Erm

OD

h

a

ARRAA

KMDRAC

1

oph

(

0h

Colloids and Surfaces A: Physicochem. Eng. Aspects 436 (2013) 343– 353

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces A: Physicochemical andEngineering Aspects

jo ur nal ho me page: www.elsev ier .com/ locate /co lsur fa

valuation of reduction kinetics of 2,2-diphenyl-1-picrylhydrazyladical by flavonoid glycoside Rutin in mixed solvent basedicellar media

yais Ahmad Chat, Muzaffar Hussain Najar, Aijaz Ahmad Dar ∗

epartment of Chemistry, University of Kashmir, Srinagar 190006, J&K, India

i g h l i g h t s

• We report effect of micellar mediain mixed solvent on DPPH reductionkinetics.

• DPPH reduction was faster in postmicellar range except in non-ionicsurfactant.

• Binary surfactant mixtures werehighly efficient compared to singlesystems.

• Partitioning and interaction modeof Rutin had great effect on DPPHkinetics.

g r a p h i c a l a b s t r a c t

We report effect of micellar aggregates on DPPH reduction kinetics by antiradical Rutin.

r t i c l e i n f o

rticle history:eceived 8 May 2013eceived in revised form 19 June 2013ccepted 21 June 2013vailable online xxx

a b s t r a c t

The work evaluates the reduction kinetics of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical by Rutin indifferent mixed solvent based micellar media comprising of various cationic, anionic, zwitterionic, non-ionic and their binary mixtures using spectrophotometric, fluorimetric and tensiometric methods. Theanalysis of kinetic measurements in micellar systems was done using models proposed by Goupy et al.and Sendra et al. applicable to initial and overall DPPH reduction kinetics respectively. The rate constants

eywords:icellesPPHutinntioxidantMC

observed in the methanol–water media containing micelles (except in cationic micellar medium) weresignificantly faster than those without micelles suggesting the use of such media as efficient systems forevaluation of DPPH activity in comparison with the pure organic solvents having environmental con-cern. Rate of DPPH reduction was dependent on nature as well as concentration of amphiphile used.The results were explained on the basis of compartmentalization and mode of interaction of Rutin withmicellar nanostructures.

© 2013 Elsevier B.V. All rights reserved.

. Introduction

Antioxidants exhibit a pivotal role in detoxifying the reactive

xygen species (ROS) produced during the normal cell aerobic res-iration [1–3]. Flavonoids are generally believed to provide certainealth benefits due to their antioxidant activity. Rutin, a flavonoid

∗ Corresponding author. Tel.: +91 194 2424900; fax: +91 194 2421357/2425195.E-mail addresses: aijaz n5@yahoo.co.in, aijazdar@kashmiruniversity.ac.in

A.A. Dar).

927-7757/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.colsurfa.2013.06.035

glycoside is reported to exhibit several beneficial pharmacologi-cal properties [4,5], due to its high radical scavenging activity andantioxidant capacity [4–6].

Among the different published methodologies [7] for deter-mining the antiradical activity, the 2,2-diphenyl-1-picrylhydrazyl(DPPH) assay has been widely used due to its simplicity. Literature[8–13] reveals the influence of the concentration of DPPH, incuba-

tion time, reaction solvent, pH of the reaction mixture and possiblerole of deprotonation of the phenolic antioxidants on the kineticsof the DPPH/antioxidant reaction. Due to the poor aqueous solu-bility of DPPH, the antioxidant capacity of potential antioxidants

3 hysicochem. Eng. Aspects 436 (2013) 343– 353

iImbowm

eeatahmlhtacmptiDantibawmmrfioa

frierfRstkbbdio

2

2

dd1mp

Fig. 1. Plots of surface tension versus logarithm of surfactant concentration in 1:1

44 O.A. Chat et al. / Colloids and Surfaces A: P

s usually evaluated in less environment friendly organic solvents.n order to maximize the employment of eco-friendly aqueous

edium in DPPH radical scavenging assay, various attempts haveeen made to solubilize the DPPH in such medium like preparationf stable water-soluble DPPH nanoparticles [14], use of bufferedater–alcohol mixtures [12] or employment of aqueous micellaredia.Increasing concern on understanding the parameters that influ-

nce the activity of antioxidants in complex or multiphase systemsncountered in food and biological assemblies [15–19], micellesnd other disperse systems have been widely used for modellinghe effects of heterogeneous environments on reaction dynamicsnd mechanism [20–23]. In this endeavour, a few authors [24,25]ave attempted to develop DPPH method in pure aqueous micellaredia. One serious problem encountered during these studies is the

imited solubilization tendency of micelles towards DPPH [24]. Weave previously reported that to get the aqueous solubility of DPPHo almost 60 �M the surfactant concentration needs to be very highlthough use of mixed surfactant systems drastically decreases thisoncentration to achieve the same level of solubilization. Since inost of the reports the concentration of 100 �M is used during the

rotocols, the use of some organic solvents is enforced to achievehe desired concentration of DPPH in the solution. Even in the stud-es by the Nopia et al. [25] in aqueous micellar systems, use of 50 �MPPH in 2 mM aqueous CTAB solution was made possible with theid of methanol though CTAB at this level of concentration doesot solubilize that much of DPPH [24]. Therefore, it is demandedhat the effect of micelles on DPPH reduction kinetics be followedn the methanol–water system where the following advantges cane envisaged: (a) the concentration of DPPH can be maintianed at

desired level due to its appreciable solubility in such systems, (b)idely used methanol during various protocols can be replaced byore environmental friendly water–methanol mixture, (c) use oficelles during DPPH assay mimics the microheterogenous envi-

onments prevalent in complex food and biological systems andnally (d) relevance to the DPPH radical scavenging assays carriedut in methanol for the extracts containing inherent water contentnd other possible amphiphilic molecules.

