The molecular basis of working mechanism of natural polyphenolic antioxidants

Food Chemistry 125 (2011) 288–306

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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

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

a r t i c l e

i n f o

Article history: Received 20 April 2010 Received in revised form 21 July 2010 Accepted 6 August 2010

Keywords: Natural antioxidants Flavonoids DFT BDE IP Acidities Metal complexes

a b s t r a c t In this review, we present a summary of the research work performed so far using high accuracy quantum chemical methods on polyphenolic antioxidant compounds. We have reviewed the different groups of polyphenols, which mostly belong to the Mediterranean food culture, i.e. phenolic acids, flavonoids and 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 elucidation of the beneficial effects on health of these substances. Ó 2010 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4.

5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Parent and radicals polyphenols structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BDE and IP evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. BDEs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. IPs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Determination of polyphenols acidity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Formation of complexes between polyphenols and transition metals ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Phenolic compounds are plant secondary metabolites commonly found in herbs and fruits such as berries, apples, citrus fruit, cocoa, grapes, vegetables like onions, olives, tomatoes, broccoli, lettuce, soybeans, grains and cereals, green and black teas, coffee beans, propolis, and red and white wines (Brit, Hendrich, & Wang, 2001; Clifford, 1999; Hertog, Hollman, Katan, & Kromhout, 1993;

⇑ Corresponding author. Tel.: +39 0984492106; fax: +39 0984493390. E-mail address: [email protected] (N. Russo). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.08.012

288 291 292 296 297 298 298 300 303 303 303

Kris-Etherton et al., 2002; Rencher, Spencer, Kuhnle, Hahn, & Rice-Evans, 2001; Rice-Evans, Spencer, Schroeter, & Rechner, 2000; Robak & Gryglewski, 1996; Ross & Kasum, 2002). Many of these phenolics are responsible for the attractive colour of leaves, fruits and flowers (Hermann, 1993). In the last decades, they have attracted growing global interest upon the discovery of the so-called ‘‘French Paradox”, i.e. the 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 the UK and Germany (Renaud & de Lorgeril, 1992). The most popular explanation has been recognised in the relatively high daily

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consumption of red wines rich in phenolic compounds, by the French, which in some way act to protect them from heart diseases (Frankel, Kanner, German, Parks, & Kinsella, 1993; Hertog, Freskens, Hollman, Katan, & Kromhout, 1993). The term phenolics encompasses approximately 8000 naturally occurring compounds, all possessing one common structural feature, a phenol (an aromatic ring bearing at least one hydroxyl substituent). A further classification divides them in polyphenols and simple phenols, depending on the number of phenol subunits (see Scheme 1). Simple phenols include phenolic acids (Robbins, 2003). Polyphenols possessing at least two phenol subunits include the flavonoids, the stilbenes, and those compounds possessing three or more phenol subunits are referred to as the tannins (King & Young, 1999). Phenolic acids are phenols that possess one carboxylic acid functionality. They contain two distinguishing constitutive carbon frameworks: the hydroxycinnamic and hydroxybenzoic structures (see Scheme 1). Hydroxycinnamic acids are more common than hydroxybenzoic acids and consist chiefly of p-coumaric, caffeic, ferulic, and sinapic acids (Robbins, 2003). The flavonoids consist of a large group of low-molecular weight polyphenolic substances, benzo-c-pyrone derivatives (see Scheme 1) (Coultate, 1990). The basic structural feature of all flavonoids is the flavane (2-phenyl-benzo-c-pyrane) nucleus, a system of two benzene rings (A and B) linked by an oxygen-containing pyrane ring (C). According to the degree of oxidation of the C ring, the hydroxylation pattern of the nucleus, and the substituent at carbon 3, the flavonoids can be categorised into the subclasses flavones, isoflavones, flavanols (catechins), flavonols, flavanones, anthocyanins, and proanthocyanidins. Flavonols differ from flava-

289

nones by a hydroxyl group at the C3 position, and by a C2–C3 double bond. Anthocyanidins differ from the other flavonoids by possessing a charged oxygen atom in the ring C. The ring C is open in the chalcones. Many flavonoids occur naturally as glycosides, and carbohydrate substitutions include D-glucose, L-rhamnose, glucorhamnose, galactose, and arabinose (Harborne, 1986; Harborne, 1988; Hodnick, Milosavljevic, Nelson, & Pardini, 1988; Kijhnau, 1976). Stilbenes family includes several compounds (Langcake & Pryce, 1976; Soleas, Diamandis, & Goldberg, 1997) among which resveratrol, pterostilbene, and piceatannol are the main representatives, characterised by a double bond connecting the phenolic rings (see Scheme 1). Polymeric compounds, called tannins, are divided into two groups, i.e. condensed and hydrolyzable. Condensed tannins are polymers of flavonoids, and hydrolyzable tannins contain gallic acid, or similar compounds, esterified to a carbohydrate (Hagerman, Zhao, & Johnson, 1997). The pharmacological, medicinal and biochemical properties of phenolics have been extensively reviewed (Cody, Middleton, & Harborne, 1986; Cody, Middleton, Harborne, & Beretz, 1988; Das, 1990; Harborne, 1986). They have been reported to have antioxidant (Kandaswami & Middleton, 1994), vasodilatory, anticarcinogenic, antinflammatory, immune-stimulating, antiallergic, antiviral (Duarte, Perez-Vizcainom, Utrilla, et al., 1993; Duarte, Perez-Vizcainom, Zarzuelo, Jiminez, & Tanargo, 1993) and estrogenic effects, and inhibition activities against phospholipase A2, cyclooxygenase, lipoxygenase (Brown, 1980; Ho, Chen, Shi, Zhang, & Rosen, 1992; Jovanovic, Jankovic, & Josimovic, 1992; Lindahl & Tagesson, 1993; Mabry, Markham, & Chari, 1982; Middleton &

Scheme 1. Structures of benzoic and hydroxycinnamic acids, flavonoids and stilbenes.

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Kandaswami, 1992; Robak, Shridi, Wolbis, & Krolikowska, 1988; Sogawa et al., 1993), glutathione reductase (Elliot, Scheiber, Thomas, & Pardini, 1992) and xanthine oxidase enzymes (Chang, Lee, Lu, & Chang, 1993). The best described property of phenolics is the antioxidant capability towards free radicals normally produced by cells metabolism or in response to external factors. Free radicals can damage biomolecules such as lipids, nucleic acids, proteins, cause cellular membranes peroxidation (De Groot, 1994; Grace, 1994) and attract various inflammatory mediators (Halliwell, 1995). Polyphenols scavenge free radicals and reacting oxygen species (ROS), that are made so inactive (De Groot, 1994; Grace, 1994). Flavonoids inhibit nitric oxide synthase that generates nitric oxide, which in turn reacts with free radicals to generate the peroxynitrite species, in addition to be itself a radical (Dehmlow, Erhard, & de Groot, 1996; Huk, Brovkovych, & Nanobash, 1998; Shoskes, 1998; Shutenko et al., 1999; van Acker, Tromp, Haenen, van der Vijgh, & Bast, 1995). Xanthine oxidase is implicated in oxidative injury, especially after ischemia–reperfusion, because it reacts with molecular oxygen and releases superoxide. Flavonoids, in particular quercetin and luteolin, are potent inhibitor of xanthine oxidase (Cos et al., 1998; Iio, Ono, Kai, & Fukumoto, 1986; Sanhueza, Valdes, Campos, Garrido, & Valenzuela, 1992; Shoskes, 1998). The mechanism of the antitumor effects of flavonoids seems to depend on their structure, with each compound displaying various biological potency and mechanism(s) of action (Di Carlo, Mascolo, Izzo, & Capasso, 1999). However, the essential feature of flavonoids is their free radical scavenging activity, partially responsible for their antitumor effects. Flavonoids have antiproliferative effects and induce apoptosis in different cancer cell lines. As free radical scavengers, flavonoids inhibit invasion and metastasis (Cipak, Rauko, Miadokova, Cipakova, & Navotny, 2003; Krol, Czuba, Threadgill, Cunningham, & Pietsz, 1995; Kuntz, Wenzel, & Daniel, 1999; Nijveldt et al., 2001; Win, Cao, Peng, Trush, & Li, 2002). Some aglycone flavonoids are potent inhibitors of oxidative modification of LDL in vitro by macrophages or copper ions (De Whalley, Rankin, Hoult, Jessup, & Leake, 1999). Platelet–blood vessel interactions are implicated in the development of thrombosis and atherosclerosis. Particular flavonoids inhibit platelet aggregation and adhesion (Beretz, Anton, & Cazenave, 1986; Beretz & Cazenave, 1988; Beretz, Cazenave, & Anton, 1982; Gryglewski, Korbut, Robak, & Swies, 1987; Mora, Paya, Rios, & Alcaraz, 1990; Robak, Korbut, Shridi, Swies, & Rzadkowska-Bodalska, 1988; Swies et al., 1984; Tzeng, Ko, Ko, & Teng, 1991). However, the antiaggregatory effects of flavonoids cannot be attributed to a single biochemical mechanism because they appear to influence several pathways involved in platelet function (Landolfi, Mower, & Steiner, 1984; Tzeng, Ko, Ko, & Teng, 1991). Flavonoids appear to increase vasodilatation by inducing vascular smooth muscle relaxation which may be mediated by the inhibition of protein kinase C, PDEs, or by decreased cellular uptake of calcium (Duarte, Vizcaino, et al., 1993). Six flavonoids have been evaluated for their ability to prevent injury in mesencephalic cultures, resulting that all protect neurons from damage by the dopaminergic toxin N-methyl-4-phenyl1,2,3,6-tetrahydropyridinium hydrochloride MPP+ (Mercer, Kelly, Horne, & Beart, 2005). Concerning their metabolism, most of flavonoids are absorbed into the intestinal cells by passive mechanisms (Barnes et al., 2003; Day et al., 2000; Sfakianos, Coward, Kirk, & Barnes, 1997; Yasuda, Kano, Saito, & Ohsawa, 1994). Once on the blood circulation, they are converted into metabolites with higher antioxidant and estrogenic activities with respect to their unmetabolized parent molecules (Adlercreutz et al., 1986; Axelson, Sjövall, Gustafsson, & Setchell, 1984; Coldham et al., 1999; Rimbach et al., 2003).

