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IN VITRO ANTIPROLIFERATIVE ACTIVITY of AQUEOUS ROOT BARK EXTRACT of Cassia Abbreviata (Holmes) Brenan

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IN VITRO ANTIPROLIFERATIVE ACTIVITY of AQUEOUS ROOT BARK EXTRACT of Cassia Abbreviata (Holmes) Brenan IN VITRO ANTIPROLIFERATIVE ACTIVITY OF AQUEOUS ROOT BARK EXTRACT OF Cassia abbreviata (Holmes) Brenan NJAGI SHADRACK MWENDA (Btech, Hons) I56/24935/2013 A Thesis Submitted in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science (Biotechnology) in the School of Pure and Applied Sciences of Kenyatta University OCTOBER, 2016 ii DECLARATION iii DEDICATION To the almighty God iv ACKNOWLEDGMENT I praise the Almighty GOD for the success of this work. The completion of this work is due to His mercies and grace throughout the walking of this journey, glory is to thee God. I would like to thank my supervisors, Dr Mathew Piero Ngugi and Dr Peter Mwitari. I will be forever grateful. I thank you for being a fountain of wisdom, inspiration and support. In addition, I thank Festus Kioko, Wyclife Arika, Robert Ouko, Dorothy Nyamai, Philip Ogola, James Kimani, Hibbert Rachuonyo, Japheth Wambani, Kimani Njoroge and Peter Ndwiga. I also recognize the support of Japheth Kipngetich, Gerald Onyancha and Nicholus Adipo. Many thanks to Dr Lucia Keter for her support during the study. Dr Antony Ireri (Cewa) thank you for your inspiration. I also recognize the support I received from Kenyatta University and KEMRI; Centre for Traditional Medicine and Drug Research, am grateful. My gratitude to my friend Amos Musyoki Mawia. I thank you for being a true friend. Rose Chemutai Lagat, I thank you you for your moral support. I thank my family for their support, inspiration and motivation. A standing ovation for my mentor Mr Richard Kimenyi, I am humbled for providing a pillar of unwavering support to me and sponsorship for my education. Thank you for cherishing my dreams and facilitating my academic life. Lastly, I appreciate everyone who has been part of my story, I am strong today because am standing on your shoulders. I will be forever grateful. v TABLE OF CONTENTS DECLARATION ............................................................................................................. ii DEDICATION ................................................................................................................. ii ACKNOWLEDGMENT ................................................................................................ iv TABLE OF CONTENTS ................................................................................................ v LIST OF TABLES ....................................................................................................... viii LIST OF FIGURES ....................................................................................................... ix LIST OF APPENDICES ................................................................................................. x ABBREVIATIONS AND ACRONYMS ...................................................................... xi ABSTRACT ................................................................................................................... xii CHAPTER ONE .............................................................................................................. 1 INTRODUCTION ........................................................................................................... 1 1.1 Background Information ............................................................................................. 1 1.2 Problem Statement and Justification ........................................................................... 4 1.3 Research Questions ..................................................................................................... 6 1.4 Objectives.................................................................................................................. 6 1.4.1 Main Objective .......................................................................................................... 6 1.4.2 Specific Objectives ................................................................................................... 6 CHAPTER TWO ............................................................................................................ 7 LITERATURE REVIEW ............................................................................................... 7 2.1 The Biology of Cell Proliferation ................................................................................ 7 2.2 Control of Cell Proliferation ..................................................................................... 7 2.3 Defective Regulation of Cell Proliferation .................................................................. 9 2.4 Cancer Metastasis ...................................................................................................... 11 2.5 Hepatocellular Carcinoma (HCC) ............................................................................. 12 2.6 Conventional Treatment of Cancer ........................................................................... 12 2.6.1 Chemotherapeutic Drugs ........................................................................................ 12 2.6.1.1 Alkylating Agents ............................................................................................... 13 2.6.1.2 Antimetabolites ................................................................................................... 13 2.6.1.3 Topoisomerase Inhibitors .................................................................................... 14 2.6.1.4 Antitumor Antibiotics ......................................................................................... 16 2.6.1.5 Metals .................................................................................................................. 17 2.6.1.5.1 Platinum Agents ............................................................................................... 18 2.6.1.5.2 Arsenic ............................................................................................................. 19 vi 2.6.2 Surgery ................................................................................................................... 20 2.6.3 Radiotherapy .......................................................................................................... 21 2.7 Economics of Maladies ............................................................................................. 22 2.8 Herbal Management of Maladies .............................................................................. 22 2.9 Methods for Determination of Cell Proliferation ...................................................... 26 2.9.1 Real-Time Luciferase Assay .................................................................................. 26 2.9.2 ATP Assay ............................................................................................................. 27 2.9.3 Lactate Dehydrogenase Assay ............................................................................... 27 2.9.4 Protease Viability Marker Assay ............................................................................ 29 2.9.5 MTT Tetrazolium Assay ........................................................................................ 29 2.10 Anticancer Associated Phytochemicals................................................................... 30 2.11 Mechanism of Activity of Anticancer Associated Phytochemicals ........................ 33 2.12 Cassia abbreviata .................................................................................................... 35 2.13.1 Plant Description .................................................................................................. 35 2.13.2 Economic Importance .......................................................................................... 36 2.13.3 Medicinal Uses ..................................................................................................... 36 2.13.3.1 Roots ................................................................................................................. 36 2.13.3.2 Stem Bark .......................................................................................................... 37 2.13.3.3 Leaves ............................................................................................................... 37 CHAPTER THREE ...................................................................................................... 38 MATERIALS AND METHODS.................................................................................. 38 3.1 Collection and Preparation of Plant Material ............................................................ 38 3.2 Aqueous Extraction ................................................................................................... 38 3.3 Determination of Antiproliferative Activity.............................................................. 39 3.3.1 Cell Lines ............................................................................................................... 39 3.3.2 Cell Culture ............................................................................................................ 39 3.3.3 Cell Treatment ........................................................................................................ 39 3.3.4 Viability MTT Test ................................................................................................ 40 3.3.4.1 Principle ............................................................................................................... 40 3.3.4.2 MTT Assay Procedure .......................................................................................
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