Department of Biotechnology

“Prevention of cardiovascular pathogenesis by Malabaricone C and 4,4’-

Dihydroxystilbene”

A Dissertation Submitted to the

University of Mumbai
And KET’s V. G. Vaze College of Arts, Science and Commerce
Towards the partial Fulfilment of the
Degree of Masters of Science in Biotechnology (By Paper)

Submitted by

KATARIYA MINAKSHI MOHANLAL

Department of Biotechnology,

KET’s V.G. Vaze College of Arts, Science and Commerce

Mithagar Road, Mulund (East), Mumbai-400081

University of Mumbai

Under the Guidance of

Dr. JITESH SINGH RATHEE

SO/F, Bio-Organic Division

Bhabha Atomic Research Centre, Trombay, Mumbai-400085

 CERTIFICATE

KET’s V.G. Vaze College of Arts, Science and Commerce

Mithagar Road, Mulund (East), Mumbai-400081

Department of Biotechnology

This is to certify that Ms. Minakshi Mohanlal Katariya has satisfactorily completed the
research project entitled “Prevention of cardiovascular pathogenesis by Malabaricone C
and 4,4’-Dihydroxystilbene ” as prescribed by the University of Mumbai towards the partial
fulfilment of the M.Sc. in Biotechnology (By Paper) during academic year 2018-2019.

The work entered in this dissertation is bonafide work of the student as carried out at Bio-
Organic Division, Bhabha Atomic Research Centre, Trombay, Mumbai-400085.

______________________ ____________________ _____________

External Examiner Signature Date

 DECLARATION

I, Katariya Minakshi Mohanlal, hereby declare that the thesis entitled “Prevention of
cardiovascular pathogenesis by Malabaricone C and 4,4’-Dihydroxystilbene” being
submitted for partial fulfilment of the requirement for the Degree of Master of Science in
Biotechnology to KET’s V. G. Vaze College of Arts, Science and Commerce is bonafide
work carried out under supervision of Dr. Jitesh Singh Rathee, SO/F, Bio-Organic Division,
Bhabha Atomic Research Centre (BARC), Trombay-85, during the course of the year 2018-
2019.

I further declare that the work reported in this thesis has not been submitted, either partially
or fully for the award of any other Degree, Diploma, Associate ship, and Fellowship in this
institute or any other institute or university.

Place: Mumbai Signature of Candidate

Date: KATARIYA MINAKSHI MOHANLAL
 ACKNOWLEDGEMENT

I would like to express my gratitude to Dr. Deepali Karkhanis, Head of the Department of
Biotechnology, KET’s V.G. VAZE College of Arts, Science and Commerce, who allowed
me to carry out the project work at the Bhabha Atomic Research Centre, Trombay-85.

I take this opportunity to express my profound gratitude and deep regards to my guide Dr.
Jitesh Singh Rathee SO/F for his exemplary guidance throughout the course of this project.
The blessing, help and guidance given by him shall carry me a long way in the journey of life
on which I am about to embark. I also take this opportunity to express a deep sense of
gratitude to Dr. B. S. Patro (SO/G), Head, Chemical Biology Section, for letting me have this
opportunity to work under his guidance.

I also sincerely thank Dr. Mahesh Subramanian, Dr. Mrityunjay Tyagi, Mr Saikat
Chakraborty, Mr Ganesh Pai B, Mr Ananda Guha Majumdar, Ms. Pooja Gupta, Ms.
Krupa Thankam Philip and Ms. Deepti Singh for the cordial support, valuable time,
information and guidance, which helped me in completing this task through various stages.

I am really grateful to my colleagues, Priyanka Sharma and Namita Bamne both of them
have been a really great company to be with. Their presence made our respective work a fun
filled experience. They have been a great moral support throughout the duration of the project
work and will be missed truly and deeply.

Last but definitely not the least; I am indebted to my parents for the values they have
implanted in me.

I once again thank the almighty for giving me a chance to meet such wonderful people, who
gave me such ever so helpful friends, my loving parents and the awe-inspiring beautiful
surroundings of the workplace. May he bless all of us with Peace and good health.

Thank you

Yours Sincerely,

Katariya Minakshi Mohanlal

 INDEX

Sr. Title Page

No. No.
I Abbreviations and Acronyms I
II List of Figures IV
III List of Graphs V
1. Abstract 2
2. Introduction 4
3. Review of Literature 18
4. Materials and Methods 24
4.1. Experimental Animals 25
4.2. Drugs and Chemicals 25
4.3. Preparation of Drugs and Chemicals 25
4.4. Experimental Design 26
4.4.1. Study the preventive effect of Malabaricone C on Hypertension 26
4.4.2. Study the preventive effect of 4,4’-Dihydroxystilbene on Myocardial Infarction 27
4.5. Anesthesia 27
4.6. Uninephrectomy (UNX) 28
4.7. Induction of Experimental CVD 28
4.7.1. Induction of Experimental Hypertension 28
4.7.2. Induction of Experimental Myocardial Infarction 28
4.8. Measurement of BP 28
4.8.1. Non-Invasive BP (NIBP): Tail cuff method 28
4.8.2. Invasive BP (IBP): Intra-Arterial Catheter Method 29
4.9. ECG 29
4.10. Weight of the Organs 30
4.11. Collection of Blood Plasma 30
4.12. Statistical Analysis: Mal C+ DOCA Experiment and DHS+ ISO Experiment 30
5. Results and Discussion 31
6. Conclusions 42
7. References 45
 ABBREVIATIONS AND ACRONYMS

LIST OF ABBREVIATIONS

CVD Cardiovascular Disease

CHD Coronary Heart Disease

IVC Inferior Vena Cava
SVC Superior Vena Cava
HT/ HTN Hypertension
BP Blood Pressure
HBP High Blood Pressure
SBP Systolic Blood Pressure
DBP Diastolic Blood Pressure
NIBP Non-Invasive Blood Pressure
IBP Invasive Blood Pressure
ECG Electrocardiogram
mmHg Millimeter of mercury
Ang II Angiotensin II
ROS Reactive Oxygen Species
SMCs Smooth Muscle Cells
ECM Extracellular Matrix
NADPH Nicotinamide Adenine Dinucleotide Phosphate Hydrogen
NOX NADPH Oxidases
SOD Superoxide Dismutase
VSMC Vascular Smooth Muscle Cells
ET Endothelium
LDL Low Density Lipoprotein
OxLDL Oxidative LDL

I
 HF Heart Failure
LV Left Ventricle
NO Nitric Oxide
ONOO- Peroxynitrite
SA Sino Atrial
AV Atrioventricular
ms milliseconds
Mal C Malabaricone C
ACEIS Angiotensin Converting Enzyme Inhibitors
ARBS Angiotensin Receptor Blockers
ESRD End stage Renal Disease
CHF Congestive Heart Failure
ACS Acute Coronary Syndrome
RAS Renin Angiotensin System
DMF Dimethylformamide
DSHR DOCA–Salt Hypertension rat
˚C Degree Celsius
IP Intraperitoneally
PBS Phosphate Buffer Saline
BPM Beats Per Minute
Hz Hertz
mV Millivolt
MI Myocardial Infarction
mL Milliliter
EDV End Diastolic Volume
ESV End Systolic Volume
HR Heart Rate
HRV Heart Rate Variability

hrs Hours

II
 min Minute
rpm Revolutions Per Minute
mg milligram
kg kilogram
g gram
ISO Isoproterenol 4- [1-hydroxy-2-(isopropyl amino) ethylbenzene-1,2-diol
hydrochloride
PUFAs Polyunsaturated Fatty Acids
DHS 4,4’-Dihydroxystilbene

III
 LIST OF FIGURES

Sr. No. Title Page No.

1 Anatomy of the Heart 6

2 Oxidative Stress in CVD 13

3 ECG wave 15

4 Membrane Potentials in Cardiac Contractile Cells 16

5 Chemical Structure of Malabaricone C 17

6 Chemical Structure of 4,4’-Dihydroxystilbene 17

IV
 LIST OF GRAPHS
1. Mal C + DOCA Experiment

Sr. No. Title Page No.

1 Effect of Mal C on Body Weight and Water Consumption 32

2 Effect of Mal C on NIBP (SBP) and IBP (SBP & DBP) 32

3 Effect of Mal C on Heart Rate, P Duration, QRS Complex and QTc 33

Interval
4 Effect of Mal C on LF/HF ratio 34

5 Effect of Mal C on Mean Pressure 34

6 Effect of Mal C on Dichrotic Notch 35

7 Effect of Mal C on Ejection Duration 35

8 Effect of Mal C on Maximum and Minimum LV Pressure 36

9 Effect of Mal C on Systolic and Diastolic Duration 36

10 Effect of Mal C on Maximum and Minimum dP/dT Pressure 37

11 Effect of Mal C on Contractility Index 37

12 Effect of Mal C on Organ Hypertrophy 38

V
 2. DHS + ISO Experiment

Sr. No. Title Page No.

