Study On Active Metabolites Of GRP13 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.In this paper,an endophytic fungus named GRP13 was chosen as the research object,which was isolated from the root of Glycyrrhiza uralensis,and the fermentation condition of GRP13 was optimized using single-factor test combined with orthogonal experimental design with antimicrobial activities as the index.Under the optimal process,fermentation broth of GRP13 was separated and purified by silica gel column chromatography and macroporous resin,Then,the structures of these compounds were identied by HPLC,MS,¹H-NMR,and ¹³C-NMR,meanwhile,the antibacterial activities and antioxidant activities were determined.Results were as follows:The optimized fermentation process of GRP13 was as follow:the glucose of carbon source was 2.0%(m/v),the peptone of nitrogen source was 1%(m/v),the inoculation amount was 5%(v/v),the initial pH value was set at 7.0,the fermentation temperature was 28?,speed of shaker was 140 r·min^-1,fermentation volumn was set at50%(v/v)and the fermentation time was 14d.and the diameter of antimicrobial activity in the best fermentation condition is increased to 22.1±0.20mm,which was better than the initial fermentation condition.10L fermentation broth of GRP13 was extracted by ether,ethyl acetate and n-butanol,respectively.The extract parts were separated and purified by silica gel column chromatography,crystal 1 and 2 were obtained from ether extract part,crystal 3was obtained from ethyl acetate extract part and crystal 4 was separated from mycelium;meanwhile,10L fermentation broth of GRP13 were separated by macroporous resin,and crystal 5 was obtained.HPLC,MS,¹H-NMR and ¹³C-NMR were used to identified the structures of the compounds,and crystal 1 was proved to be benzoic acid,crystal 3 was determined as pseurotin A and crystal 5 was liquiritigenin.But structure of crystal 4 was too complex to analysis,and structure of crystal 2 is uncertain due to the easy oxidation and the very low content.The Escherichia coli and Staphylococcus aureus were chosen as the test strains to determine the antimicrobial activity of these crystals,the result showed:crystal 1 has the strongest antibacterial activity,the diameter of inhibition zone of Escherichia coli was 33.1mm,the effect of Staphylococcus aureus was reached to 30.0mm,followed by crystal 5,inhibition zone diameters to Escherichia coli and Staphylococcus aureus of crystal 5 were 30.4mm 29.8mm respectively,the antibacterial effect of crystal 3 and 4were relatively weak.Antioxidant action of crystals were studied by(DPPH)free radical method,the results indicated that crystal 5 had scavenging effects on DPPH radicaland the scavenging percent decreases to 51.97%,close to 66.85%of Vc removal rate in the same concentration,crystal 1,3 and 4 showed weaker effects on the DPPH radical.As conclusion,benzoic acid,pseurotin A and liquiritigenin were separated from the fermentation broth of GRP13,thus proves that GRP13 strain is a Glycyrrhiza uralensis endophytic fungus that has wild application prospect in future..