Study On Pharmacodynamics And Pharmacokinetics Of Jian Pi Decoction From Zhuyun Pill

ObjectiveTo clarify the chemical composition and source of Jian Pi decoction from Zhuyun Pill quickly and comprehensively with ultra-performance liquid chromatography with quadrupole-time-of-flight mass spectrometry(UPLC/Q-TOF-MSE).The results could be used for the quality control of Jian Pi decoction.To investigate the pharmacokinetic study of lobetyolin and interaction between Chinese herb and decoction.To provide a necessary theoretical basis for clini-cal application between Chinese herbs and help to further development Jian Pi decoction.Culture the decidual cells primarily and observe the effect and acting mechanisms of the active ingredients of Jian Pi decoction on decidual cells damage model.Methods1.The genuine medicinal materials were purchased and identified.The Jian Pi decoction was prepared using the reflux condensation.Using UPLC/Q-TOF-MSE technology,the chemical composition of Jian Pi decoction were analyzed in the positive and negative ions full scan test patterns.To identificate the chemicals with standard,the retention time,accurate mass number,isotope abundance and multi-stage mass spectrogram,mass spectrometry identification cracking rulestandard from the decoction was compared with the standards by Masslynx 4.1 software.The anthers were identificated not only using the method mentioned above but also according to references.The paper classified the main chromatographic peaks too.2.Lobetyolin is one of active ingredients from the Jian Pi decoction.To determine contents of Lobetyolin in Codonopsis pilosula extraction and Jian Pi decoction,The appropriate UPLC/Q-TOF-MS method was esestablished.3.On the basis of content of lobetyolin,the rats were oraly administrated lobetyolin?Codonopsis pilosula extraction and Jian Pi decoction.24 male SD rats were randomly divided into 3 groups as mentioned above with 8 for each group.Serum of rats was got after oraly administrated drugs 5min?15min?30min?45min?60min?90min?120min?240min?480min?720min?1440 min.Liquid-liquid extraction(LLE)for the sample pretreatment was used.To establish a UPLC/Q-TOF-MS/MS method to determine contents of lobetyolin at different time.Then drug-time curve was drawn.A seriesof pharmacokinetics parameters(such as Cmax?Tmax?AUC?MRT?CL)we obtained by the functional pharmacokinetics program PK.Solutions 2.0.Compare the differences between them.4.The decidual tissues were digested by 0.25%trypsin and 0.1%collagenase?,purified by differential adhesion method and proliferation and passage in vitro.The primary culture cells were identificaed by inverted microscope?electron microscope and immunocyto-chemistry.5.Esestablish decidual cells damage model induced by mifepristone and observe the effect and acting mechanisms of the active ingredients of Jian Pi decoction on it.Compare the differences between the normal decidual cells and the decidual cells damage model by proliferation?apoptosis and apoptotic proteins expression of Bax?Bcl-2?Caspase-3?Caspase-9.We can get active ingredients from the Jian Pi decoction.Results:1.The genuine medicinal materials were identified completely.Establish an ultra high performance liquid chromatography coupled with tandem quadrupole time-of-flight mass spectrometer detection(UPLC/Q-TOF-MS/MS)comprehensive quickly clarify chemical of Jian Pi decoction.Based on the UPLC/Q-TOF-MSE analysis,37 peaks were identified on line.By using the known standard 7 representative compounds were identified.The herbal sources of these peaks were assigned.The results implied that flavonoids,saponins,lactones,and glycosides were the main components in effective part of Jian Pi decoction.2.Develop a special UPLC/Q-TOF-MS for rapid quantitative analysis of Lobetyolin in Jian Pi decoction.We had good linearity(r>0.999)with in the detection range(5-5000ng/mL),The precision(RSD),accuracy,sensitivity proved the advantage of this new method.The contents of Lobetyolin in Codonopsis pilosula extraction and Jian Pi decoction are 239.5?g/ml and 87?g/ml.3.The Lobetyolin had good linearity(r>0.99)within the detection range(5-5000ng/ml),the inter and intra day precision were good(RSD<6.0%,RSD<5.0%),and the average recovery rate reaches 70.0%,and the average recovery rate of IS reaches 70.0%?The pharmacokinetic parameters were calculated by PK.Solutions 2.0 software in the three groups were:Cmax(ng/mL):138.59 ±28.65?127.61±16.88?172.26± 94.71;Tmax(hr):172.26±94.71?1.57±0.61?1.20±0.47;AUC(Area ng-hr/mL):176.61 ± 20.59?551.07±83.26?737.57±127.97;MRT(area hr):2.27±1.80?4.46±1.86?5.37±1.94;CL(mL/hr):6791.79±1785.45?2023.76±311.44?1559.73 ± 334.67.4.The primary culture cells were identificaed to be decidual cells by inverted microscope?electron microscope and immunocytochemistry(Keratin is positive and vimentin is negative).The purity of decidual cells could reach 99%with PRL.5.We established decidual cells damage model successfully induced by 60?mol/L mifepristone.Lobetyolin Astragaloside ??Calycosin?Calycosin-7-O-?-glucosidehas?Formononetin?ononin?Atractylenolide I and Jian Pi decoction has the proliferation induction and apoptosis inhibition effect on decidual cells damage model.Conclusion1.The method established is simple and rapid for elucidation the constitunts of Jian Pi decoction and the results could be used for the quality control of Jian Pi decoction.2.The decidual cells damage model induced by RU-486 could be use as carries for screening out active ingredients from Jian Pi decoction.The active ingredients may effect decidual cells damage model on proliferation induction and apoptosis inhibition.3.Establish an accurate UPLC/Q-TOF-MS method for rapidly determining the content of Lobetyolin in Codonopsis pilosula and Jian Pi decoction.And this method could be used for rearch pharmacokinetic of Lobetyolin.4.Somethings in Codonopsis pilosula and Jian Pi decoction are good for absorpting substances Lobetyolin and increase the length of time it remains in bodies.