Study On The Immune Regulation Mechanism Of Ginseng Based On Intestinal Flora And Chromatography-mass Spectrometry

Objective:The core function of wild ginseng is to invigorate vitality.It is a commonly used qi in clinical medicine.It is used to treat various qi deficiency syndromes and is used for various diseases（such as malignant tumors）that lead to a sharp decline in immunity.The current research on Wild ginseng mainly focuses on the efficacy of a certain western medicine disease,such as the treatment of type 2 diabetes and hypertension.As for how to implement forest Senate qi effect by regulating the immune system studies rarely reported,and its mechanism is not clear.This study aims to explore the immunomodulatory effects of ginseng based on the intestinal flora.Taking spleen-qi deficiency model rats as the research object,through phenotype,body weight,immune organs,organ index,organ tissue morphology,spleen cell proliferation,CD⁴⁺,CD⁸⁺lymphocytes,immunity and inflammation and other indicators were measured to investigate the immune regulation effect of ginseng.Analyze the flora metabolites in stool samples based on chromatography-mass spectrometry technology and apply metabolomics.Combining with microbial diversity,explore the immune regulation mechanism of ginseng based on intestinal flora,which is the clinical application of ginseng Provide scientific basis.Methods:1.Establish an analytical method for ginsenosides in wild ginseng:using Ultra-performance liquid chromatography coupled with quadrupole orbitrap tandem mass spectrometry,UPLC-Q-Orbitrap MS/MS),established a qualitative analysis method for the effective components of ginseng,combined with plant metabolomics to compare the differences of the main saponins in ginseng and garden ginseng,and passed ultra performance liquid chromatography-triple quadrupole mass spectrometry(Ultra High performance liquid chromatography-mass spectrometry（UPLC-QQQ MS）combined with negative ion multiple reaction monitoring mode（MRM）was used to quantitatively analyze the difference components in wild ginseng and garden ginseng.2.The pharmacodynamic study on immune regulation of ginseng:the spleen-qi deficiency rat model was established by the compound modeling method of fatigue,spleen injury and controlled diet.The positive control group was intragastrically given Shenlingbaizhusan(3.24g drug·kg^-1),wild ginseng and garden ginseng(1.62 g crude medicinal materials·kg^-1),and garden ginseng（1.62g crude medicinal materials·kg-1）water extract for 30 days by gavage to investigate the effects of Wild ginseng on the phenotype,body weight,immune organ index,spleen organ tissue morphology,blood analysis,immune biochemical indicators,macrophage phagocytic ability,and lymphocyte proliferation ability in rats with spleen-qi deficiency syndrome after intervention.3.Study on fecal metabolomics of rats with spleen-qi deficiency syndrome:detection of feces by ultra-performance liquid chromatography with high-resolution quadrupole time-of-flight mass spectrometer（ultra-performance liquid chromatography with high-resolution quadrupole time-of-flight mass spectrometer）full scan Samples are collected by Mass Hunter software,peak alignment and normalization are performed by QA software,and then imported into SIMCA14.0 for centralization,principal component analysis（PCA）and orthogonal partial least squares discriminant analysis（OPLS-DA）Multivariate statistical analysis.Based on VIP>1 and t-test p<0.05,the biomarkers related to the immune regulation of ginseng were screened,and the KEGG（Kyoto Encyclopedia of Genes and Genomes）database was used to extract the possible metabolic pathways of the screened differential metabolites.4.Research on microbial diversity of rats with spleen deficiency syndrome:Use r RNA high-throughput sequencing technology to detect each group of fecal samples,analyze changes in microbial community structure,gene function annotations and metabolic pathways,and screen key strains closely related to spleen deficiency,And then clarify the immune regulation of ginseng and the law of intestinal flora.Results:1.Qualitative and quantitative analysis of ginsenosides in the main components of in wild ginseng:A total of 36 ginsenoside components have been identified in wild ginseng and garden ginseng,all of which are common components of two types of ginsengs.The total saponins content in the water extract of wild ginseng was determined by ultraviolet spectrophotometry to be 6.18%.Plant metabolomics analyzed 12 kinds of saponins with significant differences,namely Rg₁,Re,Rf,Rg₂,Rb₁,Rh₁,Rc,Rb₂,Rb₃,Rd,Ro,Rg₃,established a multi-reaction