Lebensm.-Wiss. u.-Technol.

, 30, 609615 (1997)

Kinetics and Mechanisms of Antioxidant Activity using the DPPH Free Radical Method
V. Bondet, W. Brand-Williams and C. Berset* Laboratoire de Chimie des Substances Naturelles, Departement Science de lAliment, E.N.S.I.A., 1, Avenue des Olympiades, 91305 Massy (France) (Received September 17, 1996; accepted January 14, 1997)

The reaction mechanisms of three antioxidants are proposed in order to explain experimental results obtained from a kinetic study using the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, previously adapted in our laboratory. In its radical form, DPPH shows an absorbance maximum at 515 nm which disappears upon reduction by an antiradical compound. BHT, a synthetic antioxidant, slowly reacts with DPPH reaching a steady state within 5 h. This 2.8-stoichiometric complete reaction follows a 1.5-order with respect to DPPH and 0.5 to BHT. The kinetic rate constant, k, is estimated to be 5.0 L/(mols) at 20 C and the energy of activation, Ea, is equal to 35 kJ/mol in methanol. Eugenol reacts with DPPH reaching a steady state within 2 h. This 1.9-stoichiometric reaction follows a 2-order with respect to both DPPH and eugenol, k and Ea are estimated to be 1010 L3/(mol3s) at 20 C and 30 kJ/mol, respectively. The eugenol mechanism may involve a dimerization between two 5.4 phenoxyl radicals. The reaction with isoeugenol is rapid and reversible, with a stoichiometry of 1.1. It is rst order with respect 102 s1 at 10 C. This reaction is consistent with a pseudo-monomolecular to isoeugenol with k (direct reaction) equal to 8.9 mechanism.

1997 Academic Press Limited Keywords: DPPH; antioxidant activity; stoichiometry; kinetic parameter; BHT; eugenol; isoeugenol

Introduction Lipid autooxidation is a radical process involved in a chain reaction including induction, propagation and termination steps. During the induction period, alkyl and peroxyl radicals are formed. These highly reactive chemical species produce hydroperoxides (ROOH) during the propagation phase. Termination is the association of two radicals together to form more stable products. The whole sequence is responsible for organoleptic and nutritional alterations due to the formation of offavour volatile compounds from degradation of ROOH and the disappearance of essential fatty acids. Furthermore, radicals formed are involved in the ageing processes of tissues and pathologies such as cancer or cardiovascular diseases (1). Therefore, it is necessary to protect food lipids and human tissues against free radicals by endogenous and exogenous antioxidants from a natural or synthetic origin. Today natural products are increasing in food consumer preferences. Antiradicalar antioxidants act by donating hydrogen atoms to lipid radicals. Radicals obtained from antioxidants with molecular structures such as phenols are

*To whom correspondence should be addressed.

stable species and will then stop the oxidation chain reaction. In order to determine antioxidant activity, many tests use accelerated oxidative conditions which provoke lipid oxidation by the means of both a high temperature and a high oxygen supply. It is well known that such tests are not always representative of the natural evolution of lipids in foods (2). Moreover, for many antioxidants, the risk of degradation with such conditions during these tests is high. In the DPPH free radical method (3), antioxidant efciency is measured at ambient temperature and thus eliminates the risk of thermal degradation of the molecules tested. However, the reactional mechanism between the antioxidant and DPPH depends on the structural conformation of the antioxidant. In a recent paper (4), modications of the operating conditions were proposed in order to adapt the DPPH method to each kinetic case. Some compounds react very quickly with DPPH, reducing a number of DPPH molecules equal to their number of available hydroxyl groups. However, for the majority of the compounds tested, the reactions are slower and the mechanisms seem to be more complex. It would therefore be useful to build plausible kinetic models in order to obtain a better understanding of the mechanisms involving DPPH and antioxidants. In this paper, we explore the kinetic behaviour of three different antioxidants.
1997 Academic Press Limited

