Introduction: Dark spots appearing on the skin caused by hyperpigmentation results from the action of tyrosinase, an enzyme whose activity leads to the production of the skin pigment melanin. Extracts of the plant Glycyrrhiza glabra, also known as liquorice, are commonly used to treat a range of conditions including skin hyperpigmentation. This study aimed at isolating and identifying compounds in extracts from South African liquorice root and assaying these compounds as to their antioxidant activity, their ability to inhibit the tyrosinase enzyme and their level of cytotoxicity. Methods: The ability of plant extracts to scavenge free radicals was tested using the 2,2-diphenyl-1-picrylhydrazyl (DPPH), [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonicacid)] (ABTS) and the ferric ion reducing power (FRAP) tests. The polyphenolic content of extract fractions was determined and extract compounds were identified using UHPLC-QToF-20 MS. In vitro anti-tyrosinase activity was also investigated as well as cytotoxicity in HepG2 liver and SK-MEL-1 melanoma cells using the MTT cell viability assay. Results: Of the four fractions prepared from the 70% methanolic extract of liquorice root, fraction 3 (F3) showed increased polyphenolic content and antioxidant properties with IC50 of 56.1 ± 6.32, 39.14 ± 1.1 and 66.34 ± 1.4 μg/ml against DPPH, ABTS and FRAP, respectively. The anti-tyrosinase activity of this fraction showed an IC50 of 358.54 μg/ml compared to Kojic acid (0.75 mM) used as the control. In addition, this fraction showed reduced liver toxicity as a higher percentage cell viability was noted in the HepG2 cells compared to the SK-MEL-1 skin melanoma cells. However, both cell types showed higher percentage viability compared to acetaminophen that was used as cytotoxic control. The LC-MS analysis revealed the presence of a wide variety of compounds including 4-azido-3-benzyl-coumarin, ferulic acid, glycyrrhizin, quercitrin, cirsilineol, gentioflavine and 4'',6,7-trihydroxyisoflavone. The literature indicates the use of these compounds regarding antioxidant and anti-tyrosinase activity. Significantly, cularidine was identified in this study, a compound not previously reported in studies involving liquorice root. Conclusion: The results from this study concur with previous reports as to the anti-tyrosinase and antioxidant activities associated with liquorice roots, activities perhaps due to the relatively high polyphenolic content in extracts from South African liquorice root. / Life and Consumer Sciences / M. Sc. (Life Sciences)

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)

See the attached abstract below

MSCAGR ( Plant Production) / Many high valued tree species of medicinal significance in the Limpopo Province, South Africa exhibit seed dormancy, and also contain aromatic oils which inhibit rooting of their stem cuttings. These plant species are under pressure due to human over-exploitation. The main objective of this study was to investigate effects of rooting hormones on the rooting ability that will help in domesticating some of the selected high valued medicinal plants, Elaeodendron transvaalense (bushveld saffron), Brackenridgea zanguebarica (yellow peeling plane), and Warburgia salutaris (pepper-bark tree). Stem cuttings of these plant species were prepared and treated with various concentrations (500, 1000 and 2000 ppm) of IBA, IAA and NAA in different growth media (Natural soil, farm soil and hygromix) at a nursery house. 180 experimental units were sown and arranged in Randomized Complete Block Design (RCBD), each treatment replicated five times and then monitored for a period of three months. Data were only obtained from Brackenridgea zanguebarica as other species dried up two weeks after sprouting. The two variables measured from the experiments were sprouted stems and number of leaves. The data obtained were subjected to analysis of variance and least significant difference (LSD) at 5% probability level was used to compare treatment using STATISTICA software analysis package. The hormone, hormone concentration, growth media and their interactions had effect on sprouted stems and number of leaves produced on Brackenridgea zanguebarica cuttings, with no record of rooting ability. IBA (500 ppm and 1000 ppm) and control (without rooting hormone) showed high significant results with natural soil and farm soil in terms of leaf production compared to the hygromix, which is significantly lower from others. IBA at the various concentrations (500, 1000 and 2000 ppm) and the control gave the highest percentage sprouted stem on both natural soil and farm soil as compared with other hormones at the same / NRF

