The Molecular Basis of Working Mechanism of Natural Polyphenolic Antioxidants ⇑ Monica Leopoldini, Nino Russo , Marirosa Toscano

Food Chemistry 125 (2011) 288–306

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Food Chemistry

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Review The molecular basis of working mechanism of natural polyphenolic antioxidants ⇑ Monica Leopoldini, Nino Russo , Marirosa Toscano

Dipartimento di Chimica and Centro di Calcolo ad Alte Prestazioni per Elaborazioni Parallele e Distribuite-Centro d’Eccellenza MIUR, Universita’ della Calabria, I-87030 Arcavacata di Rende (CS), Italy article info abstract

Article history: In this review, we present a summary of the research work performed so far using high accuracy quan- Received 20 April 2010 tum chemical methods on polyphenolic antioxidant compounds. We have reviewed the different groups Received in revised form 21 July 2010 of polyphenols, which mostly belong to the Mediterranean food culture, i.e. phenolic acids, ﬂavonoids and Accepted 6 August 2010 stilbenes. The three main proposed mechanisms through which the antioxidants may play their protective role, which is the H atom transfer, the single electron transfer and the metals chelation, have been analysed and discussed in details. This work represents a further important contribution to the elucida- Keywords: tion of the beneﬁcial effects on health of these substances. Natural antioxidants Ó 2010 Elsevier Ltd. All rights reserved. Flavonoids DFT BDE IP Acidities Metal complexes

Contents

1. Introduction ...... 288 2. Methods ...... 291 3. Parent and radicals polyphenols structures ...... 292 4. BDE and IP evaluation ...... 296 4.1. BDEs ...... 297 4.2. IPs...... 298 5. Determination of polyphenols acidity...... 298 6. Formation of complexes between polyphenols and transition metals ions ...... 300 7. Conclusions...... 303 Acknowledgements ...... 303 References ...... 303

1. Introduction Kris-Etherton et al., 2002; Rencher, Spencer, Kuhnle, Hahn, & Rice-Evans, 2001; Rice-Evans, Spencer, Schroeter, & Rechner, Phenolic compounds are plant secondary metabolites com- 2000; Robak & Gryglewski, 1996; Ross & Kasum, 2002). monly found in herbs and fruits such as berries, apples, citrus fruit, Many of these phenolics are responsible for the attractive col- cocoa, grapes, vegetables like onions, olives, tomatoes, broccoli, let- our of leaves, fruits and ﬂowers (Hermann, 1993). tuce, soybeans, grains and cereals, green and black teas, coffee In the last decades, they have attracted growing global interest beans, propolis, and red and white wines (Brit, Hendrich, & Wang, upon the discovery of the so-called ‘‘French Paradox”, i.e. the 2001; Clifford, 1999; Hertog, Hollman, Katan, & Kromhout, 1993; observation that although the French have smoking tendency and a diet rich in fats, they show much reduced rates of coronary heart disease when compared with northern European nations such as ⇑ Corresponding author. Tel.: +39 0984492106; fax: +39 0984493390. the UK and Germany (Renaud & de Lorgeril, 1992). The most pop- E-mail address: [email protected] (N. Russo). ular explanation has been recognised in the relatively high daily

consumption of red wines rich in phenolic compounds, by the nones by a hydroxyl group at the C3 position, and by a C2–C3 dou- French, which in some way act to protect them from heart diseases ble bond. Anthocyanidins differ from the other flavonoids by (Frankel, Kanner, German, Parks, & Kinsella, 1993; Hertog, Fres- possessing a charged oxygen atom in the ring C. The ring C is open kens, Hollman, Katan, & Kromhout, 1993). in the chalcones. Many flavonoids occur naturally as glycosides, The term phenolics encompasses approximately 8000 naturally and carbohydrate substitutions include D-glucose, L-rhamnose, glu- occurring compounds, all possessing one common structural fea- corhamnose, galactose, and arabinose (Harborne, 1986; Harborne, ture, a phenol (an aromatic ring bearing at least one hydroxyl sub- 1988; Hodnick, Milosavljevic, Nelson, & Pardini, 1988; Kijhnau, stituent). A further classification divides them in polyphenols and 1976). simple phenols, depending on the number of phenol subunits Stilbenes family includes several compounds (Langcake & Pryce, (see Scheme 1). Simple phenols include phenolic acids (Robbins, 1976; Soleas, Diamandis, & Goldberg, 1997) among which resvera- 2003). Polyphenols possessing at least two phenol subunits include trol, pterostilbene, and piceatannol are the main representatives, the flavonoids, the stilbenes, and those compounds possessing characterised by a double bond connecting the phenolic rings three or more phenol subunits are referred to as the tannins (King (see Scheme 1). & Young, 1999). Polymeric compounds, called tannins, are divided into two Phenolic acids are phenols that possess one carboxylic acid groups, i.e. condensed and hydrolyzable. Condensed tannins are functionality. They contain two distinguishing constitutive carbon polymers of flavonoids, and hydrolyzable tannins contain gallic frameworks: the hydroxycinnamic and hydroxybenzoic structures acid, or similar compounds, esterified to a carbohydrate (Hager- (see Scheme 1). Hydroxycinnamic acids are more common than man, Zhao, & Johnson, 1997). hydroxybenzoic acids and consist chiefly of p-coumaric, caffeic, The pharmacological, medicinal and biochemical properties of ferulic, and sinapic acids (Robbins, 2003). phenolics have been extensively reviewed (Cody, Middleton, & The flavonoids consist of a large group of low-molecular weight Harborne, 1986; Cody, Middleton, Harborne, & Beretz, 1988; polyphenolic substances, benzo-c-pyrone derivatives (see Das, 1990; Harborne, 1986). They have been reported to have Scheme 1)(Coultate, 1990). The basic structural feature of all flavo- antioxidant (Kandaswami & Middleton, 1994), vasodilatory, anti- noids is the flavane (2-phenyl-benzo-c-pyrane) nucleus, a system carcinogenic, antinflammatory, immune-stimulating, antiallergic, of two benzene rings (A and B) linked by an oxygen-containing antiviral (Duarte, Perez-Vizcainom, Utrilla, et al., 1993; Duarte, pyrane ring (C). According to the degree of oxidation of the C ring, Perez-Vizcainom, Zarzuelo, Jiminez, & Tanargo, 1993) and estro- the hydroxylation pattern of the nucleus, and the substituent at genic effects, and inhibition activities against phospholipase A2, carbon 3, the flavonoids can be categorised into the subclasses flav- cyclooxygenase, lipoxygenase (Brown, 1980; Ho, Chen, Shi, Zhang, ones, isoflavones, flavanols (catechins), flavonols, flavanones, & Rosen, 1992; Jovanovic, Jankovic, & Josimovic, 1992; Lindahl & anthocyanins, and proanthocyanidins. Flavonols differ from flava- Tagesson, 1993; Mabry, Markham, & Chari, 1982; Middleton &

Scheme 1. Structures of benzoic and hydroxycinnamic acids, ﬂavonoids and stilbenes. 290 M. Leopoldini et al. / Food Chemistry 125 (2011) 288–306

