a physicochemical examination of the free radical scavenging activity of trolox: mechanism, kinetics...

4642 Phys. Chem. Chem. Phys., 2013, 15, 4642--4650 This journal is c the Owner Societies 2013

Cite this: Phys. Chem.Chem.Phys.,2013,15, 4642

A physicochemical examination of the free radicalscavenging activity of Trolox: mechanism, kinetics andinfluence of the environment†

Marta E. Alberto,a Nino Russo,a Andre Grandb and Annia Galano*c

The free radical scavenging activity of Trolox was studied for aqueous and lipid environments using the

Density Functional Theory. Several reaction mechanisms and free radicals of different chemical nature

have been included in this study, as well as the influence of the pH. Trolox was found to be a powerful�OH and alkoxy scavenger, regardless of the conditions under which the reaction takes place. It was

also found to be very efficient as a peroxy radical scavenger in aqueous solution, while its protective

effects against this particular kind of free radicals are significantly reduced in lipid solution. Four

reaction mechanisms were found to significantly contribute to the �OH scavenging activity of Trolox in

aqueous solution: hydrogen transfer (HT), radical adduct formation (RAF), single electron transfer (SET),

and sequential proton loss electron transfer (SPLET), while in lipid media two of them are relevant: HT

and RAF. The �OCH3, �OOH, and �OOCHCH2 scavenging processes are predicted to take place almost

exclusively by HT from the phenolic OH group in lipid media, and in aqueous solution at pH o 11,

while at higher pH values the SPLET mechanism is proposed as the main one. This is also the case for

other non-halogenated alkyl or alkenyl peroxy (and alkoxy) radicals. The agreement with the available

experimental data supports the reliability of the presented calculations.

Introduction

Trolox (H2Tx; 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) is a water soluble a-tocopherol derivative thatis frequently used as a reference against which the antioxidantcapacity of other compounds is compared and expressed asTrolox equivalents. Therefore it is crucial to know the mecha-nism, or mechanisms, involved in the radical scavengingactivity of Trolox to perform fair comparisons since there areseveral antioxidant assays and the ruling mechanism of reac-tion is not the same for all of them. While in ORAC (oxygenradical absorbance capacity) and TRAP (total radical-trapping

antioxidant parameter) assays the main mechanism is thehydrogen transfer (HT); the FRAP (ferric reducing antioxidantpower) assay is governed by single electron transfer (SET); andTEAC (Trolox equivalent antioxidant capacity), or other ABTS(2,20-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) assays andDPPH (2,2-diphenyl-1-picrylhydrazyl)) assays involve both SETand HT, even though they are frequently classified as SETassays.1–3 Trolox has been used as the reference compoundfor all these assays, so it has been implicitly assumed that itsfree radical scavenging efficiency is good and similar throughboth SET and HT. However, as far as we know this has not beendemonstrated yet.

Before using any of the above-mentioned assays to evaluatethe antioxidant activity of a particular compound, relative toTrolox, it is important to know if both of them react throughthe same mechanism. For example glutathione is known forbeing an excellent antioxidant, despite the fact that it is a poorelectron donor and thus its protective effects arise almostexclusively from HT reactions.4 Accordingly, while ORAC andTRAP would be adequate to evaluate its free radical scavengingactivity, the FRAP assay would not. It is also important to notethat the relative importance of different reaction mechanismsdepends not only on the scavenger but also on the chemical

a Dipartimento di Chimica - University of Calabria, Arcavacata di Rende, CS 87036,

Italyb INAC/SCIB UMR E-3 CEA/UJF, CEA-Grenoble 1e Rue des Martyrs,

38054 Grenoble Cedex 9, Francec Departamento de Quımica, Universidad Autonoma Metropolitana-Iztapalapa,

San Rafael Atlixco 186, Col. Vicentina, Iztapalapa, C. P. 09340, Mexico D. F.,

Mexico. E-mail: [email protected]

† Electronic supplementary information (ESI) available: Thermochemical andkinetic data for the reactions of H2T in aqueous solution, and for the SETreactions involving the extended set of free radicals. Correlations between theSET DG values and the aqueous electron affinities of the free radicals. Optimizedstructures of the transition states. See DOI: 10.1039/c3cp43319f

Received 20th September 2012,Accepted 30th January 2013

DOI: 10.1039/c3cp43319f

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This journal is c the Owner Societies 2013 Phys. Chem. Chem. Phys., 2013, 15, 4642--4650 4643

nature of the radical it is reacting with.5–7 It has been reportedthat Trolox can efficiently scavenge a wide variety of freeradicals.8–17 However there are no previous estimations of therelative importance of HT, SET, or other mechanisms for suchactivity, or how much it changes depending on the reactingradical.

