Philosophiae Doctor - PhD / Dyspigmentation is one of the commonest dermatological presenting complaints from patients, particularly hyperpigmentation. Hyperpigmentation can cause dangerous psychological and emotional impact on self-perception and health quality of lives of people affected. However, of all the diseases encountered globally, epidemiological data has shown that skin diseases account for almost 34% of all the diseases and these dermatological disorders have gotten worse over time. The gold standard for treatment of hyperpigmentation is hydroquinone. Despite its efficacy, hydroquinone and other current modalities of treatments are associated with some side effects. There are a number of natural products derived from medicinal plants that have proven to be an abundant source of biologically active compounds and a lot of these have served as the basis for the development of new lead chemicals for pharmaceutical. The present study focused on screening of selected South African plants (Maclura pomifera, Otholobium fruticans, Phyylica ericoides, Psoralea aphylla, Rhynchosia villosa, and Serruria furcellata) for their antimelanogenic potentials. Methanol and ethyl acetate were used for the extraction of plant materials. Standard methods were employed for evaluation of cytotoxicity of the methanolic leaf extracts (MLE), ethyl acetate leaf extracts (ELE) and melanin synthesis potentials on B16 melanoma cells. To elucidate mechanisms of melanin reduction action, intracellular tyrosinase activity was determined by measuring the rate of L-DOPA oxidation. Tyrosinase activity was assessed further with dihydroxyphenylalanine (DOPA) staining. The mode of action was further determined by evaluating reactive oxygen species (ROS) and expressions of melanogenesis gene using qPCR. The results showed that O. fruticans and S. furcellata reduced melanin synthesis without cytotoxicity. O. fruticans inhibited tyrosinase activity, increased ROS and suppressed the expression of TYR, TRP-1, TRP-2/Dopachrome tautomerase, MITF, MC1R but upregulated β-Catenin. S. furcellata stimulated tyrosinase activity and did not increase ROS. It upregulated the expression of TYR, TRP-1, TRP-2, and MC1R while MITF and β-Catenin were suppressed. The results showed that O. fruticans reduced melanin synthesis via cAMP pathway while S. furcellata reduced the synthesis via possibly degradation of melanin pigment. The present study on O. fruticans and S. furcellata has shown that leaves of these plants are candidate anti-melanogenic agents. However, more work still needs to be done to elucidate other possible mechanisms that are relevant to antimelanogenic effects of these two plants. / 2020-08-31

Thesis (MSc. (Biochemistry)) -- University of Limpopo, 2013 / Hydroxynitrile lyases (HNLs) are enzymes that catalyse enantioselective cleavage of the substrate in a reaction and are also used as important industrial biocatalysts for the synthesis of chiral cyanohydrins. The aim of the study was to screen indigenous South African plants for potential hydroxynitrile lyase activity, purify and biochemically characterise the active hydroxynitrile lyase(s) from the selected plants. Several indigenous plants were randomly collected, identified and screened for HNL activity. The plant parts (leaves, seeds or fruits) were processed using established experimental protocols in order to obtain the crude enzyme extracts. The enzymatic conversion of benzaldehyde and potassium cyanide to mandelonitrile was optimised and consequently used for the screening of HNL activity. Enzyme activity was detected in the crude enzyme extracts of Kalanchoe spp and Senecio spp and these were then designated as Ks and Sb, respectively. Ammonium sulphate fractionation, DEAE Toyopearl 650M and Concanavalin A chromatography techniques were then used in the purification process of the active crude enzyme extracts. Subsequently, two purified active fractions were isolated from each plant species with molecular masses estimated at 64.64 kDa and 64.06 kDa for the KsHNL enzymes and 70.60 kDa and 74.04 kDa for SbHNL enzymes. The optimum temperature and pH of all the isolated enzymes were determined as 50°C and pH 5, respectively. The experimental Km and Vmax values of the enzymes were respectively determined to be 0.33 and 0.73 mM and 1.238 and 1.948 μM/min for KsHNL; while that for SbHNL enzymes were 5.86 and 0.22 mM and 9.741 and 1.905 μM/min. The effect of additives and metal ions (viz., DTT, DEP, mercury chloride, magnesium chloride and zinc chloride) was determined. The experimental data obtained alluded to the notion that both KsHNL and SbHNL enzymes may contain the cysteine and serine residues next to their active sites and that a histidine residue may be involved in the catalytic activities of both the isolated KsHNL enzymes and one of the SbHNL enzymes. All the isolated enzymes from the two plant species did not seem to contain an FAD group. These findings compared favourably to the theoretical type II HNLs, although with a slight difference in that they displayed high molecular weights. Kalanchoe spp and Senecio spp are the two indigenous South African plants that were found to contain active HNLs. The isolated HNLs from the two plants have a potential to be xv purified to homogeneity, cloned and overexpressed into robust recombinant enzymes that can be used for large scale industrial applications.

