Studies On Pharmacokinetics And Compatibility Mechanism Of Zishen Pill

Zishen Pill (ZSP) is a traditional Chinese medicinal formula widely used to treat benign prostatic hyperplasia for a long period of time. The pill contains Anemarrhenae Rhizoma, Phellodendri Amurensis Cortex and Cinnamomi Cortex with a ratio of 10: 10: 1 in weight. The pharmacokinetics of the pill was studied and utilized to elucidate its compatibility mechanism. The content includes five main parts. Firstly,A sensitive and reliable high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) has been developed and validated for simultaneous determination of active components, i.e., xanthone glycosides (neomangiferin and mangiferin), timosaponins (timosaponin E1, timosaponin B-II and timosaponin B) and alkaloids (palmatine and berberine) in rat plasma after oral administration of ZSP extract. Plasma samples were pretreated by protein precipitation with acetonitrile containing the internal standards ginsenoside Re (for xanthone glycosides and timosaponins) and tetrahydroberberine (for alkaloids). LC separation was achieved on a Zorbax SB-C18 column (150 mm×2.1 mm I.D., 3.5μm) with gradient elution using a mobile phase consisting of acetonitrile-0.1% formic acid in water at a flow rate of 0.25 mL/min. The detection was carried out by a triple-quadrupole tandem mass spectrometer in MRM mode via polarity switching between negative (for xanthone glycosides and timosaponins) and positive (for alkaloids) ionization mode. Linear calibration curves were obtained over the concentration range of 5-1000 ng/mL for mangiferin, 0.5-100 ng/mL for neomangiferin, timosaponin E1, timosaponin B-II and timosaponin B, and 0.05-10 ng/mL for palmatine and berberine.This fully validated method was successfully applied to pharmacokinetic study of the above seven compounds in rats. Neomangiferin is the derivative of mangiferin, in which the hydroxyl group attached to C-7 is substituted by glucose, but their pharmacokinetic parameters are remarkably different. Owing to the very similar structures, timosaponin E1, timosaponin B-II and timosaponin B have parallel pharmacokinetic parameters and concentration-time curves in vivo, being absorbed and eliminated with the similar rate. Notably, distinct double peaks were observed in both individual and mean plasma concentration-time curves of all these timosaponins. It is interesting to note that multiple plasma concentration peaks of palmatine and berberine were observed. This new method will be used in our ongoing pharmacokinetic interaction study of ZSP in rats. Sencondly,The HPLC-MS/MS method was further adapted for the analysis of plasma samples after rats were intragastric administered with single, herb couple or ZSP extract at different dose. Then the pharmacokinetic parameters were compared. As the results shown, after the compatibility, C_max and AUC_0-t values of neomangiferin and mangiferin were remarkably smaller than those in Anemarrhenae Rhizoma extract group. But there was no statistic differences among the parameters of timosaponins in three groups. With the effects of Anemarrhenae Rhizoma, C_max and AUC_0-t values of palmatine and berberine were promoted. The result claimed the rationality of herb couple compatibility. On the contrary, Cinnamomi Cortex inhibited the promoting effects of Anemarrhenae Rhizoma. The outcome reflected the corrigent function of Cinnamomi Cortex in the formula at some extent. Thirdly,To avoid the intense interfere of matrix effect, a HPLC-MS/MS method with pulse gradient elution was established to determine five glucosides in prostate tissue. In order to reduce the run time, another HPLC-MS/MS method with isocratic elution was developed to determine two alkaloids in tissue. Excepting for xanthone glycosides, the main active components have similar pharmacokinetic parameters and concentration-time curves. The results hinted that we can employ the normal rats instead of the model rats as the experiment object in following pharmacokinetic studies.The plasma and prostatic samples were analysed after rats were intragastric administered with single herb, herb couple or ZSP extract. The concentration of xanthone glycosides and timosaponins in prostatic tissue was less than that in plasma. Notably, the concentration of alkaloids in prostatic tissue was more than that in