1 Effect of DHS on PR Interval 39

2 Effect of DHS on Heart Rate and RR Interval 40

3 Effect of DHS on QRS Complex 40

4 Effect of DHS on QT Interval 41

5 Effect of DHS on ST Segment 41

VI
 “Prevention of cardiovascular
pathogenesis by Malabaricone C and
4,4’-Dihydroxystilbene”

1
1. ABSTRACT

2
Cardiovascular disease (CVD) is the pathological condition of the heart, blood vessels, brain
and kidney leading to insufficiency or arrest of cardiac activity. It is the leading cause of
mortality globally and in India. Incidences of CVD in India have increased by 59%, in 2010
from 1990. Available drugs to prevent and treat CVD are expensive and mostly exhibit side
effects. Hence there is a need to develop new drugs which are economical with lesser side
effects. In the current study, we have checked the CVD preventive potential of a naturally
occurring phenolic Malabaricone C from the plant Myristica malabarica in DOCA–Salt
hypertensive rat model (DSHR). The DSHR model show acute hypertension and pathological
cardiovascular remodeling in addition to the cardiac electric disturbances. In comparison to
DSHR, Mal C pre-treatment in DSHR reduced the extent of hypertension, and corrects the
electric disturbances by reducing the QTc, PR, and PQ segments, and improved cardiac
rhythmicity as revealed by ECG profiles. In addition, Mal C treatment in DSHR shows
reduction of sympathetic tones and stimulation of parasympathetic tones thus shifting the
sympathovagal balance towards parasympathetic also depicted by the reduced heart rate.
Moreover, Mal C treatment also showed improved ventricular contractility, diastolic filling,
cardiac output, and reduced diastolic and systolic pressures, along with the end diastolic
pressures measured invasively by catheterization of carotid artery and ventricle. Besides,
marked reduction in organ hypertrophy and water consumption was observed indicating
better kidney; heart functioning with reduced hemodynamic load in mal C treated DSHR. The
results indicated that Mal C prevents the onset and the extents of cardiovascular damage in
DSHR model. It acts on multi targets in cardiovascular system to achieve desired end results
in addition to being non-toxic and economical. Thus Mal Cs shows great potential to be used
as a potent cardio tonic for the prevention of CVD.

We have also studied whether the oral administration of 4,4’-Dihydroxystilbene (DHS) can
prevent the myocardial Infarction (MI) in rats. In rats, Isoproterenol 4-[1-hydroxy-2-
(isopropyl amino) ethylbenzene-1, 2-diol hydrochloride (ISO) is a synthetic catecholamine
and beta adrenergic agonist responsible for production of severe oxidative stress,
inflammation in the myocardium and results in MI in supramaximal dose. Since oxidative
stress is one of the main factors leading to myocardial infarction, DHS which is an
antioxidant with anti-inflammatory activity was used to evaluate their cardio protective
effects. DHS improved the ventricular function by correcting the intra-ventricular
contraction, rhythmic electrical propagation in cardiomyocytes and impulse conduction.

3
2. INTRODUCTION

4
2.1. CARDIOVASCULAR DISEASE

The cardiovascular system is made up of the heart and blood vessels. Cardiovascular disease
(CVD) is an abnormal condition of the heart, blood vessels, brain and kidney. CVD includes
coronary heart disease (CHD), stroke, peripheral vascular disease, congenital heart disease,
endocarditis, and many other conditions. Many cardiovascular diseases (CVDs) are
preventable. CVDs have now become the leading cause of mortality in India. A quarter of all
mortality is attributable to CVD. The Global Burden of Disease study estimate the average
death population due to CVD in India is higher than the global average population. By 2010
mortality rate due to CVD is increased by 59% in India [1].

2.2. CARDIOVASCULAR SYSTEM

The cardiovascular system is the pumping and transport system of the body. This system
consists three important parts: the heart, blood vessel and blood itself. The heart is the
system’s pump and the blood vessels are the delivery routes [2].

2.2.1. The heart

The heart is located in the chest between the lungs. The heart is a muscle about the size of a
fist, and is roughly cone-shaped. The heart is surrounded by the pericardium, a fibrous
covering. It holds the heart in place but allows it to move as it beats. The wall of the heart is
made up of a special type of muscle called cardiac myocytes [3].

2.2.1.1. Chambers of the heart

The heart has the right side and the left side, each have two chambers. The two top chambers
are known as the left and right atria. The left atrium receives blood from the lungs and the
right atrium receives blood from the rest of the body. The bottom two chambers are known as
the left and right ventricles. The right ventricle pumps blood to the lungs while the left
ventricle pumps out blood to the rest of the body. The walls of the ventricles are much thicker
than the atria which allow them to pump blood out to the whole body.

5
2.2.1.2. Blood vessels

Blood Vessels are tubes which carry blood. Blood vessels are of three types, veins, arteries
and capillaries. Veins carry deoxygenated blood from the body back to the heart. Arteries
carry oxygenated blood from the heart to the body. Capillaries connect arteries and veins
together. Capillaries are responsible for transfer gas and nutrients from the bloodstream to the
other tissues of the body. The aorta is the largest artery in our body. The left ventricle pumps
blood into the aorta which then carries it to the rest of the body through smaller arteries.
Pulmonary arteries take the blood to the lungs whereas the pulmonary veins take blood from
the lungs to the left atrium. All the other veins in our body drain into the inferior vena cava
(IVC) or the superior vena cava (SVC) which then take the blood from the rest of the body
into the right atrium.

Figure 1 Anatomy of the Heart

2.2.1.3. Valves

Valves are fibrous flaps of tissue found between the heart chambers and in the blood vessels.
They are acts as gate which prevents blood from flowing in the wrong direction. Valves
between the atria and ventricles are known as the right and left atrioventricular valves,
otherwise known as the tricuspid and mitral valves respectively. Valves between the

6
ventricles and the great arteries are known as the semilunar valves. The aortic valve is found
at the base of the aorta, while the pulmonary valve is found at the base of the pulmonary
trunk.

2.2.2. The Cardiac Cycle

The cardiac cycle is the sequence of events that occurs in one complete beat of the heart. The
pumping phase of the cycle, also known as systole, occurs when heart muscle contracts. The
filling phase, which is known as diastole, occurs when heart muscle relaxes. At the beginning
of the cardiac cycle, both atria and ventricles are in diastole. During this time, all the
chambers of the heart are relaxed and receive blood. The atrioventricular valves are open.
During atrial systole, the left and right atria contract at the same time and push blood into the
left and right ventricles, respectively. The next phase is ventricular systole. During
ventricular systole, the left and right ventricles contract at the same time and pump blood into
the aorta and pulmonary trunk, respectively. In ventricular systole, the atria are relaxed and
receive blood. The atrioventricular valves close immediately after ventricular systole begins
to stop blood going back into the atria. However, the semilunar valves are open during this
phase to allow the blood to flow into the aorta and pulmonary trunk. Following this phase, the
ventricular diastole occurs. The semilunar valves close to stop the blood from flowing back
into the ventricles from the aorta and pulmonary trunk. The atria and ventricles are again in
diastole together and the cycle begins again. The heart beats around 70 to 80 times a minute
at rest. The sound of heart beat is usually described as “lubb-dupp”. The “lubb” sound known
as the first heart sound, is caused by the closure of the atrioventricular valves. The “dupp”
sound is due to the closure of the semilunar valves when the ventricles relax [1].

2.2.3. Functions of the Cardiovascular System

A. Transport of Oxygen, Carbon Dioxide, Nutrient and Waste Product

The most essential function of the cardiovascular system is to supplying oxygen to the body.
Oxygen and nutrients enters the blood stream and are diffuses out of the blood into the cells
of the body's organs and tissues. At the same time, carbon dioxide absorbed into the blood,
transported to the lungs and through the air sacs is then exhaled. This cycle occurs with every
breath. In addition to carbon dioxide, the circulatory system picks up metabolic waste

7
products and toxins and transports them to the liver, kidneys and lungs for eventual
elimination from the body.

B. Disease Protection and Healing

The circulatory system has a number of disease-fighting cells and proteins, and acts as
messengers of the immune system. Immune system cells called white blood cells circulates in
the body in search of invading germs. If an infection occurs, these cells send chemical signals
that travel through the bloodstream, which subsequently transports infection-fighting cells to
[2]
the site of the infection . The circulatory system also carries chemical messengers that
attract cells to heal tissues that have been damaged due to injury or disease.

C. Hormone Delivery
Hormones are chemical messengers produced by endocrine glands. The cardiovascular
system serves as the transportation connection between the endocrine glands and the organs
or tissues they control via hormones. The blood-sugar-lowering hormone insulin produced in
the pancreas affects the uptake and use of blood sugar throughout the body. And thyroid
hormones affect the metabolic rate of virtually every body organ and tissue.

D. Body Temperature Regulation

Body temperature regulation is an important function of the cardiovascular system. If body
temperature begins to rise, blood vessels close to the body surface dilate, increasing in size.
This allows the body to rid itself excess heat through the skin. Conversely, if body
temperature drops, surface blood vessels constrict to conserve body heat. The cardiovascular
system works in concert with the body's sweating mechanism as the primary regulators of
body temperature [5].

2.3. RISK FACTORS FOR CVD

Risk factors are variables that predict who is most likely to develop CVD. Risk factors are of
two types modifiable and non-modifiable. Modifiable risk factors include tobacco use, high
blood pressure, physical inactivity, high blood cholesterol, obesity, heavy alcohol
consumption, and poor nutrition. Non-modifiable risk factors are age and family history [6].

8
2.3.1. Modifiable risk factors

A. High blood pressure (hypertension) is one of the most important risk factors for CVD. If
blood pressure is too high, it can damage blood vessels and kidney among other ill effects.

