Application of free radical diphenylpicrylhydrazyl

(DPPH) to estimate the antioxidant capacity of food

samples
Krystyna Pyrzynska
*
and Anna Pekal
The 2,2-diphenylpicrylhydrazyl (DPPH) assay is widely used in plant biochemistry to evaluate the properties
of plant constituents for scavenging free radicals. The method is based on the spectrophotometric
measurement of the DPPH concentration change resulting from the reaction with an antioxidant.
Several protocols have been followed for this assay using dierent conditions such as dierent reaction
times, solvents, pH and dierent compounds used as antioxidant standards. This review shows to what
extent the mentioned parameters have the inuence on the presented results.
Introduction
There is more recent evidence that free radicals induce oxidative
damage to biomolecules. This damage has been implicated in
ageing and in several human pathologies and other diseases.
1
Several studies indicate that the active dietary constituents of
fresh fruits, vegetables and beverages prevent these free radical-
induced diseases and protect foodstus against oxidative
deterioration.
2
These protective eects have been attributed to
the antioxidant species, such as polyphenolic compounds,
carotenoids, vitamins C and E, which scavenge free radicals.
3,4
Interest in natural sources of antioxidants for use in the food,
beverage and cosmetic industries has resulted in a large body of
research in recent years.
Antioxidant capacity is widely used as a parameter to char-
acterize dierent substances and food samples with the ability
of scavenging or neutralizing free radicals. This capacity is
related to the presence of compounds capable of protecting a
biological system against harmful oxidation. There are several
methods for the evaluation of antioxidant eciency of pure
compounds and plant extracts such as ORAC (oxygen radical
absorbance capacity), FRAP (ferric reducing antioxidant
power), CUPRAC (cupric reducing antioxidant capacity), the 2,2-
azinobis(3-ethyl-benzothiazoline-6-sulphonate) radical cation
(ABTS
+
_) assay and the 2,2-diphenyl-1-picrylhydrazyl radical
(DPPH_) assay.
512
The most common assays are those involving
chromogen compounds of radical nature, which in the presence
Krystyna Pyrzynska received her
PhD in Chemistry from the
University of Warsaw, Poland.
Now she is working in the
Laboratory of Flow Analysis and
Chromatography, University of
Warsaw. Her research interest is
focused on the application of
solid sorbents for preconcentra-
tion and speciation purposes of
some metal ions in environ-
mental samples as well as chro-
matographic analysis of
phenolic compounds and anti-
oxidant properties of food
samples.
Anna Pe kal received her Mas-
ter's degree in analytical chem-
istry from the University of
Warsaw. She is currently a PhD
candidate in the Department of
Chemistry at the University of
Warsaw. Her research interest
includes analytical methods for
the evaluation of antioxidant
capacity of food samples.
University of Warsaw, Department of Chemistry, Pasteura 1, 02-093 Warsaw, Poland.
E-mail: kryspyrz@chem.uw.edu.pl; Fax: +48 22 8223532
Cite this: Anal. Methods, 2013, 5, 4288
Received 7th March 2013
Accepted 11th June 2013
DOI: 10.1039/c3ay40367j
www.rsc.org/methods
4288 | Anal. Methods, 2013, 5, 42884295 This journal is The Royal Society of Chemistry 2013
Analytical
Methods
MINIREVIEW
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of an antioxidant disappear. This reaction can be easily followed
by common spectrophotometric detection.
The DPPH assay is one of the most popular and frequently
employed methods to test the ability of compounds to act as
free radical scavengers or hydrogen donors, and to evaluate the
antioxidant capacity of foods. The DPPH_ radical is a long-lived
organic nitrogen radical with a deep purple color. It is
commercially available and does not have to be generated
before assay as in other scavenging assays. When a solution of
DPPH_ radical is mixed with an antioxidant/reducing
compound, its color turns from purple to yellow of the corre-
sponding hydrazine (Fig. 1). The reducing ability of antioxi-
dants towards DPPH_ can be evaluated by monitoring the
decrease of its absorbance at 515528 nm as the formed cor-
responding hydrazine DPPH
2
yields a yellow solution
13
or by
electron spin resonance.
14
Originally, it was believed that the
DPPH assay involved a hydrogen transfer reaction, but Foti
et al.
15
have suggested a dierent mechanism. The initial elec-
tron transfer occurs very quickly and the subsequent hydrogen
transfer occurs more slowly and this depends on the hydrogen-
bond accepting solvent used such as methanol and ethanol.
