5-Lipoxygenase Inhibition

The 5-LOX inhibition was investigated according to the method of Chung et al. (2009) in which lipoxygenase from soybean (type V) (100 ng/ml) prepared in Tris-HCl (pH 7.4, 50 mM) was incubated for 5 min at room temperature with the test samples and positive control respectively. All test samples were prepared to stock concentrations of 20 mg/ml in DMSO and serially diluted to a concentration range of 400–3.125 ug/ml. A vehicle control containing 0.2% DMSO, a blank containing 5-LOX to which linoleic acid was added after the addition of the FOX reagent, positive control containing caffeic acid at the same concentration range as the extracts, and color controls containing the plant extract and Tris-HCl buffer were included. The reaction was then initiated by the addition of linoleic acid (140 μM) prepared in Tris-HCl (pH 7.4, 50 mM) and incubated for 20 min at room temperature in the dark. The reaction was terminated through the addition of freshly prepared FOX reagent (30 mM sulfuric acid; 100 µM xylenol orange; 100 µM ferrous sulphate, and methanol: water (9:1). The formation of a color complex was allowed to develop for 30 min at room temperature, and the absorbance was measured at 560 nm using a BIO-TEK Power-Wave XS multi-well plate reader (A.D.P, Weltevreden Park, South Africa) and KC Junior software. The IC₅₀ values were calculated using GraphPad Prism 4 software.