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ACKNOWLEDGEMENT
I bow my head before Almighty ALLAH, The omnipotent, the omnipresent,
The merciful, The most gracious, The compassionate, who is the entire and
only source of every knowledge and wisdom endowed to mankind.
I am highly grateful to Prof. S.W. Akhtar, Honorable Vice Chancellor of
Integral University for his kind help to provide all necessary facilities for this
work. I am also thankful to Prof. T.Usmani, P.V.C. & Chairman R.D.C. for his
valuable suggestions.
I would like to take this opportunity to convey my cordial gratitude and
appreciation to my respected supervisor Prof. (Dr) H.H. Siddiqui, Dean &
Adviser to V.C. Faculty of pharmacy, Integral University, without whose
constant help, deep interest and vigilant guidance, the completion of this thesis
was not possible. I am really thankful to him for his accommodative attitude,
thought provoking guidance, immense intellectual input, patience and loving
behavior. His sage counsel, well meaning criticism and able guidance have aided
me in many ways to write this thesis in innumerable ways.
I am highly grateful to my co-supervisor Prof. Rakesh Dixit, Professor,
Department of Pharmacology, King George Medical University, Lucknow for
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his generous guidance, constant help, deep support, continuous encouragement
and valuable suggestions in bringing this work to conclusion.
I am also extremely grateful to all of my colleagues specially Dr. Juber Akhtar,
Head, Faculty of Pharmacy, Mr. Paramdeep Bagga, Mr. Mohd. Sarfaraz
Hussain, Mr. Ranjan Kumar and Mr. Mohd. Mujahid, for his valuable
assistance, kind help and cooperation whenever it needed.
I would like to pay a heartiest thank Dr. Mohd. Sajid Khan, Department of
Biotechnology, Integral University for his kind support and help to perform
some portion of this work. I am also thankful to Prof. S.K. Maulik, Department
of Pharmacology, All India Institute of Medical Sciences, Delhi for their
valuable guidance and suggestion during this work.
I am also thankful to all the administrative and laboratory staff of the faculty
especially Mr. Mohd. Rashid Siddiqui, Mrs. Bushra Parveen, Mrs. Barnali
Bose, Mr. Suresh Kumar for their support.
I had a good fortune, to have a cheerful group of friends who often helped me at
critical junctures. I am very pleased to thanks to all of my dear friends especially
Dr. Jamal Anwar Beg, Dr. Jawed Akhtar, Mr. Naushad Ali, Mr. Mohd.
Sarfaraz, Dr. Masihuzzaman Khan, Mr. Tarique Usmani, Mr. Shah Faisal and
Mr. Ayaz Alam for their cooperation and moral support throughout the project.
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Finally, I would like to thank my family; my parents Mr. Akhtar Mahmood and
Mrs. Saeeda for all support, cooperation and blessings, my wife Zeenat Khatoon
and my son Mohd. Fuzail for their love, support and patience during my long
hours in front of the computer, my sister Eram Fatima, brother in law Mr.Tariq
Ashraf, my nephew Mohd. Hassan for their moral support, cooperation and
encouragement, which made finishing the thesis at all.
I will always remain sympathetic towards all the innocent animals that had to be
sacrificed for the completion of the project work and also for the betterment of
the human society.
Tarique Mahmood Ansari
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ABSTRACT
Introduction: Bombyx mori cocoon (Abresham) is one of the important
cardioprotective drug mentioned by Avicenna and chief constituent of two
popular and widely used Unani formulations Khamira Abresham Sada (KAS) &
Khamira Abresham Hakim Arshadwala (KAHAW) for cardiac problems. The
present study was undertaken to investigate its cardioprotective activity, clinical
correlation and comparative evaluation of its polyherbal formulations KAS and
KAHAW against various cardiac diseases. Methods: Cardioprotective activity
of Abresham was investigated by two different experimental models, myocardial
necrosis; oxidative stress and hypertrophy associated with isoprenaline induced
cardiotoxicity and High fat diet induced Dyslipidemia and obesity. In
isoprenaline induced cardiotoxicity model, Wistar strain rats were pretreated
with ethanolic extract of Bombyx mori cocoons in two doses (250 mg/kg &500
mg/kg) and its polyherbal formulations, Khamira Abresham sada (KAS) &
Khamira Abresham Hakim Arshadwala (KAHAW) in one dose each (800
mg/kg) orally for 30 days and cardiotoxicity were induced by administration of
isoprenaline (85 mg/kg, subcutaneous) given twice on 29th & 30
th day. In the
second model, Wistar strain rats were fed with High fat diet to induce
dyslipidemia and obesity and were pretreated with ethanolic extract of Bombyx
mori cocoon in two doses (250 mg/kg &500 mg/kg) and its polyherbal
formulations, Khamira Abresham sada (KAS) & Khamira Abresham Hakim
Arshadwala (KAHAW) in one dose each (800 mg/kg) orally for 28 days.
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Results: Cardioprotection was assessed by gross examination and grading of
heart damage. Estimation of various cardiac marker enzymes like aspartate
transaminase (AST), alanine transaminase (ALT), lactate dehydrogenase (LDH),
creatinine kinase (CK-MB) and specific marker of cardiac damage troponin. In
isoprenaline treated group the levels of these enzymes significantly increased.
