Thesis (MSc (Paraclinical Sciences))--University of Pretoria, 2008. / Includes bibliographical references. Also available in print format.

Thesis (PhD. (Phytomedicine)--University of Pretoria, 2006. / Includes bibliographical references. Available on the Internet via the World Wide Web.

Thesis (MSc (Paraclinical Sciences, Veterinary Science)--University of Pretoria, 2009. / Includes bibliographical references. Also available in print format.

Thesis (PhD (Phytomedicine, Paraclinical Sciences))--University of Pretoria, 2007. / Includes bibliographical references.

Thesis (MSc (Phytomedicine))--University of Pretoria, 2006. / Includes bibliographical references.

Thesis (MSc (Phytomedicine, Paraclinical Sciences))--University of Pretoria, 2007. / Includes bibliographical references.

Several investigations into the antimicrobial activity of members of the Combretaceae have been undertaken in recent years. Although the antibacterial properties of various species of Combretum, Terminalia and Pteleopsis have been investigated in depth, this is not the case for their antifungal properties. Due to the increasing importance of fungal infections the aim is to address this by focusing on antifungal activities of Combretaceae species. This was done by focusing on the following objectives: <ol> <li>Developing minimum inhibitory concentration (MIC) and bioautography procedures for fungi to be used in the laboratory in order to screen Combretum andTerminalia species for antifungal activity.</li> <li>Selecting three or four species for further investigation based on antifungal activity and availability.</li> <li>Isolating the antifungal compounds from one or more of the selected species.</li> <li>Determining the chemical structure and in vitro biological activity of the antifungal compound.</li> <li>Developing and applying a protocol and determining in vivo antifungal activity of Combretum and Terminalia extracts and isolated compounds in rats infected with different fungal pathogens.</li> </ol> Leaves of 24 Combretum and 6 Terminaliaspecies were collected in the Lowveld National Botanical Gardens (LNBG) in Nelspruit. After the dried plants were milled to a fine powder, they were extracted with hexane, dichloromethane, acetone and methanol. Chemical constituents of the 120 extracts were analyzed by thin layer chromatography (TLC). The TLC plates were developed with one of the three eluent systems developed in our laboratory that separate components of Combretaceae extracts well i.e.: Ethyl acetate/methanol/water (40:5.4:5) [EMW] (polar/neutral), Chloroform/ethyl acetate/formic acid (5:4:1) [CEF] (intermediate polarity/acidic) and Benzene/ethanol/ammonia hydroxide (90:10:1) [BEA] (non-polar/basic). To detect the separated compounds, vanillin-sulphuric acid-methanol was sprayed on the chromatograms and heated at 110 °C to optimal colour development. Methanol was the best extractant, extracting a greater quantity of plant material than any of the other solvents. There was similarity in the chemical composition of the non-polar compounds of extracts using extractants of varying polarity Qualitative analysis of antioxidant activity, the 2, 2,diphenyl-1-picrylhydrazyl (DPPH) assay on TLC plates was used as a screen test for the radical scavenging ability of the compounds present in the different 120 extracts. TLC-DPPH screening method indicated the presence of antioxidant compounds in some of the extracts tested, with C. woodii and C. hereroense showing the most prominent antioxidant activity. Methanol and acetone extracted the most antioxidant compounds based on DPPH TLC. In vitro</li> studies coupled with the phytochemical analysis confirm that the extracts had antioxidant activity. The solvent tolerance of the microorganisms was tested using the following solvents; DMSO, acetone, methanol and ethanol. In order to determine the maximum concentration at which different solvents would allow the test microorganisms to reach normal growth, different concentrations from 10 to 100% were used. Uninhibited growth was evaluated as no toxic effects of the solvent. Methanol and ethanol were found to be toxic. The growths of the fungi were not affected by DMSO and acetone concentrations up to 60%. A serial microdilution assay was used to determine the minimum inhibitory concentration (MIC) values for plant extracts using tetrazolium violet reduction as an indicator of growth. This method had previously been used only for antibacterial activities. To apply it to measuring antifungal activities, a slight modification was made to suit fungal growth conditions. The following fungal pathogens were used: yeasts (Candida albicans and Cryptococcus neoformans), thermally dimorphic fungi (Sporothrix schenckii) and moulds (Aspergillus fumigatus and Microsporum canis). To determine MIC values, growth was checked after 24 and 48 hours to determine the end point. The MIC values of most of the extracts were in the order of 0.08 mg/ml and some had values as low as 0.02 – 0.04 mg/ml after 24 hours incubation. TLC plates were loaded with 100 ㎍ (5 ㎕ of 20 mg/ml) of each of the extracts. The prepared plates were developed