2.4. Alpha-amylase inhibitory assay

The assay was performed by using a previous method with minor modifications [13]. Two hundred fifty microliters of various ME concentrations (0.125–2.0 mg/mL) were prepared in a tube. The same volume of 20 mM sodium phosphate buffer (pH 6.9) containing the α-amylase solution (0.5 mg/mL) was added. Following preincubation at 25 °C for 10 minutes, 250 µL of 1% starch solution in 20 mM sodium phosphate buffer (pH 6.9) was added and left at 25 °C for 10 minutes. The reaction was stopped by adding 500 µL of dinitrosalicylic acid reagent. Subsequently, the tube was incubated in a water bath (100 °C) for 5 minutes and cooled to 25 °C. The reaction mixture was diluted with distilled water and the absorbance was measured at 540 nm with a spectrophotometer. The inhibition of α-amylase activity was determined by the following formula:

Where, A is the absorbance of the control and B is the absorbance of the test sample. Acarbose was used for the standard reference.