Media effects on antioxidant activities of phenols and catechols

Article abstract of DOI:10.1021/ja990878u

The H-atom donating activities of 2,6-di-tert-butyl-4-methylphenol (BHT), 2,6-di-tert-butyl-4-methoxyphenol (DBHA), 2,2,5,7,8-pentamethyl-6-hydroxychroman (PMHC), and 3,5-di-tert-butylcatechol (DTBC) toward the nitrogen-centered 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical were measured by stopped flow methods in hexane, 1-propanol, tert-butyl alcohol, and acetone. Decreases in these activities on transferring from hexane to the hydrogen bond accepting (HBA) solvents, the kinetic solvent effect (KSE), are attributed to hydrogen bonding from the phenolic group. Steric hindrance accounts for a lower decrease observed for the highly hindered BHT and DBHA compared to PMHC. The catechol, DTBC, a very active H-atom donor to DPPH in hexane, showed a dramatic loss of activity in HBA solvents, especially acetone. Higher H-atom donating activities of BHT, DBHA, and PMHC were observed toward the oxygen-centered radical of 2,6-di-tert-butyl-4-(4′-methoxyphenyl)phenoxyl (DBMP), and the decreases in activity in the HBA solvents paralleled those found with DPPH. Thus the KSE was found to be independent of the nature of the abstracting radical for DPPH and DBMP. The inhibition of the oxygen uptake (IOU) method was used to determine the antioxidant activities (k_inh) of α-tocopherol, PMHC, catechol, and DTBC during free radical autoxidation of styrene and mixtures of styrene and tert-butyl alcohol. The k_inh of α-tocopherol and PMHC dropped to one-tenth of the values with increasing tert-butyl alcohol content due to the HBA activity of the alcohol compared to styrene.

Full text of DOI:10.1021/ja990878u

6226
J. Am. Chem. Soc. 1999, 121, 6226-6231
Media Effects on Antioxidant Activities of Phenols and Catechols
L. R. C. Barclay,* C. E. Edwards, and M. R. Vinqvist
Contribution from the Department of Chemistry, Mount Allison UniVersity,
SackVille, New Brunswick, Canada E4L 1G8
ReceiVed March 18, 1999
Abstract: The H-atom donating activities of 2,6-di-tert-butyl-4-methylphenol (BHT), 2,6-di-tert-butyl-4-
methoxyphenol (DBHA), 2,2,5,7,8-pentamethyl-6-hydroxychroman (PMHC), and 3,5-di-tert-butylcatechol
(DTBC) toward the nitrogen-centered 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical were measured by stopped
flow methods in hexane, 1-propanol, tert-butyl alcohol, and acetone. Decreases in these activities on transferring
from hexane to the hydrogen bond accepting (HBA) solvents, the kinetic solvent effect (KSE), are attributed
to hydrogen bonding from the phenolic group. Steric hindrance accounts for a lower decrease observed for the
highly hindered BHT and DBHA compared to PMHC. The catechol, DTBC, a very active H-atom donor to
DPPH in hexane, showed a dramatic loss of activity in HBA solvents, especially acetone. Higher H-atom
donating activities of BHT, DBHA, and PMHC were observed toward the oxygen-centered radical of 2,6-di-
tert-butyl-4-(4′-methoxyphenyl)phenoxyl (DBMP), and the decreases in activity in the HBA solvents paralleled
those found with DPPH. Thus the KSE was found to be independent of the nature of the abstracting radical
for DPPH and DBMP. The inhibition of the oxygen uptake (IOU) method was used to determine the antioxidant
activities (kinh) of R-tocopherol, PMHC, catechol, and DTBC during free radical autoxidation of styrene and
mixtures of styrene and tert-butyl alcohol. The kinh of R-tocopherol and PMHC dropped to one-tenth of the
values with increasing tert-butyl alcohol content due to the HBA activity of the alcohol compared to styrene.
The reactions of oxygen-centered radicals with biological
and nonprotic, and established two significant principles for the
kinetic solvent effects (KSE)4 on phenolic antioxidant activi-
ties: (1) The magnitude of the KSE is determined by the
strength of the interaction between the hydrogen bond donor
(e.g., the antioxidant, HBD) and the hydrogen bond accepting
solvent (HBA), protic or nonprotic, and (2) the KSE on H-atom
abstractions from hydroxyl groups are independent of the nature
of the abstracting radical.3 These principles provide a basis for
our extended studies of the effect of media on the H-atom
donating activity of various classes of antioxidants and on
antioxidant activities during radical chain peroxidation.