in the three different mobile systems used: CEF, BEA and EMW. The chromatograms were dried for a week at room temperature under a stream of air to remove the remaining solvent. The TLC plates developed were inoculated with a fine spray of the concentrated suspension containing approximately 109 organisms per ml of actively growing fungi e.g. conidia for A. fumigatus and yeast cells (blastocysts) for the other fungi in a Biosafety Class II cabinet (Labotec, SA) cupboard. The plates were sprayed until they were just wet, and after drying were sprayed with a 2 mg/ml solution of INT. White areas indicate where reduction of INT to the coloured formazan did not take place due to the presence of compounds that inhibited the growth of tested fungi. During this study we experienced a number of difficulties. Firstly I found that preparing cultures some days before spraying them makes it difficult to get good results, possibly due to quick mycelial overgrowth and blockage of the spray gun with mycelia. The new method was developed. This procedure led to reduced overgrowth of the mycelia. In the study of biologically active compounds from extracts, it was indicated that the extracts had antifungal compounds. Fractionation and bioassay-guided isolation of the antifungal compounds was undertaken on the crude extracts of C. nelsonii, based on very low MIC’s of the crude extracts on all tested pathogens, it had several compound which are active against all pathogens, lastly it is one of the Combretum species which have never being worked on. Antifungal compound was successfully isolated from the leaves of C. nelsonii. The structure was elucidated. After structure elucidation bioassays of isolated active compounds was done to confirm that the compound isolated is the one expected, and how active the compound is, on its own. The compound was very active against all tested pathogens. Cytotoxicity of the acetone extracts of C. imberbe, C. nelsonii, C. albopunctactum and T. sericea were evaluated using Brine shrimp (Artemia salina assay and tetrazolium-based colorimetric assay (MTT assay) on Vero monkey kidney cells. These four extracts were chosen because of the good in vitro antifungal activity of crude extracts and there was intention of using them in in vivo studies in animal models. The results on brine shrimps indicated that the four leaf extracts have LC50 values above 20 ㎍/ml, the recommended cut-off point for detecting cytotoxic activity. Using MTT assay it was found that the four extracts did not suppress mitochondrial respiration in monkey kidney cells. Only C. imberbe was closer to the cut-off value (200 µg/ml), which was used by other authors. In searching for cytotoxic activity to the criteria of the American National Cancer Institute, the LC50 limit to consider a crude extract promising for further purification is lower than 30 µg/ml. In vivo antifungal activity was investigated on the wound irritancy and efficacy of the four most promising, Combretum nelsonii, Combretum imberbe, Combretum albopunctactum and Terminalia sericea extracts applied topically to skin wounds in fungal infected skin wound of rat model. Wound irritancy and wound healing were evaluated by macroscopical, physical and histological methods. Aspects evaluated include wound healing, erythema, exudate formation and possible toxic effects of the extracts. Twenty rats were used in two pilot studies (Exploratory studies and Infection with different pathogens). During the pilot studies rats were not irritated by treatment of infection. The wound healed within three weeks. Only one rat was terminated due to weight loss and it was found that nasal discharge was due to external factors, which were not related to the experiment. The clinical treatment of skin infected with pathogens continues to be a major problem especially in immuno-compromised patients. Therapeutic agents selected for the treatment of infected wounds had ideally shown antifungal activity on in vitro studies. I investigated whether these agents would improve phases of wound healing without producing deleterious side effects. All the parameters showed that the crude extracts and amphotericin B were effective in decreasing formation of the exudate, increasing crust formation and that they have antifungal activities used in in vivo studies. Acetone extract of leaves of C. nelsonii, C. albopunctactum, C. imberbe and T. sericea possessed remarkable growth inhibitory activities against fungal pathogens. Acetone extracts of leaves and isolated compound demonstrated wound healing properties comparable with that of antibiotic powder (amphotericin B). The results of this study in general indicate that the Terminalia and Combretum species possess substantial antifungal properties. This explains the use of these plants in folk medicine for the treatment of various diseases related to fungal infections. / Thesis (PhD (Phytomedicine))--University of Pretoria, 2006. / Paraclinical Sciences / unrestricted

Thesis (PhD (Phytomedicine Programme) (Paravet. Studies))--University of Pretoria, 2008. / Includes bibliographical references. Also available in print format.

