Identification Of Active Compounds From Gymnopus Sp. And Their Activities In Vito And In Vitro Against Blue And Green Mold On Citrus

Jiangxi province is one of the major growing citrus fruit in China,citrus postharvest rot caused great economic loss by blue and green mold disease.Some chemical fungicides are forced to be banned or restricted because of the problems of drug residues and pathogen resistance in current chemical preservative agents,and biological source citrus preservatives have good environmental compatibility and difficult to produce resistance to insecticides which become mainstream method of postharvest preservative preservation of citrus.A wild strain of Gymnopus sp.06012-9was found by our laboratory.Its fermentation broth could effectively inhibit the growth of blue and green mold,but the structure and properties of its extracellular active substances were not clear.In this paper,using Penicillium italicum and P.digitatum as indicator bacteria,the active substances were separated by macroporous resin dynamic adsorption,silica gel column chromatography,reverse silica gel column chromatography,and semi-preparative high performance liquid phase?Pre-Semi HPLC?method.The active substances were traced using Oxford cup,Developed thin-layer chromatography-Bioautography?TLC-BA?,TLC-UV,Coloration with 10%Concentrated sulfuric acid mix with ethanol and HPLC method.Determination of the inhibitory dose and EC₅₀value of active substances against blue and green mold respectively using hyphal growth inhibition method and TLC-BA.The structure of the compounds was elucidated by ESI-MS,NMR,CD and UV spectroscopy.The results showed that the EC50 value of the crude extract of ethyl acetate against Penicillium Italiana and P.digitatum respectively was 217.13?g/mL and 145.24?g/mL.The results of citrus fruit in vivo showed that the crude extract had better control effect on Penicillium digitatum than Penicillium Italiana.Two active substances were isolated from crude extracts by silica gel column.The EC₅₀0 of the active substance?'to Penicillium Italiana and P.digitatum,respectively,was 95.717?g/mL and 137.36?g/mL,and the EC₅₀ of the active substance?'to Penicillium Italiana and P.digitatum,respectively,was 213.59?g/mL and 319.28?g/mL.The stability of the substance?'and the substance?'under the conditions of different temperatures,pH values,ultraviolet irradiation?UV?,light and oxidation showed that the substance I'was stable under the conditions of high temperature?121??,oxidation and pH?pH 2¹0?,and the antifungal effect was weakened after 7 days of light exposure.Substance?'was stable under 100?and below 100?and pH?pH 2⁷?from acid to neutral,the antifungal effect was weakened after 4 d⁹ d treatment under light and oxidation for4 h after UV treatment.Five monomers subtances,respectively I-1.1,?-1.2,?-1.2,?-2,?-3 and?were isolated and purified by ODS column chromatography and Pre-Semi HPLC.The results showed that five compounds all had good antifungal effective against Penicillium Italiana and P.digitatum by developed TLC-BA.The effective inhibitory dose of compounds?-1.1,?-2,?-3 and?againstPenicillium Italiana was 18.6?g,and the effective inhibitory dose of compounds?-1.2 against Penicillium Italiana and P.digitatum was 39.9?g.The inhibitory effect of?and?-2 on penicillium Italiana was more obvious.The retention time of each compound on analytical HPLC respectively was 6.05 min,6.18 min,7.20 min,7.05 min and 4.36min.Compounds I-1.2 and I-3 were elucidation by means of ESI-MS,NMR(¹H NMR,¹³C NMR,2D-NMR,HMBC,HSQC),CD and UV spectral data analysis shown that Compound I3 is fulvoferruginin and compound II-1.2 is5-epi-fulvoferruginin,they are isomers,both of which are Carotane sesquiterpenoids.Both of the compounds were first time isolated from the Gymnpus sp.,of which?-1.2 is a new natural compound.Based on the above results,the results show that the strain of Gymnpus sp.has great potential development value in postharvest biological preservation and preservation of citrus.