DPPH Radical-Scavenging Activity and Kinetics of Antioxidant AgentHesperidin in Pure Aqueous Micellar Solutions

Morteza Jabbari* and Azam Jabbari

School of Chemistry, Damghan University, 36716-41167 Damghan, Iran

E-mail: m_jabari@du.ac.ir

Received: March 19, 2016; Accepted: May 6, 2016; Web Released: August 15, 2016

Morteza JabbariMorteza Jabbari received B.Sc. degree from Ferdowsi University of Mashhad in 2004. He received his M.Sc.(2006) and Ph.D. (2011) degrees in physical chemistry from Shahid Beheshti University of Tehran undersupervision of Prof. Farrokh Gharib. Since 2012, he worked at Damghan University as an assistantprofessor. His research interests focus on the kinetics of antioxidant reactions and thermodynamics ofsolutions.

AbstractThe antioxidant ability of bioactive agent hesperidin was

assessed in terms of radical-scavenging activity (RSA) againstthe 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical in aqueouscolloidal media containing micelle using UV­vis spectropho-tometry. The DPPH assay was carried out at 25.0 « 0.1 °C andcationic surfactant CTAB and anionic surfactant SDS at variousconcentrations above the critical micelle concentration (CMC).The rates of the antioxidant reaction (Rs) of hesperidin werealso measured in the micelle systems. The activity and rateof the DPPH radical scavenging by hesperidin were found todepend on concentration and nature of the surfactants used, sothat both RSA and Rs values increase with increasing concen-tration of micelles CTAB and SDS. Finally, the micelle effectson the antioxidant efficiency were explained based on possi-ble interaction modes between hesperidin and the micellarsurfaces.

1. Introduction

Hesperidin (C28H34O15), (s)-7-[[6-o-(6-deoxy-α-L-manno-pyranosyl)-β-D-glucopyranosyl]oxy]-2,3-dihydro-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one, is abioflavonoid diglycoside abundantly found in citrus fruits likeorange, lemon, petitgrain, etc., such that the peel and mem-branous parts of lemons and oranges have the highest hes-peridin concentrations. This compound possesses a wide rangeof potential beneficial effects for human health such as anti-oxidant, anti-inflammatory, anticancer, hypolipidemic, anticar-cinogenic, and antimicrobial properties.1­3 Besides the protec-tive effects (most notable in the heart and brain, but extendto every organ), hesperidin may be able to reduce a lack ofappetite and have minor antiallergic properties. Hesperidin is awhite to yellow crystalline solid with poor aqueous solubility.

This limits its dissolution rate in water, which finally results inpoor in vivo bioavailability. Hesperidin has also been reportedas insoluble in most of the physiologically safe organic sol-vents useful in pharmaceutical dosage form development.1,4,5 Asketch of the molecular structure of hesperidin is shown inScheme 1.

Flavonoids or bioflavonoids are polyphenolic phytochemi-cals that are frequently found in nature and easily extractedfrom different parts of the plants, such as the roots, stems,leaves, flowers, fruits, or seeds. They are extremely safe andwith low toxicity, which makes them excellent disease-preventing dietary supplements and cancer-preventive agents.6

O

O

OH

O

OH

A C

B7

5 4

4'

2

O

OH

OH

OH

O O

OHOH

OHCH3 OCH3

HO

HO

HO

O

OH

1

23

4

5 6

Hesperidin

Gallic acid

N

O2N

O2N

NO2N

DPPH

Scheme 1. Chemical structure of bioflavonoid hesperidin,gallic acid, and DPPH radical.