Keeping in view the above facts, we report the effect of sur-actants in their single and mixed states on the kinetics of DPPHeduction by the antiradical activity of standard antioxidant Rutinn methanol–water system. Mainly the focus has been on (a) theffect of varying nature of surfactants in both pre and post-cmcegions, and (b) effect of mixed micelles of the surfactants of dif-erent hydrophilic groups on the radical scavenging activity ofutin against DPPH and its kinetics. Two different models corre-ponding to initial [26] and overall kinetics [27] were used to fithe experimental data and attempts were made to correlate theinetic parameters calculated using these models. This study maye important in understanding the reduction kinetics of DPPH iniomimetic systems using mixed solvent system and helpful ineveloping a standardized method to estimate antioxidant activity

n more relevant biological systems without using large volumes ofrganic solvents.

. Experimental

.1. Materials

The amphiphiles polyoxyethylene (4) lauryl ether (Brij30),odecyldimethylethylammonium bromide (DDEAB), sodium

odecylsulfate (SDS), N-dodecyl-N,N-dimethyl-3-ammonio--propanesulphonate (DDAPS) and spectrophotometric gradeethanol were all Aldrich products, and used as received. The

urity of the surfactants was further ensured by the absence

water–methanol medium: (a) zwitterionic and ionics, (b) Brij30 and binary surfac-tant systems.

of minima in surface tension (�) versus log [surfactant] plots(Fig. 1). The antioxidant Rutin trihydrate (Rutin, >95%) and theradical 2,2-diphenyl-1-picrylhydrazyl (DPPH, >98%) were alsoAldrich products (Scheme 1). Surfactant solutions were preparedin triple-distilled water.

2.2. Methods

2.2.1. Determination of cmcThe cmc values of single and equimolar mixed surfactant solu-

tions in 1:1 methanol–water (M–W) were determined from the plot

O.A. Chat et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 436 (2013) 343– 353 345

structu

o(pscsmpocha

2

atwiw

2

wrwJc

Scheme 1. Chemical

f surface tension (�) versus logarithm of surfactant concentrationlog Ct) (Fig. 1). Surface tension measurements were made by thelatinum ring detachment method with a Krüss-9 (Germany) ten-iometer equipped with a thermostable vessel holder. Surfactantoncentration was varied by adding solution (1:1 M–W) of knownurfactant concentration in small instalments using a Hamiltonicrosyringe to 20 cm3 of 1:1 M–W mixture in the sample vessel

laced in the vessel holder. Measurements were made after thor-ugh mixing and temperature equilibration at 25 ◦C (±0.1 ◦C) byirculating water from a HAAKE GH thermostat through the vesselolder. The accuracy of measurements was within ±0.1 dyn cm−1

nd the readings were taken in triplicate to ensure reproducibility.

.2.2. Fluorescence measurementsThe fluorescence emission spectra of Rutin were recorded with

n RF-5301PC Schmadzu spectrofluorometer (Japan) operating inhe steady-state mode at 25 ± 0.1 ◦C in the range of 400–600 nmith excitation wavelength at 445 nm. Measurements were made

n 1 cm path length quartz cuvette with excitation and emission slitidth of 5 mm.

.2.3. Kinetic measurementsThe working solutions of antiradical Rutin and radical DPPH

ere freshly prepared before analyses. The rate constant for the

eaction between DPPH and an antioxidant in the micellar systemas monitored spectrophotometrically (UV-1650PC, Schmadzu,

apan) using optical path length of 1 cm. The temperature in theell was kept at 25 ± 0.1 ◦C by circulating water from Brookfield

re of materials used.

TC 2010 thermostat through vessel holder. Typical procedure wasperformed as follows. To a 1.5 ml of a given aqueous surfactantsolution, 1.5 ml of 200 �M DPPH prepared in methanol was addedto make the final volume of 3 ml followed by the stirring for a periodof 5 min. The reaction was initiated by adding x ml (0.01–0.05 mL)of 3 mM Rutin previously prepared in methanol to the cuvette. Thefinal concentration of DPPH in the system was 100 �M and the vol-ume percentage of methanol in the medium amounts to 50 ± 1%.The kinetics for the reaction between DPPH and an antioxidantin single and equimolar mixed surfactant systems was monitoredusing a Schimadzu spectrophotometer (Model UV-1650). The decayin absorbance at 515 nm was measured for a maximum of 1500 s. Allexperiments were performed in triplicate and the kinetic parame-ters were calculated as a mean of three independent measurementscorresponding to each Rutin concentration according to the mod-els proposed by Goupy et al. [26] and Sendra et al. [27]. Finallythe kinetics parameters in each surfactant system were reportedas average of the values obtained corresponding to different Rutinconcentrations.

Erstwhile to the kinetic experiments, calibration curve ofabsorbance versus concentration of DPPH in 1:1 M–W was obtainedto determine the molar extinction coefficient (ε) of DPPH. Fromthe linear fitting of data, the values determined for ε was0.73 × 104 L/(mol cm). The value was utilized to obtain the concen-

tration of DPPH at various times wherever required. As a prototype,variation of DPPH spectrum with time after initiating reaction withRutin and the variation of concentration of DPPH (determinedfrom absorbance values at 515 nm) with time corresponding to

346 O.A. Chat et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 436 (2013) 343– 353

300 350 400 450 500 550 600 6500.0

0.5

1.0

1.5

2.0

Ab

so

rban

ce

(nm)

Without Brij 30

T= 0 mins.

