Thesis (M.Sc.) - University of KwaZulu-Natal, Pietermaritzburg, 2009. / Full text also available online. Scroll down for electronic link.

Thesis (M.S.)--The University of North Carolina at Greensboro, 2008. / Directed by Nadja Cech; submitted to the Dept. of Chemistry and Biochemistry. Title from PDF t.p. (viewed Apr. 13, 2010). Includes bibliographical references (p. 62-68).

Thesis (Ph.D.) - University of KwaZulu-Natal, Pietermaritzburg, 2009. / Full text also available online. Scroll down for electronic link.

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 343-355). Available also in a digital version from Dissertation Abstracts.

M.Sc. (Botany) / The market and public demand for medicinal plants over the past few decades has increased dramatically with more than 1 000 plant species actively traded for medicinal purposes throughout South Africa. Intensive harvesting of wild material is now acknowledged as a serious threat to biodiversity in this country. Also the substitution of a valuable commodity (medicinal plant) by a cheaper alternative (other plant species), either inadvertently due to misidentification, or deliberately to cheat consumers, raises some serious concerns as these adulterants may not be as effective or may even be toxic and cause harm to consumers. To add to the problem many species are either traded as dried leaf, root, bark products, or extracts and their identification becomes problematic. Therefore, DNA barcoding can help to provide a rapid and accurate identification tool for medicinal plants. In the current study I targeted the most commonly used medicinal plants in South Africa and produced a set of barcodes for fast and easy DNA-based species identification (rbcLa & matK). I tested the efficiency of core barcodes in the identification of medicinal plants using four main analyses, in the R package Spider 1.1-1. Here the extent of specific genetic divergence, DNA barcoding gap, BLAST test, and the ability to discriminate between species were assessed. Overall, the matK region was found to be a more useful tool for the species identification of medicinal plants in South Africa.

Mycobacterium tuberculosis and Human Immuno-Deficiency Virus (HIV) have a high prevalence in South Africa. The development and spread of drug resistant tuberculosis is a serious problem which is exacerbated by tuberculosis (TB) co-infection in HIV patients. Traditional medicinal plants like Annona muricata and Artemisia afra are used for respiratory ailments and antiviral therapies respectively. The aim of this study was to evaluate Annona muricata (ethanolic extract) and Artemisia afra (ethanolic and aqueous extracts) for inhibitory activities against M. tuberculosis and HIV. In vitro bioassays for anti-TB activity included: microplate alamar blue assay (MABA), flow cytometry and ρ-iodonitrotetrazolium chloride assays while anti-HIV activity was determined using an HIV-1 reverse transcriptase colorimetric ELISA kit and an HIV-1 integrase colorimetric immunoassay. Cytotoxicity of plant extracts were assessed by the MTT assay on Chang Liver and HepG2 cells. Potential synergistic effects were determined using the basis of Combination Index. Potential interactions of plant extracts with drug metabolic pathways were evaluated with the Glutathione-S-Transferase assay kit as well as the CYP3A4 assay kit. A. muricata ethanolic extract exhibited anti-TB activity with MIC 125 μg/mL. MABA was shown to be the most sensitive and effective method for the detection of anti-TB activity. Artemisia afra aqueous extract showed HIV-1 reverse transcriptase inhibition exhibiting ˃85 percent inhibition at 1 mg/mL while the ethanolic extracts of A. afra and A. muricata showed inhibition of HIV-1 integrase activity at ˃86.8 percent and ˃88.54 percent respectively at concentrations >0.5 - 4 mg/mL. The aqueous extract of A. afra displayed inhibition of HIV-1 integrase ˃52.16 percent at 0.5 mg/mL increasing to 72.89 percent at 4 mg/ml of the extract. A. muricata was cytotoxic at an IC50 of 30 μg/mL and 77 μg/mL on Chang Liver and HepG2 cells respectively, whilst A. afra aqueous and ethanol extracts were not cytotoxic to both cell lines. The ethanolic extract of A. muricata showed both antagonistic and synergistic properties at various IC values, when used in conjunction with rifampicin. A. afra ethanolic extract interrupted GST activity while aqueous extracts of A. afra and A. muricata had a slight effect. All extracts interrupted CYP3A4 activity, however the ethanolic extracts of A. muricata and A. afra showed greater inhibition than the aqueous extract of A. afra. These extracts should be investigated further as they could be an important source of compounds for treatment of M. tuberculosis and HIV respectively.

Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2015 / Antioxidants play an important role in living organisms to control level of free radicals and other reactive molecules in the body to reduce oxidative damage. Synthetic antioxidant compounds are used in food industries as food additives to boost our immune systems. These compounds are associated with a number of critical side effects including liver damage and carcinogenesis. Scientists are also concerned about microorganisms that have developed resistant genes against current antibiotics used in hospitals. The aim of the study was to isolate and characterize bioactive compounds from Ricinus communis leaves with activity against Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). Consequently, medicinal plants are studied and considered for their efficacy and safety, because they possess bioactive compounds with various biological activities. Leaves of R. communis were collected at the University of Limpopo, Turfloop campus in Limpopo province, South Africa. The leaves were dried and milled to a fine powder. A number of trial extraction methods were employed using various solvents of different polarities on a fine powder leaves to identify the best extraction method. Plant extracts were analyzed by thin layer chromatography (TLC) developed in four mobile phases. To detect separated phytochemical compounds, TLC plates were sprayed with vanillin- sulphuric acid in methanol and heated at 110oC for optimal colour development. Qualitative antioxidant activity was determined by using 2, 2–diphenyl-1-picrylhydrazyl (DPPH) assay on TLC plates. Quantitative antioxidant activity was determined by measuring percentages scavenging activity of DPPH and 2, 2’-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) free radical molecules by plant extracts. Antibacterial activity of all extracts was quantified by a serial microbroth dilution method while bioautography was used in qualitative analysis of the active compounds. Cytotoxicity effect of R. communis extracts was evaluated using tetrazolium-based calorimetric assay on human Caucasian skin fibroblast (Bud-8) cell line. Anti-inflammatory activity was assessed using phagoburst kit on Raw 264.7 macrophages cell line. Pure compounds were subjected to nuclear magnetic resonance spectroscopy for 1H, 13C and DEPT experiments to elucidate structures of compounds. 2 During extraction process, methanol was the best extractant, extracting greater amount of extracts than any of the other solvents. Serial exhaustive extraction method was selected as the best extraction method for extracting compounds from ground plant materials. In quantitative antioxidant assays, chloroform and methanol extracts had highest percentage scavenging activity against DPPH free radicals compared to other extracts and vitamin C. Methanol extract had the highest percentage scavenging activity of ABTS free radicals and minimum percentage scavenging activity was in hexane extract. Acetone, ethyl acetate and ethanol extracts showed strong antioxidant activity against DPPH free radicals in qualitative antioxidant assay on TLC plates. In quantitative antibacterial assay, crude extracts showed lowest minimum inhibitory concentration value of 0.13 mg/ml against all tested organisms and the highest was 1.05 mg/ml. Hexane extracts revealed potent antibacterial activity against all tested microorganisms on bioautograms. Hexane and acetone extracts also revealed anti-inflammatory activity and have ability to reduce oxidative stress. In cytotoxicity effect of plant extracts, Methanol extracts had lethal concentration for 50% of the cells (Lc50) of 784 μg/ml on Human Caucasian skin fibroblast (Bud-8) cell line while hexane extracts had Lc50 of 629 μg/ml. Plant extracts with high Lc50 are low toxic to normal cell line and preferable to work with for drug development. Bioassay-guided fractionations results in successful isolation of three antioxidant and two antibacterial compounds from R. communis using column chromatography. Isolated compounds were tested for their biological activities using qualitative DPPH assay on TLC plates for antioxidant activity and bioautography for antibacterial activity. Antioxidant compounds showed strong antioxidant activity after spraying with DPPH in methanol and antibacterial compounds showed less activity compared to the crude extracts. The study suggests the use of crude extracts to fight against pathogenic microorganisms compared to pure compounds. Compound 4 was successful identified as the mixture of stigmasterol and β-sitosterol. The present study recommends the use of R. communis leaves as the potential source of antioxidant, antibacterial and anti-inflammatory compounds. The study serves as a scientific proof for use of this plant in traditional medicine for treatment of various ailments.

