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Food Research International 41 (2008) 274285 www.elsevier.com/locate/foodres

Updated methodology to determine antioxidant capacity in plant foods, oils and beverages: Extraction, measurement and expression of results
rez-Jime nez a, Sara Arranz a, Maria Tabernero a, M. Elena D az- Rubio a, Jara Pe b b a,* Serrano , Isabel Gon Jose i , Fulgencio Saura-Calixto
a

cas (IF-CSIC), Calle Jose Antonio Novais, 10, 28040 Madrid, Spain Department of Metabolism and Nutrition, Consejo Superior de Investigaciones Cient b 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 dierent laboratories is quite dicult because of substantial dierences in sample preparation, extraction of antioxidants and expression of results. An updated methodology to determine of antioxidant capacity in plant foods, oils and beverages including extraction of antioxidants, measurement of antioxidant capacity and expression of results is presented. During sample preparation, loss of antioxidants in drying and milling steps must be minimized. Antioxidant capacity is determined in aqueous-organic extracts (combining at least two extraction cycles) and in the corresponding residues (acidic hydrolyzates to release condensed proanthocyanidins and hydrolyzable phenolics). Dierent aspects, such as type of solvent and possible interferences form non-antioxidant compounds, that may aect the results of the most common methods of antioxidant capacity (FRAP, ABTS, DPPH and ORAC) are discussed. The dierent ways of expressing antioxidant capacity results, including kinetic parameters, are described. 2007 Elsevier Ltd. All rights reserved.
Keywords: Antioxidant capacity; Antioxidants extraction; FRAP; ABTS; DPPH; ORAC

1. Introduction The antioxidant capacity of plant foods is derived from the cumulative synergistic action of a wide variety of antioxidants such as vitamins C and E and polypheAbbreviations: ABTS, 2,20 -azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid); AE, antirradical eciency; DPPH, 2,2-diphenyl-1-picrylhydrazyl; EC50, concentration of antioxidant needed to reduce the original amount of radical by 50%; FRAP, ferric/reducing antioxidant power; HAT, hydrogen atom transfer; LDL, low-density lipoprotein; ORAC, oxygen radical absorbance capacity; SET, single electron transfer; TAC, total antioxidant capacity; TEAC, Trolox equivalent antioxidant capacity; tEC50, time needed for the EC50 to reach 50% of the original amount of radical scavenged; TPTZ, 2,4,6-tri(2-pyridyl)-s-triazine. * Corresponding author. Tel.: +34 91544567; fax: +34 915492300. E-mail address: fsaura@if.csic.es (F. Saura-Calixto). 0963-9969/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2007.12.004

nols, carotenoids, terpenoids, Maillard compounds and trace minerals. These antioxidants appear to play a role in the prevention of oxidative stress-related diseases and in the reduction of total mortality associated with diets rich in plant foods, particularly fruits and vegetables (Bazzano et al., 2002; Brighenti et al., 2005; Pitsavos et al., 2005; Trichopoulou, Costacou, Bamia, & Trichopoulos, 2003). Quantitatively, the main dietary antioxidants are polyphenols, followed by vitamins and carotenoids; dietary daily intakes are about 1 g for polyphenols, 110 mg for antioxidant vitamins and 9.4 mg for carotenoids (ONeill et al., 2001; Saura-Calixto & Gon i, 2006). The antioxidant content of plant foods, and hence their associated antioxidant capacity, depends rstly on the variety and the degree of ripening (Olsson et al.,

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2004; Prakash, Upadhyay, Singh, & Singh, 2007). After harvesting, polyphenols undergo certain reactions that may cause a decrease in the antioxidant capacity of the sample (Srivastava, Akoh, Yi, Fischer, & Krewer, 2007). Also, dierent post-harvest aspects, such as the conditions of storage (time, temperature, atmosphere, etc.) and processing (cutting, time and temperature of possible treatments, addition of synthetic antioxidants, etc.) aect antioxidant capacity of foodstus (Manzocco, Anese, & Nicoli, 1998; Olsson et al., 2004; Srivastava et al., 2007). Antioxidant capacity may be a key parameter for both food science and technology and nutritional studies, and therefore there is presently a need to develop a standardized methodology to measure total antioxidant capacity (TAC) in plant foods. In the literature there are substantial dierences in sample preparation, extraction of antioxidants (solvent, temperature, etc.), selection of end-points and expression of results, even for the same method, so that comparison between the values reported by dierent laboratories can be quite dicult. There are several articles reviewing the large number of assays developed to measure antioxidant capacity in the last two decades (Frankel & Meyer, 2000; Prior, Wu, & nchez-Moreno, 2002). The most widelySchaich, 2005; Sa used procedures are ferric reducing/antioxidant power (FRAP), 2,20 -azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) or Trolox equivalent antioxidant capacity (TEAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC). Most original works and reviews on antioxidant capacity focus mainly on the characteristics of the measurement procedure such as free radical generating system, redox interactions, molecular target, end-point, lipophilic and hydrophilic solubility, etc. However, little attention has been paid to critical steps such as sample preparation (Luthria, 2006) or the procedure for extraction of antioxi rez-Jime nez & Saura-Calidants (Pellegrini et al., 2007; Pe xto, 2005). Antioxidant capacity is usually measured in food extracts obtained with chemical aqueous-organic solvents (methanol, ethanol, acetone, chloroform, etc.). However, there is no solvent that would be entirely satisfactory for extraction of all the antioxidants present in a food, especially those associated with complex carbohydrates and proteins (Bravo, Abia, & Saura-Calixto, 1994). Consequently, there is a considerable amount of antioxidants remaining in the extraction residues, which is ignored in most chemical and biological studies. And yet these nonextracted antioxidants are released from the food matrix into the human gut by the action of digestive enzymes and intestinal microora and may produce signicant biological eects. In short, nowadays there is a good understanding of how to measure antioxidant capacity in plant foods, but less attention has been paid to the complete extraction of antioxidants.

The present work is intended to provide an updated methodology for determination of antioxidant capacity in plant foods, oils and beverages, considering three essential steps: extraction of antioxidants, antioxidant capacity measurements and expression of results. The procedures selected for each step are described and applied to specic samples, considering both experimental and bibliographical data. 2. Materials and methods 2.1. Reagents 2,2-Diphenyl-1-picrylhydrazyl (DPPH), potassium persulfate, uorescein (3,60 -dihydroxy-spiro-[isobenzofuran-1[3H],90 [9H]-xanthen]-3-one) and iron III-clorure-6-hydrate s, Barcelona, Spain. from Panreac, Castellar del Valle 0 2,2 -Azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman2-carboxylic acid), catechin and gallic acid from Sigma mica, S.A., Madrid, Spain. Aldrich Qu 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ) from Fluka Chemicals, Madrid, Spain. All reagents used were of analytical grade. 2.2. Samples Red grape pomace and red grape seeds came from Cencibel variety, vintage year 2005, from Manzanares region, Spain. Commercial extract from cocoa was provided by Natraceutical S.A. (Valencia, Spain). Dark chocolate (52% cocoa), milk chocolate (34% cocoa) and cocoa paste were from Valor S.A. (Villajoyosa, Alicante, Spain) Cocoa soluble powder, Cola-Cao was from Nutrexpa S.A, Barcelona, Spain. Walnuts (Juglans regia) were from Iberic walnut Pizarro, Borges S.A. (Barcelona, Spain) and almonds (Prunus dulcis) without shell, hazelnuts (Corylis avellana), peanuts (Arachis hypogaea) without shell and pistachios s(Pistachia vera) were from Aperitivos Medina S.L. (Mo toles, Madrid, Spain). Fucoidan (99%) from Fucus vesiculosus was purchased mica, S.A. (Madrid, Spain). from SigmaAldrich Qu 2.3. Sample preparation and extraction of antioxidants 2.3.1. Plant foods Foodstus are freeze-dried and milled to a particle size of less than 0.5 mm in a centrifuge milling. Analysis should be performed preferently immediately after extraction. Alternatively, samples are stored at 20 C until analysis. The procedure followed for extraction of antioxidants is shown in Fig. 1 (Saura-Calixto, Serrano, & Gon i, 2007). The purpose of this extraction is to obtain extractable antioxidants using aqueous-organic solvents, and non-extract-

