Synthesis of 3-O-methylgallic acid a powerful antioxidant by electrochemical conversion of syringic acid

Article abstract of DOI:10.1016/j.bbagen.2013.02.012

Background A kinetic study of the electrochemical oxidation of syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) by cyclic voltammetry at treated gold disk was combined with results of electrolyses at Ta/PbO₂ anode in order to convert it into potentially high-added-value product. Methods The electrochemical oxidation of syringic acid was carried out in order to convert this compound to 3-O-methylgallic acid. This latter was identified by mass spectrophotometry using LC-MS/MS apparatus. The 3-O-methylgallic acid synthesis was controlled by cyclic volammetry, Ortho-diphenolicdeterminations and DPPH radical-scavenging activity. Results The proposed mechanism is based on the hypothesis of a bielectronic discharge of syringic acid molecule under free and adsorbed form involving two intermediate cation mesomers. Hydrolysis of the more stable of this last one leads to the formation of the 3,4-dihydroxy-5- methoxybenzoic acid (3-O-methylgallic acid) as a major product. The latter aromatic compound was synthesized by anodic oxidation of syringic acid at PbO2 electrode. The cyclic voltammogram of the electrolysis bath of syringic acid shows that the anodic peak potential of 3-O-methylgallic acid was lower (E _pa = 128 mV) than that of SA (E_pa = 320 mV). And the strongest antiradical activity was detected when the 3-O-methylgallic acid concentration was higher . Conclusion The electrochemical oxidation using PbO₂ anode is a rapid, simple and efficient method tool for a conversion of SA into 3-O-methylgallic acid, a potent antioxidant derivative General Significance The electrochemical process consists in a simple transformation of the syringic acid into 3-O-methylgallic acid having a better antioxidant capacity. This result has been justified by cyclic voltametry which shows that anodic peak of 3-O-methylgallic acid is reversible. Furthermore, its potential is lower than that of the irreversible anodic peak of syringic acid to 3-O-methylgallic acid.

Full text of DOI:10.1016/j.bbagen.2013.02.012

Biochimica et Biophysica Acta 1830 (2013) 3643–3649
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Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbagen
Synthesis of 3-O-methylgallic acid a powerful antioxidant by
electrochemical conversion of syringic acid
⁎
Olfa Dridi Gargouri, Boutheina Gargouri, Souhel Kallel Trabelsi, Mohamed Bouaziz , Ridha Abdelhédi
Laboratoire d'Electrochimie et Environnement, Ecole Nationale d'Ingénieur de Sfax, BP 1173, 3038 Sfax, Université de Sfax, Tunisia
a r t i c l e i n f o
a b s t r a c t
Article history:
Background: A kinetic study of the electrochemical oxidation of syringic acid (3,5-dimethoxy-4-hydroxybenzoic
acid) by cyclic voltammetry at treated gold disk was combined with results of electrolyses at Ta/PbO2 anode in
order to convert it into potentially high-added-value product.
Methods: The electrochemical oxidation of syringic acid was carried out in order to convert this compound to
3-O-methylgallic acid. This latter was identified by mass spectrophotometry using LC-MS/MS apparatus. The
3-O-methylgallic acid synthesis was controlled by cyclic volammetry, Ortho-diphenolicdeterminations and
DPPH radical-scavenging activity.
Received 11 October 2012
Received in revised form 2 February 2013
Accepted 11 February 2013
Available online 20 February 2013
Keywords:
Syringic acid
Results: The proposed mechanism is based on the hypothesis of a bielectronic discharge of syringic acid molecule
under free and adsorbed form involving two intermediate cation mesomers. Hydrolysis of the more stable of this
last one leads to the formation of the 3,4-dihydroxy-5-methoxybenzoic acid (3-O-methylgallic acid) as a major
product. The latter aromatic compound was synthesized by anodic oxidation of syringic acid at PbO2 electrode.
