sign in sign up
<<
Home , Cinnamic acid, Ferulic acid, Verbascoside

Send Orders of Reprints at [email protected]

92 Combinatorial Chemistry & High Throughput Screening, 2013, 16, 92-97 Redox Behaviour of and Eric de Souza Gil1,2, Teodor Adrian Enache1 and Ana Maria Oliveira-Brett*,1

1Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Coimbra, Portugal 2Faculdade de Farmácia, Universidade Federal de Goiás, Goiânia, Goiás, Brazil

Abstract: The electrochemical oxidation mechanisms of rosmarinic acid (RA) and verbascoside (VB), both esters with two catechol moieties, were investigated. The redox mechanism is associated with the oxidation of the catechol groups, and was studied over a wide pH range by cyclic, differential pulse and square wave voltammetry, using a glassy carbon electrode. The voltammetric study revealed that both molecules, RA and VB, are reversibly oxidized in two successive pH-dependent steps each with the transfer of two electrons and two protons. Moreover, it was found that the first oxidation step is associated with the caffeic acid moiety, whereas the second oxidation step corresponds to the oxidation in VB of the group and in RA of the 3,4-dihydroxyphenyl lactic acid residue. Keywords: Acteoside, caffeic acid, derivatives, electrochemical characterization, hydroxytyrosol, , voltammetry.

1. INTRODUCTION OH VBH2 The global market of natural products is driven by health OH OH and anti-aging products, which in most cases play an essential role against oxidative stress [1-4]. The antioxidant O O activity of many natural compounds has gained great OH O attention due to their promising biological relevance. O Among these compounds, and OH O derivatives, such as rosmarinic acid O OH (RA) and verbacoside (VB) (Scheme 1), are of particular OH OH OH interest due to their widespread occurrence in fruits and vegetables consumed by humans [1-4]. Due to their inherent HT antioxidant power, they have received considerable attention CA O [2, 5-7], and pharmacological studies of RA and VB have OH HO reported a large variety of important biological properties, OH such as anti-inflammatory, antibacterial, anticonvulsant, antitumour, anti-viral, anticancer, antibacterial as well as OH OH being good UV screeners, by inhibition and conversion of OH ultraviolet light into harmless low energy radiation, thus preventing tissue damage [7-15]. RA O RA, a hidroxycinnamic acid derivative (Scheme 1), found in many herbs and spices, has a large OH OH O antioxidant activity and is being used in various O biotechnological applications [2]. Structurally, it is an ester OH OH of caffeic acid (CA) (Scheme 1), with 3,4-dihydroxyphenyl lactic acid (DHPLA), thus having two electroactive catechol OH moieties, which are able to neutralize free radicals by an Scheme 1. Chemical structures: verbascoside (VB), rosmarinic acid electron/proton donor mechanism. Furthermore, the (RA), hydroxytyrosol (HT) and caffeic acid (CA). hydroxyl and carboxyl oxygen confer good metal-chelating properties to RA [3, 4]. (Aloysia triphylla), mullein (Verbascum Thapsus), Indian warrior (Pedicularis densiflora), savannah tea (Lippia VB, also named as acteoside, is another hydroxycinnamic multiflora) [8, 16-19], and olive fruits [5, 9, 20, 21]. ester derivative widely distributed in plants, including Chemically, VB is a water-soluble phenylpropanoid diverse medicinal herbs, such as echinacea, lemon verbena [17], structurally characterized by caffeic acid (CA) linked by a -(D)-glucopyranoside to a hydroxytyrosol

*Address correspondence to this author at the Departamento de Química, (HT) (Scheme 1), with higher antioxidant activity than CA Faculdade de Ciências e Tecnologia, Universidade de Coimbra, 3004-535 or HT [1]. VB was also shown to be more active in Coimbra, Portugal; Tel/Fax: +351-239-854487; E-mail: [email protected] preventing oxidative stress than CA and HT in equimolar mixtures [20], which is in agreement with the radical

