Methodology to Determine Antioxidant Capacity in Plant Foods

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Updated methodology to determine antioxidant capacity in plantfoods, oils and beverages: Extraction, measurement and

expression of results

Jara Perez-Jimenez a, Sara Arranz a, Maria Tabernero a, M. Elena Daz- Rubio a,Jose Serrano b, Isabel Goni b, Fulgencio Saura-Calixto a,*

a Department of Metabolism and Nutrition, Consejo Superior de Investigaciones Cientficas (IF-CSIC), Calle Jose Antonio Novais, 10, 28040 Madrid, Spainb Nutrition and Gastrointestinal Health Unit (CSIC/UCM), Universidad Complutense de Madrid (UCM), Spain

Received 11 October 2007; accepted 10 December 2007

Abstract

The comparison between antioxidant capacity values reported by different laboratories is quite difficult because of substantial differ-ences in sample preparation, extraction of antioxidants and expression of results. An updated methodology to determine of antioxidantcapacity in plant foods, oils and beverages including extraction of antioxidants, measurement of antioxidant capacity and expression ofresults is presented. During sample preparation, loss of antioxidants in drying and milling steps must be minimized. Antioxidant capac-ity is determined in aqueous-organic extracts (combining at least two extraction cycles) and in the corresponding residues (acidic hydroly-zates to release condensed proanthocyanidins and hydrolyzable phenolics). Different aspects, such as type of solvent and possibleinterferences form non-antioxidant compounds, that may affect the results of the most common methods of antioxidant capacity (FRAP,

ABTS, DPPH and ORAC) are discussed. The different ways of expressing antioxidant capacity results, including kinetic parameters, aredescribed. 2007 Elsevier Ltd. All rights reserved.

Keywords: Antioxidant capacity; Antioxidants extraction; FRAP; ABTS; DPPH; ORAC

1. Introduction

The antioxidant capacity of plant foods is derivedfrom the cumulative synergistic action of a wide varietyof antioxidants such as vitamins C and E and polyphe-

nols, carotenoids, terpenoids, Maillard compounds andtrace minerals. These antioxidants appear to play a rolein the prevention of oxidative stress-related diseasesand in the reduction of total mortality associated withdiets rich in plant foods, particularly fruits and vegeta-

bles (Bazzano et al., 2002; Brighenti et al., 2005; Pitsavoset al., 2005; Trichopoulou, Costacou, Bamia, & Tricho-poulos, 2003). Quantitatively, the main dietary antioxi-dants are polyphenols, followed by vitamins andcarotenoids; dietary daily intakes are about 1 g for poly-phenols, 110 mg for antioxidant vitamins and 9.4 mg forcarotenoids (ONeill et al., 2001; Saura-Calixto & Goni,2006).

The antioxidant content of plant foods, and hencetheir associated antioxidant capacity, depends firstly onthe variety and the degree of ripening (Olsson et al.,

0963-9969/$ - see front matter 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.foodres.2007.12.004

Abbreviations: ABTS, 2,20-azino-bis(3-ethylbenz-thiazoline-6-sulfonicacid); AE, antirradical efficiency; DPPH, 2,2-diphenyl-1-picrylhydrazyl;EC50, concentration of antioxidant needed to reduce the original amountof radical by 50%; FRAP, ferric/reducing antioxidant power; HAT,hydrogen atom transfer; LDL, low-density lipoprotein; ORAC, oxygenradical absorbance capacity; SET, single electron transfer; TAC, totalantioxidant capacity; TEAC, Trolox equivalent antioxidant capacity;tEC50, time needed for the EC50 to reach 50% of the original amount ofradical scavenged; TPTZ, 2,4,6-tri(2-pyridyl)-s-triazine.* Corresponding author. Tel.: +34 91544567; fax: +34 915492300.

E-mail address: [email protected] (F. Saura-Calixto).

www.elsevier.com/locate/foodres

Available online at www.sciencedirect.com

Food Research International 41 (2008) 274285
mailto:[email protected]:[email protected]


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2004; Prakash, Upadhyay, Singh, & Singh, 2007). Afterharvesting, polyphenols undergo certain reactions thatmay cause a decrease in the antioxidant capacity of thesample (Srivastava, Akoh, Yi, Fischer, & Krewer,2007). Also, different post-harvest aspects, such as theconditions of storage (time, temperature, atmosphere,

etc.) and processing (cutting, time and temperature ofpossible treatments, addition of synthetic antioxidants,etc.) affect antioxidant capacity of foodstuffs (Manzocco,Anese, & Nicoli, 1998; Olsson et al., 2004; Srivastavaet al., 2007).

Antioxidant capacity may be a key parameter for bothfood science and technology and nutritional studies, andtherefore there is presently a need to develop a standard-ized methodology to measure total antioxidant capacity(TAC) in plant foods. In the literature there are substantialdifferences in sample preparation, extraction of antioxi-dants (solvent, temperature, etc.), selection of end-pointsand expression of results, even for the same method, so that

comparison between the values reported by different labo-ratories can be quite difficult.

There are several articles reviewing the large number ofassays developed to measure antioxidant capacity in thelast two decades (Frankel & Meyer, 2000; Prior, Wu, &Schaich, 2005; Sanchez-Moreno, 2002). The most widely-used procedures are ferric reducing/antioxidant power(FRAP), 2,20-azino-bis(3-ethylbenz-thiazoline-6-sulfonicacid) (ABTS) or Trolox equivalent antioxidant capacity(TEAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and oxy-gen radical absorbance capacity (ORAC).

Most original works and reviews on antioxidant capac-

ity focus mainly on the characteristics of the measurementprocedure such as free radical generating system, redoxinteractions, molecular target, end-point, lipophilic andhydrophilic solubility, etc. However, little attention hasbeen paid to critical steps such as sample preparation(Luthria, 2006) or the procedure for extraction of antioxi-dants (Pellegrini et al., 2007; Perez-Jimenez & Saura-Cali-xto, 2005).