The cyclic voltammogram of the electrolysis bath of syringic acid shows that the anodic peak potential of
3-O-methylgallic acid was lower (Epa = 128 mV) than that of SA (Epa = 320 mV). And the strongest antiradical
activity was detected when the 3-O-methylgallic acid concentration was higher".
3-O-methylgallic acid
Anodic oxidation
Cyclic voltammetry
Antioxidant activity
Conclusion: The electrochemical oxidation using PbO2 anode is a rapid, simple and efficient method tool for a con-
version of SA into 3-O-methylgallic acid, a potent antioxidant derivative
General Significance: The electrochemical process consists in a simple transformation of the syringic acid into
3-O-methylgallic acid having a better antioxidant capacity. This result has been justified by cyclic voltametry
which shows that anodic peak of 3-O-methylgallic acid is reversible. Furthermore, its potential is lower than that
of the irreversible anodic peak of syringic acid to 3-O-methylgallic acid.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
[12,13] that the chemical structure of the side chain, as well as the num-
ber and position of free hydroxy and methoxy groups attached to the ar-
Phenolic compounds are a complex group of substances that have
attracted considerable attention due to their roles in providing flavor
and color characteristics of food and in human health [1]. Higher con-
centration of these compounds is found in olive and mainly in olive
mill wastewater. Phenolic compounds may vary in structure due to dif-
ference in number and position of the hydroxyl groups on the aromatic
ring. As a group, these naturally occurring compounds have been found
to be strong antioxidants against free radicals and other reactive oxygen
species (ROS) which constitute the major cause of many chronic human
diseases such as cancer and cardiovascular diseases [2,3].
Compounds which are antioxidants by virtue of their ability to act as
reductants in solution tend to be easily oxidized at inert electrodes. In
this context, cyclic voltammetry has been applied to characterize a
range of antioxidants, including phenolic acids and flavonoids [4–8],
ascorbic acid [9], and synthetic antioxidants [10,11]. It has been reported
omatic ring played key roles in the antioxidant activity of the phenolic
acids. Moreover, the decrease of the oxidation potential of these com-
pounds leads generally to an increase of their antioxidant activity [14].
The electrochemical oxidation mechanism of the phenolic compounds
has been well discussed [15–18].
It has been well recognized that this last one undergoes a first
monoelectronic transfer leading to the formation of phenoxy radicals.
This first step should be ascribed to the antioxidant activity of these com-
pounds. Depending on their structure and the magnitude of the anodic
potential, the phenoxy radicals can lead to the formation of polymers
and/or hydroquinonic (p-hydroquinones and catechol derivatives) and
benzoquinonic (p and o-benzoquinone derivatives) structures [17–20].
The reversible anodic oxidation of p and o-hydroquinone structures
occurs at lower potentials than those of the starting monophenols
[17,21]. In the contrary, the anodic oxidation of their oxidized forms (p
and o-benzoquinonic structures) is irreversible and more difficult than
that of the starting monophenols [14,16,17]. According to our previous
work, Table 1 shows the relative values of anodic oxidation potentials
⁎
Corresponding author. Tel.: +216 98667581.
E-mail address: mohamed.bouaziz@fsg.rnu.tn (M. Bouaziz).
0304-4165/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bbagen.2013.02.012

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O.D. Gargouri et al. / Biochimica et Biophysica Acta 1830 (2013) 3643–3649
of 300 V min−1 for a period depending on the desired oxide layer thick-
ness. The initially bright gold electrode became red brown. The electro-
chemical response of the treated electrode is presented in Fig. 1 (curve
1). The cyclic voltammogram was characterized by a large anodic peak
at +760 mV corresponding to the Au oxide formation and a cathodic
peak at +500 mV relative to the Au oxide reduction. The gold oxide
electrode was characterized by the charge Q corresponding to the elec-
trochemical formation of the gold oxides [25]. Q was considered as the
main characteristic of the electro-active surface.
Table 1
Potential values of anodic peaks of phenol, hydroquinone and
p-benzoquinone. Electrode: pre-treated Pt disk (r=1 mm), Q=4 mC,
T=25 °C [22].
