Full Paper
Hydroxyanthraquinones Carminic Acid and Chrysazin Anodic
Oxidation
Eric de Souza Gil,a, b Severino Carlos B. de Oliveira,a Ana Maria de Oliveira-Brett*a
a
Departamento de Qumica, Faculdade de CiÞncias e Tecnologia, Universidade de Coimbra, 3004-535 Coimbra, Portugal
Faculdade de Farmcia, Universidade Federal de Gois, Setor Universitrio, 74605-220, Goinia, Gois, Brasil
*e-mail: brett@ci.uc.pt
b
Received: August 7, 2012;&
Accepted: September 6, 2012
Abstract
The anodic oxidation of the hydroxyanthraquinones carminic acid (CA) and chrysazin (CR) was investigated. The
oxidation of CA proceeds in a pH-dependent cascade mechanism, concerning the hydroquinone, the catechol and
the 3-OH groups in the anthraquinone moiety. The oxidation of the hydroquinone following the catechol electrondonating groups occurs first at low positive potentials, the 3-OH group is oxidized irreversibly at a higher potential.
The oxidation of CR is pH-dependent and occurs in successive steps. Oxidation of the hydroquinone tautomer in
the CR-ring occurs first, and the symmetrical 1-OH and 8-OH groups are irreversibly oxidized at the same higher
potential.
Keywords: Hydroxyanthraquinones, Carminic acid, Chrysazin, Oxidation mechanism, Tautomerism
DOI: 10.1002/elan.201200433
1 Introduction
Hydroxyanthraquinones are one the most common
groups of phenolic compounds widespread in the vegetal
kingdom, being important chemosystematic markers of
medicinal plants, i.e. Cassia and Senna genus [1–3], and
anthraquinones present a wide range of biotechnological
potentialities [3–8].
The ethnobotanical use of anthraquinones derivatives
and plants that contain hydroxyanthraquinones can be attributed to diverse physiological activities, which are
mainly associated to gastrointestinal and immunological
functions [1–5], and the mild laxative effect exhibited by
senna (Cassia senna), aloe (Aloe vera), ruibarbo (Rheum
palmatum L) and frangula bark (Rhamnus frangula) is
one the most common used plant-derived drugs [1–7].
Moreover, anthraquinones are good DNA-intercalating
agents, usually presenting good cytostatic activity, and important in anti-AIDS, anticancer, antimalarial, antibacterial and antifungal activity [3,6–9].
Anthraquinones have also historical importance in the
colorant industry. Indeed, natural anthraquinones dyes
are generally the main choice within coloring agents for
foods and pharmaceuticals, being also chemical precursors of many synthetic dyes used in textile and paint industry [4,10,11]. Furthermore, due to their redox activity,
anthraquinones have also been used as catalysts in many
industrial processes [12,13]. The redox processes of anthraquinones occur in the phenol and quinone moieties
[3–6,12–15].
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The chemical/biochemical processes linked to the biological and industrial applications are based on the chemical properties of hydroxyanthraquinones, as they deviate
from the 9,10 quinonid structure and exit in a tautomeric
dynamic equilibrium [16–18].
Carminic acid and chrysazin (Scheme 1) are hydroxyanthraquinones that have been widely studied for their biological and chemical properties [8–18].
Scheme 1. Chemical structures of the hydroxyanthraquinones:
carminic acid (CA) and chrysazin (CR).
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Chrysazin (CR), 1,8-dihydroxyanthraquinone, exist in
a tautomeric dynamic equilibrium [16–18], is also known
under the generic name Dantron, and is a natural component of laxative medicines [1,2,19]. CR has two electroactive phenolic groups, and is an effective redox mediator
in electrochemical reactions [20].
Carminic acid (CA), 7-b-d-glucopyranosyl-9,10-dihydro-3,5,6,8-tetrahydroxy-1-methyl-9,10-dioxo-2-anthracenecarboxylic acid, is a natural red dye extracted from the
body and eggs of the insect Dactylopius coccus, popularly
known as cochineal, has low toxicity, being mostly used in
food and cosmetic red dyes [10,11,21,22]. According to its
poly-hydroxylated anthraquinone core linked to a sugar
unit, CA exhibits higher aqueous-solubility, intense redox
activity, good chemical stability and has a tautomeric interchanging equilibrium, characterized by eight possible
forms [16–18,23]. The higher redox activity of CA leads
to a higher radical scavenging activity [23–25], and CA is
an effective redox mediator in electrochemical sensors
[24,25].
The aim of this work is to investigate the electrochemical behavior of CA and CR at glassy carbon electrode in
different electrolyte conditions, using cyclic, differential
pulse and square wave voltammetry that so far is not
clearly understood.
