Comparative Pharmacological and Biological Evaluation of the Stem and Leaves of Hedera Nepalensis from District Malakand Khyber Pakhtunkhwa, Pakistan

Comparative Pharmacological and Biological Evaluation of the Stem and Leaves of Hedera nepalensis from District Malakand Khyber Pakhtunkhwa, Pakistan

Muhammad Romman

DEPARTMENT OF BOTANY ISLAMIA COLLEGE PESHAWAR KHYBER PAKHTUNKHWA, PAKISTAN

2011-2016 Comparative Pharmacological and Biological Evaluation of the Stem and Leaves of Hedera nepalensis from District Malakand Khyber Pakhtunkhwa, Pakistan

Muhammad Romman

Registration No: 2011/ICP/M.Phil. BOT-232

A Thesis Submitted to the Department of Botany, Islamia College Peshawar in Partial Fulfillment of the requirements

For the Degree of

Doctor of Philosophy (Ph.D)

BOTANY

DEPARTMENT OF BOTANY ISLAMIA COLLEGE PESHAWAR KHYBER PAKHTUNKHWA, PAKISTAN

2011-2016

Declaration

The whole of the experimental work described in this thesis has been carried out by me at the Medicinal Botanic Center, PCSIR Laboratories Complex, Peshawar, and Botany Laboratory (Pharmacognosy Section) Islamia College Peshawar. I have not previously presented any part of this work elsewhere for any other degree.

Muhammad Romman

DEDICATED TO MY LOVING PARENTS, BROTHERS & MY STUDENTS

SPECIAL THANKS TO THOSE WHO ALWAYS PRAY FOR MY SUCCESS

Table of Contents Page No. Acknowledgments I List of Tables II List of Figures VII List of Appendices X List of Abbreviations XIII Abstract XIV

Chapter: 1 Introduction 1 1.1 Biological study of H.nepalensis 2 1.2 Medicinal uses 3 1.3 Pharmacognostic evaluation of crude extract 4 1.3.1 Macroscopic and microscopic study 4 1.3.2 Physiochemical characterization 5 1.3.2.1 Extractive values 5 1.3.2.2 Ash value 5 1.3.2.3 Heavy metals analysis 5 1.3.3 Phytochemical characterization 6 1.4 Biological evaluation of the crude extract 9 1.4.1 Antioxidant activities 9 1.4.2 Antimicrobial activities 10 1.4.2.1 Antibacterial activities 10 1.4.2.2 Antifungal activities 11 1.5 Pharmacological evaluation of the crude extract 11 1.5.1 Introduction to Diabetes mellitus 11 1.5.2 Types of Diabetes 12 1.5.3 Treatment of Diabetes mellitus 13 1.5.4 Antidiabetic activity of other plants 13 1.5.5 Aims and objectives 16

Chapter: 2 Materials and Methods 17 2.1 Selection of plant 18

2.2 Collection and identification of the plant 18

2.3 Pharmacognostic evaluation of the crude extract 18 2.3.1 Macroscopic and microscopic study 18

2.3.2 Physiochemical characterization 18

2.3.2.1 Crude extract preparation 18

2.3.2.2 Fractionation procedure 19

2.3.2.3 Determination of extractive values 19

2.3.2.4 Determination of total ash value 19

2.3.2.5 Heavy metals analysis 20

2.3.3 Phytochemical characterization 20

2.3.3.1 Qualitative tests 20

2.3.3.1.1 Carbohydrates 20

2.3.3.1.2 Proteins 20

2.3.3.1.3 Saponins 20

2.3.3.1.4 Alkaloids 21

2.3.3.1.5 Tannins 21

2.3.3.1.6 Flavonoids 21

2.3.3.1.7 Terpenoids 21

2.3.3.1.8 Phenolic compounds 21

2.3.3.1.9 Phytosterols 21

2.3.3.1.10 Cardiac glycosides 22

2.3.3.2 Quantitative tests 22

2.3.3.2.1 Carbohydrates 22

2.3.3.2.2 Proteins 22

2.3.3.2.3 Saponins 23

2.3.3.2.4 Alkaloids 23

2.3.3.2.5 Tannins 23

2.3.3.2.6 Flavonoids 23

2.3.3.2.7 Terpenoids 23 2.3.3.2.8 Phytosterols 24

2.4 Biological evaluation of the crude extract 24

2.4.1 Antioxidant activity 24

2.4.1.1 DPPH free radical scavenging activity 24

2.4.1.2 Hydrogen peroxide free radical scavenging activity 24

2.4.2 Antimicrobial activities 25

2.4.2.1 Culture media used 25

2.4.2.2 Preparation of media 26

2.4.2.3 Microorganisms used 26

2.4.2.4 Stock solution of extracts used 27

2.4.2.5 McFarland 0.5 turbidity standard 28

2.4.2.6 Disc diffusion susceptibility 28

2.4.2.7 Step wise methodology of antimicrobial bioassay 28

2.5 Pharmacological evaluation of the crude extract 30

2.5.1 Toxicological studies 30

2.5.2 Acute toxicity (LD50) 30 2.5.3 Selection of animals 30

2.5.4 Grouping of rabbits 31

2.5.5 Equipments 31

2.5.6 Chemicals 31

2.5.7 Induction of Diabetes 32

2.5.8 Drug administration 33

2.5.9 Collection of blood samples 33

2.5.10 Isolation of blood serum 33

2.5.11 Biochemical analysis of the blood of rabbits 33

2.5.11.1 Glucose determination 33

2.5.11.2 Triglyceride determination 35 2.5.11.3 Cholesterol determination 36

2.5.11.4 Total Bilirubin determination 37

2.5.11.5 Total Protein determination 39

2.5.11.6 Albumin determination 39

2.5.11.7 Globulin determination 40

2.5.11.8 A/G ratio determination 41

2.5.11.9 GCI determination 41

2.5.11.10 Creatinine determination 41

2.5.11.11 ALP determination 43

2.5.11.12 GGT determination 44

2.5.11.13 ALT determination 45

2.5.11.14 AST determination 46

2.5.11.15 AST/ALT ratio 48

2.6 Data analysis 48

Chapter: 3 Results 49

3.1 Pharmacognostic evaluation of the crude extract 50

3.1.1 Morphological study 50

3.1.2 Physiochemical characterization 50

3.1.2.1 Fractionation 50

3.1.2.2 Analysis of extractive values in H. nepalensis 52

3.1.2.2.1 Extractive values for leaves 52

3.1.2.2.2 Extractive values for stem 52

3.1.2.3 Analysis of total ash values in H. nepalensis 53

3.1.2.4 Heavy metal analysis of H. nepalensis 53

3.1.2.4.1 Heavy metals analysis of the leaves 53

3.1.2.4.2 Heavy metals analysis of the stem 54

3.1.3 Phytochemical screening of H. nepalensis 55 3.1.3.1 Qualitative tests for phytochemicals in leaves and stem 55

3.1.3.2 Quantitative tests for phytochemicals in leaves 55

3.1.3.3 Quantitative tests for phytochemicals in stem 56

3.2 Biological evaluation of the crude ethanolic extract 56

3.2.1 Antioxidant activity of the leaves and stem 56

3.2.1.1 DPPH free radical scavenging activity 56

3.2.1.2 Hydrogen peroxide free radical scavenging activity 59

3.2.2 Antimicrobial activities of H. neplensis 64

3.2.2.1.1. Antibacterial activities of leaves 64

3.2.2.1.1.1 Methanol extract 64

3.2.2.1.1.2 Ethanol extract 65

3.2.2.1.1.3 n-Hexane extract 66

3.2.2.1.1.4 Petroleum ether extract 66

3.2.2.1.1.5 Chloroform extract 67

3.2.2.1.2 Antibacterial activities of stem 68

3.2.2.1.2.1 Methanol extract 68

3.2.2.1.2.2 Ethanol extract 69

3.2.2.1.2.3 n-Hexane extract 69

3.2.2.1.2.4 Petroleum ether extract 70

3.2.2.1.2.5 Chloroform extract 71

3.2.2.2.1 Antifungal activities of leaves 72

3.2.2.2.1.1 Methanol extract 72

3.2.2.2.1.2 Ethanol extract 73

3.2.2.2.1.3 n-Hexane extract 73

3.2.2.2.1.4 Petroleum ether 74

3.2.2.2.1.5 Chloroform extract 74

3.2.2.2.2 Antifungal activities of stem 75 3.2.2.2.2.1 Methanol extract 75

3.2.2.2.2.2 Ethanol extract 76

3.2.2.2.2.3 n-Hexane extract 77

3.2.2.2.2.4 Petroleum ether 77

3.2.2.2.2.5 Chloroform extract 78

3.2.2.3 Antimicrobial activities of standard antibiotics 79

3.3 Pharmacological evaluation of the ethanolic extract 81

3.3.1 Toxicological studies 81

3.3.2 Biochemical blood analysis of rabbit’s blood in response to the leaves extract 82

3.3.2.1.1 Glucose level 82

3.3.2.1.2 Triglyceride level 85

3.3.2.1.3 Cholesterol level 88

3.3.2.1.4 Total bilirubin level 90

3.3.2.1.5 Total protein level 93

3.3.2.1.6 Albumin level 95

3.3.2.1.7 Globulin level 98

3.3.2.1.8 A/G ratio level 100

3.3.2.1.9 GCI level 101

3.3.2.1.10 Creatinine level 103

3.3.2.1.11 ALP level 106

3.3.2.1.12 GGT level 109

3.3.2.1.13 ALT level 111

3.3.2.1.14 AST level 114

3.3.2.1.15 AST/ALT ratio 116

3.3.2.2 Biochemical analysis of rabbit’s blood in response of stem 118

3.3.2.2.1 Glucose level 118

3.3.2.2.2 Triglyceride level 121 3.3.2.2.3 Cholesterol level 123

3.3.2.2.4 Total bilirubin level 126

3.3.2.2.5 Total protein level 129

3.3.2.2.6 Albumin level 131

3.3.2.2.7 Globulin level 134

3.3.2.2.8 Albumin/globulin ratio 136

3.3.2.2.9 GCI category 137

3.3.2.2.10 Creatinine level 139

3.3.2.2.11 ALP level 142

3.3.2.2.12 GGT level 144

3.3.2.2.13 ALT level 147

3.3.2.2.14 AST level 150

3.3.2.2.15 AST/ALT ratio 152

Chapter: 4 Discussion 155

4.1 Pharmacognostic studies of H. nepalensis 156

4.1.1 Physiochemical studies 156

4.1.1.1 Heavy metals studies 156

4.1.2 Phytochemical screening 159

4.2 Biological activities of H. nepalensis 162

4.2.1 Antioxidant activities 162

4.2.1.1 DPPH free radical scavenging activity 162

4.2.1.2 Hydrogen peroxide free radical scavenging activity 163

4.2.3 Antimicrobial activities 164

4.2.3.1 Antibacterial activities 164

4.2.3.2 Antifungal activities 166

4.3 Pharmacological activities 170

4.3.1 Toxicological studies 170 4.3.2 Antidiabetic activity 171

4.3.2.1 Effect of multiple doses of extracts of H. nepalensis on blood glucose level 172 in diabetic Rabbits 4.3.2.2 Effect of multiple doses of extracts of H. nepalensis on blood triglyceride level 176 in Diabetic rabbits 4.3.2.3 Effect of multiple doses of extracts of H. nepalensis on blood cholesterol level 177 in Diabetic rabbits 4.3.2.4 Effect of multiple doses of extracts of H. nepalensis on kidney functions 177 4.3.2.4.1 Creatinin 177 4.3.2.5 Effect of multiple doses of extracts of H. nepalensis on liver functions 178 4.3.2.5.1 Total bilirubin 178 4.3.2.5.2 Proteins total 179 4.3.2.5.3 Albumin 179 4.3.2.5.4 Globulin 180 4.3.2.5.5 A/G ratio 180 4.3.2.5.6 GCI category 181 4.3.2.5.7 ALP 181 4.3.2.5.8 GGT 182 4.3.2.5.9 ALTor SGPT 182 4.3.2.5.10 AST or SGOT 183 4.3.2.5.11 AST/ALT ratio 185 4.4 Conclusion 184 4.5 Recommendations 184

References 185

Acknowledgement

I am very grateful to Almighty ALLAH, the great Compassionate and Beneficent to all human beings, whose grace and mercy provided me a good health and enthusiasm and other opportunities to complete my research work in time.

I offer my humblest love to Prophet Muhammad (Swallallahu Alaihe Wasallam) who is the last Messenger of ALLAH and whose principles enlighten the life of his follower.

My cordial and profound gratitude would aptly sum up my feelings towards my supervisor Dr. Samin Jan (Assistant Professor, Department of Botany, Islamia College Peshawar) and co-supervisor Dr.Muhammad Hamayun (Associate Professor, Department of Botany, Abdul Wali Khan University, Mardan) whose guidance, suggestions and instructive discussions and criticism made my work complete.

I thank Dr. Muhammad Saleem Khan, Dr. Izhar Ahmad, Dr. Naveed Akhtar, Dr. Wisal Ahmad, Khushnood ur Rahman Dr. Arshad Iqbal, (Botany department, Islamia College Peshawar) for their suggestions during the research work.

A deep appreciation is extended to Dr. Ali Muhammad and Dr. Muhammad Zahid (Department of Zoology, Islamia College Peshawar) for their suggestions and discussion during the research work.

I would like to acknowledge with gratitude Dr. Siraj u Din (Chairman, Department of Botany, University of Peshawar), Dr. Mohammd Nisar (Chairman, Deparment of Botany, Univerisity of Malakand), Dr. Ayaz Ali, Shariyatullah (University of Malakand), Dr. Mushtaq Ahmad, Dr. Shahid Farooq (Scientific Officers PCSIR, Peshawar), and my friends Mr. Saqib, and Mr. Fahad, for their co-operation during my research work.

I gratefully acknowledge my Late Grand Father Razi Mand (May ALLAH keep his soul in rest) and my loving Mother, Father Taj Muhammad Khan and other family members for their support, patience, advice, prayers, understanding and encouragement throughout my education and prayed for my success.

Muhammad Romman

I List of Tables

Table 1: Nutrient agar composition used for culturing and applying test 25

Table 2: Composition of nutrient broth 25

Table 3: Bacterial strains used for antibacterial activities 26

Table 4: Fungal species used for antifungal activities 27

Table 5: Fractionation of the leaves and stem of H. nepalensis 50

Table 6: Extractive values of the leaves and stem of H. nepalensis 52

Table 7: Heavy metals analysis of the leaves of H. nepalensis 54

Table 8: Heavy metals analysis of the stem of H. nepalensis 54

Table 9: Qualitative presence of phytochemicals of the stem of H. nepalensis 55

Table 10: Quantitative presence of phytochemicals of the leaves of H. nepalensis 55

Table 11: Quantitative presence of phytochemicals of the stem of H. nepalensis 56

Table 12: DPPH radical scavenging activity of H. nepalensis leaves at 517 nm absorbance 57 in triplicate

Table 13: DPPH radical scavenging activity of H. nepalensis leaves at 517nm 57

Table 14: DPPH radical scavenging activity of ethyl acetate, petroleum ether, DCM, 57 ethanol and methanol for leaves extracts of H. nepalensis

Table 15: DPPH radical scavenging activity of H. nepalensis stem at 517 nm with 58 absorbance in triplicate

Table 16: DPPH Radical scavenging activity of H. nepalensis leaves at 517 nm with mean, 59 standard deviation and number

Table 17: DPPH radical scavenging activity of ethyl acetate, petroleum ether, DCM, 58 ethanol, methanol for stem extracts of H. nepalensis

Table 18: Hydrogen peroxide radical scavenging activity of H. nepalensis leaves at 285nm 59 absorbance in triplicate

Table 19: Hydrogen peroxide activity of H. nepalensis leaves at 285 nm 60

II Table 20: Hydrogen peroxide radical scavenging activity of ethyl acetate, petroleum ether, 61

DCM, ethanol and methanol for leaves extracts of H. nepalensis

Table 21: Hydrogen peroxide radical scavenging activity of H. nepalensis stem at 285nm 62 with absorbance in triplicate

Table 22: Hydrogen peroxide radical scavenging activity of H. nepalensis stem at 285nm 62

Table 23: Hydrogen peroxide radical scavenging activity of ethyl acetate, petroleum ether, 63 DCM, ethanol and methanol for stem extracts of H. nepalensis

Table 24: Antibacterial activities of the methanol extract for leaves of H. nepalensis 64

Table 25: Antibacterial activities of the ethanol extract for leaves of H. nepalensis 65

Table 26: Antibacterial activities of the n-hexane extract for leaves of H. nepalensis 66

Table 27: Antibacterial activities of the petroleum ether extract for leaves of H. nepalensis 67

Table 28: Antibacterial activities of the chloroform extract for leaves of H. nepalensis 67

Table 29: Antibacterial activities of the methanol extract for stem of H. nepalensis 68

Table 30: Antibacterial activities of the ethanol extract for stem of H. nepalensis 69

Table 31: Antibacterial activities of the n-hexane extract for stem of H. nepalensis 70

Table 32: Antibacterial activities of the petroleum ether extract for stem of H. nepalensis 70

Table 33: Antibacterial activities of the chloroform extract for stem of H. nepalensis 71

Table 34: Antifungal activities of the methanol extract for leaves of H. nepalensis 72

Table 35: Antifungal activities of the ethanol extract for leaves of H. nepalensis 73

Table 36: Antifungal activities of the n-hexane extract for leaves of H. nepalensis 73

Table 37: Antifungal activities of the petroleum ether extract for leaves of H. nepalensis 74

Table 38: Antifungal activities of the chloroform extract for leaves of H. nepalensis 75

Table 39: Antifungal activities of the methanol extract for stem of H. nepalensis 75

Table 40: Antifungal activities of the ethanol extract for stem of H. nepalensis 76

III Table 41: Antifungal activities of the n-hexane extract for stem of H. nepalensis 77

Table 42: Antifungal activities of the petroleum ether extract for stem of H. nepalensis 78

Table 43: Antifungal activities of the chloroform extract for stem of H. nepalensis 78

Table 44: Antibacterial activities of the azithromycin (Am) and ciprofloxacin (Cf) 79

Table 45: Antifungal activities of the clotrimazole (Cm) 80

Table 46: LD50 of ethanolic extract of H. nepalensis in rabbits 82

Table 47: Blood Glucose (mg/dl) level of rabbits in triplicate 83

Table 48: Mean blood glucose (mg/dl) level of rabbits with standard deviation 84

Table 49: Blood triglyceride (mg/dl) level of rabbits in triplicate 86

Table 50: Mean blood triglyceride (mg/dl) level of rabbits with standard deviation 91

Table 51: Blood cholestrol (mg/dl) level of rabbits in triplicate 89

Table 52: Mean blood cholestrol (mg/dl) level of rabbits with standard deviation 89

Table 53: Blood total bilirubin (mg/dl) level of rabbits in triplicate 91

Table 54: Mean blood total bilirubin (mg/dl) level of rabbits with standard deviation 92

Table 55: Blood total protein (g/dl) level of rabbits in triplicate 94

Table 56: Mean blood total protein (g/dL) level of rabbits with standard deviation 94

Table 57: Blood albumin (g/dL) level of rabbits in triplicate 96

Table 58: Mean blood albumin (g/dl) level of rabbits with standard deviation 97

Table 59: Blood globulin (g/dl) level of rabbits 99

Table 60: Blood A/G ratio level of rabbits 101

Table 61: GCI categories for blood of rabbits 101

Table 62: Blood GCI level of rabbits 103

IV Table 63: Blood creatinine (mg/dl) level of rabbits in triplicate 104

Table 64: Mean blood creatinine (mg/dl) level of rabbits with standard deviation 105

Table 65: Blood ALP (IU/L) level of rabbits in triplicate 107

Table 66: Mean blood ALP (IU/L) level of rabbits with standard deviation 108

Table 67: Blood GGT (IU/L) level of rabbits in triplicate 110

Table 68: Mean blood GGT (IU/L) level of rabbits with standard deviation 110

Table 69: Blood ALT (IU/L) level of rabbits in triplicate 112

Table 70: Mean blood ALT (IU/L) level of rabbits with standard deviation 113

Table 71: Blood AST (IU/L) level of rabbits in triplicate 115

Table 72: Mean blood AST (IU/L) level of rabbits with standard deviation 115

Table 73: Blood AST/ALT ratio level of rabbits in triplicate 117

Table 74: Blood glucose (mg/dl) level of rabbits in triplicate ` 119

Table 75: Mean blood glucose (mg/dl) level of rabbits with standard deviation 120

Table 76: Blood triglyceride (mg/dl) level of rabbits in triplicate 122

Table 77: Mean blood triglyceride (mg/dl) level of rabbits with standard deviation 122

Table 78: Blood triglyceride (mg/dl) level of rabbits in triplicate 124

Table 79: Mean blood triglyceride (mg/dl) level of rabbits with standard deviation 125

Table 80: Blood total bilirubin (mg/dl) level of rabbits in triplicate 127

Table 81: Mean blood total bilirubin (mg/dl) level of rabbits with standard deviation 128

Table 82: Blood total protein (g/dL) level of rabbits in triplicate 130

Table 83: Mean blood total protein (g/dL) level of rabbits with standard deviation 130

Table 84: Blood albumin (g/dL) level of rabbits in triplicate 132

V Table 85: Mean blood albumin (g/dL) level of rabbits with standard deviation 133

Table 86: Blood globulin (g/dL) level of rabbits 135

Table 87: Blood A/G ratio level of rabbits 137

Table 88: GCI categories for blood of rabbits 137

Table 89: Blood GCI level of rabbits 138

Table 90: Blood creatinine (mg/dl) level of rabbits in triplicate 140

Table 91: Mean blood creatinine (mg/dl) level of rabbits with standard deviation 141

Table 92: Blood ALP (IU/L) level of rabbits in triplicate 143

Table 93: Mean blood ALP (IU/L) level of rabbits with standard deviation 143

Table 94: Blood GGT (IU/L) level of rabbits in triplicate 145

Table 95: Mean blood GGT (IU/L) level of rabbits with standard deviation 146

Table 96: Blood ALT (IU/L) level of rabbits in triplicate 148

Table 97: Mean blood ALT (IU/L) level of rabbits with standard deviation 149

Table 98: Blood AST (IU/L) level of rabbits in triplicate 151

Table 99: Mean blood AST (IU/L) level of rabbits with standard deviation 151

Table 100: Blood AST/ALT ratio level of rabbits in triplicate 153

VI List of Figures

Figure 1: Leves and stem of H. nepalensis 3

Figure 2: Weights of different fractions of the leaves of H. nepalensis 51

Figure 3: Weights of different fractions of the stem of H. nepalensis 51

Figure 4: Weights of different extracts of the leaves of H. nepalensis 52

Figure 5: Weights of different extracts of the stem of H. nepalensis 53

Figure 6: Heavy metals of the leaves of H. nepalensis 54

Figure 7: Heavy metals of the stem of H. nepalensis 54

Figure 8: Quantitative presence of phytochemicals in the leaves of H. nepalensis 56

Figure 9: Quantitative presence of phytochemicals in the stem of H. nepalensis 56

Figure 10: DPPH radical scavenging activity of H. nepalensis leaves extracts 58

Figure 11: DPPH radical scavenging activity of H. nepalensis stem extracts 59

Figure 12: Hydrogen peroxide radical scavenging activity of H. nepalensis 61

leaves extracts

Figure 13: Hydrogen peroxide radical scavenging activity of H. nepalensis stem 64

extracts

Figure 14: Antibacterial activities of the methanol extract of leaves of H. nepalensis 65

Figure 15: Antibacterial activities of the ethanol extract of leaves of H. nepalensis 65

Figure 16: Antibacterial activities of the n-hexane extract of leaves of H. nepalensis 66

Figure 17: Antibacterial activities of the petroleum ether extract of leaves of H. 67

nepalensis

Figure 18: Antibacterial activities of the chloroform extract of leaves of H. nepalensis 68

Figure 19: Antibacterial activities of the methanol extract of stem of H. nepalensis 68

Figure 20: Antibacterial activities of the ethanol extract of stem of H. nepalensis 68

Figure 21: Antibacterial activities of the n-hexane extract of stem of H. nepalensis 70

Figure 22: Antibacterial activities of the petroleum ether extract of stem of H. nepalensis 71

VII Figure 23: Antibacterial activities of the chloroform extract of stem of H. nepalensis 72

Figure 24: Antifungal activities of the methanol extract of leaves of H. nepalensis 72

Figure 25: Antifungal activities of the ethanol extract of leaves of H. nepalensis 73

Figure 26: Antifungal activities of the n-hexane extract of leaves of H. nepalensis 74

Figure 27: Antifungal activities of the petroleum ether extract of leaves of H. nepalensis 74

Figure 28: Antifungal activities of the chloroform extract of leaves of H. nepalensis 75

Figure 29: Antifungal activities of the methanol extract of stem of H. nepalensis 76

Figure 30: Antifungal activities of the ethanol extract of stem of H. nepalensis 77

Figure 31: Antifungal activities of the n-hexane extract of stem of H. nepalensis 77

Figure 32: Antifungal activities of the petroleum ether extract of stem of H. nepalensis 78

Figure 33: Antifungal activities of the chloroform extract of stem of H. nepalensis 79

Figure 34: Antibacterial activities of the azithromycin (Am) and ciprofloxacin (Cf) 80

Figure 35: Antifungal activities of the clotrimazole (Cm) 80

Figure 36: Blood glucose level of rabbits 85

Figure 37: Blood triglyceride level of rabbits 87

Figure 38: Blood cholesterol level of rabbits 90

Figure 39: Blood total bilirubin level of rabbits 92

Figure 40: Blood total protein level of rabbits 95

Figure 41: Blood albumin level of rabbits 98

Figure 42: Blood globulin (g/dl) level of rabbits 99

Figure 43: Blood A/G ratio level of rabbits 101

Figure 44: Blood GCI level of rabbits 103

Figure 45: Blood creatinine level of rabbits 106

Figure 46: Blood ALP (IU/L) level of rabbits 108

Figure 47: Blood GGT (IU/L) level of rabbits 111

Figure 48: Blood ALT (IU/L) level of rabbits 114

VIII Figure 49: Blood AST (IU/L) level of rabbits 116

Figure 50: Blood AST/ALT ratio level of rabbits 118

Figure 51: Blood glucose level of rabbits 120

Figure 52: Blood triglyceride level of rabbits 123

Figure 53: Blood cholesterol level of rabbits 126

Figure 54: Blood total bilirubin level of rabbits 128

Figure 55: Blood total protein level of rabbits 131

Figure 56: Blood albumin level of rabbits 134

Figure 57: Blood globulin (g/dL) level of rabbits 135

Figure 58: Blood A/G ratio level of rabbits 137

Figure 59: Blood GCI level of rabbits 139

Figure 60: Blood creatinine level of rabbits 141

Figure 61: Blood ALP (IU/L) level of rabbits 144

Figure 62: Blood GGT (IU/L) level of rabbits 147

Figure 63: Blood ALT (IU/L) level of rabbits 149

Figure 64: Blood AST (IU/L) level of rabbits 152

Figure 65: Blood AST/ALT ratio level of rabbits 154

IX Appendices

Appendix 1: Two-way ANOVA (multiple comparisons) table for blood glucose level 232

of rabbits

Appendix 2: Two-way ANOVA (multiple comparisons) table for blood triglyceride 232 level of rabbits

Appendix 3: Two-way ANOVA (multiple comparisons) table for blood cholesterol level of 233 rabbits

Appendix 4: Two-way ANOVA (multiple comparisons) table for blood total bilirubin 233 level of rabbits

Appendix 5: Two-way ANOVA (multiple comparisons) table for blood total protein level 234 of rabbits

Appendix 6: Two-way ANOVA (multiple comparisons) table for blood albumin level of 234

rabbits

Appendix 7: Two-way ANOVA (multiple comparisons) table for blood globulin level of 235

rabbits

Appendix 8: Two-way ANOVA (multiple comparison) table for blood A/G ratio level of 235 rabbits

Appendix 9: Two-way ANOVA (multiple comparisons) table for blood GCI level 236

of rabbits

Appendix 10: Two-way ANOVA (multiple comparisons) table for blood creatinine 236

level of rabbits

Appendix 11: Two-way ANOVA (multiple comparisons) table blood ALP (IU/L) 237

level of rabbits

Appendix 12: Two-way ANOVA (multiple comparisons) table blood GGT (IU/L) 237

level of rabbits

Appendix 13: Two-way ANOVA (multiple comparisons) table blood ALT (IU/L) 238 level of rabbits

X Appendix 14: Two-way ANOVA (multiple comparisons) table blood AST (IU/L) 238

level of Rabbits

Appendix 15: Two-way ANOVA (multiple comparisons) table blood AST/ALT 239 ratio level of rabbits

Appendix 16: Two-way ANOVA (multiple comparisons) table blood glucose level of 239 rabbits

Appendix 17: Two-way ANOVA (multiple comparisons) table blood triglyceride 240 level of rabbits

Appendix 18: Two-way ANOVA (multiple comparisons) table blood cholesterol 240

level of rabbits

Appendix 19: Two-way ANOVA (multiple comparisons) table blood total 241 bilirubin level of rabbits

Appendix 20: Two-way ANOVA (multiple comparisons) table blood total protein 241 level of rabbits

Appendix 21: Two-way ANOVA (multiple comparisons) table blood albumin 242 level of rabbits

Appendix 22: Two-way ANOVA (multiple comparisons) table blood globulin 242 level of rabbits

Appendix 23: Two-way ANOVA (multiple comparisons) table blood A/G ratio 243 level of rabbits

Appendix 24: Two-way ANOVA (multiple comparisons) table blood GCI level of 243 rabbits

Appendix 25: Two-way ANOVA (multiple comparisons) table blood creatinine 244 level of rabbits

Appendix 26: Two-way ANOVA (multiple comparisons) table blood ALP (IU/L) 244 level of rabbits

Appendix 27: Two-way ANOVA (multiple comparisons) table s blood GGT (IU/L) 245 level of rabbits

Appendix 28: Two-way ANOVA (multiple comparisons) table blood ALT (IU/L) 245 level of rabbits

XI Appendix 29: Two-way ANOVA (multiple comparisons) table blood AST (IU/L) 246 level of rabbits

Appendix 30: Two-way ANOVA (multiple comparisons) table blood AST/ALT ratio 246 level of rabbits

XII List of Abbriviations

A/G Ratio: Albumin/Globulin Ratio

GCI: Globuline Compensation Index

ALP: Alkaline Phosphatase

GGT: Gamma-Glutamyltransferase

ALT: Alanine Aminotransferase

AST: Aspartate Aminotransferase

XIII Abstract Plants are the prime source of medicine and are used for the treatment of various diseases globaly. About 70% percent of Pakistani population is dependent on medicinal plants. During this study, Hedera nepalensis K. Koch (Araliaceae) was thoroughly screened for its natural products, bioloigical and pharmacological activities. The phytochemicals analysis showed that 3 gm of crude exract from leaves of H. nepalensis contain about 3.2 mg/L of carbohydrates, 2.7 mg/L of proteins, 8.9 mg/L of saponins, 0.7 mg/L of alkaloids, 2.5mg/L of tanins, 2.1 mg/L of flavonoids, 2.8 mg/L of terpenoids, 0.3 mg/L of phenolic compounds and 1.9 mg/L of phytosterols, while 3 gm of crude exract of the stem showed 1.3 mg/L of carbohydrates, 1.9 mg/L of proteins, 0.6 mg/L of saponins, 0.7 mg/L of alkaloids, 3.4 mg/L of a tanins, 0.76 mg/L of a flavonoids, 0.31 mg/L of terpenoids, 0.9 mg/L of phenolic compounds and 0.84 mg/L of phytosterols. The highest content of ash in the leaves was calculated as 34.7% and for stem 23.5 %.

The free radical scavenging activity of extracts of stem and leaves of H. nepalensis were restrained in terms of hydrogen donating or radical scavenging ability using the stable radical 2, 2-diphenyl-1-picrylhydazyl (DPPH) and hydrogen peroxide. Higher free radical scavenging activity was confirmed by low value of absorbance. The DPPH radical scavenging for leaves showed 50, 54.54, 47.27, 60, and 70.90 scavenging activity respectively, while for stem it exhibited 50, 77, 74, 20.66 and 72 scavenging activity respectively. The hydrogen peroxide scavenging activity was studied at the absorbance of 285 nm with average time of 10 minutes incubation for leaves of methanol, ethanol, ethyl acetate, DCM, petroleum ether extracts with concentration of 0.1 mg/ml exhibited 51.81, 58.18, 42.72, 46.36 and 26.36 respectively, While the stem showed 61, 53, 34.02, 31 and 45.13 respectively.

The heavy metals analysis for the leaves of H. nepalensis revealed the presence of 140.41 mg/L of Iron (Fe), 12.09 mg/L of Manganese (Mn), 56.51 mg/L of lead (Pb), 3.7 mg/L of zinc (Zn), 1.85 mg/L of copper (Cu), 1.35 mg/L of nickel (Ni), 0.24 of Cadmium (Cd) and 0.37 mg/L of Chromium (Cr) while the stem showed 156.43 mg/L of Iron (Fe), 123.79 mg/L of Manganese (Mn), 156.43 mg/L of lead (Pb), 20.41 mg/L of zinc (Zn), 6.99 mg/L of copper (Cu), 4.51 mg/L of nickel (Ni), 0.33 mg/L of Cadmium (Cd) while the stem failed to show Chromium (Cr) presence. XIV Antibacterial activity of methanol, ethanol, n-hexane, petroleum, ether and chloroform extract of leaves and stem extracts of H. nepalensis was studied against Pseudomonas aeruginosa, Shigella sonnei, Enterococcus faecalis, Klebsiella pneumonia, Escherichia coli, Bacillus subtilis, Erwinia cartovara, Bacillus atrophaeus, Citrobacter freundii, Salmonella typhi, Staphylococcus aureus, Bacillus cereus and Agrobacterium tumefacians. Similarly, antifungal activity of methanol, ethanol, n-hexane, petroleum, ether and chloroform extract of leaves and stem extracts of Hedera nepalensis was studied against Alternaria alternate, Aspergillus flavus, Penicillium notatum, Aspergillus niger, Trichoderma harzianum and Candida albican. The highest value of 36 mm of inhibition zone was produced against Bacillus subtillus by methanol extract of H. nepalensis followed by 38 mm of inhibition zone against Klebsiella pneumonia and Escherichia coli by methanol and ethanol extract of H. nepalensis.

The ethanolic extract of H. nepalensis was found to kill rabbits at the dose rate of 7.88 g/kg body weight. The multiple oral administration of the tested doses of ethanolic leaf extracts at the dose rate of 200 mg/kg, 300 mg/kg, 400 mg/kg, Vitamin C (positive control) and glibinclamide (20 mg/kg b/wt.), at seventh, fourteenth, twenty first, and twenty eighth day post administration considerably reduced the levels of blood glucose, cholesterol, triglyceride, bilirubin, globulin, A/G ratio, GCI level, creatinine, ALP, GGT, ALT, AST, AST/ALT ratio while increased the total proteins and albumin whereas stem extract of H. nepalensis showed non significant change in blood glucose level, cholesterol, triglyceride, bilirubin, globulin, A/G ratio, GCI level, creatinine, ALP, GGT, ALT, AST, AST/ ALT ratio, total proteins and albumin. It is concluded that leaves of H. nepalensis have strong capacity to cure various infectious and non infectious diseases.

Chapter - 1 Introduction

Chapter 1 Introduction

In past plants were used for food, medicine, and shelter but later on man used plants for other purposes as well (Ali and Qaiser, 2009). Plants have been considered as the major source of primary health care (Buitron, 1999). In past folk medicines were obtained from about 70,000 plants (Lewington, 1990; Marles and Fransworth, 1985). Herbal medicnies had been the best sorce of treatment in the past 2,000 years (Thomson, 1978). About 75% percent people in Pakistan depend on plant medicines (Khan et al., 2012). Pakistan has been gifted with a wide variety of medicinal plants in which some of the identified medicinal plants includes about 1572 genera and 5521 species) and these plants are used as folk remedies (Ali, 2008). According to estimate of the World health organization, plant medicines were used by 80% for the treatment of themselves (Farnsworth et al., 1985). Plants are the major source of bioactive phytochemicals that can be used as medicine. The history of medicinal plants is as old as human civilization itself. Plants provide about 40% of medicine in the form of neutraceuticals and herbal drugs. Drug discovery and phytochemicals continue to be the source of medicinal plant’s research (Balick et al., 1996; Shulz et al., 2001; Skidmore and Roth, 2004). Higher plants were used for the purpose of extraction to obtain 119 medicines (Farnsworth et al., 1985).

1.1 Biological study of H. nepalensis

Kurie palul (Hedera nepalensis K. Koch) belongs to the family Araliaceae. In Pakistan these are found throughout the mountaineous area of Malakand, Dir, Swat, Azad Jammu and Kashmir at an altitude of 800-3000 meters. The berries are purgative and are useful in febrile disorders. The dried leaves are used to stimulate sores, stimulant, cathartic, diaphoretic and used as antidiabetic (Shinwari et al., 2000).

The family Araliaceae has 84 genera and 920 species of flowering plants. In Pakistan, there are only three genera of Araliaceae which are Hedera, Aralia and Scheflera. Hedera is a genus of 15 species (Nasir and Ali, 1975). The taxonomic consideration of the genus Hedera is ambiguous but this genus has been used for economic development (Ackerfield and Wen, 2002). The species selected in the present research work is H. nepalensis, locally known as Arbambal (Shah and Khan, 2006).

Chapter 1 Introduction

Figure 1: Leaves and stem of Hedera nepalensis

1.2 Medicinal uses Traditionally, different parts of the plants were used as folk medicines for the treatment of various diseases. Extracts of leaves and berries can be used as stimulating, diaphoretic and cathartic agents (Qureshi et al., 2007). H. nepalensis has the power of reducing blood glucose level and can be used for the cure of fever, pulmonary dieseases and rheumatism (Shah and Khan, 2006). Majester Savornin et al. (1991) explored the antileishmanial activity of Hedera helix. Cioca et al. (1978) studied the antibacterial property of the saponin from Hedera helix. The triterpenoid saponins of Hedera helix show antifungal activity (Favel et al., 1994). Hamayun et al. (2006) reported its anticancer properties as well. Inayatullah et al. (2007) screened the crude methanolic extract of H. nepalensis (leaves + stem) for different biological activities such as brine shrimp cytotoxic activity, potato disc antitumor activity and phytotoxic activity. This plant has also been evaluated for its antifungal activity by Xue et al. (2010). Α-hedrin from hedra helix show cytotoxic activity by upsetting the cell membrane which leads to cell expiry (Danloy et al. 1994; Quetin-Leclerq et al., 1992). Elias et al. (1990) found that the saponins of hedra helix show antimutagenic activity against promutagen, Benzo-(a) Pyrone. Inayatullah et al. (2007) found that H. nepalensis extract have antitumor activity. The extract of H. nepalensis is antitussive, antispasmodic and anti-infammatory activity as the research work o shows of Braudet (1967). Mehran et al. (1974) found α-hedrin, hedrasaponin B and hedrasaponin C, sterols, tannins,

Chapter 1 Introduction nitrogenous bases, oxides, and flavonoids in the hedra helix. Pant (1988) found three types of saponins which are: Nepalin-I, Nepalin-II and Nepalin-III. Twelve saponins which are: A(I), B(II), D(III), D(IV), F(VI), H(VII), I(VIII), K(X), M(XII), N(XIII) and P(XIV) were isolated by Kizu et al. (1985). Following the work of Inayatullah et al. (2007), the potato disc antitumor activity was also conducted on the fractions of H. nepalensis to find out how the different phytochemicals are separated into different fractions. The genus Hedera belongs to family Araliaceae found mainly in North Africa, Europe, Asia including Pakistan, Afghanistan. One species of the genus Hedera is found in Hazara division of Pakistan. Its saponins have been used for the treatment of human ailments, muscles relaxation, tranquilization, for killing fungi, mulluscs, leishmania (Timon et al., 1980; Julien et al., 1985). The leaf extract of Hedera helix inhibit the conidial germination of Vinturi ineaualis Bosshard (1992). Methanolic and aqueous extracts of Hedera helix were found hypoglycemic activity in rabbits to a noteworthy level (Ibrar et al., 2003), and the leaves of Hedera helix also exhibited cytotoxicity (Ibrar et al., 2001). Toxicity for the leaves and fruits of Hedera helix was checked and was found to cause irritation of gastrointestinal canal and skin (Ghias et al., 2011). Despite its multipurpose traditional uses, very little data exist on its phytochemical constituents and antibacterial activity. Hence, the current study has been intended to assess its antibacterial activity and phytochemical constituents. In the view of the medicinal importance of H. nepalensis, based on traditional knowledge and surveyed literature, the present study focused on the fractionation procedure, antioxidant properties, and plant chemicals investigation of the crude methanolic extract and fractions.

1. 3 Pharmacognostic standardized evaluation of crude extract 1.3.1 Macroscopic and microscopic study

Medicinal plants provide the basic raw materials for indigenous pharmaceuticals which are important economically (Aiyelaagbe, 2001; Augusti et al., 1996). Drug authentication is very important as Valeriana wallichii is adulterated by Acorus calamus (Ahmad et al., 2009). The evaluation of the drug includes various ways some of which are macroscopic, microscopic methods. Macroscopic methods are the size, shape, odor, taste, texture of the crude drugs. Color of the stem, leaves, roots, bark, and rhizome give the color of the drug and texture is identified from the structure of the form of the drug. Odor and taste are of utmost significance in the identification of the drug. Small scars, wrinkle, fracture etc. are also helpful in the recognition of the given drug. Microscopic methods include the

Chapter 1 Introduction features of spines, spores, and epidermal structures. Cell types are also important to the close study of the drug. Therefore it is required that the plant must be identified properly and the nature and level of the adulterants should be identified to the possible purity level.

1.3.2 Physiochemical characterization of the crude extract

1.3.2.1 Extractive values

Extraction is the separation of the active component of the drug from the inactive components. This is depending on the solvent extraction procedure. The product obtained may be in the liquid extract, infusions, decoctions, solid or powder form. Such type of preparations is called galenicals (Handa et al., 2008). Extractive values are used for the evaluation of drug. For quality study the crude drug is subjected to the extraction procedures. Various solvents are used like n-hexane, water, methanol, ethanol etc). In aqueous extraction, dissolved portion is isolated and is considered as substandard product and the undissolved portion is called marc. The alcohol used is in 90%/20% (v/v) but it depends on the type of drug. Petroleum and n-hexane extraction procedures are used to extract lipid contain portion of the plant.

1.3.2.2 Ash value

It is the residue of the incineration of the crude drug. It is used to study the powder drug as it contains inorganic salts and inorganic matter. Drugs are usually separated from these the lump of such dirt as specified by pharmacopeia. So plants parts are burnt to produce ashes. Kumar et al, (2012) determined the total ash which was 13.77, acid insoluble as 0.073 and water soluble ash 4.803 of the the stem bark of Bauhinia racemosa. It gives the value of the inorganic matter present in the plant part as impurity. The total ash value indicates carbonates, phosphates, silicates, and silica.

1.3.2.3 Heavy metals analysis

Heavy metals are toxic in nature while its excessive consumption of heavy metals for the last few decades is of significant importance (Livingstone, 2001; Yang and Rose, 2003). These elements produce reactive oxygen species (ROS) causing oxidative strain which in turn can cause cancer and can pose problems to human health and the environment (Damien et al., 2004 and Farombi et al., 2007).

Chapter 1 Introduction

The heavy metals in plants are very essential and studied for their eminence control (Arceuz et al., 2010). Their extraordinary levels are noxious. Most of the heavy metals such as Zn, Fe and Mg are vivacious for the regular performance of plants Grodner et al., 2000). Medicinal plants along with nurients absorb heavy metals which vary in in various species of the plant (Robert and Scoot, 1998). Medicinal plants are not lethal but are made lethal by the absorbed heavy metals Arpadjan et al., 2008; Itanna, 2002; Lasisi et al., 2006; Obi et al., 2006). In the two types of heavy metals, fundamental heavy metals are required to perform different functions in the body of human and in plants while noxious heavy metals are creating adverse effects in the body even in small amount Friberg et al., 1986; Abu-Darwish et al., 2009; Islam et al., 2007; Singh and Garg, 2006). Cadmium is a redundant heavy metal and is the most noxious to contaminate and is dangerous to the health of organisms (Rashed, 2001). Lead stops the formation of heme by changing the activities of its several enzymes. When lead is absorbed in stomach, it is send toward liver, kidney, heart and male gonads and affects the immune system (ATSDR, 2005). Some structural changes in various cellular systems and chromosomal abnormalities are caused by nickel (Coen et al., 2001). Chromium has two oxidation states; Cr (III) and Cr (VI). Cr (VI) is highly soluble in water so is most toxic and can cause DNA lesions on exposure (Reynolds et al. (2004). Hina et al. (2011) dogged heavy metals like Pb, Cd, Cu, Cr, Co, Fe, Ni, Zn in G.glabra, O.bracteatum, V. odorata, F. vulgare, C. cyminum, C. sativum and Z. officinalis through atomic absorption spectrophotometer. Adongo et al. (2012) used flame atomic absorption spectroscopy (FAAS) for the determination of heavy metals like Zn, Mg and Fe in eight medicnal plants. Radulescu et al. (2012) determined Cd, Cr, Co, Cu, Pb, Ni, and Zn using flame atomic absorption spectroscopy (FAAS) in plant samples.

1.3.3 Phytochemical characterization

Only 5- 10% of the total plants ranging in 250,000 to 750,000 species have been studied for the investigation of their dynamic compounds. The ancillary metabolites of the plants are in the form of plant odor (terpenoids), pigmentation (tannins and quinines) or in flavor (capsacin) form (Mallikharjuna et al., 2007). Extracts from higher plants were subjected to isolate 119 pure chemicls for medicine use throughout the world (Fransworth and Morris, 1985). The preliminary phytochemical screening is a qualitative chemical evaluation which indicates level of chemical constituents present in a plant drug. The chemical evaluation also leads to proper identification of biologicaly active groups or varieties from naturally occurring (crude) plant drugs (Gokhale, 1997). A plant is more

Chapter 1 Introduction important if it contain more active components. Phytochemicals as secondary metabolites are produced in numerous parts of the plant and is stored in several parts of the plant in the form of alkaloids, flovoinds, tannins, cyanogenic glycosides, phenolic compounds, saponins, and lignin. The purely isolated alkaloids are used against spasm and bacterial effects (Stray, 1998); Okwu, 2005). Medicinal plants are possessing biologically active components have therapeutic value (Harsha et al., 2002).

Souza et al, (2006) detected the presence of saponons, flavonoides, anthraquinones, and cardiac glycosides in the leaves of A.mellefolium. Phytochemicals like alkaloids, saponins from the leaves, bark and roots exracts of the Senna hirsute, Landolphia dulcis and Daniella oliveri were isolated by Collborn and Bolatito (2010). In response to microbial infection, plants produce flavonoids (Dixon et al., 1983). Flavonoids like swertifrancheside, glycyrrhizin and chrysin can be used against AIDS virus (Pengsuparp et al., 1995; Watanbe et al., 1996; Critchfield et al., 1996). Four compounds like bakuchiol, corylifolin, corylin, and psoralicin were sequestered from P.corylifolia by (Wand et al., 2004). Palanisamy et al. (2012) carried out the phytochemical investigation of the whole plant of Dipteracanthus prostrates nees and found alkaloids, tannins, glycosides, tannins, fats, phenolic compounds, gums, mucilage, flavonoids and carbohydrates. The flavonoids protect plants from microorganisms (Sohn et al., 2004). The plant extracts and phytochemicals have both antimicrobial and other therapeutic significance (Almagboul et al., 1985). Phytochemicals and biochemicals in plants have characteristic color, flavor, smell, and texture. Phyto- chemicals can prevent diseases (Renu, 2005). Plants secondary metabolic products have active substances like phenolic compounds, essential oils, and tannin etc (Jansen, 1986). Tannins were found to have complex pharmacological properties (Ferreira et al., 2008). Tannins-rich preparations can be used for killing of helminthes, for reducing oxidants, and against virus and bacteria, for cancr treatment and chelating dietry iron (Buzzini et al., 2008; Ketzis et al., 2006; Koleckar et al., 2008), Clauss et al., 2007; Chung et al., 1998). Tannins have a distinguishing smell and biting taste and have the ability to bind with proteins and have impact on animal nutrition, including inhibition of growth rate digestive enzymes (Bennick, 2002). Tannins are a class of polymeric phenolic bodies having molecular weight of 500 to 3,000 and have astringent property (Haslam, 1996), and they can be extracted from bark, wood, leaves, fruits and roots (Scalbert, 1991). They have two classes viz hydrolysable and condensed tannins. Hydrolysable tannins are as multiple esters with D-glucose; while condensed tannins (also known as proanthocyanidins) are obtained from flavonoid

Chapter 1 Introduction monomers. Tannins found in woody tissues of plants are molded into shapes either by condensations of flavan derivatives or polymerization of quinone units (Geissman, 1963). Tannins have been used for the treatment of diarrhoea (Yoshida et al., 1991) as diuretics, (Okuda et al., 1983; Hatano et al., 1991) against stomach and duodenal tumours (Saijo et al., 1996). Tannins also show anti-inflammatory and antiseptic effect (Haslam, 1996). Tannins cause the precipitation of heavy metals and alkaloids (except morphine) result in the production of different phytomedicines (Haslam, 1996). Tannins are used to produce cationic dyes and iron gallate ink. Tannins are added to clarify wine, beer, and fruit juices in food industries (Würdig and Woller, 1989). Some of the tannins have the property of stoping HIV replication (Quideau and Feldman, 1996).

