Study On Biological Characteristics And Some Active Metabolite Of Cordyceps Japonica

In recent years, with the decreasing of wild Cordyceps resources, domestic and foreign scholars have made a lot of research work on fermentation of Cordyceps mycelia, development of active products, and looking for new resources of Cordyceps fungus. Cordyceps japonica is one of the Cordyceps funguses which are studied by Japan and Korea. It currently has been used as a substitute for Cordyceps sinensis, and achieved related industrial fermentative production. In China, the study of Cordyceps japonica is scarce, especially in the cultivation and active metabolite. With the increasing of development and utilization of edible-medicinal fungi, further research of cultivation and active metabolite of Cordyceps japonica must be of great academic value and economic value.One strain is picked from the strains which are isolated and purified from the wild Cordyceps japonica of the Changbai Mountain. The biological characteristics and related active metabolite of Cordyceps japonica are studied by using PCR, GPC, GC, GC-MS, IR and other techniques. The results are showed below:1. Identification and life history confirmation of Cordyceps japonica: Isaria japonica is identified by means of morphology and 16S rRNA sequencing. Results of the life history confirmation of Cordyceps japonica show that Cordyceps japonica and Isaria japonica are the same species during the different growth stages.2. Optimization of culture conditions for artificial cultivation: The artificial cultured conditions are optimized according to the one-at-a-time method and orthogo- nal test, with fruitbody's quality and production for index. The best medium and best culture conditions are as follows: wheat is chosen as the major nutrition for culture under the moisture 70%, wheat 25g, optimal pH 7.0, sucrose 0.4%, temperature 19～25℃, with appropriate ventilation under high temperature, single-jar yield can reach 4.576 g (dry weight), biological conversion 18.3%.3. Preparation and analysis of polysaccharides: Exopolysaccharide (EPS), mycelium polysaccharide (IPS1) and fruitbody polysaccharide (IPS2) of Isaria japonica are analysed in physical and chemical properties, monosaccharide composit- ion and structure for the first time. EPS's sugar content 87.4%, molecular weight 74 kDa, mannose, galactose and glucose's molar ratio 12.9:2.4:1.0; IPS1's sugar content 89.2%, molecular weight of the main component is 233kDa, three monosaccharidees'molar ratio 11.7:5.8:1.0,β-pyranose; IPS2-1's sugar content 91.3%, molecular weight 265kDa, three monosaccharides'molar ratio 1.3:1.0:2.9,α-pyranose; IPS2-2's sugar content 93.2%, molecular weight 25 kDa, three monosaccharides'molar ratio 1.5:1.1:1.0,β-pyranose. The polysaccharides (including EPS and IPS) of Isaria japonica, Paecilomyces militaris, Isaria farinose, Beauveryia bassiana and Hirsutella sinensis are compared. Conclusions are showed below: EPS and IPS of Cordyceps funguses are basically of the same monosaccharide composition, including mannose, galactose and glucose. Generally, mannose accounts for a large proportion, while glucose accounts for the least; most of polysaccharides'molecular weights are about 20 to100 kDa; main-chain conformations are mostlyβ- type, especially the IPS.4. Activity assay and analysis of micromolecule metabolin: Further investigation is focus on antibacterial, anti-virus and anti-tumor activities in vitro. Antibacterial test shows that the extracts can inhibit the growth of bacteria, while can't inhibit fungal growth. The antibacterial activity of ethyl acetate extract is slightly higher than that of n-butanol extract. Anti-tumor experiments show that both the extracts of the fruitbody can effectively inhibit the growth of Hela cells and P388 cells, which could be a potential new anticancer drug resource and have the value of further purification. Anti-viral activities of the extracts are low. The main volatile components of ethyl acetate extracts are assayed by GC-MS. The results show that most of these compone- nts are ketones, amides and esters, containing phenyl and oxygroup, which have high activities and can be easily oxidized.