plasma. Consequently, it will be helpful to exert their effects on benign prostatic hyperplasia. Generally, there was no notable difference in the concentration of xanthone glycosides among three extract group. Timosaponins also had the same character. This finding told us that there was no distinct difference in concentration of these components with the coadministration of the three herbs. Similar to the situation in plasma, C_max and AUC_0-t values of palmatine and berberine were remarkably promoted with the effects of Anemarrhenae Rhizoma, but Cinnamomi Cortex slightly inhibited the promoting effects of Anemarrhenae Rhizoma. The results profoundly elucidated that the rationality of the herb couple and the corrigent effects of Cinnamomi Cortex. Fourthly,Three herb extract were tested on Caco-2 cell monolayer to systematically investigate the absorption of the main active compounds in ZSP. It was found that their apparent permeability coefficients (P_app) except for timosaponin B were all less than 10-6cm/s. It was firstly elucidated that palmatine and berberine are substrates of P-gp, and they are dramatically effused by P-gp. It could be one of important factors to decrease the oral bioavailability.The effects of ZSP and 21 bioactive constituents on CYPlA2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 enzyme activity were investigated through human liver microsomes incubation. The results displayed that neomangiferin and mangiferin had stronger inhibition on CYP2E1, and IC50 values were 0.47μM and 0.15μM, respectively. Phellodendrine showed noticeable inhibition on CYP2D6, with IC50 value being 0.56μM.To assess the potential of three kinds of extract to alter expression of six cytochrome P450s extensively involved in drug metabolism, we investigated the enzyme-inducing effects of the extract in HepG2 cells. The results showed that Phellodendri Amurensis Cortex extract increased the mRNA expression of CYP1A2 at 10μg/mL and 30μg/mL in HepG2 cells. Furthermore, we utilized HepG2 cell line to identify individual components in Phellodendri Amurensis Cortex extract as potential inducers of CYP3A4. It was found that berberine and palmatine were novel CYP1A2 inducers. A concentration-dependent increase of CYP1A2 mRNA expression was observed in HepG2 cells treated with 0.5⁴.5μg/mLberberine, which ranged from 1.5- to 16.1-fold. The mRNA expression of CYP1A2 was also significantly increased by 2.7- to 7.2-fold in HepG2 cells exposed to palmatine at 1³0μg/mL. Likewise, potent induction of CYP1A2 activity was observed after treatment with the two alkaloids, where 1.6- to 4.9-fold and 1.1- to 1.7-fold activity up-regulation resulted from the exposure of berberine and palmatine, respectively. It is noteworthy that the maximal induction of CYP1A2 by berberine at high concentration exceeded that induced by the positive controlβ-naphthoflavone (1.9-fold) markedly. Fifthly,Mangiferein was determined in the rat plasma following four oral doses (5, 15, 45 and 90 mg/kg). C_max was 37.2±17.8, 86.7±41.5, 93.7±53.2 and 151.5±76.8 ng/mL at the four dose levels. And AUC_0-t was 120.7±46.4, 414.8±252.5, 521.1±296.0 and 715.2±355.5 ng.h/mL. No proportional relationship was observed between doses and C_max or AUC_0-t. The results indicated that mangiferin fits to the features of nonlinear pharmacokinetics after oral doses.Timosaponin B-II was determined in the rat plasma following three intravenous doses (0.9, 1.8 and 5.4 mg/kg). C_max was 3976.9±188.2, 11295.3±806.6 and 49996.5±9572.3 ng/mL at the three dose levels. And AUC_0-t was 833.7±33.2, 2263.6±180.4 and 12550.6±2204.2 ng.h/mL. A proportional relationship was observed between doses and C_max or AUC_0-t. The results indicated that timosaponin B-II fits to the features of linear pharmacokinetics after intravenous doses. C_max of timosaponin B-II was 55.6±38.4, 432.6±353.3 and 1644.5±1242.8 ng/mL following oral doses of 30, 90 and 180 mg/kg, respectively. And AUC_0-t was 122.6±114.9, 330.0±218.2 and 2191.3±1194.5 ng.h/mL. No proportional relationship was observed between doses and C_max or AUC_0-t. The results indicated that mangiferin does not fit to the features of linear pharmacokinetics after oral doses. It is maybe caused by nonlinear elimination.The absolute oral bioavailability of timosaponin B-II in SD rats and Beagle dogs was only 1.1% and 0.71%, respectively.