2.7.2. Inhibition Assays for α-amylase Activity

The α-amylase inhibition was determined by 96-well microtiter plate method based on calorimetric assay as previously described by Balasubramaniam et al. [24]. Equal volume of test samples (5 mg mL−1) and α-amylase solution (0.5 mg mL−1, Sigma) prepared in 30 mM phosphate buffer (pH 7.0) was pre- incubated at 37 ◦C for 10 min. 50 μL of 0.5% starch solution was added and incubated for 10 min at 37 ◦C. 120 μL of DNS reagent (Sigma) was added to stop the reaction. The reaction mixture was incubated at 95 ◦C for 5 min, cooled to room temperature. Absorbance was measured at 540 nm in a microplate reader. Acarbose at the concentration 2 mg mL−<sup>1</sup> was taken as positive control. The inhibition percentage of amylase was determined by the formula reported in the previous paragraph.

#### *2.8. Cytotoxicity Assay*

The human cell lines HT-29 (Colon cancer), MDA (Breast cancer) and U-87 MG (Brain cancer) were procured from National Centre for Cell Science, Pune, India. The cell lines were cultured and maintained in Dulbecco's modified Eagle's medium (DMEM, Sigma) in T-flasks in the incubator at 37 ◦C and internal atmosphere of 95% air and 5% CO2. The cytotoxicity was determined by standard MTT dye assay, according to the method described by Carmichael et al. [25]. Briefly, varying

concentration of extracts were dissolved in 1% DMSO and treated to cells seeded in 96 well tissue culture plates. The plates were kept for incubation at 37 ◦C for 24 h, MTT solution (Sigma) was added and incubated for 4 h at 37 ◦C. The amount of purple formazan crystals resulting from the reduction of MTT dye by succinic dehydrogenase in mitochondria of the viable cells was determined by measuring OD at 570 nm. The IC50 value was calculated using graph pad prism. Each assay was performed in triplicate.
