2.7. Pancreatic Lipase (PL) Inhibition

The porcine pancreatic lipase (PPL) inhibitory assay was conducted using the methods from Jaradat et al. [34], Bustanji et al. [35], and Siew-Ling et al. [36] with some modifications. VOs stock solution of 1mg/mL was prepared in 10% Dimethyl sulfoxide (DMSO) and diluted with DMSO 10% to produce different concentrations (10, 50, 100, 150, 200, 400, 600, and 800 μg/mL). Orlistat was used as a reference for pancreatic lipase inhibition assay and was prepared by the same procedure of plant extract. Pancreatic lipase enzyme stock solution was prepared immediately before use by suspending in DMSO10% at concentration of 1 mg/mL. The stock solution of p-nitrophenyl butyrate (PNPB) was prepared according to manufacturer's instructions (20.9 mg of PNPB in 2 mL of acetonitrile). From each working solution of plant extract, 200 μL was mixed with 100 μL of porcine pancreatic lipase (1 mg/mL), and then the volume was brought to 1000 μL with Tris-HCl solution and incubated at 37°C in water bath for 15 min. After the incubation time, 100 μL of PNPB solution was added. The mixture was again incubated in water bath for 30 min at 37°C. A negative control solution was prepared without plant extract, by mixing 100 μL of porcine pancreatic lipase (1 mg/mL) solution with Tris-HCl solution up to 1mL. The same procedure was followed for Orlistat used as positive control. Tris-HCl buffer was used to zero UV-Vis spectrophotometer at 405 nm. Pancreatic lipase activity was determined by measuring the hydrolysis of p-nitrophenolate to p-nitrophenol at 405 nm using UV-Vis spectrophotometer. The lipase inhibition activity of M fruticosa serpyllifolia VOs or Orlistat as a reference was identified by measuring the effect on the enzyme reaction rate after adding extracts, compared with the control. I% was calculated by the using the following equation [37].

where I% is the percentage inhibition of pancreatic lipase.