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Many of the pharmacological effects are usually ascribed to theability of flavonoids to trap free radicals, an ability common toall phenols that is generally equated to “antioxidant activity”.This property of flavonoids is a biological function, importantin keeping the oxidative stress levels below a critical point inthe body and can thus help preserve neuronal health. Radicalscavenging by flavonoids occurs by electron or hydrogen dona-tion from the free hydroxyl groups on the flavonoid nucleus.This leads to the formation of a less reactive flavonoid aroxylradical that is stabilized by resonance and therefore plays amoderator role in the propagation of radical-induced damage inbiological systems.7,8 Numerous research groups have focusedon the free radical-scavenging effectiveness evaluation offlavonoids until now. Some of them have confirmed that thefree radical-scavenging activity of flavonoids depends not onlyon the environmental parameters like pH, temperature, natureof the solvent, etc.,7,9­11 but also on a number of other factorssuch as their partitioning between the different regions of thesystem.12,13 A general methodology for modeling this factoris to employ a microheterogeneous environment such as amicellar system. Surfactant unimers in aqueous solution self-assemble into micelles at a specific concentration called thecritical micelle concentration (CMC). The CMC is an importantcharacteristic, specific to each individual surfactant. Surfactantmicelles are dynamic entities and can have different shapes,such as spherical, spheroid, oblate, and prolate. Most micellesare spherical and contain between 60 and 100 surfactant mole-cules.14,15 The information obtained in the aqueous micellarsystems can play a crucial role in understanding antioxidantactivity of water-insoluble bioactive compounds.

Various methods have been designed to measure antioxidantpower of bioactive compounds so far. These assays can givedifferent results depending on the specific free radical beingused as a reactant. Among them, the one which is the mostwidely used for in vitro testing is based on employing the stablefree radical 2,2-diphenyl-1-picryhydrazyl (DPPH, Scheme 1)which has an unpaired valence electron at one atom of thenitrogen bridge.16 The DPPH assay has often been performedin pure organic or mixed aquo-organic solvents due to theinsolubility of species used in water. Therefore, the purpose ofthe present investigation was to develop a DPPH assay forevaluation of the radical-scavenging activity (RSA) of naturalpolyphenol hesperidin in pure aqueous solution using surfactantmicelles of CTAB and SDS without need of an organic co-solvent. The effects of the type of surfactant, concentration ofthe surfactant and the flavonoid as factors that can affect theantioxidant capacity are examined. Additionally, the reactionrates of DPPH radical with hesperidin are evaluated in pureaqueous micellar media to study antioxidant behavior of thisflavonoid kinetically.

2. Experimental

2.1 Chemicals. All chemicals used were of analytical gradepurity. Gallic acid (Scheme 1), hesperidin and the stable DPPHfree radical were supplied from Fluka and used without furtherpurification. The anionic surfactant SDS and cationic surfac-tant CTAB (Scheme 2) of the highest quality available werepurchased from Sigma-Aldrich. The deionized doubly distilledwater (1.2 « 0.1¯³¹1 cm¹1 conductivity) was used throughout.

2.2 DPPH Radical-Scavenging Assay (RSA). The relativestability of radical DPPH, sensitivity and the technical simplic-ity of DPPH assay execution makes this colorimetric methodpopular for measuring antioxidant capacity of food products orplant extracts. This method is based on the decay of the mainabsorption band in the visible spectrum of DPPH radical. Inthe presence of an electron- or hydrogen-donating compoundlike flavonoids, the DPPH radical would be scavenged throughelectron or hydrogen donation, and its absorbance is decreased.The decreased extent of absorbance is taken as a measure forthe antioxidant activity.17 The DPPH assay was carried out inpresence of the cationic CTAB and anionic SDS micelles asthe following procedure. The aqueous stock solutions of DPPH(0.030mM) and hesperidin (0.025mM) were daily prepared inthe same surfactant concentration and then stored in a refrig-erator until needed. To a known volume of aqueous micellesolution taken in a 5mL volumetric flask, 2mL DPPH solutionwas pipetted together with a volume of deionized water. Thereaction was started by adding an aliquot (0.16­0.60mL) ofhesperidin to the mixture to make a final volume of 3mL. Thereaction mixture was then vigorously shaken by hand andallowed to stand in the darkness for 5min at ambient tem-perature for completion of the reaction. The same procedurewas repeated for at least five different concentrations of thesurfactants tested in a range 6.50­8.50mM of CTAB and15.00­25.00mM of SDS. In each concentration of micelle, sixdifferent concentrations of hesperidin were used. The DPPH inaqueous micelle solution without an antioxidant was used ascontrol of this experiment. After 5min of starting the reaction,the samples were analyzed using a Perkin-Elmer (Lambda 25)UV­vis spectrophotometer in the wavelength range of 450 to650 nm. The aqueous micelle solutions were used as blank. Theradical-scavenging potential in each surfactant micelle solutionwas derived based on the value of the DPPH visible absorbanceat the maximum according to the following expression:

%RSA ¼ Abscontrol � Abssample

Abscontrol� 100 ð1Þ

where Abscontrol and Abssample are the absorbance at maximawavelength of the control and the sample, respectively. Allassays were performed in three independent runs and the RSAvalues in each micelle system were calculated as a mean ofthese measurements. The RSAvalues were usually compared toa reference antioxidant of which its antiradical capacity wasmeasured under the same conditions. In this work, gallic acidwas used as a reference standard compound.

