Culture And Identification Of Annmorph Of Cordyceps Sinensis And Its Anti-tumor Mechanism

In this study, the culture of Annmorph of Cordyceps sinensis was discussed because of its expense and rare resources, while Annmorph was identified, efficient component was tested, and anti-tumor mechanism of Cordyceps sinensis was analysed, thus making solid basis for substitution of crude Cordyceps sinensis to artificial medcine.Several strains of annmorph of Cordyceps sinensis were successfully isolated through the seeding of budding ascopes derived from collection of several sporophores of Cordyceps sinensis. The culture condition of mycelium was tested. The results showed that mycelium of Cordyceps sinensis are aerobic, have very slow growth rate, brown secretion and auto-fluorescence. The optimal temperature for growth is 15~18℃, with pH 5~7. These provide valued refences for culture of Cordyceps sinensis, culture medium selection and immuno-analysis.There is mycelium weight increase in 0~16 days by flasks culture. The nitrogen and carbon sources have effect on mycelium weight. The better carbon sources are glucose, starch and mannitol. Among all nitrogen sources nitrogen-compounds source was the best. Larva of the Hepialu armoricamus was successfully infected by feed soaked with hyphae and ascopes; muscardine cadaver rate was 70-76.6 percent. There is same antigen of hyphae in the anamorph and the larva of the field infected Hepialu armoricamus by immuhistochemical method.These indicated that crude and artifical Cordyceps sinensis has some contents of efficient component, mannitol, that contents are affected by culture method and nutrition, so providing valued data for substitution of crude Cordyceps sinensis to artificial medcine.In this study, the rDNA ITS and 5.8SRNA of Cordyceps sinensis and its related taxa were amplified and sequenced The sequence comparison showed that similarity is 97.5%~100% between Cordyceps sinesis and A,B,C isolates, whereas similarity between Cordyceps sinesis and Stachybotry chartarum, Cordyceps militari, Tolypocladium caledonica,, Cephalosporium maydiss, Paecilomyces sinensis,Verticillium bulbillosum,were 78.9%, 76.7%, 72.2%, 68.6%,62.8%, 59.5%, respectively. Similarity between these isolates and Hirsutella sinensis were 98.4%~99.7%. Taken together, the three isolates and Cordyceps sinesis were same organism within different life cycle stages. These isolates were therefore the anamorph of Cordyceps sinesis, namely Hirsutella sinensis, rather than other species.The mannitol content of crude Cordyceps sinensis, cultured mycelium of cordyceps sinensis and Cordyceps militaris was measured via colorimetric method. While the effects of flasks culture and nitrogen soureces on mannitol content were also analyzed. The result showed that the mannitol content in crude Cordyceps sinensis was higher than that in culture mycelium, while which slightly higher than Cordyceps militaris culture. Mannitol content in mycelium and culture liquid decreased with flasks culture. The different nitrogen sources have various effects on mannitol level in mycelium and culture liquid, Nitrogen-compounds source was the best nitrogen sources. These indicated that mannitol contents are affected by culture method and nutrition, so providing valued data for substitution of crudeCordyceps sinensis to artificial medcine.The polysaccharides in crude cordyceps sinensis and mycelium were measured using phenol-sulphuric acid method, the effect of flasks culture and nitrogen sources on mannitol content were tested. The results indicated that the content of polysaccharides in mycelium was higher than that in crude Cordyceps sinensis. The content of polysaccharides was inhibited as result of decrease of mycelium level by flasks culture. The content examination of regulatory aspects on polysaccharides by different nitrogen showed that nitrogen-compounds was appropriate source. These indicated that polysaccharide contents are affected by culture method and nutrition, so providing valued data for substitution of crude Cordyceps sinensis to artificial medcine.To investigate the anti-tumor mechanism on SP2/0 myeloma cells by water extracts and polysaccharides from mycelia of Cordyceps sinensis, the inhibitory ratio of the cells were measured by MTT assay; apoptosis by agarose gel electrophoresis, flow cytometry (FCM) and hoechst 33342 fluorescence staining; cell cycle by FCM, and the expression of p53 gene was analyzed by real time RT-PCR in both the test and control groups. The results indicated that water extract and polysaccharides suppress the growth of SP2/0 myeloma cell line, induce cell apoptosis and phase G0/G1 arrest, and the level of p53 was decreased. These suggest that the change of p53 gene expression induced apoptosis and phase arrest of SP2/0 cells. The proliferation of SP2/0 myeloma cell inhibited through apoptosis and phase arrest.