In-vitro studies of membrane stabilizing and lipoxygenase inhibitory

activities of ​Cordia dichotoma​ G. forst leaves

A Thesis Proposal
Presented to the Faculty
Department of Pharmacy
College of Allied Medical Professions
Lyceum of the Philippines University – Batangas

In Partial Fulfillment
of the Requirements of the Degree
Bachelor of Science in Pharmacy

Diane Antoinette A. Balitaan, Mar Donna Dei B. Cabrera, Jethro C. Hernandez,

Frances Lyn G. Mangubat, Leigh Danica D. Mendoza,
Aaron Dell A. Cobeng & Oliver Shane R. Dumaoal

INTRODUCTION to react with nonspecific stimuli like

exercise and cold air. Other symptoms
According to the World Health
include excessive mucus production,
Organization, asthma is one of the most
airway narrowing and airway wall
important non- communicable
remodeling, chest tightness, shortness
conditions. It is a chronic disease that
of breath and wheezing (Lambrecht &
inflames and narrows the air passages
Hammad, 2015). The cause of asthma
of the lungs. According to the most
is unknown, but there are risk factors
recent WHO estimates released in
that have been identified. One factor
December 2016, 383 000 asthma-
includes genetics and heritability ranges
related deaths occurred in 2015.
from 35% to 95%. Environmental factors
Meanwhile, the Philippines is ranked 9​th
such as exposure to pollutants, ozone,
in asthma mortality across the globe
smoke and tobacco increase the risks of
with approximately 315 million Filipinos
developing asthma. The underlying
suffering from this condition. Back in
inflammation can be asymptomatic and
2002, the prevalence of asthma was
inflammation control is central in asthma
reported at 26.7 percent according to
management. The disconnection
the National Epidemiology Survey
between inflammation and symptoms
(Jambora, 2018). Asthma is defined as
can lead to poor awareness of asthma,
a chronic inflammatory disease of the
which can lead to poor recognition and
bronchioles. Symptoms include
non-compliance treatment (Mims, 2015).
bronchial hyper-reactivity, which is
According to numerous studies, airway
described as the tendency of the cells of
inflammation is a central key to the
the smooth muscle in asthmatic patients
pathophysiology of asthma (Kudo,
Ishigatsubo, & Aoki, 2013). One of the and tumor angiogenesis. LOX is
major causes of airway inflammation is responsible for the conversion of
the production of leukotrienes via arachidonic acid to 5-hydroxy-6, 8, 11,
lipoxygenase (LOX) pathway upon 14- eicosatetraenoic acid (5-HPETE).
exposure to certain allergens (Di 5-HPETE when furthered converted
Gennaro & Haeggström, 2013). The would result to a series of leukotrienes,
liberation of potent chemical mediators which are mediators of inflammation in
from inflammatory cells resulting to asthma (Sacan & Turhan, 2014).
chronic inflammation of the airway,
Corticosteroids, B2- agonist,
airway remodeling, which is
lipoxygenase and leukotriene inhibitors
characterized by thickening of airway
have been effective in suppressing
wall compartments that may lead to the
inflammatory responses like asthma but
progression and development of the
prolonged treatment can result to
disease (Kudo et al., 2013).
notable adverse effects and drug
Membrane destabilization of the resistance. (Sullivan, Ghushchyan,
immune system cells is a physiologic Globe, & Schatz, 2018; Cazzola, Page,
indicator in the pathogenesis of several Rogliani, & Matera, 2013 & Rossi et al.,
inflammatory diseases. The secretion of 2010). Hence, discovery of natural
the lysosomal constituents (bacterial product alternative therapies that do not
enzymes and proteases) of the immune produce or lessen significant adverse
system cell system (mast cells, effects has become a trend. Sustaining
neutrophils, basophils, eosinophils and and combating diseases through the
leukocytes) in response to a stimuli may use of plant derived medicines from
lead to progression of the damage thus their specific plant source is considered
resulting in several physiologic therapeutic and the first line of defense
response which are prominent in the (Jamkhande, Barde, Patwekar, & Tidke,
usual signs of inflammation (Sinbad, 2013). However, scientific investigation
Samuel, Adewale, & Adedoyin, 2017). or studies and proper screening from
different plant kingdoms specifically
Lipoxygenases belong to a class
higher plants, which can act as both
of non-heme iron containing
potential crude drug and a source of
dioxygenases which catalyze the
developing chemotherapeutic agents
addition of oxygen molecule to fatty
should be performed. (Ali, Kala , & Khan
acids containing a cis,
, 2018).
