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Screening of Anti-Microbial Activity of Secondary Metabolites Extracted from Crinum Macowanii and Its Associated Bacterial Endophytes

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Screening of Anti-Microbial Activity of Secondary Metabolites Extracted from Crinum Macowanii and Its Associated Bacterial Endophytes COPYRIGHT AND CITATION CONSIDERATIONS FOR THIS THESIS/ DISSERTATION o Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. o NonCommercial — You may not use the material for commercial purposes. o ShareAlike — If you remix, transform, or build upon the material, you must distribute your contributions under the same license as the original. How to cite this thesis Surname, Initial(s). (2012). Title of the thesis or dissertation (Doctoral Thesis / Master’s Dissertation). Johannesburg: University of Johannesburg. Available from: http://hdl.handle.net/102000/0002 (Accessed: 22 August 2017). Screening of anti-microbial activity of secondary metabolites extracted from Crinum macowanii and its associated bacterial endophytes A Dissertation submitted to the Faculty of Science, University of Johannesburg In fulfilment of the requirement for the award of a Master’s Degree in Technology: Biotechnology By REBOTILOE FRIDAH MORARE STUDENT NUMBER: 201010153 Supervisor: Dr M.H. Serepa-Dlamini Co-supervisors: Dr E. Ubomba-Jaswa, Dr V. Mavumengwana Abstract Medicinal plants produce several secondary metabolites, which are significant in drug development. Crinum macowanii is a medicinal plant distributed in Zimbabwe, South Africa, Mozambique, Swaziland, Botswana, Zambia, Kenya and Malawi. C. macowanii is used to heal sexually transmitted diseases, backaches and stimulate lactation in women and cows. However, excessive use of medicinal plants for drug development could lead to plant species extinction and deforestation. The endophytes, bacterial and fungal species residing within the plant hosts, produce novel compounds similar to their plant hosts, which can be used in drug development. In this study, five bacterial endophytes were isolated from surface sterilised plant organs of the medicinal plant C. macowanii. Gram staining technique, scanning electron microscope and sequencing of 16S rRNA genes were used to identify the endophytes. The isolates were Staphylococcus sp. C2, Staphylococcus sp. C3, Bacillus sp. C4, Acinetobacter sp. C5 and Staphylococcus sp. C6. Antibacterial activities of each endophyte were assessed against Gram-negative strains Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and Klebsiella pneumoniae (ATCC 13182); Gram- positive Staphylococcus aureus (NCTC 6571) and Bacillus cereus (ATCC 10876) using the disk diffusion method with Streptomycin used as a positive control. The crude extracts of all the endophytes showed varying degrees of activity against all the test strains, except against Klebsiella pneumoniae. Phytochemical analysis of C. macowanii and associated bacterial endophytes varied slightly with presence of alkaloids. The Liquid Chromatography Quadrupole Time of Flight Mass Spectrometry (LCQTOF/MS) data showed that bacterial endophytes and C. macowanii have similar metabolites, which was based on having similar molecular formula and mass. However, based on few identified metabolites of bacterial endophytes and C. macowanii, the extraction methods used in this study need to be improved. This study contributed towards exploring metabolites from bacterial endophytes, which present a potential natural source for metabolites that can be used in drug discovery or applied for agricultural purposes. Keywords: Crinum macowanii, Endophytes, Secondary metabolites, Liquid Chromatography Quadrupole Time of Flight Mass Spectrometry, Antibacterial activity. ii DECLARATION I, Rebotiloe Fridah Morare declare that this study entitled “Screening of anti-microbial activity of secondary metabolites extracted from bacterial endophytes associated with Crinum macowanii” is my own work, completed under the supervision of Dr M.H Serepa- Dlamini, Dr Eunice Ubomba-Jaswa and Dr Vuyo Mavumengwana. Furthermore, I declare that this work has never been submitted elsewhere as a fulfilment for any qualification. In case where other sources have been used, acknowledgement has been fully given with citations and full references. Rebotiloe Fridah Morare iii DEDICATION I dedicate this work to the Almighty Lord and my family for the wonderful support they have given me. iv ACKNOWLEDGEMENT I would likely pass my gratitude to the following individuals and organization for their support throughout my studies I would like to give my outmost gratitude to the Almighty Lord for providing me with strength throughout my studies (Timothy 4:17). My supervisors; Dr Mahloro Hope Serepa-Dlamini, Dr Eunice Ubomba-Jaswa and Dr Vuyo Mavumengwana for their generous contribution towards the success of this work. I thank you for your directions, guidance, and constructive criticism and ensuring that all necessary materials required for this study were available. I thank my mom, Mrs Morare for being my life support structure throughout my life, to my sisters; Mmatlawa and Refiloe you have been greatly supporting throughout my academic life, your guidance and encouragement is appreciated. A big thank you To my dearest colleague, Olga Diale; you have been a great study partner with courage and passion for our study, thank you for your guidance in this project and for showing different perspective of the project. You have been more than a colleague to me by giving me personal guidance and encouragement. Dr N.E Madala thank you for your help in analysing the secondary metabolites by LC- qtof-MS and guidance on the extraction of secondary metabolites. I would also like to pass my gratitude to National Research Foundation (NRF) for funding this study. v ARTICLES PREPARED FOR PUBLICATION • Morare R.F, Ubomba-Jaswa, E and Serepa-Dlamini, M.H. (2018). Isolation and identification of bacterial endophytes from Crinum macowanii Baker in South Africa. African Journal of Biotechnology. Published. • Morare R.F, Ubomba-Jaswa, E and Mahloro H. Serepa-Dlamini. (2017). Extraction and Characterisation of secondary metabolites from Crinum macowanii and its associated bacterial endophytes. South African Journal of Botany. To be submitted. vi Table of Contents 1.1 Introduction ...................................................................................................................................... 2 1.2 Problem Statement .......................................................................................................................... 3 1.3 Aims .................................................................................................................................................. 4 1.4 Objectives ......................................................................................................................................... 4 1.5 References: ....................................................................................................................................... 4 2.1 Anti-microbial resistance .................................................................................................................. 9 2.2 Drug discovery from natural resources .......................................................................................... 10 2.2.1 Plants as source of drugs ......................................................................................................... 11 2.3 The use of medicinal plants ............................................................................................................ 11 2.4 Phytochemical studies of medicinal plants and their medical values ............................................ 12 2.4.1 Classes of phytochemical compounds ..................................................................................... 13 2.4.1.1 Phenolic compounds ......................................................................................................... 13 2.4.1.1.1 Tannins ........................................................................................................................... 14 2.4.1.1.2 Flavonoids ...................................................................................................................... 14 2.4.1.2 Saponins ............................................................................................................................ 15 2.4.1.3 Steroids ............................................................................................................................. 16 2.4.1.4 Alkaloids ............................................................................................................................ 16 2.5 The genus Crinum ........................................................................................................................... 17 2.5.1 Taxonomy ................................................................................................................................ 17 2.5.2 Phytochemistry and uses of Crinum ........................................................................................ 18 2.6 Medicinal plant, Crinum macowanii ............................................................................................... 18 Figure 2.2: Flowers and leaves characteristics of Crinum macowanii (Maroyi, 2016). ................ 19 2.6.1 Location and distribution ......................................................................................................... 19 Figure 2.3: Geographical distribution of C. macowanii in South Africa. (Obtained redlist.sanbi.org › Genera: C) ..................................................................................................
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