Full Paper
Anodic Behaviour of Flavonoids Orientin, Eriodictyol and Robinin at a Glassy Carbon Electrode
Eric de Souza Gil,a, b Adrian Teodor Enache,a Ana Maria de Oliveira-Brett*a a Departamento de Qu mica, Faculdade de CiÞncias e Tecnologia, Universidade de Coimbra, 3004–535 Coimbra, Portugal b Faculdade de Farm cia, Universidade Federal de Goi s, 74605–220, Goi nia, Goi s, Brasil *e-mail: [email protected]
Received: April 20, 2012;& Accepted: May 31, 2012
Abstract Orientin, eriodictyol and robinin are polyphenolic compounds, and their oxidation mechanism is pH-dependent, in two steps, involving a different number of electrons and protons. Orientin and eriodictyol first oxidation occurs at a lower potential, corresponding to the reversible oxidation of the catechol group, and is followed by an irreversible oxidation on the ring-A at more positive potential. Robenin oxidation is irreversible, with the formation of electro- active products, and occurs at ring-A and ring-B. The electrochemical characterization of their redox behaviour brought useful data about their chemical stability, antioxidant and pro-oxidant activity, enabling a comprehensive understanding of their redox mechanism.
Keywords: Orientin, Eriodictyol, Robinin, Oxidation, Glassy carbon electrode.
DOI: 10.1002/elan.201200211
1 Introduction Orientin is a flavone, found in passion flower, bamboo leaves, aÅai pulps and wardii berries [5–7]. Chemically is Flavonoids constitute, among other compounds, an impor- the 8-C glucoside of the widespread citrus flavone, luteo- tant class of antioxidants that inhibit the oxidative degra- lin [8]. dation of organic materials including a large number of Eriodictyol, 3’,4’,5,7-tetrahydroxyflavanone, is found in biological aerobic organisms and commercial products. several types of plants, such as yerba santa (eriodictyon As well as many polyphenols, flavonoids owe their anti- californicum), pistachio (pistacia vera), cork (quercus oxidant activity to their ability to scavenge radicals, by suber), lemon balm (melissa officinalis), rose hips (rosa hydrogen or electron transfer, in a much faster process canina), tomato and lemon species [9–13]. than the radical attack to an organic substrate [1,2]. Robinin, kaempferol 3-O-robinoside-7-O-rhamnoside, The major class of flavonoids may be divided into sev- is a glycoside of the well-studied flavonol, kaempferol eral subclasses, flavonols, flavones, flavanonols, flava- [14,15], that can be isolated from various genus of herbs, nones, isoflavonoids, catechins and anthocianidins, as well such as astragalus (flaronin, milk-vetch), pueraria a varying number of polymeric compounds [1–3]. (kudzu), vinca (periwinkle) and also from false acacia Consumption of flavonoid-rich foods, in particular (robinia pseudoacacia), a garden tree [14–16]. fruits and vegetables, is associated with a lower incidence The importance of these three distinct flavonoids is in of heart disease, ischemic stroke, cancer, and other chron- their diverse biological effects which include radioprotec- ic diseases. Additional studies also found inverse associa- tive, antitumour, antimicrobial, vasodilatory and anti-in- tions between flavonoid intake and the risk of stroke and flammatory actions [5–14]. Eriodictyol has been used as lung and colorectal cancers [2–4]. Because these chronic bitter masking in cosmetic and pharmaceutical formula- diseases are associated with increased oxidative stress and tions [13], while robinin is one of the active constituents flavonoids are strong antioxidants in vitro, it has been of the commercial drug flaronin, which has been widely suggested that dietary flavonoids exert health benefits used in the treatment of acute and chronic renal failures through antioxidant mechanisms [1–5]. Besides these [17,18]. benefits, several studies have shown that in specific condi- Regarding the structure activity relationship, the tions the flavonoids can play pro-oxidant function [3,4]. mechanism of action of flavonoids is related to the physi- Among flavonoids derivatives, orientin, eryodictiol and cochemical properties of their electroactive moieties, e.g., robenin, Scheme 1, are of particular interest due their phenol, catechol and resorcinol [2–4]. great occurrence in foodstuffs, cosmetics and pharmaceut- Due to their high sensitivity, voltammetric methods ical products. have been successfully used for the investigation of oxida- tion mechanism of biological active substituted phenols,
1576 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2012, 24, No. 7, 1576 – 1583 Anodic Behaviour of Flavonoids Orientin, Eriodictyol and Robinin at a GCE