monitoring mode（MRM）saponins quantification method to quantitatively analyze the main different saponins components.The 12 saponin components in the water extract of wild ginseng accounted for the total components The 12 saponins in the water extract of garden ginseng accounted for 2.61%of the total composition.The results showed that compared with garden ginseng,the 12 saponins in the water extract of garden ginseng were 1.37 times that of garden ginseng.2.Results of immunomodulatory pharmacodynamics of wild ginseng:（1）physical signs and weight:the phenotype of rats was significantly improved after the intervention of wild ginseng（p<0.01）,and the body weight began to increase significantly on the 15th day（p<0.05）;（2）Immune indicators:Significantly increase the phagocytic activity of macrophages（p<0.01）and lymphocyte proliferation activity（p<0.05）,the ratio of CD³⁺/CD⁴⁺T and CD⁴⁺/CD⁸⁺cells are significantly increased（p<0.01）,immune indicators The improvement of IL-6,C3 and s Ig G was not obvious（p>0.05）,and other indicators increased significantly（p<0.01）;（3）Blood indicators:the four indicators that were significantly decreased in the model group were called back by wild ginseng（p<0.05）,among which WBC,RBC,PLT increased significantly（p<0.01）;（4）Organ index and morphology:Wild ginseng can significantly increase the spleen and thymus organ index of rats with spleen-qi deficiency syndrome（p<0.01）.At the same time,the morphology of the organs was basically restored to a normal level in varying degrees,and the morphology of the rat’s organs was improved.3.Fecal metabolomics analysis of rats with spleen-qi deficiency syndrome:A total of40 potential biomarkers were found among the blank group,model group,positive control group,wild ginseng administration group and garden administration ginseng group,and wild ginseng can recall 25 biomarkers,Including Indole,Guanidoacetic acid,Cholic acid,Deoxycholic acid,Leukotriene E4,Pregnenolone,etc.,which involve 4 main metabolic pathways,namely amino acid metabolism,bile acid metabolism,steroid metabolism and fatty acid metabolism,participating in NF-κB,MAPK,MTOR,Akt and many other signal pathways.4.Analysis of the influence of wild ginseng on the intestinal flora of rats with spleen-qi deficiency syndrome:the relative abundance of Firmicutes in rats with spleen-qi deficiency syndrome decreases,Bacteroides,Soft-walled,Verrucomicrobs,Proteobacteria,The relative abundance of Planctomycetes and Actinomycetes increased,and the ginseng administration group reversed the abundance of the above-mentioned bacteria.At the genus level,the beneficial bacteria Lactobacillus,Akkermansia,and Faecalibaculum in the model group were significantly reduced,while the increase in beneficial bacteria of ginseng was more significant（p<0.01）.KEGG functional gene prediction wild ginseng mainly adjusts the intestinal microenvironment of rats with spleen-qi deficiency syndrome through Global and overview maps,Amino acid metabolism,and Energy metabolism,thereby improving the body’s immunity The role of mechanism,in which amino acid metabolism and lipid metabolism are the same as the results of fecal metabolomics research.Conclusion:In this paper,the qualitative analysis of the saponins of wild ginseng was carried out,and 12 kinds of saponins that were significantly different in content from that of garden ginseng were found through plant metabolomics.The multiple reaction monitoring（MRM）mode was used to quantitatively analyze the saponins in the wild ginseng.The content of 12 saponins is 1.37 times that of garden ginseng.The effects of wild ginseng on the phenotype,body weight,organ index,immunoglobulin,interleukin,and other immune indexes of rats with spleen deficiency,the morphological observation and pathological analysis of the spleen and other organs,confirmed that wild ginseng can increase spleen deficiency Immune activity of model rats.Subsequently,fecal metabolomics was used to confirm the effect of lindling ginseng on fecal metabolites,and 16s r RNA high-throughput sequencing technology was used to show that lindling ginseng is beneficial to increase the intestinal flora of rats with spleen-qi deficiency syndrome at the phylum and genus level species composition.The trend of reducing the relative abundance of harmful bacteria,and the improvement of this intestinal flora may play an important role in disease treatment and health care functions.This study preliminarily clarified the immune regulation mechanism of ginseng based on the axis of"intestinal flora-metabolite-immunity",which provides a scientific basis for the clinical application of ginseng.