The conditions under which the reactions take place are alsoan important factor to consider. Friaa and Brault12 found thatthe rate of the reaction between Trolox and DPPH increaseswith the pH of the solution. They rationalized this findingbased on the higher reactivity of the phenolate form of Trolox,which suggests a sequential proton loss electron transfer(SPLET) mechanism. This is in line with previous reports onthe importance of such mechanisms for the reactions ofphenolic compounds with DPPH,6,18–21 and also with differentreactive oxygen species (ROS).22–26 The role of the pH in the freeradical scavenging activity of Trolox has also been studied byMitarai et al.13 They found that when reacting with the aroxylradical in Triton X-100 micellar solution (5.0 wt%) the neutralform of Trolox and its di-anion react 2.1 and 2.6 times fasterthan the mono-anion, respectively. Based on these results, andon the fact that the di-anion has no phenolic OH, they proposedthat the reaction with the di-anion takes place via SET. Inaddition for other phenolic compounds it has been reportedthat at acid pH values, at which the phenolate anion is notformed to a significant extent, HT is the main mechanism ofreaction.26 This might also be the case for Trolox but it needs tobe proven. This is particularly important for this compound,since if that hypothesis is confirmed its activity in the above-mentioned assays would depend on the acidity of the environ-ment they are conducted in.

Based on its structure (Scheme 1), there is also anotherpossible mechanism for the radical scavenging activity ofTrolox, the radical adduct formation (RAF). If such a mechanismsignificantly contributed to the overall reactivity of Troloxtowards free radicals it would mean that when using an assaydesigned for HT of SET mechanisms such activity would beunderestimated. It should be noted that RAF is not expected tocontribute to the Trolox’s reactivity towards DPPH because forthis particular radical the addition process would be stericallyhindered. However, for smaller radicals such as many of theROS formed in biological systems RAF might be a viablemechanism. To our best knowledge this possibility has not beeninvestigated yet.

According to the above discussion it is evident that furtherinvestigations on important aspects of the radical scavenging

activity of Trolox are still needed. It is the main goal of thepresent work to address such aspects. In particular we intend toidentify the preponderant mechanism of reaction in polar andnon-polar media, as well as in aqueous solutions depending ontheir pH. To that purpose we have investigated several mechan-isms of reaction including SET, HT, RAF and SPLET. We alsoaim to establish if the main mechanism changes depending onthe radical reacting with Trolox. Detailed kinetic data includingrate constants and branching ratios are proposed for thefirst time.

Computational details

Geometry optimizations and frequency calculations have beencarried out using the M05-2X functional27 and the 6-31+G(d,p)basis set, in conjunction with the SMD continuum model28

using pentyl ethanoate and water as solvents to mimic lipid andaqueous environments, respectively. The M05-2X functionalhas been recommended for kinetic calculations by theirdevelopers,27 and it has also been successfully used byindependent authors to that purpose.29–32 It is also amongthe best performing functionals for calculating reaction energiesinvolving free radicals.33 SMD is considered a universal solvationmodel, due to its applicability to any charged or unchargedsolute in any solvent or liquid medium for which a few keydescriptors are known.28

Unrestricted calculations were used for open shell systemsand local minima and transition states were identified by thenumber of imaginary frequencies (NIMAG = 0 or 1, respectively).In the case of the transition states it was verified that theimaginary frequency corresponds to the expected motion alongthe reaction coordinate, by Intrinsic Coordinate calculations(IRC). All the electronic calculations were performed with theGaussian 09 package of programs.34 Thermodynamic correc-tions at 298.15 K were included in the calculation of relativeenergies, which correspond to 1 M standard state. In additionthe solvent cage effects have been included according to thecorrections proposed by Okuno,35 taking into account the freevolume theory.36

The rate constants (k) were calculated using the conven-tional Transition State Theory (TST).37–39 Reaction path degen-eracies and tunneling corrections have been taken intoaccount. The tunneling corrections, defined as the Boltzmannaverage of the ratio of the quantum and the classical probabil-ities, were calculated using the Zero Curvature Tunnelingcorrections (ZCT).40 For the electron transfer reactions thebarriers were estimated using the Marcus theory41 as:

DGa ¼ l4

1þ DGl

� �2

(1)

where DG is the free energy of reaction and l is areorganization term.

