1. Introduction
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are co-products of normal cellular metabolism. Some of these species play an important role in cell signalling, differentiation, survival and death. These reactive species can exist in radical forms, containing one or more unpaired, non-radical electrons. ROS include superoxide anion (O2°
-), hydroxyl radical (°OH), hydrogen peroxide (H
2O
2), and hypochlorous acid (HClO); RNS include nitric oxide (NO) and peroxynitrite (ONOO
-) [
1].
When these radicals are produced in excessive quantities, they can lead to oxidative or nitrosative stress. Oxidative stress is a physiological condition that occurs when the body’s antioxidant defence systems (enzymatic or non-enzymatic) lose their ability to neutralize excess of reactive oxygen and nitrogen species, leading to oxidation of biological macromolecules such as nucleic acids, proteins and lipids. Numerous studies have shown that they are involved in the pathophysiology of numerous chronic diseases such as cardiovascular, inflammatory, metabolic, neurodegenerative diseases, and especially cancers [
2].
In addition to endogenous antioxidant defence systems, protection against ROS/RNS involves exogenous antioxidants capable of preventing their formation or promoting their elimination. Plant-based foods and beverages are the main sources of antioxidants such as vitamins and phenolic phytochemicals. Dietary polyphenols are the most abundant antioxidants in our diet. The antioxidant activities of dietary polyphenols have been shown in some cases to be as effective or more effective than those of certain essential vitamins such as vitamins C and E [
3]. The antioxidant effects of polyphenols are due to their reducing power, by donating a hydrogen atom to a wide range of ROS, or by scavenging them. They also have the ability to chelate transition metals (Fe, Cu), thereby directly reducing the Fenton reaction and preventing oxidation caused by highly reactive hydroxyl radicals [
4,
5].
Numerous analytical methods exist in order to measure the antioxidant capacity. Those methods are based on the scavenging or reduction of free and stable radicals and are convenient to identify the various antioxidant mechanisms existing from one phenolic compound to another. These assays include the scavenging of NO, the reduction of ABTS, DPPH, or peroxide radicals in the ORAC methods, and the reduction of a ferric derivative to a ferrous iron derivative in the ferric reducing antioxidant power (FRAP) assay. In studies that aimed to understand the nature of the antioxidant activities of natural compounds, it is interesting to use a multi-methods approach to evaluate the different mechanisms of action of antioxidants [
6].
In food or nutraceuticals, compounds are present in complex mixtures and can therefore interact with each other. The resulting biological effect may then be the result of additive, synergistic or antagonistic interactions. Antioxidants can interact synergistically through a regenerative mechanism, i.e., one antioxidant regenerates the other. For example, the antioxidant synergy between vitamin C (ascorbic acid) and vitamin E (α-tocopherol) is explained by the fact that α-tocopherol, by scavenging peroxide radicals, is oxidised to a tocopheroxyl radical and this radical is immediately regenerated by ascorbate to α-tocopherol [
7]. With regard to polyphenols, Aftab and Vieira highlighted a mechanism involving resveratrol in the regeneration of the reduced form of curcumin. Resveratrol is able to regenerate oxidised curcumin, thereby increasing the antioxidant activity of curcumin [
8].
Stilbenes are polyphenols known for their antioxidant activities [
9,
10,
11]. The main compound of this family is resveratrol, whose oligomerisation can produce numerous stilbenes containing up to 8 resveratrol units. In most studies on their biological activities, these compounds are used individually. However, in plants, they are present in mixtures in variable amount and proportions and their interactions are poorly studied. The aim of our study is to measure the antioxidant activities of three natural stilbenes, resveratrol (RSV), ε-viniferin (VNF) and vitisin B (VB) (a dimer and a tetramer of resveratrol obtained from vine extracts, respectively) individually and to compare these activities when these compounds are used in combination (
Figure 1). These activities are also compared with those of a stilbene-enriched extract obtained from
Vitis vinifera vine shoots, known for its high antioxidant properties [
11]. This extract was characterized and contains, in mass, 33.7% RSV and 63.1% VNF, 3.2% VB. The antioxidant activities are measured using the FRAP, NO and DPPH methods and the interactions between these compounds are obtained using the method of Chou and Talalay using CompuSyn software [
12].
3. Discussion
Our results show that the three polyphenols studied had antioxidant activities in the DPPH, FRAP and NO assays. RSV and VNF showed the highest antioxidant activities. In the literature, the antioxidant potential of RSV is well documented by the DPPH, FRAP and NO assays. On the other hand, few studies have evaluated the antioxidant potential of VNF and VB, and no studies using the FRAP and NO assays were reported, despite their in vitro anti-inflammatory and antioxidant activities in cells culture [
13]. In the literature, the RSV IC
50 values obtained for the DPPH assay (the most widely used method for measuring its antioxidant activity) are highly variable (
Figure 7) ranged between 24.3 µM [
14], and 667.18 µM [
15]. In our study, the RSV IC
50 (83.69 µM) is similar to that reported in the study of Ha and the study of Wang who obtain IC
50 values of 81.2 and 80.5 µM, respectively [
9,
16]. Three studies reported VNF IC
50 between 52.6 µM and 92 µM, which is comparable to the VNF IC
50 of our study (82.61 µM) [
9,
10,
17]. No study is available for VB.
In the FRAP and NO antioxidant assays, RSV is the most active molecule, followed by VNF and then VB, which shows much less activity. With regard to the FRAP assay, it is difficult to compare our results with those in the literature, as they are often expressed in a different manner. Nevertheless, a few studies calculate IC
50 for RSV. In particular the studies by Lin et al., who obtained an IC
50 of 20.7 µM, which is very close to the IC
50 of 15.38 µM we obtained, but very different from the IC
50 obtained by Kurin et al. and Skroza et al., which are 162.02 µM and 335.9 µM respectively [
5,
14,
18].
