Antimicrobial activity and cytotoxicity of extracts and an isolated compound from the edible plant Grewia flava against four enteric pathogens

Abstract

Bacterial infections of the gastrointestinal tract (GIT) causes vomiting, diarrhoea and even systemic infection. There is resistance to the commonly used antibiotics used to combat bacterial infection; there is a need for development of natural products into alternative and safer medicines. Studies have been conducted on the other species of the genus Grewia and there are reports of pharmacologically active compounds. There are no published studies up to date on Grewia flava. This study evaluated the antimicrobial activity of extracts prepared from berries, leaves, bark and roots of the edible plant Grewia flava against four enteric pathogens, Escherichia coli, Bacillus cereus, Staphylococcus aureus and Salmonella Typhimurium. The genus Grewia was named after Nehemiah Grew, an English plant anatomist and physiologist. It is a member of the family Malvaceae, formerly Tiliaceae. Plant material was extracted with acetone and water. The extracts were tested against the enteric pathogens using the minimal inhibitory concentration (MIC) method with gentamicin as positive control and ƥ-iodonitrotetrazolium violet (INT) as growth indicator. Antioxidant activity of the extracts was analysed qualitatively by developing the chromatogram in three different mobile systems and spraying the plates with 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and vanillin in sulphuric acid. Quantitative determination of antioxidant activity was carried out spectrophotometrically with DPPH, a stable free radical, and Trolox as a positive control. The antimicrobial activity was further determined qualitatively by the bioautography method. The toxicity of the extracts was determined using the MTT reduction assay against Vero monkey kidney cells, with doxorubicin as a positive control. The crude extracts of the leaves, bark and roots of Grewia flava were able to inhibit the growth of the enteric pathogens with MIC values ranging from 0.03 mg/ml to >2.5 mg/ml. Different compounds were visible on the plates after spraying with vanillin-H2SO4 reagent, and some of the compounds showed antioxidant activity when the plates were sprayed with DPPH and comparing the Rf values. Bioautography proved that the extracts were able to inhibit growth of the bacteria, S. Typhimurium and B. cereus. The acetone extracts were fractionated using solvent-solvent fractionation and five fractions were obtained; hexane, chloroform, butanol, 35% water in methanol and water. The chloroform fraction was effective against B. cereus, E. coli and S. Typhimurium with MIC values ranging from 0.15 mg/ml – 0.3 mg/ml. This fraction was also not toxic with a LC50 of 509.23 ± 24.88 μg/ml. Bioassay-guided isolation was carried out with the chloroform fraction, using open column chromatography, with silica gel as the stationary phase. The compounds were eluted with hexane: ethyl acetate solvent system at different polarities. Nine fractions were collected from the first column, and these fractions were tested for antimicrobial activity. Fraction 2 was the least effective with MIC above 1 mg/ml compared to fraction 3 which was effective against B. cereus and E. coli. On the bioautographs there was a visible clear zone with corresponding Rf on the thin layer chromatogram (TLC). Compound 1 was isolated from fraction 2 of the first column and compound 2 from fraction 3 of the first column, and the compounds were tested for antimicrobial activity and cytotoxicity. Probably due to synergistic effects, the single or isolated compound was less effective than the mixture of compounds (i.e. the chloroform fraction and acetone crude extract). The compounds were less effective against the tested microorganisms than the fraction and extract. Compound 1 was the least toxic with LC50 of 39.38±3.21 μg/ml compared to LC50 3.02 ± 0.03 μg/ml of the positive control compound, doxorubicin. Compound 1 was identified as Lupeol. The indigenous edible plant, Grewia flava contains antimicrobial compounds, as indicated by the inherent free radical scavenging activity and could therefore be used as a natural product with little toxicity to host cells. Further in vivo studies have to be conducted to confirm the toxicity.