Isolation And Purification Of Polysaccharide From Cordyceps Militaris And Its Bioactivity

In this dissertation, we choose Polysaccharides from cordyceps militaris stroma as experiment materials. A study of the extraction of Polysaccharides from cordyceps militaris stroma was conducted by ultrasonic wave treatment and enzyme hydrolysis. In order to further study the activity of Polysaccharides from cordyceps militaris stroma, it was isolated, purified and determined with a series of instruments analysis technique. Polysaccharides from cordyceps militaris stroma and its acetylated-derivates’activities were investigated.1. To establish the extraction technology of cellulase assisted by ultrasonic wave of Cordyceps militaris polysaccharides. The optimization was applied with Box-Behnken center united design and the method of response surface analysis (RSM). On the base of single factor investigation, effects of cellulase content, enzymolysis time, ultrasonic wave time and ultrasonic wave temperature as well as their interactions on yield of the polysaccharides were studied. The optimal extraction conditions were obtained as follows:the amount of cellulase1.9%, enzymolysis time1.1h, ultrasonic wave temperature50℃, ultrasonic wave time41min. Under the optimal condition, the yield of Cordyceps militaris polysaccharides was up to25.45%.2. These different ways to removing protein from Polysaccharides from cordyceps militaris stroma, sevag method, TCA, activated carbon and enzyme hydrolysis, and that were compared with the clearance rate of protein and the polysaccharide retention as index, and choose the optimization way. In order to study the activity of Polysaccharides from cordyceps militaris stroma, the de-proteinated Polysaccharides were purified by DEAE-cellulose52, and it was analyzed by techniques of UV and SephadexG-100. The result showed that CMRP-1was homogeneous Polysaccharides.3. Using acetic anhydride as acetylated regent, DMF as catalyst,the acetylated CMRP-1were synthesized. The antioxidative activity of CMRP-1and prepared acetylated CMRP-1in vitro was determined, including scavenging activity against superoxide.and hydroxyl. The degree of the acetylated CMRP-1was0.468and the result showed that they were successfully acetylated by infrared spectroscopy. At the meantime,antibacterial activity test showed that the acetylated CMRP-1had a stronger antibacterial effect than CMRP-1and close to ascorbic acid. 4. In order to further study the hepatoprotective effect of acetylated CMRP-1on50%alcohol-induced acute liver injury in mice. The mice in each group were killed by cervical dislocation and blood were collected for the measurement of Superoxide dismutase (SOD) activity, Triglyceride (TG) content, alanine transaminase (ALT) and aspartate transaminase (AST) levers in serum, the liver, spleen and thymus were collected to calculate the coefficients, preparation of liver homogenate were measured the activities of Superoxide dismutase (SOD) activity, Glutathione peroxidase(GSH-PX)activity, Malondialdehyde(MDA) and glutathione(GSH) content, and pathological observation of liver tissue. Results The spleen coefficients, serum SOD activity TG content, ALT and AST activities, and SOD activity, GSH-PX activity, MDA and GSH content in the liver of mice from the150(mg/kg) acetylated CMRP-1groups were significantly different to those of the liver injury control group (P<0.05). Compare with blank control group, the indicators of150(mg/kg) acetylated CMRP-1group were extremely close to normal level, and the spleen coefficients, ALT and AST activities and GSH-PX activity, GSH content in the liver of mice from150(mg/kg) acetylated CMRP-1group were significantly advantage the positive control group.300(mg/kg) acetylated CMRP-1groups and600(mg/kg) acetylated CMRP-1groups had no protective on50%alcohol-induced acute liver injury in mice. It may be because the content of acetylated CMRP-lwas overtop. Conclusion150(mg/kg) acetylated CMRP-1has significantly hepatoprotective effect on alcohol-induced acute liver injury in mice.