Study On Separation,Purification,Structure Identification And Antioxidant Of Elaeagnus Umbellata Polysaccharide

Elaeagnus umbellata Thunb.is a wide shrub,belongs to the Elaegnaceae family.E.umbellata is well known for lycopene contents in its berries,with antioxidant and anti-inflammatory activity.Its polysaccharides physiological activity has not been explored in the past,because the contents of polysaccharides are relatively the lower than lycopene.In this paper,the polysaccharide from Elaeagnus umbellata in Shanxi Province was isolated and purified.We used various methods to analyze the structure and molecular morphology of the polysaccharide,and to study its antioxidant activity and in vivo immune activity.This study provided a theoretical basis for the development and utilization of Elaeagnus umbellata polysaccharide.1.The extraction process of polysaccharides from Elaeagnus umbellata by enzymatic and homogenate co-assisted extraction was optimized via both single-factor assays and RSM assays,with the polysaccharide yield as the target.The extracting parameters including extraction temperature 79°C,homogenate extraction time 81 s,ratio of liquid material 32:1(g/m L)were optimized by using response surface methodology design based on the single-factor experiments.The best auxiliary enzymes were cellulase and pectinase.The best auxiliary enzymes were cellulase and pectinase,the optimum dosage was 1% through the experiments,and the optimal enzyme ratio is 1:5.And the extraction rate of crude Elaeagnus umbellata polysaccharide was 0.939%,which was close to the theoretical value of0.9525%.Enzyme-homogenate extraction can significantly improve the extraction rate of polysaccharides from Elaeagnus umbellata.The results showed that the extraction rate of polysaccharides by enzyme and homogenate co-assisted extraction was 6 times higher than that by hot water extraction alone.2.The protein were hydrolyzed using neutral protease,then the protein was removed with Sevag reagent(1-butanol: chloroform,1:4).The AB-8 macroporous resin adsorption method is selected as the method of polysaccharide decolorization.After deproteinization and decolorization,crude polysaccharides(CEP)were separated into two components QEP1 and QEP2 by DEAE-52 cellulose anion exchange column chromatography.Two fractions were further purified by Sephadex G-100 gel column chromatography and isolated purified polysaccharides(EUP1 and EUP2).3.High performance gel permeation chromatography(HPGPC)was used to determine the molecular weight.The Mws of EUP1 and EUP2 were estimated as 63 k Da and 38 k Da,respectively.The result of high performance liquid chromatography(HPLC)indicate that the molar ratio of L-Rhamnose and D-Galactose were 3.9:1.9 for the polysaccharide in EUP1,the molar ratios of D-Mannose,L-Rhamnose,D-Galactose,and D-Arabinose were2.7:1.8:5.0:1.8 for the polysaccharide in EUP2 respectively.The infrared spectra showed that it had the characteristic absorption peak of polysaccharides.The Congo red test result indicated that there was a triple-helix structure in EUP2,EUP1 was not.The results of TGA suggested that EUP2 had more a stable structure than EUP1.The structure of EUP1 is more compact than EUP2 observed under the scanning electron microscope(SEM).4.The in vitro antioxidant activity of E.umbellata polysaccharides(EUP1,EUP2)and E.umbellata crude polysaccharides(CEP)were studied.The results of antioxidant activity show that the purified Elaeagnus umbellata polysaccharides are better than crude polysaccharides in scavenging free radicals.EUP1 has high scavenging activity for DPPH free radicals,hydroxyl free radicals,ABTS free radicals and superoxide anions.EUP2 is not as strong as EUP1 in these respects,but it also has a moderate level of removal.The scavenging ability of CEP is not high,but its scavenging ability of superoxide anion is stronger than EUP2.EUP possessed appreciable scavenging activities against DPPH,ABTS,superoxide anion and hydroxyl radicals.Then the mass concentration is 1 mg/m L,the scavenging ability of EUP1 on DPPH free radicals,hydroxyl free radicals,ABTS free radicals,and superoxide anions are 91.31%,95.01%,46.22%,and 94.65%,respectively;EUP1 has an effect on DPPH free radicals and hydroxyl free radicals.The scavenging abilities of CEP radicals,ABTS free radicals and superoxide anions were 69.49%,71.59%,43.01%,41.02%,respectively;the scavenging abilities of CEP on DPPH free radicals and superoxide anions were 65.34% and 65.49%,respectively.Therefore,EUP could be developed as a potential antioxidant agent for application in the field of functional foods or medicine.