Isolation And Identification Of Secondary Metabolites Of Two Aspergillus Strains And Evaluation Of Their Biological Activity

Aspergillus is a highly aerobic fungi with strong environmental adaptability and diverse metabolic pathways,capable of secreting secondary metabolites with novel structures and unique physiological effects.The structural types of Aspergillus fungal secondary metabolites include sesquiterpenoids,polyketides,diketopiperazines.These compounds have significant antibacterial,anti-inflammatory,anti-tumor cell proliferation and other physiological effects.Thus,Aspergillus spp.are an important source for researching natural products and obtaining more novel small molecule drugs with significant activities,which deserve further study.In this thesis,six small filamentous fungi were selected for optimal culture conditions by OSMAC strategy,and the results of pre-experiments were analyzed by high performance liquid chromatography and high resolution mass spectrometry to screen out Aspergillus penicillioide GDMCC 3.587 and Aspergillus aureoterreus GDMCC 3.619)for compound isolation,identification and biological activity evaluation.Firstly,the strains were fermented,their ethyl acetate extracts were collected,and the crude extracts were separated and purified by chromatographic separation techniques(CC,TLC,HPLC,ODS,Sephadex TM LH-20),and the monomeric compounds were identified by spectroscopic techniques(NMR,MS,IR,UV,ECD.).Some compounds were also tested for cytotoxic activity by the MTS method.The test cells included promyelocytic leukemia cells(HL-60),lung cancer cells(A549),liver cancer cells(Hep G-2),breast cancer cells(MCF-7)and ovarian cancer cells(SKOV-3),and the following results were obtained.A total of 26 compounds,including 14 new compounds and 12 known compounds,were isolated and identified from the culture extracts of the two Aspergillus strains.A total of 19 compounds were isolated and identified from the crude extract of Aspergillus aureoterreus GDMCC 3.619,including12 new sesquiterpenoids(A1-A12),two newα-pyrone containing polyketide derivatives(A13,A14),one known sesquiterpenoid(A15),one knownα-pyrone containing polyketide analog(A16),one known triterpenoid(A17),one known cyclopentenone(A18),and one known cinnamoyl derivative(A19),the new compounds were dominated by sesquiterpene and polyketide structures.The structures and absolute conformations of the new compounds were determined by ~1H-~1HCOSY,HMBC spectra,coupling and constant and ROESY spectral correlations and ECD calculations.A total of six known compounds(ap-1,2,3,4,6,7)and one known cyclic ether of phenolic acid(ap-5)were isolated and identified from the crude extract of Aspergillus penicillioide GDMCC3.587,and the structures of the compounds were determined by hydrogen and carbon spectroscopy and literature data search.The results of antitumor activity assay showed that compounds A1,A3,A9and A10 showed good cytotoxic activity against promyelocytic leukemia cells,hepatocellular carcinoma cells and ovarian cancer cells.Compound A1 was found to be effective in inducing apoptosis in HL-60 cells at 5μM with an apoptosis rate of 55.2%by flow cytometry study.This thesis screens the optimal culture conditions of two Aspergillus spp.,and isolates and identifies their secondary metabolites with activity analysis,enriching the chemical diversity of the secondary metabolites of Aspergillus spp.and providing a reference for the biological activity research of Aspergillus spp.