Iron complexes of dietary flavonoids: Combined spectroscopic and mechanistic study of their free radical scavenging activity

Food Chemistry 129 (2011) 1567–1577

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Iron complexes of dietary flavonoids: Combined spectroscopic and mechanistic study of their free radical scavenging activity Jasmina M. Dimitric´ Markovic´ a,⇑, Zoran S. Markovic´ b, Tanja P. Brdaric´ c, Vesna M. Pavelkic´ c, Milka B. Jadranin d a

Faculty of Physical Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia Department of Biochemical and Medical Sciences, State University of Novi Pazar, Vuka Karadzˇic´a bb, Novi Pazar 36300, Serbia Kirilo Savic´ Institute, Vojvode Stepe 51, 11000 Belgrade, Serbia d Institute of Chemistry, Technology and Metallurgy, Njegoševa 12, 11000 Belgrade, Serbia b c

a r t i c l e

i n f o

Article history: Received 15 April 2011 Received in revised form 1 June 2011 Accepted 5 June 2011 Available online 12 June 2011 Keywords: Baicalein Quercetin Iron(III) complex formation Electronic, mass and Raman spectra DPPH test B3LYP/6-31G (d, p) level of theory

a b s t r a c t Combined spectroscopic (UV/visible, Raman, MS) and theoretical approaches were used to assess interaction of iron(III) with quercetin and baicalein in aqueous buffered solutions. Obtained results implicated formation of two iron quercetin complexes, with pH-dependent stoichiometries of 1:2 and 1:1, and one iron baicalein complex with stoichiometry of 1:1. Results of vibrational analysis and theoretical calculations implicated 3-hydroxy-4-carbonyl and the 30 -hydroxy-40 -hydroxy group of catechol as chelating sites for quercetin. For baicalein 5-hydroxy-6-hydroxy group is energetically the most favourable, although 5-hydroxy-4-carbonyl and 6-hydroxy-7-hydroxy chelating sites are energetically similar. Antiradical activity, reaction stoichiometry and number of inactivated DPPH molecules per mole of antioxidant indicated quercetin as a better antioxidant than its iron complex, baicalein and iron baicalein complex. The same structural features appeared to be important both in complexation and antioxidant activity. The equilibrium geometries, optimised using the B3LYP/6-31G (d, p) level of theory, predicted structural modifications between the ligand molecules in free state and in the complex structures. Correlation between experimental and theoretical results was very good. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction The flavonoids are secondary plant metabolites, which belong to the class of polyphenolics. Structurally they are heterocyclic p-electron systems built upon a C6H5(A)C3C6H5(B) flavone skeleton. They are reported to exert a wide range of positive health attributes (prevention of oxidative stress induced by chronic diseases, antimutagenic, antibacterial, anti-inflammatory, antiallergic, antiviral, anti-thrombotic and vasodilatory actions) mainly arising from their antioxidant ability, which operates at different levels in the oxidative processes induced by reactive free radical species (Cook & Samman, 1996; Pietta, 1997; Ren, Qian, Wang, Zhu, & Zhang, 2003; Rice-Evans & Miller, 1996). Due to their unique electron-rich and highly conjugated chemical structure flavonoids generally act as very good hydrogen and electron donors which are very important determinants of antioxidant activity. The extended delocalisation and the lack of suitable sites for attack by molecular oxygen make their radical intermediates relatively stable. The distinct pathways by which flavonoid molecules (ArOH) transfer their charge distinguish several mechanisms of their antioxidant action. ⇑ Corresponding author. Fax: +381 11 2187 133. E-mail address: [email protected] (J.M. Dimitric´ Markovic´). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.06.008

The first one leads to the direct O–H bond breaking and proceeds by rapid donation of the proton and electron to a radical form (ArOH + HO ? ArO + HOH), while the second one assumes indirect H atom abstraction (ArOH + HO ? ArOH+ + OH ? ArO + HOH). Additionally, flavonoids can also chelate potentially toxic transition metal ions (Fe(II), Fe(III), Cu(II). . .) preventing metal-catalysed free radical generation reactions. Among these reactions iron chelation is of particular interest since binding of iron to the flavonoid antioxidants can reduce the accessibility of the iron to oxygen molecules and consequently diminish its high toxicity. Iron chelation can also serve as an effective tool in modulating cellular iron homoeostasis, under physiologically relevant conditions. The significance of iron is enormous because it is an essential element for most life on earth (Beard, Dawson, & Pinero, 1996). In humans after food ingestion significant concentrations of labile iron complexes, with their coordination spheres not completely saturated, can be produced in catabolism of endogenous ligands. When it is ‘‘free’’ iron binds non-specifically to many cellular components, which make it very toxic. In metal-assisted decomposition of hydroperoxides, which are the inevitable products of aerobic metabolism, poorly bound iron species can lead to the catalytic production of hydroxyl radical ðFeðIIÞ þ H2 O2 ! FeðIIIÞ þ OH þ OH Þ, very