T= 25 mins.

300 350 400 450 50 0 55 0 60 0 65 00.0

0.5

1.0

1.5

2.0

T= 25 min s.

Ab

so

rba

nc

e

(nm)

With Brij 30

T= 0 min s.

Fi

di

2

mmtcf

R

wira

2

2

Dma

0 50 0 100 0 150 0

0

20

40

60

80

100 0.01 mM

0.01 5

0.02

0.03

DP

PH

co

nc

en

tra

tio

n (

µM

)

BRIJ 30

0 50 0 100 0 150 0

20

40

60

80

100

0.01 mM

0.01 5

0.02

0.03

DP

PH

co

nc

en

tra

tio

n (

µM

)

DDEAB

0 10 0 20 0 30 0 40 0 50 0 60 0

20

40

60

80

100

0.01 mM

0.01 5

0.02

0.03

0.03 5

DP

PH

co

nc

en

tra

tio

n (

µM

)

Time (sec )

DDEAB+BR IJ 30

ig. 2. Variation of DPPH spectrum with time during its reaction with Rutin (20 �M)n 1:1 M–W medium without and with Brij30 (0.15 mM) surfactant.

ifferent concentrations of Rutin in some systems are presentedn Figs. 2 and 3, respectively.

.2.4. Evaluation of DPPH scavenging activity of RutinThe solutions were prepared as in kinetic studies. The 1:1 M–W

icelle solution was used as a blank solution. The DPPH in 1:1 M–Wicellar solution without an antioxidant was used as a control solu-

ion. The antiradical activity at each concentration of Rutin wasalculated as the percentage of DPPH decolouration [28] using theollowing equation:

SA = 100 ×(

1 − Aa

A0

)(1)

here Aa is the absorbance of DPPH in presence of Rutin and A0s the absorbance of DPPH without Rutin. The experiments wereepeated three times corresponding to each Rutin concentrationnd the resultant RSA data are presented as mean ± SE.

.3. Kinetic models

.3.1. Initial kinetics (Model 1)

In order to obtain information about pathways involved in the

PPH reduction by Rutin, the reaction parameters were deter-ined. DPPH absorbance at 515 nm decays quickly with potent

ntioxidants over 1–2 min as a result of the transfer of the most

Fig. 3. Time evolution of DPPH concentration in various surfactant systems abovecmc range.

labile H atoms of the antioxidant (fast step). This step is typicallyfollowed by a much slower decrease in visible absorbance, featu-ring the residual H-donating ability of the antioxidant degradationproducts (slow step). It is worth mentioning here that only the faststages of the reaction (100 s) were detailed in terms of kinetic analy-sis (Scheme 2), where it is commonly accepted that the stabilizationof DPPH radicals results from the transfer of the most labile H atomsof the antioxidants (�-OHs) [29].

The rate constant (k) of the reaction was determined in all thesystems at 25 ◦C by monitoring the decay of DPPH absorbance at515 nm. The rate of DPPH reduction under these conditions can bewritten as follows [26,29]:

−d[DPPH]dt

= k[�-OH][DPPH] (2)

Following the method of Goupy et al. [26], the values of k canbe calculated from Eq. (3) which is valid for second order kinetics

O.A. Chat et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 436 (2013) 343– 353 347

Scheme 2. Reduction of DPPH radical by Rutin (first step), IV reacts further [27].

-5

-4

-3

-2

-1

0

0 10 20 30 40 50 60

Rutin= 0.015 mM

ln(1

-Ab

sf/A

bs)/

(1-A

bs

f/A

bs

o)

Time(s)

FD

itr

l

n

Taiowc

2

ddop

y

Table 1Critical micelle concentration (cmc) values of various single and binary surfactantssystems in 1:1 M–W and pure aqueous (cmcaq) systems. Also cmcideal is cmc of binarysurfactant systems obtained using Clint equation in M–W system at 25 ◦C.

System cmc (mmol dm−3) cmcaq (mmol dm−3) cmcideal (mmol dm−3)

Brij30 0.056 0.0382a

DDEAB 119.8 13.2a

SDS 75.9 7.59b

DDAPS 38.6 3.4c

DDEAB + Brij30 0.54 – 0.11SDS + Brij30 0.45 – 0.11DDAPS + Brij30 0.41 – 0.11

Error limits of cmc was ±7%.

ig. 4. Prototype plot showing determination of rate constant (k) for Rutin inDAPS + Brij30 surfactant system.

n the early stages of reaction (Scheme 2). The method is also usedo calculate number of reduced DPPH during the early stages of theeaction per molecule of antioxidant (nes) using Eq. (4).

n

(1 − Absf/Abs1 − Absf/Abs0

)= −k

Absf

εt (3)

es = Abs0 − Absf

[�-OH]0ε(4)

he Abs0 and Absf correspond to initial and final (100 s) values ofbsorbance, respectively. When ln[(1 − Absf/Abs)/(1 − Absf/Abs0)]s plotted versus time, a straight line with zero intercept is obtainedver most of the duration of the fast step, divergence occurring onlyhen Abs gets close to Absf (Fig. 4 as a prototype). The slope of the

urve readily gives access to k.