This paper presents anoverviewof the publications regarding
the use of the DPPH method. Various research groups have used
dierent conditions for this assay (dierent concentrations of
DPPH_ radical, incubation times and reaction solvents). The
sample pHandthe presence of metal ions inthe studiedextracts,
as natural components of plants, are very oen ignored.
Stability of DPPH_ reagent
The DPPH_ reagent is soluble only in organic solvents. Tsimo-
giannis and Oreopoulou
16
found that the reactivity of DPPH_ is
much more enhanced in the presence of methanol than in ethyl
acetate. The stability of DPPH_ solution is inuenced by the
solvent used for its preparation. The absorbance of DPPH_ in
methanol and acetone decreased by 20% and 35%, respectively,
for 120 min at 25

C under light, while in the dark it did not
change signicantly for 150 min.
17
Deng et al.
17
reported that the eect of temperature on the
concentration of DPPH_ is more notable than that of time. The
decrease in absorbance by 6% within 90 min was observed as
the temperature changed from 4

C to 25

C. The authors
concluded that it is better to keep the reagent solution for a
short period of time at room temperature than in a refrigerator.
On the other side, some authors conducted the DPPH assay at
elevated temperatures, such as 37

C (ref. 19) or even 50

C.
20
Chemical conditions for measurement
The experiments have been performed under dierent chemical
conditions. The excess of DPPH_reagent should be used in order
to exhaust the H-donating capacity of polyphenols. However, in
accordance with Beer's law and the normal practice in spec-
trophotometry, the initial DPPH_ concentration in a cuvette
should be chosen such to give absorbance values less than 1.0
(which corresponds to the light intensity being reduced no
more than ten-fold while passing through the sample). This
implies that the nal concentration of the DPPH_ solution in the
cuvette is in the range of 2570 mM.
21
Some workers used the
DPPH_ concentration higher than 200 mM.
19,22,23
For most of the compounds which exhibit antioxidant
properties, their reaction with DPPH_ is biphasic, with a fast
decay in absorbance in the rst few minutes, followed by a
slower step in which degradation products are involved, until
equilibrium is reached. As can be seen from Fig. 2, the rate of
reaction varies widely among dierent compounds.
24
The DPPH
assay is most frequently simplied by measuring the DPPH_
concentration at the beginning of the reaction and aer a
certain incubation time. In the original assay the reaction time
of 30 min was recommended.
25
However, a shorter time in the
range of 316 min has been used,
2629
while the use of 24 h
incubation time has also been reported in some papers.
30,31
This
is one of the reasons for the diversity of the results published for
similar samples which consequently limits the comparison of
data between studies.
Fig. 1 DPPH_ radical's chemical structure and its reaction with a scavenger indicated by AH.
Fig. 2 The kinetic curves of the scavenged DPPH_ by some phenolic compounds
at the concentration of 0.1 mM (ref. 24) (reproduced with permission).
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Magalh~aes et al.
32
proposed a kinetic matching approach,
where the oxidation kinetic behavior of a standard mixture
(caeic acid, (+)-catechin, hesperetin, morin and ()-epigallo-
catechin gallate) was compared to that attained for red wines,
selected as a model for a complex food matrix. This comparison
involved the calculation of antioxidant capacity values and,
when a kinetic matching standard is found, these values are
constant along the reaction time. The second part of this
strategy consisted of converting the calculated antioxidant
capacity value (expressed as equivalents of the kinetic matching
standard) into an antioxidant capacity value expressed as
equivalents of trolox by taking into account the number of
electrons transferred by each compound. There was no statis-
tical dierence between the results obtained by the kinetic
matching approach (aer <10 min of reaction) and those
obtained under end point conditions (aer 120 min). The
sample throughput increased from <18 (end point measure-
ments) to >108 per hour using the proposed kinetic approach.
Expression of results
The results of the DPPH assay have been presented in many
ways. The majority of studies expressed the results in terms of
the reduction percentage of the DPPH_ solution, referred to
also as the percent of inhibition or quenching, and calculated as
I% [(A
0
A
t
)/A
0
] 100, where A
0
and A
t
are the absorbance
values in the absence and presence of an antioxidant. In some
papers the results are presented in the form of the percentage of
residual DPPH_ calculated in the following way: DPPH_
res

[(DPPH_)
t
/(DPPH_)
0
] 100, where DPPH_
0
and DPPH_
t
are the
concentrations at the initial and steady state, respectively,
obtained from a calibration curve.