2 Experimental
2.1 Chemicals and Solutions
All reagents were of high purity analytical grade, carminic acid (CA) was obtained from Sigma Aldrich, while
chrysazin (CR), was obtained from Extrasynthse
(Genay, France). Stock solutions of 1.0 mM of CA, in deionized water and 1.0 mM of CR, in ethanol, were prepared and stored in the darkness at 4 8C.
The 0.1 M ionic strength electrolyte solutions were:
pH = 2.0 KCl/HCl, pH = 3.5–5.5 acetate buffer, pH = 6.0–
8.0 phosphate buffer, pH = 9.0–11.0 ammonia buffer, and
were prepared using analytical grade reagents and purified water from a Millipore Milli-Q system (conductivity
0.1 mS cm 1) [26]. All experiments were done at room
temperature (25 1 8C).
Microvolumes were measured using EP-10 and EP-100
Plus Motorized Microliter Pippettes (Rainin Instrument
Co. Inc., Woburn, USA). The pH measurements were
carried out with a Crison micropH 2001 pH-meter with
an Ingold combined glass electrode.
2.2 Electrochemical Measurements
Voltammetric experiments were carried out using an Autolab PGSTAT 10 potentiostat/galvanostat running with
GPES 4.9 software, Eco-Chemie, Utrecht, The Netherlands. Measurements were carried out using a glassy
carbon working electrode (GCE) (d = 1.0 mm), a Pt wire
counter electrode, and an Ag/AgCl (3 M KCl) as reference electrode, in a 1 mL one-compartment electrochemi2080
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cal cell. The experimental conditions for differential pulse
(DP) voltammetry were: pulse amplitude 50 mV, pulse
width 70 ms and scan rate 5 mV s 1. For square wave
(SW) voltammetry the experimental conditions were frequency 50 Hz and a potential increment of 2 mV, corresponding to an effective scan rate of 100 mV s 1.
The GCE was polished with alumina (particle size
1 mm, Sigma Aldrich) before each electrochemical experiment. After polishing, the electrode was rinsed thoroughly with Milli-Q water. Following this mechanical treatment, the GCE was placed in buffer supporting electrolyte and voltammograms were recorded until steady state
baseline voltammograms were obtained. This procedure
ensured very reproducible experimental results.
2.3 Acquisition and Presentation of Voltammetric Data
All DP voltammograms presented were background-subtracted and baseline-corrected using the moving average
with a step window of 2 mV included in GPES version
4.9 software. This mathematical treatment improves the
visualization and identification of peaks over the baseline
without introducing any artefact, although the peak
height is in some cases reduced (< 10 %) when compared
to untreated curve. Nevertheless, this mathematical treatment of the original voltammograms was used in the presentation of all experimental voltammograms for a better
and clearer identification of the peaks. The values for
peak current presented in all graphs were determined
from the original untreated voltammograms after subtraction of the baseline.
3 Results and Discussion
3.1 Cyclic Voltammetry
The oxidation behavior of CA and CR at a GCE was investigated by CV, scan rate 100 mV s 1, in 0.1 M acetate
buffer pH = 5.0.
CVs in 25 mM of CA were performed in the potential
range 0.2 V till + 1.3 V (Figure 1) and three consecutive
well–separated oxidation peaks, from the hydroquinone
moiety peak 1a, at Ep1a = + 0.47 V, from the catechol
moiety peak 2a, at Ep2a = + 0.60 V and from 3-OH group
peak 3a, at Ep3a = + 1.09 V, occurred on the first scan.
Successive scans without polishing the electrode between
the cycles clearly demonstrated a decrease of the oxidation currents due to the adsorption of CA oxidation products on the GCE electrode surface (Figure 1A). CVs in
75 mM of CA were performed in the potential range 0.0 V
till + 0.7 V. On the first scan peak 1a and peak 2a were
obtained, and on the reverse scan, a very broad cathodic
peak was observed, showing that peaks 1a and 2a correspond to quasi-reversible processes (Figure 1B).
CVs in 75 mM CR showed a peak 1a, at Ep1a = + 0.46 V,
and peak 2a, at Ep2a = + 1.12 V, and the oxidation products adsorbed strongly and block the electrode surface,
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Hydroxyanthraquinones Carminic Acid and Chrysazin Anodic Oxidation
Fig. 1. CVs in 0.1 M acetate buffer pH = 5.0 in CA: (A) 25 mM
and (B) 75 mM, (—) first and (·····) second scans, v = 100 mV s 1.
