Isolation, Characterization and Biological Activities of Terpenoids from Gunnera Perpensa
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Isolation, characterization and biological activities of terpenoids from Gunnera perpensa Submitted in fulfilment for the Degree of Master of Applied Sciences in Biotechnology in the Department of Biotechnology and Food Technology, Durban University of Technology, Durban, South Africa Fitsum K. Mammo SUPERVISOR: Dr Viresh Mohanlall CO- SUPERVISORS: Professor Francis O. Shode Professor Bharti Odhav REFERENCE DECLARATION I, Miss Fitsum K. Mammo (student number: 21449777) and Dr Viresh Mohanlall do hereby declare that in respect of the following dissertation: Title: Isolation, characterization and biological evaluation of terpenoids from Gunnera perpensa. 1. As far as we ascertain: a) no other similar dissertation exists; 2. All references as detailed in the dissertation are complete in terms of all personal communication engaged in and published works consulted. Signature of student Date Signature of supervisor Date Signature of co-supervisor Date Signature of co-supervisor Date DEDICATION This thesis is dedicated to my mother, Mrs. Aberash Zeleke Kuri and my late father, Mr. Kassa Mammo Abate, who instilled in me the virtues of perseverance and commitment to strive for excellence. TABLE OF CONTENTS ACKNOWLEDGEMENTS .......................................................................................................... i ABBREVIATIONS ....................................................................................................................... ii LIST OF FIGURES ..................................................................................................................... iv LIST OF TABLES ....................................................................................................................... vi ABSTRACT .................................................................................................................................. vii CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW ............................................... 1 1.1. Introduction .......................................................................................................................... 1 1.1.1. Aim ................................................................................................................................ 3 1.1.2. Objectives ...................................................................................................................... 3 1.2. Gunnera perpensa ................................................................................................................ 4 1.2.1. Taxonomy and classification ......................................................................................... 6 1.2.2. Geographical Distribution ............................................................................................. 7 1.2.3. Ethnomedicinal uses ...................................................................................................... 9 1.2.4. Phytochemistry ............................................................................................................ 11 1.2.5. Pharmacology .............................................................................................................. 14 1.3. Plant secondary metabolites ............................................................................................... 16 1.3.1. Biosynthesis of secondary metabolites ........................................................................ 18 1.3.3. Terpenes....................................................................................................................... 22 CHAPTER 2: EXTRACTION, ISOLATION AND STRUCTURAL ELUCIDATION OF SECONDARY METABOLITES FROM Gunnera perpensa ........................ 29 2.1. Introduction ........................................................................................................................ 29 2.2. Materials and Methods ....................................................................................................... 32 2.2.1. Collection of plant material ......................................................................................... 32 2.2.2. Extraction..................................................................................................................... 32 2.2.3. Isolation and purification ............................................................................................. 35 2.3. Results ................................................................................................................................ 39 2.3.1. Extraction and isolation of secondary metabolites (Protocol 1) .................................. 39 2.3.2. Extraction and isolation of secondary metabolites (Protocol 2) .................................. 41 2.3.3. Structural elucidation of isolated compounds from the leaves .................................... 45 2.3.4. Structural elucidation of isolated compounds from the stalks ..................................... 47 1 2.4. Discussion .......................................................................................................................... 55 CHAPTER 3: BIOLOGICAL SCREENING OF EXTRACTS AND SECONDARY METABOLITES OF Gunnera perpensa .............................................................. 58 3.1. Introduction ........................................................................................................................ 58 3.2. Materials and Methods ....................................................................................................... 62 3.2.1. Antimicrobial activity using agar disc diffusion assay ................................................ 62 3.2.2. Antimicrobial activity using bio-autography ............................................................... 63 3.2.2. Anti-sickling activity using Emmel’s test ................................................................... 64 3.2.3. Anti-oxidant activity using DPPH photometric assay ................................................. 65 3.2.4. Anticancer activity using MTT assay .......................................................................... 65 3.2.5. Toxicity evaluation using Brine shrimp assay ............................................................. 69 3.3. Results ................................................................................................................................ 70 3.3.1. Agar disc diffusion assay ............................................................................................. 70 3.3.2. Bio-autography assay .................................................................................................. 72 3.3.2. Anti-sickling, Emmel’s test ......................................................................................... 73 3.3.3. DPPH radical scavenging capacity .............................................................................. 75 3.3.4. MTT assay ................................................................................................................... 76 3.3.5. Biosafety assay ............................................................................................................ 77 3.4. Discussion .......................................................................................................................... 79 CHAPTER 4: GENERAL CONCLUSION .................................................................................. 85 CHAPTER 5: REFERENCES ...................................................................................................... 87 APPENDIX I .............................................................................................................................. 106 APPENDIX II: PUBLICATIONS .............................................................................................. 115 APPENDIX III: SUBMISSION ................................................................................................. 122 2 ACKNOWLEDGEMENTS I would like to extend my sincere thanks and gratitude to: • My supervisors, Dr V. Mohanlall, Prof. B. Odhav and Prof. F. O. Shode for their patience, guidance, constructive criticism and words of encouragement throughout the course of this project. • My mum, my sisters and my brother, for their support and encouragement. • My friends and colleagues, esp. Mr Kabange Kasumbwe, for their moral support and assistance. • Prof. H. Baijnath, University of Kwa-Zulu Natal for the collection of plant material. • Prof. M. Singh and Mr C. Grimmer, University of Kwa-Zulu Natal (UKZN), for their technical assistance. • Mr D. Tshibangu, University of Kinshasa, Department of chemistry, for his technical assistance. • National Research Foundation (NRF) for funding this project. i ABBREVIATIONS TLC Thin layer chromatography PTLC Preparative Thin-Layer Chromatography CC Column chromatography HPLC High-performance liquid chromatography NMR Nuclear magnetic resonance DEPT Distortionless enhancement by polarisation transfer DPPH 2,2-diphenyl-1-picryhydrazyl MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide UV Ultra-violet MEP Methylerythritol Phosphate MVA Mevalonic acid IPP Isopentenyl pyrophosphate DMAPP Dimethylallyl pyrophosphate PCT Pentacyclic Triterpene OA Oleanolic acid UA Ursolic acid BA Betulinic acid MA Maslinic acid DCM Dichloromethane SiO2 Silica gel IR Infra-red MS Mass Spectrometry CDCl3 Deuterated chloroform DMSO Dimethyl sulfoxide TMS Tetramethylsilane ii MIC Minimum inhibitory concentration ABTS 2,2’-azino-di [3-ethylbenzthiazoline sulfonate] ORAC Oxygen radical absorbance