Food Chemistry 130 (2012) 986–993

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Extraction conditions can greatly influence antioxidant capacity assays

in plant food matrices
Jean Albert Michiels a,b, Claire Kevers b,⇑, Joel Pincemail c, Jean Olivier Defraigne c, Jacques Dommes b
a
CECOTEPE, 101, rue de Cockerill, B-4100 SERAING, Belgium
b
Plant Molecular Biology and Biotechnology Unit, B22, University of Liège, Sart Tilman, B-4000 LIEGE, Belgium
c
CREDEC, Pathology Tower B23, University of Liège, Sart Tilman, B-4000 LIEGE, Belgium

a r t i c l e i n f o a b s t r a c t

Article history: The estimated antioxidant capacity of different matrices can vary considerably between research reports.
Received 24 August 2010 Besides intrinsic factors (not studied here), our work showed that may have various causes. Firstly, dif-
Received in revised form 24 May 2011 ferent methods are used to measure antioxidant capacity. Secondly, the results obtained for a single
Accepted 31 July 2011
matrix by one method (such as ORAC) can vary with the extraction conditions. Parameters having a great
Available online 5 August 2011
impact on the amount and composition of antioxidants in extracts, and thus on the measured antioxidant
capacity, notably include the extraction solvent composition, temperature, extraction time (duration),
Keywords:
solvent-to-solid ratio, and storage conditions. Standardisation of the extraction procedure is thus neces-
Antioxidant
Extraction
sary for accurate and reproducible determination of the antioxidant capacity and phenolics in different
Phenolic compounds food matrices by different laboratories. In this study we optimised such a procedure for four fresh plant
Solvent matrices (orange, apple, leek, and broccoli). The optimised procedure requires extraction in a mixture of
Storage acetone/water/acetic acid (70/28/2, v/v/v) for 1 h at 4 °C, with a solvent-to-solid ratio of 20 mL per 1 g.
Temperature Fresh material should be used, but if this is not possible, one may lyophilise the plant matrices or store
the extracts for a few days at 20 °C before analysis.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction The antioxidant properties of food matrices, however, are due to

Plant foods are now regarded as important sources of dietary
antioxidants. A diet rich in antioxidants appears to correlate with
a reduced risk of cardiovascular disease (Genkinger, Platz,
Hoffman, Comstock, & Helzlsouer, 2004). With the development
of functional foods having specific health effects, interest in plant
antioxidants is increasing among scientists, food manufacturers,
and consumers. Consumers, furthermore, require more nutritional
information about what they eat. Although it is unclear whether
active compounds remain active against free radicals after being
absorbed and metabolised by cells in the body, radical-scavenging
assays have gained acceptance for their capacity to rapidly screen
materials of interest (Ferreira, Baptista, Vilas-Boas, & Barros, 2007).
Natural antioxidants may act as free-radical scavengers and chain
breakers, chelators of pro-oxidant metal ions, and quenchers of
singlet oxygen present in the environment (Amarowicz, Pegg,
Rahimi-Moghaddam, Barl, & Weil, 2004), thereby reducing oxida-
tive damage to lipids, DNA, and proteins.
⇑ Corresponding author. Fax: +32 43 66 38 72.
E-mail addresses: JAMichiels@ulg.ac.be (J.A. Michiels), c.kevers@ulg.ac.be
(C. Kevers), J.Pincemail@chu.ulg.ac.be (J. Pincemail), JO.Defraigne@chu.ulg.
ac.be (J.O. Defraigne), j.dommes@ulg.ac.be (J. Dommes).
the presence of a complex mixture of compounds of varying polarity,
such as vitamin C, vitamin E, carotenoids, and polyphenols. Due to
the potential synergistic action of all these bioactive compounds
present in food (Serafini, Bellocco, Wolk, & Ekstrom, 2002), the total
antioxidant capacity of some foods has increasingly become a sales
argument for professionals in the food industry. Among a wide diver-
sity of tests used to estimate this capacity, the Oxygen Radical Anti-
oxidant Capacity (ORAC) assay has emerged for different reasons.
Initially developed with the support of the US Agricultural Research
Service, this assay is based largely on work reported by Glazer’s lab-
oratory, describing the oxidation of phycoerythrins by 2,20 -azobis(2-
amidinopropane) dihydrochloride (AAPH) used as a peroxyl radical
generator (Glazer, 1990). This assay was presented as a simple but
sensitive and reliable method of quantifying the global antioxidant
capacity in serum (Cao, Alessio, & Culter, 1993) and was further ap-
plied to evaluating the total antioxidant capacity of fruits (Wang,
Cao, & Prior, 1996) and vegetables (Cao, Sofic, & Prior, 1996). In
2001, scientists at Brunswick Inc., Wareham, USA decided to replace
the fluorescent probe b-phycoerythrin with fluorescein, which has
many advantages: no interaction with antioxidants, excellent stabil-
ity, and reduced cost (Ou, Hampsch-Woodill, & Prior, 2001).
In 2004 was published a first public ORAC database providing
antioxidant capacity estimates for 171 foods (Wu et al., 2004). This

