Surface electrochemical oxidation and polymerization mechanism of epicatechin

Electrochimica Acta 90 (2013) 27–34

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Surface electrochemical oxidation and polymerization mechanism of epicatechin Shubo Han ∗ , Kaodi Umera, Xiaoyan Han, Justin W. Graham Department of Chemistry and Physics, Fayetteville State University, NC 28301, United States

a r t i c l e

i n f o

Article history: Received 28 August 2012 Received in revised form 5 November 2012 Accepted 7 November 2012 Available online 17 December 2012 Keywords: Epicatechin Cyclic voltammetry Polymerization Flavonoid Computation

a b s t r a c t Electrochemical process of epicatechin, one of the flavonoids antioxidants, was studied here by cyclic voltammetry and semiempirical molecular orbital computation (MOPAC). Electrochemical oxidation of epicatechin showed a multistep mechanism with two anodic peaks being recognized at about +0.14 V and +0.52 V (vs. Ag/AgCl). The first peak is strong concentration dependent, showing an adsorptive feature between 1 × 10−8 M and 2 × 10−7 M, a diffusion controlled feature between 2 × 10−7 M and 1 × 10−5 M, and a surface polymerization feature between 1 × 10−5 M and 1 × 10−3 M. Computation showed that the first electron was released at 4 -hydroxyl group in B-ring. No charge delocalization occurs between A- and B-rings. Higher pH medium favors oxidation. The oxidation rate is faster in strong acidic or basic medium and slower in a weak acidic medium. This research may help to explain the complexity of antioxidant activity of flavonoids and as a complement method to characterize the role of flavonoids antioxidants in treating oxidative stress diseases. © 2013 Published by Elsevier Ltd.

1. Introduction Flavonoids, a group of ubiquitous polyphenols found in plants, have been associated with reduced risk of a variety of diseases, due to their capability as potent antioxidants, as chelators of redoxactive metals and as inhibitors of lipid peroxidation [1]. Flavonoids might be particularly effective in the prevention of neurodegenerative diseases [2,3]. However, controversial results have been reported, with some positive findings, many null findings, and some suggestions of harm in certain high-risk populations, due to the intrinsic diversity of the multiple-step antioxidant reactions among the different flavonoids at varied conditions [4,5]. In addition, the variance of the methods used in oxidative process research often lead to disagreeable conclusions, too. Three assays are widely used for standardization of the antioxidant capacities measurement in foods and dietary supplements: the oxygen radical absorbance capacity assay, the Folin–Ciocalteu method, and the Trolox equivalent antioxidant capacity assay. However, it is still a challenging task to express the details of an antioxidant process (e.g., the multiple step mechanisms and multiple roles of flavonoids, the impact of pH, ionic strength and other surface or solution conditions, etc.), and in the mean time to consider the specialty of a bimolecular target. A more detailed and convenient method is needed to observe antioxidant process

∗ Corresponding author. Tel.: +1 910 672 1303. E-mail address: [email protected] (S. Han). 0013-4686/$ – see front matter © 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.electacta.2012.11.019

in vivo or in vitro. Electrochemical analysis, particularly cyclic voltammetry (CV), has been validated for quantitative analysis of the antioxidant capacity of blood plasma, tissue homogenates, and plant extracts [6–8]. Chevion et al. proposed that the area under the anodic current wave, rather than Ia , is a better parameter reflecting the antioxidant capacity [7]. Janeiro et al. investigated the electrochemical oxidation of the flavonoids (+)-catechin over a wide range of conditions, using CV, differential and square wave voltammetry [9]. Zielinska et al. evaluated the antioxidant activity of quercitin, its glucosides, and onion by using CV and spectrophotometric methods based on free radical-scavenging activities and reducing power, suggesting that CV assay is an efficient tool for describing the reducing activity of quercetin and its glucosides based on their redox properties. Meanwhile, care should be taken during analytical work to avoid the adsorption of oxidative products on the carbon electrode surface [10]. Noticeably the surface adsorption is very common in protein interactions that should play roles in neurodegenerative disease development or prevention. Therefore, further investigations on the surface phenomena of the flavonoids, along with other coexisting interactions, were discussed together with electrochemical processes in this work. Epicatechin, a flavonoid compound rich in cocoa, teas, wines and fruits, which is well recognized to be effective in lowering the risk of four common killer diseases, stroke, heart disease, cancer and diabetes, is selected as a model flavonoid in this study. Mechanisms of epicatechin antioxidant activity in solution and at surface were studied by CV and the electroactive sites are predicted by quantum chemical computation.

