J Sci Food Agric 1998, 76, 270276

A Procedure to Measure the Antiradical Efficiency of Polyphenols

Concepcion Sanchez-Moreno, Jose A Larrauri and Fulgencio Saura-Calixto*
Instituto del Fri o, Departamento de Metabolismo y Nutricion, Consejo Superior de Investigaciones Cienti cas, 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 addition of dierent standard concentrations to DPPH (0025 g litre~1), the percentage of remaining DPPH~ was determined at dierent times from the absorbances at 515 nm. The percentage remaining DPPH~ against reaction time followed a multiplicative model equation : ln [DPPH~ ] \ b ln t ] ln a. The REM slopes of these equations may be useful parameters to dene the antioxidant capacity. The steeper the slope, the lower the amount of antioxidant necessary to decrease by 50% the initial DPPH~ concentration (EC ). This parameter, EC , 50 50 is widely used to measure antioxidant power, but it does not takes into account 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 (1144) [ caeic acid (275) P gallic acid (262) [ tannic acid (057) P DL-a-tocopherol (052) [ rutin (021) P quercetin (019) [ ferulic acid (012) P 3-tert-butyl-4-hydroxyanisole, BHA (010) [ resveratrol (005). ( 1998 SCI. J Sci Food Agric 76, 270276 (1998) Key words : polyphenols ; free radical scavenging ; DPPH~ ; kinetic behaviour ; antiradical efficiency

INTRODUCTION There is a considerable amount of epidemiological evidence indicating an association between diets rich in fresh fruit and vegetables and a decreased risk of cardiovascular disease and certain forms of cancer. It is generally assumed that the active dietary constituents contributing to these protective eects are the antioxidants (vitamins, carotenoids, sterols). Recent work is highlighting the role of the polyphenolic components of the higher plants as antioxidant, antimutagenic, antiinammatory and antimicrobial (Tamura and Yamagami 1994 ; Jacob 1995 ; Lee et al 1995 ; Yen and Chen
* To whom correspondence should be addressed. Contract/grant sponsor : Comunidad de Madrid. Contract/grant number : 06G/048/96.

1995). Several studies have reported that specic polyphenols scavenge superoxide radicals and hydroxyl radicals, reduce lipid peroxyl radicals, and inhibit lipid peroxidation (Kanner et al 1994 ; Salah et al 1995 ; Vinson and Hontz 1995). Lipid autoxidation is a free radical chain reaction and could be described in terms of initiation, propagation and termination processes. According to their mode of action, antioxidants may be classied as free radical terminators, chelators of metal ions capable of catalysing lipid oxidation, or as oxygen scavengers that react with oxygen in closed systems (Shahidi and Wanasundara 1992). Oxidation is one of the most important processes of the food deterioration because it may aect food safety, colour, avour and texture. Antioxidants may protect food quality by preventing oxidative deterioration of 270

( 1998 SCI.

J Sci Food Agric 0022-5142/98/$17.50.

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Antiradical efciency of polyphenols

TABLE 1 Kinetic behaviour of the selected standards Standards Concentration (g Antioxidant kg~1 DPPH~) 10 17 50 100 171 10 30 50 75 100 50 60 141 161 181 18 30 50 80 130 177 10 40 80 100 151 201 18 54 90 181 905 1810 50 100 151 201 502 19 100 195 803 1406 1950 100 201 301 402 502 Slope (% DPPH~ min~1) [0151 [0140 [0277 [0691 [0949 [0074 [0082 [0098 [0169 [0254 [0219 [0274 [0413 [0375 [1039 [0080 [0083 [0207 [0386 [0528 [1103 [0079 [0063 [0074 [0204 [0560 [1055 [0018 [0100 [0196 [0473 [0810 [0941 [0067 [0121 [0253 [0424 [0645 [0048 [0084 [0176 [0549 [0662 [0679 [0127 [0259 [0430 [0502 [0998 Correlation coefcients [0870 [0818 [0862 [0962 [0969 [0797 [0765 [0756 [0847 [0930 [0851 [0889 [0925 [0894 [0912 [0869 [0821 [0815 [0841 [0865 [0892 [0816 [0721 [0804 [0937 [0966 [0945 [0984 [0991 [0977 [0971 [0972 [0954 [0900 [0983 [0991 [0989 [0943 [0838 [0944 [0983 [0997 [0991 [0984 [0885 [0901 [0933 [0934 [0966 Remaining DPPH~ at the steady state (%) 7517 6599 2486 696 580 8365 7031 5776 3196 1954 6917 6116 2541 2206 709 8739 8276 6441 4922 3403 1604 8379 6552 5309 2795 948 784 9522 7045 5151 1912 746 598 7236 5827 3322 1906 788 8802 6855 4222 1374 912 821 7462 5021 2487 1711 496