The molecular basis for the antioxidant properties of polyphenols is recognised into three main mechanisms, arising from the direct reaction with free radicals (Leopoldini, Marino, Russo, & Toscano, 2004a, 2004b; Leopoldini, Prieto Pitarch, Russo, & Toscano, 2004; Wright, Johnson, & DiLabio, 2001), and from the chelation of free metals, the latter involved in reactions finally generating free radicals (Jovanovic, Steenken, Simic, & Hara, 1998). As primary antioxidants, polyphenols inactivate free radicals according to the hydrogen atom transfer (HAT) (1) and to the single electron transfer (SET) (2) mechanisms (see Scheme 2). In mechanism 1, the antioxidant, ArOH, reacts with the free radical, R, by transferring to it a hydrogen atom, through homolytic rupture of the O–H bond:

ArOH þ R ! ArO þ RH

ð1Þ

The products of the reaction are the harmless RH species and the oxidised ArO radical. Even if the reaction leads to the formation of another radical, it is less reactive with respect to R because stabilized by several factors (see below). The SET mechanism (2) provides for an electron to be donated to the R:

ArOH þ R ! ArOHþ þ R

ð2Þ



The anion R is an energetically stable species with an even number of electrons, while the cation radical ArOH+ is also in this case a less reactive radical species. In particular, the ArO and ArOH+ are aromatic structures in which the odd electron, originated by the reactions with the free radical, has the possibility to be spread over the entire molecule, resulting into a radical stabilization (Leopoldini et al., 2004a, 2004b; Wright et al., 2001; Leopoldini, Prieto Pitarch, et al., 2004). In the former mechanism, the bond dissociation enthalpy (BDE) of the phenolic O–H bond is an important parameter in evaluating the antioxidant action; the lower the BDE value, the easier the dissociation of the phenolic O–H bond and the reaction with the free radicals. In the SET mechanism, the ionisation potential is the most significant parameter for the scavenging activity evaluation; the lower the IP value, the easier the electron abstraction and the reaction with free radicals. Another antioxidant mechanism (Transition Metals Chelation, TMC, see Scheme 2) arises from the possibility that transition metals ions may be chelated by polyphenols, leading to stable complexed compounds (Brown, Khodr, Hider, & Rice-Evans, 1998; Jovanovic et al., 1998; van Acker et al., 1996). The latter entrap metals and avoid them to take part in the reactions generating free radicals. In fact, some metals in their low oxidation state (mainly Fe2+) may be involved in Fenton reactions with hydrogen peroxide (Schulz, Lindenau, Seyfried, & Dichganz, 2000), from which the very dangerous reactive oxygen species (ROS) OH is formed:

H2 O2 þ Mnþ ! HO þ HO þ Mðnþ1Þþ The OH is generally accepted to be one of the most reactive radicals. It has a very short half-life (around 109 s) and a very high reactivity. With respect to the hydroperoxides that are metabolized by superoxide dismutase, hydroxyl radicals cannot be eliminated by enzymatic reactions. So they will react with every kind of substrate they encounter (Palmer & Paulson, 1997). Transition metals like copper, manganese, cobalt are able to catalyse this reaction, under certain conditions when these metal ions are not bound to proteins or chelators. Fenton-like reaction may take place and cause site specific accumulation of free radicals and initiate biomolecules damage processes. Fenton chemistry occurs in dopaminergic neurons of nervous tissue, where normally dopamine catabolism produces some levels of hydrogen peroxide. The accumulation of free radicals in these

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291

Scheme 2. Mechanisms for the antioxidant activity.

neurons may be recognised as the main aetiological agent of Parkinson disease (PD) (Schulz et al., 2000). Other neurodegenerative diseases, such as Alzheimer’s diseases (AD) and Huntington’s chorea, have as hallmark a significant increase in iron in some brain regions (Gerlach, Ben-Shachar, & Riederer, 1994; Hirsch & Faucheux, 1998; Youdim, Ben-Shachar, & Riederer, 1993). Basal ganglia ferritin iron content is increased in patients affected by AD (Bartzokis et al., 2000), whereas iron is found in higher concentration up to 35% in the substantia nigra pars compacta in PD patients (Double, Gerlach, Youdim, & Riederer, 2000). It has been proposed that hydroxyl radicals and Fe(III) are generated upon Fenton reaction that accounts for the increase of ferric ions and reactive oxygen species in these degenerating zones of the brain (Linert et al., 1996; Owen, Shapira, & Jenner, 1997; Smythies, 2000). Metal-chelating compounds remove the metals and can alter their redox potentials rendering them inactive. Moreover, the use of natural metal chelators such as flavonoids should be favored against other synthetic chelators which may present some problems of toxicity. Flavonoids with their multiple hydroxyl groups and the carbonyl group at the 4 position on ring C (see Schemes 1 and 2) may offer several available sites for metal complexation. The purpose of this review is to give an overview of the research carried out in the field of antioxidant polyphenolic compounds, employing theoretical and computational methods. It analyses in details the working mechanisms of flavonoids and polyphenols as antioxidants, covering the relevant literature on this subject. 2. Methods All the calculations reported are performed with the Gaussian03 code (Frisch et al., 2003). The principal conceptual tools used here are density functional theory (DFT) methods, employing the Becke3 (Becke, 1993) and Lee Yang Parr (Lee, Yang, & Parr, 1988) (B3LYP) hybrid functional. It can be written as:

Becke F B3LYP ¼ ð1  AÞF Slater þ AF HF þ CF LYP þ ð1  CÞF VWN c x þ BF x x c

where F Slater is the Slater exchange, F HF is the Hartree–Fock exx x is the gradient part of the exchange functional of change, F Becke x is the correlation functional of Lee, Yang and Parr, and Becke, F LYP c is the correlation functional of Volsko, Wilk and Nusair. The F VWN c A, B and C coefficients are determined by fitting experimental heats of formation (Becke, 1993). The accuracy of the DFT methods have been tested through G2 benchmark test of 55 small first- and second-row molecules (Bauschlicher, Ricca, Partridge, & Langhoff, 1997; Curtiss, Raghavachari, Trucks, & Pople, 1991), according to which B3LYP method seems to yield good results in predicting atomization energies. For geometries optimisation, all DFT means have given quite accurate results. Concerning transition-metals complexes, for which few accurate experimental data are available, systematic theoretical studies have been performed on small MR+ systems, where M is a firstrow transition metal and R is H, CH3, CH2 and OH. The average absolute error in M–R binding energies results to be in the range of 3.6–5.5 kcal/mol, as the B3LYP functional is employed (Armentrout & Kickel, 1996; Blomberg, Siegbahn, & Svensson, 1996; Ricca & Bauschlicher, 1997). Other theoretical studies on M–CO complexes binding energies have indicated B3LYP to give good agreement with experiments, being the average error 2.6 kcal/mol (Blomberg et al., 1996; Ricca & Bauschlicher, 1994). The choice of B3LYP functional in this study is dictated by its good performance in geometries optimisation, as well as by its quite accurate prediction of X–H bond energetic, and binding energies. For example, post-HF MP2 optimization of quercetin molecule and its deprotonated and semiquinone forms, converged to a planar arrangement, as found with the B3LYP method (Fiorucci, Golebiowski, Cabrol-Bass, & Antonczak, 2007). Test calculations on propene have proposed B3LYP to predict bond dissociation energies in good agreement with the values obtained by employing

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the more accurate and expensive MP2 and CCSD methods (DiLabio, 1999). However, relative evaluation of antioxidant activity is performed in this study, taking the phenol molecule as reference, in order to observe the effect of some functional groups and/or spatial disposition on the antioxidant power of these natural compounds. Other methods reported in this study are the Hartree–Fock, the semi-empirical AM1 (Anders, Koch, & Freunscht, 1993; Davis, Guidry, Williams, Dewar, & Rzepa, 1981; Dewar & Holder, 1990; Dewar & Jie, 1989; Dewar, Jie, & Zoebisch, 1988; Dewar, McKee, & Rzepa, 1978; Dewar & Merz, 1988; Dewar & Reynolds, 1986; Dewar & Thiel, 1977; Dewar & Yuan, 1990; Dewar, Zoebisch, & Healy, 1985) and PM3 (Stewart, 1989a, 1989b), and the ab initio MP2 (Frisch, Head-Gordon, & Pople, 1990a, 1990b; Head-Gordon & Head-Gordon, 1994; Head-Gordon, Pople, & Frisch, 1988; Møller & Plesset, 1934; Saebø & Almlöf, 1989) ones. The selected polyphenols molecules, and their radicals and anions, are optimised without constraints at B3LYP level, employing 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 LANL2DZ pseudopotential (Hay & Wadt, 1985), are chosen for C, O and H atoms, and for Fe2+ cation, respectively. Geometry optimisation is followed by single-point calculations using the extended 6-311++G** basis set for the non-metal atoms, in order to refine electronic energies. The B3LYP functional has been widely used for the treatment of transition metal containing molecules, for two main reasons. It has shown to be the most accurate of the DFT functionals in benchmark tests, and also to be fast enough to be able to treat rather large models, even up to a few hundred atoms (Siegbahn, 2003). This method has shown good performance for a truly wide variety of chemical systems and properties, although specific limitations and failures have also been identified. For example, metal–ligand binding energies always appear to be underestimated by B3LYP, so far never overestimated, which is helpful in the analysis of the results. Concerning the employment of the LANL2DZ effective core potential and its orbital basis set in the study of metal–flavonoids complexes, this method can handle high Z atoms. It is widely used in this kind of studies, and it gives good results in binding energies for transition metals ligands complexes (Siegbahn, 2003, 2006). Minima are identified through frequency calculations performed at the same level of theory. Zero point energy corrections, obtained from vibrational analysis, are then included in all the relative energy values. The unrestricted open-shell approach is used for polyphenols radical species. No spin contamination is found for radicals, being the hS2i values of 0.750 in all cases. Natural Bond Orbital (NBO) (Carpenter, 1987; Carpenter & Weinhold, 1988; Foster & Weinhold, 1980; Reed, Curtiss, & Weinhold, 1988; Reed & Weinhold, 1983; Reed & Weinhold, 1985; Reed, Weinstock, & Weinhold, 1985; Weinhold & Carpenter, 1988) analysis implemented in the Gaussian03 package is used to better characterise electronic structure. Solvent effects are computed in the framework of Self-Consistent Reaction Field Polarizable Continuum Model (SCRF-PCM) (Cossi, Barone, Cammi, & Tomasi, 1996; Miertus, Scrocco, & Tomasi, 1981; Miertus & Tomasi, 1982) using the Simple United Atom Topological Model (UA0) (Barone, Cossi, Menucci, & Tomasi, 1997) set of solvation radii to build the cavity for the solute in its gas-phase equilibrium geometry. The molecule is placed in a cavity, which is created via a series of overlapping spheres. In PCM method, the variation of the free energy when going from vacuum to solution is composed of the work required to build a cavity in the solvent (cavitation energy, Gcav) together with the electrostatic (Gel) and nonelectrostatic work (Gdisp + Grep).