Kandaswami, 1992; Robak, Shridi, Wolbis, & Krolikowska, 1988; The molecular basis for the antioxidant properties of polyphe- Sogawa et al., 1993), glutathione reductase (Elliot, Scheiber, Tho- nols is recognised into three main mechanisms, arising from the di- mas, & Pardini, 1992) and xanthine oxidase enzymes (Chang, Lee, rect reaction with free radicals (Leopoldini, Marino, Russo, & Lu, & Chang, 1993). Toscano, 2004a, 2004b; Leopoldini, Prieto Pitarch, Russo, & Toscan- The best described property of phenolics is the antioxidant o, 2004; Wright, Johnson, & DiLabio, 2001), and from the chelation capability towards free radicals normally produced by cells metab- of free metals, the latter involved in reactions finally generating olism or in response to external factors. Free radicals can damage free radicals (Jovanovic, Steenken, Simic, & Hara, 1998). biomolecules such as lipids, nucleic acids, proteins, cause cellular As primary antioxidants, polyphenols inactivate free radicals membranes peroxidation (De Groot, 1994; Grace, 1994) and attract according to the hydrogen atom transfer (HAT) (1) and to the single various inflammatory mediators (Halliwell, 1995). electron transfer (SET) (2) mechanisms (see Scheme 2). In mecha- Polyphenols scavenge free radicals and reacting oxygen species nism 1, the antioxidant, ArOH, reacts with the free radical, R, by (ROS), that are made so inactive (De Groot, 1994; Grace, 1994). transferring to it a hydrogen atom, through homolytic rupture of Flavonoids inhibit nitric oxide synthase that generates nitric the O–H bond: oxide, which in turn reacts with free radicals to generate the Å Å peroxynitrite species, in addition to be itself a radical (Dehmlow, ArOH þ R ! ArO þ RH ð1Þ Erhard, & de Groot, 1996; Huk, Brovkovych, & Nanobash, 1998; The products of the reaction are the harmless RH species and Shoskes, 1998; Shutenko et al., 1999; van Acker, Tromp, Haenen, the oxidised ArOÅ radical. Even if the reaction leads to the forma- van der Vijgh, & Bast, 1995). tion of another radical, it is less reactive with respect to RÅ because Xanthine oxidase is implicated in oxidative injury, especially stabilized by several factors (see below). after ischemia–reperfusion, because it reacts with molecular oxy- The SET mechanism (2) provides for an electron to be donated gen and releases superoxide. Flavonoids, in particular quercetin to the RÅ: and luteolin, are potent inhibitor of xanthine oxidase (Cos et al., Å þÅ 1998; Iio, Ono, Kai, & Fukumoto, 1986; Sanhueza, Valdes, Campos, ArOH þ R ! ArOH þ R ð2Þ Garrido, & Valenzuela, 1992; Shoskes, 1998). The anion R is an energetically stable species with an even The mechanism of the antitumor effects of flavonoids seems to Å number of electrons, while the cation radical ArOH+ is also in this depend on their structure, with each compound displaying various case a less reactive radical species. biological potency and mechanism(s) of action (Di Carlo, Mascolo, Å Å In particular, the ArO and ArOH+ are aromatic structures in Izzo, & Capasso, 1999). However, the essential feature of flavonoids which the odd electron, originated by the reactions with the free is their free radical scavenging activity, partially responsible for radical, has the possibility to be spread over the entire molecule, their antitumor effects. Flavonoids have antiproliferative effects resulting into a radical stabilization (Leopoldini et al., 2004a, and induce apoptosis in different cancer cell lines. As free radical 2004b; Wright et al., 2001; Leopoldini, Prieto Pitarch, et al., 2004). scavengers, flavonoids inhibit invasion and metastasis (Cipak, Rau- In the former mechanism, the bond dissociation enthalpy (BDE) ko, Miadokova, Cipakova, & Navotny, 2003; Krol, Czuba, Threadgill, of the phenolic O–H bond is an important parameter in evaluating Cunningham, & Pietsz, 1995; Kuntz, Wenzel, & Daniel, 1999; Nij- the antioxidant action; the lower the BDE value, the easier the dis- veldt et al., 2001; Win, Cao, Peng, Trush, & Li, 2002). sociation of the phenolic O–H bond and the reaction with the free Some aglycone flavonoids are potent inhibitors of oxidative radicals. In the SET mechanism, the ionisation potential is the most modification of LDL in vitro by macrophages or copper ions (De significant parameter for the scavenging activity evaluation; the Whalley, Rankin, Hoult, Jessup, & Leake, 1999). lower the IP value, the easier the electron abstraction and the reac- Platelet–blood vessel interactions are implicated in the devel- tion with free radicals. opment of thrombosis and atherosclerosis. Particular flavonoids in- Another antioxidant mechanism (Transition Metals Chelation, hibit platelet aggregation and adhesion (Beretz, Anton, & Cazenave, TMC, see Scheme 2) arises from the possibility that transition met- 1986; Beretz & Cazenave, 1988; Beretz, Cazenave, & Anton, 1982; als ions may be chelated by polyphenols, leading to stable com- Gryglewski, Korbut, Robak, & Swies, 1987; Mora, Paya, Rios, & Alca- plexed compounds (Brown, Khodr, Hider, & Rice-Evans, 1998; raz, 1990; Robak, Korbut, Shridi, Swies, & Rzadkowska-Bodalska, Jovanovic et al., 1998; van Acker et al., 1996). The latter entrap 1988; Swies et al., 1984; Tzeng, Ko, Ko, & Teng, 1991). However, metals and avoid them to take part in the reactions generating free the antiaggregatory effects of flavonoids cannot be attributed to radicals. In fact, some metals in their low oxidation state (mainly a single biochemical mechanism because they appear to influence Fe2+) may be involved in Fenton reactions with hydrogen peroxide several pathways involved in platelet function (Landolfi, Mower, & (Schulz, Lindenau, Seyfried, & Dichganz, 2000), from which the Steiner, 1984; Tzeng, Ko, Ko, & Teng, 1991). Å very dangerous reactive oxygen species (ROS) OH is formed: Flavonoids appear to increase vasodilatation by inducing vascular smooth muscle relaxation which may be mediated by the inhi- nþ Å ðnþ1Þþ H2O2 þ M ! HO þ HO þ M bition of protein kinase C, PDEs, or by decreased cellular uptake of calcium (Duarte, Vizcaino, et al., 1993). The OHÅ is generally accepted to be one of the most reactive rad- Six flavonoids have been evaluated for their ability to prevent icals. It has a very short half-life (around 109 s) and a very high injury in mesencephalic cultures, resulting that all protect neurons reactivity. With respect to the hydroperoxides that are metabo- from damage by the dopaminergic toxin N-methyl-4-phenyl- lized by superoxide dismutase, hydroxyl radicals cannot be elimi- 1,2,3,6-tetrahydropyridinium hydrochloride MPP+ (Mercer, Kelly, nated by enzymatic reactions. So they will react with every kind Horne, & Beart, 2005). of substrate they encounter (Palmer & Paulson, 1997). Concerning their metabolism, most of flavonoids are absorbed Transition metals like copper, manganese, cobalt are able to into the intestinal cells by passive mechanisms (Barnes et al., catalyse this reaction, under certain conditions when these metal 2003; Day et al., 2000; Sfakianos, Coward, Kirk, & Barnes, 1997; ions are not bound to proteins or chelators. Fenton-like reaction Yasuda, Kano, Saito, & Ohsawa, 1994). Once on the blood circula- may take place and cause site specific accumulation of free radicals tion, they are converted into metabolites with higher antioxidant and initiate biomolecules damage processes. and estrogenic activities with respect to their unmetabolized par- Fenton chemistry occurs in dopaminergic neurons of nervous ent molecules (Adlercreutz et al., 1986; Axelson, Sjövall, Gustafs- tissue, where normally dopamine catabolism produces some levels son, & Setchell, 1984; Coldham et al., 1999; Rimbach et al., 2003). of hydrogen peroxide. The accumulation of free radicals in these M. Leopoldini et al. / Food Chemistry 125 (2011) 288–306 291

Scheme 2. Mechanisms for the antioxidant activity. neurons may be recognised as the main aetiological agent of Par- B3LYP Slater HF Becke LYP VWN F ¼ð1 AÞFx þ AFx þ BFx þ CFc þð1 CÞFc kinson disease (PD) (Schulz et al., 2000). Other neurodegenerative diseases, such as Alzheimer’s diseases (AD) and Huntington’s cho- where FSlater is the Slater exchange, FHF is the Hartree–Fock ex- rea, have as hallmark a significant increase in iron in some brain x x change, FBecke is the gradient part of the exchange functional of regions (Gerlach, Ben-Shachar, & Riederer, 1994; Hirsch & Fauc- x Becke, FLYP is the correlation functional of Lee, Yang and Parr, and heux, 1998; Youdim, Ben-Shachar, & Riederer, 1993). Basal ganglia c FVWN is the correlation functional of Volsko, Wilk and Nusair. The ferritin iron content is increased in patients affected by AD (Bartzo- c A, B and C coefficients are determined by fitting experimental heats kis et al., 2000), whereas iron is found in higher concentration up to of formation (Becke, 1993). 35% in the substantia nigra pars compacta in PD patients (Double, The accuracy of the DFT methods have been tested through G2 Gerlach, Youdim, & Riederer, 2000). It has been proposed that hy- benchmark test of 55 small first- and second-row molecules (Bau- droxyl radicals and Fe(III) are generated upon Fenton reaction that schlicher, Ricca, Partridge, & Langhoff, 1997; Curtiss, Raghavachari, accounts for the increase of ferric ions and reactive oxygen species Trucks, & Pople, 1991), according to which B3LYP method seems to in these degenerating zones of the brain (Linert et al., 1996; Owen, yield good results in predicting atomization energies. For geome- Shapira, & Jenner, 1997; Smythies, 2000). tries optimisation, all DFT means have given quite accurate results. Metal-chelating compounds remove the metals and can alter Concerning transition-metals complexes, for which few accurate their redox potentials rendering them inactive. Moreover, the use experimental data are available, systematic theoretical studies of natural metal chelators such as flavonoids should be favored have been performed on small MR+ systems, where M is a first- against other synthetic chelators which may present some prob- row transition metal and R is H, CH ,CH and OH. The average lems of toxicity. Flavonoids with their multiple hydroxyl groups 3 2 absolute error in M–R binding energies results to be in the range and the carbonyl group at the 4 position on ring C (see Schemes of 3.6–5.5 kcal/mol, as the B3LYP functional is employed (Arment- 1 and 2) may offer several available sites for metal complexation. rout & Kickel, 1996; Blomberg, Siegbahn, & Svensson, 1996; Ricca & The purpose of this review is to give an overview of the research Bauschlicher, 1997). Other theoretical studies on M–CO complexes carried out in the field of antioxidant polyphenolic compounds, binding energies have indicated B3LYP to give good agreement employing theoretical and computational methods. It analyses in with experiments, being the average error 2.6 kcal/mol (Blomberg details the working mechanisms of flavonoids and polyphenols et al., 1996; Ricca & Bauschlicher, 1994). as antioxidants, covering the relevant literature on this subject. The choice of B3LYP functional in this study is dictated by its good performance in geometries optimisation, as well as by its 2. Methods quite accurate prediction of X–H bond energetic, and binding energies. For example, post-HF MP2 optimization of quercetin molecule All the calculations reported are performed with the Gauss- and its deprotonated and semiquinone forms, converged to a pla- ian03 code (Frisch et al., 2003). The principal conceptual tools used nar arrangement, as found with the B3LYP method (Fiorucci, Gole- here are density functional theory (DFT) methods, employing the biowski, Cabrol-Bass, & Antonczak, 2007). Test calculations on Becke3 (Becke, 1993) and Lee Yang Parr (Lee, Yang, & Parr, 1988) propene have proposed B3LYP to predict bond dissociation ener- (B3LYP) hybrid functional. It can be written as: gies in good agreement with the values obtained by employing 292 M. Leopoldini et al. / Food Chemistry 125 (2011) 288–306 the more accurate and expensive MP2 and CCSD methods (DiLabio, The O–H bond dissociation energy (BDE) is computed at 298 K 1999). However, relative evaluation of antioxidant activity is per- as the difference in enthalpy (H) between products and reactants formed in this study, taking the phenol molecule as reference, in for the reaction (1), that is: order to observe the effect of some functional groups and/or spatial Å Å BDE ðArO —HÞ¼HðArO ÞþHðH ÞHðArOHÞ disposition on the antioxidant power of these natural compounds. Other methods reported in this study are the Hartree–Fock, the The ionisation potential (IP) values are computed at 298 K as semi-empirical AM1 (Anders, Koch, & Freunscht, 1993; Davis, the enthalpy difference between products and reactants for the Guidry, Williams, Dewar, & Rzepa, 1981; Dewar & Holder, 1990; reaction (2), that is: Dewar & Jie, 1989; Dewar, Jie, & Zoebisch, 1988; Dewar, McKee, þÅ & Rzepa, 1978; Dewar & Merz, 1988; Dewar & Reynolds, 1986; IPðArOHÞ¼HðArOH ÞHðArOHÞ Dewar & Thiel, 1977; Dewar & Yuan, 1990; Dewar, Zoebisch, & The gas-phase acidity is computed at 298 K as the enthalpy dif- Healy, 1985) and PM3 (Stewart, 1989a, 1989b), and the ab initio ference between the anion (A) and its neutral species (HA): MP2 (Frisch, Head-Gordon, & Pople, 1990a, 1990b; Head-Gordon & Head-Gordon, 1994; Head-Gordon, Pople, & Frisch, 1988; Møller DHacidity ¼ HðA ÞHðHAÞ & Plesset, 1934; Saebø & Almlöf, 1989) ones. For the calculations in the condensed phase, the acidities are The selected polyphenols molecules, and their radicals and an- computed in the same way but given in terms of total free solva- ions, are optimised without constraints at B3LYP level, employing tion energies (DG). the 6-311++G** basis set (Ditchfield, Hehre, & Pople, 1971; Gordon, 1980; Hariharan & Pople, 1974; Hehre, Ditchfield, & Pople, 1972). For iron quercetin complexes, the 6-31G* basis set, and the 3. Parent and radicals polyphenols structures LANL2DZ pseudopotential (Hay & Wadt, 1985), are chosen for C, O and H atoms, and for Fe2+ cation, respectively. Geometry optimi- The knowledge of the conformational, electronic and geometri- sation is followed by single-point calculations using the extended cal features of phenolic systems is of crucial importance to under- 6-311++G** basis set for the non-metal atoms, in order to refine stand the relationship between the molecular structure and the electronic energies. antioxidant activity. It is commonly accepted that the main struc- The B3LYP functional has been widely used for the treatment of tural characteristics for a good radical scavenging activity are: transition metal containing molecules, for two main reasons. It has shown to be the most accurate of the DFT functionals in bench- – the occurrence of multiple OH groups attached to the aromatic mark tests, and also to be fast enough to be able to treat rather ring; large models, even up to a few hundred atoms (Siegbahn, 2003). – the arrangement of these hydroxyls in the ortho-dihydroxy con- This method has shown good performance for a truly wide variety formation, when possible; of chemical systems and properties, although specific limitations – the planar structure of phenolics, that allows conjugation and and failures have also been identified. For example, metal–ligand electronic delocalization, as well as resonance effects; binding energies always appear to be underestimated by B3LYP, – the presence of additional functional groups, like the carbon– so far never overestimated, which is helpful in the analysis of the carbon double bond and the C@O carbonyl group. results. Concerning the employment of the LANL2DZ effective core potential and its orbital basis set in the study of metal–flavonoids Since polyphenols are considered to react mainly with free rad- complexes, this method can handle high Z atoms. It is widely used icals by donating to them an HÅ (or an electron), the knowledge of in this kind of studies, and it gives good results in binding energies the geometrical and electronic structures of the radicals arising for transition metals ligands complexes (Siegbahn, 2003, 2006). from this interaction is relevant for the investigation of this kind Minima are identified through frequency calculations per- of mechanism. formed at the same level of theory. Zero point energy corrections, Molecules with multiple OH groups can give rise to several rad- obtained from vibrational analysis, are then included in all the rel- icals depending on which group is radicalised. The relative energies ative energy values. of the radicals of some polyphenols are reported in the Table 1. The The unrestricted open-shell approach is used for polyphenols optimised geometries of the most stable radical species are pre- radical species. No spin contamination is found for radicals, being sented in the Fig. 1. The complete geometrical parameters of all the hS2i values of 0.750 in all cases. investigated systems are available on request. Natural Bond Orbital (NBO) (Carpenter, 1987; Carpenter & Tyrosol and hydroxytyrosol are the main phenolic compounds Weinhold, 1988; Foster & Weinhold, 1980; Reed, Curtiss, & Wein- present in the virgin olive oil (see Scheme 3)(Brenes, Garcia, Garcia, hold, 1988; Reed & Weinhold, 1983; Reed & Weinhold, 1985; Reed, Rios, & Garrido, 1999). The different conformers of these two mole-

