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ORIGINAL ARTICLE
Gallic acid functions as a TRPA1 antagonist with relevant
antinociceptive and antiedematogenic effects in mice
Gabriela Trevisan &Mateus F. Rossato &Raquel Tonello &Carin Hoffmeister &
Jonatas Z. Klafke &Fernanda Rosa &Kelly V. Pinheiro &Francielle V. Pinheiro &
Aline A. Boligon &Margareth L. Athayde &Juliano Ferreira
Received: 25 August 2013 /Accepted: 30 March 2014 / Published online: 11 April 2014
#Springer-Verlag Berlin Heidelberg 2014
Abstract The transient receptor potential ankyrin 1 (TRPA1)
has been identified as a relevant target for the development of
novel analgesics. Gallic acid (GA) is a polyphenolic com-
pound commonly found in green tea and various berries and
possesses a wide range of biological activities. The goal of this
study was to identify GA as a TRPA1 antagonist and observe
its antinociceptive effects in different pain models. First, we
evaluated the ability of GA to affect cinnamaldehyde-induced
calcium influx. Then, we observed the antinociceptive and
antiedematogenic effects of GA (3–100 mg/kg) oral adminis-
tration after the intraplantar (i.pl.) injection of TRPA1 agonists
(allyl isothiocyanate, cinnamaldehyde, or hydrogen perox-
ide—H
2
O
2
) in either an inflammatory pain model (carrageen-
an i.pl. injection) or a neuropathic pain model (chronic con-
striction injury) in male Swiss mice (25–35 g). GA reduced
the calcium influx mediated by TRPA1 activation. Moreover,
the oral administration of GA decreased the spontaneous
nociception triggered by allyl isothiocyanate,
cinnamaldehyde, and H
2
O
2
. Carrageenan-induced allodynia
and edema were largely reduced by the pretreatment with GA.
Moreover, the administration of GA was also capable of
decreasing cold and mechanical allodynia in a neuropathic
pain model. Finally, GA was absorbed after oral administra-
tion and did not produce any detectable side effects. In con-
clusion, we found that GA is a TRPA1 antagonist with
antinociceptive properties in relevant models of clinical pain
without detectable side effects, which makes it a good candi-
date for the treatment of painful conditions.
Keywords Analgesic .Cold allodynia .Antioxidant .
Anti-inflammatory .Neuropathic pain
Abbreviations
AITC Allyl isothiocyanate
CCI Chronic constriction injury
DPPH 2,2-Diphenyl-1-picrylhydrazyl
GA Gallic acid
H
2
O
2
Hydrogen peroxide
4-HNE 4-Hydroxynonenal
i.p. Intraperitoneal
PWT Paw withdrawal threshold
PBS Phosphate-buffered saline
p.o. Oral route
s.c. Subcutaneously
TRPA1 Transient receptor potential ankyrin 1
G. Trevisan (*):M. F. Rossato :R. Tonello :F. Ro sa :J. Ferreira
Graduate Program in Biological Sciences: Toxicological
Biochemistry, Department of Chemistry, Center of Natural and Exact
Sciences, Federal University of Santa Maria (UFSM), Avenida
Roraima, 1000, Camobi, Santa Maria-RS 97105-900, Brazil
e-mail: gabitrev@hotmail.com
C. Hoffmeister :K. V. Pinheiro :F. V. Pinheiro:A. A. Boligon :
M. L. Athayde :J. Ferreira
Graduate Program in Pharmacology, Department of Physiology and
Pharmacology, Center of Health Sciences, Federal University of
Santa Maria (UFSM), Santa Maria, RS 97105-900, Brazil
J. Z. Klafke
Grupo Multidisciplinar de Saúde, Cruz Alta University (UNICRUZ),
Cruz Alta, RS 98020-290, Brazil
J. Ferreira
Department of Pharmacology, Center of Biological Sciences, Federal
University of Santa Catarina (UFSC), Florianópolis, SC 88049-900,
Brazil
G. Trevisan
Laboratory of Cellular and Molecular Biology, Graduate Program in
Health Sciences, University of the Extreme South of Santa Catarina
(UNESC), Criciúma, SC 88806-000, Brazil
C. Hoffmeister
Faculdade de Educação e Cultura de Vilhena, Vilhena,
RO 76980-000, Brazil
Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:679–689
DOI 10.1007/s00210-014-0978-0
Introduction
The transient receptor potential ankyrin 1 (TRPA1) is a non-
selective cation channel in the TRP superfamily (Moran et al.
2011). This channel is highly expressed in unmyelinated and
thinly myelinated sensory neurons in the dorsal root, trigem-
inal, and nodose ganglia, where it is involved in the transduc-
tion of nociceptive stimulus (Andrade et al. 2012). The
TRPA1 receptor’s unique structure is composed of a large
N-terminal cytoplasmic domain, which contains repetitive
ankyrin repeats and diverse cysteine residues and allows it to
be activated by reactive compounds that modify cysteine
residues (Cordero-Morales et al. 2011). Therefore, diverse,
exogenous, pungent, and oxidant substances are able to acti-
vate TRPA1 including allyl isothiocyanate (present in mus-
tard, wasabi, and horseradish), cinnamaldehyde (found in
cinnamon), and acrolein (present in tear gas and cigarette
smoke) (Andrade et al. 2012). Moreover, TRPA1 is gated by
endogenous reactive compounds produced after tissue dam-
age and associated painful conditions, namely, 4-
hydroxynonenal (4-HNE), prostaglandins, and reactive oxy-
genspecies,suchashydrogenperoxide(Andradeetal.2012).
