Quality Comparison Of Astragali Radix From Different Sources Based On Polysaccharide Molecular Weight Distribution And Immune Activity

Rationales: Astragali Radix(AR)is one of the Chinese herbs in Shanxi.Polysaccharides,as the main immunoactive components in AR,have not been used as quality control indicators due to structural controllability problems.With the development of membrane materials and chromatographic packing technologies in recent years,the application of ultrafiltration and next-generation gel chromatography has enabled polysaccharide components to be systematically characterized and separated according to molecular weight.The application of new technologies and new characterization methods can not only avoid the technical bottleneck of polysaccharide structure determination,but also accurately characterize the structural characteristics of polysaccharides in Chinese medicines as a whole.Characterization of molecular weight distribution of Astragalus polysaccharides(APS)based on TSK gel chromatography can identify the differences of Astragalus medicinal materials in different species,different bases,and different growth modes.APS with different molecular weights(Mw)can be prepared by ultrafiltration,which lays the foundation for screening of systemic immune activity.Therefore,in this study,TSK gel chromatography and ultrafiltration retention methods were used to characterize and prepare APS with different Mw,and to conduct structural studies(monosaccharide composition and ligation site analysis),as well as non-specific and specific immunization.Activity screening(in vitro cell activity evaluation and in vivo animal activity verification)to find the most active component in APS,establish a characteristic sugar profile of APS and combine cell immune activity evaluation methods to compare the quality of AR from different sources.The research not only lays a foundation for elucidating the pharmacological functions of astragalus polysaccharides,but also promotes drug development.Objective: To separate different components of APS according to their Mw distribution,and to screen out the components with the strongest immune activity.And by establishing Astragalus polysaccharide,oligosaccharide and monosaccharide specific chromatograms,combined with mature cell immune activity experiments,a quality evaluation method of AR with polysaccharides as quality control indicators was established.Method:(1)Extraction of polysaccharides from AR of Shanxi Hunyuan by a method of water extraction and alcohol precipitation.According to the Mw distribution,the APS was separated by ultrafiltration retention method,and the structural differences between them were determined by measuring the monosaccharide composition,infrared spectrum,methylation and nuclear magnetic resonance spectroscopy of different components.(2)The prepared APS with different Mw were tested on mouse immune cells in vitro using MTT method to measure the proliferation activity of spleen lymphocytes and ELISA method to measure the secreted Ig G level of spleen lymphocytes induced by LPS,which to detect APS specific immunity.The phagocytic function of peritoneal macrophages was measured by the phagocytosis neutral red method and the spleen NK cell activity was measured by the lactate dehydrogenase assay,which to detect the non-specific immunity of APS.In this way,the components with the strongest immune activity in APS were screened.(3)The prepared APS with different Mw were used in cyclophosphamide immunosuppressive mouse model in vivo,and blood routine,immune organ index and its effects on specific and non-specific immune function were measured to evaluate immunomodulatory activity of APS with different molecular weight,and screen out the most immunocompetent components of APS.(4)In this study,high-performance gel filtration chromatography was used to establish polysaccharide specific chromatograms of different Astragalus medicinal materials,HILIC specific chromatograms analysis based on partial acid hydrolysis was used to establish oligosaccharide specific chromatograms of different AR,and the method of acid degradation of total polysaccharides,and combined with pre-column derivatization,HPLC-UV was used to establish monosaccharide specific chromatograms of different AR,to find the similarities and differences of APS in MG from different origins and planted methods,and the immunoregulatory activity of APS in three kinds of AR and the immunomodulatory activity between each APS hydrolysate and total polysaccharide were compared by a mature and recognized method of