Fig. 2. CVs in 0.1 M acetate buffer pH = 5.0 in CR: (A) 75 mM
(—) first and (·····) second scans, v = 100 mV s 1 and (B) 300 mM
first scan, v = 10 mV s 1.
causing the rapid decrease of the CR oxidation peaks
(Figure 2A).
CVs in 300 mM CR were performed and the potential
was inverted before peak 2a, confirming the irreversibility
of peak 1a (Figure 2B). CVs were also obtained for different scan rates. Increasing the scan rate, the peak 1a current increases, but there was not a linear relationship between Ip1a and the square root of the scan rate, as expected for a diffusion-controlled oxidation process, due to the
strong adsorption of CR molecules and/or their oxidation
products on the GCE surface.
3.2 Differential Pulse Voltammetry
The oxidation of CA and CR in buffer supporting electrolyte solutions by DP voltammetry for 2.0 < pH < 12.0 (Figures 3, 4 and 5) was investigated.
The DP voltammograms in 0.1 M phosphate buffer
pH = 6.0 in 5 mM CA showed that the oxidation occurs in
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Fig. 3. DP voltammograms in 0.1 M phosphate buffer pH = 6.0
in 5 mM: CA (—) first and (·····) second scans and CR (—, gray)
first and (·····, gray) second scans, v = 5 mV s 1.
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Fig. 4. (A) 3D plot of DP voltammograms baseline corrected in
50 mM CA as a function of pH. (B) Plot of Ep1a (&), Ep2a (*) and
Ep3a (~) vs. pH.
Fig. 5. (A) 3D plot of DP voltammograms baseline corrected in
50 mM CR as a function of pH. (B) Plot of Ep1a (&) and Ep2a (*)
vs. pH.
three steps, peaks 1a, 2a and 3a (Figure 3) in agreement
with the CVs results.
The effect of pH on the anodic behavior of CA was
studied (Figure 4).
For 2.0 < pH < 9.0 the peaks 1a and 2a potential were
shifted to more negative values with increasing pH (Figure 4A). In the Epa vs. pH plot (Figure 4B) the slope of
the dotted line, 59 mV per pH unit, showed that the
mechanism of the peaks 1a and 2a, involve the same
number of electrons and protons. The number of electrons transferred was determined by the peak width at
half height W1/2 50 mV for both peaks, corresponding to
electrochemical reactions involving the transfer of two
electrons. CA peak 1a corresponds to the oxidation of the
hydroquinone group while the peak 2a is corresponds to
the oxidation of catechol group (Scheme 1) and both processes occur with the transfer of two electrons and two
protons. For pH 9.0, the oxidation peak 1a and 2a is
pH-independent indicating a mechanism involving only
one electron (Figure 4B).
CA peak 3a corresponds to an oxidation on the 3-OH
group of the CA-ring (Scheme 1). The potential shifted to
more negative values with increasing pH, and only occurs
for electrolytes with pH 10.0 (Figure 4A and B). In the
Epa vs. pH plot (Figure 4B) the slope of the dotted line,
59 mV per pH unit, shows that the mechanism involves
the same number of electrons and protons. Taking into
consideration that the width at half height of the CA oxidation peak 3a was W1/2 90 mV, it is concluded that the
oxidation with the transfer of one electron and one
proton [27].
The variation of peaks 1a, 2a and 3a current versus pH
shows that the current reaches a maximum in the 3.0 <
pH < 5.0 (Figure 4A) due to all CA OH groups being protonated in acid media and consequently increasing their
hydrophobicity and adsorption on the hydrophobic GCE
surface.
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Hydroxyanthraquinones Carminic Acid and Chrysazin Anodic Oxidation
for peak 1a, W1/2 60 mV, and for peak 2a, W1/2 95 mV,
corresponding to electrochemical reactions involving the
transfer of two electron and one electron respectively.
CR peak 1a corresponds to the oxidation processes of the
quinoidal moiety of CR with the transfer of two electrons
and two protons, while the peak 2a corresponds to the oxidation processes of the 1-OH and/or 8-OH groups with
the transfer of one electron and proton (Scheme 1). For
pH > 9.0, as the CR undergoes chemical deprotonation in
alkaline electrolytes, and the oxidation peak is pH-independent with a mechanism involving only one electron.
3.3 Square Wave Voltammetry
Fig. 6. SW voltammograms in 0.1 M acetate buffer pH = 5.0 in
50 mM: (A) CA and (B) CR, first scan. It : total current, If : forward current and Ib : backward current, veff = 100 mV s 1.
