Synthesis, electrochemical, spectral, and antioxidant properties of complexes of flavonoids with metal ions

Article abstract of DOI:10.1081/SIM-120023482

Complexes of metal ions with the flavonoids quercetin (H₂L ¹), rutin (H₂L²) and 3-hydroxyflavone (HL ³) have been synthesized. From the data obtained by elemental analyses, molar conductance, atomic absorption, thermal analyses, ¹H NMR, IR, UV-Vis and EPR spectroscopies one can propose the compositions: [Cu₂(L¹)(H₂O)₄]Cl₂ (1), [Cu₃(L²)₂(H₂O)₆]Cl ₂ (2), [Cu(L³)(H₂O)₂]Cl (3), [Fe₂ (L¹)(H₂O)₈]Cl₂ (4), [Fe₃(L²)₂(H₂O)₁₂]Cl ₂ (5) and [Fe(L³)(H₂O)₄]Cl (6). We have used the assumption that the electrochemical oxidation can be used as a model for antioxidant activities for the flavonoids. The cyclic voltammograms show irreversible oxidation processes at +0.58 V and +0.91 V for (1), +0.82 V for (2), +0.89 V for (3), +0.58 V and +0.90 V for (4), +0.78 V for (5) and +0. 86 V for (6) versus Ag/AgCl. These values are lower than those for the free flavonoids indicating a more favorable antioxidant activity. Furthermore, these complexes were also more effective than the uncomplexed flavonoids scavenging superoxide radicals generated by reduction of the oxygen-saturated solution. The superoxide radical scavenging activities of flavonoid complexes seemed to correlate with the redox potentials of the flavonoid.

Full text of DOI:10.1081/SIM-120023482

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Synthesis, Electrochemical, Spectral, and Antioxidant
Properties of Complexes of Flavonoids with Metal Ions
Rubens F. V. de Souza a , Eliana M. Sussuchi a & Wagner F. De Giovani a
a Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto,
Universidade de São Paulo, Av. Bandeirantes 3900, 14040‐901, Ribeirão Preto, SP, Brazil
Version of record first published: 15 Feb 2007.
To cite this article: Rubens F. V. de Souza , Eliana M. Sussuchi & Wagner F. De Giovani (2003): Synthesis, Electrochemical,
Spectral, and Antioxidant Properties of Complexes of Flavonoids with Metal Ions, Synthesis and Reactivity in Inorganic and
Metal-Organic Chemistry, 33:7, 1125-1144
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SYNTHESIS AND REACTIVITY IN INORGANIC AND METAL-ORGANIC CHEMISTRY
Vol. 33, No. 7, pp. 1125–1144, 2003
Synthesis, Electrochemical, Spectral, and
Antioxidant Properties of Complexes of
Flavonoids with Metal Ions
Rubens F. V. de Souza, Eliana M. Sussuchi,
*
and Wagner F. De Giovani
Departamento de Qu´ımica, Faculdade de Filosofia Cieˆncias e
Letras de Ribeira˜o Preto, Universidade de Sa˜o Paulo,
Ribeira˜o Preto, SP, Brazil
ABSTRACT
Complexes of metal ions with the flavonoids quercetin (H2L1), rutin
(H2L2) and 3-hydroxyflavone (HL3) have been synthesized. From the
data obtained by elemental analyses, molar conductance, atomic
1
absorption, thermal analyses, H NMR, IR, UV-Vis and EPR spectro-
scopies one can propose the compositions: [Cu2(L1)(H2O)4]Cl2 (1),
*
Correspondence: Wagner F. De Giovani, Departamento de Qu´ımica, Faculdade
de Filosofia Cieˆncias e Letras de Ribeira˜o Preto, Universidade de Sa˜o Paulo,
Av. Bandeirantes 3900, 14040-901, Ribeira˜o Preto, SP, Brazil; Fax: 55-16-633-8151;
E-mail: wfdgiova@usp.br.
1125
DOI: 10.1081/SIM-120023482
0094-5714 (Print); 1532-2440 (Online)
www.dekker.com
Copyright D 2003 by Marcel Dekker, Inc.

