Study On Material Basis Of Wu-Zhu-Yu Decoction In The Treatment Of Headache

Wu-Zhu-Yu decoction is a classic Traditional Chinese Medicine formula which has been extensively used for the treatment of headache and gastrointestinal disorders,etc.in both traditional and modern clinical application in China.Wu-Zhu-Yu decoction was selected as the objective of the study.Comprehensive profiling of ingredients in Wu-ZhuYu decoction,absorptive compounds and their metabolites screening in rat plasma,pharmacokinetic study of active ingredients and metabolomics study on headache rats after oral administration of Wu-Zhu-Yu decoction were achieved and deeply investigated.A rapid and reliable liquid chromatography-high resolution mass spectrometry method was established for comprehensive profiling of the chemical ingredients in WuZhu-Yu decoction.The method was achieved on a quadrupole time-of-flight mass spectrometer along with an advanced data processing procedure consisting of mass accuracy screening,mass defect filtering and fragment filtering.After eliminating interference with a filtering approach,the MS data profiling was made more distinct and accurate.With the optimized conditions of only 35 min LC separation and single sample injection of each positive or negative ion mode,a total of 168 compounds were characterized,including 23 evodiamine and its analogous alkaloids,12 limonoids,17 gingerols,38 ginsenosides,15 flavonoids,16 organic acids and 47 other compounds.The fragmentation patterns of the representative compounds were illustrated as well.A rapid and sensitive liquid chromatography-high resolution mass spectrometry method with multiple data processing algorithms was developed and applied for metabolite profiling of absorbed prototype compunds in rat plasma after administration of Wu-Zhu-Yu decoction.All samples were purified using hydrophilic-lipophilic balanced solid phase extraction cartridges and analyzed by a high resolution mass spectrometer with a 35 min liquid chromatography gradient elution.High resolution full scan mass spectrometry and information-dependent acquisition tandem mass spectrometry data were analyzed using multiple data processing approaches.Results indicated that the detected eight prototype alkaloids could be metabolized to 58 metabolites through both phase I and phase ? reactions.A fast,sensitive and reliable ultra fast liquid chromatography-tandem mass spectrometry method has been developed and validated for simultaneous quantitation of eight main active ingredients in rat plasma after oral administration of Wu-Zhu-Yu decoction.The analytes and internal standard were separated using the validated method.The validated method has been successfully applied to compare pharmacokinetic profiles of the eight active ingredients in rat plasma between normal and headache rats after administration.The results indicated that the absorption of evodiamine,rutaecarpine,gensenoside Rbl,Re and Rgl in headache group were significantly higher than those in normal group with similar concentration-time curves while no significant differences existed in limonin and ginsenoside Rd between the two groups.A comprehensive and reliable metabolomics approach was established to find potential biomarkers and to study the metabolism disturbance in headache rats model.Meanwhile,Wu-Zhu-Yu decoction was also studied to reveal the regulation mechanism of the brain homogenate and plasma metabolites in the headache rats.NO donor headache model was adopted in the study.The brain homogenates and plasma samples were analyzed using a liquid chromatography-high resolution mass spectrometry method to determine the endogenous metabolites.Principal component analysis-discriminant analysis showed that the control,model,Wu-Zhu-Yu decoction and flunarizine group were all separated distinctly,which demonstrated the metabolic network of the rats was disturbed after modeling.Accordingly,17 biomarkers were found and identified.In the model group,the levels of glutathione,oxidized glutathione,adenine,adenosine,adenosine monophosphate,nicotinamide adenine dinucleotide,glycerophosphocholine,butyryl-carnitine,valerolactam,and caprolactam increased,while the levels of cholecalciferol,arginine,carnitine,3-methylindole,hydroxyindole,indoleacetic acid,and 5-hydroxyindoleacetic acid dropped dwon.The levels of these metabolites were recovered in different degrees after administration of Wu-Zhu-Yu decoction.Furthermore,the study on the metabolic pathways of the 17 potential biomarkers was accomplished afterwards,and a metabolic network was established to reveal the attack of headache.Combining with the behavior study results,Wu-Zhu-Yu decoction could ameliorate the headache syndrome in the headache rats.