CUPRAC total antioxidant capacity assay of lipophilic antioxidants in combination with hydrophilic...

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CUPRAC total antioxidant capacity assay oflipophilic antioxidants in combination with

hydrophilic antioxidants using the macrocyclicoligosaccharide methyl b-cyclodextrin as the solubility enhancer

S.E. Celik, M. Ozyurek, K. Guc�lu, R. Apak *

Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar, 34320 Istanbul, Turkey

Available online 31 July 2007

Abstract

Antioxidants are important health beneficial compounds in regard to their ability to quench reactive O,N-speciesunder ‘oxidative stress’ conditions. We recently reported a simple, low-cost, and widely applicable total antioxidantcapacity assay for dietary polyphenols, vitamins C and E, and plasma antioxidants in alcoholic aqueous media utilizingthe copper(II)-neocuproine (Nc) reagent as the chromogenic oxidant, which we named the CUPRAC method.Although this novel method was applied to synthetic mixtures of complex nature, plant extracts, human serum, andhydroxyl radical scavengers, lipophilic antioxidants were normally treated separately from hydrophilic ones. Withoil-soluble antioxidants like butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone(TBHQ), propyl gallate (PG), lauryl gallate (LG), and vitamin E (a-tocopherol), it was necessary to apply theCUPRAC method with dichloromethane extraction, which is not useful for hydrophilic antioxidants. Thus, as animprovement to classical CUPRAC methodology, this work reports the assay both lipophilic and hydrophilic antiox-idants simultaneously, by making use of their ‘host–guest’ complexes with methyl b-cyclodextrin (M-b-CD), a cyclicoligosaccharide, in aqueous medium. The turbidity limit for a-tocopherol, BHT, PG, and LG were 1:3 (v/v) alco-hol–water solutions, but when these suspensions were mixed with equal volumes of 7% M-b-CD aqueous solution,clear solutions were obtained in which the CUPRAC assay could be directly performed. In addition to solubilityenhancement, molecular spectroscopic evidence was also provided for the formation of (M-b-CD + antioxidant) inclu-sion complexes. The calibration curves of individual (lipophilic and hydrophilic) antioxidants were constructed in suchmedia, with their molar absorptivities and linear concentration ranges determined. Testing of synthetic ternary andquaternary mixtures of lipophilic and hydrophilic antioxidants in M-b-CD–containing media yielded theoreticallyexpected CUPRAC antioxidant capacities, considering the additivity of absorbances of constituents obeying Beer’slaw. Thus M-b-CD served as the solubility enhancer host molecule to establish a modified CUPRAC method forthe total antioxidant capacity assay of a mixture of diverse antioxidants of different lipophilicity levels in solutions richin water content. The relative M-b-CD concentration used to form the inclusion complex could be restricted so as notto significantly decrease the antioxidant capacity exhibited by the tested compounds.� 2007 Elsevier Ltd. All rights reserved.

1381-5148/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.reactfunctpolym.2007.07.045

* Corresponding author. Tel.: +90 212 4737028.E-mail address: [email protected] (R. Apak).

Available online at www.sciencedirect.com

Reactive & Functional Polymers 67 (2007) 1548–1560

www.elsevier.com/locate/react

REACTIVE&

FUNCTIONALPOLYMERS


Keywords: Methyl-b-cyclodextrin; Host–guest interaction; Solubility enhancement; Lipophilic antioxidants; CUPRAC antioxidant capacityassay

1. Introduction

Antioxidants are health beneficial compoundsthat react with an excess of reactive oxygen andnitrogen species under ‘oxidative stress’ conditions,thereby preventing related diseases such as cell age-ing, cardiovascular diseases, mutagenic changes andcancerous tumor growth [1,2]. Consumption offoods naturally bearing antioxidant power is themost efficient way of combating such health haz-ards. Thus, the opportunity for improving healthby improving diet is great [3].

The chemical diversity of antioxidants makes itdifficult to separate and quantify individual antiox-idants (i.e., parent compounds, glycosides, poly-mers, and many isomers) from the vegetablematrix. Moreover, the total antioxidant power isoften more meaningful to evaluate health beneficialeffects because of the cooperative action of antioxi-dants. Therefore, methods should be establishedthat can measure the total antioxidant capacity(AOC) directly from vegetable extracts [4]. Antioxi-dant capacity assays may be broadly classified aselectron transfer (ET)- and hydrogen atom transfer(HAT)-based assays [5,6].

The HAT mechanisms of antioxidant action canbe summarized by the reaction:

ROO� þAH=ArOH! ROOHþA�=ArO� ð1Þ

where the aryloxy radical (ArO�) formed from theantioxidant phenol (ArOH) and peroxyl radical(ROO�) is stabilized by resonance. AH/ArOH denotevarious antioxidants. Since both the fluorescentprobe and antioxidants react with ROO�, the antiox-idant activity can be determined from competitionkinetics by measuring the fluorescence decay curveof the probe in the absence and presence of antioxi-dants, and integrating the area under these curves[5,6]. As an example of HAT-based assays, oxygenradical absorbance capacity (ORAC) assay [7]applies a competitive reaction scheme in which anti-oxidant and substrate kinetically compete for ther-mally generated peroxyl radicals through thedecomposition of azo compounds such as ABAP(2,20-azobis(2-aminopropane) dihydrochloride) [5,6].

On the other hand, the ET mechanism of antiox-idant action is based on the reaction:

ROO� þAH=ArOH! ROO�

þAHþ=ArOHþ $ A�=ArO� þHþ ð2Þ

where the reactions are relatively slower than those ofHAT-based assays, and are pH-dependent. In fact, inmost ET-based assays, the antioxidant action is sim-ulated with a suitable redox-potential probe, i.e., theantioxidants react with a fluorescent or coloredprobe (oxidizing agent) instead of peroxyl radicals.Spectrophotometric ET-based assays measure thecapacity of an antioxidant in the reduction of an oxi-dant, which changes colour when reduced [5,6]. Thedegree of colour change (either an increase or de-crease of absorbance at a given wavelength) is corre-lated to the concentration of antioxidants in thesample. These assays generally set a fixed time forthe concerned redox reaction, and measure thermo-dynamic conversion during that period. ET- basedassays include ABTS/TEAC [8,9], DPPH [10],Folin-Ciocalteu (FCR) [11,12], and FRAP [13], usingdifferent chromogenic redox reagents with differentstandard potentials. Although the reducing capacityof a sample is not directly related to its radical scav-enging capability, it is a very important parameterof antioxidants. The reaction equations of variousET-based assays can be summarized as follows:

Folin: MoðVIÞðyellowÞþe�ðfrom AHÞ!MoðVÞðblueÞ;kmax¼765 nm ð3Þ

where the oxidizing reagent is a molybdophospho-tungstic heteropolyacid comprised of 3H2O–P2O5 –13WO3 – 5MoO3–10 H2O, in which the hypothe-sized active center is Mo(VI).

