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ORIGINAL ARTICLE

The use of the stable free radical diphenylpicryl-


hydrazyl (DPPH) for estimating antioxidant
activity

Philip Molyneux

Abstract
Molyneux, P.
The use of the stable free radical diphenylpicrylhydrazyl (DPPH)
for estimating antioxidant activity

The use of the stable free radical diphenylpicrylhydrazyl (DPPH) to estimate the activity of antioxi-
dants is reviewed. Current applications of the method are examined, particularly the use of the parameter
EC50 (substrate concentration to produce 50% reduction of the DPPH). Some recommendations are made as
to the most suitable ways of carrying out this assay and evaluating the data produced.

Key words : DPPH, diphenylpicrylhydrazyl, free radical, antioxidant activity

Ph.D.(Polymer Chemistry), Macrophile Associates, 9 Brewery Lane, Salisbury, Wiltshire, SP1 2LJ, U.K.
E-mail: molyneux@easynet.co.uk
Received, 8 June 2003 Accepted, 15 December 2003
Use of DPPH to estimate antioxidant activity
Molyneux, P.

There is an increasing interest in antioxi- hydrazyl (,-diphenyl--picrylhydrazyl; DPPH:


dants, particularly in those intended to prevent the 1) is characterised as a stable free radical by virtue
presumed deleterious effects of free radicals in the of the delocalisation of the spare electron over the
human body, and to prevent the deterioration of molecule as a whole, so that the molecules do not
fats and other constituents of foodstuffs. In both dimerise, as would be the case with most other
cases, there is a preference for antioxidants from free radicals. The delocalisation also gives rise to
natural rather than from synthetic sources (Abdalla the deep violet colour, characterised by an absorp-
and Roozen, 1999). There is therefore a parallel tion band in ethanol solution centred at about 520
increase in the use of methods for estimating the nm.
efficiency of such substances as antioxidants When a solution of DPPH is mixed with that

(S a nchez-Moreno, 2002; Schwarz, et al., 2001). of a substance that can donate a hydrogen atom,
One such method that is currently popular then this gives rise to the reduced form (2) with the
is based upon the use of the stable free radical loss of this violet colour (although there would be
diphenylpicrylhydrazyl (DPPH). The purpose of expected to be a residual pale yellow colour from
this paper is to examine the basis of this method, the picryl group still present). Representing the

and to further examine the use of the parameter DPPH radical by Z and the donor molecule by
EC50 (equivalent concentration to give 50% AH, the primary reaction is
effect) which is currently used in the interpretation
of experimental data from the method. Z + AH = ZH + A [1]
It should be noted that the present paper is

not concerned with the correlation between the where ZH is the reduced form and A is free ra-
results of the DPPH method and the actual acti- dical produced in this first step. This latter radical
vity of the substance in autoxidation reactions will then undergo further reactions which control
(Schwarz, et al., 2001); neither is it concerned with the overall stoichiometry, that is, the number of
the actual efficiency of these substances either as molecules of DPPH reduced (decolorised) by one
antioxidants or as life-style enhancers in humans molecule of the reductant.
(Wanjek, 2001). The reaction [1] is therefore intended to
provide the link with the reactions taking place in
Basis of the Method an oxidising system, such as the autoxidation of a
lipid or other unsaturated substance; the DPPH

1. DPPH - free radical and reduced form molecule Z is thus intended to represent the free
The molecule of 1,1-diphenyl-2-picryl- radicals formed in the system whose activity is to

1: Diphenylpicrylhydrazyl (free radical) 2: Diphenylpicrylhydrazine (nonradical)


Use of DPPH to estimate antioxidant activity
213 Molyneux, P.

be suppressed by the substance AH. among other compounds active in this reaction are
glutathione, aromatic amines (such as p-phenylene
2. The original Blois method diamine and p-aminophenol), and -tocopherol
The DPPH method as summarised above (Vitamin E - 2:1 stoichiometry) and polyhydroxy
was evidently introduced nearly 50 years ago by aromatic compounds (such as hydroquinone and
Marsden Blois, working at Stanford University pyrogallol). On the other hand, monohydric phenols
(Blois, 1958). Although this paper is short (a little (such as tyrosine), simple sugars (such as glucose),
over one page in the journal Nature), it provides purines and pyrimidines, do not react, while pro-
a succinct and clear account of the method. He teins are precipitated. It was also noted that in-
used as his model antioxidant the thiol-containing organic ions in lower valence states may of course
amino acid cysteine. Representing the DPPH ra- interfere and must be eliminated or determined

dical by Z and the cysteine molecule by RSH, the separately which presumably applies most im-
initial reaction is then portantly to ferrous iron (Blois, 1958).
In the original paper, a so-called typical
Z + RSH = ZH + RS [2] calibration curve is presented; this however seems
to have been constructed artificially from the
The free radical RS evidently then reacts original experimental data, since the absorbance
with another molecule of the same kind that was values (there called by the previous name, optical
produced by a parallel reaction to [2] density) are round number values (0.6 down to
0.2), which have therefore evidently been calcu-

