Journal of Colloid and Interface Science 355 (2011) 140–149

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Effects of surfactant micelles on solubilization and DPPH radical scavenging activityof Rutin

Oyais Ahmad Chat, Muzaffar Hussain Najar, Mohammad Amin Mir, Ghulam Mohammad Rather ⇑,Aijaz Ahmad Dar ⇑Department of Chemistry, University of Kashmir, Srinagar-190006, J&K, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 17 August 2010Accepted 16 November 2010Available online 20 November 2010

Keywords:Radical scavenging activityRutinMicellesDPPHSolubilization

0021-9797/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.jcis.2010.11.044

⇑ Corresponding authors. Fax: +91 194 2421357/24E-mail addresses: gmrather2002@yahoo.com (G.

co.in (A.A. Dar).

The interaction of the antioxidant Rutin with the radical DPPH (2,2-diphenyl-1-picrylhydrazyl) in pres-ence of cationic (CTAB, TTAB, DTAB), non-ionic (Brij78, Brij58, Brij35), anionic (SDS) and mixed surfactantsystems (CTAB-Brij58, DTAB-Brij35, SDS-Brij35) has been followed by spectrophotometric and tensio-metric methods to evaluate the DPPH radical scavenging activity (RSA) of Rutin in these model self-assembled structures. The results show that the solubilization capacity of various single surfactant sys-tems for both DPPH as well as Rutin followed the order cationics > non-ionics > anionic. The radical scav-enging activity of Rutin in the solubilized form was higher within ionic micelles than in non-ionicmicelles. However, the antioxidant exhibited enhanced activity for the radical in mixed cationic–non-ionic micelles compared with any of the single component micelles. In contrast, anionic–non-ionic mixedmicelles modulated the activity of Rutin in-between that seen for pure anionic and non-ionic micellesonly.

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1. Introduction

Redox reactions represent an essential part of aerobic life andcell metabolism. Oxygen uptake inherent to aerobic systems pro-duces Reactive Oxygen Species (ROS) like H2O2, 1O2, O��2 , OH�, ROO�

and nitric oxide which have been linked with aging and manydegenerative diseases such as cancer, inflammation, immune sys-tem decline, cardiovascular diseases, neurological disorders andatherosclerosis [1–3].

Flavonoids, a group of naturally occurring benzo-c-pyronederivatives, have been reported to possess multitude of biologicalproperties and proven to be strong antioxidants and free radicalscavengers [4–7]. Rutin (Scheme 1), a kind of flavonol glycoside,shows various pharmaceutical effects like antihypertensive, anti-inflammatory, antihemorrahagic etc. that have been attributedpartly to its ability to scavenge free radicals [8]. It has beenreported that such activity of polyphenols is highly sensitive tothe environmental factors like solvent polarity, use of micellarmedia etc. [8–10].

Interest in understanding the parameters that influence theactivity of antioxidants in complex or multiphase systems isincreasing as actual food products are multicomponent matrices[11–15]. As per Frankel [16], interfacial phenomena are key to

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25195.M. Rather), aijaz_n5@yahoo.

better understanding of antioxidant action in heterogeneous foodsand biological systems. Micelles and other disperse systems havebeen extensively used for modeling the effects of heterogeneousenvironments on reaction dynamics and mechanism, and complexbehavior encountered in food and biological assemblies [17–20].Solubilization of antioxidants in different phases and environs ofmicelles results in different physicochemical interactions com-pared to homogeneous systems thereby strongly influencing theiractivity [21–23]. The knowledge of partition coefficient is of crucialimportance for understanding such differences in antioxidantactivity in various micellar media and establishing the location ofantioxidants within the micelles [14–24].

Interaction of different antioxidants with variously architec-tured surfactants in aqueous media and the consequent influenceon their antioxidant activity has been the subject of a number ofstudies [8,9,25–30]. Heins et al. [9] reported that the antioxidantactivity of phenolic compounds depends upon their interaction siteat the interface of the micellar systems. The occupation of differentenvironments by the radical and the antioxidant serves as a phys-ical barrier to the action of antioxidants. Thus, solubilization of Ru-tin within CTAB micelles makes it difficult to quench hydroxylradical [8]. On the other hand concentration of radical and the anti-oxidant in the same environment will enhance the antioxidantactivity as has been reported for Quercetin in CTAB micelles [29].Studies have also revealed that the reaction between Puerarinand DPPH in Triton X-100 micelles is much faster than in semi-aqueous solutions [30].

Scheme 1. Structure of surfactants, DPPH and Rutin used in this study.

O.A. Chat et al. / Journal of Colloid and Interface Science 355 (2011) 140–149 141

The literature reveals [8,9,25–33] that most of the studiesundertaken to understand the influence of micellar media on theantioxidant activity of flavonoids and other antioxidants have fo-cussed on a limited number of surfactants without taking into ac-count the effects arising due to changes in nature and size of thehead groups and hydrophobicity of surfactant. Moreover, no at-tempt has been made so far to study antioxidant activity of flavo-noids in mixed micellar media. Mixtures of the surfactants havereceived wide attention for several decades because of their effi-cient solubilization, suspension, dispersion, and transportationcapabilities [34].

