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By

T.T.T.T. KirithikaKirithikaKirithikaKirithika

Supervisor

Dr. K. UshaDr. K. UshaDr. K. UshaDr. K. Usha

A thesis submitted to

Avinashilingam Institute for Home Science and Higher Education for Women,

Coimbatore - 641043

In partial fulfilment of the requirement for the Degree of

Doctor of Philosophy in BiochemistryDoctor of Philosophy in BiochemistryDoctor of Philosophy in BiochemistryDoctor of Philosophy in Biochemistry

September, 2013September, 2013September, 2013September, 2013

Acknowledgement

One of the joys of completion is to look over the journey past and

remember all the friendsfriendsfriendsfriends and familyfamilyfamilyfamily who have helped and supported me

along this long but fulfilling road.

I take this opportunity to extend my heartfelt thanks to all those who

have contributed to make this Ph.D. thesis a success. I give glory and honour

to Almighty GodAlmighty GodAlmighty GodAlmighty God for his blessings, wisdom and guidance for enabling me to

complete my thesis.

I expresses my deep sense of gratitude to Dr. T.S. Avinashilingam,Dr. T.S. Avinashilingam,Dr. T.S. Avinashilingam,Dr. T.S. Avinashilingam, the

Founder and First Chancellor of Avinashilingam Institute for Home Science

and Higher Education for Women and Padmashri Dr. (Mrs.) Rajammal P. Dr. (Mrs.) Rajammal P. Dr. (Mrs.) Rajammal P. Dr. (Mrs.) Rajammal P.

DeDeDeDevadas,vadas,vadas,vadas, Former Chancellor, Avinashilingam Institute for Home Science and

Higher Education for Women, Coimbatore, for their heavenly blessings the

execution of the study.

I conveys my sincere thanks to Dr. T.K. ShanmuganandamDr. T.K. ShanmuganandamDr. T.K. ShanmuganandamDr. T.K. Shanmuganandam, the Former

Chancellor and Dr.Dr.Dr.Dr. T.S.K. Meenakshi SundaramT.S.K. Meenakshi SundaramT.S.K. Meenakshi SundaramT.S.K. Meenakshi Sundaram, Chancellor, Avinashilingam

Institute for Home Science and Higher Education for Women, Coimbatore, for

extending all possible help towards the completion of the study.

I records my sincere gratitude to Dr. Saroja PrabhakaranDr. Saroja PrabhakaranDr. Saroja PrabhakaranDr. Saroja Prabhakaran, Former Vice

Chancellor and Dr. Sheela Ramachandran, Vice Chancellor, Avinashilingam

Institute for Home Science and Higher Education for Women, Coimbatore, for

creating an atmosphere conducive for critical and analytical thought.

I sincerely thank to Dr.Dr.Dr.Dr. GowrGowrGowrGowri Ramakrishnani Ramakrishnani Ramakrishnani Ramakrishnan, Registrar,


Coimbatore, for providing the opportunity and all the amenities for the

conduct of the study.

I expresses my profound sense of faithfulness and sincere thanks to

Dr. R. ParvathamDr. R. ParvathamDr. R. ParvathamDr. R. Parvatham, former Dean of Science and Dr. S. AnnapooraniDr. S. AnnapooraniDr. S. AnnapooraniDr. S. Annapoorani, Head of

the Department of Biochemistry, Biotechnology and Bioinformatics,


Coimbatore, for her inspiration and constant encouragement rendered

throughout the study.

I would also like to thank Dr. P. Santhana Krishnan,Dr. P. Santhana Krishnan,Dr. P. Santhana Krishnan,Dr. P. Santhana Krishnan, Director of

Research and Consultancy, Avinashilingam Institute for Home Science and

Higher Education for Women, Coimbatore, for extending all possible help for

the smooth conduct of the study.

I owes my deepest admiration and gratitude for my guide, Dr. K.Dr. K.Dr. K.Dr. K. UshaUshaUshaUsha,,,,

Professor, Department of Biochemistry, Biotechnology and Bioinformatics,


Coimbatore, who is a paragon of dedication all through her years of research

work. Without her kind words, her astute knowledge and her light

admonitions, this thesis would never have taken form.

