Journal of Molecular Structure 744–747 (2005) 759–766

www.elsevier.com/locate/molstruc

Copper(II)–Quercetin complexes in aqueous solutions:

spectroscopic and kinetic properties
Armida Torreggiania,*, Maurizio Tambaa, Andrea Trincherob, Sergio Bonorab
a
Istituto ISOF (CNR), via P. Gobetti 101, 40129 Bologna, Italy
b
Dip. Biochimica, Università di Bologna, via Belmeloro 8/2, 40126 Bologna, Italy
Received 7 September 2004; revised 30 November 2004; accepted 30 November 2004
Available online 28 January 2005

Abstract
Quercetin (Querc), one of the most common dietary flavonols, was investigated in the presence of Cu(II) ions under basic conditions by
different techniques in order to obtain some elucidation on the mechanism of its beneficial action against free radical-mediated damage.
The spectroscopic studies (UV/Vis, Raman and IR) were useful to assess the relevant interaction of Querc with Cu(II) ions, the chelation
sites and the dependence of the complex structure from the metal/ligand ratio. In the presence of a slight excess of the ligand (0.5 M/L) Querc
acts as a bidentate ligand trough the catechol moiety on B ring. At M/LO1 the metal interaction also involves the CaO group and a close
hydroxyl group. Thermogravimetric analysis confirmed the stoichiometry of the proposed complexes.
Information on the reactivity of the Cu(II) chelates towards oxidizing radicals (%OH, N,3 and BrK,2 ) were obtained by the pulse radiolysis
data. The chelates appear to fast react with these oxidizing radicals, giving rise to intermediates transients, namely resonance-stabilised
phenoxyl radicals.
q 2004 Elsevier B.V. All rights reserved.

Keywords: Quercetin; Cu(II)–quercetin complex; Spectroscopic study; Complex structure; Pulse radiolysis

1. Introduction Since oxidative damage is implicated in most disease and

epidemiological processes, researches on flavonoids and
Flavonoids, also referred to as vitamin P, are a ubiquitous other antioxidants suggest their use in the prevention
group of polyphenolic substances which are present in most and treatment of these [8,9]. Although the antioxidant
plants, concentrating in seeds, fruit skin or peel, bark and activity of the polyhydroxyflavones seems to be primary a
flowers, and account for a significant percentage of function of their ability to act as free radical acceptors [10],
chemical constituents of some, e.g. dried green tea leaves the metal-complexing properties of these molecules may
contain approximately 30% flavonoids by weight [1]. give some contribution to their total antioxidant activity.
Flavonoids have been shown to have antibacterial, anti- Quercetin (Querc) may be considered as one of the most
inflammatory, antiallergic, antimutagenic, antiviral, anti- biologically active and common dietary flavonols. It is
neoplastic, antithrombotic and vasodilator activity [2–4]. present in grapes, grapefruit, onions, berries, green veggies,
The ability of flavonoids in scavenging hydroxyl radicals, and legumes as well as Blue Green Algae, and the average
superoxide anions and lipid peroxy radicals may be one of dietary intake of Querc is 16 mg/day [10–12]. Many of the
the most important functions of these compounds beneficial effects of Querc are related to its antioxidant
and underlies many of the above action in the body [5–7]. properties which may result from its ability in scavenging
free radicals, i.e. peroxyl radicals, and in chelating metal
* Corresponding author. Tel.: C39 051 639 9821; fax: C39 051 639 ions (Fe(II) and Fe(III), Cu(II), etc.) [13–16].
9844. In general, radical scavenging by flavonoids occurs via
E-mail addresses: torreggiani@isof.cnr.it (A. Torreggiani), tamba@ electron donation from the free hydroxyls on the flavonoid
isof.cnr.it (M. Tamba), andrea@chem.unibo.it (A. Trinchero), sergio.
nucleus with the formation of less reactive flavonoid aroxyl
bonora@unibo.it (S. Bonora).