South Africa has one of the richest and diverse floras in the world with over 30000 species of higher plants. There are approximately 3000 species of medicinal plants in South Africa. The discovery of active compounds in medicinal plants plays a strategic role in the phytochemical investigation of crude plant extracts. Secondary metabolites of medicinal plants are a major source of drugs for the treatment of various health disorders. Cardiac glycosides are one of the subgroups of steroids modified from terpenoids. The existence of cardiac glycosides in some plant species often indicates toxicity. Among the contagious elements, diseases caused by viruses are one of the major causes of death, disability, and social and economic disruption for millions of people. Viruses also cause many important plant diseases and are responsible for huge losses in crop production and quality in all parts of the world. According to literature, plants containing cardiac glycosides demonstrate potential for the discovery of more effective new drugs in the treatment of infection especially viral contagions. The main objectives of this study were to evaluate the antiviral (HSV-2 and PVYN), antibacterial and cytotoxic activities of South African plants containing cardiac glycosides. Furthermore, isolation and identification of compounds with an emphasis on cardiac glycosides was carried out. Eight plants known to contain cardiac glycoside compounds were selected. Leaves of Gomphocarpus fruticosus, Nerium oleander, Cotyledon orbiculata, the fruits and leaves of Strophanthus speciosus, the bulbs of Bowiea volubilis and Merwilla plumbea were selected to examine their antimicrobial activities, toxicity, antiviral and to isolate potential pure compounds. The ethanol extracts of all selected plants were screened for antibacterial activity against two Gram-positive (Staphylococcus aureus and Enterococcus faecalis) and two Gram-negative (Escherichia coli and Klebsiella pneumonia) pathogens. Plant extracts showed promising antibacterial action against Gram-positive bacteria. The most active extract against both Gram-positive bacteria was C. orbiculata with the MIC value of 1.25 mg/ml. Cell toxicity was monitored by determining the effect of the ethanolic crude extracts on human embryonic kidney cell line (HEK 293) using the XTT method. All extracts exhibited high toxic effects with IC50 < 100 μg/ml on the tested cell line. The XTT assay was used to determine of the antiviral activity of crude ethanolic extracts on the kidney epithelial cells of African Green Monkey (Vero). The results revealed that the crude ethanolic extracts of all iv selected plants exhibited a cytotoxic effect on Vero cells at concentrations lower than their EC50. Consequently, the determination of antiviral activity of the selected plant extracts was not successful. Based on the chromatographic and bioassay results from the six plants selected for this study, the fruit extract of S. speciosus, belonging to the Apocynaceae family, was chosen for the isolation of compounds, particularly cardiac glycosides. All plant extracts were tested for antibacterial activity against E. coli (Gram-negative) and E. faecalis (Gram-positive) by using bioautography. The general TLC test showed more variety of compounds in the fruit extract of S. speciosus. The results of the bioassay showed promising activity of the fruit extract of S. speciosus and the bulb extract of B. volubilis against Gram-positive bacteria. The chromatographic investigation of the S. speciosus fruit extract led to the isolation of three pure compounds including a cardiac glycoside. The three compounds were identified based on NMR (1D and 2D) and HRMS. The isolated compounds were identified as: a triterpene (ursolic acid methyl ester), a sugar: myo-inositol methyl ester and an unidentified cardiac glycoside. According to literature, it is the first report of the isolation of ursolic acid methyl ester and myo-inositol-methyl ether from S. speciosus fruit extract. The isolated cardiac glycoside exhibited no inhibitory activity at 1.25 mg/ml (the highest concentration tested) against all four tested bacteria (S. aureus, E. faecalis, E. coli and K. pneumonia). The cytotoxicity and anti-HSV-2 screening of the isolated cardenolide demonstrated the highly toxic effect of this compound on the HEK 293 cell line with 4.62 μg/ml IC50 value and < 25 μg/ml IC50 of Vero cell line. No evidence could be found in the literature of the cytotoxic activity of cardiac glycoside compounds on the HEK 293 cell line. The ethanolic extracts of all plant extracts and isolated cardenolide were tested against the PVYN in vivo and in vitro. The results revealed that the high concentration (50 mg/ml) of M. plumbea, N. oleander, B. volubilis (fresh bulb), C. orbiculata and isolated cardenolide reduced the PVYN symptoms on tobacco plants in an in vivo experiment. In in vitro analysis, the high concentration (50 mg/ml) of S. speciosus (leaves & fruits), and especially M. plumbea (dry bulb) showed significant antiphytoviral activity. In vivo and in vitro results demonstrated that M. plumbea has potential antiphytoviral activity. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Plant Science / Unrestricted