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Studies on the Beneficial Effects of Ethanolic Extract and
Total Alkaloids from whole Plant of Boerhaavia diffusa on
Isoproterenol Induced Myocardial Infarction in Wistar Rats.
Thesis submitted to
SRI KRISHNADEVARAYA UNIVERSITY
ANANTAPURAMU
In partial fulfillment for the award of the degree of
DOCTOR OF PHILOSOPHY
IN
BIOCHEMISTRY
by
S. SHAMEELA, M.Sc., B.Ed.,
Under the research supervision of
Prof. K. LAKSHMI DEVI
Department of Biochemistry
SRI KRISHNADEVARAYA UNIVERSITY Anantapuramu-515003 Andhra Pradesh, INDIA
JANUARY, 2016
SRI KRISHNADEVARAYA UNIVERSITY
ANANTAPURAMU-515003, A.P., INDIA
Prof. K. Lakshmi Devi Phone: +91 9885264125
Department of Biochemistry [email protected]
CERTIFICATE This is certify that the thesis entitled “Studies on the Beneficial Effects of
Ethanolic Extract and Total Alkaloids from whole Plant of Boerhaavia diffusa on
Isoproterenol Induced Myocardial Infarction in Wistar Rats” submitted to Sri
Krishnadevaraya University, Anantapuramu is a bonafide record of the research work
carried out by S. Shameela under my guidance and supervision for the award of the
degree of Doctor of Philosophy. This work has not been submitted in part or full for
any degree or diploma of any other University.
Place: Anantapuramu
Date: (Prof. K. Lakshmi Devi)
DECLARATION
I hereby declare that this thesis entitled “Studies on the Beneficial Effects of
Ethanolic Extract and Total Alkaloids from whole Plant of Boerhaavia diffusa on
Isoproterenol Induced Myocardial Infarction in Wistar Rats” comprises original
work done by me under the supervision of Prof. K. LAKSHMI DEVI, Department of
Biochemistry, Sri Krishnadevaraya University, Ananthapuramu, and that the thesis has
not been submitted in part or full for any degree or diploma of any other University.
Date: (S. Shameela)
Place: Anantapuramu
ACKNOWLEDGMENTS
I am greatly beholden words to express my deep sense of gratitude to my research
supervisor Prof. K. Lakshmidevi, Head, Department of Biochemistry, Sri Krishnadevaraya
University, Anatapur, for her excellent guidance, parental care, valuable suggestions and
encouragement throughout my research work.
I am grateful to Prof. D. Saralakumari, Prof. N.Ch. Varadacharyulu, Prof. C.
Suresh Kumar, Dr. P. Suresh Kumar and Dr. M. Narendra of the Dept. of Biochemistry
for their valuable suggestions and providing the necessary facilities in the laboratory for
completion of the work.
A very special thanks to Dr. Appa Rao of Sri Venkateswara University Tirupati,
Dr. P.Moulali (Retd, Lecturer in Botany, Govt College for men, Anantapur), Dr K.
Sesikala (Retd. Principal, Govt. Degree College, Mydukur) and Dr. G. Dhanunjaya (Head,
Dept of Biochemistry, Govt College for men, Anantapur) for their help in preparation of
plant extracts.
I would like to express my sincere thanks to Dr. Mohammad Aktar, Department of
Statistics, for statistical evaluation of the data.
It is my pleasure to thank my seniors Dr. M. Abdul Kareem, Dr. G. Saayi
Krushna, and my colleagues Dr. S. Althaf Hussain, V and E. Maruthi Prasad, Mrs.
Sivaranjani and Mrs. Umamaheswari for their help and cooperation throughout the study.
I extend my personal and deep appreciation Dr. P. Mallaiah & Family, N.
Sreenivasulu
I sincerely thank to Management of JVRRM PG College, Anatapurum, for
providing necessary infrastructure to conduct my research work. Also my special thanks to
Prasad (Late), (Lab Asst., JVRRM PG College, Anatapur), and my students who helped
me during the study.
Its pleasure to express my gratitude whole heartedly to my roommates Preethi,
Harini, Brahmini and Indumathi, for warm, supporting and genuine friendship enough for
helping me to carry out something that was not possible without them. My sincere thanks
to you them being reachable constantly, taking out time always from your very hectic
schedule, whenever entailed.
I wish to express my unboundful gratitude to my beloved parents Smt. S. Sufiya
and Mr. S. Mastanvali (Rtd, Asst. Director, Ground Water Dept, Kurnool) for their
dedicated effort shown on all my academics, without their invaluable moral support I
could not have reached to this stage.
My Uncle’s family members are the motive force behind me to complete Ph.D
work. Especially it is because of their wishes and blessings, that I could face and overcome
challenges. Smt. Dr S. Shamshad (Lecturer in zoology, KVR Degree college for Women,
Kurnool), Mr. S.M. Subhani (SSE, S C Rly , Hyd) and their Children. Their significance in
my life and in this thesis cannot be measured by any softwares. I am lucky enough that I
have with me what is important in life.
I am indebted to my Aunt, S. Amulu and her Daughter S. Naziya and my beloved
Sisters, Brother-in-laws, and their children for their nature and moral support during the
course or study.
I extend my personal and deep appreciation to staff of AP Model School, Gospadu,
Kurnool, Dr. D. Ishrath Begum (Principal), P.N. Mallikarjunappa (PGT Economics), S.
Thaharunnisa (TGT Hindi,) P. Sukanya (Computer Operator) and every member who
prayed for me in completion of the thesis.
Without the encouragement and participation of many people this work would
never have accomplished. My personal and very deep appreciation goes to each of the
extending hands and helping hearts. With my sincere apology, if I was not being able to
mention them all individually.
And last but not the least, my unlimited thanks to number of lab animals sacrificed
for finding the solutions to the aimed questions, in this research work.
S.Shameela
CONTENTS
S. No Topic P. No
Abbreviations i
List of figures iii
List of tables vii
1 Introduction 1
2 Aims and Objectives 74
3 Materials and Methods 76
4 Result and Discussion 108
5 Summary and Conclusion 187
6 References 191
7 Annexure 231
i
ABBREVIATIONS
% percentage
˚C Degree centigrade
ACE Angiotensin converting enzyme
ADP Adenosine diphosphate
AHA American heart association
ALP Alkaline phosphatase
ALT Alanine transaminase
AMI Acute myocardial infarction
AMP Adenosine mono phosphate
AR Analytica grade
AST Aspartate transaminase
ATP Adenosine triphosphate
BDEWP Ethanolic Extract of whole plant of Boerhaavia diffusa
BDTALK Total alkaloids of Boerhaavia diffusa
BHF British Heart Foundation
C- AMP Cyclic adenosine mono phoshate
CAT Catalase
CE Cholesterol esters
CHD Congental heart failure
CHF Congestive heart failure
CK Creatinine kinase
CM Chylomicron
CVD Cardio Vascular Diseases
DMSO Dimethyl sulfoxide
DPPH Diphenyl picrine hydroxyl
FFA Free fatty acids
GCMS Gas chromatography mass spectroscopy
GGT ɣ glutamyl transferase
GPX Glutathione peroxidase
GRX Glutathione reductase
GSH Glutathione
GST Glutathione S- Transferase
H2O2 Hydrogen peroxide
ii
HDL High density lipoprotein
HMGCR Hydroxy methyl glutamyl co A reductase
ISPH Isoproterenol
kg Kilogram
kgbw Kilogram body weight
L Cat Lecitithine cholesterol acyl transferase
LDH Lactate dehydrogenase
LDL Low density lipoprotein
LDLR Low density lipoprotein receptor
LPL Lipoprotein lipase
LPO Lipid peroxidation
LRP LDLR related protein
MDA Monaldehyde
MI Miocardial Infarction
NADP Nicotinamide adenosine diphosphate
NEFA Non esterified fatty acids
NHLBI National heart and lung institute
NO Nitric oxide
NOS Nitric oxide synthase
PL Phospholipid
PO Protein oxidation
PUFA Poly unsaturated fatty acids
RCT Reverse cholesterol transporter
RNS Reactive nitrogen species
ROS Reactive oxygen species
RT Retension time
SES Social Economic Status
SOD Super oxide dismutasae
TC Total cholesterol
TG Triglyceride
VLDL Very low density lipoprotein
WHO World Health Organisation
iii
LIST OF FIGURES
S.NO Title of Figures P.NO
1.1 Epidemiology of Diseases 2
1.2 Global burden from CVD during the period 1990 – 2020 2
1.3 Anatomy of Heart 6
1.4 Conduction System of the Heart and Cardiogram 7
1.5 Cardiac excitation–contraction–energy coupling 10
1.6 Pathways involved in cardiac energy metabolism 10
1.7 Types of cardiac disease 12
1.8 Prevalence of Cardiovascular Diseases in American Men and Women,
Ages 20 and Older
18
1.9 Overview of relationship between cardiovascular diseases and
cardiovascular risk factors.
20
1.10 Schematic overview of pathways involved in lipoprotein metabolism. 30
1.11 Schematic illustration of reverse cholesterol transport. 31
1.12 Pathogenesis of atherogenesis 35
1.13 Reduction of molecular oxygen to superoxide, and of peroxide to
hydroxyl radical
42
1.14 Protein oxidation pathways 44
1.15 Superoxide dismutase, Catalase, and the GSH peroxidase /GSSG
reductase system
50
1.16 Boerhaavia diffusa 66
1.17 Therapeutic properties of Boerhaavia diffusa 68
1.18 Main phytoconstituents of B. diffusa 70
1.19 Structure of Isoproterenol 71
1.20 Mechanism of induction of myocardial injury by ISPH 73
4.1 % Inhibition of Lipid Peroxidation 114
4.2 Hydroxyl Radical Inhibition Activity 114
4.3 Inhibition of Nitric Oxide Radical Formation 115
4.4 Scavanging Capacity Against DPPH 115
4.5 Superoxide Radical Scavenging Activity 116
iv
4.6 Reducing Ability of BDEEWP 116
4.7(A) BDTALK GCMS Parent Spectrum 119
4.7(B) BDTALK GCMS Parent Spectrum (from R. Time 16.30 to 18.20 min) 119
4.8 BDTALK GCMS signal of Methyl Valinate, m/z 182 120
4.9 BDTALK GCMS signal of 2,4, Di-tert butyl phenol, m/z 206 120
4.10 BDTALK GCMS signal of Methyl Ferulate, m/z 208 121
4.11 BDTALK GCMS signal of Syringic Acid, m/z 212 121
4.12 BDTALK GCMS signal of Loliolide, m/z 212 122
4.13 BDTALK GCMS signal of 7,9-di-tert-butyl-1-Oxaspiro (4.5) deca-6,9-
diene-2,8-dione m/z 276
122
4.14 Effect of BDEEWP on plasma lipid profile in control and experimental
rats
132
4.15 Effect of BDEEWP on plasma FFA in control and experimental rats 133
4.16 Effect of BDEEWP on plasma C/P Ratio in control and experimental rats 133
4.17 Effect of BDEEWP on myocardial lipid profile in control experimental
rats
135
4.18 Effect of BATALK on plasma lipid profile in control and experimental
rats
137
4.19 Effect of BATALK on plasma FFA in control and experimental rats 137
4.20 Effect of BATALK on plasma C/P RATIO in control and experimental
rats
138
4.21 Effect of BDTALK on myocardial lipid profile in control and
experimental rats
138
4.22 Effect of BDEEWP on Plasma lipoproteins in control and experimental
rats
140
4.23 Effect of BDEEWP on Plasma Atherogenic Index in control and
experimental rats
141
4.24 Effect of BDEEWP on cardiac lipoproteins in control and experimental
rats
141
4.25 Effect of BDTALK on Plasma lipoproteins in control and experimental
rats
143
4.26 Effect of BDTALK on Plasma Atherogenic Index in control and
experimental rats
143
v
4.27 Effect of BDTALK on cardiac lipoproteins in control and experimental
rats
144
4.28 Effect of BDEEWP treatment on tissue lipid peroxidation in control and
Experimentation rats
148
4.29 Effect of BDEEWP treatment on tissue Protein oxidation in control and
Experimentation rats
148
4.30 Effect of BDTALK treatment on tissue lipid peroxidation in control and
Experimentation rats
150
4.31 Effect of BDTALK treatment on tissue Protein oxidation in control and
Experimentation rats
150
4.32 Effect of BDEEWP treatment on Nitric oxide in cardiac tissue of control
and Experimentation rats
152
4.33 Effect of BDTALK treatment on Nitric Oxide in cardiac tissue of control
and Experimentation rats
152
4.34 Effect of in vivo treatment of BDEEWP against H2O2 induced lymphocyte
DNA damage
156
4.35 Protective ability of BDEEWP against ISPH induced lymphocyte DNA
damage
156
4.36 Effect of BDEEWP on Cardiac Tissue Antioxidant Enzyme Levels in
Control and Experimental Rats
158
4.37 Effect of BDTALK on Cardiac Tissue Antioxidant Enzyme Levels in
Control and Experimental Rats
160
4.38 Effect of BDEEWP on Cardiac GSH Levels in control and Experimental
Rats
162
4.39 Effect of BDTALK on Cardiac GSH Levels in control and Experimental
Rats
162
4.40 Effect of BDEEWP on Cardiac Vitamin - C levels in control and
Experimental Rats
164
4.41 Effect of BDTALK on Cardiac Vitamin-C Levels in control and
Experimental Rats
164
4.42 Effect of BDEEWP on serum Sodium Levels in control and Experimental
Rats
166
4.43 Effect of BDEEWP on serum Potassium Levels in control and
Experimental Rats
166
vi
4.44 Effect of BDEEWP on serum Calcium Levels in control and
Experimental Rats
167
4.45 Effect of BDEEWP on serum Iron Levels in control and Experimental Rats 167
4.46 Effect of BDTALK on serum Sodium Levels in control and Experimental
Rats
169
4.47 Effect of BDTALK on serum Potassium Levels in control and
Experimental Rats
169
4.48 Effect of BDTALK on serum Calcium Levels in control and
Experimental Rats
170
4.49 Effect of BDTALK on serum Iron Levels in control and Experimental
Rats
170
4.50 Effect of BDEEWP on myocardial membrane bound ATPases in control
and experimental rats
174
4.51 Effect of BDTALK on myocardial membrane bound ATPases in control
and experimental rats
174
4.52 Effect of BDEEWP on activity of LCAT Enzyme in control and
experimental rats
177
4.53 Effect of BDTALK on activity of LCAT Enzyme in control and
experimental rats
177
4.54 Effect of BDEEWP on activity of Lipo Protein Lipase Enzyme in
control and experimental rats
179
4.55 Effect of BDTALK on activity of Lipo Protein Lipase Enzyme in
control and experimental rats
179
4.56 Effect of BDEEWP on activity of HMGCR Enzyme in control and
experimental rats
181
4.57 Effect of BDTALK on activity of HMGCR Enzyme in control and
experimental rats
181
4.58 Hitopathological examination of the heart of control, BDEEWP treated
and ISPH treated experimental animals
183
4.59 Hitopathological examination of the heart of control, BDTALK treated
and ISPH treated experimental animals
184
4.60 Best lead molecules autodock interaction of HMG CoA reductase with
Punarnavoside
185
vii
LIST OF TABLES
S.No Title of the Table PAGE.
No
1.1 Verbal autopsy of deaths at all ages in 45 villages, Andhra Pradesh 3
1.2 Physical properties and composition of human plasma lipoproteins 28
1.3 Anti-atherogenic activities of HDL particles 37
1.4 Experimental evidence on the beneficial effects of Potassium 56
1.5 Mechanism, examples and limitations of therapeutics for CVD 61
4.1 Qualitative Phytochemical Profile of Ethanolic Extract of B. diffusa 110
4.2 Chemical Characterization of Phytochemical Constituents of
BDTALK 118
4.3 Effect of BDEEWP on body weight and heart weight of control and
experimental rats 125
4.4 Effect of BDTALK on body weight and heart weight of control and
experimental rats 125
4.5 Effect of BDEEWP on activity of marker enzymes in serum of
control and experimental groups 128
4.6 Effect of BDEEWP on marker enzymes in heart tissue of control
and experimental groups 128
4.7 Effect of BDTALK on activity of marker enzymes in serum of
control and Experimental groups 130
4.8 Effect of BDTALK on Activity of marker enzymes in heart tissue of
control and Experimental groups 130
4.9 Effect of in vivo treatment of BDEEWP against H2O2 induced
lymphocyte DNA damage 154
4.10 Protective ability of BDEEWP against ISPH induced lymphocyte
DNA damage 154
4.11 Autodock 4.0 energy values of lead molecules (Punarnavoside) with
rat HMG CoA reductase. 186
1
INTRODUCTION
1.1 Epidemiology of Cardiovascular Diseases
In the modern world, prevalence of non-communicable diseases is increasing
and cardiovascular diseases have a major share in these non-communicable diseases
(WHO Bulletin, 2005). Cardiovascular (heart and circulatory) disease causes more
than a quarter of all deaths in the UK, or around 155,000 deaths each year - an
average of 425 people each day or one every three minutes (BHF Headline Statistics,
2015). Cardiovascular disease (CVD) or diseases of the circulatory system can be
described as all diseases relating to the heart and blood vessels (Delbridge and
Bernard (editors) 1988). It is the world‟s largest killer disease, claiming 17.1 million
lives a year. About 80% of the global burden of CVD death occurs in low and middle-
income countries (WHO, 2010). CVD remain the principal cause of death in both
developed and developing countries accounting for roughly 20 % of all deaths
worldwide per year (Rajadurai and prince, 2007). Cardiovascular diseases are
increasing throughout the developing world and cause almost 16.7 million deaths each
year, 80% of which occur in low and middle-income countries (Sliwa et al, 2010). The
World Health Organization (WHO) estimates there will be about 20
million CVD deaths in 2015, accounting for 30 percent of all deaths worldwide
(Figure1.1). By 2030, researchers project that non-communicable diseases will
account for more than three-quarters of deaths worldwide; CVD alone will be
responsible for more deaths in low income countries than infectious diseases
(including HIV/AIDS, tuberculosis, and malaria), maternal and prenatal conditions,
and nutritional disorders combined (Beaglehole and Bonita, 2008).
The CVD burden afflicts both men and women, with cardiovascular deaths
accounting for 34% of all deaths in women and 28% in men in 1998 (WHO, 1999).
By 2040, women in the study countries (Russian, Brazil, India, China and South
America) will represent a higher proportion of CVD deaths than men (Integrated
Management of Cardiovascular Risk, Report of WHO Meeting, 2002). Studies have
documented those immigrants from Indian subcontinent (South Asians) living in
western countries has a higher burden in CVD than other ethnicities (Abhinav and
Salim, 2006). According to WHO (2007) the impact of CVD worldwide is as
2
follows: an estimated 17 million people die every year from CVD and there is one
death every two seconds, one heart attack every five seconds and one stroke every six
seconds. Thus, CVD is today the largest single contributor to global mortality and will
continue to dominate mortality trends in the future (WHO, 2009).
Figure 1.1: Epidemiology of Diseases
Figure 1.2: Global burden from CVD during the period 1990 – 2020.
3
India is already the „Death Capital‟ of the world and is projected to have the
highest number of individuals suffering from atherosclerotic CVD by the year 2020
(Yusuf et al., 2001). According to World Health Report 2002, CVD will be the largest
cause of death and disability by 2020 in India. In 2020 AD, 2.6 million Indians are
predicted to die due to coronary heart disease, which constitutes 54.1 % of all CVD
deaths. Nearly half of these deaths are likely to occur in young and middle aged
individuals (30-69 years). The Global Burden of Disease (GBD) study estimates that
52% of CVD deaths occur below the age of 70 years in India compared with about
22% in the west. Between 2000 and 2030, about 35% of all CVD deaths in India will
occur among those between 35 and 64, compared with only 12%in the United states
and 22% in China (Leeder et al., 2004). Figure1.2 depicts Global burden from CVD
versus Disability Adjusted Life Years (DALY) for the period 1990 – 2020.
In a survey conducted in 45 rural villages of Andhra Pradesh in India, 32% of
all deaths were due to CVD (Joshi et al., 2006) (Table 1.1). In one study, Muslim men
have been shown to have the highest CHD prevalence rates while Christian men have
been shown to have the lowest CHD prevalence rates (Gopianth, 1995). However,
another study demonstrated that the highest CHD prevalence rates in Hindu men
(Gupta, 2002) where as two other studies reported the highest rates in Gujarati men
(Chandha and Gopianth, 1992).
Table1.1: Verbal autopsy of deaths at all ages in 45 villages, Andhra Pradesh
Cause
Male
Female
Cardiovascular 34% 30%
External 15% 12%
Infectious 12% 12%
Cancers 5% 10%
Respiratory 6% 5%
Joshi et al. Int J Epidemiol 2006; 35:1522
4
1.2 Heart Anatomy and Physiology
1.2.1 Heart Anatomy
The human heart is a hollow muscular organ, nearly the size of a closed fist that
weighs approximately 300 grams in the adult male and 250 grams in the adult female.
Weight and size varies depending on age, sex, height and nutritional status. The heart is
a powerful muscular organ that pumps blood through blood vessels to body‟s cells with
the nutrients needed for survival. It beats non-stop every minute of every hour, resting
only for a fraction of a second between each contraction. Unlike skeletal muscle, which
contracts in response to nerve stimulation, specialized pacemaker cells at the entrance
of the right atrium termed as sinoatrial node display the phenomenon of automaticity
and are myogenic, meaning that they are self-excitable without a requisite electrical
impulse coming from the central nervous system. It is because of this automaticity that
an individual's heart does not stop when a neuromuscular blocker is administered, such
as during general anesthesia.
The wall and Coverings of the Heart
The heart wall has three layers. The outermost layer of the heart, the
epicardium, also known as the visceral pericardium, consists of epithelial cells that form
a serous membrane that covers the entire heart. The innermost layer of the heart is
known as the endocardium. It is a serous membrane that lines the inner surface of the
heart, its valves, and the chordae tenediae. The endocardium is continuous with the
intima (eg, the inner lining of arteries). The middle layer of the heart is the muscular
layer known as the myocardium. It is responsible for the major pumping action of the
ventricles. The myocardial cells have an intrinsic ability to contract in the absence of
stimuli (i.e, automaticity) and in a rhythmic manner (i.e, rhythmicity), and to transmit
nerve impulses (i.e, conductivity) (Berne and Levy, 2000; Mader, 2004).
Heart chambers
The heart has four hollow chambers: two superior atria and two inferior
ventricles (Figure1.3). Internally, the atria are separated by the interatrial septum, and
the ventricles are separated by the interventricular septum. Therefore, the heart has a
left and a right side. The atria have thin walls, and they send blood into the adjacent
ventricles. The ventricles are thicker, and they pump blood into blood vessels that travel
to parts to different of the body. The left ventricle has a thicker wall than the right
5
ventricle; the right ventricle pumps blood to the lungs. The left ventricle pumps blood to
all the other parts of the body. The right atrium has a thin muscle wall. It receives
deoxygenated (i.e, venous) blood from the head and upper extremities via the superior
vena cava, from the trunk and lower extremities via the inferior vena cava, and from the
coronary sinus, which drains blood from the myocardium. The coronary sinus empties
into the right atrium just above the tricuspid valve.
Most blood flow into the right atrium occurs during inspiration when right
atrium pressure drops below that in the inferior and superior vena cava, causing the
blood to flow from an area of higher to lower pressure. In the right ventricle, the cusps
of the tricuspid valve are connected to fibrous cords, called the chordae tendineae. The
chordae tendineae in turn are connected to the papillary muscles, which are conical
extensions of the myocardium. The right ventricle receives blood from the right atrium
through the tricuspid valve and ejects it through the semilunar valve into the pulmonary
artery where it travels to the lungs. This particular semilunar valve prevents blood from
flowing back into the right ventricle. The left atrium receives oxygenated (i.e, arterial)
blood from the lungs through the right and left inferior and superior pulmonary veins.
The wall of the left atrium is slightly thicker than that of the right atrium and breathing
does not affect its filling. Blood passes from the left atrium into the left ventricle
through the bicuspid (mitral) valve. The left ventricle has a thick muscular wall. It
receives blood from the left atrium through the mitral valve and ejects it through the
aortic valve to the systemic circulation via the aorta. Pressure in the left ventricle is
high. The ventricular septum, a thick muscular area that becomes membranous as it
nears the atrioventricular (AV) valves, separates the right and left ventricles. It houses
electrical conduction tissue and provides stability for the ventricles during contraction.
1.2.2 Internal conduction (Stimulation) system
Unlike skeletal muscle, which contracts in response to nerve stimulation,
specialized pacemaker cells at the entrance of the right atrium termed the sinoatrial
node display the phenomenon of automaticity and are myogenic, meaning that they are
self-excitable without a requisite electrical impulse coming from the central nervous
system. The rest of the myocardium conducts these action potentials by way of
electrical synapses called gap junctions. The conduction system coordinates the
contraction of the atria and ventricles so that the heart is an effective pump (Berne and
Levy, 2000).
6
Figure 1.3: Anatomy of Heart
7
The heartbeat is controlled by nodal tissue, which has both muscular and
nervous characteristics. This unique type of cardiac muscle is located in two regions
of the heart: The SA (sinoatrial) node is located in the upper posterior wall of the right
atrium; the AV (atrioventricular) node is located in the base of the right atrium very
near the interatrial septum (Figure 1.4).
The SA node which is called pacemaker initiates the heartbeat and
automatically sends out an excitation impulse every 0.85 second. From the SA node,
impulses spread out over the atria, causing them to contract. When the impulses reach
the AV node, there is a slight delay that allows the atria to finish their contraction
before the ventricles begin their contraction. The signal for the ventricles to contract
travels from the AV node through the two branches of the atrioventricular bundle (AV
bundle) before reaching the numerous and smaller Purkinje fibers. The AV bundle, its
branches, and the Purkinje fibers consist of specialized cardiac muscle fibers that
efficiently cause the ventricles to contract. An area other than the SA node can
become the pacemaker when it develops a rate of contraction that is faster than the SA
node. This site, called an ectopic pacemaker, may cause an extra beat, if it operates
only occasionally, or it can even pace the heart for a while. Caffeine and nicotine are
two substances that can stimulate an ectopic pacemaker.
1. Stimulus originates in the SA node and travels across the walls of the atria,
causing them to contract.
2. Stimulus arrives at the AV node and travels along the AV bundle.
3. Stimulus descends to the apex of the heart through the bundle branches.
4. After stimulus reaches the Purkinje fibers, the ventricles contract.
Figure 1.4: Conduction System of the Heart and Cardiogram.
8
A graph that records the electrical activity of the myocardium during a cardiac
cycle is called an “Electrocardiogram”, or ECG. An ECG is obtained by placing on
the patient‟s skin several electrodes that are wired to a voltmeter (an instrument for
measuring voltage). As the heart‟s chambers contract and then relax, the change in
polarity is measured in millivolts. An ECG consists of a set of waves: the P wave, a
QRS complex, and a T wave (Figure1.4). The P wave represents depolarization of the
atria as an impulse started by the SA node travels throughout the atria. The P wave
signals that the atria are going to be in systole and that the atrial myocardium is about
to contract. The QRS complex represents depolarization of the ventricles following
excitation of the Purkinje fibers. It signals that the ventricles are going to be in systole
and that the ventricular myocardium is about to contract. The QRS complex shows
greater voltage changes than the P wave because the ventricles have more muscle
mass than the atria. The T wave represents repolarization of the ventricles. It signals
that the ventricles are going to be in diastole and that the ventricular myocardium is
about to relax. Atrial diastole does not show up on an ECG as an independent event
because the voltage changes are masked by the QRS complex.
1.2.3 Pumping Action of the Heart
The pumping action starts with the simultaneous contraction of the two atria.
This contraction serves to give an added push to get the blood into the ventricles at the
end of the slow-filling portion of the pumping cycle called "diastole." Shortly after
that, the ventricles contract, marking the beginning of "systole." The aortic and
pulmonary valves open and blood is forcibly ejected from the ventricles, while the
mitral and tricuspid valves close to prevent backflow. At the same time, the atria start
to fill with blood again. After a while, the ventricles relax, the aortic and pulmonary
valves close, and the mitral and tricuspid valves open and the ventricles start to fill
with blood again, marking the end of systole and the beginning of diastole. It should
be noted that even though equal volumes are ejected from the right and the left heart,
the left ventricle generates a much higher pressure than does the right ventricle (Berne
and Levy, 2000).
1.2.4 Myocardium and mechanism of contraction
Although the myocardium is made up of individual cells with discrete
membrane boundaries, the cardiac myocytes that constitute the ventricles contract
9
almost in unison, as do those of atria. The myocardium functions as a syncytium with
an all-or-none response to excitation. Cell to cell conduction occurs through gap
junctions that connect the cytosol of adjacent cells. During the upstroke of the action
potential, voltage-gated Ca2+
channels open to admit extracellular Ca2+
into the cell.
Calcium channels underlie the electrical activity of cells and form the means by which
electrical signals are converted to responses within the cell (Figure1.5). Calcium
channels play an integral role in excitation in the heart and shaping the cardiac action
potential. In addition, calcium influx through calcium channels is responsible for
initiating contraction. Abnormalities in calcium homeostasis underlie cardiac
arrhythmia, contractile dysfunction and cardiac remodeling (Hool, 2007).
The influx Ca2+
triggers the release of Ca2+
from the sarcoplasmic reticulum.
The elevated intracellular Ca2+
produces contraction of the myofilaments. Relaxation
of the myocardial fibers is accomplished by restoration of the resting cytosolic Ca2+
level by pumping Ca2+
back into the sarcoplasmic reticulum and exchanging it for
extracellular Na+ across the sarcolemma. Contractility is increased mainly by
interventions that increase intracellular Ca2+
levels and decreased by interventions that
decrease intracellular Ca2+
levels (Berne and Levy, 2000).
1.2.5 Energetic metabolism of myocardium
The heart has continuously high energy demands related to the maintenance of
specialized cellular processes, including ion transport, sarcomeric function, and
intracellular Ca2+
homeostasis. Myocardial workload (energy demand) and energy
substrate availability (supply) are in continual flux, yet the heart has a limited capacity
for substrate storage. Thus, ATP-generating pathways must respond proportionately
to dynamic fluctuations in physiological demands and energy delivery (Berne and
Levy, 2000; Huss and Kelly, 2005).
Oxidation of fatty acids and glucose in mitochondria accounts for the vast
majority of ATP generation in the healthy adult heart (Stanley and Chandler, 2002;
Taegtmeyer, 1994). Fatty acids are the preferred substrate in the adult myocardium,
supplying about 70% of total ATP (Bing et al., 1954; Shipp et al., 1961; Wisnecki et
al., 1987). Fatty acids derived from circulating triglyceride-rich lipoproteins and
albumin bound nonesterified fatty acids are oxidized in the mitochondrial matrix by
the process of fatty acid oxidation, whereas the pyruvate-dehydrogenase (PDH)
10
complex, localized within the inner mitochondrial membrane (Figure1.6), oxidizes
pyruvate derived from glucose and lactate. Acetyl-CoA, derived from both pathways,
enters the tricarboxylic acid (TCA) cycle. Reduced flavin adenine dinucleotide
(FADH2) and NADH are generated via substrate flux through the oxidation spiral and
the TCA cycle, respectively. The reducing equivalents enter the electron transport
chain, producing an electrochemical gradient across the mitochondrial membrane that
drives ATP synthesis in the presence of molecular oxygen (oxidative
phosphorylation).
Figure 1.5: Cardiac excitation–contraction–energy coupling
Figure 1.6: Pathways involved in cardiac energy metabolism
11
1.3. Pathophysiology
According to the National Heart, Lung, and Blood institute (2006), CVD or
Heart disease is a general name for a wide variety of diseases, disorders and
conditions that affect the heart and sometimes the blood vessels as well.
Heart disease is the number one killer of women and men in the United States,
and more than a million Americans have myocardial infarctions. Some specific forms
of cardiovascular disease are illustrated in the Figureure 1.7 they are
a) Coronary Artery Disease: This is a common form of cardiovascular
disease. CAD, coronary artery disease is the leading cause of heart attacks. It
generally means that blood flow through the coronary arteries has become
obstructed, reducing blood flow to the heart muscle.
b) Heart Attack: A heart attack is an injury to the heart muscle caused by a loss
of blood supply. The medical term for heart attack is "Myocardial Infarction"
often-abbreviated MI. The term MI focuses on the heart muscle, which is called
the myocardium, and the changes that occur in it due to the sudden deprivation of
circulating blood. The word "infarction" comes from the Latin "infarcire" meaning
"to plug up or cram." It refers to the clogging of the artery, which is frequently
initiated by cholesterol piling up on the inner wall of the blood vessels that
distribute blood to the heart muscle.
The severity of MI is dependent on three factors: the level of the
occlusion in the coronary artery, the length of time of the occlusion, and the
presence or absence of collateral circulation. Generally, the more proximal the
coronary occlusion, the more extensive is the amount of myocardium at risk of
necrosis. The larger the MI, the greater is the chance of death due to a mechanical
complication or pump failure. The longer the time period of vessel occlusion, the
greater the chances of irreversible myocardial damage distal to the occlusion. The
extent of myocardial cell death defines the magnitude of the MI.
c) Cardiomyopathy: Cardiomyopathy means diseases of the heart muscle. Some
types of cardiomyopathy are genetic, while others occur for reasons that are less
well understood. There are many types of cardiomyopathies. One of the most
common types of cardiomyopathy is idiopathic dilated cardiomyopathy, an
enlarged heart without a known cause.
12
Figure1.7(a) Coronary Artery Disease Figure1.7(b) Cardiomyopathy
Figure1.7(c) Aneursyms Figure1.7(d) Pericardial effusion
Figure1.7(e) Peripheral arterial disease Figure1.7(f) Atherosclerosis
Figure 1.7: Types of cardiac disease
13
d) Congenital Heart Disease: The term congenital or hereditary heart disease refers to
heart disease that is passed down through the family, and this is considered as being a
congenital type as it is principally inevitable and unpreventable. Congenital heart
disease refers to a problem with the heart's structure and function due to abnormal
heart development before birth. Congenital heart disease is the most common type of
birth defect. It is responsible for more deaths in the first year of life than any other
birth defects.
e) Aneurysm : An aneurysm is a bulge or weakness in a blood vessel (artery or vein)
wall. Aneurysms usually get bigger over time. Aneurysms can occur in arteries in any
location in the body. The most common sites include the abdominal aorta and the
arteries at the base of the brain.
f) Valvular Heart Diseases: These are diseases of the heart valves. Four valves
within the heart keep blood flowing in the right direction. Valves may be damaged by
a variety of conditions leading to narrowing (stenosis), leaking (regurgitation or
insufficiency) or improper closing (prolapse). The valves may be damaged by such
conditions as rheumatic fever, infections (infectious endocarditis), connective tissue
disorders, and certain medications or radiation treatments for cancer.
g) Pericardial Diseases: These are diseases of the sac that encases the heart
(pericardium). Pericardial disorders include inflammation (pericarditis), fluid
accumulation (pericardial effusion) and stiffness (constrictive pericarditis). These can
occur alone or together.
h) Congestive Heart Failure (CHF): Congestive heart failure is when the heart does
not pump adequate blood to meet the needs of body‟s organs and tissues. It doesn't
mean that heart has failed and can't pump blood at all. With this less effective
pumping, vital organs don't get enough blood, causing such signs and symptoms as
shortness of breath, fluid retention and fatigue. CHF can often result from heart
problem and constricted arteries.
i) High Blood Pressure: High blood pressure (hypertension) is the excessive force of
blood pumping through blood vessels. High blood pressure also causes many other
types of cardiovascular disease, such as stroke and heart failure.
14
j) Stroke: A stroke occurs when blood flow to the brain is interrupted (ischemic stroke)
or when a blood vessel in the brain ruptures (hemorrhagic stroke). Both can cause the
death of brain cells in the affected areas. Stroke is also considered a neurological
disorder because of the many complications it causes.
k) Arrhythmias: Heart rhythm problems (arrhythmias) occur when the electrical
impulses in heart that coordinates heartbeats don't function properly, causing the heart
to beat too fast, too slow or irregularly. Other forms of cardiovascular disease can
cause arrhythmias.
l) Stable Angina: Also known as Angina pectoris is chest pain caused by myocardial
ischemia. It usually last from 3 to 5 minutes and if the blood flow is restored no
permanent change or damage results. It is usually experienced by chest discomfort
ranging from a sensation of heaviness or pressure to moderately severe pain.
Discomfort may radiate to the neck, lower jaw, left arm and left shoulder, or
occasionally to the back or down the right arm. Discomfort is commonly mistaken for
indigestion.
m) Coronary Heart Disease (Ischemic Heart Disease): Coronary heart disease or in its
medical term Ischemic heart disease is the most frequent type of heart problem of all,
and is also the leading reason of heart attacks. Coronary heart disease is a term that
refers to damage to the heart that happens because its blood supply is decreased, and
what happens here is that fatty deposits build up on the linings of the blood vessels
that provide the heart muscles with blood, resulting in them narrowing. These
narrowing decreases the blood supply to the heart muscles and causes pain that is
identified as angina.
There are a few factors which are considered as being responsible causes of
coronary heart disease. One in particular is high cholesterol that can increase fat
concentration in our blood and create the building up of fatty deposits.
n) Pulmonary Heart Disease: Pulmonary heart disease is a disease that comes from a
lung, or pulmonary, disorder, or a complication of lung problems where the blood
flow into the lungs is slowed or even totally blocked, resulting in increased pressure
on the lungs. There are a number of different symptoms that typically come with
pulmonary heart disease, such as shortness of breath, syncope, dyspnea, and chest
pain.
15
o) Rheumatic Heart Disease: Rheumatic heart disease frequently derives from strep
throat infections. This can be a reason for alarm for many because strep throat, while
often preventable, is a quite common condition that affects many people who do not
treat a minor sore throat infection in time. However, there is no reason to be because
rheumatic heart disease that comes from strep throat is fairly rare.
p) Atherosclerosis: The word Atherosclerosis is derived from the Greek athere
(porridge) and scleros (hardness). Atherosclerosis is a complex process involving a
variety of structural and functional changes in the arterial wall. It involves the
interaction of several cell types with the endothelium, including monocytes, platelets,
and smooth muscle cells. The end result is the formation of fibrous atherosclerotic
plaques in the tunica intima and media of large- and medium-sized arteries,
particularly those located in areas of high blood pressure and turbulence (Wang and
Wang, 2005). Endothelial dysfunction, inflammation, and modification of lipids are
all key early events in the initiation and progression of atherosclerosis (Ross, 1993).
During the early stages of plaque formation, oxidized low density lipoproteins (LDLs)
are taken up by macrophages in the subendothelial space, leading to the formation of
foam cells and “fatty streaks” (Ross, 1993). Abnormal proliferation and migration of
vascular smooth muscle cells from the medial layer into the intimal space of the artery
are also key early events in atherosclerosis (Ross, 1993; Nakashima et al., 2007).
