Antioxidant Features of Red Wine Pyranoanthocyanins - American

Article pubs.acs.org/JAFC

Antioxidant Features of Red Wine Pyranoanthocyanins: Experimental and Theoretical Approaches Joana Azevedo,† Joana Oliveira,† Luis Cruz,† Natércia Teixeira,† Natércia F. Brás,‡ Victor De Freitas,† and Nuno Mateus*,† †

Chemistry Investigation Centre (CIQ), Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Rua do Campo Alegre 687, 4169-007 Porto, Portugal ‡ REQUIMTE, Departamento de Quı ́mica e Bioquı ́mica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal S Supporting Information *

ABSTRACT: This work is focused on the study of the antioxidant properties of red wine anthocyanin derivatives (carboxypyranoanthocyanins, methylpyranoanthocyanins, oxovitisins, and pyranoanthocyanin-phenolics) derived from malvidin3-glucoside. Some antioxidant features were determined using the DPPH assay and the ability to delay lipid peroxidation in a liposome membrane system by monitoring oxygen consumption. The pyranoanthocyanin-phenolics have higher antioxidant potential than that of malvidin-3-glucoside, suggesting that the addition of a catechol or flavanol moiety increases the antioxidant capacity. The only derivatives that showed lower antioxidant features than malvidin-3-glucoside were oxovitisins and methylpyranomalvidin-3-glucoside. Also, the radical scavenging capacity of these pyranoanthocyanins was computationally explored using DFT methods. All pyranoanthocyanins were suggested as good candidates as antioxidant compounds because they easily donate an H atom to the free radicals, originating stable species. Altogether, these results support the fact that the antioxidant potential arising from anthocyanins is not impaired by some of their transformations during red wine aging. KEYWORDS: pyranoanthocyanins, antioxidant, liposome, red wine, malvidin-3-glucoside, DFT

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described as effective scavengers of reactive oxygen species.13 Previous works have already reported the effect of different functional groups in the antioxidant capacity of anthocyanins.14,15 Most of these studies concluded that the effect observed is dependent on the methodology used. Generally, it has been described that the antioxidant capacity of anthocyanins is related to their hydroxylation and methoxylation pattern.16 As to anthocyanin-derived pigments, some biological and antioxidant properties have been described in the literature.17−20 Previous studies have indicated that the formation of anthocyanin pyruvates, which may occur during wine aging or fruit juice processing, decreases the hydroxyl and superoxide anion scavenging and thus could decrease the antioxidant potential of these compounds.14,21 However, there are few data in the literature reporting the antioxidant properties of anthocyanin derivatives. With this in mind, the aim of the present work was to study the antioxidant properties of individual pigments belonging to different classes of pyranoanthocyanins, namely, vitisins, oxovitisins, methylpyranoanthocyanins, pyranoanthocyanin-catechins, and pyranoanthocyanin-catechols. In addition, some computational studies were performed to better assess the molecular basis of the antioxidant properties of

INTRODUCTION The color of red wine is continuously changing during wine storage and aging. These changes are associated with the chemical reactions involving anthocyanins. Anthocyanins are highly reactive and are readily involved in chemical reactions with other red wine components such as aldehydes or polyphenols (e.g., tannins), yielding new anthocyanin derivatives. The detection and identification of new pigments in wines aid in a better understanding of the color changes observed during maturation.1,2 The chemical transformations involving anthocyanins and other red wine components yield new classes of anthocyanin derivatives, especially a large group of pigments called pyranoanthocyanins.3−6 This class of anthocyanin-derived pigments has been studied as to their contribution to the overall wine color. Pyranoanthocyanins are known to be formed from the reaction of anthocyanins with small molecules such as acetaldehyde,3 acetoacetic acid,7 pyruvic acid,8 vinylphenol,4 vinylguaiacol,9 vinylcatechol,10 and vinylcatechin.11 They are thought to contribute to the orange hues observed during wine maturation and aging and are very stable because of their structural properties. Besides their color properties, these newly formed pigments may also present some antioxidant features in the same way that their anthocyanin precursors do. The high antioxidant activity of anthocyanins is generally attributed to their peculiar structure, which allows the easy donation of H atoms from aromatic hydroxyl groups, improving the capacity to bear the impaired electron via conjugation around the π-electron system.12 Hence, anthocyanins have been © 2014 American Chemical Society