MSc (Botany) / Department of Botany / Since time immemorial plants have traditionally been used for various purposes in many parts of the world including Vhembe district, Limpopo province of South Africa. The use of C. abbreviata plants at Matsa village of Vhembe district also contributes significantly to the livelihood of the community. However, little work has been made in the past to properly document and promote the knowledge. The purpose of the present study was to record and analyse the ethnobotanical knowledge of C. abbreviata within the people of Matsa village around Nzhelele in Vhembe district. Semi-structured questionnaires were administered on 50 informants from different households selected randomly to gather data regarding the local name of C. abbreviata, the uses and its availability. Significantly higher number of medicinal use was reported by elderly people including both men and women as compared to middle-aged, younger people. Other use categories included firewood, furniture and joinery production and shade for people in the area. Awareness on the importance of the species should therefore be created amongst the people, especially the young ones. Understanding the ethnobotanical knowledge of indigenous species within an area is crucial towards development of its management plan. / NRF

MSc (Microbiology) / Department of Microbiology / BACKGROUND: Antibiotic resistance amongst microbial pathogens has become a challenge over past decades, bringing about genuine and frequently deadly contaminations that can't be dealt with by ordinary means. This has led to a search on developing solutions to this problem by searching for new source of antimicrobial agents or chemically altering the existing ones. Traditional medicinal plants and nanoparticles are highly targeted as promising agents to address the challenge. Pyrenacantha grandiflora Baill from Icacenaceae family possess pharmaceutical activities and is used by Vhavenda people to cure gastrointestinal related infections, diarrhea and tooth pain. OBJECTIVES: The present study aimed to synthesize, characterize and evaluate the efficacy of Pyranacantha grandiflora extracts alone and when conjugated with selected nanoparticles against pathogenic microorganisms. Furthermore, this study investigated the efficacy of selected antibiotics when conjugated with nanoparticles against selected pathogenic microbes. METHODS: Pyrenacantha grandiflora Baill (tubers) were collected from Masisi area. Bioactive compounds were extracted using different solvents such as methanol, acetone, hot water, dichloromethane and chloroform. Preliminary phytochemical screening was done to identify different phytochemicals in the extracts and their functional groups were identified by Fourier Transform Infrared spectroscopy (FTIR). Extracts were further assessed for their total phenolic and flavonoids content. Thin layer chromatography was used to separate the compounds from the plant extracts and active compounds/group of compounds were identified by bioautography. The antioxidant ability of the extracts to scavenge free radical DPPH was also determined. Silver and gold nanoparticles were synthesized using chemical and biological methods, characterized by Ultraviolet-Visible Spectrophotometry (UV-VIS), Transmission Electron Microscopy (TEM) and Energy dispersive X-ray analysis (EDX). Plant extracts, nanoparticles and antibiotics were xix conjugated differently, and conjugates were analyzed by FTIR and their antimicrobial activities were evaluated against different bacteria and fungi. The conjugates were tested for antimicrobial activity against extended beta-lactamase producing Escherichia coli (ATCC 35218), Escherichia coli (ATCC 25922), methicillin-resistant Staphylococcus aureus (ATCC 25923), methicillin-susceptible Staphylococcus aureus (ATCC 33591) and beta-lactamase producing Klebsiella pneumonia (ATCC 700603) using agar diffusion assay and the minimum inhibitory concentrations (MIC) were determined using the microdilution method. The minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) were determined by sub-culturing from the MIC plates on Mueller-Hinton Agar. RESULTS: Pyrenacantha grandiflora was found to contain phenolics, saponins, alkaloids, tannin, steroids, terpenoids and flavonoids. FTIR spectroscopic studies revealed different characteristic peak values with various functional compounds similar in most extracts but differed with transmittance values. The total phenolic contents in the examined extracts ranged from 14.167 to 19.02 mg GA/g. The total flavonoid content in the examined extracts ranged from 26.603 to 34.621 mg QE/g. Thin-layer chromatography revealed various Rf values and when analyzed with bioautography, well-defined inhibition zones within the Rf value of 0.236 was identified against E. coli and K. pneumonia. The MICs of the extracts were determined, and all the extracts showed some antimicrobial activity against all tested strains ranging from 0.06-7.5 mg ml/g. Some extracts appeared to be fungicidal and hot water extracts were more active against Cryptococcus neoformans with the MFC value of 0.06 mg/ml. The methanol extract was also active against most tested strains including Candida tropicalis with the minimum fungicidal concentration value of 3.75 mg/ml. Pyrenacantha grandiflora tuber extracts conjugated with silver or gold nanoparticles exhibited a good antibacterial activity against all bacterial strain used and very few were able to exhibit bactericidal activity. Penicillin showed improvement of antibacterial activity xx when conjugated with compounds from the acetone extracts and vancomycin was found to be more effective when conjugated with silver nanoparticles and water extracts. CONCLUSION: The present study validated the efficacy of conjugated P. grandiflora tuber extracts which is used in traditional medicine. The results revealed that water extracts which are generally used by the traditional healers are active against most microorganisms tested as well as methanol and acetone extracts and the synergistic effect was observed when they were conjugated to gold and silver nanoparticles. The results of the present investigation clearly indicate that antimicrobial activity of Pyrenacantha grandiflora Baill tuber when conjugated with selected nanoparticles and antibiotics vary with test strain and the type of solvent used during extraction, thus giving hope for future development of drug leads. / NRF

