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Investigation of antioxidant activity of Quercetin (2-(3, 4-dihydroxyphenyl)- 3,5,7-trihydroxy-4H-chromen-4-one) in aqueous micellar media Suraya Jabeen, Oyais Ahmad Chat, Ghulam Mohammad Rather , Aijaz Ahmad Dar Department of Chemistry, University of Kashmir, Hazratbal, Srinagar-190006, J&K, India abstract article info Article history: Received 17 September 2012 Accepted 16 December 2012 Keywords: Quercetin Radical scavenging activity Surfactants Hydroxyl radical The interaction of the antioxidant Quercetin with the hydroxyl radical (generated by Fenton's reaction) in pres- ence of cationic (DDEAB, DTPB), nonionic (Brij30), anionic (SDBS, SDS) and their mixed binary and ternary sur- factant systems (DDEAB-Brij30, DTPB-Brij30, SDBS-Brij30, SDS-Brij30, DDEAB-DTPB-Brij30, SDBS-SDS-Brij30) was followed by spectrophotometric and tensiometric methods to evaluate oxidation kinetics and hydroxyl radical scavenging activity (RSA) of Quercetin. The analysis of the data was done using the rst order kinetic model. Coefcient of Determination (R 2 ) was used as a statistical measure for comparison of the experimental values with the model simulated values. The rate of oxidation of Quercetin was found to depend on nature and concentration of the surfactant used. The rate constant of oxidation reaction (k) observed in different micel- lar systems with varying surfactant concentration exhibits the same trend as that of the RSA. Both k and RSA values of Quercetin were higher within ionic micelles than in nonionic micelles. In mixed binary micelles the values were intermediate between those for single surfactant systems except in DTPB-Brij30 where the values were higher. However, these values exhibited enhancement in mixed ternary cationiccationicnonionic surfac- tant systems when compared with anionicanionicnonionic mixed systems. The results are explained on the basis of compartmentalization and orientation of Quercetin in micellar nanostructures. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction There is an increasing concern regarding the role of reactive oxygen species (ROS) in tissue damage, aging, liver injury, cancer, cardiovascu- lar diseases etc. (Arora, Nair, & Strasburg, 1998; Formica & Regelson, 1995; Gaziano, 2000). Among the ROS, OH exhibits the strongest oxida- tive activity and is the most toxic radical known, as it can nonspecically oxidize all classes of biological macromolecules at virtually diffusion- limited rates (Imlay & Linn, 1988). In addition, lipid oxidation caused by free radicals is one of the most important processes that can cause deterioration in lipid bearing foods, which leads to the development of rancidity/off-avors and hence decrease in the shelf-life and nutritive value of the food products (Akoh & Min, 2008; Min & Boff, 2002). Among large number of natural antioxidants, avonoids have gained great interest within the pharmaceutical and food industries for being strong antioxidants (Brenna & Pagliarini, 2001; Papadopoulou, Green, & Frazier, 2005) in addition to having multitude of biological, pharma- cological and medicinal properties including anti-inammatory, anti- allergic, antiviral, antithrombotic, antimutagenic, antineoplastic and cytoprotective effects (Formica & Regelson, 1995). The activity and mechanism of complex natural antioxidants are affected by many fac- tors like the type of antioxidant and solvent system used, the conditions of oxidation and the partitioning properties of the antioxidants among different phases and at their interfaces in multiphase systems (Frankel, Huang, Kanner, & German, 1994; Frankel & Meyer, 2000). The curiosity to unravel the behavior of dietary antioxidants in microheterogeneous environments found in complex food and biologi- cal systems has made scientists to use micelles, microemulsions and other colloidal disperse systems as simple biomimetic models (Carvalho & Cabral, 2000; Cuvelier, Bondet, & Bercet, 2000; Gordon, Paiva-Martins, & Almeida, 2001; Granato, Katayama, & de Castro, 2011). Quercetin (3, 3,4, 5, 7-pentahydroxyavone, Scheme 1), the most common avonoid present in nature possesses strong radical scaveng- ing property sensitive to environmental changes like that in solvent po- larity, pH, use of micellar media etc. (Aliaga, Razende, & Arenas, 2009; Guo & Wei, 2008; Heins, McPhail, Sokolowski, Stockmann, & Schwarz, 2007). The use of such avonoids has been limited due to their poor water solubility and instability under conditions encountered during food/pharmaceutical products processing (temperature, light, pH), in the gut (pH, enzymes, presence of other nutrients) or during storage (light, oxygen).These factors limit the advantages and potential health benets of these compounds in functional food or pharmaceutical prod- ucts (Pool et al., 2012). Such limitations can be overcome by use of surfactant nano-cavities, known to improve bioavailability and resist degradation of pharmacologically active molecules (Martin, 1993). Therefore, surfactant-antioxidant interactions and the consequent in- uence on the antioxidant activity has been the subject of a number of studies (Liu & Guo, 2005a, 2005b, 2006a, 2006b, 2008; Heins et al., Food Research International 51 (2013) 294302 Corresponding authors. Tel.: +91 194 2424900; fax: +91 194 2421357. E-mail addresses: [email protected] (G.M. Rather), [email protected] (A.A. Dar). 0963-9969/$ see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2012.12.022 Contents lists available at SciVerse ScienceDirect Food Research International journal homepage: www.elsevier.com/locate/foodres

Investigation of antioxidant activity of Quercetin (2-(3, 4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one) in aqueous micellar media

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Food Research International 51 (2013) 294–302

Contents lists available at SciVerse ScienceDirect

Food Research International

j ourna l homepage: www.e lsev ie r .com/ locate / foodres

Investigation of antioxidant activity of Quercetin (2-(3, 4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one) in aqueous micellar media

Suraya Jabeen, Oyais Ahmad Chat, Ghulam Mohammad Rather ⁎, Aijaz Ahmad Dar ⁎Department of Chemistry, University of Kashmir, Hazratbal, Srinagar-190006, J&K, India

⁎ Corresponding authors. Tel.: +91 194 2424900; faxE-mail addresses: [email protected] (G.M.

