| Objective:To study the secondary metabolites of two endophytic fungi of Aconitum plants,namely Alternaria alternata WT-31 and Penicillium virgatum AV-45,and to discover resource strains and active natural products.Methods:1.The endophytic fungi from Aconitum scaposum Franch.and Aconitum vilmorinianum Kom.were isolated by plate dilution method.,whose DNA were extracted for ITS gene sequence detection.The isolated endophytic fungi were identified based on the comparation of their ITS gene sequences with those in the NCBI gene database using BLAST method,which was aided by morphological identification.All of these identified endophytic fungi in the rice solid medium,and their fermentation products were ultrasonically extracted with methanol.The secondary metabolite extracts were detected by TLC,and the target endophytic fungi for further secondary metabolite research were selected according to the diversity of the chemical components of the secondary metabolites along with the growth conditions of the corresponding endophytic fungi.2.Two fungi,Alternaria alternata WT-31 and Penicillium virgatum AV-45,were further fermented with rice solid culture medium.The fermentation products were ultrasonically extracted with methanol,and then extracted by ethyl acetate,which was concentrated under reduced pressure to afford the total extracts.Separation and purification of secondary metabolites in total extracts were performed by comprehensive use of normal phase silica gel column chromatography,Sephadex column chromatography,and preparative HPLC.The purified monomeric compounds were structurally identified by MS,NMR spectroscopy.Some of the identified compounds were tested for their bacteriostatic activities.Results:1.A total of 17 endophytic fungi were identified from Aconitum scaposum Franch.and Aconitum vilmorinianum Kom.,most of belong to Ascomycota and Sordariomycetes.The endophytic fungi A.alternata WT-31 from Aconitum scaposum Franch.and the endophytic fungus P.virgatum AV-45 from the roots of Aconitum vilmorinianum Kom.were selected for further research.2.A total of 19 compounds were isolated and identified from the fermentation p roducts of these two fungi,all of which were known compounds.Among them,nine compounds were isolated from the endophytic fungi A.alternata WT-31,inc luding alternariol（1）,alternariol-5-O-monomethyl ether（2）,3-O-β-D-glucopyranosyl-alternariol-5-O-monomethyl ether（3）,altenuene（4）,cerevisterol（5）,syringaldehy de（6）,p-hydroxybenzaldehyde（7）,3,5-dihydroxybenzoic acid（8）,and linoleic aci d（9）.Compounds 6 and 8 were isolated from the genus Alternaria for the first time.In addition,ten compounds were isolated from the endophytic fungus P.vir gatum AV-45,namely（2R,3S）-1-（4-hydroxyphenyl）butane-2,3-diol（10）,cyclonerodi ol（11）,demethylincisterol A3（12）,ergosterol 5,8-peroxide（13）,cerevisterol（14）,β-sitosterol（15）,3-acetyl-β-sitosterol（16）,1-monoolein（17）,10-eicosenoic acid（18）,and methyl 12,13-epoxyoleate（19）.Among them,compounds 10,12 and 19were isolated from the genus Penicillium for the first time.The results of antib acterial activity experiments showed that compounds 1-3,5,and 9 had certain an tibacterial activities.Conclusion:1.The endophytic fungi of Aconitum scaposum Franch.were studied for the first time.And a total of 17 endophytic fungi belong to three phyla and 14 genera.The results revealed the diversity of species for the endophytic fungi in the Aconitum plants.2.A total of 19 compounds isolated from two endophytic fungi of two Aconitum plants,among which five compounds were discovered from corresponding genera for the first time.Some of compounds showed antibacterial activities.The research showed that the endophytic fungi of Aconitum possess research value and development potential,which provides basis and new ideas for the development and utilization of Aconitum plant resources. |
