Rapid and Repeatable Redox Cycling of an Insoluble Dietary

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Rapid and Repeatable Redox Cycling of an Insoluble Dietary Antioxidant: Electrochemical Analysis Morgan E. Lee,† Eunkyoung Kim,†,‡ Yi Liu,†,‡ John C. March,§ William E. Bentley,†,‡ and Gregory F. Payne*,†,‡ †

Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, United States Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, Maryland 20742, United States § Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York 14853, United States ‡

S Supporting Information *

ABSTRACT: There are many unresolved questions concerning the health benefits of dietary antioxidants due in part to the complexity of the materials and mechanisms of action. We applied a new electrochemical method and report new observations for one of the richest sources of dietary antioxidants. We observed that the insoluble fraction of clove is redox-active and can be rapidly and repeatedly switched between oxidized and reduced states. Also, the radical scavenging antioxidant properties of insoluble clove are largely independent of this reversible redox activity, which is similar to observations made with the natural phenolic melanin. In contrast to melanin, insoluble clove was observed to have little pro-oxidant activity (as measured by H2O2 generation) irrelevant to whether it was poised in an oxidized or reduced state. These results suggest that dietary antioxidants, even when insoluble and nonabsorbed, can undergo important redox interactions in the intestinal tract. KEYWORDS: clove, chitosan, redox cycling, antioxidant, pro-oxidant, electrochemistry, redox activity

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INTRODUCTION Oxidative stress has been implicated in various diseases, and while there has been persistent epidemiological evidence that diets rich in antioxidants confer health benefits, interventional trials have been less definitive.1−5 Since phenolics are among the most abundant antioxidants in our diet,6,7 considerable efforts have focused on their potential health benefits.8−11 Historically, the benefits have been attributed to the radical scavenging antioxidant properties of phenolics.9,12,13 Free radicals generated by the transfer of single electrons to O2 are capable of damaging cellular components (e.g., proteins, lipids, and DNA), and such reactive oxygen species (ROS) are prominently featured in theories of oxidative stress.3,4,14,15 In accordance with an emphasis on free radical damage, traditional chemical methods to quantify the potential benefits of dietary phenolics are based on free radical scavenging assays.16,17 Biological studies of oxidative stress often focus on the cellular signaling pathways that affect gene expression18−22 and also on the thiol redox couples (e.g., glutathione, cysteine, and thioredoxins).23,24 Interest in these thiol redox couples is motivated by the following observations: (i) the sulfur atoms in these thiol couples are potential targets of ROS action25 and can serve as an “interface” between ROS and cell signaling;26 (ii) diseased states are often associated with a greater oxidation of the small molecule redox couples;27,28 and (iii) mechanisms can be readily proposed to explain how redox information can be transmitted through cellular signaling pathways29 (i.e., cysteine residues of proteins can serve as “redox switches” whereby the formation/loss of disulfide linkages alters protein structure/function as a part of cellular signal transduction pathway).30−32 Interestingly, the oxidation of thiols to form disulfides is a two-electron process that suggests that free © 2014 American Chemical Society

radicals may not be an absolute requirement for oxidative stress. This observation suggests that oxidative stress may need to be broadened from a “free radical hypothesis” to a “redox hypothesis.”14 We have been developing a simple and rapid electrochemical method to detect redox activities of phenolic materials.33−37 Importantly, this method can be performed in the absence of O2 to probe intrinsic redox activities independent of ROS contributions. Here, we apply this method to probe the redox activity of dietary antioxidants using the spice clove as our model because it is reported to be one of the richest sources of dietary antioxidants.38,39 Specifically, we incorporated the insoluble fraction of this spice into a thin hydrogel film adjacent to an electrode and probed the redox properties of the film. We report that the insoluble fraction of clove possesses both antioxidant and redox activities and this insoluble fraction can rapidly and repeatedly exchange electrons with soluble mediators. These observations suggest that dietary antioxidants (even if insoluble) may participate in the complex set of redox interactions occurring in the intestinal tract.

