mofsp

ppeRoberto Lazzaroni b, Jean-Luc Duroux a

a

Our analysis also includes the Mulliken spin density distribution for the radicals formed after H-removal on each OH site of both

in vitro studies have established the hierarchy of avo-noids in terms of their antioxidant activities (Coset al., 1998; Rice-Evans, Miller, & Paganga, 1996) and

.

* Corresponding author. Tel.: +33 0555 435 927; fax : +33 0555 435845.

E-mail address: trouillas@unilim.fr (P. Trouillas).

Food Chemistry 97 (200

Food0308-8146/$ - see front matter 2005 Elsevier Ltd. All rights reservedavonoids. The results clearly show that the 3-OH quercetin radical possesses a large spin density on the C-2 atom, which explainsthe C-ring opening process observed in dierent redox systems, including metabolization. 2005 Elsevier Ltd. All rights reserved.

Keywords: Flavonoid; Antioxidant; DFT; BDE; Spin density

1. Introduction

Over the past decade, a number of epidemiologicalstudies (Hertog, Feskens, Hollman, Katan, & Kromh-out, 1993; Trichopoulou & Vasilopoulou, 2000) havecontributed to build the consensus that diets rich in fruitsand vegetables have benecial eects on human health.

The subsequent decrease in the risk of certain patholo-gies, including cardiovascular diseases and cancer, isattributed in part to phenolic compounds contained insuch food. Those molecules have demonstrated multiplein vitro and in vivo biological properties including anti-oxidant activities (Cadenas & Packer, 2002).

Among the natural phenolic antioxidants, the avo-noid family is the most important class. A number ofLaboratoire de Biophysique, Faculte de Pharmacie, 2 rue du Docteur Marcland, 87025 Limoges, Franceb Service de Chimie des Materiaux Nouveaux, Universite de Mons-Hainaut, Mons, Belgium

c Laboratoire de Chimie Theorique et de Modelisation Moleculaire, UMR 6517-Case 521, Faculte des Sciences de Saint-Jerome,

Universite de Provence, Av. Esc. Normandie Niemen, 13397 Marseille Cedex 20, France

Received 21 April 2005; accepted 20 May 2005

Abstract

Over the past decade, the chemical behaviour of avonoids as antioxidants has become the subject of intense experimentalresearch. In this paper, we use a quantum-chemical approach to shed light on the reactivity of two avonoids, quercetin and taxif-olin. We particularly focus on the 3-OH site and the role played by the 2,3-double bond in the reactivity of that site. In order toestablish the most ecient theoretical methodology, dierent methods, either HartreeFock-based or derived from density func-tional theory, and dierent basis sets (from 6-311G(d) to 6-311++G(2d,p)) were tested on phenol and catechol, for which experi-mental bond dissociation enthalpy (BDE) values are available. It appears that (U)B3P86/6-311+G(d,p) is the most relevant methodfor BDE prediction of these phenolic compounds and it has, therefore, been used for an extensive study of the two avonoids.

The analysis of the theoretical BDE values, for all OH sites of quercetin and taxifolin, clearly shows the importance of the B-ringand the 3-OH group only when the 2,3-double bond is present (i.e. in quercetin). We have also considered the importance of ketoenol tautomerism (present in quercetin but not in taxifolin) to rationalize the dierence in reactivity between the two compounds.Rapid com

A DFT study of the reactivitytaxifolin antioxidants: The

Patrick Trouillas a,*, Philidoi:10.1016/j.foodchem.2005.05.042unication

OH groups in quercetin andecicity of the 3-OH site

Marsal b, Didier Siri c,

www.elsevier.com/locate/foodchem

6) 679688

Chemistry

680 P. Trouillas et al. / Food Chemthe corresponding structureactivity relationships. It hasbeen clearly proved that the B-ring is the most impor-tant site for H-transfer and consequently for the antiox-idant capacity. This is particularly true when the B-ringis a catechol moiety, as in the avonol quercetin(Scheme 1). In contrast, the A-ring seems to be lessimportant. The 2,3-double bond also contributes tothe antioxidant activity, as it ensures p-electron delocal-ization between the B- and C-rings, which contributes tothe stabilization of RO, after H-abstraction (Van Ackeret al., 1996).

