Voltammetric study of the oxidation of quercetin and catechin in the presence of cyanide ion

Article abstract of DOI:10.1007/s11164-011-0312-0

The reaction of electrochemically generated o-benzoquinones from oxidation of quercetin and catechin as Michael acceptors with cyanide ion as nucleophile has been studied using cyclic voltammetry. The reaction mechanism is believed to be EC; including oxidation of catechol moiety of these antioxidants followed by Michael addition of cyanide ion. The observed homogeneous rate constants (k_obs) for reactions were estimated by comparing the experimental voltammetric responses with the digitally simulated results based on the proposed mechanism. The effects of pH and nucleophile concentration on voltammetric behavior and the rate constants of chemical reactions were also described.

Full text of DOI:10.1007/s11164-011-0312-0

Res Chem Intermed (2011) 37:1047–1055
DOI 10.1007/s11164-011-0312-0
Voltammetric study of the oxidation of quercetin
and catechin in the presence of cyanide ion
•
Lida Khalafi Mohammad Rafiee Farzaneh Yadaei
•
Received: 25 January 2011 / Accepted: 16 April 2011 / Published online: 4 May 2011
Ó Springer Science+Business Media B.V. 2011
Abstract The reaction of electrochemically generated o-benzoquinones from
oxidation of quercetin and catechin as Michael acceptors with cyanide ion as
nucleophile has been studied using cyclic voltammetry. The reaction mechanism is
believed to be EC; including oxidation of catechol moiety of these antioxidants
followed by Michael addition of cyanide ion. The observed homogeneous rate
constants (kobs) for reactions were estimated by comparing the experimental vol-
tammetric responses with the digitally simulated results based on the proposed
mechanism. The effects of pH and nucleophile concentration on voltammetric
behavior and the rate constants of chemical reactions were also described.
Keywords Catechin Á Quercetin Á o-Quinone Á Cyanide ion Á Digital simulation
Introduction
An antioxidant is defined as a molecule capable of slowing or preventing the
oxidation of other molecules [1], and flavonoids are the most widely used group of
antioxidants. The electrochemical oxidation of flavonoids is of great interest
because of their action as antioxidants with the ability to scavenge radicals by
electron transfer processes [2]. Also, many other biologically important processes
involve redox reactions, and electrochemical methods compose a collection of
L. Khalafi (&)
Department of Chemistry, Islamic Azad University, Shahr-e-Qods Branch, Tehran, Iran
e-mail: l_khalafi@yahoo.com
M. Rafiee
Department of Chemistry, Institute for Advanced Studies in Basic Sciences (IASBS), Zanjan, Iran
F. Yadaei
Department of Chemistry, Islamic Azad University, Saveh Branch, Saveh, Iran
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extremely useful tools for the analysis of their reactions. Among the characteristics
of electrochemical methods, in situ generation of reactive intermediates and
inversion in polarity by transfer of electrons at the electrode–solution interface have
found considerable attention in various areas of chemistry [3]. Molecular
electrochemistry studies the mechanistic events at or near an electrode on a
molecular level and voltammetry is convenient and also the most frequently used
technique for this purpose [4].
Quercetin and catechin as natural flavonoids were selected for this study. They
have several biological activities including radical scavenging, anti-inflammatory,
antimutagenic, and anti-cancer activities [5]. Quercetin is a frequent component of
major dietary constituents, such as onions and apples [6]. Research shows that
quercetin influences cellular mechanisms in vitro and, in animal studies, there is
evidence from human population studies that quercetin may, in a very limited
fashion, reduce the risk of certain cancers [7]. Catechin is extracted from plants and
is present in natural foods and drinks such as green tea [8]. The application of
catechin as a phenolic standard in quantization of reducing equivalents in food and
biological fluids and its oxidation mechanism are of particular interest [9]. The
interest in the quantification of catechin is consistent with its importance as being
potentially beneficial to human health. The evidence of the presence of catechin in
foodstuffs can be related to epidemiological studies that take into account the
relationships between catechin intake and diseases [10].
Quercetin and catechin can be chemically or electrochemically oxidized and their
electrochemical behaviors have been studied in some papers [11]. Both quercetin
and catechin have a catechol skeleton in their structures and electrochemical
experiments show that the first oxidation of these compounds is related to the
oxidation of their catechol group. Oxidation of catechol derivatives occurs at very
low positive potentials, and is a reversible reaction. The product of oxidation
generates o-quinone, a highly reactive species, which undergoes various chemical
reactions. It can be attacked by the side chain group of parent molecules or suitable
nucleophiles via a Michael addition reaction [12, 13].
The aim of this work is the electrochemical oxidation of catechin and quercetin in
the presence of cyanide ion and then the estimation of the rate constants of reactions
by digital simulation. Cyanide is acutely toxic to mammals by all routes of
administration, with a very steep and rate-dependent dose–response curve that
involves inactivation of cytochrome oxidase and inhibition of cellular respiration
and consequent cellular anoxia. In humans and animals, cardiovascular, respiratory,
and central nervous systems are primarily affected [14].
