Bioactivity And Metabolomics Analysis Of Metabolites Of Endophytic Fungi Isolated From Brassica Rapa L.

Objective: This study will isolate and identify the endophytic fungi of Brassica rapa L.,and identify if they have the activities of anti-tumor,anti-bacterial and antioxidant,Subsequently,metabolomics analysis was carried out for the metabolites.And provide the endophytic fungi resources for the development and utilization of microbial active products in the future.Methods: 1)Using traditional microbial isolation and culture technology to isolate the endophytic fungi from the Brassica rapa L.grown in Xinjiang.And through the ITS sequencing technology,the sequence is compared with the similar sequence in Gene Bank,the phylogenetic tree was constructed using MEGA,to characterize the isolated endophytic fungi by molecular biology.2)Human non-small cell lung cancer cell line(A549),human liver cancer cell line(Hep G2),human prostate cancer cell line(PC-3),human gastric cancer cell line(SGC-7901),cervical cancer cell line(Hela)were used to identify the activity of cytostatic effect of the ethyl acetate extracts of the isolated Brassica rapa L.endophytic fungal metabolites at 100μg/m L and 200μg/m L by the CCK8 assay.3)The ethyl acetate extract of the metabolite of endophytic fungus at 1mg/m L was identified against standard strains of Candida Albicans,Escherichia coli,Staphylococcus aureus、Pseudomonas aeruginosa by the Kindy-Bauer assay.4)The T-AOC and DPPH assays were used to identify the antioxidant activity of ethyl acetate extract of endophytic fungi.5)The ethyl acetate extracts with active strain metabolites were identified for their anti-tumor activity and research the anti-tumor mechanism in the tumor-bearing mouse model,according to results of the tumor suppression rate,Western-blot,real-time fluorescent quantitative PCR,TUNEL assays.6)The metabolites of the active strains and other strains were detected by GC-TOF-MS,then compared and analyze the specific components in the metabolites of active strains.Results: 1)A total of 40 endophytes were isolated from the leaves,tubers,and fibrous roots of Brassica rapa L.,Through ITS sequencing and analysis of the phylogenetic development tree constructed,a total of 15 endophytic fungi of Brassica rapa L.were isolated.They were Alternaria sp.,Cladosporium sp.,Corallomycetella sp.,Cryptococcus sp.,Pseudogymnoascus sp.,Rhodotorula sp.,Vishniacozyma sp.,among which Cladosporium sp.is dominant.2)The metabolites of endophytic fungi extracted with ethyl acetate,which were identified the cytostatic activities on 5 cell lines by CCK8 assay.The results showed that the ethyl acetate extracts of Pr10(Alternaria sp.)have a cytostatic effect on A549 at 200μg/m L(growth inhibition rate is 55%).3)The ethyl acetate extracts of endophytic fungi were identified the antibacterial activity against Candida Albicans,Escherichia coli,Pseudomonas aeruginosa,Staphylococcus aureus at 1mg/m L by KindyBauer assay.4)Through the T-AOC and DPPH assays,the DPPH result showed that the endophytic fungus Pr10(Alternaria sp.)has antioxidant activity compared with other fungi(82%).5)The A549 tumor-bearing mouse model was constructed,then the ethyl acetate extracts of Pr10 and Pr8 metabolites were injected intradermally at 200mg/kg daily.After 10 days,measured the tumor weight and volume.The results show that the tumor inhibition rate of Pr10 was 56%.Compared to the control group,it had the effect of inhibiting tumors(P<0.01).the HE assays showed dead cells observe in the group Pr10.TUNEL assay showed apoptosis index in group Pr10 was greater than that in the control group(P<0.05).The expression of Bcl-2 and Caspase3 protein was detected.Compared with the control group,the expression of Bcl-2 decreased in the group Pr10(P<0.05),and the expression of Caspase3 increased(P<0.05).Therefore,it is inferred that the metabolite of Pr10 could induce tumor cell apoptosis in A549-bearing mice by down-regulating Bcl-2 and upregulating the expression of Caspase3.6)Untargeted Metabolomics was utilized to research the metabolic characteristics of four endophytic fungi Pr10,Pr6,Pr7,Pr8 by GC-TOF-MS assay.The metabolites were compared and analyzed by metabolomic analysis methods.The results showed that Pr10’s metabolites are rich in amino acids and sugar derivatives such as trehalose,whose antitumor activity to the tumor cell line and antioxidant activity have been proved.Conclusion: 15 endophytic fungi were isolated from Brassica rapa L.,and they were 7 genus fungi.And the metabolites of Pr10(Alternaria sp.)has antitumor activity,according to the result of antitumor assays in vitro and in vivo,and metabolomics comparison analysis of active products,as result,the metabolites of endophytic fungus Pr10 from Brassica rapa L.have antitumor and antioxidant activity.The active ingredients of its metabolites have the potential for drug development,providing a scientific basis for the antitumor and antioxidant activity of the endophytic fungi from Brassica rapa L..