An important issue that is still under debate is therole of the 3-OH group. In vitro studies demonstratedthat 3-OH contributes to the antioxidant potential. In-deed, blocking the 3-OH group with a sugar moiety,as in rutin, or removing this group, as in luteolin, signif-icantly decrease the activity (Rice-Evans et al., 1996).However, the eectiveness of this group seems to bedependent on the presence of the 2,3-double bond andthe 4-carbonyl group. 3-OH is also thought to be in-volved in the metabolization by copper-containing quer-

5

7O1 2

43

3'4'

OOH

OH

OH

OH

OH

5

7O1 2

43

3'4'

HO

OH

OH

OH

O

H

OH

H

OH

OH

OH

CatecholPhenol

B

CA

B

CA

Quercetin Taxifolin Scheme 1.cetin 2,3-dioxygenase issued from human intestinalbacteria. Mechanisms have been proposed for the quer-cetin metabolism pathway, in which the copper-contain-ing quercetin 2,3-dioxygenase binds to the molecule(Steiner, Kalk, & Dijkstra, 2002). Quercetin is believedto coordinate to the copper atom of the dioxygenase en-zyme, via the 3-OH and 4-carbonyl groups. The conse-quence of this complex-formation is H-removal fromthe 3-OH group and some authors proposed that C-ringopening takes place after a second redox attack by O2 onthe C-2 atom (Balogh-Hergovich, Kaiser, & Speier,1997). This leads to the formation of the correspondingdepside (phenolic carboxylic acid ester) and releasing ofcarbon monoxide. Afterwards, small phenol compoundssuch as phenol acids can be formed and are more easilyabsorbed. Thus, this group is particularly important;however, to our knowledge, no fundamental study isavailable to explain its redox capacity.It is well-established that phenolic compounds(ROH) scavenge free radicals (OH in the followingexamples) according to two possible reducing pathways:

(i) H-transfer from the molecule to the radical (directOH bond breaking)

ROHOH ! RO HOH 1(ii) Electron-transfer from the molecule to the radical,

leading to indirect H-abstraction

ROHOH ! ROH OH ! RO HOH 2

The rst mechanism is governed by the OH bond dis-sociation enthalpy (BDE), while the second one is gov-erned by the ionization potential and by the reactivity ofthe ROH+ cation. In any case, the formed radical (RO)must be relatively stable, so that: (i) reactions (1) and (2)are thermodynamically favourable (in the sense that it iseasier to remove a hydrogen atom from ROH than fromHOH) and (ii) it reacts slowly with neighbouring mole-cules, without toxic eects, such as oxidative stress.

Theoretical calculations of the BDE, using either den-sity functional theory (DFT) or semi-empirical HartreeFock methods, have been useful for elucidating the highcapacity of the OH groups of phenolic antioxidants toreact by H-transfer (Leopoldini, Pitarch, Russo, & Tos-cano, 2004; Leopoldini, Marino, Russo, & Toscano,2004; Lemaska et al., 2001; Lucarini, Pedulli, & Guerra,2004; Priyadarsini et al., 2003; Russo, Toscano, & Uc-cella, 2000; Trouillas et al., 2004; Wright, Jonhson, &DiLabio, 2001; Zhang, Sun, & Chen, 2001; Zhang,Sun, & Wang, 2003; Zhang, 2004). Nevertheless quan-tum-chemical studies on avonoids are far from com-plete: most theoretical investigations have focussedonly on the B-ring (particularly the catechol moiety)and the most reliable methodology still has to be estab-lished. In the present study, we decided to focus on the3-OH group and its surroundings, in order to elucidatethe reactivity of this group and the correspondingradical formed after H-abstraction. We compare theproperties of quercetin with those of taxifolin, the corre-sponding dihydroavonol (Scheme 1), in order to gaininsight on the role of the 3-OH group and to understandthe interplay between the 2,3-double bond and the 3-OHgroup. Quercetin is a avonol predominantly derivedfrom onions, apples, and red wine. It possesses allrequirements (ortho-OH groups on the B-ring, 2,3-dou-ble bond, 3-OH group) to be a reference phenolic anti-oxidant. Taxifolin is mainly found in Citrus fruits,especially grape fruits and oranges (Bohm, 1975). Itsantioxidant activity is twice lower than that of quercetin,even though it remains quite interesting (Rice-Evanset al., 1996).

Due to the importance of p-electron delocalization in

istry 97 (2006) 679688avonoids, theoretical investigations require high-level

including metabolization.

