Phenolic standards were dis solved in water at a concentration of 1 mg/mL and indi vidually injected into the HPLC under the same conditions with the retention times compared to that of the red wine sample to indentify each phenolic compound. Statistical analyses were conducted using SPSS v19. The significance of reduction in 20S proteasome inhibitor glucuronidation activity was tested for using a directional one sample t test with the significance level set at 0. 05. Mixed model ANOVA was used to test for statistically significant difference over time and between concentra tions, including testing for interaction effect. Results Inhibitory effects of red wine on UGT2B17 The effects of increasing concentrations of red wine on the UGT2B17 mediated glucuronidation of testosterone were assessed as a function of the reduction in conversion of testosterone to testosterone glucuronide.
The results show that increasing the concentration of red wine resulted in a lower conversion of testosterone to its glucur onide conjugate. A reduction in UGT2B17 activity was observed for all, ranging between ca 10% to over 70% over two hours for additions of 2% to 8% red wine. The percentage of ethanol present in the final assay was in the range of 0. 26% 1. 04% corresponding to additions of red wine at 2% 8% respectively. It is notable that during a 2 hour period the inhibition is more pro nounced at higher concentration of the red wine. Statisti cally significant differences were evidenced between times and concentrations, with significant interaction between the two.
The effect on UGT2B17 activity by the addition of an evaporated red wine sample reconstituted with an equal volume of water at concentrations of 4% and 8% of the reaction volume resulted in a glucuronidation % of con trol at 59. 18 3. 154 and 23. 48 4. 405, respectively. These values, taken after a two hour duration, resemble those from the intact wine samples indicating minimal contributions from the ethanol content on the inhibition of UGT2B17 by red wine. The effects of increasing concentrations of ethanol on the reduction of testosterone by UGT2B17 are shown in Figure 2. The results indicated that testosterone glucuro nidation was only slightly altered by ethanol at a 1% concentration . however as the concentration of ethanol was increased to above 2% of the reaction vol ume, testosterone glucuronidation was affected as shown but not reaching a statistically significant level.
Analysis of red wine In order to identify individual inhibitors of UGT2B17, the phenolic content in the wine sample was investigated by HPLC. Analysis of the red wine confirmed the pres ence of gallic acid, chlorogenic acid, caffeic acid, p coumaric acid and quercetin, which informed subsequent experiments. The inhibitory effect of individual phenolic compounds Cilengitide Initial experiments were performed to screen the pheno lics found in red wine for their effects on UGT2B17.