Antioxidant protective effect of flavonoids on linoleic acid peroxidation induced by copper(II)/ascorbic acid system

Abstract

Antioxidants are compounds that can delay or inhibit lipid oxidation. The peroxidation of linoleic acid (LA) in the absence and presence of Cu(II) ion–ascorbate combinations was investigated in aerated and incubated emulsions at 37 °C and pH 7. LA peroxidation induced by copper(II)–ascorbic acid system followed first order kinetics with respect to hydroperoxides concentration. The extent of copper-initiated peroxide production in a LA system assayed by ferric thiocyanate method was used to determine possible antioxidant and prooxidant activities of the added flavonoids. The effects of three different flavonoids of similar structure, i.e. quercetin (QR), morin (MR) and catechin (CT), as potential antioxidant protectors were studied in the selected peroxidation system. The inhibitive order of flavonoids in the protection of LA peroxidation was: morin > catechin ≥ quercetin, i.e. agreeing with that of formal reduction potentials versus NHE at pH 7, i.e. 0.60, 0.57 and 0.33 V for MR, CT, and QR, respectively. Morin showed antioxidant effect at all concentrations whereas catechin and quercetin showed both antioxidant and prooxidant effects depending on their concentrations. The structural requirements for antioxidant activity in flavonoids interestingly coincide with those for Cu(II)-induced prooxidant activity, because as the reducing power of a flavonoid increases, Cu(II)–Cu(I) reduction is facilitated that may end up with the production of reactive species. The findings of this study were evaluated in the light of structure–activity relationships of flavonoids, and the results are believed to be useful to better understand the actual conditions where flavonoids may act as prooxidants in the preservation of heterogeneous food samples containing traces of transition metal ions.

Introduction

Lipid oxidation occurs when oxygen reacts with lipids in a series of free radical chain reactions that lead to complex chemical changes. Oxidation of lipids in foods causes quality losses. In vivo, lipid oxidation may play a role in coronary heart disease, atherosclerosis, cancer, and the ageing process (Jadhav et al., 1996). Many factors affect oxidation, including temperature, oxygen pressure, metal catalysts, fat composition and form, that may vary depending on the oxidation conditions used (Frankel, 1993). Antioxidants, when present in the medium, can delay or inhibit lipid oxidation.

The cupric complex-catalyzed oxidation of linoleic acid was studied in air-saturated solutions (Ueda et al., 1999). During lipid oxidation, the prooxidative effect was observed at micromolar Cu(II) concentrations. Such behaviour was expected in rapid peroxidation of potassium linoleate in solutions containing ascorbic acid (AA), first observed by Haase and Dunkley (1969), and recently interpreted in terms of AA-catalyzed, transition metal-induced peroxidation (Kritharides, 1999).

The autoxidation of linoleic acid and methyl linoleate was inhibited by flavonoids such as catechin, quercetin, and morin (Torel et al., 1986), morin being the most inhibitory compound. The flavonols quercetin and morin were studied as chain-breaking antioxidants for the autoxidation of linoleic acid in cetyl trimethylammonium bromide (CTAB) micelles; both flavonols exhibited antioxidant activity (Wang and Zheng, 1992). In a micellar system where the lipophilicity of antioxidants was comparable (as reflected in octanol–water partition coefficients), structural characteristics such as the presence of o-dihydroxy phenolic (o-catecholic) groups were important in antioxidant activity, because the intermediary oxidation product of such phenolics, i.e. the ortho-hydroxy phenoxyl radical, is more stable in the micellar phase due to intra-molecular hydrogen bonding (Foti et al., 1996), and stabilization of this phenoxyl radical makes that phenolic a stronger antioxidant (due to the decrease in redox potential).

The effect of quercetin on the Cu(II)-induced oxidation of isolated LDL was studied in the presence or absence of urate, and quercetin alone was reported to behave as a prooxidant toward peroxidized LDL (Filipe et al., 2004). Zhu et al. (2000) showed that flavonoids (like quercetin and morin) behave as antioxidants, protecting LDL from oxidation as a function of the number and location of hydroxyl groups.

If oxygen is passed through a linoleic acid (LA) emulsion system by adding copper(II) salt, hydroperoxides are formed in the course of oxidation, causing rancidity of lipid foods. Through reaction with acidic Fe(II)–thiocyanate, these peroxides can be colorimetrically determined by formation of the red Fe(III)–thiocyanate complex (Lea, 1952, Mihaljevic et al., 1996). Peroxide production as a function of time in a LA system was used to determine both antioxidant and prooxidant activities initiated by a metal catalyst (Cu(II)) (Fukumoto and Mazza, 2000). The antioxidant activity of protector compounds added to the medium is inversely proportional to the amount of Fe(III)–thiocyanate complex formed per unit time.

The prooxidant activity of a compound is assumed to derive from its ability to reduce metal ions (e.g., Fe³⁺ and Cu²⁺) to lower valencies that may react with O₂ or H₂O₂ to form reactive species (e.g., via Fenton-like reactions) giving rise to undesired oxidations. Similar to antioxidant potency, prooxidant behaviour of flavonoids has been correlated to the number of hydroxyl groups in a molecule, and for the same number of –OH bearing flavonoids, flavones have been reported to show a higher Cu(II)-initiated prooxidant activity than flavanones (Cao et al., 1997). As a general rule, antioxidants of plant origin are claimed to show prooxidant activity at low concentration and antioxidant activity above certain critical values (Yen et al., 1997, Wanasundara and Shahidi, 1998).

In this study, the peroxidation of linoleic acid (LA) in the absence and presence of Cu(II) ions was investigated in aerated and incubated emulsions at 37 °C and pH 7. Additionally, the peroxidation of LA was studied in the presence of Cu(II) and ascorbic acid (AA) together. The effects of three flavonoids (i.e. two flavonols bearing the 3-hydroxyflavone backbone: quercetin, morin, and a flavanol:catechin) all having five hydroxyl groups in different locations as potential antioxidant protectors were studied in the (copper(II) + AA)-induced LA peroxidation system.