Potentiation of antimicrobial activity and isolation of active compounds from Loxostylis alata

Abstract

The development of antimicrobial resistance has provided momentum to the search for potential alternatives for treatment of microbial diseases in both humans and animals. Medicinal plants are a useful alternative source of new medicines, pharmaceutical entities and bioactive compounds that may be used for not only treating human diseases but also for enhancing animal production and health (Cowman, 1999). Loxostylis alata from the family Anacardiaceae is a traditional medicinal plant which is used by indigenous people as a pain reliever during childbirth and as an immune modulator (Pooley, 1993; Pell, 2004). This species was found to have good antifungal activity against Aspergillus fumigatus in a previous study. The problem was that the same extract that had activity was also found to be toxic In vivo using broiler chicks. Hence the aim of this project was to increase the reported activity and reduce the toxicity of the extract by selective techniques, and also to isolate bioactive compounds from the plant. The objectives were to evaluate the antimicrobial activity of extracts prepared using different solvents, to use bioassay-guided fractionation to develop an extract with good antimicrobial activity, to determine in vitro toxicity of Loxostylis alata extracts, and also to isolate and identify biologically active compounds and determine their toxicity. The leaf plant material was extracted in separate aliquots with four solvents (hexane, dichloromethane or DCM, acetone and methanol). Antifungal activity was tested using a microdilution method to determine Minimum Inhibitory Concentration (MIC) values. Bioautography was also performed against three animal fungal species, namely Cryptococcus neoformans, Aspergillus fumigatus and Candida albicans. The antibacterial activity was determined against ATCC strains of the Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis and the Gram-negative Escherichia coli and Pseudomonas aeruginosa. Solvent-solvent fractionation was conducted with the aim of increasing activity of the active extract and reducing its toxicity. Bioactive compounds were isolated using bioassay-guided column chromatography. Antimicrobial activity and cytotoxicity of the bioactive compounds was evaluated. Toxicity was evaluated using the colorimetric MTT (3-(4.5- dimethylthiazolyl-2)-2, 5- diphenyltetrazolium bromide) assay on C3A human liver cells. All four extracts prepared using different solvents showed different antimicrobial activity in the microdilution assay. The acetone extract had the highest antimicrobial activity with the lowest MIC value of 0.02 mg/ml, compared to the other extracts. The acetone extract was the only extract that showed one compound with antimicrobial activity in the bioautography assay. Based on these results, acetone was used for bulk extraction for isolation of active compounds. From the bulk extract, solvent-solvent fractionation was performed and five fractions (hexane, DCM, ethyl acetate, acetone and methanol) were obtained. Antimicrobial activity of the fractions was evaluated to determine if there was a loss or a gain in activity after solvent-solvent fractionation. The hexane and DCM fractions had a high antimicrobial activity with MIC values of 0.02 mg/ml and 0.04 mg/ml respectively. However this activity was not observed with the hexane and DCM extracts. This demonstrated that solvent-solvent fractionation helped to increase the activity of the fractions. The ethyl acetate and acetone fractions had antifungal and antibacterial compounds with the same Rf values. Their lowest MIC value was <0.02 mg/ml. The ethyl acetate fraction had a high total activity followed by the hexane fraction compared to the other fractions. Based on the MIC values, the presence of bioactive compounds in bioautography and a high total activity, the hexane and ethyl acetate fractions were subjected to further column chromatography for isolation of bioactive compounds using silica gel as a stationary phase. Two compounds with antimicrobial activity were isolated from the hexane fraction. One compound was isolated from the ethyl acetate fraction. The structural elucidation of the isolated compounds was done using NMR spectroscopy and Mass Spectrometry (MS). Compound 1 from the hexane fraction was identified to be lupeol while compound 2 was a mixture of lupeol, alpha-amyrin and beta amyrin. The two compounds had antifungal activities with MIC values ranging from 63 - 125 μg/ml against the tested fungal pathogens. The compound from the ethyl acetate fraction had both antifungal and antimicrobial activity with an MIC value of 4 μg/ml. Cytotoxicity of the active fractions and compounds was tested to evaluate toxicity of fractions or compounds compared to the acetone crude extract. The compound isolated from the ethyl acetate fraction had low toxicity in vitro against C3A cells compared to the crude extract and fractions, with LC50 = 110.2834 μg/ml. This compound had the highest activity with the lowest MIC value of 4 μg/ml and selectivity index of 27.57. The increased activity of the compound compared to that of the ethyl acetate fraction from which it was isolated shows that there was a removal of inhibitory compounds from the ethyl acetate fraction during the isolation process, and a concentration of the active compound from a relatively low concentration in the fraction. Suleiman (2010) reported that the acetone extract of L. alata at a dose of 200 mg/kg was as effective as ketoconazole which was used as a positive control. The acetone extract was able to treat broiler chicks which were infected with Aspergillus fumigatus. Thus the plant showed it has therapeutic values against Aspergillus fumigatus. However the active acetone extract was also found to be toxic to the chicks. The aim of the current study was to increase the activity of the acetone extract and reduce its toxicity. This aim was fulfilled since we were able to develop ethyl acetate fraction through bioassayguided fractionation which and isolate a compound from it with a high antimicrobial activity (4 μg/ml) and a low toxicity.