0022-2860/$ - see front matter q 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2004.11.081
760 A. Torreggiani et al. / Journal of Molecular Structure 744–747 (2005) 759–766

radicals [17,18]. The phenolic groups are also responsible of are also some studies that have proposed the catechol
the metal-chelating activity. In fact, it is to be considered moiety as the major site for metal chelating [16,27].
that OK,
2 and H2O2 in the presence of transition metals such The purpose of this study was to better elucidate
as iron and copper may be converted into highly reactive molecular aspects of beneficial effect of Querc; to obtain
%
OH. In vivo most copper is ‘tightly bound’ to the plasma this, the capability of Querc in binding Cu(II) ions and the
protein caeruloplasmin but some is attached to albumin and reactivity of the Cu(II)–Querc system towards oxidizing
to amino acids such as histidine that can catalyse free radical radicals, such as %OH, N,3 and Br,K 2 , was examined under
reactions [19]. Querc may sequester these metal ions by basic conditions by different techniques. The structural
chelation and, preventing the metal-mediated generation of characterisation of Cu(II)–Querc complexes was performed
harmful oxidizing radicals, may protect the potential by vibrational spectroscopy (Raman and IR), whereas the
biological targets from oxidative stress [20,21]. Moreover, reactivity of the Cu(II)–Querc system towards radiation-
the participation of flavonoids and copper(II) in biochemical induced radicals was investigated by pulse radiolysis.
reactions has become of interest in recent years; in fact,
enzymes such as Cu-containing quercetin-2,3 dioxygenase,
occupy a special position among enzymes that catalyse
2. Experimental
redox processes involving flavonoids [22].
Some papers deal with the complex formation of Querc
Quercetin (Querc) ([2-(3,4-dihydroxyphenyl)-3,5,7-tri-
with metals, such as Al(III), Fe(II), Fe(III) and Cu(II), etc.
hydroxy-4H-1-benzopyran-4one]) by Sigma, CuCl2$2H2O
however, data on the composition, structure and complex-
by Fluka, and all other chemicals of Analar grade were used
formation features are sometimes incomplete and contra-
as supplied. All solutions were freshly prepared with triple
dictory. Two complexes of stoichiometry Al(III): Querc of
distilled water before experiments and used immediately.
1:2 and 2:1 in methanol have been showed by UV–Visible Querc was dissolved in triple distilled water and pH of
spectroscopy [23], whereas IR investigation have evidenced the solutions was adjusted by adding NaOH to 10, where the
the formation of a complex 1:1 between Mo(VI) and Querc deprotonation of the OH groups favours the solubility of
[24]. Querc acts as a bidentate ligand towards rare earth Querc. The solutions were saturated immediately prior to
metal (III) and forms a mononuclear complex with one ion irradiation with N2O. The Cu(II)–Querc complexes were
bonded to three ligands (ML3$6H2O) [25]. The formation of obtained by mixing freshly prepared solutions of CuCl2 and
a charge transfer complex between Cu(II) and Querc (mixed Querc at pH 10 in the metal/ligand (M/L) molar ratios 0.5
in the metal ligand ratio of 6:5) has been reported; this and 2.
complex was stable under anaerobic conditions but its decay UV measurements (220–600 nm) were carried out at
was accelerated by the presence of DNA [26]. More room temperature by means of a Perkin Elmer model
recently, spectroscopic studies have indicated that Querc Lambda 40 spectrophotometer. The CuCl2 and Querc (5!
chelates Cu(II) ions; however, on titrating out the bound 10K5 M) solutions were mixed in two M/L ratios (0.5 and 2)
Cu(II) ions, a new spectrum has appeared, indicating that at pH 10.
Querc has not reverted to its original form before Cu(II) Pulse radiolysis experiments were performed with a
interaction [16]. 12 MeV electron linear accelerator (LINAC). Spectral cells
There are three possible metal complexing domains that of 0.5 dm optical pathlength were used throughout. Optical
can interact with metal ions: the 3 0 ,4 0 -dihydroxy group filters were employed to minimise photochemical effects.
located on the B ring and the 3- or 5-hydroxy and 4-carbonyl Dosimetry was based on the initial yield of dithiocyanate
group in the C ring (Fig. 1). Generally, the chelating radical anion, ðSCNÞ,K 2 (GZ0.3 mmol J K1 and 3Z
2
properties of flavonoids towards metal ions have been 710 m mol K1
at 480 nm) obtained in irradiated air-
attributed to the presence of the 3- or 5-hydroxypyran-4one, saturated aqueous solution of 10K1 M KCNS. The signals
rather then the ortho hydroxyl groups in the B ring, but there from the photomultiplier were digitised on a Tektronix
ID620B and analysed by a software running on PC. In
radiolysis of aqueous diluted solution, the primary radicals
formed are solvated electrons ðeK
aq Þ, hydroxyl radicals ( OH)
%

