Structure-property-activity relationship of phenolic acids and derivatives. Protocatechuic acid alkyl esters

Article abstract of DOI:10.1021/jf100569j

The esterification of hydrophilic phenolic antioxidants is an efficient approach to enhance their solubility in apolar media. Herein, structurea property studies on the antiradical activity of a series of protocatechuic acid alkyl esters have been accomplished. The increase of the lipophilicity was shown to significantly improve the antioxidant activity of protocatechuic esters. Their efficiency as radical scavengers was evaluated using distinctive analytical methods, namely, 2,2-diphenyl-1-picrylhydrazyl (DPPH) UV/visible method, electrochemistry, and differential scanning calorimetry. All the new alkyl protocatechuate antioxidants studied possessed better radical-scavenging capacity than the natural antioxidant protocatechuic acid. This work has shown that the alkyl ester side chain markedly influences the lipophilicity of this type of phenolic system without disturbing the core of the molecule responsible for antioxidant activity. The data on the antioxidant activity obtained using the different analytical methods correlated well with each other and have revealed the interesting antioxidant potential of alkyl esters of protocatechuic acid.

Full text of DOI:10.1021/jf100569j

6986 J. Agric. Food Chem. 2010, 58, 6986–6993
DOI:10.1021/jf100569j
Structure-Property-Activity Relationship of Phenolic Acids
and Derivatives. Protocatechuic Acid Alkyl Esters
‡,§
B
RUNO
R
EIS,† MARTA
E. MANUELA
M
ARTINS,† BA´ RBARA
B
ARRETO,† NUNO
M
ILHAZES
ARRIDO,*,†,§ AND
,
G
ARRIDO
,
†,§ PAULO
S
ILVA,† JORGE
,§
G
F
ERNANDA
B
ORGES
*
†CIETI/Departamento de Engenharia Quı
´
mica, Instituto Superior de Engenharia do Porto, IPP, Porto,
^
‡
Portugal, Instituto Superior de Ciencias de Sau´ de-Norte, Gandra, PRD, Portugal, and
§CIQUP/Departamento de Quı
mica, Faculdade de Ciencias, Universidade do Porto, Porto, Portugal
^
´
The esterification of hydrophilic phenolic antioxidants is an efficient approach to enhance their solubility
in apolar media. Herein, structure-property studies on the antiradical activity of a series of proto-
catechuic acid alkyl esters have been accomplished. The increase of the lipophilicity was shown to
significantly improve the antioxidant activity of protocatechuic esters. Their efficiency as radical
scavengers was evaluated using distinctive analytical methods, namely, 2,2-diphenyl-1-picrylhydrazyl
(DPPH) UV/visible method, electrochemistry, and differential scanning calorimetry. All the new alkyl
protocatechuate antioxidants studied possessed better radical-scavenging capacity than the natural
antioxidant protocatechuic acid. This work has shown that the alkyl ester side chain markedly
influences the lipophilicity of this type of phenolic system without disturbing the core of the molecule
responsible for antioxidant activity. The data on the antioxidant activity obtained using the different
analytical methods correlated well with each other and have revealed the interesting antioxidant
potential of alkyl esters of protocatechuic acid.
KEYWORDS: Protocatechuic acid; alkyl esters; antioxidant activity; electrochemistry; DSC
INTRODUCTION
phenolics by the use of reactions that increase lipophilicity have been
suggested in order to enhance their hydrophobicity and improve
their resulting antioxidant properties by a better location at the lipid/
aqueous phase interface where the oxidation occurs (6-8).
Protocatechuic acid (3,4-dihydroxybenzoic acid) and its esters
are o-diphenols ubiquitously found in edible plants, vegetables,
and fruits. They are known to exhibit potent in vitro antioxidant
activities (9, 10). It has been found that protocatechuic acid has
preventive effects in carcinogenesis and cardiovascular diseases
that are associated with free radicals (11-13).
The radical-scavenging activity of protocatechuic acid and
derivatives toward DPPH was reported in several in vitro
studies (14-16). Despite many studies, important points regard-
ing the structure-activity of protocatechuic acid derivatives are
still poorly clarified, especially those concerning the relative
antioxidant activity in organic and aqueous environments (17).
Moreover, studies concerning the relation between lipophilicity
and antioxidant efficiency are scarce for this type of compounds.
The present study evaluates the impact of hydrophilic-lipophilic
balance on the antioxidant activity of protocatechuic acid deriva-
tives. An insight on the structure-property-activity relationship
that underlies the antioxidant activity of protocatechuic esters
(Figure 1) will be presented. With this aim protocatechuic acid (1)
and its esters, protocatechuic methyl ester (2), protocatechuic ethyl
ester (3), and protocatechuic propyl ester (4) were synthesized, and
some biological relevant physicochemical parameters, such as redox
potentials (Ep) and partition coefficients (log P), were evaluated.
Lipid oxidation is an old but still very important topic in food
stability and in human health. Many studies have been carried out to
search for and to develop antioxidants having a natural origin. The
potential of using phenolic acid compounds as natural antioxidants
has drawn more attention because of their ubiquitous occurrence in
nature. Phenolic acids have been widely investigated as potential
models for the development of new primary antioxidants, which can
prevent or delay in vitro and/or in vivo oxidation processes (1-3).
The antioxidant activity of dietary phenolic compounds has
attracted much attention in relation to their physiological functions,
namely, in the prevention of coronary heart disease, cancer, and
inflammation (4, 5). Phenolic antioxidants have also been added to
food for years to prevent oxidation processes and are widely used
today for better food preservation (1, 3).
