5L of distilled water, the cooking was done in a home pressure pan, for 30 min after the constant output of stream by the pressure valve; the CWSW sample was soaked in 1.5L of distilled water for 8 h at 25 °C, then cooked with the rest of the non absorbed soaking water as the CWS sample; the COSW sample was prepared under the same described conditions, but the soaking water was removed and then the same

volume of non absorbed water in the soaking to perform the cooking INK 128 in vivo was added. For each way of preparation, 500 g of bean were used in 1.5L of distilled water for soaking and cooking. After the cooking the broth was separated from the grain with the help of a plastic sieve (15 cm of diameter),

and then they were dried, mixed, packaged in sterile bags and stored in the dark to perform the analysis. The soaking water of the COSW samples was also packaged in sterilized containers and stored in the frozen at −18 °C to be analyzed later. The antioxidant activity assay was performed according to the method proposed by Brand-Willians, Cuvelier, and Berset (1995) modified by Sanchez-Moreno, Larrauri, and Saura-Calixto (1998), which consists in a reduction in the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) by an antioxidant or species of antioxidants resulting in the reduction of absorbance of 515 nm. For the calibration curve an aliquot of 0.1 mL of methanol containing different concentrations find more of standard was added to 3.9 mL of DPPH 2.5 mg/L diluted in methanol prepared at the time of analysis. The samples were prepared in three different dilutions in triplicate. In a dark environment, 0.1 mL of each dilution was transferred to tubes with 3.9 mL of the DPPH radical. Methanol was used as blank sample and the reading was done in a UV–Vis spectrophotometer and monitored until the stabilization does the EC50 calculation. The EC50 indicates the amount in grams of the bean sample which is necessary to decrease the activity of 1 mg of DPPH.

Thus, as lower the EC50 gets the greater the antioxidant activity will be. cAMP The results are expressed in g of sample by mg of DPPH (g sample/mg DPPH). The total phenolic content was determined according the Folin–Ciocalteu method described by Singleton, Joseph, and Rossi (1965), using gallic acid as a standard. Each 2 g of the sample was diluted in 50 mL of deionized water, later an aliquot of 1 mL of dilution was transferred to a tube with the addition of 9 mL of deionized water and 1 mL of the Folin–Ciocalteu reagent. The mixture was agitated, left to rest for 5 min for the subsequent addiction of 8 mL of the 7 g/100 mL Na2CO3. The tube was agitated again and it was left incubated for 1 h at room temperature. The reading was done with a wavelength at 765 nm in a UV–Vis spectrophotometer, being the distilled water used as a blank.