Isolation And Identification Of Sea Anemone-derived Fungi From The South China Sea And The Activated Secondary Metabolites Of A Fungus

Marine ivertebrates with physical weak or moving slowly such as sponge, coral,and sea anemone can survive in the competitive marine environment, which mainlydepend on the secondary metabolites they produced. They play an important part inallelopathy and symbiosis, fight for food and spaces and information transfering ofspecies, and will be the valuable resources for the research on medicinal leadcompounds. However, marine herbs resource from organisms has becoming a urgentproblem in the research of marine drugs. The marine microorganism withoutrestriction of natural conditions could settle the puzzle of medicinal resource throughfermentation of the mass production. In addition, the most important thing is that thereal producer of certain actived chemicals once thought produced by marineorganisms is the symbiotic fungi, which made the marine microorganism of greatactual and ecological significance.The thesis introduced the isolation, identification and antibacterial activities of48endophytic fungi including2phyla (Basidiomycota, Ascomycota),8orders(Capnodiales, Trichosphaeriales, Pleosporales, Hypocreales, Eurotiales, Agaricales,Diaporthales, Xylariales),21genera (Cladosporium sp., Pleosporaceae sp.,Phomopsis sp., Fusarium sp., Coprinellus radians, et al), from one sea anemone(TA26) collected in the South China Sea led to the discovery of7activated strainsCladosporium sp.(TA26-2, TA26-6, TA26-61), Nigrospora sp.(TA26-9), Alternariasp.(TA26-16, TA26-37), Exserohilum sp.(TA26-20), which showed strong inhibitioneffects against9pathogenic bacteria at1mg/mL comparable with the effect ofciprofloxacin at20μM. In addition, the diversity of fungi was analysed.The actived secondary metabolites of Nigrospora sp.(TA26-9) isolated from seaanemone were studied. The secondary metabolites were isolated by column chromatography on silica gel, Sephadex LH-20and preparative HPLC, and elucidatedby spectroscopy of NMR, MS, IR, UV and X-ray from the crude extraction offermentation broth and fungal mycelia.15compounds (1–15) were respectivelydetermined, including10anthraquinones,1sesquiterpene and4steroids:4a-epi-9α-methoxydihydrodeoxybostrycin (1),10-deoxybostrycin (2), igrosporin B(3),9α-hydroxydihydrodesoxybostrycin (4),9α-hydroxyhalorosellinia A (5),4-deoxybostrycin (6), bostrycin (7),3,5,8-trihydroxy-7-methoxy-2-methylanthracene-9,10-dione (8), austrocortirubin (9), compound10,cis,trans-abscisic acid (11),(22E,24R)-cerevisterol (12),(24S)-24-methyl-5α-cholestane-7,22-diene-3β,5-diol (13),(22E,24R)-24-methyl-Sa-cholesta-7,22-diene-3β,5α-diol (14), ergosta peroxide (15).Compounds1,2,10were novel compounds, and the single crystals of1,4,9wereobtained at the same time, which contributed to determine the absolute configurationof1as2S,3R,9R,1aS,4aR. Nine new acetyl derivatives (6a、7a、8a–8f、9a) and onereported derivative (8g) were synthetized to discuss the structure-activity relationshipof anthraquinones. Preliminary antibacterial activity experiment showed that, thecompounds2,3and6a were found to be most active. Compound2showed strongantibacterial activity against B. subtilis with an MIC value of625nM. Compound3showed pronounced antibacterial activity against B. subtilis and B. cereus with MICvalues of312nM, which were equivalent or stronger than those of the positive controlciprofloxacin with MIC values of312and1250nM, respectively. Additionally, thederivative6a exhibited promising inhibitory activity against B. cereus with an MICvalue of48.8nM, which was approximately25-fold more potent than that ofciprofloxacin (MIC=1250nM). The results of antibacterial experiment indicated thathydroanthraquinone analogues play a key role as antibacterial secondary metabolitesin the fungus Nigrospora sp.On the basic of allelopathy reaserch during endophytic fungi derived from seaanemone, two strains (TA26-33�'�TA26-46) were co-cultured by solid medium whichled to the isolation of3macrolide paecilomycin F (16), LL-Z1640-1(17), zeaenol(18). Comparing with the monoculture of TA26-46, the yield of compound16 changed obviously during co-culture, and the antibacterial activity against B. subtilis,S. aureus, M. tetragenus, E. coli of16was also apparently stronger than other twocompounds.The research on the diversity and antibacterial activities of fungi provided a basicfor searching activited strains. The evaluation of antibacterial activities ofanthraquinone derivatives isolated from Nigrospora sp. possessed an importantecological significance in the discovering medicinal lead compounds. The explorationof microbial coculture maybe represent a potentially important approach to discovernovel activated metabolites.