We now report on the effects of various media on the H-atom
donating ability of several classes of phenolic antioxidants
including the following: 1, the two hindered o-di-tert-butyl-
phenols, 2,6-di-tert-butyl-4-methylphenol (BHT), and 2,6-di-
tert-butyl-4-methoxyphenol (DBHA); 2, 2,2,5,7,8-pentamethyl-
6-hydroxychroman (PMHC), an antioxidant of the vitamin E
class; and 3, 3,5-di-tert-butylcatechol (DTBC). The phenols
BHT and DBHA are well-known synthetic antioxidants, and
PMHC, in homogeneous solution, is expected to mimic the
behavior of vitamin E, nature’s most active lipid-soluble
phenolic antioxidant.5 The catechol derivative DTBC was
selected because it contains the structural unit that appears to
be the main center responsible for the antioxidant activity of
the flavonoids (vide infra). These studies were carried out by
molecules such as lipid membranes are implicated in various
degenerative human diseases, and consequently the mechanism
and antioxidant activities of natural and synthetic antioxidants
continue to receive a great deal of interest.1 The reactions of
phenolic antioxidants in relevant biological systems such as
heterogeneous aqueous/lipid phases are subject to complex
differential solvation and diffusion phenomena. Consequently
the antioxidant activities of phenols are reported to be markedly
reduced in model heterogeneous phases of micelles or lipid
bilayers compared to nonprotic solvents. Such effects were
attributed in a qualitative way to hydrogen bonding of the
phenolic hydroxyl group, resulting in inhibition of the hydrogen
transfer of the phenolic hydrogen atom to peroxyl radicals in
the rate-controlling step.2 Recent reports by Ingold et al. have
provided the first clear quantitative evidence of the role that
hydrogen bonding plays on the H-atom donor activities of
phenolic antioxidants.3 These reports provide a very useful guide
for further studies of the effects of media on the activities of
antioxidants since they cover a wide range of solvents, protic
* Address correspondence to this author. Phone: (506) 364-2369.
FAX: (506) 364-2313. E-mail: rbarclay@mta.ca.
(1) For recent reports and some reviews on this subject see: (a) Barclay,
L. R. C.; Vinqvist, M. R.; Antunes, F.; Pinto, R. E. J. Am. Chem. Soc.
1997, 119, 5764-5765. (b) Barclay, L. R. C.; Antunes, F.; Egawa, Y.;
McAllister, K. L.; Mukai, K.; Nishi, T.; Vinqvist, M. R. Biochim. Biophys.
Acta 1997, 1328, 1-12.
(2) (a) Barclay, L. R. C.; Edwards, C. D.; Mukai, K.; Egawa, Y.; Nishi,
T. J. Org. Chem. 1995, 60, 2739-2744. (b) Pryor, W. A.; Strickland, T.;
Church, D. F. J. Am. Chem. Soc. 1988, 110, 2224-2229. (c) Castle, L.;
Perkins, M. J. J. Am. Chem. Soc. 1986, 108, 6382-6384. (d) Barclay, L.
R. C.; Baskin, K. A.; Dakin, K. A.; Locke, S. J.; Vinqvist, M. R. Can. J.
Chem. 1990, 68, 2258-2269.
(3) (a) Valgimigli, L.; Ingold, K. U.; Lusztyk, J. J. Am. Chem. Soc. 1996,
118, 3545-3549. (b) MacFaul, P. A.; Ingold, K. U.; Lusztyk, J. J. Org.
Chem. 1996, 61, 1316-1321. (c) Valgimigli, L.; Banks, J. T.; Ingold, K.
U.; Lustyk, J. J. Am. Chem. 1995, 117, 9966-9971.
(4) Abbreviations used in the order in which they appear in the text:
KSE, kinetic solvent effect; HBD, hydrogen bond donor; HBA, hydrogen
bond acceptor; BHT, 2,6-di-tert-butyl-4-methylphenol; DBHA, 2,6-di-tert-
butyl-4-methoxyphenol; PMHC, 2,2,5,7,8-pentamethyl-6-hydroxychroman;
DTBC, 3,5-di-tert-butylcatechol; DPPH, 1,1-diphenyl-2-picrylhydrazyl;
DBMP, 2,6-di-tert-butyl-4-(4′-methoxyphenyl)phenoxyl; IOU, inhibition of
oxygen uptake; AIBN, azo-bis-isobutyrylnitrile.
(5) (a) Burton, G. W.; Doba, T.; Gabe, E. J.; Hughes, L.; Lee, F. L.;
Prasad, L.; Ingold, K. U. J. Am. Chem. Soc. 1995, 107, 7053-7065. (b)
Burton, G. W.; Ingold, K. U. Acc. Chem. Res. 1986, 19, 194-201.
10.1021/ja990878u CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/17/1999

Antioxidant ActiVities of Phenols and Catechols
J. Am. Chem. Soc., Vol. 121, No. 26, 1999 6227
Table 2. Second-Order Rate Constants,a M-1 s-1, at 30 °C for
H-Atom Abstraction from Antioxidants by the
2,6-Di-tert-butyl-4-(4′-methoxyphenyl)phenoxy (DBMP) Radical
antioxidantb
solvent
hexane
1-propanol
tert-butyl alcohol
acetone
BHT
DBHA
PMHC
160
44.3 × 102
5.77 × 102
6.42 × 102
2.77 × 102
(93.9 × 103)6
4.40 × 103
5.64 × 103
3.28 × 103
41.1
63.4
19.3
a See Table 1, footnote a. b See Table 1, footnote b. Linear plots of
kobs versus [ArOH] gave satisfactory correlations of 0.90-0.99.
caused a decrease in the H-atom transfer, and acetone exerted
an additional retarding effect. In contrast, the H-atom donating
ability of PMHC is about the same in acetone and the two
alcohols, as shown in the similar rate constants in these media,
indicative of the same degree of solvent effect. The catechol,
DTBC, is a much more active H-atom donor than the other
phenols in hexane but exhibited a larger solvent effect, so that
DBTC appears to be in a class by itself in this regard.