Chem2. Theoretical methodology

2.1. General procedure

BDE was calculated as the dierence in total enthalpybetween the corresponding radical (formed after H-abstraction) and the avonoid, according to the follow-ing reaction:

ROH! RO H

As a starting point, we calculated the energies byusing the ab initio (U)HF method. HF theory has longbeen used for the investigation of open-shell systemswith unpaired electrons and the performance and limitsof this approach are well known (Szabo & Ostlund,1982, Chap. 3). In order to obtain a more accuratedescription of the BDE, one needs to go beyond theHF level, to take into account electron correlation.MP2 allows taking such correction into account.

As an alternative to post-HF methods, DFT has re-cently been developed and has succeeded in BDE esti-mation and in the description of radical species;nevertheless, the choice of the functional has beenshown to signicantly inuence the accuracy of the re-sults (DiLabio, Pratt, LoFaro, & Wright, 1999; Feng,Liu, Wang, Huang, & Guo, 2003; Fox & Kollman,calculations over the entire molecules, in order to takeinto account all the possible electronic eects and theirinuence on the redox reactivity of the 3-OH group. Toselect the most appropriate theoretical approach, we per-formed a comprehensive methodological study on twomodel compounds: phenol and catechol (Scheme 1).

In this paper, we rst present the results of BDE cal-culations for phenol and catechol, obtained by using dif-ferent methods (ab initio HartreeFock (HF) and DFT)and dierent basis sets. Afterwards, we develop the se-lected methodology for quercetin and taxifolin, accord-ing to the results obtained on phenol and catechol. Theresults are compared and analyzed in terms of BDE,electron distribution in the singly occupied molecularorbital (SOMO) or the a-highest occupied molecularorbital (a-HOMO), and Mulliken spin density distribu-tion in the radical species. The structureactivity rela-tionship is examined in the light of those results andwe pay particular attention to quantum-chemical inter-pretation of the reactivity of the 3-OH group in querce-tin and the radical formed after H-removal from thismolecule. Ketoenol tautomerism before H-abstractionis also discussed for explaining the role of this group. Fi-nally, the calculated electronic structures allow as to elu-cidate the chemical reaction that leads to the C-ringopening of quercetin in various oxidative systems,

P. Trouillas et al. / Food1996; Marsal, Roche, Tordo, & De Sainte Claire,1999). Following Pople, Gill, and Handy (1995), wehave decided to use an unrestricted scheme for open-shell DFT calculations, instead of a restricted open-shellscheme, and we have tested a number of exchange-correlation potential schemes. Previous calculations haveshown the interest of combining HartreeFock andBecke exchange to provide accurate BDE values (Fenget al., 2003; Marsal et al., 1999). BDE is also very sensi-tive to the correlation potential. In order to evaluate thiseect, we decided to use the LeeYangParr correlationpotential, either with the three-parameter scheme for ex-change (B3LYP), or with the one-parameter (B1LYP)developed by Adamo and Barone (Adamo & Barone,1997). We also tested the PerdewWang and the Perdewcorrelation potentials, in combination with the three-parameter scheme (B3PW91 and B3P86, respectively).

BDE values were corrected to take into account thezero point energy (ZPE) and the contributions fromtranslational, rotational, and vibrational degrees of free-dom in the heat of reaction at 298 K. The use of scalingfactors for vibrational frequencies and ZPE was notconsidered because the basis sets we used with DFT(from 6-311G(d) to 6-311++G(2d,p)) would lead tonegligible rescaling.

Concerning the general methodology, another impor-tant structural issue must be taken into account. Indeed,three of the molecules studied here (catechol, quercetinand taxifolin) possess a catechol-like moiety in which astabilizing eect exists due to H-bonding between thetwo adjacent OH groups. This structural feature mustbe taken into account in the molecule but also in theradicals originating from the catechol moiety (Russoet al., 2000; Wright et al., 2001). Therefore, in those rad-icals, we took care that H-bonding is maintained be-tween the O-atom where H-abstraction takes place andthe ortho-OH group.

