Ellagic Acid: An Unusually Versatile Protector against Oxidative Stress

Article pubs.acs.org/crt

Ellagic Acid: An Unusually Versatile Protector against Oxidative Stress Annia Galano,*,† Misaela Francisco Marquez,‡ and Adriana Pérez-González† †

Departamento de Química, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco 186, Col. Vicentina, Iztapalapa, C.P. 09340 México D. F., México ‡ Instituto Politécnico Nacional-UPIICSA, Té 950, Col. Granjas México, C.P. 08400 México D. F., México S Supporting Information *

ABSTRACT: Several aspects related to the antioxidant activity of ellagic acid were investigated using the density functional theory. It was found that this compound is unusually versatile for protecting against the toxic effects caused by oxidative stress. Ellagic acid, in aqueous solution at physiological pH, is able of deactivating a wide variety of free radicals, which is a desirable capability since in biological systems, these species are diverse. Under such conditions, the ellagic acid anion is proposed as the key species for its protective effects. It is predicted to be efficiently and continuously regenerated after scavenging two free radicals per cycle. This is an advantageous and unusual behavior that contributes to increase its antioxidant activity at low concentrations. In addition, the ellagic acid metabolites are also capable of efficiently scavenging a wide variety of free radicals. Accordingly, it is proposed that the ellagic acid efficiency for that purpose is not reduced after being metabolized. On the contrary, it provides continuous protection against oxidative stress through a free radical scavenging cascade. This is an uncommon and beneficial behavior, which makes ellagic acid particularly valuable to that purpose. After deprotonation, ellagic acid is also capable of chelating copper, in a concentration dependent way, decreasing the free radical production. In summary, ellagic acid is proposed to be an efficient multiple-function protector against oxidative stress.

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INTRODUCTION

aspect of the antioxidant activity of ellagic acid that deserves further investigation. There is rather scarce information concerning the ellagic acid way of action as an antioxidant. Most of the information gathered so far concerns its primary antioxidant activity, i.e., its role as free radical scavenger, and even for this, there are numerous aspects that have not been elucidated yet. Cozzi et al.39 proposed that the observed cytogenetic protection against hydrogen peroxide by ellagic acid can take place by scavenging reactive free radicals. Zafrilla et al.3 proposed that the antioxidant activity of ellagic acid is similar to that of kaempferol, epicatechin, gallic acid, caffeic acid, and protocatechuic acid, higher than that of ferulic acid, and lower than that of quercetin. They also proposed that the antioxidant activity of ellagic acid is mainly due to the presence of two pairs of neighbor hydroxyl groups in its structure. Barch et al.51 suggested that both hydroxyl groups are required for ellagic acid to directly detoxify the diolepoxide of benzo[a]pyrene, while only the 2a-hydroxyl groups are necessary for ellagic acid to inhibit CYPlAl-dependent benzo[a]pyrene hydroxylase activity. Accordingly, they proposed that different portions of the ellagic acid molecule (Scheme 1) are responsible for its diverse presumed anticarcinogenic activities. Moreover, they

Ellagic acid (2,3,7,8-tetrahydroxy-chromeno[5,4,3-cde]chromene-5,10-dione, H4EA) is a polyphenol present in numerous fruits and vegetables, including nuts, grapes, pomegranate, and a wide variety of berries.1−5 It is also the major phenolic constituent in distilled beverages.6 Ellagic acid has been reported to have a wide spectrum of benefits for human heath such as antiviral,7 antibacterial,8 anti-inflammatory,9,10 gastro-protective,11 and cardio-protective12 properties, cancer preventive and suppressive effects,5,13−34 inhibition of UV-induced oxidative stress,35 and protection against lipid peroxidation.36−38 These beneficial effects can be attributed, at least partially, to the antioxidant activity of ellagic acid, which has been extensively demonstrated.39−47 Moreover, it has been proposed that ellagic acid provides better protection against oxidative stress (OS) and lipid peroxidation than vitamin E succinate.37 In addition, it has been reported that ellagic acid metabolites, urolithins, also present antioxidant activity.47 This is a very appealing feature that distinguishes ellagic acid from most antioxidants. This continuous protection has been referred to as the free radical scavenging cascade for melatonin,48−50 which also presents this desirable characteristic. This behavior usually implies high efficiency in protecting against OS, and its toxic effects, even at low concentrations.50 Therefore, this is an © 2014 American Chemical Society

Received: February 25, 2014 Published: April 3, 2014 904

dx.doi.org/10.1021/tx500065y | Chem. Res. Toxicol. 2014, 27, 904−918

Chemical Research in Toxicology

Article

bond dissociation enthalpy (BDE), adiabatic ionization potential (IP), O-H proton dissociation enthalpy (PDE), proton affinity (PA), and electron transfer enthalpy (ETE). On the basis of these analyses, they were able to envisage that some of the studied compounds, not experimentally characterized yet, are promising antioxidants. They also proposed HT as the main reaction mechanism in the three investigated environments. No kinetic data were reported in this case. Markovic et al.57 also calculated the BDE, IP, PDE, PA, and ETE values of ellagic acid and its phenoxide anions. They proposed that the thermochemical feasibility of different mechanisms is influenced by the polarity of the environment and also by the deprotonation degree of ellagic acid, being the anionic species more active than the neutral one. They also proposed the SPLET as the most important mechanism in aqueous solution and HT in gas phase and benzene solution. Site 1a (Scheme 1) was suggested as the most active site for deactivating free radicals. Considering the data gathered so far on the antioxidant activity of ellagic acid, it becomes evident that further studies are still needed to fully understand the protective effects of ellagic acid against oxidative stress. For example, there is no kinetic data reported so far for its reactions with peroxyl radicals, other than CCl3OO•. There is no quantitative data on the relative importance of the different mechanisms and sites of reaction. No kinetic data have been reported for the free radical scavenging activity of ellagic acid metabolites. The possible ability for metal chelation (secondary antioxidant activity) of this compound has not been explored in detail yet. In addition, from a theoretical point of view, there is no calculated rate constant in close agreement with the experimental values. Accordingly, it is the main goal of the present work to provide information on these aspects.