0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.07.117
 J.A. Michiels et al. / Food Chemistry 130 (2012) 986–993 987

was followed by a second, more detailed one in 2007 (http://
www.ars.usda.gov/nutrientdata). Currently the ORAC value is
widely used as a sales argument. Food supplement products (e.g.
extracts of fruits with a high antioxidant capacity) have been com-
peting for the title of ‘‘Highest ORAC value’’. Yet what is largely ig-
nored by professionals in the food industry is that mastering the
ORAC assay is much more complex than expected. A solvent
extraction step (with acetone/water, methanol. . .) is needed, and
different extraction conditions can lead to major variations in the
obtained ORAC value. Comparing ORAC values from different
sources is therefore not as straightforward as it seems.
The antioxidant capacity has often been correlated with the phe-
nolic content (Cantin, Moreno, & Gogorcena, 2009). More than 8000
different phenolics have been reported in the literature, showing
large variations in polarity and in their possible biochemical modi-
fications (glycosylation, acetylation, manolynation, esterification to
organic acids. . .). Hence, optimisation of sample preparation is
essential to accurate analysis of different classes of phenolic metab-
olites present in various food matrices (Luthria, Mukhopadhyay, &
Kwansa, 2006; Pellegrini et al., 2007). Comparing the antioxidant
capacities of different food matrices will be impossible without a
standardised procedure for sample preparation.
The aim of the present work was to establish a standard antiox-
idant extraction method applicable to various matrices. To opti-
mise the extraction procedure, we focused mainly on solvent
composition, temperature, the extraction time (duration), the sol-
vent-to-solid ratio, and the storage conditions. These parameters
greatly influence the extract amount and composition, and thus
the measured antioxidant capacity. It is necessary to ensure that
the majority of phenolics are extracted. The chosen fresh matrices,
which have already been tested and compared by various authors
(Kevers et al., 2007; Wu et al., 2004) for their antioxidant capacity,
were: apple (Serra et al., 2010), orange (Duzzioni, Franco, & De
Sylos, 2009), leek (Tsouvaltzis, Gerasopoulos, & Siomos, 2007),
and broccoli (Akhlaghi & Bandy, 2010).
2. Material and methods
2.1. Materials
Apples (var. Kingjonagold), oranges (Mountain orange AIDAs),
broccoli (var. Italica), and leeks (cultivar Blue Liege) were purchased
between March and June from Belgian local supermarkets. The fresh
material was stored for no more than 3 days at 4 °C prior to analysis.
2.2. Sample preparation
Sampling and sample preparation were standardised. For each
experiment (comprising the whole set of extraction conditions
shown in Fig. 1 or 2 or 3 or 4), we used slices of one apple with
the peel but without the stalk, segments of one orange without
the peel, same-sized external heads from one broccoli, and quar-
ters of the white base of one leek (5 cm long, 2–7 cm from the
base). Each extraction was done in triplicate or more (independent
extractions on different days) and analyses were performed on the
day of extraction (or after storage when the stability was studied).
Within each experiment, all the tests on a given matrix (except the
stability tests) were done on the same day, but different matrices
were extracted and tested on different days.
Fresh material (2 g), or exceptionally frozen or lyophilised mate-
rial if required for the experiment, was immediately ground with a
blender before addition of solvent mixture (20 mL) as previously

