The Chemical Constituents And Pharmacokinetics Study Of Deng-Zhan-Xi-Xin Injection

Deng-Zhan-Xi-Xin injection?DZXXI?is a sterile aqueous solution prepared by extracting Erigeron breviscapus?Vant.?Hand.-Mazz.,which has the effect of promoting blood circulation,removing blood stasis and relieving pain.DZXXI is a famous traditional Chinese medicine?TCM?prescription for the treatment of cardiovascular and cerebral vessel diseases,such as coronary heart disease,angina pectoris,and ischemic stroke.However,there have been few reports on its chemical constituents and metabolic pathway,which has blocked its further quality control and studies on its pharmacology and mechanism of action.Therefore,it is necessary to establish comprehensive and sensive analytical method to systematically characterize the pharmacodynamic material basis of DZXXI.Our research object was DZXXI in this study.The chemical components in vitro,the prototypes and metabolites in vivo were characterized to elucidate the chemical basis of DZXXI.Besides,the HPLC fingerprint was established for the quality control of DZXXI.And the dynamic changes of multiple components in vivo were elucidated to provide scientific basis for clinical drug monitoring.The research contents are as follows:The main chemical constitutions of DZXXI were characterized by using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry combined with multiple data processing approaches in UNIFI^TMplatform.A chemical component database including 252 compounds was established by searching TCM database and related literature.Then,the chemical component database was imported into the UNIFI^TM platform to match the mass spectra of DZXXI.As a result,a total of 40 compounds were tentatively characterized by summarizing fragmentation patterns for different types of compounds,comparing with reference substance and related literature.The fingerprint of 20 batches of DZXXI was established by using HPLC-DAD technology and the similarity is greater than 0.98 according to“Similarity Evaluation System for Chromatographic Fingerprint of TCM”.Among them,28 common chromatographic peaks were established with scutellarin as the reference peak,and 13common chromatographic peaks were identified by the reference substance.The establishment of the HPLC fingerprint provides a reference for its quality control.On the basis of the chemical composition and quality control of DZXXI,UPLC-Q-TOF-MS^Ecombined with Metasense^TM and UNIFI^TM data processing platform were used to screen and analyze the prototypes and metabolites in vivo.The prototype constituents were the the common peaks between DZXXI and drug biological sample,and the different peaks were metabolites.As a result,25 prototypes were characterized in rat plasma,urine and feces.A total of 45 metabolites were identified,including 18flavonoids and 27 phenolic acids for the first time in DZXXI.The main types of metabolic reactions are methylation,glucuronic acid conjugation,hydrogenation,acetylation,hydrolysis and sulfation.Based on the above studies,the pharmacokinetic behavior of the main components of DZXXI was researched.The DZXXI was intravenously administered to the sham surgery group and middle cerebral artery occlusion?MCAO?model rats by the tail at8.4 m L/kg,and the blood was collected from the orbital venous plexus of the rats at different time points.An UFLC-ESI-MS/MS method was eatablished for the simultaneous determination of 3-caffeoylquinic acid?3-CQA?,5-caffeoylquinic acid?5-CQA?,4-caffeoylquinic acid?4-CQA?,1,3-dicaffeoylquinic acid?1,3-di CQA?,erigeside I,3,4-dicaffeoylquinic acid?3,4-di CQA?,3,5-dicaffeoylquinic acid?3,5-di CQA?,4,5-dicaffeoylquinic acid?4,5-di CQA?,scutellarin and apigenin-7-O-glucuronide in rat plasma.Detection was performed using multiple reaction monitoring?MRM?mode with an ESI source under a negative ionization condition.The lower limits of quantification for 3-CQA,5-CQA,4-CQA,1,3-di CQA,erigeside I,3,4-di CQA,3,5-di CQA,4,5-di CQA,scutellarin and apigenin-7-O-glucuronide were 0.25 ng/m L?0.25 ng/m L?0.25 ng/m L?0.20 ng/m L?0.25 ng/m L?0.17 ng/m L?0.25 ng/m L?0.50 ng/m L?0.25 ng/m L?0.50 ng/m L,respectively.The extraction recoveries of the 10 analytes from rat plasma were more than 78%,and the intra-day and inter-day precision and accuracy deviations were less than 14%.The validated method was successfully applied to a pharmacokinetic study of the two flavonoids and eight phenolic acids in rat plasma after intravenous administration of DZXXI.Compared with the sham operation group,the area under the drug-time curve of caffeoylquinic acid were significantly increased in the model group,and the average residence time of 4-O-caffeoylquinic acid,1,3-O-dicaffeoylquinic acid,3,4-O-dicaffeoylquinic acid and 4,5-O-dicaffeoylquinic acid were significantly delayed.