Special Issue: International Workshop on Anthocyanins (IWA2013) Received: Revised: Accepted: Published: 7002

October 22, 2013 January 3, 2014 January 7, 2014 January 7, 2014 dx.doi.org/10.1021/jf404735j | J. Agric. Food Chem. 2014, 62, 7002−7009

Journal of Agricultural and Food Chemistry

Article

malvidin-3-coumaroylglucoside, respectively, with vinyloxytrimethylsilane as previously reported;33 vitisin A (6) was obtained from the reaction of malvidin-3-glucoside with pyruvic acid in water at pH 2.6 and at 35 °C with a molar ratio 50:1 (pyruvic acid/malvidin-3-glucoside) over 5 days as previously reported;32,34 methylpyranomalvidin-3glucoside (MethylPyMv3gluc) (11) was synthesized and purified according to the literature;7 PyMv3gluc-(+)-catechin (4), PyMv3coumgluc-(+)-catechin (5), PyMv3gluc-catechol (2), and PyMv3coumgluccatechol (3) pigments were obtained according the procedures described elsewhere.11,35,36 Briefly, solutions containing Mv3glc or Mv3coumglc (2.5 mM) were incubated with 8-vinyl-(+)-catechin, 8vinyl-(−)-epicatechin, or 4-vinylcatechol (2−3 equiv) in 5−10% ethanol/water at pH 3.5 (adjusted with dilute HCl or NaOH) at 30 °C. Purity Evaluation of the Compounds. The purity of all pigments obtained was confirmed by MS and NMR analysis by using a 1H NMR (600.13 MHz) and 13C NMR (150.90 MHz) in DMSO/TFA (9:1) on a Bruker Avance III 600 spectrometer with a TCI CryoProbe at 293 K using TMS as internal standard. Radical Scavenging Assay (DPPH). The tested compound reacts with DPPH (2,2-diphenyl-1-picrylhydrazyl) and induces a decrease of the absorbance measured at 515 nm, which indicates the scavenging potential of the compounds. Because all tested pigments also absorb at 515 nm, their initial absorbance (t = 0) was subtracted. The reaction for scavenging DPPH radicals was performed in a microplate reader of 96well plates (Biotek Powerwave XS with software KC4). The reaction was carried out on the plate wells with a temperature of 25 °C. A solution of 60 μM DPPH was prepared in methanol, and 297 μL of this latter solution was added in each well together with 3 μL of antioxidant. The compounds to be tested were previously dissolved in methanol and used in a final concentration of 10 μM. The decrease in absorbance was measured at 515 nm, at t = 0 and every 10 min, during 30 min. For the final results, the 0−20 min reaction time window was used. Antiradical activity was expressed as micromolar Trolox equivalents. The antiradical activity was calculated from the equation determined from linear regression after plotting standard solutions of Trolox with different concentrations. Lipid Peroxidation Assay. Liposomes were prepared as described elsewhere.19 Lipid peroxidation of L-α-phosphatidylcholine soybean lipid unilamellar vesicles (LUVs) was induced by peroxyl radicals generated at a constant rate, by thermal degradation of the azo compound AAPH in the presence or absence of antioxidants, and followed by measuring the oxygen consumption. The rate of oxygen consumption was measured continuously with a Clark-type oxygen electrode (Hansatech) provided with an automatic recording apparatus. The reaction mixture containing 1220 μL of Hepes buffer (pH 7.4), 256 μL of liposomes (340 μM final concentration), and 2 μL of the antioxidant (1 mM initial concentration) tested, dissolved in methanol, was left in a 37 °C thermostated bath for 1 h. This mixture was introduced in a closed glass vessel, protected from light, thermostated at 37 °C, and provided with a stirrer, and the reaction was started by the addition of 22 μL of AAPH (10 mM final concentration).37 The induction periods (Ti) in the presence of antioxidants were determined graphically from the profiles of oxygen consumption by the coordinates of the interception of tangents to the inhibited and uninhibited rates of oxidation.19 In addition to the analysis of the anthocyanin-derived pigments, the antioxidant activity of Trolox (water-soluble analogue of vitamin E) was evaluated. The results were expressed in terms of ratio between these pigments and Trolox antioxidant capacities. Theoretical and Computational Studies. The energies of all geometries of anthocyanin-derived pigments and their respective radicals were calculated in vacuum with the density functional theory (DFT) approach, at the unrestricted B3LYP38−40 hybrid density functional level (Becke−Slater−HF exchange with Lee−Yang−Parr correlation functional) using the 6-311+G(d) basis set as implemented in the Gaussian 09 package.41 The inclusion of polarization functions correctly takes into account intramolecular H bonds. The optimized structures were confirmed as true minima by vibrational analysis performed at the same level of theory. A temperature of 298.15 K and a pressure of 1 atm were used for vibrational frequency and thermal energy correction calculations. Single-point energy calculations using