TRPA1 agonists produce evoked pain and neurogenic in-
flammation in rodents and humans (Andrade et al. 2012;
Baraldi et al. 2010; Moran et al. 2011). Interestingly, TRPA1
is also involved in the development of mechanical and cold
allodynia in neuropathic and inflammatory models of pain
(Andrade et al. 2012; Moran et al. 2011). Collectively, these
features have highlighted the TRPA1 receptor as an attractive
target for novel analgesic molecules (Andrade et al. 2012;
Baraldi et al. 2010; Moran et al. 2011).
Gallic acid (GA; 3,4,5-trihydroxybenzoic acid) is a com-
mon plant polyphenolic compound. It is found in high levels
in green tea, walnut, grapes, and different berries. The diverse
pharmacological properties of GA including antiallergic, an-
tioxidant, anti-inflammatory, neuroprotective, and anticancer
have been previously reported (Inoue et al. 1995; Kim et al.
2011; Kroes et al. 1992;Lietal.2005;Luetal.2006;Nabavi
et al. 2011;PatelandGoyal2011). These properties have been
linked to several mechanisms, including the inhibition of his-
tone acetyltransferase, COX-2, the NF-κB signaling pathway,
and the transient receptor potential canonical 5 (TRPC5) chan-
nel(Kimetal.2011; Verma et al. 2013;Nayloretal.2011).
Furthermore, some studies have reported the
antinociceptive effects of GA in models of acute pain, such
as the acetic acid-induced abdominal writhing test and
cyclophosphamide-elicited cystitis (Boeira et al. 2011;
Krogh et al. 2000). Except for GA-reduced hyperglycemia
and lipoperoxidation in streptozotocin-induced diabetes in rats
(an effect related to its antioxidant activity), the effects of GA
in diabetes-related pain has not been investigated (Boeira et al.
2011; Punithavathi et al. 2011; Stanely Mainzen Prince et al.
2011). Of note, TRPA1 antagonists are able to reduce the
nociception caused by diabetes and cystitis in rodents (Wei
et al. 2009b;Wangetal.2013;Andreetal.2008; Geppetti
et al. 2008; Meotti et al. 2013). Thus, the mechanisms involved
in the antinociceptive activity of GA and the analgesic effects of
GA in chronic pain models have not been studied until now.
Collectively, these findings lead us to hypothesize that GA
could functions as a TRPA1 antagonist. Thus, the goal of this
study was to observe the antinociceptive effects of GA in
different nociceptive models that involve TRPA1 and to iden-
tify this molecule as a novel TRPA1 antagonist.
Materials and methods
Animals
The experiments were conducted using male Swiss mice (25–
35 g). Animals were maintained with free access to food and
water and were kept in a temperature-controlled room (22 ± 2
°C) under a 12-h light/dark cycle (lights on from 6:00 a.m. to
6:00 p.m.). Mice were acclimatized to the laboratory for at
least 2 h before experiments and were used only once. All
experiments were carried out between 08:00 a.m. and 5:00
p.m. The experiments were performed with the approval of the
Ethics Committee of the Federal University of Santa Maria
(CEUA, process number 124/2011) and are in accordance
with current ethical guidelines for the investigation of exper-
imental pain in conscious animals (Zimmermann 1983). In
addition, the number of animals and the intensity of the
noxious stimuli used were the minimum necessary to demon-
strate the consistent effects of the drug treatments.
Drugs
The allyl isothiocyanate (AITC), cinnamaldehyde, capsaicin,
carrageenan, gallic acid (GA), 2,2-diphenyl-1-picrylhydrazyl
(DPPH), SB-366791, HC-030031, myricitrin, fura 2-AM, and
hydrogen peroxide (H
2
O
2
) were purchased from Sigma
Chemical Co. (St. Louis, USA). The GA was diluted in
phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM
KCl, and 10 mM phosphate buffer) before injection. The
HC-030031 was diluted in 1 % dimethyl sulfoxide (DMSO)
in PBS and the AITC and cinnamaldehyde were diluted in 0.1
% DMSO in PBS. The capsaicin was dissolved in 90 %
ethanol and 10% Tween80 and then diluted to the appropriate
concentration in PBS.
Calcium (Ca
2+
)influxassay
To evaluate the ability of GA to act as TRPA1 antagonist, we
examined its capacity to affect cinnamaldehyde-induced cal-
cium influx in synaptosomes prepared from mouse spinal
cords (Trevisan et al. 2012). Briefly, mouse spinal cords were
680 Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:679–689
homogenized in assay buffer (50 mM phosphate buffer and
320 mM sucrose, pH 7.4) and centrifuged for 5 min at
1,000×gat 4 °C. Then, the supernatant was centrifuged for
20 min at 10,000×gat 4 °C. The final pellet was resuspended
in Krebs-Ringer buffer (Ca
2+
-free) at a final protein concen-
tration of 1 mg/mL and incubated with fura 2-AM (10 μM) for
30 min at 37 °C. The samples were centrifuged for 30 s at
12,000×g, and the final pellet was resuspended in 1.5 mL
Krebs-Ringer medium (Ca
2+
-free). To start the reaction, 15
μL of 0.1 M CaCl
2
was added to each sample, and after 10
min, different concentrations of GA (1, 10, 30, and 100 μM),
HC-030031 (10 μM, TRPA1 antagonist used as a positive
control), myricitrin (100 μM, antioxidant used as a positive
control), SB-366791 (1 μM, TRPV1 antagonist used as a
positive control), or vehicle (0.1 % DMSO) were added,
followed after 1 min by the addition of 15 μL cinnamaldehyde
(5 μM) or capsaicin (20 μM). We also analyzed the possible
Ca
2+
influx induced only by addition of GA (100 μM), HC-
030031 (10 μM), myricitrin (100 μM), or vehicle (0.1 %
DMSO). Ca
2+
influx was measured by using the fluorescence
at 382 nm (excitation) and 505 nm (emission) in a spectroflu-
orometer (RF-5301 PC, Shimadzu), and the time point of
1 min after challenge was chosen based in previous experi-
ments and represents the time with better detection of fura 2-
AM fluorescence. Background fluorescence was determined
using an equivalent sample of synaptosomes that were not
loaded with fura 2-AM. Calibration was performed by record-
ing the maximum and minimum fluorescence values after
adding 15 μLof10%(w/s) Triton X-100 at the end of each
experiment. The results were expressed as the percentage of
the maximum response obtained with Triton X-100 for each
synaptosomal preparation and then are compared with the
influx caused by cinnamaldehyde or capsaicin (Klafke et al.
