Oxidation reactions of thymol: A pulse radiolysis and theoretical study

Article abstract of DOI:10.1021/jp3082358

The reactions of ^?OH and O^?-, with thymol, a monoterpene phenol and an antioxidant, were studied by pulse radiolysis technique and DFT calculations at B3LYP/6-31+G(d,p) level of theory. Thymol was found to efficiently scavenge OH radicals (k = 8.1 × 10⁹ dm³ mol^-1 s^-1) to produce reducing adduct radicals, with an absorption maximum at 330 nm and oxidizing phenoxyl radicals, with absorption maxima at 390 and 410 nm. A major part of these adduct radicals was found to undergo water elimination, leading to phenoxyl radicals, and the process was catalyzed by OH^- (or Na₂HPO₄). The rate of reaction of O^?- with thymol was found to be comparatively low (k = 1.1 × 10⁹ dm³ mol ^-1 s^-1), producing H abstracted species of thymol as well as phenoxyl radicals. Further, these phenoxyl radicals of thymol were found to be repaired by ascorbate (k = 2.1 × 10⁸ dm³ mol ^-1 s^-1). To support the interpretation of the experimental results, DFT calculations were carried out. The transients (both adducts and H abstracted species) have been optimized in gas phase at B3LYP/6-31+G(d,p) level of calculation. The relative energy values and thermodynamic stability suggests that the ortho adduct (C6-OH adduct) to be most stable in the reaction of thymol with OH radicals, which favors the water elimination. However, theoretical calculations showed that C4 atom in thymol (para position) can also be the reaction center as it is the main contributor of HOMO. The absorption maxima (λ_max) calculated from time-dependent density functional theory (TDDFT) for these transient species were close to those obtained experimentally. Finally, the redox potential value of thymol^?/ thymol couple (0.98 V vs NHE) obtained by cyclic voltammetry is less than those of physiologically important oxidants, which reveals the antioxidant capacity of thymol, by scavenging these oxidants. The repair of the phenoxyl radicals of thymol with ascorbate together with the redox potential value makes it a potent antioxidant with minimum pro-oxidant effects.

Full text of DOI:10.1021/jp3082358

Article
pubs.acs.org/JPCA
Oxidation Reactions of Thymol: A Pulse Radiolysis and Theoretical
Study
S. Venu,† D. B. Naik,‡ S. K. Sarkar,‡ Usha K. Aravind,§ A. Nijamudheen,∥,& and C. T. Aravindakumar*,⊥,#
†School of Chemical Sciences, §Advanced Centre of Environmental Studies and Sustainable Development, ⊥School of Environmental
#
Sciences, and Inter University Instrumentation Centre, Mahatma Gandhi University, Kottayam 686560, Kerala, India
‡Radiation and Photochemistry Division, Bhabha Atomic Research Centre, Mumbai 400085, India
∥Indian Institute of Science Education and Research, Thiruvananthapuram, Kerala, India
S
* Supporting Information
•
ABSTRACT: The reactions of OH and O•−, with thymol, a monoterpene
phenol and an antioxidant, were studied by pulse radiolysis technique and DFT
calculations at B3LYP/6-31+G(d,p) level of theory. Thymol was found to
efficiently scavenge OH radicals (k = 8.1 × 109 dm3 mol−1 s−1) to produce
reducing adduct radicals, with an absorption maximum at 330 nm and oxidizing
phenoxyl radicals, with absorption maxima at 390 and 410 nm. A major part of
these adduct radicals was found to undergo water elimination, leading to
phenoxyl radicals, and the process was catalyzed by OH− (or Na2HPO4). The
rate of reaction of O•− with thymol was found to be comparatively low (k = 1.1
× 109 dm3 mol−1 s−1), producing H abstracted species of thymol as well as
phenoxyl radicals. Further, these phenoxyl radicals of thymol were found to be
repaired by ascorbate (k = 2.1 × 108 dm3 mol−1 s−1). To support the
interpretation of the experimental results, DFT calculations were carried out.