2.2. Test calculations on phenol and catechol

We compare our theoretical results on avonoidswith the structureactivity relationship obtained frommany experimental studies. However, since no experi-mental values of BDE and IP are available for avo-noids, it is dicult to directly give quantitativesupport to the calculations. As we have seen above,the antioxidant activity of avonoids is attributed, inpart, to H-transfer from the B-ring. Depending on itsdegree of oxidation, the B-ring is equivalent to phenolor catechol, for which experimental data indeed exist,and can, therefore, be used for testing the theoreticalmethodology. The OH groups of A-ring and the 3-OHgroup are signicantly dierent from the OH groupsof phenol and catechol. Nonetheless we assume that, ifthe method gives a reliable evaluation of the BDE inphenol and catechol, it will give valuable results for

istry 97 (2006) 679688 681the A-ring and 3-OH as well.

tween the B-ring and the C-ring is favoured (the factthat AM1 gives a non-planar geometry is probably

ChemIn the recent literature, there are several experimentalreports on the OH BDE determination of phenols,based on a photoacoustic calorimetry technique (Borgesdos Santos & Martinho Simoes, 1998). Among the re-cent values, Wayner et al. (1995) obtained 87.0 1.0kcal/mol (Wayner et al., 1995). More recently, De Heer,Korth, and Mulder (1999) proposed correcting that va-lue by taking into account the enthalpy of intermolecu-lar hydrogen bonding with benzene (i.e., the solventused for the measurements); they obtained 86.2 kcal/mol. Concerning the BDE in catechol, there are veryfew quantitative studies. Brigati, Lucarini, Mugnaini,and Pedulli (2002) reported experimental values mea-sured by an EPR equilibration technique for variousphenolic antioxidants containing two or more hydroxylgroups. From that work, the BDE of catechol is79.3 0.3 kcal/mol. The study was performed in ben-zene solution; because of the small dielectric constantof that solvent, the authors suggested that the valuesare probably close to those expected in the gas phase.

In order to obtain an accurate description of theBDE, we tested several quantum-chemical methodsand basis sets. All the details concerning the results thatwe have obtained on phenol and catechol with (U)HF,(U)MP2, (U)B1LYP, (U)B3LYP, (U)B3P86, and(U)B3PW91 and the dierent basis sets (from 6-311G(d)to 6-311++G(2d,p)) are available as ****supplementarymaterial. Globally, it appears that, for phenol and cate-chol, the DFT/B3P86/6-311G(d,p) scheme is sucientto give an accurate description of the BDE, since it per-forms within 1 kcal/mol of the most extended schemecompared to the experimental gas phase value. Never-theless, we decided to include diuse functions forthe avonoids, in order to obtain a better descriptionof the delocalization eects that are crucial for thegeometry as well as for the electronic structure; we con-sequently used 6-311+G(d,p) in the following of thestudy.

2.3. Calculations on quercetin and taxifolin

A semi-empirical study of the conformation of quer-cetin has previously been published (Russo et al., 2000)and we recently applied the same method for taxifolin(Trouillas et al., 2004). In those papers, the potential en-ergy surfaces were investigated along the torsion angle s,dened by the C3C2C1 0C2 0 atoms (Scheme 1). Forquercetin, the energy minimum is found for a torsionangle of 153 (Russo et al., 2000). Taxifolin is adihydroavonol and it must be stressed that thedihydroavonol structure possesses two chiral centres,C-2 and C-3, leading to the existence of the four diaste-reomers 2S3S, 2R3R, 2R3S and 2S3R. The 2S3S isomershows a torsion angle of 100 at the AM1 level and theconformational behaviour of the 2S3S compound is a

682 P. Trouillas et al. / Foodmirror image of that of 2R3R. Moreover, since the anti-due to the underestimation of the p-electron delocaliza-tion at the semi-empirical level, along with the at shapeof the potential energy surface in the surroundings of180).

The BDE values are calculated with B3P86/6-311+G(d,p), according to what was shown above forphenol and catechol. We also used B3LYP/6-311+G(d,p), since it is the most common functionalused in the literature; this allows us to compare our re-sults with those that are likely to appear in the future atthe B3LYP level. Nevertheless, the discussion is essen-tially based on the B3P86 values. All calculations corre-spond to systems in vacuum, i.e., no solvent eects weretaken into account. Inuence of solvent on phenoliccompounds has been discussed by Wright et al. (2001)and, more recently, by Guerra, Amorati, and Pedulli(2004) for para-substituted phenols and they concludedthat this correction is below 2 kcal/mol.

Geometry optimizations on the radicals were per-formed, starting from the optimized structure of the par-ent molecule, after the H-atom was removed from the 3,5, 7, 3 0, or 4 0 position. In the discussion, the radicalformed by H-removal from the 3-OH group of quercetinis called 3-OH quercetin radical. The same notation isused for the other four radicals. No geometrical param-eter constraint was imposed during the optimization.We took care only that the OH group neighboring theprimary radical site in the B-ring is oriented in sucha way that H-bonding is preserved. Indeed, afterH-abstraction from 4 0-OH, the ortho-OH group wastilted to form a H-bond with the remaining O-atom.