Acetate units on aggregation produce terpenoids and have more cyclic structures and more branching so are different from fatty acids which have no such type of branching. Some of the terpenoids are camphor (monoterpenes), farnesol and artemisin (sesquiterpenoids (C- 15)). Artemisin with its derivative α-arteether also called qinghaosu. Qinghaosu are used as antimalarials (Vishwakarma, 1990). Terpenenes or terpenoids have the potential to kill bacteria (Ahmed, 1993; Amaral et al., 1998; Barre et al., 1997; Habtemariam et al., 1993; Himejima et al., 1992; Kubo et al., 1992). Capsaicin A is the component of terpenoid which is used as analgesic can affect nervous, cardiovascular, and digestive systems (Virus and Gebhart, 1979; Cordell and Araujo, 1993). Purple colored clover extract which is soluble in ethanol produce a terpenoid called petalostemumol have bacteriostatic effect on Bacillus subtilis and Staphylococcus aureus and have no potential against gram-negative bacteria as well as Candida albicans (Hufford et al., 1993). Kadota et al. (1997) elaborated that a diterpene- trichorabdal A extracted from a Japanese herb has bactericidal effect on H. pylori. Baricevic et al. (2001) reported that diterpene carnosol, triterpenes, oleanolic and ursolic acids show anti-inflammatory activities). German pharmacist ―Meissner‖ used the term alkaloid. They are naturally occurring group of Heterocyclic basic nitrogen containing compounds. More than 6000 alkaloids of different types are known and dozens of new alkaloids are being identified daily in the phyto-chemical laboratories of the world. They are basic in nature and form salt with acids. Alkaloids are the secondary metabolites distributed to certain families and genera of plant kingdom. They have been reported in 9% of over 10,000 plant genera. Among the angiosperm, they occur abundantly in certain dicotyledons, although a few are found in lower plans e.g. muscarine and ergotoxin, as well as in ferns and fungi (Cordell, 1981). In 1805, morphine an alkaloid was secluded from the opium poppy

Chapter 1 Introduction

Papaver somniferum (Fessenden and Fessenden, 1982); Morphine is a Greek Morpheus which means god of dreams. Two derivatives of morphine are Codeine and heroin. The most important alkaloid bearing families are Apocynaceae, Liliaceae, Astraceae, Berberidaceae, Fabaceae, Leguminoseae, Lonaniacaae, Meniepermaceae, Papavaraceae, Ranunculaceae, Rubiaceae, and Solanaceae (Cordell, 1981). Alkaloid may occur in various parts of plants such as in seeds (Datura, Hyosymus) in fruit (conium), in leaves (Atropa) etc. Their function in plants are still unknown and they are often regarded as nitrogenous waste product analogous to urea and uric acid in animal and their occurrence in vacuole rather than in living parts of protoplast supports such a view (Cordell, 1981). Berberine an alkaloid has the potential against trypanosomes and plasmodia (Omulokoli et al., 1997; Freiburghaus et al., 1996). Berberine and harmane both are aromatic planar quaternary alkaloids (Hopp et al., 1976) and have the ability of integration with DNA (Phillipson and Neill, 1987). Phenolic compounds, nitrogen compounds, vitamins, terpenoids and some other secondary metabolites can be used against oxidant, inflammation, tumor, cancer, bacteria and virus (Geissman, 1963). Presently 12,000 secondary metabolites have been isolated (Schultes, 1978). They are new sources of economic materials like tannin, industrial oil, gums, and other precursors (Farnsworth, 1985).

1.4 Biological evaluation of the crude extract

1.4.1 Antioxidant activities

Phenolic compounds produced by plants have redox properties and metal chelation property show antioxidant activity (Rice-Evans et al., 1995; Khanavi et al., 2009; Huda- Faujan et al., 2009). Antioxidants are produced as a consequence of metabolic activities of the plants (Ramaranthnam et al., 1995; Demo et al., 1998; Sanchez-Moreno et al., 1999; Clouladis et al., 2003; Linn et al., 2003). Oxidizing chain reactions are blocked by the oxidation process through antioxidants. In the two types of antioxidents, the synthetic antioxidants can cause carcinoma therefore is banned (Bronen, 1975) while the natural antioxidants have anticarcinogenic, anti-mutagenic, anti-aging activity (Cook and Samman, 1996); Liyana-Pathirana and Shahidi et al. 2006). David et al. (2010) used the DPPH (2, 2- diphenyl-1-picrylhydrazyl) to test for compounds with antioxidant activity in A.millefolium aqueous extract. In the activity of free radical scavengers or hydrogen donors, the violet color of the persistent free radical DPPH changes to yellow and the change is monitored through spectrophotometer. Antioxidant activity of G. sylvestre leaf extract using DPPH assay was

Chapter 1 Introduction studied by (Rach et al., 2009). According to Wand et al. (2001), the compounds metroterpine and flavonoids sequestered from the seeds of P. corylifolia acts as antioxidant activity. Inactivation of the cell may occur if free radicals attack on the unsaturated fatty acids as it causes peroxidation of the cell membrane (Dean and Davies, 1993). Therefore antioxidants are used for the treatment of different types of cellular degradation (Tutour, 1990).

1.4.2 Antimicrobial activities

1.4.2.1 Antibacterial activities

The saponins and cardiac glycosides steroids, tannins, volatile oils, phenols and flavones exhibited potent molluscicidal and antibacterial activities (Ahmad et al., 1998; Pamplona-Roger, 1999; Shariff, 2001). The tannins polyphenols and flavonoids possess good antibacterial activities (Zhentian et al., 1999; Meng et al., 2001; Hideyuki et al., 2002). According to Mitscher (1987) plants have shown the potentialities against the new bacterial agents. Plant stems, roots, leaves, bark, flowers or fruits may be used in crude form as antimicrobial agents (Beuchat et al. (1994). Bacteria like Escherichia coli, Shigella flexneri, Salmonella typhi and Staphylococcus aureus are pathogenic to human beings (Anne and Geboes, 2002). About 122 known plant species were used to stop growth of Staphylococus aureus, Escherichia coli and Aspergillus niger (Martinez, 1994; Martinez, 1996). Aldehyde and phenolic compounds have the potential of stoping the growth of microbes (Lai and Roy, 2004). Phenolic compounds which is a secondary metabolite show antimicrobial activity Jansen et al., 1978; Rahman et al., 2009). The growth of bacteria is inhibited by tannins (Chung et al., 1998). Natural aggregation of different compounds in crude extracts having enhanced antimicrobial activity than individual and the purified components (Delaquis et al., 2002). The extracts of A. calmus were found to be antibacterial (Bhuvaneswari et al., 2009). The growth of K. pneumonia and Proteus mirabilis can be inhibeted by using the Aqueous and ethanolic extracts of seeds of Fumaria indica (Parckh and Chanda, 2007). Similarly the growth of B. pumilis, B. subtilis, P. aeruginosa and S. aureus can be inhibited by using the ethanolic extract of G. sylvestre leaves (Satdive et al., 2003). The extract of Origanum vulgare was premeditated for stopping the growth of 11 different genera of gram negative bacilli (Chaudhry et al., 2007).

Chapter 1 Introduction

1.4.2.2 Antifungal activities

Biologically active compounds have been isolated which have shown antifungal activities by stopping their growth through various modes like binding to chitin or breaking the fungal membrane or cell wall (Grayer and Harborne, 1994). Parthenum argentatum was grounded and exract was obtained and compounds were isolated and were checkedin contradiction of Candida albicans, Torulopsis, Hansemula, Klebsiella pneumoniae and Pseudomonas aeruginosa was detected (Martinez, 1994; Martinez, 1996). Singh et al. (2010) determined antifungal activity of methanolic crude exract of Acorus calmus. The Psoralea corylifolia extract showed antifungal activity (Jiangning et al., 2004). The crude methanolic extracts of the the six cultivars of V. zizanioides root show antifungal activity (Gangrade et al., 1990); Hammer et al., 1999); Leupin et al., 2000). Takahashi et al., 2004) investigated the antifungal activity of the extract of Eucalyptus leaves against Trichophyton mentagrophytes. The antifugal activity of methanolic leaf extracts of Eucalyptus camaldulensis against Microsporum gypseum, Trichphyton rubrum, Tricophyton schoenleinii, tricophyton mentagrophytes, Microsporum canis and Epedermophyton floccosum. The lea extract of was vulnerable against a fungal species Candida albicans (Akinsulire et al., 2007). The essential oil display substantial antifungal activity against a broad spectrum of tested fungi (Recep et al., 2008). The alkaloids Phenanthroindolizidine from members of Asclepiadaceae, exhibit pronounced cytotoxicity against Candida albicans (Staerk et al., 2000). Fawzi et al., (2009) determined the antifungal activity of Cinnamum zeylanicum, Cymbopogon proximus, Laurus nobilis, Persea americana and Zingiber officinale against Alternaria alternate and Fusarium oxysporum by using cold distalled water which have strong antifungal effects. The antifungal activity of the essential oil of Callistemon rigidus, Callistemon citrinus, Eucalyptus saligna and Eucalyptus camadulensis against Aspergillus flavus by (Dongmo et al., 2010).

1.5 Pharmacological evaluation of the crude extract

1.5.1 Introduction to Diabetes mellitus

Diabetes mellitus is a disease of various factors which has great effect on health, quality of life and probability of patients and on the health maintenance system. From 1997 to 2010, 221 million people have been reported with diabetes (Amos et al., 2010). According to 2008 data, 230 million people have diabetes globally (Arumugam et al., 2008). This disease

Chapter 1 Introduction results from damage cells of the Langerhan islets which make the body to produce pancreatic hypoglycemic hormone called insulin. Three main signs of this disease are: excessive urine called Polyuria, glycosauria (glucose presence in urine) and hyperglycemia (high glucose level in blood) (Koffi et al., 2009). Glucose and lipid metabolism is changed due to diabetes (Rajasekaran et al., 2006). Changes in lipid metabolism have been proved experimentally (Sochar et al., 1985). Diabetus mellitus and obesity are co-related (Afridi and Khan, 2009). Liver depends on insulin and take part in oxidation and metabolic transformation of fatty acids, creation of cholesterol, phospholipids, and triglycerides (Seifter et al., 1982).

1.5.2 Types of Diabetes

Diabetes is of two types.

i. Diabetes insipidus: it is due to defect in pituitary glands. The affected patient may secrete urine of 5-10 litters a day. It’s rare. ii. Diabetes mellitus: it due to the defect in pancreas which release insulin. In diabetes mellitus low level of insulin is produced which is insufficient to counteract the high level of glucose in blood. So flucose level becomes high in blood. It has in turn two types. a. Type 1 or Insulin Dependent Diabetes mellitus (IDDM): This type of diabetes is caused when the body autoimmune destroys pancreatic islets cells. This results into absolute deficiency insulin. The initial sign include ketoacidosis with an acute illness. Other disorders like Adison disease, thyrioiditis, and pernicious anemia may also result. Type 1 diabetes is considered to be inherited. It is common in children Galloway (1988). The patient may be thin and has low weight Katzung (1993). b. Type-2 or Non-Insulin Dependent Diabetes mellitus (NIDDM): Patient of Type 2 diabetes has relative insulin deficiency and is related with obesity, age and physical indolence. Type 2 diabetes do not have ketoacidosis, not require insulin frequently, and is asympatomatic for many years (Federal Bureau of Prisons, 2009). It more common in aged persons. The muscles have low level of insulin as the β-cells of the pancreas has been destroyed (Glloway, 1998). Therapeutic insulin production do not meet demands so it has been that herbal treatment is the final remedy for type І and type ΙΙ diabetes (Arumugam et al., 2008).

Chapter 1 Introduction

1.5.3 Treatment of Diabetes mellitus The (NIDDM) can be treated by physical excersice, diet management and oral hypoglycemic drugs Herfindal (1988). Hypoglycemic drugs are: i. Sulphonylureas: these drugs stimulate the production of indegenus insuline (Herfindal, 1988). For example: Tolbutamide, acetohexamide and Glipizide. ii. Biguanides: These drugs stimulate the intake of glucose in the intestine, use of glucose in muscles and reduce appetite. For example: Metformin and Phenformin. iii. Insulin: They are genetically engineered. They are produced by GMB like E.coli. They are produced in commercial scale. They are available in different forms. 1.5.4 Antidiabetic activity of other plants More the 1200 plants species were used internationally for the cure of diabetes mellitus and many of them show effective hypoglycemic activity after laboratory testing (Eddouks et al., 2005 ). Ahmed et al. (2009) reported 23 families including three species of Liliaceae, four species of Poaceae and five species of Fabaceae, 33 gener and 37 plants were exposed for the cure of diabetes (Marles and Fransworth, 1995). The medicinal plants which are used orally to reduce blood glucose level is a supplement to current treatments (Bailey and Day, 1989). In Pakistan, Diabetes mellitus is more common in female than male (Samad, 1992). Momordica charanta is hypoglycemic action in alloxane induced diabetic rabbits (Akhtar et al., 1981); Day et al., 1990). Blood glucose lowering effect of Aegle marmelos and Saacia reticulate was revealed by Jimanz et al. (1984). Lamela et al. (1985) observed the hypoglycemic activity of Salvia lavanddulfolia in alloxane induced diabetec rabbits. Lathyrum salicaris stem and flowers also show hypoglycemic activity in rats (Lamela et al., 1985). Akhtar et al. (1986) reported the hypoglycemic effect of dried roots of Onosama echiodes in diabetic rabbits. The extract of Eriobotrya japonica shows hypoglycemic effect in already diabetic animals (Wadood et al., 1988; DeTommasi et al., 1991). The antidiabetic activity of Gymnema sylvestra was demonstrated by (Shanmugasunaram et al. (1990). Konecka and Jezierski (1997) studied White New Zealand male rabbits were nourished with a high-cholesterol (1%) diet for 7 weeks activity of Alkaline Phosphatase (ALP), Alanine aminotransferases (ALT), Aspartate Aminotransferases (AST) and level of glucose in the blood plasma of White New Zealand male rabbits were fed by high cholesterol (1%) diet for

Chapter 1 Introduction

7 weeks. Nagappa et al. (2003) used various solvents like petroleum ether, methanol, and aqueous for extraction purpose to investigate the hypoglycemic effect of Terminalia catappa Linn (combretaceae) fruit on alloxan-induced diabetic rats. Adeneye et al. (2007) reported that Diabetes mellitus can be treated with cold water decoction of Clerodendrum capitatum. Florence et al. (2007) studied the effect of methanolic extract of Dorstenia picta methanolic on blood glucose levels in normal and streptozotocin-induced diabetic rats. The extract was found to have the hypoglycemic activity.

Mallick et al. (2007) studied the glucose lowering activity of aqueous-methanolic (40:60) extract of root of Musa paradisiaca and leaf of Coccinia indica on streptozotocin-induced diabetic rats. Noor et al. (2008) deliberated the hypoglycemic effects of Aloe vera (L.) in streptozotocin-induced diabetic rats. And 300 mg/kg body wt of the drug was sufficient to reduce the blood glucose level. Nwaegerue et al. (2007) testified that the leaf extracts of Viscum album have hypoglycemic role. Jaouad et al. (2007) described that the plant Ajuga iva has both hypoglycemic and hypolipidemic effect in anesthetized normal and streptozotocin (STZ)-induced diabetic rats. Eliza et al. (2009) isolated Eremanthin compound from Costus speciosus is an antidiabetic agent while its oral use of (20 mg/kg b.w) considerably declined glycosylated hemoglobin (HbA1c), LDL-cholesterol serum total cholesterol, triglyceride and rise in HDL-cholesterol, plasma insulin, tissue glycogen and serum protein. Eremanthin also reestablished the changed plasma enzyme (lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and acid phosphatase) levels close to average. Koffi et al. (2009) studied Chrysophyllum cainito; a plant recognized by the traditional healers of Aboudé-Mandéké, as having antidiabetic properties. An aqueous decoction of plant’s leaves of C. cainito was evaluated as on rabbits induced with alloxane, a diabetogenic product. Different graded doses of this herbalmedicine were applied on postprandial blood sugar levels of diabetic rabbits. At doses of 10 g/l, C. cainito did not induce the decrease in glucose level consequence. A dose of 20 g/l reduced the hyperglycaemia from 5g/l to 1.4 g/l. A dose of 30 g/l of C. cainito produced a graded decrease in hyperglycaemia from 6.3 g/l to 3.2 g/l. After 6 weeks of treatment, the induced diabetic rabbits stopped eating and succumbed between the 8th and the 9th weeks of experimentation. It was thus concluded that C. cainito leaves have glucose lowering effect at doses > 10 g/l and appears toxic and lethal at 30 g/l. C. cainito produces its hypoglycaemic effect mainly through alkaloids, sterols or triterpens, the antidiabetic active constituents. Sivaraj et al. (2009) reported that Cassia auriculata and Aegle marmelos have anti-

Chapter 1 Introduction hyperglycemic and anti-hyperlipidemic propertyin streptozotocin induced diabetic rats. As a result β cells of islets of Langerhans were shrinked. They can be rcovered at the dose rate of 450 mg/by wt. Verma et al. (2009) worked on methanolic extract of dried leaves of Indigofera tinctoria Linn and found noteworthy drop in blood glucose level of rabbits as expected by Folin-Wu Method. In this experiment, alloxane is utilized as diabetes inducing agent. Gaamoussi et al. (2010) studied Chamaerops humilis and checked aqueous leaf extract of this plant has hypolipidemic properties in an animal model of obesity, hyperglycemia, and hyperlipidemia. The results of this study authorize the traditional use of the leaves of Chamaerops humilis in the cure of diabetes in Morocco. Since, the aqueous leaf extract also decreased total cholesterol and triglycerides, the plant may also be useful in the management of secondary hitches of diabetes (dyslipidemia). Furthermore, the plant may become a good basis of antidiabetic treatment. Jha et al. (2010) aimed to study the dried petroleum ether (60- 80°C) extracts of flower head of Sphaeranthusindicus were exposed for hypoglycemic activity in wistar rats. Alkaline phosphatase (ALP) is a membrane-bound enzyme found in liver. The serum levels of liver and bone ALP are used to diagnose hepatobiliary disease and bone disorders (Aus et al., 2006). ALP is also found in many tissues including bone, gall bladder, intestine, placenta, kidney and liver. Liver conditions cause bile stasis in liver which results into ALP elevations (Pelkonen and Hannimen, 1997). The two primary sources of Alkaline phosphatase are the liver (specifically, the biliary tree) and bones (it marks new bone formation) (Taylor et al., 1994). Alkaline phosphatase (ALP) removes phosphate groups from nucleotides, proteins, and alkaloids called dephosphorylation (Tamas et al., 2002). If bilirubin, Aspartate Aminotransferase (AST), or Alanine Aminotransferase (ALT) is elevated then it shows that ALP is coming from the liver. If calcium and phosphate levels are not normal then ALP is coming from bone. If 5’-nucleotidase is also increased, then high ALP shows liver disease. If both of the two tests are normal, then the high ALP is due to a bone disorder. In case of hepatitis, ALP is lower than AST and ALT. When bile ducts are not functioning properly then ALP and bilirubin level is increased than AST or ALT (American Association for Clinical Chemistry, 2001).

The present form of study is the evaluation of H. nepalensis by modern techniques to study their biological and pharmacological activities and to recommend if any of its effective side to pharmaceutical industries to fulfill the requirement of medicinal plants by human beings.

Chapter 1 Introduction

1.5.5 Aims and Objectives

 To standardize the evaluation of the drug.  To study the heavy metals of the test plant parts.  To develop safety protocol for the quality use of the medicinal plant.  To investigate its phyto-chemicals both qualitative and quantitatively in the  To envisage the essential oils and free amino acids of both parts of thleaves and stem of the plant.  To evaluate both parts through biological activities.  To study antimicrobial activities of both parts of the plant.  To find out the effectiveness of the acute and chronic study of the plant.  To report the outcome of plant extracts from stem and leaves on the glycemia with respect to triglyceride, cholesterol, bilirubin total, proteins total, albumin, Creatinine, ALP, GGT, ALT and AST.  To find new therapy which could be easily accessible inexpensive and efficient.  To strengthen the Pak Pharmacopoeia by reporting new uses of Hedera nepalensis.

Chapter - 2

Materials and Methods

17 Chapter 2 Materials and Methods

2.1. Selection of Plant

No antidiabetic study has yet been conducted on Hedera neplenensis of District Malakand; therefore, the said plant was selected.

2.2. Collection and identification of plant material The stem and leaves of H. nepalensis were collected in the month of March 2012 from village Kharkai, Tehsil Dargai, District Malakand Khyber Pakhtunkhwa, Pakistan. The plant was identified by Dr. Siraj u Din, Taxonomist at the Department of Botany, University of Peshawar, Khyber Pakhtunkhwa, Pakistan, and a voucher specimen ICP/HB/Pharma/010 was deposited at the herbarium of Islamia College University, Peshawar.

2.3. Pharmacognostic evaluation of crude extract Pharmacognostic studies were carried out by studying the macroscopic, microscopic, physiochemical (including extractive values, total ash value, and heavy metal analysis) and phytochemical characterization.

2.3.1. Macroscopic and microscopic study

For macroscopic and microscopic studies of the crude drug, protocol of Wallis (1976), Trease and Evanse (1982) and Mahmud et al. (2010) were used. Color, taste, odor, external margins, apices, texture, external and internal marking, fracture, shape and size was studied using magnifying glass or sensory organs in macroscopic studies. While for microscopic studies, several samples of were dissolved in the media (chloral hydrate and water). The media were mounted on clean slides and covered by cover slips gently.

2.3.2 Physiochemical characterization

Physiochemical characterization helps in the study of the pure line study and qualitative study of the crude drug. Extractive values, total ash values, and heavy metal analysis in different solvents of the extracts were carried out as follows.

2.3.2.1 Crude extract preparation

The plant collected was washed with fresh water. The washed plants were kept aside in shad for 21 days for drying. The plant material was exposed to sun light time to time to avoid fungal attack. Dry plant powder of 1400 grams was mixed with

18 Chapter 2 Materials and Methods

2450 ml methanol (Merck, D/6100 Darmstadt, and F.R Germany ). Methanol is highly flammable. It is toxic by inhalation. After mixing the powder with methanol, solution was kept for 5 days. After 5 days, the extract was sieved by filter paper. The extract was then exposed to rotary evaporator (Heidolph Laborata 4000 Efficient). After passing through the rotary evaporator, methanol was removed and filtrate accumulated in flask. Residue was again mixed with methanol for 5 days. After 5 days, the extract was separated from plant residue and exposed to rotary evaporator. Methanol was again removed and the dry filtrate accumulated in flask. The remaining residue was again mixed with methanol for 5 days. In similar way, the filtrate was separated from plant residue and by exposing to rotary evaporator. The filtrates again accumulated in flask. The extract from rotary evaporator was removed and was placed at room temperature for further dryness.

2.3.2.2 Fractionation procedure

Crude extract of 55 gm was dissolved in distilled water. This aqueous extract was then imperiled to get fractions in various solvents. Various solvents like Ethyl Acetate, Methanol, ethanol, n-Hexane, Dichloromethane and water solvents were used. The aqueous extract and organic solvents were poured into a separating funnel and were shake forcefully. The two layered were shaped. In which one was that of aqueous layer and the other was that of organic layer. Both were collected in separate flasks. The process was repeated three times and the solvent extracts were concentrated using Rotary Evaporator under reduced pressureat temperature 30-45 0C.

2.3.2.3 Determination of extractive values

Extractive values were determined by Soxhlet Extractor/apparatus.Twelve gram of the semisolid extract was heated at 45 0C for five hours in a china dish. These extracts were weighed after complete dry and the percentage of the extract was calculated by using the following formula.

%age of the extract= weight of the extract/ground material wieght × 100

2.3.2.4 Determination of total ash value

For total ash value study, the plant materials were grinded finely and 2 grams of the plant materials were poured into pre-weight crucibles. Now the crucibles were placed on flame to burn the plant materials and the ash value were determined.

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2.3.2.5 Heavy metals analysis

Two gram of ground plant material was booked in crucible. The crucible was heated on burner for half hour and it was retained in furnace at 600 0C (Elmer, 1982). The ash was obtained and thirty ml of hydrochloric acid solution (60 ml hydrochloric acid in 40 ml distilled water) was supplemented to it. It was then sieved and shifted to volumetric flask alreadycontaining fifty ml of distilled water, and level was made equal to hundred ml. After that it was analysed for heavy metals like Cu, Pb, Mn, Zn, Fe, Ni, Cd, Cr, Ag, Sr and Ba via atomic absorption spectrophotometer (AAS).

2.3.3 Phytochemical characterization

The following protocols were used for phytochemical screening of Saponins, Alkaloids, Tannins, Cardiac Glycosides, Terpenoids, Phenolic Compounds, Flavonoids, Free Amino Acids, Carbohydrates, Anthraquinones, Saponins, and Phytosterols.

2.3.3.1 Qualitative tests

2.3.3.1.1 Carbohydrates

Two ml of aqueous extract was booked in test tube. Some drops of 20% ethanolic solution of alpha napthol (20 gm in 100 ml ethanol) and one ml of concentrated sulfuric acid were added to it. The appearance of violet rings indicates the presence of carbohydrates (Trease and Evans, 1989).

2.3.3.1.2 Proteins

Aqueous extract of 3 ml was booked in test tube. Few drops of sodium hydroxide were supplemented to make it basic. Then some drops of 0.02% copper sulfate solution (0.02 gm of copper sulfate in 100 ml of distilled water) were supplemented to test tube. Brick red color seemed showing the presence of proteins (Trease and Evans, 1989).

2.3.3.1.3 Saponins A 0.2 g of each fraction was booked in test tube and 5 ml of distilled water was added and warmed to boiling. Frothing (presence of creamy miss of small bubbles) persisted for three minutes shows the presence of saponins (Sofowora, 1993).

20 Chapter 2 Materials and Methods

2.3.3.1.4 Alkaloids

Each fraction of 0.2 g and crude was heated with 2% H2SO4 for 2 min. The reaction mixtures were sieved and then a few drops of Dragendroff’s reagent were supplemented to each filtrated fraction. An orange red precipitate shows the existence of alkaloids moiety (Sofowora, 1993).

2.3.3.1.5 Tannins A 0.5 gm of each fraction was to be dissolved in water and warmed on water bath and filtered. It was shifted to a test tube and a few drops of ferric chloride were added to each of the filtrated fraction. A dark green color shows the existence of tannins (Trease and Evans, 1989).

2.3.3.1.6 Flavonoids A 0.2 g of each fraction was dissolved in diluted NaOH, a small piece of Magnesium and two drops of HCl were supplemented. A yellow solution that goes brown shows the existence of flavonoids (Sofowora, 1993).

2.3.3.1.7 Terpenoids Two ml of methanol and one ml of dichloromethane were mixed with 0.2 g of each fraction and was added which was followed by careful addition of four drops concentrated

H2SO4. The development of a reddish brown coloration shows the presence of Terpenoids (Harborne, 1973).

2.3.3.1.8 Phenolic compouds

Three gram of each extract was dissolved in 40 ml of distilled water. Three ml of from this aqueous extract was booked in test tube. Few drops of lead acetae solution (1.25 gm lead acetate in 25 ml of ditilled water) were supplementary to it. The formation of precipitates shows the presence of phenolic compounds (Trease and Evans, 1989).

2.3.3.1.9 Phytosterols Each of 2 gm extract was dissolved in twenty ml of ethanol, shakes and heated on water bath for one hour. It was then sieved and resolute on rotary evaporator. The extract was the dissolved in five ml dichloromethane, filtered and transferred to the test tube. The

21 Chapter 2 Materials and Methods

Liebermann-Buchardreagent (acetic anhydride: sulfuric acid, 9:1) was added to it. Presence of green ring confirmed the existence of phytosterols (Sofowora, 1993).

2.3.3.1.10 Cardiac glycosides For the qualitative study of cardiac glycosides, the protocol of Trease and Evans (1989) was adopted. Twenty ml of distilled water was added to 0.5 g of each fraction. The mixtures were heated and filtered. Five ml of the filtrate was booked in a test tube and one drop of 0.1% ferric chloride solution; 2 ml of glacial acetic acid and one ml of concentrated sulfuric acid were added to it. The brown colored rings appearance shows the presence of cardiac glycosides.

2.3.3.2 Quantitative tests

2.3.3.2.1 Carbohydrates

Ten gram of extract was thawed in distilled water and then filtered. The filtrate was centrifuged for ten minutes at 3,000 rpm. The floating material was together and volume was prepared up to 25 ml with water and 4 ml anthrone reagent was supplemented It was then kept in boiling water for 10 minutes. The tube was made chilled and the absorbance was measured at 530 nm. The amount of total carbohydrates was studied using the standard graph of glucose (Roe, 1965).

2.3.3.2.2 Proteins

Three grams of the extract was dissolved in 35ml of H2SO4, 5gm CuSO4 and kept in Kjeldahl unit for seven hours. The solution became clear and permitted to chill. 70ml of 4% boric acid was added in a receiving flask and 5 drops of indicator were added and was placed beneath the condenser. Then 100 ml of water and 80ml of NaOH solution were supplemented to the Kjedahl flask. When distillation was completed, 200 ml was together in receiving flak and was titrated and 0.1 N HCl was taken in burette. After titration percentage of protein was deliberated (Pearson, 1976).

22 Chapter 2 Materials and Methods

2.3.3.2.3 Saponin

Three gm of each fraction was taken in twenty ml of methanol in conical flask. It was somewhat warmed on water bath and forty ml of cold acetone was supplemented to it. The precipitates were appeared and it was kept in refrigerator for one night. Next day it was filtered and filter paper was retained in desiccator to guard it from moisture absorption. The filter paper was then kept in oven for one hour at 105 0C and weight was taken. The process was repeated for three times. The saponin content was deliberated as percentage (Obedoni and Ochuko, 2001).

2.3.3.2.4 Alkaloid

A 10 gram of the powder plant sample were weighed and put in a 250 ml beaker. After that 200 ml of 20% acetic acid in ethanol was supplemented and the mixture was shielded to stand for 4 hr. after filtration the extract was resolute using a rotary evaporator to one-quarter of the original volume. Till completion of precipitation, drops of concentrated ammonium hydroxide were added. Precipitate was sieved and weighed (Harborne, 1973 and Obadoni and Ochuko, 2001).

2.3.3.2.5 Tannins

A 0.008 M potassium ferrocyanide 10 mg of the extract and three ml of 0.1 M FeCl3 in 0.1 N HCl was mixed and the absorbance was measured through spectrophotometer at 120 nm while tannin acid was taken as standard (Van-Burden and Robinson, 1981).

2.3.3.2.6 Flavonoid

Ten gram of the plant powder was dissolved in 100 ml of 80% ethanol for 24 hours at room temperature. The filtrate was then extracted in petroleum ether (60 oC). The dried extract was weight (Boham and Kocipai, 1994).

2.3.3.2.7 Terpenoids

Five gram of the plant powder was dissolved in 100 ml of 95% ethanol for 24 hours. The filterate was then extracted in petroleum ether (60 oC). The dried extract was weight (Ferguson, 1956).

23 Chapter 2 Materials and Methods

2.3.3.2.8 Phytosterols

Three grams of the extract was dissolved in 30 ml of n.Hexane and kept on shaker for 1 hour and then sieved. The weight was booked by drying the together filtrate by using gaseous nitrogen and heat (Singh et al. 2003; Moreau et al. 1996).

2.4 Biological evaluation of the crude extract 2.4.1 Antioxidant activity

2.4.1.1 DPPH free radical scavenging activity

The antioxidant activity was dogged according to the protocol of Ahmad et al. (2010). The free radical scavenging activity of ethanolic extracts of all the plants were restrained in terms of hydrogen donating or radical scavenging ability using the stable radical 2, 2- diphenyl-1-picrylhydazyl (DPPH). The test extracts were prepared in ethanol therefore the DPPH was also prepared in ethanol independently. DPPH of 3.96 mg was dissolved in 20 ml of ethanol. 1 ml of DPPH solution 0.5 ml of sample solution was added separately. At room temperature, each solution mixture was brought to dark for 30 min and absorbance was dignified at 517 nm. Higher free radical scavenging activity was confirmed by low value of absorbance. All tests were carried out three times. Percentage scavenging DPPH activity was calculated as follows:

% scavenging DPPH free radical = (1-AE/AD) × 100

AE represents absorbance of the solution while adding extract and AD represents the absorbance of the DPPH solution with nothing added (blank).

2.4.1.2 Hydrogen peroxide free radical scavenging activity

The methanol, ethyl acetate, DCM, ethanol, petroleum ether extracts of Hedera nepalensis to the scavenge hydrogen peroxide were subjected to spectrophotometer for determination at 285nm using the protocol of Beers and Sizer (12). Hydrogen peroxide solution of 2 mm was organized in phosphate buffered saline (PBS) at pH 7.4. Various concentrations of the test extract such as 0.005 mg/ml, 0.01 mg/ml, 0.02 mg/ml, 0.05 mg/ml and 0.1 mg/ml were added to the solution of hydrogen peroxide. The absorbance was measured through spectrophotometer and was taken after 10 min against blank solution

24 Chapter 2 Materials and Methods containing test extract in phosphate buffered saline without hydrogen peroxide. All the tests and analysis were performed in triplicates and averaged.

2.4.2 Antimicrobial activities

Both antibacterial and antifungal activities were brought about by using the following techniques.

2.4.2.1 Culture media used:

For culturing and growth of all microorganisms, Nutrient agar media was used. Nutrient broth media was used for inoculation, incubation, and then standardization of the given microorganisms.

Table 1: Nutrient agar composition used for culturing and applying test

Nutrient agar modified QUELAB QB-39-3504 Composition g1-1 1. Beef extract 1 2. Yeast extract 2 3. Gelatin extract 5 4. Sodium chloride 5 5. Agar medium 15 Total: 28

Table 2: Composition of nutrient broth used for Inculcation and standardization

Composition of nutrient broth modified Composition g1-1

1. Gelatin peptone 5 2. Beef extract 1 3. Yeast extract 2 4. Sodium chloride 5 Total: 13

25 Chapter 2 Materials and Methods

2.4.2.2 Preparation of media

Nutrient agar of 2.8 g/100 ml was dissolved in 300 ml of distilled water. Nutrient broth of 1.3g was dissolved in 100 ml in distilled water and was taken in conical flask. Twenty ml of the nutrient broth was poured into test tubes. Cotton wool was used to plug media flasks and test tubes and sterilization was brought about in an autoclave machine at 1.5 pound pressure at 121˚C for half an hour. In a laminar flow hood, nutrient agar media was poured into sterilized petri plates. To avoid contamination, sterilized environment was produced. After one hour of the solidification of the nutrient agar, the petri dishes were placed inverted an incubator at 37 ˚C for 24 hours. Those plates which were uncontaminated were used for culturing of bacteria and fungi after 24 hours. Twenty ml of the nutrient broth in flasks were used for shaking incubation of micro-organisms while nutrient broths in test tubes were used for normalization of microbial cultures.

2.4.2.3 Microorganism used: The following microorganisms had been used.

Table 3: Bacterial strains used for antimicrobial activities

Bacterial species Type Source of fungal speceies/strain Escherichia coli Gram negative ATCC # 25922 Pseudomonas aeruginosa Gram negative ATCC # 9721 Salmonella typhi Gram negative Clinical Isolates obtained from micro lab PCSIR

Kleibsiella pheumonia Gram negative Clinical isolates obtained from micro lab PCSIR Erwinia cartovara Gram negative Clinical isolates obtained from micro lab PCSIR Staphylococcus aureus Gram positive ATCC # 6538 Bacillus subtilis Gram positive Clinical isolates obtained from micro lab PCSIR Bacillus atrophaeus Gram positive Clinical isolates micro lab Bacillus cereus PCSIR Gram positive Clinical isolate obtained

26 Chapter 2 Materials and Methods

from Microbiology Lab. QAU. Enterococcus faecalis Gram positive Isolate from micro lab PCSIR Shigella sonnei Gram positive Isolate from micro lab PCSIR Citrobacter freundii Gram positive Isolate from micro lab PCSIR Agrobacterium Tumefacians Gram positive Isolate from micro lab PCSIR

Table 4: Fungal species used for antimicrobial activities

Fungal species Source of fungal speceies Candida albican Isolate from micro lab PCSIR Aspergillus niger Isolate from micro lab PCSIR Aspergillus flavus Clinical Isolates obtained from micro lab PCSIR Alternaria alternate Clinical isolates obtained from micro lab PCSIR Penicillium notatum Clinical isolates obtained from micro lab PCSIR Trichoderma harzianum Clinical isolates obtained from micro lab PCSIR

2.4.2.4 Stock solution of extract used

n-Hexane, chloroform, ethyl acetate, butanol and aqueous of these were assessed for antimicrobial activity. The dehydrated crude extract of the plant were diluted and attuned to 1mg 6µlˉl in Dimethylsulfoxide (DMSO) n-hexane, chloroform, ethyl acetate, beutanol, or

27 Chapter 2 Materials and Methods aqueous depending on their solubility. The stock solution was made and each six µl of the solution fenced along one mg of the extract.

2.4.2.5 McFarland 0.5 turbidity standard

In order to prepare 0.5 McFarland, 0.5 ml of a 1.175% (wt/vol) barium chloride dihydrate (BaCl2.2H2O) solution was added to the solution of 99.5 ml of 1% (wt/vol) sulfuric acid. The turbidity standard was then relocated into test tube, and vacuum-packed it with Parafilm. McFarland standards may be hoarded for up to 6 months in the dark at room temperature (220C to 250C temperature). Before each use, shake well the McFarland standards, mixing the fine white precipitate of barium sulfate in the tube. The precision of the density of a prepared McFarland standard should be controlled by using a spectrometer. The absorbance for 0.5 McFarland standards at a wavelength of 625nm should be 0.08 to 0.1. The attuned suspension must contribute a count of 10 8 colony forming units/ ml (Baker and Thornberg, 1983).

2.4.2.6 Disc diffusion susceptibility method

Disc diffusion method was utilized according to the protocol of Aida et al., (2001) to assess the plant extracts for antimicrobial studies. 0.5 Mcfarland turbidity was taken as standards with whom the bacterial culture were attuned and inoculated onto nutrient agar plates (diameter: 15cm). The concentration of 108 cfu/ml was taken as standard to which fungus culture was adjusted. Culture of fungus were adjourned in sterile solution of 0.9% normal saline solution and vaccinated onto Sabroud dextrose Agar plates. Sterile filter paper (Whattman-1) discs (diameter 6 mm for bacteria and 13 mm for fungi) soaked with plant extracts in concentrations of 1, 2 and 3 mg disc-1 in 6, 12 and 18 µl volume were smeared on the disc earlier planted with the 0.5 Mcfarland and 10 6 cafe/ml cultures of bacteria and fungi respectively. Bacterial culture and those of fungi were then reared at 37 ˚c for 18 h (Odunbaku and Ilusanya, 2008 and olfimiahan and Fawole, 2003).

2.4.2.7 Stepwise methodology of antimicrobial bioassay

The following protocol was adopted for the assessment of antimicrobial activities.

Day 1st: Nutrient agar of 2.8 g1-1 was dissolved in 300 ml of distilled water. Nutrient broth of 1.3g was dissolved in 100 ml in distilled water and was taken in conical flask. Seven ml of the nutrient broth was poured into test tubes. Cotton wool was used to plug media flasks and

28 Chapter 2 Materials and Methods test tubes and sterilization was brought about in an autoclave machine of all the apparatus and media, viz., Petri plates, blue tips, yellow tips, Whattman filter paper disc, normal saline etc at 1.5 pound pressure at 121˚C for half an hour. In a laminar flow hood, nutrient agar media was tipped into sterilized petri plates.

Day 2nd: To avoid contamination, sterilized environment was produced. After one hour of the solidification of the nutrient agar, the petri dishes were placed inverted an incubator at 37 ˚C for 24 hours. The microbial stock cultures were ventilated by rushing with sterile inoculation loop on the nutrient agar plates in a laminar flow hood. These cultures were reared at 37˚C for 24 hrs.

Day 3rd: At 37˚C the first streaked cultures were re-reared on fresh agar media plates and again reared for 24 hrs.

Day 4th: At 37˚C for 18 hours at 200 rpm the second streaked cultures were re-reared on 25 ml broth media in flasks.

Day 5th: Turbidity (0.5 Mcfarland) was used as standard with which the diluted microbial cultures were compared. In order to produce six deep wells, 6 mm strile cork borer was used. Glass spreader was used to spread 50 µl of standardized microbial cultures on each nutrient agar. The plates were kept in refrigerator for 15 minutes for absorption.

Applying test: Sterilized forcep was used to place Whattman filter paper-1 discs (6 mm in diameter) on agar media in Laminar flow. Then the loaded the methanol, ethanol, n-hexane, petroleum ether, chloroform extract in different concentration of 1, 2 and 3 mg disc-1 in 6, 12 and 18 µl volume were applied on discs. Antibiotics (Azithromycin, Ciprofioxacin, Clotrimazole) were applied (6µl disc-1) on separates plates as positive control for gram negative bacteria, gram positive bacteria and fungi respectively. All the solvent used for creating stock solution were smeared (6µl disc-1) on the discs as negative control. These plates were nursed at 37˚C (Emeruwa, 1982).

Positive control

Azithromycine 50µg.6µl-1 was used against Gram positive bacteria

Ciprofloxacin 30µg 6µl-1 was used against Gram negative bacteria

Clotrimazole 50 µg 6 µ-1l was used against fungi

29 Chapter 2 Materials and Methods

Day 6th: Zones of inhibition around each paper discs in mm were measured for the assessment of each extract. To avoid any type of ambiguity, all tests were carried out three times for each microorganisms. The lowest concentration which stopped the growth of the respective organisms was considered as Minimum Inhibitory Concentration (MIC).

2.5 Pharmacological evaluation of the crude extract

2.5.1. Toxicological studies The toxicological studies of aqueous and ethanolic extracts of Hedera nepalenses carried out included acute toxicity.

2.5.2. Acute toxicity (LD50)

Acute toxicity (LD50) was performed in rabbits (Kärber, 1931). For this purpose five groups of 6 rabbits each having the weight of 20- 25 g b.wt. were selected and and different oral dose at the rate of (1 to 5 g/kg b.wt.) for the aqueous extract and (5-15 g/kg b.wt.) for the ethanolic extract were administered. The toxic symptoms, killing rate and postmortem findings in each group were recorded 24 hours post administration. LD50 of the tested extracts was intended according to the following formula:

LD50 = Dm - ∑ (z×d)/n Where: Dm = The largest dose which kill all animals. z = Mean of dead animals between 2 successive groups. d = The constant factor between 2 successive doses. n = Number of animals in each group. ∑ = The sum of (z×d).

2.5.3 Selection of animals

Rabbits (Oryctolagus cuniculus) were purchased locally and were reared and kept till increase in their number at the Biopark of Islamia College university Peshawar) were selected as experimental animal. A total of 120 rabbits were purchased from local market. All the rabbits were transferred to Bio Park of Islamia College University Peshawar for acclimatization. Water and fodder were provided ad libitum.

30 Chapter 2 Materials and Methods

2.5.4 Grouping of rabbits

After 10 days of rearing the rabbits in a Bio Park, a 100 number of rabbits were randomly selected. They were divided into 7 groups. The rabbits having weight in the range of (1200-1300 g) were selected. Each group had 20 numbers of rabbits.

Group No-1 =Untreated Control (Normal Control)

Group No-2 =Diabetic Control (Negative Control)

Group No-3 =Diabetic + Glibinclimide (20mg/kg) (Standard Control)

Group No-4 =Diabetic control + Vitamin C (Positive Control)

Group No-5 =Diabetic + Ethanolic extract of H.N 200 mg/kg)

Group No-6 =Diabetic + Ethanolic extract of H.N 400 mg/kg)

Group No-7 =Diabetic + Ethanolic extract of H.N 600 mg/kg)

2.5.5 Equipments

The equipment will be required during experiment;

 Rotary Evporator (Heidolph Laborata 4000 Efficient)  Centrifuge 5702 R  ERMA/Caloimeter  Filter paper (Whitemann no:1)  Laminar flow

2.5.6 Chemicals

The chemical were used during experiment;

 Bacterial and fungal growth medias Growth medias  Bacterial and fungal growth medias Growth medias  Methanol (Merck, D/6100 Dermtadt, F. R Germany)  Ethyl Alcohal  Ethyl Acetate  n-Hexane

31 Chapter 2 Materials and Methods

 Alloxane monohydrate (Applichem GmvH OhoweG 4 D_64291 Dermstadt Germany)  Daonil (Glibenclimide) Dermstadt Germany).  Glucose PAP kit (Merck, France)  Triglycerides (Merck, France)  Cholestrol (Merck, France)  Bilirubin Total (Chemilex, S A. Pol. Ind. Can Castells. Barcelona)  Proteins Total (Chemilex, S A. Pol. Ind. Can Castells. Barcelona)  Albumin Kit (Merck, France)  Creatinine Jaffe Kit (Merck, France)  ALP kit (Randox Laboratories Limited, UK)  GGT (Randox Laboratories Limited, UK)  ALT (Randox Laboratories Limited, UK)  AST (Randox Laboratories Limited, UK)

2.5.7 Induction of Diabetes mellitus

Alloxane was used for the induction of Diabetes mellitus in rabbits. Alloxane (cyclic urea compound) induces permanent diabetes in animals. Alloxane results into redox cycle and forming superoxide radicals. These radicals are changed to hydrogen peroxide. As a result highly reactive hydroxyl radicals are made by the penton reaction. Reactive oxygen act on cytosolic calcium which destroys of β-cells (Szkudelski, 2005).From the previous research work conducted, it was clear that alloxane has the potentials to cause diabetes (Hansen et al., 2007). Alloxane monohydrate was purchased on demand from local supplier. The protocol of Hansen et al., 2007 was used for induction of diabetes mellitus in all 100 rabbits. The control group of rabbits (Negative control) received citrate buffer alone. The rabbits were made diabetic with an injection of alloxane monohydrate in 0.05 mM citrate buffer (pH 4.3) at the dose of 65 mg/kg body weight in jugular vein after an overnight fast consecutively for three days. To reduce the risk of nephrotoxicity, an intravenous injection of 0.9 % saline was given immediately after the injection of alloxane. To counteract initial hypoglycemia, glucose of 4 mg/ kg body weight was set subcutaneously 5 – 6 hours after the injection of alloxane. And 5 % glucose was provided in the drinking pots ad libitum for 24 hours. Rabbits with a blood glucose level of more than 260 mg/dl were nominated for study. Before the initiation of medication, the blood samples were together randomly from all the groups. Blood

32 Chapter 2 Materials and Methods samples were kept in cold environment for the isolation of serum. Serum oozed out and was analyzed through ERMA/ Caloimeter.

2.5.8 Drug administration

Ethanol extract of both leaves and stem of Hedera nepalensis deferred in physiological saline were fed to three Diabetic groups at 200 mg/kg, 300mg/kg and 400 mg/kg for regular five days at the interval of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Vitamin C was fed to Diabetic control (Positive control) at the dose of 200 mg/kg body weight for regular five days at the interval of 0 day, 7th day, 14th day, 21st day and 28th day.

2.5.9 Collection of blood samples

The rabbits were fasted for 24 hours and blood was collected after the last dose directly from a pinna venule using a syringe A No. 26 needle at the for regular five days at the interval of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Blood glucose and other biochemical parameters like Triglyceride, Cholesterol, Bilirubin Total, Proteins Total, Albumin, Creatinine, ALP, GGT, ALT and ASTwas determined through ERMA / Caloimeter.

2.5.10 Isolation of blood serum

Serum was unruffled in falcon tubes and was centrifuge atat 4000 rpm for 8 minutes and was analyzed by ERMA/Caloimeter.

2.5.11 Biochemical analysis of the blood of rabbits

The following parameters were analyzed using serum samples of each group.

Glucose, Triglyceride, Cholesterol, Bilirubin Total, Proteins Total, Albumin, Creatinine, ALP, GGT, ALT and AST.

2.5.11.1 Glucose determination

For the determination of glucose, the protocol of Trinder, 1969; Sacks, 2001; Dods, 2003; Tietz, 1995; Burmin, 1985; Vassault, 1997; and Young, 1997 were used.

Reaction Principle: Glucose oxidase causes the addition of oxygen process of glucose into gluconic acid and hydrogen peroxide. Hydrogen peroxide reacts with phenol and 4-

33 Chapter 2 Materials and Methods aminophenazone in the presence of peroxidase which results into the production of a red- violet quinoneimine. Quinoneimine is a dye and is used as indicator.

Reaction Principle

Glucose+O2 →gluconic acid+ H2O

2 H2O + 4-aminophenazone +Phenol(POD)→ quinoneimine 4 H2O

ASSAY:

Wavelength: 500 nm, Hg 546 nm

Optical path: 1cm

Temperature: 20-25°C or 37 °C

Measurement: Against reagent blank

Pippetting scheme

Semi micro

Pipette into cuvettes [STD]/Sample Reagent blank

[STD]/Sample [RGT] 10ul 1000ul -----

1000ul

Mixed, incubated for 10 min at 20-25 °C or 5min at 37 °C. Measured the absorbance of [STD] and the sample in contradiction of the reagent blank within 60 min (∆A).

Calculation of the glucose concentration

C=100×∆A sample / ∆A [STD] mg/dl

∆A=Absorbance

[STD]=Standard Or

Glucose concentration = standard concentration/ Δ Est × Δ Esa (mg/dL), (mmol/L)

34 Chapter 2 Materials and Methods

2.5.11.2 Triglyceride determination

For the determination of glucose, the protocol of Naito, 2003; Expert Panel on Detection, 2001; Fossati and Prencipe, 1982; Tietz, 1995; Vassault et al., 1997; and Young, 2001 were used.

Reaction Principle: The triglycerides are dogged by hydrolysis using lipases (Lipoproteinlipase (LPL) converts triglyceride into glycerol and fatty acids. Glycerol gets energy in the form of ATP in the presence of GK and produce glycerol-3-phosphate and ADP. Glycerol-3-phosphate oxidation occurs in the presence of GPO and is converted into dihydroxyacetone phosphate and hydrogen peroxide. Hydrogen peroxide and 4- aminoantipyrine reacts and produce quinoneimine in the existence of peroxidase. Quinoneimine is a dye and is used as indicator.

Lipases (LPL) Triglycerides glycerol + fatty acids GK Glycerol +ATP glycerol-3-phosphate + ADP GPO Glycerol-3-phosphate +O2 dihydroxyacetone phosphate +

H2O2

POD

H2O2 +4-amino antipyrine quinoneimine + HCl + H2O

Assay: Wavelenght: 500 nm, Hg 546 nm Optical path: 1cm Temperature: 20-25 ºC or 37 ºC Measurement: against reagent blank

35 Chapter 2 Materials and Methods

Pipetting Scheme

Pipette into cuvettes Reagent blank Sample or [STD] Sample / [STD] ……………. 10 µl Reagent solution [RGT] 1000 µl Amalgam and nurse for 10 minutes, at 20-25 ºC or for 5 minutes, at 37 ºC. Size the absorbance of the sample (ΔA sampl ) and the standard (ΔA [STD] ) in contradiction of the reagent blank within 60 minutes.

Calculation: C = 200× ΔA Sample [mg/dl] ΔA [STD]

2.5.11.3 Cholesterol determination:

For the determination of cholesterol, the protocol of Rifai et al., 2001; Naito, 2003; Allain et al., 1974; Tietz, 1965; Expert Panel on Detection, 2001; Vassault et al., 1999; and Young, 1999 were used. Cholesterol is a steroid. At third carbon, a secondary hydroxyl group is attached. Liver and intestinal wall tissues produce cholesterol. One quarter is taken from food while three quarter is produced. Cholesterol analysis was studied for the first time by Liebermann in 1885, shadowed by Burchard in 1889. The Abell and Kendall method is precise for cholesterol but is exactly complex, and entail the use of corrosive agents. Roveschalue and Allian describe the fully enzymatic method.

