In The Name of Allah, the Most Beneficent, the Most Merciful

PHARMACOLOGICAL RATIONALE FOR THE USE OF AEGLE MARMELOS. L AND PYRUS CYDONIA. L IN CARDIOVASCULAR DISORDERS

ATIQ-UR-RAHMAN MBBS

DEPARTMENT OF FACULTY OF PHARMACY FEDERAL URDU UNIVERSITY OF ARTS, SCIENCE AND TECHNOLOGY GULSHAN-E-IQBAL CAMPUS, KARACHI PAKISTAN 2017

PHARMACOLOGICAL RATIONALE FOR THE USE OF AEGLE MARMELOS. L AND PYRUS CYDONIA. L IN CARDIOVASCULAR DISORDERS

Thesis submitted in partial fulfillment of the requirements for the degree of doctor of philosophy in pharmacology

By ATIQ-UR-RAHMAN MBBS

Supervised By S. INTASAR H TAQVI, Ph. D T.T.S Assistant Professor

DEPARTMENT OF PHARMACOLOGY FACULTY OF PHARMACY FEDERAL URDU UNIVERSITY OF ARTS, SCIENCE AND TECHNOLOGY, GULSHAN-E-IQBAL CAMPUS, KARACHI PAKISTAN 2017

CERTIFICATE

This is certified that ATIQ-UR-RAHMAN carried out this research work entitled “Pharmacological rationale for the use of Aegle marmelos. L and

Pyrus cydonia. L in cardiovascular disorders” under my supervision. The idea was conceived by supervisor which was well supported by the scientific literature conforming the originality and experimental evidence was provided for the doctoral thesis. I found the thesis is ready to submit for the degree of Doctor of Philosophy in Pharmacology and is up to my full satisfaction.

DR. S. INTASAR H TAQVI T.T.S Assistant Professor Department of Pharmacology, Faculty of Pharmacy Federal Urdu University of Arts, Science and Technology, Gulshan-e-Iqbal Campus, Karachi, Pakistan

DEDICATION

This work is dedicated to my beloved Parents, family members and friends

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CONTENTS

S.NO TITLE Page No.

BISMILLAH TITLE PAGE CERTIFICATE DEDICATION CONTENTS I LIST OF TABLES X LIST OF FIGURES XII LIST OF ABRREVIATIONS XXIV ACKNOWLEDGEMENT XXVIII ABSTRACT IN ENGLISH XXX URDU TRANSLATION OF ABSTRACT XXXIII 1 CHAPTER -1 1 INTRODUCTION 1.1 Brief historical background of herbal plants 2 1.2 Importance of research and scientific validation of herbal medicine 3 1.3 Importance of research in herbal medicine in Pakistan 4 1.4 Plants use in cardiovascular disorders 5 1.4.1 Hypertension 5 1.4.2 failure 5 1.4.3 Ischemic heart disease 6 1.4.4 Arrhythmia 6 1.5 Prevalence of cardiovascular diseases 7 1.5.1 Hypertension 7 1.5.2 Congestive heart failure 8 1.5.3 Ischemic heart diseases 8 1.5.4 Arrhythmia 9 1.6 Functional properties of the heart 9 1.6.1 Chronotropism 9 1.6.2 Inotropism 9

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1.6.3 Lusitropy 10 1.7 Animal models for evaluating cardiovascular activity 10 1.7.1 Isolated perfused heart preparations: 10 1.7.2 Isolated smooth muscle preparation 11 1.8 Aims and Objective 14 2 CHAPTER -2 15 LITERATURE REVIEW 2.1 Aegle Marmelos Linn 16 2.1.1 Habitat and Morphology 16 2.1.2 Traditional uses in various disorders 16 2.1.3 Pharmacological activities 18 2.1.4 Phytochemical constituents 20 2.2 Pyrus Cydonia Linn 20 2.2.1 Habitat and Morphology 21 2.2.2 Traditional uses in various disorders 21 2.2.3 Pharmacological activities 22 2.2.4 Phytochemical constituents 23 3 CHAPTER -3 24 MATERIALS AND METHODS 3.1 , standards and chemicals 25 3.2 Selection of plants 25 3.2.1 Collection and identification of plant materials 26 3.2.2 Preparation of crude extracts 26 3.2.3 Fractionation and isolation 26 3.2.4 Isolation of marmelosin 27 3.2.5 Spectral characterization of marmelosin isolated from 28 A.marmelos fruit 3.3 Animals 34 3.4 Experimentations 34 3.4.1 In vivo experiments 34

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3.4.1.1 Acute oral toxicity study of crude extract of Aegle 34 marmelosLinn. 3.4.1.2 Acute oral toxicity studies of crude extract of Pyrus 35 cydoniaLinn. 3.4.2 In vitro experiments 35 3.4.2.1 Isolated Langendorff’s heart preparation 35 3.4.2.1.1 Study of crude extracts on Isolated Langendorff’s rat heart 35 3.4.2.1.1.1 Concentrations of crude extract of A.marmelosLinn. 37 3.4.2.1.1.2 Concentrations of crude extract of P.cydoniaLinn. 37 3.4.2.2 Isolated Working heart preparation 38 3.4.2.2.1 At fixed preload and afterload 40 A. Concentrations of crude extract, fractions and marmelosin used 40 A-1 Crude extract of A. marmelos. L 40 A-2 Aqueous fraction of A. marmelos Linn 41 A-3 Butanolic fraction of A. marmelos Linn 41 A-4 Marmelosin isolated (ISD) and commercially available 41 marmelosin (STD) A-5 Digoxin 41 A-6 Verapamil hydrochloride 41 3.4.2.2.2 At variable preloads 41 B. Concentrations of crude extract, fractions and marmelosin used 42 B-1 Crude extract of A. marmelos Linn 42 B-2 Aqueous fraction of A. marmelos Linn 43 B-3 Butanolic fraction of A. marmelos Linn 43 B-4 Marmelosin isolated from A. marmelos Linn 43 B-5 Digoxin 43 B-6 Verapamil hydrochloride 43 3.4.2.2.3 Calcium paradox experiments 43 C. To study the effects of crude extract of A.marmelos on calcium 45 paradox C-1 Ca++-free KH followed by Normal KH 45

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C-2 Ca++-free KH followed by Normal KH + crude extract 30.0 mg/ml 45 C-3 Ca++-free KH + crude extract 30.0 mg/mL followed by Normal KH + 45 crude extract 30.0 mg/mL D. To study the effects of Verapamil hydrochloride on calcium 45 paradox D-1 Ca++-free KH followed by Normal KH 45 D-2 Ca++-free KH followed by Normal KH + Verapamil 1µM 46 D-3 Ca++-free KH+ Verapamil 1µM followed by Normal KH + Verapamil 46 1µM 3.4.2.3 Isolated rat thoracic aorta 46 3.4.2.3.1 Preparation of thoracic aortic ring 46 3.4.2.3.2 Effect on vascular tone 47 3.4.2.3.3 Endothelium-dependent and endothelium-independent 48 effects 3.4.2.3.4 Determination of Ca++ antagonistic activity 48 3.5 Statistical analysis 49 4 CHAPTER -4 50 RESULTS TOXICITY STUDIES 51 4.1 Acute oral toxicity studies of crude extracts 51 4.1.1 Aegle marmelosLinn 51 4.1.2Pyrus cydoniaLinn 51 LANGENDORFF’S HEART EXPERIMENTS 51 4.2 Effect of crude extracts on Langendorff’s rat heart 51 4.2.1 Aegle marmelos. L 51 4.2.1.1 Left ventricular pressure (mm Hg) 51 4.2.1.2 Systolic pressure (mm Hg) 52 4.2.2 Pyrus cydonia. L 52 4.2.2.1 Left ventricular pressure (mm Hg) 52 4.2.2.2 Systolic pressure (mm Hg) 52 WORKING HEART EXPERIMENTS 53 A. CRUDE EXTRACT OF AEGLE MARMELOS (Am.Cr) 53

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4.3 Crude extract of Aegle marmelos(Am.Cr) on working rat heart 53 4.3.1 At fixed physiological preload and afterload 53 4.3.1.1 Effect on coronary effluent, aortic outflow and cardiac output 53

4.3.1.2 Effect on dP/dt(max) and dP/dt(min) 54 4.3.1.3 Effect on systolic and diastolic pressure and heart rate 54 4.3.1.4 Effect on peak aortic systolic pressure and end diastolic 55 pressure 4.3.1.5 Effect on ejection fraction and stroke volume 55 4.3.1.6 Effect on rate pressure product and cardiac power 56 4.3.2 At variable preloads 56 4.3.2.1 Pre-treatment at the concentration of 3.0 mg/mL 56 4.3.2.2 Pretreatment at the concentration of 30.0 mg/mL 58 4.3.2.3 Pretreatment at the concentration of 100.0 mg/mL 60 4.3.3 Calcium paradox experiments 61 4.3.3.1 Effects on coronary effluent 62 4.3.3.2 Effects on aortic outflow 62

4.3.3.3 Effects on dP/dt(max) 63

4.3.3.4 Effects on dP/dt(min) 63 4.3.3.5 Effects on heart rate 64 4.3.3.6 Effects on left ventricular mean pressure 64 4.3.3.7 Effects on stroke volume 65 4.3.3.8 Effects on rate pressure product 65 EXPERIMENTS ON ISOLATED RAT AORTA 66 4.3.4 Effect of crude extract of A.marmelos L 66 4.3.4.1 At baseline tension 66 4.3.4.2 On pre-contracted rat aorta 66 4.3.4.3 At baseline tension 67 4.3.4.4 On K+80 mM pre-contracted aorta 67 4.3.4.5 Construction of Ca++ curves 68

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DISCUSSION 69 Crude extract of A.marmelos. L B. AQUEOUS FRACTION OF AEGLE MARMELOS (Aq.Fr.Cr) 124 4.4 Aqueous fraction of Aegle marmelos(Am.Cr) on working rat heart 124 4.4.1 At fixed physiological preload and afterload 124 4.4.1.1 Effect on coronary effluent, aortic outflow and cardiac output 124

4.4.1.2 Effect on dP/dt(max) and dP/dt(min) 124 4.4.1.3 Effects on systolic and diastolic pressure and heart rate 125 4.4.1.4 Effect on peak aortic systolic pressure and end diastolic 125 pressure 4.4.1.5 Effect on ejection fraction and stroke volume 126 4.4.1.6 Effect on rate pressure product and cardiac power 126 4.4.2 At variable preloads 126 4.4.2.1 Pretreatment at the concentration of 1.0 mg/mL 127 4.4.2.2 Pretreatment at the concentration of 10.0 mg/mL 128 4.4.2.3 Pretreatment at the concentration of 30.0 mg/mL 130 EXPERIMENTS ON ISOLATED RAT AORTA 132 4.4.3 Effect of Aq.Fr.Cr on rat aorta 132 4.4.3.1 At baseline tension 132 4.4.3.2 On phenylephrine pre-contracted rat aorta 132 4.4.3.3 At baseline tension 133 4.4.3.4 On K+80 mM pre-contracted rat aorta 133 4.4.3.5 At baseline tension 134 4.4.3.6 On L-NAME-incubated and PE pre-contracted rat aorta 134 DISCUSSION 135 Aqueous fraction of A.marmelos. L C. BUTANOLIC FRACTION OF AEGLE MARMELOS (But.Fr.Cr) 171 4.5 Butanolic fraction of Aegle marmelos(Am.Cr) on working rat heart 171 4.5.1 At fixed physiological preload and afterload 171 4.5.1.1 Effects on coronary effluent, aortic outflow and cardiac output 171

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4.5.1.2 Effects on dP/dt(max) and dP/dt(min) 171 4.5.1.3 Effect on systolic and diastolic pressure and heart rate 172 4.5.1.4 Effect on peak aortic systolic and end diastolic pressures 172 4.5.1.5 Effect on ejection fraction and stroke volume 173 4.5.1.6 Effect on rate pressure product and cardiac power 173 4.5.2 At variable preloads 174 4.5.2.1 Pretreatment at the concentration of 0.01 mg/mL 174 4.5.2.2 Pretreatment at the concentration of 0.1 mg/mL 176 4.5.2.3 Pretreatment at the concentration of 1.0 mg/mL 177 EXPERIMENTS ON ISOLATED RAT AORTA 179 4.5.3 Effect of But. Fr. Cr of A. marmelos 179 4.5.3.1 At baseline tension 179 4.5.3.2 On phenylephrine pre-contracted rat aorta 180 4.5.3.3 At baseline tension 180 4.5.3.4 On K+ 80 mM pre-contracted rat aorta 181 4.5.3.5 Construction of Ca++ curves 181 DISCUSSION 183 Butanolic fraction of A.marmelos D. MARMELOSIN (ISD Marm. and STD Marm.) 218 4.6 Isolated and standard marmelosin (ISD Marm and STD Marm) on 218 working rat heart 4.6.1 At fixed physiological preload and afterload 218 4.6.1.1 Effect of the ISD Marm 218 4.6.1.1.1 Effect on coronary effluent 218 4.6.1.1.2 Effect on aortic outflow 218 4.6.1.1.3 Effect on cardiac output 218

4.6.1.1.4 Effect on dP/dt(max) 219

4.6.1.1.5 Effect on dP/dt(min) 219 4.6.1.1.6 Effect on systolic pressure 219 4.6.1.1.7 Effect on diastolic pressure 219

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4.6.1.1.8 Effect on heart rate 220 4.6.1.1.9 Effect on peak aortic systolic pressure 220 4.6.1.1.10 Effect on end diastolic pressure 220 4.6.1.1.11 Effect on ejection fraction 220 4.6.1.1.12 Effect on stroke volume 220 4.6.1.1.13 Effect on rate pressure product 221 4.6.1.1.14 Effect on cardiac power 221 4.6.1.2 Effect of the STD Marm 221 4.6.1.2.1 Effect on coronary effluent 221 4.6.1.2.2 Effect on aortic outflow 222 4.6.1.2.3 Effect on cardiac output 222

4.6.1.2.4 Effect on dP/dt(max) 222

4.6.1.2.5 Effect on dP/dt(min) 222 4.6.1.2.6 Effect on systolic pressure 223 4.6.1.2.7 Effect on diastolic pressure 223 4.6.1.2.8 Effect on heart rate 223 4.6.1.2.9 Effect on peak aortic systolic pressure 223 4.6.1.2.10 Effect on end diastolic pressure 224 4.6.1.2.11 Effect on ejection fraction 224 4.6.1.2.12 Effect on stroke volume 224 4.6.1.2.13 Effect on rate pressure product 224 4.6.1.2.14 Effect on cardiac power 224 4.6.2 At variable preload 225 4.6.2.1 Pretreatment at the concentration of 1µM 225 4.6.2.2 Pretreatment at the concentration of 10µM 227 4.6.2.3 Pretreatment at the concentration of 100 µM 228 EXPERIMENTS ON ISOLATED RAT AORTA 230 4.6.3 Effect of standard marmelosin on rat aorta 230

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4.6.3.1 At baseline tension 230 4.6.3.2 On phenylephrine pre-contracted rat aorta 231 4.6.3.3 At baseline tension 231 4.6.3.4 On K+80 mM pre-contracted rat aorta 232 4.6.3.5 At baseline tension 232 4.6.3.6 On L-NAME-incubated and PE pre-contracted rat aorta 232 DISCUSSION 234 Isolated (ISD) and Standard (STD) Marmelosin 5 CHAPTER -5 275 GENERAL DISCUSSION AND CONCLUSION Aegle marmelos fruit in cardiovascular disorders REFERENCES 279 APPENDICES 306 TABLES AND FIGURES Appendix No. 1 307 DIGOXIN Appendix No.2 332 VERAPAMIL HCl

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LIST OF TABLES

S.No Titles Page No

4.1.1 Effect of oral toxicity study of crude extract of A.marmelos in Swiss 83 albino mice. 4.1.2 Effect of oral toxicity study of crude extract of P.cydonia in Swiss albino 83 mice. 4.2 Effect of crude extracts of A.marmelos and P.cydonia on left ventricular 84 pressure and systolic pressure in Langendorff’s rat heart 4.3.1 Effects of crude extract of A.marmelos on various parameters in 85 isolated rat working heart 4.3.2.1 Effect of variable pre-loads on isolated rat working with or 86 without crude extract of A.marmelos (3.0mg/mL) 4.3.2.2 Effect of variable pre-loads isolated rat working heart with or without 87 crude extract of A.marmelos (30.0mg/mL) 4.3.2.3 Effect of variable pre-loads on isolated rat working heart with or 88 without crude extract of A.marmelos (100.0 mg/mL) 4.3.3.1 Effect of calcium paradox with/or without crude extract of A.marmelos 89 (30.0mg/mL) on coronary effluent 4.3.3.2 Effect of calcium paradox with/or without crude extract of A.marmelos 89 (30.0mg/mL) on aortic outflow 4.3.3.3 Effect of calcium paradox with/or without crude extract of A.marmelos 90 (30.0mg/mL) on dP/dt(max) 4.3.3.4 Effect of calcium paradox with/or without crude extract of A.marmelos 90 (30.0mg/mL) on dP/dt(min) 4.3.3.5 Effect of calcium paradox with/or without crude extract of A.marmelos 91 (30.0mg/mL) on heart rate 4.3.3.6 Effect of calcium paradox with/or without crude extract of A.marmelos 91 (30.0mg/mL) on left ventricular mean pressure 4.3.3.7 Effect of calcium paradox with/or without crude extract of A.marmelos 92 (30.0mg/mL) on stroke volume 4.3.3.8 Effect of calcium paradox with/or without crude extract of A.marmelos 92 (30.0mg/mL) on rate pressure product 4.3.4.1 Effect of crude extract of A.marmelos on rat aorta pre-contracted by 93 phenylephrine (1 µM) 4.3.4.2 Effect of crude extract of A.marmelos on rat aorta pre-contracted by 93 high K+(80 mM) 4.4.1 Effects of aqueous fraction of A.marmelos on various parameters in 144 isolated rat working heart 4.4.2.1 Effect of variable pre-loads on isolated rat working heart with or 145 without aqueous fraction of A.marmelos (1.0 mg/mL)

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4.4.2.2 Effect of variable pre-loads on isolated rat working heart with or 146 without aqueous fraction of A.marmelos (10.0 mg/mL) 4.4.2.3 Effect of variable pre-loads on isolated rat working heart with or 147 without aqueous fraction of A.marmelos (30.0 mg/mL) 4.4.3.1 Effect of aqueous fraction of A.marmelos on rat thoracic aorta pre- 148 contracted by phenylephrine (1 µM) 4.4.3.2 Effect of aqueous fraction of A.marmelos on rat thoracic aorta pre- 148 contracted by high K+(80 mM) 4.4.3.3 Effect of aqueous fraction of A.marmelos on L-NAME-incubated and 149 PE pre-contracted rat thoracic aorta. 4.5.1 Effects of butanolic fraction of A.marmelos on various parameters in 192 isolated rat working heart 4.5.2.1 Effect of variable pre-loads on isolated rat working heart with or 193 without butanolic fraction of A.marmelos (0.01 mg/mL) 4.5.2.2 Effect of variable pre-loads on isolated rat working heart with or 194 without butanolic fraction of A.marmelos (0.1 mg/mL) 4.5.2.3 Effect of variable pre-loads on isolated rat working heart with or 195 without butanolic fraction of A.marmelos (1.0 mg/mL) 4.5.3.1 Effect of butanolic fraction of A.marmelos on rat thoracic aorta pre- 196 contracted by phenylephrine (1µM) 4.5.3.2 Effect of butanolic fraction of A.marmelos on rat thoracic aorta pre- 196 contracted by high K+(80 mM) 4.6.1.1 Effects of isolated marmelosin on various parameters in isolated rat 243 working heart 4.6.1.2 Effects of standard marmelosin on various parameters in isolated rat 244 working heart 4.6.2.1 Effect of variable pre-loads on isolated rat working heart with or 245 without marmelosin isolated from A.marmelos (1 µM) 4.6.2.2 Effect of variable pre-loads on isolated rat working heart with or 246 without marmelosin isolated from A.marmelos (10 µM) 4.6.2.3 Effect of variable pre-loads on isolated rat working heart with or 247 without marmelosin isolated from A.marmelos (100 µM) 4.6.3.1 Effect of standard marmelosin on rat thoracic aorta pre-contracted by 248 phenylephrine (1µM) 4.6.3.2 Effect of standard marmelosin on rat thoracic aorta pre-contracted by 248 high K+ (80 mM) 4.6.3.3 Effect of standard marmelosin on L-NAME-incubated and PE pre- 249 contracted rat thoracic aorta

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LIST OF FIGURES

S.No Titles Page No

Fig 4.2.1 Effect of crude extracts of A.marmelos and P.cydonia on left 95 ventricular pressure in isolated rat Langendorff's heart Fig 4.2.2 Effect of crude extract of P.cydonia and A.marmelos on systolic 95 pressure in isolated rat Langendorff's heart Fig 4.3.1.1 Effect of crude extract of A.marmelos on coronary effluent , 96 aortic out flow and cardiac output in isolated rat working heart

Fig 4.3.1.2 Effect of crude extract of A.marmelos on dP/dt(max) and dP/dt(min) 96 in isolated rat working heart Fig 4.3.1.3 Effect of crude extract of A.marmelos on systolic, diastolic 97 pressure and heart rate in isolated rat working heart Fig 4.3.1.4 Effect of crude extract of A.marmelos on peak aortic systolic 97 pressure and end diastolic pressure in isolated rat working heart Fig 4.3.1.5 Effect of crude extract of A.marmelos on ejection fraction and 98 stroke volume in isolated rat working heart Fig 4.3.1.6 Effect of crude extract of A.marmelos on rate pressure product 98 and cardiac power in isolated rat working heart Fig 4.3.2.1.1 Effect of variable preloads on coronary effluent with or without 99 pretreatment of crude extract of A.marmelos (3 mg/mL) in isolated rat working heart Fig 4.3.2.1.2 Effect of variable preloads on aortic outflow with or without 99 pretreatment of crude extract of A.marmelos (3 mg/mL) in isolated rat working heart Fig 4.3.2.1.3 Effect of variable preloads on cardiac output with or without 100 pretreatment of crude extract of A.marmelos (3 mg/mL) in isolated rat working heart Fig 4.3.2.1.4 Effect of variable preloads on dP/dt(max) with or without 100 pretreatment of crude extract of A.marmelos (3 mg/mL) in isolated rat working heart Fig 4.3.2.1.5 Effect of variable preloads on dP/dt(min) with or without 101 pretreatment of crude extract of A.marmelos (3 mg/mL) in isolated rat working heart Fig 4.3.2.1.6 Effect of variable preloads on heart rate with or without 101 pretreatment of crude extract of A.marmelos (3 mg/mL) in isolated rat working heart Fig 4.3.2.1.7 Effect of variable preloads on left ventricular pressure with or 102 without pretreatment of crude extract of A.marmelos (3 mg/mL) in isolated rat working heart

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Fig 4.3.2.1.8 Effect of variable preloads on peak aortic systolic pressure with 102 or without pretreatment of crude extract of A.marmelos (3 mg/mL) in isolated rat working heart Fig 4.3.2.1.9 Effect of variable preloads on coronary vascular resistance with 103 or without pretreatment of crude extract of A.marmelos (3 mg/mL) in isolated rat working heart Fig 4.3.2.1.10 Effect of variable preloads on myocardial work with or without 103 pretreatment of crude extract of A.marmelos (3 mg/mL) in isolated rat working heart Fig 4.3.2.2.1 Effect of variable preloads on coronary effluent with or without 104 pretreatment of crude extract of A.marmelos (30 mg/mL) in isolated rat working heart Fig 4.3.2.2.2 Effect of variable preloads on aortic outflow with or without 104 pretreatment of crude extract of A.marmelos (30 mg/mL) in isolated rat working heart Fig 4.3.2.2.3 Effect of variable preloads on cardiac output with or without 105 pretreatment of crude extract of A.marmelos (30 mg/mL) in isolated rat working heart Fig 4.3.2.2.4 Effect of variable preloads on dP/dt(max) with or without 105 pretreatment of crude extract of A.marmelos (30 mg/mL) in isolated rat working heart Fig 4.3.2.2.5 Effect of variable preloads on dP/dt(min) with or without 106 pretreatment of crude extract of A.marmelos (30 mg/mL) in isolated rat working heart Fig 4.3.2.2.6 Effect of variable preloads on heart rate with or without 106 pretreatment of crude extract of A.marmelos (30 mg/mL) in isolated rat working heart Fig 4.3.2.2.7 Effect of variable preloads on left ventricular pressure with or 107 without pretreatment of crude extract of A.marmelos (30 mg/mL) in isolated rat working heart Fig 4.3.2.2.8 Effect of variable preloads on peak aortic systolic pressure with 107 or without pretreatment of crude extract of A.marmelos (30 mg/mL) in isolated rat working heart Fig 4.3.2.2.9 Effect of variable preloads on coronary vascular resistance with 108 or without pretreatment of crude extract of A.marmelos (30 mg/mL) in isolated rat working heart Fig 4.3.2.2.10 Effect of variable preloads on myocardial work with or without 108 pretreatment of crude extract of A.marmelos (30 mg/mL) in isolated rat working heart Fig 4.3.2.3.1 Effect of variable preloads on coronary effluent with or without 109 pretreatment of crude extract of A.marmelos (100 mg/mL) in isolated rat working heart

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Fig 4.3.2.3.2 Effect of variable preloads on aortic out flow with or without 109 pretreatment of crude extract of A.marmelos (100 mg/mL) in isolated rat working heart Fig 4.3.2.3.3 Effect of variable preloads on cardiac output with or without 110 pretreatment of crude extract of A.marmelos (100 mg/mL)in isolated rat working heart Fig 4.3.2.3.4 Effect of variable preloads on dP/dt(max) with or without 110 pretreatment of crude extract of A.marmelos (100 mg/mL) in isolated rat working heart Fig 4.3.2.3.5 Effect of variable preloads on dP/dt(min) with or without 111 pretreatment of crude extract of A.marmelos (100 mg/mL) in isolated rat working heart Fig 4.3.2.3.6 Effect of variable preloads on heart rate with or without 111 pretreatment of crude extract of A.marmelos (100 mg/mL) in isolated rat working heart Fig 4.3.2.3.7 Effect of variable preloads on left ventricular pressure with or 112 without pretreatment of crude extract of A.marmelos (100 mg/mL) in isolated rat working heart Fig 4.3.2.3.8 Effect of variable preloads on peak aortic systolic pressure with 112 or without pretreatment of crude extract of A.marmelos (100 mg/mL) in isolated rat working heart Fig 4.3.2.3.9 Effect of variable preloads on coronary vascular resistance with 113 or without pretreatment of crude extract of A.marmelos (100 mg/mL) in isolated rat working heart Fig 4.3.2.3.10 Effect of variable preloads on myocardial work with or without 113 pretreatment of crude extract of A.marmelos (100 mg/mL) in isolated rat working heart Fig 4.3.3.1 Effect of crude extract of A.marmelos (30 mg/mL) on coronary 114 effluent in Ca++-paradox experiments in isolated rat working heart Fig 4.3.3.2 Effect of crude extract of A.marmelos (30 mg/mL) on aortic out 115 flow in Ca++-paradox experiments in isolated rat working heart

Fig 4.3.3.3 Effect of crude extract of A.marmelos (30 mg/mL) on dP/dt(max) in 116 Ca++-paradox experiments in isolated rat working heart

Fig 4.3.3.4 Effect of crude extract of A.marmelos (30 mg/mL) on dP/dt(min) in 117 Ca++-paradox experiments in isolated rat working heart Fig 4.3.3.5 Effect of crude extract of A.marmelos (30 mg/mL) on heart rate 118 in Ca++-paradox experiments in isolated rat working heart. Fig 4.3.3.6 Effect of crude extract of A.marmelos (30 mg/mL) on left 119 ventricular mean pressure in Ca++-paradox experiments in isolated rat working heart Fig 4.3.3.7 Effect of crude extract of A.marmelos (30 mg/mL) on stroke 120 volume in Ca++-paradox experiments in isolated rat working heart

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Fig 4.3.3.8 Effect of crude extract of A.marmelos (30 mg/mL) on rate 121 pressure product in Ca++-paradox experiments in isolated rat working heart Fig 4.3.4.1 Effect of crude extract of A.marmelos on baseline and PE pre- 122 contracted endothelium intact and denuded rat aorta Fig 4.3.4.2 Effect of crude extract of A.marmelos on baseline and K+80 mM 122 pre-contracted endothelium intact and denuded rat aorta Fig 4.3.4.3.1 Construction of Ca++ curves in the presence and absence of 123 various concentrations of crude extract of A.marmleos in endothelium denuded rat aorta showing Ca++ agonistic activity Fig 4.3.4.3.2 Construction of Ca++ curves in the presenceand absence of 123 various concentrations of crude extract of A.marmelos in endothelium denuded rat aorta showing Ca++ antagonistic activity Fig 4.4.1.1 Effect of aqueous fraction of A.marmelos on coronary effluent, 151 aortic out flow and cardiac output in isolated rat working heart Fig 4.4.1.2 Effect of aqueous fraction of A.marmelos on dP/dt(max) and 151 dP/dt(min) in isolated rat working heart Fig 4.4.1.3 Effect of aqueous fraction of A.marmelos on systolic, diastolic 152 pressure and heart rate in isolated rat working heart Fig 4.4.1.4 Effect of aqueous fraction of A.marmelos on peak aortic systolic 152 pressure and end diastolic pressure in isolated rat working heart Fig 4.4.1.5 Effect of aqueous fraction of A.marmelos on ejection fraction and 153 stroke volume in isolated rat working heart Fig 4.4.1.6 Effect of aqueous fraction of A.marmelos on rate pressure 153 product and cardiac power in isolated rat working heart Fig 4.4.2.1.1 Effect of variable preloads on coronary effluent with or without 154 pretreatment of aqueous fraction of A.marmelos (1 mg/mL) in isolated rat working heart Fig 4.4.2.1.2 Effect of variable preloads on aortic out flow with or without 154 pretreatment of aqueous fraction of A.marmelos (1 mg/mL) in isolated rat working heart Fig 4.4.2.1.3 Effect of variable preloads on cardiac output with or without 155 pretreatment of aqueous fraction of A.marmelos (1 mg/mL) in isolated rat working heart Fig 4.4.2.1.4 Effect of variable preloads on dP/dt(max) with or without 155 pretreatment of aqueous fraction of A.marmelos (1 mg/mL) in isolated rat working heart Fig 4.4.2.1.5 Effect of variable preloads on dP/dt(min) with or without 156 pretreatment of aqueous fraction of A.marmelos (1 mg/mL) in isolated rat working heart Fig 4.4.2.1.6 Effect of variable preloads on heart rate with or without 156 pretreatment of aqueous fraction of A.marmelos (1 mg/mL) in

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isolated rat working heart Fig 4.4.2.1.7 Effect of variable preloads on left ventricular pressure with or 157 without pretreatment of aqueous fraction of A.marmelos (1 mg /mL) in isolated rat working heart Fig 4.4.2.1.8 Effect of variable preloads on peak aortic systolic pressure with 157 or without pretreatment of aqueous fraction of A.marmelos (1 mg /mL) in isolated rat working heart Fig 4.4.2.1.9 Effect of variable preloads on coronary vascular resistance with 158 or without pretreatment of aqueous fraction of A.marmelos (1 mg /mL) in isolated rat working heart Fig 4.4.2.1.10 Effect of variable preloads on myocardial work with or without 158 pretreatment of aqueous fraction of A.marmelos (1 mg/mL) in isolated rat working heart Fig 4.4.2.2.1 Effect of variable preloads on coronary effluent with or without 159 pretreatment of aqueous fraction of A.marmelos (10 mg/mL) in isolated rat working heart Fig 4.4.2.2.2 Effect of variable preloads on aortic out flow with or without 159 pretreatment of aqueous fraction of A.marmelos (10 mg/mL) in isolated rat working heart Fig 4.4.2.2.3 Effect of variable preloads on cardiac output with or without 160 pretreatment of aqueous fraction of A.marmelos (10 mg/mL) in isolated rat working heart Fig 4.4.2.2.4 Effect of variable preloads on dP/dt (max) with or without 160 pretreatment of aqueous fraction of A.marmelos (10 mg/mL) in isolated rat working heart Fig 4.4.2.2.5 Effect of variable preloads on dP/dt (min) with or without 161 pretreatment of aqueous fraction of A.marmelos (10 mg/mL) in isolated rat working heart Fig 4.4.2.2.6 Effect of variable preloads on heart rate with or without 161 pretreatment of the aqueous fraction of A.marmelos (10 mg/mL) in isolated rat working heart Fig 4.4.2.2.7 Effect of variable preloads on left ventricular pressure with or 162 without pretreatment of aqueous fraction of A.marmelos (10 mg/mL) in isolated rat working heart Fig 4.4.2.2.8 Effect of variable preloads on peak aortic systolic pressure with 162 or without pretreatment of aqueous fraction of A.marmelos (10 mg/mL) in isolated rat working heart Fig 4.4.2.2.9 Effect of variable preloads on coronary vascular resistance with 163 or without pretreatment of aqueous fraction of A.marmelos (10 mg/mL) in isolated rat working heart Fig 4.4.2.2.10 Effect of variable preloads on myocardial work with or without 163 pretreatment of aqueous fraction of A.marmelos (10 mg/mL) in isolated rat working heart

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Fig 4.4.2.3.1 Effect of variable preloads on coronary effluent with or without 164 pretreatment of aqueous fraction of A.marmelos (30 mg/mL) in isolated rat working heart Fig 4.4.2.3.2 Effect of variable preloads on aortic outflow with or without 164 pretreatment of aqueous fraction of A.marmelos (30 mg/mL) in isolated rat working heart Fig 4.4.2.3.3 Effect of variable preloads on cardiac output with or without 165 pretreatment of aqueous fraction of A.marmelos (30 mg/mL) in isolated rat working heart Fig 4.4.2.3.4 Effect of variable preloads on dP/dt(max) with or without 165 pretreatment of aqueous fraction of A.marmelos (30 mg/mL) in isolated rat working heart Fig 4.4.2.3.5 Effect of variable preloads on dP/dt(min) with or without 166 pretreatment of aqueous fraction of A.marmelos (30 mg/mL) in isolated rat working heart Fig 4.4.2.3.6 Effect of variable preloads on heart rate with or without 166 pretreatment of aqueous fraction of A.marmelos (30 mg/mL) in isolated rat working heart Fig 4.4.2.3.7 Effect of variable preloads on left ventricular pressure with or 167 without pretreatment of aqueous fraction of A.marmelos (30 mg/mL) in isolated rat working heart Fig 4.4.2.3.8 Effect of variable preloads on peak aortic systolic pressure with 167 or without pretreatment of aqueous fraction of A.marmelos (30 mg/mL) in isolated rat working heart Fig 4.4.2.3.9 Effect of variable preloads on coronary vascular resistance with 168 or without pretreatment of aqueous fraction of A.marmelos (30 mg/mL) in isolated rat working heart Fig 4.4.2.3.10 Effect of variable preloads on myocardial work with or without 168 pretreatment of aqueous fraction of A.marmelos (30 mg/mL) in isolated rat working heart Fig 4.4.3.1 Effect of aqueous fraction of A.marmelos on baseline and PE pre- 169 contracted endothelium intact and denuded rat aorta Fig 4.4.3.2 Effect of aqueous fraction of A.marmelos on baseline and K+80 169 mM pre-contracted endothelium intact and denuded rat aorta Fig 4.4.3.3 Effect of aqueous fraction of A.marmelos on baseline and L- 170 NAME incubated and PE pre-contracted endothelium intact rat aorta Fig 4.5.1.1 Effect of butanolic fraction of A.marmelos on coronary effluent, 198 aortic outflow and cardiac output in isolated rat working heart Fig 4.5.1.2 Effect of butanolic fraction of A.marmelos on dP/dt(max) and 198 dP/dt(min) in isolated rat working heart Fig 4.5.1.3 Effect of butanolic fraction of A.marmelos on systolic, diastolic 199 pressure and heart rate in isolated rat working heart

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Fig 4.5.1.4 Effect of butanolic fraction of A.marmelos on peak aortic systolic 199 pressure and end diastolic pressure in isolated rat working heart Fig 4.5.1.5 Effect of butanolic fraction of A.marmelos on ejection fraction 200 and stroke volume in isolated rat working heart Fig 4.5.1.6 Effect of butanolic fraction of A.marmelos on rate pressure 200 product and cardiac power in isolated rat working heart Fig 4.5.2.1.1 Effect of variable preloads on coronary effluent with or without 201 pretreatment of butanolic fraction of A.marmelos (0.01 mg/mL) in isolated rat working heart Fig 4.5.2.1.2 Effect of variable preloads on aortic out flow with or without 201 pretreatment of butanolic fraction of A.marmelos (0.01 mg/mL) in isolated rat working heart Fig 4.5.2.1.3 Effect of variable preloads on cardiac output with or without 202 pretreatment of butanolic fraction of A.marmelos (0.01 mg/mL) in isolated rat working heart Fig 4.5.2.1.4 Effect of variable preloads on dP/dt(max) with or without 202 pretreatment of butanolic fraction of A.marmelos (0.01 mg/mL) in isolated rat working heart Fig 4.5.2.1.5 Effect of variable preloads on dP/dt(min) with or without 203 pretreatment of butanolic fraction of A.marmelos (0.01 mg/mL) in isolated rat working heart Fig 4.5.2.1.6 Effect of variable preloads on heart rate with or without 203 pretreatment of butanolic fraction of A.marmelos (0.01 mg/mL) in isolated rat working heart Fig 4.5.2.1.7 Effect of variable preloads on left ventricular pressure with or 204 without pretreatment of butanolic fraction of A.marmelos (0.01 mg/mL) in isolated rat working heart Fig 4.5.2.1.8 Effect of variable preloads on peak aortic systolic pressure with 204 or without pretreatment of butanolic fraction of A.marmelos (0.01 mg/mL) in isolated rat working heart Fig 4.5.2.1.9 Effect of variable preloads on coronary vascular resistance with 205 or without pretreatment of butanolic fraction of A.marmelos (0.01 mg/mL) in isolated rat working heart Fig 4.5.2.1.10 Effect of variable preloads on myocardial work with or without 205 pretreatment of butanolic fraction of A.marmelos (0.01 mg/mL) in isolated rat working heart Fig 4.5.2.2.1 Effect of variable preloads on coronary effluent with or without 206 pretreatment of butanolic fraction of A.marmelos (0.1 mg/mL) in isolated rat working heart Fig 4.5.2.2.2 Effect of variable preloads on aortic out flow with or without 206 pretreatment of butanolic fraction of A.marmelos (0.1 mg/mL) in isolated rat working heart

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Fig 4.5.2.2.3 Effect of variable preloads on cardiac output with or without 207 pretreatment of butanolic fraction of A.marmelos (0.1 mg/mL) in isolated rat working heart Fig 4.5.2.2.4 Effect of variable preloads on dP/dt(max) with or without 207 pretreatment of butanolic fraction of A.marmelos (0.1 mg/mL) in isolated rat working heart Fig 4.5.2.2.5 Effect of variable preloads on dP/dt(min) with or without 208 pretreatment of butanolic fraction of A.marmelos (0.1 mg/mL) in isolated rat working heart Fig 4.5.2.2.6 Effect of variable preloads on heart rate with or without 208 pretreatment of butanolic fraction of A.marmelos (0.1 mg/mL) in isolated rat working heart Fig 4.5.2.2.7 Effect of variable preloads on left ventricular pressure with or 209 without pretreatment of butanolic fraction of A.marmelos (0.1 mg/mL) in isolated rat working heart Fig 4.5.2.2.8 Effect of variable preloads on peak aortic systolic pressure with 209 or without pretreatment of butanolic fraction of A.marmelos (0.1 mg/mL) in isolated rat working heart Fig 4.5.2.2.9 Effect of variable preloads on coronary vascular resistance with 210 or without pretreatment of butanolic fraction of A.marmelos (0.1 mg/mL) in isolated rat working heart Fig 4.5.2.2.10 Effect of variable preloads on myocardial work with or without 210 pretreatment of butanolic fraction of A.marmelos (0.1 mg/mL) in isolated rat working heart Fig 4.5.2.3.1 Effect of variable preloads on coronary effluent with or without 211 pretreatment of butanolic fraction of A.marmelos (1 mg/mL) in isolated rat working heart Fig 4.5.2.3.2 Effect of variable preloads on aortic out flow with or without 211 pretreatment of butanolic fraction of A.marmelos (1 mg/mL) in isolated rat working heart Fig 4.5.2.3.3 Effect of variable preloads on cardiac output with or without 212 pretreatment of butanolic fraction of A.marmelos (1 mg/mL) in isolated rat working heart Fig 4.5.2.3.4 Effect of variable preloads on dP/dt(max) with or without 212 pretreatment of butanolic fraction of A.marmelos (1 mg/mL) in isolated rat working heart Fig 4.5.2.3.5 Effect of variable preloads on dP/dt(min) with or without 213 pretreatment of butanolic fraction of A.marmelos (1 mg/mL) in isolated rat working heart Fig 4.5.2.3.6 Effect of variable preloads on heart rate with or without 213 pretreatment of butanolic fraction of A.marmelos (1 mg/mL) in isolated rat working heart

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Fig 4.5.2.3.7 Effect of variable preloads on left ventricular pressure with or 214 without pretreatment of butanolic fraction of A.marmelos (1 mg/mL) in isolated rat working heart Fig 4.5.2.3.8 Effect of variable preloads on peak aortic systolic pressure with 214 or without pretreatment of butanolic fraction of A.marmelos (1 mg/mL) in isolated rat working heart Fig 4.5.2.3.9 Effect of variable preloads on coronary vascular resistance with 215 or without pretreatment of butanolic fraction of A.marmelos (1 mg/mL) in isolated rat working heart Fig 4.5.2.3.10 Effect of variable preloads on myocardial work with or without 215 pretreatment of butanolic fraction of A.marmelos (1 mg/mL) in isolated rat working heart Fig 4.5.3.1 Effect of butanolic fraction of A.marmelos on baseline and PE 216 pre-contracted endothelium intact and denuded rat aorta Fig 4.5.3.2 Effect of butanolic fraction of A.marmelos on baseline and K+80 216 mM pre-contracted endothelium intact and denuded rat aorta Fig 4.5.3.3.1 Construction of Ca++ curves in the presence and absence of 217 various concentrations of butanolic fraction of A.marmelos in endothelium denuded rat aorta showing Ca++ agonistic activity Fig 4.5.3.3.2 Construction of Ca++ curves in the presence and absence of 217 various concentrations of butanolic fraction of A.marmelos in endothelium denuded rat aorta showing Ca++ antagonistic activity Fig 4.6.1.1.1 Effects of isolated and standard marmelosin on coronary effluent 251 in isolated rat working heart Fig 4.6.1.1.2 Effect of isolated and standard marmelosin on aortic outflow in 251 isolated rat working heart Fig 4.6.1.1.3 Effects of isolated and standard marmelosin on cardiac output in 252 isolated rat working heart Fig 4.6.1.1.4 Effects of isolated and standard marmelosin on dP/dt(max) in 252 isolated rat working heart Fig 4.6.1.1.5 Effects of isolated and standard marmelosin on dP/dt(min) in 253 isolated rat working heart Fig 4.6.1.1.6 Effects of isolated and standard marmelosin on systolic pressure 253 in isolated rat working heart Fig 4.6.1.1.7 Effects of isolated and standard marmelosin on diastolic pressure 254 in isolated rat working heart Fig 4.6.1.1.8 Effects of isolated and standard marmelosin on heart rate in 254 isolated rat working heart Fig 4.6.1.1.9 Effects of isolated and standard marmelosin on peak aortic 255 systolic pressure in isolated rat working heart Fig 4.6.1.1.10 Effects of isolated and standard marmelosin on end diastolic 255 pressure in isolated rat working heart

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Fig 4.6.1.1.11 Effects of isolated and standard marmelosin on ejection fraction 256 in isolated rat working heart Fig 4.6.1.1.12 Effects of isolated and standard marmelosin on stroke volume in 256 isolated rat working heart Fig 4.6.1.1.13 Effects of isolated and standard marmelosin on rate pressure 257 product in isolated rat working heart Fig 4.6.1.1.14 Effects of isolated and standard marmelosin on cardiac power in 257 isolated rat working heart Fig 4.6.1.2.1 Effect of variable preloads on coronary effluent with or without 258 pretreatment of isolated marmelosin (1 µM) in isolated rat working heart Fig 4.6.1.2.2 Effect of variable preloads on aortic out flow with or without 258 pretreatment of isolated marmelosin (1 µM) in isolated rat working heart Fig 4.6.1.2.3 Effect of variable preloads on cardiac output with or without 259 pretreatment of isolated marmelosin (1 µM) in isolated rat working heart Fig 4.6.1.2.4 Effect of variable preloads on dP/dt(max) with or without 259 pretreatment of isolated marmelosin (1 µM) in isolated rat working heart Fig 4.6.1.2.5 Effect of variable preloads on dP/dt(min) with or without 260 pretreatment of isolated marmelosin (1 µM) in isolated rat working heart Fig 4.6.1.2.6 Effect of variable preloads on heart rate with or without 260 pretreatment of isolated marmelosin (1 µM) in isolated rat working heart Fig 4.6.1.2.7 Effect of variable preloads on left ventricular pressure with or 261 without pretreatment of isolated marmelosin (1 µM) in isolated rat working heart Fig 4.6.1.2.8 Effect of variable preloads on peak aortic systolic pressure with 261 or without pretreatment of isolated marmelosin (1 µM) in isolated rat working heart Fig 4.6.1.2.9 Effect of variable preloads on coronary vascular resistance with 262 or without pretreatment of isolated marmelosin (1 µM) in isolated rat working heart Fig 4.6.1.2.10 Effect of variable preloads on myocardial work with or without 262 pretreatment of isolated marmelosin (1 µM) in isolated rat working heart Fig 4.6.2.2.1 Effect of variable preloads on coronary effluent with or without 263 pretreatment of isolated marmelosin (10 µM) in isolated rat working heart Fig 4.6.2.2.2 Effect of variable preloads on aortic outflow with or without 263 pretreatment of isolated marmelosin (10 µM) in isolated rat

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working heart Fig 4.6.2.2.3 Effect of variable preloads on cardiac output with or without 264 pretreatment of isolated marmelosin (10 µM) in isolated rat working heart Fig 4.6.2.2.4 Effect of variable preloads on dP/dt(max) with or without 264 pretreatment of isolated marmelosin (10 µM) in isolated rat working heart Fig 4.6.2.2.5 Effect of variable preloads on dP/dt(min) with or without 265 pretreatment of isolated marmelosin (10 µM) in isolated rat working heart Fig 4.6.2.2.6 Effect of variable preloads on heart rate with or without 265 pretreatment of isolated marmelosin (10 µM) in isolated rat working heart Fig 4.6.2.2.7 Effect of variable preloads on left ventricular pressure with or 266 without pretreatment of isolated marmelosin (10 µM) in isolated rat working heart Fig 4.6.2.2.8 Effect of variable preloads on peak aortic systolic pressure with 266 or without pretreatment of isolated marmelosin (10 µM) in isolated rat working heart Fig 4.6.2.2.9 Effect of variable preloads on coronary vascular resistance with 267 or without pretreatment of isolated marmelosin (10 µM) in isolated rat working heart Fig 4.6.2.2.10 Effect of variable preloads on myocardial work with or without 267 pretreatment of isolated marmelosin (10 µM) in isolated rat working heart Fig 4.6.2.3.1 Effect of variable preloads on coronary effluent with or without 268 pretreatment of isolated marmelosin (100 µM) in isolated rat working heart Fig 4.6.2.3.2 Effect of variable preloads on aortic out flow with or without 268 pretreatment of isolated marmelosin (100 µM) in isolated rat working heart Fig 4.6.2.3.3 Effect of variable preloads on cardiac output with or without 269 pretreatment of isolated marmelosin (100 µM) in isolated rat working heart Fig 4.6.2.3.4 Effect of variable preloads on dP/dt(max) with or without 269 pretreatment of isolated marmelosin (100 µM) in isolated rat working heart Fig 4.6.2.3.5 Effect of variable preloads on dP/dt(min) with or without 270 pretreatment of isolated marmelosin (100 µM) in isolated rat working heart Fig 4.6.2.3.6 Effect of variable preloads on heart rate with or without 270 pretreatment of isolated marmelosin (100 µM) in isolated rat working heart

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Fig 4.6.2.3.7 Effect of variable preloads on left ventricular pressure with or 271 without pretreatment of isolated marmelosin (100 µM) in isolated rat working heart Fig 4.6.2.3.8 Effect of variable preloads on peak aortic systolic pressure with 271 or without pretreatment of isolated marmelosin (100 µM) in isolated rat working heart Fig 4.6.2.3.9 Effect of variable preloads on coronary vascular resistance with 272 or without pretreatment of isolated marmelosin (100 µM) in isolated rat working heart Fig 4.6.2.3.10 Effect of variable preloads on myocardial work with or without 272 pretreatment of isolated marmelosin (100 µM) in isolated rat working heart Fig 4.6.3.1 Effect of standard marmelosin on baseline and PE pre-contracted 273 endothelium intact and denuded rat aorta Fig 4.6.3.2 Effect of standard marmelosin on baseline and K+80 mM pre- 273 contracted endothelium intact and denuded rat aorta Fig 4.6.3.3 Effect of standard marmelosin on baseline and L-NAME 274 incubated and PE pre-contracted endothelium intact rat aorta

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LIST OF ABBREVIATIONS

Ach Acetylcholine α Alpha Am.Cr Crude extract of Aegle marmelos AoF Aortic out flow Aq Aqueous Aq.Fr.Cr Aqueous fraction of A.marmelos BP pressure But.Fr.Cr Butanolic fraction of A.marmelos οC Degree centigrade Ca++ Calcium ion [Ca2+]i Intracellular calcium or cytosolic calcium

CaCl2 Calcium chloride Ca++CRCs Calcium concentration response curve Ca++ Paradox Calcium paradox CCBs Calcium channel blockers CE Coronary effluent CHF Congestive heart failure CO Cardiac output

CO2 Carbon dioxide CP Cardiac power CPP Coronary perfusion pressure CVR Coronary vascular resistance

±dP/dt(max) First derivative of left ventricular developed pressure dP/dt(max) Rate of contraction dP/dt (min) Rate of relaxation DP Diastolic pressure E-C coupling Excitation-contraction coupling

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EDP End diastolic pressure EDHF Endothelium-derived hyperpolarizing factor EDRF Endothelium-derived relaxing factor EE Endocardial endothelium EF Ejection fraction EGTA Ethylene Glycol-bis (β-Aminoethylether)-N-N,N’N’- Tetracetic acid -veEnd Endothelium-denuded +veEnd Endothelium-intact F-S Curve Frank-starling curve g Gram hr Hour HR Heart rate i.p Intraperitoneal i.u International unit i.v Intravenous (rout of administration) IVC Inferior vena cava ISD Marm Isolated marmelosin K+ Potassium ion KCl Potassium chloride K+80mM Potassium chloride 80 milimole Kg Kilogram KH Krebs - Henseleit

KH2PO4 Potassium dihydrogen phosphate LA Left atrium L-NAME N-nitro-L-arginine methyl ester hydrochloride LV Left ventricle LVDP Left ventricular developed pressure LVEDP Left ventricular end-diastolic pressure

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LVP Left ventricular pressure LVSP Left ventricular systolic pressure mg Miligram

MgSO4.7H2O Magnesium sulphate heptahydrate min Minute mL Mililitre mm Milimeter mmHg Milimeter of mercury mM Milimolar MV Mitral valve MW Myocardial work n Number of observations Na+ Sodium ion NaCl Sodium chloride

NaHCO3 Sodium bicarbonate NCX Na+/Ca++ exchanger NE Nor epinephrine bitartarate NO Nitric oxide

O2 Oxygen PA Pulmonary artery PASP Peak aortic systolic pressure Pc.Cr Crude extract of Pyrus cydonia PE Phenylephrine p.o Per oral RA Right atrium ROCCs Receptor-operated calcium channels RPP Rate pressure product RV Right ventricle s.c Subcutaneous

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SEM Standard error of means SP Systolic pressure SR Sarcoplasmic reticulum STD Marm Standard marmelosin SV Stroke volume µ Micron µg Microgram µM Micromolar VDCCs Voltage dependent calcium channels VSMs Vascular smooth muscles VSMCs Vascular smooth muscle cells

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ACKNOWLEDGEMENT

I put down my head and heart before the Almighty, Omnipotent, Omniscient and Omnipresent ALLAH. I am very grateful to almighty ALLAH who makes me enable to complete this research work. Special praise and all the respects are due to His last messenger, the prophet (PBUH), who is the treasure of knowledge and wisdom.

I do not find enough words to express my thanks and gratitude to my great and inspiring supervisor Dr. S. Intasar H Taqvi, T.T.S Assistant Professor, Department of Pharmacology, Faculty of Pharmacy, Federal Urdu University of Science, Arts and Technology, Gulshan-e- Iqbal, Karachi, Pakistan for his valuable guidance, all-time supervision, encouraging attitude, keen interest and untiring support throughout the experiments and write-up. My supervisor, developed the idea for the research thesis, provided the financial help and instruments in his lab entitled “Dr. Intasar Cardiovascular Pharmacology and Toxicology Lab”. The lab was developed by the financial grant won by Dr. Intasar under National Research Program for Universities No: 20-1724/R&D/10, dated 31-5-11.

Special thanks and gratitude to Dr. M.A Versiani and his students Miss. Amna Khatoon and Miss. Ambreen Akram, who provided the extract, fraction and isolated compound. Not only this, the team of Dr. Versiani handed over the flow charts, characterization data of marmelosin, references and literature at the time of write-up. I remember the smiling face of Dr. Versiani whenever I approached him with a problem and he came up with the solution. His problem-solving attitude is an asset for me throughout the period of experimentation and write-up. I thank specially to Dr. Zameer Ahmad the veterinary expert and incharge of Animal House facility at Dow University of Health Sciences, Karachi for the support to provide the animals as and when required. The scientific knowledge of Dr. Zameer was found to be helpful regarding the selection of species and constant supply of the required species, feed and gender. This helped us to avoid the gender and species differences in the result. The staff of the animal house namely, Dr.Furrukh Bilal, Mr. Rehan Ahmed, Mr. Maqsood Ur Rahman also deserve our thanks.

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I owe a special debt of thanks to Dr. Mahjabeen, Chairperson, Department of Pharmacology, Faculty of Pharmacy, Federal Urdu University of Science, Arts and Technology, Gulshan-e- Iqbal, Karachi, Pakistan, who came forward to help me out whenever I approached her. Her co-operation can never be neglected. I am thankful to Dr. Arfa, Dean, Faculty of Pharmacy and all other respected teachers. I am also thankful to all office staff of the Department of Pharmacology for their help during official work.

I am very deeply thankful to my all collegesof PCSIR Mrs. Zahra Yaqeen, Dr. Zakir-ur-Rehman, Dr. Nudrat Fatima, Dr. Tehmina Sohail, Dr. Hina Imran, who co-operated me to continue my studies during my job, provide immense help and moral support for completing this assignment.

