Thesis (PhD. (Anthropology)) --University of Limpopo, 2008 / In this study, the multidisciplinary epistemology of ethnobotany is reviewed within the wider context of ethnoecology and indigenous knowledge systems. The major problems derived from this theoretical framework cover aspects of the relationship between cultural groups and their natural environment - particularly the botanical component of the latter. Problems and critical questions about this relationship were investigated in selected areas of the Bushbuckridge district, which are inhabited by a number ofTsonga-Shangana communities. Salient aspects of the natural habitat in the study area are discussed with special reference to the vegetation, climate and soil types. A proper understanding of the culturally-based exploitation of plant resources necessitates a sound knowledge of the culture concerned, including relevan t aspects of its value-system and cosmology. In this review, emphasis is placed on those aspects of culture that are directly concerned with the utilisation of plant resources. The latter include a wide range of economic, technological and medicinal uses. Specimens of 200 plants were collected and identified by the University of Limpopo Herbarium. The uses of every species are described. Systematised tables of plant uses are presented, as well as comparative lists in which selected uses are compared to those of other cultures in which the same species are used. The effects of plant utilisation, as well as indigenous forms of resource management, are also discussed. Recommendations are made on how to counter the process of environmental degradation which is caused by over-exploitation .

MSc (Microbiology) / Department of Microbiology / BACKGROUND: Medicinal plants form an important part of the Southern African cultural heritage. Indigenous populations, for example the Vha-Venda people, tend to use medicinal plants in formulations rather than western medicines for health and survival. In order to certify and give scientific credibility to the use of medicinal plants formulations used by Vha-Venda people for the treatment of diseases, several assays were carried out. The present study was aimed at assessing phytochemical content, antimicrobial, antioxidant and cytotoxic activities of four indigenous Venda medicinal plants in a formulation and compare their activity with each plant used individually. METHODS: Peltophorum africanum (roots), Pterocarpus angolensis (bark), Terminalia sericea (roots) and Ximenia caffra (roots) were collected from the Thohoyandou area. The collected plant parts were extracted with methanol and water respectively. Individual plant extracts and Five designed formulations were tested for their antimicrobial activity against Staphylococcus aureus ATCC 25923 (Methicillin Resistant), Staphylococcus aureus ATCC 33591(Methicillin Susceptible), beta lactamase producing Klebsiella pneumonia (ATCC 700603) and extended spectrum beta lactamase producing E. coli (ATCC 35218), four clinical isolates of Candida spp and Cryptococcus neoformans using the Broth dilution method. Minimum bactericidal concentration (MBC) of the extracts was determined by culturing the contents of minimum inhibitory concentration (MIC) on nutrient agar. Similarly, minimum fungicidal concentration (MFC) was also determined by culturing contents of MIC in sabouraud dextrose agar (SDA). Extracts were further assessed for their total phenolic content, total flavonoid content and Qualitative phytochemical analysis. The antioxidant ability of the plants extracts and formulations to scavenge free radical DPPH was also determined. The plant formulations were assessed for their anti-HIV activity using the reverse transcriptase colorimetric assay kit. Cytotoxicity against human lymphatic endothelial cells (HLEC) was determined using MTT assay. RESULTS: Methanolic and aqueous extracts of T. sericea exhibited the best antifungal and antibacterial activities whilst P. angolensis and X. caffra showed poor activities. Methanolic plant formulations showed good activities compared to aqueous formulations. However, Fractional Inhibition Concentration Index showed that there was 1 synergistic interaction, 25 additive interactions and 14 antagonistic interactions between the plant extracts. The methanolic formulation 3 showed the best overall phenolic content at 11.85±0.109 mgGAE/g whilst