Analytica Chimica Acta 538 (2005) 273–281

An electrochemical approach for detecting copper-chelating properties

of flavonoids using disposable pencil graphite electrodes: Possible
implications in copper-mediated illnesses
Mun’delanji Vestergaard ∗ , Kagan Kerman, Eiichi Tamiya
Department of Biological Science and Biotechnology, School of Material Science, Japan Advanced Institute of Science and Technology,
Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan

Received 27 September 2004; received in revised form 26 January 2005; accepted 30 January 2005
Available online 5 March 2005

Abstract

We have studied the electrochemistry of eight flavonoids belonging to four flavonoid sub-classes: flavone, flavonol, flavanol and antho-
cyanidin using pencil graphite electrodes (PGEs). We present the electrochemistry of delphinidin, cyanidin and catechin gallate for the first
time. The use of electrochemical methods in connection with PGE in the study of flavonoids and their interaction with copper ions has not
been previously reported. Our results compare favorably with previously reported studies, which utilised glassy carbon electrodes (GCEs) for
the detection of flavonoids. We calibrated all eight flavonoids (r2 > 0.9620), six of them at at least two peak potentials. The relative standard
deviation (R.S.D.) for peak potential was <5.0% and peak height was <10.0%; thus, this method could be used to characterise and quantify
flavonoid-containing extracts (purified).
An inverse relationship between oxidation potential and metal-chelation was established. Oxidation potential was influenced by the location
of OH groups relative to each other, the oxidation state of the pyranose ring, the presence of a C-4-oxo group and the total number of OH
groups. Further, we showed that the steric configuration of the compound influenced the reactivity. The order of flavonoid reactivity to Cu(II)
ions was myricertin = catechin gallate > quercetin > delphinidin = baicalein > cyanidin > catechin.
These findings may be significant in neuroscience and metal toxicological studies, in which copper ions have been reported to play a crucial
role in initiating and/or promoting the progression of diseases such as Alzheimer’s and Parkinson’s.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Flavonoids; Natural flavonoids; Antioxidants; Metal chelation; Pencil graphite electrodes (PGEs)

1. Introduction Antioxidant is a term generally used for molecules that,

Flavonoids are widely distributed in nature and exhibit
beneficial traits such as anti-inflammatory, anti-cancer and
antioxidant activities [1–3]. The dietary intake of flavonoids
is remarkably high in comparison with other dietary antioxi-
dants such as Vitamins C and E, carotenoids [4] and selenium
[5]. They also chelate highly redox-active metal ions [6,7]
giving them an even stronger protective effect against ox-
idative damage. The most common sources of flavonoids for
humans are green tea, red wine, fruits and vegetables [8–10].
∗ Corresponding author. Tel.: +81 761 51 1660; fax: +81 761 51 1665.
E-mail address: munde@jaist.ac.jp (M. Vestergaard).
at low concentrations relative to the target substrate, can in-
hibit or slow down the oxidation of the substrate. Antioxi-
dants protect cells against the damaging effects of reactive
oxygen species, such as singlet oxygen, superoxide, per-
oxyl radicals, hydroxyl radicals and peroxynitrite by form-
ing an antioxidant–radical complex. The antioxidant–radical
formed after scavenging should be stable enough through
intramolecular H-bonding [11]. A disparity between an-
tioxidants and reactive oxygen species may lead to oxida-
tive stress and subsequent cellular damage. Oxidative stress
has been linked to cancer, atherosclerosis, aging, inflamma-
tion and neurodegenerative diseases such as Parkinson’s and
Alzheimer’s. Flavonoids, along with antioxidant vitamins

