| Objective:1.To investigate the stability of icariin in artificial gastric/intestinal juice and in human and mouse feces.2.To establish sensitive and selective HPLC-MS/MS methods for the determination of icariin and its active metabolite -icarisideⅡ,in biological specimen after administration of icariin andⅩⅩcapsule.3. To study the pharmacokinetics ofⅩⅩcapsule in healthy Chinese volunteers after 15 doses ofⅩⅩcapsule and to calculate its pharmacokinetic parameters.4.To compare the pharmacokinetics of icariin monomer andⅩⅩcapsule in Wistar mouse after administration of 50 mg/kg icariin monomer andⅩⅩcapsule.5.To investigate metabolism of icariin in Caco-2 cells and to interpret the reasons of poor bioavailability.Methods:1.The stability of icariin in artificial gastric/intestinal juice was studied by HPLC-UV method.Icariin was separated on an Agilent Extend C18 column and eluted with the mobile phase of acetonitrile-0.1%formic acid(30:70,V/V) at a flow rate of 1 mL·min-1 and detected at 274 nm.20μL was injected to analyze.2.The plasma samples were extracted by ethyl acetate and separated on a AGT Venusil XBP-C18 column(250mm×4.6mm,5μm),eluted with the mobile phase of 0.1% formic acid -acetonitrile(30:70,V/V) at a flow rate of 1.0 mL·min-1.The temperature of column was 30℃.20μL was injected to analyze.Mass spectrometer was operated using electrospay ionization(ESI) with positive ionization mode at following parameters:nebulizer gas of 40 psi,spray gas of 9 L·min-1.The turbo ion spray source temperature was set at 350℃,and the capillary voltage was 4000V.The determination was performed by multiple reaction monitoring(MRM) mode,in order to measure the following ion transitions:m/z 677.1(M+H) to m/z 369.0(product ion) for ICA,m/z 515.1(M+H) to m/z 369.1(product ion) for ICSⅡand m/z 321.0(M+H) to m/z 275.0 (product ion) for I.S.,respectively,with a scan time of 0.2s per transition.Nitrogen was used as collision gas(28,8,20eV for ICA ICSⅡand I.S.,respectively).The fragment voltage for ICA,ICSⅡand I.S.was 135V,100V and 135V,respectively. Electron multiplier voltage(EMV) was 800V.3.8 healthy Chinese volunteers with half male and female were orally given 15 doses ofⅩⅩcapsule.The plasma samples were collected as scheduled and icariin and icarisideⅡwere analyzed by HPLC-MS/MS.The pharmacokinetic parameters of icarisideⅡwere calculated by DAS 2.0.4.16 Wistar rats were divided into two groups randomLy and received an intragastric administration of 50mg/kg icariin monomer(5mL/kg) and 1g content ofⅩⅩcapsule(containing 50 mg icariin)/kg body weight,respectively.The plasma samples were collected as scheduled and icariin and icarisideⅡwere analyzed by HPLC-MS/MS.The pharmacokinetic parameters of icariin and icarisideⅡwere calculated by non-compartment model.5.Icariin and Caco-2 cells were incubated together,200μL sample was taken from the basal compartment to determine the concentration of ICA and its metabolites.Results:1.About 10%icariin was decomposed after 2 hours in artificial gastric juice,80%icariin was left after 12h in artificial intestinal juice,which showed that icariin could be degraded in artificial intestinal juice.ICA was completely hydrolyzed into its aglycon ICT and ICSⅡ,and could not be detected one hour after incubation in human feces.ICSⅡappeared immediately as the hydrolysis of ICA,but decreased fast within 8h,then almost approached the LOQ of this method.The main metabolites of ICA in feces were icariside and aglycon.Besides,little amount of isomerizedicaritin was detected.The degradation of ICA and ICSⅡin human feces were similar with that in mouse feces,while the main differences were that the rate of degradation of ICSⅡin mouse feces was slower than that in human feces,and the amount of ICT was relatively less,especially from 4 to 10 hours.2.The linear range of the calibration curve for determination of icariin and icarisideⅡin plasma by HPLC-MS/MS method was 0.05～10 ng·mL-1,and the LOQ was 0.05ng·mL-1.The regression equations