.3.2. Overall kinetics (Model 2)Fig. 3 shows the time evolution of the concentration of DPPH

uring its reduction by different initial concentrations of Rutin inifferent surfactant media. The sets of experimental data pointsbtained from Fig. 3 were fitted using the following kinetic equationroposed by Sendra et al. [27]. In brief the equations involved were:

− ys = y1(y0 − y1)y1 − y0(1 − e(k1/�1)y1t)

+ y2(y0 − y2)y2 − y0(1 − e(�1/�2)y2t)

(5)

a Ref. [30].b Ref. [24].c Ref. [31].

With the constraints:

y2 = y0 + ys − y1 = y0 − �2a0

y0 − y1 = �1a0

y0 − ys = (�1 − �2)a0

(6)

where y is the time-dependent concentration of DPPH radical, y0 isthe initial concentration of DPPH, a0 is the initial concentration ofthe antiradical, t is the reaction time, k1 is the fast kinetics rate con-stant, y1 is the asymptote that would be reached due solely to thefast kinetics antiradical activity, �2 is the slow kinetics pseudo-rateconstant, y2 is the asymptote that would be reached due solely tothe slow kinetics antiradical activity, ys is the experimental asymp-tote of the reaction, and �1 and �2 are the stoichiometric constantsof the fast and slow kinetics, respectively.

Both the kinetic models were fitted in case of DPPH reductionby Rutin in all the micellar systems assuming that basic kineticequations are unaltered in presence of micellar aggregates whichcould also be ascertained by goodness of fit (values of regressioncoefficient), which were close to 1 (0.995–0.999) for all data sets.

3. Results and discussion

3.1. cmc of surfactants in 1:1 methanol–water

The cmc values of selected single and equimolar mixed sur-factant systems, obtained from surface tension versus logarithmof surfactant concentration plots are presented in Table 1 alongwith the ideal cmc values, cmcideal, of binary surfactant systemsbased on the Clint equation [32]. In presence of methanol, mice-llization gets delayed which is clear from the higher values of cmc

obtained for each surfactant system. It is important to mention herethat methanol has only one hydrogen bonding centre in its struc-ture and therefore incapable of forming three-dimensional solventcages as is the case of water. Instead, the methanol molecules form

348 O.A. Chat et al. / Colloids and Surfaces A: Physico

Table 2Kinetic parameters during early stages of DPPH reduction by Rutin in pre and postmicellar ranges at 25 ◦C (Model 1).

System Pre-cmc Post-cmc

k nes n total k nes n total

Methanol–water 0.30 1.3 4.1Brij30 1.00 2.0 3.9 0.99 1.7 4.1DDAPS 0.29 2.0 2.9 0.54 1.4 3.1DDEAB 0.03 2.0 4.0 0.22 1.4 3.5SDS 0.27 1.7 2.6 – – –DDAPS + Brij30 0.31 1.8 2.6 1.12 2.0 4.5DDEAB + Brij30 0.30 1.5 2.0 0.52 1.5 1.9SDS + Brij30 0.32 1.4 2.1 1.40 2.3 3.9

Wc

otsssTctfasiprtsittcpfitaSorfgaictsncm

3

aa(ccdBt

here units of k = (L mmol−1 s−1) and nes = (mmol DPPH/mmol Rutin). Error in thealculation of k, nes was ±0.02, ±0.3, respectively.

nly the two dimensional network to an extent determined byhermal agitation. Because of this, less disruption of the solventtructure is necessary to accommodate the hydrocarbon chain ofurfactant molecule making methanol–water a better solvent forurfactant molecules leading to delay in micellization process [33].he variation of surface tension (�) versus logarithm of surfactantoncentration (log Ct) depicted in Fig. 1 reveals different behaviourhan observed in pure aqueous system. Instead of decrease in sur-ace tension with addition of surfactant, it initially remains constantnd thereafter shows usual decrease. The initial constant values ofurface tension indicate the reduced surface activity of amphiphilesn this medium due to less polarity of mixed solvent system thanure water and hence higher solubility of surfactants. Therefore,elatively higher concentration of surfactant is required to saturatehe surface of solvent leading to higher values of cmc. In case ofingle surfactant systems, except for Brij30 surfactant where risen cmc value was only 1.5 times as reported for TX-100 surfac-ant in ethanol–water system [34], the cmc increases almost byen-fold compared to that in aqueous medium. Therefore, largeoncentration (two times cmc) of each surfactant was needed toroduce stable micellar aggregates in the M–W reaction mediumor making the solution micro-heterogeneous (>80 mM in case ofonic/zwitterionic surfactants). This requirement of large concen-ration of surfactants in M–W system was drastically reduced (tobout 1 mM) by using mixed surfactants in the mole ratio of 1:1.ince it is known that the self-repulsions between the head groupsf ionic surfactants in the mixed micelles are reduced by incorpo-ation of non-ionic surfactants and are simultaneously replaced byavourable ion-dipole interactions between the hydrophilic headroups of ionic and non-ionic surfactants, mixed micellization islways highly favoured in such situations. This leads to the signif-cant reduction in cmc values compared to the ionic/zwitterionicounterparts as evident from the data in Table 1 in M–W sys-em. Therefore, to produce microheterogeneous medium in M–Wystem using minimal concentration of surfactants and simulta-eously attaining the good concentration of micelles with similarharge as in case of pure ionic/zwitterionic micelles, the employ-ent of mixed micelles in the protocol was an appropriate choice.