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short-lived (half-life of approx. 109 s) and highly reactive species which can damage amino acids, lipids (causing lipid peroxidation), carbohydrates and nucleic acids (mainly causing mutations in DNA or cell death) (Fridovich, 1978; Pryor, 1976). This paper addresses in vitro experimental and theoretical research regarding the iron chelation of quercetin and baicalein (Fig. 1) and their antioxidant activities. Although extensive experimental and theoretical investigations have been performed on these reactions (Bodini, Copia, Tapia, Leighton, & Herrera, 1999; Fernandez, Lurdes, Florencio, & Jennings, 2002; Leopoldini, Pitarch, Russo, & Toscano, 2004; Leopoldini, Russo, Chiodo, & Toscano, 2006; Leopoldini, Russo, & Toscano, 2011; Mira et al., 2002; Yoshino & Murakami, 1998) there are still different perspectives on mechanisms, chelation sites, structures, stoichiometries and stabilities of complexes formed, especially in acidic aqueous solutions. Since structure–activity relationships consistently rank quercetin (Bodini et al., 1999; Hajji, Nkhili, Tomao, & Dangles, 2006) as one of the most powerful antioxidants in the flavonoid class, capable of iron chelation, it is considered once more. Baicalein is one of the major flavonoids contained in dried roots, possessing a multitude of beneficial effects, among which the most striking include the treatment of symptoms, such as insomnia, fever, and copious perspiration (Duen-Suey et al., 2007). 2. Experimental 2.1. Chemicals The following substances were used as received: quercetin, baicalein, sodium chloride, ethanol, methanol, acetic acid, ferric chloride and sodium hydroxide (Merck, Darmstadt, Germany). 2.2. Supporting electrolytes Acetate-buffered solutions of pH 3.0–8.0 were used. The ionic strength was adjusted by sodium chloride (50 mM). The solutions

were obtained by mixing acetic acid (50 mM) and sodium hydroxide (1.5 M). Stock solutions of quercetin and baicalein (5 mM) were prepared in methanol. These solutions were diluted to 50 lM concentration by addition of the buffers. During all measurements, the quercetin and baicalein concentrations were kept constant. Stock solutions of FeCl39H2O (5 mM) were prepared in water. 2.3. Electronic spectra Electronic spectra of free and complexed quercetin and baicalein at different pH values were recorded on a Cintra GB-10 UV–vis spectrophotometer (GBC Scientific Equipment Pty Ltd., Arlington Heights, IL) at room temperature. Reference solutions were acetate-buffered solutions. Each spectroscopic measurement was repeated three times. Quartz cuvettes of 10 mm optical path length were used. 2.4. Mass spectra An attempt to obtain mass spectra of the complexes in buffered solutions gave, after many trials, no meaningful spectra either by positive or negative modes. Instead, the solutions were made as follows: the ferrous chloride was dissolved in water and quercetin and baicalein in methanol. Equal volumes of those solutions were mixed and then diluted, in ration of 1:10, with methanol–water solution (1:1, with 0.1% of acetic acid) leading to equimolar solutions of iron and quercetin (or baicalein), with ligand concentrations around 50 lM. The final pH of these solutions was 3.3. Mass analysis was carried out on a 6210 Time-of-Flight LC/MS system (G1969A, Agilent Technologies, Santa Clara, CA). The mobile phase was a 50:50 mixture of a 0.2% aqueous solution of formic acid and acetonitrile. The samples (20 lL, prepared as described) were introduced via a 1200 Series HPLC system (Agilent Technologies, Waldbronn, Germany) equipped with binary pump, autosampler, column compartment (with zero dead volume cell instead of column) and DAD detector. The flow rate was 0.2 mL min1. The mass spectrometer was run in positive electrospray ionisation (ESI) mode with the mass/charge (m/z) ratio in the range 100–3200. The capillary voltage was 4000 V, gas temperature 350 °C, drying gas flow-rate 12 L min1, nebuliser pressure 45 psig and fragmentor voltage 140 V. Agilent MassHunter Workstation software was used for data acquisition and data processing. 2.5. Raman spectra A Thermo Scientific (Waltham MA) Nicolet Almega XR Raman spectrometer with a fibre optic probe was used for obtaining Raman spectra. A 780 nm laser working at a power of 140 mW was used for sample illumination, enabling a spectral resolution of 2 cm1. The spectra were collected after 30 s of exposure. The number of exposures was six for each spectrum. The buffer spectra were not subtracted from the Raman spectra. The curve-fitting analysis was performed using Peak Fit programme (Version 4). The number of curve fitting components, in a certain wave number region, was found to match, as closely as possible, the number of components in the same interval of the spectra of quercetin and baicalein powders. 2.6. Antiradical test

Fig. 1. Structural formulas of quercetin (a) and baicalein (b).

The antiradical activity was measured by the capacity of pure baicalein, quercetin and their iron complexes (in methanol) to inhibit stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. The reducing activity of the investigated flavones and their iron complexes was monitored through the decrease in DPPH absorbance at 515 nm, as a result of the formation of a colourless radical form.

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Methanol solutions containing different concentrations of quercetin and baicalein (c = 25–200 lM) were added to a methanol solution of DPPH (c = 50 lM). The total volume was 3 mL. The same procedure was repeated using ironquercetin (1:1) and ironbaicalein (1:1) complexes. Solutions were kinetically monitored by HP Agilent 8453 diode array spectrophotometer and a Photophysics stopped-flow instrument equipped with a quartz cell of 1-cm optical path length (Applied Photophysics Ltd., Leatherhead, UK). Each measurement was repeated three times. Antiradical activity is determined as the amount of antioxidant necessary to decrease initial DPPH concentration by 50% (EC50). Antiradical power (ARP) is defined in terms of 1/EC50: the larger the ARP, the more efficient the antioxidant. Reaction stoichiometry was obtained by multiplying the EC50 value of each antioxidant and its complexes by two which gives the theoretical effective concentration of each component needed to reduce 100% of the DPPH. The number of DPPH moles reduced by one mole of antioxidant is obtained as the inverse value of the reaction stoichiometry.

2.7. pH Measurements An Iskra MA 5730 (Kranj, Slovenia) pH meter with a Sentek (Braintree, UK) combined electrode was used for the pH measurements. Standard buffer solutions of pH 4.0 and 7.0 were used for pH meter calibration.