The transients (both adducts and H abstracted species) have been optimized in gas phase at B3LYP/6-31+G(d,p) level of
calculation. The relative energy values and thermodynamic stability suggests that the ortho adduct (C6_OH adduct) to be most
stable in the reaction of thymol with OH radicals, which favors the water elimination. However, theoretical calculations showed
that C4 atom in thymol (para position) can also be the reaction center as it is the main contributor of HOMO. The absorption
maxima (λmax) calculated from time-dependent density functional theory (TDDFT) for these transient species were close to
those obtained experimentally. Finally, the redox potential value of thymol•/thymol couple (0.98 V vs NHE) obtained by cyclic
voltammetry is less than those of physiologically important oxidants, which reveals the antioxidant capacity of thymol, by
scavenging these oxidants. The repair of the phenoxyl radicals of thymol with ascorbate together with the redox potential value
makes it a potent antioxidant with minimum pro-oxidant effects.
body.2 Thus, natural antioxidants as a safe alternative to synthetic
antioxidants are always of interest. In this context, phenolic
1. INTRODUCTION
Studies on natural antioxidants and their role in human health
antioxidants have high importance in the antioxidant series.
and drug design are subjects of general interest of the past several
Thymol (2-isopropyl-5-methylphenol) is a phenolic antioxidant
decades.1 The majority of the antioxidants comprise phenolic
with high relevance in the free radical scavenging processes and it
compounds that can scavenge reactive oxygen species and
reactive nitrogen species. These reactive species are generated in
the biological systems as a result of exposure to ionizing
radiations, photosensitization, enzymatic, and biochemical
is the compound of interest in the present study.
Thymol is a colorless crystalline monoterpene phenol
generally isolated from medicinal plants such as Thymus vulgaris
and Oroganum vulgare.3−7 It has a pleasant aromatic odor and is
processes. These oxidizing species have a deleterious effect on
slightly soluble in water at ambient pH. It can be considered a
living cells, mainly on their interaction with DNA, which may
derivative of cymene, containing one phenolic hydroxyl group
lead to diseases like cancer, tumors, and arthritis and also to
(OH), one methyl group, and one isopropyl group. Thymol
aging. Of the various reactive free radicals, the OH radical is
possesses antimicrobial, antifungal, and antioxidant properties
considered one of the most devastating free radicals and is among
and is a major component of the oils of thyme.8 These properties
the major reactive oxygen species generated during exposure to
are attributed to the presence of the phenolic hydroxyl group in
radiation. The importance of any antioxidant is that it has a
its structure, as phenolic compounds are known to show potent
sacrificial role in scavenging highly reactive free radicals. Both
natural and synthetic antioxidants are generally known to
scavenge free radicals. However, synthetic phenolic antioxidants,
such as butylated hydroxyanisole and butylated hydroxytoluene,
are not advisable, as they have an adverse effect on the human
Received: August 18, 2012
Revised: December 13, 2012
Published: December 14, 2012
© 2012 American Chemical Society
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H• + OH− → e− + H2O
antioxidant activity by absorbing and neutralizing free radicals
that are harmful to biomolecules.9,10 The use of thymol dates
back to the ancient Egyptians, in conjunction with its isomer,
carvacrol, for the preservation of mummies. It is also used as an
active ingredient in food flavorings, perfumes, cosmetics,
mouthwash, and a number of oils and topical ointments that
have been formulated for massaging the joints and to treat nail
fungi. According to the EPA (Environmental Protection
Agency), there are no known adverse effects of the use of
thymol when used in animals and humans.11
aq
(3)
On saturating the solution with N2O at high pH, e− is
aq
quantitatively converted to O•−, because •OH is in equilibrium
with O•− at high pH (•OH ⇔ O•− ; pKa = 11.9)
•
N2O + e− → OH + OH− + N2
aq
(4)
(5)
•OH + OH− ⇔ O•− + H2O
The bimolecular rate constant values were determined from the
rate of buildup of transients (pseudounimolecular) under varying
concentrations of thymol.
In an earlier free radical study, thymol is reported to be a good
scavenger of peroxyl radicals (CCl3O2; calculated rate constants
>106 dm3 mol−1 s−1) with less prooxidant effects.12 In this study,
a detailed investigation is carried out to understand the reaction
of OH radicals with thymol using the pulse radiolytic technique.