Antioxidant capacity is usually measured in foodextracts obtained with chemical aqueous-organic solvents(methanol, ethanol, acetone, chloroform, etc.). However,there is no solvent that would be entirely satisfactory forextraction of all the antioxidants present in a food, espe-cially those associated with complex carbohydrates andproteins (Bravo, Abia, & Saura-Calixto, 1994). Conse-quently, there is a considerable amount of antioxidantsremaining in the extraction residues, which is ignored inmost chemical and biological studies. And yet these non-extracted antioxidants are released from the food matrixinto the human gut by the action of digestive enzymesand intestinal microflora and may produce significant bio-logical effects.

In short, nowadays there is a good understanding ofhow to measure antioxidant capacity in plant foods, butless attention has been paid to the complete extraction of

antioxidants.

The present work is intended to provide an updatedmethodology for determination of antioxidant capacity inplant foods, oils and beverages, considering three essentialsteps: extraction of antioxidants, antioxidant capacity mea-surements and expression of results. The proceduresselected for each step are described and applied to specific

samples, considering both experimental and bibliographicaldata.

2. Materials and methods

2.1. Reagents

2,2-Diphenyl-1-picrylhydrazyl (DPPH), potassium per-sulfate, fluorescein (3,60-dihydroxy-spiro-[isobenzofuran-1-[3H],90[9H]-xanthen]-3-one) and iron III-clorure-6-hydratefrom Panreac, Castellar del Valles, Barcelona, Spain.

2,20-Azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid)(ABTS), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-

2-carboxylic acid), catechin and gallic acid from SigmaAldrich Qumica, S.A., Madrid, Spain.

2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ) from FlukaChemicals, Madrid, Spain.

All reagents used were of analytical grade.

2.2. Samples

Red grape pomace and red grape seeds came fromCencibel variety, vintage year 2005, from Manzanaresregion, Spain.

Commercial extract from cocoa was provided by Natra-

ceutical S.A. (Valencia, Spain). Dark chocolate (52%cocoa), milk chocolate (34% cocoa) and cocoa paste werefrom Valor S.A. (Villajoyosa, Alicante, Spain) Cocoa solu-ble powder, Cola-Cao was from Nutrexpa S.A, Barce-lona, Spain.

Walnuts (Juglans regia) were from Iberic walnutPizarro, Borges S.A. (Barcelona, Spain) and almonds (Pru-nus dulcis) without shell, hazelnuts (Corylis avellana), pea-nuts (Arachis hypogaea) without shell and pistachios(Pistachia vera) were from Aperitivos Medina S.L. (Mos-toles, Madrid, Spain).

Fucoidan (99%) from Fucus vesiculosus was purchasedfrom SigmaAldrich Qumica, S.A. (Madrid, Spain).

2.3. Sample preparation and extraction of antioxidants

2.3.1. Plant foods

Foodstuffs are freeze-dried and milled to a particle sizeof less than 0.5 mm in a centrifuge milling. Analysisshould be performed preferently immediately after extrac-tion. Alternatively, samples are stored at 20 C untilanalysis.

The procedure followed for extraction of antioxidants isshown in Fig. 1 (Saura-Calixto, Serrano, & Goni, 2007).The purpose of this extraction is to obtain extractable anti-

oxidants using aqueous-organic solvents, and non-extract-

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able antioxidants using acidic hydrolysis. 0.5 g of sample isplaced in a capped centrifuge tube; 20 mL of acidic metha-nol/water (50:50, v/v; pH 2) is added and the tube is thor-oughly shaken at room temperature for 1 h. The tube iscentrifuged at 2500g for 10 min and the supernatant isrecovered. Twenty millilitres of acetone/water (70:30, v/v)is added to the residue, and shaking and centrifugationare repeated. Methanolic and acetonic extracts were com-

bined and used to determine the antioxidant capacity asso-ciated with extractable antioxidants.

The residues of these extractions are subjected to twodifferent acidic treatments in order to release non-extract-able antioxidants, which make up a quantitatively impor-tant fraction of the dietary intake of antioxidants:

The residues are mixed with 20 mL of methanol and2 mL of concentrated sulphuric acid. Samples are placedin a water bath with constant shaking at 85 C for 20 h.Samples are then centrifuged (2500g for 10 min) andsupernatants recovered. After two washings with dis-tilled water, the final volume is taken up to 50 mL(Hartzfeld, Forkner, Hunter, & Hagerman, 2002). Theantioxidant capacity of this residue refers to hydrolysa-ble tannins and other phenolics linked to carbohydratesand proteins.

The residues are treated with HCl/buthanol/FeCl3 (5:95,v/v) at 100 C for 3 h. Samples are then centrifuged(2500g for 10 min) and supernatants recovered. Aftertwo washings with HCl/buthanol (5:95, v/v), the finalvolume is taken up to 25 mL (Porter, Hrstich, & Chan,1985; Reed, McDowell, Van Soest, & Horvarth, 1982).The antioxidant capacity of this residue refers to con-densed tannins (proanthocyanidins) not extracted by

the previous aqueous-organic procedure.

2.3.2. Beverages

Total antioxidant capacity is determined directly in bev-erages, after diluting aliquots in water when necessary.

To determine antioxidant capacity associated withhydrophilic and lipophilic compounds separately, ethylacetate is mixed with the beverage in a 1:1 ratio, and aftershaking for 1 h samples are centrifuged at 1800g for10 min. Antioxidant capacity is determined in the aqueous

and the organic phases (Pulido, Hernandez-Garca, &Saura-Calixto, 2003).

2.3.3. Oils

Total antioxidant capacity was determined directly invegetable oils, after diluting aliquots in ethyl acetate.

