The Orchidaceae makes up the largest and most diverse family of flowering plants. Orchids are popular, often expensive ornamentals, with a broad range of ethnobotanical applications. There is very limited documented information on South African medicinal orchid species; no formal pharmacopoeia outlining ethnobotanical uses; and ethnobotanical and distribution records are either scarce or inconsistent and plant populations are becoming gradually smaller. There have been significant developments in medicinal orchid research worldwide with medicinal use and corresponding pharmacological and phytochemical properties being extensively investigated. It is evident from the literature that there is no pharmacological research on South African medicinal orchids; hence the need to explore biological activity and chemical composition of South African medicinal orchid species. The ethnobotanical approach used to select the orchid species for pharmacological and phytochemical research elsewhere, yielded valuable biological compounds. Thus, a similar approach was applied to South African medicinal orchids. There are approximately 20 000 species and 796 genera of orchids distributed across the world. In southern Africa, orchids are widely represented with 55 genera and 494 species. Approximately 75% are endemic to this region. As part of the current investigation a review of available ethnobotanical literature on South African medicinal orchids was prepared. The review revealed that an estimated 49 indigenous orchid species from 20 orchid genera are currently being informally traded and used in South African traditional medicine. They are used primarily for medicinal and cultural purposes, especially by the Zulu community in South Africa. Medicinal uses of orchid species include: treatment of inflammatory, intestinal, neurological and reproductive disorders and emetics are used to cause emesis. Non-medicinal uses of orchid species include: love, fertility, protective and lethal charms. Based on their ethnobotanical uses and endemism, South African orchids were considered to be one of the untapped sources of bioactive compounds that needed to be researched. The current investigation addressed the broader aims of medicinal plant research by determining the efficacy, safety and chemical profile of seven indigenous orchid species used in South African traditional medicine and practices. The biological and toxic effects of orchid plant extracts were assessed using established pharmacological bioassays. The phytochemical evaluation of the seven orchid plant extracts provided insight into the classes of chemical compounds present and their possible role in the observed biological activities. The potential of plant extracts from seven orchid species used in South African traditional medicine, as sources of natural bioactive products, are discussed. The current investigation determined the biological activity and chemical profile of seven orchid species commonly traded in KwaZulu-Natal herbal markets: Ansellia africana Lindl., Bulbophyllum scaberulum (Rolfe) Bolus, Cyrtorchis arcuata (Lindl.) Schltr., Eulophia hereroensis Schltr., Eulophia petersii (Rchb.f.) Rchb.f., Polystachya pubescens (Lindl.) Rchb.f. and Tridactyle tridentata (Harv.) Schltr. Well established in vitro micro-dilution bioassays were used to determine the antibacterial, antifungal, anthelmintic activities of crude orchid extracts. A minimum inhibitory and/or lethal effect of organic and aqueous crude orchid extracts was observed against Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Candida albicans and Caenorhabditis elegans. Tridactyle tridentata aqueous root extract produced the most effective antibacterial activity against S. aureus (0.049 mg/ml). All T. tridentata organic root extracts produced significant inhibitory activities against B. subtilis and S. aureus. Eulophia petersii DCM pseudobulb extracts significantly inhibited all bacterial strains tested (0.39 mg/ml against S. aureus and 0.78 mg/ml against B. subtilis, E. coli, and K. pneumoniae). Eulophia hereroensis 80% EtOH root extract was the only other extract to exhibit significant inhibitory effects against K. pneumoniae (0.65 mg/ml). After 48 h C. albicans was most susceptible to P. pubescens aqueous pseudobulb extract (0.0816 mg/ml). Eulophia petersii DCM pseudobulb extract however, exhibited significant activity against C. albicans (0.65 mg/ml) over 72 h. Cyrtorchis arcuata leaf and root extracts were the most effective anthelmintic extracts with MLCs of 0.041 mg/ml for 80% EtOH leaf and root extracts; 0.049 mg/ml for aqueous leaf extracts and 0.78 mg/ml for aqueous and DCM root extracts. Caenorhabditis elegans was most susceptible to all A. africana and T. tridentata organic root extracts. A similar significant effect was observed for all E. petersii organic pseudobulb extracts, DCM extracts and organic root extracts of B. scaberulum. Only the DCM tuber and root extracts of E. hereroensis exhibited lethal effects on C. elegans. All of the P. pubescens extracts showed poor anthelmintic activity. Similarly, in vitro enzyme based cyclooxygenase (COX) 1 and 2 and acetylcholinesterase (AChE) inhibitory bioassays, revealed