2.3. Computational Method. Density functional theory
calculations were performed on the Gaussian 03 computational
package.16 As Becke’s three-parameter hybrid functional
combined with correlation functional of Lee, Yang, and Parr,
denoted as B3LYP17 has been effective for obtaining the optimal
geometries, harmonic frequencies, and electronic properties, a
similar procedure is followed for the present study. All the
geometries were optimized without any symmetry constrains,
with the 6-31+G(d,p) basis set, in gas phase. Spin unrestricted
calculations were performed for open shell systems. Optimized
structures were identified as local minima with real harmonic
vibrational frequencies and the saddle point with one imaginary
frequency. TDDFT method was used to calculate the excited
state electronic properties.18
•
In addition to OH radicals, reactions of azide radical (N3 ) and
oxide radical anion (O•−) have been carried out for a better
understanding of the reaction mechanism. The redox potential of
thymol has been measured using cyclic voltammetry to support
its radical scavenging capacity. Furthermore, to interpret the
experimental results computational calculations using Gaussian
program were also carried out. By and large, a complete oxidative
radical chemistry is investigated with the help of both
experimental and theoretical studies.
2. EXPERIMENTAL SECTION
2.4. Cyclic Voltammetry. The cyclic voltammetric experi-
ments were carried out using an Autolab Electrochemical System
(Eco Chemie, The Netherlands), PGSTAT 20 driven by GPES
software. Platinum wire of about 1 mm diameter was used as
working and counter electrode. The reference electrode used was
Ag/AgCl, 3 M KCl electrode. Working and counter electrodes
were thoroughly cleaned before each measurement. Blank
measurements were also carried out before each scan. Solutions
were deaerated by bubbling N2 for 10 min prior to recording the
cyclic voltammogram.
2.1. Chemicals. Thymol (99.5%) was from Sigma-Aldrich.
Commercially available sodium azide and 2,2′-azinobis(3-
ethylbenzothiazoline-6-sulfonate) (ABTS2−), were purchased
from Aldrich and were used without further purification. All the
reactions were carried out in aqueous medium. Solutions were
prepared in water from Millipore A-10 system having
conductivity less than 0.1 μS/cm.
Thymol is reported to aggregate in aqueous medium having a
critical micellar concentration of 4 μ mol dm−3.13 Therefore,
UV−vis spectra were recorded at varying concentrations of
thymol (0.12−500 μmol dm−3) with and without the presence of
phosphate buffer (see Supporting Information). However, we
did not observe any significant change in the spectral properties
and hence the indication of aggregation under our experimental
conditions (i.e., in pure aqueous medium).
3. RESULTS AND DISCUSSION
3.1. Reaction with OH Radicals. a. Pulse Radiolysis
Studies. Reaction of OH radicals with thymol was carried out at
pHs 5.8, 7.8, and 9.2, where thymol exists in its neutral form. A
bimolecular rate constant of 8.1 × 109 dm3 mol−1 s−1 was
determined from the pseudo-first-order buildup of the transient
at 340 nm as a function of thymol concentration at pH 5.8 (Table
1). The dependence of the pseudo-first-order rate constant
2.2. Pulse Radiolysis. Radiolysis of water produces three
major radical species e−aq, •OH, •H, along with less reactive H2
and H2O2.
•
•
H2O ⇝ e−aq, OH, H, H2, H2O2, H3O+
(1)
Table 1. Absorption Maxima of the Transients and Second-
Order Rate Constants for the Reaction of Thymol with
Selected Oxidizing Radicals
Pulse radiolysis experiments were carried out using a linear
accelerator delivering electron pulse of 7 MeV energy of 50 ns
duration. An aerated aqueous solution of KSCN (1 × 10−2 mol
dm−3) was used to monitor the dose per pulse using a Gε value of
2.6 × 10−4 m2 J−1 for (SCN)2•− at 475 nm,14 and the dose was
kept at 13.5 Gy. The details of pulse radiolysis setup have been
described elsewhere.15
radicals
pH
5.8
λmax (nm)
330
k2 (dm3 mol−1 s−1
)
•OH
O•−
8.1 × 109
1.1 × 109
2.3 × 109
>13.5
6.8
330, 390, 410
390, 410
•
N3
To study the reaction of thymol with OH radicals, eaq− formed
in the radiolysis were converted to OH radical by saturating the
solution with N2O.
against the concentration of thymol is presented in the
Supporting Information. The transient spectrum recorded at 2
μs after the pulse at pH 5.8 has a maximum at 330 nm (Figure 1).
This was found to undergo decay with respect to time. A small
hike in the spectrum at around 400 nm region (390 and 410 nm)
is also observed when the spectrum was recorded at 40 μs after
the pulse. Although the absorbance is very feeble at this region, it
was highly reproducible. When the spectrum was recorded at pH
The specific one-electron oxidant N3• radical was produced in
N2O saturated solution containing 0.02 mol dm−3 NaN3.
