Greyia radlkoferi is a South African indigenous tree, which has recently been discovered to be a source of extracts that have a potential in the development of cosmeceutical herbal products having the ability to treat hyperpigmentation disorders. For product development however, G. radlkoferi would need to be available in a commercial scale. Greyia radlkoferi grows naturally in the wild and is often available for cultivation as an ornamental plant. In order to establish this plant into cultivation, suitable propagation techniques must be established for rapid multiplication of trees and thus a sustainable leaf production. For consistent and improved leaf supply to the market, agronomic practices that will enhance leaf production were investigated in the current study. Furthermore, in order to meet market demand in terms of good quality extracts with guaranteed therapeutic efficiency, pre-harvest and post-harvest factors that affect the chemical composition of the extracts were investigated. Recently developed biotechnology techniques such as metabolomics using 1H-NMR and multivariate data analysis offered a platform to study the chemical variation of extracts. Therefore, the current study was aimed at understanding the requirements for propagation and optimum leaf production as well as conditions that favour optimum production of secondary metabolite of G. radlkoferi plant material (at pre and post-harvest) and thus assess its commercial viability. To understand the effects of temperature on seed germination of G. radlkoferi, seeds were exposed to five temperatures (10°C, 15°C, 20°C, 25°C and 30°C) in the incubators in the laboratory. Germination of G. radlkoferi by seeds was discovered to be temperature dependent. The optimum germination temperature of 81% was obtained at 25°C. In the case of vegetative propagation by stem cuttings, the effect of cutting position (basal or apical), exogenous rooting hormone (Seradix1, Seradix 2, 0.1% IBA, 0.3% IBA and 0.8% IBA) and cutting position were investigated in the glasshouse. The cutting position had a significant effect on rooting of G. radlkoferi cuttings with basal cuttings exhibiting 35% rooting as compared to 6% rooting attained for the apical cuttings. A clear trend in rooting response to application of rooting hormones was observed, with 0.1% Indole butyric acid (IBA) showing the highest rooting percentage of 63%. Considering the outcomes of the propagation studies as well as the limited material for vegetative propagation, seed propagation appears to be the most suitable technique for large-scale multiplication of G. radlkoferi. The effect of different pruning techniques as well as harvesting frequencies on fresh and dry weights of G. radlkoferi leaves were evaluated. Factors considered were four pruning treatments (‘pruned but not tipped’, ‘tipped but not pruned’, ‘not pruned nor tipped’ as well as ‘pruned and tipped’) and three harvesting periods (monthly, bimonthly and once–off). Bimonthly harvests highly increased leaf production compared to trees that were harvested monthly and once-off with higher leaf fresh weight yield of 238 g per tree or 2.38 tons/per hectare as well as dry weight yield of 83 g per tree or 0.830 tons/hectare. This outcomes of this study further suggested that a suitable pruning practice for G. radlkoferi would be to either ‘prune only’ or ‘cut back the main stem’ rather than a combination of the two treatments. The influence of seasons (summer, autumn, winter and spring) on the anti-tyrosinase activity and metabolomics profile of G. radlkoferi leaf extracts were investigated. Seasons significantly influenced the chemical composition and the efficacy of the plant extracts. Tyrosinase enzyme inhibition was investigated against monophenolase (tyrosine) with kojic acid as positive control. The highest tyrosinase inhibition concentration with IC50 (50% tyrosinase inhibition concentration) value of 30.3±1.8 μg/ml were obtained in winter harvested leaves compared to the other seasons. The lowest IC50 values were obtained in spring. Metabolomics analysis using orthogonal partial least square discriminant analysis (OPLS-DA) provided a clear class separation according to the harvest season. Extracts from winter harvested leaves contained sucrose, acetamide, alanine and a compound of the catechin group (gallocatechin-(4 alpha->8)-epigallocatechin) as revealed by 1H-NMR metabolomics with assistance of LC-MS. Since compounds of the catechin group are well-known tyrosinase inhibitors, the high tyrosinase activity exhibited in extracts of winter harvested G. radlkoferi leaves could be ascribed to the presence of gallocatechin-(4 alpha->8)-epigallocatechin. Based on the outcomes of the seasonal study, we suggest that in order to obtain extracts with high bioactivity, the best suitable time for harvesting leaf samples is in late autumn-early winter. Processing leaf material using three different drying methods (sun, oven and air drying) significantly influenced chemical composition and the efficacy of the plant extracts. Extracts prepared from air-dried leaf material showed the highest tyrosinase inhibition with IC50 value of 17.80 μg/ml compared to extracts of the other drying methods. Extracts of leaves processed with air drying preserved most metabolites during processing while extracts of sun-dried and oven-dried leaves clearly depleted some metabolites especially amino acids and some aromatic compounds. 1H-NMR metabolomics approach with the assistance of LC-MS data successfully determined a positive association of alanine, acetamide, sucrose and gallocatechin-(4 alpha->8)-epigallocatechin as the chemical constituents contributing to the variation in the air-dried leaves compared to the oven-dried leaves. A positive association of valine, alanine, leucine, isoleucine, gallocatechin-(4 alpha->8)-epigallocatechin and glucose contributed to the variation in air-dried group, compared to the sun-dried group. The highest tyrosinase inhibitory activity exhibited in air-dried samples compared to the other drying methods was associated with the presence of gallocatechin-(4 alpha->8)-epigallocatechin. Because air drying preserved most leaf metabolites compared to sun and oven drying, it was regarded as the most suitable method for processing G. radlkoferi leaf material. This study is the first scientific account that provides guidelines and recommendations to (1) establish G. radlkoferi as a cultivated plant for commercialization, (2) optimize leaf production for sustainable supply to the commercial markets and (3) optimize medicinal content of G. radlkoferi related to harvesting time and post-harvest processing (drying), for enhanced quality of extracts and its products / Agriculture, Animal Health and Human Ecology / Ph. D. (Agriculture)