.2. Reduction kinetics of DPPH in micellar media

Model 1: Table 2 presents the rate constant (k), and nes in prend post micellar regions for DPPH reduction reaction (Scheme 2)s an average corresponding to different concentrations of Rutin0.01–0.04 mM). The results show that the number of DPPH radi-als reduced by Rutin during the early stages of reaction and the

orresponding rate constants of hydrogen abstraction kinetics isependent on the type of micelles in reaction medium. Whilerij30, DDAPS, DDEAB, SDS and DDAPS + Brij30 favour transfer ofwo labile H atoms (nes = 2 ± 0.2), DDEAB + Brij30 and SDS + Brij30

chem. Eng. Aspects 436 (2013) 343– 353

favour transfer of only one labile H atom (nes = 1 ± 0.4) as observedin M–W system without surfactant. From Table 2, it can be seen thatthe values for the parameter nes are fractional suggesting presenceof additional pathway for DPPH stabilization [35]. In addition tonumber of H atoms transferred to DPPH radical, the rate constant kis a key parameter to help in understanding the underlying mecha-nism of the DPPH reduction reaction. It is apposite to mention herethat stability of DPPH radical was checked in a given surfactantmedium prior to addition of Rutin. DPPH was found to be stable inall the surfactant systems ensuring that the decay in absorbanceduring reaction was solely due to the antiradical activity of Rutin.However, in case of SDS above cmc (at 150 mM) the DPPH showedinstability and as such the reaction could not be carried out in post-cmc region of SDS, the reason of which could not be figured out. Inpre-cmc region of all the given surfactant solutions, the values of kdid not change appreciably except in presence of Brij30 and DDEABsurfactants. This reveals that the surfactants in monomeric form donot affect kinetics of DPPH reduction by Rutin. It is evident if a givensurfactant stabilizes reactants (Scheme 2), then the value of k willbe lower while stabilization of products will yield a higher valueof k. In pre-cmc conditions, the values of k among single surfac-tant systems are high in Brij30 and low in DDEAB than obtainedin pure M–W mixture. Stabilization of Rutin and DPPH by DDEABmonomers is one of the possible reasons for lower value of k inbelow cmc range. DDEAB has positively charged head groups andmay be thought to aggregate around the negatively charged Rutin[36] forming possible loose aggregates which reduce the active con-centration of antiradical in bulk, thereby decreasing rate of reaction.In case of Brij30 surfactants in pre-cmc region, the acceleration ofDPPH reduction could be due to the formation of pre-aggregatesincorporating the DPPH and Rutin thereby increasing their localconcentration. It has been reported that the cmc in most of the sys-tems marks the onset of the increase in rate of reaction but the ratescould also increase below cmc, either because reactants inducemicellization or because other species, so-called premicelles, arekinetically effective [37].

The rate of DPPH reduction in post-cmc regions gets enhancedsignificantly compared to that observed in pre-cmc regions (k val-ues in Table 2). It shows that the presence of microheterogenietyin the solvent in the form of colloidal nanoaggregates of surfac-tants plays a vital role, the effect being dependent on the nature ofsurfactant. In our earlier report [24] pertaining to the DPPH reduc-tion in pure aqueous micellar media, it was inferred that DPPHis exclusively solubilized within micelles due to its poor watersolubility while as Rutin gets distributed between micellar andaqueous phases thereby leading to their interaction only withinthe micelles. However, in the present study wherein micelles arepresent in 1:1 M–W system the reaction between radical andantiradical (Scheme 2) is expected to occur both within and out-side the surfactant assemblies. The rate of reaction highlighted inTable 2, therefore, has contribution from extent of reaction occur-ring within and outside the micro-reactors. The hydrogen-transferrate constants of different phenols get affected by the nature ofsolvent [38] irrespective of the nature of attacking free radical[39]. In this context, the contribution of reaction occurring outsidethe micelles (say R1) towards observed rate constant is expectedto be similar in every micellar systems since the solvent in eachcase is the same. However, due to the difference in the natureof micelles/micelle–solvent interface in different micellar systems,the reaction occurring inside/at the interface of the micelles (sayR2) would have different contributions towards the observed rateconstants. Hence, the variation in rate constant in different micellar

systems could be justified by the factors that affect the R2. It hasbeen reported by Noipa et al. [25] that the DPPH radical scaveng-ing rate by the antiradicals gets enhanced in CTAB micellar solutioncompared to that in methanol due to incorporation of DPPH inside

O.A. Chat et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 436 (2013) 343– 353 349

450 50 0 55 0 60 0 65 0

0

50

100

150

200

250 Butanol

Methanol

Dioxa ne

1:1 M-W

Water

em/nm

Flu

ore

sc

en

ce

In

ten

sit

y(a)

0

20

40

60

80

Flu

ore

sc

en

ce

In

ten

sit

y

475 50 0 52 5 55 0 57 5 600

0

10

20

30

40

50

60

70

80[DDEAB ] (mM)

0

12 0

17 1

22 1

31 0

35 0

Flu

ore

sc

en

ce

In

ten

sit

y

em /n m

(d)

475 50 0 525 55 0 57 5 60 0

0

10

20

30

40

50

60[DDAPS] (mM)

0

6

17

27

42

55

62

65

Flu

ore

sc

en

ce

In

ten

sit

y

em /nm

(c)

475 50 0 525 55 0 575 600

0

5

10

15

20

25

[Brij30] (mM)

0

0.04

0.23

0.40

0.57

0.72

0.86

0.99

Flu

ore

sc

en

ce

In

ten

sit

y

em/nm

(b)

F solven(

tinbhfmfeDa(of

ttDTtIsstT

ig. 5. Variation of fluorescence intensity of Rutin with emission wavelength in (a)d) DDEAB surfactants.

he hydrophobic core of the micelle consequently leading to eas-er abstraction of phenol H-atom by DPPH being favoured by theon-polar environment. Thus, it can be concluded that R2 woulde greater than R1 in present micellar systems due to favourableydrophobic environment. Further, contribution to overall rate by

aster R2 will increase if partitioning of reactants is efficient inicellar pseudophase. The reduction reaction will, therefore, be

astest in surfactant systems with greater solubilization and hencefficient partitioning capabilities. Owing to these reasons the rate ofPPH reduction, in general, is accelerated in presence of the nano-ggregates of the surfactants as revealed by the higher values of kTable 2) except in Brij30 wherein the rate of H transfer increasednly slightly than in pre-cmc conditions, a possible consequence oformation of pre-aggregates in such system below cmc[40].