The antioxidant concentration necessary to decrease the
initial DPPH_ concentration by 50% inhibition (named as inhi-
bition concentration IC
50
or eciency concentration EC
50
) is
also oen used for the comparison of the antioxidant capacities
of dierent compounds or extracts of natural samples. A lower
value of IC
50
indicates higher antioxidant activity. The IC
50
value is calculated using the graph obtained by plotting the
inhibition percentage against the extract or compound
concentration. However, the values of IC
50
parameter were
expressed in dierent units: in grams of antioxidant per kg of
DPPH_,
16
the molar or the mass ratio of antioxidant to DPPH_,
3337
as mmoles of an antioxidant
21,3840
or in a concentration unit as
mg mL
1
.
41
Moreover, dierent values of IC
50
could be found in
the literature for the same compounds. This parameter highly
depends on the reaction time and the initial DPPH_ concentra-
tion as shown in Table 1 for gallic and ascorbic acids as
examples.
The time needed to reach the steady state with IC
50
concentration dened as TIC
50
was calculated graphically by
plotting the time at the steady state against the concentration of
each antioxidant compound.
44
However, this may lead to a large
variation of TIC
50
values as there is no linear correlation
between the time at the steady state and the concentration of
antioxidants and as a consequence dierent TIC
50
values would
be obtained. Moreover, the absolute values of IC
50
and TIC
50
are
highly dependent on the units of the antioxidant concentration
(mg L
1
or mgL
1
). Sanchez-Moreno et al.
44
further proposed
another parameter to express the antioxidant activity called
antiradical eciency (AE) where AE 1/EC
50
TIC
50
. The
larger the AE the more ecient is the antioxidant. A concep-
tually similar parameter for the evaluation of the radical scav-
enging eciency (RSE) was suggested by De Beer et al.
41
RSE was
calculated as the ratio of the starting rate of DPPH_decay to IC
50
.
Recently, Scherer and Godoy
43
proposed a new antioxidant
activity index (AAI) calculated as the ratio of the nal concen-
tration of DPPH_ (in mg mL
1
) to IC
50
(in mg mL
1
). AAI takes
into consideration the mass of DPPH_and the mass of the tested
compounds in the reaction resulting in a constant for each
antioxidant, independent of the concentration of DPPH_ and
sample used. However, the concentration of DPPH_ used in this
study produced absorbance values between 2 and 3, which fell
outside the range and accuracy of spectral measures.
The change of DPPH_ absorbance could be compared to the
change induced by a reference compound. Several compounds
have been used as a standard antioxidant in the performed
experiments including ascorbic acid,
25,4547
a-tocopherol,
48,49
gallic acid,
45
butylhydroxytoluene (BHT)
46,50
and trolox.
27,31,5153
Trolox, a water soluble analogue of vitamin E, does not have any
physiological signicance and its choice as the standard for
antioxidant activity is arbitrary. However, the expression of the
results as trolox equivalents (mmol of trolox necessary to provide
the same antioxidant activity as a gram of the sample) helps for
comparison of the published data.
Eect of solvent
The antioxidant properties of extracts, estimated by the DPPH
method, obtained from the food samples using dierent
solvents (methanol, ethanol, acetone or their mixtures with
water in dierent proportions as well as ethyl acetate or chlo-
roform) are discussed in many papers.
30,5456,65
The antioxidant
properties of such extracts are largely related to dierences in
their quantitative and qualitative composition resulting from
dierent extraction ability of the used solvents. Antioxidant
compounds in food may be water soluble, fat soluble, insoluble
Table 1 IC
50
values for gallic and ascorbic acids from various reported studies
IC
50
(mM) [DPPH_]
0
a
(mM) Time (min) Ref.
Gallic acid 0.74 29.3 40 18
1.48 58.5 40 18
4.15 50 30 29
4.2 50 30 37
5.1 63 20 42
6.88 78 90 43
17.1 195 90 43
Ascorbic acid 1.35 29.3 40 18
2.74 58.5 40 18
10.2 50 30 37
50 250 30 23
55.9 85.7 30 38
629 100 10 39
a
[DPPH_]
0
the nal concentration in the cuvette.