The relative low solubility of phenolic compounds in apolar
media may be seen as a drawback when considering their use as
antioxidants for stabilizing unsaturated lipids in formulated
complex emulsified systems. Recently, some modifications of these
*To whom correspondence should be addressed. For J.G.: (e-mail)
jjg@isep.ipp.pt; (address) Departamento de Engenharia Quı
Instituto Superior de Engenharia do Porto, IPP, Rua Dr. Anto
´
mica,
nio
´
Bernardino de Almeida 431, 4200-072 Porto, Portugal; (phone) þ351
228 340 500; (fax) þ351 228 321 159. For F.B.: (e-mail) fborges@fc.up.pt;
^
(address) Departamento de Quımica, Faculdade de Ciencias, Universidade
´
do Porto, Rua Campo Alegre 687, 4169-007 Porto, Portugal; (phone)
þ351 220 402 560; (fax) þ351 220 402 009.
pubs.acs.org/JAFC
Published on Web 05/06/2010
© 2010 American Chemical Society

Article
J. Agric. Food Chem., Vol. 58, No. 11, 2010 6987
calibrated using In, Bi, Sn, Zn, and KNO3, and the enthalpy calibration
has been carried out to the heat of fusion of the same standards. The
oxidation induction temperature (OIT) of pure and spiked linoleic acid
was analyzed by heating the samples at 5 K min-1 under constant oxygen
flow (50 mL/min). A computer-generated plot of heat flow (W/g) versus
temperature was used to graphically determine OIT. OIT was determined
by the onset of the oxidation process that is characterized by an exothermic
peak in the heat flow-temperature plot.
Synthesis. General Synthetic Procedure. The alkyl esters of proto-
catechuic acid were obtained by acid catalyzed esterification following the
general procedure: a solution of the phenolic acid (1.0 g, 6.49 mmol) was
stirred at room temperature with 75 mL of the corresponding alcohol
(methanol, ethanol, or n-propanol) containing 2 mL of H2SO4 for ∼5
days. The solvent was then partially evaporated under reduced pressure,
and the solution was neutralized with aqueous 1 M Na2CO3. The mixture
was extracted with diethyl ether (3 ꢀ 150 mL). The organic phases were
combined, washed with water (3 ꢀ 100 mL), dried over Na2SO4, and
concentrated under reduced pressure. The crude product was then purified
by column chromatography using mixtures of petroleum ether/ethyl ether
(7:3 to 5:5) as eluent and recrystallized.
Figure 1. Chemical structures of (1) protocatechuic acid, (2) protocate-
chuicmethylester, (3) protocatechuicethylester, (4) protocatechuicpropyl
ester, (5) gallic acid, and (6) Trolox.
The antioxidant activity of the compounds was evaluated using
a total antioxidant capacity radical assay (DPPH•, 2,20-diphenyl-
1-picrylhydrazyl radical) and by assessment of its ability to limit
the oxidation of linoleic acid, using differential scanning calori-
metry (DSC). The antioxidant properties obtained for proto-
catechuic acid and its esters were compared with those acquired
for the reference compounds gallic acid and Trolox. Gallic acid
was chosen because of its recognized antioxidant capacity and as
representative of natural phenolics and Trolox, a water-soluble
analogue of vitamin E, mainly because of its effectiveness in both
lipophilic and hydrophilic systems.
Protocatechuic Methyl Ester (Methyl 3,4-dihydroxybenzoate)
(2). Yield 81%. FTIR υmax (cm-1): 3467, 3263, 1689, 1612, 1448, 1296,
1269, 1240, 1184, 1165, 1095, 764. UV λmax (nm) (log ε): 297 (3.8), 262
(4.0), 221 (4.2), 208 (4.2). 1H NMR δ: 3.76 (3H, s, OCH3), 6.80 (1H, d, J =
8.1, H(5)), 7.30 (1H, dd, J = 8.2, 2.1, H(6)), 7.34 (1H, d, J = 2.1, H(2)),
9.64 (2H, br s, OH). 13C NMR δ: 51.6 OCH3, 115.3 C(5), 116.2 C(2), 120.5
C(6), 121.8 C(1), 145.1 C(3), 150.4 C(4), 166.2 (CdO). EI-MS m/z (%):
168 (Mþ•, 59), 138 (15), 137 (100), 109 (27), 81 (17). Mp 133-135 °C
(diethyl ether/n-hexane).
Protocatechuic Ethyl Ester (Ethyl 3,4-dihydroxybenzoate)
(3). Yield 75%. FTIR υmax (cm-1): 3498, 3274, 1682, 1610, 1516, 1369,
1334, 1294, 1236, 1124. UV λmax (nm) (log ε): 296 (3.8), 262 (4.0), 221 (4.2),
208 (4.2). 1H NMR δ: 1.27 (3H, t, J = 7.1, CH3), 4.22 (2H, m, OCH2), 6.80
(1H, d, J = 8.2, H(5)), 7.30 (1H, dd, J = 8.2, 2.1, H(6)), 7.35 (1H, d, J =
2.1, H(2)), 9.58 (2H, br s, OH). 13C NMR δ: 14.3 CH3, 60.0 OCH2, 115.3
C(5), 116.2 C(2), 120.8 C(6), 121.7 C(1), 145.0 C(3), 150.3 C(4), 165.7
(CdO). EI-MS m/z (%): 182 (Mþ•, 50), 154 (25), 138 (17), 137 (100), 109
(22), 81 (14). Mp 131-133 °C (diethyl ether/n-hexane).
