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Electrochimica Acta 133 (2014) 359–363

Contents lists available at ScienceDirect

Electrochimica Acta

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wo oxidation pathways of bioactive flavonol rhamnazin undermbient conditions

ˇárka Ramesováa,1, Ilaria Deganob, Romana Sokolováa,∗,1

J. Heyrovsky Institute of Physical Chemistry, v.v.i., Academy of Sciences of the Czech Republic, Dolejskova 3, 18223 Prague, Czech RepublicDepartment of Chemistry and Industrial Chemistry, University of Pisa, Via Risorgimento 35, 56100 Pisa, Italy

r t i c l e i n f o

rticle history:eceived 6 January 2014eceived in revised form 9 April 2014ccepted 11 April 2014vailable online 21 April 2014

a b s t r a c t

Two pathways of the oxidation mechanism of rhamnazin under ambient conditions are proposed. Theredox potential of rhamnazin strongly depends on the presence of dissociation forms in solution. In situspectroelectrochemistry and identification of degradation products by HPLC-DAD and HPLC–ESI-MS/MSconfirmed the presence of fast subsequent chemical reactions following the electron transfer. As demon-strated, strict anaerobic conditions have to be preserved in studies of antioxidant properties and of its

eywords:xidationlavonoidshamnazin

pharmacological efficiency. In the absence of oxygen, 2,4-dihydroxy-2-(4′-hydroxy-3′-methoxybenzoyl)-6-methoxy-benzofuran-3(2H)-one was identified as the only oxidation product.

© 2014 Elsevier Ltd. All rights reserved.

lectron transferigh Pressure Liquid Chromatography

. Introduction

Rhamnazin (3,4′,5-trihydroxy-3′,7-dimethoxyflavone) 1, foundn Rhamnus disperma sp. or Elsholtzia sp., belongs to thearge group of flavonols (the inset of Fig. 1A). The oxidationroperties of flavonols are widely investigated due to their phar-aceutical importance: flavonols exhibit antioxidant activity,

nti-hepatotoxic and anti-inflammatory properties, and inhibitertain enzymes [1–3]. Their oxidation mechanism has not beenompletely clarified yet. We found that 1 is unstable when exposedo atmospheric oxygen in alkaline, neutral and also in acidic solu-ions. This behaviour makes difficulties in the identification ofhamnazin efficiency and its analytical determinations. The aim ofhis work is the determination of the oxidative degradation pro-esses of rhamnazin 1 in aqueous solutions in both, the absencend presence of oxygen. The oxidation mechanism of 1 is supportedy in situ spectroelectrochemistry and HPLC–ESI-MS/MS and HPLC-AD identification of oxidation products. The oxidation mechanismf 1 has never been studied in literature.

The inset of Fig. 1A shows the chemical structure of the flavonol

uercetin 2, which differs from rhamnazin 1 due to the absencef two methoxy- groups, which may play an important role in itsed-ox properties: the participation of protons in the oxidation

∗ Corresponding author.E-mail address: romana.sokolova@jh-inst.cas.cz (R. Sokolová).

1 ISE member.

ttp://dx.doi.org/10.1016/j.electacta.2014.04.074013-4686/© 2014 Elsevier Ltd. All rights reserved.

process of hydroxy-compounds is known [4–14]. The oxidationmechanism of the compound 2 in aqueous solution and thedifferences in number of electrons involved in its oxidation wereclarified in our recent paper [15]. The compound 1 is unstablewhen exposed to atmospheric oxygen. A poor stability of 1 inalkaline, neutral and also in acidic solutions is consistent withthat of quercetin 2: strictly deaerated conditions were proved tobe required when working with its solutions, mainly in alkalinesolutions [16]. Concerning the oxidation mechanism of 2, whichis proved in the literature [15,17,18] and also described [19],two-electron and two-proton oxidation of the ring B in molecule of2 leads to formation of o-quinone derivative in acidic and neutralsolutions. Subsequent chemical reactions such as hydroxylation ordimerization take place [15,20]. 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxybenzofuran-3(2H)-one as one of the oxidation productsof 2 was isolated and identified [19]. In our previous works thiscompound was found as the main oxidation product of 2 underinert atmosphere in aqueous and non-aqueous solution [13–16]and its presence was also confirmed by other techniques [21].

