Submitted in fulfillment for the Degree of Doctor of Technology: Biotechnology, Durban University of Technology, 2010. / Ceratatheca triloba (Bernh.) E. Mey. Ex Hook. f. is one of four species that is common to the summer rainfall areas in South Africa, especially the grasslands. It is used in traditional medicine to treat stomach cramps, nausea, fever and diarrhea. Like many other plants used in the traditional medicine system, these uses are not justified through scientific investigations. This study was undertaken to characterize the functionality of the main bioactive compounds from Ceratatheca triloba. This was achieved by isolating and identifying predominant chemicals from the non polar extracts using conventional chromatography techniques. Once identified the crude extracts and identified compounds were tested for their antimicrobial, anti-oxidant activity, anti-inflammatory activity and anticancer activity. This was followed by investigating the safety of the crude extracts and the purified compounds by the Brine shrimp lethality assay, and its toxicity to HepG2 cells and the Salmonella mutagenecity test. For large scale production, we set up a protocol to produce 9, 10 anthracenedione in a cell suspension culture system. Following the complete chemical profile of the roots, stems, flowers and leaves the predominant compounds were isolated, characterized and identified by UV-Vis, IR, EI-LCMS and NMR (COSY, HMQC, HMBC and DEPT). Three anthraquinone derivatives and one steroid, 9, 10 anthracenedione, 1-hydroxy-4-methylanthraquinone, 5, 8-dimethoxy-2, 3, 10, 10a-tetrahydro-1H-phenanthrene-4, 9-dione and androst-5-ene-3, 17, 19-triol were determine by analysis of spectral data (UV, 1H NMR, 13C NMR and EI-LC-MS) 9, 10 anthracenedione and 1 hydroxy-4-methylanthraquinone showed antibacterial activity against S.aureus, M. luteus, B cureus and E. coli. Due to the synergistic effect of the individual compounds, the crude extract exhibited good potency (>500) against S.aureus and M. luteus, medium potency against E. coli. and S. typhimurium (<100) and very low potency against B cureus (<10). Although a similar trend was observed for 9, 10 anthracenedione and 1 hydroxy-4-methylanthraquinone unlike the crude extract. A very low potency against S.aureus for 9, 10 anthracenedione and a high potency for 1 hydroxy-4-methylanthraquinone. Thus 9, 10 anthracenedione is an effective drug against E. coli and S. typhimurium and 1 hydroxy-4-methylanthraquinone is effective against S.aureus and M. luteus. The crude root extracts and 9, 10 anthracenedione, 1 hydroxy-4-methylanthraquinone, 8-dimethoxy-2, 3, 10, 10a-tetrahydro-1H-phenanthrene-4 showed a ± 50% reduction of the free radicals. No anti-inflammatory activity was observed. The purified extracts showed moderate toxicity against HepG2 cells at high concentrations and no toxicity was observed against brine shimp larvae. No mutagenecity was observed with the crude extracts using the Ames test. All purified and crude extracts showed potent inhibition of the human topoisomerase II enzyme. In conclusion, although this study does not indicate any relationship to its traditional usage it provides valuable information that paves a way for commercial exploitation of C. triloba. 9, 10 anthracenedione and 1 hydroxy-4-methylanthraquinone can be used as antibacterial agents. Their antioxidative potential can be exploited for anti-cancer as in many cancers reactive oxygen species are implicated in the aetiology of these cancers. Furthermore, in this study 9, 10 anthracenedione was produced from both callus cultures and cell suspension cultures. This compound demonstrates potent anti-topoisomerase II activity which is vital to cancer treatment. Thus, the synergistic effect of 9, 10 anthracenedione and 1 hydroxy-4-methylanthraquinone as antibacterial, anti-oxidative and anti-cancer compounds demonstrate the importance of C. triloba. / Centre for Research Capacity Development ; National Research Foundation