B. Tobacco smoke contains high levels of carbon monoxide (CO). CO affects the heart by
reducing the amount of oxygen the blood is able to carry. Nicotine causes an increase in heart
rate and blood pressure. Smoking actually triples the risk of dying from heart disease.
Cigarette smoking is a major cause of stroke by increasing clotting factors in the blood,
decreasing HDL cholesterol levels, increasing triglyceride levels, and damaging the lining of
blood vessels.

C. Cholesterol is a fatty substance found in the blood. High cholesterol causes blood vessels
to narrow and increase the risk of developing a blood clot.

D. Diabetes causes blood sugar level to become too high that can damage the blood vessels,
making them more likely to become narrowed.

2.4. HYPERTENSION

In Hypertension (HTN or HT) or high blood pressure (HBP), the blood pressure in the
arteries is persistently elevated. Long-term HBP is a major risk factor for coronary artery
disease, stroke, heart failure, atrial fibrillation, peripheral vascular disease, vision loss,
chronic kidney disease, and dementia. HBP is classified as primary (essential) HBP and
secondary HBP. Primary HBP is the most prevalent which occur due to excess salt in the diet,
excess body weight, smoking, and alcohol use. The secondary HBP occur due to chronic
kidney disease, narrowing of the kidney arteries, an endocrine disorder, or the use of birth
control pills. Blood pressure is expressed by two measurements, the systolic and diastolic
pressures, which are the maximum and minimum pressures, respectively. For most adults,
normal blood pressure at rest is within the range of 100–130 millimetres mercury (mmHg)
systolic and 60–80 mmHg diastolic. High blood pressure is present if the resting blood
[5]
pressure is at or above 130/80 or 140/90 mmHg . Some people with HBP may experience
headaches, as well as light-headedness, vertigo, tinnitus (buzzing in the ears), altered vision

9
or fainting episodes. On physical examination, hypertension may be associated with the
presence of changes in the optic fundus [6].

2.5. HYPERTENSIVE VENTRICULAR REMODELING

HBP is the single most important risk factor for heart failure. Most of the heart failure cases
have hypertension. Because terminally differentiated cardiac myocytes are inefficient at re-
entering the cell cycle, these cells respond to pressure-overload stress by enlarging. This
response, called hypertrophy, leads to ventricular wall thickening and stiffening [7].

2.5.1. Cardiac Hypertrophy

Thickening of the heart muscle refers to the hypertrophy of the ventricular myocardium due
to chronic and increased stress on the heart. The most common causes for ventricular
hypertrophy are: a) intense sports activities, b) ventricular pressure overload, and c)
ventricular volume overload. As the wall thickness of the left ventricle, it becomes stiffer,
leading to reduced elasticity (diastolic dysfunction). The first symptom of a cardiac
hypertrophy is shortness of breath, especially during strenuous physical exertion. Any form
of physical activity becomes very strenuous and even painful for the person affected. At the
same time, the longer the ventricular hypertrophy persists, the more they are at risk of
suffering a heart attack. It is commonly diagnosed by performing electrocardiogram (ECG)
which detects the pattern of abnormal function or increased heart mass [7].

2.5.2. Vascular Hypertrophy

Angiotensin II (Ang II) plays an important role in the development of vascular hypertrophy
and HT. Reactive oxygen species (ROS) such as superoxide anion and H2O2 are involved in
the hypertensive and hypertrophic responses to Ang II. In vascular smooth muscle cells
(SMCs), the NADPH oxidase is one of the major sources of superoxide and H2O2. Ang II
increases intracellular superoxide, which can be readily converted into H2O2 by superoxide
dismutase (SOD). Ang II–mediated increases in intracellular H2O2 are inhibited by
extracellular catalase, the NADPH oxidase inhibitor diphenylene. Ang II increases

10
intracellular H2O2 via mechanism involving activating the NADPH oxidase via the Ang II
type 1 receptor [8].

2.5.3. Cardiac Fibrosis

Cardiac fibrosis is the excess deposition of extracellular matrix proteins in the cardiac muscle
and causes systolic as well as diastolic dysfunction. Cardiac fibrosis involved in the
activation and transformation of cardiac fibroblasts to myofibroblasts, which participate in
extracellular matrix (ECM) production and fibrotic process and several inflammatory
pathways. The involvement of renin-angiotensin-II-aldosterone system, transforming growth
factor-β signalling and actin-linked kinase 5 in the mechanisms of cardiac fibrosis, these
pathways and the involved proteins are useful as therapeutic targets [7].

2.5.4. Endothelial Dysfunction

The inner wall of blood vessels is covered with a single-cell lining membrane called
endothelium which acts as a barrier separating the circulating blood and the vascular smooth
muscle cells. Endothelial cells synthesize and release several important factors which regulate
vascular function. Any imbalance in the production and release of these substances leads to a
variety of vascular diseases, such as hypertension, atherosclerosis and thrombosis.
Endothelial dysfunction is characterized by reduction of the bioavailability of vasodilators,
particularly nitric oxide (NO), and/or an increase in endothelium-derived contracting factors.
In endothelial dysfunction, the production of nitric oxide (NO) is down regulated and the
production of ET is up regulated. Decline in NO may be caused by decreased expression of
endothelial cell NO synthase (eNOS). The oxidatively modified form of LDL; OxLDL is
taken up into the macrophages and smooth muscle cells, and changes these cells into foam
cells. Similarly, OxLDL was also taken up by endothelial cells and binding to the LOX-1
induced production of the super-oxide anion and activated NFκB, a redox-sensitive
transcription factor. OxLDL reduced NO production from the endothelial cells via generation
of reactive oxygen. The reduction in NO production by OxLDL, ET-1 production from
endothelial cells is enhanced by OxLDL; via activation of NFκB. The endothelial cells bound
by activated platelets enhanced the production of superoxide anions and ET-1 [9].

11
2.5.5. Myocardial Infarction

Myocardial Infarction (MI) is the medical term for heart attack. The word “infarction” comes
from the Latin “infarcire” meaning “to plug up or cram”. It refers to the clogging of the
artery. MI is the damage and death of heart muscle from the sudden blockage of coronary
arteries by a blood clot. Coronary arteries are blood vessels that supply the heart muscle with
blood and oxygen. Blockage in coronary artery deprives the heart muscle of blood and
oxygen, causing injury to the heart. If blood flow is not restored to the heart muscle within
20-40 mins, irreversible death of the heart muscle will begin to occur. Muscle continues to
die for six to eight hours at which time the heart attack usually complete. The dead heart
muscle is eventually replaced by scar tissue. Atherosclerosis is the main cause of MI. Plaques
(collection) of cholesterol is deposited in the walls of arteries which cause hardening of the
arterial walls and narrowing of the lumen of the artery. Chest pain is the most common
symptom of MI. when a large amount of heart muscle cells dies, the ability of heart to pump
blood to the rest of the body is diminished, and this can result in HF and ventricular
fibrillation. MI can be diagnosed by performing ECG and blood test to check the level of
cardiac enzyme proteins like creatinine phosphokinase (CPK), troponin, sub-fractions of CPK
that are released into blood by dying heart muscle [10].

Isoproterenol 4-[1-hydroxy-2-(isopropyl amino) ethylbenzene-1, 2-diol hydrochloride (ISO)

is a synthetic catecholamine and β-adrenergic agonist responsible for production of severe
stress in the myocardium and results in MI. ISO is commonly administered in high doses to
induce experimental acute MI in rats. Several mechanisms for the cardio toxic effects of high
levels of ISO include, coronary insufficiency, intracellular Ca2+ overload, changes in
electrolyte contents and oxidative stress. Activation of β1-adrenergic receptors in the heart
induces the myocardial oxidative stress, inflammation and calcium overload which results in
MI. Persistent β-adrenergic receptor activation with ISO is associated with left ventricular
hypertrophy, increased ventricular collagen content and a reduced inotropic response to ISO.
ISO treatment directly increases cardiac expression and activity of angiotensin converting
enzymes (ACE); thus, activation of the circulatory as well as the cardiac angiotensin system
[11]
could be expected under sympathoexcitatory heart failure . ISO produces myocardial
necrosis that leads to cardiac dysfunction, increased lipid peroxidation and increased levels
of myocardial lipids, and altered cardiac enzyme and antioxidant activities. ISO generates

12
highly cytotoxic free radicals through the autoxidation of catecholamines. These free radicals
may attack to polyunsaturated fatty acids (PUFAs) within the membranes, forming peroxyl
radicals. The radicals then attack adjacent fatty acids, causing a chain reaction of lipid
peroxidation which are harmful and may contribute to increased membrane permeability and
[26]
mitochondrial Ca2+ uptake leading to the development of cardiomyopathy . ISO cause
histological, biochemical, electrolyte and membrane change. ISO induced myocardial
infarction enhances adenyl cyclase activity, resulting in increased cAMP formation, which in
turn would lead to the higher lipid accumulation in the myocardium.

2.6. ROLE OF OXIDATIVE STRESS IN CVD

Oxidative stress is an imbalance between the generation and detoxification of reactive oxygen
species (ROS). In patients with HF, oxidative stress occurs in the myocardium and plasma,
and correlates with left ventricular (LV) dysfunction. Reactive oxygen species negatively
affect disposition of myocardial calcium (Ca+2), cause arrhythmia, and contribute to cardiac
remodeling by inducing hypertrophic signalling, apoptosis, and necrosis.