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MATERIALS AND METHODS

Chemicals. The following were purchased from Sigma−Aldrich: chitosan, 1,1′-ferrocenedimethanol (Fc), Ru(NH3)6Cl3 (Ru3+), and 2,2-azobis-2-methyl-propanimidamide dihydrochloride (AAPH). A bottle of ground clove was purchased from a local grocery store. The water (>18 MΩ) used in this study was obtained from a Super Q water system (Millipore). Chitosan solutions (1%, pH 5.5) were Received: Revised: Accepted: Published: 9760

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prepared by dissolving chitosan flakes in HCl to achieve a final pH of 5−6. The solutions of catechol and mediator were prepared in phosphate buffer (0.1 M, pH 7.0). Preparation of the Clove−Chitosan Film. To obtain an insoluble clove fraction, clove (5, 30, or 60 mg/mL) was suspended in water, vortexed, and then, centrifuged (3200g for 5 min). After discarding the upper brown solution, the settled clove was suspended in fresh water and again centrifuged. This process was repeated until the upper solution showed no absorbance peaks (300−700 nm) where phenolic antioxidants would be expected (see Figure S1, Supporting Information). The insoluble clove fraction was next suspended in aqueous solutions containing chitosan (0.5%, pH 5.5), and then, small amounts of this suspension (20 μL) were spread onto a standard gold electrode (area ∼ 0.0314 cm2). The film was vacuum-dried at 37 °C for 30 min and then immersed in phosphate buffer (pH 7.0; 30 min). This buffer treatment partially deprotonates the primary amines of chitosan and converts the film into a water-insoluble form. In most experiments, films containing insoluble clove were compared against control chitosan films (lacking clove). Electrochemical Instruments. Electrochemical measurements (cyclic voltammetry and chronocoulometry) were performed using a three-electrode system (CHI Instruments 6273C electrochemical analyzer). The working electrode was the standard gold electrode (with chitosan film, area = 0.03 cm2), the reference electrode was Ag/ AgCl, and the counter electrode was a Pt wire. All electrochemical experiments were done in a mixed solution of 50 μM Fc and 50 μM Ru3+ in phosphate buffer (0.1 M, pH 7.0). Air was excluded by purging N2 during the experiment. For measurement of antioxidant and prooxidant activities, the gold-coated silicon wafer was used as a working electrode because its larger surface area (0.9 cm2) increased sensitivities of these measurements. Antioxidant Assay. Antioxidant activity was measured by adapting the standard oxygen radical absorbance capacity (ORAC) method that probes a hydrogen atom transfer mechanism. We used this method to probe if/how antioxidant properties varied depending on whether the clove−chitosan film was poised in an oxidized or reduced state. In this case, the film-coated electrode was electrochemically poised in an oxidized or reduced state (see the text for details) and then incubated in a solution (200 μL) of fluorescein (10 nM) in 0.1 M phosphate buffer (pH 7.0) followed by the rapid addition of the AAPH solution (20 mM). The mixture was incubated for a fixed time of 30 min at 37 °C, and then, the fluorescence was recorded using a fluorescence plate reader (SpectraMax M2, Molecular Devices, CA). Assay for Pro-oxidant Activity. The film-coated electrodes were electrochemically poised in either oxidized or reduced states, dried with N2, and then, incubated in air-saturated water for 15 min. The H2O2 generated in this aqueous solution was assayed using Amplex Red reagent (Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit, Invitrogen, OR), and the results were measured using a fluorescence plate reader. Statistical Analysis. All experiments were carried out in triplicate. Statistical analyses were performed using one-way analysis of variance compared with control.