anthocyanin-derived pigments. The molecular basis for the antioxidant properties mainly occurs by two different pathways: a hydrogen atom transfer or a single-electron transfer. These two mechanisms are described by eqs 1 and 2, respectively:22−25 ArOH + R• → ArO• + RH

(1)

ArOH + R• → ArOH•+ + R−

(2)

Previous computational studies have been performed on flavonoids and pyranoanthocyanins,22−29 in which the bond dissociation energy (BDE) of their hydroxyl groups and the ionization potential (IP) were determined as the better representative descriptors for the radical scavenging ability. Lower BDE and IP values favor both H abstraction and the electron-transfer reactions and, in general, the gas phase computed values of BDE and IP for flavonoids fall in the ranges of 70−90 and 160−190 kcal/mol, respectively.25 In this paper, the geometrical features and the BDE and IP thermodynamic parameters of one anthocyanin and seven pyranoanthocyanins from aged wine have been computed to corroborate the experimentally determined antioxidant capability of these compounds.

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MATERIALS AND METHODS

Chemicals. Dimethyl sulfoxide (DMSO) was purchased from Euriso-top; Trolox, 2,2-diphenyl-1-picrylhydrazyl, 2,2′-azobis[2-methylpropionamidine] dihydrochloride (AAPH), Hepes, NaCl, soybean Lα-phosphatidylcholine, iron(III) chloride (FeCl3), and 2,4,6-tripyridyls-triazine (TPTZ) were purchased from Sigma-Aldrich. TSK Toyopearl gel HW-40(S)-Tosoh (Tokyo, Japan), polyamide resin (100−120 mesh, SINOPEC, Hunan, China), acetonitrile (HPLC grade), and ethanol (absolute) were purchased from Carlo Erba (France); formic acid (98%, PA-ACS) and methanol (99.5%) were purchased from Panreac Quı ́mica Sau (Spain); hydrochloridric acid (37% v/v), sodium hydroxide, and ethyl acetate were purchased from José M. Vaz Pereira, S.A. (Portugal). Extraction of Anthocyanins. Anthocyanins were extracted from Vitis vinifera red grapes of Touriga Nacional variety by the addition of an acidulated solution of methanol/water (50% v/v) as described elsewhere.30 The obtained anthocyanin extract was concentrated using a nanofiltration system (membrane Desal 5 model DK 150−300 Da). Methanol was evaporated by rotary evaporation under vacuum at 30 °C, and the solution was frozen in water and freeze-dried. The anthocyanin extract, dissolved in water, was purified by column chromatography using a polyamide resin. The fraction recovered with a solution of 10% (v/v) aqueous methanol was analyzed by HPLC and found to contain anthocyanin-3-glucosides; another fraction recovered with a solution of water/methanol 40% (v/v) was shown to contain acylated anthocyanins. The fractions were concentrated in a rotary evaporator under vacuum and frozen in water for later lyophilization.31 The pigments were purified by preparative HPLC (Knauer K-1001) fitted with a Purospher C-18 reversed-phase column, 250 mm × 25 mm i.d., at 520 nm using a UV−vis L-2420 Elite LaChrom detector. The injection volume was 2 mL. The solvents were (A) H2O/HCOOH (9:1) and (B) HCOOH/MeOH/H2O (1:5:4). The gradient consisted of a linear gradient from 60 to 0% A in 70 min at a flow rate of 5 mL/min for the isolation of malvidin-3-coumaroylglucoside and from 65 to 15% A in 70 min at a flow rate of 10 mL/min for the isolation of malvidin-3glucoside. The column was washed with 100% B during 20 min and then stabilized at the initial conditions for another 20 min. The isolated compounds were frozen in water and freeze-dried. The purity of the compounds was confirmed by HPLC/DAD-ESI/MS and NMR. Hemisynthesis and Purification of Pyranoanthocyanins. Oxovitisin (9) and the coumaroyl derivative of oxovitisin (10) were synthesized and purified according to the procedure described elsewhere;32 vitisin B (7) and the coumaroyl derivative of vitisin B (8) were synthesized from the reaction of malvidin-3-glucoside and 7003

