Pharmacokinetic Study On Effective Part Of Caulophyllum Robustum Maxim By UPLC-MS/MS

Chinese medicine treatment of Rheumatoid arthritis(RA)has a long history,of which Caulophyllum robustum Maxim is one of the representative drugs,but the effective parts in the body change rule is not yet clear.With the development of modern technology,the study found that Caulophyllum robustum Maxim has good clinical efficacy for treatment of RA.The UPLC-MS/MS in vitro research methods of four kinds of component under test(magnoflorine,cauloside C,hederagenin and oleanolic acid)of Caulophyllum robustum Maxim were established in this topic,and it was successfully applied to the effective part of Caulophyllum robustum Maxim extract in vivo pharmacokinetic study,which lays a foundation for further study of Caulophyllum robustum Maxim.Main research contents and results in the paper:1.A content determination method was developed and verified for 4 kinds of component of Caulophyllum robustum Maxim effective part in rat plasmaThe method of content determination of 4 kinds of component in Caulophyllum robustum Maxim in rat plasma was established by ultra-performance liquid chromatography-tandem mass spectrometry technology(UPLC-MS/MS).Chromatographic separation was achieved on ACQUITY HSS T3 column(50 mm x 2.1 mm i.d.,2.1 ?m)by 90%acetonitrile-water isocratic elution at a flow rate of 0.2 mL/min in only 4 min.The retention time of magnoflorine,cauloside C,hederagenin,oleanolic acid and digoxin(I.S.)were 0.71 min,0.79 min,1.32 min,2.31 min and 0.73 min respectively.The compound was ionized in the electrospray ionization(ESI)and operated in negative mode,in addition to magnoflorine.The other component mass spectrometer on various parameters were optimized and monitored by multiple reaction monitoring(MRM)mode.The results showed that the established method had a better specialization and there were good linear relationships of 4 kinds of components under test(r>0.9926);The precision(relative standard deviation)of intra-day and inter-day were in the 1.57?10.48%and 1.31?10.68%range respectively,meanwhile the accuracy(relative error)of intra-day and inter-day were in the-2.24?9.90%and-1.97?11.17%respectively.The recovery and matrix effect of each component were 80.80?93.06%and 83.41?104.21%respectively,and the extraction rate and matrix effect of internal standard compound were 92,04%and 94.27%;The dilution effect of the component ranged from 99.42%to 102.03%;There was good stability of plasma samples of the component under test which kept at room temperature for 4h and at 4oC for 12h.After three freeze-thaw cycle and at-80? for one month,they didn't appear obvious degradation reaction.The method was rapid and easy to operation,which has high sensitivity and good selectivity.It can be used to detect four components of the plasma samples of Caulophyllum robustum Maxim effective part.2.Pharmacokinetic behavior research of Caulophyllum robustum Maxim effective part in normal rats in vivoThis part of the research will establish the UPLC/MS/MS method which was successfully applied in pharmacokinetic study of Caulophyllum robustum Maxim effective part.Rats were given orally in single dose of 1.5g/kg Caulophyllum robustum Maxim effective part,and pharmacokinetic behavior of 4 components in the normal rats in vivo was observed.Pharmacokinetic parameters of four kinds of components were calculated by DAS 2.0 software.Results showed that the kinetic parameters of the component:(1)magnoflorine:one compartment model,half-life(t1/2)of(5.15±0.26)h,peak concentration(Cmax)of(1941.69±24.83)?g/L,the peak time(Tmax)for(4.00±0.00)h,the apparent volume of distribution(V)of(442.45±22.60)L/kg,clearance(CL)of(59.53±0.50)L/h/kg,the area under the curve(AUC0?T and AUC0??)of(24259.84±486.64)?g/L*h and(25464.07±502.08)?g/L*h respectively,the average time encumbrances(MRT0?T and MRT0??)of(8.00±0.07)h and(9.10±0.15)h respectively;(2)cauloside C:two compartment model,distribution of half-life(t1/2?)of(1.53±0.19)h,phase elimination half-life(t1/2?)of(17.67±6.59)h,peak concentration(Cmax)of(1580.20±70.97)?g/L,the peak time(Tmax)for(2.33±0.52)h,the apparent volume of distribution(V)of(488.58±57.37)L/kg,elearance(CL)was(116.96±3.59)L/h/kg,the area under the curve(AUC0?T and AUC0??)of(11375.68±409.27)?g/L*h and(13196.34±350.86)?g/L*h respectively,the average time encumbrances(MRT0?T and MRT0??)of(6.94±0.09)h and(11.13±0.93)h respectively;(3)hederagenin:one compartment model,half-life(t1/2)of(2.87 ± 0.20)h,peak concentration(Cmax)of(834.56±26.72)?g/L,the peak time(Tmax)for(6.00±0.00)h,the apparent volume of distribution(V)of(2239.31±477.10)L/kg,elearance(CL)of(537.05±82.72)L/h/kg,the area under the curve(AUC0?T and AUC0??)of(4651.65±165.62)?g/L*h and(4652.21±165.99)?g/L*h respectively,the average time encumbrances(MRT0?T and MRT0??)of(6.13±0.14)h and(6.20±0.15)h respectively;(4)oleanolic acid:two compartment model,distribution of half-life(t1/2?)of(1.61±0.17)h,Phase elimination half-life(t1/2?)of(65.63±9.02)h,peak concentration(Cmax)of(782.25±40.69)?g/L,the peak time(Tmax)for(0.75±0.00)h,the apparent volume of distribution(V)of(4376.01±650.24)L/kg,elearance(CL)was(1676.14±149.60)L/h/kg,the area under the curve(AUC0?T and AUC0??)of(983.72±99.21)?g/L*h and(1017.75±126.38)?g/L*h respectively,the average time encumbrances(MRT0?T and MRT0??)of(4.03±0.14)h and(5.32±0.71)h respectively.Change and the regularity of different substances in the body were different,which may cause synergy or antagonism effect each other.The contents of four components were detected by UPLC/MS/MS method firstly at the same time in this study,and the change rule in the body was studied through the pharmacokinetic theory.The research contents provided the reference of medicinal materials and mechanism of Caulophyllum robustum Maxim effective part.