| Astragalus and ginseng are two typical qi-invigorating drugs,which were widely used in clinical practice for the treatment of TCM syndromes such as Qi-deficiency.They can be used to improve the sub-health people’s state of fatigue,hypodynamia and other immunocompromised states,and can also be used to treat the sudden decline in immunity due to critical diseases,such as malignancy.The study on the connotation of invigorating qi of astragalus and ginseng is not clear in clinical practice,and the relevant action mechanism has not yet been discussed in depth.This paper focused on the interpretation of the connotation of invigorating qi of astragalus and ginseng.Two animal models including normal model and immunosuppressive model were taken asthe research object.Immunological techniques and metabolomics techniques were used to investigate the change of pharmacological indicators and metabonomic profiles at organ level,cell level and molecular level.Furthermore,system pharmacological method was applied to find potential target between astragalus and ginseng.In this way,preliminary exploration of the connotation of invigorating qi of these two drugs was carried out.1 Quality control of astragalus and ginsengAccording to standard of the 2015 edition of the Chinese Pharmacopoeia,the quality of astragalus and ginseng was investigated.Determination of astragaloside IV in astragalus was conducted by high performance liquid chromatography(HPLC)combined with evaporation light method.Determination of Rg1,Re,Rb1 in ginseng and calycosin-7-glucoside in astragalus were concuted by HPLC with ultraviolet(UV)detector.The results showed that the content of astragaloside Ⅳ was 0.128%,the content of calycosin glycoside is 0.0315%,the content of ginsenoside Rbl,Ginsenoside Re and Rgl were 0.683%,0.412%and 0.213%,which accord with the standard of pharmacopoeia.2.Immunoregulation and metabolomics study in normal animal modelsIn order to investigate the properties difference between astragalus and ginseng,the effects of astragalus and ginseng on normal mice were analyzed by immunological and plasma metabolomic method.Normal mice were given with astragalus(low,medium and high dose),ginseng(low,medium and high dose),astragalus:ginseng =1:1(low,medium and high dose),astragalus:ginseng=4:1(low,medium and high dose),astragalus:ginseng=:1(low,medium and high dose)for 30 days.The Viscera index results showed that astragalus,ginseng and their compatibility could enhance the spleen index of mice,and the thymus index of mice could be enhanced only after compatibility.Cell experiments showed that astragalus and ginseng could improve the proliferation and transformation rate of splenic lymphocytes.But,there was no significant difference between these two groups.The effect of the 8:1 group was the best.Meanwhile,astragalus and ginseng all showed the same behavior that they could inhibited NK cell killing activity.It can be seen that astragalus and ginseng could improve the body’s immunity by enhancing viscera index,nonspecific immunity(NK cell activity)and specific immunity(lymphocyte proliferation activity).Based on the HPLC-ESI-Q-TOF-MS system,plasma metabonomics method of astragalus,ginseng and its compatibility in normal mice was established.The original profile data was converted into CEF data by MPP software,compared the difference among data by statistical analysis.Blank control group(BCG)was selected as the comparison object.Data frequency analysis,non-paired t test and analysis of variance analysis were used.p value is less than 0.05 and greater than 2 fold change were chosen as selection criteria for biomarkers.At the same time,SIMCA-P 14.1 software was used to carry out unsupervised principal component analysis(PCA)and supervised orthogonal partial least squares discriminant analysis(OPLS-DA).The final biomarkers were identified by mass fragmentography.Combined with immunological indexes,the immune metabolic pathways of astragalus and ginseng were constructed by MetPA database.Through correlation analysis,four strong correlation immune indexes of biomarkers were obtained.In astragalus group:Uracil,LysoPC(183(6Z,9Z,12Z,))and Sphinganine;In ginseng group:LPA(0:0/16:0),UDP-glucuronate,PC(14:0/18:0)and LPA(0:0/16:0);In 1:1 group:Uracil and Dihydroceramide;In group 4:1:Uracil and L-Tyrosine;In group 8:1:Uracil and Kynurenine.It is suggested that astragalus and ginseng have the same pathway,which are related to glycerophospholipid metabolism.On the other way,astragalus could regulate the pyrimidine metabolism and sphingolipid metabolism,and ginseng regulate sugar metabolism.3.Immunoregulation and metabolomics study in immunosuppressed animal modelsIn order to further investigate the properties difference between astragalus and ginseng,we studied the effects of astragalus,ginseng and their compatibility on immunosuppressed mice by immunology,hematology,plasma metabolomics and bone marrow metabonomics.From 1th to 9th day,intragastric administration once a day;from 4th to 6th day,intraperitoneal injection of cyclophosphamide once a day,an immunosuppressive model was built,and blood routine examination were performed at 1,4,7,9th day.The characteristics of the trial administration is to simulate clinical chemotherapy treatment through preventive administrationBlood cell experiment results suggested that cyclophosphamide can effectively reduce the levels of white blood cells,red blood cells,hemoglobin and platelets in the blood.Compared with the model group,the levels of white blood cells,red blood cells,hemoglobin and platelets increased significantly in astragalus group,ginseng group,1:8 group and 8:1 group,but there was no significant difference between astragalus and ginseng.So,we could know that astragalus and ginseng can enhance the immunity of mice by improving the concentration of blood cells,especially in 8:1 group.It was hard to distinguish the effects of astragalus and ginseng on blood cells.The immunological experiment suggested that the spleen index and the thymus index