α amylase from human saliva

We investigated the effect of CM3 on amylase activity using α-amylase from human saliva (Sigma-Aldrich, Steinheim, Germany). At first, 10 μl of α-amylase (8 U/ml) were incubated for 15 min with 40 μl CM3 (2 μM), amylase inhibitor (AI) (2 μM) (A1520, Sigma-Aldrich, Steinheim, Germany), acarbose (1, 10, 100 μM) (Sigma-Aldrich, Steinheim, Germany) or 66.43 μg/ml BSA in PBS in a 96 well plate at 37°C under continuous shaking. Subsequently, 30 μl of starch (1.3 mg/ml) dissolved in water dH2O was added and the enzymes were given 15 min at 37°C to catalyze hydrolyzation of the glycosidic bonds. The reaction was stopped by the addition of 20 μl of 1 M HCl. One hundred microliter of lugol solution (diluted 1:40, Carl Roth, Germany) was added to detect remaining starch. The iodine from lugol solution forms an intermolecular charge-transfer complex with the starch resulting in a blue color. Absorbance was measured at 580 nm with Spectramax id3 microplate reader (Molecular Devices, CA, USA).

All reagents were of analytical purity. The 2,2-diphenyl-1-picrylhydrazyl (DPPH), ABTS diammonium salt (2,2-azinobis-3-ethylbenzothiazoline-6-sulphonic acid), Folin–Ciocalteu’s reagent, standards of Trolox and gallic acid were purchased from Sigma-Aldrich Corp. (Milan, Italy). Bile salts were from Oxoid™ (Hampshire, U.K.). Spectrapor 3 45 MM/15 M membrane (cut-off 3.5 kDa) was purchased from Fischer Scientific (Milan, Italy). Pancreatin (from porcine pancreas: P3292-100G), pepsin (from pig gastric mucosa: ≈2500 units/mg protein), α-amylase from human saliva (A0521-500 units/mg), formic acid, potassium sorbate, sodium carbonate, trifluoroacetic acid (TFA), and peptidyl-dipeptidase were purchased from Sigma-Aldrich Corp. (Milan, Italy).
Hydroxytyrosol, tyrosol, oleuropeion, apigenin, luteolin and myricetin were purchased from Extrasynthese (Lyon, France); caffeic acid, chlorogenic acid, pinoresinol, and ligstroside were purchased from Sigma-Aldrich Corp. (Milan, Italy). Acetonitrile was purchased from Pai Acs, Panreac. All other chemicals and solvents were of the highest analytical grade from Sigma-Aldrich Co. (St. Louis, MO, USA).

GS-GN was incubated with α-amylase from human saliva (Sigma) to deglycosylate the complex. Reactions contained 4 μM GS-GN with either 500 mU or 1 U α-amylase in buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP and 5 mM CaCl₂. Reactions were incubated for 30 min, 1 h or 2 h at 37 °C and terminated by the addition of Laemmli buffer.

Fast Blue BB (FBBB) [4-(benzoylamino)-2,5-dimethoxybenzenediazonium chloride hemi-(zinc chloride), bile extract porcine, pancreatin from porcine pancreas, pepsin from porcine gastric mucosa, α-amylase from human saliva, 2,2′-diazobis-(2-aminodinopropane)-dihydrochloride (AAPH), fluorescein, and GABA (>99% purity) were purchased from Sigma-Aldrich, Co. (St. Louis, MO, USA).