Abstract Few in vitro screening assays for biological activities of plant extracts consider the potential effect of the gastrointestinal system on orally consumed plant extracts. Crude water and methanol extracts of Tarchonanthus camphoratus (wild camphor) and Agathosma betulina (‘buchu’) were prepared and exposed to simulated gastric fluid and simulated intestinal fluid during dissolution studies to address this aspect. The crude extracts and resulting simulated gastric fluid and simulated intestinal fluid products were screened for antimicrobial activity against Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922) and Proteus vulgaris (ATCC 33420). The T. camphoratus crude extract exhibited antimicrobial activity which was reduced after exposure to simulated gastric fluid. After exposure to simulated intestinal fluid no antimicrobial activity was detected, which suggests chemical alteration or degradation of the active compounds. For A. betulina, the crude water extract and simulated gastric fluid product exhibited no antimicrobial activity, while the simulated intestinal fluid product exhibited antimicrobial activity. This suggests activation of antimicrobial constituents during exposure to simulated intestinal fluid. The chemical composition profiles of the crude extracts and products were determined by means of liquid chromatography coupled to an ultraviolet detector (LC-UV) and a mass spectrometer (LC-MS) to qualitatively assess the effect of exposure to simulated gastrointestinal conditions on the chemical composition of the extracts. In many cases, the peak area of compounds decreased after exposure to simulated gastric fluid and simulated intestinal fluid, while the peak area of other compounds increased. Thus, it can be deduced that the antimicrobial activity and chemical composition was altered after exposure to intestinal conditions during dissolution studies.

South Africa has offered the world two indigenous aromatic plants from which commercially important natural products have been developed: Pelargonium graveolens (and its hybrids) the source of geranium oil and Agathosma betulina, from which ‘Buchu’ oil is produced. Despite the historical use of ‘Buchu’ and the commercial interest developed around this coveted indigenous resource the (limited) research has not been coherently assembled. This overview aims to unite aspects on the botany, traditional and modern day uses, chemistry and pharmacological data on ‘Buchu’ which is undeniably one of South Africa’s most renowned botanical assets.

Magister Scientiae Dentium - MSc(Dent) / Candida albicans is a clinical fungal isolate that is most frequently isolated from different host niches, and is implicated in the pathogenesis of several fungal infections, including oral candidiasis. The pathogenesis and antifungal resistance mechanisms of Candida species are complex and involve several pathways and genes. Oral candidiasis incidence rates are rapidly increasing, and the increase in resistance to conventional antifungals has led to the need to develop innocuous and more efficacious treatment modalities. The purpose of this study was to explore a single pot process for phytosynthesis of zinc oxide nanoparticles (GZnO NPs) and to assess their antifungal potential.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: The Mountain Fynbos biome, a division of the Cape Floristic Region (CFR), is home to round-leafed Buchu [Agathosma betulina (Berg.) Pillans], one of South Africa’s best-known endangered herbal medicinal plants. Agathosma betulina is renowned as a traditional additive to brandy or tea, which is used for the treatment of a myriad of ailments. In its natural habitat, A. betulina thrives on mountain slopes in acid and highly leached gravelly soils, with a low base saturation and low concentrations of organic matter. To adapt to such adverse conditions, these plants have formed mutualistic symbioses with arbuscular mycorrhizal (AM) fungi. In this study, the effect of indigenous AM taxa on the physiology of A. betulina is investigated. In addition, the AM taxa responsible for these physiological responses in the plant were identified using morphological and molecular techniques. Agathosma betulina