(A.A. Dar).

0963-9969/$ – see front matter © 2012 Elsevier Ltd. Allhttp://dx.doi.org/10.1016/j.foodres.2012.12.022

a b s t r a c t

a r t i c l e i n f o

Article history:Received 17 September 2012Accepted 16 December 2012

Keywords:QuercetinRadical scavenging activitySurfactantsHydroxyl radical

The interaction of the antioxidant Quercetin with the hydroxyl radical (generated by Fenton's reaction) in pres-ence of cationic (DDEAB, DTPB), nonionic (Brij30), anionic (SDBS, SDS) and their mixed binary and ternary sur-factant systems (DDEAB-Brij30, DTPB-Brij30, SDBS-Brij30, SDS-Brij30, DDEAB-DTPB-Brij30, SDBS-SDS-Brij30)was followed by spectrophotometric and tensiometric methods to evaluate oxidation kinetics and hydroxylradical scavenging activity (RSA) of Quercetin. The analysis of the data was done using the first order kineticmodel. Coefficient of Determination (R2) was used as a statistical measure for comparison of the experimentalvalues with the model simulated values. The rate of oxidation of Quercetin was found to depend on natureand concentration of the surfactant used. The rate constant of oxidation reaction (k) observed in different micel-lar systems with varying surfactant concentration exhibits the same trend as that of the RSA. Both k and RSAvalues of Quercetin were higher within ionic micelles than in nonionic micelles. In mixed binary micelles thevalues were intermediate between those for single surfactant systems except in DTPB-Brij30 where the valueswere higher. However, these values exhibited enhancement inmixed ternary cationic–cationic–nonionic surfac-tant systems when compared with anionic–anionic–nonionic mixed systems. The results are explained on thebasis of compartmentalization and orientation of Quercetin in micellar nanostructures.

© 2012 Elsevier Ltd. All rights reserved.

1. Introduction

There is an increasing concern regarding the role of reactive oxygenspecies (ROS) in tissue damage, aging, liver injury, cancer, cardiovascu-lar diseases etc. (Arora, Nair, & Strasburg, 1998; Formica & Regelson,1995; Gaziano, 2000). Among theROS, •OHexhibits the strongest oxida-tive activity and is themost toxic radical known, as it can nonspecificallyoxidize all classes of biological macromolecules at virtually diffusion-limited rates (Imlay & Linn, 1988). In addition, lipid oxidation causedby free radicals is one of the most important processes that can causedeterioration in lipid bearing foods, which leads to the development ofrancidity/off-flavors and hence decrease in the shelf-life and nutritivevalue of the food products (Akoh & Min, 2008; Min & Boff, 2002).Among large number of natural antioxidants, flavonoids have gainedgreat interest within the pharmaceutical and food industries for beingstrong antioxidants (Brenna & Pagliarini, 2001; Papadopoulou, Green,& Frazier, 2005) in addition to having multitude of biological, pharma-cological and medicinal properties including anti-inflammatory, anti-allergic, antiviral, antithrombotic, antimutagenic, antineoplastic andcytoprotective effects (Formica & Regelson, 1995). The activity andmechanism of complex natural antioxidants are affected by many fac-tors like the type of antioxidant and solvent system used, the conditions

: +91 194 2421357.Rather), [email protected]

rights reserved.

of oxidation and the partitioning properties of the antioxidants amongdifferent phases and at their interfaces in multiphase systems(Frankel, Huang, Kanner, & German, 1994; Frankel & Meyer, 2000).The curiosity to unravel the behavior of dietary antioxidants inmicroheterogeneous environments found in complex food and biologi-cal systems has made scientists to use micelles, microemulsions andother colloidal disperse systems as simple biomimetic models(Carvalho & Cabral, 2000; Cuvelier, Bondet, & Bercet, 2000; Gordon,Paiva-Martins, & Almeida, 2001; Granato, Katayama, & de Castro, 2011).

Quercetin (3, 3′, 4′, 5, 7-pentahydroxyflavone, Scheme 1), the mostcommon flavonoid present in nature possesses strong radical scaveng-ing property sensitive to environmental changes like that in solvent po-larity, pH, use of micellar media etc. (Aliaga, Razende, & Arenas, 2009;Guo & Wei, 2008; Heins, McPhail, Sokolowski, Stockmann, & Schwarz,2007). The use of such flavonoids has been limited due to their poorwater solubility and instability under conditions encountered duringfood/pharmaceutical products processing (temperature, light, pH), inthe gut (pH, enzymes, presence of other nutrients) or during storage(light, oxygen).These factors limit the advantages and potential healthbenefits of these compounds in functional food or pharmaceutical prod-ucts (Pool et al., 2012). Such limitations can be overcome by use ofsurfactant nano-cavities, known to improve bioavailability and resistdegradation of pharmacologically active molecules (Martin, 1993).Therefore, surfactant-antioxidant interactions and the consequent in-fluence on the antioxidant activity has been the subject of a numberof studies (Liu & Guo, 2005a, 2005b, 2006a, 2006b, 2008; Heins et al.,

Scheme 1. Structure of surfactants and Quercetin used in this study.