Test Principle: Cholesterol ester reacts with water to produce free cholesterol and fatty acids in the presence of Cholesterol esterases. Cholesterol is converted cholest -4 –en-3 –one and hydrogen peroxide by oxygen. Hydrogen peroxide reacts with 4-aminoanitipyrine and phenol in the presence of peroxides to produce quinoneimine of red color dye as an indicator and water.

Cholesterol ester + H2O → Cholesterol + Fatty acid

Cholesterol + O2 (CHO) → cholest-4-en-3-one + H2O2

2H2O2 + 4-Aminoantipyrine + Phenol (POD) → Quinoneimine + 4H2O

36 Chapter 2 Materials and Methods

Manual procedure

Wavelenght: 546 nm (500-550nm) Temperature: +25/+30/+37 ºC Cuvette: 1 cm light path Zero adjustment: one reagent blank per series only Blank sample/reagent R1 1000µl 1000µl Sample/standered ……... 10µl Amalgam and nurse for 5 minutes at 37 ºC or 10 minutes at 20 ºC to 25ºC. Within 60 minutes read absorbance or calibrator and sample in contradiction of reagent blank. Calculation ΔA sample Standard concentration = Cholestrol concentration ΔA =Absorbance [STD]=Standard

2.5.11.4 Total Bilirubin determination

Hemoglobin on halt produces bilirubin. Bilirubin don not dissolve in water. It is elated from spleen to the liver and evacuated into bile. Hyperbilirubinemia results from the rise of bilirubin concentrations in plasma. It causes of hyperbilirubemia:

Total bilirubin: Increase hemolysis, genetic errors, neonatal jaundice, ineffective erythropoiesis, and drugs.

Direct bilirubin: Hepatic cholestasis, genetic errors, hepatocellular damage.

For the determination of Bilirubin, the protocol of Kaplan et al., 1984; Malloy et al., 1937; Martinek, 1966; Young, 2001; Burtis et al., 1999; and Tietz, 1995 were used.

Test summary: Diazotized sufanic acid converts bilirubin into colored azobbilirubin and measured through photometer. Serum contains two types of fractions which are loosely bound to albumin. One is bilirubin-gluuromide reacts directly in aqueous solution (bilirubin direct), while free bilirubin requires solubilization with dimethylsulphoxide (DMSO) to respond (bilirubin indirect). In the fortitude of indirect bilirubin the direct is also resolute, the results are assumed as follows: The intensity of the color formed is proportional to the bilirubin concentration in the sample.

37 Chapter 2 Materials and Methods

Specimen: Serum

Stability: Bilirubin is stable at 2-80 for 4 days and 2 months at – 200C.

Test procedure

1. Assay conditions: Wavelength:………………………………555nm. (530-580) nm Cuvette:…………………………………1cm light path Temperature:……………………………15-250C 2. Adjust the instrument to zero with distilled water. 3. Pipette into a cuvette: Blank Sample R.1 (ml) 1.5 1.5 R.2 (µl) 50 Sample/ calibrator (µl) (Note 1) 100 100 4. Amalgam and nurse for exactly 5 minutes at room temperature. 5. Read the absorbance (A).

Calculations

With Calibrator

Bilirubin (mg/dl) = (A) Sample – (A) Sample Blank/ (A) Calibrator – (A) Calibrator Blank × Calibrator conc.

With Factor:Bilirubin(mg/dl) = Sampe – (A) Sample Blank × Factor* Note 2

*Theoritical factor = 19.1

Conversion factor: mg/dl × 17.1 = µmol/L

Note:

1. For bilirubin fortitude in newborns, pipette 50 µL of sample. Multiply the result by 2. Factor = concentratin of Calibrator/ (A) Calibrator – (A) Calibrator Blank

38 Chapter 2 Materials and Methods

2.5.11.5 Total protein determination Total protein is the sum of albumin and globulins. For the determination of Total Protein, the following protocol of Kollar, 1984; Young, 2001; Burtis et al., 1999; and Tietz, 1995 was used. Quantitative determination of total protein: only for in vitro use in clinical laboratory (IVD). Test Summary: Protein gives an rigorous violet-blue complex and copper salts in an alkaline medium. Iodide is added as an antioxidant agent. The intensity of the color formed is relational to the total protein concentration in the sample. Specimen: Serum Test Procedure: Assay conditions are:

Wavelength:…………………………… 530-550 nm Cuvette:…………………………………1cm light path Temperature:……………………………370C / 15-250C Adjust the instrument to zero with distilled water. Pipette into a cuvette Blank Calibrator Sample R.1 (ml) 1.0 1.0 1.0 Calibrator (Note 1-2) (µl) ----- 25 ---- Sample ----- 25

Amalgam and nurse for exactly 5 minutes at room temperature (15-250C or 30-370C. Read the absorbance (A) of the samples and calibrator, against the Blank. The color is stable for at least 30 minutes.

Calculations: Total Protein (g/dL) = (A) Sample / (A) Standard × 5 (Calibrator conc.)

Conversion factor: g/dl × 144,9 = µmol/L

2.5.11.6 Albumin determination Liver uses dietary protein for the production of albumin. Within the vascular space, fluid volume is maintained by osmotic pressure developed due to albumin presence in plasma. Low albumin is used as a sign of poor health. For the determination of albumin the protocol of Tietz, 1990 and Young, 1997 were used.

39 Chapter 2 Materials and Methods

Methodology: In 1965, Rodkey introduced a convenient, direct method for determining albumin concentrations in serum utilizing a neutral buffered solution of bromocresol green (BCG) as the dye binding indicator. In 1971, Doumas et al. increased the sensitivity of the reaction by adding a nonionic surfactant to the reagent to prevent turbidity and improve linearity. This Albumin method is a modification of the Doumas and Rodkey procedures utilizing a different buffering system. Green complex is produced when albumin reacts with bromocresol green at pH 4.2. The absorbance of the albumin-BCG complex is measured bichromatically (600/800 nm) and is relational to the albumin concentration in the sample pH 4.2. Albumin + Bromocresol Green Complex Reagents: Final concentration of reactive ingredients: 1) Succinate buffer (pH 4.2) 100 mmol/L 2) Bromocresol green 0.2 mmol/L 3) Also contains preservatives. Sensitivity: The change in absorbance for 1 g/dL of Albumin is 138 m Absorbance.

2.5.11.7 Globulin determination

Globulins are proteins that include gamma globulins (antibodies) and a variety of enzymes and carrier/transport proteins. It has four types;

1. Gamma globulins 2. Beta globulins 3. Alpha-2 globulins 4. Alpha-1 globulins

One type abnorlality represent the increase r decrease of the other. Gamma type is produced in large amount in the body. Antibodies are produced by mature B lymphocytes called plasma cells, liver produce alpha and beta types of globulins. Calculation of globulin = (total protein - albumin)

40 Chapter 2 Materials and Methods

2.5.11.8 A/G Ratio determination

The albumin globulin ratio (A/G ratio) means alteration in serum proteins. In liver diseases, globulins (G) level increase while serum albumin (SA) level decreases (Clinical Diagnosis and Management by Laboratory Methods, 1996; Dufour et al., 2000).

Liver and kidney diseases can be assessed through albumin/globulin (A/G) ratio. The ratio is calculated using the following formula: Albumin/globulin = (Total Protein – Albumin) 2.5.11.9 GCI category determination

The albumin globulin ratio (A/G ratio) is meant to represent the ratio of alterations in serum proteins. In liver disease, globulins (G) level increase while serum albumin (SA) level decreases (Clinical Diagnosis and Management by Laboratory Methods, 1996; Dufour et al., 2000). However, because of the lack of pathophysiological value, its’ use has been restricted. Whereas the globulin compensation index (GCI) is the measure of the changes in serum globulins with decrease in albumin. (Al-Joudi and Wahab, 2004). The globulin compensation index (GCI) for each patient was calculated using the following equation: GCI = G – 25 / 35 – SA, where GCI = globulin compensation index G = globulin concentration SA = serum albumin concentration 25 and 35 shows the minimum normal ranges Thus, G-25 shows the deviation of G from the inferior range of the serum G value, taken as 25 g/L, and 35-SA shows the value of reduction in serum albumin under the minimum of the normal range, taken as 35 g/L (Clinical Diagnosis and Management by Laboratory Methods, 1996).

2.5.11.10 Creatinine determination

For the determination of creatinine, the protocol of Allston, 1993; Newman and Price, 2001; Butler, 1975; Vasiliades, 1976; Tietz, 1965; Vassault et al, 1986 and Young, 1997 were used. In alkaline solution with Picric acid, Creatinine produces a yellow-orange colored

41 Chapter 2 Materials and Methods compound. Creatinine concentration is measured when dye is formed as a result. Intrusion is not developed when reaction takes place between creatinine and picric acid.

Sample Material: Serum

Stable for 2 hours at +2 to +0C or several months at -18 to -20 0C.

Reagents:

Reagent 1:R1(Picric acid) = 8.73 mmol/L

Reagent 2:R2 (Sodium hydroxide) = 312.5 mmol/L

(Disodium phosphate) = 12.5 mmol/L

Standard:Std

Creatinine: 2.0 mg/dl=20 mg/L=177µmol/L

Procedure: Mix Reagent 1 and reagent 2 in ratio of 1+1. The solution mixture is stable for 7 days when stored at +15 to +250C. Since the reaction is highly temperature sensitive, care must be taken to indorse that the solutions are preheated to an exact temperature and the reaction must proceed at a constant temperature. The reaction temperature of the standard and that of the sample must be identical. Provided these conditions are fulfilled the determination can be performed at any chosen temperature between + 20 and 370C.

Wavelength: 492, 500nm or 505. Light path:1cm.

Prepare one or two standards for each test series.

Serum 50µL ---- Standard ---- 50µL Working Reagent 500µL 500µL

Mix and Size the absorbance of the sample (AS1) and standard (ASt) after 1 min. exactly 2 min after the first measurement.

Redetermine the absorbance of the sample (AS1) and standard (ASt). If the reaction is 0 performed at a temperature above +37 C read absorbance A1 after 30 seconds.

42 Chapter 2 Materials and Methods

Calculation

Serum Creatinine concentration [mg/dL] = AS2 – AS1 /ASt2 – ASt1 × 2.00

[µmol/L] = AS2 – AS1 /ASt2 – ASt1 × 177.00

Dilution Limit: If the creatinine value is higher than 15 mg/dL(1326 µmol/L) in serum, repeat the determination with the sample diluted 1+5 with isotonic saline (9 g/L, 154 mmol/L NaCl) and multiply the result by 6.

2.5.11.11 ALP determination

For the determination of glucose, the protocol of Rec, 1972 and Englehardt et al., 1970 were used. Alkaline phosphtase (ALP) is actually a group of isoenzymes that hydrolyze monophosphate esters in alkaline medium. Optimum ph for these ALP isoforms activates is about 9-10. Alkaline phosphtase level is the highest in kidney liver, placenta, intestine and bone. Measurement of ALP isoenzymes is useful in diagnosis of these organ diseases.

Kinetic method by International Federation of Clinical Chemistry (IFCC).

2-amino-2methyl-1-propanol+p-nitrophenylophosphate+H2O (ALP) → 4-nitrophenol+2- amino-2-methyl-1-propanol phosphate.

The rate of 4-nitrophenol formation is directly proportional to the ALP activity.

Specimen: Serum

Procedure: These reagents may be used both for manual assay (sample start and reagent start method) and in several automatic analyzers. Manual Procedure: Wavelength 405nm, Temperature 30 37 °C, Cuvette 1cm Sample Start Method: Pipette into the Cuvette: Working agent 1000μL

Bring up to the temperature of determination Sample 20μL

43 Chapter 2 Materials and Methods

Mixed and incubated at adequate temperature. After about 1min.read the absorbance against air or water. Repeated the reading after exactly 1, 2 and 3 minutes.

Calculated the mean absorbance change per minute (∆A/min) = U/L.

Calculation: ALP activity [U/l] =∆A/min × F F=Standard=3442

2.5.11.12 GGT determination

Intended Use: For the determination of GGT the protocol of Tietz, 1986 and Young, 1997 were used. Summary: Gamma-glutamyltransferase measurements are used in the verdict of liver diseases like alcoholic cirrhosis and primary and secondary liver tumors. In case of liver diseases, high level serum gamma-glutamyltransferase (GGT), also called GGTP is instituted. It is more sensitive than alkaline phosphatase, the transaminases, and leucine aminopeptidase (LAP) in showing cholecystitis, obstructive jaundice and cholangitis. Moderate levels show infectious hepatitis. However, high level of GGT levels is related to chronic alcoholism, diabetes, and certain neurological disorders. GGT level is normal in skeletal diseases. Methodology: This GGT procedure is a modification of the Szasz procedure. GGT catalyzes the transmission of the gamma-glutamyl group from the substrate (gamma-glutamyl-3- carboxy-4-nitroanilid) to glycylglycine which produce 5-amino-2-nitrobenzoate. The alteration in absorbance at 410/480 nm is due to the creation of 5-amino-2-nitrobenzoate and is directly proportional to the GGT activity in the sample. γ-GT → L-γ-Glutamyl-3-carboxy-4-nitroanilide → Glyclyglycine (L-γ- Glutamylglycylglycine) → 5-Amino-2-nitrobenzoate Reagents: Final concentration of reactive ingredients: Tris Buffer, pH 7.95 (37°C) 100 mmol/L Glycylglycine 100 mmol/L L-γ-Glutamyl-3-carboxy-4-nitroanilide 4.0 mmol/L Also contains preservatives

Specimen Collection and Preparation Serum samples, free from hemolysis, are the suggested specimens. If plasma must be used, the recommended anticoagulant is EDTA. Heparinized plasma becomes turbid in the reaction mixture; citrate, oxalate and fluoride depress activity by 10 to 15%.1

44 Chapter 2 Materials and Methods

Sample Storage and Stability: The GGT determination should be performed as soon after specimen collection as possible. GGT in serum is stable for 1 month at 2 – 8°C and 1 year at ≤ -20°C.4 Interfering Substances: It has been found that some antiepileptic drugs (phenytoin, barbiturates) may result in falsely elevated GGT values.5 Heavy alcohol consumption just prior to specimen collection may falsely elevate serum GGT.6 Results of studies7 show that the following substances interfere with this GGT procedure. The criteria when 10% of the initial value recovery is considered as no significance. Bilirubin: No significant interference up to 40 mg/dL Bilirubin Hemolysis: No significant interference up to 350 mg/dL Hemolysate Lipemia: No significant interference up to 1000 mg/dL Intralipid* Results: Automatically printed out for each sample in U/L at 37°C. Expected Values Adults:4 9 – 64 U/L Sensitivity: The change in absorbance per minute for 1 U/L of Gamma-Glutamyltransferase is 0.23 m Absorbance.

2.5.11.13 ALT determination Principle Intended use: For the determination of ALT the protocols of Tietz, 1995 and Young, 2000 were used. Clinical Significance: Alanine aminotransferase measurements are used in the diagnosis and treatment of certain liver diseases (e.g., viral hepatitis and cirrhosis) and heart diseases. Methodology: The ALT- reagent is used to measure the level of alanine aminotransferase in serum or plasma by an enzymatic rate method. The reversible transamination is carried out by ALT of L-alanine and alpha-ketoglutarate to pyruvate and L-glutamine. Lactate dehydrogenase (LDH) reduces pyruvate into lactate results into the oxidation of β- Nicotinamide Adenine Dinucleotide (reduced form) (NADH) to β-Nicotinamide Adenine Dinucleotide (NAD). Chemical reaction scheme

L-Alanine + -Oxoglutarate ALT L-Glutamate + Pyruvate (primery reaction)

+ LD + Pyruvate + NADH + H L-Lactate + NAD (indicator reaction)

45 Chapter 2 Materials and Methods

Sample volume: The optimum volume should be 0.3 mL of sample. Reagents contents: Each kit contains the following items: Two Alanine Aminotransferase (ALT-) Reagent Cartridges (2 x 300 tests) or (2 x 100 tests) Volumes per test Sample Volume = 23 μL ORDAC Sample Volume = 3 μL Total Reagent Volume = 258 μL Cartridge Volumes A 250 μL B 8 μL C – – Reagent constituents

Tris buffer, pH: 7.15 (37ºC) 100 mmol/L L- Alanine 500 mmol/L -Oxoglutarate 12 mmol/L LDH  1.8 kU/L NADH 0.20 mmol/L Pyridoxal-5’-phosphate 0.25 mmol/L Reagent storage and stability: ALT- reagent when stored unopened at +2°C to +8°C Sensitivity:The lowest measurable concentration which can be notable from zero with 95% confidence is called sensitivity. Sensitivity for ALT- determination is 5 IU/L (0.08 μkat/L). Serum (in the range of 9 to 343 IU/L): Correlation coefficient (r) = 0.9980 Calculations: For SI Units (kat/L), multiply the results by 0.017.

2.5.11.14 AST determination

For the determination of Aspartate Amnotransferase, the following protocol of Reitman, 1957 was used. For the quantitative in vitro determination of Aspartae Aminotransferase (AST) in serum. Calorimetric method (Reitman and Frankel) for determination of serum aspartate aminotransferase.

Principle: Α-oxoglutarate + L-aspartate (GOT)→ L-glutamate + Oxaloaceetate

46 Chapter 2 Materials and Methods

Aspartate Aminotransferase is measured by monitoring the concentration of oxaloacetate hydrazine formed with 2,4-dinitrophenyl-hydrazine.

Sample: Serum

Procedure Note:Transaminase activities in some sera are stimulated by high concentration of aldehydes, ketones, or oxo acids.

Procedure:Measurement against sample blank

Pipette into test tubes:

Sample blank Sample

Sample ----- 0.1ml Buffer (R 1) 0.5ml 0.5ml

Mix, incubate for exctly 30 min. at 370C

2,4-dinitrophenyl-hydrazine (R2) 0.5ml 0.5ml Sample 0.1ml ------

Mix allow to stand for exactly 20 min. at 20 to 250C

Sodium Hydroxide (R3) 5.0 ml 5.0ml

Mix, read the absorbance of the sample (A sample) agaist the sample blank after 5 minutes.

Wavelength: (490-560 nm) Cuvette: 1cm light path Temperature: 20-250C

Pipette into test tubes:

Tube No Diluted pyruvate Standard (ml) Redistilled water (ml) Buffer (ml)

1 0.00 0.2 1.00 2 0.05 0.2 0.95 3 0.010 0.2 0.90 4 0.15 0.2 0.85 5 0.20 0.2 0.80 6 0.25 0.2 0.75 7 0.30 0.2 0.70 8 0.35 0.2 0.65 9 0.40 0.2 0.60 10 0.45 0.2 0.55

47 Chapter 2 Materials and Methods

Mix and pipette into each tube 1.0 ml of 2,4-dinitrophenyl-hydrazine. Mix and incubate for 20 min at 20 to 250C. add 10 ml of sodium hydroxide solutioin to each tube. Mix and read asorbance agaist blank (tube no 1) after 5 mins.The absorbance of the increasing amounts of pyruvate (0.05 – 0.45 ml Pyruvate Standard) corresponds to the following transaminase activities in U/l. Tube No. AST U/L 2 6 3 11 4 16 5 20 6 25 7 31 8 37 9 44 10 52 The standard curve is obtained by plotting the measured absorbance agaist the transaminase activities in U/L. Ordinate = absorbance, Abscissca = activity in U/L Caluculation: Obtain the activity of AST in the serum from the table: Absorbance U/L Absorbance U/L 0.020 7 0.100 36 0.030 10 0.110 41 0.040 13 0.120 47 0.050 16 0.130 52 0.060 19 0.140 59 0.070 23 0.150 67 0.080 27 0.160 76 0.090 31 0.170 89

2.5.11.15 AST/ALT Ratio determination

The Aspartate Amnotransferase Alanine aminotransferase ratio (AST/ALTratio) is obtained when AST value is divided by ALT.

2.6 Data analysis: Data obtained were analyzed statistically. Different groups were compared using online software Prism Demo version (www.gaphpad.com).The data were presented in the form of tables and graphs.

Chapter - 3

RESULTS

Chapter # 3 Results

In the present investigation, pharmacognostic, biological and pharmacological activities were performed. The two parts viz stem and leaves of H. nepalensis were studied.

3.1 Pharmacognostic evaluation of crude extract

3.1.1 Morphological description: This shrub was 30 m tall clinging with roots; leaves were, 2-15 cm long; broadly ovate or elliptic, variously lobed to entire, nerves prominent; flowersis axillary paniculat umbels, small, yellow; pedicels 7-12 mm long; pedicels and peduncles hairy, Calyx entire, Anthers 1-2 mm long, Stylar column c, 1 mm long, persistent, fruit was drup sub-globose, smooth, round berries 5-7 mm long, 5-10 mm broad.

Organoleptic: Odor was somewhat bitter

Macroscopic characters: Externally dark green, rough longitudinally, rough in touch, irregular fracture

Microscopic characters: Epidermal cells and oil cells were visible, phloem parenchyma, xylem parenchyma; wood fibers and tracheids were present.

Parts used: Stem and Leaves

3.1.2 Physiochemical characterization

3.1.2.1 Fractionation

The crude extract was subjected to fractionation in different solvents openhanded different values, as offered in table 05. The different solvents used were Ethyl acetae, Ethanol, n-Hexane, Petroleum Ether, Chloroform, DCM (Dichloromethane) and Distilled water. The utmost fraction was obtained in chloroform (16.1 gm) followed by Ethyl Acetate (14.3 gm) while the least value was given by ethanol (2.6 gm).

Table 05: Fractionation of the leaves and stem of H. nepalensis

Ethanol n-Hexane Petroleum Ether Chloroform DCM Aqueous Ethyl acetae Leaves 2.6 9.7 12.2 16.1 3.4 5.9 14.3 Stem 7.8 17.1 10.2 18.4 1.1 2.3 2.9

Chapter # 3 Results

n i

2 0

n E th y l a c e ta e

o i

t E th a n o l

1 5 c

a n -H e x a n e

f 1 0 P e tro le u m E th e r

t C h lo ro fo rm h

g 5 D C M i

e A q u e o u s

w r e e 0 l a e h m s t o r n t M u e n o a E f o c a x C e a o h e m D u l t r q y H u o E - e l h n l h A t o E r C t e P s o lv e n ts

Figure 02: Weights of different fractions of the leaves of H. nepalensis

The crude extract was subjected to fractionation in different solvents giving different values, as offered in table 06. The different solvents used were Ethyl acetate, Ethanol, n- Hexane, Petroleum Ether, Chloroform, DCM (Dichloromethane) and Distilled water. The utmost fraction was obtained in chloroform (18.4 gm) followed by n-hexane (17.1 gm) while the least va;ue was given by DCM (1.1 gm).

2 0

E th y l a c e ta e m

g E th a n o l

1 5

n i

n -H e x a n e

o i t P e tro le u m E th e r

c 1 0

r f

C h lo ro fo rm

t 5 D C M

g i

e A q u e o u s w 0

l e M s e o r m u ta n n e r C a a o e x th fo D e c th e u a E o l E H r q y - m lo A n u th h le C E o tr e P s o lv e n ts

Figure 03: Weights of different fractions of the stem of H. nepalensis

Chapter # 3 Results

3.1.2.2 Analysis of extractive values of H. nepalensis

3.1.2.2.1 Extractive values for leaves

The value of percentage extractive is one of the most important parameters in standardization and quality control of precious indigenous drug as it indicates the quantity of active constituents of the plant. For instance, the high percentage of water soluble extractive is an indication of the high carbohydrate contents. Percentage extractive analysis in different solvents (Methanol Ethanol n-Hexane Petroleum Ether Chloroform DCM, distilled water and Ethyl acetate.The extract obtained in maximum value was with ethyl acetate (2.3 and ), followed by petroleum ether (1.8 and ) and the least value was given by Distilled water (0.8 and ).

Table 06: Extractive values of the leaves of H. nepalensis

Methanol Ethanol n-Hexane Petroleum Ether Chloroform DCM Aqueous Ethyl acetae Leaves 1.1 0.9 1.3 1.8 1.7 1.2 0.8 2.3 Stem 0.7 0.3 1.3 7.1 2.5 1.2 2.1 1.7

n 6

o M e th a n o l

i t

c E th a n o l a

r 4 n -H e x a n e

f P e tro le u m E th e r

t C h lo ro fo rm

h 2

g D C M i

e D is tille d w a te r r

w r e e l e 0 l e h m t a E th y l a c e ta e o o n t r a t n E o M e e n a f w a a x C c e e e h m o d a t h e r D t u e l e H o l E - e l l y l i M n h t h o t r C s t i E e D P s o lv e n ts

Figure 04: Weights of different extracts of the leaves of H. nepalensis

3.1.2.2.2 Extractive values for stem

Chapter # 3 Results solvents (Methanol Ethanol n-Hexane Petroleum Ether Chloroform DCM, distille water and Ethyl acetae) of the said plant waere carried out. The extract obtained in maximum value was with Petroleum ether (7.1 and ), followed by Chloroform (2.5 and ) and the least value was given by Ethanol (0.3 and ).

8 m

g M e th a n o l

n i

E th a n o l

n 6 o

i n -H e x a n e

t c

a P e tro le u m E th e r

r 4

f C h lo ro fo rm

h 2 D C M

g i

e A q u e o u s w 0 E th y l a c e ta e l l r s e o e e m o n r M u ta n n h C o a a t o e a x E f D e c h h e o u t t r a e E H m o q l - u l A y M n h h le t o C E tr e P s o lv e n ts

Figure 05: Weights of different extracts of the stem of H. nepalensis

3.1.2.3 Analysis of total ash in H. nepalensis

There is a considerable difference in the ash content of the samples, depending on the particle size of the drug and therefore, it is difficult to decide which one is considered an ash standard however if we return to British Pharmacpoeia, it defines the sample to be subjected for ash determination, is to be the ground drug. Moreover, during sample preparation for moderate fine and very fine powder, a portion of the crude drug was reduced in size but not completely converted into fine powder. In present study the stem and leaf of the H. nepalensis were subjected to total ash content determination. The results indicate that the highest ash content was calculated for the leaves 34.7% and for stem it was 23.5%.

3.1.2.4 Heavy metal analysis of H. nepalensis

3.1.2.4.1 Heavy metals analysis of the leaves

The heavy metals study of Leaves of H. nepalensis revealed that the metal present in maximum amount was Iron (Fe) (140.41 mg/L) followed by lead (Pb) (56.51 mg/L), manganese (Mn) (12.09 mg/L), zinc (Zn) (3.70 mg/L), copper (Cu) (1.85 mg/L), nickel (Ni) (1.35 mg/L), Chromium (Cr) (0.37 mg/L) and Cadmium (Cd) (0.24 mg/L). Silver (Ag) was found absent.

Chapter # 3 Results

Table 7: Heavy metals of the leaves of H. nepalensis

Pb Fe Mn Zn Cu Ni Ag Cd Cr 56.51 140.41 12.092 3.701 1.851 1.357 0.0 0.24 0.370

)

/ g

1 5 0 m

( P b

n F e

o i

t 1 0 0 M n

a r

t Zn n

e C u

5 0 c

n N i o

A g C 0 C d b e n n u i g d r N P F M Z C A C C C r H e a v y M e ta ls in le a v e s o f H .n e p a le n s is

Figure 6: Heavy metals of the leaves of H. nepalensis

3.1.2.4.2 Heavy metals study of the stem

The heavy metals study of Leaves of H. nepalensis revealed that the metal present in maximum amount was Iron (Fe) (156.43 mg/L) followed by manganese (Mn) (123.79 mg/L), zinc (Zn) (20.41 mg/L), copper (Cu) (6.99 mg/L), nickel (Ni) (4.51 mg/L) and Cadmium (Cd) (0.33 mg/L) and lead (Pb) (0.11 mg/L). Chromium (Cr) and Silver (Ag) was found absent.

Table 8: Heavy metals of the stem of H. nepalensis

Pb Fe Mn Zn Cu Ni Ag Cd Cr

0.113 156.430 123.797 20.413 6.992 4.511 0.0 0.338 0.0

)

g k

/ 2 0 0

g P b m

( F e

1 5 0 n

o M n

i t

a Zn

r 1 0 0 t

n C u e

c 5 0 N i n

o A g C 0 C d b e n n u i g d N P F M Z C A C H e a v y M e ta ls in th e s te m o f H .n e p a le n s is

Figure 07: Heavy metals of the stem of H. nepalensis

Chapter # 3 Results

3.1.3 Phytochemical screening of H. nepalensis

3.1.3.1 Qualitative tests for phytochemicals in leaves and stem

The qualitative tests for different phytochemicals of the leaves and stem of H. nepalensis were carried out. The phytochemicals investigation confirmed the existence of Carbohydrates, Proteins, Saponins, Alkaloids, Tanins, Flavonoids, Terpenoids, Phenolic compounds, Phytosterols as revealed in Table 9 while cardiac glycoside were found absent.

Table 9: Qualitative presence of phytochemicals of the leaves and Stem of H. nepalensis

S.No. Tests Results 1 Carbohydrates + 2 Proteins + 3 Saponins + 4 Alkaloids + 5 Tanins + 6 Flavonoids + 7 Terpenoids + 8 Phenolic compounds + 9 Phytosterols + 10 Cardiac glycoside -

3.1.3.2 Quantitative tests for phytochemicals in leaves of H. nepalensis

As shown in Table 10, the quantitative tests revealed that 3 gm of crude extract contains maximum amount of saponins (8.9 gm), followed by alkaloid (4.7 mg), carbohydrates (3.7 gm), terpenoids (2.8 gm ), proteins (2.7 gm), tanins (2.5 gm), phenolic compounds (2.3 gm), flavonoids (2.1 gm) and phytosteols (1.9 gm).

Table 10: Quantitative presence of phytochemicals of the leaves of H. nepalensis

Carbohydrat m Proteins Saponins Alkaloids Tanins Flavonoids Terpenoids Phenolic Phytosterols g

es compounds

n i

3.2 2.7 8.9 4.7 2.5 2.10 2.80 2.3 1.90

n o

c a

r 1 0

f C a rb o h y d ra te s

f 8 P ro te in s o

S a p o n in s

t 6 s

d A lk a lo id s h 4 n T a n in s g s u i e s o l

t s s F la v o n o id s e 2 a s s p o d d r s r n d i i T e rp e n o id s i s m w d n i e i o o n o t y0 n o e l i n n P h e n o lic c o m p o u n d s c s t o h a n o e o P h y to s te ro ls o o p k a v p c t i r l r b a T a l y r P l S A eo h a F T n P C e h

P p h y to c h e m ic a ls Figure 08: Quantitative presence of phytochemicals of the leaves of H. nepalensis

Chapter # 3 Results

3.1.3.3 Quantitative tests for phytochemicals in stem of H. nepalensis

As shown in Table 13, the quantitative tests revealed that 3 gm of crude extract contains maximum amount of saponins (4.6 gm), followed by tanins (3.4 mg), proteins (1.9 gm), carbohydrates (1.3 gm), terpenoids (2.7 gm), phenolic compounds (0.9 gm), phytosteols (0.84 gm) and flavonoids (0.76 gm) and alkaloid (0.7 gm).

Table 11: Quantitative presence of phytochemicals of the stem of H. nepalensis

Carbohydrates Proteins Saponins Alkaloids Tanins 1.3 1.9 4.6 0.7 3.4 Flavonoids Terpenoids Phenolic Phytosterols 0.76 0.31 0.9 0.84

m Carbohydrates

n 4 Proteins

o Saponins i

t 3

c Alkaloids

f 2 Tanins

h Flavonoids

g 1 i

e Terpenoids w 0 Phenolic compounds s s s s s s s s s e n n d n d d d l t i i i i i i n ro Phytosterols ra te n lo n o o u e d o o a a n n o t y r p lk T o e p s h P a v rp to o S A la e m y b F T o h r c P a c C li o n e h P solvents Figure 09: Quantitative presence of phytochemicals of the stem of H. nepalensis

3.2 Biological Evaluation of methanolic extract of H. nepalensis

3.2.1 Antioxidant activity of the Leaves and Stem

3.2.1.1 DPPH free radical scavenging activity

The DPPH scavenging activity was studied at the absorbance of 517nm. The absorbance was due the number of the electrons taken up. At increase in concentration absorbance was reduced gradually. The free radical scavenging activity of leaves methanol, ethanol, ethyl acetate, DCM, petroleum ether extracts is given in the table 12. Concentration of 0.1 mg/ml exhibited 42.72, 26.36, 46.36, 58.18 and 51.81 scavenging activity respectively. While free radical scavenging activity of stem methanol, ethanol, ethyl acetate, DCM, petroleum ether extracts are given in the table 15. Concentration of 0.1 mg/ml exhibited 34.02, 45.13, 31, 53, and 61 scavenging activity respectively. However, DPPH scavenging

Chapter # 3 Results activity of ascorbic acid at this concentration exhibited marked scavenging activity 72.72 and 76 respectively.

Table 12: DPPH radical scavenging activity of H. nepalensis leaves at 517 nm with absorbance in triplicate

Compound Absorbance in triplicate

Control (.1) 1.11 1.09 1.10 Et. ac.E. (.1) 0.67 0.60 0.62 Pet. Ether.E. (.1) 0.81 0.83 0.80 DCM.E. (.1) 0.61 0.57 0.59 Ethanol.E. (.1) 0.44 0.46 0.49 Methanol.E. (.1) 0.55 0.52 0.54 Ascorbic acid (.1) 0.31 0.34 0.27

Table 13: DPPH radical scavenging activity of H. nepalensis leaves at 517 nm with mean, standard deviation and number

Compound Mean SD N

Control (.1) 1.100 0.009999991 3 Et. ac.E. (.1) 0.630 0.03605551 3 Pet. Ether.E. o(.1) 0.8133333 0.01527524 3 DCM.E. (.1) 0.590 0.02000001 3 Ethanol.E. (.1) 0.4633333 0.02516612 3 Methanol.E. (.1) 0.5366667 0.01527527 3 Ascorbic acid (.1) 0.3066667 0.03511884 3

Table 14: DPPH radical scavenging activity of ethyl acetate, petroleum ether, DCM, ethanol and methanol leaves extracts of H. nepalensis and standard ascorbic acid

Compound Concentration Absorbance at Inhibition %age (mg/ml) 517nm Control 0.1 1.10±0.009 ---- Et. ac.E. 0.1 0.63±0.0360 42.72 Pet. Ether.E. 0.1 0.81±0.0152 26.36 DCM.E. 0.1 0.59±0.0200 46.36 Ethanol.E. 0.1 0.46±0.0251 58.18 Methanol.E. 0.1 0.53±0.0152 51.81 Ascorbic acid 0.1 0.30±0.035 72.72 All the tests and analysis were run in triplicates and averaged. Each value represents the mean + standard deviation of triplicate analysis.

)

Chapterl # 3 Results

g m

( 1 .5

L e g e n d

i t

a 1 .0

n c

e 0 .5 n

o ) ) ) 1 ) . C ) 1 1 ( ) . ) 1 . 1 ( . ( 1 . 1 ( 0 .0 . ( . . . d ( ( . i . E E c l . . E . E . l a o . r E l r . o t c e o c i n a h M n n t a b o . C a r t E h h o C E . D t t t e c e E s M P A

A b s o rb a n c e a t 5 1 7 n m

Figure 10: DPPH radical scavenging activity of H. nepalensis leaves extracts

Table 15: DPPH radical scavenging activity of H. nepalensis stem at 517 nm with Absorbance in triplicate

Compound Absorbance in triplicate

Control (.1) 1.44 1.47 1.41 Et. ac.E. (.1) 0.99 0.97 0.91 Pet. Ether.E. (.1) 0.83 0.80 0.76 DCM.E. (.1) 1.02 0.98 0.98 Ethanol.E. (.1) 0.64 0.68 0.70 Methanol.E. (.1) 0.55 0.58 0.52 Ascorbic acid (.1) 0.34 0.33 0.35

Table 16: DPPH radical scavenging activity of H. nepalensis Leaves at 517 nm with mean, standard deviation and number

Compound Mean SD N

Control (.1) 1.440 0.03000003 3 Et. ac.E. (.1) 0.9566667 0.04163331 3 Pet. Ether.E. (.1) 0.7966667 0.03511884 3 DCM.E. (.1) 0.9933333 0.02309399 3 Ethanol.E. (.1) 0.6733334 0.03055051 3 Methanol.E. (.1) 0.550 0.030 3 Ascorbic acid (.1) 0.340 0.009999991 3

Table 17: DPPH radical scavenging activity of ethyl acetate, petroleum ether, DCM, ethanol and methanol stem extracts of H. nepalensis and standard ascorbic acid

Compound Concentration (mg/ml) Absorbance at Inhibition 517nm %age Control 0.1 1.44±0.03 ---- Et. ac.E. 0.1 0.95±0.04 34.02 Pet. Ether.E. 0.1 0.79±0.03 45.13

Chapter # 3 Results

DCM.E. 0.1 0.99±0.02 31 Ethanol.E. 0.1 0.67±0.03 53 Methanol.E. 0.1 0.55±0.03 61 Ascorbic acid 0.1 0.34±0.009 76

All the tests and analysis were run in triplicates and averaged. )

Eachl value represents the mean + standard deviation of triplicate analysis.

m (

2 .0

L e g e n d

o i

t 1 .5

r t

n 1 .0

c e

n 0 .5

o ) ) ) ) 1 1 . C ) 1 ( ) . ) 1 . 1 ( . ( 1 . 1 ( 0 .0 . ( . . . d ( ( . i . E E c l . . E . E r . l a o . E l r e . o t c o c n i n a h M n t a b o . C a r t E h h t o C E . D t t e c e E s M P A

A b s o rb a n c e a t 5 1 7 n m

Figure 11: DPPH radical scavenging activity of H. nepalensis stem extracts

3.2.2 Hydrogen peroxide free radical scavenging activity

The hydrogen peroxide scavenging activity was studied at the absorbance of 285nm with average time of 10 minutes incubation. The hydrogen Peroxide radical scavenging activity of leaves methanol, ethanol, ethyl acetate, DCM, petroleum ether extracts is given in the table 18. Concentration of 0.1 mg/ml exhibited 50, 54.54, 47.27, 60, and 70.90 scavenging activity respectively. The free radical scavenging activity of leaves methanol, ethanol, ethyl acetate, DCM, petroleum ether extracts is given in the table 21. Concentration of 0.1 mg/ml exhibited 50, 77, 74, 20.66 and 72 scavenging activity respectively. However, DPPH scavenging activity of ascorbic acid at this concentration exhibited marked scavenging activity 80.50 and 85.

Table 18: Hydrogen peroxide radical scavenging activity of H. nepalensis Compound absorbance in triplicate

Control 1.220 1.130 1.190 Et. ac. E. (.005) 1.080 1.070 1.060 Et. ac. E. (.01) 1.040 1.010 1.040 Et. ac.E. (.02) 1.010 1.050 1.090 Et. ac. E. (.05) 0.810 0.800 0.803 Et. ac.E. (.1) 0.610 0.600 0.570 Pet. Ether.E. (.005) 1.130 1.110 1.130 Pet. Ether.E. (.01) 1.110 1.010 1.000 Pet. Ether.E. (.02) 1.000 1.030 1.070

Chapter # 3 Results

Pet. Ether.E. (.05) 0.710 0.770 0.790 Pet. Ether.E. (.1) 0.530 0.490 0.500 DCM.E. (.005) 0.930 0.900 0.870 DCM.E. (.01) 0.860 0.890 0.880 DCM.E. (.02) 0.770 0.790 0.790 DCM.E. (.05) 0.700 0.690 0.670 DCM.E. (.1) 0.640 0.600 0.520 Ethanol.E. (.005) 0.550 0.570 0.580 Ethanol.E. (.01) 0.660 0.610 0.630 Ethanol.E. (.02) 0.600 0.670 0.660 Ethanol.E. (.05) 0.510 0.500 0.490 Ethanol.E. (.1) 0.440 0.430 0.450 Methanol.E. (.005) 0.930 0.900 0.920 Methanol.E. (.01) 1.130 1.110 1.100 Methanol.E. (.02) 0.720 0.710 0.690 Methanol.E.(.05) 0.610 0.550 0.570 Methanol.E. (.1) 0.320 0.310 0.340 Ascorbic acid (.1) 0.200 0.230 0.270

Table 19: Hydrogen peroxide activity of H. nepalensis Leaves at 285 nm with mean, standard deviation and number

Compound Mean SD N

Control 1.180 0.04582578 3 Et. ac. E. (.005) 1.070 0.01000005 3 Et. ac. E. (.01) 1.030 0.01732049 3 Et. ac.E. (.02) 1.050 0.04000002 3 Et. ac. E. (.05) 0.8043333 0.005131601 3 Et. ac.E. (.1) 0.5933334 0.02081667 3 Pet. Ether.E. (.005) 1.123333 0.011547 3 Pet. Ether.E. (.01) 1.040 0.06082764 3 Pet. Ether.E. (.02) 1.033333 0.03511887 3 Pet. Ether.E. (.05) 0.7566667 0.04163334 3 Pet. Ether.E. (.1) 0.5066667 0.02081664 3 DCM.E. (.005) 0.900 0.030 3 DCM.E. (.01) 0.8766667 0.01527524 3 DCM.E. (.02) 0.7833334 0.01154703 3 DCM.E. (.05) 0.6866667 0.01527524 3 DCM.E. (.1) 0.5866666 0.06110102 3 Ethanol.E. (.005) 0.5666667 0.01527524 3 Ethanol.E. (.01) 0.6333333 0.02516612 3 Ethanol.E. (.02) 0.6433334 0.03785939 3 Ethanol.E. (.05) 0.500 0.009999991 3 Ethanol.E. (.1) 0.440 0.009999991 3 Methanol.E. (.005) 0.9166667 0.01527527 3 Methanol.E. (.01) 1.113333 0.01527524 3 Methanol.E. (.02) 0.7066667 0.01527526 3 Methanol.E.(.05) 0.5766667 0.03055051 3 Methanol.E. (.1) 0.3233333 0.01527525 3 Ascorbic acid (.1) 0.2333333 0.03511885 3

Chapter # 3 Results

Table 20: Hydrogen peroxide radical scavenging activity of ethyl acetate, petroleum ether, DCM, ethanol and methanol leaves extracts of H. nepalensis and standard ascorbic acid

Compound Concentration Absorbance at 285nm Inhibition %age (mg/ml) Control 0.1 1.180±0.045 ----- Et. ac. E. 0.005 1.070±0.010 9.32 Et. ac. E. 0.01 1.030±0.017 12.71 Et. ac. E. 0.02 1.050±0.040 1.01 Et. ac. E. 0.05 0.804±0.0051 32.20 Et. ac. E. 0.1 0.593±0.0208 50 Pet. Ether.E. 0.005 1.12±0.011 1.81 Pet. Ether.E. 0.01 1.040±0.0608 5.45 Pet. Ether.E. 0.02 1.033±0.035 6.36 Pet. Ether.E. 0.05 0.756±0.041 31.81 Pet. Ether.E. 0.1 0.506±0.020 54.54 DCM.E. 0.005 0.900±0.030 18.18 DCM.E. 0.01 0.876±0.015 20.90 DCM.E. 0.02 0.783±0.011 29.09 DCM.E. 0.05 0.686±0.015 38.18 DCM.E. 0.1 0.586±0.061 47.27 Ethanol.E. 0.005 0.566±0.015 49.09 Ethanol.E. 0.01 0.633±0.025 42.72 Ethanol.E. 0.02 0.643±0.037 45.76 Ethanol.E. 0.05 0.500±0.009 54.54 Ethanol.E. 0.1 0.440±0.009 60 Methanol.E. 0.005 0.916±0.015 17.27 Methanol.E. 0.01 1.113±0.015 5.93 Methanol.E. 0.02 0.706±0.015 36.36 Methanol.E. 0.05 0.576±0.030 48.18 Methanol.E. 0.1 0.323±0.015 70.90 Ascorbic acid 0.1 0.23±0.035 80.50

All the tests and analysis were run in triplicates and averaged )

Eachl value represents the mean + standard deviation of triplicate analysis

/ g

m As c o r b ic a c id (.1 ) (

M e th a n o l.E .(.0 5 )

n M e th a n o l.E . (.0 1 )

o E th a n o l.E . (.1 ) i

t E th a n o l.E . (.0 2 )

a E th a n o l.E . (.0 0 5 ) r

t D C M .E . (.0 5 )

n D C M .E . (.0 1 )

c P e t. E th e r .E . (.1 )

e P e t. E th e r .E . (.0 2 )

n P e t. E th e r .E . (.0 0 5 )

o E t. a c . E . (.0 5 )

C E t. a c . E . (.0 1 ) C o n tr o l

0 5 0 5 . . . . 0 0 1 1 A b s o rb a n c e a t 2 8 5 n m

Figure 12: Hydrogen peroxide radical scavenging activity of H. nepalensis leaves extracts

Chapter # 3 Results

Table 21: Hydrogen peroxide radical scavenging activity of H. nepalensis Stem at 285nm with absorbance in triplicate

Compound Absorbance in triplicate

Control 1.530 1.480 1.500 Et. ac. E. (.005) 1.110 1.060 1.020 Et. ac. E. (.01) 0.970 0.930 0.900 Et. ac.E. (.02) 0.780 0.770 0.730 Et. ac. E. (.05) 0.670 0.660 0.680 Et. ac.E. (.1) 0.550 0.540 0.570 Pet. Ether.E. (.005) 1.030 1.050 1.000 Pet. Ether.E. (.01) 1.000 0.990 1.010 Pet. Ether.E. (.02) 0.840 0.860 0.880 Pet. Ether.E. (.05) 0.730 0.700 0.740 Pet. Ether.E. (.1) 0.310 0.350 0.370 DCM.E. (.005) 1.320 1.300 1.280 DCM.E. (.01) 1.240 1.270 1.290 DCM.E. (.02) 1.110 1.190 1.220 DCM.E. (.05) 0.710 0.680 0.670 DCM.E. (.1) 0.360 0.400 0.410 Ethanol.E. (.005) 1.430 1.410 1.460 Ethanol.E. (.01) 1.330 1.370 1.390 Ethanol.E. (.02) 1.250 1.220 1.250 Ethanol.E. (.05) 1.210 1.230 1.270 Ethanol.E. (.1) 1.130 1.170 1.290 Methanol.E. (.005) 0.950 0.970 0.920 Methanol.E. (.01) 1.190 1.220 1.260 Methanol.E. (.02) 1.010 1.070 1.080 Methanol.E.(.05) 0.910 0.940 0.970 Methanol.E. (.1) 0.420 0.410 0.400 Ascorbic acid (.1) 0.220 0.210 0.240

Table 22: Hydrogen peroxide radical scavenging activity of H. nepalensis Stem at 285 nm with mean, standard deviation and number

Compound Mean SD N

Control 1.503333 0.02516609 3 Et. ac. E. (.005) 1.063333 0.04509252 3 Et. ac. E. (.01) 0.9333333 0.03511887 3 Et. ac.E. (.02) 0.760 0.02645749 3 Et. ac. E. (.05) 0.670 0.009999991 3 Et. ac.E. (.1) 0.5533333 0.01527524 3 Pet. Ether.E. (.005) 1.026667 0.02516609 3 Pet. Ether.E. (.01) 1.000 0.009999991 3 Pet. Ether.E. (.02) 0.860 0.02000001 3 Pet. Ether.E. (.05) 0.7233334 0.02081667 3 Pet. Ether.E. (.1) 0.3433333 0.03055051 3 DCM.E. (.005) 1.300 0.02000004 3 DCM.E. (.01) 1.266667 0.02516609 3 DCM.E. (.02) 1.173333 0.05686242 3

Chapter # 3 Results

DCM.E. (.05) 0.6866667 0.02081664 3 DCM.E. (.1) 0.390 0.02645751 3 Ethanol.E. (.005) 1.433333 0.02516615 3 Ethanol.E. (.01) 1.363333 0.03055048 3 Ethanol.E. (.02) 1.240 0.01732049 3 Ethanol.E. (.05) 1.236667 0.03055048 3 Ethanol.E. (.1) 1.196667 0.08326662 3 Methanol.E. (.005) 0.9466667 0.02516612 3 Methanol.E. (.01) 1.223333 0.03511881 3 Methanol.E. (.02) 1.053333 0.03785942 3 Methanol.E.(.05) 0.940 0.030 3 Methanol.E. (.1) 0.410 0.009999991 3 Ascorbic acid (.1) 0.2233333 0.01527525 3

Table 23: Hydrogen peroxide radical scavenging activity of ethyl acetate, petroleum ether, DCM, ethanol and methanol stem extracts of H. nepalensis and standard ascorbic acid

Compound Concentration (mg/ml) Absorbance at 285nm Inhibition %age Control 0.1 1.503±0.025 ----- Et. ac. E. 0.005 1.063±0.045 29.33 Et. ac. E. 0.01 0.933±0.035 38 Et. ac. E. 0.02 0.760±0.026 49.33 Et. ac. E. 0.05 0.670±0.009 55.33 Et. ac. E. 0.1 0.553±0.015 50 Pet. Ether.E. 0.005 1.026±0.025 32 Pet. Ether.E. 0.01 1.000±0.009 33.33 Pet. Ether.E. 0.02 0.860±0.02 42.66 Pet. Ether.E. 0.05 0.723±0.02 52 Pet. Ether.E. 0.1 0.343±0.030 77 DCM.E. 0.005 1.30±0.020 13.33 DCM.E. 0.01 1.266±0.025 16 DCM.E. 0.02 1.173±0.056 22 DCM.E. 0.05 0.686±0.0208 54.66 DCM.E. 0.1 0.390±0.026 74 Ethanol.E. 0.005 1.433±0.025 4.6 Ethanol.E. 0.01 1.363±.030 9.33 Ethanol.E. 0.02 1.240±0.017 17.33 Ethanol.E. 0.05 1.236±0.030 20 Ethanol.E. 0.1 1.196±0.083 20.66 Methanol.E. 0.005 0.946±0.025 37.33 Methanol.E. 0.01 1.223±0.035 18.66 Methanol.E. 0.02 1.053±0.037 30 Methanol.E. 0.05 0.940±0.030 37.33 Methanol.E. 0.1 0.410±0.009 72 Ascorbic acid 0.1 0.223±0.015 85 All the tests and analysis were run in triplicates and averaged Each value represents the mean + standard deviation of triplicate analysis

)

Chapter # 3l Results

(

As c o r b ic a c id (.1 ) n

M e th a n o l.E .(.0 5 ) o

i M e th a n o l.E . (.0 1 ) t E th a n o l.E . (.1 )

a E th a n o l.E . (.0 2 ) r

t E th a n o l.E . (.0 0 5 )

D C M .E . (.0 5 ) n

D C M .E . (.0 1 ) c

P e t. E th e r .E . (.1 ) e P e t. E th e r .E . (.0 2 ) n P e t. E th e r .E . (.0 0 5 )

o E t. a c . E . (.0 5 )

C E t. a c . E . (.0 1 ) C o n tr o l

0 5 0 5 0 . . . . . 0 0 1 1 2 A b s o rb a n c e a t 2 8 5 n m

Figure 13: Hydrogen peroxide radical scavenging activity of the stem of H. nepalensis extracts

3.2.2 Antimicrobial activities of H.nepalensis

The antimicrobial activity of extract of stem of H. nepalensis in different solvents like methanol, ethanol, n-hexane, petroleum ether and chloroform showed different zones of inhibition against bacteria like Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi klebsiella pneumonia, Erwinia cartovara Staphylococcus aureus, Bacillus subtilis, Bacillus atrophaeus, Bacillus cereus, Enterococcus faecalis, Shigella sonnei, Citrobacter freundii and A.Tumefacians and against fungi like Candida albican, Aspergillus nige, Aspergillus flavus, Alternaria alternate, Penicillium notatum and Trichoderma harzianum.

