IJCS | Volume 32, Nº2, May/June 2019

240 Britto Junior et al. Antioxidant and vasodilatory action of grape juices Int J Cardiovasc Sci. 2019;32(3)238-246 Original Article with gallic acid. The total phenolic content was expressed as μg equivalents of gallic acid (GAE) per ml of sample. All analyses were done in triplicate. Electrochemical analyses To perform the electrochemical measurements, an μAutolab III potentiostat/galvanostat integrated with the software GPES 4.9 (Eco-Chemie, Utrecht, The Netherlands) was used. The analyses were carried out in an electrochemical cell with capacity for 3 mL of a solution operated with a three-electrode system, an Ag/ AgCl reference electrode (KCl 3 mol.L -1 ), an auxiliary platinum electrode and, as a carbon paste electrode prepared with 0.075 g of carbon and 0.035 g of mineral oil was used as a working electrode. The tests using differential pulse voltammetry (DPV) were performed with pulse amplitude of 50 mV and scanning velocity of ʋ = 10mVs -1 . The analyses were carried out in an electrochemical cell containing 1.75 ml of phosphate buffer pH 7.0 (0.1 M), adding 25 μL of the ethanolic solution of the analyte. The electrochemical index (EI) of the samples was calculated by summing up the result of each division of the current by the potential (I/E) for each anodic peak observed on the differential pulse voltammograms. Animals used and preparation of isolated arteries Male Wistar rats (200-230 g) from the UFG central vivarium were used. All experimental protocols respected the protocols approved by the UFG Research Ethics Committee (protocol: 044/17). This study is in line with the European Union Guide for the Care and Use of Experimental Animals (2010/63/EU). The rats were euthanized by exsanguination under inhalational anesthesia (n = 5-6). The thoracic aorta was isolated, separated from the connective and adipose tissues and cut into rings (± 4 mm), which were mounted between two metal hooks, one of which was connected to a power transducer to record the isometric voltage (DATAQ Instruments, Akron, OH, USA) and the other was attached to the vial for the isolated organ containing modified Krebs solution [composition in mM: NaCl, 130.0; KCl, 4.7; KH 2 PO 4, 1.2; CaCl 2 , 1.6; MgSO 4 , 1.2; NaHCO 3 , 14.9; glucose, 5.5], pH 7.4, under gasification with carbon dioxide (95% O 2 + 5% of CO 2 ) at 37°C and maintained at baseline at 1.5 g (optimal rest tension, as previously standardized in our laboratory). Experimental protocols After 60 minutes of basal tension stabilization, the arteries were pre-contracted with phenylephrine (0.1 μM) and the presence of endotheliumwas determined using ACh (1 μM). The rings were disposed of when the relaxation for ACh was smaller than 80%. The whole grape juices analyzedwere filtered on a filter paper under light and immediately divided and frozen at -20°C for future experiments. The arteries with intact precontracted endothelium (phenylephrine, 0.1 μM) were stimulated to relax by increasing the concentration of juice directly in the bath solution (0 to 30 μL/mL). As wine is popularly known throughout the world as a vasodilator, we also perform relaxation curves stimulated with Cabernet Sauvignon redwine, produced in France in 2015, in order to compare the vascular effect. In another experiment series, the vasodilatory effect of the juice samples was repeated after treatment with the NOS, L-NAME (100 μM, 30 min) inhibitor in order to determine the participation of NO in the mediated juice-stimulated vasodilation. Statistical analysis The charts were made and analyzed by the software GraphPad Prism (GraphPad Software Corporation, version 5.0) by ANOVA plus Bonferroni post-test. In the analyses, the continuous variables showed normal distribution and the results were expressed as the mean ± standard deviation of the mean of at least five experiments (n = 5-6) obtained from different animals. All analyses considered a statistical significance level of 5% (p < 0.05). Results Evaluation of antioxidant activity and determination of total phenols Antioxidant activitywas evaluated using two different methods: DPPH andABTS. Although the sample fromRJ had the best result (lower CE 50 ), we can see in Figure 1A that the four samples obtained fromRS (1) , RS (2) , RJ andGO had similar DPPH discoloration indexes (CE 50 : 18.2 ± 0.8; 22.4 ± 2.1; 17.1 ± 0.6; 20.5 ± 0.7 µL, respectively), indicating equivalent antioxidant activity. The samples fromPR and SP presented similar results comparing each other, and lower antioxidant capacity (p < 0.05) compared to the other samples (CE 50 : 29.1± 1.1; 28.6 ± 1.6, respectively).