2. Experimental

2.1. Reagents

Rhamnazin and quercetin were purchased from Sigma Aldrich.The reagents used as supporting electrolytes such as potassiumchloride and chemicals for preparation of Britton-Robinson (BR)buffers (0.04 mol l−1 stock solutions of H3PO4, CH3COOH, H3BO3

360 S. Ramesová et al. / Electrochimica

Fig. 1. Cyclic voltammogram of 3 × 10−4 mol l−1 (a) rhamnazin 1 and (b) quercetin 2iew

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n Britton Robinson buffer and 40% ethanol (v/v) on glassy carbon electrode at differ-nt pH: (A) 6.6, (B) 10.8. Scan rate was 0.25 V s−1. Cyclic voltammograms registeredhen changing the polarity behind the oxidation peak I (dotted lines).

nd 0.2 mol l−1 NaOH) were of reagent grade. BR buffers (pH.3–11.4) were prepared at constant ionic strength. The solutionsere prepared with ultrapure water (Millipore). Methanol and

cetonitrile were HPLC grade (Carlo Erba, Milan, Italy). Standardolutions for HPLC were prepared in methanol. All reagents andhemicals were used without any further purification.

.2. Methods

.2.1. Electrochemical setupElectrochemical measurements were done in Britton-Robinson

uffers or 0.1 mol l−1 KCl using an electrochemical system for cyclicoltammetry. It consisted of a fast rise-time home-made potentio-tat interfaced to a personal computer via an IEEE–interface cardAdvanTech, model PCL–848) and a data acquisition card (PCL–818)sing 12-bit precision. A three-electrode electrochemical cell wassed with an Ag|AgCl|1 mol l−1 LiCl reference electrode separatedrom the test solution by a salt bridge (0.43 V against Fc/Fc+ car-oxylic acid).

The working electrode was a glassy carbon microelectrodediameter 0.7 mm). The auxiliary electrode was a platinum net.xygen was removed from the solution by passing a stream ofrgon. The oxidation products of 1 were prepared by exhaustivelectrolysis on carbon paste electrode in the concentration rangerom 2.4 × 10−4–6.2 × 10−4 mol l−1 solutions. The exhaustive elec-rolysis and cyclic voltammetry before and after electrolysis wereerformed using a PGSTAT 12 AUTOLAB potentiostat (Metrohmutolab, Netherlands).

.2.2. UV-VIS spectroelectrochemistrySpectroelectrochemistry was performed using an optically

ransparent thin-layer electrode (OTTLE) cell [22] with a threelectrode system (platinum working and auxiliary electrode,

Acta 133 (2014) 359–363

silver quasi reference electrode) mounted in a thin layer (thickness1.7 mm) between optical windows. Sufficiently optically trans-parent platinum gauze (80 mesh) of the size 5 × 5 mm served asthe working electrode. The Ag-wire serves as quasireference elec-trode (0.35 V against Fc/Fc + -carboxylic acid). The response of thecell allows to complete electrolysis within time of several tensof seconds (20 s when tested with ferrocene in acetonitrile). Thepotential scan rate was 5 mV s−1. Spectral changes in the course ofelectrolysis were registered using Agilent 8453 diode-array UV-Visspectrometer. The 1.0 cm quartz cuvettes were used for recordingthe absorption spectra when testing the stability of compoundwhen exposed to the air oxygen.

