Alkyl esters of hydroxycinnamic acids with improved antioxidant activity and lipophilicity protect PC12 cells against oxidative stress

Article abstract of DOI:10.1016/j.biochi.2011.12.015

Hydroxycinnamic acids (HCAs) are phenolic compounds present in dietary plants, which possess considerable antioxidant activity. In order to increase the lipophilicity of HCAs, with the aim of improving their cellular absorption and expansion of their use in lipophilic media, methyl, ethyl, propyl and butyl esters of caffeic acid and ferulic acid have been synthesized. All caffeate esters had a slightly lower DPPH IC₅₀ (13.5-14.5 μM) and higher ferric reducing antioxidant power (FRAP) values (1490-1588 mM quercetin/mole [mMQ/mole]) compared to caffeic acid (16.6 μM and 1398 mMQ/mole, respectively) in antioxidant assays. In contrast, ferulate esters were less active in DPPH (56.3-74.7 μM) and FRAP assays (193-262 mMQ/mole) compared to ferulic acid (44.6 μM and 324 mMQ/mole, respectively). Redox properties of HCAs were in line with their antioxidant capacities, so that compounds with higher antioxidant activities had lower oxidation potentials. Measurement of partition coefficients disclosed the higher lipophilicity of the esters compared to parent compounds. All esters of caffeic acid significantly inhibited hydrogen peroxide-induced neuronal PC12 cell death assessed by MTT assay at 5 and 25 μM. However, caffeic acid, ferulic acid and ferulate esters were not able to protect the cells. In conclusion, these findings suggest that alkyl esterification of some HCAs augments their antioxidant properties as well as their lipophilicity and as a consequence, improves their cell protective activity against oxidative stress. These compounds could have useful applications in conditions where oxidative stress plays a pathogenic role.

Full text of DOI:10.1016/j.biochi.2011.12.015

Biochimie 94 (2012) 961e967
Contents lists available at SciVerse ScienceDirect
Biochimie
journal homepage: www.elsevier.com/locate/biochi
Research paper
Alkyl esters of hydroxycinnamic acids with improved antioxidant activity
and lipophilicity protect PC12 cells against oxidative stress
,
,
Jorge Garrido a b, Alexandra Gaspar a, E. Manuela Garrido a b, Ramin Miri c, Marjan Tavakkoli c,
**
Samaneh Pourali c, Luciano Saso d, Fernanda Borges a , Omidreza Firuzi
,
c,
*
a CIQ/Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal
b Department of Chemical Engineering, School of Engineering (ISEP), Polytechnic Institute of Porto, 4200-072 Porto, Portugal
c Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
d Department of Physiology and Pharmacology "Vittorio Erspamer", Sapienza University of Rome, Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Hydroxycinnamic acids (HCAs) are phenolic compounds present in dietary plants, which possess
considerable antioxidant activity. In order to increase the lipophilicity of HCAs, with the aim of improving
their cellular absorption and expansion of their use in lipophilic media, methyl, ethyl, propyl and butyl
esters of caffeic acid and ferulic acid have been synthesized. All caffeate esters had a slightly lower DPPH
Received 6 November 2011
Accepted 16 December 2011
Available online 23 December 2011
IC50 (13.5e14.5
quercetin/mole [mMQ/mole]) compared to caffeic acid (16.6
antioxidant assays. In contrast, ferulate esters were less active in DPPH (56.3e74.7
(193e262 mMQ/mole) compared to ferulic acid (44.6 M and 324 mMQ/mole, respectively). Redox
properties of HCAs were in line with their antioxidant capacities, so that compounds with higher anti-
oxidant activities had lower oxidation potentials. Measurement of partition coefficients disclosed the
higher lipophilicity of the esters compared to parent compounds. All esters of caffeic acid significantly
m
M) and higher ferric reducing antioxidant power (FRAP) values (1490e1588 mM
M and 1398 mMQ/mole, respectively) in
M) and FRAP assays
Keywords:
Hydroxycinnamic acid
Caffeic acid
Ferulic acid
Alkyl esters
Antioxidant
Redox properties
PC12 cells
m
m
m
inhibited hydrogen peroxide-induced neuronal PC12 cell death assessed by MTT assay at 5 and 25 mM.
Neuroprotection
However, caffeic acid, ferulic acid and ferulate esters were not able to protect the cells. In conclusion,
these findings suggest that alkyl esterification of some HCAs augments their antioxidant properties as
well as their lipophilicity and as a consequence, improves their cell protective activity against oxidative
stress. These compounds could have useful applications in conditions where oxidative stress plays
a pathogenic role.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
considerable in vitro antioxidant activities against many ROS [5,6].