RS + RS = RS SR [3] lated. The graph was also not been extended to
allow the line to meet the axis as would be ex-
This therefore leads to the observed reduc- pected to give the end-point for the titration. When
tion of two molecules of DPPH by two molecules extended down to the x-axis, the end-point would
-7
of cysteine, that is, a 1:1 stoichiometry. correspond to 2.310 moles (230 nanomoles) of
If however the molecule has two adjacent this substrate (cysteine hydrochloride). The pre-
sites for hydrogen abstraction which are internally sumed full titration if continued beyond the end
connected, as is the case with ascorbic acid (Vita- point is shown in idealised form in Figure 1; this
min C), then there may be a further hydrogen graph, however, takes no account either of any
abstraction reaction after the first one: residual yellow colour from the reduced form, or
HO OH HO O of any absorbance contribution there may be from
| | | | the added sample itself.

Z + R C = C R' = ZH + R C = C R' [4] It should be evident that the method is a
constant-volume colorimetric titration, although
HO O O O the slowness of the overall reaction (with mix-
| | || | | tures having to be left for 30 minutes before the

Z + R C = C R' = ZH + R C C R' [5] absorbance reading is taken) complicates the
experimental procedure.
This leads to a 2:1 stoichiometry, that is, two
molecules of DPPH reduced by one molecule of 3. The current situation
ascorbic acid. The same stoichiometry is shown in The original Blois method has been followed
the reaction with hydroquinone (1,4-dihydoxy- by several recent workers (Kim et al., 2002; Zhu
benzene) that leads to the production of quinone et al., 2002). The more recently introduced method
(1,4-benzoquinone) by a similar two-step mecha- of Brand-Williams and colleagues (Brand-Williams
nism. et al., 1995) has been used as a reference point by

It was noted in the original paper that several groups of workers (G O mez-Alonso et al.,
Use of DPPH to estimate antioxidant activity
Molyneux, P.

Figure 1. Idealised plots of absorbance A (left hand scale and filled circles), and percentage
reduction Q (right hand scale and open squares), versus the amount of reductant
added, for the constant-volume colorimetric titration of DPPH with cysteine
hydrochloride; adapted from Blois (1958).

2003; Lebeau et al., 2000; Yepez et al., 2002). This that causes 50% loss of the DPPH activity (col-
more recent work has indicated that the picture our).
originally suggested by Blois is somewhat over- This parameter was apparently introduced
simplified, and that because of the complexity of by Brand-Williams and his colleagues (Brand-
the reactions that follow the initial one [equation Williams et al., 1995; Bondet et al., 1997), and
1], the overall stoichiometry need not necessarily has been used subsequently by several groups of
be a whole number (integer) such as 1 or 2. workers for presenting their results (Kim et al.,
Furthermore, the initial step [equation 1] may be 2002; Lebeau et al., 2000; Leit a o et al., 2002;

reversible, as can be demonstrated by adding the Lu and Foo, 2000; S a nchez-Moreno et al., 1998;

reduced form ZH at the end of the reaction (Bon- S a nchez-Moreno et al., 1999). As a term, it was
det et al., 1997). Nevertheless, the Blois picture presumably introduced on analogy with biologi-
remains a useful one, and the original paper should cal parameters such as LD50.
be read by anyone proposing to use the DPPH However, such terminology seems to
method. obscure the true nature of the method, particularly
when used alongside such terms as the dose-
4. The parameter EC50 (efficient concentra- response curve to refer to the titration plot; for it
tion value) gives the impression that this is in itself some test
One parameter that has been introduced of biological activity, giving validation to the use
recently for the interpretation of the results from of the substrate as an antioxidant in a biological
the DPPH method, is the efficient concentration system. Indeed, if anything it is the EC100 value
or EC50 value (otherwise called the IC50 value). that we are concerned with, that is, corresponding
This is defined as the concentration of substrate to the endpoint of the titration. It should be noted
Use of DPPH to estimate antioxidant activity
215 Molyneux, P.