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Fig. 1. Dependence of first anodic peak on epicatechin concentration in PBS solution. (A) Cyclic voltammograms of epicatechin from 1.00 × 10−8 M to 1.00 × 10−3 M: from top to bottom of the anodic scans: 1.00 × 10−3 M (red), 5.00 × 10−4 M (yellow green), 2.50 × 10−4 M (purple), 1.00 × 10−4 M (green), 1.00 × 10−5 M (sky blue), 6.00 × 10−6 M (bronze green), 3.00 × 10−6 M (ruby), 2.00 × 10−6 M (navy), 1.00 × 10−6 M (turquoise), 5.00 × 10−7 M (dark blue), 1.00 × 10−7 M (red), and 1.00 × 10−8 M (navy). (B) Cyclic voltammograms of epicatechin from 1.00 × 10−8 M to 1.00 × 10−5 M: from top to bottom of the anodic scans: 1.00 × 10−5 M (red), 6.00 × 10−6 M (navy), 3.00 × 10−6 M (ruby), 2.00 × 10−6 M (blue), 1.00 × 10−6 M (yellow green), 5.00 × 10−7 M (green), 1.00 × 10−7 M (bronze green), and 1.00 × 10−8 M (purple). (C) Peak current (Ipa1 )-concentration (C) ratio correlation with concentration of epicatechin (C). (D) Linear relationship of peak current (Ipa1 ) and concentration of epicatechin (C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

2. Experimental

maintain a similar pH, osmolarity and ion strength as a physiological medium.

2.1. Materials (−)-Epicatechin and all other chemicals were purchased from Sigma–Aldrich. 2.2. Equipments CHI 440A Electrochemical Analyzer. A three-electrode system consisted of a CHI 104 glassy carbon working electrode (3 mm dia.), a CHI 115 Platinum Wire Counter Electrode, and a CHI 111 Ag/AgCl Reference Electrode (CHI Instruments Inc., USA) were used in this research. 2.3. Procedures Unless announced otherwise, all the experiments in this research were carried out in a pH 7.4 PBS buffer in order to

(1) Electrode polishing: Put a small quantity of alumina powder (0.05 ␮m) to a polishing pad and wet with distilled water. Vertically and firmly hold the working electrode with moderate force to move in a figure-of-eight motion for 5 min to ensure uniform and thorough polishing. Electrodes with rougher or scratched surface must go through 1.0, 0.3 and 0.05 ␮m alumina in a sequence in order to remove the surface defects. Once polishing has been completed, the electrode surface is fully rinsed by spraying with copious distilled water directly onto the electrode surface. Finally the polished electrode is sonicated in Millipore Type I ultrapure water (18.2 M cm at 25 ◦ C) for 3 min to ensure complete removal of the alumina particles. (2) CV and linear sweep voltammetry: Unless announced, parameters were set up as follows: initial and lower potential, −0.2 V;