Scheme 1. Ellagic Acid (H4EA), Structure, and Site Numbering

suggested that even minor modifications in the ellagic acid structure may alter its anticarcinogenic actions. In a very thorough and detailed study, Priyadarsini et al.52 also investigated the primary antioxidant activity of ellagic acid using the DPPH assay. Their results supported the proposal by Zafrilla et al.3 that the antioxidant activity of ellagic acid is mainly due to its free radical scavenging activity. Priyadarsini et al.52 also studied the •OH, •N3, •NO2, CCl3OO•, and ABTS•− scavenging activity of ellagic acid and estimated the associated bimolecular rate constants, as well as the influence of pH on their values (Table S1, Supporting Information). These authors showed that this compound is very effective, even at micromolar concentrations, in inhibiting lipid peroxidation. In addition, they proposed that the scavenging activity of ellagic acid is similar to those of other antioxidants such as vitamin E and vitamin C. Because of its low solubility in water, ellagic acid was proposed as a lipophilic antioxidant and as a peroxyl radical scavenger. In the same work, Priyadarsini et al.52 pointed out the necessity of further studies on the direct reactions between ellagic acid and free radicals, in particular on kinetics in terms of rate constants. Regarding the metal chelating activity of ellagic acid, it was confirmed by in vitro studies and proposed to be responsible for the protective action against mitochondrial damage in myocardial infarction.53 This activity was reported to be concentration dependent.54 To our best knowledge, this is the only information gathered so far on this important aspect of the antioxidant protection exerted by ellagic acid. From a theoretical point of view, there are three previous studies on the free radical scavenging activity of ellagic acid. Tiwari et al.55 studied the reactions of •OH, •OCH3, and •NO2 radicals with ellagic acid and its methyl and dimethyl derivatives in gas phase and in aqueous solution. They found that the efficiency of ellagic acid and its derivative to scavenge the studied free radicals follows the trend •OH ≫ •OCH3 > •NO2. The reported rate constants in aqueous solution were in the ranges 1013−1016 M−1 s−1, 108−1012 M−1 s−1, and 104−108 M−1 s−1, for the reactions involving •OH, •OCH3, and •NO2, respectively. Taking into account the values experimentally obtained by Priyadarsini et al.52 (Table S1, Supporting Information), together with the diffusion rate constants in aqueous solution (usually around 109 M−1 s−1), it seems that, at least for the reactions with •OH and •OCH3, the rate constants reported in ref 55 are overestimated by 3 to 6, and up to 2 orders of magnitude. Mazzone et al.56 performed a systematic investigation on the free radical scavenging activity of ellagic acid and some of its derivatives in gas phase and also in methanol and aqueous solution. On the basis of the knowledge that hydrogen transfer (HT) and sequential proton loss electron transfer (SPLET) mechanisms usually play important roles on the free radical scavenging activity of chemical compounds, they analyzed the

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METHODS

Geometry optimizations and frequency calculations have been carried out using the M05-2X functional58 and the 6-311+G(d,p) basis set, in conjunction with the SMD continuum model59 using pentyl ethanoate and water as solvents to mimic lipid and aqueous environments, respectively. The M05-2X functional has been recommended for kinetic calculations by their developers,58 and it has been also successfully used by independent authors to that purpose.60−63 It is also among the best performing functionals for calculating reaction energies involving free radicals.64 SMD is considered a universal solvation model, due to its applicability to any charged or uncharged solute in any solvent or liquid medium for which a few key descriptors are known.59 For the species including Cu and for all the related energies of reaction, the M05 functional58 has been used also with the 6-311+G(d,p) basis set and the SMD model. This functional has been chosen for this part of the study because it was parametrized including both metals and nonmetals, whereas M05-2X is a highly nonlocal functional with double the amount of nonlocal exchange (2X) that was parametrized for nonmetals. M05 has been recommended for studies involving both metallic and nonmetallic elements and has been reported to perform well not only for main-group thermochemistry and radical reaction barrier heights but also for interactions with transition metals.58 Unrestricted calculations were used for open shell systems. Local minima and transition states were identified by the number of imaginary frequencies (0 or 1, respectively). In the case of the transition states, it was verified that the imaginary frequency corresponds to the expected motion along the reaction coordinate, by intrinsic coordinate calculations. All the electronic calculations were performed with the Gaussian 09 package of programs.65 Thermodynamic corrections at 298.15 K were included in the calculation of relative energies. In addition, the solvent cage effects have been 905