271

Gallic acid

Tannic acid

Caffeic acid

Ascorbic acid

Quercetin

BHA

Rutin

Ferulic acid

DL-a-Tocopherol

272

C Sa nchez-Moreno, J A L arrauri, F Saura-Calixto

TABLE 1Continued Standards Concentration (g Antioxidant kg~1 DPPH~) 20 201 502 803 2008 Slope (% DPPH~ min~1) [0065 [0077 [0204 [0316 [0483 Correlation coefcients [0968 [0948 [0991 [0990 [0967 Remaining DPPH~ at the steady state (%) 7386 6655 3263 1814 1006

Resveratrol

lipids (Kinsella et al 1993 ; Noguchi et al 1994). Several methods to determine free radical scavenging have been reported, but those utilising the xanthine xanthine oxidase system (Hayashi et al 1988), phenazine methosulphate-NADH system (Robak and Gryglewski 1988) and the reaction with 2,2-diphenyl-1-picrylhydrazyl, DPPH~ (Yoshida et al 1989 ; Shimada et al 1992 ; Yen and Hsieh 1995 ; Yen and Chen 1995 ; BrandWilliams et al 1995), have received more attention. This last method is based on the measurement of free radical scavenging of the antioxidant compounds using DPPH~ radical, and the authors used dierent initial DPPH~ concentrations (0415 g litre~1 to 0025 g litre~1) and reaction times (30 min or time at the steady state). Nevertheless, a kinetic model for a better understanding of the antioxidant behaviour was not reported. The objective of this work was to study the kinetic behaviour of the free radical scavenging of some polyphenols common in fruits and to select kinetic parameters useful to determine their antioxidant efficiency. EXPERIMENTAL Standards and reagents The following standards were used : caeic acid, ferulic acid, gallic acid, quercetin, rutin, tannic acid, DL-atocopherol and resveratrol, all from Sigma Chemical Co (St Louis MO, USA). Ascorbic acid was from Panreac Qui mica, SA, (Barcelona, Spain). 3-tert-Butyl-4-hdyroxyanisole, BHA was from Merck Farma y Qui mica, SA, (Madrid, Spain). These substances are common in fruits, being derived from phenolic acids, avonoids, stilbenes, hydroxycinnamic acids and tannins. 2,2-Diphenyl-1-picrylhydrazyl (DPPH~) was purchased from Sigma-Aldrich Qui mica, SA, (Madrid, Spain), and methanol was from Panreac Qui mica, SA. All of the reagents used were of analytical quality. Free radical scavenging method The eect of each antioxidant on DPPH~ radical was estimated according to the procedure described by