for ICA and ICSⅡwere Y=0.2766X+0.0197,(r=0.99302),Y=0.4331 X+0.0824,(r=0.99357),respectively.The absolute recoveries were more than 65% and 55%,respectively.The relative recoveries were between 85%and 120%. Intra-day RSD and inter-day RSD were less than 10%and 15%,respectively.Icariin and icarisideⅡin plasma were stable when frozen at -20℃for two weeks and stable after three freeze-thawing cycles.3.Little ICA was detected after administration of 15 doses ofⅩⅩcapsule,but lower than LOQ of the methods.ICSⅡwas determined by this method,and double hump was appeared from concentration-time profile.Tmax were 1.5h and 4h,with 1.601 and 1.704 ng·mL-1 as corresponding Cmax.Other parameters calculated by DAS2.0 were as follows:t1/2(8.954±3.424) h,AUC0-12 (9.642±4.520) ng·mL-1·h,AUC0～∞(12.884±6.750) ng·mL-1·h,MRT0-12(6.400±0.253) h.A 1/c weighted coefficients regression analysis and one-compartmental model were used to fit the disposition of ICSⅡin human.No statistical significance of main parameters was found between male and female.4.The plasma concentration of ICA and ICSⅡafter administration of ICA monomer were all higher than that afterⅩⅩcapsule.Pharmacokinetic parameters were calculated by non-compartment model. The main parameters of ICA after ICA monomer andⅩⅩcapsule were shown as follows:t1/2(3.612±1.067) h and(6.022±3.381) h,Tmax(0.958±0.668)h and(1.219±1.129)h,Cmax(21.037±9.090)ng·mL-1and(14.904±4.492)ng·mL-1,AUC0-12 (78.488±42.653)ng·mL-1·h and(42.532±13.839)ng·mL-1·h,AUC0～∞(87.054±45.391) ng·mL-1·h and(51.214±13.043)ng·mL-1·h.The main parameters of ICSⅡafter ICA monomer andⅩⅩcapsule were shown as follows:t1/2(3.014±1.358) h and (17.171±10.170) h,Tmax(3.938±2.275)h and(0.938±0.477)h,Cmax(20.943±8.583)ng·mL-1 and(5.448±2.381)ng·mL-1,AUC0-12(87.101±24.814) ng·mL-1·h and (18.822±10.160)ng·mL-1·h,AUC0～∞(96.295±22.934) ng·mL-1·h and(35.161±8.405)ng·mL-1·h。Compared with ICA,the Tmax of ICSⅡwas lagged behind,and the Cmax and AUC were relatively lower after ICA monomer,which were coincident with the conclusions of first part.ICA was hydrolyzed into ICSⅡby bacteria in intestinal tract,therefore,Tmax of ICSⅡwas latter than ICA.Besides ICSⅡ,ICA could be further hydrolyzed into ICT,so the concentration of ICSⅡwas lower. However,no ICT was detect in rat plasma,which was possibly related to its poor absorption in intestinal epithelium cells.Cmax and AUC of ICA and ICSⅡafter ICA monomer were higher than that of ICA and ICSⅡafterⅩⅩcapsule.Statistical analysis of main pharmacokinetic parameters of ICA showed that AUC0-12 and AUC0～∞ have statistical significance,while all of the main pharmacokinetic parameters of ICSⅡhave statistical significance.5.Caco-2 cells and icariin were incubated together,which shown that icariin could be metabolized to icarisideⅡ,and further reduce the bioavailability of icariin.Conclusion:1.Icariin was relatively stable in artificial gastric juice,could be degraded to 80%in artificial intestinal juice,but quickly degraded into icarisideⅡand icaritin in human and mouse feces.2.Major icarisideⅡand minor icariin were detected in human plasma after oral administration of 15 doses ofⅩⅩcapsule.T- test indicated that no statistical significance was found between the main pharmacokinetic parameters of male and female volunteers.3.The plasma concentration of ICA and ICSⅡafter administration of ICA monomer were all higher than that afterⅩⅩcapsule. Statistical analysis of main pharmacokinetic parameters of ICA showed that AUC0-12 and AUC0～∞ have statistical significance,while all of the main pharmacokinetic parameters of ICSⅡhave statistical significance,which demonstrated that flavonoid glycoside mixture did not benefit the absorption of ICA and ICSⅡ.4.Icariin could be metabolized to icarisideⅡ,which further reduce the bioavailability of icariin.5.The trial was well done and all volunteers were in good compliance.No adverse reactions were observed during the whole study.
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