3.8. Pancreatic Lipase Inhibitory Activity Assay In Vitro

Lipase activity was measured using p-NPB (p-nitrophenyl butyrate) as substrate. The method was modified from the one previously described by Kim et al. [49]. Briefly, an enzyme buffer was prepared by the addition 20 μL of solution of porcine pancreatic lipase (20 mg/mL in TRIS buffer, pH 7) to 160 μL of Tris buffer (100 mM Tris-HCl and 5 mM CaCl2, pH 7.0). Then, increasing concentrations of various extracts (ranging from 0 to 11.25 mg/mL) dissolved in TRIS buffer were mixed with 20 μL of the enzyme buffer and incubated for 30 min at 37 °C. Twenty µL of substrate (10 mM p-NPB in dimethyl formamide) were then added. Lipase activity was determined by measuring the hydrolysis of p-NPB to p-nitrophenol at 405 nm using an ELISA reader. The inhibition of lipase activity was expressed as the percentage of absorbance decrease when porcine pancreatic lipase was incubated with the test compounds. Lipase inhibition (%) was calculated according the following formula:

where A is the lipase activity in the reaction solution without sample and B is the lipase activity in the reaction solution containing sample. The measurements were made in triplicate and the IC₅₀ (inhibitory concentration at which 50% inhibition of enzyme activity occurs) values of the test samples were determined by performing the assay as above with varying concentrations of test samples. The IC₅₀ values were determined from the regression curves of inhibition percentage vs extract concentration.