Fig. 1. Effect of extraction solvent composition on the total phenolics yield (A) and on the antioxidant capacity assayed by the DPPH (B) or ORAC (C) method. M acet
1 = acetone/water/acetic acid (70/28/2, v/v/v), M acet 2 = acetone/water/acetic acid (70/29.5/0.5, v/v/v), M acet 3 = acetone/water/acetic acid (70/29.8/0.2, v/v/v), M MeOH
1 = methanol/water (50/50, v/v), M MeOH 2 = methanol/water/acetic acid (50/49.5/0.5, v/v/v), M MeOH 3 = methanol/acetic acid (99.5/0.5, v/v) at 4 °C. For the same matrix
and assay, significant differences as determined by ANOVA (p < 0.05) are indicated by different letters. The percentages above the bars are percentages of variation of the
mean value for all four matrices with respect to the mean for the first condition.
988 J.A. Michiels et al. / Food Chemistry 130 (2012) 986–993

Fig. 2. Effect of temperature and extraction solvent composition on the total phenolics yield (A) and the antioxidant capacity assayed by the DPPH (B) or ORAC (C)
method. All four plant matrices were extracted in two solvent mixtures (M acet = acetone/water/acetic acid (70/28/2, v/v/v) and M MeOH = methanol/acetic acid (99.5/0.5,
v/v)) at four temperatures (4, 25, 50, and 70 °C). For the same matrix, significant differences as determined by ANOVA (p < 0.05) are indicated by the letter a if the value
obtained for a given temperature with M MeOH is different from that obtained with M Acet. They are indicated by the letter and b if the value obtained for the indicated
temperature was different from that obtained for 4 °C with the same solvent. Percentages of variation (of the mean for all four matrices) with respect to the first condition
are also reported.

described by Tabart et al. (2007). The mixture was shaken for 1 h at
4 °C and centrifuged at 15,000g for 15 min. If more than one step
was required, the supernatant was removed and the pellet washed
with 5 mL of the same solvent, shaken for 15 min, and centrifuged
by the same procedure (15,000g, 15 min). The supernatants were
pooled.
The supernatant was used directly to determine total phenolics
and to measure the antioxidant capacity by the DPPH and ORAC
assays.
Various extraction parameters were tested as specified in the
results section: solvent mixture (see Fig. 1), solvent volume (2–
100 mL per g fresh weight), extraction temperature (4, 25, 50,
70 °C), extraction time (20, 40, 60 min), and number of extractions
(1–3). To test the stability, extracts were stored at various temper-
atures (4, 20, and 80 °C). They were kept in liquid or lyophilised
form or as material having undergone evaporation at 50 °C for 2 h
under nitrogen.
2.3. Total phenolics
The total phenolics content was determined according to the
Folin–Ciocalteu method (Caboni et al., 1997). Although not com-
pletely specific for phenolics, this protocol gives a good idea of
the total phenolics content. Appropriately diluted extracts
(3.6 mL) were mixed with 0.2 mL Folin–Ciocalteu reagent (Merck).
After 3 min, 0.8 mL sodium carbonate (20% w/v) was added. The
mixture was heated at 100 °C for 1 min. After cooling, the absor-
bance at 750 nm was measured. Chlorogenic acid (Sigma) was used
as standard, and results were expressed in mg chlorogenic acid
equivalents (CAE) per g fresh weight. Analyses were performed
in duplicate on each extract.
2.4. Antioxidant capacity
The antioxidant capacity was determined by reduction of the
radical 2,2-diphenyl-1-picryhydrazyl (DPPH) (Tadolini, Juliano,
Piu, Franconi, & Cabrini, 2000). A stock solution was prepared by
stirring 75 mg of DPPH in 1 L methanol overnight. In the assay,
0.75 mL extract, standard (0–0.1 mM Trolox), or blank (methanol)
and 1.5 mL DPPH solution were mixed. Sample, standard, and
blank absorbances at 517 nm were determined after 6 min. For
each extract, a blank with 1.5 mL methanol instead of DPPH re-
agent was included to correct for any sample absorbance at
517 nm. The percentage of remaining DPPH was inversely propor-
tional to the antioxidant concentration.
ORAC assays were carried out on a Victor3 (Perkin Elmer) 96-
well plate reader. The temperature of the incubator was set at
37 °C. Procedures were based on the method of Wu et al. (2004).
Briefly, AAPH was used as a peroxyl radical generator, Trolox as a
standard, and fluorescein as a fluorescent probe. Filters were used
to select an excitation wavelength of 485 nm and an emission
wavelength of 535 nm. Twenty-five microlitres of diluted sample,
blank, or Trolox calibration solution (0–100 lmol) was mixed with
150 ll of 4 lM fluorescein and incubated for 15 min at 37 °C before
injection of 25 ll AAPH solution (173 mM). The fluorescence was
measured every 2 min for 4 h. All samples were analysed in dupli-
 J.A. Michiels et al. / Food Chemistry 130 (2012) 986–993 989