0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2005.01.067
274 M. Vestergaard et al. / Analytica Chimica Acta 538 (2005) 273–281
and enzymes, may help protect against these diseases. A re-
port by van Acker et al. [12] suggested that flavonoids could
replace Vitamin E as a chain-breaking antioxidant in liver mi-
crosomal membranes. The antioxidant and metal-chelating
properties of flavonoids are attributed to the presence of aro-
matic OH groups, their location relative to each other, the
oxidation state of the C-ring and the overall number of OH
groups present [6,7].
Several studies have proposed methods for determining
the antioxidant activity [13,14] and metal chelating prop-
erties of flavonoids [6,7]. Few studies have combined the
two [7,15]. Metal chelation studies using UV–vis spectropho-
tometry, based on the shift in wavelength of maximum ab-
sorbance of the flavonoid, coupled with residual metal detec-
tion by fluorescence or/and atomic absorption spectrometry,
might provide information on the relative degree of chela-
tion and enable an inference to be drawn as to the influence
of structure on the extent of chelation; however, they lack
the sensitivity to distinguish similarly-structured compounds.
UV–vis spectrophotometric measurements are also limited in
that they do not perform optimally if analytes precipitate prior
to or during the measurements. Atomic absorption methods
fail to distinguish between metal ions present in isolation or
in soluble complexes.
Electrochemical methods have been used to evaluate the
antioxidant properties (also referred to as antioxidant power)
of flavonoids and other polyphenols [9,10,16]. Detailed elec-
trochemical studies of catechol-containing flavonoids [17],
catechin oxidation mechanisms [18], kinetic analysis of cat-
echin autoxidation [19] and oxidation of quercetin [20] have
been performed, and the relationship of structure to pos-
sible antioxidant properties drawn. Studies on quercetin (a
flavonoid)–DNA interactions in the presence and absence of
Cu(II) have been carried out and have shown pro-oxidant
activities of quercetin in the presence of DNA [21,22]. How-
ever, electrochemical studies of the interactions between
flavonoids and metal ions (significant in oxidative damage)
have not been done. Brett and Disculescu [21,22] touched
on the interaction between quercetin and copper ions in their
investigation of the interaction between quercetin and DNA.
There is a need to establish the electrochemical activity of
flavonoids, proposed in literature as possible therapeutics
[23] and their interaction with very reactive metal ions, also
implicated in many diseases. Our present study investigated
these issues. Further, our method for analysing these inter-
actions employed differential pulse voltammetry (DPV) and
cyclic voltammetry (CV) at disposable pencil graphite elec-
trodes (PGEs). DPV and CV have been used for analysing
flavonoids but mostly at conventional glassy carbon elec-
trodes (GCEs) [10,16,18,20–22]. To the best of our knowl-
edge, this is the first time that disposable PGEs were em-
ployed in antioxidant and/or antioxidant interaction studies,
though they have been used, successfully, in the detection of
nucleic acids and hybridization [24,25]. PGE has a larger ac-
tive electrode surface area and is therefore able to detect low
concentrations and/or volume of the analyte. This might be
significant when only small amounts of analyte are available.
Moreover, disposable PGE significantly reduces total analy-
sis time by by-passing the cleaning and pre-treatment process,
which is essential when using GCEs. Analysis time is also
shortened because samples are analysed without deposition,
at concentrations for which deposition might be required if
GCEs were used [26]. Further, the use of PGE, with its large
active surface area, has enabled us to study the reversibil-
ity/irreversibility of oxidised functional groups and also the
fate of oxidised and later reduced groups over three wave
scans using CV.
The present study has investigated the electrochemical in-
teraction between eight flavonoids (covering four classes of
flavonoids) and the highly reactive copper ions. The infor-
mation may be crucial in understanding the role of flavonoid
structure in scavenging copper-induced oxidative damage.
The findings may have wider implications and possible ap-
plication(s) in neurodegenerative diseases (e.g., Alzheimer’s
and Parkinson’s), in which metal ions such as copper ions
have been implicated in plaque formation, one of the main
causes of the diseases [27–29]. Studies of other copper
toxicity-induced illnesses may also benefit from these find-
ings.
2. Experimental
2.1. Reagents
Flavonoids of >97% purity were purchased: cyani-
din chloride and delphinidin chloride from Extrasynthese
(SSX, Lyon, France); myricertin and catechin gallate from
Sigma–Aldrich Co. (MO, USA); baicalein from Acris An-
tibodies GmbH (Hiddenhausen, Germany); luteolin and
quercetin from Wako Pure Chemical (Tokyo, Japan) and cat-
echin from Calbiochem (CA, USA). A standard solution of
copper nitrate (1000 mg/l in 0.5 mol/l nitric acid) was sup-
plied by Wako Pure Chemical (Tokyo, Japan). All other
reagents were purchased from Wako Pure Chemical (Tokyo,
Japan) and were of analytical grade. Unless otherwise stated,
all solutions were prepared using a 50 mM sodium phosphate
buffer solution (PBS), pH 7.4. Deionised water obtained from
Millipore Milli-Q purification system (Millipore, Bedford,
MA, USA) was used for preparation of PBS and for cleaning
of electrodes.
2.2. Apparatus
Differential pulse voltammetry (DPV) and cyclic voltam-
metry (CV) were performed with an Autolab PGSTAT 12
electrochemical analysis system (Eco Chemie, The Nether-
lands) in connection with its General Purpose Electrochem-
ical System (GPES) software. The three-electrode system
consisted of a pencil graphite electrode (PGE) as the work-
ing electrode, the reference electrode (Ag/AgCl), and a plat-
inum wire as the auxiliary electrode. The pencil leads were
 M. Vestergaard et al. / Analytica Chimica Acta 538 (2005) 273–281
