (Thomas et al., 2008, Li and Shah, 2003). Among the
different isoforms of the NADPH oxidase complex, NOX4 is a 578-amino acid residue
containing protein responsible for the generation of oxidative stress in the renal environment.
NOX4is thought to be chiefly involved in hyperglycemia-induced oxidative stress through by
means of potentiating ROS production that further stimulates protein kinase B (also known as
Akt) and extracellular signal-regulated kinases 1 and 2 (ERK 1 and 2) (Zhu et al., 2008). Also,
during chronic hyperglycemia profibrotic pathways get potentiated by activation of Ang II
expression by NOX4 that leads to renal hypertrophy and fibronectin expression in early DN
(Block et al., 2009, Geiszt and Leto, 2005, Gorin et al., 2005).
NOX4 is an extensively studied isoform of NADPH oxidase especially for its role in
oxidative stress-induced DN. Also, it is reported that NOX4 inhibition is responsible for
reduced oxidative stress and prevention of renal tissue injury in experimentally induced DN
(Gorin et al., 2005).
4.2.2. Oxidative Stress in the Glomerulus

Oxidative stress-induced injury involves almost all the layers of the glomerulus that
particularly begins with functional changes associated with the interaction between glomerular
endothelial cells with their glycocalyx layer and podocytes. This is followed by deposition of
extracellular matrix in addition to increased expression/secretion of collagen IV (Gnudi et al.,
1979; Abrahamson et al., 2009). Another way by which ROS induce abnormality in glomerular
capillaries is through interaction with heparan sulfate, component glycosaminoglycans (GAGs)
that play a vital role in glomerular filtration membrane. heparan sulfate is a major target for
oxidant species especially excess H2O2 favors detachment of heparan sulfate from GAGs
and/orGAGs degradation, leading to increased glomerular permeability to macromolecules
(Jeansson and Haraldsson, 2006; Singh et al., 2007).
4.2.3. Oxidative Stress in Tubules

Increased oxidative stress is the main culprit in the early tubular injury as it initiates the
processes known to be involved in chronic renal scarring. It starts with apoptosis mediated via
multiple caspases, like caspase 3, 8 and 9. In addition, TGF-β, which is activated due to
enhanced oxidative stress in combination with increased Ang II levels, is a primary regulator
in the remodeling of extracellular matrix especially of the mesangium and tubulointerstitium
(Allen et al., 2003; Baricos et al., 1999; Lee et al., 2003).
Also, relative hypoxia of tubulointerstitial cells of the juxtamedullary region and outer
renal medulla due to limited blood supply may be worsened in the hyperglycemic state as
chronic hyperglycemia may alter the renal blood flow, causing decreased oxygen delivery and
diminished oxygen tension in this region (Palm, 2006).
In the renal medulla, during hypoxia, NO concentration is generally maintained at a high
level for controlling mitochondrial respiration. Cellular adaptations to hypoxic conditions are
governed through hypoxia-inducible factor (HIF-1α) which mediates all its effect by enhancing
vasculogenesis, betterment in oxygen availability, cellular metabolism modulation and
protection against fibrotic processes in the kidney through fibrosis-promoting genes (Palm et
al., 2008; Higgins et al., 2004).
Hyperglycemia is found to impair the stabilization of HIF-1α, thus there is a reduction in
its availability which may lead to interstitial fibrosis. HIF-1α is said to be chiefly modulated by
ROS. Also, there is involvement of ROS in the degradation of HIF-1α especially during

hypoxia (Catrina et al., 2004; Callapina et al., 2005). Sustained stimulation of HIF-1α by
enhanced ROS generation in the context of reduced NO availability may adversely affect HIF-
1α stability, leading to tubulointerstitial fibrosis (Gupte and Wolin, 2008).
Taken together, oxidative stress is a detrimental phenomenon for kidneys especially in
uncontrolled hyperglycemic conditions within the body that causes the imbalance of cellular
oxidation/reduction levels leading to the generation of free radicals. It is, therefore, necessary
to scavenge these free radicals otherwise they can lead to tissue damage leading to organ
pathophysiology.
5. AMELIORATION OF DIABETIC NEPHROPATHY

BY ANTIOXIDANT THERAPY
During normal physiological conditions, the equilibrium between promoters and inhibitors
of oxidant injury is always maintained as a result of some natural antioxidants involved in
scavenging the ROS and inhibit them to reach a significant level. These natural antioxidants
carry out this task by promoting several distinct intrinsic renoprotective and cytoprotective
factors by both enzymatically and nonenzymatically governed mechanisms (Fujita et al., 2009;
Samuni et al., 2001; Abraham and Kappas, 2005). During oxidative stress, the antioxidant
defense gets reduced due to fall in cellular antioxidant levels in addition to diminished activity
and expression of antioxidant enzymes. Some of the major antioxidant enzymes are like
superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione
reductase (GR), myeloperoxidase (MPO), heme oxygenase, biliverdin reductase etc. to
scavenge free radicals and their bad effects(Fujita et al., 2009; Samuni et al., 2001; Abraham
and Kappas, 2005).
SOD is the primary antioxidant enzyme providing physiological defense against oxidative
stress by converting superoxide to hydrogen peroxide. The H2O2 thus formed is degraded by
CAT and GPx. Glutathione, in fact, is used by GPx to form water and lipid peroxides for
degradation of H2O2 (Fujita et al., 2009; Samuni et al., 2001).
CAT has gained increased attention after the finding concerned with its overexpression in
the PCT of transgenic diabetic mice. In this study CAT attenuated interstitial fibrosis and
tubular apoptosis. This action of CAT was due to increased degradation of H2O2; resulting in
reduced oxidative stress thereby reducing the stimulation of Ang II-mediated activation of
TGF-β (Brezniceanu et al., 2008). Heme oxygenase is another antioxidative enzyme
responsible for the conversion of a pro-oxidant heme into iron, carbon monoxide (CO) and
biliverdin. Bilirubin, a potent antioxidant is formed in this reaction by transformation of
biliverdin in presence of biliverdin reductase (Abraham and Kappas, 2005).
Apart from the antioxidant enzymes, several nonenzymatic antioxidant compounds are also
proved beneficial in DN. These antioxidants compounds are capable of combating the oxidation
of a variety of intracellular molecules thereby preventing the cells from the deleterious effects
of ROS and RNS (Kashiba et al., 2002). There are several distinct mechanisms by which
antioxidants exert their good biochemical effect at multiple sites. As stated earlier,
hyperglycemia during DM plays a pivotal role in the generation of free radicals as it often
interferes with the intracellular ascorbate metabolism resulting in increased vulnerability to
oxidative injury. It is reported by several researches on experimental DM, that the concentration

of different antioxidants got reduced, both transcriptionally and post-transcriptionally, in renal

tissues (Kashiba et al., 2002; Ceriello et al., 2000; Kim et al., 2014).
Also, the ROS overproduction can be inhibited by providing antioxidants exogenously. It
is now a day’s considered as an effective strategy in curbing DN. It has been shown by various
independent research groups that there betterment the renal physiology in DN after antioxidants
treatment that acts directly against oxidative tissue damage (Agrawal et al., 2015). Moreover,
antioxidants administered exogenously are capable of providing protection against the
progression of DN. Antioxidant does this by blocking the formation of excessive ROS
effectively and scavenging the preformed intracellular ROS (Agrawal et al., 2015). Various
nonenzymatic antioxidants that can be used effectively in DN are shown in Table 2.
Table 2. Exogenous antioxidants used in diabetic nephropathy.
Class Examples
Vitamins Vitamin A, C, and E
Vitamin like substances Carotenoids, α-lipoic acid, coenzyme Q10 (CoQ10)
Trace elements Copper, selenium, zinc
Cofactors Uric acid, folic acid, albumin
The preclinical studies explored the effectiveness of whole herbs, plants or seeds and their
active ingredients in well-established animal models of DN. They ameliorate oxidative stress
induced kidney damage, enhance the antioxidant system, and decrease the inflammatory
process and fibrosis. Along with the ability to scavenge ROS, antioxidants are also responsible
for the modulation of gene expression required to improve the oxidative stress induced DN
(Malireddy et al., 2012). Several antioxidants like dietary phytochemicals exhibit
immunomodulatory effect thereby protecting pancreatic β-cells from the destruction which is
achieved through enhancing immunotolerance and suppression of autoimmunity (Gao et al.,
2014; Sun et al.,2014; Malireddy et al., 2012).
Many antioxidants are shown to attenuate oxidative stress induced renovascular
complications during DN. Numerous Studies are in fact conducted to fathom the significance
of herbs and plants with their active constituents throughout the globe. Many of these
antioxidants obtained from the natural or synthetic source were tested either preclinically in
animal models of DN or proved beneficial clinically in human studies. Some of these
antioxidants with their beneficial effects on DN and possible mechanism of action are
summarized in Table 3.
Among these antioxidants, most of the exogenous antioxidants have shown advantageous
effects in amelioration of kidney dysfunctions in DN. The active phytoconstituents responsible
for the activities of plant products have been identified in some and yet to be identified in others.
Numerous antioxidants showed their ability to prevent kidneys from bad effects of
hyperglycemia by reducing blood glucose while the others exhibited direct actions on renal
functions (Al-Waili et al., 2017). The following sections will describe these antioxidants in
detail.

5.1. Therapeutic Plants and Phytoconstituents
5.1.1. Zingiber officinale (Ginger)

Rhizomes of ginger used medicinally from ancient time for its anti-inflammatory,
anticancer, anticlotting, antihyperglycemic, diuretics, and analgesic activities. Ginger possesses
an antioxidant activity due to the presence of about 50 different antioxidants (Ojewole et al.,
2006; Masuda et al., 2004). It is found to be involved in the reduction of blood sugar levels
(BSL) in streptozotocin (STZ) induced DM in rats. Ginger when given with honey shown to
decrease SOD and CAT activities and lowers the malondialdehyde (MDA) level and increases
GSH level and GSH/GSSG ratio back to normal in diabetic rats. It is also found to increase
insulin release and sensitivity. Also in one study, ginger extract injected intraperitoneally
decreased BSL and urine protein in addition to the protection against progression of structural
nephropathy in diabetic rats (Akhani et al., 2004; Morakinyo et al., 2011; Sani et al., 2014).
Constituents isolated from ginger including gingerdione, shogaol, and hexahydrocurcumin
are showed to be beneficial in attenuating oxidative stress. Another pungent constituent from
ginger, zerumbone, obtained from Zingiber zerumbet (Zingiberaceae family), causes decreased
expression of intercellular adhesion molecules, TGF-β1, and fibronectin in the DN and
improved histological architecture in the diabetic kidney (Al-Qattan et al., 2008; Tzeng et al.,
2013).
Gingerol, the major pungent component in ginger at a dose of 100 mg/kg, significantly
decreased BSL in db/db type 2 diabetic mice and exhibited a protective effect on pancreatic β-
cells. Ginger and its derivatives might decrease inflammatory processes in DN as well as
increase antioxidants by affecting NF-κB activation (Takada et al., 2005; Singh et al., 2009).
5.1.2. Terminalia arjuna (Arjuna)

It is well recognized and extensively studied ayurvedic medicinal plants used in the
management of a variety of physiological ailments. The whole plant constitutes various
biologically active components including tannins, triterpenoid saponins, flavonoids, ellagic
acid, gallic acid, proanthocyanidins, phytosterols (Ghosh and Sil, 2013).
The bark of Terminalia arjuna contains arjunolic acid which is a pentacyclic triterpenoid
saponin. Arjunolic acid is well known free radical scavenger that possesses potent antioxidant
activity due to its abilities to donate hydrogen, to form chelates with the metal ion and the
resonance stabilization of carboxyl radical (Ghosh and Sil, 2013). Arjunolic acid is proved
efficacious in ameliorating STZ induced DM and underlying renal histological injury by
reducing both oxidative stress and nitrosative stress may be due to suppressing the polyol
pathway. It is also shown to restore the antioxidant defense by the enhancement in the activities
of antioxidant enzyme and the increment in the redox ratio of reduced glutathione to oxidized
glutathione (GSH/GSSG). These findings suggest the usefulness of arjunolic acid in
suppressing hyperglycemia-induced oxidative stress thereby preventing the initiation of DN
(Ghosh et al., 2010; Manna et al., 2007).
5.1.3. Curcuma longa (Turmeric)

Curcuma longa of family Zingiberaceae is generally known as turmeric. It is a famous
spice used in cooking and traditional medicine in India and other parts of the world. The main
bioactive constituent of turmeric is curcumin, a polyphenol (diferuloylmethane). It was found

that curcumin has many actions most of which attributed to its antioxidant property.
Curcuminoids have an antioxidant effect as tested and proved in several experimental models,
such as prevention of lipid peroxidation (Meng et al., 2013; Ak and Gülçin, 2008).
Curcumin scavenges superoxide anion, hydroxyl radical and H2O2 as well as peroxynitrite,
and peroxyl radical. It can induce the expression of SOD, CAT, GPx, etc (Ak T and Gülçin,
2008).
Curcumin is extensively studied for its renoprotective effect using several experimental
models including DN, CKD, ischemia, and reperfusion-induced renal damage, and induction
of nephrotoxicity using various chemicals. It is postulated that curcumin provides
renoprotection through inhibition of mitochondrial dysfunction, induction of Nrf2, attenuation
of the inflammatory response, restoration of antioxidant enzymes, and prevention of oxidative
stress. Another study has suggested that curcumin treatment ameliorates DN via inhibition of
inflammatory gene expression by reversing caveolin-1 Tyr14 phosphorylation as it results in
prevention of epithelial-mesenchymal transition of podocytes, proteinuria, and kidney injury
(Trujillo et al., 2013; Sun et al., 2014).
Curcumin has also demonstrated a reduction in AGE-induced oxidative stress and restores
AGE-induced mesangial cell apoptosis. Curcumin significantly decreased albuminuria in db/db
mice with DN, and attenuated glomerular sclerosis (Liu et al., 2012). Clinically, at the dose of
500 mg/day curcumin significantly attenuated microalbuminuria, reduced plasma MDA
concentration with enhanced the Nrf2 together with betterment antioxidative defense in
diabetics. Curcumin treatment results in increased IκB which is an inhibitory protein produced
during inflammation by lymphocytes of. In another study, curcumin significantly lowered
mesangial area, proteinuria, blood urea nitrogen (BUN), and serum creatinine (SCr) and
provided the protection in DKD in experimental animals (Yang et al., 2015; Wu et al., 2014).
5.1.4. Trigonella foenum-graecum (Fenugreek)

Fenugreek is a non-poisonous edible item with medicinal value especially its seeds, which
are used for the treatment of diabetes. Fenugreek seeds have proved to possess hypoglycemic,
hypocholesterolemic and hyperinsulinemic effects both clinically in the forms of DM and in
experimental diabetic animals (Haeri et al., 2012).
Diabetic rats treated for 12 weeks with powder of fenugreek seed has significantly
enhanced CAT and SOD activity with increased the concentration of GSH to reverse oxidative
damage. Levels MDA and lipid peroxides in the kidney homogenates are reduced upon
fenugreek supplementation to diabetic animals by virtue of its antioxidant potential (Xue et al.,
2011; Sayed et al., 2012). Antioxidant nature of fenugreek is responsible for restoring
antioxidant enzymes and reduced AGE production. This reduces the inflammatory response
and improves antioxidant enzymes and kidney protection from functional and histopathological
normalities of DM (Liu et al., 2012). As fenugreek lacks the toxicity, it may be included in the
diet of the patients with DN and may also be regarded as an active oral antidiabetic of the future.
5.1.5. Camellia sinensis (Green Tea)

Green tea is well established antioxidant and anti-inflammatory agent. It contains strong
antioxidative flavonoids. Many studies suggested that green tea polyphenols are advantageous
in oxidative stress of renal tissues. It protects kidney against gentamycin, cyclosporine, and
intravenous contrast media-induced nephropathy in experimental animals (Rehman et al.,
2013). Clinically, a polyphenol from green tea found effective to overcome albuminuria in

diabetic individuals. Green tea treatment significantly reduces BSL, glycosylated protein
(HbA1c), SCr, and BUN in diabetic rats. Another study showed that polyphenols enhanced
antioxidant enzymes like SOD in the nephrectomized rat kidney subjected to STZ induced
diabetes (Renno et al., 2008; Yokozawa et al., 2005; Kang et al., 2012).
Catechins in the green tea extract possess significant anti-inflammatory and antioxidant
properties. They inhibit chelate transition metals, leukocyte chemotaxis, scavenge free radicals,
and inhibit lipid peroxidation. It has been demonstrated that rats with STZ induced diabetes
treated with high doses of catechins for 4 weeks, attenuates proteinuria, hyperfiltration,
glomerulosclerosis, and tubulointerstitial fibrosis with suppression of 8-hydroxy-2′-
deoxyguanosine, lipid peroxides, and TGF-β1 (Chennasamudram et al., 2012). Renoprotective
properties of catechin have been shown by another research. The results are comparable with
renin-angiotensin inhibition and suggested that co-administration catechin with enalapril might
be useful in improving endothelial function and reducing UAE (Chennasamudram et al., 2012).
The most abundant and most active catechin polyphenol extracted from green tea is
epigallocatechin-3-gallate. It could restore renal dysfunction, and prevent unilateral ureteral
obstruction-induced oxidative stress and inflammatory responses in the obstructed kidney.
Furthermore, it could induce both NFκB and Nrf2 nuclear translocation in the unilateral ureteral
obstruction kidney and promote heme oxygenase-1 (HO-1) production (Wang et al., 2015; Ye
et al., 2015).
5.1.6. Psidium guajava (Guava)

Guava is an evergreen shrub which is widely cultivated in tropical and subtropical regions
around the globe for its fruits. The plant is not only used as food but gained importance in folk
medicine too due to its diverse pharmacologic activities (Barbalho et al., 2012). The fruit is the
chief source of vitamin C, fiber and phenolic compounds. Major phenolic constituents include
epicatechin and gallic acid which makes it superior natural antioxidants (Jiménez-Escrig et al.,
2001).
The extract obtained with leaves of guava and its fruit has conventionally been used for
anti-hyperglycemic action (Rai et al., 2009). The extract obtained with guava shows presence
of caffeic acid, myricetin, and quercetin, cinnamic, coumaric and ferulic acids. This fruit extract
attenuated the formation of ROS dose-dependently through reserved GSH content, retention of
GPx activity and decreased the formation of IL-6, TNF-α, and IL-1β in the kidney and
therefore, it is providing protective action against diabetes-induced kidney damage via its
antioxidative, anti-inflammatory, and anti-hyperglycemic properties.
Guava has renoprotective action as it is responsible to reduce ROS, MDA and lipid
peroxidation and restores antioxidant enzymes (Adesida et al., 2012).
Leaves contain total triterpenoids shown to decrease the level of BSL in diabetic rats,
increased insulin sensitivity index, and protect the kidney from injury in diabetic rats. It also
found to suppress the renal activity of aldose reductase (Kuang et al., 2012; Lin and Yin, 2012).
The levels of BUN and SCr were decreased and the pathological alteration in the kidney of
diabetic rats is attenuated by leaves extract of guava.
5.1.7. Aloe vera (Aloe)

It is one of the well-recognized plants in the management of DN. Aloe is originated from
ancient Egypt and enormously by Indians as a traditional medicine for diseases affecting the
skin, constipation, worm infestation, colic, and infections. In Chinese medicine, aloe is

considered as the remedy for fungal diseases. It consists of the high concentration of phenolic
compounds including glycosides and other chemical components responsible for its
pharmacological actions (Khan et al., 2010).
Aloe, when administered orally, stimulates β-cells of pancreas leading hypoglycemic effect
along with significant reduction in the lipid peroxides. Due to this effect, aloe is considered as
an efficient remedy in ameliorating DN. Aloe is involved in enhancing the degradation of
H2O2by CAT and GPx thereby reducing its toxic effects to a significant extent (Rajasekaran et
al., 2006). In experimental diabetes induced in rats with STZ, aloe is shown to normalize fasting
blood glucose (FBG) and insulin. Diabetic rats treated with aloe showed decreased levels of
triglycerides, cholesterol and free fatty acids in plasma, liver, and kidney (Ramachandraiahgari
et al., 2012). Taken together Aloe is the potential candidate for alleviating DN, by combating
oxidative injury and restoring antioxidant enzymes.
5.1.8. Cinnamomum zeylanicum (Cinnamon)

Cinnamon is used as a spice in almost all parts of the world. It possesses different
pharmacological activities like antimicrobial, antioxidant, antiulcerogenic, antidiabetic, and
analgesic (Lin et al., 2003; Ooi et al., 2006). Cinnamon is particularly advantageous in
individuals with T2DM (Khan et al., 2003). Its oil extract possesses a powerful antioxidant
property due to the occurrence of phenolic and polyphenolic substances which may be useful
in attenuating DN and other diabetic complications. It reduces BSL, the polyphenols from
cinnamon shown insulin-enhancing biological activity. Cinnamaldehyde, an important
constituent in the volatile oil from cinnamon possesses significant protection against alloxan-
induced renal damage. It was also found to induce Nrf2 transcriptional activity and upregulation
of levels of Nrf2, GSH levels, and HO-1 and gamma-glutamyl-cysteine synthetase (Anderson
et al., 2004; Mishra et al., 2010).
5.1.9. Rosa laevigata (Cherokee Rose)

It is Chinese medicinal plant containing total flavonols imparting the potent antioxidant,
hypolipidemic, hepatoprotective, and antithrombotic activities to the plant. It shows all these
effects through attenuating inflammation, suppression of apoptosis, and altering mitogen-
activated protein (MAP) kinase signaling. Studies carried on the plant showed significant
amelioration of renal dysfunction in diabetic rats (Zhang et al., 2013). The protection involves
increment in the SOD and total antioxidant capacity, with decreased MDA levels leading to
reduced ROS generation. It is also found to inhibit the expression of NFκB. The total flavonoids
are said to have a nephroprotective effect against ischemia/reperfusion injury by affecting
Sirt1/Nrf2/NFκB signaling cascade (Zhou et al., 2012; Zhao et al., 2016). Other useful plants
and their constituents are mentioned in Table 3 along with their utility and possible mechanism
in attenuating DN.
5.2. Vitamins and Their Analogues
5.2.1. Vitamin E
Vitamin E is a fat-soluble vitamin available as eight different forms, four tocopherols and four
tocotrienols. Tocopherols and tocotrienols possess significant physiological actions that involve
immunostimulatory activity along with their ability to inhibit DNA damage thereby preventing genetic changes

in the cells (Kuhad and Chopra, 2009). Generally, it is known for its scavenging ability of free radicals.
Vitamin E is also involved in the formation of reduced forms of vitamin A and C through the recycling process.
It is principally a chain-breaking antioxidant that inhibits lipid peroxidation. Previous studies have shown that
tocopherol has the ability to modulate cell signaling through inhibition of PKC (Thomas, 2016). Tocopherols
have been shown to carry out gene regulatory functions due to their ability to act as ligands for the peroxisome
proliferators-activated receptor-γ (PPAR-γ). Diabetic individuals showed reduced plasma concentrations of the
tocopherol and it is even at the lower level in diabetic complications including DN (Thomas, 2016).
Vitamin E and tocotrienol decrease SCr in diabetic animals. Clinically, oral administration
of vitamin E reduced HbA1C levels in T2DM. When given in combination with vitamin C,
vitamin E showed to improve renal parameters in patients with T2DM. Vitamin E in these
patients decreased microalbuminuria and showed the nephroprotective effect which is thought
to be mediated by its antioxidant potential and its ability to inhibit thromboxane A2 production
(Khan et al., 2011). Tocotrienol, a different form of vitamin E, reduced PCT injury and renal
lipid peroxidation with stimulation of CAT activity and increased GSH level. Vitamin E forms
like tocotrienol and α-tocopherol may be useful in patients with DN as it showed to inhibit the
activation of NFκB (Kuhad and Chopra, 2009; Haidara et al., 2009; Bursell et al., 1999;
Hirnerová et al., 2003).
5.2.2. Vitamin C
Vitamin C is a major player in the antioxidant defense and apoptosis. Decreased vitamin C
levels were noted in individuals with DN which is a key element in the generation of oxidative
stress. In diabetic rats, vitamin C showed protection against podocyte injury by its antioxidant
capacity (Lee et al., 2007; Qin et al., 2008). It decreases lipid peroxidation and enhances the
activities of antioxidant enzymes, SOD, CAT, and GPx. Vitamin C showed a reduction in
albuminuria and GBM thickness in the kidneys of diabetic rats. Vitamin C decreased BUN,
SCr, and UAER with an increased rate of Cr clearance in DN rats. Furthermore, the expressions
of collagen type IV were significantly downregulated in treatment groups. It was found that
vitamin C protects renal abnormalities during DN by inhibiting expression of collagen IV.
Combination therapy with vitamin C and E at therapeutic doses reduce albuminuria in T2DM
patients (Gaede et al., 2001). Metformin and vitamin C when given together shown to reduce
HbA1c levels in diabetic patients up to a significant extent in comparison to metformin alone
(Varma et al., 2014). It is also noted that administering vitamin C with antidiabetic drugs
enhances antioxidant defenses and reduces oxidative damage (Das et al., 2012).
5.2.3. Paricalcitol
Paricalcitol is an active, third generation vitamin D derivative showing similar biological
effects as that of vitamin D. The biological activity of this vitamin analog takes place through
t binding and activating vitamin D receptor leading to reduced albuminuria and retarded the
progression of kidney disease. Many studies on experimental DN have shown the improvement
of glomerular damage after paricalcitol administration. Moreover, it is evident that the
increased renin activity during DN leads to overproduction of Ang II leading to increased ROS
production and subsequent renal damage. Therefore, agents that inhibit the renin-angiotensin
system (RAS) are currently used for the treatment of DN. Vitamin D or its analogs like
paricalcitol are acting as negative regulators of RAS hence responsible for antioxidative effects
(Cheng et al., 2012; Ari et al., 2012).

5.2.4. Pyridoxamine
Pyridoxamine is one form of vitamin B6 has the ability to scavenge free radicals. It is
shown to inhibit AGEs production and has blocking effects on AGE pathway. In clinical
evaluation, the effects on SCr level were studied. It is showed that 52 weeks pyridoxamine
treatment did not affect SCr in patients with DN, but in the individuals with the normal renal
function, it is found to decrease average SCr concentration (Williams et al., 2007).
5.3. Vitamin-Like Substances
5.3.1. Alpha Lipoic Acid

Alpha lipoic acid (ALA) is octanoic acid derivative and a dithiol compound it is one of the
established antioxidants that dissolves in both aqueous and oily mediums. It is derived from both plants and
animals origins. ALA is metabolized by the cell which uptakes it. The principal metabolite of ALA is
dihydrolipoic acid (DHLA, Reduced ALA). Both ALA and DHLA are found to scavenge ROS and RNS with
the ability to restore vitamin C and E (Rosenberg and Culik, 1959). ALA and DHLA are also shown to
regenerate oxidized glutathione within the cells. Apart from these functions, ALA is serving as a cofactor for
a variety of mitochondrial enzymes that decrease lipid peroxidation thereby reducing oxidative stress. The
possible mechanism by which ALA acts as an antioxidant may be due to its capability to inhibit hexosamine
and AGEs pathways (Packer et al., 1995; Smith et al., 2004).
5.3.2. Carotenoids
These are oil-soluble phytoconstituents synthesized mainly by plants, where they are
serving as pigments and antioxidants protecting cellular components from harmful effects of
oxidative stress. Decreased serum level of carotenoids is shown to be associated with the
development of CVD and T2DM. Carotenoids are potent antioxidants and anti-inflammatory
agents found advantageous in diabetic microvascular complications like DN (Johnson, 2002).
They are also linked to several cellular signaling mechanisms and serving as modulators of
gene expression. Carotenoids can scavenge a variety of highly reactive species like ROS and
other free radicals. It is demonstrated that carotenoids or their analogs having the affinity for
cysteine residues of the IκB kinase or NF-κB subunits. This may lead to the inhibition of the
NF-κB induced inflammatory signaling cascade (Johnson, 2002; Rao et al., 2007; Ciccone et
al., 2013).
Lycopene is widely studied carotenoid for its effects against DN. Preclinically, lycopene
supplementation in mice for 8 weeks resulted in the reduction in BSL, proteinuria and renal
damage in a dose-dependent manner. Also, there is the augmentation of SOD and GPx leading
to restoration of antioxidant defense. Lycopene is responsible for fall in plasma MDA level,
therefore, reduces chances of lipid peroxidation. Lycopene intake also showed a decrease in
renal NF-κB and TNF-α expression. Reduced BUN, 24-h urinary protein, and creatinine are
also seen with lycopene treatment (Guo et al., 2015; Toyama et al., 2014).
5.3.3. N-Acetylcysteine
N-Acetylcysteine (NAC) is a cysteine and glutathione precursor in mammals that serve as
a powerful antioxidant. It also shows protective action on the β-cells. NAC administration
decreases hyperglycemia with the improvement in the glucose intolerance associated with the
glucose-induced insulin secretion (Haber et al., 2003). NAC is also found beneficial in

attenuating renal injury by protecting renal structure by decreasing the apoptosis due to ROS
thereby retaining renal functions as indicated by reduced urinary protein excretion. Treatment
with NAC resulted in attenuation and reduction in the levels urinary thiobarbituric acid reactive
substances (TBARS) which is playing a role in renal oxidative stress. Therefore, NAC is proved
advantageous in patients with DN as it acts through dual way by reducing both hyperglycemia
and renal oxidative stress (Haber et al., 2003; Ahmad et al., 2012; Shimizu et al., 2005).
5.3.4. Coenzyme Q10

Coenzyme Q10 (CoQ10), is an endogenous fat-soluble vitamin-like substance serving as a
micronutrient and natural antioxidant. CoQ10 is available in almost all living cells where it acts
as a principal player of the electron transport chain (ETC) of the mitochondria responsible for
the synthesis of ATP (Hodgson et al., 2002). Apart from the proved pharmacological actions of
CoQ10 like its antihypertensive action, it is a serving as a powerful antioxidant responsible for
its free radical scavenging action protecting the cells from oxidative stress (Hodgson et al., 2002;
Rosenfeldt et al., 2007).
Several researchers showed the relationship between reduced plasma levels of CoQ10 and
development of DKD. As CoQ10 is serving as an endogenous antioxidant, its deficiency is
thought to be involved in an imbalance of antioxidative defense system against oxidative stress
that in case of DM is induced by hyperglycemia and may lead to diabetic complications like
DN (Sourris et al., 2012; Ahmadvand et al., 2012).
Studies also showed the effectiveness of CoQ10 on glycemic control among individuals
with T2DM as its administration at a dose of 200 mg/d for 12 weeks reduced HbA1c levels.
CoQ10 is said to have a protective effect on β cells by virtue of its antioxidant potential
(Maheshwari et al., 2014). It also causes down-regulation of insulin receptors to improve
insulin action and provides protection to the kidneys in DN. CoQ10 significantly inhibited
leukocyte infiltration, glomerulosclerosis and decreased levels of MDA in serum and kidney
content (Ahmadvand et al., 2012).
CoQ10 is having the ability to overcome diabetes-induced decreases in antioxidant defense
mechanisms and this effect was potentiated by IGF-1 antagonism. Studies showed that CoQ10
increased serum levels of GSH, CAT, and SOD with decreased lipid peroxidation in alloxan-
induced diabetic animals. It was shown that coenzyme Q10 induces Nrf2 nuclear translocation,
augments the cellular antioxidant defense capacity (Li et al., 2016).
5.4. Trace Elements
5.4.1. Zinc
Zinc (Zn) is an essential trace element playing crucial physiological roles, in addition, to
serve as a cofactor for many enzymes and proteins. Zn is also an important component of
proteins participating in the oxidative defense system. In DM patients, deficiency of Zn is
common which arise because of increased urinary excretion and restricted food intake (Capdor
et al., 2013; Tang et al., 2010).
It is explored that supplementation with Zn has improved glycemic control and reduced
diabetes-induced renal pathological damage. Zn also thought to improve the effectiveness of
hypoglycemic agents and may be beneficial in decreasing BSL, triglyceride, and inflammation

in T2DMinduced nephropathy. Zn has a protective effect against diabetic damage through

stimulation of metallothionein synthesis, a potent antioxidant, and regulation of the oxidative
stress showed by a recent study (Özcelik et al., 2012; Khan et al., 2013). Zn upregulates Nrf2
function by stimulating Akt-mediated inhibition of Nrf2 nuclear exporter Fyn. Zn has
antioxidant properties that may be mediated by the upregulation of Nrf2 (Li et al., 2014).
5.5. Drugs
5.5.1. Metformin
Metformin, an oral hypoglycemic agent a biguanide, belongs to aminoguanidine class.
Metformin is the only drug from this class used in current clinical practice in the management
of NIDDM and preventing its complications, especially DN. The drug shows antioxidant
activity with reduced UAER in patients with DM. Metformin is known to reduce AGE
production thereby improving the antioxidant defense system. This is governed through the
ability of metformin to scavenge ROS (Hou et al., 2010). Renoprotective effect of metformin
is thought to be due to its ability to reduce ROS production in DM. The antioxidant nature of
metformin has been identified by a rise in the renal ATP to AMP in normal rats receiving
metformin for 8 weeks as compared to STZ induced diabetic rats. Metformin treatment also
resulted in decreased MDA level thereby reducing lipid peroxidation. Taken together,
metformin is capable of restoring the antioxidant defense proving itself as a good candidate for
the clinical use in the treatment of DN (Hou et al., 2010; Kim et al., 2012; Alhaider et al., 2011).
5.5.2. Pioglitazone
Pioglitazone is a glitazone, an antihyperglycemic drug of thiazolidinedione (TZD) group.
Significant effects of pioglitazone and other TZDs in the amelioration of antioxidant enzyme
levels in renal histopathology and renal tissue associated with DN has recently been
demonstrated by many studies (Chen et al., 2013). Pioglitazone does not alter BSL but it
prevents renal histopathological abnormalities associated with glomerulus and renal tubular
system. The drug is said to normalize bowman capsular volume and reduce endothelial
constitutive nitric oxide synthase (ecNOS) in the glomerular endothelium of rats. It also
improves the glomerular hyperfiltration (Kuru Karabas et al., 2013; Tanimoto et al., 2004).
Pioglitazone therapy reduces NF-κB expression of in renal tubules and glomeruli that is
increased during DN. This results in protection from pathophysiological alterations in renal
structures (Chen et al., 2013; Tawfik, 2012).
5.5.3. Telmisartan
Telmisartan is an Ang II type 1 receptor blocker (ARB) is proved advantageous in patients
with DN. In addition, to reduce elevated blood pressure due to overactivation of RAS in DN, it
exerts a powerful antioxidant effect. Clinically, it is considered and proved more effective in
renoprotection in comparison to other ARBs (Fujita et al., 2011). This effectiveness of
telmisartan is due to the difference in the pharmacokinetic and physiochemical properties. It
shows an excellent binding affinity for Ang II type1 receptors, the maximum plasma t1/2 and
the highest lipophilicity among all available ARBs. Therefore telmisartan by virtue of these
distinct features become a long-lasting antioxidant. The mechanisms by which it acts involve

enhancement of the antioxidant enzyme activity especially SOD and down-regulation NOX
responsible for superoxide production (Fujita et al., 2011; Kakuta et al., 2005). As Ang II is
shown to be involved in the synthesis of superoxide through the activation of NOX, Ang II
blockade by telmisartan ameliorates oxidative stress induced kidney damage eduring DN. It
telmisartan is also found to decrease albuminuria in patients with DN and retards the
progression of early form to the overt DN (Makino et al., 2007; Sugiyama et al., 2005).
5.5.4. Spironolactone
It is an aldosterone antagonist which is proved clinically and preclinically for its beneficial
effects in early stages of DN. The drug is shown to attenuate renal injury and with a reduction
in proteinuria therefore helpful in the management of DN. Spironolactone prevents diabetic
kidney from damage due to increased ROS production induced by NOX4 during DM. it
decreases NOX4 expression (Taira et al., 2008). As discussed earlier, hyperglycemia induces
glomerular TGF-β expression leading to ROS production. Treatment with spironolactone down
regulates TGF-β and therefore capable of inhibiting oxidative stress in kidneys of diabetic rats.
Due to such action spironolactone is said to possess antioxidant properties as it is also shown
to enhance antioxidative defense systems including GSH and thereby reduces oxidative stress.
Though the drug is much more effective in early DN, its utility in progressive DN is limited
(Yuan et al., 2007).
Table 3. Antioxidants in diabetic nephropathy
Active Effects on Diabetic Nephropathy References

Ingredients
/Plants/Herbs
Tempol  Suppression of albuminuria, TGF-β levels, Fujita et al., 2009
collagen synthesis, and oxidative stress
Ebselen  Amelioration of tubulointerstitial pathology, Chander et al.,2004
inflammation, and markers of nitrosative and Yamaguchi et al., 1998
oxidative stress in early DN
Ginger  Decreases BSL, increases insulin release decreases Akhani et al.,2004
(Gingerol, urinary protein reduces SOD and CAT activities, Morakinyo et al., 2011;
Gingerdione, lowers MDA level, restricts the progression of DN Sani et al., 2014;
Zerumbone) Takada et al., 2005;
Singh et al., 2009
Terminalia  Ameliorating hyperglycemia, reducing oxidative Ghosh and Sil, 2013;
arjuna and nitrosative stress, suppressing polyol pathway, Ghosh et al., 2010;
(Arjunolic acid) restore the antioxidant defense Manna et al., 2007
Turmeric  Induces expression of SOD, CAT, GR, GPx Meng et al., 2013; Ak T
(Curcumin)  Lowers mesangial area, proteinuria, BUN, and SCr and Gülçin, 2008;
 Prevents epithelial-mesenchymal transition of Trujillo et al., 2013;
podocytes and proteinuria Sun et al., 2014;
 Decreases albuminuria and attenuates glomerular Liu et al., 2012; Yang
sclerosis et al., 2015; Wu et al.,
2014