The O–H bond dissociation energy (BDE) is computed at 298 K as the difference in enthalpy (H) between products and reactants for the reaction (1), that is:

BDE ðArO —HÞ ¼ HðArO Þ þ HðH Þ  HðArOHÞ The ionisation potential (IP) values are computed at 298 K as the enthalpy difference between products and reactants for the reaction (2), that is:

IPðArOHÞ ¼ HðArOHþ Þ  HðArOHÞ The gas-phase acidity is computed at 298 K as the enthalpy difference between the anion (A) and its neutral species (HA):

DHacidity ¼ HðA Þ  HðHAÞ For the calculations in the condensed phase, the acidities are computed in the same way but given in terms of total free solvation energies (DG). 3. Parent and radicals polyphenols structures The knowledge of the conformational, electronic and geometrical features of phenolic systems is of crucial importance to understand the relationship between the molecular structure and the antioxidant activity. It is commonly accepted that the main structural characteristics for a good radical scavenging activity are: – the occurrence of multiple OH groups attached to the aromatic ring; – the arrangement of these hydroxyls in the ortho-dihydroxy conformation, when possible; – the planar structure of phenolics, that allows conjugation and electronic delocalization, as well as resonance effects; – the presence of additional functional groups, like the carbon– carbon double bond and the C@O carbonyl group. Since polyphenols are considered to react mainly with free radicals by donating to them an H (or an electron), the knowledge of the geometrical and electronic structures of the radicals arising from this interaction is relevant for the investigation of this kind of mechanism. Molecules with multiple OH groups can give rise to several radicals depending on which group is radicalised. The relative energies of the radicals of some polyphenols are reported in the Table 1. The optimised geometries of the most stable radical species are presented in the Fig. 1. The complete geometrical parameters of all investigated systems are available on request. Tyrosol and hydroxytyrosol are the main phenolic compounds present in the virgin olive oil (see Scheme 3) (Brenes, Garcia, Garcia, Rios, & Garrido, 1999). The different conformers of these two molecules arise from the flexibility of the side chain –CH2CH2OH, and in hydroxytyrosol, also from the relative disposition of the OH groups. Minimum energy conformers are characterised by the folded gauche conformation of the alkyl chain in which the alcoholic OH is oriented toward the aromatic ring, so that a hydrogen bond like interaction can be established (Leopoldini et al., 2004a, 2004b). The two OH groups in hydroxytyrosol realise a hydrogen bond in which the O4–H hydroxyl plays the H-bond donor function. Tyrosol and hydroxytyrosol radicalisation originates one and two radicals, respectively. As far as the catechol functionality is concerned, the radicalisation of the 4-OH group in hydroxytyrosol absolute minimum causes the loss of the internal hydrogen bond. This can be re-established by a free rotation around the C3–O–H bond, that requires an energetic expense of around 3 kcal/mol (Leopoldini et al., 2004a). The other 4-OH radical (radicals are indicated as the original hydroxyl group from which the hydrogen is

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M. Leopoldini et al. / Food Chemistry 125 (2011) 288–306 Table 1 Relative energies (values in kcal/mol) of some polyphenols radicals in the gas-phase. Radicals

DE

Radicals

DE

Radicals

DE

Hydroxytyrosol 3-OH 4-OH

0.0 1.4

Gallic acid 3-OH 4-OH

6.4 0.0

Caffeic acid 3-OH 4-OH

6.6 0.0

Catechin 30 -OH 40 -OH 5-OH 7-OH

0.7 0.0 8.1 7.9

Epicatechin 30 -OH 40 -OH 5-OH 7-OH

0.0 0.1 8.3 10.6

Kaempferol 40 -OH 3-OH 5-OH 7-OH

0.0 0.2 13.5 5.7

Quercetin 30 -OH 40 -OH 3-OH 5-OH 7-OH

2.5 0.0 8.4 23.2 14.3

Apigenin 40 -OH 5-OH 7-OH

0.0 23.8 5.2

Luteolin 30 -OH 40 -OH 5-OH 7-OH

2.3 0.0 31.4 12.9

Radicals

DE

Resveratrol 3-OH 5-OH 40 -OH

6.3 5.8 0.0

Cyanidin 30 -OH 40 -OH 3-OH 5-OH 7-OH

4.3 3.1 0.0 2.5 5.7

Taxifolin 30 -OH 40 -OH 5-OH 7-OH

0.6 0.0 22.4 29.7

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.

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Scheme 3. Some polyphenols studied.

removed) conformer missing this interaction is thermodynamically less favoured by 8.8 kcal/mol (Leopoldini et al., 2004a). This value can be considered as an estimation of the stabilising effect coming from the H-bond. The radical 3-OH lies at 1.4 kcal/mol with respect to the 4-OH one. This energy difference is explained by considering that in the 4-OH species, the electronic vacancy is supplied by the electron-donating effect of the –CH2CH2OH group, that does not occur in the other radical, as an analysis of the resonance structures immediately suggests. Vitamin E is one of the non enzymatic endogenous systems acting as antioxidant in living organisms. It contains a-, b-, c- and d-tocopherols that possess a phytyl tail (C16H33) ensuring to the molecule the solubility in membranes (Morris, & Evans, 2002). The radical scavenging ability is due to the OH group. The computations (Leopoldini et al., 2004a) on a model system of vitamin E, that is the 6-hydroxy-2,2,5,7,8-pentamethylchroman, HPMC (Scheme 3), confirm that this compound has the features of an aro-

matic system, with a complete electronic delocalization occurring on the aromatic ring carrying the phenolic OH, while the –CH3 substituents increase the charge density on the same ring. The group of the phenolic acids contains a lot of strong antioxidant natural compounds (Robbins, 2003). Gallic acid (Scheme 3) is present itself or as ester moiety in other polyphenols. Three OH groups are present in its minimum energy structure, arranged as to form two hydrogen bonds of 2.196 Å (Leopoldini et al., 2004a). Bond order values computations find a double bond in the C@O carbonyl group (bond order of 1.756), while the values of bond order of the carbon–carbon couple are 1.360, as a confirmation of the expected electronic delocalization typical of aromatic rings. From gallic acid it is possible to get two radicals, the 3-OH (5-OH) and the 4-OH. The latter is the absolute minimum (relative energy of the 3-OH (5-OH) is 6.4 kcal/mol), stabilized by the coupled effect of the neighbouring OH in orthoand the COOH in the para-position. In both radicals, the unpaired

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electron appears to be delocalised over the aromatic ring (Leopoldini et al., 2004a). The presence of the CH@CH bridge between the benzene and the carboxyl group in caffeic acid (Scheme 3) favours resonance and conjugation effects. A complete exploration (Leopoldini et al., 2004a; VanBesien & Marques, 2003) of caffeic acids conformers leads to fourteen isomers arising from S-cis or S-trans conformation of the carboxylic group dihedral, disposition of the two phenolic hydroxyls dihedral with respect to the ring, mutual orientation of the aromatic ring and carboxyl group. Most stable conformers are characterised by the S-cis orientation of the carboxyl group and by the mutual trans disposition of this group and the aromatic ring (VanBesien & Marques, 2003). The OH groups are involved in a hydrogen bond (2.152 Å). The stabilizing effect of this internal Hbond can be estimated roughly 4.5 kcal/mol, that represents the relative energy of the conformer missing this kind of weak interaction (VanBesien & Marques, 2003). The dihedral between the phenyl and the substituent is, in the minimum geometry, 0° (Leopoldini et al., 2004a; VanBesien & Marques, 2003). With an expense of only 6.1 kcal/mol which leads the ethylene group perpendicular to the plane of the ring, a relative minimum lying at 0.4 kcal/mol above the global one and characterised by a torsion angle of 180°, can be easily reached (Leopoldini et al., 2004a). The relative energies of these conformers and the low activation energy required indicate that the conformers may coexist (Leopoldini et al., 2004a). Caffeic acid radicalisation yields to two radicals, 3-OH and 4OH. The electronic delocalization effect of the –CH@CH–COOH is responsible for the energetic stability of the 4-OH radical (Table 1) (Leopoldini et al., 2004a). Rosmarinic acid is a phenolic compound extracted from Rosemarinus officinalis L. It contains two phenolic rings both carrying two ortho-hydroxyl groups (Petersen & Simmonds, 2003). There are a carbonyl group, an unsaturated double bond and a carboxylic acid between the two phenolic rings. Its structure is quite different from the other phenolics. Geometry minimisation indicate as preferred structure the one with the ring A coplanar with the double bond and the 9-carbonyl, and the ring B out of plane of the rest of the molecule, as expected on the basis of resonance structures (Cao et al., 2005). Upon radicalisation of rosmarinic acid, four radicals are obtained. Among them, the most stable is the 2-OH one, followed in energy by the 40 -OH (DE = 0.4 kcal/ mol) (Cao et al., 2005). Radical 1-OH is found at 2.6 kcal/mol. By looking at the molecular structure, it can be noted that in the case of the 2-OH/40 -OH species, the odd electron is better delocalised by the presence of substituents in the para-position. This possibility is missing in the case of the less stable 1-OH species (Cao et al., 2005). Resveratrol (trans-3,5,40 -trihydroxystilbene, see Scheme 3) is a natural product found in grapes, mulberries, peanuts. It is one of the main non alcoholic components in the red wines (Jang et al., 1997). Its structure is characterised by two phenolic rings, linked by a double bond. The B3LYP/6-311++G** optimisation (Leopoldini et al., 2004a) yields an absolute minimum characterised by planarity, conjugation and electronic delocalization. The mutual position of the hydroxyls does not allow the formation of any intramolecular H-bonds (Caruso, Tanski, Villegas-Estrada, & Rossi, 2004; Leopoldini et al., 2004a). Radicalisation of the 40 -OH group generates the most stable radical, while the other 3-OH and 5-OH systems lie at 6.3 and 5.8 kcal/mol, respectively (Leopoldini et al., 2004a). As pointed out for rosmarinic acid, only in the first species the resonance forms show the unpaired electron spread over the whole molecule. Flavanols lack the 2,3-double bond in the ring C, so that four stereoisomers exist, of which (+)-catechin (b-OH in ring C) and ()-epicatechin a-OH in ring C) are the most important ones (Jia,

