In most countries of subtropical Africa, fungal infections represent an increasing problem. Candida albicans is the most common fungal pathogen and has developed an extensive array of putative virulent mechanisms that allows successful colonization and infection of the host under suitable predisposing conditions.
Crude acetone, water and hexane extracts of eight plant species, used as traditional medicine to treat fungal related diseases were evaluated for antifungal activity against C. albicans standard strain (ATCC 10231) and six clinical isolates using a serial microplate dilution method. Antioxidant activity was also determined by using the TEAC and DPPH assays and cytotoxicity was determined against mouse fibroblast cells with a MTT assay.
MIC’s below 1 mg/ml were observed for the acetone and water extracts of Sclerocarya birrea (stem-bark), as well as the acetone extracts of Harpephyllum caffrum (leaves), Ptaeroxylon obliquum (leaves) and Rapanea melanophloeos (leaves). Bioautography confirmed the presence of compounds with antifungal activity in extracts of S. birrea and P. obliquum. S. birrea extracts had the highest antioxidant activity and the lowest cytotoxicity. However, P. obliquum contained the highest number of active compounds against C. albicans standard strain (ATCC 10231) and clinical isolates. MIC data also indicated that P. obliquum was the most active of all the plant species tested against C. albicans standard strain (ATCC 10231) and clinical isolates. Therefore, P. obliquum was chosen for bioassay-guided fractionation and isolation of antifungal compounds from P. obliquum acetone leaf extract.
Two compounds were isolated from the most active fraction obtained from P. obliquum acetone leaf extract, using column chromatography with silica gel as the stationary phase.
Compound 1 and Compound 2 inhibited the growth of C. albicans standard strain (ATCC 10231) at MIC 0.004 mg/ml compared to amphotericin B, MIC 0.11 mg/ml. The cytotoxicity of the compounds was determined against mouse fibroblast cells using the MTT assay. Berberine was used as a positive control and was found to be toxic with a LC50 of 9 04 μg/ml. The cytotoxicity of Compound 1 (against mouse fibroblast cells) was 0.002 μg/ml and that of Compound 2, 7.23 μg/ml. Selectivity Index (SI) of Compound 1, against both C. albicans standard strain (ATCC 10231) and clinical isolates, was 0.0004 and for Compound 2 it was 1.8 against the standard strain (ATCC 10231) and >0.03 against the clinical isolates tested.
The structures of the compounds were determined by extensive nuclear magnetic resonance (NMR) techniques and chemical methods mainly by 1D NMR (1H, 13C and DEPT) and 2D NMR (HSQC, HMBC and COSY) and by comparison with the literature data. Compound 1 is an isomeric mixture of β-amyrin and lupeol. Compound 2 is a novel compound and was identified as:
8,11-Dihydro-5-hydroxy-12-hydroxymethyl-2-methyl-4H-pyrano[2,3-g] benzoxepin-4-one 12-O-acetate. Candidal adherence to human buccal epithelial cells (HBEC) is considered the critical initial step in the pathogenesis of oral candidiasis, which may eventually lead to a systemic infection, especially in immuno-compromised individuals. Therefore, it was important to determine the ability of extracts and isolated compounds on the adherence of C. albicans standard strain (ATCC 10231) and clinical isolates to HBEC cells. P. obliquum acetone leaf extract at concentrations of 15.6 mg/ml and 7.8 mg/ml inhibited the proliferation and hyphae formation of C.albicans and thus adhesion. Natural products inhibiting adhesion of C. albicans to the oral mucosa may be beneficial in managing oral candidiasis.
The high antifungal activity of the acetone leaf extract of P. obliquum indicates that it could be considered as a potential therapeutic agent against oral Candida infections. The therapeutic effects of this extract may be limited only to low concentrations to avoid cytotoxic reactions as the cytotoxicity was less favourable (LC50 = 35.58 μg/ml against mouse fibroblast cells). However, as oral rinses are used topically and not swallowed the systemic effect may not be of critical importantance.