The Role And Modulation Of P-glycoprotein In The Absorption Of Components In The Total Flavones Of Hippophae Rhamnoides L.

The critical problems of the modernization of the Traditional Chinese Medicines(TCMs) consist in the screening of the effective substances and the fundamentalADMET studies of their effective mechanisms involved in the human body. Theabsorptive studies of the effective substances in TCMs are the first step of theirbiopharmaceutical studies and the bottleneck of the modernization of TCMs. Therevealment of the absorptive mechanisms of TCMs, the discovery of the membranetransporters involved in the absorption of TCMs and the interaction between thepharmacokinetics of various components in TCMs mediated by membranetransporters will be in favor of the establishment of the modernized methods ofpharmacodynamic researches and effective substances screening of TCMs, reveal theessentials of the characteristic activities and the compatibility/composition theory inTCMs.Hippophae Rhamnoides L. has been wildly used for medical care in Tibetian andMogolian for thousand years; Total Flavones of Hippophae Rhamnoides L. (TFH) have been developed and used for cadiavascular diseases for almost 30 years inChina. To study the role and modulation of P-glycoprotein in the absorption of TCMs,TFH was selected to be the model Chinese crude drug in this study, and this study ismainly focused on isorhamnetin, which is used for the quality control of TFHenscrolled in the national standard of TFH. In this study, multiple approaches onmolecular level (MDR1-MDCKⅡcells model), cellular level (Caco-2 cells model)and whole animal in vivo test's level were employed to investigate the absorptivemechanism of isorhamnetin in TFH, the role of P-glycoprotein in its absorption, thechange of the pharmacokinetic profiles of isorhamnetin via P-glycoproteinmodulation and the pharmacokinetic interaction of various flavonols in TFHmediated by P-glycoprotein. In the present study, the results from three researchmodels of multiple levels correlated well. The sequence of the oral absorption (Cmax,AUC) of three tested flavonols was kaempferol＞quercetin＞isorhamnetin. Theapical to basolateral apparent permeability coefficients of isorhamnetin, quercetinand kaemferol across Caco-2 cells model were 2.28±0.11, 4.97±0.25, 8.74±0.27 (×10^-6, cm.s^-1) respectively, the basolateral to apical apparent permeability coefficientsacross Caco-2 cells model were 3.09±0.08, 6.69±0.79, 9.30±0.87 (x10^-6, cm.s^-1)respectively, their ratios were 0.74, 0.74, 0.94, respectively. Based on the results of invivo tests and Caco-2 transport assay, MDR1-MDCKⅡcells model was employingto validate the interaction between isorhamnetin and P-glycoprotein. The resultscorrelated well with previous studies. The apical to basolateral apparent permeabilitycoefficient of isorhamnetin across MDR1-MDCKⅡcells model was 1.22±0.11(×10^-6, cm.s^-1), the basolateral to apical apparent permeability coefficient of isorhamnetinacross MDR1-MDCKⅡcells model was 4.96±0.03 (×10^-6, cm.s^-1), the ratio is 0.24.The results corroborated isorhamnetin as the substrate of P-glycoprotein.The intestinal efflux and bile excretion of isorhamnetin mediated byP-glycoprotein resulted in the multi-peak character of its concentration-time curve.Coadministration of verapamil (P-glycoprotein inhibitor) and digoxin(P-glycoprotein substrate) enhanced the oral absorption and bioavailability ofisorhmanetin. Bile salts excretion pumps (BSEP) inhibitor (rifampin) inhibited the bile excretion of isorhamnetin, subsequently improved its bioavailability. Otherflavonols in TFH also exhibited interactive properties with P-glycoprotein. Thus, dueto P-glycoprotein inhibition, these flavonols' pharmacokinetic profiles were affectedby each other and its oral absorption increased after coadministration. Some otherdrug effiux pumps, such as MRP2 and Bcrpl, might be also involved in thesepharmacokinetic interactions. Therefore, it is obvious that the oral dose of mixedflavonols in TFH gained a significant biopharmaceutical advantage compared tosingle use of its flavonols. After oral administration of TFH, the flavonols wouldimprove each other's oral absorption and bioavailability. The results of the presentstudy provided a biopharmaceutical base of the absorptive mechanism, clinical useand drug preparation design of TFH.Application of research on molecular level, cellular level and whole animal in vivotest's level to absorptive proceses of components in TCMs, and studies of thecorelativity of the results is the most efficient way to reveal the coincidence of themost microcosmic substance-molecule and the most macroscopical life body. Thepresent study provided a new notion and method for the microcosmicbiopharmaceutical study of effective substances in TCMs, found a new approach tointerpret the effective mechanism of TCMs based upon the microcosmic interactionbetween effective substances in TCMs. With these methods, a brand-new approachfor the modelization of TCMs could be developed.