dministration 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. 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 19151731” cells were grown in an atmosphere of 5% CO2 at 37uC and cell medium were changed every other day. 5 5 30 30 85 85 38 38 doi:10.1371/journal.pone.0035768.t005 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 Indirubin-3′-oxime 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 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 plasma concentration-time curve was calculated using the trapezoidal method. For the transport assay, the apparent permeability coefficient and efflux ratio were calculated as reported 10460232” previously. Data are expressed as mean 6 S.E.. Comparisons for betweengroups were performed using Student’s t test. For multiple comparisons, one-way analysis of variance followed by Post-Hoc test was adopted. The difference was considered to be statistically significant if the probability value was less than 0.05. Effects of 20-Rh2 and 20-Rh2 on Adriamycin Sensitivity in P-gp highly-expressed MCF-7/Adr Cells MTT colorimetric assay was used to measure the cell growth inhibition after incubation with various concentrations of adriamycin in the absence or presence of 
20-Rh2 or 20-Rh2 at 37uC for 72 h. The concentrations required to inhibit growth by 50% were calculated from survival curves using the Bliss method. The phytopathogenic oomycete Pseudoperonospora cubensis, the causative agent of cucurbit downy mildew, infects a wide range of cucurbits, including cucumber, squash, and melon. As an obligate biotroph, Ps. cubensis is dependent on i
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