Bioactivities Of The Secondary Metabolites Of Endophytes Isolated From Ginkgo Biloba Exocarp

Ginkgo biloba is a gymnosperm.The seed of Ginkgo biloba is termed as its“nut”and consists of fleshy exocarp,osseous mesocarp,and membranous endocarp.The exocarp accounts for 75%of the total weight of the seed.During the harvesting season,the abandoned exocarp pollutes streams,ponds,and weirs in Ginkgo-producing areas.To solve this problem,peasants use local methods to prepare various extracts of exocarp and spray them into fields and forests.This strategy causes poisoning in some pests.G.biloba exocarp remarkably inhibits fungi and bacteria.Endophytic bacteria in G.biloba exocarp have been barely studied.To evaluate the activity of endophytic bacteria and their secondary metabolites in G.biloba exocarp,we isolated endophytes from fresh G.biloba seed exocarp.After isolation and identification,strains were selected for fermentation on the basis of their present and previously observed characteristics.The activities of secondary metabolites were also studied.Eight endophytes were isolated from the G.biloba exocarp.These endophytes included three endophytic bacteria belonging to Bacillus,Methylobacterium,and Sphingomonas and five endophytic fungi belonging to Psathyrella,Alternaria,Penicillium,Ophiosphaerella,and Aspergillus.After fermentation and production studies,Psathyrella candolleana F1-y and Alternaria brassicae F2-wb were selected as the starting strains for product isolation,identification,and activity evaluation.P.candolleana F1-y produces yellow pigments.The organic phase extracted with ethyl acetate after fermentation showed good biological activity.The substance inhibited Staphylococcus aureus at an MIC value of 0.78 mg/mL but poorly inhibited Escherichia coli.The substance also affected the scavenging of DPPH(IC₅₀=567.53?g/mL)and hydroxyl radicals(IC₅₀=4.68 mg/mL).Eight compounds were isolated by repeatedly separating ethyl acetate through the use of macroporous resin,silica gel column,Sephadex LH-20 column,ODS column,and other columns.The eight compounds identified were ergosterol?1?,2'-hydroxychalone?2?,myristic acid?3?,cis-9-octadecanolamide?4?,quercetin?5?,benzoic acid?6?,uracil?7?,and nicotinamide?8?.Compound 5 showed excellent antioxidant activity against DPPH(IC₅₀=14.54?g/mL).Compounds 5,6,and 7 exhibited antimicrobial activity against S.aureus at MIC values of 0.39,0.78,and 6.25 mg/mL,respectively.After A.brassicae F2-wb was cultured,the media emitted an evident fruity aroma.At a time node in each period,the composition of the volatile organic compounds in the fermentation products was studied.In the lag phase,cell growth was slow and secondary metabolites showed the highest acid content,the largest amount of alkenes,and additional alcohols,of which 7H-benzocycloheptene and10-octadecenoic acid methyl ester were unique ingredients.Hence,alkenes may be used as a marker compound in the early stage of A.brassicae growth.In the logarithmic phase,the cells grew rapidly,in contrast with the lag phase.Ketone content remarkably increased with low olefin content,and alcohol content peaked.Among the alcohols,terpinen-4-ol and 6.32%of 2,5-dihydroxyacetophenone may be used as marker compounds.During the stable phase,cell content reached the maximum,alkene substances disappeared,and alcohol content decreased.However,ester content increased sharply,reaching 33.67%.Amines also began to appear,and ester content increased.Among the amines,4-isopropylbenzaldehyde was not detected in other periods.Compared with the three other periods,the decline period was characterized by few components that mainly include numerous acids,some amines of esters,and few or no other components.The appearance of numerous amines may be a sign of cell aging and death.The detection of antioxidant activity and tyrosinase inhibition showed that the volatile organic compounds produced by F2-wb had strong DPPH,hydroxyl radical scavenging and reducing ability,and tyrosinase scavenging activity.The compounds also exhibited whitening effect.After 10 days of culture,F2-wb had the strongest DPPH(IC₅₀=0.56 g/L),hydroxyl radical scavenging ability(IC₅₀=0.47 g/L),reducing ability,and tyrosinase inhibition ability(IC₅₀=5.18 g/L).These properties may be due to the large amount of ketones and alcohols produced during the logarithmic phase.