295S. Jabeen et al. / Food Research International 51 (2013) 294–302

2007; Guo &Wei, 2008; Xi & Guo, 2009; Chat, Najar, Mir, Rather, & Dar,2011; Noipa, Srijaranai, Tuntulani, & Ngeontae, 2011). Most of thesestudies are focused on a limited number of surfactants and do nottake into account the effects arising due to changes in nature and sizeof the head groups and hydrophobicity of surfactants.

Since surfactant mixtures have natural existence in different foodformulations/biological systems, due to their efficient solubilization,suspension, dispersion, and transportation capabilities (Hill, 1993;Rosen, 1989), studying the interaction of dietary flavonoid, Quercetin,with the mixed micelles of various surfactants and hence their conse-quent influence on its antioxidant properties deserves attention. Thereports regarding the effect of binary and ternary mixed micelles onthe antioxidant activity of bioactive molecules are scarce and henceone of the main objectives of the present study.

Specifically the presentwork focuses on the effect of cationic, anion-ic and nonionic surfactants and their binary and ternarymixtures on theantioxidant capacity of Quercetin toward hydroxyl radicals generatedthrough fenton reaction in aqueous solution. The main aim was tofind out the influence of compartmentalization of Quercetin withinthe micelles on its radical scavenging activity toward •OH radicalswhich may or may not accumulate at the micelle-water interface,depending on nature of the micelles. Moreover, influence of mixed mi-celles of various surfactants on RSA of Quercetin has been investigatedfor revealing the factors that affect this activity in more naturalmicroheterogeneous media. This report is expected to have relevancein understanding the antioxidant mechanism of Quercetin in real com-plex foods and biological systems.

2. Experimental section

2.1. Materials

The nonionic amphiphile polyoxyethylene (4) mono-n-dodecylether (Brij-30), cationic amphiphiles dodecyldimethylethylammoniumbromide (DDEAB) and dodecyltriphenylphosphoniumbromide (DTPB),anionic amphiphiles sodium dodecylsulfate (SDS) and sodiumdodecylbenzenesulphonate (SDBS), the antioxidant Quercetin dihydrate(>98%) were all Aldrich products, and were used as received. Methanol(Merck) was used after distillation. The purity of the surfactants wasfurther ensured by the absence of minimum in surface tension vs. thelogarithm of surfactant concentration plots. FeSO4.7H2O and H2O2 wereof analytical grade. The structures of the surfactants, and antioxidantused are presented in Scheme 1. Surfactant solutions were prepared intriple distilled water.

2.2. Methods

2.2.1. Determination of cmcThe cmc values of all surfactant solutions were determined from the

plot of surface tension (γ) vs. logarithm of surfactant concentration(logCt) as shown in Fig. 1. Surface tension measurements were madeby the platinum ring detachment method with a Krüss-9 (Germany)tensiometer equipped with a thermostable vessel holder (Dar, Rather,& Das, 2007). Surfactant concentration was varied by successive addi-tion of small installments of surfactant stock solution using a Hamiltonmicro syringe. Measurements were made after thorough mixing andtemperature equilibration at 25 °C (±0.1 °C). The accuracy ofmeasure-ments was within ±0.1 dyne cm−1 and the readings were taken intriplicate to ensure reproducibility. The cmc values of each surfactantsystem and other calculated parameters are reported as mean of thethree parallel experiments in Table 1 along with the standard error asa foot note.

2.2.2. Evaluation of hydroxyl-radical (•OH) scavenging activityHydroxyl radical scavenging potential of the antioxidant Quercetin

in each surfactant solution was determined by first dissolving the

antioxidant in surfactant solution followed by addition of Fenton'sreagent to the mixture after thorough shaking by hand at 25 °C. Thedecrease in absorbance at the absorption wavelength of Quercetin(370 nm) after 60s intervals was monitored with a Schimadzu UV-1650PC spectrophotometer (absorbance limit; ±0.001). The decay in absor-bance, an index of the oxidation of •OH radicals, wasmeasured for amax-imumof 30 min. In total 3 ml of solution, the concentrations of Quercetin,FeSO4 and H2O2 were fixed at 0.05 mM, 0.0125 mM and 0.125 mM re-spectively. Three different surfactant concentrations in the pre-micelle,micelle and post-micelle range were used for each of the single, binaryand ternary surfactant system. All the experiments were performed intriplicate. The radical scavenging activity (RSA: antioxidant activity) wascalculated using the following equation:

RSA ¼ 100� 1−A=A0� �

ð1Þ

where A is the absorbance of sample after 10 min of starting of reactionwhile A0 is the absorbance at zero time. The RSA is presented asmean±SE in the Table 2. Absorption spectra of Quercetin at differenttimes during its reaction with hydroxyl radical in different single surfac-tant systems are presented in Fig. 2.

3. Results and discussion

3.1. CMC and surfactant–surfactant interactions

The cmc values of selected single, mixed binary and ternary surfac-tant systems are presented in Table 1 along with the ideal cmc values,cmcideal, of the equimolar binary as well as ternary mixtures based onthe Clint equation (Eq. (2))(Clint, 1975). All the observed cmc valueswere found to be lower than ideal values, indicating synergistic be-havior for mixed micelle formation.

-2.530

35

40

50

45

55

60

65

70 Brij30SDSSDBSDDEABDTPB

γ [m

N m

-1]

log[Surfactant]/mM

30

35

40

45

50

55

60

65

70 Brij30+SDSBrij30+SDBSSDS+SDBSBrij30+DDEABBrij30+DTPBDDEAB+DTPB

log[Surfactant]/mM

-2.030

35

40

45

50

55

60

log[Surfactant]/mM

Brij30+SDS+SDBSBrij30+DDEAB+DTPB

γ [m

N m

-1]

γ [m

N m

-1]

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5

-2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5

-1.5 -1.0 -0.5

Fig. 1. Plots of surface tension versus logarithm of surfactant concentration for different

296 S. Jabeen et al. / Food Research International 51 (2013) 294–302

1cmcideal

¼ α1

cmc1þ α2

cmc2þ α3

cmc3þ :::::: ð2Þ

where cmci and αi are the experimental critical micellization concen-tration and mole fraction of the ith component in the bulk surfactantmixture.

