2.7. In Vitro α-Amylase Inhibition Assay

The α-amylase inhibition activity of morin was assessed using 3, 5-dinitrosalicylic acid (DNSA) assay [27]. Morin, phloroglucinol, and 1-hexyl benzene were dissolved in mixture of 10% DMSO, 0.02M Na₂HPO₄/NaH₂PO₄ buffer, and 0.006M NaCl (pH 6.9) and finally dilutions (31.05, 62.5, 125, 250, 500 and 1000 µg/mL) were prepared. In total, 200 μL α-amylase (2 units/mL) was mixed with 200 μL morin and incubated at 30 °C for 10 min. After that, 200 μL starch (1%) was added to each serial dilution and incubated for 3 min. To stop the reaction 200 μL sodium potassium tartrate tetrahydrate reagent, 2M NaOH, and 20 mL DNSA (96mM) reagent were added to the reaction mixture. The reaction mixtures were boiled for 10 min at 85–90 °C in a water bath. After cooling the mixture was diluted with 5mL distilled water and finally at 540 nm absorbance was recorded. A blank solution was prepared containing only isolated compound morin but no enzyme. Standard acarbose (100 μg/mL–2 μg/mL) was prepared as positive control. The α-amylase enzyme inhibitory activity was calculated by the following Equation (2):