dx.doi.org/10.1021/jf404735j | J. Agric. Food Chem. 2014, 62, 7002−7009


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Figure 1. Structures of the pigments studied: malvidin-3-glucoside (1), PyMv3gluc-catechol (2), PyMv3coumgluc-catechol (3), PyMv3gluc(+)-catechin (4), PyMv3coumgluc-(+)-catechin (5), vitisin A (6), vitisin B (7), coumaroyl derivative of vitisin B (8), oxovitisin (9), coumaroyl derivative of oxovitisin (10), and MethylpyMv3gluc (11).

Figure 2. Radical scavenging activity assessed by the DPPH method. Columns represent mean ± standard deviation (SD), p < 0.05. Columns with the same letter do not differ statistically. the 6-311++G(d,p) basis set were performed at the B3LYP level of theory. This basis set was specifically used because it provided very reliable results in other studies performed for polyphenolic antioxidants.22,23,27,29 The unrestricted open-shell approach was used for

radical species. No spin contamination was found for radicals; values were 0.750 in all cases. To evaluate the effect of the solvent environment, the energy of all optimized geometries (using a dielectric constant (ε) of 78.35 to mimic the water solution) was recalculated. Solvation energies 7004



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Figure 3. Inhibition of AAPH-initiated oxidation of soybean PC liposome by a 1.25 μM concentration of compound by monitoring oxygen consumption. Results are inhibition times in ratio to Trolox. Columns represent mean ± standard deviation (SD) p < 0.05. Columns with the same letter do not differ statistically. were computed with the polarizable continuum method (PCM) using a polarizable conductor calculation model.42 Gas-phase corrections were employed to calculate enthalpy values in the aqueous and alcoholic phases, as thermal corrections are expected to be similar in both gas and solvated phases. Both BDE and IP values were computed at 298.15 K. The former is the sum of the enthalpy of the radical resulting from the hydrogen atom abstraction and that of the hydrogen atom minus the enthalpy of the parent molecule, whereas the latter is the enthalpy difference between the radical cation and the parent molecule. Statistical Analysis. All tests were conducted at least in triplicate. Values are expressed as means ± standard deviation. Statistical significance was evaluated by one-way ANOVA, followed by the Bonferroni test. Differences were considered significant when p < 0.05. Columns with the same letter in the figures are not statistically different.

catechols and pyranoanthocyanin-catechins, the results are similar to those obtained with the vitisin-like pigments except for PyMv3gluc-catechol (also known as pinotin A) that displayed a 2-fold higher antiradical capacity than malvidin-3-glucoside (Figure 2). This outcome is likely to be related to the presence of an additional o-dihydroxyl group in its structure (catechol) combined with the extended conjugation of π electrons that could readily stabilize the radical scavenged. This structural feature is often associated with a higher radical scavenging capacity.15,16,44,45 However, this outcome was not obtained in the case of the two pyranoanthocyanin-catechins tested (PyMv3gluc-catechin and PyMv3coumgluc-catechin). Other structural features such as steric hindrances may partly impair the antiradical capacity of the compounds. Altogether, the general tendency of the antiradical capacity observed in this study agrees with some data already reported in the literature, although only a marginal effect of the catechol moiety was observed in another work using a different lipid substrate.46 Monitoring Oxygen Consumption during the Oxidation of Liposomes. The protection afforded by these malvidin derivatives toward lipid peroxidation was assayed by monitoring oxygen consumption during oxidation of soybean PC liposomes (Figure 3). Liposomes were used as they are usually employed to mimic biological targets (e.g., cellular membranes). The evaluation of the antioxidant capacity of the anthocyanin pigments, against oxidation of soybean PC liposomes, was performed using AAPH as a peroxidation initiator, as commonly used in this kind of study. The generation of peroxyl radicals from AAPH induces a significant oxidation of phosphatidylcholine, because they are able to remove hydrogen atoms from polyunsaturated acyl chains, yielding radicals that lead to the propagation chain.47 The data yielded from the oxygen consumption assays showed that all of the anthocyanin pigments scavenged efficiently the peroxyl radicals generated in the aqueous phase compared to the control assay (without any compound) (data not shown). The results are presented in the form of a ratio between the induction time (Ti) of the compound tested and that of Trolox. This induction time is representative of the delayed time of oxygen consumption, which remains practically constant prior to the initiation of lipid peroxidation.37