There are various hypotheses for the initiating event in atherogenesis. The
“oxidative modification hypotheses” is currently the major hypotheses for the
initiating event in atherosclerosis (Stocker and Keaney, 2004). This hypotheses
suggests that increased oxidative stress and generation of ROS in the vascular wall
leads to oxidation of LDL, which then mediates a multitude of effects on the vascular
wall. For example, oxidized LDL has been shown to attract circulating monocytes
into the intimal space and induce adhesion to the endothelium, promote foam cell
formation, injure endothelial and other cells of the vascular wall (via both apoptosis
and necrosis), interfere with endothelium-dependent vasodilation, and modulate
mitogenic signaling in vascular SMCs leading to migration and proliferation (Stocker
and Keaney, 2004). In contrast, the monoclonal hypotheses is based on the molecular
links that exist between atherosclerosis and carcinogenesis. Both diseases are
characterized by uncontrolled cellular proliferation and dedifferentiation that may
16
arise through the mutation of proto-oncogenes. The monoclonal hypotheses states that
the formation of atherosclerotic lesions result from proliferation of a transformed
SMC phenotype and can be viewed as benign smooth muscle cell tumors of the artery
wall (Ramos and Partridge, 2005). Although the oxidative modification hypotheses is
currently favoured over the monoclonal hypotheses for atherogenesis, neither has
been disproven. Furthermore, these hypotheses are not mutually exclusive and
potentially both mechanisms may contribute to atherosclerosis.
1.4 Principal Symptoms of Heart Disease
Everyone will experience different symptoms with each heart attack. Heart
attacks frequently occur from 4:00 A.M. to 10:00 A.M due to higher adrenaline
amounts released from the adrenal glands during the morning hours (Willich et al.,
1992; Brezinski et al., 1988). Because there are many possible conditions that follow
under the umbrella of heart disease, the related symptoms are numerous. But some of
the principal symptoms of the heart disease include dyspnea, chest pain or discomfort,
syncope, heart palpitations, and fatigue, lethargy or daytime sleepiness.
a) Dyspnea: Braunwald et al., (1988) defined dyspnea as “an abnormally
uncomfortable awareness of breathing; it is one of the principal symptoms of
cardiac and pulmonary disease”. It is associated with a wide variety of diseases of
the heart, lungs, chest wall and respiratory muscles as well as with anxiety.
b) Chest pain or discomfort: Although chest pain or discomfort is one of the
cardinal manifestations of the heart disease, it is critical to recognize that it may
originate not only in the heart but also in a variety of non-cardiac reasons
(Braunwald et al., 1988). Few symptoms are more alarming than chest pain. In the
minds of many people, chest pain equals heart pain.
"Chest pain" is an imprecise term. It is often used to describe any pain, pressure,
squeezing, choking, numbness or any other discomfort in the chest, neck, or upper
abdomen, and is often associated with pain in the jaw, head, or arms.
c) Syncope: According to Richard (2009), Syncope is a sudden and temporary loss of
consciousness, or fainting. It is a common symptom - most people pass out at least
once in their lives - and often does not indicate a serious medical problem. Sometimes
17
syncope indicates a dangerous or even life-threatening condition, so when syncope
occurs it is important to figure out the cause. The causes of syncope can be grouped
into four major categories: neurologic, metabolic, vasomotor and cardiac. Of these,
only cardiac syncope commonly leads to sudden death.
d) Heart Palpitations: This common symptom of the heart disease is defined as an
unpleasant awareness of the forceful or rapid beating of the heart. It may be
brought by a variety of disorders involving changes in cardiac rhythm or rate.
Most people who complain of palpitations describe them either as "skips" in the
heartbeat (that is, a pause, often followed by a particularly strong beat,) or as
periods of rapid and/or irregular heartbeats. The most common causes of
palpitations are premature atrial complexes (PACs), premature ventricular
complexes (PVCs), episodes of atrial fibrillation, and episodes of supraventricular
tachycardia (SVT). Unfortunately, on occasion, palpitations can signal a more
dangerous heart arrhythmia, such as ventricular tachycardia (Richard, 2009).
e) Fatigue, Lethargy or Daytime Sleepiness: These are very common symptoms.
Fatigue or lethargy can be thought of as an inability to continue functioning at
one's normal levels. Somnolence implies, in addition, that one either craves sleep -
or worse, finds oneself suddenly asleep, a condition known as narcolepsy - during
the daytime.
1.5 Risk Factors
Risk factors of CVD have been categorized in two major groups--modifiable
and non-modifiable risk factors. Recently, contextual factors have been added as risk
factors that contribute to CVD. (AHA, 2004, 2006; Appel et al., 2002; Grundy et al.,
1999; HSFC, 2003; WHO, 2004a; Yusuf et al., 2001).
1.5.1 Non-Modifiable Risk Factors
a) Advancing Age
Advancing age is the most potent independent risk factor for cardiovascular disease.
Increased age is the foremost risk condition for heart disease and stroke. These are
true among both men and women. Evidence showed that risk of stroke doubles every
decade after age 55 (AHA, 2004). However, it has been indicated that women aged
over 55 years or after experience menopausal have a greater risk for CVD (Figure1.8).
18
This is caused by estrogen hormone which protects women from CVD during the
younger age (AHA, 2004).
Figure1.8: Prevalence of Cardiovascular Diseases in American Men and Women,
Ages 20 and Older
b) Family History
Family history of CVD has been indicated as a non-modifiable risk factor for
CVD. In the background of most diseases lies genetic inheritance. HSFC (2003)
indicated that a family history of CVD, particularly coronary arterial disease (CAD) is
an important risk factor for CVD. It is also an independent risk factor for stroke. The
factors that contribute to this association may include familial factors, lifestyle and
molecular defects in vascular physiology, which render the vessel wall more
susceptible to atherosclerosis. Mc Sweeney et al. (2003) asserted that one of the most
frequencies of risk factors for CVD; in particular MI, among women was Family
history of CVD. Family history cannot be modified. Thus, those with a positive
family history can gain enormous advantage by moderating their fat and food intake,
by being active and by not smoking. Even though studies from the US and other
countries indicated that family history is a major risk factor for CVD
1.5.2 Modifiable Risk Factors
CVD is potentially very confusing, with a variety of similar but distinct
conditions affected by a range of overlapping but not identical risk factors, and which
can affect one another. Figure1.9 gives an overview of the main CVDs, the
aetiological relationships between them and the various risk factors that affect them.
19
1.5.2.1 Physiological Risk Factors
a) High blood pressure.
According to WHO, high blood pressure is defined as a systolic blood
pressure (SBP) above 140 mmHg and/or a diastolic blood pressure (DBP) above 90
mmHg. Although high blood pressure or hypertension is part of CVD, it is in turn a
remarkable risk for other cardiovascular diseases. AHA (2006) indicated that high
blood pressure is a leading cause of stroke. High blood pressure is one of the most
important preventable causes of premature death worldwide and has been correlated
with other cardiovascular risk factors. In most countries, up to 30% of adults suffer
from high blood pressure and a further 50% to 60% would be in better health if they
reduced their blood pressure, by increasing physical activity, maintaining an ideal
body weight and eating more fruits and vegetables. In people aged up to 50years, both
DBP and SBP are associated with cardiovascular risk; above this age, SBP is a far
more important predictor (WHO, 2004a).
Blood pressure usually rises with age, except where salt intake is low and
physical activity is high. A high intake of salt independently increases the risk of
CVD in overweight persons. In addition to lifestyle changes, effective medication is
available for control of high blood pressure (HSFC, 2003; WHO, 2004a). High blood
pressure also increases overall cardiovascular risk by 2 to 3 times. Individuals who
have excess weight, are physically inactive, use alcohol heavily, or have excessive
salt intake are more likely to develop high blood pressure. High BP is commonly
associated with other metabolic cardiovascular risk factors such as insulin resistance,
obesity, hyperuricemia, and dyslipidemia (HSFC, 2003).
b) Abnormal serum cholesterol.
Abnormal blood cholesterol has been suggested as a strong cardiovascular risk
factor. High levels of total and LDL-C, and other abnormal lipids (fats), are risk
factors for cardiovascular disease (AHA, 2006; Mosca et al., 2007; WHO, 2004a). A
high level of LDL-C can lead to clogging of the arteries, increasing the risk of heart
attack and ischemic stroke, while a high level of HDL-C reduces the risk of coronary
heart disease and stroke (AHA, 2006; Mackay and Mensah, 2004; WHO, 2004a).
20
The risk of heart attack in both men and women is highest when they have
lower HDL-C levels (below 40 mg/dL) and higher total cholesterol levels (above 240
mg/dL). Higher level of triglycerides which is the most common type of fat in the
body often go with higher levels of total cholesterol and LDL-C, lower levels of
HDL-C and increased risk of diabetes. High triglycerides may increase the risk of
CVD for women more than men (AHA, 2004; Kannel and Mc Gee, 1979).
Figure 1.9: Overview of relationship between cardiovascular diseases and
cardiovascular risk factors.
21
Diabetes mellitus
Diabetes is one of determinant risk factors of CVD. Diabetes is a disorder of
carbohydrate metabolism and a risk factor for CVD. Insulin is a hormone produced by
the pancreas and used by the body to regulate glucose. Diabetes occurs when the body
does not produce enough insulin, or cannot use it properly, leading to too much sugar
in the blood. The majority of people with diabetes have type II diabetes, in which
insulin is produced in smaller amounts than needed or is not properly effective. This
form of diabetes is preventable, because it is related to physical inactivity, excess
calorie intake and obesity (Mackay and Mensah, 2004). There are evidence relating
diabetes and mortality from CVD. As AHA (2004), two-thirds to three-fourths of
people with diabetes will die of some form of heart or blood vessel disease. Adults
with diabetes have heart disease death rates about two to four times higher than those
for adults without diabetes.
Diabetes not only increases the incidence of CVD but adversely influences
outcome as well. Individuals with diabetes have higher mortality rate from CVD.
Diabetics frequently have high blood pressure and high cholesterol and are
overweight, increasing their risk of CVD even more (AHA, 2004). Maintaining a
healthy weight through healthy diet and regular physical activity can prevent diabetes.
In addition, diabetes mellitus is associated with 3 to 7- fold increase in risk for
developing CHD in women compared to a two three-fold elevation risk in men. The
reason for this gender difference is not fully established, but it may be the result of a
more deleterious effect of diabetes on blood pressure and lipids in women (Mosca et
al., 2002). Therefore, women with type 2 diabetes have a higher risk of heart disease,
heart attack and stroke than non-diabetic same-aged women.
c) Obesity
Obesity is one of significant risk factor of CVD. According to WHO (1999;
2004), being overweight has been defined by utilizing the Body Mass Index (BMI), a
measure of weight in relation to height. BMI is commonly used for classifying
overweight and obesity. The BMI for observed risk in different Asian populations
varies from 22 to 25. BMI provides a simple, convenient measurement of obesity.
BMI has also been related to other cardiovascular risk factors such as diabetes and
high blood pressure. The risks of cardiovascular disease and type II diabetes tend to
22
increase on a continuum with increasing BMI. But for practical purposes a person
with a BMI of over 25 is considered overweight, while someone with a BMI of over
30 is obese.
WHO (2004) claimed that obese smokers live 14 fewer years than nonsmokers
of normal weight. Also, HSFC (2003) indicated that being overweight or obese
among individual aged between 18 and 64 years is one of the most common factors
influencing the development of high blood pressure and diabetes. These conditions
are, in turn, two important risk factors for the development of CVD. In general,
healthy nutrition and regular physical activity can reduce excess weight and obesity.
Thus, obesity may impact directly and indirectly through other risk factors in
contributing to CVD.
d) Menopause status
Menopause can be a modifiable risk factor of CVD among women (Bittner,
2002; Grundy et al., 1999; WHO, 2004a). Menopause is a life transition women
experience. Menopause has been correlated with CVD. Women who are over 55 or
experience menopausal periods tend to be at higher risk for CVD and women between
the ages of 40 and 59 are at greater risk for developing CVD (AHA, 2006; Mackay
and Mensah, 2004). Menopause has also been identified as a mediator associated with
stress and cardiovascular disease and may be a unique risk factor in women.
1.5.2.2 Behavioral Risk Factors
a) Cigarette smoking.
Cigarette smoking has been well documented as a major-behavioral risk factor
of CVD. Cigarette smoke contains many chemicals, including nicotine and carbon
monoxide. Some of these chemicals and/or the carbon monoxide damage the inner
layer of the arteries. That damage permits more rapid entry of cholesterol into the
artery wall. Cigarette smoking also leads to blood clotting in the arteries, leading to
heart attack (Goble, 2005). Furthermore, cigarette smokers have been defined that, the
individuals are considered to be daily smokers if they regularly smoke at least one
cigarette per day.
Tobacco use, other than smoking, and passive smoking are also implicated as
CVD risks. Smoking promotes CVD through several mechanisms. It damages the
endothelium lining of the blood vessels, increases cholesterol plaques (fatty deposits
23
in the arteries), increases clotting, raises LDL-C and lowers HDL-C, and promotes
coronary artery spasm. Nicotine accelerates the heart rate and raises blood pressure. A
gene has been discovered that increases smokers‟ risk of developing coronary heart
disease by up to four times. Around a quarter of the population carries one or more
copies of this gene (WHO, 2004a).
b) Physical inactivity.
Physical inactivity is estimated to cause two million deaths worldwide
annually. Globally, it is estimated to cause about 22% of ischemic heart disease.
Estimated attributable fractions are similar in men and women. Appropriately regular
physical activity is a major component in preventing the growing global burden of
chronic disease, e.g., CVD and diabetes.
The AHA asserted that physical inactivity increases risk of heart disease and
stroke by 50%. It has been indicated that industrialization, urbanization and
mechanized transport have reduced physical activity, even in developing countries, so
that currently more than 60% of the population worldwide are not sufficiently active.
Physical activity, even at an older age, can significantly reduce the risk of coronary
heart disease, diabetes, high blood pressure, and obesity, help reduce stress, anxiety
and depression, and improve lipid profile. It also reduces the risks of colon cancer,
breast cancer and ischemic stroke. In 1997, WHO defined physical activity as all
movements in everyday life. Inactivity is generally higher amongst girls and women
(Mackay and Mensah, 2004). Physical activity is defined as all taking at least 2.5
hours per week of moderate exercise or 1 hour per week of vigorous exercise (WHO,
2002a). The health benefits of regular physical activity are many. At least 30 minutes
of moderate physical activity, for example brisk walking, is enough to bring many of
these effects. Regular physical activity reduces the risk of dying from heart disease or
stroke and the risk of developing heart disease up to 50%. In addition, it reduces the
risk of developing type II diabetes 50%; helps to prevent/reduce hypertension;
promotes psychological well-being, reduces stress, anxiety and feelings of depression
and loneliness; and helps control weight and lower the risk of becoming obese by
50% compared to people with sedentary lifestyles (Mackay and Mensah, 2004).
24
c) Alcohol consumption.
Alcohol consumption/drinking can damage the heart and blood vessel system
and is one of significant risk factors for CVD in both developed and developing
countries (WHO, 2004a). However, drinking is not encouraged as a strategy for
reducing CVD risk due to the risks involved with alcohol use, including alcoholism,
high blood pressure, stroke, cancer, liver disease, accidents, and fetal alcohol
syndrome (Krauss et al., 1996).
1.5.2.3 Psychological Risk Factors
Hemingway and Mamot (1999) explained the contributions of psychological
factors to CVD. They claimed that evidence of mechanisms linking psychosocial
factors with coronary heart disease is important in making causal inferences and
therefore in designing preventive interventions. Psychosocial factors may act alone or
combine in clusters and may exert effects at different stages of the life course.
Broadly, three interrelated pathways may be considered. Firstly, psychosocial factors
may affect health related behaviors such as smoking, diet, alcohol consumption, or
physical activity, which in turn may influence the risk of coronary heart disease.
Secondly, psychosocial factors may cause direct acute or chronic patho-physiological
changes. Thirdly, access to and content of medical care may plausibly be influenced
by, for example, social support (but there is little direct evidence for this).
a) Stress.
Stress is a part of life. The stressor may be something actually or potentially
unpleasant. People might have not confront or cope with the stress. If stresses
continue or multiply, they may overwhelm them, inducing a sense of despair,
hopelessness or depression (Gopalan, 1998). With the growing recognition by medical
professionals, that stress management is an appropriate preventive treatment for CVD.
The scientific community has acknowledged stress as contributing risk factors to
CVD (AHA, 2006; Appel et al., 2002; Grundy et al., 1999; Mackay and Mensah,
2004; Rozanski, Blumenthal, and Kaplan, 1999; Yusuf et al., 2001). Stress and other
psychosocial factors are not considered independent CVD risk factors since the exact
role stress plays in development of CVD is not clearly understood (AHA, 2004).
The stress response is marked by the secretion of catecholamines, which
enhances the heart rate and increase blood pressure, blood sugar levels, respiration,
25
and circulation to skin and muscles. These physiologic changes are thought to ready
individuals for the Fight-or-flight response. Stress appears to have important
implications for the progression of chronic disease such as cancer, cardiovascular
disease, and other illnesses.
However, the exact role of stress on health or in those diseases is not known
yet. The cardiovascular system circulates blood to every cell delivering oxygen fuel,
hormones, and removing waste products. When excess stress becomes chronic
situation, the neural and hormonal stimulation may cause blood pressure to become
chronically elevated. Elevated of the stress hormone such as cortisol may also
damage the artery lining that attracts white blood cells attempting to repair the
problem. This causes atherosclerotic plaques, rough surface that cause platelets
leading to blood clots and more cell growth (Brehm, 1998).
b) Depression.
Depression generally involves feelings of sadness, tiredness, indecisiveness,
and worthlessness. Numerous studies have established that depression predicts the
incidence of CHD in previously healthy people (Davidson et al., 2004). In recent
years, depression has emerged in the discussion on the impact of psychological
aspects on coronary risks (AHA, 2006; Davidson et al., 2004; Rozanski et al., 1999;
Rugulies, 2002; WHO, 2004a). Several prognostic studies have shown that depression
is a predictor for survival after MI (Davidson et al., 2004; Rozanski et al., 1999;
Rugulies, 2002).
c) Coping.
Coping has been defined as “constantly changing cognitive and behavioral
efforts to manage specific external and internal demands that are appraised as taxing
or exceeding the resources of the person” (Lazarus and Folkman, 1984).
1.5.3 Contextual Risk Factors
Not only have modifiable and non-modifiable risk factors been delineated as
risk factors for CVD, but also contextual risk factors have been claimed as the factors
that contribute to CVD in human (Appel et al., 2002). Contextual factors include
socioeconomic status, SES (education level, family income) and rural context
(distance to hospital, transportation, poverty).
26
1.5.3.1 Socioeconomic Status
a) Education level.
Education or formal education level is the most widely used measure of SES
in epidemiologic studies. Of the studies of chronic disease, in which measures of SES
were used, education was used by 45% as a surrogate measure of SES (Liberatos,
Link and Kelsey, 1988). According to Edelman and Menz (1996), rural dwellers
showed lower educational attainment than their urban counterparts.
b) Family income.
There is evidence indicating that rural residents have lower income than their
urban counterparts (Edelman and Menz, 1996), which may have an effect on their health.
Measures of income are obviously an important marker of SES. Income provides access
to goods and services, including quality education and medical care that may protect
against disease. However, lower income may reflect the influence of poorer health.
Low socioeconomic position and lower educational level are known to be risk
factors for CHD. SES has also been correlated with CVD risk factors. There is
substantial evidence for an inverse relationship between SES and almost all the
cardiovascular disease risk factors, with the possible exception of cholesterol level.
Most of rural populations engage poverty worldwide. Thus, they are more likely to
develop depression which is in turn a conditional risk factor for CVD.
1.5.3.2 Rural Contexts
CVD is one of chronic illness that has been indicated high prevalence among
rural adults (Gamm and Hutchison, 2003). In general, rural people are not healthy as
urban populations. Rural adults are less likely than urban counterparts to engage in
preventive behaviors such as regular blood pressure checks, other physical check-up,
or health promotion programs.
a) Rural health care services
There are two issues of concern regarding health care services in rural
community: availability and accessibility of services. In rural areas, there are fewer
physicians and nurses in general, as well as fewer family practice physicians, nurse
practitioners, and specialists, especially obstetricians, pediatricians, psychiatrists, and
social service professionals. Long travel distances, lack of public transportation, lack
27
of telephone services, a shortage of health care providers, unpredictable weather
conditions, inability to obtain entitlements (Gamm and Hutchison, 2003). In rural
communities, both accessibility and availability issues of services and providers must
always be considered.
b) Distance to hospital
Long distance to health care is an important factor which affects access to
care. Long distance increases travel time for healthcare services. Long distance could
also be seen as a disadvantage more in an emergency situation. Lack of a convenience
of public and personal transportation affects health care access.
Moreover, distances coupled with lack of local care services and specialists
contribute to poor health of the population (Lee, 1998). Generally, there are health
centers and community hospitals that provide health care services for rural
populations. However, since those are limited to resources (specialists and health care
technology), the rural patients have to be referred to provincial hospitals located in the
city, in which farther from where they live. Distance to health care causes patients
(e.g., CVD patients) with high-risk care or follow-up care may have difficulty keeping
appointment and obtaining medication. Thus, measuring the distance to care as one of
rural context, which has an effect on health care access, is important to examine as
one of the contextual risk factors for CVD.
c) Poverty
Poverty is a significant barrier in accessing healthcare (IOM, 2004) since rural
residents have lower incomes than do their urban counterparts (Edelman and Menz,
1996). Poverty is multi-dimensional in its causes as well as in its cures. Limited
employment, low income, and low levels of literacy make rural dwellers become
poverty. Rural residents are more likely living in poverty than do their urban
counterparts. Thus, poverty has an impact on rural residents‟ health status.
1.6 Role of Lipoproteins and Lipid Metabolism in CVD’s
1.6.1 Lipoproteins
The major lipids in human plasma are cholesterol, cholesteryl esters (CE),
triglycerides (TG) and phospholipids (PL). TG and CE molecules, which are insoluble in
aqueous solutions, are carried in the core of spherical macromolecular complexes, called
28
lipoproteins (Ginsberg, 1998). Most lipoproteins share a common spheroid structure
consisting of a neutral lipid core of hydrophobic CE and TG surrounded by a surface
monolayer of PL, unesterified free cholesterol (FC) and apolipoproteins. Since
lipoproteins constitute a heterogeneous population of particles, they are traditionally
classified into six major classes according to their densities. Four of the major classes of
lipoproteins are very low density lipoprotein (VLDL), intermediate density lipoprotein
(IDL), low density lipoprotein (LDL), and high density lipoprotein (HDL). They are
derived from the liver and are present in plasma from both fasted and non-fasted subjects.
The other two major classes are chylomicrons (CMs) and CM remnants and those are
derived from the small intestine and are found in the plasma only after a fatty meal. The
characteristics of the various human plasma lipoproteins are listed in Table 1.2. As
shown, their relative contents of protein and lipid determine their hydrated density, size
and electrophoretic mobility and hence, their classification. In more detail, CMs, VLDL,
and LDL have apolipoprotein B (apoB) as their primary protein, whilst apolipoprotein A-
I (apoA-I) is the major protein constituent of HDL. CMs and VLDL are TG-rich
lipoproteins, whilst LDL and HDL contain relatively high levels of CE and PL,
respectively. Furthermore, the size of different lipoproteins is inversely correlated to their
density, with VLDL being the largest lipoprotein and HDL the smallest one. The
heterogeneity in lipoprotein size and composition induces changes in interaction with
different tissues, thereby influencing the lipoprotein metabolism.
Table 1.2: Physical properties and composition of human plasma lipoproteins
a - According to the electrophoretic mobility of plasma α and β globulins on
agarose gel electrophoresis
b - The values given for composition are expressed as percentage of total
weight Adapted from Ginsberg, 1998
29
1.6.2 Lipid metabolism
The liver plays an essential role in lipid metabolism. Lipoprotein metabolism
can be divided into three distinct pathways, based on origin, function and fate of the
lipid content of the particles involved (Havel, 1989; and Mahley and Hussain, 1991).
A condensed overview of metabolic routes is depicted below.
1) Exogenous lipid transport, describing the metabolic route of dietary lipids
after their absorption from the intestine. In detail, dietary TG and CE are hydrolyzed
in the intestine and subsequently assembled into CMs. These large TG-rich CMs are
transported from the lymph to the blood circulation (Figure 1.10). Nascent CMs
consist mainly of TG (88%), but also PL, CE, FC, and apolipoproteins (e.g. apoA-I,
apoA-II, apoA-IV, apoB-48, and apoCs) (Table 2). Upon entering the circulation,
these CMs are processed by lipoprotein lipase (LPL), which hydrolyzes TG, thereby
delivering liberated free fatty acids (FFA) to peripheral tissues such as adipose tissue,
skeletal muscle and heart (as energy source), and the liver (as storage or generation of
lipoprotein particles) (Fielding et al., 1978; Zechner, 1997). Due to hydrolysis of the
core lipids, CM particles shrink and the excess of surface material, i.e., PL, FC and
apolipoproteins, are in part transferred to HDL particles (Havel and Kane, 1989). The
CM remnants thus formed are rapidly taken up by the liver via a apoE-specific
recognition site on hepatocytes, including the LDL receptor (LDLR), LDLR-related
protein (LRP) heparan sulphate proteoglycans (HSPG) (Mahley and Ji 1999), and
possibly also scavenger receptor BI (SR-BI) (Out, 2004).
2) Endogenous lipid transport, describing the distribution of lipids from the
liver to peripheral tissues, in particular relevant during periods of fasting. In detail, the
endogenous pathway begins with the production and secretion of TG-rich lipoproteins
by the liver in the form of VLDL (Figure1.10). These lipids are either derived from
incoming CM remnants, IDL, LDL, and HDL, or from de novo synthesis. TG-rich
VLDL particles containing a single molecule of apo B provide energy-rich material to
the periphery during periods of fasting. Another important apolipoprotein on VLDL is
apoE, which plays an important role in the secretion (Kuipers et al., 1997) as well as
in the metabolic fate of these VLDL particles, the latter due to its interaction with
specific receptors (Mahley, 1988). Upon entering the blood circulation, VLDL is
30
further enriched with apoE and apoCs (e.g. apoCI, poC-II, apoC-III, and apoE) (Table
1.2). Similar to CMs, TG-rich VLDL particles can undergo lipolysis catalyzed by
LPL (Fielding, 1978), leading to formation of VLDL remnants or IDL, which is partly
cleared by the liver as mediated by apoE (Brown et al., 1981). The remainder is
extensively processed by LPL and hepatic lipase (HL) to become cholesterol-rich
LDL with apoB100 as its sole apolipoprotein, which is recognized by the LDLR on
the liver and peripheral tissues (Brown et al., 1981).
3) Reverse cholesterol transport, reflecting transport of cholesterol from
peripheral tissues to the liver. HDL is a relatively small lipoprotein, which carries
approximately one-third of the cholesterol in human plasma and, is involved in the
removal of excess cholesterol from cells (Rothblat and Phillips, 1986). There are
subclasses of HDL particles, including nascent discoidal HDL (pre-ß HDL) and
spherical HDL (HDL2 and HDL3). One of the major functions of HDL is to transport
cholesterol from peripheral tissues to the liver for elimination via the bile. This occurs
by a pathway called reverse cholesterol transport (RCT), which involves the
coordinate action of multiple cellular and plasma proteins (Figure 1.11).
Figure 1.10: Schematic overview of pathways involved in lipoprotein
metabolism.
31
Figure 1.11: Schematic illustration of reverse cholesterol transport.
1. The liver and the intestine (Gotto, 1983) form nascent HDL particles, lacking
CE and containing apoA-I as their major apolipoprotein. Cholesterol efflux from
peripheral cells can occur by passive diffusion, or it may involve HDL or apoA-I
receptors. For instance, ATP-binding cassette transporter A1 (ABCA1) may promote
cholesterol efflux from peripheral cells to lipid-poor apoA-I. In addition, ABCG1 and
SR-BI may promote cellular cholesterol efflux to mature HDL (Zannis et al., 2006).
2. Subsequent activity of lecithin: cholesterol acyltransferase (LCAT) leads to
formation of large, CE-rich HDL (Zannis et al., 2006 and Davis et al., 1982), thus
producing spherical HDL particles, converting HDL3 to larger HDL2. The latter
mature HDL particles can then be taken up by the liver by 3 pathways:
(1) As part of a holo-HDL uptake mechanism, probably involving proteoglycans
(PG), apoE, and possibly other factors;
(2) Via hepatic SR-BImediated selective uptake of CE and FC (Mahley and Ji,
1999 and Acton et al., 1996); and
32
(3) By cholesterol ester transfer protein (CETP)-mediated transfer to TG-rich
lipoproteins (TRLs) (Tall et al., 1984), with subsequent uptake of TRL remnants
in the liver, involving LDLR, PG, or LRP.
3. Upon delivery of HDL-CE to the liver, the CE‟s are hydrolyzed and reused for
lipoprotein assembly. Alternatively, cholesterol is secreted into the bile either as
neutral sterols or bile salt via ABCG 5/8 (half transporters that work together as
heterodimers) and ABCB11 (BSEP)-mediated pathways (Yu et al., 2005 and
Wakabayashi et al., 2004).
Thus, in order for the cycle of RCT to persist, new acceptors of cellular
cholesterol (ie, apoA-I, pre-HDL) must be continuously synthesized or regenerated in
reactions that are catalyzed by lipid transfer proteins (i.e., CETP, PLTP, HL and EL),
acting in conjunction with ABC-transporters (i.e., ABCA1, ABCG1, ABCG5/G8, and
ABCB11) to enhance cellular cholesterol efflux.
1.6.3 Role of lipoproteins in Atherosclerosis
Atherosclerosis is a disease characterized by the development of
atherosclerotic plaques in arterial walls. Growing of a plaque causes local arterial wall
thick and stiff and makes narrowing of arterial lumen. An atherosclerotic plaque can
block blood circulation when it drops off from the wall or even when it is too big and
becoming a blood clot. Atherosclerosis is the main cause for several fatal diseases,
including coronary artery diseases, cerebral thrombosis, and aortic aneurysm. In a part
of arterial wall, atherosclerotic plaques distribute randomly, and they have different
sizes and different shapes.
The initiation of atherosclerosis has been debated for many years and several
hypotheses have been proposed. One of the earliest, the „response to- injury‟
hypotheses stated that endothelial injury leads to an inflammatory response as part of
a healing process in the arterial wall (Ross et al., 1977). Subsequently, the „response-
to-oxidation‟ hypotheses has evolved to focus on specific pro inflammatory oxidized
phospholipids that result from the oxidation of LDL phospholipids containing
arachidonic acid and that are recognized by the innate immune system in animals and
humans, proposing that lipoprotein oxidation is the important link in atherosclerosis
(Steinberg et al., 1989). In 1995, it was suggested that retention of lipoproteins is the
33
initiating step which leads to oxidation, inflammation and endothelial dysfunction,
consistent with previous hypotheses. This so-called „response-to-retention‟
hypotheses was based on pioneering work in the 70s and 80s showing that
lipoproteins can interact with the arterial wall (Williams and Tabas, 1995). According
to Fabrizio, 2010 and McCormick, 2012, Atherosclerosis is a typical aging-associated
disease; however traditional aging theories cannot interpret the in-homogeneity of
plaques. For example, gene-controlling theory suggests that aging is a process that is
controlled completely and independently by certain genes theory. However, if such
genes exist, they should work in the same way in all cells, and aging changes should
develop homogenously. Damage-accumulation theory predicts that aging is a result of
accumulation of faults (damage) (Kirkwood, 2005). However, if the random faults are
the origins of aging, accumulation of faults should result in a homogenous distribution
of aging changes.
Atherosclerosis is a progressive phenomenon with three successive stages:
fatty streaks, simple arterial plaque and then complicated plaque. During the first step,
LDL accumulates in the intima. It is a passive phenomenon that is all the more
important due to high LDL concentrations in the bloodstream (Nievelstein et al.,
1991). This lipid infiltration phase is followed by LDL oxidative modification, of
which the first mechanism is the oxidation of a lysine molecule in Apo B (Navab et
al., 2004). LDL is oxidized mainly through the interaction of its lipid components (PL
and CE) with reactive oxygen radicals (O2 - and OH
-) present in small quantities in
the bloodstream. Lipid hydroperoxides formed in this way catalyze the non-enzymatic
formation of other oxidized PLs. This step occurs in the intima via lipoxygenase and
myeloperoxidase pathways that favor the addition of hyroperoxide lipids to LDL
(Navab et al., 2004). When the level of oxidized lipids within the LDL is sufficient,
LDL becomes oxidized and pro-inflammatory (Navab et al., 2000). They then trigger
atherosclerotic phenomena by initiating endothelial activation within the intima, an
indispensable step at the beginning of the second stage.
The second stage begins with the recruitment of circulating monocytes, their
transformation into macrophages and then into foam cells (Shibata and Glass, 2010).
Endothelial activation leads to the expression at the endothelial surface of adhesion
molecules such as vascular cell adhesion molecule-1 (VCAM-1), intercellular
adhesion molecule-1 (ICAM-1) or E-selectin that will allow the adhesion of
34
circulating monocytes (Blankenberg et al., 2003). In the bloodstream, the integrins
expressed by the monocytes are characterized by weak ligand activity. Their
activation by pro-inflammatory molecules makes it possible to increase this affinity
(Berthet et al., 2014). After adhesion, circulating monocytes penetrate the sub-
endothelial space with the help of monocyte chemotactic protein-1 (MCP-1) where
they change into macrophages under the influence of monocyte-colony stimulating
factor produced by the activated endothelium. Some of these macrophages change
into foam cells (Shibata and Glass, 2010) by capturing oxidized LDL (oxLDL) via
scavenger receptors, whereas others induce a local chronic inflammatory reaction by
secreting pro-inflammatory cytokines. The activation of the inflammatory process is
initiated by toll-like receptors expressed on the surface of the macrophages, activated
by binding with their various ligands such as heat-shock protein 60,
lipopolysaccharide and oxLDL. The activation of the macrophages by the toll-like
receptor leads to the production of pro-inflammatory cytokines (Dinasarapu et al.,
2013) protease like matrix metallo protease, vasoactive molecules such as nitric oxide
(NO) and procoagulant factors (Hansson et al., 2006). Pro-inflammatory cytokines,
such as tumor necrosis factor (TNF)-α or interleukin (IL)-1, maintain endothelial
activation, increasing the adhesion of new circulating monocytes.
The third step is the formation of adult plaque. The accumulation of plaque
lipids forms a cluster called a lipid core, which is isolated by a fibro-muscular cap.
The cap is composed of smooth muscle cells and proteins from the extracellular
matrix (collagen, elastase) that stabilize the plaque. Then, over the years, the
development of the plaque becomes complicated (rupture, thrombosis and/or
aneurism), thereby generating clinical manifestations of cardiovascular diseases such
as MI and cerebrovascular accidents. A schematic overview of atherosclerosis is
depicted in Figure 1.12. However, the anti-oxidant, anti-inflammatory and anti-
thrombotic properties of the HDL prevent the anti-atherosclerotic lesions.
35
Figure1.12: Pathogenesis of atherogenesis. The development of
atherosclerosis from the endothelium injury to the fibrous cap formation
and thinning.
36
1.6.4 Properties of HDL in Atherogenesis
The strong inverse relationship between HDL levels and atherosclerosis has
been known for more than 3 decades (Rhoads et al., 1976). Surprisingly, no effective
therapy specifically directed at HDL has yet been developed, which reflects
uncertainty about the multiple mechanisms underlying the protective effect of HDL
against the development of atherosclerosis (Calabresi et al., 2003; Barter et al., 2004
and Khovidhunkit, 2004). Almost every step in the pathogenesis of atherosclerosis
has been reported to be favorably influenced by HDL (Mackness et al., 2004 and
Cuchel et al., 2006), as summarized in Table 1.3
Experiments with transgenic animals suggest that disruption of one or more
steps in RCT results in accelerated atherosclerosis, whereas over expression of pivotal
proteins in RCT, such as apoA-I, LCAT, and SRBI exerts atheroprotective effects
(Zhang et al., 2003). This information supports the concept that cholesterol efflux and
RCT are key anti-atherogenic properties of HDL. However, the important lesson from
experimental approaches is that disruption of RCT and resulting atherosclerosis may
occur in the presence of either decreased or increased HDL cholesterol levels,
depending on which step of RCT is dysfunctional. For instance, decreased HDL levels
associated with increased accumulation of cholesterol in peripheral tissues and/or
atherosclerosis were observed in humans and animals with apoA-I or ABCA1-
deficiency (Francis et al., 1995 and Schaefer et al., 1980), in which the initial steps of
RCT are inhibited. By contrast, increased HDL concentrations combined with
enhanced arteriosclerosis were presented in animals with dysfunctional SR-BI, in
which the later step of RCT is impaired (Zannis et al., 2006). Therefore, it is not the
increase or decrease of plasma HDL cholesterol concentration per se, but rather the
concentrations of various HDL subclasses, the cellular mobilization and transport of
lipids, and the kinetics of HDL metabolism that critically determine the
atherosclerotic risk. Diagnostic measures allowing more accurate insight into the
efficacy of cholesterol flow along RCT pathways still await development.
37
Table 1.3: Anti-atherogenic activities of HDL particles
Activity Documented protective effects
1. Facilitation of RCT Efflux of cholesterol from foam cells
in artery wall
2. Anti inflammatory Inhibition of the synthesis of platelet
activating factor
Inhibition of leucocyte adhesion to the
arterial wall via attenuation of the
expression of VCAM-1 and other
cytokine- induced cell adhesion
molecules.
Inhibition of expression of MCP-1
3. Improved endothelial
function
Stimulation of endothelial NO
synthase activity
Enhanced endothelial dependent
vasodilation
Prevention of endothelial cell
apoptosis
Stimulation of prostacyclin synthase
4. Antioxidative Protection of LDL from oxidation
Via apo A1-mediated antioxidative
action
Via paroxonase mediated antioxidative
actions
5. Anti-thrombotic Protection of erythrocytes against the
generation of pro coagulant activity
Stimulation of prostacyclin synthesis
Inhibition of thrombin- induced
endothelial tissue factor expression
6. Anti infectious Reduction of the pyrogenic activity of
bacterial lipopolysacharides
Lysis of Trypanosoma brucei
38
Other mechanisms
HDL may impart anti-atherogenic effects through its function as an autonomous
protective factor for the endothelium (Moira et al., 2001). Endothelial dysfunction
characterized by decreased bioavailability of nitric oxide (NO), a potent vasodilator, and
increased affinity of the endothelial surface for leukocytes is often encountered in the
early stages of atherosclerosis. In advanced plaques, denudation of the endothelium as a
consequence of increased apoptotic cell death can be observed. Furthermore, HDL
attenuates expression of VCAM-1, ICAM-1, and E-selectin, as well as cytokines such as
IL-8 that promote leukocyte extravasation (Dimayuga et al., 1999 and Cockerill et al.,
2001). Endothelial apoptosis was prevented in the presence of HDL, and this effect was
associated with inhibition of typical apoptosis pathways such as the activation of caspases
(Kimura et al., 2001). In addition, HDL activates protein kinase Akt, a ubiquitous
mediator of anti apoptotic signaling (Nofer et al., 2001). Thus, HDL can suppress
expression of cytokine-induced endothelial cell adhesion molecules and block the
migration of macrophages into the sub endothelial space of blood vessels.