Several recent reports have also shown that HCAs isolated from
Oxidative stress is caused by the action of reactive oxygen
species (ROS) and is implicated in the pathogenesis of several
human diseases such as cancer and neurodegenerative diseases
[1,2]. Antioxidants are believed to counteract the harmful effects of
ROS and regulate the physiological defence systems and therefore
could be useful for prevention or treatment of oxidative stress-
related diseases [3,4].
Hydroxycinnamic acids (HCAs, including caffeic acid, ferulic acid
and sinapic acid among others) are phenolic compounds widely
distributed in plants and prevalently found in cereals, fruits,
vegetables and beverages [5]. These compounds have shown
plants or as purified compounds protect neuronal cells against
various types of oxidative damage [7e10]. Furthermore, a number
of studies have shown that these compounds may play a protective
role against ischemic brain injury in rats [11] and can be effective
neuroprotective agents in mice [12].
The lipophilicity of a compound is a crucial factor that deter-
mines its efficiency as an antioxidant in lipophilic compartments
and has a great impact on its ability in crossing the cell membranes
and reaching its target [13]. Therefore, various esters of HCAs that
are generally more lipophilic compared to their parent compounds
have been synthesized in the recent years and some of them have
shown some advantages over their precursor compounds [14,15].
For example, the influence of esterification on the in vitro antioxi-
dant efficiency [16,17] and neuroprotective activity [18] of ferulic
acid has been reported. Likewise, caffeic acid phenethyl ester
(CAPE) has shown promise in cell [19] and animal models of
* Corresponding author. Tel.: þ98 711 230 3872; fax: þ98 711 233 2225.
** Corresponding author. Tel.: þ351 220 402 560; fax: þ351 220 402 009.
E-mail addresses: fborges@fc.up.pt (F. Borges), firuzio@sums.ac.ir, foomid@
yahoo.com (O. Firuzi).
0300-9084/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.biochi.2011.12.015

962
J. Garrido et al. / Biochimie 94 (2012) 961e967
neurodegeneration [20], in which oxidative stress is involved. We
have recently shown that alkyl esterification of sinapic acid has
a positive impact on its partition coefficient and can improve its
antioxidant efficacy in more lipophilic media [21].
In this study, we have evaluated the antioxidant activity of 4
different esters of caffeic acid (3,4-dihydroxycinnamic acid) and
ferulic acid (4-hydroxy-3-methoxycinnamic acid). In addition, we
have measured the redox potentials and partition coefficients of
these compounds as important determinants of their physico-
chemical properties and have assessed the ability of these deriva-
tives in protecting neuronal PC12 cells against oxidative stress.
chromatography using silica gel as stationary phase and dichloro-
methane or dichloromethane/methanol as eluent.
The structural data of methyl, ethyl and propyl caffeates and
methyl, ethyl and propyl ferulates were described elsewhere
[22e25].
2.3.1. Trans-butyl-3-(4-dihydroxy)propenoate
Recrystallized of dichloromethane/light petroleum ether; Yield
70%. 1H NMR
d
(DMSO): 0.90 (3H, t, J ¼ 7.5, CH3), 1.32e1.42 (2H, m,
CH2), 1.56e1.65 (2H, m, CH2), 4.11 (2H, t, J ¼ 6.6, CH2), 6.27 (1H, d,
J ¼ 15.9, H ( )), 6.76 (1H, d, J ¼ 8.1, H(5)), 7.00 (1H, dd, J ¼ 2.0,8.13,
H(6)), 7.05 (1H, d, J ¼ 2.0, H(2)), 7.46 (1H, d, J ¼ 15.9, H( )), 9.60 (2H,
s, 3, 4-OH); 13C NMR
(DMSO): 23.3 (CH3), 28.4 (CH2), 40.0 (CH2),
73.2 (CH2), 123.8 (C5), 124.3 (C2), 125.5 (C ), 131.2 (C6), 135.2 (C1),
154.7 (C ), 155.3 (CeOH), 158.1 (CeOH), 176.5 (COOH).
b
a
d
2. Materials and methods
a
b
2.1. General
2.3.2. Trans-butyl-3-(3,4-hydroxy-3-methoxyphenyl)propenoate
Quercetin and Trolox C were purchased from Acros Organics
(Geel, Belgium). Fetal bovine serum (FBS), penicillin/streptomycin,
RPMI 1640, sterile phosphate buffered saline (PBS) and trypsin EDTA
0.05% were from Biosera (Ringmer, UK) and horse serum was
acquired from Invitrogen (San Diego, CA, USA). Acetic acid glacial,
hydrochloric acid 32%, dimethyl sulfoxide, ferrous sulfate heptahy-
drate, ferric chloride, methanol, sodium acetate and 2,4,6-tripyridyl-
s-triazine (tptz) were obtained from Merck (Darmstadt, Germany).