Successive DP voltammograms were also recorded in
5 mM CA in 0.1 M acetate buffer pH = 6.0 (Figure 3) and
was clearly demonstrated that the peaks 1a, 2a and 3a
current decreased with the number of scans due to the
decrease of the available electrode surface area owing to
the strong adsorption of CA oxidation products, in agreement with the CV results aforementioned.
The DP voltammograms in 0.1 M phosphate buffer
pH = 6.0 in 5 mM CR showed that the oxidation occurs in
two steps, peaks 1a and 2a (Figure 3), in agreement with
the CVs results.
The effect of pH on the anodic behavior of 50 mM CR
was studied (Figure 5).
For 2.0 < pH < 9.0 the peaks 1a and 2a potential were
shifted to more negative values with increasing pH (Figure 5A). In the Epa vs. pH plot (Figure 5B), the slope of
the dotted line, 59 mV per pH unit, showed that the
mechanism of peak 1a and 2a, involves the same number
of electrons and protons. The number of electrons transferred was determined by the peak width at half height
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The advantages of SW voltammetry are greater speed of
analysis, lower consumption of the electroactive species
and reduced problems with poisoning of the electrode
surface in relation with CV and DP voltammetry [27].
SW voltammograms in 50 mM of CA and CR showed similar features to DP voltammograms, i.e. the same number
of pH-depended anodic peaks (Figure 6).
Another great advantage of SW voltammetry is the
possibility to see during only one scan if the electron
transfer reaction is reversible or not. Since the current is
sampled in both positive and negative-going pulses, peaks
corresponding to the oxidation and reduction of the electroactive species at the electrode surface can be obtained
in the same scan. Thus, the reversible redox behavior of
both compounds is confirmed by plotting the equal value
of forward and backward components of the total current.
The quasi-reversibility of peaks 1a and 2a of CA (Figure 6A), and of peak 1a of CR (Figure 6B) is clearly
shown, as the backward components of the total current
are much smaller than the forward components of the
total current. The irreversibility of peak 3a of CA (Figure 6A), and peak 2a of CR (Figure 6B) was also confirmed in these experiments.
3.4 Redox Mechanism
The oxidation of OH groups occurs via the formation of
a phenoxy radical, and the oxidation potential of this process follows the order: hydroquinone < catechol ! resorcinol < phenol. The orto and para position enable reversible
oxidation with the transfer of two electrons and two protons, while the mono phenol oxidation occurs irreversibly
in a one electron one proton process [27]. However, hydroxyanthraquinones, as they deviate from the 9,10 quinonid structure and exit in a tautomeric dynamic equilibrium [16–18], their oxidation also follow other pathways
whereas the radical can initiate polymerization, leading to
adsorbed products on the electrode surface [4,20,23,27].
The SW, CV and DP voltammetric results of CA and
CR are all in agreement concerning the electrochemical
oxidation mechanisms, and it was found that the quinoidal and phenolic groups attached to the anthraquinone
were responsible for their electroactivity (Scheme 1).
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The electrochemical investigation in different pH environments enabled the determination for CA of pKa 9
from data in Figure 4B. The in vitro redox behavior of
CA is very important to predict in vivo reactions, and the
role of pKa on absorption is related with its lipophilicity
and solubility. For CA peaks 1a is related to the oxidation
of hydroquinone (5,8-di-hydroxyl) whereas peak 2a to the
oxidation of catechol (5,6-di-hydroxyl) electron-donating
groups, and peak 3a to the oxidation of 3-OH group
(Scheme 1).
For CR peak 1a is related to the oxidation of the quinoidal moiety obtained by di-hydro tautomer forms, with
the transfer of two electrons and two protons, while the
peak 2a to the oxidation processes of the 1-OH and/or
8-OH groups (Scheme 1), with the transfer of one electron and proton. Differences in the oxidation peak currents, lower for the di-hydro quinoidal group in peak 1a
and higher for the two symmetrical phenolic groups in
peak 2a, were observed in agreeing with tautomeric equilibriums.
According to spin density over hydroxyl groups the oxidation can be easier or not. Thus, electron withdrawing
substituents such as carboxyl group in the 4-OH position
of CA, as well as both carbonyl groups in CR tautomer,
hamper the peak 3a oxidation processes of the 3-OH
group of CA and of the peak 1a di-hydroquinoidal group
in CR [5,20,23–25].
4 Conclusions
Both CA and CR structures (Scheme 1) are associated
with tautomeric forms and their oxidation at the GCE
surface is a pH-dependent anodic process and their mechanism was investigated. CA oxidation occurs in a cascade
mechanism first in electron-donating groups, the hydroquinone (5,8-di-hydroxyl) moiety and the catechol (5,6di-hydroxyl) moiety, followed by the oxidation of the
3-OH group. CR oxidation processes are associated with
the tautomeric forms of the quinoidal moiety obtained by
its di-hydro tautomer form, followed by the oxidation of
the 1-OH and/or 8-OH groups.