FRAP : FeðTPTZÞ3þ2 þArOH$ FeðTPTZÞ2þ2þArO� þHþ; kmax ¼ 595 nm; ð4Þ

where TPTZ: 2,4,6-tripyridyl-s-triazine ligand.PRUSSIAN BLUE:

FeðCNÞ3�6 þArOH$ FeðCNÞ4�6 þArO� þHþ ð5ÞFeðCNÞ4�6 þ Fe3þ þKþ !

KFe½FeðCNÞ6�; kmax ¼ 700 nm ð6Þ

S.E. Celik et al. / Reactive & Functional Polymers 67 (2007) 1548–1560 1549


ABTS/TEAC:

ABTSþK2S2O8 ! ABTS�þ; kmax ¼ 734 nm ð7ÞABTS�þ þArOH$ ABTSþArO� þHþ ð8Þ

where ABTS: 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and TEAC is trolox-equivalent anti-oxidant capacity (also the name of the assay).

DPPH : DPPH� þArOH$ DPPHþArO� þHþ

ð9Þ

where DPPH� is the [2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl] stable radical with kmax = 515 nm.

Our group has developed a simple, diverselyapplicable, and low-cost antioxidant capacity(AOC) assay for dietary polyphenols, vitamins Cand E, utilizing the copper(II)-neocuproine(Cu(II)-Nc) reagent as the chromogenic oxidizingagent, named by us as the CUPRAC (cupric ionreducing antioxidant capacity) method [14]. Wehave applied the CUPRAC assay to human serum[15], ascorbic acid in complex mixtures [16,17],apricot [18], herbal teas [19], and water-solublehydroxyl radical scavengers [20]. Hydrophilic andlipophilic antioxidants are treated separately inthe CUPRAC assay. With oil-soluble antioxidantslike butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), tert-butylhydroquinone(TBHQ), propylgallate (PG), laurylgallate (LG),and vitamin E (a-tocopherol), it is necessary toapply the CUPRAC method with dichloromethane(DCM) extraction, which is not useful for hydro-philic antioxidants. Cyclodextrins (CDs) are cyclicoligosaccharides shaped as a truncated cone witha hydrophilic outer surface and a lipophilic interiorcavity [21], and can form inclusion complexes withvarious hydrophobic organic molecules enhancingtheir aqueous solubility [22]. This may be advanta-geous over solvent extraction procedures with chlo-rinated solvents which may cause toxicological,phase separation, and insufficient recovery prob-lems. The aim of this work is to develop a modifiedCUPRAC procedure for hydrophilic antioxidantswhich are not assayed along with the hydrophilicones in currently available antioxidant assays.Since lipophilic antioxidants require a hydrophobicsolvent as assay medium, it is highly probable thata combination of hydrophilic and lipophilic antiox-idants may not be assayed in the same solution. Inorder to find a unique medium capable of dissolv-ing all kinds of antioxidants regardless of their

lipophilicity level, we made use of cyclodextrinspossessing a hydrophobic cavity that has a remark-able capacity to form inclusion complexes withvarious organic molecules through host–guestinteractions [23]. This is expected to pave the wayfor the simultaneous antioxidant assay of lipophilicand hydrophilic antioxidants in a solvent mediumrich in water content.

2. Materials and methods

2.1. Chemicals, instruments, and solutions

a-Cyclodextrin, b-cyclodextrin, methyl-b-cyclo-dextrin (M-b-CD) (purum, average molecularwt.% = 1310 g mol�1), lauryl gallate (LG), andb-carotene were purchased from Fluka; butylatedhydroxyanisole (BHA), butylated hydroxytoluene(BHT), tert-butylhydroquinone (TBHQ), propylgallate (PG), quercetin (QR), ferulic acid (FRA),caffeic acid (CFA), ascorbic acid (AA; vitamin C),a-tocopherol (vitamin E), and neocuproine (2,9-dimethyl-1,10-phenanthroline), and methanol fromSigma Co.; trolox (6-hydroxy-2,5,7,8-tetrame-thylchroman-2-carboxylic acid) from Aldrich Co.;cupric chloride, ammonium acetate, sodiumhydroxide, and absolute ethanol from E. Merck.All reagents were of analytical reagent grade unlessotherwise stated. Bidistilled water was used to pre-pare and dilute the solutions.

Spectrophotometric measurements were madewith a Varian CARY 1E UV–vis spectrophotome-ter using a pair of matched quartz cuvettes of 1 cmoptical path. The pH measurements were per-formed with a E512 Metrohm Herisau pH-meterusing glass electrode. UV spectra of samples andinclusion complexes were taken in aqueous med-ium containing 60% EtOH such that the final com-plex solutions included equimolar amounts (i.e.,1 mL each of 1.0 � 10�2 M) BHA and M-b-CDin the same solvent. The alkalinized mixture solu-tions additionally contained 1 mL of 0.1 M NaOH.The final volume was 10 mL for all solutions,purged with nitrogen gas before incubation. Inthe preparation of solid host–guest complexes, aTelstar Cryodos freeze-dryer and a HeraeusRVT360 Type vacuum oven were used. FT-IRspectra of the solid samples were recorded on aDigilab Excalibur-FTS 3000MX model (USA)infrared spectrometer in the spectral range of4000–400 cm�1. The samples were prepared in

1550 S.E. Celik et al. / Reactive & Functional Polymers 67 (2007) 1548–1560


(IR-spectroscopic grade) KBr pellets (sample/KBr = 1/200, w/w).

The standard solutions at 1.0 � 10�3 M concen-tration of lipophilic antioxidants BHA, TBHQ,LG, PG, and a-tocopherol; of hydrophilic antioxi-dants AA, FRA, CFA, TR, and QR; and at1.0 � 10�2 M concentration of BHT were all pre-pared in EtOH. The cyclodextrin stock solutionswere prepared in water at 1.0 � 10�2 M concentra-tion for a-CD and b-CD, and at 7% (w/v) forM-b-CD. Cupric chloride solution, 1.0 � 10�2 M,was prepared by dissolving 0.4262 g CuCl2.2H2Oin water, and diluting to 250 mL. Ammonium ace-tate buffer at pH 7.0, 1.0 M, was prepared by dis-solving 19.27 g NH4Ac in water and diluting to250 mL. Neocuproine (Nc) solution, 7.5 � 10�3 M,was prepared daily by dissolving 0.039 g Nc in96% ethanol, and diluting to 25 mL with ethanol.