Figure 1. Structures of antioxidants and radicals.
Table 1. Second-Order Rate Constants,a M-1 s-1, at 30 °C for
H-Atom Abstraction from Antioxidants by the
1,1-Diphenyl-2-picrylhydrazyl (DPPH) Radical
antioxidantsb
solvent
hexane
1-propanol
tert-butyl alcohol
acetone
BHT
DBHA
PMHC
DTBC
1.13
0.31
0.41
0.16
37.6
5.23
4.27
1.14
6.77 × 103
2.31 × 102
2.23 × 102
1.99 × 102
20.3 × 103
Kinetic Measurements Employing the Oxygen-Centered
Radical, DBMP. The second-order rate constants, k2, for
H-atom abstraction were obtained by following the decay of
DBMP absorption at 370 nm in hexane and at 374 nm in the
alcohols and acetone on the diode array spectrometer in the
presence of excess H-atom donor under conditions of pseudo-
first-order kinetics. The k2 values were obtained from plots of
eq 1 as before. The k2 values for H-atom abstraction by DBMP
from BHT, DBHA, and PMHC are given in Table 2. The higher
rate constants for H-atom abstraction by DBMP reflect the effect
of the more reactive oxygen-centered radical, compared to the
values from the nitrogen-centered radical of DPPH. The drop
in k2 observed by BHT and DBHA on transferring from hexane
to the alcohols or acetone for H-atom abstraction by DBMP
parallels the results shown in Table 1 for abstraction by DPPH.6
Antioxidant Activities of r-Tocopherol, PMHC, and
Catechols in tert-Butyl Alcohol/Styrene. To determine the
effect of a protic solvent on the peroxyl radical trapping activity
of these phenols, we adapted the method developed by Ingold
et al., namely, the measurement of inhibited oxygen uptake
(IOU) by the antioxidants during controlled initiation of styrene
peroxidation by the azo initiator AIBN.5 In our studies, solvent
mixtures of styrene, the substrate, and the alcohol were used.
Sufficient styrene was required to ensure a kinetic chain length
of at least 10 during the inhibition periods in the presence of
antioxidants. We conducted a series of measurements with
various amounts of tert-butyl alcohol to determine the effect of
this protic solvent on the absolute rate constant for the rate-
determining step of inhibition, kinh, eq 2.
80.4
56.1
16.7
a Each rate constant is an average of two or three determinations
and the experimental error was usually within 5%. b The ratio of excess
antioxidant to DPPH varied depending on the activity found in trial
experiments (see Experimental Section). Linear plots of kobs versus
[ArOH] gave satisfactory correlations of 0.90-0.99.
using two hydrogen atom abstracting agents, 1,1-diphenyl-2-
picrylhydrazyl (DPPH), a nitrogen-centered radical, and 2,6-
di-tert-butyl-4-(4′-methoxyphenyl)phenoxy (DBMP), an oxygen-
centered radical. (See Figure 1 for structures of the antioxidants
and radicals.) The reactions were followed by monitoring the
decay of the main absorption bands in the visible spectra for
DPPH or DBMP.
In addition, the antioxidant actiVities of R-tocopherol, PMHC,
catechol, and DTBC were determined by employing the
inhibition of oxygen uptake method (IOU) during oxidation
initiated by peroxyl radicals, generated under controlled condi-
tions from azo-bis-isobutyrylnitrile (AIBN), in styrene and in
mixtures of styrene and tert-butyl alcohol, a protic solvent.
Results
Kinetic Measurements Employing the Nitrogen-Centered
Radical, DPPH. Second-order rate constants, k2, were obtained
by following the decay of DPPH absorption at 528 nm on a
diode array spectrometer in the presence of excess H-atom donor
under conditions of pseudo-first-order kinetics. The k2 values
were determined from plots of kobs versus [ArOH] according to
eq 1.
kinh
ROO• + ArOH 8 ROOH + ArO•
(2)
kobs ) k0 + k2[ArOH]
(1)
The phenoxyl radical, ArO•, will undergo rapid termination with
a second peroxyl radical, eq 3, so that the stoichiometric factor,
n, for trapping peroxyl radicals by phenolic antioxidants is 2.
The expression for suppressed oxygen uptake during the
inhibition period is given by eq 4. In calculations, we used the
reported value5 of 41 M-1 s-1 for the propagation rate constant,
kp, for peroxidation of styrene at 30 °C.
The k2 values for H-atom abstraction by DPPH from BHT,
DBHA, PMHC, and DTBC in four solvents, hexane, 1-propanol,
tert-butyl alcohol, and acetone, are given in Table 1.
The results with DPPH show that for BHT, DBHA, and
PMHC, the order of H-atom donating activity parallels the
known antioxidant activities determined earlier in styrene/
chlorobenzene mixtures,5 but the absolute values differ for the
different reactions (free radical chain autoxidation with peroxyl
radicals5 compared to H-atom abstraction by DPPH). For DBHA
and BHT, the protic solvents 1-propanol and tert-butyl alcohol
(6) The k2 value for H-atom abstraction from PMHC by DPPH in hexane
is in reasonable agreement with that reported (7.40 × 103 M-1 s-1).2b Our
value for the reaction of PMHC with DBMP in hexane is considered less
accurate since the rate of decay of the DBMP radical appeared to reach the
limit of the diode array used.

