Journal of Ethnopharmacology 157 (2014) 257–267

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Research Paper

Antinociceptive, anti-inflammatory and gastroprotective effects

of a hydroalcoholic extract from the leaves of Eugenia punicifolia
(Kunth) DC. in rodents
Rosanna T. Basting a, Catarine M. Nishijima a, Juliana A. Lopes a, Raquel C. Santos a,
Larissa Lucena Périco a, Stefan Laufer b, Silke Bauer b, Miriam F. Costa c, Lourdes C. Santos c,
Lúcia R.M. Rocha a, Wagner Vilegas d, Adair R.S. Santos e, Catarina dos Santos f,n,1,
Clélia A. Hiruma-Lima a,n,1
a
Univ. Estadual Paulista—UNESP, Departamento de Fisiologia, Instituto de Biociências, CEP 18618-970, Botucatu, SP, Brazil
b
Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Eberhard Karls University of Tübingen, Auf der Morgenstelle 8, 72076
Tübingen, Germany
c
Univ. Estadual Paulista—UNESP, Departamento de Química Orgânica, Instituto de Química, CEP 14800-900, Araraquara, SP, Brazil
d
Univ. Estadual Paulista-UNESP, Campus Experimental do Litoral Paulista, CEP 11330-900, São Vicente, SP, Brazil
e
Universidade Federal de Santa Catarina, Departamento de Ciências Fisiológicas, CEP 88040-900, Florianópolis, SC, Brazil
f
Univ. Estadual Paulista—UNESP, Departamento de Ciências Biológicas, Faculdade de Ciências e Letras, CEP 19806-900, Assis, SP, Brazil

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: An ethnopharmacological survey indicated that leaves from Eugenia punici-
Received 28 July 2014 folia (Kunth) DC. (Myrtaceae) are popularly used as a natural therapeutic agent to treat pain and inflammation.
Received in revised form Aim of the study: The overall objective of the present study was to evaluate the antinociceptive, anti-
24 September 2014
inflammatory and gastroprotective activities of a hydroalcoholic extract of leaves from Eugenia punicifolia
Accepted 28 September 2014
(HEEP) in rodents.
Available online 13 October 2014
Material and methods: The antinociceptive effects of HEEP were evaluated in mice after oral administration in
Keywords: chemical (formalin and glutamate) and thermal (hot-plate) tests. We evaluated the involvement of the
Eugenia punicifolia glutamatergic, opioidergic and nitrergic pathways in the antinociception of HEEP and the effect of HEEP on the
Myrtaceae inhibition of p38α MAPK. The anti-inflammatory effect of HEEP was evaluated in mice and rats using xylene-
Antinociceptive
induced ear edema and carrageenan-induced paw edema, respectively. Furthermore, the gastroprotective effect
Anti-inflammatory
of HEEP was evaluated in rats with acute gastric lesions induced by ethanol or indomethacin. Finally, we
Gastroprotective
p38α MAPK
performed a phytochemical analysis of HEEP.
Results: The oral administration of HEEP (125, 250 and 500 mg/kg, p.o.) significantly inhibited the neurogenic
and inflammatory phases of formalin-induced licking, and HEEP (250 mg/kg, p.o.) also significantly inhibited
the nociception caused by glutamate. The antinociceptive effects of HEEP were significantly reversed by L-
arginine (500 mg/kg, i.p.) but not by naloxone (1 mg/kg, i.p.) in the formalin test. HEEP did not affect animal
motor performance in the rotarod model. In addition, HEEP also increased the paw withdraw latency in the
hot-plate test. HEEP significantly inhibited ear edema induced by xylene (64%) and paw edema induced by
carrageenan (50%) compared to the control group. Furthermore, HEEP (3–30 mg/mL) also inhibited the
phosphorylation of p38α MAPK by approximately 90%. In addition, HEEP (125, 250 and 500 mg/kg, p.o.)
protected the rats against ethanol (88.4–99.8%) and indomethacin (53–72.3%) and increased the mucus levels
of the gastric mucosa without producing an antisecretory effect. The phytochemical profile of HEEP obtained
using HPLC-PDA showed secondary metabolites already reported for the genus, mostly flavonoids, gallotannins
and proanthocyanidins.
Conclusions: These data show for the first time that HEEP has significant antinociceptive and anti-
inflammatory effects, which appear to be related to the inhibition of the glutamatergic system, the synthesis

Abbreviations: TNF, tumor necrosis factor; HEEP, hydroalcoholic extract of leaves from Eugenia punicifolia; PGE2, prostaglandin E2; NO, nitric oxide; MAPK, mitogen-
activated protein kinase; ATF-2, activating transcription factor 2; TLC, thin layer chromatography; ELISA, enzyme linked immunosorbent assay; TBS, tris-buffered saline; BB,
blocking buffer; SPE, solid phase extraction, substrate A (solution containing hydrogen peroxide) and substrate B (solution containing 3,30 ,5,50 tetramethylbenzidine); CGRP,
calcitonin gene-related peptide; NSAIDs, non-steroidal anti-inflammatory drugs
n
Corresponding authors. Tel.: þ 55 14 38800312; fax: þ551438153744.
E-mail addresses: csantos@assis.unesp.br (C. dos Santos), hiruma@ibb.unesp.br (C.A. Hiruma-Lima).
1
C.A. Hiruma-Lima and C. Santos contributed equally to the supervision of this study.

