Isolation Purification,Structure Identification And Biological Activities Of Polysaccharide From Codonopsis Lanceolata

Codonopsis lanceolata is Campanulaceae and perennial vines.It is distributed in China,Korea,Japan,and Europe.It shows high nutritional and medicinal values.Polysaccharide and saponins are the main active ingredients.To date,it has been studied more on saponins.However,no detailed studies on purification,structural characteristics and their biological activities of Codonopsis lanceolata polysaccharide.In the present study systematic studies about the extraction,isolation and purification,physicochemical property and antioxidative activity,inhibits melanoma metastasis via regulating integrin signalling of polysaccharide from Codonopsis lanceolata.In order to provide new direction and reference for comprehensive development and utilization of Codonopsis lanceolata resources in China.The main results are as follows:(1)The yield of polysaccharides from Codonopsis lanceolata by ultrasonic combination of cellulose enzyme method was used as an index.The optimal technological parameters were as follows:enzymolysis temperature 45 ℃,amount of enzyme 0.7%,pH 4.5,enzymolysis time 3h,the extraction rate of polysaccaride reached 10.18%,ultrasonic power 147 W,ultrasonic time 15 min,ultrasonic temperature 36℃.Under the optimal conditions,the extraction rate of polysaccaride reached 11.77%.(2)Four methods were applied to compare removal rate of different deproteinization treatment through Sevag method,trichloroacetic acid method,trichlorotrifluoroethane method and Sevag method mixed with protease pretreatment.The method of Sevag mixed with protease pretreatment was the best way to get rid of protein.The best deproteinization conditions are as follows:enzymic hydrolysis pretreatment of adding 0.20%protease to the polysaccharides solution at 65 ℃ with pH 6 for 3 hours,then Sevag method was repeated for four times to get rid of protein and the deproteinization rate was 92.01%and retention rate of polysaccharides was 97.66%.Polysaccharides were separated by DEAE cellulose-52 after deproteinization,and three important fractions CLPS 1(48.30%),CLPS2(40.50%)and CLPS3(8.15%)were got seperately.Using Elution pattern of polysaccharide through Sephadex S-300 elution peak,the peak was symmetrical and the high purity of Polysaccharide was obtained.The retention rate of CLPS1,CLPS2 and CLPS3 was 94.12%,92.31%and 90.5%.(3)The antioxidant capacity of polysaccharides from Codonopsis lanceolata showed that they have good antioxidant effects and CLPS1 was particularly obvious.The physical and chemical properties of CLPS1 showed that its solubility was affected by temperature and pH.And the optimal dissulation temperature was 80℃,during alkaline conditions it would dissule faster,but NaCl had little effect on the solubility of CLPS1.With increasing contents of CLPS1,it had higer viscocity,while the temperature of CLPS1 solution increasing its viscosity would be lesser.With the increasing of duration and rate of shear the viscosity falled obviously.The polysaccharide molecular of CLPS1 showed clustered with molecular diameter 80～180nm and the branched chain was short and cross.(4)Specific rotation of CLPS1 is +44 °,the content of the total carbohydrate is 97.60%and uronic acid of CLPS1 is 13.18%.Monosaccharide component of CLPS1 contains Ara,Gal,Glc,GalA,Man with molar ratio of 2.7:3.4:2.3:1.1:0.5.Chemical composition and strucutre of polysaccharide CLPS1 was studied by spectrum and chemical analysis.The results showed as follows.Monosaccharide compositons of CLPS1 contains GluA and pyranose,while pyranose included both a-pyranoside and p-pyranoside in structure at the same time.The backbone was composed of Ara and Gal,the brancd with Gal,and the terminal residues of backbone with Glu.The molecular weight of CLPS1 is 91.7 kDa.Branch point of Gal residues is linked by Glc(1→4,6);Gal(1→3,6);Gal contains1→,1→3,1→4,1→6,1→3,6;Gal contains 1→,1→3,1→4,1→6,1→3,6;Glc contains 1→,1→4,1→4,6;Ara contains 1→5;GalA contains1→3.(5)In order to certifity the antioxidant effect of CLPS1 in vivo we constructed aging mice with D-galactose,the results showed that CLPS1 could increased GSH-Px,SOD and CAT activities in plasm,hepatic tissues,brain and skin,meanwhile it could decreased MDA content above tissues in those mice.(6)When the concentration of CLPS1 is 400 mg/kg,the Metastatic melanoma in the model mice is reduced by about 37.7%.The dose of 400 μg/mLdoes not cause cytotoxicity.The migration of B16F10 cells is strongly inhibited when the concentration of CLPS1 is 200μg/mLand 400 μg/mL.CLPS1 can reduced the adhesion-dependent formation of FA and down-regulated β1 integrin-mediated signalling via suppressing downstream phosphorylation of FAK and paxillin.The total reasons resulted in the subsequent loss of melanoma cell migration.