4.6. Pancreatic Lipase Inhibition Assay

Inhibitory activity against pancreatic lipase was measured by a slightly modified method of previous report [39]. Briefly, an enzyme buffer was prepared by the addition of a solution of porcine pancreatic lipase, 2.5 mg/mL in buffer containing 10 mM MOPS (morpholinepropanesulfonic acid) and 1 mM EDTA (pH 6.8) to 169 μL Tris buffer (100 mM Tris-HCl and 5 mM CaCl₂, pH 7.0). Then, either 20 μL of extract solution at the test concentration (100 μg/mL) or compound solution (100, 50 and 25 μM) or orlistat (1.0, 0.2, 0.04, 0.008, and 0.0016 μM) was mixed with 178 μL enzyme buffer and incubated for 15 min at 37°C with 2 μL of the substrate solution [10 mM p-NPB (p-nitrophenyl butyrate) in dimethylformamide]. The enzymatic reactions were allowed to proceed for 20 min at 37°C. Lipase activity was determined by measuring the hydrolysis of p-NPB into p-nitrophenol. An increase in light absorption at 405 nm was measured using a microplate reader (Synergy H1; BioTek Instruments, Inc., Winooski, VT, USA). Inhibition of lipase activity was expressed as the percentage decrease in optical density when porcine pancreatic lipase was incubated with the test compounds. Orlistat was used as a positive control. Lipase inhibition (%) was calculated according the following formula:

where A is the absorbance of incubated solution with substrate, a is the absorbance incubated solution with substrate and lipase, B is the absorbance of incubated solution with sample, substrate and lipase, b is the absorbance of incubated solution containing sample and substrate.