Distribution and reactivity of gallates towardgalvinoxyl radicals in SDS micellar solutions —Effect of the alkyl chain length

Raquel Bridi, Carolina Aliaga, Alexis Aspee, Elsa Abuin, and Eduardo Lissi

Abstract: In this work, we have evaluated the reactivity of methyl gallate, propyl gallate, and n-octyl gallate toward galvi-noxyl radicals in homogeneous solution (ethanol) and sodium dodecyl sulfate micelles. In ethanol, the rate of galvinoxylconsumption was independent of the gallate’s alkyl chain length. On the other hand, reaction rates in micelles were de-pendent on the length of the gallate alkyl chain. Measurements of the rate of the reaction at different pH show that, evenat pH 7.0, the reaction mainly involves the monodeprotonated anionic form of the gallates. Plots of reaction rates as afunction of the surfactant concentration allowed us to evaluate the distribution of the gallates (KGH) and an apparent rateconstant defined by the product between the intramicellar rate constant of the anion (kG� ) and its partition constant (KG� ).The values of the increment of the apparent rate constant per methylene group in the alkyl chain strongly suggest that in-tramicellar rate constant values are independent of the alkyl chain length. This points to a similar intramicellar localizationof the phenolate moiety in the three gallates considered.

Key words: galvinoxyl radical, gallates, rate-constant values, sodium dodecyl sulfate (SDS) micelles.

Resume : Dans ce travail, on a evalue la reactivite du gallate de methyle, du gallate de propyle et du gallate d’octyle vis-a-vis les radicaux galvinoxyles, en solution homogene dans l’ethanol et dans des micelles de dodecylsulfate de sodium.Dans l’ethanol, la vitesse de disparition du galvinoxyle est independante de la longueur de la chaıne alkyle des gallates.Par ailleurs, les vitesses de reaction dans les micelles dependent de la longueur de la chaıne alkyle des gallates. Des mesu-res de vitesse de reaction a divers pH montrent que meme a un pH de 7,0, la reaction implique principalement la formeanionique monodeprotonee des gallates. Des courbes des vitesses de reaction en fonction de la concentration d’agent desurface a permis d’evaluer la distribution des gallates (KGH) et une constante de vitesse apparente definie par le produit en-tre la constante de vitesse intramicellaire de l’anion (kG� ) et sa constante de partition (KG� ). Les valeurs des incrementsde la constante de vitesse apparente par groupe methylene de la chaıne alkyle suggerent fortement que les valeurs la cons-tante de vitesse intramicellaire sont independantes de la longueur de la chaıne alkyle. On a considere ces points pour unelocalisation intramicellaire semblable de la portion phenolate dans les trois gallates consideres.

Mots-cles : radical galvinoxyle, gallates, valeurs des constantes de vitesse, micelles de dodecylsulfate de sodium (DSS).

[Traduit par la Redaction]

Introduction

Oxidative stress imposed by reactive oxygen species mayplay a crucial role in the pathophysiology associated withneoplasia, atherosclerosis, and neurodegenerative diseases.The oxidative damage can be controlled by compounds thatremove active free radicals. These compounds can be en-dogenous or exogenous and most of them have phenolicgroups as active moieties. They show a wide range of activ-ities in vitro and are thought to exert protective effectsagainst major diseases such as cancer and cardiovasculardiseases.1–3 The efficiency of a compound to remove reac-tive radicals will depend on the reactivity of its phenolic

groups and the relative localization of the radical and thefree-radical scavenger.4 The synthetic n-alkyl esters of gallicacid, in particular propyl and octyl gallates, are widely em-ployed as antioxidants in food and pharmaceutical indus-tries. Besides the antioxidant activity, other biological roleshave been described for this group of molecules, such asanticancer, antibacterial, and antifungal roles.5

Several studies have shown a good correlation betweenthe inhibition of lipid oxidation of a given compound andits reactivity toward stable radicals.6,7 However, in dispersed(microheterogeneous) systems like emulsions, antioxidantactivity is related to several parameters determined by theinterface. In particular, depending on the emulsifier used, an

Received 16 March 2010. Accepted 11 June 2010. Published on the NRC Research Press Web site at canjchem.nrc.ca on 20 January2011.

This article is part of a Special Issue dedicated to Professor J. C. Scaiano.