In addition, some of the calculated rate constants (k) areclose to the diffusion-limit, thus the apparent rate constant(kapp) cannot be directly obtained from TST calculations. In the

Scheme 1 Trolox structure, and site numbering. Blue labels denote potential HTreaction sites, and red labels potential RAF reaction sites.

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present work the Collins–Kimball theory is used to thatpurpose:42

kapp ¼kDkact

kD þ kact(2)

where kact is the thermal rate constant, obtained from TSTcalculations, and kD is the steady-state Smoluchowski43 rateconstant for an irreversible bimolecular diffusion-controlledreaction:

kD = 4pRDABNA (3)

where R denotes the reaction distance, NA is the Avogadronumber, and DAB is the mutual diffusion coefficient ofthe reactants A (free radical) and B (Trolox). DAB has beencalculated from DA and DB according to ref. 44, DA and DB havebeen estimated from the Stokes–Einstein approach:45

D ¼ kBT

6pZa(4)

where kB is the Boltzmann constant, T is the temperature, Zdenotes the viscosity of the solvent, in our case water (Z = 8.91�10�4 Pa s) and pentyl ethanoate (Z = 8.62 � 10�4 Pa s); and a isthe radius of the solute.

Results and discussion

Trolox has two pKa values, 3.89 (ref. 46) and 11.92.47 The firstone involves the deprotonation of the carboxylic group and thesecond one the phenolic OH (Scheme 2). The molar fractionsof the different species estimated from these pKa values atphysiological pH (7.4) indicate that under such conditions thepopulations of the neutral and di-anionic forms of Trolox arenegligible (Fig. 1). This means that in aqueous solution atphysiological pH the dominant form of Trolox is its carboxylateanion (HTx�). Accordingly this is the key species for the freeradical activity of Trolox under such conditions and the onewe will be focused on. However some analyses will also beperformed for the other forms of Trolox in aqueous solution toassess the influence of the pH on the scavenging activity of thiscompound in such media. On the other hand, in non-polar(lipid) media only the neutral form is studied, since such anenvironment does not promote the necessary solvation tostabilize the ionic species.

As mentioned in the introduction, Trolox can scavenge freeradicals through a variety of reaction mechanisms, as it is thecase for many other scavengers.48–54 Those considered in thiswork are HT, RAF, SET and SPLET. They correspond, for thereactions of HTx� with any of the studied radicals (�R), to thefollowing processes:

HT: HTx� + �R - Tx�� + HRRAF: HTx� + �R - [HTx–R]��

SET: HTx� + �R - HTx� + R�

SPLET: (i) HTx�" H+ + Tx2� (ii) Tx2� + �R - Tx�� + R�

The relative importance of the above-detailed mechanismshas been investigated for the reactions of Trolox with fourdifferent ROS: �OH (R1), �OOH (R2), �OCH3 (R3), and�OOCHCH2 (R4). �OH has been chosen for being amongthe most electrophilic,55 and reactive of the oxygen-centeredradicals, with a half-life of B10�9 s.56 Compared to �OH, peroxyradicals are less reactive species capable of diffusing to remotecellular locations.57 Their half-lives are of the order of seconds.58

We have chosen �OOH because it is the simplest of the peroxyradical, and �OOCHCH2 to mimic larger, and unsaturated,peroxy radicals. The �OCH3 radical has been chosen to representalkoxy radicals which have intermediate reactivity, in between�OH and peroxy radicals.

The thermochemical viability of the different mechanismsand channels of reaction has been investigated first since it willdetermine if a chemical process can actually be observable. TheGibbs energies of reaction (DG) for H2Tx in non-polar mediaand for HTx� in aqueous solution are shown in Fig. 2(A) and(B), respectively. Their values are reported in Table S1 (ESI†). Inaddition the values for H2Tx in aqueous solution are providedin Table S2 (ESI†). In non-polar environments the SET mechanismis not expected to contribute to the overall reactivity of Troloxtowards free radicals since such environments do not promote thenecessary solvation of the intermediate ionic species yielded bythis mechanism. However, just to prove this point, the Gibbsenergy of reaction (DG) for the SET process was calculated andfound to be larger than 40 kcal mol�1 for all the studied radicals.

Scheme 2 Deprotonation sequence and pKa values.46,47

Fig. 1 Distribution diagram and molar fractions of neutral (H2Tx), mono-anionic(HTx�), and di-anionic (Tx2�) forms of Trolox at physiological pH.

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These values definitively rule out the viability of SET inlipid media.

