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African Potato) Evaluating the anticancer and antimicrobial properties of extracts from Hypoxis hemerocallidea (African potato) Xolani Sikhakhane Evaluating the anticancer and antimicrobial properties of extracts from Hypoxis hemerocallidea (African potato) by Xolani Sikhakhane (200606751) Dissertation Submitted in fulfilment of the requirements for the degree Magister Scientiae In Biochemistry In the Faculty of Science at the University of Johannesburg Supervisor: Prof Marianne J Cronjé Co-supervisor: Dr Jacinda T James November 2013 AFFIDAVIT: MASTER AND DOCTORAL STUDENTS TO WHOM IT MAY CONCERN This serves to confirm that I______________________________________________________ Full Name(s) and Surname ID Number/ Passport_____________________________________________________________________ Student number____________________ enrolled for the Qualification ____________________ __________________________________________________________________________ in the Faculty of Science Herewith declare that my academic work is in line with the Plagiarism Policy of the University of Johannesburg with which I am familiar. I further declare that the work presented in the ___________________________________(minor dissertation/dissertation/thesis) is authentic and original unless clearly indicated otherwise and in such instances full reference to the source is acknowledged and I do not pretend to receive any credit for such acknowledged quotations, and that there is no copyright infringement in my work. I declare that no unethical research practices were used or material gained through dishonesty. I understand that plagiarism is a serious offence and that should I contravene the Plagiarism Policy notwithstanding signing this affidavit, I may be found guilty of a serious criminal offence (perjury) that would amongst other consequences compel the University of Johannesburg to inform all other tertiary institutions of the offence and to issue a corresponding certificate of reprehensible academic conduct to whomever requests such a certificate from the institution. Signed at Johannesburg ________________ on this _______ of ____________________2013 Signature __________________________________ Print name _________________________ STAMP: COMMISSIONER OF OATHS Affidavit certified by a Commissioner of Oaths This affidavit conforms with the requirements of the JUSTICES OF THE PEACE AND COMMISSIONERS OF OATHS ACT 16 OF 1963 and the applicable Regulations published in the GG GNR 1258 of 21 July 1972; GN 903 of 10 July 1998; GN 109 of 2 February 2001 as amended. UNIVERSITY OF JOHANNESBURG FACULTY OF SCIENCE DECLARATION ON SUBMISSION OF FINAL COPIES AND CD I, (Mr / Mrs / Ms /Dr ): Student number: ID number / Passport: declare that the thesis / dissertation / minor dissertation, which I hereby submit for the qualification (e.g. PhD in Zoology, MSc Mathematics, Minor Dissertation in Aquatic Health): at the University of Johannesburg: Is the final copy and includes all corrections required by the assessors, supervisor and co-supervisors. The thesis, dissertation / minor dissertation complies with the requirements of the University of Johannesburg Policy on Plagiarism The electronic PDF format of the thesis / dissertation / minor dissertation is a true replica of the examined and corrected thesis / dissertation / minor dissertation. In the case of a MSc Minor dissertation / Dissertation: A manuscript based on a topic of the dissertation in the format of a research article meeting the requirements for publication has been submitted to the supervisor and electronically submitted to the Faculty YES / NO In the case of a PhD Thesis: An article based on the thesis accepted for publication by an accredited journal was submitted YES / NO Evidence of acceptance for publication from the editor or the editorial panel of such an accredited journal was submitted to the Faculty electronically. I am aware of the fact that I will be subject to criminal prosecution, should this declaration be false or contain a misrepresentation. SIGNATURE DATE DECLARATION BY SUPERVISOR AND CO-SUPERVISOR: The submission by the above student of the final corrected thesis/dissertation/minor dissertation, is hereby approved and an electronic manuscript/article is available and has also been submitted to the Faculty. SUPERVISOR: SIGNATURE DATE CO-SUPERVISOR: SIGNATURE DATE Title and copies are in order: FACULTY OFFICER: POSTGRADUATE STUDIES DATE “While there’s life, there’s hope” - Marcus Tullius Cicero (106 BC – 43 BC) “The key to everything is patience. You get the chicken by hatching the egg, not by smashing it” - Arnold Henry Glasow (1905 – 1998) Throughout my MSc studies I have learned an important life lesson that through hope, patience, persistence, faith and prayer, all things