The Study Of Metabolism Of Main Flavonoids Of Traditional Chinese Medicine Ingredients In Vitro And In Vivo And Qualitative And Quantitative Analysis Of Chemical Composition For Commelina Communis L.Based On LC-MS Technology

After drugs enter the body,chemical structure changes through the role of metabolic enzymes,which generate toxic or active metabolites.The liver is the main organ of the the drug producing metabolites.Furthermore,the incubation system of liver microsome is the most commonly used in vitro metabolic model.In vivo metabolism of drug more fully illustrate the metabolic pathways of drugs,which lay the foundation of clinical drug treatment and monitoring of toxic and side effects.Commelina communis Linn.trational Chinese medicine,is commonly applied to common cold,high fever,sore throat,edema oliguria,hot shower astringent pain,bloated boils poison.However,assaying of Commelina communis Linn.was not recorded in CP2015.Therefore,the purpose of this study was to establish a LC-MS method for the analysis of the metabolic profiles of the main flavonoids in Cirsium japonicum DC.extract in rats,in vitro and in vivo metabolites of fisetin,sophoricoside and liquiritin,and the chemical composition and determination of Commelina communis Linn.Part one The metabolic pathways study of major flavonoids in rats after lavage Cirsium japonicum DC.extractObjective:To develop an UHPLC-Q-TOF-MS/MS method for analysis of the metabolic pathways of major flavonoids in rats after lavage Cirsium japonicum DC.extract.Methods:The blood,urine and bile were collected after lavage Cirsium japonicum DC.extract,which were extracted by a liquid–liquid extraction?LLE?procedure with ethyl acetate.Firstly,On-line data were acquired using full-scan,and accurate MS/MS data were obtained using an effective MMDF and DBS-dependent data acquisition method.The post-acquisition data mining was performed using various data-mining tools.The structures of the metabolites of major flavonoids?pectolinarin,linarin and pectolinarigenin?were clarified based on the accurate mass measurement from the software MetabolitePilot 1.5,relevant drug bio-transformation knowledge,previously investigated fragmentation patternsof pectolinarin,linarin and pectolinarigenin and MS/MS spectra of metabolites.The useful parameter calculated log P,which was calculated using the program Chemdraw Ultra12.0,was introduced to distinguish the structural isomers.The chromatographic separation was carried on Poroshell 120 EC-C₁₈?2.1×100mm,2.7?m?with a SecurityGuard?UHPLC C₁₈ pre-column?Poroshell?.The mobile phase was consisted of water containing 0.1%formic acid and methanol.The gradient elution program was optimized for the separation.he mobile phase flow rate was set to 0.3 mL/min,and the injection volume was 5?L.Results:A total of 21 metabolites were detected in rat blood,urine and bile sample using UHPLC-Q-TOF-MS/MS.A total of 12 main absorped flavonoids were detected Hydrolysis reaction was the first step for the pectolinarin and linarin.Then oxidation and reduction take place.But phase II metabolites for pectolinarin and linarin were not detected.The main biotransformation routes of pectolinarigenin were identified as oxidation,reduction,hydrolysis,glucuronide and glucose conjugation.Conclusions:It was the first report of screening and identification of metabolites of pectolinarin,linarin and pectolinarigenin of Cirsium japonicum DC.exact by UHPLC-Q-TOF-MS/MS.Furthermore,the application of this approach will lay the foundation for further pharmacological and pharmaceutical activity studies of Cirsium japonicum DC.The method is simple and rapid,which is suitable for the analysis of metabolites in Chinese herbal medicines.Part two Metabolism study of fisetin in vitro and in vivoObjective:UHPLC-Q-TOF-MS/MS was employed to screen and identify metabolites of fisetin.Methods:The typical incubation mixture of phase I metabolism was established.The typical incubation mixture?200?L final volume?was carried out in a 0.1 mol/L K₂HPO₄ buffer including rat microsomes?1.0 mg/mL?,3.3mmol/L Mg Cl₂,1.3 mmol/L?