can be enhanced by astragalus and ginseng.At the same time,the compatibility effect was stronger than the usage of single drug.The 8:1 group showed better effect compared the other groups.On cell level,astragalus and ginseng could improve the specific immunity(enhance the proliferation and transformation rate of spleen lymphocytes),especially in 8:1 group.Astragalus and ginseng can improve the nonspecific immunity of the body(increase the killing activity of NK cells).It can be seen that astragalus and ginseng can enhance immune function by enhancing viscera index,specific immunity and non-specific immunity of immurodeficient mice.Based on the HPLC-ESI-Q-TOF-MS system,we established plasma and bone metabonomics method of astragalus,ginseng and their compatibility in immunosuppressed mice,investigating the qi-invigorating mechanism in qi-deficiency animal.Plasma metabolomics studies showed that the astragalus groups were more likely to regulate retinol metabolism.The ginseng group immune association biomarker was citric acid and it related pathway was glyoxylate and dicarboxylate metabolism,carboxylic acid metabolic tricarboxylic acid cycle.At the same time,tyrosine metabolism(biomarker:homovanillin)was also found.The immune-re la ted metabolic pathways under the 8:1 group are as followed:1.arginine and proline metabolism;2.glycerolipid metabolism;3.glycerophospholipid metabolism.It can be seen that the role of astragalus in regulating the inner environmental pathways is not the same as that of ginseng,and ginseng focuses on the tricarboxylic acid cycle and amino acid metabolism.After compatibility,the 8:1 groups were further involved in regulating the metabolic pathways of multiple amino acids in the body,while lipid metabolism(glycerolipid metabolism and glycerophospholipid metabolism)is also involved in regulating the immune system,enhancing the immunity of the body.Bore marrow metabonomics study showed that in astragalus groups,PC A profile analysis of astragalus in low dosegroup was closer to the blank group than other groups,indicating that low dose of astragalus has the effect of improving bone marrow environment.Comparing astragalus,ginseng,1:8,1:1 and 8:1 group,high dose of ginseng is farthest from the blank group in PC A.After compatibility,1:8 group and 8:1 group tend to approach the blank control group,indicating that compatibility was helpful to improve the immunosuppressive mice.According to the correlation analysis,astragalus groups participated in glycerophospholipid metabolism,and the ginseng group participated in the regulation of metabolic pathways such as tyrosine metabolism,phenylalanine,tyrosine and tryptophan biosynthesis.The 8:1 group was mainly involved in regulating metabolic pathways for sphingolipid metabolism and glycerophospholipid metabolism.4.Systemic pharmacology studyThe results of systemic pharmacology study showed that there were 87 and 190 chemical components obtained from astragalus and ginseng,respectively.These chemical components were screened by ADME(OB>30%,DL>0.18)and related literature and finally 37 and 72 potential active molecules were obtained from astragalus and ginseng,respectively.And then,WES model was used to find related targets with active compounds,a total of 152(astragalus)and 156(ginseng)targets were obtained.Using these target genes,metabolic pathways were constructed through the KEGG database,and the pathways derived from metabolomics were mapped onto the gene-building pathways.As a result,it was found that the four enzymes such as acetylcholinesterase,sphingosine kinase 1,β-galactosidase,and cytidine deaminase influenced immune-related glycerophospholipid metabolism,sphingolipid metabolism,and pyrimidine metabolism in the astragalus group.In ginseng group,aldose reductase,glycogen phosphorylase,UDP-glucuronosyltransferase 2B7,choline O-acetyltransferase,acetylcholinesterase and lysosomal alpha-glucosidase also regulate glycerolipid metabolism,glycerophospholipid metabolism and glucose metabolism(Pentose and glucuronate interconversions,starch and sucrose metabolism).To sum up,astragalus and ginseng can play a role in regulating immune by different ways.The difference of immunoregulation of astragalus and ginseng could not be distinguished in normal animal model.But,it was found that there were similaritie s and differences between astragalus and ginseng in metabonomics way.The common pathway of astragalus and ginseng for invigorating qi is glycerophospholip id metabolism.Astragalus characteristics may be related to pyrimidine metabolism and sphingolipid metabolism.The characteristics of ginseng may be related to glucose metabolism.In terms of pharmacodynamic studies,astragalus and ginseng can improve the immune function of immunosuppressed mice by improving blood indexes and immunology(specific and non-specific immunity),and the compatibility is the best.According to the analysis of plasma metabonomics and bone marrow metabolomics data,astragalus may enhance the immune system by regulating the retinol metabolism and glycerophospho lipid metabolism pathway,and ginseng regulate amino acid metabolism(arginine and proline metabolism),glyoxylate and dicarboxylate metabolism and citrate cycle.And after compatibility,and connotation of qi-invigorating reflected in the regulatory pathway such as sphingolipid metabolism and glycerophospholpid metabolism.The bone marrow metabolism is more conservative than that in plasma.In the bone marrow environment,the astragalus group mainly regulates the glycerolipid metabolism,and the ginseng group mainly controls phenylalanine,tyrosine and tryptophan biosynthesis.After compatibility,metabolic conditions shift to sphingolipid metabolism and glyceropho spho lip id metabolism.Acetylcholinesterase,sphingosine kinase 1,β-galactosidase,cytidine deaminase,aldose reductase,glycogen phosphorylase,UDP-glucuronosyltransferase 2B7,choline O-acetyltransferase,acetylcholinesterase and lysosomal alpha-glucosidase were regarded as the targets of immune regulation. |