•
N3− + OH → N3• + OH−
(2)
In basic conditions, the H• is converted to e− with a rate
aq
constant of 2.2 × 107 dm3 mol−1 s−1, according to eq 3.
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Figure 3. Time-resolved transient absorption spectra obtained by pulse
Figure 1. Time-resolved absorption spectra obtained by pulse radiolysis
of N2O saturated 5 × 10−4 mol dm−3 thymol solution at (a) pH 5.8 and
radiolysis of N2O-saturated aqueous solution containing 0.01 mol dm−3
■
■
○
NaN3 and 5 × 10−4 mol dm−3 thymol solution at pH 6.8 after ( ) 2 μs
(b) pH 7.8 at ( ) 2 μs and ( ) 40 μs after the electron pulse. Insets: (i)
Decay traces at 340 nm at pH 5.8. (ii) Absorption buildup of ABTS•− at
735 nm.
○
and ( ) 40 μs after the electron pulse. Insets: (a) absorption buildup of
ABTS•− at 735 nm obtained from the reaction of intermediate with
ABTS2− at pH 6.8; (b) decay of phenoxyl radical of thymol at 410 nm
obtained by the reaction of 5 × 10−4 mol dm−3 thymol with •N3 at pH
6.8 (very similar decay trace was obtained in the presence of N2O:O2 =
4:1).
7.8, the absorption buildup was more prominent at 400 nm
region (Figure 1). A similar trend was observed even at pH 9.2.
At pH 9.2 the transient absorption at 330 nm fully decayed after
20 μs (spectrum not shown).
The absorption traces at 330 nm obtained at pH 5.8 and 7.8
showed that the decay rate of the transient at 330 nm followed
first-order kinetics and was found to increase with pH (kΦ = 1.09
× 104 s−1 at pH 5.8 and kΦ = 2.9 × 104 s−1 at pH 7.8). The decay
was also monitored in the presence of Na2HPO4 and a significant
increase in the rate of decay is observed. Because the rate of decay
at 330 nm was found to increase with an increase in the
concentration of Na2HPO4 (Figure 2), it is clear that the rate of
transformation can also be enhanced by phosphate
oxidizing nature of the intermediate radical is demonstrated from
the reaction of ABTS2−. The absorption buildup obtained at 735
nm (Figure 3) confirms the formation of an oxidizing radical of
thymol.
Azide radical is a known specific one-electron oxidant20 (Eo =
1.33 V vs NHE). This radical is generally known to produce a
radical cation of both heterocyclic and homocyclic compounds.21
It is reported that a phenolic type radical can be formed from the
reaction of azide radical with organic compounds containing
phenolic moiety.22,23 In this context, it is very likely that the azide
radical produces a phenoxyl radical potentially formed from the
result of deprotonation of the initially formed radical cation
(reaction 6). The oxygen-centered radical can be convincingly an
oxidizing radical that can easily react with ABTS2−. A similar
oxidizing nature for oxygen-centered radical is reported.24−26
The oxidizing property of this intermediate was further
confirmed in the presence of oxygen. A typical absorption
decay at 410 nm in the presence of 20% oxygen was investigated.
A very similar decay pattern at 410 nm was observed in the
absence and presence of oxygen, which clearly indicates the
oxidizing nature of the intermediate and hence the assignment as
phenoxyl radical.
Figure 2. Dependence of the decay kinetics of the intermediate with
respect to the concentration of Na2HPO4 in the reaction of •OH with
thymol (5 × 10−4 mol dm−3) at 330 nm. Inset: decay at 330 nm without
buffer.
To study the redox property of the transients, reactions were
carried out in the presence of ABTS2−, at sufficiently low
concentrations at pH 5.8 so as to avoid direct reaction of OH
radicals with the reductant. A clear absorption buildup of
ABTS•− was monitored at 735 nm (Figure 1). This clear
absorption buildup of ABTS•− indicated the presence of an
oxidizing intermediate, as it is well-known that the one-electron
oxidation of ABTS2− by the oxidizing radicals generates ABTS•−
absorbing at 735 nm.19
As it is presumed that only the phenoxyl radical is formed from
the reaction of azide radical with thymol, the yield of ABTS•− is
taken as 100%. Based on this assumption, the yield of ABTS•− in
the case of the reaction of OH radical is calculated as 0.22 μmol
J−1 at pH 5.8, which constitutes nearly 35% of the total reaction.
