Studies On The Bioactive Constituents Of Secondary Metabolites Of Two Sponge-associated Fungi

Sponge-associated microorganism is a rich source of secondary metabolites with diverse structures and biological activities. A study is carried out to investigate the bioactive lead compounds derived from sponge-associated microorganism. This thesis describes the study of the secondary metabolites produced by two selected sponge-associated fungi with anti-tumor and anti-inflammatory activities.Two hundred and eight fungal strains were isolated from twenty seven sponge samples which were collected from the seashore of Qiong hai and Sanya in Hainan Province. Fifteen active strains were picked out based on the cytotoxic assay using the P388 cell line by the SRB method and on the HPLC and TLC chemical screening. Among them, two strains which were identified as Epicoccum sp. JJY-40 and Alternaria sp. JJY-32 by ITS sequencing also exhibited high anti-inflammatory activities. So they were selected for further study of their secondary metabolites.Large-scale fermentations of the two bioactive strains were performed to obtain the active extracts. By means of chromatography over silica gel column, Sephadex LH20, preparative TLC and semi-preparative HPLC, eighteen (1-15,29,30,31) and sixteen compounds (16-28,32,33,34) were isolated from the sponge-associated fungi JJY-40 and JJY-32, respectively.Basing on their physico-chemical properties and spectral data (IR, UV, MS, NMR, etc.), twenty eight structures were elucidated as 5-methylbenzene-1,3-diol(1), 1,3-Dihydroxy-4,5-dimethylbenzene (2), 2,4-dihydroxyl-5,6-dimethyl- benzoic acid (3), 2,4-dihydroxyl-6-methyl-benzoic acid (4), 3-indolylacetic acid methyl ester (5), cyclo-(Tyr-Pro)(6),N-acetyltyramine(7),3-hydroxy-7-methyl-6,7-dihydrocyclop-enta [c]pyridine-5-one (8), cyclo-(Pro-Leu) (9), cyclo-(Pro-Ile) (10),cyclo-(Val-Pro)(11), isomaltol-glucoside(12), Inosine(13), flazin(14), D8646-2-6'(15), Ergosterol (16), ACTG-toxins H (17), Dicycloalternarene 1b(18), Dicycloalternarene 2b(19), Dicycloalternarene 3a(20), Dicycloalternarene 3b(21), Dicycloalternarene 4b(22), Dicycloalternarene 4a(23), Phencycloalternarene 1(24), Phencycloalternarene 2(25), Tricycloalternarene 6a(26) , Tricycloalternarene 12a(27) and Tricycloalternarene 13a(28). Among the twelve new compounds, there were ten terpenoid derivatives (18-25,27,28), a hydroxy derivative of Aucubinine B (8) and a pyran derivative (15). New compounds 18-25 are a series of terpenoid derivatives with novel side-chain which have not been reported previously. New compound 28 is a rare spiro-oxindole terpenoid derivative. The known compounds were identified as four benzol derivatives (1-4), four cyclic dipeptides (6,9-11), a glucoside (12), a nucleoside (13), three alkaloids (5 , 7 , 14), a sterol (16) and two tricycloalternarene-type terpenoid derivatives (17,26), respectively.Macrophages play a crucial role in innate immunity which is the body's first line of defense against infection and LPS can activate macrophages to induce inflammatory response by stimulating the secretion of cytokines. So we used LPS- stimulated RAW264.7 cells which can simulate macrophage system in vivo perfectly as cell model to evaluate immunomodulatory activity of some compounds in vitro by ELISA, RT-PCR, Western Blot and Immunofluorescent staining screening methods. The results indicated that compounds 15 and 17 could inhibit secretion and mRNA expression of LPS-induced TNF-αand NO. They also inhibited LPS-induced iNOS and COX-2 expression obviously. The high anti-inflammatory activities of such compounds have not been reported yet. The anti-inflammatory and anti-tumor activities of the rest new compounds are being evaluated.In a word, fifteen active sponge–associated fungal strains with cytotoxic activities were obtained by combinatorial screening. Among them two strains also exhibited high anti-inflammatory activities. Twenty eight compounds were obtained from the two aimed fungi. Among them twelve compounds were new including a rare spiro-oxindole terpenoid derivative. Compounds 15 and 17 were first found to show high anti-inflammatory activities. This study provides novel structure types for natural product chemistry and bioactive template compounds for exploring new anti-inflammatory drugs. It also proves that the method we used is effective in looking for new bioactive secondary metabolites from sponge-associated fungi.