Studies On The Bioactive Constituents Of Strain F121112 From East China Sea

Twenty-one century has been recognized as the century of sea. It is well known that the marine organisms are characterized as the diversity in biologic species, genes, metabolism mechanisms and metabolites because of its particular living environments. Therefore, marine organisms have been widely thought to be a treasure of natural drug resources and become one of the hotspots for exploring and developing the new generation drugs.In the present study, F121112 was isolated from marine sediments in the East China Sea. This testing strain is a halophilic microbe and its optimum salt concentration is 2.5%. The evidence from Pyricularia oryzae model screening demonstrated that this strain powerfully inhibited the growth of Pyricularia oryzae and the antimitotic activity failed to lose (modify) after screening again. The strain F121112, which is a G~+ bacterium, has a spore in center of cell. The physiological and biochemical studies have shown that strain F121112 has the characteristics of Bacillus family. Based on 16S rDNA sequence analysis, the strain F121112 has been identified to be the genus of Bacillus subtilis.The fermentation conditions have been further examined in the current work. The results showed that the best fermentation conditions was: the optimum fermentation mediums was composed of 0.4% peptone. 0.5% yeast extraction. 0.4% beef cream, and 0.5% glucose, an initial pH of 6.0 -6.5, 4%~6%(v/v) of inoculation doses and 250-mL Erlenmeyer flasks containing 60 mL of fermentation medium, 130 r/min of the oscillation frequency, 25℃ of the culture and 120 h for the fermentation time. Under these conditions, the strain effectively produced the high concentration of the bioactive compounds.We have also tested the heat stability of fermentation. The fermentation wasfound to be stable to heating because its antimitotic activity was not markedly attenuated after treatment with 80 °C for 1 h.A total of 20 compounds were isolated from the strain F121112 by the column chromatography on silica gel, RP-Cig and Sephadex LH-20, followed by reverse-phase high performance liquid chromatography. The structures of 9 compounds were mainly elucidated by using spectroscopic methods ( 'H-NMR, 13C-NMR, EI-MS). According to the physicochemical properties, those structures were identified to be Cyclo-Phe-Ala(I), Cyclo-Phe-Val(II), Cyclo-D-Phe-L-Leu(III), Cyclo-D-Phe-D-Pro(IV), Cyclo-L-Ala-L-Leu(V), 3-methyl-6-(2-methylbutyl)-piperazine-2,5-dione(VI), Cyclo-L-Trp-L-Val(VII), Uridine(VIII), L-Tryphan(IX) and Cyclo-Ala-Pro(X). It should be noted that the compound VI is obtained firstly from the nature. The detailed structures of these compounds were described as following:Bioassay results further indicated that compound II and VI were capable of playing an inhibitory effect against the growth of Escherichia coli, the inhibition zone on plate of compound II is 19 mm and of compound VI is 14 mm.In summary, we demonstrated in the present study that eight cyclodipeptides, Uracil and L-Tryptophan were successfully isolated from F121112 fermentation broth. It is the first time to find that compound II is an inhibitor to E.coli, and compound VI is isolated firstly from the nature. The present study will provide a chemical and biological basis for generating the new antibiotics-like bioactive drugs from the secondary metabolites from marine Bacillus subtilis.