| As one of the most abundant biological resources on earth,microorganisms are demonstrated as an important source of novel natural products.Microbial metabolites always exhibit the diversity of chemical structures and complexity of chemical composition with low active ingredients content.Traditional separation methods rely mainly on off-line combination of open-column chromatography and preparative HPLC.However,the multi-step and prolonged separation procedure,as well as exposure to oxygen,may lead to the structural transformation of metabolites.In two-dimensional liquid chromatography(2D-LC),two independent chromatographic columns are connected in series for the separation,which has the advantages of small sample loss,no pollution,good reproducibility,automation and high throughput analysis.In this paper,2D-LC is applied to the separation of secondary metabolites of microbes.The main contents are as follows:1.A new strategy for separation of secondary metabolites from microorganism based on analytical and preparative two-dimensional RPLC × RPLC.The strategy was applied for the separation of Chaetomium globosum SNSHI-5.Analytical comprehensive two-dimensional liquid chromatography(2D-LC)was applied to analyze the composition of the metabolites,and to optimize the separation conditions for the preparative 2D-LC.Preparative 2D-LC separation of the crude fungus extract was then performed on a medium-pressure liquid chromatograph × preparative high-performance liquid chromatography system,under the optimized condition.In total,12 cytochalasan derivatives,including two new compounds2.The construction of a new NP × RP chromatography system:Based on the modified thermal evaporation assisted adsorption interface(TEAA)and simplified the design of the instrument interface,the ethyl acetate extract of Escherichia coli was as the research object and a good separation effect was obtained by combining NPLC with RPLC which possess different separation mechanisms.The composition of the metabolites was analyzed and amplified to the preparation,and 28 compounds were obtained.3.The new on-line two-dimensional NPLC × RPLC separation system was applied to isolate secondary metabolites of target strains,including ethyl acetate extract of fermentations from a bacterial and five fungals from different sources.From analytical 2D-LC magnification to preparative separation,114 compounds,including 22 new compounds,were obtained.4.At last,the bioactivity assays of the compounds including cytotoxic activity,anti-inflammatory activity,anti-influenza virus activity and anti-pathogenic fungi activity.As a result,4 cytotoxic compounds,7 anti inflammatory compounds,11 compounds with anti influenza virus activity were found,and 4 compounds against tea pathogenic fungi were found to be stronger than the positive drug sulphate. |
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