Isolation and characterisation of antifungal and antibacterial compounds from Combretum molle (Combretaceae) leaf extracts

Abstract

The main aim of this study was to isolate and characterise antifungal and antibacterial compounds from leaf extracts of Combretum molle which belonging to the Combretaceae family. C. molle is one of the commonly used medicinal plants in southern Africa for numerous ailments. Three animal fungal pathogens, namely, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus and five plant fungal pathogens, namely, Aspergillus niger, Aspergillus parasiticus, Fusarium oxysporum, Penicillium janthinellum, Rhizoctonia solani and four nosocomial bacteria Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa were used as test microorganisms for bioactive compounds in leaf extracts of C.molle. Experiments for phytochemical analysis were done using different C. molle leaf extracts which were made using acetone, methanol, ethanol, ethyl acetate, chloroform, butanol and hexane as extractants. Thin Layer Chromatography (TLC) fingerprints of different leaf extracts were developed in three mobile phase systems, EMW, CEF and BEA and detected with vanillin-sulphuric acid spraying agent. The different extracts of C. molle showed the presence of many different compounds with distinct retardation factors (Rf), separated according to their polarities. Bioautography was carried out to determine the number of active compounds and their Rf values. The TLC plates were developed in three mobile systems, each sprayed with either fungal or bacterial strains. In BEA bioautograms of A. fumigatus, clear zones of inhibition were observed at Rf values of 0.12, 0.23, and 0.40. In EMW bioautogram of C. albicans, clear zones of inhibition were observed at Rf value of 0.73, 0.81, 0.87. C. neoformans had weak growth inhibition. Most of the fungal and bacterial strains tested in the bioautography displayed susceptibility to the active compounds, with P. janthinellum and P. aeruginosa showing exceptional sensitivity. The minimum inhibitory concentrations (MIC) values ranged from 0.02 to 2.5 mg/ml against the tested pathogens. The acetone and ethyl acetate extracts had the best inhibitory activity against P. janthinellum with an MIC value of 0.02 mg/ml. The acetone extract of C. molle gave the highest total activity (775 ml/g) against P. janthinellum. C. albicans was the most resistant pathogen with an average MIC value of 0.56 mg/ml compared with the other tested strains. Extracts were active against both Gram-positive and Gram-negative strains. P. aeruginosa extracts had the highest average MIC value (0.24 mg/ml) among the tested bacterial strains. In general, there was good overall inhibitory activity by different extracts of C. molle. Bioassay-guided fractionation of DCM extract of the leaves of C. molle yielded 32 fractions. Further fractionation led to the isolation of five compounds (C1, C2, C3, C4 and C5). Compound C1 was selected for structure elucidation due a larger quantity isolated and higher antimicrobial activity compared with the other isolated compounds. Nuclear magnetic resonance (NMR) spectroscopy and mass spectroscopy (MS) was used to show that compound C1 was taraxerol, belonging to the taraxerane group. Antimicrobial activity of the isolated compound against P. janthinellum had an MIC value of 0.08 ug/ml. Although the compound taraxerol have been discovered in other plant species, it is reported for the first time from C. molle in the study. The results illustrate that crude extracts and compound taraxerol from C. molle can be used as either an antibacterial or antifungal, and warrants further investigation.