Study On The Secondary Metabolites Of Endophytic Fungi From Callicarpa Kwangtungensis Chun

Endophytic fungi are microorganisms that live in or colonize healthy plant tissues at a certain stage of their life cycle,but do not produce obvious disease or infection to host plants.Plant endophytic fungi are an important source for the discovery and preparation of lead compounds,which on the one hand can discover compounds of different structures and biological activities from their secondary metabolites based on activity screening,and on the other hand can be mined based on certain gene clusters of their shared host plants that share the same medicinal molecules as the host plants.The chronic diseases gout and diabetes chronically disturb human healthy life,xanthine oxidase andα-Glucosidases are effective targets for the treatment of above two diseases,respectively.Search for naturally derived xanthine oxidase inhibitors andα-Glucosidase inhibitors are of great research value.This study focused on the secondary metabolites of xanthine oxidase andα-glucosidase inhibitory activity in endophytic fungi of medicinal plant Callicarpa kwangtungensis Chun:（1）isolation and activity screening of fermentation extracts from endophytic fungi isolated from different parts of C.kwangtungensis;（2）secondary metabolites of xanthine oxidase inhibitory activity of endophytic fungus Alternaria alternata GDZZ-J6;（3）secondary metabolites of xanthine oxidase inhibitory activity of endophytic fungus Penicillium citrinum GDZZ-R13;（4）secondary metabolites ofα-glucosidase inhibitory activity of endophytic fungus Fusarium incarnatum GDZZ-G2.A total of 17 endophytic fungi were isolated from the roots,stems,leaves and fruits of C.kwangtungensis by the tissue block method,including 4 from the roots,5from the stems,4 from the leaves and 4 from the fruits.The in vitro selection model was used to screen xanthine oxidase andα-glucosidase inhibitory activity from fermented extracts of 17 endophytic fungi.Among them,the fermented extracts from the endophytic fungi GDZZ-J6 and GDZZ-R13 showed significant inhibition of xanthine oxidase with 72.19%±0.93%,75.71%±0.46%,respectively（200μg/ml）,the fermented extracts from the endophytic fungi GDZZ-G2 showed significant inhibition ofα-glucosidase with 98.22±2.14%（400μg/ml）.The endophytic fungi GDZZ-J6,GDZZ-R13 and GDZZ-G2 were identified as Alternaria alternata,Penicillium citrinum and Fusarium incarnatum by means of morphology and molecular biology.Six compounds were isolated from the fermentation extr act of endophytic fungus A.alternata GDZZ-J6 by ODS column chromatography and preparative high performance liquid chromatography.The structure of the compounds was identified asα-acetylorcinol（A1）,2-hydroxy-6-methylbenzoic acid（A2）,alternariol（A3）,alternariol9-methylether（A4）,ergosterolperoxide（A5）,（3β,22E）-ergosta-5,7,22-trien-3-ol（A6）.Among them,alternariol and alternariol9-methyl ether had strong inhibitory activity on xanthine oxidase,and the IC ₅₀ value were 0.23±0.01μM and 0.46±0.03μΜ,respectively.Their inhibitory effect was significantly stronger than that of allopurinol(IC ₅₀=2.98±0.07μM).The IC₅₀values of three dibenzo-α-pyrones,urolithins A,B,and C,against XO were further compared,and their structure-activity relationships were discussed.The enzyme inhibition kinetics showed that the inhibition type of alternariol was uncompetitive inhibition.Molecular docking showed that hydrogen bond and hydrophobic interactions were the main forces for the combination of alternariol with xanthine oxidase substrate complex.Eight compounds were isolated from the fermentation extract of endophytic fungus P.citrinum GDZZ-R13 by ODS column chromatography and preparative high performance liquid chromatography.The structure of the compounds was identified as 4-hydroxy phenylacetic（R1）,4-hydroxybenzoic acid（R2）,4-hydroxybenzaldehyde（R3）,2-hydroxy phenyl acetic acid（R4）,2-（6-hydroxy-5,7-dimethylbenzofuranone-4-yl）acetaldehyde（R5）,Penicitrinone A（R6）,Citrinin（R7）,2’,3’-Dihydrosorbicillin（R8）.The inhibitory activities of the eight compounds on xanthine oxidase were<25%（200μM）.Eight compounds were isolated from the fermentation extract of endophytic fungus F.incarnatum GDZZ-G2 by ODS column chromatography and preparative high performance liquid chromatography.The structure of the compounds was identifiedasN-isobutylacetamine（G1）,2-（2’S-hydroxypropyl）-5-methyl-7-hydroxychromone（G2）,（S）-1,3,6-trihydroxy-7-（1-hydroxyethyl）anthracene-9,10-dione（G3）,CyclonerodiolB（G4）,Equisetin（G5）,22E,24R-ergosta-7,22-diene-3β,5α,6β,9α-tetraol（G6）,Palmitic acid（G7）,N-2’-hydroxy-3’E-octadecenoyl-1-O-β-D-glucopyranosyl-9-methyl-4E,8E-sphingadi ene（G8）.Among them,（S）-1,3,6-trihydroxy-7-（1-hydroxyethyl）anthracene-9,10-dione showed strongest inhibitory activity onα-glucosidase.The IC₅₀ of（S）-1,3,6-trihydroxy-7-（1-hydroxyethyl）anthracene-9,10-dione was 77.67±0.61μM,which was much lower than acarbose（711.8±5.0μM）.Enzyme inhibition kinetics showedthattheinhibitiontypeof（S）-1,3,6-trihydroxy-7-（1-hydroxyethyl）anthracene-9,10-dione was uncompetitive.Molecular docking showed that hydrogen bond and hydrophobic interactions were the main forces for the combination of active compound withα-glucosidase-substrate complex.This study provides guidance for the development and application of endophytic fungal resources of C.kwangtungensis.