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included according to the corrections proposed by Okuno,66 taking into account the free volume theory.67 The rate constants (k) were calculated using the conventional transition state theory (TST)68−70 and 1 M standard state, following the quantum mechanics-based test for the overall free radical scavenging activity (QM-ORSA) protocol.71 This computational protocol has been validated by comparison with experimental results, and its uncertainties have been proven to be no larger than those arising from experiments.71

reported by Markovic et al.57 Therefore, this is the species chosen for modeling the monoanion of ellagic acid (H3EA−). In nonpolar (lipid) media, however, only the neutral form is used since such media do not promote the necessary solvation for ionic species to be formed to a significant extent. Primary Antioxidant Activity of Ellagic Acid. Peroxyl Radical Scavenging Activity. To investigate the free radical scavenging activity of ellagic acid, we have first chosen its reactions with peroxyl radicals because they are among those of biological relevance that can be effectively scavenged to retard OS.75 This is because their half-lives are not too short, which is a requisite for efficient interception by phenolic compounds.76 That is why ROO• have been proposed as major reaction partners for these compounds.76 Moreover, it has been proposed that the main antioxidant function of phenolic compounds is to trap ROO•.77,78 In addition, radicals of intermediate to low reactivity have been recommended for studying the relative scavenging activity of different compounds79,80 since using highly reactive radicals that usually react at diffusion-limited rates might lead to incorrectly conclude that all of the tested compounds are equally efficient as antioxidants. Among ROO• radicals, we have selected the hydroperoxyl radical (•OOH), which is the simplest member of the ROO• family and has been suggested to be central to the toxic side effects of aerobic respiration.81 It has also been pointed out that more information on the reactivity of this species is needed.81 Different reaction mechanisms have been considered since the free radical scavenging activity of ellagic acid can involve a variety of them, as is the case for many other scavengers.82−87 Those investigated in this work are

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RESULTS AND DISCUSSION Acid/Base Equilibriums. The ellagic acid conformer used in this work is the same as that reported as the lowest in energy in two previous conformational searches.55,57 According to the ellagic acid structure (Scheme 1), up to four pKa values can be expected for this compound. However, only two of them have been reported so far (Table 1). Using the average values for the Table 1. pKa Values of Ellagic Acid and Molar Fractions (mf) at pH 7.4 ref pKa1

average pKa2 average mf (H4EA) mf (H3EA−) mf (H2EA2−)

6.59 6.3 6.54 6.48 11.0 11.2 11.1 0.107 0.893 R1 > R20 > R13 > R18 > R12 > R19 > R3 > R5 > R2 > R11 > R8 > R4 > R15 > R21 > R7 > R6 > R16 > R14 > R10 > R9. Most of the reactions are fast, with rate constants larger than 103 M−1 s−1. According to the gathered kinetic results, under such conditions, ellagic acid is very efficient for deactivating R17, R1, R20, and R13, with rate constants that are within, or close to, the diffusion limit regime (kET ≥ 108 M−1 s−1). It is also efficient for scavenging R18, R12, R19, R3, R5, R2, R11, and R8, with overall ET rate constants ranging from 103 M−1 s−1 to 106 M−1 s−1. However, it is not expected to scavenge radicals R4, R15, R21, R7, R6, R16, R14, R10, and R9 fast enough, via ET. The differences in reactivity are attributed to the chemical nature of the radicals, in particular to their electron withdrawing capacities. This becomes particularly evident for the series of halogenated peroxyl radicals, i.e., the electron transfer process becomes faster as the halogenation degree of the radicals increases. In summary, it can be stated that ellagic acid, in aqueous solution, at physiological pH, is capable of deactivating a wide

(11) 910



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Table 8. Rate Constants for the Electronic Transfer Reactions from Ellagic Acid to Free Radicals, in Aqueous Solution, at Different pH Values R13(•OOCCl3) calc. exp.52 R19(•NO2) calc. exp.52 R20(•N3) calc. exp.52