The estimate of the negative deviation and hence non-ideality ofmixed binary surfactant systems has been obtained using Rubingh's

single and mixed surfactant systems.

(1979) model. This model, based on the regular solution theory andapplicable for nonideal mixing, gives the estimate of deviation of ex-perimental cmc values from cmcideal. Analysis of the cmc as a functionof net mole fraction α1 of component 1 in the mixed surfactant sys-tems in terms of micellar composition (X1

M ) at the cmc has beenmade in the light of Rubingh's equation (Eqs. (3)–(5a), (5b)):

XM1

� �2ln cmc12α1=cmc1X

M1

� �

1−XM1

� �2 ln cmc12 1−α1ð Þ=cmc2 1−XM1

� �� � ¼ 1 ð3Þ

where cmc1, cmc2, cmc12 denote the cmc values of the surfactants 1, 2and mixed system respectively. The parameter, β, of mixed micelleformation given by

β ¼ln cmc12α1=cmc1X

M1

� �

1−XM1

� �2 ¼ln cmc12α2=cmc2X

M2

� �

1−XM2

� �2 ð4Þ

is an indicator of the degree of interaction between two surfactants inmixed micelle and accounts for deviation from ideality. A negativevalue of β implies attractive interactions between two surfactants inthe mixed micelle. The activity coefficients, fi, of individual surfac-tants within the mixed micelles are related to the interaction param-eter through the equations.

f 1 ¼ exp β 1−XM1

� �2�

ð5aÞ

f 2 ¼ exp βXM2

1

� ð5bÞ

β values along with the micellar mole fraction, XiM, and activity co-

efficient, fi, of the ith surfactant within mixed micelles calculatedthrough Rubingh equations are also presented in Table 1. It is wellknown (Zhou & Rosen, 2003; Zhou & Zhu, 2004) that in ionic–nonion-ic mixed surfactant systems the electrostatic self-repulsion of ionicsand weak steric self-repulsion of non-ionics (depending on theheadgroup size) before mixing are weakened by dilution effectsafter mixing and also the electrostatic self-repulsion of ionic surfac-tants is replaced by ion–dipole interactions.

Among anionic–nonionic mixed surfactant systems, lower magni-tude of β of the SDBS-Brij30 system over SDS-Brij30 system may bedue to the presence of the benzene ring in SDBS, contributing to stericrepulsion thereby reducing stability of mixed micelles. Similarlyamong cationic–nonionic surfactant systems DDEAB-Brij30 andDTPB-Brij30 bulkier phenylated head group of DTPB, reduces themagnitude of β due to steric self-repulsion toward inter-headgroupinteractions. A small (negative) value of β and small deviation of fifrom unity in the case of anionic–anionic (SDBS-SDS) binary surfactantsystem indicates it's almost ideal behavior. However, the cationic–cationic DTPB-DDEAB mixed surfactant system exhibits significantsynergistic interaction as indicated by the magnitude and sign of β.It could be related to the presence of three phenyl rings in the headgroup of DTPB posing appreciable steric self-repulsion in its pure mi-celles which gets diluted when mixed micelles are formed. Althoughboth the surfactants in this system are similarly charged, such a nega-tive value of β is in tune with the results reported in the literature(Haque, Das, Rakshit, & Moulik, 1996; Janquera & Aicart, 2002; Ray,Chakraborty, Ghosh, Moulik, & Palepu, 2005) for other cationic–cationicsurfactant mixtures.

Holland and Rubingh (1983) model for multicomponent nonidealmixed micelle based on pseudo-phase separation approach has beensuccessfully applied to ternary surfactant systems (Holland, 1992;Holland & Rubingh, 1983) for evaluation of micellar composition, activ-ity coefficients, and cmc values. According to this model, the activitycoefficients fi, fj, … of micelle forming surfactant species i, j, …in an

Table 1Experimental and literature criticalmicelle concentration values (cmcexp and cmclit) of single surfactant systems, experimental and ideal cmc values (cmcexp and cmcideal) calculatedwithclint equation, along with the miceller mole fraction (xiM), interaction parameter (β) and activity coefficients (fi) calculated with Rubingh and Rubingh Holland methods for equimolarbinary and ternary surfactant systems at 25 °C. Also presented are predicted cmc values as per Rubingh Holland methods (cmcRH) for ternary surfactant systems.

System cmcexp(cmclit)/(mmol dm−3)

System cmcexp(cmcideal)/(mmol dm−3)

β XIM/X2

M f1/f2

Single surfactant systems Binary surfactant system

Brij30 0.039(0.0351)a Brij30-DDEAB 0.051(0.078 −6.88 0.85/0.15 0.80/0.10DDEAB 14.02(14)b Brij30-DTPB 0.057(0.076) −3.30 0.81/0.19 0.89/0.11DTPB 1.37(2)c DDEAB-DTPB 2.17(2.5) −1.22 0.82/0.18 0.96/0.44SDS 7.4(8.1)d Brij30-SDS 0.057(0.077) −5.34 0.84/0.16 0.87/0.02SDBS 2.02(2.2)e Brij30-SDBS 0.07(0.076) −2.02 0.91/0.09 0.98/0.19

SDS-SDBS 3.04(3.184) −0.25 0.24/0.76 0.87/0.99

Ternary surfactant systems

System cmcexp(cmcideal)/(mmol dm−3)

cmcRH X1M/X2

M/X3M f1/f2/f3

Brij30-DDEAB-DTPB 0.101(0.115) 0.069 0.77/0.14/0.09 0.76/0.01/0.18Brij30-SDS-SDBS 0.088(0.115) 0.082 0.82/0.15/0.03 0.85/0.02/0.37

Error limits of cmc, X1, β and f are ±7%, ±0.02, ±0.02 and ±0.02 respectively.aRef. (Kim, Park, & Kim, 2001),bRef. (Janquera & Aicart, 2002), cRef. (Ghosh & Verma, 2009), dRef. (Akbas & Taliha, 2003),eRef. (Segota, Heimer, & Tezak, 2006).