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3. Results and discussion 3.1. Complex formation reactions Flavonoids act as weak polybasic acids and therefore pH plays an important role in their basic structure. The absorption spectrum of quercetin has two bands positioned at 256 and 367 nm (pH 3). By increasing the pH, the bands belonging to the cinnamoyl (B + C ring, 367 nm) and benzoyl (A + B ring, 256 nm) moieties increase in intensity and shift slightly towards longer wavelengths, according to the supposed facilitated deprotonation.On addition of iron(III) to an acetate-buffered solution of pH 4, quercetin long-wavelength band shifts bathochromically (Fig. 2a) (Dk  50 nm), giving simultaneous rise to a new absorption band, which corresponds to the complex formed. The isosbestic point ðkiso pH4 ¼ 393 nmÞ indicates equilibrium between free and complexed quercetin. In neutral and alkaline solutions addition of iron(III) to quercetin also produces a new, bathochromically shifted, band (Dk  35 nm) kiso pH8 ¼ 400 nm, Fig. 2b) also attributed to the complex formed. At higher concentration of iron, both in acidic and alkaline solutions (up to the molar ratio Fe:Q  4), the simultaneous formation of another complex is not observed, like it was in the case of iron(III)quercetin complexes investigated

2.8. Theoretical The stability constant values and the composition of the complexes formed were obtained by molar ratio method (Martell & Hancock, 1996).

2.9. Computational method Calculations were performed with the Gaussian 09 software package (Frisch et al., 2009). The gas phase minimum energy equilibrium geometries of the complexes under investigation were calculated with the B3LYP method and 6-31G (d, p) basis set (Becke, 1993). The geometries thus obtained were verified to be minima on the potential energy surface by a normal mode analysis with no imaginary frequencies obtained. Transitions to the lowest excited singlet electronic states of the complexes were computed by using the TD-B3LYP procedure (Casida, Jamorski, Casida, & Salahub, 1998) with the 6-31G (d, p) basis set. The influence of water as solvent upon the electronic transitions was approximated by the polarised continuum model (PCM; Tomasi & Persico, 1994). UV spectral analysis was performed using Facio 3D (Suenaga, 2005) and ChemCraft 1.5 (Zhurko & Zhurko, 2008).

2.10. Bipyridyl test An indication of the iron(II) oxidation state was its reaction with 2,20 -bipyridyl which produces red-coloured [FeðIIÞ ðbipyÞ2þ 3 ] product. The 2,20 -bipyridyl (0.1 g) was dissolved in 50 mL of water. The metal–ligand solution (0.1 mM), adjusted to pH 3.0–3.5, was left for at least half an hour at room temperature to ensure completion of the complex formation. Five millilitres of this solution were mixed with 1 mL of the 2,20 -bipyridyl solution and the colour change was monitored. A 1 mM solution of pure iron(III) was also tested to ensure that no iron(II) was present in the solutions used for titrations.

Fig. 2. Titration curves of quercetin with iron(II) at pH 4. Inset: complex absorbance at 415 nm versus [Fe]/[Q] concentrations (a); titration curves of quercetin with iron(III) at pH 8. Inset: complex absorbance at 420 nm versus [Fe]/[Q] concentrations (b).

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under different experimental conditions (Bodini et al., 1999; Hajji et al., 2006). The results imply the importance of the experimental conditions, i.e., oxidising agent, pH value and solvent system in the metal chelation. The coexistence of free and bound species present in the system cause the band broadening present both in acidic and alkaline solutions. Insets on Fig. 2a and b, which present the molar ratio plots at 415 nm (pH 4) and 420 nm (pH 8), indicate the formation of 1:2 (ironquercetin) and 1:1 complexes respectively. The 1:2 stoichiometry persists up to pH 5; from pH 6 and higher the complex stoichiometry is 1:1. The reduction of iron(III) to iron(II) in acidic media, consistent with reducing ability of quercetin and the pH domain investigated, was tested by bipyridyl test and the colour change. The spectra of neutral and alkaline solutions, obtained during the later stages of the titration, show characteristics similar to the iron(II)quercetin complexation in acidic buffers, with complex bands positioned close to each other, at 415 and 420 nm. The results are suggesting that iron(III), at least a part of it, is probably reduced to iron(II), even at higher pH values. The partial reduction of iron(III) to iron(II) by quercetin was also indicated by ESI mass measurements which coincide with the published data (Hajji et al., 2006; Mira et al., 2002). This phenomenon is well known for hydroxybenzene compounds in aqueous media. Baicalein (Fig. 1b) undergoes complexation only at pH 6. Addition of iron(III) to baicalein solution produces smaller bathochromic