significant inhibition of COX-1, COX-2 and AChE enzymes by crude organic and certain aqueous orchid extracts. Out of a total of 53 evaluated extracts, 21 and 13 extracts exhibited significant anti-inflammatory activity in the COX-1 and COX-2 assays respectively. The DCM tuber extract of E. hereroensis was the only extract to significantly inhibit both COX enzymes, 100.02±0.11% and 87.97±8.38% respectively. All B. scaberulum root extracts (DCM, EtOH and water) exhibited COX-2 selective inhibitory activity (100.06±0.01, 93.31±2.33 and 58.09±3.25%). Overall, the DCM root extract of A. africana was found to be the most potent extract (EC50 0.25±0.10 mg/ml). The 80% EtOH root extract of B. scaberulum was the most potent in the COX-2 assay (EC50 0.44±0.32 mg/ml). Generally the root extracts exhibited greater AChE inhibitory activity; where the most active extract was B. scaberulum DCM root extract (EC50 0.02±0.00 mg/ml). All aqueous extracts, except that of A. africana roots and B. scaberulum pseudobulbs, showed poor or no COX-1 and COX-2 inhibition. The antioxidant capacity of crude orchid extracts was determined using: hydrogen atom transfer (HAT) (β-carotene/linoleic acid assay) and single electron transfer (SET) (2,2‟-diphenylpicrylhydrazyl (DPPH) free radical scavenging assay and ferric reducing antioxidant power (FRAP) assay) reaction-based assays. Potent antioxidant effects were observed for certain crude methanolic orchid extracts. Generally, there was a dose-dependent change in radical scavenging activities of crude extracts from which EC50 values were determined. The root extracts of all species, except that of E. petersii, had consistently more effective radical scavenging activity than that of other plant parts within each species. The pseudobulb extract of E. petersii, was the most potent extract (EC50 1.32±0.86 mg/ml). In the β-carotene-linoleic acid assay, based on the oxidation rate ratio (ORR), the leaf extract of T. tridentata and the root extracts of C. arcuata and E. hereroensis exhibited the best antioxidant effects (0.02, 0.023 and -0.15 respectively). Similarly, the average antioxidant activity (%ANT) of these samples was greater than that of BHT (95.88±6.90%) and all other samples. Bulbophyllum scaberulum leaf, pseudobulb and root extracts, E. petersii pseudobulb extract and T. tridentata root extract also exhibited a greater capacity to prevent β-carotene oxidation when compared to BHT. All crude orchid extracts tested demonstrated a general dose-dependent response in the ferric reducing power assay. The reducing power of ascorbic acid (0.08 mM) and BHT (0.05 mM), as measured as absorbance, was 1.12±0.12 and 0.73±0.08 respectively. At 6.25 mg/ml, A. africana root and E. petersii pseudobulb extracts were the most effective in reducing power activity. The short-term bacterial reverse mutation Ames Salmonella/microsome mutagenicity (ASMM) assay, which makes use of mutant histidine-dependent Salmonella typhimurium strains, was used to determine the mutagenicity and toxicity of crude orchid extracts. In the presence of a mutagen S. typhimurium TA98 strain detects frameshift events while the TA100 and TA102 strains detect base-pair substitutions. In the absence of metabolic activation, mutagenic extracts were observed against the TA98 strain only. All A. africana DCM leaf and stem extracts tested, the DCM root extract (0.5, 0.05 mg/ml) and EtOH leaf, stem and root extracts at 5 mg/ml exhibited mutagenic effects. The EtOH root extracts (5, 0.5 mg/ml) of B. scaberulum exhibited mutagenic indices (MI) comparable to that of 4NQO (17.00 and 13.00, respectively). Eulophia petersii PE pseudobulb extract demonstrated mutagenic potential at 5 mg/ml. The ethanolic root extracts of T. tridentata showed mutagenic effects at 5 and 0.5 mg/ml. The mutagenicity index (MI) with metabolic activation (S9) was determined using only the TA98 strain; where no mutagenic effects were observed. In the phytochemical evaluation of crude methanol orchid extracts, the Folin-Ciocalteu assay for total phenolics, butanol-HCl assay for condensed tannins, rhodanine assay for gallotannins and vanillin assay for flavonoids revealed a quantitative chemical profile of the tested samples. The correlation between observed biological effects and chemical compounds present was found to be generally significant. The significant antimicrobial, anthelmintic, anti-inflammatory and antioxidant activity of E. petersii pseudobulb extracts and E. hereroensis tuber and root extracts may be attributed to their high total phenolic content. Alternatively, the significant levels of gallotannin content in E. hereroensis may have contributed to the bioactivity. The flavonoid content of B. scaberulum and T. tridentata may explain the potent activity observed in the anti-inflammatory, antioxidant and acetylcholinesterase inhibitory assays; while the flavonoid content C. arcuata may have contributed to the potent anthelmintic and antioxidant activities. The significantly higher levels of gallotannin content may explain the significant anti-inflammatory and anthelmintic activity of A. africana. A number of biologically active compounds have been isolated from certain Orchidaceae species around the world on the basis of their traditional medicinal uses. The traditional uses of these orchid species were scientifically validated. No pharmacological research has been previously conducted on South African medicinal orchids; therefore the current investigation has produced novel findings on the efficacy and safety of these orchid species and promotes the continued research of medicinal orchids in South Africa. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Magister Pharmaceuticae - MPharm / The use of traditional medicine through the use of medicinal plants in Africa and especially in South Africa has long been considered an important characteristic of people's daily lives and socio-cultural heritage. Cotyledon Orbiculata is among the medicinal plants that are used by South African traditional practitioners for the treatment of epilepsy and painful conditions such as corns, warts, toothache, earache, boils and various other ailments. However, the claim of therapeutic successes of medicinal plants by traditional medicine practitioners are hardly subjected to scientific scrutiny. This study therefore, investigated the anti-epileptic property of Cotyledon Orbiculata by studying the effects of the methanol extract of the plant against chemically induced seizures by pentylenetetrazole, picrotoxin, bicuculline and N-methyl-DL-aspartic acid in mice. The study also investigated the analgestic effects of Cotyledon Orbiculata by studying the effect of the plant extract on pain induced by acetic acid and hot plate thermal stimulation. / South Africa

The use of traditional medicine through the use of medicinal plants in Africa and especially in South Africa has long been considered an important characteristic of people's daily lives and socio-cultural heritage. Cotyledon Orbiculata is among the medicinal plants that are used by South African traditional practitioners for the treatment of epilepsy and painful conditions such as corns, warts, toothache, earache, boils and various other ailments. However, the claim of therapeutic successes of medicinal plants by traditional medicine practitioners are hardly subjected to scientific scrutiny. This study therefore, investigated the anti-epileptic property of Cotyledon Orbiculata by studying the effects of the methanol extract of the plant against chemically induced seizures by pentylenetetrazole, picrotoxin, bicuculline and N-methyl-DL-aspartic acid in mice. The study also investigated the analgestic effects of Cotyledon Orbiculata by studying the effect of the plant extract on pain induced by acetic acid and hot plate thermal stimulation.

Medicinal bulbs form part of the diversified flora in South Africa. The plants are used extensively in South African traditional medicine in the treatment of various ailments. Due to the ever-increasing demand and the unrestricted collection of medicinal plants from the wild, many of these slow growing bulbous plant species are driven into over-exploitation and extinction. The main parts collected for use are the underground bulbs, leading to the destructive harvesting of the whole plant. This form of plant harvesting poses threats to the long term sustainability of these plant resources from their natural habitats. Sustainable harvesting of these plants should be within the limits of their capacity for self-renewal. However, this seldom occurs with the often inconsiderate medicinal plant gatherers. Conservation of these plants is therefore necessary. A strategy that would take into consideration the sustainable harvesting and perhaps simultaneously provide similar medicinal benefits, would be the substitution of bulbs with leaves of the same plant. This study was aimed at evaluating the seasonal pharmacological and phytochemical properties in bulbs/corms and leaves of medicinal bulbs with a view of promoting the substitution of bulbs with leaves in traditional medicinal use. Four medicinal bulbous plants, Tulbaghia violacea, Hypoxis hemerocallidea, Drimia robusta and Merwilla plumbea were evaluated for the pharmacological and phytochemical properties in their bulbs/corms and leaves in spring, summer, autumn and winter seasons, with a view of promoting the use of leaves as a conservation strategy. Dried plant materials were sequentially extracted with petroleum ether (PE), dichloromethane (DCM), 80% ethanol (EtOH) and water in each season. The extracts were tested for activities against Gram-positive (Bacillus subtilis and Staphylococcus aureus), Gram-negative (Escherichia coli and Klebsiella pneumoniae) bacteria and the fungus Candida albicans using the in vitro microdilution assays to obtain minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC). The four plant species were also evaluated for their ability to inhibit cyclooxygenase (COX-1 and COX-2) enzymes. Spectrophotometric methods were used to evaluate saponin and phenolic contents of samples from the four plant species in each season. Antibacterial activity was fairly comparable between bulbs/corms and leaves of H. hemerocallidea, T. violacea, and M. plumbea, with at least one extract showing some good activity (MIC < 1 mg/ml) in most of the seasons. Bulb extracts of D. robusta did not