Study On The Chemical Constituents Of Daldinia Eschscholtzii And Penicillium Herquei, An Endophytic Fungus From Cordyceps Sinensis

This paper mainly comprises two sections.One is about study on the chemical constituents of mycelia of Daldinia eschscholtzii.Another is to study the chemical constituents of secondary metabolites of Penicillium herquei,an endophytic fungus from Cordyceps sinensis.Section 1:With the purpose of understanding on the chemical constituents of mycelia of D.eschscholtzii,and seeking more compounds with novel structures and good bioactivities,the mycelia of this fungus had been inoculated in fermentation broth,and structures and cytotoxic activities of its constituents had been illustrated and evaluated after a large scale fermentation.In the early stage,microbiological methods were applied to inoculate the collected fungus to the solid medium,and the mycelia used in all experiments was obtained by expanded liquid fermentation of D.eschscholtzii.Then the mycelia was extracted by EtOAc and isolated by various chromatography methods.Structures of chemical compounds obtained were identified through NMR,MS and other means.MTT method was used to test the cytotoxicity of compounds.7compoundswereisolatedandidentifiedas:（E）-6-（non-3-en-1-yl）-2H-pyran-2-one（DE-1）,（R）-5-hydroxy-8-methoxy-2-methylchroman-4-one（DE-2）,ergosta-4,6,8（14）,22-tetraen-3-one（DE-3）,5-methoxycoumarin（DE-4）,5-hydroxy-2-methyl-4H-chromen-4-one（DE-5）,3,4-dihydro-5-methoxy-2-methyl-2H-1-benzopyran-4-ol（DE-6）,2,3-dihydro-5-methoxy-2-methylchromen-4-one（DE-7）.There are four chromones,four coumarins,one ergosterol,and one pyranone.Compounds DE-1 and DE-2 are new compounds.Bioactivity assay showed that compound DE-3 showed a moderate cytotoxic activity against H1299,H460,HGC-27,A549 and MNK-45 with IC₅₀0 values of 25.2±2.9μmol/L,32.3±4.2μmol/L,29.2±1.2μmol/L,33.9±3.7μmol/L,18.0±7.0μmol/L respectively..Section 2:To further exploit the medicinal value of C.sinensis and provide a new way for comprehensive utilization of resource of C.sinensis.Considering the large species,and wide variety of secondary metabolites of endophytic fungi with novel structures,they can provide another way to obtain more active ingredients.This section focused on chemicals studying of ethyl acetate extract of metabolites of an endophytic fungus isolated from C.sinensis.In this section,microbiological methods were employed to purify,separate and ferment P.herquei,the endophytic fungus from C.sinensis.In addition,chemical methods like solvent extraction,column chromatography,thin layer chromatography and preparation chromatography were used to extract and isolate the fermentation broth.Eventually,structures of chemical constituents that had been obtained were identified by means of NMR,MS,ECD and so on.14 compounds were isolated from the P.herquei fermentation broth,and they were identified as:（S）-6-（sec-butyl）-5-（hydroxymethyl）-4-methoxy-2H-pyran-2-one（PH-1）,（5S,7R）-7-ethyl-4,5-dimethoxy-7-methyl-5,7-dihydro-2H-furo[3,4-b]pyran-2-one（PH-2）,（5R,7R）-7-ethyl-4,5-dimethoxy-7-methyl-5,7-dihydro-2H-furo[3,4-b]pyran-2-one（PH-3）,2’,4’-dihydroxy-3’,5’-dimethylacetophenone（PH-4）,vanillin（PH-5）,3-oxo-α-ionone（PH-6）,3,3,5-trimethyl-4-（3-oxobut-1-en-1-yl）cyclohexan-1-one（PH-7）,p-hydroxybenzaldehyde（PH-8）,p-hydroxyacetophenone（PH-9）,methyl p-hydroxyphenylacetate（PH-10）,cyclo-（Pro-Ile）（PH-11）,cyclo-（Pro-Val）（PH-12）,4-methoxyphenylacetic acid（PH-13）,cyclo-（4-methyl-Pro-Val）（PH-14）.Among them,there are four derivatives of acetone,three cyclodipeptides,three derivatives of pyranone,two ionones,and two benzaldehydes.Compounds PH-1,PH-2 and PH-3 are new compounds.