Chemical Constituents Of Five Endolichenic Fungi And Their Biological Activities

Endolichenic fungi are nonobligate microfungi that reside asymptomatically in lichen thalli in close relationships with algae.Lichens,symbiont of fungi with green algae and/or cyanobacteria,can resist to hostile environment,such as drought,severe cold and ultraviolet radiation.Living in such a special environment,endolichenic fungi tend to form peculiar biometabolic pathways and thus produce novel structures and bioactive compounds,which is of great significance for the discovery of new lead compounds and unique targets.In this study,the chemical investigation and bioactivity evaluation on five endolichenic fungi was carried out.Based on the preliminary results of bioactivity screening and chemical analysis,five strains Eupenicillium javanicum,Floricola striata,Aspergillus versicolor,Xylaria sp.and Phialocephala fortinii were chosen for scale-up fermentation using rice medium.Besides,0.5 mM SAHA was added into the PDB medium of Phialocephala fortinii for epigenetic modification.Seperation of their EtOAc extracts using silica gel column,Sephadex LH-20 CC5 ODS,MPLC and HPLC led to the discovery of 77 compounds,including 36 new ones.The planar structure and absolute configuration of these new compounds were determined by comprehensive spectroscopic analysis combined with single-crystal X-ray diffraction,ECD calculations,GIAO NMR calculations and the helicity rule of ?,?-unsaturated ketones.In addition,the plausible biosynthetic pathways for some novel skeletons were proposed.Seperation of the secondary metabolites of endolichenic fungus Eupenicillium javanicum(No.0974a)afforded seventeen compounds,including sixteen polyketides(1-16)and one 2-pyridone alkaloid(17).Among these compounds,1-9 are new and 6-7 possess an unprecedented spiral 5/6/5/6/6 polycyclic system.These compounds were evaluated for potential anti-inflammatory activity against LPS-activated RAW 264.7 cells.Compounds 5 and 13 displayed moderate inhibitory effects on NO production,with IC50 values of 17.00 and 13.46?M,respectively.Twenty-six compounds were obtained from the extract of Floricola striata(No.8938a),including twenty-four p-terphenyls(18-41)and two pimarane diterpenes(42-43),of which 18-28 and 42 are new.Cytotoxicity assay demonstrated that 22 and 23 displayed significant inhibitory effects on the growth of A2780 cell line,with the IC50 values of 3.4 and 8.6?M,respectively.Further evaluation for the MDR reversal activity of 22 revealed it enhanced the sensitivity of MCF-7/ADR cells towards adriamycin 39-fold at 10 ?M through modulating P-gp-mediated drug exclusion.Isolation of the extract of yielded fourteen compounds,including eight diphenyl ethers(44-51),three pyrogallol ethers(52-54),two sterigmatocystins(55-56)and one polyketide(57).The phenyl ethers compounds 44-54 showed no inhibitory activities against the fungus Candida albicans SC5314,except that 49 displayed significant antifungal activity against Candida albicans DSY654(MIC80 4?g/ml).However,compounds 44-49,52 and 53 can reverse azole resistance of Candida albicans YEM13.Further mechanism research revealed compound 44 reversed azole resistance through inhibiting the drug energy-dependent efflux.Seventeen compounds,including four novel isoindoline alkaloids(58-61),two novel angucyclinone derivatives(62-63),one spiral sesquiterpene derivative(64),three 3-substituted-3-hydroxy-2-oxindole alkaloids(65-67),one polyketide(68),one isobenzofuranone(69),one carboxylated mullein(70),one phthalimide(71),two pyrrole alkaloids(72-73)and one quinoline alkaloid(74)were isolated from the endolichenic fungus Xylaria sp.(LCSS1a).Among these compounds,58-68 are new and 58-63 possess novel skeletons.Histone deacetylase inhibitor SAHA was added into the PDB medium of Phialocephala fortinii for epigenetic modification,which resulted in the emergence of new peaks in the HPLC-UV profile and increased the cytotoxity of extracts.Guided by the new UV,we obtained three new perylenequinonoids(75-77).Comparison of the retention time and UV absorption with those of control group revealed the production of perylenequinonoids increased significantly.Chemical investigation and bioactivity evaluation on five endolichenic fungi led to the discovery of abundant new compounds,some of which even possess novel skeletons or significant bioactivity.All aforementioned proves that endolichenic fungi become the important source of new bioactive natural products,which has yet to be further explored.