Figure 2 Oxidative Stress in CVD

Enzymatic sources for ROS, such as the nicotinamide adenine dinucleotide phosphate
(NADPH) oxidases (NOXs), uncoupled nitric oxide (NO) synthase, and mitochondria are all
considered relevant sources of ROS in HF, causing vascular and myocardial dysfunction.

13
Mitochondria mainly amplify ROS derived from NOXs. Specific NOX isoforms exist in
endothelial cells (ECs) and smooth muscle cells (SMCs). In the latter isoform, physiological
stretch activates the sarcolemmal and transverse tubule-localized NOX2 (X-ROS signalling),
and these ROS sensitize nearby ryanodine receptors to trigger Ca+2 release from the
sarcoplasmic reticulum. This also involves NO synthases and calmodulin-dependent protein
kinase II, raising the possibility that peroxynitrite (ONOO) and methionine oxidation are
involved. Activation of NOX2 occurs through Gq-coupled angiotensin II (Ang II) receptors.
Suppressor doses of Ang II induce cardiac hypertrophy that is abolished by NOX2 deletion
[12]
.

2.7. CARDIAC ELECTRICAL CONDUCTION SYSTEM:

The cardiac electrical conduction system determines the heart rate and coordinates the
1
beating of heart muscles. This system is very important for the function of the heart. Any
abnormality in this system can causes problem with heart rate like arrhythmia. The electrical
signal of heart is called as electrical impulse. This signal is produced by Sino Atrial (SA)
node present on the right atrium. From the SA node, the electrical signal spreads across the
right and left atria, causing both atria to contract by pushing blood into the right and left
ventricle. This process is called as atrial depolarization and it generates the P wave. The
electrical signal then passes slowly through the Atrioventricular (AV) node and generates PR
interval. Since impulse travels very slowly through AV node it creates a pause, which allows
the atria to contract fully. Then this signal from AV node passes to His bundle, bundle
branches and purkinje fibers, causing ventricle to contract and generates QRS complex. With
extreme stimulation by the SA node, the AV node can transmit impulses maximally at 220
per minute [13].

2.7.1. Electrocardiogram (ECG)

The ECG or EKG represents the electrical and muscular functions of the heart. ECG can
provide the indirect evidence of blood flow to the heart muscle. It shows P wave, QRS
complex and T wave. Normal resting heart rate of human and rats are 60-80 bpm and 250-
300 bpm respectively. Heart Rate Variability (HRV) is the variation in the time interval
between heartbeats. HRV value reflects the interaction between sympathetic and
parasympathetic regulatory activities. HRV measured by spectral analysis.
14
 P Wave- Depolarization of atria in response to the
electrical signal from SA node (0.10 sec)

PR Interval- Delay in impulse conduction through the

AV node for ventricle filling (0.12-0.20 sec)

RR segment- Interval between two successive R waves

QRS Complex- Depolarization of ventricles (0.06-0.10

sec)

ST Segment- Initiation of repolarization of ventricles

T Wave- Repolarization of ventricle (0.10-0.25 sec)

QT interval- Total duration of ventricular depolarization

and repolarization
Figure 3 ECG wave

There are two major spectral components in the power spectrum at low frequency (LF)
(0.6Hz) and high frequency (HF) (1.4Hz). On the basis of these data two frequency bands
were measured i.e. LF (0.04-1.0Hz) and HF (1.0-3.0Hz). The LF/HF ratio is important for the
symphovagal balance. The increase in LF/HF ratio reduces the parasympathetic activity [14].

2.7.2. Membrane Potentials and Ion Movement in Cardiac Contractile Cells

Cardiac cells have different electrical pattern involving the contractile cells include, rapid
depolarization, followed by a plateau phase and then repolarization. The cardiac myocytes
normally do not initiate their own electrical potential, although they are capable of doing so,
but rather wait for an impulse to reach them. Contractile cells has a much more stable resting
phase than conductive cells at approximately -80 mV for cells in the atria and -90 mV for
cells in the ventricles. When stimulated by an action potential, voltage-gated channels rapidly
open, beginning the mechanism of depolarization by rapid influx of positively charged ions
which raises the membrane potential to approximately +30 mV, at which point the sodium
channels close. The rapid depolarization period typically lasts 3–5ms. Depolarization is
followed by the plateau phase, in which membrane potential declines relatively slowly. This
is due in large part to the opening of the slow Ca2+ channels, allowing Ca2+ to enter the cell
while few K+ channels are open, allowing K+ to exit the cell.

15
 Figure 4 Membrane Potentials in Cardiac Contractile Cells

[14]
The relatively long plateau phase lasts approximately 175 ms . Once the membrane
potential reaches approximately zero, the Ca2+ channels close and K+ channels open, allowing
K+ to exit the cell. The repolarization lasts approximately 75 ms. At this point, membrane
potential drops until it reaches resting levels once more and the cycle repeats. The entire
event lasts between 250 and 300 ms. Electrocardiogram (ECG) is a reflection of these cellular
electrical events. In ventricular myocytes (i.e. QRS complex and T wave), activation of the
Na+ current causes rapid depolarization followed by a repolarization of transient outward K+
current [15].

2.8. ELECTRICAL REMODELING

Electrical remodeling can be divided into primary and secondary remodeling. Primary
electrical remodeling occurs in response to an altered sequence of electrical activation. For
example, during right ventricular pacing the normal sequence of electrical activation is
altered because the initiating electrical impulse arises from ventricular myocytes in the right
ventricle and not through the specialized purkinje system. By contrast, secondary electrical
16
remodeling develops as a result of a structural alteration such as heart failure (HF),
hypertrophy, or myocardial infarction [15].

2.9. MALABARICONE C

Malabaricone C, one of the secondary metabolites from the fruit rind of Myristica malabarica
found to be most active in terms of its antioxidant and anti-inflammatory properties.
Previously it has been shown that Mal C acts as an excellent antihypertensive and
cardiovascular remodulatory drug given therapeutically. Here, in this study we have checked
whether given in preventive doses is able to either prevent the onset of HT and cardio
vascular remodulatory activities or able to reduce anti HT and cardiovascular remodulatory
activities [16].

Figure 5 Chemical Structure of Malabaricone C

2.10. 4, 4’-DIHYDROXYSTILBENE

4, 4’-Dihydroxystilbene (DHS), are naturally found in a bark of Yucca Periculosa plant. Here
we used a synthetic analogue of resveratrol, a phytoalexin known for its biological activities.
DHS is effective in preventing or delaying pathological process-like cardiovascular diseases.
DHS is active in protecting against lipid peroxidation and ROS production. Hence, the
present investigation was aimed to assess the preventive effect of DHS on Myocardial
Infarction (MI) [27].

Figure 6 Chemical Structure of 4, 4’-Dihydroxystilbene

17
3. REVIEW OF LITERATURE

18
3.1. MEASUREMENT OF CARDIAC FUNCTIONING

Function of the heart is measured by recording Blood Pressure (BP). Most widely used
methods for recording the BP in rat are, (i) Tail cuff method (Non-Invasive), (ii) Intra-arterial
catheters method (Invasive). Intra-arterial catheter (Invasive) gives the most precise values of
basal BP, and surgery is required to use them. Sometimes in the BP fluctuation occurs due to
the anesthesia which interferes with the normal BP [17].

3.1.1. Non-Invasive BP: Tail cuff method

The systolic BP (SBP) of rats was measured by the device consists of sphygmomanometer,
pulse inducer, triway and non-collapsible rubber tubes and inflatable cuff (latex balloon). The
cuff was placed in a plastic case such a way that it remains in contact with the inner surface
of the plastic case around the central hole, so that balloon encircles the tail of the animal kept
in restrainer. One end of triway was connected to the cuff and other two were connected to
inflating-deflating pump and sphygmomanometer. It measures SBP by determining the cuff
pressure at which blood pulse to the tail was returned. This is recorded to the physiography
through suitable coupler [17,18,19].

3.1.2. Invasive BP: Intra-Arterial catheter method

Invasive BP recording is considered as gold standard. It provides a direct indication of the

[18]
effect of the investigational products on the circulatory system . The adult Wistar rat was
anesthetized. Pressure transducer was calibrated. Anesthetised animal was placed in position
and shaved midline neck. A 1-2 cm midline neck incision was made. One of the red coloured
carotid arteries was exposed and carefully separated from the vagus nerve (white in colour)
and the adjacent connective tissue. Carotid artery cleaned carefully. Once the artery has been
isolated, one bull dog clamp was placed closer to the head for complete ligation of the vessel.
Second bull dog clamp was placed closer to the heart. Warm PBS was frequently added to
keep vessel hydrated. Under a dissecting microscope, finally small incision was made at
proximal end of the artery using small incision scissor. The tip of the catheter that had been
placed in warm PBS was inserted into the carotid artery in the direction of the heart and was
secured in place by tying the knot using thread. The catheter was slowly pushed towards the

19
atrium and left ventricle chamber and systolic as well as diastolic BP were recorded. With the
help of Lab Chart software (AD instruments, Australia) pressure readings were taken which
was connected to the Power lab machine and to the catheter. Ventricle pressure was recorded,
identified with the absence of notch in the trace [20].