shift of about Dk  20 nm (Fig. 3a). The molar ratio plot at 356 nm (inset on Fig. 3a) indicates the formation of 1:1 ironbaicalein complex. Unlike some literature results (Perez, Wei, & Guo, 2009), in more acidic and alkaline solutions complexation does not occur (Fig. 3b), implying the impact of the medium. The affinity of specific flavonoid sites towards metal ions depends upon structural features, metal ions and the medium investigated (Bodini et al., 1999; Duen-Suey et al., 2007; Hajji et al., 2006). The spectral characteristics obtained at different pH values could lead to the assumption of potential chelation sites involved. Quercetin is a pentahydroxyflavone with three potential sites for the chelation of cations: the 3-hydroxy-4-carbonyl groups of the C ring, 4-carbonyl-5-hydroxy groups of the C and A rings and catechol moiety on the B ring. The results obtained here implicate the 3-hydroxy-4-carbonyl or 5-hydroxy-4-carbonyl functions in the formation of the ironquercetin complex at pH 4. Catechol function, present in quercetin, is not implied in the complex formation in acidic solutions, since its chelating ability decreases as the pH decreases and because the chelate formation requires both 30 -hydroxy and 40 –hydroxy groups to be dissociated. This is also confirmed by monitoring ironcatechin complexation in acidic buffers (Fig. S1). The catechin spectrum presents no modifications upon titration with iron(III) at these pH values. Since catechin lacks the 4-carbonyl group it is possible to infer that this chelation site, along with the 3-hydroxy group, is involved in complexation of quercetin at lower pH. The assumption of 3-hydroxy group participation in chelation prevails over the assumption of 5-hydroxy group participation because it is known that the presence of free 3-OH group is crucial for complexation, as none of luteolin, flavonol 3-methyl ether or quercetin 3-methyl ether show any spectral change on interaction with ferric iron (Guo et al., 2007). In alkaline media quercetin enters complexation through the catechol group in the ring B. This argument is also implicated by the results of ironcatechin complexation in alkaline buffers. Baicalein is a trihydroxyflavone with three potential sites for chelation: 5-hydroxy-4-carbonyl groups of the C and A rings, 5-hydroxy-6-hydroxy groups and 6-hydroxy-7-hydroxy groups of the A ring. No measurable complexation in more acidic buffers could be the consequence of either a lack of chelation ability or the instability of the complex at pH lower than 6. The reaction of iron(III) and quercetin can be accounted for according to Eq. (1):

mFe3þ þ nH5 Q ¢ ½Fem ðH5y Q Þn 3myn þ nyHþ

ð1Þ

where m and n are the numbers of iron ions and quercetin molecules bonded in the complex structure, and y is the number of H+ ions detached from one quercetin molecule upon complex formation. The corresponding equilibrium constant is:

h

c¼

i Fem ðH5y Q Þn3myn ½Hþ ny

ð2Þ

½Fe3þ m ½H5 Q n

Eq. (2) is transformed into Eq. (3) giving rise to stability constant b:

b¼

c Hþ

h ny ¼

Fem ðH5y Q Þ3myn n

i

½Fe3þ m ½H5 Q n

ð3Þ

Hydrated iron oxides are formed in the presence of both water and oxygen according to equation: Fig. 3. Titration curves of baicalein with iron(II) at pH 6. Inset: complex absorbance at 356 nm versus [Fe]/[B] concentrations (a); titration curves of baicalein with iron(II) at pH 4. Inset: titration curves of baicalein with iron(III) at pH 8 (b).

Fe3þ þ qH2 O ¢ FeðOHÞ3q þ qHþ q The iron hydrolysis constant is:

ð4Þ

J.M. Dimitric´ Markovic´ et al. / Food Chemistry 129 (2011) 1567–1577 Table 1 Relative stability constant values with iron at different pH values of acetate buffered solutions.

* **

Complex

pH

Log b

Log b*

Log b**

Iron–uercetin (1:2) Iron–uercetin (1:1) Iron–aicalein (1:1)

4.0 8.0 6.0

9.56 5.50 4.43

10.70 6.30 /

/ / 6.47,method 1 6.00method 2

Results by Guo et al. (2007). Results by Perez et al. (2009).

h

i

FeðOHÞ3q Hþ q i K¼ h Fe3þ ½H2 Oq

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q ð5Þ

Upon substituting [Fe3+] from Eq. (5) into the Eq. (3), complex stability constant becomes:

b¼

c Hþ

nyp

h

i Fem ðH5y Q Þ3myn ½H2 Oq K n ¼ h iq þ ½H5 Ln FeðOHÞ3q q ½H

ð6Þ

When exact values of iron hydrolysis and ligand dissociation constants are not known, Eq. (6) presents a relative stability constant, valid for certain experimental conditions. Calculated con-

Fig. 4. ESI-mass spectra of iron(II)quercetin complex in methanol–water mixture (pH 3.3). Inset: isotopic pattern for the peak detected at m/z = 658.00.

Fig. 5. Raman spectra of quercetin and iron(II)quercetin complex at pH 4 (a); curve fitting analysis of the 1800–1300 cm1 Raman region of quercetin spectrum (b); curve fitting analysis of the 1800–1300 cm1 Raman region of iron(II)quercetin complex spectrum at pH 4 (c).

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stants for 1:2 and 1:1 ironquercetin complexes are b = 3.6  109 dm6 mol2 and b = 3.2  105 dm3 mol1 respectively and b = 2.7  104 dm3 mol1 for 1:1 iron–baicalein complex (Table 1). Quercetin binds iron more strongly than baicalein (in 1:1 complexes) which is another proof of the importance of so called ‘‘iron-binding motifs’’ (Guo et al., 2007). Values presented in Table 1 are generally smaller, in comparison with the stability constant values of baicalein (Perez et al., 2009) and quercetin (Guo et al., 2007) complexes formed in phosphate buffers, indicating significant impact of the media.

3.2. ESI-mass spectra The ESI-mass spectrum of the iron-quercetin water–methanol (1:1) solution is shown in Fig. 4. The peak at m/z = 303.05 belongs to protonated quercetin, [Q+H]+, while the peak at m/z = 658.00 could be assigned to a 1:2 iron(III)quercetin complex, ½FeðIIIÞþ ðQ  HÞ2 þ . However, the isotopic pattern (inset in Fig. 4) implies  þ a mixture of FeðIIIÞ þ ðQ  HÞ2 ðm=z ¼ 658Þ and ½FeðIIÞ þ Q þ þ ðQ  HÞ ðm=z ¼ 659Þ ions with iron in different oxidation states. The reducing activity of quercetin is consistent with the assumption of the importance of factors related to flavonoid basic structure, the degree of glycosylation and the hydroxylation pattern (Rice-Evans & Miller, 1996). As mentioned before, the same structural features are also important for complexation and autoxidation processes as well. The 1:1 iron-quercetin complex, which is expected to be at m/z = 358.0, is not identified under these experimental conditions, most probably because it is formed in alkaline not acidic media. The ESI-mass spectra of the ironbaicalein

water–methanol (1:1) solution gave no meaningful results and could not prove or disapprove molar ratio method results, most probably because of the pH of the solution.

