The Selenizing Modification Of Codo-Nopsis Pilosula Polysaccharide And Their Antioxidative And Immune-Enhancing Activities

Codonopsis pilosula polysaccharide (CPPS) is one of the main active components of Codonopsis Pilosula. It has many actions, such as immune-enhancing, antioxidation, anti-virus, anti-fatigue, anti-tumor and so on. Selenium as a kind of microelement also has the efficacy of antioxidation, immunoenhancement, participating in the formation of coenzyme Q, reducing the metal toxicity of mercury and lead. Recently, many studies confirmed that molecular modification could greatly enhance the biological activities or produce much new medicinal value for herbal polysaccharides. In order to explore the possibility of the selenizing modification improving polysaccharide biological activity, in this study CPPS was selected as the research object, extracted, isolated, purified and selenizingly modified after the conditions was optimized, and the change of antioxidative and immune-enhancing activities of the polysaccharide before and after modification were compared. The details were divided into five parts as follows:Experiment1. Extraction, purification and isolation of CPPS The crude CPPS (CPPSc) was extracted by water decoction and ethanol precipitation, deproteinized by trichloroacetic acid (TCA) method and isolated by DEAE-52column chromatography through elution respectively with distilled water and sodium chloride solution. Two purified CPPS, CPPSpl and CPPSp2were obtained. The purity of CPPSpl was identified by Sephadex G-200column chromatography. The carbohydrate and protein contents were measured respectively by phenol-sulphuric acid method and Coomassie brilliant blue G-250method. The results showed that the extraction rate of CPPSc was9.677%, carbohydrate content was76.01%and protein content was3.67%; the yield of CPPSpl and CPPSp2were respectively3.792%and2.741%, carbohydrate content were respectively99.95%and25.77%and protein content were respectively0.02%and0.05%. The Sephadex G-200column chromatography of CPPSpl still displayed one elution peak, which indicated that CPPSpl was a uniform polysaccharide.Experiment2. Optimization of selenizing modification condition of CPPS The influence of reaction time (A), reaction temperature (B) and reagent ratio (C) in the selenizing modification conditions on the yield and selenium content of product was studied by response surface method (RSM). Regression model was built, the reliability of this model was verified, interaction between factors was analyzed and the model was verified. The structure of selenide CPPS (sCPPS) was analyzed by infrared spectrum. The results showed that in the range of5-10h of reaction time,50-90℃of temperature and0.4-1.2of reagent ratio, the quadratic polynomial regression equation about three factors to product yield (Y1) and selenium content (Y2) were Y1=94.74163-4.97620A-1.31068B-35.32375C+(5.10000E-003) A B+1.09500A C+0.28688B C+0.25288A2+(5.85125E-003) B2+21.81563C2and Y2=-45.42204+2.27043A+0.80002B+41.9153C-0.025588A B-0.77625A C+0.15172B C+0.036755A2-(4.50696E-003) B2-30.57598C2. The variance analysis showed that two equations possessed high fitting degree and could be used in control of modification conditions. The influence degrees of three factors on both yield and selenium content of the product were C> B> A. The product yield and selenium content in confirmatory test were not significant difference as compared with predictive value. The infrared spectrum of sCPPS demonstrated the bond of selenite selenium ester and selenium ester, which showed that CPPS was selenylated.Experiment3. Effects of sCPPS on serum CAT activity and MDA content in mice168mice were randomly assigned divided into14groups,12in each group. The mice in model group and high, middle and low dose groups of4polysaccharides (sCPPSpl, sCPPSc, CPPSpl, CPPSc) were intraperitoneally injected with0.1mL5%hydrogen peroxide solution. At the same time, the mice in3doses groups of each polysaccharide were intraperitoneally injected respectively with0.2mL corresponding polysaccharide solution, in model group and control group, equal-volume physiological saline, once a day for eight successive days. On second day after last injection, the blood of six mice from each group was sampled by removing eyes to separate serum. The CAT activity and the MDA content were determined with kit. The results showed that CAT activities in every polysaccharide group were significantly higher, MDA content significantly lower, than that of model group. The CAT activities in sCPPS groups were significantly higher, most MDA content significantly lower, than that of the same dose of CPPS group. In the same polysaccharide, CAT activity increased and MDA content decreased along with the increase of dose. In high dose group of sCPPSpl the CAT activity was the highest and MDA content was lowest. These results indicate that every polysaccharide had a obvious antioxidant activity, selenizing modification could further enhance the antioxidant activity of CPPS, and there was a certain dose-effect relationship, the antioxidant activity of sCPPSpl at high dose was strongest.Experiment4. Effects of sCPPS on growth and structure of spleen in mice The experimental animals and treatment were the same as Experiment3. On second day after last injection, six mice from each group were weighed, the spleens were weighed for calculating spleen index and made paraffin section to measure the thickness of capsula serosa lienis and lymphocyte density of periarterial lymphatic sheath. The results showed that all polysaccharides could increase the spleen index, thickness of capsula serosa lienis and lymphocyte density. In all dose groups of sCPPS, the spleen index, thickness of capsula serosa lienis and lymphocyte density were numberly or significantly higher than that in the same dose of CPPS group. In the same polysaccharide,3data increased along with the increase of dose. In high dose group of sCPPSpl the spleen index and lymphocyte density were the largest. These results indicated that all polysaccharide could promote the spleen growth, structure improvement and lymphocyte proliferation thus strengthening immunity, selenizing modification could further enhance the immune-enhancing activity of CPPS, and there was a certain dose-effect relationship, the immune-enhancing activity of sCPPSp1at high dose was strongest.