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DISSERTATION O Attribution 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. PhD. (Chemistry)/ M.Sc. (Physics)/ M.A. (Philosophy)/M.Com. (Finance) etc. [Unpublished]: University of Johannesburg. Retrieved from: https://ujcontent.uj.ac.za/vital/access/manager/Index?site_name=Research%20Output (Accessed: Date). DETERMINATION OF BIOLOGICAL ACTIVITY OF CELTIS AFRICANA EXTRACTS AND ITS ENDOPHYTIC MICROFLORA AND MYCOFLORA. A Dissertation submitted to the Faculty of Science, University of Johannesburg In partial fulfilment of the requirement for the award of a Master’s Degree in Technology: Biotechnology By EVONIA KANYANE NCHABELENG STUDENT NUMBER: 201182098 Supervisor: Dr. V. Mavumengwana Co-supervisor: Dr. D.T. Ndinteh Co-supervisor: Dr. N. Niemann JANUARY, 2017 ABSTRACT With the rapid rise in untreatable diseases, researchers are compelled to search for new drugs that can combat these diseases. Plants are recognized as unlimited sources of bioactive compounds that can be used to treat different ailments and also be applied in various sectors including agriculture, pharmaceutical and food. In addition, plants are also known to be associated with endophytes such as bacteria and fungi, which are also regarded as fertile sources of bioactive constituents. Celtis africana is an ornamental and medicinal plant that is used to treat different ailments. In the study reported herein, the aims were to identify and characterize secondary metabolites produced by both the plant and its endophytes and to further investigate the antibacterial activity of C. africana extracts against fourteen pathogenic bacteria. To achieve these aims, endophytes were isolated from fresh and apparently healthy aerial parts (leaves, stems and fruit) of C. africana. The isolated endophytes were then cultivated and secondary metabolites extracted sequentially with hexane, dichloromethane and ethyl acetate. Dried plant aerial parts were screened for the presence of phytochemicals, extracted successively with hexane, ethyl acetate and dichloromethane: methanol (1:1 v/v) and the crude extracts tested for antibacterial activity. The crude extracts of both the plant parts and endophytes were analyzed using two dimensional gas chromatography coupled with time of flight mass spectrometry(GC×GC-TOF/MS ) to determine their volatile secondary metabolites constituents. From the results obtained, it is evident that C. africana has endophytic bacteria and fungi in the stem and fruit, but not in the leaves. Seven bacteria (Kocuria sp., Micrococcus luteus, Staphylococcus hominis, Bacillus sp., Staphylococcus saprophyticus, Brachybacterium conglomeratum and Arthrobacter sp) were isolated and identified. Four fungal endophytes, all belonging to the same genus (Aspergillus) were also isolated and identified, of which two were identified to genus level and two were found to be A. niger and A. flavus. The plant crude extracts showed antibacterial activity against seven of the test microorganisms, viz., Escherichia coli, Proteus mirabilis, Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter aerogenes. These microorganisms are causal agents of various ailments ranging from food-borne illnesses to skin infections, urinary tract infections and pneumonia. i The profiles of volatile secondary metabolites of both the endophytes and the plants revealed the presence of numerous compounds with reported biological importance ranging from antimicrobial to antioxidant, anticancer and flavoring. Furthermore, the endophytes had similarities in the type of secondary metabolites they produce with specificity to the alkaloidal compounds (diketopiperazines). The current study has laid a groundwork on the types of secondary metabolites that C. africana and its endophytes produce, and thus both the plant and endophytes are suggested to be a potential source of compounds with therapeutic uses. Keywords: Antibacterial, bioactive compounds, Celtis africana, endophytes, secondary metabolites ii DECLARATION I, Evonia Kanyane Nchabeleng hereby declare that this study titled “Determination of biological activity of Celtis africana extracts and its endophytic microflora and mycoflora” is my own work, completed under the supervision of Dr. V. Mavumengwana, Dr. N. Niemann, and Dr. D.T. Ndinteh. Furthermore I declare that this work has never been submitted elsewhere as a fulfillment for any qualification. In case where other sources have been used, acknowledgement has been fully given with citations and full references. ______________________________ EVONIA KANYANE NCHABELENG iii DEDICATION This work is dedicated to my parents Mr. T.J. and Mrs. M.M. Nchabeleng, I am where I am today because of the immense support and sacrifices they have made. iv THANKS AND ACKNOWLEDGEMENTS My deepest gratitude goes out to the below individuals and organizations for their support throughout this study: To my supervisors Dr. V. Mavumengwana, Dr. N. Niemann, and Dr. D.T. Ndinteh, this study has been a success solely because of your guidance, constructive criticism, patience, and for providing all the needed materials throughout the course of this study. Most importantly thank you, for being helping out even when you were in your personal space. Not only have you groomed me academically but you have also grown me personally as an individual. A big thank you. To my parents, Mr. and Mrs. Nchabeleng, thank you for the support you have given me throughout my life. To my siblings, Johanna, Mpho, Thabo, and my cousin Phummy, your support, encouragement and inspiration to do better are much appreciated. You all have been my pillars of strength and source of motivation. “Kea leboga Batau ba Seokodibeng” My laboratory colleagues, Bongeka Mbambo and Letlhabile Moyaha, thank you for your guidance through the fruitful discussions we would have over the late night and weekend lab experiments. I also sincerely thank Tendani Sebola, Natasha Samongoe, Nkem Ucheokafor and Mbali Webb with whom I have also worked with. Dr. Judith Phoku thank you for the assistance with the fungal isolates identification and fermentation. Dr. S. Sekar, for your help with the bacterial fermentation and construction of the phylogenetic trees. Mr. O. Adebo, your assistance with reviewing part of this work is much valued. I would also like to thank the National Research Foundation for funding this study, the University of Johannesburg Merit bursary and also Alumni Bursary for partially funding this study. Lastly I would to thank I AM, the Omnipresent Almighty God, whom through His grace and love I am still breathing. Greater is He that is within me than he that is in the world (1 John 4:4). v ARTICLES PREPARED FOR PUBLICATION Nchabeleng, E.K., Ndinteh, D.T., Niemann, N., Mavumengwana, V. In vitro antibacterial activity testing and profiling of volatile constituents of the leaves, stem, and fruit of Celtis africana from South Africa. Prepared for submission to South African Journal of Botany. Nchabeleng, E.K., Ndinteh, D.T., Niemann, N., Mavumengwana, V. Identification and characterization of volatile secondary metabolites produced by endophytic fungi associated with Celtis africana from South Africa. Prepared for submission to Journal of Microbiology and Biotechnology. Nchabeleng, E.K., Ndinteh, D.T., Niemann, N., Mavumengwana, V. Identification and characterization of volatile secondary metabolites produced by endophytic bacteria associated with Celtis africana from South Africa. Prepared for submission to Journal of Microbiology and Biotechnology. vi TABLE OF CONTENTS ABSTRACT .............................................................................................................................. i DECLARATION..................................................................................................................... iii DEDICATION......................................................................................................................... iv THANKS AND ACKNOWLEDGEMENTS ......................................................................... v ARTICLES PREPARED FOR PUBLICATION …. ........................................................... vi TABLE OF CONTENTS ...................................................................................................... vii LIST OF FIGURES ............................................................................................................... xii LIST OF TABLES ................................................................................................................ xiii LIST OF ABBREVIATIONS AND ACRONYMS ............................................................ xiv LIST OF UNITS .................................................................................................................... .xv DISSERTATION OUTLINE ............................................................................................... xvi CHAPTER ONE ....................................................................................................................
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