Samples of each Selleckchem Crizotinib variety harvested in 2008 were supplied by different wineries (A, B and C) located in Videira, Santa Catarina state, Brazil. Five samples of Cabernet Sauvignon and five samples of Merlot were collected at winery A, five samples of Bordeaux were collected at winery B and five samples of Isabel were collected at winery C. Videira has a wet temperate climate, with well-defined seasons and mean temperatures of 35 °C in summer and 0 °C in winter. The red wine vinification technique was conducted
with daily pumping and contact of the skins and seeds with the juice for 6 days, after which the must was pressed and the pomace samples collected. The general properties of the pomaces were maintained during transportation to the laboratory by keeping them in isothermal boxes containing ice. Once in laboratory, the pomace samples were lyophilised and stored at −37 °C before analysis. After powdering in liquid nitrogen, samples (1 g) were extracted with 50 mL of acidified (0.1% HCl) methanol Neratinib price in Turrax equipment (Metabo®, Nurthigen-Germany) for 1 h (4 × 15 min) in an ice bath (∼4 °C) and under penumbra conditions.
The homogenate was filtered through Whatman No. 1 filter papers and analysed for total phenolic compounds content, and total monomeric anthocyanins contents, as well as for antioxidant activity. The extraction yield was determined as dry weight after dying in an oven at 105 °C until constant Paclitaxel chemical structure weight. To determine the individual phenolic compounds by HPLC, the filtered
extracts were evaporated under vacuum at 40 °C in a rotatory evaporator and made up to 10 mL with ultrapure water. An aliquot of 5 mL of the extract was added to a 1 g polyamide SC6 column (Macherey–Nagel Gmbh and Co., Düren, Germany) preconditioned with methanol (20 mL) and water (60 mL). The column was washed with water (20 mL) and further eluted with methanol (50 mL) to elute the neutral flavonoids, and with methanol/ammonia (99.5:0.5) to elute the acidic flavonoids. These fractions were evaporated to dryness under pressure at 40 °C, redissolved in methanol (1 mL), filtered through 0.22 μm PTFE (polytetrafluoroethylene) filters (Millipore Ltd., Bedford, MA) and analysed by HPLC. The total phenolic content of each extract was determined spectrophotometrically (Hewlett–Packard 8452 A Spectrophotometer) according to the Folin–Ciocalteau method (Singleton & Rossi, 1965). Absorbance was read at 765 nm and results were expressed, in dry weight (dw) of pomace, as mg/g gallic acid equivalent (GAE). Total anthocyanins content was determined by the pH-differential method (Giusti & Wrolstad, 2001). Absorbance was read at 520 and 700 nm. Results were expressed (on a dw basis) as concentration of monomeric pigments (mg/g) of malvidin-3-glucoside equivalent (molar extinction coefficient of 28.000 L/cm/mol and molecular weight of 463.3 g/mol).