DPPH assay, as pointed out by Moreno [35], based on the reduc-tion of absorbance at 515 nm of the stable DPPH radical by an anti-radical is easy and potentially accurate for measuring the radicalscavenging activity (RSA). However, there is no report discussingsolubilization aspects of DPPH radical in surfactant systems.

We have evaluated and compared the DPPH radical scavengingactivity of Rutin in presence of cationic, anionic, non-ionic andmixed aqueous micelles that mimic complex biological systemswith simple micro-heterogeneous environment. The work compre-hensively focuses on solubilization of Rutin and DPPH and simulta-neous assessment of RSA of the solubilized antioxidant in studiedmodel systems, emphasizing the correlation between the solubili-zation capacity of various micellar media towards these sparinglysoluble compounds and extent of the reaction in such media. Theinvestigation has relevance in understanding the antioxidantmechanism in real complex biological systems.

2. Experimental section

2.1. Materials

The non-ionic amphiphiles Polyoxyethylene(23) lauryl ether(Brij35), Polyoxyethylene(20) cetyl ether (Brij58), Polyoxyethyl-

ene(20) stearyl ether (Brij78), cationic amphiphiles, dodecyltrim-ethylamonium bromide (DTAB), tetradecyltrimethylammoniumbromide (TTAB), cetyltrimethylammonium bromide (CTAB) andanionic amphiphile sodium dodecylsulfate (SDS), and the solventsn-octane, diethylether and methanol were all Aldrich products,and used as received. The purity of the surfactants was furtherensured by the absence of minimum in surface tension c vs. log [sur-factant] plots (Supplementary material; Fig. 1S). The antioxidantRutin trihydrate (Rutin, >95%) and the radical 1,1-diphenyl-2-pic-rylhydrazyl (DPPH, >98%) were also Aldrich products. The structuresof the surfactants, antioxidant and radical used are presented inScheme 1. Surfactant solutions were prepared in triple-distilledwater.

2.2. Methods

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

from the plot of surface tension (c) vs. logarithm of surfactantconcentration (log Ct) (Supplementary material Figs. 1S and 2S).Surface tension measurements were made by the platinum ringdetachment method with a Krüss-9 (Germany) tensiometerequipped with a thermostable vessel holder. Surfactant concentra-tion was varied by adding solution of known surfactant concentra-tion in small installments using a Hamilton microsyringe to 30 cm3

of water in the sample vessel placed in the vessel holder. Measure-ments were made after thorough mixing and temperature equili-bration at 25 �C (±0.1 �C) by circulating water from a HAAKE GHthermostat through the vessel holder. The accuracy of measure-ments was within ±0.1 dyne cm�1 and the readings were taken intriplicate to ensure reproducibility.

2.2.2. Determination of solubilitySolubilities of the antioxidant Rutin and DPPH were measured

in single and equimolar binary combinations of selected surfactant

142 O.A. Chat et al. / Journal of Colloid and Interface Science 355 (2011) 140–149

systems as a function of surfactant concentration. Excess amountsof Rutin/DPPH were added to 5 mL vials containing 2 mL of the sur-factant solution and the sample vials were sealed with Teflon linedscrew caps and were then magnetically agitated for a period of 2 hin a thermostated shaker bath maintained at 25 ± 0.5 �C. The solu-tions were subsequently subjected to centrifugation at 13,000 rpmusing Eppendorf Minispin Centrifuge for 20 min to remove theundisolved compounds. The concentration of solubilized Rutinwas determined spectrophotometrically with a Schimadzu spec-trophotometer (Model UV–1650) following appropriate dilutionof an aliquot of the supernatant with the corresponding surfactantsolution. However, in case of DPPH 2 mL of the supernatantwithout dilution was analysed. Surfactant solutions of sameconcentrations as in the samples were used as references. Thesolubilities were determined from the absorbance vs. concentra-tion plots using the calculated extinction co-efficients of 8.99 �103 dm3 mol�1 cm�1 of Rutin in 50% methanol–water system and1.14 � 104 dm3 mol�1 cm�1 for DPPH in methanol at their charac-teristic wavelengths of 360 nm and 515 nm respectively. The molarextinction coefficient (e) for DPPH was within the range ofpublished values [36]. The water solubility of Rutin using thecalculated extinction coefficient was confirmed to be 1.5 �10�4 mol dm�3 which is in good agreement with the literaturevalue [37].