Scheme 1. Experimental Sample, System, and Electrochemical Redox Cyclinga

(a) Standard gold electrode and preparation of film with insoluble clove entrapped within a chitosan matrix (films with covalently grafted catechol moieties are used for illustrative and comparative purposes). (b) Electrochemical system used for cyclic voltammetry (cyclic input potentials are imposed). (c) Redox cycling of electrochemical mediators (Fc and Ru3+) and chemical mechanism proposed for the case of catechol−chitosan films.40 a

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“working” electrode is inserted into a buffered solution (0.1 M phosphate; pH 7.0) along with the reference (Ag/AgCl) and counter electrodes. As illustrated, an input potential (E; volts) can be imposed to the underlying gold electrode, and the basis of this electrochemical analysis is to infer redox activities from the observed output currents. In initial studies, this clove− chitosan film was probed by cyclic voltammetry such that the potential (E) of the electrode was controllably cycled between oxidative values (more positive values of E) and reductive values (more negative values of E). If a chemical species can access the electrode and undergo redox reactions with the electrode, then oxidation is observed as a negative current while reduction is observed as a positive current. (Note: cyclic voltammetry data is commonly displayed as a plot of current vs potential without explicitly showing time.)

RESULTS AND DISCUSSION Initial Evidence of Redox Activity. Scheme 1a illustrates the experimental approach for preparing a chitosan film containing insoluble clove. The photograph shows a standard gold electrode while the schematic illustrates that a clove− chitosan film is prepared directly adjacent to this electrode. Scheme 1a also illustrates the structure of this film with the insoluble clove particles entrapped within the chitosan network. Previously, we studied chitosan films containing insoluble melanin particles36 and chitosan films with grafted catechol moieties.40 Results from these earlier studies can be used to compare observations with insoluble clove. Scheme 1b shows the electrochemical system used to probe the redox activities of the clove−chitosan film. This film-coated 9761

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reduction currents should be observed in the CVs. The second control in Figure 1a is for a chitosan film (without clove) that was incubated in the presence of these two mediators. When the potential for this chitosan-coated electrode was cycled to oxidative voltages, a small oxidation peak current was observed near +0.27 V consistent with the expected Fc oxidation. Figure 1a shows that, when the potential for this chitosan-coated electrode was cycled to reductive voltages, a small reduction peak current was observed near −0.27 V consistent with the expected Ru3+ reduction. If the insoluble clove particles entrapped in the chitosan film can exchange electrons with the mediators, then a third expectation is that the oxidation and/or reduction currents observed in the CVs should be altered compared to those observed with the chitosan control film. The experimental sample in Figure 1a is the clove−chitosan film incubated in the presence of both Fc and Ru3+ mediators. Considerable amplification of the Fc-mediated oxidation currents and the Ru3+-mediated reduction currents are observed in Figure 1a. If the observed amplification of mediator currents is due to redox interactions with the entrapped clove particles, then a further expectation is that amplification should vary with the amount of entrapped clove. As expected, the CVs in Figure 1b show that amplification of the Fc-oxidation and Ru3+-reduction currents increases with increasing amounts of clove incorporated into the films. These results provide initial evidence that the insoluble components of clove can undergo redox interactions with the soluble Fc and Ru3+ mediators. A possible mechanistic explanation for the above observations is provided in Scheme 1c that was proposed in earlier studies with catechol-modified chitosan films.41 Specifically, the Fc mediator can diffuse from solution through the film and undergo oxidation at the electrode to generate Fc+ species. The oxidized Fc+ species can either diffuse out of the film into solution or undergo redox interactions with the insoluble clove incorporated in the film. The observed amplification of the oxidation currents suggests the clove donates electrons to the Fc+ to regenerate the Fc species that can undergo oxidative redox cycling with the electrode. Similarly, Scheme 1c illustrates that amplification of the reducing currents is indicative of reductive redox cycling of the Ru3+ mediator such that it accepts electrons at the electrode but donates these electrons to the insoluble clove. In previous studies, we proposed that the observed redox cycling involved a switching between the reduced catechols (QH2) and oxidized o-quinones (Q) as illustrated by the reactions in Scheme 1c.41 The thermodynamic plot in Scheme 1c shows that electron transfer occurs from more negative to more positive potentials. Switching the Films to Oxidized or Reduced States. A convenient feature of electrochemical methods is that arbitrarily complex input potentials can be imposed on a sample. As illustrated in Scheme 2, we use step changes in the input potential in the presence of the Fc and Ru3+ mediators to switch the redox state of the film. To switch the film to a reduced state, Scheme 2a shows that the potential is stepped to provide reducing conditions (−0.4 V vs Ag/AgCl). As illustrated by the schematic, this step change would drive the Ru3+ reductive-redox-cycling reaction to mediate the transfer of electrons from the electrode to the sample (e.g., to quinone moieties). Over time, this reduction (or charging) step should “saturate” the sample with electrons. To switch the film to an oxidized state, Scheme 2b shows that the potential is stepped to an oxidative value (+0.5 V vs Ag/AgCl) to drive the Fc