cis-1,4-pentadiene system to yield an
unsaturated fatty acid hydroperoxides. Cordia dichotoma G. Forst
This enzyme is found to play a commonly known as ​anonang,​ is
significant role in numerous disorders claimed to originate in South and
such as bronchial asthma, inflammation Southeast Asia. It grows in tropical and
subtropical regions and belongs to the antifertility, juvenomimetic biliary
Boraginaceae family. Anonang is a obstruction, nutritional food (Jamkhande
medium sized tree found widespread in et., al, 2013) and contraceptives
the thickets and secondary forests at (Bhattacharya & Saha, 2013).
low and medium altitudes in the
The need for novel compounds
Philippines. (Jamkhande, Barde,
which can acts as an alternative for the
Patwekar, & Tidke , 2013; Ragasa, et
current anti-asthma drugs is evident
al., 2015)
since drugs for this disease such as
corticosteroids, B2-agonist and
5-Lipoxygenases and leukotrienes
inhibitors are predominantly used but
these agents contains various drug
interactions and adverse effects. Herbal
medicine greatly depends on herbs for
curing numerous illnesses. Even though
plants are accepted as a product used
in developing new drugs, empirical data
from scientific studies should strongly
validate the therapeutic effects of
natural products to strengthen and
continue the development of natural
resources and medicinal compounds.
Because of these reasons, the
FIGURE 1. ​Cordia dichotoma t​ ree researchers decided to conduct a study
Retrieved from: on the leaves of ​Cordia dichotoma
https://sylviatramos.blog/ G.forst a ​ nd evaluate its anti-asthma
activity through lipooxygenase inhibition
Phytochemical analysis on
and membrane stabilization as
Cordia dichotoma ​proves that its leaves
parameters. In addition, discovery of
contains Alkaloid, carbohydrate,
new source of drug molecules for the
flavonoids, proteins, amino acids,
treatment of asthma with the main goal
phenols, tannins, glycosides and
of identifying safer and more tolerable
steroids are chemical constituents found
substitute is also one of the aims of this
in the ​Cordia dichotoma plant (Ali, Kala ,
study.
& Khan , 2018).Several scientific studies
have been made on ​Cordia dichotoma, METHODOLOGY
which proves different pharmacologic
activities of its leaves like analgesic, Plant Collection​ ​and Authentication
antipyretic (Gupta & Kaur, 2014),
 The ​Cordia dichotoma ​leaves will flavones. Other than that, formation of
be obtained from Barangay Ticub, acid labile complexes with
Laurel, Batangas and will be brought to ortho-dihydroxyl groups in the rings A or
the UST Herbarium located in España, B of flavonoids. According to studies,
Manila for authentication and quercetin is the suitable reference for
certification purposes. total flavonoid determination in the plant
sample extract. Various concentrations
of quercetin will be prepared to make
the standard calibration curve. In 100 ml
of methanol, 10 mg of quercetin will be
Preparation of Plant dissolved and will be diluted to 6.25,
The leaves of ​Cordia dichotoma 12.5, 25, 50, 80, and 100 µg/ml with
will be properly clean with water and methanol. To prepare the plant extract
shade dry under room temperature. stock solution, dissolve 100 mg
Complete drying should be observed. methanolic extract in 5ml methanol and
Dried leaves will be coarsely powdered transfer it to a 10ml volumetric flask. 1M
through a mechanical mixer (Hatware et potassium citrate and 10% aluminum
al., 2018). chloride will be prepared using distilled
water. The assay will be determined by
Extraction of Plant 0.5 ml of each dilution of standard
quercetin and each of the plant extract
A sample of 100g from the aerial
stock solution in separate test tubes. In
part will be taken in a soxhlet and
a test tube create a mixture of 1.5ml
addition of 80% methanol to the 2
methanol, 0.1ml potassium acetate
siphons which is up to 500ml shall be
solution, 0.1 ml aluminum chloride
filled. The temperature is set to 70 °C
solution and 2.8ml of distilled water.
and the extraction will be performed in a
Prepare a sample blank for the three
span of 5 hours. After that, the extract
methanolic extract and all dilution of
that will be obtain will be filtered and
standard quercetin. But instead of using
concentrated at 70 °C.(Linn, 2013).
aluminum chloride replace it with
Determination of Flavonoid Content distilled water. All solutions will be
filtered through a Whatmann filter paper
By using the aluminum chloride before absorbance is measured (Bag &
colorimetric method, the total flavoinoid Devi, 2015).
