Screening For The Anti-tumor Activity From Marine Bacteria And Research On The Active Products

Due to its special ecological environment, ocean is rich in biological species resources, and these marine organisms contain a lot of valuable biologically active substances. More and more attention have been paid onto marine microorganisms because of their higher specificity and the ease of fermentation. In this work, MTT assay was used to screen antitumor active agents from251marine bacteria. And then a preliminary study about the isolation and identification of the active metabolites from No.657ferments was also conducted. The main conclusions of the study are listed as follows:1. In this study, by using MTT assay, we screened for the antitumor activity from some marine bacterial strains which were isolated from San Juan Island, the USA. The results showed that, among251tested bacterial isolates,54isolates (about21.5%) showed better antitumor activity. Their inhibition rates against HeLa cell at the concentration of100μg/mL were larger than50%. And7of them showed stronger antitumor activity with the inhibitory rate higher than85%. Among all the active strains, bacterium No.657showed the most remarkable antitumor activities. The antitumor activities (IC50) of its metabolies extracted from liquid fermentation and solid-state fermentation are36.53μg/mL and3.19μg/mL respectively. The solid-state fermentation should be better for its active metabolites production.2. This active strain No.657was then identified as Serratia proteamaculans, according to its morphological observation and16S rDNA sequence analysis.3. The solid fermentation conditions of strain No.657were also studied. Single factor experiments showed that the optimum fermentation conditions are:temperature20-23℃, initial pH value of6-7; By Plackett-Burman (PB) design (N=12),4important factors for affecting active compounds production were firstly screened out, and orthogonal experiment was further conducted to arrange the importance of the4factors, which was NaCl> peptone> MgSO4> yeast extract. The optimum medium contained10.0g of peptone, 1.0g of yeast extract,19.45g of NaCl and0g of MgSO4.4. We conducted a preliminary study on the seperation and identification of active metabolites from the strain No.657. By thin layer chromatography and silica gel column chromatography, the organic extracts of bacterial cells collected from solid-phase ferments were isolated and purified. The mobile phase of silica gel column chromatography was dertermined to use dichloromethane (DCM)-methanol solvent system based on the TLC spectrum, and the separation conditions were gradiently eluted with:Pure DCM, the proportion of DCM and MeOH100:1,98:1,95:1,90:1,70:1,40:1,20:1and pure MeOH. Activity guided seperation showed that the fractions eluted with100:1-95:1DCM-MeOH have better antitumor activities, and among them98:1fraction showed the strongest activity with the IC50of0.248μg/mL, nearly13times higher than that of the crude extract. TLC analysis also indicated that the purity of98:1fraction was higher than other fractions.5. By gas chromatography-mass spectrometry (GC-MS) and proton nuclear magnetic resonance spectroscopy (1H-NMR) analysis for the compound identification, the main active substance in the98:1fraction was identified as prodigiosin.