Words are inadequate to express my sincere thanks to

Dr. (Mrs.) G.P. JeyanthiDr. (Mrs.) G.P. JeyanthiDr. (Mrs.) G.P. JeyanthiDr. (Mrs.) G.P. Jeyanthi, Controller of Examination and Dr. P.R. Padma,Dr. P.R. Padma,Dr. P.R. Padma,Dr. P.R. Padma,

Professor, Department of Biochemistry, Biotechnology and Bioinformatics,


Coimbatore for their valuable suggestions, enthusiastic encouragement and

timely help rendered for the study.

I express my deep felt thanks to all the faculty membersfaculty membersfaculty membersfaculty members of the

Department of Biochemistry, Biotechnology and Bioinformatics who were

always helpful, considerate and supportive.

I thank to Dr. V. Sharulatha, Assistant professor, Department of

Chemistry, Avinashilingam Institute for Home Science and Higher Education

for Women, Coimbatore for her help rendered for the study.

I sincerely thank to DrDrDrDr.Moorthy.Moorthy.Moorthy.Moorthy and Dr.NileshDr.NileshDr.NileshDr.Nilesh, Scientist, Botanical

survey of India, Coimbatore for their rendering help throughout my study.

I wishes to express my special thanks to E. Emerald Ponniyen SelvanE. Emerald Ponniyen SelvanE. Emerald Ponniyen SelvanE. Emerald Ponniyen Selvan,

Head in Charge, Blood bank, Kongunadu Hospital Coimbatore for giving the

facilities and help to carry out the study.

I also wish to express my thanks to the Lab Assistants especially

Ms. M. Lakshmi,Ms. M. Lakshmi,Ms. M. Lakshmi,Ms. M. Lakshmi, Mrs. Poongothai Mrs. Poongothai Mrs. Poongothai Mrs. Poongothai and MMMMssss.... RenuhaRenuhaRenuhaRenuha, Department of

Biochemistry, Avinashilingam Institute for Home Science and Higher

Education for Women, Coimbatore for their help rendered for the study.

I particularly wishes to put on record my gratitude to my friends

Ms.Ms.Ms.Ms. R.R.R.R. GomathiGomathiGomathiGomathi, Ms.Ms.Ms.Ms. Thoudam BhaiThoudam BhaiThoudam BhaiThoudam Bhaiggggyabatiyabatiyabatiyabati, Mrs. A. SmirithiMrs. A. SmirithiMrs. A. SmirithiMrs. A. Smirithi, Mrs. BabyMrs. BabyMrs. BabyMrs. Baby

ShakilaShakilaShakilaShakila, Mrs K.P. SreMrs K.P. SreMrs K.P. SreMrs K.P. Sreenaenaenaena, Ms. A. PoongothaiMs. A. PoongothaiMs. A. PoongothaiMs. A. Poongothai, Mrs. K.M. SharadhaMrs. K.M. SharadhaMrs. K.M. SharadhaMrs. K.M. Sharadha,

Mrs.Mrs.Mrs.Mrs. Sumaya,Sumaya,Sumaya,Sumaya, Mrs. Mrs. Mrs. Mrs. AmuthapriyaAmuthapriyaAmuthapriyaAmuthapriya and Mrs.Mrs.Mrs.Mrs. Jeba sweetlyJeba sweetlyJeba sweetlyJeba sweetly, Department of

Biochemistry, Biotechnology and Bioinformatics, Avinashilingam Institute for

Home Science and Higher Education for Women, Coimbatore, for their

camaraderie, timely help and for being a great solace in times of need.

My Special thanks to Ms.Ms.Ms.Ms. P. P. P. P. Shanmuga priyaShanmuga priyaShanmuga priyaShanmuga priya, Assistant professor and

Mr. Mr. Mr. Mr. R. R. R. R. Nantha Nantha Nantha Nantha GGGGopanopanopanopan, Assistant professor, Department of Physiotherapy,

PSGIMSR, Coimbatore who spent their valuable time and suggestions for the

successful completion of this work.

I wish to express my sincere appreciation to Mr. K. SelvakumarMr. K. SelvakumarMr. K. SelvakumarMr. K. Selvakumar,

Euro Informatics, Coimbatore for his meticulous and timely coordination in

the creation process of this thesis.

I would like to thank my well-wishers, Mr N. AnthananMr N. AnthananMr N. AnthananMr N. Anthanan and

Mrs N. NiraimathyMrs N. NiraimathyMrs N. NiraimathyMrs N. Niraimathy who have helped me emotionally through this, at times

difficult journey. I would like to thank them for their sensitive support and

advice.