http://dx.doi.org/10.1016/j.jep.2014.09.041
0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
258 R.T. Basting et al. / Journal of Ethnopharmacology 157 (2014) 257–267
of nitric oxide and the inhibition of the phosphorylation of p38α MAPK. HEEP also has interesting
gastroprotective effects related to the maintenance of protective factors, such as mucus production. These
results support the use of Eugenia punicifolia in popular medicine and demonstrate that this plant has
therapeutic potential for the development of phytomedicines with antinociceptive, anti-inflammatory and
gastroprotective properties.
1. Introduction
Eugenia punicifolia (Kunth) DC., popularly known as “pedra-
ume-caá, pedra-ume, murta or muta”, is a shrub largely distrib-
uted in the Amazon region and Savanna biome. This medicinal
species is one of the unexplored traditional medicinal plants of
Brazil; however, this species was recorded by the French naturalist
August de Saint-Hilaire in his travels to the Brazilian territories
from 1816 to 1822 (Brandão et al., 2012). The leaves of this
medicinal plant are popularly used in decoctions or aqueous
infusions as a natural therapeutic agent to treat inflammation
(Maia et al., 2001), fever and the flu (Chaves and Barros, 2012;
Silva, 1998), diabetes (Maia et al., 2001; Leite et al., 2010) and in
alcoholic infusions for the treatment of wounds and infectious
diseases (Oliveira et al., 2005).
Phytochemical studies carried out with the genus Eugenia have
demonstrated the occurrence of many classes of constituents
including flavonoids, tannins, terpenoids and essentials oils
(Oliveira et al., 2006; Galeno et al., 2014). From a pharmacological
point of view, studies using crude extracts of this genus showed its
anti-inflammatory, analgesic, antifungal, antipyretic, hypotensive,
antidiabetic and antioxidant activities (Slowing et al., 1994;
Donepudi et al., 2012; Oliveira et al., 2006). Pharmacological
studies of Eugenia punicifolia have shown antioxidant activity
(Gonzaga et al., 2007; Galeno et al., 2014), beneficial effects in
diabetic rats induced by streptozotocin (Brunetti et al., 2006),
increased neurotransmission mediated by nicotinic acetylcholine
receptors (Granjeiro et al., 2006) and the release of excitatory
catecholamines that could be used to treat patients with Alzhei-
mer’s disease (Pascual et al., 2012). Recently, Leite et al. (2014)
investigated the effects of pentacyclic triterpenes obtained from
Eugenia punicifolia on the activation of mechanisms influencing
skeletal muscle repair and in vivo muscular tissue remodeling.
In this context, because of the therapeutic properties attributed
to this medicinal plant by traditional medicine, we investigated
the antinociceptive and anti-inflammatory effects of the hydro-
alcoholic extract obtained from the leaves of Eugenia punicifolia
(HEEP) on in vivo and in vitro experimental models. As several
nonsteroidal anti-inflammatory drugs (NSAIDs) often cause muco-
sal lesions in the stomachs of humans, we also verified the effect of
HEEP on gastric lesions induced by ethanol and an NSAID.
2. Materials and methods
2.1. Preparation of the extract and phytochemical analyses
2.1.1. Extract preparation
Leaves of Eugenia punicifolia were collected in December (2009) by
Dr. Catarina dos Santos in the Instituto Florestal e Estações Experi-
mentais – Floresta Estadual de Assis at the point 221330 to 221370 Lat.
S–501210 to 501240 Long. W, Assis, State of São Paulo, Brazil. The
specimen was identified by Dr. Antônio C.G. Melo, and the voucher
specimen (no. 43.522) was deposited in the Herbarium D. Bento
Pickel, Assis, SP, Brazil, for future reference. The extract was prepared
with 10 g vegetal material (dried and triturate leaves) and 100 mL
solvent (ethanol:water 70:30 v/v). The solution was extracted using
& 2014 Elsevier Ireland Ltd. All rights reserved.
dynamic maceration for 2 h at room temperature (2572 1C). After-
wards, the solution was filtered, and the residue was re-extracted
two more times following the same procedure. The solution was
dried in a rotary evaporator at 40 1C yielding 4.49 g (45%) of extract
from the original plant material.
2.1.2. Phytochemical screening
A phytochemical screening to detect tannins, flavonoids and
saponins was performed as described in literature (Harborne,
1998). Crude plant extracts were screened for alkaloids, flavonoids,
saponins and sesquiterpene lactones on thin layer chromatogra-
phy plates (TLC aluminum sheets, 20  20 cm, Silica gel 60, Fluka)
using appropriate spray reagents and UV absorbances according to
procedures described by Wagner and Bladt (1995).
2.1.3. HPLC-PAD analyses
HPLC-grade methanol was purchased from J.T. Baker (Phillipsburg,
NJ, USA). HPLC-grade water was prepared using a Milli-Q purification
system (Millipore, Bedford, MA, USA). The Sep-Pak RP18 cartridges
(500 mg/6 mL) for the solid phase extraction (SPE) were obtained
from Waters Co. (Waters, Milford, USA). In the SPE step, HEEP (10 mg)
was dissolved in 1 mL methanol and applied to a Sep-Pak RP18
cartridge that had been preconditioned with methanol (2  6 mL). The
cartridge was eluted with methanol (6 mL) to remove chlorophyll, and
the effluent was collected and evaporated under a nitrogen stream.
The solid obtained was re-dissolved in methanol/water (1:1, v/v) and
filtered through a 0.45 μm nylon filter membrane (Sigma–Aldrich, St.
Louis, USA), then aliquots (20 μL) were submitted to HPLC analysis. All
samples and reagents were prepared immediately before use. The
chromatographic profile of the sample obtained from SPE was
established using a Jasco (Tokyo, Japan) liquid chromatography system
equipped with a PU-2089 quaternary solvent pump, an MD-2010 PAD
and a Rheodyne 7725 sample injector with a 20 μL sample loop.
The analytical column was a Phenomenex Synergi Hydro RP18
(250  4.6 mm i.d.; 4 μm) equipped with a Phenomenex security
guard column (4.0  2.0 mm i.d.). The mobile phase was composed
of water (eluent A) and acetonitrile (eluent B) both containing 0.1%
TFA. The following elution gradient was applied: 5% eluent B increas-
ing for 35 min to 100% B and then held at 100% B for 10 min. The flow
rate was 1.0 mL/min and the total run time was 55 min. The software
ChromNav (Worstation Jasco, ChromNav1.18.03) was used to control
the analytical system and to collect and process the data.
2.2. Animals
The experiments were conducted using adult male and female
Swiss mice (25–35 g; approximately 6 weeks of age) and male Wistar
rats (160–200 g; approximately 8 weeks of age) obtained from Biotério
Anilab Ltda. (Paulínia, São Paulo, Brazil). All animals were housed in
collective cages and kept in a controlled environment (2371 1C under
a 12 h light/dark cycle (lights on at 06:00 h) with free access to water
and food (Presences, Brazil). The animals were fasted prior to all
assays because the standard drugs and HEEP were always adminis-
tered orally (by gavage) using a saline solution (10 mL/kg) as the
vehicle. The animals were housed in cages with raised floors of wide
mesh to prevent coprophagy and were habituated to the laboratory
 R.T. Basting et al. / Journal of Ethnopharmacology 157 (2014) 257–267
conditions for at least 1 h before testing. All experiments were