2.2.3. High-pressure liquid chromatography with diode arraydetector

An high-pressure liquid chromatography Agilent 1200 SeriesHPLC Systems with a G1311A Quaternary Gradient Pump andG1322A degasser equipped with a G1329A autosampler and aG1315B diode array detector (Agilent Technologies) was used. Thechromatographic separation was performed on analytical reversephase C8 column (HyPurity C8, 150 × 3 mm, 5 �m, Thermo Scien-tific, Dubuque, USA) connected to C18 pre-column (HyPurity C18,10 × 3 mm, 5 �m, Thermo Scientific, Dubuque, USA). Column tem-perature was 20 ◦C. The gradient elution program used eluents (A):aqueous solution of 0.1% H3PO4 and (B): acetonitrile. The gradientwas: 0 - 2 min, 95% A; 2–30 min, linear gradient to 40% A; 30–35min, linear gradient to 0% A and 100% B; 35–40 min, 100% B. Theflow rate was 0.2 mL min−1, the injection volume was 40 �l. Diodearray detector acquisition parameters were: acquisition range 190-800 nm, 2 nm step.

2.2.4. High-pressure liquid chromatography with electro-sprayionisation tandem mass spectrometer

HPLC-ESI-MS/MS was carried out using a 1200 Infinity HPLC(Agilent Technologies, USA), coupled to a Jet Stream ESI inter-face (Agilent) with a Quadrupole-Time of Flight tandem massspectrometer 6530 Infinity Q-TOF (Agilent Technologies). The chro-matographic separation took place at 30 ◦C and was performed onan analytical reverse phase C-18 column (C18-extended 1.8 �m,50 × 2.1 mm, Agilent Technologies, USA) connected to a C-18 pre-column (TC-C18 (2) 5 �m, 12.5 × 2.1 mm, Agilent Technologies,USA).

The eluents were: A, 90% H2O with 1% formic acid, and B, 10%acetonitrile with 1% formic acid, and the flow rate was 0.2 mL/min.Injection volume was 1 �L. The programme was: 90% A and 10% Bfor 2 minutes; then to 50% A and 50% B in 9 minutes; then to 30% Ato 70% B in 3 minutes; then to 10% A to 90% B in 5 minutes, hold for4 minutes. Conditioning takes 5 minutes.

The ESI operating conditions were: drying gas (N2, purity >98%):350 ◦C and 10 L min−1; capillary voltage 4.5 kV; nebulizer gas 35 psig;sheath gas (N2, purity >98%): 375 ◦C and 11 L min−1. The collisiongas was nitrogen (purity 99.999%). The fragmentor potential was175 V and the collision energy for the MS/MS experiments rangedbetween 20 and 50 V depending on the target compound.

High resolution MS and MS/MS spectra were achieved innegative mode in the range 100-3200 m/z; the mass axis was cali-brated using the Agilent tuning mix HP0321 (Agilent Technologies)prepared in acetonitrile and was also corrected in continuo by com-parison with the m/z 121,050873 and 922,009798 from a referencesolution in acetonitrile of purine and HP0921 reference solution(Agilent Technologies).

2.2.5. Theoretical calculationsMolecular orbital calculations were performed using the density

functional theory (DFT) calculations with B3LYP/6-31G* basis set(Spartan ‘10, v.1.1.0). Calculated values of HOMO, LUMO energies

S. Ramesová et al. / Electrochimica Acta 133 (2014) 359–363 361

Fig. 2. Cyclic voltammogram of 3 × 10−4 mol l−1 rhamnazin in Britton Robinsonb6o

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The oxidation of 1 under ambient conditions was investigatedby means of UV-Vis spectrometry and separation techniques.Fig. 4 shows the absorption spectra of 1 during the exposure

uffer and 40% ethanol (v/v) on glassy carbon electrode at different pH: (a) 4.3, (b).6, (c) 7.5, (d) 8.9, (e) 9.9, (f) 10.5, (g) 11.4. Scan rate was 0.25 V s−1. The dependencef the anodic peak potential on pH is shown in inset.

nd electrostatic potentials of hydroxyl groups atoms for com-ounds 1 and 2 are summarized in SuppTable S1.