In aqueous solution the SET reactions from both H2Txand HTx� to �OH were found to be exergonic by 3.06 and7.44 kcal mol�1, respectively (Tables S1 and S2, ESI†). Incontrast, the SET reactions with the other studied ROS werefound to be endergonic by more than 10 kcal mol�1. This doesnot mean that the SET mechanism is not important for otherfree radicals. In fact the viability of such processes would bestrongly influenced by the electro-accepting nature of thereacting free radical. Since SET is usually a fast process thatcan take place at relatively large reaction distances it is usuallyvery important in biological systems. Therefore we have studiedthis particular mechanism for a wider set of free radicals andthe three forms of Trolox (Table S3, ESI†). As expected it wasfound that the thermochemical viability of the SET processincreases with the degree of deprotonation, which supports therole of the pH in the investigation of the relative importance ofthis mechanism. The SET reactions involving Tx2� were foundto be exergonic with all the studied radicals. For the speciesHTx� and H2Tx, on the other hand, only the reactions withthose free radicals with higher electrophilic character (�OH,�OCCl3, �OOCCl3, SO4

�, NO2�, N3

� and 2dG�+) were found to be

exergonic. In addition for HTx� the reactions with �OOCHCl2

and Br2�� are almost isoergonic.

The electron affinities of the studied free radicals are alsoprovided in Table S3 (ESI†). These values directly correlatewith the Gibbs energies of reaction (DG), which supports theimportance of the chemical nature of the reacting free radicalfor the viability of the SET processes. The turning points of the

aqueous adiabatic electron affinity values (AqAEA) of the freeradicals, at which SET becomes exergonic, were found to be5.39, 5.20, and 4.03 eV for the reactions with H2Tx, HTx�, andTx2�, respectively (Fig. S1, ESI†). The equivalent values for theaqueous vertical electron affinities (AqVEA) are 5.0, 4.8, and3.6 eV. The latter values are provided with fewer significantdigits because the correlation between DG and AqVEA is poorerthan that with AqAEA. This can be explained based on the factthat different species should have different reorganizationenergies, which are not necessarily in line with the electronaffinity order. In any case, both of these criteria might behelpful for predicting the viability of SET reactions with thedifferent forms of Trolox, based on very simple calculations. As longas the level of theory used for such estimation is the same as thatused in this work the only requirement for a free radical tospontaneously accept an electron from Trolox species is to have anelectron affinity larger than, or similar to, the above-proposed limits.

Regarding the HT and RAF mechanisms, the most exergonicreaction path was found to be that involving HT from site 1a,i.e. from the phenolic OH group, for all the studied radicals,both in polar and non-polar media, and regardless of thereacting form of Trolox (H2Tx or HTx�). In addition the RAFpaths are systematically less thermochemically favored than theHT paths. Accordingly mechanisms HT and RAF were notmodeled for Tx2�, since this species does not have any phenolicOH. For the reactions involving �OH all the HT and RAF pathswere found to be exergonic regardless of the polarity of theenvironment, and also of the reacting form of Trolox (Fig. 2).This is a logical outcome based on the high reactivity, i.e. lowselectivity, of this radical.

Fig. 2 Gibbs free energies of reaction (DG, kcal mol�1), at 298.15 K. (A) Pentyl ethanoate solution; (B) aqueous solution.

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For the reactions involving �OCH3, which is more selectivethan �OH but still highly reactive, all the HT paths are exergonicin aqueous solution, while in pentyl ethanoate (lipid) solutionpath 9a is almost isoergonic. The RAF reactions involving thisradical, on the other hand, were found to be endergonic in lipidmedia, except path 3, which is nearly isoergonic. In aqueoussolution most of the RAF reactions between �OCH3 and H2Txare also endergonic, the exceptions are paths 1 (Bisoergonic)and 3 (exergonic). In contrast, for the reactions of this radicalwith HTx�most RAF reactions are exergonic, with the exceptionsof paths 5 (endergonic), 2 and 4 (Bisoergonic).

Radicals �OOH and �OOCHCH2, which are the least reactiveof the studied radicals, are highly selective and react exclusivelyby the HT mechanism. All the RAF paths were found to besignificantly endergonic for those radicals, with DG valueslarger than 10 kcal mol�1. Therefore the RAF mechanism canbe ruled out for the peroxyl radical scavenging activity of Trolox.The HT path 1a was found to be the only spontaneous path forthe reactions of these radicals in lipid solution, as well as inaqueous solution when the dominant form of Trolox is HTx�,i.e. the one prevailing at physiological pH (Table S1, ESI†), whilefor the neutral form of Trolox (Table S2, ESI†) path 7 becomesslightly exergonic.