Table 3 (Continued)

Ingredients
/Plants/Herbs
Fenugreek seeds  Hypoglycemic, hypocholesterolemic and Liu et al., 2012; Xue et
hyperinsulinemic, attenuation of AGE al., 2011; Sayed et al.,
 reduction of NF-κB, decrease in the level of IL-6 2012
Green tea  Renoprotective potential Rehman et al., 2013;
(Polyphenols)  Reduced BSL, HBA1c, SCr, and BUN Renno et al., 2008;
 Attenuates proteinuria, hyperfiltration, Yokozawa et al., 2005;
glomerulosclerosis, and tubulointerstitial fibrosis Kang et al., 2012;
 Ameliorates experimentally induced kidney injury Chennasamudram et
and lupus nephritis al., 2012; Wang et al.,
2015; Ye et al., 2015
Guava  Decreases BGL and increases the insulin Jiménez-Escrig et al.,
(Total sensitivity index 2001;
triterpenoids)  Protects renal lesions Decreases BUN and SCr and Rai et al., 2009;
improves the renal structural damages Adesida et al., 2012;
Kuang et al., 2012; Lin
and Yin, 2012
Aloe  Stimulation of the pancreatic β-cells, reduction in Ramachandraiahgari et
lipid peroxide, CAT, GPx enhancement al., 2012; Rajasekaran
 Normalizing FBG and insulin et al., 2006
Cinnamon  Reduces BSL, insulin-enhancing effect, inhibits Lin et al., 2003; Khan
(Cinnamaldehyd AGE, induce Nrf2 transcriptional activity, GSH et al., 2003; Anderson
e) levels et al.,2004; Mishra et
al., 2010
Rosa laevigata  Ameliorates renal dysfunction cell injury Zhou et al., 2012; Zhao
 Decreases SCr and BUN levels et al., 2016
 Inhibits expression of NFκB, IκBα protein
 Upregulates the levels of Nrf2, Sirt1, and HO-1
 Downregulates the levels of KEAP1 and NFκB
Milk thistle  Reduces urinary excretion of albumin Khazim et al., 2013;
(Flavonolignans,  Lowers levels of BSL, HbA1c, urine volume, SCr, Sheela et al., 2013,
Silymarin) serum uric acid, and urine albumin Wang et al., 2012
 Increases SOD, GPx activity, and total antioxidant
capacity decrease MDA concentration in diabetic
patients
 Decreases the urinary albumin-creatinine ratio,
levels of TNF-α
Rosmarinus  Reduces focal glomerular necrosis, dilatation of Jiang et al., 2012;
officinalis Bowman’s capsule, degeneration of tubular Erkan et al., 2008;
(Rosmarinic epithelium
acid, Carnosic  Necrosis in tubular epithelium, and tubular
acid) dilatation
 Prevents ischemia/reperfusion injury in the kidneys
 Ameliorates glomerulosclerosis


Ingredients
/Plants/Herbs
Panax ginseng  Reduces the cisplatin-induced nephrotoxicity Baek et al., 2006; Kang
(Ginsenosides,  Ameliorates hyperglycemia and renal damage in et al., 2013
Panaxytriol) diabetic rats reduces proteinuria
 Augments creatinine clearance
Garlic  Reduces hyperglycemia Mariee et al., 2009;
(Allicin, S-allyl  Attenuates mesangial expansion Thomson et al., 2016;
cysteine, Diallyl  Glomerulosclerosis Maldonado et al., 2003
trisulfide)  Decreases urinary excretions of albumin
 Decreases BGL, BUN, and SCr
 Normalizes blood pressure and renal clearance
function
Ginkgo biloba  Reduces oxidative stress, and improves the Li et al., 2011; Tang et
structure and renal functions of renal tissue al., 2009; Lu et al.,
 Protects against glomerulosclerosis 2007
 Decreases BSL, SCr, BUN, urinary protein, and
oxidative stress
 Inhibits AGEs production
Flaxseed  Prevents heavy metals-induced nephrotoxicity and Velasquez et al., 2003;
(Omega-3 fatty oxidative stress damage Garman et al., 2009;
acid)  Increases the antioxidant defense systems An et al., 2009.
 Reduces proteinuria and glomerular and
tubulointerstitial lesions
 Protects against nephrotoxicity, and STZ-induced
type 1 and type 2 DN
Paricalcitol  Reduced albuminuria and retarded the progression Cheng et al., 2012; Ari
of kidney disease, suppresses RAS, antioxidative et al., 2012
effects
Pyridoxamine  Scavenge free radicals and carbonyl products, and Williams et al., 2007
block synthesis of AGEs
 Reduces SCr
Vitamin E  Ameliorates SCr Khan et al., 2011;
(Tocotrienol, α-  Reduces HbA1C levels Kuhad and
Tocopherol)  Decreases microalbuminuria Chopra,2009; Haidara
 Nephroprotective effect et al., 2009; Bursell et
 Reduces proximal tubular injury and renal LPO al., 1999
 Increases GSH level and CAT activity
Vitamin C  Protects podocyte by increasing antioxidative Lee et al., 2007; Qin et
capacity al., 2008; Gaede et al.,
 Reduced oxidative stress 2001
 Reduces albuminuria, and GBM thickness Das et al., 2012; Varma
 Decreases BUN, SCr, and UAER et al., 2014
 Reduces HbA1c levels in diabetic patients

Table 3 (Continued)

Ingredients
/Plants/Herbs
α-Lipoic acid  Free radical scavengers, regeneration of vitamins C Rosenberg and Culik,
and E and oxidized glutathione 1959; Packer et al.,
 Improved renal blood flow 1995; Smith et al., 2004
Carotenoids  Quenching ROS and other free radicals, inactivates Johnson, 2002; Rao et
(Lycopene) the NF-κB pathway al., 2007; Ciccone et
 Increases SOD, GPx activity, decreases MDA al., 2013; Guo et al.,
 Decreases BUN, SCr, and UAER 2015; Toyama et al.,
2014
N-  Protective effects in the β-cells, prevents Haber et al., 2003;
Acetylcysteine hyperglycemia, ameliorated renal injury, reduced Ahmad et al., 2012;
urinary protein urinary and TBARS Shimizu et al., 2005
Coenzyme Q10  Decreases HbA1c Maheshwari et al., 2014
 Attenuates diabetes-induced decreases in Sourris et al., 2012;
antioxidant defense mechanisms, increased serum Ahmadvand et al., 2012
levels of GSH, CAT, and SOD Li et al., 2016.
 Inhibits leukocyte infiltration, glomerulosclerosis,
and MDA
 Increases serum levels of GSH, CAT, and SOD
 Prevents altered mitochondrial function and
morphology, glomerular hyperfiltration, and
proteinuria
Zinc  Improves glycemic control in DM Capdor et al., 2013;
 Reduces renal pathological changes Tang et al., 2010;
 Decreases BSL, UAE, inflammation Özcelik et al., 2012;
Khan et al., 2013; Li et
al., 2014
Metformin  Reduced UAER, improving the antioxidant defense Hou et al., 2010; Kim
system, scavenge ROS, renoprotective effect et al., 2012; Alhaider et
increment in the ATP/AMP ratio decreases MDA al., 2011
Pioglitazone  Amelioration of antioxidant enzyme levels Chen et al., 2013;
prevents renal histopathological abnormalities, Tawfik, 2012; Kuru
glomerular focal necrosis tubular epithelial Karabas et al., 2013;
necrosis, normalize bowman capsular volume, Tanimoto et al., 2004
reduce ecNOS
Telmisartan  Ang II type 1 receptor antagonist, reduce elevated Fujita et al., 2011;
blood pressure, enhancement of the antioxidant Makino et al., 2007;
enzyme SOD, downregulation NOX, ameliorates Kakuta et al., 2005;
oxidative stress-induced renal injury, reduce Sugiyama et al., 2005
albuminuria, retards progression of early form to
the overt DN
Spironolactone  Aldosterone antagonist, attenuate renal injury Yuan et al., 2007; Taira
reduction in proteinuria et al., 2008; Ziaee et al.,
 Decreases NOX4 expression 2013; Pessoa et al.,
 Down regulates TGF-β expression, reduces 2012
oxidative stress


AND CHALLENGES
Both the forms of DM are widely studied organ-specific metabolic disorder. Most of the
studies on DM showed its association with oxidative stress and the generation of ROS and
RNS. However, with the advancement in the knowledge and emergence of a variety of
strategies for its prevention or cure, of DM, there is a gap the available theoretical
understanding and realistic approach in the treatment of DM and the complications arising due
to its presence. One of the different approaches to overcome DM and its complications is
through the use of a variety of antioxidant molecules which are found to be effective in
alleviating diabetic complications including nephropathy. These agents not only capable of
scavenging the free radicals activity, but some antioxidants are also shown of modulating
various signaling pathways and restoring the normal renal function. Also, T1DM, which is
associated with autoimmunity, is showed to generate oxidative stress through many different
mechanisms.
Nowadays, there is the availability of many antioxidants with an ability to overcome
autoimmune disease, inhibit inflammatory process and scavenge free radicals. These agents are
proved advantageous in diabetes-induced complications especially DN. Studies are being
undertaken on targeting different T-regulatory cells which can suppress proinflammatory
response via attenuation of respective signaling cascades. Experiments on different animal
models have demonstrated the utility of agents in treating DN induced by oxidative stress.
Clinical evaluations of a few of these agents demonstrated promising outcomes. The effects of
extracts obtained from therapeutic plants on diabetic individuals have shown to decrease ROS
production, BSL and most important an improvement in antioxidants status. Studies conducted
human volunteers showed the renoprotective effect of most of the above-mentioned drugs
possibly by decreasing AGE production, inhibition of PKC, inhibition of aldose reductase,
decreased expression of TGF-β, NFκB signaling inhibition, NOX downregulation and
upregulation Nrf2 function along with prevention of albuminuria. All these molecules are
promising but in order to improve their efficacy target, specific delivery is required which is
missed in the current scenario.
Therefore research is to be undertaken which will identify and focus a target-specific and
efficient delivery system for these antioxidant molecules which helps them to reach the targets,
especially within renal milieu with ease. This kind of approach will be beneficial for betterment
in the mode of action and enhancement of the efficacy of antioxidants in the preventive
treatment of renal complications. Moreover, understanding the relationship between
overexpression antioxidant genes and the development of strategies for their endogenous
administration may also serve as another approach in the treatment of DN.
CONCLUSION
This chapter primarily focused on understanding induction of oxidative stress following DM and
hyperglycemia by various signaling pathways leading to the formation of ROS thereby trigger a range of
downstream signaling cascade inducing structural and functional damage to the kidney. Therefore we have
pointed out the use of different antioxidant strategies and antioxidant agents in restoring the antioxidant defense
system to scavenge ROS mediated injuries to renal structures for prevention of DN. This chapter, therefore,

represented the antidiabetic, antihyperglycemic effects of different therapeutic plants, vitamins,

trace elements, and other drugs with their antioxidant potential. However, this treatment should
be combined with proper glycemic control and the correct use of medication. A detailed study,
in fact, is required to fathom the potential protective role of antioxidants for the effective
treatment of DN with the development of novel therapeutic approaches to serve mankind at
large.
REFERENCES
Abraham N. G, Kappas A. Heme oxygenase and the cardiovascular-renal system. Free Radic.
Biol. Med. 2005;39:1–25.
Abrahamson, D. R, Hudson, B. G, Stroganova, L, Borza, D. B, St John, P. B, Cellular origins
of type IV collagen networks in developing glomeruli, Journal of the American Society of
Nephrology: JASN. 2009; 20(7):1471-1479.
Adesida A, Farombi E. O. Free radical scavenging activities of guava extract in vitro. Afr J
Med Med Sci. 2012;41(Suppl):81–90.
Agrawal N, Sadhukhan P, Saha S, Sil P. C. Therapeutic Insights against Oxidative Stress
Induced Diabetic Nephropathy: A Review. J Autoimmun Disod. 2015, 1:1.
Ahmad A, Mondello S., Di Paola R et al. Protective effect of apocynin, a NADPH-oxidase
inhibitor, against contrast-induced nephropathy in the diabetic rats: a comparison with n-
acetylcysteine. Eur J Pharmacol. 2012; 674: 397–406.
Ahmadvand H., Tavafi M., Khosrowbeygi A. Amelioration of altered antioxidant enzymes
activity and glomerulosclerosis by coenzyme Q10 in alloxan-induced diabetic rats. J
Diabetes Complications. 2012;26:476–482.
Ak T, Gülçin I. Antioxidant and radical scavenging properties of curcumin. Chem Biol Int.
2008;174:27–37.
Akhani S. P, Vishwakarma S. L, Goyal R. K. Anti-diabetic activity of Zingiber officinale in
streptozotocin-induced type I diabetic rats. J Pharm Pharmacol. 2004;56(1):101–105.
Alhaider A. A, Korashy H. M, Sayed-Ahmed M. M, Mobark M, Kfoury H, et al. Metformin
attenuates streptozotocin-induced diabetic nephropathy in rats through modulation of
oxidative stress genes expression. Chem Biol Interact. 2011; 192: 233-242.
Allen D. A, Harwood S, Varagunam M, Raftery M. J, Yaqoob M. M. High glucoseinduced
oxidative stress causes apoptosis in proximal tubular epithelial cells and is mediated by
multiple caspases. FASEB J. 2003;17: 908–910.
Al-Qattan K, Thomson M, Ali M. Garlic (Allium sativum) and ginger (Zingiber officinale)
attenuate structural nephropathy progression in streptozotocin-induced diabetic rats.
ESPEN. 2008;3(2):e62–e71.
Al-Waili N, Al-Waili H, Al-Waili T, Salom K. Natural antioxidants in the treatment and
prevention of diabetic nephropathy; a potential approach that warrants clinical trials, Redox
Report, 2017;22:3, 99-118.
An W, Kim H, Cho K, et al. Omega-3 fatty acid supplementation attenuates oxidative stress,
inflammation, and tubulointerstitial fibrosis in the remnant kidney. Am J Physiol.
2009;297:F895–F903.

Anderson R, Broadhurst C, Polansky M, et al. Isolation and characterization of polyphenol

type-A polymers from cinnamon with insulin-like biological activity. J Agric Food Chem.
2004;52:65–70.
Ari E, Kedrah AE, Alahdab Y, Bulut G, Eren Z, et al. Antioxidant and renoprotective effects
of paricalcitol on experimental contrastinduced nephropathy model. Br J Radiol. 2012 85:
1038-1043.
Babazono, T, Drezgic J. K, Dlugosz J. A, Whiteside C. Altered Expression and Subcellular
Localization of Diacylglycerol-Sensitive Protein Kinase C Isoforms in Diabetic Rat
Glomerular Cells. Diabetes. 1998;47, 668-676.
Baek S. H, Piao X. L, Lee U. J, et al. Reduction of cisplatin-induced nephrotoxicity by
ginsenosides isolated from processed ginseng in cultured renal tubular cells. Biol Pharm
Bull. 2006;29:2051–2055.
Barbalho S, Farinazzi-Machado F, de Alvares G, Brun-nati A, Otoboni A. Psidium Guajava
(Guava): A plant of multipurpose medicinal applications. Med Aromat Plants. 2012; 1:
2167-0412.1000104.
Baricos W. H, Cortez S. L, Deboisblanc M, Xin S. Transforming growth factor-beta is a potent
inhibitor of extracellular matrix degradation by cultured human mesangial cells. J. Am.
Soc. Nephrol. 1999;10:790–795.
Bernobich E, Consenzi A, Campa C, Zennaro C, Sasso F, Paoletti S et al. Antihypertensive
treatment and renal damage: amlodipine exerts protective effect through the polyol
pathway. J Cardiovasc Pharmacol 2004;4:401–6.
Besarab A, Frinak S, Yee J: An indistinct balance: The safety and efficacy of parenteral iron
therapy. J Am Soc Nephrol. 1999;10:2029-2043,.
Block K, Gorin Y, Abboud H. E. Subcellular localization of Nox4 and regulation in diabetes.
Proc. Natl Acad. Sci. 2009;106:14385–14390.
Brezniceanu M. L. et al. Attenuation of interstitial fibrosis and tubular apoptosis in db/db
transgenic mice overexpressing catalase in renal proximal tubular cells. Diabetes.
2008;57:451–459.
Brownlee M. Biochemistry and molecular cell biology of diabetic complications, Nature.
2001;414:813-20.
Brownlee M. The Pathobiology of Diabetic Complications, A Unifying Mechanism (2005);
54(6):1615-1625.
Bursell S, Clermont A, Aiello L, et al. High-dose vitamin E supplementation normalizes retinal
blood flow and creatinine clearance in patients with type 1 diabetes. Diabetes Care.
1999;22(8):1245–1251.
Callapina M, Zhou J, Schmid T, Köhl R, Brüne B. NO restores HIF-1alpha hydroxylation
during hypoxia: role of reactive oxygen species. Free Radic. Biol. Med. 2005;39: 925–936.
Capdor J, Foster M, Petocz P. Zinc and glycemic control: a metaanalysis of randomized placebo
controlled supplementation trials in humans. J Trace Elem Med Biol. 2013;27:137–142.
Caramori M. L and Mauer, M. Diabetic nephropathy. In: Greenberg A, Cheung AK, Coffman
TM, Falk RJ, and Jennette JC editors. Primer on kidney diseases, 3rd ed. Florida: National
Kidney Fundation;2001.
Castro C. N, Barcala Tabarrozzi A. E, Winnewisser J, Gimeno M. L, Antunica Noguerol M, et
al. Curcumin ameliorates autoimmune diabetes. Evidence in accelerated murine models of
type 1 diabetes. Clin Exp Immunol. 2014; 177: 149-160.

Catrina S. B, Okamoto K, Pereira T, Brismar K, Poellinger L. Hyperglycemia regulates

hypoxiainducible factor-1alpha protein stability and function. Diabetes. 2004;53:3226–
3232.
Ceriello A, Morocutti A, Mercuri F, Quagliaro L, Moro M, et al. Defective intracellular
antioxidant enzyme production in type 1 diabetic patients with nephropathy. Diabetes.
200049: 2170-2177.
Ceriello A, Motz E: Is oxidative stress the pathogenic mechanism underlying insulin resistance,
diabetes, and cardiovascular disease? The common soil hypothesis revisited. Arterioscler
Thromb Vasc Biol. 2004;24(5):816-823.
Chander P. N. et al. Nephropathy in Zucker diabetic fat rat is associated with oxidative and
nitrosative stress: prevention by chronic therapy with a peroxynitrite scavenger ebselen. J.
Am. Soc. Nephrol. 2004;15:2391–2403.
Charonis A. S, Tsilbary E. C. Structural and functional changes of laminin and type IV collagen
after nonenzymatic glycation. Diabetes 1992;41:49–51.
Chen P, Chen J, Zheng Q, Chen W, Wang Y, et al. Pioglitazone, extract of compound Danshen
dripping pill, and quercetin ameliorate diabetic nephropathy in diabetic rats. J Endocrinol
Invest. 2013;36: 422-427.
Cheng D, Kuhn P, Poulev A, et al. In vivo and in vitro antidiabetic effects of aqueous cinnamon
extract and cinnamon polyphenolenhanced food matrix. Food Chem. 2012;135:2994–
3002.
Cheng J, Zhang W, Zhang X, Li X, Chen J. Efficacy and safety of paricalcitol therapy for
chronic kidney disease: a meta-analysis. Clin J Am Soc Nephrol. 2012 7: 391-400.
Chennasamudram S. P, Kudugunti S, Boreddy P. R, et al. Renoprotective effects of (+)-
catechin in streptozotocin-induced diabetic rat model. Nutr Res. 2012;32(5):347–356.
Ciccone M. M, Cortese F, Gesualdo M, Carbonara S, Zito A, Ricci G, De Pascalis F,
Scicchitano P, Riccioni G. Dietary intake of carotenoids and their antioxidant and anti-
inflammatory effects in cardiovascular care. Mediators Inflamm. 2013;2013:782137.
Cooke D. W, Plotnick L. Type 1 diabetes mellitus in pediatrics. Pediatr Rev. 2008;29: 374-
384.
Cooper M. E, and Gilbert R. E. Pathogenesis, prevention, and treatment of diabetic
nephropathy, In: Johnson RJ, and Feehally J, editors. Comprehensive clinical nephrology.
2nd ed. Edinburgh: Mosby; 2003; 439-450.
Das S. K, Vijayakumar P. A, Senthil R, et al. Antioxidant effect of vitamin C on type 2 diabetes
mellitus patients along with two different oral hypoglycemic agents for smooth glycemic
control. WJPPS. 2012;1:1113–1122.
Erkan N, Ayranci G, Ayranci E. Antioxidant activities of rosmary extract, black seed essential
oil, carnosic acid, rosmarinic acid and sesamol. Food Chem. 2008;110:76–82.
Evans J. L, Goldfine I. D, Maddux B. A., Grodsky G. M: Oxidative stress and stress-activated
signaling pathways: a unifying hypothesis of type 2 diabetes. Endocr Rev. 2002; 23(5):599-
622.
Foley R. N. et al. Cardiac disease in diabetic end-stage renal disease. Diabetologia. 1997;40,
1307–1312.
Forbes J. M, Coughlan M. T and Cooper M. E. Oxidative stress as a major culprit in kidney
disease in diabetes. Diabetes. 2008;57:1446–1454.

Fujita H, Sakamoto T, Komatsu K, Fujishima H, Morii T, et al. Reduction of circulating

superoxide dismutase activity in type 2 diabetic patients with microalbuminuria and its
modulation by telmisartan therapy. Hypertens Res. 2011; 34: 1302-1308.
Fujita, H. et al. Reduction of renal superoxide dismutase in progressive diabetic nephropathy.
J. Am. Soc. Nephrol. 2009;20:1303–1313.
Gaede P, Poulsen H. E, Parving H. H, et al. Double-blind, randomized study of the effect of
combined treatment with vitamin C and E on albuminuria in type 2 diabetic patients.
Diabetes Med. 2001;18 (9):756–760.
Gao P, Li L, Ji L, Wei Y, Li H, et al. Nrf2 ameliorates diabetic nephropathy progression by
transcriptional repression of TGFß1 through interactions with c-Jun and SP1. Biochim
Biophys Acta -Gene Regulatory Mechanisms. 2014; 1839: 1110-1120.
Garman J, Mulroney S, Manigrasso M, et al. Omega-3 fatty acid rich diet prevents diabetic
renal disease. Am J Physiol Renal Physiol. 2009;296:F306–F316.
Geiszt, M., and Leto, T. L. The Nox family of NAD(P)H oxidases: host defense and beyond.
J. Biol. Chem. 2004;279(20):51715–51718.
Geiszt M, Kopp J. B, Varnai P, et al: Identification of renox, an NADPH oxidase in kidney.
Proc Natl Acad Sci USA. 2000; 97:8010-8014.
Geraldes P, Hiraoka-Yamamoto J, Matsumoto M, Clermont A, Leitges M, et al. Activation of
PKC-delta and SHP-1 by hyperglycemia causes vascular cell apoptosis and diabetic
retinopathy. Nat Med. 2009;15: 1298-1306.
Ghosh J and Sil P. C. Arjunolic acid: a new multifunctional therapeutic promise of alternative
medicine. Biochimie. 2013; 95: 1098- 1109.
Ghosh J, Das J, Manna P, Sil P. C. Protective effect of the fruits of Terminalia arjuna against
cadmium-induced oxidant stress and hepatic cell injury via MAPK activation and
mitochondria dependent pathway. Food chemistry. 2010;123: 1062-1075.
Gnudi L, Benedetti S, Woolf A. S, Long D. A. Vascular growth factors play critical roles in
kidney glomeruli, Clinical science (London, England: 1979). 2015;129(12):1225-36.
Go Y, Jones P. Redox compartmentalization in eukaryotic cells. Biochim Biophys Acta.
2008;1780:1273–1290.
Gorin Y. et al. Nox4 NAD(P)H oxidase mediates hypertrophy and fibronectin expression in the
diabetic kidney. J. Biol. Chem. 2005;280:39616–39626.
Gugliucci A. Glycation as the glucose link to diabetic complication. J. Am. Osteopath. Assoc.
2000; 100:621-632.
Guo Y, Liu Y, Wang Y. Beneficial effect of lycopene on anti-diabetic nephropathy through
diminishing inflammatory response and oxidative stress. Food Funct 2015;6:1150–6.
Gupte SA and Wolin MS. Oxidant and redox signaling in vascular oxygen sensing: implications
for systemic and pulmonary hypertension. Antioxid. Redox Signal. 2008;10: 1137–1152.
Haber C. A, Lam T. K, Yu Z et al. N-acetylcysteine and taurine prevent hyperglycemia-induced
insulin resistance in vivo: possible role of oxidative stress. Am J Physiol Endocrinol
Meta. 2003; 285: E744–E753.
Haeri M. R, Limaki H. K, White C. J, White K. N. Non-insulin dependent anti-diabetic activity
of (2S, 3R, 4S) 4-hydroxyisoleucine of fenugreek (Trigonella foenum graecum) in
streptozotocin induced type I diabetic rats. Phytomedicine. 2012; 19: 571-574.
Haidara M. A, Mikhailidis D. P, Rateb M. A, et al. Evaluation of the effect of oxidative stress
and vitamin E supplementation on renal function in rats with streptozotocin-induced Type
1 diabetes. J Diabetes Complicat. 2009;23:130–136.

Hakim F. A and Pflueger A. Role of oxidative stress in diabetic kidney disease. Med. Sci. Monit.
2010;16: RA37–RA48..
Hao H. H, Shao Z. M, Tang D. Q, Lu Q, Chen X, et al. Preventive effects of rutin on the
development of experimental diabetic nephropathy in rats. Life Sci. 2012; 91: 959-967.
Higgins D. F. et al. Hypoxic induction of Ctgf is directly mediated by Hif-1. Am. J. Physiol.
Renal Physiol. 2004;287:F1223–F1232.
Hirnerová E, Krahulec B, Strbová L, et al. Effect of vitamin E therapy on progression of
diabetic nephropathy. Vnitr Lek. 2003;49 (7):529–534.
Hodgson J. M, Watts G. F, Playford D. A, Burke V, Croft K. D. Coenzyme Q10 improves
blood pressure and glycaemic control: A controlled trial in subjects with type 2 diabetes.
Eur J Clin Nutr. 2002;56:1137-42.
Hou X, Song J, Li X. N, Zhang L, Wang X, et al. Metformin reduces intracellular reactive
oxygen species levels by regulating expression of the antioxidant thioredoxin via the
AMPK-FOXO3 pathway. Biochem Biophys Res Commun. 2010; 396: 199-205.
Ishii H, Jirousek M. R, Koya D, Takagi C, Xia P, Clermont A, et al. Amelioration of vascular
dysfunctions in diabetic rats by an oral PKC beta inhibitor. Science, 1996;272:728-231.
Jeansson M, and Haraldsson B. Morphological and functional evidence for an important role
of the endothelial cell glycocalyx in the glomerular barrier, American journal of
physiology. Renal physiology. 2006;290(1):F111-116.
Jiang W. L., Xu Y, Zhang S. P., et al. Effect of rosmarinic acid on experimental diabetic
nephropathy. Basic Clin Pharmacol Toxicol. 2012;110(4):390–395.
Jiménez-Escrig A, Rincón M, Pulido R, et al. Guava fruit (Psidium guajava L.) as a new source
of antioxidant dietary fiber. J AgricFood Chem. 2001;49(11):5489–5493.
Johnson EJ. The role of carotenoids in human health. Nutr Clin Care. 2002;5:56–65.
Kakuta H, Sudoh K, Sasamata M, Yamagishi S. Telmisartan has the strongest binding affinity
to angiotensin II type 1 receptor: comparison with other angiotensin II type 1 receptor
blockers. Int J Clin Pharmacol Res. 2005; 25: 41-46.
Kang K, Ham J, Kim Y, et al. Heat-processed Panax ginseng and diabetic renal damage: active
components and action mechanism. J Ginseng Res. 2013;37(4):379–388.
Kang M., Park Y., Kim B., et al. Preventive effects of green tea (Camellia sinensis var.
Assamica) on diabetic nephropathy. Yonsei Med J. 2012;53:138–144.
Kashiba M, Oka J, Ichikawa R, Kasahara E, Inayama T, et al. Impaired ascorbic acid
metabolism in streptozotocin-induced diabetic rats. Free Radic Biol Med. 2002;33: 1221-
1230.
Khan A, Safdar M, Khan M, et al. Cinnamon improves glucose and lipids of people with type
2 diabetes. Diabetes Care. 2003;26:3215–3218.
Khan M, Siddique K, Ashfaq F, et al. Effect of high-dose zinc supplementation with oral
hypoglycemic agents on glycemic control and inflammation in type-2 diabetic nephropathy
patients. J Nat Sci Biol Med. 2013;4:336–340.
Khan MA, Tania M, Zhang D, Chen H. Antioxidant enzyme and cancer. Chin J Cancer
Res. 2010;22:87–92.
Khan M. R, Siddiqui S, Parveen K, et al. Nephroprotective action of tocotrienol-rich fraction
(TRF) from palm oil against potassiumdichromate (K2Cr2O7)induced acute renal injury
in rats. Chem Biol Interact. 2011;186:228–238.

Khazim K, Gorin Y, Cavaglieri RC, et al. The antioxidant silybin prevents high glucose-
induced oxidative stress and podocyte injury in vitro and in vivo. Am J Physiol Renal
Physiol. 2013;305(5):F691–700.
Kim H, Quan M, Kim J. New insights into the mechanisms of polyphenols beyond antioxidant
properties; lessons from the green tea polyphenol, epigallocatechin-3-gallate. Redox Biol.
2014;2:187–195.
Kim HS, Quon MJ, Kim JA. New insights into the mechanisms of polyphenols beyond
antioxidant properties; lessons from the green tea polyphenol, epigallocatechin 3-gallate.
Redox Biol. 2014; 2: 187-195.
Kim J, Shon E, Kim C. S, Kim J. S. Renal podocyte injury in a rat model of type 2 diabetes is
prevented by metformin. Exp Diabetes Res. 2012: 210821.
Koya D, and King G. L. Protein kinase C activation and the development of diabetic
complications. Diabetes, 1998; 47:859-866.
Kuang Q. T, Zhao J. J, Ye C. L, et al. Nephroprotective effects of total triterpenoids from
Psidium guajava leaves on type 2 diabetic rats. Zhong Yao Cai. 2012;35(1):94–97.
Kuhad A., Chopra K. Attenuation of diabetic nephropathy by tocotrienol: involvement of NF
KB signaling pathway. Life Sci. 2009;84 (9–10):296–301.
Kuru Karabas M, Ayhan M, Guney E, Serter M, Meteoglu I. The effect of pioglitazone on
antioxidant levels and renal histopathology in streptozotocin-induced diabetic rats. ISRN
Endocrinol. 2013: 858690.
Lee E. Y, Lee M. Y, Hong S. W, et al. Blockade of oxidative stress by vitamin C ameliorates
albuminuria and renal sclerosis in xperimental diabetic rats. Yonsei Med J. 2007;48:847–
855.
Lee H. B, Yu M. R, Yang Y, Jiang Z, Ha H. Reactive oxygen species-regulated signaling
pathways in diabetic nephropathy. J. Am. Soc. Nephrol. 2003;14(Suppl. 3):S241–S245.
Leeuwenburgh C., Hardy M. M., Hazen S. L., et al: Reactive nitrogen intermediates promote
low density lipoprotein oxidation in human atherosclerotic intima. J Biol Chem.
1997;272:1433-1436.
Li B., Cui W., Tan Y, et al. Zinc is essential for the transcription function of Nrf2 in human
renal tubule cells in vitro and mouse kidney in vivo under the diabetic condition. J Cell
Mol Med. 2014;18 (5):895–906.
Li J. M and Shah A M. ROS generation by nonphagocytic NADPH oxidase: potential relevance
in diabetic nephropathy. J. Am. Soc. Nephrol. 2003;14(Suppl. 3): S221–S226.
Li L, Du J, Lian Y, et al. Protective effects of Coenzyme Q10 against hydrogen peroxide-
induced oxidative stress in PC12 cell: the role of Nrf2 and antioxidant enzymes. Cell Mol
Neurobiol. 2016;36:103–111.
Li X, Yan H., Wang J. Extract of Ginkgo biloba and alpha-lipoic acid attenuate advanced
glycation end products accumulation and RAGE expression in diabetic nephropathy rats.
Zhongguo Zhong Xi Yi Jie He Za Zhi. 2011;31(4):525–531.
Lin C., Wu S., Chang C. Antioxidant activity of Cinnamomum cassia. Phytother Res.
2003;17:726–730.
Lin, C. Y, Yin, M. C. Renal protective effects of extracts from guava fruit (Psidium guajava
L.) in diabetic mice. Plant Foods Hum Nutr. 2012;67(3):303–308.
Lin, Y. C., Chang, Y. H., Yang, S. Y., Wu, K. D., Chu, T. S. Update of pathophysiology and
management of diabetic kidney disease. Journal of the Formosan Medical Association.
2018; 117: 662-675.

Liu, J, Feng, L, Zhu, M. The in vitro protective effects of curcumin and demethoxycurcumin in
Curcuma longa extract on advanced glycation end products-induced mesangial cell
apoptosis and oxidative stress. Planta Med. 2012;78:1757–1760.
Liu, Y, Kakani, R, Nair, M. G. Compounds in functional food fenugreek spice exhibit anti-
inflammatory and antioxidant activities. Food Chemistry. 2012;131: 1187-1192.
Lu, Q, Yin, X. X., Wang, J. Y., et al. Effects of Ginkgo biloba on prevention of development
of experimental diabetic nephropathy in rats. Acta Pharmacol Sin. 2007;28(6):818–828.
Maheshwari, R., Balaraman, R, Sen, A, et al. Effect of coenzyme Q10 alone and its combination
with metformin on streptozotocin-nicotinamide- induced diabetic nephropathy in rats.
Indian J Pharmacol. 2014;46:627–632.
Mahmoud, R. A, el-Ezz, S. A., Hegazy AS. Increased serum levels of interleukin-18 in patients
with diabetic nephropathy. Ital J Biochem. 2004;53: 73-81.
Maitra, A. Endocrine Disorders In: Robbins and Cotran Pathologic basis of disease 9th ed place:
pub; 2015.
Makino H, Haneda M, Babazono T, Moriya T, Ito S, et al. Prevention of transition from
incipient to overt nephropathy with telmisartan in patients with type 2 diabetes. Diabetes
Care. 2007; 30: 1577-1578.
Maldonado P. D, Barrera D, Medina-Campos O. N, et al. Aged garlic attenuates gentamicin-
induced renal damage and oxidative stress in rats. Life Sci. 2003;73:2543–2556.
Malireddy S, Kotha S. R, Secor J. D, Gurney T. O, Abbott J. L, et al. Phytochemical
Antioxidants Modulate Mammalian Cellular Epigenome: Implications in Health and
Disease. Antioxid Redox Signal. 2012;17: 327-339.
Manna P, Das J, Sil P. C. Role of sulfur containing amino acids as an adjuvant therapy in the
prevention of diabetes and its associated complications. Curr Diabetes Rev. 2013;9: 237-
248.
Manna P, Sinha M, Pal P, Sil PC. Arjunolic acid, a triterpenoid saponin, ameliorates arsenic-
induced cyto-toxicity in hepatocytes. Chemico-biological interactions. 2007;170: 187-200.
Mariee A. D, Abd-Allah G. M, El-Yamany M. F. Renal oxidative stress and nitric oxide
production in streptozotocin-induced diabetic nephropathy in rats: the possible modulatory
effects of garlic (Allium sativum L.). Biotechnol Appl Biochem. 2009;52(Pt 3):227–232.
Maritim A. C, Sanders R. A, Watkins J. B 3rd: Diabetes, oxidative stress, and antioxidants: A
review. J Biochem Mol Toxicol. 2003;17(1):24-38.
Masharani, U. Diabetes Mellitus and hypoglycemia. In: McPhee S. J, Papadakis M. A, and
Tierney L. M, editors. Current medical diagnosis and treatment, 55th ed. New York:
McGraw-Hill; 2015.
Masuda Y, Kikuzaki H, Hisamoto M, et al. Antioxidant properties of gingerol related
compounds from ginger. Biofactors. 2004;21:293–296.
Meier M, and King G. L. Protein kinase C activation and its pharmacological inhibition in
vascular disease. Vascular Medicine. 2000; 5: 173–185.
Meng B., Li J., Cao H. Antioxidant and antiinflammatory activities of curcumin on diabetes
mellitus and its complications. Curr Pharm Des. 2013;19(11):2101–2113.
Mishra A, Bhatti R, Singh A, et al. Ameliorative effect of the cinnamon oil from Cinna-momum
zeylanicum upon early diabetic nephropathy. Planta Med. 2010;76(5):412–417.
Mogensen C. E, Christensen C. K, Vittinghus E. The stages in diabetic renal disease. With
emphasis on the stage of incipient diabetic nephropathy. Diabetes. 1983;32 (Suppl. 2), 64–
78.

