Extraction?Purification And Active Function Of Protein And Polysaccharide From Binhai's Cynanchum Auriculatum Royle Ex Wigh

Cynanchum auriculatum Royle ex Wigh from Binhai is rich in protein and polysaccharide which is the important material foundation of biological activity resulting it has a good value for research and development.In this experiment,we extracted the two kinds of active substance from Cynanchum auriculatum Royle ex Wigh and thoroughly analyzed its functions,in order to provide the theoretical basis for its application in food industry.In this experiment,we mainly extracted the protein of Cynanchum auriculatum Royle ex Wigh,and evaluated its physicochemical properties and processing properties.Subsequently,we adopted the optimal response surface method to optimize the extraction conditions of polysaccharide.Finally,the crude polysaccharide was purified and analyzed physicochemical properties and antioxidant activities in vitro.The specific research contents and the experimental results are as follows:Firstly,this study compared the effects of different solvent extraction methods on the soluble protein yield of Cynanchum auriculatum Royle ex Wigh,and analyzed the extraction effect of proteins by using electrophoresis,and explored its preliminary characterization.The proteins were identified by liquid chromatography tandem mass spectrometry and analyzed the processing characteristics which the isolated soybean protein served as a control.The result showed that the PBS method is superior to the traditional alkali extraction and Tris-HCl methods.The molecular weight of the proteins are mainly distributed in about 348 kDa,and relative migration rate of another band is low,and we deduced that the electrophoretic band of near 700 kDa is aggregates and the proteins are mainly composed of a subunit?molecular weight of 28 kDa?.The protein has a good processing performance,especially the oil holding capacity,emulsification and emulsion stability and foamability,all these properties are better than soybean protein.However,the solubility of Cynanchum auriculatum Royle ex Wigh proteins and the solubility of soybean protein keep up with the pH variation tendency.In this experiment,we choose three facts?material liquid ratio,extraction temperature,extraction time?conducting single factor test.Subsequently,we adopted the response surface analysis method to confirm the optimum process parameters based on the results of single factor test which the yield of polysaccharide served as the response value.The results showed that the optimal extraction process of Cynanchum auriculatum Royle ex Wigh polysaccharide is material liquid ratio 1:17,extraction temperature was 91?,extraction time 3 h.Under the above conditions,the polysaccharide yield was 1.58%which is accordance with the expected value of the model.The crude polysaccharide which not contained nucleic acids and proteins obtained from the extracting solution of Cynanchum auriculatum Royle ex Wigh through the following processes:enzymic method removing starch,Sevge method removing protein,95%ethanol precipitation,dialysis and freeze-dried.The polysaccharide was purified by DEAE-Sepharo Fast Flow anion exchange chromatography and Sephadex G-150 gel column chromatography and obtained a purified component.The molecular mass of the component was 830.93 KDa by using HPGPC.The composition of monosaccharide was analyzed with the PMP derivatization method.The result show that the monosaccharide is mainly composed of galacturonic acid,galactose,Arabia sugar,rhamnose,mannose and glucuronic acid and the molar ratio of mannose rhamnose glucuronic acid galacturonic acid galactose and arabia sugar is 0.058:0.280:0.071:1.455:0.918:0.469.This study adopted four kinds of methods for determination of antioxidant activity to analyze and research the ability of antioxidant and scavenging free radicals of Cynanchum auriculatum Royle ex Wigh.The results show that the polysaccharide has ability to scaveng DPPH free radical,superoxide radical,ABTS free radical.Meanwhile,the clearance rate gradually increased with the concentration of polysaccharide increased.DPPH free radical scavenging,superoxide radical,ABTS free radical ability of IC₅₀ separately was 0.5543 mg/mL,0.5881 mg/mL and 0.1232 mg/mL,of which the ABTS free radical scavenging ability is close to the VC.The ORAC value of polysaccharide is 109.94 ?mol Trolox/g,indicating that it has a strong antioxidant capacity.Finally,in order to research resistance to oxidation of the polysaccharide by through establishment of TNF alpha induction of human umbilical vein endothelial cells generate oxidative stress reaction model.The results showed that it can significantly improve TNF-alpha induced by human umbilical vein endothelial cells and the activity of SOD enzyme activity,and significantly inhibited ROS in cells?ROS?.This indicated that the polysaccharide can reduce the oxidative damage of vascular endothelial cells.