performed during the light phase of the light/dark cycle. All animal
care and experimental procedures were performed in accordance with
the ethical standards established by the National Guidelines for the
Use of Experimental Animals of Brazil, and the protocols were
approved by the Committee for the Ethical Use of Animals (no. 369/
CEUA and 497/CEUA). All efforts were made to demonstrate the
consistent effects of the drug treatments and to minimize both the
number of animals used and their suffering (Zimmerman, 1983).
2.3. Acute toxicity and Hippocratic screening
Adult male and female Swiss mice (25–35 g) were divided into
groups (n¼10) that received a saline solution (10 mL/kg) or HEEP
(5000 mg/kg) orally. After oral administration, the acute toxicity and
behavioral parameters were described according to the methods of
Souza Brito (1995). The observations were performed 30, 60, 120, 240
and 360 min after the treatments, and the observed symptoms were
recorded and graded according to the method of Malone and
Robichaud (1962). For 14 days, the animals were weighed, and the
number of deaths was noted. On the 14th day, the mice were killed,
and the hearts, livers, kidneys, lungs and spleens were collected. We
compared all parameters from the HEEP-treated animals with those
obtained from their respective control groups that received the
vehicle (saline).
2.4. Formalin-induced nociception
The procedure used was essentially the same as that described
previously (Hunskaar et al., 1985). The animals received an intra-
plantarly (i.pl.) injection of 20 mL 2.7% formalin (1% formaldehyde) in
saline in the ventral surface of the right hind paw and were observed
during the first 5 min (neurogenic phase) and between the 15th and
30th min (inflammatory phase). The mice were treated with HEEP
(125, 250 and 500 mg/kg) or saline solution (10 mL/kg) via the oral
route (by gavage) 1 h before the formalin injection. After the formalin
injection, the animals were immediately placed into glass cylinders
with 20-cm diameters, and the time spent licking the injected paw
was recorded with a chronometer as an indicator of nociception. The
lower effective dose (that induced an antinociceptive effect in both
phases) was used to characterize the mechanism of action.
2.5. Hot plate test
Thermal hypersensitivity was evaluated as previously described by
Eddy and Leimbach (1953) with modifications. The temperature of the
metal surface (Insight Ltda., Ribeirão Preto, Brazil) was set at 5672 1C,
the animals were placed on the hot plate and the nociception was
recorded as the latency time (in seconds). The latency response to the
thermal stimuli consists of licking the hind paw or jumping. A cut-off
time of 20 s was chosen to avoid tissue injury. The animals were pre-
selected (24 h before starting the experiment), excluding those with a
response time of less than 4 s or greater than 11 s. The mice (n¼ 7–10)
were pretreated with saline (vehicle), morphine (2.5 mg/kg) or HEEP
(125 or 250 mg/kg) 30 min before beginning the ratings. The latency
was recorded 60, 90 and 210 min following oral administration of the
treatments. The prolongation of the latency times (s) compared with
the values of the controls was used for statistical comparison.
2.6. Locomotor performance
The effect of HEEP (250 mg/kg, p.o.) on locomotor performance
was also tested on the rotarod apparatus (Insight Ltda., Ribeirão
Preto, Brazil) as described previously (Dunham and Myia, 1957).
Twenty-four hours before the experiments, male Swiss mice were
trained on the rotarod (4 cm in diameter, 6 rpm) until they could
259
remain on the apparatus for 60 s without falling. On the day of the
experiment, the mice were treated with vehicle (saline), diazepam
(2 mg/kg, i.p.) or HEEP (250 mg/kg) and tested on the rotarod 1 h
after the administration of saline and HEEP or 30 min after the
administration of diazepam. The number of falls from the appara-
tus was recorded with a stopwatch for 180 s.
2.7. Analysis of the possible mechanisms of action of HEEP
2.7.1. Involvement of opioid system
To evaluate the participation of opioid receptors in the anti-
nociceptive activity of HEEP, male Swiss mice were pretreated
intraperitoneally (i.p.) with naloxone (1 mg/kg). After 30 min, the
animals received HEEP (250 mg/kg—the lower effective dose, p.o.),
morphine (2.5 mg/kg, s.c., an opioid analgesic whose effects can be
reversed by pretreatment with naloxone) or saline (10 mL/kg, p.o.,
control). The nociceptive response to the formalin intraplantar
injection was recorded 1 h after the administration of HEEP or
control and 30 min after the administration of morphine. Another
group of mice was pretreated with vehicle (saline 10 mL/kg, i.p.)
and received HEEP, morphine or saline 1 h, 30 min and 1 h before
the formalin injection, respectively.
2.7.2. Involvement of the L-arginine–nitric oxide pathway
To evaluate the role played by the L-arginine–nitric oxide pathway
in the antinociceptive effects of HEEP, male Swiss mice were pre-
treated with L-arginine (500 mg/kg, i.p.) or saline (10 mL/kg, i.p.,
control). After 30 min, the mice received HEEP (250 mg/kg, p.o.),
L-Name (65 mg/kg, i.p., a nitric oxide syntheses inhibitor) or saline
(10 mL/kg, p.o., control). The nociceptive response to the formalin
intraplantar injection was recorded 1 h after the administration of
HEEP or vehicle and 30 min after the administration of L-Name.
Another group of mice was pretreated with vehicle (saline) and after
30 min, received HEEP, L-Name or saline, 1 h, 30 min and 1 h before
the formalin injection, respectively.
2.7.3. Glutamate-induced nociception
The procedure used was similar to one previously described
(Beirith et al., 2002). Male Swiss mice were orally treated with saline
(control, 10 mL/kg) and HEEP (250 mg/kg). After 1 h, the animals
received an intraplantar injection in the right hind paw of 20 mL
glutamate (30 mmol/paw prepared in saline solution). The animals
were observed for 15 min, and the time they spent licking, in seconds,
was recorded and considered to be indicative of nociception.
2.8. Ear edema induced by xylene
The procedure used was similar to that described previously
(Swingle et al., 1981). Fasting mice (2 h) were treated with saline
(control, 10 mL/kg) or HEEP (250 mg/kg, v.o.) 1 h before the topical
application of xylene, applied to the anterior (20 mL) and posterior
(20 mL) surfaces of the right ear. Dexamethasone (5 mg/kg, i.p.) was
used as a positive control 2 h before xylene application. The left ear
was used as a control. After 1 h of edema induction, the mice were
killed, and circular sections (8 mm of diameter) of their ear were cut
with a puncher. The extent of edema was expressed by the difference
between the weight (in milligrams) of the left ear and the right ear.
2.9. Carrageenan-induced hind paw edema in rats
Paw edema was induced by carrageenan injection in male Wistar
rats (Henriques et al., 1987). Groups of rats were treated through the
oral route with vehicle (10 mL/kg), HEEP (250 mg/kg), or piroxicam
(30 mg/kg) 1 h prior to the carrageenan injection. To induce paw
edema, 0.1 mL carrageenan (1% in sterile saline solution) was injected
260 R.T. Basting et al. / Journal of Ethnopharmacology 157 (2014) 257–267
intradermally on the plantar side of the right hind paw. The rat paw
