Investigation of Antibacterial Effects of Hibiscus Sabdariffa L. Dried Calyx by Agar Diffusion and Bioautographic Methods

Abstract: Introduction: In this study, the antibacterial activities of methanolic and ethyl acetate extracts of calyx of Hibiscus sabdariffa L. (Malvaceae) traditionally used as Chai-Makii, against Staphylococus aureus, Staphylococus epidermidis, Escherichia coli, Bacillus subtilis, Klebsiella pneumonia and Pseudomonas aeroginosa were investigated by cylinder –plate and bioauthography methods separately. Method: In cylinder – plate method methanolic extract of the calyces were prepared by maceration and after concentrating the extracts, they were dried. Then the concentrations of 50, 25, 12.5, 6.2 and 3.1mg/ml of the methanolic solutions were used for searching antibacterial effects. The standard bacteria with certain concentration (0.5 Mac Far land) were inoculated on to the Muller – Hinton agar medium. Prepared extracts were dropped in cylinders and 18-24 hours after incubation and penetration of extract into the culture medium the antibacterial effects and inhibitory zone were observed. In bioauthography method, the ethyl acetate extract was prepared by decantation of methanolic extract and evaporating to dryness. Then this extract was separated by ethyl acetate: Chloroform: Methanol (32:53:15) by thin layer chromatography method. After placing TLC papers in culture medium with certain concentration of bacteria and incubation, spot of inhibitory zone appeared by using tetrazolium salts and indicated as Rf. Results: Methanolic and ethyl acetate extracts in cylinder – plate method showed antibacterial effects on all six bacteria. The minimum and maximum applied concentrations were respectively 3.1 and 50mg/ml. In bioauthography method, ethyl acetate extract showed antibacterial effect on Staphylococus epidermidis, Klebsiella pneumonia, Bacillus subtilis and Echerichia coli in Rf =0.15 and Rf=0.75. Conclusion: According to ultra violet spectroscopy of these two components, they could be flavones. Keywords: Bioautography, Cylinder plate, Antibacterial effect, Hibiscus sabdariff