Morakinyo A, Akindele A, Ahmed Z. Modulation of antioxidant enzymes and inflammatory

cytokines: possible mechanism of anti-diabetic effect of ginger extracts. Afr J Biomed Res.
2011;14:195–202.
Navarro-González J. F, Mora-Fernández C, Muros de Fuentes M, García-Pérez J. Inflammatory
molecules and pathways in the pathogenesis of diabetic nephropathy. Nat Rev Nephrol.
2011; 7: 327-340.
Navarro-González J. F, Mora-Fernández C. The role of inflammatory cytokines in diabetic
nephropathy. J Am Soc Nephrol. 2008; 19: 433-442.
Ojewole J. Analgesic, antiinflammatory and hypoglycaemic effects of ethanol extract of
Zingiber officinale (Roscoe) rhizomes (Zingiberaceae) in mice and rats. Phytother Res.
2006;20:764–772.
Onozato M. L, Tojo A, Goto A, et al: Oxidative stress and nitric oxide synthase in rat diabetic
nephropathy: Effects of ACEI and ARB. Kidney Int. 2002; 61:186-194.
Ooi L, Li Y, Kam S, et al. Antimicrobial activities of cinnamon oil and cinnamaldehyde from
the Chinese medicinal herb Cinnamomum cassia Blume. Am J Chin Med. 2006;34:511–
522.
Özcelik D, Nazıroglu M, Tunçdemir M, et al. Zinc supplementation attenuates metallothionein
and oxidative stress changes in kidney of streptozotocin-induced diabetic rats. Biol Trace
Elem Res. 2012;150(1–3):342–349.
Packer L, Witt EH, Tritschler HJ. Alpha-lipoic acid as a biological antioxidant. Free Radic Biol
Med. 1995;19:227- 50.
Pal P. B, Sinha K, Sil P. C. Mangiferin attenuates diabetic nephropathy by inhibiting oxidative
stress mediated signaling cascade, TNFα related and mitochondrial dependent apoptotic
pathways in streptozotocin-induced diabetic rats. PLoS One 9: e107220. 2014.
Palm F. et al. Reduced nitric oxide in diabetic kidneys due to increased hepatic arginine
metabolism: implications for renomedullary oxygen availability. Am. J. Physiol. Renal
Physiol. 2008;294:F30–F37.
Palm F. Intrarenal oxygen in diabetes and a possible link to diabetic nephropathy. Clin. Exp.
Pharmacol. Physiol. 2006;33:997–1001.
Parving H, Osterby R, Ritz E. Diabetic nephropathy. In: Brenner BM, Levine S editors, The
Kidney, Philadelphia, W. B. Saunders, 2000.
Pessoa B. S, Peixoto E. B, Papadimitriou A, Lopes de Faria J. M, Lopes de Faria J. B.
Spironolactone improves nephropathy by enhancing glucose-6-phosphate dehydrogenase
activity and reducing oxidative stress in diabetic hypertensive rat. J Renin Angiotensin
Aldosterone Syst. 2012;13: 56-66.
Qin Q. J, Deng H. C, Zhao T. F, et al. Study on the effect and mechanism of ascorbic acid on
renal podocytes in diabetes. Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2008;24(1):112–
115.
Rai P. K, Jaiswal D, Mehta S, Watal G. Anti-hyperglycaemic potential of Psidium guajava raw
fruit peel. Indian J Med Res. 2009 129:561-565.
Rajasekaran S, Ravi K, Sivagnanam K, Subramanian S. Beneficial effects of aloe vera leaf gel
extract on lipid profile status in rats with streptozotocin diabetes. Clin Exp Pharmacol
Physiol. 2006; 33: 232-237.
Ramachandraiahgari R, Somesula S, Adi P, Mannur I, Enamalaa M, et al. Protective role of
ethanolic extract of aloe vera antioxidant properties on liver and kidney of streptozotocin-
induced diabetic rats. Dig J Nanomater Bios. 2102;7: 175-184.

Ramachandran A, Snehalatha C, Shetty A. S, Nanditha A. Trends in prevalence of diabetes in

Asian countries. World J Diabetes 2012; 3: 110-117.
Ramana K. Aldose Reductase: New Insights for an Old Enzyme. Biomol Concepts. 2011;2:
103-114.
Rao AV, Rao L. G. Carotenoids and human health. Pharmacol Res. 2007;55:207–16.
Rashid K, Sil P. C. Curcumin enhances recovery of pancreatic islets from cellular stress induced
inflammation and apoptosis in diabetic rats. Toxicol Appl Pharmacol. 2015;282: 297-310.
Reddy S, Aggarwa B. Curcumin is a non-competitive and selective inhibitor of phosphorylase
kinase. FEBS Lett. 1994;341:19–22.
Rehman H, Krishnasamy Y, Haque K, et al. Green tea polyphenols stimulate mitochondrial
biogenesis and improve renal function after chronic cyclosporin a treatment in rats. PLoS
One. 2013;8:1–12.
Remacle J, Raes M, Toussaint O, Renard P, Rao G. Low levels of reactive oxygen species as
modulators of cell function, Mutat Res. 1995; 316(3):103-22.
Renno W. M, Abdeen S, Alkhalaf M, et al. Effect of green tea on kidney tubules of diabetic
rats. Br J Nutr. 2008;100:652–659.
Rizvi S, Raza S. T, Mahdi F. Association of genetic variants with diabetic nephropathy. World
J Diabetes 2014;5: 809-816.
Rosenberg H. R, Culik R, Efect of lipoic acid supplementation on vitamin C and vitamin E
deficiencies, Archives of Biochemistry and Biophysics.1959; 801:86-93.
Rosenfeldt F. L, Haas S. J, Krum H. Coenzyme Q10 in the treatment of hypertension: a meta-
analysis of the clinical trials. J Hum Hypertens. 2007;21(4):297–306.
Rubin R, Strayer D. S. Rubin’s pathology: Clinicopathologic foundations of medicine. 6th ed.
Philadelphia PA: Lippincott Williams & Wilkins; 2012.
Sahin K, Tuzcu M, Gencoglu H, et al. Epigallocatechin-3-gallate activates Nrf2/HO-1 signaling
pathway in cisplatin-induced nephrotoxicity in rats. Life Sci. 2010;87:240–245.
Samuni A. M, DeGraff W, Krishna M. C, Mitchell J. B. Cellular sites of H2O2-induced
damage and their protection by nitroxides. Biochim. Biophys. Acta. 2001;1525:70–76.
Sani N. F, Belani L. K, Sin C. P, et al. Effect of the combination of gelam honey and ginger on
oxidative stress and metabolic profile in streptozotocin-induced diabetic Sprague-Dawley
rats. Biomed Res Int. 2014;2014:160695.
Sayed A. A, Khalifa M, Abd el-Latif F. Fenugreek attenuation of diabetic nephropathy in
alloxan-diabetic rats: attenuation of diabetic nephropathy in rats. J Physiol Biochem.
2012;68: 263-269.
Schena F. P and Gesualdo L. Pathogenetic mechanisms of diabetic nephropathy. J. Am. Soc.
Nephrol., 2005;16:30-33.
Sharma V, Sharma P. Role of Different Molecular Pathways in the Development of Diabetes-
Induced Nephropathy. J Diabetes Metab. 2013;S9: 004.
Sheela N, Jose M. A, Sathyamurthy D, et al. Effect of silymarin o streptozotocin- induced
diabetic nephropathy. Iran J Kidney Dis. 2013;7(2):117–123.
Shimizu M. H, Coimbra T. M, de Araujo M et al. N-acetylcysteine attenuates the progression
of chronic renal failure. Kidney Int. 2005; 68: 2208–2217.
Singh A, Satchell S. C, Neal C. R, McKenzie E. A, Tooke J. E, Mathieson P. W. Glomerular
endothelial glycocalyx constitutes a barrier to protein permeability, J Am. Soc. Nephrol.
2007;18(11):2885-2893.

Singh A. B, Akanksha, Singh N, et al. Anti-hyperglycaemic, lipid lowering and anti-oxidant

properties of [6]-gingerol in db/db mice. Inter J Med Sci. 2009;1(12):536–544.
Singh R, Barden A, Mori T, Beilin L. Advanced glycation end-products: A review.
Diabetologia 2001;44:129–46.
Singh V. P, Baker K. M, Kumar R. Activation of the intracellular reninangiotensin system in
cardiac fibroblasts by high glucose: role in extracellular matrix production. Am J Physiol
Heart Circ Physiol. 2008;294: H1675-1684.
Smith A. R, Shenvi S. V, Widlansky M, Suh J. H, Hagen T. M, Lipoic acid as a potential
therapy for chronic diseases associated with oxidative stress, Current medicinal chemistry,
11, 2004, 1135–46.
Song, J, Eyster, K. M, Kost, C. K Jr, Kjellsen, B, Martin D. S (2010) Involvement of protein
kinase C-CPI-17 in androgen modulation of angiotensin II-renal vasoconstriction.
Cardiovasc Res. 2010; 85: 614-621.
Sourris K, Harcourt B, Tang P, et al. Ubiquinone (coenzyme Q10) prevents renal mitochondrial
dysfunction in an experimental model of type 2 diabetes. J Free Radic Biol Med.
2012;52:716–723.
Sugiyama H, Kobayashi M, Wang DH, Sunami R, Maeshima Y, et al. Telmisartan inhibits both
oxidative stress and renal fibrosis after unilateral ureteral obstruction in acatalasemic mice.
Nephrol Dial Transplant. 2005; 20: 2670-2680.
Sun L, Li W, Li W, Xiong L, Li G, et al. Astragaloside IV prevents damage to human mesangial
cells through the inhibition of the NADPH oxidase/ROS/Akt/NF-?B pathway under high
glucose conditions. Int J Mol Med. 2014;34:167-176.
Sun L. N, Yang Z. Y, Lv S. S, et al. Curcumin prevents diabetic nephropathy against
inflammatory response via reversing caveolin-1Tyr14 phosphorylation influenced TLR4
activation. Int Immunopharmacol. 2014;23:236–246.
Taira M, Toba H, Murakami M, Iga I, Serizawa R, et al. Spironolactone exhibits direct
renoprotective effects and inhibits renal renin-angiotensin-aldosterone system in diabetic
rats. Eur J Pharmacol. 2008; 589: 264-271.
Takada Y, Murakami A, Aggarwal B. Zerumbone abolishes NF-κB and IκBα kinase activation
leading to suppression of antiapoptotic and metastatic gene expression, upregulation of
apoptosis, and downregulation of invasion. Oncogene. 2005;24:6957–6969.
Tang D, Zhang Z, Gao Y, et al. Protective effects of serum containing Ginkgo biloba extract
on glomerulosclerosis in rat mesangial cells. J Ethnopharmacol. 2009 6;124(1):26–33.
Tang Y, Yang Q, Lu J, et al. Zinc supplementation partially prevents renal pathological changes
in diabetic rats. J Nutr Biochem. 2010;21 (3):237–246.
Tanimoto M, Fan Q, Gohda T, Shike T, Makita Y, et al. Effect of pioglitazone on the early
stage of type 2 diabetic nephropathy in KK/Ta mice. Metabolism. 2004;53: 1473-1479.
Tawfik M. K. Renoprotective activity of telmisartan versus pioglitazone on ischemia/
reperfusion induced renal damage in diabetic rats. Eur Rev Med Pharmacol Sci. 2012;16:
600-609.
Taylor R. Causation of type 2 diabetes—The Gordian knot unravels. N Engl J Med. 2004;
350:639-641.
Thomas M. C. Epigenetic Mechanisms in Diabetic Kidney Disease. Curr Diab Rep. 2016; 1631.
Thomas S. R, Witting P. K, Drummond G. R. Redox control of endothelial function and
dysfunction: molecular mechanisms and therapeutic opportunities. Antioxid. Redox Signal.
2008;10:1713–1765.

Thomson M, Al-Qattan K. K, Ali M. Anti-diabetic and anti-oxidant potential of aged garlic

extract (AGE) in streptozotocin-induced diabetic rats. BMC Complement Altern Med.
2016;16:17.
Thorp M. L. Diabetic nephropathy: common questions. Am Fam Physician. 2005;72:96–99.
Toyama T, Shimizu M, Furuichi K, Kaneko S, Wada T. Treatment and impact of dyslipidemia
in diabetic nephropathy. Clin Exp Nephrol. 2014;18:201–5.
Trujillo J, Chirino Y. I, Molina-Jijón E, et al. Renoprotective effect of the antioxidant curcumin:
recent findings. Redox Biol. 2013;1(1):448–456.
Tsai P. Y, Ka S. M, Chang J. M, et al. Epigallocatechin-3-gallate prevents lupus nephritis
development in mice via enhancing the Nrf2 antioxidant pathway and inhibiting NLRP3
inflammasome activation. Free Radic Biol Med. 2011;51:744–754.
Turko IV, Marcondes S, Murad F: Diabetes-associated nitration of tyrosine and inactivation of
succinyl-CoA:3-oxoacid CoA transferase. Am J Physiol Heart Circ Physiol.
2001;281(6):H2289-2294.
Tzeng T. F, Liou S. S, Chang C. J, et al. A tropical ginger sesquiterpene, ameliorates
streptozotocin-induced diabetic nephropathy in rats by reducing the hyperglycemia
induced inflammatory response. Nutr Metab (Lond). 2013;10(1):64.
Usui H., Shikata K., Matsuda M., Okada S., Ogawa D., Yamashita T., Hida K., Satoh M., Wada
J., and Makino H. HMG-CoA reductase inhibitor ameliorates diabetic nephropathy by its
pleiotropic effects in rats. Nephrol. Dial. Transplant., 2003;18:265-272.
Varma V, Varma M, Sarkar P, et al. Correlation of vitamin C with HBA1C and oxidative stress
in diabetes mellitus with or without nephropathy. Nat J Med Res. 2014;4:151–155.
Vasavada N and Agarwal R Role of Oxidative Stress in Diabetic Nephropathy. Advances in
Chronic Kidney Disease. 2005;12(2):146-154.
Vasavada N and Agarwal R. Role of oxidative stress in diabetic nephropathy. Adv Chronic
Kidney Dis. 2005;12(2):146–154.
Velasquez MT, SJ, T, et al. Dietary flaxseed meal reduces proteinuria and ameliorates
nephropathy in an animal model of type II diabetes mellitus. Kidney Int. 2003;64(6):2100–
2107.
Vissers M, C., and Winterbourn C, C. The effect of oxidants on neutrophil-mediated
degradation of glomerular basement membrane collagen. Biochim Biophys Acta.
1986;889: 277-286.
Vora J. P and Ibrahim H. A. A. Clinical manifestations and natural history of diabetic
nephropathy, In: Johnson RJ, and Feehally J editors. Comprehensive clinical nephrology,
2nd ed. Edinburgh: Mosby; 2003; 425-437.
Wang Q, Liu M, Liu WW, et al. In vivo recovery effect of silibinin treatment on streptozotocin-
induced diabetic mice is associated with the modulations of Sirt-1 expression and
autophagy in pancreatic β-cell. J Asian Nat Prod Res. 2012;14(5): 41(5):413–423.
Wang Y, Wang B, Du F, et al. Epigallocatechin-3-gallate attenuates oxidative stress and
inflammation in obstructive nephropathy via NF-κB and Nrf2/HO-1 signalling pathway
regulation. Basic Clin Pharmacol Toxicol. 2015;117(3):164–172.
Whiteside C. I and Dlugosz J. A. Mesangial cell protein kinase C isozyme activation in the
diabetic milieu. Am. J. Physiol. Renal Physiol. 2002;282, 975-980.
Williams M. E, Bolton W. K, Khalifah RG, Degenhardt T. P, Schotzinger R. J, McGill J. B.
Effects of pyridoxamine in combined phase 2 studies of patients with type 1 and type 2
diabetes and overt nephropathy. Am J Nephrol 2007;27(6):605e14.

Wolf G. New insights into the pathophysiology of diabetic nephropathy: from haemo-dynamics
to molecular pathology. European Journal of Clinical Investigation. 2004;34, 785–796.
Wu W, Geng H, Liu Z, et al. Effect of curcumin on rats/mice with diabetic nephropathy: a
systematic review and meta-analysis of randomized controlled trials. J Tradit Chin Med.
2014;34 (4):419–429.
Xue W, Lei J, Li X, Zhang R. Trigonella foenum graecum seed extract protects kidney function
and morphology in diabetic rats via its antioxidant activity. Nutr Res. 2011; 31: 555-562.
Yamaguchi, T. et al. Ebselen in acute ischemic stroke: a placebo-controlled, double-blind
clinical trial. Ebselen Study Group. Stroke. 1998;29(69):12–17.
Yang H, Xu W, Zhou Z, et al. Curcumin attenuates urinary excretion of albumin in type II
diabetic patients with enhancing nuclear factor erythroid-derived 2-like 2 (Nrf2) system
and repressing inflammatory signaling efficacies. Exp Clin Endocrinol Diabetes.
2015;123(6):360–367.
Ye T, Zhen J, Du Y, et al. Green tea polyphenol (–)-epigallocatechin-3-gallate restores Nrf2
activity and ameliorates crescentic glomerulonephritis. PLoS One. 2015.
Yokozawa T, Nakagawa T, Oya T, et al. Green tea polyphenols and dietary fibre protect against
kidney damage in rats with diabetic nephropathy. J Pharm Pharmacol. 2005;57(6):773–
780.
Yoon SP, Maeng Y. H, Hong R, et al. Protective effects of epigallocatechin gallate (EGCG) on
streptozotocin-induced diabetic nephropathy in mice. Acta Histochem. 2014;116:1210–
1215.
Yuan J, Jia R, Bao Y. Beneficial effects of spironolactone on glomerular injury in
streptozotocin-induced diabetic rats. J Renin Angiotensin Aldosterone Syst. 2007; 8: 118-
126.
Zhang S, Zheng L, Dong D, et al. Effects of flavonoids from Rosa laevigata Michx fruit against
high-fat diet-induced non-alcoholic fatty liver disease in rats. Food Chem. 2013;141:2108–
2116.
Zhao L, Xu L, Tao X, et al. Protective effect of the total flavonoids from Rosa laevigata Michx
fruit on renal ischemia-reperfusion injury through suppression of oxidative stress and
inflammation. Molecules. 2016;21(7):952.
Zheng J. M, Zhu J. M, Li L. S, Liu Z. H. Rhein reverses the diabetic phenotype of mesangial
cells over-expressing the glucose transporter (GLUT1) by inhibiting the hexosamine
pathway. Br J Pharmacol. 2008;153: 1456-1464.
Zhou Y, Liao Q, Luo Y, et al. Renal protective effect of Rosa laevigata Michx. by the inhibition
of oxidative stress in streptozotocin induced diabetic rats. Mol Med Rep. 2012;5(6):1548–
1554.
Zhu J, Sun N, Aoudjit L, Li H, Kawachi H, Lemay S, Takano T. Nephrin mediates actin
reorganization via phosphoinositide 3-kinase in podocytes. Kidney international. 2008;
73(5): 556-66.
Ziaee A, Abbas Vaezi A, Oveisi S, Javadi A, Hashemipour S, et al. Effects of additive therapy
with spironolactone on albuminuria in diabetes mellitus: A pilot randomized clinical trial.
Caspian J Intern Med. 2013 4: 648-653.
Ziyadeh F. N, Mogyorosi A, Kalluri R. Early and advanced non-enzymatic glycation products
in the pathogenesis of diabetic kidney disease. Exp Nephrol 1997;5:2–9.

Chapter 17
ANTIOXIDANTS IN GLYCATION RELATED DISEASES
Ahmad Ali1,, Additiya Paramanya1, Prairna1,

Koula Doukani2 and Sadaf Zehra3
1
University of Mumbai, Mumbai, India
2
University of IbnKhaldoun, Tiaret, Algeria
3
University of Windsor, Windsor, Canada
ABSTRACT
Diabetes has become a dangerous health threat and a socio-economic problem for the
world. Change in lifestyle and food habits have added to the increasing list of causative
factors of diabetes. Hyperglycaemia is used as the marker for the identification of this
disorder. This causes the generation of a group of harmful products commonly known as
advanced glycation end products (AGEs). These heterogeneous compounds have been
implicated in secondary complications of diabetes as well as neurodegenerative disorders.
Reactive oxygen species (ROS) are also formed during the process of glycation. These
ROS cause damage to cellular integrity by altering the structure of most of the
biomolecules. The strategies adopted for preventing the accumulation of AGEs include
inhibiting one of the steps of glycation. However, antioxidants are emerging as a major
class of drugs because of their multipotent role as antiglycating and anti-aggregating agents
in the recent reports. This chapter deals with the recent advances in glycation and the role
of antioxidants in diabetes, cancer, Alzheimer’s disease, and aging.
1. INTRODUCTION
Oxygen is one of the most indispensable elements needed for life. However, when present
in the free radical form, it can cause potential damage to the cells (Lobo et al., 2010). Free
energy is generated in the form of ATP by the oxidation-reduction or redox process inside the
mitochondria. However, this redox process generates free radicals in the form of reactive
oxygen/nitrogen species (ROS/RNS) (Kaneko, 2008).

Corresponding Author’s Email: ahmadali@mu.ac.in.

466 Ahmad Ali, Additiya Paramanya, Prairna et al.
Free radicals are produced in the body all the time simultaneously along with the metabolic
processes (Bagchi and Puri, 1998). A disruption in the rate of generation of free radicals and
their scavenging by the antioxidant system leads to a condition commonly termed as oxidative
stress. This increased level of free radicals leads to damage and structural alteration of
biomolecules which ultimately cause the disintegration of cells and various diseases (Pham-
Huy et al., 2008). According to Sies et al., (1992), oxidative stress can directly be related to
aging. Various age-related complications such as a decrease in the efficiency of the immune
system further lead to a cascade of diseases such as brain dysfunction, cardiovascular diseases,
cataract and cancer (Lobo et al., 2010).
Antioxidants have been used in our diet for a long period as they perform several functions
in the cell (Kattappagari et al., 2015). They cause the prevention of oxidative spoilage of food
items, quenching of reactive oxygen species and the breaking of a chain reaction of free-radical
induced damage to the cells. As result antioxidants have been considered by many researchers
as a mechanism by nature to control stress-related disorders like cardio- and cerebrovascular
disease, aging, and physiological stress (Frei et al., 1998; Halliwell, 1995). Plants are rich in
phytochemicals which have been known to possess potent antioxidant capacity. The major
classes of compounds which act as antioxidants include phenols, flavonoids and carotenoids
and members of these classes can detoxify the effect of most of the free radicals like superoxide
anion radical (O2•−), hydroxyl radical (OH•), or super peroxy radicals (Gutteridge and
Halliwell, 2000; Halliwell, 1994).
Natural antioxidants are present in food and various medicinal plants (Kapoor and Kaur,
2001). These natural antioxidants mainly constitute carotenoids, vitamins, and phenolic
compounds (Larson, 1998). Plant-derived antioxidants perform various functions like as
oxygen quenchers, free radical scavengers, act as peroxide decomposers, synergists and
enzyme inhibitors (Manach et al., 1998). It has been noticed that fruits and vegetables contain
vitamins (C and E) and phytonutrients, namely, carotenoids, flavonoids, tannins, phenols which
act as a potential antioxidant (Ramarantham et al., 1995). Various enzymes like superoxide
dismutase (Adachi et al., 1992), glutathione peroxidase (Baldwin et al., 1995), glutathione
reductase and metal binding proteins like ferritin, albumin, lactoferrin, ceruloplasmin which
are involved in the catalysis of free radical quenching reactions also acts as an antioxidant. This
chapter focuses on glycation and the diseases caused due to it. Emphasis has been given on four
major diseases i.e., Alzheimer’s disease, diabetes, cancer, and aging. The present chapter
further describes the way antioxidants can be used in the treatment of the above-mentioned
diseases.
2. GLYCATION
Glycation is a non-enzymatic reaction in which binding of the free amino (–NH3) group of
biological macromolecules such as proteins and nucleic acids with a reducing sugar from
carbohydrates or lipids, forms the Schiff base and then gives the more stable form of Amadori
product which undergoes dehydration, rearrangement and cyclization product and which finally
forms the AGEs (Aldini et al., 2013).
Glycation is a two-step process consisting of the early stage and the late stage. In the early
and reversible stage, a Schiff base is formed due to the interaction of glucose with the –NH3

Antioxidants in Glycation Related Diseases 467
groups of protein, which rearranges to form a stable ketoamine adduct. This process is also
termed as Maillard reaction. Within a few days, Amadori products are formed by rearrangement
Schiff bases. These products have been considered as early glycation products (Ahmad et al.,
2014). The accumulated Amadori products further lead to the late stage of glycation thereby
causing an increase in advanced glycation end products (AGEs). This is facilitated by a
combination or individual reactions involving chemical rearrangements resulting from redox
reactions and hydrations (Goldin et al., 2006) AGEs form a stable non-homogenous group of
molecules which leads to anomalies in many intracellular functions through signaling pathways
and through cross-linking of an extracellular protein (Eble et al., 1993). Number of AGEs has
been described, including 3-deoxyglucosone (3DG), pyrraline, pentosidine, and N-ɛ-
(carboxymethyl) lysine (CML). Some of these AGEs may possess a fluorophore or a
chromophore which imparts fluorescence or color to the AGE respectively. Non-fluorescent
AGE dimers include glyoxal lysine dimer (GOLD) or methylglyoxal lysine dimer (MOLD)
(Gkogkolou and Bohm, 2016). It has been observed that glycation is connected to a variety of
diseases such as diabetes mellitus, aging, and age-related disorders, myocardial infarction,
hypertrophy, neurodegenerative diseases, etc (Singh et al., 2014).
Glycation results in the chemical modifications, conformational changes and retardation of
protein function (Roy et al., 2004). Proteins have been observed to be the most affected
biomolecule. A wide array of proteins and enzymes are supposedly affected by glycation
leading to alterations in their structure and functions. This, in turn, affects the normal metabolic
processes which further causes a decline in the effectiveness of the immune system and makes
the individual more prone to diseases. Zhang et al., (2008) describe the relationship of glycated
proteins such as human serum albumin (HSA), immunoglobulins, apolipoproteins, etc with
their contributions in diseased conditions. AGEs react with their specific receptors known as
RAGEs. An interaction between AGE and RAGE further leads to several complications
(Daroux et al., 2010).
Glycation process is toxic because of its ability to inhibit and alter specific characteristics
of proteins as well as to generate reactive oxygen radicals (Ali and Sharma, 2015). Glycation
plays a major role in aging and neurodegenerative diseases because of its ability to alter protein
structure and functions and also causing mutations and other damages in DNA (Iannuzzi et al.,
2014). However, it is still not clear that which product is/are involved in these complications
and what is the mechanism.
2.1. DNA Damage
Several studies have shown that incubation of DNA with reducing sugars imparts
chromophoric or fluorophoric properties to their end product. These properties appear to be
similar to certain known AGEs that contain a chromophore or fluorophore. Glycation often
leads to conformational alterations in biomolecules. DNA often exhibits an AP site
(apurinic/apyrimidinic site) when affected by glycation (Ahmad et al., 2016). The unwinding
of the DNA double helix, strand breaks, etc. can also be an indicator of the damages in DNA
due to AGEs. Impairment of genomic integrity takes place leading to discrepancies in
transcription which in turn leads to AGE-related disorders (Ahmad et al., 2014) as discussed
below. Wuenschell et al., (2010) discuss the DNA-AGE interaction with the most studied
adduct N2(1-carboxyethyl)-2′-deoxyguanosine (CEdG). CEdG has been used as a standard to

study glycation-induced damage in DNA and has been quantified using mass spectrometry.
Jaramillo and colleagues (2017) found that CEdG levels are higher in diabetic patients which
were represented by its elevated levels in urine and tissues of mice models. The oxidative
modifications within the DNA lead to suppressing the expression of the gene and its mechanism
(Nagai et al., 2010).
2.2. Protein Modification
The irreversible non-enzymatic binding of sugar molecules to the proteins leads to the
formation of glycated proteins (Figure 1). Glycated hemoglobin A1c (HbA1c) was the first ever
glycated protein discovered and was studied by gel electrophoresis. It is formed when
hemoglobin is the protein which reacts with the surrounding reducing sugars (Kaneko, 2008).
Similarly, glycated albumin and immunoglobulins have also been studied (Ahmad and
Siddique, 2015).
These glycated proteins exhibit conformational changes thereby performing different
functions compared to their non-glycated counterpart. AGEs can trigger the aggregation of
proteins wherein the misfolded proteins tend to form amyloid fibrils. Altered functions of
glycated proteins tend the AGEs to interact with RAGE thereby leading to anomalous signaling
(Wei et al., 2012).
Protein glycation related studies have highly taken into account the glycation reactions of
an amino group of proteins especially lysine residues with monosaccharides like aldoses and
ketoses. Monosaccharides are present in an acyclic form in the body. The carbonyl (C = O)
group of the aldoses and ketoses reach with the amino (–NH3) group of the amino acid side
chain of the protein. This is a reversible reaction which leads to the unstable compound i.e.,
Schiff base. The Schiff base is thus formed by adimine bond and exists in a cyclic
glycosylamine form (Neglia et al., 1983).
Figure 1. Formation of advanced glycation end products. Glycation begins with the interaction between
sugar and proteins. Initially, Schiff bases are formed and in the later stages advanced glycation end
products are formed.

According to Kaneko (2008), the most vital component in the study of protein glycation is
the half-life of the proteins. The proteins with higher half-life have been observed to be affected
more by glycation than the ones with a shorter half-life. Moreover, with time, the concentration
of AGEs in the body increases thereby leading to significant changes in the various biological
processes. AGEs formed by proteins such as human serum albumin (HSA), immunoglobulins,
etc. have been known to give rise to a wide array of pathological conditions (Zhang et al., 2008)
which have been discussed below.
3. PATHOPHYSIOLOGY DUE TO GLYCATION

3.1. Glycation in Age-Related Diseases
Disruption in combating the formation of free radicals in the tissues has been known to be
accelerated in the glycation process. This suggests that the glycation process is involved in the
origination and development of aging and age-related disorders (Francis, 2016). As we know
that glycated products can’t be removed from the human body because of the absence of
enzymes for their removal, so we can make a statement that the aging and thus the lifespan
associated with the formation of free radicals, the antioxidative defense as well as with AGEs
(Yan and Harding, 1997).
Glycation of proteins boosts the nucleation and precipitation of proteins. It is stated that
the misfolding, aggregation and precipitation of proteins results in various neurodegenerative
diseases like Alzheimer’s disease, Parkinson’s disease. In vitro, non-enzymatic glycosylation
of amyloid and tau proteins has manifested that the glycation process raises the seeding
influence of amyloid protein (Kikuchi et al., 2003). Due to the modulations in insulin or insulin
receptor signaling and the utilization of glucose in the brain all through aging and Alzheimer’s
disease, two significant changes are revealed inhibition at the S/G2/M phases of the cell cycle
and activation of the G1 phase. These changes further lead to the accumulation of β-amyloid
protein and the hyperphosphorylation of tau protein (Kim et al., 2017). In dialysis-associated
arthropathy (DRA) patients, initiation of the local inflammatory response is a major
consequence of a hike b2 macroglobulin (b2M) and AGE b2M levels and this inflammation
may cause the destruction of bones and deterioration of connective tissue (Thorpe and Baynes,
1996).
During the early stages of Parkinson’s disease only, the imbalance between free radicals
and antioxidants dependent AGEs formation has conjointly been detected (Van Puyvelde et al.,
2014). In Alzheimer’s patient, AGEs are originated within the amyloid plaques and
neurofibrillary tangles using anti-AGE antibodies and the amount of these formed AGEs are
elevated high in AD brains. Rheumatoid arthritis and lupus erythematosus, common age-related
autoimmune diseases are described by elevated inflammatory responses that quicken the
process of aging in tissues. Elevation within the levels of plasma pentosidine implies imbalance
between free radicals and antioxidants and provides harm to collagen in rheumatoid arthritis
patients (de Groot et al., 2011).
There are a number of known receptors for AGEs, one of which is termed RAGE. RAGE
recognizes AGEs and binds with them and hence takes part in the initiation process of
intracellular signaling that distorts various cellular functions. Also, the binding of the AGEs to

RAGE results in activation of NADPH-oxidases. Furthermore, the activation of RAGE was

also associated with ERK, p38 MAP-Kinase, JAK/STAT-pathway, Rho-GTPases and
phosphoinositol-3-kinase activation (Bierhaus et al., 2005). It has been noticed that the
interaction between AGEs to RAGE existing on an array of cells (monocytes, lymphocytes)
arouse inflammatory responses (Suji and Sivakami, 2004).
Alzheimer’s disease is a chronic mental disorder that results in neuronal loss and
neuroinflammation within the brain and also results in progressive feature decline, memory
loss, and alteration in behavior and other peculiarities (Langseth, 1993). Within the cerebral
cortex and hippocampus, neuritic plaques are of the pathological peculiarity of AD. Proteolytic
cleavages of amyloid-β-protein precursor (APP) forms a 40–42 amino-acid peptide named
amyloid beta (Aβ), which is one among the most parts of neuritic plaques. This amino acid
peptide is toxic and has the capability to cause oxidative stress along with neurodegeneration
(Walsh et al., 2002). Neurofibrillary tangles (NFTs) is another personal trait of AD which
contains bundles of paired helical filaments (PHFs) (Iqbal et al., 2010), and furthermore, these
helical filaments mainly consist of MAP-tau protein which is highly phosphorylated
microtubule-associated protein (Mietelska-Porowska et al., 2014). This tau protein has an
influential role in the instigation of assembly and firmness of microtubules and ultimately
participates in axonal transport (Guo et al., 2017).
It is still ambiguous to tell about the etiology and pathogenesis of AD completely. It has
been reported that AGEs play a critical role in the origination and development of Alzheimer's
disease. As previously discussed, NFTs and senile plaques are the characterized features of AD,
which majorly contains tau and Aβ protein, respectively. It has been demonstrated that, in AD,
AGEs confer to the pathological process of AD in two completely different ways:
1. Cross-linking cytoskeletal proteins induce neuronal dysfunction and

2. Cell death and accumulating on Aβ deposits chronically activating micro- and
astroglial cells.
Non-enzymatic glycosylation and fully phosphorylated tau seem to intensify paired helical
filaments formation, and non-enzymatic glycosylation of Aβ augments the aggregation of the
protein in vitro (Ledesma et al., 1994). The accumulation of Aβ peptides is considered to be an
early ingenious event in the focalization of AD which increases as the disease progresses and
in turn leads to the production of NFTs and ultimately neuronal death (Vitek et al., 1994). It
has been suggested that in Alzheimer disease, AGEs formed in the paired helical filament of
tau can induce free radical generation and hence promotes neuronal dysfunction (Yan et al.,
1994).
3.3. Diabetes
Diabetes mellitus (DM) is possibly the world’s fastest-growing metabolic disorder which
is characterized by hyperglycemia due to irregulation of insulin secretion. Based on current

trends, more than 360 million individuals will have diabetes by the year 2030 (Inzucchi et al.,
2015). In the next two decades, the adult population in developing countries will supposedly
increase by 36% while developed countries are expected to increase by 2%. With the increase
in population the number of diabetic individuals is likely to increase by 69% in former and 20%
in the latter (Shaw et al., 2010).
Hyperglycaemia is an important characteristic of diabetes. Hyperglycaemia in diabetes
causes non-enzymatic glycosylation of free –NH3 groups of proteins (of lysine residues) and
results in their structural and functional changes leading to rising in complications of diabetes
(Tariq and Ali, 2018). It has been observed that in recent times the number of patients with
non-insulin-dependent diabetes mellitus (NIDDM) is increasing at a tremendous rate. Non-
enzymatic glycosylation of protein is one of the noteworthy consequences resulting from
hyperglycemia. The Maillard reaction, an important step in glycation eventually results in the
formation of AGEs. The complications in diabetes have been related to glycation and AGE
modification which impair the pathological changes in the structure of the proteins resulting in
impairment of their function (Tarwadi and Agte, 2010).
There is increasing affirmation that damage is occurring due to free radical generation and
additionally takes part in the progression of insulin resistance, β-cell dysfunction, impaired
glucose tolerance, and type 2 diabetes mellitus (Jay et al., 2006). Hyperglycaemia can foster
the imbalance between free radical generation and antioxidants, open-air oxidation of glucose,
AGEs formation, and polyol pathway triggering other circulating factors that are elevated in
diabetics (Figure 2). Elevated free fatty acids and leptin in diabetic patients are also responsible
for ROS extension (Jay et al., 2006). As the age increases, non-enzymatic glycosylation
increases which signifies the increment in diabetics (Wautier and Schimdt, 2004). The
deposition of AGEs enhances the microvascular lesions in diabetic retinopathy which
ultimately responsible for cardiovascular complexities in diabetic patients (Jay et al., 2006;
Wautier and Schmidt, 2004). There are other reports also which suggest the damage of
macromolecules by glycation and glycation-induced formation of free radicals (Ali et al., 2014;
Jha et al., 2018).
The risk of cardiovascular diseases has been observed to be high in diabetic individuals.
Several microvascular and macrovascular complications have been related to the fatalities in
diabetic individuals. The former can be exemplified by retinopathy and neuropathy, while
coronary heart disease (Hertog et al., 1993) is an important example of the latter. While several
clinical trials have shown the effectiveness of antioxidants in the reduction of complications
arising due to diabetes, no significant results have been noted in its use to prevent
cardiovascular diseases resulting from diabetes (Hughes et al., 1998). Since ROS have been
known to be one of the leading causes in the onset of diabetes as well as its development and
progression, use of antioxidant for ameliorating oxidative stress can be an effective therapy for
diabetes individuals. Vitamin E is one such antioxidant which has been found to be effective in
diabetes; however, it could not significantly prevent the complications (Johansen et al., 2015).
The linkage of diabetes mellitus with a higher rate of production of ROS and the
inefficacious function of antioxidants proves that diabetes complications can be prevented by
controlling the oxidative stress. The antioxidant properties of micronutrients such as vitamins
and minerals can be explored to determine its efficacy in diabetes and its related complications
(Bonnefont-Rousselot et al., 2000).