was grown under glasshouse conditions in its native rhizosphere soil containing a mixed population of AM fungi. Control plants, grown in the absence of AM fungi, were included in the experimentation. In a time-course study, relative growth rate (RGR), phosphorus (P)-uptake, P utilization cost, and carbon (C)-economy of the AM symbiosis were calculated. The data showed that the initial stages of growth were characterized by a progressive increase in AM colonization. This resulted in an enhanced P-uptake in relation to non-AM plants once the symbiosis was established. Consequently, the lower P utilization cost in AM plants indicated that these plants were more efficient in acquiring P than non-AM plants. When colonization levels peaked, AM plants had consistently higher growth respiration. This indicated that the symbiosis was resulting in a C-cost to the host plant, characterized by a lower RGR in AM plants compared to non-AM plants. Arbuscular mycorrhizal colonization decreased with increasing plant age that coincided with a decline in P-uptake and growth respiration, along with increases in RGR to a level equal to non-AM plants. Consequently, the AM benefit was only observed during the initial stages of growth. In order to identify the AM fungi in planta, morphological and molecular techniques were employed, which indicated colonization by AM fungi belonging to the genera Acaulospora and Glomus. Phylogenetic analyses of a dataset containing aligned 5.8S ribosomal RNA gene sequences from all families within the Glomeromycota, including sequences obtained during the study, supported the above mentioned identification. / AFRIKAANSE OPSOMMING: Die Fynbos bergbioom, ‘n onderafdeling van die Kaapse Floristiese Streek, huisves rondeblaar Boegoe [Agathosma betulina (Berg.) Pillans], een van Suid Afrika se bekendste bedreigde medisinale plante. Agathosma betulina is bekend vir sy gebruik as tinktuur vir die behandeling van verskeie kwale. Die plant kom voor in bergagtige streke, in suur en mineraal-arm grond, met ‘n lae organiese inhoud. Gevolglik, om aan te pas by hierdie ongunstige kondisies, vorm die plante simbiotiese assosiasies met blaasagtige, struikvormige mikorrisa (BSM). In die huidige studie is die effek van hierdie BSM op die fisiologie van A. betulina ondersoek. Die identiteit van die BSM is ook gevolglik met morfologiese en molekulêre identifikasie tegnieke bepaal. Agathosma betulina plante is onder glashuis kondisies in hul natuurlike grond gekweek, wat ‘n natuurlike populasie van BSM bevat het. Kontroles is ook in die eksperiment ingesluit en hierdie stel plante is met geen BSM geïnokuleer nie. Gevolglik is die relatiewe groeitempo, fosfor opname, fosfor verbuikerskoste asook die koolstof ekonomie van die plante bereken. Die data het getoon dat die eerste groeifase gekarakteriseer is deur toenames in BSM kolonisasie vlakke. Dit het tot ‘n hoër fosfor opname in BSM geïnokuleerde plante gelei. Die laer fosfor verbuikerskoste gedurende hierdie fase het aangedui dat die plante wat geïnokuleer is met BSM oor beter meganismes beskik het om fosfor uit die grond te bekom. Toe BSM kolonisasie vlakke gepiek het, was groei respirasie hoër in BSM geïnokuleerde plante as in die kontroles. Dit het aangedui dat die BSM kolonisasie van plante tot hoër koolstof kostes vir hierdie plante gelei het, wat weerspieël is in die laer groeitempo van die BSM geïnokuleerde plante. Die BSM kolonisasie vlakke het gedaal met toenemende ouderdom van hul gasheer plante, wat gekarakteriseer is deur ‘n laer opname van fosfor en laer groei respirasie, tesame met ‘n toename in relatiewe groeitempo tot vlakke soortgelyk aan die van die kontrole plante. Die BSM voordele vir die plant is dus net gedurende die eerste groeifase waargeneem. Die BSM wat verantwoordelik is vir hierdie fisiologiese veranderinge is gevolglik geïdentifiseer met behulp van morfologiese en molekulêre tegnieke en dit is gevind dat BSM wat behoort tot die genera Acaulospora en Glomus binne hierdie plante voorkom. Filogenetiese analise gegrond op opgelynde 5.8S ribosomale RNA geen volgordes afkomstig van al die families binne Glomeromycota asook volgordes gevind in die studie, het die bogenoemde identifikasie gestaaf.