NH3CCH3

CH3H3C

Br

CTAB

SDS

Scheme 2. Sketch of molecular structure of cationic surfac-tant CTAB and anionic surfactant SDS.

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2.3 Kinetic Measurements. The reaction betweenbioflavonoid hesperidin and the DPPH radical was followedspectrophotometrically by monitoring the absorbance changesof DPPH radical at its maxima wavelength as a function oftime. Earlier it was verified that there is negligible interfer-ence from the other reagents at this wavelength. The kineticmeasurements were carried out at different concentration ofthe micelle systems ranging from 6.50 to 8.50mM for CTABand 15.00 to 25.00mM for SDS. The progress of the reactionwas followed on a Perkin-Elmer (Lambda 25) UV­vis spec-trophotometer with thermostatizable quartz cells of 10mmoptical path. The temperature was maintained at 25.0 « 0.1 °Cby a Peltier system in conjunction with a LabTech LCB-R08thermocirculator.

The kinetic runs for each concentration of micelle solutionswere done by adding an aliquot (0.48mL) of micellar solutionof hesperidin to 2mL micelle solution of DPPH (0.030mM)into a UV cell containing a known volume of micelle stocksolution. For all runs, the concentration of DPPH was main-tained 5 times higher than hesperidin concentration. The de-crease in absorbance was monitored at 527 (in CTAB) and 531nm (in SDS) until the reaction reached a plateau or obtained asteady state of decrease in DPPH absorbance.

Before the kinetic measurements, a calibration curve ofabsorbance versus concentration of DPPH in micelle solutions(8.50mM of CTAB and 25.00mM of SDS) was obtained todetermine the exact DPPH concentration in the reaction medi-um. From the linear regression of data, the following equationswere obtained

Abs527nm ðCTABÞ ¼ 4:348� ½DPPH� þ 0:0017 ð2ÞAbs531nm ðSDSÞ ¼ 5:008� ½DPPH� þ 0:0005 ð3Þ

where [DPPH] is expressed as mM. The correlation coefficientof r2 = 0.999, indicated the goodness of fitting. The eqs 2 and3 were utilized to obtain the concentration of DPPH at varioustimes wherever required.

3. Results and Discussion

3.1 Radical-Scavenging Efficiency. Bioflavonoids belongto a group of natural products that possess potent antioxidantactivity and hence are considered strong scavengers of freeradicals. In order to evaluate the effectiveness of bioflavonoidhesperidin as antioxidant, we used DPPH assay to study itsradical-scavenging capacity. Since hesperidin and DPPH freeradical have aromatic rings, and are large in size, their solu-bility in water is too low. Therefore, this assay was carried outin surfactant micelle medium. An aqueous surfactant solution,especially at concentration higher than its CMC range, is amore suitable colloidal medium rather than some toxic organicsolution for study of biological/pharmacological effects ofbioactive substances.18

The free radical-scavenging activity (RSA) of an anti-oxidant compound can be expressed as the percentage of DPPHreduced by a known amount of that compound. The RSAvalues of bioflavonoid hesperidin in different concentrationsof micelles CTAB and SDS were calculated according to eq 1and are listed in Tables 1 and 2, respectively, together with theRSA values related to gallic acid as a reference antioxidantwhich were obtained in our previous work.19 These data are theaverages of three reproducible determinations with an uncer-tainty of less than «0.9%. As can be seen in these Tables, whenthe concentration of hesperidin increases from 1.33 to 5.00¯Min each SDS and CTAB micellar system, antioxidant activity(RSA value) increases accordingly. Moreover, in each working

Table 1. Values of RSA in different concentrations of bioflavonoid hesperidin and CTAB micellar solution at 25.0 « 0.1 °C incomparison with values of gallic acid as reference antioxidant

