Studies On The Secondary Metabolites From Three Halotolerant Fungi Quorum-sensing Inhibiting Activity Under Salt Stress

Halotolerant fungi are a group of microorganisms with unique gene types that can still grow normally in high-concentration salt media.The special living environment of high salt may activate the silent genes of halotolerant fungi and induce unique microbial metabolic pathways,thereby producing novel structural compounds.As an important source of drug discovery,halotolerant fungi have received extensive attention in recent years.The bacterial quorum sensing?QS?system regulates the expression of related virulence factors?promotes the production of extracellular enzymes and toxins of pathogenic bacteria,bacterial movement,biofilm formation,etc.?,and has been highly valued as a new target for the development of anti-infective drugs.One hundred and fifty-two strains of halotolerant fungi were isolated from roots,stems,leaves,fruits and other tissues and rhizosphere soil of Limonium sinense,Glycine soja,Vitex trifolia and Apocynum venetum,they were respectively numbered L1-L152.Through thin-layer chromatography?TLC?,high performance liquid chromatography?HPLC?and QS inhibitory activity evaluation of the rice culture fermentation products of halotolerant fungal under seawater conditions,it was found that the most of fermentation products have diverse chemical species.In this paper,different fermentation methods were used to carry out large-scale fermentation of the three selected halotolerant fungi?L129,L36,L130?.After extracting the secondary metabolites of the strain with ethyl acetate,it is comprehensively separated by column chromatography techniques such as normal phase silica gel chromatography,sephadex LH-20,medium-pressure preparative chromatography,high-pressure semi-preparative chromatography and semi-preparative high performance liquid chromatography technology for purification.The structure of the obtained monomer compound was identified by means of infrared spectroscopy?IR?,ultraviolet spectroscopy?UV?,nuclear magnetic resonance?NMR?,circular dichroism chromatography?CD?,and mass spectrometry?MS?.A total of thirty-three monomer compounds were isolated,twenty-nine of which have been identified for their structure:peniquinone A and B?1 and2?,penizofuran A?3?,quinadoline D and A?4 and 5?,3,4-dimethoxy-5-methylphenol?6?,orcinol?7?,1,3,5,6-tetrahydroxy-8-methylxanthone?8?,mucorisocoumarins A?9?,penicillic acid?10?,dihydropenicillic acid?11?,dechlorogriseofulvin?12?,griseofulvin?13?,isogriseofulvin?14?,dehydrogriseofulvin?15?,trans-capsaicin?16?,dihydrocapsaicin?17?,linoleic acid?18?,dibutylphthalate?19?,2,4-dihydroxy-5,6-dimethylbenzaldehyde?20?,4,5-dimethylbenzene-1,3-diol?21?,4,5,6-trimethylbenzene-1,3-diol?22?,2,4-dihydroxy-5,6-dimethylbenzoic acid?23?,asterric acid?24?,methyl dichloroasterrate?25?,dihydrogeodin?26?,iizukine B?27?,questin?28?,nafuredin A?29?.Compounds 1-4 are new compounds.Finally,the QS inhibitory activities of compounds were tested by hole punching method,antibacterial and antifungal activities of compounds were tested by paper disc assay,and cytotoxic activities of compounds were tested by MTT method.Biological activities test results showed that compounds 1 and 2 showed significant cytotoxicity to MCF-7,U87 and PC3 cell lines,with IC₅₀ values of 12.39,9.01,14.59?M and 25.32,13.45,19.93?M,respectively;compounds 13 and 14 have effective antifungal activities against Colletotrichum musae?ACCC 31244?,with MIC values of 0.1?g/scrip,and compound 14 also shows antifungal activity against Colletotrichum coccodes?ACCC36067?,with MIC value of 0.1?g/scrip;compounds 2 and 8 have weak QS inhibitory activities against Chromobacterium violaceum 026,with MIC values of 20?g/well.Compound 10 was identified as penicillic acid and is a classic strong quorum sensing inhibitor.