obtained from Tombo Co. Ltd., Japan. All leads had a to-
tal length of 60 mm and a diameter of 0.5 mm. The pencil
leads were used as received. The leads were (about 6 mm)
vertically dipped in the analyte solution giving a geometric
electrode surface area of 9.62 mm2 . PGEs were used only
once and disposed of after each run. The miniaturized ref-
erence electrode with 2 mm i.d. was obtained from Cypress
Systems (KS, USA).
2.3. Procedure
Flavonoid stock solutions (1 mM) were prepared in
methanol and stored in 0.1 ml vials at −20 ◦ C. Just before
analysis the vials were left to equilibrate to room temperature.
Intermediate copper nitrate solution (1 mM) was prepared in
PBS and stored at 4 ± 1 ◦ C until required for analysis.
Complexation of Cu(II) and flavonoids was carried out at
room temperature (RT, 24 ± 1 ◦ C) in PBS. A range of con-
centrations was tested: 0 and 40 ␮M for Cu(II) (prepared
from 1 mM Cu(II) intermediate solution) and 0–600 ␮M for
flavonoid solutions (prepared from the 1 mM stock solutions).
Sample solutions of 1 ml volume were mixed in 1.6 ml vials
for 10 s using an MSI Mini shaker, followed by incubation at
RT for 10 min. All experiments were carried out at RT.
All electrodes were rinsed with Milli-Q water and blot
dried before each individual analysis. Initial voltammograms
were recorded soon after insertion of the electrodes in order
to minimise adsorption of the analyte to the working elec-
trode surface prior to the run. All samples were pre-analysed
using DPV: scan range from −0.5 V to 1.2 V; step potential
5.1 mV; modulation amplitude 25.05 mV and a scan rate of
10 mV/s and without deposition. Once the peak potentials
were established, the scan range was adjusted accordingly
for each analyte. The other parameters remained unchanged.
The parameters for CV were scan rates of 10, 25, 50, 100 and
200 mV/s and a step potential of 2.4 mV. All flavonoids were
monitored with three consecutive scans.
3. Results
3.1. Electrochemistry of flavonoids using cyclic
voltammetry
The numbering system and the four sub-classes of
flavonoids analysed in this study are shown in Scheme 1.
First, we used CV to investigate the presence of re-
ducible groups after each oxidation wave (reversibil-
ity/irreversibility) and whether or not the flavonoids adsorbed
to the electrode surface. In this experiment, all flavonoids
were prepared in 50 mM PBS at 100 ␮M concentration at
RT. Cyclic voltammograms of two of the flavonoids are pre-
sented in Fig. 1. During the first oxidation wave, the same
number and position of peak potentials were observed for
all flavonoids with the exception of the anthocyanidins. For
cyanidin (Fig. 1A) and delphinidin, the third and fourth peak
275
potentials were not detected. During the second and third ox-
idation wave scans, there was a reduction in the current sig-
nals detected at each peak potential for luteolin, myricertin,
quercetin, catechin gallate (Fig. 1B), cyanidin and delphini-
din. The reduction could have been as a result of flavonoids
having adsorbed to the electrode surface and subsequently
blocking the surface after each wave scan. This is consistent
with reported literature [18,20]. Exceptionally, there was an
increase in the current for catechin and baicalein during the
second scan for the first peak potential but it dropped again
during the third scan. This could suggest that the reducible
group during the first wave scan was re-oxidised during the
second scan. The total sum of this re-oxidised group and the
natural flavonoid oxidised at the leftover/available electrode
surface was bigger than the oxidation of the natural flavonoid
group during the first scan. In fact, this result was found to be
entirely plausible since both catechin and baicalein had the
highest reduction signals compared to the other flavonoids
(as a percent of the first scan oxidation signal) at 84% and
44%, respectively.
For all flavonoids, there was a single reduction peak at a
potential slightly lower than the first peak potential. It is log-
ical to conclude that this peak was a result of the reduction
of the product of the oxidised functional group correspond-
ing to the first oxidation peak potential. This phenomenon
has been reported previously [10,17,18,20]. Brett and Ghica
[20] also reported reversibility of the third peak potential.
The behaviour of these reduction peaks, in terms of the cur-
rent detected, was more or less similar among the flavonoids.
Baicalein, luteolin, catechin, myricertin and cyanidin all had
reduction peaks in all three scans and there was a reduc-
tion in current after each scan. Delphinidin and quercetin had
very low reduction signals and we could not deduce any-
thing from the results. The reduction pattern displayed by
catechin gallate was interesting. The highest reduction signal
was manifested during the second wave scan, and the weakest
during the first. The differences in the strength of the signal
might suggest a lag period between oxidation and reduction.
A shorter scan rate might have showed a similar trend to the
rest of the flavonoids. From a sterical point of view, catechin
gallate is a bigger compound than the other flavonoids. This
might have played a role in delaying the oxidation of catechin
gallate.
The effect of scan rate (10, 25, 50, 100 and 200 mV/s)
on response was carried out on four flavonoids (catechin,
baicalein, cyanidin and quercetin) belonging to each of the
flavonoid sub-classes. For all flavonoids, there was at least a
five-fold increase in the current signal with increase in scan
rate from 50 to 200 mV/s. Fig. 2 shows the effect of scan
rate on the current signal of baicalein. The lower current sig-
nal at slower scan rates could indicate a rapid formation of a
non-electro-active film on the electrode surface in agreement
with a suggestion by Janiero and Brett [30]. The faster the
scan rate, the lesser time the film is allowed to deposit on
the electrode surface, and the higher the current signal. The
change in response due to changes in scan rates also suggests
276 M. Vestergaard et al. / Analytica Chimica Acta 538 (2005) 273–281