Caffeic acid, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT), 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferulic acid,
Recrystallized of dichloromethane/light petroleum ether; Yield
75%. 1H NMR
d
(CDCl3): 0.97 (3H, t, J ¼ 7.4, CH3), 1.39e1.49 (2H, m,
CH2), 1.65e1.72 (2H, m, CH2), 3.92 (3H, s, OCH3), 4.20 (2H, t, J ¼ 6.7,
CH2), 5.97 (1H, s, OH), 6.29 (1H, d, J ¼ 15.9, H ( )), 6.92 (1H, d, J ¼ 8.1,
H(5)), 7.03 (1H, d, J ¼ 1.8, H(2)), 7.07 (1H, dd, J ¼ 1.8,8.1, H(6)), 7.61
(1H, d, J ¼ 15.9, H( (DMSO): 14.7 (CH3), 20.1 (CH2),
)); 13C NMR
31.7 (CH2), 56.8 (CH3), 65.2 (CH2), 110.2 (C5), 115.6 (C2), 116.5 (C ),
), 147.7 (CeOH), 158.1 (CeOCH3),
b
a
d
a
123.9 (C6), 127.9 (C1), 145.6 (C
168.3 (COOH).
b
hydrogen peroxide and N-acetyl-
L-cysteine were purchased from
2.4. Measurement of antioxidant activity
SigmaeAldrich (San Louis, MO, USA). All reagents and solvents were
pro analysis grade and were used without further purification.
Deionised water (conductivity < 0.1 m
S cmꢀ1) was used in all the
2.4.1. DPPH radical scavenging activity
DPPH radical scavenging activity of caffeic acid and ferulic acid
derivatives (Scheme 1) was assessed as previously described with
some modifications [26,27]. Briefly, the test compound was dis-
solved in dimethyl sulfoxide and 4 different concentrations were
experiments.
Thin layer chromatography (TLC) analyses were performed on
aluminium silica gel sheets 60 F254 plates (Merck, Darmstadt,
Germany) and spots were detected using a UV lamp at 254 nm.
mixed with a methanolic solution of DPPH 100 mM in duplicate.
After 30 min of incubation at room temperature in the dark, the
absorbance at 517 nm was measured by a spectrophotometer (Bio-
2.2. Apparatus
Tek, Model Uvikon XL). The concentrations (in the range 1e100 mM)
1H and 13C NMR data were acquired, at room temperature, on
a Brüker BioSpin GmbH 400 spectrometer operating at 400.15 and
were carefully chosen for each compound in order to produce
a suitable doseeresponse curve. The percent inhibition of the
radical was calculated based on the absorbance of the mixture
compared to the absorbance of DPPH solution alone. IC50 values
were calculated by the software Curve-Expert (for Windows,
version 1.34).
100.62 MHz, respectively. Chemical shifts are expressed in
d (ppm)
values relative to tetramethylsilane (TMS) as internal reference;
coupling constants (J) are given in Hz. Assignments were also made
from DEPT (distortionless enhancement by polarization transfer)
(underlined values).
Microwave-assisted peptide synthesis was performed in BiotageÒ
Initiator Microwave Synthesizer.
2.4.2. Ferric reducing antioxidant power (FRAP) assay
FRAP assay was performed as described previously [28,29].
Briefly, the FRAP solution was freshly prepared by mixing 10 ml of
acetate buffer 300 mM at pH 3.6,1 ml of ferric chloride hexahydrate
20 mM dissolved in distilled water and 1 ml of 2,4,6-tris(2-pyridyl)-
Voltammetric studies were performed using an Autolab PGSTAT
12 potentiostat/galvanostat (Eco-Chemie, 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 ¼ 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).
s-triazine 10 mM dissolved in HCl 40 mM. Ten
ferulate derivatives and quercetin dissolved in dimethyl sulfoxide
were mixed with 190 l of the FRAP solution in 96-well microplates
in triplicate at the final concentrations of 10, 40 and 20 M,
ml of caffeate and
m
m
respectively. After 30 min of incubation at room temperature,
absorbance at 595 nm was measured by a microplate reader (Bio-
Rad, model 680). Light absorbance of the FRAP solution in the
presence of test compounds and quercetin were measured and
FRAP values for hydroxycinnamic acids (HCAs) were expressed as
mM equivalent of quercetin per mole of compound.