1875-5402/13 $58.00+.00 © 2013 Bentham Science Publishers Redox Behaviour of Verbascoside and Rosmarinic Acid Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 2 93 scavenging activities of catechol groups, water- and (GCE, d = 1.0 mm) was the working electrode, Pt wire the the reducing properties conferred by the sugar residues [22]. counter electrode and Ag/AgCl (3 M KCl) the reference electrode. In pharmaceutical technology, the combination of water- soluble and organic-soluble antioxidants is common practice, The GCE was always polished using diamond (particle also being important in physiological systems, by preserving size 1 m) (Kemet International, UK) before each lipid membranes and cytosolic compartments from attack by electrochemical experiment in order to ensure a clean surface free radicals [22]. In innovative cosmetic products, natural and to avoid possible problems from the adsorption of antioxidants of vegetable origin with different water- oxidation products onto the electrode surface. After solubility levels, such as RA and VB, are now used as a polishing, it was rinsed thoroughly with Milli-Q water. functional ingredient [2, 4, 7]. Following this mechanical treatment, the GCE was placed in buffer supporting electrolyte and voltammograms were It is well known that, as redox agents, antioxidants recorded until steady-state baseline voltammograms were (depending on medium conditions) can also act as pro- obtained. This procedure ensured very reproducible oxidants. Thus, the investigation of the electrochemical experimental results. oxidation mechanisms of natural antioxidants is crucial for a better understanding of their real contribution in preventing The experimental conditions for differential pulse (DP) free radical damage and oxidative stress [23-25]. voltammetry were: pulse amplitude 50 mV, pulse width 70 ms and scan rate 5 mV s-1. For square wave (SW) The present study was concerned with the investigation, voltammetry: 50 mV pulse amplitude, potential increment for the first time, of the electron transfer properties of RA, 2 mV, and frequencies ranging from 8 to 200 Hz VB and HT at a glassy carbon electrode (GCE), using cyclic corresponding to effective scan rates ranging from 15 to 400 (CV), differential pulse (DP) and square wave (SW) -1 voltammetry, in different pH conditions. The electron mV s , were used. For cyclic voltammetry (CV), scan rates from 10 to 500 V s-1 were used. transfer properties of CA and HT were probed in the same experimental conditions and the results obtained are important for clarifying the oxidation mechanism of VB and 2.3. Acquisition and Presentation of Voltammetric Data RA. All the voltammograms presented were background- subtracted and baseline-corrected using the moving average 2. MATERIAL AND METHODS with a step window of 5 mV included in GPES version 4.9 software. This mathematical treatment improves the 2.1. Chemicals and Solutions visualization and identification of peaks over the baseline Verbascoside (VB), C29H36O15 (PM = 624.61) and without introducing any artefact, although the peak height is hydroxytyrosol (HT), C8H10O3 (PM = 154.17) were in some cases reduced (< 10%) relative to that of the purchased from Extrasynthèse (Genay, France). Caffeic acid untreated curve. Nevertheless, this mathematical treatment of (CA), C9H8O4, (PM = 180.16) and rosmarinic acid (RA), the original voltammograms was used in the presentation of C18H16O8, (PM = 360.31) were obtained from Sigma Aldrich all experimental voltammograms for a better and clearer (Spain). identification of the peaks. The values for peak current Stock solutions of RA were prepared in ethanol (Merck), presented in all graphs were determined from the original untreated voltammograms after subtraction of the baseline. while the stock solutions of VB, HT and CA were prepared in water. All phenolic compound stock solutions were stored at 4 °C in the dark. The working standard solutions were 3. RESULTS AND DISCUSSION prepared daily by diluting the stock solution in supporting The anodic oxidation behaviour of RA, VB, CA and HT electrolyte. was investigated at a GCE in different experimental The supporting electrolyte solutions of 0.1 M ionic conditions using CV, and DP and SW voltammetry, over a strength were: pH 2.0 KCl/HCl, pH 3.5–5.5 acetate buffer, wide pH range between 1.0 and 12.0. The electrochemical pH 6.0–8.0 phosphate buffer, and pH 9.0–11.0 ammonia study of CA and HT at GCE was carried out in order to buffer [26]. All experiments were done at room temperature identify the redox active centres of RA and VB. (25 ± 1 ºC). All solutions were prepared using analytical -1 grade reagents and purified water (conductivity  0.1 S cm ) 3.1. Cyclic Voltammetry from a Millipore Milli-Q system (Molsheim, France). The CV behaviour of RA and VB was studied in The pH measurements were carried out with a Crison solutions of 50 M in phosphate buffer pH = 7.0 0.1 M, at a micropH 2001 pH-meter using an Ingold combined glass clean GCE surface (Fig. 1). electrode. In these conditions, the oxidation of RA occurs in two steps, first a shoulder peak 1a, at Ep1a = + 0.17 V, followed 2.2. Electrochemical Measurements by a well-defined anodic peak 2a, at Ep2a = + 0.22 V. On Voltammetric experiments were carried out using an the negative-going scan, two reduction peaks, peak 2c, at Autolab PGSTAT 10 potentiostat/galvanostat running with Ep2c = + 0.19 V, and peak 1c, at Ep1c = + 0.14 V, were GPES 4.9 software (Eco-Chemie, Utrecht, The Netherlands). observed. The differences between the anodic and cathodic Measurements were carried out using a three-electrode peak potentials, |Ep1a- Ep1c| and |Ep2a- Ep2c|, are in agreement system in a 1 mL one-compartment electrochemical cell with the theoretical value of 30 mV for a two-electron (Cypress System Inc., USA). A glassy carbon electrode reversible reaction at each peak (Fig. 1A) [27-32]. 94 Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 2 de Souza Gil et al.

