`In vitro' evaluation of the antioxidant activity and biomembrane interaction of the plant phenols oleuropein and hydroxytyrosol
Introduction
One of the major targets for oxygen radicals are undoubtedly phospholipid bilayers of cellular and subcellular membranes. The compounds which inhibit the membranous phospholipid peroxidation seem to exert a pharmacological effect in the prevention of oxygen radical-induced pathological events (Rice-Evans and Diplock, 1993, Roberfroid and Calderon, 1995). However, together with the site of radicals to be generated in the phospholipid bilayers, the localization of antioxidants should be taken into account in understanding the effectiveness of their antioxidant activities (Terao et al., 1994, Saija et al., 1995a, Bonina et al., 1996); in fact the degree of incorporation and the uniform distribution into lipid bilayers and the rate of transport into cells are particular factors influencing the efficiency of antioxidant compounds (Thomas et al., 1992, Kaneko et al., 1994).
Phenolic compounds (both natural and synthetic) are prototypic chain-breaking antioxidants; their protective effect against lipoperoxidative damage depends on the hydrogen-donating capacity of a hydroxyl group in each molecule (Kahl, 1991). Interest in the biological actions of olive phenols has recently arisen since epidemiological studies have linked high dietary intake of natural antioxidants (such as vitamins and phenolic compounds) with lower incidence of pathological conditions associated to uncontrolled production of free radicals (Martin-Moreno et al., 1994).
The secoiridoids (oleuropein and derivatives) represent one of the major classes of phenolic compounds with antioxidant activity contained in olives and olive oil (Montedoro et al., 1992, Montedoro et al., 1993). Oleuropein, the bitter principle of olives, and hydroxytyrosol (Fig. 1), derived by hydrolysis from oleuropein and responsible for the high stability of olive oil (Tsimidou et al., 1992), have been previously demonstrated to possess several biological properties, including antimicrobial, vasodilator, hypotensive and hypoglycemic activities, many of which may be related, partially at least, to their antioxidant and free radical-scavenger ability (Visioli and Galli, 1995, Visioli et al., 1995a, Visioli et al., 1995b). However, no data are known about the antioxidant activity of these compounds in biomembranes.
Hence, together with their scavenging activity against the stable 1,1-diphenyl-2-picrylhydrazyl radical, we have investigated the antioxidative effect of oleuropein and hydroxytyrosol in a model system consisting of dipalmitoylphosphatidylcholine/linoleic acid unilamellar vesicles and a water-soluble azo compound as a free radical generator. This model system helps in understanding the effectiveness of antioxidants against the attack of oxygen radicals on biomembranes from the aqueous phase (Fiorentini et al., 1994, Terao et al., 1994, Castelli et al., 1997). Furthermore, the capability of oleuropein and hydroxytyrosol to interact with dimyristoylphosphatidylcholine multilamellar vesicles, as a biological membrane model, was investigated by means of differential scanning calorimetry, a powerful and non-perturbing thermodynamic technique which allows characterization of the thermotropic phase behaviour of lipid bilayers in liposomal structures, and convenient and sensitive determination of the interaction of drugs with artificial membranes ( O'Learly et al., 1986, Jain, 1988, Seydel, 1991, Saija et al., 1995b). The possible relationship between biophenol antioxidant efficiency and membrane interaction was discussed.
Section snippets
Quenching of DPPH (DPPH test)
The free radical-scavenging capacity of oleuropein and hydroxytyrosol was tested as bleaching of the stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH; Nanjo et al., 1996). The reaction mixture (3.5 ml of ethanol) contained 86 μM DPPH and different concentrations of oleuropein, hydroxytyrosol and α-tocopherol (the last one used as a control antioxidant standard); an equal volume (30 μl) of the solvent (ethanol) employed to dissolve the compounds tested was added to control tubes. After 10 min
DPPH test
Oleuropein and, especially, hydroxytyrosol elicited a good concentration-dependent scavenging effect, allowing the calculation of the half-scavenging concentrations (SC50) reported in Table 1.
LP–LUV test
Incubation of DPPC/LA LUVs in presence of AAPH induced a large increase in the accumulation of LOOH formed from LA peroxidation. The addition of the two biophenols tested reduced the amount of LOOH formed in a concentration-dependent manner, oleuropein being more effective than hydroxytyrosol (Fig. 2). The
Discussion
A brief comment regarding the interaction of olive phenols with model membranes is needed as a prelude to the discussion of results. Between the two compounds tested, oleuropein, but not hydroxytyrosol, appears to interact with DMPC membranes; this also occurs despite the presence, in oleuropein backbone structure, of a sugar moiety, generally claimed to prevent drug access to lipid membranes (Ratty and Das, 1988). Lipophilicity of drugs has been clearly demonstrated to modulate their
Acknowledgements
This work was partially supported by the Italian MURST and C.N.R. and by Bromatheia OEVO-POP 93 funds.
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