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RESULTS AND DISCUSSION This work aimed at studying some antioxidant features of pigments derived from malvidin-3-glucoside (the major anthocyanin present in Vitis vinifera red grapes). For this, 10 malvidin-3-glucoside derivatives belonging to different classes of pyranoanthocyanins were obtained by hemisynthesis (Figure 1). All of these pigments have already been detected in red wines, especially Port red wines, although in very low amounts. The antioxidant features of these compounds were tested by assessing their antiradical potential (using the DPPH method) and their capacity to prevent lipid peroxidation using soybean Lα-phosphatidylcholine (PC) LUVs as lipid substrate. The protection against lipid peroxidation was assayed by monitoring oxygen consumption during oxidation of soybean PC liposomes. The antioxidant features of all the pyranoanthocyanins tested were compared to that of malvidin-3-glucoside. DPPH. Following the method described in the literature43 with some modifications, radical activities were determined by using DPPH as a free radical. Overall, the results indicate that all classes of pyranoanthocyanins tested displayed antiradical properties, as seen from Figure 2. The structural transformations of malvidin-3-glucoside into new pigments did not compromise its antiradical capacity, having even increased it in certain cases. Indeed, the three vitisin-like pigments tested were found to display a higher antiradical capacity. On the other hand, the oxovitisins tested and methylpyranomalvidin-3-glucoside were found to maintain an antiradical capacity similar to that of malvidin-3-glucoside (Figure 2). As to the pyranoanthocyanin7005



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Figure 4. Optimized geometry of pigments 1, 2, 3, 4, 6, 9, 10, and 11. Oxygen, carbon, and hydrogen atoms are colored in red, gray, and white, respectively.

are related to PyMv3gluc-catechin and the pyruvic adduct of malvidin-3-glucoside Mv-py, also known as vitisin A. These two latter pigments were shown to be the most effective in delaying lipid peroxidation. The available data allow only speculation that this feature may be due to the proximity/affinity of the molecules toward the lipid layer, thereby promoting a higher protection through a more efficient radical scavenging capacity. This feature is closely related to the polarity of part of or the whole molecule. Theoretical and Computational Studies. The pigments studied here were 1, 2, 3, 4, 6, 9, 10, and 11. and their optimized geometries are shown in Figure 4. All pyranoanthocyanins possess an intense π electron delocalization and conjugation that involve the rigid core composed by A−C−D rings and a less rigid B ring. In pigments 2 and 3, the presence of the E ring draws electron flow that enhances the possibility of delocalization and conjugation in this part of the molecule. A similar behavior was observed in compound 4 that possesses a catechin group and allows a major delocalization and conjugation in this moiety. Furthermore, all minimum energy structures are specially arranged to maximize the number of intramolecular H bonds

In the presence of any antioxidant, lipid peroxidation is delayed during a certain period of time that varies with the antioxidant capacity of the tested compound. The anthocyanin pigments are thought to trap AAPH-derived peroxyl radicals, inhibiting the initiation of lipid peroxidation. On the other hand, if located at the surface of the liposome, they may also quench liposome-derived peroxyl radicals, inhibiting the chain propagation.48 Bearing in mind the results obtained from the DPPH assay, one could assume that a similar trend was expected for the lipid peroxidation assay. However, some slight differences were observed. The oxovitisins tested and methylpyranomalvidin-3-glucoside were found to maintain an antioxidant potential similar to that of malvidin-3-glucoside (Figure 3), as also observed in the DPPH assay (Figure 2). Furthermore, the vitisin-like pigments, the pyranoanthocyanin-catechols and pyranoanthocyanin-catechins, were found to delay the peroxidation of the PC liposomes more than malvidin-3-glucoside (Figure 3). These results also agree with those yielded from the DPPH assay. The only differences 7006



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Table 1. Relative Energies (Kilocalories per Mole) of All Anthocyanin/Pyranoanthocyanin Radical Species radical pigment