Brand-Williams et al (1995). An aliquot of methanol (01 ml), solution containing dierent standard concentrations (see Table 1) was added to 39 ml of DPPH~ 0025 g litre~1 in methanol prepared daily. Absorbances at 515 nm were measured at dierent time intervals on a Perkin Elmer UVVis model Lambda 12 spectrophotometer until the reaction reached a plateau. The DPPH~ concentration in the reaction medium was calculated from the following calibration curve, determined by linear regression : \ 293568[DPPH~] [ 218 ] 10~3 515 nm T where [DPPH~] was expressed as g litre~1 r \ 0999 T The percentage of remaining DPPH~ (% DPPH~ ) REM was calculated as follows : A %DPPH~ \ [DPPH~] /[DPPH~] REM T T/0 The percentage of remaining DPPH~ against the standard concentration was then plotted to obtain the amount of antioxidant necessary to decrease the initial DPPH~ concentration by 50%. The time needed to ) was reach the steady state to EC concentration (T 50 EC50 calculated graphically. , aect Taking into account that both, EC and T 50 EC50 the antiradical capacity, a new parameter : Antiradical efficiency (AE), which combines these two factors, was dened : AE \ 1/EC T 50 EC50 Statistical analysis Data were analysed by an analysis of variance (P O 005) and the means separated by Duncans multiple range test. Results were processed by the computer programmes : Excel 4.0 and Statgraphics 5.0.

RESULTS AND DISCUSSION Phenolic antioxidants (PheOH) are free radical terminators. This activity depends mainly on dierent structural features such as OH bound dissociation energy, resonance delocalisation of the phenol radical (PheO~) and steric hindrance derived from bulky groups substituting

Antiradical efciency of polyphenols hydrogen in the aromatic ring (Shahidi and Naczk 1995). The rate constants of the reaction of PheOH with free radicals will indicate the order of reactivity. The main reaction would be : DPPH~ ] PheOH ] DPPHH ] [PheO~(I) % PheO~(II) % PheO~(III) . . .] (1)

273 compare the kinetic behavior of dierent antioxidants at the same concentration. The concentration of antioxidant [PheOH] needed to decrease by 50% the initial substrate concentration (EC ) is a parameter widely used to measure the anti50 oxidant power (Robak and Gryglewski 1988 ; Yoshida et al 1989 ; Cuvelier et al 1992 ; Gieseg and Esterbauer 1994 ; Kanner et al 1994 ; Vinson et al 1995a). The lower the EC , the higher the antioxidant power. The values 50 found for our standards are shown in Table 2 and they agree with those values reported by Brand-Williams et al (1995) as follows : gallic acid (34), caeic acid (50), ascorbic acid (121), BHA (110), ferulic acid (212), DL-a-tocopherol (273). The antioxidant power sequence obtained (Table 2) also agrees with the data reported by other authors : tannic acid [ quercetin [ rutin [ ascorbic acid [ DL-a-tocopherol (Robak and Gryglewski 1988 ; Yoshida et al 1989 ; Cuvelier et al 1992 ; Vinson and Hontz 1995 ; Vinson et al 1995a,b). Many attempts to explain the structureactivity relationship of some polyphenols have been reported in the literature. It is known that the monophenols are less efficient than the polyphenols, but in gallic acid the inductive eect of the three hdyroxyl groups is an important factor that may enhance the activity. Another factor that increases substantially the antioxidant power of monophenols is the methoxy substitution such as occurs in BHA. In the case of phenolic acids methoxy substitution was far from equivalent to the addition of a hydroxyl group hence ferulic acid remained substantially less efficient than caeic acid (Cuvelier et al 1992 ; Shahidi and Wanasundara 1992 ; Salah et al 1995). The accessibility of the radical centre of DPPH~ to each polyphenol could also inuence the order of the antioxidant power obtained (Yoshida et al 1989).

where (I), (II), (III) . . . are resonance structures. The residual concentration of DPPH~ will depend exclusively on the concentration and structure of the phenolic compound, because there are two theoretical termination reactions : DPPH~ ] DPPH~ ] DPPH [ DPPH DPPH~ ] PheO~ ] DPPH [ PheO (2) (3)

but eqn (2) is forbidden due to steric hindrance and eqn (3) may occur in some cases, but it also may be forbidden depending on molecular PheOH and aromatic ring substituent volumes. Equation (3) will compete with the PheO~ coupling termination reaction : PheO~ ] PheO~ ] PheO [ PheO (4)