. Results and discussion

.1. The electrochemical properties in buffered solution

Cyclic voltammetry of rhamnazin 1 yields three oxidation wavesn a glassy carbon electrode up to the potential 1.2 V in aque-us solution at pH = 6.6 (Fig. 1A). The first electron oxidation wavet 0.33 V (labelled as I) is reversible. The dependence of the peakurrent on the square root of the scan rate is linear and thus thexidation process is diffusion controlled. Oxidation wave I corre-ponds to the oxidation of the hydroxyl group 4′-OH at the ring

and occurs at higher potential than the corresponding wave inhe case of 2. This difference corresponds to the difference in theirHOMO energies, EHOMO = −515.17 kJ mol−1 and EHOMO = - 528.89 kJol−1, respectively (see Supporting information). The second wave

I is due to the oxidation of a product formed at the potential ofxidation wave I [13,15]. The third oxidation wave III belongs tohe oxidation of a hydroxyl group at the ring A, as in the case of

[13,15,17,20]. Cyclic voltammograms of 1 were measured at dif-erent pH values ranging from 4.3 to 11.4 (Fig. 2). The potentialf all three oxidation waves is pH dependent, proving that pro-ons participate in the oxidation process. The potential of eachxidation wave decreases with increasing pH of solution and thishift is accompanied with the loss of the reversibility of oxidationave I. Significant differences in the electrochemical behaviour of

and 2 were observed in alkaline solutions. Cyclic voltammetry of at pH = 10.8 showed an additional one-electron reversible waveelated to the oxidation of the dianion of 2 present in the solu-ion at this pH (see Fig. 1B) [15]. This one-electron oxidation waves not present in the case of 1, neither at the highest pH values.he pK1 or pK2 for 1 are not known, except the calculated valueK1 = 6.14 ± 0.40 [23]. We suppose that compound 2 dissociatesore easily to a dianion than compound 1 due to the presence

f two hydroxyl groups at the ring B. The inset of Fig. 2 shows theinear dependence of peak potential vs. pH and the slope is 66 mVH−1. The logarithmic analysis of anodic currents for the whole pHange measured approaches RT/F, so the results are consistent withverall two-electron and two-proton quasireversible oxidation.

.2. The UV-Vis spectroelectrochemistry

During the electrolysis, UV-Vis spectra were obtained bypectroelectrochemistry in an OTTLE cell. The spectra obtaineduring the electrolysis by at the potential of the first oxidation

Fig. 3. The in situ spectroelectrochemistry of 6 × 10−4 mol l−1 rhamnazin in solutionof Britton Robinson buffer and 40% ethanol (v/v), pH = 9.0, (A) at the potential behindthe oxidation wave I, and (B) at the potential of the oxidation wave II.

wave in solution of pH 5.2 show a decrease of the absorptionbands at 251 nm, 376 nm and the increase of the absorptionband at 296 nm (see SuppFig. S1 in Supporting information). Theabsorption spectrum of the solution after electrolysis matches tothe absorption spectrum of the benzofuranon derivative formedby oxidation of 2 [15,19] The identification of 2,4-dihydroxy-2-(4′-hydroxy-3′-methoxybenzoyl)-6-methoxy-benzofuran-3(2H)-one(R1) (Scheme 1) as the main oxidation product was supportedby HPLC-DAD and HPLC-ESI-MS analysis. The sharp isosbesticpoint indicates that the subsequent chemical reactions are fastunder these experimental conditions. Similar behaviour occurs insolution at pH 9.0 (Fig. 3) and in non buffered conditions of 0.1 moll−1 KCl and 3.6 × 10−3 mol l−1 KOH. The formation of the oxidationproduct R1 is accompanied by a decrease of absorption bands at231 nm, 266 nm and 420 nm and an increase of bands at 255 nmand 366 nm (Fig. 3A). A decomposition of R1 occurs during theoxidation at the potential of the oxidation wave II (Fig. 3B).

3.3. Oxidation under ambient conditions and the identification ofoxidation products

Fig. 4. Absorption spectra of 3.6 × 10−5 mol l−1 rhamnazin in 0.1 mol l−1 KCl and3.6 × 10−3 mol l−1 KOH (A) during exposure to atmospheric oxygen for (a) 0, (b)360, (c) 560, (d) 760, (e) 1160, (f) 1560, (g) 2520, (h) 4520 s, and (B) (h) 4520 s, (i)92 min, (j) 122 h and (k) 124 h.

