Biochem Journal Critique

Mariano Marcos State University
College of Health Sciences

Department of Pharmacy
Journal Critique
Submitted by:
Azurin, Alecs Jane
Bayongan, Maria Yvonne
Gutierrez, Kristella Millen
Pagdilao, Charmiane
Sobprepena, Neslie Lou
Tabili, Chantelle Mea
Tomogbong, Diane Rose
In partial fulfillment of the requirement in PHBS 103

(Pharmaceutical Biochemistry)
Submitted to:
Jay Vee S. Racuya, RPh
November 2019
Name of the authors:
Kathrin Arnhard, David Bernhard, Philipp Eller, Michael Grimm,
Katharina Heinz, Günther Laufer, Barbara Messner, Herbert
Oberacher Florian Pitterl, Andreas Ritsch, Bernhard Scharinger, ,
Daniela Schuster, Hermann Stuppner, Adrian Türkcan,, Anna Vuorinen
Title of the Article:

Leoligin, the major lignan from Edelweiss, inhibits 3-hydroxy-3-
methyl-glutaryl-CoA reductase and reduces cholesterol levels in
ApoE-/-mice
Title of the journal

Journal of Molecular and Cellular Cardiology
Statement of the problem or issued discussed:

A problem that was indicated in the study is that despite the
indispensable clinical importance of statin and the fact that the discovery of
statins and their widespread use is a tremendous success story, the search
for new lipid lowering drugs is still a highly relevant task.
The author’s purpose, approach or methods, hypothesis, and major

conclusions:
The aim of the study is to reveal the impact of leoligin on
cholesterol levels and to define its mechanism of action. The tests and
methods performed are Assessment of atherosclerotic plaque area,
assessment of atherosclerotic plaque area, intraperitoneal glucose
tolerance test, HMG-CoA-reductase activity assay, peroxisome
proliferative-activated receptor-gamma activity assay, in silico modelling
(PPAR-γ), immunofluorescence staining of HMG-CoA-reductase on liver
sections, hepatic protein extraction, western blotting and
immunodetection, hepatic protein extraction, oil Red O staining of frozen
liver sections and quantification, Statistical analyses.
According to the study conducted, leoligin was identified as a

potential new cholesterol lowering drug that significantly reduces total
serum cholesterol as well as LDL-cholesterol and leads to favourable
cholesterol ratios in ApoE−/− mice via the inhibition of HMGCR. Data shows
conflicts on the effects of traditional HMGCR inhibitors in ApoE−/− mice
exist- (i) statins were shown to have no effect, an increasing effect, or a
decreasing effect on cholesterol levels and ii) it was suggested that statins
decrease atherosclerotic plaque formation independent of their impact on
cholesterol levels [29–31] – the researchers then concluded that leoligin,
which is not a statin and does not have a statin-like structure, decreases
cholesterol levels through direct inhibition of HMGCR in vivo.
CHAPTER 1
Introduction
Background of the Study

In 1913, Anitschkow and Chalatov indicated that a cholesterol rich
diet in rabbits produces vascular lesions like atherosclerotic lesion in people.
Thusly, various animal, clinical, and epidemiological examinations explained
the affiliation and causality between dyslipidaemia, the development of
atherosclerosis, and coronary supply artery disease (CAD) mortality.
Treatment choices for atherosclerosis and CAD which incorporate
conservative approaches, such as physical exercise, diet, and
pharmacotherapy just as interventional choices, namely coronary artery
bypass grafting and percutaneous coronary intervention are versatile and
fundamentally relied upon a person's hereditary foundation, lifestyle, also,
the phase of the illness. Over the most recent two decades LDL-cholesterol
lowering statins have been appeared down to diminish death rates in primary
and secondary prevention of CAD.
Leoligin, a compound isolated from the roots of Leontopodium nivale
subsp. alpinum Cass. (Edelweiss), has shown promising anti-atherosclerotic
characteristics in previous studies: Duwensee et al. investigated the influence
of leoligin on cholesteryl ester transfer protein (CETP), an enzyme that
promotes the transfer of cholesteryl esters from high-density lipoprotein
(HDL) to, among other lipoproteins, LDL. Thus CETP inhibition raises HDL-
cholesterol levels. In the same study, leoligin was also revealed to inhibit
CETP in human plasma. In addition, transgenic CETP mice given leoligin orally
showed a trend towards reduced serum LDL-cholesterol levels even though
elevated CETP activity was measured, suggesting a possible lipid lowering
effect due to other mechanisms.
Statement of the Problem

A problem that was indicated is the study is that despite the
indispensable clinical importance of statin and the fact that the discovery of
statins and their widespread use is a tremendous success story, the search
for new lipid lowering drugs is still a highly relevant task.
Hypothesis
Leoligin reduces low-density (LDL) – cholesterol levels in ApoE-/-mice
independent from its suggested effects on cholesteryl ester transfer protein
(CEPT) activity and thereby inhibit the formation of atherosclerotic plaque.
Significance of the Study

