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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