Experimental
Apparatus
Cyclic voltammetry was performed using a Behpajoh Model BHP 2061-C
potentiostat/galvanostat. In the voltammetry experiments, a glassy carbon disc
(2 mm in diameter) and a platinum wire were used as working and counter
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Study of the oxidation of quercetin and catechin
1049
electrodes, respectively. The working electrode potentials were measured versus
Ag/AgCl (KCl 3.0 M). All electrodes were from Azar Electrode (Ourumieh, Iran).
Reagents and solutions
All of the experiments were performed with analytical reagent-grade chemicals
purchased from Merck. These chemicals were used without further purification. The
stock solutions of the catechin and sodium cyanide were prepared fresh daily by
dissolving the compounds in distilled water. The stock solution of quercetin was
prepared daily by dissolving it in distilled water/acetonitrile (60/40) solution.
Samples were prepared by taking the appropriate aliquots from the stock solutions
followed by dilution with buffer solutions. The 0.15 M buffered solutions were
prepared based on Kolthoff tables [15]. The homogeneous rate constants were
estimated by analyzing the cyclic voltammetric responses using the simulation
CVSIM software [16].
Results and discussion
Voltammetric study
Figure 1, curve a, shows the cyclic voltammograms of 1.0 mM quercetin in the
absence of cyanide ion at pH 6.0 in the mixture of acetonitrile/water, 20/80,
solution. The cyclic voltammograms of quercetin shows one anodic (A1) and
corresponding cathodic peak (C1) with peak current ratio nearly but less than unity,
Fig. 1 Cyclic voltammograms of 1.0 mM quercetin: a in the absence, b in the presence of 1.0 mM of
cyanide ion, at glassy carbon electrode, in aqueous/acetonitrile solution containing phosphate buffer
(pH = 6.0, c = 0.15 M), Scan rate 100 mV s-1
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within two-electron process. The electrode response of quercetin can be related to
the transformation of its catechol group to o-quinone and vice versa.
Figure 1, curve b, shows a cyclic voltammogram of 1.0 mM quercetin in the
presence of 5.0 mM cyanide ion in the same solution. In this condition, the most
important difference between the voltammograms in the presence and absence of
cyanide ion is decreasing magnitude of cathodic peak (C1) currents. As mentioned
above, the C1 peak is due to reduction of o-quinone and its currents are proportional
to the quantity of it. A decrease in the C1 peak currents is indicative of this fact that
the oxidized intermediate formed at the surface of electrode removed by a chemical
reaction.
The time scale of a voltammetric experiment is determined by the scan rate. It is
an important parameter in the study of homogeneous reactions coupled with
electrode reactions [17]. Figure 2 shows the effect of the potential scan rate on
voltammograms of quercetin in the presence of cyanide ion at pH 6.0. It is seen that
when the scan rate is decreased, the C1–A1 current peak ratio decreases. This is
reflected the low extent of chemical reaction at the period of recording the cyclic
voltammograms at high scan rates. The inset of Fig. 2 shows the peak current ratio
(IpC/IpA) versus scan rate for a mixture of quercetin and cyanide ion.
Also the voltammetric studies were performed at various concentrations of
cyanide ion (Fig. 3). By increasing the concentration of cyanide ion, the height of
the cathodic peaks and their ratio over the A1 peak is decreased.
Such behaviors are good criterions for EC mechanism consists of an electron
transfer reaction (E) followed by a chemical reaction (C) [17]. According to these
results, electrochemical oxidation of quercetin causes the formation of related
Fig. 2 Cyclic voltammograms of 1.0 mM quercetin in the presence of 5.0 mM cyanide ion at a glassy
carbon electrode in phosphate buffer solution (pH = 6.0, c = 0.15 M). Scan rates from a to e are 50, 100,
200, 400, and 800 mV s-1, respectively. Inset variation of cathodic to anodic peak current ratios versus
scan rate
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Study of the oxidation of quercetin and catechin
1051
Fig. 3 Cyclic voltammograms of 1.0 mM quercetin in the presence of different concentration cyanide
ion, pH = 6.0, Concentrations from a to d are 1.0, 2.0, 4.0, and 8.0 mM; Scan rate 100 mV s-1
o-quinone that undergoes Michael addition reaction with cyanide ion. It is faster
than other secondary reactions and leads to the related nitrile derivatives. The
oxidation of the product is more difficult than the oxidation of the parent molecules
OH
-2e- -2H+
O
O
HO
O
O
HO
O
O
OH
OH
OH
OH
OH
quercetin
O
O
NC
OH
OH
+ CN-
HO
O
HO
O
OH
O
OH
OH
O
OH
OH
NC
OH
OH
OH
HO
O
HO
O
-2e- -2H+
+ CN-
OH
OH
catechin
OH
OH
Scheme 1 Proposed mechanism for electrochemical oxidation of quercetin and catechin in the presence
of cyanide ion
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L. Khalafi et al.
and was prevented by virtue of the presence of the electron-withdrawing nitrile
group on catechol ring [18]. The proposed reaction pathway is shown in Scheme 1.