A electron-withdrawing effect (-), while alkyl groups have an  electron-releasing effect (+ ) [1, 33-35]. The electrostatic induction weakens along the chain, so the negative inductive effect of the carboxylic function in the case of DHPLA will be small, whereas the double bond keeps the deactivating 100 nA effect of the carboxyl function in the case of CA [33-35], but

0,0 0,1 0,2 0,3 0,4 0,5 the carboxylic group decreases the electron density in the E / V ( vs. Ag/AgC l) phenolic groups of CA, making the electron transfer more difficult. B CVs were also obtained at different scan rates for a solution of 50 M VB in phosphate buffer pH 7.0 0.1 M (Fig. 2). Between measurements, the electrode surface was always polished in order to ensure a clean surface and to avoid possible problems from the adsorption of VB

oxidation products onto the GCE surface. Increasing the scan rate, it was observed that the oxidation peak potentials of 1a 100 nA and 2a were shifted to slightly more positive values and the reduction peaks 1c and 2c were shifted to slightly more 0,0 0,1 0,2 0,3 0,4 0,5 negative values. However, the differences |E - E | and E / V (vs. Ag/AgC l) p1a p1c |Ep2a- Ep2c| were always close to the theoretical value of 30 mV or a reversible two-electron and two-proton

C mechanism, even for high scan rates. Increasing the scan rate, the peak current of peaks 1a and 2a increases linearly with the square root of the scan rate, consistent with diffusion-limited oxidation of a solution species [1, 32].