297S. Jabeen et al. / Food Research International 51 (2013) 294–302

n-component mixture are represented, on a general basis, by theequation.

lnf i ¼Xni ¼ 1j≠1ð Þ

βijXM2

j þXn

j ¼ 1ð Þi≠j≠kð Þ

Xj−1

k¼1

βij þ βik−βjk

� �XMj X

Mk ð6Þ

where βij represents the net (pair wise) interaction between compo-nents i and j and XjM is the mole fraction of the j-th component in themicelles. At cmc, the relation

XMi ¼ αicmcjf jX

Mj

αjcmcif ið7Þ

holds, where terms cmci and cmcj are cmc values of the i- and j-th com-ponents in their pure state, respectively. The interaction parameter, βij

can be obtained independently from binarymixtures using the Rubinghmethod. The activity coefficients for a three component system, i.e., f1,f2, and f3 at mixed cmc can be calculated from the above equations byusing the method of successive substitutions subject to the constraintthat the sum of XiM values equals unity. The values of fi so obtainedcan then be used to find the mixed micellar cmc, cmcRH, of ternary sys-tems by the equation:

1cmcRH

¼X3i¼1

αi

f icmcið8Þ

These calculations were done using solver in MS Excel and the re-sults obtained are presented in Table 1.

The mole fractions of individual amphiphiles in the mixed micellesXi are different from stoichiometric composition αi: Xionic values beingmuch lower than αionic values, but Xnonionic values are only fairly higherthan αnonionic values. The activity coefficients of ionics are very low,those for nonionics being close to unity. Among ternary systems, theBrij30-SDS-SDBS system shows a fair agreement between cmcexp andcmcRH while a deviation was observed for Brij30-DDEAB-DTPB systemwhich could be the manifestation of high steric repulsion related tothe presence of three phenyl goups in the DTPB. However, both cmcexpand cmcRH are lower than the ideal cmc, indicating synergistic nonidealnature of mixed ternary micellar systems. Close agreement betweencmcRH and cmcexp in case of Brij30-SDBS-SDS indicates fair applicabilityof the RH method for such system.

3.2. Quercetin-hydroxyl radical reaction in micellar media

Quercetin (3, 3′, 4′, 5, 7-pentahydroxyflavone, Scheme 1), a modelflavonoid is abundant in plants and foods and displays the structuralrequirements (C-2-C-3 double bond, ortho-dihydroxy substitution onring B and the presence of a 4-oxo in C ring) favorable to strong anti-oxidant activity (Spencer, Kuhnle, Williams, & Rice-Evans, 2003).

Quercetin is known to exist in the anionic state with one or twocharges in aqueous solution, because the phenolic OH groups at the3, 7 positions of the molecule can dissociate resulting in a mixtureof neutral and anionic species (Agrawal & Schneider, 1983; Kitson,Kitson, & Moore, 2001). 3′,4′ hydroxyls on the B ring are the most ac-tive antioxidant parts in the Quercetin molecule having ability toscavenge hydroxyl radicals. With the hydroxyl radicals cleared, theQuercetin molecule itself degrades and its absorption peak intensitydecreases accordingly (Fig. 2). Therefore, we studied ability of Quer-cetin in different micellar media to scavenge hydroxyl radicals gener-ated by Fenton's reaction by monitoring changes in its characteristicabsorption spectrum. After mixing with Fenton's reagent, the absorp-tion peak of Quercetin drops rapidly within the first 10 min thenslows down and finally levels off. The influence of surfactant concen-trations on the degradation of Quercetin upon attack by hydroxyl rad-icals is shown in Fig. 3.

The pseudo first order rate constant of Quercetin degradation indifferent surfactant concentrations were determined by fitting exper-imental data (initial 5 min) to the following equation.

A ¼ A0e−kt ð9Þ

where A0 is the absorbance at time t=0, A is the absorbance at time t,and k is the degradation rate constant. A few prototype plots showingvariation of A/A0 vs t for single surfactant systems are shown in Fig. 3.The values of k (Table 2) were determined by carrying out a linear re-gression analysis of − log (A/A0) vs t as shown in Fig. 4. To test thecorrelation between the experimental and simulated values as perthe Eq. (9), analysis of variance (ANOVA) for regression was done.The Coefficient of Determination (R2) and the F-values wereemployed to check how well the regression model fits to our data.The values of R2 greater than 0.96 and higher F-values with P-valueless than 0.05 for all the surfactant systems at all concentrations(Table 2) indicate that the fitted equation significantly representsour experimental data. The use of excess Fenton's reagent comparedto Quercetin justifies the use of Eq. (9). The degradation rate constant

Table 2Radical scavenging activity (RSA) and kinetic parameters of Quercetin degradation insingle, binary and ternary surfactant systems.