To determine separately antioxidant capacity associatedto polar and apolar compounds, 5 mL of oil were mixedwith 5 mL of methanol. The mixture was vigorously stirredfor 20 min and centrifuged at 2500g for 10 min and thesupernatant was recovered. Another 5 mL were added

and the same process was repeated. Antioxidant capacitywas measured directly in the methanolic fraction (thatextracts polar compounds) and in the remaining oil (apolarfraction), after dilution with ethyl acetate (Espn, Soler-Rivas, & Wichers, 2000).

2.4. Determination of antioxidant capacity. Expression of

results

Following are the protocols for the most common meth-ods for determining the antioxidant capacities of foods and

beverages:

0.5 g of sample

20 mL methanol/water (50:50 v/v, pH 2)

centrifugation

supernatant residue

20 mL acetone/water (70:30 v/v)

residue

centrifugation

supernatant

antioxidant

extract(extractable

polyphenols)

antioxidant extract

(hydrolyzable tannins)

antioxidant extract

(condensed tannins)

Antioxidant capacity (AC1)

methanol/ H2SO4


buthanol / HCl


Fig. 1. Scheme of the extraction of antioxidants from a plant food.

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FRAP assay. FRAP reagent, containing TPTZ, FeCl3and acetate buffer, is mixed with distilled water andthe test sample (aqueous-organic extract) or the blank(solvent). Maximum absorbance values at 595 nm aretaken at 37 C after 30 min, using a Beckman DU-640spectrophotometer (Beckman Instruments Inc., Fuller-

ton, CA, USA) (Benzie & Strain, 1996; Pulido, Bravo,& Saura-Calixto, 2000). Solutions of known Trolox con-centrations are used for calibration.

DPPH assay. The method described by Brand-Williams,Cuvelier, and Berset (1995) was later modified by San-chez-Moreno, Larrauri, and Saura-Calixto (1998) inorder to determine kinetic parameters. After adjustingthe blank with methanol, sample is mixed with a DDPH

methanolic solution. The absorbance at 515 nm is mea-sured until the reaction hasreached the plateau. A calibra-tion curve is plotted at that wavelength to calculate theremaining DDPH. The parameter EC50, which reflects50% depletion of the free radical, is expressed in terms

of g dry weight/g DDPH

. The time taken to reach thesteady state at EC50 (tEC 50) and the antiradical efficiency(AE = 1/EC50 tEC50) are also determined.

ABTS assay at a fixed end-point. After addition of sampleor Trolox to an ABTS+ solution (generated from a solu-tion of potassium persulphate mixed with ABTS), absor-bance readings at 658 nm are taken every 20 s using aBeckman DU-460 spectrophotometer (Beckman Instru-ments Inc., Fullerton, CA, USA) at 30 C. The reactionis monitored for 6 min. The percentage inhibition ofabsorbance vs. time is plotted and the area below the curve(06 min) is calculated. Solutions of known Trolox con-

centrations are used for calibration (Re et al., 1999). ABTS assay expressed kinetically. The ABTS radical

cation is generated as described for the ABTS assay ata fixed end-point. A recent procedure described byPerez-Jimenez and Saura-Calixto (2008) modified theoriginal method so as to determine kinetic parameters.An aliquot of the sample extract (0.1 mL) is added to3.9 mL of ABTS+ (0.044 g/L) in methanol which wasprepared daily. Absorbances at 658 nm are measuredat different time intervals on a Beckman DU-640 spec-trophotometer (Beckman Instruments Inc., Fullerton,CA, USA) until the reaction reaches a plateau. TheABTS+ concentration in the reaction medium is calcu-lated by plotting concentration vs. absorbance. EC50,tEC50 and AE are calculated as in the DPPH assay.

ORAC assay. Sample/blank is mixed with PBS, AAPHand fluorescein. Fluorescence is recorded until it reacheszero (excitation wavelength 493 nm, emission wavelength515 nm) in a fluorescence spectrophotometer PerkinElmer LS 55 at 37 C. Results are calculated using the dif-ferences of areas under the fluorescein decay curve betweenthe blank and the sample and are expressed as Troloxequivalents (Ou, Hampsch-Woodill, & Prior, 2001).

All these methods can be performed directly in bever-

ages. In the case of oils, DPPH is the most suitable meth-

ods, for both total oil and methanol extracts from it, asmentioned in Section 3.

A discussion of the applicability of the different methodsin plant foods is included. Determination of antioxidantcapacity in aqueous-organic extracts (AC1, Fig. 1) can beperformed by any of these methods. FRAP, DPPH and

ORAC can be performed in the hydrolyzates (methanol/H2SO4) of the residues of the extractions (AC3), while onlyABTS can be used to determine antioxidant capacity of thehydrolyzates (buthanol/HCl) of the residues, because ofsolvent interferences in the other procedures.

3. Results and discussion

3.1. Sample preparation

3.1.1. Drying of the sample

Determinations of antioxidant capacity of plant foodswere performed on a dry powder. Depending on the proce-

dure chosen for drying the sample, this process may resultin the retention of most of the samples antioxidant capac-ity, or in significant loss of it.

Drying by means of high-temperature and/or prolongedtreatments causes a decrease in antioxidant capacity. Thishas been observed in orange by-products or in differenttomato cultivars subjected to different conditions of air-drying (Kerkhofs, Lister, & Savage, 2005; Garau, Simal,Rossello, & Femena, 2007), as well as in other products,such as the edible seaweed Fucus vesiculosus (Jimenez-Escrig, Jimenez-Jimenez, Pulido, & Saura-Calixto, 2001).

Losses of antioxidant capacity are minimized if freeze-

drying is used. For example, a study comparing the antiox-idant capacity of strawberries subjected to convectivedrying, microwave-vacuum drying and freeze-drying(Bohm, Kunhert, Rohm, & Scholze, 2006) found that thelatter treatment was the only one in which there was nota significant loss of antioxidant capacity compared to theoriginal sample (Table 1).

