Pharmacological and Toxicological Screening of Vernonia anthelmintica Subia Jamil DEPARTMENT OF PHARMACOLOGY FACULTY OF PHARMACY AND PHARMACEUTICAL SCIENCES UNIVERSITY OF KARACHI KARACHI-75270, PAKISTAN 2016 Pharmacological and Toxicological Screening of Vernonia anthelmintica Subia Jamil Thesis submitted for the partial fulfillment for the requirement of the Ph.D. degree in the Department of Pharmacology University of Karachi, Pakistan. DEPARTMENT OF PHARMACOLOGY FACULTY OF PHARMACY AND PHARMACEUTICAL SCIENCES UNIVERSITY OF KARACHI KARACHI-75270, PAKISTAN 2016 In the name of Allah, the Most Beneficent, the most merciful CERTIFICATE This thesis is accepted in its present form by the Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical sciences, University of Karachi for the partial fulfillment of the requirements for the degree of Ph.D. in Pharmacology Research Supervisor __________________________ Dr. Rafeeq Alam Khan Professor (Meritorious) Department of Pharmacology, Faculty of Pharmacy & Pharmaceutical sciences University of Karachi. Dedicated to Mrs. Laila Zaidi, Mrs. Riaz Fatima (late), my mothers and Mr. Sabir Zaidi, my husband. CONTENTS Acknowledgement List of tables I List of Figures III List of abbreviations V SUMMARY English IX Urdu X 1. INTRODUCTION 1.1 Description of Vernonia anthelmintica 2 1.2 Plant taxonomy 2 1.3 Importance of extraction 4 1.4 Herbal derived bioactive compounds 4 1.5 Components of Vernonia anthelmintica seeds 5 1.6 Toxicological screening 7 1.6.1 Hematology and coagulation 8 1.6.2 Hepatic profile 10 1.6.3 Renal profile and electrolytes 10 1.7 Pharmacological screening 12 1.7.1 Antibacterial 12 1.7.2 Cytotoxicity 13 1.7.3 Antioxidant 14 1.7.4 Inflammation 15 1.7.5 Nociception 16 1.7.6 Phytotoxic and insecticidal activity 17 1.7.7 Antileishmanicidal activity 18 1.7.8 Diabetes 18 1.7.9 Hyperlipidemia 20 1.8 Objective of the study 21 2. MATERIALS AND METHODS 2.1 Collection and identification of seed 23 2.2 Preparation of extract 23 2.3 Experimental design 23 2.4 Invitro studies 24 2.4.1 Qualitative phytochemical analysis 24 2.4.1.1 Wagner test for alkaloids 24 2.4.1.2 Ferric chloride test for cardiac glycosides 24 2.4.1.3 Benedict test for carbohydrates 24 2.4.1.4 Xanthoprotein test for proteins 25 2.4.1.5 Ferric chloride test for flavonoids 25 2.4.1.6 Foam test for saponins 25 2.4.1.7 Lead test for terpenoids 25 2.4.1.8 Salkowaski’s test for terpenoids 25 2.4.1.9 Spot test for fixed oils and fatty acids 25 2.4.2 Antibacterial assay 26 2.4.3 Cytotoxic assay 26 2.4.4 Brine shrimp lethality Assay 28 2.4.5 Antioxidant Assay 27 2.4.6 Antiglycation Assay 29 2.4.7 Phytotoxic Assay 30 2.4.8 Insecticidal Activity 30 2.4.9 Antileismanial Assay 31 2.5 Invivo studies 32 2.5.1 Animals 32 2.5.2 Acute oral toxicity 32 2.5.3 Sub chronic studies 33 2.5.3.1 Animal grouping and experimental design 33 2.5.4 Pharmacological models 34 2.5.4.1 Hyperlipidemic model (High fat high sµgar model) 34 2.5.4.2 Diabetic models 35 2.5.4.3 Inflammatory model 36 Carrageenan induced rat paw model 36 2.5.4.4 Analgesic models 36 Tail flick method 37 Hot plate method 37 2.6 Sample collection 37 2.7 Hematological analysis 38 2.8 Coagulation studies 38 2.9 Biochemical analysis 39 2.9.1 Lipid profile analysis 40 2.9.2 Blood glucose analysis 41 2.9.3 Liver function test 42 2.9.4 Renal function test 43 2.9.5 Electrolytes analysis 44 2.10 Histopathlogical examinations 45 2.11 Statistical analysis 47 3. RESULTS 3.1 Invitro studies 48 3.1.1 Qualitative phytochemical analysis 48 3.1.2 Antibacterial bioassay 49 3.1.3 Cytotoxic bioassay 49 3.1.4 Brine shrimp lethality test 52 3.1.5 Antioxidant bioassay 53 3.1.5.1 Radical scavenging activity of EEVA 54 3.1.6 Antiglycation bioassay 55 3.1.7 Phytotoxic bioassay 56 3.1.8 Insecticidal bioassay 57 3.1.9 Antileismanial bioassay 58 3.2 Invivo studies 59 3.2.1 Acute oral toxicity 59 3.2.2 Sub chronic studies 60 3.2.2.1 Effects on body weight 60 3.2.2.2 Effects on hematologic profile 61 3.2.2.3 Effects on Coagulation 62 3.2.2.4 Effects on glucose metabolism 63 3.2.2.5 Effects on lipid profile 64 3.2.2.6 Effects on liver function 65 3.2.2.7 Effects on renal function 66 3.2.2.8 Effects on electrolytes 67 3.3 Pharmacological models 68 3.3.1 Anti-inflammatory activity 68 3.3.1.1 Carrageenan induced rat paw model 69 3.3.2 Analgesic activity 70 3.3.2.1 Hot plate method 70 3.3.2.2 Tail flick method 71 3.3.3 Hyperlipidemic model ( High fat high sµgar model) 72 3.3.3.1 Antihyperlipidemic activity 72 3.3.3.2 Cardiac risk parameters 74 3.3.3.3 Anti-diabetic profile 78 3.3.3.4 Hepatic profile 80 3.3.4 Diabetic models 81 3.3.4.1 Normoglycemic model 81 3.3.4.2 Glucose induced hyperglycemic model 83 3.3.4.3 Alloxan induced diabetic model 85 3.4 Histopathological examinations 87 3.4.1 Heart 