are possible... I dedicate this to.... My mother Ntombifikile and aunt Sibongile for raising me. ABSTRACT A rich diversity of medicinal plants is found in Southern Africa and approximately 80% of the population still relies on medicinal plants to fulfil its primary health care needs. Many of these medicinal plants are used to treat ailments such as burns, sores, urinary tract infections, colds, flu, rheumatism, gout, cancer, hypertension, diabetes, human immunodeficiency virus infections and acquired immunodeficiency syndrome. An example of such a plant is Hypoxis hemerocallidea (Fisch & CA Mey), formerly known as Hypoxis rooperi and popularly known as the African potato, from the Hypoxidaceae family. This plant is found across five of the South African provinces and corm extracts are reported to contain bioactive compounds that account for the plant’s medicinal and therapeutic properties. This study was conducted to investigate the anti-oesophageal cancer and antimicrobial potential of H. hemerocallidea. In cancer patients, the currently used cancer treatments such as radiotherapy and chemotherapy are ineffective in decreasing disease progression, prolonging survival, providing cure and are associated with side-effects such as cytotoxicity to normal body cells and tumour non-specificity. Therefore, current cancer research is aiming at searching for novel plant-based anticancer compounds that can be used for the development and manufacturing of cancer treatment drugs that will have less side-effects and less toxicity towards the normal human body cells, and ultimately provide cure for cancer. In addition to cancer, infectious diseases still contribute to most premature deaths worldwide and are now becoming more difficult to treat due to multidrug resistance developed by pathogens against many of the currently used antibiotics. This multidrug resistance of human pathogens to antibiotics has led to a search for new antimicrobial compounds from plants sources, for use in the production of new affordable antibiotic drugs to effectively treat infections without posing any unwanted toxicity and harm towards the human body. An oesophageal SNO cancer cell line was treated with H. hemerocallidea extracts and the effect of the extracts on the cancer cells were investigated with cell viability assays (trypan blue dye exclusion and AlamarBlue® viability assays), light microscopy and flow cytometrical analysis (forward and side scatter analysis). The plant extracts were also tested for antimicrobial activities against various microorganisms - Gram-positive and Gram-negative bacteria, yeast and fungi cultures by means of thin layer chromatographic bioautography (TLC-DB), microdilution assays and the BacTiter-GloTM assay. Antimicrobial compounds were then putatively identified and characterised using gas chromatography-mass spectrometry (GC-MS). No morphological changes were observed in the SNO cells and significant cell death did not occur following treatment with either water or ethanolic H. hemerocallidea extracts from fresh or dried corms or leaves. The ethanolic leaf extracts did not show any significant inhibition against any of the microorganisms tested in contrast to the ethanolic extracts from the corms, which showed microbial growth inhibition against Gram-positive bacteria and fungi and partial inhibition of the Gram-negative bacteria. The bioactive compounds responsible for the antibacterial and antifungal activities were identified as levoglucosan (as the major antimicrobial compound), pyrocatechol and hexahydro-3-(2-methylpropyl)-pyrrolo[1,2- α]pyrazine-1,4-dione. These results show that H. hemerocallidea plant extracts possessed no anticancer effects towards the SNO cell line. In addition, the corm extracts of H. hemerocallidea contain a levoglucosan compound, which may work synergistically with other antimicrobial compounds to exert antimicrobial properties. With more research, the antimicrobial compounds in H. hemerocallidea may hold promise for possible candidates for use in the development of antibiotic or antiseptic products (for example, topical creams and lozenges) to be used in the treatment of skin and soft tissue infections caused by bacterial and fungal infections. PREFACE Part of the work in this dissertation was presented at the following symposia: Biochemistry Symposium hosted by the Department of Biochemistry at the University of Johannesburg - 30th of September 2011. Cross-Faculty Postgraduate Symposium hosted by the Postgraduate Centre (Research and Innovation Division) at the University of Johannesburg - 26th of October 2011. AKNOWLWDGEMENTS AND THANKS I would like to extend a word of thanks to everyone who has contributed positively to my life and personal development. More especially, I would like
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