6228 J. Am. Chem. Soc., Vol. 121, No. 26, 1999
Barclay et al.
ROO• + ArO• fast8 nonradical products
-d[O2]/dt ) kp/kinh × [RH]Ri/n[ArOH]
(3)
(4)
The rate of radical chain initiation, Ri, is known for initiation
by the azo initiator AIBN, and can be measured by the induction
period method by using eq 5, where τ is the length of the
induction period during suppressed oxygen uptake.
Ri ) n[ArOH]/τ
(5)
The antioxidant activity, represented by the absolute rate
constant for inhibition, kinh, is determined by measuring the
oxygen uptake during the course of the inhibition period. For
calculations, the integrated form of the inhibition period, eq 6,
was used as before.7
Figure 2. Profiles of antioxidant activities of R-tocopherol (R-toc),
b, and PMHC, 2, during inhibited oxidation of styrene, initiated by
AIBN (typically 0.0235 M), showing the effects of added tert-butyl
alcohol. Each point represents the average of three experiments, with
error limits within 12.5% for each point. The concentration of styrene
varied from 8.7 (neat) to 3.8 M for experiments with R-toc ([R-toc]
was 2 to 3 µM) and 8.7 to 1.1 M for experiments with PMHC ([PMHC]
was 1.5 to 3 µM). The rate of chain initiation, Ri ) 2[ArOH]/τ, was
within the range (2.5-3.9) × 10-9 M s-1 for these experiments. The
point × is from the R-toc inhibited peroxidation of linoleate in tert-
butyl alcohol (Barclay, L. R. C.; Baskin, K. A.; Locke, S. J.; Schaefer,
T. D. Can. J. Chem. 1987, 65, 2527-2540).
∆[O2]t ) - kp/kinh[RH] ln(1 - t/τ)
(6)
The rate constants were obtained from the slopes of the plots
of ∆[O2]t versus -ln(1 - t/τ), which equal kp[RH]/kinh. The
kinh were first determined in neat styrene for the antioxidants,
R-tocopherol, PMHC, catechol, and DTBC. Then, for each
antioxidant the values were determined for known mixtures of
styrene and tert-butyl alcohol, carefully controlling the amount
of alcohol in the mixture so that the kinetic chain length was at
least 10 during the inhibition period, a necessary condition for
this method.7 The profiles of the results, indicating the effect
of the varying concentrations of tert-butyl alcohol on the kinh
values8 of R-tocopherol and PMHC, are plotted in Figure 2.
The results for catechol and TBHC are plotted in Figure 3. The
plots show that tert-butyl alcohol causes an initial sharp drop
in the kinh values, followed by a tailing off as the solvent mixture
is made richer in the protic solvent.
Discussion
As noted above (Tables 1 and 2) there are significant drops
in the second-order rate constants for H-atom abstraction on
transferring from hexane to the two alcohols or acetone. These
solvents all have about the same hydrogen bond accepting ability
(HBA).9 One would expect that they would have about the same
degree of hydrogen bond donor (HBD) interaction with the
phenolic hydroxyl group, and consequently a comparable drop
in H-atom donating activity of HBD to HBA would be expected.
What is actually observed is that the effects depend markedly
on the structure of the antioxidants. This is clear from the ratios
of second-order rate constants for transferring from hexane to
the HBA solvents, summarized in Table 3.
Figure 3. Profiles of antioxidant activities of catechol, b, and DTBC,
2, during inhibited oxidation of styrene, initiated by AIBN (typically
0.0235 M), showing the effects of added tert-butyl alcohol. Each point
represents the average of three experiments, with error limits within
15% for each point. The concentration of styrene varied from 8.7 (neat)
to 7.0 M for experiments with catechol ([catechol] was 1.1 to 3.5 µM)
and 8.7 to 6.5 M for experiments with DTBC ([DTBC] was 0.80 to
2.2 µM).
The hindered phenols BHT and DBHA exhibit similar khexane
solvent values for 1-propanol and tert-butyl alcohol, but the effect
of acetone is significantly higher. On the other hand, the three
solvents have about the same effect, although larger, on the
vitamin E model PMHC. We attribute these differences to the
/
Table 3. Ratios of H-Atom Donor Activities, khexane/ksolvent
khexane/ksolvent for
k
antioxidant
1-propanol
tert-butyl alcohol
acetone
DPPH as abstracting radical
BHT
3.6
7.2
29
253
2.8
8.8
30
362
7.1
33
34
(7) Barclay, L. R. C.; Vinqvist, M. R.; Mukai, K.; Itoh, S.; Morimoto,
H. J. Org. Chem. 1993, 58, 7416-7420.
DBHA
PMHC
DTBC
(8) The absolute inhibition rate constants (M-1 s-1)determined in neat
styrene are as follows: R-tocopherol, 2.74 × 106, PMHC, 3.20 × 106,
compared to literature values of 3.20 × 106 and 3.80 × 106, respectively.5a
For DTBC and catechol the values are 1.20 × 106 and 0.53 × 106, compared
to literature values of 1.49 × 106 and 0.55 × 106, respectively: Xi, F.;
Barclay, L. R. C. Can. J. Chem. 1998, 76, 171-182.
