| Wendan Decoction is a classic clinical prescription of traditional Chinese medicine,which is derived from the book “Qian Jin Yao Fang” written by Sun Simiao,a famous doctor in the Tang Dynasty.The prescription is composed of six Chinese herbs including Pinellia Ternata,Bamboo Shaver,Fructus Aurantii Immaturus,tangerine Peel,Ginger,Glycyrrhiza.Wendan Decotcion has efficacy of regulating qi and resolving phlegm,and benefiting stomach and gallbladder,which is a main prescription for treating insomnia after serious illness.Modern clinical study has showed that this recipe is mainly used for the treatment of insomnia,depression,schizophrenia,hypertension and so on.At present,the research on Wendan Decoction mainly focus on clinical efficacy and pharmacological action.But,its chemical compound analysis,basic research on pharmacodynamic substances and quality control research are relatively rare.In this thesis,ultra-high performance liquid chromatography plus Q-Exactive Orbitrap tandem mass spectrometry(UPLC-Q-Exactive Orbitrap-MS)was used as the main analytical method to qualitatively identify the chemical components of Wendan Decoction in vitro and the compounds of Wendan Decoction in rat plasma.Meanwhile,Ultrahigh performance liquid chromatography tandem triple quadrupole mass spectrometry(UPLCQQQ-MS)was used to analyze the content of 15 main components of Wendan Decoction and study the pharmacokinetics of 6 bioactivity components of Wendan Decoction in rat plasma,which lay a foundation and provide reference for further elucidating the pharmacodynamic substances of Wendan Decoction.The main research contents include the following parts:1 Analysis of Chemical Constituents in Wendan DecoctionA method based on UPLC-Q-Exactive Orbitrap-MS was established for chemical composition analysis of Wendan Decoction.The precursor ions and fragment ions information of Wendan Decoction compounds were extracted by Xcalibar 3.0 software,which was compared with the reference substance,combining with literature reports and mass database information.A total of 122 chemical components of Wendan Decoction were identified,including 70 flavonoids,18 coumarin,10 saponins,10 alkaloids,3 phenylpropanoids and 11 other compounds.Among these compounds,15 components were confirmed after comparing with the reference substance.UPLC-Q-Exactive Orbitrap-MS technology was used to systematically clarify the basic ingredients of Wendan Decoction,which can lay a foundation for the better research of the pharmacodynamic material basis and the reasonable formulation of quality standards.2 Rapid quantification of 15 chemical constituents in Wendan Decoction based on UPLCQQQ-MSA quantitative analysis method of 15 components of Wendan Decoction based on UPLCQQQ-MS was established by using multiple response monitoring mode(MRM).In which the compounds have high specificity,Great linear relationship(r≥0.999),favorable precision(RSD≤3%),Excellent repeatability(RSD≤3%),Great stability within 24 h at 4 ℃(RSD≤3%)and good accuracy(The recoveries of naringin,narirutin,hesperidin,neohesperidin and glycyrrhizic acid were in the range of 92% to 105%;The recoveries of synephrine,liquiritin,poncirin and eriocitrin were in the range of 90% to 108%;The recoveries of liquiritigenin,tangeretin,isoliquiritin,rhoifolin,and didymin were in the range of 85% to 110%;The recoveries of isoliquiritigenin was in the range of 80% to 115%).The method validation conforms to the standard of Chinese Pharmacopoeia 2020 Editon.This method was used to determine the content of 15 batches of Wendan Decoction.With multivariate statistical analysis,it was concluded that tangeretin,rhoifolin,eriocitrin,naringin,didymin,neohesperidin and narirutin were significantly different,which should be paid attention to in the process of quality control.The method established in this study is accurate,reliable and reproducible,which can provide reference for the quality control of Wendan Decoction.3 Preliminary study on components of Wendan Decoction in rat plasmaThe components of Wendan Decoction in rat plasma were quickly identified by UPLC-QExactive Orbitrap-MS.Xcalibar 3.0 software was used to extract the precursor ions and fragment ions information of Wendan Decoction compounds in rat plasma,and 29 prototype components were identified by comparison with MS fragment of components of Wendan Decoction in vitro.45 metabolites were predicted by Compound Discover 3.0 software and referenced to relevant literature reports,and their metabolic pathways were briefly summarized,which preliminarily clarified the metabolism rules of the main components of Wendan Decoction in vivo.4 Pharmacokinetic study of Wendan Decoction in ratsAn analytical method was established for the determination of hesperidin,liquiritigenin,neohesperidin,naringin,liquiritin and narirutin in rat plasma samples by UPLC-QQQ-MS.This method has high selectivity,geat linear relationship(r≥0.999),favourable intra-day and intraday precisiom(RSD≤15%).Matrix effect of every compound was in the range of 93.14% to113.33%,and the RSD value ≤14.15%.Extraction recovery of each compound was in the range of 84.7% to 99.80%,and the RSD value≤11.92%.And accuracy of each compound was in the range of 91.09% to 112.1%,and the RSD value ≤5.74%.Stability of compound within 24 h at4 ℃ and-80 ℃ repeated freeze-thaw met the requirements.Methodology validation basically in line with the standard of the 2020 edition of the Chinese Pharmacopoeia.The contents of narirutin,liquiritigenin,liquiritin,hesperidin,neohesperidin and naringin in every time of rat plasma were determined by UPLC-QQQ-MS.The data were processed by DAS 3.2.8 software to obtain the blood drug concentration-time curve and the pharmacokinetic parameters of the component,then to clarify the pharmacokinetic behavior of corresponding compounds combine with the result of metabolism in vivo.It provides a reference that maybe clarify the metabolic process and effective mechanism,and lays a basis for Clinical medicine which is conductive to further development and utilization. |
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