Figure 7.
Summary of IC50 for resveratrol in the DPPH assay according to literature.
Figure 7.
Summary of IC50 for resveratrol in the DPPH assay according to literature.
Very few studies of NO scavenging capacity have been carried out with RSV. Man-Ying Chan et al. showed that 50 µM RSV in solution in the presence of ethanol inhibited NO by 46.2%, unlike our study where the inhibition was lower (25.3%) [
19].
The antioxidant capacity of these three compounds was evaluated in the ORAC test by Biais et al. [
11]. The authors concluded that VNF had the greatest antioxidant capacity, three times greater than the one of RSV and 21 times greater than the one of VB. In our study, the antioxidant activity of RSV is higher than VNF in two of the three assays used (FRAP and NO). One reason for this difference could be due to the reaction mechanisms involved. According to Huang et al., two chemical processes, shared by the majority of polyphenols, are responsible for their antioxidant effectiveness, namely hydrogen atom transfer and electron transfer [
20]. The ORAC assay is based on hydrogen atom transfer, whereas the DPPH, FRAP and NO assays are based on electron transfer.
It is known that the more hydroxyl groups a molecule has, the stronger its antioxidant activity. In our study, RSV was as effective, if not more so, as compared to its dimer VNF and its tetramer VB, which possess the highest number of hydroxyl groups. This observation could be explained by the fact that RSV, by virtue of its structure, could release its protons more easily than VNF or VB. In the DPPH assay, some authors have stated that the steric accessibility of the DPPH radical is a major determinant of the reaction, so small molecules have better access to the radical site and therefore a higher antioxidant capacity. Conversely, large compounds react slowly, which could explain their lower activity [
6,
20].
A number of studies have been carried out on the interactions between RSV and other polyphenols. Most of them have shown antagonistic interactions in the DPPH assay. These antagonistic interactions have mainly been shown with polyphenols that belong to the flavonoids and phenolic acids families such as catechin, quercetin, caffeic acid, kaempferol and gallic acid [
5,
15,
18,
21]. In our study, the stilbenes were able to interact with each other in an additive manner. Those results suggest that within the same class of polyphenols, they appear to cooperate with each other and potentiate their effects, whereas polyphenols that belong to different classes can sometimes exert antagonistic effects. Concerning the FRAP assay, we observed synergistic effect for the RSV+VNF combination, whereas when VB which was a less active molecule was combined with RSV or VNF, effects were antagonistic. Here it could be assumed that VB interacted negatively with RSV or VNF by reducing their antioxidant activities. In the literature, Abraham et al. have found additive effects between RSV in combination with polyphenols such as chlorogenic acid, pelargonidin, and epigallocatechin gallate [
22]. Similarly, Skroza et al. observed synergistic effects when RSV was combined with catechin or caffeic acid [
18]. However, when it was combined with gallic acid or quercetin, the interactions led to antagonistic effects. Concerning the NO scavenging assay, combination of RSV with VNF or VB, gave additive effects. Kurin et al. observed that the interaction of RSV with caffeic acid induced synergistic effect and the combination with quercetin induced an additive one [
5].
To our knowledge, no study has been carried out to measure the interactions that involve VNF or VB, either between these two oligomers or with compounds belonging to other families. We showed that VNF that presents interesting antioxidant activities produced in combinations with RSV either additive or synergic interactions. Whereas, VB that presents much less antioxidant activities could not produce any synergic interaction but even antagonist ones. In the ternary combination, VB reduced the antioxidant activities of the mixture of RSV+VNF, e.g., the IC50 for the FRAP assay is 23.53 µM (RSV+VNF+VB) versus 14.72 µM (RSV+VNF).
Our results as well as those reported in the literature highlight that the nature of the interactions depends on the mixtures of compounds and also to the assay used to measure the antioxidant capacity. As an example, Skroza et al. have shown that interactions can be highly variable—additive, synergistic or antagonistic—depending on the compound mixed with RSV (gallic acid, caffeic acid, (+)-catechin or quercetin) [
18]. In addition, Kurin et al. also showed that the combination of RSV and quercetin produced an antagonistic effect in the SRD, FRAP, DPPH and ABTS assays, an additive effect in the NO scavenging assay, and a synergistic effect in the NRD and RP assays [
5].
The fraction, derived from a vine shoots extract, consists mainly of RSV and VNF, with a very small proportion of VB and unknown compounds. Our results have shown that the RSV+VNF combination exhibited similar antioxidant effects compared to the one observed for the fraction in the DPPH, FRAP and NO scavenging assays. However, at the highest concentrations, the fraction showed a statistically greater or less effect than the combination in the FRAP and NO scavenging assay, respectively. These observations show that the presence of some compounds, even in very low concentrations, can modify the nature of the interactions of the major compounds. Therefore, in order to estimate the nature of compound interactions within a mixture, it would appear necessary to characterize all the compounds present in the extract, even those present in very low concentrations.
Some authors have attempted to explain the mechanisms involved in the interactions. In the case of additive interactions, these authors assume that the molecules act independently of each other. Synergistic or antagonistic interactions, on the other hand, could be explained by a regeneration mechanism: either a less effective antioxidant regenerates the more effective one, which in turn exerts antioxidant activities, hence the phenomenon of synergy, or conversely, the more effective antioxidant regenerates the less effective antioxidant, which will in turn exert a weaker antioxidant activity, hence the phenomenon of antagonism [
8,
23,
24]. In our study we could hypothesize that VB could be regenerated by either RSV or VNF when in combination and therefore produce an antagonistic effect.