that in all these cases, any residual (yellow) colour the reaction to be followed, and allows adequate
from the reduced form or any non-specific ab- time for the overall reaction to go to completion
sorbance from the sample has to be taken into ac- in each individual reaction mixture ( 6).
count in defining the endpoint of the titration, or
the 50% point. 3. Solvent and pH
This EC50 parameter also has the drawback Regarding the solvent to be used, the method
that the higher the antioxidant activity, the lower seems to work equally well with methanol or
is the value of EC50. This is a disadvantage parti- ethanol, neither of which seems to interfere with
cularly when results are presented graphically as the reaction. The use of other solvent systems, such

a bar chart (S a nchez-Moreno et al., 1999) even if as almost neat extracts in water or acetone, seems
the same data are also available in numerical form to give low values for the extent of reduction (Guo

(S a nchez-Moreno et al., 1998). et al., 2001).
Regarding the pH level, in the original Blois
Recommended Methods of Measurement paper it was suggested that the system should be
and Interpretation maintained at a pH in the range 5.0 to 6.5 by using
acetate buffers; however, this precaution seems to
1. Introduction have been abandoned in current practice. Indeed
In this section, some recommendations are there is great uncertainty in the meaning of pH
made as to the methods to be used in the DPPH values in these predominantly organic (methanol
technique, and in the interpretation of the experi- or ethanol) media.
mental data. They arise in part from trying to dis-
entangle the method as used in a number of recent 4. Reagent concentration, and the use of stan-
papers. It seems that the basis of the original Blois dards
procedure has been lost sight of with the passage In accordance with the normal practice in
of time, and that some pitfalls have therefore spectrophotometry, the initial DPPH concentration
appeared in the application of this apparently in the cuvette should be chosen to give absorbance
straightforward technique. values less than 1.0 (which corresponds to the light
intensity being reduced no more than tenfold in
2. Reaction vessel passing through the sample). This implies a con-
Assuming that the measurements are carried centration for the stock solution in the range 50 to
out using standard 1-cm pathlength spectrophoto- 100 M ( 7). In the originating paper (Blois,
meter cuvettes, with a maximum working volume 1958) it was noted that the stock solutions of this
of 4 ml, then for the optimum analytical accuracy stable free radical do slowly deteriorate; it
the mixtures should of 2 mL DPPH solution and 2 was therefore recommended to use an automatic
mL of the reductant, unless the amounts available burette with a nitrogen atmosphere and covered
preclude this. The common practice of using smaller with aluminium foil, whereby the loss of free
volumes in either case (such as 0.1 mL plus 3.9 radical activity may be reduced about 2-4 per cent
mL, or vice versa) reduces the accuracy of the per week.
relative volumes. Since the absorption is well into The substrate concentrations may initially
the visible region ( 5) then it is possible to use be chosen over a wide range to scan the titration
cheap plastic disposable cuvettes, which are not plot, but when the approximate end-point has been
attacked by the solvents most commonly used found then the values should be spaced evenly up
here (methanol or ethanol) (Bondet et al., 1997). to twice the end-point value to define the two linear
Numbers of reactions, representing the points sections of the plot (Figure 1).
along the titration plot (Figure 1), can thus be It should also be noted that when the molar
carried out in parallel. This enables the progress of mass of the substrate is known, the practice of
Use of DPPH to estimate antioxidant activity
Molyneux, P.