On comparison of the spectrum obtained with azide radicals
(Figure 3) with that of the OH radical (Figure 1a,b), it is possible
to assign the absorption maxima at 390 and 410 nm to the
phenoxyl radical in both the reactions. Thus, the absorption at
390 and 410 nm observed in the case of the reaction of OH
radicals is an indication of the formation of the phenoxyl type
radical of thymol, though in a low yield (Figure 1). A close
analysis of the spectrum (Figure 1) at longer time scale shows a
•
The reaction of azide radical (N3 ) with thymol has been
carried out at pH 6.8. The time-resolved transient absorption
•
spectrum obtained from the reaction of N3 with thymol is
shown in Figure 3. A reaction rate constant of 2.3 × 109 dm3
mol−1 s−1 has also been determined. The transient spectrum
showed two clear absorption maxima at 390 and 410 nm. The
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buildup of the absorbance at 390 and 410 nm with a concomitant
decay at 330 nm.
On the other hand, the reaction of OH radical with thymol
resulted an absorption spectrum with a predominant species
having a maximum at 330 nm in the initial stage. One of the
major possibilities of •OH attack is in the benzene ring itself with
a characteristic absorption in the 310−350 nm range.22,23,27−32 It
is well-known that OH radicals add to benzene ring systems to
form cyclohexadienyl type radicals.21,22,27 It is therefore highly
likely that the initial species with 330 nm is the OH-adduct of
thymol. The decay at the 330 nm can be understood as the
dehydration of the OH-adduct to produce the stable phenoxyl
radical of thymol (reaction 7).
Figure 4. Optimized geometry of thymol at the B3LYP/6-31+G(d,p)
level of theory. Bond lengths are in Å.
Thus probable mechanism of the reaction of OH radicals with
thymol, involves the initial formation of an OH_adduct, which
then undergoes a water elimination reaction catalyzed by OH−
(or Na2HPO4) leading to a phenoxyl radical. On the other hand,
the yield of phenoxyl radical, as obtained from the yield of
ABTS•−, is only about 35%, which indicates that there could be
other intermediates formed under this conditions. It is
understandable that only those additions which are adjacent to
the −OH group can favor water elimination. It is very probable
that •OH can attack in other positions as well. As these individual
adduct isomers are closely related to their physical and chemical
properties, it is a difficult task to identify these isomers from their
spectral behavior. Computational methods have been proven
effective for predicting the probable reaction mechanisms
involving radical species.33,34 To resolve the existence of the
various adduct radicals and to clearly identify the structure and
the reaction possibilities, we have carried out a detailed DFT
calculation and the results obtained are discussed and compared
with the experimental findings.
Figure 5. MESP (Vmin) located near the C2, C6, and C4 atoms.
radical to all five sites of the aromatic ring (Scheme 1). The
adducts thus formed, viz. C2_OH, C3_OH, C4_OH, C5_OH,
and C6_OH, have been modeled and optimized. The optimized
geometries of the adduct radicals are depicted in Figure 6. Also,
their relative energy values with respect to the sum of the
energies of the thymol molecule and the OH radical are shown.
For the C2_OH adduct, the spin maxima are centered on the C5,
C1, and C3 atoms and the values are 0.58, 0.49 and 0.35,
respectively. For the C6_OH adduct, the spin maxima are on the
C3, C5, and C1 atoms and the values obtained are 0.6, 0.5, and
0.4 respectively. These correspond to the resonance forms of the
thymol_•OH adduct radical systems.
According to the relative energy values, the order of stability of
the OH adduct of thymol, computed in the gas phase is C6_OH
adduct > C2_OH adduct > C4_OH adduct. The relative
enthalpies as well as the relative free energy values of these radical
adducts also show the same trend as that of their relative energy
values (Figure 7). Furthermore, the relative enthalpies, relative
free energy values and the relative energy of these radical adduct
systems also agrees with the MESP analysis, as it shows Vmin at
C2, C4, and C6 (Figure 5).
b. Theoretical Study on the Reaction of OH Radicals.
i. Structure and Electronic Properties of Thymol. The
optimized geometry of thymol in the gas phase is depicted in
the Figure 4 with the important bond lengths in Å.
From the Figure 4 we can see that the bond lengths C1−C6,
C2−C3, and C4−C5 are 1.395, 1.398, and 1.398 Å, respectively.