[CTAB]/mM

RSAa)/%

[Hesperidin]/¯M [Gallic acid]/¯M

1.33 1.67 2.50 3.33 4.17 5.00 1.33 1.67 2.50 3.33 4.17 5.00

8.50 1.92 3.92 4.09 4.44 5.03 5.77 18.00 21.32 23.09 24.98 26.22 27.458.00 1.26 1.78 1.85 1.93 2.15 2.67 15.13 17.68 22.98 23.82 25.73 26.157.50 0.54 0.99 1.38 1.44 1.53 1.91 13.37 13.76 15.20 16.64 17.56 20.187.00 0.15 0.39 0.54 1.31 1.39 1.54 12.71 13.01 14.61 15.58 16.06 19.326.50 0.11 0.31 0.42 0.73 1.25 1.31 11.42 12.72 13.15 14.45 15.03 18.79

a) Uncertainties in the RSA values are below «0.9%.

Table 2. Values of RSA in different concentrations of bioflavonoid hesperidin and SDS micellar solution at 25.0 « 0.1 °C in comparisonwith values of gallic acid as reference antioxidant

[SDS]/mM

RSAa)/%

[Hesperidin]/¯M [Gallic acid]/¯M

1.33 1.67 2.50 3.33 4.17 5.00 1.33 1.67 2.50 3.33 4.17 5.00

25.00 3.19 5.15 6.25 7.06 8.53 9.35 44.28 53.29 63.74 81.82 85.33 90.5922.50 2.28 4.73 5.60 6.13 7.53 8.93 40.79 49.21 62.90 78.42 82.50 87.9020.00 1.22 1.92 2.80 4.90 5.77 6.99 37.52 46.76 59.58 76.92 81.88 86.1417.50 0.88 1.12 2.48 3.95 4.63 5.15 33.51 41.04 56.60 74.93 81.17 84.1615.00 0.37 0.73 2.20 3.11 3.30 4.76 31.59 40.99 55.98 68.09 78.82 83.49

a) Uncertainties in the RSA values are below «0.9%.

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concentration of hesperidin, its effectiveness as an antioxidantagent increases with increasing both CTAB and SDS content ofaqueous solution. This is probably due to increasing the extentof solubilization of hesperidin through hydrophobic inter-actions, which causes more collisions between the flavonoidhesperidin and DPPH radical within the micelle systems.

3.2 Kinetics of DPPH Free Radical Scavenging. Theknowledge of the kinetic behavior of natural antioxidants isimportant because free radicals in living organisms are short-lived species, implying that the impact of a substance as anantioxidant depends on its fast reactivity towards free radi-cals.20 Therefore, to gain a deeper insight into the antioxidantability of the flavonoid hesperidin, the kinetics of its radicalscavenging has been studied by allowing it to react with thestable free radical DPPH. To quantify the kinetic behavior ofhesperidin biomolecule, the rate of DPPH radical scavenging(Rs), was estimated. The kinetic measurements were performedusing various concentrations of CTAB (6.50 to 8.50mM) andSDS (15.00 to 25.00mM) micelles in an aqueous solutionand 25.0 « 0.1 °C to study the influence of micelles on reac-tivity of hesperidin toward DPPH radical. During the radical-scavenging period (first 10 sec), the concentration of the DPPHdecreased approximately linearly with time, and the slope ofthis line was designated as Rs, the value of which reflects theantioxidant potential of the bioflavonoid hesperidin. Generally,the linear plots obtained showed good correlation (with correla-tion coefficient of r2 ² 0.99) in all the investigated concen-trations of the micelle. The exact concentration of the DPPH inthe reaction medium containing CTAB and SDS micelles wascalculated from eqs 2 and 3, respectively. The values of the Rs

for hesperidin are given in Table 3. The kinetics of gallic acidantioxidant behavior cannot be evaluated with the usual UV­vis spectrophotometric method used here because gallic acidis one of the strongest antioxidants and reacts very fast withDPPH radical. Some typical kinetic curves obtained (absorb-ance-time and concentration-time curves) during the scaveng-ing process of DPPH in various concentrations of CTAB and

SDS micelles are given in Figures 1 and 2, respectively. Itwas observed that the rate of the radical-scavenging reactionincreases with increasing CTAB micelle concentration andby increasing the amount of SDS micelle in aqueous medi-um, as shown in Table 3. Therefore, the decay rate of DPPHradical by hesperidin (Rs) showed a similar trend as that of theDPPH radical-scavenging activity (RSA) in different concen-trations of the micellar solutions as discussed in the previoussection.