In single surfactant micellar solutions wherein the concentra-ion of surfactant was kept two times their cmc values to ensurehe presence of stable micelles inside the solution, the rate ofPPH reduction showed the order Brij30 > DDAPS > M–W > DDEAB.he reaction could not be monitored in the post-cmc concentra-ion of SDS due to the instability of DPPH as mentioned earlier.n order to explain the variation, it is important to find out the

olubilization site of Rutin and DPPH in micellar solutions. Theolubilization site of DPPH can be envisaged by the shift in absorp-ion maximum in UV-spectrum as a function of solvent polarity.he absorption maximum of DPPH shifted from 516 nm in pure

ts of different polarities and at various concentrations of (b) Brij30, (c) DDAPS and

methanol to 524 nm in 1:1 M–W system indicating that increasein polarity induced bathochromic shift in DPPH. Therefore, its sol-ubilization in the micellar core would decrease the absorptionmaximum of DPPH. However, in all the surfactant systems in theirpre- and post-cmc concentrations in 1:1 M–W system, the absorp-tion maximum remained fairly constant at 524 nm indicating thatthe DPPH mainly remains in the bulk phase and/or at the interface ofmicelles. Since the shift in wavelength of maximum emission (�em)and/or increase in the fluorescence intensity of a compound duringfluorescence measurements respond better to the microenviron-mental variation, Rutin was elucidated by fluorescence techniquesince the molecule is fluorescent. Fig. 5a shows the emission spec-tra of Rutin in solvents of different polarities and/or viscosities.Rutin presents very small fluorescence intensity in aqueous envi-ronment possibly due to the non-radiative decay processes thatare usually most prevalent in aqueous environment [41]. Rutinshows bathochromic shift in �em with the increase in polarity ofthe solvent from dioxane to 1:1 M–W system, being large fromdioxane (511 nm) to butanol (524 nm) and relatively slight shiftfrom butanol to methanol (526 nm) and then to 1:1 M–W (531 nm)solvents. However, increase in fluorescence intensity shows a negli-

gible correlation with solvent polarity but shows strong correlationwith the dynamic viscosity of the solvent. Large increase in the flu-orescence intensity of Rutin in butanol, having dynamic viscosityof 2.45 cP [42], relative to other solvents, having dynamic viscosity

350 O.A. Chat et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 436 (2013) 343– 353

Table 3Kinetic parameters of DPPH reduction by Rutin in pre and post micellar ranges at 25 ◦C (Model 2).

System Pre-cmc Post-cmc

k1 �2 �1 �2 �1 + �2 k1 �2 �1 �2 �1 + �2

Methanol–water 0.26 0.12 1.9 1.5 3.4Brij30 1.13 0.10 1.4 1.7 3.1 1.01 0.03 3.0 1.5 4.4DDAPS 0.27 0.02 1.4 1.8 3.2 0.56 0.36 2.9 0.9 3.8DDEAB 0.03 0.03 1.8 1.8 3.5 0.23 0.15 1.8 1.3 3.1SDS 0.30 0.17 1.4 0.9 2.3 0.0DDAPS + Brij30 0.30 0.14 0.8 1.7 2.5 1.15 0.06 2.0 1.0 3.0DDEAB + Brij30 0.29 0.11 0.9 1.1 2.0 0.51 0.18 1.5 0.5 2.0

2.

W , ±0.3

lpn

fltii

SDS + Brij30 0.31 0.17 1.0 1.1

here units of k and �2 = (L mmol−1 s−1). Error in the calculation of k, nes was ±0.02

ess than 1 cP, indicates that the restricted medium produces sup-ression of the vibronic modes of Rutin that provide pathways foron-radiative transitions between excited and ground states.

In order to study the effect of surfactant self-assemblies on theuorescence of Rutin and hence its possible solubilization within

he micelles, the surfactant concentration was gradually increasedn the 80 �M Rutin solution in 1:1 M–W solution. As depictedn Fig. 5, fluorescence intensity shows an exponential decay with

0.010 0.01 5 0.020 0.02 5 0.03 0 0.03 5

10

20

30

40

50

60

70

80

DP

PH

ac

tiv

ity

Rutin (mM )

Methan ol- wate r

DD EAB

DD APS

BR IJ 30

(a)

DP

PH

ac

tiv

ity

0.010 0.01 5 0.020 0.02 5 0.03 0 0.03 5

10

20

30

40

50

60 (c)

DP

PH

ac

tiv

ity

Rutin (mM )

DDEA B

BRIJ3 0

DDEAB+ BRIJ3 0

Fig. 6. DPPH scavenging activity of Rutin in (a) single, (b) binary and (c and d) in di

1 1.30 0.11 2.4 1.5 3.8

, respectively.

