Chemical Composition Analysis Of Secondary Metabolites From Two Endolichenic Fungi

Lichen is a special symbiotic complex composed of fungi and algae,which has a unique physiological form,and many fungi live in the medullary layer alternately.Common lichens live on the branches and leaves of tropical rain forests and temperate forests or attach to bark,and also can grow in extreme environments such as drought,high radiation and polar regions.It is found that there are symbiotic fungi,symbiotic algae,basidiomycetes,bacteria and other microorganisms in lichen symbiotic system,among which the species and quantity of enndolichenic fungi are rich.Endolichenic fungi derive a unique metabolic pathway,and produce a large number of compounds with complex structure,novel skeleton and rich biological activities,such as alkaloids,polyketones,terpenoids,quinones and other compounds,which become important natural medicinal chemical sources.Therefore,the secondary metabolites of endolichenic fungi are studied in order to find new antibiotics,natural drugs or specific drugs.In this paper,Flavoparmelia caperata and Usnea aciculifera were picked from Yunnan province,and endolichenic fungi were isolated and identified.Among them,8 strains of edolichenic fungi were isolated and identified from F.caperata,distributed in 1 phylum,3classes,4 orders,7 families and 8 genera;Two endolichenic fungi,Nemania diffusa and Pseudocercosporella fraxini,were isolated from U.longispinosa.By constructing the phylogenetic tree of single gene and multi-gene,it was determined that the edolichenic fungus8005b belonged to a new species of Preussia,and it was named Preussia herbisordor sp.nov.The new species has a special structure ascoma initials in the middle of hyphae,and the hyphae are connected with each other to form a pseudoparenchymatous mass surrounded by outer hyphae,but no sexual structure is formed in the end.Ten endolichenic fungi were screened with Fusarium graminearum as the pathogen by plate confrontation method.The results showed that Preussia herbisordor sp.nov.and Nemania diffusa SW034 had good activity against plant pathogenic fungi.Strains Preussia herbisordor sp.nov.and Nemania diffusa SW034 were pre-fermented to obtain crude extracts,which were tested for their antifungal（Candida albicans SC5314）,antibacterial（Staphyloccocus aureus ACTT29213,Escherichia coli ATCC51446）and cytotoxic（human hepatocellular carcinoma cell hepg2）activities.The results showed that the crude extract of strain Preussia herbisordor had antifungal activity against wild-type C.albicans SC5314,and its MIC₈₀ value was 128μg/m L.The crude extract of strain Nemania diffusa SW034 has cytotoxic activity on human hepatocellular carcinoma cell hepg2,and its IC₅₀ value is 100μg/m L.Strains 8005b and SW034 were fermented in rice medium for 60 days,and then extracted with ethyl acetate to obtain crude extracts.Silica gel column chromatography,gel column chromatography,reversed-phase column chromatography,C18 reverse Flash column chromatography and semi-preparative high performance liquid chromatography were used for separation and purification,and the structures of the secondary metabolites of the two strains were determined by spectral technology.The results were as follows:（1）After fermentating Preussia herbisordor 8005b,obtained 20.5 g crude extract.Secondary metabolites were isolated and purified,and four compounds were identified.Compound 1 is leptosphaerone B,which belongs to polyketones;Compound 2 is 1,3-bis（（e）-（2-hydroxyl-3-methyl-5-oxo-3,5-dihydro-4h-imidazol-4-ylidene）methyl）-1,3-dimethylurea,which belongs to imidazole compounds;Compound 3 is 2,4-dimethyl-5-oxo-2,5-dihydrofuran-2-carboxylic acid,which belongs to alkaloid compounds.Compound 4 is（z）-4-amino-2-methyl penta-1,3-dien-3-ol,belonging to diene compounds;Among them,compounds 2,3 and 4 are new compounds,and compound 4 is the main product of this strain,with a total yield of794.7mg.（2）After fermentating Nemania diffusa SW034,obtained 107.5 g crude extract.Secondary metabolites were isolated and purified,and two known compounds were identified.Compound5 is（2S,5R）-2-ethyl-5-methylhexaenoic acid,which belongs to fatty acids;Compound 6 is Cytochalasin Z₁₇,which belongs to cytochalasin compounds,and compound 5 is the main product of this strain.In this study,10 endolichenic fungi were isolated from two lichens by skin scraping method.Among them,strain 8005b is a new species of Preussia,and its special microstructure has been observed.The biological activity of the crude extract was evaluated,and it was found that two endolichenic fungi had antifungal and cytotoxic activities respectively.The secondary metabolites of two endolichenic fungi were analyzed and three new compounds were identified.Enriched the research on the diversity and secondary metabolites of endophytic fungi in Preussia and Nemania,and provided reference for exploring new natural drugs for endolichenic fungi.