System [Surfactant]/mM

RSA(%) k(min−1)(×10−2)

R2 F-value P-value

SingleBrij30 0 18.3 1.7 0.98 237 0.0001

0.01 10.1 1.2 0.93 76 0.00090.039 8.6 1.0 0.93 71 0.0010.1 5.2 0.6 0.92 69 0.0010 18.3 1.7 0.98 237 0.0001

SDS 2 14.8 1.8 0.99 317 b0.00017.4 11.3 1.0 0.92 50 0.002

18 8.7 0.9 0.96 138 0.00020 18.3 1.7 0.98 237 0.0001

SDBS 0.1 16.1 0.9 0.99 11354 b0.00012 58.4 6.6 0.99 1926 b0.00016 72.4 8.8 0.99 786 b0.00010 18.3 1.7 0.98 237 0.0001

DDEAB 5 19.0 2.2 0.98 190 0.000214 24.2 2.0 0.99 580 b0.000130 39.6 4.0 0.99 2869 b0.00010 18.3 1.7 0.98 237 0.0001

DTPB 0.5 17.3 1.5 0.99 2318 b0.00011.37 23.8 2.2 0.99 2172 b0.00015 40.6 3.5 0.99 2597 b0.0001

BinaryBrij30-SDS 0 18.3 1.7 0.98 237 0.0001

0.01 19.2 2.0 0.96 124 0.00030.057 9.9 0.9 0.97 173 0.00010.2 7.1 0.6 0.98 273 b0.00010 18.3 1.7 0.98 237 0.0001

Brij30-SDBS 0.01 19.7 2.1 0.98 252 b0.00010.07 8.9 0.7 0.95 94 0.00060.2 6.2 0.3 0.95 106 0.00040 18.3 1.7 0.98 237 0.0001

Brij30-DDEAB 0.01 17.2 1.6 0.98 130 0.00030.051 10.0 1.0 0.95 111 0.00040.2 9.5 0.8 0.98 239 0.00010 18.3 1.7 0.98 237 0.0001

Brij30-DTPB 0.01 18.2 1.8 0.94 83 0.00070.057 25.2 2.6 0.98 281 b0.00010.2 33.8 3.7 0.98 273 b0.0001

TernarySDS-SDBS-Brij30

0 18.3 1.7 0.98 237 0.00010.01 15.7 1.7 0.95 106 0.00040.088 9.6 1.2 0.92 54 0.0010.25 8.4 1.0 0.92 63 0.0010 18.3 1.7 0.98 237 0.0001

DTPB-DDEAB-Brij30 0.01 31.6 3.0 0.97 159 0.00020.101 43.9 4.0 0.99 363 b0.00010.3 44.5 4.5 0.98 526 b0.0001

RSA = radical scavenging activity, R2 the Coefficient of Determination and F-values arestatistical measure of goodness of linear fitting to the experimental data. Error in thecalculation of RSA and k was ±0.5 & ±0.002 respectively.

0.2

0.3

0.4

0.5 (a) [Brij30]=0.039mM

t=20min

t=0

Ab

sorb

ance

Wavelength(nm)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8 (b)t= 0 min

t=27 min

[SDS]=7.40 mM

Ab

sorb

ance

Wavelength(nm)

3000.1

320 340 360 380 400 420 440 460 480 500

320 340 360 380 400 420 440

320 340 360 380 400 420 4400.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

t=28min

t=0[DDEAB]=14mM(c)

Ab

sorb

ance

Wavelength(nm)

Fig. 2.Absorption curves ofQuercetin showing its degradationwith timeupon reactionwithOH radicals, in presence of: (a) 0.039 mM Brij30, (b) 7.40 mM SDS, (c) 14.0 mM DDEAB.

298 S. Jabeen et al. / Food Research International 51 (2013) 294–302

(k) (Table 2) of Quercetin showed a similar trend as that of the hy-droxyl radical scavenging activity (RSA) in different surfactant sys-tems as a function of varying surfactant concentrations. The resultsobtained are presented in Table 2 and plotted in Fig. 5 for differentsingle surfactant systems as a function of surfactant concentration.Both RSA and k decrease with increase in concentration of Brij30and SDS, below as well as above their cmcs, indicating that the reac-tion between Quercetin and hydroxyl radicals is partly sloweddown due to the solubilization of Quercetin within these micelles.The suggested (Liu & Guo, 2006a) possible orientation of Quercetinmolecule within SDS micelles is such that the B-ring, containing theelectroactive hydroxyl groups, is embedded within the palisadelayer of micelle while rings A and C, carrying negative charge, lieoutside the micelle to avoid unfavorable electrostatic repulsionswith similarly charged head groups. In other studies (Heins et al.,

2007; Stockmann, Schwarz, & Huynh-Ba, 2000) it has been establishedthrough spectroscopic and electrochemical studies that Rutin (glyco-side derivative of Quercetin) molecules are located in the palisadelayer of polyoxyethylene surfactant micelles involving hydrophobicand hydrogen bonding interactions thereby solubilizing it preferablywith its B-ring pointing towardmicellar core. These studies also showedthat hydrogen abstraction kinetics of Rutin by the radical is inhibiteddue to strong hydrogen bonding of electroactive hydroxyls with theOE units of nonionic surfactants. Liu & Guo, 2006a have demonstratedthat Quercetin interacts with cationic surfactants via rings A and C due

0.8

0.9

1.00 mM0.01 0.039 0.1

A/A

0

Time (min.)

(a) Brij30

00.5

0.6

0.7

0.8

0.9

1.0 0 mM51430

A/A

0

Time (min.)

(c) DDEAB

0.75

0.80

0.85

0.90

0.95

1.000 mM2.07.418

A/A

0

Time (min.)

