Study On The Bioactivity Of Secondary Metabolites Of Endophytic Fungi From Agastache Rugosus And The Chemical Constituents Of N-Butyl Alcohol Extract

In recent years,secondary metabolites of endophytic fungi in medicinal plants have become one of the important sources of new drugs and novel structural compounds.Because the endophytic fungi in medicinal plants have important ecological and economic significance,they have become the key points of research in Chinese Medicine,Microbiology and Plant Protection.Gansu Province has a wide variety of climate types and many types of ecological species,so it has nurtured a variety of wild medicinal plants.A large number of literature have reported that the chemical constituents of medicinal plants.The types of endophytic fungi and their secondary metabolites in medicinal plants are less studied in literature and reports.In this study,the Agastache rugosus which located in Gansu Province was studied.The species of endophytic fungi in the Agastache rugosus were studied.The antibacterial activity,scavenging DPPH free radical activity and chemical constituents of secondary metabolites of endophytic fungi of Agastache rugosus were studied.The content and results were as follows:Thirteen kinds of endophytic fungi were isolated and purified from the flowers of the medicinal plant Agastache rugosus by the way of surface sterilization and tissue separation.Using microscopic morphological features,identify them as Rhizoctonia sp,Nigrospora sp,Helminthosporium sp,Helminthosporium sp,Trichosporum sp,Alternaria sp,Fusarium sp and Cphalosporium Genus sp.Thirteen endophytic fungal fermentation broths were extracted with ethyl acetate and n-butanol.Mycelium was subjected to ethanol reflux extraction.The extracted fermentation broth was evaporated to dryness under reduced pressure.We got the ethyl acetate extraction part of the fermentation broth,the n-butanol extraction part of the fermentation broth,the water part and the ethanol extract of the mycelium.Antimicrobial and DPPH free radical activity of various parts of secondary metabolites of endophytic fungi were determined by micro-broth dilution method and TLC-biological auto-development method.The results of in vitro antibacterial activity test showed that the antibacterial activity was mainly concentrated in the ethyl acetate extraction and the n-butanol extraction,especially the ethyl acetate extraction.The ethyl acetate extraction of the secondary metabolite of strain H-2 had the best antibacterial activity,and the MIC value of the six tested bacteria was 250 ?g/mL.The MIC value of the ethyl acetate extraction of fermentation of strain H-9 against Streptococcus agalactiae was 31.25 ?g/mL,and the MIC value of the other five tested bacteria was 500 ?g/mL.The n-butanol extraction of the secondary metabolite also had antibacterial activity.The n-butanol extraction of the secondary metabolite of strains H-2,H-5,H-6 and H-9 had MIC values of 1000 ?g/mL for the six tested bacteria.The results of in vitro scavenging activity of DPPH free radicals showed that the substances with DPPH free radical scavenging activity were mainly concentrated in the ethyl acetate fraction of secondary metabolites of endophytic fungi.Among all the samples,the ethyl acetate extract of the strain H-9 fermentation broth had the strongest ability to scavenge DPPH free radicals.When the DPPH methanol solution concentration is 0.1%(w/v),the unit area clearance rate corresponding to the positive control 0.75 mg/mL Vc was 70.15±3.70%.The ethyl acetate fraction of strain H-13 also has a strong ability to scavenge DPPH free radicals.Its clearance rate was equivalent to 48.84±4.68% per unit area clearance of 0.75 mg/mL Vc of the positive control.Molecular biological identification of strain H-2 and strain H-9 were Nigrospora sp and Alternaria sp.A strain of the endophytic fungus Y-2,which has a good secondary DNA metabolite with good DPPH free radical activity,was used as a research object.The chromatogram was obtained by using a macroporous resin column,a silica gel column and a Sephadex LH-20 gel column.The column method was combined with thin layer chromatography to detect and purify two monomer compounds from the n-butanol fraction of the fermentation broth.The structure of the compound was identified by nuclear magnetic resonance spectroscopy and mass spectrometry.Compound 1 was identified as 5?,8?-cyclodioxomannin-6,22-diene-3?-ol.Compound 2 was not identified to have a definite structure due to its low content.