In addition, the anti-atherogenic mechanism of HDL in inhibiting LDL lipid
oxidation, especially via the 12-lipoxygenase-induced pathway, is believed to be
crucial for preventing the scavenger receptor-mediated uptake of modified LDL
particles into macrophage cells located within the subendothelial intima (Navab, et al.,
2004). The oxidation of phospholipids is largely generated by the lipoxygenase and
myeloperoxidase pathways in the formation of cell derived reactive oxygen species
(ROS) (Vance and Vance, 2002). These ROS oxidize lipoprotein phospholipids
containing arachidonic acid, which in turn, makes the particles pro-inflammatory. The
ability of HDL to inhibit oxidation and inflammation is by way of the high levels of
antioxidants inherent to this lipoprotein, such as apoA-I65, and the enzymes;
paraoxonase (PON) and platelet activating factor acetyl-hydrolase (PAF-AH)
(Watson, et al., 1995). These prevent oxidation reactions and catalyze the breakdown
of oxidized phospholipids on the LDL particle.
1.7 Lipid Metabolic enzymes
The effect of Lipoprotein lipase (LPL) was first described by Hahn who
noticed that administration of heparin to dogs abolished the turbidity in blood
associated with postprandial lipemia (Hahn, 1943). Later, Korn concluded that the
39
“clearing factor” was a heparin-releasable lipase that hydrolyzed core triglycerides of
large lipoproteins and consequently reduced the light-scattering in lipemic plasma,
and hence the enzyme was later named lipoprotein lipase (Korn, 1955). LPL is a 448
amino acid residue glycoprotein that is mainly synthesized in heart, skeletal muscle
and adipose tissue. Other tissues also produce LPL. Of special interest is LPL in
macrophages which may promote atherosclerosis. LPL is central for the metabolism
of lipids in blood. The enzyme acts at the endothelial surface of the capillary bed
where it hydrolyzes triglycerides in circulating triglyceride-rich lipoproteins (TRLs)
and thereby allows uptake of fatty acids in adjacent tissues. LPL activity has to be
rapidly modulated to adapt to the metabolic demands of different tissues.
Mature LPL is secreted to the vascular endothelium from the parenchymal
cells of adipose and muscle tissues, its major sited of synthesis, and a variety of other
tissues which have also been implicated as lesser, but significant, sources of the
enzyme (Braun and severson, 1992; Enerback and Gimble, 1993). The most recent
evidence implicating that LPL has both pro-atherogenic and anti- atherogenic roles.
The effects of LPL on atherosclerosis have been controversial. As atheromatous
plaques contain substantial amounts of LPL in situ, Zilversmit (1973) proposed that
local LPL is atherogenic. Furthermore, LPL mediates the lipolytic conversion of
triglyceride-rich lipoproteins to atherogenic cholesterol- rich lipoproteins such as
LDL-C and chylomicron remnants. Supporting this, LPL deficiency in humans, a
common genetic cause of chylomicronemia syndrome, results in very low plasma
levels of LDL-C, and is believed to cause resistance to premature atherosclerosis
(Brunzell, 1995). However, Benlian et al. (1996) have reported that several LPL-
deficient patients have developed relatively advanced atherosclerosis.
HMG- CoA reductase is the rate controlling enzyme of mevalonate pathway,
the metabolic pathway that produces cholesterol and other isoprenoids. Normally in
mammalian cells this enzyme is suppressed by cholesterol derived from the
internalization and degradation of LDL-C via the LDL receptor as well as oxidized
species of cholesterol. Competitive inhibitors of the reductase induce the expression
of LDL receptors in the liver, which in turn increases the catabolism of plasma LDL-
C and lowers the plasma concentration of cholesterol, an important determinant of
atherosclerosis.
40
Lecithin cholesterol acyltransferase (LCAT) is a HDL associated enzyme, which
is synthesized in the plasma (Francone and Fielding, 1991; Wang et al., 1997). LCAT
binds to HDL-C to catalyze the transfer of a fatty acid residue from the sn-2 position of
lecithin to cholesterol to form cholesterol ester and lysolecithin (Jimi et al., 1999). A lack
of LCAT activity leads to increase in free cholesterol and phospholipids and decrease in
esterified cholesterol. Cholesterol esterification would be a key step in transferring
cholesterol from the tissues of the body to the liver. This process, termed reverse
cholesterol transport (Glomset et al., 1966) was proposed to facilitate the removal of
cholesterol from the body. Thus the RCT is one of the several proposed mechanisms by
which HDL-C provides protection from CVD (Miller and Miller, 1975; Rhoads et al.,
1976). Traditionally LCAT activity has been considered „antiatherogenic‟ as the
cholesterol esterification apparently creates a gradient necessary for the flow of
unesterified cholesterol from tissues to plasma. However, newer data suggest that higher
plasma cholesterol esterification rate is not necessarily protective and that the protective
or conversely atherogenic role of LCAT may depend on the concentration and quality of
plasma HDL-C and LDL-C particles and on availability of lipid transferring proteins such
as cholesteryl ester transfer protein (Milada and Jiri, 1999).
1.8 Oxidative stress and lipid peroxidation in CVD:
Oxidative stress (OS) is induced by reactive oxygen species (ROS) is implicated
in the pathogenesis of a variety of vascular diseases including atherosclerosis,
hypertension and coronary artery disease. ROS are toxic to cells because they can react
with most of the cellular macromolecules, including proteins, lipids and DNA. It has been
implicated in the pathogenesis of a number of diseases and clinical conditions. These
include atherosclerosis, cancer, adult respiratory distress syndrome, alzheimers and
Parkinson diseases, ischaemia-reperfusion, injury of various organs, chemicals and
radiation induced injury, diabetes, etc (Lakshmi et al., 2005). Oxygen radicals are
continuously formed in the living organisms with deleterious effects that lead to cell
injury and death. Production of oxidative species occurs under physiological conditions at
a controlled rate, but it is dramatically increased in conditions of oxidative stress. OS
results in the imbalance between oxidant and antioxidant reaction either due to excess
free radical formation or insufficient removal by antioxidant leads to oxidative stress
(Kumar et al., 2008; Kaur et al., 2008).
41
Free radicals are highly reactive molecules, which destroy tissue by oxidizing
cell membrane lipids and damaging the body‟s genetic material (Kuhn, 2003). A free
radical is an atom, molecule or compound that is highly unstable because of its atomic
or molecular structure (i.e, the distribution of electrons within the molecule). As a
result, free radicals are very reactive as they attempt to pair up with other molecules,
atoms or even individual electrons to create a stable compound to achieve a more
stable state, free radical can steal a hydrogen atom from another molecule, bind to
another molecule, or interact in various ways with other free radicals (Goyns, 2002).
One chemical element frequently involved in free radical formation is oxygen.
Oxygen is vital to life but as a diatomic molecule it is remarkably unreactive. However,
oxygen is the substrate for the generation of a variety of ROS, some of which may be
deleterious to cell function. Molecular oxygen can accept four electrons, one at a time,
and the corresponding number of protons to generate two molecules of water. Reduction
of molecular oxygen to superoxide and of peroxide to hydroxyl radical is illustrated in
Figure1.13. During this process, different oxygen radicals are successively formed as
intermediate products including superoxide (O2.-); peroxide (O2
-2), which normally exists
in cells as H2O2 and OH-. Superoxide can react with itself to produce H2O2. Superoxide,
peroxide and the OH- are considered reactive oxygen species.
ROS are generally speaking O2 molecules in different states of oxidation or
reduction as well as compounds of O2 with hydrogen and nitrogen. ROS include
superoxide anion radical (O2-), hydrogen peroxide (H2O2), hydroxyl radical (OH
-) , nitric
oxide (NO), and peroxy nitrite (ONOO-).under physiological conditions ROS are
produced in low concentrations and act as a signaling molecule that regulate VSMC
contraction and relaxation, and participate in VSMC growth (Touyz and Sehiuffrin,
1999). A number of sites within the cell participate in the production of superoxide
including Xanthine/Xantine oxidase reaction autoxidation of catecholamines and
arachidonic acid metabolism (Kukreja and Hess, 1992).
ROS are toxic to cells because they can react with most cellular macromolecules,
including proteins, lipids, and DNA. It has been implicated in the pathogenesis of a
number of diseases and clinical conditions. These include atherosclerosis, cancer, adult
respiratory distress syndrome, Alzheimer‟s and Parkinson‟s diseases, ischaemia-
42
reperfusion injury of various organs, chemical and radiation-induced injury, diabetes, etc
(Lakshmi et al., 2005).
Figure 1.13: Reduction of molecular oxygen to superoxide, and of peroxide to
hydroxyl radical
The deep complicated structure of the animal tissues and the metabolic
relationship between the different chemical components present such as proteins, fats,
carbohydrates, hormones, enzymes as well as their intermediates and by products
represent a serious problem in understanding the action of ionizing radiation on
individual metabolic process of these compounds. So, the interference between the
cell constituents, the sensitivity of the organ itself as well as the susceptibility of the
cell constituents toward radiation levels, greatly differ and are considered dose and
time dependent (Yarmonenko, 1988).
Proteins perform numerous crucial functions in the cell, primarily in the form
of enzymes that mediate most biochemical reactions required for cellular functions.
Proteins are made up of approximately 20 different building blocks called amino
acids, which differ in their sensitivity to interactions with ROS. For example, the
amino acids cysteine, methionine, and histidine are especially sensitive to attack and
oxidation by the hydroxyl radical. Accordingly, enzymes in which these amino acids
are located at positions that are critical to the enzyme‟s activity will become
inactivated by the interaction with ROS (Toykuni, 1999).
43
Alternatively, the ROS–induced oxidation of proteins can lead to changes in the
proteins‟ three dimensional structure as well as to fragmentation, aggregation, or cross–
linking of the proteins. Finally, protein oxidation often will make the marked protein
more susceptible to degradation by cellular systems responsible for eliminating damaged
proteins from the cell (Toykuni, 1999).
Lipids that contain phosphate groups (i.e., phospholipids) are essential
components of the membranes that surround the cells as well as other cellular structures,
such as the nucleus and mitochondria. Consequently, damage to the phospholipids will
compromise the viability of the cells. The complete degradation (i.e., peroxidation) of
lipids is a hallmark of oxidative damage (Saada et al., 2001).
The polyunsaturated fatty acids (PUFA) present in the membranes‟ phospholipids
are particularly sensitive to attack by •OH and other oxidants. A single •OH can result in
the peroxidation of many PUFA molecules because the reactions involved in this process
are part of a cyclic chain reaction. In addition to damaging cells by destroying
membranes, lipid peroxidation can result in the formation of reactive products that
themselves can react with and damage proteins and DNA (Cederaum, 2001).
DNA is the cell‟s genetic material, and any permanent damage to the DNA can
result in changes (i.e., mutations) in the proteins encoded in the DNA, which may lead to
malfunctions or complete inactivation of the affected proteins. Thus it is essential for the
viability of individual cells or even the entire organism that the DNA remain intact. ROS
are a major source of DNA damage, causing strand breaks, removal of nucleotides, and a
variety of modifications of the organic bases of the nucleotides (Goyns, 2002).
1.8.1Protein Oxidation:
Free radical species can react directly with the protein or they can react with other
molecules such as lipids and carbohydrates, forming products that subsequently react
with the protein (Figure 1.14). Thus, the oxidation of proteins, peptides and amino acids
leads to altered physicochemical and functional properties, and may even result in
formation of toxic compounds (Karel et al., 1975; Rice-Evans et al., 1993). Oxidation of
proteins has also been linked to changes occurring during aging, particularly with
progression of diseases and disorders in humans (Levine et al., 2001; Levine, 2002).
44
Figure1.14: Protein oxidation pathways via A) free radical transfer, B) oxidation,
and C) crosslinking (Adapted from Karel et al., 1975, and Schaich, 2008). PH =
protein, P• = protein radical, AH = any molecule with abstractable hydrogens, A• =
non-protein radical, PO• = alkoxyl radical, POO• = peroxyl radical, POOH =
hydroperoxide, P-CH=O = secondary products such as aldehydes.
Free radical transfer
Protein radicals (P•) are formed when lipid peroxyl and alkoxy radicals arise
from lipid hydroperoxides, and transfer free radicals to proteins by abstracting
hydrogens (Karel et al., 1975) (Figure 1.14A). Protein hydroperoxides (POO•) and
other protein radicals (P•) are highly reactive, and thus oxidize to secondary
compounds (Davies et al., 1995). The peptide bond in the backbone of the protein or
the side-chains of the amino acids may be the target for amino acid modifications.
The oxidative modification can cause cleavage of the protein backbone and cross
linking of the side chains. The reactions are usually highly influenced by redox
cycling metals such as iron and copper. In addition, protein radicals can also transfer
radicals to other proteins, lipids, carbohydrates, vitamins and other molecules,
especially in the presence of metal ions. Radical transfer occurs early in lipid
oxidation, and this process underlies the antioxidant effect for lipids. Consequently, it
may appear that lipid oxidation is not proceeding whereas the radical transfer to
proteins is in its highest (Schaich, 2008).
Oxidation
Backbone fragmentation of proteins occurs via C-C or β-scission that
decarboxylates the target amino acid side-chain during exposure to radicals (radiation,
oxidizing lipids) in the presence of oxygen as shown in Figure 1.14B. For example, β-
scission of alanine, valine, leucine, and aspartic acid side chains generates free
45
formaldehyde, acetone, isobutyraldehyde, and glyoxylic acid, respectively. In each
case, cleavage of the side-chain gives α-carbon radical (-NH •CHCO-) in the
polypeptide chain. This reaction occurs via the formation and subsequent β-scission
of the alkoxyl radical (Headlam et al., 2002).
Cross linking
The general reaction for free radical crosslinking generates usually polymers
of intact protein monomers, with and without oxygen bridges (Figure 1.14C)
(Schaich, 2008). Oxidative modifications of proteins generating intra- and
intermolecular crosslinks can occur by different mechanisms: 1) direct interaction of
two carbon-centered radicals, 2) interaction of two tyrosine radicals, 3) oxidation of
cysteine sulfhydryl groups, 4) interactions of the carbonyl groups of oxidized proteins
with the primary amino groups of lysine side-chains in the same or different protein,
5) reactions of both carbonyl groups of malonaldehyde with two different lysine side-
chain in the same or two different protein molecules, 6) interactions of
glycation/glycoxidation derived protein carbonyls with either a lysine or an arginine
side-chain of the same or a different protein molecule, 7) interaction of a primary
amino group of lysine side-chain with protein aldehydes obtained via Michael
addition reactions with the lipid aldehydes such as 4-hydroxy-2-alkenal (HNE)
(Stadtman et al., 2003; Stadtman, 2006).
1.8.2 Lipid Peroxidation
Lipid peroxidation (LPO) is a natural metabolic process under normal
conditions. It plays an important role in many diseases (Tomoko and Masafumi,
2014). In particular, low-density lipoprotein (LDL) oxidation may be a key step in the
development of atherosclerosis (Stocker and Keaney, 2004). Oxidation of LDL is
essential for its accumulation within the macrophages and for the formation of foam
cells, which can upregulate proatherogenic chemokines and adhesion molecules and
induce interleukin 6 (IL-6), tumor necrosis factor alpha, and C-reactive protein
secretion (Tomoko and Masafumi, 2014). Lipid peroxides are derived from the
oxidation of polyunsaturated fatty acids (PUFA) of membranes and are capable of
further lipid peroxidation by a Free radical chain reaction. The effects of lipid
peroxides i.e, endothelial cell damage, uncontrolled lipid uptake, decreased
46
prostaglandin synthesis and associated thrombogenecity are strongly implicated in the
pathogenesis of atherosclerosis (Onvura et al., 1998).
MDA is one of the most known secondary products of LPO, and it can be
used as a marker of cell membrane injury (Esterbayer et al., 1991). It has been
observed that diabetic patients with CHD have higher levels of MDA than those
diabetic without this disease. It is shown that CVD‟s have also been related to free
radical mediated mechanisms and to LPO (Kesavulu et al., 2001). The levels of LPO
expressed as MDA, which is the product of major chain reactions leading to definite
oxidation of polyunsaturated fatty acids such as linoleic acid and linolenic acid, saves
as reliable marker of LPO (Boaz et al., 2000). The superoxide anion is known to
inactivate enzymes and initiate the damaging chain reactions of LPO (Lenaz, 1998).
There is evidence from epidemiologic studies that implicates a role for LPO in
atherogenesis (Salonen et al., 1997), superoxide and hydrogen peroxide cause
endothelial cytotoxicity and LPO (Epstein, 1998). Serum MDA levels have also been
shown to be elevated in MI. Rumley et al., (2004) reported that LPO may be one
mechanism through which several cardiovascular risk factors (Smoking, serum TG,
low HDL, and low vitamin C) may promote CVD.
LPO occurs when free radicals are generated adjacent to PUFA (such as
arachidonic acid and linolenic acid) in membrane lipids. The reactive radical will
abstract a hydrogen atom from one of the =CH- groups in the FA to generate a carbon
–centered radical within the membrane. This process is particularly easy with the
PUFA. Carbon centered radicals will combine with molecular oxygen and produce
peroxyl radicals. Therefore the net result of one very reactive radical species attack.
Upon the membrane is to convert PUFA into lipid hydro peroxides. The lipid
hydroperoxides tend to migrate away from the hydrophobic interior of the membrane
to the surface, thus disrupting membrane organization. LPO of biological membranes
increases their leakiness to ions and causes damage to transmembrane proteins such as
receptors and enzymes. Lipid hydroperoxides decomposes in the presence of iron and
copper ions to form a wide range of cytotoxic aldehydes such as malonaldehyde
(MDA) and hydroxynonenal which themselves are capable of chemically modifying
proteins and DNA.
47
1.8.3 Role of Nitric Oxide (NO) In CVD
Nitric oxide (NO) is a unique signaling gaseous molecule, which was labeled
as the “molecule of the year” in 1992. NO is a short half life molecule with 6-30sec,
produced by the enzyme known as the Nitric Oxide synthase (NOS), in a reaction
that convert organic oxygen into citrulline and NO.
Arginine +O2 NOS Citrulline + NO
NO is known as a vascular smooth muscle tone controller, it inhibits platelet
activation, modulates apoptosis and inflammatory cell aggregation and activation at
low cone. On the other hand, NO can react with super oxide anion (O2-) to form
ONOO-- which is highly auto toxic the damage of the vascular endothelial is always
followed by vaso constriction, platelet aggregation and inflammatory cell adhesion,
which lead to an increased production of NO and consequently, ONOO-. Associated
with other factors, the over production of NO is one of the most important issues
involved in the development of lipid atherosclerotic plaques (Shaw et al., 2005).
There are three isoforms of the enzyme: neuronal NOS (nNOS), inducible
NOs (iNOS), and endothelial NOS (eNOS). It is now known that each of these
isoforms and cell types. Only nNOS and eNOS are reported to be consecutive
enzymes and are calcium dependent and produce low levels of gas as a cell signaling
molecule (Parvu et al., 2005). iNOS is calcium independent, activated by cytokines
and produce large amounts of gas which can be cytotoxic. NO is released from
endothelial cells in response to stress produced by increasing blood flow and
activation of a variety of receptors, hormones, neurotransmitters and autocoids and
can be inhibited by super oxide anion (Ignarro et al., 2005; Koh et al., 2007).
NO exists as a nitrate form, nitrite and nitrate are measured in serum, plasma
and urine as markers of NO generated (Koh et al., 2007). Physiologically the most
relevant action of NO is to scavenge o2- and other free radicals and also inhibits o2
-
driven fenton reaction and liquid peroxidation and the levels of NO will be increased
under conditions of oxidant injuries. The excess NO reacts rapidly with super oxide
anion to form peroxinitrite, which may be cytotoxic by itself are easily decomposed to
highly reactive and toxic hydroxyl radical and nitrogen dioxide NO2 ( Ravikanth et
al., 2008)
48
NO + O2- → ONOO
-
ONO O- + H
+ → ONOOH
ONOOH → HO-- +NO2
-
NO has been found to exert negative inotropic and negative chronotropic
effect on cardiac muscle cell. There is also evidence for the release of NO along with
other factors from the endocardium and may be involved in the beat to beat regulation
of cardiac function (Shah, 1993). Basal level of NO regulates blood flow in the brain
(Fineman et al., 1991) and gastrointestinal tract (Iwata et al., 1992). Thus NO plays a
key role in basal blood flow regulation and vascular homeostasis (Andre et al., 2005).
Reactive nitrogen species (RNS) are produced in the body during normal
metabolism. RNS are well recognized for playing a dual role as both deleterious and
beneficial species (Marian et al., 2007). Over production of RNS is called nitro0sative
stress (Ridnour et al., 2004). This may occur when the generations of RNS in a system
exceed the system ability to neutralize and eliminate them. Nitrosative stress may
lead to nitrosylation reactions that can alter the structure of proteins and so inhibit
their normal function. Inactivation and nitration of human SOD by fluxes of RNS
have been shown (Verconica et al., 2007). NO is an important regulator and mediator
of numerous processes in the nervous, immune and cardiovascular system.
Several studies revealed that NO interacts with various important
biomolecules resulting in formation of nitrated lipids, nitrated proteins, nitrosamines,
iron nitrosyls etc. Elfering et al., (2004) indicated that the nitration process may occur
as a result of two conditions: (i) a primary sequence that allows the nitration of
tyrosine at the ortho position of the hydroxyl group and (ii) the localization of the
protein at a close to the membrane. Further, NO appears to be involved in DNA
damage, regulation of metabolism and also in several membrane dependent processes.
Membrane transport is a key process which is responsible for various specialized
properties of individual cell types and ultimately responsible for the behavior of cells.
Loss of NO signaling control results in excessive inhibition of respiratory chain
leading to bioenergetic dysfunction (i.e. decreased ATP synthesis) and increased ROS
production.
49
1.9 Antioxidants in CVD
Antioxidants are molecules capable of reducing or preventing the oxidation of
other molecules and terminate the chain reactions by removing free radical
intermediates, and inhibit other oxidation reactions by being oxidized themselves. As
a result, antioxidants are often called reducing agents. These compounds may be
synthesized in the body (endogenous) or obtained from the diet (exogenous)
(Vertuani, 2004). Natural antioxidant manufactured in the body has several cellular
mechanisms that counterbalance the production of ROS, including enzymatic and non
enzymatic pathways (Hayes and McLellan, 1999). It is widely accepted that a plant-
based diet with high intake of fruits, vegetables, and other nutrient-rich plant foods
may reduce the risk of oxidative stress-related diseases (Kensler, et al., 2007).
Endogenous antioxidants include enzymes, coenzymes and sulfur-containing
compounds. Exogenous antioxidants include vitamins C, E, bioflavonoids and
Carotenes to protect the cells, and oxygen systems of the body against ROS, humans
have evolved a highly sophisticated and complex antioxidant protection system. It
involves a variety of components which function interactively and synergistically to
neutralized free radicals (Hennekens and Gaziano, 1993).
Myocardial antioxidants are defined as substances which inhibit and delay the
oxidative damage to sub cellular proteins, carbohydrates, lipids and DNA. Preventive
antioxidant enzymes inside the cells are an important defense against free radicals; the
main enzymatic “scavengers” responsible for the prevention of ROS formation and
oxidation are superoxide dismutase, catalase and glutathione peroxidase and vitamin E.
Superoxide dismutase (SOD) is found in virtually every oxygen-based
organism, and its major function is to catalyze the dismutation of O2. -
to H2O2. This
reaction is generally considered to be the body‟s primarily antioxidant defense
because if prevents further generation of free radicals. In humans, the highest levels of
SOD are formed in the liver, adrenal gland, kidney and spleen (Halliwell, 1996). SOD
is present in the cytoplasm as well as on the endothelial cell surface with either copper
or zinc (Cu SOD, Zn SOD) and in the mitochondria manganese (Mn SOD).
Catalase (CAT) is an iron containing enzyme found primarily in the small
membrane endosed cell components called peroxisomes. It serves to detoxify H2O2
and various other molecules. One way that CAT eliminates H2O2 is by catalyzing a
50
reaction n between two H2O2 molecules, resulting in the formation of H2O and O2. In
addition can promote the interaction of H2O2 compounds, that can be serve as
hydrogen donors, so that the H2O2 can be converted to one molecule of H2O and
the reduced donor becomes oxidized (a process sometimes called the peroxdatic
activity of catalase) (Victor et al., 2006). The liver, kidney and red blood cells posses
high levels of catalase.
Glutathione peroxidase (GPx) system consists of several components
including the enzymes GPx and GSH-reductase (GRx) and the co factors reduce
glutathione (GSH) and reduced nicotinamide adenosine dinucleotide phosphate
(NADPH). Together these molecules effectively remove H2O2 with the formation of
oxidized glutathione (GSSG) (Figure1.15). GSH is an essential component of this
system and serves as a cofactor an enzyme called glutathione transferase, which helps
remove certain drugs and chemicals as well as the reactive molecules from the cells.
Moreover, GSH can interact directly with certain ROS (eg. OH.) to detoxify them, as
well as performing other critical activities in the cell (Victor, 2006).
Figure 1.15: Superoxide dismutase, Catalase, and the GSH peroxidase /GSSG
reductase system
Glutathione - S- transferase (GST) also known as glutathione transferase
found mainly in the cytosol is thought to play a physiological role in initiating the
detoxification of potential alkylating agents (Habig et al., 1974), including
pharmacologically active compounds. These enzyme catalyse the reaction of such
51
compounds with the –SH group of glutathione, there by neutralizing their
electrophilic sites and rendering the products more water- soluble.
SOD and CAT are important antioxidant enzymes in migrating free radical
induced cell injury. A decrease in the activity of SOD and CAT could result in the
decreased removal of superoxide ion and H2O2 radicals, which brings about a number
of reactions which are harmful to the myocardium (Sumitra et al., 2001).
In addition to GSH, numerous other non enzymatic antioxidants are present in
the cells; most prominently vitamin E (α-tocopherol and vitamin C (ascorbate).
vitamin E is a major antioxidant found in the lipid phase of membrane s and act
as a powerful terminator of lipid peroxidation . During the reaction between
vitamin E and a lipid radical , the vitamin E radical is formed from which from
which vitamin E can be regenerated in a reaction involving GSH and ascorbate (Nanji
and Hiller , 1997).
1.10 Diagnosis of MI
The world health organization criteria for diagnosing MI are that the patient
presents with at least two of the following three criteria
1. Clinical symptoms suggestive of Myocardial ischemia
2. Characteristic changes on the electro cardio gram (ECG) and
3. A raise and fall in biochemical markers.
The clinical symptoms can be non specific for about one third of the patients
particularly for those with diabetes and for the elderly who most frequently present a
typical symptoms of the ischemia similarly the ECG is not a perfect evaluation tool,
because its clinical sensitivity for MI is only about 50%. Monitoring changes in
cardiac markers, is consider the bench mark for the diagnosis of MI. Rapid and real
time availability of cardiac markers has become an integral part of most Chest Pain
Evaluation centers (CPEC) protocols, which have cut the rate of missed MI from
4.2% to 0.4%.
Most of diagnostic investigations pivot around hypoxia induced electrical
alteration in the heart i.e. electrocardiogram (ECG) and structural damage of cardiac
myocyte and its sub-cellular organelles with massive efflux of muscle enzymes via
52
creative kinase (CK), asparate transaminate (AST), lactate dehydrogenate (LDH),
myoglobin and troponin assays.
1.10.1 Biomarkers:
The measurement of CK and CK-MB levels has long been used for the
diagnosis of AMI. CK, an enzyme present in many tissues, including the myocardium
and skeletal muscle has three isoenzymes: MM, MB and BB. CK-MB is present in
relatively high concentration in the myocardium (roughly 20% of the total myocardial
CK), whereas the concentration of CK-MM is highest in skeletal system (98% of total
muscle CK) with only a small amount of CK-MB (usually about 2%). However,
healthy skeletal muscle can have up to 5% CK-MB, and higher levels (upto 20%) of
CK-MB can be found in patients with renal failure and chronic myopathic skeletal
muscle injury (as occurs in polymyositis and dermatomysitis) or in the muscle tissue
of trained athelets, although CK-MB constitutes about 20% of the total CK in the
myocardium, it should be noted that CK-MM is still the most abundant CK isozyme
in the myocardial tissue. Elevation of the total CK level is not cardiac specific and
may be observed in patients with skeletal muscle injury and other disorders.
Following myocardial injury, the initial CK-MB risk occurs 4-9 hours after the onset
of chest pain, peaks at 24 hours, and returns to baseline at 48-72 hours. One advantage
of CK-MB over markers that remain elevated for longer periods is that it is easier to
detect reinfarction using serial CK-MB measurement.
LDH activity has also been found reliably elevated in the presence of MI.
Human LDH consists of five distinct components having a characteristic distribution
(Wroblewski, 1963). Serum or plasma of humans contains all five isozymes, the
concentrations diminishing in the order LDH-4, LDH-5, LDH-3, LDH-2 and LDH-1.
Estimation of serum LDH isoenzymes in combination with CK is well-established
laboratory procedure for diagnosing AMI (Lott and Stang, 1980; Lee and Goldman,
1986). As the myocardium has a preponderance of LDH-I with lesser amounts of
LDH-2, necrosis of myocardium resists in release of relatively more LDH-1 than
LDH-2 into the blood, reversing the normal ratio, usually between 12 hours and 24
hours, reaching a peak 48 hours after the infarct (Lott and Stang, 1980 and Lott,
1984). Warburton et al., 1967 described an increased proportion of LDH-3 (above
20%) in serum of 10, of 50 patients with AMI. According to their investigations, the
53
source for the increased LDH-3 proportion was necrotic myocardium. MI is also
characterized by elevation of LDH-4 and LDH-5.
Myocardium contains an abundant concentration of many enzymes and once
metabolically damaged, released its contents into extracellular fluid (Gangualy et al.,
1980). Aminotransferases may be raised in cardiac diseases and hepatic diseases.
Clinical and laboratory studies performed on human subjects have shown that the
serum levels of SGOT rises significantly following AMI (ladie et al., 1954; Karmen et
al., 1955). The aminotransferases are the most frequently utilized and specific
indicators of hepatocellular necrosis. These enzymes AST and ALT catalyze the
transfer of the aminoacid of aspartate and alanine respectively to the keto group
ketoglutaric acid. ALT is primarily in the liver but the AST is present in a wide
variety of tissues like the heart, skeletal muscle, kidney, brain and liver (Rosen and
Keefe, 2000; Friedman et al., 2003). AST is elevated in the serum with hepatic cell
involvement, skeletal muscle fiber inflammation and myocardial cell injury.
Alkaline phosphates (ALP) are a family of zinc metalo enzymes with a serine
at the active center, they release inorganic phosphate from various organic
orthophosphates and are present in nearly all tissues. In liver, ALP is found
histochemically in the microvilli of bile canaliculi and on the sinusoidal surface of
hepatocytes. ALP from the liver, bone and kidneys are thought to be from the same
gene but that from intestine and placenta are derived from different genes (Rosalki
and Mlintyre, 1999). Highest levels of ALP occur in cholestic disorders.
γ -Glutamyl transpeptidase (GGT) is a unique biomarker for cardiac and
metabolic risk evaluation. It is a membrane bound glycoprotein which catalyses the
transfer of γ-glutamyl group to other peptides, aminoacids and water. GGT levels
were first associated with CVD and all cause mortality in a British Regional Heart
study by Wannamethee et al., reported in October of 1995. Large amounts are found
in the kidneys, pancreas, liver, intestine and prostate. Non hepatic causes of increases
levels of the enzymes include anorexia nervosa, gulliian barre syndrome,
hyperthyroidism, obesity and dystrophica myotonica.
54
1.10.2 Membrane bound ATPases:
ATPases are membrane-bound enzymes and any perturbation in the activities
of these enzymes affects membrane status by inflicting changes in
electrophysiological energetics and normal homeostasis. They are intimately
associated with the plasma membrane and participate in the energy requiring
translocation of Na+-K
+, Ca
2+ and Mg
2+. Determination of membrane associated
enzyme activity like ATPases indicates alternations its membrane under pathological
conditions. Na+/K
+ -ATPase is responsible for the generation of the membrane
potential through the active transport of sodium and potassium ions. It simultaneously
transports sodium ions out of the cell and potassium ions into the cell. These are
present at high concentrations in brain, consuming about 40-50% of the ATP
generated in this organ (Erecinska and Silver, 1994). Na+/K
+ -ATPase is implicated in
metabolic energy production as well as in the uptake, storage, and metabolism of
catecholamines, serotonin, and glutamate (Carageorgiou et al. 2007). Ca2+
-ATPase
activity is associated with neuronal excitability, cellular depolarization and fine
tunning of Ca2+-
channel activity (Lees, 1991). Mg2+
- ATPase activity associated with
mitochondrial membrane bound enzyme which is involved in turnover of ATP
synthesis in conjugation with oxidative phosphorylation.
The plasma membrane calcium ATPases are ATP-consuming calmodulin-
dependent pumps which eject Ca2+
into the extracellular space (Cartwright, 2005).
There are four isoforms of PMCA. PMCAs are 134 kD proteins which belong to the
P-type ATPase family of proteins. All four proteins have a similar structure
comprising 10 transmembrane (TM) domains and four major intracellular regions:
The N-terminal region is a region of low sequence similarity between the four
isoforms and its function has not been fully elucidated; the loop between TM domains
2 and 3 is involved in calcium pore function; whilst the very large loop between TM
domains 4 and 5 is the site of ATP binding and contains the aspartate residue which is
phosphorylated during the calcium transport cycle; finally the C-terminal tail contains
the calmodulin binding domain (CaM-BD) which is essential in the regulation of
pump activity. Binding of the CaM-BD to regions on the two large intracellular loops
leads to auto inhibition of the pump and this inhibitory effect is released upon binding
of calmodulin to the CaM-BD. Ca+2
ATPase is the major active Ca2+
transport protein
55
responsible for the maintenance of normal intracellular Ca2+
levels in a variety of cell
types (Rajadhurai et al., 2007).
1.10.3 Serum electrolytes
Electrolytes (electrical conductors) play an important role in many body processes,
such as controlling fluid levels, acid/base balance, nerve conduction, blood clotting and
muscle contraction (including that of the heart). Alterations in electrolyte balance have been
claimed to play a role in the pathophysiology of coronary heart disease; however, the
relationship between the electrolyte pattern and other clinical variables immediately after an
acute vascular event is unclear (Solini and Zamboni, 2006).
Calcium ion is essential for vital body functions. Calcium dependent processes
play a central role in several different cells of the cardiovascular system including
vascular smooth muscle and endothelial cells and also in monocytes, macrophages
and platelets. It is one of the essential factors for excitation- contraction coupling in
the cardiac muscle cell as well as for the conduction of electrical impulses in certain
regions of the heart particularly through AV node. Calcium is critical in the
contractile process. In the resting myocytes Ca 2+
, Na +
exchanger maintain a low level
of free intracellular Ca2+
, which contributes to relaxation but may run in the reverse
direction excitation (Murry et al., 2000). Accumulation of intracellular Ca2+
is a main
event in the final stages of cell death. Ca2+
is thought to inhibit intracellular energy
flow. When present in heart cells, it accumulates in mitochondria, causes uncoupling
of oxidative phosphorylation and leads to decreased ATP production.
Calcium antagonist drugs principally act on L- type calcium channels into the
cells of the body. Calcium antagonist drugs are able to influence a wide range of
cellular process which has been implicated in atherosclerosis, left ventricular
hypertrophy and insulin resistance.
Serum K+ and Na
+ will also play important role in cardiovascular activity
(Nurminen et al., 1998). Adrenaline stimulates the sodium-potassium-ATPase pump
via beta 2-receptors and shifts potassium intracellularly (Brown et al., 1983). The
catecholamine surge that accompanies acute MI (AMI) causes redistributional
hypokalemia and hyperpolarizes non-ischemic myocardium, producing electrical
inhomogeneity and ventricular arrhythmias. Potassium depletion produces diastolic
56
dysfunction in animal and human models (Srivastava and Young, 1995).
Hypokalemia is therefore a common, reversible factor in the natural history of
cardiovascular disease. Resting transmembrane potential difference depends on
intracellular and extracellular potassium concentrations (Table 1.4). The decrease in
the potassium ions causes cellular hyperpolarity, increases resting potential, hastens
depolarization, and increases automaticity and excitability (Gettes and Surawicz,
1968). Because cardiac repolarization relies on potassium influx, hypokalemia
lengthens the action potential and increases QT dispersion (reflecting electrical
inhomogeneity). Hypokalemic ventricular ectopy is suppressed by potassium
replacement (Surawicz and Lepeschkin, 1953). Thus, hypokalemia increases risk of
ventricular arrhythmia and sudden cardiac death.
Table1.4: Experimental evidence on the beneficial effects of Potassium
There is a relationship between body iron overload and pathogenesis of
numerous degenerative diseases, including atherosclerosis (Merono et al., 2011).
Moreover, in experimental models the infusion of trivalent iron salts was shown to
cause diffused thrombosis. It is a common belief that free blood iron, via the Fenton-
like reaction, is responsible for so-called oxidative stress that, in turn, leads to
atherosclerosis and related cardiovascular diseases (Griendling and Gerald, 2003).
According to Lipinski and Pretorius (2012), the trivalent iron ion (FeIII) generates in
aqueous solutions powerful hydroxyl radicals that subsequently modify fibrinogen
molecules converting them to insoluble fibrin-like polymer (Lipinski and Pretorius,
57
2012). It should be emphasized that such a polymer is not only resistant to fibrinolytic
dissolution, but also to proteolytic digestion, i.e. with chymotrypsin, that normally
degrades fibrin(ogen) into smaller polypeptide fragments. The resistance of fibrin
clots to enzymatic degradation can be occur in an alternative iron induced mechanism
of blood coagulation According to this concept free iron of blood (Fe III) generates
hydroxyl radicals, which in turn convert circulating FBG into an insoluble fibrin-like
material (or parafibrin) without the action of thrombin (Lipinski and Ptretorious,
2012). As a consequence such a dense fibrin polymer acquires the features of a
foreign body and attracts macrophages resulting in a permanent state of inflammation
known to be associated with atherosclerosis (Kleemann et al., 2011; Libby, 2012).
Also it is of interest to note the reports on the relationship between inflammation and
blood coagulation. Moreover there are numerous experimental and clinical studies
that indicate the relationship between inflammation, iron overload and cardiovascular
diseases (Menke et al., 2009; Lele et al., 2009).
1.10.4 Myoglobin:
Myoglobin is an oxygen binding protein found in high concentration in both
cardiac and skeletal muscle. Myoglobin, a relatively small protein molecule that is
released into serum as early as one hour after AMI, reaches a peak in the range of 4 –
12 hours and then is rapidly cleared. The major advantage of myoglobin as a cardiac
marker is that it is released earlier from damaged cells than another cardiac marker,
permitting earlier detection of AMI (Vaidya, 1994; Gibler et al., 1987; Adams et al.,
1993; Mair et al., 1995; Mercer, 1997). Rapid release of myoglobin probably reflects
its low molecular weight and cyotoplasmic location. Myoglobin as an early marker of
AMI exhibits a high negative predictive value. The main reason that myoglobin has
not been used by most hospitals for the evaluation of chest pain is its poor clinical
specificity (60% - 90%) owing to the presence of large quantities of myoglobin in
skeletal muscle. Myoglobin, therefore, is potentially useful for ruling out but not for
confirming the diagnosis of AMI. The use of myoglobin for the detection of
reinfarction is complex.
