| Endophytic fungi are a group of microorganisms that are mainly parasitic in the internal tissues or organs of plants and do not cause significant tissue damage to their hosts.Endophytic fungi have the potential to produce chemicals identical or similar to their hosts during their long-term symbiosis with the host,and are one of the hot research topics in the field of natural products.Orixa japonica Thunb is a genus of Orixa japonica in the family Rutaceae.At present,the research on Orixa japonica at domestic and international are mainly focused on the chemical composition,and the research on its endophytic fungi is relatively little or neglected.In order to explore whether the same or similar compounds as O.japonica plants can be obtained from O.japonica endophytic fungi and to seek new strategies to obtain the active ingredients of O.japonica.The present study was conducted to classify and identify an endophytic fungus from the roots of O.japonica and to systematically study its secondary metabolites to provide a basis for future research on the application of O.japonica and its endophytic fungi.The study contents and results are as follows:（1）The strain was induced to produce conidia,strain DNA was extracted and a phylogenetic tree was constructed,information was compared by BLAST with the Gene Bank database,and based on phylogenetic analysis（combining ITS and LSU sequence data）and morphological evidence,the strain was taxonomically identified as a new species belonging to the genus Amphisphaeria,named Amphisphaeria orixae.（2）The strains were subjected to bulk fermentation and the ferments were extracted using ethyl acetate:methanol（10:1）at room temperature.The metabolites were purified using various chromatographic techniques,and the compounds were identified by measuring the spectral data and combining with the physicochemical properties.23 monomeric compounds（one of which was new）were identified,and the types of compounds included alkaloids,phenylpropanoids,steroids,flavonoids,etc.Ten of these compounds were previously obtained in the roots of the plant Orixa japonica.All compounds were obtained for the first time from the fungus of the genus Amphisphaeria.（3）Some of the isolated compounds were assayed for antibacterial,antitumor and antioxidant biological activities,among which compounds 1,6,8,9 and 14 showed different degrees of inhibition against E.coli,S.aureus,P.aeruginosa and C.albicans at a concentration of 0.25 mg/m L and the alkaloids were more active than coumarins;The antitumor assay showed that the test compounds inhibited human breast cancer cells（MCF-7）better than human pancreatic cancer cells（PANC-1）and human liver cancer cells（HepG2）in the range of 0-200μmol/L,The inhibitory activity of coumarins was stronger than that of alkaloids,but the inhibitory activities were not significant,and their IC50 were all greater than 50μmol/L.Antioxidant assays showed that compound9 exhibited significant antioxidant activity with an IC50 value of 8.56μg/m L. |