3.3. Raman spectra of the complexes Much information on structures of complexes has been provided by analysing Raman spectra of the molecules, especially the bands in the 1700–1400 cm1 region, which can be associated with aromatic in-plane skeletal vibrations, the aromatic character of the pyrone ring and the double-bond character of the carbonyl group (Jurasekova, Garcia-Ramos, Domingo, & Sanchez-Cortes, 2006; Jurasekova, Torreggiani, Tamba, Sanchez-Cortes, & Garcia-Ramos, 2009; Teslova et al., 2007; Torreggiani, Jurasekova, Sanchez-Cortes, & Tamba, 2008; Torreggiani, Tamba, Trinchero, & Bonora, 2005a; Torreggiani, Trinchero, Tamba, & Taddei, 2005b; Varsanayi, 1974; Wang et al., 2007). In the Raman spectra of free quercetin at pH 4 (Fig. 5) the band positioned at 1640 cm1 represents the combination of C@O and C2@C3 stretching modes, as a consequence of a decreased double bond character of 4-carbonyl group and aromatic character of the pyrone ring. Most of the bands between 1500 and 1000 cm1 involve CC stretching, OC stretching and in-plane CCH, COH, CCO and CCC bending modes of the rings. The Raman spectrum of ironquercetin complex at pH 4 (Fig. 5a) shows rather obvious changes in number and intensities of the bands. Intense broadening of the bands characteristic both to A and C rings, and even ring B reflects the solvatochromic effect and most probably the autoxidation process present even at pH 4.

Fig. 6. Raman spectra of quercetin and iron(III)quercetin complex at pH 8 (a); curve fitting analysis of the 2100–1000 cm1 Raman region of quercetin spectrum (b); curve fitting analysis of the 2100–1000 cm1 Raman region of iron(III)quercetin complex spectrum at pH 8 (c).

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The massive band assigned to the combination of C@O and C2@C3 stretching modes in the spectrum of quercetin becomes a part of an even broader and more intense band in the spectrum of the complex, which extends over the 2200–1000 cm1 region (Fig. 5a). The indication of the possible quercetin chelation sites can be obtained from the deconvoluted spectra of quercetin and ironquercetin complex (Fig. 5b and c). The Raman bands profiles are described by Voight function. Due to complex formation, the bands at 1639 and 1588 cm1 in the deconvoluted spectrum of quercetin (Fig. 5b) shift to lower wave numbers (1619 and 1560 cm1, Fig. 5c) indicating ring C as one affected by chelation of iron. The changes are also evident in the bands positioned at 1485 cm1 (A bending modes), 1456 cm1 (3,5,7-OH in-plane (IP) bending of the rings A and C) and 1420 cm1 (3,5,7-OH IP bending of the rings A and C, CC stretching of the ring A and CC stretching of the ring B). Theoretical study, by which the chelation on the carbonyl group modifies (increases) the bond length (Table 4), supports the assumption of its involvement in iron binding in acidic media as well. The Raman spectrum of quercetin in buffer pH 8 (Fig. 6) is rather changed comparing to the spectrum at pH 4. Broad, intense bands of low definition imply the superposition of more, closely positioned bands. The Raman spectrum of the complex is also low in definition with two massive, broad and intense bands in the region of C@O and C2@C3 stretching modes, rings A and B normal modes and below 1400 cm1 (Fig. 6a). The Raman bands profiles, in the region 2200–1000 cm1, described by Voight function, also provide good indication of the possible quercetin chelation sites. Comparing the spectra of quercetin and its iron complex (Fig. 6b and c) it is evident that bands assigned to the combination of C@O and C2@C3 stretching modes (1639 and 1558 cm1, Fig. 6b) do not change significantly in position upon complexation (1640 and 1560 cm1, Fig. 6c). The same effect is observed for the bands at 1452 and 1392 cm1 (Fig. 6b), assigned to A and C ring modes, respectively. The shift of the band at 1318 cm1 (Fig. 6b) to 1298 cm1 (Fig. 6c), assigned to the catechol vibrational modes (30 -OH and 40 -OH), supports the assumption of its involvement in iron coordination at higher pH. The implication of the B-ring involvement is also in agreement with smaller bathochromic shift of the complex absorption band in the pH 8 buffer (Fig. 2b) relative to the bathochromic shift of the same band in the pH 4 buffer (Fig. 2a) and with the increased affinity of catechol unit towards iron(III) as pH rises. The assumed involvement of catechol group is also consistent with the fact that B-ring catechol-containing flavonoids have a lower pKa value than the corresponding B-ring phenol-containing flavonoids, making them dissociate more readily

Fig. 7. Raman spectra of baicalein and iron(II)baicalein complex at pH 4.