Chemical intuition suggests several expectations for this initial cyclic voltammetry experiment. First, minimal oxidation or reduction currents should be observed if the clove−chitosan film is probed in a buffer solution that lacks diffusible redoxactive species. In this case, there are no accessible electrons to exchange with the electrode: chitosan is not redox-active and cannot conduct electrons, and the insoluble clove particles are entrapped too far from the electrode surface for direct electron exchange. The first control in Figure 1a is for a chitosan film

Figure 1. CVs provide initial evidence that insoluble clove has redox activity (scan rate = 50 mV/s). (a) CVs for electrodes coated with chitosan films: (i) clove−chitosan control incubated without mediators shows neither chitosan nor clove are conducting (no peak currents are observed); (ii) chitosan control incubated with both Fc and Ru3+ mediators (50 μM each) shows that the chitosan film is permeable to these mediators (the small peak currents correspond to the diffusion of mediators); (iii) clove−chitosan incubated with both mediators shows amplified peak currents consistent with redox cycling. (b) CVs for electrodes coated with chitosan films containing varying clove levels show amplified peak currents increase with increasing clove levels.

prepared with the insoluble fraction of clove and probed in the absence of soluble mediators. As expected, the cyclic voltammograms (CVs) for this control show no oxidation or reduction peaks. In order for oxidation or reduction currents to be observed, a soluble redox mediator must be incorporated in the buffer, and this mediator must be able to diffuse through the film to access the electrode. We incorporate two electrochemical mediators in the buffer: Fc typically exists in its reduced state but can be readily and reversibly oxidized when the potential is cycled near its E° (+0.25 V vs Ag/AgCl); and Ru3+ typically exists in its oxidized state but can be readily and reversibly reduced near its E° (−0.2 V vs Ag/AgCl). A second expectation is that, if these two mediators are added into the buffer, then they should be capable of diffusing through the films to exchange electrons directly with the underlying electrode and thus oxidation and 9762

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the Fc mediator that diffuses from the bulk solution through the film to the electrode surface. The charge transfer for the clove−chitosan film (Qsample) is attributed to both Fc diffusion and Fc redox cycling in the film. The difference between these values is used to calculate the redox capacity (Nfilm) of the film as illustrated in Scheme 2c. Experimental results for samples prepared with differing levels of clove are shown in Figure 2a. As expected, the

Scheme 2. Step Changes in Input Potential Can Be Used to (a) Engage Ru3+ Reductive Redox Cycling to Charge the Film with Electrons, (b) Engage Fc Oxidative Redox Cycling to Discharge the Film, and (c) Quantify Redox Capacity by Chronocoulometry (Impose a Constant Potential and Quantify Electron Transfer)

Figure 2. Electrochemical method (chronocoulometry) to quantify the redox capacity of the films (Nfilm). (a) Experimental results for the Fcmediated oxidative charge transfer in response to a step input of oxidative potential (from an open circuit potential to +0.5 V vs Ag/ AgCl that is maintained constant for 2 min). (b) The redox capacity for clove−chitosan films increases with increasing levels of insoluble clove.