2.3. Synthesis
The cinnamic acid (1 g), the appropriate alcohol (30 mmol) and
concentrated sulfuric acid (2 drops) were put together in a glass vial
(2e5 ml) sealed with a cap and heated in the MW reactor cavity
under mechanical stirring for 5 min. The temperature was set at
20 ꢁC above the boiling point of the alcohol. After cooling to room
temperature, the crude material was purified by flash
2.5. Electrochemical measurements
Stock solutions of caffeic acid and ferulic acid and their ester
derivatives (10 mM) were prepared by dissolving an appropriate

J. Garrido et al. / Biochimie 94 (2012) 961e967
963
Scheme 1. Structures of caffeic acid and ferulic acid and their alkyl esters.
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 achieve a final
concentration of 0.1 mM.
The concentration of H2O2 in the well was 75
hour, the medium was replaced with fresh one and cells were
incubated overnight. In the end, the medium was replaced with
m
M. After another
30
ml of MTT 0.5 mg/ml dissolved in RPMI without phenol red.
The supporting electrolyte pH 7.3 used in the voltammetric
determinations was prepared by dilution of 6.2 ml of dipotassium
hydrogen phosphate 0.2 M and 43.8 ml of potassium dihydrogen
phosphate 0.2 M in 100 ml of water.
Formazan crystals were solubilised in 200
m
l DMSO after 1.5 h of
incubation at 37 ꢁC. The optical density was measured at 570 nm
with background correction at 655 nm using a Bio-Rad microplate
reader (Model 680). Each experiment was repeated 4e6 times. The
percent inhibition of cell damage was calculated with reference to
the absorbance of control wells not challenged with hydrogen
peroxide (assumed as 100% protection) and the absorbance of wells
treated with hydrogen peroxide in the absence of any antioxidants
(assumed as 0% protection). N-Acetylcysteine was also tested as
a reference antioxidant compound.
2.6. Calculation of partition coefficient (LogP)
Calculation of the LogP values, simulating partitioning of
phenols in an n-octanol/water (1:1, v/v) system, was accomplished
using the Molinspiration WebME Editor 1.16. [http://www.
molinspiration.com].
2.8. Statistical analysis
2.7. Protection of PC12 cells against damage induced by hydrogen
peroxide
Comparisons between several groups were performed by one-
way analysis of variance (ANOVA). Statistical analyses were
carried out with SPSS software for Windows version 11.5.0.
2.7.1. Cell culture
PC12 (rat pheochromocytoma) cells were a generous gift from
Professor Lloyd A. Greene (Department of Pathology and Cell
Biology, Columbia University, New York, USA). Cells were main-
tained in RPMI 1640 supplemented with 10% horse serum, 5% fetal
3. Results and discussion
3.1. Chemistry
bovine serum, 100 U/ml penicillin G and 100
mg/ml streptomycin.
Alkyl cinnamates (Scheme 1) have been previously synthesized
using one of most popular esterification techniques: the Fisher acid
catalysis esterification [22,23,25,31]. Although the process is widely
used, it still has several disadvantages including long reaction
times, tedious workup procedures, safety and waste disposal
problems and harsh reaction conditions.
They were cultured on plates coated with collagen obtained from
rat tail and were grown at 37 ꢁC in humidified air containing 5%
CO2. Two-thirds of the growing medium was changed every 2e3
days and the cells were subcultured roughly once a week.
2.7.2. Cell viability assay
Since microwave-assisted organic synthesis has several advan-
tages over conventional reactions, the alkyl cinnamic esters were
obtained by a Fisher esterification reaction using microwave irra-
diation [24,32]. The reaction was accelerated (5 min), the volume of
solvent/reagent was drastically reduced and the tedious extraction
step of the classic work-up was not needed. These reactions led to
moderate to high yields of the desired compounds, which were
identified by NMR spectroscopic techniques (1H NMR, 13C NMR).
Cell viability following exposure to hydrogen peroxide was
estimated by the MTT reduction assay [30]. MTT is absorbed by
viable cells and is converted to formazan by the enzyme succinate
dehydrogenase in the mitochondria. The amount of produced for-
mazan thus correlates with the number of living cells.