3. Discussion

3.1. BDE values for quercetin and taxifolin

The middle part of Table 1 shows the (U)B3P86/6-311+G(d,p) and (U)B3LYP/6-311 + G(d,p) calculatedBDE values for the ve radicals formed by H-abstrac-tion on quercetin and taxifolin. A systematic increasein energy of about 4 kcal/mol is observed fromB3LYP to B3P86, as in phenol and catechol. Even ifoxidant activity has only been reported for a racemicmixture of both trans-taxifolin isomers (2S3S and2R3R), we did not consider the cis-isomers (2S3R and2R3S) in this study. Therefore, the present study is re-stricted to the 2S3S isomer.

B3LYP/6-311+G(d,p) and B3P86/6-311+G(d,p)geometry optimizations were carried out and torsion an-gles, s, of 180 and 78 were found for quercetin andtaxifolin, respectively. Thus, as expected, quercetin is aplanar molecule, in which electron delocalization be-

istry 97 (2006) 679688no experimental data are available for comparison for

The present DFT BDE values clearly demonstrate

tin and taxifolin

H 40-OH 5-OH 7-OH H-2 keto form

0 74.6 99.3 88.6 64.30 76.0 99.6 92.0

6 71.1 94.7 84.4 61.69 72.7 95.0 87.8

of quercetin. The last column reports the BDE of the H-atom bound to the

P. Trouillas et al. / Food Chemistry 97 (2006) 679688 683avonoids, these DFT results appear quite realistic forphenol compounds.

Both methods give the following BDE sequence forthe OH groups: 4 0-OH < 3 0-OH < 3-OH (querce-tin) < 7-OH < 5-OH < 3-OH (taxifolin). This clearlyconrms that H-transfer from the B-ring (4 0-OH and3 0-OH groups) is easier than from the A-ring (7-OHand 5-OH groups), consistent with what is known fromthe literature concerning structureactivity relationshipsof antioxidant avonoids (Rice-Evans et al., 1996).According to the B3P86 calculations, the BDE valuesfor the OH sites on the A-ring are higher than thoseon the B-ring, by: (i) 22 kcal/mol between 3 0-OH and5-OH; (ii) l2 kcal/mol between 3 0-OH and 7-OH; (iii)25 kcal/mol between 4 0-OH and 5-OH; (iv) 14 kcal/mol between 4 0-OH and 7-OH for quercetin, and by:(i) 16 kcal/mol between the OH groups on the B-ringand 7-OH; (ii) 24 kcal/mol between the OH groups onthe B-ring and 5-OH for taxifolin. From such dier-ences, there is no doubt that the reactivity of the B-ringis higher than that of the A-ring, whatever the kind ofoxidative system involved. This hierarchy may only beovercome if the oxidation of the molecules takes placevia an enzymatic action, for which the binding congu-ration with the protein receptor governs the location ofthe redox reactions.

In quercetin, it is interesting to note that the BDE islower for 4 0-OH by 2.4 kcal/mol compared to 3 0-OH,whereas the values are identical for both groups in taxif-olin. An interpretation for this small but signicant dif-ference will be proposed later in the paper, in the light of

Table 1Calculated values of the BDE for the dierent OH positions in querce

BDE (kcal/mol)

3-OH 30-O

B3P86/6-311+G(d,p) Quercetin 83.7 77.Taxifolin 106.7 76.

B3LYP/6-311+G(d,p) Quercetin 79.7 73.Taxifolin 103.0 72.

The middle part (from column 3 to column 7) is related to the enol formC-2 atom in the keto form of quercetin.the electronic structure.As expected, the behaviour of the 3-OH group is