Leoligin’s promising effects on cholesterol levels can be of benefit in
hyperlipidaemic individuals and in the regression of coronary atherosclerosis.
A new discovery of a lipid lowering drug is significant in the reduction of
coronary heart disease in humans.
Scope and Limitation of the Study
The study was focused mainly on Leoligin, the major lignan from
Edelweiss, inhibits 3-hydroxy-3-methyl-glutaryl-CoA reductase and reduces
cholesterol levels in ApoE-/-mice.
The research study used 40 mice which is randomly divided into 4
treatment groups (n=10 per group). Female 24 weeks-old ApoE−/− mice on
C57BL/6J genetic background were obtained from Charles River (Sulzfeld,
Germany) and housed at 24 °C and a 12 h light/dark cycle in a virus/antibody-
free environment of the Core Unit for Biomedical Research at the Medical
University of Vienna. All animal experiments corresponded to the European
Union directive 2010/63/EU, were approved by Austrian veterinary
authorities, and were carried out in accordance to the “Principles of
laboratory animal care” formulated by the National Society for Medical
Research and the “Guide for the care and use of laboratory animals”,
prepared by the Institute of Laboratory Animal Resource and published by
the NIH.
Chapter 2
Methods and Procedures
Study Design
Biochemical analysis
Assessment of atherosclerotic plaque area
Intraperitoneal glucose tolerance test
HMG-CoA-reductase activity assay
Peroxisome proliferative-activated receptor-gamma activity assay
In silico modelling (HMG-CoA-reductase)
In silico modelling (PPAR-y)
Immunofluorescence staining of HMG-CoA-reductase on liver section
Hepatic protein extraction, Western blotting and immunodetection
Oil red O staining of frozen liver section and quantification
Statistical analysis
Research Locale
Female 24 weeks-old ApoE−/− mice on C57BL/6J genetic background were

obtained from Charles River (Sulzfeld, Germany) and housed at 24 °C and a 12 h
light/dark cycle in a virus/antibody-free environment of the Core Unit for Biomedical
Research at the Medical University of Vienna. All animal experiments corresponded
to the European Union directive 2010/63/EU, were approved by Austrian veterinary
authorities, and were carried out in accordance to the “Principles of laboratory
animal care” formulated by the National Society for Medical Research and the
“Guide for the care and use of laboratory animals”, prepared by the Institute of
Laboratory Animal Resource and published by the NIH.
Research Procedure
ApoE−/− mice were fed a standard chow diet V1534–300 (ssniff, Germany)
and received drinking water ad libitum. After a short period of acclimatization,
ApoE−/− mice (n = 40; age = 29.5 ± 2 weeks) were randomly divided into four
treatment groups (n = 10 per group) to receive either: vehicle control (0.1%
dimethylsulfoxide; Sigma-Aldrich, USA), 1 μM leoligin, 10 μM leoligin, or 50 μM
leoligin dissolved in drinking water (all in 0.1% dimethylsulfoxide end concentration),
respectively. Anaesthesia was carried out using 7.2 mg xylasol + 96 mg ketamine
(Bayer, Germany; Richter Pharma, Austria) per kg body weight.
After 5 and 10 weeks of treatment fasting blood samples for biochemical analyses
were collected from the tail vein using a Microvette CB300Z (Sarstedt, Germany).
After 2 and 16 weeks of treatment, intraperitoneal glucose tolerance tests (IPGTT)
were performed in fasting mice (n = 5 per treatment group).
After 16 weeks of treatment, mice were anaesthetized (240 mg xylasol + 1200 mg

ketamine/kg body weight) and final blood samples were obtained by puncture of
the inferior vena cava, mice were sacrificed by removal of the heart and
consequent terminal bleeding. Finally the aorta was extracted and carefully
cleaned of all perivascular tissue.
I.
A. Critique on the flow of thought of the headings used.
∙ The flow of the thoughts and the content of the journal are
clearly written and organized. The construction of the
sentences and paragraphs are in order and was created in
a way that it is easy to understand. The research designs
are well emphasized allowing the readers to have a better
comprehension of the study. The use of the scientific
terms is consistent and the abbreviations and their
corresponding meanings are written completely
∙ The statement of the problem is not clearly stated in the
journal.
∙ Diagram, charts and tables are well illustrated.
B. Critique the paper in terms of the references used.
C. Critique on the methodology

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Mariano Marcos State University

College of Health Sciences

In partial fulfillment of the requirement in PHBS 103

Title of the Article:

Title of the journal

Statement of the problem or issued discussed:

The author’s purpose, approach or methods, hypothesis, and major

According to the study conducted, leoligin was identified as a

Background of the Study

Statement of the Problem

Significance of the Study

Methods and Procedures

Assessment of atherosclerotic plaque area

Intraperitoneal glucose tolerance test

HMG-CoA-reductase activity assay

Peroxisome proliferative-activated receptor-gamma activity assay

In silico modelling (HMG-CoA-reductase)

In silico modelling (PPAR-y)

Immunofluorescence staining of HMG-CoA-reductase on liver section

Hepatic protein extraction, Western blotting and immunodetection

Oil red O staining of frozen liver section and quantification

Female 24 weeks-old ApoE−/− mice on C57BL/6J genetic background were

After 16 weeks of treatment, mice were anaesthetized (240 mg xylasol + 1200 mg

B. Critique the paper in terms of the references used.

C. Critique on the methodology

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