3.3 Micellar Effect. It has been proved that the presence ofa surfactant micelle in a system could play an important role inthe chemical behavior of antioxidant products.21,22 The naturalpolyphenolic compounds like bioflavonoids can usually exerttheir antioxidant action by three different mechanisms includ-ing hydrogen-atom transfer, single-electron transfer to freeradicals, and finally metal chelation. These mechanisms areaffected by antioxidant structural features, solubility, partitioncoefficient, and solvent system.23 Furthermore, the behavior ofmany antioxidants in heterogeneous micellar microenviron-ments, which are more relevant to biological system, has notbeen well elucidated yet.24,25 Therefore, these items provide abasis for our extended studies of the micelle effects on anti-oxidant activities during radical-scavenging process.

Surfactant micelles can be pictured as having a nonpolarinterior and a relatively polar interfacial region. The interior ofthe micelle is generally considered the locus of solubilizationfor nonpolar solubilizate such as aromatic compounds.11 How-ever, the site of solubilization within a micelle is closely relatedto the chemical nature of the solubilizate.14 The bioflavonoidhesperidin is a compound that has very low solubility in water(4.93¯gmL¹1 at 25 °C).26 It has been reported that the extentof solubilization of hesperidin within the micelles would beaffected by the presence of a glycoside moiety in it. The reasonis that these sugar groups inhibit hesperidin from reaching themicellar surface due to higher aqueous-phase solubility andsteric hindrance.21 It can be seen in Tables 1 and 2 that bothRSA and Rs values increase by increasing in concentration of

(b)

(b)

(a)

(a)

(c)

(c)

Figure 1. Plots of absorbance (at 527 nm) and concentration change of the DPPH radical versus time for the reaction of hesperidinwith DPPH in (a) 8.50, (b) 7.50, and (c) 6.50mM CTAB micellar solution at 25.0 « 0.1 °C, [Hesperidin] (4.00¯M) and [DPPH](20.00¯M).

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anionic SDS and cationic CTAB micelle, probably due to thesolubilization of hesperidin within these micelles and increas-ing its availability to DPPH radical. In an ionic micelle system,the antioxidant polyphenolic compounds such as hesperidinwill prefer first the Stern (hydrophilic) and then the palisade(hydrophobic) layer of micelle as a consequence of hydrogenbonding,13 electrostatic, and hydrophobic interactions.21 On theother hand, the DPPH radical is also a lipophilic species andit will be easily drawn inside the hydrocarbon core of themicelle.16,21 The possible orientation of hesperidin biomoleculewithin SDS micelles is such that the ring B, containing theelectroactive hydroxyl group (4¤-OH), binds to the Stern layerof micelle by hydrogen-bonding interactions while ring C,carrying negative charge, lies outside the micelle to avoidunfavorable electrostatic repulsions with negatively chargedSDS head groups. In addition, probably the hydroxyls on sugargroups of hesperidin are also involved in hydrogen-bondinginteraction with SDS surface. However, it seems the extent ofsolubilization increases with increasing the concentration ofSDS as a result of hydrophobic interactions. This helps hes-peridin to be drawn more into the palisade layer. All theseinteractions help to facilitate the interaction of hesperidin withthe DPPH radical inside the micelle, hence radical scavengingof this bioflavonoid increases by increasing in micelle concen-tration. The representation of possible orientation of bioflavo-noid hesperidin within SDS micelles is illustrated in Figure 3.It should be noted that in hesperidin biomolecule, two hydroxylgroups on the A and B rings (5- and 4¤-OH) are responsiblefor scavenging the DPPH free radicals. On the other hand, inaqueous solution of cationic CTAB micelle, bioflavonoid hes-peridin interacts with the micelle surface via ring C due toelectrostatic interaction between negatively charged center (4-oxo) of hesperidin and positively charged CTAB head groups,Figure 3. Probably the hesperidin-micelle hydrophobic inter-actions are strong enough to outweigh the electrostatic inter-actions between them, which cause the hesperidin to be drawninto the palisade layer. Therefore, the solubility of hesperidin

in medium would increase and reacts more with DPPH radical.For this reason, the values of RSA and Rs increase with in-crease in concentration of CTAB micelle in aqueous solution.Similar results have been reported in antioxidant effect studiesof flavonoid rutin in the microenvironment of cationic CTABmicelles.27

