2.6. Pancreatic Lipase Inhibition Assay

The pancreatic lipase inhibition assay was performed according to the procedure by Sridhar et al. [23] with slight modification. The enzyme solution was prepared immediately before use, and for this, 25 mg of porcine pancreatic lipase was suspended in 5 ml of the TrisHCl buffer (pH 7.4). The solution was then subjected to centrifugation (4000 rpm, 18°C for 10 min). The supernatant was collected and used. For all, i.e., DCME, the isolated compounds and the positive control (orlistat), different concentrations (100 μg/ml, 50 μg/ml, and 25 μg/ml) were prepared in TrisHCl buffer containing 1% dimethyl sulfoxide. The final 1000 μl reaction mixture is comprised of a preincubated mixture (5 min at 37°C) of 875 μl of buffer, 100 μl of enzyme, and 20 μl of DCME/isolated compounds/orlistat of different concentrations, followed by the addition of 5 μl of the substrate (4-nitrophenyl butyrate, 10 mM in acetonitrile).

The absorbance of the final mixture was determined using the microplate spectrophotometric reader (Spectra Max ABS Plus, USA) after 5 min at 405 nm. The assay was performed in triplicate, and the percentage inhibition was calculated using the formula

where AE is the absorbance of enzyme control (without inhibitor) and AT is the difference between the absorbance of test sample, with and without substrate.