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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,


at low concentrations relative to the target substrate, can in-
Flavonoids are widely distributed in nature and exhibit hibit or slow down the oxidation of the substrate. Antioxi-
beneficial traits such as anti-inflammatory, anti-cancer and dants protect cells against the damaging effects of reactive
antioxidant activities [1–3]. The dietary intake of flavonoids oxygen species, such as singlet oxygen, superoxide, per-
is remarkably high in comparison with other dietary antioxi- oxyl radicals, hydroxyl radicals and peroxynitrite by form-
dants such as Vitamins C and E, carotenoids [4] and selenium ing an antioxidant–radical complex. The antioxidant–radical
[5]. They also chelate highly redox-active metal ions [6,7] formed after scavenging should be stable enough through
giving them an even stronger protective effect against ox- intramolecular H-bonding [11]. A disparity between an-
idative damage. The most common sources of flavonoids for tioxidants and reactive oxygen species may lead to oxida-
humans are green tea, red wine, fruits and vegetables [8–10]. tive stress and subsequent cellular damage. Oxidative stress
has been linked to cancer, atherosclerosis, aging, inflamma-
∗ Corresponding author. Tel.: +81 761 51 1660; fax: +81 761 51 1665. tion and neurodegenerative diseases such as Parkinson’s and
E-mail address: munde@jaist.ac.jp (M. Vestergaard). 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- significant when only small amounts of analyte are available.
port by van Acker et al. [12] suggested that flavonoids could Moreover, disposable PGE significantly reduces total analy-
replace Vitamin E as a chain-breaking antioxidant in liver mi- sis time by by-passing the cleaning and pre-treatment process,
crosomal membranes. The antioxidant and metal-chelating which is essential when using GCEs. Analysis time is also
properties of flavonoids are attributed to the presence of aro- shortened because samples are analysed without deposition,
matic OH groups, their location relative to each other, the at concentrations for which deposition might be required if
oxidation state of the C-ring and the overall number of OH GCEs were used [26]. Further, the use of PGE, with its large
groups present [6,7]. active surface area, has enabled us to study the reversibil-
Several studies have proposed methods for determining ity/irreversibility of oxidised functional groups and also the
the antioxidant activity [13,14] and metal chelating prop- fate of oxidised and later reduced groups over three wave
erties of flavonoids [6,7]. Few studies have combined the scans using CV.
two [7,15]. Metal chelation studies using UV–vis spectropho- The present study has investigated the electrochemical in-
tometry, based on the shift in wavelength of maximum ab- teraction between eight flavonoids (covering four classes of
sorbance of the flavonoid, coupled with residual metal detec- flavonoids) and the highly reactive copper ions. The infor-
tion by fluorescence or/and atomic absorption spectrometry, mation may be crucial in understanding the role of flavonoid
might provide information on the relative degree of chela- structure in scavenging copper-induced oxidative damage.
tion and enable an inference to be drawn as to the influence The findings may have wider implications and possible ap-
of structure on the extent of chelation; however, they lack plication(s) in neurodegenerative diseases (e.g., Alzheimer’s
the sensitivity to distinguish similarly-structured compounds. and Parkinson’s), in which metal ions such as copper ions
UV–vis spectrophotometric measurements are also limited in have been implicated in plaque formation, one of the main
that they do not perform optimally if analytes precipitate prior causes of the diseases [27–29]. Studies of other copper
to or during the measurements. Atomic absorption methods toxicity-induced illnesses may also benefit from these find-
fail to distinguish between metal ions present in isolation or ings.
in soluble complexes.