Compounds
Potential anodic peak
(V/SHE)
Phenol
Hydroquinone
p-Benzoquinone
1.11
0.63
1.23
of phenol and its oxidation products namely the p-hydroquinone and
p-benzoquinone [22].
2.4. Electrolysis
Syringic acid (SA) (3,5-dimethoxy-4 hydroxybenzoic acid) has been
investigated by cyclic voltammetry at a glassy carbon electrode [14]. In
this context, Alaexandra et al. [14] have observed that syringic acid has a
very poor radical scavenging activity and higher oxidation potential in
comparison with protocatechuic acid and caffeic acid [14,23,24].
The present study concerns the electrochemical behavior of SA in
aqueous solutions. We were interested at first in the kinetic study of
the first stages of the oxidation mechanism of SA by cyclic voltammetry
at electrodes of Au/Au oxides. These oxidation steps are those that pre-
cede the opening of the aromatic ring. Gold is often regarded as the ideal
solid electrode for fundamental electrochemical investigation as it is dif-
ficult at this metal to completely oxidize aromatic compounds. The elec-
trocatalytic properties of Au/Au oxide electrode were characterized by
the charge Q corresponding to the electrochemical formation of the
gold oxides. Besides, we showed in a previous work that the charge Q
allows control of electrocatalytic properties of the Au/Au oxide electrode
[16,17,25]. In the second part of this work, we will use a Ta/PbO2 anode
in view to convert SA into a product having an important biological ac-
tivity. It should be noted that at low anodic potential, PbO2 behaves like
gold vis-à-vis the aromatic compound oxidation [26]. On the other hand,
the antiradical ability of SA and its oxidation products has been assessed
by measuring the ease of their reaction with the stable free radical
diphenylpicrylhydrazyl (DPPH•) leading to its reduced form (DPPH-H).
Good free radical scavengers react fast leaving small amounts of
unreduced DPPH, which indicates the presence of an effective antioxi-
dant [23].
Galvanostatic electrolysis of SA in aqueous solutions was carried out
in an isothermal reactor with two compartments. The electrolytic solu-
tion treated (V=150 cm3) initially contained SA (2 mmol L−1) in
H2SO4 (pH=1.8). The Ta/PbO2 anode was placed in front of the cath-
ode; the opposite face of plate relative to the cathode was masked
with a protective film (transparent polyethylene tape, Scotch TM 480.
3 M). The total surface area of the working electrode was 4 cm2. Exper-
imental details for the preparation of Ta/PbO2 were described in our pre-
vious work [17]. The cathode was a bar of graphite (φ=1 cm, L=6 cm)
placed in a porous ceramic cylinder (Norton, Refractaire RA 84)
containing 1 mol L−1 sulfuric acid solution. The acid solution of SA
was electrolyzed at 30 °C, with magnetic stirring and under a constant
anodic current density of 15 mA cm−2
.
2.5. LC–MS/MS analysis
Liquid chromatography–mass spectrometry (LC–MS) of the SA after
240 min of electrolysis was performed on an Agilent 1100 series
LC-MSD. The compounds were separated with a Zorbax 300 A°
Extend-C-18 column (2.1×150 mm, particle size 5 μm, Agilent Tech-
nology, INC, Wilmingtom, DE, USA). The mobile phase was a mixture
of two solvents formic acid (1%) in water (A) and methanol:acetoni-
trile:formic acid (89.5:9.5:1, v/v/v) (B). Analysis conditions were as de-
scribed before [27]. The percentage by volume of (B) varies linearly with
time as follows: from 10 to 30% for the first 10 min, then from 30 to 40%
up to 25 min after isocratic during 5 min, from 40 to 50% up to 40 min
and from 50 to 100% up to 50 min, after that back to 10% up to 55 min
and finally isocratic up to 65 min. The column outlet was coupled with
an Agilent MSD ion trap XCT mass spectrometer (Santa Clara, CA, USA)
equipped with an ESI ion source.
