The isolation and characterization of an antibacterial compound from Terminalia sambesiaca (Combretaceae)

Abstract

This was an investigation of the antimicrobial activity of the Terminalia species and isolation of the compound(s) responsible for such activity. Terminalia species are extensively used in the indigenous medicines in most parts of Africa. They are a source of many potent biologically active compounds. Terminalia sericea has been identified as one of the 51 most important African medicinal plants. There are several Terminalia species in South Africa and in this study the following species were investigated: Terminalia sericea, Terminalia phanerophlebia, Terminalia mollis, Terminalia gazensis, Terminalia brachystemma, and Terminalia sambesiaca. The leaves of six Terminalia species were sequentially extracted with hexane, dichloromethane, ethyl acetate and methanol. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay on TLC plates was used to screen for the radical scavenging ability of the compounds present in the plant extracts. All plants had antioxidant activities present in different extracts. Most of the antioxidatively active compounds were present in the ethyl acetate and methanol extracts. Minimum inhibitory concentration (MIC) was determined using a serial microdilution assay where tetrazolium violet reduction was used as an indicator of growth. This was done for both bacteria and fungi. Pathogens used included Gram-negative (Pseudomonas aeruginosa and Escherichia coli) and Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus) as well as fungi; yeasts (Candida albicans and Cryptococcus neoformans), thermally dimorphic fungi (Sporothrix schenckii) and moulds (Aspergillus fumigatus and Microsporum canis). All six Terminalia species were active against the selected pathogens. E. faecalis and E. coli were the most sensitive pathogens while S. aureus and P. aeruginosa were relatively resistant. Most extracts gave MIC values of 80 μg/ml while others (T. sambesiaca) gave values as low as 20 μg/ml after 24 hours of incubation. Terminalia sambesiaca gave the lowest average MIC value at 100 μg/ml, followed by Terminalia mollis with an MIC value of 118 μg/ml after 24 hours of incubation. The total activity of the species was calculated by dividing the quantity extracted in milligrams (mg) from 1 gram leaves by the MIC value in mg/ml. This value indicates the volume to which the biologically active compound/s present in 1 gram of the dried plant material can be diluted and still kill the bacteria. T. sambesiaca had the highest average total activity of 4312 ml/g while T. gazensis had the lowest average activity (1371 ml/g). For antifungal activity, most extracts gave MIC values of 80 μg/ml. T. sambesiaca was the most active species showing values as low as 40 μg/ml after 24 hours of incubation. Amphotericin B was used as a positive control and there was no growth in any of the wells containing the antifungal agent, indicating a MIC of less than 20 μg/ml. T. sambesiaca and T. brachystemma had the lowest average MIC value of 0.20 mg/ml followed by T. gazensis with an average MIC value of 0.21 mg/ml after 24 hours of incubation. T. sambesiaca gave the lowest MIC values against the tested pathogens and was therefore selected for in depth investigation. Isolation of compounds was undertaken on the crude extracts of Terminalia sambesiaca leaves. Open column chromatography was used to further separate compounds seen on TLC when phytochemical analysis was done. A compound was isolated from the ethyl acetate extract and its structure was elucidated. The isolated compound was identified as -sitosterol and it was subjected to bioassays to ascertain activity reported in literature. It was active against all tested pathogens. The MIC values for the isolated compound against the tested pathogens were: 280 μg/ml for E. faecalis, 400 μg/ml for E. coli and 320 μg/ml for S. aureus. E. faecalis was more susceptible to the isolated compounds than the other pathogens used. Although -sitosterol was isolated from other plant species and from Terminalia arjuna, this is the first report of this compound from Terminalia sambesiaca. The fact that -sitosterol was active against the selected pathogens confirms the findings from other studies. The excellent activity found in the Terminalia sambesiaca extracts could be a lead in the development of antimicrobial agents. It is a possibility that more compounds could still be isolated from Terminalia species that are responsible for antimicrobial activity of these plants. These findings justify the use of Terminalia species in ethnopharmacology.