3.2. RAT MODELS FOR HYPERTENSION

In cardiovascular research, animal models are used to study the disease. The aim of these
studies is to provide clear concepts for selected investigations in humans. An ideal animal
model for any cardiovascular disease in humans should follow the given criteria: They
should, (i) Mimic the human disease, (ii) Allow studies in chronic, stable disease, (iii)
Produce symptoms which are predictable and controllable, (iv) Satisfy economical, technical
and animal welfare considerations, and (v) Allow measurement of relevant cardiac,
biochemical and hemodynamic parameters. The use of rats as animal models is economical
and many techniques have been developed to measure the relevant functional parameters [21].

Different Rat Models used for Hypertension are,

1. Spontaneously Hypertensive Rats (SHRs)
2. Stroke-prone SHR (SHR-SP)
3. Mineralocorticoid (DOCA–Salt)
4. NO synthase Inhibition (L-NAME administration)
5. Transgenic [TGR (mREN2)]
6. Diabetic Hypertensive Rats (STZ-SHR, Zucker)

Mineralocorticoid DOCA–Salt Hypertension rat (DSHR) model

Most widely used rat model for HT induction is mineralocorticoid (DSHR) model. The model
is economical. Retention of salt can rapidly achieved in uninephrectomised rats by
mineralocorticoid administration, by weekly subcutaneous injections of deoxycorticosterone
acetate (DOCA), and salt loading as 1% NaCl in the drinking water. Uninephrectomised rats
given deoxycorticosterone or NaCl alone do not show any major changes in blood pressure,
and it is only the combination of DOCA and NaCl that produces blood pressure and increases
cardiac and renal weight with cardiovascular remodelling characteristic of human volume-

20
overload induced hypertension, especially hypertrophy, fibrosis, conduction abnormalities
and endothelial dysfunction. HT develops more quickly and becomes more severe in male
than female DSHR. The DSHR model shows a markedly depressed renin–angiotensin system
and thus has been used in HT research as an angiotensin-independent model in the
characterization of new antihypertensive compounds. The DSHR model relies on impairment
of kidney capacity and salt loading to rapidly induce HT and hypertrophy. These model
progress quickly to severe hypertension and hypertrophy, and therefore are not suited for
long-term studies in chronic, stable disease [21,22].

3.3. CURRENT CLINICAL ANTIHYPERTENSIVE DRUGS

Antihypertensive drugs comprise several classes of compound with the therapeutic intention
of preventing, controlling, or treating hypertension. These drugs are classified on the basis of
mechanism of the action. Currently used different antihypertensive drugs are, diuretics,
angiotensin converting enzyme inhibitors (ACEIS), angiotensin receptor blockers (ARBS), β-
blockers and calcium channel blocker. All these drugs have their different special points and
side effects [23,24,25].

3.3.1. DIURETICS (Chlorothiazide)

Special Points: They are effective in lowering blood pressure in the great majority of patients,
especially those over 60 and those with diabetes. Diuretics increase the effectiveness of all
other categories of antihypertensive. Diuretics are the original antihypertensive and hence
their efficacy and adverse effects are very well understood. Thiazide diuretics reduce calcium
excretion and have beneficial effects in preventing bone loss and fractures.

Adverse Effects: Diuretics increase the excretion of potassium and can lead to hypokalaemia
which causes irregular heartbeats, and muscular weakness. They cause a small increase in
blood glucose, but it is unclear whether this predisposes to diabetes in the long term. Over
treatment with diuretics can lead to low blood pressure, weakness, dizziness and possibly
fainting on standing and a feeling of tiredness and lethargy.

21
3.3.2. ANGIOTENSIN CONVERTING ENZYME INHIBITORS (ACEIs) (Benazepril)

Special Points: ACEIs are widely used to treat HT because they are effective, have relatively
few side effects and in reduce the complications of HT such as heart attacks and strokes.
They are specially used for patients with diabetes mellitus and patients with chronic kidney
disease (CKD). ACEIs block the action of the renin angiotensin system (RAS). Renin is
released from the kidney during low blood pressure, low salt intake or diuretic usage and
generates angiotensin II, which constricts blood vessels, retains salt and water by the kidneys
and raises blood pressure. Therefore, these drugs target important hypertensive mechanisms.
They interact very well with diuretics. ACEIs act on the kidney to retain some potassium,
thereby reducing the adverse effect of low blood potassium that can occur during diuretic
therapy.

Adverse Effects: ACEIs develop an irritant cough in 10 to 20 percent of subjects. Very rarely,
they can cause a dangerous swelling of the tongue, lips and throat, which, in extreme
circumstances, can seriously interfere with breathing and requires emergent treatment.
Patients with CKD often have a temporary worsening of kidney function. Biochemical
changes with ACEIs in those with impaired kidneys can raise the serum potassium
concentration to levels that are dangerous.

3.3.3. ANGIOTENSIN RECEPTOR BLOCKERS (ARBS) (Olmesartan)

Special Points: ARBs also block the renin angiotensin system (RAS), similar to ACEIs, but
have a different mechanism of action by blocking the actions of angiotensin II in the tissues
rather than the generation of angiotensin II. Moreover, they do not cause an irritant cough or
the rare danger of swelling of the lips, tongue and throat, that can occur with ACEIs.

Adverse Effects: These drugs do not cause irritant cough, but have a similar spectrum of
adverse actions to ACEIs.

3.3.4. BETA BLOCKERS (Atenolol)

Special Points: They act on the sympathetic nervous system. Blockade of the sympathetic

22
nervous system reduces blood pressure by relaxing blood vessels, and decreasing the rate and
force of contraction of the heart. The actions of these agents are enhanced in patients taking
diuretic drugs and therefore are a good second- or third-line selection in those patients who
are not controlled with a diuretic and an ACEI or ARB. Beta blockers are affective in lower
blood pressure and reducing its complications.
Adverse Effects: Their popularity has diminished because of a large range of annoying
adverse effects. The most frequent adverse effects of beta blockers are: slow heart rate,
depression and irritability, impaired sleep, decreased exercise capacity, wheezing and
precipitation of asthma, sexual dysfunction, and an increase in serum potassium.

3.3.5. CALCIUM CHANNEL BLOCKERS (CCBS) (Amlodipine)

Special Points: These are very effective in lowering blood pressure in elderly, obese and
diabetic patients. They act directly on the blood vessels to cause relaxation. They are used
sometimes as first line therapy but more often with diuretics or ACEIs or ARBs as second- or
third-line therapy.

Adverse Effects: They are usually well tolerated, and most patients have few side effects.
However, dihydropyridine CCBs do cause swelling of the ankles (edema). This usually does
not indicate a major problem such as heart failure but an increased passage of fluid from the
plasma into the tissues of the skin. Non-dihydropyridine CCBs cause cardiac slowing. This
typically reduces the heart rate by about 10%.

All these drugs acts on single target, have various side effects, do not give desired end point
and may be expensive. There is a need to look for the new drug to counter/prevent CVD
which acts on multi target, with no or less side effects, show the desired end point result and
may be from edible natural sources. Therefore the aim of this study is,

AIM: To analyse the effect of Mal C and DHS on cardiovascular pathogenesis in a

preventive rat model.

23
4. MATERIALS AND METHODS

24
4.1. Experimental Rats

Adult male Wistar rats (200– 300g) were used in this study. Rats were bred in the animal
housing facility of the Bhabha Atomic Research Centre (BARC), Trombay, India, were
procured after obtaining clearance from the BARC Animal Ethics Committee (BAEC). All
the experiments were conducted with strict adherence to the ethical guidelines laid down by
the European Convention for the Protection of Vertebrate Rats used for Experimental and
Other Scientific Purposes. In addition, the ethical guidelines laid down by the Committee for
the Purpose of Control and Supervision of Experiments on Rats, constituted by the Animal
Welfare Division, Government of India, on the use of rats in scientific research were
followed. The experiments were permitted by BAEC. Rats were kept in a room at room
temperature of 23 ± 2°C. Rats were individually housed in plastic cages with corn cob
bedding under a natural 12 h day-night cycle. Rats were provided with a pellet diet and water.
The body weight, food and water intake of rats were measured daily throughout the entire
experimental period.

4.2. Drugs and Chemicals

Drugs used are, Malabaricone C (Mal C) compound was isolated from the methanol extract
of dried fruit rinds of M. malabarica and Synthetic 4,4’-Dihydroxystilbene (DHS) compound.
Other chemicals used were, Deoxycorticosterone Acetate (DOCA), Heparin, Urethane,
Dimethyl Sulfoxide (DMSO), Isoproterenol Hydrochloride [Sigma Chemicals (St. Luois,
MO)], Dimethylformamide (DMF) [S d fine- Chem limited], NaCl (Thomas Baker, Mumbai
India), Edible oil, Phosphate Buffer Saline (PBS), Ketamine (Themis Medicare Ltd.,
Mumbai, India), Xylazine (Indian Immunological Ltd, Hyderabad, India), Diazepam (Svizera
Health Care, Mumbai, India), Sodium Thiopentone (Neon Lab. Ltd., Mumbai, India).

4.3. Preparation of Drugs and Chemicals

Mal C (10 mg/mL) was first dissolved in DMSO and then reconstituted using edible oil (as
DMSO is toxic in high concentration). DOCA (24 mg/400 µL) solution was prepared in DMF
by slight heating to dissolve. 1% NaCl solution was prepared using tap water. 1 X PBS was
prepared by dissolving 8.5 g NaCl, 1.41 g Na2HPO4, and 0.26 g NaH2PO4 in 1000 mL

25
distilled water. Formalin (10%) was prepared by mixing formaldehyde (37%) in PBS. DHS
(10 mg/mL) was prepared by dissolving DHS into 0.5 mL DMSO and reconstituted using
edible oil. Isoproterenol Hydrochloride (ISO) (150 mg/mL) was prepared by dissolving ISO
into 1 X PBS.

