Mangrove Endophytic Fungi Trichoderma Sp.Xy24 Terpenoid Metabolites And Microbial Transformation Of Harzianone

Previously one endophytic fungus was isolated from a mangrove plant Xylocarpus granatum by our research group, and the ethyl acetate extracts exhibited good cytotoxicity against several cell lines. Successively 10 L fermentation was performed and extracts of the filtrate and mycelia were gained.In this thesis, the extracts of the 10 L fermentation was isolated and purified by using various column chromatography techniques. Structures of the compounds were identified by MS and extensive spectroscopic analysis. Four known compounds were obtained:one Harziane type diterpene Harzianone (1), one acorane type sesquiterpene Trichoacorenol (2) and two polyketides Trichodimerol (3) and Trichocage B (4). Among them, Harzianone was one member of the scarce family of compounds which were named as Harziane type diterpenes; Trichodimerol exhibited medium antiviral activity. Considering the rareness of the Harziane diterpenes,60 L fermentation were performed aiming at the discovery of this kind of compounds and other bioactive compounds.The petroleum ether layer in which the terpenoids were rich were carefully isolated and purified, nine compounds (compounds 5-13) were obtained and characterized as: 1α-isopropyl-4α,8-dimethylspiro[4.5]dec-8-ene-2β,7α—diol (5), 1α-isopropyl-4α,8-dimethyl-spiro[4.5]dec-8-ene-3β,7α-diol (6), Cyclonerodiol (7),10,11-dihydroxy-cyclonerodiol (8), 14-hydroxy-trichoacorenol (9), (9R,10R)-dihydro-harzianone (10), Ergosterol (11), Ergokonin B (12), methyl stearate (13). Among them, compounds 5-9 were sesquiterpenes,10 was Harziane diterpene,11-12 were sterols,13 was aliphatic ester. Compounds 8-10 were new compouds.During the formentation of Trichoderma sp. Xy24, several cultures grew in an abnormal way. According to the analysis of their second metabolites, an array of new peaks was present in comparison to those of the normally growing cultures in the HPLC profiles. HPLC-UV analysis and HPLC-MS" showed that the compounds represented by the new peaks were derivatives of harzianone which was isolated as the second metabolites of Trichoderma sp. Xy24 mentioned above. So we presumed that these cultures growing abnormally were contaminated by other microbes, which led to the transformation of harzianone. This was verified by our consequent study on the transformation of harzianone by a Bacillus strain isolated from the liquid culture medium in which the abnormal cultures were growing.Nine transformation products were obtained and identified by MS and 1D NMR,2D NMR:4β-hydroxy-harzianone (14),2α-hydroxy-harzianone (15),2α,17-dihydroxy-harzianone (16),3α-hydroxy-3β,17-epoxy-harzianone (17),3β,17-epoxy-harzianone (18), Harzianone acid (19),3β,4(3-dihydroxy-harzianone (20),2α,3β-dihydroxy-harzianone (21), Harziandione (22). Among them, compounds 14-21 were new members of the scarce family of compounds known as Harziane type diterpenes. According to the characters of these transformation products, their biological synthesis pathsways were rationally presumed which included hydroxylation, oxidation, dehydration and intramolecular acetalization. This study made a contribution to the diversity of the scarce Harziane diterpene family.