pH 4.5

pH 7

pH 8.5

pH 10.7

1.57 × 1006 4.70 × 1007

1.15 × 1008 1.40 × 1008

1.48 × 1008 3.20 × 1008

1.07 × 1008 4.20 × 1008

1.03 × 1006 2.00 × 1007

1.33 × 1006 8.00 × 1007

4.95 × 1009 3.70 × 1009

6.36 × 1009 4.60 × 1009

variety of ROS and RNS, which is also a desirable behavior since several of them are expected to be present in biological systems. Influence of pH. Electron transfer processes are very sensitive to the pH of the environment because it rules the fraction of neutral and anionic species, and the latter are the most efficient for this particular kind of reaction. However, including pH in theoretical calculations, via molar fractions, is a not so common a strategy. Therefore, we have tested the reliability of the data reported in this work for ET reactions, at specific pH values (Table 8). To that purpose, we have used, as reference, the experimental data reported by Priyadarsini et al.52 The mean unsigned error (MUE), expressed as log(k) is 0.67. The larger absolute errors were found for the reactions of NO2 and for that of •OOCCl3 at the most acidic tested pH (4.5). This is a logical finding since only electron transfer reactions were included in these calculations. Thus, the differences are attributed to the role of other reaction mechanisms (mainly HT). NO2 is not expected to exclusively react via ET. However, while •OOCCl3 is expected to mainly react via ET, at low pHs the deprotonated fraction of ellagic acid is too small, i.e., its neutral form is by far the most abundant, and the relative importance of HT is expected to increase. If these three reactions are excluded from the analysis, the MUE is reduced to 0.25. In any case, the agreement with the experimental data is excellent and validates the methodology used in this work. Primary Antioxidant Activity of Ellagic Acid Metabolites via Electron Transfer. Most of the antioxidants consumed in the human diet lose their protective effects after being metabolized. This implies that their efficiency as protectors last very short periods of time and are limited to only some regions within the biological systems. Therefore, chemical compounds that retain their antioxidant activity after being metabolized are particularly important to fight oxidative stress. They can exert such an action for a longer period of time and in a wide range of regions, which means that their protective effects can be appreciated even when their initial concentrations are relatively low. There are very few studies dealing with the activity of the antioxidants’ metabolites. In the particular case of ellagic acid, there is previous evidence suggesting that its metabolites, urolithins, also exhibit antioxidant activity.47 Therefore, in an attempt to quantify such an activity we have also investigated the reactions of several ellagic acid metabolites (Scheme 3) with the set of free radicals shown in Table 6 via electron transfer. Because of the complexity of their structure, conformational studies were performed to identify the conformer with the lowest energy for each metabolite. Since orienting phenolic H

4.60 × 1009 8.10 × 1009

Scheme 3. Structures, Names, and Acronyms of the Studied Metabolites of Ellagic Acid

out of plane or pointing it toward neighbor H atoms increases the energy of the systems, conformations with such structural characteristics were not included in this analysis. The studied conformations, together with their relative energies, are provided in Figures S2 to S10 (Supporting Information). The general behavior, as expected, is that the higher the number of intramolecular H bonds, the lower the energy of the conformer. For the metabolites where such interactions are not possible (UA, UB, UM7, and IUA), the relative energies of all the conformers are very similar with variations no higher than 0.3 kcal/mol. In general, the studied conformers differ by ≤1 kcal/ mol for all of the studied metabolites. Thus, they can be considered as almost isoenergetic. However, we have selected the lowest in energy for the rest of the study. In addition, all of these metabolites, except UB, have more than one phenolic OH. Consequently, it is also necessary to identify the most likely deprotonation site for each metabolite. The different anions and their relative energies are reported in Figures S11 to S18 (Supporting Information). Those with the lowest energy are the ones used for the free radical scavenging activity toward free radicals. We have used approximated eq 12 since it was demonstrated to be valid for ellagic acid. To do so, it is crucial to know the first pKa for each metabolite (Table 9). For UA, UB, UC, and UD, they were already reported. However, for UE, UM5, UM6, UM7, and IUA there is no previous information on their pKa values. Therefore, we have calculated them using the proton exchange method, also known 911



Article

within, or close to, the diffusion limit regime (kSPLET > 108 M−1 s−1). In general, the slowest reactions are those involving IUA. This suggests that among the studied metabolites, it is the least efficient for scavenging diverse free radicals. However, UA, UB, UE, and UM7 react very fast with most of the studied radicals. In addition, all of the reactions involving R1, R12, R13, R18, and R19 are diffusion limited, indicating that all of the studied metabolites are very efficient for scavenging these radicals. According to the values in Table 10, it can be stated that the ellagic acid metabolites are capable of efficiently scavenging a wide variety of free radicals via SPLET. Moreover, most of the studied reactions are faster for the metabolites than for the ellagic acid itself (Table 7). It seems worthwhile to emphasize the fact that SPLET is only one of the possible reaction mechanisms for these compounds and that it has been investigated only for the monoanions. Therefore, the reported rate constants can be considered as lower limits for the overall rate coefficients of the studied metabolite + free radical reactions. In summary and based on these results, it is proposed that the free radical scavenging activity of ellagic acid is not reduce after metabolization. On the contrary, it provides continuous protection against oxidative stress through a free radical scavenging cascade. This is a rare and very desirable behavior, which makes ellagic acid particularly valuable to that purpose. Secondary Antioxidant Activity via Metal Chelation. There is a wide diversity of trace metals within living organisms. We have chosen copper for the present study because it is ubiquitous in the human body, and even though it is essential for the proper function of most living organisms,122 it also induces cellular toxicity. There is evidence that copper may be involved in the pathogenesis of atherosclerosis, Alzheimer’s disease, and other neurodegenerative disorders.123 One of the most accepted explanations to copper’s toxicity is its involvement in the formation of ROS,124 particularly the very reactive and very damaging OH radical, through reduction and

Table 9. First pKa Values of Ellagic Acid Metabolites and the Molar Fractions of the Monoanions (mfA−) at pH 7.4 UA UB UC UD UE UM5 UM6 UM7 IUA

ref

pKa1

mfA−

118 119 120 121 this work this work this work this work this work

7.21 7.67 7.27 7.32 5.66 4.52 4.86 6.29 5.62

0.61 0.35 0.57 0.55 0.98 ∼1.00 ∼1.00 0.93 0.98

as the isodesmic method or the relative method,117 which involves the reaction scheme: HA + Ref − ⇌ A− + HRef

where HRef/ref‑ is the acid/base pair of a reference compound that should be as structurally similar to the system of interest as possible. Within this approach, the pKa is calculated as pK a(HA) =

ΔGs + pK a(HRef) RT ln(10)

(13)

