Polygala Saponin Hydrolysate Of Pharmacokinetic Study

The Chinese herbal medicine, Radix Polygala called "Yuan-Zhi" in Chinese, is the roots of Polygala tenuifolia Willd and Polygala sibirica L. and has a long history of use as a sedative in folk medicine for the treatment of patients with insomnia, neurosis and dementia. The saponins are recognized as the main ingredients responsible for the cognition-improving effects of this herb. However, the Polygala saponins are highly toxic to animals; this toxicity seriously limits the application and development of an effective agent from the herb for cognitive improvement. The Polygala saponin hydrolysates (PSH) is a mixture of hydrolysates, which is yield by hydrolyzed Polygala saponins under an alkaline condition to cleave the ester linkage between triterpenoids and cinnamic acid derivatives. PSH, mainly composed of3,4,5-trimethoxycinnamylic acid (TMCA), p-methoxycinnamylic acid (PMCA) and tenuifolin (TF), exhibited stronger effects in improving cognition and learning memory with less toxicity compared with their original saponins. In this study, we investigated the pharmacokinetics of PSH in rat plasma and brain by microdialysis technique and LC-MS/MS method, and the absorption mechanism by using the Caco-2cell model.Firstly, a sensitive LC-MS/MS method was developed for the simultaneous determination of three active components of Polygala saponin hydrolysate (PSH), TMCA, PMCA and TF, in rat plasma using glycyrrhizic acid as an internal standard. The analytes were extracted with ethyl acetate and applied on a C18column with an acetonitrile-water solution (50:50, v/v) containing0.2%formic acid at0.4ml/min. A Q-Trap mass spectrometer with an electrospray ionization source in negative ion mode was used for analyte detection. The ion pairs for multiple reaction monitoring were set at m/z of237.0/103.0,177.0/116.6and679.5/425.3for TMCA, PMCA and TF, respectively. The results indicated that the calibration curves for the three analytes had good linearity (r2>0.99) within the tested concentration ranges. The limits of detection were1,10and0.5ng/ml for TMCA, PMCA and TF, respectively. The intra-day and inter-day precisions of the three analytes were less than14.82%and the accuracies were between93.20%and113.31%at the concentrations tested. The extraction recoveries of TMCA, PMCA and TF ranged from96.64%to111.98%.Secondly, the pharmacokinetic properties of three components TMCA, PMCA and TF were studied in rats following a single oral and intravious (i.v.) administration of PSH at100and20mg/kg, respectively, using the validated LC-MS/MS method. The results showed that all three compounds could be absorbed into the circulatory system after oral dosing. The two cinnamic acid derivatives, TMCA and PMCA were rapidly absorbed and reached peak concentrations approximately9min with relatively high bioavailabilities of90.12%and98.30%, respectively. While, TF was absorbed slowly, with Tmax of24min after oral dosing, and its oral bioavailability was only2.09%. However, the t1/2_λz value of TF is approximately5h, which is much longer than those of TMCA and PMCA. In addition, the concentrations of TMCA and PMCA were declining rapidly in rat plasma after i.v. administration of PSH at20mg/kg (equivalent to TMCA4.94mg/kg, PMCA1.54mg/kg, TF2.98mg/kg). The overall clearance rates (CL) of TMCA and PMCA were5.25L/h/kg and2.40L/h/kg, respectively. The data showed that TMCA and PMCA were rapidly clearance and the residence time of them is short in vivo. While, the CL of TF was0.22L/h/kg, which can show that TF was clear slower than TMCA and PMCA. Therefore, TF could remain active longer for in vivo activities of PSH. These results indicated that the three compounds were bioavailable active ingredients of PSH and might display their in vivo pharmacological activity at different levels and different time periods after dosing.Moreover, the bidirectional transportation of active components of PSH (TMCA, PMCA and TF) in intestinal epithelial was studied by the Caco-2cell model. The results showed that TMCA and PMCA have good absorption transport with PappAPâ†'BL values of (10.43～13.88)×10-6cm/s and (13.17～16.76)×10-6cm/s, while, the absorption transport of TF was fairly poor with PappAPâ†'BL values of (0.72～0.94)×10-6cm/s. The bidirectional studies were performed under non-gradient conditions at a concentration of5～150μg/ml. The transported amounts of the three components were all increased with the increase of concentrations, with no changes for Papp values. The Papp values of each component was not significant different between the two directional transports. So, the three active components of PSH can be transported across intestinal epithelial by passive diffusion and there is no efflux transport mechanism.Finally, the brain pharmacokinetics of TF was investigated in awake and freely-moving rats after i.v. administration of PSH at a dose of20mg/kg (equivalent to TMCA4.94mg/kg, PMCA1.54mg/kg, TF2.98mg/kg) by microdialysis technique and LC-MS/MS method. The result showed that the probe recoveries of TMCA, PMCA and TF were16.84±0.67%,22.76±0.65%and8.15±0.16%determined by increment method and they did not changed in the concentration range (50-500ng/mL). The results showed that TMCA and PMCA were not detected in all microdialysis samples after i.v. administration. TF could be detected and its concentration was rising from0to42min and reached the highest concentration (0.25μg/mL) at42min in the rat brain after i.v. administration of TF (2.98mg/kg). The t1/2_λz value of TF was approximately2.40h. The areas under the curve (AUC0-∞) of TF were0.53and10.62h*μg/mL in rat brain and blood, respectively. Therefore, the blood-brain barrier penetration of TF was4.99%. It indicated that TF could pass through blood-brain barrier and played neural activity in rats.