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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)

antioxidant extract
(extractable polyphenols) supernatant

centrifugation

residue

Antioxidant capacity (AC1) buthanol / HCl methanol/ H2SO4

antioxidant extract (condensed tannins)

Antioxidant capacity (AC2)

antioxidant extract (hydrolyzable tannins)

Antioxidant capacity (AC3)

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

able antioxidants using acidic hydrolysis. 0.5 g of sample is placed in a capped centrifuge tube; 20 mL of acidic methanol/water (50:50, v/v; pH 2) is added and the tube is thoroughly shaken at room temperature for 1 h. The tube is centrifuged at 2500g for 10 min and the supernatant is recovered. Twenty millilitres of acetone/water (70:30, v/v) is added to the residue, and shaking and centrifugation are repeated. Methanolic and acetonic extracts were combined and used to determine the antioxidant capacity associated with extractable antioxidants. The residues of these extractions are subjected to two dierent acidic treatments in order to release non-extractable antioxidants, which make up a quantitatively important fraction of the dietary intake of antioxidants: The residues are mixed with 20 mL of methanol and 2 mL of concentrated sulphuric acid. Samples are placed in a water bath with constant shaking at 85 C for 20 h. Samples are then centrifuged (2500g for 10 min) and supernatants recovered. After two washings with distilled water, the nal volume is taken up to 50 mL (Hartzfeld, Forkner, Hunter, & Hagerman, 2002). The antioxidant capacity of this residue refers to hydrolysable tannins and other phenolics linked to carbohydrates and 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. After two washings with HCl/buthanol (5:95, v/v), the nal volume is taken up to 25 mL (Porter, Hrstich, & Chan, 1985; Reed, McDowell, Van Soest, & Horvarth, 1982). The antioxidant capacity of this residue refers to condensed tannins (proanthocyanidins) not extracted by the previous aqueous-organic procedure.

2.3.2. Beverages Total antioxidant capacity is determined directly in beverages, after diluting aliquots in water when necessary. To determine antioxidant capacity associated with hydrophilic and lipophilic compounds separately, ethyl acetate is mixed with the beverage in a 1:1 ratio, and after shaking for 1 h samples are centrifuged at 1800g for 10 min. Antioxidant capacity is determined in the aqueous ndez-Garc a, & and the organic phases (Pulido, Herna Saura-Calixto, 2003).

2.3.3. Oils Total antioxidant capacity was determined directly in vegetable oils, after diluting aliquots in ethyl acetate. To determine separately antioxidant capacity associated to polar and apolar compounds, 5 mL of oil were mixed with 5 mL of methanol. The mixture was vigorously stirred for 20 min and centrifuged at 2500g for 10 min and the supernatant was recovered. Another 5 mL were added and the same process was repeated. Antioxidant capacity was measured directly in the methanolic fraction (that extracts polar compounds) and in the remaining oil (apolar n, Solerfraction), after dilution with ethyl acetate (Esp Rivas, & Wichers, 2000).

2.4. Determination of antioxidant capacity. Expression of results Following are the protocols for the most common methods for determining the antioxidant capacities of foods and beverages:

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FRAP assay. FRAP reagent, containing TPTZ, FeCl3 and acetate buer, is mixed with distilled water and the test sample (aqueous-organic extract) or the blank (solvent). Maximum absorbance values at 595 nm are taken at 37 C after 30 min, using a Beckman DU-640 spectrophotometer (Beckman Instruments Inc., Fullerton, CA, USA) (Benzie & Strain, 1996; Pulido, Bravo, & Saura-Calixto, 2000). Solutions of known Trolox concentrations are used for calibration. DPPH assay. The method described by Brand-Williams, nCuvelier, and Berset (1995) was later modied by Sa chez-Moreno, Larrauri, and Saura-Calixto (1998) in order to determine kinetic parameters. After adjusting the blank with methanol, sample is mixed with a DDPH methanolic solution. The absorbance at 515 nm is measured until the reaction has reached the plateau. A calibration curve is plotted at that wavelength to calculate the remaining DDPH. The parameter EC50, which reects 50% depletion of the free radical, is expressed in terms of g dry weight/g DDPH. The time taken to reach the steady state at EC50 (tEC 50) and the antiradical eciency (AE = 1/EC50 tEC50) are also determined. ABTS assay at a xed end-point. After addition of sample or Trolox to an ABTS+ solution (generated from a solution of potassium persulphate mixed with ABTS), absorbance readings at 658 nm are taken every 20 s using a Beckman DU-460 spectrophotometer (Beckman Instruments Inc., Fullerton, CA, USA) at 30 C. The reaction is monitored for 6 min. The percentage inhibition of absorbance vs. time is plotted and the area below the curve (06 min) is calculated. Solutions of known Trolox concentrations are used for calibration (Re et al., 1999). ABTS assay expressed kinetically. The ABTS radical cation is generated as described for the ABTS assay at a xed end-point. A recent procedure described by rez-Jime nez and Saura-Calixto (2008) modied the Pe original method so as to determine kinetic parameters. An aliquot of the sample extract (0.1 mL) is added to 3.9 mL of ABTS+ (0.044 g/L) in methanol which was prepared daily. Absorbances at 658 nm are measured at dierent time intervals on a Beckman DU-640 spectrophotometer (Beckman Instruments Inc., Fullerton, CA, USA) until the reaction reaches a plateau. The ABTS+ concentration in the reaction medium is calculated by plotting concentration vs. absorbance. EC50, tEC50 and AE are calculated as in the DPPH assay. ORAC assay. Sample/blank is mixed with PBS, AAPH and uorescein. Fluorescence is recorded until it reaches zero (excitation wavelength 493 nm, emission wavelength 515 nm) in a uorescence spectrophotometer Perkin Elmer LS 55 at 37 C. Results are calculated using the differences of areas under the uorescein decay curve between the blank and the sample and are expressed as Trolox equivalents (Ou, Hampsch-Woodill, & Prior, 2001). All these methods can be performed directly in beverages. In the case of oils, DPPH is the most suitable meth-

ods, for both total oil and methanol extracts from it, as mentioned in Section 3. A discussion of the applicability of the dierent methods in plant foods is included. Determination of antioxidant capacity in aqueous-organic extracts (AC1, Fig. 1) can be performed 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 only ABTS can be used to determine antioxidant capacity of the hydrolyzates (buthanol/HCl) of the residues, because of solvent 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 foods were performed on a dry powder. Depending on the procedure chosen for drying the sample, this process may result in the retention of most of the samples antioxidant capacity, or in signicant loss of it. Drying by means of high-temperature and/or prolonged treatments causes a decrease in antioxidant capacity. This has been observed in orange by-products or in dierent tomato cultivars subjected to dierent conditions of airdrying (Kerkhofs, Lister, & Savage, 2005; Garau, Simal, a, 2007), as well as in other products, Rossello, & Femen nezsuch as the edible seaweed Fucus vesiculosus (Jime nez-Jime nez, Pulido, & Saura-Calixto, 2001). Escrig, Jime Losses of antioxidant capacity are minimized if freezedrying is used. For example, a study comparing the antioxidant capacity of strawberries subjected to convective drying, microwave-vacuum drying and freeze-drying (Bo hm, Ku nhert, Rohm, & Scholze, 2006) found that the latter treatment was the only one in which there was not a signicant loss of antioxidant capacity compared to the original sample (Table 1). If freeze-drying is not available, drying under vacuum is another option, as long as the temperature is controlled and does not exceed 5060 C depending on the sample. For instance, in a previous study by our group (Larrauri, rez, & Saura-Calixto, 1997), it was found that drying Rupe red grape pomace at 60 C did not signicantly reduce the content of antioxidant compounds as compared to freezedried sample. Some authors have observed an increase in antioxidant capacity after certain drying processes (Cheng et al., 2006; Mrkic, Cocci, Dalla Rosa, & Sachetti, 2006) due to the formation of new antioxidant compounds (Maillard compounds, polymeric structures of polyphenols with higher antioxidant capacity). It could be useful to optimize these treatments for the development of processed foods with high antioxidant capacity, but in analysis of raw foodstus, the drying conditions must be selected to avoid such generation of new compounds, since these would not be present in the product as it is intended to be consumed.

