Dietary phenolic acids and derivatives. Evaluation of the antioxidant activity of sinapic acid and its alkyl esters

Article abstract of DOI:10.1021/jf103075r

The action of sinapic acid and its alkyl esters as potential antioxidants has been investigated. For this purpose, a series of sinapic acid ester derivatives was synthesized and their antioxidant activities were evaluated using distinctive analytical meth

Full text of DOI:10.1021/jf103075r

J. Agric. Food Chem. 2010, 58, 11273–11280 11273
DOI:10.1021/jf103075r
Dietary Phenolic Acids and Derivatives. Evaluation of the
Antioxidant Activity of Sinapic Acid and Its Alkyl Esters
†,§
,
A
LEXANDRA
ORGE
G
ASPAR,† MARTA
M
F
ARTINS,§ PAULO
S
ILVA,§ E. MANUELA
G
ARRIDO
^
J
G
ARRIDO
,
†,§ OMIDREZA
IRUZI,*,,#
R
AMIN
M
IRI,# LUCIANO
S
ASO
,
AND
,,†
*
F
ERNANDA
B
ORGES
†CIQ/Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, 4169-007
Porto, Portugal, §CIETI/Department of Chemical Engineering, School of Engineering (ISEP), Polytechnic
Institute of Porto, 4200-072 Porto, Portugal, #Medicinal and Natural Products Chemistry Research
Center, Shiraz University of Medical Sciences, Shiraz, Iran, and ^Department of Physiology and
Pharmacology “Vittorio Erspamer”, Sapienza University of Rome, Rome, Italy
The action of sinapic acid and its alkyl esters as potential antioxidants has been investigated. For
this purpose, a series of sinapic acid ester derivatives was synthesized and their antioxidant
activities were evaluated using distinctive analytical methods, namely, 2,2-diphenyl-1-picrylhydrazyl
(DPPH) and FRAP UV-vis methods and differential scanning calorimetry. The electron-donating
activity and lipophilicity of these phenolic compounds were also evaluated. From the overall results it
was concluded that alkyl ester sinapates (linear alkyl esters) present almost the same antioxidant
activity, albeit slightly lower, exhibited by the parent compound (sinapic acid). Furthermore, the
addition of an alkyl ester side chain has a positive effect on the partition coefficient of sinapic acid,
improving its utility as an antioxidant in a more lipophilic medium. The data on the antioxidant activity
obtained by different analytical methods correlated well with each other and have revealed
interesting antioxidant data of alkyl esters of sinapic acid.
KEYWORDS: Sinapic acid; alkyl esters; antioxidant activity; electrochemistry; DSC
INTRODUCTION
underlying their action are not yet completely understood. Some
data point out that it could be intrinsically linked to their
antioxidant activity and in turn strongly dependent on their
structural characteristics (10, 11).
Polyphenols are a complex group of bioactive compounds
widely spread in the plant kingdom (1). Nowadays it is believed
that these compounds are involved in the defense process against
deleterious oxidative damage due to, at least in part, their
antioxidant properties. Among polyphenolic compounds, hydro-
xycinnamic acids and derivatives are a well-known group of
phytochemicals, which are found in cereals, roasted coffee,
asparagus, green vegetables, and many other plants (2). In
addition to their primary antioxidant activity, this type of
compound also possesses other significant biological functions
such as anti-inflammatory, anticarcinogenic, and antimicrobial
effects (3-5). In fact, recent data demonstrate the beneficial
effects of these compounds as preventive and/or therapeutic
agents in several diseases, such as atherosclerosis, inflammatory
injury, and cancer (6, 7). Because of their hydrophilic nature,
hydroxycinnamic acids cannot be properly used in oil-based
processes, which is an important issue in industrial applications,
or be effective as antioxidants in biological systems. Recently,
some structural modifications have been performed on the
phenolic acid skeleton to improve their application outline (e.g.,
to increase the lipophilicity) (8, 9). Although cinnamic esters, in
particular, were shown to display remarkable growth inhibition
properties toward some human cancer cell lines, the mechanisms
Sinapic acid (3,5-dimethoxy-4-hydroxycinnamic acid; Figure 1)
is a hydroxycinnamic acid widely distributed in nature and present
in many foods, edible plants, and fruits (12-14). Rapeseed partic-
ularly contains sinapic acid as the predominant phenolic com-
pound (14). Sinapic acid has been proposed as a potent antioxidant,
its effectiveness being described as higher than that of ferulic acid
(3-methoxy-4-hydroxycinnamic acid) and sometimes comparable
to that of caffeic acid (3,4-dihydroxycinnamic acid) (15-17).
