Pancreatic α amylase from porcine pancreas

The α‐amylase inhibitory potential of AFE was determined by the previously described method.21 Briefly, pancreatic α‐amylase from porcine pancreas (Sigma‐Aldrich) or salivary α‐amylase from human saliva (Sigma‐Aldrich) was prepared in sodium acetate buffer (0.1 mol L^–1, pH 4.8). Different concentrations of AFE (1 mL, 7.8–500 μg mL⁻¹) were incubated with α‐amylase (1 mL, 5.0 U mL⁻¹) for 10 min at 37 °C. Starch (1 mL, 0.5% w/v) was added and the mixtures were incubated for 30 min at 37 °C, followed by the addition of DNS solution (2 mL, 40 mmol L^–1 DNS, 1 mol L^–1 sodium potassium tartrate, 0.4 mol L^–1 sodium hydroxide) and incubated in a boiling water bath at 100 °C for 15 min. After boiling, 9 mL of demineralized water was added and the solution cooled to room temperature. The optical density was measured at a wavelength of 540 nm using a spectrophotometer (Shimadzu Corporation, Kyoto, Japan). A sample without adding AFE served as the untreated control. Acarbose was used as an experimental reference control. Each analysis was performed in triplicate on two different occasions. The following formula was used to calculate α‐amylase inhibition:
$\begin{array}{cc}\text{Inhibition}& \left(\%\right)=\\ & 100-\left(\frac{\text{Absorbance of sample}-\text{Absorbance of blank}}{\text{Absorbance of control}}\times 100\right)\end{array}$