Pretreatment with Abresham and its formulations significantly (p<0.01)
decreases the level of these enzymes. Heart homogenate was used to estimate the
cardiac antioxidants like superoxide dismutase (SOD), catalase, glutathione
(GSH) and lipid per oxidation parameter thiobarbituric acid reactive substance
(TBARS). Pretreatment with Abresham and its formulations significantly
(p<0.01) increased the level of these antioxidants and decreases the level of
TBARS. The observed results were further confirmed by histopathological
findings. Pretreatment groups significantly (p<0.01) decreased the obesity
parameters body weight gain, atherogenic index ,Liver weight, hepatic
cholesterol and lipid profile parameters total cholesterol, LDL-C, triglycerides
and significantly increases the HDL-C against high fat diet induced
hyperlipidemia and obesity. The results were comparable with clinically
established drugs like Metoprolol, Atorvastatin and Orlistat. Conclusion: The
findings of this study indicate that Bombyx mori (500 mg/kg) exerts potent
cardioprotection similar to its polyherbal formulations Khamira Abresham
Hakim Arshadwala & Khamira Abresham Sada which were comparable with
Metoprolol, Atorvastatin and Orlistat against Isoprenaline induced cardiotoxicity
and high fat diet induced hyperlipidemia and obesity model respectively.
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Keywords: Bombyx mori, Abresham, myocardial necrosis, oxidative stress,
cardiotoxicity, Khamira Abresham, anti obesity, atherosclerosis, high fat diet,
hyperlipidemia
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LIST OF TABLES
Table No. Title Page No.
2.1
Classification of obesity according to BMI
28
2.2 Characteristics of cocoon 34
2.3 List of 64 drugs mentioned in Avicenna’s tract of
cardiac drugs
39
2.4 Composition of Khamira Abresham Sada according
to National Formulary of Unani Medicine, Part V
and Hamdard Laboratories [
40
2.5 Composition of Khamira Abresham Arshadwala
according to National Formulary of Unani
Medicine, Part V and Hamdard Laboratories
43
3.1 List of Drugs, enzymatic kits and equipments 87
3.2 Composition of high fat diet 99
4.1 Effect of of Bombyx mori, Khamira Abresham sada
and Khamira Abresham Hakim Arshadwala on
Assessment and Grading of Heart
118
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4.2 Effect of ethanolic extract of Bombyx mori,
Khamira Abresham sada and Khamira Abresham
Hakim Arshadwala on Heart weight: body weight
ratio
120
4.3
Effect of ethanolic extract of Bombyx mori,
Khamira Abresham sada and Khamira Abresham
Hakim Arshadwala on cardiac marker enzymes.
122
4.4 Effect of ethanolic extract of Bombyx mori,
Khamira Abresham sada and Khamira Abresham
Hakim Arshadwala on cardiac marker enzyme
Troponin
123
4.5 Effect of ethanolic extract of Bombyx mori,
Khamira Abresham sada and Khamira Abresham
Hakim Arshadwala on cardiac antioxidants and
lipid peroxidation.
126
4.6 Effect of HFD on body weight gain, food intake,
total cholesterol and triglycerides on 8th day after 24
hrs fasting (Baseline characteristics of obesity and
hyperlipidemia)
135
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4.7 Effect of Bombyx mori, Khamira Abresham sada
and Khamira Abresham Hakim Arshadwala on
body weight gain, total cholesterol and HDL
Cholesterol.
137
4.8 Effect of Bombyx mori, Khamira Abresham sada
and Khamira Abresham Hakim Arshadwala on
body weight gain, total cholesterol and HDL
Cholesterol.
138
4.9 Effect of Bombyx mori , Khamira Abresham sada
and Hakim Arshadwala on liver weight , hepatic
TG and hepatic TC
143
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LIST OF FIGURES
Fig. No. Title Page No.
2.1
Gross view of myocardial infarction
10
2.2 Development of myocardial necrosis 11
2.3 ECG image of STEMI and NSTEMI Evolution 13
2.4 View of normal artery & artery having
Atherosclerosis
25
2.5 Development of atherosclerotic plaque in artery 26
2.6 Larva of Bombyx mori 32
2.7 Cocoons of the Bombyx mori 32
3.1 Assembly for SDS-PAGE 111
4.1 Photographs of hearts of different experimental
group
119
4.2 Troponin –I Rapid test kit 124
4.3 Troponin –I Rapid test kit 124
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4.4 Effect of ethanolic extract of Bombyx mori,
Khamira Abresham sada and Khamira Abresham
Hakim Arshadwala on Heart weight: body weight
ratio.