Weinstock, & Weinhold, 1985; Weinhold & Carpenter, 1988) anal- cules arise from the ﬂexibility of the side chain –CH2CH2OH, and in ysis implemented in the Gaussian03 package is used to better char- hydroxytyrosol, also from the relative disposition of the OH groups. acterise electronic structure. Minimum energy conformers are characterised by the folded gauche Solvent effects are computed in the framework of Self-Consis- conformation of the alkyl chain in which the alcoholic OH is oriented tent Reaction Field Polarizable Continuum Model (SCRF-PCM) toward the aromatic ring, so that a hydrogen bond like interaction (Cossi, Barone, Cammi, & Tomasi, 1996; Miertus, Scrocco, & can be established (Leopoldini et al., 2004a, 2004b). The two OH Tomasi, 1981; Miertus & Tomasi, 1982) using the Simple United groups in hydroxytyrosol realise a hydrogen bond in which the

Atom Topological Model (UA0) (Barone, Cossi, Menucci, & O4–H hydroxyl plays the H-bond donor function. Tomasi, 1997) set of solvation radii to build the cavity for the Tyrosol and hydroxytyrosol radicalisation originates one and solute in its gas-phase equilibrium geometry. The molecule is two radicals, respectively. As far as the catechol functionality is placed in a cavity, which is created via a series of overlapping concerned, the radicalisation of the 4-OH group in hydroxytyrosol spheres. In PCM method, the variation of the free energy when absolute minimum causes the loss of the internal hydrogen bond. going from vacuum to solution is composed of the work This can be re-established by a free rotation around the C3–O–H required to build a cavity in the solvent (cavitation energy, Gcav) bond, that requires an energetic expense of around 3 kcal/mol together with the electrostatic (Gel) and nonelectrostatic work (Leopoldini et al., 2004a). The other 4-OH radical (radicals are indi- (Gdisp + Grep). cated as the original hydroxyl group from which the hydrogen is M. Leopoldini et al. / Food Chemistry 125 (2011) 288–306 293

Table 1 Relative energies (values in kcal/mol) of some polyphenols radicals in the gas-phase.

Radicals DE Radicals DE Radicals DE Radicals DE Hydroxytyrosol Gallic acid Caffeic acid Resveratrol 3-OH 0.0 3-OH 6.4 3-OH 6.6 3-OH 6.3 4-OH 1.4 4-OH 0.0 4-OH 0.0 5-OH 5.8 40-OH 0.0 Catechin Epicatechin Kaempferol Cyanidin 30-OH 0.7 30-OH 0.0 40-OH 0.0 30-OH 4.3 40-OH 0.0 40-OH 0.1 3-OH 0.2 40-OH 3.1 5-OH 8.1 5-OH 8.3 5-OH 13.5 3-OH 0.0 7-OH 7.9 7-OH 10.6 7-OH 5.7 5-OH 2.5 7-OH 5.7 Quercetin Apigenin Luteolin Taxifolin 30-OH 2.5 40-OH 0.0 30-OH 2.3 30-OH 0.6 40-OH 0.0 5-OH 23.8 40-OH 0.0 40-OH 0.0 3-OH 8.4 7-OH 5.2 5-OH 31.4 5-OH 22.4 5-OH 23.2 7-OH 12.9 7-OH 29.7 7-OH 14.3

Fig. 1. Equilibrium geometries of the most stable radicals obtained after H-atom removal from polyphenols: (a) tyrosol, (b) hydroxytyrosol, (c) tocopherol, (d) gallic acid, (e) caffeic acid, (f) resveratrol, (g) catechin, (h) epicatechin, (i) kaempferol, (l) apigenin, (m) luteolin, (n) taxifolin, (o) quercetin, (p) cyanidin. 294 M. Leopoldini et al. / Food Chemistry 125 (2011) 288–306

