Résumé
ObjectiveTo explore the material basis of bile-processed Coptidis Rhizoma clearing excessive fire of liver-gallbladder based on ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF/MS） metabolomics and molecular docking. MethodUPLC-Q-TOF/MS metabolomics was used to analyze the chemical constituents of Coptidis Rhizoma， water-processed Coptidis Rhizoma and bile-processed Coptidis Rhizoma. Chromatographic separation was achieved with 0.1% formic acid aqueous solution（A）-acetonitrile（B） as the mobile phase in gradient elution（0-2 min， 5%B； 2-20 min， 5%-65%B； 20-40 min， 65%-10%B； 40-45 min， 10%B； 45-46 min， 10%-95%B； 46-49 min， 95%B）， and electrospray ionization（ESI） was applied and operated in positive and negative ion modes， the acquisition range was m/z 80-1 200. Based on this， partial least squares-discriminant analysis（PLS-DA） and variance analysis were used to screen the differential compounds among the three products of Coptidis Rhizoma. Network pharmacology and molecular docking were used to verify the degree of association between differential compounds and excessive fire of liver-gallbladder syndrome. ResultA total of 33 chemical constituents were identified， including 2 phenolic acids， 5 binding bile acids and 26 alkaloids. And 16 differential compounds were identified by multivariate statistical analysis， including 11 alkaloids and 5 binding bile acids. Pathway enrichment analysis in the Kyoto Encyclopedia of Genes and Genomes（KEGG） database yielded 8 pathways related to excessive fire of liver-gallbladder， and the key protein phosphatidylinositol 4，5-bisphosphate 3-kinase catalytic subunit alpha isoform（PIK3CA） was obtained according to the "component-target-pathway" network analysis. Molecular docking results showed that 11 alkaloids had good binding ability with PIK3CA. ConclusionPorcine bile is unique in the processing of bile-processed Coptidis Rhizoma， which can promote the production and dissolution of 11 alkaloids， including berberine and dihydrochelerythrine. Based on the results of molecular docking and reported pharmacological experiments， it can be concluded that 16 different compounds such as berberine， dihydrochelerythrine and taurohyodeoxycholic acid are the material basis of bile-processed Coptidis Rhizoma.
Résumé
Objective:Based on UPLC-Q-Orbitrap HRMS and network pharmacology, the material basis, processing principle and molecular mechanism of bile processed Coptidis Rhizoma (BPRC) for reducing excess fire of liver and gallbladder were elucidated.Method:The chemical ingredients of BPRC were analyzed by UPLC-Q-Orbitrap HRMS. Chromatographic separation was achieved with 0.1% formic acid solution (A)-acetonitrile (B) as the mobile phase in gradient elution (0-20 min, 5%-80%B; 20-30 min, 80%-95%B; 30-30.1 min, 95%-5%B; 30.1-35 min, 95%-5%B). The flow rate was 0.2 mL·min-1, electrospray ionization (ESI) was applied and operated in positive and negative ion modes, the acquisition range was m/z 100-1 500. Based on the clinical manifestations and pathogenic factors of excess fire of liver and gallbladder, the potential effective ingredients, targets and functional characteristics of BPRC were predicted and analyzed by online database. Based on the characteristics of the new active ingredients after processing, the processing principle of BPRC was investigated by network pharmacology.Result:A total of 19 ingredients in BPRC were identified, six of which were newly added cholic acids after processing. It was determined that the alkaloids, including worenine, epiberberine, jatrorrhizine, coptisine, berberrubine, berberine, palmatine and cholic acids, including glycohyodeoxycholic acid, taurohyodeoxycholic acid, glycochenodeoxycholic acid, hyodeoxycholic acid and taurochenodeoxycholic acid, were identified as material basis of BPRC. A total of 66 targets of reducing excess fire of liver and gallbladder of BPRC were screened. There were 16 common targets and multiple same signaling pathways between cholic acids and alkaloids of BPRC, and many lesions of excess fire of liver and gallbladder were target organs of cholic acids. By acting on some targets, including albumin (ALB), Caspase-3 (CASP3), mitogen-activated protein kinase 14 (MAPK14), glucocorticoid receptor (NR3C1) and other targets and some signaling pathways, including interleukin (IL)-17, phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt), MAPK and other pathways, BPRC could reduce excess fire of liver and gallbladder.Conclusion:BPRC has the characteristics of multi-component, multi-target and multi-pathway on reducing excess fire of liver and gallbladder. Bile and Coptidis Rhizoma have synergistic effect and bile can enhance the intensity of BPRC in lesions, which confirms the processing theory that the effect of BPRC on excess fire of liver and gallbladder enhance after being processed by bile.
Résumé
Objective To establish QAMS method to determine the contents of three alkaloids in bile processed Coptidis Rhizoma; To compare the results of QAMS with those from external standard method; To prove the feasibility of QAMS.Methods An HPLC method was developed. Berberine hydrochloride was selected as the internal reference substance. 2 relative correction factors (RCF) of berberine hydrochloride to palmatine hydrochloride and to jatrorrhizine hydrochloride were established. Obtained RCFs were used to conduct content calculation (calculated value) to complete QAMS method. At the same time, the contents (measured value) of the three components were also determined by external standard method. Calculated value and measured value were compared.Results The analysis results showed that there was no significant difference between the calculated values and the measured values of the three alkaloids in 10 batches of bile processed Coptidis Rhizoma.Conclusion The QAMS method can be applied in the determination of alkaloids in bile processed Coptidis Rhizoma.
Résumé
A urinary metabonomics method based on ultra-performance liquid chromatography coupled with LTQ-Orbitrap-mass spectrometry (UPLC/LTQ-Orbitrap-MS) was developed to study the difference of action mechanism of Coptidis Rhizoma (CR) and bile processed CR on the heat syndromein rats, and reveal the scientificity of CR processing method. The heat syndrome rat models were established by intragastric administration of water decoction of Aconiti Lateralis Radix Preparata, Zingiberis Rhizoma and Cinnamomi Cortex for 15 days combined with subcutaneous injection of dry yeast suspension. After administration for 15 days, the urine of rats in each group was collected at 0-6 h after modeling, 6-12 h after modeling and 12-24 h after modeling; principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were used for data treatment. Good separation was observed between the normal groups and model group at 0-6 h and 6-12 h, but overlapped at 12-24 h with no separation trend. Obvious separation was achieved in urine samples between CR group, BCR group and model groups at 0-6 h, close to the normal group. Separation trend occurred between CR group and BCR group at 0-6 h and 6-12 h. Thirty potential biomarkers related with heat syndrome were identified by PLS-DA approach. The results showed that the overall therapeutic effect of CR for heat syndrome had been changed after being processed with pig bile. Bile processed CR has the characteristics of multiple targets, rapid onset and strong effects, mainly playing a role of antipyretic effect through regulating cholinergic neurotransmitter, amino acid metabolism and purine metabolism.