2.2. Recommended procedure

The CUPRAC method of total antioxidantcapacity assay [14,18,19] was applied with somemodifications in the following procedure forenhanced solubility of lipophilic antioxidants:

Briefly, 5 mL-aliquots of ethanolic antioxidantsolutions were taken, 15 mL water was added toeach, and 2 mL of the resulting mixture was takenfor analysis. This was completely solubilized byadding 2 mL of 7% M-b-CD solution in the aqueousmixture. The subsequent CUPRAC assay was per-formed as follows:

One mL CuCl2, 1 mL Nc solution, and 1 mLNH4Ac solution were added to x mL of the M-b-CD–containing final analyte mixture, followed by(1.1 � x) mL water. The absorbance of the finalsolution (of 4.1 mL total volume) at 450 nm wasread against a reagent blank after 30 min standingat room temperature. The calibration curves (absor-bance vs. concentration graphs) of each antioxidantwere constructed under the described conditions,and their trolox-equivalent antioxidant capacities(TEAC coefficients [14,15,18,19], found as the ratioof the molar absorptivity of each compound to thatof trolox in the modified CUPRAC assay) werecalculated.

2.3. Other experimental procedures

The preliminary experiments for testing the solu-bility of antioxidants were performed by taking

5 mL-aliquots of their ethanolic solutions and add-ing water onto them at different ratios until theappearance of turbidity. When the solutions of lipo-philic antioxidants turned turbid, suitable portionsof CD solutions were added to reobtain clearsolutions.

The solid inclusion complexes of M-b-CD withantioxidants (BHA and TBHQ) were prepared asdescribed by Calabro et al. for 3-hydroxyflavonecomplexes [26]. Briefly, kneaded products wereobtained by wetting in a mortar physical mixturesof the antioxidant and oligosaccharide (at a moleratio of 1:1) with a minimal amount of MeOH–H2O (1:1). A mixture of 1.31 g (1 � 10�3 mol)M-b-CD and 0.18 g (1 � 10�3 mol) BHA was wet-ted with 1 mL MeOH–H2O mixture (1:1, v/v), andgrounded in an agat mortar. Likewise, 1.31 g(1 � 10�3 mol) M-b-CD and 0.166 g (1 � 10�3 mol)TBHQ was wetted with 1 mL MeOH:H2O (1:1,v/v), and grounded in an agat mortar. The mixtureswere vacuum-dried (pressure: 100 mm Hg) over-night in an oven at 35 �C, and brought to constantweight to obtain the kneaded products. Thefreeze-dried products were prepared by dissolving2.62 g (2 � 10�3 mol) M-b-CD and 0.36 g (2 �10�3 mol) BHA in 100 mL of MeOH–H2O mixture(1:1, v/v), and the flasks were transferred to a freeze-dryer for 48 h standing. The same procedure wasapplied to TBHQ such that 2.62 g (2 � 10�3 mol)M-b-CD and 0.33 g (2 � 10�3 mol) TBHQ were dis-solved in 100 mL of MeOH–H2O (1:1, v/v), andfreeze-dried for 2 days.

The theoretical trolox-equivalent antioxidantcapacity of a synthetic mixture solution (expressedin the units of mM TR) was calculated by multiply-ing the TEAC coefficient of each antioxidant consti-tuting the mixture with its final concentration, andsumming up the products (e.g., 10 lM TBHQ hav-ing a TEAC coefficient of 0.9 would count as9 lM TR in such a mixture, where the total antiox-idant capacity (AOC) of the mixture should beequal to the sum of the AOCs of its constituents,considering the validity of Beer’s law for this modi-fied CUPRAC assay). The experimental trolox-equivalent AOC of the same mixture was calculatedby dividing the observed absorbance (A450) to themolar absorptivity of trolox (eTR being 1.56 �104 L mol�1 cm�1 under the selected conditions).Then the theoretically found total AOCs were com-pared to the experimentally observed ones to testthe applicability of Beer’s law (i.e., the principle ofadditivity of individual absorbances of constituents



making up a mixture). Validity of Beer’s law for amixture implies that the observed absorbance isthe sum of the individual absorbances due to con-stituents of the mixture.

To test the effect of M-b-CD concentration onantioxidant capacity, the alcoholic BHT solution(1.0 � 10�2 M) was diluted with water at a ratioof 1:4 (v/v), 2 mL of this was taken, and varyingvolumes (between 2 and 6 mL) of 7% M-b-CDaqueous solution were added with dilution to a finalvolume of 8 mL using H2O. The modifiedCUPRAC method was applied (see: recommendedprocedure) to 0.2 mL of this solution, and the vari-ation of absorbance with the volume of M-b-CDsolution was recorded.

3. Results and discussion

3.1. Solubility enhancement with cyclodextrins

The critical volume ratio of water–EtOH for thesolubility edge of lipophilic antioxidants, i.e., BHT,LG, PG, and a-tocopherol, was 3:1, meaning that5 mL ethanolic solutions of these antioxidants turnedturbid upon the addition of 15 mL water. On theother hand, the water–EtOH mixture solutions ofBHA and TBHQ were clear at the same volumeratios, due to the higher solubility of the latter lipo-philic antioxidants. The other tested antioxidantswere hydrophilic, and perfectly dissolved in theselected medium. Thus, solubility enhancement bycyclodextrin addition was mainly tested at 1:3EtOH–H2O mixture solutions, and the recommendedprocedure was applied to such solutions rich in watercontent, which may easily be encountered in realcases for certain extractions of plant phenolics.

