The Pharmacodynamic Material Basis And Pharmacokinetic Study Of Jitai Tablets

Jitai tablets (JTT), an important TCM prescription in treating opiate addiction sinceQing dynasty, has been proved to be very safe and effective in the inhibition of protractedwith-drawal symptoms with less harmful side effects, which have been already approvedfor the treatment of opiate addiction by the Chinese State Food and Drug Administration(SFDA). JTT is prepared from fifteen medicinal materials, including Rhizoma Corydalis,Radix Salviae Miltiorrhiae, Radix Angelicae sinensis, Ligusticum Chuanxiong, SemenPersicae, Flos Carthami, Radix Aconite, Radix Ginseng, Cortex Cinnamomi, RhizomaZingiberis, Semen Myristicae, Flos Daturae, RadixAucklandiae, Lignum AquilariaeResinatrm and Margarita. A great deal of advantages have been carried out by JTT foropiate detoxification, including less harmful and side effects, high safety and satisfiedeffects in the inhibition of protracted withdrawal symptoms, and effective in therehabilitation of abnormal body functions induced by chronic drug use and good for therehabilitation of abnormal body functions induced by chronic drug use.Even with so many beneficial effects, the substantial foundation and pharmacologicalmechanism of JTT are still unclear for hundreds of various chemical constituents from thecomprised drugs. And no report of the pharmacokinetic (PK) study of JTT could be foundin literature survey, for not only the complexity of the formula, but also the traceconcentration of the comprised chemical constituents in vivo. Hence, it is imperative toestablish sensitive and comprehensive analytical methods to acquire a better understandingof the material basis and further to elucidate the dynamic changes in vivo.In the present study, JTT was chosen as the research object. Take advantage of variousinnovation theory and advanced technology, the main chemical components of JTT and themultiple absorbed bioactive components and metabolites in rat plasma after oraladministration of JTT were screened and anlyzed. Besides of that, the bioactivecomponents and metabolites in rat CSF after oral administration of JTT were identified andquantified. Finally, the pharmacokinetic study of the bioactive components in rat plasmaafter oral administration of JTT was successfully clarified. The specific contents are asfollows:A universally applicable high-performance liquid chromatography/diode-arraydetection (HPLC/DAD) coupled with electrospray tandem mass spectrometry (HPLC/ESI-MS/MS) method was developed for carrying out the comprehensivecharacterization of JTT. Based on the ESI-MSnfragmentation patterns of the referencestandards, a total of101components were identified or tentatively characterized bycomparing their retention times, UV and MS spectra with those of reference standards orthrough the matching of empirical information with those of published components in thein-house library. The characteristic fragmentation pattern of alkaloids, phenolic acids,tanshinones, flavonoid glycosides, cyanogenic glycosides, ginsenosides,2-(2-phenylethyl)chromones, phthalides and gingerol-related compounds were tentatively elucidated usingstructurally-relevant product ions. It was observed that neutral losses of C9H10O3andC9H8O2were the characteristic product ions of scopola alkaloids. Neutral fragmentmandelonitrile was the characteristic ion of cyanogenic glycosides. To our knowledge,tropylium ion and C4H2O unit were the characteristic ions of2-(2-phenylethyl) chromone,which resulted from the Retro-Diels-Alder (RDA) cleavage of the C ring. The resultsindicated that the developed analysis method could be employed as a rapid, effectivetechnique for structural characterization of chemical constituents in TCM. This work isexpected to provide comprehensive information for the quality evaluation andpharmacokinetic studies of JTT.Based on the serum pharmacochemistry technique and HPLC-DAD/ESI-MS/MS, amethod for screening and analysis of the multiple absorbed bioactive components andmetabolites of JTT in orally dosed rat plasma was developed. Plasma was treated bymethanol precipitation prior to HPLC, and the separation was carried out on a SymmetryC18column, with a linear gradient (0.1%formic acid/water/acetonitrile). Mass spectrawere acquired in negative and positive ion