and hydrogen atoms (H ). By saturating solution with N2O,

%

oxidizing conditions will prevail, since essentially all the eK

aq
(R98%) are converted into %OH.
The FT-Raman spectra of the solid samples, obtained by
lyophylization of the Querc and the Cu(II)–Querc solutions,
were recorded by a Bruker IFS 66 spectrometer equipped
with a FRA-106 Raman module and a cooled Ge-diode
detector. The excitation source was a Nd3C-YAG laser
Fig. 1. Chemical structure of Quercetin (Querc). (1064 nm) and the spectral resolution was 4 cmK1.
 A. Torreggiani et al. / Journal of Molecular Structure 744–747 (2005) 759–766 761

The FT-infrared spectra were recorded with a JASCO and in the visible region of the spectrum where free Querc
FT/IR-300E using the KBr-pellet technique. The spectral does not absorb; on the contrary, the spectrum of the Cu(II)–
resolution was 4 cmK1 and the error in wave-number was Querc system with M/L 0.5 showed only minor changes in
about 0.4 cmK1. the same spectral regions (i.e. at 260 nm 5%). These results
Thermogravimetric measurements (from room tempera- suggest that at least two different Cu(II)-containing species
ture to 800 8C) were carried out by means of a Mettler are formed by changing the M/L ratio and the catechol
Toledo TGA/SDTA 851e (heating rate 10 8C/min). structure of Querc is an important chelating site implicated
in both complexes. The latter result agrees well with the
evidences that the catechol-type B ring is the most active
3. Results and discussion antioxidant moiety in flavonoids [30,31].

3.1. Spectroscopic studies 3.1.2. Raman and IR studies

To achieve a better insight into the Cu(II)–Querc
3.1.1. UV/Vis studies interactions, both Querc alone and in the presence of
The UV/Vis spectra of free Querc, CuCl2 and the Cu(II)– Cu(II) (M/L 0.5 and 2) were investigated by Raman and IR
Querc system (M/L 0.5 and 2) at pH 10 are reported in spectroscopy at pH 10. The vibrational spectra of Querc are
Fig. 2. Querc, like most flavones and flavonols, exhibits two relatively complicated because of the presence of the
major absorption bands in the ultraviolet/visible region, complex ring system. Moreover, the presence of an H-bond
Band I in the 300–500 nm range, representing the B ring network broads many bands with a consequent overlapping,
absorption (cinnamoyl system), and Band II at l!280 nm, which strongly decreases the spectra definition. However, a
due to A ring (benzoyl system) [28]. Band I appears at semi-empirical treatment by Cornard et al. has given rise to
pH 10 as two absorptions at 320 and z375 nm because of a good set of band assignments [32]; indeed, the theoretical
the deprotonation of the molecule both in 4 0 and 7 positions calculations are in good accordance with the experimental
(pK1Z6.7 and pK2Z9.0) [29]. data. Tables 1 and 2 report the spectral features of the main
At the considered M/L ratios, the addition of Cu(II) to the vibrational bands of free Querc and the Cu(II)–Querc
Querc solutions gave rise to remarkable intensity decrease system (M/L 0.5 and 2) in the 1700–1000 cmK1 range. In
of Band I (z40%), attributed to the B ring system, this region most bands of free Querc have been attributed to
suggesting that the interaction with Cu(II) at the two the A or B ring stretching; only the strong Raman doublet at
different concentrations occurs at the level of the o-dihy- 1607 and 1597 cmK1 seems to be attributable to both
droxyl phenyl functional group (Fig. 1). When the M/L ratio aromatic rings.
was 2, an intensity increase was also observed both in The first observation of the vibrational spectra indicated
Band II, attributable to the benzoyl system (at 260 nm 10%) that the major structural and electronic changes occur after
the first addition of Cu(II), whereas the increased Cu(II)
concentration induces only minor modifications; indeed,
whereas the Raman spectra of the Cu(II)–Querc system with
Table 1
Wavenumbers (cmK1) and assignments of the main Raman bands of the
Cu(II)–Querc system at different metal/ligand molar ratios and pH 10