2. Experimental
2.1. Reagents and chemicals
Sodium molybdate dehydrate and organic solvents were pur-
chased from Merck (Darmstadt, Germany). Syringic acid (SA), 2,2-
diphenyl-1-picrylhydrazyl (DPPH), and all other chemicals were pur-
chased from Sigma–Aldrich (St. Louis, MO). All reagents and chemicals
used were of analytical grade.
51
(1)
(2)
34
17
I’1
2.2. Apparatus
I1
A potentiostat type PJT Tacussel was used for electrochemical mea-
surements and the data was recorded using a GSTP4 Tacussel X-Y re-
corder. The charge Q corresponding to the electrochemical formation
of the gold oxides was measured using an IG6-N Tacussel integrator.
In this investigation, all the potentials refer to Hg/Hg2SO4/K2SO4 elec-
trode (E =+0.61 V/SHE).
0
-17
-34
-51
2.3. Treatment of the gold electrodes
The working electrode was a polycrystalline gold disk of ca.
0.0314 cm2 exposed area. This electrode was mechanically polished
with a fine alumina powder (φ=0.3 μm) and washed with ultrapure
water before transfer to the cell. The electrode was then subjected to
potential cycling conditions, in sulfuric acid (0.5 mol L−1), between
−610 and +1190 mV vs. a saturated mercurous sulfate at a scan rate
-640
-320
0
320
640
780
E (mV)/Hg/Hg2SO4
Fig. 1. Cyclic voltammograms recorded on a disk of gold (φ=1 mm). Q=0.22 mC; v=
50 mV s−1; T=20 °C; H2SO4=0.5 mol L−1; SA concentration: (1) 0; (2) 0.6 mmol L−1
.

O.D. Gargouri et al. / Biochimica et Biophysica Acta 1830 (2013) 3643–3649
3645
2.6. Ortho-diphenolic determinations
I2 at 128 mV which is ascribed to the oxidation of the majority SA oxi-
dation product.
Ortho-diphenolic content was determined following the previously
modified method described by Cert et al. [28]. This method is based
on the formation of yellow complex between ortho-diphenols and mo-
lybdate ions. Briefly, 2 mL of sample was added to 0.5 mL of sodium
molybdate dihydrate solution (0.5 g L−1, in a mixture ethanol/water,
1/1, v/v). The mixture was shaken, and after 15 min at room tempera-
ture, the absorbance was measured at 370 nm. Standards of gallic acid
were prepared similarly.
Moreover the relative importance of the two peaks I1 and I′1 de-
pends on the SA concentration, the potential scan rate and the Q
value characterizing the state of the treated gold surface.
3.1.1. Influence of SA concentration
Fig. 3 regroups some of the first cyclic voltammograms recorded at
treated gold electrode characterized by a Q value of 0.22 mC in SA
aqueous solution at concentrations ranging from 0.1 to 4 mmol L−1
.
For SA concentration less than 0.2 mmol L−1, only the peak I1 is ob-
served. With increasing SA concentration above 0.2 mmol L−1 the
peak I′1 appears. When the SA concentration increases the current in-
tensities ip(I1) (Fig. 4 curve 1) and ip(I′1) (Fig. 4 curve 2) of these
peaks increase then tend towards around 16 and 12 μA respectively.
2.7. Determination of DPPH radical-scavenging activity
The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging ef-
fect was evaluated following the procedure described in a previous
study [29]. Aliquots (50 μL) of various concentrations (5, 10, 15, 20
and 25 μg/mL) of the test extracts in methanol were added to 5 mL
of methanolic solution containing DPPH radicals (6×10−6 mol L−1).
After 30 min incubation period at room temperature and in the dark,
the absorbance was read in opposition to the control at 517 nm. The
Inhibition (IC50) of free radical DPPH (IC50%) was calculated in per-
centage: IC50%=[(Ablank−Asample)/Ablank]×100, where Ablank is the
absorbance of the control reaction (containing all reagents except
the test extract), and Asample is the absorbance of the test extract.