2.2.3. Evaluation of DPPH scavenging activity of RutinScavenging potential of the antioxidant Rutin for the DPPH rad-

ical in each surfactant solution was determined by first solubilizingboth the radical and antioxidant in solutions of same surfactantconcentration followed by addition of 10 lL of surfactant solubi-lized Rutin to a fixed volume (2 mL) of the solubilized DPPH radicalsolution. The decrease in absorbance at the characteristic absorp-tion wavelength of DPPH (515 nm) was monitored spectrophoto-metrically, 30 s after each addition (10 lL) and thorough shakingby hand at 25 �C. Surfactant concentrations for cationic surfactantswere maintained approximately five times their cmc values.However, a concentration of 20 mM for anionic SDS and non-ionicsurfactants was selected because DPPH solubilized in low concen-trations of SDS/Brijs was insufficient for convenient measurementof absorbance values. In case of ionic–non-ionic binary surfactantsystems, 10 mM of total surfactant concentration was found tobe appropriate for analyzing DPPH radical scavenging activity ofRutin. All the experiments were performed in triplicate. Theantiradical activity was calculated as the percentage of DPPHdecoloration [38] using the following equation:

RSA ¼ 100� ð1� Aa=AoÞ ð1Þ

where Aa is the absorbance of sample after appropriate addition ofsurfactant solubilized Rutin and Ao is the absorbance of samplewithout Rutin.

Table 1Experimental and literature critical micelle concentration values (cmcexp and cmclit.) ofinteraction parameter (b) and activity coefficients (fi) for equimolar binary system calcula

Single surfactant systems Mixed

System cmcexp (cmclit) mmol dm�3 System cmcex

CTAB 0.764(0.815)a CTAB-Brij58 0.010TTAB 3.8(3.7)b DTAB-Brij35 0.079DTAB 14.5(15.1)a SDS-Brij35 0.082SDS 7.59(8.1)c

Brij78 0.004(0.004)d

Brij58 0.0061(0.0081)a

Brij35 0.044(0.05)c

a Ref. [45].b Ref. [46].c Ref. [47].d Ref. [48].

Since the maximum solubility of DPPH varied in different sur-factant systems, the RSA was normalized for 50 lM concentrationof the radical by the following relation:

ðRSAÞ0 ¼ ðRSAÞo � Co

C 0oð2Þ

where (RSA)0 and (RSA) are the radical scavenging activities at DPPHconcentration of 50 lM (C0o) and a maximum solubilized concentra-tion (Co) in a given surfactant solution respectively. All these activ-ities were compared with the activity of Rutin obtained at 50 lMconcentration of DPPH at varied Rutin concentrations in methanol.

3. Results and discussion

3.1. cmc and surfactant–surfactant interactions

The cmc values of selected single and mixed surfactant systems,obtained from surface tension vs. logarithm of surfactant concen-tration plots are presented in Table 1 along with the ideal cmc val-ues, cmcideal, of binary surfactant systems based on the Clintequation [39]. All the observed cmc values were found to be lowerthan ideal values, indicating negative deviation from ideal behaviorfor mixed micelle formation. The estimate of the negative devia-tion and hence nonideality of binary surfactant systems has beenobtained from Rubingh’s model [40]. The interaction parameter,b, that accounts for deviation from ideality is an indicator of the de-gree of interaction between two surfactants in the mixed micelles.b values along with the micellar mole fraction, XM

i , and activitycoefficient, fi, of the ith surfactant within mixed micelles calculatedthrough Rubingh equations [40] are also presented in Table 1. Thenegative values of b indicate synergistic interactions. It is well-known [41,42] that in ionic–non-ionic mixed surfactant systemsthe significant electrostatic self-repulsion of ionics and weak stericself-repulsion of non-ionics (depending on the headgroup size)before mixing are weakened by dilution effects after mixing andthat the electrostatic self-repulsion of the ionic surfactant isreplaced by ion–dipole interactions. Moreover, these mixedmicelles are dominated by non-ionic surfactants as indicated byXM

i values in Table 1, in conformity with analogous results in theliterature [43,44].

3.2. Solubilization characteristics

3.2.1. Molar solubilization ratio (MSR)A measure of the effectiveness of a surfactant solution in solu-

bilizing a given solubilizate is the molar solubilization ratio(MSR) defined as the amount of solute that can be solubilized inone mole of micellized surfactant. In presence of excess of hydro-phobic organic compound MSR, given by the equation [45,49,50].

single and binary surfactant systems, along with the micellar mole fraction (XMi ),

ted by Rubingh’s method at 25 �C.

surfactant systems

p (cmcideal) mmol dm-3 b XM1 /XM

2f1/f2

9 (0.0121) �2.99 0.09/0.91 0.08/0.98(0.088) �3.98 0.08/0.92 0.03/0.98(0.087) �2.71 0.06/0.94 0.09/0.99

O.A. Chat et al. / Journal of Colloid and Interface Science 355 (2011) 140–149 143

MSR ¼ ½St � � ½Scmc�Ct � cmc

ð3Þ

is obtained from the slope of the curve that results when solubiliz-ate concentration is plotted against surfactant concentration. [St] isthe total apparent solubility of solubilizate in single/mixed surfac-tant solution at a particular total surfactant concentration, Ct, abovecmc. [Scmc] is its apparent solubility at cmc, generally taken as thewater solubility, since it changes only very slightly up to the cmc.The effectiveness of solubilization can alternatively be expressedin terms of the partition coefficient, Km [45], related to MSR, as:

0 1 2 3 4 5 6 7 8 9 10 110.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8(c)

(St-S

cmc)/m

M

(Ct-cmc)/mM

CTAB-Brij58DTAB-Brij35SDS-Brij35

0 10 20 30 40 50 60 70 80 90 1000.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0 (a)

(St-S

cmc)

/mM

CTABTTABDTABSDS

0 2 4 6 8 10 12 14 16 18 20 220.00.10.20.30.40.50.60.70.80.91.01.1

(b)

(St-S

cmc)

/mM

Brij35Brij58Brij78

Fig. 1. Solubility of Rutin vs. surfactant concentration in single and equimolarbinary surfactants systems at 25 �C.