The enhancement of water solubility of certainlipophilic antioxidants such as a-tocopherol [24],carotenoids [25], and 3-hydroxyflavones [26] bythe use of CDs were observed without significantloss of their antioxidant activity. The basic mecha-nism of solubility enhancement was proposed asencapsulation of appropriately sized hydrophobiccompounds or lipophilic portions of molecules intotheir hydrophobic cavity via hydrophobic interac-tions [27]. Preliminary experiments of this workshowed that methyl-b-cyclodextrin was much moreefficient in enhancing solubility than either a- orc-cyclodextrins (which could not eliminate the tur-bidity of 1:3 EtOH–H2O mixture solutions ofBHT, LG, PG, or a-tocopherol). Although allthe tested CD compounds had hydrophobic inte-

rior cavities as opposed to hydrophilic outer sur-faces [27], the –OH groups of the inner core ofa- or c-CDs were partly replaced by –OMe groupsin M-b-CD structure, increasing the hydrophobic-ity of the CD core and therefore its complexationability for lipophilic antioxidants (structural for-mulae of M-b-cyclodextrin as well as of all lipo-philic antioxidants tested can be seen in Fig. 1).As a result, M-b-CD was observed to have a muchgreater solubilizing power for the lipophilic indoli-nonic nitroxide radical [27] than other CDs, similarto the finding of this work.

3.2. Molecular spectroscopic evidence of complex

formation with M-b-CD

FT-IR spectroscopy allowed the analysis ofchanges in the spectral features of the ‘guest’ anti-oxidants with respect to those of the ‘host’ oligo-saccharide. As for the FT-IR spectra of theinclusion compounds, the existence of new specieswith different spectroscopic features was confirmedby the disappearance of the characteristic bands ofthe free phenolics, as well as by the appearance ofnew bands originally nonexistent in the spectra ofeither M-b-CD or phenolic antioxidant alone.Fig. 2 shows the FT-IR spectra of solid inclusioncomplexes of BHA with M-b-CD obtained byvarious methods. The BHA spectrum exhibitedpeaks at 3393 cm�1 for –OH associated phenol,2953 cm�1 assigned as vibration –CH2, 1507 cm�1

for phenyl ring, 1415 cm�1 for the bending of –CH2 adjacent to the phenyl group, 1364 cm�1 forthe adjacent –CH3, 1032 cm�1 for the strongstretching absorption of C–O, 817, 772, and683 cm�1 bands for C@C out-of-plane ring [28].Especially those characteristic bands of BHA inthe region: 2000–500 cm�1 were obscured uponinclusion–complexation, either in kneaded or infreeze-dried products. On the other hand, newpeaks emerged in the FT-IR spectrum of thefreeze-dried (BHA + M-b-CD) equimolar mix-ture, namely the 1590, 1512, 1425, 1293, 1217,and 808 cm�1 bands (see Fig. 2). Fig. 3 depictsthe FT-IR spectra of solid inclusion complexes ofTBHQ with M-b-CD obtained by various meth-ods. The TBHQ spectrum exhibited 3445 and3272 cm�1 bands for –OH associated in phenol,2963 cm�1 were assigned as unsaturated @CH–(as in aromatics), 1590, 1484 and 1442 cm�1 weredue to phenyl ring stretching and –C–OH in-planebending, 1366 and 1310 cm�1 for methylene and



methyl bending vibration, 1209 and 1184 cm�1 forgem-dimethyl group (i.e., two methyl groups of thesame carbon atom), characteristic of O–C aromaticvibration, and 808, 771 and 692 cm�1 for out-of-plane –CH and ring [29]. The characteristic bandsof TBHQ were again obscured in the 2000–500 cm�1 range, in the kneaded and freeze-driedproducts, verifying complex formation with M-b-CD. On the other hand, new peaks emerged inthe FT-IR spectrum of the freeze-dried(TBHQ + M-b-CD) equimolar mixture, namelythe 1454, 1331, 1201, 1157, and 580 cm�1 bands(see Fig. 3). These FT-IR findings confirmed thatthe nature of interaction between the antioxidantand the oligosaccharide is of chemical but not of

simply physical nature, as in the latter case, onlya superposition of the signals would have beenobserved [26].

The UV spectra of antioxidants and their M-b-CD inclusion complexes did not show distinct dif-ferences, though NaOH– added formulations uponlonger standing gave rather different absorbancesat selected wavelengths. Fig. 4 shows the changeof UV spectra of BHA solutions upon incubationand NaOH addition, and Fig. 5 gives the changeof UV spectra of an equimolar mixture of(BHA + M-bCD) upon incubation and NaOHaddition. The original minimum of (BHA + M-b-CD) mixture at 252 nm shifts to higher wave-lengths upon NaOH addition and incubation

COO-CH2-(CH2)10-CH3

OH

OH

OH

COO-CH2-CH2-CH3

OH

OH

OH

Lauryl gallate (LG) Propyl gallate (PG)

OCH3

C(CH3)3

OH OH

OCH3

C(CH3)3

OH

Butylated hydroxyanisoles (BHA) Butylated hydroxytoluene (BHT)

OHOH OH

O

tert-butyl hydroquinone (TBHQ) α-tocopherol (vitamin E)

R = H or CH3

O

O

OR

OR

O

R

Methyl-β-cyclodextrin (M-β-CD)

Fig. 1. The structural formulae of the tested lipophilic antioxidants and of M-b-cyclodextrin.



(i.e., to 261–259 nm), and the absorbancesrecorded at those minima (Fig. 5) were lower thanthe corresponding values of BHA alone (Fig. 4).M-b-CD does not give any spectra within the spec-tral range of interest. The observed differences ofabsorbance minima indicate that acid-base equilib-ria of inclusion complexes give rise to slight differ-ences in UV absorbances of complexes at roughlythe same wavelengths compared to those of pure‘guest’ molecules. Likewise, the UV spectra of freeand M-b-CD– included carotenoids (especiallyupon longer periods of incubation) were reportedto show only slight differences [25]. If the HOMO(highest occupied molecular orbital) and LUMO(lowest unoccupied molecular orbital) levels ofthe antioxidant molecule are not greatly separatedas a result of host–guest interaction (as shouldhappen when the guest molecule is included as awhole in the hydrophobic cavity of the host, andnot bound through specific functional groups),the wavelengths corresponding to absorption max-ima and minima should not be expected to varysignificantly.