3.1.2. Influence of the charge (Q)
Some cyclic voltammograms are recorded for 0.6 mmol L−1 SA
aqueous solution at treated gold electrodes characterized by Q values
in the range 0.15–0.33 mC.Fig. 5 shows that currents ip(I1) and ip(I′1)
increase linearly with Q value.
3.1.3. Influence of the potential scan rate
From voltammograms presented in Fig. 6, we have shown that the
current ip(I1) varies linearly with the square root of the potential scan
rate v (Fig. 7a). The slope of the straight line representing ip(I1) as a
function of the square root of the potential scan rate gives the diffu-
sion coefficient D of SA by application of Eq. (A.1).
The concentration of the test extract providing 50% inhibition (IC50
,
expressed in μg mL−1) was calculated from the graph plotted with in-
hibition percentage against the extract concentration. The synthetic
antioxidant reagent butylated hydroxytoluene (BHT) was used as pos-
itive control and all tests were carried out in triplicate.
105 S n3=2Dred1=2v1=2C
ðA:1Þ
3. Results and discussion
ipðI1Þ ¼ 2:69
ꢀ
3.1. Voltammetric study
where n is the number of exchanged electrons; S is the electrode sur-
face area (cm2); D is diffusion coefficient (cm2 s−1); v is the scan rate
(V s−1); and C is the SA concentration (mol cm−3).
The SA oxidation was first studied by cyclic voltammetry at treated
gold electrode.
A
typical voltammogram for the oxidation of
The diffusion coefficient of SA calculated for n=2, is
4.46·10−6 cm2 s−1 at 25 °C. Although to our knowledge this dif-
fusion coefficient has not previously been estimated, its value
found in this work is situated in the range of compounds having
a similar structure which undergo a bielectronic discharge [16].
0.6 mmol L−1 SA solution in sulfuric acid (0.5 mol L−1) at a scan rate
of 50 mV s−1 is shown in Fig. 1 (curve 2). During the first forward
positive scan, SA exhibits two anodic peaks I1 and I′1 at Epa=270 and
E′pa=320 mV respectively. The successive cyclic voltammograms of
4 mmol L−1 SA solution at the same electrode are presented in Fig. 2.
From the second cycle, the voltammograms exhibit a reversible peak
I’1
46
(5)
89
23
0
(4)
(3)
46
(1)
(2)
(3)
I1
(2)
(1)
I2
23
0
-23
-46
0
350
-320
0
320
460
E/mV(Hg/Hg2SO4)
E (mV)/Hg/Hg2SO4
Fig. 2. Successive cyclic voltammograms of SA (4 mmol L−1) at gold disk in sulfuric acid
aqueous solution at potential scan rate of 50 mV s−1; Q=0.22 mC; H2SO4=0.5 mol L−1
SA concentration 4 mmol L−1; (1): 1th, (2): 2th, (3): 3th cycle.
Fig. 3. Cyclic voltammograms of SA at a disk of gold (φ=1 mm). Q=0.22 mC; v=
;
50 mV s−1; T=20 °C; H2SO4=0.5 mol L−1; SA concentration: (1) 0.1; (2) 0.4; (3) 0.8;
(4) 2; (5) 4 mmol L−1
.

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O.D. Gargouri et al. / Biochimica et Biophysica Acta 1830 (2013) 3643–3649
20
16
12
8
I1
I’1
14
I’1
(4)
I1
(3)
(2)
7
0
(1)
4
0
0
2
4
6
8
10
12
14
16
C x 10-4 (mol L-1)
Fig. 4. Variation of the current of the peaks I1 and I′1 as function of SA concentration.
Q=0.22 mC; v=50 mV s−1; T=20°C; H2SO4=0.5 mol L−1
-7
.
0
200
400
600
E (mV)/Hg/Hg2SO4
On the other hand, the voltammograms shown in Fig. 6 show that
when the potential scan rate increases, ip(I′1) rises more than ip(I1).