2012; Trevisan et al. 2012). Moreover, we have presented the
calcium influx data as shown in Fig. 1a as the fluorescence
intensity of fura 2-AM in mouse spinal cord synaptosomes
with cinnamaldehyde (5 μM), vehicle, or gallic acid (10 μM)
plus cinnamaldehyde stimulation after different time points. In
addition, in Fig. 1a, we have indicated the baseline values
(indicated as B1) and the fluorescence values after 10 min of
0.1 M CaCl
2
addition on synaptosomes (indicated as B2).
Antioxidant activity
To demonstrate the antioxidant activity of GA, we performed a
DPPH (2,2-diphenyl-1-picryhydrazyl) assay. Briefly, we exam-
ined the ability of GA, myricitrin, and HC-030031 (1000–0.01
μM) to reduce the DPPH radical (0.15 mM) and decrease its
purple color, which was observed at 520 nm after 30 min of
incubation at room temperature in the dark. The assay was
performed in a microplate in a final volume of 200 μL. One well
was incubated only with ethanol (the vehicle of the solutions)
andconsideredtobe100%DPPHoxidation(Orhanetal.2011).
TRPA1 agonist-induced spontaneous nociceptive response
First, we evaluated the antinociceptive effect of GA in differ-
ent models of spontaneous nociception induced by pungent
substances. We used the term “nociception”in this study to
refer to the pain-like behaviors observed in experimental
models of pain. The procedures used were similar to those
previously described (Andrade et al. 2008). The doses of
gallic acid used in this study were calculated based on previ-
ous data in the literature (Boeira et al. 2011).Briefly,1hafter
GA treatment (1–100 mg/kg, oral route (p.o.)), HC-030031
(300 mg/kg, p.o.; used as positive control) or vehicle (PBS, 10
mL/kg, p.o.), 20 μL of AITC (10 nmol/paw prepared in 0.05
% DMSO in PBS), or cinnamaldehyde (100 nmol/paw pre-
pared in 0.05 % DMSO in PBS) were injected subcutaneously
(s.c.) under the surface of the right hind paw in different
groups of animals. Separate groups of animals received s.c.
injection of the appropriate vehicle solutions; however, vehi-
cle solutions did not evoke nociception behavior (data not
shown). Animals were placed individually in chambers (trans-
parent glass cylinders 20 cm in diameter) and were adapted for
20 min prior to the s.c. paw injection of algogenic substances.
After the challenge, mice were observed individually for 5
min, and the amount of time spent licking the injected paw
was timed with a chronometer and was considered to be
indicative of nociception. The dose and the time of adminis-
tration of drugs were based on pilot studies.
Spontaneous nociceptive response, mechanical allodynia,
and edema mediated by hydrogen peroxide s.c. injection
In addition, we have assessed the antinociceptive and
antiedematogenic effects of GA after the s.c. paw injection
of H
2
O
2
in mice. For this purpose, animals were pretreated
with GA (1–100 mg/kg, p.o.), HC-030031 (300 mg/kg, p.o.),
or vehicle (10 mL/kg, p.o.) 1 h before the s.c. injection of
H
2
O
2
(2 μmol/paw prepared in PBS, 20 μL) into the right
hind paw (Keeble et al. 2009). Then, the development of
spontaneous nociception was observed for 5 min after the
injection, and the amount of time spent licking the injected
paw, timed with a chronometer, was considered indicative of
spontaneous nociception.
Moreover, the mechanical allodynia mediated by H
2
O
2
was evaluated 20 min after the s.c. H
2
O
2
paw injection. For
the evaluation of mechanical allodynia, mice were placed
individually in clear Plexiglas boxes (7 × 9 × 11 cm) on
elevated wire mesh platforms to allow access to the plantar
surface of the hind paws. Prior to the test procedures, the
animals were kept in this apparatus for approximately 1 h
for adaptation. The mechanical threshold was determined
before and after s.c. paw injection of H
2
O
2
with flexible nylon
von Frey filaments using the “up-and-down”paradigm as
described previously (Trevisan et al. 2012). Briefly, the paw
Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:679–689 681
was touched with a series of seven von Frey hairs in logarith-
mic increments of force (0.02, 0.07, 0.16, 0.4, 1.4, 4.0, and
10.0 g). The von Frey hairs were applied perpendicular to the
plantar surface with sufficient force to cause a slight buckling
against the paw and held for approximately 2–4 s. The absence
of paw lifting after 5 s led to the use of the next filament with an
increased weight, whereas paw lifting indicated a positive
response and led to the use of the next weaker filament. This
continued until six measurements were collected or until four
consecutive positive or negative responses occurred. The 50 %
mechanical paw withdrawal threshold (PWT) response was
then calculated from these scores as described previously
(Dixon 1980;Trevisanetal.2012). Mechanical allodynia was
considered to be a decrease in the threshold when compared
with the same paw before the s.c. injection (basal values).