Fig. 3. Effect of extraction time and of the number of extraction steps on the total phenolics yield (A) and on the antioxidant capacity assayed by the DPPH (B) or ORAC (C)
method. The four plant matrices were extracted in acetone/water/acetic acid (70/28/2, v/v/v) at 4 °C for 20, 40, or 60 min (20, 40, 60) and this was followed or not by one or
two 20-min steps (respectively 20 + 20, 40 + 20, and 60 + 20 + 20). Letters (a–c) indicate significant differences for the same matrix as determined by ANOVA (p < 0.05). Also
indicated is the percentage of variation (of the mean for all four matrices) with respect to the first condition.

cate at three different dilutions. The final ORAC values were calcu-
lated from the net area under the fluorescence decay curves.
Trolox [(±)6-hydroxy-2,5,7,8-tetramethylchromane-2-carbox-
ylic acid; Fluka Chemie GmbH, Switzerland] was used as a standard.
The antioxidant capacity was expressed in mg Trolox equivalents
(TE) per gram fresh weight. Each analysis was performed in
duplicate.
All results presented are means (±SE) of at least three indepen-
dent experiments (extractions). Statistical analysis (ANOVA with a
statistical significance level set at P < 0.05) was carried out with
Microsoft Excel (Microsoft, Roselle, IL).
In the figures, to facilitate comparisons among different test
conditions, we also report for each condition the percentage of var-
iation of the mean result for the four matrices with respect to the
mean obtained for the first condition, taken as 100%.
tion/reduction reaction and, as such, can be viewed as another
method of antioxidant capacity determination (Prior, Wu, & Scha-
ich, 2005).
The principal factors contributing to the efficiency of extraction
are: type of solvent, pH, temperature, number of steps, volume of
solvent, and material state (Escribano-Bailon & Santos-Buelga,
2002). Many other phenomena may also influence extraction, such
as solvent saturation, solvent selectivity due to composition and
temperature, degradation of components (Hinneburg & Neubert,
2005). In many published studies, only one matrix was tested.
Here, in order to establish a general protocol for the extraction of
antioxidant compounds from plant material, various parameters
were tested on four different plant matrices: two fruits (orange
and apple) and two vegetables (leek and broccoli).