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Table 1 Antioxidant capacity of Camarossa strawberry after dierent drying treatments (Bo hm et al., 2006) ABTS (mmol/100 g) Fresh sample Convective drying Microwave-vacuum drying Freeze-drying 8.4 0.2 5.1 1.4 5.3 1.5 8.2 0.4 a b b a FRAP (mmol Fe2+/100 g) 24 2.1 17.5 3.6 17.7 2.5 20.2 1.2 a b b a,b

Dierent letters in a column imply the existence of signicant dierences (p < 0.05).

3.1.2. Milling procedure Milling conditions are another important aspect when preparing a sample for determination of antioxidant capacity. During milling, heating of the sample and time in an oxidizing atmosphere must be kept to a minimum. Milling should be performed in a centrifuge mill or in a hammer mill, but not in a shear mill, since even under a nitrogen atmosphere this produces signicant loss of antioxidant capacity (Table 2). On the other hand, it has been observed that reduction of particle size increases the in vitro antioxidant capacity of dierent samples, such as wheat, blackcurrant juice press residues or black cohosh. This is presumably because reduction of the particle size breaks up certain structures of the food matrix, releasing bound antioxidants and reducing the distance the analyte has to travel to reach the surface. At the same time, the enlargement of the particle surface improves solvent penetration (Cheng et al., 2006; Landbo & Meyer, 2001; Mukhopadhyay, Luthria, & Robbins, 2006). However, it is important to note that this reduction of the particle size also reduces thermal stability, and therefore determination of antioxidant capacity should be performed as close as possible to the milling step to avoid degradation of antioxidant compounds. 3.2. Extraction of antioxidants 3.2.1. Plant foods Several procedures for extraction of antioxidants from plant foods have been described, based mainly on mixtures of water with ethanol, methanol or acetone in dierent proportions (Antolovich, Prenzler, Robards, & Ryan, 2000;
Table 2 Antioxidant capacity (lmol Trolox/g dry matter) of red grape pomace after milling in dierent systemsa System Cutting mill Hammer mills Shear mill in N2 atmosphere FRAP 71 3 a 63 1 b 62 1 b

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 of water increases the eciency of extraction, until it reaches an optimum (Mukhopadhyay et al., 2006). Ecient extraction of antioxidants requires the use of solvents with dierent polarities: certain antioxidants require polar solvents such as methanol, while ethyl acetate or chloroform are used to extract lipophilic antioxidants. Another means of improving the eciency of extraction of antioxidants is to use acidied solvents (Awika, Rooney, & Waniska, 2005; Gorinstein et al., 2007; Iqbal, Bhanger, & Anwar, 2007). A procedure for extraction of antioxidants from plant foods should combine at least two extraction cycles performed with aqueous-organic solvents with dierent polarities in order to extract antioxidant compounds with dierent chemical structures. A general procedure is routinely used at our lab to extract antioxidants from dierent foodstus, including extraction with acidic methanol/water (50:50, v/v; pH 2), followed by acetone/water (70:30, v/v) rez-Jime nez & Saura-Cal(Fig. 1) (Larrauri et al., 1997; Pe ixto, 2005; Saura-Calixto & Gon i, 2006). It was observed in dierent samples, such as commercial extracts from cocoa or red grape seeds that, after the rst extraction cycle, the sample retained signicant antioxidant capacity, which was removed by the second extraction (Table 3). Also, when the order of the two extraction solvents was altered in the case of red grape seed, it was observed that the rst extraction solvent again produced greater extraction of antioxidant compounds (78% of antioxidant capacity by FRAP assay), but the sample retained a signicant percentage of antioxidant capacity, which was extracted with the second extraction solvent (22% of antioxidant capacity by FRAP assay). It should be noted that these determinations were performed in a normal atmosphere in a capped centrifuge tube, as well as in a nitrogen atmosphere, and no signicant dierences were found between the resulting values. The solid-to-solvent ratio also needs to be considered, since it is reported that when this ratio is increased, the

Table 3 Antioxidant capacity after applying dierent solvents in a commercial extract from cocoa and in red grape seeds (lmol Trolox/g dry matter) Sample Commercial extract from cocoa Red grape seeds Extraction solvent 1: acidic methanol/water (50:50, v/v; pH 2) 2: acetone/water (70:30, v/v) in the residue 1: acidic methanol/water (50:50, v/v; pH 2) 2: acetone/water (70:30, v/v) in the residue % antioxidant capacity extracted (FRAP) 59.7 40.3 64.3 35.7

Dierent letters in a column imply the existence of signicant dierences (p < 0.05). a Extraction with acidic methanol/water (50:50, v/v; pH 2) plus acetone/ water (70:30, v/v).

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eciency of extraction of phenolic compounds also increases, until an optimum is reached (Mukhopadhyay et al., 2006). These conditions for extraction of antioxidants have been optimized for plant foods in order to determine the total antioxidant capacity of a sample. Several extraction procedures have been developed to selectively extract certain phenolic compounds, vitamins and carotenoids if a particular compound or group of compounds are to be guez-Bernaldo de studied (Antolovich et al., 2000; Rodr s & Costa, 2006; Ueda & Igarashi, 1990). Quiro Another important aspect is that most published works on antioxidant capacity deal exclusively with antioxidants in aqueous-organic extracts. However, the residues of these extracts may retain a considerable amount of antioxidant capacity, mainly associated with hydrolysable phenolics and carotenoids associated with bre and protein. This antioxidant capacity may become bioactive in the human gut once it is released from the food matrix by the action of digestive enzymes in the small intestine and bacterial degradation in the large intestine (Jenner, Rafter, & Halliwell, 2005). Moreover, many foodstus may contain more of these non-extractable antioxidants than extractable polyphenols (Saura-Calixto et al., 2007). For example, Table 4 shows the results of antioxidant capacity associated with the extracts and the residues of dierent products derived from cocoa and from dietary fruits and pulses. It can be seen that the antioxidant capacity associated with the residues (in this case, proanthocyanidins) is of the same order or even greater than the capacity associated to the extracts. Similarly, cereals possess a higher antioxidant capacity associated to the residues (rich in hydrolysable rez-Jime nez & tannins) than associated to the extracts (Pe Saura-Calixto, 2005) This suggests that the analysis of the antioxidant capacity present in the residue of aqueous-organic extractions should be included in routine determination of the antioxidant capacity of plant foods. An alternative to the acidic hydrolysis described in this work to release hydrolysable phenolics is a basic hydrolysis, that some authors have applied to foodstus such as
Table 4 Antioxidant capacity by FRAP assay (lmol Trolox/g dry matter) associated to aqueous-organic extracts (acidic methanol/water followed by acetone/water) and their residues (hydrolysis with buthanol/HCl) in products derived from cocoa (Serrano, 2005; Tabernero et al., 2006) Aqueous-organic extracts Dark chocolate Milk chocolate Cocoa soluble powder Cocoa paste Dietary fruitsa Dietary pulsesb
a