content would be estimated. The
principle in this colorimetric method is Isolation and purification of bioactive
that AlCl3 will form stable complex with compound
keto groups of C-4 and either the C-3 or
Pack a column chromatography
C-5 hydroxyl group of flavonols and
(CC) (50 x 9 cm) starting off with 1.5 cm
fine sand layer and silica gel (particle a micro capillary to apply an aliquot of
size 63-200 mm, 430 g) at a depth of 20 each fraction to the baseline (measuring
cm. Cover again with 1.5 cm sand layer 0.7 cm from one end of the plate) of the
to protect the adsorbent surface. ​A TLC (about 7 x 5 cm for TLC) and place
volume of hexane (4 x 1 L) will pass it in the TLC chamber. Develop TLC,
through the CC and would result to leaving the solvent with capillary action
rising of bubbles and settling of silica to raise the TLC plate, until the solvent
gels allowing them to pack tightly into reaches almost the top (0.5 cm from the
the column. Organic fraction must be plate). Then view the developed TLC
dissolved in 40ml of CH​2​Cl​2 and mix it with 254 nm wavelengths under a UV
with silica gel forming a slurry. Use the lamp and stain the plates with the oleum
rotary evaporator to remove solvent and system and heat them to 80 - 100 ° C in
obtaining dry powder. Slowly and evenly a hot plate up until to the appearance of
apply the dried powder extract on the the spots (Martín-Rodríguez et al.,
top portion of the packed column and 2015).
perform a gradient elution with a mixture
Membrane Stabilization Assay
of hexane/EtOAc. The mixture of the
solvents and eluents that will be Preparation of Human Red Blood Cells
collected are of increasing polarity (HRBC) Suspension
varying in concentration. Starting off
with 100% hexane and will be followed Collect fresh whole human blood
by hexane/EtOAc mixtures of 8:2, 6:4, and mix it with equal volume of sterilized
4:6, 2:8, 1:9 and lastly with 100% ethyl Alsever solution (0.05% citric acid,
acetate. A ratio of 10:0 to 0:10 will be 0.42% sodium chloride, 0.8% sodium
observed. In round-bottom flasks (1 L) citrate and 2% dextrose in water). Blood
collect the resulting fractions and will be centrifuged at 3000 rpm for 10
remove the mobile phase in each minutes. Pack cells will be washed
fraction by using a rotary evaporator (at thrice with isosaline (0.85%, pH 7.2).
≤ 4°C). (Martín-Rodríguez et al., 2015) Volume of blood will be measured and
reconstituted as a 10% v/v suspension
Thin-Layer Chromatography with isosaline.
According to their TLC profile Heat Induced Hemolysis
similarities, incorporate the 15 fractions
into 10 sub-fractions. Add the hexane / The membrane lysis caused by
ethyl acetate solvent mixtures with hypotonicity through the stabilization of
increasing polarity from 8:2 (8 ml human red blood cell is the concept
hexane plus 2 ml ethyl acetate) to 6:4 in tangled here. 1ml of phosphate buffer
the TLC chamber at a depth of just with 7.4 pH and 0.15M, 2 ml hyposaline
under 0.5 cm to run the TLC plates. Use (0.36%), 10% v/v of 0.5 ml HRBC
suspension with extracts of plant 0.5ml DESCRIPTION
and diclofenac sodium as a standard
Asthma is defined as a chronic
with different concentrations (50, 100,
inflammatory disease of the bronchioles.
250, 500, 1000, 2000 µg/ml) and control
Symptoms include bronchial
(distilled water to produce 100 percent
hyper-reactivity, which is described as
hemolysis instead of hyposaline) are the
the the tendency of the cells of the
mixtures that comprises the assay and
smooth muscle in asthmatic patients to
will be incubated at 37°c for 30min and
react with nonspecific stimuli like
will be centrifuged correspondingly. The
exercise and cold air. Other symptoms
spectrophotometer at 560nm will be
include excessive mucus production,
used to the project the content of
airway narrowing and airway wall
hemoglobin suspension (Chippada,
remodeling, chest tightness, shortness
Volluri, Bammidi, & Vangalapati, 2011).
of breath and wheezing (Lambrecht &
Calculation of percentage hemolysis of Hammad, 2015).
HRBC membrane:​
PREVALENCE
OD of test sample
%Hemolysis= OD of control
x 100
According to the World Health
Lipoxygenase Inhibition Assay Organization, asthma is one of the most
important non- communicable
Cayman Chemical’s conditions. It is a chronic disease that
Lipoxygenase Inhibitor Screening Assay inflames and narrows the air passages
Kit will be purchased at Sapphire of the lungs. Asthma is a common
Bioscience and manufacturer’s protocol childhood disease and approximately
will be followed. Cayman’s 235 million people suffer from it. The
Lipoxygenase Inhibitor Screening Assay majority of deaths associated with
Kit detects and measures the asthma occur in countries with low
hydroperoxides produced in the medium incomes. According to the most
lipoxygenation reaction using a purified recent WHO estimates released in
LO. The detection reaction is equally December 2016, 383 000 asthma-
sensitive to hydroperoxides at various related deaths occurred in 2015.