We have also observed that edema developed 20 min after
s.c. H
2
O
2
paw injection as previously described (Trevisan
et al. 2012). The H
2
O
2
-elicited spontaneous nociception,
edema, and mechanical allodynia were observed in the same
group of animals.
The inflammatory model of nociception induced by the s.c.
paw injection of carrageenan was used to observe the possible
antinociceptive and antiedematogenic properties of GA as
described previously (Oliveira et al. 2009). In this way, mice
were s.c. injected with 20 μL of carrageenan (300 μg/paw in
PBS) 0.5 h after the administration of GA (1–30 mg/kg, p.o.),
HC-030031 (300 mg/kg, p.o.), or vehicle (10 mL/kg, p.o.).
Then, the mechanical threshold and edema formation were
measure at 0.5, 1, 2, and 4 h after the carrageenan injection.
The baseline mechanical value was evaluated before the dif-
ferent treatments. Mechanical allodynia was described as a
reduction of the threshold value after the s.c. carrageenan
injection when compared with the basal values as described
above. To assess a possible effect of GA administration alone
on mechanical thresholds, a separate group of animals was
treated only with GA (1–100 mg/kg, p.o.), HC-030031 (300
Fig. 1 a Fluorescence intensity of fura 2-AM in mouse spinal cord
synaptosomes with cinnamaldehyde (5 μM), vehicle, or gallic acid (10
μM) plus cinnamaldehyde stimulation after different time points (number
of synaptosomal preparations = 4). Baseline values were indicated as B1,
and fluorescence values after 10 min of 0.1 M CaCl
2
addition on synap-
tosomes were indicated as B2. Calcium influx was observed as the
increase in fura 2-AM fluorescence. bThe evaluation of the ability of
GA to reduce Ca
2+
influx elicited by cinnamaldehyde (5 μM) in mouse
spinal cord synaptosomes. The TRPA1 antagonist HC-030031 (HC,10
μM) was used as a positive control. cCa
2+
influx observed only in the
presence of vehicle (Ve h ), GA (100 μM), myricitrin (100 μM), or HC-
030031 (10 μM) in mouse spinal cord synaptosomes without challenge
with cinnamaldehyde (n=3–4, number of synaptosomal preparations).
(b)and(c) calibration was performed by recording the maximum and
minimum fluorescence values after adding 15 μLof10%(w/s) Triton
X-100 at the end of each experiment. dAntioxidant activity of GA and
myricitrin. HC-030031 (1000–0.01 μM) did not diminish DPPH oxida-
tion (percentage of the control, vehicle). The data are expressed as the
mean ± S.E.M. The asterisks denote the significance levels, *P<0.05
when compared with vehicle or
#
P< 0.05 when compared with
cinnamaldehyde (2-way ANOVA followed by Bonferroni’s post hoc test)
682 Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:679–689
mg/kg, p.o.), or vehicle (10 mL/kg, p.o.) and the mechanical
threshold was evaluated after 0.5, 1, 2, and 4 h. Edema
formation was also assessed 0.5, 1, 2, and 4 h after s.c.
carrageenan injection, as described above.
Neuropathic mechanical and cold allodynia induced by nerve
injury
The chronic constriction injury (CCI) of the sciatic nerve was
used as a model of neuropathic pain in mice; the procedure
was performed as described elsewhere (Sommer et al. 1998).
For this purpose, a group of animals underwent an operation
for CCI on the right sciatic nerve, while a different group
served as unoperated controls (sham). In brief, the right sciatic
nerve was exposed under intraperitoneal (i.p.) anesthesia
using a mixture of ketamine (90 mg/kg) and xylazine (30
mg/kg), and then three loosely constrictive ligatures were
placed around the right sciatic nerve with a distance of
1 mm between the ligatures. In the sham surgery, the animals
were anesthetized, and the sciatic nerve was exposed without
placing the ligatures. The development of cold allodynia after
CCI was also observed in the mice as previously described
(Materazzi et al. 2012). For this, the acute nocifensive re-
sponse to acetone (50 μL)-provoked evaporative cooling
was assessed for 1 min, and the time spent elevating the paw
and licking the plantar region was measured. The mechanical
and cold thresholds were evaluated before (basal values) and 7
days after the neuropathic or sham procedure (time point 0), as
described above. Then, the GA (30 mg/kg, p.o.), HC-030031
(300 mg/kg, p.o.), or vehicle (10 mL/kg, p.o.) was adminis-
tered, and the mechanical and cold thresholds were assessed
after0.5,1,2,and4h.
Assessment of motor performance
We examined spontaneous motor coordination using the open
field test and forced motor activity using the rotarod test as
described previously (Trevisan et al. 2012). The open field test
was performed using a wooden box measuring 40 × 60 × 50
cm. The floor of the arena was divided into 12 equal squares,
and the number of squares crossed with all paws was counted
in a 5-min session. Mice were pretreated with GA (30 mg/kg,
p.o.) or vehicle (10 mL/kg, p.o.), and the test was carried out
1 h after the treatment. The rotarod test was performed 24 h
after the open field test. All mice were trained on the rotarod
(3.7 cm in diameter, 8 rpm) until they could remain on the
apparatus for 60 s without falling. On the day of the experi-
ment, the animals were preinjected with GA (30 mg/kg, p.o.)
or vehicle (10 mL/kg, p.o.) 1 h prior to the test. The number of
falls and the latency to the first fall from the apparatus was
recorded for 240 s.