I owe sincere thanks to my respected father in law

Mr.M.BalasubramanianMr.M.BalasubramanianMr.M.BalasubramanianMr.M.Balasubramanian and all my family members their unwavering faith

in me and their understanding and support in the course of my study.

I will also take this opportunity to thank my lovable brother T.T.T.T. ArvinthArvinthArvinthArvinth,

who would always lighten up my spirits with a joke. I expresses my special

thanks to all other unseen hands during the course of the study for the help

rendered in the successful completion of the study.

I cannot find words to thank my beloved parents for shouldering days

when nothing seemed right. The mere mention of their names wouldn’t do

justice to the care and support they gave. I am highly indebted to my mother

Mrs. N.Mrs. N.Mrs. N.Mrs. N. SasikalaSasikalaSasikalaSasikala for her prayers and motivation, she has a very special place

in my life and character. I have no words to express my feel about my father

Mr.Mr.Mr.Mr. S.S.S.S. ThirunavukarasuThirunavukarasuThirunavukarasuThirunavukarasu for all his generosity and selfless support without

which pursuing my dream would have been impossible. I learned from him to

stay strong in life’s many challenges.

The best outcome from these past three years is finding my best friend,

soul-mate, it’s my ever affectionable husband Mr. Mr. Mr. Mr. MarxMarxMarxMarx Chidambara babu Chidambara babu Chidambara babu Chidambara babu.

There are no words to convey my most sincere and warmest thanks to him. He

has been a true and great supporter during my good and bad times. These

past several months have not been an easy ride, both academically and

personally. I truly thank to my Marx for sticking by my side, even when I was

irritable and depressed. The last word goes towards my dearest son M.M.M.M. AjeeshAjeeshAjeeshAjeesh

for being the sunshine of my life. They have been and are always beside me at

every stage of my life.

Finally, I would like to dedicate this work to my parentsmy parentsmy parentsmy parents, my husbandmy husbandmy husbandmy husband

and mymymymy cute soncute soncute soncute son.

T.T.T.T. KirithikaKirithikaKirithikaKirithika

C O N T E N T S

CHAPTER NO

TITLE PAGE NO.