12.2 × 102
DBMP as abstracting radical
3.9
7.7
BHT
DBHA
2.5
6.9
8.3
16
(9) The hydrogen bond accepting ability (HBA) of these solvents is
expressed as b values: tert-butyl alcohol, 0.49; 1-propanol, 0.45 (Laurence,
C.; Berthelot, M.; Sraidi, K. J. Phys. Chem. 1989, 93, 3799-3802). The
corresponding â2H values are the following: tert-butyl alcohol, 0.49; primary
alcohols, 0.45; and acetone 0.50 (Abraham, M. H.; Grellier, P. L.; Prior,
D. V.; Morris, J. J. J. Chem. Soc., Perkin 2 1990, 521-529).
steric hindrance provided by the ortho tertiary butyl groups in
BHT and DBHA, which hinder the HBD of these phenols to
HBA interaction of the alcohols. Space-filling models indicate
that the alcohols must come within contact distance to the ortho

Antioxidant ActiVities of Phenols and Catechols
J. Am. Chem. Soc., Vol. 121, No. 26, 1999 6229
tertiary butyls to approach the phenolic hydroxyl. On the other
hand, the HBA carbonyl oxygen of acetone does not show this
kind of hindrance. In the case of PMHC, the ortho methyls do
not present the same degree of steric hindrance to solvent
interaction with the phenolic hydroxyl. Steric hindrance has been
invoked before7,10 to account for differences in antioxidant
activities of hindered phenols, and these results provide new,
quantitative evidence for such effects.
Scheme 1. Stabilization of the DTBC Aroxyl Semiquinone
Radical by Hydrogen Bonding
DTBC is a useful model for studying the more complex
flavonoids. Flavonoids are widely distributed in fruits and
vegetables and are widely used as nutritional supplements. There
are several reviews on the antioxidant properties of these
polyphenols,11 and there is theoretical12 and experimental13
evidence linking their antioxidant properties to the catechol
moiety usually found in their structures. The catechol structure
is also present in natural catechol amines, such as L-dopa and
dopamine, which are reported to have both toxic and antioxidant
effects,14 and in catechol steroids, where effective antioxidant
activity was found in lipoproteins15 and rat liver microsomes.16
Many of these studies were carried out in the presence of heavy
metal ions, thus it is difficult to evaluate the activities of
polyphenols as radical chain breaking antioxidants because they
may also act as preVentatiVe antioxidants by complexing metal
ions.17 We use DTBC as a simple model system to study the
H-atom donating activity of the catechol system, including
effects of solvents, and by implication resolve some of the
uncertainties concerning the activities of the more complex
natural polyphenols as H-atom donors and antioxidants. It is
clear from our current results that DTBC is the most active
H-atom donor to DPPH in a hydrocarbon solvent (see Table
1). However, it is also clear that its activity decreases
substantially, more than any of the other phenols studied, in
the alcohols and especially in acetone, where the decrease is
more than 3 orders of magnitude from the value in hexane.
Evidence was provided earlier to show that the main effect
controlling the activity of DTBC as an antioxidant is the
(10) (a) Howard, J. A.; Ingold, K. U. Can. J. Chem. 1963, 41, 2800-
2806. (b) MacFaul, P. A.; Ingold, K. U.; Lusztyk, J. J. Org. Chem. 1996,
61, 1316-1321. (c) Noguchi, N.; Iwaki, Y.; Takahashi, M.; Komuno, E.;
Kato, Y.; Tamura, K.; Cynishi, O.; Kodawa, T.; Niki, E. Arch. Biochem.
Biophys. 1997, 342, 236-243.
(11) (a) Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Trends Plant Sci.
1997, 2, 152-159. (b) Kandaswami, C.; Middleton, E. In Natural
Antioxidants, Chemistry, Health Effects, and Applications; Shahidi, F., Ed.;
AOAC Press: Champaign, IL, 1997; Chapter 10, pp 174-203. (c) Li, W.;
Sun, G. Y. In Biological oxidants and Antioxidants. Molecular Mechanisms
and Health Effects; Packer, L., Ong, A. S. H., Eds.; AOAC Press:
Champaign, IL, 1998; Chapter 12, pp 90-103. (d) Cook, N. C.; Samman,
S. J. Nutr. Biochem. 1996, 7, 66-76. (e) Formica, J. V.; Regelson, W. Ed.
Chem. Toxic. 1995, 33, 1061-1080.
(12) van Acker, S. A. B. E.; de Groot, M. J.; van den Berg, D. J.; Tromp,
M. N. J. L.; den Kelder, G. D. O.; van der Vijh, W. J. F.; Bast, A. Chem.
Res. Toxicol. 1996, 9, 1305-1312.
(13) (a) Jovanovic, S. V.; Steenken, S.; Hara, Y.; Simic, M. G. J. Chem.
Soc., Perkin 2 1996, 2497-2504. (b) Arora, A.; Nair, M. G.; Strasburgh,
G. M. Free Radical Biol. Med. 1998, 24, 1355-1363. (c) Jovanovic, S.
V.; Steenken, S.; Tosic, M.; Marjanovic, B.; Simic, M. J. Am. Chem. Soc.
1994, 116, 4846-4952. (d) Foti, M.; Piattelli, M.; Baratta, M. T.; Ruberto,
G. J. Agric. Food Chem. 1996, 44, 497-501.
(14) (a) Hastings, T. G.; Lewis, D. A.; Zigmond, M. J. Proc. Natl. Acad.
