Studies On The Bioactive Metabolites Of Sponge-associated Fungi

Bioactive substances that interfere with microtubule function can generally inhibit the conidia growth of Pyricularia oryzae (a plant pathogenic fungus) and induce morphological deformation of conidia or mycelia of Pyricularia oryzae by swelling, curling, beads formation and hyper-divergence effect. We apply the method using conidia of Pyricularia oryzae as a primary screening assay for compounds such as antifungal antibiotics and anticancer substances from secondary metabolites of sponge-associated fungi. From 100 fungi samples isolated from sponges, we obtained 9 strains possessing better and stable activity. From the fermentation broth of strain 05JANF163/165, 05JANF151, 05JANF154, 05ABR145, 05ABR116, 98F152 and S_1-1 , twenty active compounds were isolated and purified by activity-guided fractionation. Structures of nineteen compounds were established on the basis of various NMR spectroscopic analyses, including 1D-and 2D-NMR spectroscopic analyses (¹H-¹H COSY, ROESY, HMQC, and HMBC spectra), and MS (FAB, EI and ESI). The nineteen compounds were Fusarielin A (1) , Fusarielin B (2) , Fusarielin E (3) , (-)-Oxysporidinone (4) , Roridin A (5) , Roridin H (6) , Roridin J (7) , Verrucarin A (8) , Verrucarin B (9) , Verrucarin L Acetate (10) , Verrucarin J (11) , Deoxyfusapyrone (12) , 5'-Hydroxyzearalenol (13) , Zearalenol (14) , 8'-Hydroxyzearalenone (15) , Zearalenone(16) , Zeaenone (17) ,α,β-Dehydrocurvularin (18) and Mycophenolic Acid (19) . Compounds (3) and (13) were new nature compounds, and compounds (6) and (7) were first elucidated their spectroscopic structures by X-ray single-crystal analysis.Compounds (1) and (3) inhibited the conidia growth of Pyricularia oryzae by swelling effect and induced morphological changes of the mycelia by curling effect with the minimum inhibitory concentration (MIC) 3．1μg/mL. Compounds (5) , (6) , (9) and (11) induced morphological changes of the mycelia by weak curling effect with MIC 12．5μg/mL, 3．1 μg/mL, 0．2μg/mL and 0．2μg/mL, respectively. Compound (12) induced morphological changes of the mycelia by weak curling and strong beads formation with MIC 3．1μg/mL. Compounds (4) , (7) , (8) , (10) , (15) , (17) , (18) and (19) inhibited the mycelia growth with MIC 25μg/mL, 6．2μg/mL, 1．6μg/mL, 0．2μg/mL, 3．1μg/mL, 0．1μg/mL, 25μg/mL and 25μg/mL, respectively.The activity results of our study accorded with the relevant reports and it showed that the bioassay method using conidia of Pyricularia oryzae could efficiently guide the isolation of the bioactive metabolites of marine-derived fungi and could be a primary screening for bioactive substances.