Okuda (1993) reported a coupling of alkyl gallate radicals between C-centred galloyl radicals, after scavenging DPPH~. Table 1 shows the kinetic behaviour obtained for the standards at dierent concentations and they follow a general multiplicative model : ln [DPPH~ ] \ b REM ln time ] ln a, where b is the slope and a is the intercept. High correlation coefficients were obtained. The higher the concentration, the steeper the slopes in the models and the lower the remaining DPPH~. Consequently, the slopes may be useful parameters to

TABLE 2 Standard concentration needed to decrease by 50% the initial DPPH~ concentration (EC ) and their kinetic 50 classication Standards EC (g Antioxidant 50 kg~1 DPPH~)a Ranges of times at the steady state (min) for the standard concentrations 50293 65600 40205 1015 60750 1951195 235600 80585 70193 415900 a,b (min) T EC50 Classication

Gallic acid Tannic Caffeic acid Ascorbic acid Quercetin BHA Rutin Ferulic acid DL-a-Tocopherol Resveratrol

26 ^ 1 59 ^ 3 69 ^ 7 76 ^ 7 84 ^ 6 93 ^ 6 102 ^ 9 163 ^ 10 201 ^ 11 337 ^ 12

1469 ^ 110 2955 ^ 160 526 ^ 031 115 ^ 008 6328 ^ 315 10385 ^ 721 4600 ^ 320 4974 ^ 418 952 ^ 071 6046 ^ 425

Intermediate Intermediate Rapidintermediate Rapid Slow Slow Slow Slow Intermediate Slow

a Each value is the mean ^ standard deviation. b Time needed to reach the steady state to EC concentration. 50

274 A previous antioxidant kinetic classication based on the time to reach a steady state, has been reported (Brand-Williams et al 1995) but they have not considered that the time at the steady state depends on the antioxdiant concentration as indicated in Table 2. To avoid this, we dene the parameter : T as the EC50 time needed to reach a steady state at the concentration corresponding to EC . This parameter was obtained 50 by plotting the times at the steady state against the concentration for each antioxidant compound, as is illustrated in Fig 1 for DL-a-tocopherol. Based on the T EC50 values (Table 2) we have classied the kinetic behaviour

C Sa nchez-Moreno, J A L arrauri, F Saura-Calixto of the antioxidant compound as follows : \5 min (rapid) ; 530 min (intermediate) and [30 min (slow). Figure 2 illustrates several examples of rapid, intermediate and slow kinetic behaviour of some of the compounds studied. The percentage of remaining DPPH~ concentration against antioxidant compound concentrations may be expressed by exponential models : ln [DPPH~ ] \ b REM [antioxidant] ] a in many of the cases (Table 3). Nevertheless, BHA and ferulic acid kinetics were expressed by a multiplicative model : ln [DPPH~ ] \ b REM ln [antioxidant] ] ln a. This could suggest a dierent free radical scavenging behaviour by these compounds. High correlation coefficients (r [ 093) 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 dierent. 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 antioxidant power results rst 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 time should be added to the rst condition resulting low concentration, and low time because the reaction time is also important to dene antioxidant capacity. We propose a new parameter : Antiradical efficiency (AE) which involves the potency (1/EC ) and the reac50 ). The lower the EC , the lower the tion time (T 50 EC50 T and the higher the AE. EC50 An example to illustrate the signicance of the AE can be deduced from Table 2. BHA and ascorbic acid EC were comparable nevertheless, the T of BHA 50 EC50

Fig 1. Determination of the time needed to reach the steady state to EC concentration. 50

TABLE 3 Antiradical efficiencies of the standards Standards Slope (]10~3) [17880 [15893 [12784 [9981 [13634 [0666 [5096 [0573 [5731 [1107 Correlation coefcients [0950 [0985 [0945 [0990 [0974 [0981 [0962 [0969 [0972 [0937 Antiradical efciencya (]10~3) 262 057 275 1144 019 010 021 012 052 005 Classication

Gallic acidb Tannic acidb Caffeic acidb Ascorbic acidb Quercetinb BHAc Rutinb Ferulic acidc DL-a-Tocopherolb Resveratrolb