3.1. Solvent mixture

3. Results and discussion
Numerous methods can be used to evaluate the antioxidant
capacities of natural compounds in foods or biological systems. Here
we have selected three methods for their different characteristics.
One free radical (DPPH) is commonly used to assess antioxidant
activity in vitro. Reduction of this radical by hydrogen-donating
antioxidant(s) is monitored as a decrease in optical density. In the
ORAC assay, free radicals from the thermal decomposition of AAPH
are generated at a constant rate throughout the test. These radicals
are responsible for the destruction of fluorescein, and antioxidants
delay the decay of this probe. These two methods show good repeat-
ability (Tabart, Kevers, Pincemail, Defraigne, & Dommes, 2009). The
Folin–Ciocalteu assay has been used for many years to measure total
phenolics in natural products, but the basic mechanism is an oxida-
The first step was to select the most appropriate solvent (Pinelo,
Rubilar, Jerez, Sineiro, & Nunez, 2005). In the literature, many
solvent types are used to extract antioxidant compounds from var-
ious plant materials. The most widely used solvents for extracting
phenolic substances are methanol, acetone, and their water mix-
tures, acidified or not (Pinelo, Rubilar, Sineiro, & Nunez, 2004; Rub-
ilar, Pinelo, Franco, Sineiro, & Nunez, 2003). Here, six different
mixtures (Fig. 1) were tested. The properties of the extracting sol-
vents significantly affected the measured total phenolics content
(±25% variation) and antioxidant capacity (up to 30% variation).
All the acetone-based mixtures extracted more phenolic com-
pounds than the methanol-based mixtures, especially from apples.
Furthermore, the acetic acid level decrease in the acetone mixtures
reduced the extraction yield, as observed for both phenolic com-
990 J.A. Michiels et al. / Food Chemistry 130 (2012) 986–993

Fig. 4. Effect of the volume of extraction solvent on the total phenolics yield (A) and antioxidant capacity assayed by the DPPH (B) or ORAC (C) method. One gramme of each
of matrix was extracted in acetone/water/acetic acid (70/28/2, v/v/v) at 4 °C for 60 min. The solvent volume varied from 2 to 100 mL. Letters (a–f) indicate significant
differences for the same matrix as determined by ANOVA (p < 0.05). Also reported on the figure are the percentages of variation of the means for the four matrices with
respect to the mean for the first condition.