Scheme 1. The numbering system of flavonoids and chemical structures of four sub-classes of flavonoids used in the present study.

that the electrode response was controlled by semi-finite dif-
fusion. As the scan rate was increased, there was less time
for the diffusion layer to extend into the bulk of the solution,
causing a large diffusion gradient and consequently, a higher
peak current [31].
3.2. Electrochemistry of flavonoids using differential
pulse voltammetry
We investigated the following flavonoid characteristics:
(i) the peak potential, (ii) the number and position of peak
potentials, and (iii) the current heights at each peak potential
for all eight flavonoids. The significance of these parameters
will be discussed in more detail in the following paragraphs.
All flavonoids were analysed at 40 ␮M concentrations pre-
pared in 50 mM PBS at RT using DPV with PGE. The number
and position of peak potentials and their corresponding cur-
rents are displayed in Table 1. Fig. 3 shows voltammograms
of three of the flavonoids whose electrochemistry is reported
for the first time.
All flavonoids had at least two peak potentials. With the ex-
ception of baicalein, all flavonoids had a catechol/pyrogallol
group on the B-ring. The first peak potential displayed by
these flavonoids corresponded to the oxidation of the cate-
 M. Vestergaard et al. / Analytica Chimica Acta 538 (2005) 273–281 277

Fig. 1. Cyclic voltammograms of flavonoids; 100 ␮M cyanidin (A) and catechin gallate (B) in sodium phosphate buffer, pH 7.4 (PBS) at 24 ± 1.0 ◦ C (RT)
carried out over three wave scans at 50 mV/s. The first wave scan is shown in grey, followed by the second and third scans in black and light grey, respectively.

at pH 7.0. A third peak at around 0.644 V, corresponding to

Fig. 2. Cyclic voltammograms of 200 ␮M baicalein in PBS at 50 mV/s (grey
line) and 200 mV/s (black line). Other experimental conditions were the same
as described in Fig. 1.
chol/pyrogallol group, the most redox-active group [17]. The
flavonols had a second peak potential, hardly separated from
the first, i.e., it manifested itself as a shoulder on the first
peak, just over 0.10 V higher potential than the first one. We
have characterised this peak potential as corresponding to
OH groups on C-3. Hendrickson et al. [17] reported peak po-
tentials from the oxidation of this OH group at about 0.40 V,
i.e., at 0.32 V higher potential than quercetin’s first oxida-
tion peak in 50 mM PBS/500 mM KNO3 and 50% methanol
the oxidation of the resorcinol group on the A-ring, was also
detected. This peak appeared at about 0.70 V in the study
by Hendrickson [17], and at 0.60 V in a study by Brett and
Ghica [20]. The difference in pH might have been of most
significance to the observed difference between the two sets
of data. An increase in pH lowers the oxidation potential of
flavonoids [10,18], at least between pH 3 and 9. The differ-
ence might also have arisen, in part, due to the difference
in the electrode material used. PGEs have a slightly rougher
surface and have many more different functional groups on
the electrode surface compared to GCEs [26].
The oxidation potentials displayed by catechin follow a
similar pattern to the flavonols, differing only in that catechin
functional groups were oxidised at higher potentials. This was
in agreement with the previously reported results [10,17,18].
The electrochemistry of catechin gallate, on the other hand,
was more complex to interpret. The peak potentials of cate-
chin gallate were 0.041, 0.137, 0.555 and 1.040 V. Because
of the presence of the gallate group on the C-3, we surmised
that the oxidation potential might have been increased at this
position. In order to investigate which functional group was
the most redox-active, two other flavanols: gallocatechin and
gallocatechin gallate (see Scheme 1 for structural differences
within this group) were analysed. As shown in Table 1, both
gallocatechin gallate and gallocatechin had a peak potential at

Table 1
The voltammetric behavior of flavonoids (40 ␮M) in PBS
Flavonoid Peak potential (V) and (current (␮A))