cereals or nuts (Pellegrini et al., 2006; Serpen, Capuano, Fogliano, & Go kmen, 2007). However, it should be addressed that this procedure should be complemented with the determination of condensed tannins in the residues of the aqueous-organic extracts. 3.2.2. Oils To determine total antioxidant capacity of vegetable oils it is not necessary to perform an extraction, and measurements can be performed directly on the oil after diluting rez-Jime nez, & aliquots in ethyl acetate (Arranz, Pe Saura-Calixto, in press) or in n-hexane (Pellegrini et al., 2003). It is also possible to determine antioxidant capacity associated with polar and non-polar compounds separately, for which extraction with methanol is necessary. This separation is based in the fact that antioxidant present in an oil will be present in the polar or in the apolar fraction according to their partition coecient. Table 5 shows the results for total oil and the two fractions (methanolic and non-polar) of dierent nut oils performed by DPPH assay. Only the values for total oil and the non-polar fraction should be compared, since both are measured using ethyl acetate, but in the case of the methanolic fraction, the solvent may interfere in the DPPH rez-Jime nez & Saura-Calixto, 2006). It should assay (Pe also be noted that, although in the oil remaining after methanolic extraction there are no polar compounds, the methanolic extract contains both polar compounds and other 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 interactions with lipid constituents in the total oil but not in the methanolic extracts; for example, an antagonist eect has been observed between quercetin and a-tocopherol in sunower oil (Becker, Ntouma, & Skibsted, 2007). It could also be related to the so-called polar paradox, according to which lipophilic antioxidants are more eective in polar media, for instance methanol (Schwarz et al., 2000). On this basis, the antioxidant capacities of oils should only be compared when analyses have been performed using the same extraction medium and the same measurement method. Moreover, the contradictory results obtained for dierent extracts from oils show the necessity of a further research in this topic. 3.2.2.1. Fatty foods. In the case of samples with high fat content, such as nuts, fat may interfere in the determination of antioxidant capacity of the whole sample (Arranz et al., in press, available on-line). Therefore, the procedure should be to perform prior defatting at room temperature (0.5 g of milled sample are placed in a test tube and 20 mL of petroleum ether are added; after shaking for 20 min and centrifugation at 2500g for 10 min, the supernatant is recovered). The antioxidant capacity is then determined separately in the oil as described in Section 2.3.3, and in the defatted matter including extractable and

Residues (proanthocyanidins) 144.05 1.82 84.31 0.58 51.83 3.33 246.14 5.47 60.2 37.6

149.87 8.01 61.50 1.70 71.83 0.34 606.14 42.91 25.5 0.5 9.0 0.2

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

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Table 5 Antioxidant capacity of nut oils measured by DPPH method (EC50 values, g/g DPPH) Walnut oil Total oil No polar fractionb Methanolic fractionc
a b c a

Almond oil 712.2 36 2717.5 68.8 1109.2 38.8

Hazelnut oil 478.5 8.6 1096.4 52.1 366.4 60.4

Peanut oil 1395.9 99.7 3492.8 53 190.2 48.5

Pistachio oil 377.9 31.8 863.5 41.1 8.42 1.5

1514.3 70,2 1764.1 125.1 688.8 17.5

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

non-extractable antioxidants as described in Section 2.3.1; examples of how this procedure is applied to dierent defatted nuts are shown in Table 6. These data show that the antioxidant capacity present in the residue (associated to hydrolysable tannins) is quite higher than the present in the aqueous-organic extracts (lower EC50); moreover, when these values are compared to the ones in Table 5 for the oils of these nuts, it can be seen that the contribution of oil to total antioxidant capacity of the sample is much lower than the one of the defatted fraction. 3.2.3. Beverages The antioxidant capacities of beverages common in the diet, including total antioxidant capacity and antioxidant capacity associated with hydrophilic and lipophilic extracts, are shown in Table 7. It can be seen that the hydrophilic fraction normally contributes more to the total antioxidant than the lipophilic fraction (Pulido et al., 2003). There have been specic studies on antioxidant va ri, capacity of tea, wine, coee and beer (Lugasi & Ho nchez-Gonza lez, 2003; Naithani, Nair, & Kakkar, 2006; Sa nez-Escrig, & Saura-Calixto, 2005; Sa nchez-Moreno, Jime Larrauri, & Saura-Calixto, 1999). Determination of the antioxidant capacities of beverages can be quite important from a nutritional point of view, since it has been established that they are the main contributors to the total antioxidant capacity of a whole diet such as the Mediterranean diet (Saura-Calixto & Gon i, 2006). Moreover, the antioxidant capacity present in beverages may be more bioaccessible than the capacity associated with solid plant foods, where enzymatic action is necessary to release antioxidant compounds. 3.3. Measurement of antioxidant capacity Growing interest in possible healthy eects of antioxidants has led to the development of a large number of

Table 7 Total antioxidant capacity of total beverage (ABTS assay) and antioxidant capacity associated to hydrophilic and lipophilic extracts of beverages from the Spanish diet (Pulido et al., 2003) Beverage Coee Wine Tea Beer Orange juice Milk Cola Total antioxidant capacity 13,280 51 10,932 542 6308 80 772 17 2494 34 2194 107 <100 Hydrophilic extracts 8772 26 8788 54 3932 71 714 21 1969 241 118 10 <100 Lipophilic extracts 2950 94 2084 34 2072 28 162 8 162 1 Not detected Not detected

Results are expressed as lmol Trolox/L.

assays to determine the antioxidant capacities of food extracts. Since the antioxidant capacity of a food is determined by a mixture of dierent antioxidants with dierent mechanisms of actions, among which there may be synergistic interactions, it is necessary to combine more than one method to determine in vitro antioxidant capacity of foodstus (Frankel & Meyer, 2000; Laguerre, Lecomte, & Villeneuve, 2007). FRAP, ABTS, DPPH and ORAC are the most common methods for determining in vitro antioxidant capacity. It is recommended that at least two, and preferably all of these assays be combined if possible, so as to provide comprehensive information on the total antioxidant capacity of a foodstu, taking into account the pros and cons of each assay as well as their applicability. As regards the basis of these methods, FRAP measures the ability of a sample to reduce metals, while ABTS, DPPH and ORAC measure a samples free radical scavenging capacity. From a mechanical standpoint, in FRAP and ABTS there is a SET (Single Electron Transfer) reaction, while in ORAC there is a HAT (Hydrogen Atom Transfer) reaction, while DPPH combines both (Foti, Dasquino, & Geraci, 2004; Prior et al., 2005).

Table 6 Antioxidant capacity by DPPH assay of defatted nuts (EC50, g dry whole nut/g DPPH) Walnut Aqueous-organic extracts Residuesb
a b a

Almond 401.5 78.5 22.6 1.0

Hazelnut 46.1 2.1 26.1 0.9

Peanut 277.9 12.7 53.1 3.2

Pistachio 32.9 0.3 23.9 0.7

14.3 0.1 4.0 0.2

Supernatants of acidic methanol/water and acetone/water extracts. Hydrolyzates (methanol/H2SO4) of the residues of the aqueous-organic extraction.

