ANTI-DIABETIC ACTIVITY-GUIDED STUDIES OF SYZYGIUM POLYANTHUM (WIGHT) LEAF EXTRACTS AND ELUCIDATION OF THEIR MECHANISMS OF ACTION TRI WIDYAWATI UNIVERSITI SAINS MALAYSIA 2015 ANTI-DIABETIC ACTIVITY-GUIDED STUDIES OF SYZYGIUM POLYANTHUM (WIGHT) LEAF EXTRACTS AND ELUCIDATION OF THEIR MECHANISMS OF ACTION by TRI WIDYAWATI Thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy November 2015 TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF TABLES x LIST OF FIGURES xii LIST OF PLATES xvi LIST OF ABBREVIATIONS xvii ABSTRAK xx ABSTRACT xxii CHAPTER ONE: INTRODUCTION 1.1. Diabetes mellitus 1 1.1.1. Definition and historical aspect 1 1.1.2. Epidemiology 2 1.1.3. Anatomy of the pancreas 2 1.1.4. Physiological regulation of glucose homeostasis and insulin 3 action 1.1.5. Pathophysiology 6 1.1.6. Clinical manifestation and complications 6 1.1.7. Classification 7 1.1.8. Diagnosis 8 1.1.9. Treatment 9 1.2. Medicinal plant of the Syzygium genus 11 1.3. Syzygium polyanthum Wight. 13 iii 1.3.1. Synonyms and common names 13 1.3.2. Taxonomy 13 1.3.3. Structural features 14 1.3.4. Geography 14 1.3.5. Phytochemical constituents 16 1.3.6. Traditional uses 16 1.3.7. Pharmacological activity 16 1.4. Objectives of the study 20 CHAPTER TWO: HYPOGLYCAEMIC, GLUCOSE TOLERANCE AND ANTI-HYPERGLYCAEMIC PROPERTIES OF SYZYGIUM POLYANTHUM (WIGHT) LEAF 2.1. Introduction 21 2.2. Objectives of the study 26 2.3. Material and methods 27 2.3.1. Chemicals and equipments 27 2.3.2. Plant material 27 2.4. Extraction procedure 27 2.4.1. Solvents for extraction 27 2.4.2. Extraction and fractionation 27 2.4.2.1. Preparation of the extracts 27 2.4.2.2. Preparation of the fractions 28 2.4.2.3. Preparation of the subfractions 29 2.5. Animals 33 2.6. Diabetes induction in experimental diabetic rats 33 2.7. Experimental set up 33 iv 2.7.1. Hypoglycaemic test of the four extracts in normal rats 33 2.7.2. Preliminary intra-peritoneal glucose tolerance test (IPGTT) in 34 normal rats 2.7.2.1. Intra-peritoneal glucose tolerance test (IPGTT) in 34 normal rats 2.7.3. Anti-hyperglycaemic test of single-dose oral administration of 35 the four extracts in STZ-induced diabetic rats 2.7.4. The effect of twice-daily oral administration of the four 35 extracts on the blood glucose levels of STZ-induced diabetic rats 2.7.5.The effect of twice-daily oral administration of the four fractions 35 on the blood glucose levels of STZ-induced diabetic rats 2.7.6. The effect of twice-daily oral administration of the two 36 subfractions on the blood glucose levesl of STZ-induced diabetic rats 2.7.7. The effect of different doses of methanolic extract of S. 36 polyanthum leaves on the blood glucose levels of STZ-induced diabetic rats 2.8. Phytochemical screening of methanol extract, chloroform fraction, 36 water fraction and n-hexane fraction of S. polyanthum leaves 2.8.1. Identification by chemical tests 36 2.8.1.1. Identification of free reducing sugars 36 2.8.1.2. Identification of tannins 37 2.8.1.3. Identification of steroids (Liebermann-Burchard test) 37 2.8.1.4. Identification of terpenoids 37 2.8.1.5. Identification of flavonoids (Ferric chloride test) 37 2.8.1.6. Identification of soluble starches 38 2.8.1.7. Identification of saponins 38 2.8.1.8. Identification of glycosides 38 2.8.2. Identification by gas chromatography-mass spectrometry 38 (GC-MS) v 2.9. The effect of different doses of squalene on the blood glucose level of 39 STZ-induced diabetic rats 2.10. Statistical analysis 40 2.11. Results 2.11.1. Extracts, fractions and subfractions yields 40 2.11.2. Hypoglycaemic effects of the four extracts in normal rats 41 2.11.3. Preliminary intra-peritoneal glucose tolerance test (IPGTT) in 42 normal rats 2.11.4. Intraperitoneal glucose tolerance test (IPGTT) in normal rats 44 2.11.5. The effects of single-dose administration of the four extracts on 45 the blood glucose levels of STZ-induced diabetic rats 2.11.6. The effects of twice-daily oral administration of the four 48 extracts on the blood glucose levels of STZ-induced diabetic rats 2.11.7. The effects of twice-daily oral administration of the four 49 fractions on the blood glucose levels of STZ-induced diabetic rats 2.11.8. The effects of twice-daily oral administration of the two sub 50 fractions on the blood glucose levels of STZ-induced diabetic rats 2.11.9. The effect of different doses of the methanolic extract on the 51 blood glucose levels of STZ-induced diabetic rats 