show good antibacterial activity while the leaf extracts showed good activity (0.78 mg/ml) against B. subtilis in spring, summer, and autumn and S. aureus (0.78 mg/ml) in autumn. The best antibacterial activity was recorded in winter, with MIC values as low as 0.195 mg/ml from the DCM bulb extracts of T. violacea against K. pneumoniae and S. aureus and PE corm extracts of H. hemerocallidea (0.195 mg/ml) against B. subtilis. Good antibacterial activity from water extracts were only recorded from corm extracts of H. hemerocallidea in summer, autumn and winter, H. hemerocallidea leaf extracts in autumn and winter, and M. plumbea bulb extracts in autumn. The leaf extracts of all the screened plant species demonstrated good fungicidal activity in autumn, with H. hemerocallidea corm water extracts recording an MFC value as low as 0.39 mg/ml. The leaf extracts of H. hemerocallidea (water), D. robusta (DCM) and M. plumbea (DCM) had good MFC values of 0.78 mg/ml each, in spring. The DCM leaf extracts of T. violacea also showed good fungicidal activity (0.78 mg/ml) in summer, while corm water extracts of H. hemerocallidea had an MFC value of 0.39 mg/ml in winter. There were no fungicidal activities recorded from all the bulb extracts in all the seasons. All the PE and DCM extracts in all the tested plant samples recorded between moderate (40-70%) and high (> 70%) COX-1 and COX-2 inhibition levels across all seasons. The EtOH corm extracts of H. hemerocallidea also demonstrated moderate to high inhibitory activity against COX-1 enzyme across all seasons. Bulb and leaf extracts of T. violacea showed selective inhibitory activity for COX-2 enzyme in all the seasons. The highest COX inhibitory levels were recorded in COX-2 from the PE leaf (spring) and bulb (autumn) extracts of T. violacea, with both recording 100% inhibitory activity. Phytochemical analysis revealed higher total phenolic compounds in bulbs/corms and leaves of all the analysed plant species, to be either higher in spring or winter. Plant material collected in autumn had the least levels of total phenolics. An almost similar trend to that of total phenolics was observed for flavonoids, gallotannins and condensed tannins in most plant samples, with higher levels either in spring or winter. Total saponins were consistently higher in winter than in the other seasons in all the screened plant species. There were in some cases, relationships between the peaks in the levels of some phytochemical compounds and the observed levels of bioactivity in different assays. The results obtained from this study demonstrate that the leaves of the screened plant species may substitute or complement bulbs in the treatment of certain ailments in traditional medicine. Thus, plant part substitution can be sustainably utilised in the conservation of these plant species while retaining the same medicinal benefits. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

The use of traditional medicine through the use of medicinal plants in Africa and especially in South Africa has long been considered an important characteristic of people's daily lives and socio-cultural heritage. Cotyledon Orbiculata is among the medicinal plants that are used by South African traditional practitioners for the treatment of epilepsy and painful conditions such as corns, warts, toothache, earache, boils and various other ailments. However, the claim of therapeutic successes of medicinal plants by traditional medicine practitioners are hardly subjected to scientific scrutiny. This study therefore, investigated the anti-epileptic property of Cotyledon Orbiculata by studying the effects of the methanol extract of the plant against chemically induced seizures by pentylenetetrazole, picrotoxin, bicuculline and N-methyl-DL-aspartic acid in mice. The study also investigated the analgestic effects of Cotyledon Orbiculata by studying the effect of the plant extract on pain induced by acetic acid and hot plate thermal stimulation.

M.Sc. (Botany) / A few poorly studied Cape herbal medicinal plants of historic and potential commercial interest which form part of the traditional medicine system of the Cape Region have been selected for this study. These are Centella glabrata L., Olea europaea L. subsp. africana (Mill.), Rafnia amplexicaulis (L.) Thunb. and Tulbaghia alliacea L.f. Centella C. asiatica is mainly used to treat skin conditions and wounds, tuberculosis and coughs and cooked and eaten as a vegetable in South Africa. The treatment of heartburn in the Wild Coast is a recent record. Decoctions of the leaves and stalks of C. glabrata were used for treating diarrhea and dysentery by the early Cape settlers. C. stenophylla or “klipdagga” is used in the treatment of high blood pressure and diabetes in the Still Bay area. This is also a recent new record. The anatomy of the few Centella species studied differed dramatically both in the leaf lamina and the petiole and could be used to differentiate between the species studied. A wider study would be very interesting and could be useful to distinguish between the species. This could have great potential as the ca. 45 species of Centella are often very difficult to distinguish from one another.