Sci. U.S.A. 1996, 93, 1956-1961. (b) Mytilineou, C.; Han, S. K.; Cohen,
G. J. Neurochem. 1993, 61, 1470-1478.
(15) (a) Tang, M.; Abplanalp, W.; Ayers, S.; Subbiah, M. T. R.
Metabolism 1996, 45, 411-414. (b) Taniguchi, S.; Yanase, T.; Kobayashi,
K.; Takayanagi, R.; Haji, M.; Umeda, F.; Nawata, H. Endocrinol. Jpn. 1994,
41, 605-611.
(16) (a) Lacort, M.; Leal, A. M.; Liza, A. M.; Martin, C.; Matiinez, R.;
Ruiz-Larrea, M. B. Lipids 1995, 30, 141-146. (b) Takanashi, K.; Watanabe,
K.; Yoshizawa, I. Biol. Pharm. Bull. 1995, 18, 1120-1125.
(17) For a discussion of the role of metal ion complexing agents as “metal
ion deactivators” compared to chain-breaking antioxidants see: Antunes,
F.; Barclay, L. R. C.; Ingold, K. U.; King, M.; Norris, J. Q.; Scaiano, J. C.;
Xi, F. Free Radical Biol. Med. 1999, 26, 117-128.
stabilization by intramolecular H-bonding of the aroxyl radical
semiquinone (Scheme 1, B) formed in the rate-determining
inhibition step.8 It is expected that protic solvents would interfere
with this stabilization through intermolecular H bonding so that
the H-atom donating activity is greatly reduced as found.18
However, it is not completely clear how acetone causes an
additional drop in activity of DTBC as a H-atom donor. It
appears that acetone is able to act more strongly as a HBA on
the diol, perhaps by acting as an effective HBA on both phenolic
hydroxyl groups, thus blocking the initial state of the catechol
(Scheme 1, A) from H-atom abstraction more effectively than
the alcohols do. These results with the model system, DTBC,
indicate that experiments to measure the H-atom donor or
antioxidant activities of polyphenols such as the flavonoids must
be carried out with great care, and must take into account the
possible effects of traces of metal ions and of the solvent system
used. In addition, other isolated phenolic groups in the system
may impart some prooxidant activity (e.g., start new oxidation
chains), because the phenoxyl radical is now known to possess
high reactivity19 even compared to peroxyl radicals (the main
chain-carrying radicals in lipid peroxidation).
As indicated in the Results, solvent effects on the second-
order rate constants for H-atom abstraction from the phenols
by the oxygen-centered radical, DBMP, parallel the results found
for DPPH. We have just sufficient data to confirm an important
principle, namely, that the kinetic solvent effects (KSE) on
H-atom abstractions from hydroxyl groups are independent of
the nature of the abstracting radical.3 Thus the ratios of the KSE
observed, khexane/ksolvent, for H-atom abstraction from DBHA and
BHT by the nitrogen-centered radical DPPH are approximately
equal to these ratios for H-atom abstraction by the oxygen-
centered radical DBMP for the solvents used, with the exception
of DBHA in acetone (see Table 3).
DPPH is frequently used to measure the H-atom donor
properties of antioxidants, especially the flavonoids.20 The
oxygen-centered radical, DBMP, provides the same advantage.
Although it has been used less frequently, its H-atom abstracting
activity appears to follow the antioxidant activity of the
tocopherols, at least in a relative manner.21 While studies with
stable radicals such as DPPH and DBMP provide quantitative
evidence on H-atom donating activities of antioxidants, actual
antioxidant actiVities must measure the ability of the H-atom
donors to trap peroxyl radicals. Here the H-atom donating
antioxidants must be more active than the substrate to break
the propagating chain reaction, eq 7. In other words kinh (eq 2)
(18) A Reviewer pointed out that if the solvent effect acts to destabilize
the semiquinone, this could be “reflected as a decrease in the antioxidant
activities of the parent catechol” by accelerating disproportionation or
radical-radical coupling processes.
(19) Foti, M.; Ingold, K. U.; Lusztyk, J. J. Am. Chem. Soc. 1994, 116,
9440-9447.
(20) (a) Sanchez-Moreno, C.; Larrauri, J. A.; Saura-Calixto, F. J. Sci.
Food Agric. 1998, 76, 270-276. (b) Ko, F. N.; Chu, C. C.; Lin, C. N.;
Chang, C. C.; Teng, C.-M. Biochim. Biophys. Acta 1998, 1389, 81-90. (c)
Bonet, V.; Brand-Williams, W.; Berset, C. Food Sci. Technol. 1997, 30,
609-615. (d) Soares, J.; Ninis, T. C. P.; Cunha, A. P.; Almeida, L. M.
Free Radical Res. 1997, 26, 469-478.
(21) Mukai, K.; Fukuda, K.; Ishizu, K. Chem. Phys. Lipids 1988, 46,
31-36.

6230 J. Am. Chem. Soc., Vol. 121, No. 26, 1999
must be orders of magnitude greater than kp.
Barclay et al.