The relevance of sinapic acid in cell protection and in oxidative-
related diseases was already reported owing to its peroxynitrite
(ONOO-) scavenging activity (18, 19). In addition, anxiolytic
and anti-inflammatory properties were also ascribed to this com-
pound (20, 21).
Although a huge number of investigations have been carried
out on the study of the influence of esterification on the anti-
oxidant efficiency of phenolic acids, namely, in cinnamic ana-
logues, for example, ferulic acid (22,23), the information reported
about the activity of sinapic acid and its alkyl esters is scarce.
Accordingly, an interactive project was developed aiming at
accomplishing a more reliable understanding of the antioxidant
activity of sinapic acid and its alkyl esters derivatives. Therefore,
the synthesis of a new set of lipophilic phenolic antioxidants
(Figure 1) was realized and their antioxidant profile determined
*Corresponding authors [(F.B.) e-mail fborges@fc.up.pt, phone þ351
220 402 560, fax þ351 220 402 009; (O.F.) e-mail foomid@yahoo.com].
© 2010 American Chemical Society
Published on Web 10/15/2010
pubs.acs.org/JAFC

11274 J. Agric. Food Chem., Vol. 58, No. 21, 2010
Gaspar et al.
Figure 1. Sinapic acid and its alkyl esters under study.
using different analytical methods. In addition, the redox poten-
tials and partition coefficients of sinapic acid and its derivatives
properties were also acquired to test their influence in the
antioxidant performance of the phenolic compounds.
Synthesis. General Synthetic Procedure. The alkyl esters of the phe-
nolic acid were obtained using the following general procedure: Sinapic
acid (1.0 g) was dissolved in 75 mL of the corresponding alcohol
(methanol, ethanol, n-propanol, or butanol) containing 1 mL of H2SO4,
and the solution was stirred, at room temperature, during ca. 5 days. The
solvent was partially evaporated under reduced pressure. The mixture was
then extracted with diethyl ether (3 ꢀ 75 mL), and the organic phases were
combined, washed with 10% Na2CO3 solution, and dried over Na2SO4.
The solvent was evaporatedunder reduced pressure, and the crude product
was purified by column chromatography and then recrystallized.
Methyl sinapate ((E)-methyl 3-(4-hydroxy-3,5-dimethoxyphenyl )propenoate):
yield, 79%; FTIR, 3522, 3460, 3062, 2946, 2845, 1704, 1631, 1604, 1515,
1461, 1428, 1384, 1339, 1287, 1260, 1233, 1181, 1152, 1120; 1H NMR, 8.98
(1 H, s, 4-OH), 7.57 (1H, d, J = 15.9, H ( β)), 7.03 (2H, s, H(2), H(6)), 6.54
(1H, d, J = 15.9, H(R)), 3.80 (6H, s, 2 ꢀ OCH3), 3.72 (3H, s, CH3); 13C
NMR, 167.1 (CdO), 148.0 (C(3), C(5)), 145.5 (C( β)), 138.3 (C;OH), 124.4
(C(1)), 114.6 (C (R)), 106.2 (C(2), C(6)), 56.1 (2 ꢀ OCH3), 51.3 (CH3);
EI-MS, 238 (100, M•þ), 207 (32), 175 (20), 163 (21), 135 (16), 121 (14), 119
(13), 77 (17), 65 (19), 59 (13), 56 (36), 53 (18), 51 (17).
MATERIALS AND METHODS
General. Linoleic acid (LA, cis,cis- 9,12-octadecadienoic acid, 99%),
2,2-diphenyl-1-picrylhydrazyl (DPPH•), quercetin, sinapic acid, and Tro-
lox were purchased from Sigma-Aldrich, St. Louis, MO. 2,4,6-Tripyridyl-
s-triazine (TPTZ), dimethyl-d6 sulfoxide (99.8%), and tetramethylsilane
(TMS) were obtained from Merck, Darmstadt, Germany. All other
reagents and solvents were of pro analysis grade and used without
additional purification. Deionized water (conductivity < 0.1 μS cm-1
)
was used throughout all of the experiments.
Thin-layer chromatography (TLC) was carried out on precoated silica
gel 60 F254 (Merck) with a layer thickness of 0.2 mm. For analytical
control the following systems were used: ethyl ether/petroleum ether and
chloroform/methanol. 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.