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FRAP, ABTS and DPPH are performed in a UVvis spectrophotometer, while ORAC requires a uorimeter, which is not so commonly found in many laboratories. Regarding the time needed for each assay, ABTS is reported to take 7 min (Re et al., 1999), while the FRAP assay takes 30 min (Pulido et al., 2000) and ORAC 30 40 min (Ou et al., 2001); because it measures kinetic parameters and is based on the testing of dierent concentrations of a sample, and DPPH takes longer, depending on the nchez-Moreno et al., 1998). individual sample (Sa Another aspect is the applicability of each of these assays. FRAP, ABTS and ORAC are usually used to measure the antioxidant capacity of hydrophilic compounds, although some modications have been suggested for ORAC (Wu et al., 2004) and ABTS (Pulido et al., 2003) in order to determine the antioxidant capacity of a sample associated with its lipohilic compounds. However, DDPH is the only one of these methods that has been routinely applied in both aqueous-organic extracts of plant foods s-Barbera n, (Cheng, Ling, & Hsieh, 2007; Llorach, Toma & Ferreres, 2004) and vegetable oils (Tuberoso, Kowalczyk, Sarritzu, & Cabras, 2007). Each method gives accurate, repeatable values, but antioxidant capacity gures may dier substantially between one method and another. All these are proper methods, but at the same time all of have some drawbacks; these are summarized in Table 8. Table 9 shows that these methods can be applied to samples of dierent natures, from extracts performed directly in a food sample such as walnut, to by-products of the food industry such as grape pomace, or pure compounds such as fucoidan, a sulphated polysaccharide present in certain edible seaweeds. Although the ranking of antioxidant capacity of a group of samples determined by dierent antioxidant capacity assays usually follows the same trend irrespective of the method considered, since they are based on dierent reaction mechanisms there may be certain dierences in the
Table 8 Main drawbacks associated to the methods of antioxidant capacity Method FRAP Main drawbacks

ranking of particular samples. For instance, although grape pomace presented the greatest antioxidant capacity in FRAP, ORAC and DPPH assays, in the ABTS assay walnut presented the greatest capacity. This shows that more than one method need to be combined to characterize the antioxidant capacity of a sample, and also that comparisons should only be performed between values of antioxidant capacity obtained using the same method and the same solvent. 3.3.1. Possible interferences There are some aspects that may interfere in the determination of antioxidant capacity and should be taken into account when analysing results. Firstly, the solvent in which the reaction takes place is a key factor in the results, since the polarity of the solvent aects the mechanism of the reaction. This aspect has been discussed for several foodstus or standards, such as wine ndez-Pacho n, Troncoso, & Garin ORAC (Villan o, Ferna ca-Parrilla, 2005), quercetin in DPPH (Pinelo, Manzocco, n Nu ez, & Nicoli, 2004), dietary polyphenols in FRAP (Pulido et al., 2000) or wheat bran in ABTS (Zhou & Yu, 2004). The fact that this also aects pure standards, and not only extracts from food samples, indicates that this interference is due to the solvent itself, as the reaction medium, and not to the fact that the extraction of antioxidants varies and aects the results of antioxidant capacity depending on the solvent. Table 10 shows the values of antioxidant capacity for a mixture of catechin and gallic acid in dierent solvents (methanol, water and acetone/water 50:50, v/v), where the eect that the reaction medium may have on the determination of antioxidant capacity can be clearly seen. This eect was greater in ORAC and in ABTS assays. Therefore, values of antioxidant capacity should always be compared with reference to the same method and with the same solvent as reaction medium.

 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 pH  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  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  The kinetics of reaction may vary depending on the concentration of the antioxidant, what enables this method to be used for kinetics studies  It measures the ability of antioxidants to scavenge peroxyl radicals, present in vivo; however, the procedure to generate these peroxyl radicals is not physiological  Protein may have an interfering eect

ABTS

DPPH

ORAC

pez, Mart nez, Del Valle, Ferrit, & Luque, 2003; Ou et al., 2002; Osman, Wong, Hill, & Fernyoungha, 2006; Refs.: (Arnao, 2000; Arts et al., 2004; Lo Pinelo et al., 2004; Prior et al., 2005).

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Table 9 Antioxidant capacity associated to aqueous-organic extracts (acidic methanol/water followed by acetone/water) of several samples Sample Walnut Fucoidan Red grape pomace FRAP (lmol Trolox/ g dm) 114.9 4.0 27.2 2.6 273.9 16.6 ABTS (lmol Trolox/ g dm) 153.8 16.2 17.0 0.6 124.4 0.3 ORAC (lmol Trolox/ g dm) 187.2 9.1 57.4 8.4 214.3 37.3 EC50 (g/g) 14.3 0.1 13.2 0.2 3.5 0.5 DPPH tEC50 (min) 30.5 0.6 14.0 0.8 38.1 2.6 AE (103) 2.3 5.4 7.4

Another factor to be considered in the determination of antioxidant capacity is the possible presence in the sample of certain non-antioxidant compounds, which may react in the antioxidant capacity assays, producing over estimations of antioxidant capacity. For instance, in a recent study by our group, it was established that several aminoacids may provide a false positive in antioxidant capacity rez-Jime nez & Saura-Calixto, 2006). This agrees assays (Pe with the recorded fact that when ORAC is measured in plasma, values for deproteinized plasma are much lower than for complete plasma (Ou et al., 2001) and this fact may explain the recently reported high antioxidant capacity of proteins of certain cereals and pseudocereals (Gorinstein et al., 2007). Therefore, the protein content of a sample, and in particular certain amino acids, should be considered when analysing results. 3.4. Expression of results Expression of results is the last step in the determination of a samples antioxidant capacity, but it is also a key point. Several dierent ways of expressing the results can be found in works published on antioxidant capacity, even for the same method, making it quite dicult to compare results from similar samples (Villan o et al., 2005). The expression of results of antioxidant capacity assays can be summarized in three categories: results based on measurements at a xed end-point compared to an standard, results expressed considering lag-phase, and results based on kinetic parameters. ABTS, FRAP and ORAC assays are usually performed at a xed end-point; results are interpolated in a calibration curve of Trolox (a hydrosoluble analogue of vitamin E) and are expressed as Trolox equivalents that is, the lmol of Trolox necessary to provide the same antioxidant capacity as a gram of the sample (Cao, Russell, Lischner, & Prior, 1998; Ou et al., 2001; Re et al., 1999). The higher the Trolox Equivalent value, the more antioxidant the sample is. Trolox does not have any physiological signicance and its choice as the standard for antioxidant capacity is

arbitrary; however, since its use is quite generalized, choosing it can make it easier to compare published data. For instance, the results of FRAP assay were initially expressed as equivalents of Fe2SO4 (Benzie & Strain, 1996), but this standard was later changed to Trolox so as to express results in the same form as other methods. The use of other standards such as vitamin C or vitamin E may be useful for specic nutritional studies. In any case, this way of expressing the results is quite important, since it permits direct comparison between dierent 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 xed end-point, which is commonly used for DPPH assays, is to measure the percentage of inhibition, comparing the change in the absorbance caused by a blank and by a test sample (Xu, Yang, Chen, Hu, & Hu, 2003; Yu et al., 2002). However, since this percentage of inhibition will depend on the concentration of radical and of sample taken in each case, it is not possible to compare studies that use dierent initial amounts. In any case, in assays performed at a xed end-point it should always be conrmed that the reaction was completed at the time chosen. Some antioxidant capacity methods express their results with reference to the lag-phase, which is the time during which the antioxidant is able to exert its action before the oxidation process starts. This has been employed, for example, in LDL oxidation assay (Kleinveld, Hak-Lemmers, Stalenhoef, & Demacker, 1992) or in Total Radical-Trapping Antioxidant Parameter (TRAP) assay. However, it has been noted that not all antioxidants exhibit a clearly dened lag-phase (Prior et al., 2005), and also that this parameter would not be useful when determining the antioxidant 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 was nchez-Moreno et al. (1998) in order to intromodied by Sa duce this kind of parameters; by testing dierent initial

Table 10 rez-Jime nez & Saura-Calixto, 2006) Results of ORAC, ABTS, FRAP and DPPH assays of a mixture of catechin:gallic acid 1:1 M in dierent solvents (Pe Solvent Methanol Water Acetone/water (50:50, v/v) ABTS (lmol Trolox/g dm) 11,291.4 837 a 28104 275.8 b 13,894.8 240.2 a DPPH EC50 (g/g) 0.083 0.004 a 0.067 0.001 b 0.083 0.003 a FRAP (lmol Trolox/g dm) 9642.4 977.1 a 9559.2 110.7 a 10,100.1 489.7 a,b ORAC (lmol Trolox/g dm) 20,836.6 2079.6 a 13,543.6 298.4 b 30,217.5 2100.1 c

Dierent letters in the same column imply signicant dierences.

