G5003

Alpha-glucosidase enzymatic activity was measured as previously described [18 (link)] by quantifying the amount of p-nitrophenol released from p-nitrophenyl-alpha-D-glucopyranoside. Each assay volume contained 2 mM p-4-nitrophenol glucopyranoside (p-NPGP, Sigma N1377), phosphate buffer (0.1 M, pH 6.8), alpha-glucosidases from Saccharomyces cerevisiae (0.1 U, Sigma G5003) or an intestinal crude extract (Sigma I1630), and 1 mL of the control drug acarbose or the experimental extract at concentrations ranging from 0.2 μg/mL to 1,000 μg/mL. The reaction was monitored for 480 s. Measurements were acquired every 30 s at 405 nm with a BioTek spectrophotometer (model ELx800). Assays were performed in duplicate.

The inhibitory activity of free polyphenols extracts (FP, FP-95) and polysaccharide–polyphenol conjugates (AE, PHWE-95 and PHWE-120) extracts was conducted with pancreatic porcine α-amylase (A6255, Sigma Aldrich) and α-glucosidase from Saccharomyces cerevisiae (G5003, Sigma Aldrich) using ethylidene-4-nitrophenyl-α-D-maltoheptaoside and 4-nitrophenyl-α-D-glucopyranoside as enzymes substrates (for α-amylase and α-glucosidase, respectively). All solutions were prepared in 10 mM phosphate buffer saline solution (pH 6.8), except for FP and FP-95 which were prepared in DMSO. First, α-amylase (15 U.mg⁻¹) or a-glucosidase (0.011 U.mg⁻¹), alone or with different concentrations of each fraction, was pre-incubated at 37 °C for 10 min. Then, substrate at 2.5 mM or 0.269 mM (a-amylase and a-glucosidase, respectively) was added to the reaction mixture and the reaction was followed for 50 min at 405 nm and 37 °C on a plate reader (FlexStation 3 Multi-Mode Microplate Reader). Acarbose, an oligosaccharide of microbial origin that is used as inhibitor of carbohydrate digestion for clinical management of T2DM [43 (link)], was used as the positive control. Results were expressed as inhibition percentage (Equation (1)) and a non-linear regression dose–response curve was established to calculate IC₅₀ values (µg.mL⁻¹) of each fraction.
$$Inhibition\%=100-\left(\frac{A_{control}-A_{sample}}{A_{control}}\times 100\right)$$

α‐glucosidase type 1 from baker‘s yeast (G5003; Sigma‐Aldrich, St. Louis, MO, USA), p‐nitrophenyl α‐D‐glucopyranoside (N1377, Sigma‐Aldrich), sodiumphosphatemonobasic (S3139, Sigma‐Aldrich), sodiumphosphatedibasic (S5136, Sigma‐Aldrich), andacarbose (A8980, Sigma‐Aldrich), DMSO (Dimethylsulfoxide), α‐amylase from Aspergillus oryzae (Sigma Aldrich), starch, DNS (3, 5‐dinitrosalicylic acid), sodiumpotassiumtartratetetrahydrate.

Bovine serum albumin (EC Number 232-936-2, A8806), α-glucosidase (EC Number 3.2.1.20 Saccharomyces cerevisiae, G5003), and p-nitrophenyl-α-D-glucopyranoside (pNPG, N1377) were purchased from Sigma-Aldrich (St. Louis, MO, USA), while acarbose was purchased from TCI Chemicals (Hydrabad, India). A specific spray reagent, citroborate (5 g citric acid, 5 g boric acid, and 100 mL ethanol), and 10% (v/v) H₂SO₄ in a MeOH solution were used to identify flavonoids and other compounds. Additionally, during extraction and separation, the entire solvents were distilled before use, making them of technical grade.