1.10.5 Troponin
Cardiac troponin I (cTn I) and T (cTn T) are the most sensitive and specific
biochemical markers of myocardial cell damage. A consensus document issued by the
58
European and American college of cardiology committee for the redefinition of MI
promoted cardiac troponoin I and T as the preferred markers for myocardial damage
because of their nearly absolute myocardial tissue specificity, high sensitivity, and the
ability of these marker to reflect microscopic zones of myocardial necrosis
(Anonymous, MI redefined, 2000).
Both cTnT and cTnI are stored in a 2-compartment distribution in the
myocyte, including a small cytoplasmic pool (4%-6%), with the majority of the
remaining troponin found in the sarcomere. Thus, TnT and TnI have similar release
kinetics from damaged myocardium. Both troponins increase in serum within 4 to 9
hours after AMI, peak at 12 to 24 hours, and remain elevated for up to 14 days
(Bertinchant et al., 1996 and Wu et al., 1999b). An appropriate testing strategy is
sampling for TnT or TnI at baseline, 8 and 16 hours, which has been demonstrated to
be optimal for the diagnosis of myocardial necrosis (Newby et al., 1998). With
currently available assays, cTnT and cTnI have equal myocardial tissue specificity, as
well as high sensitivity (Wu, 1999a).
1.10.6 Role of Homocysteine and CRP in CVD:
Several plasma biomarkers have been investigated to determine their use as
tools for predicting the risk of CVD including homocysteine and C-reactive protein.
Plasma homocysteine, a sulfohydryl containing a formed during the metabolism of
methionine, is associated with endothelial damage and hypertension (Tayama et al.,
2006). There is evidence that elevated plasma homocystein is associated with CVD
(Bhandari et al., 2008). A number of studies have indicated that
hyperhomocysteinemia promotes atherosclerosis through increased Oxidative Stress,
impaired endothelial function and induction of thrombosis (Bhandari et al., 2008).
Thus screening for homocysteine may be beneficial as another tool for identifying
those at risk for CVD. Homocysteine has now been implicated in increased OS, DNA
damage triggering of apoptosis and excitotoxicity all important mechanisms in neuro
degeneration (Mattson and Shea, 2003) and natural antioxidants play a protective role
in hyperhomocysteinemia (Nappo et al., 1999).
Over the last couple of decades it has been proved that CRP is the most
reliable marker of inflammation. It is 135000D non immunoglobulin protein, having
five identical sub units (Figure1.17). The name of the C- Reactive protein derives
59
from the fact that is reacts with capsular polysaccharides of streptococcus
pneumoniae. Now several reports have shown that CRP concentration are elevated in
the individuals who are at risk of developing coronary artery disease and cerebro
vascular events. Various studies have suggested that CRP levels measured also may
be useful as an independent predictor of new coronary events, including AMI and
death in patients with IHDS (Liuzzo et al., 2001). The prognostic information derived
from troponin and CRP is additive in patients with unstable angina and non – Q-
Wave AMI, negative and low levels of CRP are associated with an approximately 1 %
risk of death at 14 days Vs a 9 % risk for patients with CRP concentrations (Morrow
et al., 1998; Heeschen et al., 2000; Lindahl et al., 2000; Mueller et al., 2002). In
addition, CRP has been shown to promote the uptake of oxidized LDL which could be
relevant to the genesis of the atherosclerotic lesion including plaque instability (Jialal
et al., 2004, Jialal et al., 2006).
1.11 Therapeutics for CVD
Despite this complexity, impressive recent progress has been achieved in
advancing our understanding and appreciation of the cellular processes and
mechanistic bases underlying cardiac dysfunction associated with MI and most
significantly applying this knowledge to therapeutic interventions (Karmazyn, 1996).
As myocardial injury is irreversible in nature, most of the drugs available are effective
in the prevention of spreading or dispersal of necrotic damage to the adjacent cells.
There are a variety of drugs prescribed for patients with heart disease. It's important
for both patients living with heart disease and those who care for them to understand
the prescribed medication, to follow the directions of usage, and to be able to
recognize the possible side effects associated with the medicine. The drugs most
commonly prescribed for heart disease includes anti-coagulants, ACE (Angiotensin
Converting Enzyme) inhibitors, β-blocking agents, calcium agonists, nitrates, statins,
thrombolytic agents, etc are the drugs available for the treatment of MI. Nevertheless,
all these drugs are having their own undesirable effects and limitations which are
summarized in table 1.5
Anti-coagulants: Anticoagulants are often called blood thinners. They help prevent
blood clots from forming and growing and reduce the risk for heart attack, stroke and
blockages in arteries and veins. They cannot, however, break up blood clots that are
60
already formed. Although they are often called blood thinners, anticoagulants do not
really thin the blood, but decrease its ability to clot (coagulate). Anticoagulants are
available in several forms. They can be taken as tablets or given by injection or
intravenously (by IV). Because anticoagulants delay clotting, their major side effect is
unwanted bleeding.
ACE Inhibitors: ACE inhibitors are a type of medication that dilates (widens)
arteries to lower blood pressure and make it easier for the heart to pump blood. They
also block some of the harmful responses of the endocrine system that may occur with
heart failure.
Aldosterone Inhibitor: Spironolactone and eplerenone are potassium-sparing
diuretics. They can be prescribed to reduce the swelling and water build-up caused by
heart failure. Diuretics cause the kidneys to get rid of unneeded water and salt from
the tissues and blood into the urine. They may improve heart failure symptoms that
are still present despite use of other treatments. These drugs protect the heart by
blocking aldosterone in the body that causes salt and fluid build-up. This medication
is used to treat patients with certain types of severe heart failure.
Calcium Channel Blockers or calcium antagonists: Calcium channel blockers are
prescribed to treat angina (chest pain) and high blood pressure. Calcium channel
blockers affect the movement of calcium in the cells of the heart and blood vessels.
As a result, the drugs relax blood vessels and increase the supply of blood and oxygen
to the heart, while reducing its workload. Calcium channel blockers are only used to
treat heart failure caused by high blood pressure when other medications to lower
blood pressure are ineffective. Certain calcium channel blockers are used for certain
types of heart failure.
Beta-Blockers: Beta-blockers block the effects of adrenaline (epinephrine) which can
improve the heart's ability to perform. They also decrease the production of harmful
substances produced by the body in response to heart failure. There is an extensive
literature on the effects of beta blockers on the processes which led to death from
IHD. Thus beta blockers reduce endothelial damage (Frishman et al., 1978). The
cardioprotective role of beta blockers in post infarct patients, in particular their
capacity to reduce the risk of sudden death has been shown for three lipophilic beta
blockers, timolol (anonymous Norwegian study group, 1981), propranolol
61
(Anonymous beta blocker heart attack trail research group, 1982) and metoprolol
(Hjalmarson et al., 1981), but not for the hydrophilic sotalol (Julian et al., 1982).
Therefore, betablockers are the drugs which come closest meeting the crieteria for
being cardioprotective drugs.
Table 1.5: Mechanism, examples and limitations of therapeutics for CVD
MEDICINE MODE OF ACTION EXAMPLES ADVERSE EFFECTS
ACE
inhibitors
Prevent kidneys from
retaining sodium and
water by deactivating
angiotensin-converting
enzyme, which converts
inactive angiotensin I to
the active angiotensin II.
Angiotensin II raises
blood pressure by
triggering sodium and
water retention and
constricting the arteries.
Benazepril
(Lotensin),
Captopril,
Enalapril
(Vasotec)
Cough, rash, fluid
retention,
high potassium levels,
and loss of taste.
May cause low blood
pressure and fainting.
Can worsen kidney
impairment if narrowed
arteries feed both
kidneys.
May cause fetal
abnormalities.
Beta
Blockers
Reduces the myocardial
contractility, conduction
velocity, cardiac output &
decreases heart rate.
Propranolol,
Metoprolol,
Atenolol
Bronchoconstriction,
Bradycardia,
hypoglycemia
Calcium
Antagonist
Binds to α sub unit of
cardiac L type ca+2
channels, thereby prevents
the diffusion of Ca +2
ions
through the pores.
Verapamill,
Nifedipine,
Diltiazem
Headache,
constipation,
ankle oedema
Blood
thinners
Inhibits the platelets
aggregation & prevents
the thrombotic events in
event of MI
Aspirin,
Hypersensitivity,
Increase in bleeding
tendency
Nitrates antianginal action by
increase coronary blood
flow & increase in
content in coronary blood
Glyceryltrinit
rate,
Isosorbidemo
nonitrate
Headache,
hypotension
Statins
Decreases Cholesterol
synthesis by inhibiting
HMG – CoA reductase
Symbastatin,
Lovostatin,
Atorvastatin,
Pravastatin
Head ache,
nausea,
rashes,
insomnia
Fiber acids Incactivity of lipoprotein
lipase, decrease the
release of FA from
adipose tissue
Bezafibrate,
Ciprofibrate,
Fenofibrate,
Clofibrate
Acute renal failures,
myositis,
Myoglobinuria,
62
Cholesterol -Lowering Drugs or Statins: Cholesterol helps the body to build new
cells, insulate nerves, and produce hormones. But inflammation may lead to
cholesterol build-up in the walls of arteries, increasing the risk of heart attack and
stroke. Some people have a genetic predisposition to high cholesterol levels. These
people may need drug therapy in addition to a healthier diet to bring their cholesterol
down to a safe level. Actually said, statins are the inhibitors of 3-hydroxy-3- methyl-
glutaryl-C A reductase (HMGCR). They are the pharmacological group with highest
reduction of the serum LDL-C concentration. Thus statins have become the most
important pharmacological weapon for cardiovascular risk reduction when associated
with atherosclerosis.
Thrombolytic agents: Thrombus formation and plaque rupture are believed to be the
final step in the development of a coronary occlusion. Aspirin (Acetylsalicylic acid) is
known to reduce platelet aggregation, to prevent clotting and to increase thrombus
breakdown, which have been evaluated in clinical studies. Aspirin was reported to
reduce coronary events, since the fatal and non fatal MI were lower in those on aspirin
and anticoagulant warfarin reduced coronary mortality in post infarct patients
(Manson et al., 1991).
Nitrates: Nitrates have been used for more than a century to treat angina. They work
by dilating arteries and veins in the heart and elsewhere in the body. This reduces
stress on the heart muscle which, in turn, reduces the amount of oxygen the heart
requires. This lets the heart function longer without developing ischemia, even when
blood flow through the coronary arteries is partially blocked by atherosclerosis. Some
common side effects of nitrates include headaches, flushing, dizziness, fainting, low
blood pressure (hypotension) and irregular heart rhythms (arrhythmia).
1.12 Cardioprotective Medicinal Plants and Natural Products
In many nations, herbal therapies are among the most popular of all
„alternative‟ treatments. Herbs have been used for many centuries in different
countries for the treatment of heart disease. They use different natural products and
antioxidant vitamins to treat or prevent the hypercholesterolemia, coronary heart
disease, and congestive heart failure (CHF) (Gavagan, 2002). Herbal therapies have
shown promise in terms of efficacy. Recently, the National Institutes of Health (NIH)
63
acknowledged the importance of herbal therapies and developed a strategy regarding
research in this area for cardiovascular conditions.
Modern drugs are effective in the control of CVD, but due to their side effects
their utilization is limited (Stollberger and Finsterer, 2005). So, there is a need to
search for a medicine to treat CVD without any side effects. The prevention of CVD
has been associated with the ingestion of fresh fruits, vegetables rich in antioxidants
(Argolo et al., 2004). Demand for medicinal plants is increasing in both developing
and developed countries due to growing recognition of natural products, being non-
narcotic having no side effects, easily available at affordable price and sometime the
only source available to the poor. Among the entire flora, 35,000 to 75,000 species
have been used for medicinal purposes. In India, of the 17,000 species of higher
plants, 7,500 are known for medicinal uses. This is the highest proportion of
medicinal plants known for their medical purposes in any country of the world for the
existing flora of that respective country. The Indian herbal is one of the prominent
systems to treat diseases without any side effects. Recently, several plants of Indian
origin have been found to possess medicinal properties with their beneficial effects in
ailments like atherosclerosis, ischemia, cancer, diabetes and liver dysfunction
(Vandana and Suresh, 2008).
The traditional systems of medicines – Ayurveda, Siddha and Unani are based
on the experiences in use of plant products in amelioration of common diseases. More
than 2000 plants have been listed in traditional (Herbal/Alternative) systems of
medicine and some of these are providing comprehensive relief to the people
suffering from CVD, especially hyperlipidemia and IHD. WHO reported that around
80% global population still relies on botanical drugs and several herbal medicines
have advanced to clinical use modern times (Mahmood et al., 2010). The use of
Western medical drugs for treatment of hypertension, congestive heart failure and
post MI are widely accepted. For CVD, herbal treatments have been used in patients
with CHF, systolic hypertension, angina pectoris, atherosclerosis, cerebral
insufficiency, venous insufficiency and arrhythmia (Mashour et al., 1998).
Plants show enormous flexibility in synthesizing complex material, which
have no immediate obvious growth and metabolic functions. These complex materials
are referred to as secondary metabolites, which are also referred to as phytochemicals.
64
The phytochemicals include alkaloids, saponins, tannins, anthraquinones, terpenoids,
steroids, flavonoids, cardiac glycosides, etc.
Phytochemicals are naturally occurring and biologically active compounds and
have potential disease inhibiting capabilities. Hence, the medicinal values of these
plants lie on their component phytochemicals, which produce definite physiological
actions on the human body. Constituents of herbal medicines are found in leaves,
flowers, stems, seeds, roots, fruits and bark. Various phytoconstituents from plants
were responsible for cardioprotective activity including carotenoids (Eugenia
uniflora); triterpenes (Ganoderma lucidum); flavonoids (Anacardium occidentale,
Nelumbo nucifera); cardiac glycosides (Digitalis purpurea, Antiaris toxicaria);
alkaloids (Desomodium gangeticum, Erythroxylon coca Tinospora cordifolia);
saponins (Asparagus racemosus, vaccaria pyramidata); terpenoids (Ginkgo bibola);
fatty acids (Elaeis guineensis) (Arya and Gupta, 2011). Herbal medicines have been
given a value status and readily available products for primary health care, and WHO
has endorsed their safe and effective use (WHO Research Guidelines, 1993). Aqueous
extract of Oxalis corniculata showed effective cardio protection in experimental MI
(Abhilash et al., 2011). Saponins from Panax japanicos showed cardio protective
effect on acute myocardial ischemia against oxidative stress triggered damage and
cardiac cell death in rats (Haibo et al., 2012). The water and ethanolic extract of
Salvia miltiorrhiza showed cardio protective effect in an experimental model of MI
(Ru et al., 2012).
1.13 Boerhaavia diffusa
Boerhaavia diffusa (B. diffusa) is a medicinal plant widely used in the
Ayurvedic medicine (Lad, 1999). B. diffusa (Hogweed in English), belonging to the
family, Nyctaginaceae, is mainly a diffused perennial herbaceous creeping weed of
India (its traditional name is Punarnava) and of Brazil (known as Erva tostão).
The plant was named in honour of Hermann Boerhaave, a famous Dutch
physician of the 18th century (Chopra, 1969). B. diffusa (Figure 1.18) is up to 1 m
long or more, having spreading branches. The stem is prostrate, woody or succulent,
cylindrical, hairy, and thickened at its nodes. The plant grows profusely in the rainy
season and mature seeds are formed in October-November. It has a large root system
65
bearing rootlets. The tap root is tuberous, cylindrical to narrowly fusiform, conical or
tapering, light yellow, brown or brownish grey. It is thick, fleshy and very bitter in
taste (Capasso et al., 2000).
The leaves are simple, thick, fleshy, and hairy, arranged in unequal pairs,
green and glabrous above and usually white underneath. The shape of the leaves
varies considerably ovate - oblong, round, or subcordate at the base and smooth
above. The margins of the leaves are smooth, wavy or undulate. The upper surface of
the leaf is green, smooth and glabrous, whereas it is pinkish white and hairy beneath.
The flowers are minute, subcapitate, present 4-10 together in small bracteolate
umbrellas, mainly red or rose, but the white varieties are also known. The achene fruit
is detachable, ovate, oblong, pubescent, five-ribbed and glandular, anthocarpous and
viscid on the ribs (Goyal et al., 2010). The seeds germinate before the onset of the
monsoon.
1.13.1 Traditional Medical Use
The whole plant of B. diffusa have been employed for the treatment of various
disorders in the Ayurvedic herbal medicine (daily used by millions of people in India,
Nepal, Sri Lanka and indirectly through it being the major influence on Unani,
Chinese and Tibetan medicines). The root is mainly used to treat gonorrhea, internal
inflammation of all kinds, dyspepsia, oedema, jaundice, menstrual disorders, anaemia,
liver, gallbladder and kidney disorders, enlargement of spleen, abdominal pain,
abdominal tumours, and cancers (Kirtikar and Basu, 1956), digestive aid, laxative and
a menstrual promoter. The root powder, when mixed with mamira (Thalictrum
foliolosum), is used to treat eye diseases. It cures corneal ulcers and night blindness
(Gupta et al., 1962), and helps restore virility in men. People in tribal areas use it to
hasten childbirth (Shah et al., 1983). The juice of B. diffusa leaves serves as a lotion in
ophthalmia. It is also administered orally as a blood purifier and to relieve muscular
pain (CSIR, 1988).
66
Figure 1.16(a) B. diffusa leaves Figure 1.16(b) B. diffusa flowers
Figure 1.16(c) B. diffusa fruits Figure 1.16(d) B. diffusa roots
Figure1.16: Boerhaavia diffusa
1.13.2 Ethnobotanical Use
Various parts of B. diffusa are used for the treatment of numerous disorders in
different parts of India. The most interesting metabolites from the therapeutic point of
view are the rotenoids (known as boeravinones A - F) (Mishra and Tewari, 1971; Jain
and Khanna, 1989; Kadota et al., 1989; Lami et al., 1990; 1992). In Purulia (West
Bengal) the tribes eat this plant as a vegetable. Boerhaavia leaves are cooked and
eaten in Assam.
1.13.3 Pharmacological and Clinical Properties
The first pharmacological studies have demonstrated that the root of
Punarnava exhibits a wide range of properties: anti-inflammatory (Bhalla et al., 1968,
1971), diuretic (Gaitonde et al., 1974), laxative (Chopra et al. 1956), antiurethritis
(Nadkarni, 1976), anticonvulsant (Adesina, 1979), antinematodal (Vijayalakshmi et
67
al., 1979), antifibrinolytic (Jain and Khanna, 1989), antibacterial (Olukoya et al.,
1993), antihepatotoxic (Das and Agarwal, 2011; Chandan et al., 1991; Rawat et al.,
1997), anthelmintic, febrifuge, antileprotic, antiasthmatic, antiscabby and antistress
activities. An aqueous extract of thinner roots of B. diffusa at a dose of 2 mg/kg
exhibited the remarkable protection of various enzymes such as serum glutamic-
oxaloacetic transaminase, serum glutamic pyruvic transaminase, and bilirubin in
serum against hepatic injury in rats (Rawat et al., 1997).
Maximum diuretic and anti-inflammatory activities of Punarnava have been
observed in samples collected during the rainy season. Due to the combination of
these two activities, Punarnava is regarded therapeutically highly efficacious for the
treatment of renal inflammatory diseases and common clinical problems such as
nephritic syndrome, oedema, and ascites developing at the early onset of the liver
cirrhosis and chronic peritonitis. The root is used to treat other renal ailments
(calculations and cystitis), seminal weakness and blood pressure (Kuldeep and
Mishra, 2011) and as a diuretic (Singh et al., 1992; Anand, 1995). It is also used in the
treatment of stomach ache, anaemia, cough, and cold, and as a diaphoretic, laxative,
expectorant and a potent antidote for snake and rat bites. Punarnava is useful in the
treatment of nephritic syndrome (Singh and Udupa, 1972), hepatitis, gall bladder
abnormalities and urinary disorders (Mudgal, 1975; Cruz, 1995).
The plant was reported to be efficient for the treatment of the abdominal
tumours and cancers and as a growth promoter for the children whome fed with milk
fortified with the plant drug. In the form of a powder or an aqueous decoction, the
plant drug was proved to be beneficial in the treatment of nephritic syndrome and
compared well with corticosteroids. It was also demonstrated that the drug decreased
the albumin urea, increased the serum protein and lowered serum cholesterol level
(Ramabhimaiah et al., 1984).
Singh and Udupa (1972) reported that the dried root powder showed curative
efficiency when administered orally for one month to the children or adults suffering
from the helminth infection. The patients became worm-free within five days of the
treatment. The drug, singly or in combination with other drugs, was found to be
efficient in liver disorders, gastrointestinal disorders, heart diseases (hypertension,
angina, cardiac failure, etc.), respiratory tract infections, leukorrhea, spermatorrhea,
68
etc. The purified glycoprotein from B. diffusa exhibited strong antimicrobial activity
against RNA bacteriophages (Awasthi and Menzel, 1986).
Figure 1.17: Therapeutic properties of Boerhaavia diffusa
Chakraborti and Handa (1989) also reported a hepatoprotective activity of the
aerial parts of B. diffusa. The hepatoprotective activity of the B. diffusa root was
demonstrated by Rawat (1997) and by Chandan (1991) too. These investigators found
that the watery extract from the root of B. diffusa minimised the toxic effects
generated by the CCl4 and the thioacetamide in the liver. Further experimental studies
also evidenced a beneficial activity of the Punarnava root for the treatment of the
jaundice (Singh and Pandey, 1980; Gopal and Shah, 1985).
The treatment with the watery extract from the root of B. diffusa induced
leucocytosis with predominant neutrophils, associated to the phagocytosis ability and
it was bactericidal to the neutrophils and the macrophages (Mungantiwar et al., 1997).
The recent study carried out by Pari et al., (2004) demonstrated that the leaves of B.
diffusa reduced the levels of glucose in the blood increasing the insulin release from
the β cells of pancreas. The watery extract of B. diffusa was proved to possess
69
protective abilities to the rodents suffering from the peritonitis induced by
Escherichia coli (Hiruma-Lima et al., 2000). It was evidenced that the leaves and root
possessed antifibrinolitic and anti-inflammatory activities (Hiruma-Lima, 2000).
Mehrotra et al. (2002) reported that the etanolic extract of B. diffusa showed a
significant immunosuppressive activity on human cells and on murine cells as well.
Toxicological studies conducted on B. diffusa demonstrated the absence of teratogenic
and mutagenic effects (Singh et al., 1991).
The B. diffusa plant contains a large number of compounds such as flavonoids,
rotanoids, alkaloids, steroids, triterpenoids (Kadota et al., 1989; Lami et al., 1990;
Jain and Khanna 1989). In a preliminary screening, plants revealed presence of
sterols (Singh and Udupa, 1972), β-sitosterol (Srivastava et al., 1972) and alkaloids
(Garg et al., 1980). Presence of steroids, sugars and alkaloids were also reported
(Shukla, 1982). It contains about 0.04% of alkaloid known as Punarnavine
(C17H22N2O) (Surange and Pendse, 1972) and punarnavoside, an anti fibrinolytic
agent. It also contains about 6 % of Potassium Nitrate, an oily substance and ursolic
acid (kokate et al., 2005). Hentry acontane, a β-sitosterol and ursolic acid along with
glucose, fructose and sucrose were isolated from the root (Misra and Tiwari, 1971).
A new C-methyl flavones characterized as 5,7-dihydroxy-6-8-dimethoxy flavones
was reported from root (Gupta and Ahmed, 1984) and designated as boerhavone
(Ahmed and Yu,1992). Many rotenoids have been isolated from the roots of plant
(Kadota et al., 1989, Lami et al., 1990).
4 new compounds were isolated from B. diffusa namely (i) eupalitin 3-O- β-
D-galactopyranoside) (ii) 3,31,5-trihydroxy-7-methoxy flavones (iii) 4,7-dihydroxy-3-
methyl flavone and (iv) 3,4 dimethoxy phenyl-1-O- β-D-apiofuranosyl-D-
glucopyranoside. Two known lignans namely Liriodrin and Syringaresinol mono- β-
D-glycoside have been isolated (Lami et al 1991a). Two quinolizidine alkaloids
identified as Punarnavine-I and Punarnavine-II were isolated from roots, stem and
leaves. The alkaloidal content was initially low during commencement of pre-
reproductive phase, gradually increased in different plant parts, becoming maximum
during termination phase of reproductive stage (Nandi and Chatterjee, 1974).
70
Figure 1.18: Main phytoconstituents of B. diffusa
71
1.14 Isoproterenol induced MI
Isoproterenol (ISPH) is an L-β- (3, 4-dihydroxyphenyl)-or-isopropyl amino
ethanol hydrochloride with a molecular formula of C11H17NO3.HCl. Its molecular
weight is 247.7. The hydrochloride salt of ISPH is a white crystalline powder of
melting point 170-171 °C (Figure1.19). It is soluble in water and ethanol. It is a
synthetic catecholamine and β adrenergic agonist by its positive inotropic and
chronotropic actions that causes severe stress in the myocardium resulting in infarct
like necrosis of the heart muscle (Meenakshi, 2014). The rat model of ISPH induced
myocardial necrosis serves as well accepted standardized model to evaluate several
cardiac dysfunctions and to study the efficacy of various natural and synthetic
cardioprotective agents (Upaganlawar et al., 2011).
Figure 1.19: Structure of Isoproterenol
Mechanism of action and biological effects of ISPH
ISPH is a synthetic β-adrenergic agonist and have been used for the induction
of MI. The pathophysiological changes associated with MI induced by ISPH mimics
to a greater extent with those occurring in humans (Ravichandran et al., 1990). ISPH
is a β- adrenergic receptor agonist that increases cytosolic cAMP. In the case of B-
adrenergic agonist action, the circulating hormones or drug is the first “messenger”,
interacting with 5-adrenergic receptor on the external surface of the target cells
(Rendon and Lopez, 2001). The drug hormone receptor complex activates the enzyme
adenyl cyclase on the internal surface of the plasma membrane of the target cells. This
accelerates the intracellular formation of cyclic adenosine monophosphate (cyclic
AMP), the second “messenger” which then stimulates or inhibits various metabolic or
72
physiological processes (Robison et al., 1968; Motulsky and lnsel, 1982). Several
studies have investigated the molecular and cellular mechanism of ISPH induced cell
injury of the myocardium (Chagoya De Sanchez et al., 1997; Curti et al., 1990;
Capozza et al., 1992; Rendon and Lopez, 2000; Kondo et al., 1987). Among these, the
investigation by Chagoya De Sanchez et al. (1997) establishes a long-term, integrated
model of ISPH induced myocardial cell damage encompassing structural, biochemical
and physiological aspects.
ISPH is reported to increase lipolysis (Mohan and Bloom, 1999) and this may
play a role in ISPH induced myocardial necrosis. Hypertriglyceridemia and high
levels of ester cholesterol in serum and heart tissue are the major factors responsible
for the altered cardiovascular functions during ISPH induced MI (Freedmann et al.,
1988). Accelerated degradation of membrane phospholipids by phospholipase and
lysophospholipase has also been proposed to be related to membrane dysfunction and
irreversible ischemic injury (Farber and Young, 1981).
The administration of ISPH increases the activities of myocardial cholesterol
ester synthetase and triglyceride lipase with simultaneous decline in the activities of
cholesterol ester hydrolase and lipoprotein lipase. Accumulation of ester cholesterol
occurs when the rate of esterification by cholesterol ester synthetase exceeds the rate
of hydrolysis, which in turn results in myocardial membrane damage. Peroxidation of
endogenous lipid is a major factor in the cytotoxic action of ISPH (Namikawa et al.,
1992). A growing body of evidence is emerging which suggests that reactive oxygen-
derived free radicals play a crucial role in the pathogenesis of ISPH induced MI
(Nirmala and Puvanakrishnan, 1994).
The ISPH induced alterations in experimental animals includes increase in
heart weight, marked electrocardiographic changes, increase in the level of serum
marker enzymes and lipid peroxides and decrease in the levels of antioxidants
(Manikandan et al., 2002). lsoproterenol administration produces a marked increase in
CPK, LDH, phospholipase and significant decrease in cardiac glycogen, ATP,
creatine phosphate and phospholipid level (Kaul and Kapoor, 1989).
73
A considerable body of clinical and experimental evidence now exists
suggesting the involvement of free radical mediated oxidative process in the
pathogenesis of ISPH induced MI (Nirmala and Puvanakrishnan, 1996b). Alterations
in tissue defense systems including chemical scavengers or antioxidant molecules and
the enzymes catalase, superoxide dismutase, and glutathione peroxidase have been
reported in ISPH induced MI (Sathish et a1, 2003; Sheela and Shyamala devi, 2000).
The administration of ISPH produces necrotic lesions in the myocardium and
increases lipid peroxidation in the cardiac tissue, which plays a significant part in the
pathogenesis of MI (Noronha-Dutra et al., 1985; Singal et al., 1982 and l983). A
significant depletion of cardiac glutathione (GSH) has been reported in ISPH induced
MI in rats (Nirmala and Puvanakrishnan, 1996). Depletion of GSH is known to result
in enhanced lipid peroxidation and excessive lipid peroxidation can cause increased
GSH consumption and increase the susceptibility of the myocardial cells to reactive
oxygen metabolites (Meister, 1988). GSH and GSH-dependent antioxidant enzyme
systems are directly related to the pathogenic mechanism of ISPH induced MI
(Remiao et al., 2000).
Figure1.20: Mechanism of induction of myocardial injury by ISPH
74
AIM AND OBJECTIVES
2. Aim and Objectives
Aim and Scope
Despite the wide therapeutical use of B. diffusa in Ayurvedic medicine, there
is still no scientific data in the literature which clearly demonstrate the
cardioprotective activity. Hence it is aimed to investigate and validate the ethnical
medicinal uses of the B. diffusa (Linn) for its cardioprotective activity.
Objectives of the study:
To prepare the ethanolic extract from whole plant of B. diffusa
To isolate the total Alkaloids from whole plant of B. diffusa
To identify the phytochemical constituents of ethanolic extract from whole
plant of B. diffusa.
To estimate the in vitro antioxidant activity of the ethanolic extract of B.
diffusa
To identify the phytochemical constituents of total alkaloids of B. diffusa by
performing GC-MS
To measure the body weights and heart weights in control and experimental
rats.
To estimate the activity of cardiac marker enzymes like AST, ALT, ALP, CK,
LDH, and GGT in serum and heart tissues of normal and experimental rats.
To study lipid profile parameters like total cholesterol, phospholipids, free
fatty acids triglycerides, HDL-C, LDL-C and VLDL-C in serum and heart
tissues.
To measure the extent of lipid peroxidation, protein oxidation and NO in
serum and heart tissues of normal and experimental rats.
To confirm the genoprotective activity of B. diffusa by measuring DNA
damage using comet assay.
75
To estimate the content of antioxidant GSH, vitamin C and the activity of
antioxidant enzymes like GPx, GST, GRx, CAT and SOD in heart tissues.
To analyse the electrolytes such as sodium, potassium, calcium and iron in
serum of normal and experimental rats.
To estimate the activity of membrane bound enzymes- sodium potassium (Na+
K+) ATPase, magnesium (Mg
2+) ATPase and calcium (Ca
2+) ATPase in the
heart tissue of normal and experimental rats.
To estimate the activity of LCAT in serum
To estimate the activity of lipid metabolising enzymes such as LPL and
HMGCR in liver tissues of normal and experimental rats.
To analyse the docking studies of the B. diffusa active constituent i.e.,
punarnavoside with the regulatory enzyme HMGCR.
To carryout histopathological studies of heart tissue isolated from normal and
experimental rats by simple light microscopy.
76
MATERIALS AND METHODS
3. Materials and Methods
3.1 Collection of Plant Material
The whole plant of fresh Boerhaavia diffusa was collected in the month of
august at Bramhadevum village of Anantapur district, Andhrapradesh. The whole
plant containing fresh leaves, stem, root, flowers and fruits were thoroughly washed
for 3 times under tap water and finally with distilled water. The plants were cut into
small pieces, shade dried, and homogenized to a fine powder and stored in airtight
bottles for isolation of total alkaloids and extraction with ethanol.
3.2 Preparation of Extracts
A) Preparation of ethanolic extract
The shade dried powder from whole plant of B. diffusa Linn., (3.0kg) was
extracted with ethanol at room temperature for 16 h for 5 times (5 X 4L). The
combined extracts were evaporated under vacuum using rotavapor at 40 0C.
B) Isolation of total Alkaloids
Total alkaloids was isolated from the 5.0kg of the shade dried powder from
whole plant of B. diffusa Linn., by the modified method of Cordell (1981). The total
alkaloids obtained was 3.416gms i.e., 0.069%, stored at room temperature and
protected from sun light until use.
3.3 Qualitative Screening for Phytochemicals
The alcoholic extract was qualitatively tested for the presence of various
phytochemical constituents (Brain and Tunfer, 1975; Harborne, 1976; Sofowora,
1982; Trease and Evans, 1983).
a) Alkaloids
The alcoholic extract was tested for the presence of alkaloids using the
procedures of Smolenski et al. (1972). A portion of the alcoholic extract was made
alkaline with 10 % ammonium hydroxide and treated with ether. The ether extract
was extracted with 10 % hydrochloric acid and the acidic alcoholic solution was
collected and tested for alkaloids. The resulting acidic solution was divided into three
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portions. Of these, two portions were tested for alkaloids by adding Mayer‟s reagent
(1.35 g HgCl2 and 5 g KI in 100 ml distilled water), and Wagner‟s reagent (2 % KI in
distilled water) respectively, while the third was used as blank. The formation of a
faint turbidity or a precipitate after the addition of the above reagents indicates the
presence of alkaloids.
b) Amino acids:
To the extract, 0.25% w/v ninhydrin reagent was added and boiled for few
minutes. Formation of blue colour indicated the presence of aminoacids.
c) Anthracene glycosides
Presence of anthracene glycosides was tested by Borntrager‟s test (Peyer,
1931). To 1.0 ml of the alcoholic extract, 1.0 ml of chloroform was added and the
separated chloroform layer was collected. To this, an equal volume of 2.5 %
ammonium hydroxide solution was added. Appearance of red colour indicates the
presence of anthracene glycosides.
d) Anthraquinones
The ethonolic extract was extracted into 0.5% potassium hydroxide. To the
alkaline extract 1.0ml of hydrogen peroxide, 1.0ml of acetic acid and 1.0ml of
benzene were added. The mixture was treated with an equal amount of dilute
ammonia. Appearance of red colour in the ammonia layer indicates the presence of
anthraquinones.
e) Carbohydrates
i) Molisch’s Test: To 3 ml of extract, 2 drops of freshly prepared 20% alcoholic
solution of alpha napthol was added and mixed. To this solution, 2 ml of concentrated
sulphuric acid was added, so as to form a layer below the mixture. Formation of
reddish violet colour ring at the junction of the solution and its disappearance on
addition of excess solution indicated the presence of carbohydrates.
ii) Fehling’s test: To 2 ml of extract, 1 ml of equal parts of Fehling solution A and B
was added. The contents were boiled for few minutes. Formation of red or brick red
precipitate indicated the presence of carbohydrate.
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f) Carboxylic acids
1 ml of the alcoholic extract was treated with a few ml of Sodium Bicarbonate
solution. Effervescence due to liberation of CO2 indicates the presence of carboxylic
acids.
g) Coumarins
The ethonolic extract was tested for the presence of coumarins (Casparis,
1944). 1.0 ml of ethonolic extract was treated with alcoholic sodium hydroxide.
Appearence of dark yellow colour indicates the presence of coumarins.
h) Flavonoids
To 5.0 ml of the alcoholic extract, 1.0 ml of alcohol was added and subjected
to the following tests:
Ferric chloride test: To 1.0 ml of the above solution, 2-3 drops of
neutral ferric chloride solution was added. Appearance of blackish
red colour indicates the presence of flavonoids.
Lead acetate test: To 1.0 ml of the above alcoholic solution, a few
drops of alcoholic basic lead acetate solution was added. Formation of
reddish brown bulky precipitate indicates the presence of flavonoids.
i) Gallic-tannins and Catecholic compounds
To 1.0 ml of the alcoholic extract, 2.0 ml of ethanol and 2-3 drops of dilute
ferric chloride solution were added. Formation of bluish black colour indicates the
presence of gallic-tannins, while greenish black colour indicates the presence of
catecholic compounds.
j) Phenols
One ml of the alcoholic extract was treated with a few ml of neutral ferric
chloride solution. A dark green color indicates the presence of phenols.
k) Resins
1 ml of the alcoholic extract was treated with a few drops of acetic anhydride
solution followed by 1.0 ml of concentrated sulphuric acid. Appearance of orange to
yellow colour indicates the presence of resins.
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l) Saponins
The alcoholic extract was tested for the presence of saponins (Cambie et al.,
1961). 5 ml of the ethanol extract was mixed with 10 ml of distilled water and
agitated in a graduated cylinder for 15 min. Formation of persistent foam indicates
the presence of saponins.
m) Steroids and Triterpenoids
The alcoholic extract was tested for the presence of steroids and triterpenoids
by Libermann-Burchard test (Harborne, 1976). The ethanol extract was dissolved in
5.0 ml of chloroform and subjected to the following tests:
Salwoski test: To 1.0 ml of the above solution, 1.0 ml of concentrated
sulphuric acid was added, mixed and allowed to stand for 5 min. Appearance of
golden yellow colour in lower layer indicates the presence of steroids.
Libermann-Burchard Test: 1 ml of the above solution was treated with a few
ml of acetic anhydride and 1.0 ml concentrated sulphuric acid from the sides of the
test tube and allowed to stand for 5 min. Formation of brown ring at the junction of
the two layers and appearance of green colour in the upper layer indicates the
presence of steroids.
n) Terpenoids
1.0 ml of ethonolic extract was treated with 5 ml of 1% aqueous hydrochloric
acid. After 3-6 hours, the extract was treated with 1 ml of Trim-Hill reagent (10 ml of
acetic acid, 1.0 ml of 0.2% copper sulphate in water and 0.5ml of concentrated
hydrochloric acid) and heated in a boiling water bath. The appearance of blue colour
indicates the presence of aucubins (diterpinoids) while green colour indicates the
presence of iridoids (monoterpinoids).
3.4 Gas Chromatography– Mass Spectroscopy (GC-MS)
Gas chromatography is generally used as an analytical tool to separate small
quantities of compounds. Acquisition method is Gas Chromatography Electron
impact Mass Spectroscopy (GCEIMS). Gas Chromatograph instrument (Agilent 6890
with 5973N MSD, USA) consists of a fused silica capillary column with 30m length
and 0.25μm thickness was employed for analysis. The column is located in an
insulated oven with adjustable temperature controls. Chromatograph was produced
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maintaining by the oven temperature in the range of 50°C to 280°C at a temperature
rise of 10°c/min. helium was used as sweep was held constant at 1.0 ml/min. Total
run time was adjusted to 15min. quantification and retention time (RT) were
determined by using spectra physics SP 4290 integrator.