In our case, we have used two different HRef’s, namely UB and UC, for the compounds with higher and lower numbers of OH groups, respectively, in order to use the closest structural reference acid in each case. The values of the rate constants for the electron transfer reactions from the monoanions to the free radicals (SPLET), at physiological pH, are reported in Table 10. The values of the Gibbs free energies of reaction, reorganization energies, Gibbs free energies of activation, and the rate constant without including molar fractions, are provided in Tables S4 to S7 (Supporting Information). It was found that most of the SPLET reactions are fast, with kSPLET ≥ 103 M−1 s−1. Moreover, a large subset of them are

Table 10. Rate Coefficients for the Sequential Proton Loss Electron Transfer Reactions (kSPLET, M−1 s−1, Calculated Using Eq 12) from Ellagic Acid Metabolites, in Aqueous Solution, at 298.15 K and pH 7.4 UA R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21

4.67 4.45 4.53 3.39 4.56 4.68 2.03 3.87 1.36 2.20 3.69 4.90 4.70 5.28 1.62 4.83 3.84 4.53 4.39 4.14 2.88

× × × × × × × × × × × × × × × × × × × × ×

1009 1009 1009 1009 1009 1007 1008 1009 1005 1005 1009 1009 1009 1005 1009 1005 10−02 1009 1009 1004 1008

UB 2.80 2.02 2.52 4.90 2.52 1.73 8.54 1.01 2.66 9.24 1.35 2.75 2.68 2.54 2.23 2.19 8.88 2.59 2.43 2.98 3.19

× × × × × × × × × × × × × × × × × × × × ×

1009 1009 1009 1008 1009 1006 1006 1009 1003 1003 1009 1009 1009 1004 1008 1004 10−01 1009 1009 1006 1007

UC 4.73 1.02 2.50 6.06 1.18 1.50 8.42 3.10 6.47 1.68 1.21 1.78 4.28 6.54 3.41 4.74 6.43 3.17 1.18 4.57 7.28

× × × × × × × × × × × × × × × × × × × × ×

1009 1006 1007 1004 1007 1002 1002 1005 10−02 1000 1007 1009 1009 1000 1005 1000 1006 1009 1009 1009 1004

UD 4.53 4.10 1.12 2.48 4.84 6.24 3.54 1.35 2.38 7.74 7.27 1.35 4.06 3.12 1.86 2.22 1.08 2.77 9.16 4.37 4.20

× × × × × × × × × × × × × × × × × × × × ×

1009 1005 1007 1004 1006 1001 1002 1005 10−02 10−01 1006 1009 1009 1000 1005 1000 1007 1009 1008 1009 1004

UE 7.69 7.42 7.39 7.41 7.43 1.55 3.52 7.19 2.25 1.01 6.88 8.02 7.65 1.89 5.60 1.91 1.98 7.36 7.20 1.85 2.05 912

× × × × × × × × × × × × × × × × × × × × ×

UM5 1009 1009 1009 1009 1009 1009 1009 1009 1007 1007 1009 1009 1009 1007 1009 1007 1001 1009 1009 1008 1009

8.34 1.50 2.66 8.83 1.57 2.12 1.16 3.77 1.35 1.82 6.20 4.66 7.60 6.45 2.27 4.91 4.02 6.35 3.05 8.03 4.22

× × × × × × × × × × × × × × × × × × × × ×

1009 1007 1008 1005 1008 1003 1004 1006 1000 1001 1007 1009 1009 1001 1006 1001 1006 1009 1009 1009 1005

UM6 8.23 3.74 5.53 2.22 3.61 5.37 2.90 8.73 4.13 4.12 9.77 5.23 7.56 1.39 4.03 1.08 2.92 6.56 3.50 7.93 7.07

× × × × × × × × × × × × × × × × × × × × ×

1009 1007 1008 1006 1008 1003 1004 1006 1000 1001 1007 1009 1009 1002 1006 1002 1006 1009 1009 1009 1005

UM7 7.44 6.64 6.86 4.73 6.89 6.63 2.72 5.55 2.91 3.04 5.08 7.36 7.11 6.84 1.85 6.43 9.40 6.88 6.59 8.30 2.96

× × × × × × × × × × × × × × × × × × × × ×

1009 1009 1009 1009 1009 1007 1008 1009 1005 1005 1009 1009 1009 1005 1009 1005 1001 1009 1009 1008 1008

IUA 8.16 1.52 1.69 1.85 1.65 1.05 6.01 2.46 8.30 6.03 7.53 4.81 6.76 3.57 7.90 2.06 1.87 2.08 5.20 7.88 3.27

× × × × × × × × × × × × × × × × × × × × ×

1009 1002 1004 1001 1003 10−01 10−01 1002 10−06 10−03 1005 1008 1009 10−02 1003 10−02 1007 1009 1008 1009 1003



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oxidation processes.123 In addition, it has been proposed that metal chelation can reduce the feasibility of such processes, decreasing the ROS formation, compared to the same reactions when involving free copper ions.125,126 Logically, for this kind of protection against OS to take place it is crucial that the chelation yield stable complexes, i.e., the chelation reaction must be thermochemicaly feasible. Accordingly, the first step in the study of the secondary antioxidant activity of chemical compounds, via metal chelation, is to explore the thermochemistry of these reactions. In the present study, we have performed such an investigation for the neutral and anionic forms of ellagic acid and their chelation ability toward Cu(II) and Cu(I). We have used as reactants both free and hydrated copper ions (Figure S19, Supporting Information). Four water molecules have been used for the latter since it was previously reported that Cu(II) water complexes, in the aqueous phase, are more stable in nearly square-planar four-coordinate geometry.127 For the sake of consistency, we have modeled the hydrated Cu(I) with the same number of water molecules. However, it is interesting to note that this ion favors the linear two-coordinate configuration. The optimized geometries of the complexes involving free and hydrated Cu are reported in Figures S10 (Supporting Information) and 4, respectively. It was found, as expected, that