0023-6438/97/060609 + 07 $25.00/0/fs970240

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Materials and Methods Reagents Methanol (Code No. 412532) was of HPLC grade (999 g/kg) from Analyticals Carlo Erba (Milano, Italy). 2,2-Diphenyl-1-picrylhydrayzl (DPPH, 950 g/kg, Cat. No. D21, 140-0) and its reduced hydrazine form (DPPHH, 980 g/kg, Cat. No. 28, 168-9), butylatedhydroxy-toluene (BHT, 990 g/kg, Cat. No. D4, 740-4) and isoeugenol (990 g/kg, Cat. No. I1, 720-6) were purchased from Aldrich (Saint Quentin Fallavier, France). Eugenol (990 g/kg, Art. No. 11.911.77) was purchased from Janssen Chimica (Beerse, Belgium).

DPPH + AH DPPHH + A

Eqn [2]

The reversibility of the reaction is evaluated by adding DPPHH at the end of the reaction. If there is an increase in the percentage of remaining DPPH at the plateau, the reaction is reversible, otherwise it is a complete reaction.

Determination of the kinetic parameters of the reaction The rate (v) of a complete reaction between moles of DPPH and one mole of antioxidant (AH) as a function of time (t) is dened as follows: v = dCDPPH/( = k Cx DPPH dt) Cy AH Eqn [3]

Spectrophotometric measurements All spectrophotometric data were performed using an Uvikon 810 Kontron spectrophotometer. Disposable cuvettes (1 cm 1 cm 4.5 cm) from Muller Ratiolab (Dreieich, Germany) were used for visible absorbance measurements. Slow kinetics were studied at 20 C and rapid kinetics at 10 C. Thermodynamical parameters were studied at different temperatures from 10 to 30 C. Temperature was controlled using a thermoregulating system.

where C are the concentrations, k the rate constant, and x and y the orders with respect to the reactive forms. If this reaction is reversible, v is dened as follows: v = dCDPPH/( Cx = k1 DPPH dt) Cy k1 AH iCPi
zi

Eqn [4]

Determination of the DPPH/antioxidant reaction stoichiometry Experimental data were obtained for BHT, eugenol and isoeugenol. For each antioxidant, different molar ratios (MR), expressed as moles of antioxidant per mole of DPPH, were tested. A 0.5 mL sample of antioxidant solution in methanol was added to 3.5 mL of methanolic DPPH solution so that the initial DPPH concentration in the cuvettes was approximately 6 105 mol/L. The exact initial DPPH concentration, noted C0 DPPH (mol/L), was calculated from the linear regression (4): 105 C0 DPPH = 7.99 7 10 A515 + 2.06 Eqn [1]

where k1 and k1 are the rate constants for direct and reverse reactions, and i CPi Zi the mathematical product between the concentrations of the reaction products Pi (orders zi). Orders are representative of the mechanistic step which limits the rate of the whole reaction. The determination of the x order with respect to DPPH is possible using the method of isolation (order degeneration method) (7, 8). In practice, this method can be used only if the studied antiradical compound shows a slow kinetic behaviour. Initially, if CAH is more than 50 CDPPH, then CAH remains constant during the reaction. Using a logarithmic conversion of Eqn [3] and plotting ln (v) as a function of ln (CDPPH), x can be determined from the slope of the linear curve. The order with respect to AH (y) can be calculated if initially CAH = CDPPH/ . In this case, and after a logarithmic conversion, Eqn [3] becomes: ln (v) = ln (k/y) + (x + y) ln (CDPPH) Eqn [5]

Absorbance (A) was read at 515 nm until the reaction reached a plateau. For each MR, the remaining percentage of DPPH at the plateau was determined and graphed, and EC50 was read on the graph as the MR which reduces half of the initial DPPH concentration (4). Since some tested compounds did not lead to a complete disappearance of DPPH, the stoichiometry (, number of reduced DPPH molecules per one molecule of antioxidant), was dened as 1/(2 EC50). In this method we have considered the values at the steady state and not after 30 min as in older studies (5, 6), where the results depended on the type of kinetic behaviour.