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Table 11 Antioxidant capacity associated to aqueous-organic extracts (acidic methanol/water followed by acetone/water) of nuts considering kinetic parameters ABTS EC50 (g/g) Walnut Almond Hazelnut Peanut Pistachio 1.5 0.01 577.9 18 39.9 0.3 182.4 18.7 27.1 1.3 tEC50 (min) 12.8 0.4 17.7 0.7 9.3 0.9 19.3 2.6 16 1 AE (10 ) 51.6 0.1 2.7 0.3 2.3
3

DPPH EC50 (g/g) 14.3 0.1 401.5 78.5 46.1 2.1 277.9 12.7 32.9 0.3 tEC50 (min) 30.5 0.6 12.2 1.4 37.1 2.9 29.3 3.8 46.2 0.9 AE (103) 2.3 0.2 0.6 0.1 0.7

concentrations of the test sample, it is possible to establish EC50, that is the amount of sample needed to scavenge 50% of the original concentration of the free radical; tEC50, or the time taken by that concentration to reach equilibrium; or AE, that is the inverse of the product of EC50 and tEC50. With these parameters it is possible to gain more comprehensive information on the samples antioxidant capacity, taking into account not only their activity (dened by EC50) but also whether the antioxidant acts quickly or slowly (tEC50) and both simultaneously (AE). This approach has been successfully used by many research a-R os, Garc agroups in quite dierent samples (Femen n, Herna ndez-Gala n, Mac as-Sa nchez, & Collado, Pajo nchez-Moreno et al., 1999; Vergara-Valencia 2006; Sa et al., 2007). A similar modication was recently reported for the rez-Jime nez & Saura-Calixto, 2008). First ABTS assay (Pe of all, this approach considers the time taken by some compounds to nish reacting with the free radical, given that certain antioxidants do not do so within the 6 min normally required (Arts, Voss, Haenen, & Bast, 2004; Prior et al., 2005). Secondly, as noted earlier, the information derived with kinetic parameters is more comprehensive. Table 11 shows an example of the application of kinetic parameters to ABTS and DPPH assays on dierent nuts. One drawback of this approach is that it is more time-consuming than taking measurements at a xed end-point, and the time needed for one assay will depend on each sample. However, as can be seen in Table 11, the average time taken by antioxidants to react with the ABTS radical is shorter than the time taken to react with the DPPH radical, so that kinetic parameters may be easier to apply to this method. 4. Conclusions Determination of antioxidant capacity of foods and beverages includes three steps: sample preparation and extraction of antioxidants, measurement of antioxidant capacity, and expression of results. During sample preparation, the loss of antioxidants in the drying and milling steps must be kept to a minimum. In the extraction of antioxidants, at least two extraction cycles with mixtures of dierent polarity of water and organic solvents must be combined. Determination of total antioxidant capacity must be performed both in aqueous-organic extracts and in their corresponding residues, which may exhibit higher anti-

oxidant capacity than the aqueous-organic extracts, a fact usually ignored in the literature. Antioxidant capacity values should only be compared where the method, the solvent and the analytical conditions are the same. Possible interference from certain food constituents must also be taken into account when determining antioxidant 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 evaluation of antioxidant capacity.

Acknowledgements The present research was performed under the nancial support of the Spanish Ministry of Education and Science (Project AGL 2004-07579-C04-01/ALI). S. Arranz had an n y CienFPI scholarship from the Ministerio de Educacio a Elena D az-Rubio had a scholarship from the cia. Mar Instituto Danone. References
Antolovich, M., Prenzler, P., Robards, K., & Ryan, D. (2000). Sample preparation in the determination of phenolic compounds in fruits. The Analyst, 125, 9891009. Arnao, M. B. (2000). Some methodological problems in the determination of antioxidant activity using chromogen radicals: A practical case. Trends in Food Science and Technology, 11, 419421. rez-Jime nez, J., & Saura-Calixto, F. (in press). Antioxidant Arranz, S., Pe capacity of walnut (Juglans regia L.): Contribution of oil and defatted matter. European Food Research and Technology, doi:10.1007/s00217007-0737-2. Arts, M. J. T. J., Voss, H. P., Haenen, G. R. M. M., & Bast, A. (2004). Antioxidant capacity of reaction products limits the applicability of the Trolox equivalent antioxidant capacity (TEAC) assay. Food and Chemical Toxicology(1), 4549. Awika, S. M., Rooney, L. W., & Waniska, R. D. (2005). Anthocyanins from black sorghum and their antioxidant properties. Food Chemistry, 90(12), 293301. Bazzano, L. A., He, J., Ogden, L. G., Loria, C. M., Vupputuri, S., Myers, L., et al. (2002). Fruit and vegetable intake and the risk of cardiovascular disease in US adults: The rst National Health and Nutrition Examination Survey Epidemiologic Follow-up Study. American Journal of Clinical Nutrition, 76, 9399. Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: The FRAP assay. Analytical Biochemistry, 239, 7076.