Of these, the shortest bond distance is between C1 and C6,
suggesting a more double bond character. This may be attributed
to the presence of the phenolic group; otherwise, the bond
lengths would be the same due to the equal distribution of the π
electron cloud over the aromatic ring. Because more double bond
character is observed in the C1−C6 bond, constituent atoms of
this bond are expected to be more reactive sites for an
electrophilic addition reaction with the hydroxyl radical.
The molecular electrostatic potential (MESP) is a useful tool
to predict the reaction center, by locating the electron rich center
at the most negative valued atoms (Vmin).35 The MESP analysis
showed the most negative values of −0.402937, −0.308019, and
−0.169601 for C2, C6, and C4 atoms, respectively. The MESP
plot is shown in Figure 5. These values suggest that C2, C6, and
C4 atoms are the potential centers for the electrophilic attack of
the OH radical.
In addition, we have considered the possibility of the
formation of an intermediate complex between the thymol
ground state and the OH radical. For this we optimized the
structure of an intermediate in which the oxygen of the OH
radical pointed toward the π-face of the thymol. It is found that
the OH radical moved out from the π-face to the plane of the
ii. Addition Reactions of OH Radical with Thymol. We have
initially considered the possibility of the addition of the OH
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Scheme 1. Possible Addition Reactions of Hydroxyl Radicals
with Thymol
Figure 7. Relative enthalpy change (ΔH) and free energy change (ΔG)
for the OH addition reactions computed, in gas phase, at the B3LYP/6-
31+G(d,p) level of theory.
molecule and eventually forming a reaction-complex [thymo-
l···OH] presented in Figure 8. In this complex, the oxygen of OH
Figure 8. Complex formed as a result of OH reaction of thymol,
optimized at the B3LYP/6-31+G(d,p) level.
radical is at a distance of 2.26 Å from C4 atom of thymol and the
relative energy of this complex is −6.8 kcal/mol. The activation
energy required for the formation of the C4_OH adduct from
this complex is found to be of 0.08 kcal/mol (Scheme 2). The
highest occupied molecular orbital (HOMO) of thymol (Figure
9) shows high MO coefficient at C4 atom which favors the
electrophilic addition of the OH radical at this center. This
further supports the addition of OH radical at C4 atom as one of
the potential reaction channels.
Because the C4_OH adduct formation takes place by a
barrierless reaction, the adduct formation reactions is expected to
be a spontaneous reaction leading to a direct addition of hydroxyl
radical to thymol. However, unlike the C4_OH adduct, the two
OH groups in the thermodynamically stable, C2_OH and
C6_OH adducts, are in adjacent positions favoring the
elimination of water, thus yielding the phenoxyl radical of
thymol. The C4_OH adduct is presumed to undergo radical−
radical decay and gives rise to stable end products.
Figure 6. Optimized structures of the adducts, in gas phase, at the
B3LYP/6-31+G(d,p) level of theory. The relative energy is shown in
parentheses.
iii. TDDFT Calculations. The optical absorption maxima
(λmax) for the thermodynamically stable C2_OH and C6_OH
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Scheme 2. Representation of the Formation of the C4_OH Adduct from the Complex S1 via a Transition State (TS), Which Has
Very Low Barrier Energy
Figure 10. Time-resolved transient absorption spectra obtained by pulse
radiolysis of N2O-saturated 5× 10−4 mol dm −3 thymol solution at pH
■
○
∼14 at ( ) 2 μs and ( ) 40 μs after the electron pulse.
Figure 9. Highest occupied molecular orbital of thymol calculated at the
B3LYP/6-31+G(d,p) level of calculation.
electron oxidized species, ABTS•−. A bimolecular rate constant
of 4 × 108 dm3 mol−1 s−1 was also calculated for the reaction. This
value is almost the same as that obtained with the intermediate
formed in the azide radical reaction.
adducts were calculated using TDDFT method and values of
339.11 and 342.11 nm were observed, in the gas phase. The
TDDFT calculation at the same level for the kinetically favored
C4_OH adduct showed a transition wavelength (absorption
maximum) of 332.04 nm. These values are close to the
experimentally observed λmax of 330 nm. Though the TDDFT
calculation supports the existence of the above adducts, the
thermodynamic considerations prefers C6_OH adduct to
C4_OH adduct and the kinetic consideration favors C4_OH
adduct. From the above studies, it is clear that OH radical attacks
thymol, at the ortho (preferably at C6) position to yield adducts
that are thermodynamically more stable than that formed by the
attack at the para (C4) position of the phenolic group. The
experimental results are in line with this observation as both
water eliminated species and the stable (at the pulse radiolysis
scale) OH-adduct contribute to the observed spectrum.