Scheme 1. Chemical structures of orientin, eriodictyol and robinin. providing valuable insights into their redox behaviour 2 Experimental and their detection in various samples. The electrochemi- cal characterization of the mostly widespread flavonoids 2.1 Materials and Reagents has already been investigated at different carbon elec- trode materials: glassy carbon, carbon paste, etc. [19–23] Orientin, eriodictyol and robinin were purchased from and their oxidation mechanisms were correlated with the Extrasynth se (Genay, France) and used without further electroactive groups: phenol, resorcinol and catechol. purification. A stock solution was prepared in ethanol-de- The oxidation of phenol and resorcinol is an irreversi- ionised water (30:70, v/v) and stored at +48C. Solutions ble pH dependent process, occurring in one step, leading of different concentrations were prepared by dilution of to electroactive products [24]. The catechol oxidation is the appropriate quantity in supporting electrolyte. All also pH dependent, and occurs in a two electron two supporting electrolyte solutions, were prepared using ana- proton reversible mechanism [24]. Although the electro- lytical grade reagents and purified water from a Millipore chemical oxidation mechanisms of flavonoids is basically Milli-Q system (conductivity 0.1 mScm 1) [25]. addressed considering the position and number of hy- droxyl groups, the different types of chemical substitu- 2.2 Apparatus ents, OCH3, sugar, etc., can also have influence on their redox behaviour and oxidation products formation [19– Voltammetric experiments were carried out using a mAu- 24]. tolab running with GPES 4.9 software, Eco-Chemie, The present study is concerned with the investigation Utrecht, The Netherlands. Measurements were carried of the electron transfer reactions of the three flavonoids, out using a three-electrode system in a 0.5 mL one-com- orientin, eriodictyol and robinin, using cyclic, differential partment electrochemical cell (Cypress System Inc., pulse and square wave voltammetry at a glassy carbon USA). Glassy carbon electrode (GCE, d=1.0 mm) was electrode, and so far no electrochemical study of these the working electrode, Pt wire the counter electrode and compounds has been carried out. Therefore, owing to the Ag/AgCl (3 mol L 1 KCl) reference electrode. The their different structure, their redox mechanisms and the GCE was polished using diamond particles of 1 mm influence of non electroactive groups, like the sugar (Kemet, UK) before each electrochemical experiment. moiety and the C3=C2 double bond, will influence the After polishing, it was rinsed thoroughly with Milli-Q redox behaviour of these flavonoids, providing important water. Following this mechanical treatment, the GCE was data for the development of electroanalytical methods placed in buffer supporting electrolyte and voltammo- and also for the understanding of structure activity rela- grams were recorded until a steady state baseline voltam- tionships and antioxidant properties. mograms were obtained. This procedure ensured very re-
Electroanalysis 2012, 24, No. 7, 1576 – 1583 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.electroanalysis.wiley-vch.de 1577 Full Paper E. de Souza Gil et al. producible experimental results. The experimental condi- tions for differential pulse (DP) voltammetry were: pulse amplitude 50 mV, pulse width 70 ms and scan rate 5mVs 1. For square wave (SW) voltammetry were: pulse 50 mV, frequency 50 Hz and potential increment 2 mV, corresponding to an effective scan rate of 100 mVs 1. The pH measurements were carried out with a Crison micropH 2001 pH-meter with an Ingold combined glass electrode. All experiments were done at room tempera- ture (25 18C) and microvolumes were measured using EP-10 and EP-100 Plus Motorized Microliter Pippettes (Rainin Instrument Co. Inc., Woburn, USA).
2.3 Acquisition and Presentation of Voltammetric Data All the voltammograms presented were background-sub- tracted and baseline-corrected using the moving average application with a step window of 5 mV included in GPES version 4.9 software. This mathematical treatment improves the visualisation and identification of peaks over the baseline without introducing any artefact, al- though the peak intensity is, in some cases, reduced (<10%) relative to that of the untreated curve. Never- theless, this mathematical treatment of the original vol- tammograms was used in the presentation of all experi- mental voltammograms for a better and clearer identifica- tion of the peaks. The values for peak current presented in all plots were determined from the original untreated voltammograms after subtraction of the baseline.
