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TABLE 1
Kinetic behaviour of the selected standards
TABLE 1ÈContinued
hydrogen in the aromatic ring (Shahidi and Naczk compare the kinetic behavior of di†erent antioxidants
1995). The rate constants of the reaction of PheOH with at the same concentration.
free radicals will indicate the order of reactivity. The concentration of antioxidant [PheOH] needed to
The main reaction would be : decrease by 50% the initial substrate concentration
(EC ) is a parameter widely used to measure the anti-
DPPH~ ] PheOH ] DPPHH 50
oxidant power (Robak and Gryglewski 1988 ; Yoshida
et al 1989 ; Cuvelier et al 1992 ; Gieseg and Esterbauer
] [PheO~(I) % PheO~(II) % PheO~(III) . . .] (1)
1994 ; Kanner et al 1994 ; Vinson et al 1995a). The lower
where (I), (II), (III) . . . are resonance structures. the EC , the higher the antioxidant power. The values
50
The residual concentration of DPPH~ will depend found for our standards are shown in Table 2 and they
exclusively on the concentration and structure of the agree with those values reported by Brand-Williams
phenolic compound, because there are two theoretical et al (1995) as follows : gallic acid (34), ca†eic acid
termination reactions : (50), ascorbic acid (121), BHA (110), ferulic acid (212),
DL-a-tocopherol (273). The antioxidant power se-
DPPH~ ] DPPH~ ] DPPH [ DPPH (2) quence obtained (Table 2) also agrees with the data
reported by other authors : tannic acid [
DPPH~ ] PheO~ ] DPPH [ PheO (3) quercetin [ rutin [ ascorbic acid [ DL-a-tocopherol
(Robak and Gryglewski 1988 ; Yoshida et al 1989 ;
but eqn (2) is forbidden due to steric hindrance and eqn
Cuvelier et al 1992 ; Vinson and Hontz 1995 ; Vinson
(3) may occur in some cases, but it also may be for-
et al 1995a,b).
bidden depending on molecular PheOH and aromatic
Many attempts to explain the structureÈactivity
ring substituent volumes. Equation (3) will compete
relationship of some polyphenols have been reported in
with the PheO~ coupling termination reaction :
the literature. It is known that the monophenols are less
PheO~ ] PheO~ ] PheO [ PheO (4) efficient than the polyphenols, but in gallic acid the
inductive e†ect of the three hdyroxyl groups is an
Okuda (1993) reported a coupling of alkyl gallate rad- important factor that may enhance the activity. Another
icals between C-centred galloyl radicals, after scav- factor that increases substantially the antioxidant power
enging DPPH~. of monophenols is the methoxy substitution such as
Table 1 shows the kinetic behaviour obtained for the occurs in BHA. In the case of phenolic acids methoxy
standards at di†erent concentations and they follow substitution was far from equivalent to the addition of a
a general multiplicative model : ln [DPPH~ ] \ b hydroxyl group hence ferulic acid remained substan-
REM
ln time ] ln a, where b is the slope and a is the inter- tially less efficient than ca†eic acid (Cuvelier et al 1992 ;
cept. High correlation coefficients were obtained. The Shahidi and Wanasundara 1992 ; Salah et al 1995). The
higher the concentration, the steeper the slopes in the accessibility of the radical centre of DPPH~ to each
models and the lower the remaining DPPH~. Conse- polyphenol could also inÑuence the order of the anti-
quently, the slopes may be useful parameters to oxidant power obtained (Yoshida et al 1989).
TABLE 2
Standard concentration needed to decrease by 50% the initial DPPH~ concentration (EC ) and their kinetic
50
classiÐcation
A previous antioxidant kinetic classiÐcation based on of the antioxidant compound as follows : \5 min
the time to reach a steady state, has been reported (rapid) ; 5È30 min (intermediate) and [30 min (slow).
(Brand-Williams et al 1995) but they have not con- Figure 2 illustrates several examples of rapid, interme-
sidered that the time at the steady state depends on the diate and slow kinetic behaviour of some of the com-
antioxdiant concentration as indicated in Table 2. pounds studied.
