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

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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 By 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 By 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) In 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.
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