ORAC-Fluorescein Assay To Determine the Oxygen Radical Absorbance Capacity of Resveratrol Complexed in Cyclodextrins C. LUCAS-ABELLÁN, M. T. MERCADER-ROS, M. P. ZAFRILLA, M. I. FORTEA, J. A. GABALDÓN, AND E. NÚÑEZ-DELICADO* Dpto. de Ciencia y Tecnología de Alimentos, Universidad Católica San Antonio de Murcia, Avenida de los Jerónimos s/n. 30107 Guadalupe, Murcia, Spain The effect of the complexation of resveratrol with hydroxypropyl--cyclodextrins (HP--CDs) on the antioxidant capacity of the polyphenol is studied for the first time by means of the oxygen radical absorbance capacity (ORAC) method, using fluorescein (FL) as the fluorescent probe. The method is validated through its linearity, precision, and accuracy for measuring the ORAC of resveratrol in the absence or presence of cyclodextrins (CDs). The complexation of resveratrol in CDs increased the net area under the FL decay curve (net AUC) of resveratrol up to its saturation level, at which the polyphenol showed almost double the antioxidant activity it shows in the absence of CDs. The complexation constant (K c ) between resveratrol and HP--CDs was calculated by linear regression of the phase solubility diagram (K c ) 18048 M -1 ). The antioxidant activity of resveratrol was dependent on the complexed resveratrol because CDs acts as a controlled dosage reservoir that protects resveratrol against rapid oxidation by free radicals. In this way, its antioxidant activity is prolonged and only reaches its maximum when all the resveratrol is complexed. KEYWORDS: Resveratrol; cyclodextrin; ORAC assay; antioxidant; fluorescein INTRODUCTION Resveratrol (trans-3,5,4′-trihydroxystilbene) is a natural polyphenol found in a variety of plants, for example in grapes and peanuts (1), where it is produced during environmental stress or pathogenic attack (2). It is found principally in the skins and seeds of grape berries and therefore usually occurs in greater concentrations in red than in white wines due to skin contact during fermentation. This phytoalexin has been reported to have antiplatelet (3), anti-inflammatory (4, 5), and anticarcinogenic effects (6). The various activities of resveratrol have been attributed to its antioxidant properties (7), which contribute to controlling the intracellular redox balance by inhibiting the formation of reactive oxygen species (6). The phenolic nature of resveratrol explains its antioxidant activity. The high hydrophobicity of resveratrol and its sensitivity to external agents such us air, light, and oxidative enzymes may constitute a serious problem for its bioavailability in the formulation and manipulation of functional foods. In recent years, cyclodextrins (CDs) complexation has been successfully used to improve the solubility, chemical stability and bioavailability of a number of poorly soluble compounds. CDs are a group of naturally occurring cyclic oligosaccharides derived from starch with six R-cyclodextrins, seven -cyclo- dextrins, or eight γ-cyclodextrins glucose residues, linked by R(1f4) glycosidic bonds (8). In the pharmaceutical, cosmetics and food industries, cyclodextrins have been used as complexing agents to increase the water solubility of various compounds, such as drugs, vitamins, and food colorants, increasing the solubility, stability, and bioavailability of the guest molecule (9–11). As has been recently described by our group, CDs can be used as resveratrol complexation agent, not only to increase the total resveratrol concentration in aqueous solution, while the free resveratrol concentration remains constant, but also to decrease the free resveratrol concentration in aqueous solution, while the total concentration remains constant (12). In both cases, CDs acts as substrate reservoir in a dosage-controlled manner. The complexation of resveratrol by CDs delays resveratrol enzymatic oxidation (12), but it is not known whether entrap- ment in the internal cavity of CDs affects the antioxidant capacity of the polyphenol. There are several methods available to determine antioxidant activity in vitro (13). However, the oxygen radical absorbance capacity (ORAC) method, adapted to use fluorescein (FL) as fluorescent probe, is increasingly used for this purpose in biological samples and foods. The ORAC method is based on inhibition of the peroxyl-radical-induced oxidation initiated by thermal decomposition of azo-compounds, like 2,2′-azobis(2- amidinopropane) dihydrochloride (AAPH), and is the only * Corresponding author. Fax: #34968-278620. E-mail: enunez@ pdi.ucam.edu. 2254 J. Agric. Food Chem. 2008, 56, 2254–2259 10.1021/jf0731088 CCC: $40.75  2008 American Chemical Society Published on Web 02/27/2008