1 2 3 4
Myricertin −0.029 (6.11) 0.130 (3.05) 0.646 (0.37)
Quercetin 0.052 (13.30) 0.155 (3.16) 0.641 (0.80)
Luteolin 0.177 (12.70) 0.716 (0.62)
Baicalein 0.091 (5.82) 0.529 (0.38) 0.897 (0.15)
Delphinidin 0.057 (2.52) 0.450 (0.12) 1.101 (<0.05)
Cyanidin 0.167 (4.65) 0.485 (0.12) 1.120 (<0.05)
Catechin 0.117 (5.41) 0.444 (0.25) 0.957 (0.37)
Catechin gallate 0.041 (3.86) 0.137 (2.39) 0.555 (0.22) 1.042 (0.84)
Confirmatory flavonoids
Gallocatechin −0.029 0.424 0.811
Gallocatechin gallate −0.028 0.073 0.435
278 M. Vestergaard et al. / Analytica Chimica Acta 538 (2005) 273–281
Fig. 3. Differential pulse voltammograms for the first oxidation potentials of flavonoids at 40 ␮M with (A) delphinidin (a) and cyanidin (b); (B) catechin gallate
(a) and catechin (b) with a step potential of 5.1 mV and a modulation amplitude of 25.05 mV at 10 mV/s in PBS.
about −0.028 V, lower than the first peak potential of catechin
gallate. This would undoubtedly correspond to the oxidation
of the pyrogallol group on the B-ring. A comparison with
myricertin and quercetin peak potentials confirmed that the
0.041 V oxidation potential of catechin gallate corresponded
to its catechol group on the B-ring. The peak potential at
1.040 V corresponded to the oxidation of the OH groups on
the A-ring. A further comparison of catechin gallate with cat-
echin and gallocatechin gallate suggested that both peak po-
tentials at 0.137 and 0.550 V were a result of the oxidation of
the gallate group on C-3. It was reasonable to assume that the
potential at 0.137 V probably corresponded to the oxidation
of a catechol/pyrogallol moiety and that the 0.550 V potential
represented the oxidation of the carbonyl group alone or in
conjunction with the OH group closest to it.
The electrochemical properties of luteolin in this study
indicated that the catechol group on the B-ring oxidised at
0.177 V and the resorcinol group on the A-ring oxidised at
0.716 V, agreeing with an earlier study [17]. Baicalein peak
potential at 0.091 V might have corresponded to the oxi-
dation of OH groups on the A-ring. The oxidation of 7,8-
dihydroxyflavone in 50 mM PBS/500 mM KNO3 and 50%
methanol at pH 7 was reported as 0.20 V [17]. To be more pre-
cise, we proposed that the oxidation of 6,7-dihydroxyphenol
groups resulted in the oxidation potential at 0.091 V. The ox-
idation potential at 0.529 V might have corresponded to the
oxidation of the C-5 OH group in conjunction with the 4-oxo
group. Chelation of Cu(II) by some flavones/flavonols has
been reported to occur between C-5 OH and the 4-oxo group
[6]. The peak potential at 0.897 V might have corresponded to
a further oxidation of the redox products of the A-ring. Both
anthocyanidins displayed three oxidation potentials, the first
corresponding to the catechol/pyrogallol group, the second
to the OH group on C-3 and the third (very low signal) at
about 0.980 V, to the OH groups on the A-ring. Cyanidin had
a shoulder at about 0.016 V on the first oxidation peak. It was
difficult to clarify whether or not this was a separate peak po-
tential corresponding to the oxidation of a separate functional
group (from the one oxidised at 0.167 V). Delphinidin also
had a very small shoulder on a similar position to cyanidin.
We hypothesised that this shoulder was a separate functional
group. The structures of anthocyanidins at pH 3 and 7–8 are
different. Our sample was prepared and analysed at pH 7.4
from a stock solution at pH 3. We concluded that the shoul-
der was the oxidation potential corresponding to functional
groups (OH) present on the B-ring at pH 3, i.e., the yet un-
transformed groups. The main peak potential corresponded
to the oxidation of the redox-active groups (ketones) present
at pH 7.4.
The relationship between oxidation potential and an-
tioxidant power has been reported [9,10]. According to
these reports, our results suggest the antioxidant power
of the analysed flavonoids to be in the following or-
der: myricertin > catechin gallate > quercetin > delphinidin >
baicalein > catechin > cyanidin > luteolin. However, this may
be a rather simplistic approach to a more complex issue.
We followed on by examining the strongest peak potentials
in terms of their current levels. Interestingly, we noted that
the current signal from delphinidin (Fig. 3A-a) was lower
than that from cyanidin (Fig. 3A-b) for the oxidation of the
OH groups on the B-ring. This pattern was also shown by
quercetin > myricertin and gallocatechin > catechin (Fig. 3B-
b). In all three cases, the pyrogallol group was oxidised more
readily than the catechol group (comparison restricted to the
same sub-class of flavonoids), but the signal was lower in
the case of the pyrogallol group. We propose that although
the pyrogallol groups were more readily oxidised, their con-
formational structure was such that the group was not easily
accessible to the electrode surface. Accessibility of the func-
tional groups is highly relevant, especially when assessing the
potential of a flavonoid to oxidise/reduce another compound.
This point will be revisited after studying the electrochem-
ical reactions between the flavonoids and copper ions. The
results also showed that all flavonoids had the highest cur-
rent detected at their lowest peak potentials, in agreement
with the higher antioxidant activity corresponding to the cat-
echol/pyrogallol groups.
Calibration of all eight flavonoids (r2 > 0.9620) was car-
ried out (data not shown). Six flavonoids could be success-
fully calibrated at two oxidation potentials, at least. The cal-
ibration data also provided relevant information as to the
optimum oxidation potentials most suitable to use at spe-
cific concentrations. The relative standard deviation (R.S.D.)
for peak potential was <5.0% and peak height was <10.0%,
 M. Vestergaard et al. / Analytica Chimica Acta 538 (2005) 273–281 279

Table 2
Peak potentials of flavonoids (40 ␮M) and copper nitrate (40 ␮M) solutions before and after complexation in PBS
Flavonoid Peak potentials (V)
Before complexation After complexation

1 2 3 4 Cu(II) 1 2 3 4
Myricertin −0.029 0.130 0.646 −0.109 0.236
Quercetin 0.052 0.155 0.641 −0.109 0.168 0.295
Luteolin 0.177 0.716 −0.114 0.078 0.304
Baicalein 0.091 0.529 0.897 −0.119 0.232
Delphinidin 0.057 0.450 1.101 −0.126 0.103
Cyanidin 0.167 0.485 1.120 −0.124 0.171
Catechin 0.117 0.444 0.957 −0.129 0.138 0.460
Catechin gallate 0.041 0.137 0.555 1.042 −0.134 0.148
Cu(II) −0.104

