The ferrous oxidation-xylenol orange (FOX) assay method for determination of lipid hydroperoxides is based on that under acidic conditions Fe2+ is oxidized to Fe3+, which then oxidizes xylenol orange to a product that absorb at 550 nm. The procedure has been adapted for determination of lipoxygenase activity in plant extracts. This enzyme is responsible for generation of off-flavors in vegetal foods, bleaching of pigments, and a lot of oxidative degradations. It is of interest to check the initial lipoxygenase activity in vegetal foods before the processing, using an assay that is rapid, reproducible, and easily adaptable to high throughput format. The enzymatic assay is based on a discontinuous determination of lipoxygenase activity using the FOX reagent for colorimetric determination of hydroperoxides accumulated in the medium by a period of incubation that is established by the addition of the extract (start of the reaction) and the addition of FOX reagent (finish of the reaction). The procedure is capable of detecting lipoxygenase activity in a number of vegetable homogenates, being especially useful for a rapid visual evaluation of this enzymatic activity.
Lipoxygenase, in the presence of hydrogen peroxide, produces the oxidative decomposition of quercetin, naringenin, and resveratrol, known antioxidant molecules. Quercetin was the molecule more efficiently oxidized, followed by resveratrol and naringenin. When this molecule was incubated in the presence of GSH, a quinoid derivative was produced. This compound was not obtained in the presence of naringenin or resveratrol, suggesting that in the presence of hydrogen peroxide and lipoxygenase, quercetin may be oxidized to a prooxidant species. When hydrogen peroxide was substituted by hydroperoxy linoleic acid, the same oxidative process was observed. This means that in food products in which lipoxygenase and linoleic acid are presents, quercetin may be oxidized to prooxidant species; in contrast, naringenin and resveratrol may constitute a valid additive for the prevention of the oxidative degradation of foods. Keywords: Oxidation; polyphenolics; hydroperoxidase; lipoxygenase; linoleic acid; quercetin; naringenin; resveratrol; antioxidants
In this work the oxidative degradation of resveratrol catalyzed by lipoxygenase-1 (LOX-1) has been studied. The process has been characterized by spectroscopic and polarographic measurements. The oxidation of resveratrol was dependent on the concentration of resveratrol and the enzyme. When resveratrol was incubated in the presence of lipoxygenase at pH 9.0, the reaction displayed a k(M) value of 18.6 x 10(-)(6) M and a catalytic efficiency (k(cat)/k(M)) of 4.3 x 10(4) s(-)(1) M(-)(1). These values are close to those shown by the enzyme when linoleic acid is used as the substrate. The effect of lipoxygenase inhibitors on the lipoxygenase-catalyzed resveratrol oxidation was also evaluated. The rate of resveratrol oxidation was markedly decreased by the presence of NDGA in the incubation mixture. From HPLC measurements, it can be deduced that resveratrol is oxidatively decomposed to a complex mixture of products similar to those obtained when the molecule is oxidized by hydrogen peroxide.
Despite the abundant information about the antioxidant properties of individual components of wine, the studies about the efficiency of wine as inhibitor of enzymatic systems involved in oxidative processes are scarce. In this work we describe the effect of wine on the activity of soybean lipoxygenase and human recombinant 5-lipoxygenase. This prooxidant enzyme is involved in inflammatory processes and stress oxidative.In the presence of increased concentration of wine phenolics, a reduction of the lipoxygenase activity in a dose-dependent mode is observed for soybean and human recombinant enzymes. Our results clearly show the high efficiency of wine as inhibitor of lipoxygenases. This anti-lipoxygenase activity of wines is related with their Trolox equivalent antioxidant capacity, being the total content of phenolic compounds the main factor responsible for the antioxidant efficiency of wines. PRACTICAL APPLICATIONSIn this study we show the high efficiency of phenolic compounds present in wines as lipoxygenase inhibitors. In addition to the potential relevance of this effect at a pharmacological level, it is known that lipoxygenase activity is the origin of the off flavor generation in a number of foods from animal and vegetal sources. On the basis of the anti-lipoxygenase activity shown by phenolics present in wines, the use of these compounds as food preservatives may contribute to enhance the oxidative stability of foods and, as consequence, the phenolic extracts obtained from wines or from by products of the winemaking may be of great interest for the agrofood industry.
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