Purification,Structural Elucidation And Immune-Enhancing Activity Of Polysaccharides From Quinoa(Chenopodium Quinoa Willd.) Seeds

Chenopodium quinoa were planted in the Andes region of South America,with high food value,and has been recognized as the “whole nutrition food”.Quinoa has been popularized and planted in other parts of the world in recent years,and our country has realized large-scale planting in Shanxi,Tibet,jingle,Sichuan,Qinghai and other places.Jingle has grown rapidly since its successful trial in 2011 and is known as “the home of quinoa in China”.In this paper,the polysaccharide from quinoa jingle 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 quinoa polysaccharide.1.The extraction process of crude polysaccharides from Chenopodium quinoa(CPS)by enzymatic and ultrasound co-extraction was optimized with the polysaccharide yield as the target.The best auxiliary enzyme was cellulase and the optimum dosage was 3% through the experiments.The extracting parameters including extraction temperature 65?,ultrasonic time18 min,material liquid ratio of 1:33(g/m L)were optimized by using response surface methodology design based on the single-factor experiments,and the extraction rate was 68.08%.The enzyme has the characteristics of small amount and high catalytic efficiency,which can accelerate the polysaccharide dissolution and effectively reduce the production cost.The cavitation produced by ultrasonic waves makes the cells break up and the intracellular lysates dissolve better.Enzyme-ultrasonic extraction can significantly improve the extraction rate of polysaccharides from quinoa.The results showed that the extraction rate of polysaccharides by enzyme-assisted ultrasonic extraction was 1.5 times higher than that by ultrasonic extraction alone.2.CPS was extensively treated with ?-amylase and dialyzed to removestarch,ended up with no starch-iodine reaction.The protein were hydrolyzed using neutral protease then the protein was removed twice with Sevag reagent(1-butanol:chloroform,1:4).The procedure was then repeated.The water-soluble CPS deproteinated were separated by DEAE-cellulose anion-exchange and Sephadex G-50 gel column chromatography.The soluble non-starch polysaccharide fraction(QPS1)eluted with Na Cl solution from anion-exchange chromatography should be acidic polysaccharides.The protein removal rate was 79.85% with less polysaccharide loss.Thus,the combined method can utmost remove proteins in an efficient way.Column chromatography is a commonly used method to purify polysaccharides.After deproteinization of starch,crude polysaccharides were separated into two components QPD1 and QPD2 by DEAE-52 cellulose anion exchange column chromatography.Since the second portion were minor,only QPD1 was further studied.The water-soluble non-starch refined polysaccharides(QPS1)were further isolated and purified by Sephadex G-50 gel column chromatography.3.The structure of polysaccharides determines their biological activity,the monosaccharide composition and proportion;molecular weight and the triple helix structure determine the differences in the biological activity of polysaccharides.Structural properties and molecular morphology of QPS1 was examined by FT-IR,NMR,HPGPC,HPLC,AFM,SEM and Congo red staining.Then we elucidate the relationship between its active expression and its internal structure.High performance gel permeation chromatography(HPGPC)was used to determine the molecular weight,and the composition by HPLC.QPS1,with a molecular weight of 34.0 k Da,was mainly composed of mannose,rhamnose,galacturonic acid,glucose,galactose,xylose and arabinose at a molar ratio of 2.63:2.40:1.64:6.28:1.95:2.48:5.0.The single molecular weight distribution indicated that the high purity of polysaccharides.The infrared spectra showed that it had the characteristic absorption peak of polysaccharides,and it was a pyranoid polysaccharides with ?-,?-glycosides.NMR imaging showed that QPS1 contained ?-L-rhapresidues and ?-,?-glycosides.The results of IR,NMR imaging and HPLC were consistent.Congo red experiment shows that QPS1 has triple helix structure,which exhibits particular molecular recognition and incomparable features.The QPS1 molecular chain agglomerated into spherical particles in aqueous solution with particle diameter in the range of 8-14 nm,supporting their branched structure.The surface of the QPS1 fragments have plenty of pores,indicating that it had excellent rehydration properties.4.In this study,several in vitro antioxidant models with different reaction mechanisms were used for the comprehensive evaluation of the antioxidant activity of the QPS1.Moreover,studies have also proven that the antioxidant activities of polysaccharides mainly depend on their physicochemical features,such as monosaccharide compositions,molecular weight,chain conformation,and so on.It has been reported that there are high correlations between the antioxidant activities and monosaccharide composition,especially the contents of glucuronic acid and glucose.In addition,the molecular weight(Mw)of polysaccharides had a notable effect on their biological activities,and low-Mw polysaccharides showed better antioxidative effects than did higher-Mw ones.It had been suggested that the polysaccharides with the molecular weight in the range from 4.0 to 100.0k Da exhibited better antioxidant activity.In this study,the QPS1 exhibited potent antioxidant activity,which might be associated with its low molecular weight and monosaccharide composition,as it consisted with high glucose content.Body weight and organ index can reflect the recovery of body and immune organs in mice.Macrophages are the first barrier of the human immune system,and monocyte-macrophage phagocytosis can characterize the body's non-specific immunity.The activated macrophages play an important role in antigen delivery,resistance to infection,and inflammation.Several studies have shown that the stimulatory effect of polysaccharides starts by enhancing the phagocytosis of cells.Cytokines are mainly secreted by activated macrophages and play important roles in the cellular immune process by aiding the eliminations of abnormal cells.DTH is a type ofcytoimmunity mediated by T lymphocytes.Measurement of toe thickness is commonly used as part of a DTH assay to evaluate T cell-mediated immune responses.To evaluate the immune activation of QPS1 in vivo,the cytokine secretion and serum lysozyme activity,monocyte-macrophage phagocytosis,organ index,and delayed type hypersensitivity were measured.The results showed that administration of QPS1 significantly raised spleen and thymus indices,improved the content of IFN-?,IL-6,IFN-?,Ig M and lysozyme(LYSO)in their serum,and enhanced the phagocytic function of mononuclear macrophages and ameliorative delayed allergy in mice.