4290 | Anal. Methods, 2013, 5, 42884295 This journal is The Royal Society of Chemistry 2013
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or bound to cell wall. Certain antioxidants require polar
solvents such as methanol or ethanol, while ethyl acetate or
chloroform are used to extract lipophilic antioxidants. Hence,
extraction eciency is an important factor in the evaluation of
antioxidant capacity. It was observed that the addition of water
to alcohol increases the eciency of extraction, until it reaches
the optimum.
57,58
There is no solvent that would be entirely
satisfactory for extraction of all the antioxidants present in food,
particularly those associated with complex carbohydrates and
proteins. Consequently, there is a considerable amount of
bioactive compounds with antioxidant properties in the
extraction residues, which are ignored in most chemical and
biological studies. Perez-Jimenez et al.
55
proposed at least two
extraction cycles from plant foods performed with aqueous
organic solvents with dierent polarities in order to extract
antioxidant compounds with dierent chemical structures.
Although higher extract yields were obtained by the reux
extraction technique, in general higher amounts of total
phenolic contents and better antioxidant activity were found in
the extract prepared from some medicinal plants using a
shaker. It has been reported that thermal extraction conditions
might result in the loss of natural antioxidants and the eect of
degradation of avonoids depends on the extraction conditions
and their chemical structure.
59
The high number of hydroxyl
groups promotes degradation of avonoids, whereas sugar
moiety and methoxyl groups protect them. The smallest
decomposition was observed by the heated reux extraction
procedure within 30 min in water bath and by microwave
assisted extraction (160 W during 1 min).
59
On the other hand,
Dutra et al.
60
reported that among dierent extraction tech-
niques (reux, maceration, ultrasound, heating plate) extrac-
tion performed under reux using ethanolwater (70 : 30, v/v)
oered the highest polyphenols level from plant material.
Dawidowicz et al.
61
reported that the type and the amount of
solvent used for antioxidant dissolution signicantly inuenced
the dierence in the amount of unreacted DPPH_. The presence
of ethyl acetate or dioxane in the system decreases the kinetics
of the reaction between DPPH_ and BHT in relation to its
kinetics in the system containing only methanolic solutions.
This eect was linear for the ethyl acetate volume in the
examined range of 501000 mL, while the increase of the
dioxane volume caused almost constant suppression of the
DPPH_BHT reaction kinetics. According to Musialik and Lit-
winienko,
62
aprotic solvents (such as ethyl acetate or dioxane)
suppress the kinetics of DPPH_antioxidant reaction. On the
other side, small volumes (up to 440 mL in the total volume of 3
mL) of chloroformic solution of BHT accelerated the reaction
kinetics, whereas large volumes (up to 1000 mL) decelerated the
reaction rate. While the latter observed a decrease of the reac-
tion rate, which is unstable (chloroform is also an aprotic
solvent), the eect at small volumes of this solvent is intriguing.
The authors explained that it might be connected with the
structural changes of bulk methanol caused by the addition of
chloroform to the measuring system.
The increase in water content in the mixture of methanol
generally leads to the acceleration of the DPPH_antioxidant
reaction kinetics in comparison to the kinetics of the system
without water and that dierence was greater in the case of
small water content (up to 30 mL with 910 mL of methanol).
61
Stasko et al.
33
studied the limits for water as a component of the
mixed waterethanol solvent for the DPPH radical assay. They
concluded that 50% (v/v) aqueousethanol solution is a suitable
choice for lipophilic and hydrophilic antioxidants and the
reaction rate increases considerably with the increasing water
ratios. However, at the water content over 60% (v/v), the anti-
oxidant capacity decreases, since a part of DPPH_coagulates and
it is not easily accessible in the reaction with antioxidants.
The evaluation of the antioxidant capacity of the mixtures
obtained aer mixing equal volumes of catechin and gallic acid
standard solutions (at a concentration of 1 mol L
1
each) in
dierent solvents (water, methanolwater (30 : 70 v/v),
methanolwater (50 : 50 v/v), methanol and acetonewater
(50 : 50 v/v)) showed a weak inuence on the DPPH assay.
54
The
greatest signicant dierence was between water (IC
50
0.067)
and acetonewater mixture and pure methanol (IC
50
0.083 for
both systems).
Noipa et al.