3.2.2.1.1. Antibacterial activities of leaves of H. nepalensis

3.2.2.1.1.1 Methanol extract of leaves

Methanol extract of leaves of H. nepalensis had shown best inhibition against Pseudomonas aeruginosa (20 mm) followed by Shigella sonnei (19 mm), Enterococcus faecalis (18 mm), klebsiella pneumoniae (17 mm), Escherichia coli (15 mm), Bacillus subtilis (15 mm), Erwinia cartovara (13 mm), Bacillus atrophaeus (11 mm), Citrobacter freundii (10 mm) and the methanol extract of leaves of H. nepalensis has no effect against Salmonella typhi, Staphylococcus aureus, Bacillus cereus and Agrobacterium Tumefaciens.

Table 24: Antibacterial activities of the methanol extract of leaves of H. nepalensis

E.coli P. S. typhi K. E. cartovara S. B. Bacillus aeruginosa pneumoni aureus subtilis atrophaeus ae 15 20 00 17 13 00 15 11 B.cereus E. S. C. A.Tumefacians faecalis sonnei freundii 00 18 19 10 00

Chapter) # 3 Results

( e

n 2 5

E .c o li o

z 2 0

P . a e ru g in o s a

n S . ty p h i

o 1 5

i K . p n e u m o n ia e

t i

1 0 E . c a rto v a ra b

s h u S . a u re u s

n 5 e a e i a s i a a i B . s u b tilis r s s i i i n s ih s i o a l l e d i h u ip 0 l n o v u a n B a c illu s a tro p h a e u s i e t n o p o e c o r br n u g y m t r c t e o . u t u r u u e e B .c e re u s a a r . a s s r E e a c f f . . e S c . .s . . n E . fa e c a lis a B S p . S Bu E . l C E l . i P S . s o n n e i K c a C . fre u n d ii B te s te d b a c te r ia

Figure 14: Antibacterial activities of the methanol extract of leaves of H. nepalensis

3.2.2.1.1.2 Ethanol extract of leaves

Methanol extract of leaves of H. nepalensis had shown best inhibition against Bacillus atrpenses (27 mm) followed byBacillus subtilis (25 mm), Staphylococcus aureus (22 mm), Erwinia cartovara (17 mm), Pseudomonas aeruginosa (15 mm), Salmonella typhi (14 mm), Bacillus cereus (14 mm), Citrobacter freundii (13 mm) and Escherichia coli (11 mm) and and the methanol extract of leaves of H. nepalensis has no effect against Enterococcus faecalis, Shigella sonnei and Agrobacterium Tumefacians.

Table 25: Antibacterial activities of the ethanol extract of leaves of H. nepalensis

E.coli P. S. K. E. cartovara S. B. Bacillus aeruginos typhi pheumoni aureu subtili atrophaeu a a s s s 11 15 14 10 17 22 25 27 B.cereu E. S. C. A.Tumefacian s faecalis sonne freundii s i 14 00 00 13 00

) 3 0

m E .c o li m

( P . a e ru g in o s a e

2 0 S . ty p h i

o z

K . p h e u m o n ia

n o

t E . c a rto v a ra i 1 0

b S . a u re u s

h n i B . s u b tilis

0 B . a tro p h a e u s a i a a s i i i i s s s is i l s h n r li u l e o o a u i e u d p o e t a n n c n y v r a e c n B .c e re u s . i t o b r u m t u u h e e o E g . u p e u r a s c a s r S e a . o . f . f r . r . . E . fa e c a lis e h c S t B S p . B E C a a . . E . P K B S . s o n n e i te s te d b a c te r ia C . fre u n d ii

Figure 15: Antibacterial activities of the ethanol extract of leaves of H. nepalensis

Chapter # 3 Results

3.2.2.1.1.3 n-Hexane extract of leaves

Methanol extract of leaves of H. nepalensis had shown best inhibition against Bacillus subtilis (27 mm) followed by Staphylococcus aureus (25 mm), Escherichia coli (22 mm), Bacillus atrpenses (19 mm), Citrobacter freundii (18 mm), Bacillus cereus (17 mm), klebsiella pneumonia (11 mm) and the methanol extract of leaves of H. nepalensis has no effect against Pseudomonas aeruginosa, Erwinia cartovara, Enterococcus faecalis, Shigella sonnei and Agrobacterium Tumefacians.

Table 26: Antibacterial activities of the n-hexane extract of leaves of H. nepalensis

E.coli P. S. K. E. cartovara S. B. Bacillus aeruginos typhi pheumoni aureu subtili atrophaeu a a s s s 22 00 11 00 25 27 19 B.cereu E. S. C. A.Tumefacian s faecalis sonne freundii s i 17 00 00 18 00

E.coli )

m P. aeruginosa

m (

e 20 S. typhi

n o

z K. pheumonia

o E. cartovara

i t

i 10

b S. aureus

h n i B. subtilis 0 B. atrophaeus li a i a a s s s s s i ii o s h i r u li u u li e d c o p n a e ti e e a n n B.cereus . n ty o v r b a r c n u E i . m to u u h e e o e g S u r a s p .c fa s fr ru e a . . o B . . . E. faecalis e h c S B tr E S a p . a C . . E . P K B S. sonnei tested bacteria C. freundii

Figure 16: Antibacterial activities of the n-hexane extract of leaves of H. nepalensis

3.2.2.1.1.4 Petroleum ether extract of leaves

Methanol extract of leaves of H. nepalensis had shown best inhibition against Pseudomonas aeruginosa (17 mm) followed by Salmonella typhi (17 mm), Shigella sonnei (17 mm), followed by Escherichia coli (15 mm), Erwinia cartovara (14 mm), klebsiella pneumonia (11 mm), Bacillus atrpenses (11 mm) and the methanol extract of leaves of H. nepalensis has no effect against Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Enterococcus faecalis,Citrobacter freundii and Agrobacterium Tumefacians.

Chapter # 3 Results

Table 27: Antibacterial activities of the petroleum ether extract of leaves of H. nepalensis

E.coli P. S. K. E. cartovara S. B. Bacillus aeruginos typhi pheumoni aureu subtili atrophaeu a a s s s 15 17 17 11 14 00 00 11 B.cereu E. S. C. A.Tumefacian s faecalis sonne freundii s i 00 00 17 00 00

E.coli )

m P. aeruginosa

m (

e S. typhi

n o

z K. pheumonia

10 n

o E. cartovara

t i

b 5 S. aureus

h n i B. subtilis 0 B. atrophaeus li a i a a s s s s s i o s h i r u li u u li e c o p n a e ti e e a n B.cereus . n ty o v r b a r c n E i . m to u u h e e o g S u r a s p .c fa s ru e a . . o B . . E. faecalis e h c S B tr E S a p . a . . E . P K B S. sonnei tested bacteria

Figure 17: Antibacterial activities of the petroleum ether extract of leaves of H. nepalensis

3.2.2.1.1.5 Chloroform extract of leaves

Methanol extract of leaves of H. nepalensis had shown best inhibition against Enterococcus faecalis (27 mm) followed by Escherichia coli (26 mm), Pseudomonas aeruginosa (17 mm), Citrobacter freundii (15 mm), Bacillus cereus (14 mm), Salmonella typhi (13 mm) and Staphylococcus aureus (12 mm) and the methanol extract of leaves of H. nepalensis has no effect against klebsiella pneumonia, Erwinia cartovara, Bacillus subtilis, Bacillus atrpenses, Shigella sonnei and Agrobacterium Tumefacians.

Table 28: Antibacterial activities of the chloroform extract of leaves of H. nepalensis

E.coli P. S. typhi K. E. cartovara S. B. Bacillus aeruginosa pheumonia aureus subtilis atrophaeus 26 17 13 00 00 12 00 00 B.cereus E. S. C. A.Tumefacians faecalis sonnei freundii 14 27 00 15 00

Chapter # 3 Results

E.coli )

m P. aeruginosa

m (

e 20 S. typhi

n o

z K. pheumonia

o E. cartovara

i t

i 10

b S. aureus

Figure 18: Antibacterial activities of the chloroform extract of leaves of H. nepalensis

3.2.2.1.2 Antibacterial activities of stem

3.2.2.1.2.1 Methanol extract of stem

Methanol extract of leaves of H. nepalensis had shown best inhibition against Bacillus subtilis (38 mm) followed by klebsiella pneumonia (36 mm), Escherichia coli (32 mm), Salmonella typhi (19 mm), Agrobacterium Tumefacians (17 mm), Staphylococcus aureus (16 mm), Bacillus atrpenses (14 mm), Erwinia cartovara (13 mm), Citrobacter freundii (13 mm), Bacillus cereus (11 mm) and Pseudomonas aeruginosa (11 mm) and the methanol extract of leaves of H. nepalensis had no effect against Enterococcus faecalis and Shigella sonnei.

Table 29: Antibacterial activities of the methanol extract of stem of H. nepalensis

E.coli P. S. typhi K. E. cartovara S. B. Bacillus aeruginosa pheumonia aureus subtilis atrophaeus 32 11 19 36 13 16 38 14 B.cereus E. S. C. A.Tumefacians faecalis sonnei freundii 11 00 00 13 17

E.coli )

m P. aeruginosa

m (

e S. typhi

n o

z K. pheumonia

20 n

o E. cartovara

t i

b 10 S. aureus

h n i B. subtilis 0 B. atrophaeus i i i i l a h ia ra s is s s is e i s o s p n a u il u u l n d n .c o y e t e e a n ia B.cereus n t o v r b a r c n u c E i . m to u u h e e o e a g S u r a s p .c fa s fr f ru e a . . o B . . . e E. faecalis e h c S B tr E S m a p . a C . . E . u P .T K B A S. sonnei tested bacteria C. freundii A.Tumefacians Figure 19: Antibacterial activities of the methanol extract of stem of H. nepalensis

Chapter # 3 Results

3.2.2.1.2.2 Ethanol extract of stem

Methanol extract of leaves of H. nepalensis had shown best inhibition against Escherichia coli (36 mm) followed by Bacillus subtilis (29 mm), Shigella sonnei (28 mm), Bacillus cereus (25 mm), Enterococcus faecalis (22 mm), Pseudomonas aeruginosa (16 mm), Staphylococcus aureus (13 mm) and Salmonella typhi (10 mm) and the methanol extract of leaves of H. nepalensis has no effect against klebsiella pneumonia, Erwinia cartovara, Bacillus atrpenses, Citrobacter freundii and Agrobacterium tumefacians.

Table 30: Antibacterial activities of the ethanol extract of stem of H. nepalensis

E.coli P. S. K. E. cartovara S. B. Bacillus aeruginos typhi pheumoni aureu subtili atrophaeu a a s s s 36 16 10 00 00 13 29 00 B.cereu E. S. C. A.Tumefacian s faecalis sonne freundii s i 25 22 28 00 00

E.coli )

m P. aeruginosa

m (

e S. typhi

n o

z K. pheumonia

20 n

o E. cartovara

t i

b 10 S. aureus

Figure 20: Antibacterial activities of the ethanol extract of stem of H. nepalensis

3.2.2.1.2.3 n-Hexane extract of stem

Methanol extract of leaves of H. nepalensis had shown best inhibition against Shigella sonnei (37 mm) followed by Enterococcus faecalis (36 mm), Erwinia cartovara (17 mm), Bacillus subtilis (17 mm), Bacillus cereus (17 mm), Agrobacterium tumefacians (15 mm), Pseudomonas aeruginosa (13 mm), Citrobacter freundii (11 mm)and Salmonella typhi (10 mm), and the methanol extract of leaves of H. nepalensis has no effect against Escherichia coli, klebsiella pneumonia, Staphylococcus aureus and Bacillus atrpenses.

Chapter # 3 Results

Table 31: Antibacterial activities of the n-hexane extract of stem of H. nepalensis

E.coli P. S. K. E. cartovara S. B. Bacillus aeruginos typhi pheumoni aureu subtili atrophaeu a a s s s 00 13 10 00 17 00 17 00 B.cereu E. S. C. A.Tumefacian s faecalis sonne freundii s i 17 36 37 11 15

E.coli )

m P. aeruginosa

m (

e S. typhi

n o

z K. pheumonia

20 n

o E. cartovara

t i

b 10 S. aureus

Figure 21: Antibacterial activities of the n-hexane extract of stem of H. nepalensis

3.2.2.1.2.4 Petroleum ether extract of stem

Methanol extract of leaves of H. nepalensis had shown best inhibition against Bacillus cereus (36 mm) followed by Bacillus atrpenses (33 mm), Escherichia coli (22 mm), Bacillus subtilis (21 mm), Staphylococcus aureus (19 mm), Pseudomonas aeruginosa (17 mm), Citrobacter freundii (16 mm) Enterococcus faecalis (15 mm) Salmonella typhi (13 mm), and the methanol extract of leaves of H. nepalensis has no effect against klebsiella pneumonia, Erwinia cartovara and Shigella sonnei.

Table 32: Antibacterial activities of the petroleum ether extract of stem of H. nepalensis

E.coli P. S. K. E. cartovara S. B. Bacillus aeruginos typhi pheumoni aureu subtili atrophaeu a a s s s 22 17 13 00 00 19 21 33 B.cereu E. S. C. A.Tumefacian s faecalis sonne freundii s i 36 15 00 16 12

Chapter # 3 Results

4 0

E .c o li )

m P . a e ru g in o s a

3 0

m (

e S . ty p h i

n o

z K . p h e u m o n ia

2 0 n

o E . c a rto v a ra

t i

b 1 0 S . a u re u s

h n i B . s u b tilis

0 B . a tro p h a e u s i i l a a a s s s s i ii s h i r i s i e o s p n u il u u l d n c o a e t e e a n n a B .c e re u s . ty o v r r c n i E in b a u c . m to u u h e e o e a g S u r a s c a s r f u . p . f . f e r e a . o B . . E . fa e c a lis h c S r S e . B t E C m a p a u . . E . T P K . S . s o n n e i B A te s te d b a c te r ia C . fre u n d ii A .T u m e fa c ia n s

Figure 22: Antibacterial activities of the petroleum ether extract of stem of H. nepalensis

3.2.2.1.2.5 Chloroform extract of stem

Methanol extract of leaves of H. nepalensis had shown best inhibition against Pseudomonas aeruginosa (35 mm) followed by Bacillus subtilis (22 mm), Salmonella typhi (17 mm), Staphylococcus aureus (16 mm), klebsiella pneumonia (13 mm), Shigella sonnei (12 mm), Erwinia cartovara (11 mm), Bacillus subtilis (22 mm), Bacillus atrpenses (11 mm), Agrobacterium Tumefacians (10 mm)and the methanol extract of leaves of H. nepalensis has no effect against Escherichia coli, Bacillus cereus, Enterococcus faecalis and Citrobacter freundii.

Table 33: Antibacterial activities of the chloroform extract of stem of H. nepalensis

E.coli P. S. K. E. cartovara S. B. Bacillus aeruginos typhi pheumoni aureu subtili atrophaeu a a s s s 00 35 17 13 11 16 22 11 B.cereu E. S. C. A.Tumefacian s faecalis sonne freundii s i 00 00 12 00 10

Chapter # 3 Results

)

m ( 4 0

e E .c o li n

o P . a e ru g in o s a

z 3 0

S . ty p h i n

o K . p h e u m o n ia

i 2 0 t

i E . c a rto v a ra b

h 1 0 S . a u re u s n

i s a a s B . s u b tilis i a i n s s u s i i i n r s i s i a 0 i o a l e l e d i l h o u i u B . a tro p h a e u s n t a a n c o i p v e n h e c n a y m o r b r u c g t f . t u u u p e o e B .c e re u s u r e e E . e a o a s r r a s c f f . r . . m e S h c . t . . E . fa e c a lis a p S B S u . B a E C . T . E . . P K S . s o n n e i B A te s te d b a c te r ia C . fre u n d ii A .T u m e fa c ia n s

Figure 23: Antibacterial activities of the chloroform extract of stem of H. nepalensis

3.2.2.2.1 Antifungal activities of H. nepalensis

3.2.2.2.1.1 Methanol extract of leaves

Methanol extract of leaves of H. nepalensis had shown best inhibition against Alternaria alternate (35 mm) followed by Aspergillus flavus (19 mm), Penicillium notatum (14 mm) Aspergillus niger (13 mm), Trichoderma harzianum (10 mm) and the methanol extract of leaves of H. nepalensis has no effect against Candida albican.

Table 34: Antifungal activities of the methanol extract of leaves of H. nepalensis

Candida Aspergillus Aspergillus Alternaria Penicillium Trichoderma albican niger flavus alternata notatum harzianum 00 13 19 35 14 10

)

m m ( 4 0

e C a n d id a a lb ic a n n

o A s p e rg illu s n ig e r

z 3 0

A s p e rg illu s fla v u s n

o A lte rn a ria a lte rn a ta

i 2 0 t

i P e n ic illiu m n o ta tu m b

h 1 0 T ric h o d e rm a h a rz ia n u m m n r s a m i t u n e u u a a t n g v n a c i a i i a r t 0 n l z b f e l t o r s l a s n a u a l u h a l l a m i l i a d i u i g r i r g m d r a l e l r n e n i p r c e a p i s e d C s t n o A l A e h A P c i r T te s te d fu n g i Figure 24: Antifungal activities of the methanol extract of leaves of H. nepalensis

Chapter # 3 Results

3.2.2.2.1.2 Ethanol extract of Leaves

Methanol extract of leaves of H. nepalensis had shown best inhibition against Penicillium notatum (39 mm) followed by Aspergillus flavus (33 mm), Aspergillus niger (17 mm), Candida albican (14 mm) and Trichoderma harzianum (13 mm) and the methanol extract of leaves of H. nepalensis has no effect against Alternaria alternate.

Table 35: Antifungal activities of the ethanol extract of leaves of H. nepalensis

Candida Aspergillus Aspergillus Alternaria Penicillium Trichoderma albican niger flavus alternata notatum harzianum 14 17 33 00 39 13

)

m 5 0

m C a n d id a a lb ic a n

( e

4 0 A s p e rg illu s n ig e r

n o

z A s p e rg illu s fla v u s 3 0

n A lte rn a ria a lte rn a ta

o i

t 2 0

i P e n ic illiu m n o ta tu m b

h T ric h o d e rm a h a rz ia n u m

1 0 n

i r a m s t m n e u 0 a u a u g t n c i v n i a a a l r t i b n l f e o z s t r a s l n u a l u a a l l h i l a m d i i i g r u a r g i d a l e r l m n e n i r p a p r c e s e i C s t d A l n A e o A P h c i r T te s te d fu n g i

Figure 25: Antifungal activities of the ethanol extract of leaves of H. nepalensis

3.2.2.2.1.3 n-Hexane extract of leaves

Methanol extract of leaves of H. nepalensis had shown best inhibition against Aspergillus niger (36 mm) followed by Candida albican (31 mm) and Trichoderma harzianum (15 mm) and the methanol extract of leaves of H. nepalensis has no effect against Aspergillus flavus, Alternaria alternat and Penicillium notatum.

Table 36: Antifungal activities of the n-hexane extract of leaves of H. nepalensis

Candida Aspergillus Aspergillus Alternaria Penicillium Trichoderma albican niger flavus alternata notatum harzianum 31 36 00 00 00 15

Chapter) # 3 Results

( e 4 0

n C a n d id a a lb ic a n o

z A s p e rg illu s n ig e r 3 0

n A s p e rg illu s fla v u s o

i A lte rn a ria a lte rn a ta

t 2 0 i

P e n ic illiu m n o ta tu m b m h a u 1 0 r s t m T ric h o d e rm a h a rz ia n u m n n e u u n a t i a a g v n c i a i i a r t z n l b f e o r 0 l t s l n a a s u a h l u a l l i l a m a i i d r u i g i m r g l d r a l r e i n e n e p r c a p i d s e C s t n o A l A e h A P c i r T te s te d fu n g i Figure 26: Antifungal activities of the n-hexane extract of leaves of H. nepalensis

3.2.2.2.1.4 Petroleum ether extract of Leaves

Methanol extract of leaves of H. nepalensis had shown best inhibition against Trichoderma harzianum (19 mm) followed by Penicillium notatum (16 mm), Aspergillus niger (14 mm), Candida albican (11 mm) and Alternaria alternate (11 mm) and the methanol extract of leaves of H. nepalensis has no effect against Aspergillus flavus.

Table 37: Antifungal activities of the petroleum ether extract of leaves of H. nepalensis

Candida Aspergillus Aspergillus Alternaria Penicillium Trichoderma )

albicanm niger flavus alternata notatum harzianum m

11( 14 11 16 19 e

n 2 0

C a n d id a a lb ic a n

o z

A s p e rg illu s n ig e r 1 5

n A s p e rg illu s fla v u s

o i

t 1 0 A lte rn a ria a lte rn a ta i

b P e n ic illiu m n o ta tu m m

h a m u 5 r s t T ric h o d e rm a h a rz ia n u m n e u a u n n a t a i g v n i c i a i a r t z n l f e o r b t 0 l s l a s n a h u a l u a l l a m a i l i d i u i g r i m r g l d r a l r e i e n e n p r c a p i d s e n o C s t A l A e h A c P i r T te s te d fu n g i

Figure 27: Antifungal activities of the petroleum ether extract of leaves of H. nepalensis

3.2.2.2.1.5 Chloroform extract of leaves

Methanol extract of leaves of H. nepalensis had shown best inhibition against Penicillium notatum (27 mm) followed by Trichoderma harzianum (26 mm), Aspergillus niger (23 mm) and Candida albican (14 mm) and the methanol extract of leaves of H. nepalensis has no effect against Aspergillus flavus and Alternaria alternate.

Chapter # 3 Results

Table 38: Antifungal activities of the chloroform extract of leaves of H. nepalensis

Candida Aspergillus Aspergillus Alternaria Penicillium Trichoderma albican niger flavus alternata notatum harzianum 14 23 27 26

) m

3 0 m

( C a n d id a a lb ic a n e

n A s p e rg illu s n ig e r o

z 2 0 A s p e rg illu s fla v u s

n A lte rn a ria a lte rn a ta

i t

i P e n ic illiu m n o ta tu m

b 1 0

h T ric h o d e rm a h a rz ia n u m

n i a m r s t m n e u a u a u g t n 0 c i v n i a a a n l r t i b e z l f o s t r a s l n u a a l u a l l h i l a m d i i a i g r u r g i d a l m e r l n e n i r p r c e a p i s e d C s t n A l o A e A h P c i r T te s te d fu n g i

Figure 28: Antifungal activities of the chloroform extract of leaves of H. nepalensis

3.2.2.2.2 Antifungal activities of stem

3.2.2.2.2.1 Methanol extract of stem

Methanol extract of leaves of H. nepalensis had shown best inhibition against Trichoderma harzianum (17 mm)followed by Alternaria alternate (13 mm), Penicillium notatum (10 mm) and the methanol extract of leaves of H. nepalensis has no effect against Candida albican, Aspergillus niger and Aspergillus flavus.

Table 39: Antifungal activities of the methanol extract of stem of H. nepalensis

Candida Aspergillus Aspergillus Alternaria Penicillium Trichoderma albican niger flavus alternata notatum harzianum 00 00 00 13 10 17

Chapter # 3 Results

2 0

C a n d id a a lb ic a n )

m A s p e rg illu s n ig e r

1 5

m (

e A s p e rg illu s fla v u s

n o

z A lte rn a ria a lte rn a ta

1 0 n

o P e n ic illiu m n o ta tu m

t i

b 5 T ric h o d e rm a h a rz ia n u m

n i

n r s a a e u t m m c g v a u i i n t u b n la r a n l f t a a s te o i u s l n z a ll u a r d i ll a i g i ia m h d r g r u r i a n e a ll a p e n i m s p r c r C s e i e A lt n A e d A o P h ic r T te s te d fu n g i

Figure 29: Antifungal activities of the methanol extract of stem of H. nepalensis

3.2.2.2.2.2 Ethanol extract of stem

Methanol extract of leaves of H. nepalensis had shown best inhibition against Trichoderma harzianum (32 mm)followed by Alternaria alternate (14 mm) and the methanol extract of leaves of H. nepalensis has no effect against Candida albican, Aspergillus niger, Aspergillus flavus and Penicillium notatum.

Table 40: Antifungal activities of the ethanol extract of stem of H. nepalensis

Candida Aspergillus Aspergillus Alternaria Penicillium Trichoderma albican niger flavus alternata notatum harzianum 00 00 00 14 00 32

Chapter # 3 Results

)

m (

e 4 0

n C a n d id a a lb ic a n

o A s p e rg illu s n ig e r

z 3 0

A s p e rg illu s fla v u s

n o

i 2 0 A lte rn a ria a lte rn a ta

t i

P e n ic illiu m n o ta tu m b

h 1 0 m T ric h o d e rm a h a rz ia n u m a n r s t m u

i n e u a u n a t g v a c i n a i i a r t 0 n l z b f e o r l t s l n a a s u a h l u a l l a m i l i a d i u i g r i r g l m d r a l r e i n e n e p r c a p i d s e C s t n o A l A e h A P c i r T te s te d fu n g i Figure 30: Antifungal activities of the ethanol extract of stem of H. nepalensis

3.2.2.2.2.3 n-Hexane extract of Stem

Methanol extract of leaves of H. nepalensis had shown best inhibition against Aspergillus flavus (35 mm) followed by Trichoderma harzianum (17 mm), Alternaria alternate (13 mm), Penicillium notatum (11 mm) and the methanol extract of leaves of H. nepalensis has no effect against Candida albican and Aspergillus niger.

Table 41: Antifungal activities of the n-hexane extract of stem of H. nepalensis

Candida Aspergillus Aspergillus Alternaria Penicillium Trichoderma albican niger flavus alternata notatum harzianum

00 00 35 13 11 17 )

( e

4 0 n

C a n d id a a lb ic a n o

z A s p e rg illu s n ig e r 3 0

n A s p e rg illu s fla v u s o i A lte rn a ria a lte rn a ta

t 2 0 i

Figure 31: Antifungal activities of the n-hexane extract of stem of H. nepalensis

3.2.2.2.2.4 Petroleum ether extract of Stem

Methanol extract of leaves of H. nepalensis had shown best inhibition against Alternaria alternate (37 mm), Trichoderma harzianum (22 mm) and Aspergillus flavus (13

Chapter # 3 Results mm), and the methanol extract of leaves of H. nepalensis has no effect against Candida albican, Aspergillus niger and Penicillium notatum.

Table 42: Antifungal activities of the petroleum ether extract of stem of H. nepalensis

Candida Aspergillus Aspergillus Alternaria Penicillium Trichoderma albican niger flavus alternata notatum harzianum 00 00 13 37 00 22

)

m ( 4 0

e C a n d id a a lb ic a n n

o A s p e rg illu s n ig e r

z 3 0

A s p e rg illu s fla v u s n

o A lte rn a ria a lte rn a ta

i 2 0 t

i P e n ic illiu m n o ta tu m b

h 1 0 T ric h o d e rm a h a rz ia n u m m

n a r s m u

i t n e u u a a t n g v a c i n a i i a r t 0 n l z b f e o l t r s l n a a s u a h l u a l l a m i l i a d i u i g r i r g l m d r a l e i r n e n p r c e a p i s e d C s t n o A l A e h A P c i r T te s te d fu n g i Figure 32: Antifungal activities of the petroleum ether extract of stem of H. nepalensis

3.2.2.2.2.5 Chloroform extract of stem

Methanol extract of leaves of H. nepalensis had shown best inhibition against Candida albican (26 mm)followed by Alternaria alternate (16 mm) and Trichoderma harzianum (12 mm) and the methanol extract of leaves of H. nepalensis has no effect against Aspergillus niger, Aspergillus flavus and Penicillium notatum.

Table 43: Antifungal activities of the chloroform extract of stem of H. nepalensis

Candida Aspergillus Aspergillus Alternaria Penicillium Trichoderma albican niger flavus alternata notatum harzianum 26 16 12

Chapter # 3 Results

3 0 )

C a n d id a a lb ic a n

m m

( A s p e rg illu s n ig e r e

n 2 0 A s p e rg illu s fla v u s

o z

A lte rn a ria a lte rn a ta

n o

i P e n ic illiu m n o ta tu m t

i 1 0

b T ric h o d e rm a h a rz ia n u m

n i

0 r s a n e t m m a u a u u c ig v t i a n a n n l r t a lb f e i s t o z a s l n r lu a a l lu a d i l a m h i g i i d r g r iu a n e r a ll e n i m a p r r s p ic C s e e A lt n d A e A o P h ic r T te s te d fu n g i

Figure 33: Antifungal activities of the chloroform extract of stem of H. nepalensis

3.2.2.3 Antimicrobial activities of standard antibiotics:

The standard antibiotic tested against the Gram positive bacteria was Azithromycin (Am) in a concentration of 55µg/6µL, Ciprofloxacin (Cf) was used in the concentration of 35µg/6µL against Gram negative bacteria, while Clotrimazole (Cm) was used in the concentration of 55µg/6µL against fungi. These anitbiotics were applied 6µL/disc on separate plates as positive control for the said organisms respectively. Azithromycin showed 30 mm, 22 mm, 27 mm, 23 mm, 18 mm, 26 mm, 33 mm, 41 mm and 34 mm against Erwinia cartovara, Staphylococcus aureus, Bacillus subtilis, Bacillus atrpenses, Bacillus cereus, Enterococcus faecalis, Shigella sonnei, Citrobacter freundii and Agrobacterium Tumefacians respectively.

While Clotrimzole showed 33 mm, 42 mm, 35 mm, 30 mm, 24 mm and 28 mm against Candida albican, Aspergillus niger, Aspergillus flavus, Alternaria alternate, Penicillium notatum and Trichoderma harzianum respectively.as shown in Table and graph.

Table 44: Antibacterial activities of the czithromycin (Am) and ciprofloxacin (Cf)

E.coli P. S. typhi K. E. cartovara S. B. B. aeruginos pheumoni aureu subtili atrpense a a s s s 44 32 55 25 30 22 27 23 B.cereu E. S. C. A.Tumefacian s faecalis sonne freundii s i 18 26 33 41 34

Chapter # 3 Results

)

m m

( 6 0

e C f n

C f

o z

4 0 C f n

o C f

i t i A m

b 2 0

h A m n i A m s a a i a s i n s i s s i i 0 n r s i e s i a i o l l e d i A m l h o a u i s u n v t a n n c oi p e n e m r b c n a c y o e r u f . g t t u e o A m u r u p e e e E u . a a s r r e a s r c f f S . t . . e h c . . . m a B A m a p . S B S u . E C . . T E B . P K A m A te s te d B a c te r ia A m

A m

Figure 34: Antibacterial activities of the Azithromycin (Am) and Ciprofloxacin (Cf)

Table 45: Antifungal activities of the clotrimazole (Cm) )

Candidam Aspergillus Aspergillus Alternaria Penicillium Trichoderma

albicanm niger flavus alternata notatum harzianum (

33e 42 35 30 24 28 n

o 5 0

z C m

4 0 C m n

o C m i

3 0 t

i C m m u b 2 0 a m r s t C m n n u u h e a t a a g v n i C m i a n 1 0 c a r t z i

i l r n e f o b t a l s l n s h a a 0 u u l a a l l a m i l i i d u g r i m i g r a l r d r l e i e e n n r c d p i a p s e o s t n C l A e h A A c P i r T T e s te d F u n g i

Figure 35 : Antifungal activities of the clotrimazole (Cm

Chapter 3 Results Results

3.3 Pharmacological Evaluation of the Crude extract of H. nepalensis

Diabetes mellitus is a disease of various factors which has great impact on health, quality of life and prospect of patients and on the health maintenance system. From 1997 to 2010, 221 million people have been reported with diabetes (Amos et al., 2010). According to 2008 data, 230 million people have diabetes globally (Arumugam et al., 2008). This disease results from damage cells of the Langerhan islets which make the body to produce pancreatic hypoglycemic hormone called insulin. Three main signs of this disease are: excessive urine called Polyuria, glycosauria (glucose presence in urine) and hyperglycemia (high glucose level in blood) (Koffi et al., 2009). Glucose and lipid metabolism is changed due to diabetes (Rajasekaran et al., 2006). Changes in lipid metabolism have been proved experimentally (Sochar et al., 1985). Diabetus mellitus and obesity are co-related (Afridi and Khan, 2009). Liver depends on insulin and take part in oxidation and metabolic transformation of fatty acids, creation of cholesterol, phospholipids, and triglycerides (Seifter et al., 1982). H. nepalensis leaves were taken to check anti diabetic activity and hepatoprotective activity. So Glucose, Triglyceride, Cholesterol, Bilirubin Total, Proteins Total, Albumin, Creatinine, ALP, GGT, ALT and AST were studied for this purpose. The project aims to reduce Diabetes mellitus by herbal medicines. This is because of the issuing problem in our society.

3.3.1 Toxicological Studies:

Determination of LD50 of Hedera nepalensis: a- Aqueous Extract: No mortalities in mice following oral administration of aqueous extract of H. nepalensis in doses up to 5 g/kg b.wt. b- Ethanolic Extract: The following mwthod was used and the symptoms of toxicity were pigeonholed by rapid and shallow respiration (gasping), high heart rate, shrinkages in abdominal wall, tremors and convulsions followed by recumbence, coma and death (Buck et al., 1976).

Chapter 3 Results Results

Table 46: LD50 of ethanolic extract of Hedera nepalensis in rabbits Group number of Dose Number of z d ∑ (z × d) animals/ (mg/kg dead group b.wt.) animals/ group

1 9 4000 0 0 0 0 2 9 6000 2 1 2000 2000

3 9 8000 5 3.5 2000 7000

4 9 10000 7 6 2000 12000

5 9 12000 9 8 2000 16000

Total 37000

z = Mean of dead animals between 2 successive groups. d = Difference between 2 successive doses.

LD50 = Dm - ∑ (z×d)/n

LD50 = 12000-37000/9 = 7888.88 mg/kg b.wt. = 7.88 g/kg b.wt.

3.3.2 Biochemical blood analysis of rabbit in respect of leaves

3.3.2.1.1 Glucose level

Glucose normal value in the blood is 65-105 mg/dl. Alloxane was administered to all the groups except untreated group. Untreated group’s blood glucose level was recorded (98.00±1.0 — 92.66±2.51) as normal. Initially there was hypoglycemia which was countered by infusion of normal saline through I/V and administration of glucose in water. A significant increase (p<0.0001) was recorded in glucose level at day 3. After induction of diabetes, group B was kept untreated diabetic control (normal control). The glucose level remained high (287±1.0 — 430.66±7.02) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (109.33±1.52) was recorded at the end of the treatment. Group D was given with Vitamin C

Chapter 3 Results Results at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of medication the glucose level was high (275.33±5.68 mg/dl). These were a significant increase in glucose level (432.66±0.52 mg/dl) at the end of administration period showing that Vitamin C has no counter effect on glucose. Group E was medicated with extract of H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the glucose level was high (283±1.00 mg/dl). These were a significant decrease in glucose level (110.33±1.52 mg/dl) at the end of administration period. Group F was given with 300mg/dl plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of glucose (280±1.00 mg/dl). The glucose level tended to decrease towards normal as the administration period extended. There was a significant decrease in glucose level (111.33±1.52 mg/dl) at the end of administration with plant extract. A 400mg/kg plant extract was administered orally to group G. Initially there was a high level of glucose (275±1.52 mg/dl). A noteworthy fall in glucose level (101±1.00 mg/dl) was recorded at the end of administration. The result revealed that the extract has significant antidiabetic activity at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 47.

Table 47: Blood glucose (mg/dl) level of rabbits in triplicate

0 day 7th day Untreated control 98.0000 97.0000 99.0000 97.0 98.0 96.0 Diabetic control 287.0000 289.0000 285.0000 350.0 345.0 352.0 Diabetic control + Glibenclamide (10mg/kg) 280.0000 279.0000 281.0000 165.0 164.0 166.0 Diabetic control + Vitamin C (Positive Control) 269.0000 277.0000 280.0000 300.0 307.0 310.0 Diabetic + Ethanolic extract of H.N 200mg/kg 284.0000 282.0000 283.0000 230.0 228.0 227.0 Diabetic + Ethanolic extract of H.N 300mg/kg 281.0000 280.0000 279.0000 250.0 248.0 251.0 Diabetic + Ethanolic extract of H.N 400mg/kg 274.0000 275.0000 277.0000 210.0 211.0 208.0

Chapter 3 Results Results

14th day 21st day 28th day 96. 95. 93. 94. 91. 92. 93. 95. 90.

380. 378. 385. 410. 412. 410. 430. 424. 438. 140. 144. 139. 110. 114. 111. 109. 108. 111. 342. 341. 344. 397. 399. 400. 432. 433. 433. 150. 147. 151. 115. 117. 115. 112. 110. 109.

140. 138. 142. 112. 110. 109. 111. 110. 113. 120. 117. 119. 109. 109. 110. 101. 100. 102.

Table 48: Mean blood glucose (mg/dl) level of rabbits with standard deviation

0 day 7th day

Untreated control 98.000 1.000 3 97.000 1.000 3

Diabetic control 287.000 2.000 3 349.000 3.605551 3

Diabetic control + Glibenclamide (10mg/kg) 280.000 1.000 3 165.000 1.000 3

Diabetic control + Vitamin C (Positive Control) 275.3333 5.686241 3 305.6667 5.131601 3

Diabetic + Ethanolic extract of H.N 200mg/kg 283.000 1.000 3 228.3333 1.527525 3

Diabetic + Ethanolic extract of H.N 300mg/kg 280.000 1.000 3 249.6667 1.527525 3

Diabetic + Ethanolic extract of H.N 400mg/kg 275.3333 1.527525 3 209.6667 1.527525 3

14th day 21st day 28th day 94.66666 1.527525 3 92.33334 1.527525 3 92.66666 2.516612 3 381.000 3.605551 3 410.6667 1.154701 3 430.6667 7.023769 3 141.000 2.645751 3 111.6667 2.081666 3 109.3333 1.527525 3 342.3333 1.527525 3 398.6667 1.527525 3 432.6667 0.5773503 3 149.3333 2.081666 3 115.6667 1.154701 3 110.3333 1.527525 3 140.000 2.000 3 110.3333 1.527525 3 111.3333 1.527525 3 118.6667 1.527525 3 109.3333 0.5773503 3 101.000 1.000 3

Chapter 3 Results Results

From the graph, it is clear that glucose level significantly increased at day 28. The graph has

been shown in figure 36.

)

d /

g 5 0 0 m

( 0 d a y r

a 7 th d a y

g 4 0 0 u

s 1 4 th d a y

d 3 0 0 2 1 s t d a y

o o

l 2 8 th d a y b

2 0 0

i t

s 1 0 0

a 0 e y y y y y M a a a a a d d d d d h t h 0 th t s t 7 4 1 8 1 2 2 T im e (d a y s )

Figure 36: Blood glucose level of rabbits

3.3.2.1.2 Triglyceride level

Triglyceride normal value in the blood is 60-73 mg/dl. Group A (Untreated group) blood triglyceride level war recorded (73.00±2.00 — 86.33±.57) as normal. Group B was kept untreated diabetic control (Normal control). The triglyceride level remained high (91.00±1.00 — 105.33±1.52) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (75.66±1.52) was recorded at the end of the treatment. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the triglyceride level was high (89.33±0.57 mg/dl). These were a significant increase in triglyceride level (99.33±0.57 mg/dl) at the end of administration period showing that Vitamin C has no counter effect on triglyceride. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the triglyceride level was high (81.33±1.15 mg/dl). These were a significant decrease in triglyceride level (78.00±1.00 mg/dl) at the end of administration period. Group F was given

Chapter 3 Results Results with 300 mg/dl plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of triglyceride (77.66±2.51 mg/dl). The triglyceride level tended to decrease towards normal as the administration period extended. There was a noteworthy dfall in triglyceride level (72.66±3.05 mg/dl) at the end of administration with plant extract. A 400mg/kg plant extract was administered orally to group G. Initially there was a high level of triglyceride (76.00±1.00 mg/dl). A significant decrease in triglyceride level (66.00±1.00 mg/dl) was recorded at the end of administration. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 49.

Table 49: Blood triglyceride (mg/dl) level of rabbits in triplicate

0 day 7th day

Untreated control 73.0000 75.0000 71.0000 71.0 70.0 72.0

Diabetic control 90.0000 92.0000 91.0000 97.0 98.0 98.0 Diabetic control + Glibenclamide (10mg/kg) 85.0000 80.0000 83.0000 86.0 85.0 84.0 Diabetic control + Vitamin C (Positive Control) 89.0000 89.0000 90.0000 88.0 88.0 87.0 Diabetic + Ethanolic extract of H.N 200mg/kg 82.0000 80.0000 82.0000 84.0 81.0 83.0

Diabetic + Ethanolic extract of H.N 300mg/kg 78.0000 75.0000 80.0000 76.0 77.0 78.0

Diabetic + Ethanolic extract of H.N 400mg/kg 75.0000 76.0000 77.0000 74.0 75.0 74.0

14th day 21st day 28th day 70. 69. 68. 72. 70. 71. 68. 68. 69. 99. 100. 101. 104. 100. 102. 107. 105. 104. 84. 82. 81. 81. 79. 78. 76. 74. 77. 94. 94. 94. 98. 95. 96. 99. 99. 100. 81. 80. 84. 80. 78. 79. 78. 77. 79. 75. 78. 70. 75. 70. 75. 72. 70. 76. 72. 70. 71. 71. 70. 68. 67. 66. 65.

Chapter 3 Results Results

Table 50: Mean blood triglyceride (mg/dl) level of rabbits with standard deviation

0 day 7th day

Untreated control 73.000 2.000 3 71.000 1.000 3 Diabetic control 91.000 1.000 3 97.66666 0.5773503 3 Diabetic control + Glibenclamide (10mg/kg) 82.66666 2.516612 3 85.000 1.000 3 Diabetic control + Vitamin C (Positive Control) 89.33334 0.5773503 3 87.66666 0.5773503 3 Diabetic + Ethanolic extract of H.N 200mg/kg 81.33334 1.154701 3 82.66666 1.527525 3 Diabetic + Ethanolic extract of H.N 300mg/kg 77.66666 2.516612 3 77.000 1.000 3 Diabetic + Ethanolic extract of H.N 400mg/kg 76.000 1.000 3 74.33334 0.5773503 3

14th day 21st day 28th day 69.000 1.000 3 71.000 1.000 3 68.33334 0.5773503 3 100.000 1.000 3 102.000 2.000 3 105.3333 1.527525 3 82.33334 1.527525 3 79.33334 1.527525 3 75.66666 1.527525 3 94.000 0.000 3 96.33334 1.527525 3 99.33334 0.5773503 3 81.66666 2.081666 3 79.000 1.000 3 78.000 1.000 3 74.33334 4.041452 3 73.33334 2.886751 3 72.66666 3.05505 3 71.000 1.000 3 69.66666 1.527525 3 66.000 1.000 3

From the graph, it is clear that triglyceride level significantly increased at day 28. The graph

has been shown in figure 37.

)

g m

( 1 5 0 e

d 2 8 th d a y

i r

e 2 1 s t d a y

c y

l 1 4 th d a y

1 0 0

g i

r 7 th d a y

d 0 d a y

o o

l 5 0

s a

f 0

n y y y y y a a a a a a e d d d d d 0 h h t h

M t t s t 7 4 1 8 1 2 2 T im e (d a y s )

Figure 37: Blood triglyceride level of rabbits

Chapter 3 Results Results

3.3.2.1.3 Cholesterol level

Cholesterol normal value in the blood is 60-73 mg/dl. Group A (untreated group) blood cholesterol level war recorded (110.33±2.51 — 119.33±2.08) as normal. Group B was kept untreated diabetic control (normal control). The cholesterol level remained high (211.33±2.08 — 281.66±2.08) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (117.00±5.56) was recorded at the end of the treatment. Group D was given with Vitamin Cat the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the cholesterol level was high (210.66±3.05 mg/dl). These were a significant increase in cholesterol level (251.66±8.32 mg/dl) at the end of administration period showing that Vitamin C has no counter effect on cholesterol. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of medication the cholesterol level was high (186.33±3.78 mg/dl). These were a noteworthy fall in cholesterol level (184.00±4.58 mg/dl) at the end of administration period. Group F was given with 300mg/dl plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of cholesterol (173.33±3.05 mg/dl). The cholesterol level tended to decrease towards normal as the administration period extended. There was a significant decrease in cholesterol level (116.33±3.05 mg/dl) at the end of administration with plant extract. A 400mg/kg plant extract was administered orally to group G. Initially there was a high level of cholesterol (163.33±2.51 mg/dl). A significant decrease in cholesterol level (105.33±2.51 mg/dl) was recorded at the end of administration. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 51.

Chapter 3 Results Results

Table 51: Blood triglyceride (mg/dl) level of rabbits in triplicate

0 day 7th day 110.000 108.000 113.000 Untreated control 0 0 0 114.0 112.0 115.0 213.000 209.000 212.000 Diabetic control 0 0 0 224.0 226.0 227.0 161.000 155.000 156.000 Diabetic control + Glibenclamide (10mg/kg) 0 0 0 154.0 152.0 155.0 Diabetic control + Vitamin C (Positive 214.000 208.000 210.000 Control) 0 0 0 201.0 200.0 203.0 Diabetic + Ethanolic extract of H.N 188.000 182.000 189.000 200mg/kg 0 0 0 171.0 174.0 177.0 Diabetic + Ethanolic extract of H.N 170.000 174.000 176.000 300mg/kg 0 0 0 152.0 156.0 157.0 Diabetic + Ethanolic extract of H.N 166.000 163.000 161.000 400mg/kg 0 0 0 144.0 140.0 147.0

14th day 21st day 28th day

117. 113. 115. 113. 110. 111. 120. 117. 121.

234. 237. 240. 265. 262. 261. 284. 280. 281.

134. 131. 130. 121. 119. 129. 111. 118. 122.

211. 214. 211. 240. 233. 234. 261. 245. 249.

167. 169. 167. 181. 180. 185. 180. 183. 189.

143. 140. 139. 123. 119. 120. 113. 117. 119.

134. 131. 134. 120. 118. 116. 108. 105. 103.

Table 52: Mean blood triglyceride (mg/dl) level of rabbits with standard deviation

0 day 7th day 110.333 2.51661 113.666 1.52752 Untreated control 3 2 3 7 5 3 211.333 2.08166 225.666 1.52752 Diabetic control 3 6 3 7 5 3 157.333 153.666 1.52752 Diabetic control + Glibenclamide (10mg/kg) 3 3.21455 3 7 5 3 Diabetic control + Vitamin C (Positive 210.666 201.333 1.52752 Control) 7 3.05505 3 3 5 3 186.333 3.78593 Diabetic + Ethanolic extract of H.N 200mg/kg 3 9 3 174.000 3.000 3 173.333 2.64575 Diabetic + Ethanolic extract of H.N 300mg/kg 3 3.05505 3 155.000 1 3 163.333 2.51661 143.666 3.51188 Diabetic + Ethanolic extract of H.N 400mg/kg 3 2 3 7 5 3

Chapter 3 Results Results

14th day 21st day 28th day 115.000 2.000 3 111.3333 1.527525 3 119.3333 2.081666 3 237.000 3.000 3 262.6667 2.081666 3 281.6667 2.081666 3 131.6667 2.081666 3 123.000 5.291503 3 117.000 5.567764 3 212.000 1.732051 3 235.6667 3.785939 3 251.6667 8.326664 3 167.6667 1.154701 3 182.000 2.645751 3 184.000 4.582576 3 140.6667 2.081666 3 120.6667 2.081666 3 116.3333 3.05505 3 133.000 1.732051 3 118.000 2.000 3 105.3333 2.516612 3

From the graph, it is clear that Cholesterol level significantly increased at day 28. The graph

has been shown in figure 38.

)

(

o r

t 3 0 0

s 0 d a y

e l

o 7 th d a y h 1 4 th d a y

C 2 0 0

d 2 1 s t d a y

o o

l 2 8 th d a y b

1 0 0

a f

0 n

a y y y y y a a a e a a d d d d d t M h h h 0 t t s t 7 4 1 8 1 2 2 T im e (d a y s )

Figure 38: Blood cholesterol level of rabbits

3.3.2.1.4 Total Bilirubin level

Total Bilirubin normal value in the blood is 8.16-8.56 mg/L. Group A (untreated group) blood Total Bilirubin level war recorded (8.16±0.20 mg/L — 8.2±0.15 mg/L) as normal. Group B was kept untreated diabetic control (normal control). The Total Bilirubin level remained high (24.13±1.53 mg/L — 29.66±.60 mg/L) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (6.43±.32 mg/L) was recorded at the end of the treatment. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the Total Bilirubin level was high (19.56±0.37 mg/L). These were

Chapter 3 Results Results a significant decrease in Total Bilirubin level (17.63±0.72 mg/L) at the end of administration period showing that Vitamin C has counter effect on Total Bilirubin. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the Total Bilirubin level was high (22.03±0.60 mg/L). These were a significant decrease in Total Bilirubin level (15.63±1.23 mg/L) at the end of administration period. Group F was given with 300 mg/L plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of Total Bilirubin (16.76±0.80 mg/L). The Total Bilirubin level tended to decrease towards normal as the administration period extended. There was a noteworthy fall in Total Bilirubin level (7.90±0.59 mg/L) at the end of administration with plant extract. A 400 mg/kg plant extract was administered orally to group G. Initially there was a high level of Total Bilirubin (14.06±0.55 mg/L). A significant decrease in Total Bilirubin level (5.36±0.20 mg/L) was recorded at the end of administration. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 53.