Thesis (MScAgric (Agronomy)--University of Stellenbosch, 2007. / An increase in the demand of buchu (Agathosma betulina) oil has lead to an increase in the commercial cultivation of buchu in fields and also in hydroponic systems. A nutrient solution for hydroponically grown buchu is still required to ensure optimal growth and yield. ASNAPP (Agribusiness in Sustainable Natural African Plant Products) South Africa has done some trials to achieve optimal EC and pH in the nutrient solution. Phosphate concentrations in the nutrient solution might play a significant role due to reports by a variety of researchers on the sensitivity of Protea plants to phosphate. Buchu and Proteas are both part of the Fynbos biome and are found in regions with similar soil (sandy soils with a low pH and mineral contents) and climatic conditions. Two separate experiments were conducted to determine the effect of increasing phosphate concentrations (ranging from 0.00 to 1.40 me L-1) in the nutrient solution on buchu growth. The first experiment was done in a plastic covered structure with a pad and fan and the objective of this trial was to determine the effect of increasing phosphate concentrations in the nutrient solution on the general growth, biomass production, oil composition, mortality rate and chemical composition of the buchu plants. The second experiment was done in a glasshouse with mechanical temperature control and the aim of this trial was to determine the response of buchu to increasing concentrations of P at two different root temperatures. A chemical analysis of the plants was done and the general growth, yield and root mass were recorded to determine the response of buchu plants to the phosphate and temperature treatments. In the greenhouse experiment an optimum growth and yield response of buchu plants was found at a phosphate concentration of 0.7 me L-1 in the nutrient solution. Phosphate concentrations lower or higher than 0.7 me L-1 lead to a decrease in growth and yield. An increase in the phosphate concentration in the nutrient solution lead to a general increase in N, P, K, Ca, Mg and B content in the buchu plants and a decrease in Fe content. The mortality rate of the buchu plants increased with an increase in the phosphate concentration from 0.0 to 1.4 me L-1 in the nutrient solution. The phosphate concentration in the nutrient solution only made a significant difference on one major component of the buchu oil which was Ψ-Diosphenol, but no general trend with Ψ-Diosphenol content and P concentration could be found and the significant difference in Ψ-Diosphenol observed in this trial may only have been due to genetic variation between the plants. The effect of the different root temperatures in the glasshouse experiment was very clear. The buchu plants grown at the high root temperature (20°C) produced a higher yield and better overall growth than the plants grown at lower (10°C) temperatures. The buchu plants grown at 20°C had a significantly higher N, K, Na and B content than plants grown at 10°C. Buchu plants grown at 10°C showed no significant response in terms of growth and yield to the phosphate concentration in the nutrient solution, but plants grown at 20°C exhibited growth and yield peaks at phosphate concentrations of 0.35 and 1.4 me L-1. The peak observed in the plants growth at high phosphate concentrations is unexplainable and can possibly be ascribed to the limitation of the plants per experimental unit and/or amount of replications. The increase in P concentration in the nutrient solution caused a general increase in N, P and K content in the buchu plants. A significant interaction between the phosphate concentration and root temperature was observed for the P, Mn en Zn contents of the plants which meant that the buchu plants respond differently towards phosphate concentrations at different root temperatures.

Thesis (MScAgric (Agronomy)--University of Stellenbosch, 2007. / The Cape Floristic Region is a highly distinctive phytogeographical unit which is recognized as a floral Kingdom on its own. Buchu (Agathosma betulina) plants fall under this important Kingdom. Buchu is one of the traditional medicinal plants originating in the Western Cape province of South Africa and the essential oil derived from the leaves is exported in large volumes. Due to high demand, under supply, restrictions of wild harvesting and high prices for Buchu essential oil, growers have started to introduce and commercialize this species as a crop. This commercialization of Buchu necessitated agronomic research to optimize production techniques. The objective of this study was to determine the optimum pH range for the cultivation of high yielding Buchu with acceptable essential oil quality under controlled conditions and compare this with the conditions in the natural habitat. Plant, soil and climatic data were gathered from eleven sites in the natural habitat of Buchu (A. betulina) in the Cederberg Mountains. At all sites most rainfall occurred from May to September, while high temperatures were recorded in summer. Soil analyses indicated low levels of nutrients and low soil pH, ranging from 3.7 to 5.3 at all the sites studied. Low levels of nutrients were also obtained from foliar analysis collected from plants at each of the different sites. Chemical analyses of the essential oil indicated that the plants were from a high quality diosphenol chemotype. In the greenhouse experiment, five different pH levels (pH 33.99, 4-4.99, 5-5.99, 6-6.99 and 7-7.99) were evaluated to determine the effect on growth, yield and quality of A. betulina. Complete nutrient solutions were used to irrigate the plants grown in pots filled with a sand and coco peat mixture. Although the plants subjected to the pH treatment of 4-4.99 tended to have the highest growth rate and yield, this did not differ significantly (P=0.05) from plants subjected to pH values between 3 and 6.99. In contrast, the pH 7-7.99 treatment lead to reduced growth and lower vegetative yields. Levels of nutrients obtained from the leaf mineral analysis differed significantly with different pH treatments. High pH levels resulted in high nitrogen, phosphorus, sodium, manganese and boron contents, but lower contents of copper. Nitrogen, phosphorus, calcium and zinc were higher than those recorded for plants from their natural habitat, but still within the norm reported for most plants. Levels of manganese, sodium, magnesium and copper were found to be more or less similar to the values obtained in plants from the natural habitat. No significant differences were found in essential oil quality in response to the pH treatments. However, high pulegone levels (10.8 to 13.2 %) were obtained from all the treatments in the greenhouse experiment. The high levels of this essential oil constituent could have a negative effect on the marketability of the oil and this aspect may need some attention in future studies.

Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Horticultural Sciences in the Faculty of Applied Sciences at the CAPE PENINSULA UNIVERSITY OF TECHNOLOGY Supervisor: Dr L Kambizi Co-supervisor: Dr NP Makunga Cape Town December 2013 / Agathosma betulina (Berg.) Pillans, previously known as Barosma betulina, is a member of the Rutaceae family, and indigenous to the fynbos botanical biome of the Western Cape of South Africa. It is commonly known as buchu. Extracts as well as powdered leaves have traditionally been used for the treatment of various ailments. The increase in the international demand for A. betulina for health as well as food and beverage benefits, have raised concerns over exploitation of wild populations and the lack of horticultural information necessitates this study to evaluate the propagation of this economical important species. The main objective of this study was to establish a simple and highly productive micropropagation protocol for A. betulina through experimenting with nodal explants. Testing of the effect of various treatments (physical scarification, chemical scarification, GA, stratification, smoke and combinations thereof) on the in vitro germination of A. betulina seeds was done to elucidate the factors which control seed germination. The study revealed that the physical scarification and smoke-induced germination had a significant effect on germination percentages. In terms of germination rate, the radical generally started to appear after approximately 10 days in the physical scarification with smoke treatment. Initial decontamination methods with the exposure of various concentrations of NaOCl gave fatal results, however 1.5% NaOCl had more phenolic reactions rather than fungal or bacterial contamination. Interestingly, contamination rates of explants were influenced by the stage of maturity of the explant material. This plant material was used to test different strengths of regeneration media, to ensure that the explants receive ample nutrients. Results made exhibited that ½ MS was the best strength for growing A. betulina nodal explants. Compared comparison between in vitro derived explants and ex vitro collected explants showed that the ex vitro derived explants had significant results, but the explants lost vigour soon after the initial exponential growth leading to the explants dying off. Furthermore, ex vitro decontaminated plant material was not economically viable to continue with. Seedlings derived from germinated seeds appeared to be the preferred method of propagation as this spent the least time in culture and produced a stable plant with an established root system, which is essential during the hardening off process after in vitro growth. When exposing nodal explants to phytohormone 2,4-D it responds best to dosages 0.5mg Lˉ¹ and 1mg Lˉ¹. Phytohormone BA was very effective in producing soft friable callus. The best results were shown when 0.5mg Lˉ¹ BA was applied to ½ MS media. For both shoot length and multiple shoot production, a combination of phytohormones BA-NAA (1: 0.5mgLˉ¹) had the most significant results. Interestingly, a higher phytohormone concentration of NAA is necessary to develop multiple adventitious roots. The effect of 3mg Lˉ¹ was significant in that it resulted in multiple adventitious roots, but fewer calli was observed in this treatment. Micropropagation becomes valuable as little attention between subcultures is needed; making it less labour intensive compared to conventional nursery propagation systems where weeding watering and spraying of plants are labour intensive. In the traditional world of medicine, more so in Southern Africa, extracts are prepared by adding boiling water to the plant material; however commercial ethanol is used as an extractant. Establishment of the essential oil quality of the in vitro cultures post exposure to various treatments was done. Analysis of essential oils from A. betulina resulted in the identification of twenty one compounds. The results showed qualitative as well as quantitative differences amongst the samples used in the study. The highest relative concentration of limonene was observed in the callus of nodal explants after it was exposed to 0.5mg lˉ¹ NAA. No pulegone was found in this treatment making it ideal for limonene production. This suggests that liquid culture with the same treatment may produce more calli making it ideal for the production of limonene.