Electrochemical methods have been used to evaluate the
antioxidant properties (also referred to as antioxidant power) 2. Experimental
of flavonoids and other polyphenols [9,10,16]. Detailed elec-
trochemical studies of catechol-containing flavonoids [17], 2.1. Reagents
catechin oxidation mechanisms [18], kinetic analysis of cat-
echin autoxidation [19] and oxidation of quercetin [20] have Flavonoids of >97% purity were purchased: cyani-
been performed, and the relationship of structure to pos- din chloride and delphinidin chloride from Extrasynthese
sible antioxidant properties drawn. Studies on quercetin (a (SSX, Lyon, France); myricertin and catechin gallate from
flavonoid)–DNA interactions in the presence and absence of Sigma–Aldrich Co. (MO, USA); baicalein from Acris An-
Cu(II) have been carried out and have shown pro-oxidant tibodies GmbH (Hiddenhausen, Germany); luteolin and
activities of quercetin in the presence of DNA [21,22]. How- quercetin from Wako Pure Chemical (Tokyo, Japan) and cat-
ever, electrochemical studies of the interactions between echin from Calbiochem (CA, USA). A standard solution of
flavonoids and metal ions (significant in oxidative damage) copper nitrate (1000 mg/l in 0.5 mol/l nitric acid) was sup-
have not been done. Brett and Disculescu [21,22] touched plied by Wako Pure Chemical (Tokyo, Japan). All other
on the interaction between quercetin and copper ions in their reagents were purchased from Wako Pure Chemical (Tokyo,
investigation of the interaction between quercetin and DNA. Japan) and were of analytical grade. Unless otherwise stated,
There is a need to establish the electrochemical activity of all solutions were prepared using a 50 mM sodium phosphate
flavonoids, proposed in literature as possible therapeutics buffer solution (PBS), pH 7.4. Deionised water obtained from
[23] and their interaction with very reactive metal ions, also Millipore Milli-Q purification system (Millipore, Bedford,
implicated in many diseases. Our present study investigated MA, USA) was used for preparation of PBS and for cleaning
these issues. Further, our method for analysing these inter- of electrodes.
actions employed differential pulse voltammetry (DPV) and
cyclic voltammetry (CV) at disposable pencil graphite elec- 2.2. Apparatus
trodes (PGEs). DPV and CV have been used for analysing
flavonoids but mostly at conventional glassy carbon elec- Differential pulse voltammetry (DPV) and cyclic voltam-
trodes (GCEs) [10,16,18,20–22]. To the best of our knowl- metry (CV) were performed with an Autolab PGSTAT 12
edge, this is the first time that disposable PGEs were em- electrochemical analysis system (Eco Chemie, The Nether-
ployed in antioxidant and/or antioxidant interaction studies, lands) in connection with its General Purpose Electrochem-
though they have been used, successfully, in the detection of ical System (GPES) software. The three-electrode system
nucleic acids and hybridization [24,25]. PGE has a larger ac- consisted of a pencil graphite electrode (PGE) as the work-
tive electrode surface area and is therefore able to detect low ing electrode, the reference electrode (Ag/AgCl), and a plat-
concentrations and/or volume of the analyte. This might be inum wire as the auxiliary electrode. The pencil leads were
M. Vestergaard et al. / Analytica Chimica Acta 538 (2005) 273–281 275

obtained from Tombo Co. Ltd., Japan. All leads had a to- potentials were not detected. During the second and third ox-
tal length of 60 mm and a diameter of 0.5 mm. The pencil idation wave scans, there was a reduction in the current sig-
leads were used as received. The leads were (about 6 mm) nals detected at each peak potential for luteolin, myricertin,
vertically dipped in the analyte solution giving a geometric quercetin, catechin gallate (Fig. 1B), cyanidin and delphini-
electrode surface area of 9.62 mm2 . PGEs were used only din. The reduction could have been as a result of flavonoids
once and disposed of after each run. The miniaturized ref- having adsorbed to the electrode surface and subsequently
erence electrode with 2 mm i.d. was obtained from Cypress blocking the surface after each wave scan. This is consistent
Systems (KS, USA). with reported literature [18,20]. Exceptionally, there was an
increase in the current for catechin and baicalein during the
2.3. Procedure second scan for the first peak potential but it dropped again
during the third scan. This could suggest that the reducible
Flavonoid stock solutions (1 mM) were prepared in group during the first wave scan was re-oxidised during the
methanol and stored in 0.1 ml vials at −20 ◦ C. Just before second scan. The total sum of this re-oxidised group and the
analysis the vials were left to equilibrate to room temperature. natural flavonoid oxidised at the leftover/available electrode
Intermediate copper nitrate solution (1 mM) was prepared in surface was bigger than the oxidation of the natural flavonoid
PBS and stored at 4 ± 1 ◦ C until required for analysis. group during the first scan. In fact, this result was found to be
Complexation of Cu(II) and flavonoids was carried out at entirely plausible since both catechin and baicalein had the
room temperature (RT, 24 ± 1 ◦ C) in PBS. A range of con- highest reduction signals compared to the other flavonoids
centrations was tested: 0 and 40 ␮M for Cu(II) (prepared (as a percent of the first scan oxidation signal) at 84% and
from 1 mM Cu(II) intermediate solution) and 0–600 ␮M for 44%, respectively.