modes, respectively. As a result,26bioactivecomponents originated from JTT and5metabolites were tentatively identified in orallydosed rat plasma by comparing their retention time and MS spectra with those of authenticstandards and literature data. It is concluded that an effective and reliable analyticalmethod was set up for screening the bioactive components of Chinese herbal medicine,which provided a meaningful basis for further pharmacology and active mechanismresearch of JTT.Take advantage of the combination of liquid chromatography tandem of time-of-flightmass spectrometry (LC-Q-TOF-MS/MS) and liquid chromatography tandam of triplequaduapole mass spectrometry (LC-QqQ-MS) technique, a method for fast quantification and quantitation of the multiple bioactive components and metabolites of JTT in orallydosed rat cerebrospinal fluid (CSF) was developed. CSF was treated by methanolprecipitation prior to liquid chromatography, and the separation was carried out on a HSST3column, with a linear gradient (0.1%formic acid/water/acetonitrile). Mass spectra wereacquired in both full scan mode and targeted MS/MS mode. By comparing the retentiontime and the accurate mass and the MS/MS spectrum of blank CSF, dosed CSF and JTT,11prototype components and3metabolites were tentatively identified. In order to confirm theidentification results, the6target components in rat CSF were further quantitativelydetermined by the LC-QqQ-MS. This experiment provides a rapid method of highsensitivity for the detection of trace component, provides a strategy to study the materialbasis for the efficacy of TCM and will provide some reference for the further study ofmechanism of drug action.A specific and reliable LC-ESI-MS/MS method was developed for the simultaneousdetermination of tetrahydropalmatine (THP), tetrahydroberineper (THB),tetrahydrocoptisine (THC) and corydaline (CDL) in rat plasma using jatrorrhizine asinternal standard. Plasma samples were pretreated by protein precipitation with acetonitrile.Chromatographic separation was performed on a Zorbax Eclipse Plus C18column (3.0mm×100mm,1.8μm) with gradient elution using acetonitrile–0.1%formic acid water asmobile phase at a flow rate of0.3mL/min. The detection was carried out by atriple–quadrupole tandem mass spectrometer in multiple-reaction monitoring mode. Themass transition ion-pair was followed as m/z35â†'6.1292.1, m/z324.1â†'176.0, m/z340.1â†'176.0, and m/z370.3â†'192.1for THP, THC, THB and CDL. Linear calibrationcurves were obtained over the range of0.2–2000ng/mL, and the method limits were2.0ng/mL for THC,0.2ng/mL for THB,1.0ng/mL for THP, and0.4ng/mL for CDL,respectively. The mean recovery of the analytes ranged from85.51%to93.99%. The intra-and inter-day precisions were in the range of1.21–4.73%and the accuracies were between92.23%and98.23%. The validated method was successfully applied to a pharmacokineticstudy of THP, THB, THC and CDL in rat plasma following oral administration of Jitaitablets.A LC-ESI-MS/MS method was developed and validated for the simultaneousdetermination of amygdalin (ADL), danshensu (DSS), ferulic acid (FA), hydroxysaffloryellow A (HSYA), salvianolic acid A (SAA) and salvianolic acid B (SAB) in rat plasma. Plasma samples were pretreated by protein precipitation with acetonitrile. LC separationwas performed on a Zorbax Eclipse Plus C18column (3.0mm×100mm I.D,1.8μm)with gradient elu-tion using a mobile phase consisting of acetonitrile-0.1%formic acid inwater at a flow rate of0.3mL/min. ESI-MS spectra was acquired in negative ion multiplereaction monitoring mode. The mass transition ion-pair was followed as m/z456.â†'0323.1, m/z197.3â†'178.8, m/z193.0â†'133.9, m/z611.1â†'325.2, m/z493.0â†'295.0,and m/z717.0â†'519.0for ADL, DSS, FA, HSYA, SAA and SAB, respectively. Allana-lytes showed good linearity over a wide concentration range (r>0.99). The lower limitof quantification was7ng/mL,2ng/mL,4ng/mL,1ng/mL,2ng/mL, and4ng/mL forADL, DSS, FA, HSYA, SAA and SAB, respectively. The mean recovery of the analytesranged from86.29%to93.16%. The intra-and inter-day precisions were in the range of1.50–9.98%and the accuracies were between91.17%and99.46%. The validated methodwas successfully applied to a pharmacokinetic study of the six hydrophilic components inrat plasma after oral administration of Jitai tablets.