1126
de Souza, Sussuchi, and De Giovani
[Cu3(L2)2(H2O)6]Cl2 (2), [Cu(L3)(H2O)2]Cl (3), [Fe2(L1)(H2O)8]Cl2 (4),
[Fe3(L2)2(H2O)12]Cl2 (5) and [Fe(L3)(H2O)4]Cl (6). We have used the
assumption that the electrochemical oxidation can be used as a model for
antioxidant activities for the flavonoids. The cyclic voltammograms
show irreversible oxidation processes at + 0.58 V and + 0.91 V for (1),
+ 0.82 V for (2), + 0.89 V for (3), + 0.58 V and + 0.90 V for (4), + 0.78
V for (5) and + 0.86 V for (6) versus Ag/AgCl. These values are lower
than those for the free flavonoids indicating a more favorable antioxidant
activity. Furthermore, these complexes were also more effective than the
uncomplexed flavonoids scavenging superoxide radicals generated by
reduction of the oxygen-saturated solution. The superoxide radical scav-
enging activities of flavonoid complexes seemed to correlate with the
redox potentials of the flavonoid.
Key Words: Flavonoids; Flavonoid complexes; Quercetin; Rutin;
3-Hydroxyflavone; Antioxidants.
INTRODUCTION
Flavonoids constitute a group of natural antioxidant substances which
have been studied intensively due, in part, to their therapeutical properties,
such as antibacterial, anti-inflammatory, antiallergic, antimutagenic, anti-
viral and anticancer.[1–3] The capacity of flavonoids to act as antioxidants
depends on their molecular structure (Figure 1). It has been demonstrated
Figure 1. Common structure of the flavone subclass and the division of bands I and
II related to the UV-Vis absorption bands.

Complexes of Flavonoids with Metal Ions
1127
that the flavonoids can coordinate to transition metal ions like Zn(II) and
Al(III).[4–6] In this work we report the synthesis, characterization and
antioxidant activities of quercetin (H2L1), rutin (H2L2) and 3-hydroxy-
flavone (HL3) complexed with Cu(II) and Fe(II) ions. The antioxidant
activities of the compounds were evaluated by their oxidation potentials
assuming that both the electrochemical oxidation (Fl–OH ! Fl–OÁ +
e– + H + ) and the antioxidant activity (Fl–OH !Fl–OÁ + HÁ) involve
the breaking of the same O–H bond;[7] the smaller the oxidation potential
the bigger will be the antioxidant activity of the compound. This suggests
that the electrochemical properties of the flavonoids may be associated with
their biological activities.
EXPERIMENTAL
Materials
The compounds CuCl2Á2H2O, FeCl2Á4H2O, quercetin, rutin and 3-
hydroxyflavone were purchased from Aldrich Chemical Co. and were used
as received. Other chemicals, from different manufacturers, were of AR
grade and were purified by the usual procedures.[8]
Instrumentation and Measurement
Elemental analyses were performed using a CHNS-O CE Instruments
model EA 1110 elemental analyzer. Copper and iron were determined by
a model V-85 B. Braun ICP-AES-atomic emission spectrometer. Molar
conductances, in MeOH, were determined in a DSS-11A Metrohm con-
ductivity meter. The 1H NMR spectra in DMSO-d6 were obtained on a
RMN Bruker DR X 400 9.4T spectrometer. EPR spectra at room
temperature were recorded in the solid state on a K-band Bruker EP 200D
spectrometer, with a microwave frequency of 24.06 GHz and a modulation
frequency of 51 KHz. The IR spectra were recorded on a Nicolet FTIR
5ZDX spectrometer using KBr pellets. UV-Vis. spectra in MeOH were
registered on a Hewlett-Packard 8453 Diode Array UV-Vis spectropho-
tometer using standard 1.00 cm quartz cells. The e values represent the
average obtained from three different solutions. Thermogravimetric ana-
lysis measurements were performed by a TA Instruments SDT 2960-
Simultaneous DTA-TGA-Thermal Analysis, between room temperature
and 800 °C at a heating rate of 10 °C min–1. The cyclic voltammograms
were obtained on a 273A EG&G Potentiostat/Galvanostat, using a one-
compartment electrochemical cell equipped with a glassy carbon working