Quantification of GA in different tissues of mice
by high-performance liquid chromatography with diode-array
detection
To quantify the presence of GA in different tissues, animals
were administered with gallic acid (30 mg/kg, p.o.) and
sacrificed 1 h later to assess its disponibility in the spinal cord
and plasma. Spinal cords were collected, homogenized in 300
μL of PBS, and centrifuged at 5,000×gfor 10 min at 4 °C.
Then, acetone (1:1) was added to the supernatant or plasma for
deproteinization, followed by centrifugation at 10,000×gfor
10 min at 4 °C. The supernatant was used to quantify the
presence of gallic acid by high-performance liquid chroma-
tography diode-array detection (HPLC-DAD) as described
previously (Watabiki et al. 2011;Zhaoetal.2010). We used
a Shimadzu Prominence Auto Sampler (SIL-20A) HPLC
system (Shimadzu, Kyoto, Japan) equipped with Shimadzu
LC-20AT reciprocating pumps connected to a DGU 20A5
degasser with a CBM-20A integrator, a SPD-M20A diode
array detector, and LC solution 1.22 SP1 software. Reverse-
phase chromatographic analyses were carried out under
isocratic conditions using a C18 column (4.6 × 150 mm)
packed with 5-μm diameter particles; the mobile phase was
methanol/acetonitrile/water (40:15:45, v/v/v) containing 1.0 %
acetic acid (Boligon et al. 2009). The chromatography peak
was confirmed by comparing its retention time and the spectra
at 271 nm with those of the reference standard. The flow rate
was 0.5 mL/min, and the volume injected was 40 μL. All
samples and mobile phases were filtered through a 0.45-μm
membrane filter (Millipore) and then degassed using an ultra-
sonic bath prior to use. Stock solutions of GA standard were
prepared in the HPLC mobile phase at a concentration range
of 0.025–0.250 mg/mL. The calibration curve for GA was: Y=
4,054.1x + 78,865 (r= 0.9997). All chromatography opera-
tions were carried out at ambient temperature and in triplicate.
Statistical analysis
The results are presented as the mean ± S.E.M. or S.D.
(Fig. 4), except for the ID
50
and IC
50
values, which are
reported as the geometric means accompanied by their respec-
tive 95 % confidence limits. The ID
50
and IC
50
values were
determined by nonlinear regression analyses with a sigmoid
dose-response equation using the GraphPad Software 5.0
(GraphPad, USA). The percentages of maximal inhibition
(I
max
) are reported as the mean ± S.E.M. of inhibition obtained
in each individual experiment in relation to the control values
(vehicle-treated mice for the in vivo nociception tests, 100 %
response obtained with Triton X-100 for the Ca
2+
influx assay,
or vehicle of the DPPH test). The significance level was set at
P< 0.05. The data were analyzed using Student’sttest and a
1-way or 2-way analysis of variance (ANOVA) followed by
Bonferroni’s post hoc test. Data in Fig. 4were analyzed using
Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:679–689 683
repeated ANOVA followed by multiple Bonferroni’sadjusted
group ttests post hoc for each time point, which is a test able
to perform the correction for alpha for multiple testing.
Results
GA functions as a TRPA1 antagonist
Figure 1a shows the baseline values and also the fluorescence
values after calcium addition, and we have indicated the
values for calcium influx as the fluorescence for fura 2-AM
(10 μM) detected at 382 nm (excitation) and 505 nm
(emission) in a spectrofluorometer. We have also showed the
fluorescence detection when we incubated the synaptosomal
suspension with gallic acid (10 μM) before cinnamaldehyde
stimulation. As indicated in the Fig. 1a,wehaveobservedthat
GA is largely able to reduce the calcium influx mediated by
cinnamaldehyde a synaptosomal suspension, with a maximal
inhibition of 51 ± 2 % and an IC
50
of 11 μM(witharangeof
6–21 μM) (Fig. 1b). The selective TRPA1 antagonist and HC-
030031 (10 μM) inhibited 43 ± 8 % of the cinnamaldehyde-
mediated calcium influx. The plant oxidant myricitrin (up to
100 μM) was not able to decrease the calcium influx induced
by cinnamaldehyde in a synaptosomal suspension (Fig. 1b).
Moreover, neither gallic acid nor HC-030031 was able to
reduce the calcium influx mediated by capsaicin, which is
largely reduced (60 ± 7 %) by the TRPV1 antagonist SB-
366791 (1 μM) (data not show). In addition, gallic acid,
myricitrin, and HC-030031 alone did not induce a calcium
influx in a synaptosomal suspension (Fig. 1c).
We also evaluated the antioxidant activity of GA by the
DPPH method. The GA produced antioxidant activity, with an
IC
50
value of 5.4 μM (with a range of 4.1–7.1 μM). Myricitrin
(used as positive control) also reduced DPPH oxidation, with
an IC
50
value of 14.1 μM(witharangeof11.7–17.0 μM). On
the other hand, HC-030031 did not elicit antioxidant activity
in this assay (Fig. 1d).