-NADPH,3.3 mmol/L glucose-6-phosphate,1.0U/mL glucose-6-phosphate dehydrogenase and 100?mol/L fisetin?dissolved in 50%methanol solution?methanol solution?1%of final volume?.The typical incubation mixture of phase II metabolism was established.The reaction mixture?total volume 200?L?contained 1.0 mg/mL rat liver microsome,2 mmol/L UDPGA,8 mmol/L MgCl₂,25?g/mL alamethicin in PBS?pH 7.4?and 100?mol/L fisetin dissolved in 50%methanol solution?1%of final volume?.The blood,urine and bile were collected after lavage fisetin suspension.Liquid–liquid extraction,protein precipitation by methyl alcohol and protein precipitation by methyl alcohol were applied for phase I metabolism in vitro,phase II metabolism in vitro and in vivo metabolism,respectively.Results:A total of 53 metabolites?11 phase I metabolites and 42 phase II metabolites,18 from the blood,49 from the urine,34 from the bile and 34from the faeces?in vivo and 14 metabolites in vitro were recognized in this study.Metabolic pathways of fisetin mainly included oxidation,reduction,hydrogenation,methylation,sulfation and glucuronidation.From view of metabolism of fisetin,the reactions of oxidation and loss of oxygen were the basis for other metabolic reactions,which contained methylation,hydrogenation,sulfation and glucuronidation.Conclusions:UHPLC-Q-TOF-MS/MS were first explored for identification of the metabolites of fisetin in vitro and in vivo.UHPLC-Q-TOF-MS/MS was a powerful and effective tool to detect and characterize drug metabolite in biological samples,which led to a better understanding of biotransformation of drug and could provide a basis for the clinical application and pharmacological study.Part three Metabolism study of sophoricoside in vitro and in vivoObjective:A strategy was firstly developed to identify the metabolites and metabolic pathways of sophoricoside in vitro and in vivo using UHPLC-Q-TOF-MS/MS.Methods:The typical incubation mixture of phase I and phase II metabolism were established,respectively.The blood,urine,bile and feces were collected after administration sophoricoside suspension.Liquid–liquid extraction was chose for sample pretreatment of phase I metabolism in vitro,while protein precipitation by methyl alcohol was applied for phase II metabolism in vitro and in vivo metabolism.The chromatographic separation was carried on Poroshell 120 EC-C₁₈?2.1×100 mm,2.7?m?with a SecurityGuard?UHPLC C₁₈ pre-column?Poroshell?.The mobile phase flow rate was set at 0.3 mL/min,and the injection volume was 3?L.Results:The present study explored a powerful analytical strategy for fast screening and identification the metabolites of sophoricoside using MMDF and DBS dependent on-line data acquisition combined with multiple post acquisition data processing.Based on the proposed method,60metabolites were structurally characterized in vivo including 22 phase I and 38phase II metabolites,and 4 metabolites in vitro were detected containing 2phase I and 2 phase II metabolites.Sophoricoside mainly underwent conjugation reaction,which included methylation,sulfate,glucuronide,acetylation,glutamine and glycine conjugation.Furthermore,oxidation,reduction and hydrolysis metabolites were detected in blood,urine,bile and feces samples.However,oxidation and hydrolysis metabolites were only found in vitro metabolism.Conclusions:It was the first time that UHPLC-Q-TOF-MS/MS combining various data mining method was established to characteristic metabolites of sophoricoside in vitro and in vivo.Our study of the metabolism in vitro and in vivo will lead to a better understanding of the biotransformatios and pharmaceutical application of sophoricoside.Moreover,these results will lay the foundation of pharmacology and toxicology research.Part four Metabolism study of liquiritin in vitro and in vivoObjective:Metabolites of liquiritin in vitro and in vivo were recognized by the method