pounds (from 100% to 83%) and the antioxidant capacity (ORAC,
from 100% to 71%). Among the methanol-based mixtures, the third
(MeOH 3, without water) was the most efficient, with higher val-
ues obtained in all three assays. The acetone/water/acetic acid
(70/28/2, v/v/v) and methanol/acetic acid (99.5/0.5, v/v) mixtures
appeared generally to be best for extracting phenolics and antiox-
idant compounds from the four tested matrices.
In keeping with these results, Gonzalez-Montelongo, Lobo, and
Gonzalez (2010) have shown that a mixture of acetone and water
both extracts total phenolics very efficiently from banana peel and
produces extracts with a high antioxidant capacity. The observed
non-correlation between total extracted phenolic compounds and
the antioxidant capacity of apple extracts may be due to the pres-
ence of other, non-phenolic antioxidant compounds.
3.2. Temperature
Different extraction temperatures have been used in the past,
varying from 4 °C (Tabart et al., 2007) to room temperature (Kuri-
lich, Jeffery, Juvik, Wallig, & Klein, 2002) or higher temperature
(Hinneburg & Neubert, 2005; Hu & Skibsted, 2002). Four different
temperatures were tested: 4, 25, 50, and 70 °C (Fig. 2) with the
two solvent mixtures previously selected: acetone/water/acetic
acid (70/28/2, v/v/v) and methanol/acetic acid (99.5/0.5, v/v).
As previously observed, both solvent mixtures extracted the
same quantity of phenolic compounds from orange, broccoli, and
leek, but for apple, the acetone-based mixture was better than
the methanol mixture (between 20% at 25 °C and 34% at
50 °C, Fig. 2). A higher temperature (70 °C) led to an increased
extraction yield from orange (+21%) and to a decreased extraction
yield from leek (10%). In orange, heat treatment might produce
changes in phenol extractability because of disruption of the plant
cell wall, as suggested by Choi, Lee, Chun, Lee, and Lee (2006). In
the case of leek, a higher temperature may be responsible for par-
tial destruction of its phenolic compounds, as previously reported
for various plant materials (Barreira et al., 2009).
The antioxidant capacities measured by the DPPH and ORAC as-
says were higher for most acetone-based extracts than for metha-
nol-based extracts. One exception was leek, for which methanol
extracts obtained at 50 °C and 70 °C yielded higher ORAC values.
An increase in temperature led to a decreased antioxidant capacity
(DPPH and ORAC) in apple acetone extracts. For broccoli, the DPPH
assay gave the highest values at 70 °C in both the acetone and the
methanol mixtures. Baumgertel, Grimm, Eisenbeiss, and Kreis
(2003) have shown that a higher temperature and a higher meth-
anol concentration can decrease the hydrolytic activity, this lead-
ing to better stability of some flavonoids in solution. This may be
the case for broccoli.
Since there was no indication that one extraction condition was
really better than the others for all the matrices and all the assays,
we decided to continue testing with the acetone mixture at 4 °C, as
this condition nearly always yielded the highest values.
3.3. Incubation duration and successive extractions
The time during which the extraction solvent and matrix are in
contact can influence the progressive release of solute from matrix
to solvent, thus the extraction efficiency. We tried three incubation
times: 20, 40, and 60 min, followed or not by one or two additional
20-min extractions. The aim was to determine the adequate
extraction time for obtaining a maximum of antioxidants without
making the procedure too long for routine use.
 J.A. Michiels et al. / Food Chemistry 130 (2012) 986–993
Whatever the duration and number of extraction steps, the
quantity of phenolic compounds extracted from apple or broccoli
remained the same (Fig. 3A). In the case of leek, two successive
extraction steps led to a better yield than a single extraction
(+40% with 40 + 20 min), and the same was true for orange (+12%
with 20 + 20 min). When the means of the four matrices were con-
sidered, the two-step extraction (40 + 20 min) showed the best re-
sults. In the case of apple, the DPPH antioxidant capacity increased
with the extraction time (as compared to a 20-min extraction, the
increase observed after 40 or 60 min, respectively was 41% or 51%).
The antioxidant capacity was higher after a three-step extraction
from apple (+77%), broccoli (+57%), or orange (+16%) (Fig. 3B).
Increasing the extraction time or the number of extraction steps
had no effect or a small negative effect on the ORAC values for
apple and leek (12% and 21% after 60 and 40 + 20 min respec-
tively, as compared to 20 min), and a small positive effect on those
for orange and broccoli (+12% and +13% after 20 and 60 min,
respectively as compared to 20 min). Thus for the ORAC assay, a
60-min incubation appeared adequate for optimal extraction of
antioxidants from all these plant matrices. Additional extraction
steps did not improve the extraction yield. A 60-min extraction
time was also optimal for assaying total phenolics (except in the
case of orange, where successive extractions led to a 20% higher
yield). For evaluation of the antioxidant capacity by the DPPH
assay, a single 60-min extraction was almost optimal, except for
