Supplementary Materialsnutrients-11-00061-s001. in vitro, and compares the outcome with the earlier

Supplementary Materialsnutrients-11-00061-s001. in vitro, and compares the outcome with the earlier in vivo findings. The results demonstrate that all blueberry varieties as well as the blueberryCapple juice were more effective in reducing oxidative stress as compared to the single compounds (e.g., DNA strand break reduction: EC50: Elliot 8.3 mg/mL, Aurora and Draper 11.9 mg/mL, blueberryCapple juice 12.3 mg/mL, and Bluecrop 12.7 mg/mL; single compounds). In addition, the gene expression profiles (consisting of 18 selected genes from Fulvestrant irreversible inhibition the in vivo study) induced by the blueberry varieties were more similar to the profile of the human intervention study (range 44C78%). The blueberry variety Elliot showed the strongest and most similar effects, almost 80% of gene expression modulations were similar compared to the in vivo results. From the single compounds (range 17C44%), quercetin induced the most comparable gene expression changes, i.e., 44%. This approach could be useful in agriculture for identifying crop varieties containing combinations of phytochemicals which show optimal preventive capacities. 0.01). In order to investigate which of the blueberry extracts and single compounds possessed the highest chemopreventive properties, linear log regression was applied. From the log linear regression equation, the EC50 was estimated which is usually shown in the legend. Open in a separate window Open in a separate NR4A3 window Physique 2 Radical formation in Caco-2 cells as measured by ESR spectroscopy. Results are expressed as percentage of solvent control levels. AUC: area under the curve of radical specific signals. Error bars indicate standard deviations. Caco-2 cells were pre-incubated for 2 h with different concentrations of the extract of blueberryCapple juice or extracts of four different blueberry varieties (a), or single compounds (b) and subsequently exposed to 150 M tert-butylhydroperoxide (TBH) for 30 min. Pre-incubation for 2 h with medium, Fulvestrant irreversible inhibition solvent control (0.5% end concentration of 70% methanol/0.1% formic acid), the maximal concentration of the different extracts (i.e., 7 mg/mL), or 100 M of single compounds did not induce significant levels of radical formation. ** 0.01; * 0.05, significantly different from Caco-2 cells exposed to solvent control for 2 h and challenged with 150 M TBH for 30 min. The 4 single compounds were tested in a concentration range of 0, 25, 50, and 100 M and pre-incubated for 2, 6, 24, and 48 h. The final concentration of the solvent in the medium was 0.5%. After pre-incubation, a subset of cells was challenged with the oxidant tert-butylhydroperoxide (TBH) (Sigma Aldrich, Zwijndrecht, The Netherlands). For Comet assay experiments, Caco-2 cells were challenged with 100 M TBH for 1 h, as this exposure condition resulted in cell viability levels 80%, and a moderate increase in oxidative DNA damage (Physique S1). The optimal exposure condition of Caco-2 cells in the ESR spectroscopy measurements was decided at 150 m TBH for 30 min as at this condition cell viability levels were 80% and a significant increase in free radical Fulvestrant irreversible inhibition formation was observed. Experiments were carried out in triplicate (Physique S2). After exposure, cells had been cleaned with 1 mL Hanks Well balanced Sodium Option double, without Ca and Mg (HBSS, Lifestyle Technologies, Leusden, HOLLAND), isolated by trypsinization, resuspended in 1 PBS and positioned on snow subsequently. For gene appearance experiments, cells had been lysed in the lifestyle dish using TRIzol? Reagent (Invitrogen, Breda, HOLLAND), and kept at ?20 C until make use of. 2.4. Cytotoxicity Assay Cytotoxicity from the blueberry ingredients, the one substances, and TBH was assessed using the trypan blue exclusion assay. Fifteen L cell suspension system was blended with 15 L 0.4% trypan blue option (Life Technology, Leusden, HOLLAND) and incubated for 1 min at 37 C. The blend was used in a Brker keeping track of chamber (Sigma Aldrich, Zwijndrecht, HOLLAND). The real amount of practical colorless cells and the amount of useless blue cells had been counted, and viability was computed as percentage practical cells..