100 nA 500 nA

0,0 0,1 0,2 0,3 E / V ( vs. A g /A g C l) 2

Fig. (1). CVs: in 50 M solutions of (A) RA and (B) VB () first, 1  (- - -) second and (•••) third scans; (C) 15 M solutions () HT 0 (nA) I and (- - -) CA second scans; in phosphate buffer pH = 7.0 0.1 M, -1 scan rate  = 50 mV s-1. 150 300 450  (mV.s-1) The CV recorded in a solution of VB shows the same behaviour as for RA, with small differences in the peak 0.0 0.1 0.2 0.3 0.4 0.5 0.6 potentials, the first anodic peak 1 , is at E = + 0.18 V whereas E / V (vs. Ag/AgCl) a p1a the second peak 2a, is at Ep2a = + 0.24 V. Reversing the scan Fig. (2). CVs in 50 M VB in phosphate buffer pH = 7.0 0.1 M, direction, two cathodic peaks, peak 1a, at Ep1a = + 0.14 V and first scan for different scan rates. Inset: Ip vs . peak 2a, Ep2a = + 0.20 V, are seen (Fig. 1B). The chemical structures of RA and VB presented two 3.2. Differential Pulse Voltammetry catechol moieties, in RA one corresponding to CA and the other to DHPLA, and in VB one to CA and the other to HT. The electrochemical oxidation of RA, VB, HT and CA in In order to clarify the mechanisms of the two successive phosphate buffer pH 7.0 0.1 M using DP voltammetry was anodic oxidations occurring in RA and VB, an studied. In the first DP voltammogram VB showed two electrochemical study of CA and HT was undertaken. oxidation peaks, whereas RA, CA and HT, showed only one (Table 1, Figs. 3, 4). CVs recorded in solutions of 15 M HT and CA in phosphate buffer pH = 7.0 0.1 M, at a clean GCE Table 1. DP Voltammetry Oxidation Potentials in 15 M of surface, showed a similar behaviour with only one VB, RA, CA and HT, in Phosphate Buffer pH 7.0 0.1 M well-defined reversible peak (Fig. 1C). For HT peak 1 Solutions a occurred at Ep1a = + 0.16 V, whereas for CA peak 1a was at

Ep1a = + 0.21 V, and reversing the scan direction, the HT Compounds Ep1a (V) Ep2a (V) peak 1c occurred at Ep1c = + 0.13 V, and the CA peak 1c at verbascoside (VB) 0.14 0.20 Ep1c = + 0.16 V. rosmarinic acid (RA) 0.16 The difference in the oxidation peak potentials of CA and HT is due to electron density and inductive effects, caffeic acid (CA) 0.16 considering that carboxylic and ester groups have an hydroxytyrosol (HT) 0.13 Redox Behaviour of Verbascoside and Rosmarinic Acid Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 2 95

A that the DHPLA of RA may have an opposite effect on the electron density, and the oxidation occurs at a higher potential.

The effect of pH on the redox behaviour of RA and VB 25 nA was studied using DP voltammetry over a wide pH range between 1 and 12. 10 It was found for RA and VB that the oxidation peak 8 0,6 potential shifted linearly to less positive values until pH 9.0, 6 pH 0,4 with increasing pH, showing that deprotonation is involved, 4 l) the oxidation being pH-independent at higher values 0,2 AgC . Ag/ (Figs. 3, 4). The break point of the slopes at ~ pH 9 observed 2 0,0 (vs E / V in Figs. (3B, 4B), indicates a pKa value between 9.0 and 10.0 for VB and RA, in agreement with the values of 9.0 for B 0,6 caffeic acid and 9.5 for hydroxytyrosol moieties [36].

) 0,5 The slopes, for RA and VB, of Epa vs pH of 60 mV/pH unit and the width at half-height of W1/2 ~ 60 mV, indicate a 0,4 two-electron and two-proton transfer in each oxidation step, 0,3 consistent with the mechanism involving catechol moieties vs. Ag/AgCl ( 0,2 [29, 32]. Considering the cyclic voltammetry results, the / V global oxidation reaction involves a reversible four electron 0,1

pa P 2a E transfer mechanism. Moreover, the low oxidation potential 0,0 P 1a of all electrode reaction steps it is in agreement with the high 02468101214 antioxidant activity exhibited by both compounds [4, 7]. pH