Protocatechuic Propyl Ester (Propyl 3,4-dihydroxybenzoate)
(4). Yield 71%. FTIR υmax (cm-1): 3492, 3328, 1684, 1608, 1442, 1292,
1230, 1161, 1099, 984, 771. UV λmax (nm) (log ε): 296 (3.8), 262 (4.0), 221
(4.2), 208 (4.2). 1H NMR δ: 0.94 (3H, t, J = 7.4, CH3), 1.67 (2H, m, CH2),
4.13 (2H, t, J = 6.6, OCH2), 6.80 (1H, d, J = 8.2, H(5)), 7.31 (1H, dd, J =
8.3, 2.1, H(6)), 7.36 (1H, d, J = 2.0, H(2)), 9.57 (2H, br s, OH). 13C NMR
δ: 10.4 CH3, 21.7 CH2, 65.5 OCH2, 115.3 C(5), 116.2 C(2), 120.8 C(6),
121.7 C(1), 145.1 C(3), 150.4 C(4), 165.7 (CdO). EM-IE m/z (%): 196
(Mþ•, 33), 154 (72), 138 (17), 137 (100), 109 (24), 81 (16). Mp 113-115 °C
(diethyl ether/n-hexane).
Electrochemical Measurements. Stock solutions of protocatechuic
acid and its alkyl esters (10 mM) were prepared by dissolving an appropriate
amount in ethanol. The voltammetric working solutions were prepared, in the
electrochemical cell, by diluting 0.1 mL of the stock solution in 10 mL of
supporting electrolyte to get a final concentration of 0.1 mM.
The pH 7.3 supporting electrolyte used in the voltammetric determina-
tions was prepared by dilution to 100 mL of 6.2 mL of 0.2 M dipotassium
hydrogen phosphate and 43.8 mL of 0.2 M potassium dihydrogen
phosphate.
MATERIALS AND METHODS
General. Protocatechuic acid, gallic acid, Trolox, DPPH• (1,1-diphenyl-
2-picrylhydrazyl), and linoleic acid (LA, cis,cis-9,12-octadecadienoic acid,
99%) were purchased from Sigma-Aldrich Quımica S.A. (Sintra,
´
Portugal). Dimethyl sulfoxide-d6 (99.8%) and tetramethylsilane (TMS)
were obtained from Merck (Lisbon, Portugal). All other reagents and
solvents were pro analysis grade and were acquired from Merck and used
without additional purification. Deionized water (conductivity of <0.1
μS cm-1) was used throughout all the experiments.
Thin-layer chromatography (TLC) was carried out on precoated silica
gel 60 F254 (Merck) with layer thickness of 0.2 mm. For analytical control
the following systems were used: ethyl acetate/petroleum ether, ethyl
acetate/methanol, chloroform/methanol in different proportions. The
spots were visualized under UV detection (254 and 366 nm) and iodine
vapor. Normal-phase chromatography was performed using Merck silica
gel 60, 0.2-0.5 or 0.040-0.063 mm.
€
Solvents were evaporated in a Buchi rotavapor (Flawil, Switzerland).
Apparatus. 1H and 13C NMR data were acquired at room temperature
€
on a Bruker AMX 300 spectrometer operating at 300.13 and 75.47 MHz,
respectively. Dimethyl sulfoxide-d6 was used as a solvent. Chemical shifts
are expressed in δ (ppm) values relative to tetramethylsilane (TMS) as
internal reference, and coupling constants (J) are given in Hz. Electron
impact mass spectrometry (EI-MS) was carried out on a VG AutoSpec
instrument. The data are reported as m/z (% of relative intensity of the
€
most important fragments). Melting points were obtained on a Kofler
DSC Measurements. Samples of linoleic acid (2.5 to 3.0 mg) were
placed in standard aluminum pans. An appropriate amount of each tested
antioxidant was dissolved in methanol to produce a 1 mM solution. Linoleic
acid was then spiked, in the aluminum pan, with 10 μL of the antioxidant
solution. Small variations in sample size in this weight range had no
noticeable effect on the oxidation induction temperature (OIT) measured.
Samples were then kept at room temperature for a time period to allow the
removal of excess of solvent. Each sample test was run in triplicate.
Radical-Scavenging Activity (DPPH Assay). The radical scaven-
ging activity of protocatechuic acid and its esters was determined using the
free radical DPPH• in ethanol (0.25 mM) (18). Monitoring of the DPPH•
concentration depletion was accomplished by absorbance measurement at
517 nm. Different concentrations were used, expressed as (moles of
microscope (Reichert Thermovar) and are uncorrected.
Voltammetric studies were performed using an Autolab PGSTAT 12
potentiostat/galvanostat (Eco-Chemie, The Netherlands) and a one-
compartment glass electrochemical cell. Voltammetric curves were re-
corded at room temperature using a three-electrode system. A glassy
carbon working electrode (GCE) (d = 2 mm), a platinum wire counter
electrode, and an Ag/AgCl saturated KCl reference electrode were used
(Metrohm, Switzerland). A Crison pH meter with glass electrode was used
for the pH measurements (Crison, Spain).
Spectrophotometric measurements were carried out using a Helios
Gamma (Unicam, Portugal) UV/visible spectrophotometer.
A Netzsch DSC 204 calorimeter (Netzsch, Germany) was employed
for the thermoxidation stability measurements. The temperature scale was

6988 J. Agric. Food Chem., Vol. 58, No. 11, 2010
Reis et al.
