ASSESSMENT OF THE GENOTOXICITY AND MUTAGENIC POTENTIAL OF TWO MEDICINAL PLANTS, PARINARI CURATELLIFOLIA (PLANCH. EX BENTH.) KUNTZE AND AZADIRACHTA INDICA A. JUSS, USING THE ALLIUM CEPA ASSAY AND THE AMES TEST KATABALE MASSAMA KOSSI MASTER OF SCIENCE (Molecular Biology and Biotechnology) 2017 i Assessment of the Genotoxicity and Mutagenic Potential of Two Medicinal Plants, Parinari Curatellifolia (Planch. Ex Benth.) Kuntze and Azadirachta indica A. Juss, Using the Allium cepa Assay and the Ames Test Katabale Massama Kossi A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in Molecular Biology and Biotechnology at the Pan African University Institute for Basic Sciences, Technology and Innovation 2017 ii DECLARATION This thesis is my original work and has not been presented for a degree in any other university. Signature Date Katabale Massama Kossi PAUISTI This thesis has been submitted for examination with my/our approval as university supervisors Signature Date Prof. Daniel Kariuki JKUAT, Kenya Signature Date Dr. Esther N. M. Maina University of Nairobi, Kenya iii TABLE OF CONTENTS DECLARATION ....................................................................................................................iii TABLE OF CONTENTS ........................................................................................................ iv ACKNOWLEDGEMENT ....................................................................................................... x LIST OF TABLES .................................................................................................................. xi LIST OF FIGURES ............................................................................................................... xii LIST OF PLATES ................................................................................................................ xiv ABBREVIATIONS AND ACCRONYMS ........................................................................... xv ABSTRACT ......................................................................................................................... xvii RESUME (Français) ............................................................................................................. xix CHAPTER ONE ...................................................................................................................... 1 INTRODUCTION ................................................................................................................... 1 1.1 General Introduction .......................................................................................................... 1 1.2 Background Information .................................................................................................... 2 1.3 Statement of the Problem ................................................................................................... 3 1.4 Justification of the Study ................................................................................................... 4 1.5 Research Questions ............................................................................................................ 5 1.6 Hypothesis.......................................................................................................................... 5 1.6.1 Null Hypothesis .............................................................................................................. 5 1.7. General Objective ............................................................................................................. 5 iv 1.7.1 Specific Objectives ......................................................................................................... 5 CHAPTER TWO ..................................................................................................................... 7 LITERATURE REVIEW ....................................................................................................... 7 2.1 Description and importance of Parinari curatellifolia (Planch. ex Benth.) Kuntze and Azadirachta indica (A. Juss) in herbal medicine ..................................................................... 7 2.1.1 Biology and medical benefits of Parinari curatellifolia (Planch. ex Benth.) Kuntze. ... 7 2.1.1.1 Taxonomy .................................................................................................................... 7 2.1.1.2 Ethnobotanical use of Parinari curatellifolia (Planch. ex Benth.) Kuntze. ................. 7 2.1.1.3 Scientific studies and biological activities ................................................................... 8 2.1.2 Biology and medical uses of Azadirachta indica (A. Juss) ............................................ 9 2.1.2.1 Taxonomy .................................................................................................................... 9 2.1.2.2 Ethnobotanical use of Azadirachta indica (A. Juss) .................................................... 9 2.1.2.3 Compounds and scientific studies in A. indica .......................................................... 10 2.2 Genotoxicity and mutagenicity ........................................................................................ 12 2.2.1 Guidelines on the genotoxicity assessment in medicinal plants ................................... 13 2.2.3 Medicinal Plant Extracts and induction of Chromosomal Alterations ......................... 14 2.3 Some plant compounds associated with genotoxic or mutagenic effects ........................ 20 2.3.1 Aristolochic acid ........................................................................................................... 20 2.3.2 Integerrimine ................................................................................................................. 21 2.3.3 Estragole ....................................................................................................................... 22 v 2.3.4 Trans-anethole............................................................................................................... 23 2.3.5 Lasiocarpine .................................................................................................................. 23 2.3.6 Methyleugenol .............................................................................................................. 24 2.3.7 Heliotrine ...................................................................................................................... 25 2.3.8 Myristicin ...................................................................................................................... 25 2.3.9 Quercetin ....................................................................................................................... 26 2.4.10 Reserpine..................................................................................................................... 27 2.3.11 Riddeliine .................................................................................................................... 28 2.3.12 Safrole ......................................................................................................................... 28 2.4.13 Senkirkine ................................................................................................................... 29 CHAPTER THREE ............................................................................................................... 31 MATERIAL AND METHODS ............................................................................................. 31 3.1 Sampling site for the medicinal plants ............................................................................. 31 3.2. Transport of plant samples .............................................................................................. 33 3.3 Preparation of the medicinal plant extracts ...................................................................... 33 3.3.1 Pretreatment of the plants ............................................................................................. 33 3.3.2 Preparation of methanol extracts .................................................................................. 34 3.3.3 Preparation of aqueous extracts .................................................................................... 34 3.4 Phytochemical analysis .................................................................................................... 34 3.4.1 Phytochemical screening .............................................................................................. 34 vi 3.4.1.1 Detection of saponins ................................................................................................. 34 3.4.1.2 Detection of tannins ................................................................................................... 35 3.4.1.3 Detection of flavonoids .............................................................................................. 35 3.4.1.4 Detection of glycosides .............................................................................................. 35 3.4.1.5 Detection of alkaloids ................................................................................................ 35 3.4.1.6 Detection of phytosterols ........................................................................................... 36 3.4.1.7 Detection of terpenoids .............................................................................................. 36 3.4.2 Quantitative analysis ....................................................................................................