HO-4′

HO-5′

HO-7′

1 2 3 4 6 9 10 11

0.0 10.5 2.1 3.1 0.0 0.0 0.0 0.0

4.4

12.7 19.9 11.7 13.7 10.6 5.9

HO-2″

HO-3″

HO-4″

HO-5″

HO-7″

24.4

16.6 9.4 3.9

0.0 0.0 0.0

8.9

4.9

HO-4‴

6.1

5.9

4.6 9.9

within the B, E, and sugar rings. It was observed that in all molecules, the HO-4′ establishes a H bond to the adjacent 3′OCH3 group, whereas an additional H bond is established between the 3′-OH and 4′-OH groups of pigments 2 and 3. Pigments 3 and 10 possess another H bridge established between the carbonyl group of the p-coumaroyl moiety and the 4-OH of glucose unit. Compound 4 shows two more specific H bonds, one within the catechin unit and another between an OH group of the catechin B ring and the OH group of the pyranoanthocyanin A ring. Furthermore, there are several H bonds established between the various hydroxyl groups of the pyranoanthocyanin sugar ring. However, they should not influence the reactivity of the polyphenol molecules, although these energetic H bonds can stabilize the whole systems. Radicals from Hydrogen Abstraction. By abstracting an H atom from every HO group present on the eight molecules studied, two (for 6, 9, and 11), three (for 1 and 10), four (for 2), five (for 3), and seven (for 4) radical species are obtained. The relative energies of all radicals are presented in Table 1, and the optimized geometries of all radicals are given in Figure SI-1 in the Supporting Information (SI). As observed, the HO-4′ radicals of pigments 1, 6, 9, 10, and 11 are the most stable, probably due to resonance effects around the radical center. For pigments 2, 3, and 4, the absolute minimum of radical species is the HO-4″. Radical species 2 and 3 are particularly stabilized by the presence of the previously mentioned intramolecular H bond within the OH groups of catechol moiety, whereas the radical of compound 4 is also stabilized by the intermolecular H bond in the B ring of its catechin group. Figure SI-1 also shows the highest values (colored in green) of atomic spin density distribution of the most stable radicals of all eight pigments. As seen, radical species of pigments 1, 6, 9, 10, and 11 have the spin density distribution more concentrated at O-4′, suggesting that the unpaired electron remains on the radical oxygen atom and that there is some involvement of the adjacent −OCH3 groups and atoms of pyranoanthocyanin B ring due to the possibility of resonance effects. Similarly, the atomic spin density of pigments 2 and 3 shows that the unpaired electrons are located mostly on the O4″ atoms at E rings, as well as the unpaired electron of radical 4-O4″ is mainly placed on the O4″ atom. It was also observed that a certain delocalization around all atoms of the B ring of catechin group exists. Radicals from Electron Abstraction. The optimized geometries of the eight radical cations are shown in Figure SI-2 in the SI. All cation radical species retain the intermolecular H bonds established in the parent stable molecule. Figure SI-2 also indicates that the atomic spin density of all cation radicals is mainly found at the pyranoanthocyanin B ring. Both cation radicals of 9 and 10 have a high atomic spin density concentrated

at the oxygen atom of ring D, due to the high capacity of the carbonyl group to retain the unpaired electron. Bond Dissociation Energy (BDE) and Ionization Potential (IP) Values. Table 2 shows the BDE and IP Table 2. Bond Dissociation Energies (BDE) and Ionization Potentials (IP) in the Gas Phase and Water Environment BDE

