The Studies On Screening, Isolation And Identification Of Metabobites Of Microbes For Inducing Differention Of HUC-MSCs In Vitro

A great variety of microbes and complex chemical structures of metabolites provide abundant supply for research and development of new drugs.Recently, although more and more active compounds were discovered from microbes metabolites,which also promote a major advancement on developing of microbes, the most active compounds based on anti‐tumor were researched and few studies focused on other potential activites of microbes metabolites,such as anti‐inflammatory, anti‐angiocardiopathy and so on. So we attempt to find some new active compounds inducing adipogenic or osteogenic differentiation of human umbilical cord‐derived mesenchymal stem cells (hUC‐MSCs) from microbes metabolites,and we also hope that some compounds with neoteric structures may be discovered.To screen the microbes metabolites for inducing adipogenic or osteogenic differentiation of human umbilical cord‐derived mesenchymal stem cells (hUC‐MSCs), a screening method based on96‐well plate was developed. The hUC‐MSCs cells were seeded into the wells and cultured with metabolite extracts. Cell phenotypic changes were observed and adipogenesis or osteogenesis were identified by Oil red O staining or alkaline phosphatase staining respectively during cell culture. From more than400metabolite samples, one extract of Streptomyces sp.3418showed potential activity inducing adipogenic differentiation of hUC‐MSCs.To find the active compounds in metabolites of Streptomyces sp.3418, the strain were fermented in50L fermentation tank,ensuring that we could gain abundant metabolites for compound isolation.In fermentation process,the3418strain grew stably in fermentation tank by assaying OD600、wet weight and revertose of the strain.And we finally raped30L fermentation broth.The fermentation broth were extracted by organic solvent, and the activities of crude extracts were detected by cell experiments. The active extracts were separated further by tracing the active compent,and8compounds were isolated or purified by serial process，such as silica chromatography, sephadex LH‐20,preparative TLC and so on. The structure of5compounds were identified,one is new compound. And we detect initially that one compound can induce adipogenic differentiation of hUC‐MSCs by TLC.The structure of5compounds were determined by1H‐NMR，13C‐NMR，HSQC，HMBC,ESI‐MS etc.Those compounds were Uracil,Thymine,Cytosine,Heptadecane acid and1‐(3,5‐Dihydroxy‐2‐imino‐pyrrolidin‐1‐yl)‐ethanone.And one active compound is Nerve amine,the other2compounds are Aliphatic series.