Ethyl sinapate ((E)-ethyl 3-(4-hydroxy-3,5-dimethoxyphenyl )propenoate):
yield, 83%; FTIR, 3654, 3496, 3090, 2938, 2841, 1690, 1636, 1600, 1515,
1461, 1425, 1373, 1340, 1287, 1259, 1222, 1187, 1151, 1116; 1H NMR, 8.97
(1 H, s, 4-OH), 7.55 (1H, d, J = 15.9, H ( β)), 7.02 (2H, s, H(2), H(6)), 6.53
(1H, d, J = 15.9, H(R)), 4.16 (2H, q, J = 7.0, CH2), 3.79 (6H, s, 2 ꢀ OCH3),
1.24 (3H, t, J= 7.0, CH3); 13C NMR, 166.6 (CdO), 148.0 (C(3), C(5)), 145.2
(C( β)), 138.2 (C;OH), 124.4 (C(1)), 115.0 (C (R)), 106.2 (C(2), C(6)), 59.7
(CH2), 56.1 (2 ꢀ OCH3), 14.3 (CH3); EI-MS, 252 (100, M•þ), 207 (34), 180
(48), 175 (22), 163 (13), 121 (16), 119 (13), 91 (14), 77 (15), 65 (20), 58 (20), 53
(15), 51 (14).
€
Solvents were evaporated in a Buchi Rotavapor (Flawil, Switzerland).
Apparatus. 1H and 13C NMR data were acquired, at room tempera-
€
ture, on a Bruker AMX 300 spectrometer operating at 300.13 and 75.47
MHz, respectively. Dimethyl-d6 sulfoxide was used as a solvent; chemical
shifts are expressed in δ (ppm) values relative to TMS as internal reference;
coupling constants (J ) are given in hertz. Assignments were also made
from distortionless enhancement by polarization transfer (DEPT)
(underlined values). Electron impact mass spectra (EI-MS) were carried
out on a VG AutoSpec instrument; the data are reported as m/z (% of
relative intensity of the most important fragments). Infrared spectra were
recorded on an ATI Mattson Genesis series FTIR spectrophotometer
using potassium bromide disks; only the most significant absorption bands
are reported (νmax, cm-1).
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
recorded 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. A
Crison pH-meter with glass electrode was used for the pH measurements
(Crison, Spain).
Spectrophotometric measurements of the absorbance of DPPH free
radical were carried out using a UV-vis spectrophotometer (Bio-Tek,
model Uvikon XL). The absorbance of Fe2þ/TPTZ complex in the FRAP
assay was determined by a microplate reader (Bio-Rad, model 680).
A Netzsch DSC 204 calorimeter (Netzsch, Germany) was employed for
the thermoxidation stability measurements. The temperature scale was
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.
Propyl sinapate ((E)-propyl 3-(4-hydroxy-3,5-dimethoxyphenyl )propenoate):
yield, 82%; FTIR, 3532, 3425, 2968, 2938, 2880, 2840, 1682, 1634, 1598,
1513, 1459, 1424, 1378, 1340, 1314, 1283, 1222, 1193, 1152, 1118; 1H NMR,
8.95 (1 H, s, 4-OH), 7.55 (1H, d, J = 15.9, H ( β)), 7.03 (2H, s, H(2), H(6)),
6.53 (1H, d, J = 15.9, H(R)), 4.08 (2H, t, J = 6.6, CH2), 3.79 (6H, s, 2 ꢀ
OCH3), 1.68-1.63 (2H, m, CH2), 0.93 (3H, t, J = 7.4, CH3); 13C NMR,
166.7 (CdO), 148.0 (C(3), C(5)), 145.3 (C( β)), 138.2 (C;OH), 124.4 (C(1)),
114.9 (C (R)), 106.2 (C(2), C(6)), 65.2 (CH2), 56.1 (2 ꢀ OCH3), 21.7 (CH2),
10.4 (CH3); EI-MS, 266 (100, M•þ), 224 (55), 207 (42), 180 (51), 175 (26), 147
(13), 121 (13), 119 (14), 91 (12), 65 (12).