positions within the fatty acid, and will
work with fatty acids of any carbon On the other hand, the
length. It is thus a general detection Philippines rank No. 9 in asthma
method for LO, and can be used to mortality with approximately 315 million
screen libraries of compounds for those suffering from the ailment globally. The
which inhibit LO enzymes. prevalence of asthma in the Philippines
was reported at 26.7 percent back in
REVIEW OF RELATED LITERATURE
2002 based on the National Several possible risk factors
Epidemiology Survey (Jambora, 2018). related to the development of asthma
have been studied. Atopy is often
ETIOLOGY
identified as a strong risk factor for the
The cause of asthma is unknown, development of asthma. Some studies
but risk factors have been identified and have shown that early sensitization of
interactions between gene and mites and maternal asthma are
environment are important. Genetics is important predictors of asthma. Parental
a factor in asthma and heritability smoking is a major risk factor for acute,
ranging between 35% and 95%. lower respiratory infections in infants
Hundreds of genetic variants is related and wheezing and asthma development
to an increased risk of asthma have in children. However, children who are
been identified in large genetic studies. not exposed to tobacco smoke can also
Epigenetic variations in the transition of have a high incidence of asthma. Active
the genetic code have also been smoking in adults is linked to the later
demonstrated to play a role in the development of asthma. Air pollutions
development of asthma. Respiratory and viral infections are well known
infections particularly early life viral irritant for exacerbations of asthma, but
infections increase the risk of there are conflicting data as to whether
developing asthma, especially when these factors contribute to the
symptoms are severe. Airborne development of asthma. Microbial
exposures to the environment increase exposure is inversely linked to the
the risk of asthma tobacco, smoke, development of asthma and atopy and
ozone and pollutants. The development may account for the disparate
of asthma is also associated with atopic prevalence of asthma in urban versus
conditions and sensitization to inhalant rural environments. Ultimately, asthma
allergens. Other factors are also is likely to develop in genetically
involved in developing asthma like sensitive people by combining complex
microbiome effects, vitamin D, chemical environment exposures (Maslan &
exposure, dietary changes, stress and Mims, 2014).
metabolites. Current understanding of
In most cases, the inflammation
asthma involves a wide range of genetic
underlying asthma is chronically
diversity, which is variable and
present, but asthma often occurs
environmentally influenced by
clinically in attacks or episodes. The
epigenetic and transcriptional factors,
underlying inflammation can be present
leading to less diverse histopathological
with a lack of symptoms and
characteristics with resulting cardinal
inflammation control is central in the
asthmatic symptoms (Mims, 2015).
asthma management. The
disconnection between inflammation recruited by dendritic cells. Multiple
and symptoms can lead to a poor factors such as C-C chemokine receptor
awareness of asthma that can cause types 7 (CCR7), CCR8, and CCRL2
poor recognition and non-compliance mediate the migration of dendritic cells
treatment. The inflammation and from the lymphatics to the regional
associated changes in pathological lymph nodes. T cell differentiation is
tissue leading to a constellation of affected by dendritic cells and under
symptoms like shortness of breath, certain circumstances produce T-helper
coughing, and tightness of the chest. type 2 (TH2) response seen in atopic
The bronchial obstruction is mainly due asthma (Olin & Wechsler, 2014).
to the production of mucus, tissue
In allergic sensitization, the
edema and smooth muscle constriction.
dendritic cell (DCs), which processes
Smooth muscle constriction in the
the antigenic molecules, absorbs
bronchi usually responds to inhaled β2
allergen. Once the allergen is taken up
agonists and creates a reversible
by the dendritic cell, it is presented to
component for episodes of asthma
the naïve T helper cells. These inhaled
(Mims, 2015).
allergens stimulate cell proliferation of
T- helper type 2( Th2), subsequently
Th2 cytokines, interleukin( IL)-4, IL-5
and IL-13 production and release (Kudo
et al., 2013). Through the signaling of
IL-4 and IL-13 to eosinophils and B
PATHOPHYSIOLOGY OF ASTHMA cells, it promotes inflammation.
Dendritic cells, like airway Remodeling by signaling of the
epithelial cells, are also directly exposed interleukins to fibroblasts, airway
to the external environment. Pulmonary smooth muscle, dendritic cells and
dendritic cells act as antigens epithelial cells (Olin & Wechsler, 2014).
presenting cells and express on their The leading candidates in conferring
cell surface a variety of pattern susceptibility to asthma are the genes of
recognition receptors. In response to the IL-4-IL-13-IL-4R cytokine pathways.