Experimental Section
Materials. The highest grade of solvents was used for all analyses
and they were distilled just before use. The stable radical, 1,1-diphenyl-
2-picrylhydrazyl (DPPH), was purchased from Sigma and used as
received. The 2,6-di-tert-butyl-4-(4′-methoxyphenyl)phenol, provided
by Dr. Kazuo Mukai,21 was converted to the radical (DBMP) by
oxidation with an excess of lead dioxide25 under argon in the solvent
of interest immediately before use. The antioxidants 2,6-di-tert-butyl-
4-methylphenol (BHT) and 2,6-di-tert-butyl-4-methoxyphenol (DBHA)
were purchased from Aldrich and stored at -23 °C. The catechol
antioxidant, 3,5-di-tert-butylcatechol (DTBC), was also purchased from
Aldrich and stored at room temperature. The vitamin E model, 2,2,5,7,8-
pentamethyl-6-hydroxychroman (PMHC), was synthesized as de-
scribed26 and stored at -23 °C. It was recrystallized twice from hexane
prior to use. Solutions of antioxidants were prepared in known
concentrations in the solvent of choice just prior to use. Azo-bis-
isobutyrylnitrile (AIBN) from Eastman was recrystallized from metha-
nol and stored at -23 °C. Solutions of known concentration were
prepared in chlorobenzene or tert-butyl alcohol to initiate reaction in
the autoxidation experiments. Styrene from Aldrich was passed through
a column of neutral alumina under argon to remove commercial
inhibitor and traces of hydroperoxides just before use.
Kinetic Measurements. 1. Diode Array. Experiments were con-
ducted with an HP-8452A diode array spectrophotometer at 30 °C under
an argon atmosphere. The equipment was flushed with the solvent of
choice prior to the experiment, and a blank baseline obtained. Solutions
were prepared just before use under argon and the concentration of the
radical was checked by UV. The decay of the radical with time in the
absence of antioxidant was monitored in the solvent prior to each
experiment, to ensure the kobs value was not being affected by radical
decay. Samples were rapidly mixed in the reaction cuvette with use of
an Applied Photophysics Model RX1000 stop flow assembly. The
loading syringes were filled with the radical (syringe 1) and the
antioxidant (syringe 2), and the cuvette was flushed with an excess
volume of these for each measurement to ensure no contamination from
the previous injection. Syringe 2 was rinsed with solvent each time
the antioxidant concentration was changed. A minimum of five different
concentrations of antioxidant were used to obtain the second-order rate
constant in each solvent system, with a minimum of three measurements
at each antioxidant concentration. At least one duplicate experiment
was conducted. The R2 for the determinations of kobs and k2 was 0.90-
0.99.
(a) Measurements with DPPH Radical. The molar absorptivity
was calculated to be ꢀ ) 1.34 × 103 (λmax ) 515 nm in methanol),
assuming 90% purity of the DPPH sample, compared to the literature
value of ꢀ ) 1.43 × 103 (λmax ) 525 nm in chloroform).27 The literature
value was used in all calculations. The actual concentration ratio of
[antioxidant]/[DPPH] used in the experiments depended upon the
antioxidant used and the solvent: with DBHA, average [DPPH] ) 6.66
× 10-5 M, and [DBHA]/[DPPH] varied from 12 to 1684; with BHT,
average [DPPH] ) 6.47 × 10-5 M, and [BHT]/[DPPH] varied from
47 to 1394; with PMHC, average [DPPH] ) 9.46 × 10-5 M, and
[PMHC]/[DPPH] varied from 2 to 60; and with DTBC, average [DPPH]
) 8.81 × 10-5 M, and [DTBC]/[DPPH] varied from 1 to 101.
(b) Measurements with DBMP Radical. An excess of lead dioxide
was placed in a test tube with 2 mL of the solvent of interest under
argon. A known amount of 2,6-di-tert-butyl-4-(4′-methoxyphenyl)-
phenol was added and the sample was vortex mixed for 1 min, then
centrifuged on a benchtop centrifuge for 2 min. The solvent was
removed and filtered into a 50 mL volumetric with use of a Millex-SR
0.5 mm syringe tip filter. This was repeated with a second aliquot of
solvent to obtain all the radical, the volumetric was prepared to the
mark under argon, and the concentration of the DBMP radical was
determined by using UV. The molar absorptivity was determined to
be ꢀ ) 2.41 × 104 (λmax ) 370 nm in hexane). The actual concentration
kp
Rs-H (substrate) + ROO• 98 Rs• + ROOH
(7)
Since protic solvents show a remarkable effect on the H-atom
donating ability of common antioxidants, it is important to
quantitatively determine the effect of a protic solvent on the
peroxyl radical trapping efficiency of antioxidants. The effect
of tert-butyl alcohol on the kinh of PMHC, R-tocopherol, and
catechols was determined under controlled conditions during
peroxidation of styrene. The protic solvent decreases the kinh of
both R-tocopherol and PMHC in a similar manner (Figure 2).