Injecting the sample
1.0μL sample dissolved in equal volumes methanol and dichloromethane is
injected using micro hypodermic syringe through the silicone rubber septum into the
vaporizing chamber.
Quantitative analysis by GC
GC analysis shows the number of components in a sample, their retention time
and approximate boiling points. The time taken for a particular component to pass
through the column is called the component RT. The RT is a function of the physical
properties of the compound, the rate of gas flow, the temperature, the liquid phase and
the length and diameter of the column. RT is measured from the point of the injection
of the sample to the appearance of peak and is usually reported in minutes.
Mass Spectroscopy (MS)
Mass spectral analysis was run by Electron impact Ionization (EI) at 70ev and
Mass selective detector (MSD). Mass spectrometer separates the individual atoms or
molecules on the basis of the difference in their masses. Mass Spectroscopy is used to
characterize organic molecules in two principle ways:
i. To measure exact molecular weights and from this exact molecular formulae can
be determined.
ii. To indicate within a molecule the points at which it prefers to fragment; from
this, the presence of certain structural units in the organic compounds can be
recognized.
The graphic representation of the mass spectrum of a compound is constructed
by plotting mass/charge ratio (m/z) versus relative abundance or percentage of base
peak, where the base peak is the most intense peak in the spectrum. Mass spectrum
contains of peaks and each peak corresponds to a set of ions of particular m/z value.
In the mass spectrum, the parent peak is the peak of highest mass number
(except for the isotope peaks). This parent peak gives the exact molecular weight of
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the sample from which the molecular formula can be inferred. Since, the intensity
i.e., the height of the peak is proportional to the concentration of the ion giving that
peak and, the parent peak in the spectrum is, often less intense or quite small.
Components identification and quantification
Identification and quantification of the individual components were
accomplished with the aid of various interpretative techniques as well as by
interpretation of the spectra. The computer techniques include a comparison of
unknown spectra with collection of authentic and spectra and plotting of relative
intensities of significant masses of components, mass chromatograms to structural
isomers of compounds or to detect some minor components and the compounds
hidden by other components in a completely separated gas chromatographic peak.
The intensities of different peaks in mass spectrum of a mixture of compounds
are compared with that of pure compounds to get the concentration of each
component i.e. compound of the mixture and thus, the composition of mixture can be
determined quantitatively. The different constituents were identified by matching their
spectral and retention indices data with those reported in the authentic spectra
(Stenhagan et al., 1974; Masada, 1976; Adams, 1989).
3.5 In Vitro Antioxidant Studies
3.5.1 Screening for Inhibition of Lipid Peroxide Formation
Lipid peroxidation (LPO) was induced by Fe3+
-ADP-ascorbate system in rat
liver homogenate by the method of Sujioka et al. (1987) and extent of LPO was
estimated by the method of Utley et al. (1967).
Rat liver homogenate preparation: 10% liver homogenate is prepared in 0.15
M KCl from normal control rats.
The reaction mixture, containing 1.0 ml of rat liver homogenate, 0.3 ml of 1
mM ferric chloride, 0.3 ml of 17 mM ADP-Na2, 0.4 ml of 3.75 mM ascorbic acid, 0.2
ml of BDEEWP at different concentrations (150 to 3000 µg) and 0.8 ml of 0.15 M
KCl, was incubated at 37°C for 20 min. A system with distilled water, devoid of
extract, served as the control. One ml from each of test, and control was treated with
4.0 ml of 0.67 % TBA, 2.0 ml of 10 % TCA and heated in a boiling water bath for 30
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min. After cooling, the absorbance of supernatant was read at 535 nm and the per
cent inhibition of LPO was determined by comparing with control.
The ability of BDEEWP to inhibit LPO was compared with Curcumin as a
standard. Curcumin, a powerful antioxidant of plant origin, is an orange-yellow
crystalline powder which is the most important fraction responsible for the biological
activities of turmeric. Turmeric is a spice which is obtained from rhizomes of plant
Curcuma longa, a member of the family Zingaberaceae.
3.5.2 Hydroxyl Radical Scavenging Activity
Hydroxyl radicals are generated by a mixture of Fe3+
, ascorbate, and H2O2 in
the presence of slight molar excess of EDTA over the Fe3+
salt (Gutteridge, 1981;
1987). The hydroxyl radicals attack the deoxyribose and set off a series of reactions
that eventually result in the formation of MDA, measured as a pink MDA-TBA
chromogen at 535 nm. Hydroxyl radical scavenging was measured by studying the
competition between deoxyribose and the test compounds for hydroxyl radicals
generated as adapted by Halliwell and Gutteridge (1981).
To 0.5 ml of 40 mM Potassium phosphate buffer (pH 7.4), 0.3 ml of freshly
prepared Fenton reaction mixture (16.2 mg of FeCl3, 38.7 mg of EDTA, 17.2 mg of
ascorbic acid dissolved in 99.96 ml of deaerated distilled water and 0.34 ml of H2O2),
0.1 ml of 28 mM 2-Deoxy-D-ribose and 0.1 ml of extract with different
concentrations (200 to 1000 µg) were added and incubated at 37°C for 1 h. Control
was maintained by substituting BDEEWP in distilled water. One ml each from test
and control were treated with 1.0 ml of 0.67 % TBA and heated in a boiling water
bath for 30 min. After cooling, absorbance was read at 535 nm and per cent inhibition
of hydroxyl radical formation was determined by comparing the optical density of
treatments with that of the control. The hydroxyl radical scavenging activity of
BDEEWP was compared with curcumin as the standard.
3.5.3 Superoxide Radical Scavenging Activity
Measurement of superoxide anion scavenging activity of BDEEWP was based
on the method described by Liu et al. (1997) as modified by Oktay et al. (2003).
Superoxide radicals are generated in PMS-NADH systems by oxidation of NADH
and assayed by the reduction of NBT.
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The reaction mixture contained 1.5 ml of 32 mM Tris-HCl buffer (pH 8.0), 0.5
ml of 300 µM NBT, 0.5 ml of 468 µM NADH and 0.5 ml of extract at different
concentrations (50-500 µg). The reaction was initiated by the addition of 1.0 ml of
30 µM PMS and incubated at 25°C for 5 min. The absorbance was measured at 560
nm against buffer blank. System devoid of the extract served as control. Decrease in
absorbance of the reaction mixture indicates the presence of superoxide anion
scavenging activity. The percentage of inhibition of superoxide radicals was
determined by comparing the optical density of treatments with that of the control.
Superoxide radical scavenging activity of BDEEWP was compared with curcumin as
the standard.
3.5.4 Nitric Oxide Radical Scavenging Activity
Nitric oxide, generated from sodium nitroprusside in aqueous solution at
physiological pH, interacts with oxygen to produce nitrite ions which were measured
by Griess reaction as adapted by Green et al. (1982) and Marcocci (1994).
The reaction mixture containing 1.0 ml of 30 mM sodium nitroprusside, 1.5
ml of 10 mM Tris-HCl buffer (pH 7.4) prepared in saline and 0.5 ml of extract with
different concentration (10 to 1000 µg) was incubated at 25°C for 150 min. After
incubation 1.0 ml of the reaction mixture was treated with 1.0 ml of Griess reagent
(250 mg of sulfanilamide and 25 mg of naphthylethylene diamine dihydrochloride
dissolved in a few ml of water. To which, 0.5 ml of orthophosphoric acid (H3PO4)
was added and the volume was made up to 25 ml with water) and the absorbance of
the chromophore was measured at 546 nm against the buffer blank. The system
devoid of the extract was served as control and the percentage of inhibition of nitric
oxide radicals was determined by comparing the optical density of treatments with
that of the control. The nitric oxide radical scavenging activity of BDEEWP was
compared with curcumin as the standard.
3.5.5 Hydrogen Peroxide Scavenging Activity
The ability of the extract to scavenge H2O2 was determined according to the
method of Ruch et al. (1989).
Different concentrations (10 to 1000 µg) of the extract in 1.5 ml of distilled
water were added to 0.6 ml of buffered H2O2 (40 mM H2O2 in 20 mM Phosphate
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buffer, pH 7.4) solution. Absorbance of H2O2 at 230 nm was measured after 10 min
against buffer blank. System devoid of the extract was chosen as control. The
percentage of scavenging of H2O2 was determined by comparing the absorbance of
treatments with that of the control. Hydrogen peroxide scavenging activity of
BDEEWP was compared with curcumin as the standard.
3.5.6 Reducing Power
The reducing power of extract was determined according to the method of
Oyaizu (1986). Potassium ferricyanide complex gets reduced to reddish brown
colored ferrous form with absorbance maxima at 700 nm.
The different concentrations of the BDEEWP (50 to 500 µg) in 1.0 ml of
distilled water were mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6), 2.5 ml of
1 % potassium ferricyanide and incubated at 50°C for 20 min. 2.5 ml of 10 % TCA
was added, centrifuged at 1000 x g for 10 min. To 2.5 ml of upper layer, 2.5 ml of
distilled water and 0.5 ml of 0.1 % FeCl3 were added. Systems devoid of extract were
chosen as control. The absorbance was measured at 700 nm. The reducing power was
determined by comparing the absorbance of treatment with that of the control.
3.5.7 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) Radical Scavenging Capacity
The free radical scavenging activity of BDEEWP was measured by using
DPPH, following the method of Okada and Okada (1998). Decrease in absorbance of
the reaction mixture indicates the presence of free radical scavenging activity.
Different concentrations of the extract (50 to 600 µg) in 0.3 ml of distilled
water mixed with 2.7 ml of 1.0 mM DPPH (prepared in 99% ethanol) solution and
shaken vigorously were allowed to stand at room temperature for 30 min. Systems
devoid of extract were considered as control. The absorbance was measured at 517
nm. The free radical scavenging capacity was determined by comparing the
absorbance of treatment with that of the control. DPPH radical scavenging activity of
BDEEWP was compared with curcumin as the standard.
3.6 Animal Ethical Committee Clearance
Local Institutional Animal Ethical Committee of our University obtained
ethical clearance for conducting experiments on animals from the Committee for the
85
Purpose of Control and Supervision of Experiments on Animals (CPCSEA) (Regd.
no. 470/01/a/CPCSEA, dt.24th
Aug 2001). The present work was carried out with a
prior permission from the Local Institutional Animal Ethical Committee.
3.7 Procurement of Animals and Maintenance
Male albino Wistar rats of 1 to 2 months of age and weighing 150 to 180 g
were procured from Sri Venkateswara Enterprises, Bangalore. Animals were
maintained as per the guidelines of NIN Animal User‟s Manual. Animals were
acclimatized to our animal house, maintained at temperature of 22 to ± 2οC for 7 days.
The light source in the animal room was regulated with 12 h light period followed by
12 h dark schedule. Two to three animals were housed per cage sized (41x28x14 cm).
Paddy husk was used for bedding and on every alternative day the bedding was
changed and washed thoroughly with water along with Domex, a disinfectant and a
detergent. The rats were fed on a standard pellet diet purchased from Sai Durga Feeds
and Foods, Bangalore, and water ad libitum before dietary manipulation.
3.8 Experimental design:
Total 48 male Wistar rats with average weight of 150 - 180g were used for the
present study and were divided into following 8 groups with six rats in each
Group -I: CONTROL (20% Dimethyl sulfoxide is given to the animals fed on
normal diet for 45 days through oral intubation).
Group-II: ISPH (20% Dimethyl sulfoxide is given for 45 days through oral
intubation and on 44th
and 45th
day ISPH (85mg of ISPH/Kg body weight) dissolved
in physiological saline is given i.p. with an interval of 24 hrs).
Group III: BDEEWP (Ethanolic extract from whole plant of B.diffusa dissolved in
20℅ DMSO administered at the dose of 150mg/Kg body weight/day for 45 days
through oral intubation).
Group-IV: BDEEWP + ISPH (Ethanolic extract from whole plant of B.diffusa
dissolved in 20℅ DMSO administered at the dose of 150mg/Kg body weight/day for
45 days through oral intubation and on 44th
and 45th
day ISPH (85mg of ISPH/Kg
body weight) dissolved in physiological saline is given i.p. with an interval of 24 hrs.
86
Group -V: CONTROL (20% Dimethyl sulfoxide is given to the animals fed on
normal diet for 15 days through oral intubation).
Group-VI: ISPH (20% Dimethyl sulfoxide is given for 15 days through oral
intubation and on 14th
and 15th
day ISPH (85mg of ISPH/Kg body weight) dissolved
in physiological saline is given i.p. with an interval of 24 hrs).
Group -VII: BDTALK (Total alkaloids of B.diffusa dissolved in 20℅ DMSO
administered at the dose of 50mg/Kg body weight/day for 15 days through oral
intubation).
Group-VIII: BDTALK + ISPH (Total alkaloids of B.diffusa dissolved in 20℅
DMSO administered at the dose of 50mg/Kg body weight/day for 15 days through
oral intubation and on 14th
and 15th
day ISPH (85mg of ISPH/Kg body weight)
dissolved in physiological saline is given i.p. with an interval of 24 hrs ).
During experimental period all groups of rats have free access to tap water and
feed ad libitum. The animals were maintained at standard conditions of temperature,
relative humidity and with a 12-h light/dark cycle in their particular groups for
respective period of days. After 15 days of experimentation, the rats from Group V,
Group VI, Group VII and Group VIII (total of 24) were scarified by cervical
dislocation after 12 hours starvation and organs/tissues were collected, weighed and
stored at -800C for further biochemical and histological analysis. At the end of 45
days, the remaining animals (Group I, II, III and IV) were sacrificed and tissues were
collected in the same manner for analysis of various biochemical parameters.
3.9 In vivo studies
3.9.1 Cardiac Marker Enzymes
(A) Aspartate Transaminase (AST EC 2.6.1.1)
The activity of the enzyme AST was estimated by the method of Teitz (1976) using
Aspen Diagnostic Kit.
Principle: The enzyme aspartate transaminase or aspartate amino transferase (AST)
also called serum glutamate oxaloacetate transaminase (SGOT) is a tissue enzyme
that catalyses the transfer of an amino and keto group between ∞-keto acids. It is
87
widely distributed in tissues mainly heart, liver, muscle and kidney and injury to these
tissuses result in the realease of SGOT in to the circulation.
α-keto glutatate + L- alanine L-glutamate + oxoalo acetate.
Procedure: To 100μl of test sample, 1ml of reconstituted reagent was added, mixed
and incubated at 370C for 1min. after incubation; change in the optical density was
measured upto 3 minutes at an interval of 1 min against reagent blank at 340nm. The
activity of AST was expressed as U/L of serum.
(B) Alanine Transaminase (ALT, EC 2.6.1.2)
The activity of the enzyme ALT was estimated by the method of Teitz (1976) using
Aspen Diagnostic Kit.
Principle: the enzyme alanine transaminase or alanine amino transferase (ALT) also
called as serum glutamate pyruvate transaminase (SGPT), is widely distributed in a
variety of tissue sources. The major source of ALT is of hepatic origin and elevated
levels are found in hepatitis, obstructive jaundice and in myocardial infarction. The
enzymatic reaction sequence in the assay is as follows:
α-keto glutatate + L- alanine L-glutamate + pyruvate.
Procedure: To 100μl of test sample, 1ml of reconstituted reagent was added, mixed
and incubated at 370C for 1min. after incubation, change in the optical density was
measured upto 3 minutes at an interval of 1 min against reagent blank at 340nm. The
activity of ALT was expressed as U/L of serum.
(C) Alkaline Phosphotase (ALP, EC 3.1.3.1)
The activity of alkaline phosphotase was estimated by the method of Teitz
(1976) using Robonik Diagnostic Kit.
Principle: Alkaline phosphatase catalyses the hydrolysis of p-Nitrophenyl phosphate
to ortho phosphoric acid and p-nitro phenol. p-nitro phenol has maximum absorbance
at 405nm. The rate of increased absorbance at 405nm is proportional to the enzyme
activity.
88
Procedure: This is based on the kinetic method. 20 μl of test sample 1ml of reagent,
mixed well and incubated for 1 min at 370C. After 1min absorbance was taken at
405nm. The activity of alkaline phosphate was expressed as U/L.
(D) Creatine Kinase (CK, EC 2.7.3.2)
The activity of CK was estimated by the method of Roaslki (1967) using
Aspen Diagnostic Kit.
Principle: CK catalyzes the conversion of creatine phosphate and ADP to creatine
and ATP. The ATP and glucose are converted to ADP and glucose – 6- phosphate.
Glucose-6- phosphate dehydrogenase (G-6-PDH) oxidises D-glucose-6-phosphate and
reduces the nicotnamide adenine dinucleotide (NAD). The rate of NADH formation,
measured at 340 nm, is directly proportional to serum CK activity.
Procedure: one ml working reagent was added to 50μl of test sample, mixed and
incubated at 370C for 1 min. after incubation, change in the optical density was
measured for 3 minutes at an interval of 1 min against reagent blank at 340nm. The
activity of CK expressed as U/L.
(E) Lactate Dehydrogenase (LDH, EC 1.1.1.27)
The activity of the enzyme LDH was estimated by the method of Teitz (1976)
using agape Diagnostic Kit.
Principle: The enzyme LDH is distributed in tissues particularly in heart, muscles
and kidney. LDH catalysis the oxidation of lactate to pyruvate in the presence of
NAD which is subsequently reduced to NADH. The rate of NADH formation
measured at 340nm is directly proportional to LDH activity.
Procedure: one ml working reagent was added to 10μl of test sample, mixed and
incubated at 370C for 1 min. after incubation, change in the optical density was
measured for 3 minutes at an interval of 1 min against reagent blank at 340nm. The
activity of LDH was expressed as U/L.
(F) γ-Glutamyl Transferase (GGT, EC 2.3.2.2)
The activity of γ-glutamate transpeptidase was estimated according to the
method of Young (1995) using Robonic Diagnostic Kit.
89
Principle: γ-glutamate transferase (γ-GT) catalyses the transfer of the gamma
glutamyl group from gamma glutamyl peptides to suitable acceptors. In the reaction
rate analysis method, the artificial substrate γ-glutamyl-p-nitroanilide is used.
Procedure: 50ml of test sample was added to 1ml of reagent, mixed well and
incubated for 1 min. After incubation at 370C, the samples were measured at 405nm.
The activity of γ-glutamyl transferase was expressed as U/L.
3.9.2 Studies on Lipid Metabolism
Isolation and Estimation of Tissue Total Lipids
Total tissue lipids were extracted in purified form according to the method of
Folch et al. (1957) which involved two successive operations.
Extraction of Lipids: The tissue (250 mg) was homogenized with 2:1
chloroform-methanol mixture (v/v) to a final volume of 5 ml. After temperature
equilibration and final volume adjustment, the homogenate was filtered through a fat-
free paper into a glass-stoppered vessel.
Washing of Crude Extract: The crude extract was mixed thoroughly with 1 ml
of water and the mixture was allowed to separate into two phases, without interfacial
fluff, either by standing or by centrifugation. As much of the upper phase as possible
was removed and removal of its solutes was completed by rinsing the interface three
times with small amounts (1.5 ml) of pure solvent upper phase in such a way not to
disturb the lower phase. Pure solvent upper phase consists of 3:48:47 of chloroform-
methanol-water. Finally, the lower phase and the remaining rinsing fluid were made
into one phase by the addition of methanol, and the resulting solution was diluted to
final volume of 10 ml by the addition of 2:1 chloroform-methanol mixture. This water
washing entails the loss of about 1 % of the tissue lipids and completely removes non-
lipid substances. Triglycerides, total cholesterol free fatty acids and phospholipids in
this extract were estimated.
Plasma cholesterol and triglycerides were estimated using Accurex enzymatic
diagnostic kit and HDL- cholesterol was estimated by using Autozyme cholesterol
diagnostic kit.
90
(A) Total Cholesterol (TC)
Principle: Cholesterol esterase hydrolyses cholesterol esters into free cholesterol
and fatty acids. Cholesterol oxidase converts cholesterol to cholest-4-en-3-one and
hydrogen peroxide. In presence of peroxidase, hydrogen peroxide oxidatively couples
with 4 – aminoantipyrine and phenol to produce red quinoneimine dye which has
absorbance maximum at 510nm. The intensity of the red colour is proportional to the
amount of total cholesterol (Allain, 1974).
Procedure: To 0.01ml of serum, 1.0ml of the reagent was added, mixed and
incubated at 370 C for 10min. Cholesterol standard and water blank were also treated
in a similar manner. After incubation, absorbance was read at 510nm and values are
expressed as mg/dL.
(B) Triglycerides (TG)
Principle: Glycerol released from hydrolysis of triglycerides by lipoprotein lipase is
converted by glycerol kinase into glycerol-3-phosphate, which is oxidized by glycerol
phosphate oxidase to dihydroxyacetone phosphate and hydrogen peroxide. In
presence of the peroxidase, hydrogen peroxide oxidizes phenolic chromogen to a red
coloured compound which is measured at 510nm (Foosati et al., 1982).
Procedure: To 0.01ml of serum, 1.0ml of the reagent was added, mixed and
incubated at 37˚ C for 10min. Triglyceride standard and water blank were also
treated in a similar manner. After incubation, absorbance of the standard and serum
was read at 510nm against and values are expressed as mg/dL
(C) High density lipoprotein cholesterol (HDL)
Priniciple: Phosphotungstate/Mg2+
precipitate chylomicrons, Low density lipoprotein
(LDL) fractions. High density lipoprotein (HDL) fraction remains uneffected in
supernatant. Cholesterol content of HDL fraction is assayed using Autozyme
cholesterol diagnostic kit (Assmann, 1983).
Procedure: To 0.2ml of serum, 0.2ml of precipitant reagent was added, mixed and
centrifuged at 4,000rpm for 10min to obtain a clear supernatant. To 0.05ml of
supernatant, 1.0ml of Autozyme of HDL cholesterol working solution was added,
91
incubated for 10min at 37˚C and color developed was read at 510nm against a blank
and a standard (50mg%) was run simultaneously. Values are expressed as mg/dL.
(D) Very Low Density Lipoprotein Cholesterol (VLDL-C) and Low Density
Lipoprotein Cholesterol (LDL-C)
VLDL and LDL were calculated using the Friedewald et al (1972) formulas as
follows:
VLDL = TG/5
LDL = Total CHL - TG/5 +HDL.
(E) Atherogenic index
Atherogenic index (AI) is the ratio of total cholesterol and HDL cholesterol
and was calculated by the following formula: AI = TC/HDL-C
(F) Free Fatty Acids
The free fatty acids form a complex with cupric ions when mixed with copper
reagent which is soluble in chloroform. Diethyl dithiocarbamate is used as a colour
developer (Itaya and Ui 1965).
To one ml of tissue lipid extract placed in a centrifuge tube, 4 ml of
chloroform solution and 2.5 ml of copper reagent (this consists of 9 volumes of
aqueous 1 M triethanolamine, 1 volume of 1 N acetic acid and 10 volumes of 6.45%
Cu(NO3)2.3H2O) were added. The tubes were stoppered and shaken vigorously for 1
min and centrifuged for a few minutes. The supernatant aqueous phase was removed
by suction with a fine hypodermic needle. The surface of the chloroform phase can
easily be left clean with only traces of aqueous phase adhering to the wall of the tube.
Chloroform layer (2.5 ml) was taken into a clean dry tube. Care was taken such that
the pipette did not touch the inner wall of either tube, as traces of copper containing
aqueous phase might be transferred. Then 0.5 ml of diethyl dithiocarbamate reagent
(0.1% (w/v) of sodium diethyl dithiocarbamate in redistilled secondary butanol, stored
in refrigerator, was added to the chloroform solution and mixed. The extinction was
read at 440 nm against blank in a spectrophotometer. A standard curve was prepared
by taking myristic acid as standard fatty acid in the range of 10 to 100 µM. The values
of tissue free fatty acids were expressed as mg/g tissue.
92
(G) Phospholipids
Principle: Ammonium molybdate under acidic conditions reacts with phosphorus to
form phosphomolybdate complex which is reduced to blue colour complex by 2,4
aminonapthosulphonic acid (ANSA). The absorbance of the colour developed is
proportional to the inorganic phosphorus concentration (Connerty, et al., 1961).
Procedure: Phospholipids, as precipitate, were obtained in 0.2ml of serum by the
addition of 5ml trichloroacetic acid(5%w/v), 1ml of digestion mixture (50ml distilled
water, 25ml of 70% Perchloric acid, 25ml conc.H2SO4 ) and heated gently for about
30-45 minutes until the liquid becomes colourless. It was cooled and then, 1ml of
distilled water was added, allowed to boil for 15 seconds to convert pyrophosphate to
orthophosphate. 1ml of 50% sodium acetate was added and phosphorus was estimated
by Fiske Subbarow (1925) method. To the above digest,1ml of molybdate-II reagent
(2.5% ammonium molybdate in 3N H2SO4 ) , 0.4ml of amino napthosulphonic acid
(ANSA) reagent (0.5gms of α-ANSA in 195ml of 15% sodium bisulphate and 5ml of
20% sodium sulphate) was added and volume was made up to 10ml with distilled
water and absorbance was read in spectrophotometer at 680nm.
Potassium di hydrogen phosphate is used as a standard, concentration ranging
from 4-80μg, to which 1ml of molybdate-I reagent (2.5% of ammonium molybdate in
5N H2SO4 ), 0.4ml of ANSA reagent were added and the volume was made up to
10ml with distilled water and the absorbance was read in spectrophotometer at
680nm.
3.9.3 In vivo Studies on Antioxidant and Oxidative Stress
(A) Lipid Peroxidation
Lipid peroxidative extent was measured by the formation of Malondialdehyde (MDA)
by using the method of Okhawa et al. (1979).
Principle: Malondialdehyde formed from the breakdown of polyunsaturated fatty
acids, serves as a convenient index for determining the extent of peroxidation
reaction. Malondialdehyde has been identifies as the product of lipid peroxidation that
reacts with TBA to give a chromogen absorbing at 535nm.
93
Procedure: 1ml of the test sample was mixed with 600μl of distilled water and 200μl
of 8.1% (w/v) sodium dodecyl sulphate (SDS), vortexed and incubated at room
temperature for 5 min. After adding 1.5ml of 20% w/v acetic acid (pH 3.5) and 1.5 ml
of 0.8% (w/v) TBA, the mixture was heated at 950C for 60min. After heating 1ml of
distilled water 5ml of a mixture of n-butanol and pyridine (15:1 v/v) were added and
vortxed. Then after centrifugation at 800g for 15 min, the absorbance of the upper
layer was measured at 532 nm using 1,1,3,3 tetra ethoxypropane as the standard.
(B) Protein Oxidation
Protein carbonyl content was measured by forming labeled hydrazone derivatives
using 2,4-DNPH which were quantified spectrophotometrically at 370 nm according
to the method of Levine et al. 2001).
To 0.1 ml of the sample (supernatant obtained from 10% homogenate in 0.15
M KCl), 500 µl of 20% TCA was added and protein was precipitated by
centrifugation at 12000 ×g for 15 min at 4oC. Then 500 µl of 10 mM 2,4-DNPH in 2
N HCl was added to protein pellets and kept at 37oC for 1 h with vortexing for every
10 to 15 min. After 1 h, protein was reprecipitated from sample with 500 µl of 20%
TCA and then washed three times with ethanol: ethyl acetate (1:1) solution to remove
free DNPH and lipid carbonyl DNPH derivatives. Then protein was dissolved in 600
µl of 6 M guanidine HCl in 2 mM potassium phosphate buffer pH 6.5 and insoluble
debris was removed by centrifugation at 12000 ×g for 15 min and the absorbance was
read at 370 nm against the HCl treated blank. The protein carbonyl products were
quantified using extinction co-efficient of 22 cm-1
mmol-1
.
(C) Estimation of Nitric oxide
Nitric oxide (NO) being a free radical is highly unstable and converted to an
equimolar ratio of its stable metabolites, nitrite and nitrate. The estimation of nitric
oxide is therefore done by estimating the stable metabolite. The amount of
nitrite/nitrate formed is an index for nitric oxide in the samples. Nitrite is measured
by the method of Greiss (Lepoivre 1990).
Assay procedure:
0.5 ml of serum was precipitated with 50 µl of 70% Sulfo salicylic acid, mixed
well for 5 minutes, vortexed and then centrifuged at 3000 rpm for 20 minutes. 200 µl
94
of supernatant was taken and 30 µl of 10% NaOH, 300 µl of Tris HCl buffer and 530
µl of Greiss reagent were added and incubated for 10 minutes in dark. The
absorbance was read against water blank at 540 nm. The concentration of nitrite in
serum was determined using the standard curve. The standard curve was prepared
using known amounts of sodium nitrite (NaNO2).
Units : µM of nitrite.
(D) Estimation of DNA Damage by Single Cell Gel (SCG) Assay or Comet Assay:
The SCGE assay combines the simplicity of biochemical techniques for
detecting DNA single strand breaks and/or alkali labile sites with the single cell
approach typical of cytogenetic assays. Thus, the power of the assay lies in its ability
to evaluate DNA damage and repair in proliferating or nonproliferating cells and to
provide insight into intercellular differences in response. The technique is simple,
sensitive and reproducible to cost effective. As very few cells are actually required
for analysis, it is possible to conduct a damage/repair kinetic study using the
leucocytes in a single drop of human blood or to evaluate organ specific levels of
DNA damage in vivo using small samples from plants, animals or humans. The slides
can be stained wither by fluorescent stains like Ethidium bromide, by non fluorescent
staining by silver nitrate (Ahuja and Saran, 1999).
I. Preparation of Reagents
Materials required
Normal melting agarose (NMA) (40-420C)
Low melting agarose (LMA) (370C)
Ethylene diamine tetra acetic acid disodium salt (Na2 EDTA)
Triton X
Tris – HCl
Sodium hydroxide (NaOH)
Sodium chloride (NaCl)
Dimethy sulphoxide (DMSO)
Sodium N-Lauryl Sarcosinate
95
PBS (Ca++
, Mg++
Free)
Silver nitrate
Sodium carbonate
Ammonium nitrate
Procedure:
A. Phosphate Buffered Saline (Ca + Mg Free) for 1000 ml
8 gm of sodium chloride, 0.2 gm of potassium chloride, 1.15 gm of disodium
orthophosphate and 0.2 gm of potassium dihydrogen phosphate was weighed and
dissolved in 500 ml of distilled water and then made upto 1000 ml. The pH was
adjusted to 7.4 filtered and stored at 40C.
B. Low melting agarose (LMA)
To prepare 0.5% low melting agarose, 125 mg of agarose was mixed with 25ml PBS
and heated until near boiling to dissolve the agarose. The gel was stored in 5ml
aliquots at 40C.
C. Normal melting agarose (NMA)
To prepare 0.67% nomal melting agarose, 167 mg agarose was mixed with 25ml PBS.
The gel was heated until boiling to dissolve the agarose. The melted gel was Stored
in 5ml aliquots at 40C.
D. Lysing solution
The stock (890 ml) was prepared by dissolving 146.1g NaCl, 37.2g EDTA and 1.2 g
Tris in 700 ml double distilled water and stirred 10 gm sodium lauryl sarcosinate was
added and the contents stirred again. To dissolve the contens, 12 g NaOH was then
added. The pH was adjusted to 10. The lysing stock solution was filtered and stored
at room temperature. To prepare the working lysing solution 1% Triton X-100 and
10% dimethyl sulphoxide (DMSO) were added to the fresh stock solution and
refrigerated 1 hour prior to use.
96
E. Electrophoresis buffer (alkaline)
To prepare the 10N NaOH of stock solution 200 mg of NaOH was dissolved in 500
ml of double distilled water and preserved at room temperature in drak bottle. To
prepare 20Mm (EDTA stock solution) 14.89 g of EDTA was dissolved in 200ml
double distilled water, filtered and preserved at 40C.
The pH of this solution is adjusted to 10. For the working buffer, which is made fresh
before each run, 7.5 ml of NaOH stock solution was mixed with 1.25ml of EDTA stock
solution and the volume adjusted to 250 ml with double distilled water (pH > 13).
F. Neutralizing buffer
To prepare this solution, 14.55gm of TRIS was dissolved in 300 ml of distilled water,
filtered and the pH was adjusted to 7.5. The solution was stored at 40C.
G. Fixing solution
To prepare 500 ml of fixing solution about 75 g of trichloroaceticacid, 25 g of zinc
sulphate and 25 g of glycerol was mixed in 500 ml of double distilled water.
H. Staining solution
Staining solution (A): To prepare 500 ml of staining solution (A) 25 g of sodium
carbonate was dissolved in 500 ml double distilled water and stirred vigorously for 20
– 30 min.
Staining solution (B): This solution was prepared by mixing the following (in the
giver order) 100mg ammonium nitrate, 100mg silver nitrate, 500 mg tungstosilicylic
acid and 250µl formaldehyde in 500ml double distilled water. The solution was
stirred to dissolve the contents.
Staining solution (C): 32ml of solution A and 68 ml of solution B (made fresh).
I. Stopping solution
This solution consists of 1% glacial acetic acid (1ml glacial acetic acid made upto
100ml with double distilled water).
97
Preparation of slides:
Slides were performed in duplicate for each experiment
(a) First layer: Dust free, plain slides were covered with a layer of 140µl of 0.67%
NMA and allowed to dry for 10 minutes in hot oven. This layer serves as an
anchor for additional layers to prevent the slippage.
(b) Second layer: about 110µl of NMA was layered as second layer and was
immediately covered with a cover slip and was kept at 40C for 10 minutes to allow
the agarose to gel.
(c) Third layer: 20 µl of test sample was mixed with 110µl of warm 0.5%LMA and
this mixture was layered as third additional layer and gelled at 40C for 10 minutes.
(d) Fourth layer: A fourth additional layer of 110µl of LMA was added on top and
gelled in the similar way as mentioned above, to sandwitch the middle sample
layer and to prevent the loss of sample.
(e) Lysing: After the fourth layer of gel was set ,the slides were treated overnight in
freshly prepared, chilled lysis buffer solution at 40C.With this treatment the cell
membrane at 40C and nuclear membrane were lysed and the majority of proteins
were removed to expose the nucleiods.
(f) Alkali treatment: The slides were then removed from the lysing solution, drained
and placed in a horizontal gel electrophoresis thank side by side avoiding spaces
and with agarose end facing the anode. The tank was filled carefully with fresh
electrophoretic buffer to a level approximately 0.25 cm above the slides. The
slides were left in the high pH (pH > 13) buffer for 20 minutes to allow unwinding
of DNA and expression of alkali labile sites before electrophoresis.
(g) Electrophoresis: Electrophoresis was carried out at room temperature for 40
minutes at 300mA, 20v. The current was adjusted to 300mA(milliamperes) by
raising or lowering the buffer level in the tank DNA fragments, if any, due to
DNA damage migrate in to gel.
(h) Neutralization: After electrophoresis, the slides were flooded 3 times gently with
chilled neutralizing solution (Tris pH 7.5) for 5 minutes so as to remove any traces
of detergents and alkali which would otherwise interfare with staining the slides
were washed thrice with distilled water and air dried completely.
(i) Silver staining: The slides were silver stained by the method of Ahuja and saran
(1999). Briefly the air-dried slides were immersed in the fixing solution for 10
98
minutes and washed gently with double distilled water several times. The washed
slides were allowed to air dried for about 1 hour before staining. Just before
staining. 68 ml of staining solution (B) was mixed with 32 ml of staining solution
(A) and poured over the dried slides so as to cover the slides uniformly. This step
was repeated with a fresh mixture of staining until a grayish color developed on
the slides no need of stopping solution.
Note: The whole procedure was carried out in dim light to minimize artificial
DNA damage.
For screening the slides a bright field transmission light microschope (Leitz
Laborlux) was used. Comet tail length was measured in each case using an ocular
micrometer fitted in the eyepiece. Randomly 100 cells were selected from duplicated
slides at 400 X magnification. Mean tail length was calculated for each sample.
Quantification of the DNA damage was done in terms of
Comet tail length (µm) = (total length of comet) – (head diameter).
Photographs were taken on soft or glossy grade photographic papers.
3.9.4. Studies on Nonenzymatic Antioxidants
(A) Reduced Glutathione
Principle: Total reduced glutathione content was measured by following method
Ellman`s method (1959). This method was based on the development of a yellow
color, when 5,51 –dithio-2-nitro benzoic acid (DTNB) reacts with the compounds
containing sulphydryl groups with a maximum absorbance at 412nm.
Procedure: 0.5ml of heart tissue homogenate was deproteinized with 3.5ml of 5%
TCA and centrifuged. To 0.5ml of supernatant, 3.0ml phosphate buffer and 0.5ml of
Ellman`s reagent were added and the yellow colour developed was read at 412nm. A
series of standards (4-20µg) were treated in a similar manner along with a blank.
Values are expressed as µg GSH/mg protein.
99
(B) Ascorbic Acid (Vit-C)
Ascorbic acid was estimated by the method of Omaye et al. (1979).
The tissue homogenate (0.5 ml) was mixed thoroughly with 1.5 ml of 6%
TCA and centrifuged for 20 min at 3500 g. To 0.5 ml of the supernatant, 0.5 ml of
DNPH reagent (2.0 g of DNPH and 4.0 g thiourea in 100 ml of 9N sulphuric acid)
was added and mixed well. The tubes were allowed to stand at room temperature for
an additional 3 hrs. Removed, placed in ice-cold water and added 2.5 ml of 85%
sulphuric acid and allowed to stand for 30 min. A set of standards (10 to 50 g) was
treated in a similar manner along with a blank, containing 0.5 ml 4% TCA. The colour
developed was read at 530 nm.
3.9.5 Assay of Antioxidant Enzymes
Sample preparation
10% tissue homogenate in 0.1M Tris HCl was prepared using Potter-Elvehjem
homogenizer at 0oC and centrifuged in cold (0-4
oC) at 12,000rpm for 45min.The
supernatant thus obtained was distributed into eppendorf tubes, labeled and stored at -
20 oC and all the antioxidant enzymes were assayed at the earliest.
a) Glutathione Peroxidase (EC 1.11.1.9)
Principle: A known amount of the enzyme preparation was allowed to react with
H2O2 in the presence of GSH for a specified time period according to the method of
Rotsruck (1973) and remaining GSH was measured by Ellman`s method (1959).
Procedure: To 0.5ml buffer, 0.2ml enzyme source, 0.2ml GSH and 0.1ml H2O2 were
added, incubated at room temperature for 10min along with the control tube
containing all reagents except enzyme source. The reaction was arrested by adding
0.5ml of 10% TCA, centrifuged at 4000rpm for 5min. and GSH content in
0.5ml of supernatant was estimated. The activity was expressed as µg of GSH
consumed/min/mg protein.
100
b) Glutathione-S-transferase (EC 2.5.1.18)
Principle: Glutathione-S-transferase activity was measured by monitoring the
increase in the absorbance at 340nm using 1-chloro-2,4-dinitrobenzene (CDNB) as a
substrate according to the method of Habig et al (1974).