Scheme 3. Some polyphenols studied. removed) conformer missing this interaction is thermodynami- matic system, with a complete electronic delocalization occurring cally less favoured by 8.8 kcal/mol (Leopoldini et al., 2004a). This on the aromatic ring carrying the phenolic OH, while the –CH3 sub- value can be considered as an estimation of the stabilising effect stituents increase the charge density on the same ring. coming from the H-bond. The radical 3-OH lies at 1.4 kcal/mol with The group of the phenolic acids contains a lot of strong antiox- respect to the 4-OH one. This energy difference is explained by idant natural compounds (Robbins, 2003). considering that in the 4-OH species, the electronic vacancy is sup- Gallic acid (Scheme 3) is present itself or as ester moiety in plied by the electron-donating effect of the –CH2CH2OH group, that other polyphenols. Three OH groups are present in its minimum does not occur in the other radical, as an analysis of the resonance energy structure, arranged as to form two hydrogen bonds of structures immediately suggests. 2.196 Å (Leopoldini et al., 2004a). Bond order values computations Vitamin E is one of the non enzymatic endogenous systems find a double bond in the C@O carbonyl group (bond order of acting as antioxidant in living organisms. It contains a-, b-, 1.756), while the values of bond order of the carbon–carbon couple c- and d-tocopherols that possess a phytyl tail (C16H33) ensuring are 1.360, as a confirmation of the expected electronic delocaliza- to the molecule the solubility in membranes (Morris, & Evans, tion typical of aromatic rings. From gallic acid it is possible to get 2002). The radical scavenging ability is due to the OH group. The two radicals, the 3-OH (5-OH) and the 4-OH. The latter is the abso- computations (Leopoldini et al., 2004a) on a model system of vita- lute minimum (relative energy of the 3-OH (5-OH) is 6.4 kcal/mol), min E, that is the 6-hydroxy-2,2,5,7,8-pentamethylchroman, HPMC stabilized by the coupled effect of the neighbouring OH in ortho- (Scheme 3), confirm that this compound has the features of an aro- and the COOH in the para-position. In both radicals, the unpaired M. Leopoldini et al. / Food Chemistry 125 (2011) 288–306 295 electron appears to be delocalised over the aromatic ring (Leopol- Zhou, Yang, Wu, & Liu, 1998). The OH group in ring C is an alcoholic dini et al., 2004a). group to which cannot be ascribed any antioxidant capability. The presence of the CH@CH bridge between the benzene and Catechin (Scheme 3) lowest energy structure is characterised by the carboxyl group in caffeic acid (Scheme 3) favours resonance a torsional C20 –C10–C2–C3 of 77.4° (Leopoldini, Russo, & Toscano, and conjugation effects. A complete exploration (Leopoldini et al., 2007), that indicates that the rings B and C are almost perpendic- 2004a; VanBesien & Marques, 2003) of caffeic acids conformers ular. The hydroxyls in ring B establish an H-bond of 2.152 Å. The leads to fourteen isomers arising from S-cis or S-trans conformation electronic delocalization occurs separately on rings B and A. of the carboxylic group dihedral, disposition of the two phenolic In the case of ()-epicatechin (Scheme 3), the same torsion is hydroxyls dihedral with respect to the ring, mutual orientation of 92.49°, that indicates that in the latter the ring B is more twisted. the aromatic ring and carboxyl group. Most stable conformers The other conformer of epicatechin, with torsion of 270.14°, lies are characterised by the S-cis orientation of the carboxyl group at only 0.3 kcal/mol. The energy required to pass from one con- and by the mutual trans disposition of this group and the aromatic former to another is computed to be 1.1 kcal/mol (Leopoldini ring (VanBesien & Marques, 2003). The OH groups are involved in a et al., 2004a), so they may coexist. hydrogen bond (2.152 Å). The stabilizing effect of this internal H- Radicalisation of catechin and epicatechin molecules gives as bond can be estimated roughly 4.5 kcal/mol, that represents the most stable radical species the isoenergetic 30-OH and 40-OH in relative energy of the conformer missing this kind of weak interac- both cases (Leopoldini et al., 2004a; Leopoldini et al., 2007). In tion (VanBesien & Marques, 2003). the absence of conjugation with ring C, the main stabilizing factor The dihedral between the phenyl and the substituent is, in the is the internal hydrogen bond at the ring B. The 5-OH and the 7-OH minimum geometry, 0° (Leopoldini et al., 2004a; VanBesien & Mar- are found at 8.1 and 8.3 kcal/mol, and at 7.9 and 10.6 kcal/mol, for ques, 2003). With an expense of only 6.1 kcal/mol which leads the catechin and epicatechin, respectively. ethylene group perpendicular to the plane of the ring, a relative The absence of ortho-diphenolic structure on ring B in kaempf- minimum lying at 0.4 kcal/mol above the global one and character- erol could determine its lesser efficiency as hydrogen donor (Rice- ised by a torsion angle of 180°, can be easily reached (Leopoldini Evans, Miller, & Paganga, 1996). In kaempferol absolute minimum et al., 2004a). The relative energies of these conformers and the (Scheme 3), the hydrogen bonds are established between the 3- low activation energy required indicate that the conformers may OH/5-OH and the C4@O carbonyl oxygen (Leopoldini et al., coexist (Leopoldini et al., 2004a). 2004a). The molecule is completely planar as the dihedral value Caffeic acid radicalisation yields to two radicals, 3-OH and 4- indicates, at both B3LYP (Leopoldini et al., 2004a) (180°) and RHF OH. The electronic delocalization effect of the –CH@CH–COOH is (van Acker et al., 1996) (179.86°) levels. Radical 40-OH and 3-OH responsible for the energetic stability of the 4-OH radical (Table 1) have practically the same stability, being their energetic gap only (Leopoldini et al., 2004a). 0.2 kcal/mol (Leopoldini et al., 2004a). B3LYP bond order analysis Rosmarinic acid is a phenolic compound extracted from Rose- and the value of the torsional angle (U = 180°) indicate that for marinus officinalis L. It contains two phenolic rings both carrying both, a broad delocalization of the odd electron contributes to two ortho-hydroxyl groups (Petersen & Simmonds, 2003). There the radical stability (Leopoldini et al., 2004a). Planar arrangement are a carbonyl group, an unsaturated double bond and a carbox- of the kaempferol radicals is found also as far as RHF computations ylic acid between the two phenolic rings. Its structure is quite dif- are performed (van Acker et al., 1996). ferent from the other phenolics. Geometry minimisation indicate Apigenin and luteolin (Scheme 3) differ by a hydroxyl on the 30 as preferred structure the one with the ring A coplanar with the position in the ring B. Both, in their B3LYP/6-311++G** equilibrium double bond and the 9-carbonyl, and the ring B out of plane of geometries, are planar molecules with torsional angles between the rest of the molecule, as expected on the basis of resonance rings C and B (C3–C2–C10–C20) of 0.0° (Leopoldini, Prieto Pitarch structures (Cao et al., 2005). Upon radicalisation of rosmarinic et al., 2004). The conformers with a dihedral of 180.0° are found acid, four radicals are obtained. Among them, the most stable is at 0.1 (apigenin) and 0.2 (luteolin) kcal/mol. The transition states the 2-OH one, followed in energy by the 40-OH (DE = 0.4 kcal/ in going from 0.0° to 180.0° are found at 4.0 and 3.7 kcal/mol mol) (Cao et al., 2005). Radical 1-OH is found at 2.6 kcal/mol. and characterised by a dihedral of 90.9° and 90.8°, for apigenin By looking at the molecular structure, it can be noted that in and luteolin, respectively (Leopoldini, Prieto Pitarch et al., 2004). the case of the 2-OH/40-OH species, the odd electron is better RHF/STO-3G computations find for both flavones a non planar con- delocalised by the presence of substituents in the para-position. formation, with dihedral of 16.5° and 16.3°, respectively (van Acker This possibility is missing in the case of the less stable 1-OH spe- et al., 1996). An explanation to these findings (van Acker et al., cies (Cao et al., 2005). 1996) is recognised in the lack of the 3-OH group in ring C, that Resveratrol (trans-3,5,40-trihydroxystilbene, see Scheme 3)isa establishing hydrogen like interaction with the ring B, should force natural product found in grapes, mulberries, peanuts. It is one of the system in a planar disposition. So, flavones lacking the 3-OH the main non alcoholic components in the red wines (Jang et al., group should be slightly twisted (luteolin, apigenin, diosmin) 1997). Its structure is characterised by two phenolic rings, linked (van Acker et al., 1996). HF/6-31G(d) method also predicts for flav- by a double bond. The B3LYP/6-311++G** optimisation (Leopoldini ones a non planar conformation, as well as B3LYP/6-31G(d) ones, et al., 2004a) yields an absolute minimum characterised by planar- that find for apigenin and luteolin a torsional angle of 16.4° and ity, conjugation and electronic delocalization. The mutual position 18.1° (Martins, Leal, Fernandez, Lopes, & Cordeiro, 2004). The dis- of the hydroxyls does not allow the formation of any intramolecu- crepancies between HF and DF approaches can be ascribed to the lar H-bonds (Caruso, Tanski, Villegas-Estrada, & Rossi, 2004; Leo- fact that the former (as well as AM1 and PM3) methods underesti- poldini et al., 2004a). Radicalisation of the 40-OH group generates mate the stabilizing p-electrons delocalization contributions with the most stable radical, while the other 3-OH and 5-OH systems respect to DF. Concerning the comparison between B3LYP results lie at 6.3 and 5.8 kcal/mol, respectively (Leopoldini et al., 2004a). (Leopoldini, Prieto Pitarch, et al., 2004; Martins et al., 2004), it As pointed out for rosmarinic acid, only in the first species the res- should be noted that the use of an extended basis set including dif- onance forms show the unpaired electron spread over the whole fuse functions (Leopoldini, Prieto Pitarch, et al., 2004) should im- molecule. prove the electronic structure description. Flavanols lack the 2,3-double bond in the ring C, so that four The H-atom removal from apigenin and luteolin molecules orig- stereoisomers exist, of which (+)-catechin (b-OH in ring C) and inates radicals of which the 40-OH is the most stable (Leopoldini, ()-epicatechin a-OH in ring C) are the most important ones (Jia, Prieto Pitarch, et al., 2004). In contrast to apigenin 40-OH radical, 296 M. Leopoldini et al. / Food Chemistry 125 (2011) 288–306 in which the odd electron leaves the radicalised oxygen, in luteolin absolute minimum (DE = 5.6 kcal/mol) because of its full planarity, 40-OH the unpaired electron remains on the radicalisation site, due while the steric hindrance in the s-trans one causes a certain devi- to the intramolecular H-bond. RHF computations (van Acker et al., ation from the planarity (torsion angle O–C–Ca–Cb of approxi- 1996) also yield planar radicals, despite the parent molecules show mately 142°)(Kozlowski et al., 2007). The energy cost to pass twisted rings B. from one conformer to another is found to be 8.3 kcal/mol. Compu- Saturated taxifolin (Scheme 3) does not present planar confor- tations on the other chalcones also lead to planar configuration, 0 mation because of the absence of the C2–C3 double bond in the ring even in the absence of the 2 -OH group that is supposed to be in C. B3LYP/6-311++G** computations indicate a C3–C2–C10–C20 tor- part responsible for the planarity (Kozlowski et al., 2007). Radicali- sion of 101.0° (Leopoldini, Prieto Pitarch, et al., 2004). RHF/STO- sation of the 20,40,60,3,4-pentahydroxycalchone involves the forma- 3G also find a non planar conformation for taxifolin (152.4°)(van tion of five radicals, the most stable one is again characterised by Acker et al., 1996), as well as for hesperetin and naringenin. From an internal H-bond and by delocalization and conjugation effects taxifolin, four radicals exist, and among them the 30-OH and 40-OH (Kozlowski et al., 2007). are the most stable ones (Leopoldini, Prieto Pitarch, et al., 2004). Their stability depends on the same factors indicated for saturated catechin and epicatechin. 4. BDE and IP evaluation Quercetin (Scheme 3) is a flavonol on which many biochemical, epidemiologic, medical as well as theoretical works exist. B3LYP Antioxidants may play their protective role by donating an H- optimisations (Leopoldini et al., 2004b) yield as preferred structure atom or a single electron, so the bond dissociation enthalpies

(I) a planar conformation (C3–C2–C10–C60 U 180° characterised by (BDEs) for the O–H bonds and the ionisation potentials (IPs) are three intramolecular hydrogen bonds, established between the 3- of particular interest to evaluate their potentiality. BDEs and IPs 0 0 OH/5-OH and the C4@O, and between the 3 -OH and 4 -OH in the for polyphenols of Scheme 3 (except for rosmarinic acid, chalcones ring B. A relative minimum (II) with a U 0° is found lying at and myricetin) are collected in the Tables 2 and 3. In the same 0.5 kcal/mol with respect to the former, and it can be reached with tables, the values for phenol are also reported, with the purpose an energetic expense of only 5.6 kcal/mol (Leopoldini et al., 2004b). to quantitatively estimate the effect of OH groups and substituents AM1 (Russo, Toscano, & Uccella, 2000) and RHF/6-31G* (Vasilescu on the basic activity of phenol. & Girma, 2002) computations yield as preferred structure a slightly twisted conformation (U = 153.3° and 162.3°, respectively), also identifying a relative minimum lying at 0.2 kcal/mol that is Table 2 Bond dissociation energies (BDE) for reached through a barrier of 2.5 and 4.0 kcal/mol, respectively. polyphenols in the gas-phase. Values Even if some differences can be found in the absolute values, all are given in kcal/mol. theoretical data indicate that quercetin may exist into two con- Compound BDE formers that easily may interconvert (Leopoldini et al., 2004b). Starting from the two B3LYP quercetin minima I and II, ten radicals Phenol 82.9 Tyrosol 82.0 0 0 are obtained breaking the 3-, 3 -, 4 -, 5- and 7-O–H bonds, all char- Hydroxytyrosol 73.5 0 acterised as planar species. Among them, the 4 -OH(I) species is the Gallic acid 72.2 most stable, followed by the 40-OH(II) one (DE = 0.2 kcal/mol at Caffeic acid 73.6 B3LYP/6-311++G** level) (Leopoldini et al., 2004b). The energetic HPMC 71.7 Resveratrol 77.3 gaps among the radicals arising from the radicalisation of the rings Catechin 74.2 B and C fall within 8 kcal/mol, while radicalisation occurring at Epicatechin 73.7 the ring A produces radicals very high in energy (range of 13– Kaempferol 80.9 25 kcal/mol). These latter radicals exhibit a spin distribution that Cyanidin 79.4 leaves the odd electron on the radicalisation site, probably because Quercetin 72.3 @ Apigenin 82.2 of the presence of the –C O and –O-moieties in the adjacent ring C Luteolin 74.5 (Leopoldini et al., 2004b). Taxifolin 74.7 Cyanidin (Scheme 3) minimum energy structure is a completely planar system (U =0°) since the bond order average values are