I am also thankful to Mr. Irshadullah, computer operator who helped and guided me during the writing and lab staff of Department of Pharmacology, Mr. Fraz Ahmed, Mr. Ashfaq Choudhri and Mr. Sohail Sattar for their prayer and moral support during research work.

Finally, I wish to express my greatest thanks and gratitude’s to my family for their continuous encouragement, support and prayers that enable me to work with new zeal and confidence.

ATIQ UR RAHMAN

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ABSTRACT

The crude extracts, fractions and isolated marmelosin were investigated to rationalize the therapeutic potentials in cardiovascular disorders. Heart and aorta were isolated from Wistar rat for working and Langendorff’s heart and aortic ring preparations. Preliminarily, Langendorff’s heart determined the following studies. Firstly, working heart was performed at fixed preload of 15 cmH2O and afterload of 80 cmH2O. Secondly, variable preload 5, 10, 15,

20 and 25 cmH2O was employed, whereas, afterload was fixed at 80 cmH2O. In Ca++ paradox; Ca++ free KH perfused the heart, afterwards, firstly normal KH, secondly normal KH plus Am.Cr and thirdly Ca++ free KH plus Am.Cr and normal KH plus Am.Cr.

In Langendorff’s heart Am.Cr and Pc.Cr increased the left ventricle (LVP) and systolic pressures (SP). This determined the path to study in more detail.

Am.Cr; exhibited biphasic effect on dP/dt(max) dose-dependently in working heart. Am.Cr increased the AoF and EF dose-dependently. Am.Cr increased the dP/dt(min), DP and EDP. The SV and CP were increased at higher doses. CE was decreased while SP, PASP and RPP were unaffected. In aorta Am.Cr inhibited PE and high K+-induced contractions in both +veEnd and -veEnd, showing VDCCs and ROCCs blocking effect and release of Ca++ from sarcoplasmic reticulum.Am.Cr shifted the Ca++CRCs to the left at lower and to the right at higher doses showing agonists and antagonistic effect on Ca++ channels. At variable preload the heart was pretreated by 3.0, 30.0 and 100 mg/mL of Am.Cr. The AoF, LVP and CVR were increased by acute preload reduction that followed by load-dependent increase. The CE was decreased dose- and load-dependently. The dP/dt(max) and HR were decreased, whereas dP/dt (min) showed dose- and load-dependent increase. The CO increased variably while PASP and MW were unaffected. In calcium paradox experiments; Ca++ free perfusion decreased the contractility whereas, Am.Cr increased the contractility whether it was added in Ca++ free or normal KH solution at various series of experiments.

Aq.Fr.Cr decreased the dP/dt(max) and RPP significantly in working heart. It increased AoF, DP,

CO and CP. The dP/dt(min) was reduced.Aq.Fr.Cr caused dose-dependent minimal reduction in

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CE. The EDP, SP, PASP, EF and SV were unaffected. Aq.Fr.Cr induced dose-dependent relaxation of PE- and high K+-induced contractions in +veEnd aortic rings. In -veEnd rings; it inhibits PE-induced contraction only, whereas in high K+-induced contraction Aq.Fr.Cr failed to relax. The cumulative addition to the L-NAME pre-incubated and PE pre-contracted +veEnd rings did not show relaxation. This explains endothelium-dependent vasorelaxation through NO/cGMP pathway. At variable preloads; pretreatment ofthe heart with 1.0, 10.0 and 30.0 mg/ml of Aq.Fr.Cr; increased the AoF, LVP and CVR by acute preload reduction while preload increment caused load-dependent increase. The dP/dt(max) and HR were decreased by acute preload reduction and was not affected by preload increase. The dP/dt(min) was decreased by acute preload reduction while increasing preload caused load-dependent increase. CE was decreased at all changes in preload. The PASP, MW and CO were minimally affected during preload changes.

But.Fr.Cr increased dP/dt(max), dP/dt(min), AoF, SP, CP and CO in working heart. The PASP, EF, SV and RPP were decreased. CE was decreased significantly and dose-dependently. In aorta; But.Fr.Cr induced inhibitory effect in both +veEnd and -veEnd ring pre-contracted by PE and high K+. This shows blockade of ROCCs, VDCCs and release of Ca++ from sarcoplasmic reticulum. But.Fr.Cr at lower concentrations caused leftward and at higher concentrations rightward shift of Ca++ CRCs showing calcium channel agonists and antagonistic activities. At variable preload; the heart was pretreated by 0.01, 0.1 and 1.0 mg/mL of But.Fr.Cr. The AoF, LVP and CVR were increased by acute preload reduction and continued by increasing preload.

The dP/dt(max) and dP/dt(min) were decreased by preload reduction and not affected by increasing preload. HR was decreased by acute preload reduction and continued during increase in preload. The PASP, MW and CE were decreased at all preloads. The CO was less affected.

ISD Marm increased the AoF, CO and CP in working heart. The dP/dt(max) was decreased minimally while dP/dt(min) and HR were decreased significantly at higher doses. CE decreased dose-dependently. The SP, DP, PASP, EDP, EF and RPP were not affected. STD Marm reduced the CE and increased the AoF, CO, CP, SV and EF comparatively more than ISD Marm. The

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dP/dt(max), dP/dt(min), HR, SP, DP, EDP and RPP were reduced whereas SV was not affected. In aorta; STD Marm induced dose-dependent relaxation in both +veEnd and -veEnd PE pre- contracted rings. This was significant at higher doses in +veEnd rings. Whereas in high K+ pre- contracted +veEnd and -veEnd rings it showed insignificant effect. The cumulative addition to L-NAME pre-incubated and PE pre-contracted +veEnd rings it showed partial but significant dose-dependent relaxation. At variable preloads; pretreatment of the heart by 1µM, 10 µM and 100 µM of ISD Marm; increased the AoF, LVP and CVR by acute preload reduction while preload escalation showed load-dependent increase. The CO and MW were increased by acute preload reduction, whereas preload increment led to attenuation of CO and MW. The dP/dt(max), dP/dt(min), HR, CE and PASP were decreased by acute preload reduction and continued during preload increment.

Thus, this study may rationalize the therapeutic potential of A.marmelos and P.cydonia possesses cardiotonic effect. The crude extract of A.marmelos, its fractions and isolated marmelosin exhibited numerous effects on the heart and aortic muscle mediating through multiple pathways that pointed out that though crude extract showed most of the activities, but it appears as no single compound can be a true representative of the plant. Because, it constitutes multiple compounds with different properties which can be exploited for various therapeutic purposes. Further studies would establish the clinical significance of A.marmelos in the management of cardiovascular disorders and promising chemical agent may be identified.

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1

CHAPTER 1 INTRODUCTION

Chapter 1: Introduction 2

1.1 Brief historical background of herbal plants The term "herbs" or “medicinal plants” refers to whole plant or their parts including grasses, flowers, berries, seeds, leaves, nuts, stems, stalks and roots, which are being used for their therapeutic and health enhancing properties. Medicinal plants have been exploited as a source of bioactive metabolite for human benefit and prior to the development of synthetic drugs plant extracts were the only source of organo-chemical and medicinal compounds (Lahlou., 2016). This led to the development of Greco-Arab-Arab system of herbal medicine and individualized treatment which grew up the concept of pharmacogenetics (Gilani and Atta-ur-Rahman., 2005). It has been estimated that 30%–50% of all drugs on the market have their origin from medicinal plants (Aslam and Sial., 2014).

Evidence of extensive use of plants as drugs can be found in traditional Chinese, Indian ‘ayurveddic’, and Pakistani ‘unani’ literature (Mahady., 2002; Pemberton., 1999), functioning with the combination of “Hakeems” and herbal dealers (Usmanghani et al., 1986). Some factors that facilitate the abundant usage of medicinal plants and plant derived drugs include their ease in availability, cultural significance, history of known efficacy and most importantly, inexpensive procurement compared to synthetic drugs (Aslam and Sial., 2014). Herbal medicinal dosage forms (medicines / drugs) contain synergistic compounds and /or substances that are associated with attenuation of side-effects which is of clinical interest (Thomford et al., 2015).

According to WHO estimates, 60 % of the world’s population depend on traditional medicine, and 80 % of the population in developing countries depend almost completely on traditional medical practices for primary health care needs. Whereas, 70–80 % population in the developed countries use some form of complementary and alternative medicine (Zhang., 2000; Duke., 2002; Calvo and Cavero., 2014). Traditional healers treat 65 % patients in Srilanka, 85 % in Burma, 80 % in India, 60 % in Indonesia, 75 % in Nepal, 90 % in Bangladesh and more than 60% in Pakistan respectively (Inamul-Haq., 1983). Ethnopharmacology has played important role in the development of modern conventional medicine and thus expected to play more significant role in the years to come (Gilani and Atta-ur-Rahman.,

Chapter 1: Introduction 3

2005). Presently, most of the drugs manufactured are of synthetic origin. Approximately 25 % of drugs which have been used worldwide are / or were derived from plants and used either in their pure form, (e.g. morphine, codeine, atropine, ephedrine, digoxin, quinine, reserpine, tubocurarine, vincristine and vinblastine etc) (Gilani and Atta-ur-Rahman., 2005) or as they appear in nature (Kutchan., 1995). Some of these could not be substituted despite the enormous advancement in synthetic chemistry.

Plants have also served as models from which wholly synthetic drugs are designed. Examples include; tropicamide derived from atropine an alkaloid obtained from Atropa belladonna, chloroquine derived from quinine, and procaine and tetracaine derived from . Revival of interest in herbal products and conventional medicine at the global level resulted in the dramatic increase in the sales of herbal products in the world worth surprisingly over 100 billion dollars a year (Gilani and Atta-ur-Rahman., 2005). The benefits of treatment may be achieved by using botanicals, for examples, use of Silybum marianum to prevent liver damage from poisoning by death cap mushroom and infectious hepatitis, use of immune from coneflower for viral , and the controversial use of Cannabis sativa to treat pain and nausea in cancer and cancer (Kutchan., 1995; Zollman and Vickers., 1999). There is an increasing trend in North America and Europe to incorporate the complementary and alternative medicine in the medical curriculum (Wetzel et al., 2003; Harrison., 1998).

1.2 Importance of research and scientific validation of herbal medicine The natural products have provided the basis for the discovery of many clinically successful drugs by trial and error over many years in different cultures and system of medicines but still providing useful therapeutic agents like antihypertensive, cardiac glycosides, , antibacterial and anticancer drug therapies (Gilman et al., 1985; Gordon et al., 1997). The major advantage of natural products for random screening is the structural diversity, whereas the clinical, pharmacological and chemical studies provided the basis of development of medicines such as aspirin, digitoxin, quinine and pilocarpine (Grabley et al., 2000). Plants also have been employed as markers in the explanation of some physiological processes like

Chapter 1: Introduction 4 determination of muscarinic and nicotinic receptors in the tissues (Williamson et al., 1996). Now-a-days the medicinal plants are being used as an imperative tool for research and development of new drugs and pharmacological and biochemical investigation (Gilani et al., 1998).

According to World Health Organization, the use of incorrect species is a threat to consumer safety (WHO., 2011; Palhares et al., 2015). Furthermore, there is little or no information on animal experiments or clinical trials for most herbal drugs presently in circulation (Tomassoni and Simone., 2001). Moreover, there is no comprehensive data on pharmacokinetics, drug interactions, and toxicities of traditional herbal medicines. Hence, the quantity and quality of the safety and efficacy data on traditional medicine so far are not enough to support its use worldwide. Research is being carried out on traditional medicines in various countries because the present shift of WHO towards the incorporation of traditional medicines in the essential drug list demands an extensive chemical and pharmacological study to determine the safety, efficacy and quality of these drugs (Zhang., 2000; Gilani and Atta-ur-Rahman., 2005; WHO., 2011). Need of research is also encouraged because it is estimated that of the 300,000 plant species that exist in the world, only 15% have been evaluated to determine their pharmacological potential (Luca et al., 2012; Ahmad et al., 2015). Therefore, more attention should be paid to determine their possible side effects, toxicities and even interactions. There is no regulatory body to oversee the standards of traditional medicines and to evaluate the rational use of traditional medicines. A lot of life-threatening and even lethal interactions and adverse reactions by these drugs go unnoticed.

1.3 Importance of research in herbal medicine in Pakistan Pakistan has a rich heritage of the medicinal plants and herbal medicines based on folklore are still being widely used. It has been reported that about 6000 species of plants with potential medicinal activities are widely distributed in the country. In most of the regions of Pakistan, people have old knowledge about the medicinal uses of indigenous plants which is based on hit and trial method (Ikram et al., 2015). Medicinal plants are living resources, exhaustible if overused, but sustainable if used with care and wisdom because 60 %

Chapter 1: Introduction 5 population of Pakistan from villages rely on medicinal plants for their drug related needs (Qureshi et al., 2010). Therefore, we thought to contribute from our end by selecting Aegle marmelos and Pyrus cydonia for the present research work to validate the cardiovascular therapeutic use.

1.4 Plants use in cardiovascular disorders Herbal treatments have been found since long for treatment of hypertension, congestive heart failure, atherosclerosis, cerebral insufficiency, venous insufficiency, arrhythmia and ischemic heart diseases (Mashour et al., 1998). One of the important areas in which natural compounds have contributed successfully is the research for the development of safer and more effective and curative cardiovascular agents (Gilani., 1998). The scientist discovered the efficacy of herbal plants and their compounds in different cardiovascular disorder to aim better control and management.

1.4.1 Hypertension Discovery of therapeutic effects of reserpine in the management of hypertension brought to the attention of the modern world (Gilani and Atta-ur-Rahman., 2005d). Stephania tetrandra and root of Lingusticum wallichii used for hypertension, their active constituents, tetrandrine and tetramethyl pyrazine cause through inhibition of calcium channel (Sutter and Wang., 1993; Ody., 1993). Lingusticum wallichii also used as circulatory and aggregation inhibitor. Uncaria rhynchophylla, its indole alkaloids, rhynchophylline, hirsutine, and rutaecarpine from Evodia rutaecarpa are vasodilatory, act endothelium- dependently via nitric oxide (NO) release and inhibit platelet aggregation (Kuramochi et al., 1994; Sutter and Wang, 1993; Mashour et al., 1998; Mahady., 2002; Chiou et al., 1997). Syzygium guineense, Pennisetum glaucum and Sonchus asper reported to produce decreases in blood pressure in hypertensive rats (Yohannes et al., 2010; Mushtaq et al., 2015).

1.4.2 Heart failure Plant derived cardioactive glycosides, which have positive inotropic actions like digitoxin, derived from either Digitalis purpurea (foxglove) or Digitalis lanata, and digoxin, from Digitalis lanata alone, has been used in the treatment of congestive heart failure for many

Chapter 1: Introduction 6 decades. Some common plant are sources of cardiac glycosides include ; Apocynum cannabinum (black Indian emp), Asclepias curassavica (red headed cotton bush), Asclepias friticosa (balloon cotton), Calotropis precera (king's crown), Carissa spectabilis (winter sweet), Cerebra manghas (sea mango) Cheiranthus cheiri (wallflower), Convallaria majalis (lily of the valley, convallaria), Cryptostegia grandiflora (rubber vine), Helleborus niger (black hellebore) and Nerium oleander (oleander) etc., (Awang., 2009; Stuhlemmer et al., 1993; Radford et al., 1986; Mashour et al., 1998).

1.4.3 Ischemic heart disease Crataegus hawthorn, and its species (such as Crataegus oxyacantha and Crataegus monogyna in the West and Crataegus pinnatifida in China) reputed as a tonic for the cardiovascular system. Extract of Crataegus leaves, flowers, and fruits containing biologically active substances oligomeric procyanins, flavonoids, and catechins having antioxidant properties, inhibit thromboxane formation and are useful in ischemic heart disease (Bahorun., 1994; Vibes., 1994). In high concentrations, showing cardioprotective effect on ischemic-reperfused hearts without causing an increase in coronary blood flow (Nasa., 1993). In essence, Crataegus increases coronary perfusion, produces mild hypotension, antagonizes atherogenesis, with positive inotropic and negative actions (Shanthi., 1994). Panaxnoto ginseng (pseudo ginseng) a selective calcium ion channel blocker in vascular tissue, used in the treatment of angina and coronary artery disease. It does not interact with the L-type calcium ion channel but rather interact with the receptor-operated calcium ion channel (Kwan., 1995; Ody., 1993). Panaxnoto ginseng and Salvia miltiorrhiza demonstrate dilatation of coronary arteries in all concentrations showing protective action on ischemic myocardium and enhancing the recovery of contractile force on reoxygenation in animal studies (Yagi., 1989). Salvia miltiorrhiza has also shown to protect myocardial mitochondrial membranes from ischemia-reperfusion injury and lipid peroxidation because of its free radical-scavenging effects (Zhao., 1996).

1.4.4 Arrhythmia

Chapter 1: Introduction 7

Leonurus cardiac (mother wort), has been used as a remedy for non-pathologic arrhythmias occur as a functional heart complaint due to autonomic imbalance (Weiss., 1988). Scutellaria lateriflora, (Skullcap) found useful for regulating the conductive activity of the heart and treating minor arrhythmias (Felter., 1922). Cytisus scoparius (Scotch broom), containing quinolizidine alkaloids, particularly sparteine acts very similarly to quinine and quinidine and recommended to regulate heart rhythm in congestive heart failure and to improve venous return (Weiss., 1988). Rauvolfia serpentine contains alkaloids; ajmaline has shown efficacy particularly in preventing arrhythmias caused by cardiac ischemia (Obayashi., 1976) and for preventing supraventricular arrhythmias (Kel’man., 1977).

1.5 Prevalence of cardiovascular diseases The major cardiovascular diseases are hypertension, heart failure, ischemia and arrhythmias, which lead to further complications if left untreated. 17.5 million deaths have occurred worldwide in 2012 due to CVDs, which represents 31 % of all global deaths and three quarters of these deaths were in developing countries (WHO., 2015). Of these deaths, an estimated 7.4 million were due to coronary heart disease and 6.7 million were due to stroke. Per survey report, deaths have been further increased due to CHD in Pakistan (Aziz et al., 2008) except the rheumatic heart disease which is still prevalent in different countries including Pakistan (Rizvi et al., 2004).

1.5.1 Hypertension A cutoff of value 140/90 mm Hg is accepted as a threshold (Williams., 2001). Hypertension has been reported to be the fourth contributor to premature deaths in the developed and seventh in the developing countries. The prevalence of hyper and pre-hypertension is higher in males as compared to females and the trend is increasing in both sexes with increasing age (Ghelani et al., 2014). It accounts for 12.8 % of all the deaths. WHO has estimated the prevalence in adults aged 25 years and above to be around 40 % and the value for uncontrolled hypertension was approximatelyfound to one billion (Dharmarajan and Dharmarajan., 2015). Approximately 970 million people all over the world have been suffering from hypertension and out of these about 640 million people are belonging to the

Chapter 1: Introduction 8 developing world while 330 million people live in the developed countries. Recently, it has been reported that about 1 billion people worldwide have hypertension and this number is likely to increase to 1.56 billion by the year 2025 (Ahmad et al., 2015). The National Health Survey of Pakistan has estimated that 33% of all adults above the age of 45 years and 18% of all adults have high blood pressure. In another report, it was shown that 18% of people of Pakistan suffer from hypertension with every third person over the age of 40 are susceptible to an extensive range of diseases (Ahmad et al., 2015).

1.5.2 Congestive heart failure Coronary artery disease and hypertension are responsible to develop congestive heart failure in most of the cases and still increasing in developing countries. Myocardial infarction is also playing an important role to develop heart failure in recent years (Teerlink et al., 1991). Hypertension increases the occurrence of myocardial infarction finally increasing the risk of heart failure five to six-fold. Heart failure related mortality remains unacceptably high (Kannel., 2000). In Europe over 10 million patients are suffering from CHF. In the United States the prevalence of clinically documented CHF was 27.1% (Goff et al., 2000), 78% of men and 85% of women hospitalized with heart failure were aged >/= 65years (Haldeman et al., 1999). Ischemic heart disease was the most common etiologic factor to develop heart failure in all regions except Africa, where hypertensive heart disease was most common (Dokainish et al., 2016).

1.5.3 Ischemic heart diseases Over the last 25 years, age-standardized ischemic heart disease (IHD) mortality has fallen by more than half in high income countries, but the trend is flat or increasing in low-and-middle income countries which now account for more than 80% of global IHD deaths (Finegold et al., 2013). IHD presents a high mortality and morbidity rate in Leon with the percentage in men 5.66% and among women, 4.63%, respectively, (Vega-Alonso et al., 2000). In Korean adult men have 75.2% coagulopathies, small artery occlusive disease 17.4%, cardio-embolism 18.1%, large artery atherosclerosis 20.8 % and undetermined etiologies 16.8 % and other determined etiologies 26.8% (Kwon et al., 2000). In New Zealand, Ischemic heart disease

Chapter 1: Introduction 9

(IHD) in admitting patients is 11 % (Stewart et al., 1999). In Pakistan, ischemic heart disease was the leading cause of death, killing 111.4 thousand people (8.4%) in 2012 (Pakistan: WHO statistical profile., 2015).

1.5.4 Arrhythmia Atrial fibrillation is the most common sustained arrhythmia. This increases the risk of stroke by 5-fold. The features that predict higher risk of stroke in atrial fibrillation are: prior stroke, hypertension, advancing age, diabetes and congestive heart failure (Ryder and Benjamin., 1999). Atrial fibrillation affects an estimated 2.2 million adults in the United States, the median age is 75 years and its effects 8.8 % population of US >80 years of age. In Europe prevalence is same but in Asia may be lower. The ventricular tachycardia in hospitalized patients was 28.4 % in US (Brady et al., 1999). The most serious of all cardiac arrhythmias is ventricular fibrillation, which, if not stopped within 1 to 3minutes, is almost invariably fatal (Guyton and Hall., 2016).

1.6 Functional properties of the heart The focus of the present studies is the heart and vessel, so it seems rational to write a few lines regarding the functional properties of the heart muscle. The heart contracts rhythmically to pump blood through the . The amount of blood pumped is determined by rate and force of contraction defining chronotropism and inotropism (Van

Putte et al., 2016; Guyton and Hall., 2016).

1.6.1 Chronotropism This refers to the rate at which the heart beats or contracts. The heart is an autorhythmic pump, whose rhythmicity can be affected by external influences. Parasympathetic stimulation leads to slowing of the heart rate, a negative chronotropic effect, while sympathetic stimulation leads to increase in heart rate, a positive chronotropic effect.

1.6.2 Inotropism Inotropism refers to the force produced by the contracting ventricular muscle fibers. When heart pumped blood out of the heart and into circulation, it should develop enough

Chapter 1: Introduction 10 intraventricular pressure to overcome the aortic pressure. It is determined by different factors, the most important being the length of ventricular muscle fibers at the end of ventricular filling; the Frank-Starling law. Parasympathetic stimulation affects to a lesser degree than sympathetic stimulation. Sympathetic stimulation increased the force of contraction, a positive inotropic effect, while parasympathetic stimulation decreases the force of contraction, a negative inotropic effect.

1.6.3 Lusitropy It is the rate of myocardial relaxation. An increase in the rate of myocardial relaxation is known as positive lusitropy. Increased catecholamine levels promote positive lusitropy. Whereas decreasing in rate of myocardial relaxation is known as negative lusitropy.

1.7 Animal models for evaluating cardiovascular activity Animal models used to evaluate cardiovascular activities may be in-vivo or in-vitro and ex- vivo preparations. In in-vivo model whole animals are used as a test subject, and results obtained are representative of the action of the test substance in humans. In in-vitro model isolated organs or parts of organs are used in simulated physiological environment. The isolated organ perfusion technique is used to maintain an organ in vascular isolation from the rest of the tissues and organs of the body, by mechanically assisted circulation of suitable physiological solution through its vascular beds (Mehendale., 1989). In this preparation, concentrations of endogenous or exogenous stimulatory substances and other factors are under the experimental control. It provides direct actions of test substances on the tissues without confounding effects of other systems and neurohormonal activity. The isolated tissue, organ technique can be used effectively to collect information about the drug and drug receptors, which may become useful for classification purpose and general statements about the structure and activity (Kenakin., 1984). The following preparation/technique was employed in the present studies.

1.7.1 Isolated perfused heart preparations: 1) Langendorff’s heart preparation

2) Isolated working heart preparation

Chapter 1: Introduction 11

1.7.2 Isolated smooth muscle preparation: 1) Isolated aorta

1.7.1 Isolated perfused rat heart preparations We selected the healthy adult male Wistar albino rat for this study. Isolated perfused rat heart preparation is a recognized experimental model. This represents the best compromise between the quantity and quality of data which can be obtained from an experimental model as against the relevance of such results to human populations (Sutherland and Hearse., 2000). Isolated working and non-working heart models have proven invaluable in studying the pharmacological activity of a test substance on myocardial function, , and vascular reactivity. Moreover, in the investigation of clinically relevant disease states such as ischemia-reperfusion injury, diabetes, obesity, and heart failure (Aronson et al., 1976, 1977) this model enjoys the validity till now.

The low cost, high reproducibility and quick results add the advantage over the other techniques.We can measure a broad spectrum of different indices without confounding effects of: (1) the influence of other organs (2) systemic circulation, (3) circulating neurohormonal factors (4) changing in loading parameters and (5) anesthesia (6) controlled dose-response studies of metabolic or pharmacological interventions.

1) Langendorff’s rat heart preparation

It is the most extensively used preparation and currently it has become the mainstay of models and very useful tool in translational cardiovascular, physiological, pathological, and pharmacological research (Skrzypiec-Spring et al., 2007; Bell et al., 2011; Mersmann et al., 2011; Anderson et al., 1990; Schechter et al., 2014). It represents a highly reproducible model for studying broad spectrum of biochemical, physiological, morphological, and pharmaceutical parameters, including analysis of intrinsic heart mechanics, metabolism, and coronary vascular response (Olejnickova et al., 2015). It allows measuring the direct effect of drugs on the heart, contractile functions by measuring LV pressure (in terms of LVDP by the balloon), electrical activity, heart rate, ischemia/reperfusion injury, infarct size and myocyte isolation etc. (Lateef et al., 2015). This method has the advantages of long viability duration,

Chapter 1: Introduction 12 simple preparation, low cost and reliable data collection in quality and quantity. The present studies are aimed to investigate specifically left ventricular function in isolated Langendorff’s rat heart preparation.

2) Working rat heart preparation The working heart model is set up in such a way that it mimics the physiological circulation. This is more complex preparation with ventricular filling via the left atrium and ejection in the normal direction via the aorta. It has an advantage of ability to permit the heart to generate pressure-volume work by measuring pump function of the heart with different preloads and afterloads. Rat hearts are the most frequently used species for working heart preparations, but all species can be used even the dog or pig (Sutherland and Hearse., 2000). It has advantages over Langendorff’s preparation as it allows the heart to generate pressure- volume work and to yield more information on functional performance of the heart to measure pump function with variable preloads and afterloads. The isolated working heart permits the studies under highly controlled conditions in which the function of the heart as an ejection pump, myocardial mechanics and myocardial metabolism can all be evaluated simultaneously. The advantage of the system is that the heart rate can be controlled; studies can be performed in which preload and afterload, stroke volume, ventricular volume, oxygen consumption, substrate utilization and lactate and pyruvate production can all be measured. Derivatives of these in terms of myocardial mechanics and overall ventricular function and metabolism can be calculated (James Scheuer., 1977). This preparation provides an opportunity to use the same heart as a “working” or “non-working” model for investigating the functions of the heart (Igic., 1996). We used working rat heart model in our studies to find out the effects of crude extract, fractions and marmelosin isolated from dry fruit of Aegle marmelos on functional performance of the heart in a normal physiological state, load dependent effects of constructing the Frank-Starling curve and therapeutic and /or protective effects in Ca++ paradox experiments.

1.7.2 Isolated smooth muscle preparation

Chapter 1: Introduction 13

Studies on smooth muscle preparations are common both in fundamental research and for pharmacological screening purpose. As the phylogenetic differentiation and specialization of smooth muscle cells is minimal, they do not have highly specific oxygen and substrate requirements, therefore easy to use. In addition, isolated tissue is free from the nervous and humoral regulatory influences which complicate the function in vivo. This means that in certain standardized test condition, the typical reaction can be obtained and reproduced reliably by relatively simple means. Conditions under which mechanical muscle activity can be measured are:

1) Isotonic Measurements: Changes in length can be measured in a muscle to which a constant predetermined force is applied.

2) Isometric Measurements: Changes in force can be measured in a muscle to which a constant predetermined length is applied.

3) Auxotonic Measurement: Changes in length and force can be measured simultaneously, if the length of the muscle and the force applied is predetermined.

Studies in smooth muscle preparations usually distinguish three different types of drugs:

a) Agonists; substances which elicit, reversibly, contractions.

b) Antagonists; substances which relax smooth muscle and prevent contraction or reverse contraction.

c) Potentiating substances; substances which elicit no visible reaction when given alone, but which increases the effect of certain agonist or antagonists.

There are number of smooth muscle preparations used for study purpose, like guinea pig ileum, rat uterus, rat aorta, rabbit ileum / jejunum, rabbit aorta, trachea and spleen etc. These are amongst the most frequently used preparations. We used rings of rat descending thoracic aorta in our studies to elucidate the mechanism of action of our test substances produced in vascular smooth muscles.

Chapter 1: Introduction 14

1.8 Aims and Objectives These studies are aimed to provide pharmacological rationale for the use of Aegle marmelos and Pyrus cydonia Linn in cardiovascular disorders. The activity guided fractions and isolated compound which was identified as Marmelosin were further characterized. Specifically, this study provides the basis for the use as a therapeutic agent and a lead to conduct further research.

Models, tissues, extracts, fractions and compounds:

- At first, to determine the cardioactive effects of crude extracts of Aegle marmelos Linn and Pyrus cydonia Linn in isolated perfused Langendorff’s rat heart.

- To separate different activity directed-fractions and isolation and concentration of the active constituents of crude extract of A.marmelos and to test them on isolated rat working heart preparation for evaluation of their inotropic, chronotropic, pressure volume relationship and vasoactive effects. Keeping the preload and afterload constant at the physiological level.

- To find out the effects on load dependent effect on the working heart by constructing the Frank-Starling curve.

- Ca++ paradox experiments determine the therapeutic and /or prophylactic effects.

- To elucidate the possible mechanism of action of crude extract, fractions and marmelosin on isolated rat aorta.

15

CHAPTER 2 LITERATURE REVIEW

Chapter 2: Literature Review 16

2.1 Aegle marmelos L. Aegle marmelos belongs to the family; Rutaceae, genus; Aegle and species marmelos. The botanical name is Aegle marmelos. Different parts of A.marmelos plant are used for a wide variety of disorders like fruit; ripe and un-ripe, leaves and branches, flower, root and bark. In addition to medicinal uses fruit is also used for nutritional purposes. Here we are focused on medicinal uses, especially the cardiovascular disorders.

2.1.1 Habitat and Morphology Bael is a subtropical plant, growing up to an altitude of 1,200 m above sea level. It grows in dry forests in hilly and plain areas. It is the native of India. It is found in India, Pakistan, Sub- Himalayan forests, Ceylon, China, Nepal, Sri Lanka, Myanmar, Bangladesh, Nepal, Vietnam, Laos, Cambodia, Thailand, Indonesia, Andaman, Malaysia, Nicobar Islands, Tibet, Sri Lanka, Java, Philippines and Fiji (Dhankhar et al., 2011; Hameed et al., 2011; Lambole et al., 2010).

A.marmelos is a slow-growing, medium sized tree, up to 12 to 15 m tall with short trunk, thick, soft, flaking bark and spreading spiny branches, the lower ones are drooping. Fruit which is our focus of study is spherical or oval with a diameter of 2-4 inch. The shell is thin, hard and woody in nature. Fruiting occurs in the month of May and June. Unripe fruit is greenish and turns into yellowish color upon ripening. It takes about 11 months. The pulp is yellow, soft, pasty, sweet, resinous and fragrant composed of 8-15 segments. Seeds are embedded in the pulp of the fruit. These are small (nearly 1 cm in length), hard, flattened- oblong, bearing woolly hairs and each enclosed in a sac of adhesive, transparent mucilage that solidifies on drying (Lambole et al., 2010; Dhankar et al., 2011).

2.1.2 Traditional use in various disorders According to Chopra “No drug has been longer and better known, nor more appreciated by the inhabitants of India than the Bael fruit” (Chopra., 2006). A.marmelos is one of the most important medicinal plants of Pakistan, India, Burma and Ceylon (Srivastva et al., 1996). Traditionally it is used against various diseases. The effectiveness of fruit in the treatment of diarrhea and dysentery results its entry into the British pharmacopoeia (Chopra., 2006).

Chapter 2: Literature Review 17

A.marmelos is commonly used to treat jaundice, constipation, chronic diarrhea, dysentery, stomachache, stomachic, fever, sprue, asthma, inflammations, febrile delirium, acute bronchitis, snakebite, abdominal discomfort, acidity, burning sensation, epilepsy, indigestion, leprosy, myalgia, smallpox, spermatorrhoea, leucoderma, eye disorders, ulcers, mental illnesses, nausea, sores, swelling, thirst, thyroid disorders, tumors, ulcers and upper respiratory tract infections (Sekar et al., 2011; Chopra., 2006). Almost each and every part of A.marmelos is used in different forms as a remedy for treating various ailments by herbal drug healers.

Poultice of leaves is found efficacious to heal inflamed parts and unhealthy ulcers. Young leaves are eaten to cause sterility or even abortion. Fresh juice is , useful in asthmatic complaints, ophthalmia and other eye affections. The decoction is used as a febrifuge and expectorant. Oil prepared from leaves gives relief from recurrent cold and respiratory infections. It is used in Abscess, backache, abdominal disorders, vomiting, cut and wounds, dropsy, beriberi, weakness of heart, as cardiotonic, cholera, diarrhea, animal bites, nervous disorders, hair tonic, acute bronchitis, in childbirth.

As veterinary medicine for wound, killing worms, fodder for sheep, goat and cattle, stimulation of respiration, contraction of denervosed nictitating membrane in anesthetized cats (George et al., 2003; Gaur., 1999; Dhankhar et al., 2011).

The root bark is used in heart disorders, intermittent fever, fish poison, dog’s bite, gastric troubles and palpitation of heart. Bark Juice with a little cumin in milk as remedy for poverty of seminal fluid. It is used as antiamoebic, hypoglycemic, and antirheumatic. Root decoction is used to treat melancholia (Veerappan et al., 2000; Dhankhar et al., 2011; Gavimathet al., 2012; Sekar et al., 2011). Distillation of flowers used as a tonic for stomach and intestine, as antidysenteric, antidiabetic, diaphoretic, expectorant and local . It is also used in epilepsy (Rahman and Ahmad., 1986). Fruit is eaten during convalescence after diarrhea. The fruit is used as a remedy for diarrhea and dysentery, gastric troubles and constipation. Useful as laxative, digestive, stomachic, brain and heart tonic, antiviral, in cases of intestinal parasites, tuberculosis, colitis, gonorrhea and epilepsy (Parmar and Kaushal., 1982;

Chapter 2: Literature Review 18

Veerappan et al., 2000; Kaushik and Dhiman., 1999; Kumar et al., 2012). In Ayurvedic system ripe fruit as a mild astringent used in chronic diarrhea and dysentery, as tonic for the heart and brain and as an adjuvant. It promotes digestion and helpful in treating inflammation of the rectum. It showed antiviral activity against Ranikhet disease virus. Pulp of ripe fruit is sweet, cooling, aromatic and nutritive when taken fresh. Marmalade prepared from the pulp is used as preventive during cholera epidemics, to inhibit the growth of piles, useful in chronic dysentery characterized by alternate diarrhea and constipation. Ripe fruit relieves flatulent colic in chronic gastrointestinal catarrh (Vyas et al., 1979; Dhankhar et al., 2011). Fine powder of the unripe fruit is effective against intestinal parasite, Entamoeba histolytica and Ascaris lumbricoides. Decoction of the unripe fruit is astringent, useful in diarrhea and chronic dysentery and given in piles. It is used as tonic, restorative, digestive, demulcent and cardiacal (Trivedi et al., 1978). Seed oil exhibits antibacterial activity against different strains of Vibrio’s and inhibits the growth of Vibrio cholerae, staphylococcus aureus and Escherichia coli. Essential oil exhibits activity against fungi Physalospora tucumanesis, Eeratocystis paradoxa, Selerotium ralfsii, Curvularia lunata, elminthosporium sacchari, Fusarium monthforme and cephalosporium sacchari (Banerji and Kumar., 1949; Jain., 1977).

2.1.3 Pharmacological activities A.marmelos is rich in biologically active constituents showing a variety of pharmacological activities. Extracts of leaf, root, fruit, seed and oils showed , antifungal, antibacterial, antiviral and antiseptic activities (Sharma et al., 2011; Gupta et al., 2011; Badam et al., 2002; Huang., 1993). Different organic extracts of leaves and fruit exhibited the significant , anti-inflammatory and antipyretic effects (Arul et al., 2005; Sharma et al., 2011; Dhankhar et al., 2011; Ghangale et al., 2008; Rahman et al., 2015). Seed, leaf and fruit showed significant antiulcer activity against pylorus-ligated and aspirin-induced gastric ulcers, cold restraint stress-induced gastric ulcers and absolute ethanol induced gastric mucosal damage (Goel et al., 1997; Dhuley., 2007; Phoolsingh et al., 2010). Leave extract showed anticancer activity against Ehrlich ascites carcinoma, tumor cell lines in brine shrimp lethality assay and methyl thiazolyl tetrazolium (MTT) based assay, human ovarian cancer cell line and prevent chemically induced skin carcinogenesis and antiproliferative (Dhankhar et

Chapter 2: Literature Review 19 al., 2011; Maity et al., 2009; Lambol et al., 2010; Agarwal et al., 2011), antioxidant and hepatoprotective against CCl4 and alcohol induced liver injury and prevent ethanol and platelet activating factor induced gastric ulcer, radioprotective and cytoprotective in fresh water fish exposed to heavy metals (Maity et al., 2009; Singanan et al., 2007; Sekar et al., 2011; Khan and Sultana., 2009), antispermatogenic and antisperm motility (Remya et al., 2009 ; Akram et al., 2012), antithyroid (Panda and Kar., 2006), antidiabetic (Upadhya et al., 2004; Kamalakkanan and Prince., 2005; Sharma et al., 2011; Dhankhar et al., 2011), antihyperglycemic (Upadhya et al., 2004; Murlidharan et al., 2014; Sachdeva et al., 2001), antihyperlipidaemic (Rajadurai and Prince., 2005; Vijaya et al., 2009), antiasthmatic, antihistaminic and act as immune stimulant (Arul etal., 2004; Megrajet al., 2011), , , anticonvulsive, , and having potential by providing significant neuroprotection and memory enhancement (Kothari et al., 2010; Rastogi and Mehrotra., 1993; Gavimath et al., 2012). It exhibited cardiotonic effects on frog’s heart (Haravey., 1968) and act as a potent cardioactive agent with hypoglycemic and antiinflammatory effects. It is cardioprotective; protects the heart from isoproterenol induced myocardial infarction by ameliorating effect on cardiac enzymes, lipid peroxidases, serum lipids and lipoproteins (Prince et al., 2005; Rajadurai and Prince., 2005). obtained from leaves showed cardioprotective effects by inhibiting enzymes CK-MB and SGPT like other cardiac glycosides and prevent doxorubicin induced cardiomyopathy (Panda and Kar., 2009). Wound healing and antigenotoxic effects have been noted in animals (Arunachalam et al., 2012). It has antimicrofilarial activity (Lambole et al., 2010). An essential oil from leaves possesses insect controlling and repellent properties. Seed extract has significant protective effects against diabetes induced spatial learning and memory deficits (Farshchi et al., 2011).

Seeds and leaves extract exhibited antimalarial activity against the NK65 strain of Plasmodium berghei in vivo and in vitro experiments. Root bark extract exhibits a potent inhibitory effect on spontaneously beating heart and reduces the damage of myocardium in calcium-paradox, reduces palpitation, used in heart disorders, rheumatism and anti-dog bite (Kakiuchi et al., 1991; Patel et al., 2012). Fruit extract augmented the immune activity by

Chapter 2: Literature Review 20 cellular and humoral mediated mechanism (Patel and Asdaq., 2010; Govinda and Asdaq., 2011), promote wound healing by enhancing connective tissue formation and showed potent antioxidant activity (Gautam et al., 2014; Rahman et al., 2016), unripe fruit extract reduces the intraocular pressure and thus prevent glaucoma (Agarwal et al., 2009; Dama et al., 2010), antibacterial, antiviral against human coxackie virus, anthelminthic, antispasmodic, prevent diabetes and its complications, artemicidal, cytotoxic, antidiarrheal, reduces chemical induced diarrhea, antidysentric and astringent (Sharmila and Devi., 2011; Rajan et al., 2011; Gheisariet al., 2011). It prevents inflammatory bowel disease, radiation-sickness and mortality, act as antiulcer, anticancer and chemoprotective agent (Maity et al., 2009; Baliga et al., 2011). Inhibiting the chronotropic effect on cardiac tissue and useful in the treatment of hypertension. Fruit juice showed cardiotonic activity in the hypodynamic frog’s heart. It increases the rapidity and force of contraction (Dama et al., 2010).

2.1.4 Phytochemical constituents A.marmelos is rich in phytochemical compounds. Various chemical constituents have been isolated and identified from different parts of the tree like alkaloids, coumarins and steroids, etc. Coumarins; Marmelosin, marmesin, imperatorin, marmin, alloimperatorin, methyl ether, xanthotoxol, scopoletin, scoparone, umbelliferone, psoralen and marmelide (Farooq., 2005).

Alkaloids; Aeglin, aegelenine, dictamine, fragrine (C13H11O3N), Omethyl halfordinine, isopentenyl halfordinol (Farooq., 2005). Saponin glycosides and cardiac glycosides (Rajan et al., 2011; Sekar et al., 2011; Sarkozi et al., 1996; Sridhar et al., 2014). Polysaccharides; Galactose, arabinose, uronic acid and L-rhamanose are obtained on hydrolysis (Basak et al., 1982). Seed oil; Composed of palmitic, stearic, oleic, linoleic and linolenic acid (Farooq., 2005). Tannins; The maximum tannin content in bael fruit was recorded as much as 9 %. Carotenoids: are responsible for the pale color of fruit.

2.2 Pyrus cydonia Linn Pyrus cydonia belongs to the family; Rosaceae, genus; Cydonia, species; oblonga bearing the botanical name of cydonia oblonga Miller.

Chapter 2: Literature Review 21

2.2.1 Habitat and Morphology Quince tree is cultivated in gardens under warm temperature and grows up to 8 m high and 4 m wide. It was cultivated in Mesopotamia, now called Iraq. It was called “Golden Apple” in ancient Biblical history. It grows in Trans-Caucasian region, including Armenia, Azerbaijan, Southwestern Russia, Turkmenistan, Middle East, Greece, around the Mediterranean like Iran, Iraq, Turkey, China, Japan and North-West India (Nadkarni., 1982; Westwood., 1978; USDA., 2009; Postman., 2008; Postman., 2009; Gholgholab., 1961). Fruits are bright yellowish and usually pear shaped. Three kinds of quince fruit are commonly found that is sweet, sour and subacid.

2.2.2 Traditional Uses in various disorders Literature revealed that P.cydonia has been traditionally used as cardiac, cephalic, demulcent, astringent, restorative, tonic; carminative, gastric tonic; , antiinflammatory, in uterine and hemorrhoid bleeding, , vulnerary, antialcoholic, anticancer, anticatarrhal and antipyretic specially fever of warm origin (Duke., 2002; Usmanghani., 1997; Nadkarni., 1982; Chopra et al., 2006; Oliveira et al., 2008; Yildrim et al., 2001; Aslam and Sial., 2014). As remedy for cough, bronchitis, nausea, diarrhea, dysentery, cystitis, constipation, hemorrhoids, diabetes, hypertension (Khoubnasabjafari and Jouyban., 2011). Also used in the treatment of Phthisis, blenorrhalgia, skin cracking, whooping cough and as antiemetic (Saganuwan., 2010). Fruit is used in the treatment of cardiovascular diseases, hemorrhoids, bronchial asthma, cough, as a tonic, appetitive; lessen thirst, astringent, diuretic, expectorant, antiulcer and demulcent. Dried fruit is regarded as refrigerant (Usmanghani., 1997; Nadkarni., 1982; Yildrim et al., 2001). Seed is regarded as medicinal part of the plant and useful as antitubercular, expectorant, good against cough, sore throat, bronchitis, asthma, stomatitis, in treating gonorrhea, fever and dysentery with inflammation of the mucus membrane, burning sensation and ulcers. Also, used as sedative, antidiarrheal, antitussive, antidiabetic, in the treatment of various skin diseases, provides health promoting compounds with strong antioxidant effects. Decoction used in cases of migraine, nausea, common cold and influenza (Duke., 2002; Usmanghani., 1986; Nadkarni., 1982; Chopra et al., 2006; Oliveira et al., 2008; Yildrim et al., 2001; Khoubnasabjafari and

Chapter 2: Literature Review 22

Jouyban., 2011). The leaf is used as sedative, antipyretic, antidiarrheals, antitussive, in skin diseases, in cardiovascular diseases, haemorrhoids, bronchial asthma, cough, as nephron protective and hepatoprotective, (Ganopoulos et al., 2011; Yildrim et al., 2011; Saganuwan., 2010; Jouyban et al.,2011).

2.2.3 Pharmacological activities Pyrus cydonia is an important medicinal plant possessing pharmacologically active potentials to use successfully in the treatment of different disorders. It possesses antioxidant, antimicrobial, antiviral, anticancer activities. It has suppressive and antiproliferative effects against human renal and colon cancer cell line. Quince extract inhibits the growth of adenocarcinoma of the human colon. It prevents drug-induced myocardial necrosis. It is used as an because it has sexual behavior enhancing effects (Fattouch et al., 2007; Al- Khazraji., 2013; Aslam and Sial., 2014; Carvalho et al., 2010; Boivin et al., 2009; Alesiani et al., 2010; Riahi-Chebbi etal., 2016; Goyal et al., 2010). Fruit has antidiabetic, antiallergic, antiviral activities. It inactivates the influenza viruses. It inhibits the immunoglobulin E (IgE) - dependent late-phase immune reactions of mast cells and it is immunomodulatory (Tahraoui et al., 2007; Shinomiya et al., 2009; Hamauzu et al., 2005; Silva et al., 2004; Kawahara and Iizuka., 2011 and Aslam and Sial., 2014). Quince fruit has antiradical, antimicrobial, antibacterial and potent antioxidant activities, act as laxative, demulcent and emollient for the skin, used in inflammatory bowel disease, dysentery and cystitis (Rahimi et al., 2010; Agelet et al., 2003; Pieroni et al., 2004; Sezik et al., 2001; Bonjar., 2004; Fattouch et al., 2007; Al-Khazraji., 2013; Khoubnasabjafari and Jouyban., 2011 and Aslam and Sial., 2014). Antiradical activity of seed extract is much higher than standard antioxidants (Alesiani et al., 2010). Possesses antimicrobial, antiproliferative, antiulcerative effects, used in cough, bronchitis, sore throat, constipation, fever, gonorrhea, dysentery, diarrhea and stomach ulcers, conjunctivitis, migraine, nausea, common cold and influenza, as healer of skin lesions, Scald, burns and blister (Siddiqui et al., 2002; Ghanadi et al., 2003; Usmanghani., 1986; Fattouch et al., 2007; Hemmati et al., 2010; Saric-Kundalic et al., 2011; Al-Khazraji., 2013; Aslam and Sial., 2014; Khoubnasabjafari and Jouyban.,2011; Alesiani et al., 2010; Riahi- Chebbi et al., 2016). The leaves have antidiabetic, antioxidant, antiradical, antihemolytic and

Chapter 2: Literature Review 23 free radical scavenging properties. Leave showed antihyperglycemic and antihyperlipidemic activities. Lipid lowering activity is comparable to Atorvastatin (Khademi et al., 2013). It is used in cardiovascular disorders, hypertension, hemorrhoids, cough, bronchitis and bronchial asthma, diarrhea, dysentery and stomach ulcers. It is diuretic, nephroprotective, gastroprotective and protect testes and spermatogenesis from hypercholesterolemia (Saric- Kundalic et al., 2011; Aslan et al., 2010; Palmese et al., 2001; Costa et al., 2009; Teresa et al., 2001; Yildrim et al., 2001; Camejo-Rodrigues et al., 2003; Tuzlaci and Tolon., 2000; Tuzlaci and Aymaz., 2001; Kültür., 2007; Ashrafi et al., 2013; Jouyban et al., 2011).

2.2.4 Phytochemical constituents Phytochemical studies revealed that P.cydonia has a significant number of phytochemical compounds. Various phenolic compounds, organic acids and volatile compounds have been isolated from different parts of quince plant. Phenolic compounds isolated from P. cydonia were 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 3,5-O- dicaffeoylquinic acid, quercetin-3-O-galactoside , quercetin-3-O-rutinoside, kaempferol-3-O- glycoside, kaempferol-3-O-rutinoside, 6-C-Glucosyl-8-C-pentosyl chrysoeriol, Isoschaftoside, Lucenin-2, Schaftoside, Stellarin-2, and Vicenin-2 etc. Total phenolic content of methanolic extract of leaf was thehighest (27.96 g/kg dried) followed by peel (7.41 g/kg), pulp (1.17 g/kg) and seed (0.52 g/kg) respectively. The major phenolic compoundin leaf extract was 5-O caffeoylquinicacid. Quince seed contains fat soluble bioactive compounds like tocopherols, phytosterols and phenolic acids. Tocopherols consisted of α-tocopherol (16.03 mg/100 gdry seed), β-tocopherol (0.15), γ-tocopherol (0.32) andtotal tocopherol of 16.49 mg/100 g dry seed were found.The phytosterols were campestrol (0.32 mg/g fat), stigmasterol (0.20 mg/g), sitosterol (2.60mg/g), avanasterol (0.44 mg/g) and total phytosterols of 3.56 mg/g fat. Fruit possesses the highest amount of hydroxycinnamic acid derivatives i.e 30.9 mg/100 g, (Magalhaes et al., 2009; Costa et al., 2009; Carvalho et al., 2010; Alesiani et al., 2010; Khoubnasabjafari and Jouyban., 2011).

24

CHAPTER 3 MATERIALS AND METHODS

Chapter 3: Material And Method 25

3.1 Drugs, standards and chemicals

All the drugs, standards and chemicals used, were of analytical grade, purchased from the source given: Acetylcholine (ACh), butanol, digoxin, dimethyl sulfoxide (DMSO), ethyl acetate, ethanol, hexane, methanol, marmelosin, norepinephrine bitartarate (NE), N-Nitro-L-arginine methyl ester hydrochloride (L-NAME), phenylephrine hydrochloride (PE), polysorbate 80 (Tween-80) and verapamil hydrochloride (Sigma chemical company, St. Louis, MO, USA). Pentobarbitone sodium (Abbot Laboratories Karachi, Pakistan) and heparin (Leo Pharmaceutical Product, Denmark).

The chemicals used for the preparation of Krebs-Henseleit solution (physiological salt solutions) included sodium chloride, potassium chloride, magnesium sulphate 7H2O, potassium dihydrogen phosphate, glucose, sodium bicarbonate, calcium chloride (Merck- Schuchard, Muenchen, Germany) and EGTA were purchased from (Sigma chemical company, St. Louis, MO, USA).

Stock solutions of drugs, standards and physiological salt solution were made in distilled water. The dilutions were made with the distilled water, fresh on the day of experiments. DMSO, Tween-80 and ethanol was used to make some fractions completely soluble before use.

3.2 Selection of plants

The most common strategy for selection of plant is careful observation of the use of natural resources in folk medicine in different cultures (Rates., 2001). One major criterion for the selection of the plants for such type of studies is the claims of traditional healers or prescribers for its therapeutic usefulness (Williamson et al., 1996). Therefore, plants were selected based on literature available regarding their traditional uses and scientific work. Only two plants were selected which were traditionally reputed to use in cardiovascular disorders namely:

i) Aegle marmelos Linn (Dry fruit). ii) Pyrus cydonia Linn (seeds).

Chapter 3: Material And Method 26

3.2.1 Collection and identification of plant materials

The dry and ripe fruit of the Aegle marmelos commonly known as “bael” and seeds of Pyrus cydonia known as “Behidana” were purchased from a famous store in the local herbal market of Karachi, Pakistan. The plant materials were identified and authenticated by Dr. Bina Naqvi; taxonomist / botanist of Plant tissue culture lab, FMRRC, PCSIR labs; complex, Karachi according to Flora of Pakistan (1980) and voucher specimens (No. AM/pharm/001/2012 and PC/pharm/002/2012) were deposited in the herbarium of Federal Urdu University, Karachi respectively for future reference.

3.2.2 Preparation of crude extract

The crude extracts of the test plant materials were prepared by using the cold maceration process. The plant materials were cleaned of adulterants and then coarsely grounded. These materials (475g A.marmelos fruit and 500g P.cydonia seed) were soaked in the methanol: water (70:30) mixture (aqueous-methanolic mixture) at room temperature (23-25oC) for 72 hours with occasional shaking. It was then filtered through Whatman qualitative grade 1 filter paper. The process of soaking and filtration was repeated thrice. Finally, all the filtrates were combined and concentrated in a rotary evaporator (Buchi Rotavapor R-210) accompanied with a Recirculation chiller (Buchi Distillation Chiller B-741), Eyela Aspirator (A-3S) and a heating water bath (B-491). The process of evaporation was carried out at 40-50oC under a reduced pressure of about-760 mm Hg. This produced (250g, AMFM) thick semi solid mass of dark brown color of dry fruit of A.marmelos and (107.11g, PCSM) thick, pasty mass of brown color of P.cydonia seeds. Both crude extracts were stored and preserved at -4oC until use.

The crude extract was studied to explore the cardiovascular activity which led to make fractions and isolation further. We found the crude extract of A.marmelos more potent than the crude extract of P.cydonia.

3.2.3 Fractionation and isolation

A.marmelos. L was selected for more fractions based on activity to separate or concentrate the cardiovascular active constituents in one of the corresponding fraction (Williamson et al.,

Chapter 3: Material And Method 27

1998). To isolate the active chemical constituents, the crude extract of A.marmelos. L (AMFM), the fractions were prepared from non-polar to polar by using hexane, ethyl acetate, butanol, methanol and water and obtained the soluble fractions AMFMH, AMFMEA,

AMFMBut, AMFMM and AMFMH2O respectively (Scheme-1). The crude extract of P.cydonia was decided not to prepare further fractions.

3.2.4 Isolation of marmelosin

To get the marmelosin the hexane soluble fraction (AMFMH) was further treated by hexane, chloroform and ethyl acetate and obtained AMFMHH, AMFMHC and AMFMHEA respectively. The sub-fraction AMFMHH found to be the mixture of fourteen compounds through GC and sub-fraction AMFMHC found to be mixture of five compounds through GC/GCMS. Marmelosin was present in hexane soluble fraction AMFMHH, chloroform soluble sub- fraction AMFMHC of AMFMH and was the major constituents in ethyl acetate soluble sub- fraction AMFMHEA (Scheme-2).