127
4.5 Effect of Bombyx mori, Khamira Abresham sada
and Khamira Abresham Hakim Arshadwala on
cardiac marker enzymes
129
4.6 Effect of Bombyx mori, Khamira Abresham sada
and Khamira Abresham Hakim Arshadwala on
cardiac antioxidants and lipid peroxidation
132
4.7 Photomicrographs of histopathological studies 134
4.8 Effect of ethanolic extract of B. mori, Khamira
Abresham sada and Khamira Abresham Hakim
Arshadwala on body weight gain, total cholesterol,
HDL-C , Atherogenic index , triglycerides and
LDL-C
141
4.9 Effect of Bombyx mori , Khamira Abresham sada
and Hakim Arshadwala on liver weight , hepatic
TG and hepatic TC
145
4.10 SDS- Polyacrylamide Gel Electrophoresis (SDS-
PAGE)
147
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List of Symbols and Abbreviations
α Alpha
β Beta
γ Gama
δ Delta
μg Microgram
μl Micro liter
μm Micrometer
% Percentage
ΔA Difference in absorbance
× Multiplication
° C Degree Celsius
° F Degree Fahrenheit
+ve Positive/ Presence
–ve Negative/Absence
kDa kilo daltons
mM mili moles
mA mili ampere
M Molal
v/v volume by volume
w/v weight by volume
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ACE-inhibitors Angiotensin Converting Enzyme Inhibitors
ACS Acute Coronary Syndrome
AST Aspartate amino transferase
ALT Alanine amino transferase
ALLIANCE Aggressive Lipid-Lowering Initiation
Abates New Cardiac Events
AMI Acute myocardial infarction
APS Ammonium per sulphate
ASVD Atherosclerotic vascular disease
ATO Atorvastatin
BMI Body Mass Index
BNP B-type natriuretic peptide
BP Blood pressure
BSA Bovine serum albumin
BW body weight
BWG body weight gain
CABG Coronary artery bypass grafting
CAD Coronary artery disease
CAM Complementary and alternative medicine
CAT Catalase
CCB Calcium Channel Blocker
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CCRUM Central Council for Research in Unani medicine
CDRI Central Drug Research Institute
CT Scan Computerized tomography
CHD Coronary Heart Disease
CHF Congestive heart failure
CK Creatinine kinase
CK-MB Creatinine kinase Myoglobulin
CPCSEA Committee for the Purpose of Control and
Supervision of Experiments on Animals
CO 2 Carbon dioxide
CVD Cardio vascular disease
DNA Deoxyribonucleic acid
DPPH Diphenyl picryhydrazylene
DVT Deep venous thrombosis
DW Distilled water
ECG Electrocardiogram
FAS Fatty acid synthase
FPG Fasting Plasma Glucose
FTIR Fourier transform infrared
GSH Tissue/reduced Glutathione
GR Glutathione reductase
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GPx Glutathione peroxidase
Gm gram
HBA 1C Glycocylated Heamoglobin 1C
HDL High density lipoprotein
HDL -C High density lipoprotein cholesterol
Hrs Hours
HMG-CoA Hydroxy-3-methyl glutaryl coenzyme A
HF Heart failure
HFD High fat diet
HFD-C High fat diet Control
HW Heart weight
i.e. that is
i.p. Intraperitonial
IAEC Institutional Animal Ethical Committee
IHD Ischaemic Heart Disease
ISO Isoprenaline
ISO-C Isoprenaline control
IGT Impaired glucose tolerance
IU International unit
Kg Kilogram
K+ Potassium
KAHAW Khamira Abresham Hakim Arshad wala
KAS Khamira Abresham Sada
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LA Linoliec acid
LDH Lactate dehydrogenase
LDL Low Density Lipoprotien
LDL-C Low Density Lipoprotien cholesterol
LVH Left ventricular hypertophy
LVEDP Left ventricular end-diastolic pressure
LPL Lipo Protein Lipase
LPO Lipid Peroxidation
MDA Malondialdehyde
MI Myocardial infarction
MRI Magnetic Resonance Imaging
Mg milligram
min. Minute
ml Millileter
MUFA Monounsaturated fatty acid
NAFLD Non alcoholic fatty liver diseases
NC Normal control
NCEP National cholesterol education program
NFUM National formulary of Unani medicine
nm Nanometer
NPD Normal pellet diet
NSTEMI Non-ST segment elevation myocardial infarction
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NO Nitric oxide
O 2 Oxygen
o.d. Once daily
OD Optical density
PAD Peripheral Artery Disease
PAGE Polyacrylamide gel electrophoresis
PCI Percutaneous coronary intervention
PGL Plasma glucose
p.o. Per Oral
PUFA polyunsaturated fatty acid
PPG Post-Prandial Plasma Glucose
PGL Plasma glucose
RAS Renin angiotensin system
RSA Radical scavenging Activity
s.c. Subcutaneous
SDS Sodium dodecyl sulphate
SEM Standard error of mean
SOD Superoxide dismutase
SFA Saturated fatty acid
STEMI ST Segment elevation myocardial infarction
STZ Streptozotocin
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TBARS Thiobarbituric acid reactive substances
TC Total cholesterol
TCA Trichloroacetic acid
TEMED Tetramethyl ethylene diamine
TG Triglycerides
TM Traditional medicine
TXA2 Thromboxane A2
TIA Transient Ischemic attack
UV Ultraviolet
U.S.A. United States of America
VLDL Very Low Density Lipoprotein
WBC White Blood Corpuscles
WHO World Health Organization
Wk week