Km ¼ MSRScmc :Vm ð1þMSRÞ ð4Þ

where Vm is the molar volume of water (0.01805 L/mol) at 25 �C.

3.2.2. Solubilization of RutinSolubilities of Rutin as a function of micellized surfactant con-

centration in single and equimolar binary surfactant systems areplotted in Fig. 1. The aqueous solubilities increase linearly overthe range of the single and mixed surfactant concentrations abovecmc indicating solubility enhancement in aqueous micellar media,presumably due to micellar solubilization [51]. The MSR values cal-culated from these plots using Eq. (3) are given in Table 2 for allsurfactant systems used in the study.

Rutin contains aromatic rings and its solubility in water is low(0.2 mM). However, it can be drawn into the micelles throughhydrophobic interactions. Another factor influencing the Rutin sol-ubility in micellar solution is the interaction between its polar orionized OH groups and the charged head groups or polar oxyethyl-ene groups of surfactants. The most acidic phenolic OH group ofRutin is at the 7th position of the molecule, which dissociates inaqueous solutions as per the following ionization equilibrium [8].

O

O

OR

OH

OH

OH

O

O

OR

O-

OH

OH

pKa = 7.1

It might, therefore, be concluded that the antioxidant will preferthe palisade layer of micelle as a consequence of hydrophobic aswell as electrostatic interactions [8,29,52]. Among single ionicsurfactant systems, the solubilization is least efficient in SDS asevident from the values of MSR, a possible consequence of electro-static repulsion between anionic Rutin and negatively charged SDShead groups. Similar results have been reported in solubilizationstudies of Ibuprofen [53], Sulfanilamide [54] and Naproxen [55].However, the extent of solubilization is observed to increase withconcentration of SDS through hydrophobic interactions due to in-crease in micelle concentration. The possible orientation of Rutinmolecule within SDS micelles is shown in Scheme 2a, which indi-cates that the B ring would, on average, be embedded within thepalisade layer of micelle, while rings A and C close to the negativelycharged center lie outside the micelle away from the negativelycharged head groups to avoid unfavorable electrostatic repulsionas reported in case of Quercetin [29].

The solubilizing power of cationic surfactants is in the orderCTAB > TTAB > DTAB (Table 2), a noticeable manifestation of the

Table 2Molar solubilization ratio (MSR) of Rutin and DPPH insingle and mixed surfactant systems at 25 �C.

Surfactant system MSR (�10�2)

DPPH Rutin

CTAB 0.751 6.74TTAB 0.372 5.85DTAB 0.124 4.66SDS 0.151 2.33Brij78 0.523 3.09Brij58 0.394 3.07Brij35 0.172 4.35CTAB-Brij58 2.51 11.3DTAB-Brij35 0.423 7.69SDS-Brij35 0.261 5.45

--

-

-

-

-

-

-

---

-

-

(a)

O

OH

OHOH

O

O

OHOH

CH3

OH

O

OO

O-

OH

OHOH

ACB

O

OH

OH

OH

OO

OHOH

CH3

OH

O

OO

O-

OH

OHOH

ACB

(c)

+ +

+

+

+

+

+

+

+

++

+

+

(b)

OO

O

O-OH

OH

OH

AC

B

O

OH

OHOH

O

O

OH

OH

CH3

OH

Scheme 2. Average location of Rutin in: (a) anionic, (b) cationic and (c) non-ionic micelles.

144 O.A. Chat et al. / Journal of Colloid and Interface Science 355 (2011) 140–149

decrease in hydrocarbon chain length, hence micellar space withinthe palisade layer in the same order [56]. This result is opposite tothat expected on the basis of electrostatic interactions on accountof the variation in the micellar surface charge density in thereverse order. Probably the Rutin-micelle hydrophobic interac-tions, in the present case, are strong enough to outweigh theelectrostatic interactions between them. However, the electrostaticattraction between the cationic surfactant and negatively chargedRutin would additionally explain the higher MSR values of the

cationics (Table 2), also supported by the solubilization of cationicdrug trifluoperazine in SDS micelles [57]. Liu and Guo [29], usingcyclic voltammetry, demonstrated that Quercetin interacts withCTAB micelles via rings A and C while as it interacts via ring B withanionic SDS micelles. Therefore, in analogy, the favorable electro-static attraction between negatively charged center of Rutin andpositively charged head groups of surfactants would possibly lead,on average, to solubilization of the antioxidant through rings A andC leaving ring B away from the micellar surface (Scheme 2b). It has

0 2 4 6 8 10 12 14 16 18 20 220.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07 (a)

(St-S

cmc)

/mM

Brij35Brij58Brij78

0 10 20 30 40 50 60 70 80 900.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14 (b)

(St-S

cmc)

/mM

CTABTTABDTABSDS

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08(c)

(St-S

cmc)

/mM

(Ct-cmc)/mM

CTAB-Brij58DTAB-Brij35SDS-Brij35

0 1 2 3 4 5 6 7 8 9 10 11

Fig. 2. Solubility of DPPH vs. surfactant concentration in single and equimolarbinary surfactant systems at 25 �C.