The oxide radical anion (O•−) is known to react by one-
electron oxidation. Hence it reacts with the monoanionic form of
thymol by one-electron oxidation mainly from the anionic center
located at the oxygen atom, leading to the production of the
oxygen-centered phenoxyl radical. From the earlier discussions,
the peaks at 390 and 410 nm were identified as that of the
phenoxyl radicals. These phenoxyl radicals are mostly oxidizing
in nature. The yield of the oxidizing radicals was calculated to be
62.8% of the total radical reaction (assuming that the yield of O•−
is 0.6 μmol J−1).
However, the reaction of thymol anion with O•− radicals can
also proceed via H-abstraction mostly from the −HC (CH3)2
and/or −CH3 groups. Both these reactions lead to the
production of carbon-centered radicals, which are reducing in
nature. As addition of O•− radicals to the aromatic ring is not a
common reaction, it is likely that O•− radicals undergo H-
abstraction reactions and the peak at 330 nm has been assigned
to these H-abstracted species. H-abstraction reaction of O•− is
already reported in a number of compounds.36
3.2. Reaction of O•− with Thymol. a. Pulse Radiolysis. The
reaction of oxide radical anion with thymol has been carried out
at pH ∼ 14. The absorption spectrum obtained for the transient
species as a result of O•− reaction with thymol, recorded at 2 and
40 μs after the electron pulse (Figure 10), is characterized by
absorption maxima at 330, 390, and 410 nm. As the pKa of
thymol is determined to be 10.45, the deprotonated form
(anionic form) of thymol is the species that reacts with O•−
radicals. The bimolecular rate constant for the reaction of O•−
radical was determined by following the buildup of the transient
absorption at 390 nm as a function of thymol concentration and
is given in Table 1.
Thus, it is clear that the reaction of O•− radicals with thymol
produces a mixture of oxidizing and reducing radicals. To
distinguish the two major reaction pathways such as electron
transfer and H-abstraction reaction as well as the various H-
abstracted species, we have carried out DFT calculations as in the
case of the reaction of OH radicals.
b. Theoretical Study on the Reaction of O•− Radicals. The
oxide radical anion (O•−) is known to undergo one-electron
oxidation as well as H-abstraction reactions, compared to
addition reactions, and in this study we have presented the
reaction possibilities of H-abstraction reactions as well as one-
The oxidizing property of the transients was monitored by its
reaction with the reductant, ABTS2−, by monitoring the
absorption buildup at 735 nm, which corresponds to the one-
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electron oxidation reaction from the monoanionic form of
thymol.
is found that this oxygen-centered radical is the most stable one
(with a relative energy of −361.25 kcal mol−1) in the gas phase.
Thus the above thermodynamic considerations show that the
oxygen-centered radical is the most stable radical when
compared to the carbon-centered radicals formed by the H-
abstraction reactions. The probable carbon-centered radicals
formed are mainly IV/I.
i. H-Abstraction Reactions of O•− with Thymol. The H-
abstraction reactions occur mainly from the methyl and isopropyl
substituents of the monoanionic form of thymol. The H-
abstraction results in the carbon-centered radicals and the various
possible radicals are depicted in the Scheme 3. The radicals are
These theoretical studies are in line with the experimental
results, which showed a maximum yield of phenoxyl type radical
as the major transient and the carbon-centered radicals as the
minor transient.
Scheme 3. Possible H-Abstracted Species Formed from the
Reaction of the O•− Radical
3.3. Scavenging of the Phenoxyl Radical of Thymol by
Ascorbate Ions. The phenoxyl radical formed from the above-
described oxidation reactions might in turn attack proteins,
lipids, and other delicate molecules like DNA. Thus in this
context it is relevant to study the scavenging of phenoxyl radicals
with endogenous antioxidant. Vitamin C (ascorbic acid) is an
endogenous water-soluble physiological antioxidant. It has a pKa
at 4.1 and 11.8 and therefore it exists as a monoanion (AscH−) at
neutral pH.37 The one-electron oxidized species of AscH is
characterized by the absorption maximum at 360 nm (ε = 3300
dm3 mol−1 cm−1).38 The experiment was performed with 2 ×
10−3 mol dm−3 of thymol with varying concentrations of ascorbic
acid [(0.1−0.06) × 10−3 mol dm−3]. It was observed that in the
presence of ascorbic acid, the decay of the phenoxyl radicals of
thymol was much faster than in its absence. Also it is found that
there is a simultaneous absorption buildup at 360 nm.