Table 11. Enthalpies (ΔH, kcal/mol) and Gibbs Free Energies (ΔG, kcal/mol) for the Chelation Reaction, in Aqueous Solution, at 298.15 K free Cu H4EA-Cu(II) H4EA-Cu(I) H3EA−-Cu(II) H3EA−-Cu(I) 2H3EA−-Cu(II) 2H3EA−-Cu(I)

hydrated Cu

ΔH

ΔG

ΔH

ΔG

−7.31 −3.64 −31.03 −5.48 −50.85 −17.16

−2.89 −0.71 −27.32 −2.14 −38.72 −6.50

10.38 2.11 −5.26 −3.32 −8.56 −0.24

7.80 1.27 −8.09 −4.71 −15.03 −3.44

activity of ellagic acid but also its secondary antioxidant activity via metal chelation. In addition, the chelation reactions are more exergonic for Cu(II) than for Cu(I), but they are viable in both cases. At this point, it seems worthwhile to note that Cu ions, when present in the aqueous phase of biological systems, are expected to be hydrated. Therefore, the data corresponding to such a case is the most relevant regarding the secondary antioxidant activity of ellagic acid by chelating these ions. In this particular case, only the reactions involving the anions are exergonic. As commented at the beginning of this work, at physiological pH, most of the ellagic acid (89%) is expected to be in its deprotonated form. Therefore, under such conditions, it is expected to successfully chelate Cu ions. The previous discussion is related to complexes with 1:1 (ellagic acid/Cu) stoichiometry. However, to investigate the role of the ellagic acid concentration on its chelating ability, we have also modeled the 2:1 complexes. To that purpose, we have modeled only those involving the ellagic acid anion since it was previously demonstrated that they are the relevant ones (Figure 5).

Figure 4. Optimized geometries of the 1:1 complexes of hydrated copper with ellagic acid (H4EA) and its anion (H3EA−). Distances are reported in Å.

the shortest Cu−O distance is reduced when the chelation involves the anion of ellagic acid instead of its neutral form. This shows that H3EA− yields tighter Cu complexes than H4EA. In addition, the Cu(II)-O distances were found to be systematically shorter than the Cu(I)-O distances, suggesting that the binding in Cu(II) complexes is stronger than that in Cu(I) complexes. Regarding the hydrated models, the shortest Cu−O distance corresponds to a Cu−water interaction for the complexes involving H4EA, while it corresponds to a Cu− ellagic acid interaction when the ligand is H3EA−. This applies for both Cu(II) and Cu(I), and indicates that it is crucial for ellagic acid to be deprotonated for replacing coordinated water molecules and binding with these ions. The energies of the chelation reactions (Table 11) are in line with the above-described geometrical features of the Cu complexes. They are systematically lower for the anion than for the neutral form of ellagic acid. Moreover, when the reactions involved hydrated Cu ions, they are exergonic for H3EA− but endergonic for H4EA. Therefore, it is proposed that deprotonation increases not only the primary antioxidant

Figure 5. Optimized geometries of the 2:1 complexes of ellagic acid anion (H3EA−) with Cu. Distances are reported in Å.

It was found that the shortest Cu−O distances are reduced, with respect to the 1:1 complexes, indicating that the chelation becomes stronger to some extent. The energies in Table 11 (two last lines) also show that the relative stability of the complexes increases with the number of H3EA− ligands. However, the data in this table was calculated with respect to the isolated fragments, while the most likely process for the formation of the 2:1 complexes is from the 1:1 complexes. The ΔG values for the second chelation step are −11.4 and −4.4 kcal/mol for Cu(II) and Cu(I), respectively, when the calculations are performed without hydration of water 913



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molecules. However, considering them, the ΔG values become −6.9 and 1.3 kcal/mol for Cu(II) and Cu(I), respectively. These latter values, which are the most relevant ones for biological systems, indicate that increasing the ellagic acid concentration will increase Cu(II) chelation but is not expected to have a significant effect for Cu(I). In order to quantify the effect of chelation on the reduction (or oxidation) processes involving the Cu ions, which are assumed to be involved in the ROS production, we have estimated the reduction potential (E0) of Cu(II). To that purpose, a strategy similar to that previously proposed by Fu et al.128 has been used, which involves the reaction

protective effects against OS in a concentration dependent way. The structures of the complexes, the corresponding quelation energies, the importance of the deprotonation for the quelation processes, and the quantification of the influence of chelation on the E0 values for Cu are provided here for the first time.

