Studies On Secondary Metabolites And Metabolic Regulation Of Paraconiothyrium Brasiliense

Insect had an abundant biodiversity, a very wide range of distribution, and was far in excess of the plant in species and amount. The insect's intestinal tract was alkaline, besides, it was rich in a variety of enzymes, which built a special environment that might breed special new microbial species. These microorganism might also have some unique metabolic pathways, which could produce some new skeleton compounds. Thus, in the recent years, in order to improve the chances of finding novel active compounds, the microbial research object had been extending from land and ocean to insects. However, at present, people still do relatively few research on the insect endophytes, the study of its second level metabolic product is also in a preliminary stage. Predictably, the research of insect endophytes as a new point of innovative drug research and development has a board prospect, moreover, it will also develop a new field for the development of natural product as well as provide important source of the development of novel active leading compounds.The fungus Paraconiothyrium brasiliense of the study was isolated from the gut of healthy Acrida cinerea, and the insect sample collected from the Shennongjia Forestry District (Dajiuhu national wetland park) in Hubei Province. The main studies include isolation, purification and configuration determination of the secondary metabolites, metabolic regulation for the strain and biological activities of some isolated compounds.The main results can be described as follows:1.71 compounds were isolated from the rice fermentation extracts by various chromatographic methods including normal phase silica gel column, reversed phase silica gel column, Sephadex LH-20 and HPLC. Then, their structures were elucidated by the chemical and modern spectroscopic methods, such as IR, UV, MS,1H-NMR, 13C-NMR, DEPT, HMBC, HSQC,'H-'H COSY, NOESY, CD and X-ray. Among them, there were 22 meroterpenoids (1-22),20 furanones (23-42), one pyrone (43), two spiroketals (44-45),10 dipeptides (46-55) and 16 other compounds (56-71). Of the above compounds, there were 41 new compounds, including 20 meroterpenoids (2-10,12-22),18 furanones (24-31,33-42), one pyrone (43), two spiroketals (44-45). In the study, the absolute configurations of compounds 24-26,28,32,33,37 and 43 were established by analyzing the CD spectrum. Additionally, the absolute configuration of compound Craterellins C was determined by single crystal X-ray diffraction (Cu Ka) for the first time, and then, the absolute configurations of compounds 2-16 were established by NOESY and coupling constants. The study provided spectroscopic evidences for the structure identification of meroterpenoids.2.The study expanded the diversities of secondary metabolites of the genus by OSMAC (One Strain Many Compounds) strategy, including changing the medium composition, salt stress, adding high concentration of sugar and high concentration of phosphate, epigenetic modification, adding precursors and alkali stress. The differences of fermentation products were apparently under the conditions of adding 3% NaCl or 3% NaBr in the medium by TLC and HPLC analyses. Finally, five eremophilanes (72-76) were obtained from the strain under the condition of adding 3% NaBr in the medium by repeated column chromatography, and their structures were elucidated by the modern spectroscopic methods. Additionally, the relative configutations of compound (72) were established by X-ray. Among the above five compounds, three (73,74,75) were found to be new compounds.It was the first isolation of eremophilanes (72-76) from the genus Paraconiothyrium sp., and we postulated that salt stress can effectively activate the silent metabolic pathway of the fungus. By epigenetic modification, two pyridinones (77-78) were isolated from Paraconiothyrium sp. that cultured with histone deacetylase inhibitor SBHA, which including one new compound (78). The pyridinones (77-78) were isolated from the genus for the first time, which could be due to the activation of silent gene by adding the SBHA in the medium.3. Some of the isolated compounds were screened for their cytotoxic activities in vitro, anti-R5 HIV-1 infection activities and 20S proteasome inhibition activities. Unfortunately, none of the tested compounds showed effective biological activities. The neuroprotective activities of the isolated compounds were evaluated by PC 12 cell apoptosis induced by hydrogen peroxide. Compounds 14 and 28 exhibited significant neuroprotective activity against H2O2-induced PC 12 cell damage.Summarily,78 compounds were isolated from Paraconiothyrium sp. in this study, and they were identified as meroterpenoids, furanones, dipeptides, and eremophilanes etc. Among them,45 compounds were new ones. The results showed that the secondary metabolites of the genus were abundant and diversified in structure types, which provided structure supports for the further research of the genus. Furthermore, the secondary metabolites of the genus were expanded by OSMAC strategy in the study. Besides the eremophilanes and pyridinones were isolated from the genus for the first time, which supplied experimental evidences for the application and popularization of OSMAC strategy in the research of microorganism.