Journal of Ethnopharmacology 157 (2014) 257–267

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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 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 b

art ic l e i nf o

a b s t r a c t

Article history: Received 28 July 2014 Received in revised form 24 September 2014 Accepted 28 September 2014 Available online 13 October 2014

Ethnopharmacological relevance: An ethnopharmacological survey indicated that leaves from Eugenia punicifolia (Kunth) DC. (Myrtaceae) are popularly used as a natural therapeutic agent to treat pain and inflammation. Aim of the study: The overall objective of the present study was to evaluate the antinociceptive, antiinflammatory and gastroprotective activities of a hydroalcoholic extract of leaves from Eugenia punicifolia (HEEP) in rodents. Material and methods: The antinociceptive effects of HEEP were evaluated in mice after oral administration in chemical (formalin and glutamate) and thermal (hot-plate) tests. We evaluated the involvement of the glutamatergic, opioidergic and nitrergic pathways in the antinociception of HEEP and the effect of HEEP on the inhibition of p38α MAPK. The anti-inflammatory effect of HEEP was evaluated in mice and rats using xyleneinduced ear edema and carrageenan-induced paw edema, respectively. Furthermore, the gastroprotective effect of HEEP was evaluated in rats with acute gastric lesions induced by ethanol or indomethacin. Finally, we 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 Larginine (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 antiinflammatory effects, which appear to be related to the inhibition of the glutamatergic system, the synthesis

Keywords: Eugenia punicifolia Myrtaceae Antinociceptive Anti-inflammatory Gastroprotective p38α MAPK

Abbreviations: TNF, tumor necrosis factor; HEEP, hydroalcoholic extract of leaves from Eugenia punicifolia; PGE2, prostaglandin E2; NO, nitric oxide; MAPK, mitogenactivated 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: [email protected] (C. dos Santos), [email protected] (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.

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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. & 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Eugenia punicifolia (Kunth) DC., popularly known as “pedraume-caá, pedra-ume, murta or muta”, is a shrub largely distributed 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 Alzheimer’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 hydroalcoholic 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 mucosal lesions in the stomachs of humans, we also verified the effect of HEEP on gastric lesions induced by ethanol and an NSAID.

dynamic maceration for 2 h at room temperature (2572 1C). Afterwards, 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 chromatography 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 increasing 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. Materials and methods 2.2. Animals 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 Experimentais – 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

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 administered 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

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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 intraplantarly (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 preselected (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.

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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 apparatus 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 antinociceptive 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 pretreated 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.

2.6. Locomotor performance

2.9. Carrageenan-induced hind paw edema in rats

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

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

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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 temperature. 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

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 diaphragm. 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 measured in a spectrometer at 598 nm, and the results expressed as mg of Alcian Blue/g of tissue. 2.15. Statistical analyses

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.

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.

2.12. Evaluation of antiulcer activity in the gastric ulcer model induced by indomethacin

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).

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 carbonate 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),

3. Results and discussion

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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 HEEP

13.84 7 0.17 13.62 7 0.29

3.99 70.05 3.84 70.07

4.46 7 0.07 4.50 7 0.11

6.29 7 0.08 6.127 0.05

3.99 7 0.18 3,687 0.13

0 0

♀

Control HEEP

12.86 7 0.14 12.90 7 0.13

3.99 70.07 3.89 70.16

4.737 0.09 5.63 7 0.74

5.727 0.07 6.117 0.27

3.56 7 0.10 3.79 7 0.13

0 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 20

*** *

15 10 5 0

Latency (seconds)

Latency (seconds)

20

90 minutes

15

**

*

10 5 0

120 minutes Latency (seconds)

20 15

***

**

Control Morphine 2.5 mg/kg HEEP 125 mg/kg HEEP 250 mg/kg

10 5 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

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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.

Time of licking (s)

150

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 glutamateinduced 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.

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. Therefore, we evaluated the integrity of motor coordination on the basis of locomotor performance of the mice on the rotarod apparatus.