As above, y can be determined from the slope of this linear curve since x is known. Determination of the rate constant k is also possible since ln (k/y) is the intersection between the linear curve (Eqn [5]) and the ordonate axis. However, using this method leads to a very large variance factor. So, k was calculated using the two following methods. The rst consists of integrating Eqn [3] with respect to time. The absorbance of disappeared DPPH (A) during the reaction is plotted as a function of time (t) and ke is deduced from the slope of the linear curve (Eqn [6]) when CAH = CDPPH/: A1 x y = (ke 1 x y) t + C1 x y Eqn [6] 0 DPPH where is the molar extinction coefcient and C0 DPPH the initial concentration of DPPH. k is then calculated from ke using Eqn [7]: ke = (x + y 1) k/y Eqn [7]

Determination of the reaction kinetic types DPPHH is a product of the reaction between DPPH and an antioxidant (AH):

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The determination of k can also be obtained with a good variance factor using the reaction half-life method (8). The half-life (t1/2) of the reaction is dened as the time required for DPPH concentration to decrease to one-half its initial value. If initially CAH = CDPPH/, at t1/2 Eqn [6] becomes: t1/2 = ((2x + y 1 1)/ke) C1 x y DPPH Eqn [8]

and c = ln(2/1) 2 = e C0 DPPH CO AH/(e (C0 DPPH + C0 AH) C0 DPPH C0 AH) Eqn [13]

If the reaction is reversible, the determinations of x + y and the rate constant are possible (8). In a graph showing the rate (v) as a function of the disappeared concentration of DPPH (named , and equal to A/), the tangent at the origin of the curve intercepts the abcisse axis at 0 (Fig. 1). The global order of the reaction, x + y, is determined graphically from 0: x + y = C0 DPPH/0 Eqn [9]

Eqn [14]

k1 keq = (2 1) (e (C0 DPPH + C0 AH) C0 DPPH C0 AH)/2 e

Eqn [15]

In practical cases, the initial rate of the reaction is so great that the tangent cannot be obtained before approximately 5 s. k1 can then be calculated from Eqn [10]: v0 = (k1/y)
+ y Cx DPPH 0

where e is the value at the equilibrium state, and C0 are the initial concentrations of the reactive compounds.

Eqn [10]

where v0 can be graphically determined from the intersection between the tangent and the ordonate axis. For a better correlation factor, as described above, Eqn [4] can also be integrated for the rst times of the reaction, and k1 calculated by linear regression.

Determination of the rate-determining step of the reaction The rate-determining step (RDS) is the slowest phase of a kinetic mechanism and commands the rate of the whole reaction. The RDS is described by the orders with respect to the reactive forms. All the antioxidants studied react rst with DPPH as described in Eqn [2] (9, 10). In order to determine if the reaction (Eqn [2]) may or may not be the RDS, we have compared the experimental rate constant k previously calculated for the global reaction with the rate constant k1, calculated by integrating Eqn [4] as a function of time: ln ((2 )/(1 )) = keq 1 = e
Rate of DPPH disappearance 9 (10 mol/(L.s))

Determination of thermodynamic parameters The activation energy (Ea) is calculated with the Arrhenius equation after the determination of k at different temperatures. The enthalpy of reaction (H) can be calculated as the difference between the energies of activation for the reverse (Ea1) and direct (Ea1) reactions. For that, and only for antioxidants showing an RDS described by Eqn [2], k1 can be obtained by Eqn [11], and k1 from k1 using Eqn [4] at the equilibrium state where v = 0.

Statistical recovery of the results In order to determine correlation and variance factors, at least ve repetitions were done for each reaction.

