Studies On Fermentation Optimization And Preparation Methods Of Ophiobolins

Microorganisms from extraordinary environments with unique adaptation mechanisms and metabolic pathways can produce secondary metabolites with novel structures and various activities, which have become an important source of lead compounds. "Talented strains" which can produce rich and active secondary metabolites have attracted widespread attention in the scientific community and have become great potential for drug development resources. Aspergillus ustus 094102 isolated from the rhizosphere soil of the mangrove plant Bruguiera gymnorrhiza grown in Wenchang, Hainan Province produces Ophiobolin O and Ophiobolin O reversed MCF-7/ADR resistance to adriamycin in the early pharmacological study. But due to small yield of Ophiobolin O, it becomes a problem for the further study of the compound. In order to solve the problem of drug supply, we carried out the research on optimization of fermentation and preparation methods of ophiobolins. At the same time, the ophiobolins produced by the strain and the secondary metabolites of one actinomycete strain from the South China Sea sponge sample were studied to find the active lead compounds. The main contents of the dissertation are listed as follows:1. The dissertation focuses on qualitative and quantitative detection, fermentation optimization, extraction, separation and preparation methods of ophiobolins which were produced by Aspergillus ustus 094102 from extraordinary environments.2. Basing on a variety of chromatographic separation and purification methods, such as TLC, reverse phase silica gel column chromatography, gel column chromatography, Sephadex LH-20 and HPLC, seventeen compounds were isolated from Aspergillus ustus 094102 and three compounds were isolated from actinomycete NO.71, thus twenty compounds were obtained. By means of spectral analysis (IR, UV, MS, NMR) and chemical reaction methods, the structures (Fig.1) of the twenty pure compounds were determined, including four new compounds. Among them there are fourteen ophiobolins (1～14), three drimane sesquiterpenes (15～17), two cyclic peptide (18～19) and an indolocarbazole compound (20).3. The biological activity screening models were used to test the compounds separated from Aspergillus ustus 094102 and actinomycete No.71. The activities of the compounds against human cancer cells were evaluated by the MTT assay method. Compounds 4-5 and 7-11 exhibited significant activity against G3K, MD-MAB-231, MCF-7 and MCF/3K cell lines and compound 18 showed significant activity against K562 cell line.4. The new compounds from marine fungi (except Penicillium sp. and Aspergillus sp.) have been summarized. A total of 1123 new compounds from marine fungi (except Penicillium sp. and Aspergillus sp.) from 1950s when Cephalosporins were discovered to August 2014 have been summarized, and the environmental source categories, structural categories and activites of these natural products were also described.In summary, this dissertation describes the optimization of fermentation conditions for production of ophiobolins by Aspergillus ustus 094102 from extraordinary environments and qualitative and quantitative detection, extraction and preparation methods of ophiobolins, which provides an idea about how to solve the drug source in drug research. Fourteen ophiobolins were isolated and identified on further studies of Aspergillus ustus 094102, four of them were new compounds. In addition, three drimane sesquiterpenes were also isolated from Aspergillus ustus 094102 and three compounds were isolated from actinomycetes No.71. Ophiobolins and echinomycin exhibit significant cytotoxic activity, providing lead compounds for the study of antitumor drugs. The environmental source categories, structural categories and activites of 1123 new compounds from marine fungi (except Penicillium sp. and Aspergillus sp.) have been summarized, which provides an important reference for the development of marine fungus.