IP

pigment

gas phase

water

gas phase

water

1 2 3 4 6 9 10 11

73.3 64.6 71.2 68.8 72.9 68.9 72.9 72.9

70.5 62.7 70.0 67.0 70.3 67.6 69.6 69.8

228 210 204 208 226 159 147 222

142 134 139 138 142 131 123 140

computed values for the eight pigments in the gas and aqueous media. The BDE gas phase values ranged between 64.6 and 73.3 kcal/mol, which slightly decrease in a water environment. Pigment 2 shows the lowest value of BDE in both vacuum and condensed phases. This pigment possesses two hydroxyl groups at rings B and E, and the high electron delocalization involving both aromatic rings causes a stabilization of radicals formed upon H abstraction, which affects and decreases the BDE value. In addition, the presence of two O-methyl groups around the OH group at the B ring favors the radical formation and allows low BDE values. Compared with pigments 2 and 3, the addition of the bulky cathechin and p-coumaroyl groups increases the BDE value by approximately 4 and 6 kcal/mol (BDE values of 68.8 and 71.2 kcal/mol in vacuum, as well as 67.0 and 70.0 kcal/mol in water, respectively). Although pigments 6, 9, and 11 differ in the substituent group at C12 (COOH, CO, and CH3 groups, respectively), their BDE values are very similar, which indicates the absence of specificity of this carbon position to BDE values. The addition of an extra p-coumaroyl group in pigment 10 increases the BDE value by approximately 2 kcal/mol, in relation to pigment 9. According to the IP values computed for the eight anthocyanin/pyranoanthocyanins studied, the condensed media also decrease the energy required to extract a single electron. However, the drop in condensed IP values is more highlighted due to the occurrence of electrostatic interactions with the double-charged radicals. The lowest IP values (159 and 147 kcal/ mol in the gas phase; 131 and 123 kcal/mol in water) exhibited by pigments 9 and 10, respectively, indicate that they are better in donating a single electron to oxidant molecules in solution. These pigments are followed by pigments 2, 4, and 3, in which their extra E rings/B catechin ring contribute to the lowering of 7007



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the IP values because it allows a better distribution of the unpaired electron. The Mv-3-gluc molecule has the highest BDE and IP values in both gas and condensed phases, revealing a lesser ability of this molecule to act as a good antioxidant. Therefore, all of these results are in agreement with previous computational studies that suggested that the presence of D and E rings in pyranoanthocyanins contributes to the lowering of BDE values compared to anthocyanin molecules. In addition, an extended delocalization and conjugation of the π-electrons and the resonance phenomena greatly contributes to the mechanism of the electron transfer, decreasing the IP values.23 Gas phase BDE values of the HO-4′ group in malvidin (96.0 kcal/mol)49 and malvidin-3-O-β-glucoside (88.9 kcal/mol)50 were computed at B3LYP/6-31G(d,p) and B3LYP/6-31G(d)//6-311G(2d,2p) levels. More recently, the gas and condensed phase BDE values of pigments 2 (78.5 and 89.9 kcal/mol), 6 (81.7 and 94.2 kcal/ mol), and 9 (78.2 and 94.5 kcal/mol) were also computed at the B3LYP/6-311+G(d)//6-311++G(d,p) level.26−29 The BDE values for these three molecules presented in this work are not exactly equal to the previous computational studies because the latter have substituted the glucose unit by a methyl group to reduce the computational effort. However, the ranking of the relative BDE and IP values of these molecules is similar, validating all of the other values. In conclusion, the present experimental and computational data suggest that all classes of pyranoanthocyanins displayed antiradical properties, in particular the pigments 2, 9, 3, 6, and 11. The only exceptions are the higher antiradical potential and antioxidant ability observed computationally for pigments 9, 10, and 11 when compared with experimental values. This unexpected result may be due to some approximations within the DFT method to describe BDE and IP values of uncharged molecules or some experimental conditions such as solubility problems associated with these specific molecules that can influence their antiradical potential. The antioxidant potential of anthocyanin derivatives, especially pigments derived from Mv-3gluc, maintains the same level or is even increased. It was verified that the inclusion of a catechol or catechin moiety increases the antioxidant potential of the anthocyanin derivatives, which agrees with the key role of catechol proposed previously for the antioxidant capacity.28,50

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Figures SI-1 and SI-2 show the optimized geometries of radicals from H abstraction and cation radicals, respectively, of compounds 1, 2, 3, 4, 6, 9, 10, and 11; atomic spin density values of some atoms are also shown. This material is available free of charge via the Internet at http://pubs.acs.org.

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Article

AUTHOR INFORMATION

Corresponding Author

*(N.M.) E-mail: [email protected]. Fax: +351.220402562. Phone: +351.220402659. Funding

We thank FCT (Fundaçaõ para a Ciência e Tecnologia) (POCI, FEDER, Programa Comunitário de Apoio) for two postdoctoral grants (SFRH/BPD/72652/2010 and SFRH/BPD/65400/ 2009), a Ph.D. grant (SFRH/BD/70053/2010), and a project grant (PTDC/AGR-TEC/2227/2012. Notes

The authors declare no competing financial interest. 7008



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Antioxidant Features of Red Wine Pyranoanthocyanins - American

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