If freeze-drying is not available, drying under vacuum isanother option, as long as the temperature is controlledand does not exceed 5060 C depending on the sample.For instance, in a previous study by our group (Larrauri,Ruperez, & Saura-Calixto, 1997), it was found that dryingred grape pomace at 60 C did not significantly reduce thecontent of antioxidant compounds as compared to freeze-dried sample.

Some authors have observed an increase in antioxidantcapacity after certain drying processes (Cheng et al.,2006; Mrkic, Cocci, Dalla Rosa, & Sachetti, 2006) due tothe formation of new antioxidant compounds (Maillardcompounds, polymeric structures of polyphenols withhigher antioxidant capacity). It could be useful to optimizethese treatments for the development of processed foodswith high antioxidant capacity, but in analysis of raw food-stuffs, the drying conditions must be selected to avoid suchgeneration of new compounds, since these would not be

present in the product as it is intended to be consumed.



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3.1.2. Milling procedure

Milling conditions are another important aspect whenpreparing a sample for determination of antioxidant capac-ity. During milling, heating of the sample and time in anoxidizing atmosphere must be kept to a minimum. Millingshould be performed in a centrifuge mill or in a hammermill, but not in a shear mill, since even under a nitrogen

atmosphere this produces significant loss of antioxidantcapacity (Table 2).

On the other hand, it has been observed that reductionof particle size increases the in vitro antioxidant capacityof different samples, such as wheat, blackcurrant juice pressresidues or black cohosh. This is presumably becausereduction of the particle size breaks up certain structuresof the food matrix, releasing bound antioxidants andreducing the distance the analyte has to travel to reachthe surface. At the same time, the enlargement of the par-ticle surface improves solvent penetration (Cheng et al.,2006; Landbo & Meyer, 2001; Mukhopadhyay, Luthria,

& Robbins, 2006).However, it is important to note that this reduction ofthe particle size also reduces thermal stability, and there-fore determination of antioxidant capacity should be per-formed as close as possible to the milling step to avoiddegradation of antioxidant compounds.

3.2. Extraction of antioxidants

3.2.1. Plant foods

Several procedures for extraction of antioxidants fromplant foods have been described, based mainly on mixturesof water with ethanol, methanol or acetone in different pro-portions (Antolovich, Prenzler, Robards, & Ryan, 2000;

Dalla Valle, Mignani, Spinardi, Galvano, & Ciappellano,2007; Gray, Clarke, Baux, Bunting, & Salter, 2002;Mukhopadhyay et al., 2006; Yu, Perret, Davy, Wilson, &Melby, 2002). It has been observed that the addition ofwater increases the efficiency of extraction, until it reachesan optimum (Mukhopadhyay et al., 2006).

Efficient extraction of antioxidants requires the use of sol-vents with different polarities: certain antioxidants requirepolar solvents such as methanol, while ethyl acetate or chlo-roform are used to extract lipophilic antioxidants. Anothermeans of improving the efficiency of extraction of antioxi-dants is to use acidified solvents (Awika, Rooney, &Waniska, 2005; Gorinstein et al., 2007; Iqbal, Bhanger, &Anwar, 2007).

A procedure for extraction of antioxidants from plantfoods should combine at least two extraction cycles per-formed with aqueous-organic solvents with different polar-ities in order to extract antioxidant compounds withdifferent chemical structures. A general procedure is rou-

tinely used at our lab to extract antioxidants from differentfoodstuffs, including extraction with acidic methanol/water(50:50, v/v; pH 2), followed by acetone/water (70:30, v/v)(Fig. 1) (Larrauri et al., 1997; Perez-Jimenez & Saura-Cal-ixto, 2005; Saura-Calixto & Goni, 2006). It was observed indifferent samples, such as commercial extracts from cocoaor red grape seeds that, after the first extraction cycle, thesample retained significant antioxidant capacity, whichwas removed by the second extraction (Table 3).

Also, when the order of the two extraction solvents wasaltered in the case of red grape seed, it was observed thatthe first extraction solvent again produced greater extrac-

tion of antioxidant compounds (78% of antioxidant capac-ity by FRAP assay), but the sample retained a significantpercentage of antioxidant capacity, which was extractedwith the second extraction solvent (22% of antioxidantcapacity by FRAP assay). It should be noted that thesedeterminations were performed in a normal atmospherein a capped centrifuge tube, as well as in a nitrogen atmo-sphere, and no significant differences were found betweenthe resulting values.

The solid-to-solvent ratio also needs to be considered,since it is reported that when this ratio is increased, the

Table 1Antioxidant capacity of Camarossa strawberry after different dryingtreatments (Bohm et al., 2006)

ABTS(mmol/100 g)

FRAP(mmol Fe2+/100 g)

Fresh sample 8.4 0.2 a 24 2.1 aConvective drying 5.1 1.4 b 17.5 3.6 b

Microwave-vacuum drying 5.3 1.5 b 17.7 2.5 bFreeze-drying 8.2 0.4 a 20.2 1.2 a,b

Different letters in a column imply the existence of significant differences(p < 0.05).

Table 2Antioxidant capacity (lmol Trolox/g dry matter) of red grape pomaceafter milling in different systemsa

System FRAP

Cutting mill 71 3 aHammer mills 63 1 bShear mill in N2 atmosphere 62 1 b

Different letters in a column imply the existence of significant differences(p < 0.05).a Extraction with acidic methanol/water (50:50, v/v; pH 2) plus acetone/

water (70:30, v/v).