Procedure: The assay system contained 1.7 ml of buffer, 0.2ml GSH and 0.04ml
enzyme source (40µg protein). The reaction was initiated by 0.06ml CDNB. The
change in absorbance was recorded at 1 minute intervals at 340nm for 5min and the
activity was calculated using an extinction coefficient of CDNB-GSH conjugate as
9.6 mM-1
and expressed as mmoles of CDNB-GSH conjugate formed/min/mg protein.
c) Glutathione reductase (EC 1.6.4.2)
Principle: Glutathione reductase catalyses the reduction of oxidized glutathione
(GSSG) by NADPH to GSH. The activity of the enzyme was measured by following
the oxidation of NADPH spectrophotometrically at 340nm according to the method of
Pinto and Bartley (1969).
Procedure: The assay system contained 0.50ml of buffer, 0.10ml of EDTA, 0.10ml
of NADPH, 0.96ml of distilled water and 0.10ml of enzyme source (150 µg proteins).
The reaction was initiated by addition 0.24ml of GSSG. The change in absorbance
was recorded at 1min intervals at 340 nm for 5min. The specific activity was
expressed as mol of NADPH oxidized/min/mg protein using an extinction
coefficient for NADPH of 6.22 cm-1
µmol-1
.
d) Catalase (EC 1.11.1.6)
Principle: Catalase catalyses the break down of H2O2 to H2O and O2 and the rate of
decomposition of H2O2 was measured spectrophotometrically at 240nm following the
method of Beers and Sizer (1952).
Procedure: The assay system contained 1.9ml buffer, and 1.0ml H2O2. The reaction
was initiated by the addition of 0.1ml enzyme source (45µg protein). The decrease in
absorbance was monitored at 1 min interval for 5 min at 240nm and activity was
calculated using a molar absorbance index of H2O2 43.6. The activity was expressed
as mmoles of H2O2 decomposed/min/mg protein.
101
e) Superoxide dismutase (EC 1.15.1.1)
Principle: SOD activity was measured based on the ability of the enzyme to inhibit
the autoxidation of pyrogallol. A modified procedure described by Marklund and
Marklund (1974).
Procedure: The assay system contained 2.1ml of buffer, 0.02ml of enzyme source
(35µg protein) and 0.86ml of distilled water. The reaction was initiated with 0.02ml
of pyrogallol and change in absorbance was monitored at 420nm. The per cent
inhibition was calculated on the basis of comparison with a blank assay system. One
unit of SOD was defined as that amount of enzyme required to inhibit the auto-
oxidation of pyrogallol by 50% in standard assay system of 3 ml. The specific
activity was expressed as units/min/mg protein read in spectrophotometer.
3.9.6 Serum Electrolytes
(A) Sodium (Na+)
The content of sodium was estimated by the method of Trinder (1951) using excel
diagnostic kit.
Principle: The sodium and proteins were precipitated simultaneously by means of a
reagent containing magnesium uranyl acetate containing alcohol. The precipitate is
separated by centrifugation. The content of sodium is calculated from the loss in the
concentrated of magnesium uranyl acetate in the reagent solution in comparison to a
standard sodium solution treated similarly. The residual amount of magnesium uranyl
acetate is estimated by forming brown (dark) ferrous uranyl acetate which is read in a
colorimeter.
Procedure: 1ml of sodium precipitating reagent was taken in to two test tubes labeled
as standard and test. 0.02ml of standard sodium was added to the first tube and 0.02ml
of serum was added to the second tube. The tubes were mixed well, incubated for 5
minutes at room temperature and then centrifuged for 1 minute at 3000rpm. 0.05ml of
the supernatant was separated into another set of test tubes. 3ml of distilled water and
0.2ml of sodium colour reagent was added, mixed well and incubated for 5 minutes at
room temperature. Then the absorbance was measured at 540nm.
102
(B) Potassium (K+)
The content of potassium was estimated by the method of Jocabs and Hoffmann
(1931) using excel diagonistic kit.
Principle: Potassium can be determined by the number of different methods. It can be
directly estimated by Flame photometry and colorimetry. It can also be measured by
the use of ion selective method electrode. The method is based on the measurement of
turbidity of the reaction mixture containing sodium tetra phenyl boron, alkaline
EDTA, formaldehyde and sample containing potassium or standard potassium salt.
Procedure: 1ml of potassium reagent was taken in to 2 test tubes labeled as standard
and test. 0.05ml of standard potassium was added to the first tube and 0.05ml of
serum was added to the second tube. The tubes were mixed well and incubated for 5
minutes at room temperature. Then the absorbance was measured at 620nm.
(C) Calcium (Ca2+
)
The content of calcium was estimated by the method of Tietz (1995) using Robonik
Diagnostic Kit.
Principle: Calcium with Arsenazo, at neutral pH yields a blue colored complex,
whose intensity is proportional to the calcium concentration. Interference by
magnesium is eliminated by addition of 8-hydroxyquinoline-5-sulfonic acid.
Procedure: 1 ml of reagent was added to 10μl of standard and 10μl of test samples.
After mixing well, the samples were incubated for 10 min and then the absorbance
was read at 630nm.
(D) Iron
Serum iron content was estimated by the method of Ramsay (1958).
Procedure: equal volumes of serum (50µl), 0.1 M sodium sulphite and dipyridyl
reagent (50µl) were mixed in glass Stopper centrifuge tubes. The tubes were heated in
a boiling water bath for 5 min. The contents were cooled and 12 ml of chloroform was
added in each tube. The tubes were stopper again and mixed vigorously for 30s and
centrifused for 5 min at 1000rpm. The colour intensity was measured at 520nm.
103
3.9.7 Membrane-bound ATPases
ATPases transport electrolytes against concentration gradient at the cost of ATP
molecules liberating inorganic phosphate (Pi). The liberated Pi is estimated by Fiske
and Subbarow (1925).
(A) Estimation of inorganic phosphorus
Inorganic Phosphorus was estimated by the method of Fiske and Subbarow
(1925). The method is based on the formation of phoshomolybdic acid by the reaction
between a phosphate and molybdic acid and its subsequent reduction to a dark blue
phosphomolybdic acid, the intensity of which is proportional to the phosphate ion
concentration.
Procedure
To suitable aliquots of the supernatant, 1.0 ml of ammonium molybdate reagent was
added. 0.4 ml of ANSA was added after 10 min incubation at room temperature.
Standards and blank were also treated in the above manner. The blue colour
developed was read after 20 min at 640 nm in a Shimadzu-UV-1601
Spectrophotometer. The values were expressed as μg /mg protein.
(B) Na+/K
2+-dependent ATPase (EC 3.6.3.9)
Na+/K+ ATPase activity was measured from the amount of Pi released according to
the method of Bonting (1970).
Procedure
The reaction mixture consisted of 100μl tissue homogenate incubated in the medium
containing Tris-HCl (75mM, pH-7.5), NaCl (600mM), KCl (50mM), MgCl2 (5mM),
EDTA (1mM) and ATP (3mM) for 30 min at 300C. The reaction was arrested by the
addition of TCA. The results were expressed as μM of phosphorus liberated/min/mg
of protein.
(C) Ca2+ ATPases
The activity of Ca2+ ATPases was estimated according to the method of Hjerten and
Pan (1983).
Procedure: The incubation mixture contained 0.1ml of buffer, 0.1ml of calcium
chloride, 0.1ml of ATP, 0.1ml of distilled water and 0.1ml of tissue homogenate. The
contents were incubated at 370C for 15min. the reaction was then arrested by the
104
addition of 0.5mlof ice – cold 10% TCA. The results were expressed in μM of
phosphorus liberated/min/mg of protein.
(D) Mg2+ ATPases
The activity of Mg2+ ATPases was estimated according to the method of Ohnishi
(1982).
Procedure: The reaction mixture consisted of 75mM Tris – HCl, pH 7.6, MgCl2 and
3mM Na2-ATP in the total of 0.15ml of 30%TCA. The results were expressed as μM
of phosphorus liberated/min/mg of protein.
3.9.8 Assay of Key Enzymes in Lipid Metabolism
(A) Lecithin Cholesterol Acyl Transferase (LCAT)
LCAT activity was determined according to the self-substrate method explained by
Ngasaki and Akanuma (1977).
Procedure: Lecithin suspension (0.25%) and 150mM sodium iodo-acetic acid
solution was prepared using 50mM phosphate buffer (PH 7.4). The subject serum was
prepared by adding 20ml of 0.25% lecithin suspension and 20ml of 50mM phosphate
buffer (PH 7.4) to 40ml of serum. Meanwhile, the control serum was prepared by
adding 20ml of 0.25% lecithin suspension and 20ml of 50mM sodium iodo-acetic
acid solution to 40ml of the serum. After incubation of subject serum and control
serum for 40 min at 370C, 20ml of 150mM sodium iodoacetic acid solution was
added to the subject serum and 20ml of 50mM phosphate buffer (PH 7.4) was added
to the control serum, and the free cholesterol in both was determined using a
commercial assay kit. LCAT activity was calculated as the decreased level (mg) of
free cholesterol in 1 ml of serum per 1 hr by subtraction of the free cholesterol level
of subject from that of control serum.
(B) Lipoprotein Lipase (E.C. 3.1.1.34)
Ten percent tissue homogenate was prepared in 100 mM sodium phosphate
buffer. The enzyme hydrolyzes p-nitrophenyl butyrate into p-nitrophenol and butyric
acid. The assay was done by continuous spectrophotometric rate determination of
formation of p-nitrophenol at 400 nm (Quinn et al., 1982).
105
To 0.9 ml of 100 mM sodium phosphate buffer (with 150 mM NaCl and 0.5%
(v/v) triton x-100, pH 7.2), 0.1 ml of tissue homogenate was added and the reaction
was initiated by the addition of 0.01 ml of 50 mM p-nitrophenyl butyrate in
acetonitrile. The increase in the absorbance at 400 nm was monitored for 10 min. The
activity was expressed as µmol of P-nitrophenol released/min/mg protein using
extinction co-efficient as 0.0148 cm-1
µmol-1
.
(C)HMG -CoA Reductase
The ratio between 3-hydroxy-3-methylglutaryl-CoA and mevalonate
concentrations in tissues in terms of absorbance was taken as an index of the activity
of HMG-CoA reductase as described by Rao and Ramakrishnan (1975). HMG-CoA
was deterimed by reaction with hydroxyl amine at pH 5.5 and subsequent colorimetric
measurement of the resulting hydroxamic acid by formation of complexes with ferric
salts. Because mevalonate interferes in this estimation at acid or neutral pH, alkaline
hydroxylamine was used to estimate specifically HMG-CoA only. Possible
interference by coenzyme A is also minimal when readings are taken at 540 nm.
Mevalonate was estimated by reaction with the same reagent, but at pH 2.1. At this
pH, the lactone form of mevalonate readily reacts with hydroxylamine to form the
hydroxamate.
Equal volumes of fresh 10 % tissue homogenate in saline and diluted
perchloric acid (50 ml/L) were mixed and centrifuged at 2000 rpm for 10 min. One ml
of supernatant was treated with 0.5 ml of freshly prepared hydroxylamine reagent
{alkaline hydroxylamine reagent in the case of HMG-CoA (1:1 ratio of 2 M
hydroxylamine HCl and 4.5 M NaOH)} and (1 M hydroxyl amine HCl for
mevalonate), after 5 min, 1.5 ml of ferric chloride reagent was added mixed and after
10 min, absorbance was measured at 540 nm against similarly treated saline blank.
3.10 Histopathological studies
After sacrificing the rats, hearts were removed and cut into small pieces and
preserved in buffered formalin for histo morphological examination.
Fixation
Tissue blocks with 3 mm thickness were cut from small pieces of heart tissue
and were placed in a fixative solution (pH 7.0), prepared by adding 100ml of 37-40%
106
formaldehyde, 900ml of distilled water, in which 4g of Na2HPO4 and 6.5g of
NaH2PO4 were dissolved.
The tissue block was processed through a series of solvents: alcohol 80%-1hr,
alcohol 90%-1hr, alcohol 95%-2 changes-1hr each, isopropyl alcohol-1hr, acetone-2
changes-1hr each, chloroform-3 changes-1hr each and paraffin-2 changes-1hr each as
per scheduled for dehydration, clearing and paraffin infiltration. This block was then
ready for embedding. During the process of embedding, the tissue blocks were
oriented so that sections were cut in the desired plane of the tissue. Two L-shaped
metal moulds were laid on metal plate so as to enclose a rectangular or square space.
This is then partly filled with melted paraffin and the tissue was placed in it in the
desired position. The container was then filled with melted paraffin and allowed to
cool until reasonably firm so that the set block of paraffin with the tissue can be
removed from the moulds. The block was trimmed to a suitable size and fixed on a
metal objects holder. The block was further trimmed so that paraffin overlaying the
piece of tissue is excluded and an adequate area of the tissue facing the knife is
exposed. The block was then kept for cooling at 0˚C.
Section Cutting
The section were cut at 5μm thickness and floated in a water bath between 38-
49˚C. The sections from the water were mounted on clean glass slides, which have
been smeared with a drop of Mayer‟s egg albumin. They were then dried on a hotplate
at about 50˚C for 30 min and the sections on the slides were then subjected to
staining.
Staining
The slide containing the section was processed serially as follows-xylol 1- 3
min, xylol II-3 min, acetone-3 min, 95% alcohol-3 min, running water-3 min,
hematoxylin stain-20 min, wash in running tap water-20 min, eosin working solution-
2 min-15 sec, 95% alcohol-2 to 3 dips, 95% alcohol-2 changes 1 to 2 min each,
acetone-2 changes-3 min each , xylol-2 changes-3 min each and mounted in D.P.X.
and viewed under microscope. The nuclei stained with blue and cytoplasm in various
shades of pink (Raghuramulu et al., 1983).
107
Statistical analysis
All results were expressed as means ± SE of a six individual observations
Duncan's Multiple Range (DMR) test was performed to know the level of significance
among all the experimental groups (Duncan, 1955).
3.10 Docking Studies
A binding interaction between a small molecule ligand and an enzyme protein
may result in activation or inhibition of the enzyme. If the protein is a receptor, ligand
binding may result in agonism or antagonism. Docking is most commonly used in the
field of drug design - most drugs are small organic molecules, and docking may be
applied to: hit identification – docking combined with a scoring function can be used
to quickly screen large databases of potential drugs in silico to identify molecules that
are likely to bind to protein target of interest (see virtual screening). Lead
optimization – docking can be used to predict in where and in which relative
orientation a ligand binds to a protein (also referred to as the binding mode or pose).
Keeping the aim of constructing novel ligand complexes for H3, a library of
10 molecules was synthesized. The Auto Dock 4.0/ADT (Laskowski et al., 2005)
program was used to investigate ligand binding to structurally refined H3 model using
a grid spacing of 0.375 Å and the grid points in X, Y and Z axis were set to
60×60×60. The search was based on the Lamarckian genetic algorithm (Oprea et al.,
2001) and the results were analyzed using binding energy. For each ligand, a docking
experiment consisting of 100 stimulations was performed and the analysis was based
on binding free energies and root mean square deviation (RMSD) values. Substrate
docking with synthesized substrates was also performed on to H3 model with same
parameters and PMV 1.4.5 viewer was then used to observe the interactions of the
docked compounds to the H3 model.
The present work all the calculations were performed on a workplace by AMD
64 bits dual processing hi end server machines. Molecular docking calculations were
performed with AutoDock 4.0.
108
RESULTS AND DISCUSSION
4. Results and Discussion
Nature has been a source of medicinal agents for thousands of years and an
impressive number of modern drugs have been isolated from natural sources. The plant
based drugs continue to play an important role in the primary health care of about 80-85%
of the world‟s population (Deepika et al., 2014). Plants have the ability to make various
chemical constituents like flavonoids, proteins, alkaloids, and steroids (Naveen et al., 2009),
glycosides, phytosterols, phenolic compounds, carbohydrates, amino acids and saponins
(Saraswathi et al., 2011), which are in turn used to alleviate many diseases like as body
ache, bronchitis, jaundice, toothache, piles, diabetes, fever, leprosy, cystitis, ulcers,
gonorrhea, diarrhea, cough, urine output, lung disease (Naveen et al., 2009). Many clinical
discomforts are associated with the effect of free radicals as they react with important
cellular components such as proteins, DNA and cell membrane. Free radicals are required
for immune system responses. However, an overload of these molecules had been linked to
certain chronic diseases like cancer, liver and heart diseases (Prakash et al., 2007).
MI is a life threatening heart disease and a major public health problem all over the
world. Cardiovascular diseases encompass an immense category of disorders, because
many things can go wrong with the heart and blood vessels. Cardiovascular diseases are
leading cause of 17.1million fatalities each year and it will reach up to 20 million in 2020
(Upaganlawar et al., 2011). The risks of cardiovascular diseases among the Indians are four
times higher than that of White Americans, six times more than that of Chinese and 20
times greater than that of Japanese (Jain and Jain, 1998). Nowadays, herbal medicines are in
great demand in the developed as well as developing countries for primary health care,
because of their wide range of biological activities, higher safety margins and lesser costs.
Herbal therapy is one of the best practices to overcome the illness. The medicinal properties
of plants could be based on the antioxidant, antimicrobial, antipyretic effects of the
phytochemicals (Adesokan et al., 2008). Presently, people are in demand of modestly
processed fruits and vegetables (herbal medicine) as modern (chemical based) medicines
are having more side effects. Hence, more attention is to be needed to search for naturally
occurring substances to triumph over the diseases.
109
As there were no systematic scientific reports in literature on the cardioprotective
effect of B. diffusa and its extracts against ISPH induced MI in rats, the present
investigation was undertaken to study the cardioprotective effect of BDWEEP and
BDTALK. In the present study ISPH at a dose of 85 mg/Kg body weight for two
consecutive days was used to induce MI in rats. The cardioprotective effect of BDWEEP
and BDTALK was calculated in ISPH administered rats by measuring various biochemical
parameters in plasma and heart tissues.
4.1 Phytochemical profile of BDEEWP
The phytochemical screening of ethanolic extract of B. diffusa revealed the
presence of alkaloids, glycosides, flavonoids, tannins, phenols, saponins, steroids,
terpenoids, lignins, carbohydrates and proteins (Table4.1). Numerous epidemiological
studies suggest that herbs/diets rich in phytochemicals and antioxidants execute a protective
role in health and disease (Vinson et al., 2001).
Higher contents of saponins and alkaloids than flavonoids were observed in the extract.
Saponin is a known anti-nutritional factor, which reduces the uptake of certain nutrients
including glucose and cholesterol at the gut through intra-lumenal physicochemical
interaction and thus may aid in lessening the metabolic burden that would have been placed
on the liver. Alkaloids are beneficial chemicals to plants with predator and parasite
repelling effects. However, they inhibit certain mammalian enzymatic activities such as
those of phosphodiesterase, prolonging the action of cyclic AMP. They also affect
glucagons and thyroid stimulating hormones, while some forms have been reported to be
carcinogenic. It is noteworthy that at the concentration of these chemicals in edible
vegetables are usually non-toxic.
This plant also contains flavonoids, which are phenolic compounds that serve as
flavoring ingredients of spices and vegetables. They have been found to have anti-oxidation
effects in animals. Polyphenolic compounds also possess a variety of other biological
activities such as reduction of plasma lipids, which might be due to up regulation of LDL
receptor expression (Kuhn et al., 2004) and inhibition of hepatic lipid synthesis (Theriault et
al., 2000).
Since the extract contains the above mentioned phytochemicals, it may be
beneficial for treatment or control of metabolic alterations observed against ISPH induced
cardiac damage.
110
Table 4.1: Qualitative Phytochemical Profile of Ethanolic Extract of B. diffusa
Phytochemicals Presence/Absence Phytochemicals Presence/Absence
Phenols Carboxylic acids -
Flavanoids + Aminoacids +
Saponins Coumarins -
Alkaloids Starch -
Carbohydrates + Terpenoids +
Proteins + Volatile acids -
Glycosides + Resins -
Antraquinones - Steroids
Lignins + Tannins +
Where “+” denotes presence and “–” denotes absence.
111
4.2 In vitro free radical scavenging and antioxidant screening
Nowadays, there is fairly enough evidence that antioxidants present in foods
or plants play a relevant role in the prevention of disease and maintenance of health
(Cantuti-Castelvetri et al., 2000). It was demonstrated that free radicals may adversely
affect cell survival because of membrane damage through the oxidative damage of
lipid, protein and irreversible DNA modification. Antioxidants are agents which
scavenge the free radicals and stop the damage caused by them. They can greatly
reduce the damage due to oxidants by neutralizing the free radicals before they can
attack the cells and prevent damage to lipids, proteins, enzymes, carbohydrates and
DNA (Fang et al., 2002). Antioxidants may function as free radical scavengers,
complexes of pro-oxidant metals, reducing agents and quenchers of singlet oxygen
formation (Andlauer and Furst, 1998). Many antioxidant compounds, naturally
occurring from plant sources, were identified as free radical or active oxygen
scavengers. Recently, interest has increased considerably in finding naturally
occurring antioxidant for use in foods or medicinal materials to replace synthetic
antioxidants, which are being restricted due to their side effects such as
carcinogenicity. Natural antioxidants especially phenolics and flavanoids from tea,
wine, fruits, vegetables and spices are already exploited commercially either as
antioxidant additives or as nutritional supplements (Schular, 1990).
The present in vitro antioxidant studies with BDEEWP also confirmed its
antioxidant potential from the following results.
The extract inhibited lipid peroxides generated by the induction of
Fe3+
/ADP/ascorbate in rat liver homogenate by 7.26 to 68.12 % from 25 to 1000 µg
/ml concentration of BDEEWP in a dose dependent manner as presented in the Figure
4.1. This effect of BDEEWP was compared with well known natural isolated
antioxidant curcumin as a standard. The IC50 values of BDEEWP and Curcumin for
inhibition of LPO are 717.36 and 76 µg/ml respectively. Thus, BDEEWP showed
protection against LPO induced by Fe3+
-ADP-ascorbate system in rat liver
homogenate. This protection could be due to several mechanisms, such as (a)
chelation of iron (b) by reducing the formation of free radicals (c) by scavenging the
˙OH radicals, ˙O2 radicals and other reactive oxygen molecules, (d) by supplying a
competitive substrate for unsaturated lipids in the membrane which are responsible
112
for LPO. In order to understand the protective role of BDEEWP against LPO, its
capacity to scavenge ROS such as hydroxyl radicals, hydrogen peroxide, superoxide
radicals, nitric oxide, DPPH radical and its reducing power were assessed in the
present study. Results from this study revealed that BDEEWP has scavenging activity
against above said free radicals with efficient reducing capacity.
Hydroxyl radicals are capable of abstracting hydrogen atoms from membrane
and bring about peroxide reactions of lipids (Kitada et al., 1979). Hydroxyl radicals
are generated by a mixture of Fe 3+
, ascorbate and H2O2 in the presence of a slight
molar excess of EDTA over the Fe 3+
salt (Gutteridge, 1987). Hydroxyl radical is
another damaging radical with a half life of 10 sec. In the present study, BDEEWP at
a concentration of 25 to 1000 μg/ml showed 11.6 to 75.75 % inhibition of hydroxyl
radical formation with IC50 of 326.9 μg/ml. Curcumin at different concentrations
ranging in between 5 to 150 μg/ml showed 7.65to 85.13 % inhibition of hydroxyl
radical formation with IC50 of 53.13 μg/ml (Figure 4.2).
Nitric oxide (NO) radical generation at physiological pH from sodium
nitroprusside was inhibited by BDEEWP from 25 to 1000 μg/ml. The scavenging of
NO˙ increased gradually from 7.95 to 67.06 % with an IC50 of 408.5 μg/ml (Figure
4.3). Curcumin at different concentrations ranging from 5 to 150 μg/ml showed 11.38
to 85.13 % inhibition of nitric oxide radical generation with an IC50 of 37.9 μg/ml
(Figure 4.3). Of the reactive nitrogen species (RNS), NO is the most important
mediator of oxygen radical injury because it contains an unpaired electron that readily
combines with many free radicals. Many tissues are routinely exposed to substantially
higher concentrations of NO˙
than oxygen radicals (Beckman et al., 1994).
Furthermore, pathological conditions can greatly stimulate the inducible form of nitric
oxide synthase in many types of tissues. The target molecules of NO are intracellular
thiol metal containing proteins and low molecular weight thiol like GSH and cysteine.
1,1-Diphenyl-2-picrylhydrazyl (DPPH˙) is a free radical compound widely
used to test the free radical scavenging ability of various samples (Hatano et al.,
1997). The effect of antioxidants on DPPH˙ scavenging was thought to be due to their
hydrogen donating ability. DPPH˙ is a stable free radical and accepts an electron or
hydrogen radical to become a stable diamagnetic molecule (Soares et al., 1997). The
data presented in the Figure 4.4 shows remarkable decrease in the concentration of
113
DPPH˙ due to scavenging ability of BDEEWP. The free radical scavenging activity
gradually increased (11.58 to 78.56 %) with increase in concentration of BDEEWP
(25 to 1000 μg/ml) and showed IC50 at 200.2 μg/ml, whereas the standard curcumin
showed IC50 at 48.4.0 μg/ml.
Superoxide is a free radical that can arise from physiological processes.
Superoxide radical is known to be generated during several metabolic processes in the
respiring cells. This is due to the leakage of electrons from the respiratory chain that
will reduce oxygen (Halliwel, 1991). In majority of cases, these radicals are generated
enzymatically. This, besides being itself toxic, generates more toxic species such as
˙OH which promote LPO. Superoxide radical generated in PMS-NADH systems by
oxidation of NADH was assayed by the reduction of Nitro blue tetrazolium. In
superoxide scavenging test, (Figure 4.5) inhibition of ˙O2 formation was observed
(from 19.59 to 70.31 %) at 25 to 1000 µg/ml of BDEEWP, while curcumin showed
8.49 to 66.69% inhibition at concentrations of 5 to 150 µg/ml. These results indicate
that BDEEWP has reasonable scavenging capacity of O2 radicals.
The scavenging capacity of both ˙O2 and NO˙ by BDEEWP reveals preventive
role against highly toxic peroxynitrite (ONOO-) radical formation. The ONOO
- is
another powerful oxidant that interacts with a wide range of targets to cause tyrosine
nitration, thiol oxidation, LPO, DNA strand break, guanosine nitration/oxidation and
ultimately cell death (Irshad and Chaudhuri, 2002). During its decomposition at
physiological pH, ONOO- can produce some of the strongest oxidants known in a
biological system, initiating reactions characteristic of ˙OH radicals, nitronium ion,
and nitrogen dioxide. The unusual stability of ONOO- as an anion contributes to its
toxicity by allowing it to diffuse far from its site of formation while being selectively
reactive with cellular targets.
The reducing capacity of a compound may serve as a significant indicator of
its potential antioxidant activity. Reducing power of BDEEWP increased (23.42 to
78.78 %) with increase in concentration (from 25 to 1000 μg/ml) and showed 50 % of
reducing power at 500.0 μg/ml, whereas curcumin showed 50 % of reducing power at
15.0 μg/ml (Figure 4.6).
114
Figure 4.1:% Inhibition of lipid peroxidation
Figure 4.2: Hydroxyl radical inhibition activity
0
10
20
30
40
50
60
70
80%
In
hib
itio
n
Concentration (µg/ml)
BDEEWP
CURCUMIN
IC50-717.36
0
10
20
30
40
50
60
70
80
90
% I
nh
ibit
ion
Concentration in μg/mL
BDEEWP
CURCUMIN
IC50-53.13
IC50-326.9
115
Figure 4.3: Inhibition of Nitric oxide radical formation
Figure 4.4: Scavanging capacity against DPPH
0
20
40
60
80
100
120
5
10
15
20
25
30
40
50
60
70
80
100
125
150
200
300
400
500
600
800
100
0
% I
nh
ibit
ion
Concentration (µg/ml)
BDEEWP
Curcumin
IC50-37.87
0
10
20
30
40
50
60
70
80
90
100
% I
nh
ibit
ion
Concentration (µg/ml)
BDEEWP
Curcumin
IC50-48.4
IC50-200.2
116
Figure 4.5: Superoxide radical scavenging activity
Figure 4.6: reducing ability of BDEEWP
0
10
20
30
40
50
60
70
80
5
10
15
20
25
30
40
50
60
70
80
100
125
150
200
300
400
500
600
800
100
0
% I
nh
ibit
ion
Concentration (µg/ml)
BDEEWP
Curcumin
IC50-59.3
IC50-201.5
0
20
40
60
80
100
120
140
51
01
52
02
53
04
05
06
07
08
01
00
12
51
50
20
03
00
40
05
00
% In
hib
itio
n
Concentration (µg/ml)
BDEEWP
Curcumin
IC50-15.0
IC50-500.0
117
The significant antioxidant activity of BDEEWP against various varieties of
ROS and free radicals observed in the study might be due to the presence of
polyphenols, saponins, flavonoids and alkaloids (Table 4.1). It has been mentioned
that the antioxidant activity of plant might be due to their phenolic compounds. Plant
products including phenols, flavonoids, and tannins in the plants extracts were
reported to be radical scavengers and inhibitors of LPO. The antioxidant properties of
phenolic acids and flavonoids are due to their redox properties, ability to chelate
metals and quenching of singlet oxygen (Rice-Evans et al., 1996). When
phytochemical compounds react with a free radical, it is the delocalization of the
gained electron over the phenolic antioxidant and the aromatic nucleus that prevents
the continuation of the free radical chain reaction. This is often called “Radical
Scavenging”. But polyphenolic compounds inhibit oxidation through a variety of
mechanisms (Cuvelier et al., 1992).
4.3 Qualitative fingerprint of BDTALK by GC – MS
Several phytochemical screening studies have been carried out in different
parts of the world using GC-MS (Sangeetha and Vijayalakshmi, 2011). In the present
study, the chemical profile of BDTALK fractions of B. diffusa was identified using
GC- MS. The gas chromatogram shows the relative concentrations of various
compounds getting eluted as a function of retention time. The mass spectrometer
analyses the compounds eluted at different times to identify the nature and structure
of the compounds. The large compound fragments into small compounds giving rise
to appearance of peaks at different m/z ratios. These mass spectra are fingerprint of
that compound which can be identified from the data library.
Figures 4.7 to 4.13 show the mass spectrum and structures of the major phyto
compounds. Six active components were identified in BDTALK fraction. Viz, Methyl
Valinate, 2,4, Di Tert Butyl Phenol, Methyl Ferulate, Syringic Acid, Loliolide and
7,9-Di-Tert-Butyl-1-Oxaspiro(4,5)Deca-6,9-Diene-2,8-Dione. The active principles
with their retention time (RT), molecular formula, molecular weight (MW), nature of
the compound and their biological activities have been tabulated in Table 4.2. The
biological activities listed are based on Dr. Dukes Phytochemical and Ethnobotanical
Data bases by Dr. Jim Duke of the Agricultural Research Service, USDA. The
presence of various bioactive compounds confirms the therapeutic applications of B.
118
diffusa for various ailments by traditional practitioners. However, isolation of
individual phytochemical constituents may be undertaken to find a novel drug.
Table 4.2: Chemical Characterization of Phytochemical Constituents of
BDTALK
Peak Retention
Time
Molecular
Formula
Molecu
lar
Weight
Name Of The
Compound
Compound
Nature Activities
1 14.91 C9H10O4 182.17
Methyl Valinate (4-
Hydroxy, 3- Methoxy
Benzoic Acid Ester)
Aromatic
Methyl
Esters
Anti Cancer,
Antibacterial
2 14.96 C14H22O 206.32 2,4,Di Tert Butyl
Phenol
Alkylated
Phenol Antioxidant
3 17.13 C11H12O4 208.21
METHYL
FERULATE (2
Propenoic Acid 3-(4-
Hydroxy-3-Methoxy
phenyl) Methyl Ester
Aromatic
Methyl
Esters
Anti
Microbial,
hypoglycemic
Antioxidant
4 17.78 C9H10O5 198.17
SYRINGIC ACID (4-
Hydroxy-3,5-Di
Methoxy benzoic
Acid)
Aromatic
Methyl
Esters
Antioxidant
5 17.95 C11H16O3 196.24
LOLIOLIDE (6-
hydroxy-4,4,7a- Tri
methyl-6,7-Dihydro-
5H-1- Benzofuran-2-
One)
Mono
terpenoid
Antioxidant
Cell
protective
6 19.33 C17H24O3 276.00
7,9-Di-Tert-Butyl-1-
Oxaspiro(4,5)Deca-
6,9-Diene-2,8-Dione
Ketone Antimicrobi
al
119
Figure 4.7(a): BDTALK GC-MS Parent Spectrum
Figure 4.7(b): BDTALK GC-MS Parent Spectrum (Enlarged R. Time 16.30 to
18.20 min)
Normal spectrum
120
Figure 4.8: BDTALK GC-MS signal of Methyl Valinate, m/z 182
Peak: 1 R.Time: 14.91
Figure 4.9: BDTALK GC-MS signal of 2,4, Di-tert butyl phenol, m/z 206
Peak: 2 R.Time: 14.96
121
Figure 4.10: BDTALK GC-MS signal of Methyl Ferulate, m/z 208
Peak: 3 R.Time: 17.13
Figure 4.11: BDTALK GC-MS signal of Syringic Acid, m/z 212
Peak: 4 R.Time: 17.78
122
Figure 4.12: BDTALK GC-MS signal of Loliolide, m/z 212
Peak:5 R.Time: 17.95
Figure 4.13: BDTALK GC-MS signal of 7,9-di-tert-butyl-1-Oxaspiro (4.5) deca-
6,9-diene-2,8-dione m/z 276
Peak: 6 R.Time: 19.33
123
Phytochemicals exhibit a wide range of biological effects as a consequence of
their antioxidant properties. Several types of polyphenols (phenolic acid, hydrolysable
tannins and flavonoids) show anticarcinogenic and antimutagenic effects (Uruquiaga
and Leighton, 2000). Polyphenols might interfere in several steps that lead to the
development of malignant tumors, inactivating carcinogens, inhibiting the expression
of mutagens .Several studies have shown that in addition to their antioxidant effect,
polyphenols, particularly flavonoids inhibit the initiation, promotion and progression
of tumors (Okwu, 2004). Alkaloids protect against chronic diseases. Saponins protect
against hypercholesterolemia and possess antibiotic properties. Steroids and
triterpenoids show analgesic properties. The steroids and saponins are responsible for
proper functioning of central nervous system (Amin et al., 2013). It was earlier
recorded that bitter leaf contains an alkaloid which is capable of reducing headaches
associated with hypertension (Amin et al., 2013).
4.4 General Observations
Mortality was not observed in any of the experimental groups throughout the
experimental period. No visible side effects and variation in animal behaviour
(respiratory distress, abnormal locomotion and catalepsy) were observed in pretreated
(BDEEWP and BDTALK) groups representing the non-toxic nature of ethanolic
extract and alkaloids of B. diffusa. The plant has drawn lot of attention due to its uses
in Indian traditional medicine, various parts of the plant were used in the treatment of
cancer, jaundice, dyspepsia, inflammation, enlargement of spleen, abdominal pain and
as an anti-stress agent (Kirtikar and Basu, 1956; Chakraborti and Handa, 1989; Leslie
Taylor, 2005). The Acute and subchronic toxicity studies of Orish Ebere Orisakwe et
al. (2003) demonstrated the non-toxic nature of aqueous leaf extract of B. diffusa with
LD50 value as greater than 2000mg/kg body weight. The animals treated with the plant
extracts showed increase in body weights. This could be due to increased food and
water intake of these animals. The fact that B. diffusa extracts increase body weight of
experimental animals has been reported (Wheeler, 1997).
124
4.6 Effect of B. diffusa on Heart weight
Table 4.3 represents the effect of BDEEWP on the heart weight in normal
control and ISPH treated rats. We observed a significant (P< 0.05) increase (36.85%)
in the heart weight in ISPH administered rats as compared to control rats. Pre-
treatment with BDEEWP significantly decreased (79.08%) the heart weight in ISPH-
administered rats. There was a decrease (12.55%) in the final body weight of ISPH
administered groups compare to control group. The BDEEWP pre-treated group
showed a significant increase (100.49%) in the final body weight when compare to
ISPH administered group. The rats administered with ISPH showed significant
increase (57.02%) in heart weight to body weight ratio compared to control rats
whereas rats pre-treated with BDEEWP showed a significant decrease (85.71%) than
ISPH administered rats. Our present results are in agreement with the previous reports
of Nayira et al (2009) who finds that the administration of amlodipine markedly
reduced the heart to body weight ratio in ISPH induced MI rats. The rats treated with
BDEEWP maintained normal heart weight, and heart weight to body weight ratio
when compared to control group.
Table 4.4 represents the effect of BDTALK on the heart weight, body weight
and relative heart weight i.e., heart to body weight ratio in normal control and ISPH
treated rats. We observed a significant (P<0.05) increase (42.86%) in the heart weight
in ISPH administered rats as compared to control rats. Pre-treatment with BDTALK
significantly decreased (62.50%) the heart weight in ISPH-administered rats. There
was a decrease of 9.25% in the final body weight of ISPH administered groups
compare to control group. The BDTALK pre-treated group showed a significant
increase (100.00%) in the final body weight when compare to ISPH administered
group. The rats administered with ISPH showed significant increase (58.82%) in
relative heart weight compared to control rats whereas rats pre-treated with BDTALK
showed a significant decrease (70.00%) than ISPH administered rats. The rats treated
with BDTALK maintained normal heart weight, body weight and heart weight to
body weight ratio when compared to control group.
125
Table 4.3: Effect of BDEEWP on body weight and heart weight of control and
experimental rats
Parameter CONTROL ISPH BDEEWP ISPH+BDEEWP
Body weight (g) 269.5 ± 5.13a 235.67± 12.18
c 291.67 ± 8.06
b 269.67 ± 5.57
a
Heart weight (g) 1.00 ± 0.01a 1.36 ± 0.01
c 1.11 ± 0.02
b 1.07 ± 0.01
a,b
H.W/B.W Ratio 0.37 ± 0.00a 0.58 ± 0.01
b 0.38 ± 0.01
a 0.40 ± 0.00
a
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
Table 4.4: Effect of BDTALK on body weight and heart weight of control and
experimental rats
Parameter CONTROL ISPH BDTALK ISPH+BDTALK
Body weight (g) 164.00±5.42a 148.83±2.74
b 167.33±2.31
a 164.00±1.84
a
Heart weight (g) 0.56±0.01a 0.80±0.03
c 0.61±0.02
a,b 0.65±0.02
b
H.W/B.W Ratio 0.34±0.01a 0.54±0.02
c 0.37±0.01
a,b 0.40±0.01
b
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
126
The Significant increase in the heart weight of ISPH-treated rats has been
attributed to increased water content, formation of oedematous intramuscular spaces,
extensive necrosis of cardiac muscle fibres and invasion by inflammatory cells
(Thounaojam et al., 2010). The decrease in the body weight might be due to the
reduced intake of food and water. BDEEWP and BDTALK pretreatment prevented
the ISPH toxicity and responsible for increasing the body weight of rats, which further
supported by histopathalogical studies of the heart tissues of control and experimental
rats. These results were in agreement with those of Nayira et al., (2009) and Aman et
al., (2009). Our results were also consistent with the previous reports of Gayathri et
al., (2002); Nirmala & Puvanakrishnan (2004) and Chauhan (2005). They stated that,
the rats injected with ISPH showed significant increase in heart weight accompanied
by decrease in body weight. This increase in heart weight to body weight ratio by
ISPH administration may be due to increased proliferation of non-contractile protein
collagen in heart muscle (Kumar & Sharma, 2007).