1128
de Souza, Sussuchi, and De Giovani
electrode, a platinum wire auxiliary electrode and a Ag/AgCl reference
electrode; the voltammograms were recorded in MeOH (1 Â 10–3 molar
solutions) with LiClO4 as supporting electrolyte. Electrolysis were per-
formed at fixed applied potentials, at 25
plate working electrode.
1 °C, using a glassy carbon
Stoichiometric Ratios of the Complexes
Job’s method[9] was used to determinate in methanol the stoichiometric
ratio for the reaction between the flavonoid and the metal ion. The solutions
were prepared by mixing solutions of both components of equal molar
concentration (1.1 Â 10–4 molar) in ratios varying from 1:9 to 9:1.
Absorbances were measured at 413 nm.
Superoxide Anion Assay
The superoxide ion was generated by reduction of the oxygen-saturated
solution and its concentration was determined by the anodic current in the
cyclic voltammogram. Knowing that an oxygen-saturated solution in
DMSO is 2.1 Â 10–3 molar, we have assumed this superoxide concentra-
tion at the surface of the electrode after the reduction process. This
assumption is based on the fact that the cathodic peak current for oxygen
reduction has the same value as the anodic peak current for superoxide
oxidation in the cyclic voltamogramm of an oxygen-saturated solution.
Preparation of the Metal Complexes
The complexes were prepared by mixing, in methanol, stoichiometric
amounts of the flavonoid and metal ion, to reach the M:L molar ratio,
previously determined by Job’s method.
Quercetin Complexes. 0.3383 g (1 mmol) of quercetin; 0.3410 g (2
mmol) of CuCl2Á2H2O or 0.3976 g (2 mmol) of FeCl2Á4H2O; 100 mL of
methanol; reflux 2 h.
Rutin Complexes. 0.6105 g (1 mmol) of rutin; 0.2557 g (1.5 mmol) of
CuCl2Á2H2O or 0.2982 g (1.5 mmol) of FeCl2Á4H2O; 200 mL of methanol;
reflux 5 h.
3-Hydroxyflavone Complexes. 0.2382 g (1 mmol) of 3-hydroxyflavone;
0.1705 g (1 mmol) of CuCl2Á2H2O or 0.1988 g (1 mmol) of FeCl2Á4H2O;
70 mL of methanol; reflux 2 h.
In all preparations, pale yellow precipitates were formed immediately
when the cooled mixtures were poured into H2O (500 mL). The crystals

Complexes of Flavonoids with Metal Ions
1129
were washed thrice with 1:3 EtOH/H2O mixtures and then several times
with H2O and dried under vacuum.
RESULTS AND DISCUSSION
The syntheses of the complexes can be represented by the equations:
H2L1 þ 2MCl2ÁxH2O ! ½M2ðL1ÞðH2OÞ2xŠCl2 þ 2HCl
2H2L2 þ 3MCl2ÁxH2O ! ½M3ðL2Þ2ðH2OÞ3xŠCl2 þ 4HCl
HL3 þ MCl2ÁxH2O ! ½MðL3ÞðH2OÞxŠCl þ HCl
where: M = Cu(II), x = 2; M = Fe(II), x = 4.
The solid amorphous complexes are pale yellow, stable in the air and
can be stored without change of composition. They are soluble in MeOH,
EtOH and DMSO, slightly soluble in Me2CO, but scarcely soluble in H2O
and CCl4. Table 1 shows the analytical data for the complexes. The molar
conductance data in MeOH for the complexes are given in Table 2.
Stoichiometric Ratios of the Complexes
In pure methanol, results clearly show the three possible complex-
ing sites: 3’–4’, 3–4 and 7-OH groups, in Figure 1. Figure 2 shows the
absorption spectra of 3-hydroxyflavone, in methanolic solutions, with
different concentrations of CuCl2Á2H2O. The disappearance of the band
at 343 nm, associated with the appearance of a band at 413 nm as the
mole ratio is increased, is consistent with the formation of a new
species in the system. The maximum of Job’s curve (Figure 3) appears
at the 0.5 mole fraction indicating 1:1 stoichiometry for the reaction of
Cu(II) and 3-hydroxyflavone. Table 3 summarizes the stoichiometries of
the complexes.
1H NMR of the Fe(II)-Flavonoid Complexes
The 1H NMR spectrum of the Fe(II)-complexes has fully resolved
spin–spin couplings and lies completely within the diamagnetic region,