thus this method could be used to characterise and quantify
flavonoid-containing extracts (purified).
3.3. Electrochemical detection of the interaction
between Cu(II) and flavonoids
First, we performed the voltammetry of copper nitrate so-
lution with PGE at 40 ␮M in PBS at RT. This exhibited a
single reduction potential at −0.104 V. The calibration data
gave a linear range between 0 and 40 ␮M (r2 = 9623). The
R.S.D. of the peak currents and peak potentials were 3.2%
and 1.7%, respectively. We calculated the number of electrons
(n) transferred during the oxidation process from the equa-
tion of a semi-finite diffusion-controlled electrode response
[31]:
w = 3.53 RT/nF
where w is half-width of the semi-derivative peak. Since w
was 0.101 V (analysed in triplicate), n was computed as 2.07
which corresponded to two electrons. Thus, we concluded
that the copper ion oxidation peak corresponded to the oxi-
dation of Cu(II).
The interactions between flavonoids and copper ions were
studied at equimolar concentrations (40 ␮M) under the same
conditions as described earlier. Table 2 and Fig. 4 show the
Fig. 4. Differential pulse voltammograms of 40 ␮M Cu(II) alone (a, black
line) and 40 ␮M catechin gallate alone (b, black line); and after complexation
of the flavonoid and copper ions (a + b, grey line) in PBS. Other experimental
conditions were the same as described in Fig. 3.
changes in the current and the oxidation potential(s) after the
chelation of copper ions by the flavonoids.
There was a slight increase in the peak potential of cop-
per ions upon complexation. This change (<30 mV) might
be attributed to the changes in the pH of the media. There
were varying changes in the peak potentials of the flavonoids.
All eight flavonols had a significant decrease (of at least
50% of the control) in the current detected, indicating a de-
crease in the availability of functional groups to be oxidised.
In the case of myricertin, the peak potentials at −0.029,
0.130 and 0.646 V disappeared completely to be replaced
by a new peak potential at 0.236 V. This suggested that all
the redox-active ‘natural’ functional groups had been com-
plexed and that the complexed product(s) of at least one of
the groups was redox-active at 0.236 V. The peak potential
at 0.646 V reappeared when the concentration of myricertin
was increased to 120 ␮M. The re-appearance of the A-ring
functional groups at a higher flavonoid:Cu(II) molar ratio
suggested that the chelation of copper ions occurred, pref-
erentially, at the B-ring and the C-3 OH group. This is in
agreement with other reported literature using different an-
alytical techniques [6,7]. The other flavanol, quercetin, also
lost its oxidation potentials at 0.052 V and 0.641 V. However,
the group at 0.155 V was still redox-active at 0.168 V. Addi-
tionally, a new group emerged and was oxidised at 0.295 V.
This group could be similar to the redox-active product of
myricertin interaction with copper ions (0.236 V). The fact
that the C-3 OH group of quercetin was still redox-active
and that of myricertin was inert might be because the cat-
echol group had a higher current (better exposure of func-
tional groups to the molecular surface) than the pyrogallol
group of myricertin, thus attracting more copper ions with
a consequent reduction of copper ions available to complex
with the C-3 OH groups on quercetin. The new oxidation
potential at 0.236, 0.295 V reflected the oxidation of the re-
dox products either on the B-ring or at a different site. We
doubt that it corresponded to the oxidation of oxidised prod-
ucts of the OH group at C-3 in the presence of copper ions
since these have previously been reported to be redox-inactive
[7].
280 M. Vestergaard et al. / Analytica Chimica Acta 538 (2005) 273–281
Upon chelating copper ions, the C-3 OH group and the
resorcinol group of both delphinidin and cyanidin were no
longer redox-active. The oxidation potential of the functional
groups on the B-ring shifted to slightly higher potentials. Cat-
echin lost the peak potential corresponding to the oxidation
of OH groups on the A-ring. The rest were unchanged. Cat-
echin gallate lost its oxidation potentials at 0.041, 0.555 and
1.042 V but retained the peak potential at 0.137 V (Fig. 4).
The potential at 0.137 V represented the oxidation of a func-
tional group at C-3.
The changes in the oxidation potentials of the flavones
were more complex. In the presence of copper ions, luteolin
had two oxidation potentials at 0.078 and 0.304 V. The latter
potential was a new functional group, probably a similar func-
tional group to the one formed by myricertin and quercetin.
The first peak potential most likely corresponded to a shift
in the first peak potential of the natural luteolin. Baicalein
upon complexation with copper ions, lost two peaks and a
new one was observed at 0.232 V. We were inclined to con-
clude that this new peak was a result of an oxidised new
functional group similar to the one observed with quercetin,