284

rez-Jime nez et al. / Food Research International 41 (2008) 274285 J. Pe Iqbal, S., Bhanger, M. I., & Anwar, F. (2007). Antioxidant properties and components of some commercially available varieties of rice bran in Pakistan. Food Science and Technology(2), 361367. Jenner, A. M., Rafter, B., & Halliwell, B. (2005). Human fecal water content of phenolics: The extent of colonic exposure to aromatic compounds. Free Radical Biology and Medicine, 38, 763772. nez-Escrig, A., Jime nez-Jime nez, I., Pulido, R., & Saura-Calixto, F. Jime (2001). Antioxidant activity of fresh and processed edible seaweeds. Journal of the Science of Food and Agriculture, 81, 530534. Kerkhofs, N. S., Lister, C. E., & Savage, G. P. (2005). Changes in colour and antioxidant content of tomato cultivars following air-force drying. Plant Foods for Human Nutrition, 60(30), 117121. Kleinveld, H. A., Hak-Lemmers, H. L. M., Stalenhoef, A. F. H., & Demacker, P. N. M. (1992). Improved measurement of low-densitylipoprotein susceptibility to copper-induced oxidation: Application of a short procedure for isolating low-density lipoprotein. Clinical Chemistry, 38, 20662072. Laguerre, M., Lecomte, J., & Villeneuve, P. (2007). Evaluation of the ability of antioxidants to counteract lipid oxidation: Existing methods, new trends and challenges. Progress in Lipids Research, 46, 244282. Landbo, A. K., & Meyer, A. S. (2001). Enzyme assisted extraction of antioxidative phenols from blackcurrant juice press residues (Ribes nigrum). Journal of Agricultural and Food Chemistry, 49(7), 3169 3177. rez, P., & Saura-Calixto, F. (1997). Eect of drying Larrauri, J. A., Rupe temperature on the stability of polyphenols and antioxidant activity of red grape pomace peels. Journal of Agricultural and Food Chemistry, 45(4), 13901393. s-Barbera n, F., & Ferreres, F. (2004). Lettuce and Llorach, R., Toma chicory byproducts as a source of antioxidant phenolic extracts. Journal of Agricultural and Food Chemistry, 52(16), 51095116. pez, M., Mart nez, F., Del Valle, C., Ferrit, M., & Luque, R. (2003). Lo Study of phenolic compounds as natural antioxidants by a uorescence method. Talanta, 60, 609616. va ri, J. (2003). Antioxidant properties of commercial Lugasi, A., & Ho alcoholic and nonalcoholic beverages. Nahrung/Food, 47(2), 7986. Luthria, D. (2006). Signicance of sample preparation in developing analytical methodologies for accurate estimation of bioactive compounds in functional foods. Journal of the Science of Food and Agriculture, 86(14), 22662272. Manzocco, L., Anese, M., & Nicoli, M. C. (1998). Antioxidant properties of tea extracts as aected by processing. LWT Food Science and Technology, 31, 694698. Mrkic, V., Cocci, E., Dalla Rosa, M., & Sachetti, G. (2006). Eect of drying conditions on bioactive compounds and antioxidant activity of broccoli (Brassica oleracea L.). Journal of the Science of Food and Agriculture, 86, 15591566. Mukhopadhyay, S., Luthria, D. L., & Robbins, R. J. (2006). Optimization of extraction process for phenolic acids from cohosh (Cimicifuga racemosa) by pressurized liquid extraction. Journal of the Science of Food and Agriculture, 86, 156162. Naithani, V., Nair, S., & Kakkar, P. (2006). Decline in antioxidant capacity of Indian herbal teas during storage and its relation to phenolics content. Food Research International, 39(2), 176181. Niki, E. (2002). Antioxidant activity: Are we measuring it correctly? Nutrition(6), 524525. Olsson, M. E., Ekvall, J., Gustavsson, K. E., Nilsson, J., Pillald, D., Sjoho lm, I., et al. (2004). Antioxidants, low molecular weight carbohydrates, and total antioxidant capacity in strawberries (Fragaria ananassa): Eects of cultivar, ripening, and storage. Journal of Agricultural and Food Chemistry, 52(9), 24902498. ONeill, M. E., Carroll, Y., Couridan, B., Olmedilla, B., Granado, F., Blanco, I., et al. (2001). A European carotenoid database to assess carotenoid intakes and its use in a ve-country comparative study. British Journal of Nutrition, 85, 499507. Osman, A. M., Wong, K. K. Y., Hill, S. J., & Fernyoungha, A. (2006). Isolation and the characterization of the degradation products of the mediator ABTS-derived radicals formed upon reaction with polyphe-

Becker, E. M., Ntouma, G., & Skibsted, L. H. (2007). Synergism and antagonism between quercetin and other chain-breaking antioxidants in lipid system of increasing structural organisation. Food Chemistry, 103, 12881296. Bo hm, V., Ku nhert, S., Rohm, H., & Scholze, G. (2006). Improving the nutritional quality of microwave-vacuum dried strawberries: A preliminary study. Food Science and Technology International, 12, 6775. Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. Lebensmittel Wissenchaft und Technologie, 28, 2530. Bravo, L., Abia, R., & Saura-Calixto, F. (1994). Polyphenols as dietary ber associated compounds. Comparative study on in vivo and in vitro properties. Journal of Agricultural and Food Chemistry, 42(7), 14811487. Brighenti, F., Valtuen a, S., Pellegrini, N., Ardigo, D., Del Rio, D., Salvatore, S., et al. (2005). Total antioxidant capacity of the diet is inversely and independently related to plasma concentrations of highsensitive C-reactive protein in adult Italian subjects. British Journal of Nutrition, 93(5), 619625. Cao, G., Russell, M., Lischner, N., & Prior, R. L. (1998). Serum antioxidant capacity is increased by consumption of strawberries, spinach, red wine or vitamin C in elderly women. Journal of Nutrition, 128(12), 23832390. Cheng, Z., Su, L., Moore, J., Zhou, K., Luther, M., Yin, J. J., et al. (2006). Eects of postharvest treatment and hest stress on availability of wheat antioxidants. Journal of Agricultural and Food Chemistry, 54(15), 56235629. Cheng, H. Y., Ling, Y. C., & Hsieh, C. L. (2007). Evaluation of antioxidant activity of aqueous extract of some selected nutraceutical herbs. Food Chemistry, 104(4), 14181424. Dalla Valle, A. Z., Mignani, I., Spinardi, A., Galvano, F., & Ciappellano, S. (2007). The antioxidant prole of three dierent peaches cultivars (Prunus persica) and their short-term eect on antioxidant status in human. European Food Research and Technology, 225(2), 167 172. n, J. C., Soler-Rivas, C., & Wichers, H. J. (2000). Characterization of Esp the total free radical scavenger capacity of vegetable oils and oil fractions using 2,2-diphenyl-1-picrylhydrazyl radical. Journal of Agricultural and Food Chemistry, 48, 648656. a-R os, M., Garc a-Pajo n, C. M., Herna ndez-Gala n, R., Mac asFemen nchez, A. J., & Collado, I. G. (2006). Synthesis and free radical Sa scavenging activity of a novel metabolite from the fungus Colletotrichum gloeosporioides. Bioorganic and Medicinal Chemistry Letters, 16(22), 58365839. Foti, M. C., Dasquino, C., & Geraci, C. (2004). Electron-transfer reaction of cinnamic acids and their methyl esters with the DPPH radical in alcohol solutions. Journal of Organic Chemistry, 69, 23092314. Frankel, E. N., & Meyer, A. S. (2000). Review. The problems of using one-dimensional methods to evaluate multifunctional food and biological antioxidants. Journal of the Science of Food and Agriculture, 80, 19251941. a, A. (2007). Eect of airGarau, M. C., Simal, S., Rossello, C., & Femen drying temperature on physico-chemical properties of dietary bre and antioxidant capacity of orange (Citrus aurantium v. Canoeta) byproducts. Food Chemistry, 104(3), 10141024. Gorinstein, S., Vargas, O. J. M., Jaramillo, N. O., Salas, I. A., Ayala, A. vila, P., et al. (2007). The total polyphenols and the L. M., Arincibia-A antioxidant potentials of some selected cereals and pseudocereals. European Food Research and Technology, 225(34), 321328. Gray, D. A., Clarke, M. J., Baux, C., Bunting, J. P., & Salter, A. M. (2002). Antioxidant activity of oat extracts added to human LDL particles and in free radical trapping assays. Journal of Cereal Science, 36, 209218. Hartzfeld, P. W., Forkner, R., Hunter, D. M., & Hagerman, A. E. (2002). Determination of hydrolysable tannins (gallotannins and ellagitannins) after reaction with potassium iodate. Journal of Agricultural and Food Chemistry, 50, 17851790.