MSc (Botany) / Department of Botany

MAAS / Department of African Studies / Medicinal plants are now used as a livelihood activity by the marginalized urban poor communities in various places around the world. Indigenous knowledge in medicinal plants is owned and practiced by the knowledge holders for different purposes. It entails the passing of skills and knowledge from one generation to the other within a specific geographical area. Vhembe district is well known to be rich in plants and the people own the rich knowledge in medicinal plants. However, much debate has emerged around the effectiveness of indigenous knowledge in alleviating poverty levels amongst the communities. Despite the wealth and abundance of indigenous knowledge in medicinal plants, Limpopo Province is still rated as one of the poorest provinces in South Africa. Consequently, this study sought to investigate the utilization of indigenous knowledge by medicinal plant vendors in Thohoyandou, Vhembe District. The study has used the qualitative research approach by means of an interview schedule and semi-structured interviews to collect data from a sample of 10 respondents, who were selected using the purposive and snowballing non-probability sampling techniques. The data collected was analyzed thematically. The findings in this study revealed that the sale of medicinal plants by vendors is a source of employment done mostly by men than women who have been engaged in this form of street trading for more than 23 years. The CBD in Thohoyandou is deemed preferably by the medicinal plant vendors as it is busy and attracts more customers. Although the medicinal plant vendors make a living out of selling their practice, they are not fully supported by key stakeholders. The research therefore concluded that the use of indigenous knowledge by medicinal plant vendors has an important role to play in creating employment for indigenous knowledge holders and as such should be invested in. The study recommends that key stakeholders such as the municipality, private companies, business support groups and the government should take the initiative to upgrade, develop and invest in indigenous knowledge v holders of medicinal plants to reduce unemployment in the province and avoid the risk of extinction of the knowledge. Lastly, more research should be conducted on a much bigger scale / NRF

