Porcine pancreatic lipase

Approximately 20 mg of liver was placed in 500 μL of chilled PBS (pH 7.4) and homogenized. The homogenate was centrifuged at 2000×g for 10 min at 4 °C and 100 μL of the supernatant was then removed for free glycerol and total protein analysis. Triglyceride content was measured using a method adapted from Starke et al. [25 (link)]. The remaining liver homogenate was incubated with 100 μL of lipase (porcine pancreatic lipase, MP Biomedicals, Santa Ana, CA) for 16 h at 37 °C, and glycerol content was then determined by an enzymatic glycerol phosphate oxidase method (Sigma-Aldrich Co.). Triglyceride content was calculated based on the difference between glycerol concentrations before and after lipase digestion. Total protein content of the original homogenate was analyzed by a Coomassie blue [26 (link)] colorimetric method (Thermo Scientific, Pierce, Rockford, IL). Liver TG concentration was normalized by protein concentration, which is unaltered in fatty liver [27 (link)], to avoid potential bias introduced by differences in water content of liver samples.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) (Sigma Aldrich, Germany) and ((s)-(-)-6 hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid) (Sigma Aldrich, Denmark). n-hexane (Frutarom Ltd, Israel). Methanol (Lobachemie, India), The solvents used for the extraction of plant material were of HPLC grade. Sigma-Aldrich (USA) provided porcine pancreatic lipase, starch, acarbose, DPPH (2,2-diphenyl-1-picrylhydrazyl), PNPP (p-nitrophenyl palmitate), and orlistat, while MP Biochemicals (Illkirch, France) provided porcine pancreatic α-amylase. All the chemicals used in the experiments were of analytical grade. O. basilicum seeds were obtained from different regions in Palestine. The plant seeds were characterized in the Pharmacy Department at An-Najah National University. The plant material was collected in accordance with the WHO Guidelines for the Assessment of Herbal Medicines and Legislation.
The rotary evaporator (Heidolph OB2000, VV2000, Germany), the grinder (Moulinex model, Uno, China), the balance (Radwag, AS 220/c/2, Poland), and the filter papers (Machrery-Nagel, MN 617, Whatman no. 1, USA) were all used.