R. Bridi.1 Universidade Federal de Rio Grande do Sul, Av. Ipiranga 2752 Porto Alegre, Rio Grande do Sul 90610-000, Brazil.C. Aliaga, A. Aspee, E. Abuin, and E. Lissi. Facultad de Quımica y Biologıa, Universidad de Santiago de Chile, Av. L. B. O’Higgins3363, Casilla 40, Correo 33, Santiago, Region Metropolitana, Chile.

1Corresponding author (e-mail: raquel.bridi@ufrgs.br).

181

Can. J. Chem. 89: 181–185 (2011) doi:10.1139/V10-114 Published by NRC Research Press

Can

. J. C

hem

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

CL

EM

SON

UN

IVE

RSI

TY

on

11/1

1/14

For

pers

onal

use

onl

y.

accumulation of antioxidants at the interface can beachieved. Although lipidic domains are the main sites oflipid peroxidation, association of antioxidants at the inter-face does not necessarily result in an improvement of theirantioxidant activity. This efficiency can be determined byinteractions between antioxidants and emulsifiers at the in-terface, which may be hydrophobic or due to hydrogenbonding.6,7 The hydrophobicity of the substrate will stronglyinfluence its distribution between the microphase and the ex-ternal medium and can also determine the intraparticle sub-strate distribution, affecting its reactivity toward thehydrophobic radical.6,8 In this work, we have evaluated thereactivity of methyl gallate (MG), propyl gallate (PG), andn-octyl gallate (OG) toward galvinoxyl radicals in homoge-neous solution and sodium dodecyl sulfate (SDS) micelles.Galvinoxyl, a stable phenoxyl radical, has a strong absorp-tion peak at 428 nm in ethanol. In the presence of phenoliccompounds, it is reduced by electron and (or) hydrogen-atom transfer. This last process has been related to the O–Hbond dissociation energy of the donor group.6,9

Results and discussionFigure 1A presents the depletion of galvinoxyl radicals

(3 mmol/L) in ethanol solution in the presence of 100 mmol/Lmethyl, propyl, or octyl gallate. This figure shows that thecourse of the reaction, and in particular the initial rate ofthe process, is barely modified by the length of the alkylchain. This result suggests an identical reactivity of the phe-nol moiety in ethanol for methyl, propyl, or octyl gallate.10

On the other hand, different rates are observed for the dif-ferent gallates in SDS solutions. Figure 1B represents, as anexample, the depletion of galvinoxyl radicals (3 mmol/L) inSDS (0.1 mol/L) in the presence of 100 mmol/L MG, PG, orOG. In particular, the data obtained in these systems showthat the rate of the process increases when the length of thegallate alkyl chain increases.

For a given gallate, at fixed gallate and galvinoxyl con-centrations, the rate of the process depends on the surfactantconcentration. Initial rates measured in these conditions areshown in Fig. 2. The data of this figure show that (i) overthe whole surfactant concentration range, the initial rate ofgalvinoxyl consumption follows the order OG > PG > MG(observed differences are larger at low surfactant concentra-tions); and (ii) the rate of the process decreases when thesurfactant concentration increases.

These results can be due to the following: (i) distributionof galvinoxyl between the external solvent and the micellarensemble; (ii) distribution of the gallates between the exter-nal solvent and the micelles; and (or) (iii) dependence of theintramicellar rate constant on the length of the gallate alkylchain.

To assess the localization and environment of galvinoxylradicals, electron paramagnetic resonance (EPR) spectrawere recorded in buffer and SDS solutions (0.025 and0.1 mol/L). The spectra are shown in Fig. 3.

Galvinoxyl EPR spectra in buffer showed a broad singletcharacteristic of solid microaggregates.11 On the other hand,well-resolved spectra were observed in micellar solutions,with a hyperfine structure that is very little dependent onthe surfactant concentration. This would indicate that, in the

tested SDS range, all the radicals are incorporated to themicelles and that their surroundings are similar, independentof the surfactant concentration.6

Fig. 1. Depletion of galvinoxyl radicals (3 mmol/L) by 100 mmol/Lmethyl gallate (*), propyl gallate (~), and octyl gallate (&) inethanol (A) and in 0.1 mol/L SDS in phosphate buffer, pH 7.0 (B).

Fig. 2. Initial rate of galvinoxyl consumption elicited by the addi-tion of 100 mmol/L methyl gallate (*), propyl gallate (~), and oc-tyl gallate (&) as a function of SDS concentration.