This study was carried out to determine alternative approaches used by resource-limited farmers to enhance fertility and in the treatment of reproduction ailments of goats in Mount Frere area, Eastern Cape Province of South Africa. Furthermore, information on farmer perceptions pertaining to these practices was also gathered. A questionnaire survey was conducted to determine materials used by resource-limited farmers to enhance fertility and treat reproductive ailments. The survey revealed 10 plant species that were commonly used for reproductive health in goats. Elephantorrhizza elephantina (37.1%) and Rhoicissus tomentosa (25.7%) were the most frequently cited plant species. Elephantorrhiza elephantina and R. tomentosa recorded the highest Fidelity Level (FL) values of 92.86% and 75% respectively, for their use in the treatment of reproductive ailments and these were the plants on which further studies were conducted. The study also revealed that farmers had different perceptions on the effectiveness of the traditional medicines. An in-vivo assessment was done to determine efficacy of E. elephantina and R. tomentosa using serum estradiol and progesterone profiles. The highest (P<0.05) serum estradiol and progesterone concentration was observed in animals administered with aqueous extracts of E. elephantina and R. tomentosa mixture while the least (P<0.05) concentrations were observed in the negative control group. Does administered with a mixture of aqueous E. elephantina and R. tomentosa extract had the highest (P<0.05) body weight (65 kg) as well as body condition scores, while the negative control which were administered with distilled water had the least body weights (54 kg) and body condition scores. Does that were administered with a mixture of E.elephantina and R. tomentosa were all pregnant with twins (100%), confirmed by a doppler ultrasound scan.

Ticks are the most common external parasites of economic importance in the livestock farming sector of Southern Africa. They are vectors of tick-borne diseases and are commonly controlled using conventional acaricides, which are expensive and not readily available to resource-limited farmers. As a result many resource-limited farmers resort to use of alternative materials to control ticks on their livestock. Tick resistance to acaricide is an increasing problem in Southern Africa and poses a real economic threat to livestock and veterinary pharmaceutical industries. The objective of this study was to document and validate the ethno-veterinary remedies used as alternative methods of controlling cattle ticks by resource-limited farmers. A questionnaire survey was conducted in May 2007, at Qolora by-Sea and Nontshinga in Centane district, to determine tick control methods used by resource limited farmers in the Amathole District Municipality of the Eastern Cape Province, South Africa. Furthermore, in vitro and in vivo experiments were done to validate the acaricidal properties of ethnoveterinary remedies used by resource-limited farmers. Ticks were reported to be a major problem, transmitting diseases like anaplasmosis (89.8%), babesiosis (55.9%) and ehrlichiosis (16.9%), they also cause wounds that predispose cattle to screwworm infestation, tick worry and teat damage. All farmers used acaricides and dipping tanks provided by the government to control ticks, as the main tick control method. However, the majority (94.9%) were of the opinion that the dip wash was not effective in controlling ticks. As a result, farmers complemented the government dipping service with use of own initiatives like spraying with conventional acaricides (22%), household disinfectants such as Jeyes fluid (18.6%), used engine oil (10.2%), chickens feeding on ticks (5.1%), manual iv removal (5.1%), and pouricides (1.7%). In addition, some farmers also use plants (6.8%), mainly the leaf of Aloe ferox and the bark of Ptaeroxylon obliquum. The in vitro repellency models showed Jeyes fluid (76.8 and 100% concentrations) and P. obliquum (40%) had repellent properties that lasted 6 hours and 40 minutes respectively while that of, Tabard (35% diethyltoluamide) positive control, lasted for 4 hours when applied against nymphal stages of Rhipicephalus sanguineus ticks. In the contact bio-assay, undiluted used engine oil, T. minuta oil (50%), Ektoban® (Cymiazol 17.5% and cypermethrin 2.5%) and Jeyes fluid (76.8%) caused higher tick mortality of more than 86%. Other materials (A. ferox, Lantana camara and Tagetes minuta) had no repellence and acaricidal properties. In the in vivo study, Jeyes fluid at a concentration of 76.8% and used engine oil displayed efficacy of 71 and 76.4% respectively which was similar to that of the positive control Ektoban® at 97.8% tick load reduction. Among plant materials L. camara at 40% concentrations had an efficacy of 57% while A. ferox, P. obliquum and T. minuta were not effective at all. The remedies used by resource-limited farmers to control ticks vary in efficacy. Their ability to reduce tick load on cattle appears to be promising and a welcome development as their use could probably reduce tick burdens in cattle with less expenditure. However, further investigations need to be done before they are recommended for use. Despite being effective some of the materials have potential toxic effects in animals and also cause environmental contamination and I recommend used engine oil, Jeyes fluid and L. camara for further testing as they showed some efficacy compared to other remedies.