4 nitrophenyl α d glucopyranoside pnp g

Ascorbic acid, 1,1-diphenyl-2-picrylhydrazyl (DPPH^●), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS^●+), neocuproine, trichloroacetic acid (TCA), and potassium ferricyanide were used for the determination of the antioxidant activities and were purchased from Sigma Chemical Co. (Sigma-Aldrich GmbH, Stern-heim, Germany). The following chemicals were used for the enzyme inhibition assays: α-glucosidase from Saccharomyces cerevisiae, 4-nitrophenyl α-D-glucopyranoside (pNPG), α-amylase from porcine pancreas, β-nicotinamide adenine dinucleotide (β-NADH), phenazinemethosulphate (PMS), nitrotetrazolium blue chloride (NBT) and Ascorbic acid were obtained from Sigma (St. Louis, MO, USA), while acarbose and potato starch were purchased from Fluka (Bucharest, Romania) and Fisher (Pittsburgh, PA, USA), respectively. Sodium nitroprusside, sulfanilamide, and 3,5-dinitrosalicylic acid (DNS) were obtained from Acros Organics (Hampton, NH, USA). Folin–Ciocalteu reagent, Na₂CO₃, and gallic acid were purchased from Panreac (Barcelona, Spain). Standards used for the elucidation of the phenolic compounds and for elaboration of the calibration curves were obtained from EXTRA SYNTHESE (Genay Cedex, France). Acetonitrile HPLC-grade and formic acid were purchased from Panreac (Barcelona, Spain). All other chemicals were of analytical grade.

The α-glucosidase inhibition was assessed according to the slightly modified method of Ma et al. [30 (link)]. All samples were dissolved in DMSO at a concentration of 50 μM. The α-glucosidase (Sigma Chemical Co., St. Louis, MO, USA) and substrate (4–Nitrophenyl α-D-glucopyranoside, PNPG, Sigma Chemical Co., St. Louis, MO, USA) were dissolved in potassium phosphate buffer (0.1 M, pH 6.7). The samples were preincubated with α-glucosidase at 37 °C for 10 min. Then, PNPG was quickly added to the 96-well enzyme label plate to start the reaction, and the plate was incubated at 37 °C for 50 min. At the same time, a blank control without samples and a positive control of quercetin (10 mM) was set up. All samples were thoroughly mixed and analyzed in triplicate. The OD value was measured at 405 nm using a microplate reader. The inhibition percentage (%) was calculated by the following equation: Inhibition (%) = (1 − OD _sample)/OD _{control blank} × 100.

Ascorbic acid (L(+)-ascorbic acid powder) was purchased from AppliChem (Darmstadt, Germany). Glucose (D-glucose) was obtained from Winkler (Santiago, Chile). The De Man, Rogosa, and Sharpe (MRS) agar was purchased from OXOID (Basingstoke, Hampshire, England). The Folin–Ciocalteu reagent, sodium carbonate (Na₂CO₃), monopotassium phosphate (KH₂PO₄), dipotassium phosphate (K₂HPO₄), methanol, sodium chloride (NaCl), starch, and sodium and potassium tartrate tetrahydrate (C₄H₄KNaO₆·4H₂O) were obtained from Merck (Darmstadt, Germany). Gallic acid (3,4,5-trihydroxybenzoic acid) was purchased from Acros Organics by Thermo Scientific (Waltham, MA, USA). The DPPH reagent (2,2-diphenyl-1-picrylhydrazil), fluorescein sodium salt, 2,2′-azo-bis(2-amidino-propane) dihydrochloride (AAPH), Trolox, α-amylase from porcine pancreas enzyme, 3,5-dinitrosalicylic acid (DNS), sodium hydroxide (NaOH), acarbose, α-glucosidase enzyme, and 4-nitrophenyl α-D-glucopyranoside (PNPG) were obtained from Sigma-Aldrich (Saint Louis, MO, USA).

α-Glucosidase (EC3.2.1.20) from S. cerevisiae, 4-nitrophenyl-α-D-glucopyranoside (PNP-G) as a synthetic substrate, alloxan, and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO, USA), acarbose was biological reagent, and K₂HPO₄·3H₂O, NaOH, and others were of reagent grade. Norathyriol and mangiferin were provided by Research Department of Kunming Pharmaceutical Corporation (Kunming, China).