Chemical Investigations Of Three Strains Of Endolichenic Fungi And Their Biological Activities

Endolichenic fungi are diverse groups of predominantly filamentous fungi that reside asymptomatically in the interior of lichen thalli.They are one of the ideal candidates of the study for bioactive natural products,due to their rich biodiversity,easy cultivation,short growth cycle,structural diversity,as well as resource-conservation.Furthermore,the chemical studies on fungi derived from the special habitats,contribute to the discovery of natural products with unique architectures or promising pharmacological properties and have great significance for the discovery and development of new drugs.In this study,the chemical investigation on three endolichenic fungi was carried out.Based on the preliminary cytotoxity assay and chemical analysis,three endolichenic fungi Ophiosphaerella korrae,Aspergillus unguis,Alternaria alternata were selected for scale-up fermentation in the solid sterile rice medium.The ethyl acetate crude extracts,which had been obtained from the above three fungi,were fractionated utilizing column chromatography on silica gel,ODS,Sephadex LH-20,Medium Pressure Liquid Chromatography,as well as reversed-phase high performance liquid chromatography.In total,a number of separations and purifications led to 81 compounds,40 of which are new.Their structures were established on the basis of various spectroscopic data,ECD comparison,ECD calculations,single-crystal X-ray diffraction measurement,NMR calculations,calculations of spin-spin coupling constants,J-Based Configurational Analysis and biogenetic considerations.Separation of the extract of Ophiosphaerella korrae(No.20140207b)afforded twenty-nine compounds,including nine bicyclo[4.1.0]heptenones(1-9),two oxaspiro[4.4]nonenones(10-11),two furopyran-3,4-diones(12-13),two benzo-y-pyrones(14-15),four isocoumarins(16-19),two isobenzofuranones(20-21),one sesquiterpenoid(22),five perylenequinones(23-27)and two steroids(28-29),of which compounds 1-16 and 22 are new.Taking the unique structure features into consideration,plausible biogenetic pathways for compounds 1-13 were proposed.It turned out that the five known perylenequinones(23-27),as the major components,are responsible for the cytotoxic activity of the extract.Preliminary test with thin-layer chromatography bioautography found that 3 showed moderate acetylcholinesterase(AChE)inhibitory effects(the minimum quantity is 1.25 ?g);further phytotoxic studies indicated that 22,as an analogue of the well-known phytohormone abscisic acid,inhibited root elongation in the germination of Arabidopsis thaliana with an IC50 value of 18.06 mg/mL.Separation of the extract of Aspergillus unguis(No.20141257a)afforded thirty-seven compounds,containing seven asperane-type sesterterpenoids(30-36),nineteen varitriol-like polyketides(37-55),one tetrahydronaphthalene derivative(56),one 5-alkenylresorcinol(57),one pyrano[4,3-b]pyran-7-one(58),one coumarin(59),one isocoumarin(60),two steroids(61-62),one anthraquinone(63),one aflatoxin(64),dihydroterrein(65)and p-hydroxybenzaldehyde(66),of which compounds 30-35,37-46,51,56 and 58 are new.Bioassay of the isolated compounds found that 32 showed cytotoxicity against the human cancer cell line A549 with an IC50 value of 6.2?M.Further investigation revealed that the observed cell death was a result of G0/G1 cell cycle arrest via DNA damage followed by cellular apoptosis.Inhibition on LPS-induced NO production in RAW 264.7 murine macrophages was investigated,and the results showed that 45 and 46 exhibited weak anti-inflammatory activity.Separation of the extract of Alternaria alternata(No.20111721-4)afforded fifteen compounds,including four tenuazonic acid derivatives(67-70),one oxazine alkaloid(71),four alternariol-like mycotoxins(72-75),four biosynthetic relatives(76-79),one meroterpenoid(80)as well as one simple chromone(81),of which compounds 67-70 are new and exist as two tautomeric forms differing in the configurations of the enamine.The cytotoxic activities were preliminarily evaluated,and the results show that alternariol-like mycotoxins(72-75)should be responsible for the cytotoxic activity of the extract.Chemical studies of these three endolichenic fungi and their biological activities have led to numerous new and bioactive structures as well as many solutions to the determination of spatial configurations of new compounds.These results indicated that maybe we can conduct a thorough study into the potential fungi utilizing several biotechniques and lay a strong foundation for the discovery and development of new drugs.