List of Tables

List of Figures

List of Plates

List of Appendices

List of Abbreviations

1.0 Introduction 1

2.0 Review of Literature 9

3.0 Experimental Procedure 46

4.0 Results and Discussion 62

5.0 Summary and Conclusion 146

Bibliography 154

Appendices 179

Publications 210

List of Tables

Table No

Title Page No

1 Functions of lipoproteins 26

2 Details of the assessment of biochemical parameters in

selected diabetic cardiomyopathy patients 48

3 Solvent system for HPTLC analysis 53

4 Experimental design of the study 59

5 Prevalence of diabetic cardiomyopathy in selected hospitals 64

6 Age wise distribution of diabetic cardiomyopathy patients in

selected hospitals 65

7 Genderwise prevalence of diabetic cardiomyopathy in


8 The prevalence of familial restrictive cardiomyopathy in


9 Signs and symptoms of diabetic cardiomyopathy patients in


10 Levels of HbA1C, lipid profile and cardiac enzymes in

selected diabetic cardiomyopathy patients 73

11 Levels of HbA1C, lipid and cardiac profile in selected diabetic

cardiomyopathy men (n=50) during the treatment 85

12 HbA1C level, lipid and cardiac profile in selected diabetic

cardiomyopathy women (n=50) during the treatment 87

13 Phytochemical constituents of various extracts of A. saman

leaves 93

14 Phytochemical constituents of various extracts of white

N. nucifera flower 94

15 DPPH radical scavenging activity (%) of various extracts of

A. saman leaves 96


white N. nucifera flowers 98

Table No

Title Page No

17 Total antioxidant activity of various extracts of A. saman

leaves 100

18 Total antioxidant activity of various extracts of white

N. nucifera flowers 101

19 HPTLC phenolic profile of the methanolic extract of A. saman

and Quercetin 104

20 HPTLC flavonoid profile of the methanolic extract of A. saman

and rutin 106

21 HPTLC cardiac glycoside profile of the methanolic extract of

A. saman and Stevioside 108

22 HPTLC phenolic profile of the methanolic extract of

N. nucifera and Quercetin 110

23 HPTLC flavonoid profile of the methanolic extract of

N. nucifera and Rutin 112

24 HPTLC glycoside profile of the methanolic extract of

N. nucifera and Stevioside 114

25 FT-IR peak values and their functional groups of A. saman

leaves 119

26 FT-IR peak values and their functional groups of N. nucifera

flowers 120

27 Changes in body weight and heart weight in experimental

groups 130

28 Levels of blood glucose in different experimental groups 131

29 Activities of serum cardiac marker enzymes in experimental

groups 133

30 Lipid profile in different experimental groups of rats 136

31 Antioxidant activities in experimental animals 138

32 Histopathological changes on the effect of MeAsL and MeNnF

in the hearts of Isoproterenol induced rats 143

List of Figures

Figure No

Title Page No

1 Development of diabetic cardiomyopathy 11

2 Structure of normal and DCM heart 12

3 Formation of atherosclerosis in blood vessels 15

4 Abnormalities associated with diabetic cardiomyopathy 16

5 Overview of leptin resistance and hyperleptinemia in obesity-

related cardiovascular disease

17

6 Pathophysiology of obesity and cardiomyopathy 18

7 Structure of cardiac muscle 20

8 Organization of the troponin-tropomyosin complex in cardiac

muscle fibre

21

9 Schematics showing the degeneration of a healthy cardiac

muscle cell to necrotic death

23

10 Development of heart disease 28

11 Foods and Flavonoids 31

12 Mechanisms of antioxidant effects of flavonoids 32

13 Protection by Polyphenols 33

14 Metabolism of Cardiac glycosides 34

15 Involvement of ROS in membrane lipids in pathophysiology of

cells

39

16 HbA1C level in diabetic cardiomyopathy patients 74

17 Levels of total cholesterol, triglycerides, LDL-C, HDL-C and

VLDL-C in diabetic cardiomyopathy patients

75

18 Activities of the cardiac enzymes in selected diabetic

cardiomyopathy patients

80

Figure No

Title Page No

19 C-reactive protein levels in selected diabetic cardiomyopathy

patients

81

20 Levels of Apolipoprotein A and B in selected diabetic

cardiomyopathy patients

83


A. saman leaves

97


white N. nucifera flowers

97

23 Total antioxidant activity of various extract of A. saman leaves 100

24 Total antioxidant activity of various extract of white N. nucifera

flowers

102

25 HPTLC finger print of the methanolic extract of A. saman and

Quercetin

105

26 Peak densitogram of the methanolic extract of A. saman and

Quercetin

105


Rutin

107


Rutin

107


Stevioside

109


Stevioside

109

31 HPTLC fingerprint of the methanolic extract of N. nucifera and

Quercein

111

32 Peak densitogram of the methanolic extract of N. nucifera and

Quercetin

111


Rutin

113


Rutin

113

Figure No

Title Page No


Stevioside

115


Stevioside

115

37 HPLC profile of the methanolic extract of A. saman leaves 117

38 HPLC profile of the methanolic extract of white

N. nucifera flower

117

39 FT-IR spectra of A. saman (methanolic extract) leaves 119

40 FT-IR spectra of N. nucifera (methanolic extract) flowers 120

41 GC-MS chromatogram of the methanolic extract of A. saman 121

