Extraction And Isolation And Purification And Structural Analysis Of AmPR10-16kDa And HQGP-2 Protein From Astragalus Monholicus And Its Therapeutic Effect And Mechanism On The Experimental AutoimmuneEncephalomyelitis In Rats

Objective:Huangqi（Radix Astragali）is the most common Chinese herbal medicines.Astragalus has been immunosuppressive effects by Modern pharmacologic-al studies.Early results of this research groupshowed that Radix Astragali protein had been immunosuppressive effects.In this paper,protein of Mongolian astragalus as raw material has been extracted and isolated and purified,in the mean,whose nature of the structure and activity in vitro immunosuppressive has been studied,whose the therapeutic mechanism of EAE model mice has been explored.So as to the comprehensive utilization of resources of Astragalus and its value-added comprehensive development and enrich the active protein types and functions will be been created in the good conditions.Methods:1.The key factors of extraction process of Astragalus protein on buffer solution method have been optimized by the key advantage of single factor and orthogonal test.immunosuppresive activities of the rough protein extracted by buffer solution method at different solutions were tested by CCK-8 way.2.Based on the immunosuppressive activity test,two novel proteins AmPR10-16-kDa and HQGP-2 with highest immunosuppressive activities was obtained from Mo-ngolia Astragalus,which are detected by a series of qualitative criteria.3.The structure message of AmPR10-16kDa and HQGP-2 has been obtained from amino acid ingredients experiment,moonosaccharide coomposition experiment,β-elimination response,infrared spectrometry,circular dichroism spectroscopy.4.They are divided into both treatment groupa with Radix Astragali protein and EAE control group,which clinical scores and body mass changes are recorded and observed by every other day.Spinal cord tissue inflammatory cell infiltration is detected by HE staining and immunofluorescence.Cell activity is detected by CCK-8assay.Release of nitric oxide（NO）is detected by Griess assay.Cytokines are detected by ELISA assay.Variety of CD4⁺T cell subsets is detected by flow cytometry.Results:1.Extraction conditions ptimized are the extraction time 60 min,extracting saltcon centration 25 mmol·L^-11 Tris-HCl（pH value 8.00）included by 10 mmol·L^-11 NaCl and ratio of liquid to solid 16 mL·g^-1,by which protein yield was 6.3%.Crude protein from Mongolia Astragalus express a certain inhibitory effect on Spleen lymphocytes ftom ICR mice and SD raats,the hiighest iinhibitory actiivity was revealed on Spleen lymphocytes from ICR mice,which value was 64.10%.The degree of cell damage and sample concentration is positively correlated.2.Both protein of AmPR10-16kDa and HQGP-2 yield were both 40.12 and 17.12mg from Mongolia Astragalus 30 g powder（through No.4）.Based on the immunosup-pressive activity test,a glycoprotein HQGP-2 with highest immunosuppressive activ-ity was obtained from Mongolia Astragalus.SDS-PAGE eleectrophoresis and HPGPC test revealed that AmPR10-16kDa and HQGP-2 both were single band and single peak,which weight-aveerage moolecular weight respectively were 16 578 and30 575 Da.Periodic acid staining-Schiff showed that AmPR10-16kDa are not glycoproteins.Am-PR10-16kDa is a new Pathogenesis-related proteins PR10 in accordance with its weight-average molecular weight,PI 4.35 and homology associated with Pathogenesis related proteins AmPR-10.3.The results displayed that the protein component of HQGP-2 is 28.48%.The Sugar component of HQGP-2 is 72.29%,the glycan of HQGP-2 principaly constituted of arabinose,glucose.The glycopeptide bond is an O-glycopeptide bond,the glycan of HQGP-2 is pyranose withα-glycosidic bond constructure.Circular dichroism-data showed that there is 29%α-helix,22.9%β-strand,1.1%β-turns and47%unordered in HQGP-2,and 34%α-helix,24.6%β-strand,1.4%β-turns and40%unordered in AmPR10-16kDa.4.AmPR10-16kDa and HQGP-2 treatment can reduce symptoms of EAE,inhib-it inflammatory cell infiltrationin Central Nervous System,splenic lymphoid mono-nuclear cell activity and secretion of NO,TNF-αand IL-6,and promote the secretion of IFN-γ,and increase the proportion of cell subsetsof CD4⁺IFN-γ⁺、CD4⁺IL-10⁺和CD4⁺CD25⁺T.Each index showed that AmPR10-16kDa is superior to HQGP-2 in terms of efficacy.Conclusion:1.AmPR10-16kDa and HQGP-2 with the highest inhibitory effects on splenic lymphocytes were produced by extraction buffer solution optimized and AKTA avant25 protein purification system,which provides a viable techniques for extracting the component in the production process of medium level.2.AmPR10-16kDa and HQGP-2 can inhibit releaseing of inflammatory cytokine and relieve inflammation of EAE by regulating proportion of T cell subsets.