3 Results and Discussion
3.1 Cyclic Voltammetry Orientin and eriodictyol have a catechol moiety at ring-B, and two hydroxyl groups at ring-A similar to resorcinol and were studied by cyclic voltammetry. The cyclic voltammogram recorded from +0.00 V to +1.20 V in a 75 mM orientin solution, in pH 7.0 0.1 M phosphate buffer, showed two anodic peaks, peak 1a, at
Ep1a =+0.28 V, and peak 2a, at Ep2a =+0.90 V, reversing Fig. 1. Cyclic voltammograms in pH 7.0 0.1 M phosphate buffer the scan direction, cathodic peak 1c, at Ep1c =+0.22 V of 75 mM: (A) orientin and (B) eriodictyol; (—) first, (––––) (Figure 1A) appeared. second and (····) third scans at v=50 mVs 1. The second cyclic voltammogram scanning in the posi- tive direction, in the same solution and without cleaning the GCE surface, showed both anodic peaks 1a and 2a. C O-8-glucoside moiety has no influence in the oxidation The differences between the anodic and the cathodic mechanism. peak potentials of the first peak jEp1a Ep1c j =30 mV cor- The reversible peak 1a–1c corresponds to the catechol respond to a two-electron reversible reaction [26]. moiety oxidation whereas peak 2a is due to the resorcinol The cyclic voltammograms recorded for eriodictyol in moiety oxidation [24]. the same experimental conditions of orientin showed also The effect of scan rate, on orientin and eriodictyol re- two anodic peaks, peak 1a, at Ep1a =+0.25 V, and peak versible redox pair 1a–1c, was investigated, (Figures 2A 2a, at Ep2a =+0.89 V, and in the reverse scan one catho- and 2B). The cyclic voltammograms were obtained in dic peak 1c, at Ep1c =+0.19 V, Figure 1B. pH 7 0 0.1 M phosphate buffer for solution of 75 mMat These experiments showed that the oxidation of orien- GCE. Increasing the scan rate was observed that the oxi- tin and eridictyol occurs by the same redox mechanism, dation potential of peak 1a was slightly shifted to more and the absence of the C2 C3 double bond and the positive potentials and the reduction potential of peak 1c
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Fig. 2. Cyclic voltammograms in pH 7.0 0.1 M phosphate buffer of 75 mM: (A) orientin and (B) eriodictyol at scan rates 10 to 500 mVs 1.
was slightly shifted to negative potentials. The value of Comparing this results with those obtained for eridoc- jEp1a Ep1c j 30 mV was found for all scan rates, corre- tyol and orientin, the robinin peak 1a is attributed to the sponding to a reversible two-electron two-proton mecha- oxidation of the phenol moiety in ring-B while peak 2a nism. Moreover, increasing the scan rate, the current of corresponds to the oxidation of the OH group in ring-A. peaks 1a and 1c increased linearly with the square root of This behaviour is in agreement with the differences in the scan rate, consistent with a diffusion-limited oxidation the chemical structure of molecules (Scheme 1). The ab- of a solution species [26]. sence in robinin of a catechol moiety in ring-B explains The voltammetric results obtained for robinin, in simi- the absence of the peak 1c. Furthermore, the inherent lar experimental conditions, were different from those ob- steric hindrance of the large C-3-O-robinoside and C-7- tained for orientin and eriodictyol. The cyclic voltammo- O-rhamnoside sugar moieties in robinin structure has an grams recorded for 75 mM robinin in pH 7.0, 0.1 M phos- impeditive effect on the electron transfer reactions, and phate buffer presented two irreversible anodic peaks, all possible intramolecular H-bond interactions lead to peak 1a, at Ep1a =+0.77 V, and peak 2a, at Ep2a = minor effects causing the peak potential differences [15]. +1.02 V (Figure 3) and no new peaks were observed on the second scan.
Electroanalysis 2012, 24, No. 7, 1576 – 1583 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.electroanalysis.wiley-vch.de 1579 Full Paper E. de Souza Gil et al.
Fig. 3. Cyclic voltammograms in 75 mM robinin in pH 7.0 0.1 M phosphate buffer: (—) first, (----) second and (····) third scans at v=50 mVs 1.
3.2 Differential Pulse Voltammetry
The electrochemical oxidation of orientin, eriodictyol and robinin, Scheme 1, was studied by DP voltammetry in pH 7.0 0.1 M phosphate buffer (Figure 4), and between 0.00 V and +1.20 V successive DP voltammograms for each compound in the same solution, were recorded. The first DP voltammograms in 10 mM orientin and eriodictyol solutions showed two consecutive oxidation peaks, peak 1a, at Ep1a =+0.25 V, and peak 2a, at Ep2a = +0.83 V (Figures 4A and 4B). On successive recorded scans, in the same solution and without cleaning the GCE surface, the oxidation potential of peak 1a, due to the oxidation of catechol moiety in ring-B, remained constant but its current varied, due to the adsorption of the catechol-moiety oxidation product formed on peak 2a [24], that corresponds to the resorci- nol-moiety oxidation in ring-A. Moreover, the current of peak 2a also decreased slight- ly on successive scans due to the decrease of the available electrode surface area owing to adsorption of orientin or eriodictyol oxidation products. Successive DP voltammograms were also recorded in a50mM robinin solution in pH 7.0 0.1 M phosphate buffer (Figure 4C). The first oxidation peak 1a occurred Fig. 4. DP voltammograms base-line corrected in pH 7.0 0.1 M at a more positive value, E =+0.75 V, and correspond- phosphate buffer: (A) orientin, (B) 10 mM eriodictyol and (C) 50 p1a mM robinin; (—) first, (––––) second, and (····) third scans. ing to the oxidation of phenol moiety in ring-B. The rob- inin peak 2a, at Ep2a =+0.95 V, corresponds to the oxida- tion of the OH group in ring-A. The second DP voltam- after adsorption of robinin oxidation products (Fig- mogram, in the same solution and without cleaning the ure 4C).
