Study On Immune Mechanism And Its Active Components Of The Part Of Radix Astragali By Membrane Separation

Objective:The research object of this study was Radix Astragali,a Shanxi geo-authentic medicinal herb.The study focused on the mechanism of the immune regulatory effect of the small molecule active components(M4,mainly saponins and flavonoids)obtained by membrane separation technology,in order to provide new thoughts for the application of membrane separation technology in the development of small molecular components from Radix Astragali.Methods:Using Polyethersulfone Membranes(10 kDa)to separate and purify the small molecular components of Radix Astragali.Taking the transmittance of Radix Astragali total saponin as the evaluation index,the single factor analysis was carried out on the temperature of the drug solution,the ratio of material to liquid,the pH,and the operation time.The orthogonal experimental design(three factors and three levels)was adopted,and the multi-stage membrane separation process of Radix Astragali was optimized to obtain the M4 part(mainly composed of saponins and flavonoids).The immunocompromised rat model was made by using Cyclophosphamide(CTX)and Hydrocortisone(HC)to evaluate the immunoregulatory effect of M4.The chemical components of M4 were identified by UPLC-MS.Methods of network pharmacology were used to study the mechanism of the immunoregulatory effect of M4 by predicting the signaling pathways related.By the aid of public databases such as TCMSP,PubChem,and SWISS,the active components and disease targets were selected,and The PPI networks of component-targets and disease-targets were constructed.KEGG pathway enrichment analysis was conducted to study the signal pathway related to the immunoregulatory effect of M4.Method of serum metabonomics and NMR were used to study serum metabolites.Principal Component Analysis(PCA),Univariate statistical Analysis(UVA),Orthogonal Partial Least Square method Discriminant Analysis(OPLS-DA)were performed to identify differential metabolite,to explore the metabolic pathways of the immunoregulatory effect of M4.Results:The optimal membrane separation process of Radix Astragali was as follows(the loading quantity of the sample in each step was 2L).(1)Pre-treatment of Radix Astragali drug solution with Polyethersulfone Membrane(30 kDa);(2)Separation was conducted with Polyethersulfone Membrane(10 kDa)under the condition of the material liquid ratio of 1:25,solution pH of 4.85,the operation time of 9 min and pressure of 8.5 MPa;(3)A second separation with Polyamide Membrane(2.5 kDa)under the condition of the pressure of 8.5 MPa and operation time of 8.5 min;(4)The concentration treatment was carried out with Polyamide Membrane(600 Da).Under the optimal conditions,the purity of the total saponins of M4 freeze-dried powder was 206.76 mg/g,which is 2.67 times higher than that of the Radix Astragali solution freeze-dried powder.The Pharmacology experiments showed that M4 significantly improved the spleen index(P<0.01),the serum levels of IL-2,IFN-?and TNF-?(P<0.05)and the ratio of activated CD4~+/CD8~+in spleen lymphocytes(P<0.05)in CTX induced rats model.In HC induced rats model,M4 significantly increased the serum levels of IL-2 and IFN-?(P<0.05),and the ratio of activated CD4~+/CD8~+in spleen lymphocytes(P<0.01).M4 has a protective effect on the spleen and can regulate the immune capacity of the two models by regulating the levels of IL-2,IFN-?,and TNF-?in serum.UPLC-MS analysis showed that there were24 main components in M4,including fourteen flavonoids(Rutin,Calycosin-7-glucoside,Kumatakenin,etc.)and eight saponins(Astragaloside I,Astragaloside II,Acetylastragaloside I,etc.).Seventy-three core targets were obtained from the mapping of24 components(47 primary targets)and 37 immune-related primary targets.The results of the KEGG pathway enrichment analysis showed that 16 signal pathways with high reliability(P<0.01)were identified.The immunoregulatory effect of M4 may be related to signaling pathways such as PI3K-Akt,NF-?B,Jak-STAT,Fc epsilon RI signaling pathway,B cell receptor signaling pathway,T cell receptor signaling pathway,chemokine signaling pathway,and other immune-related signaling pathways.In OPLS-DA analysis,differential metabolites were identified by VIP>1 and P<0.01.The immunoregulatory effect of M4may be associated with regulating the level of lactic acid,creatine,glycerin,lysine,glycine,glucose,ketisohexanoic acid and other substances,mainly through the regulation of amino acid metabolism(Glycine,serine,and threonine metabolism,glutathione metabolism,valine,leucine,and isoleucine degradation,valine,leucine,and isoleucine biosynthesis)and energy metabolism(glycerol metabolism,glyoxylate and dicarboxylic acid metabolism,pyruvate metabolism,starch and sucrose metabolism,galactose metabolism).In CTX induced rats model,amino acid metabolism was mainly regulated,and in HC induced rats model,energy metabolism was mainly regulated.Conclusion:The M4 obtained from Radix Astragali by the optimized membrane separation process has an immunoregulatory effect on two types of the immunocompromised rat models induced by CTX and HC.The chemical components of M4 obtained by UPLC-MS analysis made the results of network pharmacology prediction more reliable.The metabolic pathways obtained by the analysis of serum metabolomics better explained the mechanism of the immunoregulatory effect of M4.This study adopted methods of pharmacology experiments,network pharmacology,and serum metabolomics,from multiple perspectives,to study the effect and mechanism of M4,small molecular components from Radix Astragali by membrane separation,in regulating the immune system.This study is conducive to the further development and utilization of small molecular components of Radix Astragali,which are mainly saponins and flavonoids,and provides a basis for the application and development of membrane separation technology.