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Radioprotective Effects of Rooibos Herbal Tea on The RADIOPROTECTIVE EFFECTS OF ROOIBOS HERBAL TEA ON THE DEVELOPING CENTRAL NERVOUS SYSTEM OF WISTAR RATS By Milod M Ahmed Alrtemi (3483079) A thesis submitted in fulfilment of the requirements for the degree of Magister Scientiae (MSc) Department of Medical Biosciences Faculty of Natural Sciences Supervisor Dr. Okobi Ekpo https://etd.uwc.ac.za DECLARATION I declare that the thesis titled: “Radioprotective effects of Rooibos herbal tea on the developing central nervous system of Wistar rats” is my own work submitted for the Master of Science degree in Medical Biosciences at the University of the Western Cape, South Africa, and that this work has not been submitted for any degree or examination in any other university, and that all the sources I have used or quoted have been indicated and acknowledged by complete references Full name... Milod M Ahmed Alrtemi Signed....................................... ii https://etd.uwc.ac.za DEDICATION This study is dedicated to my parents and my family iii https://etd.uwc.ac.za ACKNOWLEDGEMENTS First of all, I will like to thank the Almighty Allah for giving me faith, strength, good health, wisdom, strength and perseverance to go through the Masters programme and complete the writing of this thesis. I would like to express my appreciation to the following individuals and organizations that made my research experience formative and memorable: • My supervisor, Dr. Okobi Ekpo for giving me the opportunity to be accepted as a student in his Research Group and to carry out this study in his laboratory. I am grateful for his guidance, help, support, advice and encouragement throughout my M.Sc. programme, and for his painstaking proofreading of all versions of my thesis. • Fundi of the UWC Animal house for all his help with animal work. • Mr. Philip Beukes of iThemba Labs, Cape Town for helping with irradiation of my animals. • My colleagues at the Experimental Neuroscience Lab: Sylvester, Adaze, Khalid, Bushra and Taahirah, for all their diverse assistance. • My family and friends for their immense support. • The Libyan Embassy in South Africa for all the assistance during my stay in Cape Town, South Africa. iv https://etd.uwc.ac.za LIST OF ABBREVIATIONS % Percent °C Degree Celsius AAF Acetylaminofluorence ACE Angiotensin converting enzymes AFB Aflatoxin B ANOVA Analysis of variance ATP Automated tissue processor CAT Catalase CHO Chromosome CNS Central nervous system CV Cresyl Violet D Day DCM Diabetic cardiomyopathy DEN Diethyl nitrosamine dH2O Distilled water DNA Deoxyribonucleic acid DPX: Distyrene Plasticizer Xylene DV Daily value EGCG Epigllocatechin gallate ETBR Ethidium bromide FFPE Formalin-fixed, paraffin-embedded v https://etd.uwc.ac.za FRAP Ferric reducing antioxidant power FRHT Fermented Rooibos herbal tea FSH Follicle stimulating hormone G Gram GPX Glutathione peroxide GR Glutathione reductase GSH Glutathione Gy Gray H Hour H&E: Haematoxylin and Eosin. HCl Hydrochloric acid IHC Immunohistochemistry LPO Lipid peroxidation M Molar Mg/Ml Milligram per millilitre MMC Mitomycin c MNRET Micro nucleated reticulocytes N Number of samples Na2CO3 Sodium carbonate Na2HPO4 Disodium hydrogen phosphate NaCl Sodium chloride NAH2PO4 Anhydrous monobasic sodium phosphate NaOH Sodium hydroxide vi https://etd.uwc.ac.za NOR Novel Object Recognition NS Normal saline O2- Superoxide anion OF Open Field OH Hydroxyl radical OSS Oxidative stress status PBS Phosphate buffered saline PCR Polymerase chain reaction PFDB Paraformaldehyde pH Protons of hydrogen PND Postnatal days ROS Reactive oxygen species RT Room temperature Sec Second SOD Superoxide dismutase UV Ultraviolet vii https://etd.uwc.ac.za KEYWORDS Gamma Radiation Brain Fermented rooibos tea Ionizing radiation Neurobehavioural Central Nervous System Hippocampus Cerebellum viii https://etd.uwc.ac.za LIST OF TABLES Table 2.1: Bioactive compounds present in Rooibos and their antioxidant activity…………………………………………………… 20 Table 3.1: Animal grouping…………………………………………………… 27 Table 3.2: List of chemical and supplier………………………………………… 29 Table 3.3: List of equipment used and supplier………………………………… 29 Table 3.4: Tissue processing protocol…………………………………………… 31 Table 3.5: Haematoxylin and Eosin staining protocol…………………………… 33 Table 3.6: IHC staining procedure………………………………………………. 34 ix https://etd.uwc.ac.za LIST OF FIGURES Figure 2.1: Diagram showing stages of penetration all ionizing radiation. ................................... 7 Figure 2.2: Figure showing the stages of radiation-induced brain injury .................................... 12 Figure 2.3: Rooibos plantation in the Clanwilliam area (Joubert and de Beer, 2011). ................ 16 Figure 2.4: Production areas of Aspalathus linearis in and around the Greater Cederberg Biodiversity Corridor ................................................................................................. 16 Figure 4.1: Average total distance travelled inside the Open Field apparatus………………….40 Figure 4.2: Representative trajectory maps of changes in locomotor activity inside the open field apparatus. ................................................................................................................... 40 Figure 4.3: Average central square entry in the Open Field apparatus.. ...................................... 41 Figure 4.4: Average number of times offspring rats stood on hind limbs with raised forelimbs. 42 x https://etd.uwc.ac.za TABLE OF CONTENTS DECLARATION ............................................................................................................................ ii DEDICATION ............................................................................................................................... iii ACKNOWLEDGEMENTS ........................................................................................................... iv LIST OF ABBREVIATIONS ......................................................................................................... v KEYWORDS ............................................................................................................................... viii LIST OF TABLES ......................................................................................................................... ix LIST OF FIGURES ........................................................................................................................ x TABLE OF CONTENTS ............................................................................................................... xi ABSTRACT .................................................................................................................................. xv CHAPTER ONE ............................................................................................................................. 1 INTRODUCTION .......................................................................................................................... 1 1.1. Background .......................................................................................................................... 1 1.2. Significance of Study ........................................................................................................... 4 1.3. Hypothesis............................................................................................................................ 4 1.4. General Aim ......................................................................................................................... 5 1.5. Objectives ............................................................................................................................ 5 CHAPTER TWO ............................................................................................................................ 6 2.1. RADIATION ....................................................................................................................... 6 2.2. Types of Radiation ............................................................................................................... 6 2.2.1. Non-ionizing radiation ..................................................................................................... 6 2.2.2. Ionizing Radiation ............................................................................................................ 6 2.2.2.1. Alpha particle ................................................................................................................... 7 2.2.2.2. Beta particles .................................................................................................................... 8 2.2.2.3. Neutrons ........................................................................................................................... 8 2.2.2.4. Gamma rays...................................................................................................................... 8 2.2.2.5. X-rays ............................................................................................................................... 9 2.3. Biological Effects of Radiation ............................................................................................ 9 xi https://etd.uwc.ac.za 2.3.1. Direct Effects of Radiation ............................................................................................. 10 2.3.2. Indirect Effect of Radiation ............................................................................................ 10 2.4. Mechanism of Radiation-induced Brain Injury ................................................................
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