Fig. (3). (A) 3D plot of first scan DP voltammograms in 15 M For VB, it was also found that Ip1a/Ip2a ~ 1, for neutral and -1 alkaline pH, becoming higher than 1.4 for pH < 5.0 (Fig. 3). VB; and (B) Plot of Ep1a () and Ep2a () vs pH. Scan rate 5 mV s . This behaviour corroborates the influence of the secondary intra and intermolecular interactions on the oxidation A process, mainly hydrogen bonds [1]. For both molecules, VB and RA, a decrease of the peak currents with increase of

supporting electrolyte pH was observed. This is explained by the effect of pH on the ionization of OH groups. In very acid 10 nA media the OH groups increase the molecules hydrophobicity and their adsorption on the hydrophobic GCE surface. At 10 8 0,6 alkaline pH, the OH groups are almost or fully deprotonated, 0,4 6 ) thus increasing the VB and RA hydrophilicity and

pH 4 0,2 AgCl consequently decreasing their adsorption on the electrode Ag/ 2 0,0 s. (v surface. Nevertheless, the decrease of VB oxidation current V E / is higher than RA. B 0,6 0,5 3.3. Square Wave Voltammetry 0,4 The advantages of SW voltammetry are greater speed of 0,3 analysis, lower consumption of the electroactive species in relation with DP voltammetry, and reduced problems with 0,2 poisoning of the electrode surface [32]. Since the current is / V (vs. Ag/AgCl) 0,1 pa P sampled in both positive and negative-going pulses, peaks E 2a 0,0 P corresponding to the oxidation and reduction of the 1a electroactive species at the electrode surface can be obtained 02468101214 pH in the same experiment and SW voltammetry enables the possibility to see during only one scan if the electron transfer Fig. (4). (A) 3D plot of first scan DP voltammograms in 15 M -1 reaction is reversible or not. RA; and (B) Plot of Ep1a () and Ep2a () vs pH. Scan rate 5 mV s . SW voltammograms were recorded at frequencies in the The two oxidation peaks of VB correspond to the range 8 - 200 Hz, with 5 M RA and VB, in phosphate oxidation of the two catechol moieties, CA and HT. buffer pH 7.0 0.1 M, always using a clean GCE surface However, RA, that also has two catechol moieties, CA and (Fig. 5). For a frequency of 8 Hz only one oxidation peak DHPLA, only presents one very wide anodic peak with was observed for RA and VB, peak 1a, at Ep1a ~ + 0.2 V width at half-height W1/2 ~ 100 mV in acidic and mild basic (Fig. 5A). Increasing the frequency peak, 1a becomes wider conditions, suggesting that the oxidation potentials of each and the current increases. For frequencies > 40 Hz, two small catechol group are very close, the peaks becoming peaks appeared for higher potentials that are due to double indistinguishable. Nevertheless, in alkaline medium RA bond oxidation of the CA moiety and oxidation of presents two anodic peaks, that can be explained considering 96 Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 2 de Souza Gil et al. dimerization products. Plotting the forward (If) and aromatic ring, lowering the potential of the electrochemical backward (Ib) components of the total (It) current, showed oxidation. the identical value of the potential of forward and backward HO R - + O R peaks, and the reversible redox behaviour of RA and VB was -2e , -2H confirmed (Fig. 5). +2e-, +2H+  HO O A I R = t O + If OH OH O-RVB  - O-RVB/RA I b CA moiety HT moiety OH OH 500 nA   +  + 0,0 0,2 0,4 0,6 0,8 1,0 1,2 OH - O-RRA OH O-RRA E / V (vs. Ag/AgCl) - + - OH O OH OH O O B DHPLA moiety I DHPLA moiety t "alkaline medium" "acid medium"

If Scheme 2. Oxidation mechanism of the catechol moiety, in RA and VB. In RA, there occurs a stronger electron withdrawing effect of the carboxylate present in the alkyl chain, the I b influence of the double bond extension on the inductive effects on aromatic rings is reinforced. In alkaline medium the ionization of the carboxylic group on the DHPLA 500 nA moiety, causes inversion of the inductive effect and the clear peak separation. The electron donor effect on the alkyl 0,0 0,2 0,4 0,6 0,8 1,0 1,2 chain linked to the catechol moieties follows the sequence E / V (vs. Ag/AgCl) HT > ionized DPHLA > non-ionized DPHL > CA. It can be concluded that the RA and VB first redox pair C It corresponds to the oxidation of the CA residue, while the

If second redox pair in RA corresponds to DHPLA and in VB corresponds to HT.