volume up to the ankle joint was measured with a plethysmometer
(Insight Ltda., Ribeirão Preto, Brazil) at 1, 2 and 3 h after the injection
of carrageenan. The anti-inflammatory effect of the extract was
measured by the amount of decrease in the difference in paw edema
volume between the left (without carrageenan) and right (with
carrageenan) hind paw (mL).
2.10. Inhibition of the phosphorylation of p38α MAPK
An ELISA was performed as described previously (Goettert et al.,
2012). Briefly, 96-well plates were coated with 0.5 mg ATF-2/50 mL
(1:192 in TBS buffer pH 7.8) and incubated overnight at 4 1C. Next, the
plate was blocked with blocking buffer for 30 min at room tempera-
ture. The extract (stock solution) was diluted in Cremophor-ELR EtOH
(77:23). The stock solution (30, 10 and 3 mg/mL) was diluted using
kinase buffer, which contains ATP [10 mM], phosphatase-inhibitors
and activated p 38αMAPK. The kinase buffer was used to dilute the
HEEP at 1:1000 to generate a final concentration of 300 pg/mL. This
was the inhibitor dilution and transferred to the plate with ATF-2. The
reaction was incubated for 50 min at 37 1C. Then, the plate was
blocked with BB for 15 min. The antibody was diluted 1:5000 in BB
adjusted to pH 6.5. Then, 50 mL antibody solution was added to each
well and incubated for 1 h at 37 1C. After each incubation step, the
wells were washed three times with double-distilled water and dried
for 5 min at 37 1C. To detect the anti-phospho-ATF-2 complex, TMB
substrate (50 mL/well) was added, and after incubation for 5–10 min at
room temperature, a dark blue color developed. The reaction was
stopped with 25 mL 2 N H2SO4 per well. The absorbance was measured
at 450 nm with an ELISA reader equipped with SOFTmax PRO
software.
2.11. Evaluation of antiulcer activity in the model of gastric ulcer
induced by ethanol
Rats were divided into five groups and treated, after fasting for
16 h, with vehicle (saline, 10 mL/kg), carbenoxolone (positive control,
100 mg/kg) or HEEP (125, 250 or 500 mg/kg). After 1 h, gastric lesions
were induced by administering 1 mL absolute ethanol as described by
Robert et al. (1979). The animals were killed 1 h after induction, and
their stomachs were removed to count and classify the lesions. To
quantify the gastric lesions, the stomachs were placed between glass
plates and scanned for analysis using the AVSoft BioView s (mm2)
application.
2.12. Evaluation of antiulcer activity in the gastric ulcer model
induced by indomethacin
Rats were separated into groups and deprived of food for 16 h with
water ad libitum. Saline (control, 10 mL/kg), lansoprazole (30 mg/kg),
and HEEP (125, 250 and 500 mg/kg) were administered orally, and
after 30 min, indomethacin (50 mg/kg, solubilized in sodium carbo-
nate 0.5%, pH 7.4) was administered orally to all animals as described
by Guidobono et al. (1997). All animals were killed 6 h after the
administration of the injurious agent (indomethacin). The stomachs
were removed and opened along the greater curvature, and the area
of damage (mm2) was determined using the program AVSoft BioView
s
(mm2).
2.13. Shay ulcer
To evaluate the antisecretory effect of the extract given through the
oral route, male rats (n¼7/group) were randomly divided into three
groups and fasted for 16 h with free access to water. Thirty minutes
after the oral administration of HEEP (125 mg/kg), lansoprazole
(30 mg/kg, positive control) or vehicle (saline, negative control),
pylorus ligature was performed as described by Shay (1945). Four
hours later, the animals were killed, their abdomens were opened and
another ligature was placed around the esophagus near the dia-
phragm. The stomach was removed, and the gastric juice volume (mL)
and hydrogenion concentration (in milliequivalents per milliliter per
4 h) were recorded. The assay was performed according to the method
described by Shay (1945) with slight modification. To evaluate the
antisecretory effect of HEEP given through the intraduodenal route, all
groups of male rats (n¼7/group) were fasted for 16 h with free access
to water. Immediately after a pylorus ligature, HEEP (125 mg/kg, the
lower effective dose), lansoprazole (30 mg/kg, positive control) or
vehicle was administered via the intraduodenal route. The animals
were killed 4 h later, the abdomen was opened and another ligature
was placed around the esophagus near the diaphragm. The stomachs
were removed, and the gastric content was collected to determine the
total amount of gastric-juice acid (in milliliters). Distilled water was
added, and the resultant solution was centrifuged at 3000  g for
10 min. The hydrogen ion concentrations (in milliequivalents per
milliliter per 4 h) in the gastric secretion were recorded by adjusting
the supernatant volume by titration to pH 7.0 with 0.01 N NaOH.
2.14. Determination of gastric mucus
Following the method described by Rafatullah et al. (1990),
after 16 h of fasting, the rats were randomly divided into groups
(n ¼7/group) and treated with HEEP (125 mg/kg), carbenoxolone
(200 mg/kg) or vehicle through oral administration. After 4 h, the
animals were killed, and the glandular portion of their stomachs
were separated, weighed and immersed in Alcian Blue solution for
the mucus quantification procedure. The absorbencies were mea-
sured in a spectrometer at 598 nm, and the results expressed as mg
of Alcian Blue/g of tissue.
2.15. Statistical analyses
The results were expressed as the mean 7 standard error of the
mean (S.E.M.) of the parameters obtained. The mean values were
analyzed using one-way ANOVA followed by Dunnett’s test to
compare three or more groups or by Student’s t-test to compare
two groups. In the statistical analysis of the results, p o0.05 was
considered to be the minimum significance level.
3. Results and discussion
As a part of this pharmacological evaluation, HEEP was first
investigated for acute toxicity in male and female Swiss mice. A
single oral dose of HEEP (5 g/kg, p.o.) did not produce any visible
signs or symptoms of toxicity in mice of either sex. We did not
observe any behavioral changes in the male or female mice using a
Hippocratic screening (data not shown). In the fourteen days after
the administration of HEEP, no animals died, and no significant
changes in organ weights (Table 1) or daily body weights were
observed (data not shown).
To investigate whether HEEP has analgesic and anti-inflammatory
effects, the formalin test was a useful tool for the initial screening
(Hunskaar and Hole, 1987). This test has a distinct response (biphasic)
to pain; the neurogenic or the initial phase, which lasts five min after
the formalin injection, consists of pain that is the result of a direct
irritant effect on nociceptors activating primary afferent fibers and
resulting in the release of substance P, CGRP and neuropeptide in the
central and peripheral terminals (Tjolsen et al., 1992; Hunskaar and
Hole, 1987). In the second stage, the inflammatory phase, which starts
15 min after the formalin-induced pain and remains for the following
45 min, peripheral sensitization is characterized by inflammatory
mediators modulated through the spinal cord (Le Bars et al., 2001).
 R.T. Basting et al. / Journal of Ethnopharmacology 157 (2014) 257–267 261