observed charge transfer during this 2 min discharging step was greater when the sample contained more insoluble clove incorporated into the chitosan films. Figure 2b shows the calculated values of redox capacity and demonstrates that the insoluble clove possesses considerable redox capacity. To provide some estimate of the significance of these measured redox capacities, we consider a clove−chitosan film prepared by applying 20 μL of a 30 mg/mL solution to a working electrode area of 0.03 cm2 to obtain ≈20 mg clove/cm2. If the redox capacity is attributed to a two-electron transfer involving a quinone−catechol couple, then the measured value of 13 nmole e−/cm2 corresponds to ≈0.7 μg/cm2 catechol. Thus, the observed redox capacity could be the result of ≈0.04 μg/mg catechol equivalence in this insoluble clove sample. It is important to emphasize that, while the above estimate is stoichiometric, redox cycling can act catalytically for transferring electrons from a reductant to an oxidant.40 There are two important caveats to the method for calculating Nfilm. First, particulate samples (e.g., insoluble clove) can be difficult to completely charge or discharge because of limitations in the abilities of the mediators to diffuse into and access redox-active moieties within the particles.36 Thus, the measured redox capacities are operational measures

oxidative-redox-cycling reaction to mediate electron transfer from the sample to the electrode (i.e., to discharge electrons from the sample). Quantifying Redox Capacity. We used such step changes in input potential to quantify the redox capacity of the clove entrapped within the chitosan film. Initially, the film was charged with electrons using a reducing potential step (−0.4 V; 2 min). After charging, the potential was stepped to an oxidative value (+0.5 V; 2 min) to allow Fc to discharge the film. During discharging, the electrode current (i) was measured, and the electron transfer (Q) was calculated by integration of the measured current as illustrated in Scheme 2c (this method of imposing a constant potential and measuring Q is referred to as chronocoulometry). The redox capacity of the film is determined by comparing an experimental sample (clove−chitosan film) against a control chitosan film. As illustrated in Scheme 2c, the charge transfer observed for the control chitosan film (Qcontrol) is attributed to the oxidation of 9763

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useful for comparison but an underestimation of the true ability of the sample to accept, store, and donate electrons. Second, our method probes a comparatively small window of redox potentials that is most relevant to the reversible oxidation and reduction of catecholic moieties. We believe this potential region is important given the redox gradients in the gut42 and the ability of reversible redox cycling in this milieu to provide a means for redox interactions among the microbiota and the epithelium.43 We do not expect these imposed potentials are sufficiently aggressive to oxidize monophenolics (e.g., eugenol is a major component of clove39) that can undergo irreversible reactions important to radical scavenging activities.44 Again, while this narrow potential region allows us to focus on reversible redox interactions, we are underestimating the true redox capacity of a sample. Repeatable Redox Switching. In addition to using cyclic voltammetry to provide initial evidence for redox activities (Scheme 1 and Figure 1) and chronocoulometry to provide semiquantitative measure of redox capacity (Scheme 2 and Figure 2), we are developing a third electrochemical method to assess the reversibility of these redox interactions. Specifically, we collected CVs for numerous oxidation and reduction cycles to observe the repeatability of redox cycling. In principle, this approach is analogous to approaches used in control theory in which oscillating inputs are used to impose to small perturbations to a system and the resulting oscillating outputs provide information on the response of the system to these inputs. If the response of the system is fully reversible, then the oscillating outputs are expected to be “steady”; the amplitude and frequency should remain constant over time (provided the oscillating input remained the same). Experimentally, we tested film-coated electrodes (either chitosan or clove−chitosan films) in solutions containing both the Fc and Ru3+ mediators with the imposed cyclic input potentials indicated in Figure 3a. Visual inspection of the output currents shows no noticeable changes over the eight cycles of this experiment and provides initial evidence that the output is steady. Steady output currents would indicate that the redox activities being probed by this method are fully reversible (i.e., the oxidation and reduction reactions of clove are reversible). A more subtle implication of steady outputs is that, during each cycle, the Fc-mediated oxidative redox cycling of clove is nearly balanced by the Ru3+-mediated reductive redox-cycling reduction of clove. To more rigorously analyze the results in Figure 3a, we focus on the current versus potential curves (i.e., the CVs) for each cycle as illustrated in Scheme 3. A CV from an individual cycle serves as the “signal” that is divided into four quadrants based on whether the currents are oxidizing or reducing and how the currents are assigned, to the oxidative redox cycler (e.g., Fc) or reductive redox cycler (e.g., Ru3+). (We should note that the assignment of each quadrant to activities of a single chemical entity is useful for processing the information although such an assignment is a simplification of the underlying chemistries.) As indicated by the upper equation in Scheme 3b, the charge transfer for each quadrant is calculated by integration of the current for that quadrant (Q = ∫ i dt). If the redox reactions being detected by this method are fully reversible, then one expectation is that the charge-transfer values should remain constant over time. Figure 3b shows the calculated Q for the two largest quadrants of Scheme 3a were nearly constant for the eight cycles of the experiment.