GA effects on endogenous and exogenous TRPA1
agonist-mediated spontaneous nociception and edema
formation
To demonstrate the antinociceptive effect of GA in different
nociceptive tests involving TRPA1, we initially used sponta-
neous nociception induced by the exogenous TRPA1 agonists
AITC and cinnamaldehyde. Orally administered GA (3, 10,
30, or 100 mg/kg) decreased the spontaneous nociceptive
response stimulated by the s.c. paw injection of either AITC
or cinnamaldehyde (30 mg/kg) with mean ID
50
values (and its
95 % confidence limits) of 2.6 mg/kg (1.5–4.4) and 2.9 mg/kg
(1.2–6.8), respectively, and maximal inhibition values of 69 ±
6 and 63 ± 3 %, respectively (Fig. 2a and b). In addition, the
selective TRPA1 antagonist HC-030031 (300 mg/kg, p.o.)
diminished the AITC-induced (73 ± 4 % decrease) and
cinnamaldehyde-induced (86 ± 2 % decrease) nociceptive
responses.
Pretreatment with GA (3, 10, 30, or 100 mg/kg, p.o.) or
HC-030031 (300 mg/kg, p.o.; 86 ± 2 % decrease) reduced the
spontaneous nociceptive response induced by the endogenous
TRPA1 agonist H
2
O
2
(2 μmol/paw); GA had a mean ID
50
value (and its 95 % confidence limits) of 5.2 mg/kg (3.8–7.0)
and an I
max
value of 91 ± 3 % at a dose of 100 mg/kg (Fig. 3a).
In addition, the s.c. injection of H
2
O
2
(2 μmol/paw) induced
mechanical allodynia (mechanical thresholds diminished from
2.06 ± 0.35 g at baseline to 0.11 ± 0.01 g 20 min after H
2
O
2
administration; P< 0.001, Student’sttest) and edema forma-
tion (paw thickness increased from 2.90 ± 0.04 mm at baseline
to 3.58 ± 0.07 mm 20 min after H
2
O
2
administration; P<
0.001, Student’sttest). Treatment with GA or HC-030031
was also able to prevent the edema and the mechanical
allodynia induced by H
2
O
2
(2 μmol/paw); GA (100 mg/kg)
684 Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:679–689
Fig. 2 The antinociceptive effects of orally administered gallic acid (GA)
against exogenous TRPA1 agonist-induced spontaneous nociceptive re-
sponse in mice. The GA (1–100 mg/kg, p.o.), HC-030031 (HC, 300 mg/
kg, p.o.), or vehicle (Ve h , 10 mL/kg, p.o.) was administered 1 h prior to the
test. Nociception time was measured for 5 min after s.c. hind paw
injection of aAITC (10 nmol/paw) or bcinnamaldehyde (100
nmol/paw). Each point represents the mean ± S.E.M. for six or seven
animals. The asterisks denote the significance levels, *P<0.05when
compared with vehicle-pretreated group (1-way ANOVA followed by
Bonferroni’s post hoc test)
had a mean ID
50
value (and its 95 % confidence limits) of 12.2
mg/kg (7.7–19.7) and 23.7 mg/kg (12.9–43.5) and an I
max
value of 99 ± 1 and 81 ± 26 %, for the antiedematogenic and
antiallodynic effects, respectively. The HC-030031 (300 mg/
kg, p.o.) administration produced an inhibition of 61 ± 6 and
86 ± 12 % for the antiedematogenic effect and the
antiallodynic effect, respectively (Fig. 3b and c).
Mechanical allodynia and edema in an inflammatory pain
model were attenuated by orally administered GA
To determine the capacity of GA to decrease the mechanical
hypersensitivity and edema induced by an inflammatory pain
model, we performed the carrageenan test. First, the s.c. paw
injection of carrageenan induced mechanical allodynia mea-
sured by a reduction in the mechanical threshold (the mechan-
ical threshold was reduced from 2.03 ± 0.32 g at baseline to
0.16±0.04g1hafters.c.carrageenaninjection;P<0.001,
Student’sttest). The s.c. carrageenan injection also produced
edema formation (paw thickness increased from 2.88 ±
0.05 mm at baseline to 4.03 ± 0.07 mm 1 h after s.c. carra-
geenan injection; P< 0.001, Student’sttest). The inflamma-
tory mechanical allodynia and edema formation that occurs
from 0.5 to 2 h after the s.c. injection of carrageenan was
largely reduced by pretreatment with GA (30 mg/kg, p.o.)
with an inhibition of 53 ± 7 and 30 ± 2 %, respectively,
observed at 1 h after the carrageenan injection (Fig. 4a and
b). In addition, orally administered HC-030031 (300 mg/kg,
p.o.) was also capable of preventing the mechanical allodynia
and edema formation induced from 0.5 to 2 h after carrageen-
an injection, with an inhibition of 81 ± 16 and 41 ± 5 %
observed at 1 h, respectively (Fig. 4a and b). Moreover, the
administration of GA (30 mg/kg, p.o.) or HC-030031 (300
mg/kg, p.o.) did not alter the mechanical threshold or paw
thickness of control mice not injected with carrageenan (data
not shown).
GA was able to decrease mechanical allodynia and cold
allodynia in a neuropathic pain model
Sciatic nerve injury in mice produced a pronounced decrease
in the mechanical thresholds (mechanical thresholds were
reduced from 1.87 ± 0.25 g at baseline to 0.097 ± 0.03 g 7
days after the CCI procedure; P< 0.001, Student’sttest) and
provoked cold allodynia (the nociceptive response to acetone
increased from 9 ± 1 s at baseline to 18 ± 1 s 7 days after the
CCI procedure; P< 0.001, Student’sttest). The administra-
tion of GA (30 mg/kg, p.o.) was capable of decreasing the
mechanical allodynia (75 ± 10 % inhibition at 2 h) and cold
allodynia (82 ± 5 % inhibition at 1 h) from 0.5 to 2 h (Fig. 4c
and d). Similarly, HC-030031 (300 mg/kg, p.o.) treatment was
able to decrease the mechanical and cold allodynia from 0.5 to
2 h after treatment, with inhibition values of 59 ± 7 and 91 ± 8
%, respectively, observed at 1 h (Fig. 4c and d). In addition,
treatment with GA (30 mg/kg, p.o.) or HC-030031 (300 mg/
kg, p.o.) was not able to modify the mechanical threshold or
the cold perception of the sham animals (data not shown).