Butyl sinapate ((E)-butyl 3-(4-hydroxy-3,5-dimethoxyphenyl )propenoate):
yield, 94%; FTIR, 3592, 3511, 2968, 2937, 2895, 2873, 1692, 1636, 1601,
1515, 1456, 1424, 1376, 1343, 1286, 1261, 1223, 1184, 1151, 1117; 1H NMR,
8.94 (1 H, s, 4-OH), 7.55 (1H, d, J = 15.9, H ( β)), 7.03 (2H, s, H(2), H(6)),
6.53 (1H, d, J = 15.9, H(R)), 4.12 (2H, t, J = 6.4, CH2), 3.80 (6H, s, 2 ꢀ
OCH3), 1.64-1.56 (2H, m, CH2), 1.42-1.33 (3H, m, CH2), 0.91 (3H, t, J =
7.3, CH3); 13C NMR, 166.7 (CdO), 148.0 (C(3), C(5)), 145.3 (C( β)), 138.3
(C-OH), 124.4 (C(1)), 115.0 (C(R)), 106.2 (C(2), C(6)), 63.4 (CH2), 56.1
(2 ꢀ OCH3), 30.4 (CH2), 18.8 (CH2), 13.6 (CH3); EI-MS, 280 (88, M•þ), 225
(13), 224 (100), 209 (15), 207 (45), 181 (12), 180 (58), 175 (27), 167 (17), 165
(12), 163 (12), 149 (16), 147 (16), 135 (12), 133 (12), 121 (21), 119 (17), 91
(22), 77 (21), 65 (26), 58 (46), 57(13), 55 (15), 53 (19), 51 (16).
Electrochemical Measurements. Stock solutions of the sinapic acid
and derivatives (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.

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J. Agric. Food Chem., Vol. 58, No. 21, 2010 11275
Figure 2. Differential pulse voltammograms for 0.1 mM solutions of (a) sinapic acid, (b) methyl sinapate, (c) ethyl sinapate, (d) propyl sinapate, and (e) butyl
sinapate, in physiological pH 7.3 supporting electrolyte. Scan rate = 5 mV s-1
.
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.
was measured with or without methanol, and no significant variation was
seen between the two measurements. Samples were kept at room tem-
perature for a time period to allow the removal of excess of solvent. Each
sample test was run in triplicate.
DSC Measurements. Samples of linoleic acid (2.5-3.0 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. Some experiments were performed to check whether the
methanol had any effect upon the OIT results. The OIT of linoleic acid
Calculation of Partition Coefficient (LogP). Calculation of the log
P values, simulating partitioning of tested compounds in an n-octanol/
water (1:1, v/v) system, was based on Crippen’s fragmentationmethod(24)
and was accomplished using the Molinspiration program (25).
DPPH Radical Scavenging Activity. The radical scavenging activity
of sinapic acid and its derivatives against the stable free radical DPPH was
measured as described previously, with some modifications (26). Briefly,
the test compounds were dissolved in dimethyl sulfoxide and incubated

11276 J. Agric. Food Chem., Vol. 58, No. 21, 2010
Gaspar et al.
with a methanolic solution of DPPH 100 μM. The test concentrations (in
the range of 1-100 μM) were carefully chosen for each compound to
produce a suitable dose-response curve. After 30 min of incubation at
room temperature in the dark, the absorbanceat 517 nm was measuredin a
spectrophotometer. The percent inhibition of DPPH radical was calcu-
lated on the basis of the reduction of light absorbance. The dose-response
curve was plotted by using the software SigmaPlot for Windows version
8.0, and IC50 values were calculated by the software curve-expert (for
Windows, version 1.34). Trolox was used as reference standard.
Ferric Reducing Antioxidant Power (FRAP Assay). The FRAP
assay was performed as described (27,28). Briefly, the FRAP solution was
freshly prepared by mixing 10 mL of acetate buffer 300 mM (pH 3.6), 1 mL
of ferric chloride hexahydrate 20 mM in distilled water, and 1 mL of TPTZ
10 mM in HCl 40 mM. Ten microliters of the test compound dissolved in
dimethyl sulfoxide was mixed with 190 μL of the FRAP solution.
Absorbance was determined at 595 nm in a microplate reader, after 30
min of incubation at room temperature. Sinapic acid derivatives were
tested at the final concentration of 40 μM, whereas reference compounds,
Trolox and quercetin, were tested at the final concentration of 20 μM.
Absorbance in the presence of test compounds was compared with
absorbance in the presence of quercetin, and the FRAP value for test
compounds was expressed as milliequivalents of quercetin per mole of
compound.