4.4. Experimental Design

4.4.1. Study the preventive effect of Malabaricone C on Hypertension

After a one-week of acclimation period, male Wistar rats weighed 200-300 g were randomly
divided into 4 groups each containing 6 rats and were treated as followed: All rats underwent
uninephrectomy surgery.

a) UNX Control: After uninephrectomy on 4th day rats received the vehicle, edible oil
by oral gavaging and normal tap water for drinking.

b) Mal C Control: Rats were administered Mal C (10 mg/kg) daily by orally gavaging
and received normal tap water.

c) DSHR: Rats were received subcutaneous injection with DOCA (24 mg/kg) at an
interval of 4 days and 1% NaCl water.

d) Mal C+ DOCA: Rats of this group were uninephrectomised on 4th day and pre-
treated with Mal C (10 mg/kg) daily by gavage needle, from 4 days prior to UNX
surgery, followed by subcutaneous injection with DOCA (24 mg/kg) at an interval of
4 days throughout the experimental period and received 1% NaCl water.

Effect of Mal C on DSHR model was measured by observing followed parameters: ECG and
NIBP of all the grouped rats were recorded on 4th, 15th and 32nd day using three lead systems
and tail cuff method respectively. On the 32nd day, the rats from all the groups were
anesthetized and invasive blood pressure (IBP) were recorded using intra-arterial catheter
method. The rats were then euthanized using 100 mg/kg Sodium Thiopentone (IP), blood and
organs were collected. The organs were suspended in PBS. Weight of the kidney, adrenal

26
gland, spleen, right ventricle and left ventricle were taken. Organs were then preserved in
10% formalin for further studies.

4.4.2. Study the preventive effect of 4,4’-Dihydroxystilbene on Myocardial Infarction

After a one-week acclimation period, male Wistar rats weighed 350-400 g were randomly
divided into 3 groups each containing 3 rats and were treated as followed:

a) Normal Control: Rats were administered the vehicle, edible oil by orally gavage.

b) ISO Control: Rats were administered ISO (150 mg/kg) subcutaneous injection on 8th
and 9th day.

c) DHS + ISO: Rats were administered DHS (10 mg/kg) by oral gavage for 10 days,
followed by ISO (150 mg/kg) subcutaneous injection on 8th and 9th day.

Effect of DHS on rats with ISO induced MI was measured by observing followed parameters:
ECG and NIBP of all the grouped rats were recorded on 10th day using three lead systems and
tail cuff method respectively. Rats were then euthanized using 100 mg/kg Sodium
Thiopentone (IP), blood and organs were collected. The organs were preserved in 10%
formalin for further studies.

4.5. Anesthesia

Different anesthetic drugs were used in different combinations for different purpose in this
study. For UNX surgery, rats were anesthetized Intraperitoneally (IP) using combination of
drugs, ketamine 70 mg/kg (as a dissociative, analgesic and hypnotic agent), xylazine 5 mg/kg
(as a muscle relaxing and pain killing agent) and diazepam 2 mg/kg (as an early and deep
sleep-inducing agent). Rats were kept inside the cage and cage was covered with cloth for
making rats anesthetic early. Rats were kept under 60W bulb to keep them warm. Rats to be
euthanized after surgery were injected with combination of drugs, ketamine 25 mg/kg,
xylazine 5 mg/kg, and urethane 1.3 mg/kg (produces long-lasting steady level of surgical
anesthesia).

27
4.6. Uninephrectomy (UNX)

Animal was first anesthetized with combination of drugs and then placed on a heated mat.
The left flank was shaved and swabbed with surgical disinfectant. A small incision was made
in the left flank using scissor to gain access to the left kidney. The kidney was ligated with
non-absorbable thread (placed in 70% ethanol) and was then cut with surgical scissor. The
incision site was sutured with absorbable thread and metal suture clips were applied to close
the wound. Metal suture clips were removed spontaneously after 7-14 days indicated healing.

4.7. Induction of Experimental CVD

4.7.1. Induction of Experimental Hypertension

Rats from the group of Mal C+ DOCA and DSHR were anesthetized IP on 4th day using 70
mg/kg ketamine (as a dissociative, analgesic and hypnotic agent), 5 mg/kg xylazine (as a
muscle relaxing and pain killing agent) and 2 mg/kg diazepam (as an early and deep sleep-
inducing agent). Electrocardiogram (ECG) and Non-Invasive BP (NIBP) of the rats were
recorded using three lead systems and tail cuff method respectively. UNX was performed on
anesthetized rats. Dissected kidneys of the rats were dried and weighed. Uninephrectomised
rats were received subcutaneous injection with DOCA (24 mg/kg) at an interval of 4 days and
1% NaCl water.

4.7.2. Induction of Experimental Myocardial Infarction

Rats from the group of ISO and DHS+ ISO were subcutaneously injected with ISO (150
mg/kg) at 24 h intervals for 2 days to induce experimental MI.

4.8. Measurement of BP

4.8.1. Non-Invasive BP (NIBP): Tail cuff Method

The systolic BP (SBP) of rats was measured using a device consists of inflatable-deflatable
cuff (latex balloon) connected to the NIBP machine. This further connected to the LAB chart

28
software and AD instruments (Sydney, Australia). The rats were kept in restrainer after
acclimatizing them, the tail was passed through the hole of the cuff and the pulse transducer
was tied around the vein. As the system was switched on the pulse was recorded on, the cuff
was inflated by the pump and the pressure in the cuff was raised until the pulse was
eliminated. The pulse was recorded as an electrical signal in the Power Lab data acquisition
unit on computer. The pressure at which the pulse signal was returned recorded as the systolic
BP of the animal. In such a way 8-10 readings were taken for each rat.

4.8.2. Invasive BP (IBP): Intra-Arterial Catheter Method

IBP was measured by using Millar micro tip pressure sensitive transducer SPR 320. Catheter
was calibrated and tip of the catheter was placed into warm PBS to maintain the temperature
of 37°C. Anesthetised animal was placed in position and shaved midline neck. A 1-2 cm
midline neck incision was made. One of the red coloured carotid arteries was exposed and
carefully separated from the vagus nerve (white in colour) and the adjacent connective tissue.
Carotid artery cleaned carefully. Once the artery has been isolated, one bull dog clamp was
placed closer to the head for complete ligation of the vessel. Second bull dog clamp was
placed closer to the heart. Warm PBS was frequently added to keep vessel hydrated. Under a
dissecting microscope, finally small incision was made at proximal end of the artery using
small incision scissor. The tip of the catheter that had been placed in warm PBS was inserted
into the carotid artery in the direction of the heart and was secured in place by tying the knot
using thread. The catheter was slowly pushed towards the atrium and left ventricle chamber
and systolic as well as diastolic BP were recorded. With the help of Lab Chart software (AD
instruments, Sydney, Australia) pressure readings were taken connected to the Power lab
machine and to the catheter. Ventricle pressure was recorded, identified with the absence of
notch in the trace.

4.9. ECG

The ECG was recorded using 3 lead systems. This system consists of 3 electrodes viz.
positive (red), negative (black), and reference (green) electrodes and was subcutaneously
inserted in the left arm, right arm, and right leg, respectively. Electrodes were connected to
the Animal Bio-amp FE136 (AD instruments, Australia) and data was obtained by a Power

29
lab data acquisition system PL3508/P (AD Instruments, Sydney, Australia). Rat ECG module
was selected in Lab Chart Pro software 8.1 (AD Instruments, Sydney, Australia) to analyse
the data.

4.10. Weight of the Organs

The rats were euthanized using IP injection of Sodium Thiopentone (100 mg/kg). The organs,
kidney, liver, spleen, adrenal gland, right ventricle and left ventricle were dissected out and
suspended in PBS. Organs were dried using tissue paper and weighed.

4.11. Collection of Blood Plasma

The rats were injected with heparin (100 IU) in femoral artery. Blood was collected in tubes,
centrifuged at 4000 rpm for 15 mins. The supernatant (yellow coloured plasma) was collected
in another tube and stored at -20⁰C for further use.

4.12. Statistical Analysis

4.12.1. Statistical analysis of Mal C+ DOCA experiment

Values are means ± SE. The results were analyzed by one-way ANOVA. *P ≤ 0.05
compared to UNX. # P ≤ 0.05 compared to DSHR.

4.12.2. Statistical analysis of DHS+ ISO experiment

Values are means ± SE. The results were analyzed by one-way ANOVA. *P ≤ 0.05 compared
to normal control. # P ≤ 0.05 compared to ISO control.

30
5. RESULTS AND DISCUSSION

31
 A) STUDY OF THE EFFECT OF Mal C ON CARDIOVASCULAR REMODELING

1. Body Weight and Water Consumption of DSHR during the experiment

A) Body Weight B) Water Consumption

UNX UNX
420 DOCA-treated Mal C+ DOCA
Mal C + DOCA-treated 300
DSHR
400 Mal C Mal C

250

Water consumption (ml)

380
Body Weight (gms)

360 200

340 150

320
100

300
50

280
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Days Days

Figure 6 Effect of Mal C on Body Weight and Water Consumption

Body Weight and Water Consumption of all the grouped rats were taken daily. DSHR
showed increase in water intake indicate the DOCA model was developed successfully. Mal C
reduced the water intakes, without affecting the body weight by acting as diuretics.