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CONCLUSIONS Several aspects related to the ellagic acid protection against oxidative stress and its toxic effects were investigated using the density functional theory. It is predicted that the HOO• radical scavenging activity of ellagic acid takes place exclusively by the HT mechanism regardless of the polarity of the environment. However, the environment influences the relative importance of the different reaction paths. Ellagic acid is not expected to be efficient for preventing peroxyl oxidation of lipids in nonpolar media. On the contrary, in aqueous solution, at physiological pH, it is predicted to be capable of exerting protection against this process. Under such conditions, ellagic acid is predicted to be efficiently, and continuously, regenerated after scavenging two free radicals (one ROO• and one O2•−) per cycle, until a different reaction consumes some of the intermediates. This is a desirable and unusual behavior for an antioxidant that contributes to increase its protective effects at low concentrations. Ellagic acid, in aqueous solution, at physiological pH, is capable of deactivating a wide variety of ROS and RNS, which is also a desirable behavior since several of them are expected to be present in biological systems. Its metabolites are also capable of efficiently scavenging a wide variety of free radicals, at least via SPLET. Moreover, most of the studied reactions are faster for the metabolites than for ellagic acid itself. Accordingly, it is proposed that the free radical scavenging activity of ellagic acid is not reduced after metabolization. On the contrary, it provides continuous protection against oxidative stress through a free radical scavenging cascade. This is a rare and very desirable behavior, which makes ellagic acid particularly valuable to that purpose. After deprotonation, ellagic acid is also capable of chelating copper in aqueous solution, yielding stable complexes. These reactions are expected to decrease free radical production, especially OH production, in a concentration dependent way. Thus, metal chelation is another way for ellagic acid to exert its protection against OS. The anionic form of ellagic acid is proposed as the key species for its manifold antioxidant activity.

reduced form ⇌ oxidized form + e−

The value of E0 is usually measured relative to a reference electrode, the normal hydrogen electrode (NHE) in this work. Therefore, E0 has been calculated as

E0 =

ΔG 0 F

(14)

where F is the Faraday constant (23.06 kcal/mol V), and ΔG0 is estimated as ΔG 0 = G(oxidized form) − G(reduced form) − 4.44

(15)

with the last term in this equation (−4.44 eV) being the free energy change associated with the NHE half-reaction: + H(aq) + e− ⇌

1 H 2(g) 2

If such a calculation is performed for free Cu, i.e., without hydration, the deviation from the experimental value (E0Cu(II)/Cu(I) = 0.16 eV) is very large (1.25 eV). However, for the hydrated model used in this work, which includes four explicit water molecules, it is reduced to 0.35 eV. Obtaining a better agreement with the experiment for E0Cu(II)/Cu(I) escapes the purposes of this work, but it can probably be achieved by including a larger number of water molecules when mimicking Cu ions in solution. It seems reasonable to expect a systematic error for the calculated E0 values of the Cu(II)/Cu(I) pair when coordination involves different ligands but an identical number of them. Accordingly, we have corrected eq 14 as follows: E0 =

ΔG 0 − 0.35 F

■

(16)

The E0 values obtained this way for hydrated Cu, the 1:1 H3EA− complex, and the 2:1 H3EA− complex are 0.16, 0.01, and −0.34 eV, respectively. They indicate that chelation by H3EA− significantly reduces the feasibility of the Cu(II) to Cu(I) reduction. Moreover, for the 2:1 complex this is expected to be an unlikely process. In biological systems, Cu is essentially in its oxidized form; thus, the production of OH radicals is expected to follow a two step process, the Haber Weiss reaction yielding Cu(I), followed by the Fenton reaction. Accordingly, it can be concluded that chelation by ellagic acid will decrease the Cu related OH radical production, in a concentration dependent way since it will dramatically reduce Cu(I) formation. In summary, after deprotonation, ellagic acid is capable of chelating both Cu(II) and Cu(I) in aqueous solution, yielding stable complexes. These reactions are expected to decrease the free radical production and thus increase the ellagic acid

ASSOCIATED CONTENT

S Supporting Information *

Experimental rate constants for the bimolecular reactions of ellagic acid with different radicals; Gibbs free energies of reaction, reorganization energies, and activation energies for the electron transfer reactions from ellagic acid and its metabolites; optimized geometries of the OH radical and anion with 4 explicit water molecules; structures of the different neutral conformers, and anions, of ellagic acid metabolites and their relative energy; and optimized geometries of the hydrated Cu(II) and Cu(I), and the 1:1 complexes of free copper with ellagic acid and its anion. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 52 55 5804 4675. E-mail: [email protected]. 914