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in aqueous solutions. General intense broadening of the bands characteristic to both A and C rings normal modes reflects, most probably, the autoxidation process present in alkaline media and the solvatochromic effect as well. Raman spectra of baicalein and ironbaicalein complex at pH 4 (Fig. 7) show no changes in position or intensity of the bands. Accordingly, it is possible to conclude that neither 5-hydroxy-4carbonyl group, nor any other hydroxyl group (C5, C6 or C7) of the molecule, is participating in the complex formation at this particular pH. The Raman spectra of baicalein and ironbaicalein complex at pH 6 are also spectra of low definition with two massive, broad, intense bands in the region of carbonyl and C2@C3 stretching modes and other modes of the rings A and C (Fig. S2). The Raman bands profiles, in the 1800–1100 cm1 region, are fitted by Voight function as well. The bands at 1628 and 1553 cm1 in the deconvoluted spectrum of baicalein (Fig. S2a), attributed to the combination of C@O and C2@C3 stretching modes, do not undergo a significantly high shift (to 1632 and 1551 cm1 respectively), indicating ring C as one not affected by chelation. The shift of the band at 1455 cm1, characteristic of the A ring modes, to 1431 cm1 (Figs. S2a and b), indicates its involvement in iron chelation, which is also confirmed by theoretical study.

Fig. 8. Decay of the visible absorbance (515 nm) of DPPH solution in methanol (50 lM) induced by different quercetin concentrations (25, 50, and 100 lM). Inset: decay of DPPH (50 lM) visible absorbance at 515 nm induced by quercetin and ironquercetin complex (mole ratio 0.5).

Fig. 9. Percent of DPPH remaining as a function of Q/DPPH ratio. Inset: percent of DPPH remaining as a function of (Q–Fe)complex/DPPH mole ratio.

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Table 2 Classification of antiradical efficiencies, stoichiometry and number of reduced DPPH molecules.

*

Compound

EC50

AR power

Stoich. value

Number of reduced DPPH mol.

Quercetin Fe–Q complex* Baicalein Fe–B complex

0.11 1.31 0.15 1.05

9.09 0.76 6.58 0.95

0.220 2.614 0.304 2.10

4.54 0.38 3.29 0.48

Results referring iron–quercetin complex are valid for mole ratios (Fe–Q)complex/DPPH 6 2.

Fig. 10. Geometry optimised structure of the 1:2 iron(II)quercetin complex and the calculated electronic spectra for the supposed complex structure.

3.4. DPPH test An H-transfer reaction from flavonoid antioxidants to DPPH has two distinguished steps: the first one during which the DPPH visible absorbance (kmax = 515 nm measured in methanol) decays usually very quickly (within 1–2 min) and a second step during which the DPPH visible absorbance decays slowly until reaching a plateau. The fast step refers to the abstraction of the most labile H atoms and the second one to the residual activity of the degradation products. Fig. 8 presents DPPH visible absorbance decay after addition of different quercetin concentrations. The transfer of the most labile protons is longer, around 300 s (trace h, Fig. 8), for smaller mole ratios of the components. As the concentration of quercetin rises the period is much shorter, less than 100 s (traces and). The plateau which is reached after 400–450 s, for all quercetin concentrations, indicates that quercetin degradation products have no more

residual H-transfer ability. Ironquercetin complexes do not behave the same across the whole range of mole ratios investigated. For smaller mole ratios decay of the visible absorbance of DPPH reaches a plateau after less than 100 s (inset on Fig. 8 and S3). For mole ratios (FeQ)/DPPH > 2, DPPH visible absorbance is decreasing slowly without reaching saturation even after longer kinetic runs (Fig. S4), indicating prolonged H-transfer, which is probably caused by a more complex mechanism of the reaction. The percentage of the remaining DPPH molecules, calculated at the steady state approximation (Brand-Williams, Cuvelier, & Berset, 1995), against the quercetin/DPPH mole ratio, is presented in Fig. 9. Exponential decay indicates increased quercetin activity for the bigger mole ratios. Ironquercetin complexes (mole ratios (FeQ)complex/DPPH 6 2) show quite opposite behaviour, exponential rise of the percentage of the remaining DPPH in the system (inset in Fig. 9), as a consequence of the decreased quercetin activity when bonded to iron (i.e., increased stability

J.M. Dimitric´ Markovic´ et al. / Food Chemistry 129 (2011) 1567–1577

of the complex formed). Calculations for higher (QFe)complex/ DPPH mole ratios have not been performed, due to steady state not being reached, even after kinetic runs which lasted a couple of hours. Calculated antiradical activity (Brand-Williams et al., 1995) (see experimental section for the explanation), EC50, and antiradical power (Table 2) indicate quercetin as a better antioxidant than its iron complex. This is also confirmed by stoichiometric number and the number of reduced DPPH molecules by one quercetin molecule, which is 4.5. This number could correspond to the number of hydrogen atoms available for the reduction of DPPH. After complex formation, H-donating ability of quercetin reduces significantly (Table 2) emphasising the same structural features, 3-OH, carbonyl and 30 -OH-40 -OH groups, as important both in complexation and antioxidant activity of flavonols. Baicalein shows similar behaviour to quercetin. The transfer of the most labile protons becomes faster as concentration rises (Figs. S5 and S6). The plateau is reached after approximately 300 s, for all baicalein concentrations. The same applies to baicalein complex, i.e. for all (FeB)/DPPH mole ratios investigated, which is opposite to higher (FeQ)/DPPH mole ratios (Fig. S4). The percentage of the remaining DPPH molecules, calculated at the steady state approximation (Brand-Williams et al., 1995), against baicalein/DPPH mole ratios is presented in Fig. S7. Baicalein shows increased activity as the concentration rises while the complex shows decrease in activity, which (Table 2; inset in Fig. S7) is consistent with the lower number of hydrogens available for transfer after complex formation. Compared to quercetin baicalein is a less potent antioxidant. The results are in agreement with the structure–activity relationship of the free radical-scavenging behaviour of flavonoids, which emphasise the importance of the hydroxylation pattern and consequently the increased conjugation and delocalistion of the p electronic system (Rice-Evans & Miller, 1996).