 rez-Jime nez et al. / Food Research International 41 (2008) 274285 J. Pe nols. Biochemical and Biophysical Research Communication, 340, 597603. Ou, B., Hampsch-Woodill, M., & Prior, R. L. (2001). Development and validation of an improved oxygen radical absorbance capacity assay using uorescein as the uorescent probe. Journal of Agricultural and Food Chemistry, 49, 46194626. Ou, B., Huang, D., Hampsch-Woodill, M., Flanagan, J. A., & Deemer, E. K. (2002). Analysis of antioxidant activities of common vegetables employing oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays: A comparative study. Journal of Agricultural and Food Chemistry, 50, 31223128. Pellegrini, N., Serani, M., Colombi, B., Del Rio, D., Salvatore, S., Bianchi, M., et al. (2003). Total antioxidant capacity of plant foods, beverages and oils consumed in Italy assessed by three dierent in vitro assays. Journal of Nutrition, 133, 28122819. Pellegrini, N., Serani, M., Salvatore, S., Del Rio, D., Bianchi, M., & Brighenti, F. (2006). Total antioxidant capacity of spices, dried fruits, nuts, pulses, cereals and sweets consumed in Italy assessed by three dierent in vitro assays. Molecular Nutrition and Food Research, 50, 10301038. Pellegrini, N., Colombi, B., Salvatore, S., Brenna, O. V., Galaverna, G., Del Rio, D., et al. (2007). Evaluation of antioxidant capacity of some fruit and vegetable foods: Eciency of extraction of a sequence of solvents. Journal of the Science of Food and Agriculture, 87, 103111. rez-Jime nez, J., & Saura-Calixto, F. (2005). Literature data may Pe underestimate the actual antioxidant capacity of cereals. Journal of Agricultural and Food Chemistry, 53, 50365040. rez-Jime nez, J., & Saura-Calixto, F. (2006). Eect of solvent and certain Pe food constituents on dierent antioxidant capacity assays. Food Research International, 39, 791800. rez-Jime nez, J., & Saura-Calixto, F. (2008). Antioxidant capacity of Pe dietary polyphenols determined by ABTS assay: A kinetic expression of the results. International Journal of Food Science and Technology, 43, 185191. n Pinelo Manzocco, L., Nu ez, M. J., & Nicoli, M. C. (2004). Solvent eect on quercetin antioxidant capacity. Food Chemistry, 88(2), 201 207. Pitsavos, C., Panagiotakos, D. B., Tzima, N., Chrysohoou, C., Economou, M., Zampelas, A., et al. (2005). Adherence to the Mediterranean diet is associated with total antioxidant capacity in healthy adults: The ATTICA study. American Journal of Clinical Nutrition, 82(3), 694 699. Porter, L., Hrstich, L., & Chan, B. (1985). The conversion of procyanidins and prodelphinidins to cyaniding and delphinidin. Phytochemistry, 25, 223230. Prakash, D., Upadhyay, G., Singh, B. N., & Singh, H. B. (2007). Antioxidant and free radical-scavenging activities of seeds and agriwastes of some varieties of soybean (Glycine max). Food Chemistry, 104(2), 783790. Prior, R. L., Wu, X., & Schaich, K. (2005). Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. Journal of Agricultural and Food Chemistry, 53(10), 42904302. Pulido, R., Bravo, L., & Saura-Calixto, F. (2000). Antioxidant activity of dietary polyphenols as determined by a modied ferric reducing/ antioxidant power assay. Journal of Agriculture and Food Chemistry, 48, 33963402. ndez-Garc a, M., & Saura-Calixto, F. (2003). ContriPulido, R., Herna bution of beverages to the intake of lipophilic and hydrophilic antioxidants in the Spanish diet. European Journal of Clinical Nutrition, 57, 12751282. Re, R., Pellegrini, N., Preoteggente, A., Pannala, A., Yang, M., & RiceEvans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine(9/10), 121137. Reed, J., McDowell, R. E., Van Soest, P. J., & Horvarth, P. J. (1982). Condensed tannins: A factor limiting the use of cassava forage. Journal of the Science of Food and Agriculture, 33, 213220.

285

guez-Bernaldo de Quiro s, A., & Costa, H. S. (2006). Analysis of Rodr carotenoids in vegetables and plasma samples: A review. Journal of Food Composition and Analysis, 19(2-3), 97111. nchez-Gonza lez, I., Jime nez-Escrig, A., & Saura-Calixto, F. (2005). In Sa vitro antioxidant activity of coees brewed using dierent procedures (italian, espresso and lter). Food Chemistry, 90(1-2), 133139. nchez-Moreno, C., Larrauri, J. A., & Saura-Calixto, F. (1998). A Sa procedure to measure the antiradical eciency of polyphenols. Journal of the Science of Food and Agriculture, 76, 270276. nchez-Moreno, C., Larrauri, J. A., & Saura-Calixto, F. (1999). Free Sa radical scavenging capacity of selected red, rose and white wines. Journal of the Science of Food and Agriculture, 79(10), 13011304. nchez-Moreno, C. (2002). Review: Methods used to evaluate the free Sa radical scavenging activity in foods and biological systems. Food Science and Technology International, 8(3), 121139. Saura-Calixto, F., & Gon i, I. (2006). Antioxidant capacity of the Spanish Mediterranean diet. Food Chemistry, 94(3), 442447. Saura-Calixto, F., Serrano, J., & Gon i, I. (2007). Intake and bioaccessibility of total polyphenols in a whole diet. Food Chemistry, 101(2), 492501. Schwarz Bertelsen, G., Nissen, L. R., Gardner, P. T., Heinonen, M. I., Hopia, A., et al. (2000). European Food Research and Technology, 21, 319328. Serpen, A., Capuano, E., Fogliano, V., & Go kmen, V. (2007). A new procedure to measure the antioxidant activity of insoluble food components. Journal of Agricultural and Food Chemistry, 55(19), 76767681. Serrano, J. (2005). Disponibilidad intestinal de compuestos bioactivos de la dieta (Polifenoles y carotenoides). PhD thesis. Department of Nutrition, Faculty of Pharmacy, Universidad Complutense de Madrid, Spain. Srivastava, A., Akoh, C. C., Yi, W., Fischer, J., & Krewer, G. (2007). Eect of storage conditions on the biological activity of phenolic compounds of blueberry extract packed in glass bottles. Journal of Agricultural and Food Chemistry, 55, 27052713. Tabernero, M., Serrano, J., & Saura-Calixto, F. (2006). The antioxidant capacity of cocoa products: Contribution to the Spanish diet. International Journal of Food Science and Technology, 41(Suppl. 1), 2832. Trichopoulou, A., Costacou, T., Bamia, C., & Trichopoulos, D. (2003). Adherence to a Mediterranean diet and survival in a Greek population. New England Journal of Medicine, 348, 25992608. Tuberoso, C. I. G., Kowalczyk, A., Sarritzu, E., & Cabras, P. (2007). Determination of antioxidant compounds and antioxidant activity in commercial oilseeds for food use. Food Chemistry, 103(4), 14941501. Ueda, T., & Igarashi, O. (1990). Determination of vitamin E in biological specimens and foods by HPLC-pre-treatment of samples and extraction of tocopherols. Journal of Micronutrient Analysis, 7(2), 7996. rez, A., Agama-Acevedo, E., Touvar, Vergara-Valencia, N., Granados-Pe rez, A. (2007). Fibre concentrate from mango J., Ruales, J., & Bello-Pe fruit: Characterization, associated antioxidant capacity and application as a bakery product ingredient. Food Science and Technology, 40(4), 722729. ndez-Pacho n, M. S., Troncoso, A. M., & Garc aVillan o, D., Ferna Parrilla, M. C. (2005). Comparison of antioxidant activity of wine phenolic compounds and metabolites in vitro. Analytica Chimica Acta, 538, 391398. Wu, X., Beecher, G. R., Holden, J. M., Haytowitz, D. B., Gebhardt, S. E., & Prior, R. L. (2004). Lipophilic and hydrophilic antioxidant capacities of common foods in the United States. Journal of Agricultural and Food Chemistry, 52, 40264037. Xu, J., Yang, F., Chen, L., Hu, Y., & Hu, Q. (2003). Eect of selenium on increasing the antioxidant activity of tea leaves harvested during the early spring tea producing season. Journal of Agricultural and Food Chemistry, 51(4), 10811084. Yu, L., Perret, J., Davy, B., Wilson, J., & Melby, C. L. (2002). Antioxidant properties of cereal products. Journal of Food Science, 67(7), 26002603. Zhou, K., & Yu, L. (2004). Eect of extraction solvent on wheat bran antioxidant activity estimation. Food Science and Technology, 37(7), 717721.