182 Can. J. Chem. Vol. 89, 2011

Published by NRC Research Press

Can

. J. C

hem

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

CL

EM

SON

UN

IVE

RSI

TY

on

11/1

1/14

For

pers

onal

use

onl

y.

Campos et al.10 have developed a data treatment to evalu-ate the antioxidant partition constant and the intramicellarsecond-order rate constant for the reaction of gallates and2,2-diphenyl-1-picrylhydrazyl (DPPH). The procedure as-sumes that the reaction takes place from the gallate in itsphenolic form (non-ionized). Nevertheless, under our exper-imental conditions (phosphate buffer, pH 7 in bulk solution)gallates are partially deprotonated (pKa = 8.03). The reactiv-ity of gallates and their ionized forms toward galvinoxylradicals can be different, as can their contribution to theoverall reaction rate due to differences in their partition con-stants.

The galvinoxyl consumption rate in micelles was stronglydependent on the pH of the phosphate media. The data givenin Fig. 4 show that, for MG, the reaction rate increases ~20times when the pH changes from 5 to 9. These resultsclearly indicate a much faster reaction of the ionized gallate(G–) than the protonated form (GH).

Measurements in SDS micelles at pH 7.0 showed a de-crease in rate when SDS concentration increases (Fig. 5).The following mechanism can explain the reaction rate ob-served in the presence of micelles:

½1� GH ¼ G� þ Hþ

½2� GHþ SDS ¼ GHmic

½3� G� þ SDS ¼ G�mic

½4� Galv�mic þ GHmic ! Products

½5� Galv�mic þ G�mic ! Products

where GH and G– represent the non-ionized and ionizedforms of gallates, respectively; Galv� is the galvinoxyl radi-cal and the subscript mic indicates the micellar pseudophase.

The galvinoxyl depletion rate (n), due to eqs. [4] and [5],can be written as

½6� v�Galv� ¼ ðkGHGHmic þ kG�G�micÞ=f

where f is the fraction of volume of micelles, defined as f =Vo[SDS]/1000, and V8 is the surfactant molar volume.

The previous reaction can be expressed in terms of theconcentration in aqueous phase if partition constants of thegallate (KGH) and the ionized form (KG� ) are incorporatedin eq. [6].

½7� v�Galv� ¼

1000

VoðkGHKGHGHþ kG�KG�G�Þ

From the data given in Fig. 4, it can be concluded that thecontribution of the kGHKGH factor is much smaller than thatof the ionized gallate (kG�KG�). The overall rate can then bewritten as

½8� Galv�½G��v ¼ aþ b½SDS�

where [G] represents the analytical gallate concentration,[G] = GH + G– + GHmic + G�mic, and

½9� a ¼ 1þ Ka

½Hþ�

� ��1000

VoKG�

Ka

½Hþ� kG�

Fig. 3. Electron paramagnetic resonance spectra of 20 mmol/L gavi-noxyl in phosphate buffer at pH 7 (A), 0.025 mol/L SDS (B), and0.1 mol/L SDS (C).

Fig. 4. Observed first-order rate constant of galvinoxyl consump-tion by 100 mmol/L methyl gallate in 0.1 mol/L SDS at differentpH values.

Bridi et al. 183

Published by NRC Research Press

Can

. J. C

hem

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

CL

EM

SON

UN

IVE

RSI

TY

on

11/1

1/14

For

pers

onal

use

onl

y.

½10� b ¼ KGH þ KG

Ka

½Hþ�

� ��1000

VoKG�

Ka

½Hþ� kG�

If the dissociation constant of the gallates (Ka) and thecharge of the micelles are considered, it can be concludedthat in our experimental conditions the concentration of theionized gallate in the aqueous solution and in the micelle ismuch lower than the analytical gallate concentration. Thisconsideration allows simplification of the values of a and bin this expression to

½11� a ¼ Vo½Hþ�=1000KG�KakG�

½12� b ¼ KGHVo½Hþ�=1000KG�KakG�

A plot of the left-hand side of eq. [8] against the free sur-factant concentration (Fig. 6) allows the evaluation of KGH(from the slope and intercept). It is interesting to note that

in spite of the fact that the reactive species is the deproto-nated gallate, the rate of the process is determined by thepartition of GH. This apparent anomaly is due to the factthat the partition of GH regulates the amount of free G– andthat this determines the amount of the reactive species asso-ciated to the micelles.