The ethyl acetate soluble fraction (AMFMEA) of crude extract was further treated by using hexane, hexane-ethyl acetate (1:1), ethyl acetate and methanol and obtained the AMFMEAH, AMFMEAHEA, AMFMEAEA, AMFMEAM sub-fractions respectively. Out of all these sub- fractions AMFMEAHEA and AMFMEAEA found to be very potent antioxidants active sub- fractions (Scheme-3).

The butanol soluble fraction (AMFMBut) was sub-fractionated by using hexane, hexane-ethyl acetate (1:1), ethyl acetate and methanol and obtained the sub-fractions, AMFMButH, AMFMButHEA, AMFMButEA and AMFMButM respectively and insoluble white shiny crystalline compound. The hexane ethyl acetate soluble sub-fraction AMFMButHEA afforded a pure colorless crystalline compound by recrystallized with ethyl acetate, which was identified as marmelosin (VIII) (Chakthong et al., 2012; Waight et al., 1987; Miyakado et al., 1978) (Rf = 0.57, hexane: ethyl acetate, 7.0:3.0).

The hexane: ethyl acetate sub-fraction (AMFMEAHEA) of the main ethyl acetate fraction (Scheme-4) and ethyl acetate soluble sub-fraction (AMFMButEA) of main butanol soluble

Chapter 3: Material And Method 28 fractions were merged on the basis of thin layer chromatography (silica gel, hexane: ethyl acetate, 7:3) and obtained a combined fraction AM (0.9443 g) (Scheme-5).

To obtain the pure marmelosin the combined fraction AM (0.9443 g) was subjected to flash column chromatography (silica gel, 100 g, E-Merck 1.07553, packing 5 inches, diameter = 2.5 cm, length of column = 7.5 inches) by using hexane, ethyl acetate and methanol by increasing polarity (5 %) which gave 95 fractions (volume of each eluent 50 ml). Out of these, the fractions; 6, 13 and 94 were found to be pure and identified as marmelosin (VIII) (Chakthong et al., 2012; Waight et al., 1987; Miyakado et al., 1978), xanthotoxin (IV) and tricontain-2-one (XI) respectively. To elucidate the structure of all pure compounds extensive spectral technique such as UV, IR, EIMS andH-NMR were carried out.

3.2.5 Spectral characterization of isolated marmelosin from A.marmelos fruit

Marmelosin (VIII)

Physical State: colorless crystalline solid, Yield: 260 mg m. p. 103oC, Rf: 0.57 (Hexane: Ethylacetate, 7:3)

UV λmaxnm: 243, 249, 263, 299nm

-1 max cm : 1704-1719 (C=O), 1585 (C=C), 1026 -1080 (C-O).

1 H-NMR in CDCl3 at 400 MHz (δ in ppm and J in Hz): δ 1.71(3H, s, H-17); 1.69 (3H, s, H-16,); 4.99 (2H, d, J = 7.2, H-13); 5.58 (1H, tt, H-14); 6.35 (1H, d, J = 9.6, H-3); 6.79 (1H, d, J = 2.4, H- 11); 7.33, (1H, s, H-5); 7.66 (1H, d, J =2.4, H-12); 7.74 (1H, d, J =9.6, H-4).

EIMS (m/z): m/z 69 (C5H9, 53 %), 146 (C9H6O2, 24 %), 162 (C9H6O3, 52 %) 174 (C10H6O3, 58 %),

202 (C11H6O4, 100 %), 216 (0.5 %), 238 (0.5 %), 270.1 (C16H14O4, 0.5 %).

The crude extract of A.marmelos (AMFM), butanol soluble fraction (AMFMBut), water soluble fraction (AMFMH2O) finally dried with a lyophilizer and Marmelosin isolated from dry fruit of Aegle marmelos. Linn was selected for further cardiovascular studies.

Chapter 3: Material And Method 29

Chapter 3: Material And Method 30

Chapter 3: Material And Method 31

Chapter 3: Material And Method 32

Chapter 3: Material And Method 33

Chapter 3: Material And Method 34

3.3 Animals

Animals used for in-vivo and in-vitro studies were Swiss albino mice weighing (20-25 g) and male Wistar albino rats weighing (250-350 g) respectively, and were purchased from the animal house facility of Dow University of Health Sciences, Karachi, Pakistan. Mice of either sex were used in in-vivo experiments to evaluate the acute toxicity of crude extracts. Whereas in in-vitro experiments rats were used because they provided the best compromise between size and heart rate, as against rabbits which have a heart rate that are quite high. Only male Wistar rats were used, because, male species of the rat are recommended and commonly used than females (Hill et al., 2005) to ensure the uniformity in the results. Whereas some researchers had indicated that there were no gender specific differences in cardiovascular parameters (Livius et al., 2000).

The selected rats were healthy, adult and normotensive. They were housed in the controlled environment room with 12-hours light and 12- hour dark cycles, at room temperature of 22 ± 2oC. All the experimental animals were provided recommended nutritional and housing conditions as guided by National Research Council, USA.

All animals received humane care in compliance with the “National Research Council, Guide for the Care and Use of Laboratory Animals, Washington, DC: National Academy Press (NRC.,2011)” and the institutional guidelines for animal care and use approved by the ethical committee of Federal Urdu University of Arts, Science and Technology, Karachi, Pakistan.

3.4 Experimentations

3.4.1 In vivo experiments 3.4.1.1 Acute oral toxicity study of crude extract of Aegle marmelos Linn.

Acute oral toxicity study of crude extract of dry fruit of A.marmelos was performed by using the method previously described (Iqbal et al., 2014) with some modifications. Swiss albino mice of either sex were randomly divided into six groups of five animals each. Group-1 served as negative control and received normal saline (10 ml/kg) while other groups (Group-2, 3, 4, 5 and 6) serving as test groups (Sanmugapriya and Venkataraman., 2006) were given increasing

Chapter 3: Material And Method 35 doses of crude extract of A.marmelos Linn (500, 1000, 2500, 5000 and 7500 mg/kg, orally) in 10 mL/kg volume by gastric intubation respectively and provided free access to food and water ad libitum. All the test animals were continuously observed for changes in behavior, physiological parameters, restlessness, and seizures for 6 hours, and then intermittently monitored for any morbidity and mortality up to 24 hours (Khan and Gilani et al., 2009).

3.4.1.2 Acute oral toxicity studies of crude extract of Pyrus cydonia Linn

Acute oral toxicity study of crude extract of seeds of Pyrus cydonia Linn was performed by using the method previously described (Iqbal et al., 2014) with some modifications. Swiss albino mice of either sex were randomly divided into six groups of five animals each. Group-1 served as negative control and received normal saline (10 ml/kg) while other groups (Group- 2, 3, 4, 5 and 6) serving as test groups (Sanmugapriya and Venkataraman., 2006) received increasing doses of crude extract of Pyrus cydonia Linn (250, 500, 1000, 2500 and 5000 mg/kg, orally) in 10 mL/kg volume by gastric intubation respectively and provided free access to food and water ad libitum. All the test animals were continuously observed for changes in behavior, physiological parameters, restlessness, and seizures for 6 hours, and then intermittently monitored for any morbidity and mortality up to 24 hours (Khan and Gilani et al., 2009).

3.4.2 In vitro experiments The experiments were aimed to study the cardiovascular activity and the mode of action of the test materials. Therefore, cardiovascular activity was studied on isolated Langendorff’s and working rat heart models and mode of action was on the smooth muscles of rat aorta.

3.4.2.1 Isolated Langendorff’s heart preparation

3.4.2.1.1 Study of crude extracts on Isolated Langendorff’s rat heart

To study the cardiovascular effect of crude extracts of A.marmelos (AMFM) and P.cydonia (PCSM), the Langendorff’s rat heart (Non-working model) was used. The procedure of isolation and mounting of the heart in Langendorff’s apparatus was followed as described by

Chapter 3: Material And Method 36

(Sirfraz and Lab., 1996; Sutherland and Hearse., 2000). Animals used were male Wistar rats weighing between 250-350g. The food was withdrawn and water was allowed ad libitum for the selected rats for 12 hours before the experiments. Heparin (1500 units kg-1 i.p) was injected to prevent the formation of intracoronary thrombi (Grupp et al., 1993) and anesthetized with pentobarbitone sodium (60 mg kg−1, i.p.) one hour prior to excision of the heart Langendorff’s apparatus and recording instruments were calibrated prior to experimentation. All procedures were performed in sterilized conditions. After cervical dislocation, the diaphragm was accessed by a midline trans-abdominal incision and cut carefully to expose the thoracic cavity, median sternotomy done and the heart was removed immediately by means of a pair of scissors, cut across the great vessels about 5 mm from the heart. The pericardium and other adjacent tissues were removed quickly and aorta was cut at the point of bifurcation, so that about 1.0 cm of aorta remained intact. The isolated heart, then immediately placed in a beaker containing ice cold Krebs–Henseleit (KH) buffer solution, well aerated by carbogen during preparation at the temperature of 37 °C. After gentle squeezing, the heart was mounted on the stainless-steel cannula of the modified Langendorff’s perfusion apparatus model ML870B2; of ADInstruments, Australia, via ascending aorta. The heart was enclosed in a double wall jacket (heart chamber); the temperature was maintained at 37 °C± 2 °C by circulating warm water. The coronary flow was re-established retrogradely with Krebs–Henseleit (KH) solution saturated with carbogen (95

% O2 and 5 % CO2). The composition of Krebs-Henseleit solution was (in mM): NaCl 117, KCl

1.41, MgSO4.7 H2O 1.2, KH2PO4 1.1, glucose 5.0, NaHCO3 2.5, and CaCl2 2.5. The temperature of the perfusating fluid and the heart chamber was kept constant at 37 °C ± 2 °C which was controlled thermostatically. A probe was inserted into the heart to record the temperature of the heart during experiments. It was ensured that temperature of the perfusate in the reservoirs and in the heart should not fluctuate or decrease so that no positive inotropic effect of hypothermia might be implied. Perfusion pressure and left ventricular pressure were recorded through a physiological pressure transducer (MLT844). A pulmonary arteriotomy was performed to allow free drainage of coronary sinus effluent. A water filled thin walled latex balloon size 3 (0.03 ML) was inserted in the left ventricle cavity through a small incision

Chapter 3: Material And Method 37 in the left atrium, and connected to a physiological pressure transducer (AD Instruments) via the metal cannula (Rahman et al., 2012). The balloon volume was adjusted to produce a 10 mm Hg of diastolic pressure. The balloon volume, which remained unchanged throughout the experiment, was adjusted to achieve a left ventricle end-diastolic pressure (LVEDP) of 4-6 mm Hg at the beginning of perfusion. So, any change in the force of contraction of the left ventricle should be applied on the wall of the balloon efficiently this could be transmitted through the physiological pressure transducer, connected to bridge amplifier and chart recorder. This recorded the isovolumetric pressure of the left ventricle. The coronary flow (CF) was kept constant (Dutta et al., 1968) throughout the experimental period. The constant flow in the coronary vessels in Langendorff’s preparation allowed to measure the effect of drugs adequately on both ventricular conduction (QRS-interval) and contractility (left ventricular pressure and dP/dt(max)) (Simon et al., 2004). Perfusion pressure and left ventricular pressure were recorded directly by physiological pressure transducers. The heart was not driven electrically and allowed to beat on its own spontaneous sinus nodal rhythm. The heart wasallowed to stabilize for 30 minutes at a constant flow rate, before administration of any drug and the LVP, PP and CF were recorded at baseline. The test samples were infused through three-way stopcock placed in aortic flow line near to the stainless-steel cannula. After stabilization of the hearts, different doses of the test samples were added in ascending order; the next dose was added after ensuring that the previous dose has induced its maximum and persistent effect, it usually took 5 minutes. The test dose was infused at the rate 0.1 mL in 10 seconds (Sakai and Shiraki., 1978). The left ventricular pressure, heart rate and perfusion pressure were recorded before and after administration of each dose. The data extracted from experimenst of five animals.

3.4.2.1.1.1 Concentrations of crude extract of A.marmelos

The doses and dilutions of crude extract of A.marmelos (AMFM) were freshly prepared on the day of experiments in the distilled water to infuse into the heart at the concentration of 1.0, 3.0, 10.0, 30.0 100.0 and 300.0 mg/mL, respectively in equal volume.

3.4.2.1.1.2 Concentrations of crude extract of P.cydonia

Chapter 3: Material And Method 38

The doses and dilutions of crude extract of P.cydonia (PCSM) was freshly prepared in the distilled water on the day of experiments and infused into the heart at the concentration of 1.0, 3.0, 10.0, 30.0, 100.0 and 300.0 mg/mL respectively in equal volume.

3.4.2.2 Isolated Working heart preparation

This is a more complex preparation with ventricular filling via the left atrium and ejection in the normal direction via the aorta. This preparation offers the advantage of an ability to measure pump function with different preloads and afterloads. Rat hearts are the most frequently used species for working heart preparations with Neely’s modification (Sutherland and Hearse., 2000).

Male Wistar albino rats weighing between 250-350g were used. The food was withdrawn 12 hours before the experiments and water was allowed ad libitum for the selected rats. Heparin (1500 units kg-1 i.p) was injected to prevent the formation of intracoronary thrombi and anesthetized with pentobarbitone sodium (60 mg kg−1, i.p) one hour prior to excision of the heart. Working heart apparatus and recording instruments were calibrated prior to experimentation. All procedures were performed in sterilized conditions. The hearts were excised and cut from ascending aorta at the point of bifurcation, so that about 1.0 cm of aorta remained intact, which is long enough to cannulate easily in aortic cannula. Then, isolated heart immediately placed in a beaker containing Krebs–Henseleit (KH) buffer solution, well aerated by carbogen during preparation at the temperature of 37 °C. After gentle squeezing, the heart was mounted on aortic glass cannula of working heart instrument (ADInstruments, Australia) and tied by thread within one minute after isolating from the body of the rat. The heart was enclosed in a double jacketed heart chamber. The temperature was controlled by thermocirculator (Randnoti; heater circulating pump 170051B) maintained the temperature within the physiological range (36-37 °C). Epicardial surface temperature was recorded by thermoprobe (Phystemp, NJ07013, USA 973-779-5577) continuously. Retrograde perfusion was initiated in Langendorff’s heart mode, by warm Krebs-Henseleit solution (composition in mM:NaCl 118, KCl 4.7, MgSO₄.7H₂O 1.2, CaCl₂ 1.8, KH₂PO4 1.2, D-Glucose

11.1, NaHCO₃ 25.0) equilibrated with carbogen (95 % O2 and 5 % CO2) to provide pH 7.4. Care

Chapter 3: Material And Method 39 was taken to supply gas in such amount to avoid the formation of microscopic precipitation (Umbreit et al., 1949). The Krebs-Henseliet perfusing solution was driven by a peristaltic motor (Radnoti, peristaltic pump drive 170100 A) at the rate of 10-12 mL/m. Perfusion pressure was 70 cmH2O. Thirty minutes were allowed to perfuse the heart in Langendorff’s mode; meanwhile left atrium was cannulated via one of the orifices of the pulmonary vein. Once aortic and left atrial cannulation was accomplished, the Langendorff’s perfusion is closed and perfusion initiated by opening the perfusion line from the left atrium to allow the perfusion in non-recirculating working heart mode according to Neely, (1967) calculated at the rate of 25 mL/min for the rat of 250 g i.e 0.1 mL/g (Sutherland and Hearse., 2000). In this way the perfusion fluid entered the left atria through the pulmonary vein, passed through the left ventricle and pumped out through aorta. Compliance chamber; located in aortic outflow line, partially filled with 15 mL of air, which provided an aortic Windkessel to compensate for the lack of elasticity of the outflow tubing. The perfusion pressure was set at 15 cmH2O for the preload and 80 cmH2O for afterload (Sasaki et al., 2007; Rahman et al., 2016) to ensure an adequate coronary flow. The heart was allowed to perfuse in the working heart mode for 30 minutes for stabilization and consistent values at baseline. Coronary effluent was collected in graduated cylinders placed exactly below the heart chamber. All hearts were perfused under normoxic, non-ischemic conditions and the preload and afterload pressures were also kept constant. Aortic pressure and arterial pressure were measured with pressure transducers (MLT844; Physiological Pressure Transducer, ADInstruments, Australia) attached to aortic and atrial flow lines. The temperature, aortic pressure, atrial pressure, left ventricular pressure, dP/dt(max) and dP/dt(min), coronary effluent per minute and heart rate from LVP was directly recorded using Lab Chart Pro v7.3.7 (ADInstruments, Australia) during the experiment. The rest of the parameters were calculated offline by using different applications provided in the Lab Chart Pro. Aortic outflow and coronary effluent were timely collected and measured in graduated cylinders per five minutes, cardiac output was calculated as the sum of aortic outflow and coronary effluent (CO = AoF + CE). Systolic and diastolic pressure was calculated from arterial pressure signals. The aortic outflow was used an index of stable work performance (Chain et al., 1969). Aortic outflow was measured per

Chapter 3: Material And Method 40 five minutes and consistent values showed the stability of the heart. The hearts which did not show stability were discarded.The data extracted from experimenst of five animals.

To study the cardiovascular effects of test materials in the isolated perfused rat working heart, the following sets of experiments were performed:

3.4.2.2.1 At fixed preload and afterload

These experiments were used to study the effects of test materials on heart functions at constant atrial filling pressure (preload, 15 cmH2O) and aortic pressure (afterload, 80 cmH2O), remained constant throughout the period of experiments. After 30 minutes of stabilization the baseline measurements (control) noted within the duration of 5 minutes. Freshly prepared dilutions of test materials infused into the perfusate by means of 1 cc tuberculin syringe (B.D) at the rate 0.1 mL in 10 seconds (Sakai and Shiraki., 1978) through in-line injection port attached to the atrial flow line near the cannula. The volume of all doses of different test materials remained same in all experiments. All the experiments began and ended in the state of beating heart. The load independent effects of test materials noted at the intervals of 5 minutes on the following parameters; coronary effluent, aortic outflow, cardiac output, dP/dt(max), dP/dt(min), systolic pressure, diastolic pressure, heart rate, peak aortic systolic pressure, end diastolic pressure, ejection fraction & stroke volume obtained from the Lab chart pro. The two parameters, i.e rate pressure product (RPP = Heart rate x peak aortic systolic pressure) and cardiac power (CP = Cardiac output x Mean aortic pressure) were calculated.

A Concentrations of crude extract, fractions and marmelosin used

A-1 Crude extract of A.marmelos

The doses and dilutions of crude extract of A.marmelos (AMFM) were prepared fresh on the day of experiments in the distilled water and infused into the heart at the concentration of 1.0, 3.0, 10.0, 30.0 100.0 and 300.0 mg/mL respectively in equal volume.

Chapter 3: Material And Method 41

A-2 Aqueous fraction of A.marmelos

The doses and dilutions of aqueous fraction of A.marmelos (AMFMH2O) were prepared fresh on the day of experiments and infused into the heart at the concentration of 0.01, 0.1, 1.0, 10.0, 100.0 and 300.0 mg/mL respectively in equal volume.

A-3 Butanolic fraction of A.marmelos The doses and dilutions of butanolic fraction of A.marmelos (AMFMBut) were prepared fresh on the day of experiments and infused into the heart at the concentration of 0.01, 0.1, 0.3, 1.0, 3.0, 10.0, and 30.0 mg/mL respectively in equal volume.

A-4 Marmelosin isolated (ISD) and commercially available marmelosin (STD)

The dilutions of marmelosin isolated (ISD) from the fruit of A.marmelos and standard marmelosin (STD) were prepared fresh on the day of experiments and infused into the heart at the concentration of 0.0001, 0.01, 1.0, 100.0 and 10000.0 µM/mL respectively. The standard marmelosin was used to compare the effects of isolated one.

A-5 Digoxin

The dilutions of digoxin were freshly prepared on the day of experiments and infused into the heart at the concentration of 0.001, 0.01, 0.1, 1.0 and 10.0 µM/mL respectively.

A-6 Verapamil hydrochloride

The dilutions of verapamil hydrochloride were freshly prepared on the day of experiments and infused into the heart at the concentration of 0.01, 0.1, 1.0 and 10.0 µM/mL respectively.

3.4.2.2.2 At variable preloads

Chapter 3: Material And Method 42

To construct the Frank Starling curves, these experiments were designed to study the effects of variable preloads on cardiac performance in isolated rat working heart, whereas at constant afterload. The measurements were taken before treatment referred as control and after treatment with test sample referred as pretreated. The test sample was added in the perfusate reservoir to treat the heart continuously. In this preparation perfusion fluid enters the coronary system with variable hydrostatic pressure.

At first the heart was allowed to stabilize for 30 minutes in working mode with fixed preload

(15 cmH2O) and afterload (80 cm H2O). The baseline measurement of effect of variable preloads from 5-25 cm H2O in ascending order without any change in afterload were noted with time intervals of 2 minutes. The heart was allowed for further 5-10 minutes to stabilize again after the preload change, after that the prepared test dose in calculated volume of test material was added in the remaining perfusion fluid present in the main reservoir to get the required concentration to achieve the pretreatment status of the heart. The heart was allowed for 2-3 minutes to get the stable effect of test material prior to the measurement of effect of variable preloads. The different parameters were noted at the interval of 2 minutes. All the experiments ended in the beating state of the heart.

The parameters studied were coronary effluent (CE), aortic outflow (AoF), cardiac output

(CO), dP/dt(max), dP/dt(min), heart rate (HR), left ventricular pressure (LVP) and peak aortic systolic pressure (PASP). The other two parameters, i.e. coronary vascular resistance (CVR = Mean aortic pressure/coronary flow) and myocardial work (MW = Cardiac output x Peak aortic systolic pressure) were calculated.

B Concentration of crude extract, fractions and marmelosin used

B-1 Crude extract of A.marmelos

The concentrations of crude extract of A.marmelos (AMFM) used to pretreat the heart for evaluating the effects of variable preloads were 3.0, 30.0 and 100.0 mg/mL respectively.

Chapter 3: Material And Method 43

B-2 Aqueous fraction of A.marmelos

The concentrations of aqueous fraction of A.marmelos (AMFMH2O) used to pretreat the heart for evaluating the effects of variable preloads were 1.0, 10.0 and 30.0 mg/mL respectively.

B-3 Butanolic fraction of A.marmelos

The concentrations of butanolic fraction of A.marmelos (AMFMBut) used to pretreat the heart for evaluating the effects of variable preloads were 0.01, 0.1 and 1.0 mg/mL respectively.

B-4 Marmelosin isolated from A.marmelos

The concentrations of marmelosin isolated from dry fruit of A.marmelos used to pretreat the heart for evaluating the effects of variable preloads were 1 µM, 10 µM and 100 µM respectively.

B-5 Digoxin

The concentrations of digoxin used to pretreat the heart for evaluating the effects of variable preloads were 0.0001 µM, 0.01 µM and 1 µM respectively.

B-6 Verapamil hydrochloride

The concentrations of verapamil hydrochloride used to pretreat the heart for evaluating the effects of variable preloads were 0.01 µM, 0.1 µM and 1 µM respectively.

3.4.2.2.3 Calcium paradox experiments

It is well known that the reperfusion of isolated perfused hearts with calcium-containing solution after a short period of calcium-free perfusion results in irreversible cell damage. The calcium paradox has therefore been regarded as an important experimental model for studying the morphological, electrophysiological and biochemical basis of myocardial injury associated with calcium overload (Kojima., 2010). A series of experiments were undertaken to study the effect of crude extract of A.marmelos and Verapamil HCl during and after the

Chapter 3: Material And Method 44 development of calcium paradox (on the occurrence of the calcium paradox) in the isolated rat working heart at constant atrial filling pressure (preload, 15 cmH2O) and aortic pressure

(afterload, 80 cmH2O) which remained constant throughout the experiments. Male Wistar albino rat (250-350g) were used, food withdrawn 12 hours before the experiments but water was allowed ad libitum. Working heart apparatus and recording instruments were calibrated before experimentation. All the glass apparatus was exclusively cleaned off and carefully used during preparation of solutions and dilutions to avoid contaminations particularly with calcium. All procedures were performed in sterilized environments. In the set of these experiments three perfusion fluid reservoirs were used and connected to the atrial filling line by means of a three way stopcock allowing alternate use of the reservoirs.

Composition of Perfusion fluids:

1) Calcium-free KH (Krebs-Henseliet solution)

The composition of Calcium-free KH was (in mM: NaCl 118, KCl 4.7, MgSO₄.7H₂O 1.2, KH₂PO4 1.2, D-Glucose 11.1, NaHCO₃ 25.0), equilibrated with carbogen (95 % O2 and 5 % CO2) to provide pH 7.4. The Calcium chloride was replaced by a chelating agent EGTA (1.0 mM) instead of EDTA as it is a specific calcium chelating agent and does not chelate other minerals.

2) Calcium-containing KH (Krebs-Henseliet solution)

The composition of normal Calcium-containing KH solution was (in mM : NaCl 118, KCl 4.7, MgSO₄.7H₂O 1.2, CaCl₂ 1.8, KH₂PO4 1.2, D-Glucose 11.1, NaHCO₃ 25.0) equilibrated with carbogen (95 % O2 and 5 % CO2) to provide pH 7.4.

Perfusion time sequence:

After 30 minutes of stabilization period, during which the hearts were perfused with normal Krebs-Henseliet solution, the baseline (control) measurements were noted with the time duration of 2 minutes. The methods and perfusion time sequence used in these experiments are as follows:

Chapter 3: Material And Method 45

C To study the effects of crude extract of A.marmelos on calcium paradox

The three sets of experiment composed of three animals each were carried out toevaluate the effects of crude extract of A.marmelos on calcium paradox in the isolated rat working heart by using the following perfusion time sequence:

C-1 Perfusion of heart initiated with calcium-free Krebs-Henseliet solution for 10 minutes, followed by reperfusion with normal Krebs-Henseliet solution for 10 minutes. The measurement of parameters was noted at the intervals of 2 minutes. This produces calcium paradox. In this set of experiments the values are calculated with reference to Ca++-free perfusion.

C-2 Perfusion of heart initiated with calcium-free Krebs-Henseliet solution for 10 minutes, followed by reperfusion with Krebs-Henseliet solution added crude extract of A.marmelos at the concentration of 30.0 mg/mL for 10 minutes. The parameters were noted at the time intervals of 2 minutes. This showed the therapeutic effect of A.marmelos on calcium paradox. In this set of experiments the values are calculated with reference to Ca++-free perfusion.

C-3 Perfusion of heart initiated with calcium-free Krebs-Henseliet solution added crude extract of A.marmelos at the concentration of 30.0 mg/mL for 10 minutes, followed by reperfusion with normal Krebs-Henseliet solution having crude extract of A.marmelos at the concentration of 30.0 mg/mL for 10 minutes. The parameters were noted at the time intervals of 2 minutes. This showed the protective and therapeutic effects of A.marmelos on calcium paradox. In this set of experiments the values are calculated with reference to Ca++- free perfusion with crude extract of A.marmelos.

D To study the effects of Verapamil hydrochloride on calcium paradox

Another sets of same experiments composed of three animals each were also carried outto evaluate the effects of verapamil hydrochloride on calcium paradox in the isolated rat working heart by using the same perfusion time protocol as earlier;

D-1 Perfusion of heart initiated with calcium-free Krebs-Henseliet solution for 10

Chapter 3: Material And Method 46

minutes, followed by reperfusion with normal Krebs-Henseliet solution for 10 minutes. The measurement of parameters was noted at the intervals of 2 minutes. This produces the calcium paradox. In this set of experiments the data are calculated with reference to Ca++-free perfusion.

D-2 Perfusion of heart initiated with calcium-free Krebs-Henseliet solution for 10 minutes, followed by reperfusion with Krebs-Henseliet solution added verapamil HCl at the concentration of 1 µM for 10 minutes. The measurement of parameters was noted at the intervals of 2 minutes. This showed the therapeutic effect of verapamil HCl on calcium paradox. In this set of experiments the values are calculated with reference to Ca++-free perfusion.

D-3 Perfusion of heart initiated with calcium-free Krebs-Henseliet solution having verapamil HCl at the concentration of 1 µM for 10 minutes, followed by reperfusion with normal Krebs-Henseliet solution having verapamil HCl at the concentration of 1 µM for 10 minutes. The measurements of parameters were noted at the intervals of 2 minutes. This showed the protective and therapeutic effects of verapamil HCl on calcium paradox. In this set of experiments the data are calculated with reference to Ca++-free perfusion with verapamil HCl.

All the experiments began and ended in the spontaneously beating state of the heart. The parameters used to evaluate the effects of crude extract of A.marmelos and verapamil

HCl on calcium paradox are coronary effluent (CE), aortic outflow (AoF), dP/dt(max),

dP/dt(min), heart rate (HR) and left ventricular mean pressure (LVMP). The two parameters stroke volume (SV = cardiac output x heart rate) and rate pressure product (RPP = Peak aortic systolic pressure x heart rate) were calculated.

3.4.2.3 Isolated rat thoracic aorta

3.4.2.3.1 Preparation of thoracic aortic ring

The procedure of Furchgott and Zawadskai (1980) was followed with some modifications.

Chapter 3: Material And Method 47

Male Wistar rats (250-350 g,) were sacrificed by cervical dislocation and thoracic aorta was dissected out. Care was taken to avoid any damage to the endothelium. The isolated aorta was then transferred into carbogen aerated normal Krebs-Heseliet solution. It was cleaned of fats and adherent connective tissues and then made into rings of 3-4 mm wide. In some rings the endothelium was deliberately removed by gentle rubbing of the intimal surface with forceps (Xu et al., 2010). Both the ends of stainless steel hook attached to glass rod and the end of the open triangular wire holder were inserted through the lumen of the rings carefully and rings were suspended horizontally in the organ baths containing 10 mL normal Calcium- containing KH solution, the composition of which was (in mM: NaCl 118, KCl 4.7, MgSO₄.7H₂O

1.2, CaCl₂ 1.8, KH₂PO4 1.2, D-Glucose 11.1, NaHCO₃ 25.0) maintained at 37 °C and bubbled through with 95 % O2 and 5 % CO2 to provide pH 7.4. One hook was anchored to glass rod and the other was attached to an isotonic (Malis et al., 1991; Ko et al., 2010) force transducers (MLT 0202) coupled through Bridge Amplifier (Model: FE221) to the PowerLab data acquisition system (AD Instruments, Sydney, Australia). Each aortic ring was allowed to equilibrate for 60 minutes under a basal resting tension (preload) of 2.0 g. Before each experiment, the contractile function of the rings was tested thrice. In aortic rings the contraction was induced by P.E (1 µM) and KCl (80 mM) and rinsed several times with Krebs- Henseleit solution to attain their basal tension. The rings were then allowed to equilibrate further for 30 minutes. To confirm intact endothelium, each ring was contracted with PE (1 µM) and then exposed to ACh (10 µM); an endothelium-dependent vasodilator. Only those rings which exhibited more than 80 % relaxation of the PE contraction were selected for further studies (Xu et al., 2010). The absence of endothelium was confirmed by the lack of responses to ACh in PE-contracted rings. Rings exhibited ≤ 20 % relaxation of the PE contraction were included in studies. Ca++-free Krebs-Henseleit solution was prepared by replacing the CaCl2 with EGTA (0.1 mM). The data extracted from experimenst of five animals.

3.4.2.3.2 Effect on vascular tone

As described previously (Ghayur and Gilani., 2005), a series of experiments were conducted to assess the effect of test materials on PE and high K+-induced contractions. When the tension was at resting state or reached, a plateau induced by PE (1µM), test material was

Chapter 3: Material And Method 48 added in the organ bath in cumulative fashion (Taqvi et al., 2008; Roy et al., 1999; Jespersen., 2015). High K+ (80 mM) was also used to produce sustained contractions, allowing studying the effects mediated through voltage dependent calcium channels (VDCCs), the test material was then added cumulatively and relaxation was expressed as percent of the contractions induced by PE and high K+ (Godfraind et al., 1986; Qin et al., 2014).

3.4.2.3.3 Endothelium-dependent and endothelium-independent effects

To determine the underlying mechanism of test materials in intact endothelium rings pre- contracted with PE (1 µM), some rings were incubated with L-NAME, an inhibitor of nitric oxide synthase (10 µM) for 30 minutes before the addition of PE (1 µM). The test materials were added cumulatively on the sustained contractions induced by PE (1 µM) and the CRCs were reconstructed and compared (Taqvi et al., 2008; Jaffe., 1985; Vanhoutte., 1986; Tirapelli., 2006; Bruder-Nascimento et al., 2015).

3.4.2.3.4 Determination of Ca++ antagonistic activity

To confirm the calcium antagonistic activity of the test materials, the tissue was allowed to stabilize in normal Krebs-Henseleit solution which was then replaced with K+ normal Ca++-free Krebs-Henseleit solution containing EGTA (0.1 mM) for thirty minutes in order to remove Ca++ from the tissues. This solution was further replaced with a K+-rich and Ca++-free Krebs- Henseleit solution, having the following composition: (in mM: KCl60, MgSO₄.7H₂O 1.2, KH₂PO4 1.2, D-Glucose 11.1, NaHCO₃ 25.0 and EGTA, 0.1) bubbled through with carbogen (95 % O2 and 5 % CO2) to provide pH 7.4 and wash out by K+ normal Ca++-free Krebs-Henseleit solution. This process repeated twice and after incubation of tissue with K+-rich, Ca++free Krebs-Henseleit solution for 30 minutes, control concentration-response curves (CRCs) of Ca++ were obtained. When the control CRCs of Ca++was found superimposable usually after 2-3 cycles) the tissue was pretreated with the test material/extract for 60 minutes to test a possible calcium channel blockade CCB) effect. The CRCs of Ca++were developed in the presence of different concentrations of the test material in ascending order (Taqvi et al., 2008; Hamilton., 1986; Bruder-Nascimento et al., 2015).

Chapter 3: Material And Method 49

3.5 Statistical analysis

The data are expressed as mean ± standard error of the mean (± SEM). The median effective concentrations (EC50 values) were obtained by using Graph Pad Prism version 6.0 (http://www.graphpad.com). Data was analyzed by one way ANOVA followed by the Tukey- Kramer Multiple Comparisons Test, when significant difference was present, p <0.05 was considered statistically significant. Concentration response curves were analyzed by nonlinear regression using Graph PAD prism; graphing software (GraphPAD, San Diego, CA). In the Ca++-paradox experiments the graphs are kept in the original form as generated by the software.

50

CHAPTER 4 RESULT

Chapter 4: Result 51

TOXICITY STUDIES 4.1 Acute oral toxicity studies of crude extracts

4.1.1 Aegle marmelos Linn The crude extract of dried fruit of A.marmelos was screened for acute toxicity in Swiss albino mice irrespective of sex. The increasing concentrations of crude extract of A.marmelos were administered orally to the animals of the test groups. The crude extract did not show any sign of toxicity at the concentrations of 500, 1000, 2500, 5000 and 7500 mg/kg body weight respectively. There was no change in the behavioral and the physiological parameters of the animals during the observation period of 24 hours. No mortality of the animal among the test groups determined the safety of the test sample at the given doses so LD50 dose was not calculated. The collected data demonstrate that the crude extract of dried fruit of A.marmelos have a high margin of drug safety (Table 4.1.1)

4.1.2 Pyrus cydonia Linn The crude extract of seed of P.cydonia was screened for acute toxicity in Swiss albino mice irrespective of sex. The oral administration of crude extract in increasing concentrations of 250, 500, 1000, 2500 and 5000 mg/kg body weight did not produce any sign of untoward effects. Not a single animal showed any change in the behavioral and the physiological parameters of the animals during the observation period of 24 hours. No mortality was observed as well, so LD50 dose was not calculated. P.cydonia has a high margin of safety (Table 4.1.2).

LANGENDORFF’S RAT HEART EXPERIMENTS 4.2 Effect of crude extracts on Langendorff’s rat heart

4.2.1 Aegle marmelos.L

4.2.1.1 Left ventricular pressure (mm Hg) The crude extract of A.marmelos was administered in Langendorff’s rat heart at the concentrations of 1.0, 3.0, 10.0, 30.0, 100.0 and 300.0 mg/mL respectively. The extract

Chapter 4: Result 52 exhibited concentration-dependent increase in left ventricular pressure; at the concentrations (mg/mL) of 1.0 (103.06 ± 5.413), 3.0 (110.96 ± 5.198), 10.0 (114.12 ± 5.480), 30.0(122.93 ± 6.038), 100.0 (142.60 ± 6.118) and 300.0 (160.55 ± 13.903) respectively as compared to control measurements (99.667 ± 0.3434) (Fig. 4.2.1). The increase in left ventricular pressure was statistically significant at the concentrations (mg/mL) of 100 (p

<0.05) and 300 (p <0.001) respectively (Table 4.2). The EC50 value of crude extract ofA.marmelos was 60.53 mg/mL (95% CI, 17.46 to 209.9).

4.2.1.2 Systolic pressure (mm Hg) The crude extract of A.marmelos showed a concentration-dependent increase in the systolic pressure. It showed increase at the concentrations (mg/mL) of 1.0 (134.91 ± 26.178), 3.0 (141.02 ± 24.367), 10.0 (156.56 ± 14.087), 30.0 (178.12 ± 12.480), 100.0 (216.49 ± 16.010) and 300.0 (256.03 ± 27.752) respectively as compared to the control measurements (99.667 ± 0.343) (Fig. 4.2.2). The increase in systolic pressure was statistically significant at the higher concentrations (mg/mL) of 100.0 (p <0.05) and 300.0 (p <0.001) respectively (Table 4.2). The

EC50 value for crude extract of A.marmelos was 55.39 mg/mL (95 % CI 11.88 to 258.4).

4.2.2 Pyrus cydonia. L

4.2.2.1 Left ventricular pressure (mm Hg) The crude extract of P.cydonia was administered in Langendorff’s rat heart at the concentrations of 1.0, 3.0, 10.0, 30.0, 100.0 and 300.0 mg/mL respectively. The crude extract produced a steady increase in the left ventricular pressure at the concentrations (mg/mL) of 1.0 (109.39 ± 3.948), 3.0 (109.21 ± 4.274), 10.0 (108.77 ± 4.815), 30.0 (109.45 ± 6.057), 100.0 (110.56 ± 8.654) and 300.0 (109.28 ± 10.780) respectively as compared to control measurements (99.741 ± 0.2038) (Fig. 4.2.1). The increase in left ventricular pressure was statistically insignificant at all concentrations (Table 4.2). The EC50 value of the crude extract was 22.87 mg/mL (95% CI 0.0 to +infinity).

4.2.2.2 Systolic pressure (mm Hg) The crude extracts of P.cydonia increased the systolic pressure, concentration dependently. It increased the systolic pressure at the concentrations (mg/mL) of 1.0 (120.18 ± 8.095), 3.0

Chapter 4: Result 53

(123.20 ± 10.059), 10.0 (129.86 ± 10.330), 30.0 (135.48 ± 14.676), 100.0 (148.46 ± 18.206) and 300.0 (171.77 ± 27.307) respectively as compared to control measurements (100.00 ± 0.3114) (Fig. 4.2.2). The increase in systolic pressure was statistically insignificant at all concentrations (Table 4.2). The EC50 value of crude extract of P.cydonia was 93.08 mg/mL (95% CI 4.559 to 1901).

The effects of crude extract of seeds of Pyrus cydonia on pressure parameters in Langendorff’s rat heart preparations were statistically insignificant. Therefore, further work on P.cydonia was abandoned.

WORKING RAT HEART EXPERIMENTS

A. CRUDE EXTRACT OF AEGLE MARMELOS (Am.Cr)

4.3 Crude extract of Aegle marmelos (Am.Cr) on working rat heart 4.3.1 At fixed physiological preload and afterload

The crude extract of A.marmelos was administered at the concentrations of 1.0, 3.0, 10.0, 30.0, 100.0 and 300.0mg/mL respectively. The table (4.3.1) indicated the effects of crude extract on different parameters in rat working hearts.

4.3.1.1 Effect on coronary effluent, aortic outflow and cardiac output The results are shown in (Fig 4.3.1.1). The crude extract decreased the coronary effluent concentration-dependently; at the concentrations (mg/mL) of 1.0 (94.362 ± 3.217), 3.0 (90.524 ± 3.037), 10.0 (83.997 ± 3.659), 30.0 (79.622 ± 3.314), 100.0 (74.242 ± 4.057) and 300.0 (67.414 ± 6.516) respectively as compared to control (99.741 ± 0.2038). The crude extract at the concentrations (mg/mL) of 30.0 (p <0.05), 100.0 (p <0.01) and 300.0 (p <0.001) exhibited statistically significant effect on coronary effluent and showing EC50 value of 20.34 mg/mL (95% CI, 4.382 to 94.38).

The crude extract increased the aortic outflow continuously in a concentration-dependent manner. The crude extract at the concentrations (mg/mL) of 1.0 (102.09 ± 3.491), 3.0 (102.59 ± 3.930), 10.0 (107.55 ± 5.364), 30.0 (108.53 ± 5.324), 100.0 (109.81 ± 5.235) and 300.0

Chapter 4: Result 54

(113.88 ± 6.120) respectively increased the aortic outflow as compared to control (99.741 ±

0.203). The effect was statistically non-significant with the EC50 value of 14.47 mg/mL (95% CI, 0.1877 to 1116).

The crude extract did not affect the cardiac output, which remained almost same throughout the experiments. The effect of crude extract on cardiac output was at the concentrations (mg/mL) of 1.0 (99.335 ± 2.907), 3.0 (99.948 ± 2.947), 10.0 (99.343 ± 3.147), 30.0 (99.742 ± 3.143), 100.0 (99.927 ± 2.905) and 300.0 (99.909 ± 2.831) as compared to control (99.741 ±

0.203). The effect was statistically non-significant. The EC50 value for cardiac output is 38.94 mg/mL (95 % CI, 0 to 6.397e+028).

4.3.1.2 Effect on dP/dt(max) and dP/dt(min)

The results are shown in (Fig. 4.3.1.2). The dP/dt(max) was increased concentration- dependently till the concentration of 30.0mg/mL, then decreased. The values were at the concentrations (mg/mL) of 1.0 (101.48 ± 2.318), 3.0 (104.55 ± 3.228), 10.0 (107.64 ± 3.878), 30.0 (110.74 ± 3.694), 100.0 (104.55 ± 3.288) and 300.0 (99.584 ± 5.708) as compared to control (99.741 ± 0.203). The effect of crude extract on dP/dt(max) was statistically insignificant. The software could not converge the data to get EC50 value.

The dP/dt(min)was increased, at the concentrations (mg/mL) of 1.0 (100.47 ± 1.442), 3.0 (101.16 ± 1.361), 10.0 (101.71 ± 1.448), 30.0 (102.29 ± 1.867), 100.0 (102.96 ± 1.457) and

300.0 (102.98 ± 2.277) respectively as compared to control (99.741 ± 0.203) with EC50 value of 8.785 mg/mL (95 % CI, 0.01230 to 6275). The value was statistically non-significant.

4.3.1.3 Effect on systolic and diastolic pressure and heart rate The results of crude extract on systolic and diastolic pressure and heart rate are shown in (Fig. 4.3.1.3). The systolic pressure remained steady at all concentrations; at the concentrations (mg/mL) of 1.0 (98.811 ± 3.059), 3.0 (98.981 ± 2.833), 10.0 (99.010 ± 3.042), 30.0 (99.349 ± 2.905), 100.0 (100.04 ± 2.921) and 300.0 (100.52 ± 3.085) respectively as compared to control (99.741 ± 0.203). The effect was statistically non-significant. The EC50 value was 60.08 mg/mL (95 % CI, 1.219e-005 to 296013047).

Chapter 4: Result 55

The diastolic pressure increased till the concentration of 100.0 mg then decreased; at the concentrations (mg/mL) of 1.0 (98.719 ± 3.961), 3.0 (100.36 ± 3.973), 10.0 (102.00 ± 3.697), 30.0 (102.44 ± 3.988), 100.0 (103.12 ± 3.765) and 300.0 (100.31 ± 3.842) respectively as compared to control (99.741 ± 0.203). The effect was statistically non-significant. The EC50 value was 0.3537 mg/mL (95 % CI, 0 to 1.246e+017).

The heart rate decreased initially and then remained steady at all concentrations; at the concentrations (mg/mL) of 1.0 (98.316 ± 3.758), 3.0 (96.583 ± 3.499), 10.0 (98.044 ± 4.003), 30.0 (97.279 ± 3.829), 100.0 (97.812 ± 3.571) and 300.0 (97.702 ± 3.751) respectively as compared to control (99.741 ± 0.203). The effect was statistically non-significant. The EC50 value was 127.6 mg/mL (95 % CI, 0 to 1.55e+105).

4.3.1.4 Effect on peak aortic systolic pressure and end diastolic pressure The results are shown in (Fig. 4.3.1.4). A.marmelos decreased the peak aortic systolic pressure till the concentration of 100.0 mg/mL, then showed slight increase, but remained below the control level; at the concentrations (mg/mL) of 1.0 (96.178 ± 4.670), 3.0 (94.351 ± 4.203), 10.0 (93.564 ± 3.985), 30.0 (96.161 ± 1.702), 100.0 (97.921 ± 3.033) and 300.0 (97.178 ± 1.651) respectively as compared to control (99.741 ± 0.203). The effect was statistically non-significant. The EC50 value was 62.15 mg/mL (95 % CI, 0.0008046 to 4801276).

The end diastolic pressure was increased till the concentration of 100.0 mg/mL, then decreased; at the concentrations (mg/mL) of 1.0 (99.596 ± 2.390), 3.0 (100.15 ± 1.933), 10.0 (102.08 ± 1.824), 30.0 (103.59 ± 2.290), 100.0 (103.82 ± 2.307) and 300.0 (99.749 ± 4.814) respectively as compared to control (99.741 ± 0.203). The effect was statistically non- significant. The EC50 value was 1.286 mg/mL (95 % CI, 5.334e-007 to 3099051).

4.3.1.5 Effect on ejection fraction and stroke volume The results are shown in (Fig. 4.3.1.5). The ejection fraction was increased; at the concentrations (mg/mL) of 1.0 (105.26 ± 6.261), 3.0 (104.66 ± 6.477), 10.0 (105.09 ± 6.177), 30.0 (105.97 ± 8.245), 100.0 (106.59 ± 7.008) and 300.0 (109.21 ± 11.428) respectively against control (99.741 ± 0.203). The effect was statistically non-significant. The EC50 value was 182.5 mg/mL (95 % CI, 5.734e-007 to 58087328356).

Chapter 4: Result 56

The stroke volume was decreased initially followed by anincrease. The values are at the concentrations (mg/mL) of 1.0 (99.103 ± 2.624), 3.0 (99.328 ± 2.573), 10.0 (99.616 ± 2.469), 30.0 (101.57 ± 1.804), 100.0 (103.08 ± 1.465) and 300.0 (101.53 ± 2.966) respectively as compared to control measurement (99.741 ± 0.203) with EC50 values of 12.82 mg/mL (95 % CI, 0.0232 to 7081). The effect was statistically non-significant.

4.3.1.6 Effect on rate pressure product and cardiac power The results of calculated parameters are shown in (Fig. 4.3.1.6). The rate pressure product showed decrease-increase pattern; at the concentrations (mg/mL) of 1.0 (96.035 ± 5.074), 3.0 (95.030 ± 4.219), 10.0 (96.772 ± 6.748), 30.0 (100.10 ± 4.363), 100.0 (98.429 ± 2.881) and 300.0 (95.951 ± 2.753) respectively as compared to control (99.741 ± 0.203). The effect was statistically non-significant. The EC50 value was 3.915 mg/mL (95 % CI, 1.291e-008 to 1187837839).

The cardiac power showed steady effects except a slight increase at the concentrations of 30.0, 100.0 and 300.0 mg/mL. The values at the concentrations (mg/mL) of 1.0 (98.957 ± 2.998), 3.0 (99.395 ± 3.213), 10.0 (99.998 ± 2.952), 30.0 (100.13 ± 3.152), 100.0 (102.24 ± 3.087) and 300.0 (102.33 ± 3.375) respectively as compared to control (99.74 ± 0.203) were statistically non-significant. The EC50 value of cardiac power was 34.54 mg/mL (95 % CI, 0.01135 to 105064).

4.3.2 At variable preloads

The effect of various preloads on control and pre-treated isolated rat working heart by different concentrations of crude extract of A.marmelos was studied to construct the Frank- Starling curve, while keeping the afterload fixed at the physiological level.

4.3.2.1 Pre-treatment at the concentration of 3.0 mg/mL

The table (4.3.2.1) indicated the effects of changing preload in control and pre-treated rat hearts at different parameters.

Effect on coronary effluent

Chapter 4: Result 57

At 5 cmH2O (control 107.519, pretreated 54.2727), 10 cmH2O (control 101.203, pretreated

36.7656), 15 cmH2O (control 104.938, pretreated 32.5812), 20 cmH2O (control 99.3966, pretreated 30.5287) and 25 cmH2O (control 99.8731, pretreated 29.2432) respectively (Fig. 4.3.2.1.1)

Effect on aortic outflow

At 5 cmH2O (control 110.9, pretreated 124.324), 10 cmH2O (control 104.655, pretreated

128.912), 15 cmH2O (control 106.575, pretreated 131.655), 20 cmH2O (control 103.399, pretreated 137.382) and 25 cmH2O (control 101.403, pretreated 137.566) (Fig. 4.3.2.1.2)

Effect on cardiac output

At 5 cmH2O (control 111.397, pretreated 103.048), 10 cmH2O (control 103.982, pretreated

100.186), 15 cmH2O (control 105.818, pretreated 100.858), 20 cmH2O (control 102.029, pretreated 102.603) and 25 cmH2O (control 100.473, pretreated 102.315) (Fig. 4.3.2.1.3)

Effect on dP/dt(max)

At 5 cmH2O (control 98.1089, pretreated 70.9175), 10 cmH2O (control 98.3764, pretreated

71.3824), 15 cmH2O (control 100.56, pretreated 71.8428), 20 cmH2O (control 100.835, pretreated 71.5276) and 25 cmH2O (control 100.31, pretreated 71.7635) (Fig. 4.3.2.1.4)

Effect on dP/dt(min)

At 5 cmH2O (control 99.4329, pretreated 85.9233), 10 cmH2O (control 98.7107, pretreated

86.8023), 15 cmH2O (control 100.695, pretreated 87.9524), 20 cmH2O (control 102.11, pretreated 87.9485) and 25 cmH2O (control 102.988, pretreated 88.4529) (Fig. 4.3.2.1.5)

Effect on heart rate

At 5 cmH2O (control 99.7575, pretreated 50.6507), 10 cmH2O (control 100.105, pretreated

50.6476), 15 cmH2O (control 100.911, pretreated 50.6476), 20 cmH2O (control 100.957, pretreated 50.6473) and 25 cmH2O (control 106.054, pretreated 50.6457) (Fig.4.3.2.1.6)

Effect on left ventricular pressure

At 5 cmH2O (control 99.2514, pretreated 102.077), 10 cmH2O (control 98.781, pretreated

Chapter 4: Result 58

102.995), 15 cmH2O (control 98.9481, pretreated 103.725), 20 cmH2O (control 98.8579, pretreated 102.957) and 25 cmH2O (control 98.5915, pretreated 103.718) (Fig. 4.3.2.1.7)

Effect on peak aortic systolic pressure

At 5 cmH2O (control 99.2392, pretreated 90.9298), 10 cmH2O (control 98.0634, pretreated

91.1748), 15 cmH2O (control 99.4897, pretreated 91.6928), 20 cmH2O (control 97.8702, pretreated 91.3356) and 25 cmH2O (control 97.8068, pretreated 91.3045) (Fig. 4.3.2.1.8)

Effect on coronary vascular resistance

At 5 cmH2O (control 93.3561, pretreated 177.389), 10 cmH2O control 97.0304, pretreated

264.263), 15 cmH2O (control 94.5507, pretreated 306.527), 20 cmH2O (control 97.1238, pretreated 340.221) and 25 cmH2O (control 97.9305, pretreated 378.029) (Fig. 4.3.2.1.9)

Effect on myocardial work

At 5 cmH2O (control 111.057, pretreated 93.778), 10 cmH2O (control 103.104, pretreated

92.6191), 15 cmH2O (control 105.076, pretreated 92.4144), 20 cmH2O (control 102.577, pretreated 94.4637) and 25 cmH2O (control 99.8077, pretreated 95.3179) (Fig. 4.3.2.1.10)

4.3.2.2 Pretreatment at the concentration of 30.0 mg/mL The table (4.3.2.2) indicated the effects of changing preload in control and pre-treated hearts at different parameters.

Effect on coronary effluent

At 5 cmH2O (control 105.678, pretreated 32.8049), 10 cmH2O (control 100.963, pretreated

33.3737), 15 cmH2O (control 95.4399, pretreated 31.6844), 20 cmH2O (control 96.119, pretreated 32.1996) and 25 cmH2O (control 95.622, pretreated 29.2447) (Fig. 4.3.2.2.1)

Effect on aortic outflow

At 5 cmH2O (control 106.782, pretreated 132.009), 10 cmH2O (control 103.684, pretreated

133.208), 15 cmH2O (control 106.923, pretreated 135.108), 20 cmH2O (control 107.276, pretreated 138.742) and 25 cmH2O (control 106.879, pretreated 140.273) (Fig. 4.3.2.2.2)

Effect on cardiac output

Chapter 4: Result 59

At 5 cmH2O (control 104.576, pretreated 101.845), 10 cmH2O (control 101.532, pretreated

101.847), 15 cmH2O (control 100.835, pretreated 102.717), 20 cmH2O (control 101.119, pretreated 102.266) and 25 cmH2O (control 100.862, pretreated 102.157) (Fig. 4.3.2.2.3)

Effect on dP/dt(max)

At 5 cmH2O (control 98.1565, pretreated 83.0488), 10 cmH2O (control 99.5969, pretreated

82.7323), 15 cmH2O (control 97.9936, pretreated 82.9136), 20 cmH2O (control 94.4049, pretreated 83.6462) and 25 cmH2O (control 96.5641, pretreated 83.5362) (Fig. 4.3.2.2.4)

Effect on dP/dt(min)

At 5 cmH2O (control 99.4329, pretreated 85.9233), 10 cmH2O (control 99.1107, pretreated

86.8023), 15 cmH2O (control 102.095, pretreated 87.9524), 20 cmH2O (control 103.71, pretreated 87.9485) and 25 cmH2O (control 102.988, pretreated 88.4529) (Fig. 4.3.2.2.5)

Effect on heart rate

At 5 cmH2O (control 99.5235, pretreated 65.5641), 10 cmH2O (control 101.516, pretreated

65.5637), 15 cmH2O (control 104.763, pretreated 65.5639), 20 cmH2O (control 108.337, pretreated 65.5632) and 25 cmH2O (control 106.086, pretreated 65.6856) (Fig. 4.3.2.2.6)

Effect on left ventricular pressure

At 5 cmH2O (control 99.4361, pretreated 104.373), 10 cmH2O (control 99.2319, pretreated

104.606), 15 cmH2O (control 99.7267, pretreated 104.673), 20 cmH2O (control 99.2839, pretreated 104.808) and 25 cmH2O (control 99.5063, pretreated 105.00) (Fig. 4.3.2.2.7)

Effect on peak systolic aortic pressure

At 5 cmH2O (control 99.5916, pretreated 91.158), 10 cmH2O (control 99.8737, pretreated

91.1333), 15 cmH2O (control 99.3108, pretreated 91.6285), 20 cmH2O (control 102.087, pretreated 91.5731) and 25 cmH2O (control 99.2782, pretreated 91.9219) (Fig. 4.3.2.2.8)

Effect on coronary vascular resistance

At 5 cmH2O (control 125.673, pretreated 767.02), 10 cmH2O (control 128.85, pretreated

735.798), 15 cmH2O (control 131.87, pretreated 804.087), 20 cmH2O (control 130.366,

Chapter 4: Result 60

pretreated 836.225) and 25 cmH2O (control 133.298, pretreated 843.901) (Fig. 4.3.2.2.9)

Effect on myocardial work

At 5 cmH2O (control 104.09, pretreated 93.5263), 10 cmH2O (control 102.21, pretreated

92.0183), 15 cmH2O (control 100.108, pretreated 92.8369), 20 cmH2O (control 102.804, pretreated 94.258) and 25 cmH2O (control 99.9641, pretreated 94.3082) (Fig. 4.3.2.2.10)

4.3.2.3 Pretreatment at the concentration of 100.0 mg/mL The table (4.3.2.3) indicated the effects of changing preload in control and pre-treated rat hearts at different parameters.