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

The treatment of tuberculosis (TB) is currently a challenge due to multi- and extensively drug resistant strains of Mycobacterium tuberculosis. Mycobacterium bovis and M. tuberculosis cause clinically indistinguishable tuberculosis in humans. Both M. bovis and M. tuberculosis have been isolated from humans and animals. Plant species contain antimicrobial compounds that may lead to new anti-TB drugs. To conduct in vitro antimycobacterial assays, it is important to include current clinical isolates as new strains of bacteria might be circulating under the ongoing climate change environment. The overall goal and objectives of this study were to isolate and characterize mycobacteria species from South Africa, to test some selected plant species of the Anacardiaceae family for antimycobacterial activity using some of the newly isolated and reference strains of mycobacteria followed by cytotoxicity evaluation of the most active plant species, and finally the isolation and characterization of at least one compound from the most active and least toxic plant. This study led to the discovery of a new isolate of Mycobacterium Avium Complex species from black wildebeest. Other non-tuberculous mycobacteria and M. bovis isolates were identified from other animal species. Five out of 15 plant species screened showed good activity against Mycobacterium species. Five antimycobacterial compounds were isolated from Searsia undulata, the most active plant species. Two out of the five compounds were identified, and one compound appears to be novel, but both compounds have been isolated for the first time from Searsia undulata. An incidental finding was the potential anticancer property of extracts of Searsia undulata. Recommended future activities include isolation and identification of more active compounds from Searsia undulata which were visible in bioautography analysis, as well as synergy evaluation of antimycobacterial activities of the different compounds with current anti-tubercular drugs. / Environmental Sciences / Ph. D. (Environmental Science)

This study focuses on the use of the Athrixia phylicoides plant in Tshwane, Gauteng, South Africa. The plant is used within the domestic domain as a beverage, for medicinal purposes, as well as for the manufacturing of domestic brooms. The aim of the study was to investigate the use and market of Athrixia phylicoides in two of the Tshwane markets, namely; Denneboom and Marabastad. Case study design, participant observation and interviews were employed to collect data. A literature review was conducted to construct a theoretical framework. The study reveals that traditional healers, broom makers and customers have a remarkable knowledge about Athrixia phylicoides, which has been untapped thus far. In addition, the study indicates that possible extinction of the Athrixia phylicoides plant, due to over-harvesting and lack of conservation, will pose a challenge to the market. / Anthropology / M.A. (Anthropology)