1.300 for all couples of atoms, except for the C2–O1 and C9–O1 (Leo- poldini et al., 2004b). A relative minimum for U = 180° is found at 0.7 kcal/mol, after overcoming an energetic barrier of 10.1 kcal/mol Table 3 (Leopoldini et al., 2004b). This barrier seems to be higher than Ionisation Potentials (IP) for polyphenols in the gas-phase. Values are those computed for the other polyphenols, so probably the second given in kcal/mol. minimum cannot be easily reached. Cyanidin radicals energies fall within 6 kcal/mol, being the gas-phase stability order 3-OH > 5- Compound IP OH > 40-OH > 30-OH > 7-OH (Leopoldini et al., 2004b). The forma- Phenol 192.0 tion of these species does not entail the breaking of any H-bond Tyrosol 181.7 Hydroxytyrosol 175.1 so that their relative energies are very close. Gallic acid 189.1 Chalcones (or 1,3-diaryl-2-propen-1-one) are open-chain flavo- Caffeic acid 181.1 noids (see Scheme 1), in which two aromatic rings are linked by a HPMC 154.9 three-carbon a,b-unsaturated carbonyl system. The absence of the Resveratrol 161.3 central C ring and the presence of a a,b-unsaturated bond are two Catechin 169.7 Epicatechin 170.8 specific characteristics of chalcones, making them chemically dif- Kaempferol 168.0 ferent from the other flavonoids. Chalcones are always considered Cyanidin 246.2 to be in trans conformation as the a,b-double bond is concerned. Quercetin 166.1 Then, two conformers arise, the s-cis and s-trans, that correspond Apigenin 176.0 Luteolin 174.4 to two different orientations of the double bond and the carbonyl Taxifolin 182.8 group. The s-cis conformer of 20-hydroxy chalcone represents the M. Leopoldini et al. / Food Chemistry 125 (2011) 288–306 297

4.1. BDEs activity of the former as radical scavenger can be ascribed to the tri-hydroxy functionality. The gas-phase BDE value for phenol is computed to be Nenadis et al. (Nenadis, Zhang, & Tsimidou, 2003) have com- 82.9 kcal/mol at B3LYP/6-311++G** (Table 2)(Leopoldini et al., puted the BDE as B3LYP single point energies on AM1 optimised 2004a), 82.3 kcal/mol at B3LYP/6-311++G**//B3LYP/6-31G** geometries of ferulic acid and its derivatives. Ferulic acid is charac-

(Himo, Eriksson, Blomberg, & Siegbahn, 2000), 82.8 kcal/mol at terised by the presence of a –OCH3 in ortho to the phenolic OH and B3LYP/6-31G** (Zhang, Sun, & Wang, 2003), 82.8 kcal/mol by a –CH@CH–COOH chain in para. BDE of ferulic acid and its ethyl (de Heer, Korth, & Malder, 1999), 83.9 kcal/mol at B3LYP/6- ester are computed to be 84.3 and 83.9 kcal/mol, respectively 311++G(3df, 3pd) (Thavasi, Leong, & Bettens, 2006). All these val- (Nenadis et al., 2003). Coniferyl aldehyde and alcohol, in which ues fall in the range of 82-84 kcal/mol. the –COOH is replaced by a –CHO and –CH2OH group in the side The B3LYP/6-311 + G(2d,2p) different value of 87.1 kcal/mol chain, respectively, show a BDE of 84.4 and 81.5 kcal/mol (Nenadis computed by Wright (Wright et al., 2001) is obtained as a single et al., 2003). The lower value of the latter with respect to the oth- point energy on geometries optimised at AM1 level. The most ers, can be due to the fact that the –CH2OH does not subtract elec- recent experimental values for phenol are 87.0 ± 1 kcal/mol tron density from the side chain as the –CHO does, so when the (Wayner et al., 1995), 88.3 ± 0.8 kcal/mol (Pedulli, Lucarini, & radical is formed upon breaking of the phenolic O–H, the electron Pedrielli, 1997) and 88.7 ± 0.5 kcal/mol (Dos Santos & Simoes, vacancy is better stabilized in the case of coniferyl alcohol. The 1998), being the value of 88.7 kcal/mol retained as the more reli- same occurs for isoeugenol (BDE = 81.1 kcal/mol) that possesses a able one. Bakalbassis et al. (Bakalbassis, Lithoxoidou, & Vaﬁadis, –CH@CH–CH3 side chain (Nenadis et al., 2003). 2003) have computed the gas-phase B3LYP BDE of phenol with Resveratrol BDE is 5.6 kcal/mol (Leopoldini et al., 2004a) lower several basis set. Results show that the biggest conventional ba- than the corresponding one computed for phenol (Table 2). Here, sis set, 6-311 + G(2d,2p), gives a BDE which is still over 5.1 kcal/ there is no possibility of intramolecular hydrogen bonds, so its mol lower that the experimental value of 88.7 kcal/mol, while antioxidant activity may be mainly given in terms of a good delo- the basis set 6-31 + G (3p,d), derived upon addition of a third calization of the radical unpaired electron through the aromatic p and a fourth d polarisation function on the hydrogen atoms rings and the –CH@CH– bridge. basis set, leads to a BDE value of 88.5 kcal/mol (Bakalbassis Flavanols catechin and epicatechin show a BDE of 74.2 (Leopol- et al. 2003), which seems to be nearer to the experimental dini et al., 2007) and 73.7 (Leopoldini et al., 2004a) kcal/mol, indication. respectively (Table 2). These diastereoisomers are characterised B3LYP catechol BDE is found to be 72.6 (Himo et al., 2000), 72.8 by the catechol functionality in the ring B, while the saturated ring (Zhang et al., 2003) and 74.7 (Thavasi et al., 2006) kcal/mol. This C does not allow conjugation between rings. The factor affecting means that the effect of an OH group in ortho position is to de- the antioxidant ability in terms of H donation is again the intramo- crease the BDE by 9–10 kcal/mol with respect to phenol. The rea- lecular hydrogen bond established between the radicalised oxygen son can be found in the fact that the radical arising from H-atom and the adjacent OH. removal is stabilized by the formation of the intramolecular H- The relevance of this functional group is also underlined by the bond with the vicinal hydroxyl. BDE of 74.7 kcal/mol (Leopoldini, Prieto Pitarch, et al., 2004) for ﬂa- The para substitution of catechol molecule with –COOH, – vanone taxifolin, reported in the Table 2.

CH2COOH and –CH2CH2COOH groups entails the lowering of the BDE values for apigenin and luteolin, from the class of flavones, BDE (compared to catechol) in the last two cases and a slight in- are 82.2 and 74.5 kcal/mol, respectively (see Table 2)(Leopoldini, crease in the former (Ordoudi, Tsimidou, Vafiadis, & Bakalbassis, Prieto Pitarch, et al., 2004). Molecules differ in the ortho-diphenolic 2006). The insertion of the carboxylic group to the catechol ring re- moiety in the ring B, so that in the case of luteolin the H-atom sults in a less favourable H-radical elimination by around 2 kcal/ abstraction is easier because the derived radical can be stabilized mol, while the insertion of methylene and ethylene groups be- by the intramolecular hydrogen bond. tween the catechol ring and the carboxylic group favours the H- The BDE of flavonols kaempferol and quercetin is evaluated to radical elimination (Ordoudi et al., 2006). be 80.9 (Leopoldini et al., 2004a) and 72.3 (Leopoldini et al., The BDE values of guaiacol (2-methoxyphenol) of 80.4 (Himo 2004b) kcal/mol at B3LYP/6-311++G** level (Table 2). Because et al., 2000) and 82.7 kcal/mol (Bosque & Sales, 2003) indicate that the only difference between them is the 30-OH group in the ring the presence of –OCH3 in the ortho-position has a slightly stabiliz- B, the presence of this group lowers the energy required for the ing effect on the radicalised molecule due to the compromise be- H abstraction by 8.6 kcal/mol. tween the electron-donor and electron-withdrawing capabilities Charged cyanidin shows a value of BDE of 79.4 kcal/mol (Leo- of this group. poldini et al., 2004a)(Table 2). Since it is a completely planar Tyrosol BDE of 82.0 kcal/mol (Table 2) indicates that the stabi- and conjugated system, the H-bonding becomes less important lizing effect of a –CH2CH2OH chain in para-position with respect than in the other flavonoids. to the OH is about 1 kcal/mol as compared to the phenol BDE of Chalcones show BDEs values that fall in a range of 74.4– 82.9 kcal/mol, and it depends on the electron-donating ability of 84.5 kcal/mol (relative to the most stable radicals) (Kozlowski the substituent (Leopoldini et al., 2004a). Indeed, in the case of et al., 2007). Also for these compounds, the important role of the hydroxytyrosol, whose BDE is 73.5 kcal/mol (Leopoldini et al., catechol moiety in the B ring is confirmed.