362 S. Ramesová et al. / Electrochimica Acta 133 (2014) 359–363

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Scheme 1. The oxidatio

o atmospheric oxygen. 1 is fully degraded in one hour and itsxidation product is also not stable (Fig. 4A and 4B, respectively).he formation of oxidation products in solutions kept under inerttmosphere and in solutions exposed to the air is shown in Fig. 5.

ig. 5. HPLC-DAD chromatogram of a solution of 1 in the absence of oxygen (A)efore the electrolysis and (B) after the electrolysis. (C) and (D): chromatograms ofolutions collected during and before electrolysis and exposed to the air oxygen,espectively.

chanism of rhamnazin.

The cyclic voltammogram after the exhaustive electrolysis at thepotential behind the first oxidation wave under inert atmospherecontains oxidation wave II belonging to the oxidation of R1, andthe oxidation wave III (not shown). The electric charge required forthe electrolysis corresponds to the consumption of two electrons.The presence of R1 in solution after the electrolysis and its poorstability are confirmed by HPLC analysis (see Fig. 5B and 4 C,respectively). R1 and the other compounds in the chromatogramswere characterised by acquiring their ESI-Q-ToF spectra. Themain m/z values of R1 are: 345.06 and 183.03. Compound R1decomposes under ambient conditions to R2 (m/z values: 167.03),R3 (m/z values: 195.03 and 123.04) and R4 (m/z values: 211.02and 167.03). The second oxidation pathway of 1 under ambientconditions leads to the formation of 2-hydroxy-6-(4-hydroxy-3-methoxy benzoyloxy)-4-methoxyphenyl-2-oxoacetic acid R7(m/z values: 361.06 and 183.03). Compound R7 is not stable anddecomposes to R2 and R6 (m/z values: 211.02 and 167.03).

4. Conclusion

To summarise the results, the potential of oxidation of rham-nazin depends on pH of the solution and the oxidation processinvolves two electrons until the pH 11.4. Scheme 1 shows two oxi-dation pathways, which explain the oxidation mechanism of rham-nazin under ambient conditions. One oxidation process is preferredin the absence of oxygen. The two electron oxidation is coupledwith deprotonation reactions dependently on the pH of solution.The in situ spectroelectrochemistry and identification of degrada-tion products by HPLC-MS/MS and HPLC-DAD confirmed the pres-ence of fast subsequent chemical reactions following the electron

transfer and 2,4-dihydroxy-2-(4′-hydroxy-3′-methoxybenzoyl)-6-methoxy- benzofuran-3(2H)-one (R1) was identified as the mainoxidation product of rhamnazin under inert atmosphere. The rham-nazin oxidation products are unstable in the presence of oxygen

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nd the oxidation leads to the formation of 2-(2,6-dihydroxy--methoxyphenyl)-2-oxoacetic acid, 2,6-dihydroxy-4-methoxy-enzoic acid and 4-hydroxy-3-methoxy-benzoic acid. The minorxidation pathway under ambient conditions leads to theormation of 2-hydroxy-6-(4-hydroxy-3-methoxybenzoyloxy)-4-

ethoxyphenyl-2-oxoacetic acid (R7), which also decomposes. Theormation of low molecular weight hydroxybenzoic acids were con-rmed by several authors as oxidation products of 2 [16,24–27]. Asemonstrated by the described experiments, the handling of theolutions of rhamnazin under common laboratory conditions canake difficulties and probably not get a reliable data in the amount

f analyte, which decomposes in solutions within minutes. The pre-iously degassed solvents have to be used in studies of antioxidantroperties of rhamnazin and of its pharmacological efficiency.

cknowledgements

This work is supported by the Academy of Sciences of the Czechepublic (M200401201).

ppendix A. Supplementary data

Supplementary data associated with this article can be found,n the online version, at http://dx.doi.org/10.1016/j.electacta.014.04.074.

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