(b) SDS

5 10 15 20

0 5 10 15 20

0 5 10 15 20

Fig. 3. Degradation of Quercetin as a function of time during its reaction with hydroxylradicals at pre-cmc, cmc, post-cmc surfactant concentrations in (a) non-ionic Brij30,(b) anionic SDS and (c) cationic DDEAB surfactant media.

0 1 2 3 4

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40 SDBS=2mM(k=0.066min-1,R2=0.99) DDEAB=14mM(k=0.020min-1,R2=0.99) SDS-Brij30=0.057mM(k=0.009min-1,R2=0.97) DTPB-Brij30=057mM(k=0.026min-1,R2=0.98) DTPB-DDEAB-Brij30=0.101mM(k=0.045min-1,R2=0.99)

-Lo

g(A

/A0 )

Time (min)

Fig. 4. First order degradation of Quercetin in some single, binary and ternary surfactantsystems using Fenton's reagent.

0.0 0.1 5 10 15 20 25 300

10

20

30

40

50

60

70

80 Brij30 SDS SDBS DDEAB DTPB

Rad

ical

Sca

ven

gin

g A

ctiv

ity

[Surfactant]/mM

Fig. 5. Hydroxyl radical scavenging activity of Quercetin in different single surfactantsystems.

299S. Jabeen et al. / Food Research International 51 (2013) 294–302

to favorable interaction between negatively charged center of Quercetinand positively charged head groups of surfactants. The possible interac-tion modes of Quercetin with cationic, anionic & nonionic micelles areshown in Scheme 2 (Chat et al., 2011). As such, the OH radical scavengingactivity of Quercetin and hence k (degradation rate constant) in nonionicsurfactants is lowest compared to that in cationic micelles since OH radi-cals are mainly partitioned in the aqueous phase. Decrease in RSA/k inBrij30 surfactant system below its cmc indicates the role of hydrogenbonding in premicellar concentration that may slow the hydrogen

abstraction kinetics in contrast to that in SDS wherein the change issmall. The strong hydrogen bonding tendency along with the orientationeffect of polyoxyethylene groups of Brij30 on Quercetin reduces its RSA/keven more than in SDS micelles (Fig. 5). In SDBS micellar system RSA/kinitially decreases slightly in the premicellar region as observed in caseof SDS, followed by a large increase above the cmc. This observation isquite opposite to that in SDS micelles although both the surfactantshave samehydrocarbon chain length and charge onhead groups. This un-usual behavior could not be explained. In cationic surfactant systems,DDEAB and DTPB the radical scavenging activity and degradation con-stant of Quercetin remains almost constant in premicellar region butincrease with increase in the surfactant concentration above the cmc.Moreover, the magnitude of the two parameters was more in cationicsurfactant systems than in nonionic Brij30 and anionic SDS. This can beattributed to the favorable interaction of Quercetin via rings A andCwith-in these micelles which increases the accessibility of hydroxyl radicals inthe aqueous phase to electroactive hydroxyl groups present on ring B inthe Quercetin molecules. The hydrogen atom transfer from 3′, 4′ hy-droxyls to OH radical present in the aqueous phase can thus be facilitatedleading to higher RSA/k in cationics than in anionic/nonionic micelles inwhich Quercetin interacts via ring B.

Fig. 6 & Table 2 give a comparison of the RSA/k of Quercetin in scav-engingOH radical in equimolar nonionic–anionic andnonionic–cationic

Scheme 2. Probable location of Quercetin in: (a) anionic, (b) cationic and (c) non-ionicmicelles.

300 S. Jabeen et al. / Food Research International 51 (2013) 294–302

mixed binary surfactant systems respectively with that in their singlecomponent systems. As observed from the figure & table the RSA/k ofQuercetin in the equimolar binary nonionic–cationic, nonionic–anionicsurfactant systems lies in-between the values observed in their corre-sponding single counterparts except in equimolar binary DTPB andBrij30 system wherein a higher RSA/k than the corresponding singlesurfactant systems was observed.

It is known from the literature (Rosen & Zhou, 2001; Zhou & Rosen,2003) that in an aqueous anionic–nonionic binary surfactant solutionthe weakly basic POE head groups get protonated to acquire positivecharge, even at neutral pH. Therefore, owing to higher mole fractionof Brij30 within the mixed Brij30-SDS and Brij30-SDBS micelles(Table 1) the presence of slight positive charge would increase its in-teraction with Quercetin via rings A and C, such that their 3′,4′electroactive hydroxyls point outwards facilitating their RSA/k. Howev-er, due to combined effect of weak hydrogen bonding and presence ofsome negative charge the oxidation of Quercetin is slowed downresulting in intermediate RSA/k within binary anionic–nonionic mi-celles compared to their single component systems. In the Brij30-DDEAB binary system, the antioxidant activity of Quercetinwas also ob-served to be intermediate between that in single surfactant systems.Nonionic–cationic mixed micelles being predominantly made up ofthe nonionic component (Table 1), most of the Quercetin moleculeswould interact in such mixed micelles via ring B having 3′, 4′electroactive hydroxyls pointed inwards, thereby reducing the RSA/k.Moreover, strong hydrogen bonding effect of nonionics would alsoreduce the antioxidant activity of Quercetin. However, due to positivecharge on Brij30-DDEAB mixed micelles some Quercetin moleculeswould also interact via ring A and C with micelles making 3′ 4 ′

electroactive hydroxyls point toward aqueous phase. Both these oppo-site effects taken together are responsible for the intermediate RSA/kvalues of Quercetin between that in pure Brij30 and DDEAB micelles.On the contrary, in Brij30-DTPB binary system the values are higherthan in either of the single surfactant systems, due to delocalization of