Querc Cu(II)/Querc molar ratio Assignments

0.5 2
1649 w 1646 w 1622 w nCaO
1607 s – – n ring (A and B)
1597 s, sh – – n ring (A and B)
– 1577 s 1575 s n ring (A and B)
1547 m – – n ring B
– 1483 w 1478 m, br n ring B, nC4 0 –OK, nC3 0 –OK
1435 m 1429 s 1435 w n ring A, nC7–OH, nC5–OH
1407 m, sh 1414 w, sh – nC9–O1, nC2–C1 0 , nC5–OH,
n ring B
1363 m 1360 w 1358 w, br n ring B, nC2–C1 0 , nC9–C10
1319 m 1318 m 1320 w, br n ring A
1305 m, sh – – n ring B, nC3–C4, nC4–C10
1194 m, br 1194w, br 1192 w n ring A, D (C2 0 –C3 0 –C4 0 ),
Fig. 2. UV/Vis absorption spectra of free Querc (50 mM), the Cu(II)–Querc D (C5 0 –C6 0 –C1 0 )
mixtures at different metal/ligand (M/L) ratio, and CuCl2 (100 mM). The
absorption of free Cu(II), eventually present, has been subtracted from the Assignments: n, stretching; D, skeleton in-plane bending. Intensities: w,
two Cu(II)–Querc spectra (Querc 50 mM–Cu(II) 25 mM; Querc 50 mM– weak; m, medium; s, strong; v, very; sh, shoulder; br, broad. A and B are
Cu(II) 100 mM) in the corresponding concentration (25 and 100 mM). related to the benzenic rings.
762 A. Torreggiani et al. / Journal of Molecular Structure 744–747 (2005) 759–766

Table 2
Wavenumbers (cmK1) and assignments of the main IR bands of the Cu(II)–
Querc system at different metal/ligand molar ratios and pH 10

Querc Cu(II)/Querc molar ratio Assignments

0.5 2
1650 w 1649 w – nCaO
1598 w, sh 1610 1610 n ring (A and B)
vw, br vw, br
1567 m 1565 w 1564 vw n ring B
– 1455 s, br 1456 s, br n ring B, nC4 0 –OH, nC3 0 –OH
1368 m 1358 w, br 1350 w n ring B,, nC2–C1 0 , nC9–C10
1268 w 1265 m 1265 m n ring B, nC3–C4, nC4–C10
1202 m 1196 m 1195 w, br n ring A, D (C2 0 –C3 0 –C4 0 ),
D (C5 0 –C6 0 –C1 0 )
1176 w, sh 1173 w, sh 1169 w, sh n ring B
1111 w 1117 1116 nC3–C4, nC10–C5, nC8–C7
vw, br vw, br

Assignments: n, stretching; D, skeleton in-plane bending. Intensities: w,

weak; m, medium; s, strong; v, very; sh, shoulder; br, broad. A and B are
related to the benzenic rings.