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Fig. 2. Surface characters of epicatechin on cyclic voltammograms. (A) Cyclic voltammograms of repeat scans (20 segments) in PBS solution with a glassy carbon working electrode soaked in 1.0 × 10−5 M epicatechin for 30 min, washed with distilled water at least 10 times. (B) Cyclic voltammograms of repeat scans (20 segments) in 1.00 × 10−4 M epicatechin at pH 7.4. (C) Cyclic voltammograms of repeat scans (20 segments) in 1.00 × 10−5 M epicatechin at pH 7.4 from −0.2 V to 0.8 V (vs. Ag/AgCl) (red) and from −0.2 V to 0.28 V (vs. Ag/AgCl) (blue). (D) Peak current change in repeat scans of cyclic voltammograms showed in (C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

high potential, 0.8 V; initial scan polarity, positive; scan rate, 10 mV/s; quiet time, 60 s. (3) Electrode imaging and energy dispersive spectrum (EDS) measurement: The inverted microscope and the field emission electron gun equipped on JEOL JXA-8530F Hyperprobe were used to take the picture of the electrode surface and to measure the EDS, respectively. (4) Methodology for quantum chemical calculation: The electronic energy, charge distribution and molecular geometry epicatechin and its phenoxyl radical were computed at minimized energy levels by CS MOPAC Pro 8.0 under CHEM 3D Ultra interface. 3. Results and discussions 3.1. Dependence of peak currents on concentration of epicatechin Electrochemical behavior of epicatechin was tested by CV in a wide concentration range from 1.00 × 10−8 M to 1.00 × 10−3 M (Fig. 1A) and from 1.00 × 10−8 M to 1.00 × 10−5 M (Fig. 1B). Two consecutive anodic peaks at about +0.14 V and +0.52 V (vs. Ag/AgCl) were observed from CV curves at a rate of 10 mV/s. The first peak was strongly concentration dependent in the tested concentration range (Fig. 1C and D). Currents of the first anodic peak and the epicatechin concentration showed a nonlinear relationship between 1.00 × 10−8 and 1.00 × 10−3 M, suggesting an adsorption phenomenon or an EC mechanism: the electron transfer step

followed by a coupled chemical reaction. From 10−8 M to 2 × 10−7 M, Ip/C was decreasing with the concentration, proposing a typical surface adsorption at a lower concentration (Fig. 1C). The proportion of adsorption current to diffusion current became smaller with concentration, leading to a decline of Ip/C. It turned to be a nearly steady value of Ip/C while the concentration was higher than 2 × 10−7 M. Adsorption was accordingly negligible and Randles–Sevcik equation applied to the process, suggesting a diffusion-controlled process. In fact, Ip presented an approximately linear relationship with the concentration of epicatechin between 2 × 10−7 and 1 × 10−5 M (Fig. 1D), as expressed in the following equation: Ip = 35.76C + 0.0674 with a correlation coefficient, 0.9966, where Ip is the peak current (␮A) and C is the concentration (mM) of epicatechin. This linear relationship may be used for quantification of trace epicatechin in a biological sample. When the concentration was raised to the range of 1 × 10−5 M, this correlation curve fell slightly off the straight line again, suggesting an electrode reaction accompanied by a succeeding reaction (EC) between 1 × 10−5 M and 1 × 10−3 M. The decreasing sensitivity might result from a surface polymerization triggered by phenoxyl radicals. Generated through the electrochemical oxidation, the radicals reacted with epicatechin molecules that transported to the surface, forming dimeric or even polymeric products at electrode surface. The polymerization led to an insulating film that sticks to

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the electrode surface, hampering the further oxidation of epicatechin. Similar phenomena were also observed in other flavonoids compounds [11–13]. Consequently the first anodic peak became broader and poorly defined and the second peak was covered up by the broader first peak caused by polymerization process. The polymerization was negligible in a low concentration because the mass transportation of epicatechin is slower than further electrochemical oxidation of the radicals formed in the first step. Electrochemical processes of flavonoids account for their antioxidant activity in human body. Researchers have reported controversial conclusions because the circumstance and concentration they tested were different [14–18], which concurs with our assumption: The concentration-caused electrochemical mechanism shift might lead to very diverse consequences when flavonoids worked as an antioxidant at different circumstances or concentration levels.