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to 415 nm, is predicted at 412.0 nm. This is essentially a HOMO-3 ? LUMO transition (50%), accompanied with HOMO2 ? LUMO + 1 transition (41%). This band is assigned, mostly, to metal-to-ligand charge transfer (MLCT) transition. The shapes of the corresponding orbitals, shown in Fig. S8, confirm that the transition is associated with significant charge transfer from the metal to the ligand moieties. Theoretically calculated structure of the 1:1 iron(III)quercetin complex is in better agreement with the obtained experimental results compared to the theoretically calculated iron(II)quercetin complex structure. Formation of the 1:1 iron(III)quercetin complex proceeds with the loss of both catechol group protons, and the destruction of the hydrogen bonds (Table 4). Coordination of iron(III) slightly changes the structure of the quercetin ring B. The bonds C30 – O30 and C40 – O40 become shorter, while only C30 –C40 bond becomes longer in comparison to the isolated quercetin molecule. Since the 1:1 complex is formed in alkaline medium, another OH group from solution is bonded to iron to fulfil its coordination (Fig. S9). The complex is not planar (Table 3) with the dihedral angle between rings B and C of about 9°. There is generally a satisfactory agreement between the experimentally obtained and theoretically predicted wavelengths and intensities of the absorption maxima. The maximum of the complex band, whose experimental value is close to 420 nm, is predicted with two peaks at 397.4 and 404.7 nm. The first one is a HOMO ? LUMO transition (17%), HOMO-1 ? LUMO (18%) and HOMO ? LUMO+5 transition (40%), while the second peak represents HOMO ? LUMO (25%), HOMO-1 ? LUMO+5 (23%), and HOMO-1 ? LUMO (27%) transitions (Fig. S10). A not completely accurate correlation of the experimental and theoretical spectra is most probably influenced by the fact that in alkaline media iron hydroxyl complexes are participating in complex formation. Another reason may be the fact that the complex formed in alkaline media is most probably, due to iron oxidation change, a mixture of iron(II) and iron(III) 1:1 complex.

3.5. Conformational analysis The main geometrical parameters of quercetin and ironquercetin complexes, obtained after energy minimisation, are presented in Tables 3 and 4. The results show that the molecule of quercetin in its isolated state adopts planar conformation with the h (O1–C2–C10 –C20 ) angle value of 0.0°. The molecular structure of isolated quercetin is stabilised with three intra-molecular hydrogen bonds: O3–H–O4 = 2.005 Å, O5–H–O4 = 1.733 Å, and O30 –H–O40 = 2.125 Å (Table 4) with the first and the second bonds stronger, allowing the formation of a five-member ring coplanar to the chromone part of the molecule, h(C2–C3–O3–H) = 179.9°. Quercetin structure is affected upon complexation and, bearing in mind its bond dissociation enthalpy values (BDE) (Trouillas, Marsal, Siri, Lazzaroni, & Duroux, 2006), it is clear that positions O30 , O40 , and O3 can be reactive sites. Besides destruction of the hydrogen bonds (Table 4) the bonds C3–C4, C4–C10, and C3O3 become shorter, while C2–C3 and C4–O4 become longer, in comparison to the isolated quercetin molecule. The reaction of complexation occurs with the loss of the hydroxyl groups protons. Calculation shows that both iron(II) and iron(III) can form two types of complexes with quercetin, in 1:2 stoichiometry formed via 3-hydroxy-4-carbonyl group, both in cis and trans configuration, and the other in 1:1 stoichiometry, formed via catechol structural unit. The trans form of the 1:2 complex is found to be, by 10.64 kcal mol1, more stable than the cis form. For this reason trans form was used for further investigation. The computed electronic spectra of the 1:2 complex and the optimised complex structure are presented in Fig. 10. The correlation between the observed and predicted wavelengths and intensities is very good. The maximum of the complex band, whose experimental value is close

Fig. 11. Geometry optimised structure of the 1:1 iron(II)baicalein (O5–O6) complex and the calculated electronic spectra for the supposed complex structure.

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Table 3 Bond angles and dihedral angles (in °) calculated using B3LYP/6-31G for quercetin and iron–quercetin complexes.

Table 4 Bond lengths (in Å) calculated using B3LYP/6-31Ga for quercetin and iron–quercetin complexes.

Property

Quercetin

Iron–quercetin (1:2, trans)

Iron–quercetin (1:1)

Property

Quercetin

Iron–quercetin (1:2 trans)

Iron–quercetin (1:1)

A (O1–C2–C3) A (C2–C3–C4) A (C2–C3–O3) A (C3–O3–H3) A (C3–C4–O4) A (C3–C4–C10) A (C4–C10–C9) A (C10–C9–O1) A (C10–C5–O5) A (C5–O5–H5) A (C10–C5–C6) A (C5–C6–C7) A (C6–C7–C8) A (C6–C7–O7) A (C7–C8–C9) A (C8–C9–C10) A (C10 –C20 –C30 ) A (C30 –C40 –C50 ) A (C40 –C50 –C60 ) A (C50 –C60 –C10 ) A (C6’–C1’–C2’) A (C60 –C10 –C2) A (C40 –O40 –H40 ) A (C30 –O30 –H30 ) A (C7–O7–H7) A (C3–O3–Fe)1 A (C4–O4–Fe)1 A (O3–Fe–O4)1 A (O3(1)–Fe–O3(2)) A (O4(1)–Fe–O4(2)) A (O4(1)–Fe–O3(2)) A (O3(1)–Fe–O4(2)) A (O3–Fe–O4)2 A (C3–O3–Fe)2 A (C4–O4–Fe)2 t(O1–C2–C10 –C60 )1 t(O1–C2–C10 –C60 )2