dramatically dierent in quercetin and taxifolin. The 3-OH-BDE of quercetin (83.7 kcal/mol with B3P86) isintermediate between those on the B-ring and those onthe A-ring. It is only 7 and 9 kcal/mol higher than the4 0-OH and 3 0-OH values, 5 and 16 kcal/mol lower than7-OH and 5-OH. In strong contrast, OH bond dissoci-ation from the 3-OH group is highly thermodynamicallyunfavourable in taxifolin (BDE = 106.7 kcal/mol withB3P86), which could explain, in part, the marked dier-ence in antioxidant activity between quercetin andtaxifolin.that H-transfer is more energetically favourable fromthe B-ring and that H-transfer from the 3-OH group isalso possible in quercetin, depending on the oxidativesystem. This conrms several in vitro studies that haveshown the participation of that group in redox reactions(Balogh-Hergovich et al., 1997; Marfak, Trouillas, Al-lais, Calliste, & Duroux, 2004). At this stage, we alsoconsidered another possible mechanism for explainingthe reactivity of the 3-OH group and the 2,3-doublebond in quercetin. Indeed ketoenol tautomerism cantake place in this molecule (Scheme 2), which is notthe case for taxifolin. We have, therefore, calculatedthe BDE of H-transfer from the C-2 atom in the ketoform for both enantiomers and we have obtained a valueclose to 64 and 62 kcal/mol with B3P86 and B3LYP,respectively (right part of Table 1). This very low BDEindicates the high capacity of H-transfer from the C-2site in the keto form. However, it must be noted thatthe keto form of quercetin is markedly less stable thanthe enol form, the latter being stabilized by p-conjuga-tion from the B-ring to the C-4 carbonyl group throughthe 2,3-double bond. The dierence in stability is such(about 20 kcal/mol) that the contribution of the ketoform can be considered negligible for quercetin as a freemolecule. Nevertheless, one may speculate that, in someenzymatic environments, the stability dierence betweenthe two forms is reduced (for instance, upon the loss ofthe molecular planarity), so that the keto form becomesrelevant, and then H-abstraction from the C-2 atom canO

OOH

OH

OH

OH

OH

O

OH

OH

O

OH

OH

O

H

Enol form of quercetin

Keto form of quercetin

Scheme 2.

tin radical. Consistently, one can observe some delocal-ization beyond the ring bearing the O-atom (smallerspin densities on the surrounding atoms). As a conse-quence, the BDE is lower in the B-ring than in theA-ring.

Nevertheless, dierences in the BDE cannot be ex-plained only on the basis of the spin density value onthe O-atom where H-abstraction occurred: the BDE of5-OH is about 10 kcal/mol higher than that of 7-OH,whereas the spin density on the O-atom of the 5-OHradical is lower than that on the O-atom of the 7-OHradical. That dierence is related to the fact that a H-bond exists between the 5-OH group and the carbonylgroup on C-4. As a consequence, the BDE on that siteis higher because H-removal also implies the breakingof the H-bond.

On the basis of the spin density description, let usnow compare quercetin and taxifolin. In quercetin, theBDE of 4 0-OH (74.6 kcal/mol) is lower than that of3 0-OH (77.0 kcal/mol) by 2.4 kcal/mol, whereas thetwo values are the same in taxifolin (76.0 kcal/mol).

Fig. 1. a-HOMO of the 4-OH and the 7-OH radicals formed by H-removal from quercetin. The a-HOMO for the other radicals issuedfrom quercetin (not shown here) are quite similar (i.e. delocalized overthe entire molecule).

Chemistry 97 (2006) 679688take place easily, thereby contributing to the antioxidantproperties.

3.2. Importance of spin densities for the description of

avonoid radicals

3.2.1. General

The dierence in antioxidant activity between querce-tin and taxifolin, which is reected in the BDE valuescalculated above, is often attributed to p-electron delo-calization, which leads to the stabilization of the radicalsobtained after H-abstraction. This conclusion is drawnassuming that, if p electron delocalization exists in theparent molecule, it also exists in the corresponding rad-ical. In order to understand the relationship between theelectron delocalization and the reactivity of the radicals,one can examine the electron distribution in the singlyoccupied molecular orbital (SOMO), also called, in thiscase, the a-highest occupied molecular orbital (a-HOMO). For quercetin, the a-HOMO is delocalizedover the entire molecule (we have checked that the a-HOMO indeed corresponds to the orbital containingthe unpaired electron, by comparing its shape to thatof the rst unoccupied beta orbital (Ohta, 2002)). Itsshape is quite similar for the ve radicals (correspondingto H-removal from one of the ve OH groups) and itdoes not exhibit sucient variations for explaining thedierences in activity between those OH groups(Fig. 1). Therefore, the shape of the a-HOMO is not areliable indicator for the reactivity of avonoids.

