| Objective:Pharmacokinetic-pharmacodynamic(PK-PD)model,in vitro and in vivo analysis,network pharmacology and untargeted metabolomics were used to explore the mechanism of Wendan Decoction(WDD)in the treatment of hyperlipidemia,so as to provide a scientific basis for the further study of WDD.Methods:The chemical components and plasma components of WDD were identified by ultraperformance liquid chromatography-quadrupole-electrostatic field orbital high resolution mass spectrometry(UPLC-QE-Orbitrap-MS).The contents of 30 chemical components in WDD were determined by ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry(UPLC-QQQ-MS/MS).Combined with principal component analysis(PCA),cluster analysis(CA)and orthogonal partial least squares discriminant analysis(OPLS-DA),the quality difference markers of WDD were determined.The model rats of foodborne hyperlipidemia were established by feeding with high-fat feed,and the lipid-lowering effect of WDD was explored.The plasma drug concentrations of rats at different doses were measured by UPLC-QQQ-MS/MS technology,and PK parameters were calculated using Phoenix WinNonlin 8.1 software.The level of lipid peroxide(LPO)in plasma at different time points was measured by enzyme labeling instrument.Finally,the PK-PD model was established by using Phoenix WinNonlin 8.1 software to explore the lipid-lowering effect of WDD and the relationship between the dynamic changes of chemical components and antioxidant effect.The lipid-lowering mechanism of the plasma components of WDD was preliminarily predicted by network pharmacology.The plasma metabolites of blank group,model group and WDD group were analyzed by ultra-performance liquid chromatography tandem Fourier transform mass spectrometry(UPLC-Q Exactive HF-X),The results of PCA,partial least squares discriminant analysis(PLS-DA)and OPLS-DA judged whether there were significant differences between the groups,determined the potential metabolites by using VIP value and P value.Finally,Cytoscape 3.7.2 software was used for comprehensive analysis to determine the possible lipid-lowering mechanism of WDD.Results:In the analysis and identification of the extract of WDD,186 chemical components were obtained,including organic acids,flavonoids,phenylpropanoids and alkaloids.During the analysis and identification of the transitional components in the plasma of WDD,97 compounds were obtained,including 75 prototype components and 22 metabolites.The content of 30 components in the extract of WDD was determined by internal standard method,and OPLS-DA and other methods were used.It was found that synephrine,glycyrrhizic acid,ammonium glycyrrhizinate,aesculin,isoferulic acid,naringin and nobiletin were the main effective components of WDD.The anti-vascular endothelial cell injury(AVECI)action of WDD was studied.First,the damaging impact of palmitic acid on HUVECs was authenticated.Then,the protective effect of WDD on endothelial cells was assessed.The results indicated that,the extracts of WDD significantly increased the proliferation of endothelial cells and significantly protected the endothelial cells.However,the interaction between different WDD components and mechanism remains unclear.Statistical data further demonstrated that,ten WDD compounds(trigonelline,liquiritin,hesperidin,etc.)have a great advancing impact on pharmacodynamic parameters in vitro.Further,the in vivo intervention of WDD on hyperlipidemia rats was analyzed.It was found that,WDD can reduce the levels of total cholesterol(TC),triglyceride(TG)and low density lipoprotein cholesterol(LDL-C)in plasma,and high density lipoprotein cholesterol(HDL-C)was related to the dosage.UPLC-QQQ-MS/MS technique was used for the first time to determine plasma drug concentration in hyperlipidemia rats at different time points after WDD administration.According to the results of PK parameters,it was found that 10 components could be detected in 0.25 h,and the concentrations of the substances to be tested reached the highest in 1.5 h,indicating that it was rapidly absorbed in rats plasma.The results of software analysis showed that,the components fit well under the two compartment models.The PD results showed that,the level of LPO was lower when rats were administered with high dose of WDD compared with the model group,and the IC50 of quercetin,tangeretin,naringenin and hesperetin were lower in the PK-PD parameters fitted by the experiment.Ke0 value was lower than that of other compounds,and its drug effect lag behind the time of drug concentration.Network pharmacology was used to explore the lipid-lowering mechanism of transitional components in WDD.It was finally found that,PPAR signaling pathway,fatty acid degradation,glycine,serine and threonine metabolisms were potential pathways.The plasma metabolites of rats between groups were analyzed.According to the data of PLS-DA and OPLS-DA,the hyperlipidemia observed can be viably and dependably connected to the forecast of potential biomarkers.According to VIP value>1 and P value<0.05,60 differential metabolites were screened in blank group,model group and WDD group.With P<0.05 as the screening condition,the KEGG enrichment analysis of differential metabolites showed that,glyceride metabolism and PPAR signaling pathway were the key pathways of metabolic set.Through comprehensive analysis,22 key targets(CAT,CPT1A and ACOX1,etc.)were obtained.Three chemical components of WDD(quercetin,naringin and coumarin),two potential metabolites(phosphatidylcholine and lysophosphatidylcholine)and three key pathways(glycerol phospholipid metabolism pathway,pyrimidine metabolism and PPAR signal pathway)may be related to the lipid-lowering effect of WDD.Conclusion:The chemical components and plasma components of WDD were analyzed and identified by LC-MS/MS technology,and its multi-component content was determined.It was more scientific and strictly governed to provide help for the formulation of the quality standard of WDD,which enriched the chemical component database of the WDD.Compared different doses of WDD PK-PD,the absorption,distribution,metabolism and excretion of the tested substance in rats have changed,and the dosage can affect the absorption of the tested substance to varying degrees.Moreover,WDD may regulate the level of LPO and play a role in reducing blood lipid through the synergistic effect of various components in hyperlipidemia model rats.For the first time,we established a method based on network pharmacology and non-targeted metabolomics to analyze the lipid-lowering mechanism of WDD.And,finally identified 60 potential biomarkers related to hyperlipidemia and the changes of metabolites between groups,including pyrimidine metabolism,triglyceride metabolism,PPAR signaling pathway and other metabolic disorders.The results showed that,most of the differential metabolic compounds in WDD treatment group were altered to the same as normal group rats,indicating that WDD could regulate metabolic disorders.This experiment provided a new method and idea for exploring the potential pharmacological mechanism of Chinese herbal compound. |
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