different M/L ratio are rather similar, the comparison of

these spectra with that of Querc show relevant spectral
Fig. 3. Proposed molecular structures of the Cu(II)–Querc complexes
changes, notably in the 1500–1610 cmK1. In fact, with the
existing at pH 10 and M/L ratio 0.5 (a) and 2 (b). The geometry
complex formation, the 1607–1597 cmK1 doublet of free optimalization was performed by using Hyper-Chem v. 4, semi-empirical
Querc became single and shifted to lower wave numbers method ZINDO/1.
(1577 cmK1) at both M/L ratios. This spectral change
indicates a modification occurring on the A or/and B rings the B ring (Fig. 3a). In fact, at pH 10 the ligand is mainly
and suggests that all Querc molecules present in the two present in its di-anionic form and the dissociation of the 4 0 -
systems are affected in some way by the presence of the OH group on the B ring should increase the complexing
Cu(II) ions, since the initial stretching bands are no more ability of this catechol-type ring. This result is in agreement
clearly visible upon the addition of the metal (Table 1). with the literature, where the presence of ortho-3 0 ,4 0 -
Therefore, two molecules of Querc seem to complex one dihydroxy groups in the B ring is considered to be important
Cu(II) ion. for the Cu(II) chelation by flavonoids [16,27].
At the M/L ratio 0.5, significant intensity decreases were By increasing the metal concentration (M/L 2), other
visible in the Raman bands due to vibrational modes of the significant modifications took place in the Raman and IR
B ring (i.e. 1547, 1407 and 1305 cmK1), as expected on the spectra (Tables 1 and 2). In particular, the intensity of some
basis of our UV/Vis results. In addition, a weak band, Raman bands attributed to the A ring vibrations (i.e. 1435
attributable to the stretching of C–OH and/or C–OK groups and 1319 cmK1) [32], sensitively decreased in intensity and
of the catechol moiety [32], appeared at 1483 cmK1 the nCaO band, visible at z1650 cmK1 in the Raman and
(Table 1). On the contrary, the bands mainly attributed to IR spectrum of the free ligand, shifted to lower wave
the A ring, which appear at 1435, 1319, and 1194 cmK1 numbers (in the IR spectrum the nCaO band was
were not affected as a consequence of the complex overlapped with the broad band at 1610 cmK1 and was no
formation. more visible). The latter behaviour can be interpreted as a
Also in the IR spectrum relevant intensity changes were direct evidence of the involvement of the carbonyl group in
visible in the bands mainly attributable to the B ring the metal binding.
vibration modes (i.e. z1565, 1368, 1176 and 827 cmK1) In conclusion, from the vibrational data discussed above,
and a new strong band, attributable to the catechol moiety, Querc appears to coordinate Cu(II) as a bi-dentate ligand
appeared at z1455 cmK1 (Table 2). through the catechol moiety in the presence of a slight
As regards the band attributed to the nCaO mode, it excess of the ligand (0.5 M/L) at pH 10 (Fig. 3a). In contrast,
remained unaffected at z1650 cmK1 in both the Raman and when the M/L ratio is O1, the metal interaction also
IR spectra, supporting the idea that the carbonyl group is not involves the carbonyl function and a close hydroxyl group
involved in the Cu(II) co-ordination under these exper- (Fig. 3b). The most probable chelation site could be between
imental conditions. the 5-hydroxyl and 4-oxo group since a six-membered ring
These data further support the conclusion that, when the can be formed compared with a less stable five-membered
ligand is present in a slight excess (M/LZ0.5), chelation of ring chelate, in agreement with the higher stability of
the Cu(II) ions takes place by the ortho hydroxyl groups in the complex found in case of chelation via 5-OH [13].
 A. Torreggiani et al. / Journal of Molecular Structure 744–747 (2005) 759–766 763