4. Surface adsorption of epicatechin To confirm the surface adsorption, we soaked the electrode in 1.0 × 10−5 M epicatechin for 30 min, rinsed the electrode with distilled water at least 10 times, and then observed the CV signature in pH 7.4 PBS buffer. A clear adsorption peak was found on CV curve in a charge density about 8.79 × 10−8 C, corresponding to a surface density of epicatechin molecules 1.29 × 10−11 mol/cm2 (Fig. 2A). Interestingly soaking time did not have clear impact to the peak current, the charge densities of soaking 5, 30, 60 and 120 min were in same level, implying a fast adsorption process. The second peak became undistinguishable in repeat scans for 10−4 M (Fig. 2B) but not for 10−5 M epicatechin solution (Fig. 2C, red lines). This suggests again the increased epicatechin molecules that diffused to the electrode surface would react with epicatechin radicals – products of the first step. If polymerization occurred, the further oxidation would be blocked by the formed polymer membrane. While at a low concentration, the rate of the second oxidation is higher than the diffusion rate, dominating the surface process over the polymerization. Because of chemical reaction between epicatechin radicals and epicatechin, repeat scan showed a sharply declining peak current (Fig. 2D, lines of solid and empty circle). CV scan between −0.2 V and 0.28 V (vs. Ag/AgCl), instead, showed almost no change of the peak current in repeat scans (dashed line in Fig. 2D), 1:1 current ratio (Ipa/Ipc), and diffusion-controlled character (Fig. 2C, blue lines). Additional evidences of surface adsorption were also found in linear sweep voltammetry by changing the scan rate from 10 mV/s to 250 mV/s (Fig. 3A). A typical surface adsorption signature, the current of first peak is linear to the scan rate (v), has been observed in Fig. 3B. Although adsorption is significant at a lower concentration, the CV curves showed no separate adsorption peak probably due to the minor difference in free energy of oxidation between adsorbed and free epicatechin, showing a weak adsorption character. The first anodic peak is much higher than the corresponding cathodic peak, suggesting the weak adsorption was mainly caused by the reactant-reduced form of epicatechin. This is not surprised because the surface oxidation of epicatechin caused an increased polarity and thus would diminish the adsorption at a non-polar glassy carbon electrode surface. One hypothesis for the inhibition mechanism of ␣-synuclein fibrillization is that the increased polarity caused by oxidation enhanced the hydrophilic property of the protein molecules and minimized the chance of aggregation epimers [19]. When epicatechin combined with ␣-synuclein, a reaction between this flavonoids compound with ROS would lead to same outcome that would be helpful for the prevention of ␣-synuclein aggregation, a character in Parkinson’s disease.

Fig. 3. Linear sweep voltammograms (second scan) of 1.00 mM epicatechin in pH 7.4 PBS solution at 250 (red), 100 (blue), 50 (purple), 25 (navy) and 10 mV/s (green) (A), and peak current and scan rate correlation (B). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

In all the tested concentrations, the first anodic peak current increased with the concentration. However, the second anodic peak and the only detectable cathodic peak, which corresponds to the first anodic peak, showed a nonlinearly proportional growth with the concentration of epicatechin. At a lower concentration, the charge ratio of the first anodic peak and the cathodic peak were close; while at a higher concentration, this ratio increased to as high as 16:1 at concentration 1.0 × 10−3 M, showing a clearly EC process feature. The second cathodic peak did not build up with the concentration growth, suggesting that the radical formed in first oxidation will be consumed in the dimerization or polymerization at high concentration. The CV was also tested at various initial potentials, −0.2, −0.1, 0.0, 0.1, and 0.2 V (vs. Ag/AgCl). Positively shifting Ei was found to diminish the first anodic peak but be almost impertinent to the peak in the repeat scan. Second peak was still observable and the cathodic peak was not affected by Ei , too. The second scan always started from 0.2 V (vs. Ag/AgCl) no matter what Ei in the first scan was. This suggests again the surface adsorption character of epicatechin and the multistep mechanism. The formed thin membrane on the surface may be the determining factor which controls further surface electron exchange rate.

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Fig. 4. Picture (A) and energy dispersive spectrum (EDS) of the glassy carbon working electrode after repeated scanning for 20 cycles between −0.2 V and 0.8 V (vs. Ag/AgCl), rinsing with deionized water and drying with nitrogen gas.