For the kinetic study we have not included the reactionpaths described above as endergonic because, even if they takeplace at a significant rates, they would be reversible and there-fore the formed products will not be observed. However, theymight still represent significant channels if their productsrapidly react further. This would be particularly important ifthese latter stages are sufficiently exergonic to provide a drivingforce, and if their barriers of reactions are low.

The fully optimized geometries of the transition states (TSs)are shown in Fig. S2–S14 (ESI†). Transition states for thephenolic abstractions (site 1a) from H2Tx in aqueous and pentylethanoate solution as well as from HTx� in aqueous solution byradicals �OOH and �OOCHCH2 were located and characterized(Fig. S12–S14, ESI†). However for �OH and �OCH3 it was notpossible to locate the TSs corresponding to path 1a using fulloptimizations, except for the reaction of neutral Trolox with

�OCH3 in lipid media (Fig. S8, ESI†). Using partial optimiza-tions with frozen O� � �H and H� � �OH bond distances, we wereable to obtain structures that present a single imaginaryfrequency corresponding to the desired transition vector.Unfreezing these two distances, during a saddle point optimi-zation, invariably leads to an increase of the H� � �OH distance,and the corresponding decrease of the imaginary frequency andgradient, yielding the separated reactants. A relaxed scan,obtained by decreasing the H� � �OH distance, produces a simi-lar result, i.e. the energy decreases until the H is completelytransferred. This means that the reaction is barrierless andstrictly diffusion-controlled. In other words, every encounter iseffective producing the conversion of reactants into products.Following a suggestion from a reviewer the same search wasperformed by including two explicit water molecules and usingthe wB97XD functional, in addition to M05-2X, for the TSscorresponding to the Trolox (1a) + OH radical reaction. In bothcases the outcome was the same as that when the search wasperformed without including explicit water molecules, i.e.unfreezing the distances leads to separated reactants, orproducts depending on the frozen distances, while the energycontinuously decreases. The charge on the O atom of the OHfragment was found to be about �1.13 in all cases, while it isabout �0.5 for the HT TSs that were located. This means that atthe distance necessary for the TS to be formed, an electron isalready transferred to the OH. This explains why the HTtransition state cannot be located for path 1a, and is supportedby the negative value of DG for the SET process.

The Gibbs free energies of activation (DGa) for the reactionsof the dominant forms of Trolox in lipid and aqueous solution,at physiological pH, are reported in Table 1, while thosecorresponding to the reactions of H2Tx in aqueous solutionare provided in ESI† (Table S4). In general the lowest DGa

values correspond to the reactions with �OH followed by thoseof the �OCH3 reactions, while the highest values are those ofthe reactions with the peroxyl radicals. This is in line with therelative reactivity of the studied radicals.

For the reactions with �OH path 1a has the lowest barrieramong the HT reactions, while the RAF path with the lowest

Table 1 Gibbs free energies of activation (DGa, kcal mol�1), at 298.15 K

Lipid solution Aqueous solution

�OH �OOH �OCH3�OOCHQCH2


SET 0.02HT-1a B0.00 12.63 8.23 10.88 B0.00 11.98 B0.00 8.06HT-2a 6.34 14.86 5.40 14.87HT-3a 4.85 11.76 3.46 12.76HT-6a 6.38 13.23 6.01 12.35HT-7 6.05 12.59 3.71 11.44HT-8 6.86 16.78 5.19 15.53HT-9a 8.84 6.60 18.25RAF-1 2.40 1.32 9.87RAF-2 3.94 0.61 11.88RAF-3 4.25 10.02 0.78 10.01RAF-4 4.21 0.15RAF-5 4.33 3.12RAF-6 3.43 1.43 12.00

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barriers changes depending on the environment. In lipidsolution path 1 has the lowest barrier, while in aqueous solutionat acid pH values (H2Tx is the dominant form of Trolox) thebarrier of path 4 is very similar to that of path 1, and atphysiological pH (HTx� prevails) paths 2 and 4 are those withthe lowest barriers, which are so low that they may be consideredalmost barrierless. However, regardless of the reaction condi-tions the barriers of all HT and RAF paths are rather low for theTrolox reactions with �OH, which indicates that most of themshould have significant contributions to the overall reactivity ofTrolox towards this very reactive free radical.