The a-HOMO is the highest-occupied molecular orbi-tal of spin a. In that, it does not describe the global elec-tronic behaviour of the radical. Within an unrestrictedscheme, the spin density is often considered to be a morerealistic parameter and provides a better representationof the reactivity (Szabo & Ostlund, 1982, Chap. 3). Theimportance of the spin density for the description ofavonoids has been pointed out by the recent paper ofLeopoldini et al. (2004). We have, therefore, decidedto analyze the spin density on the various quercetinand taxifolin radicals, in order to rationalize the dier-ences in reactivity of the OH sites in avonoids and con-sequently the dierences in BDE.

3.2.2. Comparison between the spin densities of the

radicals formed from the B- and A-ringsIt must be stressed that the more delocalized the spin

density in the radical, the easier is the radical formed,and thus the lower is the BDE (Parkinson, 1999). Thespin population (on the remaining O-atom after H re-moval and on the neighbouring C-atoms) appears tobe slightly more delocalized for radicals issued fromthe B-ring (3 0-OH and 4 0-OH) than for those locatedon the A-ring (5-OH and 7-OH) (Fig. 2). For example,the spin density is 0.28 on the O-atom in the 4 0-OH

684 P. Trouillas et al. / Foodquercetin radical whereas it is 0.37 for the 7-OH querce- The spin density is actually more delocalized in the 4 0-

P. Trouillas et al. / Food Chemistry 97 (2006) 679688 685OH quercetin radical than in its 3 0-OH counterpart, inthe 3 0-OH taxifolin, and in the 4 0-OH taxifolin radicals.Indeed, in the 4 0-OH quercetin radical, the spin densityon the remaining O-atom is 0.28, whereas it is 0.33 forthe other three systems. This is a consequence of delocal-ization eects due to the presence of the 2,3-doublebond, which allows for spin presence on the C-2 andC-3 atoms: 0.13 of b type and 0.17 of a type, respec-tively. This eect can be rationalized by using the classi-cal resonance eects occurring in the phenoxy radical.Such a scheme explains the presence of the radical (highspin density) on the C-1 0 atom in para-position for the4 0-OH radicals, and the subsequent possible delocaliza-

Fig. 2. Distribution of spin densities in the radicals formed by H-removaltion eect due to the presence of the 2,3-double bondin quercetin. It is to be noted that such delocalizationcannot happen for the 3 0-OH radical of quercetin andfor the 3 0-OH and 4 0-OH taxifolin radicals.

Despite these clear dierences, we believe that thedelocalization eect acting for the 4 0-OH quercetin rad-ical is not sucient to explain why the overall antioxi-dant activity is twice higher for quercetin than fortaxifolin. Both in terms of BDE and spin density distri-bution, the redox action of the B-ring appears to be al-most the same for both molecules. We have also checkedthat the ionization potentials (IP) of quercetin and taxif-olin are very similar (the dierence is only 0.3 eV at the

from the B-and the A-rings, for quercetin (left) and taxifolin (right).

(U)B3P86 level), so that the dierence in antioxidantactivity cannot be ascribed to the predominance of theelectron transfer mechanism, which is governed by theIP value. This leads us to the hypothesis that the higherantioxidant activity of quercetin is related to the con-comitant presence of the 3-OH group and the 2,3-doublebond, which confers high reactivity to that OH site.

3.2.3. The specicity of the 3-OH site

As mentioned above, the a-HOMO of the 3-OH rad-ical is almost the same as that of the other radicals forquercetin. Thus, the a-HOMO localization cannot ex-plain the reactivity of this site. In contrast, we nd a verystrong dierence in the spin density distribution of the 3-OH radical: in taxifolin, the spin density is very high onthe O-atom at C-3 (spin density = 0.81) and concomi-tantly the delocalization is weak (Fig. 3). Since spin den-sity delocalization is related to the easiness of radicalformation; this clearly conrms that, for taxifolin, H-re-moval from 3-OH is not favoured. For quercetin, thecorresponding radical could be formed, either from theketo form or from the enol. It is nonetheless importantto note that, whatever the mechanism of H-transfer, theradical formed is the same. Calculations show that thespin density on the O-3 atom is only 0.32, conrmingthe very high capacity for H-removal from the enol-quercetin 3-OH group or from the C-2 atom of keto-

quercetin. This low value is a consequence of delocaliza-tion via the 2,3-double bond (after or before H-re-moval): the spin density is 0.44 on C-2, 0.13 on C-6 0,0.13 on C-4 0 and 0.09 on C-1 0 (Fig. 3). The presenceof a high spin density (0.44) localized on the C-2 atomimplies a high reactivity for that site, a feature that ap-pears only upon formation of the radical. This is notthe case for taxifolin (the spin density on C-2 is 0.07),due to the absence of the 2,3-double bond.