In addition, the dissociation pattern of individual hydroxyl considered the presence of two ClK ions in the two Cu(II)/
groups occurs in the sequence 7-OHO4 0 -OHO5-OH, Querc complex, whereas two NaC ions should be taken into
predicting a more favourable dissociation for the 5-OH account in the other complex (Cu(II)/Querc 0.5), according
than for 3-OH group [33]. However, the precise chelating to the proposed charges of the complexes. In addition, the
site (5-OH or 3-OH) remains questionable since the two obtained chelates exhibited different thermal behaviour.
behaviour of flavonoids presenting only one chelating sites By considering the similarity of the thermograms of the
towards metals such as Al(III) have indicated that in complex with stoichiometry 2:1 and the CuCl2 crystallised
alkaline medium (methanol containing methanoate) the at pH 10 (Fig. 4d and e), the presence of chloride in the
3-hydroxy-4-carbonyl site is preferred to the 5-hydroxy-4- complex was confirmed and could explain the thermal
carbonyl site [23]. stability in the 650–800 8C temperature range. All
considerations were made by assuming that oxides are
mainly present at 800 8C, after the time of isothermal
3.2. Thermogravimetric analysis heating; the decomposition temperatures of CuCl2$2H2O
were about 100 and 630 8C: the first step of the
In order to define the stoichiometry of the Cu(II)- decomposition is a dehydration of the salt and the second
containing species, thermogravimetric analysis of Querc a degradation of the chloride. The thermogravimetric
and the two complexes (M/L 0.5 and 2) was carried out. analysis substantially confirms the hypothesed structures
Fig. 4 shows the thermograms of Querc, Querc and suggested by the spectroscopical data.
CuCl 2$2H2O re-crystallised at pH 10, and the two
considered complexes in the 100–800 8C temperature
range. The last part of the thermograms was obtained with 3.3. Pulse radiolysis
an isothermal heating at 800 8C for 30 min. The decompo-
sition of the partially neutralised molecule in which two In order to monitor both the electron donor ability of the
hydroxyl groups are salified (Querc–Na2), took place at a Cu(II)–Querc complexes, factor linked to the antioxidant
temperature lower than that of the untreated molecule activity of Querc, and the nature of the resulting oxidized
(Fig. 4a and b). The minimal residual mass found at 800 8C transients, the azide radicals ðN,3 Þ were used as
in Querc (about 0.1 mg) was considered not significant and specific electron acceptor. The N,3 radicals were generated
due to the characteristic of the calorimeter. On the other by pulse irradiating N2O-saturated solutions containing a
hand, the residual mass in the Querc–Na2 salt is probably large excess of NaN3 (5x10K2 M) with respect to Querc
due to sodium oxides or chloride. As regard to the (5!10K5–10K4 M) and CuCl2 (M/L 0.5 and 2); pulse
complexes, the binding with Cu(II) appears to stabilise the radiolysis technique was employed to follow the fast
organic ligand; indeed, the decomposition temperature of reactions of the complexes with N,3 and the formation and
the two complexes (Fig. 4c and d) was higher than those of decay of the derived complex radicals.
Querc and Querc–Na2. Similar results have been obtained
for other Cu(II) chelates [34]. By considering the weight
loss observed in Querc, Querc–Na2 and in the two
considered complexes, the stoichiometry of the chelate
species was well confirmed within the experimental error.
It’s worth pointing out that in our calculation we have

At pH 10 the oxidation of the Cu(II)–Querc complexes

by N,3 radical ðE7 ðN,3 =NK 3 ÞZ 1:33 VÞ [35] shows the
formation of similar weak transient spectra characterised
by two bands in the 460–480 and 600–620 nm spectral
region at both M/L ratios (Fig. 5A). These spectra are
considerably different from those obtained in the absence of
Cu(II) ions (Fig. 5B); indeed, the latter showed an
absorption maximum at z520 nm, consistent with the
formation of a mono-anion radical, (OK)Querc(OH)3O%,
due to the direct attack of N,3 towards one deprotonated
hydroxyl group. The strong difference in the intensity of the
transient spectra in the absence and presence of Cu(II) ions
can be explained both by the major absorption of the
mono-anionic radical compared with the phenoxyl radical
Fig. 4. Thermograms of Querc (a), Querc–Na2 (b), the Cu(II)–Querc 0.5 (c) on the complexes and by the very rapid self reaction of N,3
and 2 (d) complexes, and CuCl2$2H2O re-crystallised at pH 10 (e). (kZ9!109 MK1sK1) [36] that makes considerably less
764 A. Torreggiani et al. / Journal of Molecular Structure 744–747 (2005) 759–766

Fig. 5. (A) Transient spectra in pulse radiolysis of N2O-saturated solutions containing 50 mM NaN3, Querc and CuCl2 in two different M/L ratio at pH 10, dose
z18 Gy. (B) Transient spectra in pulse radiolysis of N2O-saturated solutions containing 50 mM NaN3 and 0.1 mM Querc at pH 10, dose z18 Gy.

than complete the scavenging of the azide radicals by the (ZINDO/1), that evidenced the planarity loss of the three
Cu(II)–Querc system. aromatic ring of Querc upon Cu(II) addition (Fig. 3). In
With respect to the Cu(II) chelates, the oxidising attack addition, also catechin, a flavonoid with the same number
of N,3 towards the free phenolic groups, as happen for free and arrangement of hydroxyl group of Querc but without the
Querc, is limited by the Cu(II) chelation that should mainly 2,3 double bound and the 4-oxo function, which are
involve the hydroxyl groups in the 3 0 ,4 0 positions and, in the responsible for electron delocalization from the B ring,
excess of the metal, the 5-OH group. Thus, at the beginning showed a transient spectrum characterized by two absorp-
the electron transfer reaction due to the oxidation by N,3 tions at lz400 and 500 nm after the N,3 radical attack
should mainly involve the aromatic rings of Querc. [37,38].