For the reactions involving �OCH3, path 1a (HT) is also theone with the lowest barrier among the viable reactions. All the otherpaths, except RAF-1, were found to have DGa values higher than 10kcal mol�1. Regarding the SET processes with the expanded set offree radicals the reaction barriers are provided in ESI† (Table S5).Some of these reactions have high barriers, despite the fact that theyare highly exergonic. This is particularly evident for the reactions ofTx2� with the most electrophilic free radicals, for which the DGvalues of the SET reactions are significantly negative. Since it wouldbe expected that the electron-donor capability of the anions wouldbe higher than that of the corresponding neutral compound, thisbehavior is contra-intuitive. However it can be explained based onthe fact that these reactions correspond to the inverted region of theMarkus theory (DG o �l)59 and have the characteristic that DG isnot only lower, but much lower, than �l (bold letters, Table S6,ESI†). There are other reactions that, while located in the invertedregion, are close enough to the vertex of the Marcus parabola andtherefore their barrier remains low. This corresponds to the caseswhere the difference (�l) � DG is positive but relatively small.

The rate constants for the SET reactions (Table S8, ESI†)indicate that Trolox is an excellent electron donor. Dependingon the pH, i.e. on its dominant form, it is capable of scavengingall the tested free radicals through this mechanism at very fastrates. Those that are larger than 106 M�1 s�1 have been high-lighted in bold letters in the table. When reacting with the mostelectrophilic radicals (�OH, �OCCl3, SO4

�, N3�, and 2dG

�+) thescavenging efficiency of Trolox via SET is very high at pH values

lower than 11, at which the form H2Tx or HTx� prevails. Incontrast when reacting with the least electrophilic radicals(�OOH, �OOCH3, �OOCH2Cl, �OOCHCH2, �OOCH2CHCH2,and ArO�) the only form of Trolox that has high SET rates isTx2�. Since this is the dominant form at pH > 11 this mechanismis not expected to significantly contribute to the scavengingactivity of Trolox towards these radicals under physiologicalconditions. This should also be the case for other alkyl or alkenylperoxy radicals. For the particular case of the Trolox + ArO�

reaction the experimental rate constant has been reported to be3.07 � 104 M�1 s�1, at pH = 7.13 At this pH Trolox exists almostexclusively as HTx�, thus comparing our result for its SETreaction with ArO� (1.70 � 104 M�1 s�1) it can be stated thatthe agreement is excellent, which not only supports our resultsbut also suggests that SET is the main mechanism of reaction inthis case. For the reaction with DPPH�, which is also frequentlyused in antioxidant assays, as well as for �OCH3 and�OCH2CHCH2 (non-halogenated, non-aromatic alkoxy radicals),the SET process becomes important at pH > 5, i.e. at which theprevailing species are the Trolox’s anions. On the other hand for�OOCHCl2, OOCCl3, Br2

��, and NO2� this mechanism was found

to be important for all the Trolox forms, i.e. regardless of the pHof the environment. Accordingly it can be stated that Trolox is anefficient and versatile free radical scavenger via SET.

The rate constants for the different channels of reaction, inaqueous and lipid solutions, for the reactions of �OH, �OCH3,�OOH and �OOCHCH2, are reported in Table 2, together withthe overall rate coefficients, which have been calculated as thesum of the rate constants of each path. For example for theHTx� + �OH reaction, in aqueous solution:

koverall ¼ kSETapp þ kHTapp þ kRAF

app

where:

kHTapp ¼ kp1aapp þ kp2aapp þ kp3aapp þ kp6aapp þ kp7app þ kp8app þ kp9aapp

kRAFapp ¼ kp1app þ kp2app þ kp4app þ kp5app þ kp6app

Table 2 Rate constants at 298.15 K, in M�1 s�1




SET 8.16 � 109 1.37 � 107

HT-1a 1.91 � 109 3.40 � 103 5.77 � 106 6.56 � 104 1.91 � 109 8.96 � 104 1.72 � 109 7.62 � 106

HT-2a 3.46 � 108 2.05 � 103 9.90 � 108 2.03 � 103

HT-3a 1.39 � 109 2.24 � 105 1.84 � 109 4.18 � 104

HT-6a 3.23 � 108 2.23 � 104 5.28 � 108 9.85 � 104

HT-7 3.70 � 108 3.17 � 104 1.77 � 109 2.24 � 105

HT-8 1.66 � 108 4.87 � 101 9.63 � 108 3.99 � 102

HT-9a 1.93 � 107 2.36 � 108 1.28 � 101

RAF-1 1.89 � 109 1.91 � 109 7.16 � 105

RAF-2 1.71 � 109 1.91 � 109 2.42 � 104

RAF-3 1.59 � 109 5.58 � 105 1.91 � 109 5.66 � 105

RAF-4 1.61 � 109 1.91 � 109

RAF-5 1.55 � 109 1.85 � 109

RAF-6 1.82 � 109 1.91 � 109 1.99 � 104

Overall 1.47 � 1010 3.40 � 103 6.61 � 106 6.56 � 104 2.78 � 1010 8.96 � 104 1.72 � 109 7.62 � 106