If V8 is taken as –250 mL/mol, the intercept of the plotallows determination of the product between the intramicel-lar rate constant of the ionized gallate and its partition con-stant (KG�kG�). These values are shown in Table 1, wherethe results obtained in homogeneous (ethanol) solution arealso included.

The data treatment employed to obtain kG�KG� values im-plies that KG� is independent of SDS concentration, in spiteof the change in surface potential that could be elicited bythe concomitant increase in free sodium concentration. Totest the pertinence of this assumption, we measured the rateof galvinoxyl consumption by MG, employing a fixed SDSconcentration (50 mmol/L) in the absence and presence of30 mmol/L NaCl. No significant differences were observed(data not shown), indicating that under the present experi-mental conditions, we can assume that the factor kG�KG�

can be considered to be nearly independent of the free so-dium concentration.

The data collected in Table 1 show that, as expected, thegallate association constant to SDS micelles increases withthe length of the alkyl chain. A plot of ln(KGH) against thenumber of methylene groups in the gallate alkyl chain ren-ders a contribution to the free energy of transfer (DDGo) ofnearly 1 kJ per methylene group. This value can be subjectto a large error given the uncertainty in the OG distributionconstant, but is similar to those reported in other systemscomprising ionic micelles.12,13 Furthermore, it is consider-ably larger than that obtained for the same gallates in TritonX-100 micelles.9 This difference can be related to a morehydrophobic environment sensed by the gallates’ alkylchains in SDS than in Triton X-100 micelles.

Fig. 5. (A) Depletion of galvinoxyl radicals by 100 mmol/L propylgallate at different concentrations of SDS: 0.1 mol/L (*),0.075 mol/L (~), and 0.025 mol/L (&). (B) Initial rate of galvi-noxyl consumption elicited by the addition of octyl gallate in0.1 mol/L (&) and 0.05 mol/L (*) SDS. Bar lines represent thestandard deviation of three independent measurements.

Fig. 6. Inverse initial rate of galvinoxyl consumption elicited by theaddition of 100 mmol/L methyl gallate (*), propyl gallate (~), andoctyl gallate (&) as a function of SDS concentration. Bar lines re-present the standard deviation of three independent measurements.The critical micelle concentration was taken as 8 mmol/L.

184 Can. J. Chem. Vol. 89, 2011

Published by NRC Research Press

Can

. J. C

hem

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

CL

EM

SON

UN

IVE

RSI

TY

on

11/1

1/14

For

pers

onal

use

onl

y.

The factor KG�kG� shows the same trend as the partitioncoefficient of the protonated gallate (KGH) with the length ofthe gallate alkyl chain (Table 1). In agreement with this, aplot of log(KG�kG�) versus the number of methylene groupsrenders a slope of 0.18, a value that is very close to that ob-tained for the change of KGH with the number of methylenegroups. If it is assumed that the length of the alkyl chain in-fluences KG� and KGH in similar ways, the similar slopesimply that kG� is nearly independent of the number of meth-ylene groups. These results would suggest a similar intrami-cellar location of the reactive moiety.7 Therefore, thedifferences observed (Fig. 1B) would be mainly a conse-quence of the different partition constants for the three de-protonated gallate alkyl esters.

A similar conclusion has been obtained regarding the re-action of gallates with DPPH in neutral Triton X-100 mi-celles.10 The slower rates observed in SDS micellescompared with the rates observed in ethanol (Fig. 1) can bemostly ascribed to the small G– fraction associated to theanionic micelles. This fraction can lead to an electron-transfer process at the micellar surface between the phenolategroup of the gallate and the micelle-associated galvinoxylradicals.

ConclusionsDifferent rates observed for the reaction of gallates with

different alkyl chain lengths with galvinoxyl radicals inSDS micelle solutions are mainly due to partitioning of theanionic gallates. In fact, the results indicate similar intrami-cellar rate constants for the three compounds considered, in-dicating that the intramicellar localization of the reactivegroup is independent of the length of the alkyl chain.