Table 1
Toxicological parameters after the acute administration (5 g/kg) of a hydroalcoholic extract from the leaves of Eugenia punicifolia (HEEP) in Swiss mice through the oral route
(n¼ 8–10).

Sex Treatment (p.o.) Liver Heart Lung Kidney Spleen Deaths

♂ Control 13.84 7 0.17 3.99 70.05 4.46 7 0.07 6.29 7 0.08 3.99 7 0.18 0
HEEP 13.62 7 0.29 3.84 70.07 4.50 7 0.11 6.127 0.05 3,687 0.13 0

♀ Control 12.86 7 0.14 3.99 70.07 4.737 0.09 5.727 0.07 3.56 7 0.10 0
HEEP 12.90 7 0.13 3.89 70.16 5.63 7 0.74 6.117 0.27 3.79 7 0.13 0

Results are the mean 7S.E.M. of the ratio. Student’s t-test. p 40.05.The results were expressed as mean and standard error of the relative organ weight in relation to total
weight of the animals. This ratio was converted into arcsine for statistical adjustment.

Fig. 1. Effect of a hydroalcoholic extract from the leaves of Eugenia punicifolia (HEEP)(125, 250 and 500 mg/kg) in the formalin test (first phase, panel A and second phase,
panel B). Each column represents the mean of 10 animals, and the vertical lines indicate the S.E.M. The asterisks denote the significance levels compared with the control
group C. np o 0.05, nnp o 0.01 and nnnp o 0.001 using one-way ANOVA followed by Dunnett’s test.

60 minutes 90 minutes
20 20
***
Latency (seconds)
Latency (seconds)

15 * 15
** *
10 10

5 5

0 0

120 minutes
20
Control
Morphine 2.5 mg/kg
Latency (seconds)

15 ***
** HEEP 125 mg/kg
HEEP 250 mg/kg
10

0
Fig. 2. Effect of a hydroalcoholic extract from the leaves of Eugenia punicifolia (HEEP)(125 and 250 mg/kg) on the response latency at 60, 90 and 120 min after the
administration of drugs in the hot-plate test in mice. Data are reported as the mean 7 S.E.M. for n¼ 7–10 per group. The asterisks denote the significance levels compared
with the control group C. np o 0.05, nnp o 0.01 and nnnp o0.001 using one-way ANOVA followed by Dunnett’s test.

The results depicted in Fig. 1A and B show that HEEP administered
orally at doses of 125, 250 and 500 mg/kg caused a significant
antinociceptive effect in both phases (neurogenic and inflammatory)
of the formalin test. In the neurogenic phase, the inhibition was 25, 35
and 20%, respectively, and in the inflammatory phase, the inhibition
was 41, 63 and 36%, respectively, when compared to the group treated
with vehicle. The optimal antinociceptive effect was obtained from
HEEP at doses of 125 and 250 mg/kg and these both doses were
investigated in another experimental model of pain, the hot plate test.
This assay investigates the central effects of analgesic drugs. According
to Le Bars et al. (2001) the hot-plate test is known to involve the
activation of supraspinal structures, and the tail-flick response is more
262 R.T. Basting et al. / Journal of Ethnopharmacology 157 (2014) 257–267
Fig. 3. Evaluation of the involvement of the opioid system on the antinociceptive activity of a hydroalcoholic extract from the leaves of Eugenia punicifolia (HEEP 250 mg/kg)
in the formalin test (first phase, panel A and second phase, panel B). Each column represents the mean of 10–12 animals, and the vertical lines indicate the S.E.M. The
asterisks denote the significance levels compared with the control groups C. npo 0.05, nnp o0.01, nnnp o0.001 and n.s.—not significantly different using one-way ANOVA
followed by Dunnett’s test.
Fig. 4. Involvement of the L-arginine–nitric oxide pathway in the antinociceptive
activity of a hydroalcoholic extract from the leaves of Eugenia punicifolia (HEEP
250 mg/kg) in the formalin test (first phase, panel A and second phase, panel B).
Each column represents the mean of 10–12 animals, and the vertical lines indicate
the S.E.M. The asterisks denote the significance levels compared with the control
groups C. nnp o 0.01, nnnp o 0.001 and ns—not significantly different using one-way
ANOVA followed by Dunnett’s test.
related to a spinal reflex triggered by C fibers when it is elicited by
heat. Pretreatment (60, 90 and 120 min) with HEEP (250 mg/kg, p.o.)
significantly increased the latency in the nociceptive response induced
by heat (Fig. 2). However, a lower dose of HEEP (125 mg/kg) did not
induce an antinociceptive effect (p40.05). Thus, the subsequent
experiments with HEEP were carried out at a dose of 250 mg/kg.
Sometimes myorelaxant drugs or sedatives can promote
changes in the motor performance of mice resulting in false
positive results for new drugs with antinociceptive action. There-
fore, we evaluated the integrity of motor coordination on the basis
of locomotor performance of the mice on the rotarod apparatus.
150
Time of licking (s)
100
**
50
0
C 250
Eugenia punicifolia (mg/kg, p.o.)
Fig. 5. Effect of the antinociceptive effect of oral treatment with a hydroalcoholic
extract from the leaves of Eugenia punicifolia (HEEP 250 mg/kg) in glutamate-
induced nociception. Each column represents the mean of 10–12 animals, and the
vertical lines indicate the S.E.M. The asterisks denote the significance levels
compared with the control group C. nnp o 0.01 using Student’s t-test.
Their performance was not significantly affected (p40.05) by the oral
administration of HEEP at a dose of 250 mg/kg (2.12740.61 falls).
However, diazepam (2 mg/kg) significantly decreased (po0.01) the
endurance time on the rotating rod (7.6271.79 falls) when compared
with the animals from the control group (3.0670.30 falls).
Based on the results shown in Fig. 2, we investigated the possible
role of the opioid system in the antinociceptive mechanism of action
of HEEP (250 mg/kg). Naloxone (non-selective antagonist of opioid
receptor) has been used to block opioid receptors and to determine
whether HEEP loses its previously observed antinociceptive effect
(Santos et al., 2005). The opioid system is an endogenous mechanism
of the organism to manage pain and inflammation. As shown in
Fig. 3A and B, naloxone completely reversed the antinociceptive effect
caused by morphine but not the effect of HEEP in the formalin model,
confirming that the opioid system does not seem to participate in the
antinociception caused by HEEP.
Saragusti et al. (2012) described experimental evidence of the
participation of nitric oxide, among other inflammatory mediators,
in the second phase of the formalin-induced pain test. Considering
the important inhibitory effect of HEEP on the second phase of the
formalin test, the involvement of the L-arginine–nitric oxide system
in the antinociceptive response produced by HEEP was evaluated.
To investigate the possible role of nitric oxide (NO) in the anti-
nociceptive mechanism of action of this extract, we used L-arginine
(a substrate for NO synthesis which causes the reversal of the
antinociceptive effect on formalin test) to determine the possible
effect of reversing the extract through this drug. As shown in Fig. 4A
and B, there was a significant reversal of the antinociceptive effect
of the extract after pretreatment with L-arginine compared to
pretreatment with the vehicle in both phases of the formalin test.
 R.T. Basting et al. / Journal of Ethnopharmacology 157 (2014) 257–267 263