Table 3Antioxidant capacity after applying different solvents in a commercialextract from cocoa and in red grape seeds (lmol Trolox/g dry matter)

Sample Extraction solvent % antioxidant capacityextracted (FRAP)

Commercialextract fromcocoa

1: acidic methanol/water(50:50, v/v; pH 2)

59.7

2: acetone/water (70:30,v/v) in the residue

40.3

Red grape seeds 1: acidic methanol/water(50:50, v/v; pH 2)

64.3

2: acetone/water (70:30,v/v) in the residue

35.7



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efficiency of extraction of phenolic compounds alsoincreases, until an optimum is reached (Mukhopadhyayet al., 2006).

These conditions for extraction of antioxidants havebeen optimized for plant foods in order to determine thetotal antioxidant capacity of a sample. Several extraction

procedures have been developed to selectively extract cer-tain phenolic compounds, vitamins and carotenoids if aparticular compound or group of compounds are to bestudied (Antolovich et al., 2000; Rodrguez-Bernaldo deQuiros & Costa, 2006; Ueda & Igarashi, 1990).

Another important aspect is that most published workson antioxidant capacity deal exclusively with antioxidantsin aqueous-organic extracts. However, the residues of theseextracts may retain a considerable amount of antioxidantcapacity, mainly associated with hydrolysable phenolicsand carotenoids associated with fibre and protein. Thisantioxidant capacity may become bioactive in the humangut once it is released from the food matrix by the action

of digestive enzymes in the small intestine and bacterialdegradation in the large intestine (Jenner, Rafter, & Halli-well, 2005). Moreover, many foodstuffs may contain moreof these non-extractable antioxidants than extractablepolyphenols (Saura-Calixto et al., 2007). For example,Table 4 shows the results of antioxidant capacity associatedwith the extracts and the residues of different productsderived from cocoa and from dietary fruits and pulses. Itcan be seen that the antioxidant capacity associated withthe residues (in this case, proanthocyanidins) is of the sameorder or even greater than the capacity associated to theextracts. Similarly, cereals possess a higher antioxidant

capacity associated to the residues (rich in hydrolysabletannins) than associated to the extracts (Perez-Jimenez &Saura-Calixto, 2005) This suggests that the analysis ofthe antioxidant capacity present in the residue of aque-ous-organic extractions should be included in routinedetermination of the antioxidant capacity of plant foods.

An alternative to the acidic hydrolysis described in thiswork to release hydrolysable phenolics is a basic hydroly-sis, that some authors have applied to foodstuffs such as

cereals or nuts (Pellegrini et al., 2006; Serpen, Capuano,Fogliano, & Gokmen, 2007). However, it should beaddressed that this procedure should be complementedwith the determination of condensed tannins in the residuesof the aqueous-organic extracts.

3.2.2. OilsTo determine total antioxidant capacity of vegetable oilsit is not necessary to perform an extraction, and measure-ments can be performed directly on the oil after dilutingaliquots in ethyl acetate (Arranz, Perez-Jimenez, &Saura-Calixto, in press) or in n-hexane (Pellegrini et al.,2003). It is also possible to determine antioxidant capacityassociated with polar and non-polar compounds sepa-rately, for which extraction with methanol is necessary.This separation is based in the fact that antioxidant presentin an oil will be present in the polar or in the apolar frac-tion according to their partition coefficient.

Table 5 shows the results for total oil and the two frac-

tions (methanolic and non-polar) of different nut oils per-formed by DPPH assay. Only the values for total oil andthe non-polar fraction should be compared, since bothare measured using ethyl acetate, but in the case of themethanolic fraction, the solvent may interfere in the DPPHassay (Perez-Jimenez & Saura-Calixto, 2006). It shouldalso be noted that, although in the oil remaining aftermethanolic extraction there are no polar compounds, themethanolic extract contains both polar compounds andother compounds of intermediate polarity.

Paradoxically, antioxidant capacity is usually greater(lower EC50) in the polar fraction (methanolic extract) than

in the total oil. This could be because there are interactionswith lipid constituents in the total oil but not in the meth-anolic extracts; for example, an antagonist effect has beenobserved between quercetin and a-tocopherol in sunfloweroil (Becker, Ntouma, & Skibsted, 2007). It could also berelated to the so-called polar paradox, according to whichlipophilic antioxidants are more effective in polar media,for instance methanol (Schwarz et al., 2000).

On this basis, the antioxidant capacities of oils shouldonly be compared when analyses have been performedusing the same extraction medium and the same measure-ment method. Moreover, the contradictory resultsobtained for different extracts from oils show the necessityof a further research in this topic.

3.2.2.1. Fatty foods. In the case of samples with high fatcontent, such as nuts, fat may interfere in the determina-tion of antioxidant capacity of the whole sample (Arranzet al., in press, available on-line). Therefore, the procedureshould be to perform prior defatting at room temperature(0.5 g of milled sample are placed in a test tube and20 mL of petroleum ether are added; after shaking for20 min and centrifugation at 2500g for 10 min, the super-natant is recovered). The antioxidant capacity is then deter-mined separately in the oil as described in Section 2.3.3,

and in the defatted matter including extractable and

Table 4Antioxidant capacity by FRAP assay (lmol Trolox/g dry matter) associ-

ated to aqueous-organic extracts (acidic methanol/water followed byacetone/water) and their residues (hydrolysis with buthanol/HCl) inproducts derived from cocoa (Serrano, 2005; Tabernero et al., 2006)

Aqueous-organicextracts

Residues(proanthocyanidins)

Dark chocolate 149.87 8.01 144.05 1.82Milk chocolate 61.50 1.70 84.31 0.58Cocoa soluble powder 71.83 0.34 51.83 3.33Cocoa paste 606.14 42.91 246.14 5.47Dietary fruitsa 25.5 0.5 60.2Dietary pulsesb 9.0 0.2 37.6

a Mean value from a pulp of the 21 most consumed fruits in the Spanishdiet.b Mean value from a pulp of the three most consumed pulses in the

Spanish diet.



