Porcine pancreatic lipase inhibition assay

Stock solutions of 500 μg/ml were made from each plant fraction in 10% DMSO. From these, a dilution series of five concentrations of 50, 100, 200, 300, and 400 μg/ml were made. A 1 mg/ml stock solution of porcine pancreatic lipase in Tris-HCl buffer was prepared freshly just before use. The substrate, p-nitrophenyl butyrate (PNPB) was prepared by dissolving 20.9 mg in 2 ml acetonitrile.

For each working solution, 0.1 ml porcine pancreatic lipase was mixed with 0.2 ml plant fraction from each member of the dilution series. Tris-HCl was added to make the final volume of the working solutions 1 ml, and they were incubated at 37 °C for 15 min. After incubation, 0.1 ml p-nitrophenyl butyrate solution was added to each test-tube. The mixture was then incubated for a further 30 min at 37 °C. Pancreatic lipase activity was determined by measuring the hydrolysis of PNPB into p-nitrophenolate at 410 nm, using a UV spectrophotometer. The same procedure was repeated using orlistat as a standard reference compound. Percentage lipase inhibition by plant fractions was calculated with the following equation:

where, A_B is the recorded absorbance of the blank solution, and A_ts is the recorded absorbance of the tested sample solution [24].