Fig. 3 Gallic acid (GA) was able to decrease the aspontaneous
nociception, bmechanical allodynia, and cedema formation induced by
a s.c. hind paw injection of H
2
O
2
(an endogenous TRPA1 agonist, 2
μmol/paw). The gallic acid (GA,1–100 mg/kg, p.o.), HC-030031 (HC,
300 mg/kg, p.o.), or vehicle (Veh , 10 mL/kg, p.o.) was administered 1 h
prior to the nociceptive test. Nociception time was measured for 5 min
after the s.c. paw injection of H
2
O
2
(2 μmol/paw); the mechanical
allodynia and edema formation were assessed 20 min after the s.c.
injection of H
2
O
2
(2 μmol/paw). Each point represents the mean ±
S.E.M. for six animals. The asterisks denote the significance levels, *P
< 0.05 when compared with vehicle-pretreated group or
#
P<0.05when
compared with baseline values (1-way ANOVA followed by Bonferroni’s
post hoc test in (a) and (c) or 2-way ANOVA followed by Bonferroni’s
post hoc test in (b))
Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:679–689 685
GA oral administration did not induce any detectable motor
side effects and it was easily detected in plasma and the spinal
cord
GA (30 mg/kg, p.o.) administered to mice was not able to alter
their motor performance in the open field test or the rotarod
test when compared with vehicle-treated animals (Table 1).
Moreover, GA was readily detected in plasma (3.0 ± 0.23
μmol/mL, n= 5) and the spinal cord (3.4 ± 0.31 μmol/mg
of protein, n= 5) 1 h after oral administration (30 mg/kg).
Discussion
In this study, we have featured GA as a novel TRPA1 antag-
onist producing antinociceptive and antiedematogenic effects
in models of acute and chronic nociception that involve
TRPA1 activation. First, we have observed that GA functions
as a TRPA1 receptor antagonist capable of reducing the cal-
cium influx mediated by cinnamaldehyde in mouse
synaptosomes. Furthermore, we have observed that GA pos-
sesses a relevant antioxidant capacity as described previously
(Li et al. 2005). To show that the antioxidant properties and
the TRPA1 antagonism are unrelated, we tested another anti-
oxidant compound (myricitrin) with previously described
antinociceptive activity (Cordova et al. 2011;Meottietal.
2006) in the calcium influx experiment, and only the GA
was able to reduce the TRPA1 agonist-mediated calcium
Fig. 4 The administration of GA produced antiallodynic and
antiedematogenic effects in a carrageenan-induced inflammatory
nociception model. aand bThe gallic acid (GA, 30 mg/kg, p.o.), HC-
030031 (HC, 300 mg/kg, p.o.), or vehicle (Ve h , 10 mL/kg, p.o.) was
administered 0.5 h prior to the s.c. injection of carrageenan (Cg,300μg/
paw, 20 μL) for the time-response curve (measured at 0.5, 1, 2, and 4 h
after the s.c. carrageenan injection or not). Gallic acid (GA) was able to
decrease the mechanical hyperalgesia and cold allodynia induced by a
neuropathic pain model in mice. cand dThe GA (30 mg/kg, p.o.), HC
(300 mg/kg, p.o.), or vehicle (10 mL/kg, p.o.) was administered 7 days
after the neuropathic induction procedure. Basal values were measured
prior to the procedure. Data are expressed as the mean ± S.D. in mechan-
ical painful hypersensitivity, the 50 % mechanical paw withdrawal thresh-
old (g) in (a) and (c), the variation in paw thickness in (b), or the
nociceptive time to cold stimulus (acetone test) in (d). Each point repre-
sents the mean ± S.D. for seven–eight animals. The asterisks denote the
significance levels, *P< 0.05 when compared with the vehicle-pretreated
group (repeated ANOVA followed by multiple Bonferroni’sadjusted
group ttests post hoc for each time point)
Tabl e 1 Effects of gallic acid (GA, 30 mg/kg, p.o.) or vehicle (10 mL/kg)
on the spontaneous and forced locomotor activity in mice 1 h after
treatment
Treatment (p.o.) Open field Rotarod
Crossing Rearing First fall N° fall
Vehicle (10 mL/kg) 82 ± 5 57 ± 6 214 ± 20 0.5 ± 0.3
GA(30mg/kg) 107±7 69±11 200±26 0.6±0.5
No significant differences were observed between groups (Student’st
test). Results are expressed as the mean ± S.E.M (n=6)
686 Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:679–689
influx. Thus, the TRPA1 antagonist ability of GA seems not to
be related to its antioxidant action.
The TRPA1 channel is found in the soma and peripheral
nerve endings of a subpopulation of TRPV1-positive primary
peptidergic sensory fibers, but the expression of the TRPA1
receptor in the spinal cord is not well-documented
(Story et al. 2003; Bautista et al. 2005). Previously, it
has been shown that TRPA1 is expressed in the spinal cords
of rats by immunohistochemical analysis and that the presence
of the TRPA1 receptor is high in the terminals of peptidergic
primary fibers (Andersson et al. 2011). However, although
there are indications that TRPA1 is present in the spinal cord
(Kim et al. 2010), the spinal cord synaptosomes of mice are
not frequently used to detect TRPA1 activity. Our group
observed that spinal cord synaptosomes’treatment with
cinnamaldehyde, a TRPA1 agonist, leads to an increase in
[Ca(2+)](i) and a rapid release of glutamate (Klafke et al.