Acknowledgements
Financial supports from Fundażo para a CiÞncia e Tecnologia (FCT), Post-Doctoral Grant SFRH/BPD/71965/2010
(S. C. B. Oliveira), Project PTDC/QUI/098562/2008,
POPH (co-financed by the European Community Funds
FSE e FEDER/COMPETE), CEMUC-R (Research Unit
285), and Coordenażo de AperfeiÅoamento de Pessoal de
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Ensino Superior (CAPES, Brasil) Post-Doctoral Grant
(E. S. Gil), are gratefully acknowledged.
References
[1] R. G. Ayo, J. Med. Plants Res. 2010, 4, 1339.
[2] A. T. J. Ogunkunle, T. A. Ladejobi, J. Biotechnol. 2006, 5,
2020.
[3] D. Kremer, I. Kosalec, M. Locatelli, F. Epifano, S. Genovese, G. Carlucci, M. Z. Koncic, Food Chem. 2012, 131,
1174.
[4] A. M. Faouzi, B. Nasr, G. Abdellatif, Dyes Pigments 2007,
73, 86.
[5] M. Shamsipur, A. Siroueinejad, B. Hemmateenejad, A. Abbaspour, H. Sharghi, K. Alizadeh, S. Arshadi, J. Electroanal.
Chem. 2007, 600, 345.
[6] H. Zhou, Z. Y. Sun, T. Hoshi, Y. Kashiwagi, J. Anzai, G. X.
Li, Biophys. Chem. 2005, 114, 21.
[7] R. W. Winter, K. A. Cornell, L. L. Johnson, L. M. Isabellec,
D. J. Hinrichsa, M. K. Riscoe, Bioorg. Med. Chem. Lett.
1995, 5, 1927.
[8] Z. Saddiqe, I. Naeem, A. Maimoona, J. Ethnopharmacol.
2010, 131, 511.
[9] S. Uzma, H. Nusrat, N. Jawed, Pakistan J. Botany 2011, 43,
2231.
[10] M. Squin-Frey, J. Chem Ed. 1981, 58, 301.
[11] D. W. Shaw, Dermatitis 2009, 20, 292.
[12] J. C. Forti, R. S. Rocha, M. R. V. Lanza, R. Bertazzoli, J.
Electroanal. Chem. 2007, 601, 63.
[13] M. A. Nkansah, A. A. Christy, T. Barth, Chemosphere 2011,
84, 403.
[14] E. Tutem, R. Apak, K. Sozgen, J. Inorg. Biochem. 1996, 61,
79.
[15] Q. Wang, F. Gao, X. Yuan, W. Li, A. Liu, K. Jiao, Dyes Pigments 2010, 84, 213.
[16] V. Y. Fain, B. E. Zaitsev, M. A. Ryabov, Chem. Nat. Comp.
2006, 42, 269.
[17] V. Y. Fain, B. E. Zaitsev, M. A. Ryabov, Russ. J. Gen. Chem.
2007, 77, 1769.
[18] V. Y. Fain, B. E. Zaitsev, M. A. Ryabov, Chem. Nat. Comp.
2005, 41, 501.
[19] M. Bennett, H. Cresswell, Palliative Med. 2003, 17, 418.
[20] A. Vuorema, P. John, A. T. A. Jenkins, F. Marken, J. Solid
State Electrochem. 2006, 10, 865.
[21] M. J. Greenhawt, J. L. Baldwin, Ann. Allerg. Asthma Im.
2009, 103, 73.
[22] O. Reyes-Salas, M. Juarez-Espino, J. Manzanilla-Cano, M.
Barcelo-Quintal, J. Mex. Chem. Soc. 2011, 55, 89.
[23] G. X. Li, Z. Q. Liu, D. Wu, J. Phys. Org. Chem. 2009, 22,
883.
[24] W. Sun, Y. Y. Han, K. Jiao, J. Serb. Chem. Soc. 2006, 71,
385.
[25] A. P. Periasamy, Y. H. Ho, S. M. Chen, Biosens. Bioelectron.
2011, 29, 151.
[26] R. Beynon, J. Easterby, The Basics Buffer Solutions, Oxford
Science University Publications, Oxford 1996.
[27] T. A. Enache, A. M. Oliveira-Brett, J. Electroanal. Chem.
2011, 655, 9.
2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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