The results presented above may be interpreted by assuming that
the electrochemical oxidation of SA molecules takes place on the
treated gold electrode by electron transfer for both free and adsorbed
forms. The free form corresponds to the first peak I1 while the strongly
adsorbed form, which is consequently stabilized, is oxidized at a more
anodic potential and corresponds to the “post-peak” I′1.
The adsorption aspect of the peak I′1 is confirmed according to the
procedure already undertaken in previous studies in the case of the
oxidation of phenol at platinum [21]. The treated gold electrode is im-
mersed into SA aqueous solution then rinsed in ultrapure water and
immediately subjected to potential cycling in 0.5 mol L−1 sulfuric
acid aqueous solution. The voltammogram shows only a single anodic
peak which appears towards 320 mV. This peak which results from
the oxidation of the adsorbed SA coincides with the peak I′1. Its cur-
rent intensity varies linearly with potential scan rate v (Fig. 7(b))
according to Eq. (A.2).
Fig. 6. Cyclic voltammograms of SA at a disk of gold (φ=1 mm).Q=0.22 mC; T=20 °C;
H2SO4=0.5 mol L−1; SA concentration: 0.6 mmol L−1; potential scan rate: (1) 10; (2) 25;
(3) 50; (4) 70 mV s−1
.
3.2. Electrochemical oxidation of SA at PbO2 anode
The electrochemical oxidation of 2 mmol L−1 SA aqueous solution
at PbO2 anode under 15 mA cm−2 was followed by cyclic voltammetry
at treated gold electrode. Fig. 8(a) shows cyclic voltammograms at
different electrolysis time. The initial voltammogram for untreated so-
lution (t=0 min) showed only the oxidation peak I′1 of SA at E′pa
=
320 mV/(Hg/Hg2SO4).
The following voltammograms, exhibit the reversible anodic peak
I2 which has been previously observed during the cyclic voltammetric
study (Fig. 2). The current of this peak reaches its maximum at about
5 μA after 240 min of oxidation, then decreases. However, the current
of the peak I′1 decays until zero after 360 min (Fig. 8(b)).
To elucidate the structures of SA aromatic products obtained after
240 min electrolysis, a sample was analyzed by liquid chromatogra-
phy with electrospray mass spectrometry in the negative mode. The
structure assignment of these products was based on UV absorbance
spectra with a systematic search for molecular ions using extracted
ion mass chromatograms and comparing the MS/MS spectra with
those in the literature [30].
n2F2
4RT
ꢀ
ꢁ
ip I′1
¼
SvΓ
ðA:2Þ
ads
where Γ is the superficial concentration (mol cm−2) of adsorbed SA.
Compound 1 was identified to SA retained at 16.8 min (Fig. 9). The
spectrum MS1 exhibits a molecular ion at m/z 197 [M–H]−. The MS2
spectrum obtained by fragmentation of this ion presents the following
m/z values: 182, 153, 138 and 121. The fragmentation of the pseudo mo-
lecular ion [M–H]− at m/z 197 yields a fragment at m/z 182 by the loss
of 15 mass units. The ion m/z 153 by the loss (−44) of a carboxylic acid
group, providing an anion of [M–H–COO]−. The ions at m/z 138 and 121
are fragments of ion m/z 153 by the loss of methyl radical (−15) and
water molecule. This profile is compatible with syringic acid that has
been described by Gruz et al. [30]. The LC–MS analysis of compound 2
(Fig. 10) produced a negative ion of m/z 183, corresponding to the
[M–H]− precursor ion. Subsequent product ion scan (MS2) of this pre-
cursor ion produced a fragmentation pattern dominated by an ion at
m/z 168 [M–CH3–H]−. The loss (−44) of a carboxylic acid group, pro-
viding an anion (m/z 139) of [M–H–COO]−. In addition, we detected
an ion at m/z 124 explained by the loss of methyl radical (−15) from
methoxylated derivatives of compound 2 leading to a very stable
anion radical structure of [M–H–COO-CH3]−. In accordance with our
findings, Gruz et al. [30], noted analogous collision-induced fragmenta-
tion of deprotonated (poly-)methoxylated phenolic acid and flavonoids,
20
16
(1)
(2)
12
8
4
0
0
0,1
0,2
0,3
0,4
Q (mC)
Fig. 5. Variation of the current of the peaks I1 and I′1 as a function of the charge Q. v=
50 mV s−1, v=50 mV s−1, SA concentration: 0.6 mmol L−1. (1) ip(I1); (2) ip(I′1).

