South African medicinal orchids : a pharmacological and phytochemical evaluation.

Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

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.