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J Sci Food Agric 1998, 76, 270È276

A Procedure to Measure the Antiradical Efficiency


of Polyphenols
Concepcio n Sa nchez-Moreno, Jose A Larrauri and Fulgencio Saura-Calixto*
Instituto del Fri o, Departamento de Metabolismo y Nutricion, Consejo Superior de Investigaciones
Cienti Ðcas, Ciudad Universitaria, 28040-Madrid, Spain
(Received 24 April 1997 ; accepted 24 June 1997)

Abstract : The kinetic behaviour of polyphenols common in fruits as free radical


scavengers was studied using 2,2-diphenyl-1-picrylhydrazyl (DPPH~). After addi-
tion of di†erent standard concentrations to DPPHÕ (0É025 g litre~1), the per-
centage of remaining DPPH~ was determined at di†erent times from the absorb-
ances at 515 nm. The percentage remaining DPPH~ against reaction time
followed a multiplicative model equation : ln [DPPH~ ] \ b ln t ] ln a. The
REMdeÐne the antioxidant
slopes of these equations may be useful parameters to
capacity. The steeper the slope, the lower the amount of antioxidant necessary to
decrease by 50% the initial DPPH~ concentration (EC ). This parameter, EC ,
is widely used to measure antioxidant power, but it does 50 not takes into account50
the reaction time. Time needed to reach the steady state to the concentration
corresponding at EC (T ) was calculated, and antiradical efficiency (AE) was
50 EC50
proposed as a new parameter to characterise the antioxidant compounds where
AE \ 1/EC T . It was shown that AE is more discriminatory than EC .
50 EC50 50
AE values are more useful because they also take into account the reaction time.
The results have shown that the order of the AE (]10~3) in the compounds
tested was : ascorbic acid (11É44) [ ca†eic acid (2É75) P gallic acid (2É62) [ tannic
acid (0É57) P DL-a-tocopherol (0É52) [ rutin (0É21) P quercetin (0É19) [ ferulic
acid (0É12) P 3-tert-butyl-4-hydroxyanisole, BHA (0É10) [ resveratrol (0É05).
( 1998 SCI.

J Sci Food Agric 76, 270È276 (1998)

Key words : polyphenols ; free radical scavenging ; DPPH~ ; kinetic behaviour ;


antiradical efficiency

INTRODUCTION 1995). Several studies have reported that speciÐc poly-


phenols scavenge superoxide radicals and hydroxyl rad-
There is a considerable amount of epidemiological evi- icals, reduce lipid peroxyl radicals, and inhibit lipid
dence indicating an association between diets rich in peroxidation (Kanner et al 1994 ; Salah et al 1995 ;
fresh fruit and vegetables and a decreased risk of cardio- Vinson and Hontz 1995).
vascular disease and certain forms of cancer. It is gener- Lipid autoxidation is a free radical chain reaction and
ally assumed that the active dietary constituents could be described in terms of initiation, propagation
contributing to these protective e†ects are the anti- and termination processes. According to their mode of
oxidants (vitamins, carotenoids, sterols). Recent work is action, antioxidants may be classiÐed as free radical ter-
highlighting the role of the polyphenolic components of minators, chelators of metal ions capable of catalysing
the higher plants as antioxidant, antimutagenic, anti- lipid oxidation, or as oxygen scavengers that react with
inÑammatory and antimicrobial (Tamura and Yama- oxygen in closed systems (Shahidi and Wanasundara
gami 1994 ; Jacob 1995 ; Lee et al 1995 ; Yen and Chen 1992).
Oxidation is one of the most important processes of
* To whom correspondence should be addressed. the food deterioration because it may a†ect food safety,
Contract/grant sponsor : Comunidad de Madrid. colour, Ñavour and texture. Antioxidants may protect
Contract/grant number : 06G/048/96. food quality by preventing oxidative deterioration of
270
( 1998 SCI. J Sci Food Agric 0022-5142/98/$17.50. Printed in Great Britain
Antiradical efÐciency of polyphenols 271

TABLE 1
Kinetic behaviour of the selected standards

Standards Concentration Slope Correlation Remaining


(g Antioxidant (% DPPH~ min~1) coefÐcients DPPH~ at the
kg~1 DPPH~) steady state (%)