GCE surface, showed a new peak 3a, at Ep3a =+0.30 V, The potential pH dependence of the three compounds and its current increased on successive scans. This peak in different electrolytes with 0.1 M ionic strength was in- corresponds to the oxidation of catechol-moieties in A- vestigated. For each compound the DP voltammograms ring and B-ring oxidation products [24], and the peaks 1a using a clean GCE surface, in a fresh solution of 10mM and 2a current decreased gradually on successive scans for orientin and eriodictyol and 50 mM for robinin, were due to a decrease of the available electrode surface area recorded.
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Fig. 5. 3D plot of DP voltammograms base-line corrected and plot of Epa vs. pH: (A) 10 mM orientin, (B) 10 mM eriodictyol and (C) 50 mM robinin.
For 2.0
Electroanalysis 2012, 24, No. 7, 1576 – 1583 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.electroanalysis.wiley-vch.de 1581 Full Paper E. de Souza Gil et al. dation mechanism of peak 2a corresponds to an electro- chemical reaction involving the transfer of one electron and one proton. For 2.0
3.3 Square Wave Voltammetry SW voltammograms recorded in pH 7 0.1 M phosphate buffer at GCE in 10 mM orientin and eriodictyol, and 50 mM robinin solutions, showed similar features to CV and DP voltammetry. For all compounds the oxidation occurred in two steps. A greater advantage of SW voltammetry is the possibil- ity to see during only one scan if the electron transfer re- action is reversible or not. Since the current is sampled in both positive and negative-going pulses, peaks corre- sponding to the oxidation and reduction of the electroac- tive species at the electrode surface can be obtained in the same experiment [26]. Thus, the reversibility of orientin and eriodictyol oxida- tion peak 1a was confirmed by plotting the forward and backward components of the total current (Figures 6A and 6B). Moreover, peak 2a total current occurs at the same potential and equal to the forward component, and on the backward component no cathodic peak appeared, in agreement with an irreversible reaction. The first SW voltammogram in robinin showed the two irreversible oxidation peaks 1a and 2a. On the third SW voltammogram, obtained in the same solution without cleaning the electrode surface, a new reversible peak 3a appeared at a lower potential, Ep3a =+0.30 V (Figure 6C) corresponding to the oxidation of robinin oxidation prod- Fig. 6. SW voltammograms in pH 7.0 0.1 M phosphate buffer: ucts, catechol to ortho-quinone. (A) 10 mM orientin and (B) 10 mM eriodictyol first scans, and (C) 1 50 mM robinin third scan, f=50 Hz, DEs =2mV,veff =100 mVs ; It : total, If : forward and Ib : backward. 4 Conclusions
The electrochemical behaviour of orientin, eriodictyol mechanism at lower potential while the resorcinol moiety and robinin has been studied in a wide pH range by in ring-A undergo oxidation at ~1 V more positive poten- means of cyclic, differential pulse and square wave vol- tial, with the transfer of one electron and one proton. tammetry using a GCE and the effect of molecular struc- In the robinin structure, the catechol moiety in ring-B tural differences was evaluated. It has been shown that is substituted by a phenol. The phenol group is irreversi- the oxidation mechanism occur in two steps, at ring-A ble oxidized at more positive potential than the catechol, and -B, with different number of electrons and protons in- and robinin second step oxidation occurred in the OH volved. group in ring-A. Both robinin oxidation steps occur with The catechol moiety in ring-B of orientin and eriodic- the transfer of one electron and one proton and the for- tyol was reversible oxidized in a two electron two proton mation of reversible electroactive products.
1582 www.electroanalysis.wiley-vch.de 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2012, 24, No. 7, 1576 – 1583 Anodic Behaviour of Flavonoids Orientin, Eriodictyol and Robinin at a GCE
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