Along the same lines, we have recently shown that the3-OH group is the primary site of reaction with radicalspecies derived from alcohols (Marfak et al., 2004).The two following radicals: CH3

CHOH (a-hydroxy-ethyl radical or HER) and CH3O (a-hydroxymethylradical or HMR), were produced in radiolyzed alcoholsolutions (ethanol and methanol, respectively) in thepresence of oxygen. We demonstrated that those radi-cals stereospecically attack the 3-OH group, yieldingdepside formation according to reaction Scheme 3. Thismechanism is similar to that described for avonoid deg-radation by dioxygenase, except that there is no COreleasing.

Our calculations, which show a high spin density onthe C-2 atom (0.44), thus indicate that C-2 is indeedthe key site for redox reaction on the intermediateavonoxy radical formed after H-removal on the 3-OH group of quercetin. Under certain experimental con-

emov

O

O

dox at2)

iate ra

686 P. Trouillas et al. / Food Chemistry 97 (2006) 679688Fig. 3. Distribution of spin densities in the radicals formed by H-r

O

OH

OH

OH

OH

OH O OH

OH

First redox attack (radical issued fromsolvent radiolysis)

Second re(O

Quercetin IntermedScheme 3al from the 3-OH group, for quercetin (left) and taxifolin (right).

*

OH

OH

O

O

O

O

OH

OH

OHOH

R

O

tack

Depside

R=OCH3 (in methanol) R=OCH2CH3 (in ethanol)

dical RO.

two possible chemical pathways. The rst one is H-abstraction from the 3-OH group of the enol form ofquercetin, which induces spin delocalization to the C-2

Acknowledgements

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Chemistry 97 (2006) 679688 687atom. The second one is H-abstraction from the C-2atom of the keto form of quercetin, which can form asa consequence of keto-enol tautomerism via the 2,3-double bond.

While the enol form is the most stable tautomer in thefree molecule, the keto form, which possesses a muchlower BDE for H-abstraction on the C-2 atom, mightplay a signicant role in the real system, depending onthe molecular environment of quercetin and on the oxi-dative system acting on the molecule. In any case, thehigh spin density found on the C-2 atom of the radicalcan open the way to C-ring opening. This reaction cas-cade is essential for avonol degradation in radiolyzedsolutions and for degradation during metabolization.Even though experimental data are missing for radicalsditions, O2 is able to bind to C-2, thanks to the presenceof the unpaired electron, to form a peroxide. After-wards, the C-ring can open by the mechanism summa-rized on Scheme 3 (Marfak et al., 2004).

The absence of the 2,3-double bond in taxifolin pre-cludes spin delocalization to the C-2 atom. Even thoughthere is no available experimental data for that avo-noid, one can conclude that the metabolism pathwayis most probably dierent from that of quercetin. Thesubsequent small phenol acids, formed after C-ringopening in quercetin, would hardly form, indicating thattaxifolin would be less easily absorbed than quercetin. Inaddition, it must be stressed that such a mechanism (C-ring opening) has not been observed for taxifolin inradiolyzed alcohol solutions (Marfak et al., 2004).

4. Conclusion

Finding a reliable quantum-chemical method fordescribing the avonoids is of particular interest becausetheoretical insight can contribute signicantly to theunderstanding of the reactivity of those molecules in dif-ferent oxidative systems. From the present results, DFTmethods appear to be the most relevant for the descrip-tion of phenolic compounds. Among the dierent func-tionals tested, B3P86 gave the best agreement withexperimental data; for avonoids, the 6-311+G(d,p) ba-sis set appears as a good compromise between the qual-ity of the results and the computational cost.

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A DFT study of the reactivity of OH groups in quercetin and taxifolin antioxidants: The specificity of the 3-OH siteIntroductionTheoretical methodologyGeneral procedureTest calculations on phenol and catecholCalculations on quercetin and taxifolin

DiscussionBDE values for quercetin and taxifolinImportance of spin densities for the description of flavonoid radicalsGeneralComparison between the spin densities of the radicals formed from the B- and A-ringsThe specificity of the 3-OH site

ConclusionSupporting information availableAcknowledgementsReferences