pathogens and allergens, dendritic cells Th2-dependent responses like
can also be recruited to the airway. The modulation of lymphocyte and
dendritic cells can be directly stimulated antigen-presenting cell function,
by binding of allergens or infectious stimulation of IgE synthesis and
agents on the surface or stimulated induction of allergic inflammation are
indirectly by epithelial cells in the airway stimulated by these functionally
(by mediators like IL-25, IL-33, GM-CSF overlapping cytokines. Upon exposure
and TSLP). Locally, eosinophils can be to antigen, allergic airway inflammation
will develop in the presence of IL-4R Arachidonic acid (AA) is a
chain expression in the resident airway precursor to two different metabolic
tissues. Events such as induction of pathways, cyclooxygenase (COX) and
airway hyperresponsiveness (AHR), lipoxygenase (LOX), for several families
mucin overproduction, and goblet cell of lipid mediators such as
metaplasia are reliant on the signaling of prostaglandins, thromboxane,
IL-4R signaling in the airway epithelial leukotrienes and lipoxins. These
cells. Moreover, atopy and asthma compounds, commonly referred to as
includes coding genetic polymorphisms eicosanoids, are important lipid
in the human IL-4R chain gene mediators involved in maintaining
(Tachdjian et al., 2009). normal homeostasis, blood pressure
regulation, reproduction and host
B cells play an important role in
defense. Of all the eicosanoids, the
atopic asthma since they produce IgE.
leukotrienes (LTs) play a significant role
Activating factors such as IL-5 and B
in the inflammatory process.
cell promotes the survival of B cell. B
Leukocytes, which are myeloid cells,
cells must bind to T cells under the
particularly the granulocytes,
influence of IL-4 or IL-13 (through CD40
macrophages/monocytes, and mast
and CD40 ligand respectively) to
cells are responsible for the synthesis of
produce IgE, usually within regional
the leukotrienes. Leukotriene is a
lymph nodes. High affinity FC receptors
product of AA metabolism through the
on basophils and mast cells are
5-lipoxygenase (5-LO) pathway. In a
primarily bound with the secreted IgE,
paracrine signaling, they exert their
and these cells will undergo
activity at nanomolar concentrations in
degranulation when crosslinked by
the local cellular milieu targeting cells
aeroallergens thereby causing the
implicated with receptors that transduce
release of their mediators. Mast cells
the intracellular signaling and
play an important role in the
specifically recognize LTs. Leukotrienes
pathogenesis of asthma. These cells are
can be classified into two major classes,
maintained on distinct mucosal surfaces
namely the dihydroxy acid leukotriene
by IL-9 and can be stimulated by binding
B4 (LTB4) and the
of the surface receptor c-kit to the stem
cysteinyl-leukotrienes (cys-LT). LTB4 is
cell factor, binding of tyrosine kinase or
a potent chemoattractant which has an
by crosslinking of IgE. Cysteinyl
activity on immune cells such as
leukotrienes, prostaglandins and
macrophages eosinophils and
histamine are released when the mast
neutrophils. And the cys-LT possesses
cells are activated (Olin & Wechsler,
a powerful spasmogenic effect,
2014).
especially in the respiratory tract. There
are two enzymes involved in the
committed step in the LTB4 and cys-LTs Air-way smooth muscle cell hyperplasia,
biosynthesis. Leukotriene A4 hydrolase eosinophilic inflammation, mucus
(LTA4H) catalyzes LTB4 biosynthesis hypersecretion, mucus gland
and leukotriene C4 synthase (LTC4S) hyperplasia, and collagen accumulation
catalyzes cys-LTs biosynthesis, both underneath the epithelial layer and in
from LTA4 which is the common the lung interstitium at leucocyte
substrate. The AA has both infiltration sites, are involved in the
pro-inflammatory and anti-inflammatory process of airway remodeling where
mediators. Examples of those included cysLTs may participate in. LTB4 is a
in the biosynthesis of anti-inflammatory factor that may contribute to the
and pro-resolving mediators are lipoxins narrowing of the airway by increasing
(LXs) such as LXA4, LXB4 and aspirin mucus secretion and production of local
triggered lipoxins. With LTA4 as an edema. But it has no bronchoconstrictor
intermediate and through the effect in asthmatic patients and in
consecutive actions of 12-LOX, healthy subjects. In neutrophilic
acetylated COX-2 or 5-LO via phenotype of asthma, LTB4 plays and
transcellular routes, lipoxins are formed important role because of its highly
(Di Gennaro & Haeggström, 2013). potent chemoattractant activity for
neutrophils that may lead to asthma
Leukotrienes such as cys-LTs
exacerbations (Montuschi &
and LTB4, which are derived from
Peters-Golden, 2010).
arachidonic acid via the 5-lipoxygenase
(5-LO) pathway, is a potent lipid According to numerous studies,
mediator. Different types of airway inflammation is a central key to
inflammatory cells implicate the the pathophysiology of asthma.