In fact, the formation of a very weak and broad As far as the reactivity of the Cu(II) chelates is
absorption at lO500 nm, 12 ms after the pulse, may be concerned, both complexes showed to fast react with N,3
attributed to radical-cation species, known to generally (4.5!109 MK1sK1), although oxidation by an electrophilic
absorb in this spectral region (Fig. 5A). These short-lived agent like N.3 becomes easier when Querc is free and present
radical-cations undergo an unimolecular transformation in its di-anionic form (1.0!1010 MK1 sK1).
reaction (kZ4.0!104 sK1), leading to different radical(s), In order to obtain further information on the structure and
sited on A or B ring and responsible for the transient charge of the Cu(II)–Querc complex, a charged oxidising
absorption spectra developing 140 ms after the pulse radical, such as BrK, 2 , was used. In addition to the redox
property ðE7 ðBrK,2 =2Br ÞZ 1:65 VÞ [35], the reactivity of
K
(Fig. 5A). Also in this case phenoxyl-type radicals may be
hypothesized as responsible for the observed transient this radical with Querc or its metal complexes, should be
spectrum. Probably, the structure and geometry of the influenced by the presence or absence on the above species
Cu(II)-containing complexes may invalidate the extensive of charges of different signs. At pH 10 Querc is doubly
delocalization of the unpaired electron over all three rings of negatively charged and the Cu(II)–Querc complex is
Querc, so modifying the spectral characteristics of the hypothesized, by spectroscopic studies, to exist in two
resulting phenoxyl-type radical. different charged structures depending on M/L ratio: one
This conclusion is in agreement with the geometry structure having two negative charges (M/L 0.5) and another
optimisation of the proposed Cu(II)–Querc complexes, two positive charges (M/L 2). Consequently, the unfavour-
obtained by a semi-empirical method of calculation able electrostatic effect should decrease the reactivity of
 A. Torreggiani et al. / Journal of Molecular Structure 744–747 (2005) 759–766 765

BrK,
2 towards the negative-charged complex as compared the molecular mechanisms by which Querc will certainly
with the other. In fact, from the kinetic analysis of the fast prove beneficial for the cell protection from free radical- and
build-up of the absorption at 520 nm, a decrease in BrK, 2 metal-induced deleterious effects.
reactivity (k) of about a factor of 10 was observed by going
from 2 to 0.5 M/L ratio (k ranging from 2.6!109 to 2.3!
108 MK1sK1, respectively), confirming the different charges
hypothesed for the two Cu(II)-containing structures. Acknowledgements
,
Differently from BrK,2 , N3 radical is uncharged so that
electrostatic effect should be absent, as confirmed by its We thank Prof. G. Fini for the critical reading of
invariable reactivity towards the metal complexes in both the paper and the useful suggestions, Ing. A. Martelli and
M/L ratios. Mr A. Monti for their assistance to the LINAC accelerator,
Also the reaction of hydroxyl radicals (E7(%OH/OHK)Z and Dr M. D’Angelantonio for the thoughtful suggestions in
1.9 V) [35] with Querc in the presence of Cu(II) ions was the geometry optimization of the Cu(II) complexes.
investigated in order to explore the scavenging ability of the
Cu(II)–Querc complexes towards oxidizing radicals. %OH
radicals, produced by metabolic functions, carcinogens or
radiations, are very harmful free radicals since, reacting References
with cellular components, are able to cause a damage that
[1] E. Middleton, C. Kandaswami, in: J.B. Harborne (Ed.), The
can initiate chain of events resulting in disease Flavonoids: Advances in Research Since 1986, Chapman & Hall,
development. London, 1994.
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