flavonoid solutions (prepared from the 1 mM stock solutions). For all flavonoids, there was a single reduction peak at a
Sample solutions of 1 ml volume were mixed in 1.6 ml vials potential slightly lower than the first peak potential. It is log-
for 10 s using an MSI Mini shaker, followed by incubation at ical to conclude that this peak was a result of the reduction
RT for 10 min. All experiments were carried out at RT. of the product of the oxidised functional group correspond-
All electrodes were rinsed with Milli-Q water and blot ing to the first oxidation peak potential. This phenomenon
dried before each individual analysis. Initial voltammograms has been reported previously [10,17,18,20]. Brett and Ghica
were recorded soon after insertion of the electrodes in order [20] also reported reversibility of the third peak potential.
to minimise adsorption of the analyte to the working elec- The behaviour of these reduction peaks, in terms of the cur-
trode surface prior to the run. All samples were pre-analysed rent detected, was more or less similar among the flavonoids.
using DPV: scan range from −0.5 V to 1.2 V; step potential Baicalein, luteolin, catechin, myricertin and cyanidin all had
5.1 mV; modulation amplitude 25.05 mV and a scan rate of reduction peaks in all three scans and there was a reduc-
10 mV/s and without deposition. Once the peak potentials tion in current after each scan. Delphinidin and quercetin had
were established, the scan range was adjusted accordingly very low reduction signals and we could not deduce any-
for each analyte. The other parameters remained unchanged. thing from the results. The reduction pattern displayed by
The parameters for CV were scan rates of 10, 25, 50, 100 and catechin gallate was interesting. The highest reduction signal
200 mV/s and a step potential of 2.4 mV. All flavonoids were was manifested during the second wave scan, and the weakest
monitored with three consecutive scans. 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
3. Results rest of the flavonoids. From a sterical point of view, catechin
gallate is a bigger compound than the other flavonoids. This
3.1. Electrochemistry of flavonoids using cyclic might have played a role in delaying the oxidation of catechin
voltammetry gallate.
The effect of scan rate (10, 25, 50, 100 and 200 mV/s)
The numbering system and the four sub-classes of on response was carried out on four flavonoids (catechin,
flavonoids analysed in this study are shown in Scheme 1. baicalein, cyanidin and quercetin) belonging to each of the
First, we used CV to investigate the presence of re- flavonoid sub-classes. For all flavonoids, there was at least a
ducible groups after each oxidation wave (reversibil- five-fold increase in the current signal with increase in scan
ity/irreversibility) and whether or not the flavonoids adsorbed rate from 50 to 200 mV/s. Fig. 2 shows the effect of scan
to the electrode surface. In this experiment, all flavonoids rate on the current signal of baicalein. The lower current sig-
were prepared in 50 mM PBS at 100 ␮M concentration at nal at slower scan rates could indicate a rapid formation of a
RT. Cyclic voltammograms of two of the flavonoids are pre- non-electro-active film on the electrode surface in agreement
sented in Fig. 1. During the first oxidation wave, the same with a suggestion by Janiero and Brett [30]. The faster the
number and position of peak potentials were observed for scan rate, the lesser time the film is allowed to deposit on
all flavonoids with the exception of the anthocyanidins. For the electrode surface, and the higher the current signal. The
cyanidin (Fig. 1A) and delphinidin, the third and fourth peak 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- for all eight flavonoids. The significance of these parameters
fusion. As the scan rate was increased, there was less time will be discussed in more detail in the following paragraphs.
for the diffusion layer to extend into the bulk of the solution, All flavonoids were analysed at 40 ␮M concentrations pre-
causing a large diffusion gradient and consequently, a higher pared in 50 mM PBS at RT using DPV with PGE. The number
peak current [31]. and position of peak potentials and their corresponding cur-
rents are displayed in Table 1. Fig. 3 shows voltammograms
3.2. Electrochemistry of flavonoids using differential of three of the flavonoids whose electrochemistry is reported
pulse voltammetry for the first time.
All flavonoids had at least two peak potentials. With the ex-
We investigated the following flavonoid characteristics: ception of baicalein, all flavonoids had a catechol/pyrogallol
(i) the peak potential, (ii) the number and position of peak group on the B-ring. The first peak potential displayed by
potentials, and (iii) the current heights at each peak potential 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


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
Fig. 2. Cyclic voltammograms of 200 ␮M baicalein in PBS at 50 mV/s (grey functional groups were oxidised at higher potentials. This was
line) and 200 mV/s (black line). Other experimental conditions were the same
in agreement with the previously reported results [10,17,18].
as described in Fig. 1.