4.6 Effect of B. diffusa on Biochemical Marker Enzymes
The serum enzymes AST, ALT, ALP, LDH, CK, GGT and cardiac specific
proteins like troponins serve as sensitive indices to assess the severity of MI (Nigam,
2007). In this study, significant decline was shown in the activities of cardiac markers
such as AST, ALT, ALP, LDH, CK and GGT in the heart of acute ISPH-treated rats.
Decreased activities of these enzymes were due to the leakage from the damaged
heart tissues into the blood stream as a result of necrosis induced by ISPH in rats.
Senthil et al., (2007) observed that these cardio-specific marker enzymes are released
from the heart into the blood during myocardial damage due to myofibril degeneration
and myocyte necrosis. Significant increase was noticed in the activities of these
markers (AST, ALT, ALP, LDH, CK and GGT) in serum of ISPH treated rats might
be due to enhanced susceptibility of myocardial cell membrane to the ISPH mediated
peroxidative damage, resulting in increased release of these diagnostic marker
enzymes into the systemic circulation, which is in consistent with earlier reports
(Karthikeyan et al., 2007; Rajadurai & prince, 2007 and Raju et al., 2008).
127
The cardiotoxicity in the male albino wistar rats induced by ISPH
administration in the present study was evaluated primarily, by measuring
biochemical marker enzymes. Table 4.5 represents the effect of BDEEWP on the
activities of the marker enzymes in the serum of control and ISPH treated rats. The
levels of biochemical marker enzymes such as AST, ALT, ALP, LDH, CK and GGT
increased significantly by 79.03%, 94.44%, 90.66%, 65.24%, 71.03% and 62.56%
respectively in serum of ISPH administrated rats when compared to control rats. Upon
pre-treatment of rats with BDEEWP, the condition was reversed by decreasing the
levels of these marker enzymes significantly in serum of ISPH induced myocardial
infracted rats. BDEEWP decreased the activities of enzymes significantly (P<0.05) by
69.65%, 83.32%, 74.60%, 67.52%, 79.18% and 77.83% when compared to ISPH
administered rats.
Data represented in Table 4.6 show the effect of BDEEWP on the activities of
AST, ALT, ALP, LDH, CK and GGT in heart tissues of control and ISPH treated rats.
ISPH administration showed significant (P<0.05) decrease in the activities of AST,
ALT, ALP, LDH, CK and GGT by 30.27%, 28.90%, 29.74%, 25.69%, 44.37% and
63.36% respectively in heart tissue. Pre-treatment with BDEEWP increased the
activities of these enzymes significantly (P<0.05) by 55.93%, 58.53%, 82.69%,
26.03%, 57.24 % and 62.69% in heart tissue.
128
Table 4.5 Effect of BDEEWP on activity of marker enzymes in serum of control
and experimental groups
Marker
enzymes
(IU/L)
CONTROL ISPH BDEEWP ISPH+BDEEWP
AST 158.06 ± 0.45a 282.97 ± 1.14
c 159.93 ± 0.33
a 195.97 ± 0.94
b
ALT 44.94 ± 0.32a 87.39 ± 0.42
c 46.42 ± 0.29
a 52.02 ± 0.15
b
ALP 147.29 ± 0.45a 280.83 ± 1.09
c 150.57 ± 0.33
a 181.21 ± 0.36
b
LDH 620.93 ± 0.94a 1026.01 ± 1.09
c 622.71 ± 1.17
a 752.49 ± 0.51
b
CK 541.76 ± 1.22a 926.59 ± 0.72
c 543.21 ± 0.93
a 621.87 ± 1.47
b
GGT 7.28 ± 0.03a 11.84 ± 0.14
c 7.14 ± 0.01
a 8.29 ± 0.04
b
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.0, Duncan‟s Multiple range Test).
Table 4.6: Effect of BDEEWP on marker enzymes in heart tissue of control and
experimental groups
Marker
Enzyme
(U/mg
Protein)
CONTROL ISPH BDEEWP ISPH+BDEEWP
AST 46.70 ± 0.32a 32.57 ± 0.44
c 46.19 ± 0.40
a 40.47 ± 0.23
b
ALT 48.74 ± 0.38a 35.63 ± 0.46
c 47.98 ± 0.27
a 43.31 ± 0.23
b
ALP 102.54 ± 0.40a 72.04 ± 0.35
b 102.50 ± 0.29
a 97.26 ± 0.59
b
LDH 366.11 ± 0.85a 272.07 ± 2.27
d 353.99 ± 0.57
b 296.55 ± 0.56
c
CK 89.99 ± 0.34a 50.07 ± 0.48
c 90.90 ± 0.42
a 72.92 ± 0.25
b
GGT 6.55 ± 0.07a 2.40 ± 0.05
c 6.53 ± 0.02
a 5.00 ± 0.03
b
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-d)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
129
Table 4.7 represents the effect of BDTALK on the activities of the marker
enzymes in the serum of control and ISPH treated rats. The levels of marker enzymes
such as AST, ALT, ALP, LDH, CK and GGT increased significantly by 107.34%,
101.98%, 84.36%, 94.88%, 110.69% and 77.58% respectively in serum of ISPH
administrated rats when compared to control rats. Upon pretreatment of rats with
BDTALK, the condition was reversed, where the levels of these cardiac marker
enzymes decreased significantly in the serum of ISPH induced myocardial infracted
rats. BDTALK decreased the activities of enzymes significantly (P<0.05) by 96.17%
48.00% 76.55% 70.18% 46.84% and 82.49% respectively when compared to ISPH
administered rats.
Table 4.8 shows the effect of BDTALK on the activities of AST, ALT, ALP,
LDH, CK and GGT in heart tissues of control and ISPH treated rats. ISPH
administration showed significant (P<0.05) decrease in the activities of AST, ALT,
ALP, LDH, CK and GGT 32.40%, 39.63%, 37.59%, 41.18%, 56.04% and 57.58%
respectively in heart. Pre-treatment with BDTALK increased the activities of these
enzymes significantly (P<0.05) by 88.93%, 70.65%, 140.62% 45.29%, 42.41% and
63.74% respectively in heart tissue.
The serum enzymes namely AST, ALT, ALP, LDH, CK and GGT serve as
sensitive indices to assess the severity of MI. In the present investigation, we
observed a decrease in the activities of AST, ALT, ALP, LDH, CK and GGT in hearts
of ISPH administered rats indicate the myocardial damage which is in consistent with
earlier reports (Kurian et al., 2005). When myocardial cells are damaged due to the
deficient oxygen supply or glucose, the integrity of cell membrane gets disturbed and
it might become more porous that result s in the leakage of these enzymes (Arya et al.,
2006). As a result of necrosis and leakage, the levels of diagnostic indicators of MI
will increase in serum.
130
Table 4.7: Effect of BDTALK on activity of marker enzymes in serum of control
and Experimental groups
Marker
Enzyme
(IU/L)
CONTROL ISPH BDTALK ISPH+BDTALK
AST 116.02 ± 2.02a 240.56 ± 0.76
b 118.24 ± 1.78
a 120.79 ± 0.79
a
ALT 33.28 ± 0.78a 67.22 ± 0.53
c 34.54 ± 0.53
a 50.93 ± 0.90
b
ALP 133.39 ± 1.05a 245.92 ± 3.98
c 131.69 ± 1.15
a 159.78 ± 1.07
b
LDH 507.69 ± 1.41a 989.39 ± 5.05
c 501.45 ± 3.63
a 651.33 ± 1.10
b
CK 346.36 ± 4.63a 727.67±2.43
c 348.80±1.23
a 549.07±1.17
b
GGT 6.11 ± 0.30a 10.85±0.13
c 6.36±0.10
a 6.94±0.03
b
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
Table 4.8: Effect of BDTALK on Activity of marker enzymes in heart tissue of
control and Experimental groups
Marker
Enzyme
(U/mg
protein)
CONTROL ISPH BDTALK ISPH+BDTALK
AST 32.07 ± 0.60a 21.68 ± 0.45
c 34.61 ± 0.73
b 30.92 ± 1.14
a
ALT 37.57 ± 0.80a 22.68 ± 0.36
c 37.80 ± 0.53
a 33.20 ± 0.80
b
ALP 81.41 ± 0.43a 50.81 ± 0.22
c 83.10 ± 1.01
a 93.84 ± 1.01
b
LDH 259.48 ± 1.56a 152.63 ± 0.48
c 257.05 ± 1.96
a 201.02 ± 0.85
b
CK 78.73 ± 0.55a 34.61 ± 0.28
c 78.27±0.92
a 53.32±0.74
b
GGT 6.30 ± 0.06a 2.66 ± 0.09
a 6.09±0.06
b 4.98±0.15
c
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
131
Oral pre-treatment of BDEEWP and BDTALK restored the activities of
myocardial marker enzymes. This could be due to the protective effect of BDEEWP
and BDTALK on the myocardium, reducing the myocardial damage and there by
restricting the leakage of AST, ALT, ALP, LDH, CK and GGT. Our present results are
in agreement with that of Rawat et al., (1997), who also reported the protective role of
B. diffusa on liver by measuring marker enzymes. Similar observation was reported by
Kubavat and Asdaq (2009). BDEEWP decreased the activities of marker enzymes in
the serum and increase the activities of marker enzymes in the heart tissue of ISPH
administered rats which could be due to free radical scavenging property of the extract
due to the presence of phytochemicals such as Alkaloids, Flavonoids, Anthraquinones,
Tanins, Steroids, Saponins, Anthocyanidins and terpenoids (Kameswara rao et al., 2003
and Malaya gupta et al., 2002). These phytochemicals have been proved for their
antioxidant activity which may be responsible for the cardioprotective effect of extract
against ISPH induced MI.
4.7 Effect of B. diffusa on lipid metabolism
Lipids play a very important role in CVD by the way of hyperlipidemia,
development of atherosclerosis, modifying the cellular membrane composition,
structure and stability (Rajadurai & Prince, 2005). Hyperlpidemia leads to progressive
atherosclerosis, which results in blockage of coronary arteries, interruption of blood
supply to parts of heart, leading some heart cells to die which is known as MI (damage
and death of heart muscles) and ischemia (restriction in blood supply and oxygen
shortage) (Radhika et al., 2011). Hypercholesterolemia, high concentration of LDL
cholesterol, hyper triglyceridemia and low concentration of HDL cholesterol are
reported as the independent risk factors for atherosclerotic cardiovascular disease and
mortality (Wood et al., 1998; Gotto et al., 2000).
4.7A Effect of BDEEWP and BDTALK on Lipid Profile of Plasma and Heart
Figure 4.14 to 4.16 represents the levels of TC, TG, FFA, PL and C/P ratio in
plasma of control and experimental rats. Intraperitoneal administration of ISPH caused
a significant (P<0.05) rise in the levels of cholesterol (71.28%), TG (59.47%), FFA
(28.54%), PL (16.38%) and C/P ratio (47.38%) in plasma of ISPH alone administered
rats when compared to control rats. In BDEEWP pre-treated rats, alteration was
minimized in the levels of cholesterol (90.89%), TG (50.15%), FFA (175.67%), PL
132
(101.91%) and C/P ratio (84.83%) when compared to ISPH alone injected rats.
BDEEWP treatment maintained these levels near normal and found to be non-
significant.
Figure 4.14: Effect of BDEEWP on plasma lipid profile in control and
experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
133
Figure 4.15: Effect of BDEEWP on plasma FFA in control and experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
Figure 4.16: Effect of BDEEWP on plasma C/P Ratio in control and
experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
134
Figure 4.17 shows the effect of BDEEWP on myocardial lipid profile. The
TC (61.84%), TG (79.84%), FFA (42.46%) except PL showed, a significant (P<0.05)
increase. whereas PL showed a significant decrease (37.71%) in the ISPH alone
treated rats when compared to control rats.. BDEEWP pretreatment blocked the
amendment of heart lipids (FFA 87.56%, PL 60.56%, TC 82.13%, TG 90.18%) to
moderate level when compared to ISPH administered group, whereas near normal
values were maintained with in rats treated BDEEWP alone.
High levels of circulatory cholesterol and its accumulation are well associated
with cardiovascular damage (Joan et al., 1984). The increased levels of plasma
cholesterol and triglycerides observed in ISPH injected rats could be due to enhanced
lipid biosynthesis by cardiac cyclic adenosine monophosphate (Paritha and Devi
1987). Plasma hyper triglyceridaemia, which was observed in ISPH-treated rats, is
due to decrease in the activity of lipoprotein lipase, resulting in decreased uptake of
triglycerides from circulation. Hypertriglyceridaemia and increased levels of
cholesterol in plasma might be responsible for altered cardiovascular functions which
are often reported in ISPH induced MI (Freedman et al., 1988). Sreepriya et al, (1998)
reported that increased peroxidation of membrane lipids, causing release of PL, FFA
via phospholipase-A2, is responsible for increase in plasma PL and FFA. It is
proposed that ISPH like catecholamines produce ROS which increase peroxidation of
membrane lipids. The increased level of free fatty acids in plasma of animals treated
with ISPH is due to increased lipolysis.
135
Figure 4.17: Effect of BDEEWP on Cardiac lipid profile in control and
experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
136
Figure 4.18 to 4.20 represents the levels of TC, TG, FFA, PL and C/P ratio in
plasma of control and experimental rats. Intraperitonial administration of ISPH caused
a significant (P<0.05) rise in the levels of cholesterol (63.36%), TG (50.78%), FFA
(53.93%), PL (34.36%) and C/P ratio (21.30%) in plasma of ISPH alone administered
rats when compared with control rats. In BDTALK pre-treated rats alteration was
minimized in the levels of cholesterol (77.29%), TG (29.39%), FFA (129.94%), PL
(64.30%) and C/P ratio (91.30%) when compared with ISPH alone injected rats.
BDTALK treatment maintained these levels near normal and found to be non-
significant.
The levels of myocardial lipids, TC (66.48%), TG (82.34%), FFA (42.86%)
except PL showed a significant (P<0.05) increase, where as PL showed a significant
decrease (33.51%) in the ISPH alone treated rats when compared to control rats.
BDTALK pre-treatment blocked the amendment of heart lipids (FFA 80.75%, PL
44.27%, TC 74.36%, and TG 92.74%) to moderate level when compared to ISPH
administered group, whereas near normal values were maintained with in rats treated
BDTALK alone (Figure 4.21).
Antilipidemic properties have been present in many plant species and this
property in plants is confirmed by many scientific studies (Devi and Sharma 2004;
Sivakumar et al., 2007; Jain et al., 2007, Radhika et al., 2011). Many plants have
shown potent antilipidemic effect. Terminalia arjuna significantly lower the elevated
level of cholesterol (Ram et al., 1997; Tiwari 1990). The hypolipidemic effects of
BDTALK might be due to the presence of punarnavine alkaloid, since alkaloids act at
both endothelium and the underlying vascular smooth muscle to induce relaxation.
Our results are collaborated with the vasorelaxant and antiproliferative effects of
alkaloid, berberine in the vascular system (Ko et al., 2000) and could play a major
role in the management of metabolic diseases associated with high CVD risk (Cicero
and Ertek, 2009).
137
Figure 4.18 Effect of BATALK on plasma lipid profile in control and
experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
Figure 4.19 Effect of BATALK on plasma FFA in control and experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
138
Figure 4.20 Effect of BATALK on plasma C/P RATIO in control and
experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
Figure 4.21: Effect of BDTALK on Cardiac lipid profile in control and
experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
139
Our findings suggest that the extracts had an effective hypolipidemic agent
and may protect against cardiovascular diseases that result from hyperlipedimia. The
observed properties of BDEEWP and BDTALK could be due to the presence of
polyphenolic compounds like flavonoids and alkaloids. The pretreatment of total
alkaloids and ethanolic extract of B. diffusa significantly prevented the degradation of
PL thereby increasing PL levels and decreasing the ratio of cholesterol to
phospholipids (C/P). It might be due to decrease in membrane lipolysis by decreasing
the action of phospholipase A2 through the depletion of intracellular calcium levels.
The results clearly demonstrate the Antiatherogenic nature of BDEEWP and
BDTALK against ISPH action.
4.7B Effect of BDEEWP and BDTALK on lipoproteins
Lipids are being insoluble in water need a transport system made up of
lipoproteins such as chylomicrons, VLDL-C, LDL-C and HDL-C. Estimation of these
lipoproteins is used as an index to measure the levels of lipids present in the plasma.
Figure 4.22 shows significant changes in the plasma lipoprotein levels between the
normal and ISPH treated rats. In ISPH administered rats, significant (P<0.05) increase
in the levels of LDL-C and VLDL-C by 170.04% and 40.22% followed by parallel
decrease in HDL –C by 48.47% were observed when compared to control rats.
Nevertheless, the levels were statistically similar in control and BDEEWP alone
treated rats. In BDEEWP pretreated rats, a fall in the HDL-C level is partially
prevented (63.10%) and further increase in the levels of LDL-C and VLDL-C is
inhibited by 87.42% and 26.28% respectively when compared to ISPH administered
rats. Prior treatment with 45 days restored the levels of AI to control levels, but
administration of ISPH enhanced this level by 386.94% which is statistically
significant (P<0.05). Rats pre-treated with BDEEWP followed by ISPH
administration showed a significant decrease in this level by 87.37% compared to
control (Figure 4.23).
Figure 4.24 represents the effect of BDEEWP on heart lipoprotein levels in
normal control and ISPH treated rats. ISPH administered rats showed a significant
increase (P<0.05) in the concentrations of heart LDL-C and VLDL-C (295.29% and
79.46%), whereas a significant decrease (P<0.05) in the concentration of heart HDL-
C (44.31%) when compared with those of normal rats. Pre-treatment with BDEEWP
140
to ISPH treated rats decrease the levels of heart LDL-C and VLDL-C (78.95% and
180.35%) significantly (P<0.05), whereas the levels of HDL-C in heart (78.87%) of
ISPH induced myocardial infracted rats increased when compared with untreated
ISPH induced myocardial infarcted rats.
Figure 4.22: Effect of BDEEWP on Plasma lipoproteins in control and
experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
141
Figure 4.23: Effect of BDEEWP on Plasma Atherogenic Index in control and
experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
Figure 4.24: Effect of BDEEWP on cardiac lipoproteins in control and
experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
142
Figure 4.25 shows significant changes in the plasma lipoprotein levels
between the normal and ISPH treated rats. In ISPH administered rats, significant
(P<0.05) increase in the levels of LDL-C and VLDL-C by 138.29% and 50.76%
followed by parallel decrease in HDL –C by 41.05% were observed with control rats.
Nevertheless, the levels were statistically similar in control and BDTALK alone
treated rats. In BDTALK pre-treated rats, a fall in the HDL-C level is partially
prevented (60.06%) and further increase in the levels of LDL-C and VLDL-C is
inhibited by 77.20% and 29.40% respectively when compared to ISPH administered
rats. Treatment with 15 days restored the levels of AI to control levels, but
administration of ISPH enhanced this level by 278.61% which is statistically
significant (P<0.05). Rats pre-treated with BDTALK followed by ISPH
administration showed a significant decrease in this level by 79.05% compared to
control (Figure 4.26).
Figure 4.27 represents the effect of BDTALK on heart lipoprotein levels in
normal control and ISPH treated rats. ISPH administered rats showed a significant
increase (P<0.05) in the concentrations of heart LDL-C and VLDL-C (306.02% and
81.08%), whereas a significant decrease (P<0.05) in the concentration of heart HDL-
C (41.03%) when compared to those of normal rats. Pretreatment with BDTALK to
ISPH treated rats decrease the levels of heart LDL-C and VLDL-C (78.74% and
93.33%) significantly (P<0.05), whereas the levels of HDL-C in heart (102.50%) of
ISPH induced myocardial infracted rats increased when compared with untreated
ISPH induced myocardial infarcted rats .
143
Figure 4.25: Effect of BDTALK on Plasma lipoproteins in control and
experimental rats
Figure 4.26: Effect of BDTALK on Plasma Atherogenic Index in control and
experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
a
a
a
d b
b
b
c
c
c
b
d
0
20
40
60
80
100
120
HDL VLDL LDL
mg/
10
0m
l
CONTROL
ISPH
BDTALK
ISPH+BDTALK
a
d
b
c
0
1
2
3
4
5
6
7
8
CONTROL ISPH BDTALK ISPH+BDTALK
ATH
ERO
GEN
IC I
ND
EX
EXPERIMENTAL GROUPS
144
Figure 4.27: Effect of BDTALK on cardiac lipoproteins in control and
experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-d)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
a
a a
c c
d
b
b
b
a
b
c
0
0.5
1
1.5
2
2.5
3
3.5
4
HDL VLDL LDL
mg/
g W
et
tiss
ue
CONTROL
ISPH
BDTALK
ISPH+BDTALK
145
Lipoproteins are independent risk factor for CVD. LPO plays a fundamental
role in lipoprotein modifications, which makes them susceptible to atherosclerosis.
This could be the reason for AMI mediated cardio toxicity by ISPH (Zern et al.,
2003). LDL molecules become oxidized (LDL-OX) by free radicals particularly
reactive oxygen free radicals (ROS), when this oxidised LDL come in contact with
artery walls, a series of reactions started which damage the artery wall. High
concentration of LDL concentration is a big risk factor for heart diseases (Odetola et
al., 2004; Attar, 2006). ISPH induced myocardial necrosis had been shown to elevate
plasma TC, TG, LDL-C, VLDL-C levels and decreased HDL-C levels (Prince et al.,
2008). These changes in the lipid levels might be due to enhanced lipid biosynthesis
by cardiac C-AMP. Also higher levels of LDL has a positive correlation, where as
high level of HDL has a negative correlation with myocardial necrosis (Buring et al.,
1992). Plasma concentration of atherogenic LDL-C is regulated by the production of
rate of VLDL and the utilization of LDL-cholesterol by LDL receptors. ISPH causes
hyperlipidemia and increases the levels of LDL-C in the blood, which inturn leads to
the build up of harmful deposits in the arteries thus favouring CHD (Rajadurai and
Stanely, 2006).
High plasma levels of VLDL cholesterol is a risk factor for cardiovascular
disease (Ademuyiwa et al., 2005; Lichtenstein et al., 2006) and, often accompanies
diabetes mellitus (Brunzell et al., 2008; Shen, 2007; Rang et al., 2005). VLDL
contains high concentration of TGs and moderate concentration of cholesterol, PLs
and Apo lipoproteins. The breakdown of VLDL leads to the formation of cholesterol
rich particle, LDL (Draznin et al., 1993). Oxidation of lipoproteins is a characteristic
event in the oxidative stress caused by the oxidative damage to PUFAs (Nirmala and
Puvanakrishnan, 1996). Oxidative modification of LDL expresses the chemotactic
and adhesion molecules on the surface of endothelial cells. Macrophages in the sub
endothelial cells intake modified LDL via scavenger receptors, leading to the
formation of lipid loaded foam cells, the hallmark of early atherosclerotic lesions
(Catanzaro and Suen, 1996). HDL –C is involved in the transportation of cholesterol
from peripheral tissue to the liver and it acts as a protective factor against CVD
(Korytar et al., 2002). It also participates in the regulation of TG metabolism and
cholesterol ester synthesis by providing Apo C II for activation and Apo C III for
146
inhibition of lipoprotein lipase activity (Catanzaro and Suen, 1996). Alterations in
lipid metabolism directly reflect the composition of lipoproteins in ISPH treated rats.
In the present study, we observed increased levels of the plasma and heart
LDL-C, VLDL-C and decreased levels of HDL-C in the ISPH treated rats is in
agreement with a previous report of Ananthi et al., (2014). Changes in the membrane
cholesterol content affects its fluidity, permeability to ions, activities of membrane
bound enzymes, and increased degradation of phospholipids. In this study, BDEEWP
and BDTALK pretreated rats showed decreased concentration of plasma and Heart
LDL-C, VLDL-C and increased concentration of HDL-C indicating the beneficial
effects of BDEEWP and BDTALK in reducing hyperlipidemia caused by ISPH
administration by their ability to inhibit cAMP thereby maintaining the normal
fluidity and less alterations in the structure and function of the tissue membranes.
Increased levels of Plasma HDL-C observed in rats pretreatment with BDEEWP and
BDTALK may facilitates the transports of cholesterol from peripheral tissues to the
liver for catabolism and excretion from the body in ISPH treated rats. The
antihyperlipidemic effect of B. diffusa is atributed to the presence of phytochemicals
such as alkaloids, flavonoids, saponins and plant sterols.
4.8 Effect of B. diffusa on Oxidative Stress
4.8 (A) Lipid Peroxidation and Protein Oxidation:
Lipid peroxidation has been implicated in the pathogenesis of a number of
diseases include atherosclerosis, cancer etc., It is now generally accepted that lipid
peroxidation and its product play an important role in liver, kidney, heart and brain
toxicity (Lakshmi et al., 2005). Lipid peroxidation has probably been the most
extensively investigated process induced by free radicals. Lipid peroxides are derived
from the oxidation of polyunsaturated fatty acids of membranes and are capable of
further LPO by a free radical chain reaction (Das et al., 2002). All bio
macromolecules are faced with oxidative stress including proteins. Protein oxidation
is defined as the covalent modification of a protein induced either directly by reactive
ROS or indirectly by reactions with secondary by products of oxidative stress (Sermin
et al., 2007).
147
The effect of BDEEWP on the extent of LPO in heart of control and
experimental groups is shown in Figure 4.28. After the end of the experiment, ISPH
induced group showed significantly enhanced LPO in heart (59.84 %) when compared
to control group. Oral pre administration of BDEEWP to ISPH induced myocardial
infracted rats prevented the increased tissue LPO to almost normal. However,
administration of BDEEWP to normal rats showed slight decrease in tissue LPO
levels when compared to the corresponding values of control group. ISPH induced
group showed significant increase in the level of protein oxidation in heart (27.45%)
compared to normal control group (Figure 4.29). BDEEWP pre supplementation for
45 days prevented the increase in PO and maintained normal values in these tissues.
However, BDEEWP treated control group showed no deviation in the extent of
protein oxidation in heart.
148
Figure 4.28: Effect of BDEEWP treatment on tissue lipid peroxidation in control
and Experimentation rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
Figure 4.29: Effect of BDEEWP treatment on tissue Protein oxidation in control
and Experimentation rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
149
The effect of BDTALK on the extent of LPO and protein oxidation in heart of
control and experimental groups are shown in Figures 4.30 and Figure 4.31. After the
end of the experiment, ISPH induced group showed significantly enhanced LPO in
heart (72.26 %) when compared to corresponding values of control group. Oral pre
administration of BDTALK to ISPH induced myocardial infracted rats prevented the
increased tissue LPO to almost normal bringing the values to normal were observed in
the Figure 4.30. However, administration of BDTALK to normal rats showed slight
decrease in tissue LPO levels when compared to the corresponding values of control
group. ISPH induced group showed significant increase in the level of protein
oxidation in heart (27.24%) compared to normal control group (Figure 4.31).
BDTALK pre supplementation for 15 days prevented the increase in PO and
maintained normal values in these tissues. However, BDTALK treated control group
showed no deviation in the extent of protein oxidation in heart.
150
Figure 4.30: Effect of BDTALK treatment on tissue lipid peroxidation in
control and Experimentation rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-d)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
Figure 4.31: Effect of BDTALK treatment on tissue Protein oxidation in control
and Experimentation rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-d)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
151
4.8 (B) NITRIC OXIDE:
Nitric oxide (NO) is an important bioregulatory molecule in the nervous,
immune and cardiovascular system. The physiological involvement of NO in neural
transmission, immune response-induced cytosis, control of vascular tone and the
maintenance of vascular integrity (Moncada et al., 1991). NO is synthesized from L -
arginine (Palmer et al., 1988a,) by several types of NO synthase in pulmonary cells,
including inflammatory, endothelial and airway epithelial cells. Excess production of
NO, however, has also been implicated as a cause of diverse physiological conditions
such as inflammation, cardiovascular disorders and neuro degenerative diseases.
Nitric oxide is a free radical and has a very short life. Nitric oxide forms
nitrite in solution, a stable metabolite of Nitric oxide. Hence, measurement of nitrite
concentrations in the serum can be used as a marker of the amount of NO formed or
released. Nitrite concentrations in the serum were determined by using Griess reagent
(Lepoivre et al 1990). NO2 reacts with 3% sulfanilamide in 0.3% N – 1 – naphthyl –
ethylenediamine dihydrochloride forming a chromophore (Green et al, 1982).
Figure 4.32 and 4.33 represents the effect of BDEEWP and BDTALK on the
levels of serum NO in control and Experimental rats. The NO levels increased
(76.19%) and 68.57% significantly (P<0.05) in ISPH administered rats when
compared to control rats. In ISPH administered BDEEWP and BDTALK pretreated
rats the NO levels decreased by 21.62 % and 18.27% when compared to ISPH alone
administered rats.
NO is locally acting vasodilator thathas a central role in the regulation of
vascular smooth muscle tone. In addition, it inhibits leukoocytes and platelet adhesion
to the endothelium, leukocyte activation and platelet aggregation, and endothelial
permeability (Fleming and Busse, 1999). It thus optimizes blood flow regulation in
the microcirculation. In the heart, endothelium derived NO modulates myocardial
relaxation and diastolic function and reduces oxygen consumptiom independent of
effects on contractile functions (Shen et al., 1995). NO generated with in cardiac
myocytes by eNOS and posibily also nNOS may modulate excitation-contraction
coupling via effects on sarcolemmal Ca channels and sarcoplasmic reticular function
(Xu et al., 1999).
152
Figure 4.32: Effect of BDEEWP treatment on Nitric oxide in cardiac tissue of
control and Experimentation rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
Figure 4.33: Effect of BDTALK treatment on Nitric Oxide in cardiac tissue of
control and Experimentation rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-d)
in the row differ significantly from each other (p<0.05,Duncan‟s Multiple range Test).
a
c
b b
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
CONTROL ISPH BDTALK ISPH+BDTALK
µM
of
nit
rite
.
EXPERIMENTAL GROUPS
153
Kojda et al., (1996) reported that NO at low concentrations can increase
contractility of cardiac myocytes while high concentrations can attenuate. It remains
to be determined why normal physiological production of NO is protective in the
cardiovascular system and may prevent atheroma formation (Woditsch and Schror,
1994) where as excess NO produced after expression of iNOS and under conditions of
inflammation is potentially harmful. In the present study pretreatment BDEEWP and
BSDTALK decreased the levels of NO in ISPH administered rats which may be
responsible for the production against ISPH induced MI. this protective effect of
BDEEWP and BDTALK may be due to their antioxidant activity which prevents the
formation of superoxide radicals. So these superoxide radicals are not available to
react with NO to produce peroxynitrite which is cytotoxic. Our results are in
accordance with previous report of Peter et al., (2009).
4.8(C) DNA Damage
Oxidative stress can cause to all types of biomolecules, including DNA,
proteins and lipids. In many situations it is unclear that the most important target for
oxidative stress, since injury mechanisms overlap widely. The primary cellular target
of oxidative stress can be varying depending on the cell, the type of stress imposed
and how severe the stress is. For eg Carbon tetra chloride injures cells primarily by
lipid peroxidation. By contrast, DNA is an important early target of damage when
H2O2 is added to many mammalian cells increased DNA strand breakage occurs
before detectable lipid peroxidation or oxidative damage.
In the present study the in vivo treatment of BDEEWP has not resulted in
lymphocyte DNA damage (Table 4.9). A significant protection against H2O2 induced
DNA damage was observed in the lymphocytes of BDEEWP treated rats compared to
normal rats. 82.6% decreased in number of damaged cells and 77.8 % decreased in
tail length of comets (Figure 4.34).
In vitro anti genotoxic effects of BDEEWP against ISPH induced DNA
damage are presented in table 4.10. BDEEWP pretreated lymphocytes showed
decrease in the number of damage cells 99.3% and length of the comets (71.3%)
against ISPH induced DNA damage respectively (Figure 4.35).
154
Table 4.9: Effect of in vivo treatment of BDEEWP against H2O2 induced
lymphocyte DNA damage
parameter
Control rat lymphocytes BDEEWP treated rat
lymphocytes
Normal H2O2 treated normal H2O2 treated
Percentage of
cells showing
migration
1.75 ±0.71a 60.57± 0.65
b 1.50±0.81
a 10.52±0.24
b
Tail length (µm) 0.78±0.12a 5.49±0.91
b 0.75±0.06
a 1.22±0.08
b
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-b)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
Table 4.10: Protective ability of BDEEWP against ISPH induced lymphocyte
DNA damage
Parameter Control rat
lymphocytes
ISPH treated rat
lymphocytes
BDEEWP pretreated
ISPH induced rat
lymphocytes
Percentage of cells
showing migration 0.60±0.5
a 99.5±1.03
b 0.62±0.6
a
Tail length (µm) 0.58±0.21a 4.82±0.19
c 1.38±0.05
b
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
155
Excessive DNA strand breakage is associated with depletion of cellular ATP
and NAD+ levels the latter often occurs because a chromatin bound enzyme poly ADP
ribose polymerase (PARP) splits the NAD+
molecule and transfers the ADP–ribose
portion on to nuclear proteins, including itself (auto modification). ADP– ribosylation
of proteins is thought to facilitate DNA repair. However excessive activation of PARP
an deplete the NAD+ pool, interfering with ATP synthesis and perhaps even leading to
cell death (Szabo et al., 1996). The first demonstration of comets was by Ostling and
Johanson (1984) who describe the tails in terms of DNA with relaxed super coiling
and referred to the nucleiod model of Cook et al., 1976. The comet assay is most
commonly applied to animal cells, whether in culture or isolated from the organism.
Migliore et al., (2004) tested the role of endogenous oxidative stress which is
believed to play a key role in the pathogenesis of mitochondrial diseases, by comet
assay. The comet assay is ideal for investigating nutrient or micronutrient effect at the
level of DNA damage in humans. Diets differing in lipid content lead to changes in
oxidative DNA damage in lymphocytes (Jenkinson et al., 1999). On the other hand,
the protective effects of in vivo supplements of antioxidants, or of foods rich in
antioxidants, are very readily demonstrated in lymphocytes as either a decrease in
endogenous base oxidation (measured with endonuclease III or FPG) or a decreased
sensitivity of H2O2 induced damage in vivo (Pool Zobel et al., 1997 and Duthie et al.,
1996). The results of the present study the antigenotoxic effect of BDEEWP are in
accordance with the previous reports of Karuna et al., (2009) who reported that, the
pretreatment of aqueous extract of P. amarus decreased the DNA damage in
streptozotocin induced rats.
156
Figure 4.34: Effect of in vivo treatment of BDEEWP against H2O2 induced
lymphocyte DNA damage
4.34a Normal Lymphocytes 4.34b Normal Lymphocytes exposed to H2O2
4.34c BDEEWP treated lymphocytes 4.34d BDEEWP treated lymphocytes exposed to H2O2
Figure 4.35: Protective ability of BDEEWP against ISPH induced lymphocyte
DNA damage
4.35a Normal lymphocytes 4. 35b ISPH induced lymphocytes 4.35c BDEEWP Pretreated
lymphocytes
157
4.9 Effect of B. diffusa on Antioxidants
4.9A Effect of BDEEWP and BDTALK on enzymatic Antioxidants
Antioxidants can eliminate free radicals and other reactive oxygen and
nitrogen species, and these reactive species contribute to most chronic diseases. It is
widely accepted that a plant-based diet with high intake of fruits, vegetables, and
other nutrient-rich plant foods may reduce the risk of oxidative stress-related diseases
(Kensler et al., 2007). Reactive oxygen species are generated from the leakage of
electrons into oxygen from various systems (Polidori et al., 2002). Free radical
scavenging enzymes such as catalase, SOD, and GPx are the first line of cellular
defense against oxidative injury, decomposing O2 and H2O2 before their interaction to
form the more reactive hydroxyl radical.
Figure 4.36 indicates the activities of glutathione dependent antioxidant
enzymes (GPx, GST and GRx) and anti peroxidative enzymes (SOD and CAT) in
cardiac tissue of control and experimental rats. The significant (P<0.05) decrease was
observed in the activities of GSH dependent enzymes, GPx (49.41%), GST (56.88%)
and GRx (47.19%) in heart tissue. Further it is also observed that a significant
(P<0.05) decrease in the antiperoxidative enzymes, SOD, CAT by 60.43% and
62.09% respectively is noted in ISPH administered rats compared to controls.
Treatment with BDEEWP increased the activities of antioxidant enzymes by 89.97%,
85.83%, 46.54%, 99.99% and 124.13% in heart respectively for GPx, GST and GRx,
CAT and SOD (Figure 4.36).
The rise in the activities of first line cellular defensive enzymes such as SOD,
CAT and GSH dependent antioxidants in pretreated ISPH challenged group highlight
the protection against the oxidative stress by increasing the removal of super oxide
radicals. Our results are in accordance with previous reports of saayi krushna et al
(2011) and Meenakshi et al (2014) who reported the antioxidant property of aqueous
extract of Aegle marmelos and the ethonolic extract of Microcosmus exasperates.
However, BDEEWP treatment alone does not show much significant change
compared with the control groups in heart tissue.
158
Figure 4.36: Effect of BDEEWP on Cardiac Tissue Antioxidant Enzyme Levels
in Control and Experimental Rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-d)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
159
Figure 4.37 indicates the effect of BDTALK on activities of glutathione
dependent antioxidant enzymes (GPx, GST and GRx) and antiperoxidative enzymes
(SOD and CAT) in hearts of control and experimental rats. There is a significant
(P<0.05) decrease in the activities of GSH dependent enzymes, GPx, GST and GRx
by 52.19%, 57.98% and 29.54% in heart tissue. Further it is observed that a
significant (P<0.05) decrease in the antiperoxidative enzymes, SOD, CAT by 54.50%
and 54.57% in heart tissues is noted in ISPH administered rats compared to controls.
During MI, SOD and catalase are structurally and functionally impaired by free
radicals resulting in myocardial damage. The decrease in SOD and catalase may be
due to the involvement of superoxide and hydrogen peroxide free radicals in
myocardial cell damage mediated by ISPH.
Normal activities of GSH-dependent enzymes such as GPx, GRx, and GST
are vital for maintaining the antioxidant status. The decreased activity of these
enzymes in the heart is due to increased lipid peroxidation in ISPH induced rats.
Increased lipid peroxidation resulted in decreased levels of GSH. Thus, the decreased
levels of GSH resulted in decreased activities of GPx, GRx, and GST in ISPH induced
rats. pre-treatment with BDTALK increased the activities of antioxidant enzymes by
104.93%, 95.59%, 8.73%, 77.23% and 54.08% in heart respectively for GPx, GST
and GRx, CAT and SOD. The results of the present study are in accordance with
previous reports of Brindha et al (2012), who reported that phytic acid significantly
increased the activity of GSH dependent antioxidant and peroxidative enzymes.
