| It is really a great challenge to investigate the active constituents and the pharmacological mechanism of traditional Chinese medicine(TCM)due to the complexity of TCM.It is well known that TCM decoction is often taken by oral administration.After oral administration the multicomponents of TCM must go through the intestine and liver before being absorbed into the systemic blood stream.During this process,not all components can be absorbed into the systemic blood stream.Some components can be absorbed as prototypes;others may be metabolized by the enzymes in the gut wall or liver.Therefore,the components in the blood are really complex.In recent years,with the rapid development of analytical tools,liquid chromatography coupled with mass spectrometry has become a powerful and reliable tool to analyze the multicomponents and metabolites of TCM in biological samples due to its high sensitivity and rapidness.However,only identification of the components in the blood is far away to illustrate the pharmacological mechanism of TCM,we need to construct the relationship between the constituents and the therapeutical effects.In recent years,network pharmacology has become a useful tool to illustrate the pharmacological mechanism of TCM.Therefore,we propose a "Chemical Constituents-Sequential Metabolism-Network targets"method to investigate the active constituents and the pharmacological mechanism of Buddleja officinalis Extract(BOE)for the treatment of conjunctival congestion.Our study can be divided into three parts,detailed results are shown below.1.Identification of the major components of BOE by UHPLC-LTQ-Orbitrap.Results:A total of 20 compounds,including 9 flavonoids and 11 phenylethanoid glycosides,were confirmed or tentatively identified from BOE.2.Sequential metabolism of BOE in rats(1)Study on the metabolic patterns of phenylethanoid glycosidesWe selected two single compounds(Salidroside and isoacteoside)to describe the metabolic patterns of phenylethanoid glycosidesThe results of sequential metabolism of salidroside:A total of 4 metabolites of salidroside have been detected and identified in different plasma samples.Our results indicated that glucuronidation,sulfation,and deglycosylation were the major metabolic pathways of salidroside.The contribution of each metabolic site to the overall metabolism was also discussed.The results showed that salidroside was stable in digestive juice,and it was metabolized primarily by the liver and the intestinal flora and to a lesser extent by the gut wall.The results of in vivo metabolism of isoacteoside:A total of 10 metabolites were observed and characterized in rat urine.The metabolic transformation pathways of isoacteoside included hydrolyzation,dehydroxylation,glucuronide conjugation,sulphate conjugation,and methyl conjugation.Overall,the major metabolic pathways of phenylethanoid glycosides included hydrolyzation,deglycosylation,dehydroxylation,glucuronide conjugation,sulphate conjugation,and methyl conjugation.The results are really helpful for us to infer the metabolites of other compounds with similar chemical structures.(2)Study on the metabolic patterns of flavonoidsWe selected two single compounds(linarin and luteolin)to describe the metabolic patterns of flavonoids.The results of in vivo metabolism of linarin:A total of 4 metabolites were observed and characterized in rat urine and feces.The metabolic transformation pathways of linarin included hydrolyzation,glucuronide conjugation,demethylation,and hydroxylation.The results of in vivo metabolism of luteolin:A total of 6 metabolites were observed and characterized in rat urine and feces.The metabolic transformation pathways of linarin included hydrolyzation,glucuronide conjugation,and methyl conjugation.Overall,the major metabolic pathways of flavonoids included hydrolyzation,glucuronide conjugation,demethylation,hydroxylation,methyl conjugation.The results are really helpful for us to infer the metabolites of other compounds with similar chemical structures.(3)Identification of major metabolites of BOE in rat urine by UHPLC-LTQ-OrbitrapIn this study,BOE dissolved in saline was orally administered to the rats at high dose,and then we collect the rat urine to analyze the potential metabolites during the sequentical process.Results:In vivo,34 components,including 5 prototypes and 29 metabolies,were confirmed or tentatively identified in rat urine samples.(4)Sequential metabolism of BOE in ratsThe study on sequential metabolism of BOE in rats has four parts:I.Enzymatic transformation in digestive juice;?.Biotransformation by intestinal flora tract;?.Gut wall metabolism;?.Hepatic metabolism.Results:BOE was metabolized primarily by the liver and the intestinal flora and to a lesser extent by the gut wall.Research on the sequential metabolism of multicomponents would provide helpful information on how the biological activities of these components are changed before these components are delivered to the target sites.3.Network pharmacology study of BOEThe putative target profile of components and metabolite of BOE was predicted using MedChem Studio,and known therapeutic targets of conjunctival congestion were collected as well.Then an interaction network was constructed using links between putative BOE targets and known therapeutic targets of conjunctival congestion based on Protein and Protein Interaction Databases(PPI).Following the calculation of topological features of each node,major targets were identified and used to perform the pathway enrichment analysis.The results indicated the methylated metabolites of caffeic acid might be the main active constituents acting on conjunctival congestion.And the major putative targets of BOE were demonstrated to be mostly involved in pathways related to anti-inflammation processes and the immune system,which may be crucial elements during the attenuation of conjunctival congestion.However,further experimental validations of these results are required in future studies to expound the molecular mechanism of BOE for the treatment of conjunctival congestion comprehensively.In conclusion,the results of this study have elucidated the pharmacodynamic material basis and pharmacological mechanism of BOE for the treatment of conjunctival congestion.Meanwhile,the strategy of "Chemical Constituents-Sequential Metabolism-Network targets"method can provide evidence for investigating pharmacodynamic material basis and pharmacological mechanism of other materia medica. |
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