Immune Activity Screening And Preliminary Structure Study Of Oligosaccharide Fragments Degraded By Trifluoroacetic Acid For Active Astragalus Polysaccharides APS-Ⅱ

Rationales:Astragalus is the dried root of the perennial legume Astragalus membranaceus or Astragalus membranaceus.It’s praised as a tonic medicine in the research of Chinese medicine.It has the pharmacological functions of strengthening the body and strengthening the body,and has a history of thousands of years in clinical research.Astragalus polysaccharides（APS）is a natural component with rich content and strong activity.The research team found that the polysaccharide with a molecular weight of 10 k Da has the strongest immunological activity.However,the molecular weight of polysaccharides is relatively large,the structural branches are complex,and the lack of polysaccharide standards also poses a huge challenge to its structural characterization.In recent years,with the promotion of the theory of polysaccharide receptors and the guidance of top-down research strategies,the research on oligosaccharides with relatively small molecular weights has received extensive attention.The research of oligosaccharides can not only promote the research of polysaccharide structure,but also provide new ideas for the development of carbohydrate drugs.However,research on Astragalus oligosaccharides is still very limited.Therefore,this article uses trifluoroacetic acid to degrade the active Astragalus polysaccharides APS-Ⅱinto Astragalus oligosaccharides,and studing the immunologically active center fragments of APS-Ⅱfrom the oligosaccharide level.This will help break the technical bottleneck of APS-Ⅱstructure research,lay the foundation for the research and development of Astragalus carbohydrate drugs and their structure-activity relationship,and further enrich the theory of polysaccharide receptors.Objective:The active Astragalus polysaccharides APS-Ⅱwas determined by single factor experiment and orthogonal experiment to determine the optimal acidolysis conditions,and different oligosaccharide fragments were separated and prepared by preparative liquid chromatography according to the distribution of polymerization degree.The structural composition of different oligosaccharide fragments was analyzed and the oligosaccharide fragments with stronger immunological activity were screened.Method:（1）Astragalus polysaccharides in Astragalus are extracted by water extraction and alcohol precipitation.The active Astragalus polysaccharide APS-Ⅱis prepared by the ultrafiltration method.Then,the optimal acid hydrolysis conditions for the degradation of APS-Ⅱby trifluoroacetic acid were optimized by single factor experiment and orthogonal experiment.The effects of acidolysis temperature,acidolysis time and acid concentration on degradation products were mainly investigated.After ensuring the repeatability and stability of the optimal degradation conditions,the Astragalus oligosaccharides（APOS）were repeatedly prepared.（2）The APOS prepared above was separated and prepared by purification preparation chromatograph to Astragalus oligosaccharide fragments with different degreesof polymerization（DP）,and the structural characteristics of APOS were studied by a combination of chemical and instrumental analysis methods.The mass spectrometry information of Astragalus oligosaccharide was analyzed by ultra performance liquid chromatography-electrospray ionization high resolution time-of-flight mass spectrometry（UPLC-ESI-QTOF-MS）.The degree of polymerization of the oligosaccharide fragments was analyzed by high performance liquid chromatography.The monosaccharide composition of oligosaccharide fragments was studied by the method of complete acid hydrolysis combined with derivatization.Fourier transform infrared spectroscopy（FT-IR）was used to determine the structure of special functional groups in the oligosaccharide fragments.The methylation analysis of glycosidic bonds in Astragalus oligosaccharide fragments was carried out by means of gas-gas-mass spectrometry（GC-MS）detection.（3）A variety of in vitro immune cells were used for activity evaluation,and immunoglobulin G（IgG）kits were used to study the effects of different polymerization degrees of Astragalus oligosaccharide fragments on immune activity.The phagocytic activity of macrophages and the cytotoxic activity of natural killer cells（NK）can be used to understand the influence of Astragalus oligosaccharide fragments with different degrees of polymerization on innate immunity.The proliferation of T lymphocytes in splenic lymphocytes induced by lipopolysaccharide（LPS）and the proliferation of B lymphocytes in splenic lymphocytes induced by Concanavalin A（ConA）can be used to understand the influence of Astragalus oligosaccharide fragments with different degrees of polymerization on adaptive immunity.The intervention of Astragalus oligosaccharide fragments with different degrees of polymerization on mouse spleen lymphocytes can detect the level of IgG secreted by spleen cells,so as to analyze the effect of Astragalus oligosaccharides on specific immunity.Results:1.The optimal conditions for the partial hydrolysis of APS-II（purity greater than 98.5%）into oligosaccharides with trifluoroacetic acid are the hydrolysis temperature of 80℃,the trifluoroacetic acid concentration of 1.0 mol?L^-1,and the hydrolysis time of 1h.This condition has good repeatability and stability（RSD%﹤3%）.Repeated batch preparation of APOS found that it’s yield was about 75%.2.APOS was separated by preparative liquid chromatography to obtain six oligosaccharide fragments.APOS-1 is mainly based on DP2,APOS-2 includes DP3～DP7,APOS-3 includes DP5～DP9,APOS-4 includes DP9～DP14,APOS-5 includes DP12～DP17,and APOS-6 includes DP16～DP19.A series of chemical instrument analysis shows that the sugar chain breakage of APOS is regular,which is represented by C_i,^0,2A_i,^2,5A_i,^2,4A_itype fragment ions.The structure of APOS is based on→4)-Hex-(1→linked six-carbon sugars as the main chain,including glucose,galactose and mannose,with glucose as the main component.In addition,there are branched structures at the C2,C3,and C6 positions of the main chain sugar ring,and there are more branches at the C6position.The main chain sugar ring of APOS-5 and APOS-6 has a more complex multi-branched structure.3.Oligosaccharide fragments with different degrees of polymerization have immune enhancement effects on immune cells in vitro,and components with larger average molecular weights show stronger immune activity.Compared with APS-Ⅱ,the differential activity of APOS-4/5/6 is statistically significant.At 200μg?m L^-1,APOS reaches the strongest activity.It is speculated that the fragments of immunologically active centers are more likely to exist in the Astragalus oligosaccharide fragments above DP9.Conclusion:In this study,the active Astragalus polysaccharide in Astragalus was used as the research object to be partially acid hydrolyzed with trifluoroacetic acid,through a"top-down"research strategy.The degradation products obtain oligosaccharides with different degrees of polymerization,and the oligosaccharides are divided into six fragments by the preparation liquid phase.The structural differences between different oligosaccharide fragments are analyzed and compared,It showed that APOS is based on the main chain of a six-carbon sugar connected with→4)-Hex-(1→,and there are branched sugar chain structures at the C2,C3 and C6 positions of the main chain sugar ring.The in vitro immune activity test was done to screen out that oligosaccharides with a degree of polymerization of DP9 or higher have stronger immune activity.It is speculated that the immune"active center"in sugar APS-Ⅱmay be located in the sugar chain structure above DP9.This study lays a foundation for further exploring the structure-activity relationship between different structures of APOS and immunological activity,and also provides research ideas for the study of oligosaccharides of other plant polysaccharides.