O.A. Chat et al. / Journal of Colloid and Interface Science 355 (2011) 140–149 145

been reported that the introduction of a sugar moiety in theflavonols inhibits them from reaching the micellar surface due tohigher aqueous phase solubility and steric hindrance [58]. Thiswould affect the extent of solubilization of Rutin within the mi-celles without any appreciable change in its orientation comparedto Quercetin supported by our DPPH radical scavenging activitydata discussed later.

Among non-ionic surfactant systems, Brij78 and Brij58 showedsimilar MSR values while higher solubilization efficiency was ob-served for Brij35 system. Though the hydrocarbon chain length de-creases in the order Brij78 > Brij58 > Brij35, the solubilizationcapacity could be attributed to the number of oxyethylene (OE)groups present in their head groups. Spectroscopic and electro-chemical studies have shown that Rutin molecules are located inthe palisade layer of non-ionic Triton X-100 micelles involvinghydrophobic and hydrogen bonding interactions [52] making itto solubilize preferably with its B ring pointing towards the coreof non-ionic micelle as depicted in Scheme 2c.

Comparison between cationic and non-ionic single surfactantsystems shows that CTAB has higher solubilizing power than Brij58both having the same hydrocarbon chain length. Probably the elec-trostatic interactions between Rutin and cationic surfactant out-weigh the hydrogen bonding interactions within non-ionicsurfactant. However, solubilizing power of DTAB and Brij35, as evi-dent from their MSR values, is similar even though DTAB is cat-ionic. This might be due to higher number of OE groups in Brij35than in Brij58 thereby increasing the effect due to hydrogen bond-ing. Since the interaction between anionic Rutin and SDS micellesis repulsive as explained earlier, SDS exhibits lower solubilizingpower than DTAB and Brij35, all having same (C12) hydrocarbonchain length.

All the equimolar cationic–non-ionic binary surfactant mixturesshow greater MSR values compared to the single surfactant sys-tems indicating synergism in Rutin solubility enhancement. Thesolubilization of Rutin in cationic–non-ionic mixed micelles wouldbe assisted by both hydrogen bonding as well as electrostaticattraction. The increased solubilizing power of mixed micelles isa result of synergistic contribution of interactions characteristicof cationics and non-ionics present in the same mixed micelle. Asindicated by data in Table 1, since cationic–non-ionic mixed mi-celles are predominantly made up of the non-ionic component(higher Xnonionic), most of the Rutin molecules solubilized in suchmixed micelles would be oriented such that their B ring points to-wards micellar core while rings A and C lie outside the micelles.However, a few Rutin molecules may be oriented as in pure cat-ionic micelles. Again due to larger micellar size, CTAB-Brij58 sys-tem exhibits higher solubilizing power than DTAB-Brij35 system.The anionic–non-ionic SDS-Brij35 system also shows synergisticsolubilization of Rutin, being more than in either of the surfactantsas indicated by MSR values (Table 2). It is known from literature[42,59] that in aqueous anionic surfactant solution, even at neutralpH, the weakly basic POE surfactant head group gets protonated,acquiring positive charge. Therefore, owing to high micellar molefraction of Brij35 within the mixed SDS-Brij35 system (Table 2),the presence of slight positive charge would increase its interactionwith Rutin in addition to having hydrogen bonding effect charac-teristic of pure non-ionic micelles. Such micelles would presum-ably exhibit, on average, both types of orientation effects presentin pure cationics and anionics with similar probability.

3.2.3. Solubilization of DPPHThe solubility enhancements of DPPH in single and equimolar

mixed surfactant systems are plotted as a function of micellarsurfactant concentration in Fig. 2. The MSR values calculated fromthese plots using Eq. (3) are given in Table 2 for all surfactantsystems used in the study.

In non-ionic surfactant systems, solubilizing power towardsDPPH follows the order Brij78 > Brij58 > Brij35 reflecting theincrease in hydrocarbon chain length and hence micelle size(Fig. 2a). DPPH radicals are practically insoluble in water but freelysoluble in non-polar solvents [60]. Therefore, due to this DPPHwould preferably be solubilized in the micellar core and the extentof its solubilization would thus be proportional to the hydrocarbonchain length of the surfactant.

Among ionic surfactants, the solubilizing power is in orderCTAB > TTAB > DTAB = SDS (Fig. 2b), in which hydrocarbon chainlength of the surfactants decreases, and independent of the natureof charge on head groups. For the anionic SDS, the MSR value of

146 O.A. Chat et al. / Journal of Colloid and Interface Science 355 (2011) 140–149

DPPH was found equal to that in cationic DTAB a manifestation ofsimilar hydrocarbon chain length (C12).

