Antimicrobial activities of marine sponge using bioassay-direct fractionation and contact bioauthography technique

Previous report indicated that sponge voucher number (1L82008) possessed the highest antimicrobial activity among four sponges and three corals species investigated. A large amount of sponge tissue (287 g dry weight) was extracted with methanol and followed with water. The methanol and water extracts were examined against two Gram-Positive bacteria (Staphylococcus aureus and Bacillus anthracis), two Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and two fungi strain (Candidal albicans and Cryptococcus neoformans) using disc diffusion method. The results showed that methanol extract had stronger antimicrobial activity tha water extract (maximum activity was 19.5 mm against B. anthracis, 400 ug/disc). Obviously, Gram positive bacetria were more sensitive than Gram -negative bacteria and fungi strain. B. anthracis (19.5 mm, 400 ug/disc)was the most sensitive bacterial while P. aeruginosa (0 mm) was the most resistant bacteria. Since the methanol extract was found to be active, it was used for further bioassay-directed fractionation in order to isolate the active compounds. From the bioassay results, dichloromethane (24 mm, 400ug/disc) and n-butanol ( 18 mm, 400 ug/disc) fraction were found to be active. Purification of n-butanol fraction was done by using normal phase column chromatography (silica gel 70-230 mesh) eluted with n-butanol:acetic acid:water (12:3:5). Based on thin layer chromatography (TLC) profiles, the same fractions were collected, concentrated and assayed for antimicrobial activity against B. anthracis. The results showed that fraction (87-123) and fraction (124-183)were the most active fractions. The TLC of the active fractions were then developed with the same mobile phase and subjected to contact bioautography against B. anthracis, which showed inhibition zone at different retention time, indicating the presence of antimicrobial components. Based on the observation, it could be concluded that the bioassay-guided fractionation and bioautography of 1P82008 marine sponge may come up with potent bioactive antibiotics. research is still in progress to isolate the active compound from n-butanol fraction as well as from dichloromethane fraction