Table 53: Blood total bilirubin (mg/dl) level of rabbits in triplicate

0 day 7th day

A. Untreated control (Normal control) 8.4000 8.1000 8.0000 8.8 8.2 8.5 B. Diabetic control (Negative control) 24.7000 22.4000 25.3000 27.3 25.0 26.4 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 19.3000 18.5000 19.1000 17.5 17.0 17.4 D. Diabetic control + Vitamin C (Positive control) 20.0000 19.4000 19.3000 19.0 18.4 17.8 E. Diabetic + Ethanolic extract of H.N 200mg/kg 22.6000 21.4000 22.1000 20.7 19.3 19.0 F. Diabetic + Ethanolic extract of H.N 300mg/kg 17.6000 16.0000 16.7000 13.6 12.5 12.0 G. Diabetic + Ethanolic extract of H.N 400mg/kg 14.1000 13.5000 14.6000 12.7 11.0 12.5

14th day 21st day 28th day 9.0 8.8 7.9 8.3 8.0 8.7 8.2 8.4 8.1 28.6 28.2 28.0 29.2 26.0 27.4 30.3 29.6 29.1 13.2 12.0 13.0 9.1 10.5 9.0 6.8 6.3 6.2 20.3 17.0 16.7 17.2 16.3 16.0 18.1 16.8 18.0 18.4 18.0 17.4 16.7 15.3 15.8 15.3 14.6 17.0 11.8 11.0 12.3 9.4 8.4 8.5 7.3 7.9 8.5 10.5 10.0 10.6 7.3 7.0 7.1 5.6 5.3 5.2

Chapter 3 Results Results

Table 54: Mean blood total bilirubin (mg/dl) level of rabbits with standard deviation

0 day 7th day

A. Untreated control (Normal control) 8.166667 0.2081663 3 8.500 0.3000002 3 B. Diabetic control (Negative control) 24.13333 1.530795 3 26.23333 1.159022 3 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 18.96667 0.416333 3 17.300 0.2645751 3 D. Diabetic control + Vitamin C (Positive control) 19.56667 0.3785943 3 18.400 0.6000004 3 E. Diabetic + Ethanolic extract of H.N 200mg/kg 22.03333 0.6027718 3 19.66667 0.9073778 3 F. Diabetic + Ethanolic extract of H.N 300mg/kg 16.76667 0.8020808 3 12.700 0.8185355 3 G. Diabetic + Ethanolic extract of H.N 400mg/kg 14.06667 0.5507572 3 12.06667 0.9291573 3

14th day 21st day 28th day 8.566667 0.5859465 3 8.333333 0.3511884 3 8.233334 0.1527522 3 28.26667 0.3055052 3 27.53333 1.604162 3 29.66667 0.6027708 3 12.73333 0.64291 3 9.533334 0.8386496 3 6.433333 0.3214552 3 18.000 1.997498 3 16.500 0.6245003 3 17.63333 0.7234184 3 17.93333 0.5033223 3 15.93333 0.7094602 3 15.63333 1.234234 3 11.700 0.655744 3 8.766666 0.5507569 3 7.900 0.5999999 3 10.36667 0.3214552 3 7.133333 0.1527526 3 5.366667 0.2081666 3

From the graph, it is clear that Total Bilirubin level significantly increased at day 28. The graph has been shown in figure 39.

4 0

0 d a y

) L / 7 th d a y

g 3 0 m

( 1 4 th d a y

i 2 1 s t d a y b

u 2 0

r 2 8 th d a y

a 1 0

o T

y y y y y a a a a a d d d d d 0 t th th s th 7 4 1 8 1 2 2 T im e (d a y s )

Figure 39: Blood total bilirubin level of rabbits

Chapter 3 Results Results

3.3.2.1.5 Total protein level

Total Protein normal value in the blood is 6.1-6.5 g/dL. Group A (untreated group) blood Total Protein level war recorded (6.00±0.09 g/dL — 6.60±0.09 g/dL) as normal. Group B was kept untreated diabetic control (normal control). The Total Protein level remained high (4.10±0.09 g/dL — 1.63±0.15 g/dL) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant increase of (6.66± 0.05 g/dL) was recorded at the end of the treatment showing that allopathic medicine (Glucophage Metformin HCl has no counter effect on Total Protein. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the Total Protein level was high (3.40±0.10 g/dL). These were a significant increase in Total Protein level (4.80±0.10 g/dL) at the end of administration period showing that Vitamin C has no counter effect on Total Protein. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the Total Protein level was high (4.2±0.10 g/dL). These were a noteworthy rise in Total Protein level (5.03±0.05 g/dL) at the end of medication period. Group F was given with 300 mg/L plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of Total Protein (5.43±0.15 g/dL). The Total Protein level tended to decrease towards normal as the administration period extended. There was a significant decrease in Total Protein level (6.20±0.10 g/dL) at the end of administration with plant extract. A 400mg/kg plant extract was administered orally to group G. Initially there was a high level of Total Protein (6.00±0.19 g/dL). A significant decrease in Total Protein level (6.63±0.15 g/dL) was recorded at the end of administration. The result revealed that the extract has no significant role even at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 55.

Chapter 3 Results Results

Table 55: Blood total protein (g/dl) level of rabbits in triplicate

0 day 7th day

Untreated control 6.1000 6.0000 5.9000 6.5 6.6 6.7 Diabetic control 4.1000 4.0000 4.2000 3.8 3.5 3.7 Diabetic control + Glibencamide (10mg/kg) 6.0000 6.1000 6.3000 6.2 6.5 6.4 Diabetic + Ethanolic extract of H.N 200mg/kg 4.2000 4.1000 4.3000 4.3 4.4 4.5 Diabetic + Ethanolic extract of H.N 300mg/kg 5.3000 5.4000 5.6000 5.5 5.5 5.6 Diabetic + Ethanolic extract of H.N 400mg/kg 6.0000 5.8000 6.2000 6.2 6.0 6.1

14th day 21st day 28th day 6.3 6.4 6.2 6.0 5.9 6.2 6.6 6.7 6.5 3.1 3.3 3.2 2.6 2.5 2.6 1.5 1.6 1.8 6.3 6.5 6.6 6.5 6.5 6.6 6.7 6.6 6.7 4.5 4.6 4.6 4.9 4.8 5.0 5.0 5.0 5.1 5.8 5.7 5.6 6.0 6.1 6.4 6.3 6.1 6.2 6.5 6.6 6.8 6.6 6.8 6.6 6.8 6.5 6.6

Table 56: Mean blood total protein (g/dl) level of rabbits with standard deviation

0 day 7th day

A. Untreated control 6.000 0.09999991 3 6.600 0.09999991 3

B. Diabetic control 4.100 0.09999991 3 3.666667 0.1527525 3 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 6.133333 0.1527526 3 6.366667 0.1527526 3 D. Diabetic control + Vitamin C (Positive control) 3.400 0.100 3 3.566667 0.05773497 3 E. Diabetic + Ethanolic extract of H.N 200mg/kg 4.200 0.1000001 3 4.400 0.09999991 3 F. Diabetic + Ethanolic extract of H.N 300mg/kg 5.433333 0.1527524 3 5.533333 0.05773497 3 G. Diabetic + Ethanolic extract of H.N 400mg/kg 6.000 0.1999998 3 6.100 0.09999991 3

Chapter 3 Results Results

14th day 21st day 28th day 6.300 0.1000001 3 6.033333 0.1527524 3 6.600 0.09999991 3 3.200 0.100 3 2.566667 0.05773497 3 1.633333 0.1527525 3 6.466667 0.1527524 3 6.533333 0.05773497 3 6.666667 0.05773497 3 3.600 0.3605551 3 4.666667 0.4163334 3 4.800 0.1000001 3 4.566667 0.05773497 3 4.833334 0.305505 3 5.033333 0.05773497 3 5.700 0.1000001 3 6.166667 0.2081667 3 6.200 0.1000001 3 6.633333 0.1527526 3 6.666667 0.1154702 3 6.633333 0.1527526 3

From the graph, it is clear that Total Protein level significantly increased at day 28. The graph

has been shown in figure 40.

)

(

n 8 i

e 0 d a y

t o

r 7 th d a y P

l 1 4 th d a y

a t

o 2 1 s t d a y T 4

d 2 8 th d a y

l b

s a

f 0

n y y y y y a a a a a a e d d d d d 0 h h t h M t t s t 7 4 1 8 1 2 2 T im e (d a y s )

Figure 40: Blood total protein level of rabbits

3.3.2.1.6 Albumin level

Albumin normal value in the blood is 4.1-4.4 g/dL. Group A (untreated group) blood Albumin level war recorded (4.03±0.57 g/dL — 4.06±0.05 g/dL) as normal. Group B was kept untreated diabetic control (normal control). The Albumin level remained high (2.20±0.17 g/dL — 1.10±0.10 g/dL) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero

Chapter 3 Results Results day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant increase of (4.50± 0.09 g/dL) was recorded at the end of the treatment showing that allopathic medicine (Glucophage Metformin HCl has no counter effect on Albumin. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the Albumin level was high (3.23±0.15 g/dL). These were a significant increase in Albumin level (4.20±0.26 g/dL) at the end of administration period showing that Vitamin C has counter effect on Albumin. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the Albumin level was high (3.70±0.87 g/dL). These were a significant decrease in Albumin level (3.2±0.10 g/dL) at the end of administration period. Group F was givenwith 300 mg/L plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day day after induction of diabetes. Initially there was a high level of Albumin (2.63±0.15 g/dL). The Albumin level tended to decrease towards normal as the administration period extended. There was a noteworthy rise in Albumin level (3.86±0.15 g/dL) at the end of administration with plant extract. A 400mg/kg plant extract was administered orally to group G. Initially there was a high level of Albumin (2.93±0.05 g/dL). A significant increase in Albumin level (4.76±0.11 g/dL) was recorded at the end of administration. The result revealed that the extract has no significant role even at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 57.

Table 57: Blood albumin (g/dl) level of rabbits in triplicate

0 day 7th day A. Untreated control (Normal control) 4.1000 4.0000 4.0000 4.0 4.2 3.9 B. Diabetic control (Negative control) 2.3000 2.3000 2.0000 2.0 2.2 2.1 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 3.1000 3.0000 3.0000 3.3 3.1 3.1 D. Diabetic control + Vitamin C (Positive control) 3.4000 3.1000 3.2000 3.4 3.0 3.4 E. Diabetic + Ethanolic extract of H.N 200mg/kg 2.7000 4.1000 4.3000 2.8 2.0 2.3 F. Diabetic + Ethanolic extract of H.N 300mg/kg 2.8000 2.5000 2.6000 3.0 3.1 3.1 G. Diabetic + Ethanolic extract of H.N 400mg/kg 3.0000 2.9000 2.9000 3.7 3.3 3.0

Chapter 3 Results Results

14th day 21st day 28th day 4.0 4.0 4.4 4.3 4.0 4.0 4.0 4.1 4.1 1.7 1.5 1.7 1.3 1.4 1.6 1.1 1.0 1.0 3.4 3.0 3.0 3.9 3.4 3.5 4.5 4.4 4.6 3.3 3.3 3.0 3.9 3.7 3.7 4.1 4.0 4.5 2.8 2.7 2.3 3.0 2.7 2.7 3.3 3.1 3.2 3.0 2.8 2.7 3.3 3.0 3.0 3.7 3.9 4.0 3.9 3.7 3.7 4.3 4.0 4.0 4.9 4.7 4.7

Table 58: Mean blood albumin (g/dl) level of rabbits with standard deviation

0 day 7th day

A. Untreated control (Normal control) 4.033333 0.05773497 3 4.033333 0.1527524 3

B. Diabetic control (Negative control) 2.200 0.1732051 3 2.100 0.100 3

C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 3.033333 0.05773497 3 3.166667 0.1154701 3

D. Diabetic control + Vitamin C (Positive control) 3.233333 0.1527526 3 3.266667 0.2309402 3

E. Diabetic + Ethanolic extract of H.N 200mg/kg 3.700 0.8717798 3 2.366667 0.4041452 3

F. Diabetic + Ethanolic extract of H.N 300mg/kg 2.633333 0.1527525 3 3.066667 0.05773497 3

G. Diabetic + Ethanolic extract of H.N 400mg/kg 2.933333 0.05773497 3 3.333333 0.3511885 3

14th day 21st day 28th day 4.133333 0.2309402 3 4.100 0.1732052 3 4.066667 0.05773497 3 1.633333 0.1154701 3 1.433333 0.1527526 3 1.100 0.100 3 3.133333 0.2309402 3 3.600 0.2645752 3 4.500 0.09999991 3 3.200 0.1732051 3 3.766667 0.1154701 3 4.200 0.2645752 3 2.600 0.2645752 3 2.800 0.1732051 3 3.200 0.100 3 2.833333 0.1527525 3 3.100 0.1732051 3 3.866667 0.1527525 3 3.766667 0.1154701 3 4.100 0.1732052 3 4.766666 0.1154702 3 From the graph, it is clear that Albumin level significantly increased at day 28. The graph has been shown in figure 41.

Chapter 3 Results Results

6 0 d a y 7 th d a y

l 1 4 th d a y

d 4

/ g

2 1 s t d a y

n i

m 2 8 th d a y

b l

l 2 A

y y y y y a a a a a d d d d d 0 t th th s th 7 4 1 8 1 2 2 T im e (d a y s )

Figure 41: Blood albumin level of rabbits

3.3.2.1.7 Globulin level

Globulin normal value in the blood is 2.9 - 4.9 g/dL. Group A (untreated group) blood Albumin level war recorded (1.97 g/dL — 2.54 g/dL) as normal. Group B was kept untreated diabetic control (normal control). The Albumin level remained high (1.90 g/dL — 0.60 g/dL) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (2.14 g/dL) was recorded at the end of the treatment showing that allopathic medicine (Glucophage Metformin HCl has no counter effect on Albumin. Group D was given with Vitamin Cat the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the Albumin level was high (0.17 g/dL). These were a significant increase in Albumin level (0.60 g/dL) at the end of administration period showing that Vitamin C has no counter effect on Albumin. Group E was medicated with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the Albumin level was high (0.50 g/dL). These were a significant decrease in Albumin level (1.83 g/dL) at the end of administration period. Group F was given with 300 mg/L plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of Albumin (2.80 g/dL). The Albumin level tended to decrease towards normal as the administration period extended. There was a significant

Chapter 3 Results Results decrease in Albumin level (2.34 g/dL) at the end of administration with plant extract. A 400mg/kg plant extract was administered orally to group G. Initially there was a high level of Albumin (3.07 g/dL). A noteworthy fall in Albumin level (1.84 g/dL) was recorded at the end of administration. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day c have been given in table 59.

Table 59: Blood globulin (g/dl) level of rabbits

0 day 7th day 14th day 21st day 28th day

A. Untreated control (Normal control) 1.97 2.57 2.20 1.93 2.54

B. Diabetic control (Negative control) 1.90 1.56 1.60 1.13 0.60 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 3.10 3.20 3.33 1.93 2.14

D. Diabetic control + Vitamin C (Positive control) 0.17 0.30 0.40 0.90 0.60

E. Diabetic + Ethanolic extract of H.N 200mg/kg 0.50 2.04 1.96 2.03 1.83

F. Diabetic + Ethanolic extract of H.N 300mg/kg 2.80 1.87 2.87 3.06 2.34

G. Diabetic + Ethanolic extract of H.N 400mg/kg 3.07 2.77 2.87 2.56 1.84

From the graph, it is clear that Globulin level significantly increased at day 28. The graph has been shown in figure 42.

)

d /

g 3

(

i l

u 2

o l

G 1

0 y y y y y a a a a a d d d d d t h h h 0 t t s t 7 4 1 8 1 2 2 T im e (d a y s )

Figure 42: Blood globulin (g/dl) level of rabbits

Chapter 3 Results Results

3.3.2.1.8 A/G Ratio

Normally the albumin/globulin (A/G) ratio is greater than 1. Group A (untreated group) blood Albumin level war recorded (2.04 — 1.59) as normal. Group B was kept untreated diabetic control (normal control). The Albumin level remained high (1.15 — 1.71 ) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (2.08) was recorded at the end of the treatment showing that allopathic medicine (Glucophage Metformin HCl has no counter effect on Albumin. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the Albumin level was high (4.07). These were a significant increase in Albumin level (1.08) at the end of administration period showing that Vitamin C has no counter effect on Albumin. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the Albumin level was high (1.60). These were a significant increase in Albumin level (1.74) at the end of administration period. Group F was given with 300 mg/L plant extract for regular five days at the interval of 0 day, 7th day, 14th day, 21st day and 28th day after induction of diabetes. Initially there was a high level of Albumin (0.92). The Albumin level tended to decrease towards normal as the administration period extended. There was a significant increase in Albumin level (1.64) at the end of medication with plant extract. A 400mg/kg plant extract was administered orally to group G. Initially there was a high level of Albumin (0.95). A noteworthy rise in Albumin level (2.58) was recorded at the end of administration. The result revealed that the extract has no significant role even at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 60.

100

Chapter 3 Results Results

Table 60: Blood A/G ratio level of rabbits

0 day 7th day 14th day 21st day 28th day

A. Untreated control 2.04 1.56 1.86 2.12 1.59 B. Diabetic control 1.15 1.34 0.00 1.26 1.71 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 0.97 0.98 0.93 1.22 2.08 D. Diabetic control + Vitamin C (Positive control) 4.07 2.15 2.27 1.33 1.08 E. Diabetic + Ethanolic extract of H.N 200mg/kg 1.60 1.15 1.32 1.37 1.74 F. Diabetic + Ethanolic extract of H.N 300mg/kg 0.92 1.95 0.98 1.01 1.64 G. Diabetic + Ethanolic extract of H.N 400mg/kg 0.95 1.20 1.30 1.60 2.58 From the graph, it is clear that A/G Ratio level significantly increased at day 28. The graph has been shown in figure 43.

i t

a 3

/ 2 A

0 y y y y y a a a a a d d d d d t h h h 0 t t s t 7 4 1 8 1 2 2 T im e (d a y s )

Figure 43: Blood A/G ratio level of rabbits

3.3.2.1.9 GCI category

Table 61: GCI Categories for Blood of Rabbits

101

Chapter 3 Results Results

Group A (untreated group) blood GCI level war recorded (1.20 — -5.0) as normal. Group B was kept untreated diabetic control (normal control). The GCI level remained high (0.46 — -0.79 ) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (-.40) was recorded at the end of the treatment showing that allopathic medicine (Glucophage Metformin HCl has no counter effect on GCI. Group D was given with Vitamin Cat the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of medication the GCI level was high (-8.00). These were a significant increase in GCI level (2.70) at the end of administration period showing that Vitamin C has counter effect on GCI. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of medication the GCI level was high (10.00). These were a significant decrease in GCI level (- 2.33) at the end of administration period. Group F was given with 300 mg/L plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of GCI (0.33). The GCI level tended to decrease towards normal as the administration period extended. There was a significant decrease in GCI level (0.66) at the end of administration with plant extract. A 400mg/kg plant extract was administered orally to group G. Initially there was a high level of GCI (0.83). A momentous fall in GCI level (0.58) was recorded at the end of administration. The result revealed that the extract has little bit significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 62.

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Chapter 3 Results Results

Table 62: Blood GCI level of rabbits

14th 28th 0 day 7th day day 21st day day

A. Untreated control (Normal control) 1.20 -5.00 0.50 0.00 -5.00 B. Diabetic control (Negative control) 0.46 -0.70 -0.47 -0.66 -0.79 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 1.20 0.75 2.00 6.00 -0.40 D. Diabetic control + Vitamin C (Positive control) -8.00 -7.33 -7.00 7.00 2.70 E. Diabetic + Ethanolic extract of H.N 200mg/kg 10.00 -0.41 -0.66 -0.71 -2.33 F. Diabetic + Ethanolic extract of H.N 300mg/kg 0.33 -1.40 0.42 1.25 0.66 G. Diabetic + Ethanolic extract of H.N 400mg/kg 0.83 1.00 -1.50 0.00 0.58

From the graph, it is clear that GCI level significantly increased at day 28. The graph has been shown in figure 44.

0 d a y 2 8 th d a y 7 th d a y

) 2 1 s t d a y 1 4 th d a y s

y 2 1 s t d a y

a d

( 1 4 th d a y

2 8 th d a y

m i

T 7 th d a y

0 d a y

-1 0 -5 0 5 1 0 1 5 G C I

Figure 44: Blood GCI level of rabbits

3.3.2.1.10 Creatinine level

Creatinine normal value in the blood is 2.3-3.0 mg/dl. Group A (untreated group) blood Creatinine level war recorded (2.26±0.05 mg/dl — 3.06±0.05 mg/dl) as normal. Group B was kept untreated diabetic control (normal control). The Creatinine level remained high (5.30±0.20 mg/dl — 7.50±0.19 mg/dl) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of 0 day, 7th day, 14th day, 21st day and 28th day. A significant decrease of (2.66±0.05 mg/dl) was recorded at the end of the treatment. Group D was given with Vitamin C at the

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Chapter 3 Results Results dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of medication the Creatinine level was high (4.46±0.11 mg/dl). These were a significant decrease in Creatinine level (3.06±0.11 mg/dl) at the end of administration period showing that Vitamin C has no counter effect on Creatinine. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the cholesterol level was high (4.40±0.36 mg/dl). These were a significant decrease in Creatinine level (3.86±0.15 mg/dl) at the end of administration period. Group F was given with 300mg/dl plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of Creatinine (5.00±0.09 mg/dl). The Creatinine level tended to decrease towards normal as the administration period extended. There was a significant decrease in Creatinine level (2.73±0.20 mg/dl) at the end of administration with plant extract. A 400 mg/kg plant extract was administered orally to group G. Initially there was a high level of Creatinine (3.73±0.15 mg/dl). A significant decrease in Creatinine level (2.00±0.09 mg/dl) was recorded at the end of m administration. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 63.

Table 63: Blood creatinine (mg/dl) level of rabbits in triplicate

0 day 7th day

Untreated control 2.3000 2.2000 2.3000 2.6 2.6 2.5

Diabetic control 5.1000 5.3000 5.5000 6.3 6.3 6.3

Diabetic control + Glibenclamide (10mg/kg) 4.0000 4.0000 3.8000 3.3 3.0 3.1

Diabetic control + Vitamin C (Positive Control) 4.4000 4.4000 4.6000 4.3 4.2 4.5

Diabetic + Ethanolic extract of H.N 200mg/kg 4.7000 4.5000 4.0000 4.5 4.4 4.1

Diabetic + Ethanolic extract of H.N 300mg/kg 5.1000 5.0000 4.9000 5.6 5.6 5.3

Diabetic + Ethanolic extract of H.N 400mg/kg 3.9000 3.7000 3.6000 3.7 3.0 3.2

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14th day 21st day 28th day 2.7 2.7 2.7 2.9 2.8 2.6 3.1 3.0 3.1 6.7 6.5 6.7 7.2 7.0 7.1 7.7 7.3 7.5 3.1 3.0 3.4 2.9 2.7 2.7 2.7 2.7 2.6 4.2 4.5 4.0 3.2 3.3 3.4 3.0 3.2 3.0 4.5 4.0 4.4 4.3 4.0 4.1 4.0 3.7 3.9 3.4 3.1 3.0 3.1 3.0 2.9 2.9 2.8 2.5 3.2 3.0 3.0 2.9 2.0 2.5 2.1 2.0 1.9

Table 64: Mean blood creatinine (mg/dl) level of rabbits with standard deviation

0 day 7th day

Untreated control 2.266667 0.05773497 3 2.566667 0.05773497 3 Diabetic control 5.300 0.2000001 3 6.300 0.000 3 Diabetic control + Glibenclamide (10mg/kg) 3.933333 0.1154701 3 3.133333 0.1527525 3 Diabetic control + Vitamin C (Positive Control) 4.466667 0.1154699 3 4.333334 0.1527526 3 Diabetic + Ethanolic extract of H.N 200mg/kg 4.400 0.3605551 3 4.333334 0.2081667 3 Diabetic + Ethanolic extract of H.N 300mg/kg 5.000 0.09999991 3 5.500 0.1732049 3 Diabetic + Ethanolic extract of H.N 400mg/kg 3.733333 0.1527526 3 3.300 0.3605551 3

14th day 21st day 28th day 2.700 0.000 3 2.766667 0.1527526 3 3.066667 0.05773497 3 6.633333 0.1154699 3 7.100 0.09999991 3 7.500 0.1999998 3 3.166667 0.2081667 3 2.766667 0.1154701 3 2.666667 0.05773511 3 4.233333 0.2516612 3 3.300 0.100 3 3.066667 0.1154701 3 4.300 0.2645752 3 4.133333 0.1527526 3 3.866667 0.1527525 3 3.166667 0.2081667 3 3.000 0.09999991 3 2.733333 0.2081666 3 3.066667 0.1154701 3 2.466667 0.450925 3 2.000 0.09999996 3

From the graph, it is clear that glucose level significantly increased at day 28. The graph has been shown in figure 45.

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Chapter 3 Results Results

)

( e

n 1 0 i

n 0 d a y

i t

a 8 7 th d a y e

r 1 4 th d a y C 6

d 2 1 s t d a y o

o 2 8 th d a y l

b 4

n i

t 2

a f

0 n

a y y y y y a a a e a a d d d d d t M h h 0 th t s t 7 4 1 8 1 2 2 T im e (d a y s )

Figure 45: Blood creatinine level of rabbits

3.3.2.1.11 ALP level

ALP normal value in the blood is 4-22 IU/L. Group A (untreated group) blood ALP level was recorded (53.00±1.00 IU/L — 50.66±2.08 IU/L) as normal. Group B was kept untreated diabetic control (normal control). The ALP level remained high (253.33±2.51 IU/L — 302±2.64 IU/L) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (75.66±3.05 IU/L) was recorded at the end of the treatment. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the ALP level was high (212.00±1.00 IU/L). These were a significant decrease in ALP level (243.66±4.04 IU/L) at the end of administration period showing that Vitamin C has no counter effect on ALP. Group E was medicated with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the ALP level was high (236.33±4.61 IU/L). These were a significant decrease in ALP level (175.66±0.57 IU/L) at the end of administration period. Group F was given with 300mg/dl plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of ALP (194.66±1.52

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Chapter 3 Results Results

IU/L). The ALP level tended to decrease towards normal as the administration period extended. There was a significant decrease in ALP level (108±1.0 IU/L) at the end of administration with plant extract. A 400 mg/kg plant extract was administered orally to group G. Initially there was a high level of ALP (171.33±1.52 IU/L). A significant decrease in ALP level (54.33±2.08 IU/L) was recorded at the end of administration. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 65.

Table 65: Blood ALP (IU/L) level of rabbits in triplicate

0 day 7th day

A. Untreated control (Normal control) 53.0000 52.0000 54.0000 56.0 55.00 56.0 B. Diabetic control (Negative control) 256.0000 253.0000 251.0000 273.0 271.00 275.0 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 179.0000 173.0000 170.0000 153.0 151.00 157.0 D. Diabetic control + Vitamin C (Positive control) 213.0000 211.0000 212.0000 226.0 224.00 229.0 E. Diabetic + Ethanolic extract of H.N 200mg/kg 239.0000 231.0000 239.0000 219.0 213.00 215.0 F. Diabetic + Ethanolic extract of H.N 300mg/kg 196.0000 195.0000 193.0000 171.0 173.00 176.0 G. Diabetic + Ethanolic extract of H.N 400mg/kg 173.0000 171.0000 170.0000 139.0 138.00 137.0

14th day 21st day 28th day

51.0 50.0 48.0 57.0 56.0 55.0 53.0 50.0 49.0

279.0 276.0 273.0 291.0 289.0 290.0 301.0 300.0 305.0

123.0 122.0 119.0 96.0 93.0 91.0 79.0 75.0 73.0

229.0 227.0 225.0 231.0 229.0 230.0 239.0 231.0 234.0

203.0 200.0 201.0 192.0 191.0 191.0 176.0 176.0 175.0

156.0 153.0 151.0 133.0 133.0 131.0 109.0 108.0 107.0

107.0 103.0 101.0 79.0 76.0 81.0 56.0 55.0 52.0

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Chapter 3 Results Results

Table 66: Mean blood ALP (IU/L) level of rabbits with standard deviation

0 day 7th day

A. Untreated control (Normal control) 53.000 1.000 3 55.66667 0.5773503 3

B. Diabetic control (Negative control) 253.3333 2.516612 3 273.000 2.000 3 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 174.000 4.582576 3 153.6667 3.05505 3 D. Diabetic control + Vitamin C (Positive control) 212.000 1.000 3 226.3333 2.516612 3 E. Diabetic + Ethanolic extract of H.N 200mg/kg 236.3333 4.618802 3 215.6667 3.05505 3 F. Diabetic + Ethanolic extract of H.N 300mg/kg 194.6667 1.527525 3 173.3333 2.516612 3 G. Diabetic + Ethanolic extract of H.N 400mg/kg 171.3333 1.527525 3 138.000 1.000 3

14th day 21st day 28th day 49.66667 1.527525 3 56.000 1.000 3 50.66667 2.081666 3 276.000 3.000 3 290.000 1.000 3 302.000 2.645751 3 121.3333 2.081666 3 93.33334 2.516612 3 75.66666 3.05505 3 227.000 2.000 3 230.000 1.000 3 234.6667 4.041452 3 201.3333 1.527525 3 191.3333 0.5773503 3 175.6667 0.5773503 3 153.3333 2.516612 3 132.3333 1.154701 3 108.000 1.000 3 103.6667 3.05505 3 78.66666 2.516612 3 54.33333 2.081666 3

From the graph, it is clear that glucose level significantly increased at day 28. The graph has been shown in figure 46.

4 0 0 0 d a y 7 th d a y

) 3 0 0

L 1 4 th d a y

/ U

I 2 1 s t d a y

(

2 0 0 2 8 th d a y

L A 1 0 0

0 y y y y y a a a a a d d d d d t h h h 0 t t s t 7 4 1 8 1 2 2 T im e (d a y s )

Figure 46: Blood ALP (IU/L) level of rabbits

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Chapter 3 Results Results

3.3.2.1.12 GGT level

GGT normal value in the blood is 4-22 IU/L. Group A (untreated group) blood GGT level was recorded (12.93±0.81 IU/L — 12.20±1.311 IU/L) as normal. Group B was kept untreated diabetic control (normal control). The GGT level remained high (29.36±2.60 IU/L — 55.13±2.01 IU/L) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (10.73±0.46 IU/L) was recorded at the end of the treatment. Group D was given with Vitamin Cat the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the GGT level was high (30.80±0.62 IU/L). These were a significant increase in GGT level (45.56±0.51 IU/L) at the end of administration period showing that Vitamin C has no counter effect on GGT. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the GGT level was high (26.56±1.28 IU/L). These were a significant decrease in GGT level (13.16±1.04 IU/L) at the end of administration period. Group F was medicated with 300 mg/dl plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of GGT (23.13±0.51 IU/L). The GGT level tended to decrease towards normal as the administration period extended. There was a significant decrease in GGT level (9.33±0.30 IU/L) at the end of administration with plant extract. A 400mg/kg plant extract was administered orally to group G. Initially there was a high level of GGT (17.80±1.65 IU/L). A significant decrease in GGT level (5.73±1.00 IU/L) was recorded at the end of administration. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 67.

109

Chapter 3 Results Results

Table 67: Blood GGT (IU/L) level of rabbits in triplicate

0 day 7th day

A. Untreated control (Normal control) 13.5000 12.0000 13.3000 12.0 11.00 11.0

B. Diabetic control (Negative control) 31.6000 30.0000 26.5000 39.0 33.00 36.0 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 22.4000 20.0000 19.9000 20.0 17.70 16.0 D. Diabetic control + Vitamin C (Positive control) 30.1000 31.3000 31.0000 33.2 32.00 32.0 E. Diabetic + Ethanolic extract of H.N 200mg/kg 28.0000 26.2000 25.5000 23.0 42.24 24.0 F. Diabetic + Ethanolic extract of H.N 300mg/kg 23.7000 22.7000 23.0000 17.0 15.00 14.0 G. Diabetic + Ethanolic extract of H.N 400mg/kg 19.7000 16.7000 17.0000 13.0 12.40 13.1

14th day 21st day 28th day 11.0 10.0 9.0 14.2 13.0 13.0 13.6 12.0 11.0 46.0 42.0 47.0 51.0 50.6 49.0 57.0 55.4 53.0 17.0 17.0 18.0 13.0 12.0 13.1 10.2 11.0 11.0 33.3 31.0 31.0 40.1 41.0 42.0 46.0 45.7 45.0 21.0 20.0 21.0 17.0 16.7 15.0 14.0 13.5 12.0 13.0 13.0 12.0 11.0 11.0 14.0 9.6 9.0 9.4 11.0 11.5 10.7 8.3 7.3 7.8 6.7 4.7 5.8

Table 68: Mean blood GGT (IU/L) level of rabbits with standard deviation

0 day 7th day

A. Untreated control (Normal control) 12.93333 0.8144528 3 11.33333 0.5773503 3 B. Diabetic control (Negative control) 29.36667 2.60832 3 36.000 3.000 3 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 20.76667 1.415391 3 17.900 2.007486 3 D. Diabetic control + Vitamin C (Positive control) 30.800 0.6244993 3 32.400 0.6928208 3 E. Diabetic + Ethanolic extract of H.N 200mg/kg 26.56667 1.289703 3 29.74667 10.83109 3 F. Diabetic + Ethanolic extract of H.N 300mg/kg 23.13333 0.5131602 3 15.33333 1.527525 3 G. Diabetic + Ethanolic extract of H.N 400mg/kg 17.800 1.652271 3 12.83333 0.3785943 3

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Chapter 3 Results Results

14th day 21st day 28th day 10.000 1.000 3 13.400 0.6928202 3 12.200 1.311488 3 45.000 2.645751 3 50.200 1.0583 3 55.13334 2.013289 3 17.33333 0.5773503 3 12.700 0.6082764 3 10.73333 0.4618803 3 31.76667 1.327905 3 41.03333 0.9504392 3 45.56667 0.5131602 3 20.66667 0.5773503 3 16.23333 1.078579 3 13.16667 1.040833 3 12.66667 0.5773503 3 12.000 1.732051 3 9.333333 0.3055052 3 11.06667 0.4041453 3 7.800 0.500 3 5.733333 1.001665 3

From the graph, it is clear that glucose level significantly increased at day 28. The graph has been shown in figure 47.

8 0 0 d a y 7 th d a y 6 0

1 4 th d a y

L /

U 2 1 s t d a y

I (

4 0

T 2 8 th d a y

G G 2 0

y y y y y a a a a a d d d d d 0 t th th s th 7 4 1 8 1 2 2 T im e (d a y s )

Figure 47: Blood GGT (IU/L) level of rabbits

3.3.2.1.13 ALT level

ALT normal value in the blood is 7-23 IU/L. Group A (untreated group) blood ALT level was recorded (17.10±0.26 IU/L — 17.27±0.55 IU/L) as normal. Group B was kept untreated diabetic control (normal control). The ALT level remained high (34.53±0.56 IU/L — 38.66±0.28 IU/L) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (31.66±1.55 IU/L) was recorded at the end of the treatment. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day,

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Chapter 3 Results Results fourteenth day, twenty first day and twenty eighth day. At the start of medication the ALT level was high (33.73±4.08 IU/L). These were a significant decrease in ALT level (33.80±0.70 IU/L) at the end of administration period showing that Vitamin C has no counter effect on ALT. Group E was medicated with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the ALT level was high (36.50±0.78 IU/L). These were a significant decrease in ALT level 37.64±0.36 IU/L) at the end of administration period. Group F was given with 300 mg/dl plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of ALT (26.36±0.65 IU/L). The ALT level tended to decrease towards normal as the administration period extended. There was a significant decrease in ALT level (26.63±1.60 IU/L) at the end of administration with plant extract. A 400mg/kg plant extract was administered orally to group G. Initially there was a high level of ALT (19.46±0.55 IU/L). A significant increase in ALT level (22.80±0.75 IU/L) was recorded at the end of administration. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 69.

Table 69: Blood ALT (IU/L) level of rabbits in triplicate

0 day 7th day

A. Untreated control (Normal control) 17.0000 17.4000 16.9000 17.6 17.00 17.3

B. Diabetic control (Negative control) 35.0000 34.7000 33.9000 37.1 34.80 36.5 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 21.0000 20.3000 22.7000 19.0 18.33 17.3 D. Diabetic control + Vitamin C (Positive control) 32.0000 30.8000 38.4000 31.3 30.60 30.7 E. Diabetic + Ethanolic extract of H.N 200mg/kg 36.0000 36.1000 37.4000 34.7 33.50 33.7 F. Diabetic + Ethanolic extract of H.N 300mg/kg 27.0000 26.4000 25.7000 22.5 23.50 25.3 G. Diabetic + Ethanolic extract of H.N 400mg/kg 20.0000 19.5000 18.9000 17.4 17.70 17.3

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Chapter 3 Results Results

14th day 21st day 28th day 18.2 17.60 17.5 19.1 19.4 18.7 17.8 17.32 16.70 38.3 35.70 35.0 38.7 38.6 38.7 38.5 38.50 39.00 21.0 23.10 14.3 29.0 27.7 27.5 30.4 31.20 33.40 29.8 29.30 28.4 32.3 31.4 32.6 33.1 34.50 33.80 33.8 32.56 34.1 36.6 36.0 35.6 37.9 37.80 37.22 21.6 20.70 21.2 25.7 25.4 24.7 27.3 27.80 24.80 13.2 12.60 11.5 16.7 15.3 14.8 23.5 22.00 22.90

Table 70: Mean blood ALT (IU/L) level of rabbits with standard deviation

A. Untreated control (Normal control) 17.100 0.2645751 3 17.300 0.3000002 3 B. Diabetic control (Negative control) 34.53333 0.5686233 3 36.13333 1.193035 3 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 21.33333 1.234235 3 18.210 0.8563298 3 D. Diabetic control + Vitamin C (Positive control) 33.73333 4.085749 3 30.86667 0.3785932 3 E. Diabetic + Ethanolic extract of H.N 200mg/kg 36.500 0.7810262 3 33.96667 0.6429103 3 F. Diabetic + Ethanolic extract of H.N 300mg/kg 26.36667 0.6506403 3 23.76667 1.418919 3 G. Diabetic + Ethanolic extract of H.N 400mg/kg 19.46667 0.5507572 3 17.46667 0.2081674 3

14th day 21st day 28th day 17.76667 0.3785943 3 19.06667 0.3511879 3 17.27333 0.5514821 3 36.33333 1.738773 3 38.66667 0.05773635 3 38.66667 0.2886751 3 19.46667 4.596013 3 28.06667 0.8144526 3 31.66667 1.553492 3 29.16667 0.7094597 3 32.100 0.6244993 3 33.800 0.7000008 3 33.48667 0.8164135 3 36.06667 0.5033222 3 37.640 0.3671509 3 21.16667 0.4509248 3 25.26667 0.51316 3 26.63333 1.607275 3 12.43333 0.8621678 3 15.600 0.9848861 3 22.800 0.7549834 3

From the graph, it is clear that glucose level significantly increased at day 28. The graph has been shown in figure 48.

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Chapter 3 Results Results

5 0 0 d a y

4 0 7 th d a y

) L

/ 1 4 th d a y

U 3 0

I 2 1 s t d a y

(

T 2 8 th d a y

L 2 0 A

1 0

0 y y y y y a a a a a d d d d d t h h h 0 t t s t 7 4 1 8 1 2 2 T im e (d a y s )

Figure 48: Blood ALT (IU/L) level of rabbits

3.3.2.1.14 AST level

AST normal value in the blood is 11-34 IU/L. Group A (untreated group) blood AST level was recorded (101.66±1.52 IU/L — 106.00±3.00 IU/L) as normal. Group B was kept untreated diabetic control (uormal control). The AST level remained high (155.00±1.00 IU/L — 172.00±1.00 IU/L) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (83.66±0.57 IU/L) was recorded at the end of the treatment. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of medication the AST level was high (110.33±1.52 IU/L). These were a significant decrease in AST level (97.66±2.08 IU/L) at the end of administration period showing that Vitamin C has counter effect on AST. Group E was medicated with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the AST level was high (134.00±2.64 IU/L). These were a significant decrease in AST level (131.33±2.51 IU/L) at the end of administration period. Group F was given with 300 mg/dl plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of AST (127.66±0.57 IU/L). The AST level tended to decrease towards normal as the administration period extended. There was a significant decrease in AST level (121.33±1.0 IU/L) at the end of medication with plant extract. A 400 mg/kg plant extract was administered orally to group G. Initially there was a high level of AST (124.33±2.51 IU/L). A significant decrease in AST level

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(99.66±1.52 IU/L) was recorded at the end of administration. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 71.

Table 71: Blood AST (IU/L) level of rabbits in triplicate

0 day 7th day A. Untreated control (Normal control) 103.0000 102.0000 100.0000 105.0 104.00 102.0 B. Diabetic control (Negative control) 156.0000 155.0000 154.0000 161.0 158.00 157.0 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 107.0000 103.0000 102.0000 101.0 100.00 99.0 D. Diabetic control + Vitamin C (Positive control) 112.0000 109.0000 110.0000 110.0 107.00 106.0 E. Diabetic + Ethanolic extract of H.N 200mg/kg 132.0000 133.0000 137.0000 133.0 130.00 128.0 F. Diabetic + Ethanolic extract of H.N 300mg/kg 128.0000 127.0000 128.0000 127.0 126.00 122.0 G. Diabetic + Ethanolic extract of H.N 400mg/kg 124.0000 122.0000 127.0000 116.0 113.00 114.0

14th day 21st day 28th day 104.0 103.00 102.0 107.0 105.0 103.0 109.0 106.00 103.00 167.0 166.00 166.0 172.0 170.0 168.0 173.0 171.00 172.00 96.0 93.00 94.0 91.0 93.0 90.0 84.0 83.00 84.00 107.0 107.00 105.0 103.0 101.0 100.0 100.0 97.00 96.00 134.0 133.00 134.0 131.0 132.0 135.0 134.0 131.00 129.00 128.0 124.00 126.0 126.0 123.0 122.0 123.0 120.00 121.00 112.0 110.00 109.0 107.0 106.0 107.0 101.0 100.00 98.00

Table 72: Mean blood AST (IU/L) level of rabbits with standard deviation

0 day 7th day A. Untreated control (Normal control) 101.6667 1.527525 3 103.6667 1.527525 3 B. Diabetic control (Negative control) 155.000 1.000 3 158.6667 2.081666 3 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 104.000 2.645751 3 100.000 1.000 3 D. Diabetic control + Vitamin C (Positive control) 110.3333 1.527525 3 107.6667 2.081666 3 E. Diabetic + Ethanolic extract of H.N 200mg/kg 134.000 2.645751 3 130.3333 2.516612 3 F. Diabetic + Ethanolic extract of H.N 300mg/kg 127.6667 0.5773503 3 125.000 2.645751 3 G. Diabetic + Ethanolic extract of H.N 400mg/kg 124.3333 2.516612 3 114.3333 1.527525 3

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14th day 21st day 28th day 103.000 1.000 3 105.000 2.000 3 106.000 3.000 3 166.3333 0.5773503 3 170.000 2.000 3 172.000 1.000 3 94.33334 1.527525 3 91.33334 1.527525 3 83.66666 0.5773503 3 106.3333 1.154701 3 101.3333 1.527525 3 97.66666 2.081666 3 133.6667 0.5773503 3 132.6667 2.081666 3 131.3333 2.516612 3 126.000 2.000 3 123.6667 2.081666 3 121.3333 1.527525 3 110.3333 1.527525 3 106.6667 0.5773503 3 99.66666 1.527525 3

From the graph, it is clear that glucose level significantly increased at day 28. The graph has been shown in figure 49.

2 0 0 0 d a y 7 th d a y

) 1 5 0

L 1 4 th d a y

/ U

I 2 1 s t d a y (

1 0 0

T 2 8 th d a y

S A 5 0

0 y y y y y a a a a a d d d d d t h h h 0 t t s t 7 4 1 8 1 2 2 T im e (d a y s )

Figure 49: Blood AST (IU/L) level of rabbits

3.3.2.1.15 AST/ALT ratio

AST/ALT ratio normal value in the blood is 3.6-7.9. Group A (untreated group) blood AST/ALT ratio was recorded (5.94 — 6.13) as normal. Group B was kept untreated diabetic control (normal control). The AST/ALT Ratio remained high (4.55— 4.44) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was medicated with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (2.64) was recorded at the end of the treatment. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the AST/ALT ratio was high (3.27). These were a significant decrease in AST/ALT Ratio (2.88) at the end of administration period showing that Vitamin C has counter effect on AST/ALT Ratio. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day,

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Chapter 3 Results Results seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the AST/ALT Ratio was high (3.67). These were a significant decrease in AST/ALT ratio (3.48) at the end of administration period. Group F was given with 300mg/dl plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high AST/ALT ratio (4.85). The AST/ALT Ratio tended to decrease towards normal as the administration period extended. There was a significant decrease in AST/ALT Ratio (4.55) at the end of administration with plant extract. A 400mg/kg plant extract was administered orally to group G. Initially there was a high level of AST (6.38). A significant decrease in AST/ALT Ratio (4.37) was recorded at the end of administration. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 73.

Table 73: Blood AST/ALT ratio level of rabbits in triplicate

0 day 7th day 14th day 21st day 28th day

A. Untreated control (Normal control) 5.94 5.990000 5.80 5.50 6.13 B. Diabetic control (Negative control) 4.55 4.390000 4.57 4.39 4.44 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 4.87 5.490000 4.84 3.25 2.64 D. Diabetic control + Vitamin C (Positive control) 3.27 3.480000 3.64 3.15 2.88 E. Diabetic + Ethanolic extract of H.N 200mg/kg 3.67 3.820000 3.99 3.67 3.48 F. Diabetic + Ethanolic extract of H.N 300mg/kg 4.85 5.260000 5.95 4.89 4.55 G. Diabetic + Ethanolic extract of H.N 400mg/kg 6.38 6.500000 8.87 6.83 4.37

From the graph, it is clear that glucose level significantly increased at day 28. The graph has been shown in figure 50.

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Chapter 3 Results Results

1 0

)

s y

a 6

(

e 4

i T 2

y y y y y a a a a a d d d d d 0 t th th s th 7 4 1 8 1 2 2 A S T /A L T R a tio

Figure 50: Blood AST/ALT ratio level of rabbits

3.3.2.2 Biochemical Analysis of the blood of rabbit in response of stem

3.3.2.2.1 Glucose level

Glucose normal value in the blood is 65-105 mg/dl. Alloxane was administered to all the groups except untreated group. Untreated group’s blood glucose level was recorded (93.00±1.0 — 98.33±0.57) as normal. Initially there was hypoglycemia which was countered by infusion of normal saline through I/V and administration of glucose in water. A significant increase (p<0.0001) was recorded in glucose level at day 3. After induction of diabetes, group B was kept untreated diabetic control (Normal control). The glucose level remained high (264.33±3.78 — 391.00±1.00) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (94.00±1.00) was recorded at the end of the treatment. Group D was given with Vitamin C at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the glucose level was high (250.66±1.52 mg/dl). These were a significant increase in glucose level (289.66±2.51 mg/dl) at the end of administration period showing that Vitamin C has no counter effect on glucose. Group E was given with extract of H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of

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Chapter 3 Results Results administration the glucose level was high (271.33±1.52 mg/dl). These were a significant decrease in glucose level (180.00±7.00 mg/dl) at the end of administration period. Group F was given with 300 mg/dl plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of glucose (258.66±3.21 mg/dl). The glucose level tended to decrease towards normal as the administration period extended. There was a significant decrease in glucose level (157.66±7.37 mg/dl) at the end of administration with plant extract. A 400 mg/kg plant extract was administered orally to group G. Initially there was a high level of glucose (228.00±2.64 mg/dl). A significant decrease in glucose level (142.00±1.73 mg/dl) was recorded at the end of administration. The result revealed that the extract has significant antidiabetic activity at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 74.

Table 74: Blood glucose (mg/dl) level of rabbits in triplicate

0 day 7th day Untreated control 92.0000 93.0000 94.0000 90.0 95.0 93.0 Diabetic control 260.0000 266.0000 267.0000 287.0 290.0 292.0 Diabetic control + Glibenclamide (10mg/kg) 280.0000 279.0000 278.0000 151.0 152.0 155.0 Diabetic control + Vitamin C (Positive Control) 249.0000 251.0000 252.0000 260.0 267.0 270.0 Diabetic + Ethanolic extract of H.N 200mg/kg 271.0000 273.0000 270.0000 251.0 257.0 260.0 Diabetic + Ethanolic extract of H.N 300mg/kg 260.0000 255.0000 261.0000 222.0 226.0 231.0 Diabetic + Ethanolic extract of H.N 400mg/kg 226.0000 227.0000 231.0000 210.0 203.0 207.0

14th day 21st day 28th day 92. 91. 90. 95. 96. 97. 98. 98. 99. 326. 331. 331. 371. 374. 378. 391. 390. 392. 145. 141. 140. 105. 103. 104. 93. 95. 94. 279. 271. 273. 287. 288. 288. 287. 290. 292. 231. 244. 255. 192. 200. 202. 173. 180. 187. 215. 211. 222. 166. 176. 177. 152. 155. 166. 171. 174. 181. 153. 165. 161. 143. 140. 143.

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Table 75: Mean blood glucose (mg/dl) level of rabbits with standard deviation

0 day 7th day Untreated control 93.000 1.000 3 92.66666 2.516612 3 Diabetic control 264.3333 3.785939 3 289.6667 2.516612 3 Diabetic control + Glibenclamide (10mg/kg) 279.000 1.000 3 152.6667 2.081666 3 Diabetic control + Vitamin C (Positive Control) 250.6667 1.527525 3 265.6667 5.131601 3 Diabetic + Ethanolic extract of H.N 200mg/kg 271.3333 1.527525 3 256.000 4.582576 3 Diabetic + Ethanolic extract of H.N 300mg/kg 258.6667 3.21455 3 226.3333 4.50925 3 Diabetic + Ethanolic extract of H.N 400mg/kg 228.000 2.645751 3 206.6667 3.511885 3

14th day 21st day 28th day 91.000 1.000 3 96.000 1.000 3 98.33334 0.5773503 3 329.3333 2.886751 3 374.3333 3.511885 3 391.000 1.000 3 142.000 2.645751 3 104.000 1.000 3 94.000 1.000 3 274.3333 4.163332 3 287.6667 0.5773503 3 289.6667 2.516612 3 243.3333 12.01388 3 198.000 5.291503 3 180.000 7.000 3 216.000 5.567764 3 173.000 6.082763 3 157.6667 7.371115 3 175.3333 5.131601 3 159.6667 6.110101 3 142.000 1.732051 3

From the graph, it is clear that glucose level significantly increased at day 28. The graph has

been shown in figure 51.