Figure 2. Role of advanced glycation end products in diabetes. The interaction between AGE and
RAGE can cause a cascade of reactions leading to diabetes. AGE, Advanced glycation end products;
RAGE, Receptors for AGE; ROS, Reactive oxygen species.
3.4. Cancer
Cancer can be defined as uncontrolled multiplication or division of cells which transforms

the healthy or normal cells into abnormal cells. Disturbances in the gene expression pattern, a
cascade of mutations resulting from carcinogens, etc. initiates the multi-stage process of
carcinogenesis. The factors influencing the increase in the risk of cancer have been known to
increase with age. Various environmental factors, an individual’s diet or inherited traits, alcohol
consumption play a vital role in the initiation of carcinogenesis. ROS can affect all the three
stages of carcinogenesis, namely, initiation, promotion, and progression (Klaunig and
Kamendulis, 2004).
As observed in Figure 3, several factors contribute to the increase in ROS levels. The high
level of ROS triggers a homeostatic imbalance of cells which eventually leads to cell death. It
is a well-established fact that ROS induces carcinogenesis, however certain researches have
used the properties of non-cancer promoting ROS for cancer therapy (Yang et al., 2018).
Promotion of anoikis is one such property of ROS. Anoikis is the process wherein anchorage-
dependent cells are subjected to apoptosis, i.e., programmed cell death due to their detachment
from substratum (Douma et al., 2004). Tumor cells are usually clumped. ROS have been known
to promote anoikis thereby leading to unattachment amongst cancer cell and their subsequent
apoptosis (Yang et al., 2018).

Figure 3. Relationship between cancer and reactive oxygen species. Mutations in the cell lead to the
first stage of carcinogenesis. Oxidative stress is observed due to the changes caused by oncogenic
alterations. ROS, Reactive oxygen species.
3.5. Aging
Aging is a genetically defined unavoidable biological phenomenon characterized by

senescence. It affects all organisms, multicellular and unicellular. There have been many
theories related to the mechanisms of aging out of which the most common theory states the
accumulation of molecular damage with respect to time determines the longevity of the cells.
Free radical theory of aging or the oxidative damage theory of aging is one such theory which
proposes a hypothesis stating the oxidative stress formed due to unavoidable disruptions in
molecular mechanisms and accumulation of the damages caused by ROS can cause aging.
Further senescence may also give rise to various age-related disorders (Sadowska-Bartosz and
Bartosz, 2014).
4. ANTIOXIDANTS
Antioxidants refer to molecules capable of scavenging or neutralizing free radical and
prevent them from attacking the cells. Human physiological systems have extremely
complicated antioxidant systems (enzymatic and non-enzymatic), that work together to guard
the cells and organ systems of the body against free radical harm. The antioxidants are often
endogenous or obtained through diet or supplements. Some dietary components do not
scavenge free radicals rather they support endogenous antioxidants may additionally be termed
as antioxidants (Rehman, 2007).
Moreover, antioxidants are able to exhibit free radical scavenging and in turn, they inhibit
the oxidative pathway wherein they usually get oxidized. They are used in very fewer amounts.

Endogenous and exogenous antioxidants function by maintaining the redox balance in the body
(Valko et al., 2007).
An efficient antioxidant is easily absorbed, scavenge free radicals and chelate metal ions
at physiological levels. They function in an aqueous medium as well as membrane regions and
affect gene expression. Endogenous antioxidants participate in maintaining optimum cellular
functions and therefore maintain good health. However, when they undergo oxidative stress,
an endogenous antioxidant may not fully suffice and additional dietary antioxidant is needed to
fulfill the additional requirement to keep up the cellular functions (Rehman, 2007). Table 1
represents the different criteria for the classification of antioxidants.
Table 1. Classification of antioxidants (Adapted from Sen and Chakraborty, 2011)
Classification Based on Nature

Enzymatic Antioxidant Non-Enzymatic Antioxidant
Superoxide dismutase Metabolic Antioxidants Nutrient Antioxidants
(SOD) Reduced glutathione, lipoic acid, L- Vitamin E, viamin C,
Catalase(CAT) arginine, CoQ10, melatonin, uric acid, carotenoid, flavonoids,
Glutathione peroxidase bilirubin, metal chelating proteins, omega-3. Omega-6
(GPx) transferring, etc. fatty acids, trace
Glutathione reductase (GR) elements (selenium,
zinc, manganese), etc.
Classification Based on Source
Endogenous Antioxidants Dietary Antioxidants Metal Binding Proteins
Bilirubin, glutathione, Vitamin E, vitamin C, beta-carotene and Albumin (copper),
lipoic acid, N-acetyl other carotenoids, and oxycarotenoids ceruloplasmin (copper),
cysteine, NADPH and (lycopene and lutein), polyphenols metallothionein
NADH, CoQ10, uric acid, (flavonoids, flavones, flavonols) and (copper), ferritin (iron),
enzymes (SOD, CAT, GPx, proanthocyanidins myoglobin (iron),
GR) transferrin (iron)
Classification Based on Mechanism of Action
Catalytic systems-neutralize Binding/inactivation Self-suicidal and Quenching ROS
ROS of metal ions- chain breaking
prevents ROS antioxidant-
production by destroy ROS
Haber-Weiss
reaction
SOD, CAT, GPx Ferritin, Vitamin E, Carotenoids,
ceruloplasmin, vitamin C, uric anthocyanidins
catechins acid, glutathione
Researchers have confirmed that foods rich in natural antioxidants play a major role in the
prevention and treatment of many disorders, particularly diabetes, cancers, and cardiovascular
diseases (Mishra, 2013). In traditional medicine, Anethum graveolens has been used as an
anticancer, antimicrobial, antidiabetic, anti-gastric irritation, anti-inflammatory, and
antioxidant (Jana and Shekhawat, 2010). Administration of dill in diabetic animals has
considerably reduced blood glucose and cholesterol levels (Jana and Shekhawat, 2010).
Inhibitory properties of dill extract on glycation of protein have not been reported yet. Some
studies reported that aqueous or hydroalcoholic extracts of dill reduce blood glucose and shows

antioxidant activity more than other fractions (Bahramikia et al., 2008). The exact
hypoglycemic and hypolipidemic mechanisms of dill are not clearly understood so far. The
reported studies failed to survey all of the antioxidant and antiradical tests for Anethum
graveolens. On the other hand, differences in cultivation area of the plant and the method of
extraction lead to different antioxidant ability.
4.1 Antioxidants in Diet
Antioxidants are very well known for providing protection against diseases and developing
strong immunity. Evidence suggests that nutrition could play a significant role to sustain the
normal metabolic processes which might be disrupted due to oxidative damage. Thus,
antioxidants present in food or supplements can prove to be promising candidates for promoting
defense against diseases.
Under normal condition, our body is equipped with a well-developed system to combat
disorders arising by oxidative stress (Bouayed and Bohn, 2010). Vitamin C, vitamin E, N-acetyl
cysteine, carotenoids, coenzyme Q10, alpha-lipoic acid, beta-carotene, lycopene, selenium,
flavonoids etc. are popularly used as a dietary and nutritive antioxidant (Bouayed and Bohn,
2010; Yadav et al., 2016). They either act as antioxidants or support the endogenous
antioxidants to neutralize the free radicals which are released as a result of oxidative reactions
in the body.
Vitamins C and E are important antioxidants among the nonenzymatic category. They
attack free radicals such as peroxyl and convert them into weaker and harmless radicals.
Vitamin E provides protection to the cell membrane from lipid peroxidation of its fatty acids
by blocking the propagation of the peroxyl radical chain reaction. Phenolic hydrogen from
vitamin E attaches itself to a peroxyl free radical of a peroxidized PUFA. Vitamin E constitutes
a group called tocopherols, however, only α-tocopherol or vitamin E is the most active form in
humans. The vital role of vitamin E includes protection against various types of cancers, some
cardiovascular diseases, ischemia, cataract, arthritis and some neurological disorders.
Vitamin C is important for the biosynthesis of fibrous and connective tissues and acts as a
coenzyme for various metabolic reactions. Vitamin C is also a reducing agent and helps in
regenerating vitamin E and itself gets oxidized. These vitamins may be used up in lipid
peroxidation induced by oxygen radicals in reoxygenation injury to prevent the tissue damage.
Vitamin C also prevents atherogenesis and cancer, immune modulator, reduces the chances of
developing stomach cancer, prevents lung and colorectal cancer (Pham-Huy et al, 2010; Sen
and Chakraborty, 2011; Valko et al, 2007).
5. ROLE OF ANTIOXIDANTS IN THE PREVENTION OF GLYCATION

ROS or free radicals are produced by our body due to oxidation reactions which take place
in the body under normal conditions. They may damage the healthy cells and affect the cell
membranes of the body. The sources of free radicals are smoking, UV exposure, alcohol,
exposure to large amounts of heavy metals and any inflammatory response.

Over the years, demand for the intake of antioxidant supplements increased with a view of
its effect in keeping the body healthy (Benzie, 2003; Carlsen, et al., 2010). The effect of
antioxidants supplements and cosmetics is far over skin deep. Antioxidants operate in several
physiological processes and it is important to delineate the real analysis versus common
perception (Benzie, 2003).
Vitamin E is the major lipid-soluble non-enzymatic antioxidant defense systems. Most
studies in human diabetes targeted vitamin E (Pazdro and Burgess, 2010; Tribe and Poston,
1996). In streptozotocin-induced diabetes in the rat, high levels of vitamin E have been found
in the plasma, liver, and heart (Tribe and Poston, 1996).
Heterogenous results have been obtained in platelets indicating no correlation between the
vitamin E levels in platelets and diabetes. Reports of low levels or unchanged levels of vitamin
E are available (Kunisaki et al., 1990).
Another important water-soluble antioxidant, vitamin C helps in the regeneration of
vitamin E from the α-tocopheroxyl radical (Kagan et al., 1992). Some reports indicate that
plasma concentrations of vitamin C remain unchanged in type I and II diabetic patients
(Cerellio et al., 1997; Hugesh, 1998). However, Asayama et al., (1993) reported increased
vitamin C levels in type I diabetic subjects, whereas other reports (Armstrong et al., 1996)
described markedly lower values in diabetes. Low levels of serum vitamin C in type II diabetic
patients can be linked to low inflammation and renal dysfunction (Lino et al., 2005). Another
study suggests the role of non-dietary factors in diabetic retinopathy and no role of vitamin E
or C from food or supplements (Millen et al, 2004).
Moreover, a study shows that the requirement of vitamin C in more cigarettes smoked, in
new patients diagnosed with diabetes, history of smoking from those, with no diabetes (Wilson
et al., 2012). Low level of cellular glutathione has been found to be important in animal diabetic
models as well as in diabetic patients (Jain and McVie, 1994). Similarly, reduced glutathione
inside the platelet was considerably low in diabetes patients with high glycated hemoglobin
than in those with low glycated hemoglobin (Bonnefont-Rousselot et al., 2000; Muruganandam
et al., 1994).
In an, in vitro study, Banan and Ali (2016) have shown the antiglycating activities of
phenolic acids, which are well-known antioxidants. These compounds were also found to have
preventive role DNA damage induced by glycation. Ali et al., (2014) have reported the
protecting roles of several natural compounds in glycation-induced structural alteration of
proteins and DNA. In another study mannitol, the inhibitory role in the process of glycation
and glycoxidation by some established antioxidants like mannitol, sodium acetate, and eugenol
have been reported recently (Jha et al., 2018). Similarly, several reports suggest the antioxidant
and antiglycating role of thymoquinone and aqueous extracts of Nigella sativa (Pandey et al.,
2018).
5.1. Antioxidants in Alzheimer’s Disease
Alzheimer’s disease is a collective name of diseases involving several cellular and

metabolic features like changes in mitochondrial function, uncontrolled protein aggregation
and inflammation. A higher rate of mental illnesses and loss of cognition was observed in a
group of old dementia patients with higher serum levels of methylglyoxal (MG) (Beeri et al.,

2011). The relation between MG and MG-derived AGE neurotoxicity has been described using
several mechanisms.
As stated earlier AGEs lead to the pathogenesis of AD in two completely different ways
(Krautwald and Munch, 2010). Glyoxalase pathway is an effective endogenous antioxidant
defense pathway of the body. Methylglyoxal is the main target of neutralization by the
glyoxalase pathway (Allaman et al., 2015; Rabbani and Thornalley, 2015). This is important
because the accumulation of MG develops inflammatory reactions and oxidative stress. The
brain also contains highly oxidizable substrates and susceptible to oxidative stress. Disturbance
in the levels of prooxidative substrates and the antioxidant capacity to scavenge them causes
oxidative stress. This supports the progression of neurodegenerative diseases.
Since mitochondrial dysfunction is the main source and site of oxidative damage and it is
the causal agent in AD. Studies demonstrated that MG strongly affects mitochondrial
respiration and therefore the oxidative state of the cells (De Arriba, 2007). Specifically, MG
promotes ROS and lactate production in neuroblastoma cells. Huang et al., (2008) observed the
events of MG-induced Neuro-2A neuroblastoma cell line apoptosis. He reported changes in
mitochondrial membrane potential and ratio of Bax/Bcl-2, the activity of caspase-3 and poly
ADP-ribose polymerase (PARP) splitting. Investigation of the mechanism behind MG-induced
neuronal cell apoptosis revealed that MG activates MAPK signaling pathways. High caspase
activity regulated by MG is also reported by Chen et al., (2006) which has been detected in AD.
Hence, high caspase-3 activity could be a factor in the progression of cognitive decline in AD.
Figure 4. Role of MG and MG-derived advanced glycation end products in Alzheimer’s disease. MG
and MG derived AGEs lead to AD via senile plaque and diffused plaque formation respectively (AD,
Alzheimer’s disease; AGE, Advanced glycation end products; APP, Amyloid precursor protein; MG,
Methylglyoxal, PP2A; Protein phosphatase 2A).

Apoptotic toxicity to the cell is facilitated by AGEs. Yin et al., (2012) observed that AGEs
increase intracellular ROS due to high NADPH oxidative activity in SH-SY5Y cells and rat
cortical neurons. AGEs cause to facilitate oxidation in the endoplasmic reticulum, leading to
the C/EBP homologous protein (CHOP) and caspase-12 activity resulting in cell death. Tau
phosphorylation is regulated by the balance between tau phosphatase(s) and tau kinase(s), and
therefore the hyperphosphorylation of tau within the AD brain may be due to the overactive
protein kinases and/or inhibition of tau phosphatases. Further reports on the role of MG in
causing tau hyperphosphorylation to reveal a dual role of MG by activating the enzymes and
reducing the phosphate level (Li et al, 2012). MG activates GSK-3β and p38-MAPK to cause
tau hyperphosphorylation. MG is also reported to inhibit PP2 in SH-SY5Y cells exposed to
oxidative stress due to MG.
Evidence suggests a high level of MG leads to AGEs and ROS production leading to AD
progression. This is aggravated by further cross-linking proteins forming Aβ aggregates by
AGEs. Moreover, MG mediates the activation of several signaling pathways leading to
apoptosis and irregular cell functions as shown in Figure 4.
5.2. Antioxidant Micronutrients in the Prevention of Diabetes
Although micronutrients help our body in maintaining the normal physiological activities,
their increased intake, however, cannot protect the body from diseases. The influx of
micronutrients is tightly regulated in illness also. Hence, an optimum level is always required
by the body. Extensive studies have been carried out for pyridoxamine and ascorbic acid for
the prevention of protein glycation (Shenkin, 2006 and Singh et al. 2001). Inadequate research
has been carried out on the effect of micronutrients on AGE formation and dicarbonyl
modifications of proteins. Tarwadi and Agte (2010), studied nine vitamins, viz. vitamin C,
vitamin E, vitamin A, vitamins B1, B2, B3 and B6, β-carotene and folic acid, and four trace
metals, (zinc, iron, manganese, and selenium) for their in vitro effect on MGO-mediated
glycation using bovine serum albumin (BSA).
Enzymatic endogenous antioxidant systems include SOD and glutathione reductase. SOD
catalyzes the dismutation of superoxide radical to either O2 or hydrogen peroxide (H2O2). H2O2
also has oxidizing effect so this is immediately degraded by catalase/glutathione peroxidase
into the water. Glutathione peroxidase is regenerated by glutathione reductase during the
process. Maritim and colleagues (2003) observed that in diabetes the enzymes which enhance
the oxidative stress get activated and also suppress the antioxidant defense systems. Low SOD
and glutathione and high catalase activity were recorded in the heart in experimental diabetes
models. However, in patients with chronic heart failure, these enzymes exhibited low activity
in the cardiac muscle. Regular exercise has been shown to be promising in upregulating the
expression and activity of antioxidant enzymes. Hence, it is important to control the activity of
these enzymes for the proactive management of heart and kidney failure in diabetics.
Non-enzymatic antioxidants include vitamins A, C and E; glutathione; α-lipoic acid;
carotenoids; trace elements like copper, zinc and selenium; coenzyme Q10 (CoQ10); and
cofactors like folic acid, uric acid, albumin, and vitamins B1, B2, B6, and B12. Effect of the
antioxidant defenses has been reviewed in diabetes (Table 2). Glutathione (GSH) neutralizes
the radicals directly and acts as a co-substrate for GSH peroxidase. α-Tocopherol or vitamin E
is a water-insoluble vitamin that protects the cell membranes from lipid peroxidation. Hydroxyl

radical reacts with vitamin E forming a stabilized phenolic radical which is reduced back to the
phenol by ascorbate and NAD(P)H dependent reductase enzymes. CoQ10 is an electron carrier
in the complex II of the mitochondrial electron transport chain. Brownlee (2001) reported that
at this site CoQ10 acts as an endogenous antioxidant when O2-• is generated at high glucose
levels and regulates endothelial function in diabetes.
Table 2. Role of antioxidants in diabetes
Antioxidants Role in Diabetes

Total α-tocopherol, γ-tocopherol, β-tocotrienol, and carotenoids Control of type 2 diabetes risk
Phytochemicals like cinnamic acids, coumarins, diterpenes, Prevention of diabetes or diabetic
flavonoids, lignans, monoterpenes, phenylpropanoids, tannins complications
and triterpenes
α-Lipoic acid, repaglinide, caffeic acid phenethyl ester Cure of diabetes and diabetic
complications
Ascorbic acid can react with peroxyl radicals and also indirectly helps in vitamin E
regeneration. Hence, it participates in the control of lipid peroxidation of the cellular as well as
organellar membranes. α-Lipoic acid shows antioxidant effects in both hydrophilic and
hydrophobic environments. α-Lipoic acid itself gets reduced to dihydrolipoate which in turns
helps to regenerate nonenzymatic scavengers such as vitamin C, vitamin E, and glutathione
(Johansen et al. 2015).
Cardiovascular dysfunction is a major complication and responsible for death in diabetic
patients. There are several causes leading to this condition such as glucotoxicity, lipotoxicity,
fibrosis, and mitochondrial uncoupling. Oxidative stress arises from disturbances in the
production of reactive oxygen and nitrogen species (ROS and RNS) and the ability of the
physiological system to neutralize/scavenge them. Some reports have shown good therapeutic
effects of trace elements and other antioxidants, against the cardiovascular dysfunction arising
due to diabetes (Hemmeryckx et al., 2016) Antioxidants use different mechanisms to prevent
oxidative damages; they can control the generation of ROS, neutralize ROS, or interfere with
ROS-induced damages. Regulating mitochondrial activity may help in the control of ROS
production. Hence, the use of PPAR agonist to reduce fatty acid oxidation and of trace elements
such as selenium as an antioxidant and other non-enzymatic antioxidants, facilitate the
prevention of diabetes-induced abnormal cardiovascular function. However, inhibiting the
overproduction of radicals by using the antioxidant vitamins could not be explained. Hence,
strong and active antioxidant defense systems are important for the prevention of diabetic
damage (Turan and Belma, 2010).
According to Taheri et al., (2012) diabetes mellitus is a major problem resulting from the
weak antioxidant capacity and the higher oxidative stress in the human body. Both types of
diabetes have been associated with increased oxidative stress. In diabetes, the major source of
ROS generation is NADPH oxidase along with sources like oxidative phosphorylation, glucose
oxidation, lipooxygenase, cytochrome P450 monooxygenases, and nitric oxide synthase
(NOS). Matough et al., (2010) present various literature relating the decrease in enzymatic
oxidants to diabetes. Oxidative stress has also been shown to promote other diabetic problems
such as cardiovascular disease, neuropathy, kidney and retina damage, and erectile dysfunction.

5.3. Antioxidants against Cancer
Cancerous cells show redox imbalances due to oxidative stress caused by free radical
generation. Oxidative damage may influence oncogenic stimulation of DNA. Carcinogenesis
is initiated with mutations in DNA and is thus considered a vital step. High degree of DNA
glycoxidation has been observed in tumors suggesting its linkage with cancer. Reactive
nitrogen species have also been reported to facilitate DNA damage and cancer. Progression of
cancer occurs through a series of events wherein initiation occurs when ROS causes activation
of AP-1 (activator protein) and NF-κB (nuclear factor kappa B) signal transduction pathways.
These changes lead to alteration in the transcription of genes in the cell cycle (Rehman, 2007).
So far several studies on the role of various antioxidants in cancer prevention have shown
mixed results. The first study to look into the effect of antioxidants on cancer risks was on
several Chinese men and women with high cancer risk. They were given a combination of
supplements. The group taking a combination of β-carotene, vitamin E and selenium showed
lower death rates and lower risk of cancer after 1-2 years (Blot et al, 1993). Another finding
from an 8 yearlong study on the risk of lung cancer in male smokers revealed that the men
taking β-carotene were more likely to develop lung cancer (Heinonen et al., 1994). A study on
men and women with higher lung cancer risk also exhibited a 17% higher risk in the group
taking antioxidants (Omenn et al., 1996). This was supported by another clinical study which
proved that men and women (healthy and stable phase of disease) had more chances of dying
if they were taking β-carotene, vitamin E and vitamin A (Bjelakovic et al., 2012). Researchers
found that large doses of vitamin C supplements reduced the effectiveness of several anti-
cancer drugs including methotrexate, doxorubicin and imatinib, and 30-70% fewer cancer cells
killed. They concluded that vitamin C may actually be helping to stabilize cancer cells by
protecting the cells’ power source (Fukumura et al., 2012; Heaney et al., 2008).
5.4. Antioxidants against Aging
Aging is an unavoidable process leading to a decline in the physiological function and well-
being of an individual. It usually eventuates after the reproductive phase of life. The progression
of aging and lifespan is different in individuals, which may depend upon several factors such
as genetic, environmental and nutritive. One important factor affecting the rate of aging is
nutrition and oxidative stress. Improper diet and unhealthy food intake aggravate oxidative
stress in the body which in turn damages different cell components. These cells then get
impaired in their functions leading to gradual deformities in the normal metabolic pathways.
Several biological theories have been given so far to explain the process of aging. These
include stochastic theories of aging, the free radical theory of aging, the mitochondrial theory
of aging, decline theory of ubiquitin-proteasome system, genetic theory of aging (Peng et al.,
2014). Among these free radical theory states the progression of senescence to be a result of
the random deleterious effects of free radicals produced leading to DNA damage, lipids
peroxidation, and proteins oxidation if unchecked by the body defenses and accumulate over
time (Kregel and Zhang, 2007; Masaki, 2010). However, some scientists have the opinion that
controlling the free radicals by antioxidants may not be the only approach in delaying the aging
process.

Management of other disorders associated with oxidative stress can be achieved by

antioxidants. Other benefits of antioxidants suggest that the intake of such compounds may
help in improving the quality of life and prevent disease pathogenesis (Sen and Chakraborty,
2010). However, the antioxidants are required in small amounts consumption of high doses
may be toxic and cause other problems over a period of time (Buoayed and Bohn, 2010).

AND CHALLENGES
Glycation can be said to be a precursor of a wide array of diseases including certain major
complications such as diabetes and neurological disorders like AD. Similarly, free radicals have
been well-documented in the pathophysiology of glycation-related diseases. Their involvement
in aging has also been well-documented by Gkogkolou and Bohm (2012).
Antioxidants are produced by the body endogenously. However, its supplementation is
required through diet during oxidative stress. Natural antioxidants are introduced in the body
through diet. However, their bioavailability may vary even amongst the same species of plant
or plant products (Younus and Anwar, 2016). They also exhibit lower solubility and may be
expelled from the body if not digested properly. Moreover, standardization of natural products
is a tedious process and it does not provide assurance for stability and consistency (Pham-Huy
et al., 2008; Younus and Anwar, 2016).
It can be observed that antioxidants play an indispensable part in maintaining the
homeostasis of the immunity system in an individual’s body. Although oxidative stress is not
the main cause of the above-discussed diseases, its involvement in their manifestation cannot
be overruled. Antioxidants have been a part of our diet since historical time, however, their
involvement in treatments and therapy has now found its application in the health industry
(Thyagarajan and Sahu, 2018). Being non-toxic in nature, the natural antioxidants appear to be
a safer alternative to the available synthetic drugs. Additional supplementation of antioxidants
may not be required by individuals who follow a healthy lifestyle and include an appropriate
amount of antioxidants in their diet. However, the efficiency of the antioxidants can only be
assured in its purified form (Sadowska-Bartosz, and Bartosz, 2015).
The available data suggests antioxidants to be a strong contender in therapy for glycation-
related disorders. Nevertheless, it is very important to develop newer methods to test the
efficiency of antioxidants (Gulumian et al., 2018). The problem associated with natural
antioxidants can be rectified by extracting them and using them in a purified form.
CONCLUSION
Glycation can damage and alter the functions of important biomolecules such as protein
and nucleic acids. A glycated biomolecule can further induce oxidative stress which leads to
various chronic diseases. These complications can be avoided by the use of antioxidant
supplementations. The chapter takes into account the mechanism of glycation, complications
and the role of antioxidants in the prevention of these glycation induced disorders. It can be

concluded that the efficacy of antioxidants in glycation related disorder promises a healthy life
with proper intake of antioxidants in an individual’s diet.
REFERENCES
Adachi T, Ohta H, Hayashi K, Hirano K, Marklund SL. The site of non-enzymic glycation of
human extracellular-superoxide dismutase in vitro. Free Radic Biol Med. 1992; 13: 205-
210.
Ahmad R, Sah AK, Ahsan H. Biochemistry and Pathophysiology of Glycation of DNA:
Implications in Diabetes. Arch Clin Biomed Res 2016; 1 (1): 32-47.
Ahmad S, Khan M, Akhter F, Khan M. S, Khan A, Ashraf J. M. et al., Glycoxidation of
biological macromolecules: A critical approach to halt the menace of glycation. Glycobiol.
2014; 24(11):979-990. doi:10.1093/glycob/cwu057.
Ahmad S, Siddiqui Z. Protein Glycation: A Firm Link to Cause Metabolic Disease and their
Complications. J Glycomics Lipidomics. 2015; 4:127. doi: 10.4172/2153-0637.1000127.
Aldini G, Vistoli G, Stefek M, Chondrogianni N, Grune T, Sereikaite J, et al., Molecular
strategies to prevent, inhibit, and degrade advanced glycoxidation and advanced
lipoxidation end products. Free Radic Res. 2013;47(sup1):93-137. doi:
10.3109/10715762.2013.792926.
Ali A, Sharma R. A Comparative study on the role of lysine and BSA in Glycation-induced
damage to DNA. Biosci Bioeng Commun. 2015; 1: 38-39.
Ali A, Sharma R. Sivakami S. Role of natural compounds in the prevention of DNA and
proteins damage by Glycation. Bionano Frontiers. 2014; 7(12): 25-30.
Allaman I, Belanger M, Magistretti PJ. Methylglyoxal, the dark side of glycolysis. Front.
Neurosci. 2015; 9:23.
Angeloni C, Zambonin L, Hrelia S. Role of methylglyoxal in Alzheimer’s disease. Biomed Res
Int. 2014; 2014:1-12. doi:10.1155/2014/238485
Armstrong A. M, Chestnutt J. E, Gorley M. J, Young I. S. The effect of dietary treatment on
lipid peroxidation and antioxidant status innewly diagnosed non-insulin dependent
diabetes. Free Radic Biol Med. 1996; 21: 719-726.
Asayama K, Uchida N, Nakane T. Antioxidants in serum of children with insulin-dependent
diabetes mellitus. Free Radic Biol Med, 1993; 15:597-602.
Bagchi K, Puri S. Free radicals and antioxidants in health and disease. East Mediterranean
Health Jr. 1998; 4:350–60.
Bahramikia S, Yazdanparast R. Antioxidant and free radical scavenging activities of different
fractions of Anethum graveolens leaves using in vitro models. Pharmacology Online. 2008;
2:233-19.
Baldwin J, Lee L, Leung T, Muruganandam A, Mutus B. Identification of the site of non-
enzymatic glycation of glutathione peroxidase: rationalization of the glycation-related
catalytic alterations on the basis of three-dimensional protein structure. Biochim Biophys
Acta. 1995;1247(1):60-64. doi:10.1016/0167-4838(94)00202-r.
Beeri M, Silverman J, Moshier E, Godbold J, Uribarri J, Reddy S et al., Serum concentration
of an inflammatory glycotoxin, methylglyoxal, is associated with increased cognitive

decline in elderly individuals. Alzheimer’s & Dementia. 2011; 7(4):S144-S145.

doi:10.1016/j.jalz.2011.05.388.
Benzie IF. Evolution of dietary antioxidants. Comp Biochem Physiol A Mol Integr Physiol.
2003; 136(1), 113-26.
Bierhaus A, Humpert PM, Morcos M, Wendt T, Chavakis T, Arnold B, et al., Understanding
RAGE, the receptor for advanced glycation end products. J Mol Med. 2005; 83: 876-886.
Bjelakovic G, Nikolova D, Gluud L. L, Simonetti R. G, Gluud C. Antioxidant supplements for
prevention of mortality in healthy participants and patients with various diseases. Cochrane
Database Syst Review. 2012; 14 (3). doi: 10.1002/14651858.CD007176.pub2
Blot W. J., Li J, Taylor P. R., et al., Nutrition intervention trials in Linxian, China:
supplementation with specific vitamin/mineral combinations, cancer incidence, and
disease-specific mortality in the general population. J Natl Cancer Inst. 1993; 85(18):1483-
1491.
Bonnefont-Rousselot D, Bastard J. P., Jaudon M, Delattre J. Consequences of the diabetic status
on the oxidant/Antioxidant balance. Diabetes Metab, 2000; 26: 163-168.
Bouayed J, Bohn T. Exogenous Antioxidants—Double-Edged Swords in Cellular Redox State:
Health Beneficial Effects at Physiologic Doses versus Deleterious Effects at High Doses.
Oxid Med Cell Longev. 2010; 3: 228-237.
Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature. 2001;
414 (6865): 813-820.
Carlsen M. H., Halvorsen B. L, Holte K, Bøhn S. K, Dragland S, Sampson L, et al., The total
antioxidant content of more than 3100 foods, beverages, spices, herbs and supplements
used worldwide. Nutr J. 2010; 9:3. doi: 10.1186/1475-2891-9-3.
Cerellio A, Bortolotti N, Pirisi M. Total plasma antioxidant capacity predicts thrombosis-prone
status in NIDDM patients. Diab Care. 1997; 20:1589-1593.
Chen K, Maley J, Yu P. H. Potential implications of endogenous aldehydes in β-amyloid
misfolding, oligomerization and fibrillogenesis. J Neurochem. 2006; 99(5); 1413–1424.
Daroux M, Prevost G, Maillard-Lefebvre H, Gaxatte C. Advanced glycation end-products:
implications for diabetic and non-diabetic nephropathies, Diabetes Metab, 2010; 36: 1-10.
De Arriba S. G, Stuchbury G, Yarin J, Burnell J, Loske C, and Munch, G. Methylglyoxal
impairs glucose metabolism and leads to energy depletion in neuronal cells–protection by
carbonyl scavengers. Neurobiol. Aging. 2009; 28: 1044-1050.
de Groot L, Hinkema H, Westra J, Smit A. J, Kallenberg C. G, Bijl M,et al., Advanced glycation
end products are increased in rheumatoid arthritis patients with controlled disease. Arthritis
Res Ther. 2011; 13(6), R205.
Douma S, Van Laar T, Zevenhoven J, Meuwissen R, Van Garderen, E., Peeper D. S.
Suppression of anoikis and induction of metastasis by the neurotrophic receptor TrkB.
Nature. 2004; 430:1034–1039. doi: 10.1038/nature02765.
Eble A, Thorpe S, Baynes J. Nonenzymatic glycation and glucose-dependent cross linking of
protein. J Biol Chem. 1983; 258:9406-9412.
Francis F. A. How advanced glycation end-products affect chronic diseases and aging.
Pharmanews. 2016; 38:1-2
Frei B., Stocker R, Ames B. N. Antioxidant defenses and lipid peroxidation in human blood
plasma. Proc Natl Acad Sci U.S.A. 1988; 85: 9748-9752.

Fukumura H, Sato M, Kezuka K. Effect of ascorbic acid on reactive oxygen species production
in chemotherapy and hyperthermia in prostate cancer cells. J Physiol Sci. 2012; 62:251-
257.
Gkogkolou P, Bohm M. Advanced glycation end products, Dermato-Endocrinology. 2012;
4(3): 259-270.
Goldin A, Beckman J. A, Schmidt A. M, Creager M. A. Advanced glycation end products:
sparking the development of diabetic vascular injury. Circulation. 2006; 114(6): 597-605.
Gulumian M, Yahaya E. S, Steenkamp V. African Herbal Remedies with Antioxidant Activity:
A Potential Resource Base for Wound Treatment. Evid Based Complement Alternat Med.
2018; 2018:4089541. Published 2018 Nov 22. doi:10.1155/2018/4089541.
Guo T, Noble W, Hanger D. P. Roles of tau protein in health and disease. Acta
Neuropathologica, 2017; 133(5), 665-704.
Gutteridge J. M. C, Halliwell B. Free radicals and antioxidants in the year 2000. A historical
look to the future. Ann N Y Acad Sci. 2000; 899: 136-147.
Halliwell B, Aeschbach R, Loliger J, Aruoma O. I. The characterization of antioxidants. Food
Chem Toxicol. 1995; 33(07):601-617.
Halliwell B. Free Radicals, Antioxidants, and Human Disease: Curiosity, Cause, or
Consequence? Lancet. 1994; 344:721-724.
Harman D. The free radical theory of aging. Antioxid Redox Signal.2003. 5:557–61.
Heaney M. L, Gardner J. R, Karasavvas N, et al., Vitamin C antagonizes the cytotoxic effects
of antineoplastic drugs. Cancer Res. 2008;68(9):8031-8038.
Heinonen O. P, Huttunen J. K, Albanes D, et al., The effect of vitamin E and beta carotene on
the incidence of lung cancer and other cancers in male smokers. New Engl J Med. 1998;
330(15):1029-1035.
Hemmeryckx B, Hohensinner P, Swinnen M, Heggermont W, Wojta J, Lijnen H. R.
Antioxidant treatment improves cardiac dysfunction in murine model of premature aging.
J Cadiovasc Pharmacol. 2006; 68(5): 374-382.
Hertog M. G. L. Feskens E. J. M. Hollman P. C. H. Katan M. B. Krombhout D. Dietary
antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study.
Lancet, 1993; 342: 1007-1011.
Huang S. M, Chuang H. C, Wu C. H, Yen G. C. Cytoprotective effects of phenolic acids on
methylglyoxal-induced apoptosis in Neuro-2A cells. Mol Nutr Food Res. 2008; 52(8):940-
9.
Hughes K, Choo M, Kuperan P, Ong C. N, Aw T. C. Cardiovascular risk factors in non-diabetic
controls: a population-based survey among Asians in Singapore. Atherosclerosis. 1998;
136:25-31.
Iannuzzi C, Irace G, Sirangelo I. Differential effects of glycation on protein aggregation and
amyloid formation, Front Mol Biosci. 2014; 1(9): 1-8.
Iqbal K, Liu F, Gong C. X, Grundke-Iqbal I. Tau in Alzheimer disease and related tauopathies.
Curr Alzheimer Res. 2010; 7(8): 656-64.
Jain S. K., McVie R. Effect of glycemic control, race (white versus black), and duration of
diabetes on reduced glutathione content in erythrocytes of diabetic patients. Metab. 1994;
43(3): 306-309.
Jana S, Shekhawat G. S. Anethum graveolens: An Indian traditional medicinal herb and spice.
Pharmacogn Rev Pubmed. 2010; 4:179-84.doi: 10.4103/0973-7847.70915.