Table 1. Redox Potentials (Ep) Obtained for Sinapic Acid and Its Alkyl Esters
logP a,b
compound
Ep (mV)
sinapic acid
183; 298
219
189
1.29/1.26
1.55/1.88
1.89/2.26
2.38/2.76
2.79/3.32
methyl sinapate
ethyl sinapate
propyl sinapate
butyl sinapate
182
190
a Determined with ChemDraw software (ChemDraw Ultra 11.0, Cambridge Soft
Corp.). b Determined with Molinspiration Calculation service (www.molinspiration.com).
also shown two convolved anodic peaks (Figure 3a). For the alkyl
ester derivatives only one anodic wave is seen (Figure 3b-e).
All of these anodic peaks appear to correspond to irreversible
processes.
The results found herein for sinapic acid and derivatives
corroborate the scarce data available in the literature for sinapic
acid (31, 32). Moreover, the voltammetric behavior observed is
consistent with the oxidation mechanism suggested in the litera-
ture for ferulic acid, a phenolic antioxidant with an analogous
molecular structure. Therefore, the oxidation mechanism for
sinapic acid and its alkyl esters should involve a one-electron
transfer from the phenolate ion followed by one irreversible
dimerization process due to a radical-radical coupling reaction
between two phenoxyl radicals (29, 33).
RESULTS AND DISCUSSION
Chemistry. Sinapic acid derivatives (Figure 1) were synthesized
straightforwardly by Fisher acid catalysis esterification (9). These
reactions led to high yields of the desired compounds, which were
identified by the following spectroscopic techniques: NMR (1H
NMR, 13C NMR), FTIR, and MS-EI.
The differential pulse voltammetric study of the reference
compound Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-
2-carboxylic acid) revealed the presence of a well-defined anodic
peak, Ep = 110 mV, at physiological pH (9).
Electrochemical Measurements. The measurement of the reduc-
ing capacity and electrochemical behavior of phenolic compounds
can provide important information concerning their free radical
scavenging activity. In fact, the antioxidant activity of different
species is closely related to their redox properties and, consequently,
knowledge of their redox behavior is a very important basis to
obtain better explanations of their properties. Thus, the study of the
electrochemical properties of sinapic acid and its synthesized ester
derivatives was accomplished. The oxidative behavior of the com-
pounds 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 sinapic acid
(Figure 1) revealed the presence of two convolved anodic peaks,
P1 and P2, at 183 and 298 mV, respectively (Figure 2a; Table 1).
These oxidation peaks are related with the oxidation of the
phenolic group present in the molecular structure. The appear-
ance of the two waves may be interpreted by assuming that the
electrochemical oxidation of sinapic acid takes place on the glassy
carbon electrode by electron transfer for both free and adsorbed
forms. The free form corresponds to the first peak (P1), whereas
the strongly adsorbed form is oxidized at a more anodic potential
(P2). The occurrence of an adsorption peak has been also
described in the literature for voltammetric studies involving
related compounds, namely, ferulic acid (29, 30).
For all of the synthesized sinapic alkyl esters only one anodic
wave was observed at physiological pH using differential pulse
voltammetry (Figure 2b-e; Table 1). These oxidation waves could
also be ascribed to the presence of a phenolic group in the
structure of the molecules (Figure 1). The appearance of a single
wave for the sinapic alkyl esters could be interpreted as a result of
a less significant adsorption propensity of these compounds when
compared with sinapic acid. In fact, the peak observed for alkyl
esters occurs at a potential similar to that obtained for wave P1
attributed to the oxidation of the free form of sinapic acid.
Cyclic voltammograms were recorded at different sweep
rates. The cyclic voltammograms obtained for sinapic acid have
From the voltammetric data it could be concluded that the
introduction of an alkyl ester group in the ethylenic side chain of
sinapic acid did not significantly change the oxidation potentials,
when compared to the precursor. The results obtained strongly
suggest that the alkyl groups have modest or even no effect on the
electron density of the phenolic group. Hence, the introduction of
an alkyl group did not significantly influence the energy of the
electron transfer process as can be deduced from the similarity of
the Ep values for these ester derivatives.
DSC Measurements. Lipid peroxidation has long been recog-
nized as a problem either in the food industry or in biological
systems (34, 35). Unsaturated fatty acids may undergo oxidation
processes that in turn result in the formation of a wide range of
products. Biological systems with a higher concentration of
polyunsaturated fatty acids will, of course, oxidize more quickly
and, as a result, lead to a rapid development of rancid off-flavors.
