ere collected before the P-gp substrate dosing and at 0.08, 0.17, 0.25, 0.5, 1, 2, 3, 6, 8 h post-digoxin-dosing. Plasma was obtained by centrifugation at 5000 “7901789 g for 10 min and stored at 220uC before analysis. Plasma concentrations of digoxin were determined as described previously. Pharmacokinetic Studies of PP-242 web 20-Rh2 and 20-Rh2 in Rats To investigate the differences of pharmacokinetic characteristics between 20-Rh2 and 20-Rh2, rats were divided into 2 groups with five rats for each group. One received a single dose of 20-Rh2 intragastrically at 25 mg/kg suspended in 0.5% CMCNa, while the other received 20-Rh2 at the same dosage. Blood samples were collected at 0, 30, 60, 180, 240, 300, 360, 480, 660 and 840 min after oral administration into heparinized tubes. Plasma was obtained by centrifugation at 5000 g for 10 min and stored at 220uC before analysis. Stereoselective Regulations of P-Glycoprotein Metabolism of 20-Rh2 and 20-Rh2 in Rat Fecal Microflora Fresh feces of healthy rats were collected and suspended in anaerobic medium. After filtration, the rat intestinal microflora suspension was ready for anaerobic incubation of ginsenoside. An aliquot of 1 ml rat intestinal microflora suspension was spiked with 20-Rh2 or 20-Rh2, and then was incubated under anaerobic condition. At designated time, samples were taken for analysis. Time 0 7.5 8.0 10.0 10.5 14.4 14.8 20.0 Methanol 30 30 Acetonitrile 38 38 99.5 99.5 0.1% formic acid 32 32 0.5 0.5 10 10 32 32 Flow rate 750 750 700 700 800 800 750 750 Cell culture Caco-2 cells, obtained from American Type Culture Collection, were routinely cultured 
in DMEM supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 1 mM sodium pyruvate, and 100 U/ ml penicillin and streptomycin. MCF-7/ Adr cells were obtained from Institute of Hematology and Blood Diseases Hospital, and cultured in RPMI 1640 supplemented with 10% fetal bovine serum, and 100 U/ml penicillin and streptomycin. The cells were grown in an atmosphere of 5% CO2 at 37uC and cell medium were changed every other day. Effects of 20-Rh2, 20-Rh2, 20-Ppd and 20-Ppd on P-gp Mediated Bidirectional Transport of Digoxin across Caco-2 cell Monolayers The Caco-2 cell transport model was established as described previously. Then, Hank’s balanced salt solution containing 20-Rh2, 20-Rh2, 20-Ppd, 20-Ppd or 0.1% DMSO was loaded into both apical and basolateral chambers. After incubation at 37uC for 1 h, 5 mM digoxin was added to either apical or basolateral side to evaluate the transport in absorptive and secretory direction respectively. After incubation for just another 2 h, samples were taken from the receiving chamber for analysis. Verapamil was used as a positive control. Digoxin was determined by LC-MS/MS. All experiments were conducted in triplicate. and autosampler tray temperatures were 40 and 4uC, respectively. The mobile phase was consisted of methanol, acetonitrile and 0.1% formic acid with gradient elution. Mass spectrometer was operated in positive ESI mode. MS parameters were as follows: spray “9357531 voltage, 5.0 kV; sheath gas/auxiliary gas, nitrogen; sheath gas pressure, 356105 Pa; auxiliary gas pressure, 206105 Pa; ion transfer capillary temperature, 300uC. Quantification was performed using SIM mode with peak: m/z 645.4 for Rh2; m/z 483.3 for Ppd; m/z 787.5 for digitoxin. Data analysis The pharmacokinetic parameters of digoxin, 20-Rh2 and 20-Rh2 in rats were obtained by noncompartmental analysis using DAS. The area under the plas
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