Gallic acid 10 [0É151 [0É870 75É17


17 [0É140 [0É818 65É99
50 [0É277 [0É862 24É86
100 [0É691 [0É962 6É96
171 [0É949 [0É969 5É80
Tannic acid 10 [0É074 [0É797 83É65
30 [0É082 [0É765 70É31
50 [0É098 [0É756 57É76
75 [0É169 [0É847 31É96
100 [0É254 [0É930 19É54

Caffeic acid 50 [0É219 [0É851 69É17


60 [0É274 [0É889 61É16
141 [0É413 [0É925 25É41
161 [0É375 [0É894 22É06
181 [1É039 [0É912 7É09

Ascorbic acid 18 [0É080 [0É869 87É39


30 [0É083 [0É821 82É76
50 [0É207 [0É815 64É41
80 [0É386 [0É841 49É22
130 [0É528 [0É865 34É03
177 [1É103 [0É892 16É04

Quercetin 10 [0É079 [0É816 83É79


40 [0É063 [0É721 65É52
80 [0É074 [0É804 53É09
100 [0É204 [0É937 27É95
151 [0É560 [0É966 9É48
201 [1É055 [0É945 7É84

BHA 18 [0É018 [0É984 95É22


54 [0É100 [0É991 70É45
90 [0É196 [0É977 51É51
181 [0É473 [0É971 19É12
905 [0É810 [0É972 7É46
1810 [0É941 [0É954 5É98

Rutin 50 [0É067 [0É900 72É36


100 [0É121 [0É983 58É27
151 [0É253 [0É991 33É22
201 [0É424 [0É989 19É06
502 [0É645 [0É943 7É88

Ferulic acid 19 [0É048 [0É838 88É02


100 [0É084 [0É944 68É55
195 [0É176 [0É983 42É22
803 [0É549 [0É997 13É74
1406 [0É662 [0É991 9É12
1950 [0É679 [0É984 8É21

DL-a-Tocopherol 100 [0É127 [0É885 74É62


201 [0É259 [0É901 50É21
301 [0É430 [0É933 24É87
402 [0É502 [0É934 17É11
502 [0É998 [0É966 4É96
272 C Sa nchez-Moreno, J A L arrauri, F Saura-Calixto

TABLE 1ÈContinued

Standards Concentration Slope Correlation Remaining


(g Antioxidant (% DPPH~ min~1) coefÐcients DPPH~ at the
kg~1 DPPH~) steady state (%)

Resveratrol 20 [0É065 [0É968 73É86


201 [0É077 [0É948 66É55
502 [0É204 [0É991 32É63
803 [0É316 [0É990 18É14
2008 [0É483 [0É967 10É06

lipids (Kinsella et al 1993 ; Noguchi et al 1994). Brand-Williams et al (1995). An aliquot of methanol