Comparing data at the same hesperidin concentration inTables 1­3, it was obvious that both RSA (Tables 1 and 2) andRs (Table 3) at highest concentration of CTAB (8.50mM) werealways larger than the corresponding values at lowest concen-tration of SDS (15.00mM). This means that CATB at lowerconcentration than SDS contributed more effectively to theradical-scavenging process. On the other hand, the CMC valueof CTAB is far less than that of SDS (0.81mM versus 7.40mM).18,28 With these points in mind, it could be concluded thatthe micellar effect of CTAB on the radical-scavenging behav-iors (the enhancements of both RSA and Rs) is more effectivethan that of SDS. This might to be due to weak hydrogen-bonding interactions between hesperidin and SDS micelle sur-face against the strong electrostatic interactions between hes-peridin and positively charged CTAB head groups (Figure 3),which enhances its chance of being drawn into the micellesystem. Moreover, DPPH radical can interact with positivelycharge head groups of CTAB via electrostatic interactions.These attractive interactions allowed DPPH to remain insidethe hydrophobic core of the CTAB micelle.16

(a) (b) (c)

(a) (b) (c)

Figure 2. Plots of absorbance (at 531 nm) and concentration change of the DPPH radical versus time during scavenging of DPPHradical by hesperidin in (a) 25.00, (b) 20.00, and (c) 15.00mM SDS micellar solution at 25.0 « 0.1 °C, [Hesperidin] (4.00¯M) and[DPPH] (20.00¯M).

Table 3. Rate of DPPH radical scavenging by hesperidinin different concentrations of aqueous CTAB and SDSmicelle solution

[CTAB]/mM Rs/¯Ms¹1 [SDS]/mM Rs/¯Ms¹1

8.50 0.21 « 0.02 25.00 0.28 « 0.048.00 0.19 « 0.03 22.50 0.23 « 0.037.50 0.17 « 0.04 20.00 0.18 « 0.017.00 0.10 « 0.03 17.50 0.12 « 0.026.50 0.08 « 0.03 15.00 0.10 « 0.02

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In a previous work,19 we study the antioxidant activity ofwater-insoluble flavonoid naringenin in aqueous micellar solu-tions of CTAB and SDS. Based on the above discussion, it couldalso be concluded that the micellar role of CTAB on the radical-scavenging ability of naringenin (both RSA and Rs) is moreeffective than that of SDS. Hereby, we now correct our previousconclusion about the comparison of the radical-scavengingability of naringenin in CTAB and SDS media. However, com-parison of these two works indicates that antioxidant capacity(RSA) and rate (Rs) of naringenin are more than those ofhesperidin in both micellar media under study. It may be due toseveral reasons: (a) The structural differences existing betweennaringenin and hesperidin especially the number of hydroxylgroups on the phenolic rings of the flavonoids. (b) Smaller sizeof naringenin compared to hesperidin which has sugar groups.It has been pointed out that the introduction of a sugar moietyin the flavonoids inhibits them to reach the micellar surface dueto higher aqueous solubility and steric hindrance.21

4. Conclusion

The use of aqueous micellar systems as reaction mediumis wide and varied. These media can have significant effectson the radical-scavenging behavior of the polyphenolic com-pounds such as flavonoids. Our results of the spectrophoto-metric studies on the DPPH radical scavenging by hesperidin,and also kinetics of DPPH-hesperidin reaction in aqueoussolutions of micelles CTAB and SDS support this hypothesisthat the antioxidant reaction rate and consequently the RSAincrease as the amount of micelle increases in the medium. Thisis due to the predominant factors of controlling the radical-scavenging activity that are the hydrogen-bonding, hydropho-bic, and electrostatic interactions of the bioflavonoid with themicellar surface and its interior layers. These interactions causeincreasing solubility of hesperidin in aqueous solution, whichimprove its antioxidant properties (RSA and Rs).

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Stern layerPalisade layer

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OO

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OHA

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OHO

OHHO

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HOOH

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(a) (b)

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