positive charge over the three phenyl rings. This would lead to interac-tion of Quercetin via ring A and C with micelles forcing 3′, 4′electroactive hydroxyls to point toward the aqueous phase thereby en-hancing its RSA/k. Mixed systems are found to be efficient solubilizersfor non-polar and semi-polar compounds (Bhat, Rather, & Dar, 2009;Dar et al., 2007). Presence of steric interactions would, however restrictthe deeper intercalation of Quercetin within Brij30-DTPB mixed mi-celles and may opt for outer palisade layer as its solubilization site.Such an effect could have been observed in Brij30-DDEAB system aswell, but the mixed micelles would be more compact as a result oflower steric hindrance of DDEAB head group, leading to betterpartitioning of semi-polar Quercetin and hence lesser accessibility tohydroxyl radicals decreasing the RSA/k values, in addition to other ef-fects already discussed. The RSA/k is found to be greater in cationic–nonionic binary surfactant systems compared to nonionic–anionic mix-tures because of favorable orientation effect.

Fig. 7 gives a comparison between the activity of Quercetin in scav-enging OH radicals in equimolar nonionic–cationic–cationic and non-ionic–anionic–anionic ternary surfactant systems. As revealed by theprofiles, the RSA/k of Quercetin in the ternary anionic–anionic–nonionicsurfactant system decreases with increase in the total surfactant con-centration but exhibits the opposite behavior in the other system.Since the micellar mole fraction of Brij-30 is higher than the sum ofmole fraction of the SDS and SDBS in the former system (Table 1), there-fore, the RSA/k profile would be practically the same as observed in an-ionic–nonionic binary surfactant systems due to the reasons explainedearlier. The increase in the •OH scavenging activity of Quercetin in thepost-micellar region of Brij30-DTPB-DDEAB even more than theBrij30-DTPB system might be due to the higher combined micellarmole fraction of the two cationics in the ternary surfactant systemresulting in higher positive charge density on their mixed micelles, al-though for the increase in premicellar region the reason could not befigured out. Since the calculated cmcRH of this system is slightly lessthan the experimental value, it indicates that the combined micellarmole fraction of cationics would be even more than shown in theTable 1. Hencemost of the Quercetinmolecules are expected to interactwith ternary nonionic–cationic–cationic surfactant system via ring Aand C pointing toward the micelle core and ring B with 3′ 4′ hydroxylspointing toward the aqueous phase thereby increasing the proximity ofthe hydroxyls toward Quercetin and consequent increase in the RSA/k.In addition, the steric factor of DTPB would also localize Quercetin inouter micellar palisade layer contributing to enhanced activity asexplained earlier.

4. Conclusion

The present study reports the influence of single, binary and ternarysurfactants on antioxidant activity of Quercetin, in studiedmicellar sys-tems. The antioxidant activity of Quercetinwasmore in the cationic sur-factant (DDEAB, DTPB) systems than nonionic (Brij30) and anionic(SDS) surfactant systems due to their favorable orientation effect onQuercetin within their micelles and increased with increase in surfac-tant concentration above their cmc. However, the activity of Quercetinin nonionic Brij micellar system was observed to be lower than that inSDS micellar system attributable to stronger H-bonding effect in thesemicelles that hampers H-abstraction kinetics. The activity of Quercetinin the binary nonionic–cationic and nonionic–anionic surfactant sys-tems was in between the values observed in anionic, cationic and non-ionic single surfactant system except in equimolar binary DTPB andBrij30 surfactant system. However, the activity of Quercetin in ternarysurfactant systemwas more in nonionic–cationic–cationic than in non-ionic–anionic–anionic surfactant system. This is attributed to the favor-able orientation effect in the former for reaction between Quercetinand •OH.

In conclusion, the results presented in the paper give the importanceof simplemicro heterogeneous environments on the antioxidant activity

0.0 0.1 0.2 5 10 15 20 25 30 350

10

20

30

40

Brij30DDEAB

Brij30-DDEAB

Rad

ical

Sca

ven

gin

g A

ctiv

ity

[Surfactant]/mM

(c)

0.0 0.1 1 2 3 4 5 6 70

10

20

30

40 Brij30 DTPB Brij30- DTPB

Rad

ical

Sca

ven

gin

g A

ctiv

ity

[Surfactant]/mM

(d)

0.0 0.1 0.2 2 4 6 80

10

20

30

40

50

60

70

80

(b) Brij30 SDBSBrij30-SDBS

Rad

ical

Sca

ven

gin

g A

ctiv

ity

[Surfactant]/mM0.0 0.1 0.2 5 10 15 20

10

20

(a) Brij30SDS

Brij30-SDSR

adic

al S

cave

ng

ing

Act

ivit

y

[Surfactant]/mM

Fig. 6. Hydroxyl radical scavenging activity of Quercetin in mixed binary surfactant systems and their comparison with that of single surfactant systems.

301S. Jabeen et al. / Food Research International 51 (2013) 294–302

of Quercetin having some correlation with the complex biological sys-tems and are expected to contribute significantly in understanding andcontrolling the antioxidant activity at interfaces present in wide rangeof foods, cosmetics, pharmaceuticals and biological membranes.

0.00 0.05 0.10 0.15 0.20 0.25 0.300

10

20

30

40

50

SDS-SDBS-Brij30DDEAB-DTPB-Brij30

Rad

ical

Sca

ven

gin

g A

ctiv

ity

[Surfactant]/mM

Fig. 7. Hydroxyl radical scavenging activity of Quercetin in mixed ternary surfactantsystems.

Acknowledgment

We are thankful to the Head, Department of Chemistry, Universityof Kashmir, for providing the laboratory facilities and his constantencouragement and inspiration.

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