Hydrogen peroxide (8.8 M solution) stored at 4 ꢁC was first
diluted in PBS to prepare a 100 mM solution on the day of the
experiment. This was further diluted in growth medium to prepare
the final working solution. PC12 cells were plated in collagen-
coated 96-well microplates at a density of 5 ꢂ 105 cells/ml
3.2. Antioxidant activity
(100 ml per well). Blank wells contained only growth medium for
background correction. After 48 h of incubation to allow for cell
attachment, 20 l of growth medium supplemented with different
concentrations of HCAs were added in triplicate wells and pre-
incubated for 1 h. Maximum concentration of DMSO in the wells
Antioxidant activities of hydroxycinnamic acids (HCAs) and
their derivatives were measured by DPPH and FRAP assays
(Table 1). Caffeic acid and its esters appeared to be potent antiox-
idants since they had higher DPPH scavenging and FRAP activities
compared to Trolox C, a water soluble form of vitamin E, which is
m
was kept below 0.2%. Afterwards, 20 ml of H2O2 solution was added.

964
J. Garrido et al. / Biochimie 94 (2012) 961e967
Table 1
A
Antioxidant activity of hydroxycinnamic acids and their alkyl esters.
Compound
IC50 DPPH scavenging (
m
M)
FRAP value (mMQ/mole)a
500 nA
Caffeic acid
16.6 ꢄ 1.9
14.0 ꢄ 2.3
13.5 ꢄ 2.2
14.5 ꢄ 1.4
14.1 ꢄ 1.2
44.6 ꢄ 9.3
74.7 ꢄ 14.9
66.7 ꢄ 16.9
64.1 ꢄ 11.5
56.3 ꢄ 10.5
18.5 ꢄ 1.3
1397.7 ꢄ 274.0
1536.7 ꢄ 347.1
1588.0 ꢄ 324.9
1490.4 ꢄ 293.5
1553.5 ꢄ 339.0
324.0 ꢄ 46.9
193.3 ꢄ 33.6
250.3 ꢄ 10.2
218.3 ꢄ 32.4
261.7 ꢄ 36.6
427.4 ꢄ 86.2
Methyl caffeate
Ethyl caffeate
Propyl caffeate
Butyl caffeate
Ferulic acid
Methyl ferulate
Ethyl ferulate
Propyl ferulate
Butyl ferulate
Trolox C
a
mM equivalent of quercetin/mole. Values represent the mean of 3e5
experiments ꢄ S.D.
a known antioxidant. They also had FRAP values more than
1000 mMQ/mole, which indicates that they are more active than
quercetin, a strong flavonoid antioxidant. It was also observed that
caffeic acid and its derivatives were more active than ferulic acid
and its esters. The superior antioxidant activity of caffeic acid
compared to ferulic acid has been observed also in previous studies
[6,16,33] and is most probably due to the presence of the catechol
group that contributes to the stabilization of the phenoxyl radical
intermediate [34].
-0.2 0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
E/V vs Ag/AgCl
B
50 nA
Esters of caffeic acid had similar activities in both methods and
were all slightly more active than the parent compound. In
contrast, ferulate esters were all less active than their precursor.
Therefore, it appears that esterification has opposite effects on the
antioxidant activities of caffeic acid and ferulic acid. Effect of
esterification on the antioxidant activity has been addressed in
some studies; Previous work from our group has shown that caffeic
acid esters have higher DPPH and ABTS radical scavenging activities
compared to caffeic acid itself [25,31]. Other investigators have
shown that ethyl esters of caffeic acid and ferulic acid have
increased lipophilicity and a considerable antioxidant activity in
LDL peroxidation model system [35]. However, our recent study
on the alkyl esters of sinapic acid, another HCA, showed that
esterification reduced the antioxidant efficiency [21].
Overall, our present findings and previous reports seem to
suggest that the effect of alkyl esterification on the antioxidant
activity may differ from one HCA to another. This can be explained
by the fact that HCAs have different mechanisms of action, which
are mainly determined by their ring substitutions. In other words,
the impact of alkyl esterification on the antioxidant profile of HCAs
may be intrinsically related to their aromatic substitution pattern,
since they follow dissimilar oxidation reactions.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
E/V vs Ag/AgCl
Fig. 1. Differential pulse voltammograms for 0.1 mM solutions of (A) (d) caffeic acid,
(——) methyl caffeate, ( ) butyl caffeate and (B) (d) ferulic acid, (——) methyl ferulate
ꢃꢃꢃꢃ
and ( ) butyl ferulate, in a supporting electrolyte with the physiological pH of 7.3.