2004a), the simultaneous presence of both the –CH2CH2OH in para BDEs in water solution have the same general trend of those and the OH in ortho groups causes a decrease of the BDE of 9.4 kcal/ computed in the gas-phase for the same molecules, except for epi- mol (with respect to phenol). catechin that becomes the most reliable system acting through HÅ HPMC model of vitamin E shows a BDE of 71.7 kcal/mol at donation (Leopoldini et al., 2004a). The same is found for the com- B3LYP/6-311++G** level (Leopoldini et al., 2004a), with a decrease putations in benzene medium (Leopoldini et al., 2004a). of the BDE of phenol of 11.2 kcal/mol (Table 2). Here, the main fac- Results on BDEs indicate that the most efficient systems acting tor favouring the hydrogen removal is represented by the electron as hydrogen donors are those characterised by the dihydroxy func- releasing effect of the three methyls and the saturated ring. tionality, for which the values of the BDE are smaller than that of Gallic and caffeic acids BDEs, reported in the Table 2, are com- phenol reference system. Upon the radicalisation of the OH groups puted to be 72.2 and 73.6 kcal/mol, respectively (Leopoldini in these compounds, radical species arise, stabilized by resonance, et al., 2004a). If one considers their molecular structures, the better conjugation and delocalization effects. Internal H-bonds 298 M. Leopoldini et al. / Food Chemistry 125 (2011) 288–306 involving radicalised oxygen atoms further contribute to radical 5. Determination of polyphenols acidity stability. The third antioxidant mechanism by which polyphenols may 4.2. IPs perform their protective role, arises from the capability of these systems to sequester transition metals ions by chelation. Metals The ionisation potentials give different trends of reactivity (Leo- are entrapped in these polyphenols–metal complexes so they poldini et al., 2004a, 2004b; Leopoldini, Prieto Pitarch, et al., 2004) cannot participate in reactions involving production of free radicals for polyphenols with respect to the bond dissociation energies (see species. Table 3). IP value for phenol is computed to be 192.0 kcal/mol at Because chelation of metals often occurs through deprotonated B3LYP/6-311++G** (Leopoldini et al., 2004a, 2004b). hydroxyls in the polyphenols, the determination of the acidity of Tyrosol and hydroxytyrosol IP values are computed to be 181.7 these compounds is an important thermodynamic parameter that and 175.1 kcal/mol (Leopoldini et al., 2004a), in the gas-phase must be taken into account. The smaller the energy required to (Table 3). deprotonate the OH groups (acidity), the easier the metals chelation The a-tocopherol shows an IP value that is 37.1 kcal/mol lower will be. than that calculated for phenol (Leopoldini et al., 2004a). This The anions formed upon deprotonation of polyphenols means than the presence of several alkyl groups increases the hy- considered, share with the parent molecules the planar per-conjugation and stabilises the cation radical originating from disposition, that in principle allows a complete delocalization the electron removal. of the negative charge over the entire system. Exceptions to this For gallic acid, the influence of the trihydroxy moiety on the IP finding are the anions of epicatechin (Leopoldini, Russo, & value is small (189.1 kcal/mol, Table 3). Toscano, 2006), catechin, taxifolin (Martins et al., 2004), In the case of caffeic acid, the enhancement of the conjugation hesperetin, diadzein and naringenin (Zhang & Brodbelt, 2004), through the –CH@CH–COOH affects mostly the IP value that are non planar systems as well as their parent molecules (181.1 kcal/mol, Table 3)(Leopoldini et al., 2004a). (see Fig. 2). DFT/B3LYP IPs for ferulic acid and its derivatives are 167.5 For all of them, the most stable anion is that characterised by (ferulic acid), 165.5 (ethyl ferulate), 169.8 (coniferyl aldehyde), internal hydrogen bonds, especially those involving the deprotona- 155.1 (coniferyl alcohol) and 159.9 (isoeugenol) kcal/mol (Nenadis tion site oxygen (Leopoldini et al., 2006; Martins et al., 2004; Zhang & et al., 2003). Among them, coniferyl alcohol and isoeugenol are the Brodbelt, 2004). For flavonoids, the 40-position in the ring B is the compounds that can be more easily oxidised through an electron most favoured deprotonation site, followed by the 7-OH in the ring transfer mechanism. As encountered for BDEs of these compounds, A(Leopoldini et al., 2006). the electron-donor ability of substituents entails the lowering of IP Also for the acidities, the gas-phase value of phenol is com- values (Nenadis et al., 2003). puted and used as reference compound. Phenol B3LYP/6- The IP for resveratrol is computed to be 161.3 kcal/mol (Leopol- 311++G** acidity is found to be 345.1 kcal/mol (Leopoldini dini et al., 2004a), that is 30.7 kcal/mol lower than phenol (see et al., 2006), that seems to be in good agreement with the exper- Table 3). The molecular structure of this phenolic compound imental value of 346.9 kcal/mol, obtained by gas-phase proton underlines as an extended p-electrons delocalization particularly transfer equilibria. favours the electron transfer process with respect to the reference On the basis of B3LYP/6-311++G** increasing acidity values, an compound. order can be given: cyanidin (237.7 kcal/mol) > myricetin (312.5 Epicatechin and catechin show values of IP of 170.8 (Leopoldini kcal/mol) > quercetin (316.5 kcal/mol) > gallic acid (317.9 kcal/ et al., 2004a) and 169.7 (Leopoldini et al., 2007) kcal/mol, respec- mol) > caffeic acid (318.0 kcal/mol) > apigenin (321.3 kcal/mol) > tively (Table 3). kaempferol (322.7 kcal/mol) > epicatechin (327.2 kcal/mol) > res- Apigenin, luteolin, taxifolin and kaempferol show IP values of veratrol (327.5 kcal/mol) (see Table 4)(Leopoldini et al., 2006). 176.0, 174.4, 182.8 (Leopoldini, Prieto Pitarch, et al., 2004) and Other acidity values obtained as MP2/6-311 + G(d,p) (Zhang & 168.0 (Leopoldini et al., 2004a, 2004b) kcal/mol, in the order, as Brodbelt, 2004) single point energies on HF optimised geometries collected in the Table 3. are 328.1 kcal/mol, for hesperetin, 323.8 kcal/mol, for luteolin, Cyanidin appears to be less active as single electron-donor with 331.1 kcal/mol, for acacetin, 328.8 kcal/mol, for naringenin and respect to the other flavonoids (IP = 246.2 kcal/mol, Table 3)(Leo- 329.7 kcal/mol, for daidzein. poldini et al., 2004a, 2004b). This is not surprising because cyani- The value for cyanidin is the smallest one (237.7 kcal/mol) (Leo- din is just a charged molecule (charge = +1), so it is very poldini et al., 2006). This finding is not surprising because cyanidin unreliable to generate another positive charge. is a positively charged system so that deprotonation of the OH DFT/B3P86 IP values for chalcones fall in a range of 153.1– groups leads to very stable neutral species. 160.3 kcal/mol (Kozlowski et al., 2007), so that the electron trans- By looking at their molecular structure, one can argue that the fer mechanism is also important for this flavonoids. most acidic systems are those characterised by an high delocal- As far as the solution IPs are concerned, the presence of the ization of p-electrons, as the values for cyanidin, myricetin, quer- water medium involves a decrease of the absolute values. IP of cetin, and gallic and caffeic acids confirm. For the class of HPMC, that is one of the most active, changes from 154.3 to flavonoids, the delocalization in the anion involves the rings B 130.1 kcal/mol, in going from the gas-phase to the water solution (where deprotonation occurs) and C, while for the phenolic acids (Leopoldini et al., 2004a). the negative charge is delocalised over the aromatic ring and the Theoretical results show that within the mechanism of the elec- substituents. tron transfer, the main factors affecting the value of IP are the ex- Further contributions to the acidity values arise from the H-bond tended delocalization and conjugation of the p-electrons, formation occurring between the negative oxygen and the adjacent enhanced by resonances phenomena, rather than the presence of hydroxyl in systems having the ortho-dihydroxy moiety. The small- particular functional groups such as additional hydroxyls. So, est value is obtained for myricetin, for which all these functionalities resveratrol, tocopherol, quercetin, kaempferol and chalcones are are present. good candidates to work also through the second antioxidant MP2/6-311 + G(d,p) (Zhang & Brodbelt, 2004) and B3LYP/6- mechanism. 311 + G(2p,2d) (Martins et al., 2004) calculations give the same M. Leopoldini et al. / Food Chemistry 125 (2011) 288–306 299

Fig. 2. Equilibrium geometries of the most stable anions obtained after H+ removal from polyphenols: (a) gallic acid, (b) caffeic acid, (c) resveratrol, (d) epicatechin, (e) kaempferol, (f) cyaniding, (g) apigenin, (h) myricetin, (i) quercetin. trend of acidities of that obtained at B3LYP/6-111++G** (Leopoldini drawn by Himo et al. (Himo et al., 2000), for ortho-substituted et al., 2006) for kaempferol, apigenin and quercetin, even if some phenols. differences in the absolute value can be found (the latter are gen- Experimental relative acidities (Martins et al., 2004) revealed erally smaller). These discrepancies in the absolute energies can be that ﬂavones are the more acidic ﬂavonoids, with the following explained by considering that the formers are single point energies relative order: catechin > apigenin > kaempferol > taxifolin > quer- on HF optimised geometries, that often are found as non planar cetin > luteolin > myricetin. The involvement of p electron delocal- conformations. ization and conjugation, and of the catechol functionality is also The in water solution trend is to some extent different from the experimentally highlighted. gas-phase one: cyanidin (285.2 kcal/mol) > gallic acid (292.2 kcal/ The results on the gas-phase acidities match those relative to mol) > myricetin (292.6 kcal/mol) > caffeic acid (293.8 kcal/mol) > the BDE for the same systems. It is due to the fact that the release apigenin (296.4 kcal/mol) > kaempferol (296.5 kcal/mol) > querce- of a hydrogen atom (occurring in the H-atom transfer) can be con- tin (298.3 kcal/mol) > epicatechin (299.7 kcal/mol) > resveratrol sidered as the simultaneous loss of a proton and an electron. So, (301.9 kcal/mol). It is worth to note that the absolute acidity values the factors affecting the BDEs can be recognised also in determin- for every compound are very smaller than the corresponding ones ing the acidities values. in gas-phase. That is, solvent favours the deprotonation process by

30–40 kcal/mol. Of course, the trends of DpKa relative values 6. Formation of complexes between polyphenols and transition reﬂects that of acidities in terms of DG and depends on the same metals ions effects, that is the delocalization of the negative charge (with the resulting stabilization of the anion) and the formation of the Transition metals ions in their low oxidation state (Fe2+,Cu+) intramolecular hydrogen bonds. Similar conclusions were can catalyse reactions that involve formation of free radicals. 300 M. Leopoldini et al. / Food Chemistry 125 (2011) 288–306

Table 4 Gas phase and in water acidities of polyphenols. Values are in kcal/mol.

