| Xiao-Qing-Long-Tang(XQLT),was firstly recorded in the Treatise on Febrile Disease,written by Zhang Zhongjing,and is a classical prescription to treat many diseases,such as common cold,bronchial asthma,and allergic rhinitis.Modern pharmacological research has proved that XQLT possesses the effects of anti-inflammation,anti-allergy,and anti-asthma.In contemporary clinical practices,it is effective for the treatment of influenza,bronchitis,asthma.In the present study,the chemical constituents of XQLT and the in vivo and in vitro metabolism of its major components in rats were systematically investigated by using LC-MS method,which would elucidate the interactiona mechanism of XQLT,as well as provide scientific foundation for its clinical application.The investigations were as follows.1.Established a comprehensive chemical profiling of 52 marker compounds by using UHPLC-Q/TOF-MS method with an analysis run of 14.5 min,which is validated for the rapid determination of 16 components in XQLT commercial products.Among them,the quantification of asarinin,2-methoxy-4,5-methylenedioxybenzene acetone,6-gingerol,6-shogoal,albiflorin,gallic acid,schisandrin A,adenosine,phenylalanine,and isoliquiritin in XQLT commercial products is reported for the first time.The method is practical for comprehensive standardization of XQLT and holistic comparison of its commercial products from different manufacturers.2.An UHPLC/Q-TOF MS technology was developed for the identification of 12 prototype and 15 related metabolites in rat plasma and urine after oral administration of XQLT(6g*kg-1),by direct comparison of the retention time,UV spectrum with those of authentic sample or analysis fragmentation regularities of mass spectra.Among them,six ephedrine related alkaloids were derived from Ephedrae Herba,two monoterpene glycosides were derived from Radix Paeoniae Alba,two triterpenoids and eight flavonoids were derived from Clycyrrhyzae Radix et Rhizoma Praeparata Cum Melle,nine lignans were derived from Schisandrae Chinensis Fructus.Hydrolysis,demethylation,epoxidation,glucuronidation were the major metabolic pathways of the components in XQLT in vivo.This study clarified the bioactive components of XQLT and provided experimental basis for pharmacokinetic study in the future.3.An UPLC-MS/MS method was established for the first time to quantitative four prototypes ephedrine,methylephedrine,paeoniflorin,albiflorin and three metabolites liquiritigenin-7-O-glucuronide,isoliquiritigenin-7-O-glucuronide,glycyrhizin in rat plasma.Linear calibration curves of ephedrine,methylephedrine,paeoniflorin,albiflorin,liquiritigenin-7-O-glucuronide,isoliquiritigenin-7-O-glucuronide and glycyrhizin were obtained in the concentration range of 0.50～250.0,0.50～125.0,1.00～500.0,1.00～500.0,0.50～125.0,0.50～125.0 and 0.50～125.0 nM,,respectively.The lower limits of quantitation(LLOQ)were 0.50,0.50,1.00,1.00,0.50,0.50 and 0.50 nM,respectively.RSD was found to be less than 15%for both inter-day and intra-day precision assay.RE ranged from-10.4%to 11.2%for the accuracy assay.The extraction recovery of seven analytes,as well as formononetin were ranged from 93.4%to 111.4%.Seven analytes shown stable when kept at-20℃ for 20 days,during three freeze/thaw cycles,at room temperature for 24 h,and at room temperature for 24 h in sample manager,respectively.The pharmacokinetics parameters of seven analytes were investigated in rats by the UPLC-MS/MS method.The data obtained were fitted with a non-compartment model and validated by WinNonlin 6.3(Mountain View,CA,USA).The pharmacokinetic parameters for ephedrine,methylephedrine,paeoniflorin,albiflorin,liquiritigenin-7-O-glucuronide,isoliquiritigenin-7-O-glucuronide and glycyrhizin were summarized as follows:Tmax,26.25± 7.50,30.00 ± 0.00,150.00 ± 34.64,150.00 ± 77.46,348.00±107.33,330.00 ± 151.00 and 528.00 ± 107.33min.AUC0-t,2732.66 ± 97.17,2504.64 ± 266.92,13831.60 ± 1541.06,5484.51 ± 1120.77,88374.13 ± 4640.52,12701.46 ± 236.26 and 81183.46± 38211.92 nmol*min.Furthermore,we performed a comparative pharmacokinetics study that employs Wistar rat models to characterize the absorption and metabolism characteristics of seven actives compounds in XQLT with oral administration of XQLT and mixture if ephedrine,liquiritin,paeoniflorin and glycyrrhizic acid.The results indicated that there are significant differences in pharmacokinetic characteristics of ephedrine,paeoniflorin,liquiritigenin-7-O-glucuronide and glycyrrhizin between the two groups.The Cmax and AUC0-t of ephedrine in XQLT group were lower than those in mixture standards group,while the Cmax and AUC0-t of paeoniflorin,liquiritigenin-7-O-glucuronide and glycyrrhizin in XQLT group were higher than those in mixture standards group.4.Based on the Caco-2 cell monolayer model,an UPLC-MS/MS method was established to investigate the absorption and metabolism of the major components ephedrine,pseudoephedrine,methylephedrine,paeoniflorin,albiflorin,liquiritin,isoliquiritin,liquiritigenin,isoliquiritigenin,glycyrrhizic acid and glycyhhizin in XQLT.The result showed that,among all the analytes,ephedrine,pseudoephedrine and methylephedrine had the highest permeability,all the glycosides had a bad absorption with the permeability lower than 10-6.With the appearance of XQLT,the permeability of paeoniflorin,albiflorin,liquiritin,isoliquiritin,liquiritigenin,isoliquiritigenin,glycyrrhizic acid and glycyhhizin increased and the permeability of ephedrine,pseudoephedrine and methylephedrine decreased.XQLT had the same effect by decreasing the efflux ratios of liquiritin and isoliquiritin as the appearance of the MRP2 transporter inhibitor MK571,which indicated that XQLT can decrease the efflux ratios of liquiritin and isoliquiritin by inhibiting the MRP2 transporter.5.An UPLC-MS/MS method was established to determine the hydrolysis fates of different glycosides in XQLT in small intestinal S9 and fecal S9.The result showed that the main glycosides including liquiritin,isoliquiritin,paeoniflorin,albiflorin and glycyrrhizic acid were mainly degraded by the intestinal flora.Based on LC-MS technology,the present study focuses on the chemical profiles,metabolism and absorption mechanism of the components in XQLT,the results illustrated the mechanism and characters of compatibility of XQLT,which can provide scientific evidences for the safety use of XQLT in clinic. |
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