3.3. Advantages of the CUPRAC assay and means of

improvement

In previous publications regarding the CUPRACassay [14–20], its basic advantages such as low-cost,simplicity, versatility, flexibility, capability torespond to thiol-type antioxidants and to slowlyreacting weak reductants of antioxidant nature werepronounced. Perhaps the most important feature ofthe CUPRAC assay is its versatility, i.e., applicabil-ity to both hydrophilic and lipophilic antioxidantsby a simple change of solvent [14,15]. The Folinand FRAP antioxidant assays in their current formsare not applicable to lipophilic antioxidants [5].Many antioxidants are lipophilic, and it is alsoknown that the antioxidant capacity (AOC) of acompound is dependent upon reaction media [30].The original ORAC assay was also inapplicable tolipophilic antioxidants (i.e., these antioxidantsshowed very little antioxidant activity due to theaqueous nature of the ORAC assay), and had tobe modified by using an acetonated aqueous solu-

2000 1800 1600 1400 1200 1000 800 600 400

b

a

c

d

Wavenumber (cm-1)

Tra

nsm

itta

nce

(%)

Fig. 2. The FT-IR spectra of solid inclusion complexes of BHAwith M-b-CD obtained by various methods: (a) freeze-driedBHA�, M-b-CD complex, (b) kneaded BHA�, M-b-CDcomplex (vacuum-dried), (c) BHA alone, and (d) M-b-CD alone.

2000 1800 1600 1400 1200 1000 800 600 400

a

b

c

d

Tra

nsm

itta

nce

(%)

Wavenumber (cm-1)

Fig. 3. The FT-IR spectra of solid inclusion complexes of TBHQwith M-b-CD obtained by various methods: (a) freeze-driedTBHQ�, M-b-CD complex, (b) kneaded TBHQ�, M-b-CDcomplex (vacuum-dried), (c) TBHQ alone, and (d) M-b-CD alone.



tion of randomly methylated b-CD so as to includesuch antioxidants [31]. Actually in the 50% acetone-water solvent mixture used by these authorstogether with methylated-b-CD [31], the CUPRACassay could work for most oil-soluble antioxidantswithout requiring the cyclodextrin compound asthe additional solubility enhancer. Thus the ideahere is to further improve the CUPRAC assay forapplicability to aqueous extracts of higher watercontent by using M-b-CD. As a distinct improve-ment over other similar antioxidant assays, it wasintended to show that the CUPRAC assay is capa-ble of measuring both lipophilic and hydrophilic

antioxidants in the same aqueous solution contain-ing M-b-CD as the solubility enhancer.

3.4. Structure-activity relationships and TEACcoefficients of antioxidants in the improved CUPRAC

assay

Table 1 depicts the linear equations, molarabsorptivities, linear concentration ranges andTEAC coefficients of the tested antioxidants. Ascan be seen from Table 1, all antioxidants, regard-less of their hydrophobic character, could beassayed with the CD-modified CUPRAC assay in

Fig. 4. Change of UV spectra of BHA solutions upon incubation and NaOH addition: (a) BHA, (b) BHA + NaOH (initial), (c)BHA + NaOH (after 2 h), (d) BHA + NaOH (after 24 h), and (e) BHA + NaOH (after 48 h). (Solvent and reagent blank: 60% EtOH;BHA concn. = 10�3 M; NaOH concn. = 10�2 M).

Fig. 5. Change of UV spectra of an equimolar mixture solution of (BHA + M-bCD) upon incubation and NaOH addition: (a) BHA +M-bCD, (b) BHA + M-b-CD + NaOH (initial), (c) BHA + M-b-CD + NaOH (after 2 h), (d) BHA + M-b-CD + NaOH (after 24 h), and(e) BHA + M-b-CD + NaOH (after 48 h). (Solvent and reagent blank: 60% EtOH; BHA concn. = M-b-CD concn. = 10�3 M; NaOHconcn = 10�2 M).



the same solvent medium. The calibration curves ofselected antioxidants, BHT, BHA, TBHQ, and PG,are shown in Fig. 6. The oxidation of a polyphenolAr(OH)n with the CUPRAC reagent can be repre-sented by the following reaction equation:

nCuðNcÞ2þ2 þArðOHÞn$ nCuðNcÞþ2 þArð¼OÞnþnHþ

ð10Þ

where CuðNcÞþ2 is the chromogen. Among thehydrophilic antioxidants, quercetin showed thehighest antioxidant capacity, with a molar absorp-tivity of 8.06 � 104 L mol�1cm�1 and a TEAC coef-ficient of 5.16. Since the CUPRAC reagent oxidizesthe phenolic compounds to the corresponding qui-nones, the total antioxidant capacity of an antioxi-dant polyphenol is roughly proportional to thetotal number of Ar–OH groups in the molecule,and is dependent on the position of these phenolichydroxyls together with the overall conjugation ofthe molecule enabling rapid electron-transfer[14,32]. In addition, the antioxidant potency offlavonoids of similar conjugation level and roughlythe same number of –OH groups is positivelyaffected by the presence of o-dihydroxy moiety inthe B-ring [33]. Since quercetin possesses all theproperties required for high antioxidant power, ithas the largest TEAC coefficient in the modifiedCUPRAC assay. The TEAC coefficients of troloxand ascorbic acid were close to 1, and the TEACof caffeic acid was higher than that of ferulic acid(the latter having one less –OH group), just like inthe original CUPRAC assay [14]. The TEAC coeffi-cients of lipophilic antioxidant phenolics, reportedfor the first time in this work with respect to theCUPRAC method, also need some comments. Bothpropyl gallate and lauryl gallate contained the 3,4,5-trihydroxy benzoic acid moiety in their molecularstructures, and therefore they exhibited greater anti-oxidant potency than BHA, BHT, or TBHQ, inproportion to the number of their –OH groups.

Table 1The molar absorptivity (e), linear concentration range, and trolox-equivalent antioxidant capacity (TEAC coefficients) of variouslipophilic and hydrophilic antioxidants in 7% M-b-CD aqueous medium using the modified CUPRAC method

Antioxidants Linear equation and correlationcoefficient

e(L mol�1 cm�1) Linear range (M) TEACCUPRAC

Butylatedhydroxyanisole(BHA)

A = 2.33 � 104c + 0.0004, r = 0.9998 2.33 � 104 2.12 � 10�6–6.0 � 10�5 1.50

Butylatedhydroxytoluene(BHT)

A = 1.07 � 104c � 0.005, r = 0.9957 1.07 � 104 5.15 � 10�6–1.32 � 10� 4 0.68

Tert-butylhydroquinone(TBHQ)