Several methods to determine free radical scavenging (0É1 ml), solution containing di†erent standard concen-
have been reported, but those utilising the xanthineÈ trations (see Table 1) was added to 3É9 ml of DPPH~
xanthine oxidase system (Hayashi et al 1988), phenazine 0É025 g litre~1 in methanol prepared daily. Absorb-
methosulphate-NADH system (Robak and Gryglewski ances at 515 nm were measured at di†erent time inter-
1988) and the reaction with 2,2-diphenyl-1-pic- vals on a Perkin Elmer UVÈVis model Lambda 12
rylhydrazyl, DPPH~ (Yoshida et al 1989 ; Shimada et al spectrophotometer until the reaction reached a plateau.
1992 ; Yen and Hsieh 1995 ; Yen and Chen 1995 ; Brand- The DPPH~ concentration in the reaction medium
Williams et al 1995), have received more attention. was calculated from the following calibration curve,
This last method is based on the measurement of determined by linear regression :
free radical scavenging of the antioxidant compounds
A \ 2935É68[DPPH~] [ 2É18 ] 10~3
using DPPH~ radical, and the authors used di†erent 515 nm T
initial DPPH~ concentrations (0É415 g litre~1 to where [DPPH~] was expressed as g litre~1 r \ 0É999
T
0É025 g litre~1) and reaction times (30 min or time at The percentage of remaining DPPH~ (% DPPH~ )
REM
the steady state). Nevertheless, a kinetic model for a was calculated as follows :
better understanding of the antioxidant behaviour was
%DPPH~ \ [DPPH~] /[DPPH~]
not reported. REM T T/0
The objective of this work was to study the kinetic The percentage of remaining DPPH~ against the
behaviour of the free radical scavenging of some poly- standard concentration was then plotted to obtain the
phenols common in fruits and to select kinetic param- amount of antioxidant necessary to decrease the initial
eters useful to determine their antioxidant efficiency. DPPH~ concentration by 50%. The time needed to
reach the steady state to EC concentration (T ) was
50 EC50
calculated graphically.
EXPERIMENTAL Taking into account that both, EC and T , a†ect
50 EC50
the antiradical capacity, a new parameter : Antiradical
Standards and reagents efficiency (AE), which combines these two factors, was
deÐned :
The following standards were used : ca†eic acid, ferulic
acid, gallic acid, quercetin, rutin, tannic acid, DL-a- AE \ 1/EC T
50 EC50
tocopherol and resveratrol, all from Sigma Chemical Co
(St Louis MO, USA). Ascorbic acid was from Panreac Statistical analysis
Qui mica, SA, (Barcelona, Spain). 3-tert-Butyl-4-hdy-
roxyanisole, BHA was from Merck Farma y Qui mica, Data were analysed by an analysis of variance
SA, (Madrid, Spain). These substances are common in (P O 0É05) and the means separated by DuncanÏs multi-
fruits, being derived from phenolic acids, Ñavonoids, ple range test. Results were processed by the computer
stilbenes, hydroxycinnamic acids and tannins. programmes : Excel 4.0 and Statgraphics 5.0.
2,2-Diphenyl-1-picrylhydrazyl (DPPH~) was pur-
chased from Sigma-Aldrich Qui mica, SA, (Madrid,
Spain), and methanol was from Panreac Qui mica, SA. RESULTS AND DISCUSSION
All of the reagents used were of analytical quality.
Phenolic antioxidants (PheOH) are free radical termina-
Free radical scavenging method tors. This activity depends mainly on di†erent structural
features such as OÈH bound dissociation energy, reso-
The e†ect of each antioxidant on DPPH~ radical was nance delocalisation of the phenol radical (PheO~) and
estimated according to the procedure described by steric hindrance derived from bulky groups substituting
Antiradical efÐciency of polyphenols 273

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

Standards EC (g Antioxidant Ranges of times at T a,b (min) ClassiÐcation


50 EC50
kg~1 DPPH~)a the steady state (min)
for the standard
concentrations

Gallic acid 26 ^ 1 5É0È29É3 14É69 ^ 1É10 Intermediate


Tannic 59 ^ 3 6É5È60É0 29É55 ^ 1É60 Intermediate
Caffeic acid 69 ^ 7 4É0È20É5 5É26 ^ 0É31 RapidÈintermediate
Ascorbic acid 76 ^ 7 1É0È1É5 1É15 ^ 0É08 Rapid
Quercetin 84 ^ 6 6É0È75É0 63É28 ^ 3É15 Slow
BHA 93 ^ 6 19É5È119É5 103É85 ^ 7É21 Slow
Rutin 102 ^ 9 23É5È60É0 46É00 ^ 3É20 Slow
Ferulic acid 163 ^ 10 8É0È58É5 49É74 ^ 4É18 Slow
DL-a-Tocopherol 201 ^ 11 7É0È19É3 9É52 ^ 0É71 Intermediate
Resveratrol 337 ^ 12 41É5È90É0 60É46 ^ 4É25 Slow

a Each value is the mean ^ standard deviation.


b Time needed to reach the steady state to EC concentration.
50
274 C Sa nchez-Moreno, J A L arrauri, F Saura-Calixto

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

Standards Slope Correlation Antiradical ClassiÐcation


(]10~3) coefÐcients efÐciencya (]10~3)

Gallic acidb [17É880 [0É950 2É62 Medium


Tannic acidb [15É893 [0É985 0É57 Low
Caffeic acidb [12É784 [0É945 2É75 Medium
Ascorbic acidb [9É981 [0É990 11É44 Very high
Quercetinb [13É634 [0É974 0É19 Low
BHAc [0É666 [0É981 0É10 Low
Rutinb [5É096 [0É962 0É21 Low
Ferulic acidc [0É573 [0É969 0É12 Low
DL-a-Tocopherolb [5É731 [0É972 0É52 Low
Resveratrolb [1É107 [0É937 0É05 Low

a Antiradical efÐciency \ 1/EC T .


50 EC50
b Exponential model, ln [DPPH~ ] \ b [Antioxidant] ] a.
REM
c Multiplicative model, ln [DPPH~ ] \ b ln [Antioxidant] ] ln a.
REM
Antiradical efÐciency of polyphenols 275

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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