The remaining testosterone from the reactions was detected by UV detection at 246 nm using a diode array detection system. The results Palbociclib cell cycle represent the SD of dupli cate values. To assay the effects of quercetin at low concentrations, an alternate highly sensitive HPLC method was adopted to analyze testosterone. Testosterone was dissolved in acetonitrile and added as 1% v/v. The mobile phase was acetonitrile/water at a flow rate of 1 mL/min. The injection volume was 50uL and detec tion at 245 nm. The results represent the SD of triplicate values. The testosterone glucuronidation assay, described in the BD biosciences data sheet for the human UGT2B17 supersomes, employs a standard incuba tion mixture containing UDPGA, alamethi cin, magnesium chloride and pH 7.

5 Tris HCl buffer and deionised water comprising 50% of the overall reaction volume. Following incubation at 37 C for five minutes, the reaction was initiated by the addition of 0. 2 mg/mL ice cold UGT2B17 supersomes. The reactions were stopped by the transfer of 100 uL aliquots to 100 uL ice cold acetonitrile, vortex mixed with samples stored on wet ice. The samples were centrifuged at 10,000 x g for 5 minutes. The aliquots of the super natants were then analyzed by HPLC. In order to study the inhibitory effects of red wine, various volumes ranging from 2 8% of red wine were added to the reaction. The reactions were stopped after one or two hours and the remaining testosterone was analyzed to determine any increase in testosterone through the inhibition by red wine.

The red wine sample had been evaporated to dry residue to remove the etha nol and reconstituted with the same volume of water and filtered by a Millex 0. 45 uM filter device. The phenolic compounds gallic acid, caffeic acid and quercetin that are present in red wine were analyzed for the inhibition of UGT2B17. The phenolic standards were dissolved in ethanol and heated and mixed to aid dissolving where necessary and added to the reaction as 1% v/v of the reaction. The phenolic compound 4 ethylphenol was dissolved in ethanol and added to the reaction at 1% of the overall reaction vol ume at 750 uM overall concentration. The reaction dur ation was for 1 hour. The red wine sample used in the glucuronidation assays was analyzed to determine the phenolic com pounds present in the wine.

The wine sample for HPLC analysis was prepared by evaporating Anacetrapib the sample to dry ness with the remaining dry residue dissolved in water to restore the original volume of the sample. The sample was then filtered by a Millex 0. 45 uM filter device and injected into the HPLC. Quantification was performed as previously described on an Agilent 1260 HPLC using a Kromasil C18 column, 250 mm x 406 mm 5 uM with detection at 280 nm. The mobile phase consisted of 0. 1% orthophosphoric acid in water and methanol. The mobile phase gradient elusions were 0 minutes, 10 minutes, 20 minutes, 30 minutes, 50 minutes.