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non-extractable antioxidants as described in Section2.3.1; examples of how this procedure is applied to differentdefatted nuts are shown in Table 6. These data show thatthe antioxidant capacity present in the residue (associatedto hydrolysable tannins) is quite higher than the presentin the aqueous-organic extracts (lower EC50); moreover,when these values are compared to the ones in Table 5for the oils of these nuts, it can be seen that the contribu-tion of oil to total antioxidant capacity of the sample ismuch lower than the one of the defatted fraction.

3.2.3. Beverages

The antioxidant capacities of beverages common in thediet, including total antioxidant capacity and antioxidantcapacity associated with hydrophilic and lipophilicextracts, are shown in Table 7. It can be seen that thehydrophilic fraction normally contributes more to the totalantioxidant than the lipophilic fraction (Pulido et al.,2003). There have been specific studies on antioxidantcapacity of tea, wine, coffee and beer (Lugasi & Hovari,

2003; Naithani, Nair, & Kakkar, 2006; Sanchez-Gonzalez,Jimenez-Escrig, & Saura-Calixto, 2005; Sanchez-Moreno,Larrauri, & Saura-Calixto, 1999).

Determination of the antioxidant capacities of beveragescan be quite important from a nutritional point of view,since it has been established that they are the main contrib-utors to the total antioxidant capacity of a whole diet suchas the Mediterranean diet (Saura-Calixto & Goni, 2006).Moreover, the antioxidant capacity present in beveragesmay be more bioaccessible than the capacity associatedwith solid plant foods, where enzymatic action is necessaryto release antioxidant compounds.

3.3. Measurement of antioxidant capacity

Growing interest in possible healthy effects of antioxi-dants has led to the development of a large number of

assays to determine the antioxidant capacities of foodextracts. Since the antioxidant capacity of a food is deter-mined by a mixture of different antioxidants with differentmechanisms of actions, among which there may be syner-gistic interactions, it is necessary to combine more thanone method to determine in vitro antioxidant capacity of

foodstuffs (Frankel & Meyer, 2000; Laguerre, Lecomte,& Villeneuve, 2007).

FRAP, ABTS, DPPH and ORAC are the most commonmethods for determining in vitro antioxidant capacity. It isrecommended that at least two, and preferably all of theseassays be combined if possible, so as to provide compre-hensive information on the total antioxidant capacity ofa foodstuff, taking into account the pros and cons of eachassay as well as their applicability.

As regards the basis of these methods, FRAP measuresthe ability of a sample to reduce metals, while ABTS,DPPH and ORAC measure a samples free radical scav-

enging capacity. From a mechanical standpoint, in FRAPand ABTS there is a SET (Single Electron Transfer) reac-tion, while in ORAC there is a HAT (Hydrogen AtomTransfer) reaction, while DPPH combines both (Foti, Das-quino, & Geraci, 2004; Prior et al., 2005).

Table 5Antioxidant capacity of nut oils measured by DPPH method (EC50 values, g/g DPPH)

Walnut oil Almond oil Hazelnut oil Peanut oil Pistachio oil

Total oila 1514.3 70,2 712.2 36 478.5 8.6 1395.9 99.7 377.9 31.8No polar fractionb 1764.1 125.1 2717.5 68.8 1096.4 52.1 3492.8 53 863.5 41.1Methanolic fractionc 688.8 17.5 1109.2 38.8 366.4 60.4 190.2 48.5 8.42 1.5

a

Determined in oil solved in ethyl acetate.b Antioxidant capacity determined in methanolic extract.c Antioxidant capacity determined in the remaining oil after methanolic extraction.

Table 6Antioxidant capacity by DPPH assay of defatted nuts (EC50, g dry whole nut/g DPPH)

Walnut Almond Hazelnut Peanut Pistachio

Aqueous-organic extractsa 14.3 0.1 401.5 78.5 46.1 2.1 277.9 12.7 32.9 0.3Residuesb 4.0 0.2 22.6 1.0 26.1 0.9 53.1 3.2 23.9 0.7

a Supernatants of acidic methanol/water and acetone/water extracts.b

Hydrolyzates (methanol/H2SO4) of the residues of the aqueous-organic extraction.

Table 7Total antioxidant capacity of total beverage (ABTS assay) and antioxi-dant capacity associated to hydrophilic and lipophilic extracts ofbeverages from the Spanish diet (Pulido et al., 2003)

Beverage Total antioxidantcapacity

Hydrophilicextracts

Lipophilicextracts

Coffee 13,280 51 8772 26 2950 94Wine 10,932 542 8788 54 2084 34Tea 6308 80 3932 71 2072 28

Beer 772 17 714 21 162 8Orange juice 2494 34 1969 241 162 1Milk 2194 107 118 10 Not detectedCola


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FRAP, ABTS and DPPH are performed in a UVvisspectrophotometer, while ORAC requires a fluorimeter,which is not so commonly found in many laboratories.Regarding the time needed for each assay, ABTS isreported to take 7 min (Re et al., 1999), while the FRAPassay takes 30 min (Pulido et al., 2000) and ORAC 30

40 min (Ou et al., 2001); because it measures kinetic param-eters and is based on the testing of different concentrationsof a sample, and DPPH takes longer, depending on theindividual sample (Sanchez-Moreno et al., 1998).

Another aspect is the applicability of each of theseassays. FRAP, ABTS and ORAC are usually used to mea-sure the antioxidant capacity of hydrophilic compounds,although some modifications have been suggested forORAC (Wu et al., 2004) and ABTS (Pulido et al., 2003)in order to determine the antioxidant capacity of a sampleassociated with its lipohilic compounds. However, DDPHis the only one of these methods that has been routinelyapplied in both aqueous-organic extracts of plant foods

(Cheng, Ling, & Hsieh, 2007; Llorach, Tomas-Barberan,& Ferreres, 2004) and vegetable oils (Tuberoso, Kow-alczyk, Sarritzu, & Cabras, 2007).