Jaramillo R, Shuck S. C, Chan YS, Liu X, Bates S. E, Lim P. P, et al., DNA Advanced Glycation
End Products (DNA-AGEs) Are Elevated in Urine and Tissue in an Animal Model of Type
2 Diabetes. Chem Res Toxicol. 2017; 30(2):689-698.
Jay D, Hitomi H, Griendling K. K. Oxidative stress and diabetic cardiovascular complications.
Free Radic Biol Med. 2006; 40: 183–192.
Jha P, Momin A. R, Kumar D, Ali A. Reversal of glycoxidative damage of DNA and protein
by antioxidants. Annals Phytomed. 2018; 7(1): 101-105.
Johansen J. S, Harris K. A, Jeanette S. J, Alex K. H, David J. R. Cardiovascular Diabetology.
Bio Med central, 2015;187:4-8.
Kagan V. E, Shvedova A, Serbinova E, Khan S, Swanson C, Powell R, et al., Dihydrolipoic
acid--a universal antioxidant both in the membrane and in the aqueous phase. Reduction of
peroxyl, ascorbyl and chromanoxyl radicals. Biochem Pharmacol. 1992; 44(8):1637-49.
Kaneko J. J. Carbohydrate metabolism and its diseases. Clinical Biochemistry of domestic
animals 6th edn. Academic Press/Elsevier, 2008.
Kattappagari K. K, Ravi Teja C. S, Kommalapati R. K, Poosarla C, Gontu S. R, Reddy B. V.
Role of antioxidants in facilitating the body functions: A review. J Orofac Sci 2015;7:71-
5.
Kaur C, Kapoor H. C. Antioxidants in Fruits and Vegetables—The Millennium's Health. Int J
Food Sci Technol. 2001; 36: 703-725.
Kikuchi S, Shinpo K, Takeuchi M, Yamagishi S, Makita Z, Sasaki N et al., Involvement of
advanced glycation end-products (AGEs) in Alzheimer's disease. Brain Res Brain Res Rev.
2003; 41(2-3):306-23.
Kim C. S, Park S, Kim J. The role of glycation in the pathogenesis of aging and its prevention
through herbal products and physical exercise. J Exerc Nutrition Biochem. 2017;21(3):55-
61.
Klaunig J. E, Kamendulis L. M. The role of oxidative stress in carcinogenesis, Ann Rev
Pharmacol Toxicol. 2004; 239–267.
Krautwald M, Munch G. Advanced glycation end products as biomarkers and gerontotoxins -
A basis to explore methylglyoxal-lowering agents for Alzheimer's disease? Exp Gerontol.
2010; 45(10): 744-51.
Kregel KC, Zhang H. An integrated view of oxidative stress in aging: basic mechanisms,
functional effects, and pathological considerations. J. Am. J. Physiol. Regul. Integr. Comp.
Physiol. 2007; 292: 18-36.
Kuhla B, Haase C, Flach K, Luth H. J, Arendt T, Munch G. Effect of Pseudophosphorylation
and Cross-linkin by Lipid Peroxidation and Advanced Glycation End Product Precursors
on Tau Aggregation and Filament Formation. J Biol Chem. 2007; 282, (10): 6984–699.
Kunisaki M, Umeda F, Inoguchi T, Watanabe J, Nawata H. Effects of vitamin E administration
in diabetes mellitus. Diabetes Res. 1990; 14:37-42.
Langseth L. From the Editor: Antioxidants and Diseases of the Brain. Antioxidant Vitamins
Newslett. 1993; 4:3.
Larson R. A. The antioxidants of higher plants. Phytochemistry. 1998; 4: 969-978.
Ledesma M. D, Bonay P, Colaço C, Avila J. Analysis of microtuble-associated protein tau
glycation in paired helical filaments. J Biol Chem. 1994; 269:21614-21619.
Li X. H, Xie J. Z, Jiang X, Lv B. L, Cheng X. S, Du L. L, et al., Methylglyoxal induces tau
hyperphosphorylation via promoting AGEs formation. Neuromolecular Med. 2012; 14(4):
338-48.

Lino K, Fukui T, Anzai K, I wase M, Kogawa K, Ogimoto M, et al., Serum vitamin C levels in
Type 2 diabetic nephropathy. Diabetes Care. 2005; 28(11): 2808-2809.
Lobo V, Patil A, Phatak A and Chandra N. Free radicals, antioxidants and functional foods:
Impact on human health. Pharmacogn Rev. 2010; 4(8): 118-126.
Manach C, Morand C, Crespy V, Demigne C, Texier O, Regerat F, et al., Quercetin is recovered
in human plasma as conjugated derivatives which retain antioxidant properties. FEBS Lett.
1998; 426: 331-336.
Maritim A. C, Sanders R. A, Watkins J. B. Diabetes, Oxidative Stress, and Antioxidants: A
Review. J Biochem Mol Toxicol. 2003; 17(1): 24-38.
Masaki H. Role of antioxidants in the skin: anti-aging effects. J Dermatol Sci. 2010; 58: 85-90.
Matough, F. A, Budin S. B, Hamid Z. A, Alwahaibi N, Mohamed J. The role of oxidative stress
and antioxidants in diabetic complications. Sultan Qaboos Univ Med J, 2010; 12(1): 5-18.
Mietelska-Porowska A, Wasik U, Goras M, Filipek A, Niewiadomska G. Tau protein
modifications and interactions: their role in functionn and dysfunction. Int J Mol Sci.
2014;15(3): 4671-713. doi:10.3390/ijms15034671.
Millen A. E., Klein R, Folson A. R., Stevens J, Palta M, Mares J. A. Relation between intake
of vitamins C and E and risk of diabetic retinopathy in the Atehrosclerosis. Risk in
Communities Study. Am J Clin Nutr. 2004; 79(5):865-73.
Mishra N. Haematological and hypoglycemic potential Anethum graveolens seeds extract in
normal and diabetic Swiss albino mice. Vet World. 2013; 26:502-507.
Muruganandam A, Tannous M, Mutus B. ELISA for in vivo assessment of non-enzymatically
glycated platelet glutathione peroxidase. Clin Biochem. 1994; 27:293-298.
Nagai R, Mori T, Yamamoto Y, Kaji Y, Yonei Y. Significance of advanced glycation end
products in aging-related disease. J Anti-Aging Med. 2010; 7(10): 112-119.
Nawale R. B, Mourya V. K, Bhise S. B. Non-enzymatic glycation of proteins: a cause for
complications in diabetes. Indian J Biochem Biophys. 2006; 337–344.
Neglia CI, Cohen H. J, Garber A. R, Ellis P. D, Thorpe S. R, Baynes J. W. 13C NMR
investigation of non-enzymatic glucosylation of protein. Model studies using RNase A. J
Biol Chem. 1983; 258(23): 14279-14283.
Omenn G. S., Goodman G. E, Thornquist M. D, et al., Effects of a combination of beta carotene
and vitamin A on lung cancer and cardiovascular disease. New Engl J Med. 1996;
334:1150-1155.
Pandey R. Kumar D, Ali A. Nigella sativa seed extracts prevent the glycation of protein and
DNA. Curr Pers Med Aromat Plants (CUPMAP). 2018; 1: 1-7.
Pazdro R, Burgess R. J. The vitamin E and oxidative stress in diabetes complications. Mech
Ageing Dev. 2010; 131(4):276-286.
Peng C, Wang X, Chen J. Biology of Ageing and Role of Dietary Antioxidants, Bio Med Res
Intl, 2014; 2014: 831841.
Pham-Huy L. A., He H., Pham-Huy C. Free radicals, antioxidants in disease and health. Int J
Biomed Sci. 2008; 4: 89–96.
Rabbani N, Thornalley P. J. Dicarbonyl stress in cell and tissue dysfunction contributing to
ageing and disease. Biochem Biophys Res Commun. 2015; 458(2):221–226.
Rahman K. Studies on free radicals, antioxidants, and co-factors. Clin Interv Aging.
2007;2(2):219-36.
Ramarathnam N, Osawa T, Ochi H, Kawakishi S. The contribution of plant food antioxidants
to human health. Trends Food Sci Technol. 1995; 6:7582.

Roy A, Sen S, Chakraborti A. S. In vitro nonenzymatic glycation enhances the role of

myoglobin as a source of oxidative stress, Free Radic Res. 2004; 38 (2): 139-46.
Sadowska-Bartoszi, Bartosz G. Effect of antioxidants supplementation on aging and longevity.
Bio Med Res Intl. 2014; 404680.
Sakurai T, Matsuyama M, Tsuchiya S. Glycation of erythrocyte superoxide dismutase reduces
its activity. Chem Pharm Bull. 1987; 35:302-307.
Sen S, Chakraborty R, Sridhar C, Reddy Y. S. R, De B. Free radicals, antioxidants, diseases
and phytomedicines: current status and future prospect. Int. J. Pharma. Sci. Rev. Res. 2010;
3: 91-100.
Sen S, Chakraborty R. The role of antioxidant in human health. In: Silvana Andreescu, Maria
Hepel editors. Oxidative Stress: Diagnostics, Prevention, and Therapy ACS Symposium
Series, Vol. 1083 Pages 1-37. DOI:10.1021/bk-2011-1083.ch001.
Shenkin A. The key role of micronutrients. Clin Nutr. 2006; 25:1–13.
Sies H, Stahl W, Sundquist A. R. Antioxidant Functions of Vitamins: Vitamins E and C, Beta‐
Carotene, and Other Carotenoids. Ann NY Acad Sci. 1992; 669:7-20.
Sing R, Barden A, Mori T. Advanced glycation end products: a review. Diabetologia. 2001;
44:129–146.
Singh V. P, Bali A, Singh N, Jaggi A. S. Advanced glycation end products and diabetic
complications. Korean J Physiol Pharmacol. 2014; 18(1):1-14.
Suji G, Sivakami S. Glucose, glycation and aging. Biogerontology. 2004; 5: 376–378.
Taheri E, Djalali M, Saedisomeolia A, Moghadam A. M, Djazayeri A, Qorbani M. The
relationship between the activation of antioxidant enzymes in red blood cells and body
mass index in Iranian type 2 diabetes and healthy subjects. J Diabetes Metab Disord, 2012;
11(1): 3.
Tariq M, Ali A. Plant derived natural products as antiglycating agents. In: Mahapatra DK,
Bharti SK, editors. Pharmacological perspectives of low molecular ligands. Florida: Apple
Academic press, 2018.
Tarwadi KV, Agte VV. Effect of Micronutrients on Methylglyoxal-Mediated In Vitro
Glycation of Albumin. Biol Trace Elem Res. 2011; 143:718.
Thorpe SR, Baynes JW. Role of the Maillard reaction in diabetes mellitus and diseases of aging.
Drugs Aging. 1996; 9: 69-77.
Thyagarajan A, Sahu R. P. Potential Contributions of Antioxidants to Cancer Therapy:
Immunomodulation and Radiosensitization. Integr Cancer Ther. 2017; 17(2):210-216.
Tribe R. M, Poston L. Oxidative stress and lipids in diabetes: a role in endothelium vasodilator
dysfunction. Vasc Med. 1996; 1: 195-206.
Turan, Belma. Role of Antioxidant in Redox Regulation of Diabetic Cardiovascular
complications. Curr Pharm Biotechnol. 2010; 11:819.
Uzel N, Sivas A, Uysal M, Oz H. Erythrocyte lipid peroxidation and glutathione peroxidase
activities in patients with diabetes mellitus. Horm Metab Res. 1987; 19:89-90.
Valko M, Leibfritz D, Moncol J, Cronin M. T. D, Mazur M, Telser J. Free radicals and
antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol.
2007; 39: 44–84.
Van Puyvelde K, Mets T, Njemini R, Beyer I, Bautmans I. Effect of advanced glycation end
product intake on inflammation and aging: a systematic review Nutr Rev. 2014; 72: 638-
650.

Vitek MP, Bhattacharya K, Glendening J. M, Stopa E, Vlassara H, Bucala R, et al., Advanced

glycation end products contribute to amyloidosis in Alzheimer disease. Proc Natl Acad Sci
USA. 1994; 91:4766-4770.
Walsh D. M, Klyubin I, Fadeeva J. V, Cullen W. K, Anwyl R, Wolfe M. S, et al., Naturally
secreted oligomers of amyloid β protein potently inhibit hippocampal long term
potentiation in vivo. Nature. 2002; 416(6880):535-9.
Wautier J. L, Schmidt A. M. Protein glycation: a firm link to endothelial cell dysfunction. Circ
Res. 2004; 95: 233–238.
Wilson R, Willis J, Gearry R, Skidmore P., Fleming E, Frampton C, Carr A. Inadequate
Vitamin C Status in Prediabetes and Type 2 Diabetes Mellitus: Associations with
Glycaemic Control, Obesity, and Smoking. Nutrients. 2012; 9(9): 997.
Wuenschell G. E., Tamae D, Cercillieux A, Yamanaka R, Yu C, Termini J. Mutagenic potential
of DNA glycation: Miscoding by (R)-and (S)-N 2-(1-carboxyethyl)-2′-deoxyguanosine.
Biochemistry. 2010; 49: 1814-1821.
Yadav A, Kumari R, Yadav A, Mishra J. P., Srivastva S, Prabha S. Antioxidants and its
functions in human body- A Review. RELS. 2016; 9 (11): 1328-1331.
Yan H, Harding J. J. Glycation-induced inactivation and loss of antigenicity of catalase and
superoxide dismutase. Biochem J. 1997; 328:599-605.
Yan S. D., Chen X, Schmidt A. M., Brett J, Godman G, Zou Y. S., et al., Glycated tau protein
in Alzheimer disease: a mechanism for induction of oxidant stress. Proc Natl Acad Sci
USA. 1994; 91(16): 7787-91.
Yang H, Villani R. M., Wang H, Simpson M. J., Roberts M. S., Tang M, et al., The role of
cellular reactive oxygen species in cancer chemotherapy. J Exp Clin Cancer Res. 2018;
37(1): 266. doi:10.1186/s13046-018-0909-x.
Yin Q. Q, Dong C. F, Dong S. Q, Dong X. L, Hong Y, Hou X. Y, et al., AGEs induce cell death
via oxidative and endoplasmic reticulum stresses in both human SH-SY5Y neuroblastoma
cells and rat cortical neurons. Cell Mol Neurobiol. 2012; 32(8):1299-309.
Younus H, Anwar S. Prevention of non-enzymatic glycosylation (glycation): Implication in the
treatment of diabetic complication. Int J Health Sci (Qassim). 2016; 10(2):261-77.
Zhang Q. B, Tang N, Schepmoes A. A, Philips L. S. Proteomic profiling of non-enzymatically
glycated proteins in human plasma and erythrocyte membranes, J. Proteome Res. 2008; 7:
2025-2032.

SECTION VII: ANTIOXIDANT DEFENSE IN
SEXUAL HEALTH AND PREGNANCY

Chapter 18
OXIDATIVE STRESS AND ANTIOXIDANT THERAPY IN

ERECTILE DYSFUNCTION
Yogesh Chand Yadav*

Uttar Pradesh University of Medical Sciences, Saifai, India
ABSTRACT
Erectile dysfunction (ED) is a common male sexual dysfunction which develops due
to imbalance among vascular, hormonal, neurological, cavernosal and psychological,
factors. It has been shown that oxidative stress plays an important role in the progression
of ED. Oxidative stress can be decreased by antioxidant enzymes. Antioxidants defense
against ED includes almost all therapeutic categories such as the antioxidant enzyme,
natural antioxidant, and their future recent development, future opportunities and
challenges. Numerous researchers well documented that antioxidants have significant
protective and also curative in ED via improving neurohormonal and endothelium
functions. It can be concluded antioxidant, flavonoids and vitamins are well defending
against erectile by significantly improving and maintaining the integrity of the endothelium
function lead to normal penile erection. Present chapter attempts to illustrate preclinical
and clinical reports of antioxidant defense in ED. Further, it also highlights specific
antioxidant mechanisms and their response.
1. INTRODUCTION
ED is the persistent incapability to develop and uphold erections adequate for pleasing
sexual interaction. Erectile dysfunction symptoms consist of the problem in developing an
erection, problem maintaining an erection and decreasing sexual desire. It can happen due to
stress, self-confidence, contribute to the relationship, pathophysiological status or health
condition of the person (Latini et al., 2002; Corona et al., 2004). Central and peripheral various
phenomena are involved in the response to develop normal penile erectile. Normal erection is
a neurohormonal and cardiovascular phenomenon which induces to dilate blood vessel of the
Corresponding Author’s Email: drycy31@gmail.com.

*

492 Yogesh Chand Yadav
penis, relax of endothelium smooth muscle and enhance blood flow in the penis (Azadzoi et
al., 1992). ED is the widespread sexual trouble, facing millions of people Worldwide. Whereas
3 billion men in the US, 2–3 billion men in the UK suffer from ED (Faysal, 2016). The standard
scientific studies reports suggest 52% frequency of ED. The frequency of ED increases with
respect to increasing age in men such as nearly 40% of men suffers at the age of forty years,
nearly seventy percent of men are suffering at age seventy. The frequency of ED augments
from five percent to fifteen percent at age nearly seventy (Feldman et al., 1994).
Erectile dysfunction may develop due to destruction blood flow lead to occur hypoxia,
ischemia, deficiency in nutrient, reduces clearance of metabolic in penile tissues (Greinand
Schubert, 2002). Many studies have been recommended that buildup of endogenous nitric oxide
synthetase (NOS) inhibitors leading to reduces in blood flow penis which is induced problem
in erection and structural damage (Azadzoi et al., 2005; Zhang et al., 2010). Nitric oxide (NO)
is produced in the endothelium of blood vessel which plays a key role in normal penile erections
(Burnett, 1997). There are multiple factors affected endothelial function resulting in impaired
releasing NO cause to develop ED.
NO is impartment role to normal panicle erection but some other factors may be also
affected erectile dysfunction (Bello Klein et al., 2001). In this connection oxidative stress has
also impartment role to develop ED. Oxidative stress develops due to inequity between
provokes oxidants and the scavenging ability of the antioxidants. It is well documented as
oxidative stress induces fertility problems in animals. The extremely develops of reactive
oxidative species which create oxidative stress leading to significant decreases antioxidant
enzymes and nitric oxide free radical loaded tissues be predisposed to come together to
appearance peroxynitrite which produces more oxidative stress than superoxide and NO
(Helmut, 2005).
Several oxidative stresses significantly increased malondialdehyde formation, protein
deterioration, DNA decomposition and declined synthesis and availability of NO in endothelial
causes to develops to inflammatory factors like cytokines, tissue-specific receptors, etc (Qutub
and Popel, 2008). Enzymes and antioxidant defense system prevent the initiation of oxidative
damage and repair oxidative damage leading to target damaged molecules for devastation and
replacement. Various preliminary studies reports show a major involvement between the free
radicals and erectile problems in diabetic rodent models (Agarwal et al., 2006).
Vitamins E and C are good antioxidants which are widely used in scientific studies which
significantly protect the body from destructive oxidative damage (Kinlay et al., 2004). Several
studies are previously reported that natural products like polyphenols and flavonoids prevent
cardiovascular oxidative injury and effective in protecting the cardiovascular system
(Fernández et al., 2001; Fuhrman et al., 1995). Several fruits like pomegranate juice, blueberry
juice, and green tea have a rich source of polyphenols and flavonoids which inhibited oxidative
stress and prevent free radical injury (Aviram and Dornfeld, 2001). Therefore antioxidants are
significantly decreased oxidative stress induced erectile dysfunction and it is good protective
and curative agents who are capable to maintain endothelial function loads normal penile
erectile function which is called as the antioxidant defense against oxidative stress-mediated
erectile dysfunctions. The present chapter represents the antioxidant defense against oxidative
stress-mediated erectile dysfunctions.

Oxidative Stress and Antioxidant Therapy in Erectile Dysfunction 493
2. ETIOLOGY OF ERECTILE DYSFUNCTION

Oxidative stress develops due to excess generation of reactive oxygen species (ROS) like
superoxide and peroxynitrite which are contributed in the development of many cardiovascular
disorders (Moreland et al., 1995) and natural aging process (Ferrara et al., 1995). These are
directly leading to develop ED.
2.2. Pathophysiological Status
Many cardiovascular diseases and their pathophysiological status induce erectile

dysfunctions which are discussed as follow:
2.2.1. Heart Disease

Cardiovascular disease develops arteriogenic erectile dysfunction. Many scientific reports
indicate that erectile dysfunction develops due to coronary heart disease (Kaiser et al., 1988;
Dong et al., 2011).
2.2.2. Atherosclerosis
Almost 40% men who have more than 50-year-old suffering from erectile dysfunction due
to atherosclerotic disease (Vlachopoulos et al., 2013)
2.2.3. Diabetes
Erection problem develops due to diabetes. Endothelial dysfunction of arterioles,
atherosclerosis, peripheral neuropathy, and even hypogonadism are contributed as diabetes-
related sexual dysfunction (Corona et al., 2014 and 2012).
2.2.4. Obesity
The most important cause of erectile dysfunction is obesity because it alters to insulin
resistance, endothelial, hormonal imbalances, and psychological factors of sexual dysfunction.
Poland studies indicate to 79 percent of men found with erectile disorders have a BMI of 25 or
higher. In the United States alone, 8 million cases of erectile dysfunction due to diabetes and
obesity and it risk increases with increasing BMI (Giovanni e al., 2014; Skrypnik et al., 2014).
2.2.5. Psychological or Mental Health Situation

The brain plays a main role in triggers pain erections which is beginning with feelings of
sexual stimulation. But the person who suffers from a psychological problem like anxiety, stress
or emotions which interferes sexual stimulus leads to erectile dysfunction. Psychogenic is an
adrenaline-mediated erectile dysfunction. It includes stress, depression, and anxiety which
suppress to the stimulus of the normal erection (McCabe and Althof, 2014).

2.3. Imbalance Neurohormonal and Psychological Coordination

Penile erection is a multifaceted progression which includes neurohormonal and
psychological and their physiological functions on smooth muscles and blood vessels. If any
trouble among of neurohormonal and psychological and their physiological functions which
arises erectile dysfunction.
3. SYMPTOMS OF ERECTILE DYSFUNCTION

Erectile dysfunction symptoms appear in men due to the effect of pathophysiological status
and psychogenic factors which are mentioned in Table 1.
Table 1. Signs and symptoms of erectile dysfunction (McCabe and Althof, 2014)
Symptoms of Erectile Dysfunction

Psychological Unexpected start
Unbalanced effectiveness (inconsistency, conditions)
Dissatisfaction of supporting potential
Lack of admirable penile erection in night
Pathophysiological status Slow start
Recurrently progressive
Constantly deprived erection
4. RISK FACTORS CONTRIBUTES TO ERECTILE DYSFUNCTION

As we get older, erections might take longer to develop and might not be as firm. Various
risk factors can contribute to erectile dysfunction which is including as follow:
4.1. Lifestyle Factors
Alcohol and smoking habit without fail effect erectile function. Various studies are
reported to an optimistic dose response in the connection between dose and time of smoking
leads threat to erectile dysfunction (Cao et al., 2004) whereas drinking alcohol exploitation has
found the similar outcome in term of an erectile problem as smoking (Valentina et al., 2010).
While various meta-analysis reveals reasonable and extra recurrent physical exercises reduces
the risk of erectile dysfunction (Cheng et al., 2007).
4.2. Medical Conditions
Various medications develop erectile dysfunction. Whereas diuretics (thiazide) and β-

blockers reduce sexual libido and leading erectile problem. In addition to many drugs like anti-
ulcer drugs, anti-androgens, psychotherapeutics, opiates, and digoxin develop erectile
dysfunction. Chlorthalidone significantly effects on sexual function at more than 2 years its
treatment (Francis et al., 2007).

4.3. Tobacco Abuse
Tobacco affects sexual life in three ways; erectile dysfunction diminishes sexual libido and
induces infertility. Smoking develops cardiovascular disorders which are leading impairment
in blood circulations. It is well known that during erection, the blood vessel of penis dilated
and enlarge leads to filling blood. But smoking impaired normal physiology of blood vessel
dilation and also proper filling the blood in the blood vessel of penis resulting develops erectile
dysfunction (McVary et al., 2001; Brackett et al., 2010).
5. PHYSIOLOGY AND PATHOPHYSIOLOGY MECHANISM

OF ERECTILE DYSFUNCTION
It is a complex neurovascular phenomenon which includes neurohormonal, cardiovascular

and psychosomatic factors. This phenomenon collectively works together intended for making
a regular penile erection.
5.1. Neurogenic Erectile Dysfunction
It is due to insufficiency in impulse conduction into the penis. It occurs due to diabetes,
traumatic brain injury, radical pelvic surgery, spinal cord injury, etc. If lesions at above spinal
nerve T10 may slow down the impulse conduction arbitrate managing erection and spinal cord
lesions sacral (S2-S4) is characteristically accountable in favor of reflex to go up erections
results reduction in NO concentration resulting veno-occlusive impaired functions (Ferrini et
al., 2006; Ferrini et al., 2009).
5.2. Vasculogenic Erectile Dysfunction
Cardiovascular problems decrease blood supply leads to an erectile problem. It also occurs
due to reduced flexibility of arterial wall which causes enhance blood pressure. Further much
cardiovascular disorder and smoking enhance risk and decreases blood supply and also
oxygenation in penile regions lead to decreases level of prostaglandin E which promotes
collagen deposition, resulting in decreased elasticity of the penis (Moreland et al., 1995).
5.3. Role of Nitric Oxide Synthase and Nitric Oxide in Penile Erection
L-Arginine is participated to formation NO by the L-arginine NO pathway (Palmer et al.,

1987). It is catalyzing by the help of nitric oxide synthetase (NOS) to the manifestation of NO
from (Palmer et al., 1988). NO implicates in many physiological functions, including regulation
of immune system function and neural transmission in vascular tissue (Moncada et al., 1991).
Endothelial-derived relaxing factor (EDRF) like nitric oxide arbitrates vasodilator effects by
relaxation smooth-muscle leads penile erection (Burnett, 1997). Penile cavernosal nerve
endings liberate neurotransmitter acetylcholine which stimulates the nitric oxide synthetase in

neuron and it helps to discharge of NO from the blood vessel and it plays a most impotent role
in erectile response (Burnett et al., 1992). The normal erectile response is arbitrated via the
mutual contribution of neuronal and endothelial NOS (Boushey et al., 1995). It is well-
documented steroids, cholesterol, hypertension, and cytokines are down-regulated the
appearance of endothelium Nitric oxide synthetase (Maas et al., 2002).
5.3.1. Arginase Downregulates Nitric Oxide Production

L-Arginine is a precursor to the synthesis of nitric oxide synthetase. Kidney failure patients
who have increased concentration of uric acid and creatinine in the blood, they suffer from
erectile dysfunction because arginine neither transport into the cell nor participate in the
synthesis of nitric oxide (Xiao et al., 2001; Bivalacqua et al., 2001).
5.3.2. Mechanism of Nitric Oxide and Phosphodiesterase Type 5

Contraction smooth muscle of penis is harmonized by collective effect of sympathetic
control, native neuromuscular control, endothelins and prostaglandin (Figure 1) (Andersson
and Wagner, 1995; Saenz et al., 1989; Lue, 2000). Non-adrenergic noncholinergic (NANC)
nerve fibers release NO whether sexual stimulation occurs and parasympathetic cholinergic
nerve fibers release acetylcholine (Figure 2). Both are ensuing signaling pathways lead to
increases concentration cGMP and decreases Ca2+transport in the cell results penile muscle
relaxation caused by blood supply is enhanced which leads penile erection. The concentration
of cyclic GMP (cGMP) is decreased by the catalytic effect phosphodiesterase type 5 (PDE-5)
leads to erectile dysfunction due to decreases penile blood flow (Lue and Tanagho, 1987).
Figure 1. Physiological mechanism of normal erection. In endothelium nitric oxide is synthesized with
the presence of nitric oxide synthase (NOS). It diffuses into the smooth muscle and stimulates Guanylyl
cyclase resulting increase concentration cyclic GMP (cGMP) leads to potassium channels to open and
closed the calcium channels and develops hyperpolarization leading to relaxation in smooth muscle.

5.3.3. Nitric Oxide Defense Mechanism against Reactive Oxygen Species

Jones et al., (2002) has reported that vasodilatation effect of NO is overcome with the
interaction of ROS leading to erectile dysfunction. However, NO reacts with ROS to make
peroxynitrite which plays a central role in atherogenesis process (Beckman and Koppenol,
1996). Zou et al., 1997 reported that Peroxynitrite inactivates superoxide dismutase and
decreases the deduction of superoxide. It is promoted to the development of peroxynitrite and
significantly decreases concentration nitric oxide. Khan et al., (2001) reported that NO is short
lived and immediate pharmacological action like vasodilatation whereas on the behalf of nitric
action and frequent termination of vasodilatation effect, however, peroxynitrite has very slow
and extended onset action resulting blood vessel unable to recover their normal tension. That
is why the interaction of nitric oxide with ROS has ultimately produced an ineffective
relaxation in cavernosal tissue and leads to erectile dysfunction. It is well documented that if
increases reactive oxygen species interactions which significantly decrease to release nitric
oxide from endothelium leading to pathological mechanism of erectile dysfunction and
additional free radical potentiates vasoconstriction leads to reduces blood flow in penile tissue.
Thus these are two concept free radicals produce a synergistic effect on developing erectile
dysfunction (Katusic and Vanhoutte, 1989; Jeremy et al., 2000) shows in Figure 2.
Figure 2. Correlation between reactive oxygen species and erectile dysfunction. Superoxide radicals
form peroxynitrite, stimulates apoptosis, and develops endothelial dysfunction and peroxynitrite also
stimulates superoxide dismutase (SOD), leading to ineffective smooth muscle relaxation, and enhance
platelet adhesion to endothelium. NO, Nitric oxide; H2O2, Hydrogen peroxide
6. ANTIOXIDANTS DEFENSES AGAINST OXIDATIVE STRESS-

MEDIATED ERECTILE DYSFUNCTION
Antioxidants have good ability to scavenge the free radicals and protect body organs but it
is decreased in body organs resulting also decreases protection activity and haphazard damage

occurs due to overactive of oxygen free radicals. On the basis of availability, antioxidants are
endogenous and exogenous.
Endogenous antioxidants are products of the body’s metabolism, may be enzymatic or non-
enzymatic. This antioxidant enzyme protest against erectile dysfunction and occurs due to
oxidative stress. There is mainly three types of cellular antioxidant enzyme which is as follow:
6.1.1. Glutathione
Glutathione (GSH) is an endogenous antioxidant. It plays as a vital cofactor for nitric oxide
synthetase and further help to the synthesis of nitric oxide (Harbrecht et al., 1997). GSH
concentration decreases during defense against free radical oxidative stress in diabetes resulting
in synthesis and release of nitric oxide are significant decreases which lead to erectile
dysfunction (Tagliabue et al., 2005). So that if glutathione maintains a normal level which
prevents to develops erectile dysfunction due to it has a significant antioxidant defense against
oxidative stress (Tagliabue et al., 2005).

Superoxide dismutase (SOD) protects body organs from oxidative stress which is initiated
by free radicals (Lobo et al., 2010). It can be used for the treatment of many diseases like
inflammations, renal disease, gout, cancer, etc. It is also protected and develops resistance
against radiotherapy, renal transplantation, and decreases cardiac impairment. It is significantly
produced a protective effect against reactive oxygen species which leads to organs impairments
and further organs disorders (De Benito et al., 2001; Flaherty et al., 1994; Land et al., 1994).
Superoxide dismutase significantly restores erectile dysfunction in diabetic animals
mediated impaired endothelium functions and also impaired penile muscle relaxation (Khan et
al., 2001; Moncada et al., 1991).
6.1.3. Catalase
Catalase (CAT) has a powerful antioxidant property. It is acting as a super antioxidant and
is protects from reactive oxygen species-mediated oxidative stress (Giordano et al., 2015).
Erectile dysfunction drugs induce the accelerates SOD and catalase resulting decreases MDA
level and promotes the erection mechanism via increases nitric oxide-cGMP level (Sheweita et
al., 2015).
6.2. Natural Antioxidants
Natural antioxidants can be found in fruits and vegetables. Carotenoids, flavonoids,

vitamins are most used in the prevention and restore erectile dysfunction in the completion of
free radical-mediated oxidative stress. Some of them mentioned as follows:

6.2.1. Tocopherol
It is a potent antioxidant and significantly decreases free radical induces oxidation of lipid
(Esterbauer et al., 1991). Whereas it reacts more rapidly with epoxy radicals than an unsaturated
lipid, therefore it’s called a lipid phase. Keegan et al., (1995) reported as tocopherol enhances
nitric oxide-mediated vasodilatations and enhance endothelial function tocopherol (vitamin E)
reverses protein C kinase establishment and improve endothelial function (Keegan et al., 1995).
PDE-5 inhibitors are managed cGMP and have a good ability to maintaining the erection. So it
is mostly used by diabetes erectile dysfunction patient (Rendell et al., 1999). Tocopherol
improves PDE-5 inhibitor response in diabetes rodents. So that vitamin E uses is more
preferable than those who have not managed treatment PDE-5 inhibitors (De et al., 2003).
Therefore, vitamin E repair endothelium function and significantly enhances nitric oxide
release and also synergic effect with a PDE-5inhibitor in the treatment of ED. So, vitamin E as
a potent antioxidant as well as defense against erectile dysfunction.
6.2.2. α-Linolenic Acid

In the animal diabetic model, it has been reported that α-linolenic acid (ALA) significantly
prevents penile erectile trouble (Ben et al., 2017). It has good antioxidant properties so that it
makes a complex with metals and also diminished oxidative stress. It has been also found that
ALA conserves smooth muscle relaxation in diabetic rodents. Diabetes mediated erectile
dysfunction occur due to diminished endothelium functions resulting also minimized nitric acid
release leads to decreases relaxation of penile smooth muscle. Linolenic acid prevents
cavernosal dysfunction because o their significant antioxidant defense (Keegan et al., 1999).
6.2.3. Vitamin C
Antioxidant vitamin C improves blood circulation in type 2 diabetes people. Vitamin C
proves as a potent competitor against diabetes-induced vascular disease like retinopathy,
nephropathy, and atherosclerosis (Ting et al., 1996). Thus vitamin C can help to treatment in
erectile dysfunction of diabetic patients.
6.2.4. Resveratrol
Resveratrol is an active ingredient of many natural products of many fruits and has
polyphenols and flavonoids antioxidant which protects from cardiac vascular disorders. It
diminishes oxidative stress-mediated erectile dysfunction which is developed during radiation
therapy in prostate carcinoma. Radiotherapy decrease neuronal and endothelium nitric oxide
synthetase and sirtuin gene like protein expressions which are reversed by resveratrol.
Therefore, resveratrol is a potent antioxidant which has a significant defense mechanism
against erectile dysfunction (Tarik et al., 2018).
6.2.5. Quercetin
It is flavonoids which are obtained from green leaf, fruits, and various medicinal plants. It
is a potent antioxidant (Anand et al., 2016). It is not produced any side effect when it is eating
at a high dose level. It has significant protective as well as curative activities against
cardiovascular disorders, allergic reaction, cancer, systemic inflammations, neuropathy,
diabetes, etc. Zhang et al., (2015) reported that quercetin protects to appearance and functions
of endothelium nitric oxide synthetase in penile endothelial cells, and restores normal functions

of NO-cGMP passageway which leads to of penile erection. It has well antioxidant defense to
significantly prevent erectile dysfunction.
6.2.6. Curcumin
It is a yellow color crystalline which obtained from rhizomes of the Curcuma longa, has
been traditionally used in medicine and culinary practices in India (Krup et al., 2013). It
possesses various pharmacological effect viz antioxidant, hepatoprotective, anti-inflammatory,
anti-thrombosis and anti-apoptosis. It has been reported that nanoformulation of curcumin
applied as tropically which can be treated to erectile dysfunction (Draganski et al., 2018)
Curcumin boosted to release NO a leading to significant improves in erectile dysfunctions.

AND CHALLENGES
Cardiovascular disease is major cause of erectile dysfunction as compared psychological

factor because cardiovascular diseases effect on endothelium functions and also nitric oxide
release leads to erectile dysfunction whereas psychological factor which is created erectile
dysfunction can be cured by suitable counseling or nominal treatment as compared
cardiovascular diseases (Goldstein, 2000; Loprinzi and Nooe, 2015). It well documented that
with 2-year study in men suffered erectile dysfunction makes significant improve by eating
protein and fiber-rich food, green vegetables, and fruits, and vegetative oil or ghee because it
improves high-density lipoprotein and good cholesterol (Esposito et al., 2006). Various studies
from 10 years back on more than 25000 men, it has been found that maximum eating of fruits
can reduce approximately 15% risk of an erectile problem because fruits have potent
antioxidants like polyphenols and flavonoids are a diminished risk of erectile dysfunction. It
has been reported that to reduced risk of an erectile problem by eating a sufficient amount of
blueberries and citrus fruits (Cassidy et al., 2016).
Gene therapy is a new and good option as a treatment of erectile dysfunction. Many
scientists are trying to enhance the synthesis of endothelial nitric oxide leads to cure erectile
dysfunction by the help of gene therapy. Actually, gene codon which expresses to the synthesis
of endothelium nitric oxide enzyme transfers to aged rodent by the help of vector-like
adenovirus and furthers their gene expression enhances erectile response (Bivalacqua et al.,
2003). Gene therapy is a new and most significant therapeutic concept to be used for treating
erectile dysfunction. Gene encoding extracellular SOD incorporates rats resulting in the
significant synthesis of extracellular messenger RNA, cGMP, SOD and their activities which
are acting as very good monitoring leads to treating erectile dysfunctions. This is a novel
technique can be used to repair erectile response. Gene therapy will be used as a significant
future therapeutic choice for the management of erectile dysfunction.
Major challenges, the antioxidant preclinical studies results against erectile dysfunction
which is not accurately reproducible in clinical studies. Antioxidants increase the release of
nitric oxide but the measurement of NO concentration is not possible because it has a very short
half-life. So that effectiveness of nitric oxide measures indirectly on the behalf of degree
relaxation of cavernosal smooth muscle.