broccoli, in which case successive extractions led to higher yields
(+57%). From the comparison of all these results, a single 1-h
extraction thus emerged as a good compromise between extraction
efficacy and assay duration for routine use. With other non-acidi-
fied solvents, Luthria et al. (2006) have shown that 88% of the phe-
nolic compounds can be extracted in a single extraction step.
Extraction times of 1 h or less have been used by various authors
(Barreira et al., 2009). According to some authors (Liyana-Pathirana
& Shahidi, 2005), the extraction time has less effect than the
extraction solvent and temperature. In the following experiments,
a single 60-min extraction step was used.
3.4. Solvent-to-solid ratio
Various ratios of plant tissue weight to extraction solvent vol-
ume were tested (from 1 g:2 mL to 1 g:100 mL). The mean extrac-
tion efficiency for phenolic compounds was highest when the
volume used was equal to (+141%) or higher than (+132%)
40 mL (Fig. 4). The highest antioxidant capacities were recorded
with 40 mL solvent, except in the case of leek (highest DPPH va-
lue with 100 mL extraction solvent) and orange (highest ORAC va-
lue with 100 mL extraction solvent). Similar results regarding the
effect of the solvent-to-solid ratio on the extraction of phenolic
compounds have been reported by Pinelo et al. (2005). Generally
speaking, the higher the solvent-to-solid ratio, the higher the to-
tal phenolics content and antioxidant capacity obtained. This is
consistent with the principles of mass transfer; the driving force
during mass transfer is the concentration gradient between the
Table 1
Time (in days) that the total phenolics content and antioxidant capacity (DPPH and ORAC) remain stable in plant materials stored at 20 °C after rapid freezing or lyophilisation.
The plant material was considered stable if the measured loss was less than 20% of the initial value.
Apple Orange
Freezing Lyop. Freezing
ORAC 8 8 >25
DPPH 21 19 15
Phenolics >25 >25 >25
991
solid and the bulk of the liquid, which is greater when a higher
solvent-to-solid ratio is used (Cacace & Mazza, 2003).
As a compromise between maximising the extraction yield and
minimising the cost of extraction (which increases with the solvent
volume used), we chose to use 20 mL extraction solvent per 1 g
(fresh weight) plant material. Using a greater solvent volume gen-
erally had only a small positive effect on the extraction yield, while
increasing the assay cost and introducing the need of a concentra-
tion step for some matrices (because the detection limit was
reached).
3.4.1. Stability of the antioxidant capacity during storage of plant
material
A sample of each plant material was frozen in liquid nitrogen
before being stored at 20 °C for up to 25 days. Another fresh sam-
ple was directly lyophilised before storage at 20 °C for up to
25 days. Samples were regularly assayed for total phenolics and
antioxidant capacity (DPPH and ORAC assays) (Table 1). The frozen
material was used directly in the extraction solvent as indicated
above for fresh material. The antioxidant capacity evaluated by
the DPPH assay generally appeared more stable in lyophilised
material than in material that was simply frozen. Lyophilisation
did not appear as a better means of ensuring the stability of phen-
olics or of the ORAC value. In all three assays used, orange emerged
as the most stable material and leeks as the least stable. Likewise,
Murcia, Jimenez, and Martinez-Tome (2009) observed a rapid de-
crease in antioxidant capacity in leek during frozen storage.
The ORAC value did not change during the first 25 days except for
apple under both storage conditions. In contrast, the antioxidant
capacity measured by the DPPH assay began to decrease after
12 days (Table 1). The phenolic compound content was generally
stable, as previously observed in various materials (Poiana, Moigra-
dean, Raba, Alda, & Popa, 2010), except in leek, where freezing led to
a major decrease. The observed differences between matrices may
be due to modification of the concentration and/or oxidation state
of the different types of phenolic compounds (Wojdylo, Figiel, &
Oszmianski, 2009).
3.4.2. Stability of the antioxidant capacity during storage of plant
extracts
In parallel, the antioxidant capacity and total phenolics were
determined in extracts of fresh material, both before and after
either a few days of storage in the extraction solvent at different
temperatures (4, 20, and 80 °C) or lyophilisation or evaporation.
The results obtained differed according to the material and stor-
age form (Table 2). Liquid extracts were generally more stable than
lyophilised and evaporated extracts. Considering all three assays
used (total phenolics, DPPH, and ORAC), apple and broccoli extracts
emerged as the most stable and leek as the least stable. These dif-
ferences are likely due to the nature of the antioxidant compounds
extracted from the various matrices. For example, some flavonols
(e.g. quercetin) appear to delay degradation of other phenolic com-
pounds such as anthocyanins (Shrikhande & Francis, 1974).
Broccoli Leek
Lyop. Freezing Lyop. Freezing Lyop.
>25 >25 >25 >25 >25
>25 15 >25 12 18
>25 20 5 0 0
992 J.A. Michiels et al. / Food Chemistry 130 (2012) 986–993

