| Hawthorn leaves comes from Crataegus pinnatifida Bge.Var Major N.E.Br and Crataegus pinnatifida Bge..It has the effect of invigorating blood and dissolving stasis,regulating qi and dredging veins,turbid and lipid-lowering.The extract was extracted from Hawthorn leaves by 50%ethanol and eluted by D101 macroporous resin,which was first included in Chinese Pharmacopoeia 2005.At present,four flavonoids vitexin glucoside,vitexin rhamnoside,vitexin and hyperoside have been shown in the characteristic fingerprints of Hawthorn leaf extract in Chinese Pharmacopoeia,but only the content of vitexin rhamnoside was determined,and the contents of the other three components have not been determined.Flavonoids are the main active components in Hawthorn leaves.The content determination of active components and their blood concentration in vivo are beneficial to the rational use of drugs in clinic and the control of therapeutic effects.In this study,a rapid and accurate HPLC method was established for the content determination of vitexin glucoside,vitexin rhamnoside,vitexin and hyperoside in Hawthorn leaf extract.The method of UPLC-MS/MS for the determination of four flavonoids in biological samples was established to further study the content changes of four flavonoids in rat plasma after intravenous administration of each monomer and the quantitative behavior of the four flavonoids in rat plasma after oral administration of Hawthorn leaf extract.The first part Determination of four flavonoids in Hawthorn leaf extract based on QAMSObjective:A method of QAMS for the determination of four flavonoids vitexin glucoside,vitexin rhamnoside,vitexin and hyperoside in the Hawthorn leaf extract was established to improve the quality standard of extract from Hawthorn leaves.Methods:The chromatographic run with injection volume of 10μL were carried out on a ZORBAX SB-C18 column（250×4.6 mm,5μm）by maintaining oven temperature 30℃.The detection wavelength was set at 350 nm.The mobile phase consisted of acetonitrile,0.6% acetic acid aqueous solution and tetrahydrofuran in gradient elution mode,and the flow rate was 1.0 mL/min.The relative correction factors（RCFs）of vitexin rhamnoside,vitexin and hyperoside were calculated by using vitexin glucoside as internal reference,and the method for simultaneous determination of four components was established.The relative error method was used to compare the results of QAMS method with that of traditional external standard method,and the accuracy of QAMS method was investigated.Results:The Hawthorn leaf extract was separated by HPLC system and each target component was effectively separated（R>1.5）.The methodological investigation met the requirement of quantitative analysis.The relative correction factors of vitexin rhamnoside,vitexin and hyperoside were 0.963,1.116 and 1.034 by using vitexin glucoside as internal reference.The range of RSD for RCF in different chromatographic column,different chromatographic equipment and different laboratory was 0.34%～1.92%.The RCF showed a good durability.The relative retention values of vitexin rhamnoside,vitexin and hyperoside were 0.518,0.864 and 0.957,respectively.The range of RSD for RCF under different chromatographic columns was0.11%～1.89%.The chromatographic peak location was accurate.The RE of the determination results between QAMS method and traditional external standard method was less than 5.0,and the accuracy of the method met the requirements.The average content of vitexin glucoside,vitexin rhamnoside,vitexin and hyperoside in ten batches of Hawthorn leaf extract were 2.5%,12.0%,0.2%and 0.07%,respectively.Conclusion:The method established in this study was simple,accurate and suitable for simultaneous determination of four flavonoids vitexin glucoside,vitexin rhamnoside,vitexin and hyperoside in Hawthorn leaf extract,which can provide a reference for improving the quality standard of Hawthorn leaf extract.At the same time,the content of vitexin rhamnoside met the limit requirement of the Chinese Pharmacopoeia 2015（≥8.8%）,which could provide a basis for further pharmacokinetic study.The second part:Determination of four flavonoids in Rat Plasma after intravenous Administration of each monomer by UPLC-MS/MS and the Pharmacokinetic studyObjective:A UPLC-MS/MS method was established for the determination of vitexin glucoside,vitexin rhamnoside,vitexin and hyperoside in plasma samples.The applicability of the method was verified and the contents of the above four flavonoid compounds after intravenous administration of each monomer were compared.Then the pharmacokinetic characteristics of the four flavonoids were discussed.This part would provide a scientific basis for the development of flavonoids in Hawthorn leaf extract and the design of rational drug delivery scheme.Methods:Twenty-four Wister rats were randomly divided into 4 monosomic intravenous injection groups.The rats were injected with vitexin glucoside of2.5 mg/kg,vitexin rhamnoside of 5 mg/kg,vitexin of 2 mg/kg,hyperoside of 1 mg/kg via tail vein respectively.The blood samples were collected at 24 h before and after administration,and the protein was precipitated with acetonitrile.The run was carried out on Waters ACQUITY-AB SCIEX QTRAP 5500 system maintaining gradient elution mode with acetonitrile and 0.02% formic acid water（containing 2 mmol/L ammonium formate）.The internal standard was baicalin（200 ng/mL）,and the detection mode was multiple reaction monitoring（MRM）.The methodology was validated according to the guiding principles of quantitative analysis of biological samples in Chinese Pharmacopoeia 2015.The data of drug concentration and time were calculated by DAS 3.0 software with the statistical moments of