Studies On The Indolalkaloids From Two Strains Of Marine-derived Microorganisms

Because of the special ecological environment, marine-derived microorganisms possess distinct and complex metabolic capabilities, resulting in wide diversity of their secondary metabolites in chemical structure and biological activity. Thus, bacterium and actinomycetes become important resources of active lead compounds. This thesis describes the bioactive constituents from marine-derived bacterium E. tarda and Streptomycetes sp. FMA. The work includes screening aimed strains, fermentation studies, bioassay-guided fractionation, structural elucidation, preliminary evaluation for anti-tumor and anti-bacterial activities of pure compounds.A variety of marine sources including sponges, tunicates, red alga, acorn worms, and symbiotic bacteria have been shown to generate indole alkaloids, which represent the largest number and most complicated of the marine alkaloids (1/4 of total alkaloids). The alkaloids obtained from marine organisms frequently possess novel frameworks while in other cases terrestrially related compounds clearly exist. Marine metabolites often possess complexities such as halogen substituents. Their structure elucidation, chemical modification, stereochemistry, synthesis, and pharmacology have received a great deal of interdisciplinary attention from areas of research other than chemistry and include pharmacology, physiology, and medicine.Four active strains were picked out based on integrated screening that combines the cytotoxicity against P388 cells with chemodiversity (HPLC and TLC properties) of the metabolites from 126 marine-derived actinomycetes and bacterias. Among them, Streptomycetes sp. FMA was selected for further study of their secondary metabolites exhibited high anti-tumor activities and a series of HPLC peaks with similar UV absorption to those of indolocarbazoles. Besides, the gram-negative bacterium E. tarda, pathogen of fish, animal and human was also found to display a series of HPLC peaks with similar UV absorption to those of indolalkaloids. In order to investigate the potential of pathogens for drug discovery, the secondary metabolites and AHLs of E. tarda were studied as well.140L-scale fermentation of the E. tarda and 100L-scale fermentation of the Streptomycetes sp. FMA were performed to obtain the active extracts, respectively. By means of chromatography over silica gel column, Sephadex LH20, preparative TLC and semi-preparative HPLC, fifteen (1-15) and thirteen compounds (16-28) were isolated from E. tarda and Streptomycetes sp. FMA, respectively. Basing on their physico-chemical properties and spectral data (IR, UV, MS, NMR, etc.), twenty eight compounds were elucidated as 1,1-(3,3'-diindolyl)-2-phenylethane (1),3-(1H-indol-3-yl)-2,3'-bi-(1H-indole) (2),2,2-di(3-indolyl)-3-indolone (3), tri(1H-indol-3-yl) methane (4),2-[2,2-bis(1H-indol-3-yl)]ethylphenylamine (5), bis-(1H-indol-3-yl) phenylmethane (6),4-di(1H-indol-3-yl) methylphenol (7), (2S)-3,3-bis-(1H-indol-3-yl) propane-1,2-diol (8), Indole (9),2-(1H-indol-3-yl) ethanol (10), genistein (11), (2-Naphthyl)phenylamine (12), benzeneethanol (13), uracil (14), thymine (15), streptocarbazole A (16), streptocarbazole B (17),3'-(S)-epi-K252a (18),3'-(S)-K252a (19), RK 286c (20), K252c (21),4-bis(3-indolyl)-1H-pyrrole-2,5-dione (22),1H-indole-3-carboxylic acid (23), vanillic acid (24),4-hydroxybenzoic acid (25), 1-(2-phenyl)-1,2-ethandiol (26), 1-(2-furyl)-1,2-ethandiol (27) and 4-methoxy-3-methyl-2H-pyran-2-one (28), respectively. Among them, compound 1 and 2 were isolated from the natural sources for the first time. The other known compounds were identified as six indolecarbazole alkaloids (18-22), ten simple indole derivatives (2-10, 23), a isoflavonoid derived (11), a-naphthalene alkaloid (12),five benzene derivates (12-13,24-26) and four other kinds compounds (14-15,27-28). Besides, five N-acyl homoserine lactones (AHLs) signaling molecules of E.tarda were identified using gas chromatography-mass spectrometry (GC-MS) including C4-HSL, C6-oxo-HSL, C8-HSL, C8-oxo-HSL and C10-HSL.Cytotoxic activities of these compounds against several cancer cell lines were assayed by MTT and SRB methods, and three compounds (2,16,18) showed appreciated cytotoxicity. The anti-bacterial activities of compounds 1-10,12 against Clostridium perfringens, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans were tested by drug sensitive slips method and double dilution. The result indicated that compound 12 showed moderate inhibitions against C. perfringens and E. coli with MIC values of 31.25 and 37.5μg/mL, respectively.In a word, two new natural indolealkaloids, two novel indolecarbazole alkaloids together with twenty-four known compounds were obtained from the two marine-derived microorganisms. Studies mentioned above provide novel structures for searching new leading compounds and microbial resources for further study and it proves the method we used is effective in finding new active compounds.