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Zhou, Yang, Wu, & Liu, 1998). The OH group in ring C is an alcoholic group to which cannot be ascribed any antioxidant capability. Catechin (Scheme 3) lowest energy structure is characterised by a torsional C20 –C10 –C2–C3 of 77.4° (Leopoldini, Russo, & Toscano, 2007), that indicates that the rings B and C are almost perpendicular. The hydroxyls in ring B establish an H-bond of 2.152 Å. The electronic delocalization occurs separately on rings B and A. In the case of ()-epicatechin (Scheme 3), the same torsion is 92.49°, that indicates that in the latter the ring B is more twisted. The other conformer of epicatechin, with torsion of 270.14°, lies at only 0.3 kcal/mol. The energy required to pass from one conformer to another is computed to be 1.1 kcal/mol (Leopoldini et al., 2004a), so they may coexist. Radicalisation of catechin and epicatechin molecules gives as most stable radical species the isoenergetic 30 -OH and 40 -OH in both cases (Leopoldini et al., 2004a; Leopoldini et al., 2007). In the absence of conjugation with ring C, the main stabilizing factor is the internal hydrogen bond at the ring B. The 5-OH and the 7-OH are found at 8.1 and 8.3 kcal/mol, and at 7.9 and 10.6 kcal/mol, for catechin and epicatechin, respectively. The absence of ortho-diphenolic structure on ring B in kaempferol could determine its lesser efficiency as hydrogen donor (RiceEvans, Miller, & Paganga, 1996). In kaempferol absolute minimum (Scheme 3), the hydrogen bonds are established between the 3OH/5-OH and the C4@O carbonyl oxygen (Leopoldini et al., 2004a). The molecule is completely planar as the dihedral value indicates, at both B3LYP (Leopoldini et al., 2004a) (180°) and RHF (van Acker et al., 1996) (179.86°) levels. Radical 40 -OH and 3-OH have practically the same stability, being their energetic gap only 0.2 kcal/mol (Leopoldini et al., 2004a). B3LYP bond order analysis and the value of the torsional angle (U = 180°) indicate that for both, a broad delocalization of the odd electron contributes to the radical stability (Leopoldini et al., 2004a). Planar arrangement of the kaempferol radicals is found also as far as RHF computations are performed (van Acker et al., 1996). Apigenin and luteolin (Scheme 3) differ by a hydroxyl on the 30 position in the ring B. Both, in their B3LYP/6-311++G** equilibrium geometries, are planar molecules with torsional angles between rings C and B (C3–C2–C10 –C20 ) of 0.0° (Leopoldini, Prieto Pitarch et al., 2004). The conformers with a dihedral of 180.0° are found at 0.1 (apigenin) and 0.2 (luteolin) kcal/mol. The transition states in going from 0.0° to 180.0° are found at 4.0 and 3.7 kcal/mol and characterised by a dihedral of 90.9° and 90.8°, for apigenin and luteolin, respectively (Leopoldini, Prieto Pitarch et al., 2004). RHF/STO-3G computations find for both flavones a non planar conformation, with dihedral of 16.5° and 16.3°, respectively (van Acker et al., 1996). An explanation to these findings (van Acker et al., 1996) is recognised in the lack of the 3-OH group in ring C, that establishing hydrogen like interaction with the ring B, should force the system in a planar disposition. So, flavones lacking the 3-OH group should be slightly twisted (luteolin, apigenin, diosmin) (van Acker et al., 1996). HF/6-31G(d) method also predicts for flavones a non planar conformation, as well as B3LYP/6-31G(d) ones, that find for apigenin and luteolin a torsional angle of 16.4° and 18.1° (Martins, Leal, Fernandez, Lopes, & Cordeiro, 2004). The discrepancies between HF and DF approaches can be ascribed to the fact that the former (as well as AM1 and PM3) methods underestimate the stabilizing p-electrons delocalization contributions with respect to DF. Concerning the comparison between B3LYP results (Leopoldini, Prieto Pitarch, et al., 2004; Martins et al., 2004), it should be noted that the use of an extended basis set including diffuse functions (Leopoldini, Prieto Pitarch, et al., 2004) should improve the electronic structure description. The H-atom removal from apigenin and luteolin molecules originates radicals of which the 40 -OH is the most stable (Leopoldini, Prieto Pitarch, et al., 2004). In contrast to apigenin 40 -OH radical,

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in which the odd electron leaves the radicalised oxygen, in luteolin 40 -OH the unpaired electron remains on the radicalisation site, due to the intramolecular H-bond. RHF computations (van Acker et al., 1996) also yield planar radicals, despite the parent molecules show twisted rings B. Saturated taxifolin (Scheme 3) does not present planar conformation because of the absence of the C2–C3 double bond in the ring C. B3LYP/6-311++G** computations indicate a C3–C2–C10 –C20 torsion of 101.0° (Leopoldini, Prieto Pitarch, et al., 2004). RHF/STO3G also find a non planar conformation for taxifolin (152.4°) (van Acker et al., 1996), as well as for hesperetin and naringenin. From taxifolin, four radicals exist, and among them the 30 -OH and 40 -OH are the most stable ones (Leopoldini, Prieto Pitarch, et al., 2004). Their stability depends on the same factors indicated for saturated catechin and epicatechin. Quercetin (Scheme 3) is a flavonol on which many biochemical, epidemiologic, medical as well as theoretical works exist. B3LYP optimisations (Leopoldini et al., 2004b) yield as preferred structure (I) a planar conformation (C3–C2–C10 –C60 U 180° characterised by three intramolecular hydrogen bonds, established between the 3OH/5-OH and the C4@O, and between the 30 -OH and 40 -OH in the ring B. A relative minimum (II) with a U 0° is found lying at 0.5 kcal/mol with respect to the former, and it can be reached with an energetic expense of only 5.6 kcal/mol (Leopoldini et al., 2004b). AM1 (Russo, Toscano, & Uccella, 2000) and RHF/6-31G* (Vasilescu & 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 reached through a barrier of 2.5 and 4.0 kcal/mol, respectively. Even if some differences can be found in the absolute values, all theoretical data indicate that quercetin may exist into two conformers that easily may interconvert (Leopoldini et al., 2004b). Starting from the two B3LYP quercetin minima I and II, ten radicals are obtained breaking the 3-, 30 -, 40 -, 5- and 7-O–H bonds, all characterised as planar species. Among them, the 40 -OH(I) species is the most stable, followed by the 40 -OH(II) one (DE = 0.2 kcal/mol at B3LYP/6-311++G** level) (Leopoldini et al., 2004b). The energetic gaps among the radicals arising from the radicalisation of the rings B and C fall within 8 kcal/mol, while radicalisation occurring at the ring A produces radicals very high in energy (range of 13– 25 kcal/mol). These latter radicals exhibit a spin distribution that leaves the odd electron on the radicalisation site, probably because of the presence of the –C@O and –O-moieties in the adjacent ring C (Leopoldini et al., 2004b). 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 (Leopoldini 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 (Leopoldini et al., 2004b). This barrier seems to be higher than those computed for the other polyphenols, so probably the second minimum cannot be easily reached. Cyanidin radicals energies fall within 6 kcal/mol, being the gas-phase stability order 3-OH > 5OH > 40 -OH > 30 -OH > 7-OH (Leopoldini et al., 2004b). The formation of these species does not entail the breaking of any H-bond so that their relative energies are very close. Chalcones (or 1,3-diaryl-2-propen-1-one) are open-chain flavonoids (see Scheme 1), in which two aromatic rings are linked by a three-carbon a,b-unsaturated carbonyl system. The absence of the central C ring and the presence of a a,b-unsaturated bond are two specific characteristics of chalcones, making them chemically different from the other flavonoids. Chalcones are always considered to be in trans conformation as the a,b-double bond is concerned. Then, two conformers arise, the s-cis and s-trans, that correspond to two different orientations of the double bond and the carbonyl group. The s-cis conformer of 20 -hydroxy chalcone represents the

absolute minimum (DE = 5.6 kcal/mol) because of its full planarity, while the steric hindrance in the s-trans one causes a certain deviation from the planarity (torsion angle O–C–Ca–Cb of approximately 142°) (Kozlowski et al., 2007). The energy cost to pass from one conformer to another is found to be 8.3 kcal/mol. Computations on the other chalcones also lead to planar configuration, even in the absence of the 20 -OH group that is supposed to be in part responsible for the planarity (Kozlowski et al., 2007). Radicalisation of the 20 ,40 ,60 ,3,4-pentahydroxycalchone involves the formation of five radicals, the most stable one is again characterised by an internal H-bond and by delocalization and conjugation effects (Kozlowski et al., 2007).

4. BDE and IP evaluation Antioxidants may play their protective role by donating an Hatom or a single electron, so the bond dissociation enthalpies (BDEs) for the O–H bonds and the ionisation potentials (IPs) are of particular interest to evaluate their potentiality. BDEs and IPs for polyphenols of Scheme 3 (except for rosmarinic acid, chalcones and myricetin) are collected in the Tables 2 and 3. In the same tables, the values for phenol are also reported, with the purpose to quantitatively estimate the effect of OH groups and substituents on the basic activity of phenol.

