Secondary Metabolites And Anti-inflammatory Activities Of Two Mangrove Endophytic Fungi

Mangrove ecosystems were composed of a large number of salt-tolerant mangrove,semi-mangrove plants,as well as animal and microorganisms.Mangrove ecosystems were the second-largest marine ecosystem in terms of resource richness after coral reefs.Mangroves were rich in microbial resources,with fungi occupying the majority of the proportions.The unique ecological environment of mangroves,characterized by high temperature,high salinity,soil hypoxia,and periodic inundation by seawater,results in a distinctive metabolic mechanism of mangroves fungi.These secondary metabolites showed promising activities in anti-tumor,anti-inflammatory,antibacterial and hypoglycemic aspects and might be potential lead compounds for new drugs.These structurally novel active compounds may contribute to the discovery of new disease targets,provide new inspiration for chemical synthesis,and offer additional options for new drug development.In summary,it is of great significance to search for active compounds with novel structures from the treasure troves of mangrove fungi for new drug development.This article used HPLC analysis and activity screening to select two endophytic fungi form Zhanjiang Mangrove Forest Nature Reserve of Guangdong in China.:Sporormiaceae sp.QQJ-5 from the leaves of the Kandelia candel and Daldinia eschscholtzii KBJYZ-1 from the root tissue of the Plucheaindica Less.,for the study of secondary metabolites and anti-inflammatory activity.The secondary metabolite was isolated and purified by normal phase silica column chromatography,Sephadax LH-20 column chromatography and HPLC after amplification of the two endophytic fungi in rice solid culture.Their structure was identified by 1D/2D NMR,UV,IR,MS,CD and ECD calculations.In the study,44 secondary metabolites were isolated and identified,including 14 new compounds.The new compounds accounted for 31.8%.By LPS induced RAW264.7 cells were used to construct an in vitro inflammatory model,and the anti-inflammatory activities of some compounds were screened.The anti-inflammatory mechanism of some active compounds was explored by Western blot.The following were the specific sections of the paper:First of all,in the content of chapter one,we reviewed the research progress of terpenoids derived from mangrove fungi during 2014-2023,summarizing the types,structures and pharmacological activities of terpenoids to provide a reference for the future comprehensive development and utilization of this class of compounds.In the second chapter,a variety of chromatographic separation methods were comprehensively applied,combined with spectral identification technology,to isolate and identify 14 compounds（2-1～2-14）from the strain Sporormiaceae sp.QQJ-5.These compounds including eight new isopimarane diterpenes sporormiaces A-H（2-1～2-8）and six known compounds:asperolide A（2-9）,sphaeropsidin C（2-10）,wentilactone A（2-11）,asperolide A（2-12）,5-O-methylsulochine acid（2-13）and yicathin B（2-14）.In the third chapter,the same methods and techniques were used to isolate and identify twenty-nine polyketides and a sesquiterpene from the fungus Daldinia eschscholtzii KBJYZ-1,including six new compounds:dalditone B（3-1）,daldinia A-B（3-2～3-3）,eschscholin B（3-4）,（1S,4S）-5-methoxy-1,2,3,4-tetrahydronaphthalene-1,4-dio（3-5）,daldilene A（3-6）.The remaining 24 known compounds consist of 4,9-dihydroxy-1,2,11,12-tetrahydroperylene-3,10-quinone（3-7）,（-）-regiolone（3-8）,（4S）-4,8-dihydroxy-5-methoxy-α-tetralone（3-9）,（4S）-4,5-dihydroxy-α-tetralone（3-10）,5-O-methylsclerone（3-11）,5,8-dihydroxy-2-methylchromon（3-12）,5-hydroxy-8-methoxy-2-methyl-4H-1-benzopyran-4（3-13）,7-hydroxy-2,5-dimethylc-hromone（3-14）,2,3-dihydro-5-methoxy-2-methylchromen-4-one（3-15）,（2R,4R）-3,4-dihydro-5-methoxy-2-methyl-2H-1-benzopyran-4-ol（3-16）,1,8-dimethoxynaphthalene（3-17）,helicascolide A（3-18）,helicascoli-des D（3-19）,（1R,4S,5S,7R,10R,11R）-guaiane-10,11,12-triol（3-20）,1-（benzofuran-2-yl）ethan-1-one（3-21）,2-hydroxyphenylacetic acid（3-22）,1-phenylethane-1,2-diol（3-23）,6-methoxycyclohex-3-en-1-one（3-24）,R-mevalonolactone（3-25）,5-methyl-2-vinyltetra-hydrofuran-3-ol（3-26）,（1R,2R,3S,4R）-4-[（S）-1-acetoxyethyl]cyclopentane-1,2,3-triyltriace tate（3-27）,（2R,4S,5S）-hept-6-en-2,4,5-triol（3-28）,dalditone A（3-29）and（3E,6S）-3-heptene-1,6-diol（3-30）.Finally,In the four chapters,LPS induced RAW264.7 cells were used to construct an in vitro inflammatory model,and the anti-inflammatory activities of some compounds were screened.The results showed that the new compounds sporormiaces E（2-5）,eschscholin B（3-4）and daldilene A（3-6）had good anti-inflammatory activity,could inhibit LPS-induced NO release in RAW264.7 cells,with IC₅₀ of 24.5,19.3 and 12.6μM,respectively.The new compound with good activity and higher yield,eschscholin B（3-4）,was selected for further anti-inflammatory mechanisms research.Western blot results showed that eschscholin B（3-4）effectively inhibited LPS-induced expression of iNOS and COX-2 in RAW264.7 cells and might exert anti-inflammatory effects by inhibiting MAPK and NF-κB signaling pathways.