1130
de Souza, Sussuchi, and De Giovani

Complexes of Flavonoids with Metal Ions
1131
Table 2. Molar conductance data in MeOH for the complexes.
Complex [1 Â 10–3 molar]
LM (O–1 cm2 mol–1)
Electrolyte type[10]
[Cu2(L1)(H2O)4]Cl2
[Fe2(L1)(H2O)8]Cl2
[Cu3(L2)2(H2O)6]Cl2
[Fe3(L2)2(H2O)12]Cl2
[Cu(L3)(H2O)2]Cl
[Fe(L3)(H2O)4]Cl
204.21
194.13
208.85
191.56
102.48
99.25
1:2
1:2
1:2
1:2
1:1
1:1
1
indicating a low-spin Fe(II) center. The H NMR data for the complexes, as
well as those for the free ligands are listed below.
Quercetin (H2L1): d 12.50 (s, 1H, 5-OH), 10.76 (s, 1H, 7-OH), 9.60 (s,
1H, 3-OH), 9.39 (s, 1H, 4’-OH), 9.31 (s, 1H, 3’-OH), 7.68 (d, JH2’/H6’ = 1.8
Hz, 1H, 2’-H), 7.55 (dd, 1H, 6’-H), 6.89 (d, JH5’/H6’ = 8.5 Hz, 1H, 5’-H),
6.43 (d, JH8/H6 = 1.5 Hz, 1H, 8-H), 6.22 (d, 1H, 6-H).
[Fe2(L1)(H2O)4]Cl2: d 12.48 (s, 1H, 5-OH), 10.74 (s, 1H, 7-OH), 9.27
(s, 4’-OH), 9.21 (s, 1H, 3’-OH), 7.63 (d, JH2’/H6’ = 2.0 Hz, 1H, 2’-H), 7.46
(dd, 1H, 6’-H), 6.81 (d, JH5’/H6’ = 8.2 Hz, 1H, 5’-H), 6.41 (d, JH8/H6 = 1.7
Hz, 1H, 8-H), 6.21 (d, 1H, 6-H).
Rutin (H2L2): d 12.63 (s, 1H, 5-OH), 10.82 (s, 1H, 7-OH), 9.69 (s, 1H,
4’-OH), 9.28 (s, 1H, 3’-OH), 7.49 (d, JH2’/H6’ = 1.9 Hz, 1H, 2’-H), 7.52 (d,
Figure 2. UV-Vis spectra of 3-hydroxyflavone in the presence of Cu(II) ions in
methanol. The molar ratios M/L are indicated for each spectrum.

1132
de Souza, Sussuchi, and De Giovani
Figure 3. Job’s curve: plot of absorbance (at 413 nm) versus mole fraction (X) of 3-
hydroxyflavone.
JH5’/H6’ = 8.8 Hz, 1H, 5’-H), 7.85 (dd, 1H, 6’-H), 6.41 (d, JH8/H6 = 1.5 Hz,
1H, 8-H), 6.24 (d, 1H, 6-H).
[Fe3(L2)2(H2O)12]Cl2: d 12.58 (s, 1H, 5-OH), 9.57 (s, 4’-OH), 9.17 (s,
1H, 3’-OH), 7.43 (d, JH2’/H6’ = 1.6 Hz, 1H, 2’-H), 7.39 (d, JH5’/H6’ = 8.4
Hz, 1H, 5’-H) ,6.79 (dd, 1H, 6’-H), 6.36 (d, JH8/H6 = 1.8 Hz, 1H, 8-H), 6.18
(d, 1H, 6-H).
3-Hydroxyflavone (HL3): d 9.81 (s, 1H, 3-OH), 8.37 (s, 1H, 5-H), 6.82 (d,
JH6/H7 = 8.2 Hz, 1H, 6-H), 7.55 (dd, 1H, 7-H), 7.00 (d, JH8/H7 = 8.4 Hz, 1H,
8-H), 8.30 (m, 2H, 2’, 6’-H), 7.63 (m, 2H, 3’, 5’-H), 7.54 (m, 1H, 4’-H).
[Fe(L3)(H2O)4]Cl: 8.32 (s, 1H, 5-H), 6.77 (d, JH6/H7 = 7.4 Hz, 1H, 6-
H), 7.47 (dd, 1H, 7-H), 6.97 (d, JH8/H7 = 7.5 Hz, 1H, 8-H), 8.28 (m, 2H, 2’,
6’-H), 7.61 (m, 2H, 3’, 5’-H), 7.51 (m, 1H, 4’-H).
Quercetin and rutin are able to chelate metal ions via the phenolic
groups 3’ and 4’. Upon complexation, the metal ion displaces 1 proton from
Table 3. Stoichiometries (M:L) of the complexes of metal
ions with quercetin, rutin, and 3-hydroxyflavone in methanol.
Flavonoids
Cu(II)
Fe(II)
Quercetin
Rutin
3-Hydroxyflavone
2:1
3:2
1:1
2:1
3:2
1:1