Table 2 Bond dissociation energies (BDE) for polyphenols in the gas-phase. Values are given in kcal/mol. Compound

BDE

Phenol Tyrosol Hydroxytyrosol Gallic acid Caffeic acid HPMC Resveratrol Catechin Epicatechin Kaempferol Cyanidin Quercetin Apigenin Luteolin Taxifolin

82.9 82.0 73.5 72.2 73.6 71.7 77.3 74.2 73.7 80.9 79.4 72.3 82.2 74.5 74.7

Table 3 Ionisation Potentials (IP) for polyphenols in the gas-phase. Values are given in kcal/mol. Compound

IP

Phenol Tyrosol Hydroxytyrosol Gallic acid Caffeic acid HPMC Resveratrol Catechin Epicatechin Kaempferol Cyanidin Quercetin Apigenin Luteolin Taxifolin

192.0 181.7 175.1 189.1 181.1 154.9 161.3 169.7 170.8 168.0 246.2 166.1 176.0 174.4 182.8

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4.1. BDEs The gas-phase BDE value for phenol is computed to be 82.9 kcal/mol at B3LYP/6-311++G** (Table 2) (Leopoldini et al., 2004a), 82.3 kcal/mol at B3LYP/6-311++G**//B3LYP/6-31G** (Himo, Eriksson, Blomberg, & Siegbahn, 2000), 82.8 kcal/mol at B3LYP/6-31G** (Zhang, Sun, & Wang, 2003), 82.8 kcal/mol (de Heer, Korth, & Malder, 1999), 83.9 kcal/mol at B3LYP/6311++G(3df, 3pd) (Thavasi, Leong, & Bettens, 2006). All these values fall in the range of 82-84 kcal/mol. The B3LYP/6-311 + G(2d,2p) different value of 87.1 kcal/mol computed by Wright (Wright et al., 2001) is obtained as a single point energy on geometries optimised at AM1 level. The most recent experimental values for phenol are 87.0 ± 1 kcal/mol (Wayner et al., 1995), 88.3 ± 0.8 kcal/mol (Pedulli, Lucarini, & Pedrielli, 1997) and 88.7 ± 0.5 kcal/mol (Dos Santos & Simoes, 1998), being the value of 88.7 kcal/mol retained as the more reliable one. Bakalbassis et al. (Bakalbassis, Lithoxoidou, & Vafiadis, 2003) have computed the gas-phase B3LYP BDE of phenol with several basis set. Results show that the biggest conventional basis set, 6-311 + G(2d,2p), gives a BDE which is still over 5.1 kcal/ mol lower that the experimental value of 88.7 kcal/mol, while the basis set 6-31 + G (3p,d), derived upon addition of a third p and a fourth d polarisation function on the hydrogen atoms basis set, leads to a BDE value of 88.5 kcal/mol (Bakalbassis et al. 2003), which seems to be nearer to the experimental indication. B3LYP catechol BDE is found to be 72.6 (Himo et al., 2000), 72.8 (Zhang et al., 2003) and 74.7 (Thavasi et al., 2006) kcal/mol. This means that the effect of an OH group in ortho position is to decrease the BDE by 9–10 kcal/mol with respect to phenol. The reason can be found in the fact that the radical arising from H-atom removal is stabilized by the formation of the intramolecular Hbond with the vicinal hydroxyl. The para substitution of catechol molecule with –COOH, – CH2COOH and –CH2CH2COOH groups entails the lowering of the BDE (compared to catechol) in the last two cases and a slight increase in the former (Ordoudi, Tsimidou, Vafiadis, & Bakalbassis, 2006). The insertion of the carboxylic group to the catechol ring results in a less favourable H-radical elimination by around 2 kcal/ mol, while the insertion of methylene and ethylene groups between the catechol ring and the carboxylic group favours the Hradical elimination (Ordoudi et al., 2006). The BDE values of guaiacol (2-methoxyphenol) of 80.4 (Himo et al., 2000) and 82.7 kcal/mol (Bosque & Sales, 2003) indicate that the presence of –OCH3 in the ortho-position has a slightly stabilizing effect on the radicalised molecule due to the compromise between the electron-donor and electron-withdrawing capabilities of this group. Tyrosol BDE of 82.0 kcal/mol (Table 2) indicates that the stabilizing effect of a –CH2CH2OH chain in para-position with respect to the OH is about 1 kcal/mol as compared to the phenol BDE of 82.9 kcal/mol, and it depends on the electron-donating ability of the substituent (Leopoldini et al., 2004a). Indeed, in the case of hydroxytyrosol, whose BDE is 73.5 kcal/mol (Leopoldini et al., 2004a), the simultaneous presence of both the –CH2CH2OH in para and the OH in ortho groups causes a decrease of the BDE of 9.4 kcal/ mol (with respect to phenol). HPMC model of vitamin E shows a BDE of 71.7 kcal/mol at B3LYP/6-311++G** level (Leopoldini et al., 2004a), with a decrease of the BDE of phenol of 11.2 kcal/mol (Table 2). Here, the main factor favouring the hydrogen removal is represented by the electron releasing effect of the three methyls and the saturated ring. Gallic and caffeic acids BDEs, reported in the Table 2, are computed to be 72.2 and 73.6 kcal/mol, respectively (Leopoldini et al., 2004a). If one considers their molecular structures, the better

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activity of the former as radical scavenger can be ascribed to the tri-hydroxy functionality. Nenadis et al. (Nenadis, Zhang, & Tsimidou, 2003) have computed the BDE as B3LYP single point energies on AM1 optimised geometries of ferulic acid and its derivatives. Ferulic acid is characterised by the presence of a –OCH3 in ortho to the phenolic OH and by a –CH@CH–COOH chain in para. BDE of ferulic acid and its ethyl ester are computed to be 84.3 and 83.9 kcal/mol, respectively (Nenadis et al., 2003). Coniferyl aldehyde and alcohol, in which the –COOH is replaced by a –CHO and –CH2OH group in the side chain, respectively, show a BDE of 84.4 and 81.5 kcal/mol (Nenadis et al., 2003). The lower value of the latter with respect to the others, can be due to the fact that the –CH2OH does not subtract electron density from the side chain as the –CHO does, so when the radical is formed upon breaking of the phenolic O–H, the electron vacancy is better stabilized in the case of coniferyl alcohol. The same occurs for isoeugenol (BDE = 81.1 kcal/mol) that possesses a –CH@CH–CH3 side chain (Nenadis et al., 2003). Resveratrol BDE is 5.6 kcal/mol (Leopoldini et al., 2004a) lower than the corresponding one computed for phenol (Table 2). Here, there is no possibility of intramolecular hydrogen bonds, so its antioxidant activity may be mainly given in terms of a good delocalization of the radical unpaired electron through the aromatic rings and the –CH@CH– bridge. Flavanols catechin and epicatechin show a BDE of 74.2 (Leopoldini et al., 2007) and 73.7 (Leopoldini et al., 2004a) kcal/mol, respectively (Table 2). These diastereoisomers are characterised by the catechol functionality in the ring B, while the saturated ring C does not allow conjugation between rings. The factor affecting the antioxidant ability in terms of H donation is again the intramolecular hydrogen bond established between the radicalised oxygen and the adjacent OH. The relevance of this functional group is also underlined by the BDE of 74.7 kcal/mol (Leopoldini, Prieto Pitarch, et al., 2004) for flavanone taxifolin, reported in the Table 2. BDE values for apigenin and luteolin, from the class of flavones, are 82.2 and 74.5 kcal/mol, respectively (see Table 2) (Leopoldini, Prieto Pitarch, et al., 2004). Molecules differ in the ortho-diphenolic moiety in the ring B, so that in the case of luteolin the H-atom abstraction is easier because the derived radical can be stabilized by the intramolecular hydrogen bond. The BDE of flavonols kaempferol and quercetin is evaluated to be 80.9 (Leopoldini et al., 2004a) and 72.3 (Leopoldini et al., 2004b) kcal/mol at B3LYP/6-311++G** level (Table 2). Because the only difference between them is the 30 -OH group in the ring B, the presence of this group lowers the energy required for the H abstraction by 8.6 kcal/mol. Charged cyanidin shows a value of BDE of 79.4 kcal/mol (Leopoldini et al., 2004a) (Table 2). Since it is a completely planar and conjugated system, the H-bonding becomes less important than in the other flavonoids. Chalcones show BDEs values that fall in a range of 74.4– 84.5 kcal/mol (relative to the most stable radicals) (Kozlowski et al., 2007). Also for these compounds, the important role of the catechol moiety in the B ring is confirmed. BDEs in water solution have the same general trend of those computed in the gas-phase for the same molecules, except for epicatechin that becomes the most reliable system acting through H donation (Leopoldini et al., 2004a). The same is found for the computations in benzene medium (Leopoldini et al., 2004a). Results on BDEs indicate that the most efficient systems acting as hydrogen donors are those characterised by the dihydroxy functionality, for which the values of the BDE are smaller than that of phenol reference system. Upon the radicalisation of the OH groups in these compounds, radical species arise, stabilized by resonance, conjugation and delocalization effects. Internal H-bonds

