Identification and characterisation of compounds with antimycobacterial activity from stomatostemma monteiroae

Abstract

The emergence of drug resistance to the first line drugs complicates the treatment of tuberculosis (TB), especially in parts of sub-Saharan Africa where accessibility to quality health care is limited. The search for alternative medication has been the centre of research for years due to challenges posed by infectious organisms including drug resistance, lengthy treatment periods and lack of quality health care in developing countries. Stomatostemma monteiroae is used in traditional medicine to treat TB and related symptoms. The aim of this study was to isolate and characterise compounds with antimycobacterial activity from Stomatostemma monteiroae. The plant materials were collected from Ga-Madiga village in Limpopo province of South Africa. Different plant parts namely: leaves, twigs, roots, tuber and tuber-peels were separated, washed, dried and milled to a fine powder. Several solvents (n-hexane, dichloromethane, acetone and methanol) were used to extract the plant material using various extraction methods such as maceration, defatting, and extract enrichment procedure and phytochemical analysis was done using standard chemical tests and thin layer chromatography. The qualitative antioxidant activity was determined by the thin layer chromatography (TLC) based 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical scavenging activity and quantitative antioxidant activity was determined using colorimetric DPPH free radical scavenging and ferric reducing power assay. Antimycobacterial activity of the extracts was assessed using bioautography and micro dilution method tested on Mycobacterium smegmatis (ATCC 1441), Mycobacterium tuberculosis (ATCC 25177) and M. tuberculosis H37Rv (ATCC 27294). The cytotoxic effects of the extracts were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on Vero monkey kidney cells. The compounds with antimycobacterial activity were isolated using bioassay-guided fractionation and purified using preparative thin layer chromatography and thereafter identified using NMR spectroscopy to elucidate the structure. Various phytochemical constituents were detected in different plant parts, with the leaves and twigs possessing more of the phytoconstituents analysed. The TLC profile of S. monteiroae indicated that more compounds are non-polar to intermediate in polarity. The antioxidant activity analysis on TLC plates indicated that all the plant parts have low antioxidant activity, this was also confirmed by xxii

quantitative tests. The leaves of S. monteiroae had antimycobacterial activity when analysed using bioautography, while other plant parts had no active bands. The minimum inhibitory concentration values were much higher than the positive control rifampicin and the roots (0.31 mg/mL) followed by the leaves (0.83 mg/mL) had lower inhibitory concentrations when tested against M. smegmatis. The MIC values of extracts against TB causing strains varied greatly, the leaves and the roots had even higher MIC value. Toxicity analysis indicated that all plant parts were non-toxic towards Vero cells (LC50 > 0.02 mg/mL). Bioassay-guided fractionation enabled isolation of one antimycobacterial pure compound from the leaves extracts. The isolated compound was identified using NMR and was found to be a sitosterol derivative 8,9-dehydro-4-methyl-24-vinylobtusifoliol. This compound had a noteworthy activity against M. smegmatis. The present study validates the use of S. monteiroae in the treatment of TB related symptoms traditionally. Further studies are required to analyse the cytotoxic effects of the isolated compound and also testing the antimycobacterial activity of the isolated compound on TB causing pathogens.