Figure 3. Repeatability of the redox cycling of clove with the Fc−Ru3+ system (scan rate = 50 mV/s; 60 mg/mL clove). (a) The cyclic potential input allows the Fc and Ru3+ mediators (50 μM each) to sequentially oxidize and reduce the clove (see Scheme 3 for signal analysis). (b) Charge transfer (Q) for Fc oxidation and Ru3+ reduction with clove−chitosan remains relatively constant over the eight cycles tested. (c) Amplification ratios for Fc oxidation and Ru3+ reduction with clove−chitosan exceed 2 and are nearly constant. (d) Rectification ratios for Fc oxidation and Ru3+ reduction with clove− chitosan are large (>10) and nearly constant.

Previous studies have shown characteristic signatures of redox cycling emerge by considering the two sets of ratios indicated in Scheme 3b.37 The amplification ratios (ARs) compare the charge transfer (Q) in a given quadrant for the clove−chitosan film to an equivalent Q for the unmodified chitosan film (control). The AR contains information on redox cycling, since a film that can redox cycle (e.g., clove−chitosan) is expected to have an amplified output current compared to a 9764

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In summary, the results in Figure 3 demonstrate that the electrochemical mediators Fc and Ru3+ can repeatedly oxidize and reduce components in insoluble clove; that is, insoluble clove residue is redox-active and can be repeatedly and reversibly cycled between oxidized and reduced states. Antioxidant Behavior. A method commonly used to examine antioxidant radical scavenging activities is to incubate a sample with the radical generator AAPH (E° = +0.8 V vs Ag/ AgCl) and fluorescein. Fluorescein is readily damaged by the resulting free radical while a sample with free radical scavenging activities protects fluorescein. In the next study, we used this method to test whether the radical scavenging antioxidant properties of clove depended on whether it was poised in a reduced or oxidized state. As illustrated in Scheme 2, it is possible to use an electrochemical method to poise the film to either an oxidized or reduced state to investigate this question. In these experiments, we used a gold-coated silicon wafer as our electrode to allow a larger sample area to be probed (0.9 vs 0.03 cm2 for the standard gold electrode of Scheme 1a) and to facilitate comparison with previous studies. To poise the sample in an oxidized or reduced state, the clove-containing film is incubated in the presence of both the Fc and Ru3+ mediators. To reduce the sample (i.e., to charge it with electrons), a reducing potential is imposed to the underlying electrode (−0.4 V; 30 min) to allow Ru3+ reductive redox cycling to transfer electrons from the electrode to the insoluble clove. To oxidize the sample (i.e., to discharge its electrons), an oxidizing potential was imposed to the underlying electrode (+0.5 V; 30 min) to allow Fc oxidative redox cycling to transfer electrons from the insoluble clove. (Note: we used longer charging and discharging times to facilitate comparison with previous studies36 and repeated chronocoulometric determinations of Nfilm under these conditions.) These films were then incubated with fluorescein and AAPH for 30 min after which the fluorescence of fluorescein was measured. The first two entries in Figure 4a are for the negative and positive controls that show no or complete loss of fluorescence of fluorescein. The third entry in Figure 4a shows that chitosan (without insoluble clove) offers little protection from free radical damage. The final two entries in Figure 4a show that insoluble clove offers substantial antioxidant protection and the oxidized and reduced states of insoluble clove offer comparable protection. This result demonstrates the well-known antioxidant properties of clove but further demonstrates that this antioxidant property is not sensitive to whether clove is in an oxidized or reduced state. As mentioned above, the redox window used to poise clove in its oxidized or reduced state is likely too mild to alter the redox state of eugenol that is believed to be a major component of the antioxidant activity of clove.39 This result suggests that at least some of the moieties that confer radical scavenging antioxidant activities (e.g., eugenol) are not responsible for the reversible redox activities being observed by our electrochemical method. Figure 4b shows results for samples prepared with differing levels of clove and further indicates that the radical scavenging antioxidant properties are insensitive to redox state. Previous studies with other insoluble phenolic matrices (i.e., melanin) show comparable results: the radical scavenging antioxidant properties are insensitive to the redox state at least for the moieties changed by our electrochemical method.36 For comparison with results from studies with other phenolic materials,36,40 we replot our antioxidant activities in terms of redox capacity as shown in Figure 4c. This normalization