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involving radicalised oxygen atoms further contribute to radical stability. 4.2. IPs The ionisation potentials give different trends of reactivity (Leopoldini et al., 2004a, 2004b; Leopoldini, Prieto Pitarch, et al., 2004) for polyphenols with respect to the bond dissociation energies (see Table 3). IP value for phenol is computed to be 192.0 kcal/mol at B3LYP/6-311++G** (Leopoldini et al., 2004a, 2004b). Tyrosol and hydroxytyrosol IP values are computed to be 181.7 and 175.1 kcal/mol (Leopoldini et al., 2004a), in the gas-phase (Table 3). The a-tocopherol shows an IP value that is 37.1 kcal/mol lower than that calculated for phenol (Leopoldini et al., 2004a). This means than the presence of several alkyl groups increases the hyper-conjugation and stabilises the cation radical originating from the electron removal. For gallic acid, the influence of the trihydroxy moiety on the IP value is small (189.1 kcal/mol, Table 3). In the case of caffeic acid, the enhancement of the conjugation through the –CH@CH–COOH affects mostly the IP value (181.1 kcal/mol, Table 3) (Leopoldini et al., 2004a). DFT/B3LYP IPs for ferulic acid and its derivatives are 167.5 (ferulic acid), 165.5 (ethyl ferulate), 169.8 (coniferyl aldehyde), 155.1 (coniferyl alcohol) and 159.9 (isoeugenol) kcal/mol (Nenadis et al., 2003). Among them, coniferyl alcohol and isoeugenol are the compounds that can be more easily oxidised through an electron transfer mechanism. As encountered for BDEs of these compounds, the electron-donor ability of substituents entails the lowering of IP values (Nenadis et al., 2003). The IP for resveratrol is computed to be 161.3 kcal/mol (Leopoldini et al., 2004a), that is 30.7 kcal/mol lower than phenol (see Table 3). The molecular structure of this phenolic compound underlines as an extended p-electrons delocalization particularly favours the electron transfer process with respect to the reference compound. Epicatechin and catechin show values of IP of 170.8 (Leopoldini et al., 2004a) and 169.7 (Leopoldini et al., 2007) kcal/mol, respectively (Table 3). Apigenin, luteolin, taxifolin and kaempferol show IP values of 176.0, 174.4, 182.8 (Leopoldini, Prieto Pitarch, et al., 2004) and 168.0 (Leopoldini et al., 2004a, 2004b) kcal/mol, in the order, as collected in the Table 3. Cyanidin appears to be less active as single electron-donor with respect to the other flavonoids (IP = 246.2 kcal/mol, Table 3) (Leopoldini et al., 2004a, 2004b). This is not surprising because cyanidin is just a charged molecule (charge = +1), so it is very unreliable to generate another positive charge. DFT/B3P86 IP values for chalcones fall in a range of 153.1– 160.3 kcal/mol (Kozlowski et al., 2007), so that the electron transfer mechanism is also important for this flavonoids. As far as the solution IPs are concerned, the presence of the water medium involves a decrease of the absolute values. IP of HPMC, that is one of the most active, changes from 154.3 to 130.1 kcal/mol, in going from the gas-phase to the water solution (Leopoldini et al., 2004a). Theoretical results show that within the mechanism of the electron transfer, the main factors affecting the value of IP are the extended delocalization and conjugation of the p-electrons, enhanced by resonances phenomena, rather than the presence of particular functional groups such as additional hydroxyls. So, resveratrol, tocopherol, quercetin, kaempferol and chalcones are good candidates to work also through the second antioxidant mechanism.

5. Determination of polyphenols acidity The third antioxidant mechanism by which polyphenols may perform their protective role, arises from the capability of these systems to sequester transition metals ions by chelation. Metals are entrapped in these polyphenols–metal complexes so they cannot participate in reactions involving production of free radicals species. Because chelation of metals often occurs through deprotonated hydroxyls in the polyphenols, the determination of the acidity of these compounds is an important thermodynamic parameter that must be taken into account. The smaller the energy required to deprotonate the OH groups (acidity), the easier the metals chelation will be. The anions formed upon deprotonation of polyphenols considered, share with the parent molecules the planar disposition, that in principle allows a complete delocalization of the negative charge over the entire system. Exceptions to this finding are the anions of epicatechin (Leopoldini, Russo, & Toscano, 2006), catechin, taxifolin (Martins et al., 2004), hesperetin, diadzein and naringenin (Zhang & Brodbelt, 2004), that are non planar systems as well as their parent molecules (see Fig. 2). For all of them, the most stable anion is that characterised by internal hydrogen bonds, especially those involving the deprotonation site oxygen (Leopoldini et al., 2006; Martins et al., 2004; Zhang & Brodbelt, 2004). For flavonoids, the 40 -position in the ring B is the most favoured deprotonation site, followed by the 7-OH in the ring A (Leopoldini et al., 2006). Also for the acidities, the gas-phase value of phenol is computed and used as reference compound. Phenol B3LYP/6311++G** acidity is found to be 345.1 kcal/mol (Leopoldini et al., 2006), that seems to be in good agreement with the experimental value of 346.9 kcal/mol, obtained by gas-phase proton transfer equilibria. On the basis of B3LYP/6-311++G** increasing acidity values, an order can be given: cyanidin (237.7 kcal/mol) > myricetin (312.5 kcal/mol) > quercetin (316.5 kcal/mol) > gallic acid (317.9 kcal/ mol) > caffeic acid (318.0 kcal/mol) > apigenin (321.3 kcal/mol) > kaempferol (322.7 kcal/mol) > epicatechin (327.2 kcal/mol) > resveratrol (327.5 kcal/mol) (see Table 4) (Leopoldini et al., 2006). Other acidity values obtained as MP2/6-311 + G(d,p) (Zhang & Brodbelt, 2004) single point energies on HF optimised geometries are 328.1 kcal/mol, for hesperetin, 323.8 kcal/mol, for luteolin, 331.1 kcal/mol, for acacetin, 328.8 kcal/mol, for naringenin and 329.7 kcal/mol, for daidzein. The value for cyanidin is the smallest one (237.7 kcal/mol) (Leopoldini et al., 2006). This finding is not surprising because cyanidin is a positively charged system so that deprotonation of the OH groups leads to very stable neutral species. By looking at their molecular structure, one can argue that the most acidic systems are those characterised by an high delocalization of p-electrons, as the values for cyanidin, myricetin, quercetin, and gallic and caffeic acids confirm. For the class of flavonoids, the delocalization in the anion involves the rings B (where deprotonation occurs) and C, while for the phenolic acids the negative charge is delocalised over the aromatic ring and the substituents. Further contributions to the acidity values arise from the H-bond formation occurring between the negative oxygen and the adjacent hydroxyl in systems having the ortho-dihydroxy moiety. The smallest value is obtained for myricetin, for which all these functionalities are present. MP2/6-311 + G(d,p) (Zhang & Brodbelt, 2004) and B3LYP/6311 + G(2p,2d) (Martins et al., 2004) calculations give the same

M. Leopoldini et al. / Food Chemistry 125 (2011) 288–306

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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 et al., 2006) for kaempferol, apigenin and quercetin, even if some differences in the absolute value can be found (the latter are generally smaller). These discrepancies in the absolute energies can be explained by considering that the formers are single point energies on HF optimised geometries, that often are found as non planar conformations. The in water solution trend is to some extent different from the gas-phase one: cyanidin (285.2 kcal/mol) > gallic acid (292.2 kcal/ mol) > myricetin (292.6 kcal/mol) > caffeic acid (293.8 kcal/mol) > apigenin (296.4 kcal/mol) > kaempferol (296.5 kcal/mol) > quercetin (298.3 kcal/mol) > epicatechin (299.7 kcal/mol) > resveratrol (301.9 kcal/mol). It is worth to note that the absolute acidity values for every compound are very smaller than the corresponding ones in gas-phase. That is, solvent favours the deprotonation process by 30–40 kcal/mol. Of course, the trends of DpKa relative values reflects that of acidities in terms of DG and depends on the same effects, that is the delocalization of the negative charge (with the resulting stabilization of the anion) and the formation of the intramolecular hydrogen bonds. Similar conclusions were

drawn by Himo et al. (Himo et al., 2000), for ortho-substituted phenols. Experimental relative acidities (Martins et al., 2004) revealed that flavones are the more acidic flavonoids, with the following relative order: catechin > apigenin > kaempferol > taxifolin > quercetin > luteolin > myricetin. The involvement of p electron delocalization and conjugation, and of the catechol functionality is also experimentally highlighted. The results on the gas-phase acidities match those relative to the BDE for the same systems. It is due to the fact that the release of a hydrogen atom (occurring in the H-atom transfer) can be considered as the simultaneous loss of a proton and an electron. So, the factors affecting the BDEs can be recognised also in determining the acidities values. 6. Formation of complexes between polyphenols and transition metals ions Transition metals ions in their low oxidation state (Fe2+, Cu+) can catalyse reactions that involve formation of free radicals.

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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

a b

Acidities a

MP2/6-311 + G(d,p)

b

Relative acidities

Relative pKa in solution

B3LYP/6-311++G**

B3LYP/6-311++G**

B3LYP/6-311++G**

B3LYP/6-311 + G(d,p)

Phenol OH

345.1 (310.0)

–

–

0.0 (0.0)

Gallic acid 4-OH 5-OH

317.9 (292.2) 327.4 (297.9)

– –

– –

27.2 (17.8) 17.7 (12.1)

13.0 8.8

Caffeic acid 3-OH 4-OH

323.8 (297.2) 318.0 (293.8)

– –

– –

21.3 (12.8) 27.1 (16.2)

9.3 11.8

Resveratrol 30 -OH 50 -OH 6-OH

336.2 (303.7) 335.6 (303.8) 327.5 (301.9)

– – –

– – –

8.9 (6.3) 9.5 (6.2) 17.6 (8.1)

Epicatechin 30 -OH 40 -OH 0-OH 7-OH

327.2 (299.7) 327.6 (300.0) 337.5 (304.0) 339.8 (304.4)

– – – –

– – – –

17.9 17.5 7.6 5.3

(10.3) (10.0) (6.0) (5.6)

7.5 7.3 4.4 4.1

Kaempferol 40 -OH 3-OH 5-OH 7-OH

323.0 (298.6) 333.0 (299.8) 337.5 (302.3) 322.7 (296.5)

327.1 338.0 342.2 328.1

328.9 336.3 340.3 327.3

22.1 12.1 7.6 22.4

(11.4) (10.2) (7.7) (13.5)

8.3 7.4 5.6 9.8

Cyanidin 30 -OH 40 -OH 3-OH 5-OH 7-OH

251.8 (294.6) 238.3 (285.9) 240.6 (288.7) 237.7 (285.3) 238.9 (285.2)

– – – – –

– – – – –

93.3 106.8 104.5 107.4 106.2

(15.4) (24.1) (21.3) (24.7) (24.8)

11.2 17.6 15.5 18.0 18.1

Apigenin 40 -OH 5-OH 7-OH

321.3 (297.1) 346.3 (304.7) 327.6 (296.4)

324.4 349.6 328.8

327.0 346.2 330.3

23.8 (12.9) 1.2 (5.3) 17.5 (13.6)

9.4 3.9 -9.9

Myricetin 30 -OH 40 -OH 50 -OH 3-OH 5-OH 7-OH

323.2 (298.4) 312.5 (292.6) 324.2 (298.6) 334.0 (299.8) 338.3 (302.3) 323.3 (296.6)

326.5 314.8 326.9 334.8 340.9 327.3

– – – – – –

21.9 32.6 20.9 11.1 6.8 21.8

(11.6) (17.4) (11.4) (10.2) (7.7) (13.4)

8.5 12.7 8.3 7.4 5.6 9.8

Quercetin 30 -OH 40 -OH 3-OH 5-OH 7-OH

323.3 (298.3) 316.5 (299.9) 333.1 (299.8) 337.4 (302.3) 322.5 (296.6)

326.6 319.4 337.0 343.5 328.2

339.8 321.8 336.3 340.8 327.8

21.8 28.6 12.0 7.7 22.6

(11.7) (10.1) (10.2) (7.7) (13.4)

8.5 7.4 7.4 5.6 .8

0.0

4.6 4.5 5.9

38. 42.