Each method gives accurate, repeatable values, but anti-oxidant capacity figures may differ substantially betweenone method and another. All these are proper methods,but at the same time all of have some drawbacks; theseare summarized in Table 8.

Table 9 shows that these methods can be applied to sam-ples of different natures, from extracts performed directlyin a food sample such as walnut, to by-products of the foodindustry such as grape pomace, or pure compounds such as

fucoidan, a sulphated polysaccharide present in certain edi-ble seaweeds. Although the ranking of antioxidant capacityof a group of samples determined by different antioxidantcapacity assays usually follows the same trend irrespectiveof the method considered, since they are based on differentreaction mechanisms there may be certain differences in the

ranking of particular samples. For instance, althoughgrape pomace presented the greatest antioxidant capacityin FRAP, ORAC and DPPH assays, in the ABTS assaywalnut presented the greatest capacity. This shows thatmore than one method need to be combined to characterizethe antioxidant capacity of a sample, and also that compar-

isons should only be performed between values of antioxi-dant capacity obtained using the same method and thesame solvent.

3.3.1. Possible interferences

There are some aspects that may interfere in the deter-mination of antioxidant capacity and should be taken intoaccount when analysing results.

Firstly, the solvent in which the reaction takes place is akey factor in the results, since the polarity of the solventaffects the mechanism of the reaction. This aspect has beendiscussed for several foodstuffs or standards, such as winein ORAC (Villano, Fernandez-Pachon, Troncoso, & Gar-

ca-Parrilla, 2005), quercetin in DPPH (Pinelo, Manzocco,Nunez, & Nicoli, 2004), dietary polyphenols in FRAP(Pulido et al., 2000) or wheat bran in ABTS (Zhou &Yu, 2004). The fact that this also affects pure standards,and not only extracts from food samples, indicates that thisinterference is due to the solvent itself, as the reaction med-ium, and not to the fact that the extraction of antioxidantsvaries and affects the results of antioxidant capacitydepending on the solvent.

Table 10 shows the values of antioxidant capacity for amixture of catechin and gallic acid in different solvents(methanol, water and acetone/water 50:50, v/v), where

the effect that the reaction medium may have on the deter-mination of antioxidant capacity can be clearly seen. Thiseffect was greater in ORAC and in ABTS assays. There-fore, values of antioxidant capacity should always be com-pared with reference to the same method and with the samesolvent as reaction medium.

Table 8Main drawbacks associated to the methods of antioxidant capacity

Method Main drawbacks

FRAP Other compounds may absorb at 595 nm Any compound with a redox potential lower than 0.77 V, although it does not behave in vivo as an antioxidant, may reduce iron

It is performed at a non-physiological pHABTS Antioxidants, besides reacting with the radical to yield the original molecule, generate other compounds

Reaction is not over at the usually taken 6 min The free radical used is not present in vivo

DPPH Other compounds may absorb at 515 nm

There may be an steric hindrance for molecules with a higher molecular weight The free radical used is not present in vivo and it is quite stable, unlike radicals present in living organisms

ORAC The kinetics of reaction may vary depending on the concentration of the antioxidant, what enables this method to be used for kineticsstudies

It measures the ability of antioxidants to scavenge peroxyl radicals, present in vivo; however, the procedure to generate these peroxylradicals is not physiological

Protein may have an interfering effect

Refs.: (Arnao, 2000; Arts et al., 2004; Lopez, Martnez, Del Valle, Ferrit, & Luque, 2003; Ou et al., 2002; Osman, Wong, Hill, & Fernyoungha, 2006;

Pinelo et al., 2004; Prior et al., 2005).



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Another factor to be considered in the determination ofantioxidant capacity is the possible presence in the sampleof certain non-antioxidant compounds, which may react inthe antioxidant capacity assays, producing over estima-tions of antioxidant capacity. For instance, in a recentstudy by our group, it was established that several amino-acids may provide a false positive in antioxidant capacityassays (Perez-Jimenez & Saura-Calixto, 2006). This agreeswith the recorded fact that when ORAC is measured inplasma, values for deproteinized plasma are much lower

than for complete plasma (Ou et al., 2001) and this factmay explain the recently reported high antioxidant capac-ity of proteins of certain cereals and pseudocereals (Gorin-stein et al., 2007). Therefore, the protein content of asample, and in particular certain amino acids, should beconsidered when analysing results.

3.4. Expression of results

Expression of results is the last step in the determinationof a samples antioxidant capacity, but it is also a keypoint. Several different ways of expressing the results canbe found in works published on antioxidant capacity, evenfor the same method, making it quite difficult to compareresults from similar samples (Villano et al., 2005).

The expression of results of antioxidant capacity assayscan be summarized in three categories: results based onmeasurements at a fixed end-point compared to an stan-dard, results expressed considering lag-phase, and resultsbased on kinetic parameters.

ABTS, FRAP and ORAC assays are usually performedat a fixed end-point; results are interpolated in a calibrationcurve of Trolox (a hydrosoluble analogue of vitamin E)and are expressed as Trolox equivalents that is, the lmolof Trolox necessary to provide the same antioxidant capac-

ity as a gram of the sample (Cao, Russell, Lischner, &Prior, 1998; Ou et al., 2001; Re et al., 1999). The higherthe Trolox Equivalent value, the more antioxidant the sam-ple is. Trolox does not have any physiological significanceand its choice as the standard for antioxidant capacity is

arbitrary; however, since its use is quite generalized, choos-ing it can make it easier to compare published data. Forinstance, the results of FRAP assay were initially expressedas equivalents of Fe2SO4 (Benzie & Strain, 1996), but thisstandard was later changed to Trolox so as to expressresults in the same form as other methods. The use of otherstandards such as vitamin C or vitamin E may be useful forspecific nutritional studies. In any case, this way of express-ing the results is quite important, since it permits directcomparison between different published results as long as

the method, the solvent and the assay time are the same.Another way to express the results of assays performed

at a fixed end-point, which is commonly used for DPPHassays, is to measure the percentage of inhibition, compar-ing the change in the absorbance caused by a blank and bya test sample (Xu, Yang, Chen, Hu, & Hu, 2003; Yu et al.,2002). However, since this percentage of inhibition willdepend on the concentration of radical and of sample takenin each case, it is not possible to compare studies that usedifferent initial amounts.