29
have developed a modied DPPH assay using
surfactant aggregates or micelles, which do not require an
organic solvent. Among three types of surfactants studied such
as nonionic (Triton X-100), anionic (sodium dodecyl sulphate,
SDS) andcationic (cetyltrimethylammoniumbromide, CTAB), in
the last medium gallic acid and protocatechuic acid (used as the
model compounds) showed higher percent of inhibition than
TritonX-100 andSDS. The optimummicelle systemwas found to
be 2 mMCTABin0.1 Macetate buer. The reactionof DPPH_with
gallic acid observed in the CTAB solution was faster than other
micelle systems and reached the steady state absorbance within
1 min. The rate constant inthis systemwas about 30 times higher
than in methanol. It was assumed that the direct abstraction of
phenol H-atom by DPPH_ occurred inside the micelle and the
electrostatic interactions allowedthe radical toremaininside the
hydrophobic core of the CTAB micelle. This allowed the use of
shorter analysis time and to evaluate the antioxidant capacity
from aqueous extracted plant samples.
pH of sample
In the original Blois paper
12
regarding the DPPH method, it was
suggested that the system should be maintained at the pH
range of 5.06.5 by using acetate buers. However, these
conditions were abandoned in the later current practice, as
there is a great uncertainty in the meaning of pH values for
methanol or ethanol used mainly as reaction media.
Organic acids alone (such as acetic, malonic and citric acids)
exhibited no scavenging activity towards DPPH_, but when
added to the solution of ascorbic acid its action enhanced
towards DPPH_.
35
Thus, the antioxidant action of ascorbic acid
could be enhanced by some components that are not directly
involved in the free radical scavenging activity. The greatest
eect was observed in the presence of citric acid in relation to
water. This medium had the lowest pH value of 3.28.
Sharma and Bhat
21
examined the DPPH radical scavenging
activity of ascorbic acid, BHT and propyl gallate using the
reagent prepared in methanol and in buered methanol,
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which was prepared by mixing 0.1 Macetate buer (pH 5.5) with
methanol (2 : 3, v/v). They found that the radical scavenging
proles of ascorbic acid and propyl gallate were comparable in
both reagent media, while for BHT it was markedly higher in
buermethanol solution in comparison to that in methanol
alone (Fig. 3). Dawidowicz et al.
61
reported that the increase of
hydrogen ion concentration in phosphoric buermethanol
solution (in the pH range of 1.64.1) slowed down the kinetic
reaction between BHT and DPPH_ in relation to pure solvent.
The application of buer with pH 5.5 gave the similar eect to
the case when pure water was applied, leading to the accelera-
tion of this reaction kinetics.
Generally, the increase of hydrogenionconcentrationleads to
the decrease of the reaction rate of chromogen radical scav-
enging, whereas under basic conditions proton dissociation of
polyphenolics would enhance the reducing capacity of
compounds. Following,
62,63
the reaction between phenolic anti-
oxidants and DPPH radical occurs by a combination of Proton
Coupled-Electron Transfer (PC-ET) and Sequential Proton Loss
Electron Transfer (SPLET) mechanisms. The rst is slower and
dominates innon-polar solvents of lowdielectric constant andof
low basicity, whereas the second one is faster and is character-
istic for solvents of high dielectric constant and of high basicity
supporting antioxidant ionization. The SPLET mechanism is
favored in methanol, which possesses high dielectric constant
and high ability to solvate phenolic anions. In non-ionizing
solvents, the SPLET mechanism can be ever eliminated.
It was found that the kind of the used buer system also
aected the measurements.
64
Phosphate buer showed a
decrease in DPPH_ radical scavenging activity at increasing
concentration (0.050.3 mM), while the acetate buer concen-
tration did not cause a signicant dierence. Therefore, careful
monitoring of these aspects is essential for the successful use
of the DPPH assay and the comparison of the antioxidant
activity of food samples tested under varying conditions is not
appropriate.
The presence of metal ions
Metal ions are natural components of plants and their content
is inuenced by the type of plant, the soil composition and local
environment. Their concentration in the obtained extracts of
food samples depends on the metal, plant type as well as
extraction conditions.
As transition metal ions play a vital role in the initiation of
free radical processes (via the Fenton reaction), metal chelation
by polyphenolic compounds is widely considered as another
mechanism of their antioxidant activity.
65
For steric reasons the
complexes usually include no more than two avonoid mole-
cules. In addition, some avonoids exhibit the ability to reduce
Fe(III) and Cu(II) ions.