Results and Discussion All the results of this study are summarized in Table 1. With values of 2.8 for BHT, 1.9 for eugenol and 1.1 for isoeugenol, our results conrm previous studies (4).

t + c

Eqn [11]

where keq, c, 1 and 2 are dened as follows: Eqn [12]

150

Global order = initial DPPH concentration/0 100

Reaction kinetic types Adding DPPHH at the end of the reaction regenerates DPPH only in the case of isoeugenol (data not shown). The higher the DPPHH concentration added, the higher the DPPH concentration at the steady state. Therefore BHT and eugenol reactions are complete while the isoeugenol reaction is reversible.

50 Graphic determination of 0 0 5.1 5.3 5.4 5.5 5.6 5.2 5.7 5 Disappeared DPPH concentration (10 mol/L)

Fig. 1 Determination of the global order of the isoeugenol reaction

Reaction stoichiometry It must be emphasized that the stoichiometric value does not explain all the aspects of antioxidant efciency: BHT appears to be the best antioxidant because 1 mol of BHT reduces about 3 mol of DPPH, even though it reacts very slowly (plateau reached after 5 h at 20 C). Conversely, isoeugenol reduces only 1 mol of

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DPPH per mol; it may react as described in model [2], but with a high rate (plateau reached after 0.5 min at 20 C). Eugenol has an intermediate behaviour, with a stoichiometry of about 2 and a plateau reached after 120 min. This inverse relation between reaction stoichiometry and rate indicates that the slower the reaction rate, the more complex the mechanism. Brand-Williams et al. (4) and Cuvelier (11) suggested three possible pathways for BHT/DPPH reactions
Table 1 Parameters of the antioxidant/DPPH reactions BHT Stoichiometric study Plateau time (min) Stoichiometry (DPPH/AH) Kinetic study Reaction type Antioxidant order DPPH order Rate constant k (20C) Standard deviation of k Rate-determining step Thermodynamic study Energy of activation (kJ/mol) Enthalpy of reaction (kJ/mol) 300 (at 20C) 2.8 (d =0.4) Complete reaction 0.4 (s= 0.25) 1.5 (s= 0.13) 5.0 L/(mols) 0.26 L/(mols) First (or second) step 35 (r = 0.999) 13 (r =0.963)

(Fig. 2). After a rst reaction of type [2], the BHT radical species can form a kenolic compound (step a, = 2) by donation of a second hydrogen atom, or, after a regeneration of the hydroxyl group by dimerization it can react a second time with DPPH to produce a bi-quinonoid structure (step b, = 3). This pathway has been studied previously for other antioxidants (5, 9, 1214). The third pathway (step c, = 2) involves a complexation as described by Russell (15). The 2.8

Eugenol 120 (at 20C) 1.9 (d = 0.2) Complete reaction 2.4 (s=0.18) 1.9 (s =0.12) 5.4 1010 L3/(mol3s) 0.881010 L3/(mol3s) End steps 30 (r= 0.997) n.d.

Isoeugenol 15 (at 10C) 1.1 (d = 0.1) Reversible reaction 0.8 (s=0.03) 0 8.9 102 s1 (10 C) 1.4 102 s1 (10 C) First step n.d. n.d.

r =correlation factor; s=variance factor; d =difference between extreme values. n.d. =not determined.
OH (CH3)3C C(CH3)3 (CH3)3C O C(CH3)3 (a) Donation of a second hydrogen atom

CH3

DPPH DPPHH O (CH3)3C

.
DPPH

CH2 DPPHH

.
OH OH C(CH3)3 (CH3)3C C(CH3)3

.
C(CH3)3 (CH3)3C H delocalization Rate-determining step

CH3

CH2

. .