aqueous X. caffra extract showed the least content at 4.546±0.104 mgGAE/g. Higher total flavonoid contents were seen in methanolic formulation 4 at 2.75±0.02 mgQE/g. Qualitative phytochemical analysis revealed the presence of flavonoids, phenolics, terpenoids, tannins, saponins and steroids in 80% of the tested plant extracts and formulations. All plant extracts and formulations exhibited good antioxidant activity against DPPH. The methanolic formulation showed the best antioxidant activity with IC50 of 0.094 ± 0.33μg/ml. For anti- HIV inhibition, all formulations at 200μg/ml exhibited higher percentage of HIV-1 reverse transcriptase inhibition with methanolic mixture 3 being the best overall at 97.5% activity whilst aqueous mixture5 was the least active with 63.03% inhibition activity. Moreover, the best anti-HIV activity at 100μg/ml was exhibited by methanolic mixture 3 at 71% inhibition. Furthermore, aqueous X. caffra, mixture 2 inhibited 26% and 51% at 12.5mg/ml and 3.125mg/ml respectively. Peltophorum africanum and mixture 5 inhibited 34%, 54% and 43% at 3.125mg/ml, 6.25mg/ml and 12.5 mg/ml respectively of Human Lymphatic Endothelial cells growth. CONCLUSIONS: The results from the study indicated that most of the commonly used traditional medicinal Plants in the Venda region when mixed together have merit for use in traditional medical practice as they have shown good antimicrobial activities, good antioxidant xviii activities, good phytochemical activities and good cell proliferation activity. However some formulations showed antagonistic interaction against bacteria. Some Individual medicinal plants showed toxicity at higher concentrations against immune cells. Whereas formulations promoted cell proliferation, therefore, the use of such individual plants in the treatment of infections should be highly monitored as they may pose a health threat to normal immune cells. Generally, plants are potential pharmacological agents which needs to be preserved and harvested with care. / NRF

PhD (Chemistry) / Department of Chemistry / Terminalia sericea Burch. ex. DC (Combretaceae) is one of the 50 most popular medicinal plants in Africa. The fruit, leaves, stems and roots are commonly used for the treatment of cough, skin infections, diabetes, diarrhoea, venereal diseases and tuberculosis. However, the roots are most commonly used in the preparation of traditional medicines. Pharmacological studies have revealed that the crude root extracts display antibacterial activity against several Gram-positive and Gram-negative bacteria. Anolignan b, termilignan b and arjunic acid are reported to be the major antibacterial constituents present in the roots. Other compounds isolated from the roots include resveratrol-3-rutinoside, sericic acid, sericoside and arjunglucoside I. Authorities worldwide, including the Medicines Control Council of South Africa, have begun to regulate herbal drugs sold in the form of commercial formulations. Quality control of herbal drugs is challenging, since the chemical profiles of the raw materials may vary, depending on the origin of the plant material and the way that it was handled and processed. The chemistry, in turn, impacts on the safety and efficacy of the plant material. To date, there are no available data on parameters that can be used to standardise the quality of T. sericea raw materials. The aim of this study was therefore to provide information on the variation of the chemical constituents that contribute to the biological effects of the roots of T. sericea and also establish its safety. Since the compounds previously isolated from the roots were not commercially available, isolation of the major constituents of the roots was undertaken to obtain analytical standards. A crude dichloromethane:methanol (1:1) extract was initially fractionated using silica gel column chromatography, where after, some of the fractions were further purified using silica gel and Sephadex LH-20 column chromatography. Final purification of the enriched fractions was achieved using preparative high performance liquid chromatography coupled with mass spectrometry (prep-HPLC-MS). The structures of these compounds were subsequently elucidated using one- and two- dimensional nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry and identified as sericic acid (340 g), sericoside (500 g), resveratrol-3-rutinoside (240 mg) and