42 Mass spectrum of GC-MS chromatogram of the methanolic

extract of A. saman

123

43 GC-MS Chromatogram of the methanolic extract of N.nucifera 125

44 Mass fragmentation pattern of GC-MS Spectrum of

methanolic extract of white N.nucifera

126

45 Blood glucose levels in experimental animals 132

46 Activities of serum cardiac enzymes in different experimental

groups of rats

134

47 Troponin enzyme activity in different experimental groups

of rats

134

48 Lipid profile in different experimental groups of rats 137

49 Activities of catalase, superoxide dismutase and glutathione

peroxidase in the heart homogenate

139

50 Activities of reduced glutathione (GSH) and glutathione

S- transferase (GST) in the heart homogenate

140

51 Activities of lipid peroxidase (LPO) in the heart homogenate 141

List of Plates

Plate No

Title Page No

1 (I) Drugs used for the induction of diabetes and

cardiomyopathy in experimental animals

(II) Drugs used for the treatment of diabetes and

cardiomyopathy in experimental animals

55

55

2 Intraperitoneal injection on experimental animals 56

3 Dissection of the experimental animals 57

4 Dissected heart from different experimental groups 142

5 Histopathological changes in the hearts of different

experimental groups of rats

144

List of Appendices

Appendix No

Title Page No

I Initial Proforma 179

II Final Proforma 180

III Estimation of HbA1c 182

IV Estimation of total cholesterol 183

V Estimation of triglycerides 184

VI Estimation of LDL cholesterol 185

VII Estimation of HDL cholesterol 186

VIII Estimation of glutamic oxaloacetic transaminase 187

XI Assay of alkaline phosphatase 188

X Estimation of creatine kinase 189

XI High Sensitive C- reactive protein 190

XII Estimation of apolipoprotein- A 191

XII Estimation of apolipoprotein B 192

XIV Estimation of creatinine kinase-MB 192

XV Assay of troponin T 193

XVI Soxhlet extraction 194

XVII Phytochemical screening 197

XVIII DPPH assay 201

XIX Total antioxidant activity 202

XX GC-MS spectrum 203

XXI Assay of catalase (CAT) 204

XXII Assay of superoxide dismutase (SOD) 204

XXIII Estimation of glutathione peroxidase 205

XXIV Assay of glutathione-S- transferase (GST) 206

XXV Estimation of reduced glutathione 207

XXVI Assay of lipid peroxidation 207

XXVII Histological examination 208

List of Abbreviations

1,1-Diphenyl-2-picrylhydrazyl - DPPH

2, 4 -dinitrophenyl hydrazine - DNPH

4-amino phenazone - 4-AP

Adenosine triphosphate - ATP

Alkaline phosphatase - ALP

Angiotensin-converting-enzyme inhibitor - ACE

Aspartate aminotransferase - AST

Blood pressure - BP

Body mass index - BMI

Cardiac troponin T - cTnT

Cardiac troponins I - cTnI

Cardio vascular - CV

Cardiovascular disease - CVD

Catalase - CAT

Cholesterol esterase - CHE

Cholesterol oxidase - CHOD

CK isoenzyme in brain - CK-BB

CK isoenzyme in skeletal muscle - CK-MM

CK isoenzymes in heart (Membrane bound) - CK-MB

Committee for the purpose of control and supervision

of experiments on animals - CPCSEA

Coronary artery disease - CAD

Coronary heart disease - CHD

C-reactive protein - CRP

Creatine kinase - CK

Deoxyribonucleic acid - DNA

Diabetes mellitus - DM

Diabetic cardiomyopathy - DCM

Diabetic heart disease - DHD

Dihydroxy acetone phosphate - DAP

Dipeptidyl peptidase 4 -DPP-4

Duncan's multiple range test - DMRT

Ethylenediaminetetraacetic acid - EDTA

Fasting blood glucose - FBG

Fourier transform infrared spectroscopy - FTIR

Fructosyl valine oxidase - FVO

Gas chromatography–mass spectrometry - GC-MS

Glutathione peroxidase - GPx

Glutathione reductase (NADPH) - GSSGR

Glutathione reductase - GR

Glutathione S transferase - GST

Glycated hemoglobin - HbA1c

Glycerol phosphate dehydrogenase - GPO

Glycerol-3-phosphate - G3P

Half inhibitory concentration - IC50

Heart failure - HF

High density lipo protein cholesterol - HDL-C

High performance thin layer chromatography - HPTLC

High-performance liquid chromatography - HPLC

Horse radish peroxidase - POD

Hypertension - HTN

Impaired glucose tolerance - IGT

Insulin-like growth factor 1 - IGF-1

Isoproterenol - ISO

Lactate dehydrogenase - LDH

Left ventricular hypertrophy - LVH

Left ventricular - LV

Lipid peroxides - LPO

Lipoprotein lipase - LPL

Low-density lipoprotein cholesterol - LDL-C

Methanolic extract of Albizia saman leaves - MeAsL

Methanolic extract of Nelumbo nucifera flower - MeNnF

Nicotinamide adenine dinucleotide phosphate hydrogen - NADPH

Peripheral vascular disease - PVD

Polyethylene Glycol - PEG

Reactive oxygen species - ROS

Reactive species - RS

Reduced glutathione - GSH

Renin angiotensin system - RAS

Retention factor - Rf

Serum glutamic oxaloacetic transaminase -SGOT

Streptozotocin - STZ

Superoxide dismutase - SOD

Tetra methyl benzidine - TMB

Thiobarbituric acid - TBA

Total hemoglobin - THb

Traditional Chinese medicine - TCM

Trichloroacetic acid - TCA

Troponin C - TnC

Two-dimensional - 2D

Very low density lipoprotein cholesterol -VLDL-C

World Health Organization - WHO

biochemical changes in pppatients with diabetic...

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