Effect on coronary effluent

At 5 cmH2O (control 103.718, pretreated 17.7979), 10 cmH2O (control 105.219, pretreated

15.5602), 15 cmH2O (control 104.373, pretreated 11.5144), 20 cmH2O (control 101.021, pretreated 10.5297) and 25 cmH2O (control 98.7582, pretreated 9.06821) (Fig. 4.3.2.3.1)

Effect on aortic outflow

At 5 cmH2O (control 99.8944, pretreated 129.054), 10 cmH2O (control 102.053, pretreated

130.658), 15 cmH2O (control 101.759, pretreated 125.844), 20 cmH2O (control 102.373, pretreated 128.841) and 25 cmH2O (control 100.666, pretreated 131.796) (Fig. 4.3.2.3.2)

Effect on cardiac output

At 5 cmH2O (control 99.3739, pretreated 102.354), 10 cmH2O (control 102.87, pretreated

102.86), 15 cmH2O (control 102.293, pretreated 98.1574), 20 cmH2O (control 102.579, pretreated 100.822) and 25 cmH2O (control 100.765, pretreated 102.101) (Fig. 4.3.2.3.3)

Effect on dP/dt(max)

At 5 cmH2O (control 97.9139, pretreated 66.0797), 10 cmH2O (control 97.8028, pretreated

67.0018), 15 cmH2O (control 96.4172, pretreated 67.5009), 20 cmH2O (control 96.0737, pretreated 67.9416) and 25 cmH2O (control 94.7185, pretreated 67.5148) (Fig. 4.3.2.3.4)

Effect on dP/dt(min)

At 5 cmH2O (control 98.148, pretreated 107.217), 10 cmH2O (control 97.8253, pretreated

Chapter 4: Result 61

109.91), 15 cmH2O (control 97.4122, pretreated 110.614), 20 cmH2O (control 96.6304, pretreated 111.378) and 25 cmH2O (control 96.3684, pretreated 111.451) (Fig. 4.3.2.3.5)

Effect on heart rate

At 5 cmH2O (control 97.5399, pretreated 54.7865), 10 cmH2O (control 98.136, pretreated

54.926), 15 cmH2O (control 99.6932, pretreated 53.3102), 20 cmH2O (control 100.056, pretreated 53.4079) and 25 cmH2O (control 103.016, pretreated 53.28) (Fig. 4.3.2.3.6)

Effect on left ventricular pressure

At 5 cmH2O (control 100.458, pretreated 112.507), 10 cmH2O (control 101.087, pretreated

113.71), 15 cmH2O (control 103.039, pretreated 113.615), 20 cmH2O (control 103.853, pretreated 113.51) and 25 cmH2O (control 104.237, pretreated 113.628) (Fig. 4.3.2.3.7)

Effect on peak systolic aortic pressure

At 5 cmH2O (control 102.794, pretreated 98.7733), 10 cmH2O (control 102.868, pretreated

102.868), 15 cmH2O (control 99.4486, pretreated 99.2596), 20 cmH2O (control 99.4061, pretreated 99.162) and 25 cmH2O (control 100.761, pretreated 99.3006) (Fig. 4.3.2.3.8)

Effect on coronary vascular resistance

At 5 cmH2O (control 210.464, pretreated 412.63), 10 cmH2O (control 215.461, pretreated

463.139), 15 cmH2O (control 215.146, pretreated 933.097), 20 cmH2O (control 219.512, pretreated 1037.3) and 25 cmH2O (control 222.162, pretreated 1370.64) (Fig. 4.3.2.3.9)

Effect on myocardial work

At 5 cmH2O (control 101.825, pretreated 100.736), 10 cmH2O (control 104.412, pretreated

99.8856), 15 cmH2O (control 101.026, pretreated 97.9949), 20 cmH2O (control 100.313, pretreated 99.7967) and 25 cmH2O (control 101.699, pretreated 101.487) (Fig. 4.3.2.3.10)

4.3.3 Calcium paradox experiments These experiments were designed to study the effects of Ca++-paradox un-treated and pre- treated with crude extract of Am.Cr at the concentration of 30 mg/mL on different parameters of cardiac contractile functions at three series like C-1, C-2 and C-3.

Chapter 4: Result 62

4.3.3.1 Effects on coronary effluent The table (4.3.3.1) indicated the effects on coronary effluent (mL/min) in experimental series C-1, C-2 and C-3 respectively. In series C-1: hearts when reperfused by normal KH solution after exposure to Ca++-free KH solution showed decrease in coronary effluent (mL/min) by 21.065 % initially and at the end of 10 minutes by 25.759 % maximally as compared to the measurements during the exposure to Ca++-free medium respectively. In series C-2: hearts when reperfused by a normal KH solution containing a crude extract after exposure to Ca++- free KH solution the coronary effluent (mL/min) was decreased by 81.018 % initially and at the end of 10 minutes by 94.765 % maximally as compared to the measurements during the exposure to Ca++-free medium respectively. In series C-3: hearts when reperfused by a normal KH solution containing a crude extract after exposure to Ca++-free KH solution containing the crude extract showed decrease in coronary effluent (mL/min) by 34.444 % initially and at the end of 10 minutes by 67.222 % maximally as compared to the measurements during the exposure to Ca++-free medium containing crude extract of Am.Cr respectively. Graphically the results are presented in (Fig. 4.3.3.1).

4.3.3.2 Effects on aortic outflow The table (4.3.3.2) indicated the effects on aortic outflow (mL/min) in experimental series C- 1, C-2 and C-3 respectively. In series C-1: hearts when reperfused by normal KH solution after exposure to Ca++-free KH solution the aortic outflow (mL/min) was increased by 5.996 % initially and at the end of 10 minutes by 6.618 % maximally as compared to the measurements during the exposure to Ca++-free medium respectively. In series C-2: hearts when reperfused by a normal KH solution containing a crude extract after exposure to Ca++- free KH solution the aortic outflow (mL/min) was increased by 20.112 % initially and at the end of 10 minutes by 22.991 % maximally as compared to the measurements during the exposure to Ca++-free medium respectively. In series C-3: hearts when reperfused by a normal KH solution containing a crude extract after exposure to Ca++-free KH solution containing the crude extract the aortic outflow (mL/min) was increased by 0.888 % initially and at the end of 10 minutes by 3.490 % maximally as compared to the measurements during the exposure to Ca++-free medium containing crude extract of Am.Cr respectively (Fig.

Chapter 4: Result 63

4.3.3.2).

4.3.3.3 Effects on dP/dt(max)

The table (4.3.3.3) indicated the effects on dP/dt(max) (mm Hg/s) in experimental series C-1, C- 2 and C-3 respectively. In series C-1: hearts when reperfused by normal KH solution after ++ exposure to Ca -free KH solution the dP/dt(max) (mm Hg/s) was increased by 0.415 % initially and at the end of 10 minutes by 9.208 % maximally as compared to the measurements during the exposure to Ca++-free medium respectively. In series C-2: hearts when reperfused by a normal KH solution containing a crude extract after exposure to Ca++-free KH solution the dP/dt(max) (mm Hg/s) was increased by 10.056 % initially and at the end of 10 minutes by 15.695 % maximally as compared to the measurements during the exposure to Ca++-free medium respectively. In series C-3: hearts when reperfused by a normal KH solution containing a crude extract after exposure to Ca++-free KH solution containing the crude extract the dP/dt(max) (mm Hg/s) was decreased for 2 minutes by 1.402 % initially followed by increase by 0.052 % and at the end of 10 minutes by 4.035 % maximally as compared to the measurements during the exposure to Ca++-free medium containing crude extract of Am.Cr respectively (Fig. 4.3.3.3).

4.3.3.4 Effects on dP/dt(min)

The table (4.3.3.4) indicated the effects on dP/dt(min) (mm Hg/s) in experimental series C-1, C- 2 and C-3 respectively. In series C-1: hearts when reperfused by normal KH solution after ++ exposure to Ca -free KH solution showed decrease in dP/dt(min) (mm Hg/s) initially up to 2 minutes by 1.393 %, followed by increase by 0.389 % initially and at the end of 10 minutes increased by 3.088 % maximally as compared to the measurements during the exposure to Ca++-free medium respectively. In series C-2: hearts when reperfused by normal KH solution ++ containing crude extract after exposure to Ca -free KH solution the dP/dt(min) (mm Hg/s) was increased by 6.333 % initially and at the end of 10 minutes by 11.649 % maximally as compared to the measurements during the exposure to Ca++-free medium respectively. In series C-3: hearts when reperfused by normal KH solution containing crude extract after ++ exposure to Ca -free KH solution containing the crude extract the dP/dt(min) (mm Hg/s) was

Chapter 4: Result 64 decreased by 1.572 % initially which continued up to the end of 6 minutes by 1.572 % maximally, followed by an initial increase by 0.642 % which continued at the end of 10 minutes by 1.235 % maximally as compared to the measurements during the exposure to Ca++-free medium containing crude extract of Am.Cr respectively (Fig. 4.3.3.4).

4.3.3.5 Effects on heart rate

The table (4.3.3.5) indicated the effects on heart rate (BPM) in experimental series C-1, C-2 and C-3 respectively. In series C-1: hearts when reperfused by normal KH solution after exposure to Ca++-free KH solution the heart rate (BPM) was decreased by 1.325 % initially and at the end of 10 minutes by 1.322 % maximally as compared to the measurements during the exposure to Ca++-free medium respectively. In series C-2: hearts when reperfused by normal KH solution containing crude extract after exposure to Ca++-free KH solution the heart rate (BPM) was decreased by 0.654 % initially and at the end of 10 minutes by 0.671 % maximally as compared to the measurements during the exposure to Ca++-free medium respectively. In series C-3: hearts when reperfused by a normal KH solution containing a crude extract after exposure to Ca++-free KH solution containing the crude extract the heart rate (BPM) was decreased by 1.326 % initially and remained almost same at the end of 10 minutes by 1.321 % as compared to the measurements during the exposure to Ca++-free medium containing crude extract of Am.Cr respectively (Fig. 4.3.3.5).

4.3.3.6 Effects on left ventricular mean pressure The table (4.3.3.6) indicated the effects on left ventricular mean pressure (mm Hg) in experimental series C-1, C-2 and C-3 respectively. In series C-1: when hearts were reperfused by normal KH solution after exposure to Ca++-free KH solution the left ventricular mean pressure (mm Hg) was increased by 1.552 % initially and at the end of 10 minutes by 2.084 % maximally as compared to the measurements during the exposure to Ca++-free medium respectively. In series C-2: hearts when reperfused by a normal KH solution containing a crude extract after exposure to Ca++-free KH solution the left ventricular mean pressure (mm Hg) was increased by 4.279 % initially and at the end of 10 minutes by 4.996 % maximally as compared to the measurements during the exposure to Ca++-free medium respectively. In

Chapter 4: Result 65 series C-3: hearts when reperfused by a normal KH solution containing a crude extract after exposure to Ca++-free KH solution containing the crude extract the left ventricular mean pressure (mm Hg) was decreased by 0.421 % initially and at the end of 10 minutes by 0.120 % maximally as compared to the measurements during the exposure to Ca++-free medium containing crude extract of Am.Cr respectively (Fig. 4.3.3.6).

4.3.3.7 Effects on stroke volume The table (4.3.3.7) indicated the effects on stroke volume (mL/beat) in experimental series C-1, C-2 and C-3 respectively. In series C-1: when hearts were reperfused by normal KH solution after exposure to Ca++-free KH solution showed anincrease in stroke volume (mL/beat) by 0.271 % initially and at the end of 10 minutes by 0.458 % maximally as compared to the measurements during the exposure to Ca++-free medium respectively. In series C-2: hearts when reperfused by normal KH solution containing crude extract after exposure to Ca++-free KH solution the stroke volume (mL/beat) was decreased by 0.794 % initially and at the end of 10 minutes by 1.266 % maximally as compared to the measurements during the exposure to Ca++-free medium respectively. In series C-3: hearts when reperfused by normal KH solution containing crude extract after exposure to Ca++-free KH solution containing the crude extract, the stroke volume (mL/beat) was increased by 0.134 % initially and at the end of 10 minutes by 1.242 % maximally as compared to the measurements during the exposure to Ca++-free medium containing crude extract of Am.Cr respectively (Fig. 4.3.3.7).

4.3.3.8 Effects on rate pressure product The table (4.3.3.8) indicated the effects on rate pressure product (mL/beat) in experimental series C-1, C-2 and C-3 respectively. In series C-1: hearts when reperfused by normal KH solution after exposure to Ca++-free KH solution the rate pressure product (mL/beat) was increased by 1.175 % initially and at the end of 10 minutes by 1.567 % maximally as compared to the measurements during the exposure to Ca++-free medium respectively. In series C-2: hearts when reperfused by a normal KH solution containing a crude extract after exposure to Ca++-free KH solution the rate pressure product (mm Hg/min) was increased by

Chapter 4: Result 66

3.615 % initially and at the end of 10 minutes by 4.896 % maximally as compared to the measurements during the exposure to Ca++-free medium respectively. In series C-3: hearts when reperfused by a normal KH solution containing a crude extract after exposure to Ca++- free KH solution containing the crude extract the rate pressure product (mm Hg/min) was increased by 0.035 % initially and at the end of 10 minutes 0.319 % maximally as compared to the measurements during the exposure to Ca++-free medium containing crude extract of Am.Cr respectively (Fig. 4.3.3.8).

EXPERIMENTS ON ISOLATED RAT AORTA

4.3.4 Effect of crude extract of A.marmelos

4.3.4.1 At baseline tension In this set of experiments, the crude extract of A.marmelos was tested on resting basal tension at the concentrations of 0.01, 0.1, 1.0, 3.0, 5.0 and 10.0 mg/mL respectively. The cumulative addition of crude extract did not show either the stimulating or vasoconstrictor + effect up to the concentration of 10.0 mg/mL with EC50 value of 6.590 e 006 mg/mL (95 % CI, very wide) (Fig. 4.3.4.1) (Table. 4.3.4.1). None of the value was statistically significant.

4.3.4.2 On phenylephrine pre-contracted rat aorta In endothelial intact aortic rings which were pre-contracted by PE (1µM), the cumulative addition of the crude extract of A.marmelos caused inhibition of PE-induced sustained contraction in a concentration-dependent manner. A.marmelos caused inhibition of contraction at the concentrations (mg/mL) of 0.01 (97.315 ± 3.799), 0.1 (96.738 ± 3.530), 1.0 (95.123 ± 3.976), 3.0 (91.433 ± 4.188), 5.0 (73.023 ± 7.024) and 10.0 (37.707 ± 5.984) respectively as compared to control (100.00 ± 0.311). The statistically significant effects were observed at the concentrations (mg/mL) of 5.0 (p <0.01) and 10.0 (p <0.001). The respective

EC50 value of crude extract in the endothelium-intact aortic ring was ~11258 mg/mL (95 % CI, very wide) (Fig. 4.3.4.1) (Table. 4.3.4.1).

Chapter 4: Result 67

In endothelium-denuded and PE pre-contracted aortic rings the crude extract of A.marmelos caused inhibition of contraction at the concentrations (mg/mL) of 0.01 (100.71 ± 2.508), 0.1 (98.747 ± 3.328), 1.0 (95.080 ± 4.804), 3.0 (86.653 ± 6.730), 5.0 (57.202 ± 11.092) and 10.0 (33.238 ± 7.355) respectively as compared to control (100.00 ± 0.311). The statistically significant effects were observed at the concentrations (mg/mL) of 5.0 (p <0.001) and 10.0

(p <0.001). The respective EC50 value of crude extract in endothelium-denuded rings was 11.93 mg/mL (95 % CI, 1.483 to 95.95) (Fig. 4.3.4.1) (Table. 4.3.4.1).

4.3.4.3 At baseline tension In this set of experiments, the crude extract of A.marmelos was tested on baseline resting tension at the concentrations of 0.01, 0.1, 1.0, 3.0, 5.0 and 10.0 mg/mL in cumulative fashion. The crude extract did not exhibit either the stimulating or vasoconstrictor effect up to the concentration of 10.0 mg/mL. The software could not converge the data to get EC50 value (Fig. 4.3.4.2) (Table. 4.3.4.2).

4.3.4.4 On K+80 mM pre-contracted aorta In high K+ pre-contracted endothelium-intact aortic rings the crude extract of A.marmelos exhibited inhibitory effects in a concentration-dependent manner. The crude extract when added in cumulative fashion it caused the inhibition of high K+ pre-contracted aortic rings at the concentrations (mg/mL) of 0.01 (93.095 ± 3.565), 0.1 (90.235 ± 4.432), 1.0 (73.23 ± 7.461), 3.0 (78.800 ± 3.699), 5.0 (62.203 ± 4.541) and 10.0 (24.562 ± 1.598) respectively as compared to control (99.741 ± 0.203). It showed a statistically significant effect at the concentrations (mg/mL) of 1.0 (p <0.05), 3.0 (p <0.01), 5.0 (p <0.001) and 10.0 (p <0.001) respectively. The respective EC 50 value for endothelium-intact aortic rings was 7.565 mg/mL (95 % CI, 2.385 to 24) (Fig. 4.3.4.2) (Table. 4.3.4.2).

In high K+ pre-contracted endothelium-denuded aortic rings the crude extract of A.marmelos exhibited the inhibitory effect. The crude extract when added in cumulative fashion it caused the inhibition of high K+-induced contraction at the concentrations (mg/mL) of 0.01 (99.364 ± 3.588), 0.1 (97.762 ± 1.899),1.0 (85.23 ± 3.954), 3.0 (78.800 ± 3.699), 5.0 (62.203 ± 4.541) and 10.0 (24.562 ± 1.598) respectively as compared to control (99.74 ± 0.203). It produced

Chapter 4: Result 68 statistically significant inhibitory effect at the concentrations (mg/mL) of 1.0 (p <0.05), 3.0

(p <0.01), 5.0 (p <0.001) and 10.0 (p <0.001) respectively. The respective EC 50 value for endothelium-denuded aortic rings was ~ 14959 mg/mL (95 % CI, very wide) (Fig. 4.3.4.2) (Table. 4.3.4.2).

4.3.4.5 Construction of Ca++ curves Ca++ channel blocking activity of the crude extract was confirmed by constructing the Ca++ curves. At first the Ca++concentration response curves (Ca++CRCs) were prepared in the absence of crude extract of A.marmelos. The addition of the increasing concentration of CaCl2 was found to cause a stepwise increase in the tone of endothelium-denuded aortic rings served as control. Subsequently Ca++concentration response curve was prepared in the presence of different concentrations of crude extract of A.marmelos. The endothelium- denuded aortic rings when pretreated by crude extract of A.marmelos at the lower concentrations (mg/mL) of 0.0001, 0.001 and 0.003 respectively caused leftward shift of the Ca++CRCs concentration-dependently (Fig. 4.3.4.3.1) showing the calcium agonistic activity. The endothelium-denuded aortic rings when pretreated by crude extract of A.marmelos at higher concentrations (mg/mL) of 0.1, 0.3 and 1.0 respectively it produced the rightward shift of Ca++CRCs concentration-dependently (Fig. 4.3.4.3.2) showing the calcium antagonistic activity.

Chapter 4: Result 69

DISCUSSION Crude extract of A.marmelos. L

Chapter 4: Result 70

The present study was designed to investigate the pharmacological rationale of the use of both A.marmelos and P.cydonia because the literature of the traditional medicines is showing the tremendous use of both the plants; lacking the scientific rationale. Especially of our interest is cardiotonic and or cardioprotective effects (Dama et al., 2010; Prince et al., 2005; Rajadurai and Prince., 2005; Panda and Kar., 2009) and reducing the damage of myocardium in calcium-paradox (Kakiuchi et al., 1991; Patel et al., 2012). The dry fruit of A.marmelos and the seeds of P.cydonia were focused in the form of crude extract, various fractions and isolated compounds. Initially, the crude extract was studied in Langendorff’s model of spontaneously beating isolated perfused rat heart preparation to understand the potent effects on various parameters this helped to determine the direction of further studies. The crude extract of seed of P.cydonia (PC) when tested in Langendorff’s rat heart preparations showed a small increase in the left ventricular pressure and persistent increase in the systolic pressure, concentration-dependently which is indicative of a positive inotropic effect without any statistically significant effect, therefore, this led to abandon further studies. This positive inotropic effect in the seeds of PC may be due to the presence of some bio-active compound (s) like polyphenols, glycosides, flavonoids etc (Andriantsitohaina et al., 2012; Alesiani et al., 2010; Costa et al., 2009; Carvalho et al., 2010; Magalhaes et al., 2009) in the crude extract that exerted positive inotropic effect through various mechanisms.

The crude extract of Am.Cr showed concentration-dependent increase in left ventricular pressure with statistically significant effect. The effect on systolic pressure was also found statistically significant which is indicative of a positive inotropic effect. These effects indicate the presence of certain cardio-active compound (s) in the fruit. This led us to investigate A.marmelos, further pharmacologically and chemically.

The results of load independent experiments showed that the crude extract of Am.Cr has considerable, but the insignificant excitatory effect on myocardial contractility at lower concentrations by increasing the dP/dt(max), the first derivative of left ventricular pressure followed by an inhibitory effect at higher concentrations in isolated working rat heart. This shows the possibility of different mechanisms involved to produce agonistic and antagonistic

Chapter 4: Result 71 effects of Am.Cr on the myocardium. It is well known that in cardiac muscles, there is no receptor–operated Ca++ channels, but only voltage-dependent Ca++ channels are present (Yamahara et al, 1989). It has been reported that calcium channel blockers have week positive inotropic action at lower concentrations and a negative inotropic action at higher concentrations. (CCB) (such as nifedipine, felodipine, verapamil and (+)-cis-diltiazem) produced a positive inotropic effect in isolated perfused heart caused by vasodilatation which results in an increased fiber tension of myocardium leading to increase in myocardial contractile strength (Van Amsterdam et al., 1987; Punt et al., 1988a; Nasa et al., 1992).

Recent studies on isolated rat cardiomyocytes indicated that NO alters the contractility of isolated cardiomyocytes in a biphasic manner (Kojda et al., 1996). Low concentrations of NO enhanced the contractility of cardiomyocytes while high concentrations induced the opposite effect. Both changes are known to be dependent on cGMP, which alter L-type calcium current in ventricular myocytes in a biphasic manner (Ono and Trautwein., 1991; Shirayama and Pappano., 1996). This might be suggested that Am.Cr caused the liberation of NO in the coronary circulation, which alters slow channel calcium current in cardiac myocytes and produce biphasic effects on myocardial contractility (Muller-Strahl et al., 2000).

Phytochemical studies revealed that fruit of A.marmelos is rich in phytochemical compounds, specially saponin glycosides and cardiac glycosides might be responsible for the positive inotropic effect (Rajan et al., 2011; Sarkozi et al., 1996; Sridhar et al., 2014) present in Am.Cr increase cardiac contraction by inhibiting the enzyme Na+, K+ -ATPase (Na+ pump). In addition to pumping ions across the membrane, this enzyme is composed of both catalytic α and regulatory β subunits, serve as a functional receptor for digitalis/cardiac glycoside in the cell membrane of the cardiac muscle fibers (Xie and Cai., 2003). This inhibition lead to increase in intracellular Na+ which increases the efflux of Na+ in exchange for the Ca++ via Na+/Ca++ exchanger (NCX) in the cell membrane. Binding of cardiac glycosides to the catalytic α- subunit leads to inhibition of sodium pump and increases the intracellular availability of Ca++ for contractile protein with stimulation of the Na+/Ca+ exchanger (NCX), resulting in an

Chapter 4: Result 72 increase in myocardial contractility. This effect is further strengthened by concentration- dependent increase in aortic outflow and ejection fraction. The stroke volume and cardiac power were increased slightly at higher concentrations. Digoxin did not increase the cardiac contractility in the rat due to species differences (Repke et al., 1965; Chevalier et al., 1987; Capogrossi et al., 1986; Serikov et al., 2001).Whereas, AoF, EF and stroke volume were increased and it is comparable with digoxin (Appendix No. 1).

The systolic pressure, peak aortic systolic pressure and rate pressure product were not affected by Am.Cr. The dP/dt(min) is slightly increased concentration-dependently in association with an increase in diastolic pressure and end diastolic pressure showing an increase in diastolic relaxation whereas, heart rate and cardiac output were remained unaffected. Nitric oxide facilitates myocardial relaxation as is shown by a slight increase in dP/dt(min) which confirms the previous reports that myocardial contractility is affected by factors released from endocardium such as nitric oxide (NO) (Muller-Strahl et al., 2000; Grocott-Mason et al., 1994; Shah., 1996). In load independent experiments Ca++-myofilament interaction may have important influence on early phase of relaxation, whereas sarcoplasmic reticulum Ca++ uptake may be more important during late phase of relaxation in isolated working heart (Grocott-Mason et al., 1994; Smith et al., 1991). The selective effects of nitric oxide (NO) on early relaxation may be explained by a cGMP-mediated reduction in myofilament response to Ca++ with minimal effects on sarcoplasmic reticulum function. Thus cGMP, an intracellular mediator of nitric oxide activity caused enhancement of myocardial relaxation in isolated cardiomyocytes by reducing the myofilament response to Ca++ independent of changes in Ca++ transient kinetics (Shah et al., 1994; Anning et al., 1995).

Am.Cr significantly induced contraction of vascular smooth muscle of coronary vessels which may be due to increase in intracellular free Ca++ concentration. Ca++ plays essential roles in regulating the vascular tone (Somlyo and Himpens., 1989). For Ca++-influx through the sarcolemma, two types of Ca++ channels are involved in active influx of calcium, namely voltage-dependent Ca++channels (VDCs), activated by membrane depolarization and receptor-operated Ca++ channels (ROCC), activated by activation of receptors not necessarily

Chapter 4: Result 73 accompanied with depolarization (Bolton., 1979). Passive influx of calcium also occurs (Bolton., 1979). Depolarizing stimuli elicit contraction in coronary artery by promoting the Ca++ entry via membrane L-type Ca++ channels(Kalsner., 1994) while activation of receptors elicits contraction by increasing the intracellular Ca++ through release from sarcoplasmic reticulum and influx through ROCCs (Karaki et al., 1997). Ca++ influx into the cell can also be guided through Ca++ release from internal stores, like IP3-sensitive sarcoplasmic reticulum as well (Hall et al., 2006; Burt., 2005). The vasoconstrictor effect of Am.Cr may be mediated through these pathways in coronary vessels.

We are focused to understand the underlying mechanism of the crude extract of A.marmelos and relate the effects on the heart muscles. Therefore, the results of the aorta are being discussed here contrary to the pattern of result. The rat aorta was selected; (1) to evaluate the effect of Am.Cr on K+- and -PE-induced contractions, (2) to determine whether the vasomodulator effect is endothelium-dependent or -independent. Rat aorta is a prototype tissue used for evaluating the underlying pharmacodynamic mechanism of blood pressure (BP) lowering effect (Ghayur and Gilani., 2005).

Vascular resistance is an important determinant of blood pressure (Johansen., 1992) and vascular endothelium plays an important and pivotal role in modulating the vascular tone through the release of a variety of substances (Jaffe., 1985; Taqvi et al., 2008). Vasorelaxation can be divided into endothelium-dependent and -independent relaxation. In case of endothelium-dependent relaxation, vasodilators need the presence of endothelial layer and the secretion of NO (nitric oxide), prostacyclin and EDHF (endothelium-derived hyperpolarizing factor) (Bryan et al., 2005). Whereas in case of endothelium-independent relaxation the possible mechanisms involve the blockade of extracellular Ca++ influx through transmembrane calcium channels, prevention of agonist-mediated release of Ca++ from intracellular stores, opening of K+ channels and inhibition of the contractile apparatus (Xia et al., 2008). Ca++ plays essential role in regulating the vascular tone and reduction in Ca++ can lead to relaxation in vascular smooth muscle cells (VSMCs). Such reduction may be caused by prevention of Ca++entry from extracellular fluid or a decrease of Ca++ release from the

Chapter 4: Result 74 intracellular Ca++ stores (Qin et al., 2014).

Phenylephrine (PE), an α-adrenoceptor agonist causes aortic contraction by Ca++ influx through ROCCs and by release of Ca++ from sarcoplasmic reticulum (SR) (Buraei and Yang., 2010). Whereas high concentration of extracellular KCl leads to depolarization of the cell membrane which induces the increase in transmembrane Ca++ influx at the opening of the VDCCs and subsequently leads to vascular smooth muscle contraction (Xia et al., 2008; Karaki and Weiss., 1984; Qin et al., 2014). The ability of the crude/fraction/synthetic compound to attenuate the high K+ (80mM)-induced contractions would indicate L-type voltage-dependent calcium channel blockade (CCB) mode of vasodilation while inhibition of the PE-induced peak responses would signify the blockade of the Ca++ influx through the receptor-operated Ca++ channels (Karaki and Weiss., 1988; Taqvi et al., 2006).

In isolated rat aorta preparations, the cumulative addition of Am.Cr to the endothelium- intact and-denuded aortic rings pre-contracted by PE (1µM) caused concentration-dependent attenuation of PE-induced sustained contraction, significant at higher concentrations. The onset of relaxation appeared immediately in endothelium-intact rings than in endothelium- denuded rings. The relaxation of PE-induced contraction was found slightly more in endothelium-denuded rings. This indicates that Am.Cr may act simultaneously to block ROCCs and Ca++ release from SR stores to decrease intracellular concentration of Ca++ and relax aorta. Am.Cr showed endothelium-independent effect on vascular tone as well. Interestingly, the crude extract was devoid of any vasoconstrictor activity on resting baseline tension in rat aorta.

The Am.Cr when tested in high K+ (80mM) pre-contracted endothelium-intact and-denuded aortic rings followed by cumulative addition, exhibited relaxation of high K+-induced sustained contraction concentration-dependently with significant response at higher concentrations. This may indicate inhibition of VDCCs. A substance that can inhibit the High K+-induced contraction is therefore considered to be a calcium channel blocker (Godfraind et al., 1986). The inhibitory response initiated early in endothelium-intact rings finally the relaxant effect was found to be similar in both aortic rings. The vascular relaxant effect was

Chapter 4: Result 75 independent of endothelium and more pronounced in high K+-induced contraction than PE- induced contraction at a similar concentration range. Whereas the crude extract was devoid of any vasoconstrictor activity on resting baseline tension in rat aorta.

To confirm the CCB activity of Am.Cr calcium concentration response curves (Ca++CRCs) was constructed. Interestingly, Am.Cr when tested in pretreated endothelium-denuded rings in Ca++-free medium caused a leftward shift of the Ca++CRCs at lower concentrations showing the calcium channel agonistic activity. While at higher concentrations it exhibited calcium channel antagonistic activity in Ca++-free medium by causing a rightward shift of the Ca++ CRCs. These results indicated that Am.Cr has non-specific Ca++ channel agonistic and antagonistic activities (Kalsner., 1994) possessing two or more compounds. The fruit of A.marmelos is reported to contain cationic calcium in addition to other elements (Zehra et al., 2015). The calcium antagonistic activity of A.marmelos is comparable with verapamil (Appendix No. 2)

These results showed that Am.Cr caused relaxation of PE and high K+-induced contraction, through blockade of voltage-dependent calcium channels (VDCCs) and receptor-operated Ca++ channels (ROCCs) as well, suggesting that the Am.Cr possesses non-specific Ca++ antagonistic activity.

We performed some set of experiments to determine the therapeutic role of Am.Cr in some clinical situations after understanding the calcium agonistic and antagonistic effect in cardiac and vascular muscle cells. In this regard, we selected the load-dependent experiments and constructed the Frank-Starling curves. Moreover, the calcium paradox experiments were designed to determine the cardio-protective and therapeutic effects on various cardiovascular pathologies.

Preload is considered to be a significant physiological factor influencing on the cardiac muscle performance beat-to-beat (Olivier et al., 1987). The resting (diastolic) state of the heart could influence the subsequent contractile (systolic) state was initially described by Frank, Starling and their associates more than 70 years ago (Frank., 1956; Starling., 1918; Patterson et al., 1914). The Frank-Starling Law of the heart stated that “Mechanical energy set free on

Chapter 4: Result 76 passage from the resting to the contracted state depends on the length of the muscle fibers” (Olivier et al., 1987). The ability of the heart to change its force of contraction and therefore stroke volume in response to changes in venous return is called the Frank-Starling mechanism or Starling's Law of the heart (Klabunde., 2012). Within limits, increasing the preload on resting muscle length resulted in an increase of the force of isometric contraction and the shortening velocity of an isotonic contraction. This property of heart is the intrinsic characteristic of the cardiac muscles to respond against filling pressures. When venous return is increased, there is increased filling of the ventricle along its passive pressure curve leading to an increase in end-diastolic volume. If the ventricle has now contracted at this increased preload, and the afterload and inotropy are held constant, the ventricle empties to the same end-systolic volume, thereby increasing its stroke volume, which is defined as end-diastolic minus end-systolic volume. Conversely decreasing venous return decreased the stroke volume (Klabunde., 2012).

The contractile functions of the myocardium were investigated by changing preloads started from acute reduction to increase in ascending order to make a Frank-Starling curve. The Frank-Starling curves were constructed in the absence and presence of the test samples. Acute decrease in preload lead to less filling of the ventricle and therefore less stretch to cardiac muscle resulting in shorter sarcomere length. This causes the decrease in ventricular end-diastolic volume and end-diastolic pressure ultimately resulting in reduced stroke volume (Klabunde., 2012). While raising the preload increases the end-diastolic pressure of left ventricle causing the chamber to dilate before contraction. Therefore, a series of graded changes in preload, translated into a series of changes in left ventricular end-diastolic volume. The resulting effect on cardiac performance can be predicted from Frank-Starling relationship, i.e cardiac performance increases with increasing preload (Wonderjem et al., 1991).

Pre-treatment by 3.0, 30.0 and 100.0 mg/mL of Am.Cr caused an increase in the aortic outflow, left ventricular pressure and coronary vascular resistance by an acute reduction in preload followed by a consistent increase showing dose- and load-dependent effects. The coronary effluent showed dose- and load-dependent decreasing effect. The cardiac output

Chapter 4: Result 77

was found to be slightly affected. The dP/dt(max) and heart rate showed dose-dependent decrease whereas dP/dt(min) showed dose- and load-dependent increase. The peak aortic systolic pressure and myocardial work did not show dose- or load-dependent effect.

Hemodynamic variables often known as indices of preload include myocardial segment length, end-diastolic volume and end-diastolic pressure within the ventricle. An acute reduction in preload, as in hypovolemic shock, is an important negative influence on ventricular function. This led to reduced ventricular performance associated with inappropriate hypertrophy of the ventricles known as hypertrophic cardiomyopathy (Gaasch et al., 1972). Preload and its effects on systolic performance play a more important role in the normal heart, helping to adjust beat-to-beat alterations in venous return and the cardiovascular response to exercise (Olivier et al., 1987). Frank-Starling mechanism is due to changes in the number of overlapping actin and myosin units within the sarcomere. Preload changes are associated with altered calcium handling and troponin-C affinity for calcium. By increasing the sarcomere length the calcium sensitivity of troponin-C also increases, which leads to increase in the rate of cross-bridge attachments and detachments and amount of tension developed by the muscle fiber (Klabunde., 2012). This mechanism is representing the basic cardiac contractility as a length-tension relationship ex vivo and LV function curve in vivo, in which myocyte stretching to an optimal length immediately increases the contractile force with little alteration of intracellular calcium [Ca++]i, preceding to a stretch-induced slowly developing an increase in contractile force associated with an increase in [Ca++]i mobilization (Endoh., 2008). The role of calcium in cardiac excitation-contraction coupling (E- C coupling) has been established by simultaneous measurements of contractility and Ca++ transients. The process of central mechanism, i.e Ca++ binding to troponin-C and downstream mechanism, i.e thin filament regulation and cross bridge cycling are playing a significant role. Although cardiac contractile regulation is achieved by dynamic modulation of [Ca++]i mobilization i.e upstream mechanism, it has been proved that the process subsequent to elevation of [Ca++]i central and downstream mechanism is also an important target of cardiotonic agents and the contractile modulation induced by physiological and pathological intervention in the heart (Endoh., 2008). Those agents elicit a positive inotropic effect by

Chapter 4: Result 78 increasing [Ca++]i are generally associated with risk of [Ca++]i overload as in case of digitalis or cardiac glycosides. Most probably, the presence of some calcium modulating compounds in the extract is inducing the inotropic effect in the cardiac muscles. Glycosides inhibits Na+/K+ ATPase that provide energy for the Na+ pump to extrude Na+, resulting in [Na+]i accumulation, and thereby increases the [Ca++]i through suppression of the forward mode or facilitation of the reverse mode Na+/Ca++ exchanger (NCX) (Endoh., 2008). Cardiac glycoside is also reported in Am.Cr which may be mediating the said effect.

In isolated heart, nitric oxide (NO), continuously released from the cardiac endothelium under basal conditions and exert a prominent role in the regulation of Ca++ handling in cardiac myocytes. It has been reported that endogenous NO augments the Frank-Starling response (Prendergast et al., 1997). The involvement NO/cGMP signaling pathway plays a key role in endothelium-dependent myocardial contractility. NO alters the contractility of cardiomyocytes in a biphasic manner, contractility increased at low concentrations and decreased at high concentrations (Kojda et al., 1996; Mohan et al., 1996; Muller-Strahl., 2000). NO and cGMP induced biphasic myocardial contractile response concentration- dependently. These effects are modulated by the integrity or status of endocardial endothelium and by concomitant cholinergic or adrenergic stimulation. When cGMP is elevated to non-physiologically high level, it produced an inhibitory effect by direct action on contractile proteins while at lower level cGMP enhances the contractility (Mohan et al., 1996). cGMP activates the cGMP-dependent protein kinase, which induce phosphorylation of various proteins like troponin-I, and subsequent depression of the myofilament response to calcium. The decrease of cytosolic calcium level and dephosphorylation of myosin light chain results in a reduction of force and rate of contraction (Xu et al., 2010). The cGMP is responsible to alter the L-type calcium current biphasically in ventricular myocytes (Muller- Strahl., 2000) and modifies the Ca++ influx via L-type sarcolemmal Ca++ channel. It regulates the sarcoplasmic reticulum Ca++ release channel. It may cause inhibition of Ca++-ATPase mediated sarcoplasmic reticulum re-uptake of Calcium as well (Hare., 2003; Salahdeen et al., 2012). Therefore, it is more likely that the bioactive compound present in Am.Cr may exert effects on endocardial endothelium via NO/cGMP pathway. Marmelosin was isolated from

Chapter 4: Result 79 the extract of fruit of A.marmelos. The results showed the NO dependent effect. Therefore, we may understand that marmelosin present in the extract is most probably mediating its effect through the cardiac endothelium.

Calcium paradox experiments were designed to determine the cardioprotective and therapeutic effects in more detail after extracting Ca++ from the heart muscles to mimic various cardiovascular pathologies.

The reperfusion of Ca++ through normal KH solution after depletion of Ca++ caused vasoconstriction in the coronary vessels in the absence of Am.Cr. In the presence of Am.Cr at the time of reperfusion with a normal KH solution caused more vasoconstriction showing the aggravating effect of Am.Cr. When perfused with Ca++ free KH and Am.Cr which was reperfused by Ca++ containing normal KH and Am.Cr the vasoconstriction was found to be more reduced. This shows that the presence of Am.Cr at the time of Ca++ depletion and reperfusion with Ca++ containing KH, the constriction of the vessels was more attenuated.

The aortic outflow showed a maximum increase when the heart was first depleted by Ca++ free KH and reperfused by normal KH and Am.Cr. The Ca++ depletion in the absence of Am.Cr showed considerable decrease in dP/dt(max); the contractile force of the ventricles, whereas, the reperfusion by normal KH brought to near normal. The reperfusion by Am.Cr along with normal-KH following Ca++ depletion showed the recovery of the contractile force more than the control. The Ca++ depletion in the presence of Am.Cr caused a minimum reduction in the contractile force. Whereas reperfusion of heart by normal KH and Am.Cr caused improvement in contractile force up to the control values. The contractile force in the presence of the Am.Cr was found to be minimally affected while depleting the Ca++. Whereas the Am.Cr showed maximum improvement in contractile force when reperfused by normal ++ KH. The Ca depleted heart showed considerable decrease in dP/dt(min); the diastolic relaxation of the ventricle whereas the reperfusion by normal KH solution brought it to near normal level. The reperfusion by normal KH and Am.Cr following Ca++ depleting showed recovery of diastolic relaxation slightly more than control values. The Ca++ depletion in the presence of Am.Cr produced a minimum reduction in the diastolic relaxation, whereas,

Chapter 4: Result 80 reperfusion by normal KH and Am.Cr caused improvement up to the control values. The diastolic relaxation of ventricles in the presence of Am.Cr was found to be minimally affected during Ca++ depletion. Whereas the Am.Cr showed maximum improvement while reperfused by normal KH. Perfusion of heart by Ca++ free KH and reperfusion by normal KH solutions in the absence or presence of Am.Cr did not affect considerably the heart rate, left ventricular mean pressure, stroke volume and rate pressure product in any experimental series.

Calcium has been associated with cell injury in the calcium-depleted heart after re-exposure to normal calcium containing medium (Ashraf., 1979; Zimmerman and Hulsmann., 1966; Hearse et al., 1978). The loss of intracellular enzymes after calcium paradox suggest that the permeability of cell membrane is altered, allowing the accumulation of extracellular calcium in the cells. The calcium channel blocking agents like diltiazem appears to exert a protective effect by blocking the calcium entry into the cells (Fleckenstein., 1977; Kaumann and Uchltel., 1976; Golenhofen and Lammel., 1972) causing a reduction in loss of intracellular enzymes by maintaining the sarcolemmal integrity and to prevent Ca++ flux across the sarcolemma in hearts undergoing calcium paradox (Ashraf et al., 1982). It is reported that calcium entry blockers (CCB) like nifedipine, verapamil, diltiazem, D600, terodiline and fendiline are able to reduce protein leakage by up to one third (Hearse et al., 1980; Hearse and Baker., 1981; Baker and Hearse., 1981). Calcium entry blockers may be protective in a sub-maximal or mild form of the calcium paradox. Verapamil has a marked protective effect on the mild calcium paradox probably due to an inhibition of sudden calcium influx into the cardiac cells in early calcium repletion period. While Ashraf and associates (Ashraf et al., 1982) found, complete inhibition of calcium paradox injury by the combination of diltiazem treatment and hypothermia. Protection by verapamil in calcium paradox requires presence of the both verapamil and trace amounts of calcium in myocardium before the onset of reperfusion by calcium containing medium (Oskendal and Jynge., 1986). Fruit of A.marmelos contains cationic calcium in addition to other elements (Zehra et al., 2015). Therefore Am.Cr may also protect the heart in calcium paradox when perfused with the Ca++ free medium by providing cationic calcium in trace amounts and exerting effects as calcium channel blocker before reperfusion with Ca++ containing medium.

Chapter 4: Result 81

The reperfusion of heart by normal Ca++ containing KH and Am.Cr showed a beneficial therapeutic effect on myocardium by increasing myocardial contractility after perfusion by Ca++ free KH. This beneficial effect is further increased when Am.Cr is administered during both Ca++ free KH perfusion and Ca++ containing KH reperfusion.

To maintain the structural and functional integrity of cell membrane presence of calcium is necessarily required (Ashraf et al., 1982). Am.Cr may likely to exert its effects by preventing calcium loss from membrane during Ca++ free perfusion, preventing cell separation at the intercalated disc and preserve the structural integrity of gap junctions. Therefore Am.Cr may preserve enough Ca++ in the cell membrane to maintain calcium regulatory mechanisms that are lost during perfusion with Ca++ free KH medium (Alto and Dhalla., 1979).

Chapter 4: Result 82

TABLES

Chapter 4: Result 83

Table 4.1.1 Effect of oral toxicity study of crude extract of A.marmelos in Swiss albino mice. No. of Dose No. of No. of % Mortality % Survival S. No Groups animals mg/kg surviving dead animals animals 1 Group-1 05 N/S 05 Nil 0 % 100 %

2 Group-2 05 500 05 Nil 0 % 100 %

3 Group-3 05 1000 05 Nil 0 % 100 %

4 Group-4 05 2500 05 Nil 0 % 100 %

5 Group-5 05 5000 05 Nil 0 % 100 %

6 Group-6 05 7500 05 Nil 0 % 100 %

Table 4.1.2 Effect of oral toxicity study of crude extract of P.cydonia in Swiss albino mice. No. of Dose No. of No. of % Mortality % Survival S. No Groups animals mg/kg surviving dead animals animals 1 Group-1 05 N/S 05 Nil 0 % 100 %

2 Group-2 05 250 05 Nil 0 % 100 %

3 Group-3 05 500 05 Nil 0 % 100 %

4 Group-4 05 1000 05 Nil 0 % 100 %

5 Group-5 05 2500 05 Nil 0 % 100 %

6 Group-6 05 5000 05 Nil 0 % 100 %

Chapter 4: Result 84

Table 4.2 Effect of crude extracts of A.marmelos and P.cydonia on left ventricular pressure and systolic pressure in Langendorff’s rat heart Test Concentration (mg/mL) substance Parameters Control 1.0 3.0 10.0 30.0 100.0 300.0 Aegle Left 99.667 103.06 110.96 114.12 122.93 142.60* 160.55*** marmelos Ventricular ± 0.343 ± 5.413 ± 5.198 ± 5.480 ± 6.038 ± 6.118 ± 13.903 Pressure Systolic 99.667 134.91± 141.02 156.56 178.12 216.49* 256.03*** Pressure ± 0.343 26.178 ±24.367 ±14.087 ±12.480 ±16.010 ± 27.752 Pyrus Left 99.741 109.39 109.21 108.77 109.45 110.56 109.28 cydonia Ventricular ± 0.204 ± 3.948 ± 4.274 ± 4.815 ± 6.057 ± 8.654 ± 10.780 Pressure Systolic 100.00 120.18 123.20 129.86 135.48 148.46 171.77 Pressure ± 0.311 ± 8.095 ±10.059 ±10.330 ±14.676 ±18.206 ± 27.307 *p<0.05,***p<0.001

Chapter 4: Result 85

Table 4.3.1 Effects of crude extract of A.marmelos on various parameters in isolated rat working heart Parameters Concentration (mg/mL) Control 1.0 3.0 10.0 30.0 100.0 300.0 Coronary effluent 99.741 94.362 90.524 83.997 79.622* 74.242** 67.414*** (mL/min) ± 0.203 ±3.217 ± 3.037 ± 3.659 ± 3.314 ± 4.057 ± 6.516 Aortic out flow 99.741 102.09 102.59 107.55 108.53 109.81 113.88 (mL/min) ± 0.203 ±3.491 ± 3.930 ± 5.364 ± 5.324 ± 5.235 ± 6.120 Cardiac output 99.741 99.335 99.948 99.343 99.742 99.927 99.909 (mL/min) ± 0.203 ±2.907 ± 2.947 ± 3.147 ± 3.143 ± 2.905 ± 2.831

dP/dt(max) 99.741 101.48 104.55 107.64 110.74 104.55 99.584 (mm Hg/s) ± 0.203 ±2.318 ± 3.228 ± 3.878 ± 3.694 ± 3.288 ± 5.708

dP/dt(min) 99.741 100.47 101.16 101.71 102.29 102.96 102.98 (mm Hg/s) ± 0.203 ±1.442 ± 1.361 ± 1.448 ± 1.867 ± 1.457 ± 2.277 Systolic Pressure 99.741 98.811 98.981 99.010 99.349 100.04 100.52 (mm Hg) ± 0.203 ±3.059 ± 2.833 ± 3.042 ± 2.905 ± 2.921 ± 3.085 Diastolic pressure 99.741 98.719 100.36 102.00 102.44 103.12 100.31 (mm Hg) ±0.203 ±3.961 ± 3.973 ± 3.697 ± 3.988 ± 3.765 ± 3.842 Heart rate 99.741 98.316 96.583 98.044 97.279 97.812 97.702 (BPM) ± 0.203 ±3.758 ± 3.499 ± 4.003 ± 3.829 ± 3.571 ± 3.751 Peak aortic 99.741 96.178 94.351 93.564 96.161 97.921 97.178 systolic pressure ± 0.203 ±4.670 ± 4.203 ± 3.985 ± 1.702 ± 3.033 ± 1.651 (mm Hg) End diastolic 99.742 99.596 100.15 102.08 103.59 103.82 99.749 pressure (mm Hg) ±0.203 ±2.390 ± 1.933 ± 1.824 ± 2.290 ± 2.307 ± 4.814 Ejection fraction 99.741 105.26 104.66 105.09 105.97 106.59 109.21 (%) ±0.203 ±6.261 ± 6.477 ± 6.177 ± 8.245 ± 7.008 ± 11.428 Stroke volume 99.741 99.103 99.328 99.616 101.57 103.08 101.53 (mm Hg) ± 0.203 ±2.624 ± 2.573 ± 2.469 ± 1.804 ± 1.465 ± 2.966 Rate pressure 99.741 96.035 95.030 96.7 72 100.10 98.429 95.951 product (mm ± 0.203 ±5.074 ± 4.219 ± 6.748 ± 4.363 ± 2.881 ± 2.753 Hg/min) Cardiac power 99.741 98.957 99.395 99.998 100.13 102.24 102.33 ± 0.203 ±2.998 ± 3.213 ± 2.952 ± 3.152 ± 3.087 ± 3.375 *p<0.05, ** p<0.01, *** p<0.001

Chapter 4: Result 86

Table 4.3.2.1 Effect of variable pre-loads on isolated rat working hearts with or without crude extract of A.marmelos (3.0 mg/mL)

Pre-loads (cmH2O) Parameters 5 10 15 20 25

Coronary effluent Control 107.519 101.203 104.938 99.3966 99.8731 (mL/min) Pretreated 54.2727 36.7656 32.5812 30.5287 29.2432 Control 110.900 104.655 106.575 103.399 101.403 Aortic out flow (mL/min) Pretreated 124.324 128.912 131.655 137.382 137.566 Control 111.397 103.982 105.818 102.029 100.473 Cardiac output (mL/min) Pretreated 103.048 100.186 100.858 102.603 102.315 Control 98.1089 98.3764 100.56 100.835 100.31 dP/dt(max) (mm Hg/s) Pretreated 70.9175 71.3824 71.8428 71.5276 71.7635 Control 99.4329 98.7107 100.695 102.11 102.988 dP/dt(min) (mm Hg/s) Pretreated 85.9233 86.8023 87.9524 87.9485 88.4529 Control 99.7575 100.105 100.911 100.957 106.054 Heart rate (BPM) Pretreated 50.6507 50.6476 50.6476 50.6473 50.6457

Left ventricular Control 99.2514 98.781 98.9481 98.8579 98.5915 pressure (mm Hg) Pretreated 102.077 102.995 103.725 102.957 103.718

Peak aortic systolic Control 99.2392 98.0634 99.4897 97.8702 97.8068 pressure (mm Hg) Pretreated 90.9298 91.1748 91.6928 91.3356 91.3045 Coronary vascular Control 93.3561 97.0304 94.5507 97.1238 97.9305 resistance (mm Pretreated 177.389 264.263 306.527 340.221 378.029 Hg/mL x min) Control 111.057 103.104 105.076 102.577 99.8077 Myocardial work (mm Hg x mL/min) Pretreated 93.778 92.6191 92.4144 94.4637 95.3179

Chapter 4: Result 87

Table 4.3.2.2 Effect of variable pre-loads on isolated rat working heart with or without crude extract of A.marmelos (30.0 mg/mL) Pre-loads (cmH2O) Parameters 5 10 15 20 25

Coronary effluent Control 105.678 100.963 95.4399 96.119 95.622 (mL/min) Pretreated 32.8049 33.3737 31.6844 32.1996 29.2447 Control 106.782 103.684 106.923 107.276 106.879 Aortic out flow (mL/min) Pretreated 132.009 133.208 135.108 138.742 140.273

Cardiac output Control 104.576 101.532 100.835 101.119 100.862 (mL/min) Pretreated 101.845 101.847 102.717 102.266 102.157 Control 98.1565 99.5969 97.9936 94.4049 96.5641 dP/dt(max) (mm Hg/s) Pretreated 83.0488 82.7323 82.9136 83.6462 83.5362 Control 99.4329 99.1107 102.095 103.71 102.988 dP/dt(min) (mm Hg/s) Pretreated 85.9233 86.8023 87.9524 87.9485 88.4529

Heart rate Control 99.5235 101.516 104.763 108.337 106.086 (BPM) Pretreated 65.5641 65.5637 65.5639 65.5632 65.6856 Control 99.4361 99.2319 99.7267 99.2839 99.5063 Left ventricular pressure (mm Hg) Pretreated 104.373 104.606 104.673 104.808 105.00

Peak aortic systolic Control 99.5916 99.8737 99.3108 102.087 99.2782 pressure (mm Hg) Pretreated 91.158 91.1333 91.6285 91.5731 91.9219 Coronary vascular Control 125.673 128.85 131.87 130.366 133.298 resistance (mm Pretreated 767.02 735.798 804.087 836.225 843.901 Hg/mL x min) Control 104.09 102.21 100.108 102.804 99.9641 Myocardial work (mm Hg x mL/min) Pretreated 93.5263 92.0183 92.8369 94.258 94.3082

Chapter 4: Result 88

Table 4.3.2.3 Effect of variable pre-loads on isolated rat working heart with or without crude extract of A.marmelos (100.0 mg/mL)

Pre-loads (cm H2O) Parameters 5 10 15 20 25 Control 103.718 105.219 104.373 101.021 98.7582 Coronary effluent (mL/min) Pretreated 17.7979 15.5602 11.5144 10.5297 9.06821

