| Marine derived microorganism has increasingly become the focus of the object of natural product research, a large quantity of new natural product has been discovered, the fast development with an increasing trend appearing every year. Marine derived endophytic microorganisms has a special value of its research, because of the nutrition competition with host and may produce some outstanding biologically active compounds, also providing a good direction of the new drug research and development. In order to find lead compounds that are much-needed source material for drug research, the Qingdao Enteromorpha and other samples as the source of endogenous microorganisms were collected and isolated. And then through the comprehensive survey of chemical and biological activity, we carried out the work of microbial secondary metabolites of Enteromorpha prolifera -erived endophytic. The main contents include:the isolation of actinomycetes and fungal from Enteromorpha prolifera, and preliminary screening of microbial fermentation, talented strains found and further optimization of fermentation, separation and purification of compound, comprehensive analysis and identification of monomer compound structure, biological activity test of monomer compound.Enteromorpha prolifera samples were collected from Qingdao’s four big beach (the first beach, the second beach, Zhanqiao beach, Shilaoren Beach), and 200 strains of microbes were by isolated by gradient dilution method and sucrose density gradient method. Using MTT method and SRB method, the cell lines MCF-7, K562, Hela, A549 a-glucosidase activity were screened. Then through the TLC, HPLC studied, eventually two strains of Enteromorpha prolifera were identified as "talented strains" for further research-actinomycetes OUCMDZ3434 and fungi OUCMDZ3416. Actinomyces Strain OUCMDZ3434 were identified by 16sRNA as Streptomyces sp. fungi OUCMDZ3416 has not been identified.The effect of different fermentation conditions (fermentation time, pH, salinity days, etc.) were investigated, after a comprehensive analysis of the changes of the secondary metabolites of strains under different conditions, the best performing method was ultimately selected in the conditions that has been investigated for further scale-up fermentation. After the scale-up fermentation, the fermentation liquid was extracted by ethyl acetate to obtain crude extract. Then the different chromatographic techniques (silica gel column chromatography, gel chromatography, macroporous resin and high performance liquid chromatography) were took place on the fermentation product. After the separation and purification,12 monomer compounds were obtained from actinomycetes OUCMDZ3434 (1-12),6 compounds from fungi OUCMDZ3416 (13-18). Then the structure of these compounds were elucidated by physico-chemical properties and modern pop technology, including 10 new compounds. The obtained compounds all have polyketide origin.The compounds from actinomycete OUCMDZ3434 were tested with glycosidase activity, the results showed that the activity of compound 1-6 were better than the positive control drug acarbose. And in addition, compounds 1 and 2 showed very good activity with IC50 19.7 and 8.31 M,respectly, while the acarbose is 1.12 mM.In summary, the marine derived endophytic microorganisms were studied in this article. We obtain a lot of strains with very good activity. In addition, the secondary metabolites of two endophytic microorganisms of Enteromorpha prolifera were investigated, and a total of 18 compounds were isolated and identified, including 10 new compounds with two new skeleton compound; And through the screening of glycosidase activity, two very good compounds were found. These have fully demonstrated that it is valuable to research the endophytic microorganisms and has a very broad prospects. |
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