Table 1. Redox Potentials (Ep) and Partition Coefficients (log P) Obtained for
Protocatechuic Acid Its Alkyl Esters and Reference Compounds
compd
Ep (mV)
log P
protocatechuic acid (1)
protocatechuic methyl ester (2)
protocatechuic ethyl ester (3)
protocatechuic propyl ester (4)
gallic acid (5)
188, 317
244
237
0.81
1.08
1.41
1.90
0.42
4.29
242
110, 544
113
Trolox (6)
esterification wherein the carboxylic acid is treated with the alkyl
alcohol in the presence of a sulfuric acid as catalyst. In this
condensation reaction, which proceeds by a nucleophilic acyl
substitution mechanism, the equilibrium was influenced by the
use of an excess of alcohol. The reaction products were afterward
purified by multiple liquid-liquid extraction, column chromato-
graphy, and/or crystallization. The reactions led to moderate/high
yields of the desired compounds, which were identified by spectro-
scopic techniques: NMR (1H NMR, 13C NMR), FT-IR, and
EI-MS.
Electrochemical Studies. The antioxidant activity of different
species is closely related to their redox properties, and consequently,
knowledge of their electrochemical behavior is a very important
basis to obtain better explanations of their properties. To contribute
to the understanding of the structure-property-activity relation-
ship of hydroxybenzoic acids and to comprehend the role that redox
potentials exert on this relation, the study of the electrochemical
properties of protocatechuic acid and its synthesized derivatives was
done.
Thus, the oxidative behavior of the phenolic compounds was
studied, at physiological pH 7.3, by differential pulse and cyclic
voltammetry, using a glassy carbon working electrode.
The differential pulse voltammetric study of protocatechuic
acid (1) (Figure 1) revealed the presence of two convolved anodic
peaks at physiological pH (Figure 2A). The oxidation peaks at
Ep = þ0.188 V, E0p = þ0.317 V (Table 1) are related to the
oxidation of the catechol group present in the structure. The
results may be interpreted by assuming that the oxidation takes
place by electron transfer for both free and adsorbed forms. The
free form corresponds to the first peak, Ep, while the strongly
adsorbed form, which is consequently stabilized, is oxidized at a
more anodic potential, E0p. The appearance of adsorption peaks,
E0p, has already been described in the literature for electroche-
mical studies involving cinnamic acids (22).
For all the protocatechuic acid alkyl esters (2-4) (Figure 1),
only one anodic wave was observed at physiological pH using
differential pulse voltammetry (Figure 2A). The oxidation waves
observed at Ep ≈ þ0.24 V (Table 1) for these derivatives are
intrinsically related to the oxidation of catechol group present in
the molecule’s structure. The occurrence of a single voltammetric
wave for the alkyl esters derivatives could indicate a higher
superimposition of the peaks from both free and adsorbed forms
or might indicate a lesser adsorption propensity of these com-
pounds related to the parent acid (22).
Cyclic voltammograms were recorded at different sweep rates.
The cyclic voltammogram obtained for protocatechuic acid (1)
shows two convolved anodic peaks corresponding to formation of
o-quinone (Figure 3). On the reverse scan, two reduction peaks
were observed. Cyclic voltammograms for the protocatechuic acid
alkyl esters (2-4) show single anodic and cathodic peaks
(Figure 3). Studies at different scan rates involving the peak current
ratio Ipc/Ipa and the difference between cathodic and anodic peaks
potential values (ΔEp) for the different compounds under study
indicate an irreversible electrode process (data not shown).
Figure 2. Differential pulse voltammograms of 0.1 mM solutions of (A)
(-) protocatechuic acid, (- - -) protocatechuic methyl ester, ( ) proto-
3 3 3
catechuic ethyl ester, and (- -) protocatechuic propyl ester and (B) (-)
3
gallic acid and ( ) Trolox in physiological pH 7.3 buffer electrolyte. Scan
3 3 3
rate was 5 mV s-1
.
antioxidant AH)/(mole of DPPH•), and for each one the reaction kinetics
was plotted. From these graphs the percentage of [DPPH•] remaining at
the steady state was determined. These values were used to determine the
efficient concentration (EC50), the amount of antioxidant necessary to
decrease the initial [DPPH•] by 50%. Moreover, the antiradical power
(ARP), defined as 1/EC50, the reaction time needed to reach the steady
state for EC50 (TEC50), and the antiradical efficiency, AE = (1/EC50)-
(TEC50), were also calculated (19).
Despite the widespread use of the DPPH• method, the lack of standardi-
zation of the results makes it difficult to compare the antioxidant strengths of
the different compounds. To establish an appropriate relationship of the
antioxidant strength of the new synthesized esters, protocatechuic acid, and
reference compounds with results available from literature, the antioxidant
activity index (AAI) was also evaluated (20).
For comparison, the radical-scavenging activity of gallic acid and
Trolox was also tested. All tests were performed in triplicate.
Calculation of Partition Coefficient (log P). Calculation of the
log P values, simulating partitioning of tested compounds in an n-octanol/
water (1:1, v/v) system, was based onCrippen’s fragmentation method (21)
and was accomplished using the ChemDraw software (ChemDraw Ultra
12.0, Cambridge Soft Corp.).
RESULTS AND DISCUSSION
Chemistry. Protocatechuic acid alkyl ester derivatives (Figure 1)
were synthesized straightforwardly by Fisher acid catalysis

Article
J. Agric. Food Chem., Vol. 58, No. 11, 2010 6989
Figure 3. Cyclic voltammograms for 0.1 mM solutions of (a) protocatechuic acid, (b) protocatechuic methyl ester, (c) protocatechuic ethyl ester, and (d)
protocatechuic propyl ester in physiological pH 7.3 buffer electrolyte. Scan rate was 20 mV s-1
.
The gathered results are in agreement with the literature that
concerns the oxidative behavior of the lead compound (23). Electro-
chemical studies on the oxidation mechanisms of parent phenolic
acids (22) have shown that the first oxidation step involves two
electrons per molecule which likely corresponds to the formation of a
o-quinone as the first oxidation product. The formation of a number
of coupling products, namely, polymers, is also likely especially for
protocatechuic acid because of the presence of a -COOH group
with electron-withdrawing character linked to the aromatic ring.