The electrochemistry of catechin gallate, on the other hand,
chol/pyrogallol group, the most redox-active group [17]. The was more complex to interpret. The peak potentials of cate-
flavonols had a second peak potential, hardly separated from chin gallate were 0.041, 0.137, 0.555 and 1.040 V. Because
the first, i.e., it manifested itself as a shoulder on the first of the presence of the gallate group on the C-3, we surmised
peak, just over 0.10 V higher potential than the first one. We that the oxidation potential might have been increased at this
have characterised this peak potential as corresponding to position. In order to investigate which functional group was
OH groups on C-3. Hendrickson et al. [17] reported peak po- the most redox-active, two other flavanols: gallocatechin and
tentials from the oxidation of this OH group at about 0.40 V, gallocatechin gallate (see Scheme 1 for structural differences
i.e., at 0.32 V higher potential than quercetin’s first oxida- within this group) were analysed. As shown in Table 1, both
tion peak in 50 mM PBS/500 mM KNO3 and 50% methanol 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 different. Our sample was prepared and analysed at pH 7.4
gallate. This would undoubtedly correspond to the oxidation from a stock solution at pH 3. We concluded that the shoul-
of the pyrogallol group on the B-ring. A comparison with der was the oxidation potential corresponding to functional
myricertin and quercetin peak potentials confirmed that the groups (OH) present on the B-ring at pH 3, i.e., the yet un-
0.041 V oxidation potential of catechin gallate corresponded transformed groups. The main peak potential corresponded
to its catechol group on the B-ring. The peak potential at to the oxidation of the redox-active groups (ketones) present
1.040 V corresponded to the oxidation of the OH groups on at pH 7.4.
the A-ring. A further comparison of catechin gallate with cat- The relationship between oxidation potential and an-
echin and gallocatechin gallate suggested that both peak po- tioxidant power has been reported [9,10]. According to
tentials at 0.137 and 0.550 V were a result of the oxidation of these reports, our results suggest the antioxidant power
the gallate group on C-3. It was reasonable to assume that the of the analysed flavonoids to be in the following or-
potential at 0.137 V probably corresponded to the oxidation der: myricertin > catechin gallate > quercetin > delphinidin >
of a catechol/pyrogallol moiety and that the 0.550 V potential baicalein > catechin > cyanidin > luteolin. However, this may
represented the oxidation of the carbonyl group alone or in be a rather simplistic approach to a more complex issue.
conjunction with the OH group closest to it. We followed on by examining the strongest peak potentials
The electrochemical properties of luteolin in this study in terms of their current levels. Interestingly, we noted that
indicated that the catechol group on the B-ring oxidised at the current signal from delphinidin (Fig. 3A-a) was lower
0.177 V and the resorcinol group on the A-ring oxidised at than that from cyanidin (Fig. 3A-b) for the oxidation of the
0.716 V, agreeing with an earlier study [17]. Baicalein peak OH groups on the B-ring. This pattern was also shown by
potential at 0.091 V might have corresponded to the oxi- quercetin > myricertin and gallocatechin > catechin (Fig. 3B-
dation of OH groups on the A-ring. The oxidation of 7,8- b). In all three cases, the pyrogallol group was oxidised more
dihydroxyflavone in 50 mM PBS/500 mM KNO3 and 50% readily than the catechol group (comparison restricted to the
methanol at pH 7 was reported as 0.20 V [17]. To be more pre- same sub-class of flavonoids), but the signal was lower in
cise, we proposed that the oxidation of 6,7-dihydroxyphenol the case of the pyrogallol group. We propose that although
groups resulted in the oxidation potential at 0.091 V. The ox- the pyrogallol groups were more readily oxidised, their con-
idation potential at 0.529 V might have corresponded to the formational structure was such that the group was not easily
oxidation of the C-5 OH group in conjunction with the 4-oxo accessible to the electrode surface. Accessibility of the func-
group. Chelation of Cu(II) by some flavones/flavonols has tional groups is highly relevant, especially when assessing the
been reported to occur between C-5 OH and the 4-oxo group potential of a flavonoid to oxidise/reduce another compound.
