Study Of The Group Of Active Ingredients Of Zhu Riheng Dropping Pills Based On The Correlation Model Of "Intestinal Absorption-Foaming Macrophage" In Vitro

Objective:Zhu Riheng Dropping Pill（ZRH）is a modern Mongolian preparation for the treatment of"heyi"on heart.ZRH is composed of nine herbal medicines,including Guang Zao（Choerospondiatis Fructus,emperor,GZ）,Rou Doukou（Myristica fragrans Houtt,minister,RDK）,Cao Guo（Amomum Tsao-ko,CG）,Hong Hua（Carthamus tinctorius L.,HH）,et.al.To explore the active ingredients of ZRH,we builded an association model of"intestinal absorption-foaming macrophage"in vitro.Methods:1.Identification and attribution analysis of the inherent components of ZRH:In this study,high-performance liquid chromatography tandem quadrupole-electrostatic field orbitrap high-resolution mass spectrometry（HPLC-Q-Exactive-MS/MS）was used to analyze ZRH.Chromatographic separation was performed on an ACE EXCEL3 C18-PFP column（3μm,100mm×3mm）,The mobile phase consisted of methanol containing0.2%formic acid（A）-0.2%formic acid aqueous solution（B）with gradient elution.Dectection was carried out in positive and negative ionization modes with ESI ion source.Comparing with the accurate relative molecular mass,the MS fragments,the reference substance,literature,and the self-built database of ZRH,the components in the ZRH were identified.2.Study of the intestinal absorption characteristics of ZRH based on the rat everted intestinal sac model:Using the everted intestinal sac model of rat,the intestinally absorbed solution（IAS）treated with 0.176,0.252,0.378,0.504g·m L^-1ZRH at different phase were collect.HPLC-Q-Exactive-MS/MS intestinal absorption fingerprints were established to quantitatively analyze quinic acid,taurine,citric acid,gallic acid,chlorogenic acid,geniposide,hydroxy red quinic acid,3,3’-Di-O-methylellagic acid,citric acid,gallic acid,safrole,myristicin,eugenol,elemicin,methyleugenol,dehydrodiisoeugenol,geniposide,kaempferol,hydroysafflor-yellow-A,agarotetrol,umbelliferone,ferulic acid,borneol,citral,taurine,cholic acid,deoxycholic acid,quercetin,rutin,chlorogenic acid in intestinal absorption samples.Methods of validation were performed,including linearity,lower limit of quantitation（LLOQ）stability,precision,accuracy,specificity,recovery and matrix effect.Absorption profile of 24 ingredients of ZRH in rat IAS were evaluated with the accumulative absorbed dose（Q）,and the absorption rate coefficient（Ka）.3.Study of active ingredient groups of ZRH based on RAW246.7 macrophages foaming model:A foam cell formation model of raw264.7macrophage driven by Ox-LDL in vitro was established to assess the efficacy of ZRH intestinally absorbed solution with oil red O staining and the detection of total cholesterol,free cholesterol and cholesterol ester.The time-effect correlation and the different intestinal tube-effect correlation were evaluated.At the same time,the spectrum-effect analysis was carried out using Gray relational analysis（GRA）,and Orthogonal projections to latent structures-discriminant analysis（OPLS-DA）,the correlation between the group of intestinal absorption components in vitro and the group of pharmacological components was established.4.The material base research of ZRH against macrophages foaming:With the standard blending,the assessment of the efficacy the intestinally absorbed solution and the two kinds of analogs of ZRH,including“the analog of intrinsic components”and“the analog of intestinal absorption components”,against macrophages foaming were assessed.Subsequently,the inflammatory factor IL-1βand NF-κB,the regulatory factors of macrophages foaming,PPARγ,ABCA1,RXRA,and SR-A1 genes were further analyzed to explore the material basis of ZRH against the macrophages foaming.Results:1.A total of 130components in ZRH were identified by HPLC-Q-Exactive-MS/MS,including 18phenylpropanoids,24 lignans,26 flavonoids,10 coumarin,13 iridoids,15 organic acids,14 phenolic acids,8 monoterpenes,and 2 other components.2.A HPLC-Q-Exactive-MS/MS method was established for quantitative analysis of 24 constituents in intestinal absorption samples of ZRH.The methodological investigation shows that the method is sensitive and feasible.24 analytes have a good linear relationship in the linear range,and the correlation coefficients are all greater than 0.9950.The intra-day and inter-day precisions of 24 analytes ranged at 0.45%～6.95%,and 0.29%～8.80%,the accuracy at93.81%～108.15%,and 94.22%～108.94%,the recovery at 91.2%～108.25%,and the matrix effect at 86.51%～112.56%.The method conformed to the biological sample analysis standards.The components of nutmeg,safrole,eugenol,dehydrodiisoeugenol,and methyleugenol are best absorbed in the ileum or colon,and the duodenum is the best absorption site for other components.The transport mode of taurine,bile acid,citric acid,deoxycholic acid,borneol and citral belong to active transport,and the others are mostly passive diffusion.3.ZRH intestinal fluid inhibited macrophage foaming,and The intestinal fluid treated with 0.378 g·m L^-1and 0.504 g·m L^-1ZRH for 2h had the more significant effects comparing with other dose,and the intestinal fluid of 90min and 120min groups treated with 0.378 g·m L^-1ZRH shown best,meanwhile,the duodenum samples at same concentration has higher activity comparing with the other bowel samples.The spectrum-effect analysis shows that the components of ZRH mostly contributed to the inhibition of foaming,among which methyleugenol,ferulic acid,quinic acid,agarotetrol and 3,3’-Di-O-methylellagic acid are prominent.4.The intestinal absorption solutions of ZRH and both"Analogs"significantly reduced macrophage foaming,increased the expression of Abca1,Pparγ,and Rxra genes,and inhibited the expression of Sr-a1 gene,inflammatory factor IL-1βand NF-κB expression..Conclusion:the study established an effective linkage between the"intestinal absorption and active components"of ZRH,which inhibits macrophage foaminess and promotes inflammation regression by regulating the expression of genes related to cholesterol endocytosis and efflux.In addition,the 24 compounds selected largely reflected the activity of ZRH.This study provides a scientific basis for the establishment of subsequent quality markers and the clinical precision of ZRH.