ꢃꢃꢃꢃ
Scan rate: 5 mV sꢀ1
.
related to the oxidation of catechol group present in the structure of
these molecules.
Cyclic voltammograms of caffeic acid and their derivatives were
also recorded at different sweep rates and were characteristic of an
electrochemical reversible reaction showing only one anodic peak
and one cathodic peak on the reverse scan (Fig. 2A). The ratio of the
3.3. Electrochemical measurements
Table 2
The use of voltammetry as an instrumental tool for the evalu-
ation of the total antioxidant capacity of low molecular weight
antioxidants (LMWA) and the existence of a correlation between
antioxidant profile and redox properties is well established
[14,33,36,37]. In fact, electrochemistry has been employed to
evaluate antioxidant activity since it can provide direct and detailed
information on the oxidation of LMWA compounds. Thus, the
oxidative behaviour of the HCAs was studied at the physiological
pH of 7.3 by differential pulse and cyclic voltammetry, using
a glassy carbon working electrode.
The differential pulse voltammograms of caffeic acid and its
alkyl ester derivatives, present only one well-defined anodic peak
at physiological pH (Fig. 1A). The oxidation peaks of these
compounds occurred at Ep ¼ þ217 mV for the acid and at ca.
Ep ¼ þ170 mV for the esters (Table 2). The observed waves are
Redox potentials (Ep) and partition coefficients (LogP) of hydroxycinnamic acids and
their alkyl esters.
Compound
Ep (mV)
217
LogD/LogPa
Caffeic acid
0.94
1.56
1.93
2.44
2.99
1.25
1.86
2.24
2.74
3.30
e
Methyl caffeate
Ethyl caffeate
Propyl caffeate
Butyl caffeate
Ferulic acid
Methyl ferulate
Ethyl ferulate
Propyl ferulate
Butyl ferulate
Trolox C
165
170
173
167
344; 437
375
370
364
343
110
a
Determined with Molinspiration calculation service (www.molinspiration.
com).

J. Garrido et al. / Biochimie 94 (2012) 961e967
965
For ferulic acid alkyl esters, only one anodic wave was observed
at physiological pH using differential pulse voltammetry (Fig. 1B,
Table 2). The observed oxidation peaks are also related to the
oxidation of the phenolic group present in their structure. The
appearance of a single wave for the ferulic alkyl esters could be
interpreted as a result of a less significant adsorption propensity of
these compounds when compared with ferulic acid. In fact, the
peak observed for alkyl esters occurs at a potential similar to that
obtained for the first wave attributed to the oxidation of the free
form of ferulic acid.
A
100 nA
The cyclic voltammogram obtained for ferulic acid also shows
two overlapped anodic peaks, while only one anodic wave is seen
for the alkyl ester derivatives (Fig. 2B). The anodic peaks appear to
correspond to an irreversible process. The gathered data corrobo-
rate the results found by other studies regarding ferulic acid
behaviour during oxidation. The proposed mechanism involves
a one-electron transfer from the phenolate ion followed by one
irreversible dimerization process due to a radicaleradical coupling
reaction between two phenoxyl radicals [40,41].
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
E/V vs Ag/AgCl
B
The differential pulse voltammetric study of the reference
compound Trolox C (6-hydroxy-2,5,7,8-tetramethylchroman- 2-
carboxylic acid) revealed the presence of a well-defined anodic
peak, Ep ¼ þ110 mV, at physiological pH [14].
250 nA
The voltammetric results reinforce the proposition previously
established by us in other studies [33,37] that the electrochemical
potential is closely correlated to the number of hydroxyl substit-
uents on the aromatic ring. Redox potentials of caffeic acid and its
esters are lower than those of ferulic acid series (Table 2), a fact
that could be ascribed to the presence of the catechol moiety in
caffeic acid. In other words, the methoxylation of the meta-
hydroxy group of the cinnamic derivatives in ferulic acid, shifts the
peak potential towards more positive values (Table 2). The lower
redox potential of caffeic acid has been observed also in other
studies [42].
-0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
E/V vs Ag/AgCl
From the voltammetric data it could be stated that esterification
of the carboxylic acid group in this type of cinnamic acids does not
dramatically change the oxidation potential. Nevertheless, the
slight changes observed could be sufficient to explain the antioxi-
dant activity differences found between HCAs and their esters.
