Study On Active Metabolites Of Endophytic Fungus GRE9 In Licorice

Glycyrrhiza uralensis,an endangered medicinal plant,which is native to northeast of China.In this work,endophytes in Glycyrrhiza uralensis were chosen as an effective alternative or novel source for producing the same,similar or new active secondary metabolites that are also biosynthesized by their host plants.The production of bioactive compounds by endophytes,especially those exclusive to their host plants,is not only important from an ecological perspective but also from a new medicine and food standpoint.The endophytic fungus GRE9 was selected as the research object,and its morphological and molecular biological identification was carried out to determine the species of the strain.Meanwhile,the composition of GRE9 strains fermented broth was analyzed by HPLC,and the volatile substances of GRE9 strains fermented broth and its mycelium were analyzed by GC-MS;Afterwards,the fermentation broth of endophytic fungus GRE9 was separated and purified by silica gel column chromatography and macroporous resin.Then,the structures of these compounds were identied by TLC,HPLC,MS,¹H-NMR,and ¹³C-NMR,and the antibacterial activities and antioxidant activities of these compounds were also determined.Results were as follows:The endophytic fungus GRE9 was identified as Penicillium polonicum by the methods of Morphological observation and ITS r DNA sequence analysis.The fermentation broth of GRE9 strains was extracted by n-butanol,ethyl acetate and ethyl ether,then,the composition of three extractive site were analyzed by HPLC respectively.The results showed that among all fractions with different polarities,the ether fraction and ethyl acetate fraction were effective fractions for extracting and separating active metabolites subsequently.The GC-MS was used to isolate and identify the volatile compounds of the endophytic fungus GRE9 fermented broth and its mycelium,the results indicated that the volatile components in the GRE9 fermented broth and its mycelium not only contained the same with the host crude drugs,but also contained the differences substances.The extractive fractions of GRE9 fermented broth were separated and purified by silica gel column chromatography,and compounds A and B were obtained from ether extracted fraction,compound C was obtained from ethyl acetate extracted fraction.Then,the fermentation broth of GRE9 was separated by D₁₀₁ macroporous resin and eluted by graded ethanol,compound D was obtained from 90%ethanol eluate fraction and compound E was obtained from 70%ethanol eluate fraction.TLC,HPLC,MS,¹H-NMR and ¹³C-NMR were used to identified the structures of these compounds,and compound A was proved to be glycyrrhetinic acid,compound B was identified as umbelliferone,compound C was determined as ferulic acid,comp-ound D was determined as liquiritigenin and compound E was liquiritin.The activity of each compound was studied,the antimicrobial results showed that five compounds showed some different antibacterial activities,and compound D had the most obvious antibacterial activity than others.The antioxidant results showed that compound C had the strongest antioxidant activities and the clearance rate of(DPPH)free radical was reached at 86.4%.As conclusions,this project obtained five compounds from the fermentation broth of GRE9,thus proves that GRE9 strain is a Glycyrrhiza uralensis endophytic fungus which has wild application prospect in future.