Complexes of Flavonoids with Metal Ions
1133
the flavonoid nucleus. In solution, the flavonoids are in a relatively rapid
equilibrium between structures (A) and (B) (Eq. 1).[11] Both structures have
an easily removable H atom, whereas the other H atom is intramolecularly
1
bonded. From the integration of the H NMR signals, it was found that in
each complex, the ratio of structures (C) and (D) is 3:1. The 1H NMR
spectra of the Fe(II) complexes also reveal that the resonances of the
hydrogen of the 3-OH groups are not present in quercetin and 3-
hydroxyflavone complexes and that the hydrogens of the 7-OH groups
are not present in the rutin complexes indicating the loss of the OH protons
due to complexation. The proton signals of the complexes are shifted to
lower frequency relative to the free flavonoids. This result is probably due
to the increase of the conjugation caused by the effect of coordination when
the complex is formed.
Quercetin and rutin deprotonation and complexation of the 3’ and 4’ groups.
IR Spectra
Ligands and complexes IR spectra present evidence of the coordination
between the transition metal ions and flavonoids. The data are summarized
in Table 4. It can be noted that: 1) n(C–O–C) and nring(C=C)
Table 4. IR dataa (cm–1) for ligands and complexes.
Compound
n(M–O) n(C=O) n(C–O–C) n(C=C)
n(O–H)
Quercetin
–
1661 s
1609 s
1606 s
1653 s
1651 s
1649 s
1649 s
1598 s
1596 s
1260 s
1258 s
1256 s
1257 s
1252 s
1254 s
1259 s
1261 s
1260 s
1589 s 3625–3375 b, m
1587 s 3630–2390 b, m
1586 s 3672–2397 b, m
1590 s 3667–3030 b, m
1586 s 3690–2896 b, m
1588 s 3640–2896 b, m
1589 s 3324–2987 b, m
1589 s 3344–3035 b, m
1587 s 3300–2983 b, m
[Cu2(L1)(H2O)4]Cl2
[Fe2(L1)(H2O)8]Cl2
Rutin
608 w
561 w
–
[Cu3(L2)2(H2O)6]Cl2
[Fe3(L2)2(H2O)12]Cl2 559 w
610 w
3-Hydroxyflavone
[Cu(L3)(H2O)2]Cl
[Fe(L3)(H2O)4]Cl
–
610 w
562 w
ab = Broad, m = medium, s = strong, w = weak.

1134
de Souza, Sussuchi, and De Giovani
frequencies[12] are slightly shifted upon complexation indicating that the
ring oxygen (position 1, Figure 1) is not involved in the complexation, 2)
the characteristic band of carbonyl groups in the quercetin and 3-
hydroxyflavone complexes appears at 1600 cm–1, while in the free
flavonoids[13] these bands are at 1660–1650 cm–1. The ꢀ 50 cm–1
displacements suggest that the coordination of quercetin and 3-hydroxy-
flavone involves the oxygen atoms of the carbonyl and 3-OH groups, 3) for
the rutin complexes no shifts of the carbonyl bands were observed,
presumably because the 3-OH group of the rutin is blocked by the sugar
moiety (disaccharide glucorhamnoside), 4) n(M–O) frequencies appear in
the 560–610 cm–1 range,[13] 5) n(O–H) frequencies appear as broad bands
(3690–2390 cm–1) indicating the presence of water. The presence of
coordinated water is also supported by thermal analyses.
EPR Spectra
The K-band EPR spectrum of the [Cu2(L1)(H2O)4]Cl2 complex is
shown in Figure 4. The g tensor values, presented in Table 5, can be used
to derive the ground state. The g > g > 2.0023 values and the g /A
k
?
k
k
ratio suggest square-planar geometry of the complexes. Square-planar
complexes have a g /A quotient[14] ranging from 113 to 150 cm; this ratio
k
k
increases with the increase of tetrahedral distortion. This geometry is also
supported by the fact that the unpaired electron lies predominantly in the
2
dx2 À y2 orbital with B1g as the ground state (see discussion of UV-Vis
Figure 4. The K-band EPR spectrum of [Cu2(L1)(H2O)4]Cl2.

