Study On Antitumor Metabolites From Three Endophytic Fungi Of Huperzia Serrata

Huperzia serrata,a traditional Chinese medicine Qian Ceng Ta,is a fern and belongs to Huperzia of Huperziaceae.Finding active compounds from endophytes in Huperzia serrata can provide a new way for further exploitation resources of medicinal plants of Huperzia serrata.Many kinds of endophytic fungi associated widely with various plants.However,there are a little systemic researches focusing on secondary metabolites of endophytes in fern.However,the research of endophyte in fern is rare.The purpose of this dissertation was to search for antitumor active compounds from endophytic fungi of Huperzia serrata.We investigated the chemical composition of cytotoxic activity from endophytic fungi of Huperzia serrata.Methods:Three endophytic fungi were identified the fungus species by the part ITS sequences which were amplified by ITS4 and ITS5 universal primers using a modified CTAB method to extract total DNA and then BLAST to GenBank of NCBI.The identified endophytic fungi of Huperzia serrata were fermented on solid medium in a large scale.The compounds of the fermented extracts were separated and purified by column chromatography methods involving normal-phase silica gel,Sephadex LH-20,preparative thin layer chromatography(TLC)and high performance liquid chromatography(HPLC).Their structures of compounds were elucidated by their physical and chemical properties,nuclear magnetic resonance(NMR)spectroscopy,electrospray ionization mass spectrometry(ESI-MS)and with reference to the reported literatures.Antitumor activities were tested by the MTT assay.The antibacterial activities of these compounds were assayed by enzyme-labeling instrument.This paper was composed of four chapters.The first chapter is a brief review of the secondary metabolites of the Huperzia serrata endophytes.The second chapter studied the secondary metabolites of Chaetomium sp.M336,while the third chapter focused on the chemical compounds of Colletotrichum sp.MF453 and the last chapter focusd on the chemical compounds of Fusarium sp.MS545.29 compounds including 9 new compounds were identified from aforementioned three endophytic fungi of Huperzia serrate.The major types of compounds belonged to polyketides,steroid and pyridine deriveratives.100 endophytic fungi strains were fermented on PDA,SDA,and modified Fries medium rsepectively.Three endophytic fungi strains which showed strong anti-tumor activity were selected and the secondary metabolites were investigated.In the study of significant cytotoxic Chaetomium sp.M336,14 compounds including 5 new ones were isolated and identified.The strutures mainly belonged polyketide.5 new compounds were tested for anti-tumor activity.The compoud 5 exhibited strong activities against breast cancer cell line HeLa,lung cancer cell line A549 and gastric carcinoma cell line SGC 7901,the IC50 values were 0.016 ?g/ml,0.020 ?g/ml,0.017 ?g/ml.The anti-bacterial activity test of 5 new compounds showed that 1,2,3,4,5 have a certain activity against E.coli,Staphylococcus aureus,Salmonella typhimurium ATCC 6539,Enterococcus faecali.The compound 5 displayed a significant anti-bacterial activity(MIC value of 0.78 ?g/mL).8 compounds were obtained from Colletotrichum sp.MF453 including 4 new ones and 7 compounds were obtained from Fusarium sp.MS545.