Table 2
Evaluation of the anti-inflammatory activity of a hydroalcoholic extract from the leaves of Eugenia punicifolia (HEEP 250 mg/kg) on ear edema induced by xylene and paw
edema induced by carrageenan.

Model Treatment Dose (mg/kg) Volume difference

Ear edema induced by xylene (wt/mg) Control – 9.95 7 1.44

Dexamethasone 5 3.217 0.68***
HEEP 250 3.617 0.53***

Paw edema induced by carrageenan (vol/mL) Control – 0.417 0.06

piroxicam 30 0.22 7 0.06**
HEEP 250 0.20 7 0.04**

Data represent the mean 7 S.E.M. of 8–10 animals. The asterisks denote the significance levels compared with the control group.
nn
p o0.01.
nnn
p o 0.001 Using one-way ANOVA followed by Dunnett’s test.

Table 3
Evaluation of a hydroalcoholic extract from the leaves of Eugenia punicifolia (HEEP 500
30, 10 and 3 mg/mL and the respective dilutions) in a p38α MAPK assay.

Lesion area (mm 2)

400
Samples Concentration Inhibition (%)

Control (SB 203580) 10 mM 96.157 1.65 300

1 mM 88.86 7 2.00
0.1 mM 63.84 7 1.29 200
0.01 mM 12.0 7 3.51

HEEP 30 mg/mL 3  10  4 mg/mL 103.377 1.20 100

3  10  5 mg/mL 91.377 0.35 ***
3  10  6 mg/mL 17.727 1.56 *** *** ***
0
3  10  7 mg/mL 7.45 7 1.53 C Carbenoloxone 125 250 500
HEEP 10 mg/mL 1  10  4 mg/mL 97.88 7 0.23 Eugenia punicifolia (mg/kg, p.o.)
1  10  5 mg/mL 51.377 2.83
1  10  6 mg/mL 18.50 7 0.67
1  10  7 mg/mL 7.84 7 1.90 15
HEEP 3 mg/mL 3  10  6 mg/mL 90.50 7 0.75
Lesion area (mm 2)

3  10  7 mg/mL 33.807 4.57

3  10  8 mg/mL 20.39 7 2.92 10
3  10  9 mg/mL 23.137 4.05

Values represent the mean7 S.E.M. **

5 **
Thus, the antinociceptive effect of HEEP may be related to the ***
release or inhibition of nitric oxide synthesis in vivo.
To provide further evidence of the involvement of the gluta- 0 ***
C Lansoprazole 125 250 500
matergic system in antinociception caused by HEEP, the effect of
Eugenia punicifolia (mg/kg, p.o.)
HEEP in nociception caused by intraplantar injection of glutamate
was investigated. Furthermore, it has been shown that intraplantar
injection of glutamate releases excitatory amino acids, such as Control

PGE2, NO, kinins, protons, glutamate and substance P in the dorsal Carbenoxolone 200 mg/kg
(ug Alcian Blue/g of tissue)

2000
Eugenia punicifolia 125 mg/kg
horn, and that glutamate is involved in the transmission of
mucus concentration

***
nociceptive information from the periphery to the supraspinal 1500 **

regions (Beirith et al., 2002). In the nociception induced by the

administration of glutamate, a dose of 250 mg/kg HEEP inhibited
nociception by 35% compared to the group treated with vehicle, as
shown in Fig. 5. This result indicates that the antinociceptive effect
of HEEP involves the glutamatergic system. Moreover, our data
shows that HEEP inhibited nociception in both the glutamate and
formalin tests, but was more effective against the inflammatory
(second phase) pain caused by formalin; these data led us to
investigate the anti-inflammatory effect of this extract.
To characterize the anti-inflammatory activity of HEEP in neuro-
genic inflammation, the model of ear edema induced by xylene was
used. In this test, HEEP significantly reduced the edema only at a
dose of 250 mg/kg, which inhibited edema by 64% compared to the
group treated with the vehicle (saline). The positive control dex-
amethasone inhibited edema by 68%, as shown in Table 2. The anti-
edematogenic effect of HEEP was also observed in paw edema
induced by carrageenan injection at the 1th hour after treatment.
The positive control, piroxicam, inhibited edema by 46%, and HEEP
inhibited edema by 50% compared to the group treated with the
1000
500
0
Fig. 6. Evaluation of the antiulcer activity of a hydroalcoholic extract from the
leaves of Eugenia punicifolia in the models of gastric ulcer induced by ethanol
(panel A) or indomethacin (panel B), and the quantification of the adherent mucus
in gastric mucosa of rats (panel C). Each column represents the mean of 7–10
animals, and the vertical lines indicate the S.E.M. The asterisks denote the
significance levels compared with the control group C. nnpo 0.01 and nnnp o0.001
using one-way ANOVA followed by Dunnett’s test.
vehicle (saline), as shown in Table 2. These results enhance our
previous results on the effects of HEEP in the second stage, i.e., the
inflammatory phase, of the formalin test.
p38 MAPK plays a decisive role in the regulation of the production
of proinflammatory cytokines. Therapeutic protein kinase inhibitors
are in clinical development for diseases such as rheumatoid arthritis,
264 R.T. Basting et al. / Journal of Ethnopharmacology 157 (2014) 257–267