5. CONCLUSION The electrochemical oxidation mechanisms of RA and Ib VB, both CA esters with two catechol moieties were investigated and compared with the electrochemical 500 nA behaviour of CA and HT. The anodic behaviour of RA and VB follows a pH-dependent mechanism, associated with

0,0 0,2 0,4 0,6 0,8 1,0 1,2 each catechol moiety, and which occurs in two successive E / V (vs. Ag/AgCl) reversible two electron two proton electron transfers. The adsorption of oxidized products formed on the GCE surface, Fig. (5). SW voltammograms in 5 M in phosphate buffer pH = 7.0  -1 the highest adsorption being for HT and the lowest for VB, is 0.1 M: (A) RA, frequency 8 Hz, eff = 15 mV s , (B) RA and (C) dependent on molecular size, hydrophobicity and VB, frequency 200 Hz,  = 400 mV s-1. eff concentration. The first step in RA and VB oxidation is related to oxidation of the CA moiety, whereas the second 4. OXIDATION MECHANISM step corresponds to DHPLA in RA and to HT in VB. The electrochemical oxidation of CA, RA and VB follows a mechanism of that of catechol compounds, CONFLICT OF INTEREST pH-dependent and a two proton two electron process Authors declare that they have no conflict of interest. (Scheme 2). However, RA and VB have two catechol moieties with ACKNOWLEDGEMENTS different electron densities due to the groups attached. Since the carboxylic group is not directly bound to the aromatic Financial support from Coordenação de Aperfeiçoamento ring in RA or VB, the electron-withdrawing effect will be de Pessoal de Ensino Superior (CAPES), Post-Doctoral sustained by double bond stretching; the positive inductive Grant (E.S. Gil), and Fundação para a Ciência e Tecnologia effect of the alkyl chain increases the electron density on the (FCT, Portugal), Post-Doctoral Grant SFRH/BPD/80195/ Redox Behaviour of Verbascoside and Rosmarinic Acid Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 2 97