PhD (Botany) / Department of Botany / Brackenridgea zanguebarica Oliv. (Ochnaceae) commonly known as “Yellow Peeling Plane”, is an important medicinal plant species, and one of the most threatened tree species that is endemic to Thengwe village in Vhembe District, South Africa. It is widely harvested for traditional purposes and difficult to propagate sexually and asexually. Nowadays, it has become threatened and this unique plant population is believed to be declining in the Brackenridgea Nature Reserve (BNR). According to the Red List of South African plants, B. zanguebarica is considered a critically endangered medicinal plant. All these facts combined with a lack of knowledge on the conservation of this species prompted this study on its conservation. To ascertain the effective conservation and proper management of this multipurpose tree, this study was conducted with the aim to improve its conservation strategy by investigating current threats to the existence of B. zangueberica, the chemical constituents from the different plant parts, the propagation methods of the species, and the factors delaying the germination of its seeds. In this study, an assessment of the current population threats was important as a first step in conservation; this would update the status of the species in its natural habitat. The current status of Brackenridgea zanguebarica was conducted through an extensive field survey in the Brackenridgea Nature Reserve (BNR), where a belt transect method was deployed to record quantitative information, while the harvesting damage was estimated using a sliding scale from 0 to 5. The structure of the populations in terms of the stem diameter size classes showed a bell shape vi pattern. About 60% of individuals showed some signs of plant parts extraction as the stem bark was reported to be the main plant part harvested. The population structure, however, was dominated by juvenile plants due to the selective harvesting of mature individuals; this hinders fruit production leading to poor or slow seed production. The present study aimed to compare the chemical constituents and the antimicrobial activities of some parts of this plant species. The extracts were screened for phytochemicals using standard methods. Thin-layer chromatography (TLC) was carried out to compare the chemical constituents using various solvent systems of varying polarity. Agar well diffusion and broth microdilution methods were used to evaluate the antimicrobial activity and the minimum inhibitory concentrations (MIC) of plant extracts respectively, against three bacteria and four fungi. The phytochemical analysis revealed a close similarity of various phytoconstituents of the three plant parts (stem bark, twigs and leaves). TLC analysis showed a slight difference in the acetone extract of different plant parts with more bands on the leaves than the stem bark and twigs. Acetone extracts of the three plant parts exhibited varying degrees of antimicrobial activity against Staphylococcus aureus and Candida glabrata, while none of the extracts showed any activity against Escherichia coli, Klebsiella pneumonia, Candida albicans, C. krusei, and C. parapsilosis. However, analysis of the antibacterial activity of various extracts revealed that the best inhibitory activity was produced by the stem bark extract compared to the leaf and twig extract. vii The sustainable way to meet the growing demand of Brackenridgea zanguebarica is to increase its availability through propagation practices. Unfortunately, the conservation of B. zanguebarica is hampered by lack of information on how it can be propagated. Sexual and asexual propagations were conducted to determine an efficient method to increase the number of individuals of this plant species. Seed propagation was carried out to study the effect of various pre-treatments on germination, in order to understand the germination requirements; vegetative propagation was conducted to assess if B. zanguebarica could be successfully propagated via stem cuttings if the appropriate growth hormones and growing media were applied. Brackenridgea zanguebarica seed did not germinate at all under any of the conditions tested. However, the results, showed the potential of propagating this species from stem cutting despite the poor results obtained. Thus B. zanguebarica was found to be difficult to propagate sexually and asexually. Seeds serve as a means of reproduction and a vital element to ensure the survival of plant species. Seed germination is controlled by a number of factors. The propagation of B. zanguebarica via seed is very difficult and research has not been undertaken to understand possible factors that may delay its germination. This study was aimed to investigate and describe both the morphological and anatomical features of B. zanguebarica seed, in order to identify structural features implicated in its poor germination. To achieve this goal, a morphological and anatomical study was conducted based on the observation of seeds under a scanning electron microscope (SEM) and light microscopy (LM). Water uptake potential was assessed by the increase in seed mass. The morpho-anatomical analysis indicated that some structures of the seed, such as seed coat, and the presence of endosperm viii surrounding the embryo that consisted of two prominent cotyledons, seem to greatly hinder the germination of this species. The research findings provided information regarding the seed structure which is one important step in identifying useful techniques to improve seed germination for conservation purposes. / NRF