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Funding

(9) Ihantola-Vormisto, A., Summanen, J., Kankaanranta, H., Vuorela, H., Asmawi, Z. M., and Moilanen, E. (1997) Anti-inflammatory activity of extracts from leaves of Phylllanthus emblica. Planta Med. 63, 518− 524. (10) Rogerio, A. P., Fontanari, C., Borducchi, E., Keller, A. C., Russo, M., Soaresm, E. G., Albuquerque, D. A., and Faccioli, L. H. (2008) Anti-inflammatory effects of Lafoensia pacari and ellagic acid in a murine model of asthma. Eur. J. Pharmacol. 580, 262−270. (11) Martins, D., Beserra, A., Calegari, P., Souza, M., dos Santos, R., Lima, J., Silva, R., and Balogun, S. (2011) Gastroprotective and ulcer healing mechanisms of ellagic acid in experimental rats. J. Agric. Food Chem. 59, 6957−6965. (12) Lakovleva, L. V., Ivakhnenko, A. K., and Buniatian, N. D. (1998) The protective action of ellagic acid in experimental myocarditis. Eksp. Klin. Farmakol. 61, 32−34. (13) Mukhtar, H., Das, M., and Bickers, D. R. (1986) Inhibition of 3methylcholanthrene-induced skin tumorigenicity in BALB/c mice by chronic oral feeding of trace amounts of ellagic acid in drinking water. Cancer Res. 46, 2262−2265. (14) Mandal, S., Ahuja, A., Shivapurkar, N. M., Cheng, S. J., Groopman, J. D., and Stoner, G. D. (1987) Inhibition of aflatoxin B1 mutagenesis in Salmonella typhimurium and DNA damage in cultured rat and human tracheobronchial tissues by ellagic acid. Carcinogenesis 8, 1651−1656. (15) Dixit, R., and Gold, B. (1986) Inhibition of N-methyl-Nnitrosoureainduced mutagenicity and DNA methylation by ellagic acid. Proc. Natl. Acad. Sci. U.S.A. 83, 8039−8043. (16) Wood, A. W., Huang, M.-T., Chang, R. L., Newmark, H. L., Lehr, R. E., Yagi, H., Sayer, J. M., Jerina, D. M., and Conney, A. H. (1982) Inhibition of mutagenecity of bay-region diol epoxides of polycyclic aromatic hydrocarbons by naturally occurring plant phenols: exceptional activity of ellagic acid. Proc. Natl. Acad. Sci. U.S.A. 79, 5513−5517. (17) Rao, C. V., Tokumo, K., Rigotty, J., Zang, E., Kelloff, G., and Reddy, B. S. (1991) Chemoprevention of colon carcinogenesis by dietary administration of piroxicam, α-difluoromethylornithine, 16αfluoro-5-androsten-17-one, and ellagic acid individually and in combination. Cancer Res. 51, 4528−4534. (18) Boukharta, M., Jalbert, G., and Castonguay, A. (1992) Biodistribution of ellagic acid and dose-related inhibition of lung tumorigenesis in A/J mice. Nutr. Cancer 18, 181−189. (19) Kaur, S., Grover, I. S., and Kumar, S. (1997) Antimutagenic potential of ellagic acid isolated from Terminalia arjuna. Indian J. Exp. Biol. 35, 478−482. (20) Loarca-Pina, G., Kuzmicky, P. A., de Mejia, E. G., and Kadoa, N. Y. (1998) Inhibitory effects of ellagic acid on the direct-acting mutagenicity of aflatoxin B1 in the salmonella microsuspension assay. Mutat. Res. 398, 183−187. (21) Khanduja, K. L., Gandhi, R. K., Pathania, V., and Syal, N. (1999) Prevention of n-nitrosodiethylamine induced tumorigenesis by ellagic acid and quercetin in mice. Food Chem. Toxicol. 37, 313−318. (22) De Mejia, E. G., Castano Tostado, E., and Lorca Pina, G. (1999) Antimutagenic effects of natural phenolic compounds in beans. Mutat. Res. 441, 1−9. (23) Narayanan, B. A., Geoffroy, O., Willingham, M. C., Re, G. G., and Nixon, D. W. (1999) p53/p21(WAF1/CIP1) expression and its possible role in G1 arrest and apoptosis in ellagic acid treated cancer cells. Cancer Lett. 136, 215−221. (24) Koyamangalath, K., Mack, T. R., IV, and Dean, E. B. (2000) Chemo-prevention for colorectal cancer. Crit. Rev. Oncol.: Hematol. 33, 199−219. (25) Mertens-Talcott, S. U., Talcott, S. T., and Percival, S. S. (2003) Low concentrations of quercetin and ellagic acid synergistically influence proliferation, cytotoxicity and apoptosis in MOLT-4 human leukemia cells. J. Nutr. 133, 2669−2674. (26) Whitley, A. C., Stoner, G. D., Darby, M. V., and Walle, T. (2003) Intestinal epithelial cell accumulation of the cancer preventive polyphenol ellagic acid-extensive binding to protein and DNA. Biochem. Pharmacol. 66, 907−915.

This work was partially supported by a grant from DGAPA UNAM (PAPIIT- IN209812) and projects SEP-CONACyT 167491 and 167430. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the Laboratorio de Visualización y Cómputo Paralelo at Universidad Autónoma MetropolitanaIztapalapa.

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ABBREVIATIONS H4EA, ellagic acid in its neutral form; H3EA−, ellagic acid monoanion; OS, oxidative stress; DPPH, 1,1-diphenyl-2picrylhydrazyl radical; TEAC, Trolox equivalent antioxidant capacity; ABTS, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid; PCM, polarizable continuum model; CPCM, conductor polarizable continuum model; HT, hydrogen transfer; SPLET, sequential proton loss electron transfer; BDE, bond dissociation enthalpy; IP, ionization potential; PDE, O-H proton dissociation enthalpy; PA, proton affinity; ETE, electron transfer enthalpy; SMD, solvation model density; TST, transition state theory; QM-ORSA, quantum mechanics based test for overall free radical scavenging activity; mf , molar fraction; ROO •, peroxyl radicals; RAF, radical adduct formation; SET, single electron transfer; ET, overall electron transfer; PE, pentyl ethanoate; ROS, reactive oxygen species; RNS, reactive nitrogen species; MUE, mean unsigned error; UA, urolithin A; UB, urolithin B; UC, urolithin C; UD, urolithin D; UE, urolithin E; UM5, urolithin M5; UM6, urolithin M6; UM7, urolithin M7; IUA, isourolithin A

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