Porcine pancreatic α amylase enzyme

The new benzophenone derivatives (6 and 7) and the known xanthone (5) were evaluated for their AAI potential at concentrations of 5, 10, 20, and 40 µM [2 (link),3 (link),4 (link),35 (link),36 ]. The method is based on the assay of α-amylase using EnzChek^® Ultra-Amylase Assay Kit (E33651) (Thermo-Fisher Scientific Inc., Waltham, MA, USA). The provided stock solution of dye quenching (DQ^TM) starch and porcine pancreatic α-amylase enzyme (Sigma-Aldrich, Hamburg, Germany) were diluted with the reaction buffer (pH 6.9) according to the reported protocol [36 ]. To the microplate wells, the tested compound (10 µL) in DMSO, diluted enzyme (50 µL), and 40 µL of the reaction buffer were added and allowed to stand at room temperature for 5 min, then DQ^TM starch (100 µL) was added. The fluorescence intensity of the digestion products from the DQ^TM starch (with or without compounds) was measured using a Tecan Genios microplate reader at λ_max 485 ± 10 nm starting from zero min to 60 min at 10 min intervals. All experiments were performed in triplicate. Acarbose was utilized as positive control. The IC₅₀ values were calculated by linear regression analysis [2 (link),3 (link),4 (link),35 (link),36 ].

Benedict^’s and Millon’s reagents were purchased from Gadot (United States). Ninhydrin solution, Molish^’s reagent, H₂SO₄, chloroform, magnesium ribbon, HCl, FeCl_3, NaOH, and iodine solution were obtained from Alfa Aesar (England). Porcine pancreatic α-amylase enzyme, dimethyl sulfoxide (DMSO), Trolox ((s)-(−)-6 hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid), Folin-Ciocalteu’s reagent, porcine pancreatic lipase enzyme, dinitrosalicylic acid (DNSA), Doxorubicin, and 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (Germany). Gallic acid, porcine pancreatic lipase enzyme, methanol, 1% L-glutamine, Dragendroff reagent, 1% Penicillin/Streptomycin, and sodium carbonate were brought from Merck (Darmstadt, Germany).

The RDS, SDS and RS contents were determined following the method of Englyst et al. (1992) with some modifications: Porcine pancreatic α-amylase enzyme (6.0 g, 8 × USP activity/g), (No. 7545, Sigma-Aldrich, St. Louis, MO) was dispersed in water (40.0 mL) by magnetic stirring for 10 min. Subsequently it was centrifuged at 15000 x g. The supernatant (32.0 mL) was mixed with the amyloglucosidase enzyme solution (2.0 mL, activity; 5,000–8,000 units/mL) (No. 9913, Sigma-Aldrich) and deionized water (3 mL). This enzyme solution was prepared fresh for each digestibility analysis. For the samples of native or hydrolyzed pigeon pea flour, 1 g was used in a test tube and 10 mL of guar gum solution (5 g/L in 0.05 M HCl) and 5 mL of 0.5 M sodium acetate solution (pH 5.2) were added. Only the blank was gelatinized to increase digestibility. Each was then transferred to a 50 mL test tube. The total starch content in the starch samples was measured according to Sandhu and Lim (2008) . The content of RDS, SDS and RS was calculated using 30 min as incubation time. Classification of the starch, based on its digestibility was: RDS as the starch that was hydrolyzed within 30 min of incubation, RS as the starch not hydrolyzed within 120 min, and SDS as the starch digested during the period between 30 and 120 min.