| Lycium barbarum is a kind of traditional Chinese medicine,which has anti-oxidation,anti-inflammation and anti-tumor effects.In recent years,the preparation of active drug lead molecules from endophytic fungi of medicinal plants has become a research hotspot in the field of natural products.In this study,endophytic fungi of Lycium barbarum were isolated and identified,and secondary metabolites of a strain with good activity were studied.The results are as follows.（1）Endophytic fungi were isolated by tissue block separation method.A total of 17 endophytic fungi were isolated,among which 2 strains were isolated from the root,6 strains from the stem and 9 strains from the leaf.According to the ITS sequences identification,the17 strains belonged to 9 genera,including Aspergillus,Talaromyces,Penicillium,Dothiorella,Cladosporium,Alternaria,Exserohilum,Pithomyces and Fusarium.Aspergillus was the largest genus,accounting for 23.53%.（2）The strains were subjected to small-scale liquid fermentation.The fermented liquid was extracted by ethyl acetate and concentrated to obtain the crude extracts.The antibacterial activities of the crude extracts against two pathogenic fungi（F.graminearum,F.moniliforme）and two pathogenic bacteria（Staphylococcus aureus,Salmonella enteritidis）,the cytotoxic activities of the crude extracts against human hepatoma cell line Hep G2,human lung cancer cell line A549 and human breast cancer cell line MDA-MB-231,as well as scavenging ability of 1-diphenyl-2-picrylhydrazine（DPPH）free radical were determined.The results showed that GQ-6 and GQ-16 had good activity against two kinds of pathogenic fungi and two kinds of pathogenic bacteria.GQ-6 and GQ-9 showed good cytotoxic activity against three kinds of cancer cells,and GQ-5 showed strong scavenging ability against DPPH free radical.GQ-6had both good antibacterial activity and strong cytotoxic activity,which was used for the study of secondary metabolites.Based on morphological observation and molecular biology,GQ-6 was identified as Cladosporium and named Cladosporium sp.GQ-6.（3）The target strain GQ-6 was fermented on a large scale,and its crude extracts were verified by activity screening.Then,the crude extract of GQ-6 was isolated and purified by various chromatographic techniques.Finally,six compounds were identified,including two known compounds α-acetylorcinol（1）and cladosporester B（2）,two compounds with new spatial configuration,cladosporester B2（3）and seco-Patulolide C2（4）,as well as two new compounds cladosporacid F（5）and cladosporester D（6）.The results of activity determination of the six compounds showed that these compounds had weak antibacterial activity but no obvious tumor cytotoxic activity.The results showed that when the concentration was512μg/m L,compounds 2,5 and 6 displayed more than 50% inhibition rates against F.graminearum,F.moniliforme,Botrytis cinereal and Verticillium dahlia.Compounds 3 and 4had inhibition rates of more than 50% against F.graminis,F.moniliforme and B.cinerea.In addition,at the concentration of 512μg/m L,the inhibition rates of compound 2 against Escherichia coli,S.aureus,S.enteritidis and Pseudomonas aeruginosa were all above 50%. |
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