Study On Quality Standard Improvement And Pharmacokinetics Of Dangefentong Capsules

Objective:（1）To improve the quality standard of Dangefentong Capsules,its HPLC fingerprints was established,and the contents of nine components were simultaneously determined.（2）To investigate the prototype components and metabolites of Dangefentong extract in rats plasma by UPLC-MS/MS,so as to provide reference for the basic research of pharmacokinetics and the material basis of pharmacodynamics in vivo.（3）To establish a method for the concentration analysis of eight main components of the Dangefentong extract in rat plasma,to conduct pharmacokinetic studies,to reveal the compatibility mechanism of Danshen-Gegen drug pair with its main pharmacokinetic parameters,so as to provide reference for clinical administration.Methods:（1）The HPLC fingerprints of Dangefentong Capsules of ten batches were performed on an Eclipse XDB-C₁₈column（4.6mm×250 mm,5μm）,and the mobile phase comprised of 0.05%formic acid（A）and acetonitrile（B）with the gradient elution at a flow rate of 1.0m L·min^-1.The detection wavelength was set at 280 nm;the column temperature was maintained at 30℃and the injection volume was 10μL.Similarity Evaluation System for Chromatographic Fingerprint of traditional Chinese medicine（2012 edition）was used to evaluate the fingerprints.At the same time,the contents of nine active components were determined.（2）Rat plasma was collected by single gavage of Dangefentong extract for 30 min.The analysis was carried out on a Agilent Eclipse Plus C₁₈（2.1 mm×50 mm,1.8μm）column,with the mobile phase of 0.05%formic acid solution（A）-acetonitrile（B）at a flowing rate of 0.2 m L·min^-1,the column temperature was 35℃.The prototypes components and metabolites in plasma samples were analyzed by Electrospray Ionization source in both positive and negative ion mode.（3）Rats were given Gegen extract,Danshen extract and Dangefentong extract by single gavage,plasma samples of different time were collected.The chromatographic separation was performed on a Eclipse C₁₈（2.1 mm×50 mm,1.8μm）column using 0.1%formic acid-0.1%formic acid acetonitrile as mobile phase for gradient elution.A triple-quadrupole tandem mass spectrometry equipped with an Electrospray Ionization source was used as detector operating on multiple-reaction monitoring scanning in positive or negative ion mode in different time periods.All pharmacokinetic parameters were processed by non-compartmental analysis with DAS 3.2.2 software.Results:（1）The HPLC fingerprint of ten batches of Dangefentong Capsules was established,the similarities of which were more than 0.99.Twenty-eight common peaks were selected in the fingerprints of ten batches of samples,Sixteen components were identified by comparison with the reference substances.The linearity,precision,stability,repeatability,and recovery were all in line with quantitative requirements.The content of 3′-hydroxy puerarin,puerarin,3′-methoxy puerarin,puerarin celery glycoside,daidzin,salvianolic acid B,cryptotanshinone,tanshinone I and tanshinone II_Ain ten batches of samples were in the range of 9.01-12.44,53.98-65.39,13.89-15.51,13.24-19.43,14.15-18.44,58.24-75.68,2.03-2.63,1.09-1.45,2.93-3.72 mg/capsule,respectively.（2）Based on the retention time,ion fragmentation information of the blank biological samples,herb extract,and reference compounds,a total of thirty-three components,including thirteen prototype components and twenty-one metabolites mainly through metabolic pathways of hydroxylation,methylation,glucuronide conjugation,and sulfate conjugation,etc.（3）The results showed that the linear correlation coefficient of the eight components were all greater than 0.99,indicating that the method had good linearity in their respective concentration ranges.Post-preparative stability（25℃,12 h）,short-term stability（25℃,4 h）,long-term stability（-80℃,10d）,and freeze and thaw stability（3-cycles）of the eight constituents were examined to evaluate the stability of methodology.The results of the inner and inter-day relative standard deviations were both less than 11%,indicating legitimate precise and accuracy to the requirement of biological sample analysis.The assay method is proved to be sensitive,accurate and convenient.It can be applied to the pharmacokinetic study of the eight analytes.The main pharmacokinetic parameters of Pueraria flavonoids and tanshinone in plasma of rats were significantly different after intragastric administration of Dangefentong extract.After the administration of Dangefentong extract,the AUC and C_maxresults of five constituents of Pueraria isoflavones and two constituents Tanshinone measured in Dangefentong extract were increased evidently than Danshen extract and Gegen extract.There was no significant difference in pharmacokinetic parameters of salvianolic acid B.（P>0.05）.Conclusion:（1）The combination methods of HPLC fingerprint and simultaneous determinations of multiple components is feasible,simple and reproducible,which can improve the quality standard of Dangefentong capsule.（2）This experimental method is simple and reliable,which can provide a analytical method for the pharmacokinetics and the material basis of pharmacodynamics of Dangefentong extract.（3）The compatibility of effective components of Dangefentong has obvious advantages and characteristics,which can significantly improve the blood concentration of flavonoids and tanshinone components in Dangefentong extract.The results of this study not only provided a scientific explanation for the theory of drug compatibility of Danshen-Gegen drug pair,but also provided a reference for the clinical research and application of Dangefentong capsule.