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 antinociceptive 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 Dexamethasone HEEP

– 5 250

9.95 7 1.44 3.217 0.68*** 3.617 0.53***

Paw edema induced by carrageenan (vol/mL)

Control piroxicam HEEP

– 30 250

0.417 0.06 0.22 7 0.06** 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. p o 0.001 Using one-way ANOVA followed by Dunnett’s test.

nnn

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

10 mM 1 mM 0.1 mM 0.01 mM

96.157 1.65 88.86 7 2.00 63.84 7 1.29 12.0 7 3.51

3  10  4 mg/mL 3  10  5 mg/mL 3  10  6 mg/mL 3  10  7 mg/mL

103.377 1.20 91.377 0.35 17.727 1.56 7.45 7 1.53

HEEP 10 mg/mL

1  10  4 mg/mL 1  10  5 mg/mL 1  10  6 mg/mL 1  10  7 mg/mL

97.88 7 0.23 51.377 2.83 18.50 7 0.67 7.84 7 1.90

HEEP 3 mg/mL

3  10  6 mg/mL 3  10  7 mg/mL 3  10  8 mg/mL 3  10  9 mg/mL

90.50 7 0.75 33.807 4.57 20.39 7 2.92 23.137 4.05

HEEP 30 mg/mL

Values represent the mean7 S.E.M.

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 glutamatergic system in antinociception caused by HEEP, the effect of 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 PGE2, NO, kinins, protons, glutamate and substance P in the dorsal horn, and that glutamate is involved in the transmission of nociceptive information from the periphery to the supraspinal 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 neurogenic 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 dexamethasone inhibited edema by 68%, as shown in Table 2. The antiedematogenic 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

Lesion area (mm 2)

Control (SB 203580)

Inhibition (%)

400 300 200 100 0

***

*** C

Carbenoloxone

125

***

***

250

500

Eugenia punicifolia (mg/kg, p.o.)

15

Lesion area (mm 2)

Concentration

10

**

**

5

0

*** C

*** Lansoprazole

125

250

500

Eugenia punicifolia (mg/kg, p.o.)

Control

mucus concentration (ug Alcian Blue/g of tissue)

Samples

500

2000 1500

Carbenoxolone 200 mg/kg Eugenia punicifolia 125 mg/kg *** **

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,

R.T. Basting et al. / Journal of Ethnopharmacology 157 (2014) 257–267

positive control. This result demonstrates that HEEP is effective in inhibiting the p38α MAPK pathway. Meotti et al. (2007) described the involvement of p38α MAPK in the antinociceptive action of myricitrin in mice. The authors demonstrated that the mechanism of antinociceptive action of myricitrin involves the blockage of p38α MAPK. Interestingly, this article also showed that myricitrin is a flavonoid commonly present in plant of the genus Eugenia. Thus, even though we do not have a quantitative profile of the presence of myricitrin in HEEP, our qualitative profile demonstrates the presence of this flavonoid in our extract. All of these data therefore corroborate the characterization of the antinociceptive and anti-inflammatory effects of HEEP through the inhibition of p38α MAPK. 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 epithelial hemorrhage, exfoliation and cellular infiltration of inflammatory cells that may contribute to the induction of mucosal damage (Kvietys and Beveleign, 1990). The results show that HEEP at the doses of 125, 250 and 500 mg/kg inhibited the gastric lesions induced by an injurious agent by 88.4, 97.6 and 99.8%, respectively, compared to the control group, as shown in Fig. 6A. The positive control group (carbenoxolone) inhibited the lesions by 99.7%. This result demonstrates a strong protective action on the gastric mucosa by HEEP. We also evaluated the possible gastroprotective effect of this extract using the experimental model of gastric ulcer induced by indomethacin. Indomethacin is an anti-inflammatory non-steroidal indole derivative widely used in the clinic to treat various types of inflammatory diseases. Experimentally, it has become a drug of choice for the induction of gastric lesions due to its greater potential ulcerogenic effects compared to other drugs of its class, and this effect

diabetes and cardiovascular disease (Zuccotto et al., 2010). It is expected that screening for inhibitors of protein kinases will become increasingly important in the future, and one essential prerequisite for the development of new drugs and validation of new therapeutic compounds is a test that is valid, fast and easy to use (Laufer and Koch, 2008). To assess the possible effect of HEEP on the inhibition of protein kinases, the p38α MAPK test was performed because this protein is involved in the inhibition or regulation of the release of proinflammatory cytokines such as TNF-α and Interleukin 1. In the p38α MAPK assay, the hydroalcoholic extract from the leaves of Eugenia punicifolia was used at concentration of 30, 10 and 3 mg/mL. Table 3 shows that HEEP inhibited the phosphorylation of p38α MAPK at all concentration tested. The samples with higher concentrations had a greater inhibitory activity, approximately 90%, as did all concentrations of the