concentration of Brij30 (Fig. 5b) while it remains constant upto cmcand then abruptly increases with the concentration of both DDAPS(Fig. 5c) as well as DDEAB (Fig. 5d). Moreover a blue shift in theemission maximum of Rutin is observed in Brij30 surfactant systemwhile as emission maximum is red-shifted in both DDEAB as well as

in DDAPS relative to that of in 1:1 M–W solvent. Since it in known[43] that oxyethylene groups possess high affinity for water andundergo hydration, the Rutin might be getting solubilized within

0.010 0.01 5 0.020 0.02 5 0.03 0 0.03 5

10

20

30

40

50

60

70 (b)

Rutin (mM )

Methan ol- wate r

DDE AB+BR IJ3 0

DDA PS+BR IJ30

SDS +BRIJ 30

0.010 0.01 5 0.02 0 0.025 0.03 0

10

20

30

40

50

60

70

80 (d)

DP

PH

ac

tiv

ity

Rutin (mM )

DDAPS

BR IJ30

DDA PS+BR IJ30

fferent single and binary surfactant systems in 1:1 methanol–water solvent.

O.A. Chat et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 436 (2013) 343– 353 351

0.010 0.015 0.020 0.025 0.030

0

10

20

30

40

50

60

0.010 0.015 0.020 0.025 0.030

0

10

20

30

40

50

60

70

80

0.010 0.015 0.020 0.025 0.030

0

10

20

30

40

50

60

70

80

90

0.01 0.02 0.03 0.04 0.05 0.06

0

10

20

30

40

50

60

0.01 0.02 0.03 0.04 0.05

0

10

20

30

40

50

60

70

0.01 0.02 0.03 0.04 0.05

0

10

20

30

40

50

60

70

DP

PH

acti

vit

y

Rutin (mM)

DDEAB pre cmc DDEAB post cmc

DP

PH

acti

vit

y

Rutin (mM)

DDAPS pre cmc

DDAPS post cmc

DP

PH

ac

tiv

ity

Rutin (mM)

BRIJ 30 pre cmc

BRIJ 30 post cmc

DP

PH

ac

tiv

ity

Rutin (mM)

DDEAB+BRIJ 30 pre cmc

DDEAB+BRIJ 30 post cmc

DP

PH

ac

tiv

ity

SDS+BRIJ 30 pre cmc

SDS+BRIJ 30 post cmc

DP

PH

ac

tiv

ity

DDAPS+BRIJ 30 pre cmc

DDAPS+BRIJ 30 post cmc

micell

otottpw

Rutin (mM)

Fig. 7. DPPH scavenging activity of Rutin in pre- and post

r near the oxyethylene groups in both pre-aggreates as well as inhe micelles of Brij30 leading to the quenching of its fluorescencewing to greater percentage of water in the headgroup region of

he surfactant—a usual phenomenon in aqueous medium. In addi-ion slight blue shift indicates that the Rutin is solubilized in lessolar environment than M–W system which is the palisade layerithin the micelles as micelle–solvent interface has less polarity

Rutin (mM)

ar range in different single and binary surfactant systems.

than the bulk solvent [44]. However, in case of DDEAB abrupt andrelatively more increase in the fluorescence intensity above cmccompared to that in DDAPS correlates to the solubilization of Rutin

to the more restricted environment having less access of water. We,therefore, infer the predominant solubilization site of Rutin to beslightly deeper into the palisade layer of micelles in case of DDEABwhile as within the palisade layer in case of DDAPS micelles.

3 hysico

stslofrthiozwfssimabitBfltra

tbtogiiotAoi(aiprSawtrceteicmtci

ssF

52 O.A. Chat et al. / Colloids and Surfaces A: P

Keeping in view the location of DPPH and Rutin in the micellarystems, we attempt to address the kinetic data of DPPH reduc-ion reaction. It is evident from data in Table 2 that among singleurfactant systems the rate of DPPH reduction is faster in Brij30 fol-owed by DDAPS and DDEAB. The slowest rate in DDEAB is a resultf acquisition of different solubilization sites by Rutin and DPPHormer being intercalated deep into the palisade layer while latteresiding in bulk and/or at interface, resulting in segregation of reac-ants and hence appreciable decrease in the rate of reaction. Theigher reduction rate in DDAPS compared to DDEAB is a direct man-

festation of palisade layer solubilization of Rutin within micellesf former in contrast to slightly deeper palisade layer solubili-ation within DDEAB micelles, thereby facilitating its interactionith DPPH lying in the bulk and/or at the interface of micelles. The

astest rate of reduction in Brij30 micelles correlates well to theolubilization sites preferred by DPPH and Rutin molecules withinuch micelles. From the fluorescence data as stated above, Rutins solubilized within water rich oxyethylene head groups of Brij30

icelles having therefore easy access to DPPH molecules presentt the micelle–solvent interface leading to the maximum contactetween the two solubilizates and hence concomitant increase

n rate of DPPH reduction. It is pertinent to mention here thathe reaction between DPPH and Rutin is enhanced in DDAPS andrij30 micelles relative to that in simple M–W system due to the

avourable solubilization and hence catalytic action of such micel-ar nano-containers. However, DDEAB reduces the reaction relativeo that in M–W system due to separation of two reactants by favou-ing different solubilization of two reactants resulting in inhibitoryction of such micelles.

