The Studies On Isolation,Identification And Its Secondary Metabolites Bioactivity Of Endophytic Fungi From Four Medicinal Plants

Endophytic fungus is a class of huge system and special habitat microbial resources,which shows its life in plant healthy organizations and internal organs on a certain stage or allstages. Meanwhile, endophytic fungus does not cause obvious symptoms of disease, and therelationship with the host plants is a structure of mutualistic symbiosis. Endophytic fungiconstitute a large, special fungal group with abundant secondary metabolites, includinganti-tumor, anti-microbial and insecticidal agents. They are important resources for newanti-microbial and anti-cancer drugs. In view of this characteristic, four medicinal plants werechosen to carry some preliminary researches on their fungi, their bioactivities and thecompounds from the secondary metabolites. The main contents and results were as follows:1.53strains endophytic fungi were isolated from4medicinal plants:14strains fromEuphorbia pulcherrima,14strains from Pinellia ternate (Thunb.) Breit,13strains fromAsarum heterotropoides var. mandshuricum,12strains from Stellera chamaefasmeendophytes. The culture broth was filtered to separate the mycelium and the filtrate. Themycelium and the filtrate were extracted to afford3kinds of extract parts were gained asfollows: ethyl acetate extract, n-butyl alcohol extract and ethanol extract of mycelium.2. Modified Ellman’s method, MTT method and filter diffusion method were used toevaluate the bioactivities of extracts. The result showed that,7endophytic extracts hadremarkable AChE inhibitory activity, of which the inhibition rates were more than50%. Then-butyl alcohol extract of XX-10was the most potent extract with inhibition rate of79%.Anti-tumor results showed that a total of56samples had different levels inhibitory activitiesto Hela cells,4samples were higher than50%, in which,3samples were from Euphorbiapulcherrima,1sample was from Asarum heterotropoides var. mandshuricum. Theantibacterial results showed that58.1%of Euphorbia pulcherrima,40%of Asarumheterotropoides var. mandshuricum,37.8%of Pinellia ternate (Thunb.)Breit,57.1%ofStellera chamaefasme displayed inhibition activities against at least one of six indicatororganisms. The sample BX-8(s) showed obvious inhibition against Escherichia coli. andBacillus licheniformis.3. Using different separation techniques including re-crystallization, columnchromatography and thin layer chromatography,4compounds were separated from strainBX-9and BX-8, which were identified to (+)-BrefeldinA(1),2-Hydroxy-14-tricosenvicacid(2), ergosterol(3), ergosterol peroxide(4). Among them,(+)-BrefeldinA (1) is ahydrophobic macrolide antibiotic; moreover,(+)-BrefeldinA and its derivatives are antitumordrug candidates. It is worth mentioning that the yield of (+)-BrefeldinA was high (100mg/L)which showed large business prospects.4. The antibacterial activity and the antioxidant activity of the compounds1-4were also tested. The results showed that ergosterol(3) inhibited Staphylococcus aureus in a strongelevel. Studing the cytotoxicity of compounds, we found the inhibition of compound1againstHela、HepG2and A549had a threshold. When the concentration was less than1μg/mL, thecytotoxicity was very weak, while the concentration ranged from1μg/mL to50μg/mL, thecytotoxicity was strong. In a final concentration of100μg/mL, the inhibitory effect on cancercells of compounds3and4were very remarkable. When the final concentration became to50μg/mL, the cytotoxicity of compound3was still strong, and the cytotoxicity of compound4was significantly reduced.5. By using molecular biology method, including analyzing the ITS rDNA assay,contrasting with other high homology of gene in the Blast system and observing the color andthe mycelia of colony morphology, strain BX-9and BX-8were identified as Penicillium sp.and Colletotrichum sp., respectively.