In any case, in assays performed at a fixed end-point itshould always be confirmed that the reaction was com-pleted at the time chosen.

Some antioxidant capacity methods express their resultswith reference to the lag-phase, which is the time duringwhich the antioxidant is able to exert its action before theoxidation process starts. This has been employed, forexample, in LDL oxidation assay (Kleinveld, Hak-Lem-mers, Stalenhoef, & Demacker, 1992) or in Total Radi-cal-Trapping Antioxidant Parameter (TRAP) assay.However, it has been noted that not all antioxidants exhibita clearly defined lag-phase (Prior et al., 2005), and also thatthis parameter would not be useful when determining theantioxidant capacity of a complex mixture of compounds,such as plasma (Niki, 2002), which could also apply to

plant foods and beverages.Another way to express antioxidant capacity results is to

consider kinetic parameters. The DPPH assay wasmodified by Sanchez-Moreno et al. (1998) in order to intro-duce this kind of parameters; by testing different initial

Table 9Antioxidant capacity associated to aqueous-organic extracts (acidic methanol/water followed by acetone/water) of several samples

Sample FRAP (lmol Trolox/g dm)

ABTS (lmol Trolox/g dm)

ORAC (lmol Trolox/g dm)

EC50 (g/g) DPPH tEC50(min)

AE (103)

Walnut 114.9 4.0 153.8 16.2 187.2 9.1 14.3 0.1 30.5 0.6 2.3Fucoidan 27.2 2.6 17.0 0.6 57.4 8.4 13.2 0.2 14.0 0.8 5.4Red grape

pomace

273.9 16.6 124.4 0.3 214.3 37.3 3.5 0.5 38.1 2.6 7.4

Table 10Results of ORAC, ABTS, FRAP and DPPH assays of a mixture of catechin:gallic acid 1:1 M in different solvents ( Perez-Jimenez & Saura-Calixto, 2006)

Solvent ABTS (lmol Trolox/g dm) DPPH EC50 (g/g) FRAP (lmol Trolox/g dm) ORAC (lmol Trolox/g dm)

Methanol 11,291.4 837 a 0.083 0.004 a 9642.4 977.1 a 20,836.6 2079.6 aWater 28104 275.8 b 0.067 0.001 b 9559.2 110.7 a 13,543.6 298.4 bAcetone/water (50:50, v/v) 13,894.8 240.2 a 0.083 0.003 a 10,100.1 489.7 a,b 30,217.5 2100.1 c

Different letters in the same column imply significant differences.



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concentrations of the test sample, it is possible to establishEC50, that is the amount of sample needed to scavenge50% of the original concentration of the free radical;tEC50, or the time taken by that concentration to reach equi-librium; or AE, that is the inverse of the product of EC50 andtEC50. With these parameters it is possible to gain morecomprehensive information on the samples antioxidantcapacity, taking into account not only their activity (definedby EC50) but also whether the antioxidant acts quickly or

slowly (tEC50) and both simultaneously (AE). Thisapproach has been successfully used by many researchgroups in quite different samples (Femena-Ros, Garca-Pajon, Hernandez-Galan, Macas-Sanchez, & Collado,2006; Sanchez-Moreno et al., 1999; Vergara-Valenciaet al., 2007).

A similar modification was recently reported for theABTS assay (Perez-Jimenez & Saura-Calixto, 2008). Firstof all, this approach considers the time taken by some com-pounds to finish reacting with the free radical, given thatcertain antioxidants do not do so within the 6 min nor-mally required (Arts, Voss, Haenen, & Bast, 2004; Prior

et al., 2005). Secondly, as noted earlier, the informationderived with kinetic parameters is more comprehensive.Table 11 shows an example of the application of kinetic

parameters to ABTS and DPPH assays on different nuts.One drawback of this approach is that it is more time-con-suming than taking measurements at a fixed end-point, andthe time needed for one assay will depend on each sample.However, as can be seen in Table 11, the average time takenby antioxidants to react with the ABTS radical is shorter thanthe time taken to react with the DPPH radical, so that kineticparameters may be easier to apply to this method.

4. Conclusions

Determination of antioxidant capacity of foods and bev-erages includes three steps: sample preparation andextraction of antioxidants, measurement of antioxidantcapacity, and expression of results.

During sample preparation, the loss of antioxidants inthe drying and milling steps must be kept to a minimum.

In the extraction of antioxidants, at least two extractioncycles with mixtures of different polarity of water andorganic solvents must be combined.

Determination of total antioxidant capacity must beperformed both in aqueous-organic extracts and in their

corresponding residues, which may exhibit higher anti-

oxidant capacity than the aqueous-organic extracts, afact usually ignored in the literature.

Antioxidant capacity values should only be comparedwhere the method, the solvent and the analytical condi-tions are the same.

Possible interference from certain food constituentsmust also be taken into account when determining anti-oxidant capacity.

At least two assays should be performed to determine

antioxidant capacity. Expression of kinetic parameters such as EC50, tEC50

and AE may provide a more comprehensive evaluationof antioxidant capacity.

Acknowledgements

The present research was performed under the financialsupport of the Spanish Ministry of Education and Science(Project AGL 2004-07579-C04-01/ALI). S. Arranz had anFPI scholarship from the Ministerio de Educacion y Cien-

cia. Mara Elena Daz-Rubio had a scholarship from theInstituto Danone.

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Methodology to Determine Antioxidant Capacity in Plant Foods