Table 4 Effects of a hydroalcoholic extract from the leaves of Eugenia punicifolia (HEEP) administered through the oral (p.o.) or intraduodenal (i.d.) route on the biochemical parameters of gastric juice obtained from pylorus-ligature rats. Treatment

Route Dose N (mg/kg) – 30 125 – 30 125

Control p.o. Lansoprazole HEEP Control i.d. Lansoprazole HEEP

9 10 9 10 10 10

[H þ ] (μEq/L/4 h)

pH (unit)

Gastric juice (mL)

1.277 0.04 5.36 7 0.55*** 1.497 0.09 1.317 0.05 5.63 7 0.30*** 1.617 0.22

7.25 7 0.59 10.17 70.34 2.017 0.26*** 5.37 70.69*** 4.94 7 0.61** 8.46 70.81 5.63 7 0.47 6.63 70.60 2.707 0.30** 1.72 70.26*** 5.94 7 1.03 6.73 70.38

Data represent the mean 7 S.E.M. The asterisks denote the significance levels compared with the control group. nn

p o0.01. p o0.001 Using one-way ANOVA followed by Dunnett’s test.

18000 17000 16000 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0

600

600

400

400

200

200

mv

uAU

nnn

mv

264

0 0

5

10

15

20

25

30

35

0 0

5

10

Time (min)

15

20

25

30

35

Minutes

PDA-371 nm EHAEP_spe1.1

150

50

50

0

0

mv

18

20

22

24

26 Minutes

28

30

32

PDA-254 nm EHAEP_spe95mehoh

600

400

400

200

200

mv

100 mv

100

600

mv

150

0

0 5

10

15

20

25

30

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

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 modification 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 EHAEP_spe95mehoh

600

18

600

1417 1

21

13

400

400

mv

mv

20

6

200

5

2 -3

9 7-8

4

200

19 11 12 10

0

0 5

10

15

20

25

30

Minutes

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. For the UV data (peaks 1–21), see Table 5.

265

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 description of the chemical composition of HEEP showed the presence of groups of secondary metabolites that may be involved in its pharmacological activities. The features of the UV spectra with absorption maxima bands at 205–215 nm and 270–285 nm suggested the presence of polymeric proanthocyanidins. The UV spectra of the compounds 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 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 these compounds was based on the comparison of the UV spectra of the peaks with those available in the literature. The flavonoids present characteristic bands at 263–273 and 303–330 nm. Based on their fragmentation patterns in the MS/MS and UV experiments, we suggest the presence of myricetin derivatives with galloyl and pentoses units. The position of these substituent groups cannot be determined using mass spectrometry. However, the loss of 132 units and 146 units suggest the presence of arabinose and rhaminose, and the loss of 152 units suggests the presence of a galloyl group. Another compound class identified in HEEP is the gallotannin derivatives (4, 6, 8, 9, 13, and 14) with the UV spectrum (λmáx ¼271, 272 nm). The ESI-MS 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

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

2.99 6.00 6.12 7.56 8.84 11.52 12.63 13.10 13.40 14.44 14.50 15.04 20.63 20.72 21.04 21.34 21.69 22.04 23.44 24.77 29.74

λmáx (nm)

275,

255, 258, 267, 254, 265, 254, 269, 258, 221,

274 276 344 274 274 264 263 274 273 272 272 279 366 348 353 360 349 356 354 348 278

Phenolic compound assignment

Reference

Gallotannin or Gallic acid Proanthocyanidin Unknown flavonoid Gallotannin or Gallic acid Gallotannin or Gallic acid Galloyl HHDP glucose Galloyl HHDP glucose Gallotannin or Gallic acid Gallotannin or Gallic acid Gallotannin or Gallic acid Gallotannin or Gallic acid Proanthocyanidin Flavonol derivative quercetin Myricetin derivative Myricetin Quercetin derivative Rutin Myricetin derivative quercetin di-HHDP glucose

derivative

Laguari et al. (2011) Ramirez et al. (2014)

derivative derivative

Laguari et al. (2011) Laguari et al. (2011) Romani et al. (2012) Romani et al. (2012) Laguari et al. (2011) Laguari et al. (2011) Laguari et al. (2011) Laguari et al. (2011) Ramirez et al. (2014)

derivative derivative derivative derivative

Ramirez et al. (2014) Simirgiotis (2013) Simirgiotis (2013) Ramirez et al. (2014) Simirgiotis (2013), Ola et al. (2009) Simirgiotis (2013)

266

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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 antiinflammatory, 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.

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