Millipore membrane 0.45 m

Phenolic compounds extraction was carried out according to method of [15] . 500 mg callus sample from each treatment was placed in 10 mL of methanol (96 %). The mixture was then incubated in the dark for 10 h at 4 ° C. After centrifugation at 5000 rpm during 10 min, the supernatant obtained was filtered through a Millipore membrane (0.45 µm) and formed the crude phenolic extract.

The assay was made in 96-wells microplates according to Kiho and coworkers [17, (link)19] .
The enzyme solution was prepared by dissolving 3 mg of rat intestinal acetone powder (Sigma®, USA) in 1 mL of buffer phosphate (pH 6.9). The solution was centrifuged at 5000 rpm for 15 min, filtered by a Millipore® membrane (0.45 µm), and the supernatant was used for the assay. The standard and the tested substances were prepared in concentrations of 2.5, 5, 10, 15, 20, 25, 50, and 100 μg/mL. In the microplate wells were placed 30 μL of DMSO (used as the control), the standard solution of Acarbose®, and the SDE solutions. To each well was added 170 µL of enzyme solution. Microplates were incubated protected from light, for 5 min, at 37 °C. After that, 100 μL of 4-nitrophenyl α-d-glucopyranoside was added to each well and incubated again for 20 min. The analysis of the percent of inhibition (%I) was performed in triplicate and it was determined using equation 5:
A a/p = Absorbance of the Sample-Absorbance of the blank, A C = Absorbance of the negative control-Absorbance of the Blank.
The blank solution was prepared as described above for evaluating the antioxidant effect. The IC 50 values were estimated by Probit analysis using the GraphPad® Software (USA), at a level of significance of p=0.05.

The radical scavenging activity was evaluated by spectrophotometry (Shimadzu®, Japan), using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical [10, (link)17, (link)18] . For biological assays, the blank solution was prepared by dissolution of 0.25 g of colloidal silicon dioxide plus 5 g of lactose monohydrate in 100 mL of PBS buffer (pH 7.4). The mixture was sonicated for 10 min, filtered by a Millipore membrane (0.45 µm), and diluted at the same concentrations used for the SDE (2.5, 5, 10, 15, 20, 25, 50 and 100 µg/mL).