Isolation and characterisation of antibacterial secondary metabolites from odontonema strictum (acanthaceae)

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Luhata, Lokadi
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The University of Zambia
It has been noted that an increasing number of bacteria are now developing resistance to commercial antibiotics. This problem necessitates the discovery of new classes of anti-microbial drug leads. Plants have always played an important role in the discovery of new medicines. Secondary metabolites which are present in almost all plants represent a special group of molecules in drug research. Some of them have anti-microbial properties. The aim of this study was to evaluate, in the preliminary stage, the anti-bacterial potential of four plants (Odontonema strictum (OSM) (Acanthaceae), Solanum torvum, Symphytum officinale L, and Aphelandra squarrosa) from tropical regions. Five bacteria, among the nine bacterial strains of international concern, were used as test organisms. These include Klebsiella pneumoniae, Shigella dysenteriae, Salmonella typhi, Escherichia coli and Staphylococcus aureus. All the bacterial strains were locally isolated organisms (LIO) from the University Teaching Hospital (UTH) in Lusaka, Zambia. Leaf extracts were prepared by solvent extraction using a 1:1 (v/v) mixture of methanol and dichloromethane for 24 hours at room temperature. Dried crude extracts were kept in air tight containers and stored at 4 °C. For testing, extracts were dissolved in 80% acetone (in water) giving a stock concentration of 100 mg/mL and the working concentrations were prepared by a ten-fold serial dilution technique ranging from 1 to 100 mg/mL. The agar disk diffusion technique was used to screen for anti-bacterial activity of the solvent extracts. Extracts from OSM were the most potent with largest inhibition zones at a concentration of 100 mg/mL (37±1.5 mm for S. aureus, 25±1 mm for K. pneumoniae, 12±0.7 mm for S. typhi, 20±0.9 mm for E. coli and 18±1.3 mm for S. dysenteriae). Based on these preliminary results, OSM was selected for further investigation. There was no statistically significant difference (P > 0.05) in the potency of the four plant extracts and that of the antibiotics which served as positive controls. Phytochemical screening of OSM leaf extracts indicated the presence of flavonoids (type of flavones), saponins, glycosides, tannins, steroids and terpenoids. Bio-guided fractionation using chromatographic separation led to the identification of an active fraction which showed the presence of steroidal nucleus when subjected to Liebermannn-Bourchard reaction. Further column chromatographic separation on the active fraction led to the isolation of pure active compounds, beta-sitosterol and stigmasterol which were characterized by physical (melting point and thin layer chromatography (TLC)) and spectroscopic methods (infrared spectroscopy (IR) and one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy). The Minimum Inhibitory Concentration (MIC) of the active pure compounds and the crude extracts were determined by the broth dilution method using S. aureus as a test organism. The active phytosterols (beta-sitosterol and stigmasterol) were found to have MIC values in the range 1.84 to 3.68 mg/mL. Compared to the total activity (TA) (MIC = 3.83 to 7.66 mg/mL) of the crude extract, these MIC values mean that 1 g of the plant material can be diluted 10.52 to 21.4 times and it will still inhibit the growth of S. aureus, implying that they are not the only active compounds or possibly there is a synergistic effect.
Drug Resistance, Microbial , Anti-microbial drug