)

l d

/ 5 0 0 g

0 d a y

( r

a 4 0 0 7 th d a y g

u 1 4 th d a y

d 3 0 0 2 1 s t d a y

o o

l 2 8 th d a y b

2 0 0

i t

s 1 0 0

a 0 e

M y y y y y a a a a a d d d d d 0 t th th s th 7 4 1 8 1 2 2 T im e (d a y s )

Figure 51: Blood glucose level of rabbits

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Chapter 3 Results Results

3.3.2.2.2 Triglyceride level

Triglyceride normal value in the blood is 60-73 mg/dl. Group A (untreated group) blood triglyceride level war recorded (77.00±2.00 — 72.66±1.52) as normal. Group B was kept untreated diabetic control (normal control). The triglyceride level remained high (83.00±1.00 — 130.00±2.00) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (49.66±1.52) was recorded at the end of the treatment. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of medication the triglyceride level was high (63.00±1.00 mg/dl). These were a significant increase in triglyceride level (48.66±0.57 mg/dl) at the end of administration period showing that Vitamin C has no counter effect on triglyceride. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the triglyceride level was high (77.33±1.52 mg/dl). These were a significant decrease in triglyceride level (72.66±1.52 mg/dl) at the end of administration period. Group F was medicated with 300 mg/dl plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of triglyceride (79.00±2.51 mg/dl). The triglyceride level tended to decrease towards normal as the administration period extended. There was a significant decrease in triglyceride level (66.00±8.71 mg/dl) at the end of administration with plant extract. A 400 mg/kg plant extract was administered orally to group G. Initially there was a high level of triglyceride (74.66±1.52mg/dl). A significant decrease in triglyceride level (46.00±2.64 mg/dl) was recorded at the end of administration. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 76.

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Table 76: Blood triglyceride (mg/dl) level of rabbits in triplicate

0 day 7th day Untreated control 75.0000 77.0000 79.0000 70.0 73.0 75.0 Diabetic control 84.0000 83.0000 82.0000 90.0 93.0 91.0 Diabetic control + Glibenclamide (10mg/kg) 72.0000 75.0000 77.0000 73.0 72.0 71.0 Diabetic control + Vitamin C (Positive Control) 63.0000 64.0000 62.0000 55.0 56.0 58.0 Diabetic + Ethanolic extract of H.N 200mg/kg 77.0000 76.0000 79.0000 83.0 85.0 85.0 Diabetic + Ethanolic extract of H.N 300mg/kg 73.0000 84.0000 80.0000 69.0 70.0 71.0 Diabetic + Ethanolic extract of H.N 400mg/kg 73.0000 75.0000 76.0000 75.0 74.0 73.0

14th day 21st day 28th day 72. 71. 73. 72. 71. 70. 71. 73. 74. 93. 92. 93. 109. 110. 111. 128. 130. 132. 62. 60. 63. 55. 56. 57. 48. 50. 51. 53. 54. 55. 52. 50. 51. 49. 48. 49. 77. 78. 79. 75. 74. 74. 74. 73. 71. 63. 66. 65. 75. 60. 61. 72. 70. 56. 63. 65. 61. 57. 56. 55. 45. 44. 49.

Table 77: Mean blood triglyceride (mg/dl) level of rabbits with standard deviation

0 day 7th day Untreated control 77.000 2.000 3 72.66666 2.516612 3 Diabetic control 83.000 1.000 3 91.33334 1.527525 3 Diabetic control + Glibenclamide (10mg/kg) 74.66666 2.516612 3 72.000 1.000 3 Diabetic control + Vitamin C (Positive Control) 63.000 1.000 3 56.33333 1.527525 3 Diabetic + Ethanolic extract of H.N 200mg/kg 77.33334 1.527525 3 84.33334 1.154701 3 Diabetic + Ethanolic extract of H.N 300mg/kg 79.000 5.567764 3 70.000 1.000 3 Diabetic + Ethanolic extract of H.N 400mg/kg 74.66666 1.527525 3 74.000 1.000 3

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14th day 21st day 28th day 72.000 1.000 3 71.000 1.000 3 72.66666 1.527525 3 92.66666 0.5773503 3 110.000 1.000 3 130.000 2.000 3 61.66667 1.527525 3 56.000 1.000 3 49.66667 1.527525 3 54.000 1.000 3 51.000 1.000 3 48.66667 0.5773503 3 78.000 1.000 3 74.33334 0.5773503 3 72.66666 1.527525 3 64.66666 1.527525 3 65.33334 8.386498 3 66.000 8.717798 3 63.000 2.000 3 56.000 1.000 3 46.000 2.645751 3

From the graph, it is clear that triglyceride level significantly increased at day 28. The graph

has been shown in figure 52.

)

l d

/ 1 5 0 g

0 d a y

( r

a 7 th d a y g

u 1 4 th d a y

s 1 0 0

d 2 1 s t d a y

o o

l 2 8 th d a y

g 5 0

a 0 e

M y y y y y a a a a a d d d d d 0 t th th s th 7 4 1 8 1 2 2 T im e (d a y s )

Figure 52: Blood triglyceride level of Rabbits

3.3.2.2.3 Cholesterol level

Cholesterol normal value in the blood is 60-73 mg/dl. Group A (untreated group) blood cholesterol level war recorded (109.00±1.00 — 113.00±3.60) as normal. Group B was kept untreated diabetic control (normal control). The cholesterol level remained high (209.66±7.02 — 303.00±2.64) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (114.33±3.51) was recorded at the end of the treatment. Group D was given with Vitamin Cat the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the cholesterol level was high (171.00±1.00 mg/dl). These were a significant increase in cholesterol level (148.33±1.52 mg/dl) at the end of administration period showing that

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Chapter 3 Results Results

Vitamin C has no counter effect on cholesterol. Group E was medicated with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the cholesterol level was high (196.33±2.51 mg/dl). This was a significant decrease in cholesterol level (171.66±1.52 mg/dl) at the end of administration period. Group F was given with 300 mg/dl plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of cholesterol (180.00±1.00 mg/dl). The cholesterol level tended to decrease towards normal as the administration period extended. There was a significant decrease in cholesterol level (135.66±2.51 mg/dl) at the end of administration with plant extract. A 400mg/kg plant extract was administered orally to group G. Initially there was a high level of cholesterol (170.66±2.51 mg/dl). A significant decrease in cholesterol level (139.66±3.21 mg/dl) was recorded at the end of administration. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 78.

Table 78: Blood triglyceride (mg/dl) level of rabbits in triplicate

0 day 7th day

Untreated control 110.0000 108.0000 109.0000 114.0 112.0 110.0 Diabetic control 209.0000 217.0000 203.0000 227.0 233.0 234.0 Diabetic control + Glibenclamide (10mg/kg) 161.0000 155.0000 181.0000 154.0 152.0 171.0 Diabetic control + Vitamin C (Positive Control) 171.0000 172.0000 170.0000 165.0 163.0 164.0 Diabetic + Ethanolic extract of H.N 200mg/kg 196.0000 194.0000 199.0000 190.0 196.0 198.0 Diabetic + Ethanolic extract of H.N 300mg/kg 179.0000 181.0000 180.0000 174.0 173.0 170.0 Diabetic + Ethanolic extract of H.N 400mg/kg 168.0000 171.0000 173.0000 158.0 162.0 163.0

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Chapter 3 Results Results

14th day 21st day 28th day 117. 113. 107. 113. 110. 104. 116. 109. 114. 241. 245. 247. 276. 271. 273. 306. 302. 301. 134. 131. 152. 121. 119. 122. 111. 118. 114. 157. 160. 161. 148. 150. 152. 148. 147. 150. 172. 177. 180. 173. 176. 176. 172. 173. 170. 158. 160. 162. 145. 146. 147. 133. 136. 138. 157. 155. 160. 152. 150. 155. 136. 142. 141.

Table 79: Mean blood triglyceride (mg/dl) level of rabbits with standard deviation

0 day 7th day Untreated control 109.000 1.000 3 112.000 2.000 3 Diabetic control 209.6667 7.023769 3 231.3333 3.785939 3 Diabetic control + Glibenclamide (10mg/kg) 165.6667 13.61372 3 159.000 10.44031 3 Diabetic control + Vitamin C (Positive Control) 171.000 1.000 3 164.000 1.000 3 Diabetic + Ethanolic extract of H.N 200mg/kg 196.3333 2.516612 3 194.6667 4.163332 3 Diabetic + Ethanolic extract of H.N 300mg/kg 180.000 1.000 3 172.3333 2.081666 3 Diabetic + Ethanolic extract of H.N 400mg/kg 170.6667 2.516612 3 161.000 2.645751 3

14th day 21st day 28th day 112.3333 5.033223 3 109.000 4.582576 3 113.000 3.605551 3 244.3333 3.05505 3 273.3333 2.516612 3 303.000 2.645751 3 139.000 11.35782 3 120.6667 1.527525 3 114.3333 3.511885 3 159.3333 2.081666 3 150.000 2.000 3 148.3333 1.527525 3 176.3333 4.041452 3 175.000 1.732051 3 171.6667 1.527525 3 160.000 2.000 3 146.000 1.000 3 135.6667 2.516612 3 157.3333 2.516612 3 152.3333 2.516612 3 139.6667 3.21455 3

From the graph, it is clear that Cholesterol level significantly increased at day 28. The graph has been shown in figure 53.

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Chapter 3 Results Results

)

l d

/ 4 0 0 g

0 d a y

( r

a 7 th d a y

g 3 0 0

u 1 4 th d a y

d 2 1 s t d a y o

o 2 0 0

l 2 8 th d a y

n i

t 1 0 0

a 0 e

M y y y y y a a a a a d d d d d 0 t th th s th 7 4 1 8 1 2 2 T im e (d a y s )

Figure 53: Blood cholesterol level of rabbits

3.3.2.2.4 Total Bilirubin level

Total Bilirubin normal value in the blood is 8.16-8.56 mg/L. Group A (untreated group) blood Total Bilirubin level was recorded (7.36±0.05 mg/L — 6.33±0.15 mg/L) as normal. Group B was kept untreated diabetic control (normal control). The Total Bilirubin level remained high (22.93±0.68 mg/L — 35.60±1.96 mg/L) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (9.56±0.37 mg/L) was recorded at the end of the treatment. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the Total Bilirubin level was high (17.33±0.15 mg/L). These were a significant decrease in Total Bilirubin level (19.43±0.40 mg/L) at the end of administration period showing that Vitamin C has no counter effect on Total Bilirubin. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the Total Bilirubin level was high (20.46±0.32 mg/L). These were a significant increase in Total Bilirubin level (23.30±0.19 mg/L) at the end of administration period. Group F was medicated with 300 mg/L plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of Total Bilirubin (17.30±0.36 mg/L).

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Chapter 3 Results Results

The Total Bilirubin level tended to decrease towards normal as the administration period extended. There was a significant decrease in Total Bilirubin level (16.10±0.10 mg/L) at the end of administration with plant extract. A 400 mg/kg plant extract was administered orally to group G. Initially there was a high level of Total Bilirubin (15.23±0.40 mg/L). A significant decrease in Total Bilirubin level (12.43±0.15 mg/L) was recorded at the end of administration. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 80.

Table 80: Blood total bilirubin (mg/dl) level of rabbits in triplicate

0 day 7th day A. Untreated control (Normal control) 7.4000 7.3000 7.4000 7.4 7.7 7.9 B. Diabetic control (Negative control) 22.4000 23.7000 22.7000 26.3 26.4 26.0 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 14.8000 14.4000 14.0000 22.9 21.6 22.0 D. Diabetic control + Vitamin C (Positive control) 17.3000 17.5000 17.2000 14.6 15.5 16.3 E. Diabetic + Ethanolic extract of H.N 200mg/kg 20.7000 20.6000 20.1000 20.7 20.1 20.0 F. Diabetic + Ethanolic extract of H.N 300mg/kg 17.0000 17.2000 17.7000 16.4 16.4 17.1 G. Diabetic + Ethanolic extract of H.N 400mg/kg 14.8000 15.6000 15.3000 14.0 14.4 14.3

14th day 21st day 28th day 8.1 8.0 8.3 7.5 7.3 7.0 6.3 6.2 6.5 28.2 28.7 28.0 34.0 34.4 34.0 33.4 36.2 37.2 11.5 11.1 11.0 13.1 13.2 13.0 9.3 9.4 10.0 25.8 25.3 25.1 17.6 17.0 17.7 19.5 19.0 19.8 19.7 19.4 19.4 24.6 25.1 25.2 23.1 23.3 23.5 18.6 18.8 18.9 20.9 20.6 20.4 16.0 16.1 16.2 14.0 14.2 14.8 13.5 13.3 13.0 12.6 12.3 12.4

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Chapter 3 Results Results

Table 81: Mean blood total bilirubin (mg/dl) level of rabbits with standard deviation

0 day 7th day A. Untreated control (Normal control) 7.366667 0.05773497 3 7.666667 0.2516611 3 B. Diabetic control (Negative control) 22.93333 0.6806864 3 26.23333 0.2081663 3 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 14.400 0.4000001 3 22.16667 0.6658325 3 D. Diabetic control + Vitamin C (Positive control) 17.33333 0.1527523 3 15.46667 0.8504895 3 E. Diabetic + Ethanolic extract of H.N 200mg/kg 20.46667 0.3214552 3 20.26667 0.3785943 3 F. Diabetic + Ethanolic extract of H.N 300mg/kg 17.300 0.3605554 3 16.63333 0.4041456 3 G. Diabetic + Ethanolic extract of H.N 400mg/kg 15.23333 0.4041453 3 14.23333 0.2081665 3

14th day 21st day 28th day 8.133333 0.1527526 3 7.266667 0.2516612 3 6.333334 0.1527526 3 28.300 0.3605554 3 34.13334 0.230941 3 35.600 1.969771 3 11.200 0.2645751 3 13.100 0.09999991 3 9.566667 0.3785939 3 25.400 0.3605546 3 17.43333 0.3785943 3 19.43333 0.4041449 3 19.500 0.1732057 3 24.96667 0.3214552 3 23.300 0.1999998 3 18.76667 0.1527521 3 20.63333 0.2516611 3 16.100 0.1000004 3 14.33333 0.4163334 3 13.26667 0.2516612 3 12.43333 0.1527527 3

From the graph, it is clear that Total Bilirubin level significantly increased at day 28. The

graph has been shown in figure 54.

)

(

b u

r 4 0

i l

i 0 d a y

l 7 th d a y a

t 3 0

1 4 th d a y

o t

2 1 s t d a y d

o 2 0

o 2 8 th d a y

g 1 0

a f

0 n y y y a y y a a a a a e d d d d d t 0 h h h M t t s t 7 4 1 8 1 2 2 T im e (d a y s )

Figure 54: Blood total bilirubin level of rabbits

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Chapter 3 Results Results

3.3.2.2.5 Total Protein level

Total Protein normal value in the blood is 6.1-6.5 g/dL. Group A (untreated group) blood Total Protein level war recorded (6.53±0.05 g/dL — 7.16±0.15 g/dL) as normal. Group B was kept untreated diabetic control (normal control). The Total Protein level remained high (2.43±0.15 g/dL — 1.53±0.25 g/dL) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (4.43± 0.25 g/dL) was recorded at the end of the treatment showing that allopathic medicine (Glucophage Metformin HCl has no counter effect on Total Protein. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the Total Protein level was high (5.16±0.20 g/dL). This was a significant decrease in Total Protein level (4.70±0.20 g/dL) at the end of administration period showing that Vitamin C has no counter effect on Total Protein. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the Total Protein level was high (3.00±1.04 g/dL). These were a significant increase in Total Protein level (3.83±0.05 g/dL) at the end of administration period. Group F was medicated with 300 mg/L plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of Total Protein (3.63±1.56 g/dL). The Total Protein level tended to increase towards normal as the administration period extended. There was a significant decrease in Total Protein level (4.16±0.15 g/dL) at the end of medication with plant extract. A 400mg/kg plant extract was administered orally to group G. Initially there was a high level of Total Protein (4.30±0.10 g/dL). A significant increase in Total Protein level (4.70±0.20 g/dL) was recorded at the end of administration. The result revealed that the extract has no significant role even both at the dose rate of 300 mg/kg and 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 82.

129

Chapter 3 Results Results

Table 82: Blood total protein (g/dl) level of rabbits in triplicate

0 day 7th day A. Untreated control 6.5000 6.6000 6.5000 6.5 6.4 6.0 B. Diabetic control 2.4000 2.3000 2.6000 2.4 2.2 2.1 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 6.3000 6.0000 6.3000 5.4 5.5 5.5 D. Diabetic control + Vitamin C (Positive control) 5.1000 5.4000 5.0000 4.7 4.4 4.8 E. Diabetic + Ethanolic extract of H.N 200mg/kg 4.2000 2.3000 2.5000 2.8 3.1 3.2 F. Diabetic + Ethanolic extract of H.N 300mg/kg 2.4000 5.4000 3.1000 3.5 3.3 3.0 G. Diabetic + Ethanolic extract of H.N 400mg/kg 4.3000 4.4000 4.2000 4.5 4.7 4.4

14th day 21st day 28th day 7.4 7.1 7.3 6.7 6.6 6.5 7.3 7.0 7.2 2.8 3.0 3.3 2.4 2.2 2.6 1.3 1.5 1.8 5.1 5.3 5.2 5.2 5.3 5.0 4.2 4.4 4.7 4.4 4.2 4.6 4.1 4.6 4.3 4.7 4.9 4.5 3.1 3.0 3.0 3.7 3.8 3.8 3.8 3.9 3.8 3.2 3.1 3.0 3.7 3.3 3.4 4.0 4.2 4.3 4.3 4.6 4.5 4.8 4.2 4.7 4.5 4.7 4.9

Table 83: Mean blood total protein (g/dl) level of rabbits with standard deviation

0 day 7th day A. Untreated control 6.533333 0.05773497 3 6.300 0.2645752 3 B. Diabetic control 2.433333 0.1527525 3 2.233333 0.1527526 3 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 6.200 0.1732052 3 5.466667 0.05773497 3 D. Diabetic control + Vitamin C (Positive control) 5.166667 0.2081667 3 4.633333 0.2081666 3 E. Diabetic + Ethanolic extract of H.N 200mg/kg 3.000 1.044031 3 3.033333 0.2081666 3 F. Diabetic + Ethanolic extract of H.N 300mg/kg 3.633333 1.569501 3 3.266667 0.2516612 3 G. Diabetic + Ethanolic extract of H.N 400mg/kg 4.300 0.1000001 3 4.533333 0.1527524 3

130

Chapter 3 Results Results

14th day 21st day 28th day 7.266667 0.1527526 3 6.600 0.09999991 3 7.166667 0.1527526 3 3.033333 0.2516612 3 2.400 0.1999999 3 1.533333 0.2516612 3 5.200 0.1000001 3 5.166667 0.1527526 3 4.433333 0.2516611 3 4.400 0.2000001 3 4.333334 0.2516611 3 4.700 0.2000001 3 3.033333 0.05773497 3 3.766667 0.05773497 3 3.833333 0.05773511 3 3.100 0.100 3 3.466667 0.2081666 3 4.166667 0.1527526 3 4.466667 0.1527524 3 4.566667 0.3214552 3 4.700 0.2000001 3 From the graph, it is clear that Total Protein level significantly increased at day 28. The graph

has been shown in figure 55.

)

g (

n 8 i

e 0 d a y

t o

r 7 th d a y p

l 1 4 th d a y

t o

t 2 1 s t d a y

d 4 2 8 th d a y

g 2

a f

0 n

a y y y y y

e a a a a a d d d d d

M 0 t th th s th 7 4 1 8 1 2 2 T im e (d a y s )

Figure 55: Blood total protein level of rabbits

3.3.2.2.6 Albumin level

Albumin normal value in the blood is 4.1-4.4 g/dL. Group A (untreated group) blood Albumin level war recorded (2.43±0.11 g/dL — 3.30±0.30 g/dL) as normal. Group B was kept untreated diabetic control (normal control). The Albumin level remained high (2.06±0.11 g/dL — 0.86±0.05 g/dL) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (3.00± 0.00 g/dL) was recorded at the end of the treatment showing that allopathic medicine (Glucophage Metformin HCl has no counter effect on Albumin. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the Albumin level was high (1.56±0.05 g/dL). These were a

131

Chapter 3 Results Results significant increase in Albumin level (3.26±0.30 g/dL) at the end of administration period showing that Vitamin C has counter effect on Albumin. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the Albumin level was high (2.06±0.05 g/dL). These were a significant increase in Albumin level (2.23±0.25 g/dL) at the end of administration period. Group F was medicated with 300 mg/L plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of Albumin (2.13±0.15 g/dL). The Albumin level tended to increase towards normal as the administration period extended. There was a significant increase in Albumin level (3.10±0.10 g/dL) at the end of administration with plant extract. A 400 mg/kg plant extract was administered orally to group G. Initially there was a low level of Albumin (2.56±0.11 g/dL). A significant increase in Albumin level (3.80±0.10 g/dL) was recorded at the end of administration. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 84.

Table 84: Blood albumin (g/dl) level of rabbits in triplicate

0 day 7th day

A. Untreated control (Normal control) 2.3000 2.5000 2.5000 3.0 2.6 2.4 B. Diabetic control (Negative control) 2.0000 2.2000 2.0000 1.7 1.5 1.8 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 2.1000 2.0000 2.2000 2.2 2.0 2.0 D. Diabetic control + Vitamin C (Positive control) 1.6000 1.5000 1.6000 2.2 2.2 2.2 E. Diabetic + Ethanolic extract of H.N 200mg/kg 2.0000 2.1000 2.1000 2.4 2.0 2.1 F. Diabetic + Ethanolic extract of H.N 300mg/kg 2.0000 2.1000 2.3000 2.5 2.7 2.5 G. Diabetic + Ethanolic extract of H.N 400mg/kg 2.5000 2.5000 2.7000 3.0 3.2 3.0

132

Chapter 3 Results Results

14th day 21st day 28th day 3.4 4.0 3.0 4.0 2.7 2.9 3.3 3.6 3.0 1.7 1.1 1.1 1.0 0.9 1.1 0.8 0.9 0.9 2.1 2.5 2.7 2.9 2.6 2.7 3.0 3.0 3.0 2.4 2.4 2.2 2.7 2.5 2.6 3.0 3.2 3.6 2.4 2.0 2.0 2.8 3.0 3.1 3.2 3.0 3.5 2.0 2.2 2.6 2.0 2.1 2.0 3.0 3.1 3.2 3.1 3.1 3.1 3.6 3.8 3.5 3.9 3.8 3.7

Table 85: Mean blood albumin (g/dl) level of rabbits with standard deviation

0 day 7th day A. Untreated control (Normal control) 2.433333 0.1154701 3 2.666667 0.305505 3 B. Diabetic control (Negative control) 2.066667 0.1154701 3 1.666667 0.1527525 3 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 2.100 0.100 3 2.066667 0.1154701 3 D. Diabetic control + Vitamin C (Positive control) 1.566667 0.05773504 3 2.200 0.000 3 E. Diabetic + Ethanolic extract of H.N 200mg/kg 2.066667 0.05773497 3 2.166667 0.2081667 3 F. Diabetic + Ethanolic extract of H.N 300mg/kg 2.133333 0.1527525 3 2.566667 0.1154701 3 G. Diabetic + Ethanolic extract of H.N 400mg/kg 2.566667 0.1154701 3 3.066667 0.1154701 3

14th day 21st day 28th day 3.466667 0.5033223 3 3.200 0.700 3 3.300 0.300 3 1.300 0.3464102 3 1.000 0.100 3 0.8666667 0.05773501 3 2.433333 0.3055051 3 2.733333 0.1527526 3 3.000 0.000 3 2.333334 0.1154701 3 2.600 0.100 3 3.266667 0.305505 3 2.133333 0.2309402 3 2.966667 0.1527525 3 3.233333 0.2516612 3 2.266667 0.305505 3 2.033333 0.05773497 3 3.100 0.100 3 3.100 0.000 3 3.633333 0.1527525 3 3.800 0.100 3

From the graph, it is clear that Albumin level significantly increased at day 28. The graph has been shown in figure 56.

133

Chapter 3 Results Results

)

d /

g 5 (

0 d a y

n i

m 4 7 th d a y u

b 1 4 th d a y l a 3

d 2 1 s t d a y

o o

l 2 8 th d a y b

i t

s 1

a 0 e y y y y y M a a a a a d d d d d 0 t th th s th 7 4 1 8 1 2 2 T im e (d a y s )

Figure 56: Blood albumin level of rabbits

3.3.2.2.7 Globulin level

Globulin normal value in the blood is 2.9 - 4.9 g/dL. Group A (untreated group) blood Albumin level war recorded (2.10 g/dL — 3.80 g/dL) as normal. Group B was kept untreated diabetic control (normal control). The Albumin level remained high (0.37 g/dL — 0.67 g/dL) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (1.43 g/dL) was recorded at the end of the treatment showing that allopathic medicine (Glucophage Metformin HCl has no counter effect on Albumin. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the Albumin level was high (3.60 g/dL). These were a significant increase in Albumin level (1.50 g/dL) at the end of medication period showing that Vitamin C has counter effect on Albumin. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the Albumin level was high (0.94 g/dL). These were a significant decrease in Albumin level (0.60 g/dL) at the end of administration period. Group F was given with 300 mg/L plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of Albumin (1.50 g/dL). The Albumin level tended to decrease towards normal as the administration period extended. There was a significant decrease in Albumin level (1.06 g/dL) at the end of administration with plant extract. A 400 mg/kg plant extract

134

Chapter 3 Results Results was administered orally to group G. Initially there was a high level of Albumin (1.74 g/dL). A significant decrease in Albumin level (0.90 g/dL) was recorded at the end of administration. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 86.

Table 86: Blood globulin (g/dl) level of rabbits

0 day 7th day 14th day 21st day 28th day A. Untreated control (Normal control) 2.10 3.64 3.80 3.40 3.80 B. Diabetic control (Negative control) 0.37 0.57 1.73 1.40 0.67 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 4.10 3.40 2.77 2.37 1.43 D. Diabetic control + Vitamin C (Positive control) 3.60 2.43 2.07 1.70 1.50 E. Diabetic + Ethanolic extract of H.N 200mg/kg 0.94 0.87 0.90 0.80 0.60 F. Diabetic + Ethanolic extract of H.N 300mg/kg 1.50 0.70 0.84 1.43 1.06 G. Diabetic + Ethanolic extract of H.N 400mg/kg 1.74 1.47 1.36 0.93 0.90

From the graph, it is clear that Globulin level significantly increased at day 28. The graph has been shown in figure 57.

)

/ g

( 3

i l

u 2

l G 1

y y y y y a a a a a d d d d d 0 t th th s th 7 4 1 8 1 2 2 T im e (d a y s )

Figure 57: Blood globulin (g/dl) level of rabbits

135

Chapter 3 Results Results

3.3.2.2.8 Albumin/Globulin Ratio

Normally the albumin/globulin (A/G) ratio is greater than 1. Group A (untreated group) blood Albumin level war recorded (1.15 — 0.86) as normal. Group B was kept untreated diabetic control (normal control). The Albumin level remained high (5.56 — 1.28) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (2.09) was recorded at the end of the treatment showing that allopathic medicine (Glucophage Metformin HCl has no counter effect on Albumin. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the Albumin level was high (0.43). These were a significant increase in Albumin level (2.17) at the end of medication period showing that Vitamin C has no counter effect on Albumin. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the Albumin level was high (2.19). These were a significant increase in Albumin level (5.38) at the end of medication period. Group F was given with 300 mg/L plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of Albumin (1.42). The Albumin level tended to decrease towards normal as the administration period extended. There was a significant increase in Albumin level (2.92) at the end of administration with plant extract. A 400 mg/kg plant extract was administered orally to group G. Initially there was a high level of Albumin (1.47). A significant increase in Albumin level (5.22) was recorded at the end of administration. The result revealed that the extract has no significant role even at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 87.

136

Chapter 3 Results Results

Table 87: Blood A/G ratio level of rabbits

0 day 7th day 14th day 21st day 28th day A. Untreated control 1.157 0.730 0.910 0.940 0.860 B. Diabetic control 5.560 2.912 0.751 0.714 1.280 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 0.512 0.605 0.877 1.151 2.090 D. Diabetic control + Vitamin C (Positive control) 0.433 0.900 1.125 1.529 2.170 E. Diabetic + Ethanolic extract of H.N 200mg/kg 2.191 2.410 2.810 3.700 5.380 F. Diabetic + Ethanolic extract of H.N 300mg/kg 1.420 3.650 2.690 2.118 2.924 G. Diabetic + Ethanolic extract of H.N 400mg/kg 1.470 2.080 2.270 4.548 5.220

From the graph, it is clear that A/G Ratio level significantly increased at day 28. The graph has been shown in figure 58.

o i

t 4

G /

A 2

0 y y y y y a a a a a d d d d d t h h h 0 t t s t 7 4 1 8 1 2 2 T im e (d a y s )

Figure 58: Blood A/G ratio level of rabbits

3.3.2.2.9 GCI category

Table 88: GCI categories for blood of rabbits

Parameter Categories GCI 1. Negative compensation with negative GCI values (<0.0) = all G values were below 25 g/L. 2. Partial compensation with GCI ranging from 0.0 to< 1.0 = all G values were >25 g/L, but the TP values did not rise to 60 g/L. 3. Full compensation with GCI values >1.0 = all G values were > 25 g/L, and the TP values raised to normal ranges, above 60 g/L. Group A (untreated group) blood GCI level war recorded (-0.36 — 6.50) as normal. Group B was kept untreated diabetic control (normal control). The GCI level remained high (-1.46 — -0.70) during the interval of zero day, seventh day, fourteenth day, twenty first day

137

Chapter 3 Results Results and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (-2.20) was recorded at the end of the treatment showing that allopathic medicine (Glucophage Metformin HCl has no counter effect on GCI. Group D was given with Vitamin Cat the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the GCI level was high (1.30). These were a significant increase in GCI level (-3.33) at the end of administration period showing that Vitamin C has no counter effect on GCI. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the GCI level was high (0.33). These were a significant decrease in GCI level (-8.00) at the end of administration period. Group F was given with 300 mg/L plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of GCI (-0.71). The GCI level tended to decrease towards normal as the medication period extended. There was a significant decrease in GCI level (-4.75) at the end of administration with plant extract. A 400 mg/kg plant extract was administered orally to group G. Initially there was a high level of GCI (-0.80). A significant decrease in GCI level (3.60) was recorded at the end of administration. The result revealed that the extract has no significant role at the dose rate of 200 mg/kg and 300 mg/kg while the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 89.

Table 89: Blood GCI level of rabbits

0 day 7th day 14th day 21st day 28th day

A. Untreated control (Normal control) -0.36 1.00 13.00 3.00 6.50 B. Diabetic control (Negative control) -1.46 -1.21 -0.36 -0.44 -0.70 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 1.14 0.60 0.18 -0.25 -2.20 D. Diabetic control + Vitamin C (Positive control) 1.30 -0.07 -0.41 -0.88 -3.33 E. Diabetic + Ethanolic extract of H.N 200mg/kg 0.33 -1.21 -2.00 -4.16 -8.00 F. Diabetic + Ethanolic extract of H.N 300mg/kg -0.71 -1.80 -1.30 -4.20 -4.75 G. Diabetic + Ethanolic extract of H.N 400mg/kg -0.80 -2.20 -3.00 3.28 3.60

138

Chapter 3 Results Results

From the graph, it is clear that GCI level significantly increased at day 28. The graph has been shown in figure 59.

0 d a y 2 8 th d a y 7 th d a y

) 1 4 th d a y

s 2 1 s t d a y y

a 2 1 s t d a y

d (

1 4 th d a y 2 8 th d a y

m i

T 7 th d a y

0 d a y

-1 0 -5 0 5 1 0 1 5 G C I

Figure 59: Blood GCI level of rabbits

3.3.2.2.10 Creatinine level

Creatinine normal value in the blood is 2.3-3.0 mg/dl. Group A (untreated group) blood Creatinine level war recorded (2.03±0.05 mg/dl — 2.96±0.25 mg/dl) as normal. Group B was kept untreated diabetic control (normal control). The Creatinine level remained high (5.96±0.15 mg/dl — 7.20±0.10 mg/dl) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (3.16±0.15 mg/dl) was recorded at the end of the treatment. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of medication the Creatinine level was high (5.10±0.09 mg/dl). These were a significant decrease in Creatinine level (3.56±0.15 mg/dl) at the end of administration period showing that Vitamin C has counter effect on Creatinine. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the cholesterol level was high (5.86±0.15 mg/dl). These were a significant decrease in Creatinine level (4.36±0.23 mg/dl) at the end of administration period. Group F was given with 300 mg/dl plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of Creatinine (5.56±0.20 mg/dl). The Creatinine level tended to decrease towards normal as the administration period extended. There was a

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Chapter 3 Results Results significant decrease in Creatinine level (4.60±0.09 mg/dl) at the end of administration with plant extract. A 400 mg/kg plant extract was administered orally to group G. Initially there was a high level of Creatinine (5.26±0.25 mg/dl). A significant decrease in Creatinine level (3.83±0.15 mg/dl) was recorded at the end of administration. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 90.

Table 90: Blood creatinine (mg/dl) level of rabbits in triplicate

0 day 7th day Untreated control 2.1000 2.0000 2.0000 2.1 2.0 2.1 Diabetic control 5.8000 6.0000 6.1000 6.0 6.0 6.2 Diabetic control + Glibenclamide (10mg/kg) 4.3000 4.5000 4.4000 4.0 4.2 4.0 Diabetic control + Vitamin C (Positive Control) 5.1000 5.0000 5.2000 4.6 4.7 4.8 Diabetic + Ethanolic extract of H.N 200mg/kg 5.7000 5.9000 6.0000 5.4 5.4 5.7 Diabetic + Ethanolic extract of H.N 300mg/kg 5.4000 5.5000 5.8000 5.3 5.5 5.7 Diabetic + Ethanolic extract of H.N 400mg/kg 5.0000 5.3000 5.5000 4.7 5.0 5.0

14th day 21st day 28th day 2.2 2.1 2.0 2.5 2.3 2.4 3.0 3.2 2.7 6.3 6.0 6.5 7.0 7.3 7.0 7.2 7.1 7.3 4.4 4.0 4.0 3.0 3.2 3.3 3.3 3.2 3.0 4.7 4.6 4.3 4.3 4.2 4.0 3.6 3.4 3.7 4.3 4.8 5.1 4.1 4.6 5.0 4.5 4.1 4.5 5.0 5.2 5.5 5.0 5.2 5.0 4.6 4.5 4.7 4.4 4.6 4.8 4.6 4.7 4.5 3.8 3.7 4.0

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Chapter 3 Results Results

Table 91: Mean blood creatinine (mg/dl) level of rabbits with standard deviation

0 day 7th day

Untreated control 2.033333 0.05773497 3 2.066667 0.05773497 3 Diabetic control 5.966667 0.1527524 3 6.066667 0.1154699 3 Diabetic control + Glibenclamide (10mg/kg) 4.400 0.09999991 3 4.066667 0.1154699 3 Diabetic control + Vitamin C (Positive Control) 5.100 0.09999991 3 4.700 0.1000001 3 Diabetic + Ethanolic extract of H.N 200mg/kg 5.866667 0.1527526 3 5.500 0.1732049 3 Diabetic + Ethanolic extract of H.N 300mg/kg 5.566667 0.2081667 3 5.500 0.1999998 3 Diabetic + Ethanolic extract of H.N 400mg/kg 5.266667 0.2516612 3 4.900 0.1732052 3

14th day 21st day 28th day 2.100 0.100 3 2.400 0.100 3 2.966667 0.2516612 3 6.266667 0.2516612 3 7.100 0.1732052 3 7.200 0.1000001 3 4.133333 0.2309402 3 3.166667 0.1527525 3 3.166667 0.1527525 3 4.533333 0.2081664 3 4.166667 0.1527526 3 3.566667 0.1527525 3 4.733334 0.4041451 3 4.566667 0.450925 3 4.366667 0.2309402 3 5.233333 0.2516612 3 5.066667 0.1154699 3 4.600 0.09999991 3 4.600 0.2000001 3 4.600 0.09999991 3 3.833333 0.1527525 3

From the graph, it is clear that glucose level significantly increased at day 28. The graph has

been shown in figure 60.

)

m (

e 8 n

i 0 d a y

n i

t 7 th d a y a

e 6

r 1 4 th d a y C

2 1 s t d a y d

o 4 2 8 th d a y

n 2

a f

0 n

a y y y y y

e a a a a a d d d d d t M h h 0 th t s t 7 4 1 8 1 2 2 T im e (d a y s )

Figure 60: Blood creatinine level of rabbits

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Chapter 3 Results Results

3.3.2.2.11 ALP level

ALP normal value in the blood is 4-22 IU/L. Group A (untreated group) blood ALP level was recorded (53.33±3.51 IU/L — 60.33±2.51 IU/L) as normal. Group B was kept untreated diabetic control (normal control). The ALP level remained high (270.00±2.64 IU/L — 355.66±5.68 IU/L) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (85.00±2.00 IU/L) was recorded at the end of the treatment. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of medication the ALP level was high (257.33±2.51 IU/L). These were a significant decrease in ALP level (183.00±3.00 IU/L) at the end of administration period showing that Vitamin C has no counter effect on ALP. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the ALP level was high (256.00±5.56 IU/L). These were a significant decrease in ALP level (218.33±2.08 IU/L) at the end of administration period. Group F was medicated with 300 mg/dl plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of ALP (219.33±2.08 IU/L). The ALP level tended to decrease towards normal as the medication period extended. There was a significant decrease in ALP level (162.00±1.73 IU/L) at the end of administration with plant extract. A 400 mg/kg plant extract was administered orally to group G. Initially there was a high level of ALP (204.66±3.21 IU/L). A significant decrease in ALP level (143.00±1.00 IU/L) was recorded at the end of administration. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 92.

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Chapter 3 Results Results

Table 92: Blood ALP (IU/L) level of rabbits in triplicate

0 day 7th day

A. Untreated control (Normal control) 53.0000 50.0000 57.0000 54.0 58.0 51.0 B. Diabetic control (Negative control) 267.0000 271.0000 272.0000 287.0 284.0 289.0 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 195.0000 200.0000 201.0000 182.0 180.0 183.0 D. Diabetic control + Vitamin C (Positive control) 257.0000 255.0000 260.0000 238.0 240.0 241.0 E. Diabetic + Ethanolic extract of H.N 200mg/kg 250.0000 257.0000 261.0000 245.0 241.0 243.0 F. Diabetic + Ethanolic extract of H.N 300mg/kg 217.0000 220.0000 221.0000 201.0 205.0 203.0 G. Diabetic + Ethanolic extract of H.N 400mg/kg 207.0000 201.0000 206.0000 182.0 184.0 187.0

14th day 21st day 28th day 55.0 57.0 53.0 51.0 55.0 59.0 58.0 60.0 63.0 289.0 290.0 291.0 218.0 320.0 323.0 362.0 351.0 354.0 170.0 173.0 171.0 122.0 125.0 123.0 83.0 85.0 87.0 215.0 216.0 217.0 207.0 201.0 205.0 180.0 183.0 186.0 227.0 231.0 231.0 215.0 213.0 216.0 216.0 219.0 220.0 178.0 182.0 186.0 173.0 176.0 178.0 160.0 163.0 163.0 160.0 157.0 165.0 150.0 155.0 154.0 143.0 142.0 144.0

Table 93: Mean blood ALP (IU/L) level of rabbits with standard deviation

0 day 7th day A. Untreated control (Normal control) 53.33333 3.511885 3 54.33333 3.511885 3 B. Diabetic control (Negative control) 270.000 2.645751 3 286.6667 2.516612 3 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 198.6667 3.21455 3 181.6667 1.527525 3 D. Diabetic control + Vitamin C (Positive control) 257.3333 2.516612 3 239.6667 1.527525 3 E. Diabetic + Ethanolic extract of H.N 200mg/kg 256.000 5.567764 3 243.000 2.000 3 F. Diabetic + Ethanolic extract of H.N 300mg/kg 219.3333 2.081666 3 203.000 2.000 3 G. Diabetic + Ethanolic extract of H.N 400mg/kg 204.6667 3.21455 3 184.3333 2.516612 3

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Chapter 3 Results Results

14th day 21st day 28th day 55.000 2.000 3 55.000 4.000 3 60.33333 2.516612 3 290.000 1.000 3 287.000 59.77458 3 355.6667 5.686241 3 171.3333 1.527525 3 123.3333 1.527525 3 85.000 2.000 3 216.000 1.000 3 204.3333 3.05505 3 183.000 3.000 3 229.6667 2.309401 3 214.6667 1.527525 3 218.3333 2.081666 3 182.000 4.000 3 175.6667 2.516612 3 162.000 1.732051 3 160.6667 4.041452 3 153.000 2.645751 3 143.000 1.000 3

From the graph, it is clear that glucose level significantly increased at day 28. The graph has been shown in figure 61.

4 0 0 0 d a y 7 th d a y 3 0 0

) 1 4 th d a y

L /

U 2 1 s t d a y

I (

2 0 0 2 8 th d a y

L A 1 0 0

y y y y y a a a a a d d d d d h t h 0 th t s t 7 4 1 8 1 2 2 T im e (d a y s )

Figure 61: Blood ALP (IU/L) level of Rabbits

3.3.2.2.12 GGT level

GGT normal value in the blood is 4-22 IU/L. Group A (untreated group) blood GGT level was recorded (14.66±0.57 IU/L — 15.00±1.00 IU/L) as normal. Group B was kept untreated diabetic control (normal control). The GGT level remained high (28.33±1.15 IU/L — 66.00±1.00 IU/L) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (11.33±1.52 IU/L) was recorded at the end of the treatment. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the GGT level was high (26.00±1.00 IU/L). These were a significant decrease in GGT level (22.00±1.00 IU/L) at the end of administration period showing that Vitamin C has counter effect on GGT. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for

144

Chapter 3 Results Results consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the GGT level was high (27.66±1.15 IU/L). These were a significant decrease in GGT level (21.00±1.00 IU/L) at the end of administration period. Group F was given with 300mg/dl plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of GGT (23.00±1.00 IU/L). The GGT level tended to decrease towards normal as the administration period extended. There was a significant decrease in GGT level (16.66±0.57 IU/L) at the end of administration with plant extract. A 400 mg/kg plant extract was administered orally to group G. Initially there was a high level of GGT (20.00±0.00 IU/L). A significant decrease in GGT level (13.33±1.52 IU/L) was recorded at the end of administration. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 94.

Table 94: Blood GGT (IU/L) level of rabbits in triplicate

0 day 7th day

A. Untreated control (Normal control) 14.0000 15.0000 15.0000 15.0 13.0 14.0

B. Diabetic control (Negative control) 29.0000 27.0000 29.0000 37.0 37.0 38.0 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 25.0000 26.0000 25.0000 23.0 22.0 23.0 D. Diabetic control + Vitamin C (Positive control) 27.0000 25.0000 26.0000 26.0 26.0 25.0 E. Diabetic + Ethanolic extract of H.N 200mg/kg 29.0000 27.0000 27.0000 25.0 26.0 26.0 F. Diabetic + Ethanolic extract of H.N 300mg/kg 22.0000 23.0000 24.0000 22.0 21.0 22.0 G. Diabetic + Ethanolic extract of H.N 400mg/kg 20.0000 20.0000 20.0000 19.0 20.0 18.0

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Chapter 3 Results Results

14th day 21st day 28th day 14.0 15.0 15.0 13.0 12.0 13.0 14.0 15.0 16.0 55.0 54.0 55.0 59.0 60.0 62.0 66.0 65.0 67.0 18.0 18.0 19.0 12.0 13.0 14.0 13.0 11.0 10.0 26.0 24.0 25.0 23.0 22.0 23.0 23.0 22.0 21.0 23.0 24.0 25.0 23.0 22.0 23.0 20.0 21.0 22.0 25.0 23.0 22.0 18.0 20.0 20.0 16.0 17.0 17.0 17.0 18.0 16.0 12.0 11.0 13.0 15.0 13.0 12.0

Table 95: Mean blood GGT (IU/L) level of rabbits with standard deviation

0 day 7th day A. Untreated control (Normal control) 14.66667 0.5773503 3 14.000 1.000 3 B. Diabetic control (Negative control) 28.33333 1.154701 3 37.33333 0.5773503 3 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 25.33333 0.5773503 3 22.66667 0.5773503 3 D. Diabetic control + Vitamin C (Positive control) 26.000 1.000 3 25.66667 0.5773503 3 E. Diabetic + Ethanolic extract of H.N 200mg/kg 27.66667 1.154701 3 25.66667 0.5773503 3 F. Diabetic + Ethanolic extract of H.N 300mg/kg 23.000 1.000 3 21.66667 0.5773503 3 G. Diabetic + Ethanolic extract of H.N 400mg/kg 20.000 0.000 3 19.000 1.000 3

14th day 21st day 28th day 14.66667 0.5773503 3 12.66667 0.5773503 3 15.000 1.000 3 54.66667 0.5773503 3 60.33333 1.527525 3 66.000 1.000 3 18.33333 0.5773503 3 13.000 1.000 3 11.33333 1.527525 3 25.000 1.000 3 22.66667 0.5773503 3 22.000 1.000 3 24.000 1.000 3 22.66667 0.5773503 3 21.000 1.000 3 23.33333 1.527525 3 19.33333 1.154701 3 16.66667 0.5773503 3 17.000 1.000 3 12.000 1.000 3 13.33333 1.527525 3

From the graph, it is clear that glucose level significantly increased at day 28. The graph has been shown in figure 62.

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Chapter 3 Results Results

8 0 0 d a y 7 th d a y 6 0

) 1 4 th d a y

L /

U 2 1 s t d a y

I (

4 0 2 8 th d a y

G G 2 0

y y y y y a a a a a d d d d d 0 t th th s th 7 4 1 8 1 2 2 T im e (d a y s )

Figure 62: Blood GGT (IU/L) level of rabbits

3.3.2.2.13 ALT level

ALT normal value in the blood is 7-23 IU/L. Group A (Untreated group) blood ALT level was recorded (21.00±1.50 IU/L — 17.60±0.69 IU/L) as normal. Group B was kept untreated diabetic control (Normal control). The ALT level remained high (44.50±0.34 IU/L — 60.80±2.40 IU/L) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (17.86±0.85 IU/L) was recorded at the end of the treatment. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the ALT level was high (39.56±0.90 IU/L). These were a significant decrease in ALT level (31.20±1.06 IU/L) at the end of administration period showing that Vitamin C has counter effect on ALT. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the ALT level was high (40.76±1.65 IU/L). These were a significant decrease in ALT level 40.16±0.28 IU/L) at the end of administration period. Group F was given with 300 mg/dl plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of ALT (36.36±1.50 IU/L). The ALT level tended to decrease towards normal as the administration period extended. There was a significant decrease in ALT level (30.46±0.40 IU/L) at the end of administration with plant extract. A 400 mg/kg plant extract was administered orally to group G. Initially

147

Chapter 3 Results Results there was a high level of ALT (32.06±1.55 IU/L). A significant increase in ALT level (25.43±0.35 IU/L) was recorded at the end of medication. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 96.

Table 96: Blood ALT (IU/L) level of rabbits in triplicate

0 day 7th day

A. Untreated control (Normal control) 19.5000 22.5000 21.0000 18.7 19.0 19.1 B. Diabetic control (Negative control) 44.3000 44.9000 44.3000 48.7 50.2 51.0 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 29.8000 30.7000 31.2000 25.8 26.7 28.4 D. Diabetic control + Vitamin C (Positive control) 39.5000 38.7000 40.5000 35.4 37.8 38.7 E. Diabetic + Ethanolic extract of H.N 200mg/kg 39.4000 40.3000 42.6000 38.7 40.2 41.7 F. Diabetic + Ethanolic extract of H.N 300mg/kg 34.8000 36.5000 37.8000 35.9 38.5 39.8 G. Diabetic + Ethanolic extract of H.N 400mg/kg 30.3000 32.7000 33.2000 29.4 28.6 30.7

14th day 21st day 28th day 17.5 16.8 18.7 17.30 18.8 19.5 17.3 17.1 18.4 55.6 54.7 57.3 7.90 60.1 61.7 58.4 60.8 63.2

24.7 25.3 24.7 24.10 22.3 20.5 17.0 17.9 18.7 37.6 38.5 37.4 30.47 30.4 33.8 32.4 30.3 30.9 40.2 39.7 40.5 40.20 39.7 40.0 40.0 40.5 40.0 33.9 34.8 36.5 33.00 30.4 32.0 30.7 30.0 30.7

27.6 28.5 28.9 28.10 26.8 26.3 25.8 25.1 25.4

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Chapter 3 Results Results

Table 97: Mean blood ALT (IU/L) level of rabbits with standard deviation

0 day 7th day A. Untreated control (Normal control) 21.000 1.500 3 18.93333 0.2081663 3 B. Diabetic control (Negative control) 44.500 0.3464115 3 49.96667 1.167618 3 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 30.56667 0.7094607 3 26.96667 1.320354 3 D. Diabetic control + Vitamin C (Positive control) 39.56667 0.9018496 3 37.300 1.705872 3 E. Diabetic + Ethanolic extract of H.N 200mg/kg 40.76667 1.650251 3 40.200 1.500 3 F. Diabetic + Ethanolic extract of H.N 300mg/kg 36.36667 1.504438 3 38.06667 1.985782 3 G. Diabetic + Ethanolic extract of H.N 400mg/kg 32.06667 1.55027 3 29.56667 1.059875 3

14th day 21st day 28th day 17.66667 0.9609031 3 18.53333 1.123981 3 17.600 0.6999998 3 55.86667 1.320353 3 43.23333 30.61002 3 60.800 2.400 3 24.900 0.3464093 3 22.300 1.800 3 17.86667 0.8504904 3 37.83333 0.5859463 3 31.55667 1.943099 3 31.200 1.081667 3 40.13334 0.4041449 3 39.96667 0.2516611 3 40.16667 0.2886751 3 35.06667 1.320353 3 31.800 1.311488 3 30.46667 0.4041456 3 28.33333 0.6658325 3 27.06667 0.929158 3 25.43333 0.3511879 3

From the graph, it is clear that glucose level significantly increased at day 28. The graph has been shown in figure 63.