6567
Several experimental data indicate
that these complexes are more eective free radical scavengers
than the corresponding free avonoids.
6870
The naringinCu
complex exhibits a much stronger DPPH_ scavenging than nar-
ingin alone and is higher than vitamin C (Fig. 4).
69
Dawidowicz et al.
61
studied the dierence between BHT
DPPH_ reaction rates in the systems with Fe(III) and Cu(II) and
Fig. 3 Scavengingof DPPH_radical by (A) ascorbic acid(1 mM) and(B) BHT (1 mM)
prepared in methanol or buered methanol
21
(reproduced with permission).
Fig. 4 Inhibition of DPPH by rutin, vitamin C, naringin and its complex with
copper. All compounds at 10 mM concentration
69
(reproduced with permission).
4292 | Anal. Methods, 2013, 5, 42884295 This journal is The Royal Society of Chemistry 2013
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without these metal ions. The increase of metal concentration
(up to 1 mg mL
1
and 20 mg mL
1
for Cu and Fe, respectively)
caused an almost linear deceleration of the reaction kinetics.
Cu(II) suppressed BHTDPPH_ reaction kinetics more than
Fe(III). The authors concluded that the observed changes in the
reaction kinetics can be attributed to the formation of metal
complexes with the components of the measuring system.
However in their next paper,
71
it was reported that the dierence
between the percent of remaining DPPH_with and without Cu(II)
or Fe(III) aer 60 min of the BHTDPPH_ reaction is almost the
same. In both studies, the experiments were conducted in non-
buered solutions. As metal ions can behave as acids in protic
solvents (protons are released in the solution from the OH
groups due to complexation reaction), the presented data
interpretation becomes spectaculative.
Espinoza et al.
72
evaluated the change in radical scavenging
activity against DPPH_ when copper or iron compounds were
added to four dierent types of wines. For Cabernet Sauvignon
2004 red wine, the addition of 0.6 mg L
1
and 1.2 mg L
1
copper
resulted in 22% and 46%, respectively, DPPH_ peak area
increase with respect to native wine. In the same way, the
addition of 3 mg L
1
and 6 mg L
1
of iron produced 46% and
69% peak area increase with respect to native wine. These
results show that the formation of redox inactive complexes
between polyphenols of wine and studied metal ions resulted in
reduction of their antioxidant activity. The authors mentioned
that these interactions require a certain time to be established
as in the other case it produced erratic and inconsistent results
in all the cases, however, this interval time was not specied.
As certain inorganic salts occurring in extracts of plants
aect the DPPH_ radical scavenging activity, Al-Dabbas et al.
64
have proposed the desalting method using cationic or anionic
exchange resins. The anionic euent of the crude water extract
of Varthemia iphionoides plant, commonly used in folk medi-
cine, exhibited signicantly higher antioxidant properties. This
may be related to the actual DPPH_ radical scavenging activity of
antioxidants present in this extract as all the inorganic matter
was removed.
The reported dierences in the antioxidant properties of the
extracts obtained from the same plant material (growing for
example under dierent conditions) can be caused not only by
the dierent contents of polyphenolic compounds but also by
dierences in the kinds of metal ions and their concentration in
the nal extract. The results of the DPPH assay for samples
polluted with metal ions can be dierent in relation to the
samples free of these metal ions. Thus, this can be a source of
erroneous conclusions in comparison to dierent samples.
Comparison with other assays
There are several dierent methods to assess the relative anti-
oxidant capacity of dierent food samples. The ORAC method
utilizes uorescence detection, while CUPRAC, FolinCiocalteu
(FC), FRAP, DPPH, and ABTS are applied with spectrophoto-
metric measurements. Electrochemical detection has also been
used for the analysis of the anodic current waveform in cyclic
voltammetry and for the potentiometric measurement of redox
potential. Spectrophotometric methods are still the most widely
used because the reagents used are easy to get, results are given
relatively quick and the experiments are convenient.
The relative antioxidant capacity of many compounds
present in food samples varies widely according to dierent
testing assays, which dier from each other in terms of
substrates, reaction conditions and quantitation methods. The
FC method, very popular and widely used for the determination
of the so-called total polyphenol content, is based on a non-
specic phenol oxidation reaction by the two strong inorganic
oxidants (phosphotungstic and phosphomolibdic acids). This
assay is conducted in alkaline medium and gives dierent
responses to dierent phenolic compounds, depending on their
chemical structures.