Indeed, electrochemical results were in good agreement with
antioxidant assay data. Anodic potentials of caffeate esters were all
lower than caffeic acid itself (ca. 50 mV), which shows that these
compounds are more readily oxidized and therefore is in accor-
dance with their higher antioxidant capacity (Tables 1 and 2). On
the other hand, redox potentials of ferulate esters are all higher
than ferulic acid, a finding that confirms their lower antioxidant
capacity observed in FRAP and DPPH assays compared to the parent
compound (Tables 1 and 2).
Fig. 2. Cyclic voltammograms for 0.1 mM solutions of (A) (d) caffeic acid, (——) methyl
caffeate, (
) propyl caffeate and (B) (d) ferulic acid, (——) methyl ferulate and (
)
ꢃꢃꢃꢃ
ꢃꢃꢃꢃ
propyl ferulate, in a supporting electrolyte with the physiological pH of 7.3. Scan rate:
50 mV sꢀ1
.
anodic to cathodic peak heights gradually rises with increasing
potential scan rate from 10 to 100 mV sꢀ1 and the calculated
current functions (Ip n
ꢀ1/2) gradually diminish with the potential
scan rate, showing the classical behaviour of an oxidation process
coupled with a slow subsequent chemical reaction. These results
are in agreement with previous reports about the oxidative
behaviour of caffeic acid [33,37e39]. Electrochemical studies on the
caffeic acid oxidation mechanisms have shown that the first
oxidation step involves two electrons per molecule, which probably
correspond to the formation of caffeic acid ortho-quinone, an
intermediate that is quickly decomposed at pH higher than 7.4
[38,39].
The differential pulse voltammetric study of ferulic acid,
revealed the presence of two overlapped anodic peaks at physio-
logical pH (Fig. 1B). The oxidation peaks at Ep ¼ þ344 mV and
E’p ¼ þ437 mV (Table 2) are related to the oxidation of the phenolic
group present in the structure. The appearance of the two waves
may be interpreted by assuming that the electrochemical oxidation
of ferulic acid takes place on the glassy carbon electrode by electron
transfer from both free and adsorbed forms. The free form corre-
sponds to the first peak whereas the strongly adsorbed forms get
oxidized at a more anodic potential. The occurrence of an adsorp-
tion peak has also been described in previous voltammetric studies
involving ferulic acid and related compounds [21,33,37,40].
3.4. Estimation of partition coefficients (LogP)
For a better understanding of the overall physicochemical
properties of the test compounds, the lipophilicity, expressed as the
octanolewater partition coefficient and herein called LogP, was
calculated (Table 2).
The examination of the data allows concluding that the intro-
duction of an alkyl group to the structure leads to a significant
increase in the lipophilicity of the parent HCA, in both series.
Partition coefficients calculated for the HCA derivatives are well
correlated with their structural features: the longer is the alkyl
ester side-chain, the higher is the partition coefficient. Considering
that compounds, which have a LogP ꢅ1, are not able to effectively
cross the membranes, it appears that esterification of these
phenolic acids improves their membrane crossing ability. Further-
more, higher lipophilicity can also influence the ADME (absorption,

966
J. Garrido et al. / Biochimie 94 (2012) 961e967
distribution, metabolism, and excretion) properties of hydrox-
ycinnamic antioxidants, and increase their concentration at the
waterelipid interface.
Nakajima and colleagues have done an extensive study on caf-
feic acid and one of its derivatives, 3,4-dihydroxybenzalacetone
(DBL) isolated from Chaga plant. They found that caffeic acid and
DBL could effectively lower intracellular ROS levels in H2O2-chal-
lenged PC12 cells, however, they observed that the efficiency of
caffeic acid in preventing cell death was much lower than DBL [8].
Other authors have shown that ethyl ferulate increases resis-
tance to oxidative damage in rat neurons [43] and protect hippo-
3.5. Protection of PC12 cells against hydrogen peroxide-induced
damage
The capacity of HCAs in protecting neuronal PC12 cells against
oxidative stress-induced cell death caused by H2O2 was measured
by MTT reduction assay. Hydrogen peroxide in the absence of
antioxidant, typically reduced cell viability to 60e70% compared
to the control non-oxidized cells. The percent inhibition of cell
death calculated for different concentrations of antioxidants are
shown in Fig. 3. Caffeic acid was not active in this assay; however,
all caffeate esters were able to significantly inhibit cell damage at
campal neuronal cells against amyloid
b-protein toxicity [18].
