MSc (Botany) / Department of Botany / See the attached abstract below

Thesis (Ph.D. (Biochemistry)) --University of Limpopo, 2009 / Commelina benghalensis Linn is used in traditional medicine in several Asian and African countries for the treatment of various ailments such as stomach irritations, burns, sore throat and feet, diarrhoea and as an anti-inflammatory agent. Recently, our laboratory showed that the crude methanolic extract of Commelina benghalensis L (CMECB) exhibits growth inhibitory and proapoptotic effects in Jurkat T and Wil-2 NS cancer cell lines. In this study, the precise molecular mechanism(s) associated with CMECB-induced growth inhibitory and apoptosis inducing effects in Jurkat T and Wil-2 NS cell lines were investigated. This was achieved by investigating the effects of the extract on the cell division cycle distribution profile as well as its effects on various cell division cycle and apoptosis regulatory genes. Ground stems of C. benghalensis L were extracted with absolute methanol to obtain a crude extract. To assess the effect of CMECB on cancer cell growth, experimental cell cultures were exposed to various concentrations (0 to 600 μg/ml) of CMECB for up to 72 hours. The results demonstrated a significant reduction in cell viability and inhibition of proliferation of experimental cell cultures as determined by the trypan blue dye exclusion assay and the Coulter counter method, respectively. Analysis of nuclear morphological changes in cells stained with Hoechst 33258 confirmed apoptosis as the mode of cell death that is associated with the growth inhibitory effects of CMECB in both the Jurkat T and Wil-2 NS cell lines. This assertion was based on the observed presence of nuclear morphological changes such as chromatin condensation and fragmentation and apoptotic bodies in cells exposed to CMECB. In order to get an insight on the pro-apoptotic mechanisms of CMECB, Western blot xxi and quantitative real-time PCR (qrt-PCR) were used to investigate the expression profiles of various apoptosis and cell division cycle regulatory genes. Qrt-PCR results showed a lack of a clear up- and/or down-regulatory effects of CMECB on the mRNA expression levels of bax and bcl-2 in both Jurkat T and Wil-2 NS cells. Western blot analyses demonstrated that CMECB induced apoptosis by facilitating Bax protein translocation from the cytosol to the mitochondria in both Jurkat T and Wil-2 NS cells. In addition, CMECB down-regulated Bcl-2 protein expression which, as a result, led to the shift in the Bax/Bcl-2 protein ratio at certain time points and concentration in both Jurkat T and Wil-2 NS cells. The modulation of the Bcl-2 family members led to mitochondrial cytochrome c release into the cytosol and activation of caspases-9 and -3; this was also confirmed by caspase activity assays and eventual degradation of PARP. Furthermore, CMECB induced Jurkat T and Wil-2 NS cell division cycle arrest at the G2/M phase as determined by flow cytometric analysis. Western blot analyses of G2/M phase regulatory proteins demonstrated that the CMECB-induced cell division cycle arrest was associated with the downregulation of cyclin B1 and Cdc2 protein expression levels. Western blot analyses results further revealed that the arrest of Wil-2 NS cells at the G2/M phase was independent of p21 protein activity. However, Jurkat T cell division cycle arrest was found to be mediated, in part, by p21. Quantitative real-time PCR results did not show a clear trend in terms of the down- or up-regulatory effects of the extracts on the G2/M phase regulatory genes. The CMECBinduced apoptosis and G2/M arrest was found to occur in a p53-independent xxii manner due to the lack and down-regulation of p53 protein levels in both Jurkat T and Wil-2 NS cells, respectively. In conclusion, CMECB induces its anticancer activity by inducing G2/M phase arrest and mitochondrial-mediated apoptosis independent of p53 protein activity. Although the study did not perform in vivo experiments to ascertain the efficacy of extracts of CMECB against specific tumour types in animal models, the present findings somehow validate the traditional use of C. benghalensis L as an anticancer agent. A more definitive study needs to be done to ascertain this assertion. / National Research Foundation and the University of Limpopo research office

MSc (Botany) / Department of Botany / See the attached abstract below

MSc (Botany)

MA (Tshivenda) / Senthara ya M. E.R. Mathivha ya Nyambo dza Afrika, Vhutsila na Mvelele / See the attached abstract below

