The two plates were incubated for 1 h at 37 °C then 160 μl of
the second plate was added to the first plate to initiate the reaction. To calculate the percentage of lipase inhibition, the reagent blanks were subtracted from the corresponding controls or samples and the following formula was applied: Percentage of Lipase Inhibition=1-((Polymer Sample-Inhibition Control)/(Lipase Control-Inhibition Control))×100Percentage of Lipase Inhibition=1-((Polymer Sample-Inhibition Control)/(Lipase Control-Inhibition Control))×100 The olive oil assay system uses a modified version of the method of Vogel and Zieve (1963). The turbidimetric method measures this website the reduction in turbidity that occurs following the breakdown of TAGs to free fatty acids by lipase. Olive oil, with a specific viscosity of 72.5 (±10), (specific viscosity used here is unitless as it is derived from a ratio of the oil to check details that of water) was used throughout the series of experiments. The olive oil was passed through aluminium oxide (80 × 15 mm deep in a glass chromatography column) to remove free fatty acids. 10.0 g of the olive oil free from fatty acids was made up to 100 ml with acetone giving a 10% solution. This is turn was diluted 1 in 10 with acetone to achieve a 1% olive oil stock solution. The stock solution was stored at 4 °C for up to four weeks and was used over the entire series of experiments. For use in the assay, an olive oil
substrate solution was prepared by adding 4 ml of 1% stock olive oil solution to a heated solution (70 °C) of 100 ml 0.05 M Tris buffer at pH 8.3 containing 0.35% sodium deoxycholate. This solution was maintained at 70 °C and homogenised for 10 min. Once the froth had settled and the solution had returned to room temperature, the substrate solution could be used in the assay for up to 6 h. The enzyme solution contained
1.29 mg/ml lipase and 18 μg/ml colipase in deionised water. Orlistat was added (0.025 mg/ml) to the enzyme solution as an inhibition control. Biopolymers were added to the freshly prepared substrate solution containing the olive oil to give 3.6, 0.9 and 0.23 mg/ml. The samples were incubated at 37 °C for 15 min. After the incubation the substrate solution was added to the solution containing the Flavopiridol (Alvocidib) enzyme solution or deionised water. The assay was maintained at 37 °C and read every 5 min at 405 nm for 35 min. To calculate the percentage of lipase inhibition, the blanks were subtracted from their respective controls and the following equation was applied Percentage of lipase inhibition=1-((Inhibition Control-Polymer Sample)/(Inhibition Control-Lipase Control))×100Percentage of lipase inhibition=1-((Inhibition Control-Polymer Sample)/(Inhibition Control-Lipase Control))×100 All data were analysed using GraphPad Prism 4 statistical software. The comparison of inhibition levels with seaweed species was made by using a two way ANOVA.