Isolation,Identification And Characterization Of An Endophytic Fungus From Ageratina Adenophora With Antioxidant Activity

As a new microbial resource,endophytes have many biological activities,such as antibacterial,antitumor,antioxidant,insecticidal and so on,because of their mutually beneficial symbiotic relationship with host plants.As a poisonous plant,Ageratina adenophora's special internal environment is more conducive to the growth of some special active endophytic fungi.Therefore,A.adenophora is an ideal plant for finding supplementary materials fungi which can synthesize special metabolites.In order to provide theoretical basis for the development and utilization of A.adenophora and the screening of new antioxidants,the A.adenophora was selected as the research object,and the endophytic fungi in the leaf tissue were isolated and the activity of its metabolites was determined.The results are as follows:Methods:Endophytic fungi were isolated from leaves of A.adenophora by surface disinfection and tissue isolation methods.The best antioxidant strains were screened by scavenging DPPH free radical activity of fermentation broth and identified by molecular biology.The fermentation time was divided into 5,7,9,11,13,15 and 17 days.The initial pH of fermentation was divided into 3,4,5,6,7,8,9,10 and 11.The pH,ultraviolet light and temperature stability of fermentation broth were determined.Finally,silica gel column chromatography,gel column chromatography and high performance liquid chromatography were used to separate the metabolites.The structures were identified by IR,MS and NMR.Finally,the antioxidant capacity and the toxicity of mouse embryonic fibroblasts were tested.Results:1.Eight endophytic fungi of A.adenophora were isolated by tissue isolation and purified PDA medium.DPPH free radical scavenging rate of metabolites of eight endophytic fungi was determined,and the strain which with the best antioxidant activity was screened out and named F-8.The strain F-8 was identified by using classical morphological classification and molecular biology methods.Finally the results showed that F-8 was an endophytic fungus of the genus Coniochaeta sp.F-8.2.Optimization of fermentation conditions of F-8.The endophytic fungus F-8 of A.adenophora had the best antioxidant activity in the fourth group of N4 medium by homogeneous design,and the EC₅₀of DPPH free radicals in its fermentation broth was 175?g/m L.The antioxidant activity of DPPH free radicals in fermentation broth of strain F-8was determined at different fermentation time and under different pH conditions.The results showed that the antioxidant activity of fermentation broth of strain F-8 was the best when fermentation time was 11 days and pH=7.The EC₅₀of DPPH free radical was 328?g/m L and 266?g/m L,respectively.3.The results of fermentation broth stability of endophytic fungus F-8 from A.adenophora showed that the antioxidant active ingredients had good thermal stability,which had good thermal stability at about 70?.And the stability was the best in neutral alkaline environment.Ultraviolet light had a great influence on fermentation broth.With the extension of ultraviolet light time,the antioxidant activity of fermentation broth decreased.And the antioxidant activities of endophytic fungus F-8 fermentation broth were studied by testing the scavenging capacity of DPPH radical,superoxide anion(O^2-),hydroxyl radical(·OH)and reducing power.The results showed that compared with the positive control group,the fermentation broth had a better antioxidant activity,and its half scavenging concentration of DPPH radical,superoxide anion and hydroxyl radical were21.14?g/m L,1110.20?g/m L and 883.71?g/m L,respectively.4.Isolation and identification of metabolites of F-8 The compound 1 and compound 2were purified from the extract and identified by IR and NMR as phomopsichalasin A and Penialidin A,respectively.The antioxidant activities of compound 1 and compound 2 were determined.The results showed that the half scavenging concentration of compound 1 on DPPH radical,O^2-and·OH were 156.38?g/m L,1094.54?g/m L and 997.79?g/m L,separately,while the half scavenging concentration of compound 2 was 34.63?g/m L,847.44?g/m L and 683.31?g/m L,respectively.The results also showed that compound 2had better antioxidant activity than substance 1,while the reducing power of substance 1and compound 2 were both weaker than the positive control group.The cytotoxicity of compound 1 and compound 2 were determined by CCK-8 method.The results showed that there were significant differences in cell viability between compound 1 and blank control group at concentrations of 100?g/m L,200?g/m L and 400?g/m L(P<0.05),while compound 2 showed significant differences in cell viability at concentrations of 200?g/m L and 400?g/m L compared with the blank control group(P<0.05).The results showed that compound 1 had a stronger cytotoxicity than compound 2.Conclusion:Eight endophytic fungi were isolated from the leaf tissues of A.adenophora,of which strain F-8 had the best antioxidant activity.The ethyl acetate extract from fermentation broth also showed that strain F-8 could produce antioxidant substances.Two substances phomoxanthone A and Penialidin A were isolated and identified from the metabolites.Penialidin A had a better antioxidant activity than phomoxanthone A and also less cytotoxicity.It suggested that Penialidin A had the potential to become a natural antioxidant.