Isolation, characterisation and cytotoxicity of antifungal compounds present in medicinal plants used against crytococcus neoformans in Vhembe District, Limpopo Province

Abstract

The use of medicinal plants as a source of treatment for various ailments including fungal infections is still practised in South Africa and across the globe. Fungal infections especially of Cryptococcus, Candida and Aspergillus species are the main cause of morbidity and mortality worldwide, particularly in developing countries. Traditional medicine is used as a source of remedies worldwide and has contributed extensively towards the development of modern medicine. Twelve selected medicinal plants (Kleinia longiflora DC. Berchemia discolor (Klotzsch) Hemsl., Persea americana Mill., Sansevieria hyacinthoides (L.) Druce, Dichrostachys cinerea (L.) Wright &Arn, Withania somnifera Dunal (Ashgandh), Momordica balsamina L., Lonchocarpus capassa, Pappea capensis, Rhus lancea L. fil, Peltophorum africanum, Maytenus heterophylla (Eckl. & Zeyh.) Robson) were analysed qualitatively for antifungal activities against Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans. The plant materials were extracted with solvents of various polarities such as acetone, dichloromethane, methanol, hexane, and water. Methanol extracted the highest amount of crude extracts from all the plant species as compared to other organic solvents. Chemical components of the extracts were analysed using aluminum backed Thin Layer Chromatography (TLC) plates and developed using three different eluent systems: Ethyl acetate: methanol: water [EMW], Chloroform: ethyl acetate: formic acid [CEF] and Benzene: ethanol: ammonia hydroxide [BEA]. CEF was the best eluent solvent system since it separated more compounds from plant extracts. This indicates that the active compounds were relatively non-polar. More chemical compounds were observed in TLC chromatograms separated with CEF, followed by BEA and EMW. All plant extracts had shown different chemical components when separated from the three solvent systems. The bioautography and serial dilution assays were used to determine the biological activity of plant extracts against the tested microorganisms, respectively. All the tested plant extracts revealed some varying degrees of fungal inhibition, with minimum inhibitory concentrations (MIC) values ranging between 0.02 mg/ml and 2.5 mg/ml. The aqueous extracts had shown some activity against the tested microorganisms. Noteworthy, antifungal activity was observed in acetone, DCM, hexane, and methanol root extracts of D. cinerea against the three tested microorganisms with MIC values ranging between 0.02 mg/ml and 0.04 mg/ml. Furthermore, acetone extracts of D. cinerea and P. africanum had excellent activity against three fungal pathogens with MIC values of 0.02 mg/ml and 0.08 mg/ml. Active compounds were observed in dichloromethane extracts of W. somnifera with Rf values of 0.40 and 0.64. In TLC chromatograms separated with BEA, active compounds were observed in acetone, hexane, and methanol leaf extract of P. americana, this indicates that the fungal compounds were relatively non-polar. No active compounds were observed in plant extracts of K. longiflora. Active compounds were visible in all extracts of P. capensis in TLC chromatograms developed in CEF and EMW. The antioxidant present in plants prevents the free radicals from causing various diseases in humans by inhibiting the oxidation of free radicals at the cellular level. The qualitative and quantitative 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods were used to determine the antioxidant activities of plant extracts. The presence of antioxidant compounds was indicated by yellow bands against the purple background on the TLC plates. More antioxidant compounds were observed in acetone and dichloromethane extracts of S. hyacinthoides developed in BEA compared to other plant species tested. Methanol, hexane, and water extracts of L. capassa revealed good antioxidant activity against DPPH by having a high percentage of inhibition compared to other solvents. Noticeably, extracts of P. africanum possess strong antioxidant activity as compared to other plant species. Solvent-solvent fractionation using column chromatography of the acetone extract led to the isolation of six compounds. The biological activity of the isolated compounds of L. capassa was investigated against the tested pathogenic fungi. The isolated compounds revealed some varying degrees of inhibition to the fungal pathogens. The largest quantity was isolated from compound 1 (80 mg), compound 4 (39 mg), compound 3 (27 mg), compounds 2 and 5 (14 mg) and the least was compound 6 (4.8 mg). However only three compounds were successfully identified as Lupeol (compound 1), Friedelin (compound 3) and 6-(γ,γ-Dimethylallyl)-3’,4’-dimethoxy-6”,6”- dimethylpyrano-[2”,3”:7,8]-flavanone (compound 4). Compounds 2, 5 and were not identified due to some impurities. More importantly, the isolated compounds exhibited good antioxidant activity in qualitative and quantitative scavenging assays, which indicates that isolated compounds of L. capassa can scavenge the free radicals causing fungal infections in humans. The results support the traditional use of the selected plants to combat fungal infections and related ailments by the local people and traditional health practitioners in Vhembe District, Limpopo Province. The (3-(4,5-dimethylthiazol) -2,5-diphenyltetrazoliumbromide) (MTT) assay was used to determine the toxic effects of the plant crude extract and isolated compounds. Lupeol and 6-(γ,γ-Dimethylallyl)-3’,4’-dimethoxy-6”,6”-dimethylpyrano-[2”,3”:7,8]- flavanone revealed the same degree of cytotoxicity against the Vero monkey kidney cells. All the compounds were not toxic with an LC50 value of ˃ 0.2 mg/ml.