All equimolar binary systems studied revealed greater solubiliz-ing efficiency (higher MSR) as compared to single surfactant sys-tems indicating synergism in DPPH solubility enhancement(Table 2). In conformity with our earlier findings for other non-po-lar solubilizates [45], the solubilization of DPPH in binary cationic–non-ionic systems is significantly higher in C16–C16 system thanin C12–C12 system due to higher non-polar content of the former(Fig. 2c). However, the lower MSR value of DPPH in SDS-Brij35 sys-tem as compared to that in DTAB-Brij35 system, both having thesame non-polar content, may be credited to electrostatic repulsionbetween electron rich DPPH radicals and negatively charged mixedmicelle, in addition to more stable (more negative value of b)mixed micelles of DTAB-Brij35 system (Table 1).

3.3. DPPH radical scavenging activity (RSA) of Rutin

In order to understand the effect of surfactant structure on thereaction between DPPH and Rutin, it is important to consider theirstability in such media. Fig. 3 and 3S show the UV-visible spectra ofRutin and DPPH respectively in some prototype surfactant solu-tions viz. cationic CTAB, anionic SDS and non-ionic Brij35 in addi-tion to that in methanol–water and methanol systems. Asdescribed in the experimental section, the concentration of DPPHand Rutin in these surfactant systems was equal to their maximumsolubility in the respective surfactant solutions.

As evident from Fig. 3, the spectra of Rutin in micellar media aresimilar to that in methanol, except for some bathochromic shift,indicating that Rutin is stable in the studied micellar media as re-ported by Guo and Wei [8]. Rutin exhibits two main absorptionbands in the range 200–500 nm due to p–p* transition in B–C rings(at about 353 nm in methanol) and A–C rings (at about 260 nm inmethanol). Moreover, in all the systems it was found that theabsorption intensities corresponding to these peaks do not changewith time. Similarly Fig. 3S clearly reveals that UV–visible spectraof DPPH are unaffected when methanol is replaced by variousmicellar media. However, in case of cationic surfactants in additionto its usual strong peak at around 515 nm an extra peak at around425 nm is observed which might be due to some interaction be-tween DPPH and these micelles. Time evolution of absorbance ofDPPH in various surfactant systems did not show any change indi-cating its stability in these systems. The reaction between DPPHand Rutin was monitored at 515 nm after thorough mixing andreduction in absorbance was recorded as antioxidant activity.

250 275 300 325 350 375 400 425 450 475 5000.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Abs

orba

nce

Wavelength (nm)

Methanol20mM SDS5mM CTAB10mM Brij5850-50 W/M

Fig. 3. UV–visible spectra of Rutin in Methanol, 50% methanol–water system andaqueous SDS, CTAB and Brij58 solutions.

Absorbance ranges of DPPH (515 nm) and Rutin (358 nm) beingdifferent, Rutin does not interfere with the absorbance of DPPHat 515 nm. Fig. 4S depicts the time evolution of the absorbanceof DPPH at 515 nm during its reduction by Rutin in some selectedmedia. As can be seen, the kinetics was extremely fast, even forsmall initial concentrations of Rutin, the asymptote was reachedin a few seconds in all curves especially after shaking thoroughlyand starting readings after 30 s. This shows that the radicalscavenging activity could be easily calculated 30 s after additionof Rutin to DPPH solution and thorough mixing. Fig. 4 representsthe absorbance curves of DPPH at increasing concentration of Rutinin methanol, Brij35, CTAB and SDS as different solubilizing media.It is observed that absorbance of DPPH at 515 nm continuously de-creases with increase in Rutin concentration indicating consump-tion of the radical by the antioxidant. However, in CTAB micellarmedium after an initial decrease due to radical consumption absor-bance increases due to some complex formation in addition tomodifying the spectra. This was also observed in TTAB and DTABmicellar media though instead of an increase, constant absorbancewas noted above certain concentration in the range of 0–0.05 mMRutin.

The DPPH radical scavenging activity of Rutin was determinedas described in experimental section. Fig. 5 depicts the results invarious surfactant systems. It is clear that RSA of Rutin in all the se-lected micellar systems is lower, compared to that in methanol.Due to free solubility of both Rutin and DPPH in methanol, theinteraction between them is not hindered.

However, in aqueous micellar media, DPPH is mainly solubi-lized within micelles due to its solubility in less polar solventsand insolubility in water while as Rutin gets distributed betweenmicellar and aqueous phases. The interaction between Rutin andDPPH, therefore, can occur only within the micelles, suggestingthereby that DPPH scavenging activity of Rutin in such systemswould be related to: (a) its partition coefficient between aqueousand micellar phases; (b) the solubilization sites of both Rutin andDPPH within the micelles; (c) the orientation of these solubilizatesfor effective interaction between the two; and finally (d) the extentof interaction of Rutin hydroxyl groups with the head groups ofsurfactants since it mainly solubilizes in the palisade layer of mi-celles, thereby affecting the hydrogen radical transfer towardsDPPH. Reduced activity of Rutin is a consequence of the role ofall these factors compared to that in methanol where no such fac-tors prevail.