To minimize oxidative rancidity, antioxidants can be added. To
deal with some of these consequences, one must recognize the
mechanisms by which antioxidants of either natural or synthetic
origin control oxidation (36).
Many techniques have been developed and used to detect and
measure the extent of oxidation in fats and fats-containing
systems and to ascertain their resistance to oxidative deteriora-
tion. As the transfer of an oxygen molecule to an unsaturated
fatty acid requires energy, the oxidative stability of lipid systems
can be established by using thermal analysis. Of all the thermal
analytical methods available, differential scanning calorimetry
(DSC) is one of the most versatile and is suitable for a vast range
of applications. A simple method to compare the efficiency of
several antioxidants and/or stabilizing systems is the determina-
tion of the OIT. Particularly for polyolefins, OIT tests are well
established for quality control purposes as a quick screening
method to check the activity of the stabilization systems (9, 37).
Therefore, the antioxidant activity of sinapic acid and its alkyl
esters was evaluated by DSC using pure linoleic acid (LA) as a
lipid model system. Extrapolated OITs were obtained from the

Article
J. Agric. Food Chem., Vol. 58, No. 21, 2010 11277
Figure 3. Cyclic voltammograms for 0.1 mM solutions of (a) sinapic acid, (b) methyl sinapate, (c) ethyl sinapate, (d) propyl sinapate, and (e) butyl sinapate,
in physiological pH 7.3 supporting electrolyte. Scan rate = 50 mV s-1
.
first exothermal peak of nonisothermal oxidation of LA
(Figure 4) (9, 38). The DSC curve shapes are similar to that
obtained in the thermogram for the uninhibited LA oxidation
(Figure 4A). Under the described experimental conditions the
OIT obtained for pure LA was 108.1 ꢀC. The addition of
sinapic acid modifies the OIT of linoleic acid from 108.1 to
150.3 ꢀC (Table 2). This phenolic acid acts as an antioxidant,
slowing, significantly, linoleic acid oxidation. This behavior is
consistent with the data found in the literature for related
phenolic acids (9, 39).
The sinapic alkyl esters also exhibit antioxidant capacity
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 ability correspondent to that
ascribed to sinapic acid. The same stabilization pattern is also
reported in the literature for other alkyl ester derivatives of
phenolic antioxidants (9, 39).
DSC first-order derivative thermograms were also evaluated to
provide sharper and better defined peaks facilitating OIT data
reading and acquisition (Figure 4B; Table 2).

11278 J. Agric. Food Chem., Vol. 58, No. 21, 2010
Gaspar et al.
Table 3. FRAP and DPPH Scavenging Activities of Sinapic Acid and Its Alkyl
Esters
compound
DPPH (IC50 μM)
FRAP (IC50, μM/mol)a
sinapic acid
methyl sinapate
ethyl sinapate
propyl sinapate
butyl sinapate
Trolox
32.2 ( 6.2
48.7 ( 7.0
51.9 ( 6.3
50.6 ( 6.1
50.1 ( 3.8
38.4 ( 8.0
482.6 ( 61.1
355.5 ( 66.1
338.2 ( 53.4
341.9 ( 46.3
347.3 ( 75.7
427.4 ( 86.2
a μM quercetin equiv/mol. Values represent the mean of four to seven experi-
ments ( SD.
In conclusion, nonisothermal DSC data show that sinapic acid
and its alkyl ester derivatives are good antioxidants regarding the
inhibition of lipid oxidation. Moreover, the gathered results allow
one to conclude that the sinapic alkyl ester derivatives studied
exhibit an antioxidant capacity that closely matches that found
for Trolox.
Estimation of Partition Coefficients (Log P). In view of better
correlation of the overall properties of the antioxidant com-
pounds, the lipophilicity, expressed as the octanol-water parti-
tion coefficient and herein called log P, was calculated either
theoretically, according to Crippen’s fragmentation method, or
using the Molinspiration property calculation program (see
Table 1) (24, 25).
The data examination allows one to conclude that the intro-
duction of an alkyl group leads to a significant increase of the
sinapic acid lipophilicity (Table 1). It must be stressed that the
partition coefficients calculatedfor the sinapic acidderivativesare
well correlated with their structural features.
