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PHYTOCHEMICAL SCREENING, ELEMENTAL ANALYSIS and ANTIBACTERIAL INVESTIGATION of Rhoicissus Tomentosa: a MEDICINAL PLANT USED in SOUTH AFRICAN TRADITIONAL MEDICINE

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PHYTOCHEMICAL SCREENING, ELEMENTAL ANALYSIS and ANTIBACTERIAL INVESTIGATION of Rhoicissus Tomentosa: a MEDICINAL PLANT USED in SOUTH AFRICAN TRADITIONAL MEDICINE 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 [Unpublished]: University of Johannesburg.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). PHYTOCHEMICAL SCREENING, ELEMENTAL ANALYSIS AND ANTIBACTERIAL INVESTIGATION OF Rhoicissus tomentosa: A MEDICINAL PLANT USED IN SOUTH AFRICAN TRADITIONAL MEDICINE By NKEMDINMA C. UCHE-OKEREAFOR Student number: 215041255 DISSERTATION IN FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE MAGISTER TECHNOLOGIAE (M. Tech) BIOTECHNOLOGY In the FACULTY OF SCIENCE Of the UNIVERSITY OF JOHANNESSBURG SUPERVISOR: DR. VUYO MAVUMENGWANA CO-SUPERVISORS: DR. DEREK NDINTEH DECEMBER, 2016 i EXECUTIVE SUMMARY Globally medicinal plants play a very significant role in health-promotion and the reduction of disease burden in our communities. Over 80,000 species of such plants and their secondary metabolites including tannins, terpenoids, alkaloids, terpenes, phenolic compounds, glycosides and flavonoids have demonstrated excellent antimicrobial properties in vitro. Phytochemicals constitute over 25 – 50% of all pharmaceutical drugs prescribed globally. In addition, most phytochemicals have antioxidant and anti-carcinogenic properties which are beneficial to humans. Furthermore, the vitamins and other substances in plants play a significant role in protecting the human body against diabetes and heart diseases. Many plants have been the source of many pharmacologically active principles that are now used in medicine, for example, vincristine and vinblastine found in Catharanthus roseus are prescribed for hypertension and can also act as chemotherapeutic drugs against leukemia, Hodgkin’s disease, etc. Nature has provided us with a variety of very useful and important compounds but these have somehow been over looked in recent times and more research attention needs to be given to natural products to explore their many positive therapeutic attributes which is the purpose of this study on Rhoicissus tomentosa. R. tomentosa is a medicinal plant indigenous to Southern Africa. It is a vigorous, evergreen tendril climber with ornamental, vine-like leaves and bunches of purple grape-like fruits. The plant is mainly used by traditional healers to treat fertility related ailments. The rhizomes Rhoicissus tomentosa were analyzed in this study. The dry powdered plant material was first screened for the presence of phytochemicals and the findings revealed the presence of alkaloids, flavonoids, saponins, steroids, reducing sugars and tannins. The ground plant material was subsequently extracted using 100% ethyl acetate and methanol/chloroform (1:1) and pressurized hot water extraction (PHWE) technique. The extracts were analyzed for antimicrobial activity against 14 common human bacterial pathogens using the disc diffusion method and the micro titer plate method and the findings revealed that the extracts showed moderate to high inhibitory activity against most of the test organisms. While different bacterial species were investigated, the most susceptible ones to the rhizome organic solvent extracts were Staphylococcus aureus (MIC 0.063 mg/mL) and Bacillus subtilis (MIC 0.125 mg/mL) and the ones most susceptible to the PHWE extract were Staphylococcus aureus (MIC 1.0 mg/mL) and ii Bacillus cereus (MIC 4.0 mg/mL). A comprehensive Two-Dimensional Gas Chromatography coupled with Time-of-Flight (GC×GC-TOFMS) analysis of the different crude extracts revealed the presence of over 100 known phytochemical constituents and numerous unknown constituents. Inductively coupled plasma atomic emission spectroscopy (ICP-OES) was also done to ascertain the trace metal composition of this plant and consequently ascertain whether the plant contains any heavy metals of concern. The findings revealed that calcium and iron among other metals contained in the plant have known health benefits. They were identified in amounts which are within the World Health Organization permissible limits for metals in medicinal plants. The findings of this study support the use of this plant in traditional medicine as this study showed that the plant contains bioactive phytochemical compounds, has interesting antibacterial properties and contains metals in trace amounts. Keywords: Antibacterial