To avoid this, we deÐne the parameter : T as the The percentage of remaining DPPH~ concentration
EC50
time needed to reach a steady state at the concentration against antioxidant compound concentrations may be
corresponding to EC . This parameter was obtained expressed by exponential models : ln [DPPH~ ] \ b
50 REM
by plotting the times at the steady state against the con- [antioxidant] ] a in many of the cases (Table 3).
centration for each antioxidant compound, as is illus- Nevertheless, BHA and ferulic acid kinetics were
trated in Fig 1 for DL-a-tocopherol. Based on the T expressed by a multiplicative model : ln [DPPH~ ] \ b
EC50 REM
values (Table 2) we have classiÐed the kinetic behaviour ln [antioxidant] ] ln a. This could suggest a di†erent
free radical scavenging behaviour by these compounds.
High correlation coefficients (r [ 0É93) were obtained
for all models. The steeper the slope, the lower the EC
50
and the higher the antiradical power. BHA and ferulic
acid slopes were not in agreement with the rest because
their models were di†erent.
There is little information on the kinetic behaviour of
the antioxidant compounds in the oxidation process
(Robak and Gryglewski 1988 ; Gieseg and Esterbauer
1994). Thus, Halliwell (1990) reported that the anti-
oxidant power results Ðrst from the capacity to prevent
the autoxidation of free radical-mediated oxidation of
the substrate in low concentration and second, that the
resulting radical after scavenging must be stable. In our
model we consider that “low timeÏ should be added to
the Ðrst condition resulting “low concentration, and low
timeÏ because the reaction time is also important to
deÐne antioxidant capacity.
We propose a new parameter : “Antiradical efficiencyÏ
(AE) which involves the potency (1/EC ) and the reac-
50
tion time (T ). The lower the EC , the lower the
EC50 50
T and the higher the AE.
EC50
An example to illustrate the signiÐcance of the AE
Fig 1. Determination of the time needed to reach the steady can be deduced from Table 2. BHA and ascorbic acid
state to EC concentration. EC were comparable nevertheless, the T of BHA
50 50 EC50
TABLE 3
Antiradical efficiencies of the standards
Fig 2. Several examples of kinetic behaviour of three standards (concentrations expressed as g antioxidant kg~1 DPPH~) :
(a) ascorbic acid (rapid) ; (b) DL-a-tocopherol (intermediate) ; and (c) rutin (slow).
was 90É3 times higher than that for ascorbic acid. This CONCLUSIONS
fact shows that the AE is a more adequate parameter
than the widely used EC , which is not completely dis- Kinetics of polyphenols followed a multiplicative model
50
criminatory to select the antioxidant compound. and slopes were related to their antiradical activity.
According to the following classiÐcation Time needed to reach the steady state to the concen-
(AE O 1 ] 10~3 low ; 1 ] 10~3 \ AE O 5 ] 10~3 tration corresponding at EC (T ) and “antiradical
50 EC50
medium ; 5 ] 10~3 \ AE O 10 ] 10~3 high and efficiencyÏ AE were proposed as new parameters to
AE [ 10 ] 10~3 very high), ascorbic acid AE was very characterise the antioxidant compounds.
high, ca†eic and gallic acids AE were medium and the AE was very high for ascorbic acid, medium for gallic
rest were low. The order of the AE for the compounds and tannic acids and low for quercetin, BHA, rutin,
tested was : ascorbic acid [ ca†eic acid P gallic acid [ ferulic acid, DL-a-tocopherol and resveratrol.
tannic acid P DL-a-tocopherol [ rutin P quercetin [
ferulic acid P BHA [ resveratrol. ACKNOWLEDGEMENT
Further research on the radical-scavenging of natural
plant extracts by using the methodology proposed in One of the authors (C S-M) wishes to thank the Com-
this paper is needed. unidad de Madrid for the concession of a predoctoral
276 C Sa nchez-Moreno, J A L arrauri, F Saura-Calixto
fellowship. The sponsorship of the project by the Com- Salah N, Miller N J, Paganga G, Tijburg L, Bolwell G P,
unidad de Madrid : 06G/048/96 is also acknowledged. Rice-Evans C 1995 Polyphenolic Ñavanols as scavengers of
aqueous phase radicals and as chain-breaking antioxidants.
Arch Biochem Biophys 332 (2) 339È346.
Shahidi F, Naczk M 1995 Food phenolics : an overview. In :
Food Phenolics : Sources, Chemistry, E†ects and Applica-
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