11
Moreover, the FC reagent could simul-
taneously oxidize several nonphenolic organic compounds as
well as some inorganic substances to give an elevated apparent
phenolic content, thus it can be used for the measurement of
the total reducing capacity of samples. The CUPRAC method is
based on the reduction of Cu(II) to Cu(I) at neutral pH by anti-
oxidants present in a sample utilizing the copper(II)neo-
cuproine reagent as the chromogenic oxidant. Slow reacting
antioxidants needed an increased temperature incubation to
complete their oxidation.
73
The FRAP method utilizes the
reduction of the ferric tripyridyltriazine complex at low pH to its
ferrous form which has an intense blue colour (l
max
593 nm).
The ABTS
+
_radical cation during its reaction with an antioxidant
at pH 7.4 is converted to its colorless neutral form and the
decrease in absorbance is monitored. This assay is oen
referred to as the trolox equivalent antioxidant capacity (TEAC)
assay. Thus, there is no single, widely acceptable assay appli-
cable to a reasonable variety of compounds in food matrices. It
is essential to use a minimum of two methods depending on the
expected antioxidant potential and/or on the origin of the
substance.
Usually linear correlations between the results obtained by
dierent assays have been checked. However, dierent results
have beenreportedevenfor the similar kindof foodsamples. For
example the DPPH assay showed similar trends for studied tea
infusions to the FC and CUPRAC methods,
73
but contrasting
results were reported for green tea preparation.
74
The total
quantities of polyphenols in tea samples as well as the
percentage of the individual compounds variedwiththe varieties
andthe change of tea plant living conditions. Besides, it couldbe
also the result of the synergies or antagonisms of tea infusion
components in a given assay.
75
It should be remembered that all
the used assays are intended for the estimation of the anti-
oxidative potential of food. As for the antioxidative actionof food
constituents in real biological systems, this will depend also on
their availability and food antioxidant metabolism.
Conclusions
The DPPH assay is considered as an easy and useful spectro-
photometric method with regards to screening or measuring
the antioxidant activity of model compounds and extracts of
food samples. It can be used for solid and liquid samples. The
DPPH_ radical is stable, commercially available, and does not
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have to be generated before assay like ABTS
+
_. It is not specic to
any particular antioxidant component, but applies to the overall
antioxidant capacity of the sample. The steric accessibility of
the DPPH_ radical is a major determinant of the reaction, since
small molecules that have better access to the radical site have
relatively higher antioxidant capacity. On the other hand, many
large antioxidant compounds that react quickly with peroxyl
radicals may react slowly or may even be inert in this assay. The
inexistence of DPPH_ or similar radicals in biological systems is
also a shortcoming.
Spectrophotometric measurements can be aected by
compounds, such as carotenoids, which can absorb at the
wavelength of determination.
76
The interactions between the
polyphenols and the food constituents have also been
found.
35,54
Most of them produced no eect by themselves but
altered the original polyphenol IC
50
value. The glucidic
compounds such as glucose, galacturonic acid and pectin
reduced the IC
50
, indicating a greater antioxidant capacity.
Cysteine is an amino acid that mostly reduced the IC
50
and
simultaneously increased the AE values.
54
The main problem is the compatibility of the obtained values
between several laboratories due to dierent reaction times,
solvents, pH and dierent antioxidant standard compounds,
which are frequently applied. This paper shows the complexity
of the problem. The DPPH assay should be validated and
standardized with a large body of comparable data available in
the literature. It was postulated to express the H-donating
capacity as the amount of DPPH_, which may be scavenged by
the tested sample (the stoichiometric coecient for individual
antioxidants or the mass of sample).
77
However, it should be
taken into consideration that the parameter increases with the
incubation time. The trolox activity could be used as the base-
line unit of measurement to allow comparison between
dierent studies as well as between dierent methods used for
evaluation of the antioxidant activity in food, beverages, food
ingredients and dietary supplements.
The HPLC on-line method using DPPH_ in a post-column
derivatization step, which allows simultaneous separation of
compounds and evaluation of free-radical scavenging capacity
of individual compounds, has been proposed.
20,7880
The aim of
such an approach is not only the rapid measurement of the total
capacity of a given sample, but also the proling of antioxidants
in complex mixtures following their chromatographic separa-
tion from the matrix.
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