Submitted in complete fulfillment for the Degree of Doctorate of Philosophy in Biotechnology, Durban University of Technology, Durban, South Africa, 2016. / Plants have provided a source of medicine from the beginning of human history and are the core of modern medicine. Moreover, plant based drug discovery has led to the development of various anticancer drugs (such as vincristine, vinblastine, etoposide, paclitaxel, camptothecin, topotecan and irinotecan). The use of botanical, phytochemical, biological and molecular techniques have facilitated the discovery of anthraquinones from Ceratotheca triloba that can inhibit the human topoisomerase II enzyme (target for anticancer drugs) and kill cancer cells. However, the C. triloba plant has not been extensively studied for its anticancer activity. Therefore, the aim of this study was to further investigate the anticancer activity of C. triloba and determine the classes of compounds that contributed towards its activity. In this study the leaf and root extracts were prepared by using hexane, DCM, hexane: DCM (1:1), methanol and/or water. These extracts were examined for their growth inhibitory potential on three cancer cell lines (A375 [melanoma], MDA-MB-231[breast] and WHCO1 [esophageal]) by using the MTT assay. Then, different mobile phases were prepared for optimizing the separation of the compounds of the active extract by TLC. Column chromatography was performed with the active extract by using five mobile phases (hexane : DCM [60 : 40, 40 : 60], DCM, DCM : ethyl acetate [90 : 10, 70 : 30, 60 : 40, 50 : 50, 50 : 60, 30: 60, 20 : 80], ethyl acetate and ethyl acetate: methanol [80 : 20, 70 : 30, 50 : 50]). The fractions collected from the column were examined for their growth inhibitory potential on two melanoma cell lines (A375 and UACC-62). The IC50 and TGI (total growth inhibition) values of the active fractions were determined. Also, the apoptosis inducing effects of the active fractions and standards (camptothecin and doxorubicin) were determined by using flow cytometer based assays (FITC annexin assay, PE active caspase 3 assay and BD MitoScreen assay). Subsequently, the chemical structures of the compounds that contributed towards the activity of these fractions were obtained by EI-LC-MS analysis. The results demonstrated that the hexane root extract exhibited the best percentage of growth inhibition (%GI) on all three cancer cell lines. The separation of the compounds of the hexane root extract was optimized on TLC plates by using different ratios of hexane and DCM. Column chromatography allowed for fractionation of this extract. Purified compounds were not obtained due to co-elution. Further research would have to be conducted to obtain purified compounds. This may involve the use of mini-column chromatography and PTLC. Overall a total of ten combined fractions were collected from the column. Four of these fractions (F2, F4, F5 and F8) displayed a high %GI on the A375 and UACC-62 cell lines. Moreover, fraction F4 was the most active fraction as it had the lowest IC50 (0.70 µg.ml-1 [A375] and 0.39 µg.ml-1 [UACC-62]) and TGI (12.50 µg.ml-1[A375] and 25 µg.ml-1 [UACC-62]) values in comparison to the other fractions. All four fractions induced depolarization of the mitochondria membrane potential (ΔΨ), caspase 3 activation, early apoptosis (phospholipid phosphatidylserine exposure) and/or late apoptosis in the melanoma cells. The results also revealed that fraction F4 (25 µg.ml-1) induced depolarization of the ΔΨ in a higher percentage of A375 (78.11%) and UACC-62 (87.4%) cells than the other fractions and standards. This fraction also induced caspase 3 activation in a high percentage of A375 (90.56%) and UACC-62 (96.78%) cells. Therefore fraction F4 was also the most active fraction in terms of apoptosis activity. Based on our results and literature findings we can deduce that the active fractions induced the intrinsic or extrinsic (type II) apoptosis pathway in the melanoma cells. Six classes of compounds were identified from the four active fractions. These were: benzothiophenones, benzopyranones, naphthoquinones, anthraquinones, androstanes and quinazolines. In conclusion, this is the first study that evaluated the growth inhibition potential of the leaf and root extracts of C. triloba on a panel of cancer cells. This research indicated that the hexane root extract displayed the best levels of cell growth inhibition. The active constituents of this extract were isolated into four fractions which elicited apoptosis inducing effects that promoted the extrinsic (type II) or intrinsic apoptosis pathway in the melanoma cells. Furthermore, fraction F4 contained the most active compounds from C. triloba as it had the lowest IC50 and TGI values (in comparison to the other fractions) and induced depolarization of the ΔΨ in the highest percentage of melanoma cells. It was confirmed that six classes of compounds were accountable for the anticancer activity of these fractions. Thus, the C. triloba plant is a rich source of anticancer compounds. / D