ANTIMYCOBACTERIAL ACTIVITIES OF SELECTED PLANTS USED IN THE MANAGEMENT OF TUBERCULOSIS IN SEKHUKHUNE (LIMPOPO PROVINCE), SOUTH AFRICA. By Jacobus Kori Madisha (2014116681) Dissertation submitted in fulfilment of the requirements for the Degree Magister Scientiae in the Faculty of Natural and Agricultural Sciences, Department of Plant Sciences, University of the Free State Supervisor: Dr. A.O.T. Ashafa CO-Supervisor: Dr. A.O. Aiyegoro i DECLARATION I, the undersigned, hereby declare that the work contained in this dissertation supervised by Dr AOT Ashafa and Dr OA Aiyegoro is my original work and that I have not previously in its entirety or in part submitted at any university for a degree. I furthermore cede copyright of the dissertation in favour of the University of the Free State. Signature :…………………….. Date :……………………... Dr AOT Ashafa’s Signature:………………………. Date:……………………….. Dr OA Aiyegoro’s Signature……………………………….. Date:………………………………… ii DEDICATION To my late brother Moitswadi Elias Madisha who passed on this world on 09 October 2014 when I was busy with the studies, may his soul rest in peace. He will always be in our memories. iii Acknowledgement To Dr AOT Ashafa for his encouragement, patience, and most of all, his belief in me. My fellow students who assisted me in one way or the other during the program. The traditional healers and friends who helped with the collection of plant species. My sister and my brother in law, Mrs RM Mametja and Mr RH Mametja for their loving support. iv RESEARCH OUTPUTS Conference Poster J.K.Madisha, A.O.Aiyegoro, A.O.T.Ashafa Antimycobacterial activities of selected plants used in the management of Tuberculosis in Limpopo Province, South Africa. South African Association of Botanist Conference 2016 v TABLE OF CONTENTS Title and Supervisors vi Declaration vi Dedication vi Acknowledgements vi Research outputs vi Table of contents vi List of figures xi List of tables xii Abbreviations xiii List of symbols xiv Abstract xv vi TABLE OF CONTENTS CHAPTER ONE BACKGROUND 1 1.1 Introduction 1 1.2 Herbal medicine 3 1.3 Traditional African medicine 4 9 1.4 Traditional medicine in South Africa 1.5 The value of medicinal plants in drug discovery 10 1.6 Plants derived Antimicrobial compounds 11 1.7 Tuberculosis and burden of Disease 12 1.8 Tuberculosis in South Africa 12 1.9 TB Treatment 13 1.10 MDR TB and XDR TB 14 1.11 Alternative treatment of TB 15 1.12 Diagnosis use by Traditional healer for TB 16 1.13 Conservation of Bapedi medicinal plants 17 1.14 Bapedi medicinal plants for Tuberculosis 17 1.15 Literature review on Bapedi anti TB plants 18 1.15.1 Aloe marlothii A.Berger 18 1.15.2 Drimia elata Jacq. 20 1.15.3 Elephantorrhiza elephantina (Burch.) 22 1.15.4 Maerua angolensis (PROTA) 24 1.16 Microbial Isolates 26 1.16.1 Mycobacterium smegmatis 26 vii 1.16.2 Mycobacterium haemophilum 27 1.16.3 Mycobacterium peregrinum 27 1.16.4 Mycobacterium tuberculosis 28 1.17 Combination Study 28 1.18 Statement of research problem 29 1.19 Rationale 29 1.20 Aims and objectives 31 1.20.1 Aim 31 1.20.2 Objectives 31 CHAPTER TWO Ethnobotanical survey 32 2.0 Methods 32 2.1 Study area and population 32 2.2 Sampling procedure 35 2.3 Data collection 36 2.4 Ethnobotanical information obtained on some of the plants 37 2.4.1 Aloe marlothii (A.Berger) 37 2.4.2 Drimia elata (Jacq.) 37 2.4.3 Elephantorrhiza elephantine 38 2.4.4 Maerua angolensis 38 2.5.0Antimicrobial activity 38 2.5.1 Collection of plant materials 38 2.5.2 Extraction of plant material 39 2.6 Antibacterial Assay (sensitivity test) 39 viii 2.7 Streak plate disc diffusion (SPDD) 40 2.8.1 Preparation of Mycobacterium tuberculosis stocks 40 2.8.2 Bacterial strains and inoculum preparation 41 2.8.3 Storage and Maintenance of Bacterial Strains 41 2.9 Minimum Inhibitory concentration (MIC) test 41 2.10 Combination assays 42 2.11 Data analysis 43 CHAPTER THREE Results and Discussion Ethnobotanical survey 44 3.1 Results 44 3.1.1 Gender, age and years in practice of traditional healers 44 3.1.2 Level of education 45 3.1.3 Plant parts used 46 3.1.4 Preparation of Traditional medicine 47 3.2 Discussion 47 Antimicrobial activity 48 3.3.1 Sensitivity and MIC results and discussion 48 3.3.2 Minimum Inhibitory Concentration (MIC) of the plant extracts 59 3.4 Results and discussion for combination study of the plants 61 CHAPTER FOUR Conclusion and recommendations 64 4.1 Significance of the ethnobotanical survey and antibacterial 64 4.2 Correlation between traditional use and antimicrobial screening 65 ix 4.3 Conclusions 66 4.4 Limitations and future recommendations 67 4.4 REFERENCES 69 4.5.APPENDIX A 78 4.6.APPENDIX B 83 x LIST OF Figure Figure Title Page Figure 1.1 Structural formula of Rifampicin 6 Figure 1.2 Structural formula of Isoniazid 6 Figure 1.3 Structural formula of Fluoroquinolones 7 Figure1. 