Ginseng Saponin Pharmacokinetic Study

Based on their chemical structures, ginsenosides are divided into three groups:protopanaxatriol (PPT), protopanaxadiol (PPD) and oleanane types. Ginsenosides had been found a wide range of biological activities, including immunomodulatory, anti-tumor, learning and memory improving, anti-aging, antiinflammatory, antidementia, antifatigue, hemopoietic function promotion and so on. However, ginsenosides themselves can not easily be absorbed and their bioavailabilities were poor. Many studies demonstrated that ginsenosides can be hydrolyzed in the gastrointestinal (GI) tract to form their metabolites including PPT and PPD, which are more easily absorbed into the body and displayed more potent activities than ginsenosides. Dammarane Sapogenins (DS-1226), which was prepared by proprietary technology to alkaline hydrolysis of total ginsenosides derived from the stem and leaf of Panax ginseng. This production and technology has already won patent rights at eight countries or regions, including China, USA, Canada, European Union, Taiwan, etc. DS-1226was mainly composed of33%PPT and16%PPD, and had been found with significant activities in improving learning and memory on cognitive dysfunction caused by sleep interruption in mice, and also decreasing chemotherapy-induced myelosuppression. In present study, we investigated the pharmacokinetic properties of PPT and PPD in rat plasma and brain using a LC-MS method when DS-1226was administrated to rats. The permeability through the small intestine and the blood-brain-barrier (BBB) were studied by using the Caco-2cell model and in vitro blood-brain barrier model, respectively. Finally, potentially drug-drug interaction of DS-1226was investigated.1. A sensitive LC-MS method was developed for the simultaneous determination of PPT and PPD in rat plasma and brain tissue. The analytes were extracted with dichloromethane and using OA as an internal standard. In order to achieve the abundant and stable signals,0.05mmol/L ammonium chloride was added to the mobile phase, and the chloride adducted molecule ions [M+C1]-at m/z511.6,495.9and455.5were used for monitoring PPT, PPD and OA, respectively. The results indicated that the limits of detection and quantification for both PPT and PPD in rat plasma and hippocampal were10.0,20.0ng/ml, respectively. And both the limits of detection, quantification for PPT and PPD in rat cortex were3.3ng/ml,6.7ng/ml, respectively. The intra-day and inter-day precisions and the accuracies were satisfactory and indicated that the LC-MS method was suitable for biological analysis.2. The pharmacokinetics of PPT and PPD in rats were studied after intravenously (i.v.,30mg/kg), oral (75mg/kg) and portal vein (p.v.,75mg/kg) administration of DS-1226. Their in vitro stabilities in the gastrointestinal (GI) tract were also investigated. The results showed that PPT was rapid absorption and eliminated rapidly from the body with averagetmax value of0.58h and t1/2,λz of0.95h after oral administration, while PPD was absorption and eliminated relatively slowly with tmax value of1.82h and t1/2,λz of3.76h. The absolute bioavailabilities of PPT and PPD were estimated as3.69%and48.12%, respectively.DS-1226was administrated to rats through the hepatic portal vein for exploring the hepatic first-pass effect of PPT and PPD. The results showed that the bioavailabilities of PPT and PPD were101.27%and100.54%, which indicated that hepatic first-pass should not be the reason for its low bioavailabilitiy. Both PPT and PPD could be detected in brain after i.v. administration, and their concentrations in hippocampal were relatively higher than that in cortex. The stability test found that PPT was instable both in pH1.2buffer and in the stomach content solution, indicated that the instability in the stomach might be one of the reasons for PPT poor bioavailabilitiy.3. Their intestinal transport absorption were investigated by using the Caco-2cell monolayer model. The results showed that PPT and PPD at three concentrations (10,20and40μM) have good absorption transport with Papp AP→BL value of (12.40～15.35)×10-6cm/s and (22.71～25.80)×10-6cm/s. The transported amounts of PPT and PPD were all increased with the increase of loading concentrations, with no changes for Papp values, the ratios of Papp BL→AB/Papp AB→BL for PPD and PPT were less than1.5. Furthermore, the Papp values for PPD and PPT were determined in the presence of100μM verapamil, and were found similar with that of absence of verapamil. This suggests that passive diffusion is the main transport mechanism of both PPD and PPT.4. In vitro BBB model by co-culture primary cultured brain endothelial cells and astrocytes was established, and permeability of PPT and PPD through BBB were investigated. The results showed that PPT and PPD at three concentrations (10,20and40μM) both could penetrate the BBB, and with PappAP→BL value of (30.30～34.39)×10-6cm/s and (4.09～5.08)×10-6cm/s. The Papp values for PPT was about eight times higher than that for PPD, which indicated that PPT could be penetrate the BBB more easily.5. Finally, the inhibitory effect of DS-1226on the activities of hepatic microsomal CYP450enzymes were investigated by cocktail probe drugs, and the effect of DS-1226on the activity of P-gp was investigated by uptake of Rho-123. The results showed that DS-1226have significant inhibiting effects on the activity of CYP3A4, CYP2C9and CYP2C19, with the IC50value of7.58,9.69and13.28μg/mL, while the inhibiting effects on the activity of CYP2E1and CYP2D6were relatively weakly, with the IC50value of99.48μM and114.90μM. The results indicate that DS-1226might cause significant drug interactions by inhibiting hepatic CYP450enzymes in vivo. DS-1226has weak inhibition on P-gp, but there was no significant difference between DS-1226groups and negative control group (P>0.05).In conclusion, the pharmacokinetic properties of PPT and PPD after i.v., oral and p.v. administration of DS-1226as well as the factors on their bioavailabilities were investigated. Meanwhile, the permeability through the small intestine and the BBB were also studied by using the Caco-2cell model and in vitro BBB model, respectively. Finally, potentially drug interaction of DS-1226was investigated. PPT, which structure only differs from PPD by a hydroxyl group at the C-6position, showed quite different pharmacokinetic properties from PPD, the bioavailability of PPD was relatively higher than PPT both in plasma and brain. The present study could provide ideas for further research and development of ginsenoside aglycones, so as to further study their mechanisms and to evaluate their druggabilities.