working in terms of masses (grams, milligrams, et al., 1997; Brand-Williams et al., 1995; Gomez-
etc) rather than molar units completely obscures Alonso et al., 2003; Lebeau et al., 2000; S a nchez-
the interpretation of the data on a molecular basis Moreno et al., 1999), 516 nm (Schwarz et al., 2001),
(S a nchez-Moreno et al., 1998; S a nchez-Moreno 517 nm (Blois, 1958; Lu and Foo, 2000; Zhu et al.,
et al., 1999), and requires the results to be re- 2002), 518 nm (Leit a o et al., 2002), and 520 nm
calculated using the relative molar mass Mr, for (Kim et al., 2002). However, in practice, given that
DPPH (C18H12N5O6: Mr = 394.33). The use of a only the peak is a maximum, that is, round topped,
a single mass-in-volume concentration does not and that the absolute absorbance values are not
help to elucidate the structural basis of the antio- important, the wavelength can be set to that giving
xidant activity, since it provides at most only two the maximum absorbance in the instrument that is
points on the titration curve (Yepez et al., 2002). used.
Likewise, to work in terms of numbers of free Similarly, although it is general practice to
radicals (Schwarz et al., 2001) necessitates the use use a spectrophotometer to determine the absorb-
of the Avogadro number at some stage to bring the ance, it should be possible to use a simpler and
values on to a mole basis. cheaper colorimeter with the filter chosen to give
In the case of mixtures of defined substances, the maximum absorbance with DPPH solutions.
the end-point result will be the sum of that from
the individual components. In the case of complex 6. Reaction time
mixtures such as plant extracts, the results should In the original method a reaction time of 30
be expressed as DPPH equivalents per gram of minutes was recommended, and this has been
material; this would be similar to the expression followed in more recent work (Kim et al., 2002).
of the capacity values for ion-exchange resins. Shorter times have also been used, such as 5 mi-
In all these titrations, it is good practice to nutes (Lebeau et al., 2000), or 10 minutes (Schwarz,
use standards or positive controls alongside the et al., 2001). However, in view of the fact that the
main sample under study. Suitable standards that rate of reaction varies widely among substrates
are widely used are ascorbic acid (Vitamin C) (Brand-Williams et al., 1995; Bondet et al., 1997),
(Brand-Williams et al., 1995; Kim et al., 2002; the best practice seems to be to follow the reaction
Lu and Foo, 2000; S a nchez-Moreno et al., 1998; until it has gone to completion (plateau) (Lu et
S a nchez-Moreno et al., 1999) and -tocopherol al., 2000; S a nchez-Moreno et al., 1999; Yepez et
(Vitamin E) (Guo et al., 2001; Lu and Foo, 2000; al., 2002). The rate of reaction has also been pro-
S a nchez-Moreno et al., 1998; S a nchez-Moreno posed as a further parameter to characterise the
et al., 1999). These serve to check that the pro- antioxidant activity (S a nchez-Moreno et al., 1998;
cedures are working correctly. Thus, the fact that, S a nchez-Moreno et al., 1999).
in studies of the antioxidative activity of oolong
tea extracts (Zhu et al., 2002), the titration plots 7. Plotting the data
(percent quenching Q versus sample concentra- The simplest approach in interpreting the
tion - see 7 below) obtained with both with data is to plot absorbance against substrate con-
ascorbic acid and with the main samples do not centration, extending the concentration range
pass through the origin, casts some doubt on the beyond the end-point to define the subsequent
results obtained with the tea extracts themselves. section of the plot so that the intersection point
may be defined most accurately (Figure 1); this
5. Absorbance measurements - wavelength and would allow for any residual colour from the re-
instrument used duced DPPH, as well as any inherent absorbance
The working wavelength of maximum ab- from the substrate itself at the working wavelength.
sorbance, max, to be used for the absorbance mea- The substrate concentrations used should, for
surements is given variously as 515 nm (Bondet definiteness, be those that would be in the reaction
Use of DPPH to estimate antioxidant activity
217 Molyneux, P.

cuvette in the absence of any DPPH. Alternatively, (Bondet et al, 1997; Brand-Williams et al., 1995;
the amount (moles) of substrate added to the re- Lebeau et al., 2000; S a nchez-Moreno et al., 1999),
action vessel may be used (Figure 1). an additive constant invariably creeps into the
An alternative method that is commonly Beers law relation (eqn [7]), which is thus pre-
used is to work in terms of the percentage re- sented in the form:
duction of the DPPH, Q, sometime referred to as
inhibition or quenching, which is defined by A = AI + c L [8]