Medium Low Medium Very high Low Low Low Low Low Low

a Antiradical efciency \ 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 efciency 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 903 times higher than that for ascorbic acid. This fact shows that the AE is a more adequate parameter than the widely used EC , which is not completely dis50 criminatory to select the antioxidant compound. According to the following classication (AE O 1 ] 10~3 low ; 1 ] 10~3 \ AE O 5 ] 10~3 medium ; 5 ] 10~3 \ AE O 10 ] 10~3 high and AE [ 10 ] 10~3 very high), ascorbic acid AE was very high, caeic and gallic acids AE were medium and the rest were low. The order of the AE for the compounds tested was : ascorbic acid [ caeic acid P gallic acid [ tannic acid P DL-a-tocopherol [ rutin P quercetin [ ferulic acid P BHA [ resveratrol. Further research on the radical-scavenging of natural plant extracts by using the methodology proposed in this paper is needed.

CONCLUSIONS Kinetics of polyphenols followed a multiplicative model and slopes were related to their antiradical activity. Time needed to reach the steady state to the concen) and antiradical tration corresponding at EC (T 50 EC50 efficiency AE were proposed as new parameters to characterise the antioxidant compounds. AE was very high for ascorbic acid, medium for gallic and tannic acids and low for quercetin, BHA, rutin, ferulic acid, DL-a-tocopherol and resveratrol. ACKNOWLEDGEMENT One of the authors (C S-M) wishes to thank the Comunidad de Madrid for the concession of a predoctoral

276 fellowship. The sponsorship of the project by the Comunidad de Madrid : 06G/048/96 is also acknowledged.

C Sa nchez-Moreno, J A L arrauri, F Saura-Calixto

Salah N, Miller N J, Paganga G, Tijburg L, Bolwell G P, Rice-Evans C 1995 Polyphenolic avanols as scavengers of aqueous phase radicals and as chain-breaking antioxidants. Arch Biochem Biophys 332 (2) 339346. Shahidi F, Naczk M 1995 Food phenolics : an overview. In : Food Phenolics : Sources, Chemistry, Eects and Applications. Technomic Publishing Co, Pennsylvania, USA, pp 1 4. Shahidi F, Wanasundara P K J P D 1992 Phenolic antioxidants. Crit Rev Food Sci Nutr 32 (1) 67103. Shimada K, Fujikawa K, Yahara K, Nakamura T 1992 Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. J Agric Food Chem 40 (6) 945948. Tamura H, Yamagami A 1994 Antioxidative activity of monoacylated anthocyanins isolated from muscat bailey a grape. J Agric Food Chem 42 (8) 16121615. Vinson J A, Hontz B A 1995 Phenol antioxidant index : comparative antioxidant eectiveness of red and white wines. J Agric Food Chem 43 (2) 401403. Vinson J A, Dabbagh Y A, Serry M M, Jang J 1995a Plant avonoids, especially tea avonols, are powerful antioxidants using an in vitro oxidation model for heart disease. J Agric Food Chem 43 (11) 28002802. Vinson J A, Jang J, Dabbagh Y A, Serry M M, Cai S 1995b Plant polyphenols exhibit lipoprotein-bound antioxidant activity using an in vitro oxidation model heart disease. J Agric Food Chem 43 (11) 27982799. Yen G-C, Chen H-Y 1995 Antioxidant activity of various tea extracts in relation to their antimutagenicity. J Agric Food Chem 43 (1) 2732. Yen G-C, Hsieh P-P 1995 Antioxidative activity and scavenging eects on active oxygen of xylose-lysine Maillard reaction products. J Sci Food Agric 67 (3) 415420. Yoshida T, Mori K, Hatano T, Okumura T, Uehara I, Komagoe K, Fujita Y, Okuda T 1989 Studies on inhibition mechanism of autoxidation by tannins and avonoids. V. Radical-scavenging eects of tannins and related polyphenols on 1,1-diphenyl-2-picrylhydrazyl radical. Chem Pharm Bull 37 (7) 19191921.

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