myricertin and luteolin. These four flavonoids share charac-
teristics, such as a 2,3-double bond and an oxo group on C-4,
which are absent in the other four flavonoids. This new group
might be formed at the site between 4-oxo and 5-hydroxyl.
This site has been suggested, previously, for the chelation of
copper ions by these two sub-classes of flavonoids [6]. The
commonality of OH groups on the B-ring for seven of the
eight flavonoids and the lack of this new functional group in
the other four flavonoids ruled out the B-ring as a potential
source of the new peak potential. Another probable explana-
tion could be that the pyranose ring opened up at position C-1,
creating an OH group that was subsequently oxidised at this
potential.
With the exception of baicalein, in the presence of copper
ions, the oxidation potentials corresponding to the oxidation
of resorcinol groups on the A-ring disappeared, i.e., they were
unavailable for oxidation. Increasing the ratio of flavonoid
to copper ions, in some cases, enabled the re-appearance of
some functional groups. At 120 ␮M myricertin concentra-
tion, the peak potential at 0.646 V reappeared, suggesting
that the resorcinol group is the least attractive to Cu(II) [6,7].
The peak potentials at 0.897 and 0.529 V reappeared at 120
and 160 ␮M concentrations of baicalein, again suggesting
that the higher the peak potential, the lower the affinity of
the oxidisable group to Cu(II). At 80 ␮M cyanidin, the peak
potential at 0.485 V reappeared. The corresponding peak po-
tential for delphinidin reappeared at 160 ␮M delphinidin con-
centration, perhaps indirectly confirming that the reactivity
of the pyrogallol group is higher than that of the catechol
moiety. The concentration of flavonoids was increased un-
til the copper signal disappeared (<20 nA), i.e., until all the
copper ions were chelated. These concentrations are listed in
Table 3.
The possible stoichiometry of the flavonoids and Cu(II),
under the described experimental conditions, is shown
Table 3
Flavonoid concentrations at which no Cu(II) (40 ␮M) were detectable after
incubation for 10 min in PBS
Flavonoid Concentration (␮M) Possible stoichiometry
flavonoid:Cu ions
Myricertin 160 4:1
Quercetin 200 5:1
Baicalein 240 6:1
Cyanidin 400 10:1
Delphinidin 240 6:1
Catechin 600 15:1
Catechin gallate 160 4:1
in Table 3. Stoichiometry studies provide information
about how reactive a compound/molecule is with re-
spect to another compound/molecule. These results sug-
gested the order of reactivity of flavonoid to Cu(II) to
be myricertin = catechin gallate > quercetin > delphinidin =
baicalein > cyanidin > catechin. The peak potentials (Table 2)
and current in the presence and absence of copper ions pro-
vided us information on the reactivity of each flavonoid,
which functional groups were more redox-active and some
understanding of the possible conformational structure of the
flavonoids. As mentioned earlier, peak potentials have been
used to assign the relative antioxidant power of flavonoids:
the lower the peak potential, the higher its antioxidant power
[7,9,10]. The copper chelation studies have confirmed that
there was indeed an inverse relationship between peak poten-
tial and antioxidant power. Here we have equated antioxidant
power to copper ion chelation because copper ions have been
reported to be involved in causing oxidative damage [27].
Chelation of these copper ions would reduce/inhibit copper-
induced oxidative damage. Additionally, we have shown that
the location of OH groups relative to each other is of ut-
most importance in determining copper ion (metal) chelating
ability. The pyrogallol group is more potent than the catechol
group, which in turn is more potent than the resorcinol group.
The presence of the 4-oxo group in conjunction with the ox-
idation state of the C-ring also affects the redox potential of
the flavonoids. Chelation of copper on the site between the
4-oxo group and C-5 OH group in flavonols and flavones has
already been proposed [6,7]. The number of OH groups is also
important, the higher the number, the higher their chelating
ability is. The relationship between the number of OH groups
and antioxidant activity of flavonoids has also been suggested
[23]. Further, this study has shown that the conformational
structure of the flavonoid also has an influence on the reactiv-
ity/antioxidant activity of the flavonoids. The number of OH
groups on the B-ring was shown to affect the current detected.
Although the pyrogallol group, was more redox-active than
the catechol moiety, its configuration somehow affected the
accessibility of the OH groups to the electrode surface. The
importance of steric configuration and its effect on reactivity
of a compound has been demonstrated, electrochemically, in
DNA studies [32,33].
 M. Vestergaard et al. / Analytica Chimica Acta 538 (2005) 273–281
4. Conclusions
Our study carried out the electrochemical analysis of eight