8 0 0 d a y 7 th d a y 6 0

) 1 4 th d a y L

/ 2 1 s t d a y

I (

4 0 2 8 th d a y

L A 2 0

y y y y y a a a a a d d d d d 0 t th th s th 7 4 1 8 1 2 2 T im e (d a y s )

Figure 63: Blood ALT (IU/L) level of rabbits

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Chapter 3 Results Results

3.3.2.2.14 AST level

AST normal value in the blood is 11-34 IU/L. Group A (untreated group) blood AST level was recorded (107.66±0.57 IU/L — 112.33±2.51 IU/L) as normal. Group B was kept untreated diabetic control (normal control). The AST level remained high (170.33±0.57 IU/L — 215.00±4.00 IU/L) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant decrease of (94.66±1.15 IU/L) was recorded at the end of the treatment. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of medication the AST level was high (154.00±1.00 IU/L). These were a significant decrease in AST level (124.00±2.64 IU/L) at the end of administration period showing that Vitamin C has counter effect on AST. Group E was given with H. neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the AST level was high (150.66±1.15 IU/L). These were a significant decrease in AST level (134.66±1.52 IU/L) at the end of administration period. Group F was given with 300mg/dl plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high level of AST (127.66±0.57 IU/L). The AST level tended to decrease towards normal as the administration period extended. There was a significant decrease in AST level (133.66±2.08 IU/L) at the end of administration with plant extract. A 400 mg/kg plant extract was administered orally to group G. Initially there was a high level of AST (127.33±0.57 IU/L). A significant decrease in AST level (123.66±2.08 IU/L) was recorded at the end of administration. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 98.

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Chapter 3 Results Results

Table 98: Blood AST (IU/L) level of rabbits in triplicate

0 day 7th day

A. Untreated control (Normal control) 108.0000 107.0000 108.0000 107.0 108.0 105.0 B. Diabetic control (Negative control) 170.0000 170.0000 171.0000 188.0 186.0 187.0 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 100.0000 101.0000 102.0000 132.0 133.0 133.0 D. Diabetic control + Vitamin C (Positive control) 154.0000 153.0000 155.0000 142.0 143.0 144.0 E. Diabetic + Ethanolic extract of H.N 200mg/kg 150.0000 150.0000 152.0000 144.0 143.0 147.0 F. Diabetic + Ethanolic extract of H.N 300mg/kg 127.0000 128.0000 128.0000 147.0 148.0 145.0 G. Diabetic + Ethanolic extract of H.N 400mg/kg 127.0000 128.0000 127.0000 142.0 143.0 140.0

14th day 21st day 28th day 101.0 100.0 104.0 111.0 110.0 109.0 115.0 112.0 110.0 194.0 193.0 192.0 207.0 200.0 201.0 215.0 219.0 211.0 109.0 110.0 111.0 102.0 106.0 101.0 96.0 94.0 94.0 138.0 143.0 140.0 137.0 133.0 134.0 127.0 123.0 122.0 148.0 144.0 145.0 141.0 142.0 140.0 133.0 135.0 136.0 144.0 143.0 140.0 133.0 135.0 138.0 136.0 132.0 133.0 136.0 136.0 133.0 137.0 130.0 131.0 123.0 122.0 126.0

Table 99: Mean blood AST (IU/L) level of rabbits with standard deviation

0 day 7th day 0.577350 A. Untreated control (Normal control) 107.6667 3 3 106.6667 1.527525 3 0.577350 B. Diabetic control (Negative control) 170.3333 3 3 187.000 1.000 3 C. Diabetic control + Glibenclamide 0.577350 (10mg/kg)(Standard Control) 101.000 1.000 3 132.6667 3 3 D. Diabetic control + Vitamin C (Positive control) 154.000 1.000 3 143.000 1.000 3 E. Diabetic + Ethanolic extract of H.N 200mg/kg 150.6667 1.154701 3 144.6667 2.081666 3 F. Diabetic + Ethanolic extract of H.N 0.577350 300mg/kg 127.6667 3 3 146.6667 1.527525 3 G. Diabetic + Ethanolic extract of H.N 0.577350 400mg/kg 127.3333 3 3 141.6667 1.527525 3

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14th day 21st day 28th day 101.6667 2.081666 3 110.000 1.000 3 112.3333 2.516612 3 193.000 1.000 3 202.6667 3.785939 3 215.000 4.000 3 110.000 1.000 3 103.000 2.645751 3 94.66666 1.154701 3 140.3333 2.516612 3 134.6667 2.081666 3 124.000 2.645751 3 145.6667 2.081666 3 141.000 1.000 3 134.6667 1.527525 3 142.3333 2.081666 3 135.3333 2.516612 3 133.6667 2.081666 3 135.000 1.732051 3 132.6667 3.785939 3 123.6667 2.081666 3

From the graph, it is clear that glucose level significantly increased at day 28. The graph has been shown in figure 64.

2 5 0 0 d a y

2 0 0 7 th d a y

) 1 4 th d a y L

/ 1 5 0 2 1 s t d a y

I ( 2 8 th d a y

T 1 0 0

S A

5 0

y y y y y a a a a a d d d d d 0 t th th s th 7 4 1 8 1 2 2 T im e (d a y s )

Figure 64: Blood AST (IU/L) level of rabbits

3.3.2.2.15 AST/ALT Ratio

AST/ALT ratio normal value in the blood is 3.6-7.9. Group A (untreated group) blood AST/ALT ratio was recorded (5.12 — 6.38) as normal. Group B was kept untreated diabetic control (normal control). The AST/ALT Ratio remained high (3.82— 3.53) during the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. Group C was given with locally available allopathic medicine (Glucophage Metformin HCl) for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. A significant increase of (5.30) was recorded at the end of the treatment. Group D was given with Vitamin C at the dose rat of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the AST/ALT Ratio was high (3.89). These were a significant increase in AST/ALT Ratio (3.97) at the end of administration period showing that Vitamin C has counter effect on AST/ALT Ratio. Group E was given with H.

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Chapter 3 Results Results neplenensis at the dose rate of 200 mg/kg for consecutive five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day. At the start of administration the AST/ALT Ratio was high (3.69). These were a significant decrease in AST/ALT Ratio (3.35) at the end of administration period. Group F was given with 300 mg/dl plant extract for regular five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day after induction of diabetes. Initially there was a high AST/ALT Ratio (3.51). The AST/ALT Ratio tended to increase towards normal as the administration period extended. There was a significant increase in AST/ALT Ratio (4.38) at the end of administration with plant extract. A 400 mg/kg plant extract was administered orally to group G. Initially there was a high level of AST (3.85). A significant increase in AST/ALT ratio (4.86) was recorded at the end of administration. The result revealed that the extract has significant role at the dose rate of 400 mg/kg body weight in rabbits. The complete data of five days at the interval of zero day, seventh day, fourteenth day, twenty first day and twenty eighth day administration have been given in table 100.

Table 100: Blood AST/ALT ratio level of rabbits in triplicate

0 day 7th day 14th day 21st day 28th day A. Untreated control (Normal control) 5.12 5.630000 5.75 5.93 6.38 B. Diabetic control (Negative control) 3.82 3.740000 3.45 4.68 3.53 C. Diabetic control + Glibenclamide (10mg/kg)(Standard Control) 3.30 4.920000 4.41 4.61 5.30 D. Diabetic control + Vitamin C (Positive control) 3.89 3.830000 3.70 4.26 3.97 E. Diabetic + Ethanolic extract of H.N 200mg/kg 3.69 3.590000 3.62 3.52 3.35 F. Diabetic + Ethanolic extract of H.N 300mg/kg 3.51 3.850000 4.05 4.25 4.38 G. Diabetic + Ethanolic extract of H.N 400mg/kg 3.85 4.790000 4.76 4.90 4.86

From the graph, it is clear that glucose level significantly increased at day 28. The graph has been shown in figure 65.

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o 6

L 4

T S

A 2

y y y y y a a a a a d d d d d 0 t th th s th 7 4 1 8 1 2 2 T im e (d a y s )

Figure 65: Blood AST/ALT ratio level of rabbits

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Chapter 3 Results Results

Chapter-4 DISCUSSION

155

Chapter # 4 Discussion

4.1 Pharmacognostic studies Pharmacognostic study is intended to assure the quality of the crude drug at various stages of processing. Identification markers were used for this purpose. These characters were conformed by comparing with the data of flora of Pakistan (Qaiser, 2002; Hedge, 1990). The collection, identification, standardization and authentication of plant drug are called pharmacognosy. Microscopic and macroscopic markers help in the identification of crude drug. Different parts of the plant like fruit, leaves, bark, whole stem and roots may be used as a crude drug. In present study the leaves and whole stem of the H. nepalensis were reported as crude drug. Its microscopic and macroscopic characters were confirmed as Hyde (1976) also studied the macroscopic characters like small flowers, fragments of leaves and stem pieces while the microscopic markers were trichomes, pointed terminal cells, glandular trichomes, anomocytic stomata, brown colored bark.

4.1.1 Physiochemical studies Standardization of the crude drug needs determination of the extractive values and ash values. Extractive value assure the purity of the crude drug and shows the quantity of the active constituents of the plants whereas total ash value depend on the size of the particles of the crude drug. Ash value exclude drug which is adulterated by chalk or any other materials (Wallis, 1985). According to the Hyde (1979), 6 % of the total ash is allowed. The highest content of ash for the leaves was calculated as 34.7% and for stem it was 23.5 %. The average ash value for Hedera helix was reported as 8.3 % which contain silica calcium oxalate and other metallic salts (Ibrar, 1998). Unwanted material like scleroids, bark of Glycerrihzae and unpeeled pericarp of colocynth increase the value of ash (Brain and Turner, 1971).

4.1.1.1 Heavy Metals studies Heavy metals constitute a group of metals and metalloids with atomic density greater than 4 g/cm3 or 5 times or more greater than water (Nagajyoti et al., 2010). In present study the leaves and stem of Hedera nepalensis were tested for heavy metals like Iron (Fe), lead (Pb), Manganese (Mn), zinc (Zn), copper (Cu), nickel (Ni) , Chromium (Cr), Cadmium (Cd) and Silver (Ag). Plants use to accumulate heavy metals is a very less cost source called phytoextraction is a great source in reasonable time frame (Nagajyoti et al., 2010; Ajibola and Funtua, 2001; Pehlivan et al., 2009; Wang and Demshar, 1992; Kos et al, 2003; Inuwa et

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Chapter # 4 Discussion al., 2007 and Chaney et al., 1997).Various plants have been reported to store high quantity of metals in their tissue. The heavy metal analysis of the leaves of H. nepalensis reavealed that it contains about 140.41 mg/L of Iron (Fe) while the stem shows 156.43 mg/L of Iron (Fe). Eucalpytus camaldulensis root were found to have iron in sufficient amount (Zavleta-Mancera et al., 2007). Iron is found in enzymes and other proteins (Wood and Ronnenberg, 2006) Heme portion of the hemoglobin is keeping 60% of the Iron whileferritin have 20%, enzymes have 10 % of Iron and 10% of the iron is found in hemosiderin. The heavy metal analysis of the leaves of H. nepalensis shown that it contains about 12.09 mg/L of Manganese (Mn) while the stem shows 123.79 mg/L of Manganese (Mn). Manganese has been reported as a constituent in proteins and enzymes (Schafer, 2004). Wang et al. (2015) found that Manganese accumulation in roots of rice plants is reduced due to alleviation of Aluminium. Hazra et al. (2015) found different level of accumulation of manganese in the leaves, root and stem of Typha latifolia, Eichornia crassipes and Monochoria hastate. The heavy metal analysis of the leaves of Hedera nepalensis revealed that it contains about 56.51 mg/L of lead (Pb) while the stem shows 156.43 mg/L of lead (Pb). Gubrelay et al. (2015) reported that at 10, 30 and 60 mM concentration of lead can reduce phosphatase and power assay of Triticum aestivum.

The heavy metal analysis of the leaves of H. nepalensis displayed that it contains about 3.7 mg/L of zinc (Zn) while the stem shows 20.41 mg/L of zinc (Zn). Zin is required for the synthesis of proteins, cell and genetic material (Sizer and Whitney, 2003). Deepmala et al. 2014) found that heavy metals get stored in dvarious parts of paddy plant (Oryza sativa L.) including the grains. Concentrations of extra toxic heavy metals (Cd, Cr, and Pb) and the micronutrients (Cu, Mn, and Zn) were measured in the paddy field soil and plant parts. Mn and Cd are found to be stored more in shoot than in root. The metal transfer factors from soil to rice plant were high for Pb, Cd, Cu, Cr, Mn, and Zn.The heavy metal analysis of the leaves of H. nepalensis shown that it contains about 1.85 mg/L of copper (Cu) while the stem shows 6.99 mg/L of copper (Cu). Copperi as biocatalyst and is necessary for body pigmentation (Akinyele and Osibnajo, 1982). Jiang et al. (2012) found that under the Cu stress, the Cu concentration and accumulation in the seedling organs were in the order of root > leaf > stem > petiole.

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The heavy metal analysis of the leaves of H. nepalensis displayed that it contains about 1.35 mg/L of nickel (Ni) while the stem shows 4.51 mg/L of nickel (Ni). Those componds which are water soluble are considered as hepatotoxic and nephrotoxic while insoluble are carcinogenic in nature (Sunderman, 2004). Nishida et al. (2015) found that zinc (Zn) deficient conditions resulted in increased accumulation of Ni in plants, particularly in roots, in Arabidopsis thaliana. The heavy metal analysis of the leaves and stem of Hedera nepalensis revealed that it contains no silver accumulation. The heavy metal analysis of the leaves of H. nepalensis displayed that it contains about 0.24 mg/L of Cadmium (Cd) while the stem shows 0.33 mg/L of Cadmium (Cd). Deepmala et al., (2014) reported that Mn and Cd are found to be stored more in shoot than in root of paddy plants.The heavy metal analysis of the leaves of H. nepalensis displayed that it contains about 0.37 mg/L of Chromium (Cr) while the stem shows no Chromium (Cr) accumulation. Chand et al. 2015 found that among heavy metals, magnitude of chromium accumulation was higher than nickel, iron and lead in shoot as well as in root. Roots retardation is perhaps due to high level of chromium (Svetkova and Fargasova, 2007).

Sing et al. (2011) reported that the leaves of Zingiber officinale can synthesize gold and silver nanoparticles of 10 nm in size whereas Mirabilis jalapa flower can synthsize gold particles of 100 nm having spherical shape. Hypoglycemic and hypolipidimic effects have been reported for the aquoes extracts of Cichorium endivia, Sesbania sesban, Scoparia dulcis, Trichosanthes dioica and Withania coagulans (Vankar and Bajpai, 2010; Mishra et al., 2010; Huang et al., 2007; Jaiswal et al., 2009; Kamel et al., 2011; Pandhare et al., 2011; Das and Chakraborty 2011 and Diga et al. 2010). Daisy and Saipriya (2012) reported that the stem bark of Cassia fistula have the ability to synthsize gold nanoparticles which has promising hypoglycemic role in the treatment of Diabetes meilitus. Sidhu and Sharma (2014) investigated that glibenclamide and leaf powder of Ficus krishnae has some common absorption spectra revealing that they have a molecule similar in structure with that of the glibenclamide. Wavelength Dispersive X-Ray Fluorescence spectroscopy shows the similar cellulose, Ca, Si, K, Cl, Mg, P, S, Al, Fe, Na, Sr, Pd, Zn, Mn, Cr, Mo, Br, Ni, Rb and Zr. This shows that the hypoglycemic activity may be due to these smilaities in the leaves of Ficus krishnae.

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4.1.2 Phytochemical Screening The phytochemical screening revealed the presence of carbohydrates, alkaloids, saponins, phenolic compounds, terpenoids, phytosterols, flavonoids, tanins, proteins and amino acids while cardiac glycosides were found absent. The phytochemical analysis of the 3 gm of crude exract of leaves of H. nepalensis displayed that it contains about 3.2 mg/L of carbohydrates while the 3 gm of crude exract of the stem shows 1.3 mg/L of carbohydrates. Syiem and Warjri, 2015) found that the extract of Ixeris gracilis contained carbohydrate (558.189 ± 0.002 mg/g dry wt). Glucose a carbohydrate is the only of energy fro brain, nerve cells, muscles, RBCs and for the whole body (Whitney and Rolfes, 2005; US Department of Agriculture, 2010). The phytochemical analysis of the 3 gm of crude exract of leaves of H. nepalensis revealed that it contains about 2.7 mg/L of proteins while the 3 gm of crude exract of the stem shows 1.9 mg/L of proteins. Syiem and Warjri, 2015) found that the extract of Ixeris gracilis contained protein (4.368 ± 8.916 mg/g dry wt). Breakdown of amino acids results into the availability of nitrogen to the body (Layman et al. 2003). The phytochemical analysis of the 3 gm of crude exract of leaves of H. nepalensis displayed that it contains about 8.9 mg/L of saponins while the 3 gm of crude exract of the stem shows 4.6 mg/L of saponins. Yu et al., 2015 found that the total saponins from Dioscorea nipponica Makino (TSDN) against type 2 diabetes mellitus was used while reducing the fasting blood glucose level. Saponins produce antimicrobials (Margineanu et al., 1976; Okwu 2004). The phytochemical analysis of the 3 gm of crude exract of leaves of H. nepalensis displayed that it contains about 4.7 mg/L of alkaloids while the 3 gm of crude exract of the stem shows 0.7 mg/L of alkaloids. Some type of alkaloids have bactericidal role while other have analgesic and antispasmodic effects (Okwu and Josiah, 2006). The alkaloids have the the ability to stop the growth of Giardia and Entamoeba species (Ghoshal et al., 1996). The phytochemical analysis of the 3 gm of crude exract of leaves of H. nepalensis disclosed that it contains about 2.5mg/L of tanins while the 3 gm of crude exract of the stem shows 3.4 mg/L of a tanins. Phytochemical analysis of Lippia methanolic extract revealed the presence of sterols, saponins, coumarins, quinones, tannins, flavonoids, and reducing sugars (Terblanché and Kornelius, 1996). Tannins have the ability of killing microbes and some type of viruses (Moreman, 1998). Tanins may be used as a remedy for the heavy metals poisoning and in wounds healing (Ogunleye and Ibitoye, 2003).

The phytochemical analysis of the 3 gm of crude exract of leaves of H. nepalensis displayed that it contains about 2.1 mg/L of flavonoids while the 3 gm of crude exract of the

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Chapter # 4 Discussion stem shows 0.76 mg/L of a flavonoids. Hoda et al. (2015) reported the effects of quercetin in cultured hepatocytes in order to expand the understanding of the antidiabetic potential of the flavonoid. Syiem and Warjri (2015) found that the extract of Ixeris gracilis contained flavonoid (70.070 ± 0.626 mg rutin equivalent/g dry wt). The total amount of flavonoids of the extract was estimated to be 1.8% (g/g) on the basis of quercetin content (Pouraboli et al., 2015). These flavonoids have previously been reported to have anti-hyperglycemic activity (Coskun et al., 2005). Flavonoids act on various molecular marks and control different signaling pathways in pancreatic β-cells, hepatocytes, adipocytes, and skeletal myofibers. Flavonoids may exert valuable possessions in diabetes by (i) increasing insulin secretion and reducing apoptosis and promoting proliferation of pancreatic β-cells, (ii) improving hyperglycemia through regulation of glucose metabolism in hepatocytes, (iii) reducing insulin confrontation, inflammation and oxidative stress in muscle and fat, and (iv) increasing glucose uptake in skeletal muscle and white adipose tissue. This review highlights recent findings on the anti-diabetic effects of dietary flavonoids, including flavan-3-ols, flavanones, flavonols, anthocyanidins, flavones, and isoflavones, with particular emphasis on the studies that investigated the cellular and molecular mechanisms involved in the beneficial effects of the compounds (Pon et al., 2013). Due to their antioxidant properties, flavonoids are continually indicated to possess anti-diabetic potential (Rauter et al., 2010). Lupinifolin which is a flavonoid compound has been found to be a major compound of D. reticulata Craib (Chivapatet al., 2009). Phytochemical studies of other plants in Derris genus have been reported. Pyranoflavanones, epoxylupinifolin and dereticulatin were isolated from the stem of D. reticulata Benth (Mahidol et al., 1997). Furanoflavanoids were also isolated from D. indica (Ranga et al., 2009). Scandenin A, scandenin B and isoflavone derivatives have been found in D. scandens (Raoet al., 2007; Mahabusarakam et al., 2004). Total phenolic content determined by Folin-Ciocalteu method was 78.84 ± 0.01 mg GAE/g extract. Antioxidant compounds of D. reticulata extract presumably were tannins and flavonoids found in preliminary phytochemical analysis. Total flavonoid content in the extract, as measured by aluminium chloride colorimetric method was 54.72 ± 1.81 mg catechin/g extract. Several studies have reported that flavonoids possess antioxidant property (Quezada et al., 2004; Zhang et al., 2010). And the hydroxyl groups in flavonoids are responsible for the free radical scavenging activity of these compounds (Kiranmai et al., 2011). The phytochemical analysis of the 3 gm of crude exract of leaves of H. nepalensis displayed that it contains about 2.8 mg/L of terpenoids while the 3 gm of crude exract of the stem shows 0.31 mg/L of terpenoids. A new triterpenoid was also isolated from the methanolic extract of

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Chapter # 4 Discussion the Lippia (Siddiqui et al., 2007). D. laxiflora has revealed that it contains some triterpenoids (Chiu et al., 2008). Terpenoids may used for the treatment of cancer, malaria, ulcer and can be effective against microbes (Bertea et al., 2005; Haudenschild and Croteau, 1998; Lin et al., 2005; MacCaskill and Croteau, 1998; Rodriguez-Concepcion, 2004). The phytochemical analysis of the 3 gm of crude exract of leaves of H. nepalensis revealed that it contains about 2.3 mg/L of phenolic compounds while the 3 gm of crude exract of the stem shows 0.9 mg/L of phenolic compounds. Research have shown that bioactive natural products produced potential physiological activities, due to their polyphenols which act as cytoprotective, anti- inflammatory and hepatoprotective agents, forms an important source for human health research (Arumanayagam and Arunmani, 2015). Hence, these polyphenols present in Lippia nodiflora can act as an antioxidant decreasing the production of reactive oxygen species, thus protect the liver against LPS-induced liver toxicity and oxidative stress (Pietta, 2000).Syiem and Warjri, 2015) found that the extract of Ixeris gracilis contained polyphenol (76.269 ± 0.204 mg GAE/g dry wt). Patra et al., 2015) found that the partial characterization of the methanol extracts of leaf and bark revealed the presence of phenolics as the lead compound responsible for studied bioactivities of the plant extracts. Phenolic compounds are secondary metabolites of plants, which widely distributed throughout the plant kingdom. Phenolics are gaining attention because their antioxidant activities have shown health benefits (Imeh and Khokhar, 2002; Parr and Bolwell, 2000). Plant phenolics have exhibited health protective effects in many ailments, for example inflammation, cancer and hypertension (Middleton et al., 2000). The antioxidant activity of plant materials is well linked with their content of phenolic compounds (Velioglu et al., 1998; Misbah et al., 2013). The phytochemical analysis of the aqueous extract of D. reticulata stem revealed several phenolic constituents which could have potential antidiabetic property as shown in some other herbs. For example, it has been reported that Solanum torvum Swartz extract containing phenolic compounds (rutin, caffeic acid, Gallic acid and catechin) exhibits hypoglycemic activity and is known for their ability to promote β-cell regeneration (Gandhi et al., 2011). The phytochemical analysis of the 3 gm. of crude extract of leaves of H. nepalensis displayed that it contains about 1.9 mg/L of phytosterols while the 3 gm. of crude exract of the stem shows 0.84 mg/L of phytosterols. They have been found in leaves (Jouki and Khazaei, 2010; Babayi et al., 2004) and sterols in fruits of Eucalyptus camaldulensis (Edriss et al., 2012).

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Chapter # 4 Discussion

4.2 Biological Activities of the Plant

4.2.1 Antioxidant activities

4.2.1.1 DPPH free radical scavening activity

The DPPH scavenging activity was studied at the absorbance of 517nm. The absorbance was due the number of the electrons taken up. At increase in concentration absorbance was reduced gradually. The free radical scavenging activity of leaves methanol, ethanol, ethyl acetate, DCM, petroleum ether extracts were more prominent. Concentration of 0.1 mg/ml exhibited 42.72, 26.36, 46.36, 58.18 and 51.81 scavenging activity respectively. Concentration of 0.1 mg/ml exhibited 34.02, 45.13, 31, 53, and 61 scavenging activity respectively. However, DPPH scavenging activity of ascorbic acid at this concentration exhibited marked scavenging activity 72.72 and 76 respectively.

ABTS radical scavenging, DPPH radical scavenging and ferric reducing antioxidant power (FRAP) assays were performed to determine the antioxidant activity of D. reticulata extract. ABTS radical scavenging activity of the extract was 515.05 ± 0.13 μg/ml, whereas that of DPPH scavenging activity was 239.85 ± 0.13 μg/ml. In addition, the FRAP value of the extract was 0.23 ± 0.05 μmol Fe2+/mg dried extract. Together, the results indicate that D. reticulata extract possessed a moderate degree of radical scavenging activities (Pakarang et al., 2015). Barapatre et al. (2015) reported that a deciduous plant Acacia nilotica showed antioxidant activity. Pérez-Ramírez et al. (2015) Hibiscus sabdariffa L. showed DPPH scavenging activity. Patra et al. (2015) found four solvent extracts (acetone, ethanol, methanol and aqueous) of leaf and bark of Sonneratia apetala possess strong antioxidant properties. Juárez-Reyes et al., 2015) found that FM-AE has also antioxidant action effectively trapping ONOO(-) and ROO(•) radicals. Juárez-Reyes et al. (2015) reported thatthe major flavonoids isolated from the plant, namely acacetin (1) and diosmetin (2), caused significant hypoglycemic effect and possessed antioxidant activity. Human bodies keep enzymatic and non-enzymatic antioxidative mechanisms which minimize the cohort of reactive oxygen species, responsible for many deteriorating diseases including diabetes (Patel et al., 2011). In addition, methanolic extract of Origanum vulgare scavenged reactive oxygen and nitrogen species and, therefore, alleviated the need for the up-regulation of antioxidant enzymes (Vujicic et al., 2015). The effect of antioxidants on DPPH radicals is thought to be due to their hydrogen donating ability (Choi et al., 2000). Anup et al. (2014) found that in

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Chapter # 4 Discussion

DPPH free radical scavenging study, almost all the extract showed the positive result. Among them MeOH and water extract showed highest radical scavenging activity. This may be due to the presence of polar compounds like phenols, polyphenols, flavonoids etc which are soluble in water and methanol (Ebrahimzadehet al., 2009). Bibave et al. (2012) have also documented free radical scavenging activity (Antioxidant) with aqueous and methanolic extract of Holarrhena pubescens (200 – 1000 ug/ml) in DPPH assay with ascorbic acid as standard references. Rashid et al., (2014) investigated that the screening for antioxidant activity deduces the malevolent effects of diabetes that have been associated with mediation through the oxidation stress. Rashid et al. (2014) investigated that the methanolic extract from areal parts of Otostigea aucheri (OA). 2,2-Diphenyl-1 -picrylhydrazyl (DPPH) revealed significantly the antioxidant activity. Total phenolic content determined by Folin- Ciocalteu method was 78.84 ± 0.01 mg GAE/g extract. Antioxidant compounds of D. reticulata extract presumably were tannins and flavonoids found in preliminary phytochemical analysis. Total flavonoid content in the extract, as measured by aluminium chloride colorimetric method was 54.72 ± 1.81 mg catechin/g extract. Several studies have reported that flavonoids possess antioxidant property (Quezada et al., 2004; Zhang et al., 2010) and the hydroxyl groups in flavonoids are responsible for the free radical scavenging activity of these compounds (Kiranmai et al., 2011). During liver damage, inflammatory cytokines, such as TNF α, IL1β, and reactive oxygen intermediates through the nuclear factor-kappa β (NF-kB)/COX2 pathway are produced (Kim et al., 2006).

4.2.1.2 Hydrogen Peroxide Free Radical Scavenging Activity Shivapriya et al. (2015) found that the results of the study demonstrated that Hippophae rhamnoides (L.) extract possesses potential free radical scavengingactivity. The IC50 value for DPPH and OH radical scavenging assay was 70.92 μg/ml and 0.463 mg/ml, also the extract was also originate to have considerable level of lipid peroxidation activity. Fernando, C.D. and P.Soysa (2015) used a simple and rapid colorimetric assay where plant extracts are introduced to H2O2, phenol and 4-aminoantipyrine reaction system in the presence of horseradish peroxidase (HRP). This reaction yields a quinoneimine chromogen which can be measured at 504 nm. Decrease in the colour intensity reflects the H2O2 scavenged by the plant material. Optimum conditions determined for this assay were 30 min reaction time, 37 °C, pH 7, enzyme concentration of 1 U/ml and H2O2 concentration of 0.7 mM. The limit of detection (LOD) and limit of quantitation (LOQ) were 136 μM and 411 μM, respectively. The hydrogen peroxide scavenging activity was studied at the

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Chapter # 4 Discussion absorbance of 285nm with average time of 10 minutes incubation. The hydrogen Peroxide radical scavenging activity of leaves methanol, ethanol, ethyl acetate, DCM, petroleum ether extracts was prominent. Concentration of 0.1 mg/ml exhibited 50, 54.54, 47.27, 60, and 70.90 scavenging activity respectively. Concentration of 0.1 mg/ml exhibited 50, 77, 74, 20.66 and 72 scavenging activity respectively. However, DPPH scavenging activity of ascorbic acid at this concentration exhibited marked scavenging activity 80.50 and 85. Khorasani et al. (2105) found that total phenolic and total flavonoid content of some subtropical plants revealed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, superoxide anion radical scavenging and hydrogen peroxide scavenging compared to the other tested extracts. Prihantini et al. (2014) found that Elaeocarpus sylvestris extract had the highest activities on all the antioxidant assays performed such as DPPH scavenging activity (IC50 12.7 ± 0.5 μg mL(-1)), reducing power (491.1 ± 6.3 mg QE g(-1) dry extract), hydrogen peroxide (IC50 65.6 ± 0.4 μg mL(-1)) and β-carotene bleaching assays (IC50 5.1 ± 1.9 μg mL(-1)). Ghate et al. (2015) reported that the antioxidant activity of 70% methanolic extract of D. burmannii (DBME) was evaluated. DBME showed excellent DPPH, hydroxyl, hypochlorous, superoxide, singlet oxygen, nitric oxide, peroxynitrite radical and hydrogen peroxide scavenging activity. A substantial iron chelation (IC50 = 40.90 ± 0.31 μg/ml) and supercoiled DNA protection ([P]50 = 50.41 ± 0.55 μg) were observed. Munazir et al., (2015) found 2,2' diphenyl-1-picrylhydrazyl (DPPH) scavenging and hydrogen peroxide scavenging in aerial parts extracts (O.D. of 2.38) at a concentration of 100μg/ml for the methanolic root and arial parts of Jixueteng plant.

4.2.3 Antimicrobial Activities of H. neplensis 4.2.3.1 Antibacterial Activities of H. neplensis The present work revealed that the stem and leaves of Hedera nepalensis was effective against Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Kleibsiella pheumonia, Erwinia cartovara, Staphylococcus aureus, Bacillus subtilis, BacillB. atrophaeus, Bacillus cereus, Enterococcus faecalis, Shigella sonnei and Citrobacter freundii has no effect. Patra et al. (2015) the extracts of Sonneratia apetala possess strong antibacterial activity against the selected pathogenic bacteria (minimal inhibitory concentration ranging from 1.25-5.00 mg/mL).

Maria et al. (2015) found that all extracts inhibited the growth of Gram-positive bacteria such as Bacillus cereus and Staphylococcus aureus with MICs of 3.3-6.0 mg/ml.

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Gram-negative organisms such as Gram-negative organisms: Escherichia coli, domonas aeruginosa were unaffected by these extracts. Umbellulariacalifornicaextracts (leaves and bark) had the lowest MIC values. Chemical profiling detected the presence of quinones, alkaloids, flavonoids, cardenolides, tannins and saponins in these extracts. Escherichia coli was inhibited by methanol, ethanol, n-hexane, petroleum ether and chloroform extract of leaves while the stem eaxtract of methanol, ethanol and petroleum ether has no effect. Bacillus subtilis is inhibited by methanol, ethanol, n-hexane and extract of leaves while the stem eaxtract of methanol, ethanol, n-hexane, petroleum ether and chloroform has no effect. Kleibsiella pheumonia is inhibited by methanol, ethanol, n-hexane and petroleum ether and extract of leaves while the stem eaxtract of methanol and chloroform has no effect. Arumanayagam and Arunmani (2015) noticed that in agar disc diffusion method, S. aureus was highly inhibited, followed by E. coli, B. subtilis and K. pneumonia using methanolic extract of Lippia nodiflora L. leaves and there was no inhibition zone around the control DMSO4.

Chang et al. (2015) use commercial rice wine extracts obtained from different plant parts of Allium fistulosum againstgram-negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa giving a fuitful result. Pseudomonas aeruginosa is inhibited by methanol, ethanol, petroleum ether and chloroform extract of leaves while the stem eaxtract of methanol, ethanol, n-hexane, petroleum ether and chloroform has no effect. Salmonella typhi is inhibited by ethanol, petroleum ether and chloroform extract of leaves while the stem eaxtract of methanol, ethanol, n-hexane, petroleum ether and chloroform has no effect. Kuppusamy et al. (2015) studied that the aqueous crude extract of Commelina nudiflora L. shows the highest inhibitory activity against Pseudomonas aeruginosa, Escherichia coli and Salmonella typhi.

All fractions (Butanol, ethyl acetate, Hexane, methanol, water and chloroform) of Catharanthus roseus were effective against plant pathogen i.e. E. carotovora (Bakht et al., 2015). Erwinia cartovara is inhibited by methanol, ethanol and petroleum ether and extract of leaves while the stem eaxtract of methanol, n-hexane and chloroform has no effect. Zare et al., (2012) also reported that S. aureus was inhibited by the methanolic extract of leaf followed by B. subtilis. These two bacteria are principle among other bacterial populations causing infections in cirrhosis related death. Packer et al. (2015) found that the extracts of Corymbia intermedia, Lophostemon suaveolens and Syncarpia glomulifera had promising levels of antimicrobial activity (MIC 31-1,000 µg/mL) against both antibiotic sensitive and

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Chapter # 4 Discussion resistant Staphylococcus aureus. Staphylococcus aureus is inhibited by ethanol, n-hexane and chloroform extract of leaves while the stem eaxtract of methanol, ethanol, petroleum ether and chloroform has no effect (Wong et al., 2005). Thus, these results confirmed that Lippia nodiflora L. can be used as a source to improve the treatment of infection caused by these organisms. Khan et al. (2015) investigated that Alkanna tinctoria leaves extract showed potential activity against S. aureus as compare to positive control antibiotic (Imipenem).

Bacillus atrophaeusis inhibited by methanol, ethanol, n-hexane, petroleum ether and extract of leaves while the stem eaxtract of methanol, petroleum ether and chloroform has no effect. Bacillus cereus is inhibited by methanol, n-hexane and chloroform extract of leaves while the stem eaxtract of methanol, ethanol, n-hexane and petroleum ether has no effect. Van et al. (2015) found that the organic extract of Terminalia sericea showed the most prominent noteworthy antibacterial activity (mean MIC value of 0.04 mg/mL) with Bacillus cereus (ATCC 11778), and Enterococcus faecalis. Enterococcus faecalis is inhibited by methanol and chloroform extract of leaves while the stem eaxtract of ethanol, n-hexane and petroleum ether. Shigella sonnei is inhibited by methanol and petroleum ether an extract of leaves while the stem extract of ethanol, n-hexane and chloroform has no effect. de Castilho et al. (2014) found that three extracts obtained of Ipomoea alba (MIC < 40 μg/mL), Diclinanona calycina (MIC ≤ 40 μg/mL) and Moronobea coccinea (40 < MIC < 80 μg/mL; MBC = 80 μg/mL) showed noteworthy bactericidal activity against Enterococcus faecalis and four extracts obtained from I. alba (14.04 ± 0.55 mm diameter) Symphonia globulifera (14.43 ± 0.33 mm and 12.18 ± 0.28 mm diameter) and Connarus ruber var. ruber (13.13 ± 0.18 mm diameter) against Enterococcus faecalis.Citrobacter freundii is inhibited by methanol, ethanol, n-hexane and chloroform extract of leaves while the stem eaxtract of methanol, n-hexane and petroleum ether has no effect. Agrobacterium tumefacians has not been affected by any of the plant extracts.

4.2.3.1 Antifungal Activities of H. neplensis The present work revealed that the stem and leaves of Hedera nepalensis was effective against Candida albican, Aspergillus niger, Aspergillus flavus, Alternaria alternate, Penicillium notatum and Trichoderma harzianum.

Packer et al. (2015) found that the extracts of Corymbia intermedia, Lophostemon suaveolens and Syncarpia glomulifera had promising levels of antimicrobial activity (MIC 31-1,000 µg/mL) against both antibiotic sensitivefungus Candida albicans (clinical isolate).

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Chapter # 4 Discussion

Candida albican was inhibited by ethanol, n-hexane, petroleum ether and chloroform extract of leaves while the stem eaxtract of chloroform. Satyal et al. (2015) found that Nardostachys grandiflora rhizome oil showed in-vitro antimicrobial activity against Bacillus cereus, Escherichia coli, and Candida albicans (MIC = 156 μg/mL). Zubrická et al. (2015) reported highest xanthone contents in Hypericum pulchrum and H. annulatum untransformed roots. The best anti- Candida activity was obtained for hairy roots extracts of H. tetrapterum clone 2 ATCC 15834. Extracts of root cultures, hairy roots and cell suspensions of selected Hypericum spp. were screened for the presence of xanthones and tested for their antifungal activity against Candida albicans. Two xanthones, cadensin G and paxanthone, were identified in cell suspension cultures of H. perforatum. The anti-Candida activity of the obtained extracts ranged from MIC 64 to >256 µg ml-1. Among the extracts of Hypericum untransformed roots, the best antifungal activity was obtained for extracts of H. annulatum grown under CD conditions. Extracts of hairy roots clones A4 and 7 ATCC15834 of H. tomentosum and clone 2 ATCC15834 of H. tetrapterum displayed inhibition of 90 % of Candida growth with 256 μg ml-1. Buchenavia tomentosa extracts showed antifungal activity on Candida species. Candida non-albicans were more susceptible than Candida albicans. Low cytotoxicity for extract was observed. The isolated compounds presented antifungal activity at least against one Candida spp. and all compounds presented antifungal effect on Candida glabrata (Teodoro et al., 2015). Abdullah et al. (2015) conducted the antimicrobial activities of Pittosporum tetraspermum and found that the compound isolated was active against bacteria such as Staphylococcus epidermidis (125 µg/mL), Staphylococcus aureus (125 µg/mL), and Klebsiella peumoniae (62.5 µg/mL). The MIC of the compound against Candida albicans, Aspergillus niger and Trichophyton mentagrophytes was 62.5, 125, and 500 µg/mL, respectively. Antimicrobial activity of the pure compounds were identified through physico-chemical, NMR (1D, 2D) and mass spectrometric studies that consequences against Staphylococcus aureus, Escherichia coli and Candida albicans strains. From our literature survey, it appears that all pure compounds except 2 were isolated and reported for the first time in H. scabrum (Jiang et al., 2015). Ratnaweera et al. (2015) found that the most bioactive fungus was identified as Fusarium sp. and the second most active as Aspergillus niger. Methanolic extracts of Vinca rosea of antifungal activity against (Asprgillus niger, Alternariasolani and Rhizopus oryzae) was maximum zone of inhibition (34.6mm ± 0.57(a) was formed by in vitro leaf callus extract and MIC value is 6.0mg/ml against A. niger (Naz et al., 2015). Aspergillus niger was inhibited by methanol, ethanol, n-hexane, petroleum ether and chloroform extract of leaves while the stem eaxtract has no effect against these fungi.

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Chapter # 4 Discussion

Golubović et al. (2014) used disk diffusion method for the estimation of the antimicrobial activities of the extracts of Acinos miller species.

Aspergillus flavus was inhibited by methanol, ethanol extract of leaves while the stem eaxtract of n-hexane and petroleum ether. The antimicrobial potential of 5 Ganoderma lucidum (the lingzhi or reishi medicinal mushroom) samples against 5 fungal pathogens (Fusarium oxysporum, Aspergillus niger, A. flavus, Penicillium sp. and Alternaria alternata) was observed. The lowest biomass reduction (7%) was observed in 1% and 2% concentrations of a methanolic extract and 6% in the case of a water extract. Major inhibition was observed using higher concentrations of the methanolic extract (3% and 4%). These extracts significantly suppressed fungal biomass up to 38% and 56% in A. niger, 47% in A. flavus, 58% in, i> Penicillium sp., 46% in A. alternaria, and 45% in F. oxysporum compared with the control. It was concluded from these studies that methanolic extracts of G. lucidum showed better activity against all plant fungal pathogens when compared with the water extracts (Baig et al., 2015). Alternaria alternata was inhibited by methanol and petroleum ether extract of leaves while the stem eaxtract of methanol, ethanol, n-hexane, petroleum ether and chloroform. Rahman et al. (2015) reported that the crude, n-hexane, and chloroform fractions of Oxalis corniculata were also found to have momentous activity against fungal strains including Fusarium solani, Aspergillus flexneri, and Aspergillus flavus. Singh et al., (2003) reported that the inhibition zones of isolated terpenoids from Trichodesma amplexicaule Roth were also recorded and the activity index was calculated and it was found that hexacosane was more vigorous (IZ = 13.39) against E. coli and hexacosanoic acid had greater activity against A. flavus (IZ = 11.56). Patil et al., (2015) reported production of bioactive flavonoid by endophytic Aspergillus flavus obtained from A. marmelos and its pharmaceutical potential. Rashid et al., (2014) evaluated the antifungal and cytotoxic activities of the Nannorrhops ritchiana (Mazari Palm) 80% methanol extract (NR- M) and its four crude extracts i.e., petroleum ether (NR-A), dichloromethane (NR-B), ethyl acetate (NR-C) and butanol (NR-D). The antifungal activity was determined by agar tube dilution method against nine fungal strains; Aspergillus flavus, Trichophyton longifusis, Trichophyton mentagrophytes, Aspergillus flavus and Microsporum canis were susceptible to the extracts with percentage inhibition of (70-80%). Extracts exhibited significant and good antifungal activity against various fungal strains. Chitarrini et al., (2014) reported that the viable achenes of two buckwheat species, Aspergillus tataricum (var. Golden) and F. esculentum (var. Aelita) were inoculated with an AFB1-producing A. flavus NRRL 3357 to

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Chapter # 4 Discussion analyze their relative performances against fungal invasion and toxin contamination. Soliman et al. (2014) reported the (MIC) of volatile oil of the juvenile leaves against Escherichia coli, Pseudomonas aeruginosa, Streptococcus faecalis, Candida albicans, and Aspergillus flavus were 5.2, 5.6, 4, 4.8, and 12.8 μg/ml, respectively. The potential applications of biosynthesized nanoparticles using fresh extracts of Tridax procumbens linn as antimicrobial (antibacterial and antifungal) against pathogens Escherichia coli, Vibrio cholerae, Aspergillus niger and Aspergillus flavus were demonstrated (Bhati-Kushwaha et al., 2014). Ahmad et al. (2011) found that the crude methanolic extract and various fractions of Zizyphus jujuba were screened for antifungal, cytotoxic, antitermite and insecticidal activities. Low activity was shown by the crude methanolic extract (12%), n-hexane (9%), chloroform (20%) and ethyl acetate (14%) fraction against Penicillium notatum. Low activity was shown by the n-hexane fraction against Aspergillus niger (10%) and Trichoderma harzianum (13%) and inactive against Aspergillus flavus, Fusarium oxysporum and Rhizopus stolonifer. Trichoderma harzianum was inhibited by methanol, ethanol, n-hexane, petroleum ether and chloroform extract of leaves while the stem eaxtract of methanol, ethanol, n- hexane, petroleum ether and chloroform. Lanzotti et al. (2012) reported that isolated compounds from the bulbs of garlic, Allium sativum L., var. Voghiera which were active aginst Trichoderma harzianum. Both fungi were stopped significantly by Allium sativum L. var. Voghiera. As an inhibitor of mycelial growth, a coumarin from Mammea longifolia showed strongest activity against Rhizoctonia solani and Botrytis cinerea. Inhibitory effects were less pronounced in Alternaria dauci, Fusarium oxysporum and Penicillium sp. and absent in Trichoderma harzianum (Deng et al., 2005).

4.3 Pharmacological activities 4.3.1 Toxicological studies

Acute toxicity (LD50 ) was performed in mice (Kärber, 1931). Giordani et al. (2015) reported that the extract showed low acute toxicity. Ezeja et al., 2015) found the LD50 of G. longipetala was found to be > 4000 mg/kg. Arumanayagam and Arunmani (2015) studies showed that the crude extract of Lippia nodiflora above 800 mg/L was found to be fatal, while 400 mg/L was found to be a protective effect and observed that oral direction of the ethanolic extract of Lippia nodiflora in the doses less than 7.5 g/kg b.wt. failed to kill rabbits

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Chapter # 4 Discussion within 24 hours. This is similar to the lethal dose studied by Salwa et al. (2009) on Artemisia judiaca which kill mice at the dose rate of 9.17 g/kg b.wt.

4.3.2 Antidiabetic activity

Diabetus mellitus is a metabolic disease that damages cells of the Langerhan islets, as a result the body become unable to produce a pancreatic hypoglycemic hormone, insulin. This disorder is pigeonholed by three symptoms: Polyuria (more urine); glycosauria (urine having glucose) and hyperglycemia (glucose rate on an empty stomach higher than normal (Koffi et al., 2009). Diabetes is characterized by hyperglycemia together with biochemical alteration of glucose and lipid metabolism (Rajasekaran et al., 2006). Diabetes mellitus is pigeonholed by distorted lipid metabolism, with increase in blood lipids. A considerable reduction was observed in groups treated with P. guajava (100, 250 mg/kg) compared to glibenclamide group and thus reductions with other groups when compared with the diabetic untreated group (Victor et al., 2014). Diabetes mellitus, the endocrine disorder may cause death even in sever case hitches and even death of the patient (Patel et al., 2011). The effect of diabetes mellitus on lipid break down is well established. It has been found in experimentally induced diabetic animals that significant changes in lipid metabolism and structure are associated with diabetes (Sochar et al., 1985). It is known that alloxan induces diabetes mellitus by generating free radical, resulting in its toxic action on pancreatic β-cells (Frode and Medeiros; Lenzen, 2008). The results suggest that the cytoprotective effect of D. reticulata extract against alloxan may be partly derived from the free radical scavenging activity of its antioxidant compounds (Pakarang et al., 2015). The aqueous extract of D. reticulata stem was previously reported to possess potential antidiabetic property in rats (Kumkrai et al., 2014). Flavonoids and triterpenoids, the two major types of the compounds found in Potentilla discolor extract have protective effects on β-cells in diabetic rats (Zhang et al., 2010). Barapatre et al. (2015) reported a deciduous plant Acacia nilotica showed antidiabetic activity. Jesus et al. (2014) reported Genista tenera as glucose lowering plant. Vujicic et al., (2015) found that the MOE reduced diabetes incidence and preserved normal insulin secretion. Mohamed et al., (2015) fenugreek, Nigella, and termis seeds were found to ehibit a definite improvement in the number and diameter of β-cells in the diabetic group was observed (Tahira and Hussain, 2014). To investigate hypoglycaemic, hypolipidaemic and pancreatic beta cell regeneration activities of Momordica charantia L fruits. Alloxan induced diabetic rabbits were treated with methanolic and ethanolic Momordica charantia extract.

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Chapter # 4 Discussion

Effects of plant extracts and the drug glibenclamide on serum glucose, lipid profile and pancreatic beta cell were determined after two weeks of treatment. Serum glucose and lipid profiles were assayed by kit methods. Pancreatic tissue histopathology was performed to study pancreatic beta cells regeneration. Momordica charantia extracts produced significant hypoglycaemic effects (p < 0.05). Hypolipidaemic activity of Momordica charantia was negligible. Momordica charantia supplementations were unable to normalize glucose and lipid profiles. Glibenclamide, a standard drug, not only lowered the hyperglycaemia and hyperlipidaemia but also restored the normal levels. Regeneration of pancreatic beta cells by MC extracts was minimal, with fractional improvement produced by glibenclamide. The most significant finding of the present study was a 28% reduction in hyperglycaemia by Momordica charantia ethanol extracts. Antidiabetic potentials of Momordica charantia, identification of the relevant antidiabetic components and underlying mechanisms were studied. C. umbellata has been used as a traditional medicinal plant for treating stomach disorders and abdominal pains (Ramesh et al., 1999). Insulin and oral used agents like sulfonylureas, glinides and biguanides are the most effective therapies for the treatment of diabetes (Saxena and Vikram, 2004).However other traditional claims and possible pharmacological benefits of C. umbellata were not been explored. Other species from the same genus were reported to have different biological properties, which suggest the therapeutic potential of caralluma genus. Shi et al., 2014) The roots, fruits, seeds, rinds and leaves ethnolic, methanolic, or aqueous extracts of Citrullus colocynthis showed antihyperglycemic activity. C. umbellata showed promising antidiabetic properties, especially the methanolic extract of whole plant (MCU) exhibited potential glucose uptake enhancement in myotubes, and hence this extract may regulate the blood glucose level (Kawanoa et al., 2009). Comparable significant activities were also reported from other medicinal plants such as A. cepa, Z .officinale, G. lucidumshowing promising activity up to 95% glucose uptake over control, hence suggesting the potential use of medicinal plants for diabetic treatment (Noipha et al., 2010; Jung et al., 2010). Polyphenols have been reported to have antidiabetic effect and such polyphenols have been found highly in methanolic extracts as suggested by various authors (Khoddami et al., 2013). However the exact chemical constituents which may be responsible for hypoglycemic effect of extracts still remains speculative. Previous studies also support this data where alcoholic extracts from different species of similar genus, C. attenuata, C. fimbriataand C. sinaica, had shown potent antidiabetic properties (Latha et al., 2004: Sousa et al., 2004). The antidiabetic effect of Psidium guajava leaves is connected to their phytochemical constituent such as the various

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Chapter # 4 Discussion flavonoids, terpenoids, and glycosides as reported by (Okigbo et al., 2005; Ranilla et al., 2010; Cheng and Yang, 1983; Kaneto et al., 2005). The antidiabetic effect of Psidium guajava leaves is connected to their phytochemical constituent such as the various flavonoids, terpenoids, and glycosides as reported by (Okigbo et al., 2005; Ranilla et al., 2010; Cheng and Yang, 1983; Kaneto et al., 2005). Traditional agents of antidiabetic are more effective than the new drugs (Jung et al., 2006).

