| ObjectiveThis study is the preliminary of the pharmacokinetics model and PK-PD research of Sini Decoction.Based on the research of "HPLC analysis method of Sini Decoction and the establishment of the fingerprint" carried out by our group,the feasibility of applying internal standard microdialysis method to collecting samples of components in Sini Decoction was investigated.The high performance liquid chromatography(HPLC)and liquid chromatography coupled with mass spectrometry(UPLC-TOF-MS/MS)were used to analyse the components in microdialysis samples of blood and heart.This research was to provide a reference of analysing the dynamic changes of the multi-constituents in Sini Decoction in vivo with internal standard microdialysis method,and to provide an evidence of the basic pharmacodynamics research of Sini Decoction.Method1.Establishment of a HPLC analysis method and a fingerprint of Sini DecoctionEstablish a HPLC method for determination of liquiritin,benzoylmesaconine,and 6-gingerol in Sini Decoction.Specificity,standard curve,the precision,stability,limit of quantitation and detection were confirmed.Build the fingerprint of Sini Decoction.2.Screening the best perfusate modifier for microdialysis20%,30%,40%ethanol,0.5%,1.0%,2.0%glycerol,5%,10%,15%β-cyclodextrin(β-CD)were used as perfusate modifiers.Benzoylmesaconine and lappaconitine hydrobromide were used as index components.The probe recovery rates at flow rates of 0.5,1.0,1.5 and 2.0 μL/min were investigated,respectively.Screen the best perfusate modifier which has a promoting and stable effect on the recovery rate.3.Feasibility of the method of internal standard microdialysis for Sini DecoctionThe liquiritin,benzoylmesaconine,and 6-gingerol in Sini Decoction were used as the index components,and lappaconitine hydrobromide was used as the internal standard.Four flow rates(0.5,1.0,1.5,2.0 μL/min),concentrations(C1,C2,C3,C4)and temperatures(30℃,33℃,37℃,40℃)were used as different conditions on investigating the recovery rate and P value of index components in blood probe and linear probe.The stability of P’value was verified in vitro and in vivo within 10 h.4.Analysis of constituents in Sini Decoction in blood and heart of the ratsThe microdialysis samples of blood and heart in the rats within 10 h were collected after the administration of Sini Decoction.The constituents and dynamic changes of Sini decoction in rats were analyzed by UPLC/Q-TOF-MS/MS,and the differences of the components in blood and heart were also analyzed.The possible functional subcetances in Sini Decoction that take effect were analyzed.Results1.HPLC method and fingerprint of Sini DecoctionHPLC conditions were as follows:Shimadzu LC-20A high performance liquid chromatography;Kromasil 100-5-C18 chromatographic column(4.6 mm × 150 mm,5μm);mobile phase:acetonitrile-0.1%phosphoric acid;flow rate:0.8 mL/min;detection wavelength:230 nm;column temperature:30℃;elution gradient:0.01-18 min,95%-81%(A);18-42 min,81%-78%(A);42-52 min,78%-65%(A);52-80 min,65%-35%(A);80-85 min,35%-35%(A);A is 0.1%phosphoric acid water.Fingerprint of Sini Decoction was established and 24 common peaks were found.The stability and repeatability of this method were satisfied.2.30%ethanol can be used as the best perfusate modifierWith 20%,30%,40%ethanol,0.5%,1.0%,2.0%glycerol,5%,10%,15%β-CD as perfusate modifiers,recovery rates of benzoylmesaconitine and the internal standard lappaconitine hydrobromide were determined,respectively.The recovery rates of the two components were enhanced by the 9 modifiers,and 30%ethanol possessed the most significant and stable promotion effect.3.Feasibility of internal standard microdialysis method for collecting Sini Decoction in vitro and in vivoThe modified Ringer’s solution containing lappaconitine hydrobromide as the internal standard was used as the perfusate.The flow rate was 1.0 μL/min,the collection interval was 30 min,and each sample was 30 μL.At the flow rate of 0.5、1.0、1.5、2.0μL/min,in blood probe,the determination by incremental method and the subtraction method of recovery rates of liquiritin were 86.71%、94.39%,61.48%、64.21%,44.20%、42.95%,27.08%、32.42%;of benzoylmesaconine were 80.24%、86.45%,62.02%、52.65%,42.51%、37.66%,25.90%、29.30%;of 6-gingerol were 77.00%、79.75%,70.31%、78.22%,62.61%、58.54%,44.55%、43.23%;of lappaconitine hydrobromide were 87.50%、85.30%,71.53%、63.77%,45.16%、40.15%,31.04%、26.32%.At the flow rate of 0.5、1.0、1.5、2.0 μL/min,in linear probe,the determination by incremental method and the subtraction method of recovery rates of liquiritin were 87.19%、77.22%,71.50%、61.31%,51.62%、42.55%,43.37%、33.24%;of benzoylmesaconine were 71.80%、60.12%,66.10%、55.86%,51.86%、40.47%,41.71%、33.17%;of 6-gingerol were 86.95%、74.80%,73.56%、67.22%,52.03%、42.38%,41.51%、31.33%;of lappaconitine hydrobromide were 62.27%、56.79%,42.71%、53.51%,30.10%、37.91%,25.44%、30.36%.The influence of the flow rate,temperature and concentration on the P(P’)value was within the stable range.The P’value of the probes maintained steadily within 10 h in vitro and in vivo.4.Analysis of componennts of Sini Decoction in blood and heart of the rats38 compounds were identified by UPLC-TOF-MS/MS in the dialysate of Sini decoction.Within 10 hours,25 original constituents of Sini decoction were identified in the blood;21 original components were identified in the heart.The constituents showed different change regulation with time in blood and heart.Conclus ion1.Under the established chromatographic conditions,the main component peaks are well separated,the peak shape is symmetrical,and the linear range,precision and stability are in line with the experimental requirements.2.30%ethanol can be used as the best perfusate modifier in this study to improve the recovery rate of lipophilic components in Sini Decoction.3.It is feasible to carry out microdialysis method to collect the constituents in Sini Decoction with lappaconitine hydrobromide as internal standard.4.The constituents in Sini decoction showed different changes regulation with time in rats.It is feasible and suitable to use microdialysis to analyze the dynamic changes of constituents in Sini decoction... |
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