The differential pulse voltammetric study of the reference com-
pounds gallic acid and Trolox (Figure 1) revealed the presence of
well-defined anodic peaks at physiological pH (Figure 2B). The
main oxidation waves observed for these compounds occurred at
Ep = þ0.113 V and Ep = þ0.110 V, respectively (Table 1).
The electrochemical data revealed that esterification of proto-
catechuic acid does not cause significant changes in the oxidation
potentials, when compared to the lead compound. The results
obtained strongly suggest that the alkyl ester group has a modest
effect on the electron density of the catechol ring. Hence, the
introduction of an alkyl group and the increase of its length do
not significantly influence the energetic of the electron transfer, as
can be ascribed from the similarity of the Ep values for these
derivatives. However, the introduction of alkyl groups to the
carboxylic group of protocatechuic acid produced a raise of
protocatechuate radicals, as observed in the increase of the
voltammetric signal (Figure 2).
the experimental conditions used. Considering that the com-
pounds that exert strong scavenging capabilities are those pre-
senting the lowest reducing potential, it may be concluded that
both compounds used as reference must show the highest anti-
oxidant activity. However, the most powerful reducing agents are
phenolics with low reduction potential and they can, by auto-
xidation, exert prooxidant activity (23, 24). The frontier between
antioxidative and prooxidative effects is therefore very faint.
Consequently, conclusions based exclusively on voltammetric
data should be drawn cautiously.
DSC Measurements. Foods containing long-chain polyunsa-
turated fatty acids are especially labile with respect to oxidation,
which causes formation of undesirable flavors and rancid odors,
production of potentially toxic compounds, and loss of the health
beneficial and essential fatty acids. One industrially acceptable
method to overcome the oxidative instability of fatty products
and to simultaneously increase nutritional, therapeutic, or food
quality preservation is the application of natural or synthetic
antioxidants.
Because autoxidation is a process in which heat is released,
thermal analysis is a simple and direct analytical method used
to follow the reaction course by continuous monitoring of total
thermal effect of lipid oxidation occurring in the microcalori-
meter vessel. Differential scanning calorimetry (DSC) has been
increasingly used in several studies of edible oils autoxida-
tion (25).
From the voltammetric data found it is evident that all the
studied compounds investigated present antioxidant activity under
A simple method to compare the efficiency of several antioxi-
dants and/or stabilizing systems is by determination of the

6990 J. Agric. Food Chem., Vol. 58, No. 11, 2010
Reis et al.
Table 2. Oxidation Induction Temperature (OIT) and Peak Temperature
of the Derivative Curve (PTDC) Obtained for Pure and Inhibited Linoleic
Acid (LA)
compd
OIT (°C)
PTDC (°C)
pure LA
105.7
159.7
152.4
164.9
168.9
156.7
139.5
125.2
170.6
166.6
175.6
181.5
182.0
151.1
LA þ protocatechuic acid
LA þ protocatechuic methyl ester
LA þ protocatechuic ethyl ester
LA þ protocatechuic propyl ester
LA þ gallic acid
LA þ Trolox
DSC curves obtained for oxidation of LA inhibited by proto-
catechuic acid and its alkyl esters are presented in Figure 4. Their
shapes are similar to the uninhibited LA oxidation curve, but
oxidation kinetics appears slightly different (Figure 4A). By use of
the described experimental conditions (see Materials and Meth-
ods), the oxidation induction temperature (OIT) obtained for
pure LA was 105.7 °C. The addition of protocatechuic acid
modifies the OIT of linoleic acid from 105.7 to 159.7 °C
(Table 2). This phenolic acid acts as an antioxidant, slowing
down significantly linoleic acid oxidation, this behavior being
consistent with the data found in literature for related com-
pounds (28). Nevertheless, LA oxidation pattern seems to be
affected upon protocatechuic acid addition. In fact, the DSC
curve presents, near the OIT for pure LA, a slight increase in the
energy flow characteristic of the occurrence of an oxidation
reaction. However, this oxidative process seems to be quickly
counterbalanced, permitting only the main oxidation to occur at
higher temperatures (Figure 4A, Table 2). The apparent inability
of protocatechuic acid to initially block the oxidation of LA can
be linked to the difficulty of its diffusion across the lipid matrix or
to the occurrence of a rather different oxidation kinetics mechan-
ism from that occurring for pure LA. The fact of this behavior
being less evident for protocatechuic alkyl esters, compounds
with higher lipophilicity (logP), could indicate that the diffusion
assumption is morelikely. Yet the data found for Trolox highlight
another possibility. This subject will be clarified below.
To make the OIT readings from DSC thermograms simpler
and faster, the data found were re-evaluated in terms of the
thermogram first-order derivative (Figure 4B). These differential
thermograms were found to provide sharper and better defined
peaks, facilitating data reading and acquisition (Table 2).
The protocatechuic alkyl esters also exhibit antioxidant capa-
city toward LA oxidation, as shown by the increase observed in
OIT (Figure 4A, Table 2). From the results it can be concluded
that the alkyl esters display an antioxidant capacity comparable
to that ascribed to the acid. However, an increasing antioxidant
trend is observed with an increase of the number of methylenic
groups on the alkyl ester chain. This same pattern is also
described in the literature for some hindered phenol antioxi-
dants (29).
