Study On Secondary Metabolites Induced By Adding Enzyme Inhibitors And Precursor Compound Strategies In Pholiota Nameko

Pholiota nameko is a kind of higher fungi of the family Strophariaceae and the genus Pholiota,also known as Pholiota glabra.P.nameko has a bright appearance,a delicious taste,and rich nutrition.It can not only be isolated and cultivated,but also achieve artificial cultivation,and is the main cultivated edible fungi.In recent years,scholars at home and abroad have conducted a large amount of research on the chemical composition and pharmacological effects of this genus of fungi.However,there has been little research on the metabolites of P.nameko,and only a small amount of research has been conducted by our research group.We previously found that P.nameko is sensitive to cultural conditions and can produce different secondary metabolites under different cultural conditions,which is because under traditional and single cultural conditions,many biosynthetic gene clusters of secondary metabolites are in a silent state,making it difficult to produce a large number of secondary metabolites.It is particularly important to explore the resources of P.nameko to produce as many secondary metabolites as possible under different culture conditions.It will break the situation of inhibiting the diversity of fungal compounds to a certain extent,and is of great significance for the discovery of lead compounds.In order to obtain more natural products with novel structures from P.nameko,this study conducted the following research:1.Using a One Strain Many Compounds（OSMAC）strategy,the cultivation conditions of Pleurotus ostreatus were screened by adding enzyme inhibitors（SAHA,5-AZA）,precursor compounds（caryophyllene）,and changing the culture medium.The chemical composition diversity of the fermentation products was analyzed by HPLC,Finally,three fermentation conditions with abundant metabolites were selected from 42fermentation conditions,namely:（1）GP culture medium,adding P.nameko seed liquid,enzyme inhibitor 0.3 m M/L 5-AZA for 14 days,add 40μL/100 m L of caryophyllene and continue to cultivate for 14 days.（2）GP medium,add P.nameko seed solution and enzyme inhibitor 0.3 m M/L SAHA for 14 days,then add 20μL/100m L caryophyllene for further 14 days.（3）GP medium,add mus P.nameko seed solution for 14 days,then add 20μL/100 m L of caryophyllene for further cultivation for 14 days.2.The selected fermentation conditions were used to expand the fermentation of the P.namekos,and the obtained extract was subjected to chemical component separation,purification,and structural identification.A total of 22 compounds were isolated,including a new compound Paneolic A（1）and 21 known compounds:Caryophyllene oxide（2）、n-butyl（S）-2-hydroxybutanoate（3）、p-hydroxyacetophenone（4）、24-methycholest-4-en-3-one（5）、（22E,24R）-ergosta-5,7,22-trien-3β-ol（6）、4-{[（4R,5R）-4,5-dimethyl-1,3-dioxolan-2-yl]methyl}phenol（7）、Donacinol（8）、4-methoxybenzeneacetic acid（9）、（1R,4R,6R,10S）-4,12,12-trimethyl-5-oxatricyclo[8.2.0.0^4,6]dodecan-9-one（10）、Cladosporine A（11）、β-sitosterol methyl ether（12）、（22E,24R）-ergosta-7,22-dien-3β,5α,6β-triol（13）、β-sitosterol（14）、4-hydroxyphenethyl alcohol（15）、Caryophyllene（16）、5-hydroxymethyl-2-furancarboxaldehyde（17）、（22E,24R）-ergosta-4,6,8,22-tetraen-3-one（18）、（22E,24R）-5α,8α-epidioxy-ergosta-6,22-dien-3β-ol（19）、TAG（20）、p-hydroxybenzaldehyde（21）、（1R,2S）-1-phenyl-1,2-propanediol（22）。3.The scavenging activities of DPPH free radical,hydroxyl radical and superoxide anion of the fermented extract of P.namekos under different culture conditions were tested in vitro.At the same time,the effects of Caryophyllene and its transformation products on the ROS content in H₂O₂ induced mouse Raw macrophages were studied,and the enzyme activities of SOD,CAT,and POD were measured.The results showed that compounds 2,10,and 16 had certain antioxidant activity on H₂O₂ induced mouse Raw 264.7 cells.