2.11.10. Phytochemical study of S. polyanthum leaf methanol extract 52 (ME), chloroform fraction (CF), water fraction (WF) and n- hexane fraction (SF-1) 2.11.10.1. Identification by chemical tests 52 2.11.10.2. Identification by gas chromatography-mass spectrometry (GC-MS) 53 2.11.11. Anti-hyperglycaemic test of squalene in STZ- induced diabetic rats 57 2.12. Discussion and conclusions 58 vi CHAPTER THREE: ANTI-DIABETIC MECHANISMS OF ACTION OF SYZYGIUM POLYANTHUM WIGHT LEAF EXTRACTS 3.1. Introduction 70 3.2. Objectives 72 3.3. Materiasl and methods 73 3.3.1. Preparation of S. polyanthum extracts 73 3.3.2. Animals 73 3.3.3. Streptozotocin-induced diabetic rats 74 3.4. The effect of twice-daily oral administration of S.polyanthum (Wight) 74 leaf extracts on the blood glucose levels, insulin levels, body weights and lipid profiles of STZ-induced diabetic rats 3.4.1. Experimental set up 74 3.4.1.1. Blood glucose level assay 74 3.4.1.2. Insulin measurement 74 3.4.1.3. Body weight measurements 77 3.4.1.4. Lipid profiles measurement 77 3.5. Histological assessment of the islets of Langerhans of diabetic rats after 77 twice-daily oral administration of S.polyanthum (Wight) leaf extracs for 12 days 3.5.1. Experimental set up 77 3.5.2. Specimen collection and preparation 77 3.5.3. Specimen preparation 78 3.5.4. Immunohistochemical staining method 78 3.5.5. Quantifying method 79 3.6. In vitro -glucosidase inhibition assay 82 3.6.1. Experimental set up 82 3.7. In vitro -amylase inhibition assay 83 vii 3.7.1. Experimental set up 84 3.8. Glucose absorption from the averted jejunal sac preparation 85 3.8.1.Experimental set up 85 3.9. In vivo enzyme inhibition studies 86 3.9.1. In vivo enzyme inhibition studies in normal rats 86 3.9.1.1. Oral starch tolerance test in normal rats 86 3.9.1.2. Oral sucrose tolerance test in non diabetic rats 87 3.9.1.3. Oral glucose tolerance test in non diabetic rats 87 3.9.2. In vivo enzyme inhibition studies in STZ-induced diabetic rats 87 3.9.2.1. Oral starch tolerance test in STZ-induced 87 diabetic rats 3.9.2.2. Oral sucrose tolerance test in STZ-induced 88 diabetic rats 3.9.2.3. Oral glucose tolerance test in STZ-induced 88 diabetic rats 3.10. Glucose uptake by isolated rat abdominal muscle 88 3.10.1. Experimental set up 88 3.11. Glucose uptake by isolated rat soleus muscle 89 3.11.1. Experimental set up 90 3.12. Statistical analysis 90 3.13. Results 91 3.13.1. The effects of twice daily oral administration for 12 days of S. 91 polyanthum extracts (ME, CF, WF, SF-1) and squalene on blood glucose level, insulin level, body weight, and lipid profiles of STZ-induced diabetic rats 3.13.2. Histological assesment 95 3.13.3. In vitro -glucosidase inhibition assay 98 viii 3.13.4. In vitro -amylase inhibition assay 99 3.13.5. Glucose absorption via averted jejunal sac preparations 100 3.13.6. Enzyme inhibition in vivo 101 3.13.6.1. Oral starch tolerance test in normal and 102 STZ-induced diabetic rats 3.13.6.2. Oral sucrose tolerance test in normal and 103 STZ-induced diabetic rats 3.13.6.3. Oral glucose tolerance test in normal and 104 STZ-induced diabetic rats 3.13.7. Glucose uptake by isolated abdominal muscle preparation 105 3.13.8. Glucose uptake by isolated soleus muscle preparation 106 3.14. Discussion and conclusions 108 CHAPTER FOUR: GENERAL DISCUSSION AND CONCLUSION 117 REFERENCES 124 LIST OF PUBLICATIONS & WORKSHOP 141 ix LIST OF TABLES Page Table 1.1 Criteria for the diagnosis of diabetes. 8 Table 2.1 The yields of S. polyanthum (Wight) leaf extraction. 40 Table.2.2 Phytochemical screening of ME, CF, WF and SF-1 of 52 Syzygium polyanthum (Wight) leaf. Table 2.3 Phytochemical components identified in the methanolic extract 53 (ME), chloroform fraction (CF) and n-hexane subfraction (SF-1) with squalene as standard by gas chromatography-mass spectrometry (GC-MS). Table 2.4 The percentage of squalene in methanol extract (ME), 56 chloroform fraction (CF) and n-hexane fraction (SF-1). Table 3.1 Dehydration scheme of fixed pancreatic specimen. 80 Table 3.2 Imunohistochemical staining procedure. 81 Table 3.3 The effects of 12-days twice-daily oral administration of S. 94 polyanthum methanol extracts (ME) (1 g/kg), chloroform fractions (CF) (500 mg/kg), water fractions (WF) (500 mg/kg), n-hexane fractions (SF-1) (250 mg/kg), squalene (SQ) (160 mg/kg), metformin (M) (500 mg/kg) and saline (DC) (10 ml/kg) on the lipid profiles of streptozotocin-induced diabetic rats.