complete synthesis of CysLTs pathway Existence of chronic inflammation of the
and become stimulated during airway has been acknowledged for a
inflammation of the allergic airway. long time. The liberation of potent
Several cell types that lack the complete chemical mediators from inflammatory
synthetic pathway like endothelial cells cells resulting to chronic inflammation of
and platelets induce CysLTs through the airway, airway remodeling, which is
intercellular transfer mechanism from characterized by thickening of airway
the reactive mediator LTA4. LTs play a wall compartments that may lead to the
central role in asthma, but their progression and development of the
importance may vary in patients with disease (Kudo et al., 2013).
asthma. CysLTs causes diverse
ENZYME ROLE
pathophysiological responses that
contribute to asthma, while LTB4’s role Lipoxygenases belong to a class
in disease expression is more restricted. of non-heme iron containing
dioxygenases which catalyze the response which are prominent in the
addition of oxygen molecule to fatty usual signs of inflammation.(Sinbad et
acids containing a cis, al., 2017) Glucocorticoid receptor can
cis-1,4-pentadiene system to yield an be inactivated by the released
unsaturated fatty acid hydroperoxides. lysosomal enzymes through
These LOX products have been found antagonizing the interaction between
to play an important role in various glucocorticoids and GR and by altering
disorders, including inflammation, the size of the receptor complex.
bronchial asthma, and tumor Moreover, the release of lysosomes can
angiogenesis. LOX is expressed in regulate this immune response by
platelet, neutrophils, eosinophils, degradation or secretion of inflammatory
synovial fluid, colonic tissues, cytokines. On a case-to-case basis,
monocytes, lungs, and bone marrow lysosomes can either have
cells in human tissues. 5-LOX found in anti-inflammatory or pro-inflammatory
the neutrophils, converts the arachidonic mechanisms. This negative and positive
acid to to 5-hydroxy-6, 8, 11, 14- regulation of inflammation by lysosomes
eicosatetraenoic acid 5-HPETE. has been defined as “flexible” and is
5-HPETE is converted into a series of significant in maintaining this balance.
leukotrienes and the tissue varies However, lysosomes contain a
depending on the nature of the final substantial number of pro-inflammatory
product. Leukotrienes are allergic membrane protein which acts as targets
reactions and inflammation mediators. for the discovery of new drugs.
Leukotriene receptor antagonists and Lysosomes contribute to the
5-LOX inhibitors are used in asthma progression of autoimmune diseases
treatment (Sacan & Turhan, 2014). through the increase production of
autoantigens by the lysosomal
MEMBRANE DESTABILIZATION
enzymes. Cathepsins (also known as
ROLE IN INFLAMMATION
lysosomal proteases) were recognized
Membrane destabilization of the to have an important role in the
immune system cells is a physiologic pathogenesis of some autoimmune
indicator in the pathogenesis of several diseases namely rheumatoid arthritis
inflammatory diseases. The secretion of (RA), psoriasis, systemic lupus
the lysosomal constituents (bacterial erythematosus (SLE) and multiple
enzymes and proteases) of the immune sclerosis (MS) (Ge, Li, Gao, & Cao,
system cell system (mast cells, 2015). In preventing initiation of
neutrophils, basophils, eosinophils and inflammation, limiting the release of
leukocytes) in response to a stimuli may lysosomal constituents by stabilizing the
lead to progression of the damage thus lysosomal membrane is significantly
resulting in several physiologic evident. HRBC membrane stabilization
has been introduced as a technique to published studies about asthma and
observe anti-inflammatory for it has other conditions. These adverse effects
been analogous to the lysosomal are considered chronic conditions
membrane in which the component can because of its development over time.
modify the stabilization of the said Acute adverse effects (e.g.,
membrane. NSAIDS stabilize lysosomal gastrointestinal ulcers/bleed) may also
membranes or may inhibit the effects of be present with its long-term use
these lysosomal enzymes. (Sullivan et al., 2018).
(Leelaprakash & Mohan Dass, 2011).
BETA AGONIST
TREATMENT
β2-Agonists are effective
CORTICOSTEROIDS bronchodilators mainly because of their
ability to relax airway smooth muscles
The most effective
(ASM). They initiate a signaling effect
anti-inflammatory therapy for asthma
that arises to a several events leading to
are inhaled corticosteroids, these drugs
the relaxation of ASM by binding to the
works in almost every patient. But then,
active site of β2-adrenoceptors on ASM.
inhaled corticosteroids have the
Some differences exist between
potential systemic side effects because
β2-agonists. The inhaled traditional
of its lungs absorption which has placed
short-acting β2-agonists, albuterol,
an effort for the researchers to find safer
terbutaline and fenoterol afford rapid
ICs with reduced absorption from the
symptom relief and short- term
lungs and oral bioavailability or
prophylactic protection against
circulation inactivation (P. Barnes,
bronchoconstriction induced by exercise
2012). Down regulation arises by the
or other stimuli. The β2-agonists,
pro-inflammatory proteins and the
formoterol and salmeterol comprise an
asthma-induced structural changes
important advance due to its twice-daily
(airway remodeling) are reversed with
administration. Significant clinical
the increased vascularity of the
benefits are offered because of their
bronchial wall (Hossny et al., 2016).
bronchodilating effect and long-term
Adverse effects associated with improvement in lung functions to
corticosteroid are reportedly Metabolic patients with asthma (Cazzola et al.,
syndrome, osteoporosis Hypertension, 2013).