As seen in Fig. 5a the DPPH scavenging activity of Rutin in cat-ionic surfactant systems follows the order CTAB > TTAB > DTAB indirect correlation with their solubilizing efficiency (MSR) for bothRutin and DPPH. High MSR values of Rutin as well as DPPH in cat-ionic surfactants in the above order indicate high local concentra-tions of the reactants within the micelles, thus facilitating theircollisions. Castle et al. [61] have observed that the low effective-ness of Trolox as chain breaking inhibitor of lipid oxidation relativeto lipophilic antioxidants may be due to lower partitioning of Trol-ox into SDS micelles. In a similar way, since MSR and hence mi-celle-water partition coefficient (Km) of Rutin decreases fromCTAB to DTAB, the antioxidant activity also decreases in the samefashion. In the exceptional case of CTAB, above 0.016 mM Rutinconcentration, the activity begins to decrease probably due tosome complex formation with these surfactants as noted inFig. 4b. However in TTAB and DTAB, activity remained almost con-stant at higher concentrations of Rutin. Perhaps in much higherconcentration ranges similar behavior as that of CTAB could havebeen observed. Actual reason for this type of behavior could notbe figured out.

Fig. 5a reveals that the activity of Rutin (at low concentration)in SDS is higher than in DTAB, even though having the same hydro-phobic chain length in spite of having lower MSR. However, the

250 300 350 400 450 500 550 600 250 300 350 400 450 500 550 600

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(a) MethanolA

bsor

banc

e

wavelength(nm)

Fig. 4. Prototype plots of variation of DPPH absorbance as a function of wavelength with different additions of Rutin.

O.A. Chat et al. / Journal of Colloid and Interface Science 355 (2011) 140–149 147

said activity of Rutin in SDS is observed to be lower than in CTABand TTAB. Since higher MSR value of Rutin would assist higherRSA, the orientation of Rutin molecule within the micelle musthave a significant effect. If solubilization of Rutin occurs via ringB which contains the electroactive 3,4-hydroxyl groups, they willget closer to the DPPH molecule lying within the micellar coreand hence will favor higher activity. In contrast, solubilization ofRutin molecules via rings A and C will decrease the chances ofelectroactive 3,4-hydroxyl groups to transfer H-radical to DPPH,leading to lower DPPH RSA. Therefore, magnitude of MSR andorientation of Rutin within micelle need to be taken into accountsimultaneously to explain the activity in micellar systems. Ashypothesized in an earlier section as well as taking into consider-ation the experimental evidence of orientation of Quercetin (degly-coside form of Rutin) within micelles [29], orientation of Rutin inSDS micelles is favorable for its enhanced reaction with DPPH.We are of the opinion that the orientation effect of Rutin in SDS mi-celles more than compensates for its lower MSR value, leading tohigher DPPH RSA than in DTAB. However, in TTAB and more soin CTAB greater contribution of MSR values, though having onaverage unfavorable orientation of Rutin within the cationic sur-factants, leads to higher DPPH RSA of Rutin.

The activity of Rutin (Fig. 5b) in non-ionic Brij micellar systemsis observed to be lower than that in SDS even though having higherMSR values and, on average, similar orientation effect on Rutin(Scheme 2a and c) as that of SDS system. In non-ionic Brijs, thestrong hydrogen bonding of Rutin hydroxyl groups with large

number of polyoxyethylene units in surfactant head groups wouldinhibit the hydrogen abstraction kinetics [9,17,23] leading toreduced activity of Rutin. Comparison of the activity of Rutin inCTAB and Brij58 or DTAB and Brij35 shows again that Rutin is lesseffective as antioxidant in Brij surfactants than in comparablechain length trimethylammonium bromide surfactants. Thoughthe MSR value in cationics is either greater or comparable to thatof corresponding Brij surfactants, in addition to having orientationof Rutin, on average, not suited for enhanced DPPH RSA, it seemsthat the strong hydrogen bonding tendency of Rutin with polyoxy-ethylene groups has a deciding role to play in reducing the activityof Rutin in the Brij systems. Although, there is not much differencebetween the DPPH RSA of Rutin in the three non-ionic surfactantsystems, the lower activity in Brij78 than Brij35 could be attributedto the large micellar size of the former which imposes larger sep-aration between DPPH and Rutin within micelles thereby reducingcollision frequency between them.

This effect could also be seen in the activities in Brij58 andBrij35, but owing to higher number of oxyethylene groups in thelatter, the extent of hydrogen bonding must be relatively higherresulting in slightly less activity (Fig. 5b). Therefore, it can be con-cluded that higher MSR in Brij35 is due to larger number of OEgroups in its head group, but this factor also increases the extentof hydrogen bonding in them leading to reduced activity.

Fig. 6a–c gives a comparison of the activity of Rutin in scaveng-ing DPPH in equimolar cationic–non-ionic or anionic–non-ionicmixed binary surfactant systems with the corresponding single

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Fig. 5. DPPH scavenging activity of Rutin in (a) ionics, (b) non-ionics micellarmedia.