Table 2
Duration of stability in extracts stored at 20 °C in extraction solvent (Solv.), after evaporation (Evap.), or after lyophilisation (Lyop.). An extract was considered stable if the
recorded loss was less than 20% of the initial value.

Apple Orange Broccoli Leek

Solv. Evap. Lyop. Solv. Evap. Lyop. Solv. Evap. Lyop. Solv. Evap. Lyop.
ORAC 4 °C 4 0 3 4 0 3 3 0 4 0 0 0
20 °C >7 0 3 0 0 3 4 0 4 0 0 0
80 °C >7 0 3 >7 0 3 >7 0 4 0 0 0
DPPH 4 °C 8 >7 3 2 0 3 8 0 >9 1 4 4
20 °C >7 >7 3 >7 0 3 >7 >7 >9 >7 0 0
80 °C >7 >7 3 >7 0 3 >7 >7 >9 >7 >7 0
Phenolics 4 °C >9 >7 3 >9 >7 3 7 >7 4 0 0 0
20 °C >7 >7 3 >7 >7 3 >7 >7 4 0 0 0
80 °C >7 >7 3 >7 >7 3 >7 >7 4 0 0 0

Variations in storage temperature had little effect. Generally,
the results obtained at 20 and 80 °C were similar, while at
4 °C, the stability was slightly reduced or increased depending on
the method and material. In blueberry extracts, Srivastava, Akoh,
Yi, Fischer, and Krewer (2007) likewise observed a loss of phenolics
and antioxidant capacity after 15 days at 6° and no variation at
20 °C after up to 30 days. It is well known that some antioxidant
compounds, such as anthocyanins and ascorbic acid, are mutually
destructive in the presence of oxygen (Sondheimer & Kertesz,
1953). Considering the oxygen-radical-absorbing capacity of
anthocyanins, which makes them good antioxidants (Wang, Cao,
& Prior, 1997), this supports the notion that oxidation reactions
involving ascorbic acid as an activator of molecular oxygen pro-
duce free radicals (Garcia-Viguera & Bridle, 1999).
An obstacle to comparing the antioxidant capacities of different
products is the fact that the antioxidant capacity reported for a gi-
ven matrix can differ according to the study considered. One rea-
son for this is the use of different methods to measure
antioxidant capacity. In the present experiments, the ORAC assay
always yielded higher antioxidant levels than the DPPH assay. Sec-
ondly, the results obtained for a given matrix by a single method
such as ORAC can vary with the extraction method. Parameters
such as solvent, temperature, extraction time, solvent-to-solid ra-
tio, and storage conditions have a great impact on the measured
antioxidant capacity. All the parameters tested in this paper are
crucial for optimisation of sample preparation procedures. This
study also clearly demonstrates that it is essential to optimise sys-
tematically the extraction in order to maximise the yield of antiox-
idant compound extraction from each food matrix.
The antioxidant capacity often appears to correlate with the
phenolic content (Kevers et al., 2007). As indicated in Table 3, the
correlation between the phenolic content and antioxidant capacity
measured by the two assays varied from matrix to matrix. It was
better with DPPH for apple (r2 = 0.82) and broccoli (r2 = 0.94) and
better with ORAC for orange (r2 = 0.99) and leek (r2 = 0.55). The
extraction performance differed probably because each matrix
contained a different set of different antioxidant compounds.
Standardisation of sample preparation and extraction is neces-
sary to allow comparing the antioxidant capacities and phenolic
contents of different food matrices, as determined by different lab-
Table 3
Correlation coefficients (r2) between total phenolics and antioxidant capacity (DPPH
or ORAC), calculated from the results presented in Fig. 4.
Phenolics/DPPH Phenolics/ORAC
Apple 0.8178 0.2422
Orange 0.0013 0.9877
Broccoli 0.9425 0.0009
Leek 0.2409 0.5470
oratories. On the basis of the present results, we propose the
following conditions: an acetone/water/acetic acid mixture (70/
28/2, v/v/v) as extraction solvent; extraction for 1 h at 4 °C; a sol-
vent-to-solid ratio of 20 mL per 1 g; use fresh material unless this
is not possible, in which case one may resort to lyophilisation, or
the extracts can possibly be stored for a few days at 20 °C before
analysis. This should make it possible to attribute differences in
antioxidant capacity for a specific matrix to real (e.g. genetic, envi-
ronmental) factors.
Acknowledgements
We thank G. Tabourga (student at the University of Dijon) for
technical assistance. J.A.M. gratefully acknowledges the support
of the ‘‘Ministère des Technologies nouvelles’’ (First Grant). The
skilful assistance of the APE staff (provided to CEDEVIT by the re-
gional government of Wallonia) was greatly appreciated.
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