Obesity, Type 2 Diabetes Mellitus,
β2-agonists have an acceptable
Cataracts, Avascular Necrosis,
safety profile. But it possesses adverse
Dyslipidemia, Gastrointestinal
effects such as palpitations, increased
Ulcers/Bleeds, fractures, Tuberculosis
heart rate, transient decrease in PaO2
and Glaucoma in relation to the
and tremor. β2-adrenoceptors
desensitization during the first few days disadvantages like hepatic toxicity and
of treatment is responsible for the its opposing pharmacokinetic profile
observed tenacity most of these adverse from its short half-life (Rossi et al.,
effects. Nonetheless β2-agonists 2010).
bronchoprotective effects can also
Zileuton is a benzothiophene
induce tolerance and reduce the
N-hydroxyurea and is known to interfere
bronchodilator sensitivity to them
with inflammation and allergic diseases
(Cazzola et al., 2013).
via inhibition of leukotriene (LT)
Short-acting beta agonists has a short biosynthesis. Zileuton chelates the
half-life confines them as maintenance active site of iron of the enzyme with its
treatment. They are currently used as weak reducing properties asserting that
rescue medications. In contrast, it belongs to 5-lipoxygenase iron
sustained bronchodilation provided by ligand-type inhibitors. Knowing the
Long-acting beta agonists and importance of leukotrienes in the airway
ultra-long-acting beta agonists are inflammation, this drug has been added
incapable to influence to decline the to provide a therapeutic option in the
accelerated lung function characteristics therapy of persistent and chronic
in chronic obstructive pulmonary asthma. However, the clinical use of
disease (COPD) (Cazzola et al., 2013). Zileuton was limited because of its liver
toxicity thus leading to the need of
The complementary actions of
monitoring the hepatic enzyme levels.
corticosteroids with long-acting beta
Nonetheless this hepatic injury caused
agonists like salmeterol and formoterol
by Zileuton is not related to the inhibition
are now the most effective available
of 5-LOX direct toxic effect (Rossi et al.,
therapy for asthma. LABAs are
2010).
bronchodilators and important for
negating and relieving Leukotrienes (LTs), including
bronchoconstriction which last for over LTB4, cysteinyl LTs (CysLTs) has a role
12 hours (P. J. Barnes, 2010). in pathophysiology of asthma that is a
potent mediator of lipids. Increased
5-LIPOXYGENASE INHIBITORS/
airway smooth muscle activity, airway
LEUKOTRIENE INHIBITOR
secretion and microvascular
Zileuton the only approved permeability are the pathophysiological
5-lipoxygenase inhibitor for the effect of the CysLTs activation in
prevention and treatment of asthma in asthma. Montelukast, Zafirlukast and
adults and children 12 years old and Pranlukast are selective CysLTs1
above (Montuschi & Peters-Golden, receptor antagonist approved for
2010). Zileuton has various
treatment of asthma (Montuschi & using LOX-inhibitor screening assay kit
Peters-Golden, 2010). (Catalog No. ab133087, abcam, UK).
The extracts presented a
Leukotrienes are 5-lipoxygenase
15-lipoxygenase inhibitory effect at a
products synthesized by mast cells,
certain level. 88.81% of ethyl acetate
eosinophils and other inflammatory cells
leaf extract yield the inhibitory activity of
of arachidonic acid. LTB4 and cysteinyl
15-LOX inhibitor and the bark extract of
leukotrienes (cysLT: LTC4, LTD4, LTE4)
acetone with IC50 value of 41± 1.3
were both involved in asthma
µg/mL was the highest (Isa, A. I., Saleh,
pathogenesis. Potent
M. I. A., Abubakar, A., Dzoyem, J. P.,
bronchoconstrictors like cysLT also
Adebayo, S. A., Musa, I. Sani, U. F.,
have a blood vessel, mucociliary
Alexandra, 2016).
clearance and eosinophilic effect in
inflammation. LTB4 is a potent Fresh leaves of ​Bambusa
chemoattractant for neutrophils and spinosa, Amaranthus viridis ,
eosinophils and a cell activator. Commelina diffusa, Eclipta alba,
Leukotriene synthesis is inhibited by Crataeva religiosa, Euphorbia hirta,
5-lipoxygenase. CysLT1 receptors are Isotoma longiflora, Monochoria
block by leukotriene receptor vaginalis, Plumeria rubra, , Pistia
antagonists on smooth muscle in the stratiotes a ​ nd Premna odorata was
airway and other cells. The assayed with 15-lipoxygensae inhibitor
anti-inflammatory and bronchodilator activity. 51.3% of inhibition ​from C.
effects of these drugs are modest diffusa and 48.5% from ​E. hirta of LOX
(Marinho & Custovic, 2008). inhibition activity showed the highest
effects at a concentration of 167µg/ml.
