Study On Quality Control And Metabolism Of Pharmacodynamic Constituents In Vivo For Apocynum Vetenum L.

Chinese Traditional Herbal Medicine (CTHM) is a typical complex system, and the study pharmacodynamics components in CTHM is one of the key issues of its modernization. In this study,we used modern analytical techniques,established a series of research method for the study of pharmcodynamics components in Apocynum venetum L.(LBM). Identification and determination of multi-component can strictly control the contents of key ingredients in CTHM, and screen the marker components.Combining modern analytical techniques and multivariate statistical analysis techniques can help evaluate the quality of CTHM. Utilizing the modern analysis technologies like HPLC-TOF/MS, GC-MS, HPLC-MS etc. to identify chemical components and further identify relevant components in rat urine, helping us to screen pharmacodynamics components and do mechanism study. Pharmacokinetics study of traditional Chinese medicine get the dynamic change process of major components in the body, and provided perspective for the pharmacodynamics study of CTHM, and provide a good guide for the mechanism study.HPLC-TOF/MS technology was applied for the identification of chemical composition of LBM. The accurate molecular weight measurement of time of flight mass spectrometry helped us to identify a total of21ingredients,16of which were belonged to flavonoids.GC-MS was used for qualitative and identification of volatile oils in LBM. Since electron impact ion source used the standard voltage as70eV, we could obtain highly reproducible mass spectra. Then we got the identification results through the computer which matched with the corresponding database automatically. Finally we identified a total of80volatile oils in LBM. The study provided the basis for more comprehensive understanding of the chemical constituents and further quality control of LBM.LBM from different geographical locations were analyzed and classified via HPLC-TOF/MS and GC-MS combing with multivariate statistical analysis technique for the first time. Principle component analysis (PCA) and partial least squares discriminatory analysis (PLS-DA) showed that these samples could be roughly separated according to their sources, and through the loading plot, we found chemical markers such as chlorogenic acid, hyperoside,isoquercetin,trifolin and some volatile components such as (E)-2-one-3-penten2-ethyl-furan,1-ethylcyclopropanol,benzaldehyde,2-pentyl-furan,which are important for the classification of LBM from different sources. Results of this study indicated that it was an effective and novel approach to identify CTHM from different sources, while performing quantity determination of corresponding marker compounds could optimize the quality control of CTHM.According to the results of identification of LBM ingredients in vitro, we established an HPLC-DAD method for the quantitative determination of four flavonoids including lutin, hyperoside, isoquercetin and quercetin. And the method was methodology validated and its results showed that it could be used for the determination of the four compounds. Then,9batches of LBM were determined. This method was of high sensitivity, good stability, low cost, versatility, could be used for routine quality control of LBM and provided a strong base for further pharmacological studies.Then, an HPLC-TOF/MS method was developed and validated for the identification of the chemical constituents and metabolites of LBM in rat urine.Based on the method,8parent components and34metabolites were screened and identified.30of the metabolites were flavonoids and the main metabolic pathways were methylation,sulfation and glucuronidation.To further study the major components’absorption, metabolism process in the body, we established a HPLC-MS method for the quantitative determination of two flavonoids including hyperoside and isoquercitrin in rat plasma, and pharmacokinetic study was completed. Two components’linear range were of5～200ng-mL-1,5～200ng-mL-1respectively; The low, middle and high concentration levels’absolute recoveries were all between98%and108%; Precision RSD were less than15%; The results of stability experiment also meeted the requirements. Endogenous compounds didn’t obstruct the determination of targeted components; this method was simple and reliable for determination of the components in biological samples. This method had been successfully applied to SD rats who orally administered LBM extract solution as well as hyperoside and isoquercitrin monomer solution. We couldn’t detect hyperoside in20min after oral administration, it demonstrated that hyperoside had a rapid metabolic process and low bioavailability in vivo.For isoquercitrin,we compared its concentration-time curves, and found that the pharmacokinetic parameters (AUC0-t, AUC0-∞,MRT,CL and Vd) of rats who orally administered LBM extract were significantly different from who had isoquercitrin monomer solution (P<0.05). The concentration-time curves of rats who orally administered LBM extract solution and isoquercitrin monomer solution had two peaks,,these results suggested that there exsited transform traction when the compositions were absorbed into blood and there maybe exsited enterohepatic circulation. To control the quality of LBM, it is necessary to determine hyperoside and isoquercitrin at the same time. This study guides the clinical medication and further pharmacological studies.