Thesis (Ph.D. Agriculture (Plant Production)) -- University of Limpopo, 2022 / The unique phytochemical composition of the medicinal plant cancer bush (Sutherlandia frutescens) have made its foliage to gain much attention in South Africa due to its health benefits. In situ harvesting of the plant parts of this important species serve as one potential strategy to avert its extinction through whole plant harvesting, a common practice by rural communities. However, such a strategy is limited by lack of information on the agronomic requirements of the plant species and its susceptibility to root-knot (Meloidogyne species) nematodes. The objectives of the study were four-fold, namely, to: (1) identify nodulation bacteria associated with wild S. frutescens using morphological and biochemical techniques, (2) assess the efficacy of the nodulation isolates from different centres of biodiversity of S. frutescens in Limpopo Province, South Africa (3) test the compatibility of cucurbitacin-containing phytonematicides on S. frutescens for managing population densities of Meloidogyne species and (4) determine the nutritional water productivity (NWP) of S. frutescens in association with water scarcity of the region where the plant species originated. In achieving Objective 1, nodules from S. frutescens roots were washed in distilled water and healthy, undamaged, firm and pink nodules were sterilised. Aseptic nodules from S. frutescens roots and commercial strains were transferred into a smasher biomerieux polythene bag containing 10 ml distilled water and crashed to produce a milky suspension the milky suspension was streaked on Yeast extract mannitol agar (YEMA). After gram reaction, colony characterisation includes the investigation of shape, colour, configuration, elevation and margin of bacterial colony as observed in colonies on nutrient agar plates of overnight grown microorganisms using a microscope. The medium for biochemical test was prepared, inoculated with 5 μl purified xxv bacterial cultures and incubated at 37°C for 48 h. Identification of the bacterial isolates was performed using VITEK 2 Systems (bioMérieux, Inc., North Carolina, USA). Using morphological and biochemical techniques, the bacterial species associated with roots of S. frutescens in the wild were assayed primarily those in the genera Raoutella ornithinolytica and Enterobacter cloacae species dissolvens. The VITEK 2 Systems confirmed the identification of the bacterial species from 80 to 96% of the samples. Three species were confirmed from another sampling area, Sphingomonas paucimobills, Raoutella ornithinolytica and Enterobacter cloacae species dissolvens from by 86 to 96% of the samples. In achieving Objective 2, the five treatments, namely, Bradyrhizobium spp. (Arachis) strain, Rhizobium leguminosarum strain, Tubatse strain, Sebayeng strain and untreated control, were laid-out in a randomised complete block design, with seven replications during the first season (Experiment 1) and with eight replications during the second season (Experiment 2). The seasonal interactions (Experiment 1 × Experiment 2) on plant and nutrient elements were not significant (P ≤ 0.05) and data for the two seasons were pooled (n = 75). Relative to untreated control, commercial (Bradyrhizobium and Rhizobium strain) and native strains (Tubatse and Sebayeng strain) significantly increased plant height by 31, 33, 44 and 40%, respectively, root length by 30, 41, 40 and 42%, respectively and dry shoot mass by 48,195 and 17%, respectively. Similarly, rhizobia strains significantly contributed to the increase in nitrogen assimilation by 7, 25 and 80%, respectively, protein synthesis by 13, 10, 24, 69%, respectively, and symbiotic efficiency by 31, 133, 292 and 82%, respectively. However, rhizobia inoculants had no significant effects on potassium and phosphorus in leaf tissues. In achieving Objective 3, in Mean Concentration Stimulation Point (MCSP) experiments, seven treatments, xxvi namely, 0, 2, 4, 8, 16, 32 and 64% for each phytonematicide, were arranged in a randomised complete block design (RCBD), with 8 replicates. In application interval experiments, treatments, based on “weeks-per-month-of-30 days” for M. javanica, which translated to 1, 2, 3 and 4 weeks, were arranged in a RCBD, with 10 replicates. Nemarioc-AL and Nemafric-BL phytonematicides had MCSP values of 3.43 and 4.03%, respectively, with the plant having high tolerance level to the products. The respective application interval of the two products for managing population densities of Meloidogyne species were 29 and 17 days. The dosage models for Nemarioc-AL and Nemafric-BL phytonematicides were 6.62 and 13.26%, respectively. In achieving Objective 5, the study used nine treatments designated as T1, T2, T3, T4, T5, T6, T7, T8 and T9, respectively, consisting of 1, 2, 3, 4, 5, 6, 7, 8 and 9 seedlings/hole of drip irrigation transplanted using a 3S planter under field conditions, arranged in randomised complete block design (RCBD) with 9 replications (n = 81) in two seasons. The NWP of total flavonoids, total tannin and total phenol exhibited positive quadratic relations in varied planting density suggesting that this cultural practices could be manipulated to improve NWP of cancer bush. In conclusion, the wild bacterial isolates, sampled from S. frutescens plant grown in the field, outperformed the commercial bacterial strains in enhancing the productivity of the test plants. The empirically established dosage model for Nemarioc-AL and Nemafric-BL phytonematicides could be used to control Meloidogyne species in cancer bush production. There is a need to further investigate the responses of the identified strains to the test phytonematicides. Findings of the study openend new frontiers in the development and commercialisation of the observed native bacterial strains for the cultivation of S. frutescens, which has excellent medicinal importance as a cure or management for cancer. / Agricultural Research Council-Universities Collaboration Centre, the National Research Foundation (NRF) and the Flemish Inter-University Council of Belgium