Aortic out flow Control 99.8944 102.053 101.759 102.373 100.666 (mL/min) Pretreated 129.054 130.658 125.844 128.841 131.796 Control 99.3739 102.87 102.293 102.579 100.765 Cardiac output (mL/min) Pretreated 102.354 102.86 98.1574 100.822 102.101 Control 97.9139 97.8028 96.4172 96.0737 94.7185 dP/dt(max) (mm Hg/s) Pretreated 66.0797 67.0018 67.5009 67.9416 67.5148 Control 98.148 97.8253 97.4122 96.6304 96.3684 dP/dt(min) (mm Hg/s) Pretreated 107.217 109.91 110.614 111.378 111.451 Control 97.5399 98.136 99.6932 100.056 103.016 Heart rate (BPM) Pretreated 54.7865 54.926 53.3102 53.4079 53.28

Left ventricular Control 100.458 101.087 103.039 103.853 104.237 pressure (mm Hg) Pretreated 112.507 113.71 113.615 113.51 113.628 Control 102.794 102.868 99.4486 99.4061 100.761 Peak aortic systolic pressure (mm Hg) Pretreated 98.7733 98.9361 99.2596 99.162 99.3006

Coronary vascular Control 210.464 215.461 215.146 219.512 222.162 resistance (mm 412.63 463.139 933.097 1037.3 1370.64 Hg/mL x min) Pretreated Control 101.825 104.412 101.026 100.313 101.699 Myocardial work (mm Hg x mL/min) Pretreated 100.736 99.8856 97.9949 99.7967 101.487

Chapter 4: Result 89

Table 4.3.3.1 Effect of calcium paradox with/or without crude extract of A.marmelos (30.0 mg/mL) on coronary effluent (n=3) Series Solutions Time (minutes) 2 4 6 8 10 Series Ca++-free KH 97.2413 95.131 90.9256 90.5273 85.0436 C-1 ↓2.759 % ↓4.868 % ↓9.074 % ↓9.473 % ↓14.956 % Normal KH 78.9348 80.4721 79.5623 79.183 74.2414 ↓21.065 % ↓19.52% ↓20.438 % ↓20.817% ↓25.759 % Series Ca++-free KH 93.7269 92.0507 90.1851 87.4272 89.1694 C-2 ↓6.273 % ↓7.949 % ↓9.815 % ↓12.573% ↓10.831 % Normal KH + 18.9816 11.6232 9.06783 6.92271 5.2351 crude extract ↓81.018 % ↓88.37% ↓90.932 % ↓93.077% ↓94.765 % Series Ca++-free KH + 58.4112 26.3063 16.9854 13.0123 9.68973 C-3 crude extract ↓41.589 % ↓73.69% ↓83.015 % ↓86.988% ↓90.310% Normal KH + 65.5556 64.1435 51.5278 43.4028 32.7778 crude extract ↓34.444 % ↓35.85% ↓48.472 % ↓56.597% ↓67.222 %

Table 4.3.3.2 Effect of calcium paradox with/or without crude extract of A.marmelos (30.0 mg/mL) on aortic outflow (n=3) Series Solutions Time (minutes) 2 4 6 8 10 Series Ca++-free KH 100.485 103.318 103.182 103.182 103.308 C-1 ↑0.485 % ↑3.318 % ↑3.182 % ↑3.182 % ↑3.308 % Normal KH 105.996 106.819 105.782 106.354 106.618 ↑5.996 % ↑6.819 % ↑5.782 % ↑6.354 % ↑6.618 % Series Ca++-free KH 100.119 102.033 102.423 101.86 101.589 C-2 ↑0.119 % ↑2.033 % ↑2.423 % ↑1.86 % ↑1.589 % Normal KH + 120.112 122.159 122.296 121.856 122.991 crude extract ↑20.112 % ↑22.159 % ↑22.296 % ↑21.856 % ↑22.991% Series Ca++-free KH + 122.526 130.797 135.343 136.428 138.598 C-3 crude extract ↑22.526 % ↑30.797 % ↑35.343 % ↑36.428 % ↑38.598% Normal KH + 100.888 102.346 102.496 102.633 103.49 crude extract ↑0.888 % ↑2.346 % ↑2.496 % ↑2.633 % ↑3.490 %

Chapter 4: Result 90

Table 4.3.3.3 Effect of calcium paradox with/or without crude extract of A.marmelos (30.0 mg/mL) on dP/dt(max)(n=3) Series Solutions Time (minutes) 2 4 6 8 10 Series Ca++-free KH 35.0073 34.353 33.6324 33.4284 33.3039 C-1 ↓64.993 % ↓65.647 % ↓66.368 % ↓66.572 % ↓66.696% Normal KH 100.415 103.266 104.999 106.435 109.208 ↑0.415 % ↑3.266 % ↑4.999 % ↑6.435 % ↑9.208 % Series Ca++-free KH 37.4367 37.1276 36.9299 36.9124 36.9881 C-2 ↓62.563 % ↓62.872 % ↓63.070 % ↓63.088 % ↓63.012% Normal KH + 110.056 114.46 115.202 114.471 115.695 crude extract ↑10.056 % ↑14.460 % ↑15.202 % ↑14.471 % ↑15.695 % Series Ca++-free KH + 48.9353 50.0118 50.5113 49.9471 50.1653 C-3 crude extract ↓51.065 % ↓49.988 % ↓49.489 % ↓50.053 % ↓49.835% Normal KH + 98.5981 100.052 101.695 102.937 104.035 crude extract ↓1.402 % ↑0.052 % ↑1.695 % ↑2.937 % ↑4.035 %

Table 4.3.3.4 Effect of calcium paradox with/or without crude extract of A.marmelos (30.0 mg/mL) on dP/dt(min) (n=3) Series Solutions Time (minutes) 2 4 6 8 10 Series Ca++-free KH 55.2122 54.8795 54.8305 54.8586 54.8312 C-1 ↓44.788 % ↓45.120 % ↓45.169 % ↓45.141 % ↓45.169 % Normal KH 98.6073 100.389 101.149 101.417 103.088 ↓1.393 % ↑0.389 % ↑1.149 % ↑1.417 % ↑3.088 % Series Ca++-free KH 57.6885 57.8831 57.9478 58.250 58.4121 C-2 ↓42.311 % ↓42.117 % ↓42.052 % ↓41.750 % ↓41.588 % Normal KH + 106.333 107.823 109.279 110.733 111.649 crude extract ↑6.333 % ↑7.823 % ↑9.279 % ↑10.733 % ↑11.649 % Series Ca++-free KH + 66.9371 68.572 69.222 69.1637 69.6686 C-3 crude extract ↓33.063 % ↓31.428 % ↓30.778 % ↓30.836 % ↓30.331% Normal KH + 98.428 99.1814 99.9985 100.642 101.235 crude extract ↓1.572 % ↓0.819 % ↓1.572 % ↑0.642 % ↑1.235%

Chapter 4: Result 91

Table 4.3.3.5 Effect of calcium paradox with/or without crude extract of A.marmelos (30.0 mg/mL) on heart rate (n=3) Series Solutions Time (minutes) 2 4 6 8 10 Series Ca++-free KH 91.4115 91.3365 91.3349 91.4996 91.4951 C-1 ↓8.588 % ↓8.663 % ↓8.665 % ↓8.500 % ↓8.505 % Normal KH 98.6747 98.6693 98.6784 98.6761 98.6784 ↓1.325 % ↓1.331 % ↓1.322 % ↓1.324 % ↓1.322 % Series Ca++-free KH 95.4085 95.4177 95.4124 95.4181 95.4071 C-2 ↓4.591 % ↓4.582 % ↓4.588 % ↓4.582 % ↓4.593 % Normal KH + 99.3458 99.3457 99.3321 99.3344 99.3292 Crude extract ↓0.654 % ↓0.654 % ↓0.668 % ↓0.666 % ↓0.671 % Series Ca++-free KH + 86.0905 86.2715 86.087 86.2851 86.3012 C-3 crude extract ↓13.909 % ↓13.728 % ↓13.913 % ↓13.715 % ↓13.699% Normal KH + 98.6742 98.6614 98.6934 98.6721 98.6794 crude extract ↓1.326 % ↓1.339 % ↓1.307 % ↓1.328 % ↓1.321 %

Table 4.3.3.6 Effect of calcium paradox with/or without crude extract of A.marmelos (30.0 mg/mL) on left ventricular mean pressure (n=3) Series Solutions Time (minutes) 2 4 6 8 10 Series Ca++-free KH 99.5475 99.6235 99.780 99.9934 99.9196 C-1 ↓0.452 % ↓0.376 % ↓0.220 % ↓0.007 % ↓0.080 % Normal KH 101.552 101.81 101.866 101.948 102.084 ↑1.552 % ↑1.810 % ↑1.866 % ↑1.948 % ↑2.084 % Series Ca++-free KH 99.1465 99.270 99.269 99.3434 99.2804 C-2 ↓0.853 % ↓0.730 % ↓0.731 % ↓0.657 % ↓0.720 % Normal KH + 104.279 104.55 104.741 104.920 104.996 crude extract ↑4.279% ↑4.550 % ↑4.741 % ↑4.920 % ↑4.996 % Series Ca++-free KH + 98.3607 99.552 100.093 100.294 100.917 C-3 crude extract ↓1.639 % ↓0.448 % ↑0.093 % ↑0.294 % ↑0.917 % Normal KH + 99.5786 99.552 99.7278 99.8197 99.8795 crude extract ↓0.421 % ↓0.448 % ↓0.272 % ↓0.180 % ↓0.120 %

Chapter 4: Result 92

Table 4.3.3.7 Effect of calcium paradox with/or without crude extract of A.marmelos (30.0 mg/mL) on stroke volume (n=3) Series Solutions Time (minutes) 2 4 6 8 10 Series Ca++-free KH 94.2498 95.0545 93.2316 93.8891 93.0971 C-1 ↓5.750 % ↓4.945 % ↓6.768% ↓6.111 % ↓6.903 % Normal KH 100.271 100.648 101.547 102.127 99.5416 ↑0.271% ↑0.648 % ↑1.547% ↑2.127 % ↑0.458 % Series Ca++-free KH 94.9948 96.1504 95.1204 95.9749 94.9967 C-2 ↓5.005% ↓3.850 % ↓4.880 % ↓4.025 % ↓5.003 % Normal KH + 99.2061 99.6399 99.463 98.6854 98.7342 crude extract ↓0.794 % ↓0.360 % ↓0.537% ↓1.315 % ↓1.266 % Series Ca++-free KH + 89.2997 87.846 87.3908 86.6413 85.7625 C-3 crude extract ↓10.700 % ↓12.154% ↓12.609% ↓13.359 % ↓14.23% Normal KH + 100.134 101.144 101.655 100.723 101.242 crude extract ↑0.134 % ↑1.144 % ↑1.655 % ↑0.723 % ↑1.242%

Table 4.3.3.8 Effect of calcium paradox with/or without crude extract of A.marmelos (30.0 mg/mL) on rate pressure product (n=3) Series Solutions Time (minutes) 2 4 6 8 10 Series Ca++-free KH 89.8131 89.6821 89.9806 90.0023 90.3813 C-1 ↓10.187 % ↓10.318% ↓10.019% ↓9.998% ↓9.619% Normal KH 101.175 101.314 101.323 101.455 101.567 ↑1.175% ↑1.314% ↑1.323% ↑1.455% ↑1.567% Series Ca++-free KH 87.4963 87.681 87.5981 87.8033 88.0748 C-2 ↓12.504 % ↓12.319 % ↓12.402 % ↓12.197 % ↓11.92% Normal KH + 103.615 104.732 104.588 104.96 104.896 crude extract ↑3.615% ↑4.732% ↑4.588% ↑4.960 % ↑4.896% Series Ca++-free KH + 84.0893 85.9852 86.4376 86.6423 86.6932 C-3 crude extract ↓15.911 % ↓14.015 % ↓13.562 % ↓13.358 % ↓13.30 % Normal KH + 100.035 100.155 100.196 100.390 100.319 crude extract ↑0.035% ↑0.155% ↑0.196% ↑0.390 % ↑0.319%

Chapter 4: Result 93

Table 4.3.4.1 Effect of crude extract of A.marmelos on rat aorta pre-contracted by phenylephrine (1µ M) Concentration (mg/mL) Parameters Control 0.01 0.1 1.0 3.0 5.0 10.0 Baseline 99.667 94.972 95.018 95.336 97.45 95.565 88.438 tension ± 0.343 ± 2.835 ± 3.014 ± 3.139 ± 3.976 ± 3.220 ± 3.053 Endothelium- 100.0 97.31 96.738 95.123 91.433 73.023** 37.707*** intact rings ± 0.311 ± 3.799 ± 3.530 ± 3.976 ± 4.188 ± 7.024 ± 5.984 Endothelium- 100.00 100.71 98.747 95.080 86.653 57.202*** 33.238*** denuded rings ± 0.311 ± 2.508 ± 3.328 ± 4.804 ± 6.730 ± 11.092 ± 7.355 **p<0.01, *** p<0.001

Table 4.3.4.2 Effect of crude extract of A.marmelos on rat aortapre-contracted by high K+(80 mM) Concentration (mg/mL) Parameters Control 0.01 0.1 1.0 3.0 5.0 10.0 Baseline 99.667 95.305 95.33 97.454 96.943 95.565 90.772 tension ± 0.343 ± 4.533 ± 3.139 ± 3.976 ± 3.817 ± 3.220 ± 3.649 Endothelium- 99.741 93.095 90.235 73.23* 57.297*** 42.347*** 18.971*** intact rings ± 0.203 ± 3.565 ± 4.432 ± 7.461 ± 6.721 ± 6.546 ± 6.316 Endothelium- 99.74 99.364 97.762 85.23* 78.800** 62.203*** 24.562*** denuded rings ± 0.203 ± 3.588 ± 1.899 ± 3.954 ± 3.699 ± 4.541 ± 1.598 * p<0.05, ** p<0.01, *** p<0.001

Chapter 4: Result 94

FIGURES

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B. AQUEOUS FRACTION OF AEGLE MARMELOS (Aq.Fr.Cr)

4.4 Aqueous fraction of Aegle marmelos (Am.Cr) on working rat heart

4.4.1 Atfixed physiological preload and afterload The aqueous fraction of A.marmelos was injected at the concentrations of 0.01, 0.1, 1.0, 10.0, 100.0 and 300.0 mg/mL respectively. The table (4.3.2) indicated the effects of aqueous fraction of A.marmelos on different parameters in isolated rat working hearts.

4.4.1.1 Effect on coronary effluent, aortic outflow and cardiac output The results are shown in (Fig. 4.4.1.1). The aqueous fraction decreased the coronary effluent; at the concentrations (mg/mL) of 0.01 (89.817 ± 3.600), 0.1 (88.657 ± 3.034), 1.0 (83.530 ± 3.740), 10.0 (80.237 ± 5.275), 100.0 (79.033 ± 5.528) and 300.0 (79.075 ± 8.179) as compared to control (99.741 ± 0.2038) with EC50 value of 0.7060 mg/mL (95 % CI, 0.007325 to 68.06). The values were statistically non-significant.

The fraction increased aortic outflow; at the concentrations (mg/mL) of 0.01 (102.90 ± 3.697), 0.1 (105.33 ± 3.766), 1.0 (108.61 ± 3.700), 10.0 (108.23 ± 3.707), 100.0 (104.50 ± 3.802) and

300.0 (108.27 ± 4.400) as compared to control (99.741 ± 0.2038) with EC50 value of 0.04617 mg/mL (95 % CI, 2.745e-006 to 776.5). The values were statistically non-significant.

It increased the cardiac output; at the concentrations (mg/mL) of 0.01 (99.067 ± 2.784), 0.1 (101.34 ± 3.699), 1.0 (101.27 ± 3.449), 10.0 (100.23 ± 3.682), 100.0 (100.02 ± 2.801) and

300.0 (100.68 ± 3.468) as compared to control (99.741 ± 0.2038) with EC50 value of 10.94 mg/mL (95 % CI, 0.0 to +infinity). None of the value was statistically significant.

4.4.1.2 Effect on dP/dt(max) and dP/dt(min)

The results are shown in (Fig. 4.4.1.2). The aqueous fraction decreased the dP/dt(max); at the concentrations (mg/mL) of 0.01 (88.371 ± 3.012), 0.1 (85.901 ± 3.021), 1.0 (82.287 ± 2.980), 10.0 (80.757 ± 3.674), 100.0 (77.274 ± 4.727) and 300.0 (70.750 ± 3.904) as compared to control (99.741 ± 0.2038) respectively. The values at the concentrations (mg/mL) of 1.0 (p <0.05), 10.0 (p <0.01), 100.0 (p <0.001) and 300.0 (p <0.001) respectively were statistically significant. The EC50 value of dP/dt(max) was101.8 mg/mL (95 % CI, 2.594 to 3994).

Chapter 4: Result 125

It decreased the dP/dt(min) concentration dependently with statistically significant effect on

1.0, 10.0, 100.0 and 300.0 mg/mL. The aqueous fraction decreased dP/dt(min); at the concentrations (mg/mL) of 0.01 (97.260 ± 3.742), 0.1 (96.659 ± 3.770), 1.0 (94.428 ± 3.819), 10.0 (93.922 ± 3.838), 100.0 (91.829 ± 3.849) and 300.0 (87.854 ± 6.357) as compared to control (99.741 ± 0.2038) with EC50 values of 189.7 mg/mL (95 % CI, 0.04626 to 777824). The values were statistically non-significant.

4.4.1.3 Effects on systolic and diastolic pressure and heart rate The results are shown in (Fig. 4.4.1.3). It decreased the systolic pressure; at the concentrations (mg/mL) of 0.01 (97.953 ± 2.790), 0.1 (97.513 ± 2.784), 1.0 (96.890 ± 2.952), 10.0 (96.583 ± 2.813), 100.0 (95.948 ± 2.773) and 300.0 (95.834 ± 2.886) as compared to control (99.741 ± 0.2038) with EC50 value of 1.168 (95 % CI, 4.501e-007 to 3029529). None of the value was statistically significant.

The diastolic pressure was increased; at the concentrations (mg/mL) of 0.01 (100.55 ± 4.016), 0.1 (101.90 ± 3.816), 1.0 (102.56 ± 3.750), 10.0 (103.45 ± 3.776), 100.0 (103.60 ± 3.753) and

300.0 (104.15 ± 4.248) as compared to control (99.741 ± 0.2038) with EC50 value of 0.3754 mg/mL (95 % CI, 3.395e-007 to 415063). None of the value was statistically significant.

The heart rate was decreased till the concentration of 10.0 mg/mL, then it showed increase- decrease pattern; at the concentrations (mg/mL) of 0.01 (92.575 ± 4.510), 0.1 (88.518 ± 5.688), 1.0 (87.413 ± 5.523), 10.0 (85.616 ± 6.591), 100.0 (90.394 ± 4.320) and 300.0 (86.276

± 9.165) against the control (99.741 ± 0.2038) with EC50 value of 0.01376 mg/mL (95 % CI, 1.954e-014 to 9423520755). None of the value was statistically significant.

4.4.1.4 Effect on peak aortic systolic pressure and end diastolic pressure The results are shown in (Fig. 4.4.1.4). The aqueous fraction decreased the peak aortic systolic pressure; at the concentrations (mg/mL) of 0.01 (98.646 ± 1.556), 0.1 (99.032 ± 1.449), 1.0 (95.497 ± 2.404), 10.0 (93.505 ± 3.204), 100.0 (95.334 ± 2.222) and 300.0 (96.351

± 1.663) against the control (99.741 ± 0.2038) with EC50 value of 0.3159 mg/mL (95 % CI, 0.001199 to 83.24). None of the value was statistically significant.

Chapter 4: Result 126

The end diastolic pressure was slightly increased; at the concentrations (mg/mL) of 0.01 (99.555 ± 2.803), 0.1 (100.19 ± 2.895), 1.0 (100.14 ± 2.824), 10.0 (101.10 ± 2.765), 100.0 (100.30 ± 2.780) and 300.0 (99.058 ± 3.664) against the control (99.741 ± 0.2038). The aqueous fraction did not produce a statistically significant effect on end diastolic pressure.

The software could not converge the data to get EC50 value.

4.4.1.5 Effect on ejection fraction and stroke volume The results are shown in (Fig. 4.4.1.5). It decreased the ejection fraction; at the concentrations (mg/mL) of 0.01 (96.253 ± 1.385), 0.1 (95.308 ± 1.238), 1.0 (95.310 ± 1.710), 10.0 (96.014 ± 1.236), 100.0 (95.382 ± 2.362) and 300.0 (96.233 ± 1.926) as compared to + control (99.741 ± 0.2038). The EC50 value was 10.38 mg/mL (95 % CI, 0 to 2.232e 033). None of the value was statistically significant.

The stroke volume was decreased; at the concentrations (mg/mL) of 0.01 (97.081 ± 2.955), 0.1 (96.132 ± 2.761), 1.0 (95.953 ± 2.849), 10.0 (94.940 ± 3.104), 100.0 (93.595 ± 2.889) and

300.0 (93.809 ± 3.035) as compared to control (99.74 ± 0.2038) with EC50 value of 6.453 mg/mL (95 % CI, 0.0003954 to 105315). None of the value was statistically significant.

4.4.1.6 Effect on rate pressure product and cardiac power The results of calculated parameters are shown in (Fig. 4.4.1.6). The rate pressure product was decreased; at the concentrations (mg/mL) of 0.01 (88.672 ± 11.432), 0.1 (87.728 ± 12.020), 1.0 (78.666 ± 10.477), 10.0 (74.962 ± 10.575), 100.0 (75.436 ± 10.508) and 300.0

(66.575 ± 15.056) as compared to control (100.00 ± 0.3813). The EC50 value was 0.8123 mg/mL (95 % CI, 0.001388 to 475.3). None of the value was statistically significant.

The cardiac power showed slight increase; at the concentrations (mg/mL) of 0.01 (99.680 ± 2.807), 0.1 (100.61 ± 2.932), 1.0 (101.45 ± 2.892), 10.0 (101.86 ± 2.865), 100.0 (101.40 ±

2.852) and 300.0 (102.32 ± 2.772) as compared to control (100.00 ± 0.3813) with EC50 value of 0.1268 mg/mL (95 % CI, 2.158e-007 to 74479). None of the value was statistically significant.

4.4.2 At variable preloads

Chapter 4: Result 127

The effect of various preloads on control and pre-treated rat heart by different concentrations of the aqueous fraction of A.marmelos was studied to construct the Frank- Starling curve, while keeping the afterload fixed at the physiological level.

4.4.2.1 Pretreatment at the concentration of 1.0 mg/mL The table (4.4.2.1) indicated the effects of changing preload in control and pretreated hearts at different parameters.

Effect on coronary effluent

At 5 cmH2O (control 98.5078, pretreated 64.1026), 10 cmH2O (control 98.5316, pretreated

55.5281), 15 cmH2O (control 98.3588, pretreated 48.3474), 20 cmH2O (control 96.948, pretreated 44.0948) and 25 cmH2O (control 98.5087, pretreated 40.0264) (Fig. 4.4.2.1.1)

Effect on aortic outflow

At 5 cmH2O (control 99.3131, pretreated 118.156), 10 cmH2O (control 99.1354, pretreated

121.29), 15 cmH2O (control 99.5104, pretreated 126.739), 20 cmH2O (control 99.4169, pretreated 129.124) and 25 cmH2O (control 99.203, pretreated 130.121) (Fig. 4.4.2.1.2)

Effect on cardiac output

At 5 cmH2O (control 98.772, pretreated 98.3052), 10 cmH2O (control 99.2048, pretreated

97.9171), 15 cmH2O (control 99.1288, pretreated 98.8636), 20 cmH2O (control 99.5545, pretreated 95.6034) and 25 cmH2O (control 99.8025, pretreated 96.9927) (Fig.4.4.2.1.3)

Effect on dP/dt(max)

At 5 cmH2O (control 102.72, pretreated 69.9812), 10 cmH2O (control 102.288, pretreated

71.2953), 15 cmH2O (control 102.279, pretreated 72.2451), 20 cmH2O (control 104.104, pretreated 73.5708) and 25 cmH2O (control 105.408, pretreated (73.8216) (Fig. 4.4.2.1.4)

Effect on dP/dt(min)

At 5 cmH2O (control 99.6603, pretreated 69.9812), 10 cmH2O (control 101.427, pretreated

71.2953), 15 cmH2O (control 101.763, pretreated 72.2451), 20 cmH2O (control 104.22,

Chapter 4: Result 128

pretreated 73.5708) and 25 cmH2O (control 101.231, pretreated 73.8216) (Fig. 4.4.2.1.5)

Effect on heart rate

At 5 cmH2O (control 99.3001, pretreated 77.4119), 10 cmH2O (control 99.4553, pretreated

71.4205), 15 cmH2O (control 98.891, pretreated 68.9162), 20 cmH2O (control 99.6639, pretreated 69.031) and 25 cmH2O (control 100.905, pretreated 69.046) (Fig. 4.4.2.1.6)

Effect on left ventricular pressure

At 5 cmH2O (control 100.204, pretreated 102.335), 10 cmH2O (control 100.921, pretreated

103.279), 15 cmH2O (control 100.941, pretreated 103.533), 20 cmH2O (control 101.444, pretreated 104.026) and 25 cmH2O (control 101.872, pretreated 103.388) (Fig. 4.4.2.1.7)

Effect on peak aortic systolic pressure

At 5 cmH2O (control 99.8034, pretreated 89.4774), 10 cmH2O (control 101.717, pretreated

87.7166), 15 cmH2O (control 100.203, pretreated 88.4887), 20 cmH2O (control 100.925, pretreated 90.3245) and 25 cmH2O (control 103.283, pretreated 88.6925) (Fig. 4.4.2.1.8)

Effect on coronary vascular resistance

At 5 cmH2O (control 100.953, pretreated 136.115), 10 cmH2O (control 102.423, pretreated

169.756), 15 cmH2O (control 102.861, pretreated 209.305), 20 cmH2O (control 103.765, pretreated 238.373) and 25 cmH2O (control 102.008, pretreated 271.567) (Fig. 4.4.2.1.9)

Effect on myocardial work

At 5 cmH2O (control 99.4243, pretreated 90.0504), 10 cmH2O (control 100.878, pretreated

86.9677), 15 cmH2O (control 99.3134, pretreated 87.5606), 20 cmH2O (control 100.197, pretreated 86.7275) and 25 cmH2O (control 102.102, pretreated 86.5921) (Fig. 4.4.2.110)

4.4.2.2 Pretreatment at the concentration of 10.0 mg/mL The table (4.4.2.2) indicated the effects of changing preload in control and pretreated hearts at different parameters.

Chapter 4: Result 129

Effect on coronary effluent

At 5 cmH2O (control 101.303, pretreated 56.7689), 10 cmH2O (control 100.961, pretreated

48.9826), 15 cmH2O (control 99.5571, pretreated 44.7159), 20 cmH2O (control 98.6063, pretreated 38.4242) and 25 cmH2O (control 99.6475, pretreated 35.3413) (Fig. 4.4.2.2.1)

Effect on aortic flow out

At 5 cmH2O (control 100.604, pretreated 120.588), 10 cmH2O (control 101.582, pretreated

119.196), 15 cmH2O (control 101.924, pretreated 122.354), 20 cmH2O (control 102.268, pretreated 124.885) and 25 cmH2O (control 102.217, pretreated 127.634) (Fig. 4.4.2.2.2)

Effect on cardiac output

At 5 cmH2O (control 101.079, pretreated 102.216), 10 cmH2O (control 100.956, pretreated

101.055), 15 cmH2O (control 100.618, pretreated 99.8108), 20 cmH2O (control 100.444, pretreated 99.5803) and 25 cmH2O (control 100.907, pretreated 101.342) (Fig. 4.4.2.2.3)

Effect on dP/dt(max)

At 5 cmH2O (control 99.2082, pretreated 42.0966), 10 cmH2O (control 99.4322, pretreated

42.1363), 15 cmH2O (control 99.4704, pretreated 41.8244), 20 cmH2O (control 98.8977, pretreated 41.9917) and 25 cmH2O (control 97.3143, pretreated 42.0683) (Fig. 4.4.2.2.4)

Effect on dP/dt(min)

At 5 cmH2O (control 99.3693, pretreated 58.495), 10 cmH2O (control 99.1726, pretreated

58.7306), 15 cmH2O (control 100.592, pretreated 58.8333), 20 cmH2O (control 98.8482, pretreated 59.1935) and 25 cmH2O (control 98.4626, pretreated 59.5078) (Fig. 4.4.2.2.5)

Effect on heart rate

At 5 cmH2O (control 101.034, pretreated 59.3273), 10 cmH2O (control 101.201, pretreated

58.9182), 15 cmH2O (control 100.946, pretreated 58.7189), 20 cmH2O (control 100.249, pretreated 58.7219) and 25 cmH2O (control 101.026, pretreated 58.6932) (Fig. 4.4.2.2.6)

Chapter 4: Result 130

Effect on left ventricular pressure

At 5 cmH2O (control 100.173, pretreated 100.741), 10 cmH2O (control 100.818, pretreated

101.841), 15 cmH2O (control 100.667, pretreated 101.833), 20 cmH2O (control 101.065, pretreated 102.134) and 25 cmH2O (control 100.451, pretreated 102.782) (Fig. 4.4.2.2.7)

Effect on peak aortic systolic pressure

At 5 cmH2O (control 98.9634, pretreated 85.5338), 10 cmH2O (control 99.3417, pretreated

86.1223), 15 cmH2O (control 100.407, pretreated 86.0452), 20 cmH2O (control 99.1957, pretreated 86.5611) and 25 cmH2O (control 100.085, pretreated 86.407) (Fig. 4.4.2.2.8)

Effect on coronary vascular resistance

At 5 cmH2O (control 105.085, pretreated 191.209), 10 cmH2O (control 107.852, pretreated

264.595), 15 cmH2O (control 104.876, pretreated 331.941), 20 cmH2O (control 106.98, pretreated 365.563) and 25 cmH2O (control 105.633, pretreated 422.208) (Fig. 4.4.2.2.9)

Effect on myocardial work

At 5 cmH2O (control 99.2301, pretreated 87.4921), 10 cmH2O (control 100.295, pretreated

85.8963), 15 cmH2O (control 100.404, pretreated 86.8089), 20 cmH2O (control 98.8552, pretreated 86.0496) and 25 cmH2O (control 100.596, pretreated 86.816) (Fig. 4.4.2.2.10)

4.4.2.3 Pretreatment at the concentration of 30.0 mg/mL The table (4.4.2.3) indicated the effects of changing preload in control and pretreated hearts at different parameters.

Effect on coronary effluent

At 5 cmH2O (control 103.759, pretreated 64.8869), 10 cmH2O (control 102.084, pretreated

52.9934), 15 cmH2O (control 102.26, pretreated 46.8904), 20 cmH2O (control 103.141, pretreated 41.5454) and 25 cmH2O (control 102.304, pretreated 36.7238) (Fig. 4.4.2.3.1)

Effect on aortic flow out

Chapter 4: Result 131

At 5 cmH2O (control 100.291, pretreated 109.315), 10 cmH2O (control 98.9794, pretreated

109.989), 15 cmH2O (control 100.51, pretreated 114.159), 20 cmH2O (control 101.232, pretreated 113.853) and 25 cmH2O (control 100.809, pretreated 115.375) (Fig. 4.4.2.3.2)

Effect on cardiac output

At 5 cmH2O (control 101.488, pretreated 100.187), 10 cmH2O (control 99.9554, pretreated

99.352), 15 cmH2O (control 99.8968, pretreated 99.5526), 20 cmH2O (control 100.234, pretreated 98.7569) and 25 cmH2O (control 100.391, pretreated 99.5423) (Fig. 4.4.2.3.3)

Effect on dP/dt(max)

At 5 cmH2O (control 99.0437, pretreated 51.5206), 10 cmH2O (control 99.3412, pretreated

52.2343), 15 cmH2O (control 98.9928, pretreated 52.0504), 20 cmH2O (control 100.266, pretreated 52.2412) and 25 cmH2O (control 99.058, pretreated 51.8288) (Fig. 4.4.2.3.4)

Effect on dP/dt(min)

At 5 cmH2O (control 100.618, pretreated 60.8269), 10 cmH2O (control 100.355, pretreated

61.6486), 15 cmH2O (control 100.107, pretreated 61.8292), 20 cmH2O (control 100.207, pretreated 62.0815) and 25 cmH2O (control 99.042, pretreated 62.2836) (Fig. 4.4.2.3.5)

Effect on heart rate

At 5 cmH2O (control 100.145, pretreated 90.4685), 10 cmH2O (control 99.9966, pretreated

90.4751), 15 cmH2O (control 100.306, pretreated 90.486), 20 cmH2O (control 100.451, pretreated 90.4844) and 25 cmH2O (control 100.507, pretreated 90.4964) (Fig. 4.4.2.3.6)

Effect on left ventricular pressure

At 5 cmH2O (control 99.9652, pretreated 100.709), 10 cmH2O (control 100.954, pretreated

101.141), 15 cmH2O (control 100.528, pretreated 101.386), 20 cmH2O (control 99.9141, pretreated 101.372) and 25 cmH2O (control 99.8396, pretreated 102.33) (Fig. 4.4.2.3.7)

Effect on peak aortic systolic pressure

Chapter 4: Result 132

At 5 cmH2O (control 100.203, pretreated 90.7115), 10 cmH2O (control 100.069, pretreated

91.27), 15 cmH2O (control 99.5566, pretreated 91.5182), 20 cmH2O (control 99.8191, pretreated 91.7948) and 25 cmH2O (control 99.7784, pretreated 92.029) (Fig. 4.4.2.3.8)

Effect on coronary vascular resistance

At 5 cmH2O (control 95.2781, pretreated 138.097), 10 cmH2O (control 97.2649, pretreated

174.019), 15 cmH2O (control 97.817, pretreated 207.361), 20 cmH2O (control 95.6781, pretreated 248.287) and 25 cmH2O (control 97.8107, pretreated 298.588) (Fig. 4.4.2.3.9)

Effect on myocardial work

At 5 cmH2O (control 100.098, pretreated 91.423), 10 cmH2O (control 98.2263, pretreated

89.1226), 15 cmH2O (control 98.649, pretreated 98.649), 20 cmH2O (control 99.038, pretreated 89.5097) and 25 cmH2O (control 99.1601, pretreated 89.4693) (Fig. 4.4.2.3.10)

EXPERIMENTS ON ISOLATED RAT AORTA 4.4.3 Effect of Aq.Fr.Cr on rat aorta

4.4.3.1 At baseline tension In this set of experiments, the aqueous fraction of A.marmeloswas tested on resting basal tension at the concentrations of 0.001, 0.01, 0.1, 1.0, 5.0 and 10.0 mg/mL respectively. The cumulative addition of aqueous fraction did not show either the stimulating or vasoconstrictor effect up to the concentration of 10.0 mg/mL. None of the value is statistically significant. The EC50 value was ~7643 mg/mL (95 % CI, Very Wide) (Fig. 4.4.3.1) (Table. 4.4.3.1).

4.4.3.2 On phenylephrine pre-contracted rat aorta The aqueous fraction of A.marmelos was tested in endothelium intact aortic rings pre- contracted by PE (1µM). The cumulative addition of aqueous fraction at the concentrations of 0.001, 0.01, 0.1, 1.0, 5.0 and 10.0 mg/mL caused inhibition of PE-induced sustained contraction concentration dependently. It caused inhibition of contraction at the

Chapter 4: Result 133

concentrations (mg/mL) of 0.001 (95.547 ± 3.671), 0.01 (93.213 ± 3.730), 0.1 (89.591 ± 3.789), 1.0 (84.717 ± 4.588), 5.0 (59.046 ± 5.192) and 10.0 (32.889 ± 4.044) respectively as compared to control (100.05 ± 0.3140). The aqueous fraction showed statistically significant inhibitory effects at the concentrations (mg/mL) of 5.0 (p <0.001) and 10.0 (p <0.001) respectively. The respective EC50 value for the endothelium intact aortic rings was 0.01475 mg/mL (95 % CI, 2.028e-007 to 1073) (Fig. 4.4.3.1) (Table. 4.4.3.1).

In endothelium denuded aortic rings pre-contracted by PE, the cumulative addition of the aqueous fraction caused inhibition of contraction. It showed inhibitory effect at the concentrations (mg/mL) of 0.001 (97.320 ± 3.095), 0.01 (95.360 ± 2.862), 0.1 (94.513 ± 2.846), 1.0 (92.141 ± 2.815), 5.0 (81.808 ± 2.970) and 10.0 (62.823 ± 4.288) respectively as compared to control (100.00 ± 0.3813). The aqueous fraction showed statistically significant inhibitory effect at the concentrations (mg/mL) of 5.0 (p <0.01) and 10.0 (p <0.001) in endothelium-denuded aortic rings. The respective EC50 value for the endothelium-denuded aortic rings was 29.20 mg/mL (95 % CI, 2.421 to 352.2) (Fig. 4.4.3.1) (Table. 4.4.3.1).

The inhibitory effect was initiated early and prominently in endothelium-intact rings as compared to endothelium-denuded rings.

4.4.3.3 At baseline tension In this set of experiments, the aqueous fraction of A.marmeloswas tested on resting basal tension at the concentrations of 0.001, 0.01, 0.1, 1.0, 5.0 and 10.0 mg/mL respectively. The cumulative addition of aqueous fraction did not show either the stimulating or vasoconstrictor effect up to the concentration of 10.0 mg/mL. The effect was statistically insignificant. The EC50 value of baseline tension was 0.04679 mg/mL (95 % CI, 0.00941 to 2.327) (Fig. 4.4.3.2) (Table. 4.4.3.2).

4.4.3.4 On K+80 mM pre-contracted rat aorta The aqueous fraction of A.marmelos when tested in endothelium intact aortic rings pre- contracted by high K+(80mM), the cumulative addition of aqueous fraction at the concentrations of 0.001, 0.01, 0.1, 1.0, 5.0 and 10.0 mg/mL caused inhibition of high K+- induced contraction. It caused inhibition of contraction at the concentrations (mg/mL) of

Chapter 4: Result 134

0.001 (95.570 ± 3.987), 0.01 (95.398 ± 4.174), 0.1 (92.291 ± 4.170), 1.0 (86.765 ± 3.671), 5.0 (77.251 ± 3.729) and 10.0 (61.777 ± 4.520) respectively as compared to control (99.741 ± 0.203). The statistically significant effects were observed at the concentration (mg/mL) of 5.0

(p <0.01) and 10.0 (p <0.001) respectively. The respective EC50 value was 29.64 mg/mL (95 % CI, 0.2919 to 3009) (Fig. 4.4.3.2) (Table. 4.4.3.2).

The aqueous fraction of A.marmelos was tested in endothelium denuded aortic ring pre- contracted by high K+ (80mM). The cumulative addition of aqueous fraction at the concentrations of 0.001, 0.01, 0.1, 1.0, 5.0 and 10.0 mg/mL showed a minimal inhibitory effect on high K+ pre-contracted endothelium denuded rings. It produced an inhibitory effect at the concentrations (mg/mL) of 0.001 (98.910 ± 5.145), 0.01 (99.181 ± 4.575), 0.1 (98.737 ± 4.799), 1.0 (98.491 ± 4.900), 5.0 (96.346 ± 4.427) and 10.0 (90.691 ± 4.269) respectively as compared to control (100.00 ± 0.311). None of the value was statistically significant. The software could not converge the data to get EC50 value for endothelium denuded aortic rings (Fig. 4.4.3.2) (Table. 4.4.3.2).

4.4.3.5 At baseline tension In this set of experiments, the aqueous fraction of A.marmelos was tested on resting basal tension. The cumulative addition of aqueous fraction at the concentrations of 0.001, 0.01, 0.1, 1.0, 5.0 and 10.0 mg/mL did not show either the stimulating or vasoconstrictor effect up to the concentration of 10.0 mg/mL. None of the value was statistically significant. The EC50 value was 0.01475 mg/mL (95 % CI, 2.028e-007 to 1073) (Fig. 4.4.3.3) (Table. 4.4.3.3).

4.4.3.6 On L-NAME-incubated and PE pre-contracted rat aorta The aqueous fraction of A.marmelos was tested against L-NAME-incubated and PE pre- contracted endothelium intact aortic rings. The cumulative addition of aqueous fraction at the concentrations (mg/mL) of 0.001 (98.674 ± 3.642), 0.01 (98.121 ± 3.530), 0.1 (99.870 ± 3.710), 1.0 (101.06 ± 3.715), 5.0 (95.714 ± 3.866) and 10.0 (87.908 ± 3.851) respectively as compared to control (100.00 ± 0.311) did not show inhibition of contraction. None of the + value was statistically significant. The EC50 value was 2.168 e 006 mg/mL –Interrupted (Fig. 4.4.3.3) (Table. 4.4.3.3).

Chapter 4: Result 135

DISCUSSION Aqueous fraction of A.marmelos

Chapter 4: Result 136

The results of the load independent experiments demonstrated that the aqueous fraction of Am.Cr (Aq.Fr.Cr) exhibited the highly significant inhibitory effect on myocardial contractility which attracted our interest the most. At higher concentrations, the reduction was occurred in dP/dt(max), the first derivative of left ventricular pressure in a concentration-dependent manner. This shows the antagonistic effect of aqueous fraction on the contractility of myocardium. In cardiac muscles, only voltage-dependent calcium channels are present (Yamahara et al, 1989). The reduction in myocardial contractility is the result of inhibition of transmembrane Ca++ influx through L-type Ca++ channels (Taqvi et al., 2006; Fleckenstin., 1977; Conti et al., 1985). It has been reported that calcium channel blockers (CCB) have a negative inotropic action at higher concentrations. The CCB agents also caused AV block and cardiac slowing associated with a negative inotropic effect, which results from the inhibition of the slow inward Ca++ current during the action potential plateau (Rang et al., 2012) similar to verapamil; an L-type Ca++ channel inhibitor. Aq.Fr.Cr. caused cardiac slowing by producing considerable reduction in heart rate. This shows that the constituents responsible for the calcium channel inhibitory effect; which are concentrated in Aq.Fr.Cr.

Nitric oxide (NO) has a prominent role in the regulation of Ca++in cardiac myocytes. High concentration of NO caused reduction in contractility of cardiomyocytes and is dependent on cGMP (Muller-Strahl et al., 2000). NO modifies the Ca++ influx via L-type sarcolemmal Ca++ channel, it regulates the sarcoplasmic reticulum(SR) Ca++ release channel and it may inhibit Ca++-ATPase mediated SR re-uptake of calcium (Hare., 2003; Salahdeen et al., 2012). Aqueous fraction of crude extract of the fruit may act as i) NO donor or ii) enhanced NO release from the endocardial endothelium (EE) of cardiac myocytes. The NO and CCB activity seems to cause the more negative effect on the contractility of the myocardium synergistically. The extension of this effect was observed by a reduction in rate pressure product. Whereas end-diastolic pressure, systolic pressure, peak aortic systolic pressure, ejection fraction and stroke volume were unaffected. The aqueous fraction produced a slight decrease in the dP/dt(min) whereas it causes a considerable increase in diastolic pressure. Aortic outflow was increased by the aqueous fraction while cardiac output and cardiac power showed small and variable increasing effect. The aqueous fraction caused a slight reduction

Chapter 4: Result 137 in coronary effluent concentration-dependently. The increase in coronary vascular smooth muscle tone may be due to increase in intracellular free Ca++ concentration. Ca++-influx through the sarcolemma occurs by two types of Ca++ channels which are involved in active influx of calcium; i) voltage-dependent Ca++ channels (VDCs) which are activated by membrane depolarization and ii) receptor-operated Ca++ channels (ROCCs), activated by activation of receptors not necessarily accompanied with depolarization (Bolton., 1979). The passive influx of calcium also occurs (Bolton., 1979). Ca++ influx into the cell can also be guided through Ca++ release from internal stores, like IP3-sensitive sarcoplasmic reticulum as well (Hall et al., 2006; Burt., 2005). The vasoconstrictor effect of aqueous fraction may be mediated through these pathways in coronary vessels.

We are focused to understand the underlying mechanism of aqueous fraction and relate the effects on the heart muscles and the coronary vessels. Therefore, the results of the aorta are being discussed here contrary to the pattern of result. Rat aorta, is a prototype tissue routinely used for evaluating the underlying pharmacodynamics of blood pressure lowering effect (Ghayur and Gilani., 2005). We used rat aorta to evaluate the effect of Aq.Fr.Cr on PE- and K+-induced contractions and to elucidate whether the vasodilator effect of aqueous fraction is endothelium-dependent or –independent. Substances attenuating the high K+ (80mM)-induced contractions would indicate L-type voltage-dependent calcium channel blockade (CCB) while inhibition of the PE-induced peak responses would signify the blockade of the Ca++ influx through the receptor-operated Ca++ channels and release of Ca++ from sarcoplasmic reticulum (SR) (Karaki and Weiss., 1988; Taqvi et al., 2006; Buraei and Yang., 2010). Presence of intact endothelium plays pivotal role in modulating the vascular tone through the release of a variety of substances like (NO) nitric oxide (Jaffe., 1985; Taqvi et al., 2008).

The application of Aq.Fr.Cr in cumulative fashion to isolated rat aortic preparation was devoid of any vasoconstrictor effects on resting baseline tension. Whereas, the cumulative addition of aqueous fraction to the endothelium-intact and-denuded aortic rings pre-contracted by PE (1 µM) produced a concentration-dependent relaxation, statistically significant at higher

Chapter 4: Result 138 concentrations in both the preparations. This may indicate that aqueous fraction acts by blocking the receptor-operated calcium channels (ROCCs) and Ca++ release from SR stores to decrease intracellular Ca++ concentration and thus relax aorta. The relaxant effect was more potent in endothelium-intact than –denuded aortic rings.

The Aq.Fr.Cr when added cumulatively to high K+ (80mM) pre-contracted endothelium-intact and-denuded aortic rings, exhibited concentration-dependent relaxation of high K+-induced contraction significantly at higher concentrations only in endothelium-intact rings. Whereas in case of endothelium-denuded rings; aqueous fraction did not produce relaxant effect. In endothelium-intact aortic rings the aqueous fraction of Am.Cr produced more potent and pronounced vasorelaxant effect both in PE- and high K+-induced contractions. While in case of endothelium-denuded rings aqueous fraction produced significant, but less potent relaxation in PE-induced contraction. Whereas in case of high K+-induced pre-contracted endothelium-denuded rings aqueous fraction was unable to produce a relaxant effect. This indicated the significance of endothelium which plays an essential role in producing vascular relaxant effect induced by aqueous fraction.

To confirm the endothelium-dependent vasorelaxant effect endothelium-intact rings pre- incubated with L-NAME; nitric oxide synthase inhibitor was used. The cumulative addition of aqueous fraction to the L-NAME pre-incubated and PE pre-contracted endothelium-intact rings did not show a relaxation of aortic rings. It is generally known that NO/cGMP signaling pathway plays an essential role to generate vascular smooth muscle relaxation endothelium- dependently (Suzuki et al., 2007).The endothelial cells lining blood vessels produced a relaxing factor, named endothelium-derived relaxing factor (EDRF) originally termed by Furchgott and Zawadzki (1980), in response to many types of stimuli like chemical agents and mechanical stimulation (Moncada and Higgs., 1993; Chang et al., 1996). NO formed in the endothelium by activating NOS is chemically involved and its diffusion to VSMCs appears to activate soluble guanylate cyclase (sGC) to enhance the intracellular production of cGMP (Chang et al., 1996). cGMP activates protein kinase G which activates phosphatases that inactivate myosin light chains by inhibiting Ca++ influx and reducing the sensitivity of the

Chapter 4: Result 139 contractile element to Ca++(Sato et al., 1988). Myosin light chains are involved in muscle contraction (Ignarro et al., 1986). The end result is vascular smooth muscle relaxation and allowing vessels to dilate (Ignarro et al., 1986). Endothelium-dependent involvement was also confirmed by the complete inhibition of relaxation in L-NAME-incubated rings (Salahdeen et al., 2012; Furchgott and Zawadzki., 1980). In our study, L-NAME, a nitric oxide synthase inhibitor, completely abolished the relaxation effect of aqueous fraction. Therefore, it is more likely that the active components in the aqueous fraction of Am.Cr may act on vascular endothelium via NO/cGMP pathway.

Frank Starling curve was constructed to elaborate the effects of Aq.Fr.Cr in the cardiac muscle cells in the absence and presence of the Aq.Fr.Cr. The contractile functions of myocardium were investigated by changing preloads; reducing from acute reduction to increasing at the integral of 5 cmH2O. While the afterload was remain fixed throughout the experiments.

Frank-Starling and associates describe that the resting state of the heart could influence the subsequent contractile state (Frank., 1956; Starling., 1918). Heart changes its force of contraction and therefore the stroke volume in response to changes in venous return is called Frank-Starling mechanism (Klabunde., 2012). Cardiac muscles have the intrinsic property to respond against filling pressures which tend to influence on beat-to-beat performance of the myocardium (Olivier et al., 1987). Acute reduction in preload lead to less filling of the ventricle and therefore less stretch to cardiac muscle resulting in a decrease in sarcomere length. This causes the reduction in ventricular end-diastolic volume and end-diastolic pressure ultimately resulting in reduced stroke volume (Klabunde., 2012). Change in preload causes changes in left ventricular end-diastolic volume. The performance of the heart can be predicted from Frank-Starling relationship as the cardiac performance increases in response to increase in the preload (Wonderjem et al., 1991).

Pre-treatment of isolated perfused heart by 1.0, 10.0 and 30.0 mg/mL of Aq.Fr.Cr increased the aortic outflow, left ventricular pressure and coronary vascular resistance by an acute reduction in preload while a graded increase in preload caused load dependent increase at all

Chapter 4: Result 140

doses. The dP/dt(max) was found to decrease as we reduced the preload acutely. This decrease was not affected by increasing the preload. The dP/dt(min) was found to decrease by acute preload reduction at all doses and increase in preload showed load dependent increase as well. The heart rate was decreased at 5.0 cmH2O at all doses, then increasing preload did not affect considerably. The coronary effluent was decreased by reduction in preload at all doses which remained continued during graded increase in preload at all doses. The peak aortic systolic pressure was found to decrease at all preloads and doses. Whereas at the dose of

30.0 mg graded increase in preload at the integral of 5 cmH2O showed a slight load- dependent increase as well. The myocardial work showed a slight decreasing effect at all preloads. The increase in preload variably affects the myocardial work. The cardiac output was found to be less affected at all preloads. The dose of 10.0 mg showed a slight increase in cardiac output.

The systolic performance of the heart is influenced by preload to adjust beat-to-beat alterations in venous return and the cardiovascular response to stimuli like exercise (Olivier et al., 1987). Changes in preload are associated with altered calcium handling and troponin-C affinity for calcium. As the sarcomere length increases, the calcium sensitivity of troponin-C also increases. This leads to increase in the rate of cross-bridge attachments and detachments and ultimately the amount of tension developed by the muscle fiber (Klabunde., 2012). Myocytes stretching to an optimal length immediately increases the contractile force with little alteration of intracellular calcium, preceding to a stretch-induced slowly developing an increase in contractile force associated with an increase in intracellular calcium mobilization (Endoh., 2008). The role of calcium in cardiac excitation-contraction coupling (E-C coupling) has been established by simultaneous measurements of contractility and Ca++ transients (Endoh., 2008). It is most probable, the presence of some calcium modulating compound in the Aq.Fr.Cr which is responsible to increase left ventricular pressure and aortic outflow by inducing the inotropic effect in the cardiac muscles. Cardiotonic agents elicit a positive inotropic effect by increasing intracellular calcium[Ca++]i are generally associated with risk of [Ca++]i overload as in case of cardiac glycosides. Glycosides inhibits Na+/K+ ATPase that provide energy for the Na+ pump to extrude Na+,

Chapter 4: Result 141 resulting in accumulation of [Na+]i which leads to increase in the [Ca++]i through suppression of the forward mode or facilitation of the reverse mode Na+/Ca++ exchanger (NCX) (Endoh., 2008). Cardiac glycoside, is also reported in crude extract of A.marmelos being water soluble may be concentrated in this fraction as well.

Aq.Fr.Cr exhibited the inhibitory response on the rate and force of contraction. It is well known that reduction in rate of contraction is due to reduction in trans-sarcolemmal Ca++influx (Malecot and Trautwein., 1987) while a decrease in the force of contraction is the result of inhibition of transmembrane Ca++ influx through L-type Ca++ channels (Fleckenstein., 1977; Conti et al., 1985). The calcium channel blockers (CCBs) like verapamil at higher concentrations caused depression of contractile function, heart rate and AV conduction in isolated hearts (Buljubasic et al., 1991). Aq.Fr.Cr may be mediating its inhibitory effects through blocking of the Ca++ channels.

The regulation of calcium handling in myocardium is also affected by NO; continuously released from endocardial endothelium and cardiac myocytes and hence influences the contractile activity of myocardium (Paulus and Shah., 1999). It has been reported that endogenous NO augments the Frank-Starling response (Prendergast et al., 1997). NO and cGMP induced biphasic myocardial contractile response concentration-dependently; contractility increased at low concentrations and decreased at high concentrations (Kojda et al., 1996; Mohan et al., 1996; Muller-Strahl., 2000). cGMP produced an inhibitory effect by direct action on contractile proteins; by activating cGMP-dependent protein kinase and phosphorylation of various proteins like troponin-I; cGMP produces depression of the myofilament response to calcium (Mohan et al., 1996). The force and rate of myocardial contraction are attenuated by reducing the calcium level and dephosphorylating of myosin light chain. In ventricular myocytes, cGMP alter the L-type calcium current biphasically and modifies the Ca++ influx via L-type sarcolemmal Ca++ channel (Xu et al., 2010; Muller-Strahl., 2000). It regulates the sarcoplasmic reticulum Ca++ release channel. It may cause inhibition of Ca++-ATPase mediated sarcoplasmic reticulum re-uptake of calcium as well (Hare., 2003; Salahdeen et al., 2012). Aq.Fr.Cr may enhance NO release or production in cardiac myocytes

Chapter 4: Result 142 due to stepwise increase in preloads.

Paulus and Shah (1999) described that NO has significant effects on the relaxation phase of cardiac contraction, sometime even in the absence of changes in systolic function (Paulus and Shah., 1999). An acute reduction in active diastolic tone in the absence of changes in diastolic Ca++ level; leads to increase in diastolic myocardial length which is induced by NO or cGMP. These effects may be due to reduction in myofilament responsiveness to Ca++ (Shah et al,. 1994). NO release from endocardial endothelium has a distinct effect on basal diastolic LV function by hastening of LV relaxation and increase in LV distensibility. NO may also induce both chronotropic and dromotropic effects (Kelly et al., 1996; Musialek et al., 1997; Paulus and Shah., 1999). It may be possible that the bioactive compounds concentrated in Aq.Fr.Cr may mediate this effect on endocardial endothelium and cardiac myocyte via NO/cGMP pathway.