Compound Acidities Relative acidities Relative pKa in solution B3LYP/6-311++G** B3LYP/6-311 + G(d,p)a MP2/6-311 + G(d,p)b B3LYP/6-311++G** B3LYP/6-311++G** Phenol OH 345.1 (310.0) – – 0.0 (0.0) 0.0 Gallic acid 4-OH 317.9 (292.2) ––27.2 (17.8) 13.0 5-OH 327.4 (297.9) – – 17.7 (12.1) 8.8 Caffeic acid 3-OH 323.8 (297.2) – – 21.3 (12.8) 9.3 4-OH 318.0 (293.8) ––27.1 (16.2) 11.8 Resveratrol 30-OH 336.2 (303.7) – – 8.9 (6.3) 4.6 50-OH 335.6 (303.8) – – 9.5 (6.2) 4.5 6-OH 327.5 (301.9) ––17.6 (8.1) 5.9 Epicatechin 30-OH 327.2 (299.7) ––17.9 (10.3) 7.5 40-OH 327.6 (300.0) – – 17.5 (10.0) 7.3 0-OH 337.5 (304.0) – – 7.6 (6.0) 4.4 7-OH 339.8 (304.4) – – 5.3 (5.6) 4.1 Kaempferol 40-OH 323.0 (298.6) 327.1 328.9 22.1 (11.4) 8.3 3-OH 333.0 (299.8) 338.0 336.3 12.1 (10.2) 7.4 5-OH 337.5 (302.3) 342.2 340.3 7.6 (7.7) 5.6 7-OH 322.7 (296.5) 328.1 327.3 22.4 (13.5) 9.8 Cyanidin 30-OH 251.8 (294.6) – – 93.3 (15.4) 11.2 40-OH 238.3 (285.9) – – 106.8 (24.1) 17.6 3-OH 240.6 (288.7) – – 104.5 (21.3) 15.5 5-OH 237.7 (285.3) – – 107.4 (24.7) 18.0 7-OH 238.9 (285.2) ––106.2 (24.8) 18.1 Apigenin 40-OH 321.3 (297.1) 324.4 327.0 23.8 (12.9) 9.4 5-OH 346.3 (304.7) 349.6 346.2 1.2 (5.3) 3.9 7-OH 327.6 (296.4) 328.8 330.3 17.5 (13.6) -9.9 Myricetin 30-OH 323.2 (298.4) 326.5 – 21.9 (11.6) 8.5 40-OH 312.5 (292.6) 314.8 – 32.6 (17.4) 12.7 50-OH 324.2 (298.6) 326.9 – 20.9 (11.4) 8.3 3-OH 334.0 (299.8) 334.8 – 11.1 (10.2) 7.4 5-OH 338.3 (302.3) 340.9 – 6.8 (7.7) 5.6 7-OH 323.3 (296.6) 327.3 – 21.8 (13.4) 9.8 Quercetin 30-OH 323.3 (298.3) 326.6 339.8 21.8 (11.7) 8.5 40-OH 316.5 (299.9) 319.4 321.8 28.6 (10.1) 7.4 3-OH 333.1 (299.8) 337.0 336.3 12.0 (10.2) 7.4 5-OH 337.4 (302.3) 343.5 340.8 7.7 (7.7) 5.6 7-OH 322.5 (296.6) 328.2 327.8 22.6 (13.4) .8

a 38. b 42.

During the Fenton reaction, hydroxyl radicals are produced from ESI–MS studies (Satterfield & Brodbelt, 2000) have indicated hydrogen peroxide in the presence of a metal in a low oxidation that among 3,7-dihydroxyflavone, 5,7-dihydroxyflavone, luteolin, state: 7,30,40-trihydroxyflavone, 7,40-dihydroxyflavone, 7-hydroxyflavone, catechin, and quercetin, flavone possessing no hydroxyl groups nþ Å ðnþ1Þþ H2O2 þ M ! HO þ HO þ M does not form stable M(II) complexes. This is presumably due to the lack of a suitable acidic hydrogen. The lowest intensities in This reaction may occur in biological areas where accumulation the MS spectra are given by catechin, 7,30,40-trihydroxyflavone, 7- 0 of H2O2 is important, for example in the dopaminergic neurons of hydroxyflavone and 7,4 -dihydroxyflavone, each of which lacks a nervous tissue. Here, normal dopamine catabolism produces some pair of chelating oxygen atoms at the carbon 3 and carbon 4, or car- levels of hydrogen peroxide (Brown et al., 1998; Palmer & Paulson, bon 4 and carbon 5 positions. 5,7-dihydroxyflavone, luteolin, and 1997; Schulz et al., 2000; van Acker et al., 1996). 3,7-dihydroxyflavone, possessing the favourable chelating oxygen Polyphenols may offer several chelating sites, such as multiple atoms at the carbon 3 and carbon 4, or carbon 4 and carbon 5 posi- hydroxyls and carbonyl groups. tions, generated intense signals. These results have highlighted the In flavonoids possessing the 4-carbonyl group and hydroxyls at- importance of the carbonyl and hydroxyl chelating pair in metal tached to 30,40, 3 and 5, there are three potential chelating sites: coordination, and the preference for metal coordination between the catechol moiety, the 4-keto and the 3-OH, and the 4-keto and the deprotonated hydroxyl group at carbon 3 or carbon 5 and the the 5-OH groups (see Schemes 2 and 3). carbonyl group carbon 4. M. Leopoldini et al. / Food Chemistry 125 (2011) 288–306 301

Among the different 1:1, 1:2, 2:2, 2:3 stoichiometries that the 3-OH deprotonated (500.2 kcal/mol) and the 5-OH deproto- metal:flavonoid complexes can exhibit, the 1:2 is the preferred nated (499.8 kcal/mol) quercetin. one (Fernandez, Mira, Florêncio, & Jennings, 2002). In a DF-B3LYP study on the chelation of iron(II) by quercetin Formation of stable complexes between caffeic acid and Al(III) (Leopoldini, Russo, Chiodo, & Toscano, 2006), the complexes with (Cornard & Lapouge, 2006) and Pb(II) (Boilet, Cornard, & Lapouge, both neutral and deprotonated quercetin with bare and hydrated 2005) are investigated at B3LYP level of theory, considering as Fe2+ cation, are considered. putative chelating site the negative carboxylate and the catechol Among the possible chelates arising from the neutral forms, the moiety. The carboxylate group presents the greater power to gen- global minimum is that in which Fe(II) coordinates to the 4-keto erate complex with Pb(II) (Boilet et al., 2005). The behaviour of (Fe–O distance is 1.869 Å) and to the 5-OH (Fe–O distance is Pb(II) (Boilet et al., 2005) completely differs from that of Al(III) 2.096 Å) groups. Its equilibrium geometry (1) is reported in the (Cornard & Lapouge, 2006), that preferentially coordinates the cat- Fig. 3. echol group of the caffeic acid. When deprotonated forms are involved, Fe2+ may attack not DFT and molecular dynamics calculations (Kazazicä, Butkovicä, only on the deprotonation sites but also on the other positions Srzicä, & Klasinc, 2006) are performed and the elucidatation of the originating from resonance effects, thus the complexed species coordination of Fe+ and Cu+ to some representative flavonoids is gi- are quite numerous (Leopoldini et al., 2006). B3LYP computations ven. Apigenin, quercetin, and naringenin indicate a preference for indicate as global minimum the adduct in which the cation is coor- metal complexation at the 4-carbonyl oxygen when there is no dinated to the carbonyl oxygen on ring C and to the deprotonated substituent in position 3. The difference in energy with the next 5-OH hydroxyl (coordination bonds are of 1.869 and 1.813 Å, favourable attachment site of the Cu+ ion (catechol moiety) of respectively). For both neutral and ionised quercetin, relative ener- 14.2 kcal/mol indicates the C ring as the favourite chelating site gies of the chelates (Leopoldini et al., 2006) indicate a scarce che- (Kazazicä et al., 2006). lating ability of the catechol toward Fe2+ (see Fig. 3). Lapouge et al. (Lapouge, Dangleterre, & Cornard, 2006) have ex- Usually, iron(II) forms in physiological liquids hydrated com- plored the complexation of Zn(II) by 3-hydroxyflavone, 5-hydrox- plexes in which the preferred coordination around the cation is yflavone and 30,40-dihydroxyflavone, so that indications about the of the octahedral type. So, four water molecules are considered chelating power of the three possible sites 3-OH-carbonyl, 5-OH- in the coordination sphere of Fe2+ (Leopoldini et al., 2006). Among carbonyl and catechol are drawn, together with information about the complexes of hydrated ion, the global minimum (see Fig. 3)is 2+ the structural modification eventually occurring in the ligand after represented by the adduct in which [Fe(H2O)4] coordinates to the complexation. All of these ligands lead to the formation of complex carbonyl and deprotonated 3-OH group (distances are 2.015 and of 1:1 stoichiometry. The complexation by 30-40-hydroxyflavone 2.073 Å, respectively). However, this species appears to be almost does not modify the A and C rings features that remain more or less isoenergetic (DE = 1.8 kcal/mol) with the relative minimum in the same computed for the free ligand, while the ring B geometry which the oxygen atoms of the A and C rings are involved. The undergoes slight modifications in the internal bonds. The five- presence of the water molecules in the coordination sphere of membered ring formed with the Zn cation seems to be not Fe2+ seems to reduce the energy separations between the coplanar with the ring B (20°). In the case of 5-OH flavone, the complexes. 2+ coordination of Zn(II) at the C4@O, results in an electronic redistri- NBO analysis suggested that upon interaction of hydrated Fe bution mostly on the C ring (Lapouge, Dangleterre, & Cornard, with quercetin anions, the natural net charge on cation is very sim- 2006). Contrary to what encountered in the formation of Zn- ilar to that computed for free aquocomplex (1.52 vs 1.62 |e|), so 3040diOH–flavone, the chelate ring is planar, and the zinc atom is that a ionic bond is present, in this case. This finding differs from located in the chromone part plane. Similar conclusions are drawn that concerning the complexes in which the coordination sphere for the Zn-3-OH–flavone complex. The comparison of the forma- of cation is lacking in water molecules. However, this is not sur- tion constants for the three flavones allows the following classifica- prising since the orbital availability to form covalent bonds de- tion on the basis of the chelating power of the three binding sites: creases gradually as coordination number of cation increases. 3-hydroxy-carbonyl > 5-hydroxy-carbonyl > catechol (Lapouge, Since experimental findings have indicated the possible exis- Dangleterre, & Cornard, 2006). tence of chelates between two quercetin molecules and Fe(II), Cornard, Dangleterre, and Lapouge (2005) have investigated the formation of chelates made up of two deprotonated quercetin theoretically the complexation of Pb(II) by flavonol quercetin. molecules with bare and hydrated Fe2+ has been investigated. Con- Quercetin offers all the possible chelating sites existing in flavo- sidering that the most stable complexes of stoichiometry 1:1 are noids. Among them, the catechol group presents the greater chelat- obtained as the oxygen atoms linked to carbon 3-carbon 4 and car- ing power toward Pb(II). After complexation, the ligand remains bon 4-carbon 5 pairs are involved, the complexes arising from the totally planar, and the lead ion, coordinated to the catecholate interaction with the 3-OH and 5-OH anions of quercetin are taken group, is coplanar to the plane of quercetin. into account, as well as the ‘‘cis” and ‘‘trans” orientations of the two The behaviour of Pb(II) (Cornard et al., 2005) completely quercetin anions around the cation (Leopoldini et al., 2006). differs from that of Al(III) one (Cornard & Merlin, 2002), which As far as the interaction with bare Fe2+ is concerned, the global preferentially coordinates the 3-hydroxy-chromone part of quer- minimum is represented by the complex originated by the interac- cetin. tion with the 5-OH anions in ‘‘cis” position (Fig. 3). The coordina- Several quercetin activated forms involved in antioxidant tion geometry around the cation is found as tetrahedral, mechanisms have been studied by Fiorucci et al. (2007). They have involving two carbonyl O12 (2.035 and 2.032 Å) and two hydroxyl computed many thermodynamic parameters such as BDE, IP and O13 (1.924 and 1.925 Å) atoms. Charge on iron ion is 1.41 |e|. This acidity, and determined electronic and structural features of the value suggests a charge transfer from ligand to cation. NBO analy- complexes between these activated forms and Cu2+ cation. In these sis indicated the presence of two covalent bonds between Fe2+ and species, a significant electron transfer from the flavonol to the me- the oxygen atom of both deprotonated 5-OH groups. Overlap in- tal occurs, especially in the case of deprotonated forms rather than volves the two hybrid s(11.30%)p(88.7%) and s(19.00%)d(81%) orbi- the semiquinone. Complexation of Cu2+ on each of the quercetin tals of oxygen and iron, respectively. activated forms is thermodynamically favoured, as reflected by Computations also show that the ‘‘cis” arrangement of quercetin the values of the binding energies (Fiorucci et al., 2007). The higher anions is energetically favoured with respect to the ‘‘trans” one values are obtained for the complexation between copper(II) and owing to major stability of the tetrahedral coordination that 302 M. Leopoldini et al. / Food Chemistry 125 (2011) 288–306