2011 (T.A. Enache), project PTDC/QUI/098562/2008, [17] Funes, L.; Laporta, O.; Cerdan-Calero, M.; Micol, V.; Effects of POPH (co-financed by the European Community Funds FSE verbascoside, a phenylpropanoid glycoside from lemon verbena, on phospholipid model membranes. Chem. Phys. Lipids., 2010, 163, e FEDER/COMPETE), CEMUC-R (Research Unit 285), are 190-199. gratefully acknowledged. [18] Speranza, L.; Franceschelli, S.; Pesce, M.; Reale, M.; Menghini, L.; Vinciguerra, I.; Antiinflammatory Effects in THP-1 cells Treated with Verbascoside. Phytother. Res., 2010, 24, 1398-1404. REFERENCES [19] Arthur, H.; Joubert, E.; De , D.; Malherbe, C.J.; [1] Born, M.; Carrupt, P.A.; Zini, R.; Bree, F.; Tillement, J.P.; Phenylethanoid glycosides as major antioxidants in Lippia Hostettmann, K.; Testa, B.; Electrochemical behaviour and multiflora herbal infusion and their stability during steam antioxidant activity of some natural . Helv. Chim. pasteurisation of plant material. Food Chem., 2011, 127, 581-588. Acta., 1996, 79, 1147-1158. [20] Obied, H.K.; Prenzler, P.D.; Robards, K.; Potent antioxidant [2] Park, S.U.; Uddin, M.R.; Xu, H.; Kim, Y.K.; Lee, S.Y.; biophenols from olive mill waste. Food Chem., 2008, 111, 171- Biotechnological applications for rosmarinic acid production in 178. plant. Afr. J. Biotechnol., 2008, 7, 4959-4965. [21] Pereira, J.A.; Pereira, A.P.G.; Ferreira, I.C.F.R.; Valentao, P.; [3] Psotová, J.; Lasovsk, J.; Viar, J.; Metal-chelating properties, Andrade, P.B.; Seabra, R.; Table olives from Portugal: Phenolic electrochemical behavior, scavenging and cytoprotective activities compounds, antioxidant potential, and antimicrobial activity. J. of six natural phenolics. Biomed. Papers, 2003, 147, 147-153. Agric. Food Chem., 2006, 54, 8425-8431. [4] Gonçalves, D.; Couto, R.O.; Conceição, E.C.; Reis, N.S.; Gil, E.S.; [22] Rice-Evans, C.A.; Miller, N.J.; Paganga, G.; Structure-antioxidant Voltametria de pulso diferencial (VPD) em estado sólido de activity relationships of flavonoids and phenolic acids. Free Rad. manchas de cromatografia de camada delgada (CCD): Um novo Biol. Med., 1996, 20, 933-956. método de análise para fitoativos antioxidantes. Quim. Nova, 2011, [23] Salvi A; Bruhlmann C; Migliavacca E; Carrupt, P.A.; Hostettmann, 34, 330-334. K.; Testa, B.; protection by antioxidants: Development of a [5] Maramaldi G.; Artaria C.; Ikemoto T.; Haratake A.; Estratto convenient assay and structure-activity relationships of natural standardizzato di frutti di Olea europaea. Cosmet. Technol., 2006, polyphenols. Helv. Chim. Acta, 2002, 85, 867-881. 9, 9-13. [24] Reis, N.S.; Serrano, S.H.P.; Meneghatti, R.; Gil, E.S.; Métodos [6] Al-Sereiti, M.R.; Abu-Amer, K.M.; Sen, P.; Pharmacology of eletroquímicos usados para avaliação da atividade antioxidante de rosemary (Rosmarinus officinalis Linn.) and its therapeutic produtos naturais. Lat. Am. J. Pharm., 2009, 28, 949-953. potentials. Indian J. Exp. Biol., 1999, 37, 124-130. [25] Diculescu, V.C.; Enache, T.A.; Oliveira-Brett, A.M.; [7] Vertuani, S.; Beghelli, E.; Scalambra, E.; Malisardi, G.; Copetti, S.; Electrochemical oxidation at a glassy carbon electrode of the anti- Dal Toso, R.; Baldisserotto, A.; Manfredini, S.; Activity and arrhythmia drug disopyramide. Anal. Lett., 2007, 40, 2860-2871. stability studies of verbascoside, a novel antioxidant, in dermo- [26] Beynon, R.; Easterby, J. The basics buffer Solutions, Oxford cosmetic and pharmaceutical topical formulations. Molecules, University Press, Oxford, 1996. 