The OIT for linoleic acid stabilized with the two reference
compounds, gallic acid and Trolox, has also been measured and
compared with the OIT results obtained for protocatechuic acid
and its synthesized alkyl esters (Figure 5). An increase of the OIT
to higher values, 139.5 and 156.7 °C for LA inhibition with gallic
acid and Trolox, respectively, was observed indicating a clear
delaying of lipid oxidation.
Figure 4. (A) DSC and (B) first-order derivative curves of nonisothermal
oxidation of (a) linoleic acid (LA), (b) LA þ protocatechuic acid, (c) LA þ
protocatechuic methyl ester, (d) LA þ protocatechuic ethyl ester, and (e)
LA þ protocatechuic propyl ester.
oxidation induction temperature (OIT) of the material after
processing. Especially for polyolefins, OIT tests are well establi-
shed for quality control purposes as a quick screening method to
check the activity of the stabilization systems (26).
Therefore, the antioxidant activity of protocatechuic acid and
its alkyl esters was also evaluated by DSC using pure linoleic acid
(LA) as a lipid model system. Because of the presence of double
bonds in LA, its oxidation starts at relatively low temperature in
comparison with saturated fatty acids, and clear exothermic
effects due to oxidation can be easily monitored (27). Studies
reported in the literature showed that linoleic acid autoxidation
processes shouldbe assigned to initial exothermal effects recorded
on DSC curves (25). Hence, extrapolated oxidation induction
temperatures were obtained from the first exothermal peak of
nonisothermal oxidation of LA.
The DSC curve obtained for LA oxidation inhibited by Trolox
presents, near the OIT for pure LA, a slight increase in the energy
flow characteristic of the occurrence of an oxidation reaction.
This same pattern had already been observed for protocatechuic
acid inhibition of LA. Since this behavior was less evident for the

Article
J. Agric. Food Chem., Vol. 58, No. 11, 2010 6991
Table 3. Radical-Scavenging Activity of Protocatechuic Acid, Its Alkyl Esters
Derivatives, and Reference Compounds Using DPPH Assay
EC50
(μM)
TEC50
(min)
AE
(ꢀ10-3)
compd
ARP
AAI
protocatechuic acid (1)
protocatechuic methyl ester (2)
protocatechuic ethyl ester (3)
protocatechuic propyl ester (4)
gallic acid (5)
15.0
6.0
0.067
0.17
300
175
175
175
70
0.22
0.95
1.14
1.43
1.19
8.06
22.19
50.74
56.32
65.71
25.12
6.61
5.0
4.0
0.20
0.25
12.0
31.0
0.083
0.032
Trolox (6)
4
imbalance could therefore be the reason for the lesser propensity
shown by gallic acid to inhibit LA oxidation. Further studies are
currently in progress in order to clarify this subject.
In conclusion, nonisothermal DSC data showed that proto-
catechuic acid and its alkyl ester derivatives are good antioxidants
regarding the inhibition of lipid oxidation. Moreover, the gath-
ered results allow the conclusion that the protocatechuic alkyl
ester derivatives studied exhibit an antioxidant capacity higher
than gallic acid and that closely matches that found for Trolox.
Radical-Scavenging Activity. The main mechanism of action of
phenolic antioxidants is free radical scavenging. Since the DPPH
assay is a simple method that enables measurement of the ability
of antioxidants to trap free radicals, the reactivity of the proto-
catechuic acid and its alkyl esters toward this radical was
evaluated. Two reference compounds, gallic acid and Trolox,
were also included in these studies. The data obtained (Table 3)
clearly show that protocatechuic acid has a lower DPPH radical-
scavenging activity than its derivatives. All the different indexes
considered for the potency expression of the antioxidant activity,
namely, the antiradical power (ARP), the antiradical efficiency
(AE), and the antioxidant activity index (AAI), revealed this
trend. In fact, on the basis of AE values, protocatechuic propyl
ester was 6 times more efficient than protocatechuic acid. More-
over, according to antiradical efficiency classification, proto-
catechuic propyl ester presents an AE similar to gallic acid but
lower than Trolox. On the other hand, considering the AAI index,
it is possible to conclude that all the compounds under study exert
a strong antioxidant activity. Again, protocatechuic propyl ester
emerges as the most effective of the synthesized compounds
and even much more efficient than the gallic acid and Trolox
standards.
This behavior is consistent with the antiradical activity de-
scribed in the literature for related phenolic esters (16, 30). It has
been demonstrated that catechols bearing a strong electron-
withdrawing substituent at C(1) exhibit high DPPH-radical-
scavenging activity in alcoholic solvents, since electron-with-
drawing substituents enhance the electrophilicity of theo-quinone
intermediate (15). It was also suggested that the low reactivity of
the protocatechuic acid compared to its methyl ester is due to the
dissociation of the electron-withdrawing carboxylic acid function
to the electron-donating carboxylate ion which decreases the
electrophilicity of the o-quinone (16). Because the electron-
transfer mechanism is the most important pathway in DPPH•
stabilization by hydroxycinnamic acid and its methyl esters, (31)
it is expected that there is a relation between the increase of the
length of the alkyl chain and the radical-scavenging capacity. In
fact, it was observed that the increase in the length of the alkyl
chain increases the radical-scavenging capacity. This effect is
probably related to the electron-donating enhancement caused
by the alkyl chain increase, from which a well-stabilized phenoxyl
radical is achieved. The assumption is consistent with the data
obtained during the electrochemical studies. The introduction
of alkyl groups to the carboxylic group caused an increment of
Figure 5. (A) DSC and (B) first-order derivative curves of nonisothermal
oxidation of (a) linoleic acid(LA), (b) LA þ gallic acid, and (c) LA þ trolox.