CONCLUSION
This chapter discusses the role of oxidative stress in ED and antioxidants defense. The
etiology of ED is due to an imbalance of neurohormonal and psychological coordination, the
pathophysiological status of a person which also appears as symptoms of erectile dysfunction.
The risk factors of erectile dysfunction consist of lifestyles of person and, drugs abuses and
drugs used for the treatment of the patient. The mechanism of ED is a complex neurovascular
phenomenon which manifestations may be due to neurogenic or vasculogenic or abnormality
of synthesis of NOS or release of NO. Whereas antioxidants, flavonoids, vitamins are well as
defense against erectile dysfunctions by significantly improves and maintains the integrity of
the endothelium function to releasing NO leads significantly contributes pick up erectile
function.
REFERENCES
Agarwal, A., Nandipati, K.C., Sharma, R.K., Zippe, C.D. and Raina, R. (2006). Role of
oxidative stress in the pathophysiological mechanism of erectile dysfunction. J Androl,
27(3):335-47.
Anand David, A.V., Arulmoli, R. and Parasuraman, S. (2010). Overviews of Biological
Importance of Quercetin: A Bioactive Flavonoid. Pharmacogn Rev, 10(20):84-89.
Andersson, K.E. and Wagner, G. (1995). Physiology of penile erection. Physiol Rev, 75:191-
236.
Aviram, M. and Dornfeld, L. (2001). Pomegranate juice consumption inhibits serum
angiotensin converting enzyme activity and reduces systolic blood pressure. Atheroscleros,
158: 195-98.
Azadzoi, K. M., Schulman, R., Michael, A. and Siroky, M.B. (2005). Oxidative stress in
arteriogenic erectile dysfunction: prophylactic role of antioxidants. J Urol, 174, 386-93.
Azadzoi, K.M., Kim, N., Brown, M.L., Goldstein, I., Cohen, R.A. and Saenz de Tejada I.
(1992). Endothelium‐derived nitric oxide and cyclooxygenase products modulate corpus
cavernosum smooth muscle tone. J Urol, 147: 220-225.
Beckman, J.S. and Koppenol, W.H. (1996). Nitric oxide, superoxide, and peroxynitrite: the
good, the bad, and ugly. Am J Physiol, 271:1424-1437.
Bello‐Klein, A., Bock, P.M., Travacio, M., Senna, S.M., Llesuy, S., de Bittencourt, Pl Jr,
Irigoyen, M.C., Bello, A.A., Kumar D. and Singal P.K. (2001). Myocardial oxidative stress
and antioxidants in hypertension as a result of nitric oxide synthase inhibition.
CardiovascToxicol, 1: 43-50.
Ben Khedher, M.R., Bouhajja, H., Haj, A. S., Abid, M., Jamoussi, K. and Hammami, M.
(2017). Role of disturbed fatty acids metabolism in the pathophysiology of diabetic erectile
dysfunction. Lipids Health Dis, 16(1):241.
Bivalacqua, T.J., Armstrong, J.S., Biggerstaff, J., Abdel-Mageed, A.B., Kadowitz, P.J.,
Hellstrom, W.J. and Champion H.C. (2003). Gene transfer of extracellular SOD to the
penis reduces O2-* and improves erectile function in aged rats. Am J Physiol Heart Circ
Physio, 284:1408-1421.

Bivalacqua, T.J., Hellstrom, W.J., Kadowitz, P.J. and Champion, H.C. (2001). Increased
expression of arginase II in human diabetic corpus cavernosum: in diabetic-associated
erectile dysfunction. BiochemBiophys Res Commun, 283:923-927.
Boushey, C.J., Beresford, S.A., Omenn, G.S. and Motulsky, A.G. (1995). A quantitative
assessment of plasma homocysteine as a risk factor for vascular disease: probable benefits
of increasing folic acid intakes. JAMA, 274:1049-1057.
Brackett, N.L., Lynne, C.M., Ibrahim, E., Ohl D.A. and Sonksen, J. (2010). Treatment of
infertility in men with spinal cord injury. Nat Rev Urol, 7:162-172.
Burnett, A.L. (1997). Nitric oxide in the penis: physiology and pathology. J Urol,157:320-324.
Burnett, A.L., Lowenstein, C.J., Bredt, D.S., Chang, T.S. and Snyder, S.H. (1992). Nitric oxide:
a physiologic mediator of penile erection. Science,257:401-403.
Cao, S., Gan, Y., Dong, X., Liu, J. and Lu, Z. (2004). Association of quantity and duration of
smoking with erectile dysfunction: a dose–response meta-analysis. J Sex Med, 11:2376-
2384.
Cassidy, A., Franz, M. and Rimm, E.B. (2016). Dietary flavonoid intake and incidence of
erectile dysfunction. Am J ClinNutr, 103(2):534-541.
Cheng, J.Y., Ng, E.M., Ko, J.S. and Chen, R.Y. (2007). Physical activity and erectile
dysfunction: meta-analysis of population-based studies. Int J Impot Res, 19:245-252.
Corona, G., Giorda, C.B., Cucinotta, D., Guida, P., Nada, E. and Gruppo di S. (2014). Sexual
dysfunction at the onset of type 2 diabetes: the interplay of depression hormonal and
cardiovascular factors. J Sex Med, 11:2065-73.
Corona, G., Mannucci, E., Mansani, R., Petrone, L., Bartolin, M., Giommi, R., Mancini, M.,
Forti, G. and Maggi, M. (2104). Aging and pathogenesis of erectile dysfunction. Int J Impot
Res, 16: 395-402.
Corona, G., Rastrelli, G., Balercia, G., Lotti, F., Sforza, A., Monami, M., Forti, G., Mannucci,
E. and Maggi, M. (2012). Hormonal association and sexual dysfunction in patients with
impaired fasting glucose: a cross-sectional and longitudinal study. J Sex Med, 9:1669-1680.
De Benito, V., de Barrio, M., de Lopez-Saez, M.P., Ordoqui, E., Prieto-García, A., Sainza, T.
and Baeza M.L. (2001). Anaphylactic shock caused by impurities in orgotein preparations.
AllergolImmunopathol (Madr), 29:272-275.
De Young, L., Yu, D., Freeman, D. and Brock, G.B. (2003). Effect of PDE5 inhibition
combined with free oxygen radical scavenger therapy on erectile function in a diabetic
animal model. Int J Impot Res, 15:347-54.
Dong, J.Y., Zhang, Y.H. and Qin, L.Q. (2011). Erectile dysfunction and risk of cardiovascular
disease: metaanalysis of prospective cohort studies. J Am CollCardiol, 58:1378-1385.
Draganski, A., Tar, M.T., Villegas, G., Friedman, J.M. and Davies, K. (2018). Topically
Applied Curcumin-Loaded Nanoparticles Treat Erectile Dysfunction in a Rat Model of
Type-2 Diabetes. J Sex Med, 15:645-653.
Esposito, K., Ciotola, M., Giugliano, F., De Sio, M., Giugliano, G., Darmiento, M. and
Giugliano, D. (2006). Mediterranean diet improves erectile function in subjects with the
metabolic syndrome. Int J Impot Res, 18:405-410.
Esterbauer, H., Dieber-Rotheneder, M., Striegl, G. and Waeg, G. (1991). Role of vitamin E in
preventing the oxidation of low-density lipoprotein. Am J ClinNutr, 53:314-21.
Feldman, H.A., Goldstein, I., Hatzichristou, D.G., Krane, R.J. and McKinlay, J.B. (1994)
Impotence and its medical and psychosocial correlates: Results of the Massachusetts Male
Aging Stud. J Urol, 151:54-61.

Fernandez‐Arjona, M., Díaz, J., Carbonell, L., Oteros, R., Zarca, M.A. and Cortes, I. (2001)
Antioxidative state and lipid peroxidation in erectile dysfunction: preliminary study. Arch
Esp Urol. 54:361-366.
Ferrara, N., Abete, P., Ambrosio, G., Landino, P., Caccese, P., Cirillo, P., Oradei, A., Littarru,
G.P., Chiariello, M., Rengo, F. (1995) Protective role of chronic ubiquinone administration
on acute Cardiac oxidative stress. J PharmacolExp Therapy. 274:858-865.
Ferrini, M.G., Davila, H.H., Kovanecz, I., Sanchez, S.P., Gonzalez-Cadavid, N.F. and Rajfer,
J. (2006). Vardenafil prevents fibrosis and loss of corporal smooth muscle that occurs after
bilateral cavernosal nerve resection in the rat. Urology, 68:429-435.
Ferrini, M.G., Kovanecz, I., Sanchez, S., Umeh, C., Rajfer, J. and Gonzalez-Cadavid, N.F.
(2009). Fibrosis and loss of smooth muscle in the corpora cavernosa precede corporal veno-
occlusive dysfunction (CVOD) induced by experimental cavernosal nerve damage in the
rat. J SexMed, 6:415-428.
Flaherty, J.T., Pitt, B., Gruber, J.W., Heuser, R.R., Rothbaum, D.A., Burwell, L.R., George,
B.S., Kereiakes, D.J., Deitchman, D. and Gustafson, N. (1994). Recombinant human
superoxide dismutase (h-SOD) fails to improve recovery of ventricular function in patients
undergoing coronary angioplasty for acute myocardial infarction. Circulation, 89:1982-
1991.
Francis, M.E., Kusek, J.W., Nyberg, L.M. and Eggers, P.W. (2007). The contribution of
common medical conditions and drug exposures to erectile dysfunction in adult males. J
Urol, 178:591-596.
Fuhrman, B., Lavy, A. and Aviram, M. (1995). Consumption of red wine with meals reduces
the susceptibility of human plasma and LDL to undergo lipid peroxidation. Am J ClinNutr,
61:549-554.
Giordano, C.R., Roberts, R., Krentz, K.A., Bissig, D., Talreja, D., Kumar, A., Terlecky, S.R.
and Berkowitz, B.A. (2015). Catalase therapy corrects oxidative stress-induced
pathophysiology in incipient diabetic retinopathy. NvestOphthalmol Vis Sci, 56(5):3095-
3102.
Giovanni, C., Giulia, R., Sandra, F., Linda, V., Edoardo, M. and Mario, M. (2014). Erectile
dysfunction and central obesity: an Italian perspective. Asian J Androl, 16:581-591.
Goldstein, I. (2000). Male sexual circuitry. Working Group for the Study of Central
Mechanisms in Erectile Dysfunction. Scientific American, 283:70-75.
Grein, U. and Schubert, G.E. (2002). Arteriosclerosis of penile arteries: histological findings
and their significance in the treatment of erectile dysfunction. Urol Int, 68: 261-264.
Harbrecht, B.G., Di Silvio, M., Chough, V., Kim, Y.M., Simmons, R.L. and Billiar, T.R.
(1997). Glutathione regulates nitric oxide synthase in cultured hepatocytes. Ann Surg.
225(1): 76-87.
Helmut, S. (2005). Strategies of antioxidant defense. Eur J Biochem, 215, 213-219.
Jeremy, J.Y., Angelini, G.D., Khan, M., Mikhailidis, D.P., Morgan, R.J., Thompson, C.S.,
Bruckdorfer, K.R. and Naseem, K.M. (2000). Platelets, oxidant stress and erectile
dysfunction: an hypothesis. Cardiovasc Res, 46:50–54.
Jones, R.W., Rees, R.W., Minhas, S., Ralph, D., Persad, R.A. and Jeremy, J.Y. (2002). Oxygen
free radicals and the penis. Expert Opin Pharm, 3:889-897.
Kaiser, F.E., Viosca, S.P., Morley, J.E., Mooradian, A.D. and Davis, S.S. (1988). Korenman,
S.G. Impotence and aging: clinical and hormonal factors. J Am Geriatr Soc, 36: 511-519.

Katusic, Z.S. and Vanhoutte, P.M. (1989). Superoxide anion is an endotheliumderived

contracting factor. Am J Physiol, 257:33-37.
Keegan, A., Cotter, M.A. and Cameron, N.E. (1999). Effects of diabetes and treatment with the
antioxidant alpha-lipoic acid on endothelial and neurogenic responses of corpus
cavernosum in rats. Diabetologia, 42:343-350.
Keegan, A., Walbank, H., Cotter, M.A. and Cameron, N.E. (1995). Chronic vitamin E treatment
prevents defective endothelium-dependent relaxation in diabetic rat aorta. Diabetologia,
38:1475-1478.
Khan, M.A., Thompson, C.S., Jeremy, J.Y., Mumtaz, F.H., Mikhailidis, P. and Morgan, R.J.
(2001). The effect of superoxide dismutase on nitric oxide-mediated and electrical field-
stimulated diabetic rabbit cavernosal smooth muscle relaxation. BJU Int, 87(1):98-103.
Khan, M.A., Thompson, C.S., Mumtaz, F.H., Mikhailidis, D.P., Morgan, R.J., Bruckdorfer,
R.K. and Naseem, K.M. (2001). The effect of nitric oxide and peroxynitrite on rabbit
cavernosal smooth muscle relaxation. World J Urol, 19:220-224.
Kinlay, S., Behrendt, D., Fang, J.C., Delagrange, D., Morrow, J., Witztum, J.L., Rifai, N.,
Selwyn, A.P., Creager, M.A. and Ganz, P. (2004). Long term effect of combined vitamins
E and C on coronary and peripheral endothelial function. J AmCollCardiol, 43: 629-635.
Krup, V., Prakash, L.H. and Harini, A. (2013). Pharmacological Activities of Turmeric
(Curcuma longa linn): A Review. J HomeopAyurv Med, 2:133.
Land, W., Schneeberger, H., Schleibner, S., Illner, W.D., Abendroth, D., Rutili, G., Arfors,
K.E. and Messmer K. (1994). The beneficial effect of human recombinant superoxide
dismutase on acute and chronic rejection events in recipients of cadaveric renal transplants.
Transplant, 57:211-218.
Lobo, V., Patil, A., Phatak, A. and Chandra, N. (2010). Free radicals, antioxidants and
functional foods: Impact on human health. Pharmacogn Rev, 4(8): 118-126.
Loprinzi, P.D. and Nooe, A. (2015). Erectile Dysfunction and Mortality in a National
Prospective Cohort Study. The journal of sexual medicine, 12:2130-133.
Lue, T.F. (2000). Erectile dysfunction. N Engl J Med, 342:1802-1813.
Lue, T.F. and Tanagho E.A. (1987). Physiology of erection and pharmacological management
of impotence. J Urol,137:829-36.
Maas, R., Schwedhelm, E., Albsmeier, J. and Boger, R.H. (2002). The pathophysiology of
erectile dysfunction related to endothelial dysfunction and mediators of vascular function.
Vasc Med, 7:213-225.
McCabe, M.P. and Althof, S.E. (2014). A systematic review of the psychosocial outcomes
associated with erectile dysfunction: does the impact of erectile dysfunction extend beyond
a man’s inability to have sex. J Sex Med, 11:347-363.
McVary, K.T., Carrier, S. and Wessells, H. (2001). Subcommittee on Smoking and Erectile
Dysfunction Socioeconomic Committee, Sexual Medicine Society of North America.
Smoking and erectile dysfunction: evidence based analysis. J Urol, 166:1624-1632.
Moncada, S., Palmar, R.M. and Higgs, E.A. (1991). Nitric Oxide:physiology, pathophysiology,
and pharmacology. Pharmacol Rev. 43:109-142.
Moreland, R.B., Traish, A., McMillin, M.A., Smith, B., Goldstein, I. and Saenz de Tejada, I.
(1995). PGE1 suppresses the induction of collagen synthesis by transforming growth
factor-β 1 in human corpus cavernosum smooth muscle. J Urol, 153:826-834.
Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity
of endothelium-derived relaxing factor. Nature, 1987; 327:524-26.

Palmer, R.M., Ashton, D.S. and Moncada, S. (1988). Vascular endothelial cells synthesize
nitric oxide from L arginine. Nature, 333:664-666.
Qutub, A.A. and Popel, A.S. (2008). Reactive oxygen species regulate hypoxia‐inducible factor
1 differentially in cancer and ischemia. Mol Cell Biol, 28:5106-5119.
Rendell, M.S., Rajfer, J., Wicker, P.A. and Smith, M.D. (1999). Sildenafil for treatment of
erectile dysfunction in men with diabetes: a randomized controlled trial. Sildenafil
Diabetes Study Group. JAMA, 281:421-426.
Saenz de Tejada, I., Kim, N., Lagan, I., Krane, R.J. and Goldstein, I. (1989). Regulation of
adrenergic activity in penile corpus cavernosum. J Urol, 142:1117-1121.
Sener, T.E., Tavukcu, H.H., Atasoy, B.M., Cevik, O., Kaya, O.T., Cetinel, S., Dagli Degerli,
A. Tinay, I., Simsek, F., Akbal, C., Buttice S. and Sener, G. (2018). Resveratrol treatment
may preserve the erectile function after radiotherapy by restoring antioxidant defence
mechanisms, SIRT1 and NOS protein expressions. Int. J Impot Res, 30: 179-188.
Sheweita, S., Salama, B. and Hassan, M. (2015). Erectile dysfunction drugs and oxidative
stress in the liver of male rats. Toxicol Rep, 6:2:933-938.
Skrypnik, D., Bogdański, P. and Musialik, K. (2014). Obesity-significant risk factor for erectile
dysfunction in men. Pol MerkurLekarski, 36(212):137-141.
Tagliabue, M., Pinach, S., Di Bisceglie, C., Brocato, L., Cassader, M., Bertagna, A., Manieri,
C. and Pescarmona, G.P. (2005). Glutathione levels in patients with erectile dysfunction,
with or without diabetes mellitus. Int J Androl, 28(3):156-62.
Ting, H.H., Timimi, F.K., Boles, K.S., Creager, S.J., Ganz, P. and Creager, M.A. (1996).
Vitamin C can improve circulation Journal of Clinical Investigation, 97:22-28.
Valentina, B., Giovanni, C., Matteo, M., Alessandra, D.F. and Elisa, B. (2010). Priapus is
happier with Venus than with Bacchus. J Sex Med, 2010; 7:2831-2841.
Vlachopoulos, C. V., Terentes-Printzios, D. G., Ioakeimidis, N. K., Aznaouridis, K. A. and
Stefanadis, C. I. (2103). Prediction of cardiovascular events and all-cause mortality with
erectile dysfunction: a systematic review and meta-analysis of cohort studies.
CircCardiovascQual Outcomes, 2013; 6:99-109.
Xiao, S., Wagner, L., Mahaney, J. and Baylis, C. (2001). Uremic levels of urea inhibits L-
arginine transport in cultured endothelial cells. Am J Physiol Renal Physiol, 280: 989-995.
Yafi, F.A., Jenkins, L., Albersen, M., Corona, G., Isidori, A.M., Goldfarb, S., Maggi, M.,
Nelson, C.J., Parish, S., Salonia, A., Tan, R., Mulhall, J.P. and Hellstrom, W.J. (2016).
Erectile dysfunction. Nat Rev Dis Primers, 2:1-47.
Zhang, Q., Radisavljevic, Z.M., Siroky, M.B. and Azadzoi, K.M. (2010). Dietary antioxidants
improve arteriogenic erectile dysfunction. INT JAndrol, 34:225-235.
Zhang, Y., Huang, C., Liu, S., Bai, J., Fan, X., Guo, J., Jia, Y., Zhang, Z., Chen, X., Jia, Y.,
Zhang, P., Wang, B. and Zhang, X. (2015). Effects of quercetin on intracavernous pressure
and expression of nitrogen synthase isoforms in arterial erectile dysfunction rat model. Int
J ClinExp Med, 15; 8(5):7599-7605.
Zou, M., Martin, C. and Ullrich, V. (1997). Tyrosine nitration as a mechanism of selective
inactivation of prostacyclin synthase by peroxynitrite. Biol Chem, 378:707-713.

Chapter 19
OXIDATIVE STRESS AND ANTIOXIDANT STATUS

IN PREGNANCY
Parag A. Pathade* and Vinod A. Bairagi

KBHSS Trust’s Institute of Pharmacy, Malegaon, India
ABSTRACT
During earlier stages of pregnancy, oxidative stress is generated; however, when the
supply of antioxidant micronutrients is inadequate, exaggerated oxidative stress within
both maternal circulation and placenta occurs, resulting in intrauterine, fetal and neonatal
infections, premature birth, and preeclampsia. So pregnancy is a physiological state
characterized by an oxidative disturbance that contributes to the initiation and progression
of complications associated with pregnancy. Elements capable of preventing or stopping
the effects of oxidants are called antioxidants. A number of micronutrients including trace
elements like selenium, copper, zinc, manganese, and vitamins A, C, E function as essential
cofactors for many cellular enzymes, endogenous antioxidants or themselves acting as
antioxidants such as superoxide dismutase (SOD), and glutathione peroxidase and catalase.
Supplementation of such antioxidants in the daily diet of pregnant women over the period
of pregnancy may reduce the pathological complications in pregnancy. Micronutrient plays
an important role in upholding pregnancy and made central attention for forthcoming
health approaches in refining pregnancy consequences. This chapter represents the role of
antioxidants in pregnancy.
1. INTRODUCTION
In the period of prior to pregnancy, the generation of oxidative stress is common; though,
as soon as source of antioxidant micronutrients is insufficient, overstated oxidative stress inside
both maternal circulation and placenta arises, subsequent in intrauterine, fetal and neonatal
infections, premature birth, and preeclampsia (Ogbodo et al., 2012). Pregnancy is a
physiological state characterized by an oxidative disturbance that contributes to the initiation
*
Corresponding Author’s Email: paragapathade@gmail.com.

508 Parag A. Pathade and Vinod A. Bairagi
and progression of complications related to pregnancy. Oxygen which is responsible for many
harmful effects those are might be because of formation and activity of reactive oxygen species
(ROS) serve as oxidants, which is a substance with an affinity to contribute oxygen as
additional constituents (Droge, 2002). Most of the reactive oxygen species are serve as free
radicals. Any chemical substances that have one or more unpaired electrons are called free
radicals. Furthermost free radicals are unbalanced and extremely responsive. Reactive oxygen
species are those that contain reactive oxygen ions and peroxides and cause harmful effects at
high concentrations to biomolecules like nucleic acids (DNS, RNA), protein and lipids, leading
to pathological conditions in humans (Droge, 2002). In normal conditions, ROS are produced
as necessary intermediates and involve as secondary messengers in cell signaling in picomolar
concentrations. Excessive production of ROS and its uncontrolled regulation lead to
detrimental effects (Droge, 2002).
Unsaturated fatty acids are primarily oxidized to give lipid hydroperoxides. Production of
free radicals between body is the outcome of various metabolic processes (Droge, 2002). ROS
are one of the reactive free radical species, so their production in the body should be in control
to stop damage to cells and that control is provided by antioxidants and whenever balancing of
ROS and antioxidants get disturbed it results in tension to cell/oxidative stress (Aurousseau et
al., 2006). In terms of events of gestation like embryo implantation makeover of uterine tissue
settlement of villi development of villi ROS having great influence (Aurousseau et al., 2006).
During pregnancy different biochemical changes are on its paramount as a result of which
oxidative stress is high which alone can’t handle by antioxidants (Casanueva and Viteri, 2003).
If process of formation of free radicals from the pro-oxidants is not controlled rate of DNA,
lipid, and protein destruction is getting enhanced that has led to apoptosis (programmed cell
death) thus this pressure produced throughout pregnancy which is to be neutralized and for this
there is need of powerful mechanism of free radical scavenging (antioxidants) which a
counteract earlier apoptosis (Miller et al., 1993; Poston and Raijmakers, 2004). There is a
number of mechanisms meant for protection contrary to free radicals and further reactive
oxygen species different defense systems are active in different cellular sections e.g.,
glutathione peroxidases, superoxide dismutases and catalase which act defense coordination
system reduce the concentration of maximum damaging reactive species (Miller et al., 1993).
Superoxide dismutases are family of antioxidant enzymes which are significant in the catalytic
breakdown of superoxide radical leads production of bi-product. Breakdown of hydrogen
peroxide to water and oxygen proceed due to enzyme catalase whereas glutathione peroxidases
contain selenium which having importance in minimizing lipid oxidation by decreasing
formation of hydroperoxides (Poston and Raijmakers, 2004). The present chapter emphases
precisely on the importance of antioxidants in pregnancy, whereas the supply of these
antioxidants micronutrients is inadequate resulted in different complications in pregnancy. It
also focuses on those micronutrients which are associated with antioxidant activity due to the
importance of oxidative stress in both normal pregnancy and pathological pregnancy
consequences.

Oxidative Stress and Antioxidant Status in Pregnancy 509
2. OXIDANTS IN PREGNANCY
For the initial phase of pregnancy, it requires low levels of oxygen which is medically also
called physiological hypoxia of early gestational sac and that is a useful phenomenon in terms
of protection of developing fetus against cancerous effects of ROS (Shu and Ogbodo, 2005).
In order to grow fetus body demand for different vitamins, minerals micro and
macronutrients get increased consequence of speedily physiological changes, so body gets
depleted of vitamins and nutrients especially antioxidant vitamins too. Naturally, during the
gestational period, there is a need of such antioxidant vitamins, nutrients along with hemoglobin
(Shu and Ogbodo, 2005; Nwagha and Ejezie, 2005; Ogbodo et al., 2012). The placenta is rich
in mitochondria to fulfill energy needs, an electron transport chain is working in vicinity with
mitochondria, so it is obvious that production of ROS gets facilitated during kind of redox
reactions.
As the second trimester of pregnancy ends it peaks ROS production during this period there
are dynamic changes of multiple organ systems which leads to an increase in basal oxygen
consumption. Consequently during this period oxidative stress markers can be visible as
indicators of pregnancy-induced hypertension (PIH) called preeclampsia (Ogbodo et al., 2012).
That’s why it’s mandatory to pay attention to excessive oxidative damage during pregnancy
complications, including breakdown of syncytiotrophoblast. Therefore associated imbalance of
production of free radicals and antioxidant is hazardous to fetus and mother as well. It is seen
that maternal dendritic cells are responsible for catabolism of tryptophan as result maternity
immunity get lowered because of suppression of T-lymphocyte. Tryptophan metabolites are
responsible for apoptosis of T cells (Munn et al., 1998). This lowered immunity both beneficial
as well as disadvantageous to pregnant women, easily susceptible to infections in immune
deficiency. Therefore all set of related complications, fetal abnormalities and disease conditions
in pregnancy are linked mainly with free radicals (Munn et al., 1998; Kudo and Boyd, 2001;
Greenwood, 2004; Yip, 2006 Hung et al., 2010).
2.1. Oxidative Stress and Ovarian Functions
In physiological developments inside the ovary, free radicals play a very vital role whereas
oxygen is a keynote drive for gametes. Numerous literature as well studies have confirmed the
contribution of ROS in folliculogenesis and follicular-fluid atmosphere (Shiotani, 1991). Stress
has been generated by oxidation exposed to disturb midluteal corpus luteum and steroidogenic
capability in vitro as well in vivo. By means of utlizing corpora lutea gathered from pregnant
and nonpregnant patients, through usual circumstances copper, zinc superoxide dismutase (Cu–
Zn SOD) manifestation equals the levels of progesterone, through increase since initial luteal
to midluteal phase and reduction for period of deterioration of corpus luteum was perceived
(Mouatassim, 1999). The mRNA expression, yet, Cu–Zn SOD in corpus luteum during
pregnancy was greatly developed instead of midcycle corpora lutea. This elements improved
SOD expression in between pregnancy, probably due to amplified human chorionic
gonadotropin (HCG) levels, and may possibly reason of cell death of corpora lutea. Likewise,
antioxidant enzymes glutathione peroxidase and manganese superoxide dismutase (MnSOD)

is measured markers for cytoplasmic development, as these are articulated simply in metaphase
II oocytes (Mouatassim, 1999).
Poorly vascularized follicles outcome since the declined developmental potential of
oocytes has also been accredited to short intrafollicular oxygenation (Chui, 1997). Studies
validate strengthened lipid peroxidation in preovulatory Graafian follicle and glutathione
peroxidase may support to maintain reduce levels of hydroperoxides inside the follicle,
proposing an imperative part of oxidative stress generated in ovarian function. Oxidative stress
and inflammatory procedure must parts in pathophysiology of polycystic ovarian disease and
drugs such as Rosiglitazone possibly active by subsiding the levels of oxidative stress
(Sabuncu, 2001).
2.2. Oxidative Stress and the Endometrium
Recurring deviations between endometrium are attended by variations in countenance of

numerous antioxidants amongst endometrium. Enzymes, like thioredoxin, having advanced
appearance between early secretory stage and that might be essential for establishment
(Maruyama, 1997). It is remarkable to mention that targeted interruption because of thioredoxin
gene in mice has been connected through harmful impacts going on early embryo. In count,
improved manifestation of SOD occur in endometrium was perceived in late secretory stage
only earlier to menstruation. Tumour necrosis element alpha (TNF-α)-induced MnSOD
expression in human endometrial stromal cells were observed through nuclear factor kappa B
(NFκB) stimulation (Sugino, 2002). Estrogen or progesterone withdrawal led to increased
expression of cyclooxygenase-2 (COX-2). Such studies recommend role for ROS facilitated
NFκB initiation in endometrial functioning as associated with implantation and/or menstruation
(Hung et al., 2010).
2.3. Oxidative Stress and Embryo Development
During the pregnancy of mother development of embryo can be affected by imbalance of

antioxidant vitamin status is due to pregnancy where the growth of mother embryo can occur
(Shiotani, 1991). ROS behave as primary or secondary messengers on organized growth and
cell death. Some studies have been able to establish significant and straight role of ROS on
growth since redox status acts on regulation of certain transcription factors that affect
pathological cellular signaling due to production and erroneous differentiation developing near
to apoptosis (Baker, 2005).
Defective embryo growth and obstacle of embryo developing occur due to oxidative stress
which is accredited to tempted obliteration of DNA, destruction of cell-membrane and
apoptosis (Baker, 2005). Poor fertility outcomes from apoptosis consequences in split embryos,
with restricted potential for the implant. In addition, in vitro blastocyst establishment through
assisted reproductive technology (ART) is blastocyst development might be progress by
antioxidants. Deprived reproduction rates, impaired embryo growth, and increased chances of
pregnancy loss have been related with enlarged oxidative stress in male germ line (Baker,
2005). The reproduction and embryo growth in vivo takes place in an atmosphere of little

oxygen strain. Low oxygen strain progresses implantation and pregnancy rate in the period of
culture. Likewise, chances of clinical pregnancy rates are stated as increased after intake of
supplements of antioxidants used for ART instead of standard medium without antioxidants
(Greenwood, 2004).
In Haber–Weiss reaction •OH (hydroxyl radicals) produces as H2O2 (hydrogen peroxide)
and superoxide radical (O2•−). Oxidative stress results this reaction can monitor in cells. As
speed of this reaction is quite gentle, which is catalyzed by iron (Yip, 2006). In the initial phase
of catalytic cycle contains reduction of ferric ion to ferrous:
Fe3+ + O2−• → Fe2+ + O2
Additional Stage in Fenton Reaction:
Fe2+ + H2O2 → Fe3+ + OH− + OH•
Net Reaction:
O2−• + H2O2 → OH• + OH− + O2
Through this mechanism of Haber–Weiss reaction metal ions have also been encouraged
to creation ROS due to induction of metal ions. Metal ions can also boost formation of ROS
(Catt, 2000). Reduction of oxidants has been proceeding due to metal agent which serve as
chelating agent been added to media and also beneficial in fruitful development of embryo and
of course pregnancy (Catt, 2000). Literature revealed that by adding ascorbate throughout
cryopreservation decrease the hydrogen peroxide concentration and stop oxidation of embryos.
Antioxidants might show an important through ART trials but it also beneficial in stopping
consequent defeat or injury to the embryo.
2.4. Placental Oxidative Stress and Abortions
Implantation is such complex process in which relationships among the embryo and uterine
atmosphere is very well arranged, might be premature pregnancy is taking place due to
oxidative stress of placenta through the establishment of maternal circulation (Catt, 2000). As
due to significant disturbance of prooxidant and antioxidant equilibrium unprompted abortion
is occurred. Patient with recurring abortions with not exactly recognized etiology, in such
patient oxidative stress could have a role. Polymorphonuclear leucocytes (PMNL) number will
be increased during pregnancy due to which amplify generation of superoxide ions (Agarwal
and Gupta, 2005).
The moderate countenance of cytokine receptors is might occur due to oxidative stress in
cytotrophoblasts, placenta, and smooth muscle cells. Still, it is doubtful, though, if stress due
to oxidation can impact on posttranslational alteration that consequences in disappointment to
exciting development and distinction (Agarwal and Gupta, 2005). Two types of cytokine
responses, TH1 or TH2 production affected by T helper cells, which are controlled by
antioxidant glutathione (Agarwal and Gupta, 2005). Miscarriages in such patients having past
of repeated miscarriage connected through raised levels of TH1 and glutathione, although GSH

reduction signal to stoppage of TH1-type cytokines. The part of oxidative stress and
antioxidants in recurrent pregnancy loss in women is an attention-grabbing area for advanced
studies.
2.5. Oxidative Stress and Placenta
The placenta, a hormone-rich tissue, is an important source of pro-oxidizing agents, but

also of antioxidant enzymes, nevertheless, it is capable for maintaining lipid peroxidation under
control which increases during normal pregnancy (Jansson and Powell, 2007). In addition,
disturbances during maternal section can affect the placental gene methylation state plus a rise
in oxidative stress of placenta, causing alterations role of placenta (Jansson and Powell, 2007).
3. ANTIOXIDANTS IN PREGNANCY
Elements capable of preventing or stopping the effects of oxidants are called antioxidants.
Some metalloenzymes like SOD, and glutathione peroxidase and catalase, as well as, some
vitamins like vitamins A, C and E, and trace elements like copper, manganese, zinc, and
selenium, iron found to improve immune functions in human beings. Antioxidants reduce cell
damage by interfering the series of reactions involve in cellular damage or by donating
electrons or directly binding to free radicals (Ogbodo et al., 2006).
Natural antioxidant enzymes superoxide dismutase and glutathione peroxidase have the
ability to eliminate the reactive oxygen species which are playing a significant role in
inflammation. Metal ions are necessary for activation of these antioxidant enzymes for e.g.,
antioxidant property of superoxide dismutase (CuZnSOD) confers to enzyme due to presence
of copper in its structure. Activation of SOD and other enzymes needs manganese which in
turn helps in utilization of numerous key nutrients, e.g., vitamin E (Blaurock-Busch, 2010).
Pregnancy however it is complicated or uncomplicated, produces a large number of free
radicals which in turn increases oxidative stress, unfortunately, makes the pregnant women
susceptible to infections, maternal illnesses, congenital deformities, etc. (Bedwal and
Bahuguna, 1994).
Clinical studies have proven that use of vitamin supplements such as A, D, and E over a
period of pregnancy reducing intrauterine, fetal and neonatal infections, as well as reduces
complications of pregnancy such as preterm birth, premature membrane rupture and
preeclampsia (Brigelius-Flohe et al., 2002; Romero et al., 2003). During gestation period it has
been proved that serum levels of vitamin C and iron has a direct relationship and also many of
clinical studies proved that, continues the reduction in vitamin C level over gestation period,
lead to iron-deficiency anemia (Romero et al., 2003).
Many clinical studies show that pregnancy complications such as preeclampsia were due
to low levels of lipid-soluble antioxidants, also lack of antioxidants in daily diet during
gestation period in women believed to affect abnormal fetal and childhood growth (Evans and,
Halliwell, 2001). Deficiency trace elements like zinc in females lead to complications of
pregnancy such as recurrent abortion, stillbirth, preeclamptic toxaemia, extended gestation,
teratogenicity, difficulty in parturition, underweights in infants. Another trace element

selenium whose deficiency remarkably affects complication in pregnancy such as retention of

placenta, infertility, abortion because selenium is very important for normal functioning of
glutathione peroxidase (natural antioxidant) activities (Bedwal and Bahuguna, 1994).
Selenium-containing natural antioxidant enzymes like glutathione peroxidase and
thioredoxin reductase, are having ability to kill pathogens and reduces chances of infections
indirectly by protecting macrophages, neutrophils from free radical-induced damage which in
turn kill pathogen and fight against inflammation (Sappey et al., 1994).
Leafy vegetables, fruits contain natural antioxidant such as flavonoids, carnitine, carnosine
and polyphenols, consumption of such food shows great benefits to maintain a healthy lifestyle
during pregnancy, lower rates of cardiovascular disease and vice-versa. Epidemiological
studies suggest an intake of vegetable and fruits in daily diet reduces chances of development
of different types of oxidative stress induce infections during pregnancy (Chappell et al., 2002).
Regrettably peoples from rural areas mainly farmers who cultivate fruits and vegetables
not consume it but transport to metro cities for worthless of financial profit (Ogbodo, et al.,
2013). Hence, pregnancy care programmes must include consumption of fleshy fruits,
vegetable, and antioxidants to reduce production of free radical and increase antioxidants levels
during pregnancy period.
4. ANTIOXIDANT MICRONUTRIENTS IN PREGNANCY

In the period of pregnancy enlarged loads has to be on mother to fulfill satisfactory
nourishment demand of developing conceptus. Numerals of micronutrients role as vital
cofactors for or which itself performing as antioxidants (Miller, 1993). A pregnant woman and
her growing fetus have been influenced by micronutrients and vitamins which play an important
role in their health. Satisfactory maternal micronutrient status is serious throughout the period
of pregnancy which is a foremost reason for problems (Poston and Raijmakers, 2004). As can
be predictable, lack of sufficient antioxidant defense during the course of prompt metabolic
variation/development can prompt the growing fetus to suboptimal consequence. Essential
micronutrients comprise selenium because of its fundamental part in these defense tools
(Poston and Raijmakers, 2004).
Significantly developed levels are necessary throughout pregnancy. The pregnant mother
must have to deliver a source of nutrition and gaseous altercation to permit most embryonic
and fetal growth to take place, at the same time organizing her body for labor and parturition
and later demands of lactation. In such case where inadequate provisions of crucial vitamins
and micronutrients can lead to a state of biological struggle amongst the mother and conceptus,
which can be harmful to wellbeing prominence of both (Miller, 1993). Insufficiency of trace
elements in the period of pregnancy is strictly associated with death and morbidity amongst
newborn (Poston and Raijmakers, 2004). Insufficiencies of particular antioxidant actions
related through micronutrients like selenium, copper, zinc, and manganese have consequence
in deprived pregnancy conclusions, comprising fetal growth constraint (Shu and Obgodo,
2005), preeclampsia (Obgodo et al., 2012). There are many of micronutrients and vitamins have
been functioning as antioxidants or can be vital cofactors for antioxidant enzymes like
selenium, copper, zinc, manganese, and vitamins C and E. The present title recapitulates recent
knowing of significance of these micronutrient antioxidants in pregnancy.