A = 1.41 � 104c � 0.005, r = 0.9985 1.41 � 104 3.91 � 10�6–9.98 � 10�5 0.90

Lauryl gallate (LG) A = 3.09 � 104c + 0.004, r = 0.9982 3.09 � 104 1.49 � 10�6–4.52 � 10�5 1.97Propyl gallate (PG) A = 4.30 � 104c + 0.005, r = 0.9998 4.30 � 104 1.05 � 10�6–3.25 � 10�5 2.75Vitamin E A = 1.40 � 104c � 0.003, r = 0.9997 1.40 � 104 3.78 � 10�6–1.0 � 10�4 0.90Trolox (TR) A = 1.56 � 104c + 0.01, r = 0.9999 1.56 � 104 2.56 � 10�6–8.9 � 10�5 1.00Ascorbic acid (AA) A = 1.41 � 104c � 0.004, r = 0.9999 1.41 � 104 3.82 � 10�6–9.93 � 10�5 0.91Quercetin (QR) A = 8.06 � 104c + 0.016, r = 0.9998 8.06 � 104 4.22 � 10�7–1.72 � 10�5 5.16Caffeic acid (CFA) A = 4.32 � 104c � 0.005, r = 0.9997 4.32 � 104 1.27 � 10�6–3.25 � 10�5 2.77Ferulic acid (FRA) A = 1.84 � 104c � 0.01, r = 0.9994 1.84 � 104 3.26 � 10�6–7.67 � 10�5 1.18

0

CAntioxidant x106, M

0.0

0.2

0.4

0.8

Abs

orba

nce

PGTBHQBHABHT

10 20 30 40

0.6

Fig. 6. The calibration curves of PG, TBHQ, BHA and BHT in7% M-b-CD aqueous medium with respect to the modifiedCUPRAC method.



The TEAC for BHT was lower than that of BHA,although they contained the same number of pheno-lic –OH groups. This may be explained by the sterichindrance of the two bulky butyl groups in BHTcompared to the single butyl group in BHA, andby the presence of a para-methoxy group (relativeto phenolic –OH) in BHA, increasing the electrondensity in the para-position thereby stabilizing thearyloxy radical when the parent compound (BHA)loses one electron. It should be stated here thatsome structural properties such as steric hindrance

of butyl side groups in butylated hydroxytoluenesare not accounted for in the Folin assay sensitiveto phenolic groups, while ORAC test shows sucha preference [34]. Similar to our findings, the EC50

(i.e., the antioxidant concentration necessary to de-crease the initial amount of DPPH by 50%) valuesin DPPH scavenging assay of quercetin, BHA,and BHT were 0.052, 0.092, and 0.321 g g�1 DPPH,respectively, yielding an antioxidant activity orderof QR > BHA > BHT [35].

3.5. Applicability of Beer’s law to mixtures

Synthetic mixtures obeyed Beer’s law fairly well,as depicted in Table 2. Ternary and quaternary mix-tures of lipophilic and hydrophilic antioxidants,assayed in the same aqueous medium in the pres-ence of M-b-CD, exhibited the theoreticallyexpected antioxidant capacity (AOC) within ±9%,meaning that chemical deviations from Beer’s lawessentially did not exist and the CUPRAC absor-bances of constituents were additive. The originalCUPRAC method was previously shown to be freefrom chemical deviations from Beer’s law, as dem-onstrated on synthetic mixtures of hydrophilic phe-nolic compounds [14], but this fact wasdemonstrated for the first time here for mixturesof hydrophilic and hydrophobic compounds in thesame solvent medium.

If the observed absorbance of a complex mixtureat a given wavelength is the sum of the absorbancesof individual constituents of the mixture, then it canbe assumed that apparent chemical deviations fromBeer’s law do not exist, confirming the principle ofadditivity of absorbances. The validity of this prin-ciple implies that antioxidant capacities measuredwith the improved CUPRAC method are additive,i.e., the experimentally measured capacities shouldgenerally lie within (+/�) 5–10% interval of the the-oretically computed values using the formula:

Capacitytotal ¼ TEAC1concn:1 þ TEAC2concn:2

þ TEAC3concn:3þ ð11Þ

where 1,2, . . ., i denote the corresponding constitu-ents of the synthetic mixture. This is a very impor-tant property of real antioxidant mixtures in thesense that their total antioxidant power could beobjectively evaluated by the given assay (as opposedto analogous kinetic assays in which 50% inhibitiveconcentrations, known as IC50 values, of antioxi-dants are not additive).

Table 2Comparison of the theoretically expected and experimentallyfound trolox (TR)–equivalent antioxidant capacities (in mM TRunits) of synthetic mixtures of lipophilic and hydrophilic antiox-idants (with respect to the modified CUPRAC method)

Composition ofmixture

Capacity expected(as mM TR-equivalent)

Capacity foundexperimentally(as mM TR-equivalent)

0.2 mL 0.125 mMVitamin E

3.09 � 10�2 3.05 � 10�2

20 lL 1.25 mM BHT0.1 mL 0.125 mM QR0.2 mL 0.125 mM AA

0.2 mL 0.125 mMTBHQ

3.21 � 10�2 3.41 � 10�2

20 lL 1.25 mM BHT0.2 mL 0.125 mM

CFA0.2 mL 0.125 mM AA

0.2 mL 0.125 mM LG 2.78 � 10�2 2.82 � 10�2

20 lL 1.25 mM BHT0.2 mL 0.125 mM TR0.2 mL 0.125 mM AA

0.2 mL 0.125 mM TR 3.28 � 10�2 3.41 � 10�2


0.2 mL 0.125 mMTBHQ

0.1 mL 0.125 mM QR

0.2 mL 0.125 mMFRA

2.18 � 10�2 2.40 � 10�2


0.2 mL 0.125 mMBHA

0.2 mL 0.125 mMCFA

2.79 � 10�2 3.04 � 10�2


0.2 mL 0.125 mMTBHQ



3.6. Optimizing M-b-CD concentration for preserving

antioxidant capacity

Decrease of modified CUPRAC absorbance ofBHT as a function of increasing concentration ofM-b-CD in final solution is shown in Fig. 7. BHTwas chosen as the model compound for all otherlipophilic phenolics both in this test and in settingthe aqueous solubility limit of lipophilic antioxi-dants, because it had the least solubility in water(i.e., 0.6–1.1 mg L�1 at 20–25 �C) [36]. In addition,BHT losses have been reported during heating andfiltration stages [37]. The data in Fig. 7 show thatAOC of lipophilic antioxidants is reduced whenthese compounds are encapsulated in inclusion com-plexes with M-b-CD. Thus, M-b-CD should beadded to aqueous solution at optimal concentration(as described in the ‘recommended procedure’) inorder to solubilize the lipophilic antioxidant whilepreserving its AOC. Although contradictory reportsexist in literature about the antioxidant status ofencapsulated phenolics, Polyakov et al. [25]observed a considerable decrease in the antioxidantactivity of CD-encapsulated phenolics for scaveng-ing OOH radicals. This may arise from the relativestability of the inclusion complex, e.g., the forma-tion constant (Kf) for the 1:1 quercetin (QR) –b-CD complex was 129 M�1 [26]. The morehydrophobic flavones may be expected to formmore stable inclusion complexes with M-b-CD thanQR, due to the more hydrophobic character of theinner core of the latter cyclodextrin compound. Inour case, decrease in the CUPRAC absorbance ofthe M-b-CD-encapsulated phenolic antioxidants