The prominent hepatic enzyme
On the other hand, not all samples
elevation incidence in Zileuton was not
exhibited higher activity compared with
seen in Montelukast and its shorter
quercetin the positive control at 62.4%
half-life that requires an administration
at 17µg/ml concentration. The traditional
of four times daily (Montuschi &
use of these plant extracts as
Peters-Golden, 2010).
respiratory remedy in inflammation was
OTHER PLANTS WITH ANTI-LOX ACT validated via the spectrophotometric
assay related to oxygen and
Cordia Africana ​(Boraginaceae) a​ polyunsaturated fatty acid
folkloric plant medicine is used in 1,4-diene-type structure of 15-LOX
inflammation and other related reaction. Flavonoids and terpenoids
infectious diseases and conditions​. The contents were both positive in ​C. diffusa
stem and bark of ​Cordia Africana and ​E. hirta.​ (D. Paguigan &
anti-inflammatory activity was evaluated Chichioco-Hernandez, 2014)
Studied natural compounds comprises low and medium altitudes in thickets and
isolated flavonoids from orange peels, secondary forests.(Gupta & Kaur,
and from the leaves of ​Orthosiphon 2015)(Jamkhande et al., 2013)
spicatus​. The leaves and seeds extract
of ​Coriandrum sativum correlatedly
showed its effect because of its phenolic
content (D. Paguigan &
Chichioco-Hernandez, 2014)

ACTIVITIES OF FLAVONOIDS

Flavonoids are plant extracted PLANT TAXONOMY

products and belong to an essential
classification of natural products; they
belong in a particular class of secondary
plant metabolites with a polyphenolic
structure commonly found in certain
drinks, fruits, and vegetables. There are
several subgroups of flavonoids,
including flavones, chalcones,
isoflavones, and flavonols. Flavonoids
possess anti-inflammatory, antioxidant,
anti-carcinogenic and anti-mutagenic
properties coupled with their ability to
regulate key cellular enzyme functions
such as cyclooxygenase (COX),
xanthine oxidase (XO), lipoxygenase
and phosphoinositide 3-kinase. FIGURE 1. ​Cordia dichotoma t​ ree
(Panche, Diwan, & Chandra, 2016) Retrieved from:
FAMILY OF CORDIA DICHOTOMA https://sylviatramos.blog/

The genus ​Cordia b​ elongs to the Kingdom: Plantae

Boraginaceae family, with around 300 Division: Magnoliophyta
species worldwide, mainly in the warmer
regions of the world. The species is Class: Dicotyledons
common in the Philippines and found in
 Subclass: Astaridae The presence of pyrrolizidine
alkaloids, coumarins, flavonoids,
Order: Lamiales
terpenes and sterols was demonstrated
Family: Boraginaceae by chemical screening of both the
leaves and fruit. Four flavonoid
Genus: Cordia glycosides (robinin, rutin, rutoside,
Fragrant: Manjack datiscoside and hesperidine) and two
phenolic derivatives (chlorogenic acid
Species: C. dichotoma and caffeic acid) have been isolated
from fruits and leaves. The fruits have
astringent, anthelminthic, diuretic,
demulcent and expectorant activity. The
fruit’s mucilage treats cough and other
chest complaints. It is also used to treat
DESCRIPTION OF CORDIA uterine disorders and urethra. The
DICHOTOMA powder form the fruit kernels are mixed
with oil to heal tinea. The plant is also
Boraginaceae is a family of a used as laxative and diuretic. The
small to moderate deciduous tree with a leaves are used as tonic, stimulant, and
short bole, short crooked trunk and febrifuge. In cough and cold, leaf
spreading crown. The stem bark is decoction is used. The leaves are also
smooth and longitudinally wrinkled in used for headache relief and ulcer
grayish brown. Leaves are simple, treatment. The traditional use of ​C.
completely and slightly dented, dichotoma ​G. Forst leaves methanol
elliptical-lanceolate to wide ovate with a extract as analgesic, antipyretic, and
round and cordate base. The flowers anti-inflammatory drug is also evident.
are short, bisexual and white to pink and (Gupta & Kaur, 2015).
appear in loose cymes of corymbose.
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