Study On The Immune Regulation Mechanism Of Astragalus Active Polysaccharide Aps-Ⅱ And The Structure And Activity Of Enzymatic Hydrolyzed Oligosaccharide Fragments

Rationales:Astragalus polysaccharides have a wide range of biological activities.Most of the current researches use total Astragalus polysaccharides as the main research object.However,the relative molecular weight distribution of Astragalus polysaccharides is wide,which limits the in-depth study of its biological activity mechanism,and it is impossible to accurately clarify the immunomodulatory effect and its relative.The relationship between the immunomodulatory effect and its relative molecular weight cannot be accurately elucidated,and it is difficult to reveal the immunomodulatory mechanism of Astragalus polysaccharides at the molecular level.Our previous study showed that APS Ⅱ was the main immunoregulatory substance in Astragali Radix.The structure of the polysaccharide is difficult to accurately determine,which limits the in-depth study of the molecular mechanism of action of Astragalus polysaccharides."Polysaccharide receptor theory"believes that there are one or more oligosaccharide fragment"active centers"in immunologically active polysaccharide molecules.Therefore,the degradation of Astragalus polysaccharides into oligosaccharides and the study of the active centers of polysaccharides at the oligosaccharide level provide a new idea for breakthroughs in the study of the structure and mechanism of Astragalus polysaccharides.Objective:Astragalus polysaccharides of different molecular weights were separated by ultrafiltration technology.The"cyclophosphamide immunosuppressive"method was used to model,and the immune enhancement mechanism of Astragalus polysaccharides was explored through mouse serum metabolomics technology.The immunologically active polysaccharide APS-Ⅱ（10 k Da）prepared by our research group was determined by single factor test and orthogonal test to determine the best degradation conditions,and oligosaccharide components with different polymerization degrees were prepared by polyacrylamide gel chromatography.Oligosaccharide components are subjected to structural analysis and immune activity screening.Method:（1）Astragalus polysaccharides with different molecular weights were obtained by ultrafiltration,APS-I（>2000 k Da）,APS-Ⅱ（1.02×10⁴ Da）,and APS-ⅡI（286 Da）,they were applied to cyclophosphamide immunosuppressive model mice respectively,and collected mouse serum for UPLC-HRMS detection,and combine multivariate statistical analysis to explore the immune enhancement mechanism of Astragalus polysaccharides.（2）The immunologically active polysaccharide APS-Ⅱ is digested with endo-α-1,4-glucanase,and the optimal degradation conditions are determined through single factor test and orthogonal test.The degraded oligosaccharide mixture is coagulated by polyacrylamide.Gel chromatography to prepare oligosaccharide components with different degrees of polymerization.Oligosaccharides with different degree of polymerization were prepared.The structure of different oligosaccharides was analyzed by high performance liquid chromatography.The composition of monosaccharide was determined by HPLC UV and the monosaccharide linkage was determined by methylation method.The characteristic functional groups and sugar chain configurations of oligosaccharides were determined by IR.The glycosidic bond configuration of oligosaccharide fragments was determined by nuclear magnetic resonance spectroscopy（NMR）.The fine structure of oligosaccharide fragments was determined by MS/MS.（3）The activity of different oligosaccharide fragments was studied from two aspects of specific immunity and non-specific immunity,and explore their effects on the body’s non-specific immune function through the effects of different oligosaccharide components on the phagocytic activity of Raw 264.7 cells and NK cells;Different oligosaccharide components synergize the effects of Con A and LPS on mouse splenic lymphocytes to explore their effects on the body’s specific immune function.Results:1.Different molecular weights of Astragalus polysaccharides can increase the number of immune cells in mouse serum to varying degrees and improve immune organ damage.APS-Ⅱ has the best effect.Compared with the blank group,29metabolites in the serum of the model group changed significantly.APS,APS-I,APS-Ⅱ,and APS-ⅡI could significantly adjust 24,13,25,and 19 metabolites to normal level.Metabonomics analysis revealed that APS-Ⅱ in Astragalus polysaccharides is the main component that mainly exerts immunomodulatory activity.Its mechanism may be related to the regulation of phenylalanine metabolism,cysteine and methionine metabolism,TCA cycle,arginine and proline metabolism.2.The optimal degradation conditions of APS-Ⅱ were determined through single