Scheme 3. Signal Analysis of Results from the Individual CV Cyclesa

Scan rate = 50 mV/s; 60 mg/mL clove. (a) The “signal” for each cycle is analyzed by dividing the CVs into quadrants, calculating the charge transfer (Q) for each quadrant, and assigning these values to activities of a single mediator. (b) Calculation procedure for determining charge transfer (Q), amplification ratios (ARs), and rectification ratios (RRs). a

relevant control film that cannot redox cycle (e.g., unmodified chitosan film). Figure 3c shows a 2−3-fold amplification of both Fc oxidation and Ru3+ reduction currents for the clove− chitosan, and these ARs are nearly constant over the course of the eight cycles. The second ratios in Scheme 3b are the rectification ratios (RRs) that are more subtle and consider how (a) electrode currents in the Fc region are primarily oxidative (Fc+ reduction by redox cycling in the film decreases the amount of Fc+ reduction occurring at the electrode) and (b) electrode currents in the Ru3+ region are primarily reductive (Ru2+ oxidation by redox cycling in the film decreases the amount of Ru2+ oxidation occurring at the electrode). In essence, the RR considers the linkage between Fc-mediated oxidative redox cycling and Ru3+-mediated reductive redox cycling; reductive redox cycling charges the sample with electrons that are subsequently removed during the oxidative redox-cycling portion of the cycle. Thus, samples that can sequentially engage these oxidative and reductive redox-cycling reactions would be expected to have large and constant RRs.37 Figure 3d shows that large RRs are observed for the clove−chitosan film and again these ratios remain nearly constant over the course of the eight cycles. 9765

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Pro-oxidant Activity: H2O2 Generation. In a final experiment, we prepared films on gold-coated silicon wafers (as described in Figure 4), poised the films in either an oxidized or a reduced state, exposed them to air, and examined their abilities to spontaneously generate H2O2. For these studies, the poised films were exposed to air-saturated water for 15 min, after which the solution was assayed for H2O2. Figure 5a shows

Figure 5. Pro-oxidant activities (as measured by H2O2 generation) for insoluble clove are low and do not depend on the redox state. (a) Clove−chitosan films poised in either an oxidized or a reduced state showed limited ability to generate H2O2 upon a 15 min exposure to air. (b) Comparison of H2O2 generation and redox capacities for chitosan films with either entrapped melanin or clove or chemically modified with catechol (data for melanin- and catechol-modified chitosan reproduced from prior work).36