4 Structural formula of Capreomycin 7 Figure 1.5 Structural formula of Kanamycin 8 Figure 1.6 Structural formula of Amikacin 8 Figure 1.7 Habitat/Vegetative morphology of Aloe marlothii (A.Berger) 19 Figure 1.8 Habitat/Vegetative morphology of Drimia elata (Jacq.) 21 Figure 1.9 Habitat/Vegetative morphology of Elephantorrhiza Elephantina 23 (Burch.) Figure 1.10 Habitat/Vegetative morphology of Maerua angolensis (PROTA) 24 Figure 2.1 Map of South Africa 34 Figure 2.2 Map of Sekhukhune District Municipality 35 Figure 3.1 Part of plants use 49 xi LIST OF TABLES Table 1.1 South African records (2008 – 2013) of deaths from TB 13 Table 1.2 Medicinal plants with investigated antimycobacterial activities 26 Table 3.1 Gender, age and years in practice traditional healers 45 Table 3.2 Zone of inhibition (mm) Aloe Marlothii extract on bacteria isolates 49 Table 3.3 Zone of inhibition (mm) Maerua angolensis extract on bacteria isolates. 51 Table 3.4 Zone of inhibition (mm) Drimia elata extract on bacteria isolates 52 Table 3.5 Zone of inhibition (mm) Elephantorrhiza elephantina extract on bacteria 53 isolates Table 3.6 MIC results for Aloe marlothii against susceptible bacteria isolates 55 Table 3.7 MIC results for Maerua angolensis (Mogogwane) against susceptible 56 bacteria isolates Table 3.8 MIC results for Drimia elata against susceptible bacteria isolates 58 Table 3.9 MIC results for Elephantorrhiza elephantina against susceptible bacteria 60 isolates Table 3.10 MIC results for combination of Aloe Marlothii, Maerua angolensis, 61 Drimia elata, Elephantorrhiza Elephantina Table 3.11 MIC results for A. Marlothii (Ethanol) and E. Elephantina (Hydroethanol) 63 xii ABBREVIATIONS AIDS Acquired Immunodeficiency Syndromes CDC - Centres for Disease Control and Prevention CFU - Colony Forming Units CLSI - Clinical and Laboratory Standards Institute COPD - Chronic Obstructive Pulmonary Disease DMSO- Dimethyl Sulfoxide HIV - Human Immuno Virus INT- p-iodonitrotetrazolium violet M. tuberculosis- Mycobacterium tuberculosis MBC - Minimum Bactericidal Concentration MDR - Multi Drug Resistant MHA - Mueller-Hinton Agar MHB - Mueller-Hilton broth MIC - Minimal Inhibitory Concentration STD - Sexually Transmitted Diseases STI - Sexually Transmitted Infection TB -Tuberculosis WHO- World Health Organisation XDR -Extensively Drug Resistant TB xiii SYMBOLS - Negative + Positive °C - Degrees Celsius g - Gram h - Hour(s) L - Litre mg - Milligram mL - Millilitres mm - Millimetres μg/ml- Microgram per millilitre μl -Microlitre μm -Micrometer xiv A B S T R A C T Tuberculosis (TB) continues to be a devastating disease of the world affecting more than two million people annually with one-third of the world’s populations suffering from the menace. The management of TB was in the use of orthodox medicines which are not only expensive but presents severe side effects. Thus, efforts are recently geared towards the use of alternative therapy from natural sources which could offer a lasting solution to the treatment of the diseases with little or no side effects. The study investigated the antimicrobial potentials of four medicinal plants used by Bapedi tribe of Sekhukhune area, Limpopo Province of South Africa. The antimycobaceterial efficacy of Aloe marlothii, Maerua angolensis, Drimia elata and Elephantina elephantorrhiza which were selected based on ethnobotanical study carried-out in the study was tested in four solvents such as ethanol, methanol, hydroethanol and dichlrormethane against four mycobacterium species such as M. tuberculosis, M. smegmatis, M. peregrinum, M. haemophilus and other gram positive and gram negative bacteria isolates using agar well dilution method and streak plate disc diffusion assay as a way of validating the anti-tuberculosis potentials of the plants. The results revealed the anti TB activity of the four plants particularly M. angolensis, D. elata and E. elephantorrhiza which were reported for the first time in this study. Similarly, the results revealed varied degrees of antimycobacterial activities of most of the screened extracts (particularly ethanolic and methanol) going by the zone of inhibition values (10 – 32 mm) as well as minimum inhibitory concentration (MIC) values that fell within the range of 0.098 - 1.56 µg/mL and as such, could be adjudged to possess anti TB potentials. xv Conclusively, the anti TB activity witnessed by the four plants could be attributed to the presence of the secondary metabolites which are responsible for the elicited effect. The study also validates the use of these plants in the management of tuberculosis by the Sekhukhune people of Limpopo Province, South Africa. xvi CHAPTER ONE Background 1.1 Introduction Tuberculosis (TB) is one of the leading causes of morbidity and mortality globally (WHO, 2008a). The global mortality rate as at ten years ago stands at two million per year with one third of the world‟s population infected with the bacilli (Sanjay, 2004; Centre for Disease Control 2005; WHO, 2007).