Professional Documents
Culture Documents
The Use of The Stable Free Radical Diphenylpicryl-Hydrazyl (DPPH) For Estimating Antioxidant Activity
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
Songklanakarin J. Sci. Technol., 2004, 26(2) : 211-219
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.
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
Songklanakarin J. Sci. Technol. Use of DPPH to estimate antioxidant activity
Vol. 26 No. 2 Mar.-Apr. 2004 212 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.,
Songklanakarin J. Sci. Technol. Use of DPPH to estimate antioxidant activity
Vol. 26 No. 2 Mar.-Apr. 2004 214 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
Songklanakarin J. Sci. Technol. Use of DPPH to estimate antioxidant activity
Vol. 26 No. 2 Mar.-Apr. 2004 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
Songklanakarin J. Sci. Technol. Use of DPPH to estimate antioxidant activity
Vol. 26 No. 2 Mar.-Apr. 2004 216 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
Songklanakarin J. Sci. Technol. Use of DPPH to estimate antioxidant activity
Vol. 26 No. 2 Mar.-Apr. 2004 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.
Songklanakarin J. Sci. Technol. Use of DPPH to estimate antioxidant activity
Vol. 26 No. 2 Mar.-Apr. 2004 218 Molyneux, P.