At a molar ratio of alcohol to styrene of 1.0 to 1.2, the initial
differences in kinh values between PMHC and R-tocopherol are
“leveled”22 and reduced to about one-quarter of their values in
styrene. This indicates a strong HBA effect of tert-butyl alcohol
at the phenolic hydroxyl as it displaces the hydrocarbon as
solvent. The kinh of PMHC and R-tocopherol continues to drop
as the molar ratio of alcohol to styrene is increased to 7.4 (Figure
2), where one can assume that the solvent effect approximates
that of neat tert-butyl alcohol. At this point the kinh has decreased
by a factor of 10 times less than that in neat styrene. This factor
is more than the estimated decrease of 3.9 times for H-atom
abstraction from R-tocopherol in water compared to benzene,3a
and probably reflects a larger difference in the HBA values in
our experiments (between styrene and tert-butyl alcohol)
compared to the HBA difference between benzene and water.23
These results now indicate clearly that the drop in kinh for
R-tocopherol observed earlier in heterogeneous phases of
phospholipid bilayers, which is several orders of magnitude
compared to homogeneous solution,2d must be due to a
combination of various effects. The R-tocopherol may in part
be physically inaccessible3a to the lipid peroxyl radicals due to
nonuniform distribution and restricted diffusion in the bilayer.2c
The hydrogen bond acceptor (HBA) effect alone, although it is
significant, accounts for only a portion of the dimunition in
antioxidant activity.
The profiles of antioxidant activities of the catechols also
exhibit a drop of kinh with increased tert-butyl alcohol concen-
tration (Figure 3). We could follow the effect on kinh to mole
ratios of tert-butyl alcohol to styrene of only 0.03 to 0.04. At
higher ratios, there were not satisfactory inhibition periods to
measure τ values, since in alcohol the catechols appeared to
behave like “retarders” rather than true antioxidants.17 The
“antioxidant activities” of polyphenols such as the flavonoids
are often reported in alcohols in terms of the concentration of
flavonoid required to decrease the concentration of a monitoring
radical (i.e., DPPH) by 50%, the EC50 value,20a which is different
from the determination of the absolute rate constant for H-atom
transfer, kinh, which is a quantitative measure of the antioxidant
actiVity. Even in chlorobenzene, the solvent commonly used
5,7
for quantitative measurements of kinh
,
the flavonoids do not
behave as classical phenolic antioxidants.24 Our results on the
catechols provide a basis for additional studies in more
biologically relevant systems such as phospholipid bilayers.
(22) The leveling effect of antioxidant activity of R-tocopherol toward
that of BHT noted before in phospholipid membranes compared to
homogeneous solutions2d is now recognized to be partly or even mainly
due to restricted diffusion of lipophilic molecules, like R-tocopherol between
or within the bilayer or micellar structures.1,2c,3c
(23) The â2H values of water and benzene are 0.38 and 0.14, respectively,9
giving a difference of 0.24; using the log KBH ) - 0.27 for styrene9 gives
(25) Rieker, A.; Scheffler, K. Ann. Chem. 1965, 689, 78-92.
(26) Smith, L. L.; Ungrade, H. E.; Hoehn, H. N.; Wawzonek, S. J. Org.
Chem. 1939, 4, 311-317.
(27) Poirier, R. H.; Kahler, E. J.; Bennington, F. J. Org. Chem. 1952,
17, 1437-1445.
H
a â2 of 0.18 and a difference between tert-butyl alcohol and styrene of
0.49 - 0.18 ) 0.31.
(24) Roginsky, V. A.; Barsukova, T. K.; Remorova, A. A.; Bors, W. J.
Am. Oil Chem. Soc. 1996, 73, 777-786.

Antioxidant ActiVities of Phenols and Catechols
J. Am. Chem. Soc., Vol. 121, No. 26, 1999 6231
ratio of [antioxidant]/[DBMP] used in the experiments again depended
upon the antioxidant used and the solvent: with DBHA, average
[DBMP] ) 6.11 × 10-5 M, and [DBHA]/[DBMP] varied from 1 to
712; with BHT, average [DBMP] ) 8.79 × 10-5 M, and [BHT]/
[DBMP] varied from 4 to 308; and with PMHC, average [DBMP] )
7.32 × 10-5 M, and [PMHC]/[DBMP] varied from 1 to 15.
reaction was initiated by injection of a known amount of AIBN in
chlorobenzene. In cases where combinations of styrene and tert-butyl
alcohol were used, the AIBN was injected in a known volume of the
alcohol. When a constant uptake of oxygen was reached, a known
amount of antioxidant was injected and the suppressed oxygen uptake
measured until the rate of oxidation returned to the uninhibited rate.
The length of the inhibition period was measured to check the rate of
chain initiation (Ri, see text). This procedure was generally repeated
three times for determinations of inhibition rate constants for each
solvent mixture used.
2. Autoxidation/Inhibition Procedures. Autoxidations were carried
out at 30 °C/760 Torr under oxygen in a calibrated sensitive dual-
channel pressure transducer apparatus that is described elsewhere.28
The procedure for initiation of the oxidation of styrene and measuring
oxygen uptake during inhibition periods reported before7 was generally
followed. The apparatus was first “conditioned” with styrene or
combinations of styrene and tert-butyl alcohol in both the sample side
and reference side. The reference side was injected with antioxidant to
prevent self-initiated oxidation. For reactions in neat styrene, the
Acknowledgment. This manuscript is dedicated to Dr. Keith
Ingold on the occasion of his 70th birthday. Support of this
research by a grant from the Natural Sciences and Engineer-
ing Research Council of Canada (NSERC) is gratefully
acknowledged.
(28) Wayner, D. D. M.; Burton, G. W. Handbook of Free Radicals and
Antioxidants in Biomembranes; CRC Press: Boca Raton, FL, 198X; Vol.
III, pp 223-232.
JA990878U