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The Trolox reactions with �OH were found to be diffusion-controlled with overall rate coefficients >1010 M�1 s�1, regard-less of the environment’s polarity and also of the pH in aqueoussolution (Table 2 and Table S7, ESI†). At physiological pH (7.4),where HTx� prevails, the overall rate coefficient was found to be2.8 � 1010 M�1 s�1, which is in good agreement with theexperimental value estimated by Aruoma et al.9 at this pH(8.1 � 1010 M�1 s�1). This agreement also supports the relia-bility of the kinetic data calculated in the present work. Inaddition, it has been previously demonstrated that the kineticresults obtained from M05-2X calculations agree well with theexperimental data available for the reactions of phenols withOH24 and with other free radicals.26,60 The high value of theoverall rate coefficient of the Trolox + �OH reactions is in linewith the high reactivity of this radical and with the proposal ofAruoma et al.9 that Trolox is a powerful scavenger of �OH.

The overall rate constants of the Trolox + �OCH3 reactionswere found to correspond to the diffusion-limited regime inaqueous solution for both H2Tx (Table S7, ESI†) and HTx�

(Table 2) with koverall values equal to 1.3 � 109 and 1.7 � 109

M�1 s�1, respectively. Therefore in such media Trolox is pre-dicted to be an excellent �OCH3 scavenger. In lipid solution it ispredicted to still be very efficient with koverall = 6.6� 106 M�1 s�1,although less efficient than in aqueous solution. This indicatesthat for this radical, that is less reactive than �OH, the conditionsunder which the reaction takes place might significantly influ-ence the scavenging activity of Trolox. Since it is a water solublea-tocopherol derivative it seems important that its activity isincreased in such media. This is also the case for the peroxylradicals. However for them the pH does play an important role inthe overall scavenging activity of Trolox, which is significantlyincreased when HTx� is the reacting species, instead of H2Tx.Moreover since the SET reactions of �OOH and �OOCHCH2 withTx2� were also found to be very fast, it can be established that atpH > 5 this compound is very efficient as a peroxyl scavenger. Onthe other hand, Trolox is predicted to be a moderate protectoragainst the peroxyl oxidation of lipids. In such media the overallrate coefficient has been estimated to be 3.4 � 103 M�1 s�1, andthe rate constants corresponding to the �OOH damage to

unsaturated fatty acids are in the range of 1.18 � 103–3.05 �103 M�1 s�1.61

Regarding the rates of the different reaction paths, for thereactions involving �OH, all of them were found to take placevery fast. For the reactions with �OCH3, on the other hand, HTthrough path 1a is the fastest reaction while for the peroxylradicals the scavenging activity of Trolox was found to takeplace exclusively through this path. To investigate in detail therelative importance of the different mechanisms of reaction inthe free radical scavenging of Trolox we have estimated thebranching ratios of the different reaction paths for its reactionswith �OH, OCH3, �OOH and �OOCHCH2 (Table 3 and Table S9,ESI†). They represent the percent contribution of the differentchannels to the overall reaction, and have been calculated as:

Gi ¼ki

koverall� 100 (5)

where ki represents each reaction path.As expected based on its higher reactivity a very wide

distribution of products is expected for the Trolox + �OHreaction, regardless of the environmental conditions. It wasfound that the contributions of the HT and RAF mechanisms,in lipid solution, are 30.8% and 69.2%, respectively (Table 3).Among the HT paths that contributing the most to the overall�OH scavenging activity of Trolox in such media is 1a (13.0%),followed by 3a (9.5%). The contributions of the six RAF pathsare very similar and range from 10.6% to 12.9%. According tothe estimated branching ratios, products yielded by HT paths1a and 3a, as well as by all the RAF paths, are expected to beformed to a significant extent. In aqueous solution, for the�OH + H2Tx reaction, the contributions of the SET, HT, andRAF mechanisms are 34.5%, 18.4%, and 47.1% respectively(Table S9, ESI†). Path 1a is the one contributing the most to theHT mechanism, while paths 1 and 5 are the major RAFchannels. When the reactions take place in aqueous solutionat higher pH values, such as the physiological one, i.e. whenHTx� is the preponderant form of Trolox, the contributions tothe overall reactivity of this compound towards �OH are 29.4%,29.6%, and 41.0% for SET, HT, and RAF mechanisms respectively.