[6]. The peak potential at 0.897 V might have corresponded to This point will be revisited after studying the electrochem-
a further oxidation of the redox products of the A-ring. Both ical reactions between the flavonoids and copper ions. The
anthocyanidins displayed three oxidation potentials, the first results also showed that all flavonoids had the highest cur-
corresponding to the catechol/pyrogallol group, the second rent detected at their lowest peak potentials, in agreement
to the OH group on C-3 and the third (very low signal) at with the higher antioxidant activity corresponding to the cat-
about 0.980 V, to the OH groups on the A-ring. Cyanidin had echol/pyrogallol groups.
a shoulder at about 0.016 V on the first oxidation peak. It was Calibration of all eight flavonoids (r2 > 0.9620) was car-
difficult to clarify whether or not this was a separate peak po- ried out (data not shown). Six flavonoids could be success-
tential corresponding to the oxidation of a separate functional fully calibrated at two oxidation potentials, at least. The cal-
group (from the one oxidised at 0.167 V). Delphinidin also ibration data also provided relevant information as to the
had a very small shoulder on a similar position to cyanidin. optimum oxidation potentials most suitable to use at spe-
We hypothesised that this shoulder was a separate functional cific concentrations. The relative standard deviation (R.S.D.)
group. The structures of anthocyanidins at pH 3 and 7–8 are 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 changes in the current and the oxidation potential(s) after the
flavonoid-containing extracts (purified). chelation of copper ions by the flavonoids.
There was a slight increase in the peak potential of cop-
3.3. Electrochemical detection of the interaction per ions upon complexation. This change (<30 mV) might
between Cu(II) and flavonoids be attributed to the changes in the pH of the media. There
were varying changes in the peak potentials of the flavonoids.
First, we performed the voltammetry of copper nitrate so- All eight flavonols had a significant decrease (of at least
lution with PGE at 40 ␮M in PBS at RT. This exhibited a 50% of the control) in the current detected, indicating a de-
single reduction potential at −0.104 V. The calibration data crease in the availability of functional groups to be oxidised.
gave a linear range between 0 and 40 ␮M (r2 = 9623). The In the case of myricertin, the peak potentials at −0.029,
R.S.D. of the peak currents and peak potentials were 3.2% 0.130 and 0.646 V disappeared completely to be replaced
and 1.7%, respectively. We calculated the number of electrons by a new peak potential at 0.236 V. This suggested that all
(n) transferred during the oxidation process from the equa- the redox-active ‘natural’ functional groups had been com-
tion of a semi-finite diffusion-controlled electrode response plexed and that the complexed product(s) of at least one of
[31]: the groups was redox-active at 0.236 V. The peak potential
at 0.646 V reappeared when the concentration of myricertin
w = 3.53 RT/nF was increased to 120 ␮M. The re-appearance of the A-ring
where w is half-width of the semi-derivative peak. Since w functional groups at a higher flavonoid:Cu(II) molar ratio
was 0.101 V (analysed in triplicate), n was computed as 2.07 suggested that the chelation of copper ions occurred, pref-
which corresponded to two electrons. Thus, we concluded erentially, at the B-ring and the C-3 OH group. This is in
that the copper ion oxidation peak corresponded to the oxi- agreement with other reported literature using different an-
dation of Cu(II). alytical techniques [6,7]. The other flavanol, quercetin, also
The interactions between flavonoids and copper ions were lost its oxidation potentials at 0.052 V and 0.641 V. However,
studied at equimolar concentrations (40 ␮M) under the same the group at 0.155 V was still redox-active at 0.168 V. Addi-
conditions as described earlier. Table 2 and Fig. 4 show the 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-
Fig. 4. Differential pulse voltammograms of 40 ␮M Cu(II) alone (a, black ucts of the OH group at C-3 in the presence of copper ions
line) and 40 ␮M catechin gallate alone (b, black line); and after complexation since these have previously been reported to be redox-inactive
of the flavonoid and copper ions (a + b, grey line) in PBS. Other experimental [7].
conditions were the same as described in Fig. 3.
