Flavonoid–flavonoid interaction and its effect on their antioxidant activity

Abstract

Flavonoids are polyphenols widely distributed in fruits and vegetables, and have been shown to be good antioxidants in different models. However, studies undertaken with the aim of predetermining the antioxidant power of a given food on the basis of its flavonoid content have, in most cases, failed due to differences between the theoretical and the calculated antioxidant power of that given product. In the present work, the antioxidant activity of eleven flavonoids (cyanidin-3-O-glucoside, malvidin-3-O-glucoside, delphinidin-3-O-glucoside, peonidin-3-O-glucoside, pelargonidin-3-O-glucoside, catechin, epicatechin, kaempferol, myricetin, quercetin and quercetin-3-β-glucoside) was measured by two different in vitro tests: DPPH radical scavenging activity and ferric reducing antioxidant power (FRAP). In order to evaluate the effect of flavonoid interactions on their antioxidant power we compared the capacity of individual flavonoids with that obtained by mixing them with another flavonoid. The majority of DPPH scavenging activities in these combinations promoted antagonistic effects, except for some synergistic interactions such as kaempferol paired with myricetin. In the FRAP assay, the interaction between epicatechin and quercetin-3-β-glucoside showed the highest synergistic effect, whereas myricetin with quercetin resulted in an antagonistic effect.

It can be concluded, therefore, that there are synergistic and antagonistic interactions between flavonoids that may explain the results obtained when measuring the antioxidant effect of whole food extracts. The present results may also assist in the future design of functional foods or ingredients based on their antioxidant activity.

Introduction

Flavonoids are among the most studied phytochemicals found in plant foods and include a large number of different molecules which may result in diverse biological activities. Numerous studies have focused on determining flavonoid antioxidant activity, many of which have used pure compounds, calculating their individual antioxidant power and performing structure–activity relationship studies (Plumb et al., 1998, Rice-Evans et al., 1996). In other studies the antioxidant power of a given food sample, mainly oils (Mateos, Domínguez, Espartero, & Cert, 2003), fruits and vegetables (García-Alonso et al., 2004, Plumb et al., 1996) or tea and wine (Leung et al., 2001, Sánchez-Moreno et al., 1999) has been characterised in depth and, in some cases, the correlation between flavonoid composition and antioxidant power has been examined (Fernández-Pachón, Villano, García-Parrilla, & Troncoso, 2004).

When in 1936 Szent-Györgyi (Rusznyák & Szent-Györgyi, 1936) reported the presence of what he first called “vitamin P” in citrus fruits, he had already hypothesized that flavonoids and vitamin C worked synergistically to strengthen capillaries (Rusznyák & Szent-Györgyi, 1936). Subsequently, some studies showed that biological interactions took place between flavonoids and some vitamins in in vitro and in vivo models. Lotito and Fraga (1998) described the protective effect of catechin against α-tocopherol depletion in plasma. More recently, Kadoma, Ishihara, Okada, and Fujisawa (2006) demonstrated that there was a synergic antioxidant effect between δ-tocopherol and epicatechin and epigallocatechin gallate in an in vitro model. Frank et al. (2006) showed that the inclusion of quercetin, catechin or epicatechin in the diet of rats gave rise to an increase in α-tocopherol concentrations in blood plasma and liver.

Many studies on the antioxidant potential of flavonoids in fruits, vegetables, wine or tea have concluded that it is impossible to predict the antioxidant power of a given product by studying just one type of flavonoid or other kind of antioxidants contained in the product, such as vitamin C or E. In some cases the possible existence of synergic or antagonistic effects between the various antioxidants present in plant foods and derived products has been postulated (García-Alonso et al., 2004, Vinson et al., 2001). However until now very few studies have focused on the assessment of flavonoid–flavonoid interactions in terms of antioxidant activity. Heo, Kim, Chung, and Kim (2007) did not find any synergistic effect between the assayed flavonoids by using the ABTS method and expressing results as a vitamin C equivalent. However, Pinelo, Manzocco, Nuñez, and Nicoli (2004) found an antagonistic effect when phenols interacted at three different temperatures using the DPPH method and several studies showed a synergistic antioxidant effect of flavonoids on free-radical-initiated peroxidation of linoleic acid (Rossetto et al., 2002). An antioxidant effect was observed by Pignatelli et al. (2000) with the flavonoids quercetin and catechin, indicating that these components of red wine act synergistically to inhibit platelet adhesion to collagen and collagen-induced platelet aggregation by virtue of their antioxidant effect.

In general, all results for isolated flavonoids indicate high antioxidant activity. Although the in vitro antioxidant properties for isolated polyphenols have already been well documented (Rivero-Pérez, Muñiz, & González-Sanjosé, 2008), in this paper we will extend this knowledge to include the antioxidant properties produced by the interaction of two flavonoids, in terms of synergistic or antagonistic effects.

To accomplish this objective we have measured flavonoid antioxidant activity and flavonoid–flavonoid interactions by employing the following two methods: scavenging of the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and ferric reducing antioxidant power (FRAP). In particular, we have compared the antioxidant capacity of a system containing a mixture of two flavonoids with that of each single flavonoid measured individually, in order to better understand the global antioxidant capacity of flavonoid rich products such as red wine or fruit juices. The flavonoids studied were: cyanidin-3-O-glucoside chloride, malvidin-3-O-glucoside chloride, delphinidin-3-O-glucoside chloride, peonidin-3-O-glucoside chloride, pelargonidin-3-glucoside chloride, (+)catechin, (−)epicatechin, kaempferol, myricetin, quercetin and quercetin-3-β-glucoside.