| Objective The endophytes resources with antioxidant,anticancer and bacteriostatic activities were extracted from the medicinal and food homologous plant P.thomsonii,and their active secondary metabolites were isolated and identified.Methods The endophytes were isolated by tissue homogenization method,purified by dilution coating method and line culture method,and identified by morphology combined with ITS and 16S r DNA sequence alignment and constructed phylogenetic tree;Extracts of different polarity of endophytic bacteria were obtained by small-scale liquid extraction with different solvents of methanol,ethyl acetate and petroleum ether,and their antibacterial activity against 12 strains of test bacteria,cytotoxicity of human lung cancer cell A549,and scavenging ability of 1,1-diphenyl-2-picrylhydrazine（DPPH）free radicals were measured.The strains with significant activity were screened for large-scale liquid fermentation,and the secondary metabolites were separated by silica gel column chromatography,gel chromatography,semi preparative liquid and other methods.Nuclear magnetic resonance spectroscopy,high-resolution mass spectrometry and other technologies were used to identify the structure of monomer compounds,and further through activity tracking,the monomer compounds with anti-cancer and antibacterial activities were screened.Results For the first time,20 strains of endophytic fungi were isolated from the root of P.thomsonii,including 11 strains of endophytic fungi namely:Penicillium Sp.FGZ4;Debaryomyces hansenii FGZ5;Phoma macrostoma FGZ6;Schizophyllum sp.FGZ9;Fusarium oxysporum FG1Z2;Porostereum crissum FGZ15;Bionectria Sp.FGZ20;Apiospora marii FGZ21;Clonostachys rosea FGZ35;Phoma herbarum FGZ36;Trametes sp.FGZ41,Seven strains of Gram-negative bacteria are Pseudomonas Sp.FGX4;Pseudomonas Sp.FGX8;Pseudomonas Sp.FGX20;Pseudomonas veronii FGX35;Enterobacter asburiae FGX1;Rahnella aceris FGX2;Lelliottia Sp.FGX18,Two strains of Gram-positive bacteria are Staphylococcus Sp.FGX48 and Paenibacillus Sp.FGX50.The preliminary screening results showed that the activity of the ethyl acetate extract of the endophytic fungus was generally good,and the endophytic fungus A Marii FGZ21 has the most significant bacteriostatic and cytotoxic activities.It has a strong inhibitory effect on multidrug resistant Enterococcus faecalis and methicillin-resistant Staphylococcus aureus.The inhibition circle is 23.76±0.04 mm,19.61±0.16 mm,MIC value is 1.563 mg/m L,0.098 mg/m L,MBC value is 1.563 mg/m L,0.195 mg/m L,respectively.At the same time,it has a significant inhibitory effect on human lung cancer A549 cells,IC50 value is 36.03μg/m L.The DPPH free radical scavenging effect of Penicillium Sp.FGZ4,an endophytic fungus of P.thomsonii is the best,with IC50 value of 0.085 mg/m L.Further from A Eight monomer secondary metabolites were isolated and identified from the ethyl acetate extract of A.marii FGZ21:p-Hydroxybenzoic acid（1）;2-Acetamidobenzoic acid（2）;Benzoic acid（3）;Dibutyl phthalate（4）;3,4-dimethoxybenzoic acid（5）;Schizostatin（6）;Tetrahydro-5-oxo-2-furancaboxylic acid ethyl ester（7）,2-Aminobenzoic acid（8）.In the assay of activity,eight monomer compounds have inhibitory effects on multidrug resistant Enterococcus faecalis and MRSA,the diameter of the inhibition circle is 9.21±0.01 mm～24.73±0.55 mm,and Dibutyl phthalate（4）has highly sensitive inhibitory effect on Streptococcus faecalis,the inhibition circle is as high as 24.73±0.55 mm;p-Hydroxybenzoic acid（1）?2-Acetamidobenzoic acid（2）?Benzoic acid（3）?Dibutyl phthalate（4）have significant inhibitory effects on the growth of A549 lung cancer cells,and Dibutyl phthalate（4）has the best cytotoxic activity,with IC50 value of 50.94μg/Ml.Conclusion For the first time,the endophytic bacteria of P.thomsonii were systematically isolated and identified,and 20 endophytic bacteria resources were obtained,which greatly enriched the endophytic bacteria resource pool of Pueraria.20 strains of endophytic bacteria have significant antioxidant,anticancer and bacteriostatic activities,and 8 monomeric compounds with anticancer and bacteriostatic activities have been isolated and identified.In addition,as a medicinal and edible homologous plant,Pueraria lobata has the advantages of safety,low toxicity and easy scale-up production.This provides a scientific reference for the development and industrial production of endophytic resources of P.thomsonii in functional food and natural food preservatives with antioxidant and anticancer properties. |