Table 3 Branching ratios (G, %)*, at 298.15 K




SET 29.36 0.79HT-1a 12.99 100.0 87.33 100.0 6.87 100.0 99.11 100.0HT-2a 2.35 0.03 3.56 B0.00HT-3a 9.48 3.38 6.64 B0.00HT-6a 2.20 0.34 1.90 0.01HT-7 2.52 0.48 6.36 0.01HT-8 1.13 B0.00 3.47 B0.00HT-9a 0.13 0.85 B0.00RAF-1 12.88 6.86 0.04RAF-2 11.62 6.87 B0.00RAF-3 10.82 8.44 6.87 0.03RAF-4 10.94 6.87RAF-5 10.56 6.67RAF-6 12.37 6.86 B0.00

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This analysis indicates that the RAF mechanism plays a veryimportant role in the �OH scavenging activity of Trolox, regard-less of the environmental conditions, although HT and SET (inaqueous solution) also have large contributions to the overallactivity. It is important to notice that SET from HTx� actuallycorresponds to SPLET. Therefore for the particular case of itsreactions with �OH it is proposed that at least four mechanismsare involved in the scavenging activity of Trolox in aqueoussolution: HT, RAF, SET and SPLET, while in lipid media onlytwo of them are relevant: HT and RAF.

For the Trolox + �OCH3 reaction in pentyl ethanoate (lipid)solution the main mechanism is HT, which was found to beresponsible for 91.6% of the overall activity (Table 3). Inaddition path 1a is by far the one contributing the most to thismechanism (G1a = 87.3%). In aqueous solution the reactiontakes place almost exclusively via HT from site 1a for bothspecies, H2Tx and HTx� (Table 3 and Table S9, ESI†). Thismeans that at pH o 11 this is the only important mechanismfor the �OCH3 scavenging activity of Trolox. In contrast, at pH >11 with Tx2� being the prevailing form of Trolox, there is nophenolic OH group susceptible to HT and the electron transferbecomes the main mechanism of reaction, taking place atdiffusion-limited rates (Table S8, ESI†). This process can beconsidered as a sequential double proton loss electron transfer(SDPLET) with respect to neutral Trolox or as SPLET withrespect to the mono-anion, which is the dominant format physiological pH. This should also be the case for othernon-halogenated alkyl or alkenyl alkoxy radicals.

The �OOH and �OOCHCH2 radical scavenging of Trolox wasfound to take place almost exclusively by HT from site 1a whenits reacting form is H2Tx or HTx�, i.e. in lipid solution and inaqueous solution at pH o 11. However at higher pH values,when this path is no longer possible since the phenolate ion isformed, the SDPLET mechanism is predicted to be the crucialone to such activity.

Conclusions

The free radical scavenging activity of Trolox was studied indetail for aqueous and lipid environments using the DensityFunctional Theory. Several reaction mechanisms and freeradicals of different chemical nature have been included inthis study.

Trolox was found to be a powerful �OH and alkoxy radical(RO�) scavenger, regardless of the conditions under which thereaction takes place. It was also found to be a very good peroxyradical (ROO�) scavenger in aqueous solution, while itsprotective effects against this particular kind of free radicalsare only moderate in lipid solution when R is a non-halogenatedalkyl or alkenyl group.

Four reaction mechanisms were found to be involved in the�OH scavenging activity of Trolox in aqueous solution: HT, RAF,SET and SPLET, while in lipid media two of them are relevant:HT and RAF.

The �OCH3 scavenging process was found to take placealmost exclusively by HT from site 1a in lipid media, and in

aqueous solution at pH o 11, while at higher pH values theSDPLET is the important one. A similar situation was found for�OOH and �OOCHCH2 and is also predicted for other non-halogenated alkyl or alkenyl peroxy (and alkoxy) radicals.

In addition it was found that Trolox is an efficient andversatile free radical scavenger, capable of deactivating a widevariety of free radicals via electron transfer in aqueous solution.However the efficiency of such activity for each particularradical is influenced by the pH of the environment.

The new physicochemical insights on the free radicalscavenging activity of Trolox provided in the present work areexpected to contribute to a better understanding of the reactivityof this compound. It is also expected that these new data helpdesigning, or interpreting, antioxidant assays for which Trolox isused as the reference compound.

Acknowledgements

A.G. thanks Laboratorio de Visualizacion y Computo Paralelo atUAM – Iztapalapa for the access to its computer facilitiesand projects SEP-CONACyT 167491 and 167430 for financialsupport.

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a physicochemical examination of the free radical scavenging activity of trolox: mechanism, kinetics...

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