| The extraction and purification technique of polysaccharides from the culture medium of cordyceps militaris is investigated. The structures and antioxidantion of the polysaccharides from cordyceps and culture medium of cordyceps militaris are compared. Meantime, the extraction and separation of cordycepin in the culture medium of cordyceps militaris are investigated. The HPLC analysis of cordycepin has been established.1. The technology of polysaccharide extraction from the culture medium of cordyceps militaris with water, ultrasound and microwave was developed. The extraction efficiencies of polysaccharides by water bath, ultrasonic and microwave are 81.21%,67.94% and 89.64%, respectively. The process parameters of extraction by microwave are dry powder samples with 15 times the mass of water in a microwave oven 60W 1min, adding 20mL ethanol, precooled 0℃for 24h, crude polysaccharides.2.The structures of the polysaccharides from the culture medium and fruiting body of cordyceps militaris are compared. The result is that they both have the same structures and characteristic absorption peaks. The scavenging ratio by 02-·and [·OH] of polysaccharides from the culture medium increase with polysaccharides concentration.The antioxidantion ability of this purified polysaccharides is slightly enhanced and similar with the one from fruiting bodies. When the polysaccharides concentration is 1.0mg/mL,the scavenging ratios to 02-·and [·OH] are 45.19% and 22.62% before purification,and the scavenging ratios to O2-·and [·OH] are 49.06%and 24.69% after purification.3.The determination method of cordycepin and adenosine using HPLC is optimized. The cordycepin and adenosine from the culture medium of cordyceps militaris and cordyceps are separated by microwave extraction. Their concentrations are determined by using high performance liquid chromatography. The chromatographic separation was achieved by using Inertsil ODS-SP (150×4.6mm,5μm)column, using acetonitrile-water as mobile phase with a flow rate of 1mL/min at 35℃under DAD detector with 260nm and a injection volume 10μL. The calibration curves of cordycepin and adenosine are linear over the concentration range from 2-10μg/mL, and the correlation coefficiencies were 0.9998 and 0.9992. The standard recoveries were 63.30%-67.11% and 63.97%-70.39% with the RSDs were 0.56%-1.97% and 0.94%-1.97%, and the LODs were 0.19μg/mL and 0.02μg/mL.4. The technology of cordycepin extracting from the culture medium of cordyceps militaris by water bathing, ultrasonic, microwave and supercritical fluid extraction was investigated. The extraction rates of polysaccharide by water bathing, ultrasonic,microwave and SPE are 58.20%,69.05%,85.65% and 9.63%, respectively. The parameters of extraction by microwave are dry powder samples with 10 times the mass of water in a microwave oven 40W 3min.5. The adsorption and desorption of cordycepin by 732 cation exchange resin,717 anion exchange resin and silica gel are investigated. The elution rate of cordycepin is 91.14%, when 732 cation exchange resin is used with 0.15mol/L ammonia as eluent. The elution rate of cordycepin is 82.83%, when 717 anion exchange resin is used with distilled water as eluent. The elution rate of cordycepin is 89.35%, when silica gel column is used with chloroform:methanol:petroleum ether(v:v:v)=9:1:4 as eluent. Cordycepin from the culture medium of cordyceps militaris purified by 732 cation exchange resin is determined by HPLC with less impurity peak and cordycepin retention rate of 88.10%.
|
PDF Full Text Request