Study On The Isolation And The Secondary Metabolites Of Fungus And Rare Actinomycetes From Wetland Soil

Microorganisms are important sources of drug lead compounds,among which fungi and actinomycetes play an irreplaceable role.Penicilliums,aspergillus and rare actinomycetes are attracting more and more attention because of the significant diversity of chemistry and biological activity.However,with continuous research on the secondary metabolites of microbes,it has become difficult to find novel compounds and drug lead compounds from common microbes.This dilemma drives researchers to turn their eyes to the microbes from some special environments.Soil is an excellent natural habitat for microbes.Wetland,because of its unique ecological environment and abundant biodiversity,is not only an important source of new microbe species,but also a potential pool for discovering novel microbial secondary metabolites.In the present study,we investigated the secondary metabolites of fungus and rare actinomycetes isolated from different wetland soils.The research included the isolation,identification and screening of fungus and rare actinomycetes,purification and structural elucidation of the secondary metabolites and bioactivity assay of compounds.The contents were as follows:1.The isolation,purification,identification and screening of microbes strain from wetland soil157 strains of fungus were isolated from 4 wetland soil samples.By the chemical diversity analysis of 153 fungal strains using LC-MS,25 fungal strains were sesqueced and further identified.Combined with sequencing results,chemical diversity and literature investigation,two fungal strains（Penicillium oxalicum GY1 and Aspergillus Niger GY1）were selected for the next study;228 strains of bacteria were isolated from 3 wetland soil samples.By the chemical diversity analysis of 158 strains of bacteria based on LC-MS,33 strains of bacteria were sesquenced,and 11 strains of rare actinomycetes were identified.Combined with sequencing results,chemical diversity and literature investigation,one strain of rare actinomycete（Nocardia soli W1）was selected for the next study.2.The isolation and identification of secondary metabolitesBy the chemical investigation of the culture broth and mycelia of strains,9compounds（C-1～C-9）were obtained from Penicillium oxalicum GY1,including a new azo compound（C-1）,a new isochroman carboxylic acid（C-3）and two new natural products（C-2,C-4）by column chromatography（macroporous resin chromatography,reversed phase silica gel,Sephadex LH-20 and silica gel）.The structures were identified by extensive spectroscopic evidences such as ESIMS,NMR,and CD spectrum.The absolute configuration of new natural product C-2 was established firstly by X-ray diffraction.Meanwhile 15 compounds（H-1～H-15）were isolated from Aspergillus Niger GY1.Those compounds included polyketones,dimeric naphthopyrones,diketopiperazine heterodimers and amides.25 compounds（N-1～N-25）were obtained from Nocardia soli W1 including simple benzene substitutions,nucleosides,cyclic peptides and 13-ring polyketoethers.3.Biological activities of the secondary metabolitesThe secondary metabolites were evaluated for their cytotoxic activities against human tumor cell lines.The results showed that compound C-4 displayed potent cytotoxic activity against human esophageal carcinoma cell OE19 with the IC₅₀ value of 5.50μM.At the same time,Compound H-1 exhibited significant cytotoxicity aganist human lung adenocarcinoma cell A549,human colon cancer cell HCT116 and human breast cancer cell MCF-7 with IC₅₀ values of 4.29,1.77 and 1.84μM respectively.And H-2 also showed potent selective cytotoxicity against MCF-7(IC₅₀=0.44μM).The further cell viability assay demonstrated that H-1 displayed significantly selective cytotoxicity against human triple negative breast cancer MDA-MB-231 cells with an IC₅₀ value of 0.97μM and low cytotoxicity against human breast epithelial cells MCF-10A(IC₅₀>50μM).The pilot mechanism study revealed H-1 could arrest cell cycle of MDA-MB-231 cells at the G₂/M phase and significantly induce cell apoptosis.The apoptosis-inducing effect of pyoluteorin was related with reduction of mitochondrial membrane potential,accumulation of reactive oxygen species and change of apoptosis-related protein expressions.Compounds H-2,H-9 and H-10 displayed significant inhibition against NO release in LPS-induced BV2 cells with IC₅₀vales of 8.88,7.23 and 7.30μM,respectively.In conclusion,157 fungal stains and 228 strains of bacteria including 11 strains of rare actinomycetes were isolated from wetland soil samples.49 secondary metabolites,including two new compounds and two new nature products,were obtained from Penicillium oxalicum GY1,Aspergillus niger GY1 and Nocardia soli W1.The compounds were evaluated for their cytotoxic activities against human tumor cells and the inhibition activities against NO release in LPS-induced BV2 cell.The results demonstrated that compounds C-4,H-1 and H-2 exhibited significant cytotoxic activities.While compounds H-2,H-9 and H-10 could inhibit NO release in LPS-induced BV2 cells significantly.At last,the mechanism of H-1 inducing MDA-MB-231 cells apoptosis was investigated.The results revealed that H-1 had obvious cytotoxicity against MDA-MB-231 cell through inducing cell cycle arrest and apoptosis by the mitochondrial pathway.This paper enriched the chemical constituents and biological activities of wetland soil microorganisms,and also provided strategies for the systematic study of microbial secondary metabolites.