Study On Active Metabolites Of GRP10 Endophytic Fungi Of 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 heir host plants,is not only important from an ecological perspective but also from a new medicine and food standpoint.The endophytic fungus GRP10 was selected as the research object,and its morphological and molecular biological identification was carried out to determine the species of the strain.The fermentation condition of GRP10 was optimized using single-factor test combined with orthogonal experimental design with antimicrobial activities as the index.Under the optimal process,the secondary metabolites in fermentation broth of GRP10 were separated and purified by silica gel column chromatography.Then,the structures of these compounds were elucidated by HPLC,MS,¹H-NMR and ¹³C-NMR.meanwhile,the antibacterial activities and antioxidant activities were determined.Results were as follows:The endophytic fungus GRP10 was identified as Fusarium oxysporum by the methods of Morphological observation and 18S r DNA sequence analysis.The optimized fermentation process of GRP10 was as follow:the glucose of carbon source was 2.0%(m/v),the inoculation amount was 5%(v/v),the initial p H value was set at 7.0,the fermentation temperature was 28?,speed of shaker was 140 r·min^-1,fermentation volumn was set at 50%(v/v)and the fermentation time was 15d.And the diameter of antimicrobial activity in the best fermentation condition is increased to22.6±0.30mm,which was better than the initial fermentation condition;The content of vanillic acid in the fermentation broth of endophytic fungi GRP10 was determined by high performance liquid chromatography(HPLC),When the fermentation time reached15 days,the content of vanillic acid in fermentation broth reached 2.0090?g·ml^-1,The experiment further confirmed the correct fermentation conditions of the process.10L fermentation broth of GRP10 was extracted by ether,ethyl acetate and n-butanol,respectively.The extract parts were separated and purified by silica gel column chromatography and crystal A,B and C were obtained from ether extract part,crystal D and E was obtained from ethyl acetate extract part.HPLC,MS,¹H-NMR and ¹³C-NMR were used to identified the structures of the compounds,and crystal A was proved to be trans-cinnamic acid,crystal B was proved to be benzoic acid,crystal C was determined as glycyrrhetinic acid,and crystal D was vanillic acid.The antimicrobial activity of each monomer was studied,it was proved that each crystal showed some antibacterial activity.Antioxidant activities of crystals were studied by(DPPH)free radical method,the results indicated that crystal D had scavenging effects on DPPH radical and the scavenging percent decreases to 70.81%,close to 97.22%of Vc removal rate in the same concentration.As conclusion,trans-cinnamic acid,benzoic acid,glycyrrhetinic acid and vanillic acid were separated from the fermentation broth of GRP10,thus proves that GRP10strain is a Glycyrrhiza uralensis endophytic fungus which has wild application prospect in future.