may indicate either decreased AOC of phenolic orincreased interaction of the resulting chromogen,i.e., bis(neocuproine) copper(I) chloride, with thecyclodextrin. A significant decrease of AOC uponinclusion–complexation is not expected, as theredox equilibrium constant of the CUPRAC reac-tion [14] is much greater than that of the formationof antioxidant-M-b-CD complex. Since differentsites are responsible for interaction of variousantioxidant compounds with free radicals and oxi-dizing agents [25], recognition of the exact trendsin the change of antioxidant properties (e.g.,enhancement, maintenance, or weakening) uponCD-complexation may not be theoretically esti-mated, and require the performance of suitableexperiments with a given AOC assay. In any case,the useful concentration of M-b-CD should be opti-mized in the assay. The proposed method was suc-cessful for all the tested lipophilic antioxidants butb-carotene (results not reported). Possibly b-caro-tene does not fit into the cavity of M-b-CD, orhigher aggregates form between the host and theguest compounds. Polyakov et al. [25] reported asimilar aggregation, where divergent spectral char-acteristics of b–carotene and hydroxypropyl b-CDcombinations indicated either increased ordecreased solubility of b–carotene in the presenceof cyclodextrins.

3.7. The relative independence of the improved

CUPRAC assay from solvent effects

The ‘antioxidant polarity paradox’ states thathydrophilic antioxidants are often less effective inoil-in-water emulsions than lipophilic antioxidants,whereas lipophilic antioxidants are less effective inbulk oils than hydrophilic antioxidants [38,39].BHT and BHA (the latter to a less extent) were poorantioxidants in the Rancimat induction period andDPPH scavenging antioxidant assays [40,41], whileBHT acted as the best and caffeic acid the poorestinhibitor of b-carotene bleaching antioxidative assay[42]. The ability of lipophilic antioxidants to concen-trate at the interphases of small oil droplets in a lin-oleic acid emulsion and their poor solubility in theaqueous phase may partly explain these contradic-tory results. The single solvent medium containingM-b-CD of this work is expected to partly solve the‘antioxidant polarity paradox’ in the sense of devel-oping an antioxidant assay relatively independentof solvent effects so that each antioxidant, disregard-ing its lipophilicity level, may exhibit its characteris-

0.0CM-B-CD x 103 (g/mL)

0.2

0.3

0.4

0.5

Abs

orba

nce

0.5 1.0 1.5 2.0 2.5 3.0

Fig. 7. Decrease of modified CUPRAC absorbance of BHT as afunction of increasing concentration of M-b-CD in final solution.


https://www.researchgate.net/publication/230140305_Voltammetric_Behavior_and_Simultaneous_Determination_of_the_Antioxidants_Propyl_Gallate_Butylated_Hydroxyanisole_and_Butylated_Hydroxytoluene_in_Acidic_Acetonitrile-Water_Medium_Using_PLS_Calibration?el=1_x_8&enrichId=rgreq-2d475d2df5a03a485ac50071eeb4785c-XXX&enrichSource=Y292ZXJQYWdlOzIzMjkwNjU1MztBUzoxMDI4MDI2MDQ5NTM2MTBAMTQwMTUyMTQ1OTg0Mg==


tic antioxidant capacity merely arising from its struc-tural property, i.e., electron-transfer capability, in anaqueous-rich medium. It should also be borne inmind that although BHA, BHT, and TBHQ typeoil-soluble antioxidants have found wide use in thefood industry as synthetic antioxidants, regulatorymeasures have been taken for the restriction of theiruse in human food [43] due to probable health risksabove a critical dose such as carcinogenicity [44].

4. Conclusion

Since our recently reported CUPRAC (cupric ionreducing antioxidant capacity) assay as a simple,low-cost, and diversely applicable total antioxidantcapacity measurement for dietary polyphenols, vita-mins C and E, and plasma antioxidants did notcover oil-soluble antioxidants like butylatedhydroxyanisole (BHA), butylated hydroxytoluene(BHT), tert-butylhydroquinone (TBHQ), propylgallate (PG), lauryl gallate (LG), and vitamin E(a-tocopherol), this work reports the assay bothlipophilic and hydrophilic antioxidants simulta-neously in the same aqueous solvent medium, bymaking use of their ‘host–guest’ complexes withmethyl b-cyclodextrin (M-b-CD), a solubilityenhancer cyclic oligosaccharide. In addition to solu-bility enhancement, molecular spectroscopic (UVand IR) evidence was also provided for the forma-tion of inclusion complexes of M-b-CD with antiox-idants. The cyclodextrin concentration wasoptimized, and the calibration curves, molarabsorptivities, and linear concentration ranges ofeach antioxidant was established in the proposedsystem. The trolox-equivalent antioxidant capacities(TEAC coefficients) of hydrophilic antioxidants didnot differ significantly from those reported in theoriginal CUPRAC method, while TEAC values oflipophilic antioxidants were reported for the firsttime in this modified CUPRAC assay. Syntheticmixtures comprised of lipophilic and hydrophilicantioxidants gave the theoretically expectedCUPRAC antioxidant capacities, indicating thatchemical deviations from Beer’s law were basicallyabsent, and the observed CUPRAC absorbanceswere additive. The single solvent medium containingM-b-CD of this work is expected to partly solve the‘antioxidant polarity paradox’ in the sense of devel-oping an antioxidant assay relatively independent ofsolvent effects so that each antioxidant, disregardingits lipophilicity level, may exhibit its characteristic

antioxidant capacity merely arising from its struc-tural property, i.e., electron-transfer capability.

Acknowledgements

The authors would like to express their gratitudeto Istanbul University Research Fund, BilimselArastirma Projeleri Yurutucu Sekreterligi, for thefunding of Project YOP-4/27052004, to State Plan-ning Organization of Turkey (DPT) for theAdvanced Research Project of Istanbul University(2005K120430), and to TUBITAK (Turkish Scien-tific and Technical Research Council) for theresearch projects 105T402 and 106T514. Theauthors also wish to thank Professor Saadet Pabu-ccuoglu, Head of the Division of Chemical Technol-ogies, Department of Chemical Engineering ofIstanbul University, for her contribution in obtain-ing the IR spectra.