Figure 4. Radical scavenging antioxidant activities do not depend on the redox state of insoluble clove. (a) Films (60 mg/mL insoluble clove) poised in either oxidized or reduced states were observed to have similar abilities to protect fluorescein from free radical oxidative damage by APPH. (b) Films prepared with differing contents of insoluble clove have greater free radical scavenging antioxidant activities, and these activities are observed irrelevant to whether the films are poised in an oxidized or reduced state. (c) Comparison of antioxidant activities and redox capacities for chitosan films with either entrapped melanin or clove or chemically modified with catechol (data for melanin- and catechol-modified chitosan are reproduced from prior work).36

relatively low levels of H2O2 were generated in these experiments and H2O2 generation was independent of whether samples were in an oxidized or reduced state. This result differs from previous observations with catechol-modified films or melanin-containing films that showed considerable H2O2generating abilities but only when the films had been poised in a reduced state.36,40 Finally, we compare the H2O2 generation of clove−chitosan against results previously observed with other phenolic materials.36,40 In comparison to melanin−chitosan and catechol−chitosan, Figure 5b shows that clove is unusual in that its reversible redox activity is not accompanied by an equivalent ability to generate H2O2. Presumably, this difference reflects differences in the chemical components in the insoluble clove sample. Possibly the high antioxidant and low pro-oxidant activities of clove may be important in putative health beneficial effects. In conclusion, electrochemical methods were used to probe the redox activities of a model insoluble dietary antioxidant (i.e., clove), and we report two immediately interesting observations. First, we observed that insoluble clove possesses redox activities and can be rapidly and repeatedly switched between oxidized and reduced states. Recent literature indicates that considerable amounts of dietary antioxidant phenolics pass through the small intestine in an insoluble form and suggest that standard analytical methods may not fully account for their contribution

assesses how the antioxidant activities correlate to redox activities among various materials (note: the comparatively large error bars for the insoluble clove presumably reflects the particulate nature of these samples). While the incorporation of greater amounts of materials (e.g., phenolics) in the film increases both redox and antioxidant activities, the slopes of the curves vary among the different materials. The large slope for the clove−chitosan sample reflects its high antioxidant activities while the low slope for the catechol−chitosan sample indicates that its high redox activity is not accompanied by high radical scavenging antioxidant activities. Presumably, differences in the behavior between clove−chitosan and catechol−chitosan reflect differences in chemical componentsthat clove has different chemical moieties that confer radical scavenging antioxidant activity. The important observation is that antioxidant and redox activities are not exclusively linked to each other. 9766

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to health benefits.45−47 The observation that insoluble clove can undergo redox reactions with soluble mediators (electron shuttles) suggests that dietary antioxidants may participate in redox interactions in the intestine and thus affect redox homeostasis and health even in the absence of solubilization or absorption.48 Second, we observed that insoluble clove (even when poised in a reduced state) has considerably less ability to generate H2O2 compared to other catechol-based matrices (e.g., melanin). This observation suggests that insoluble clove may possess little pro-oxidant activities although we should note that (i) we only measured the most stable ROS (H2O2) while other pro-oxidant species may have been generated; and (ii) we used relatively mild oxidation conditions to focus on reversible redox activities while the literature indicates that, under more aggressive oxidation conditions, eugenol can behave as a prooxidant.49,50 Overall, we suggest that electrochemical methods may provide new and important opportunities to study redox homeostasis and oxidative stress.

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ASSOCIATED CONTENT

S Supporting Information *

Additional description of the insoluble clove fraction including the UV−visible spectra of the filtrates. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (301) 405-8389; fax: (301) 314-9075; e-mail: [email protected]. Funding

The authors gratefully acknowledge financial support from the Robert W. Deutsch Foundation and the Defense Threat Reduction Agency (HDTRA1-13-0037). Notes

The authors declare no competing financial interest.

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