However, BDTALK treatment alone does not show much significant change
compared with the control group heart tissues.
160
Figure 4.37: Effect of BDTALK on Cardiac Tissue Antioxidant Enzyme Levels
in Control and Experimental Rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-d)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
161
4.9B Effect of BDEEWP and BDTALK on Non enzymatic Antioxidants
The second line of defense consists of the non-enzymatic scavengers, Viz,
ascorbic acid, α-tocopherol, ceruloplasmin, and sulfhydryl containing compounds,
which scavenge the residual free radicals escaping from decomposition by the
antioxidant enzymes. Reduced glutathione is one of the most abundant non-enzymatic
antioxidant bio-molecules present in the body (Vinay kumar et al., 2015). It protects
the cells from free radical mediated damage caused by drugs and ionizing radiation. It
forms an important substrate for GPx, GRx and GST and several other enzymes,
which is involved in the free radical scavenging action (Khalid et al., 2014).
Figure 4.38 indicate the levels of GSH in cardiac tissue of control and
experimental rats. The significant (P<0.05) decrease was observed in the level of
GSH (47.36%) in heart tissue is noted in ISPH administered rats compared to
controls. Treatment with BDEEWP increased the levels of GSH (52.27%) in heart
tissue. However, BDEEWP treatment alone does not show much significant change
compared with the control groups in heart tissue.
Decreased in the level of GSH (51.33%) in heart tissue is noted in ISPH
administered rats compared to controls (Figure 4.39). Depletion of GSH results in
enhanced LPO and excessive LPO. Pre-treatment with BDTALK increased the levels
of GSH (60.25%) in heart tissue. However, BDTALK treatment alone does not show
much significant change compared with the control group heart tissues.
162
Figure 4.38: Effect of BDEEWP on Cardiac GSH Levels in control and
Experimental Rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
Figure 4.39: Effect of BDTALK on Cardiac GSH Levels in control and
Experimental Rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
163
Vitamin C is a primary antioxidant, water-soluble vitamin that can directly
scavenge singlet oxygen, superoxide and hydroxyl radicals. It has been suggested to
reduce the risk of CVD by reducing blood pressure, blood cholesterol and the
formation of oxidized low-density lipoprotein–cholesterol.
The concentration of Vitamin-C in heart of control and experimental groups
are summarized in the Figure 4.40. A significant (P<0.05) decrease in the tissue
Vitamin-C content was observed in heart (45.24 %) of ISPH injected rats compared
to normal group at 45 days of experimentation. pre-administration of BDEEWP
minimises the ISPH induced depletion of Vitamin-C in heart tissues. However,
BDEEWP alone treated control rats showed a slight increase in the Vitamin-C content
of heart (1.72 %) compared with the corresponding values of control rats.
The effect of BDTALK on the concentration of Vitamin-C in hearts of control
and experimental groups are summarized in the Figure 4.41. A significant (P<0.05)
decrease in the tissue Vitamin -C content was observed in heart (52.1 %) in ISPH
injected rats compared to normal group at 15 days of experimentation. pre-
administration of BDTALK minimises the ISPH induced depletion of Vitamin-C in
heart tissues. our results are in agreement with previous study of Khalid et al., (2014)
who demonstrated that the morin, a flavanoid enhances the Vitamin–C levels in heart
by preventing the ISPH induced lipid peroxidative system. However, BDTALK alone
treated rats do not show a slight increase in the Vitamin-C content of heart (2.01 %)
compared with the corresponding values of control rats.
164
Figure 4.40: Effect of BDEEWP on Cardiac Vitamin - C levels in control and
Experimental Rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
Figure 4.41: Effect of BDTALK on Cardiac Vitamin-C Levels in control and
Experimental Rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
165
4.10 Effect of B. diffusa on serum electrolytes
Electrolytes are ionized molecules found throughout the blood, tissues and
These molecules, which are either positive (cations) or negative (anions), play an
important role in many body processes, such as controlling fluid levels, acid-base
balance (pH) (Damodara et al., 2007), blood clotting, nerve conduction and muscle
contraction. Besides these, they involved in various metabolisms as co factors in most
cellular enzyme catalysed reactions.
Serum electrolytes changes in ISPH induced MI have not been studied
extensively and there is paucity of information in the literature in this regard. Scanty
information is available in the literature about prognostic value of serum electrolytes
in ischemic heart disease. Hence we assessed the alterations in the electrolytes and
minerals during ISPH induced MI. Figure 4.42 to Figure 4.45 represents the effect of
BDEEWP on serum electrolytes such as sodium, potassium, calcium and iron. Serum
of ISPH induced myocardial infracted rats showed significant (P<0.05) increase by
61.31% in the levels of sodium and 77.32% in levels of iron. Whereas a significant
(P<0.05) decrease by 34.37% in the levels of calcium and 27.22% in levels of
potassium. Pre-treatment with BDEEWP significantly (P<0.05) restored the
alteration levels of sodium (69.91%), iron (58.50%), calcium (64.37%) and potassium
(100.60%) respectively in ISPH administered rats when compared to ISPH alone
administered rats. There is no significant difference in the levels of electrolytes with
treatment of BDEEWP alone.
166
Figure 4.42: Effect of BDEEWP on serum Sodium Levels in control and
Experimental Rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
Figure 4.43: Effect of BDEEWP on serum Potassium Levels in control and
Experimental Rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
167
Figure 4.44: Effect of BDEEWP on serum Calcium Levels in control and
Experimental Rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
Figure 4.45: Effect of BDEEWP on serum Iron Levels in control and
Experimental Rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
168
Figure 4.46 to Figure 4.49 represents the effect of BDTALK on serum
electrolytes such as sodium, potassium, calcium and iron. Serum of ISPH induced
myocardial infracted rats showed significant (P<0.05) increase by 61.16% in the
levels of sodium and 77.96% in levels of iron, whereas a significant (P<0.05)
decrease by 27.57% in the levels of calcium and 35.01% in levels of potassium. Pre-
treatment with BDTALK significantly (P<0.05) restored the alteration levels of
sodium (100.11%), iron (52.58%), calcium (61.27%) and potassium (127.81%)
respectively in ISPH administered rats when compared to ISPH alone administered
rats. There is no significant effect on the levels of electrolytes with treatment of
BDTALK alone.
Na+ and K
+ play a fundamental role in cardiovascular activity (Nurminen et
al., 1998). These ions are greatly required in the transportation of substances across
membranes (Marsano and Clain, 1989). In addition, Na+ together with K
+ assist in the
maintenance of body‟s electrolyte and water balance (Nguyen and Kurtz, 2004).
Sodium is associated with blood pressure and in many hypertensive patients; a
reduction in sodium intake lowers blood pressure. On the other hand, potassium,
which is in the intra-cellular fluid, has been reported to be among the protective
electrolytes against hypertension (Akpanabiatu et al., 2005). The sodium - potassium-
adenosine triphosphatase (Na+- K
+ ATPase) pump is principally responsible for
regulating potassium entry into cells. It preserves a high intracellular K+ concentration
despite an adverse concentration gradient. Catecholamines, insulin, aldosterone and
hyperkalemia stimulate the activity of Na+- K
+ ATPase (Clausen and Everts, 1989).
The increased Na+
and reduced K+ in the serum of ISPH induced rats in the present
study could be due to decreased activity of Na+- K
+ ATPase.
169
Figure 4.46: Effect of BDTALK on serum Sodium Levels in control and
Experimental Rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
Figure 4.47: Effect of BDTALK on serum Potassium Levels in control and
Experimental Rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
170
Figure 4.48: Effect of BDTALK on serum Calcium Levels in control and
Experimental Rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
Figure 4.49: Effect of BDTALK on serum Iron Levels in control and
Experimental Rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
171
Calcium is essential to many bodily functions, including bone metabolism,
neuromuscular activity, coagulation, exocrine and endocrine functions and electrical
conduction of smooth muscle and heart. The decreased level of serum Ca2+
in ISPH
administered rats observed might be due to pronounced positive inotropic effects
mediated by an increased transmembrane Ca2+
influx to the myocardial cells.
Extracellular Ca2+
is required for maintenance of normal structure and functional
integrity of myocardial cell membrane and muscle contraction. The positive inotropic
effects of ISPH are mediated by c-AMP, when ISPH binds to β- adrenergic receptor on
myocyte and causes activation of adenylate cyclase which converts ATP to c-AMP. In
turn, c-AMP activates c-AMP dependent protein kinase, causes phosphorylation of Ca2+
channels of sarcolemma which could lead to enhanced Ca2+
influx.
The elevated Ca2+
in cytosol triggers Ca2+
release from sarcoplasmic reticulum
which further increases Ca2+
concentration in the cytosol. This Ca2+
causes actin
myosin interactions through ca2+
binding to troponin-C and brings about contraction.
Pre-treated rats with BDEEWP and BDTALK showed decreased Na+ levels and
increased K+ and Ca
2+ levels which might be due to the stabilising membrane bound
ATPases or could be due to the presence of alkaloids, saponins and Ca2+ antagonist
in B. diffusa. This observation is in agreement with those of Kaur and Goel (2011)
who reported the anticonvulsant activity of methonolic extract of B. diffusa on against
PTZ-induced convulsions particularly the liriodendrin-rich fraction of crude
methonolic extract showed significant protection against seizures induced by BAY k-
8644, a calcium channel agonist. Besides, results from the present study are also in
support with the report of Akpanabiatu et al., (2005) who worked with Nauclea
latifoliaon on rat serum electrolytes, lipid profile and cardiovascular activity.
Although atherosclerosis is also known to be associated with the inhibition of
fibrinolysis, no specific mechanism or agents have been identified. Studies by Undas
etal., (2008) shed some light on this problem by showing that the susceptibility of
fibrin clot to lysis is affected by the structure and permeability of fibrin network .
Another important fact is that there is a relationship between body iron overload and
pathogenesis of numerous degenerative diseases, including atherosclerosis (Ahluwalia
et al., 2010; Hahalis et al., 2011; Merono et al., 2011). In our present study, the
elevated levels of serum iron in ISPH administered rats might be due to release of free
iron from heme dependent proteins like haemoglobin and myoglobin which occurs
172
during ischemia (Rajadurai and Prince, 2007). It is a common belief that free blood
iron is responsible for so-called oxidative stress that, in turn, leads to atherosclerosis
and related cardiovascular diseases (Griendling and Fitzgerald 2003).
Boguslaw and Etheresia (2013) states that, the excess of blood free iron is
responsible for the non-enzymatic generation of insoluble fibrin-like material
(parafibrin) that, when deposited on the arterial wall, initiates inflammatory reactions.
Besides, tissue hypoxia leads to inadequate oxidation of blood circulation through
lungs resulting in erythrocytosis and consequent increased production of
erythropoietin (Elzayadi et al., 2002). This erythropoietin ultimately increases the red
cell mass, thereby increased the number of destroyed red blood cells in the normal
turnover process which subsequently increases iron overload (Sago and Blacerzak,
1973). In the present study the serum iron levels were restored in the ISPH induced
pretreated rats compared to the ISPH induced group. This could be due to
polyphenolic substances of B. diffusa which acts as iron chelating and free radical
scavenging agents that may be neither oxidants nor antioxidants (Lipinski 2011). Our
results are also in agreement with an earlier reports of Sheela sashikumar and
Shyamala devi, (2000).
4.11 Effect of B. diffusa on Inotropic Enzymes
Membrane bound ion transport enzymes play a significant role in the
cardiac cycle by maintaining normal ion concentrations within the cardiac tissue.
These enzymes are participating in the energy requiring translocation of sodium,
potassium, calcium and magnesium. Any alterations in the activities of these enzymes
affect the function of heart. Determination of membranes associated enzyme activities
like ATPases indicate alterations in membrane under pathological conditions.
The effect of BDEEWP on the activities of membrane bound ATPases in the
cardiac tissues of control and experimental rats are presented in Figure 4.50 There
was a significant (P< 0.05) decrease (61.66%) in heart membrane Na+-K
+ ATPase and
significant increase (73.04% and 71.43%) in heart respectively in Mg+2
ATPase and
Ca+2
ATPase activities were observed in rats treated with ISPH when compared to
control rats. Pre-treatment with BDEEWP significantly (P< 0.05) increased (92.40%)
in the activity of membrane Na+-K
+ ATPase and significantly decreased (86.34% and
173
93.33%) activities of Mg+2
ATPase and Ca+2
ATPase activities respectively in heart
tissues of ISPH administered rats. The rats treated with BDEEWP maintained near
normal activities of membrane bound ATPases when compared to control group.
The activities of membrane bound ATPases in heart tissues of control and
BDTALK treated experimental rats are presented in Figure 4.51. There was a
significant (P< 0.05) decrease (55.37%) in membrane Na+-K
+ ATPase and significant
increase (75.72% and 68.10%) in Mg+2
ATPase and Ca+2
ATPase activities
respectively were observed in hearts of rats treated with ISPH when compared to
control rats. Pre-treatment with BDTALK significantly (P< 0.05) increased (74.01%)
in the activity of membrane Na+-K
+ ATPase and significant decreased (92.34% and
80.35%) in activities of Mg+2
ATPase and Ca+2
ATPase activities respectively in
ISPH administered rats. The rats treated with BDTALK maintained near normal
activities of membrane bound ATPases when compared to control group.
Na+-K
+ATPase is the „SH‟ group containing enzymes responsible for the
active transport of Na+and K
+ across cell membrane, while Ca
2+ATPase is responsible
for the maintenance of normal intracellular calcium levels (Rajdurai, & Prince, 2007)
and Mg2+
-ATpase is sensitive to membrane fatty acid composition which is related to
the activity of intracellular Ca2+ stimulated fatty acid synthase suggesting a possible
synergistic relationship between Mg2+
-ATPase and Ca2+
ATPase.
174
Figure 4.50: Effect of BDEEWP on myocardial membrane bound ATPases in
control and experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
Figure 4.51: Effect of BDTALK on myocardial membrane bound ATPases in
control and experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
175
Currently, we observed a significant decrease in Na+-K
+, Ca
2+ and Mg
2+
ATPase activity levels in ISPH-treated rats. This is in line with previous findings
(Nirmala & Puvanakrishnan, 1996b). ATPases are integral membrane proteins which
require thiol groups and phospholipids to maintain their structure and function. ISPH
administration is known to produce reactive oxygen species, which modifies
membrane phospholipids and proteins, leading to lipid peroxidation and oxidation of
thiol groups, Thus ultimately results in the inactivation of the lipid-dependent
membrane bound ATPase. This loss of ATPase activities may be responsible for
deterioration of membrane ion gradients, opening of selective and unselective ion
channels and equilibration of intra and extra cellular ions. As a consequence of this
anoxic depolarisation K+ ions leave the cells and Na
+ and Ca
2+ ions enter. Cellular
accumulation of ions causes formation of cytotoxic edema that leads to myocardial
necrosis. In our study, pre-supplementation with the extracts of B. diffusa could resist
to a greater extent the perturbations in Na+-K
+, Ca
2+ and
Mg
2+ ATPase caused due to
ISPH. It is likely that the protective effect of B. diffusa could be due to its ability to
preserve „SH‟ group and inhibit membrane lipid peroxidation and consequent
alterations in the activity of various ATPases.
4.12 Effect of B. diffusa on Lipid metabolic enzymes
4.12 (A)Effect of BDEEWP and BDTALK on activity of serum LCAT
Lecithin cholesterol acyltransferase is the enzyme producing most plasma
cholesteryl esters and a key participant in the process of reverse cholesterol transport.
LCAT activity is necessary for the formation of mature high density lipoprotein
(HDL) and for remodelling of HDL lipoprotein particles, which are known to offer
protection against CVD. Decreased activity of LCAT inhibits the esterification of
cholesterol in ISPH treated rats. This leads to the high concentration of lipids and
lipoproteins in circulation, which are at high risk of atherosclerosis. Increased
oxidative stress resulted in the deficiency of LCAT in ISPH treated rats (Punithavathi
and Prince, 2009).
Figure 4.52 shows the effect of BDEEWP on the levels of serum LCAT in control
and ISPH administered rats. Serum LCAT level decreased significantly (P<0.05) by
54.37 % in ISPH administered rats when compared with control rats, while the oral
176
pretreatment of BDEEWP for 45 days increased the levels of LCAT significantly
(P<0.05) by 28.68 % as compared to ISPH induced myocardial infracted rats.
Figure 4.53 shows the effect of BDTALK on the levels of serum LCAT in control
and ISPH administered rats. Serum LCAT level decreased significantly (P<0.05) by
59.02 % in ISPH administered rats when compared with control rats, while the oral
pretreatment of BDTALK for 15 days increased the levels of LCAT significantly
(P<0.05) by 34.50% as compared to ISPH induced myocardial infracted rats.
BDEEWP and BDTALK increased the activity of LCAT which increases the
concentration of good cholesterol (HDL) in ISPH treated rats. HDL is heterogenous
and contain several lipoprotein particles.in man, the HDL containing apo A-1 has
been proposed to be more effective in reverse cholesterol transport than particles
containing both apo A-1 and apo A-2. Apo A-1 is a potent co factor enhancing LCAT
activity and the modulation of apo A-1 size is sensitive to the presence of apo B
containing particles and LCAT activity. BDEEWP and BDTALK pretreatment
increased the acticity of LCAT in ISPH administered rats. Thus the observed increase
in LCAT activity might be due to the blocking of LPO in BDEEWP and BDTALK
pretreated ISPH induced rats. Our results are in agreement with the previous report of
Prince et al., (2011), who reported the cardioprotection by increasing the activity of
LCAT with the treatment of Quercetin in combination with alpha tocopherol and
Vanillic acid in ISPH induced myocardial infracted Wistar rats.
177
Figure 4.52: Effect of BDEEWP on activity of LCAT Enzyme in control and
experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
Figure 4.53: Effect of BDTALK on activity of LCAT Enzyme in control and
experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
178
4.12 (B) Effect of BDEEWP and BDTALK on Lipoprotein Lipase:
Lipoprotein lipase (LPL) is a glycoprotein located on the luminal surface of
capillary endothelial cells. LPL plays a pivotal role in lipids and the metabolism of
lipoprotein (Goldberg, 1996). Major functions of LPL include the hydrolysis of TG-
rich lipoproteins and release of non-esterified fatty acid (NEFA), which are taken up
and used for metabolic energy in peripheral tissue such as muscle. LPL plays a pivotal
role in lipids and the metabolism of lipoprotein (Goldberg, 1996). This is an enzyme
responsible for the hydrolysis of triacylglycerols from plasma lipoproteins, mainly
chylomicrons and VLDL, responsible for the release of FFAs and glycerol and its
activity is influenced by nutritional and hormonal status and by environmental
conditions. When LPL hydrolyzes long-chain triglyceride substrates, the activity
becomes markedly reduced with time unless a fatty acid acceptor is present (e.g.
albumin) due to product inhibition. In the absence of fatty acid acceptors, the LPL
reaction may seemingly come to a stop, but on addition of albumin the inhibition is
immediately relieved (Bengtsson and Olivecrona, 1980). The authors suggested that
LPL forms a complex with fatty acids that prevents further hydrolysis of triglycerides,
and that this could serve as a feed-back mechanism to prevent excessive fatty acid
delivery to cells and tissues in vivo.
Figure 4.54 represents the effect of BDEEWP on the activity of LPL in liver
of control and ISPH administered rats. The rats injected with ISPH exhibited
significant (P<0.05) decrease (92.94%) in the activity of LPL when compared to
control rats. Oral pretreatment with BDEEWP to ISPH induced myocardial infracted
rats increased (31.84%) the activity of LPL significantly (P<0.05) when compared to
ISPH alone administered rats.
Figure 4.55 represent the effect of BDTALK on the activity of LPL in liver of
control and ISPH administered rats. The rats injected with ISPH exhibited significant
(P<0.05) decrease (81.72) in the activity of IPL when compared to control rats. Oral
pretreatment with BDTALK to ISPH induced myocardial infracted rats increased
(33.11%) the activity of LPL significantly (P<0.05) when compared to ISPH alone
administered rats.
179
Figure 4.54 Effect of BDEEWP on activity of Lipo Protein Lipase Enzyme in
control and experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-d)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
Figure 4.55 Effect of BDTALK on activity of Lipo Protein Lipase Enzyme in
control and experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
180
There are three potential pathways by which LPL may enhance cellular
uptake of lipoproteins. The first is receptor mediated uptake of lipoproteins bound to
the cell surface via LPL (Eisenberg et al., 1992). Secondly, LPL may act directly as a
ligand for receptors and finally the entire (heparin sulphate proteoglycans) HSPG-
LPL-Lipoprotein complex may be internalized by a receptor-independent pathway
(Obunike et al., 1994). Benlian et al., (1996) reported that several clinical studies have
shown that fibric acid induce LPL activity, lower plasma triglycerides and suppress
atherosclerosis (Frick et al., 1987; Mannien et al., 1992). In the present study
pretreatment with BDEEWP and BDTALK enhanced the activity of LPL in ISPH
administered rats. These results are in agreement with the previous reports of Kareem
et al., (2009) who worked on the aqueous extract of nutmeg and confirmed the
protective effect by accelerating the activity of LPL against ISPH induced MI in rats.
4.12 (c) Effect of BDEEWP and BDTALK on HMG Co A –reductase
HMGCR is the rate limiting enzymes in the cholesterol biosynthesis. An
increase in the activity of HMGCR inhibitors are known to decrease the secretion of
VLDL and LDL levels. Cellular cholesterol homeostasis is an important factor in the
prevention of CVD such as MI. The concentration of cholesterol can be regulated by
cholesterol biosynthesis, removal of cholesterol from circulation, absorption of
dietary cholesterol and excretion via bile and feaces.
Figure 4.56 showed the effect of BDEEWP on the activity of HMGCR in the
liver of rats. A significant increase in HMGCR (52.24%) in ISPH administered rats
when compared to control groups. Pretreatment with BDEEWP to ISPH administered
group showed the significant decrease in the activity of HMGCR (30.79%) when
compared with ISPH administered group.
Figure 4.57 reveals the effect of BDTALK on the activity of HMGCR activity
in liver of rats. A significant increase in HMGCR (47.60%) in ISPH administered rats
when compared to control groups. Pre-treatment with BDTALK to ISPH administered
group showed the significant decrease in the activity of HMGCR (27.76 %) when
compared with ISPH administered group.
181
Figure 4.56: Effect of BDEEWP on activity of HMGCR Enzyme in control and
experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
Figure 4.57: Effect of BDTALK on activity of HMGCR Enzyme in control and
experimental rats
Values are expressed as mean ± S.D (n = 6). Values do not share a common superscript (a-c)
in the row differ significantly from each other (p<0.05, Duncan‟s Multiple range Test).
182
Our findings clearly show that pre-treatment with BDEEWP and BDTALK
regulates cholesterol biosynthesis by inhibiting HMGCR in ISPH treated rats. Thus,
the decreased cholesterol levels in extract and compound treatment might be
correlated to the decreased activity of HMGCR in ISPH treated rats. The antioxidant
property indirectly helps to decrease the levels of lipids, by reducing or inhibiting
LPO. Punarnavoside, one of the active compounds of BDTALK ameliorated the
HMGCR in normal and ISPH induced MI in male wistar rats (Hansi and Prince,
2010). The results of present study are in accordance with the previous reports of
Prince and Satya (2010) and Altaf Hussain (2012) who reported that Quercetin,
Vanillic acid and Maslinic acid significantly increased the activity of HMGCR in
ISPH treated cardiotoxic male Wistar rats respectively.
4.13 Effect of BDEEWP and BDTALK on Histopathological studies:
The present study revealed the biochemical changes which are the responsible
risk factors for heart tissue damage under ISPH induced conditions. Hence, the study
was further extended to investigate the structural and histological changes in heart
muscle of ISPH induced rats and also to assess the cardioprotective role of BDEEWP
and BDTALK, histological studies were conducted in control and experimental groups.
Figure 4.58 shows the effect of BDEEWP on the extent of histopathological
changes in myocardial tissues in normal and ISPH treated rats. Figure 4.58A shows
the light micrograph of control heart showing normal architecture. Figure 4.58B
shows the light micrograph of ISPH administered group showing focal confluent
necrosis of muscle fibres with inflammatory cell infiltration, edema with fibroblastic
proliferation and myophagocytosis along with extravasation of red blood cells. The
degree of myocardial damage in BDEEWP 150 mg kg-1
bw + ISPH was less in edema
and muscle necrosis and with less inflammatory cells (Figure 4.58D). BDEEWP
150mg kg-1 administration alone did not lead to any histopathological changes in the
myocardium (Figure 4.58C).
183
Figure 4.58A Control Figure. 4.58B (ISPH induced)
Figure 4.58C (BDEEWP) Figure 4.58D (BDEEWP + ISPH)
Figure 4.58: Hitopathological examination (10X) of the heart of control,
BDEEWP treated and ISPH treated experimental animals
Figure 4.59 shows the effect of BDTALK on the extent of histopathological
changes in myocardial tissues in normal and ISPH treated rats. Figure 4.59A shows
the light micrograph of control heart showing normal architecture. Figure 4.59B
shows the light micrograph of ISPH administered group showing focal confluent
necrosis of muscle fibres with inflammatory cell infiltration, edema with fibroblastic
proliferation and myophagocytosis along with extravasation of red blood cells. The
degree of myocardial damage in BDTALK (50 mg kg-1
bw + ISPH) was less in edema
and muscle necrosis and with less inflammatory cells (Figure 4.59D). BDTALK
(50mg kg-1) administration alone did not lead to any histopathological changes in the
myocardium (Figure 4.59C).
184
Figure 4.59A Control Figure. 4.59B (ISPH induced)
Figure 4.59C (BDTALK) Figure 4.59D (BDTALK + ISPH)
Figure 4.59 Histopathological examination (40X) of the heart of control,
BDTALK treated and ISPH treated experimental animals
The histopathological studies of the control group revealed normal
architecture of the myocardium, with intact muscle fibres. Heart tissue of ISPH
induced rat showed severe infracted area with edema, inflammatory cells and
separation of cardiac muscle fibres. BDTALK pretreated and BDEEWP pretreated
rats showed near normal architecture of cardiac fibres with less edema and low
inflammatory cells. This protective effect may be due to the anti-inflammatory
phytochemicals of extracts. Our results are in accordance with previous reports of
Saayi krushna et al., (2011) and Kannan and Quine (2011).
185
Docking Studies
Docking techniques, designed to find the correct conformation of a ligand and
its receptor, have now been used for decades. The process of binding a small molecule
to its protein target is not simple; several entropic and enthalpic factors influence the
interactions between them. The mobility of both ligand and receptor, the effect of the
protein environment on the charge distribution over the ligand and their interactions
with the surrounding water molecules, further complicate the quantitative description
of the process. The idea behind this technique is to generate a comprehensive set of
conformations of the receptor complex, and then to rank them according to their
stability. The most popular docking programs include DOCK, AutoDock, FlexX,
GOLD, and GLIDE among others.
Figure 4.60 illustrate the molecular docking studies of the active compond,
punarnavoside, an alkaloid of B. diffusa with the enzyme HMG CoA reductase. After
collecting the crystal model, the possible binding sites of HMG CoA reductase were
searched with the CASTP server. From the binding site analysis of HMG CoA
reductase it was identified that, the binding pockets are identical in all chains and the
largest binding pocket was taken for further docking studies. The crystal structures of
HMG CoA reductase are similar and we have therefore taken 1HW9 (chain A) as
representative structure for docking studies. The docking compounds into the active
sites of HMG CoA reductase was performed using the GOLD software and the
docking evaluations were made on the basis of Gold score fitness functions. We
preferred Gold fitness score then Chemscore fitness as Gold fitness score is
marginally better than Chemscore fitness functions.
The result obtained showed that punarnavoside and HMG CoA reductase is an
excellent enzyme-ligand complex forming three hydrogen bonds tightly with Lys 633,
Glu 700 and Ser 705 with bond lengths of 2.96 oA, 3.18
oA and 3.14
oA respectively.
From this study it is evident that cholesterol biosynthesis can inhibited by
Punarnavoside due to loss of activity of HMG CoA reductase. The result obtained
from the docking studies are most useful to further studies for drug development.
186
Table 4.9: Autodock 4.0 energy values of lead molecules (Punarnavoside) with
rat HMG CoA reductase.
Clus | Lowest | Run | Mean | Num | Histogram
-ter | Binding | | Binding | in |
Rank | Energy | | Energy | Clus| 5 10 15
20 25 30 35
_____|___________|_____|___________|_____|____:____|____:____|
1 | -7.44 | 38 | -7.44 | 1 |#
2 | -6.86 | 30 | -5.08 | 3 |###
3 | -6.72 | 5 | -6.72 | 1 |#
4 | -6.55 | 28 | -2.05 | 5 |#####
5 | -6.14 | 22 | -6.14 | 1 |#
6 | -6.01 | 50 | -6.01 | 1 |#
7 | -5.53 | 46 | -4.02 | 2 |##
Binding amino acids of protein with punarnavoside ligand
Lys 633 wit 2.96 angstroms
Glu 700 with 3.18 angstroms
Ser 705 with 3.14 angstroms
Figure 4.60: Best lead molecules autodock interaction of HMG CoA reductase
with Punarnavoside
187
SUMMARY AND CONCLUSION
CVD is the leading cause of morbidity and mortality in many parts of the
world. Although modern drugs are effective in preventing cardiovascular disorders,
there use is often limited because of their side effects and high cost.
Since the beginning of human civilization, herbs have been an integral part of
society, valued for both their culinary and medicinal properties. Phytochemicals, the
natural products are the starting point for the discovery of many important modern
drugs. Recently, several plants of Indian origin have been found to possess medicinal
properties with their beneficial effects in ailments like atherosclerosis, ischemia,
cancer, diabetes and liver dysfunction.
Boerhaavia diffusa, commonly known as Punarnava is a medicinal plant
widely used in the Ayurvedic medicine, belonging to the family, Nyctaginaceae, is
mainly a diffused perennial herbaceous creeping weed of India. The phytochemical
screening of BDEEWP revealed the presence of alkaloids, glycosides, flavonoids,
tannins, phenols, saponins, steroids, terpenoids, lignins, carbohydrates and proteins,
which have multiple beneficial effects in CVD. The literature also indicated B diffusa
credits with antidiabetic, antioxidant, cardiotonic, anti-inflammatory and other
beneficial properties. Hence the present study has been carried to claim the protective
effect of BDEEWP and BDTALK on ISPH induced cardiac stress in rats.
The in vitro studies of BDEEWP indicated its ability to neutralize various free
radicals like hydroxyl radical, superoxide radical, nitric oxide radical and hydrogen
peroxide production. Studies carried out to find inhibition of lipid peroxidation,
reducing power and DPPH radical scavenging effect of BDEEWP obviously indicated
its ability and usefulness to use it as cardioprotective compound.
The ISPH induced myocardial stress is considered as one of the most widely
used experimental model to study the beneficial effects of many drugs, phytochemical
and cardiac function. ISPH is a potent, non selective β-adrenergic agonist. ISPH
elicits its pharmacological actions by stimulation of the adenylate cyclase-cAMP
system. Increase in the formation of ROS during myocardial ischemia and the adverse
effects of oxyradicals on myocardium have been well established. Over the years, a
188
number of mechanisms have been proposed to explain the pathogenesis of
catecholamine cardiomyopathy.
A significant increase in heart weight and a significant decrease in body weight were
observed in ISPH treated rats when compared to the control rats. Pretreatment of
ISPH administered rats with BDEEWP (150 mg/kg bw) and BDTALK (50 mg/kg bw)
effectively prevents the increased heart weights and promotes the body weights
compared to ISPH – alone treated rats.
A significant decline and a significant increase was shown in the activities of
cardiac markers such as ALT, AST, ALP, LDH, CK, and GGT in the heart tissues and
in the serum of acute ISPH treated rats. These changes were due to the leakage of
enzymes from the damaged heart tissues into the blood stream because of necrosis
induced by ISPH in rats. Significant increase in the activities of the same markers in
plasma of ISPH treated rats might be due to increased permeability of cell membranes
to the isoproterenol mediated peroxidative damage. The prior administration of
extracts (BDEEWP and BDTALK) of B diffusa significantly prevented the ISPH
induced elevation in the levels of diagnostic marker enzymes in serum, indicating the
cytoprotective activity of B diffusa. Thus, it is possible that B diffusa will also
prolong the viability of cell membrane stabilizing action.
Hyperlipidemia is one of the major factors responsible for the occurance of
MI. it is postulated that ISPH induces MI by its lipolytic action and increasing
circulatory lipids and lipoprotein levels. BDEEWP pretreatment for 45 days ad
BDTALK pretreatment for 15 days significantly cut back these elevated lipid levels in
their respective groups clearly indidates the hypolipidemic property of B. diffusa.
A significant increase in the levels of Lipid peroxidation, Protein oxidation
with concomitant decline in the content of GSH and Vit C in the heart tissue was
observed in ISPH administered rats as compared to that of control rats. Also a
significant reduction in the activities of glutathione dependent antioxidant enzymes
GPx, GRx, GST and antiperoxidative enzymes SOD and CAT was observes in ISPH
administered rats. These altered parameters were maintained near normal in
BDEEWP (150 mg/kg bw) pretreated and BDTALK (50 mg/kg bw) pretreated rats
administered with ISPH.
189
The levels of serum NO were elevated significantly in ISPH administrated rats
when compared with control rats. The values were restored to near normal in ISPH
administered BDEEWP (150 mg/kg bw) pretreated and BDTALK (50 mg/kg bw)
pretreated rats.
The in vivo treatment of BDEEWP shows a significant protection against
H2O2 induced DNA damage in the lymphocytes of BDEEWP treated rats compared to
normal rats. BDEEWP pretreated lymphocytes showed decrease in the number of
damage cells and length of the comets against ISPH induced DNA damage.
Serum of ISPH induced myocardial infracted rats showed significant increase
in the levels of sodium and iron, and significant decrease in the levels of potassium
and calcium. Pretreatment with BDEEWP (150 mg/kg bw) and BDTALK (50 mg/kg
bw) restored the altered levels of electrolytes significantly in ISPH induced rats.
Significant decrease in the activities of membrane-bound ATPases (Na+, K
+-ATPase,
Mg2+
-ATPase and Ca2+
-ATPase) in isoproterenol-induced myocardial infarction
indicated a severe derangement of subcellular metabolism and structural alterations of
cardiac cell membranes. Administration of isoproterenol significantly altered the
mineral metabolism. This might be due to increased lipid peroxidative damage of cell
membranes. In the present study, BDEEWP and BDTALK administration
significantly prevented the isoproterenol induced alterations in the activities of
membrane-bound ATPases and maintained the levels of minerals by its membrane
stabilizing property.
The activities of L CAT, LPL decreased significantly along with the increased
activity of HMGCR in ISPH induced rats, as compared to control rats. BDEEWP (150
mg/kg bw) pretreatment for 45 days ad BDTALK (50 mg/kg bw) pretreatment for 15
days to ISPH administered rats restored the activities of L- CAT, LPL and HMGCR
significantly in ISPH induced rats.
The molecular docking studies Punarnavoside with HMGCR indicated the
beneficial effects of B. diffusa on ISPH induced cardiac stress in rats. The data
showed that the HMGCR was inhibited with punarnavoside. The histopathological
studies of the control group revealed normal architecture of the myocardium, with
intact muscle fibres. Heart tissue of ISPH induced rat showed severe infracted area
with edema, inflammatory cells and separation of cardiac muscle fibres. BDEEWP
190
(150 mg/kg bw) pretreated and BDTALK (50 mg/kg bw) pretreated rats showed near
normal architecture of cardiac fibres with less oedema and low inflammatory cells.
Conclusion
The present results clearly emphasize the beneficial action of B. diffusa against
ISPH induced myocardial injury in rats, as the concentration of 150 mg/kg bw
(BDEEWP) and 50 mg/kg bw (BDTALK) showed almost all the same results. Hence
this may be useful towards the development of new antiatherogenic drugs. The GCMS
analysis of BDTALK also supports the presence of various phytochemicals like Methyl
Valinate, 2,4, Di Tert Butyl Phenol, Methyl Ferulate, Syringic Acid, Loliolide and 7,9-
Di-Tert-Butyl-1-Oxaspiro(4,5)Deca-6,9-Diene-2,8-Dione, which could be useful in the
prevention of CVD as these components aree having antioxidant properties.
Histopathalogical studies and molecular docking studies also supported the biochemical
studies to show the cardioprotective effect of BDEEWP and BDTALK against ISPH
induced MI in rats.Current investigation for the first time shows that the preventive
effects of B. diffusa against ISPH induced cardiotoxicity. This model might be useful to
investigators who are interested in exploring the various molecular, cellular and
physiological mechanisms associated with cardiac pathobiology, free radical
metabolism and inflammation in vivo by using ISPH.
191
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231
LIST OF CHEMICALS PROCURED
Sigma Chemical Co. St. Louis, MO, USA
Acetyl-CoA
Adenosine 51- monophosphate sodium salt (AMP)
Adenosine-51-triphosphate di sodium salt (ATP)
Ammonium molybdate
bovine serum albumin (BSA)
isoproterenol hydro chloride (ISPH)
Nitro blue tetrazolium (NBT)
Thiobarbituric acid (TBA)
Tri chloro acetic acid (TCA)
SISCO Research Laboratories Private Ltd., Mumbai, Maharashtra, India
1-Amino-2-naphtho-4-sulphonic acid (ANSA)
1-Chloro-2, 4-dinitro benzene (CDNB)
Glutathione oxidised (GSSG), Glutathione reduced (GSH)
Hydroxylamine hydrochloride
Perchloric acid
Phosphoenol pyruvate
tricyclohexylammonium salt
Potassium sodium tartarate
Pyrogallol
Sodium arsenate
Sodium pyruvate
Sodium disulphite
Sodium sulphite
Triethanolamine hydrochloride
α-Ketoglutaric acid
Qualigens, Mumbai, Maharashtra, India
2,4-Dinitro phenyl hydrazine (DNPH)
Ammonium ferrous sulphate
Ammonium molybdate
Ammonium sulphate
232
Cholesterol
Citric acid
Dipotassium hydrogen ortho phosphate
Disodium hydrogen ortho phosphate
Ethylene diamine tetra acetic acid disodium salt (EDTANa2)
Ferric chloride
Folin & Ciocalteu reagent (2N)
Hydrogen peroxide
Magnesium sulphate
Ortho phosphoric acid
Phenolphthalein indicator solution
Picric acid
Potassium chloride
Potassium dichromate
Potassium dihydrogen ortho phosphate
Potassium hydroxide
Potassium thiocyanate
Quercetin
Sodium carbonate
Sodium chloride
Sodium dihydrogen ortho phosphate
Sodium fluoride, Sodium hydroxide
Sodium nitroprusside
Sodium sulphate
Trichloro acetic acid
Trisodium citrate
Sd-fine Chemicals, Maharashtra, India
Di methyl sulphoxide (DMSO)
Ether, Hexane, ortho phosphoric acid