4.1. Surface polymerization of epicatechin induced by free radicals

electronic energy of neutral parent molecule (RH). The IP for the neutral molecule (IP(RH)) was calculated as the electronic energy of the radical monocation (RH+ ) subtracting the electronic energy of the neutral parent molecule. Similarly, the IP for phenoxylate monoanion (IP(R− )) was calculated as the electronic energy of the most stable phenoxyl radical (R) subtracting the electronic energy of this most-stable phenoxylate monoanion (R− ). BDE and IP show the ease of hydrogen atom donation and the ease of electron donation, respectively. Reflected by the BDE(RH) value (Table 1), the hydrogen–atom donation of epicatechin in their neutral form may need lowest energy at B ring (3 and 4 position), higher energy at A ring (5 and 7 position) and highest energy at C ring (3 position). IP(RH) and IP(R− ) value also show that electrochemical oxidation is become more difficult in the order of B, A and C rings. Then, the geometry and the stability of epicatechin and its phenoxyl radical were computed at minimized energy levels. The epicatechin neutral molecule, phenolate and its radical shared similar geometry at minimized energy (Fig. 5). The angle between the chromane moiety and the B-ring is approximately same, i.e., O1C2C1 angle 106.9◦ for neutral molecule, 106.9◦ for 3 radical, and 107.0 for 4 radical, indicating the two moieties of the molecule are roughly perpendicular and mainly independent with each other – no charge delocalization occurs between A- and B-rings, implying the existing coordination of one ring with metals may not be affected by the oxidation of the other ring. Further computation of charge distribution of epicatechin and the phenoxyl radicals showed that the charge distribution did not change after deprotonation or H-abstraction occurs in the phenolic cycle, A or B, supporting above moiety independent assumption (Table 2). The hydroxyl group with highest negative charge, which

To further confirm the formation of polymer film induced by free radicals at a higher flavonoid concentration, the glassy carbon electrode surface was observed by an inverted optical microscope. After CV scan (20 segments) in 1.0 × 10−4 M epicatechin solution with an operation condition as described above, electrode surface was dried and picture was taken. Observed by naked eyes and shown in Fig. 4A, the electrode surface was covered by a brown membrane on the originally black glassy carbon surface. This brown membrane might be the polymer film formed during EC process. EDS analysis for the dried electrode surface showed a clear peak of oxygen, suggesting the surface accumulation of epicatechin which are rich on C and O (Fig. 4B). 4.2. Computation of electrochemical reaction Comparison of the cyclic voltammogram of 5 × 10−6 M epicatechin with that of same concentration of ferrocyanide using same working electrode suggested two consecutive single electron transfer process. However, there are five oxidizable hydroxyl groups at position, 3, 5, 7, 3 , and 5 . To predict the position of the first oneelectron electrochemical oxidation, electronic parameters such as bond dissociation energies (BDE) for homolytic OH bond cleavage and ionization potentials (IP) were estimated by semi-empirical molecular orbital simulation method at 3 , 4 , 3, 5 and 7 carbon position, respectively. The BDE for homolytic OH bond cleavage in the neutral molecule (BDE(RH)) was calculated as the sum of electronic energy of the phenoxyl (R) and hydrogen radical (H) subtracting the

Table 1 Electronic energy (EE), bond dissociation energy (BDE) and ionization energy (IP) calculated by MOPAC for neutral (RH), phenoxyl radical (R), phenolate (R− ), phenoxyl radical acid (RH+ ). Position

4

3

5

7

3

H atom

EE (RH) EE (R) EE (R− ) EE (RH+ ) BDE (RH) (eV) IP (RH) (eV) IP (R− ) (eV) BDE (RH) (kcal/mol) IP (RH) (kcal/mol) IP (R− ) (kcal/mol)

−25,325.2 −24,990 −24,956.2 −25,330.7 323.80 −5.5 −33.8 7466.911 −126.83 −779.428

−25,325.2 −24,971.8 −24,958.5 −25,330.7 342.00 −5.5 −13.3 7886.603 −126.83 −306.698

