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Biochemical changes in Biochemical changes in Biochemical changes in Biochemical changes in ppppatients with diabetic cardiomyopathy during atients with diabetic cardiomyopathy during atients with diabetic cardiomyopathy during atients with diabetic cardiomyopathy during
drug therapy and the cardio protective effect ofdrug therapy and the cardio protective effect ofdrug therapy and the cardio protective effect ofdrug therapy and the cardio protective effect of selected selected selected selected
medicinal plants in Isoproterenolmedicinal plants in Isoproterenolmedicinal plants in Isoproterenolmedicinal plants in Isoproterenol induced induced induced induced
Swiss albino ratsSwiss albino ratsSwiss albino ratsSwiss albino rats
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,
Avinashilingam Institute for Home Science and Higher Education for Women,
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,
Avinashilingam Institute for Home Science and Higher Education for Women,
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,
Avinashilingam Institute for Home Science and Higher Education for Women,
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,
Avinashilingam Institute for Home Science and Higher Education for Women,
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
selected hospitals 67
8 The prevalence of familial restrictive cardiomyopathy in
selected hospitals 68
9 Signs and symptoms of diabetic cardiomyopathy patients in
selected hospitals 70
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
16 DPPH radical scavenging activity (%) of various extracts of
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
21 DPPH radical scavenging activity (%) of various extracts of
A. saman leaves
97
22 DPPH radical scavenging activity (%) of various extracts of
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
27 HPTLC finger print of the methanolic extract of A. saman and
Rutin
107
28 Peak densitogram of the methanolic extract of A. saman and
Rutin
107
29 HPTLC finger print of the methanolic extract of A. saman and
Stevioside
109
30 Peak densitogram of the methanolic extract of A. saman and
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
33 HPTLC fingerprint of the methanolic extract of N. nucifera and
Rutin
113
34 Peak densitogram of the methanolic extract of N. nucifera and
Rutin
113
Figure No
Title Page No
35 HPTLC fingerprint of the methanolic extract of N. nucifera and
Stevioside
115
36 Peak densitogram of the methanolic extract of N. nucifera and
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
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