The cancer bush (Lessertia frutescens L.) is an important leguminous perennial native to southern Africa and has been used for centuries in traditional medicine by the continent’s diverse cultural groups. Like many other legumes, the seeds of this species exhibit dormancy. Moreover, woody plants are typically difficult to propagate in in vitro culture systems. But in vitro shoot cultures are valuable in providing an alternative means of deriving desired secondary metabolites or phytocompounds, under controlled conditions. This study describes novel protocols for breaking seed dormancy, rapid and efficient in vitro propagation, bioreactor culture, and comprehensive phytochemical data following screening and analysis of in vitro and field extracts of L. frutescens. Experiments using physical, mechanical and chemical pre-sowing treatments were conducted to determine the germination response of this species. The results indicated that seeds of L. frutescens exhibited exogenous dormancy due to the inhibitory effect of the hard coat on germination. Seed dormancy was released by mechanical scarification in which 100 % germination was achieved. In vitro propagation studies using single node explants in Murashige and Skoog (MS) medium supplemented with combinations of different concentrations of benzyladenine and naphthaleneacetic acid revealed a maximum number of 10 shoots per explant in solid medium, and 12.9 shoots per explant in liquid medium inside a temporary immersion bioreactor. Indirect shoot organogenesis and plant regeneration using rachis and stem segments was achieved with the highest percentage of explants forming shoots (88.8 %) from rachis explants cultured onto MS medium supplemented with thidiazuron. Direct shoot organogenesis from hypocotyl and cotyledon segments was also achieved in L. frutescens. The highest shoot regeneration using hypocotyls (83 %) was obtained in MS medium supplemented with kinetin whilst the highest shoot regeneration using cotyledons (46 %) was obtained in MS medium supplemented with kinetin in combination with benzyladenine. Successful rooting (up to 80 %) and acclimatization (up to 90 % survival rate) was attained. Spectrophotometric and gravimetric methods indicated that saponins were the most abundant, followed by phenolics, flavonoids and then alkaloids in in vitro leaf extracts then in field leaf extracts and seed extracts, respectively. After qualitative analysis these extracts were also found to contain tannins, phlobatannins and cardiac glycosides of medicinal interest. By using gas and liquid chromatography the presence of the medicinally important L-canavanine, gamma amino-butyric acid and D-pinitol was verified in in vitro leaf, field leaf and seed extracts. In vitro leaves had higher quantities of all compounds, except for D-pinitol. Phytocompound analysis of shoots derived from several of the cytokinin-enhanced media showed that these organs contained higher quantities of L-canavanine compared to the control. This study, therefore, highlights the potential techno-economic production of medicinal phytocompounds from in vitro leaves of L. frutescens following large scale production using the protocols described in this study. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2011.

M. Tech. (Department of Chemistry and Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Context: Pelargonium alchemilliodes L L' Her is an evergreen shrub, cultivated principally for the medicinal essence and decoction in Southern Africa for the treatment of skin problems, and wounds. Objective: the aim of the study was to optimize the extraction of phenolics and flavonoids from P. graveolens by response surface methodology with particular attention on the proliferative and cytotoxic effects on human keratinocytes, as well as the antioxidant and antibacterial activities and also to isolate active compounds. Materials and Methods: The optimization was achieved by Box-Behnken design. Extract, metabolite yields, and minimal inhibitory concentrations (MIC) were determined by gravimetric, spectrophotometric, and microdilution methods, respectively. The antiradical potentials were evaluated using the phosphomolybdate. 2,2-diphenyl-1-picrylhydrazyl, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and lipid peroxidation assays, the diterpenoids were isolated and purified using open column chromatography, PTLC, and characterized with FTIR, NMR. The kinetics of the lipid protective activity was studied and fitted into models. The proliferative and cytotoxic effects were evaluated using the CellTiter® Blue cell viability and lactate dehydrogenase assay. Results: The regression coefficient r2 ≥ 0.9775 indicated a close correlation between actual and predicted values of the responses. The ideal parameter for the extraction of phenolics and flavonoids by macerations was determined as an extraction time: 9.63-12.01 h, material mass: 2.62-3.00 g, agitation speed: 143.11-197.11 rpm, and solvent volume: 68.06-69.87 mL. The optimal extractable acetone and methanol crude, flavonoids, and phenolic are (28.87±2.15%, 24.11±1.15%), (7.11±1.03 mg QE/g, 5.98±0.87 mg QE/g) and (58.08±0.88 mg GAE/g, 55.91±1.15 mg GAE/g), respectively. The detected different chemical groups of polyphenolic compounds such as alkaloids, saponins , sterols, terpenoids, flavonoids, tannins, phenols and cardiac glycosides from methanol and acetone extracts were in correlation with optimized yields. Two triterpenoids compounds 1-hydroxy-30-norlanosta-6, 8-diene and 1 2,3,4a,8,9,10,10a-octahydro-2-(2-hydroxypent-4-enyl)-4a-vinyl-1H-benzo[c]chromen-6(10bH)-one were isolated form methanol extracts. The main components of essential oils were citronellal (5.99%), citranellol (26.2%), geraniol (8.56%), citronellyl butyrate (20.3%), trans-farnesol (9.53%) and they were characterized by high amounts of oxygenated hydrocarbons (67.6%), followed by sesquterpene hydrocarbons and oxygenated sesquiterpene (9.32%) and the least being mornoterpene hydrocarbons (5.20%). Total antioxidant capacity and reducing power were comparable to standard gallic acid, while the antiradical activity has IC50 value of 0.18±0.03-8.98±0.15 mg/mL. Further, the lipid protective revealed a dose-dependent activity fitting into a pseudo-second-order kinetic model. MIC value of 1.56 mg/mL for extracts was registered against Staphylococcus aureus and salmonella typhi comparable to chloramphenicol. There was a significant (P < 0.05) increase in cell proliferation and viability when the extract was administered at concentrations of ≤50 μg/mL. However, at ≥100 μg/mL concentrations at ≤ 1000 μg/mL for essential oil exhibited a significnt cytotoxicity in comparison to the untreated cell. Conclusion: These biological activities are confirmation of the phytomedicinal application and possible source of pharmaceutical compounds. However, administration of the decoction should take into cognizance the antiproliferative effect at doses ≥100 μg/mL as well as the potential to induce and maintain keratinocyte proliferation at low concentration with an eye on the antiproliferative effect at concentrations ≥100 μg/mL, except the P. Alchemilliodes essential oils at ≤ 1000 μg/mL.