alkyl esters derivatives, this conduct was ascribed to differences in
the compound’s lipophilicity. However, this assumption could
not be valid with respect to Trolox, since it presents the highest
log P of the molecules under study. Therefore, the occurrence of
this pattern could be simultaneously related to a molecule’s
property other than lipophilicity. As was stressed during the
electrochemical studies, besides its scavenging capabilities, phe-
nolics with low reduction potentials could also exert prooxidant
activity, mainly due to autoxidation. Protocatechuic acid, gallic
acid, and Trolox present the lowest redox potentials of the
compounds under study. Therefore, this could be the key to
understanding the occurrence of a different oxidation pattern for
inhibited LA. The occurrence of prooxidative effects could be
rapidly counterbalanced by the antioxidative capacity of the
compounds, resulting in the highest OIT values. Although gallic
acid exhibits one of the smallest redox potential, it presents
also the lowest log P, meaning that is the less lipophilic of the
compounds under study. This redox potential-lipophilicity

6992 J. Agric. Food Chem., Vol. 58, No. 11, 2010
Reis et al.
the voltammetric signal, a feature that could be related to the
increased ability of stabilization of protocatechuate radicals
produced during the oxidative process.
Estimation of partition coefficients (log P). The failure of
promising candidates as viable drugs can be traced to diffi-
culties in their transport across membranes. Current approaches
for predicting permeation through lipid barriers rely on highly
generic physicochemical parameters such as the partition co-
efficients.
esters in a heterophasic system: a mechanistic insight. J. Agric. Food
Chem. 2004, 52, 6425–6432.
(7) Figueroa-Espinoza, M. C.; Villeneuve, P. Phenolic acids enzymatic
lipophilization. J. Agric. Food Chem. 2005, 53, 2779–2787.
(8) Decker, E. A.; Warner, K.; Richards, M. P.; Shahidi, F. Measuring
antioxidant effectiveness in food. J. Agric. Food Chem. 2005, 53,
4303–4310.
(9) Robbins, R. J. Phenolic acids in foods: an overview of analytical
methodology. J. Agric. Food Chem. 2003, 51, 2866–2887.
(10) Natella, F.; Nardini, M.; Felice, M. D.; Scaccini, C. Benzoic and
cinnamic acid derivatives as antioxidants: structure-activity rela-
tion. J. Agric. Food Chem. 1999, 47, 1453–1459.
The activity of an antioxidant is mainly governed by lipophi-
licity, which determines partitioning of compounds in the lipid
phase. In view of better understanding the overall properties of
the compounds, the lipophilicity, expressed as the octanol-water
partition coefficient and herein called log P, was calculated
according to Crippen’s fragmentation method (21). The exami-
nation of the data obtained allows the conclusion that esterifica-
tion leads to a clear increase of the log P compared to the acid.
Moreover, an increase of lipophilicity is also attained with the
introduction of additional methylenic groups in the ester alkyl
chain (Table 1).
The order of increasing lipophilicity on the basis of calculated
partition coefficient values (logP) was gallic acid < protocate-
chuic acid < protocatechuic methyl ester < protocatechuic ethyl
ester < protocatechuic propyl ester < Trolox (Table 1).
In conclusion, the alkyl protocatechuate compounds synthe-
sized demonstrated a higher radical-scavenging activity than the
natural antioxidant protocatechuic acid. The increase of the
length of ester alkyl chain attached to the catecholic ring seems
to influence the stabilization of the radicals formed in the
oxidation process. Moreover, the introduction of alkyl groups
in the carboxylic acid led to a significant increase of lipophilicity
which seems to be positively influenced by the antioxidant activity
of protocatechuic acid derivatives.
(11) Tanaka, T.; Kojima, T.; Kawamori, T.; Mori, H. Chemoprevention
of digestive organs carcinogenesis by natural product protocatechuic
acid. Cancer 1995, 75, 1433–1439.
(12) Tseng, T. H.; Hsu, J. D.; Lo, M. H.; Chu, C. Y.; Chou, F. P.; Huang,
C. L.; Wang, C. J. Inhibitory effect of Hibiscus protocatechuic acid
on tumor promotion in mouse skin. Cancer Lett. 1998, 126, 199–207.
(13) Nakamura, H.; Nishikawa, A.; Furukawa, F.; Kasahara, K.;
Miyauchi, M.; Son, H. Y.; Hirose, M. Inhibitory effects of proto-
catechuic acid on the post-initiation phase of hamster pancreatic
carcinogenesis induced by N-nitrosobis(2-oxopropyl)amine. Anti-
cancer Res. 2000, 20, 3423–3427.
(14) Saito, S.; Okamoto, Y.; Kawabata, J. Effects of alcoholic solvents on
antiradical abilities of protocatechuic acid and its alkyl esters.
Biosci., Biotechnol., Biochem. 2004, 68, 1221–1227.
(15) Saito, S.; Kawabata, J. Effects of electron-withdrawing substituents
on DPPH radical scavenging reactions of protocatechuic acid and its
analogues in alcoholic solvents. Tetrahedron 2005, 61, 8101–8108.
(16) Saito, S.; Kawabata, J. DPPH (=2,2-diphenyl-1-picrylhydrazyl)
radical-scavenging reaction of protocatechuic acid (=3,4-dihydroxy-
benzoic acid): difference in reactivity between acids and their esters.
Helv. Chim. Acta 2006, 89, 1395–1407.
(17) Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Structure-antioxidant
activity relationships of flavonoids and phenolic acids. Free Radical
Biol. Med. 1996, 20, 933–956.
(18) Nenadis, N.; Boyle, S.; Bakalbassis, E. G.; Tsimidou, M. An
experimental approach to structure-activity relationships of caffeic
and dihydrocaffeic acids and related monophenols. J. Am. Oil Chem.