CH2

DPPH

.
OH

DPPH

(b) Dimerization

(c) Complexation

(CH3)3C

C(CH3)3

CH2-DPPH

(CH3)3C O (CH3)3C C(CH3)3 O C(CH3)3 4 DPPH

(CH3)3C HO (CH3)3C C(CH3)3 OH C(CH3)3

Fig. 2 Proposed mechanism for BHT/DPPH reaction

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value found for BHT shows that it could follow these three pathways. With a stoichiometry of 1.9, eugenol could follow the steps (a) and (c) (Fig. 3), and perhaps step (b) if a stable mono-quinonoid structure is produced by this pathway. Indeed, eugenol can not produce bi-quinonoid structures as BHT can because such a structure is not stabilized by resonance over the whole molecule. Isoeugenol can not participate in these reactions because of its conjugated group in the para position which stabilizes the radical formed A:
OH DPPHH OCH3 Active form Rate-determining step CH2 CH CH3 DPPH CH CH CH3 OCH3 Eqn [16] O

rapid kinetics). The rst hypothesis is x = y = 0.5. As for BHT, this does not agree with the structures of isoeugenol and DPPH molecules. A global order of 1 could also be the sum of partial orders of 1 with respect to isoeugenol and 0 to DPPH. This second hypothesis is strengthened by the increase of the reaction rate with increasing initial isoeugenol concentration (data not shown). These considerations are compatible with a pseudo-monomolecular kinetic model: in the RDS, isoeugenol is converted into a reactive form, which then reacts with DPPH [16]. This activation step could also exist for eugenol and BHT, but it may not be the RDS because these compounds showed slower kinetics.

Eqn [16] Moreover, rst, the reverse reaction observed after adding DPPHH at the end of the reaction would implicate a reversible dimerization mechanism, which is unlikely. Second, such a dimer could react with DPPH leading to a stoichiometry greater than 1.

Reaction kinetic orders BHT, eugenol and isoeugenol show three different behaviours with respect to reaction orders. Partial orders of 0.5 and 1.5 with respect to BHT and DPPH were obtained. In classical chemical kinetics (8) such fractional orders implicate the dissociation of the reactive forms (0.5) and their complexation (1.5) prior to the rate-determining step (RDS). Such mechanisms do not agree with the structures and reactions of BHT and DPPH molecules. Moreover, the orders determined evolve during the reaction, indicating several different reaction steps and a feedback action of the reaction products (810, 12, 16): BHT follows a pseudosecond order kinetic model whose orders are, respectively, less and more than 1 for BHT and DPPH. So, the RDS is rst the bimolecular reaction [2], and then, a more complex mechanism involving numerous forms of DPPH and few BHT species at the end. This order variation could be explained by a regeneration of BHT at the end of the rst reaction time. The reversibility of the (a) and/or (c) pathways (Fig. 2) allows future DPPH consumption by the (b) pathway without involving BHT forms. This is not possible with eugenol because of a better resonance stability of the (a) and (c) products. For eugenol, a global order of 4 (x + y) indicates the production of a reactive species prior to the RDS (8). This form could be the A radical. Partial orders equal to 2 for both AH and DPPH (Table 1) may be related to the dimerization of these initial reactive molecules (Fig. 3) and the reaction with 2 DPPH during the RDS. For isoeugenol, the global order of the reaction is equal to 1. Determination of the orders x and y with respect to each reactive form was practically impossible (very

Study of the rate-determining step As shown above, the isoeugenol and eugenol orders determined the rate-determining steps of the reaction (RDS) with DPPH. Therefore, this study only concerned BHT for which the RDS did not appear very clearly. k1 was found to be 4.2 L/(mols) with a standard deviation of 0.44, while k was 5.0 L/(mols) with a standard deviation of 0.26, at 20 C in our working conditions. Moreover, evolution of these rate constants with temperature showed the same behaviour. So, the RDS of the BHT mechanism must be step [2]. This result agrees with that of Hogg et al. (9) and Ayscough and Russell (10). The important steric hindrance of the two ortho positioned tert-butyl groups on BHT could explain why this rst step is the RDS. But this does not explain why the RDS was changed at the end of the reaction. It is possible to consider the H delocalization as the RDS of the whole BHT/DPPH reaction (Fig. 2). Because the rst step is reversible, it can adapt its rate to this H transfer reaction. Therefore, by only measuring the absorbance of DPPH, the rst step appears to be the RDS. These RDS considerations give a central position to the methyl radical species of BHT in the kinetics of BHT with DPPH.