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MethanolSDSBrij35SDS+Brij35

Fig. 6. Comparison of DPPH radical scavenging activity of Rutin in different singleand binary mixed surfactant systems.

148 O.A. Chat et al. / Journal of Colloid and Interface Science 355 (2011) 140–149

component systems. As observed from the figures the activity ofRutin is greater in the binary cationic–non-ionic surfactantsystems than in single cationic or non-ionic systems while as inequimolar binary anionic–non-ionic mixture, the activity lies in-between the values observed in anionic and non-ionic single sys-tems. The binary mixtures were chosen such that the hydrocarbonchain lengths of component ionic and non-ionic surfactants werethe same.

In case of CTAB-Brij58 and DTAB-Brij35 binary systems, theMSR values of Rutin were observed to be much higher than ineither of the single surfactant systems (Table 2). As explained inearlier section, increased efficiency of solubilization in such sys-tems is attributed to both hydrogen bonding and favorable electro-static interactions between surfactant head groups and solubilizatemolecules. Therefore, owing to higher local concentration of Rutinwithin such mixed micelles, its activity to scavenge DPPH presentinside the micelles is sufficiently increased compared to that in thesingle surfactant systems. Thus incorporation of small amount ofcationic surfactant within non-ionic micelles of same hydrocarbonchain lengths increases the activity of the antioxidant. Comparisonof CTAB-Brij58 and DTAB-Brij35 systems reveals larger activity inthe former than latter due to higher MSR values of both Rutinand DPPH. In case of SDS-Brij35 system, activity of Rutin wasobserved to be intermediate between that in single surfactantsystems even though its MSR value in binary system is higher thaneither of the single surfactant systems. As mentioned earlier, poly-oxyethylene surfactant head groups acquire slight positive charge

in the presence of anionic surfactants leading to their enhancedelectrostatic attraction for negatively charged Rutin moleculesthereby assisting higher MSR value. However, due to solubilizationof Rutin via rings A and C in cationics (Scheme 2b), there might, onaverage, be more number of Rutin molecules having 3,4-electroac-tive hydroxyls pointed outwards than those pointing inwards. Inaddition, the hydrogen bonding effect of non-ionics in such mixedmicelles would be lowered because of acquiring positive chargehelping in hydrogen abstraction kinetics. Both these effects takentogether lower the activity of antioxidant compared to that in

O.A. Chat et al. / Journal of Colloid and Interface Science 355 (2011) 140–149 149

SDS micelles even though the MSR values are higher relative tosingle surfactant systems as they are independent of orientationof molecule within the micelles. Since in DTAB-Brij35 mixed sys-tem hydrogen bonding extent of Rutin with OE groups would notbe affected as observed in SDS-Brij35 system, the former possesseshigher MSR value for Rutin than latter. Moreover, orientation effectin former would be favorable for reaction between Rutin and DPPHwhich is highly affected the latter, thus reducing the activity of Ru-tin compared to that in DTAB-Brij58 system.

4. Conclusion

Although it is widely recognized that micellar media couldinfluence the reducing action of flavonoids, only a few studies havebeen devoted towards understanding surfactant-flavonoid interac-tions [25–33]. In the present work, an attempt has been made toexplore a correlation between the extent of solubilization of Rutinin single/mixed binary surfactant systems and its antioxidantactivity against DPPH in such micro-heterogeneous media, whichhas been neglected so far.

Hydrocarbon chain length and oxyethylene content have beenfound to be key factors responsible for Rutin solubilization withincationic and non-ionic micelles respectively. However, electro-static repulsion makes anionic micelles least efficient for Rutinsolubilization. Mixed surfactant systems exhibited better solubili-zation capacity than single component systems indicating a favor-able mixing effect on solubilization of Rutin as well as DPPH.

DPPH scavenging activity of Rutin in all the selected micellarsystems was smaller as compared to that in methanol. The activitywas found to be in direct correlation with the solubilizing effi-ciency of cationic surfactants of varying chain length towards bothRutin as well as DPPH. However, higher activity of Rutin in SDSthan in DTAB having comparable chain length was attributed tomore favorable orientation of Rutin within SDS micelles. StrongerH-bonding effect of Rutin with non-ionic Brij micellar systemswas observed to be a key factor for their low RSA within such sys-tems. The activity in binary cationic–non-ionic surfactant systemscorrelated well with the solubilizing efficiency of these surfactantsfor both Rutin and DPPH. However, in the equimolar SDS-Brij35surfactant system, the activity was in-between that seen for theirsingle component systems.

In conclusion, the results presented in the paper are expected tocontribute significantly in understanding interactions of naturalflavonoid antioxidants with interfaces modeled by different surfac-tant systems and interpreting reactions with free radicals in micro-heterogeneous environments present in a wide range of foods, cos-metics, pharmaceuticals, etc.

Acknowledgments

We are thankful to Head, Department of Chemistry, Universityof Kashmir, for providing the laboratory facilities and his constantencouragement and inspiration. OAC (JRF) acknowledges the finan-cial support, from the University Grants Commision, India underRFMS.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jcis.2010.11.044.

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