Complexes of Flavonoids with Metal Ions
Table 5. EPR spectral properties of Cu(II) complexes.
1135
A
A
Aiso
k
?
(10–4
cm–1)
(10–4
cm–1)
g /A
(10–4
cm–1)
k
(cm)
k
Complex
[Cu2(L1)-
g
g
giso
a2
k
?
2.27 2.06
2.24 2.05
2.25 2.10
163
168
157
72
76
71
139
2.13
102
107
100
0.81
(H2O)8]Cl2
[Cu3(L2)2-
(H2O)12]Cl2
[Cu(L3)-
133
143
2.11
2.15
0.76
0.66
(H2O)4]Cl
spectra). The molecular coefficients a2 were calculated by using the
following equation.[15]
a2 ¼ ÀðAk=0:036Þ þ ðgk À 2:0023Þ þ 3=7ðg? À 2:0023Þ þ 0:04
The a2 values (Table 5) indicate the fairly covalent nature of the metal-
ligand bonding. No EPR signals appeared for the iron complexes at room
temperature as expected for low-spin Fe(II) complexes.
UV-Vis Spectra
The flavonoids exhibit two major absorption bands in the ultraviolet/
visible region (Figure 1); The absorptions in the 320–385 nm range
Table 6. UV-Vis data of the ligands and complexes.
lmax (nm), (e in dm3 mol–1 cm–1)
Band I
(p ! p*)
Band II
(p ! p*)
Compound [50 Â 10–6 molar]
d–d
Quercetin
372 (14414)
451 (11054)
430 (21794)
358 (17700)
354 (18760)
365 (10200)
343 (22410)
418 (7168)
400 (29950)
256 (13706)
268 (31036)
268 (31394)
259 (21400)
267 (27472)
265 (14400)
238 (26512)
248 (23160)
246 (42256)
[Cu2(L1)(H2O)4]Cl2
[Fe2(L1)(H2O)8]Cl2
Rutin
590 (147)
605 (115)
[Cu3(L2)2(H2O)6]Cl2
[Fe3(L2)2(H2O)12]Cl2
3-Hydroxyflavone
[Cu(L3)(H2O)2]Cl
[Fe(L3)(H2O)4]Cl
598 (128)
602 (102)
590 (178)
601 (118)

1136
de Souza, Sussuchi, and De Giovani

Complexes of Flavonoids with Metal Ions
1137
correspond to the B ring portion (cinnamoyl system, band I), and the
absorptions in the 240–280 nm range correspond to the A ring portion
(benzoyl system, band II).[16,17] The spectra are related to the p! p*
transitions in the ligand molecules. These bands are shifted to higher wave
length in the spectra of the complexes as shown in Table 6. The d–d bands
around 590 nm (e ꢀ 150 M–1 cm–1) of the Cu(II) complexes can be
2
2
2
2
assigned to B1g ! A1g and B1g ! Eg transitions as observed for copper
compounds having square-planar geometry;[18] the band at ꢀ 605 nm
(e ꢀ 115 M–1 cm–1) in the Fe(II) complexes can be attributed to
1
1A1g ! T1g for a Fe(II) center with S=0.[18] The spectra give significant
information about the structures of the complexes. As the 3-hydroxy group
has a more acidic proton,[19] the 3-OH and 4-oxo groups are the first sites
involved in the complexation process. The 3’,4’-dihydroxy groups bind a
second metal ion. The 5-OH group is not involved due to lesser proton
acidity and the steric hindrance caused by the first complexation.
Thermal Analysis
Thermal data for the dehydration and decomposition of the complexes
are given in Table 7. These data show that all complexes contain coor-
dinated water. The dehydration temperature (120–220 °C) and the
corresponding weight loss indicate the number of water molecules. The
complexes decompose in the 381–589 °C temperature range causing
Figure 5. DTA and TGA diagram of the [Cu2(L1)(H2O)4]Cl2 complex.

1138
de Souza, Sussuchi, and De Giovani
ca. 80% weight loss. The final products are the metal oxides. The
thermogravimetric curve of the [Cu2(L1)(H2O)4]Cl2 complex is shown in
Figure 5. From these sets of data one can propose the complex structures
shown in Figure 6.
Electrochemical Study
The cyclic voltammograms of ligands and complexes show well-
defined oxidation peaks and practically no reverse reduction peaks indi-
cating EC processes involving two electrons.[20,21] The Epa data are shown
in Table 8.
Figure 6. Tentative structures of the complexes: (A) quercetin complexes, (B) rutin
complexes and (C) 3-hydroxyflavone complexes. M = Cu(II) or Fe(II), x = 2 for
Cu(II) and x = 4 for Fe(II).