Because of the dependence of the quercetin oxidation pathway and the acid base
equilibrium of cyanide ion on pH, the voltammetric studies were performed at
various pHs (Fig. 4).
In basic solutions, the height of quercetin reduction peak decreases due to some
considerable side reactions. They consist of the intramolecular reaction of hydroxy
groups and coupling of anionic forms of quercetin with o-quinones, but the peak
current ratio near unity at neutral and acidic solutions can be considered as relative
stability of the o-quinone [12]. Therefore, the effect of pH has been studied at pHs
lower than 7.0. At the desired solutions and presence of cyanide ion, the peak
current ratio is less than unity and decreases with increasing pH. The plot of the
current peak ratio versus solution pH is shown in the inset of Fig. 4. Cyanide ion is a
weak base and variations of reaction rates with pH are due to protonation of cyanide
ion and variation of its percentage [15, 22]. The electrochemical study of catechin
has been studied in aqueous solution using the method described for quercetin. The
results of the investigation of catechin in the presence of cyanide ion are the same as
those obtained for quercetin.
Kinetic evaluation
The schemes for the electrochemical oxidation of quercetin and catechin in the
presence of cyanide ion were proposed and tested by diagnostic criteria of cyclic
voltammograms. One of the successful methods for obtaining kinetic parameters of
the coupled homogeneous reaction is digital simulation [19]. The simulation was
Fig. 4 Cyclic voltammograms of 1.0 mM quercetin in the presence of 5.0 mM cyanide ion, at glassy
carbon electrode, at various pH values a 3.7, b 4.5, c 5.2 and d 6.0; Scan rate 200 mV s-1
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Study of the oxidation of quercetin and catechin
1053
carried out based on proposed EC mechanism and assuming semi-infinite one-
dimensional diffusion on a planar electrode. The experimental parameters entered
for digital simulation consist of the following: starting potential (Estart), switching
potential (Eswitch), scan rate (v), half wave potential (E1/2), and analytical
concentration of species. The formal potentials were obtained experimentally as
midpoint potential between the anodic and cathodic peaks (Emid). The transfer
coefficients (a) and heterogeneous rate constants for oxidation of quercetin and
catechin were estimated by experimental working curves [20, 21]. All of these
parameters were kept constant and the observed rate constant of chemical reaction
kobs was allowed to change during the fitting processes. The fitting consists of
finding a rate constant for which the differences between the digitally simulated and
the experimental data reach to its minimum [22]. The rate constants of the reaction
of produced quinones from oxidation of quercetin and catechin with cyanide ion
were estimated for various pHs, cyanide ion concentrations and scan rates. As is
shown in Fig. 5, there are good agreements between the simulated voltammograms
with those obtained experimentally.
The observed rate constant of coupling reaction of oxidized quercetin with
cyanide ion at various pHs are presented in Table 1. Based on these results, the rate
of reaction enhances at high pH values, which are related to increasing the cyanide
ion percentages.
Also, the observed rate constants of reactions were obtained by digital simulation
at various concentrations of cyanide ion. Figure 6 shows the plots of kobs as a
function of cyanide ion concentration.
The observed rate constants (kobs) increase linearly as a function of cyanide ion
concentrations, which is expected based on voltammetric results. Each point is the
average of five independent simulations at various scan rates. Since kobs = k [CN-],
where k is the rate constant of reaction, therefore, the value of rate constant can be
obtained from the slope of the obtained plot in Fig. 6. The values of the obtained
rate constants (k) at pH 6.0 are 37.6 and 34.4 M-1 -1
for catechin and quercetin,
s
respectively. The standard deviations were obtained for four independent scan rates
for each concentration of cyanide ion. The standard deviations are 9.3% for catechin
and 7.4% for quercetin.
Conclusions
The results of this work show that quercetin and catechin are oxidized to their
respective o-quinones derivative at very mild conditions. Electrochemically
generated o-quinones are quiet reactive toward the nucleophilic attack of cyanide
ion. The Michael addition reaction of this nucleophile with o-quinones leads to the
formation the nitrile derivatives of parent flavonoids. Based on voltammetric and
simulation results, the chemical reaction, which leads to the formation of
corresponding nitrile derivatives of flavonoids, has considerable homogeneous rate
constants. This is a good example of in situ generation of reactive species by
electrochemical methods and kinetic study of coupled homogeneous reaction by the
analysis of voltammetric responses.
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L. Khalafi et al.
Fig. 5 Simulated (doted) and experimental (line) cyclic voltammograms of quercetin (I) and catechin
(II) at various scan rates a 100, b 200, and c 400 mV s-1
Table 1 Homogeneous rate constants (kobs) for quercetin at various pHs
pH
3.7
4.5
5.2
6.0
kobs (s-1 a
)
0.002
0.004
0.014
0.054
a
The observed rate constants in the presence of 5.0 mM cyanide ion and scan rate of 200 mV s-1
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Study of the oxidation of quercetin and catechin
1055
Fig. 6 The plot of observed rate constant (kobs) versus cyanide ion concentration for Michael addition
reaction of oxidized quercetin at pH 5.2
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