4.3.2.1 Effect of Multiple doses of extracts of H. neplensis on blood glucose level in Diabetic Rabbits The obtained results showed that multiple oral administration of the tested doses of ethanolic leaves extract at the dose rate of 200 mg/kg, 300 mg/kg, 400 mg/kg, Vitamin C as positive control and glibinclamide (20 mg/kg b/wt.), significantly (p < 0.05) fall the blood glucose level at seventh, fourteenth, twenty first, and twenty eighth day after amedication; when compared to diabetic non-treated group. Whereas the ethanolic stem extract has also reduced glucose level less significantly as compared to ethanol leaves extract. The same results are supported by the results obtained by Pouraboli et al. (2015) using the Dracocephalum polychaetum extract (300 mg/kg) which decreased serum glucose level (27.1%) significantly at 120 min in OGTT. Pre-treatments with D. reticulata extract at the doses of 50–500 μg/ml provided a significant protective effect from alloxan-induced RINm5F cell death (Pakarang et al., 2015). RINm5F cells treated with glibenclamide showed a significant increase in insulin secretion (Pakarang et al., 2015). In contrast, D. reticulata extract at the doses of 250 and 500 μg/ml did not possess a stimulatory effect on insulin release. This result suggests that the extract may have some advantages over glibenclamide or other insulin secretagogues in terms of causing fewer clinical events of hypoglycemia (Pakarang et al., 2015). Victor et al. (2014) the effect of plants extracts on blood glucose concentration, which records glucose level relative to time and dose as seen in the trend of decline. This was also studied by Florence et al. (2007) that there is momentous drop in the blood glucose level and biochemical parameters in normal and streptozotocin induced rats, treated with Dorstenia picta methanolic extract. The aqueous and methanolic extract of root of Musa paradisiaca and leaf of Coccinia indica had shown a significant result when Mallick et al. (2007) given at a concentration of 80 mg/100g body weight/day in separate as well as in combined form in a streptozotocin-induced diabetic rat. Similar results have been investigated by Nagappa et al. (2003) while studying the anitdiabetic activity of methanolic and aqueous extracts of Terminalia catappa Linn. (combretaceae) which showed

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Chapter # 4 Discussion a momentous drop in glucose level. Leaf extract of Viscum album in normal and streptozotocin induced Diabetic rats reported by Nwaegerue et al. (2007). Glibenclamide, an insulin secretagogue, was selected as positive control. Glibenclamide stimulates insulin secretion by blocking ATP-sensitive potassium channels of the β-cell membrane, thereby causing depolarization calcium influx, and rising in cytoplasmic calcium concentration (Nolte et al., 2012). Boudjelal et al. (2015) used 30 mg/kg b. wt. aqueous infusions of A. herba- alba and A. iva which has reduced the blood glucose level as compared to glibenclamide significantly. Pakarang et al. (2015) found the results revealed that the extract, which consisted of terpenoids, saponins, tannins and flavonoids, possessed moderate radical scavenging activities. Pre-treatment of RINm5F cells with the extract was also found to exert moderate, but significant, in vitro protection against alloxan, an oxidative stress producing agent. Unlike glibenclamide, the extract did not stimulate insulin secretion. However, the extract was found to inhibit α-glucosidase activity similar to acarbose. The same results are supported by the results obtained by Pouraboli et al. (2015) using the Dracocephalum polychaetum extract (300 mg/kg) which decreased serum glucose level (27.1%) significantly at 120 min in OGTT. Serum levels of creatinine, triglycerides, cholesterol, alanine amino transferase and MDA levels in plasma, RBCs, and pancreas significantly decreased in treated (300 mg/kg) diabetic rats. The blood sugar levels in Group SA100, Group SA 200 and Group SA 400 were significantly (p < 0.05) reduced after 5, 10, 15 days of treatment. Group SA 200 and Group SA 400 rats showed significant glucose lowering efficacy between days 10–15 and the effects were dose-dependent and comparable to the effect of diabetic standard metformin. The report for OGTT investigation of same extract showed that the ethanolic extract of S. anacardium reduced blood sugar meaningfully in glucose loaded hyperglycemic rats and produced more forceful hypoglycemia in alloxan induced diabetic rats (Ali et al., 2012). Oral Administration of extract significantly (p < 0.05) reduced the total cholesterol, triglycerides and LDL-CH levels and increased HDL-CH in Groups SA 100, SA 200 and SA 400 and all the effects were dose-dependent. Among the treatment groups Group SA400 significantly improved dyslipidemia. The ethanol extract of the plant S. anacardium bark exhibited significant antihyperglycemic and antihyperlipidemic activity in alloxan-induced diabetic rats, and the activity was comparable to that of the standard antidiabetic drug, Metformin HCl (150 mg/kg b. wt.). Ali et al. (2012) and Supkamonseni et al. (2014) found that the C. asiatica extract (1000 and 2000 mg/4 mL/kg) significantly declined plasma glucose level in lipid emulsion-induced hyperlipidemic rats at 3 h efectively. Rashid et al., (2014) the methanolic extract (1.25 g/kg) significantly lowered (p < 0.005) serum glucose

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Chapter # 4 Discussion level in type II diabetic (NIDDM) models when simultaneous glucose was administered. Sidhu, M.C and T. Sharma (2014) the petroleum ether leaf extract of Ficus krishnae was administered continuously for 21 days orally at a dose of 200 mg/kg. It has been observed that the leaves of Ficus krishnae possess antidiabetic activity and it reduces the blood glucose level significantly. Ezeja et al. (2015) The extract significantly (p < 0.0001) decreased the fasting blood sugar levels of treated rats from 16.2 ± 2.03 to 6.5 ± 1.52 mM/L at 150 mg/kg within 24 h. Soussi et al. (2009) examined the antihyperglycemic/hypoglycemic effect of Eucalyptus globulus in diabetic mice, whereby dietary administration of these plants ameliorated loss of body weight, polydipsia, and blood glucose level in streptozotocin- diabetic mice. This effect is linked to the protection or regeneration of pancreatic β-cell following the exposure to the diabetogen-streptozotocin and its action by modulation of insulin secretion. Also in the works of Soussi et al. (2009) the pancreatic effects of Eucalyptus globulus extract and their blood glucose lowering ability were reported. Victor et al., (2014) reported that the antioxidant activity along with antihyperglycemic effects of H. pubescens can reduce the oxidative stress and lower blood glucose and these studies were related to the studies conducted by (Bandawaneet al., 2013; Bibave et al., 2012). Bandawane et al. (2013) documented that methanolic extract of H. pubescens bark (200, 400 mg/kg) significantly (p<0.01) reduced the increase in blood glucose levels at 60, 90, and 120 min in glucose loaded rats when compared with diabetic control rats. Similar findings of antihyperglycemic effect were documented by Bibave et al. (2012) used aqueous and methanolic extract of H. pubescens bark which reduced the blood glucose leve in diabetic rats. Hypoglycemic plants are found to contain polysac- charides, polyphenols and the various experimental results indicate that these compounds increase the levels of serum insulin, reduce the blood glucose levels and improve tolerance of glucose (Sah et al., 2010). In the recent years, the positive antioxidant effects of some antidiabetic herbal products are also established (Tabatabaei-Malazy et al., 2012). Victor et al. (2014) found that the fruits of Xylopia aethiopica have been reported to possess hypoglycemia ability as well as other biochemical activities, thus confirming its usage as an antidiabetic agent (Ameyaw and Owusu-Ansah, 1998). This was further supported by the polyherbal formula of Shen et al., 2008) which composed of X. aethiopica as one of its component, which showed a significant reduction in plasma blood glucose upon the administration of alcoholic extracts of polyherbal formula. This was found to have done better than glibenclamide. Also, in 2008 Shen et al. found a momentous drop in plasma glucose of diabetic Wister rats upon the administration/treatment with plant extracts where X. aethiopica was one of the extracts. The

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Chapter # 4 Discussion reduction in blood glucose was found to be dose dependent. It showed greater reduction in plasma blood glucose with synergic formulations of extracts A. occidentale + E. globulus compared to that of P. guajava + X. aethiopica and glibenclamide. This is further buttressed by the synergetic works of Shen et al., 2008, on the polyherbal formulation that showed effective reductions in plasma glucose concentrations of diabetic rats than that of glibenclamide. A factor responsible for this could be the synergistic interaction of some phytochemical components such as polyphenols and tannins. Victor et al. (2014). STZ causes hyperglycemia after 2 h of injection, hypoglycemia in 6 h and finally hyperglycemia by declining the insulin levels through the inhibition/destruction of pancreatic beta cell function (Rabbani et al., 2010; Szkudelski, 2001). Shewamene et al. (2015, Inter-group analysis revealed that O. integrifolia at 100 mg/kg and 200 mg/kg reduced the 4(th) hour fasting blood BGL significantly (p < 0.001) compared to the control group. The intra-group analysis result has shown O. integrifolia at 200 mg/kg produced a significant (p < 0.05) reduction in BGL at the 1(st), 2(nd), 3rd and 4th hours of post treatment compared to their respective initial levels. Moreover, in the hypoglycemicand OGTT models, O. integrifolia extract at 200 mg/kg, has shown a significant reduction in blood glucose levels compared to negative controls and across all time points. The hydroalcoholic extract of O. tenuiflorum exhibited significant antidiabetic and anti-hyperlipidemic activities against STZ– and NIC– induced diabetic rats at the dose levels of 250 and 500 mg/kg b.wt. The effect was comparable with glibenclamide but not superior to it. Glibenclamide reduced the glucose levels from 226.40 ± 8.33 to 112.80 ± 5.75, whereas O. tenuiflorum 500 mg/kg reduced the glucose levels form 229.80 ± 10.00 to 129.00 ± 13.20. O. tenuiflorum exhibited significant anti-diabetic effect but the effect was not superior than glibenclamide. This may be due to the amount of active phytoconstituents present in the plant. Giordani et al., 2015) HeECo exhibited inhibitory activity against α- glucosidase and caused a lowering in the peak levels of blood glucose in animals that received glucose overload by 36.7% and 24.1%. Syiem D, Warjri P, 2015) found that the extract displayed varying hypoglycemic activity. The dose of 250 mg/kg body weight exhibited potent antihyperglycemic activity and improved glucose tolerance. Syiem D and P. Warjri, 2015, Ixeris gracilis at the dose rate of 250 mg/kg body weight exhibited potent antihyperglycemic activity and improved glucose tolerance. Juárez-Reyes et al. (2015) The results demonstrated that the extracts and compounds from Anoda cristata were effective for reducing blood glucose levels in healthy and NA-STZ-hyperglycemic mice when compared with vehicle groups (p<0.05). Tahira and Hussain, 2014) investigated hypoglycaemic, hypolipidaemic and pancreatic beta cell regeneration activities of Momordica charantia L.

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Chapter # 4 Discussion fruits. Alloxan induced diabetic rabbits were treated with methanolic and ethanolic Momordica charantia extract. Effects of plant extracts and the drug glibenclamide on serum glucose, lipid profile and pancreatic beta cell were determined after two weeks of treatment. Serum glucose and lipid profiles were assayed by kit methods. Pancreatic tissue histopathology was performed to study pancreatic beta cells regeneration. Momordica charantia extracts produced significant hypoglycaemic effects (p < 0.05). Hypolipidaemic activity of Momordica charantia was negligible. Momordica charantia supplementations were unable to normalize glucose and lipid profiles. Glibenclamide, a standard drug, not only lowered the hyperglycaemia and hyperlipidaemia but also restored the normal levels. Regeneration of pancreatic beta cells by Momordica charantia extracts was minimal, with fractional improvement produced by glibenclamide. The most significant finding of the present study was a 28% reduction in hyperglycaemia by Momordica charantia ethanol extracts. Reliable antidiabetic potentials of MC, identification of the relevant antidiabetic components and underlying mechanisms were studied. Moreover, in the hypoglycemic and OGTT models, O. integrifolia extract at 200 mg/kg, has shown a noteworthy decline in blood glucose levels compared to negative controls and across all time points (Shewamene et al., 2015). However, the individual phytoconstituents of O. tenuiflorum such as ursolic acid (derivatives) and rosmarinic acid are known to have antidiabetic activities (Hasanein et al., 2014; Wu et al., 2014). Assad et al. (2014) The methanol extract of Brassica oleracea decreased significantly the fasting blood glucose 106.6 mg/dL as compared to diabetic control.

4.3.2.2 Effect of Multiple doses of extracts of H. neplensis on blood triglyceride level in Diabetic Rabbits The obtained results showed that multiple oral administration of the verified doses of ethanolic leaves extract at the dose rate of 200 mg/kg, 300 mg/kg, 400 mg/kg, Vitamin C as positive control and glibinclamide (20 mg/kg b/wt.), significantly (p < 0.05) decreased the blood triglyceride level at seventh, fourteenth, twenty first, and twenty eighth day after administration; when compared to diabetic non-treated group whereas the ethanolic stem extract has also reduced triglyceride level less significantly as compared to ethanol leaves extract. The same results are supported by the results obtained by Pouraboli et al. (2015) using the Oral Administration of Dracocephalum polychaetum extract (300 mg/kg) which significantly (p < 0.05) reduced the triglycerides level in alloxan-induced diabetic rats showing antihypertriglycedemic activity of the plant. Supkamonseni et al. (2014) reported

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Chapter # 4 Discussion that the C. asiatica extract (1000 and 2000 mg/4 mL/kg) significantly decreased plasma triglyceride level in rats. Inhibiting pancreatic lipase reflects in reduced absorption of monoglycerides and fatty acids (Lowe et al., 1994; Kim et al., 2011).

4.3.2.3 Effect of Multiple doses of extracts of H. neplensis on blood Cholesterol level in Diabetic Rabbits The attained results disclosed that multiple oral administration of the tested doses of ethanolic extract and glibinclamide (20 mg/kg b/wt.), significantly decreased the blood cholestrol level at seventh, fourteenth, twenty first, and twenty eighth day post administration; when compared to diabetic non-treated group whereas the ethanolic stem extract has also reduced glucose level less significantly as compared to ethanol leaves extract. The same results are supported by the results obtained by Pouraboli et al. (2015) using the Oral Administration of Dracocephalum polychaetum extract (300 mg/kg) which meaningfully (p < 0.05) reduced the cholestrol level in alloxan-induced diabetic rats showing antihypercholestrolemic activity of the plant. Similar observations were obtained by Sharma et. Al (1955) with nuts of the same plant in cholesterol fed rabbits. Pouraboli et al., (2015) using the Oral Administration of Dracocephalum polychaetum extract (300 mg/kg) which meaningfully (p < 0.05) reduced the cholesterol level in alloxan-induced diabetic rats showing hypocholestrolemic activity of the plant. Supkamonseni et al., 2014) reported that the C. asiatica extract (1000 and 2000 mg/4 mL/kg) significantly decreased plasma total cholesterol level in hyperlipidemic rats.

4.3.2.4 Effect of Multiple doses of extracts of H. neplensis on Kidney functions 4.3.2.4.1 Creatinin Singh et al. (2013) found that oral administration of assia sopherain extract inhibited STZ-induced increase in lipid peroxidation (LPO), aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), bilirubin, creatinine and urea in liver of diabetic rats. The attained results displayed that multiple oral administration of the tested doses of ethanolic extract and glibinclamide (20 mg/kg b/wt.), significantly decreased the blood creatinine level at seventh, fourteenth, twenty first, and twenty eighth day post administration; when compared to diabetic non-treated group. The same results reported as that there were no momentous increase in serum creatinine in rats after daily oral administration of ethanolic extracts of Artemisia dracunculus (Ribnicky et al., 2004). Honmore et al. (2015) reported that pretreatment with Artemisia pallens extract at the dose

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Chapter # 4 Discussion rate of (200 and 400 mg/kg) significantly (p < 0.01 and p < 0.001) decreased aspartate transaminase (AST), alanine transaminase (ALT), bilirubin, blood urea nitrogen (BUN), and serum creatinine as compared with APAP-treated rat. Pouraboli et al. (2015) using the Oral Administration of Dracocephalum polychaetum extract (300 mg/kg) which significantly (p < 0.05) reduced the creatinine level in alloxan-induced diabetic rats showing hypo- creatinine activity of the plant. Arulselvanet al. (2006) reported that ethanolic extract of M. koenigii when used at the dose rate of 200 mg/kg/b.w./day for a period of 30 days can reduce blood creatinine level significantly.

4.3.2.5 Effect of Multiple doses of extracts of H. neplensis on liver functions The increased absolute organ weight of liver was observed in diabetic animals, and this may be due to cellular damage in the liver because of increasing resistance to insulin signaling pathways in hepatocytes (Kohl et al., 2013). There was increased kidney weight: BW ratio (results were not significant) found in diabetes mellitus animals. This may be due to glomerular damage, changes in bradykinin system and increased gene expression of fibronectin and collagen I (Ma et al., 2004; Kakoki et al., 2004). In vivo study revealed that hydroalcoholic extract of O. tenuiflorum possesses the anti-diabetic and anti-hyperlipidemic activities but not superior to it., but the effect was not dose dependently. This may be due to the time of assemblage of the plant parts, and the amount of phytoconstituents present in the plant.

4.3.2.5.1 Total Bilirubin Ktari et al. (2013) repoted Salaria basilisca extract on multiple oral administration of the tested doses of ethanolic extract and glibinclamide (20 mg/kg b/wt.), significantly decreased the blood total bilirubin level at seventh, fourteenth, twenty first, and twenty eighth day post administration; when compared to diabetic non-treated group. Honmore et al. (2015) testified that pretreatment with Artemisia pallens extract at the dose rate of (200 and 400 mg/kg, p.o.) significantly (p < 0.01 and p < 0.001) shrunk aspartate transaminase (AST), alanine transaminase (ALT), bilirubin, blood urea nitrogen (BUN), and serum creatinine as compared with APAP-treated rat. Singh et al. (2013) found that oral administration of assia sopherain extract inhibited STZ-induced increase in lipid peroxidation (LPO), aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), bilirubin, creatinine and urea in liver of diabetic rats. Zaruwa et al. (2013)

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found that the methanolic leaf extract of Anisopus mannii at the dose rate of 400 mg/kg bw showed significantly (p<0.05) displayed fall in blood urea nitrogen and creatinine level, boosts in aspartate transaminase, alanine transaminase and total bilirubin levels, as well as the body weights.

4.3.2.5.2 Proteins Total Zhang et al. (2014) found that the oral direction of MLP at 50- 200mg/kgbodyweight daily for 5weeks significantly reduced the levels of fasting blood glucose (FBG), glycosylated serum protein (GSP), serum total cholesterol (TC), and serum triglyceride (TG), and increased the body weight, fasting insulin (FINS), C-peptide (C-P), liver glycogen, liver glucokinase, and serum high-density lipoprotein cholesterol (HDL-C). The obtained results showed that multiple oral administration of the tested doses of ethanolic extract and glibinclamide (20 mg/kg b/wt.), significantly decreased the blood totl proteins level at seventh, fourteenth, twenty first, and twenty eighth day after administration as cpompared to the group having no diabetes.

4.3.2.5.3 Albumin The obtained results showed that multiple oral administration of the definite doses of ethanolic extract and glibinclamide (20 mg/kg b/wt.), meaningfully declined the blood albumin level at seventh, fourteenth, twenty first, and twenty eighth day post administration; when compared to diabetic non-treated group. In diabetes mellitus control animals, liver and renal dysfunctions were observed. The surge in aminotransferase level may be due to the demolition of hepatocytes caused by STZ (Zafar et al., 2009). Honmore et al., (2015) reported that decreased level of serum albumin, serum uric acid, and HDL were significantly (p < 0.01 and p < 0.001) refurbished by Artemisia pallens extract (200 and 400 mg/kg, p.o.). Decrease in serum albumin levels was detected in diabetes mellitus animals and this may be due to corrosion of kidney function (Zafar et al., 2009). Park et al. also testified that decreased levels of albumin in marginal blood of STZ-induced diabetic rats (Park et al., 2014). Alteration in serum albumin: creatinine ratio was detected in diabetes mellitus control animal and O. tenuiflorum 150 mg/kg treated animals, and this may be due to the revision in renal functions (Viswanathan et al., 2004).

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4.3.2.5.4 Globulin Allicin, the main biologically active component of garlic clove extracts was used in freshwater Nile tilapia; Oreochromis niloticus showing that deltamethrin subacute intoxication (1.46 µg/L for 28 days) increased serum AST, ALT, ALP, cholesterol, urea, uric acid, creatinine and tissue MDA. At the same time, serum total protein and albumin was dropped (Abdel-Daim et al., 2015). The obtained results showed that multiple oral administration of the established doses of ethanolic extract and glibinclamide (20 mg/kg b/wt.), significantly decreased the blood globulin level at seventh, fourteenth, twenty first, and twenty eighth day post administration; when compared to diabetic non-treated group. M. malabathricum leaf can reduce bloog globulin level in diabetic albino rats when compared to non-diabetic control rats (Balamurugan et al., 2014).

4.3.2.5.5 A/G Ratio The obtained results showed that multiple oral administration of the tested doses of ethanolic extract and glibinclamide (20 mg/kg b/wt.), significantly decreased the blood A/G ratio level at seventh, fourteenth, twenty first, and twenty eighth day post administration; when compared to diabetic non-treated group. The albumin globulin ratio (A/G ratio) is meant to denote the ratio of amendments in serum proteins, since, in liver disease, globulins (G) rise following serum albumin (SA) decrease (Al-Joudi et al., 2004).decrease in serum albumin (SA) results in the rise in serum globulins (G)1 which is co-related with liver diseases (Clinical Diagnosis and Management by Laboratory Methods, 1996; Dufour et al., 2000). The A/G ratio is not a consistent marker of serum protein alteration because some time any environmental effects increase the protein synthesis speed (Pisters, P.W., and M.F.Brennen, 1990; A. Inui, 2002). Liver for compensation produce more proteins when level of protein reduce in other parts of the body (Bruille et al., 1994). SA levels are reduced when liver do not produce proteins normally (Clinical Diagnosis and Management by Laboratory Methods, 1996).

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4.3.2.5.6 GCI The obtained results disclosed that multiple oral administration of the tested doses of ethanolic extract and glibinclamide (20 mg/kg b/wt.), pointedly shrunk the blood glucose level at seventh, fourteenth, twenty first, and twenty eighth day post administration; when compared to diabetic non-treated group. Clinical and pathophysiological values of A/G ratio are not clear (Podolsky, P and J. Feldman, 1997). In higher diseases like hepatitis A/G ratio does not represent the clear cut value (Goldwasser, P. and J. Feldman, 1997; Feldman et al., 2000; Reuben et al., 2000 and Rzany et al., 2002). The globulin compensation index (GCI) based on the decreased serum albumin and the globulin levels give more clear cut value for non-hepatic systemic disorders. GCI is grouped into fully, partially, or negatively compensated GCIs (Bruille et al., 1994; Pisters and Brennen; Inui, 2002; Tisdale, 2001; Wigmore et al., 2000; Mercier et al., 2002 and Cogo et al., 2002) . At the same time, the liver fails, in these cases, to maintain normal SA levels, mainly due to stringency in nutritional requirements, and not due to reduced protein synthesizing capacity (Barber et al., 2000. Thus, the presence of reduced SA, that is not accompanied by a significant rise in the level of serum globular proteins, often accompanies, more closely, a state of fatigue or cachexia (Pisters and Brennen; Inui, 2002; Tisdale, 2001; Wigmore et al., 2000; Mercier et al., 2002 and Cogo et al., 2002). GCI represent reduction in globulins and albumins properly (Al-Joudi, 2005). GCI may also be a potentially useful tool in the measurement of protein-synthesizing capacity, in assessing the nutritional status of the body, in evaluating the response of patients to therapy, and in predicting the outcome of disease (Al-Joudi and Wahab, 2004). The GCI is a source for globulin compensation (Walsh et al., 2003).

4.3.2.5.7 ALP The obtained results showed that multiple oral administration of the tested doses of ethanolic extract and glibinclamide (20 mg/kg b/wt.), meaningfully shrunk P < 0.05 the blood ALP level at seventh, fourteenth, twenty first, and twenty eighth day post administration; when compared to diabetic non-treated group. This finding was also mentioned by Comfort et al., while studying effects of aqueous extract of Emelia sonchifolia on some liver enzymes in Dithizone induced diabetes in rabbits. The extract of Emelia sonchifolia significantly reduced the alkaline phosphatase activity after 24, 48 and 72 hrs of administration when compared to the two groups of

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rabbits. Saleem et al. (2008) after studying hepatoprotective activity of Annona squamosa (custard apple) Linn onexperimental animal model. It showed that the extract can reduce ALP value of different groups at different doses.Singh et al., (2013) found that oral administration of Assia sopherain extract repressed STZ- induced rise in lipid peroxidation (LPO), aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), bilirubin, creatinine and urea in liver of diabetic rats. In 2010 Uboh et al. showed that the aqueous extracts of P. guajava infer the same hepatocellular protection in rats that is linked to flavonoids, a phytochemical component. Flavonoids have been reported to possess antioxidant activity (Middleton, 1996), thus defensive cell membranes from peroxidative actions of free radicals. Alkaline phosphatase roles as a biochemical marker enzyme for maintaining membrane integrity (Akanji et al., 1993). 4.3.2.5.8 GGT Nemat et al., (1985) reported that investigated that serum gamma-glutamyl transpeptidase is untiringly eminent in organisms with having obvious liver injury. The attained results showed that multiple oral administration of the tested doses of ethanolic extract and glibinclamide (20 mg/kg b/wt.), significantly decreased the GGT glucose level at seventh, fourteenth, twenty first, and twenty eighth day post administration; when matched to diabetic non-treated group. Honmore et al. (2015) testified that pretreatment with Artemisia pallens extract at the dose rate of (200 and 400 mg/kg, p.o.) significantly (p < 0.01 and p < 0.001) decreased aspartate transaminase (AST), alanine transaminase (ALT), bilirubin, blood urea nitrogen (BUN), and serum creatinine as compared with APAP-treated rat. Nemat et al.,(1985) investigated that serum gamma-glutamyl transpeptidase is persistently elevated in organisms with having obvious liver harm (Ikewuchi et al., 2011).

4.3.2.5.9 ALT or SGPT Honmore et al. (2015) reported that pretreatment with Artemisia pallens extract at the dose rate of (200 and 400 mg/kg, p.o.) significantly (p < 0.01 and p < 0.001) decreased aspartate transaminase (AST), alanine transaminase (ALT), bilirubin, blood urea nitrogen (BUN), and serum creatinine as compared with APAP-treated rat. Singh et al. (2013) found that oral administration of assia sopherain extract inhibited STZ-induced increase in lipid peroxidation (LPO), aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase

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(ALP), bilirubin, creatinine and urea in liver of diabetic rats. Choudhari et al. (2007) found that the stem barks extract of Semecarpus anacardium protected liver which may be partially explained by the diminution of SGPT level.The obtained results showed that multiple oral administration of the tested doses of ethanolic extract and glibinclamide (20 mg/kg b/wt.), meaningfully declined the blood ALT level at seventh, fourteenth, twenty first, and twenty eighth day post administration; when compared to diabetic non-treated group. Pouraboli et al. (2015) using the Oral Administration of Dracocephalum polychaetum extract (300 mg/kg) which significantly (p < 0.05) reduced the alanine amino transferase level in alloxan-induced diabetic rats showing anti- alanine amino transferase activity of the plant. Victor et al., 2014 reported that the reductions in ALT and AST levels are said to be triggered by the hepatocellular and cardiac protection offered by these extracts. Victor et al. 2014, Hepatic, cardiac tissues release alanine aminotransferase; therefore the raise of plasma concentrations of these enzymes is a sign of hepatic and cardiac damage (Crook, 2006), as in the case of hitches in diabetes mellitus. Selda et al. (2010) found that Aspartate aminotransferase, urea and cholesterol levels were pointedly reduced by usage with extracts of Cinnamon Bark and Olive Leaf, and alanine aminotransferase by usage with olive leaf. Cinnamon bark also caused a significant decrease in platelet counts.

4.3.2.5.10 AST or SGOT Singh et al. (2013) found that oral administration of assia sopherain extract inhibited STZ-induced increase in lipid peroxidation (LPO), aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), bilirubin, creatinine and urea in liver of diabetic rats. The obtained results showed that multiple oral administration of the verified doses of ethanolic extract and glibinclamide (20 mg/kg b/wt.), significantly decreased the AST glucose level at seventh, fourteenth, twenty first, and twenty eighth day post administration; when matched to diabetic non-treated group. Choudhari et al. (2007) found that the stem barks extract of Semecarpus anacardiumsafe liver which may be partly elucidated by the attenuation of SGOT level. Victor et al. (2014) found that the decreases in ALT and AST levels are said to be caused by the hepatocellular and cardiac safety accessible by these extracts. Honmore et al. (2015) reported that pretreatment with Artemisia pallens extract at the dose rate of (200 and 400 mg/kg) significantly (p < 0.01 and p < 0.001) decreased

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aspartate transaminase (AST), alanine transaminase (ALT), bilirubin, blood urea nitrogen (BUN), and serum creatinine as compared with APAP-treated rat. Hepatic, cardiac tissues release aspartate aminotransferase therefore the raise of plasma concentrations of these enzymes is a sign of hepatic and cardiac damage as in the case of difficulties in diabetes mellitus (Victor et al., 2014; Crook, 2006).

4.3.2.5.11 AST/ALT Ratio The attained results displayed that multiple oral administration of the tested doses of ethanolic extract and glibinclamide (20 mg/kg b/wt.), significantly decreased the AST/ALT ratio glucose level at seventh, fourteenth, twenty first, and twenty eighth day after administration; when coordinated to diabetic non-treated group. In non-obese subjects, the best marker of insulin resistance was alanine aminotransferase (ALT)/aspartate aminotransferase (AST) ratio of 0.70 (95% confidence interval (CI), 0.63-0.77). ALT/AST ratio of ≥ 0.82 in non-obese subjects and ≥ 1.02 in overweight subjects. In non-obese subjects, the positive probability ratio was paramount for ALT/AST ratio (Kawamoto et al., 2012).

4.4 Conclusion

Methanolic extract of Hedra neplenensis leaves exhibited significant antihypohlycemic and hepatoprotective activities in alloxane induced rabbits. So the present investigation reveals the importance of leaves of Hedra neplenensis as an anti-diabetic agent. The plant bears potentials for further research to isolate anti-diabetic principle.

4.5 Recommendations

3 The plant may be cultivated for commercial drug production. 4 Pharmaceutical scientists can extract its pure line while using procedure of extraction. 5 Pharmaceutical industries can produce its pure product for medication in human beings against diabetes mellitus.

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Appendices

Appendix 1: Two-way ANOVA (Multiple Comparison) Table Showing Blood Glucose level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day 24.90 23.04 to 26.77 Yes **** 0 day vs. 14th day 58.81 56.94 to 60.67 Yes **** 0 day vs. 21st day 61.43 59.56 to 63.29 Yes **** 0 day vs. 28th day 55.81 53.94 to 57.67 Yes **** Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 254.1 229.2 24.90 0.7463 0 day vs. 14th day 254.1 195.3 58.81 0.7463 0 day vs. 21st day 254.1 192.7 61.43 0.7463 0 day vs. 28th day 254.1 198.3 55.81 0.7463

Appendix 2: Two-way ANOVA (Multiple Comparison) Table Showing Blood triglyceride level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day -0.6190 -1.860 to 0.6221 No ns 0 day vs. 14th day -0.1905 -1.432 to 1.051 No ns 0 day vs. 21st day 0.04762 -1.194 to 1.289 No ns 0 day vs. 28th day 0.8095 -0.4317 to 2.051 No ns Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 81.57 82.19 -0.6190 0.4967 0 day vs. 14th day 81.57 81.76 -0.1905 0.4967 0 day vs. 21st day 81.57 81.52 0.04762 0.4967 0 day vs. 28th day 81.57 80.76 0.8095 0.4967

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Appendix 3: Two-way ANOVA (Multiple Comparison) Table Showing Blood Cholesterol level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day 6.524 4.131 to 8.917 Yes **** 0 day vs. 14th day 10.81 8.417 to 13.20 Yes **** 0 day vs. 21st day 8.476 6.083 to 10.87 Yes **** 0 day vs. 28th day 5.333 2.940 to 7.726 Yes **** Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 173.2 166.7 6.524 0.9576 0 day vs. 14th day 173.2 162.4 10.81 0.9576 0 day vs. 21st day 173.2 164.8 8.476 0.9576 0 day vs. 28th day 173.2 167.9 5.333 0.9576

Appendix 4: Two-way ANOVA (Multiple Comparison) Table Showing Blood Total Bilirubin level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day 1.262 0.6586 to 1.865 Yes **** 0 day vs. 14th day 2.305 1.701 to 2.908 Yes **** 0 day vs. 21st day 4.281 3.678 to 4.884 Yes **** 0 day vs. 28th day 4.690 4.087 to 5.294 Yes **** Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 17.67 16.41 1.262 0.2414 0 day vs. 14th day 17.67 15.37 2.305 0.2414 0 day vs. 21st day 17.67 13.39 4.281 0.2414 0 day vs. 28th day 17.67 12.98 4.690 0.2414

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Appendix 5: Two-way ANOVA (Multiple Comparison) Table Showing Blood Total Protein level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day -0.1333 -0.2354 to -0.03130 Yes ** 0 day vs. 14th day -0.1667 -0.2687 to -0.06463 Yes *** 0 day vs. 21st day -0.1667 -0.2687 to -0.06463 Yes *** 0 day vs. 28th day -0.1500 -0.2520 to -0.04797 Yes ** Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 5.311 5.444 -0.1333 0.04067 0 day vs. 14th day 5.311 5.478 -0.1667 0.04067 0 day vs. 21st day 5.311 5.478 -0.1667 0.04067 0 day vs. 28th day 5.311 5.461 -0.1500 0.04067

Appendix 6: Two-way ANOVA (Multiple Comparison) Table Showing Blood Albumin level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day 0.06190 -0.1159 to 0.2397 No ns 0 day vs. 14th day 0.06667 -0.1111 to 0.2444 No ns 0 day vs. 21st day -0.1619 -0.3397 to 0.01587 No ns 0 day vs. 28th day -0.5619 -0.7397 to -0.3841 Yes **** Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 3.110 3.048 0.06190 0.07114 0 day vs. 14th day 3.110 3.043 0.06667 0.07114 0 day vs. 21st day 3.110 3.271 -0.1619 0.07114 0 day vs. 28th day 3.110 3.671 -0.5619 0.07114

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Appendix 7: Two-way ANOVA (Multiple Comparison) Table Showing Blood Globulin level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day -0.1143 -0.8202 to 0.5916 No ns 0 day vs. 14th day -0.2457 -0.9516 to 0.4602 No ns 0 day vs. 21st day -0.004286 -0.7102 to 0.7016 No ns 0 day vs. 28th day 0.2314 -0.4745 to 0.9374 No ns Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 1.930 2.044 -0.1143 0.2700 0 day vs. 14th day 1.930 2.176 -0.2457 0.2700 0 day vs. 21st day 1.930 1.934 -0.004286 0.2700 0 day vs. 28th day 1.930 1.699 0.2314 0.2700

Appendix 8: Two-way ANOVA (Multiple Comparison) Table Showing Blood A/G Ratio level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day 0.1957 -0.6979 to 1.089 No ns 0 day vs. 14th day 0.4343 -0.4593 to 1.328 No ns 0 day vs. 21st day 0.2557 -0.6379 to 1.149 No ns 0 day vs. 28th day -0.1029 -0.9965 to 0.7908 No ns Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 1.671 1.476 0.1957 0.3418 0 day vs. 14th day 1.671 1.237 0.4343 0.3418 0 day vs. 21st day 1.671 1.416 0.2557 0.3418 0 day vs. 28th day 1.671 1.774 -0.1029 0.3418

235

Appendix 9: Two-way ANOVA (Multiple Comparison) Table Showing Blood GCI level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day 2.730 -2.208 to 7.668 No ns 0 day vs. 14th day 1.819 -3.120 to 6.757 No ns 0 day vs. 21st day -0.9800 -5.918 to 3.958 No ns 0 day vs. 28th day 1.514 -3.424 to 6.453 No ns Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 0.8600 -1.870 2.730 1.889 0 day vs. 14th day 0.8600 -0.9586 1.819 1.889 0 day vs. 21st day 0.8600 1.840 -0.9800 1.889 0 day vs. 28th day 0.8600 -0.6543 1.514 1.889

Appendix 10: Two-way ANOVA (Multiple Comparison) Table Showing Blood Creatinine level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day -0.05238 -0.1930 to 0.08821 No ns 0 day vs. 14th day 0.2619 0.1213 to 0.4025 Yes **** 0 day vs. 21st day 0.5095 0.3689 to 0.6501 Yes **** 0 day vs. 28th day 0.6000 0.4594 to 0.7406 Yes **** Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 4.157 4.210 -0.05238 0.05626 0 day vs. 14th day 4.157 3.895 0.2619 0.05626 0 day vs. 21st day 4.157 3.648 0.5095 0.05626 0 day vs. 28th day 4.157 3.557 0.6000 0.05626

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Appendix 11: Two-way ANOVA (Multiple Comparison) Table Showing Blood ALP (IU/L) level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day 8.429 6.633 to 10.22 Yes **** 0 day vs. 14th day 23.19 21.40 to 24.99 Yes **** 0 day vs. 21st day 31.86 30.06 to 33.65 Yes **** 0 day vs. 28th day 41.95 40.16 to 43.75 Yes **** Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 185.0 176.5 8.429 0.7184 0 day vs. 14th day 185.0 161.8 23.19 0.7184 0 day vs. 21st day 185.0 153.1 31.86 0.7184 0 day vs. 28th day 185.0 143.0 41.95 0.7184

Appendix 12: Two-way ANOVA (Multiple Comparison) Table Showing Blood GGT (IU/L) level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day 0.8314 -0.8919 to 2.555 No ns 0 day vs. 14th day 1.838 0.1147 to 3.561 Yes * 0 day vs. 21st day 1.143 -0.5805 to 2.866 No ns 0 day vs. 28th day 1.357 -0.3662 to 3.081 No ns Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 23.05 22.22 0.8314 0.6897 0 day vs. 14th day 23.05 21.21 1.838 0.6897 0 day vs. 21st day 23.05 21.91 1.143 0.6897 0 day vs. 28th day 23.05 21.70 1.357 0.6897

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Appendix 13: Two-way ANOVA (Multiple Comparison) Table Showing Blood ALT (IU/L) level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day 1.618 0.6041 to 2.631 Yes *** 0 day vs. 14th day 2.745 1.731 to 3.758 Yes **** 0 day vs. 21st day -0.8286 -1.842 to 0.1850 No ns 0 day vs. 28th day -2.778 -3.792 to -1.765 Yes **** Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 27.00 25.39 1.618 0.4056 0 day vs. 14th day 27.00 24.26 2.745 0.4056 0 day vs. 21st day 27.00 27.83 -0.8286 0.4056 0 day vs. 28th day 27.00 29.78 -2.778 0.4056

Appendix 14: Two-way ANOVA (Multiple Comparison) Table Showing Blood AST (IU/L) level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day 2.476 1.091 to 3.862 Yes *** 0 day vs. 14th day 2.429 1.043 to 3.814 Yes *** 0 day vs. 21st day 3.762 2.376 to 5.148 Yes **** 0 day vs. 28th day 6.476 5.091 to 7.862 Yes **** Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 122.4 120.0 2.476 0.5545 0 day vs. 14th day 122.4 120.0 2.429 0.5545 0 day vs. 21st day 122.4 118.7 3.762 0.5545 0 day vs. 28th day 122.4 116.0 6.476 0.5545

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Appendix 15: Two-way ANOVA (Multiple Comparison) Table Showing Blood AST/ALT Ratio level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary

0 day vs. 7th day -0.2000 -1.155 to 0.7555 No ns 0 day vs. 14th day -0.5900 -1.545 to 0.3655 No ns 0 day vs. 21st day 0.2643 -0.6912 to 1.220 No ns 0 day vs. 28th day 0.7200 -0.2355 to 1.675 No ns

Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 4.790 4.990 -0.2000 0.3655 0 day vs. 14th day 4.790 5.380 -0.5900 0.3655 0 day vs. 21st day 4.790 4.526 0.2643 0.3655 0 day vs. 28th day 4.790 4.070 0.7200 0.3655

Appendix 16: Two-way ANOVA (Multiple Comparison) Table Showing Blood Glucose level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day 22.19 18.99 to 25.39 Yes **** 0 day vs. 14th day 24.81 21.61 to 28.01 Yes **** 0 day vs. 21st day 36.05 32.85 to 39.25 Yes **** 0 day vs. 28th day 41.76 38.56 to 44.96 Yes **** Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 235.0 212.8 22.19 1.281 0 day vs. 14th day 235.0 210.2 24.81 1.281 0 day vs. 21st day 235.0 199.0 36.05 1.281 0 day vs. 28th day 235.0 193.2 41.76 1.281

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Appendix 17: Two-way ANOVA (Multiple Comparison) Table Showing Blood triglyceride level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day 1.143 -0.8988 to 3.185 No ns 0 day vs. 14th day 6.095 4.054 to 8.137 Yes **** 0 day vs. 21st day 6.429 4.387 to 8.470 Yes **** 0 day vs. 28th day 6.143 4.101 to 8.185 Yes **** Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 75.52 74.38 1.143 0.8171 0 day vs. 14th day 75.52 69.43 6.095 0.8171 0 day vs. 21st day 75.52 69.10 6.429 0.8171 0 day vs. 28th day 75.52 69.38 6.143 0.8171

Appendix 18: Two-way ANOVA (Multiple Comparison) Table Showing Blood Cholesterol level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day 1.143 -2.305 to 4.591 No ns 0 day vs. 14th day 7.667 4.219 to 11.11 Yes **** 0 day vs. 21st day 10.86 7.409 to 14.31 Yes **** 0 day vs. 28th day 10.95 7.504 to 14.40 Yes **** Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 171.8 170.6 1.143 1.380 0 day vs. 14th day 171.8 164.1 7.667 1.380 0 day vs. 21st day 171.8 160.9 10.86 1.380 0 day vs. 28th day 171.8 160.8 10.95 1.380

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Appendix 19: Two-way ANOVA (Multiple Comparison) Table Showing Blood Total Bilirubin level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day -1.090 -1.460 to -0.7210 Yes **** 0 day vs. 14th day -1.514 -1.884 to -1.145 Yes **** 0 day vs. 21st day -2.252 -2.622 to -1.883 Yes **** 0 day vs. 28th day -1.105 -1.474 to -0.7353 Yes **** Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 16.43 17.52 -1.090 0.1478 0 day vs. 14th day 16.43 17.95 -1.514 0.1478 0 day vs. 21st day 16.43 18.69 -2.252 0.1478 0 day vs. 28th day 16.43 17.54 -1.105 0.1478

Appendix 20: Two-way ANOVA (Multiple Comparison) Table Showing Blood Total Protein level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day 0.2571 -0.02394 to 0.5382 No ns 0 day vs. 14th day 0.1095 -0.1716 to 0.3906 No ns 0 day vs. 21st day 0.1381 -0.1430 to 0.4192 No ns 0 day vs. 28th day 0.1048 -0.1763 to 0.3858 No ns

Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 4.467 4.210 0.2571 0.1125 0 day vs. 14th day 4.467 4.357 0.1095 0.1125 0 day vs. 21st day 4.467 4.329 0.1381 0.1125 0 day vs. 28th day 4.467 4.362 0.1048 0.1125

241

Appendix 21: Two-way ANOVA (Multiple Comparison) Table Showing Blood Albumin level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day -0.2095 -0.3825 to -0.03659 Yes * 0 day vs. 14th day -0.3000 -0.4729 to -0.1271 Yes *** 0 day vs. 21st day -0.4619 -0.6348 to -0.2890 Yes **** 0 day vs. 28th day -0.8048 -0.9777 to -0.6318 Yes **** Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 2.133 2.343 -0.2095 0.06920 0 day vs. 14th day 2.133 2.433 -0.3000 0.06920 0 day vs. 21st day 2.133 2.595 -0.4619 0.06920 0 day vs. 28th day 2.133 2.938 -0.8048 0.06920

Appendix 22: Two-way ANOVA (Multiple Comparison) Table Showing Blood Globulin level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day 0.1814 -0.7097 to 1.073 No ns 0 day vs. 14th day 0.1257 -0.7654 to 1.017 No ns 0 day vs. 21st day 0.3314 -0.5597 to 1.223 No ns 0 day vs. 28th day 0.6271 -0.2639 to 1.518 No ns Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 2.050 1.869 0.1814 0.3408 0 day vs. 14th day 2.050 1.924 0.1257 0.3408 0 day vs. 21st day 2.050 1.719 0.3314 0.3408 0 day vs. 28th day 2.050 1.423 0.6271 0.3408

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Appendix 23: Two-way ANOVA (Multiple Comparison) Table Showing Blood A/G Ratio level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day -0.07771 -1.759 to 1.603 No ns 0 day vs. 14th day 0.1871 -1.494 to 1.868 No ns 0 day vs. 21st day -0.2796 -1.961 to 1.402 No ns 0 day vs. 28th day -1.026 -2.707 to 0.6553 No ns Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 1.820 1.898 -0.07771 0.6430 0 day vs. 14th day 1.820 1.633 0.1871 0.6430 0 day vs. 21st day 1.820 2.100 -0.2796 0.6430 0 day vs. 28th day 1.820 2.846 -1.026 0.6430

Appendix 24: Two-way ANOVA (Multiple Comparison) Table Showing Blood GCI level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day 0.6186 -3.495 to 4.733 No ns 0 day vs. 14th day -0.9529 -5.067 to 3.161 No ns 0 day vs. 21st day 0.4414 -3.673 to 4.555 No ns 0 day vs. 28th day 1.189 -2.925 to 5.303 No ns Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day -0.08000 -0.6986 0.6186 1.574 0 day vs. 14th day -0.08000 0.8729 -0.9529 1.574 0 day vs. 21st day -0.08000 -0.5214 0.4414 1.574 0 day vs. 28th day -0.08000 -1.269 1.189 1.574

243

Appendix 25: Two-way ANOVA (Multiple Comparison) Table Showing Blood Creatinine level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day 0.2000 0.05291 to 0.3471 Yes ** 0 day vs. 14th day 0.3714 0.2243 to 0.5185 Yes **** 0 day vs. 21st day 0.4476 0.3005 to 0.5947 Yes **** 0 day vs. 28th day 0.6429 0.4958 to 0.7899 Yes ****

Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 4.886 4.686 0.2000 0.05886 0 day vs. 14th day 4.886 4.514 0.3714 0.05886 0 day vs. 21st day 4.886 4.438 0.4476 0.05886 0 day vs. 28th day 4.886 4.243 0.6429 0.05886

Appendix 26: Two-way ANOVA (Multiple Comparison) Table Showing Blood ALP (IU/L) level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day 9.524 1.445 to 17.60 Yes * 0 day vs. 14th day 22.10 14.02 to 30.17 Yes **** 0 day vs. 21st day 35.19 27.11 to 43.27 Yes **** 0 day vs. 28th day 36.00 27.92 to 44.08 Yes ****

Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 208.5 199.0 9.524 3.233 0 day vs. 14th day 208.5 186.4 22.10 3.233 0 day vs. 21st day 208.5 173.3 35.19 3.233 0 day vs. 28th day 208.5 172.5 36.00 3.233

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Appendix 27: Two-way ANOVA (Multiple Comparison) Table Showing Blood GGT (IU/L) level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day -0.1429 -0.8686 to 0.5829 No ns 0 day vs. 14th day -1.714 -2.440 to -0.9885 Yes **** 0 day vs. 21st day 0.3333 -0.3924 to 1.059 No ns 0 day vs. 28th day -0.04762 -0.7734 to 0.6781 No ns Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 23.57 23.71 -0.1429 0.2904 0 day vs. 14th day 23.57 25.29 -1.714 0.2904 0 day vs. 21st day 23.57 23.24 0.3333 0.2904 0 day vs. 28th day 23.57 23.62 -0.04762 0.2904

Appendix 28: Two-way ANOVA (Multiple Comparison) Table Showing Blood ALT (IU/L) level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day 0.5476 -3.546 to 4.641 No ns 0 day vs. 14th day 0.7190 -3.375 to 4.813 No ns 0 day vs. 21st day 4.340 0.2457 to 8.433 Yes * 0 day vs. 28th day 3.043 -1.051 to 7.137 No ns Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 34.98 34.43 0.5476 1.638 0 day vs. 14th day 34.98 34.26 0.7190 1.638 0 day vs. 21st day 34.98 30.64 4.340 1.638 0 day vs. 28th day 34.98 31.93 3.043 1.638

245

Appendix 29: Two-way ANOVA (Multiple Comparison) Table Showing Blood AST (IU/L) level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day -9.095 -10.60 to -7.594 Yes **** 0 day vs. 14th day -4.190 -5.692 to -2.689 Yes **** 0 day vs. 21st day -2.952 -4.454 to -1.451 Yes **** 0 day vs. 28th day 0.09524 -1.406 to 1.597 No ns Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 134.1 143.2 -9.095 0.6008 0 day vs. 14th day 134.1 138.3 -4.190 0.6008 0 day vs. 21st day 134.1 137.0 -2.952 0.6008 0 day vs. 28th day 134.1 134.0 0.09524 0.6008

Appendix 30: Two-way ANOVA (Multiple Comparison) Table Showing Blood AST/ALT Ratio level of Rabbits

Compare column means (main column effect) Number of families 1 Number of comparisons per family 4 Alpha 0.05 Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary 0 day vs. 7th day -0.4529 -0.9742 to 0.06845 No ns 0 day vs. 14th day -0.3657 -0.8870 to 0.1556 No ns 0 day vs. 21st day -0.7100 -1.231 to -0.1887 Yes ** 0 day vs. 28th day -0.6557 -1.177 to -0.1344 Yes *

Test details Mean 1 Mean 2 Mean Diff. SE of diff.

0 day vs. 7th day 3.883 4.336 -0.4529 0.1994 0 day vs. 14th day 3.883 4.249 -0.3657 0.1994 0 day vs. 21st day 3.883 4.593 -0.7100 0.1994 0 day vs. 28th day 3.883 4.539 -0.6557 0.1994

246