Coronary vascular contraction was increased considerably by increasing preloads in ascending order. Ca++ plays essential roles in regulating the vascular tone and fruit of A.marmelos contains cationic calcium in addition to other elements (Somlyo and Himpens., 1989; Zehra et al., 2015). Increase in intracellular free Ca++ concentration resulted by membrane depolarization to elicit contraction in coronary artery by promoting the Ca++ entry via membrane L-type Ca++ channels (Kalsner., 1994) while receptors activation elicit contraction by increasing the intracellular Ca++ through release from sarcoplasmic reticulum and influx through ROCC (Karaki et al., 1997). Inhibition of the Na+/K+-ATPase pump; a property of cardiac glycosides is also liable to cause depolarization in vascular smooth muscles which produce vasoconstriction in coronary vessels (Klabunde., 2012). Aq.Fr.Cr induced enhancement of vascular tone produced by narrowing of coronary vessels in isolated hearts may be mediated through these pathways.

Chapter 4: Result 143

TABLES

Chapter 4: Result 144

Table 4.4.1 Effects of aqueous fraction of A.marmelos on various parameters in isolated rat working heart Parameters Concentration (mg/mL) Control 0.01 0.1 1.0 10.0 100.0 300.0 Coronary effluent 99.741± 89.817 88.657 83.530 80.237 79.033 79.075 (mL/min) 0.2038 ± 3.600 ± 3.034 ± 3.740 ± 5.275 ± 5.528 ± 8.179 Aortic out flow 99.741± 102.90 105.33 108.61 108.23 104.50 108.27 (mL/min) 0.2038 ± 3.697 ± 3.766 ± 3.700 ± 3.707 ± 3.802 ± 4.400 Cardiac output 99.741 99.067 101.34 101.27 100.23 100.02 100.68 (mL/min) ±0.2038 ± 2.784 ± 3.699 ± 3.449 ± 3.682 ± 2.801 ± 3.468

dP/dt(max) 99.741 88.371 85.901 82.287* 80.757** 77.274*** 70.750*** (mm Hg/s) ±0.2038 ± 3.012 ± 3.021 ± 2.980 ± 3.674 ± 4.727 ± 3.904

dP/dt(min) 99.741 97.260 96.659 94.428 93.922 91.829 87.854 (mm Hg/s) ±0.2038 ± 3.742 ± 3.770 ± 3.819 ± 3.838 ± 3.849 ± 6.357 Systolic pressure 99.741 97.953 97.513 96.890 96.583 95.948 95.834 (mm Hg) ±0.2038 ± 2.790 ± 2.784 ± 2.952 ± 2.813 ± 2.773 ± 2.886 Diastolic pressure 99.741 100.55 101.90 102.56 103.45 103.60 104.15 (mm Hg) ±0.2038 ± 4.016 ± 3.816 ± 3.750 ± 3.776 ± 3.753 ± 4.248 Heart rate 99.741 92.575 88.518 87.413 85.616 90.394 86.276 (BPM) ±0.2038 ± 4.510 ± 5.688 ± 5.523 ± 6.591 ± 4.320 ± 9.165 Peak aortic 99.741 98.646 99.032 95.497 93.505 95.334 96.351 systolic pressure ±0.2038 ± 1.556 ± 1.449 ± 2.404 ± 3.204 ± 2.222 ± 1.663 (mm Hg) End diastolic 99.741 99.555 100.19 100.14 101.10 100.30 99.058 pressure (mm Hg) ±0.2038 ± 2.803 ± 2.895 ± 2.824 ± 2.765 ± 2.780 ± 3.664 Ejection fraction 99.741 96.253 95.308 95.310 96.014 95.382 96.233 (%) ± 0.2038 ± 1.385 ± 1.238 ± 1.710 ± 1.236 ± 2.362 ± 1.926 Stroke volume 99.741 97.081 96.132 95.953 94.940 93.595 93.809 (mm Hg) ± 0.2038 ± 2.955 ± 2.761 ± 2.849 ± 3.104 ± 2.889 ± 3.035 Rate pressure 100.00 88.672 87.728 78.666 74.962 75.436 66.575 product (mm Hg/ ± 0.3813 ± 11.432 ±12.020 ± 10.477 ± 10.575 ± 10.508 ± 15.056 min) Cardiac power 100.00 99.680 100.61 101.45 101.86 101.40 102.32 ± 0.3813 ± 2.807 ± 2.932 ± 2.892 ± 2.865 ± 2.852 ± 2.772 * p<0.05, ** p<0.01, *** p<0.001

Chapter 4: Result 145

Table 4.4.2.1 Effect of variable pre-loads on isolated rat working heart with or without aqueous fraction of A.marmelos (1.0 mg/mL)

Pre-loads (cmH2O) Parameters 5 10 15 20 25 Control 98.5078 98.5316 98.3588 96.948 98.5087 Coronary effluent (mL/min) Pretreated 64.1026 55.5281 48.3474 44.0948 40.0264 Control 99.3131 99.1354 99.5104 99.4169 99.203 Aortic out flow (mL/min) Pretreated 118.156 121.29 126.739 129.124 130.121

Cardiac output Control 98.772 99.2048 99.1288 99.5545 99.8025 (mL/min) Pretreated 98.3052 97.9171 98.8636 95.6034 96.9927 Control 102.72 102.288 102.279 104.104 105.408 dP/dt(max) (mm Hg/s) Pretreated 68.7256 66.6533 65.6264 66.5401 65.3463 Control 99.6603 101.427 101.763 104.22 101.231 dP/dt(min) (mm Hg/s Pretreated 69.9812 71.2953 72.2451 73.5708 73.8216 Control 99.3001 99.4553 98.891 99.6639 100.905 Heart rate (BPM) Pretreated 77.4119 71.4205 68.9162 69.031 69.046

Left ventricular Control 100.204 100.921 100.941 101.444 101.872 pressure (mm Hg) Pretreated 102.335 103.279 103.533 104.026 103.388 Control 99.8034 101.717 100.203 100.925 103.283 Peak aortic systolic pressure (mm Hg) Pretreated 89.4774 87.7166 88.4887 90.3245 88.6925 Coronary vascular Control 100.953 102.423 102.861 103.765 102.008 resistance (mm Pretreated 136.115 169.756 209.305 238.373 271.567 Hg/mL x min) Control 99.4243 100.878 99.3134 100.197 102.102 Myocardial work (mm Hg x mL/min) Pretreated 90.0504 86.9677 87.5606 86.7275 86.5921

Chapter 4: Result 146

Table 4.4.2.2 Effect of variable pre-loads on isolated rat working heart with or without aqueous fraction of A.marmelos (10.0 mg/mL)

Parameters Pre-loads (cmH2O) 5 10 15 20 25 Control 101.303 100.961 99.5571 98.6063 99.6475 Coronary effluent (mL/min) Pretreated 56.7689 48.9826 44.7159 38.4242 35.3413 Control 100.604 101.582 101.924 102.268 102.217 Aortic out flow (mL/min) Pretreated 120.588 119.196 122.354 124.885 127.634

Cardiac output Control 101.079 100.956 100.618 100.444 100.907 (mL/min) Pretreated 102.216 101.055 99.8108 99.5803 101.342 Control 99.2082 99.4322 99.4704 98.8977 97.3143 dP/dt(max) (mm Hg/s) Pretreated 42.0966 42.1363 41.8244 41.9917 42.0683 Control 99.3693 99.1726 100.592 98.8482 98.4626 dP/dt(min) (mm Hg/s) Pretreated 58.495 58.7306 58.8333 59.1935 59.5078 Control 101.034 101.201 100.946 100.249 101.026 Heart rate (BPM) Pretreated 59.3273 58.9182 58.7189 58.7219 58.6932

Left ventricular Control 100.173 100.818 100.667 101.065 100.451 pressure (mm Hg) Pretreated 100.741 101.841 101.833 102.134 102.782 Control 98.9634 99.3417 100.407 99.1957 100.085 Peak aortic systolic pressure (mm Hg) Pretreated 85.5338 86.1223 86.0452 86.5611 86.407 Coronary vascular Control 105.085 107.852 104.876 106.98 105.633 resistance (mm Pretreated 191.209 264.595 331.941 365.563 422.208 Hg/mL x min) Control 99.2301 100.295 100.404 98.8552 100.596 Myocardial work (mm Hg x mL/min) Pretreated 87.4921 85.8963 86.8089 86.0496 86.816

Chapter 4: Result 147

Table 4.4.2.3 Effect of variable pre-loads on isolated rat working heart with or without aqueous fraction of A.marmelos (30.0 mg/mL)

Pre-loads (cmH2O) Parameters 5 10 15 20 25 Control 103.759 102.084 102.26 103.141 102.304 Coronary effluent (mL/min) Pretreated 64.8869 52.9934 46.8904 41.5454 36.7238 Control 100.291 98.9794 100.51 101.232 100.809 Aortic out flow (mL/min) Pretreated 109.315 109.989 114.159 113.853 115.375

Cardiac output Control 101.488 99.9554 99.8968 100.234 100.391 (mL/min) Pretreated 100.187 99.352 99.5526 98.7569 99.5423 Control 99.0437 99.3412 98.9928 100.266 99.0583 dP/dt(max) (mm Hg/s) Pretreated 51.5206 52.2343 52.0504 52.2412 51.8288 Control 100.618 100.355 100.107 100.207 99.042 dP/dt(min) (mm Hg/s) Pretreated 60.8269 61.6486 61.8292 62.0815 62.2836 Control 100.145 99.9966 100.306 100.451 100.507 Heart rate (BPM) Pretreated 90.4685 90.4751 90.486 90.4844 90.4964

Left ventricular Control 99.9652 100.954 100.528 99.9141 99.8396 pressure (mm Hg) Pretreated 100.709 101.141 101.386 101.372 102.33 Control 100.203 100.069 99.5566 99.8191 99.7784 Peak aortic systolic pressure (mm Hg) Pretreated 90.7115 91.27 91.5182 91.7948 92.029 Coronary vascular Control 95.2781 97.2649 97.817 95.6781 97.8107 resistance (mm Pretreated 138.097 174.019 207.361 248.287 298.588 Hg/mL x min) Control 100.098 98.2263 98.649 99.038 99.1601 Myocardial work (mm Hg x mL/min) Pretreated 91.423 89.1226 90.9104 89.5097 89.4693

Chapter 4: Result 148

Table 4.4.3.1 Effect of aqueous fraction of A.marmelos on rat thoracic aorta pre-contracted by phenylephrine (1 µM) Concentration (mg/mL) Parameters Control 0.001 0.01 0.100 1.000 5.00 10.00 Baseline 100.00 94.397 93.678 91.704 91.734 92.073 91.707 tension ± 0.381 ± 3.388 ± 3.053 ± 3.057 ± 2.994 ± 2.732 ± 2.970 Endothelium- 100.05 95.547 93.213 89.591 84.717 59.046*** 32.889*** intact rings ± 0.314 ± 3.671 ± 3.730 ± 3.789 ± 4.588 ± 5.192 ± 4.044 Endothelium- 100.00 97.320 95.360 94.513 92.141 81.808** 62.823*** denuded rings ± 0.381 ± 3.095 ± 2.862 ± 2.846 ± 2.815 ± 2.970 ± 4.288 ** p<0.01, *** p<0.001

Table 4.4.3.2 Effect of aqueous fraction of A.marmelos on rat thoracic aorta pre-contracted by high K+ (80 mM) Concentration (mg/mL)

Parameters Control 0.001 0.01 0.100 1.000 5.00 10.00 Baseline 100.00 96.436 94.486 91.389 90.852 89.778 89.218 tension ± 0.311 ± 3.490 ± 3.515 ± 3.323 ± 3.398 ± 3.283 ± 2.720 Endothelium- 99.741 95.570 95.398 92.291 86.765 77.251** 61.777*** intact rings ± 0.203 ± 3.987 ± 4.174 ± 4.170 ± 3.671 ± 3.729 ± 4.520 Endothelium- 100.00 98.910 99.181 98.737 98.491 96.346 90.691 denuded rings ± 0.311 ± 5.145 ± 4.575 ± 4.799 ± 4.900 ± 4.427 ± 4.269 **p<0.01, *** p<0.001

Chapter 4: Result 149

Table 4.4.3.3 Effect of aqueous fraction of A.marmelos on L-NAME-incubated and PE pre-contracted rat thoracic aorta. Parameters Concentration (mg/mL) Control 0.001 0.01 0.100 1.000 5.00 10.00 Baseline 99.741 94.397 93.678 91.704 91.734 92.073 91.707 ± 0.2038 ± 3.388 ± 3.053 ± 3.057 ± 2.994 ±2.732 ±2.970 L-NAME incubated and 100.00 98.674 98.121 99.870 101.06 95.714 87.908 PE pre-contracted ± 0.311 ± 3.642 ± 3.530 ± 3.710 ± 3.715 ±3.866 ±3.851 endothelium-intact rings

Chapter 4: Result 150

FIGURES

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C. BUTANOLIC FRACTION OF AEGLE MARMELOS (But.Fr.Cr)

4.5 Butanolic fraction of Aegle marmelos (But.Fr.Cr) on working rat heart

4.5.1 At fixed physiological preload and afterload The butanolic fraction of A.marmelos was administered at the concentrations of 0.01, 0.1, 0.3, 1.0, 3.0, 10.0 and 30.0 mg/mL respectively. The table (4.5.1) indicated the effects of butanolic fraction on different parameters in rat working hearts.

4.5.1.1 Effects on coronary effluent, aortic outflow and cardiac output The results are shown in (Fig. 4.5.1.1). The butanolic fraction decreased the coronary effluent concentration dependently; at the concentrations (mg/mL) of 0.01 (87.669 ± 3.799), 0.1 (85.630 ± 4.280), 0.3 (82.029 ± 4.800), 1.0 (80.831 ± 3.882), 3.0 (79.526 ± 3.364), 10.0 (76.177 ± 2.964) and 30.0 (71.655 ± 3.811) respectively as compared to control (100.00 ± 0.3114). The concentrations (mg/mL) of 0.3 (p <0.05), 1.0 (p <0.05), 3.0 (p <0.01), 10.0 (p <0.001) and 30.0

(p <0.001) caused statistically significant decreasing effect with EC50 value of 2.541 mg/mL (95 % CI, 0.1712 to 37.72).

The aortic outflow was increased in a concentration-dependent manner; at the concentrations (mg/mL) of 0.01 (104.28 ± 1.498), 0.1 (105.26 ± 1.338), 0.3 (106.26 ± 1.573), 1.0 (106.59 ± 1.858), 3.0 (107.19 ± 2.211), 10.0 (108.21 ± 1.751) and 30.0 (108.53 ± 2.543) respectively as compared to control (100.00 ± 0.3114). The concentrations (mg/mL) of 10.0 (p <0.05) and 30.0 (p <0.05) produced statistically significant increase in aortic outflow. The

EC50 value was 0.6255 mg/mL (95 % CI, 0.008828 to 44.32).

The cardiac output showed a minimal and insignificant increase; at the concentrations (mg/mL) of 0.01 (100.98 ± 0.4672), 0.1 (101.46 ± 0.5156), 0.3 (101.63 ± 0.6715), 1.0 (101.54 ± 0.8269), 3.0 (101.67 ± 1.054), 10.0 (101.80 ± 0.6086) and 30.0 (101.13 ± 1.171) respectively as compared to control (100.00 ± 0.3114). The fraction did not induce statistically significant effect on cardiac output. The software could not converge the data to get EC50 value.

4.5.1.2 Effects on dP/dt(max) and dP/dt(min)

Chapter 4: Result 172

The results are shown in (Fig. 4.5.1.2). The dP/dt(max) showed the steady effect; at the concentrations (mg/mL) of 0.01 (99.986 ± 5.668), 0.1 (99.697 ± 5.083), 0.3 (100.07 ± 5.312), 1.0 (101.10 ± 5.741), 3.0 (103.20 ± 4.787), 10.0 (101.26 ± 5.553) and 30.0 (102.17 ± 5.535) respectively as compared to control (100.00 ± 0.3114). The EC50 value was 0.6352 mg/mL (95 % CI, 2.251e-008 to 17924694). None of the value was statistically significant.

The dP/dt(min) increased insignificantly; at the concentrations (mg/mL) of 0.01 (102.47 ± 5.303), 0.1 (101.86 ± 4.594), 0.3 (102.12 ± 4.926), 1.0 (103.20 ± 5.051), 3.0 (103.46 ± 4.941), 10.0 (103.48 ± 5.039) and 30.0 (104.32 ± 4.929) respectively as compared to control (100.00 ±

0.3114) with EC50 value of 1.711 mg/mL (95 % CI, 7.448e-010 to 3931241873). None of the value was statistically significant.

4.5.1.3 Effect on systolic and diastolic pressure and heart rate The results are shown in (Fig. 4.5.1.3). The butanolic fraction increased the systolic pressure; at the concentrations (mg/mL) of 0.01 (107.91 ± 5.030), 0.1 (108.12 ± 5.425), 0.3 (109.52 ± 6.234), 1.0 (109.55 ± 6.279), 3.0 (109.95 ± 5.908), 10.0 (108.90 ± 5.295) and 30.0 (109.41 ±

5.605) respectively as compared to control (100.00 ± 0.311). The EC50 value was 0.1016 mg/mL (95 % CI, 4.441e-014 to 228958658873). None of the value was statistically significant.

The diastolic pressure was increased; at the concentrations (mg/mL) of 0.01 (103.54 ± 3.037), 0.1 (103.50 ± 2.696), 0.3 (102.26 ± 3.021), 1.0 (102.44 ± 2.941), 3.0 (101.36 ± 2.672), 10.0 (101.45 ± 2.428) and 30.0 (101.43 ± 2.646) respectively as compared to control (100.00 ±

0.311). The EC50value was 0.4130 mg/mL (95 % CI, 1.431e-005 to 11922). None of the value was statistically significant.

The heart rate was decreased; at the concentrations (mg/mL) of 0.01 (90.910 ± 9.072), 0.1 (88.013 ± 8.775), 0.3 (87.889 ± 7.568), 1.0 (88.216 ± 7.259), 3.0 (83.477 ± 8.382), 10.0 (83.483 ± 9.575) and 30.0 (85.118 ± 8.209) respectively as compared to control (100.00 ± 0.3114) concentration-dependently with EC50 value of 0.6891 mg/mL (95 % CI, 5.962e-006 to 79656). None of the value was statistically significant.

4.5.1.4 Effect on peak aortic systolic and end diastolic pressures

Chapter 4: Result 173

The results are shown in (Fig. 4.5.1.4). It decreased the peak aortic systolic pressure; at the concentrations (mg/mL) of 0.01 (97.626 ± 3.695), 0.1 (98.937 ± 3.473), 0.3 (97.813 ± 5.385), 1.0 (92.294 ± 4.792), 3.0 (93.030 ± 3.200), 10.0 (93.767 ± 3.369) and 30.0 (96.725 ± 4.957) respectively as compared to control (100.00 ± 0.3114) with EC50 value of 0.2998 mg/mL (95 % CI, 0.0001428 to 629.3). None of the value was statistically significant.

It increased the end diastolic pressure; at the concentration (mg/mL) of 0.01 (106.87 ± 5.581), 0.1 (106.67 ± 5.196), 0.3 (104.65 ± 5.045), 1.0 (104.34 ± 4.088), 3.0 (103.84 ± 4.156), 10.0 (103.74 ± 4.412) and 30.0 (102.72 ± 3.873) respectively as compared to control (100.00 ±

0.3114) with EC50 value of 0.3191 mg/mL (95 % CI, 1.111e-005 to 9163). None of the value was statistically significant.

4.5.1.5 Effect on ejection fraction and stroke volume The results are shown in (Fig. 4.5.1.5). It decreased the ejection fraction; at the concentration (mg/mL) of 0.01 (94.638 ± 5.896), 0.1 (92.341 ± 4.870), 0.3 (93.396 ± 7.150), 1.0 (93.492 ± 6.450), 3.0 (95.613 ± 6.586), 10.0 (93.671 ± 6.159) and 30.0 (93.990 ± 6.964) respectively as compared to control (100.00 ± 0.3114). The EC50 value was 1.012 mg/mL (95 % CI, 0 to 2.998e+028). None of the value was statistically significant.

It decreased the stroke volume; at the concentration (mg/mL) of 0.01 (97.536 ± 5.290), 0.1 (95.642 ± 4.871), 0.3 (96.940 ± 6.696), 1.0 (97.342 ± 5.852), 3.0 (99.223 ± 5.724), 10.0 (97.567 ± 5.487) and 30.0 (98.075 ± 6.079) respectively as compared to control (100.00 ± 0.3114) with

EC50 value of 0.8137 mg/mL (95 % CI, 3.375e-013 to 1965917183575). None of the value was statistically significant.

4.5.1.6 Effect on rate pressure product and cardiac power The results are shown in (Fig. 4.5.1.6). These are the calculated parameters. Butanolic fraction decreased the rate pressure product initially, then showed an increased-decreased pattern; at the concentration (mg/mL) of 0.01 (89.002 ± 6.873), 0.1 (88.951 ± 9.842), 0.3 (86.718 ± 7.475), 1.0 (88.246 ± 13.811), 3.0 (89.693 ± 10.829), 10.0 (80.158 ± 10.7010 and

30.0 (86.329 ± 13.523) respectively as compared to control (100.00 ± 0.3114). The EC50 value was 4.214 mg/mL (95 % CI, 3.931e-009 to 4517568306). None of the value was statistically

Chapter 4: Result 174 significant.

The cardiac power was increased in a concentration dependent manner; at the concentration (mg/mL) of 0.01 (101.98 ± 2.797), 0.1 (102.51 ± 2.855), 0.3 (102.35 ± 2.876), 1.0 (102.62 ± 2.879), 3.0 (102.92 ± 2.844), 10.0 (102.96 ± 2.828) and 30.0 (103.26 ± 3.050) respectively as compared to control (100.00 ± 0.3114) with EC50 value of 1.091 mg/mL (95 % CI, 2.235e-011 to 53224925818). None of the value was statistically significant.

4.5.2 At variable preloads The effect of various preloads on control and pre-treated isolated rat working heart by/with different concentrations of butanolic fraction of A.marmelos was studied to construct the Frank-Starling curve, while keeping the afterload fixed at the physiological level.

4.5.2.1 Pretreatment at the concentration of 0.01 mg/mL The table (4.5.2.1) indicated the effects of changing preload in control and pre-treated rat hearts at different parameters.

Effect on coronary effluent

At 5 cmH2O (control 102.453, pretreated 54.6948), 10 cmH2O (control 97.845, pretreated

49.1958), 15 cmH2O (control 95.8165, pretreated 46.2), 20 cmH2O (control 95.8978, pretreated 40.6061) and 25 cmH2O (control 95.6273, pretreated 36.6857) (Fig. 4.5.2.1.1)

Effect on aortic flow out

At 5 cmH2O (control 101.119, pretreated 114.76), 10 cmH2O (control 100.69, pretreated

116.015), 15 cmH2O (control 100.951, pretreated 117.636), 20 cmH2O (control 101.01, pretreated 117.71) and 25 cmH2O (control 100.063, pretreated 116.254) (Fig. 4.5.2.1.2)

Effect on cardiac output

At 5 cmH2O (control 100.862, pretreated 100.011), 10 cmH2O (control 99.6889, pretreated

99.3619), 15 cmH2O (control 99.9345, pretreated 99.9112), 20 cmH2O (control 99.3087, pretreated 100.627) and 25 cmH2O (control 98.7765, pretreated 98.6904) (Fig. 4.5.2.1.3)

Chapter 4: Result 175

Effect on dP/dt(max)

At 5 cmH2O (control 100.564, pretreated 46.2768), 10 cmH2O (control 103.422, pretreated

44.8218), 15 cmH2O (control 103.479, pretreated 44.4061), 20 cmH2O (control 103.09, pretreated 44.8011) and 25 cmH2O (control 102.688, pretreated 44.3009) (Fig. 4.5.2.1.4)

Effect on dP/dt(min)

At 5 cmH2O (control 100.583, pretreated 63.4639), 10 cmH2O (control 101.285, pretreated

64.2353), 15 cmH2O (control 102.636, pretreated 63.2122), 20 cmH2O (control 100.157, pretreated 62.8196) and 25 cmH2O (control 101.025, pretreated 62.8817) (Fig. 4.5.2.1.5)

Effect on heart rate

At 5 cmH2O (control 99.8539, pretreated 99.5904), 10 cmH2O (control 100.582, pretreated

99.1539), 15 cmH2O (control 99.615, pretreated 98.9387), 20 cmH2O (control 100.641, pretreated 97.5673) and 25 cmH2O (control 99.1635, pretreated 97.7882) (Fig. 4.5.2.1.6)

Effect on left ventricular pressure

At 5 cmH2O (control 99.4459, pretreated101.439), 10 cmH2O (control 99.3215, pretreated

102.731), 15 cmH2O (control 99.5791, pretreated 103.956), 20 cmH2O (control 101.029, pretreated 103.766) and 25 cmH2O (control 101.075, pretreated 102.583) (Fig. 4.5.2.1.7)

Effect on peak aortic systolic pressure

At 5 cmH2O (control 100.65, pretreated 96.8441), 10 cmH2O (control 99.095, pretreated

97.1532), 15 cmH2O (control 101.153, pretreated 96.7957), 20 cmH2O (control 100.489, pretreated 97.27) and 25 cmH2O (control 99.9772, pretreated 97.1202) (Fig. 4.5.2.1.8)

Effect on coronary vascular resistance

At 5 cmH2O (control 110.605, pretreated 204.4), 10 cmH2O (control 114.582, pretreated

238.177), 15 cmH2O (control 115.352, pretreated 255.875), 20 cmH2O (control 115.04, pretreated 293.905) and 25 cmH2O (control 117.645, pretreated 322.355) (Fig. 4.5.2.1.9)

Chapter 4: Result 176

Effect on myocardial work

At 5 cmH2O (control 102.117, pretreated 96.4517), 10 cmH2O (control 99.576, pretreated

96.1175), 15 cmH2O (control 100.666, pretreated 95.4963), 20 cmH2O (control 101.16, pretreated 95.1364) and 25 cmH2O (control 99.9247, pretreated 95.0447) (Fig. 4.5.2.1.10)

4.5.2.2 Pretreatment at the concentration of 0.1 mg/mL The table (4.5.2.2) indicated the effects of changing preload in control and pretreated hearts at different parameters.

Effect on coronary effluent

At 5 cmH2O (control 100.15, pretreated 62.7469), 10 cmH2O (control 99.0728, pretreated

55.9023), 15 cmH2O (control 98.6396, pretreated 46.2812), 20 cmH2O (control97.959, pretreated 42.1647) and 25 cmH2O (control 98.21, pretreated 36.1384) (Fig. 4.5.2.2.1)

Effect on aortic flow out

At 5 cmH2O (control 98.9774, pretreated 114.157), 10 cmH2O (control 99.5378, pretreated

117.063), 15 cmH2O (control 99.6098, pretreated 118.871), 20 cmH2O (control 100.077, pretreated 120.592) and 25 cmH2O (control 100.116, pretreated 124.061) (Fig. 4.5.2.2.2)

Effect on cardiac output

At 5 cmH2O (control 100.872, pretreated 101.052), 10 cmH2O (control 100.191, pretreated

100.533), 15 cmH2O (control 99.768, pretreated 98.7895), 20 cmH2O (control 100.538, pretreated 99.2676) and 25 cmH2O (control 100.852, pretreated 99.6498) (Fig. 4.5.2.2.3)

Effect on dP/dt(max)

At 5 cmH2O (control 99.259, pretreated 42.0688), 10 cmH2O (control 99.4819, pretreated

41.901), 15 cmH2O (control 99.5085, pretreated 41.7928), 20 cmH2O (control 98.7874, pretreated 42.2022) and 25 cmH2O (control 98.6621, pretreated 41.9098) (Fig. 4.5.2.2.4)

Effect on dP/dt(min)

Chapter 4: Result 177

At 5 cmH2O (control 101.021, pretreated 60.1738), 10 cmH2O (control 99.8799, pretreated

60.8349), 15 cmH2O (control 99.7078, pretreated 60.9864), 20 cmH2O (control 100.193, pretreated 61.2484) and 25 cmH2O (control 99.0286, pretreated 61.3017) (Fig. 4.5.2.2.5)

Effect on heart rate

At 5 cmH2O (control 101.321, pretreated 100.22), 10 cmH2O (control 101.454, pretreated

99.7891), 15 cmH2O (control 101.367, pretreated 99.9355), 20 cmH2O (control 101.538, pretreated 99.3704) and 25 cmH2O (control 100.68, pretreated 98.686) (Fig. 4.5.2.2.6)

Effect on left ventricular pressure

At 5 cmH2O (control 98.906, pretreated 101.06), 10 cmH2O (control 98.9858, pretreated

102.155), 15 cmH2O (control 99.435, pretreated 102.192), 20 cmH2O (control 100.274, pretreated 103.145) and 25 cmH2O (control 100.299, pretreated 104.047) (Fig. 4.5.2.2.7)

Effect on peak aortic systolic pressure

At 5 cmH2O (control 99.5717, pretreated 95.8999), 10 cmH2O (control 100.681, pretreated

97.633), 15 cmH2O (control 101.304, pretreated 97.5526), 20 cmH2O (control 101.632, pretreated 97.2508) and 25 cmH2O (control 101.692, pretreated 97.537) (Fig. 4.5.2.2.8)

Coronary vascular resistance

At 5 cmH2O (control 103.649, pretreated 153.804), 10 cmH2O (control 105.291, pretreated

184.068), 15 cmH2O (control 105.533, pretreated 225.443), 20 cmH2O (control 106.474, pretreated 255.127) and 25 cmH2O (control 106.779, pretreated 296.606) (Fig. 4.5.2.2.9)

Myocardial work

At 5 cmH2O (control 102.055, pretreated 97.5048), 10 cmH2O (control 101.917, pretreated

97.0331), 15 cmH2O (control 102.31, pretreated 95.7724), 20 cmH2O (control 102.813, pretreated 96.1391) and 25 cmH2O (control 102.357, pretreated 96.824) (Fig. 4.5.2.2.10)

4.5.2.3 Pretreatment at the concentration of 1.0 mg/mL

Chapter 4: Result 178

The table (4.5.2.3) indicated the effects of changing preload in control and pretreated hearts at different parameters.

Effect on coronary effluent

At 5 cmH2O (control 100.42, pretreated 34.1206), 10 cmH2O (control 99.526, pretreated

28.0825), 15 cmH2O (control 99.2466, pretreated 22.8726), 20 cmH2O (control 97.2516, pretreated 19.9029) and 25 cmH2O (control 90.6936, pretreated 16.1644) (Fig. 4.5.2.3.1)

Effect on aortic flow out

At 5 cmH2O (control 100.974, pretreated 120.385), 10 cmH2O (control 99.8114, pretreated

119.959), 15 cmH2O (control 101.489, pretreated 122.437), 20 cmH2O (control 101.274, pretreated 123.413) and 25 cmH2O (control 100.366, pretreated 123.736) (Fig. 4.5.2.3.2)

Effect on cardiac output

At 5 cmH2O (control 99.987, pretreated 99.5666), 10 cmH2O (control 99.0355, pretreated

99.3268), 15 cmH2O (control 100.364, pretreated 99.4475), 20 cmH2O (control 99.9804, pretreated 98.4441) and 25 cmH2O (control 98.0076, pretreated 97.082) (Fig. 4.5.2.3.3)

Effect on dP/dt(max)

At 5 cmH2O (control 98.9171, pretreated 26.9572), 10 cmH2O (control 98.8858, pretreated

26.6164), 15 cmH2O (control 99.5195, pretreated 26.507), 20 cmH2O (control 94.8583, pretreated 26.5218) and 25 cmH2O (control 91.9778, pretreated 26.5463) (Fig. 4.5.2.3.4)

Effect on dP/dt(min)

At 5 cmH2O (control 100.55, pretreated 46.7282), 10 cmH2O (control 100.352, pretreated

46.7129), 15 cmH2O (control 100.332, pretreated 46.8098), 20 cmH2O (control 100.761, pretreated 46.8745) and 25 cmH2O (control 100.622, pretreated 46.8455) (Fig. 4.5.2.3.5)

Effect on heart rate

At 5 cmH2O (control 99.9844, pretreated 78.1387), 10 cmH2O (control 100.154, pretreated

Chapter 4: Result 179

77.8172), 15 cmH2O (control 99.2995, pretreated 77.7394), 20 cmH2O (control 98.3332, pretreated 77.7399) and 25 cmH2O (control 97.6044, pretreated 77.7342) (Fig. 4.5.2.3.6)

Effect on left ventricular pressure

At 5 cmH2O (control 99.4894, pretreated 101.962), 10 cmH2O (control 100.581, pretreated

103.08), 15 cmH2O (control 100.876, pretreated 103.52), 20 cmH2O (control 101.131, pretreated 101.131) and 25 cmH2O (control 101.197, pretreated 102.818) (Fig. 4.5.2.3.7)

Effect on peak aortic systolic pressure

At 5 cmH2O (control 102.918, pretreated 93.8207), 10 cmH2O (control 102.091, pretreated

93.9095), 15 cmH2O (control 101.477, pretreated 94.3758), 20 cmH2O (control 102.222, pretreated 93.924) and 25 cmH2O (control 93.924, pretreated 93.8859) (Fig. 4.5.2.3.8)

Effect on coronary vascular resistance

At 5 cmH2O (control 135.134, pretreated 135.134), 10 cmH2O (control 139.293, pretreated

472.621), 15 cmH2O (control 141.707, pretreated 6.487), 20 cmH2O (control 145.745, pretreated 610.412) and 25 cmH2O (control 154.294, pretreated 782.137) (Fig. 4.5.2.3.9)

Effect on myocardial work

At 5 cmH2O (control 103.572, pretreated 103.572), 10 cmH2O (control 102.104, pretreated

92.3759), 15 cmH2O (control 102.63, pretreated 92.7694), 20 cmH2O (control 101.61, pretreated 94.044) and 25 cmH2O (control 100.028, pretreated 93.1662) (Fig. 4.5.2.3.10)

EXPERIMENTS ON ISOLATED RAT AORTA

4.5.3 Effect of But.Fr.Cr of A.marmelos

4.5.3.1 At baseline tension In this set of experiments, the butanolic fraction of A.marmelos was tested on resting basal tension at the concentrations of 0.0001, 0.001, 0.01, 0.1, 1.0 and 5.0 mg/mL respectively. The

Chapter 4: Result 180 cumulative addition of butanolic fraction did not show either the stimulating or vasoconstrictor effect up to the concentration of 5.0 mg/mL. None of the value is statistically significant. The EC50 value on basal tension was 1.223 mg/mL (95 % CI, 0.7213 to 2.073) (Fig. 4.5.3.1) (Table. 4.5.3.1).

4.5.3.2 On phenylephrine pre-contracted rat aorta The butanolic fraction of A.marmelos was tested in endothelium-intact aortic ring preparations, pre-contracted by PE (1µ M). The cumulative addition of butanolic fraction caused the inhibition of contraction at the concentrations (mg/mL) of 0.0001 (96.103 ± 3.851), 0.001 (93.838 ± 3.679), 0.01 (89.853 ± 4.023), 0.1 (79.392 ± 4.009), 1.0 (51.025 ± 7.541) and 5.0 (12.372 ± 4.963) respectively as compared to control (99.741 ± 0.203). The significant inhibitory effects were observed at the concentrations (mg/mL) of 0.1 (p <0.05),

1.0 (p <0.001) and 5.0 (p <0.001) respectively. The respective EC50 value of butanolic fraction in endothelium-intact aortic rings was 8.039 mg/mL (95 % CI, 3.947e-008 to 16373866) (Fig. 4.5.3.1) (Table. 4.5.3.1).

The butanolic fraction of A.marmelos was tested in endothelium-denuded aortic rings pre- contracted by PE (1µM). The cumulative addition of butanolic fraction exhibited inhibitory effects on PE pre-contracted endothelium-denuded rings at the concentrations (mg/mL) of 0.0001 (97.726 ± 2.813), 0.001 (95.010 ± 2.817), 0.01 (90.835 ± 2.836), 0.1 (77.475 ± 3.268), 1.0 (48.946 ± 5.664) and 5.0 (8.862 ± 3.098) respectively as compared to control (100.01 ± 0.228). The significant inhibitory effects were observed at the concentrations (mg/mL) of 0.1 (p <0.001), 1.0 (p <0.001) and 5.0 (p <0.001) respectively in endothelium-denuded aortic preparations. The respective EC50 value of butanolic fraction in the endothelium-denuded aortic rings was 1.337 mg/mL (95 % CI, 0.6404 to 2.793) (Fig. 4.5.3.1) (Table. 4.5.3.1).

4.5.3.3 At baseline tension In this set of experiments, the butanolic fraction of A.marmelos was tested on resting basal tension at the concentrations of 0.0001, 0.001, 0.01, 0.1, 1.0 and 5.0 mg/mL respectively. The cumulative addition of butanolic fraction did not show either the stimulating or vasoconstrictor effect up to the concentration of 5.0 mg/mL. None of the value was

Chapter 4: Result 181

statistically significant. The EC50 value of baseline resting tension was 2.280 mg/mL (95 % CI, 0.8072 to 6.439) (Fig. 4.5.3.2) (Table. 4.5.3.2).

4.5.3.4 On K+ 80 mM pre-contracted rat aorta The butanolic fraction of A.marmelos when tested in endothelium-intact aortic rings pre- contracted by high K+ (80 mM) showed inhibitory effect concentration-dependently. The cumulative addition of butanolic fraction caused inhibition of high K+-induced sustained contraction at the concentrations (mg/mL) of 0.0001 (95.930 ± 3.788), 0.001 (93.307 ± 3.980), 0.01 (88.652 ± 4.294), 0.1 (78.088 ± 5.863), 1.0 (45.730 ± 7.596) and 5.0 (11.834 ± 3.739) respectively as compared to control (100.00 ± 0.381). It showed a statistically significant inhibitory effect at the concentrations (mg/mL) of 0.1 (p <0.05), 1.0 (p <0.001) and

5.0 (p <0.001) respectively in endothelium-intact aortic rings. The respective EC50 value for endothelium-intact rings was 0.01251 mg/mL (95 % CI, 6.488e-005 to 2.41) (Fig. 4.5.3.2) (Table. 4.5.3.2).

The butanolic fraction of A.marmelos when tested in endothelium-denuded aortic rings pre- contracted by high K+ (80 mM) showed inhibitory effect in a concentration-dependent manner. The cumulative addition of butanolic fraction caused inhibition of contraction at the concentrations (mg/mL) of 0.0001 (95.909 ± 2.813), 0.001 (94.689 ± 3.156), 0.01 (79.178 ± 7.514), 0.1 (79.178 ± 7.514), 1.0 (59.610 ± 9.174) and 5.0 (14.811 ± 5.099) respectively as compared to control (99.741 ± 0.203). It showed a statistically significant inhibitory effect at the concentrations (mg/mL) of 1.0 (p <0.001) and 5.0 (p <0.001). The respective EC50 value for endothelium-denuded rings was 0.9593 mg/mL (95 % CI, 0.4563 to 2.017) (Fig. 4.5.3.2) (Table. 4.5.3.2).

4.5.3.5 Construction of Ca++ curves Ca++ channel blocking activity of the butanolic fraction of A.marmelos was confirmed by constructing the Ca++-concentration response curves (Ca++CRCs). At first the concentration response curves were prepared in the absence of butanolic fraction. The addition of increasing concentration of CaCl2 was found to cause a stepwise increase in the tone of endothelium- denuded aortic rings served as control. Subsequently Ca++CRCs were prepared

Chapter 4: Result 182 in the presence of different concentrations of butanolic fraction. The endothelium-denuded aortic rings when pretreated by a butanolic fraction of A.marmelos at the lower concentrations (mg/mL) of 0.00001, 0.0001 and 0.001 respectively it caused the leftward shift of Ca++CRCs concentration-dependently (Fig. 4.5.3.3.1) showing the calcium agonistic activity. The endothelium-denuded aortic rings when pretreated by a butanolic fraction of A.marmelos at higher concentrations (mg/mL) of 0.003, 0.01, 0.1 and 0.3 respectively it produced the rightward shift of Ca++CRCs concentration-dependently (Fig. 4.5.3.3.2) showing the calcium antagonistic activity.

Chapter 4: Result 183

DISCUSSION Butanolic fraction of A.marmelos

Chapter 4: Result 184

Load independent experiments were designed to elaborate the effects of butanolic fraction of Am.Cr (But.Fr.Cr) in the isolated ejecting rat heart. The result showed the excitatory effect of But.Fr.Cr on cardiac contractility by inducing an increase in dP/dt(max); an index of cardiac contractility (Itoh et al., 1995). Cardiac contractility is the inherent property of myocardium to contract independently according to changes in the preload or afterload. Calcium channel blockers are the potent modulator of functional activity of cardiac and vascular smooth muscles. It has been reported that CCBs such as nifedipine, verapamil etc. produced weak positive inotropic action in isolated perfused hearts at lower concentrations while induced inhibitory effect at higher concentrations (Yamahara et al, 1989; Van Amsterdam et al., 1987; Punt et al., 1988). It seems some calcium agonist compounds are concentrated in the butanolic fraction which caused the positive inotropic effect. This is not verapamil like effect. The synergistic effect of marmelosin-induced NO may be considered as well.

Butanolic fraction may act as NO donor or caused the liberation of NO in coronary circulation resulting in alteration of slow channel calcium current in ventricular myocytes and produce a biphasic effect on myocardial contractility (Muller-Strahl et al., 2000). Kojda and associates (Kojda et al., 1996) described that nitric oxide (NO) alters the contractility of cardiomyocytes. Low concentrations of NO increased the contractility while high concentrations reduced the contractility through cGMP which alter L-type calcium current in ventricular myocytes in biphasic manners (Ono and Trautwein., 1991; Shirayama and Pappano., 1996). Cardiac glycosides increase myocardial contractility by inhibiting the enzyme Na+, K+-ATPase is resulting in an increase in intracellular Na+ which enhances the efflux of Na+ in exchange for Ca++ via Na+/Ca++ exchanger (NCX) in the cell membrane (Klabunde., 2012). Cardiac glycosides are reported in fruit of A.marmelos (Rajan et al., 2011; Sarkozi et al., 1996; Sridhar et al., 2014). The effect of increased contractility is further supported by the statistically significant increase in aortic outflow (AoF) at higher concentrations, considerable increase in systolic pressure (SP), cardiac power (CP) and a slight increase in cardiac output (CO) at all doses. The peak aortic systolic pressure, ejection fraction and stroke volume were decreased slightly, whereas rate pressure product showed considerable decreasing effect.

Chapter 4: Result 185

The But.Fr.Cr caused an increase in dP/dt(min) concentration-dependently, indicating enhancing effects on ventricular diastolic relaxation. Nitric oxide (NO) plays an important role in myocardial relaxation by selective effects on early relaxation of Ca++-myofilaments mediated by cGMP; an intracellular mediator of nitric oxide. cGMP causes enhancement of myocardial relaxation in isolated cardiomyocytes by reducing the myofilament response to calcium (Shah et al., 1994; Anning et al., 1995). The effect of increased diastolic relaxation is supported by an increase in diastolic pressure and end diastolic pressure. Calcium antagonists cause inhibition of voltage dependent L-type of calcium channel and decrease the frequency of opening in response to depolarization. Decrease in transmembrane calcium current leads to long lasting relaxation of vascular smooth muscle, reduction in myocardial contractility, decrease in pacemaker activity in the SA node and decrease in conduction velocity in the AV node (Sudhakar et al., 2013). There is a slight reduction in heart rate, demonstrating the slowing effect of But.Fr.Cr similar to CCBs. But.Fr.Cr caused a statistically significant reduction in coronary effluent in a concentration-dependent manner. The contraction of the smooth muscles of coronary vessels may occur by/with an increase in intracellular free Ca++ level. The vasoconstrictive effect of butanolic fraction may be mediated through Ca++entry into the cells via L-type Ca++ channels (VDCs), receptor-operated Ca++ channels (ROCCs) or guided through Ca++ release from internal stores (Bolton., 1979; Karaki et al., 1997; Hall et al., 2006; Burt., 2005).

To understand the underlying mechanism of action of butanolic fraction and to relate the effects in the cardiac myocytes and the coronary vessels, the results obtained by the experiments on rat aorta are being discussed here contrary to the pattern of result. These experiments investigated the effect of But.Fr.Cr on PE- and high K+-induced contractions and to reveal whether the vasodilator effect of butanolic fraction is endothelium-dependent or – independent. Intact vascular endothelium plays pivotal role in modulating the vascular tone through the release of a variety of substances and hence control the blood pressure and cardiac performance (Jaffe., 1985; Taqvi et al., 2008; Ghayur and Gilani., 2005; Paulus and Shah., 1999). In cardiovascular system, the agents attenuating the high K+ (80mM)-induced contractions would indicate L-type voltage-dependent calcium channel blockade (CCB) mode

Chapter 4: Result 186 of vasodilatation while inhibition of the PE-induced peak responses would signify the blockade of the Ca++ influx through the receptor-operated Ca++ channels and release of Ca++ from sarcoplasmic reticulum (SR) (Karaki and Weiss., 1988; Taqvi et al., 2006; Buraei et al., 2010).

The cumulative addition of the But.Fr.Cr in isolated rat aortic ring preparations was devoid of any vasoconstrictor effects on resting baseline tension. The But.Fr.Cr when added cumulatively to both the endothelium-intact and-denuded aortic rings pre-contracted by PE (1µ M) caused concentration-dependent relaxation statistically significant at higher concentrations. The relaxant effect was equipotent in both endothelium-intact and –denuded rings. This indicates that But.Fr.Cr may acts by blocking the receptor-operated calcium channels (ROCCs) and Ca++ release from SR stores to decrease intracellular Ca++ concentration and relax aorta. This also showed endothelium-independent effect of But.Fr.Cr on PE-induced vascular tone.

In high K+ (80mM) pre-contracted endothelium-intact /-denuded aortic rings the cumulative addition of But.Fr.Cr in ascending order, exhibited relaxation of high K+-induced sustained contraction. This effect was concentration-dependent and statistically significant at higher concentrations in both endothelium-intact and-denuded aortic rings. This indicates inhibition of voltage-dependent calcium channels (VDCCs) and independent of vascular endothelium. Any substance that can inhibit the high K+-induced contraction is therefore considered to be a calcium channel blocker (Godfraind et al., 1986).

These results suggest that But.Fr.Cr caused relaxation of both PE- and high K+-induced contractions, through blockade of voltage-dependent calcium channels (VDCCs), receptor- operated Ca++ channels (ROCCs) and release of Ca++ from sarcoplasmic reticulum as well, suggesting that the But.Fr.Cr possesses non-specific Ca++ antagonistic effect.

For confirmation of the CCBs activity of But.Fr.Cr Ca++ CRCs were constructed. Endothelium- denuded aortic rings were used to construct Ca++CRCs. Interestingly, at lower concentrations But.Fr.Cr pre-treated rings in Ca++-free medium caused a leftward shift of the Ca++CRCs showing the calcium channel agonistic activity. While at higher concentrations it showed

Chapter 4: Result 187 calcium channel antagonistic activity (CCB) in Ca++-free medium by causing a rightward shift of the Ca++CRCs. These results indicate that But.Fr.Cr also has non-specific Ca++ channel agonistic and antagonistic activities (Kalsner., 1994) may be due to the presence of two or more compounds.

To elaborate the effects of But.Fr.Cr in the cardiac muscle cells Frank-Starling curve was constructed. Frank-Starling relationship can be successfully used to predict the cardiac performance in accordance with the changes in preloads (Wonderjem et al., 1991). To determine the cardiac performance beat to beat; preload plays a significant role (Olivier et al., 1987; Frank., 1956; Starling., 1918; Patterson et al., 1914). The myocardial contractile functions were investigated by producing changes in preloads starting from acute reduction to increasing in ascending order in the absence and presence of the But.Fr.Cr, while keeping the afterload fixed throughout the experiments. Starling’s Law of the heart describes the potential of the heart to change its force of contraction and stroke volume due to the effect of changes in preload (Klabunde., 2012). When venous pressure is high due to increased venous return, the ventricle will fill to a greater extent resulting in increased end-diastolic volume and end-diastolic pressure. As the ventricle contracts; it will develop bigger pressure and eject blood more rapidly due to activation of Frank-Starling mechanism. The net effect will be an increase in stroke volume and ejection fraction. Whereas an acute reduction in preload lead to less filling of the ventricle which in turn cause less stretch to cardiac muscle resulting in shorter sarcomere length. This produces decrease in ventricular end-diastolic volume and end-diastolic pressure ultimately resulting in reduced stroke volume (Klabunde., 2012).

Isolated perfused hearts pre-treated by 0.01, 0.1 and 1.0 mg/mL of But.Fr.Cr showed increase aortic outflow and left ventricular pressure by an acute reduction in preload and graded increase in preload showed load-dependent increasing effect in But.Fr.Cr pre-treated hearts. The coronary vascular resistance was found to be increased by acute preload reduction and remained continued with increasing the preload whereas the dose of 1.0 mg caused to increase maximally. The dP/dt(max) and dP/dt(min) were found to be decreased by acute

Chapter 4: Result 188 reduction in preload. This decrease was not affected by a stepwise increase in the preload. Heart rate was decreased by acute preload reduction and increase in preload continued the decreasing effect. The dose of 1.0 mg caused to decrease maximally. The peak aortic systolic pressure and myocardial work were found to decrease at all preloads and doses. The cardiac output was found to be less affected at all preloads and doses. The coronary effluent was reduced by acute preload reduction and increasing preload showed load dependent decrease. The increase in dose caused dose-dependent decrease in coronary effluent.

Increase in preload influences the contractile force by increasing the sarcomere length, which increases the sensitivity of troponin-C for calcium; consequently the rate of cross bridge attachment and detachment is increased. Ultimately the amount of tension developed by muscle fiber is increased (Klabunde., 2012) which explains the Frank-Starling mechanism. Stretching of myocytes to an optimal length immediately increases the contractile force with little alteration in intracellular calcium [Ca++]i, preceding to a stretch-induced slowly developing increase in contractile force associated with an increase in [Ca++]i mobilization. In cardiac excitation-contraction coupling (E-C coupling) calcium plays an important role. (Endoh., 2008).

Most probably the presence of some calcium modulating compounds; like CCBs and or cardiac glycosides, concentrated in the butanolic fraction increased LVP and aortic outflow (AoF) in the cardiac muscles. Calcium channel inhibitors such as nifedipine, verapamil and (+) diltiazem also have a weak positive inotropic effect at lower concentrations in isolated perfused hearts (Nasa et al., 1992). Those cardio-active agents which exert their effects by increasing [Ca++]i are generally associated with risk of [Ca++]i overload like digitalis or cardiac glycosides. Cardiac glycosides produce an inotropic effect by inhibiting the Na+/K+ ATPase that provide energy for the Na+ pump to extrude Na+, resulting in [Na+]i accumulation, and thereby increases the [Ca++]i through suppression of the forward mode or facilitation of the reverse mode Na+/Ca++ exchanger (NCX) (Endoh., 2008). Cardiac glycoside is also reported in the fruit of A.marmelos (Sridhar et al., 2014; Rajan et al., 2011) which may be responsible for the increase in LVP, CVR and AoF.

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But.Fr.Cr caused a reduction in dP/dt(max), dP/dt(min) and heart rate. This may be due to reduction in transsarcolemmal Ca++influx leads to decrease in rate of contraction and inhibition of transmembrane Ca++ influx through L-type Ca++ channels causes decrease in the force of contraction (Malecot and Trautwein., 1987; Fleckenstein., 1977; Conti et al., 1985). The calcium channel blockers (CCBs) like verapamil at higher concentrations caused depression of contractile function and heart rate in isolated hearts (Buljubasic et al., 1991). Activation of cGMP-dependent protein kinase by cGMP induces the phosphorylation of various proteins like troponin-I and subsequent depression of the myofilament response to calcium. The decline in cytosolic calcium level and dephosphorylation of myosin light chain results in a reduction of force and rate of contraction (Xu et al., 2010). The cGMP is responsible to alter the L-type calcium current biphasically in ventricular myocytes (Muller- Strahl., 2000) and modifies the Ca++ influx via L-type sarcolemmal Ca++ channel. It regulates the sarcoplasmic reticulum Ca++ release channel. It may cause inhibition of Ca++-ATPase mediated sarcoplasmic reticulum re-uptake of calcium as well (Hare., 2003; Salahdeen et al., 2012). Therefore, it is more likely that the bio-active compounds concentrated in butanolic fraction of Am.Cr mediated inhibitory effects through blocking the Ca++ channels and or through NO/cGMP pathway.

But.Fr.Cr decreased the coronary effluent by inducing contraction of coronary vascular smooth muscles because vasoconstriction was enhanced by increasing preload. In vascular smooth muscle Ca++ plays essential role in regulating the muscle tone (Somlyo and Himpens., 1989).Interestingly the fruit of A.marmelos contains cationic calcium in addition to other elements (Zehra et al., 2015). Depolarization of cell membrane generating the rise in intracellular free Ca++ concentration which elicit the contraction of coronary vessel by promoting the Ca++ entry via membrane L-type Ca++ channels. While in case of receptor activation; the contraction is elicited by the intracellular increase of Ca++ through the sarcoplasmic reticulum (SR) release and influx through ROCC (Kalsner., 1994; Karaki et al, 1997). Cardiac glycosides act by inhibition of the Na+/K+-ATPase, which is also responsible for depolarization in vascular smooth muscles. The depolarization of vascular smooth muscle resulted in vasoconstriction (Klabunde., 2012). The vasoconstricting effect of But.Fr.Cr may

Chapter 4: Result 190 be mediated through these pathways in coronary vessels of isolated heart preparations.

