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

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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