During the Fenton reaction, hydroxyl radicals are produced from hydrogen peroxide in the presence of a metal in a low oxidation state:

H2 O2 þ Mnþ ! HO þ HO þ Mðnþ1Þþ This reaction may occur in biological areas where accumulation of H2O2 is important, for example in the dopaminergic neurons of nervous tissue. Here, normal dopamine catabolism produces some levels of hydrogen peroxide (Brown et al., 1998; Palmer & Paulson, 1997; Schulz et al., 2000; van Acker et al., 1996). Polyphenols may offer several chelating sites, such as multiple hydroxyls and carbonyl groups. In flavonoids possessing the 4-carbonyl group and hydroxyls attached to 30 , 40 , 3 and 5, there are three potential chelating sites: the catechol moiety, the 4-keto and the 3-OH, and the 4-keto and the 5-OH groups (see Schemes 2 and 3).

ESI–MS studies (Satterfield & Brodbelt, 2000) have indicated that among 3,7-dihydroxyflavone, 5,7-dihydroxyflavone, luteolin, 7,30 ,40 -trihydroxyflavone, 7,40 -dihydroxyflavone, 7-hydroxyflavone, catechin, and quercetin, flavone possessing no hydroxyl groups does not form stable M(II) complexes. This is presumably due to the lack of a suitable acidic hydrogen. The lowest intensities in the MS spectra are given by catechin, 7,30 ,40 -trihydroxyflavone, 7hydroxyflavone and 7,40 -dihydroxyflavone, each of which lacks a pair of chelating oxygen atoms at the carbon 3 and carbon 4, or carbon 4 and carbon 5 positions. 5,7-dihydroxyflavone, luteolin, and 3,7-dihydroxyflavone, possessing the favourable chelating oxygen atoms at the carbon 3 and carbon 4, or carbon 4 and carbon 5 positions, generated intense signals. These results have highlighted the importance of the carbonyl and hydroxyl chelating pair in metal coordination, and the preference for metal coordination between the deprotonated hydroxyl group at carbon 3 or carbon 5 and the carbonyl group carbon 4.

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Among the different 1:1, 1:2, 2:2, 2:3 stoichiometries that metal:flavonoid complexes can exhibit, the 1:2 is the preferred one (Fernandez, Mira, Florêncio, & Jennings, 2002). Formation of stable complexes between caffeic acid and Al(III) (Cornard & Lapouge, 2006) and Pb(II) (Boilet, Cornard, & Lapouge, 2005) are investigated at B3LYP level of theory, considering as putative chelating site the negative carboxylate and the catechol moiety. The carboxylate group presents the greater power to generate complex with Pb(II) (Boilet et al., 2005). The behaviour of Pb(II) (Boilet et al., 2005) completely differs from that of Al(III) (Cornard & Lapouge, 2006), that preferentially coordinates the catechol group of the caffeic acid. DFT and molecular dynamics calculations (Kazazicä, Butkovicä, Srzicä, & Klasinc, 2006) are performed and the elucidatation of the coordination of Fe+ and Cu+ to some representative flavonoids is given. Apigenin, quercetin, and naringenin indicate a preference for metal complexation at the 4-carbonyl oxygen when there is no substituent in position 3. The difference in energy with the next favourable attachment site of the Cu+ ion (catechol moiety) of 14.2 kcal/mol indicates the C ring as the favourite chelating site (Kazazicä et al., 2006). Lapouge et al. (Lapouge, Dangleterre, & Cornard, 2006) have explored the complexation of Zn(II) by 3-hydroxyflavone, 5-hydroxyflavone and 30 ,40 -dihydroxyflavone, so that indications about the chelating power of the three possible sites 3-OH-carbonyl, 5-OHcarbonyl and catechol are drawn, together with information about the structural modification eventually occurring in the ligand after complexation. All of these ligands lead to the formation of complex of 1:1 stoichiometry. The complexation by 30 -40 -hydroxyflavone does not modify the A and C rings features that remain more or less the same computed for the free ligand, while the ring B geometry undergoes slight modifications in the internal bonds. The fivemembered ring formed with the Zn cation seems to be not coplanar with the ring B (20°). In the case of 5-OH flavone, the coordination of Zn(II) at the C4@O, results in an electronic redistribution mostly on the C ring (Lapouge, Dangleterre, & Cornard, 2006). Contrary to what encountered in the formation of Zn30 40 diOH–flavone, the chelate ring is planar, and the zinc atom is located in the chromone part plane. Similar conclusions are drawn for the Zn-3-OH–flavone complex. The comparison of the formation constants for the three flavones allows the following classification on the basis of the chelating power of the three binding sites: 3-hydroxy-carbonyl > 5-hydroxy-carbonyl > catechol (Lapouge, Dangleterre, & Cornard, 2006). Cornard, Dangleterre, and Lapouge (2005) have investigated theoretically the complexation of Pb(II) by flavonol quercetin. Quercetin offers all the possible chelating sites existing in flavonoids. Among them, the catechol group presents the greater chelating power toward Pb(II). After complexation, the ligand remains totally planar, and the lead ion, coordinated to the catecholate group, is coplanar to the plane of quercetin. The behaviour of Pb(II) (Cornard et al., 2005) completely differs from that of Al(III) one (Cornard & Merlin, 2002), which preferentially coordinates the 3-hydroxy-chromone part of quercetin. Several quercetin activated forms involved in antioxidant mechanisms have been studied by Fiorucci et al. (2007). They have computed many thermodynamic parameters such as BDE, IP and acidity, and determined electronic and structural features of the complexes between these activated forms and Cu2+ cation. In these species, a significant electron transfer from the flavonol to the metal occurs, especially in the case of deprotonated forms rather than the semiquinone. Complexation of Cu2+ on each of the quercetin activated forms is thermodynamically favoured, as reflected by the values of the binding energies (Fiorucci et al., 2007). The higher values are obtained for the complexation between copper(II) and

301

the 3-OH deprotonated (500.2 kcal/mol) and the 5-OH deprotonated (499.8 kcal/mol) quercetin. In a DF-B3LYP study on the chelation of iron(II) by quercetin (Leopoldini, Russo, Chiodo, & Toscano, 2006), the complexes with both neutral and deprotonated quercetin with bare and hydrated Fe2+ cation, are considered. Among the possible chelates arising from the neutral forms, the global minimum is that in which Fe(II) coordinates to the 4-keto (Fe–O distance is 1.869 Å) and to the 5-OH (Fe–O distance is 2.096 Å) groups. Its equilibrium geometry (1) is reported in the Fig. 3. When deprotonated forms are involved, Fe2+ may attack not only on the deprotonation sites but also on the other positions originating from resonance effects, thus the complexed species are quite numerous (Leopoldini et al., 2006). B3LYP computations indicate as global minimum the adduct in which the cation is coordinated to the carbonyl oxygen on ring C and to the deprotonated 5-OH hydroxyl (coordination bonds are of 1.869 and 1.813 Å, respectively). For both neutral and ionised quercetin, relative energies of the chelates (Leopoldini et al., 2006) indicate a scarce chelating ability of the catechol toward Fe2+ (see Fig. 3). Usually, iron(II) forms in physiological liquids hydrated complexes in which the preferred coordination around the cation is of the octahedral type. So, four water molecules are considered in the coordination sphere of Fe2+ (Leopoldini et al., 2006). Among the complexes of hydrated ion, the global minimum (see Fig. 3) is represented by the adduct in which [Fe(H2O)4]2+ coordinates to the carbonyl and deprotonated 3-OH group (distances are 2.015 and 2.073 Å, respectively). However, this species appears to be almost isoenergetic (DE = 1.8 kcal/mol) with the relative minimum in which the oxygen atoms of the A and C rings are involved. The presence of the water molecules in the coordination sphere of Fe2+ seems to reduce the energy separations between the complexes. NBO analysis suggested that upon interaction of hydrated Fe2+ with quercetin anions, the natural net charge on cation is very similar to that computed for free aquocomplex (1.52 vs 1.62 |e|), so that a ionic bond is present, in this case. This finding differs from that concerning the complexes in which the coordination sphere of cation is lacking in water molecules. However, this is not surprising since the orbital availability to form covalent bonds decreases gradually as coordination number of cation increases. Since experimental findings have indicated the possible existence of chelates between two quercetin molecules and Fe(II), the formation of chelates made up of two deprotonated quercetin molecules with bare and hydrated Fe2+ has been investigated. Considering that the most stable complexes of stoichiometry 1:1 are obtained as the oxygen atoms linked to carbon 3-carbon 4 and carbon 4-carbon 5 pairs are involved, the complexes arising from the interaction with the 3-OH and 5-OH anions of quercetin are taken into account, as well as the ‘‘cis” and ‘‘trans” orientations of the two quercetin anions around the cation (Leopoldini et al., 2006). As far as the interaction with bare Fe2+ is concerned, the global minimum is represented by the complex originated by the interaction with the 5-OH anions in ‘‘cis” position (Fig. 3). The coordination geometry around the cation is found as tetrahedral, involving two carbonyl O12 (2.035 and 2.032 Å) and two hydroxyl O13 (1.924 and 1.925 Å) atoms. Charge on iron ion is 1.41 |e|. This value suggests a charge transfer from ligand to cation. NBO analysis indicated the presence of two covalent bonds between Fe2+ and the oxygen atom of both deprotonated 5-OH groups. Overlap involves the two hybrid s(11.30%)p(88.7%) and s(19.00%)d(81%) orbitals of oxygen and iron, respectively. Computations also show that the ‘‘cis” arrangement of quercetin anions is energetically favoured with respect to the ‘‘trans” one owing to major stability of the tetrahedral coordination that