2011, 16, 7068-7080. [27] Petrucci, R.; Astolfi, P.; Greci, L.; Firuzi, O.; Saso, L.; Marrosu, [8] Shi, Y.; Yao, S.; Jia, Z.; Yao, S.D.; Jia, Z.J.; Lin, N.Y.; Dietary G.; A spectroelectrochemical and chemical study on oxidation of phytophenols act as scavengers of reducing radicals. Food Chem., hydroxycinnamic acids in aprotic medium. Electrochim. Acta, 2011, 124, 1322-1327. 2007, 52, 2461-2470. [9] Cardinali, A.; Cicco, N.; Linsalata, V.; Minervini, F.; Pati, S.; [28] Salas-Reyes, M.; Hernández, J.; Domínguez, Z.; González, F.J.; Pieralice, M.; Tursi, N.; Lattanzio, V.; Biological activity of high Astudillo, P.D.; Navarro, R.E.; Martínez-Benavidez, E.; molecular weight phenolics from olive mill wastewater. J. Agric. Velázquez-Contrerase, C.; Cruz-Sánchez, S.;, Electrochemical Food Chem., 2010, 58, 8585-8590. Oxidation of Caffeic and Derivatives in Aprotic [10] Zhou, Y.C.; Zheng, R.L.; Phenolic-compounds and an analog as Medium. J. Braz. Chem. Soc., 2011, 22, 693-701. superoxide anion scavengers and antioxidants. Biochem. [29] Enache, T.A.; Oliveira-Brett, A.M.; Phenol and para-substituted Pharmacol., 1991, 42, 1177-1179. phenols electrochemical oxidation pathways. J. Electroanal. [11] Gutteridge, J.M.; Free radicals in disease processes: A compilation Chem., 2011, 655, 9-16. of cause and consequence. Free Radic. Res. Commun., 1993, 19, [30] Hapiot, P.; Neudeck, A.; Pinson, J.; Fulcrand, H.; Neta, P.; 141-158. Rolando, C.; Oxidation of caffeic acid and related [12] Li, J.; Zheng, Y.; Zhou, H.; Su, B.N., and Zheng, R.L.; hydroxycinnamic acids. J. Electroanal. Chem., 1996, 405, 169-176. Differentiation of human gastric adenocarcinoma cell line MGc80- [31] Giacomelli, C.; Ckless, K.; Galato, D.; Miranda, F.S.; Spinelli, A.; 3 induced by verbascoside. Planta Med., 1997, 63, 499-502. Electrochemistry of Caffeic Acid Aqueous Solutions with pH 2.0 to [13] Liu, M.J.; Li, J.X.; Guo, H.Z.; Lee, K. M., Qin, L., Chan, K.M.; 8.5. J. Braz. Chem. Soc., 2002, 13, 332-338. The effects of verbascoside on plasma lipid peroxidation level and [32] Brett, C.M.A.; Oliveira-Brett, A.M. Electrochemistry: Principles, erythrocyte membrane fluidity during immobilization in rabbits: a Methods and Applications, Oxford Science Publications, Oxford, time course study. Life Sci., 2003, 73, 883-892. 1993. [14] Korkina, L.G.; as naturally occurring [33] IUPAC. Compendium of Chemical Terminology, 2nd ed., antioxidants: from plant defense to human health. Cell. Mol. Biol., Blackwell Scientific Publications, Oxford, 1997. 2007, 53, 15-25. [34] Takahata Y.; Chong D.P.; Estimation of Hammett sigma constants [15] De-Souza P.A.; Rodrigues C.; Santiago A.P.S.A.; de Lucas, N.C.; of substituted benzenes through accurate density-functional Leitão, G.G.; Filho, A.G.; Antioxidant activity of natural calculation of core-electron binding energy shifts. Int. J. Quantum compounds of Stachytarpheta cayennensis by scavenger of Chem., 2005, 103, 509-515. mitochondrial reactive oxygen species. Braz. J. Pharmacog., 2011, [35] Gil, E.S.; Andrade, C.H.; Barbosa, N.L.; Braga, R.C.; Serrano, 21, 420-426. S.H.P.; Cyclic Voltammetry and Computational Chemistry Studies [16] Sloley, B.D.; Urichuk, L.J.; Tywin, C.; Coutts, R. T., Pang, P. K.; on the Evaluation of the Redox Behavior of Parabens and other Comparison of chemical components and antioxidant capacity of Analogues. J. Braz. Chem. Soc., 2012, in press. different Echinacea species. J. Pharm. Pharmacol., 2001, 53, 849- [36] Romero, C.; Brenes, M.; García, P.; and Garrido A.; Optimization 857. of simulated ripe olive darkening in presence of manganese. J. Food. Sci., 2000 65 254-258.

Received: April 13, 2012 Revised: July 16, 2012 Accepted: August 3, 2012