Q = 100 (A0 - Ac)/A0 [6] where the value of the intercept AI lies in the range
-3
1-310 absorbance units. This presumably arises
where A0 is the initial absorbance and Ac is the value from a too literal interpretation of the results of a
for added sample concentration c. This value of computer program for linear regression on the data.
Ac should be that in the cuvette (or other mixing There should be a strict proportionality [equation
vessel) in the absence of any DPPH, and should 7] between A and c so long as, following standard
take into account the dilution of the original practice, the instrument is zeroed with solvent in
sample solution by the added DPPH solution. a matching cuvette for each sample reading. The
Sometimes the A0 value is referred to as that of the origin (c = 0, A = 0) is thus a multiple experimental
control, that is, in the absence of any sample, point (once for each sample absorbance reading);
such as may be used to confirm the stability of the which justifies forcing the linear regression line to
measuring system. It is also presumed that the go through the origin, with AI = 0.
total concentration of DPPH is kept constant in
the measurement sequence. 8. Presenting the results
In some cases the results are presented in Insofar as the reaction between the DPPH
the form of residual concentrations of DPPH as and the substrate may be expected to be stoichio-
obtained from a calibration curve. Strictly speak- metric, the end-point may then be represented in
ing, this is an unnecessary complication in view of terms of nDPPH, the number of DPPH molecules
the fact the DPPH obeys Beers law in this con- reduced by one molecule of the substrate. This
centration region (Blois, 1958), so that absorb- form of notation also serves as a reminder that the
ances are accurately representative of concentra- result may be expected to depend on the nature of
tions in these comparative measurements. The the scavenging molecule, whether this is DPPH or
combined Beer-Lambert relation takes the stand- another similar molecule.
ard form In cases where the substrate does not have
a defined molar mass, as with plant extracts, the
A = cL [7] results may be presented in equivalences of DPPH
per gram of the extract; this would be analogous to
where is the extinction coefficient, c is the solute the manner in which the activities of ion-exchange
concentration, and L is the path length (conven- resins are quoted.
tionally, 1 cm). The value of for DPPH (in Where, to conform with current practice
-1
methanol or ethanol at 515 nm, with c in mol L ) the EC50 value is used, this value should represent
4
is given variously in the literature as 1.0910 the concentration of the substrate in the reaction
4
(Lebeau et al., 2000), 1.1610 (correcting an error vessel (cuvette) in the absence of DPPH, and the
by a factor of 100) (S a nchez-Moreno et al., 1998; initial DPPH concentration should also be speci-
S a nchez-Moreno et al., 1999), and 1.2510
4
fied; while the stoichiometry value, nDPPH, should
(Bondet et al, 1997; Brand-Williams et al., 1995). also be quoted when the molar mass of the sub-
It is also notable that, in the literature strate is known.
Use of DPPH to estimate antioxidant activity
Molyneux, P.

9. Case study but its applications should to be carried out bear-


The application of the notes above may be ing in mind the basis of the method, and the need
illustrated by studies on the antioxidative activity wherever possible to establish the stoichiometry
of tribromodihydroxybenzyl methyl ether (TDB) for the quenching reaction, so that the antioxidant
(Kim et al., 2002). The method used was stated to activity may be related to the structure of the sub-
follow that of Blois (1958), using ascorbic acid as strate molecule. Likewise, in the case of complex
the standard, and mixing 1 mL of the DPPH solu- mixtures, at least the presumed presence of active
tion with 4 mL of the substrate solution. The results sites in the material should be recognised by work-
were presented as values of IC50, (that is, EC50), ing in terms of equivalences of the DPPH mole-
giving 28.4 M for ascorbic acid and 7.8 M for cule. Finally, the originating paper (Blois, 1958)
TDB. The conclusions were drawn that TDB should be consulted by all who use the method,
has a higher antioxidant activity as compared but read in conjunction with the more recent work
with L-ascorbic acid and that TDB had strong ... of Brand-Williams and colleagues (Bondet et al.,
DPPH radical scavenging activity. 1997; Brand-Williams et al., 1995) which indicates
It would be desirable to put this on a more that the situation may not always be as simple as
quantitative basis, but this is complicated by the that originally presented.
fact that stock DPPH solution was quoted as
having a concentration of 1.5 M, which evidently Acknowledgements
in error ( 4). In addition, the values quoted evi-
dently relate to the sample concentration before This paper was prepared during my tenure
dilution by the factor of 4/5 in the cuvette, which of a Visiting Professorship at the Faculty of Phar-
give the actual concentrations in the cuvette as maceutical Sciences, Prince of Songkla University.
22.7 M and 6.24 M respectively. I am grateful to the Prince of Songkla University
For ascorbic acid, this corresponds to an for financial support, and to Dean Niwat Keaw-
end-point concentration of 45.4 M in the cuvette, pradub and his colleagues in the Faculty of Phar-
and with a 2:1 stoichiometry for this substrate this maceutical Sciences for their hospitality and kind-
requires a corresponding DPPH starting concen- ness, during this time.
tration in the cuvette of 90.8 M, and hence a
stock DPPH concentration (diluted in the cuvette References
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( 4). use of a stable free radical, Nature, 181: 1199-
Regarding the TDB, the ratio of the IC50 1200.
values is 3.64, and with a 2:1 stoichiometry for Bondet, V., Brand-Williams, W. and Berset, C. 1997.
ascorbic acid, this corresponds to that for TDB of Kinetics and mechanisms of antioxidant activity
7.3. This is an anomalously high value, but at least using the DPPH free radical method, Leben-
it gives a quantitative result to augment the purely smittel-Wissenschaft und -Technologie/Food
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Use of a free radical method to evaluate antio-
Conclusions xidant activity, Lebensmittel-Wissenschaft und
-Technologie/Food Science and Technology, 28:
The DPPH method has been widely applied 25-30.
for estimating antioxidant activity in recent years,
Use of DPPH to estimate antioxidant activity
219 Molyneux, P.

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