4.1. Selenium
Selenium (Se) is amongst one of the necessary elements of vital importance in human
being. The daily requirement of selenium is get fulfilled by diet. One should take a diet rich in
selenium as it has great importance such as a constituent in different biomolecules for e.g.,
metalloproteins such as selenoproteins, an antioxidant enzymes glutathione peroxidase,
thioredoxin reductase, etc (Beckett and Arthur, 2005).
Also, selenium is needed for normal functioning of thyroid gland as production of active
thyroid hormones is highly depends upon the availability of selenium and thereby is important
for regular thyroid function (Beckett and Arthur, 2005). Because of altered dynamically during
period of pregnancy selenium concentrations from pregnant mother and glutathione peroxidase
activity get reduced, the concentration of selenium in 1st trimester & 3rd trimester is 65 μg/L &
50 μg/L respectively. Globally there is variance in valuation of selenium supplies, suitability,
and consumptions (Table 1). Maximum of these standards been measured by considering
essential intake to maximize the process related to antioxidant glutathione peroxidase in plasma
(Zachara et al., 1993) and whereas antioxidants such as selenium, vitamins C and E have been
established ample consideration in recent years. On an average minor concentration of selenium
has been observed in babies when it been compared to the mother selenium concentration,
whereas selenium concentration in mother 58.4 μg/L; as compared to umbilical cord selenium
concentration of baby is 42.1 μg/L. (Gathwala et al., 2000) which is predictable as selenium
transportation through the placenta wide over a concentration gradient by an anion exchange
path.
Table 1. Recommended dietary allowance for selenium, copper, zinc, manganese,

vitamin C, and vitamin E (Institute of Medicine, 2001)
Recommended Vitamin Vitamin

Selenium Copper Zinc Manganese
Dietary Category C E
(μg/d) (μg/d) (mg/d) (mg/d)
Allowance (mg/d) (mg/d)
Mature
54 900 8 1.8 76 16
female
Pregnancy 60 1,000 11 2 84 15
Upper
400 10,000 40 - 2,000 1,000
limit
Selenium is having importance in different metabolic and defense mechanism. Though it

having role in different antioxidant systems the precise biochemical mechanism is still a matter
of mystery; particularly related to critical growth and growing phases of baby as in gestation.
It is noteworthy; however, that selenium does play an essential part in general host defense
strategy. As such, further difficult and strongly measured studies are necessary to entirely clear
the purpose of this exclusive element in physiology as well as in pathophysiology (Gathwala et
al., 2000).

4.2. Copper
Copper (Cu) works as a cofactor for many enzymes which are engaged in biochemical
reactions such as angiogenesis, oxygen transportation, and antioxidant defense, different
enzymes utilizing copper includes catalase, SOD and cytochrome oxidase etc (Izquierdo et al.,
2007). Plasma concentrations of copper expressively rise in between period of pregnancy, and
come back to its usual range after delivery (concentration of copper in 1st trimester: 33.5 μg/L
and in 3rd trimester 41.6 μg/L) (Izquierdo et al., 2007). As copper concentration is increased in
pregnancy is related to protein which serves as a copper binding for the synthesis of
ceruloplasmin, because of changed levels of estrogen (Fattah et al., 1976). Near about 96%
plasma copper is bind to copper binding protein ceruloplasmin which has antioxidant property.
Since investigation has been establishing that according to the Institute of Medicine the dietary
consumption of copper in women aged amongst 19 and 24 years is generally below the
recommended levels (Table 1) which could be complications throughout pregnancy once
supplies of copper rise (Institute of Medicine, 2001).
Copper is needed for the development of embryo (Masters et al., 1983). Dietary deficiency
of element in pregnant mother can result in both short and long-term consequences, with initial
embryonic death and gross structural defects, and long-term consequences like an augmented
threat of heart-related disease and reproduction level also get reduced; current
recommendations on intakes are summarised in Table 1. The recommended intake of copper
should be clearly followed as severe copper insufficiency is thoughtful concern and can cause
reproductive failure and initial embryonic death, whereas slight or moderate insufficiency has
a minute consequence on neonatal weight (Masters et al., 1983).
Generally, Cu/Zn SOD antioxidant expressed in both maternal and fetal tissues. The level
of copper has been shown to be higher in maternal plasma as compared to umbilical cord plasma
(Krachler et al., 1999). The placenta is work like barricade in transmission of copper from
mother to fetus (Krachler et al., 1999). A recent Turkish study on 61 placentae from healthy
pregnancies between 37- and 40-week gestation found that copper concentrations positively
correlated with neonatal weight. A high affinity of copper transporter (CTR1) system is
necessary for transport of copper across the placenta and has been expressed early in pregnancy,
and there is connection between the placental copper transport and iron transportation
nevertheless exact mechanism is unidentified (McArdle et al., 2008).
4.3. Zinc
Zinc (Zn) is a crucial integral part of more than 200 metalloenzymes which are involve in
protein plus carbohydrate metabolism, synthesis of DNA and RNA, in antioxidant roles by
Cu/Zn SOD, and many more important functions such as cell multiplication, cell distinction,
also for productive embryogenesis (Harley et al., 2005). Zinc element with an abundant
significance for fetal development restriction which has been used to increase fetal
development (Harley et al., 2005). Fetus brain development is assisted by zinc through the
pregnancy similarly zinc plays a vital role in mother labor.
In between the period of the third trimester of pregnancy requirement of zinc is increased
near about twice than that of nonpregnant women. Also, it has been open the keynote, as
pregnant women should not consume more than 50% of the day-to-day requirement of zinc

(Harley et al., 2005). Near about 100 mg amount of zinc retained in pregnant women’s which
indicates its importance in pregnancy, also blood zinc concentrations decrease as pregnancy is
growths (mean ± SD–1st trimester: 71.2 ± 12.8 μg/L; 3rd trimester: 57.4 ± 11.5 μg/L) (Swanson
and King, 1987).
It has been reported that if zinc homeostasis is altered in pregnancy then it might produce
various complications including labor prolongation, restriction in fetal growth, sometimes
embryonic or fetal death (Harley, 2005). A randomized double-blind placebo-controlled trial
was conducted by Goldenberg et al., on zinc supplementation (25mg per day) over pregnancy
from 19-weeks gestation in African-American women (294 in supplemented and 286 in the
placebo group). It has been reported that those women’s on zinc supplementation had shown
expressively higher birth weight and a head edge of new-borns as related to placebo group
which emphasizing significance of sufficient supply of zinc throughout pregnancy (King et al.,
2000).
Zinc supplementation trials are conducted in many countries which shown that zinc
insufficiency is high, and these women are frequently selected as they are less well-nourished
or have low plasma zinc levels; assistance of supplementation are reduced occurrence of
pregnancy-induced hypertension or underweights in newborns (Goldenberg et al., 1995).
Concentrations of zinc during initial stage of pregnancy in relative to progress of difficulties in
pregnancy remains to be recognized.
4.4. Manganese
The enzymes are required for increasing rate of reactions in all types of living cell-mostly
cells which are undergoing continues cell division i.e., growing fetus. For the activation of
enzymes, cofactors are required, one of the important cofactors which required by many of
enzymes is manganese (Mn) and thus this element must be supplied in trace amount for healthy
functioning in all existing creatures. Manganese is a cofactor for numeral of enzymes, such as
antioxidant manganese superoxide dismutase Mn-SOD, which plays a vital part in defense of
placenta from free radicals by detoxifying superoxide anions. Pintsized is known about effects
of excess or deficiency of manganese on growing fetus or pregnancy as very fewer studies has
been done on manganese compared to other micronutrients. Another reason for very fewer data
about effects of manganese is unavailability of biomarkers for it (Wood, 2009).
One of the clinical trial study shown that blood level of manganese was found to be minor
in women with fetal growth restriction as related to fit controls (mean ± SD: 16.7 ± 4.8 versus
19.1 ± 5.9 μg/L) which signifying that manganese is very important micronutrient in sustaining
fetal growth (Vigeh, et al., 2008). Some trial also stated that concentration of manganese was
higher in samples of umbilical cord than from fetal development constraint circumstances as
associated to controls signifying that manganese ions are producing dissimilar impacts on birth
weight in mothers (Vigeh, et al., 2008).
4.5. Vitamin C and Vitamin E
Vitamin C is water-soluble vitamin mainly originate in fleshy fruit and vegetables. Vitamin
C plays a very crucial role in wound healing, synthesis of collagen, preventing the anemia

(Buettner, 1993). It has been reported that decreased in vitamin C level in blood of pregnant
women indicate that amplified consumption of same by pregnant women to balance level of
reactive oxygen species homeostasis by using vitamin C to clean up surplus reactive oxygen
species produced in pregnancy. Serum vitamin C concentration was low in third trimester of
pregnancy, which shows that oxidative stress is highest during third trimester of pregnancy;
therefore adequate amount of vitamin C supplementation is required in between duration of
pregnancy (Garba and Amodu, 2006).
Deficiency of vitamin C in pregnant women has been reported that it alters structure of
placenta and make placenta vulnerable to infection which may ultimately lead to increasing
threat of early rupture of placental membrane and early births. Vitamin C supplement during
pregnancy may support to stop the growth obstacles like gestational hypertension, gestational
diabetes an intrauterine development obstruction which mainly due to oxidative stress (Garba
and Amodu, 2006).
Lipid-soluble vitamin E (α-tocopherol) work along with lipid membrane also has combine
interactions with vitamin C (Figure 1). Vitamin E having role chiefly as a chain-breaking
antioxidant that stops spreading of lipid peroxidation (Poston, 2011).
Figure 1. Synergistic mechanism of vitamin C and vitamin E to prevent lipid peroxidation. O2 •, Oxygen
free radical.
5. ANTIOXIDANTS FOR PREVENTING PREECLAMPSIA

Preeclampsia is a complication of pregnancy which is defined as increased blood pressure
more than 140/90 mm Hg and proteinuria. Preeclampsia touches near about 3 to 5% of
pregnancies and having a major influence on the health of pregnant mother (Baum, 2007). As
preeclampsia is generally detected late in pregnancy but there is nearby relationship amongst
lipid peroxidation and preeclampsia, still the cause of this is not recognized.
Preeclampsia, a life-threatening barrier of pregnancy normally starts after the 5th month of
pregnancy whereas in that period concentration of protein found in mother’s urine is at peak
level (urinary albumin ≥ 300 mg/24 h). The indications of preeclampsia sort from minor to
severe. Though, the occurrence of preeclampsia differs in dissimilar populations and in

different ethnic groups (Roberts, 2002). Usually, no symptoms are shown in women with severe
preeclampsia, or show an increase in blood pressure, through indications like disturbances in
vision, headache, upper abdominal pain (Duley, 1992).
Disorders related to hypertension during pregnancy are utmost common reason of maternal
death in Latin America and Caribbean accounting for 25.7% of all maternal deaths; in
developed countries, the consistent proportion is lower, yet still significant: 16.1% (Khan,
2006). Though numerous theories have been suggested, still the reason of preeclampsia remains
unclear. Placental insufficiency and the pathophysiology of preeclampsia have been a close
relationship. There is significant part played by placental oxidative stress in appearances of
preeclampsia (Raijmakers, 2004). Women with preeclampsia and other hypertensive disorders
have been a manifestation of dysfunction in the blood vessels of the endothelial cell which is
accompanied by oxidative stress and lipid peroxidation (Orphan, 2004). Antioxidants might be
main for the stoppage of lipid peroxidation and, theoretically, for stoppage of preeclampsia;
still, the confirmation of antioxidants effectiveness for avoiding preeclampsia has not been
established up till now.
CONCLUSION
Free radicals and oxidative stress have significant parts in controlling numerous biological
utilities over the period of pregnancy, leading to complications such as intrauterine, fetal and
neonatal infections, preterm birth, premature membrane rupture, and preeclampsia.
Supplements of antioxidants may reduce the rate of lipid peroxidation during the late phase of
pregnancy which will be beneficial to prevent preeclampsia-like complications in pregnancy.
In order to maintain successful pregnancy adequate knowledge about this specific
micronutrient plays a crucial part in determining the health of both mother and baby and made
crucial attention for forthcoming wellbeing approaches in refining pregnancy consequences.
REFERENCES
Agarwal, A., Gupta, S. and Sharma, R. K. (2005). Role of oxidative stress in female
reproduction. Reprod Biol Endocrinol, 3 (28): 1-21.
Aurousseau, B., Dominique, G. and Durand, D. (2006). Gestation linked radical ROS fluxes
and vitamins and trace element deficiencies in the rudiment. Reprod Nutr Dev, 46: 601-
620.
Baker, M. A. and Aitken, R. J. (2005). Reactive oxygen species in spermatozoa: methods for
monitoring and significance for the origins of genetic disease and infertility. Reprod Biol
Endocrinol, 3 (67): 1-9.
Baum, W. S. and Karumanchi, A. S. (2007). Pre-eclampsia: Clinical manifestataions and
molecular mechanism. Nephron Clin Prac, 106 (2): C72-81.
Beckett, G. J. and Arthur, J. R. Selenium and endocrine systems. (2005). J Endocrinol, 184 (3):
455-465.
Bedwal, R. S. and Bahuguna, A. (1994). Zinc, copper and selenium in reproduction.
Experientia, 50 (7): 626-640.

Blaurock-Busch, E. The Clinical effects of manganese. 2009. Accessed 18 January, 2010

Available at: http://www.drkaslow.com.
Brigelius-Flohe, R., Kelly, F., Salonen, J.T., Neuzil, J., Zingg, J. M. and Azzi, A. (2002). The
European perspective on vitamin E: current knowledge and future research. Am J Clin Nutr,
76 (4): 703-716.
Casanueva, E. and Viteri, F. E. Iron and oxidative stress in pregnancy. (2003). J Nutr, 133 (5)
: 1700S-1708S.
Catt, J. W. and Henm, M. Toxic effects of oxygen on human embryo development. (2002).
Hum Reprod, 15 (2): 199-206.
Chappell, L. C., Seed, P. T., Kelly, F. J., Briley. A., Hunt, B. J. and Charnock-Jones, D. S.
(2002). Vitamin C and E supplementation in women at risk of preeclampsia is associated
with changes in indices of oxidative stress and placenta function. Am J Obstet Gynecol,
187 (3): 777-784.
Chui, D. K., Pugh, N. D., Walker, S. M., Gregory, L. and Shaw, R. W. Follicular vascularity
the predictive value of transvaginal power Doppler ultrasonography in an in-vitro
fertilization programme: a preliminary study. (1997). Hum Reprod, 12 (1): 191-196.
Droge, W. Free radicals in the physiological control of cell function. (2002). Physiol Rev, 82
(1): 47-95.
Duley, L. Maternal mortality associated with hypertensive disorders of pregnancy in Africa,
Asia, Latin America and the Caribbean. (1992). Br J Obstet Gynaecol, 99 (7): 547-553.
Evans, P. and Halliwell, B. Micronutrients: oxidant/antioxidant status. (2001). Brit J Nutr, 85
(2): 567-574.
Fattah, M., Ibrahim, F. K., Ramadan, M. A. and Sammour, M. B. Ceruloplasmin and copper
level in maternal and cord blood and in the placenta in normal pregnancy and in pre
eclampsia. (1976). Acta Obstet Gynecol Scand, 55 (5): 383-385.
Garba, I. H. and Amodu, B. O. Total serum vitamin C concentration in pregnant women:
implications for a healthy pregnancy. (2006). Rev Bras Saúde Matern Infant Recife, 6 (3):
293-296.
Gathwala, G., Yadav, O. P., Singh, I. and Sangwan, K. Maternal and cord plasma selenium
levels in full term neonates. (2000). Indian J Pediatr, 67 (10): 729-731.
Goldenberg, R. L., Tamura, T., Neggers, Y., Copper, R. L, Johnston, K. E. and DuBard, M. B.
The effect of zinc supplementation on pregnancy outcome. (1995). J Am Med Assoc, 274
(6): 463-468.
Greenwood, B. The use of anti-malarial drugs to prevent malaria in the population of malaria-
endemic areas. (2004). Am J Trop Med Hyg,70 (1): 1-7.
Harley, K., Eskenazi, B. and Block, G. The association of time in the US and diet during
pregnancy in low-income women of Mexican descent. (2005). Paediatr Perinat Ep, 19 (2):
125-134.
Hung, T. H., Lo, L. M., Chiu, T. H., Li, M. J., Yeh, Y. L. and Chen, S. F. A longitudinal study
of oxidative stress and antioxidant status in women with uncomplicated pregnancies
throughout gestation. (2010). Reprod Sci, 17 (4): 401-409.
Institute of Medicine (US) Panel on Micronutrients. Dietary Reference Intakes for Vitamin A,
Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum,
Nickel, Silicon, Vanadium, and Zinc, National Academy Press, Washington, DC, USA,
2001.

Izquierdo, A. S., Castanon, S. G., Ruata, M. L. C., Aragues, E. F., Terraz, P. B. and Irazabal,
Y. G. Updating of normal levels of copper, zinc and selenium in serum of pregnant women.
(2007). J Trace Elem Med Biol, 21 (1): 49-52.
Jansson, T. and Powell, T. Role of the placenta in fetal programming: underlying mechanisms
and potential interventional approaches. (2007). Lin Sci Lond,113 (1): 1-13.
Khan, K. S., Wojdyla, D., Say, L., Gulmezoglu, A. M. and Van, P. F. Look WHO analysis of
causes of maternal death: a systematic review. (2006). The Lancet, 367 (9516): 1066-1074.
King, J. C. Determinants of maternal zinc status during pregnancy. (2000). Am J Clin Nutr, 71
(5): 1334S-1343S.
Krachler, M., Rossipal, E. and Micetic-Turk, D. Trace element transfer from the mother to the
newborn-investigations on triplets of colostrum, maternal and umbilical cord sera. (1999).
Eur J Clin Nutr, 53 (6): 486-494.
Kudo, Y. and Boyd, C. A. Characterization of L-tryptophan transporters in human placenta: A
comparison of brush border and basal membrane vesicles. (2001). J Physiol, 531 (2): 405-
416.
Maruyama, T., Kitaoka, Y., Sachi, Y., Nakanoin, K., Hirota, K., Shiozawa, T., Yoshimura, Y.,
Fujji, S. and Yodoi, J. Thioredoxin expression in the human endometrium during the
menstrual cycle. (1997). Mol Hum Reprod, 3 (11): 989-993.
Masters, D. G., Keen, C. L., Lonnerdal, B. and Hurley, L. S. Comparative aspects of dietary
copper and zinc deficiencies in pregnant rats. (1983). J. Nutr, 113 (7): 1448-1451.
McArdle, H. J., Andersen, H. S., Jones, H. and Gambling, L. Copper and iron transport across
the placenta: regulation and interactions. (2008). J Neuroendocrinol. 20 (4): 427-431.
Miller, J. K., Brzezinska-Slebodzinska, E. and Madsen, F. C. Oxidative stress, antioxidants,
and animal function. (1993). J Dairy Sci, 76 (9): 2812-2823.
Mouatassim, S., Guerin, P. and Menezo, Y. Expression of genes encoding antioxidant enzymes
in human and mouse oocytes during the final stages of maturation. (1999). Mol Hum
Reprod, 5 (8): 720-725.
Munn, D. H., Zhou, M., Attwood, J. T., Bondarev, I., Conway, S. J. and Marshall, B. Prevention
of allogenic fetal rejection by tryptophan catabolism. (1998). Sci, 281 (5350): 1191-1193.
Nwagha, U. I. and Ejezie, F. E. Serum ascorbic acid levels during pregnancy in Enugu, Nigeria.
(2005). J Coll Med, 10 (1): 43-45.
Ogbodo, S. O., Nwagha, U. I., Okaka, A. N. C., Okeke, A. C., Chukwurah, F. E. and Ezeonu,
P. O. Low levels of some nutritional parameters of pregnant women in a rural community
of South East Nigeria: implication for the attainment of millennium developmental goals
(MDGs). (2012). Ann Med Health Sci Res, 2 (1): 49-55.
Ogbodo, S. O., Okaka, A. N. C. and Nwagha, U. I. Anti-infective antioxidant minerals levels
in uncomplicated pregnancy in some rural communities of South East Nigeria. (2013). J
Med Nutr Nutraceut, 2 (1): 52-57.
Ogbodo, S. O., Shu, E. N. and Okeke, A. C. Vitamin antioxidants may prevent drug induced
haemolysis of G6PD-deficient erythrocytes. (2006). Pharmacologyonline, 1: 90-99.
Poston, L., Chappell, L., Seed, P. and Shennan, A. Biomarkers of oxidative stress in
preeclampsia. (2011). Pregnancy Hypertens, 1 (1): 22–27.
Poston, L. and Raijmakers, T. M. Trophoblast oxidative stress, antioxidants and pregnancy
outcome – a review. (2004). Placenta, 25 (Suppl. A): S72-S78.
Raijmakers, M. T., Dechend, R. and Poston, L. Oxidative stress and preeclampsia: rationale for
antioxidant clinical trials. (2004). Hypertension, 44 (4): 374-380.

Roberts, J. M. and Lain, K. Y. Recent insights into the pathogenesis of pre-eclampsia. (2002).
Placenta. 23 (5): 359-372.
Romero, R., Chaiworapongsa, T. and Espinoza, J. Micronutrients and intrauterine infection,
preterm birth and fetal inflammatory response syndrome. (2003). J Nutr, 133 (2): 1668s-
1673s.
Sabuncuaalow, T., Vural, H. and Harma, M. Oxidative stress in polycystic ovary syndrome and
its contribution to the risk of cardiovascular disease. (2001). Clin Biochem, 34 (5): 407-
413.
Sappey, C., Legrand–Pods, S., Best-Belpomme, M., Farier, A., Reutiev, B. and Piette, J.
Stimulation of glutathione peroxidase activity decreases HIV type 1 activation after
oxidative stress. (1994). Aids Res Human Retro, 19 (11): 1451-1461.
Shiotani, M., Noda, Y., Narimoto, K., Imai, K., Mori, T. and Fujimoto, K. Immuno
histochemical localization of superoxide dismutase in the human ovary. (1991). Hum
Reprod, 6 (10): 1349-1353.
Shu, E. N. and Ogbodo, S. O. Role of ascorbic acid in the prevention of iron-deficiency anaemia
in pregnancy. (2005). Biomed Res, 16 (1): 40-44.
Sugino, N., Karube-Harada, A., Sakata, A., Takiguchi, S. and Kato, H. Nuclear factor kappa B
is required for tumor necrosis factor-alpha-induced manganese superoxide dismutase
expression in human endometrial stromal cells. (2002). J Clin Endocrinol Metab, 87 (8):
3845-3850.
Swanson, C. A. and King, J. C. Zinc and pregnancy outcome. (1987). Am J Clin Nutr, 46 (5):
763-771.
Vigeh, M., Yokoyama, K., Ramezanzadeh, F., Dahaghin, M., Fakhriazad, E., Seyedaghamiri,
Z. and Araki, S. Blood manganese concentrations and intrauterine growth restriction.
(2008). Reproductive Toxicol, 25 (2): 219-223.
Wood, R. J. Manganese and birth outcome. (2009). Nutr Rev. 67 (7): 416-420.
Yip, L., McCluskey, J. and Sinclair, R. Immunological aspects of pregnancy. (2006). Clin
Dermatol, 24 (2): 84-87.
Zachara, B. A., Wardak, C., Didkowski, W., Maciag, A. and Marchaluk, E. Changes in blood
selenium and glutathione concentrations and glutathione peroxidase activity in human
pregnancy. (1993). Gynecol Obstet Invest, 35 (1): 12-17.

ABOUT THE EDITORS
Md. Sahab Uddin

Md. Sahab Uddin is a Registered Pharmacist and a Research Scholar in the Department of
Pharmacy at Southeast University, Dhaka, Bangladesh. He has published more than 90 articles
in peer-reviewed international scientific journals. He has also authored and edited several
books, including Advances in Neuropharmacology: Drugs and Therapeutics; Handbook of
Research on Critical Examinations of Neurodegenerative Disorders; Pharmakon
Comprehensive Pharmaceutical Pharmacology; Tools of Pharmacy: Getting Familiar with the
Regular Terms, Words and Abbreviations; and Quality Control of Pharmaceuticals:
Compendial Standards and Specifications. Md. Uddin also serves as an editorial and reviewer
board member of more than 80 scholarly journals. He is a member of many national and
international scientific societies. He has developed Matching Capacity, Dissimilarity
Identification and Sense Making tests for the estimation of memory, attention, and cognition.
Md. Uddin has also established Numeral Finding and Typo Revealing tests for the
determination of attention. He received his BPharm in 2014 securing a first position from the
Department of Pharmacy, Southeast University, Bangladesh. Md. Uddin’s research interest is
how neuronal operation can be restored to abate Alzheimer’s dementia.

524 About the Editors
Aman B. Upaganlawar
Aman B. Upaganlawar is presently working as an Associate Professor in the Department

of Pharmacology at SNJBs SSDJ College of Pharmacy, Maharashtra, India. He has completed
his master and PhD in Pharmacy from the MS University of Baroda, India. He has 14 years of
teaching and research experience. He has more than 70 scientific publications in national and
international journals with good citation index. He has published 16 book chapters in reputed
publishers. He is also active as a reviewer and editorial board member for several national and
international journals. He has honored with various prestigious awards and is a life member of
several professional organizations. His area of interest includes the preclinical screening of
bioactive molecules for various disorders, cardiovascular pharmacology, metabolic syndrome,
toxicology, and antioxidant study. Till date, he has guided 26 post-graduate students for their
research work.

LIST OF CONTRIBUTORS
Abhilasha Ahlawat
Ahmad Ali
Mansour Alturki
Auburn University, Alabama, USA; Abdulrahman Bin Faisal University, Damman, Saudi
Arabia
Sneha Ambwani
Sophia Naa Ayikailey Ankrah

Komfo Anokye Teaching Hospital, Kumasi, Ghana
Chandrasekar S. B.
Hi-Tech Lab, Drug Testing Laboratory, Drug Control Department, Bangalore, India
Vinod A. Bairagi
KBHS College of Pharmacy, Malegaon, India
Muralikrishnan Dhanasekaran
Auburn University, Alabama, USA
Vipin Dhote
Gayatri Devi Donepudi

J. J. M. Medical College, Davanagere, India
Koula Doukani
University of IbnKhaldoun, Algeria, India

526 List of Contributors
Ayaka Fujihashi
Hemavathi G.
Aditya Ganeshpurkar
Shri Ram Institute of Technology-Pharmacy, Jabalpur, India
Munish Garg
Manoj Govindarajulu
Sameer Goyal
Vishal S. Gulecha
Vivek Kumar Gupta

Seetha Harilal
Tanuj Joshi
Rajesh K.
Meenakshi Kaira
Priyanshi Kapoor
Tyler Lynd
Manojkumar S. Mahajan

Avinash Singh Mandloi

Shalini Mani
Arup Kumar Misra

Timothy Moore
David Darko Obiri

Newman Osafo
Eyitayo Adeyemi Oyindamola

Rashmi Pandey
Additiya Paramanya
Parag A. Pathade
KBHS College of Pharmacy, Malegaon, India
Anil T. Pawar
MAEER’s Maharashtra Institute of Pharmacy, Pune, India
Prairna
Ved Prakash
M. S. Lokesh Prasad
Sindhu Ramesh

528 List of Contributors
Archana Negi Sah

Joseph Owusu-Sarfo
Bechan Sharma
Pramod Kumar Sharma

Abdulla Sherikar
Durgesh Nandan Shukla

Aarushi Singh
Surjit Singh
Vishnu Suppiramaniam
Priyanka Tiwari
Vaishali R. Undale
Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Maharshtra, India
Aman Upaganlawar
Chandrashekhar Upasani
Chandrashekhar D. Upasani
Sushil Kumar Varma

Mahatma Gandhi Institute of Medical Sciences, Sevagram, India

Hrushikesh E. Velis
Amrutvahini College of Pharmacy, Sangamner, India
Vaibhav Walia
Yogesh Chand Yadav

Uttar Pradesh University of Medical Sciences, Saifai, India
Sadaf Zehra
University of Windsor, Windsor, Canada

INDEX
acute ischemic stroke, 366, 374, 382, 388, 389, 414,

α 463
adaptive immunity, 233, 252
α-Lipoic acid (ALA), 35, 38, 51, 52, 111, 122, 176,
adrenocorticotropic hormone (ACTH), 135, 136,
177, 182, 196, 197, 264, 307, 311, 370, 396, 421,
137, 138, 139, 142, 146, 151, 155, 162, 163
438, 444, 450, 478, 479, 499
advanced glycation end product(s), 29, 46, 48, 98,
α-synuclein, 30, 40, 42, 47, 48, 52, 96, 100, 101,
128, 400, 401, 402, 403, 404, 405, 407, 408, 420,
112, 113, 116, 117, 122, 123, 128, 129, 144
425, 430, 431, 432, 433, 435, 444, 449, 457, 458,
α-Synuclein, 30, 113, 117
465, 466, 467, 468, 469, 470, 471, 472, 477, 478,
483, 484, 485, 486, 487, 488
β age-related disease, 9
age-related macular degeneration (AMD), 62, 70
β-carotene, 17, 64, 65, 151, 177, 178, 179, 193, 196, aging, vii, xi, 3, 4, 5, 6, 7, 9, 10, 11, 12, 14, 15, 16,
246, 263, 278, 287, 299, 307, 316, 409, 410, 411, 18, 19, 20, 21, 22, 26, 31, 32, 34, 37, 39, 41, 43,
478, 480 44, 46, 47, 49, 50, 51, 79, 89, 109, 112, 114, 115,
β-Carotene, 179, 193, 196, 307 117, 125, 132, 134, 149, 157, 158, 163, 165, 168,
β-Secretase, 98, 115, 120, 170, 183 184, 187, 188, 192, 193, 196, 197, 198, 203,
β-Tocopherol, 64, 65 220,226, 231, 251, 253, 279, 337, 338, 387, 407,
414, 419, 433, 465, 466, 467, 469, 473, 480, 481,
483, 484, 485, 486, 487, 493, 502, 503
γ aldose reductase (AR), 30, 37, 42, 115, 116, 127,
129, 382, 400, 402, 403, 407, 415, 419, 422, 431,
γ-Secretase, 98, 170, 183, 197 441, 451
allergy, 154, 253, 255, 259, 267, 268, 269, 270, 271,
δ 272, 273, 336, 376
Allium cepa, 14, 410, 424
δ-Tocopherol, 277, 412 Allium sativum, 14, 410, 416, 417, 452, 458
Aloe vera, 14, 67, 313, 441, 442, 448
alpha-tocopherol (α-Tocopherol), 32, 45, 52, 64, 65,
A 66, 84, 150, 177, 179, 185, 211, 213, 263, 277,
284, 306, 333, 370, 412, 415, 416, 443, 449, 475,
abortion(s), 511, 512 478, 479, 517
acetylcholine, 98, 133, 140, 153, 154, 164, 339, 401, amyloid beta (Aβ), 28, 29, 40, 96, 98, 99, 107, 108,
405, 495, 496 109, 110, 115, 117, 119, 122, 125, 126, 127, 129,
acquired immunodeficiency syndrome (AIDS), viii, 132, 156, 159, 168, 170, 171, 172, 173, 174, 177,
297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 178, 179, 181, 182, 183, 184, 187, 188, 191, 192,
307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 194, 196, 200, 201, 470, 478
317, 318, 319, 320, 321 amyloid plaque(s), 191, 469
acute inflammation, 234, 235, 238, 364

532 Index
amyloid precursor protein (APP), 98, 99, 107, 109, ascorbic acid, 11, 36, 37, 43, 46, 50, 58, 63, 64, 65,
112, 113, 114, 120, 122, 126, 127, 129, 170, 173, 70, 71, 83, 85, 88, 89, 105, 108, 115, 119, 145,
174, 183, 185, 188, 191, 193, 194, 196, 197, 199, 150, 151, 178, 211, 213, 224, 244, 245, 250, 277,
470, 477 281, 284, 287, 294, 308, 326, 327, 333, 339, 362,
anthocyanidines, 247 408, 409, 410, 411, 456, 459, 478, 479, 484, 520,
antiasthmatic(s), 257, 258, 261, 264, 265, 266, 268, 521
270, 272 asthma, viii, 143, 146, 257, 258, 259, 260, 261, 262,
anticancer therapy, 283, 284, 285, 286 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,
antidiuretic hormone (ADH), 138 273, 274
anti-inflammatory mediator(s), 61 asthmatic symptom(s), 259
antioxidant defense, xi, 4, 5, 9, 10, 12, 15, 18, 23, 27, asthmatic(s), 258, 259, 260, 261, 262, 263, 265, 268,
31, 51, 60, 62, 73, 74, 80, 86, 89, 110, 132, 154, 269, 270, 272
157, 169, 170, 174, 175, 176, 185, 186, 187, 200, atherosclerosis, 50, 58, 62, 64, 146, 231, 232, 241,
207, 231, 244, 271, 276, 298, 301, 303, 304, 305, 242, 245, 250, 251, 252, 253, 255, 276, 326, 330,
307, 309, 312, 336, 340, 362, 389, 427, 431, 434, 338, 339, 347, 401, 405, 423, 484, 493, 499
437, 439, 443, 444, 445, 446, 447, 449, 450, 451, attention deficit disorder (ADD), 132
476, 477, 478, 479, 483, 491, 492, 498, 499, 500, autacoid(s), 233, 234, 237, 238
503, 513, 515
antioxidant enzyme(s), 11, 14, 15, 20, 27, 31, 33, 34,
37, 49, 62, 73, 76, 81, 83, 96, 105, 108, 140, 149, B
160, 168, 175, 180, 185, 200, 203, 206, 219, 248,
Bacopa monnieri, 163, 180, 200
261, 279, 280, 297, 299, 300, 301, 303, 309, 330,
bay leaf, 410
347, 361, 372, 378, 380, 437, 439, 440, 441, 442,
Berberine, 332
443, 446, 447, 450, 452, 454, 456, 457, 459, 478,
biomarker(s), 17, 41, 49, 50, 57, 58, 63, 64, 65, 68,
487, 491, 492, 498, 508, 509, 512, 513, 514, 520
70, 80, 81, 88, 91, 114, 186, 210, 227, 263, 269,
antioxidant marker, 74
270, 305, 315, 341, 342, 360, 364, 373, 379, 485,
antioxidant mixture, 85
516, 520
antioxidant process, 34
biomolecule(s), xi, 24, 42, 74, 79, 85, 147, 169, 240,
antioxidant status, 66, 89, 186, 199, 281, 282, 286,
248, 258, 261, 264, 299, 465, 466, 467, 481, 508,
290, 291, 293, 294, 295, 314, 482, 519
514
antioxidant supplement(s), 33, 35, 41, 43, 58, 62, 65,
black pepper, 411, 424
85, 86, 89, 152, 186, 275, 277, 285, 286, 287,
black tea, 14, 20, 247
288, 289, 302, 314, 337, 345, 347, 476, 481, 483
blood-brain barrier (BBB), 44, 102, 108, 153, 182,
antioxidant vitamin(s), 45, 87, 88, 89, 110, 195, 248,
185, 193, 217, 299, 313, 359, 369, 372, 381, 383
253, 293, 294, 306, 315, 479, 499, 509, 510
BN82451, 213, 216, 224
antioxidative genes, 263
Bovine serum albumin (BSA), 478, 482
anxiety, 27, 28, 34, 41, 45, 47, 48, 50, 103, 131, 132,
Brassica nigra, 411
137, 139, 140, 143, 153, 156, 157, 162, 199, 493
breath pentane output (BPO), 64, 66
apocynin, 27, 331, 341, 342, 452
apoptosis, 25, 36, 96, 111, 121, 125, 128, 137, 146,
147, 152, 172, 173, 177, 181, 184, 189, 190, 192, C
193, 200, 205, 206, 213, 216, 249, 256, 261, 282,
297, 298, 300, 301, 302, 304, 305, 312, 317, 319, caffeine, 179, 188, 192, 196, 266, 336
320, 335, 336, 339, 340, 353, 359, 360, 369, 373, Camellia sinensis, 15, 332, 440, 456
376, 379, 380, 381, 382, 383, 386, 388, 389, 396, cancer, viii, 14, 17, 19, 21, 22, 32, 33, 40, 43, 45, 47,
402, 403, 407, 412, 413, 420, 422, 423, 425, 436, 49, 52, 60, 65, 68, 69, 70, 72, 110, 124, 146, 147,
437, 440, 442, 443, 445, 452, 453, 455, 458, 460, 148, 149, 159, 162, 164, 187, 193, 194, 231, 235,
461, 472, 477, 478, 484, 497, 500, 508, 509, 510 250, 252, 254, 255, 270, 275, 276, 278, 279, 280,
arginase, 265, 401, 405, 408, 496, 502 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,
arginine vasopressin (AVP), 138, 139, 160, 162 291, 292, 293, 294, 295, 296, 310, 315, 330, 336,
Arjuna, 439 341, 456, 465, 466, 472, 473, 475, 480, 483, 484,
arthrospira platensis, 311, 321 486, 487, 488, 498, 499, 505