280 M. Vestergaard et al. / Analytica Chimica Acta 538 (2005) 273–281

Upon chelating copper ions, the C-3 OH group and the Table 3
resorcinol group of both delphinidin and cyanidin were no Flavonoid concentrations at which no Cu(II) (40 ␮M) were detectable after
incubation for 10 min in PBS
longer redox-active. The oxidation potential of the functional
groups on the B-ring shifted to slightly higher potentials. Cat- Flavonoid Concentration (␮M) Possible stoichiometry
flavonoid:Cu ions
echin lost the peak potential corresponding to the oxidation
of OH groups on the A-ring. The rest were unchanged. Cat- Myricertin 160 4:1
Quercetin 200 5:1
echin gallate lost its oxidation potentials at 0.041, 0.555 and Baicalein 240 6:1
1.042 V but retained the peak potential at 0.137 V (Fig. 4). Cyanidin 400 10:1
The potential at 0.137 V represented the oxidation of a func- Delphinidin 240 6:1
tional group at C-3. Catechin 600 15:1
The changes in the oxidation potentials of the flavones Catechin gallate 160 4:1
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. in Table 3. Stoichiometry studies provide information
The first peak potential most likely corresponded to a shift about how reactive a compound/molecule is with re-
in the first peak potential of the natural luteolin. Baicalein spect to another compound/molecule. These results sug-
upon complexation with copper ions, lost two peaks and a gested the order of reactivity of flavonoid to Cu(II) to
new one was observed at 0.232 V. We were inclined to con- be myricertin = catechin gallate > quercetin > delphinidin =
clude that this new peak was a result of an oxidised new baicalein > cyanidin > catechin. The peak potentials (Table 2)
functional group similar to the one observed with quercetin, and current in the presence and absence of copper ions pro-
myricertin and luteolin. These four flavonoids share charac- vided us information on the reactivity of each flavonoid,
teristics, such as a 2,3-double bond and an oxo group on C-4, which functional groups were more redox-active and some
which are absent in the other four flavonoids. This new group understanding of the possible conformational structure of the
might be formed at the site between 4-oxo and 5-hydroxyl. flavonoids. As mentioned earlier, peak potentials have been
This site has been suggested, previously, for the chelation of used to assign the relative antioxidant power of flavonoids:
copper ions by these two sub-classes of flavonoids [6]. The the lower the peak potential, the higher its antioxidant power
commonality of OH groups on the B-ring for seven of the [7,9,10]. The copper chelation studies have confirmed that
eight flavonoids and the lack of this new functional group in there was indeed an inverse relationship between peak poten-
the other four flavonoids ruled out the B-ring as a potential tial and antioxidant power. Here we have equated antioxidant
source of the new peak potential. Another probable explana- power to copper ion chelation because copper ions have been
tion could be that the pyranose ring opened up at position C-1, reported to be involved in causing oxidative damage [27].
creating an OH group that was subsequently oxidised at this Chelation of these copper ions would reduce/inhibit copper-
potential. induced oxidative damage. Additionally, we have shown that
With the exception of baicalein, in the presence of copper the location of OH groups relative to each other is of ut-
ions, the oxidation potentials corresponding to the oxidation most importance in determining copper ion (metal) chelating
of resorcinol groups on the A-ring disappeared, i.e., they were ability. The pyrogallol group is more potent than the catechol
unavailable for oxidation. Increasing the ratio of flavonoid group, which in turn is more potent than the resorcinol group.
to copper ions, in some cases, enabled the re-appearance of The presence of the 4-oxo group in conjunction with the ox-
some functional groups. At 120 ␮M myricertin concentra- idation state of the C-ring also affects the redox potential of
tion, the peak potential at 0.646 V reappeared, suggesting the flavonoids. Chelation of copper on the site between the
that the resorcinol group is the least attractive to Cu(II) [6,7]. 4-oxo group and C-5 OH group in flavonols and flavones has
The peak potentials at 0.897 and 0.529 V reappeared at 120 already been proposed [6,7]. The number of OH groups is also
and 160 ␮M concentrations of baicalein, again suggesting important, the higher the number, the higher their chelating
that the higher the peak potential, the lower the affinity of ability is. The relationship between the number of OH groups
the oxidisable group to Cu(II). At 80 ␮M cyanidin, the peak and antioxidant activity of flavonoids has also been suggested
potential at 0.485 V reappeared. The corresponding peak po- [23]. Further, this study has shown that the conformational
tential for delphinidin reappeared at 160 ␮M delphinidin con- structure of the flavonoid also has an influence on the reactiv-
centration, perhaps indirectly confirming that the reactivity ity/antioxidant activity of the flavonoids. The number of OH
of the pyrogallol group is higher than that of the catechol groups on the B-ring was shown to affect the current detected.
moiety. The concentration of flavonoids was increased un- Although the pyrogallol group, was more redox-active than
til the copper signal disappeared (<20 nA), i.e., until all the the catechol moiety, its configuration somehow affected the
copper ions were chelated. These concentrations are listed in accessibility of the OH groups to the electrode surface. The
Table 3. importance of steric configuration and its effect on reactivity
The possible stoichiometry of the flavonoids and Cu(II), of a compound has been demonstrated, electrochemically, in
under the described experimental conditions, is shown DNA studies [32,33].
M. Vestergaard et al. / Analytica Chimica Acta 538 (2005) 273–281 281

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