Isolation,Purification And Bioactivity Of Polysaccharide From Cordyceps Militaris Fruit Bodies And Its Quality Evaluation

Cordyceps militaris(L.)Link contains many active components such as polysaccharide,nucleosides,mannitol,etc,which has function of anti-tumor,anti-oxidant,anti-aging,regulating the immune system and so on.C.militaris was approved by the Food and Drug Administration of China in 2009 as a new resource food,which can be used in the production of food or health products.It promotes the produce of C.militaris and its application in the field of health product development.However,there are still some problems in the study of C.militaris.Only a few study on structure and activity of homogeneous polysaccharides have been reported.Fruit body quality was evaluated mainly by agronomic traits of fruit body size,color etc.,and lacking of effective evaluation methods for evaluating active components as well as lacking of systematic study on factors that influencing the active components.There are many products of caterpillar fungi on the market,however there are only a few reports on the chemical components of C.militaris and other caterpillar fungi products,and effective method for identification different caterpillar fungi is required.In view of the above problems,this paper was mainly focused on the following three aspects.(1)Isolation and purification polysaccharide with high molecular weight from C.militaris fruit bodies and identify the structure and evaluated the activities.A homogeneous polysaccharide(CP2-S)was purified from C.militaris fruit bodies by hot water extraction,ethanol precipitation,DEAE-Sepharose Fast Flow and Sephacryl S-400High-Resolution Chromatography.The polysaccharide had a molecular weight of1.09×10~6 and was mainly composed of glucose.Methylation and NMR analysis indicated this polysaccharide was consisted of a main backbone composed of?4)-?-D-Glcp-(1?4)-?-D-Glcp(1?with(1?6)D-Glcp–branches.Experiment in vitro indicated that CP2-S could stimulate morphogenetic changes of RAW264.7 macrophages cells and promoted nitric oxide production,phagocytosis,respiratory burst activity and secretion of IL-1?and IL-2.The anti-tumor activity of CP2-S was carried out by using Lewis lung cancer mice as model.The mice were intraperitoneally injected with CP2-S every day at different dosages for 14 d.Experiment in vivo indicated that CP2-S could inhibited the Lewis lung tumor growth in mice and increased the spleen index and thymus index of mice significantly as well as the secretion of Ig M and Ig G in the serum.(2)Establish methods for the determination of active components of polysaccharide,nucleosides and alditol.Distribution of polysaccharide molecular weight was determined by HPSEC-MALLS-RI,monosaccharide composition was determined by high performance anion exchange chromatography.HPSEC spectrum,monosaccharide composition and content of polysaccharide were combined to evaluated the polysaccharide in C.militaris.A method for simultaneous determination 16 kinds of nucleosides using HPLC and a method for simultaneous analysis of free alditol and small molecular sugars by high performance anion exchange chromatography with pulsed amperometric detector were established in this study.The effect of different strains,culture media and culture time on the indices of polysaccharide,nucleosides,and mannitol of C.militaris fruit bodies were evaluated.The results showed that strain had the greatest effect on the three kinds of active compounds,followed by medium and culture time,among which cordycepin,was the most active component that affected by strain,with the biggest content difference of more than 10 folds between two different strains.When C.militaris was cultured on different media,the highest contents of polysaccharide and nucleosides were recorded on treatment of silkworm chrysalis.The contents of polysaccharide and nucleosides were increased when the media of rice or wheat supplemented with silkworm powder.With the increase of culture time,the content of polysaccharide increased first and then decreased,while the cordycepin content increased with the increase of culture time.(3)C.militaris samples and the other main products of caterpillar fungi on market were analyzed by using the new establish methods of determination for nucleosides,alditols and polysaccharides.Results indicated that C.militaris,Ophiocordyceps sinensis,C.cicada could be clearly distinguished by HPLC spectrum of nucleosides.Cordycepin is the symbolic component of C.militaris and C.militaris fruit bodies containing 5 major peaks including uridine,guanosine,adenosine,cordycepin and N~6-(2-hydroxyethyl)adenosine,submerged culture mycelia of C.militaris containing 4 major peaks including uridine,guanosine,adenosine,cordycepin,and mycelia of solid-state culture of C.militaris containing 1 major peak of cordycepin.O.sinensis contains 4 major peaks including uridine,guanosine,inosine and adenosine.C.cicada and its mycelia of submerged culture and solid-state culture contain 4 major peaks inculding uridine,guanosine,adenosine and N~6-(2-hydroxyethyl)adenosine.Mycelia products of Bailing,Ningxingbao and Jinshuibao contained 3 main peaks including uridine,guanosine and adenosine.On the basis of nucleosides analysis,the HPAEC spectrum of free alditols and small molecular sugar can be used to distinguish O.sinensis,Bailing,Jinshuibao and Ningxinbao.On HPAEC spectrum,there is no erythritol peak from O.sinensis,but there is obvious erythritol peak from Bailing,Jinshuibao and Ningxinbao.The erythritol peak was the highest from Bailing capsules,which was a mycelia product of Hirsutella sinensis and the mannitol peak was the highest from Jinshuibao,a mycelia product of CS-4 strain,and both high mannitol and arabitol peaks from Ningxinbao.The HPSEC spectrum of polysaccharide can be used to identify O.sinensis,C.militaris,C.cicada and mycelia products combined with the analysis of nucleosides and alditols.