Index 533
cancer therapy, 275, 276, 283, 284, 285, 288, 289, clinical dementia rate (CDR), 174
291, 293, 294, 472 Coenzyme Q10 (CoQ), 116, 119, 173, 187, 190, 200,
carcinogenesis, 9, 17, 19, 33, 275, 276, 280, 281, 220, 222, 224, 228, 244, 309, 334, 339, 340, 347,
292, 293, 296, 336, 407, 422, 472, 473, 480, 485 371, 377, 379, 382, 383, 388, 438, 445, 450, 452,
carcinogenesis, 17, 280, 293, 422, 480 456, 457, 458, 460, 461, 475, 478
cardiovascular diseases (CVDs), 14, 35, 43, 57, 62, cognitive decline, 97, 119, 172, 188, 191, 196, 477,
73, 110, 151, 169, 184, 231, 247, 291, 293, 325, 483
329, 332, 335, 336, 337, 338, 340, 341, 342, 346, copper (Cu), 12, 29, 34, 46, 76, 83, 99, 104, 105,
350, 375, 401, 405, 413, 419, 422, 428, 454, 466, 111, 112, 114, 117, 121, 124, 149, 169, 178, 200,
471, 474, 475, 479, 486, 493, 500, 502, 513, 521 207, 280, 303, 310, 311, 327, 328, 330, 352, 366,
cardiovascular dysfunction, 302, 348, 479 368, 379, 389, 406, 438, 474, 478, 507, 509, 512,
cardiovascular system (CVS), 42, 46, 62, 87, 145, 513, 514, 515, 518, 519, 520
328, 394, 492 coronary heart diseases (CHD), 346, 350
carotene, 39, 40, 47, 65, 70, 74, 85, 110, 151, 178, corticotropin-releasing factor (CRF), 135, 136, 137,
198, 246, 263, 278, 281, 284, 287, 288, 290, 293, 138, 140, 142, 153, 154, 155, 156, 158, 159, 160,
306, 307, 309, 316, 409, 410, 474, 475, 480, 484, 161, 162, 163, 164, 165
486, 487 creatine, 105, 119, 210, 212, 213, 215, 217, 220,
carotenoid(s), 13, 21, 32, 65, 71, 105, 150, 151, 178, 222, 224, 225, 226, 227, 228, 363, 378, 381, 384,
195, 196, 198, 201, 211, 246, 247, 277, 278, 284, 386, 388
292, 303, 306, 328, 408, 438, 444, 450, 454, 456, creatine kinase, 105, 210, 217, 225, 363, 381, 384,
460, 466, 474, 475, 478, 479, 487, 498 386, 388
catalase (CAT), 5, 9, 11, 13, 14, 15, 19, 24, 26, 32, culinary herbs, 408, 409, 410, 415, 417, 418
33, 34, 44, 66, 67, 74, 81, 105, 132, 144, 149, cumin, 15, 410, 418, 421, 424
150, 157, 158, 168, 175, 176, 184, 188, 211, 215, Cuminum cyminum, 410, 418, 420, 424
239, 244, 261, 263, 269, 276, 277, 299, 302, 303, Curcuma domestica, 411
304, 305, 308, 320, 326, 327, 351, 352, 361, 367, Curcuma longa, 15, 35, 121, 152, 165, 180, 331,
373, 377, 380, 406, 437, 439, 440, 442, 443, 445, 366, 384, 439, 458, 500, 504
447, 448, 449, 450, 453, 474, 478, 488, 498, 503, curcumin, 14, 19, 35, 38, 41, 44, 45, 107, 112, 117,
507, 508, 512, 515 118, 121, 123, 126, 129, 152, 179, 180, 188, 198,
catechin, 14, 67, 332, 336, 338, 410, 441, 454 200, 282, 290, 292, 293, 294, 311, 316, 331, 335,
catecholamine, 140, 144, 159, 326, 414 336, 338, 340, 341, 373, 388, 411, 419, 439, 440,
CDDO-methyl amide (CDDO-MA), 212, 216 447, 452, 453, 458, 460, 461, 462, 463, 500, 502
cellular signaling, 125, 254, 275, 276, 444, 510 curcuminoid(s), 121, 180, 282, 331, 411, 440
cerebral injury, 362, 376 cytochrome oxidase, 7, 76, 173, 174, 195, 353, 515
cerebral reperfusion injury, 346, 353, 362, 363 cytochrome oxidase, 7
cerebrovascular diseases (CVD), 151, 346, 350, 428, cytochrome P450, 24, 58, 63, 75, 147, 170, 258, 299,
444 301, 315, 317, 320, 321, 479
chemokine(s), 61, 232, 243, 261, 265, 300, 306, 358, cytochrome P450, 170
405, 431 cytokine(s), 61, 109, 129, 198, 231, 232, 233, 234,
cholecystokinin (CCK), 143, 154, 155, 159, 162 238, 241, 243, 244, 248, 250, 251, 253, 260, 261,
cholinergic neuron(s), 98, 112, 116, 178, 179, 201, 265, 300, 301, 306, 331, 358, 359, 361, 376, 377,
405 396, 400, 401, 403, 405, 412, 430, 431, 432, 433,
cholinesterase(s), 60, 119, 181 435, 459, 492, 496, 511
chronic inflammation, 232, 235, 242, 252, 254, 261, cytosine-adenine-guanine (CAG), 104, 105, 118,
265, 273 121, 123, 130, 203, 204, 205, 210, 221, 226, 228
chronic obstructive pulmonary disease (COPD), 60,
61, 70, 72, 146, 241, 263, 265, 269, 271, 274
cigarette smokers, 57, 61, 64, 65, 68, 69, 70, 71, 284 D
cigarette smoking, xi, 57, 58, 59, 60, 61, 62, 63, 64,
DA quinone, 100, 101
65, 66, 67, 68, 69, 70, 71, 72, 335
dehydroascorbic acid (DHA), 37, 83, 145, 213
Cinnamomum zeylanicum, 442
dementia, 41, 45, 97, 98, 102, 112, 115, 120, 125,
cinnamon, 15, 442, 448, 453, 454, 456, 458, 459
128, 129, 139, 140, 144, 155, 159, 167, 168, 174,
citrus flavonoids, 409

534 Index
177, 178, 183, 186, 190, 191, 192, 196, 197, 199, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503,
204, 298, 299, 303, 414, 476, 483, 528 504, 505
dementia, 120, 144, 168, 190, 192, 483 estrogen, 17, 144, 163, 389, 412, 510, 515
depression, 27, 28, 41, 43, 46, 47, 102, 103, 131, exercise, vii, xi, 22, 32, 50, 73, 74, 75, 76, 77, 78, 79,
132, 139, 140, 142, 163, 235, 293, 414, 493, 502 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 135,
diabetes, viii, 15, 30, 35, 36, 41, 42, 43, 45, 46, 49, 165, 254, 258, 259, 260, 338, 377, 401, 405, 478
51, 52, 73, 80, 88, 146, 169, 198, 233, 235, 334,
336, 340, 375, 380, 393, 394, 395, 396, 397, 398,
399, 400, 401, 403, 405, 406, 407, 408, 409, 411, F
412, 413, 414, 415, 416, 417, 418, 419, 420, 421,
F2-isopentanes, 64
422, 423, 424, 425, 427, 428, 429, 432, 435, 440,
Fenugreek, 440, 448, 455, 458, 460
441, 442, 445, 450, 451, 452, 453, 454, 455, 456,
Ferulic acid (FA), 36, 38, 44, 45, 107, 113, 120, 124,
457, 458, 459, 460, 461, 462, 463, 465, 466, 467,
127, 129, 130, 441
470, 471, 472, 474, 476, 478, 479, 481, 482, 483,
FK-506, 212, 215, 224
484, 485, 486, 487, 488, 493, 495, 498, 499, 502,
flavanone glycoside, 37, 108, 109
504, 505
Flavanone(s), 37, 108, 109, 246, 247, 279
diabetes ketoacidosis, 399, 400
Flavone(s), 246, 247, 279, 333, 474
diabetes mellitus (DM), 30, 36, 45, 118, 119, 121,
flavonoid(s), 11, 15, 19, 32, 41, 42, 44, 67, 105, 108,
123, 126, 129, 169, 375, 379, 393, 394, 395, 396,
109, 115, 119, 123, 126, 132, 151, 152, 161, 163,
397, 398, 399, 400, 401, 405, 408, 411, 412, 416,
180, 181, 198, 211, 246, 247, 248, 251, 253, 254,
417, 419, 421, 422, 423, 424, 425, 427, 428, 429,
264, 279, 285, 289, 293, 294, 328, 337, 338, 340,
430, 432, 433, 435, 437, 439, 440, 445, 446, 447,
408, 409, 410, 413, 414, 423, 424, 439, 440, 442,
450, 451, 454, 458, 462, 463, 467, 470, 471, 479,
463, 466, 474, 475, 479, 484, 491, 492, 498, 499,
482, 485, 487, 505
500, 501, 502, 513
diabetic complication(s), 36, 42, 399, 400, 411, 415,
flavonol(s), 36, 107, 108, 246, 247, 279, 442, 474
417, 421, 423, 433, 442, 443, 445, 451, 453, 455,
free radical theory of aging (FRTA), 4, 5, 11, 14, 18,
457, 479, 483, 486, 487, 488
41, 473, 480, 484
diabetic retinopathy, 403, 404, 415, 425, 428, 455,
free radicals, 17, 18, 22, 31, 43, 46, 49, 52, 60, 74,
471, 476, 486, 503
75, 86, 87, 88, 89, 96, 145, 147, 148, 156, 159,
dietary antioxidant(s), 3, 13, 16, 17, 71, 90, 178, 179,
161, 164, 169, 196, 200, 220, 222, 227, 232, 241,
249, 288, 290, 291, 293, 425, 474, 483, 484, 505
250, 251, 252, 253, 254, 272, 275, 276, 285, 290,
dopamine (DA), 30, 31, 45, 47, 100, 101, 108, 111,
291, 292, 293, 295, 318, 339, 340, 341, 357, 422,
120, 121, 122, 123, 124, 130, 133, 144, 189, 215,
466, 482, 484, 486, 487, 504, 518, 519
218, 292
fruits, 15, 16, 38, 58, 65, 70, 83, 108, 109, 215, 246,
Drosophila, 6, 14, 17, 18, 19, 20, 21, 172, 189, 218,
247, 277, 278, 289, 306, 328, 414, 441, 455, 466,
221
485, 492, 498, 499, 500, 513
Fujimycin, 215
E
electron transport chain (ETC), 5, 7, 8, 12, 23, 25, G

29, 74, 79, 99, 100, 101, 102, 105, 181, 208, 329,
gamma-amino butyric acid (GABA), 28, 37, 133,
334, 445, 479, 509
136, 205, 210, 212, 214, 218, 397
embryo development, 519
garlic, 14, 279, 312, 313, 410, 416, 417, 421, 449,
endogenous antioxidants, 14, 96, 106, 152, 176, 222,
452, 458, 462
332, 335, 385, 473, 474, 475, 498, 507
genetic polymorphism, 48, 263, 290
endometrium, 510, 520
genetic theory of aging, 480
endoplasmic reticulum, 7, 16, 19, 21, 22, 44, 258,
genistein, 17, 264, 283
302, 303, 329, 340, 397, 478, 488
gestational diabetes, 394, 416, 417, 418, 517
endoplasmic reticulum, 7, 19
ginger, 410, 411, 418, 421, 439, 447, 452, 458, 459,
epigallocatechin gallate, 332
460, 462
erectile dysfunction (ED), xi, 113, 128, 401, 405,
414, 415, 418, 421, 422, 425, 479, 491, 492, 493,

Index 535
Ginkgo biloba, 152, 164, 180, 186, 194, 196, 199, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317,
313, 321, 333, 337, 339, 342, 366, 413, 414, 416, 318, 319, 320, 321, 521
419, 421, 422, 423, 424, 449, 457, 458, 461 Huntingtin, 105, 221, 223, 225, 226, 228
glomerulus, 428, 436, 446 Huntington’s disease (HD), 95, 96, 102, 103, 104,
glucocorticoids (GCs), 27, 81, 119, 135, 138, 141, 105, 115, 118, 121, 125, 203, 204, 205, 206, 207,
153, 154, 160, 162, 396 210, 211, 214, 215, 216, 217, 218, 219, 227
glutamate, 27, 36, 37, 48, 50, 108, 111, 118, 133, hydrogen peroxide, 5, 7, 8, 26, 51, 59, 60, 67, 74, 75,
158, 211, 213, 218, 224, 225, 310, 349, 355, 365, 76, 77, 78, 98, 114, 119, 147, 149, 150, 169, 183,
368, 385 187, 198, 209, 232, 239, 240, 247, 260, 261, 276,
glutamate antagonists, 368 277, 299, 303, 304, 325, 326, 327, 329, 351, 352,
glutathione disulfide (GSSG), 5, 12, 34, 82, 83, 85, 356, 361, 363, 366, 367, 371, 378, 406, 433, 434,
144, 174, 212, 215, 216, 251, 263, 299, 305, 351, 437, 457, 478, 497, 508, 511
362, 439 hydroxytyrosol, 38, 42, 44
glutathione peroxidase (GPx), 5, 11, 12, 13, 33, 34, hyperglycemia, 142, 396, 400, 401, 403, 407, 411,
66, 67, 74, 83, 85, 105, 121, 132, 144, 149, 150, 427, 428, 429, 430, 432, 433, 435, 436, 437, 438,
174, 175, 176, 183, 184, 211, 214, 219, 244, 250, 439, 444, 445, 447, 449, 450, 451, 454, 455, 462,
261, 263, 270, 276, 277, 279, 281, 299, 300, 302, 470, 471
303, 304, 305, 308, 309, 315, 320, 326, 327, 351, hyperglycemic hyperosmolar syndrome, 400
361, 362, 367, 373, 377, 380, 384, 406, 413, 437, hypertension, 17, 326, 328, 335, 336, 339, 341, 342,
440, 441, 442, 443, 444, 447, 448, 450, 466, 474, 375, 400, 405, 430, 520
478, 482, 486, 487, 507, 508, 509, 510, 512, 513,
514, 521
glutathione peroxidase 1 (GPx-1), 219, 304, 305 I
glutathione reductase (GR), 5, 27, 33, 45, 82, 83, 87,
idebenone, 212, 214, 225
112, 118, 141, 149, 158, 174, 175, 183, 276, 300,
immunity, 4, 137, 141, 145, 146, 169, 233, 275, 276,
305, 315, 316, 326, 327, 351, 362, 431, 437, 447,
283, 295, 296, 305, 307, 308, 312, 313, 475, 481,
466, 474, 478
509
glutathione S-transferase (GST), 34, 150, 174, 175,
immunomodulation, 295, 388, 487
263, 304, 313
immunosuppression, 282, 283
glutathione system, 305
Inducible nitric oxide synthase (iNOS), 51, 184, 212,
glycation, viii, 115, 407, 432, 455, 465, 466, 467,
216, 240, 243, 249, 256, 273, 299, 301, 331, 356,
468, 469, 475, 481, 482, 485, 487, 488
358, 361, 367, 372, 377, 388
glyoxalase system, 34
inflammation, viii, 16, 24, 38, 40, 46, 48, 52, 57, 61,
grape seed phenolic extract (GSPE), 211, 213, 214,
72, 101, 107, 111, 130, 143, 148, 162, 185, 200,
217
231, 232, 233, 234, 235, 236, 237, 238, 239, 240,
green tea, 14, 15, 19, 248, 253, 264, 312, 336, 340,
241, 242, 243, 244, 248, 249, 250, 251, 252, 253,
343, 440, 441, 448, 456, 457, 460, 463, 492
254, 255, 256, 258, 260, 261, 263, 264, 265, 266,
guava, 441, 448, 452, 453, 456, 457
268, 269, 270, 271, 272, 273, 274, 279, 303, 307,
337, 346, 358, 359, 364, 373, 377, 381, 386, 389,
H 395, 407, 409, 418, 431, 440, 442, 445, 447, 450,
452, 456, 460, 462, 463, 469, 476, 487, 512, 513
heat shock proteins, 387 inflammatory responses, 188, 251, 284, 390, 441,
herbs, 66, 67, 68, 181, 246, 297, 302, 307, 311, 312, 469, 470
313, 332, 335, 336, 350, 366, 409, 413, 415, 417, innate immunity, 116, 233, 254, 256
438, 447, 448, 449, 450, 483 interleukins, 61, 265, 361, 369
hesperidin, 45, 109, 114, 117, 119, 122, 123, 125, intracellular pH, 357
409 ischemia, 21, 49, 62, 146, 193, 199, 226, 235, 239,
hexosamine pathway, 400, 401, 402, 405, 407, 408, 249, 251, 252, 326, 346, 347, 348, 349, 351, 352,
430, 432, 435, 463 353, 354, 355, 356, 357, 358, 359, 360, 361, 362,
histamine, 188, 236, 237, 247, 248, 249, 253 363, 364, 366, 368, 369, 370, 371, 372, 373, 375,
honokiol, 185, 187, 189, 190, 193, 195, 197, 199 376, 377, 378, 379, 380, 381, 382, 383, 384, 385,
human immunodeficiency virus (HIV), viii, 53, 297, 386, 387, 388, 389, 390, 400, 401, 405, 440, 442,
298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 448, 461, 463, 475, 492, 505

536 Index
ischemic reperfusion injury, 117, 346, 363 142, 143, 144, 146, 151, 152, 154, 155, 156, 157,
ischemic stroke, 345, 348, 349, 350, 353, 365, 366, 158, 160, 162, 163, 164, 177, 178, 180, 181, 183,
367, 374, 376, 380, 382, 384, 385, 388, 389, 390, 189, 190, 191, 194, 196, 198, 211, 214, 279, 470,
405, 413, 414, 463 528
isosilybin, 330, 339 memory dysfunction, 117, 132, 138, 143, 152, 154,
isosilychristin, 330 158
isosilydianin, 330 memory impairment, 49, 107, 131, 142, 144, 158,
181, 190, 194, 211, 214
Mentha x piperita, 409
J metal chelator, 29, 38, 99, 214
metalloporphyrins, 211, 212
juice, 64, 68, 69, 72, 492, 501
metalloproteinase, 350, 384
metformin, 424, 443, 446, 450, 452, 456, 457, 458
K micronutrients, 286, 294, 298, 303, 305, 308, 310,
311, 312, 313, 314, 316, 319, 471, 478, 487, 507,
Kynurenine-3-monooxygenase, 213, 218 508, 513, 516, 519, 521
microvascular complications, 400, 407, 427, 429,
444
L mild cognitive impairment (MCI), 48, 168, 174, 175,
183, 191, 196, 198
L-Arginine, 334, 495, 496 mini-mental state examination (MMSE), 174
Laurel, 410 mitochondria, 5, 7, 8, 10, 12, 18, 20, 24, 25, 30, 31,
Laurus nobilis, 410, 418 35, 41, 43, 44, 48, 50, 51, 52, 74, 76, 80, 82, 87,
L-Carnitine, 213, 217, 319 88, 90, 96, 98, 101, 102, 112, 119, 141, 147, 173,
lipid hydroperoxides (LOOHs), 58, 69, 79, 81, 197, 174, 181, 182, 184, 188, 189, 191, 194, 195, 198,
328, 405, 508 199, 206, 207, 208, 209, 210, 211, 213, 216, 217,
lipid peroxidation (LP), 9, 19, 58, 71, 79, 86, 90, 218, 219, 258, 261, 300, 303, 328, 329, 331, 336,
148, 149, 251, 294, 361 339, 340, 342, 357, 359, 370, 371, 395, 400, 402,
low-density lipoprotein (LDL), 37, 38, 60, 63, 64, 406, 407, 445, 455, 465, 509
70, 81, 182, 233, 241, 244, 245, 246, 247, 251, mitochondrial biogenesis, 10, 22, 188, 460
333, 413, 425, 502, 503 mitochondrial decline theory of aging, 5
lycopene, 15, 16, 39, 47, 49, 65, 212, 215, 224, 246, mitochondrial dysfunction, 11, 12, 26, 28, 29, 30, 47,
278, 279, 281, 291, 295, 340, 444, 450, 455, 474, 52, 96, 101, 104, 105, 123, 124, 130, 164, 167,
475 168, 172, 174, 181, 182, 186, 189, 203, 206, 218,
221, 224, 299, 433, 440, 461, 477
mitochondrial electron transport chain, 5, 25, 26, 479
M
mitochondrial function, 46, 48, 116, 123, 124, 173,
malondialdehyde (MDA), 9, 17, 19, 58, 62, 69, 79, 181, 184, 207, 209, 216, 271, 302, 379, 381, 450,
81, 84, 85, 105, 109, 139, 148, 168, 185, 209, 476
210, 212, 215, 216, 217, 240, 262, 263, 298, 363, mitochondria-targeted antioxidants, 182
379, 385, 405, 407, 439, 440, 441, 442, 444, 445, mitochondriogenesis, 11
446, 447, 448, 450, 492, 498 mitochondriogenic pathway, 11
Manganese (Mn), 15, 83, 102, 121, 130, 149, 170, MitoQ, 181, 191, 194
187, 190, 212, 290, 303, 305, 327, 328, 381, 384, monoamine oxidase metabolizes, 357
474, 478, 507, 509, 512, 513, 514, 516, 519, 521 Morinda citrifolia, 68
Manganese superoxide dismutase (MnSOD), 14, mustards, 411
170, 173, 187, 190, 193, 290, 361, 366, 381, 384, myeloperoxidase (MPO), 240, 242, 248, 255, 262,
509, 510, 516, 521 342, 363, 364, 373, 376, 377, 383, 387, 435, 437
melatonin, 33, 140, 149, 161, 177, 184, 190, 191,
192, 196, 197, 200, 201, 212, 214, 227, 244, 277, N
333, 336, 341, 342, 474
memory, vii, 19, 49, 97, 98, 103, 107, 109, 117, 122, N-Acetyl-L-cysteine (NAC), 83, 182, 183, 212, 264,
125, 127, 131, 132, 133, 134, 136, 138, 139, 141, 287, 288, 304, 310, 312, 313, 315, 444

Index 537
nadph oxidase, 21, 24, 30, 43, 77, 222, 223, 239, nonflavonoid, 30
248, 258, 301, 328, 329, 331, 332, 333, 335, 336, noradrenaline, 75, 133, 135, 153
338, 339, 341, 342, 367, 407, 412, 434, 435, 436, norepinephrine (NE), 9, 37, 135, 140, 141, 142, 157,
455, 457, 461, 479 158, 164, 165, 183, 215, 396, 407
NADPH oxidase, 21, 24, 30, 43, 77, 222, 223, 239, nutraceuticals, 14, 16, 305, 307, 312, 313, 314, 320,
248, 258, 301, 328, 329, 331, 332, 333, 335, 336, 338
338, 339, 341, 342, 367, 407, 412, 434, 435, 436, nutrient antioxidants, 277
455, 457, 461, 479
naringin, 37, 38, 108, 118, 121, 122, 125, 129
natural products, 66, 124, 249, 268, 415, 481, 487, O
492, 499
oestrogen, 144
neonatal infections, 507, 512, 518
oestrogen progestin therapy (EPT), 144
nephropathy, viii, 42, 400, 403, 404, 405, 407, 411,
omega-3, 70, 188, 277, 279, 293, 449, 452, 455, 474
413, 418, 427, 428, 429, 430, 431, 432, 434, 437,
omega-6 fatty acids, 474
438, 439, 440, 446, 447, 448, 449, 450, 451, 452,
onion, 14, 247, 279, 410, 424
453, 454, 455, 456, 457, 458, 459, 460, 461, 462,
oregano, 409
463, 486, 499
Origanum vulgaris, 409
neurodegeneration, vii, xi, 21, 25, 28, 29, 30, 36, 44,
oxidants, vii, viii, 16, 17, 21, 24, 34, 41, 43, 57, 58,
48, 93, 95, 96, 97, 99, 100, 101, 106, 112, 115,
60, 62, 73, 86, 88, 90, 187, 219, 231, 241, 243,
117, 118, 120, 121, 122, 123, 126, 127, 129, 130,
244, 297, 317, 385, 388, 390, 406, 419, 431, 462,
163, 167, 170, 172, 178, 179, 189, 191, 195, 198,
479, 492, 507, 508, 509, 511, 512
203, 209, 211, 213, 214, 218, 221, 222, 227, 228,
oxidative damage (OD), 4, 10, 15, 18, 23, 24, 31, 32,
470
33, 35, 37, 38, 43, 50, 51, 58, 62, 63, 66, 69, 71,
neurodegenerative disorders, 95
73, 81, 82, 83, 84, 86, 95, 96, 99, 101, 110, 113,
neurofibrillary tangles (NFTs), 28, 99, 100, 113, 120,
114, 117, 125, 132, 139, 145, 149, 157, 168, 173,
126, 167, 168, 172, 173, 175, 469, 470
175, 177, 181, 183, 191, 194, 200, 201, 203, 207,
neurohormone, 142, 160
208, 209, 210, 211, 214, 221, 255, 258, 261, 263,
neurological deficit, 349, 353, 386
269, 281, 299, 302, 303, 306, 307, 308, 309, 313,
neuronal degeneration, 28, 95, 110, 225
327, 351, 361, 362, 371, 379, 406, 437, 440, 443,
neuronal loss, 101, 103, 121, 186, 209, 470
450, 473, 475, 477, 478, 479, 480, 492, 497, 507,
neuropeptide Y (NPY), 142, 156
509
neuropeptides, 133, 154, 156, 158, 159, 162, 163
oxygen radical absorbance capacity (ORAC), 19, 85
neurosteroids, 133
neurotoxicity, 111
neutrophils, 61 P
nitric oxide (NO), 8, 10, 20, 24, 27, 28, 30, 34, 43,
48, 50, 51, 57, 59, 60, 62, 75, 76, 88, 98, 107, Pancreatic beta cell(s), 30, 395, 396
108, 117, 129, 147, 148, 163, 169, 170, 172, 176, paraoxonase hydrolyzes paraoxon, 60
184, 205, 208, 209, 211, 216, 220, 224, 232, 237, paricalcitol, 443, 449, 453, 454
240, 243, 245, 249, 251, 252, 253, 254, 256, 258, penile erection, 491, 492, 494, 495, 496, 500, 501,
259, 260, 261, 262, 263, 268, 271, 273, 285, 293, 502
299, 308, 317, 325, 326, 328, 329, 331, 332, 333, penumbra, 349, 354, 355, 357, 358, 362, 365, 369,
334, 337, 338, 339, 340, 341, 352, 353, 356, 357, 376, 379, 382, 387, 388
364, 367, 370, 372, 375, 376, 377, 379, 380, 382, peppermint, 409, 416, 419
384, 386, 388, 389, 401, 402, 403, 405, 406, 407, peripheral blood lymphocytes (PBL), 58, 68, 69
412, 413, 414, 417, 418, 419, 432, 433, 434, 436, peripheral vascular diseases (PVD), 405, 414
437, 446, 453, 458, 459, 479, 492, 495, 496, 497, peroxisomes, 8, 24
498, 499, 500, 501, 502, 503, 504, 505 phenylpropanoids, 247
nitric oxide synthase (NOS), 108, 148, 170, 216, phosphorylated tau, 29, 98, 125, 470
232, 240, 243, 254, 256, 258, 328, 329, 331, 337, physical exercise, 49, 73, 74, 76, 86, 87, 88, 90, 259,
339, 356, 367, 375, 379, 382, 388, 389, 406, 412, 485, 494
433, 446, 459, 479, 492, 495, 496, 501, 503, 505 phytochemicals, 14, 69, 246, 265, 266, 279, 289,
non-adrenergic noncholinergic (NANC), 496 307, 311, 328, 342, 438, 466, 479

538 Index
picrorhiza kurroa, 331 reduced glutathione (GSH), 5, 7, 8, 9, 12, 13, 27, 30,
pioglitazone, 179, 219, 223, 446, 450, 454, 457, 461 31, 34, 42, 59, 62, 81, 82, 83, 90, 102, 106, 108,
piper nigrum, 411 109, 114, 144, 149, 150, 168, 174, 175, 176, 177,
placental oxidative stress, 518 180, 182, 183, 184, 210, 211, 215, 216, 264, 277,
plasma fibrinogen, 364, 387 281, 288, 299, 302, 303, 304, 305, 307, 309, 310,
Plectranthus barbatus, 307, 311, 317 311, 314, 351, 361, 362, 372, 373, 407, 431, 439,
poly (ADP-ribose) polymerase (PARP), 177, 360, 440, 441, 442, 443, 445, 447, 448, 449, 450, 474,
367, 368, 402, 477 476, 478, 484, 498, 511
Polygonum cuspidatum, 332 reperfusion injury, 350, 375, 379
polyol pathway, 400, 402, 403, 407, 430, 431, 435, Resveratrol, 30, 37, 38, 42, 43, 44, 45, 46, 48, 51,
439, 447, 453, 471 184, 188, 189, 193, 194, 247, 283, 332, 336, 341,
polyphenolic antioxidants, 13 342, 411, 424, 499, 505
polyphenols, 13, 14, 15, 20, 46, 52, 69, 105, 151, retinopathy, 400, 403, 412
188, 227, 246, 247, 248, 252, 314, 408, 440, 442, Rosa laevigata, 442, 448, 463
448, 457, 460, 463, 474, 492, 499, 500, 513 rutin, 36, 38, 45, 108, 111, 118, 124, 129
preeclampsia, 517
pregnancy, ix, 421, 489, 507, 509, 512, 513, 514,
520 S
proopiomelanocortin (POMC), 137, 159, 162
Salvia miltiorrhiza, 412, 413
pro-oxidants, 9, 34, 35, 110, 231, 275, 288, 298, 303,
Salvia officinalis, 152, 409
508
selenium (Se), 25, 49, 50, 85, 91, 150, 183, 187, 191,
prostaglandins, 76, 147, 179, 232, 235, 237, 238,
192, 193, 212, 214, 269, 273, 279, 290, 292, 294,
251, 255, 260, 413
307, 308, 320, 513, 514, 518
protein aggregation, 96, 104, 476, 484
Serotonin, 133, 136, 139, 155
protein kinase C (PKC), 30, 138, 400, 401, 402, 403,
Silibinin, 109, 121, 127, 194, 330
404, 405, 407, 408, 415, 430, 431, 432, 433, 435,
Silybum marianum, 109, 181, 312, 341
443, 451, 455, 456, 457, 458
Silymarin, 330, 338, 448
Psidium guajava, 441, 456, 457, 459
Sonothrombolysis, 350
psychological coordination, 501
sorbitol dehydrogenase (SDH), 30, 431
psychosomatic stressors, 135
spices, 410, 422, 424
Pueraria lobate, 412
spironolactone, 447, 450, 459, 461
puerarin, 412, 425
SS peptides, 181, 182
pyridoxamine, 444, 449
substance P (SP), 143, 144, 156, 161, 163, 164, 165,
pyruvate, 213, 214, 216
190, 193, 205, 381, 463
superoxide dismutase (SOD), 5, 11, 13, 14, 15, 16,
Q 19, 22, 26, 32, 33, 34, 61, 62, 66, 74, 76, 81, 85,
102, 105, 121, 132, 139, 144, 149, 159, 168, 175,
Quercetin, 37, 38, 107, 120, 179, 189, 247, 285, 333, 176, 180, 184, 188, 195, 200, 211, 214, 215, 219,
409, 414, 418, 486, 499, 501 239, 244, 248, 261, 263, 271, 276, 277, 281, 290,
302, 303, 304, 305, 308, 313,314, 315, 319, 326,
327, 336, 351, 352, 361, 362, 366, 373, 379, 380,
R 381, 384, 385, 386, 387, 389, 406, 415, 437, 439,
440, 441, 442, 443, 444, 445, 447, 448, 450, 455,
reactive nitrogen species (RNS), 7, 9, 18, 28, 34, 36,
466, 474, 478, 482, 487, 488, 497, 498, 500, 501,
38, 107, 147, 148, 149, 169, 176, 181, 192, 207,
503, 504, 507, 508, 509, 510, 512, 515, 516, 521
210, 212, 232, 245, 254, 258, 261, 264, 272, 276,
synthetic triterpenoids, 213, 217
337, 352, 402, 406, 407, 413, 423, 433, 437, 444,
451, 465, 479, 480
reactive oxygen and nitrogen species (RONS), 75, T
76, 77, 78, 79, 80, 81, 90, 479
reactive species (RS), 4, 41, 51, 123, 127, 147, 148, tacrolimus, 215
191, 232, 239, 240, 276, 375, 406, 412, 419, 444, tau, 29, 41, 47, 96, 98, 99, 100, 112, 113, 119, 123,
508 168, 172, 178, 179, 184, 187, 189, 190, 191, 194,

Index 539
197, 199, 201, 265, 469, 470, 478, 484, 485, 486, vegetable(s), 15, 16, 17, 38, 52, 58, 65, 70, 83, 215,
488 246, 277, 278, 279, 289, 306, 328, 414, 424, 466,
tauroursodeoxycholic acid (TUDCA), 212, 213, 216, 485, 498, 500, 513, 516
223 vitamin A, 178, 278, 292, 306, 307, 315, 321, 438,
telmisartan, 446, 450, 456, 461 519
Terminalia arjuna, 389, 439, 447, 455 vitamin B, 178, 179, 197, 201
terpenoids, 180, 333, 414 vitamin B12, 178, 179, 197
thiazolidinedione (TZD), 219, 446 vitamin C, 13, 18, 36, 37, 38, 43, 44, 82, 83, 108,
thiobarbituric acid reactive substances (TBARS), 58, 123, 124, 150, 153, 158, 161, 178, 179, 193, 195,
59, 64, 79, 81, 177, 263, 445, 450 196, 198, 244, 254, 277, 284, 293, 307, 308, 315,
thrombolytics, 349 333, 443, 449, 475, 484, 488, 499, 505, 514, 516,
tobacco, 72, 169, 258, 262, 270, 294, 495 517, 519
tocopherol, 36, 39, 40, 42, 47, 64, 65, 66, 71, 83, 84, vitamin E, 13, 20, 21, 36, 38, 82, 83, 84, 90, 91, 107,
85, 89, 90, 107, 123, 126, 150, 151, 154, 192, 113, 118, 124, 130, 177, 179, 191, 192, 193, 244,
243, 245, 246, 247, 252, 263, 277, 281, 287, 288, 245, 253, 277, 284, 306, 307, 318, 319, 333, 414,
290, 318, 326, 327, 335, 412, 443, 479, 499 442, 443, 449, 471, 474, 475, 476, 514, 516, 517
tocotrienol(s), 42, 82, 84, 107, 123, 177, 179, 188, vitamins, 82, 87, 89, 164, 198, 201, 218, 293, 307,
190, 277, 306, 318, 442, 443, 449, 456, 457, 479 319, 320, 328, 341, 342, 415, 438, 442, 475, 485,
total antioxidant capacity (TAC), 85, 263, 286, 442, 487, 492
448 Vratrum grandiflorum, 332
total antioxidant status (TAS), 66, 67, 311
transcriptional dysregulation, 121
transcriptional dysregulation, 121 W
Trigonella foenum-graecum, 440
white matter, 114, 348
trimetazidine (TMZ), 371, 372, 377, 380, 381, 382
tubular injury, 436, 449
turmeric, 15, 180, 282, 311, 316, 331, 338, 410, 411, X
439, 447, 504
type 1 diabetes mellitus (T1DM), 394, 420, 424, 428, xanthine, 77, 121, 329, 342, 356, 366
451, 454 xanthine oxidase, 121, 329, 342
type 2 diabetes mellitus (T2DM), 43, 45, 49, 393,
400, 417, 418, 420, 421, 424, 425, 428, 442, 443,
444, 445, 454, 471 Z
zinc (Zn), 29, 46, 83, 99, 104, 105, 114, 121, 149,
U 178, 200, 281, 303, 305, 307, 309, 310, 313, 314,
315, 316, 318, 319, 328, 361, 366, 379, 389, 438,
ubiquitin-proteasome system (UPS), 6, 480 445, 450, 453, 456, 457, 459, 461, 474, 478, 507,
509, 512, 513, 514, 515, 516, 518, 519, 520, 521
Zingiber officinale, 411, 418, 421, 439, 452, 459
V
vasoactive intestinal polypeptide (VIP), 141, 142,

153, 154, 159, 160, 161, 162
vasospasm, 346, 348, 389


Oxidative Stress and Antioxidant Defense

Uploaded by

Copyright:

Available Formats

Oxidative Stress and Antioxidant Defense

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Oxidative Stress and Antioxidant Defense

Uploaded by

Copyright:

Available Formats

Complimentary Contributor Copy

Complimentary Contributor Copy