Secondary Metabolites Of Endophytic Fungi From Two Medicinal Plants And Their Bio Activities

Endophytic fungi are a special and important group of microorganisms withtaxonomic diversity, which show high potential as sources of antimicrobial,antiviral, anticancer, antioxidant, and insecticidal compounds. In order toinvestigate the potential bioactive compounds derived from endophytic fungi,this dissertation described the discovery of several antimicrobial compoundsisolated from four endophytic fungi of medicinal plants by bioassay-guidedfractionation. Studies include isolating of endophytic fungi, seleeting aimedstrains, fermentation studies, bioassay-guided fractionation, structural elucidation,preliminary evaluation of antimicrobial and anti-tumor activities of purecompounds. The results are concluded as follows:Sixty-two strains isolated from roots, stems and leaves of Eucommiaulmoides, were classified, identified, and belonged to fifteen genera, ninefamilies, and seven orders according to their morphological and micro-structurecharacters. Alternaria sp., Fusrium sp. and Alternaria sp. are dominantendophytic fungi population in Eucommia ulmoides. Fifty-seven strains fromFicus carice were classified, identified, and belonged to twenty-one genera, tenfamilies, and seven orders while Fusrium sp., Penicillium sp. and Alternaria sp.are dominant endophytic fungi population. It showed that the quantities, species,and distribution of the endophytic fungus varied in different parts of Eucommiaulmoides and Ficus carice.The endophytic fungi isolated from Eucommia ulmoides and Ficus carice,were first screened by antimicrobial assay to obtain20strains which showedstrong antimicrobial activities, and possessed16.8%of total119strains tested.The20strains were subjected to the flow antimicrobial secondary screening and10strains were found to have more strong activities, which possessed10.1%oftotal119strains tested. On the basis of antimicrobial activities, the results of TLC and HPLC analysis, four ested strains, ER12, EL09, FR02and FL10, werechosen as the aimed strains to investigate their bioactive metabolites in thisstudy.According to their morphology, cultural characteristics,18S rDNA and ITSsequences, the strains ER12, EL09, FR02and FL10were indentified asLecanicillium psalliotae, Aspergillus niger, Aspergillus tamari and Fusariumtricinctum, respectively. On the basis of mycelia biomass, weight of fermentationextracts, antimicrobial activities, the results of TLC and HPLC analysis,stability-tests for the bioactive components and fermentation studies perfomredon the aimed strains. The time course experiments for the fermentation of fourstrains were then carried out, followed by solvent extraction tests for the activecomponents. Then, large-scale fermentation and preparation of the activefractions were perfomred to obtain the active fractions of the four strains.Ninety compounds were isolated from the four active strains by repeatedcolumn chromatography on silica gel, Sephadex LH-20, preparative thin layerchromatography (PTLC) and recrystallization. The structures of thesecompounds were elucidated by means of spectroscopic methods includingESI-MS, HR-ESI-MS,1D-NMR (1H NMR,13C NMR, DEPT) and2D-NMR(1H-1H COSY, HSQC, HMBC) as well as chemical method. Among them therewere three new compounds, malformin E (19), cyclo-(N-methyl-Trp-Leu)(62)and helovlic acid ester (83).Thirty-four compounds were isolated from fungus ER12, and the chemicalstructure types of compounds were involved in cyclic dipeptides (3-10,13-16),malformin E (19), fumiquinazolines (17,18,22,23), diphenyl ketones (20,21),imidazoles (12), sterols (1,2), benzene derivatives (11,24-28,31,32) andnucleosides (29,30,33,34). Eighteen compounds were isolated from fungusEL09, and the structure types of compounds were involved in sterols (38-44,1,2), fatty acids (35-37), furan derivatives (45,46), polyhydric alcohols (47,48)and indole derivatives (49,50). Twenty-two compounds were isolated fromfungus FR02, and the structure types of compounds were involved in indolyldiketopiperazine analogs (53-65), terpenes (51,52), sterols (1,39),polycarboxylic acids (67,68) and polyhydric alcohols (66). Thirty-threecompounds were isolated from fungus Fl10, and the structure types of compounds were involved in gliotoxins (74-80), helvolic acid derivatives(81-83), cyclic dipeptides (13,14,84), fatty acids (69-71,36,37), sterols (1,39,72,73), benzene derivatives (87-89), nucleosides (29,30,85,86), polyhydricalcohols and polycarboxylic acids (47,48,68,90).Ninety compounds were evaluated for their antimicrobial activities againseleven tested strains. The results indicated that twenty-six compounds showedstrong antimicrobial activities and the structure types of bioactive compoundswere involved in indolyl diketopiperazines, gliotoxins, quinazoline alkaloids,cyclic dipeptides and sterols. Remarkably, malformin E and helovlic acid estershow strong antimicrobial activities against different tested strains. The results ofantibacterial activity test indicated bioactive substances of four active fungi.Active substances of ER12were mainly fumiquinazoline alkaloids and cyclicpeptide compounds. Active substances of EL09were sterols and furans. Activesubstances of FR02were indolyl diketopiperazines. Active substances of FL10were gliotoxins and helvolic acid derivatives. Three new compounds and isolatedalkaloids were evaluated for their cytotoxicities against three cancer cell lines,MCF-7, HepG2and A549, by the MTT method. The results indicated that four ofthem showed significant cytotoxicities against different cancer cell lines andothers were weak. Among them, new compond, malformin E, showed significantinhibitory activities against MCF-7and HepG2cell lines with IC500.65,2.42μMrespectively. Fumiquinazoline C and fumiquinazoline J exhibited moderateactivities against HepG2and A549. Verruculogen showed significant inhibitoryactivities against HepG2and A549with IC501.95,2.56μM.Summarily, this work obtained four active fungi from two medicinal plantsand ninety compounds, including three new compounds and a new naturalproduct, were isolated from the fungi. The bioactivities results indicated thattwenty-six compounds showed significant antimicrobial activities and fourcompounds showed significant cytotoxicities against different cancer cell lines.This thesis indieated that endophytic fungi, as an important part of plant system,which contain novel and bioactive compounds in its secondary metabolisms, isan important source for searching lead compounds for drugs.