Complexes of Flavonoids with Metal Ions
Table 8. Electrochemical data of the ligands and complexes.
1139
Compound [1 Â 10–3 molar]
Epa versus Ag/AgCl (V)
Quercetin
+ 0.62 and + 0.93
+ 0.58 and + 0.91
+ 0.58 and + 0.91
+ 1.10
[Cu2(L1)(H2O)4]Cl2
[Fe2(L1)(H2O)8]Cl2
Rutin
[Cu3(L2)2(H2O)6]Cl2
[Fe3(L2)2(H2O)12]Cl2
3-Hydroxyflavone
[Cu(L3)(H2O)2]Cl
[Fe(L3)(H2O)4]Cl
+ 0.82
+ 0.78
+ 0.91
+ 0.89
+ 0.86
Quercetin by itself presents two oxidation processes at +0.62 V and
+0.93 V (Figure 7). Controlled-potential electrolysis of a solution of
quercetin at + 0.75 V shows that residual current is attained when a number
of coulombs equivalent to a two-electron reaction is reached; successively,
under a fixed applied potential of +1.2 V, the second anodic peak disap-
pears when additional number of coulombs corresponding to a two-electron
reaction is reached. These processes are outlined in Figure 8.
Figure 7. Cyclic voltammograms of quercetin (H2L1) and Cu(II) and Fe(II)
complexes (1 Â 10–3 molar); 0.1 molar LiClO4/MeOH; scan rate 0.1 V.s–1
.

1140
de Souza, Sussuchi, and De Giovani
Figure 8. Oxidized structures proposed for quercetin.
Figure 9. Cyclic voltammograms of [Fe2(L1)(H2O)8]Cl2, L1 = quercetin, 1 Â 10–3
molar, in the absence (—) and presence (---) of 1 equivalent superoxide ions; 0.1
.
molar Bu4NClO4/DMSO; scan rate 0.1 V.s–1

Complexes of Flavonoids with Metal Ions
1141
The data in Table 8 show that there was an appreciable difference in
the oxidation potentials of the flavonoids investigated, and that, as
expected, extended conjugation and an increasing number of hydroxyl
groups lowered the oxidation potential. The cyclic voltammetric data also
show a considerable decrease of the oxidation potentials of the
complexes related to that of the free flavonoids. Thus, flavonoid-metal
complexes have been found to be more effective antioxidant than the
free flavonoids.
Superoxide Ion Scavenging
Various low-molecular-weight superoxide dismutase (SOD)-like com-
plexes of iron and copper have also been reported to have SOD mimic
activity.[22,23] The complexation with metal ions decreases the oxidation
potential of the flavonoid/semiquinone redox pair (see Table 8). As a
consequence, the complexed flavonoids are relatively more readily oxi-
dized by superoxide ion than the uncomplexed ones. The interaction be-
tween [Fe2(L1)(H2O)8]Cl2 and 1 equivalent of superoxide ion was studied
by cyclic voltammetry (Figure 9). The absence of the oxidation peaks of
Figure 10. Mechanisms of superoxide scavenging by flavonoid complexes.

1142
de Souza, Sussuchi, and De Giovani
the [Fe2(L1)(H2O)8]Cl2 complex and of the superoxide ion reveals the
ability of these compounds to scavenge radicals. The dismutation of su-
peroxide ion by flavonoid complexes could be explained by the redox
cycling of the metal and the ligand as shown in the Figure 10: the metal
complex can be oxidized to a Fe(II)-semiquinone complex by superoxide
radical, which is oxidized by another superoxide radical to form the
Fe(III)-semiquinone complex.
CONCLUSION
The results obtained in this work show that metal ion-flavonoid
complexes, in the solid state, contain coordinated water. The spectro-
scopic data show the importance of the 3-OH group as coordination site.
The electrochemical data clearly show that the antioxidant activity de-
pends on the number and positions of OH groups in the flavonoid struc-
tures. Metal ions increase the flavonoids antioxidant activities as shown
by the lower complexes oxidation potentials relative to those of the free
flavonoids. In addition, the free radical scavenging activities are main-
tained in the complexes.
ACKNOWLEDGMENTS
The authors thank the Conselho Nacional de Desenvolvimento
Cient´ıfico e Tecnolo´gico (CNPq) and Fundac¸a˜o de Amaparo a Pequisa do
Estado de Sa˜o Paulo (FAPESP) for financial support.
REFERENCES
1. Harbone, J.B.; Williams, C.A. Advances in flavonoid research since
1992. Phytochemistry 2000, 55, 481.
2. Nijveldt, R.J.; van Nood, E.; Van Hoorn, D.E.C.; Boelens, P.G.; van
Norren, K.; van Leeuwen, P.A.M. Flavonoids: a review of probable
mechanisms of action and potentials applications. Am. J. Clin. Nutr.
2001, 74, 418.
3. Pietta, P.-G. Flavonoids as antioxidant. J. Nat. Prod. 2000, 63, 1035.
4. Santos, J.P.; Zaniquelli, M.E.D.; De Giovani, W.F.; Galembeck, S.E.
Aluminum ion complex formation with 3-hydroxyflavone in Langmuir
and Langmuir–Blodgett films. Colloids Surf., A. Physicochem. Eng.
Asp. 2002, 198–200, 569.

