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Food Research International 41 (2008) 274285

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Updated methodology to determine antioxidant capacity in plant

foods, 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 Cientcas (IF-CSIC), Calle Jose Antonio Novais, 10, 28040 Madrid, Spain
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 & Goni,
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.,

J. Perez-Jimenez et al. / Food Research International 41 (2008) 274285

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, &
Schaich, 2005; Sanchez-Moreno, 2002). The most widelyused 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 antioxidants (Pellegrini et al., 2007; Perez-Jimenez & Saura-Calixto, 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.

275

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
from Panreac, Castellar del Valles, Barcelona, Spain.
2,20 -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
Aldrich Qumica, S.A., Madrid, Spain.
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
(Pistachia vera) were from Aperitivos Medina S.L. (Mostoles, Madrid, Spain).
Fucoidan (99%) from Fucus vesiculosus was purchased
from SigmaAldrich Qumica, S.A. (Madrid, Spain).
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, & Goni, 2007).
The purpose of this extraction is to obtain extractable antioxidants using aqueous-organic solvents, and non-extract-

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J. Perez-Jimenez et al. / Food Research International 41 (2008) 274285

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)
centrifugation

antioxidant
extract
(extractable
polyphenols)

supernatant

residue

Antioxidant capacity (AC1)

buthanol / HCl

antioxidant extract
(condensed tannins)
Antioxidant capacity (AC2)

methanol/ H2SO4

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
and the organic phases (Pulido, Hernandez-Garca, &
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
fraction), after dilution with ethyl acetate (Espn, SolerRivas, & 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:

J. Perez-Jimenez et al. / Food Research International 41 (2008) 274285

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,
Cuvelier, and Berset (1995) was later modied by Sanchez-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
Perez-Jimenez and Saura-Calixto (2008) modied the
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-

277

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,
Rossello, & Femena, 2007), as well as in other products,
such as the edible seaweed Fucus vesiculosus (JimenezEscrig, Jimenez-Jimenez, Pulido, & Saura-Calixto, 2001).
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
(Bohm, Kunhert, 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,
Ruperez, & Saura-Calixto, 1997), it was found that drying
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.

J. Perez-Jimenez et al. / Food Research International 41 (2008) 274285

278

Table 1
Antioxidant capacity of Camarossa strawberry after dierent drying
treatments (Bohm et al., 2006)

Fresh sample
Convective drying
Microwave-vacuum drying
Freeze-drying

ABTS
(mmol/100 g)

FRAP
(mmol Fe2+/100 g)

8.4 0.2
5.1 1.4
5.3 1.5
8.2 0.4

24 2.1
17.5 3.6
17.7 2.5
20.2 1.2

a
b
b
a

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

FRAP

Cutting mill
Hammer mills
Shear mill in N2 atmosphere

71 3 a
63 1 b
62 1 b

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).

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)
(Fig. 1) (Larrauri et al., 1997; Perez-Jimenez & Saura-Calixto, 2005; Saura-Calixto & Goni, 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

Extraction solvent

% antioxidant capacity
extracted (FRAP)

Commercial
extract from
cocoa

1: acidic methanol/water
(50:50, v/v; pH 2)
2: acetone/water (70:30,
v/v) in the residue

59.7

Red grape seeds

1: acidic methanol/water
(50:50, v/v; pH 2)
2: acetone/water (70:30,
v/v) in the residue

64.3

40.3

35.7

J. Perez-Jimenez et al. / Food Research International 41 (2008) 274285

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
studied (Antolovich et al., 2000; Rodrguez-Bernaldo de
Quiros & Costa, 2006; Ueda & Igarashi, 1990).
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
tannins) than associated to the extracts (Perez-Jimenez &
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)

Dark chocolate
Milk chocolate
Cocoa soluble powder
Cocoa paste
Dietary fruitsa
Dietary pulsesb
a

Aqueous-organic
extracts

Residues
(proanthocyanidins)

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

144.05 1.82
84.31 0.58
51.83 3.33
246.14 5.47
60.2
37.6

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.