Mixing of surfactants drastically reduced the amount of surfac-ants used owing to severe reduction in cmc values for equimolarinary mixtures in M–W system (Table 1). Rate of DPPH reduc-ion in binary systems could be explained in analogy with thatf single surfactant systems having characteristic of the sin-le surfactant micelles. It is pertinent to mention that binaryonic + non-ionic mixtures are predominantly composed of non-onic component [30]. We attempted to investigate the influencef adding charge (cationic, zwitterionic and anionic surfactants)o Brij30 micelles and consequent effect on DPPH reduction rate.ll the binary surfactant mixtures studied showed enhanced ratef reaction compared to the ionic/zwitterionic counterpart follow-ng the order SDS + Brij30 > DDAPS + Brij30 > DDEAB + Brij30 > M–WTable 2). Additionally, it could be envisaged that the cat-lytic impact of Brij30 micelles gets modified in presence ofonic/zwitterionic surfactants. The reduction kinetics decreases inresence of DDEAB while as it is accelerated by DDAPS and SDSelative to that in presence of pure Brij30 micelles. In case ofDS + Brij30 system, the presence of SDS in mixed micelles cre-tes slight negative charge at the micelle–solvent interface. Thisould obviously lead to pushing of negatively charged Rutin more

owards the micelle–solvent interface leading to its enhancedeaction with DPPH at the interface. However, the solubilizationapability and depth of intercalation display slightly differentffect on reaction rate in DDAPS + Brij30 and DDEAB + Brij30 sys-ems. Although reaction rate should have increased to largextent in cationic/zwitterionic + non-ionic systems but the greaterntercalation capability of this system results in decreased rateompared to anionic + non-ionic mixed binary system. The highericelle–solvent positive charge density on DDEAB + Brij30 system

han DDAPS + Brij30 results in greater intercalation of negativelyharged Rutin explaining the consequent lowest reduction rate int among binary systems.

Radical scavenging activity (RSA), the rate at which antiradicalcavenges the radicals, was calculated using Eq. (1) for the earlytages of reaction and are depicted in the activity profiles shown inig. 6. The order of DPPH RSA of Rutin was same as that of the rate

chem. Eng. Aspects 436 (2013) 343– 353

of DPPH reduction, i.e. k calculated using model-1. The comparisonof RSA in single and binary systems (Fig. 7) was also supported bythe kinetic data. RSA in post micellar range was greater than in premicellar range with the exception of Brij30 as detailed in the kineticstudies.

Model 2: The nonlinear parametric fitting using equation 5 wasdone using solver in MS Excel-2010 and were excellent (r2 = 0.99).The kinetic parameters so obtained are given in Table 3. Averagestoichiometric constants (�1 and �2) reveal that number of DPPHmolecules reduced by a single molecule of Rutin is decreased inmicellar media compared to pure methanol. No correlation wasfound between type of surfactant system and values of �1 and �2.The average rate constant for the fast step (k1) calculated usingModel 2 (Table 3) were similar to the values of rate constant kcalculated using Model 1 (Table 2), applied to early stages of thereduction reaction, the differences in values may be due to differentset of approximations involved in the derivation of kinetic models.

The slow kinetics pseudo-rate constant (�2) obtained by fittingEq. (5) to the experimental data are presented in Table 3. In alco-holic solvents �2 is directly correlated with the rate constant k2 offormation of the first adduct with solvent corresponding to the sec-ond step of the reaction [27]. It is known that the solvent influencesthe value of the fast kinetics rate constant (k1), different mech-anisms have been proposed to explain this influence, while somerelate it to a differential stabilization of the charge in the DPPH rad-ical by hydrogen bonding from the solvent [38], others [9] proposethat this influence is due to the dielectric constant of the solventwhich modifies the pKa of phenols. The mechanism of the influ-ence of the solvent on the value of the slow kinetics pseudo-rateconstant (�2) is not so documented, but the work on the antiradicalactivity of the p-catechol group in protocatechuic acid alkyl estergives convincing information [45]. The addition of alcoholic solventto o-quinone intermediate regenerates the p-catechol group, andallows the reaction to go on. The values of �2 depend on the solventnature, interaction among solvents, nucleophilicity of solvent, sta-bilization of intermediate quinone by solvent, some other factorsmay come into play in presence of micellar assemblies like accessi-bility of nucleophile to quinone form which in turn is correlated topartitioning and orientation of substrate, all these factors taken intoconsideration may help to reason the values presented in Table 3.

4. Conclusion

The kinetic parameters of DPPH reduction by Rutin in 1:1 M–Wsolvent obtained using two models were function of both natureand concentration of surfactant. The compartmentalization of Rutinwithin nano-aggregates in mixed solvent system proved decisivein determining rate of DPPH reduction in bulk/interface region.Reduction in binary systems, viz. SDS + Brij30 and DDAPS + Brij30was highly efficient compared to their individual componentswhile as the rate constant in DDEAB + Brij30 mixed micelles wasobserved to be intermediate between the individual components.This result reveals the importance of hydrophobic and electro-static interactions of antiradicals of biological importance with themicelles that modulate the reduction kinetics of DPPH. Therefore,it can be concluded that binary combinations of proper surfactants,owing to their potential catalytic and transportation capabilities,could be a proper choice in such mixed solvents systems for study-ing such reactions and promise to aid in optimization of conditionsfor universal applicability of DPPH assay with advantages of min-imal use of toxic organic chemicals and amphiphilic molecules.

Finally, it is anticipated that the presence of such self-assembledsurfactant systems in the protocol may help to provide some insightinto the mechanism of antiradical action of Rutin and other relatedflavonoids in more relevant multiphase systems.

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O.A. Chat et al. / Colloids and Surfaces A: P

cknowledgments

OAC (SRF-UGC) acknowledges the financial support from theniversity Grants Commission, India under Research Fellowshipcheme in Science for Meritorious Students (RFSMS). Head Depart-ent of Chemistry is acknowledged for his constant support and

ncouragement.

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