87 3.4.2 Kidney 91 3.4.3 Liver 95 4. DISCUSSION 100 5. CONCLUSION 120 6. REFERENCES 121 ACKNOWLEDGEMENTS Almighty Allah is the one whose likehood towards knowledge and mankind compelled me to work for the safety and betterment of mankind. The increased use of herbal medicine in our population without authentic evidence made me to go for this piece of work. This thesis constitute of thorough screening of Vernonia anthelmintica seeds on pharmacological and toxicological grounds. It is my honor that I got the chance to express my greatest depth of gratitude to my research supervisor, Dr. Rafeeq Alam Khan, Professor Meritorious, Chairperson Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical sciences, University of Karachi, for his selfless support, thorough guidance and sincere advices. His new insights and valued experience helped me at every step of my work starting from selection of topic till the submission of thesis. I would like to express my appreciation to Prof. Dr. Rahela Najm, Professor, Department of Pharmacology for teaching us Ph.D. courses and all other cooperations she offered to me. No doubt she is always an inspiration to me. I am also thankful to Prof. Dr. Iqbal Azhar, Dean, Faculty of Pharmacy and pharmaceutical sciences, University of Karachi, Prof. Dr. Talat Mirza, Head of Department, Pathology, Dow University of health science and Prof. Dr. Iqbal Choudry, Head of H.E.J department for facilitating me in their capacities. I really want to mention thanks from the depth of my soul to my father, Mr. Jamil Akhter and mother Mrs. Riaz Fatima (late). Also wanted to thank Mrs. Laila Zaidi and Mr. Sabir Zaidi, who kept me motivated whenever I felt exhausted during my work. I also want to express my love to my children. My special thanks to Mr. Shadab Ahmed, lecturer, department of Pharmacology for his continued support and being a kind research fellow of mine. Sincere thanks are due for the technical staff specially Mr. Muhammad Kashif Ali, animal technician and Mr. Muhammad Ibrahim, computer technician Department of Pharmacology for the successful completion of my work. My heartily thanks to my friends and for all those who helped and motivated me in one or the other way and supported me whenever needed. Subia Jamil LIST OF TABLES Table-1 Cancer cell lines and their specifications 27 Table-2 Qualitative Phytochemical analysis of HEVA, EEVA AND WDVA 48 Table-3 Antibacterial bioassay of HEVA 49 Table-4 Antibacterial bioassay of EEVA 49 Table-5 MTT bioassay of HEVA and EEVA against 3T3 cell lines 50 Table-6 MTT bioassay of HEVA and EEVA against Hela cell lines 50 Table-7 MTT bioassay of HEVA and EEVA against PC3 cell lines 51 Table-8 MTT bioassay of HEVA and EEVA against MDA-MB-231cell lines 51 Table-9 MTT bioassay of HEVA and EEVA againstMCF-7 cell lines 51 Table-10 Brine shrimp lethality assay of HEVA 52 Table-11 Brine shrimp lethality assay of EEVA 52 Table-12 Antioxidant bioassay of HEVA and EEVA 53 Table-13 Antiglycation assay of HEVA and EEVA 55 Table-14 Phytotoxic bioassay of HEVA 56 Table-15 Phytotoxic bioassay of EEVA 56 Table-16 Insecticidal bioassay of HEVA 57 Table-17 Insecticidal bioassay of EEVA 57 Table-18 Leishmanicidal bioassay of HEVA and EEVA 58 Table-19 Oral acute toxicity of HEVA,EEVA and WDVA 59 Table-20 Effects of HEVA,EEVA and WDVA on body weight 60 Table-21 Effects of Vernonia anthelmintica on haematological profile 61 Table-22 Effects of Vernonia anthelmintica on coagulation 62 Table-23 Effects of Vernonia anthelmintica on diabetes marker 63 Table-24 Effects of Vernonia anthelmintica on lipid profile 64 Table-25 Effects of Vernonia anthelmintica on liver function 65 Table-26 Effects of Vernonia anthelmintica on renal function 66 Table-27 Effects of Vernonia anthelmintica on electrolytes 67 Table-28 Anti-inflammatory activity of Vernonia anthelmintica in rat paw 69 Table-29 Analgesic activity of Vernonia anthelmintica by hot plate method 70 I Table-30 Analgesic activity of Vernonia anthelmintica by tail flick method 71 Table-31 Effects of Vernonia anthelmintica on hyperlipidemia in HFHS rat model 73 Effects of Vernonia anthelmintica on cardiac risk parameters in HFHS rat Table-32 75 model Effects of Vernonia anthelmintica on fasting blood glucose in HFHS rat Table-33 78 model Table-34 Effects of Vernonia anthelmintica on liver function in HFHS rat model 80 Effects of Vernonia anthelmintica on blood glucose levels of Table-35 82 normoglycemic rat Effects of Vernonia anthelmintica on blood glucose levels of Table-36 84 hyperglycemic rat Effects of Vernonia anthelmintica on blood glucose levels of alloxan Table-37 86 induced diabetic rat II LIST OF FIGURES pg Figure- 1 Vernonia anthelmintica plant grown at botanical garden Department 3 of Botany, University of Karachi.