279

cereals or nuts (Pellegrini et al., 2006; Serpen, Capuano,

Fogliano, & Gokmen, 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
aliquots 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 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
assay (Perez-Jimenez & Saura-Calixto, 2006). It should
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

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280

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

Total oil
No polar fractionb
Methanolic fractionc
a
b
c

Walnut oil

Almond oil

Hazelnut oil

Peanut oil

Pistachio oil

1514.3 70,2
1764.1 125.1
688.8 17.5

712.2 36
2717.5 68.8
1109.2 38.8

478.5 8.6
1096.4 52.1
366.4 60.4

1395.9 99.7
3492.8 53
190.2 48.5

377.9 31.8
863.5 41.1
8.42 1.5

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
capacity of tea, wine, coee 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 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 & Goni, 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

Total antioxidant
capacity

Hydrophilic
extracts

Lipophilic
extracts

Coee
Wine
Tea
Beer
Orange juice
Milk
Cola

13,280 51
10,932 542
6308 80
772 17
2494 34
2194 107
<100

8772 26
8788 54
3932 71
714 21
1969 241
118 10
<100

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)
a

Aqueous-organic extracts
Residuesb
a
b

Walnut

Almond

Hazelnut

Peanut

Pistachio

14.3 0.1
4.0 0.2

401.5 78.5
22.6 1.0

46.1 2.1
26.1 0.9

277.9 12.7
53.1 3.2

32.9 0.3
23.9 0.7

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

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

J. Perez-Jimenez et al. / Food Research International 41 (2008) 274285

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
individual sample (Sanchez-Moreno et al., 1998).
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
(Cheng, Ling, & Hsieh, 2007; Llorach, Tomas-Barberan,
& 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

281

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
in ORAC (Villano, Fernandez-Pachon, Troncoso, & Garca-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 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.

Table 8
Main 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 pH

ABTS

 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 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

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).

J. Perez-Jimenez et al. / Food Research International 41 (2008) 274285

282

Table 9
Antioxidant 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
Fucoidan
Red grape
pomace

114.9 4.0
27.2 2.6
273.9 16.6

153.8 16.2
17.0 0.6
124.4 0.3

187.2 9.1
57.4 8.4
214.3 37.3

14.3 0.1
13.2 0.2
3.5 0.5

30.5 0.6
14.0 0.8
38.1 2.6

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
assays (Perez-Jimenez & Saura-Calixto, 2006). This agrees
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 (Villano 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
modied by Sanchez-Moreno et al. (1998) in order to introduce this kind of parameters; by testing dierent initial

Table 10
Results of ORAC, ABTS, FRAP and DPPH assays of a mixture of catechin:gallic acid 1:1 M in dierent 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
Water
Acetone/water (50:50, v/v)

11,291.4 837 a
28104 275.8 b
13,894.8 240.2 a

0.083 0.004 a
0.067 0.001 b
0.083 0.003 a

9642.4 977.1 a
9559.2 110.7 a
10,100.1 489.7 a,b

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.

J. Perez-Jimenez et al. / Food Research International 41 (2008) 274285

283

Table 11
Antioxidant capacity associated to aqueous-organic extracts (acidic methanol/water followed by acetone/water) of nuts considering kinetic parameters
ABTS

Walnut
Almond
Hazelnut
Peanut
Pistachio

DPPH
3

EC50 (g/g)

tEC50 (min)

AE (10 )

EC50 (g/g)

tEC50 (min)

AE (103)

1.5 0.01
577.9 18
39.9 0.3
182.4 18.7
27.1 1.3

12.8 0.4
17.7 0.7
9.3 0.9
19.3 2.6
16 1

51.6
0.1
2.7
0.3
2.3

14.3 0.1
401.5 78.5
46.1 2.1
277.9 12.7
32.9 0.3

30.5 0.6
12.2 1.4
37.1 2.9
29.3 3.8
46.2 0.9

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
groups in quite dierent samples (Femena-Ros, GarcaPajon, Hernandez-Galan, Macas-Sanchez, & Collado,
2006; Sanchez-Moreno et al., 1999; Vergara-Valencia
et al., 2007).
A similar modication was recently reported for the
ABTS assay (Perez-Jimenez & Saura-Calixto, 2008). First
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
FPI scholarship from the Ministerio de Educacion y Ciencia. Mara Elena Daz-Rubio had a scholarship from the
Instituto Danone.
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