2. Blood Pressure: Non-Invasive BP (NIBP) and Invasive BP (IBP)

NIBP was recorded on 4th, 15th and 32nd day by using tail cuff method. IBP was recorded on
32nd day using intra-arterial catheter method. All the results are an average of 8-10 readings.

A) NIBP B) IBP
UNX
Mal C
240 * DSHR 220 *
*
Mal C + D 160
220 200

140
200
Systolic Blood Pressure

180
(Systolic blood pressure

(mmHg) (Invasive)
Diastolic blodd pressure

180 #
(mmHg) (Invasive)

* 120 #
160
#
160
mmHg)

100 140
140
#
80 120
120

100 100
* 60

80 80
40 UNX Mal C DSHR Mal C + D
0 day 15 d 32 day UNX Mal C DSHR Mal C + D
Animal Groups
Days of experiment Animal Groups

Figure 7 Effect of Mal C on NIBP (SBP) and IBP (SBP & DBP)
32
DSHR showed increase in NIBP and IBP due to volume overload in body caused by UNX
surgery & 1% NaCl. Increase in BP indicates the walls of the arteries are constantly under too
much force and poor contraction and relaxation of the heart. Mal C restricted the rate of
increase and attenuates hypertension, correcting contraction and relaxation of the heart.

3. ECG

ECG was recorded on the 4th, 15th and 32nd day by 3 lead systems. All the results are an
average of 20 mins readings.

A) Heart Rate B) P Duration

UNX
UNX
450 Mal C
0.045 Mal C
DSHR
400 * * DSHR
Mal C+ D 0.040 Mal C+ D
350
# 0.035

300
(Heart Rate BPM)

0.030 #
P Duration (s)

250 0.025

200 0.020

150 0.015

100 0.010

50 0.005

0 0.000
Animal Groups Animal Groups

C) QRS Complex D) QTc Interval

UNX UNX
0.06 Mal C Mal C
DSHR DSHR
0.25
Mal C+ D * Mal C + D
0.05 *
0.20
0.04
# #
0.15
QTc
QRS (s)

0.03

0.10
0.02

0.05
0.01

0.00
0.00
Animal Groups
Animal Groups

Figure 8 Effect of Mal C on Heart Rate, P Duration, QRS Complex and QTc Interval

DSHR showed maximum increase in the Heart Rate, P Duration, QRS Complex and QTc
Interval. Increase in the heart rate indicates problem in relaxation of the heart. Prolonged P
wave & QRS complex indicates atrial and intra-ventricular conduction defect respectively.
Prolonged QTc interval has been associated with the arrhythmias. Mal C improved rhythmic
33
electrical propagation in cardiomyocytes with better arterial and ventricular conduction by
decreasing the Heart Rate, P Duration, QRS Complex and QTc Interval.

4. Heart Rate Variability (HRV)

The HRV was determined by spectral analysis. ECG was recorded as a base reading.

UNX
0.25 Mal C
* DOCA
Mal C + D
0.20

0.15
LF/HF

0.10

0.05

0.00
Animal Groups

Figure 9 Effect of Mal C on LF/HF ratio

A Low Frequency (LF) denotes sympathetic tone and High Frequency (HF) denotes
parasympathetic tone. Higher LF/HF ratio represents increased sympathetic tones. Compared
to the normal rats, subcutaneous injection of DOCA increased the LF/HF ratio. Oral
administration of Mal C decreased the LF/HF ratio by shifting the sympathetic tone toward
parasympathetic tone like beta blocker does.

5. Intra-Arterial Measurement

Readings were measured by using Millar micro tip pressure sensitive transducer SPR 320
inserted into carotid artery. All results are an average of 5 mins readings.
UNX
200 Mal C
* DSHR
180 Mal C + D
160
Mean Pressure (mmHg)

#
140

120

100

80

60

40

20

0
Animal Groups

Figure 10 Effect of Mal C on Mean Pressure

34
DSHR showed increase in mean pressure which indicates more pressure in the arteries. This
can lead to blood clots or damage to the heart muscle, which has to work a lot harder. Oral
administration of Mal C reduced mean pressure by relaxing the arterial pressure.

UNX
Mal C
180 * DOCA
Mal C + D
160

Dichrotic Notch (mmHg)

140
#
120

100

80

60

40

20

0
Animal Groups

Figure 11 Effect of Mal C on Dichrotic Notch

Dichrotic notch represents closure of the aortic or pulmonic valve at the onset of ventricular
diastole. In DSHR model dichrotic notch increased due to elevated hemodynamic load
characterized by low cardiac output and low stroke volume. This indicates severe functional
impairment of the myocardium. Compared to DSHR model, Mal C reduced the dichrotic
notch by reducing hemodynamic load.
Ejection Durations

0.10

0.09
Ejection Duration (s)

0.08

0.07

0.06

0.05

UNX mal C DSHR Mal C + D

Animal Groups

Figure 22 Effect of Mal C on Ejection Duration

Ejection duration represents the period of blood flow across the aortic valve. Increased in
arterial stiffness prolongs ejection duration. Mal C reduced the ejection duration by reducing
arterial stiffness in Mal C fed DSHR model.
35
6. Intra-Ventricular Measurements

Readings were measured by using Millar micro tip pressure sensitive transducer SPR 320
inserted into carotid artery. All results are an average of 5 mins readings.

A) Maximum LV pressure B) Minimum LV Pressure

Min LV Pressure (mmHg)
140
220
*
*

Min LV Pressure (mmHg) (mm Hg)

120
200
Max LV Pressure (mmHg)

100
180
#
80

160 #
60

140
40

120 20

100 0

UNX Mal C DSHR Mal C + D UNX Mal C DSHR MAl C + D

Animal Groups Animal Groups

Figure 13 Effect of Mal C on Maximum and Minimum LV Pressure

DSHR showed increase in maximum and minimum LV pressure. Increase in maximum and
minimum LV pressure indicates abnormal pressure volume function of ventricle. Mal C
reduced the maximum and minimum LV pressure by modulating the LV pressure function.

A) Systolic Duration B) Diastolic Duration

Systolic Duration Diastolic Duration

0.24 0.50
*
0.22
0.45
0.20
0.40
0.18
Systolic Duration (s)

Diastolic Duration (s)

0.35
0.16 #
0.14 0.30

0.12 0.25
0.10
0.20
0.08
0.15
0.06
0.10
0.04

0.02 0.05
UNX Mal C DSHR Mal C + D UNX Mal C DSHR Mal C + D

Animal groups Animal Group

Figure 14 Effect of Mal C on Systolic and Diastolic Duration

When compared to DSHR, Mal C fed rats showed decrease in both Diastolic and Systolic
duration.
36
 A) Maximum Differential Pressure B) Minimum Differential Pressure

Max dP/dt Min dP/dt

0
*
10000
-1000

8000 # -2000
Max dP/dt (mmHg/s)

Min dP/dt (mmHg/s)

-3000
6000
#
-4000

4000
-5000
*
2000 -6000

-7000
0
UNX Mal C DSHR Mal C + D UNX Mal C DSHR Mal C + D
Animal groups Animal Groups

Figure 15 Effect of Mal C on Maximum and Minimum Differential Pressure

Improper ventricular contraction causes increased in minimum differential pressure and

decreased in maximum differential pressure. Mal C increased maximum differential pressure
and decreased minimum differential pressure by improving ventricular contraction.
.

200 Contractility Index

180

160
Contractility Index (1/s)

140
#
120

100

80

60
*
40

20
UNX Mal C DSHR Mal C+ D
Animal Groups

Figure 16 Effect of Mal C on Contractility Index

Contractility index represents the contractile properties of the myocardium that are
independent of the loading condition. DOCA decreased contractility index. Reduced
contractility index indicates improper ventricular contraction and heart function. Mal C fed
rats showed improved ventricular contraction and thus better heart function.

37
7. Organ Weights

After drying with tissue paper weight of the organs, kidney, left and right ventricle and
adrenal were taken.

UNX
1.0 Mal C
DSHR
*
Mal C +D
0.8
#
Organ weight g/Kg bw

0.6

0.4
*
#
0.2

* #
* #
0.0
RV kidney Left Ventricle Adrenal
Organs

Figure 17 Effect of Mal C on Organ Hypertrophy

Thickening of the heart muscle refers to the hypertrophy of the ventricular myocardium due
to chronic and increased stress on the heart, pressure overload stress and oxidative stress in
muscles of organs. In DSHR organ hypertrophy was seen. Mal C reduced hypertrophy by
acting as an antioxidant and antihypertensive in Mal C fed DSHR.

38
 B) STUDY THE PREVENTIVE EFFECT OF 4,4’-DIHYDROXYSTILBENE ON
MYOCARDIAL INFARCTION

1. ECG

ECG was recorded on the 10th day by 3 lead systems. All the results are an average of 10
mins readings.