References

[1] B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biologyand Medicine, Oxford University Press, Oxford, UK,1989.

[2] B. Halliwell, O.I. Aruoma, FEBS Lett. 281 (1991) 9.[3] B.N. Ames, M.K. Shigenaga, T.M. Hagen, Proceedings of

the National Academy of Sciences of the USA 90 (1993)7915.

[4] B. Ou, D. Huang, M. Hampsch-Woodill, J.A. Flanagan,E.K. Deemer, J. Agric. Food Chem. 50 (2002) 3122.

[5] D. Huang, B. Ou, R.L. Prior, J. Agric. Food Chem. 53(2005) 1841.

[6] R.L. Prior, X. Wu, K. Schaich, J. Agric. Food Chem. 53(2005) 4290.

[7] G. Cao, C.P. Verdon, A.H.B. Wu, H. Wang, R.L. Prior,Clin. Chem. 41 (1995) 1738.

[8] N.J. Miller, C.A. Rice-Evans, M.J. Davies, V. Gopinathan,A. Milner, Clin. Sci. 84 (1993) 407.

[9] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang,Free Radic. Biol. Med. 26 (1999) 1231.

[10] C. Sanchez-Moreno, J.A. Larrauri, F.A. Saura-Calixto, J.Sci. Food Agric. 76 (1998) 270.

[11] O. Folin, V. Ciocalteu, J. Biol. Chem. 73 (1927) 627.[12] V.L. Singleton, R. Orthofer, R.M. Lamuela-Raventos,

Meth. Enzymol. 299 (1999) 152.[13] I.F.F. Benzie, J.J. Strain, Anal. Biochem. 239 (1996) 70.[14] R. Apak, K. Guc�lu, M. Ozyurek, S.E. Karademir, J. Agric.

Food Chem. 52 (2004) 7970.[15] R. Apak, K. Guc�lu, M. Ozyurek, S.E. Karademir, M.

Altun, Free Rad. Res. 39 (2005) 949.[16] K. Guc�lu, K. Sozgen, E. Tutem, M. Ozyurek, R. Apak,

Talanta 65 (2005) 1226.[17] M. Ozyurek, K. Guc�lu, B. Bektas�oglu, R. Apak, Anal.

Chim. Acta 588 (2007) 88.[18] K. Guc�lu, M. Altun, M. Ozyurek, S.E. Karademir, R.

Apak, Int. J. Food Sci. Technol. 41 (2006) 76.



[19] R. Apak, K. Guc�lu, M. Ozyurek, S.E. Karademir, E. Erc�ag,Int. J. Food Sci. Nutr. 57 (2006) 292.

[20] B. Bektas�oglu, S.E. Celik, M. Ozyurek, K. Guc�lu, R. Apak,Biochem. Biophys. Res. Commun. 345 (2006) 1194.

[21] T. Loftsson, M.E. Brewster, J. Pharm. Sci. 85 (1996) 1017.[22] A. Braibanti, E. Fisicaro, A. Ghiozzi, C. Compari, G. Bovis,

React. Funct. Polym. 36 (1998) 251.[23] G. Crini, M. Morcellet, J. Separat. Sci. 25 (2002) 789.[24] A. Iaconinoto, M. Chicca, S. Pinamonti, A. Casolari, A.

Bianchi, S. Scalia, Pharmazie 59 (2004) 30.[25] N.E. Polyakov, T.V. Leshina, T.A. Konovalova, O.E. Hand,

L.D. Kispert, Free Radic. Biol. Med. 36 (2004) 872.[26] M.L. Calabro, S. Tommasini, P. Donato, D. Raneri, R.

Stancanelli, P. Ficarra, R. Ficarra, C. Costa, S. Catania,C. Rustichelli, G. Gamberini, J. Pharm. Biomed. Anal. 35(2004) 365.

[27] E. Damiani, R. Tursilli, A. Casolari, P. Astolfi, L. Greci, S.Scalia, Free Radic. Res. 39 (2005) 41.

[28] W. Ammawath, Y.B. Che Man, B.S. Baharin, R.B. AbdulRahman, J. Food Lipids 12 (2005) 198.

[29] W. Ammawath, Y.B. Che Man, B.S. Baharin, R.B. AbdulRahman, J. Food Lipids 11 (2004) 266.

[30] M. Foti, G. Ruberto, J. Agric. Food Chem. 49 (2001) 342.[31] D. Huang, B. Ou, M. Hampsch-Woodill, J.A. Flanagan,

E.K. Deemer, J. Agric. Food Chem. 50 (2002) 1815.

[32] C.A. Rice-Evans, N.J. Miller, G. Paganda, Trends Plant Sci.2 (1997) 152.

[33] K. Robards, P.D. Prenzler, G. Tucker, P. Swatsitang, W.Glover, Food Chem. 66 (1999) 401.

[34] B. Ou, D. Huang, M. Hampsch-Woodill, J.A. Flanagan,FASEB J. 17 (2003) 127.

[35] P. Siddhuraju, K. Becker, J. Agric. Food Chem. 51 (2003)2144.

[36] S.-S. Chang, J.R. Maurey, J. Chem. Eng. Data 30 (1985)384.

[37] T. Galeano-Diaz, A.G. Cabanillas, M.F.A. Franco,F. Salinas, J.-C. Vire, Electroanalysis 10 (1998) 497.

[38] W.L. Porter, E.D. Black, A.M. Drolet, J. Agric. FoodChem. 37 (1989) 615.

[39] E.N. Frankel, S.-W. Huang, R. Aeschbach, E. Prior, J.Agric. Food Chem. 44 (1996) 131.

[40] M. Mittelbach, S. Schober, J. Am. Oil Chem. Soc. 80 (2003)817.

[41] M.H. Gordon, L. Kourimska, J. Sci. Food Agric. 68 (1995)347.

[42] A. Von Gadow, E. Joubert, C.F. Hansmann, J. Agric. FoodChem. 45 (1997) 632.

[43] D.V. Parke, D.F.V. Lewis, Food Addit. Contam. 9 (1992) 561.[44] P.M. Ekanayake, G.T. Park, Y.D. Lee, S.J. Kim, S.C.

Jeong, J. Lee, J. Food Lipids 12 (2005) 34.




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