arjunglucoside I (74 mg). The chemical variation within the crude root extracts of samples (n = 42) from ten populations in the Limpopo Province of South Africa, was determined using ultra performance liquid chromatography-mass spectrometry (UPLC-MS). A method was developed for the simultaneous determination of sericic acid, resveratrol-3-rutinoside, sericoside and arjungluicoside I in the extracts using UPLC with photodiode array detection (PDA). The method was validated according to the guidelines of the International Council for Harmonisation (ICH). A regression coefficient (R2) of 0.998 was obtained for sericic acid, resveratrol-3- rutinoside and arjunglucoside I, while the R2 value for sericoside was 0.999, indicating a linear relationship between the concentration and the detector response. Satisfactory limits of detection for sericic acid (25.2 ng/mL), sericoside (11.6 ng/mL), resveratrol-3-rutinoside (23.3 ng/mL) and arjunglucoside I (8.81 ng/mL) were determined. Recoveries of 98 % and 80% were obtained for samples spiked with 12.5 μg/mL and 25 μg/mL of resveratrol-3-rutinoside, respectively, indicating that the method is accurate. The intra- and inter-day variation in resveratrol-3-rutinoside concentration, measured over three days, indicated excellent analytical precision, since all the relative standard deviations were below 0.70 %. The quantitative data revealed that sericic acid (1.59 to 8.45 mg/g), sericoside (2.07 to 20.17 mg/g), resveratrol-3-rutinoside (0.65 to 29.82 mg/g) and arjunglucoside I (0.86 to 8.44 mg/g dry weight) were the major constituents of the root samples, but their concentrations were highly variable. Chemometric analysis of the aligned UPLC-MS data was used to investigate similarities and differences in the chemical profiles of the samples using an untargeted approach. A principal component analysis (PCA) model was constructed and subsequently hierarchical cluster analysis (HCA) indicated the presence of two main groups, which were found to be independent of the populations to which the samples belong. Classes, based on the HCA class identifiers, were subsequently assigned to the samples, and an orthogonal projection to latent structures-discriminant analysis (OPLS-DA) model was then constructed, (R2 cum = 0.996 and Q2 cum = 0.967). The corresponding loadings plot allowed sericic acid, sericoside and resveratrol-3-rutinoside to be identified as biomarkers associated with the first group. Quantitative, rather than qualitative differences were responsible for the observed clustering pattern. Techniques that could be applied in quality control protocols for T. sericea root were investigated. High performance thin layer chromatography (HPTLC) analysis of the root extracts was optimised by testing different developing solvents and visualization reagents. The presence of the sericic acid (Rf = 0.80), sericoside (Rf = 0.49) and resveratrol-3-rutinoside (Rf = 0.36) were clearly visible on the plates. There were visible variations in the concentrations of resveratrol-3-rutinoside in representative samples from the 10 populations, corresponding to the UPLC results. The powdered samples were then analysed by mid-(MIR) infrared spectroscopy. Chemometric analysis of the data revealed no definitive clustering pattern. Partial least squares-discriminant analysis (PLS-DA) calibration models were established from the MIR spectral data combined with the accurate UPLC-values, for the prediction of the sericoside (R2Y = 0.848, Q2 = 0.757, RMSEP = 2.70 mg/g) and resveratrol-3-rutinoside (R2Y = 0.794, Q2 = 0.695, RMSEP = 4.37 mg/g) concentrations in powdered root samples. The antibacterial activities of the root extracts, column fractions and isolated compounds were determined using three Gram-positive and five Gram-negative bacteria, all selected due to their ability to cause intestinal and skin disorders. Extracts and fractions containing high concentrations of sericic acid exhibited the highest activities against Klebsiella pneumoniae (ATCC 13883), Pseudomonas aeruginosa (ATCC 27858), Salmonella typhimurium (ATCC 14028), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12223) and Shigella sonnei (ATCC 9292). The pure compound (sericic acid) was highly active against S. sonnei (MIC 0.078 μg/mL), a Gram- negative bacterium. There were no variations in the activity of the crude extracts against B. cereus and