UHPLC-Q-TOF-MS/MS.Methods:Incubation system of phase I and II metabolism were applied to screen and characteristic in vitro metabolites.The blood,urine,bile and feces samples were collected after intragastric administration liquiritin suspension,respectively.Phase I and II in vitro and in vivo biological samples were treated by the method of liquid-liquid extraction,methanol precipitation and methanol precipitation,respectively.Data were gained online using UHPLC-Q-TOF-MS/MS technique,and accurate masses of metabolites,secondary mass spectra and fragmentation pattern of parent drug liquiritin were determined by MetabolitePilot 1.5 and PeakView 1.2 software to infer possible metabolites.The chromatographic separation was carried on Poroshell 120 EC-C₁₈?2.1×100 mm,2.7?m?with a SecurityGuard?UHPLC C₁₈ pre-column?Poroshell?.The mobile phase flow rate was set at 0.3 mL/min,and the injection volume was 3?L.Results:As a result,56 in vivo metabolites?27 phase I and 29 phase II metabolites?and 15 in vitro metabolites were structurally characterized by UHPLC-Q-TOF-MS/MS.Based on the preceding data acquisition and mining strategy,twenty,fifty-four,forty-eight,thirty-seven and fifteen metabolites?11 phase I and phase II metabolites?were detected in rat blood,urine,bile,feces and liver microsomes,respectively.The metabolic pathways of liquiritin were determined to be oxidation,reduction,hydrolysis,methylation,acetylation,and sulfate and glucuronide conjugation.Conclusions:UHPLC-Q-TOF-MS/MS method was first established to screen and identify the metabolites of LQ in vitro and in vivo.The results lay the foundation for active screening studies.In addition,this study demonstrated a powerful strategy for rapid screening and identifying metabolites and chemical constituents of traditional Chinese medicines.Part five:Qualitative and quantitative study of Commelina communis Linn.Objective:UHPLC-Q-TOF-MS was used to analysis the chemical constituents of Commelina communis Linn.HPLC-MS/MS method was developed and validated for determination of thirteen components of Commelina communis Linn.,which included orientin,isoorientin,vitexin,isovitexin,rutin,apigenin,protocatechuate,vanillic acid,caffeic acid,ferulic acid,luteolin,quercetin and isorhamnetin.Methods:Qualitative study:The chromatographic separation was carried on Poroshell 120 SB-C₁₈?2.1 mm×100 mm,2.7?m?.The column temperature was maintained at 25°C.The mobile phase consisted of water containing 0.1%formic acid and acetonitrile,which was used to gradient elution.The mobile phase flow rate was set to 0.3 mL·min^-1,and the injection volume was 1?L.Ion sources operating in the positive and negative electrospray ionization mode was used for the detection.Quantitative study:Chromatographic separation was performed on a Column Wonda Cract ODS-2 C₁₈?150 mm×4.6 mm,5?m?;mobile phase A?0.1%formic acid-2mmol·L^-11 ammonium formate?-B?methanol?,which was used to gradient elution.The flow rate was set to 0.8 mL·min^-1.Injection volume was 10?L.The multiple-reaction monitoring scanning?MRM?was employed for the quantification with switching electrospray ion source polarity in negative mode.The ion spray voltage was set at-4500 V and the turbo spray temperature was maintained at 650°C.Results:59 chemical constituents were detected,22 of which had been compared with the retention time and secondary mass spectrometry of the reference substance.37 chemical constituents were deduced according to secondary mass spectrometry.The regression equations showing linear relationships between peak areas and contents of each compound were obtained?r?0.9991?.The recoveries ranged from 98.64%to 100.8%.The intra-and inter-day precisions?RSD%,relative standard deviation%?were within 1.04%and 0.92%.Conclusions:The method is simple,rapid,and sensitive.It could be used as a quantitative determination method for the nine components in Commelina communis L.which lays a foundation for further pharmacokinetics,pharmacology and toxicology of Commelina communis L.