the phagocytosis neutral red methodResults: 1.In this study,the total polysaccharide content of APS is 74.6%,the protein content is 0.42%,and the Mw distribution is 300 Da~2 MDa.The monosaccharide composition is rhamnose,galacturonic acid,glucose,galactose and arabinose.APS was divided into three parts by ultrafiltration retention method,namely APS-I,APS-Ⅱ and APS-Ⅲ.The monosaccharide composition and linking information of three different molecular weight components in APS were compared,and the structural differences between the three were found.Although they have the same monosaccharide composition,the amount ratio of each monosaccharide is different.In APS-I the ratio of Rha,Gal A,Gal,Glu,and Ara is about 0.1:0.39:13.4:17.2:1,in APS-Ⅱ it is about 0.14:0.14:9.6:24.04:1,and in APS-Ⅲ it is about 0.375:0.375:18.8:90.5:1.It was determined by IR that they havesimilar functional groups.Methylation and NMR analysis showed that the three monosaccharide residues had different attachment sites: APS-I monosaccharide residues were connected in the manner β-L-Ara-(1 →,→ 2)-α-D-Gal-(1 →,→ 4)-α-L-Rha-(1 →,→ 6)-α-D-Glu-(1 →,→ 6)-β-D-Gal-(1 →,→ 4)-α-D-Glu-(1 →.The connection mode of APS-Ⅱ monosaccharide residues is → 2,3)-α-L-Rha-(1 →,→ 5)-α-L-Ara-(1 →,→ 3,4)-β-D-Gal-(1 →,→ 6)-β-D-Gal-(1 →,→ 4)-α-D-Glu-(1 →,→ 3,5)-β-D-Glu-(1 →.APS-Ⅲ monosaccharide residues are connected in the manner of→ 5)-α-L-Ara-(1 →,→ 6)-β-D-Gal-(1 →,→ 4)-α-D-Glu-(1→.2.The prepared three kinds of APS with different Mw act on mouse immune cells in vitro,and the results show that: APS of different Mw can enhance mouse spleen lymphocyte proliferation activity and spleen lymphocyte secretion Ig G levels to different degrees To enhance the specific immunity of APS;to enhance the non-specific immunity of APS by enhancing the phagocytic function of peritoneal macrophages and the activity of spleen NK cells.Finally,the main contributor to APS immune activity was screened for APS-II(about 10 k Da).This indicates that the biological activity of APS is related to its relative molecular mass.3.The active polysaccharides were screened by preparing three different Mw APS in mice with cyclophosphamide-induced immune depression in vivo.The results show that APS-II can improve the body weight and immune organs of cyclophosphamide immunosuppressed mice.Weight has the most pronounced effect.It has the strongest ability to increase its specific and non-specific immunity.This is mutually verified with the results of in vitro immune cell activity screening experiments,and it is more favorable to prove that the main contributor of APS immune activity is APS-II,indicating that the relative molecular mass of APS has a significant effect on biological activity.In addition to sugars or disaccharides,low-Mw APS has better immunomodulatory effects than high-Mw APS.4.Through analysis of 36 batches of Astragalus polysaccharides,oligosaccharides and monosaccharides specific chromatograms,the results show that the content of polysaccharides and the ability to enhance the phagocytic activity of macrophages of wild-simulated Astragali Radix(WAR)from Shanxi Hunyuan are higher than those of transplanted Astragali Radix(TAR).The three kinds of APS have similar Mw distributions,but the peak area of each part has a significant difference in the percentage of the total peak area.The part with a Mw of about 10 k Da in Shanxi APS is higher than that in Gansu.It can be seen from the oligosaccharide map that the similarity between the same APS partial acidhydrolysates is very high,and the differences between the three different APS partial acid hydrolysates are large.The main difference is the types and relative proportions of oligosaccharides with different degree of polymerization(DP).It can be seen from the monosaccharide map that all three APS are composed of 5 kinds of monosaccharides.However,the APS from the three origins have different amounts of monosaccharides.Conclusion: This paper analyzes and compares the structural differences between three polysaccharide components of different Mw in APS.And through in vivo and in vitro immune activity screening experiments to find the most immunologically active fragments in APS.It laid the foundation for further studying the relationship between APS with different structures and immune activity,and provided guidance for the further development of APS products.And used a combination of fingerprint of carbohydrates and the effects of APS on cellular immune function to provide a basis for quality evaluation and quality control of different habitats or planting methods.