P. aeruginosa, while the MIC values obtained against S. typhi were variable and ranged from 0.25 to 1.0 mg/mL. Sericoside and resveratrol-3-rutinoside did not display any activity. The anti-oxidant activities were evaluated using DPPH (2,2-diphenyl-1-picrylhydrazyl) and reducing power assays. The anti-oxidant assays revealed that resveratrol-3- rutinoside exhibited lower activity (DPPH = 186 μg/mL; RP = 184 μg/mL) compared to the crude extract (DPPH = 22.3 μg/mL; RP = 24.4 μg/mL) and ascorbic acid (DPPH = 11.3 μg/mL, RP = 145 μg/mL). Sericic acid and sericoside did not display any anti- oxidant activities. The variation in the anti-oxidant activities (4.58 to 26.0 μg/mL) of the samples from different populations was an indication of chemical variability. A toxicity study of the raw powdered plant material was conducted using vervet monkeys (Chlorocebus pygerythrus). Biochemical analysis (liver function tests, kidney function tests and hematology), physical and physiological examinations were conducted. The subjects were fed a normal diet supplemented with T. sericea root powder (2.14 g/kg per day) for 120 days, where after the diet was returned to normal (washout) for another 30 days. The treatment groups presented with elevated serum enzymes at Week 4, followed by the reduction of the elevated serum enzymes levels at Week 12. These results indicate short-term hepatotoxic effects, followed by hepatoprotective activity. Reduction of the serum glucose at Week 4 suggests hypoglycemic potential. However, elevated serum creatinine levels indicated possible nephrotoxicity. In conclusion, this study has indicated the variability in the chemical constituents of the roots of T. sericea, which affects the antibacterial and anti-oxidant activities. Sericic acid, resveratrol-3-rutinoside, and sericoside were, for the first time, identified as biomarkers that can be used for the quality control of raw root material to be used in herbal products. Sericic acid was also found to be the main antibacterial constituent of the roots. The hepatoprotective, nephrotoxic and hematotoxic effects observed in monkeys to which the root powder had been administered is cause for concern. / NRF

MSc (Chemistry) / Department of Chemistry / Rauvolfia caffra Sond, a species of evergreen trees and shrubs in the dogbane family, (Apocynaceae), is used as a medicinal plant among traditional communities in many countries for the treatment of malaria, diabetes, coughs, gastrointestinal disturbances, skin infections, impotence, insomnia, diarrhoea, dysentery, scabies, worm infections, and both parasitic and microbial infections. Phytochemical studies have revealed that indole alkaloids are the major constituents of the stem bark. However, there are limited studies linking the compounds with the ethnomedicinal uses. The aim of this study is to isolate and characterize bioactive compounds from Rauvolfia caffra Sond. The highest phenolic content found in a fraction was 16.06±0.125 mg GAE/g, while the highest flavonoid content measured was 9.453±0.081 mg QE/g. In the DPPH free radical scavenging activity and reducing power tests, a lowest IC50 value of 0.022±0.003 μg/mL and IC0.5 value 0.518±0.044 μg/mL, respectively, was found. Six compounds were isolated from the stem bark, including lupeol, a pentacyclic tri-terpenoid isolated for the first time from the genus Rauvolfia; raucaffricine, a rare glycoalkaloid of the monoterpenoid indole class; N-methylsarpagine, an indole alkaloid isolated for the time from R. caffra and spegatrine, an indole alkaloid isolated for the first time from R. caffra, respectively. Concerning antimicrobial activity, the highest activity of a fraction was against B. cereus with MIC values as low as 12.5 mg/mL. One fraction at the tested concentration (250 μg/mL) decreased the viability of Plasmodium falciparum (4.149±6.979 %) with an IC50 value of 6.533 μg/mL. The crude extract and some fractions affected the viability of the Trypanosomes at the tested concentration (250 μg/mL), giving -0.133 ± 0.206 %, 11.334 ± 2.692 %, 1.026 ± 0.143 % and 20.769 ± 9.054 % with IC50 values of 18.50 μg/mL, 14.15 μg/mL, 15.58 μg/mL and 34.71 μg/mL, respectively. Furthermore, the fractions did not show significant cytotoxic effects at a concentration of 50 μg/mL. / NRF

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

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