Higher lipophilicity is often required in the antioxidant re-
search field, because it could modify the absorption and distribu-
tion properties of hydroxycinnamic antioxidants, increasing their
ability to interact with the polar head groups of the membrane or
to achieve local concentration at the water-lipid interface,
preventing the initial reaction between aqueous radicals and
lipids (40).
Antioxidant Activity. Antioxidant activity data of sinapic acid
and its alkyl esters, measured by DPPH and FRAP assays, are
shown in Table 3. A good correlation was observed between both
methods (r2 = 0.998, data not shown). From the DPPH and
FRAP results one can conclude that sinapic acid itself has a
higher activity when compared to that obtained from alkyl esters.
All of the sinapic alkyl esters have similar antioxidant activities, a
fact that is in accordance with electrochemical and DSC data.
It may be stated that the examined phenolic compounds have
significant reducing capacity. The reducing abilities of the alkyl
esters are similar and lower than those of sinapic acid and Trolox.
Trolox has been shown to possess good reducing capacity in the
FRAP assay (27), and therefore a reducing ability at the level of
Trolox should be considered a remarkable activity.
Figure 4. (A) DSC and (B) first-order derivative curves of nonisothermal
oxidation of (a) linoleic acid (LA), (b) LA þ sinapic acid, (c) LA þ methyl
sinapate, (d) LA þ ethyl sinapate, (e) LA þ propyl sinapate, and (f) LA þ
butyl sinapate.
Table 2. Oxidation Induction Temperature (OIT) and Peak Temperature of
the Derivative Curve (PTDC) Obtained for Pure and Inhibited Linoleic Acid
(LA)
compound
OIT (ꢀC)
PTDC (ꢀC)
Insertion of an alkyl ester side chain has been reported to have
different effects on the antioxidant activity on phenolic acid
systems (9, 30, 40, 41). Previous works has shown that caffeic
acid (3,4-dihydroxycinnamic acid) alkyl esters have lower DPPH
and ABTS radical scavenging activities than caffeic acid itself,
data that seem to be dependent on the extension, or type, of the
ester side chain (30, 40-43). Other investigators have reported
that alkyl esterification of ferulic acid (4-hydroxy-3-methoxycin-
namic acid) decreases its activity against DPPH (22, 44). From
these results one can assume that the effect of the alkyl ester side
chain in hydroxycinnamic systems is strongly related to the
number of hydroxyl groups and the aromatic substitution pat-
tern. In fact, it seems to be intimately connected with the different
pure LA
108.1
150.3
161.1
159.4
159.3
162.2
156.7
128.7
176.2
171.7
169.4
167.0
177.1
151.1
LA þ sinapic acid
LA þ methyl sinapate
LA þ ethyl sinapate
LA þ propyl sinapate
LA þ butyl sinapate
LA þ Trolox
The OIT for linoleic acid stabilized with the reference com-
pound, Trolox, has also been measured. For the LA inhibition
with Trolox an increase of the OIT to higher values was observed,
evidencing a clear delay of lipid oxidation (Table 2).

Article
J. Agric. Food Chem., Vol. 58, No. 21, 2010 11279
oxidation mechanisms/capacity of stabilization of intermediates
associated phenolic acid systems. Caffeic acid and its derivatives
operate via quinone intermediate and ferulic and sinapic deriva-
tives via semiquinone intermediates. The last intermediates are
additionally stabilized by the inductive and mesomeric effects of
methoxyl functions. These overall effects can surpass the meso-
meric/inductive influence of the ethylenic side chain.
After the studies performed in this research area, one can say
that a general antioxidant relationship statement cannot be
performed for phenolic acids derivatives, in general, and, in
particular, for hydroxycinnamic acid derivatives: each family is
a study case, and each structural modification could affect the
activity in a particular way as consequence of the steric, meso-
meric, and inductive effects on the system.
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From the overall results it can be concluded that alkyl sinapates
present almost the same radical scavenging activity as well as
reducing capacity. These activities were slightly lower than the
activity displayed by the parent compound, sinapic acid. How-
ever, the synthesized antioxidants possess higher lipophilicity,
which is a considerable advantage for antioxidants that are
intended to function as membrane protectors in vivo and may
compensate for their slightly lower antioxidant activity.
The electrochemical and DSC data showed that sinapic acid
and its alkyl ester derivatives are good antioxidants regarding the
inhibition of lipid oxidation. Moreover, the gathered results
allow one to conclude that the sinapic alkyl ester derivatives
exhibit an antioxidant capacity that closely matches that found
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