Isolation, Purification And Biological Characterization Of The Secondary Metabolites From Marine Myxobacteria

Myxobacteria are unique gram-negative bacteria with uncommon behavior. One special aspect of their life cycle is their morphological differentiation culminating in the development of complex fruting bodies, which is reminiscent of eukaryotic myxomycetes or fungi rather than prokaryotic bacteria. Myxobacteria are common in many types of terrestrial habitats including soil, vegetarian dung, decayed plant material, and bark of living or dead trees.Only a part of microorganisms had been chemically studuied in millions of microorganisms in the world. Most of research aimed to screen some microorganisms which never been studied before in order to obtain the novel natural products. Recently, myxobacteria are increasingly recognized as a rich source of novel natural products with potent biological activities because of their potential to produce a variety of metabolites which have novel structures. More than 100 basic structures with nearly 500 structural variants have been identified so far, and some of them are being developed for medical applications.In the couse of screening, we found that the culture of several myxobacteria can inhibited the hyphal growth of pyricularia oryzae, furthermore, it showed characteristic swelling effect to mycelia. In these myxobacteria, the strain 8#,11#,13# can inhibit the growth of pyricularia oryzae strongly.Compounds extracted with methanol from the fermentation broth of the strain 8# (Myxococcus xanthus 095B06) were separated by silica chromatography, gel filtration, thin preparation layer chromatography, as well as High-performance liquid chromatography. The chemical structures of the compounds were identified by 1HNMR, 13CNMR, ESI-MS, HMQC, HMBC, DQF-COSY, NOESY and EI-MS techniques. The methanol extract was separated to yield nine known products, avermectin A1a, avermectin B1a, avermectin A2a, avermectin B2a, cerevisterol, cycle(L-Pro-D-Lea), 4-quinolinercarboxylic acid, 22E,24R-5α,6β-Epoxygergosta- 8(14),22-diene-3β,7α-diol, uridine as well as a new product N((2R,3S,E)–1,3- dihydroxy-17-methyloctadec-8-en-yl)acetamide. The structures of the known compounds were determined by a combination of spectrosopic analysis and comparison with the reported data. The structures of other compounds are under elucidation at present.The bioactive effects of these separated compounds were evaluated by Kirby-Bauer Disc Diffusion method, MTT method and a pyricularia oryzae bioassay method.Avermectins have potent anti-parasirasitic and broad-spectrum activity against nematode and arthropod parasites. In this study, avermectin B1a and avermectin A1a were tested against Hela and SMMC-7721 cells, exhibiting maderate activity, while avermectin B2a and avermectin A2a inhibited the growth of Hela and SMMC-7721 cells strongerly with IC50 values of 5μg/ml. Since these avermectins showed different antitumor activity, the free C23-OH group plays a crucial role for their antitumor activity.Compound 8, N((2R,3S,E)-1,3-dihydroxy-17-methyloctadec-8-en-yl)acetamide is a new natural product, it could inhibit the hyphal growth of pyricularia oryzae, furthermore, it showed characteristic swelling effect to mycelia. This new product was found to inhibit the proliferation of HeLa cells in a dose-dependent manner as determined by the MTT assay. Its IC50 value was 15μg/ml. We also found that it could inhibit the growth of LoVo and MCF-7 cells with IC50 values of 25μg/ml and 20μg/ml respectively. Colchicine, which is known as an inhibitor of the proliferation of tumor cells, was used as positive control. The progress of apoptosis is regulated in an orderly way by a series of signal cascades under certain circumstances. The caspase-cascade system plays vital roles in the induction, transduction and amplification of intracellular apoptotic signals. To determined whether the decrease in human Hela cell viability observed after treatment with compound 8 occurs by apoptosis, we performed caspase-3, caspase-8, caspase-9 activity assay. The cells were incubated in the absence (control) or presence of 15μg/ml of compound 8 for 24 h, and total cell lysates were assayed for caspases activity. The levels of caspase-3, caspase-8 and caspase-9 activity were elevated after the exposure to compound 8 by comparision with the level of the uninduced control. Hela cells underwent apoptosis after treatment with compound 8.The detailed chemical structures of these compounds were described as following:...