119.0 121.4 123.7 104.0 118.7 117.0 122.0 120.3 119.9 106.7 120.0 119.5 122.0 121.5 117.8 122.2 120.5 119.2 120.6 120.6 118.5 122.2 107.9 110.5 109.9

119.3 119.2 126.0

118.8 121.6 123.6 103.8 118.8 117.0 122.0 120.3 119.9 106.5 119.9 119.5 122.0 121.5 117.8 122.2 120.1 120.4 120.0 121.0 119.0 121.5

D(O1–C2) D(C2–C3) D(C3–C4) D(C4–C10) D(C5–C10) D(C5–C6) D(C6–C7) D(C7–C8) D(C8–C9) D(C9–O1) D(C9–C10) D(C10 –C20 ) D(C20 –C30 ) D(C30 –C40 ) D(C40 –C50 ) D(C50 –C60 ) D(C60 –C10 ) D(C2–C10 ) D(O3–C3) D(O4–C4) D(O5–C5) D(O7–C7) D(O40 –C40 ) D(C30 –O30 ) D(Fe–O3)1 D(Fe–O4)1 D(Fe–O3)2 D(Fe–O4)2 D(Fe–O30 ) D(Fe–O40 ) D(Fe–OH) D(O3–H) D(O5–H) D(O7–H) D(O30 –H) D(O40 –H) DH(O5–H–O4) DH(O3–H–O4) DH(O30 –H–O40 )

1375 1372 1449 1432 1424 1389 1407 1399 1390 1360 1407 1414 1385 1410 1393 1393 1409 1464 1358 1266 1345 1355 1359 1370

1374 1384 1436 1420 1425 1387 1408 1398 1390 1357 1411 1413 1386 1410 1393 1393 1409 1460 1339 1293 1347 1355 1365 1365 1851 1929 1851 1929

1376 1374 1447 1432 1423 1389 1407 1399 1390 1360 1407 1416 1388 1426 1397 1394 1412 1462 1360 1270 1345 0968 1340 1350

180.0

116.5 120.0 117.9 120.1 120.4 107.7 119.9 119.7 121.9 121.4 117.8 122.2 120.7 119.8 120.4 120.4 118.8 121.4 110.7 107.8 110.0 112.3 110.6 85.8 180.0 180.0 94.2 94.2 85.83 112.3 110.6 180.0 180.0

110.0 110.7 110.3 87.00 139.8 133.1 170.4 8857

Calculations show that the molecule of baicalein in its isolated state retains the same planar structure as quercetin with the h (O1–C2–C10 –C20 ) angle value of 0.0° (Table S1). The molecule of baicalein has also three hydrogen bonds that additionally stabilise the molecule: O5–H–O4 = 1.696 Å, O6–H–O5 = 2.281 Å and O7– H–O6 = 2.166 Å. The smallest BDE value for O6H bond indicates this hydroxyl group as a possible reactive site (results accepted for publication). The structure of baicalein is also affected by iron coordination. The structural changes in the rings A and C are manifested through the change of the bond lengths. The bonds C2–C3, C6–C7, O4–C4, and O5–C5 are shorter while C3–C4, C4–C10, C5–C10, C5–C6, C9–O1, and C9–C10 are longer in comparison with the isolated molecule (Table S2). The reaction between iron(II) and baicalein occurs with the loss of one or two protons of hydroxyl groups, and leads to the destruction of the corresponding hydrogen bonds. Optimisation of all three possible complex structures is performed and the transitions to the lowest excited singlet electronic states of optimised complexes are computed. The closest correlation between experimentally obtained and theoretically calculated electronic spectra exists with the complex involving iron(II) ligated to baicalein via O6 and O5 atoms in the ring A (Fig. 11). The other two possible complexes are not in such good correlation. The maximum of the complex band, whose experimental value is close to 356 nm, is predicted with the band at 367 nm (Fig. S10). This band represents a HOMO-2 ? LUMO+1 transition (33%), accompanied with HOMO-2 ? LUMO (25%) and HOMO-1 ? LUMO+3 (14%) transitions. The shapes of the orbitals are shown in Fig. S11.

0980 0993 0968 0967 0971 1.733 2.005 2.125

0985 0968 0970 0968 1.796

1.883 1.900 1.811 0993 0968

1.727 2.003

2.132

4. Conclusion With respect to the structure of the molecules, stoichiometric composition of the complexes formed, experimental spectra and theoretical calculations, it is possible to implicate 3-hydroxy-4carbonyl and catechol structural units as those with chelating power for quercetin, and 5-hydroxy-6-hydroxy group for baicalein. Relative stability constant values calculated for complexation in acetate buffers are smaller in comparison with the stability constant values of quercetin and some other flavonoids in phosphate buffers, indicating the impact of the media. The DPPH test proved much better antioxidant activity for quercetin and baicalein in comparison to their iron complexes. In terms of EC50 values, reaction stoichiometry and the number of deactivated DPPH molecules per mole of the antioxidant, quercetin is a better antioxidant than baicalein. This fact can be rationalised by taking into account the importance of the structural features governing antioxidant behaviour of the flavonoids. The theoretical treatment performed using the B3LYP/6-31G (d, p) level of theory reproduces the experimental results very well. The closest correlation between experimentally obtained and theoretically calculated electronic spectra exists with the quercetin complexes involving iron(II) and iron(III) (in 1:2 and 1:1 complexes, respectively). The closest correlation for baicalein exists with the 1:1 complex involving iron(II). Although the results obtained in vitro can not be simply extrapolated to the conditions in vivo, presented results could generally demonstrate the behaviour of naturally-occurring flavonols under

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