diabetes and cardiovascular disease (Zuccotto et al., 2010). It is positive control. This result demonstrates that HEEP is effective in
expected that screening for inhibitors of protein kinases will become inhibiting the p38α MAPK pathway.
increasingly important in the future, and one essential prerequisite for Meotti et al. (2007) described the involvement of p38α MAPK in
the development of new drugs and validation of new therapeutic the antinociceptive action of myricitrin in mice. The authors demon-
compounds is a test that is valid, fast and easy to use (Laufer and Koch, strated that the mechanism of antinociceptive action of myricitrin
2008). To assess the possible effect of HEEP on the inhibition of protein involves the blockage of p38α MAPK. Interestingly, this article also
kinases, the p38α MAPK test was performed because this protein is showed that myricitrin is a flavonoid commonly present in plant of
involved in the inhibition or regulation of the release of proinflam- the genus Eugenia. Thus, even though we do not have a quantitative
matory cytokines such as TNF-α and Interleukin 1. In the p38α MAPK profile of the presence of myricitrin in HEEP, our qualitative profile
assay, the hydroalcoholic extract from the leaves of Eugenia punicifolia demonstrates the presence of this flavonoid in our extract. All of
was used at concentration of 30, 10 and 3 mg/mL. Table 3 shows that these data therefore corroborate the characterization of the anti-
HEEP inhibited the phosphorylation of p38α MAPK at all concentration nociceptive and anti-inflammatory effects of HEEP through the
tested. The samples with higher concentrations had a greater inhibi- inhibition of p38α MAPK.
tory activity, approximately 90%, as did all concentrations of the We also evaluated the gastroprotective effect of HEEP through the
model of gastric ulcer induced by ethanol and NSAIDs. The oral
administration of absolute ethanol causes tissue edema, sub epithe-
Table 4 lial hemorrhage, exfoliation and cellular infiltration of inflammatory
Effects of a hydroalcoholic extract from the leaves of Eugenia punicifolia (HEEP) cells that may contribute to the induction of mucosal damage
administered through the oral (p.o.) or intraduodenal (i.d.) route on the biochem- (Kvietys and Beveleign, 1990). The results show that HEEP at the
ical parameters of gastric juice obtained from pylorus-ligature rats.
doses of 125, 250 and 500 mg/kg inhibited the gastric lesions
Treatment Route Dose N pH Gastric [H þ ] induced by an injurious agent by 88.4, 97.6 and 99.8%, respectively,
(mg/kg) (unit) juice (mL) (μEq/L/4 h) compared to the control group, as shown in Fig. 6A. The positive
control group (carbenoxolone) inhibited the lesions by 99.7%. This
Control p.o. – 9 1.277 0.04 7.25 7 0.59 10.17 70.34
result demonstrates a strong protective action on the gastric mucosa
Lansoprazole 30 10 5.36 7 0.55*** 2.017 0.26*** 5.37 70.69***
HEEP 125 9 1.497 0.09 4.94 7 0.61** 8.46 70.81 by HEEP.
Control i.d. – 10 1.317 0.05 5.63 7 0.47 6.63 70.60 We also evaluated the possible gastroprotective effect of this
Lansoprazole 30 10 5.63 7 0.30*** 2.707 0.30** 1.72 70.26*** extract using the experimental model of gastric ulcer induced by
HEEP 125 10 1.617 0.22 5.94 7 1.03 6.73 70.38 indomethacin. Indomethacin is an anti-inflammatory non-steroidal
Data represent the mean 7 S.E.M. The asterisks denote the significance levels
indole derivative widely used in the clinic to treat various types of
compared with the control group. inflammatory diseases. Experimentally, it has become a drug of choice
nn
p o0.01. for the induction of gastric lesions due to its greater potential
nnn
p o0.001 Using one-way ANOVA followed by Dunnett’s test. ulcerogenic effects compared to other drugs of its class, and this effect

18000
17000
16000
15000 600 600
14000
13000
12000
11000
400 400
10000
uAU

mv
mv

9000
8000
7000
6000 200 200
5000
4000
3000
2000
1000 0 0
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Time (min) Minutes

PDA-371 nm
EHAEP_spe1.1
150 150 PDA-254 nm
600 EHAEP_spe95mehoh 600

100 100
400 400
mv
mv

mv

mv

50 50 200 200

0 0 0 0

18 20 22 24 26 28 30 32 5 10 15 20 25 30
Minutes Minutes

Fig. 7. Analytical HPLC-PAD chromatograms. (a) A hydroalcoholic extract from the leaves of Eugenia punicifolia recorded at 254 nm, (b) fraction SPE MeOH:H2O 8:2 (v/v)
recorded at 254 nm, (c) fraction SPE MeOH:H2O, 1:1 (v/v) recorded at 371 nm and (d) fraction SPE MeOH:H2O, 95:5 (v/v) recorded at 254 nm.
 R.T. Basting et al. / Journal of Ethnopharmacology 157 (2014) 257–267 265

