Studies On The Active Secondary Metabolites Of Three Marine Fungal Strains And The Effects Of Alkaline Stress On Fungal Secondary Metabolites

Marine fungi are a kind of important source for active marine natural products.Because of its specificity of living oligotrophic, weak alkaline environment, marinefungi may have unique secondary metabolic pathways and produce the novelsecondary metabolites. In recent years, marine fungi have gradually become one ofthe hot. By integrated biological and chemical screening methods, three talentedstrains were obtained from different marine-derived samples. Optimizations offermentation condition for talented strains were systematically studied. Furthermore,we attempted to culture one talented strain under alkaline stress, and studied thedifferences in the compounds skeleton of fermentation products compared to thenormal pH culture conditions.Our study focused on the new secondary metabolites of marine fungi underalkaline stress, and includes the following three parts:1. Screening for the marine-derived talented strains.10strains of marine fungi from coral-associated sample in South China Sea, plantsof mangrove forest in Hainan and seaside plants in the Yellow River delta have beenscreened. The EtOAc extracts of these fungi were evalassesed with the combinatorymethod based on chemical and bioactive screening. Three of them were confirmed asmarine-derived talented strains with cytotoxicity or antibiotic or anti-inflammatoryactivities, while one talented strain was chosen to culture under alkaline stress. Inaddition, we carried out the preliminary study of the fermentation under alkalinestress.2. Study on secondary metabolites of three talented strains under weak alkalineenvironmentOptimizations of fermentation condition for talented strains were determinedbased on the research of effect of different conditions, such as medium, fermentationtime, alkali source and salinity. Three strains were fermented separately, and then thewhole broths were extracted with EtOAc in order to get active extracts. These extractswould be subjected to extensive silica gel column chromatography, Sephadex LH-20and HPLC purifications. After processing,76compounds were isolated from these three marine-derived talented strains and identified by means of detailedspectroscopic analysis (such as UV, IR, NMR, MS, X-Ray, etc) and chemicalmethods. Among them,25compounds were new ones, including twelve new aromaticpolyketides compounds (1–12) obtained from P. commune518#, one new phenolicbisabolane sesquiterpenoid (25) and one new diphenyl ether derivative (29) from P.expansum091006, Four new meroterpenoids,(40–43) from A. alternata007#. Theknown compounds refer to phenolic compounds, meroterpenoids, indole alkaloids andnucleosides. Compounds21–24exhibited moderate cytotoxicity against HL-60cellline with IC50values of15.7,5.4,18.2and20.8μM, respectively; Compound22alsohad significant cytotoxicity against A549cell line with an IC50value of1.9μM.Compounds4,44and47exhibited weak anti-inflammatory on the transcriptionalactivity of NF-κB in stimulated RAW264.7cells with IC50values of45.9,52.0and41.8μM, respectively. Compounds1,4–6,9and12showed antibiotic activity againstC. albicans, E. coli, P. aeruginosa or S.aureus with MIC values ranged from2.6μMto29.1μM.3. Preliminary evaluation of the effect of alkaline stress on chemical diversity ofsecondary metabolites of marine fungal strainsTo further research the effect of alkaline stress on chemical diversity of secondarymetabolites of marine fungal strains, the secondary metabolites of A. alternata007#under alkaline stress culture condition were studied by comparing the retention timeof their production. And we found that the alkali sources and the steps of sterilizationplayed an important part in the formation of secondary metabolites of this fungus. Thestrain A. alternata007#cultured under alkaline stress was found to produce6different meroterpenoids, five of which (61–65) were new ones. More importantly, itwas established that marine fungi could increase the chemical diversity of secondarymetabolites under alkaline stress.Summarily,23new compounds and53known ones were obtained in this work, and21of these compounds showed cytotoxic, anti-inflammatory and antibiotic activities.