Experimental

MaterialsThestable free-radical galvinoxyl (2,6-di-tert-butyl-a-(3,5-

di-tert-butyl-4-oxo-2,5-cyclohexadiene-1-ylidene), anionicSDS (sodium dodecyl sulfate, 99%), and the gallates methylgallate (98%), propyl gallate, and n-octyl gallate (98%) wereobtained from Sigma-Aldrich. Stock solutions of the gallatesand galvinoxyl were prepared daily in analytical grade etha-

nol (Merck). The reactions carried out in micelles contained1% ethanol. Micellar solutions of SDS were prepared in10 mmol/L sodium phosphate buffer, pH 7.0.

InstrumentsUV–visible spectra were recorded using a UV–vis spec-

trophotometer (Hewlett Packard 8453; Palo Alto, California,USA). A stopped-flow apparatus from Applied Photophysicswas used for kinetic measurements. The rate of the reactionwas followed by registering the absorbance of galvinoxyl at428 nm. EPR spectra were obtained on a Bruker EMX 1572spectrometer that operates in the X-band (9.2–9.9 GHz),equipped with a thermal cavity at 25 8C.

AcknowledgementsRaquel Bridi gratefully acknowledges the Universidade

Federal do Rio Grande do Sul for supporting a postdoctoralstay at the Universidad de Santiago de Chile. Financial sup-port from FONDECYT 1085177 is also acknowledged.

References(1) Halliwell, B.; Gutteridge, J. Free Radicals in Biology and

Medicine; Oxford University Press Inc.: New York, 2007;Chapter 9.

(2) Rice-Evans, C. A.; Miller, N. J.; Bolwell, P. G.; Bramley, P.M.; Pridham, J. B. Free Radic. Res. 1995, 22 (4), 375.doi:10.3109/10715769509145649. PMID:7633567.

(3) Boudet, A.-M. Phytochemistry 2007, 68 (22–24), 2722.(4) Barclay, L. R.; Vinqvist, M. R. Free Radic. Biol. Med. 1994,

16 (6), 779. doi:10.1016/0891-5849(94)90193-7. PMID:8070681.

(5) Kratz, J. M.; Andrighetti-Frohner, C. R.; Leal, P. C.; Nunes,R. J.; Yunes, R. A.; Trybala, E.; Bergstrom, T.; Barardi, C.R. M.; Simoes, C. M. O. Biol. Pharm. Bull. 2008, 31 (5),903. doi:10.1248/bpb.31.903. PMID:18451516.

(6) Heins, A.; McPhail, D. B.; Sokolowski, T.; Stockmann, H.;Schwarz, K. Lipids 2007, 42 (6), 573. doi:10.1007/s11745-007-3052-6. PMID:17473943.

(7) Stockmann, H.; Schwarz, K.; Huynh-Ba, T. J. Am. OilChem. Soc. 2000, 77 (5), 535. doi:10.1007/s11746-000-0085-6.

(8) Abuin, E.; Lissi, E.; Ortiz, P.; Henriquez, C. Bol. Soc. Chil.Quim. 2002, 47, 145.

(9) Shi, H.; Niki, E. Lipids 1998, 33 (4), 365. doi:10.1007/s11745-998-0216-8. PMID:9590623.

(10) Campos, A. M.; Ponce, E.; Lissi, E. A. J. Phys. Org. Chem.2009, 22 (12), 1208. doi:10.1002/poc.1580.

(11) Stasko, A.; Brezova, V.; Biskupic, S.; Misık, V. Free Radic.Res. 2007, 41 (4), 379. PMID:17454120.

(12) Sepulveda, L.; Lissi, E.; Quina, F. Adv. Colloid InterfaceSci. 1986, 25 (1), 1. doi:10.1016/0001-8686(86)80001-X.PMID:3333129.

(13) Ulloa, Y.; Rubio, M. A.; Lissi, E. A.; Aspee, A. Bol. Soc.Chil. Quim. 1994, 39, 129.

Table 1. Experimentally determined KGH and KG�kG� values inSDS micellar solutions.

KGH

(L mol–1)aKG�kG�

(L2 mol–2 s–1)bkEtOH

(L mol–1 s–1)Methyl gallate 8.7 550 4900Propyl gallate 41.7 2400 4600Octyl gallate 157 11000 6500

aValues calculated in terms of molar surfactant concentrations.bValues assuming V8= 250 mL/mol.

Bridi et al. 185

Published by NRC Research Press

Can

. J. C

hem

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

CL

EM

SON

UN

IVE

RSI

TY

on

11/1

1/14

For

pers

onal

use

onl

y.