2+ 2+ 2+ Fig. 3. Equilibrium geometries of absolute minima of neutral quercetin–Fe (1), deprotonated quercetin–Fe (2), deprotonated quercetin–[Fe(H2O)4] (3), two 2+ 2+ deprotonated quercetin–Fe (4) and two deprotonated quercetin–Fe(H2O)2] (5) complexes.

originates from this disposition with respect to the square planar (Leopoldini et al., 2006) for the global minima belonging to all dif- geometry obtainable from the trans one. ferent categories of examined systems. Values obtained are 431.9, 2+ When the hydrated [Fe(H2O)2] is involved, the most stable 517.0, 601.6, and 615.2 kcal/mol, for the ionised quercetin-bare complexes originate from the complexes characterised by the pla- iron, ionised quercetin-hydrated iron, two ionised quercetins-bare nar disposition of ligand atoms (Leopoldini et al., 2006). This can be iron, and two ionised quercetins-hydrated iron, respectively. Thus, explained by considering that the final octahedral geometry is as expected, also from a theoretical point of view, the preference more easily reachable starting from planar complexes. In other for the 2:1 ratio is confirmed. words, the rearrangement required to pass from a tetrahedral to Both neutral and deprotonated quercetin form with iron(II) sta- an octahedral coordination entails a larger energy expense. Thus, ble complexes. Among the 1:1 and 1:2 metal:ligand stoichiome- in the case of hydrated cation the global minimum (see Fig. 3)is tries, the latter is favored as binding energy values confirm. represented by the system in which the iron cation is coordinated Among the available positions present on neutral or anionic quer- by the oxygen atoms attached to C3 and C4 carbon atoms of two 3- cetin, oxygen atoms at the 3 and 4, and 5 and 4 carbons, seem to be OH anions in ‘‘cis” position and by two water molecules. The li- the favoured coordination sites for iron cation. 2+ gands are completely coplanar while the axial H2O molecules are The behaviour of Fe (Leopoldini et al., 2006) is found similar to so arranged that a slight deviation from ideal octahedral geometry that shown by Al(III) (Cornard & Merlin, 2002), but different from can be observed. that of Pb(II) (Cornard et al., 2005), which preferentially coordi- To verify the agreement with the experimental observation nates to the ortho-dihydroxy functionality. (Fernandez et al., 2002) concerning the predominance of the com- The high binding energy values (Leopoldini et al., 2006) indicate plexes having the 2:1 ligand:metal ion stoichiometry with respect that quercetin is a powerful chelating agent that can sequester ir- to those with 1:1 one, binding energy (BE) values are computed on(II) in such a way to prevent its involvement in Fenton reactions. M. Leopoldini et al. / Food Chemistry 125 (2011) 288–306 303

7. Conclusions substituents, as it occurs for gallic acid, caffeic acid and resveratrol. For flavonoids, again the 40-position on ring B and the 7-position on We have presented a brief review concerning the antioxidant ring A are the most suitable deprotonation sites because of the bet- capabilities of naturally occurring polyphenolic systems. It sum- ter possibility to delocalize the electron pair. In addition, the for- marises the current status of the research on this subject per- mer site is favoured when H-bonds between adjacent hydroxyls formed by means of quantum chemical methods. are present. The main mechanisms proposed in the literature for the anti- The most acidic polyphenolic compounds are those character- oxidant action of polyphenols, consisting in the H-atom transfer, ised by a high degree of p-electrons delocalization, for which the electron transfer and the metals chelation, have been deprotonation yields to anionic species stabilized by resonances discussed. phenomena; their stability is enhanced by the presence of an H- The OH groups acidity values and the binding energies for the bonding pattern. complexation process have been determined in order to better Polyphenols are able to chelate transition metals through their examine the chelation mechanism. multiple OH groups and the carbonyl moiety, when present. With All polyphenols investigated are planar systems characterised regard to the flavonol quercetin, it originates stable complexed by an extended conjugation and delocalization of the p-electrons species with the Fe2+ cation, both in the neutral and ionised forms, that involve the aromatic ring(s) and the substituents. and also with Cu(II). Among the possible chelating sites for iron They give rise to stable radical species upon the removal of an and copper, the 3-OH/4-keto and the 5-OH/4-keto positions are hydrogen atom or an electron; the odd electron appears to be delo- those showing the greater complexation ability, while the catechol calised over the entire molecule thanks to the planar geometry seems to be a poor chelating agent. Binding energies values dem- conformation. onstrate that the preferred iron(II):quercetin stoichiometry is the The stability of radicals is enhanced by the possibility to estab- 1:2 one. lish internal H-bonds between the radicalised oxygen atom and Further insights on the working mechanisms of antioxidant sys- vicinal hydroxyl. tems require the detailed study of their reactions with the biolog- The most efficient systems that may work through the ical molecular targets. These studies, that are currently in progress H-atom transfer mechanism display the ortho-dihydroxy in different research groups, could give new informations not only functionality. In these cases, the BDE values are very small with on the thermodynamical data but also on the kinetic ones. respect to the phenol reference compound because the radicalisation of their hydroxyls generates species stabilized by Acknowledgements intramolecular H-bonds other than by resonance effects of the substituents. So, hydroxytyrosol, gallic acid, caffeic acid, epicate- The University of Calabria, the Food Science and Engineering chin, quercetin are excellent free radicals scavenger by H atom Interdepartmental Center of University of Calabria and L.I.P.A.C., donation. Calabrian Laboratory of Food Process Engineering (Regione Cala- Within the one electron transfer mechanism, good candidates bria APQ – Ricerca Scientifica e Innovazione Tecnologica I atto inte- are those compounds that show planar conformation and wide grativo, Azione 2 laboratori pubblici di ricerca mission oriented electronic delocalization, so that their IP values are lower that interfiliera, and Azione 3 sostegno alla domanda di innovazione the reference phenol, as occurring in tocopherol, reseveratrol, nel settore agroalimentare) are gratefully acknowledged. quercetin. Most of polyphenols seem to scavange free radicals through the References hydrogen atom transfer mechanism since higher energies are involved in the single electron transfer process. However, these con- Adlercreutz, H., Fotsis, T., Bannwart, C., Wahala, K., Makela, T., Brunow, G., et al. clusions could have a more truly firm basis through computations (1986). Determination of urinary lignans and phytoestrogen metabolites, potential antiestrogens and anticarcinogens in urine of women on various of the transition state(s) and intermediate(s) for the particular habitual diets. Journal of Steroid Biochemistry, 25(5B), 791–797. reaction pathways. Anders, E., Koch, R., & Freunscht, P. (1993). Optimization and application of lithium Results presented here confirm the experimental radical scav- parameters for PM3. Journal of Computational Chemistry, 14(11), 1301–1312. enging property of natural polyphenolic compounds based on the Armentrout, P. B., & Kickel, B. L. (1996). Gas-phase thermochemistry of transition metal ligand systems: Reassessment of values and periodic trends. In B. S. 0 ability to scavenge the radical cation chromophore of 2,2 -azin- Freiser (Ed.), Organometallic ion chemistry (pp. 1–45). Dordrecht: Kluwer. obis(3-ethylbenzothiazoline-6-sulphonic acid (ABTSÅ+) in relation Axelson, M., Sjövall, J., Gustafsson, B. E., & Setchell, K. D. R. (1984). Soya – A dietary to that of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic source of the non-steroidal oestrogen equal in man and animals. Journal of Endocrinology, 102(1), 49–56. acid (Trolox), an aqueous soluble vitamin E analogue (the Trolox Bakalbassis, E. G., Lithoxoidou, A. T., & Vafiadis, A. P. (2003). Theoretical calculation equivalent antioxidant capacity (TEAC) is defined as the concentra- of accurate absolute and relative gas- and liquid-phase OH bond dissociation tion of Trolox with the same antioxidant capacity as a 1 mM con- enthalpies of 2-mono- and 2,6-disubstituted phenols, using DFT/B3LYP. The Journal of Physical Chemistry A, 107(41), 8594–8606. centration of the antioxidant under investigation). Thus, flavonols Barnes, S., D’Alessandro, T., Kirk, M. C., Patel, R. K., Boersma, B. J., & Darley-Usmar, V. and flavones containing a catechol group in ring B are found to M. (2003). The importance of in vivo metabolism of polyphenols and their be highly active, with flavonols more potent than the correspond- biological actions. In M. S. Meskin, W. R. Bidlack, A. J. Davies, D. S. Lewis, & R. K. Randolph (Eds.), Phytochemicals mechanism of action (pp. 51–59). Boca Raton: ing flavones because of the presence of the 3-hydroxyl group. 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