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The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant
activity
Philip Molyneux
Abstract
Molyneux, P.
212
[1]
where ZH is the reduced form and A is free radical produced in this first step. This latter radical
will then undergo further reactions which control
the overall stoichiometry, that is, the number of
molecules of DPPH reduced (decolorised) by one
molecule of the reductant.
The reaction [1] is therefore intended to
provide the link with the reactions taking place in
an oxidising system, such as the autoxidation of a
lipid or other unsaturated substance; the DPPH
2: Diphenylpicrylhydrazine (nonradical)
213
[2]
RS + RS = RS SR
[3]
This therefore leads to the observed reduction of two molecules of DPPH by two molecules
of cysteine, that is, a 1:1 stoichiometry.
If however the molecule has two adjacent
sites for hydrogen abstraction which are internally
connected, as is the case with ascorbic acid (Vitamin C), then there may be a further hydrogen
abstraction reaction after the first one:
HO OH
HO O
| |
| |
HO O
O O
|| | |
| |
214
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).
215
216
et al., 1997; Brand-Williams et al., 1995; GomezAlonso et al., 2003; Lebeau et al., 2000; S a nchezMoreno et al., 1999), 516 nm (Schwarz et al., 2001),
517 nm (Blois, 1958; Lu and Foo, 2000; Zhu et al.,
2002), 518 nm (Leit a o et al., 2002), and 520 nm
(Kim et al., 2002). However, in practice, given that
the peak is a maximum, that is, round topped,
and that the absolute absorbance values are not
important, the wavelength can be set to that giving
the maximum absorbance in the instrument that is
used.
Similarly, although it is general practice to
use a spectrophotometer to determine the absorbance, it should be possible to use a simpler and
cheaper colorimeter with the filter chosen to give
the maximum absorbance with DPPH solutions.
6. Reaction time
In the original method a reaction time of 30
minutes was recommended, and this has been
followed in more recent work (Kim et al., 2002).
Shorter times have also been used, such as 5 minutes (Lebeau et al., 2000), or 10 minutes (Schwarz,
et al., 2001). However, in view of the fact that the
rate of reaction varies widely among substrates
(Brand-Williams et al., 1995; Bondet et al., 1997),
the best practice seems to be to follow the reaction
until it has gone to completion (plateau) (Lu et
al., 2000; S a nchez-Moreno et al., 1999; Yepez et
al., 2002). The rate of reaction has also been proposed as a further parameter to characterise the
antioxidant activity (S a nchez-Moreno et al., 1998;
S a nchez-Moreno et al., 1999).
7. Plotting the data
The simplest approach in interpreting the
data is to plot absorbance against substrate concentration, extending the concentration range
beyond the end-point to define the subsequent
section of the plot so that the intersection point
may be defined most accurately (Figure 1); this
would allow for any residual colour from the reduced DPPH, as well as any inherent absorbance
from the substrate itself at the working wavelength.
The substrate concentrations used should, for
definiteness, be those that would be in the reaction
217
[6]
[7]
[8]
218
9. Case study
The application of the notes above may be
illustrated by studies on the antioxidative activity
of tribromodihydroxybenzyl methyl ether (TDB)
(Kim et al., 2002). The method used was stated to
follow that of Blois (1958), using ascorbic acid as
the standard, and mixing 1 mL of the DPPH solution with 4 mL of the substrate solution. The results
were presented as values of IC50, (that is, EC50),
giving 28.4 M for ascorbic acid and 7.8 M for
TDB. The conclusions were drawn that TDB
has a higher antioxidant activity as compared
with L-ascorbic acid and that TDB had strong ...
DPPH radical scavenging activity.
It would be desirable to put this on a more
quantitative basis, but this is complicated by the
fact that stock DPPH solution was quoted as
having a concentration of 1.5 M, which evidently
in error ( 4). In addition, the values quoted evidently relate to the sample concentration before
dilution by the factor of 4/5 in the cuvette, which
give the actual concentrations in the cuvette as
22.7 M and 6.24 M respectively.
For ascorbic acid, this corresponds to an
end-point concentration of 45.4 M in the cuvette,
and with a 2:1 stoichiometry for this substrate this
requires a corresponding DPPH starting concentration in the cuvette of 90.8 M, and hence a
stock DPPH concentration (diluted in the cuvette
by a factor of 5) of 454 M. On the basis of the
extinction coefficient data already listed ( 7),
this cuvette concentration gives an initial absorbance value of about 1, which is a reasonable value
( 4).
Regarding the TDB, the ratio of the IC50
values is 3.64, and with a 2:1 stoichiometry for
ascorbic acid, this corresponds to that for TDB of
7.3. This is an anomalously high value, but at least
it gives a quantitative result to augment the purely
qualitative conclusions that were originally drawn
by the authors (Kim et al., 2002).
Conclusions
The DPPH method has been widely applied
for estimating antioxidant activity in recent years,
but its applications should to be carried out bearing in mind the basis of the method, and the need
wherever possible to establish the stoichiometry
for the quenching reaction, so that the antioxidant
activity may be related to the structure of the substrate molecule. Likewise, in the case of complex
mixtures, at least the presumed presence of active
sites in the material should be recognised by working in terms of equivalences of the DPPH molecule. Finally, the originating paper (Blois, 1958)
should be consulted by all who use the method,
but read in conjunction with the more recent work
of Brand-Williams and colleagues (Bondet et al.,
1997; Brand-Williams et al., 1995) which indicates
that the situation may not always be as simple as
that originally presented.
Acknowledgements
This paper was prepared during my tenure
of a Visiting Professorship at the Faculty of Pharmaceutical Sciences, Prince of Songkla University.
I am grateful to the Prince of Songkla University
for financial support, and to Dean Niwat Keawpradub and his colleagues in the Faculty of Pharmaceutical Sciences for their hospitality and kindness, during this time.
References
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Kinetics and mechanisms of antioxidant activity
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Science and Technology, 30: 609-615.
Brand-Williams, W., Cuvelier, M.E. and Berset, C. 1995.
Use of a free radical method to evaluate antioxidant activity, Lebensmittel-Wissenschaft und
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