Thermodynamic parameters The results presented in Table 1 only concern BHT and eugenol. Isoeugenol kinetics were too rapid for practical determinations. Activation energies of antioxidant/DPPH reactions. Our results agree with the activation energies of the BHT/DPPH reaction reported in the literature as 25 kJ/mol in benzene (9), and between 21 and 42 kJ/mol in carbone tetrachloride (10, 16). These low values show that the antiradicalar reaction is thermodynamically easy. The activation energy of the eugenol/DPPH reaction is somewhat lower than the BHT/DPPH reaction. Indeed, the approach of the two reactive molecules prior to reaction is more difcult with BHT due to an important steric hindrance around the OH reactive site.

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Enthalpy of reaction for the BHT/DPPH reaction. Using the RDS model [11], we have determined the direct (Ea1) and reverse (Ea1) activation energies: Ea1 = 44 kJ/mol (regression coefcient r = 0.990) and Ea1 = 57 kJ/mol (r = 0.963). The enthalpy of the rst step of BHT/DPPH reaction is 13 kJ/mol. This result shows a spontaneous direct step and a small temperature inuence on this reversible reaction. Ayscough and Russell (10) found 7.1 kJ/mol for the tri-tertbutylphenol in benzene.

Conclusion Use of DPPH provides an easy and rapid way to evaluate the antiradicalar activities of antioxidants, but also to build plausible kinetic models of reactions. From the results obtained in the present study, the reaction mechanisms for BHT, eugenol and isoeugenol all showed a hydrogen atom transfer reversible step which reduces the DPPH and forms the antioxidant radical (A). The hypothesis of an activation step prior to any reaction with DPPH must not be eliminated. This step appears to be the rate-determining step for isoeugenol (order 0 for DPPH and 1 for isoeugenol).
OH OCH3

However, if the formed radical A is not sufciently stabilized, many different reactions are then possible. In this case, the second reaction site may be in ortho- or para- positions, due to a weak and nonsymetric delocalization of the single electron on the antioxidant radical by mesomeric or inductive effects. Indeed, with two mesomeric substituants, isoeugenol cannot show a second reactivity (stoichiometry of 1.1). However, the CH3 substituant on the BHT molecule or the nonsubstituted ortho carbon on the eugenol ring could also react. Eugenol appears to react only in a dimerization mechanism (second order kinetics), leading to a monoquinonoid species (stoichiometry of 1.9). BHT appears to follow three different pathways (complex orders) and BHT-DPPH species could be formed by complexation. Quinonoid structures could be produced by hydrogen atom delocalization and dimerization followed by a new reaction with DPPH. This last model forms bi-quinonoid structures and explains a stoichiometry of 2.8. To support our hypotheses, it would be interesting to characterize the reaction products using liquid chromatograph coupled with a mass spectrometer, and to study some intermediate antiradical compounds by para electronic resonance.
O OCH3

CH2

CH

DPPH DPPHH O

CH2

CH

CH

CH2 (a) Donation of a second hydrogen atom

DPPH

. .
O

.
OCH3 CH CH2

OCH3

.
CH2

O OCH3

CH2

CH2

CH

CH2

CH

CH2

DPPH (c) Complexation DPPH

Rate determining step

OH OCH3 (b) Dimerization CH2 CH CH2

CH2

CH O

CH2

CH2

CH OH

CH2

CH3O OH CH

OCH3 2 DPPH CH CH2

CH3O OH CH2

OCH3

CH

CH2

Fig. 3 Proposed mechanism for eugenol/DPPH reaction

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Acknowledgements The authors wish to thank Mrs F. Maguin, from the University of Orleans, and Mr H. Richard from E.N.S.I.A. for their helpful scientic contributions.
7 8 9

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