However, our data showed that caffeic acid esters were even more
effective in the cell model and in the cell-free antioxidant assays,
compared to ferulic acid esters.
These findings can also be viewed in relation to the emerging
concept of adaptive cellular stress response and vitagene network
that have been suggested to be involved in oxidative stress-related
conditions such as neurodegenerative diseases [44,45]. Plant
derived antioxidants may therefore exert their protective effect by
activating the cellular stress response pathways, including tran-
scription factors like NF-E2-related factor 2 (Nrf2), that induce the
expression of genes encoding antioxidant enzymes and other
antioxidant defence systems [44,45]. In this regard, the inhibitory
effect of ethyl ferulate on neuronal oxidative damage has been
ascribed to the ability of this compound to induce the expression of
heme oxygenase-1 (HO-1), an important antioxidant enzyme [43].
It was interesting to note that caffeic acid esters, which were
able to protect PC12 cells had higher antioxidant activity as well as
improved lipophilicity as evidenced by their higher partition
coefficients compared to caffeic acid. Other investigators have also
shown that esterification of HCAs increases the lipophilicity and
promotes their incorporation into the membranes [13]. On the
other hand, ferulate esters that could not reduce oxidative stress-
induced cell death, had lower antioxidant capacity and the mere
fact of them being more lipophilic could not render them neuro-
protective. These data put together, seem to suggest that high
antioxidant activity and lipophilicity are both essential for neuro-
protective effect. These observations show that multiple factors
should be considered for development of biologically active anti-
oxidants and demonstrate the validity of our rational in designing
more lipophilic HCAs in order to generate more efficient
antioxidants.
the concentrations of 5 and 25 mM. On the other hand, ferulic acid
and its esters were not able to protect cells against oxidative
stress. N-Acetylcysteine, used as a reference compound, signifi-
cantly protected cells against damage at concentrations higher
than 250 mM.
100
80
60
40
20
0
**
**
**
**
**
**
**
*
C
MC
EC
PC
BC
4. Conclusions
100
80
60
40
20
0
In this study, methyl, ethyl, propyl and butyl esters of caffeic acid
and ferulic acid were synthesized and their antioxidant activity
(DPPH and FRAP assays), redox potentials and partition coefficients
were examined. The capacity of these compounds in protecting
neuronal PC12 cells against oxidative damage induced by hydrogen
peroxide was also studied. Caffeic acid and its esters were generally
more active than ferulic acid and its derivatives in all assays. Esters
of caffeic acid had higher antioxidant activities, lower redox
potential and higher lipophilicity compared to their parent
compound and had the ability of protecting PC12 cells against
oxidative damage in a dose-dependent manner. On the other hand,
ferulic acid esters were weaker antioxidants and although their
lipophilicity was higher than ferulic acid, they did not show neu-
roprotective effect. These findings showed that optimization of the
antioxidant activity and lipophilicity of HCAs can lead to the
production of more efficient antioxidants. The gathered data allow
concluding that caffeic acid alkyl esters represent good candidates
for further development of therapeutic agents against oxidative
stress-related diseases.
F
MF
EF
PF
BF
Fig. 3. Protective effect of hydroxycinnamic acids (HCAs) against cell death induced by
hydrogen peroxide in PC12 cells. PC12 cells were preincubated with caffeic acid (C) and
its methyl (MC), ethyl (EC), propyl (PC) and butyl esters (BC) as well as ferulic acid (F)
and its methyl (MF), ethyl (EF), propyl (PF) and butyl esters (BF) at 0.2, 1, 5 and 25
and then challenged with hydrogen peroxide 75 M. Cell viability was assessed by MTT
mM
m
assay and percent neuronal cell protection was calculated as described in the text. *, **
Significant versus oxidized control (in the absence of antioxidant), p < 0.05, p < 0.01,
respectively.
Conflict of interest
Authors declare that they have no conflict of interest.

J. Garrido et al. / Biochimie 94 (2012) 961e967
967
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Acknowledgements
The authors wish to thank Professor Lloyd A. Greene for
providing PC12 cells. This study was supported by Shiraz University
of Medical Sciences, Vice-chancellor for Research, Iran (Grant 88-
4925) and by the Foundation for Science and Technology (FCT),
Portugal (project PTDC/QUI-QUI/113687/2009). The financial
support from the Italian Ministry for Education, University and
Research, General Management for the internationalization of
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