−25,325.2 −24,939.8 −24,961.1 −25,326.6 374.00 −1.4 21.3 8624.523 −32.284 491.178

−25,325.2 −24,976.2 −24,980.5 −25,326.2 337.60 −1.0 4.3 7785.139 −23.06 99.158

−25,325.2 −24,876.5 −24,879.5 −25,322.7 437.30 2.5 3.0 10,084.221 57.65 69.18

−11.3964

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Fig. 5. Optimized geometries calculated for epicatechin (A), its 3 -phenoxyl radical (B) and 4 -phenolate (C).

should facilitate hydrogen bonding, is found at C-3 position, suggesting the electrochemical reaction and hydrogen bonding tend to choose different hydroxyl group. Belonging to flavonoids group, epicatechin contains a diphenylpropane skeleton. However, the saturated heterocyclic C ring between A and B rings blocked the electron delocalization between A and B rings because of the lack of 4-oxo group. The electron donation is slowed down and the phenoxyl radicals formed as intermediates may be stabilized. The oxidation of the multiple hydroxyl groups is fairly considered to be the main reason of the ROS scavenging activity. Both IP and BDE values suggested that the epicatechin oxidation firstly occurs through the B-ring at C-4 position. The oxidation of epicatechin is perturbed by a following chemical reaction between formed radical and epicatechin, leading to the formation of a

Table 2 Mulliken charge distribution at optimized geometries for epicatechin and its phenoxyl radicals.

semiquinonic radical that dimerizes to an electroinactive compounds. As in other polyphenols, the epicatechin concentration is the determining factors in the competition between dimerization and quinone reduction and that causes the difference in charge ratio between oxidation and reduction when epicatechin concentration increases [11,20]. This dimerization leads to the separation between anodic and cathodic peaks at a higher concentration, increased in the irreversibility of the system and hindered the further antioxidant activity in a biological system. The second electron transfer is accompanied by the formation of epicatechin polymer. This polymer results from repeated condensation reactions between the A ring of one unit and the B ring of another unit through a mechanism known as head to tail polymerization. The orthoquinone is formed prior to the condensation products and is involved in the formation of these products. The polymer remains on the electrode surface and is the cause of the surface fouling occurring for potentials higher than 0.75 V. The proposed mechanism has been shown in Scheme 1.

Position

Epicatechin

Epicatechin-3 -phenoxyl radical

Epicatechin-4 -phenoxyl radical

5. pH-dependent radical scavenging capacity of epicatechin

O1 C2 C3 C4 C5 C6 C7 C8 C9 C10 O3 O5 O7 C1 C2 C3 C4 C5 C6 O3 O4

−0.24158 −0.00243 −0.03315 −0.24943 0.1529 −0.2614 0.1371 −0.32215 0.15027 −0.2736 −0.35746 −0.29832 −0.29785 −0.09896 −0.20487 −0.013 0.08456 −0.19416 −0.29832 −0.32489 −0.30018

−0.25061 −0.01841 −0.02815 −0.24318 0.1519 −0.25568 0.13566 −0.31842 0.14714 −0.2583 −0.36395 −0.29872 −0.29765 −0.07821 −0.24696 0.23988 0.0328 −0.22104 −0.11761 −0.33758 −0.26515

−0.24393 −0.0166 −0.03218 −0.24954 0.15419 −0.25938 0.13859 −0.32031 0.14837 −0.27253 −0.35585 −0.29663 −0.29632 −0.04975 −0.20864 0.02426 0.24226 −0.25975 −0.1324 −0.26924 −0.3367

Epicatechin presents polyacidic features due to the dissociation of the five hydroxyl groups in the molecule. The antioxidant activity of epicatechin is accordingly affected by acidity. Many researchers have shown that the radical scavenging capacity of catechins increases with the increasing of the pH. Our CV experiments at different pH showed similar trends to oxidation potential. The first peak and the second peak for both first scan and repeat scan showed 59 mV/pH slope, suggesting one proton donation process for both steps. The pH range in different compartments of human body varies from 1 to 9.3, thus a higher pH range favors the antioxidant activity of epicatechin. This is also true that the radical scavenging capability varies with pH at surface and in the fluids. Interestingly influence of pH on anodic current displayed a different feature. The peak current showed an inverted bell curve between peak current and pH with a minimum value at pH 4 in the first scan and at pH 6 in the second scan, implying different mechanisms between first and second scans that affected the reaction rate (Fig. 6).