Complexes of Flavonoids with Metal Ions
1143
5. Bravo, A.; Anacona, J.R. Metal complexes of the flavonoid quercetin:
antibacterial properties. Transit. Met. Chem. 2001, 26 (1–2), 20.
6. Boudet, A.C.; Cornard, J.P.; Merlin, J.C. Conformational and
spectroscopic investigation of 3-hydroxyflavone-aluminium chelates.
Spectrochim. Acta Part A 2000, 56, 829.
7. van Acker, S.A.B.E.; van den Berg, D.; Tromp, M.N.J.L.; Griffioen,
D.H.; van Bennekom, W.P.; van de Vijgh, W.J.F.; Bast, A. Structure
aspects of antioxidant activity of flavonoids. Free Radic. Biol. Med.
1996, 20 (3), 331.
8. Perrin, D.D.; Armarengo, W.L.F.; Perrin, D.P. Purification of
Laboratory Chemicals, 3rd Ed.; Pergamon Press: New York, 1992.
9. Bulatov, M.I.; Kalinkin, I.P. Practical Manual of Photometric Analysis.
Chemistry, 5th Ed.; Leningrad, 1986.
10. Geary, W.J. The use of conductivity measurements in organic solvents
for the characterisation of coordination compounds. Coord. Chem. Rev.
1971, 7, 81.
11. Barclay, L.R.C.; Edwards, C.E.; Vinqvist, M.R. Media effects on
antioxidant activities of phenols and catechols. J. Am. Chem. Soc.
1999, 121, 6226.
12. Chen, Y.Z. Organic Analysis; Advanced Education Publishers: Beijing,
1983.
13. Nakamoto, K. Coordination compounds. III. In Infrared and Raman
Spectral of Inorganic and Coordination Compounds, 5th Ed.; Wiley
Interscience: New York, 1997.
14. Sakaguchi, U.; Addison, A.W. Spectroscopic and redox studies of some
copper(II) complexes with biomimetic donor atoms - implications for
protein copper centers. J. Chem. Soc. Dalton Trans. 1979, 4, 600.
15. Kivelson, D.; Neiman, R. ESR studies on the bonding in copper
complexes. J. Chem. Phys. 1961, 35 (1), 149.
16. Mabry, T.J.; Markham, K.R.; Thomas, M.B. The Systematic Identifica-
tion of Flavonoids; Springer: New York, 1970.
17. Markham, K.R. Flavones, flavonols and their glycosides. In Methods in
Plant Biochemistry Vol. I, Plant Phenolics; Academic Press: New York,
1989.
18. Lever, A.B.P. Electronic spectra of dn ions. In Inorganic Electronic
Spectroscopy, 2nd Ed.; Elsevier: New York, 1984.
19. Jovanovic, S.V.; Steenken, S.; Tosic, M.; Marjanovic, B.; Simic, M.
Flavonoids as antioxidants. J. Am. Chem. Soc. 1994, 116, 4846.
20. Bard, A.J.; Faulkner, L.R. Electrochemical Methods. Fundamentals and
Applications; Wiley: New York, 2001.
21. Jorgensen, L.V.; Cornett, C.; Justesen, U.; Skibsted, L.H.; Dragsted,
L.O. Two-electron electrochemical oxidation of quercetin and

1144
de Souza, Sussuchi, and De Giovani
kaempferol changes only the flavonoid C-ring. Free Radic. Res. 1998,
29, 339.
22. Moridani, M.Y.; O’Brien, P.J. Iron complexes of deferiprone and
dietary plant catechols as cytoprotective superoxide radical scavengers.
Biochem. Pharmacol. 2001, 62, 1579.
23. Buttafava, A.; Fabrizzi, L.; Perotti, A.; Poggi, A.; Seghi, B. Ring-size
effects on the formation of dicopper(II) and dicopper(III) complexes
with bimacrocycles containing 13- and 14-membered tetraaza subunits.
Inorg. Chem. 1977, 99, 2195.
Received December 27, 2002
Accepted March 17, 2003
Referee I: F. A. Schultz
Referee II: D. G. Tuck