flavonoids belonging to four flavonoid sub-classes. The re-
port of the electrochemistry of three of the flavonoids: cat-
echin gallate, delphinidin and cyanidin, is for the first time.
We have studied and discussed the flavonoids’ copper ion
chelating properties and we have elucidated the relationship
of chelation ability to flavonoid structure. To the best of our
knowledge, this is the first study to report the interaction
between copper ions and flavonoids using electrochemistry.
Our study has analysed the determinants at disposable pencil
graphite electrodes, also for the first time in flavonoid studies.
The use of PGE shortened total analysis time by eliminating
working electrode cleaning and pretreatment procedure in
between each analysis. The relatively large available active
electrode surface area enabled us to study flavonoids at low
concentrations without the need for deposition, thus cutting
down the analysis time even further. Moreover, using cyclic
voltammetry, we were able to study the oxidation and reduc-
tion potentials of the flavonoids over three scans because of
the increased sensitivity of the PGE. Reversibility of the first
peak potentials of all flavonoids was demonstrated. The other
oxidation potentials appeared to be irreversible.
The flavonoid structure plays a crucial role in the chelation
of copper ions. These findings might be beneficial in neu-
rodegenerative studies of diseases such as Alzheimer’s and
Parkinson’s in which copper ions have been reported to play
an important role in initiating and/or promoting the progres-
sion of the diseases. Further, an understanding of the redox
properties of each functional group might aid in the synthesis
of new compounds with enhanced desirable properties.
Acknowledgments
K.K. acknowledges the Monbukagakusho scholarship for
research students from the Japan Ministry of Education, Cul-
ture, Sports, Science and Technology (MEXT).
References
[1] C.A. Rice-Evans, L. Parker, Flavonoids in Health and Disease, Mar-
cel Dekker, New York, 1998.
[2] A.K. Samhan-Aris, F.J. Martin-Romero, C. Gutierrez-Merino, Free
Radic. Biol. Med. 37 (2004) 48.
281
[3] J.B. Harbone, C.A. Williams, Phytochemistry 55 (2000) 481.
[4] T. Hirvonen, J. Virtamo, P. Korhonen, D. Albanes, P. Pietinen, Stroke
10 (2000) 2301.
[5] E. Monsen, J. Am. Diet. Assoc. 100 (2000) 1008.
[6] L. Mira, M.T. Fernandez, M. Santos, R. Rocha, M.H. Florencio,
K.R. Jennings, Free Radic. Res. 36 (2002) 1199.
[7] J.E. Brown, H. Khodr, R.C. Hider, C.A. Rice-Evans, Biochem. J.
330 (1998) 1173.
[8] J. Hong, H. Lu, X. Meng, J. Ryu, Y. Hara, C.S. Yung, Cancer Res.
62 (2002) 7241.
[9] S. Mannino, O. Brenna, S. Buratti, M.S. Cosio, Electroanalysis 10
(1998) 908.
[10] A.J. Blasco, M.C. Gonzalez, A. Escarpa, Anal. Chim. Acta 511
(2004) 71.
[11] F. Shadidi, P.K.J. Wanasundra, Cri. Rev. Food Sci. Nutr. 32 (1992)
67.
[12] F.A.A. van Acker, O. Schouten, G.R.M.M. Haenen, W.J.F. van der
Vijgh, A. Bast, FEBS Lett. 473 (2002) 145.
[13] S. Chevion, M.A. Roberts, M. Chevion, Free Radic. Biol. Med. 28
(2000) 860.
[14] B. Yang, A. Kotan, K. Ari, F. Kusu, Anal. Sci. 17 (2001) 599.
[15] I.B. Afanas’ev, A.I. Dorozhko, A.V. Brodskii, A.I. Potapovitch,
Biochem. Pharmacol. 38 (1989) 1763.
[16] H. Hotta, H. Sakamoto, S. Nagano, T. Osakai, Y. Tsujino, Biochim.
Biophys. Acta 1526 (2001) 159.
[17] H.P. Hendrickson, A.D. Kaufman, C.E. Lunte, J. Pharm. Biomed.
Anal. 12 (1994) 325.
[18] P. Janeiro, A.M.O. Brett, Anal. Chim. Acta 518 (2004) 109.
[19] M. Mochizuki, S. Yamazaki, K. Kano, T. Ikeda, Biochim. Biophys.
Acta 1569 (2002) 35.
[20] A.M.O. Brett, M.E. Ghica, Electroanalysis 15 (2003) 1745.
[21] A.M.O. Brett, V.C. Disculescu, Bioelectrochemistry 64 (2004) 133.
[22] A.M.O. Brett, V.C. Disculescu, Bioelectrochemistry 64 (2004) 143.
[23] K. Ono, Y. Yoshiike, A. Takashima, K. Hasegawa, H. Naiki, M.
Yamada, J. Neurochem. 87 (2003) 172.
[24] J. Wang, A.N. Kawde, Anal. Chim. Acta 431 (2001) 219.
[25] M. Ozsoz, A. Erdem, K. Kerman, D. Ozkan, B. Tugrul, N.
Topcuoglu, H. Ekren, M. Taylan, Anal. Chem. 75 (2003) 2181.
[26] J. Wang, A. Kawde, E. Sahlin, Analyst 125 (2000) 5.
[27] X. Huang, M.P. Cuajungco, C.S. Atwood, M.A. Hartshorn, J.A.D.
Tyndall, G.R. Hanson, K.C. Stokes, M. Leopold, G. Multhaup, L.E.
Goldstein, R.C. Scarpa, A.J. Saunders, J. Lim, R.D. Moir, C. Glabe,
E.F. Bowden, C.L. Masters, D.P. Fairlie, R.E. Tanzi, A.J. Bush, J.
Biol. Chem. 274 (1999) 37111.
[28] C.A. Rottkamp, A.K. Raina, X. Rhu, E. Gaier, A.I. Bush, C.S. At-
wood, M. Chevion, G. Perry, M.A. Smith, Free Radic. Biol. Med.
30 (2001) 447.
[29] S. Mandel, M.B.H. Youdim, Free Radic. Biol. Med. 37 (2004) 304.
[30] P. Janiero, A.M.O. Brett, Electroanalysis, in press.
[31] A.J. Bard, L.R. Faulkner, Electrochemical Methods, Fundamentals
and Applications, second ed., John Wiley & Sons, Inc., 2001.
[32] B. Meric, K. Kerman, D. Ozkan, P. Kara, M. Ozsoz, Electroanalysis
14 (2002) 1245.
[33] K. Kerman, D. Ozkan, P. Kara, A. Erdem, B. Meric, P. Nielsen, M.
Ozsoz, Electroanalysis 15 (2003) 667.