is due to its ability to inhibit both COX-1 and COX-2 (Suleyman et al.,
2010). The results of this study show that HEEP at the doses of 125,
250 and 500 mg/kg significantly inhibited indomethacin-induced
ulcers by 53, 72.3 and 53%, respectively compared to the control
group, as shown in Fig. 6B. This demonstrates that the mechanism of
action of the extract is not dependent on the cyclooxygenase pathway.
In the positive control group (lansoprazole), the inhibition was 100%.
The gastric juice parameters of the rats treated with HEEP
administered through different routes (Table 4) demonstrated that
oral treatment with HEEP decreased the gastric volume without
modifying the pH and H þ concentration. The systemic evaluation
of the intraduodenal administration of HEEP showed no modifica-
tion of gastric juice parameters compared with the control group
treated with vehicle. These results indicate therefore that HEEP
does not exert its gastroprotective effects in an antisecretory manner.
The success of gastric pharmacological treatment for ulcers
relies not only on the blockage of acid secretion but also on the
augmentation of the protective factors on the gastric mucosa, such
as mucus secretion. Gastric mucus is the first line of defense
against acid, and our results show that animals treated with HEEP
through the oral route had approximately 2 times the amount of
mucus adhered to the gastric mucosa (Fig. 6C).
The phytochemical profile of HEEP confirmed by TLC showed that
this extract also contains secondary metabolites already reported for
PDA-254 nm
18
600 EHAEP_spe95mehoh
14-
17
1
13
21
400
20
mv
the genus (flavonoids, tannins, terpenoids and saponins) as described
by Oliveira et al. (2006). HPLC-PAD analyses of the compounds present
in HEEP detected flavonoid derivates and the presence of gallotaninns
(Figs. 7and 8 and Table 5). The poor resolution of the chromatogram in
Fig. 7 and the UV data of peaks 4–12 shown in Table 5 suggest the
presence of proanthocyanidins in HEEP. In an attempt to improve the
resolution of the chromatogram, HEEP was cleaned up with SPE using
1:1 MeOH:H2O (v/v), Fig. 7B; 8:2 MeOH:H2O (v/v), Fig. 7C and finally
95:5 MeOH:H2O (v/v), Fig. 7C. It was evident that the best resolution
without damaging the content of the substances in HEEP was using
the conditions of the chromatogram in Fig. 7C. The detailed descrip-
tion of the chemical composition of HEEP showed the presence of
groups of secondary metabolites that may be involved in its pharma-
cological activities. The features of the UV spectra with absorption
maxima bands at 205–215 nm and 270–285 nm suggested the pre-
sence of polymeric proanthocyanidins. The UV spectra of the com-
pounds show absorption maxima bands characteristic of flavonoids
(255–269 and 348–366 nm) (de Pascual-Teresa et al., 2000; Rohr et al.,
2000). Compounds 14,15, 16 and 19 of HEEP are in a class of
substances commonly known as flavonoids and were detected at
the greater retention times of 6.71, 13.91, 14.52, and 23.66 min,
respectively (Fig. 8). The identification of these compounds was based
on the comparison of the UV spectra of the peaks with those available
in the literature (Table 5). The flavonoids have characteristic bands at
263–273 and 303–330 nm. Another compound class identified in
HEEP is the proanthocyanidin derivatives (1–5 and 8–11) with the
UV spectrum (λmáx ¼271, 272 nm). Compounds 2, 5, 7, and 10–12 of
600
HEEP are a class of substances commonly known as flavonoids. These
were detected in greater quantities in the leaves at the retention times
of 6.71, 13.91, 14.52, and 23.66 min, respectively. The identification of
400
these compounds was based on the comparison of the UV spectra of
the peaks with those available in the literature. The flavonoids present

mv

6
characteristic bands at 263–273 and 303–330 nm. Based on their
200 9 19 200
5 11 12 fragmentation patterns in the MS/MS and UV experiments, we suggest
2 -3 7-8
10 the presence of myricetin derivatives with galloyl and pentoses units.
4
The position of these substituent groups cannot be determined using
0 0
mass spectrometry. However, the loss of 132 units and 146 units
5 10 15 20 25 30 suggest the presence of arabinose and rhaminose, and the loss of 152
Minutes units suggests the presence of a galloyl group. Another compo-
und class identified in HEEP is the gallotannin derivatives (4, 6, 8, 9,
Fig. 8. Analytical HPLC-PAD chromatogram of the SPE Fraction 95% MeOH from a
hydroalcoholic extract from the leaves of Eugenia punicifolia recorded at 254 nm.
13, and 14) with the UV spectrum (λmáx ¼271, 272 nm). The ESI-MS
For the UV data (peaks 1–21), see Table 5. spectrum shows a desprotonated molecule at m/z 783 [M–H]  with

Table 5
UV spectra data of the peaks from the HPLC-PDA chromatogram of the SPE fraction 95% MeOH from a hydroalcoholic extract from the leaves of Eugenia punicifolia.

Peak no. tr λmáx (nm) Phenolic compound assignment Reference

1 2.99 274 Gallotannin or Gallic acid derivative Laguari et al. (2011)

2 6.00 276 Proanthocyanidin Ramirez et al. (2014)
3 6.12 275, 344 Unknown flavonoid
4 7.56 274 Gallotannin or Gallic acid derivative Laguari et al. (2011)
5 8.84 274 Gallotannin or Gallic acid derivative Laguari et al. (2011)
6 11.52 264 Galloyl HHDP glucose Romani et al. (2012)
7 12.63 263 Galloyl HHDP glucose Romani et al. (2012)
8 13.10 274 Gallotannin or Gallic acid derivative Laguari et al. (2011)
9 13.40 273 Gallotannin or Gallic acid derivative Laguari et al. (2011)
10 14.44 272 Gallotannin or Gallic acid derivative Laguari et al. (2011)
11 14.50 272 Gallotannin or Gallic acid derivative Laguari et al. (2011)
12 15.04 279 Proanthocyanidin Ramirez et al. (2014)
13 20.63 255, 366 Flavonol derivative
14 20.72 258, 348 quercetin Ramirez et al. (2014)
15 21.04 267, 353 Myricetin derivative Simirgiotis (2013)
16 21.34 254, 360 Myricetin Simirgiotis (2013)
17 21.69 265, 349 Quercetin derivative Ramirez et al. (2014)
18 22.04 254, 356 Rutin Simirgiotis (2013), Ola et al. (2009)
19 23.44 269, 354 Myricetin derivative
20 24.77 258, 348 quercetin Simirgiotis (2013)
21 29.74 221, 278 di-HHDP glucose
266 R.T. Basting et al. / Journal of Ethnopharmacology 157 (2014) 257–267
the retention times of 12.44, 14.41 and 18.91 min., which suggests that
these compounds are isomers.
The presence of proanthocyanidins in this extract is particularly
significant because Iwasaki et al. (2004) recently reported that
proanthocyanidins inhibit gastric mucosal injury by strengthening
mucosal protection, similar to what was observed in our study. The
HEEP phenol content was found to be 0.2070 mg gallic acid/g of
extract (or 0.0207%). Comparatively, the results show that despite a
phytochemical profile similar to the genus, HEEP has lower amounts of
phenols than the species studied by Magina et al. (2010). This study
also found that HEEP contains 0.1957 mg quercetin/g of extract (or
0.01957%). Quercetin, used in the quantification flavonoid standard, is
widely distributed in the plant kingdom and has significant anti-
inflammatory, antioxidant, antitumor, and antiviral properties, among
others (Kim et al., 2004). Additionally, the presence of myricetin
derivatives (Table 5, compounds 15 and 16) could explain the effect
of HEEP on the inhibition of p38α MAPK but we cannot dismiss the
role of another phenolic contents on the final pharmacological effect
of HEEP.
Based on the results of this study, we can conclude that a
hydroalcoholic extract from the leaves of Eugenia punicifolia has a
significant anti-nociceptive effect that involves glutamatergic
systems and related to the release or inhibition of nitric oxide
synthesis and an anti-inflammatory effect through the inhibition
of the phosphorylation of p38α MAPK. Our results also indicated
that HEEP has a gastroprotective effect related to an increase in the
gastric production of mucus.
Acknowledgments
This work was supported by grants from Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de
Amparo à Pesquisa do Estado de São Paulo (Fapesp) at process
numbers 2011/20145-8 and 2009/52237-9.
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