Typical traces that were obtained from the pulse radiolysis of
N2O saturated 2 × 10−3 mol dm−3 thymol containing 1 × 10−4
mol dm−3 ascorbic acid and 0.01 mol dm−3 NaN3 are given in
Figure 11. This suggests that the phenoxyl radicals of thymol,
optimized at the B3LYP/6-31+G(d,p) level of calculation. The
relative energies of these radicals (with respect to the least stable
radical species) are shown in Table 2. From the table it is clear
that both the relative energy values and relative free energy values
shows the same trend.
Table 2. B3LYP/6-31+G(d,p)-Calculated (in the Gas Phase)
Relative Energy Changes of the Various Possible Radicals
with the Reaction of O•− with Thymol
radical
species
enthalpy change (ΔH, kcal/ free energy change (ΔG, kcal/
mol)
mol)
I
−12.8
−10.7
Figure 11. Absorption traces obtained on pulse radiolysis of N2O
saturated 2 × 10−3 mol dm−3 thymol solution containing 1 × 10−4 mol
dm−3 ascorbic acid and 0.01 mol dm−3 NaN3. (a) Absorption trace at
360 nm in the absence of ascorbic acid. (b) Formation of ascorbate
radical anion at 360 nm in the presence of 1 × 10−4 mol dm−3 ascorbic
acid.
II
III
IV
V
0.00
0.00
−0.07
−16.4
−372
−0.25
−16.3
−361.25
ii. One-Electron Oxidation of O•− with Thymol. The oxide
radical anion is known to undergo one-electron oxidation, as
shown in eq 8.
which are oxidizing, are scavenged by the ascorbic acid anion,
giving ascorbate anion radical (eq 9). The rate constant for this
reaction was determined by following the pseudo-first-order
buildup at 340 nm as 2.1 × 108 dm3 mol−1 s−1.
The radical resulting from one-electron oxidation is the
oxygen-centered radical (V). This radical is also optimized as in
the case of carbon-centered radicals, and the relative energy is
compared with that of the least stable carbon-centered radical. It
This high value of the rate constant indicates that the phenoxyl
radicals of thymol can be efficiently scavenged by the ascorbic
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acid, and this may lead to a potential repair reaction. This
highlights the ability of thymol to protect cellular damage by
scavenging reactive radical species and possess minimum
prooxidant activity.
3.4. Cyclic Voltammetry of Thymol. Cyclic voltammetry is
one of the methods extensively used in determining the redox
potential of phenolic antioxidant molecules in solution.39 The
antioxidant potential and antioxidant capacity of the sample is a
function of the oxidation potential. To determine the redox
potential values, the CV experiments of thymol were carried out.
The cyclic voltammogram of thymol is given in Figure 12.
attack at the C6 atom is energetically more favorable, though the
attack at the para position is also likely to occur. Consequently,
the additions at the ortho positions are assumed to occur without
the formation of any precomplex. The redox potential of the
thymol•/thymol couple together with its nontoxicity makes it a
promising antioxidant.
ASSOCIATED CONTENT
* Supporting Information
■
S
UV−vis spectra of thymol at varying concentrations in aqueous
medium with and without the presence of phosphate buffer,
pseudo-first-order rate constants against the concentration of
thymol for the calculation of the second-order rate constant for
•
•
the reaction of OH, N3, and O•− with thymol, and the full
expansion of ref 16. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: cta@mgu.ac.in. Fax: 91 481 2731009.
■
Present Address
&Department of Spectroscopy, Indian Association for the
Cultivation of Science, Jadavpur, Kolkata 700032, India.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank the operating staff of LINAC in RPCD,
BARC, for their help and Dr. Shilpa Tawade of Chemistry
Division, BARC, Mumbai, for the assistance with cyclic
voltammetric measurements. Experimental help at NCFRR,
Pune, is also acknowledged. The authors also thank Dr. Ayan
Datta, IISER, Trivandrum, for extending the computational
facility. C.T.A. is thankful to BRNS, Mumbai. and DST (under
PURSE programme) for financial support.
Figure 12. Cyclic voltammogram of thymol at ambient pH in aqueous
medium with Ag/AgCl as reference electrode.
A maximum at 0.7715 V vs Ag/AgCl (0.98 V vsNHE) is
observed, which is assigned to the thymol•/thymol couple (eq
10).
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