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4' OH HO 7

O

3' OH

3

OH

-e-, -H+

HO

O

O

OH

5

OH

OH

OH

O

OH HO

O

O

-e-, -H+

HO

O

O

OH

OH OH

OH

OH O

HO

OH

OH HO

O

O

OH

OH OH

Dimerization Polymerization

High Concentration

OH Scheme 1. Mechanism of epicatechin electrochemical oxidation on glassy carbon electrode.

A 0.55 0.5 0.45

1st scan

1.6

2nd scan

1.4

0.4

1.2

0.35 Ip1, µA

Peak Potenal (Ag/AgCl)

B 1.8

EPCA 1st scan CA 1st scan EPCA 2nd scan CA 2nd scan

0.3

1 0.8

0.25 0.6

0.2

0.4

0.15

0.2

0.1

0

0.05 2.5

3.5

4.5

5.5 pH

6.5

7.5

8.5

2

4

6

8

10

pH

Fig. 6. pH-dependent profile of (A) peak potential of 0.100 mM epicatechin: first scan (blue) and second scan (green), and 0.100 mM catechin: first scan (red) and second scan (black). (B) Peak current of 0.1 mM epicatechin: first scan (blue) and second scan (red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Because of deprotonation, electrochemical reaction was accelerated at a higher pH value, which increased the peak current and narrowed the potential window (Ep = Epa − Epc ), an indicator for the reversibility of an electrode process. When pH was lower than 4 in first scan or 6 in second scan, raising acidity resulted in the surface enrichment of epicatechin and the hiking of peak current without improving reversibility owing to the enhanced hydrophobicity during protonation. The possible surface polymerization after first scan will inhibit the further adsorption and lower the anodic current in second scan. Catechin, the epimer of epicatechin, on other hand showed similar trends to the pH influences. However, peak potentials of catechin electrochemical oxidation is more positive than that of epicatechin, showing that epicatechin is of stronger antioxidant capability. This may explain the unequal role the two epimers in antioxidant treatment. 6. Conclusion Electrochemical oxidation of epicatechin was found to be a multistep process occurred at ring B. Two one-electron transferring peaks was recognized at about +0.14 V and +0.52 V (vs. Ag/AgCl). The first peak is a strong concentration dependent, showing an adsorptive feature between 10−8 M and 2 × 10−7 M, a diffusion controlled feature between 2 × 10−7 M and 1 × 10−5 M, and a surface polymerization (EC process) feature between 1 × 10−5 M and 1 × 10−3 M. Computation suggested the most favorite site to release electron to be 4 -hydroxyl group in ring B. Rings A, B and C do not show electron delocalization. Rings A and C do not participate in the oxidation in the first step, neither changed geometry and charge density. This helps to understand why the hydrogen binding and coordination of flavonoids are not strongly affected by oxidation and some oxidized flavonoids are still effective in resisting protein aggregation in neurodegenerative diseases. Higher pH medium was found to lower the peak potential and make the oxidation easier, but reaction rate did not show same trends, which was slowest at weak acidic medium and faster in strong acidic or basic medium. The complexity of electrochemical reaction of epicatechin showed in our research may explain the reported mixed pictures of flavonoids antioxidants as a defender of oxidative stress diseases, helping to recognize a safe and effective of flavonoids treatment. Acknowledgements This work was supported by Center for Promoting STEM Education and Research (CPSER) funded by Title III at Fayetteville State University (FSU), FSU RISE Program funded by NIH, and FSU OPTIMUM Summer Internship Program funded by National Science Foundation.

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