After cooling, the extracts were centrifuged at 8000 × g for 20 min. The collected supernatants were filtered with qualitative filter papers (Whatman) and transferred to glass flasks at 40 °C until solvent was completely evaporated (approximately 72 h). The dry glucosinolate-containing precipitate was reconstituted with 1 mL of 0.2 mol L−1 HEPES–KOH buy GDC-0449 (pH 7.0) in the same container. An extract aliquot (10 μL), which was previously reconstituted in 0.2 mol L−1 HEPES–KOH (pH 7.0), was incubated with 5 μL of a thioglucosidase solution

(0.12 U). The thioglucosidase solution contained myrosinase purified from Sinapis alba L. (Sigma–Aldrich), which was buffered in 0.2 mol L−1 HEPES–KOH (pH 7.0) at 37 °C for 24 h; this procedure was in accordance with the methodology of Li and Kushad (2005) which was performed in 3 mL test tubes. In agreement with the degradation reaction of glucosinolates by thioglucosidase, the measurement is accomplished on glucose produced upon glucosinolate hydrolysis. Glucosinolate content was quantified according to the stoichiometry proposed by Palmieri, Iori, and Leoni (1987), which states that 1 mol of released glucose is

equivalent to 1 mol of see more total glucosinolate. The enzymatic catalysis was stopped with the addition of 5 μL of 18 mmol L−1 perchloric acid solution (HClO4). To detect the background levels of glucose in the samples, a control was prepared. The control contained buffered extract (10 μL) with 18 mmol L−1 HClO4 (5 μL), and 5 μL of the thioglucosidase solution was rapidly added. The liberated total glucose was assayed enzymatically by using a glucose oxidase/peroxidase kit (CELM, Brazil). Sinigrin, an allyl-glucosinolate (Sigma), was used

as a calibrant and as a positive control. The sample extraction procedure was identical to the one described for total glucosinolates (n = 3, each in triplicate). The extracts were filtered on Millex™ polyvinylidene fluoride (PVDF) membranes (0.45 μm, Millipore) prior to HPLC injection. The methodology used for the determination of benzylglucosinolate was described by Kiddle et al. (2001) and modified by Rossetto et al. (2008). The calibration curve for benzylglucosinolate and the internal standardization for the sample recovery test were carried out according to Rossetto et al. (2008). A single chromatographic Racecadotril run with an internal standard (50 μL of 12 nmol L−1sinigrinin 1 mL of 70:30 MeOH (mL):water (mL) that also contained 1.49 g L−1 TFA) was also completed to determine the sinigrin (allyl-glucosinolate) retention time. Benzylglucosinolate was isolated by HPLC, which was coupled to an automatic injector and a quaternary pump (HP 1100). The substance was detected by a diode array (PDA) detector at a spectral range of 200–400 nm. A reverse phase column (Luna C18, 250 × 4.6 mm, 5 μm) developed by Phenomenex was used, and the column was coupled to a Security Guard pre-column (Phenomenex). The column temperature was maintained at 25 °C.