2012). We decided to use the spinal cord synaptosomes of
mice in our studies, because there are indications of the
participation of TRPA1 in the spinal cord (Andersson et al.
2011; Kim et al. 2010). The mechanical allodynia following
cinnamaldehyde treatment is consistent with recent studies
showing that a TRPA1 agonist induces mechanical allodynia
when injected intrathecally in mice and rats (Raisinghani et al.
2011; Wei et al. 2011;Gregusetal.2012; Sisignano et al.
2012).
The TRPA1 agonists are highly capable of causing spon-
taneous pain and neurogenic inflammation in both rodents and
humans (Andrade et al. 2008; Namer et al. 2005). It has been
reported that the overt nociception induced by AITC or
cinnamaldehyde in animals was reduced by TRPA1 antago-
nists and TRPA1 antisense oligodeoxynucleotide treatment
and is largely reduced in TRPA1 knockout mice (Andrade
et al. 2008;Bautistaetal.2006; Jordt et al. 2004;Petrusetal.
2007).Here,wehaveobservedthatGAprofoundlyreduced
AITC- and cinnamaldehyde-mediated nociception in a dose-
dependent manner, similar to the TRPA1-selective antagonist
HC-030031. Moreover, TRPA1 has been highlighted as an
important sensor of oxidative substances produced after tissue
injury, such as H
2
O
2
(Andersson et al. 2008;Keebleetal.
2009;Khattab2006; Sawada et al. 2008;Wangetal.2004).
The injection of H
2
O
2
into a rodent’s paw produced
nociception, edema formation, and mechanical allodynia
(Keeble et al. 2009). We have shown that GA is able to
efficaciously reduce the H
2
O
2
-mediated nociceptive and
edematogenic responses in mice. These findings confirm the
effects of GA in TRPA1-mediated responses.
Moreover, we have shown that GA reduced mechanical
allodynia and edema formation in a model of inflammatory
pain; the TRPA1 antagonist HC-030031 had similar effects.
This is in accordance with a previous study that showed the
antinociceptive and anti-inflammatory effect of GA in a model
of hemorrhagic cystitis induced by cyclophosphamide, a
model where the principally oxidative metabolite produced
is acrolein, a TRPA1 agonist (Boeira et al. 2011). The partic-
ipation of TRPA1 channels in the development and mainte-
nance of mechanical and cold allodynia in inflammatory pain
models was observed (Andrade et al. 2012; Baraldi et al.
2010; Moran et al. 2011), and these responses were reduced
by TRPA1 antagonists (da Costa et al. 2009; Petrus et al.
2007). In addition, carrageenan-induced edema was largely
reduced in TRPA1 knockout mice (Moilanen et al. 2012).
In addition, the role of the TRPA1 channel in neuropathic
pain model-associated mechanical and cold allodynia has
been shown in diverse studies (Chen et al. 2011; Eid et al.
2008; Katsura et al. 2006; Obata et al. 2005; Petrus et al.
2007). Here, we observed that GA is largely able to decrease
the nociception induced by a mechanical or cold stimulus in
the CCI model of neuropathic pain, similar to the effects of
HC-030031. This is an important finding because neuropathic
pain is still difficult to treat; thus, there is a high need for novel
drugs able to reduce this type of pain (von Hehn et al. 2012).
In this study, we have shown that GA did not alter the
motor performance of mice after a single dose, which pro-
duced antinociceptive effect; this is a relevant finding
because some drugs that induce motor impairment can
cause false positive results in nociceptive tests in animals
(Negus et al. 2006). Moreover, previously, it has been
showed that the acute administration of GA at a high
dose (5 g/kg) did not show any toxicity (evaluated as a
change in hematological parameters) or induce mortality;
also, the subacute administration of GA (1 g/kg for 28
days) was not associated with hematological alteration or
cumulative toxicity (Rajalakshmi et al. 2001).
Initially, we administered the GA orally because it is a low-
cost, safe, and easy route of administration (Buxton 2006).
Here, we found that GA was detectable in the plasma and in
thespinalcord1hafterasingleadministration,atimepoint
where we have also observed its antinociceptive effect in our
pain models. The capacity of GA to pass through the blood-
brain barrier is important because it could block the actions of
some recently identified endogenous TRPA1 ligands in spinal
cord that are involved in the maintenance of mechanical
allodynia observed in animal models and reduce the allodynia
(Buxton 2006; Sisignano et al. 2012;Gregusetal.2012).
Therefore, TRPA1 antagonists that are able to reach the spinal
cord seem to have efficacy in reducing the mechanical
allodynia associated with neuropathic and inflammatory pain
(Chen et al. 2011; da Costa et al. 2009; Wei et al. 2009a,
2012).
In conclusion, we have identified GA as a TRPA1 antago-
nist with antinociceptive and antiedematogenic effects in rel-
evant models of clinical pain. Furthermore, this molecule did
not induce any detectable side effects and is found in large
quantities in diverse natural products, a characteristic that
encourages its use for the treatment of painful conditions.
Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:679–689 687
Acknowledgments Fellowships from Conselho Nacional de
Desenvolvimento Científico (CNPq), Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior (CAPES), and Fundação de Amparo à
Pesquisa do Estado do Rio Grande do Sul (FAPERGS) (Brazil) supported
this work.
Conflict of interest The authors declare no competing financial
interests.
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