O.D. Gargouri et al. / Biochimica et Biophysica Acta 1830 (2013) 3643–3649
3647
a
a
16
12
8
30
R² = 0,9749
0h
20
10
2h
4h
5h
4
6h
0
0
0
2
4
6
8
10
v1/2 (mV1/2 s-1/2
)
-10
b
20
16
12
8
R² = 0,9869
0
195
390
585
E/mV(Hg/Hg2SO4)
b
50
40
30
20
10
0
4
0
I’1
0
20
40
60
80
v (mV s-1)
Fig. 7. Variation of the current of the peaks I1 and I′1 as function of potential scan rate, SA
concentration: 0.6 mmol L−1; Q=0.22 mC; T=20 °C; H2SO4: 0.5 mol L−1; v: 10; 25; 50
I2
and 70 mV s−1
.
0
50
100 150 200 250 300 350 400
Time (min)
respectively, distinguished by specific losses of methyl radicals from
their respective [M–H]− anions. This suggested that 2 might be a 3-O-
methylgallic acid. The mass spectrum of compound 3 (retention time
4.1 min) exhibited a base peak at m/z 167 [M–H]− in negative ion
mode and strong peaks at m/z 152 [M–H-CH3]−. This observation can
be a diagnosis of synergic acid quinone derivatives. The combined
results of the MS and UV spectra suggest that compound 3 could be
2,6-dimethoxybenzoquinone. The latter was confirmed by chromatog-
raphy with authentic standards as 2,6 dimethoxybenzoquinone.
The principal aromatic compounds, identified by LC/MS, are 3-O-
methylgallic acid and 2,6-dimethoxybenzoquinone. These results are
in good agreement with the proposed SA oxidation mechanism.
Fig. 8. (a) Cyclic voltammograms of 2 mmol L−1 SA at gold disk in sulfuric acid aqueous
solution at potential scan rate of 50 mV s−1 as a function time electrolysis of SA at PbO2.
(b) Variation of current ip(I2) and ip(I′1) during anodic oxidation of SA, japp=15 mA cm2,
pH=1.8, T=30 °C.
high concentration of o-diphenol such as 3-O-methylgallic acid. The an-
tioxidant activity and the level of the orthodiphenol content in the SA so-
lution treated suggest that the radical scavenging effect in the solution
can be attributed to hydroxylated phenolic compounds, in particular,
the number of hydroxyl substituents in the aromatic ring and the nature
2
3.3. Antioxidant activity
1
2
The DPPH radical-scavenging activities of SA solution treated by an-
odic oxidation at PbO2 were investigated during electrolysis (Fig. 11).
The low IC50 values designated potent radical-scavenging effects, as
low concentrations were adequate to inhibit the DPPH radicals [13,31].
All samples exhibited antioxidant activity, which showed correla-
tion between orthodiphenol content and DPPH radical-scavenging ac-
tivity over all anodic oxidation of SA.
1
3
0
The SA which presents a single hydroxyl group has IC50 =90 mg L−1
,
0.0
2.0
4.0
6.0
8.0
10.0 12.0 14.0 16.0 18.0 20.0 22.0
confers a limited amount of antioxidant activity. On the other hand, the
lower IC50 values (16 mg L−1), which indicated higher antioxidant po-
tential, were observed for the samples harvested at 240 min of electrol-
yses. This is probably due to the significant radical inhibition caused by a
Time (min)
Fig. 9. HPLC chromatograms at 280 nm after 240 min SA anodic oxidation at PbO2. 1:
syringic acid; 2: 3-O-methylgallic acid; 3: 2,6 dimethoxybenzoquinone.