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Fig. 3. Equilibrium geometries of absolute minima of neutral quercetin–Fe2+ (1), deprotonated quercetin–Fe2+ (2), deprotonated quercetin–[Fe(H2O)4]2+ (3), two deprotonated quercetin–Fe2+ (4) and two deprotonated quercetin–Fe(H2O)2]2+ (5) complexes.

originates from this disposition with respect to the square planar geometry obtainable from the trans one. When the hydrated [Fe(H2O)2]2+ is involved, the most stable complexes originate from the complexes characterised by the planar disposition of ligand atoms (Leopoldini et al., 2006). This can be explained by considering that the final octahedral geometry is more easily reachable starting from planar complexes. In other words, the rearrangement required to pass from a tetrahedral to an octahedral coordination entails a larger energy expense. Thus, in the case of hydrated cation the global minimum (see Fig. 3) is represented by the system in which the iron cation is coordinated by the oxygen atoms attached to C3 and C4 carbon atoms of two 3OH anions in ‘‘cis” position and by two water molecules. The ligands are completely coplanar while the axial H2O molecules are so arranged that a slight deviation from ideal octahedral geometry can be observed. To verify the agreement with the experimental observation (Fernandez et al., 2002) concerning the predominance of the complexes having the 2:1 ligand:metal ion stoichiometry with respect to those with 1:1 one, binding energy (BE) values are computed

(Leopoldini et al., 2006) for the global minima belonging to all different categories of examined systems. Values obtained are 431.9, 517.0, 601.6, and 615.2 kcal/mol, for the ionised quercetin-bare iron, ionised quercetin-hydrated iron, two ionised quercetins-bare iron, and two ionised quercetins-hydrated iron, respectively. Thus, as expected, also from a theoretical point of view, the preference for the 2:1 ratio is confirmed. Both neutral and deprotonated quercetin form with iron(II) stable complexes. Among the 1:1 and 1:2 metal:ligand stoichiometries, the latter is favored as binding energy values confirm. Among the available positions present on neutral or anionic quercetin, oxygen atoms at the 3 and 4, and 5 and 4 carbons, seem to be the favoured coordination sites for iron cation. The behaviour of Fe2+ (Leopoldini et al., 2006) is found similar to that shown by Al(III) (Cornard & Merlin, 2002), but different from that of Pb(II) (Cornard et al., 2005), which preferentially coordinates to the ortho-dihydroxy functionality. The high binding energy values (Leopoldini et al., 2006) indicate that quercetin is a powerful chelating agent that can sequester iron(II) in such a way to prevent its involvement in Fenton reactions.

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7. Conclusions We have presented a brief review concerning the antioxidant capabilities of naturally occurring polyphenolic systems. It summarises the current status of the research on this subject performed by means of quantum chemical methods. The main mechanisms proposed in the literature for the antioxidant action of polyphenols, consisting in the H-atom transfer, the electron transfer and the metals chelation, have been discussed. The OH groups acidity values and the binding energies for the complexation process have been determined in order to better examine the chelation mechanism. All polyphenols investigated are planar systems characterised by an extended conjugation and delocalization of the p-electrons that involve the aromatic ring(s) and the substituents. They give rise to stable radical species upon the removal of an hydrogen atom or an electron; the odd electron appears to be delocalised over the entire molecule thanks to the planar geometry conformation. The stability of radicals is enhanced by the possibility to establish internal H-bonds between the radicalised oxygen atom and vicinal hydroxyl. The most efficient systems that may work through the H-atom transfer mechanism display the ortho-dihydroxy functionality. In these cases, the BDE values are very small with respect to the phenol reference compound because the radicalisation of their hydroxyls generates species stabilized by intramolecular H-bonds other than by resonance effects of the substituents. So, hydroxytyrosol, gallic acid, caffeic acid, epicatechin, quercetin are excellent free radicals scavenger by H atom donation. Within the one electron transfer mechanism, good candidates are those compounds that show planar conformation and wide electronic delocalization, so that their IP values are lower that the reference phenol, as occurring in tocopherol, reseveratrol, quercetin. Most of polyphenols seem to scavange free radicals through the hydrogen atom transfer mechanism since higher energies are involved in the single electron transfer process. However, these conclusions could have a more truly firm basis through computations of the transition state(s) and intermediate(s) for the particular reaction pathways. Results presented here confirm the experimental radical scavenging property of natural polyphenolic compounds based on the ability to scavenge the radical cation chromophore of 2,20 -azinobis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS+) in relation to that of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), an aqueous soluble vitamin E analogue (the Trolox equivalent antioxidant capacity (TEAC) is defined as the concentration of Trolox with the same antioxidant capacity as a 1 mM concentration of the antioxidant under investigation). Thus, flavonols and flavones containing a catechol group in ring B are found to be highly active, with flavonols more potent than the corresponding flavones because of the presence of the 3-hydroxyl group. An additional hydroxyl group in ring B (pyrogallol group) seems to enhance further the antioxidant capacity, as exemplified by myricetin. On the contrary, the presence of only one hydroxyl in ring B diminishes the activity. Anthocyanidins and their glycosides (anthocyanins) are revealed to be equipotent to quercetin and catechin gallates, provided that a catechol structure is present in ring B (like in cyanidin), on the basis of TEAC values (Rice-Evans et al., 1996). As far as the calculated acidity values are concerned, the hydroxyl groups showing the greater acidity are those in para-position to

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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 ring A are the most suitable deprotonation sites because of the better possibility to delocalize the electron pair. In addition, the former site is favoured when H-bonds between adjacent hydroxyls are present. The most acidic polyphenolic compounds are those characterised by a high degree of p-electrons delocalization, for which deprotonation yields to anionic species stabilized by resonances phenomena; their stability is enhanced by the presence of an Hbonding pattern. Polyphenols are able to chelate transition metals through their multiple OH groups and the carbonyl moiety, when present. With regard to the flavonol quercetin, it originates stable complexed species with the Fe2+ cation, both in the neutral and ionised forms, and also with Cu(II). Among the possible chelating sites for iron and copper, the 3-OH/4-keto and the 5-OH/4-keto positions are those showing the greater complexation ability, while the catechol seems to be a poor chelating agent. Binding energies values demonstrate that the preferred iron(II):quercetin stoichiometry is the 1:2 one. Further insights on the working mechanisms of antioxidant systems require the detailed study of their reactions with the biological molecular targets. These studies, that are currently in progress in different research groups, could give new informations not only on the thermodynamical data but also on the kinetic ones. Acknowledgements The University of Calabria, the Food Science and Engineering Interdepartmental Center of University of Calabria and L.I.P.A.C., Calabrian Laboratory of Food Process Engineering (Regione Calabria APQ – Ricerca Scientifica e Innovazione Tecnologica I atto integrativo, Azione 2 laboratori pubblici di ricerca mission oriented interfiliera, and Azione 3 sostegno alla domanda di innovazione nel settore agroalimentare) are gratefully acknowledged. References Adlercreutz, H., Fotsis, T., Bannwart, C., Wahala, K., Makela, T., Brunow, G., et al. (1986). Determination of urinary lignans and phytoestrogen metabolites, potential antiestrogens and anticarcinogens in urine of women on various habitual diets. Journal of Steroid Biochemistry, 25(5B), 791–797. Anders, E., Koch, R., & Freunscht, P. (1993). Optimization and application of lithium parameters for PM3. Journal of Computational Chemistry, 14(11), 1301–1312. Armentrout, P. B., & Kickel, B. L. (1996). Gas-phase thermochemistry of transition metal ligand systems: Reassessment of values and periodic trends. In B. S. Freiser (Ed.), Organometallic ion chemistry (pp. 1–45). Dordrecht: Kluwer. Axelson, M., Sjövall, J., Gustafsson, B. E., & Setchell, K. D. R. (1984). Soya – A dietary source of the non-steroidal oestrogen equal in man and animals. Journal of Endocrinology, 102(1), 49–56. Bakalbassis, E. G., Lithoxoidou, A. T., & Vafiadis, A. P. (2003). Theoretical calculation of accurate absolute and relative gas- and liquid-phase OH bond dissociation enthalpies of 2-mono- and 2,6-disubstituted phenols, using DFT/B3LYP. The Journal of Physical Chemistry A, 107(41), 8594–8606. Barnes, S., D’Alessandro, T., Kirk, M. C., Patel, R. K., Boersma, B. J., & Darley-Usmar, V. M. (2003). The importance of in vivo metabolism of polyphenols and their 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: CRC Press. Barone, V., Cossi, M., Menucci, B., & Tomasi, J. (1997). A new definition of cavities for the computation of solvation free energies by the polarizable continuum model. The Journal of Chemical Physics, 107(8), 3210–3221. Bartzokis, G., Sultzer, D., Cummings, J., Holt, L. E., Hance, D. B., Henderson, V. W., et al. (2000). In vivo evaluation of brain iron in Alzheimer disease using magnetic resonance imaging. Archives of General Psychiatry, 57(1), 47–53. Bauschlicher, C. W., Jr., Ricca, A., Partridge, H., & Langhoff, S. R. (1997). Chemistry by density functional theory. In D. P. Chong (Ed.), Recent advances in density functional methods, part II (pp. 165–227). Singapore: World Scientific Publishing Co.. Becke, A. D. J. (1993). Density-functional thermochemistry 3. The role of exact exchange. Chemical Physics, 98(7), 5648–5652. Beretz, A., Anton, R., & Cazenave, J. (1986). The effect of flavonoids on cyclic

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