Normal control
40 Isopreterenol
DHS
35

#
30

25
*
ms

20

15

10

0
PR (ms)

Figure 18 Effect of DHS on PR Interval

The PR Interval is the distance between the onsets of the P-wave to the onset of the QRS
complex. The PR-Interval is assessed in order to determine whether impulse conduction from
atria to the ventricles is normal. Short PR interval represents accelerated conduction in the
AV node and associated with atrial fibrillation, which is responsible for fetal and non-fetal
cardiovascular outcomes in patient with MI. Patients with short PR interval are more at the
risk of HF. In MI, the short PR interval reflects the impaired left ventricular function. DHS
reduced the PR interval by improving impulse conduction.

39
 A) Heart Rate B) RR Interval

Normal control Normal control

450 Isopreterenol 200 Isopreterenol
* DHS DHS
400 180

160
350
140
Beats Per Minute

300 #
120 #
250

m sec
100
*
200
80
150
60
100
40

50 20

0 0
Heart Rate (BPM) RR (ms)

Figure 19 Effect of DHS on Heart Rate and RR Interval

Heart rate is inversely related to RR interval. RR interval is the interval between two successive R
waves. Increase in the heart rate indicates problem in relaxation of the heart. In MI, necrotic
myocardium does not generate electrical potentials and hence there is a loss of R-wave. DHS
fed rat shows improved rhythmic electrical propagation in cardiomyocytes with the increased
RR interval and decreased heart rate.

Normal control
Isopreterenol
DHS
500

#
400
*
m sec

300

200

100

0
QRS (ms)

Figure 30 Effect of DHS on QRS Complex

Reduced QRS complex indicates rapid intra-ventricular conduction could be due to

hypertrophy (confirmation is needed) i.e. the ventricular depolarization is more than normal.
ISO control rats showed reduced QRS complex i.e. rapid current flow. DHS corrected
ventricular conduction by increasing the QRS complex.

40
 Normal control
Isopreterenol
* DHS
100

#
80

m sec
60

40

20

0
QT (ms)

Figure 21 Effect of DHS on QT Interval

QT Interval represents the total time for depolarization and repolarization of ventricle. It
measured from the beginning of the QRS complex to the end of the T-wave. Prolonged QT
duration indicates ventricular arrhythmias. ISO control rats showed increased QT interval
while DHS fed rats showed decreased QT interval and hence improved ventricular
contraction and relaxation.
Normal control
Isopreterenol
0.40 * DHS

0.35

0.30
#
0.25
m volt

0.20

0.15

0.10

0.05

0.00
ST (mv)

Figure 22 Effect of DHS on ST Segment

The ST Segment represents the interval between depolarization and repolarization of the
ventricles and plateau phase of the action potential. ST segments altered in a wide range of
conditions hence studied carefully. In the MI, ST segment represent a current of injury from
damaged cells that are partially depolarized to the healthy myocardium. The ST segment
deviated (elevation) in the acute myocardial infarction may be due to formation of thrombus
in coronary vessel and modification in an action potential which is life threatening. DHS fed
rat shows reduced ST segment and hence corrects function of ventricle.

41
6. CONCLUSIONS

42
Methanol extraction of Malabaricone C from fruit rind of plant Myristica malabarica was
used in this study. The DOCA- salt hypertensive rat (DSHR) model rapidly induces
cardiovascular remodeling similar to chronic hypertension in humans. Hence natural and
synthetic compounds with antioxidants or anti-inflammatory responses were tested by using
DSHR model.

Increased water intake of the rats after DOCA and salt administration was an indirect
indication of the model being successfully prepared. The results showed that the pre-
treatment of Mal C in DSHR reduced the diastolic and systolic pressure. Anesthetic agents
have an ability to decrease BP. However anesthesia was used to measure the SBP of all
groups of rats to nullify the effect of anesthesia. The possible interaction between the
anesthetic agent and the Mal C cannot be totally excluded.

Mal C reduced the Heart Rate, P Duration, QRS Complex, and QTc Interval and hence extent
of hypertension, and corrects the electric disturbances, and improved cardiac rhythmicity
with better arterial and ventricular conduction. Mal C treatment in DSHR showed reduction
in LF/HF ratio which indicates reduction of sympathetic tones and stimulation of
parasympathetic tones thus shifting the sympathovagal balance towards parasympathetic. Mal
C treatment also showed improved ventricular contractility, diastolic filling, cardiac output,
and mean pressure. In DSHR, hypertrophy was observed in the kidney, adrenal glands, left
ventricle and right ventricle indicated by increased wet weights of the organs relative to their
body weights. Reduction in organs hypertrophy and water consumption was observed in Mal
C treated DSHR indicating better kidney; heart functioning with reduced hemodynamic load.

DSHR serves as a well-accepted standardized model to evaluate several cardiac dysfunctions

and to study the efficacy of various natural and synthetic cardio protective agents. The results
indicated that Mal C has cardio protective activities like antioxidant and anti-inflammatory
which prevent the onset and the extents of cardiovascular damage in DSHR model. It acts on
multi targets in cardiovascular system to achieve desired end results in addition to being non-
toxic and economical. Thus Mal Cs shows great potential to be used as a potent cardio tonic
for the prevention of CVD.

Intraperitoneal injection of Isoproterenol (150mg/kg) for two consecutive days at an interval

of 24 hours was successfully induced acute myocardial infarction in the rat models as
43
indicated by the characteristic ST elevation seen in ECG trace. Acute MI caused septal
infarction with necrosis involving both bundle branches. Unlike DOCA-salt hypertensive
rats, the blood pressure of rat models with ISO is not affected. Thus hypertension is not a
contributing factor to heart attack in these models. Oral administration of 10 mg/kg DHS
decreased the Heart Rate, QT Interval and ST Segment in the ISO-induced myocardial
infarction rat models and hence corrects the ventricular function. DHS also corrects intra-
ventricular conduction, impulse conduction and rhythmic electrical propagation in
cardiomyocytes by improving QRS Complex PR Interval and RR Interval. Thus DHS
exhibits cardio protective properties. However, it could not restore the heart function to the
level of normal rats.

Isoproterenol- induced myocardial necrosis serves as a well-accepted standardized model to

evaluate several cardiac dysfunctions and to study the efficacy of various natural and
synthetic cardio protective agents. Free radical scavenging and stabilization of reactive
oxygen species is the chief mechanism by which DHS act as cardio protective agent.

Still several problems remain inherent with the natural products. Requirement of a high dose,
characterization of chemical constituents and the difficulty to extrapolate the findings to
clinical research are the prime bottlenecks that prevent natural products from being brought
into the market. Nevertheless, much remains to be done and the studies mentioned above
need to be supplemented with molecular, biochemical, histological and clinical studies. This
natural product hold great potential as a first-line therapy for cardiovascular diseases
provided that the drawbacks are overcome.

44
7. REFERENCES

45
1) Anatomy and Physiology II, Module 3: The Cardiovascular System: The Heart,
https://courses.lumenlearning.com/suny-ap2/chapter/cardiac-muscle-and-electrical-
activity/.
2) Beers et al., aging and the cardiovascular system, the Merck Manual of Geriatrics.
Merck and co. inc.2006. https://www.myvmc.com/anatomy/cardiovascular-system-
heart/.
3) Journal of the American college of cardiology vol. 70, no. 2, 2017ª 2017 by the
American college of cardiology foundation, ISSN 0735-1097.
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Mediated by Vascular Smooth Muscle Cell–Derived H2O2, Hypertension, 19 Sep
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Academy. Series B, Physical and Biological Sciences, 2006 Mar; 82 (1); 17-24.
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isoproterenol-induced myocardial infarction in experimental models, Therapeutic
Advances in Cardiovascular Disease, May 9,2104, May 9,
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14) Kuwahara M et al., Power spectral analysis of heart rate variability as a new method
for assessing autonomic activity i9n the rat, J Electrocardiol.1994 Oct; 27(4):333-7.

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15) Michael J. Cutler et al., Cardiac electrical remodeling in health and diseases, Trends
in Pharmacological Sciences, 2011 Mar; 32 (3):174-180.
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occurring phenolic, Malabaricone C in DOCA-salt rats, Free Radical Research, 50:1,
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in conscious rats: feasibility for heart rate variability analysis, Biomedical and
Medical Sciences, Anais da Academia Brasileira de ciencias, Vol.82 no. 2 Rio de
Janerio June 2010.
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novel technique, Indian Journal of Pharmacology, 2014 May-Jun; 46(3): 351-352.
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Frontiers in Medicine, 2017; 4:231.
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47
FIGURES:

1) Figure 1 Anatomy of the Heart: http://en.m.wikipedia.org/wiki/heart diagram en.svg.

2) Figure 2 Oxidative Stress in CVD: Tomoh masaki et al., endothelin and endothelial
dysfunction, Proceedings of the Japan Academy. Series B, Physical and Biological
Sciences, 2006 Mar; 82 (1); 17-24.

3) Figure 3 The ECG wave: S. R. Dikondwar et al., cardiac protection in diabetes using
SVM and Cooperative Wireless Network, researchgate, 27 January 2017.

4) Figure 4 Membrane Potentials in Cardiac Contractile Cells: Eric Wong et al.,

physiology of cardiac conduction and contractility, physiol Rev. 2005 Oct; 85(4):1205-53.

5) Figure 5 Chemical Structure of Malabaricone C: U.S. National Library of

Medicine. https://nlm.nih.gov/chemidplus/rn/63335-25-1.
6) Figure 6 Chemical Structure of Dihydroxy Stilbene:
https://www.sielc.com/44-dihydroxystilbene.

48