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O.D. Gargouri et al. / Biochimica et Biophysica Acta 1830 (2013) 3643–3649
6
5
-MS, Line, 11.9min (#465), 100%=5058574
x10
182.7
4
3
2
1
0
338.4
307.5
112.8
96.7
218.6
280.8
50
100
150
200
250
300
350
400 m/z
5
x10
-MS2(182.8), Line, 11.8min (#462), 100%=290201
167.7
3.0
2.5
2.0
1.5
138.8
1.0
0.5
0.0
123.7
386.7
50
100
150
200
250
300
350
400 m/z
Fig. 10. LC–MS/MS spectrum (MS1and MS2) of SA after 240 min SA anodic oxidation at PbO2, japp=15 mA cm−2; pH=1.8; T=30 °C.
of the substituents at the para or ortho position [13]. These compounds
react with free radicals formed during autoxidation and generate a
new radical that is stabilized by the resonance effect of the aromatic nu-
cleus [24]. So 3-O-methylgallic acid exhibited antioxidant activity at a
level similar to that of BHT (IC50 =17 mg L−1).
Thus, SA (monophenol) have a higher potential (Epa=320 mV) than
3-O-methylgallic acid (Epa =128 mV).
3.4. Oxidation mechanism of the SA
Cyclic voltammetry has been applied to characterize the reducing
ability of natural phenolics [6,32] and good correlations have been ob-
served between redox potentials and antioxidant properties [33,34].
In accordance with the global mechanism proposed for SA oxidation,
the hydroxyl groups are oxidized via two electron transfer leading to
The preceding results allow to schematize the oxidation mechanism of
SA in Fig. 12. SA molecules undergo a first electron transfer under free and
adsorb forms leading to the formation of phenoxy radicals for which the
electronic charge distribution can be represented by three mesomeric
forms A, B and C. By considering the electronic effects of methoxy and
carboxy groups, the radical B would be the most probable. The formation
of 3-O-methylgallic acid (B3) could be explained assuming that the radical
B undergoes a second electron transfer leading to the carbocation B1
which gives simultaneously the 3-methoxy-4,5-dioxobenzoic acid (B2)
and a methanol molecule by hydrolysis. During the reverse scan, the
3-methoxy-4,5-dioxobenzoic acid (B2) is reduced to 3-O-methylgallic
acid (B3) at the potential of the reversible peak I2. Moreover, the propor-
tions of SA oxidation product formed via the intermediary of the less
probable radical C would be insignificant. The oxidation of this last one
leads to the formation of 2,6-dimethoxy-p-hydroquinone (C2) which is
oxidized to 2,6-dimethoxy-p-benzoquinone (C2).
the generation of a correspondent quinone after liberation of 2H+
.
These results indicated that the higher number of hydroxyl substituents
on the aromatic ring, correspond to the lower electrochemical potential.
250
200
150
100
50
400
300
200
100
0
Orthodiphenols
(1) Cothodiphenols
IC50
(2) IC50
(2)
(1)
4. Conclusion
From these studies, it was concluded that electrochemical oxida-
tion using PbO2 anode is a rapid, simple and efficient method tool for
conversion of SA into a potent antioxidant derivative. Using LC–MS,
it was possible to determine the structure of 3-O-methylgallic acid
and the other phenolics produced during electrochemical treatment.
The measured oxidation potential was closely related to the struc-
tures. The phenolic structure influences also antioxidant activity. The
ortho-diphenol compound has lower anodic peak potential and higher
0
0
200
400
600
Time (min)
Fig. 11. Free radical scavenging activity and ortho-diphenol tests during anodic oxidation
of SA at PbO2. Other conditions as in Fig. 8.

O.D. Gargouri et al. / Biochimica et Biophysica Acta 1830 (2013) 3643–3649
3649
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