Soc. 2003, 80, 451–458.
DSC measurements on the oxidation of linoleic acid with
added protocatechuic acid and its alkyl esters enabled the assess-
ment of the influence of the alkyl group on the catecholic ring and
on the antioxidant activity of protocatechuic acid derivatives. The
use of this technique, particularly of differential thermograms,
was revealed to be a simple and to expedite evaluation and
comparison of the efficiency of antioxidants.
Electrochemical measurements showed its ability for the study
and elucidation of the oxidation process and its effectiveness
for the understanding of structure-property-activity relations.
The cooperative use of different analytical methods for the anti-
oxidant activity determination has shown the interesting anti-
oxidant potential of protocatechuic acid alkyl esters.
(19) Sanchez-Moreno, C.; Larrauri, J. A.; Saura-Calixto, F. A procedure
to measure the antiradical efficiency of natural polyphenols. J. Sci.
Food Agric. 1998, 76, 270–276.
(20) Scherer, R.; Godoy, H. T. Antioxidant activity index (AAI) by
the 2,2-diphenyl-1-picrylhydrazyl method. Food Chem. 2009, 112,
654–658.
(21) Ghose, A. K.; Crippen, G. M. Atomic physicochemical parameters
for three-dimensional-structure-directed quantitative structure-
activity relationships. 2. Modeling dispersive and hydrophobic
interactions. J. Chem. Inf. Comput. Sci. 1987, 27, 21–35.
(22) Gaspar, A.; Garrido, E. M.; Esteves, M.; Quezada, E.; Milhazes, N.;
Garrido, J.; Borges, F. New insights into the antioxidant activity of
hydroxycinnamic acids: Synthesis and physicochemical characteri-
zation of novel halogenated derivatives. Eur. J. Med. Chem. 2009, 44,
2092–2099.
LITERATURE CITED
(1) Food Antioxidants;Technological, Toxicological and Health Per-
spectives; Madhavi, D. L., Desphande, S. S., Salunkhe, D. K., Eds.;
Marcel Dekker Inc.: New York, 1996.
(2) Bors, W.; Heller, W.; Michel, C.; Stettmaier, K. Flavonoids and
Polyphenols: Chemistry and Biology; Marcel Dekker Inc.: New York,
1996.
(3) Rice-Evans, C. Screening of Phenolics and Flavonoids for Anti-
oxidant Activity. In Antioxidants Food Supplements in Human Health;
Packer, L., Hiramatsu, M., Yoshikawa, T., Eds.; Academic Press: London,
1999; pp 239-253.
(4) Surh, Y. J. Molecular mechanisms of chemopreventive effects of
selected dietary and medicinal phenolic substances. Mutat. Res.
1999, 428, 305–327.
(23) Simic, A.; Manojlovic, D.; Segan, D.; Todorovic, M. Electrochemi-
cal behaviour and antioxidant and prooxidant activity of natural
phenolics. Molecules 2007, 12, 2327–2340.
(24) Andersen, M. L.; Lauridsen, R. K.; Skibsted, L. H. Optimizing the
Use of Phenolic Compounds in Foods. In Phytochemical Functional
Foods; Johnson, I., Williamson, G., Eds.; Woodhead Publishing Ltd.:
Cambridge, U.K., 2003; pp 315-346.
(25) Litwinienko, G. Analysis of Lipid Oxidation by Differential Scan-
ning Calorimetry. In Analysis of Lipid Oxidation; Kamal-Eldin, A.,
Pokorny, J., Eds.; JAOCS Press: Champaign, IL, 2005; pp 152-193.
(26) Peltzer, M.; Jimenez, A. Determination of oxidation parameters
´
by DSC for polypropylene stabilized with hydroxytirosol (3,4-
dihydroxy-phenylethanol). J. Therm. Anal. Calorim. 2009, 96, 243–248.
(27) Musialik, M.; Litwinienko, G. DSC study of linolenic acid auto-
xidation inhibited by BHT, dehydrozingerone and olivetol. J. Therm.
Anal. Calorim. 2007, 88, 781–785.
(5) Galati, G.; O’Brien, P. J. Potential toxicity of flavonoids and other
dietary phenolics: significance for their chemopreventive and anti-
cancer properties. Free Radical Biol. Med. 2004, 37, 287–303.
(6) Anselmi, C.; Centini, M.; Granata, P.; Sega, A.; Buonocore, A.;
Bernini, A.; Facino, R. M. Antioxidant activity of ferulic acid alkyl

Article
J. Agric. Food Chem., Vol. 58, No. 11, 2010 6993
(28) Litwinienko, G.; Kasprzycka-Guttman, T.; Jamanek, D. DSC study
of antioxidant properties of dihydroxyphenols. Thermochim. Acta
1999, 331, 79–86.
chain characteristics groups. J. Agric. Food Chem. 2003, 51, 1874–
1879.
(31) Foti, M. C.; Daquino, C.; Geraci, C. Electron-transfer reaction of
cinnamic acids and their methyl esters with the DPPH• radical in
alcoholic solutions. J. Org. Chem. 2004, 69, 2309–2314.
ꢀ
(29) Breese, K. D.; Lamethe, J.-F.; DeArmitt, C. Improving synthetic
hindered phenol antioxidants: learning from vitamin E. Polym.
Degrad. Stab. 2000, 70, 89–96.
Received for review February 10, 2010. Revised manuscript received
April 22, 2010. Accepted April 26, 2010.
(30) Nenadis, N.; Zhang, H. Y.; Tsimidou, M. Z. Structure-antioxidant
activity relationship of ferulic acid derivatives: effect of carbon side