screening, GC×GC-TOFMS, ICP-OES, PHWE, Phytochemical screening, R. tomentosa rhizomes. iii DECLARATION I, Nkemdinma C. Uche-Okereafor hereby declare that the composition of this dissertation and the work herein described was carried out entirely by myself unless otherwise cited or acknowledged. It has not been submitted for degree purposes at any other University or institution. Every other source(s) used have been duly cited in text and acknowledged by complete references. _______________________ Nkemdinma C. Uche-Okereafor iv DEDICATION I dedicate this work to God Almighty, for His grace. Also to my husband and family for their love and support. v ACKNOWLEDGEMENTS Firstly, I sincerely want to thank God for his grace and favour upon my life. I want to express my deepest gratitude to my supervisors Dr. V. Mavumengwana and Dr. D. Ndinteh for their support and provision of a conducive environment for this research. Especially, Dr. V. Mavumengwana for giving me an opportunity no one else was willing to give me. I also wish to thank Dr. N. Niemann for her assistance and supervision. Besides my supervisors, I wish to thank Dr. P.B. Njobeh for his encouragement and financial support; Mr. Eric Van Zyl, the Head of Department of Biotechnology and Food Technology for all his assistance and Mrs. Malie King the secretary of Department of Biotechnology and Food Technology for her kindness, support and encouragement. I wish to thank Dr. Edwin Madala for assisting in carrying out some of the experiments for this project. I also wish to thank Dr. & Mrs. C. Yah, Dr. & Mrs. O.A. Ozumba, Mr Alastair Campbell and Dr. Obinna Nwinyi for their immeasurable support and assistance when needed. I would like to extend my profound gratitude to my lovely and wonderful friends and colleagues who always cheered me up and made my stay worthwhile: Ms. Tendani Sebola, Ms. Mbali Webb, Ms. Sharon Pelo, Ms. Jade Nephawa (The Big five), Ms. Bongeka Mbambo, Ms. Evonia Nchabeleng, Mr. Terrence Malatjie, Ms. Livhuwani Nemandava, Mr. Femi Adebo, Mr. Sefater Gbashi, Ms. Siphosanele Moyo, Mrs. Ifeoluwa Adekoya, Mr. Thierry Fonkui, Dr. Martha Fosting, Mrs. Grace Daji thank you for your assistance, companionship and guidance. God bless you richly. In particular, I want to thank Mr. Femi Adebo and Mr. Sefater Gbashi for assisting during my registration and assisting with the write up of this dissertation. I also want to thank Ms. Tendani Sebola for giving me a very warm welcome to Lab 2313 and for being an amazing friend. I wish to thank Dr. R.K. Obi for his advice and words of encouragement that have carried me through the last two years. I’m indeed very grateful. I want to also thank Mr. Kevin Kasangana for his assistance and for being a good friend. vi I would like to especially thank my parents Chief Sir & Lady L.O. Dike for their love, support and prayers. I also wish to thank my siblings: Mrs. Ogechi Chijioke-Ibe, Mrs. Ozioma Gilbert, Engr. Odinaka Dike, Ms. Amarachi Dike and Ms. Nzubechi Dike for their best wishes and support. I also wish to thank my in-laws for their support. I wish to render my deepest appreciation to my husband Mr. G.U. Okereafor for his love and support, for being my biggest fan and for not stressing me out when I come back late from school; I am really grateful. I wish to also thank my family away from home: The Nkulus’- Pastor Franly, Mimi, Imani, Akila and Eric for their love, prayers and support. I also wish to thank Pastor Alfred Enitayo for his support and prayers. I wish to thank my friends: Kozuru Aladum, Olachi Emezuru, Gift Nwanma, Sopulu Ozoike, Olufunke Owonipa, Chibuzo Onu and Emmanuel Nwankwo for their prayers, love and encouragement. Lastly but not least, I wish to thank the Faculty of Science University of Johannesburg and the Gauteng Department of Agriculture and Rural Development (GDARD) for financial support. vii RESEARCH OUTPUTS Nkemdinma Uche-Okereafor, Derek Ndinteh, Nicolette Niemann, Vuyo Mavumengwana (2016). Phytochemical screening, GC×GC TOF-MS analysis and antibacterial properties of crude Rhoicissus tomentosa rhizome extract. International Conference on Advances in Science, Engineering, Technology and Natural Resources (Nov 24-25th, 2016). http://ia- e.org/images/proceedings_pdf/IAE1116436.pdf Nkemdinma Uche-Okereafor, Derek Ndinteh, Nicolette Niemann, Vuyo Mavumengwana. 2016 Autumn International Scientific Conference on Food Safety and Security, Poster Presentation: Phytochemical Screening, Antibacterial and Possible Antioxidant Potential of Rhoicissus tomentosa rhizomes. Nkemdinma Uche-Okereafor, Derek Ndinteh, Nicolette Niemann, Vuyo Mavumengwana. Women in Science, Technology,
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