non-atrioventricular model,and the pharmacokinetic parameters of each target component were obtained.Results:There was no obvious interference of endogenous substances near the retention time of the target components and the internal standard in each ion channel,and the specificity of the method was good.The linear range of the components vitexin glucoside,vitexin rhamnoside,vitexin and hyperoside in rat plasma was 10～10000 ng/mL,10～15000 ng/mL,6～6000 ng/mL,4～3600ng/mL,respectively.The RSD of intra-day and inter-day accuracy was less than 13.61%,the RE of accuracy is between-5.70% and 3.79%,and the recovery is between 75.27% and 91.97%.After intravenous administration of vitexin glucoside,vitexin rhamnoside,vitexin and hyperoside in rats,the prototype components were detected at 2 min.The half time(t1/2)of four components was 1.20±0.66 h,1.17±0.41 h,1.59±0.34 h,0.20±0.13 h,respectively.All of them were eliminated rapidly,and the elimination of hyperoside was the fastest.The mean residence time（MRT）of the four flavonoids was 3.73 h,1.45 h,1.36 h,1.25 h,respectively.Vitexin could not be detected after 10 h of blood entry,but the others could still be detected at 24 h.In addition,the apparent volume of distribution（Vz）of hyperin was larger than that of the other three components（4.60 L/kg）and mainly distributed in tissues.Conclusion:The UPLC-MS/MS analysis method established in this part showed strong specificity and high sensitivity,and all of methodology validations met the requirements of biological sample determination.This method was applicable to the determination of four flavonoids vitexin glucoside,vitexin rhamnoside,vitexin and hyperoside in plasma,and has been successfully used to characterize the pharmacokinetic behavior of the four flavonoids after intravenous administration of each monomer.In addition,by comparing the main pharmacokinetic parameters of four flavonoids,it was found that the four flavonoids could be eliminated rapidly in the blood,and the elimination of hyperoside was the fastest.The third part:Simultaneous determination of four flavonoids in plasma of rats after oral administration of Hawthorn leaf extract by UPLC-MS/MS and the Pharmacokinetic studyObjective:A UPLC-MS/MS method for simultaneous determination of vitexin glucoside,vitexin rhamnoside,vitexin and hyperoside in plasma samples was established to compare the quantitative behavior of the four flavonoids in Hawthorn leaf extract after oral administration in rats,as well as to study the pharmacokinetic characteristics of the four flavonoids.This part would be helpful to evaluate the blood absorption of the active components of Hawthorn leaf extract and to explain the mechanism of its action.Methods:Six Wister rats were given Hawthorn leaf extract orally at a dose of 1g/kg.The blood samples were collected at 24 h before and after administration,and the pretreatment process was as follows:“acetonitrile precipitated protein-nitrogen blowing（50℃）-initial mobile phase resolution”.The run was carried out on Waters ACQUITY-AB SCIEX QTRAP 5500 system maintaining gradient elution mode with acetonitrile and 0.02% formic acid water（containing 2 mmol/L ammonium formate）.The internal standard was baicalin（100 ng/mL）,and the detection mode was MRM.The methodology was validated according to the guiding principles of quantitative analysis of biological samples in Chinese Pharmacopoeia 2015.The data of drug concentration and time were calculated by DAS 3.0 software with the statistical moments of non-atrioventricular model,and the pharmacokinetic parameters of each target component were obtained.Results:The linear correlation coefficient of vitexin glucoside,vitexin rhamnoside,vitexin and hyperoside was between 0.9939 and 0.9987.The RSD of intra-day and inter-day accuracy was≤11.73%.The RE of accuracy was between-3.40%and 2.29%.The recovery was≥76.40%.The average of matrix effect was between 96.98% and 110.05%.And the RSD range of stability was 0.97%～13.76%.All specificity,linearity,precision,stability,recovery and matrix effect of the four flavonoids in rat plasma met the requirements of quantitative analysis of biological samples.After oral administrationat of Hawthorn leaf extract in rats,the pharmacokinetic characteristics of vitexin glucoside and vitexin rhamnoside were similar.The maximal blood concentration(Cmax)was obtained at 1.33 h and 1.21 h,t1/2 was about 2.8 h,and clearance（CLz）was 13.47 and 15.20L/h/kg.The Cmax of vitexin was obtained at 0.49±0.15 h,t1/2 was 4.45±2.02 h,Vz was 219.71 L/kg.The t1/2 and Vz of vitexin were larger than those of the other three flavonoids,and it was eliminated slowly and distributed mainly in tissues.The Cmax of hyperoside was obtained at 0.51±0.13 h,t1/2 was 0.89±0.15 h,and CLz was 62.66 L/h/kg.Hyperoside was rapidly absorbed into blood,and the speed and extent of elimination was greater.Vitexin glucoside,vitexin rhamnoside and vitexin were absorbed into the blood for the second time at 12 h,and there may be hepatic and intestinal circulation.Conclusion:On the basis of the UPLC-MS/MS method established in the second part,the pretreatment method of plasma sample was optimized successfully,and the response to the components vitexin glucoside,vitexin rhamnoside,vitexin and hyperoside was enhanced.The method was successfully applied to the determination of four flavonoids after oral administration of Hawthorn leaf extract.After the rats were given Hawthorn leaf extract orally,the pharmacokinetic characteristics of vitexin glucoside and vitexin rhamnoside were similar.Vitexin was eliminated slowly than the other three flavonoids and was distributed mainly in tissues.Hyperoside can be rapidly absorbed into blood and be eliminated quickly. |