MSc (Botany) / Department of Botany / Diabetes and arthritis are the most common chronic diseases. Arthritis is the leading cause of global disability and diabetes has become a major health problem which is increasing rapidly. The purpose of the study was to document medicinal plants that are used to treat and manage diabetes and arthritis by traditional medicinal practitioners around the Vhembe District Municipality as well as to evaluate their in vitro efficacy. Traditional practitioners were interviewed using semi-structured questionnaires. Seventeen plant species belonging to fourteen different families were found to be used in the treatment of diabetes as well as arthritis. Fabaceae family was dominating. Antioxidant, anti-inflammatory, cytotoxicity, alphaamylase and alpha-glucosidase) of five plant species, (Bridellia mollis, Elephantorrihiza burkei, Elaeodendron transvaalense, Senna petersiana and Searsia lancea) used traditionally to manage diabetes were investigated using the standard in vitro procedures. All extracts showed a good nitric oxide inhibition, with highest percentage inhibition found in the highest concentration of 100 μg/ml. They all had good percentage cell viability at lowest concentration which was comparable to quercetin. Only two plant extracts B. mollis (T2) and E. transvaalense (T3) had lower than inhibition of quercetin at 25 μg/ml than at 12.5 μg/ml. In vero cells low toxicity effect was observed at lowest concentration tested, and toxicity increased with the increase in concentration. In bovine dermis cell line all plant extracts had more cell viability at lower concentration than doxorubicin. Ethanol extracts of B. mollis and S. petersiana, and ethyl extract of E. transvaalense had a good alpha-amylase inhibitory activity with IC50 values 58.6, 81.9 and 131.5 mg/ml respectively. Hydro-ethanol, ethyl acetate and ethanol extracts of E. burkei exhibited a significant alpha-glucosidase inhibitory activity with IC50 values 56.9, 52.2 and 129.7 mg/ml respectively. Kinetic analysis revealed non-competitive and un-competitive inhibitions of the plant extracts on alpha-amylase and alpha-glucosidase enzymes respectively. The information obtained showed that people in Vhembe District Municipality still rely on medicinal plants to treat and manage diabetes and arthritis. All plant extracts were toxic to both bovine dermis and vero cell lines. S. lancea (T5) was found to be the most toxic plant extract. The observed good inhibitions of both alpha-amylase and alpha-glucosidase enzymes by plant extracts of B. mollis, S. petersiana, E. transvaalense and E. burkei validate their use in the traditional treatment of diabetes in the region to some extent. Aqueous extracts of these medicinal plants should also be investigated because water is the main solvent which is used by traditional practitioners in the preparation of their herbal medicines.

MSc (Botany) / Department of Botany / Understanding the uses of indigenous plants that are of economic importance to local communities is very much important in rural development strategies. The Marula (Sclerocarya birrea) Anacardiaceae family is widely used. More information on this tree species would enhance its value in agricultural landscapes, by helping farmers improve their livelihoods and ensuring environmental sustainability. Understanding how a community uses a resource and what influences the level of its use is crucial for developing a framework for its sustainable use based on local demands. Sclerocarya birrea is a species with multiple uses, which is recognized as commercially, medicinally and culturally important in Africa. Almost all parts of this species are useful. The study presented the findings of a survey of the indigenous knowledge, uses and management of S. birrea in Matshena village, Limpopo Province, South Africa. Different people of various ages were interviewed using a semi-structured questionnaire. Thirty percent of respondents indicated that they utilize the marula for beer and juice-making, the highest use category. In the sampled area the population of S. birrea is dominated by larger trees with no seedlings and juveniles. This is a sign of a population that will not be viable, since there are no younger individuals to replace the older trees when they die.

PhD (Microbiology) / Department of Microbiology / See the attached abstract below

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 (Microbiology) / Department of Microbiology / Background: The medicinal value of plants lies in some chemical substances that produce a definite physiological action in the human body. The secondary metabolites help the plants to survive hash conditions and could be used by humans as supplements of their health, as foods additives or for medicinal purposes. This bioactive compounds are not always beneficial to human beings, and some of this plants bioactive compounds can be toxic or genotoxic to human cells. This study used several methods to evaluate of phytochemical constituents and mutagenic properties of Coccinia rehmanni and Jatropha zeyheri plant extracts. Methodology: Methanol was used for extraction of the bioactive compounds from the two selected plants, filtered with Whatman filter paper and evaporated with rotary evaporator. The extracts were fractionated using open column chromatography. Chemical and TLC methods were used to determine phytochemicals of the study plants extracts and fractions. The plants extracts and fractions were tested against Vero cell lines in order to evaluate cytotoxicity and genotoxicity of the plants. NucRed and LTR Hoechst 33342 dyes were used for cytotoxicity and genotoxicity respectively. For the evaluation of cytotoxicity and genotoxicity Quantification of live and dead cells for the screening assay was performed using the ImageXpress Micro XLS Widefield Microscope and acquired images analyses using the MetaXpress software and Multi-Wavelength Cell Scoring Application Module. Antimutagenicity of plants extracts was observed using PARP universal colorimetric assay kit. Acquired data was transferred to an EXCEL spreadsheet and data was analyzed. Results and discussion: C. rehmanni (12.03%) yielded more extract than J. Zeyheri (8.20%). the two plants had different compound composition and were in different stages of maturity. The study revealed the domination of Terpenoids, Cardiac glycosides, Phenolic and tannis. With an exception of two fraction fractions all the fractions was found to be toxic to an extent were genotoxicity of such fraction could not be concluded. The reason for such extreme toxicity could be due to the influence of the retained alcohol during rotary evaporation. xvi | P a g e Conclusion: this study provides and add to existing knowledge on the phytochemicals mutagenicity and anti-mutagenicity of C. rehmanni and J. Zeyheri medicinal plants. The study serves as scientific proof that extensive use of this plant in traditional medicine for treatment of various ailments may lead to some irreversible damages. / NRF