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TABLES

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Table 4.5.1 Effects of butanolic fraction of A.marmelos on various parameters in isolated rat working heart

Parameters Concentration (mg/mL) Control 0.01 0.1 0.3 1.0 3.0 10.0 30.0 Coronary effluent 100.00 87.669 85.630 82.029* 80.831* 79.526** 76.177*** 71.655*** (mL/min) ±0.3114 ±3.799 ±4.280 ±4.800 ±3.882 ±3.364 ±2.964 ±3.811 Aortic out 100.00 104.28 105.26 106.26 106.59 107.19 108.21* 108.53* flow(mL/min) ±0.3114 ±1.498 ±1.338 ±1.573 ±1.858 ±2.211 ±1.751 ±2.543 Cardiac 100.00 100.98 101.46 101.63 101.54 101.67 101.80 101.13 output(mL/min) ±0.3114 ±0.4672 ±0.5156 ±0.6715 ±0.8269 ±1.054 ±0.6086 ±1.171

dP/dt(max) 100.00 99.986 99.697 100.07 101.10 103.20 101.26 102.17 (mm Hg/s) ±0.3114 ±5.668 ±5.083 ±5.312 ±5.741 ±4.787 ±5.553 ±5.535

dP/dt(min) 100.00 102.47 101.86 102.12 103.20 103.46 103.48 104.32 (mm Hg/s) ±0.3114 ±5.303 ±4.594 ±4.926 ±5.051 ±4.941 ±5.039 ±4.929 SystolicPressure 100.00 107.91 108.12 109.52 109.55 109.95 108.90 109.41 (mm Hg) ±0.3114 ±5.030 ±5.425 ±6.234 ±6.279 ±5.908 ±5.295 ±5.605 DiastolicPressure 100.00 103.54 103.50 102.26 102.44 101.36 101.45 101.43 (mm Hg) ±0.3114 ±3.037 ±2.696 ±3.021 ±2.941 ±2.672 ±2.428 ±2.646 Heart rate 100.00 90.910 88.013 87.889 88.216 83.477 83.483 85.118 (BPM) ±0.3114 ±9.072 ±8.775 ±7.568 ±7.259 ±8.382 ±9.575 ±8.209 Peak aortic systolic 100.00 97.626 98.937 97.813 92.294 93.030 93.767 96.725 pressure (mm Hg) ±0.3114 ±3.695 ±3.473 ±5.385 ±4.792 ±3.200 ±3.369 ±4.957 End diastolic pressure 100.00 106.87 106.67 104.65 104.34 103.84 103.74 102.72 (mm Hg) ±0.3114 ±5.581 ±5.196 ±5.045 ±4.088 ±4.156 ±4.412 ±3.873 Ejection fraction (%) 100.00 94.638 92.341 93.396 93.492 95.613 93.671 93.990 ±0.3114 ±5.896 ±4.870 ±7.150 ±6.450 ±6.586 ±6.159 ±6.964 Stroke volume 100.00 97.536 95.642 96.940 97.342 99.223 97.567 98.075 (mm Hg) ±0.3114 ±5.290 ±4.871 ±6.696 ±5.852 ±5.724 ±5.487 ±6.079 Rate pressure product 100.00 89.002 88.951 86.718 88.246 89.693 80.158 86.329 (mm Hg/min) ±0.3114 ±6.873 ±9.842 ±7.475 ±13.811 ±10.829 ±10.701 ±13.523 Cardiac power 100.00 101.98 102.51 102.35 102.62 102.92 102.96 103.26 ±0.3114 ±2.797 ±2.855 ±2.876 ±2.879 ±2.844 ±2.828 ±3.050 * p<0.05, ** p<0.01, *** p<0.001

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Table 4.5.2.1 Effect of variable pre-loads on isolated rat working heart with or without butanolic fraction of A.marmelos (0.01 mg/mL)

Pre-loads (cm H2O) Parameters 5 10 15 20 25

Coronary effluent Control 102.453 97.845 95.8165 95.8978 95.6273 (mL/min) Pretreated 54.6948 49.1958 46.2 40.6061 36.6857

Aortic out flow Control 101.119 100.69 100.951 101.01 100.063 (mL/min) Pretreated 114.76 116.015 117.636 117.71 116.254

Cardiac output Control 100.862 99.6889 99.9345 99.3087 98.7765 (mL/min) Pretreated 100.011 99.3619 99.9112 100.627 98.6904 Control 100.564 103.422 103.479 103.09 102.688 dP/dt(max) (mm Hg/s) Pretreated 46.2768 44.8218 44.4061 44.8011 44.3009 Control 100.583 101.285 102.636 100.157 101.025 dP/dt(min) (mm Hg/s) Pretreated 63.4639 64.2353 63.2122 62.8196 62.8817

Heart rate Control 99.8539 100.582 99.615 100.641 99.1635 (BPM) Pretreated 99.5904 99.1539 98.9387 97.5673 97.7882

Left ventricular Control 99.4459 99.3215 99.5791 101.029 101.075 pressure (mm Hg) Pretreated 101.439 102.731 103.956 103.766 102.583

Peak aortic systolic Control 100.65 99.095 101.153 100.489 99.9772 pressure (mm Hg) Pretreated 96.8441 97.1532 96.7957 97.27 97.1202 Coronary vascular Control 110.605 114.582 115.352 115.04 117.645 resistance (mm Pretreated 204.4 238.177 255.875 293.905 322.355 Hg/ mL x min)

Myocardial work Control 102.117 99.576 100.666 101.16 99.9247 (mm Hg x mL/min) Pretreated 96.4517 96.1175 95.4963 95.1364 95.0447

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Table 4.5.2.2 Effect of variable pre-loads on isolated rat working heart with or without butanolic fraction of A.marmelos (0.1 mg/mL)

Pre-loads (cm H2O) Parameters 5 10 15 20 25

Coronary effluent Control 100.15 99.0728 98.6396 97.959 98.21 (mL/min) Pretreated 62.7469 55.9023 46.2812 42.1647 36.1384

Aortic out flow Control 98.9774 99.5378 99.6098 100.077 100.116 (mL/min) Pretreated 114.157 117.063 118.871 120.592 124.061

Cardiac output Control 100.872 100.191 99.768 100.538 100.852 (mL/min) Pretreated 101.052 100.533 98.7895 99.2676 99.6498 Control 99.259 99.4819 99.5085 98.7874 98.6621 dP/dt(max) (mm Hg/s) Pretreated 42.0688 41.901 41.7928 42.2022 41.9098 Control 101.021 99.8799 99.7078 100.193 99.0286 dP/dt(min) (mm Hg/s) Pretreated 60.1738 60.8349 60.9864 61.2484 61.3017

Heart rate Control 101.321 101.454 101.367 101.538 100.68 (BPM) Pretreated 100.22 99.7891 99.9355 99.3704 98.686

Left ventricular Control 98.906 98.9858 99.435 100.274 100.299 pressure (mm Hg) Pretreated 101.06 102.155 102.192 103.145 104.047

Peak aortic systolic Control 99.5717 100.681 101.304 101.632 101.692 pressure (mm Hg) Pretreated 95.8999 97.633 97.5526 97.2508 97.537 Coronary vascular Control 103.649 105.291 105.533 106.474 106.779 resistance (mm Hg/ Pretreated 153.804 184.068 225.443 255.127 296.606 mL x min)

Myocardial work Control 102.055 101.917 102.31 102.813 102.357 (mm Hg x mL/min) Pretreated 97.5048 97.0331 95.7724 96.1391 96.824

Chapter 4: Result 195

Table 4.5.2.3 Effect of variable pre-loads on isolated rat working heart with or without butanolic fraction of A.marmelos (1.0 mg/mL)

Pre-loads (cm H2O) Parameters 5 10 15 20 25

Coronary effluent Control 100.42 99.526 99.2466 97.2516 90.6936 (mL/min) Pretreated 34.1206 28.0825 22.8726 19.9029 16.1644

Aortic out flow Control 100.974 99.8114 101.489 101.274 100.366 (mL/min) Pretreated 120.385 119.959 122.437 123.413 123.736

Cardiac output Control 99.987 99.0355 100.364 99.9804 98.0076 (mL/min) Pretreated 99.5666 99.3268 99.4475 98.4441 97.082 Control 98.9171 98.8858 99.5195 94.8583 91.9778 dP/dt(max) (mm Hg/s) Pretreated 26.9572 26.6164 26.507 26.5218 26.5463 Control 100.55 100.352 100.332 100.761 100.622 dP/dt(min) (mm Hg/s) Pretreated 46.7282 46.7129 46.8098 46.8745 46.8455

Heart rate Control 99.9844 100.154 99.2995 98.3332 97.6044 (BPM) Pretreated 78.1387 77.8172 77.7394 77.7399 77.7342

Left ventricular Control 99.4894 100.581 100.876 101.131 101.197 pressure (mm Hg) Pretreated 101.962 103.08 103.52 104.449 102.818

Peak aortic systolic Control 102.918 102.091 101.477 102.222 101.082 pressure (mm Hg) Pretreated 93.8207 93.9095 94.3758 93.924 93.8859 Coronary vascular Control 135.134 139.293 141.707 145.745 154.294 resistance (mm Hg/ Pretreated 373.935 472.621 606.487 610.412 782.137 mL x min)

Myocardial work Control 103.572 102.104 102.63 101.61 100.028 (mm Hg x mL/min) Pretreated 93.8733 92.3759 92.7694 94.044 93.1662

Chapter 4: Result 196

Table 4.5.3.1 Effect of butanolic fraction of A.marmelos on rat thoracic aorta pre-contracted by phenylephrine (1µ M) Parameters Concentration (mg/mL) Control 0.0001 0.001 0.01 0.1 1.0 5.0 Baseline 100.26 98.107 96.415 96.136 95.970 95.507 92.161 tension ± 0.364 ± 2.800 ± 2.787 ± 3.159 ± 3.608 ± 3.379 ± 3.061 Endothelium- 99.741 96.103 93.838 89.853 79.392* 51.025*** 12.372*** intact rings ± 0.203 ± 3.851 ± 3.679 ± 4.023 ± 4.009 ± 7.541 ± 4.963 Endothelium- 100.01 97.726 95.010 90.835 77.475*** 48.946*** 8.862*** denuded rings ± 0.228 ± 2.813 ± 2.817 ± 2.836 ± 3.268 ± 5.664 ± 3.098 * p<0.05, *** p<0.001

Table 4.5.3.2 Effect of butanolic fraction of A.marmelos on rat thoracic aorta pre-contracted by high K+ (80 mM) Parameters Concentration (mg/mL) Control 0.0001 0.001 0.01 0.1 1.0 5.0 Baseline 99.676 96.718 94.245 92.176 90.705 89.250 86.651 tension ± 0.249 ± 3.471 ± 3.620 ± 3.507 ± 3.809 ± 4.323 ± 5.596 Endothelium- 100.00 95.930 93.307 88.652 78.08* 45.730*** 11.834*** intact rings ± 0.381 ± 3.788 ± 3.980 ± 4.294 ± 5.863 ± 7.596 ± 3.739 Endothelium- 99.741 95.909 94.689 90.711 79.178 59.610*** 14.811*** denuded rings ± 0.203 ± 2.813 ± 3.156 ± 3.967 ± 7.514 ± 9.174 ± 5.099 * p<0.05, *** p<0.001

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D. ISOLATED AND STANDARD MARMELOSIN (ISD Marm and STD Marm)

4.6 Isolated and standard marmelosin (ISD Marm and STD Marm) on working rat heart

4.6.1 At fixed physiological preload and afterload

4.6.1.1 Effect of the ISD Marm The ISD Marm was administered in rat working heart at the concentrations of 0.0001, 0.01, 1.0, 100.0 and 10000.0 µM respectively. The table (4.6.1.1) is indicating the effects of ISD Marm at different parameters.

4.6.1.1.1 Effect on coronary effluent The result is shown in (Fig. 4.6.1.1.1). The isolated marmelosin decreased the coronary effluent in a concentration-dependent manner; at the concentrations (µM) of 0.0001 (96.966 ± 2.346), 0.01 (96.966 ± 2.346), 1.0 (88.761 ± 2.461), 100.0 (86.635 ± 2.901) and 10000.0 (83.227 ± 5.077) respectively as compared to control (99.741 ± 0.2038). It exhibited the statistically significant effect on the coronary effluent at the concentrations (µM) of 100.0

(p <0.05) and 10000.0 (p <0.01) respectively. The EC50 value was 0.5825µM (95 % CI, 0.02356 to 14.41).

4.6.1.1.2 Effect on aortic outflow The result is shown in (Fig. 4.6.1.1.2). The isolated marmelosin increased the aortic outflow concentration-dependently; at the concentrations (µM) of 0.0001 (100.66 ± 2.775), 0.01 (101.51 ± 3.033), 1.0 (102.82 ± 2.810), 100.0 (103.39 ± 2.760) and 10000.0 (103.15 ± 2.938) respectively as compared to control (99.741 ± 0.2038). All of the values were statistically non- significant. The EC50 value was 0.01999 µM (95 % CI, 5.686e-008 to 7026).

4.6.1.1.3 Effect on cardiac output The result is shown in (Fig. 4.6.1.1.3). The cardiac output was slightly increased by isolated

Chapter 4: Result 219 marmelosin; at the concentrations (µM) of 0.0001 (99.452 ± 3.758), 0.01 (100.42 ± 3.507), 1.0 (101.21 ± 3.488), 100.0 (101.59 ± 3.480) and 10000.0 (101.88 ± 3.479) respectively as compared to control (99.741 ± 0.2038). None of the value was statistically significant. The

EC50 value was 0.01224 µM (95 % CI, 1.443e-009 to 103867).

4.6.1.1.4 Effect on dP/dt(max)

The result is shown in (Fig. 4.6.1.1.4). The dP/dt(max) was decreased by isolated marmelosin; at the concentrations (µM) of 0.0001 (97.531 ± 3.109), 0.01 (97.282 ± 2.824), 1.0 (97.334 ± 3.345), 100.0 (97.334 ± 3.345) and 10000.0 (91.564 ± 4.975) respectively against the control of (100.00 ± 0.3813). None of the value was statistically significant. The EC50 value was 135.0 µM (95 % CI, 0.4252 to 42850).

4.6.1.1.5 Effect on dP/dt(min)

The result is shown in (Fig. 4.6.1.1.5). The isolated marmelosin decreased the dP/dt(min) concentration-dependently; at the concentrations (µM) of 0.0001 (98.753 ± 2.988), 0.01 (92.102 ± 10.038), 1.0 (81.567 ± 6.814), 100.0 (74.059 ± 8.082) and 10000.0 (63.521 ± 6.279) respectively as compared to control (100.00 ± 0.3813). It showed a statistically significant effect at the concentration of 10000.0 µM (p <0.01). The EC50 value was 0.9309 µM (95 % CI, 0.07649 to 11.33).

4.6.1.1.6 Effect on systolic pressure The result is shown in (Fig. 4.6.1.1.6). The isolated marmelosin produced a negligible effect on systolic pressure; at the concentrations (µM) of 0.0001 (100.52 ± 3.128), 0.01 (100.26 ± 3.175), 1.0 (100.53 ± 3.294), 100.0 (100.17 ± 3.379), and 10000.0 (98.900 ± 3.442) respectively against the control (100.00 ± 0.3813). None of the value was statistically + significant. The EC50 value was 498.6 µM (95 % CI, 4.992e-013 to 4.978e 017).

4.6.1.1.7 Effect on diastolic pressure The result is shown in (Fig. 4.6.1.1.7). The isolated marmelosin showed minimal effect on diastolic pressure; at the concentrations (µM) of 0.0001 (99.653 ± 2.841), 0.01 (99.753 ± 2.770), 1.0 (99.408 ± 2.902), 100.0 (100.12 ± 2.944) and 10000.0 (98.926 ± 3.718) respectively as compared to control (100.00 ± 0.3813). None of the value was found statistically

Chapter 4: Result 220

+ significant. The EC50 value was 0.08671 µM (95 % CI, 0.0 to infinity).

4.6.1.1.8 Effect on heart rate The result is shown in (Fig. 4.6.1.1.8). The isolated marmelosin decreased the heart rate concentration-dependently. Heart rate decreased at the concentrations (µM) of 0.0001 (97.819 ± 2.973), 0.01 (94.492 ± 5.668), 1.0 (89.694 ± 5.563), 100.0 (81.447 ± 4.030) and 10000.0 (55.753 ± 6.985) respectively as compared to control (100.00 ± 0.3813). It decreased the heart rate statistically significant at the concentration of 10000.0 µM (p <0.001). The EC50 value was 207.1 µM (95 % CI, 49.06 to 874.5).

4.6.1.1.9 Effect on peak aortic systolic pressure The result is shown in (Fig. 4.6.1.1.9). The isolated marmelosin showed decrease-increase effect on peak aortic systolic pressure; at the concentrations (µM) of 0.0001 (101.20 ± 3.729), 0.01 (97.850 ± 3.225), 1.0 (99.641 ± 1.674), 100.0 (101.09 ± 1.633) and 10000.0 (101.29 ± 2.872) respectively as compared to control (100.00 ± 0.3813). None of the value was found + statistically significant. The EC50 value was 14.48 µM (95 % CI, 4.086e-014 to 5.224e 015).

4.6.1.1.10 Effect on end diastolic pressure The result is shown in (Fig. 4.6.1.110). The isolated marmelosin showed minimal change in end diastolic pressure; at the concentrations (µM) of 0.0001 (98.423 ± 2.933), 0.01 (98.855 ± 3.005), 1.0 (99.835 ± 2.787), 100.0 (98.066 ± 3.088) and 10000.0 (96.255 ± 4.622) respectively as compared to control (99.741 ± 0.2038). None of the value is found statistically significant. + The EC50 value was 198.2 µM (95 % CI, 0.0009825 to 3.997e 007).

4.6.1.1.11 Effect on ejection fraction The result is shown in (Fig. 4.6.1.1.11). The isolated marmelosin showed a negligible effect on ejection fraction; at the concentrations (µM) of 0.0001 (99.822 ± 2.812), 0.01 (99.402 ± 2.928), 1.0 (99.976 ± 3.226), 100.0 (98.823 ± 3.010) and 10000.0 (98.478 ± 3.005) respectively as compared to control (99.741 ± 0.2038). None of the value was found statistically + significant. The EC50 value was 43.99 µM (95 % CI, 5.190e-011 to 3.729e 013).

4.6.1.1.12 Effect on stroke volume

Chapter 4: Result 221

The result is shown in (Fig. 4.6.1.1.12). The isolated marmelosin decreased the stroke volume concentration-dependently; at the concentrations (µM) of 0.0001 (97.257 ± 1.960), 0.01 (92.505 ± 9.072), 1.0 (87.126 ± 6.859), 100.0 (79.335 ± 8.059) and 10000.0 (65.425 ± 5.280) respectively as compared to control (99.741 ± 0.2038). It showed a statistically significant effect at the concentration of 10000.0 µM (p <0.01). The EC50 value was 105.0 µM (95 % CI, 9.797 to 1126).

4.6.1.1.13 Effect on rate pressure product The result is shown in (Fig. 4.3.4.13). The isolated marmelosin showed a slight increase in rate pressure product at the concentration of 0.0001 µM followed by a decrease. The effect was at the concentrations (µM) of 0.0001 (100.77 ± 3.148), 0.01 (99.278 ± 3.053), 1.0 (96.971 ± 2.857), 100.0 (97.558 ± 2.787) and 10000.0 (96.033 ± 3.450) respectively as compared to control (99.741 ± 0.2038). None of the value was found statistically significant. The EC50 value was 0.01626 µM (95 % CI, 5.547e-006 to 47.65).

4.6.1.1.14 Effect on cardiac power The result is shown in (Fig. 4.3.4.14). The isolated marmelosin increased the cardiac power till the concentration of 1.0 µM followed by a decrease: at the concentrations (µM) of 0.0001 (102.02 ± 2.928), 0.01 (102.08 ± 2.741), 1.0 (102.08 ±2.859), 100.0 (101.70 ± 2.890) and 10000.0 (100.29 ± 2.554) respectively as compared to control (99.741 ± 0.2038). None of the value was found statistically significant. The EC50 value was 406.3 µM (95 % CI, 7.222e-008 to 2.285e+012).

4.6.1.2 Effect of the STD Marm The STD Marm was administered in rat working heart at the concentrations of 0.0001, 0.01, 1.0, 100.0 and 10000.0 µM respectively. The table (4.6.1.2) is indicating the effects of standard marmelosin on different parameters.

4.6.1.2.1 Effect on coronary effluent The result is shown in (Fig. 4.6.1.1.1). The standard marmelosin decreased the coronary effluent concentration-dependently; at the concentrations (µM) of 0.0001 (89.932 ± 2.785),

Chapter 4: Result 222

0.01 (87.252 ± 3.201), 1.0 (77.305 ± 3.329), 100.0 (2.466 ± 2.658) and 10000.0 (64.360 ± 2.411) respectively as compared to control (99.741 ± 0.2038). It produced significant effect at the concentrations (µM) of 0.01 (p <0.05), 1.0 (p <0.001), 100.0 (p <0.001) and 10000.0

(p <0.001). The EC50 value was 0.7999 µM (95 % CI, 0.1929 to 3.317).

4.6.1.2.2 Effect on aortic outflow The result is shown in (Fig. 4.6.1.1.2). The standard marmelosin increased the aortic outflow concentration-dependently. The aortic outflow was increased at the concentrations (µM) of 0.0001 (104.06 ± 3.567), 0.01 (104.93 ± 3.504), 1.0 (106.98 ± 3.638), 100.0 (107.11 ± 3.476) and 10000.0 (108.40 ± 3.658) respectively as compared to control (99.741 ± 0.2038). None of the value was found statistically significant. The EC50 value was 0.2580 µM (95 % CI, 5.727e- 008 to 1.162e+006).

4.6.1.2.3 Effect on cardiac output The result is shown in (Fig. 4.6.1.1.3). The standard marmelosin increased the cardiac output; at the concentrations (µM) of 0.0001 (100.74 ± 3.728), 0.01 (100.93 ± 3.769), 1.0 (100.97 ± 3.935), 100.0 (101.54 ± 3.819) and 10000.0 (101.64 ± 3.868) as compared to control (99.741 ± + 0.2038) with EC50 value 5.211 µM (95 % CI, 0.0 to 1.596e 037). None of the value was found statistically significant.

4.6.1.2.4 Effect on dP/dt(max)

The result is shown in (Fig. 4.6.1.1.4). The standard marmelosin decreased the dP/dt(max) concentration-dependently. The dP/dt(max) was decreased at the concentrations (µM) of 0.0001 (91.842 ± 5.732), 0.01 (90.752 ± 6.379), 1.0 (90.550 ± 7.296), 100.0 (88.865 ± 7.275) and 10000.0 (85.325 ± 6.760) as compared to control (100.00 ± 0.3813). None of the value was found statistically significant. The EC50 value was 163.8 µM (95 % CI, 0.002082 to 1.289e+007).

4.6.1.2.5 Effect on dP/dt(min)

The result is shown in (Fig. 4.6.1.1.5). The standard marmelosin decreased the dP/dt(min) which remained almost steady at all concentrations. The effect was at the concentrations (µM) of 0.0001 (96.364 ± 3.841), 0.01 (96.119 ± 3.829), 1.0 (96.212 ± 4.223), 100.0 (95.712 ±

Chapter 4: Result 223

3.818) and 10000.0 (94.758 ± 3.805) respectively as compared to control (100.00 ± 0.3813).

None of the value was found statistically significant. The EC50 value was 188.0 µM (95 % CI, 6.864e-010 to 5.151e+013).

4.6.1.2.6 Effect on systolic pressure The result is shown in (Fig. 4.6.1.1.6). The standard marmelosin decreased the systolic pressure; at the concentrations (µM) of 0.0001 (95.212 ± 4.134), 0.01 (95.013 ± 3.922), 1.0 (95.083 ± 3.869), 100.0 (94.525 ± 3.841) and 10000.0 (94.072 ± 3.859) respectively against the control (100.00 ± 0.3813). None of the value was found statistically significant. The EC50 value was 79.01 µM (95 % CI, 1.243e-014 to 4.755e+017).

4.6.1.2.7 Effect on diastolic pressure The result is shown in (Fig. 4.6.1.1.7). The standard marmelosin decreased the diastolic pressure; at the concentrations (µM) of 0.0001 (89.259 ± 9.559), 0.01 (88.940 ± 9.603), 1.0 (89.042 ± 9.636), 100.0 (89.523 ± 9.840) and 10000.0 (89.668 ± 9.786) respectively as compared to control (100.00 ± 0.3813). None of the value was found statistically significant. + The EC50 value was 36.08 µM (95 % CI, 0.0 to infinity).

4.6.1.2.8 Effect on heart rate The result is shown in (Fig. 4.6.1.1.8). The standard marmelosin decreased the heart rate; at the concentrations (µM) of 0.0001 (88.722 ± 9.330), 0.01 (87.545 ± 9.823), 1.0 (87.273 ± 9.632), 100.0 (86.544 ± 10.193) and 10000.0 (85.939 ± 9.928) respectively as compared to control (100.00 ± 0.3813). None of the value was found statistically significant. The EC50 value was 1.180 µM (95 % CI, 0.0 to 1.522e+020).

4.6.1.2.9 Effect on peak aortic systolic pressure The result is shown in (Fig. 4.6.1.1.9). The standard marmelosin showed minimal change in peak aortic systolic pressure; at the concentrations (µM) of 0.0001 (96.628 ± 1.748), 0.01 (98.951 ± 3.192), 1.0 (99.195 ± 1.647), 100.0 (99.066 ± 2.297) and 10000.0 (99.915 ± 2.909) respectively as compared to control (99.741 ± 0.2038). None of the value was found statistically significant. The EC50 value was 0.001806 µM (95 % CI, 3.947e-010 to 8262).

Chapter 4: Result 224

4.6.1.2.10 Effect on end diastolic pressure The result is shown in (Fig. 4.6.1.1.10). The standard marmelosin decreased the end diastolic pressure; at the concentrations (µM) of 0.0001 (96.926 ± 8.365), 0.01 (93.128 ± 7.103), 1.0 (92.163 ± 6.365), 100.0 (91.728 ± 7.144) and 10000.0 (91.459 ± 6.217) respectively as compared to control (99.741 ± 0.2038). None of the value was found statistically significant.

The EC50 value was 0.003451 µM (95 % CI, 3.278e-010 to 36333).

4.6.1.2.11 Effect on ejection fraction The result is shown (Fig. 4.6.1.1.11). The standard marmelosin increased the ejection fraction insignificantly till the concentration of 1.0 µM followed by slight decrease. The effect was at the concentrations (µM) of 0.0001 (100.93 ± 3.743), 0.01 (102.33 ± 3.621), 1.0 (103.37 ± 3.677), 100.0 (102.77 ± 4.191) and 10000.0 (101.13 ± 3.773) respectively as compared to control (99.741 ± 0.2038). The effect of standard marmelosin on ejection fraction was not significant. The software could not converge the data to get EC50 value.

4.6.1.2.12 Effect on stroke volume The result is shown (Fig. 4.6.1.1.12). The standard marmelosin showed insignificant increase- decrease pattern; at the concentrations (µM) of 0.0001 (99.103 ± 3.923), 0.01 (99.817 ± 3.700), 1.0 (100.32 ± 3.702), 100.0 (99.895 ± 3.717) and 10000.0 (97.423 ± 3.782) respectively as compared to control (99.741 ± 0.2038). The effect of standard marmelosin on stroke volume was statistically not significant. The software could not converge the data to get EC50 value.

4.6.1.2.13 Effect on rate pressure product The result is shown in (Fig. 4.6.1.1.13). The standard marmelosin decreased the rate pressure product; at the concentrations (µM) of 0.0001 (84.343 ± 9.104), 0.01 (85.352 ± 10.903), 1.0 (86.534 ± 10.417), 100.0 (83.311 ± 10.553) and 10000.0 (84.561 ± 11.206) respectively as compared to control (99.741 ± 0.2038). The values were statistically non-significant. The EC50 value was 13.25 µM (95 % CI, 0.0 to +infinity).

4.6.1.2.14 Effect on cardiac power The result is shown in (Fig. 4.6.1.1.14). The standard marmelosin increased the cardiac power

Chapter 4: Result 225 till the concentration of 0.01 µM followed by decrease concentration-dependently. The effect was at the concentrations (µM) of 0.0001 (102.47 ± 4.046), 0.01 (103.11 ± 4.023), 1.0 (102.87 ± 3.877), 100.0 (102.23 ± 3.912) and 10000.0 (100.90 ± 3.674) respectively as compared to control (99.741 ± 0.2038). The effect was statistically non-significant. The EC50 value was 233.1 µM (95 % CI, 7.704e-008 to 7.051e+011).

4.6.2 At variable preload The effect of various preloads on control and pre-treated isolated rat working heart by different concentrations of ISD Marm was studied to construct the Frank-Starling curve, while keeping the afterload fixed at the physiological level.

4.6.2.1 Pretreatment at the concentration of 1µM The table (4.6.2.1) indicated the effects of changing preload in control and pretreated hearts at different parameters;

Effect on coronary effluent

At 5 cmH2O (control 100.499, pretreated 45.4596), 10 cmH2O (control 91.0963, pretreated

39.168), 15 cmH2O (control 83.0041, pretreated 83.0041), 20 cmH2O (control87.3716, pretreated 41.0921) and 25 cmH2O (control 81.3716, pretreated 81.3716) (Fig. 4.6.2.1.1)

Effect on aortic flow out

At5 cmH2O (control 113.648, pretreated 144.178), 10 cmH2O (control 111.651, pretreated

140.212), 15 cmH2O (control 140.212, pretreated 141.137), 20 cmH2O (control112.759, pretreated 143.057) and 25 cmH2O (control 115.053, pretreated 142.745) (Fig. 4.6.2.1.2)

Effect on cardiac output

At 5 cmH2O (control 117.883, pretreated 115.425), 10 cmH2O (control 112.276, pretreated

110.518), 15 cmH2O (control 113.296, pretreated 109.957), 20 cmH2O (control112.233, pretreated 111.25) and 25 cmH2O (control 113.213, pretreated 111.217) (Fig. 4.6.2.1.3)

Effect on dP/dt(max)

Chapter 4: Result 226

At 5 cmH2O (control 97.6024, pretreated 66.8883), 10 cmH2O (control 96.3947, pretreated

61.3715), 15 cmH2O (control 94.5057, pretreated 57.4483), 20 cmH2O (control92.6902, pretreated 52.4999) and 25 cmH2O (control 91.2197, pretreated 50.7116) (Fig. 4.6.2.1.4)

Effect on dP/dt(min)

At 5 cmH2O (control 98.8074, pretreated 73.8194), 10 cmH2O (control 99.2004, pretreated

70.5162), 15 cmH2O (control 97.9108, pretreated 67.9064), 20 cmH2O (control 97.9585, pretreated 68.0889) and 25 cmH2O (control 97.4722, pretreated 67.8669) (Fig. 4.6.2.1.5)

Effect on heart rate

At 5 cmH2O (control 90.0129, pretreated 64.2234), 10 cmH2O (control 87.5864, pretreated

60.4152), 15 cmH2O (control 85.5394, pretreated 60.2133), 20 cmH2O (control 83.8857, pretreated 60.3677) and 25 cmH2O (control 80.9595, pretreated 60.041) (Fig. 4.6.2.1.6)

Effect on left ventricular pressure

At 5 cmH2O (control 99.0662, pretreated 104.024), 10 cmH2O (control 99.1419, pretreated

101.439), 15 cmH2O (control 98.8914, pretreated 100.426), 20 cmH2O (control 99.6879, pretreated 101.049) and 25 cmH2O (control 98.9223, pretreated 99.7899) (Fig. 4.6.2.1.7)

Effect on peak aortic systolic pressure

At 5 cmH2O (control 103.86, pretreated 96.47), 10 cmH2O (control 102.855, pretreated

96.7366), 15 cmH2O (control 101.939, pretreated 94.7637), 20 cmH2O (control 98.7873, pretreated 93.4523) and 25 cmH2O (control 100.705, pretreated 92.4295) (Fig. 4.6.2.1.8)

Effect on coronary vascular resistance

At 5 cmH2O (control 104.458, pretreated 202.662), 10 cmH2O (control 107.166, pretreated

234.891), 15 cmH2O (control 109.287, pretreated 250.223), 20 cmH2O (control 106.739, pretreated 106.739) and 25 cmH2O (control 114.221, pretreated 269.807) (Fig. 4.6.2.1.9)

Effect on myocardial work

Chapter 4: Result 227

At 5 cmH2O (control 116.657, pretreated 113.727), 10 cmH2O (control 115.313, pretreated

115.313), 15 cmH2O (control 113.561, pretreated 105.067), 20 cmH2O (control 108.318, pretreated 105.676) and 25 cmH2O (control 110.752, pretreated 104.688) (Fig. 4.6.2.1.10)

4.6.2.2 Pretreatment at the concentration of 10µM The table (4.6.2.2) indicated the effects of changing preload in control and pretreated hearts at different parameters;

Effect on coronary effluent

At 5 cmH2O (control 97.3148, pretreated 57.5795), 10 cmH2O (control 96.2963, pretreated

51.6342), 15 cmH2O (control 98.0159, pretreated 54.4049), 20 cmH2O (control92.5053, pretreated 47.5407) and 25 cmH2O (control 94.1799, pretreated 50.2982) (Fig. 4.6.2.2.1)

Effect on aortic flow out

At5 cmH2O (control 99.938, pretreated 123.67), 10 cmH2O (control 99.5498, pretreated

124.682), 15 cmH2O (control 100.479, pretreated 127.215), 20 cmH2O (control99.8389, pretreated 127.124) and 25 cmH2O (control 100.577, pretreated 126.276) (Fig. 4.6.2.2.2)

Effect on cardiac output

At 5 cmH2O (control 99.9422, pretreated 101.043), 10 cmH2O (control 100.266, pretreated

101.136), 15 cmH2O (control 100.765, pretreated 101.276), 20 cmH2O (control100.576, pretreated 101.05) and 25 cmH2O (control 100.451, pretreated 99.994) (Fig. 4.6.2.2.3)

Effect on dP/dt(max)

At 5 cmH2O (control 100.046, pretreated 58.8826), 10 cmH2O (control 101.977, pretreated

52.0556), 15 cmH2O (control 99.5847, pretreated 48.942), 20 cmH2O (control 98.6191, pretreated 46.6483) and 25 cmH2O (control 95.9197, pretreated 45.6044) (Fig. 4.6.2.2.4)

Effect on dP/dt(min)

At 5 cmH2O (control 102.472, pretreated 65.7206), 10 cmH2O (control 103.438, pretreated

Chapter 4: Result 228

60.394), 15 cmH2O (control 102.762, pretreated 58.999), 20 cmH2O (control 101.289, pretreated 57.4144) and 25 cmH2O (control 99.3273, pretreated 57.2083) (Fig. 4.6.2.2.5)

Effect on heart rate

At 5 cmH2O (control 91.9059, pretreated 84.7519), 10 cmH2O (control 90.1294, pretreated

79.2734), 15 cmH2O (control 88.3575, pretreated 79.2152), 20 cmH2O (control 86.8271, pretreated 76.8909) and 25 cmH2O (control 85.0944, pretreated 76.8908) (Fig. 4.6.2.2.6)

Effect on left ventricular pressure

At 5 cmH2O (control 100.509, pretreated 100.931), 10 cmH2O (control 100.642, pretreated

101.546), 15 cmH2O (control 100.336, pretreated 102.14), 20 cmH2O (control 100.985, pretreated 102.024) and 25 cmH2O (control 99.1992, pretreated 100.687) (Fig. 4.6.2.2.7)

Effect on peak aortic systolic pressure

At 5 cmH2O (control 99.4277, pretreated 90.03), 10 cmH2O (control 96.8674, pretreated

87.4152), 15 cmH2O (control 99.4221, pretreated 86.0118), 20 cmH2O (control 98.623, pretreated 86.2894) and 25 cmH2O (control 99.9426, pretreated 86.1887) (Fig. 4.6.2.2.8)

Effect on coronary vascular resistance

At 5 cmH2O (control 97.4439, pretreated 192.862), 10 cmH2O (control 98.9815, pretreated

224.337), 15 cmH2O (control 99.123, pretreated 216.402), 20 cmH2O (control 106.233, pretreated 268.121) and 25 cmH2O (control 101.885, pretreated 256.885) (Fig. 4.6.2.2.9)

Effect on myocardial work

At 5 cmH2O (control 96.9534, pretreated 90.3525), 10 cmH2O (control 95.7753, pretreated

86.1653), 15 cmH2O (control 94.775, pretreated 87.8462), 20 cmH2O (control 98.0506, pretreated 88.9824) and 25 cmH2O (control 93.7316, pretreated 86.258) (Fig. 4.6.2.2.10)

4.6.2.3 Pretreatment at the concentration of 100 µM The table (4.6.2.3) indicated the effects of changing preload in control and pretreated hearts

Chapter 4: Result 229 at different parameters;

Effect on coronary effluent

At 5 cmH2O (control 103.329, pretreated 35.1279), 10 cmH2O (control 96.0131, pretreated

30.3112), 15 cmH2O (control 96.25, pretreated 29.0859), 20 cmH2O (control90.9809, pretreated 26.3735) and 25 cmH2O (control 88.9916, pretreated 25.3982) (Fig. 4.6.2.3.1)

Effect on aortic flow out

At 5 cmH2O (control 103.269, pretreated 135.119), 10 cmH2O (control 103.195, pretreated

137.103), 15 cmH2O (control 104.381, pretreated 134.236), 20 cmH2O (control 102.961, pretreated 138.51) and 25 cmH2O (control 1406.114, pretreated 140.255) (Fig. 4.6.2.3.2)

Effect on cardiac output

At 5 cmH2O (control 102.932, pretreated 101.519), 10 cmH2O (control 102.941, pretreated

101.414), 15 cmH2O (control 103.125, pretreated 100.767), 20 cmH2O (control 101.44, pretreated 100.124) and 25 cmH2O (control 101.49, pretreated 100.849) (Fig. 4.6.2.3.3)

Effect on dP/dt(max)

At 5 cmH2O (control 98.3488, pretreated 63.4318), 10 cmH2O (control 96.4019, pretreated

61.879), 15 cmH2O (control 97.367, pretreated 61.2629), 20 cmH2O (control 95.9738, pretreated 59.809) and 25 cmH2O (control 95.834, pretreated 59.0155) (Fig.4.6.2.3.4)

Effect on dP/dt(min)

At 5 cmH2O (control 100.95, pretreated 90.1012), 10 cmH2O (control 100.965, pretreated

89.1464), 15 cmH2O (control 102.004, pretreated 87.5005), 20 cmH2O (control 101.971, pretreated 87.1541) and 25 cmH2O (control 102.329, pretreated 86.9305) (Fig. 4.6.2.3.5)

Effect on heart rate

At 5 cmH2O (control 99.9689, pretreated 75.6922), 10 cmH2O (control 101.098, pretreated

75.6894), 15 cmH2O (control 101.014, pretreated 75.6962), 20 cmH2O (control 100.612,

Chapter 4: Result 230

pretreated 75.706) and 25 cmH2O (control 99.3203, pretreated 75.702) (Fig. 4.6.2.3.6)

Effect on left ventricular pressure

At 5 cmH2O (control 100.333, pretreated 102.196), 10 cmH2O (control 99.7675, pretreated

102.371), 15 cmH2O (control 100.135, pretreated 102.788), 20 cmH2O (control 100.599, pretreated 103.482) and 25 cmH2O (control 100.928, pretreated 103.217) (Fig. 4.6.2.3.7)

Effect on peak aortic systolic pressure

At 5 cmH2O (control 103.794, pretreated 92.9946), 10 cmH2O (control 102.051, pretreated

96.1031), 15 cmH2O (control 103.381, pretreated 93.6457), 20 cmH2O (control 100.233, pretreated 94.5598) and 25 cmH2O (control 100.124, pretreated 93.4403) (Fig. 4.6.2.3.8)

Effect on coronary vascular resistance

At 5 cmH2O (control 130.542, pretreated 241.51), 10 cmH2O (control 133.96, pretreated

320.475), 15 cmH2O (control 137.287, pretreated 345.418), 20 cmH2O (control 138.171, pretreated 396.228) and 25 cmH2O (control 140.362, pretreated 422.954) (Fig. 4.6.2.3.9)

Effect on myocardial work

At 5 cmH2O (control 107.379, pretreated 93.137), 10 cmH2O (control 104.00, pretreated

95.7991), 15 cmH2O (control 106.856, pretreated 94.1925), 20 cmH2O (control 101.717, pretreated 94.2831) and 25 cmH2O (control 100.359, pretreated 94.8838) (Fig. 4.6.2.3.10)

EXPERIMENTS ON ISOLATED RAT AORTA

4.6.3 Effect of standard marmelosin on rat aorta

4.6.3.1 At baseline tension In this set of experiments, the STD Marm was tested on resting basal tension at the concentrations of 0.0001, 0.001, 0.01, 0.1, 1.0 and 10.0 µM respectively. The cumulative addition of STD Marm did not show either the stimulating or vasoconstrictor effect up to the concentration of 10.0 µM. None of the value was statistically significant. The EC50 value of

Chapter 4: Result 231 standard marmelosin on basal tension was 0.003819 µM (95 % CI, 2.567e-006 to 5.682) (Fig. 4.6.3.1) (Table. 4.6.3.1)

4.6.3.2 On phenylephrine pre-contracted rat aorta The STD Marm was tested on endothelium-intact aortic ring preparations, pre-contracted by PE (1 µM). The cumulative addition of marmelosin caused inhibition of PE-induced sustained contraction in endothelium-intact aortic rings concentration-dependently. It caused inhibition of contraction at the concentrations (µM) of 0.0001 (94.549 ± 3.803), 0.001 (87.167 ± 4.213), 0.01 (80.715 ± 5.547), 0.1 (72.575 ± 6.361), 1.0 (62.245 ± 5.240) and 10.0 (44.531 ± 3.740) respectively as compared to control (100.00 ± 0.3114). It showed a statistically significant inhibitory effect at the concentrations (µM) of 0.1 (p <0.01), 1.0 (p <0.001) and

10.0 (p <0.001) respectively. The EC50 value of STD Marm in endothelium-intact rings was 0.2575 µM (95 % CI, 0.06268 to 1.058) (Fig. 4.6.3.1) (Table. 4.6.3.1)

The STD Marm was tested on endothelium-denuded aortic rings pre-contracted by PE (1 µM). The cumulative addition of STD Marm showed an initial minimal stimulant effect up to the concentration of 0.01 µM followed by inhibition of contraction till the maximum concentration of 10.0 µM in a concentration-dependent manner. It showed a stimulant effect at the concentrations (µM) of 0.0001 (100.08 ± 3.298), 0.001 (101.26 ± 3.704) and inhibitory effect at the concentrations (µM) of 0.01 (99.839 ± 4.593), 0.1 (93.475 ± 7.476), 1.0 (87.466 ± 9.579) and 10.0 (81.555 ± 9.804) respectively as compared to control (99.741 ± 0.204). None of the value was statistically significant. The EC50 value for endothelium-denuded rings was 0.2077 µM (95 % CI, 0.004279 to 10.08) (Fig. 4.6.3.1) (Table. 4.6.3.1)

4.6.3.3 At baseline tension In this set of experiments, the STD Marm was tested on rat aorta at resting basal tension at the concentrations of 0.0001, 0.001, 0.01, 0.1, 1.0 and 10.0 µM respectively. The cumulative addition of STD Marm did not show either the stimulating or vasoconstrictor effect up to the concentration of 10.0 µM. None of the value was statistically significant. The EC50 value was 0.01273 µM (95 % CI, 6.766e-007 to 239.5) (Fig. 4.6.3.2) (Table. 4.6.3.2).

Chapter 4: Result 232

4.6.3.4 On K+80 mM pre-contracted rat aorta The STD Marm when tested on endothelium intact aortic rings pre-contracted by high K+ (80mM), the cumulative addition caused inhibition of contraction in a concentration- dependent manner. It produced an inhibitory effect at the concentrations (µM) of 0.0001 (98.070 ± 3.834), 0.001 (96.689 ± 3.895), 0.01 (94.612 ± 4.024), 0.1 (89.432 ± 5.267), 1.0 (86.434 ± 5.220) and 10.0 (82.396 ± 4.462) respectively as compared to control (100.00 ±

0.3813). The inhibition of contraction was statistically insignificant. The EC50 value for endothelium-intact rings was 0.07304 µM (95 % CI, 0.003037 to 1.756) (Fig. 4.6.3.2) (Table. 4.6.3.2).

The STD Marm was tested on endothelium-denuded aortic rings pre-contracted by high K+ (80mM). The cumulative addition of STD Marm produced inhibition of contraction in a concentration-dependent manner. It inhibits the contraction of endothelium-denuded rings at the concentrations (µM) of 0.0001 (98.114 ± 3.053), 0.01 (95.990 ± 3.030), 0.1 (93.438 ± 3.093), 1.0 (91.244 ± 3.962) and 10.0 (88.335 ± 5.324) respectively as compared to control

(100.00 ± 0.3813). None of the value was statistically significant. The respective EC50 value was 0.1347 µM (95 % CI, 0.001756 to 10.33) (Fig. 4.6.3.2) (Table. 4.6.3.2).

4.6.3.5 At baseline tension In this set of experiments, the STD Marm when tested on rat aorta at resting basal tension, the cumulative addition at the concentrations of 0.0001, 0.001, 0.01, 0.1, 1.0 and 10.0 µM respectively did not show either the stimulating or vasoconstrictor effect up to the concentration of 10.0 µM. None of the value was statistically significant. The EC50 value was 0.001928 µM (95 % CI, 4.510e-006 to 0.8241) (Fig. 4.6.3.3) (Table. 4.6.3.3).

4.6.3.6 On L-NAME-incubated and PE pre-contracted rat aorta The STD Marm was tested against L-NAME-incubated and PE pre-contracted endothelium- intact aortic rings. The cumulative addition of STD Marm showed inhibition of L-NAME- incubated and PE-induced contraction in endothelium-intact aortic rings concentration- dependently. It inhibits the contraction at the concentrations (µM) of 0.0001 (93.186 ±

Chapter 4: Result 233

3.893), 0.001 (89.467 ± 3.591), 0.01 (86.820 ± 3.796), 0.1 (81.862 ± 4.751), 1.0 (80.549 ± 3.791) and 10.0 (75.545 ± 4.062) respectively as compared to control (100.00 ± 0.3813). The statistically significant inhibitory effect was observed at the concentrations (µM) of 0.1

(p <0.05), 1.0 (p <0.05) and 10.0 (p <0.01) respectively. The EC50 value was 0.02458 µM (95 % CI, 0.001179 to 0.5124) (Fig. 4.6.3.3) (Table. 4.6.3.3).

Chapter 4: Result 234

DISCUSSION Isolated and Standard Marmelosin

Chapter 4: Result 235

Isolated marmelosin (ISD Marm) Load independent experiments were designed to demonstrate the effect of marmelosin isolated from fruit of A.marmelos in isolated working rat heart. The results of experiments showed that the ISD Marm exhibited dose-dependent reduction in coronary effluent. The vasoconstricting effect on coronary vessels was highly significant at higher concentrations. Enhancement of vascular tone may be due to rise in intracellular free Ca++ level. The active influx of Ca++occurs by two types of calcium channels; i) voltage-dependent Ca++channels (VDCs), activated by membrane depolarization and ii) receptor-operated Ca++channels (ROCCs), activated by activation of receptors which is not necessarily accompanied with depolarization. In vascular smooth muscle calcium also influxes passively (Bolton., 1979). Influx of calcium also guided through calcium release from internal stores as well (Hall et al., 2006; Burt., 2005). The vasoconstrictor effect of isolated marmelosin may be mediated through these pathways in coronary vessels.

The aortic outflow (AoF), cardiac output (CO) and cardiac power (CP) were found to be slightly increased. This may be due to the calcium modulating activity of marmelosin in myocardium. Nasa and associates described that calcium channel blockers like nifedipine, verapamil and (+) diltiazem also induce weak positive inotropic effect at lower concentrations in isolated perfused heart (Nasa et al., 1992). It is more likely that the calcium channel blocking activity of marmelosin may be responsible for the increase in AoF, CO and CP.

The dP/dt(max) was decreased slightly and dose-dependently while dP/dt(min) and heart rate were decreased promptly and dose-dependently with a highly significant reduction at higher concentration. Reduction in receptor activated sarcolemmal Ca++influx leads to decrease in rate of contraction and inhibition of membrane activated Ca++ influx through L-type Ca++ channels causes decrease in force of contraction in isolated hearts (Malecot and Trautwein., 1987; Fleckenstein., 1977; Conti et al., 1985). Depression of contractile function and reduction in heart rate also produce by calcium channel inhibitors such as verapamil at higher concentrations in isolated hearts (Buljubasic et al., 1991). cGMP is responsible for activation of cGMP-dependent protein kinase and induces phosphorylation of various proteins like

Chapter 4: Result 236 troponin-I. This produces subsequent depression of the myofilament response to calcium and decreases the intracellular calcium level and dephosphorylation of myosin light chain leading to decrease in rate and force of contraction (Xu et al., 2010). cGMP alters the L-type calcium current biphasically in ventricular myocytes and modifies the Ca++ influx via L-type sarcolemmal Ca++channel. It may cause inhibition of Ca++-ATPase mediated sarcoplasmic reticulum re-uptake of calcium as well (Hare., 2003; Salahdeen et al., 2012; Muller-Strahl., 2000). This effect was also reflected by the significant reduction in stroke volume. Therefore, it is more likely that isolated marmelosin mediated inhibitory effects through blocking of the Ca++ channels and through NO/cGMP pathway. ISD Marm did not affect the systolic pressure, diastolic pressure, peak aortic systolic pressure, end diastolic pressure, ejection fraction and rate pressure product considerably.

Standard marmelosin (STD Marm) STD Marm was studied to compare the effects of isolated one. Coronary effluent was decreased at lower doses so found to be more potent. It increased the aortic outflow considerably. Cardiac output, cardiac power and peak aortic systolic pressure were increased similarly. dP/dt(max) was found to be more reduced, whereas dP/dt(min) and heart rate showed less reduction. STD Marm slightly increased the ejection fraction, whereas stroke volume was not affected. Systolic pressure, diastolic pressure and end diastolic pressure showed decreasing effect. Effect on the rate pressure product was slightly more . There is no contradictory effect was found in isolated and standard marmelosin. The differentiating effect of ISD Marm may be attributed to some organic moiety attached to.

To elucidate the mode of action of marmelosin isolated aortic rings were employed. STD Marm was studied to explain the mechanisms involved inducing the effects on the myocardium and coronary vessels. ISD Marm was not used because it is not as pure as commercially available. Therefore, the aorta is discussed here deviating from the pattern of result. These experiments were designed to investigate the effect of marmelosin on PE- and high K+-induced contractions and to elucidate whether the vasodilator effect of marmelosin is endothelium-dependent or -independent. Rat aorta: is a prototype tissue (Ghayur and

Chapter 4: Result 237

Gilani.,2005) commonly employed in pharmacodynamics experiments to explore the blood pressure decreasing effect. In cardiovascular system, the substances diminishing the high K+- induced contractions would indicate L-type voltage-dependent CCBs while inhibition of the PE-induced peak responses would signify the blockade of the Ca++ influx through the ROCCs and release of Ca++ from SR (Karaki and Weiss., 1988; Taqvi et al., 2006; Buraei and Yang., 2010). Presence of intact endothelium plays pivotal role in modulating the vascular tone through the release of a variety of substances like (NO) nitric oxide (Jaffe., 1985; Taqvi et al., 2008).

The STD Marm did not show any vasoconstrictor effects on resting baseline tension when added cumulatively in isolated rat aortic preparations. The cumulative addition of marmelosin to the endothelium-intact and -denuded aortic rings (+veEnd and -veEnd) pre- contracted by PE (1 µM) induced concentration-dependent relaxation in both preparations. The relaxing effect of marmelosin was more potent and statistically significant at higher concentrations only in endothelium-intact rings. This explains that marmelosin accomplish the vasorelaxation by blocking the ROCCs and Ca++ release from SR stores to decrease intracellular Ca++ concentration. The marmelosin when added cumulatively to high K+ (80mM) pre-contracted endothelium-intact and-denuded aortic rings exhibited a concentration- dependent weak and insignificant relaxation in both preparations comparatively.

To confirm the endothelium-dependent vasorelaxant effect, L-NAME; nitric oxide synthase inhibitor was used to pre-incubate endothelium-intact rings. The cumulative addition of standard marmelosin to the L-NAME pre-incubated and PE pre-contracted endothelium- intact rings showed partial but dose-dependent and statistically significant relaxation in aortae.

NO/cGMP signaling pathway plays an essential role to generate vascular smooth muscle relaxation endothelium-dependently (Suzuki et al., 2007). The endothelial cells lining blood vessels produced a relaxing factor, named endothelium-derived relaxing factor (EDRF) originally termed by Furchgott and Zawadzki (1980), in response to many types of stimuli like chemical agents and mechanical stimulation (Moncada and Higgs., 1993; Chang et al., 1996).

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Endothelium-derived NO is mainly formed by endothelial nitric oxide synthase (eNOS)- mediated oxidation of L-arginine to L-citruline (Palmer et al., 1988; Xu et al., 2010) and its diffusion to VSMCs appears to activate soluble guanylate cyclase (sGC) to enhance the intracellular production of cGMP (Chang et al., 1996). cGMP activates protein kinase-G which activates phosphatases that inactivate the contractile element (myosin light chains) by inhibiting Ca++ influx and reducing the sensitivity of the contractile element to Ca++(Ignarro et al., 1986; Sato et al., 1988). The end result is vascular smooth muscle relaxation and allowing vasodilatation (Ignarro et al., 1986) suggesting that relaxation response of marmelosin is dependent on physiologically intact-endothelium. Endothelium-dependent involvement may also confirm by the inhibition of relaxation in L-NAME-incubated rings (Salahdeen et al., 2012; Furchgott and Zawadzki., 1980). In our study; L-NAME, a nitric oxide synthase inhibitor, inhibited the relaxing effect of marmelosin. The relaxation of aortae by marmelosin through EDRF may be one of the potent mechanisms.

To reveal the effect of changes in preload on myocardial contractile functions and to express the therapeutic role of marmelosin; Frank-Starling curve was constructed. Frank-Starling relationship can be used to predict cardiac performance with respect to preload changes (Wonderjem et al., 1991). The cardiac resting state influences the subsequent contractile state as the heart has potential to change its force of contraction and stroke volume in response to changes in preload (Frank., 1956; Starling., 1918; Patterson et al., 1914; Klabunde., 2012). Preload was first reduced acutely followed by stepwise increasing in ascending order in the absence and presence of marmelosin while after load kept fixed throughout the experiments.

Isolated marmelosin (ISD Marm) was studied instead of standard marmelosin (STD Marm) to construct the F-S curves. The dosage forms which are used in the Subcontinent of A.marmelos somehow contain the raw form of marmelosin in the preparations. The experiments of working heart, the isolated marmelosin showed quantitatively different potential effects on various parameters; indicating the presence of some organic or inorganic moiety attached to; showing more effectiveness of the isolated marmelosin than standard

Chapter 4: Result 239 marmelosin. So, this is worthwhile to understand the effects of isolated marmelosin at selected doses (1 µM, 10 µM and 100 µM) and compare by control values on myocardium under varying preload.

Pre-treatment of hearts by of ISD Marm caused an increase in aortic outflow (AoF), left ventricular pressure (LVP) and coronary vascular resistance (CVR) by decreasing the preload acutely while escalation in preload exerted load-dependent increasing effect at all doses. Cardiac output (CO) was increased by the acute reduction in preload at all doses, whereas a stepwise increase in preload caused slight attenuation in cardiac output. dP/dt(max), dP/dt(min) and heart rate (HR) were found to decrease by an acute reduction in preload and graded preload increment produced load-dependent decreasing effect at all doses. Acute preload reduction decreased the coronary effluent (CE) which continued during stepwise increase in preload. Peak aortic systolic pressure (PASP) was decreased by acute preload reduction and continued as preload was increased irrespective of increasing the concentrations. Myocardial work (MW) was increased by the acute reduction in preload at the dose of 1µM only, whereas increasing the preload decreased MW irrespective of the increasing concentrations.

The cardiac systolic performance was influenced by changing preloads which adjust beat-to- beat alteration in venous return and the cardiovascular response to stimuli (Olivier et al., 1987). Altered calcium handling and calcium sensitivity of troponin-C; increased sarcomere length resulted in increased calcium sensitivity of troponin-C is the outcome of preload changes. This augments the rate of cross-bridge cycling and eventually the amount of tension developed by the muscle fiber. Cardiac Ca++ signaling exhibits crucial role in contractile regulation (Klabunde., 2012; Endoh., 2008). An optimal myocardial expansion increases the contractile force with little alteration of intracellular calcium; prior to stretch-induced slowly developing enhancement in contractile force coupled with arise in intracellular Ca++mobilization (Endoh., 2008). Endogenous NO augments the Frank-Starling response in isolated heart (Prendergast et al., 1997); continuously release from the endocardial endothelium (EE) under physiological conditions to regulate the Ca++ handling in cardiac myocytes; hence the contractile function of the myocardium (Paulus and Shah., 1999). It is

Chapter 4: Result 240 most likely that calcium channels modulating activity of marmelosin in cardiac muscles may influence to enhance LVP, AoF, CO and MW respectively through NO release from EE.