factor test and orthogonal test,the endo-α-1,4-glucanase concentration was 0.5 U·ml^-1,the enzymatic hydrolysis temperature was 60℃,and the enzymatic hydrolysis time was 90 minutes,the number of oligosaccharides obtained by degradation of APS-Ⅱ is the largest,the content is the highest,and the degradation consistency is good.The oligosaccharide mixture is separated by polyacrylamide gel chromatography to obtain four oligosaccharide components,P1:1-3 sugars,P2:3-6 sugars,P3:7-14 sugars,P4:10-18 sugars.3.APS-Ⅱ is degraded by endo-α-1,4-glucanase to form oligosaccharides with a degree of polymerization of 1-18.Four components are separated by polyacrylamide gel chromatography.The oligosaccharides are analyzed.The main component of 1-3sugars,3-6 sugars and 7-14 sugars produced after APS-Ⅱ enzymatic degradation is glucose,which contains traces of the remaining monosaccharides,while 10-18 sugars and enzymes degrade the total In addition to glucose,there are other monosaccharides in the oligosaccharide mixture.Among them,the content of galactose and arabinose is more,and the proportion of each monosaccharide is different.Among them,the main structure of the polymerization degree 1-3 sugar and 3-6 sugar is linear 1,4-linked glucan.The main structure of the degree 7-14 sugar is linear 1,4-linked glucan,but compared to the 1-6 sugar,the number of glucose monosaccharides with high degree of branching in the sugar chain structure increases,in addition to a small amount of arabinose and galactose;In the oligosaccharide component with a degree of polymerization of 10-18,the main sugar chain connection methods are 1→2,4-Glcp and 1→4,6-Glcp.Compared with the first three oligosaccharide components,The content of 1→4-Glcp and 1-Glcp is significantly reduced,the content of galactose and arabinose is increased,and glucose with a high degree of branching becomes the main component in the sugar chain.Comprehensive enzymatic hydrolysis product structure can infer the structure of APS-Ⅱ,withα-D-1,4-linked glucan as the main chain,the branched structure at C2,C3 and C6.In addition,there are a small amount of 1,5-linked arabinose,1-linked arabinose,2,3,4-linked galactose,and 2,4,6-linked galactose in the polysaccharide structure.4.APS-Ⅱ and its enzymatic degradation products can enhance the phagocytic function of macrophages,enhance the killing activity of NK cells,and promote non-specific immune function.It can also cooperate with Con A to promote T lymphocyte proliferation and LPS to promote B lymphocytes.Proliferate and secrete cytokine Ig G to enhance specific immune function.Among the different oligosaccharide components,the most active component is the oligosaccharide component with a degree of polymerization of 10-18.Conclusion:Metabonomics analysis revealed that APS-Ⅱ in Astragalus polysaccharides is the main component of immunomodulatory activity,and its mechanism may be related to the regulation of phenylalanine metabolism,cysteine and methionine metabolism,tricarboxylic acid circulation,arginine and proline metabolism.Therefore,it is speculated that Astragalus polysaccharides may exert the immune activity in vivo by regulating the activity of phagocytes and dendritic cells,directly affecting the body’s non-specific immunity,and indirectly affecting the body’s specific immunity through related metabolites,and finally showing a strong overall immune function repair activity.Under the guidance of the theory of"polysaccharide receptor theory",the method of enzymatic degradation of active astragalus polysaccharide APS-Ⅱ to obtain oligosaccharides was preliminarily established,and the degraded oligosaccharides were separated,and the immune activity of mixtures of oligosaccharides with different polymerization degrees was preliminarily explored.As a result,it was found that compared with the immunologically active polysaccharide APS-Ⅱ,different oligosaccharide components have significant differences in different immunological activities.Among them,the oligosaccharide with a degree of polymerization of 10-18 has the highest immunological activity.It can be seen from the structural analysis results that it is compared with the low degree of polymerization.Oligosaccharides,there are more branched structures in the 10-18sugar structure,indicating that the branchedα-D-1,4-linked glucan structure has strong immunological activity,and it also shows that the immunologically active polysaccharide APS-Ⅱ.The"active center"of the oligosaccharide fragment in the polysaccharide molecule may be located in the 10-18 sugar structure of the oligosaccharide.The results of this study laid the foundation for further elucidation of the structure and function of Astragalus polysaccharides,enriched the theory of polysaccharide receptors,and provided new ideas for the development of Astragalus polysaccharides.