Estabilización de antioxidantes fenólicos bioactivos de origen natural: modificación de tocoferoles y resveratrol mediante acilación y glicosilación enzimática

Abstract

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 22-05-2009Antioxidants protect cells against the effects of harmful free radicals and play an important role in preventing many human diseases (e.g. cancer, atherosclerosis, neurodegeneration, inflammatory disorders, etc.) and aging itself. In addition, antioxidant molecules are employed to prevent unsaturated oil products from becoming rancid during storage, thus extending oil life. The modification of natural antioxidants in order to increase their miscibility and/or stability towards the action of light and/or oxygen renders a series of “semisynthetic” antioxidants with great value-added in the food, cosmetics and pharmaceutical industries. These antioxidant derivatives may have impact not only as food preservatives but also as components of functional foods and nutraceuticals. Vitamin E (acting as a free radical scavenger) enhances the oxidative stability of polyunsaturated fatty acids towards peroxidation and, to increase its stability, is generally administered in the form of all-rac-α-tocopheryl acetate. Several approaches have been described for the enzyme-catalysed synthesis of vitamin E acetate, e.g. the regioselective hydrolysis of α-isophorone followed by reaction with isophytol. We have investigated the modification of antioxidant vitamin E by acylation with vinyl acetate catalyzed by lipases. Several parameters have been probed: (1) the source of the enzyme and its water content; (2) the effect of the carriers; and (3) the composition of the reaction medium. Resveratrol (trans-3,5,4'-trihydroxystilbene) is a polyphenolic phytochemical found in grapes and wine that is biosynthesized in response to pathogenic attack or stress conditions. It possesses a variety of antioxidant, anti-inflammatory, antitumour, cardioprotective and immunomodulatory bioactivities. As part of our interest in regioselective biotransformations of polyhydroxylated compounds, we have investigated the one-step enzymatic synthesis of acylated or glycosylated resveratrol by enzyme-catalyzed processes. Such modifications could render derivatives bearing different acyl- or glucosyl- groups in various positions, which may beneficially affect their bioavailability and/or pharmacological properties. Although the three phenolic groups of resveratrol at positions 3-, 5- and 4’- exhibit very similar chemical reactivity, our results suggest that the regioselectivity of the acylation can be controlled by an adequate selection of the biocatalyst. Regarding the glucosylation, we have investigated the modification of resveratrol by glucosyltransferases, with starch as glucosyl donor. We studied several parameters: (1) source of the enzyme, (2) presence of cosolvents, (3) reaction temperature, (4) weight ratio donor (starch) : acceptor (resveratrol). We obtained 14 new resveratrol glucosides, which were further characterized by HPLC-MS and NMR. Using Rancimat test and Trolox Equivalent Antioxidant Capacity assay (TEAC), we studied the antioxidant capability of all new derivatives of tocopherol and resveratrol. To know the surfactant properties of resveratrol glucosides, we measured the variation of the surface tension vs concentration in order to determinate the value of critical micellar concentration. Apart from this, we have characterized a new group of epoxy-activated supports, called DilbeadsTM, based on methacrylate, to immobilize enzymes (Dilbeads TA, DVK, SZ, EZ, NK and RS). Scanning electron microscopy, mercury intrusion porosimetry analysis and nitrogen adsortion isotherms were employed. We have studied the distribution of a sterol sterase within Dilbeads using fluorescence confocal microscopy and a FITC-labelled enzyme. Most of the enzyme molecules are confined in a surface layer of 10.5 μm width. There is an apparent diffusional limitations of enzyme molecules to reach the center of the bead particle, which can be caused by several factors including: (1) the tortuosity of the pore structure; (2) the steric hindrance exerted by the enzyme molecules that are immobilized in the shell of the particle. 1