ISD Marm reduced dP/dt(max), dP/dt(min), heart rate and peak aortic systolic pressure (PASP). It is well known that NO and cGMP elicit a contractile response in myocardium biphasically in a concentration-dependent manner. NO may be involved in the endothelial modulation of myocardial performance through cGMP regulation in the cardiac myocytes (Meulemans et al., 1988; Mohan et al., 1996). The NO plays important biological role in vascular and endocardial endothelium-dependent activity in cardiac and vascular muscle cells. NO increases the intracellular cGMP level by activation of soluble guanylate cyclase (sGC) which catalyzes the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP) (KOH et al., 2007). NO at higher concentrations caused a reduction in contractility of cardiomyocytes through increased intracellular cGMP concentration (Muller-Strahl et al., 2000; Mohan et al., 1996). cGMP produces inhibitory effect by: i) direct action on contractile proteins, ii) activating cGMP-dependent protein kinase and iii) phosphorylation of various proteins like troponin-I. cGMP produces depression of the myofilament response to calcium (Mohan et al., 1996). cGMP may cause inhibition of Ca++- ATPase mediated sarcoplasmic reticulum re-uptake of calcium as well (Hare., 2003; Salahdeen et al., 2012). cGMP alters the L-type calcium channel current in biphasic manner; the stimulatory effect at lower and inhibitory effect at higher concentrations and modifies the Ca++ influx via L-type sarcolemmal Ca++ channel in cardiac ventricular myocytes (Xu et al., 2010; Muller-Strahl., 2000). It is more likely that ISD Marm may act through the release of NO mediating cGMP subsequently one of the pathways in cardiac myocytes or converging the ultimate effect through all three pathways.

Profound decrease in coronary effluent by acute preload reduction reflecting the enhancement in coronary vascular tone which was reinforced by increasing preloads in ascending order. Role of Ca++ in regulation of vascular tone is obvious because the modulation of calcium channels resulted in increased calcium influx through L-type calcium channels at lower concentrations and produce calcium agonistic activity. High intracellular

Chapter 4: Result 241 free Ca++ level through membrane depolarization elicit contraction in coronary artery by promoting the Ca++ entry via membrane L-type Ca++ channels (Somlyo and Himpens., 1989; Kalsner., 1994). While activation of membrane receptors provokes contraction of vessels by increasing the intracellular Ca++ through release from the sarcoplasmic reticulum and influx through ROCCs (Karaki et al, 1997). Raise intracellular Ca++ level leading to the enhancement of vascular smooth muscle contraction in coronary vessels. This effect is also reflected by an increase in CVR. The increase in coronary vascular tone by ISD Marm may be mediated through these pathways in coronary vessels.

Chapter 4: Result 242

TABLES

Chapter 4: Result 243

Table 4.6.1.1 Effects of isolated marmelosin on various parameters in isolated rat working heart Parameters Concentration (µM) Control 0.0001 0.01 1.0 100.0 10000.0 Coronary effluent 99.741 96.966 93.007 88.761 86.635* 83.227** (mL/min) ± 0.2038 ± 2.346 ± 2.696 ± 2.461 ± 2.901 ± 5.077 Aortic out flow 99.741 100.66 101.51 102.82 103.39 103.15 (mL/min) ± 0.2038 ± 2.775 ± 3.033 ± 2.810 ± 2.760 ± 2.938 Cardiac output 99.741 99.452 100.42 101.21 101.59 101.88 (mL/min) ± 0.2038 ± 3.758 ± 3.507 ± 3.488 ± 3.480 ± 3.479

dP/dt(max) 100.00 97.531 97.282 97.334 94.882 91.564 (mm Hg/s) ± 0.3813 ± 3.109 ± 2.824 ± 3.345 ± 3.328 ± 4.975

dP/dt(min) 100.00 98.753 92.102 81.567 74.059 63.521** (mm Hg/s) ± 0.3813 ± 2.988 ± 10.038 ± 6.814 ± 8.082 ± 6.279 Systolic pressure 100.00 100.52 100.26 100.53 100.17 98.900 (mm Hg) ± 0.3813 ± 3.128 ± 3.175 ± 3.294 ± 3.379 ± 3.442 Diastolic pressure 100.00 99.653 99.753 99.408 100.12 98.926 (mm Hg) ± 0.3813 ± 2.841 ± 2.770 ± 2.902 ± 2.944 ± 3.718 Heart rate 100.00 97.819 94.492 89.694 81.447 55.753*** (BPM) ± 0.3813 ± 2.973 ± 5.668 ± 5.563 ± 4.030 ± 6.985 Peak aortic systolic 100.00 101.20 97.850 99.641 101.09 101.290 pressure (mm Hg) ± 0.3813 ± 3.729 ± 3.225 ± 1.674 ± 1.633 ± 2.872 End diastolic 99.741 98.423 98.855 99.835 98.066 96.255 pressure (mm Hg) ± 0.2038 ± 2.933 ± 3.005 ± 2.787 ± 3.088 ± 4.622 Ejection fraction 99.741 99.822 99.402 99.976 98.823 98.478 (%) ± 0.2038 ± 2.812 ± 2.928 ± 3.226 ± 3.010 ± 3.005 Stroke volume 99.741 97.257 92.505 87.126 79.335 65.425** (mm Hg) ± 0.2038 ± 1.960 ± 9.072 ± 6.859 ± 8.059 ± 5.280 Rate pressure 99.741 100.77 99.278 96.971 97.558 96.033 product mm Hg/min) ± 0.2038 ± 3.148 ± 3.053 ± 2.857 ± 2.787 ± 3.450 Cardiac power 99.741 102.02 102.08 102.08 101.70 100.29 ± 0.2038 ± 2.928 ± 2.741 ± 2.859 ± 2.890 ± 2.554 * p<0.05, ** p<0.01, *** p<0.001

Chapter 4: Result 244

Table 4.6.1.2 Effects of standard marmelosin on various parameters in isolated rat working heart Parameters Concentration (µM) Control 0.0001 0.01 1.0 100.0 10000.0 Coronary effluent 99.741 89.932 87.252* 77.305*** 72.466*** 64.360*** (mL/min) ± 0.2038 ± 2.785 ± 3.201 ± 3.329 ± 2.658 ± 2.411 Aortic out 99.741 104.06 104.93 106.98 107.11 108.40 flow(mL/min) ± 0.2038 ± 3.567 ± 3.504 ± 3.638 ± 3.476 ± 3.658 Cardiac output 99.741 100.74 100.93 100.97 101.54 101.64 (mL/min) ± 0.2038 ± 3.728 ± 3.769 ± 3.935 ± 3.819 ± 3.868

dP/dt(max) 100.00 91.842 90.752 90.550 88.865 85.325 (mm Hg/s) ± 0.3813 ± 5.732 ± 6.379 ± 7.296 ± 7.275 ± 6.760

dP/dt(min) 100.00 96.364 96.119 96.212 95.712 94.758 (mm Hg/s) ± 0.3813 ± 3.841 ± 3.829 ± 4.223 ± 3.818 ± 3.805 Systolic pressure 100.00 95.212 95.013 95.083 94.525 94.072 (mm Hg) ± 0.3813 ± 4.134 ± 3.922 ± 3.869 ± 3.841 ± 3.859 Diastolic pressure 100.00 89.259 88.940 89.042 89.523 89.668 (mm Hg) ± 0.3813 ± 9.559 ± 9.603 ± 9.636 ± 9.840 ± 9.786 Heart rate 100.00 88.722 87.545 87.273 86.544 85.939 (BPM) ± 0.3813 ± 9.330 ± 9.823 ± 9.632 ± 10.193 ± 9.928 Peak aortic systolic 99.741 96.628 98.951 99.195 99.066 99.915 pressure(mm Hg) ± 0.2038 ± 1.748 ± 3.192 ± 1.647 ± 2.297 ± 2.909 End diastolic pressure 99.741 96.926 93.128 92.163 91.728 91.459 (mm Hg) ± 0.2038 ± 8.365 ± 7.103 ± 6.365 ± 7.144 ± 6.217 Ejection fraction 99.741 100.93 102.33 103.37 102.77 101.13 (%) ± 0.2038 ± 3.743 ± 3.621 ± 3.677 ± 4.191 ± 3.773 Stroke volume 99.741 99.103 99.817 100.32 99.895 97.423 (mm Hg) ± 0.2038 ± 3.923 ± 3.700 ± 3.702 ± 3.717 ± 3.782 Rate pressure product 99.741 84.343 85.352 86.534 83.311 84.561 (mm Hg/min) ± 0.2038 ± 9.104 ± 10.903 ± 10.417 ± 10.553 ± 11.206 Cardiac power 99.741 102.47 103.11 102.87 102.23 100.90 ± 0.2038 ± 4.046 ± 4.023 ± 3.877 ± 3.912 ± 3.674 * p<0.05,*** p<0.001

Chapter 4: Result 245

Table 4.6.2.1 Effect of variable preloads on isolated rat working heart with or without marmelosin isolated from A.marmelos (1 µM)

Pre-loads (cmH2O) Parameters 5 10 15 20 25

Coronary effluent Control 100.499 91.0963 83.0041 87.3716 81.3716 (mL/min) Pretreated 45.4596 39.168 38.5331 41.0921 37.5269

Aortic out flow Control 113.648 111.651 114.735 112.759 115.053 (mL/min) Pretreated 144.178 140.212 141.137 143.057 142.745

Cardiac output Control 117.883 112.276 113.296 112.233 113.213 (mL/min) Pretreated 115.425 110.518 109.957 111.25 111.217 Control 97.6024 96.3947 94.5057 92.6902 91.2197 dP/dt(max) (mm Hg/s) Pretreated 66.8883 61.3715 57.4483 52.4999 50.7116 Control 98.8074 99.2004 97.9108 97.9585 97.4722 dP/dt(min) (mm Hg/s) Pretreated 73.8194 70.5162 67.9064 68.0889 67.8669

Heart rate Control 90.0129 87.5864 85.5394 83.8857 80.9595 (BPM) Pretreated 64.2234 60.4152 60.2133 60.3677 60.041

Left ventricular Control 99.0662 99.1419 98.8914 99.6879 98.9223 pressure (mm Hg) Pretreated 104.024 101.439 100.426 101.049 99.7899

Peak aortic systolic Control 103.86 102.855 101.939 98.7873 100.705 pressure (mm Hg) Pretreated 96.47 96.7366 94.7637 93.4523 92.4295 Coronary vascular Control 104.458 107.166 109.287 106.739 114.221 resistance (mm Pretreated 202.662 234.891 250.223 237.944 269.807 Hg/mL x min)

Myocardial work Control 116.657 115.313 113.561 108.318 110.752 (mm Hg x mL/min) Pretreated 113.727 106.482 105.067 105.676 104.688

Chapter 4: Result 246

Table 4.6.2.2 Effect of variable pre-loads on isolated rat working heart with or without marmelosin isolated from A.marmelos (10 µM)

Pre-loads (cmH2O) Parameters 5 10 15 20 25

Coronary effluent Control 97.3148 96.2963 98.0159 92.5053 94.1799 (mL/min) Pretreated 57.5795 51.6342 54.4049 47.5407 50.2982

Aortic out flow Control 99.938 99.5498 100.479 99.8389 100.577 (mL/min) Pretreated 123.67 124.682 127.215 127.124 126.276

Cardiac output Control 99.9422 100.266 100.765 100.576 100.451 (mL/min) Pretreated 101.043 101.136 101.276 101.05 99.994 Control 100.046 101.977 99.5847 98.6191 95.9197 dP/dt(max) (mm Hg/s) Pretreated 58.8826 52.0556 48.942 46.6483 45.6044 Control 102.472 103.438 102.762 101.289 99.3273 dP/dt(min) (mm Hg/s) Pretreated 65.7206 60.394 58.999 57.4144 57.2083

Heart rate Control 91.9059 90.1294 88.3575 86.8271 85.0944 (BPM) Pretreated 84.7519 79.2734 79.2152 76.8909 76.8908

Left ventricular Control 100.509 100.642 100.336 100.985 99.1992 pressure (mm Hg) Pretreated 100.931 101.546 102.14 102.024 100.687

Peak aortic systolic Control 99.4277 96.8674 99.4221 98.623 99.9426 pressure (mm Hg) Pretreated 90.03 87.4152 86.0118 86.2894 86.1887 Coronary vascular Control 97.4439 98.9815 99.123 106.233 101.885 resistance (mm Pretreated 192.862 224.337 216.402 268.121 256.885 Hg/mL x min)

Myocardial work Control 96.9534 95.7753 94.775 98.0506 93.7316 (mm Hg x mL/min) Pretreated 90.3525 86.1653 87.8462 88.9824 86.258

Chapter 4: Result 247

Table 4.6.2.3 Effect of variable pre-loads on isolated rat working heart with or without marmelosin isolated from A.marmelos (100 µM)

Pre-loads (cmH2O) Parameters 5 10 15 20 25

Coronary effluent Control 103.329 96.0131 96.25 90.9809 88.9916 (mL/min) Pretreated 35.1279 30.3112 29.0859 26.3735 25.3982

Aortic out flow Control 103.269 103.195 104.381 102.961 1406.114 (mL/min) Pretreated 135.119 137.103 134.236 138.51 140.255

Cardiac output Control 102.932 102.941 103.125 101.44 101.49 (mL/min) Pretreated 101.519 101.414 100.767 100.124 100.849 Control 98.3488 96.4019 97.367 95.9738 95.834 dP/dt(max) (mm Hg/s) Pretreated 63.4318 61.879 61.2629 59.809 59.0155 Control 100.95 100.965 102.004 101.971 102.329 dP/dt(min) (mm Hg/s) Pretreated 90.1012 89.1464 87.5005 87.1541 86.9305 Control 99.9689 101.098 101.014 100.612 99.3203 Heart rate (BPM) Pretreated 75.6922 75.6894 75.6962 75.706 75.702

Left ventricular Control 100.333 99.7675 100.135 100.599 100.928 pressure (mm Hg) Pretreated 102.196 102.371 102.788 103.482 103.217

Peak aortic systolic Control 103.794 102.051 103.381 100.233 100.124 pressure (mm Hg) Pretreated 92.9946 96.1031 93.6457 94.5598 93.4403

Coronary vascular Control 130.542 133.96 137.287 138.171 140.362 resistance (mm Pretreated 241.51 320.475 345.418 396.228 422.954 Hg/mL x min)

Myocardial work Control 107.379 104.00 106.856 101.717 100.359 (mm Hg x mL/min) Pretreated 93.137 95.7991 94.1925 94.2831 94.8838

Chapter 4: Result 248

Table 4.6.3.1 Effect of standard marmelosin on rat thoracic aorta pre-contracted by phenylephrine (1 µM) Parameters Concentration (µM) Control 0.0001 0.001 0.01 0.1 1.0 10.0 Baseline 99.764 96.736 94.387 92.379 91.012 90.380 89.294 tension ±0.261 ± 3.155 ± 2.914 ± 3.293 ± 4.831 ± 5.346 ± 5.713 Endothelium- 100.00 94.549 87.167 80.715 72.575** 62.245*** 44.531*** intact rings ± 0.311 ± 3.803 ± 4.213 ± 5.547 ± 6.361 ± 5.240 ± 3.740 Endothelium- 99.741 100.08 101.26 99.839 93.475 87.466 81.555 denuded rings ± 0.204 ± 3.298 ± 3.704 ± 4.593 ± 7.476 ± 9.579 ± 9.804 **p<0.01, *** p<0.001

Table 4.6.3.2 Effect of standard marmelosin on rat thoracic aorta pre-contracted by high K+ (80 mM) Parameters Concentration (µM) Control 0.0001 0.001 0.01 0.1 1.0 10.0 Baseline 99.908 97.321 96.009 95.369 94.585 94.023 93.047 tension ± 0.129 ± 2.986 ± 2.992 ± 2.844 ± 3.467 ± 3.501 ± 3.945 Endothelium- 100.00 98.070 96.689 94.612 89.432 86.434 82.396 intact rings ± 0.381 ± 3.834 ± 3.895 ± 4.024 ± 5.267 ± 5.220 ± 4.462 Endothelium- 100.00 98.114 96.728 95.990 93.438 91.244 88.335 denuded rings ± 0.381 ± 2.948 ± 3.053 ± 3.030 ± 3.093 ± 3.962 ± 5.324

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Table 4.6.3.3 Effect of standard marmelosin on L-NAME-incubated and PE pre-contracted rat thoracic aorta Parameters Concentration (µM) Control 0.0001 0.001 0.01 0.1 1.0 10.0 Baseline tension 99.676 97.986 94.887 92.379 91.012 90.380 89.294 ± 0.249 ± 3.249 ± 2.793 ± 3.293 ± 4.831 ± 5.346 ± 5.713 L-NAME-incubated and 100.00 93.186 89.467 86.820 81.862* 80.549* 75.545** PE pre-contracted ± 0.381 ± 3.893 ± 3.591 ± 3.796 ± 4.751 ± 3.791 ± 4.062 endothelium-intact rings * p<0.05, ** p<0.01

Chapter 4: Result 250

FIGURES

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Chapter 5: General Discussion 275

CHAPTER 5 GENERAL DISCUSSION Aegle marmelos fruit in cardiovascular disorders

Chapter 5: General Discussion 276

Since we studied the A.marmelos in detail so this is raison d’être to discuss the pharmacological rationale for the use in cardiovascular disorders comprehensively. The crude extract of the fruit of A.marmelos was found safe with a wide window of safety in acute oral toxicity testing studies in mice. Preliminary, the Langendorff’s heart model paved the way to study in detail. The working heart preparations showed more detailed results affecting different parameters related to clinical interest. The crude extract of A.marmelos, different fractions and isolated marmelosin were studied further to justify the use of the fruit scientifically. The agonistic and antagonistic effect on the vessel and the heart at lower and higher doses reflect the use of this fruit in various cardiovascular disorders accordingly.

Am.Cr has potential to reduce heart rate without imposing bradycardia in conditions like heart palpitation or tachycardia as it possesses the moderate negative chronotropic property. In conditions like hypotension and specifically postural hypotension; it acts as a circulatory stimulant and provoke a vasoconstrictive effect at lower doses to improve and maintain mean arterial pressure specially and body circulation generally will be regulated. In the case of hypertension, Am.Cr induces its beneficial effect at higher doses by reducing vascular resistance through two pathways first calcium channel blockade secondly nitric oxide induced vasodilatation. Additionally the cardiodepressant effect of Am.Cr reduces the damaging effect of high blood pressure.

In myocardial infarction and ischemia; reduction in ventricular contractile force and heart rate are the prerequisite to minimize further damage and progress of complications. Acute myocardial infarction can lead to serious complications like stroke, irregular heartbeats, heart failure and aneurysm in weakened heart chamber. This objective and goal can be achieved by Am.Cr at higher doses through its negative inotropic and chronotropic effects. Am.Cr through its cardiosuppressant activity minimize further damages next to tachycardia, hypertension and increased contractility and maintaining the functioning heart without growing demand of oxygen and energy.

In heart failure; decreased or compromised pumping functions; Am.Cr at lower doses improves cardiac performance by increasing aortic outflow, cardiac output, left ventricular

Chapter 5: General Discussion 277 pressure and myocardial work. The positive inotropy of Am.Cr; accompanied by an acceleration of the rate of contraction, relaxation of myocardium and slowing effect on heart rate without triggering bradycardia shows an additional advantage. This may explain its effective use in left-ventricular that is both systolic and diastolic heart failure. Moreover, by controlling hypertension; an etiologic and aggravating factor in cases of heart failure, it may prevent further deterioration of the condition.

In calcium paradox study the hearts when reperfused by calcium containing KH solution and

Am.Cr diminished the reduction and recuperating the parameters like dP/dt(max) and dP/dt(min) with retaining the heart rate near normal levels it showed therapeutic potential. The addition of Am.Cr in calcium free KH perfusion reduced the harmful effect of calcium paradox. The reperfusion by calcium and A.marmelos containing KH resulted in more beneficial effect. This indicates the prominent cardio-protective effect leading to the therapeutic outcome if the patient is taking the A.marmelos already in the therapeutic regimen.

These studies provide the basis of pharmacological rationale for the use of Am.Cr in treating various cardiovascular disorders like hypertension, hypotension, heart palpitation and/or tachycardia. It can be carefully advised in heart failure and myocardial infarction as well. More considerably, Am.Cr induces vasoconstriction in coronary vessels which may be harmful by encouraging the deleterious effect in ischemic heart disease and coronary artery disease. This restricted the usage of Am.Cr in these conditions and requires more careful application in ischemic conditions. This opens the avenue for further research possibilities in vivo and in vitro preparations.

Conclusion

The results showed cardiotonic, cardiosuppressant effects on heart muscle, vasoconstrictive and vasorelaxant activities in the aorta and coronary vessel at lower and higher doses which justify the use in various cardiovascular disorders. Therefore, A.marmelos can be used effectively in the management of hypotension, hypertension and heart palpitation or tachycardia. It can be used with cautions in heart failure and ischemic and infracted heart at different doses. The calcium agonistic and antagonistic and endothelium-dependent relaxant

Chapter 5: General Discussion 278 effect of the bioactive compounds present in the fruit of A.marmelos converges the therapeutic benefits in clinical settings. The result of this thesis may lead to new and attractive alternative for the safe and effective management of heart diseases / cardiovascular disorders.

279

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305

List of papers published or accepted form our recent project/thesis

1. Pharmacological rational of dry ripe fruit of Aegle marmelos L as an antinociceptive agent in different painful conditions. Atiq ur Rahman, hina Imran, S.Intasar H Taqvi,Tehmina Sohail, Zahra Yaqeen, Zakir Ur Rahman and Nudrat Fatima, Pak. J. Pharm. Sci., 2015; Vol. 28, No.2, pp.515-519.

2. Dry and ripe fruit of Aegle marmelos. L: A potent source of antioxidant, lipoxygenase inhibitors and free radical scavenger.

Atiq-ur-Rahman, Hina Imran, Lubna Iqbal, Syed Intasar Hussain Taqvi, Nudrat Fatima and Zahra Yaqeen. Pak. J. Pharm. Sci., 2016; Vol. 29, No.4, pp. 1127-1131.

3. Inotropic and Chronotropic Effects of Crude Extract and its Butanol Fraction of Dry Fruit of Aegle marmelos Linn. in Isolated Working Rat Heart. Atiq-ur-Rahman, Syed Intasar Husain Taqvi, Muhammad Ali Versiani and Amna Khatoon. J. Chem. Soc. Pak., 2016; 38, No. 04, pp. 693-699.

4. Studies to determine antidiarrhoeal and spasmolytic activities of extract of Aegle marmelos.L fruit. Syed Intasar Husain Taqvi, Atiq-ur-Rahman, Mohammad Ali Versiani, Hina Imran, Amna Khatoon and Tehmina Sohail. Bangl. J. Med. Sci., 2018. (Accepted)

306

APPENDICES Tables and Figures

Appendix No 1: Digoxin 307

Appendix No 1 Digoxin

Appendix No 1: Digoxin 308

Appendix No 1

Table 1.1 Effects of digoxin on various parameters in isolated rat working heart Parameters Concentration (µM) Control 0.001 0.01 0.1 1.0 10.0 Coronary effluent 99.741 90.284 88.063 86.642* 82.475** 80.346*** (mL/min) ± 0.2038 ± 3.527 ± 3.136 ± 3.036 ± 2.902 ± 2.961 Aortic out 99.741 104.32 107.07 107.34 108.24 108.71 flow(mL/min) ± 0.2038 ± 3.999 ± 4.069 ± 3.885 ± 3.892 ± 4.062 Cardiac 99.741 99.096 98.727 98.504 99.896 98.755 output(mL/min) ± 0.2038 ± 3.923 ± 3.649 ± 3.838 ± 3.520 ± 3.795 dP/dt(max) 99.741 94.796 95.625 95.855 93.830 91.850 (mm Hg/s) ± 0.2038 ± 2.783 ± 2.982 ± 2.767 ± 2.809 ± 2.767 dP/dt(min) 99.741 97.251 97.556 97.588 95.464 96.641 (mm Hg/s) ± 0.2038 ± 3.870 ± 3.789 ± 3.973 ± 3.966 ± 4.022 Systolic Pressure 99.741 97.649 97.893 97.925 97.609 97.597 (mm Hg) ± 0.2038 ± 2.978 ± 2.831 ± 3.188 ± 2.912 ± 2.764 Diastolic pressure 99.741 97.039 96.727 95.636 95.313 94.820 (mm Hg) ± 0.2038 ± 3.693 ± 3.543 ± 3.846 ± 4.022 ± 4.065 Heart rate 99.741 96.591 95.430 91.566 91.215 90.768 (BPM) ± 0.2038 ± 3.813 ± 4.650 ± 7.772 ± 8.109 ± 8.269 Peak aortic systolic 99.741 96.969 98.126 97.752 97.120 97.358 pressure (mm Hg) ± 0.2038 ± 2.967 ± 2.869 ± 2.976 ± 3.021 ± 3.058 End diastolic 99.741 98.041 97.579 97.688 97.812 98.327 pressure (mm Hg) ± 0.2038 ± 3.188 ± 2.939 ± 3.048 ± 3.064 ± 3.114 Ejection fraction 99.741 99.795 101.33 103.29 102.69 102.74 (%) ± 0.2038 ± 1.596 ± 3.035 ± 4.747 ± 4.734 ± 4.678 Stroke volume 99.741 98.455 100.53 103.09 102.78 102.94 (mm Hg) ± 0.2038 ± 2.747 ± 2.975 ± 3.418 ± 3.402 ± 3.313 Rate pressure 99.741 97.400 95.209 91.194 89.324 89.186 product ± 0.2038 ± 3.373 ± 5.050 ± 7.926 ± 8.091 ± 8.366 (mm Hg/min) Cardiac power 99.741 99.343 98.825 99.522 99.389 99.355 ± 0.2038 ± 2.800 ± 3.018 ± 2.796 ± 2.810 ± 2.846 * p<0.05, ** p<0.01, *** p<0.001

Appendix No 1: Digoxin 309

Table 1.2.1 Effect of variable pre-loads on isolated rat working heart with or without digoxin (0.0001 µM) Pre-loads (cmH2O) Parameters 5 10 15 20 25

Coronary effluent Control 98.625 99.2296 97.6335 97.3283 97.6463 (mL/min) Pretreated 59.4091 48.5434 38.3475 32.4877 26.2794

Aortic out flow Control 100.979 101.405 102.507 101.53 103.125 (mL/min) Pretreated 124.953 133.709 136.656 139.715 142.099

Cardiac output Control 99.3426 99.5892 99.9967 98.6671 98.8439 (mL/min) Pretreated 100.22 101.109 101.532 101.409 99.5239 Control 99.8163 100.608 99.9337 100.056 100.22 dP/dt(max) (mm Hg/s) Pretreated 50.8044 43.9995 39.2744 35.5974 32.3459 Control 101.037 100.833 100.412 99.729 99.2444 dP/dt(min) (mm Hg/s) Pretreated 48.2838 46.3651 45.0228 43.7304 43.1975

Heart rate Control 103.283 103.893 108.082 106.8 107.873 (BPM) Pretreated 71.3535 66.0618 58.2935 57.428 66.143

Left ventricular Control 98.9548 99.5609 99.7358 100.084 100.328 pressure (mm Hg) Pretreated 101.171 101.361 101.086 101.701 102.833

Peak aortic systolic Control 99.4475 100.196 100.353 100.141 99.2702 pressure (mm Hg) Pretreated 88.2313 86.7654 85.1072 84.2188 83.5606 Coronary vascular Control 98.8448 101.447 100.629 100.616 100.981 resistance (mm Pretreated 129.911 175.944 241.432 301.186 347.407 Hg/mL x min)

Myocardial work Control 100.369 100.265 100.323 99.9703 99.8593 (mm Hg x mL/min) Pretreated 88.8816 87.5005 85.973 85.2618 85.0138

Appendix No 1: Digoxin 310

Table 1.2.2 Effect of variable pre-loads on isolated rat working heart with or without digoxin (0.01 µM)

Pre-loads (cmH2O) Parameters 5 10 15 20 25

Coronary effluent Control 98.6992 97.0763 96.7311 95.1288 95.2014 (mL/min) Pretreated 60.2127 51.1186 48.1106 42.5084 39.237

Aortic out flow Control 100.552 100.616 100.935 101.513 103.149 (mL/min) Pretreated 118.791 120.128 123.48 124.297 127.551

Cardiac output Control 99.7442 99.4905 99.8073 99.8533 99.9185 (mL/min) Pretreated 100.436 100.803 101.49 101.562 100.685 Control 100.901 101.305 102.534 102.375 102.339 dP/dt(max) (mm Hg/s) Pretreated 61.6127 55.6012 51.7701 49.0169 47.8926 Control 99.2989 100.042 100.714 100.104 99.9461 dP/dt(min) (mm Hg/s) Pretreated 61.621 60.0326 59.2822 58.9493 58.4035

Heart rate Control 99.8619 99.1026 98.7126 85.8141 97.5382 (BPM) Pretreated 84.5653 80.4554 80.7044 80.0525 79.6795

Left ventricular Control 99.239 100.296 100.716 100.752 100.725 pressure (mm Hg) Pretreated 101.164 102.142 101.975 102.624 102.722

Peak aortic systolic Control 99.9406 100.76 100.747 101.319 101.007 pressure (mm Hg) Pretreated 90.4577 90.0576 88.578 88.8877 85.9384 Coronary vascular Control 100.992 102.72 101.696 102.542 104.282 resistance (mm Pretreated 158.985 204.07 218.341 253.393 274.969 Hg/mL x min)

Myocardial work Control 101.085 100.65 100.943 100.346 101.953 (mm Hg x mL/min) Pretreated 89.5861 87.3833 87.7413 87.1239 86.4394

Appendix No 1: Digoxin 311

Table 1.2.3 Effect of variable pre-loads on isolated rat working heart with or without digoxin (1 µM)

Pre-loads (cmH2O) Parameters 5 10 15 20 25

Coronary effluent Control 100.096 98.7109 99.4323 98.8932 98.1741 (mL/min) Pretreated 54.0693 48.4708 46.5898 42.5276 40.0895

Aortic out flow Control 97.0611 96.1189 97.4808 97.6945 97.9816 (mL/min) Pretreated 133.807 131.362 132.693 135.56 135.45

Cardiac output Control 99.8612 99.2395 99.5119 99.3043 99.272 (mL/min) Pretreated 104.378 102.824 102.914 102.06 101.732 Control 99.1206 100.95 101.343 102.068 107.667 dP/dt(max) (mm Hg/s) Pretreated 52.2408 47.5064 47.0654 46.5753 45.9336 Control 101.463 99.9524 100.789 101.643 100.063 dP/dt(min) (mm Hg/s) Pretreated 59.0751 58.9953 58.3671 59.0539 58.6575

Heart rate Control 98.6525 98.4514 99.6544 99.5623 99.531 (BPM) Pretreated 84.7115 83.8787 83.5398 83.5318 83.006

Left ventricular Control 99.1896 100.085 100.159 100.689 100.439 pressure (mm Hg) Pretreated 101.107 101.616 101.751 101.552 102.522

Peak aortic systolic Control 97.8867 98.0879 98.1445 98.1898 97.0213 pressure (mm Hg) Pretreated 86.3966 86.449 86.7206 84.3711 86.182 Coronary vascular Control 97.8896 97.8661 97.7591 98.0118 98.562 resistance (mm Pretreated 192.72 231.283 235.776 246.29 286.101 Hg/mL x min)

Myocardial work Control 98.118 98.3298 97.9425 97.8238 97.2131 (mm Hg x mL/min) Pretreated 90.9316 89.9984 88.3903 86.894 86.5892

Appendix No 1: Digoxin 312

Table 1.3.1 Effect of digoxin on rat thoracic aorta pre-contracted by PE (1 µM) Parameters Concentration (µM) Control 0.0001 0.001 0.01 0.1 1.0 10.0 Baseline 100.00 96.860 97.226 97.151 96.017 95.681 94.533 tension ± 0.3114 ± 3.113 ± 2.868 ± 2.970 ± 2.914 ± 2.867 ± 2.864 Endothelium- 100.01 97.135 96.061 95.250 93.731 93.155 93.406 intact rings ± 0.2632 ± 3.722 ± 3.786 ± 3.747 ± 3.677 ± 3.770 ± 3.879 Endothelium- 99.741 99.611 98.114 97.169 95.485 93.551 89.416 denuded rings ± 0.2038 ± 2.912 ± 3.078 ± 3.276 ± 3.903 ± 3.892 ± 3.906

Table 1.3.2 Effect of digoxin on rat thoracic aorta pre-contracted by high K+ (80 mM) Parameters Concentration (µM) Control 0.0001 0.001 0.01 0.1 1.0 10.0 Baseline 99.741 96.098 94.987 93.887 93.314 92.260 90.672 tension ± 0.204 ± 3.032 ± 2.852 ± 2.803 ± 2.863 ± 3.328 ± 3.228 Endothelium- 100.00 99.094 99.522 98.512 97.650 96.538 94.372 intact rings ± 0.311 ± 3.822 ± 3.916 ± 3.886 ± 3.835 ± 4.170 ± 3.966 Endothelium- 99.741 99.058 97.964 96.347 95.494 94.125 90.303 denuded rings ± 0.204 ± 3.243 ± 3.437 ± 3.446 ± 3.378 ± 3.514 ± 3.717

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Appendix No 1: Digoxin 314

Appendix No 1: Digoxin 315

Appendix No 1: Digoxin 316

Appendix No 1: Digoxin 317

Appendix No 1: Digoxin 318

Appendix No 1: Digoxin 319

Appendix No 1: Digoxin 320

Appendix No 1: Digoxin 321

Appendix No 1: Digoxin 322

Appendix No 1: Digoxin 323

Appendix No 1: Digoxin 324

Appendix No 1: Digoxin 325

Appendix No 1: Digoxin 326

Appendix No 1: Digoxin 327

Appendix No 1: Digoxin 328

Appendix No 1: Digoxin 329

Appendix No 1: Digoxin 330

Appendix No 1: Digoxin 331

332

Appendix No 2 Verapamil HCl

Appendix No 2: Verapamil Hcl 333

Appendix No 2

Table 2.1 Effects of verapamil on various parameters in isolated rat working hearts Parameters Concentration (µM) Control 0.01 0.1 1.0 10.0 Coronary effluent 99.741 94.079 91.164 91.440 89.326 (mL/min) ± 0.2038 ± 6.941 ± 7.352 ± 8.060 ± 7.647 Aortic out flow 99.741 107.84 107.92 109.49 108.93 (mL/min) ± 0.2038 ± 3.860 ± 3.976 ± 4.251 ± 4.304 Cardiac output 99.741 99.473 100.17 100.38 100.62 (mL/min) ± 0.2038 ± 2.887 ± 2.825 ± 3.138 ± 3.018

dP/dt(max) 99.741 101.72 100.82 93.599 85.560* (mm Hg/s) ± 0.2038 ± 2.820 ± 3.340 ± 3.473 ± 4.300

dP/dt(min) 100.00 98.069 97.523 96.493 94.477 (mm Hg/s) ± 0.3813 ± 2.915 ± 3.452 ± 2.967 ± 3.687 Systolic pressure 100.00 100.22 99.649 98.416 98.745 (mm Hg) ± 0.3813 ± 2.996 ± 2.923 ± 2.817 ± 2.800 Diastolic pressure 100.00 97.881 97.249 96.379 95.345 (mm Hg) ± 0.3813 ± 3.661 ± 3.700 ± 3.999 ± 3.844 Heart rate (BPM) 99.741 95.677 97.997 94.373 84.062* ± 0.2038 ± 2.827 ± 3.397 ± 2.805 ± 5.353 Peak aortic systolic 99.741 100.63 100.38 99.290 99.019 pressure(mm Hg) ± 0.2038 ± 5.335 ± 6.208 ± 4.732 ± 3.114 End diastolic pressure 99.741 96.975 97.595 96.291 92.808 (mm Hg) ± 0.2038 ± 3.236 ± 3.711 ± 4.318 ± 4.922 Ejection fraction 100.00 99.109 99.198 99.265 102.18 (%) ± 0.3813 ± 2.807 ± 3.005 ± 3.111 ± 3.276 Stroke volume 100.00 99.681 98.950 96.758 95.747 (mm Hg) ± 0.3813 ± 2.773 ± 2.836 ± 2.906 ± 3.172 Rate pressure product 100.00 97.404 98.093 95.935 84.246* (mm Hg/min) ± 0.3813 ± 3.018 ± 3.978 ± 3.378 ± 5.260 Cardiac power 100.00 100.77 100.65 100.77 100.63 ± 0.3813 ± 2.764 ± 2.969 ± 2.890 ± 2.786 * p<0.05

Appendix No 2: Verapamil Hcl 334

Table 2.2.1 Effect of variable pre-loads on isolated rat working heart with or without Verapamil (0.01 µM)

Pre-loads (cm H2O) Parameters 5 10 15 20 25

Coronary effluent Control 102.405 100.526 103.287 101.087 102.197 (mL/min) Pretreated 90.9401 87.1515 83.9115 82.352 80.8901

Aortic out flow Control 100.507 99.909 100.326 98.9123 98.9785 (mL/min) Pretreated 106.667 105.441 105.817 106.408 107.169

Cardiac output Control 100.418 99.5112 100.571 99.4792 99.7297 (mL/min) Pretreated 100.351 100.022 98.6197 98.9016 98.6163 Control 99.7852 99.4781 99.69 100.551 100.548 dP/dt(max) (mm Hg/s) Pretreated 76.9183 74.4939 73.1479 71.4677 67.8801 Control 100.26 99.4457 99.8346 99.6713 99.5106 dP/dt(min) (mm Hg/s) Pretreated 86.3474 86.848 87.1604 85.6525 84.3367

Heart rate Control 100.817 101.042 100.992 101.973 100.564 (BPM) Pretreated 83.6089 83.0674 82.9794 82.3484 81.5033

Left ventricular Control 98.4114 99.5525 99.6128 100.663 99.5237 pressure (mm Hg) Pretreated 99.5146 99.0965 99.0972 98.3898 97.7134

Peak aortic systolic Control 99.6231 100.459 99.5802 99.0333 101.853 pressure (mm Hg) Pretreated 96.344 94.0622 93.3626 92.1376 90.9085 Coronary vascular Control 97.004 99.2843 97.25 96.9567 96.8348 resistance (mm Pretreated 111.474 118.515 124.005 126.667 129.78 Hg/mL x min)

Myocardial work Control 101.399 100.806 100.898 101.707 104.668 (mm Hg x mL/min) Pretreated 97.653 95.5249 94.3399 93.8218 90.4011

Appendix No 2: Verapamil Hcl 335

Table 2.2.2 Effect of variable pre-loads on isolated rat working heart with or without Verapamil (0.1 µM)

Pre-loads (cm H2O) Parameters 5 10 15 20 25

Coronary effluent Control 100.481 99.688 99.0847 98.7124 98.708 (mL/min) Pretreated 81.6674 78.3453 73.3428 71.8546 66.2094

Aortic out flow Control 101.449 100.772 101.428 101.383 103.109 (mL/min) Pretreated 112.852 112.348 113.967 116.6 119.333

Cardiac output Control 101.341 100.351 100.678 101.057 100.762 (mL/min) Pretreated 100.661 99.8841 99.2675 100.552 100.773 Control 101.296 102.5 102.409 103.183 104.479 dP/dt(max) (mm Hg/s) Pretreated 82.7988 78.8829 74.8365 73.0703 69.7506 Control 100.134 100.925 100.469 100.18 100.307 dP/dt(min) (mm Hg/s) Pretreated 78.9401 77.3201 75.7897 74.4876 74.1362

Heart rate Control 98.3427 97.6139 97.4037 98.0331 99.9961 (BPM) Pretreated 75.6949 72.6061 69.7001 68.5948 69.0849

Left ventricular Control 99.2447 99.6992 100.346 101.539 99.6037 pressure (mm Hg) Pretreated 99.8693 101.688 101.93 100.316 100.702

Peak aortic systolic Control 99.4198 99.8267 99.7528 98.5992 98.4942 pressure (mm Hg) Pretreated 96.8853 94.8724 93.1498 92.9396 92.0359 Coronary vascular Control 99.119 99.6339 99.4535 99.454 99.1829 resistance (mm Pretreated 126.123 132.719 146.838 153.197 164.198 Hg/mL x min)

Myocardial work Control 100.93 100.977 100.746 100.52 100.344 (mm Hg x mL/min) Pretreated 98.2708 97.3913 94.1437 93.706 93.6401

Appendix No 2: Verapamil Hcl 336

Table 2.2.3 Effect of variable pre-loads on isolated rat working heart with or without Verapamil (1 µM)

Pre-loads (cm H2O) Parameters 5 10 15 20 25

Coronary effluent Control 101.28 100.084 100.101 99.5139 99.0825 (mL/min) Pretreated 74.667 68.703 65.324 61.7934 58.9478

Aortic out flow Control 100.579 99.5165 99.4394 99.2339 99.6118 (mL/min) Pretreated 117.831 119.079 121.891 122.979 125.629

Cardiac output Control 99.6121 99.2861 99.7631 99.0293 99.4841 (mL/min) Pretreated 99.9567 100.332 99.7334 99.8385 99.1598 Control 100.556 100.823 102.087 102.643 103.642 dP/dt(max) (mm Hg/s) Pretreated 74.879 69.7805 66.6032 64.8206 64.894 Control 99.2098 98.9337 100.929 100.543 100.321 dP/dt(min) (mm Hg/s) Pretreated 87.8364 86.9742 86.424 85.6068 87.8673

Heart rate Control 102.015 103.928 109.00 111.225 111.703 (BPM) Pretreated 72.9844 72.6327 72.4709 72.0248 71.789

Left ventricular Control 100.86 101.127 99.9613 99.8731 99.5414 pressure (mm Hg) Pretreated 102.45 102.702 102.667 103.598 103.852

Peak aortic systolic Control 99.5221 100.927 99.6715 99.626 100.508 pressure (mm Hg) Pretreated 93.9237 92.4193 92.2628 91.3532 90.4718 Coronary vascular Control 108.411 110.933 107.699 106.139 103.286 resistance (mm Pretreated 113.723 140.88 161.663 176.293 193.877 Hg/mL x min)

Myocardial work Control 101.684 102.13 101.334 100.543 100.422 (mm Hg x mL/min) Pretreated 95.7602 92.4404 92.6737 90.8169 90.5705

Appendix No 2: Verapamil Hcl 337

Table 2.3.1 Effect of calcium paradox with/or without verapamil (1 µM) on coronary effluent (n=3) Series Solutions Time (minutes) 2 4 6 8 10 Series Ca++-free KH 95.2494 94.277 91.9786 88.7204 82.2218 D-1 ↓4.751 % ↓5.722 % ↓8.021 % ↓11.280 % ↓17.778% Normal KH 98.5385 96.22 96.0174 97.0192 94.4777 ↓1.461 % ↓3.78 % ↓3.983 % ↓2.981 % ↓5.522 % Series Ca++-free KH 90.9146 88.667 86.4395 84.5327 80.506 D-2 ↓9.085 % ↓11.33 % ↓13.560 % ↓15.467 % ↓19.494 % Normal KH + 78.953 76.3023 72.5758 69.735 68.1724 Verapamil ↓21.047 % ↓23.69 % ↓27.424 % ↓30.265 % ↓31.828 % Series Ca++-free KH + 93.7653 92.1216 90.0867 86.845 84.5512 D-3 Verapamil ↓6.235 % ↓7.878 % ↓9.913 % ↓13.155 % ↓15.449 % Normal KH + 73.9842 71.8067 69.9106 67.2641 61.2926 Verapamil ↓26.016 % ↓28.19 % ↓30.089 % ↓32.736 % ↓38.707 %

Table 2.3.2 Effect of calcium paradox with/or without verapamil (1 µM) on aortic outflow (n=3) Series Solutions Time (minutes) 2 4 6 8 10 Series Ca++-free KH 101.3642 102.059 101.948 101.396 102.193 D-1 ↑1.364 % ↑2.059 % ↑1.948 % ↑1.396 % ↑2.193 % Normal KH 100.682 100.802 100.803 100.958 100.882 ↑0.682 % ↑0.802 % ↑0.803 % ↑0.958 % ↑0.882 % Series Ca++-free KH 103.029 105.004 104.491 106.333 104.957 D-2 ↑3.029 % ↑5.004 % ↑4.491 % ↑6.333 % ↑4.957 % Normal KH + 107.132 108.206 108.048 108.124 109.137 Verapamil ↑7.132 % ↑8.206 % ↑8.048 % ↑8.124 % ↑9.137 % Series Ca++-free KH + 103.83 104.824 104.925 106.119 107.564 D-3 Verapamil ↑3.83 % ↑4.824 % ↑4.925 % ↑6.119 % ↑7.564 % Normal KH + 104.336 104.975 107.018 108.513 108.074 Verapamil ↑4.336 % ↑4.975 % ↑7.018 % ↑8.513 % ↑8.074 %

Appendix No 2: Verapamil Hcl 338

Table 2.3.3 Effect of calcium paradox with/or without verapamil (1 µM) on dP/dt(max) (n=3) Series Solutions Time (minutes) 2 4 6 8 10 Series Ca++-free KH 39.702 39.4383 39.0922 39.0282 39.0207 D-1 ↓60.298 % ↓60.562 % ↓60.908 % ↓60.972 % ↓60.979 % Normal KH 102.034 106.01 107.68 108.927 109.512 ↑2.034 % ↑6.01 % ↑7.68 % ↑8.927 % ↑9.512 % Series Ca++-free KH 41.8263 41.3252 41.2651 41.1878 41.2691 D-2 ↓58.174 % ↓58.675 % ↓58.735 % ↓58.812 % ↓58.730 % Normal KH + 102.745 104.581 106.855 107.339 108.35 Verapamil ↑2.745 % ↑4.581 % ↑6.855 % ↑7.339 % ↑8.35 % Series Ca++-free KH + 26.7679 26.4714 26.3433 26.2209 26.1961 D-3 Verapamil ↓73.232 % ↓73.529 % ↓73.657 % ↓73.779 % ↓73.803 % Normal KH + 101.985 106.281 106.761 106.239 105.29 Verapamil ↑1.985 % ↑6.281 % ↑6.761 % ↑6.239 % ↑5.29 %

Table 2.3.4 Effect of calcium paradox with/or without verapamil (1 µM) on dP/dt(min) (n=3) Series Solutions Time (minutes) 2 4 6 8 10 Series Ca++-free KH 51.6289 51.6578 51.8976 52.0424 52.2406 D-1 ↓48.37 % ↓48.342 % ↓48.102 % ↓47.958 % ↓47.759 % Normal KH 97.9593 101.468 102.69 103.324 103.03 ↓2.040 % ↑1.468 % ↑2.69 % ↑3.324 % ↑3.03% Series Ca++-free KH 57.4591 57.7305 57.981 58.2068 58.4257 D-2 ↓42.54 % ↓42.269 % ↓42.019 % ↓41.793 % ↓41.574 % Normal KH + 101.415 103.167 103.736 105.06 105.448 Verapamil ↑1.415 % ↑3.167 % ↑3.736 % ↑5.06 % ↑5.448 % Series Ca++-free KH + 37.6461 37.6878 37.8124 37.9504 38.0685 D-3 Verapamil ↓62.35 % ↓62.312 % ↓62.188 % ↓62.050 % ↓61.931 % Normal KH + 101.281 102.993 103.86 104.455 105.257 Verapamil ↑1.281 % ↑2.993 % ↑3.86 % ↑4.455 % ↑5.257 %

Appendix No 2: Verapamil Hcl 339

Table 2.3.5 Effect of calcium paradox with/or without verapamil (1 µM) on heart rate (n=3) Series Solutions Time (minutes) 2 4 6 8 10 Series Ca++-free KH 94.6716 94.5846 94.5797 94.7691 94.7667 D-1 ↓5.328 % ↓5.415 % ↓5.420 % ↓5.231 % ↓5.233 % Normal KH 99.3376 99.3379 99.3395 99.3367 99.3362 ↓0.662 % ↓0.662 % ↓0.660 % ↓0.663 % ↓0.664 % Series Ca++-free KH 99.4136 99.0146 98.9673 98.8463 98.8111 D-2 ↓0.586 % ↓0.985 % ↓1.033 % ↓1.154 % ↓1.189 % Normal KH + 99.4136 99.3681 99.3382 99.3213 99.3264 Verapamil ↓0.586 % ↓0.632 % ↓0.662 % ↓0.679 % ↓0.674 % Series Ca++-free KH + 98.2091 98.2072 98.2047 98.2079 98.2067 D-3 Verapamil ↓1.791 % ↓1.793 % ↓1.795 % ↓1.792 % ↓1.793 % Normal KH + 99.9236 99.9708 100.013 100.057 100.104 Verapamil ↓0.076 % ↓0.029 % ↓0.013 % ↓0.057 % ↓0.104 %

Table 2.3.6 Effect of calcium paradox with/or without verapamil (1 µM) on left ventricular mean pressure (n=3) Series Solutions Time (minutes) 2 4 6 8 10 Series Ca++-free KH 92.4728 92.6222 92.777 92.9324 93.0732 D-1 ↓7.527 % ↓7.378 % ↓7.223 % ↓7.068 % ↓6.927 % Normal KH 100.083 100.145 100.238 100.27 100.409 ↓0.083 % ↓0.145 % ↓0.238 % ↓0.27 % ↓0.409 % Series Ca++-free KH 96.1318 96.3778 96.5786 96.7497 96.9106 D-2 ↓3.868 % ↓3.622 % ↓3.421 % ↓3.250 % ↓3.089 % Normal KH + 101.66 101.828 102.025 102.212 102.499 Verapamil ↑1.66 % ↑1.828 % ↑2.025 % ↑2.212 % ↑2.499 % Series Ca++-free KH + 94.2264 94.7733 94.9809 95.1872 95.3853 D-3 Verapamil ↓5.774 % ↓5.227 % ↓5.019 % ↓4.813 % ↓4.615 % Normal KH + 100.998 101.104 101.355 101.653 101.881 Verapamil ↑0.998 % ↑1.104 % ↑1.355 % ↑1.653 % ↑1.881 %

Appendix No 2: Verapamil Hcl 340

Table 2.3.7 Effect of calcium paradox with/or without verapamil (1 µM) on stroke volume (n=3) Series Solutions Time (minutes) 2 4 6 8 10 Series Ca++-free KH 94.5438 94.8761 93.7322 94.4664 94.3308 D-1 ↓5.456 % ↓5.124 % ↓6.268 % ↓5.534 % ↓5.669 % Normal KH 100.516 100.358 100.308 100.408 100.24 ↑0.516 % ↑0.358 % ↑0.308 % ↑0.408 % ↑0.24 % Series Ca++-free KH 98.7072 99.6511 98.5911 99.0625 98.3401 D-2 ↓1.293 % ↓0.349 % ↓1.409 % ↓0.937 % ↓1.660 % Normal KH + 100.418 100.635 100.53 99.9515 100.209 Verapamil ↑0.418 % ↑0.635 % ↑0.53 % ↓0.0485 % ↑0.209 % Series Ca++-free KH + 97.8914 98.3598 97.9602 97.7949 97.5696 D-3 Verapamil ↓2.109 % ↓1.640 % ↓2.040 % ↓2.205 % ↓2.430 % Normal KH + 99.6858 99.84 99.6982 99.1865 98.9836 Verapamil ↓0.314 % ↓0.16 % ↓0.302 % ↓0.813 % ↓1.0164 %

Table 2.3.8 Effect of calcium paradox with/or without verapamil (1 µM) on rate pressure product (n=3) Series Solutions Time (minutes) 2 4 6 8 10 Series Ca++-free KH 89.9513 90.0005 90.2032 90.5527 90.7009 D-1 ↓10.049 % ↓9.999 % ↓9.797 % ↓9.447 % ↓9.299 % Normal KH 100.424 100.434 100.419 100.575 100.593 ↑0.424 % ↑0.434 % ↑0.419 % ↑0.575 % ↑0.593 % Series Ca++-free KH 86.966 87.0614 87.244 87.0729 87.3787 D-2 ↓13.034 % ↓12.938 % ↓12.756 % ↓12.927 % ↓12.621 % Normal KH + 101.737 102.288 102.213 102.277 102.403 Verapamil ↑1.737 % ↑2.288 % ↑2.213 % ↑2.277 % ↑2.403 % Series Ca++-free KH + 88.2071 88.3284 88.4558 88.7457 88.9859 D-3 Verapamil ↓11.793 % ↓11.672 % ↓11.544 % ↓11.254 % ↓11.014 % Normal KH + 100.976 101.132 101.5 101.981 102.176 Verapamil ↑0.976 % ↑1.132 % ↑1.5 % ↑1.981 % ↑2.176 %

Appendix No 2: Verapamil Hcl 341

Table 2.4.1 Effect of verapamil on rat thoracic aorta pre-contracted by PE (1 µM) Parameters Concentration (µM) Control 0.01 0.03 0.1 0.3 1.0 3.0 10.0 Baseline 99.764 96.682 94.924 92.782 90.992 89.452 88.713 88.181 tension ± 0.2614 ± 2.893 ± 2.757 ± 2.924 ± 2.789 ± 2.791 ± 2.804 ± 2.862 Endothelium- 99.741 98.742 97.869 97.028 96.214 95.575 94.100 90.915 intact rings ± 0.204 ±3.913 ± 3.804 ± 3.993 ± 3.945 ± 3.737 ± 3.881 ± 3.829 Endothelium- 099.956 96.974 95.746 93.396 91.581 88.353 82.267* 73.958*** denuded rings ± 0.272 ± 3.756 ± 3.856 ± 3.896 ± 3.714 ± 3.754 ± 3.522 ± 4.315 * p<0.05,*** p<0.001

Table 2.4.2 Effect of verapamil on rat thoracic aorta pre-contracted by high K+ (80 mM) Parameters Concentration (µM) Control 0.01 0.03 0.1 0.3 1.0 3.0 10.0 Baseline 99.789 95.044 93.145 91.291 90.749 90.124 88.228 87.852 tension ± 0.173 ±2.917 ±2.752 ± 2.775 ± 3.015 ± 2.932 ± 3.023 ± 3.063 Endothelium- 99.956 94.396 84.909 60.925*** 33.307*** 21.743*** 13.435*** 12.233*** intact rings ± 0.272 ±3.723 ±3.874 ± 3.854 ± 4.150 ± 3.634 ± 3.672 ± 3.632 Endothelium- 99.956 96.348 87.364 70.731** 48.601*** 29.650*** 17.283*** 8.297*** denuded rings ± 0.272 ±2.860 ±3.112 ± 5.745 ± 7.701 ± 6.535 ± 4.974 ± 3.556 **p<0.01, *** p<0.001

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