Central and peripheral antinociceptive effects of ellagic acid in different animal models of pain

European Journal of Pharmacology 707 (2013) 46–53

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Neuropharmacology and analgesia

Central and peripheral antinociceptive effects of ellagic acid in different animal models of pain Mohammad Taghi Mansouri a,b,n, Bahareh Naghizadeh c, Behnam Ghorbanzadeh d, Yaghoub Farbood e a

Department of Pharmacology, School of Medicine, Atherosclerosis Research Center, Ahvaz Jundishapur University of Medical Sciences (AJUMS), Ahvaz, Iran Physiology Research Center, Ahvaz Jundishapur University of Medical Sciences (AJUMS), Ahvaz, Iran c Department of Pharmacology, Pain Research Center, School of Medicine, Ahvaz Jundishapur University of Medical Sciences (AJUMS), Ahvaz, Iran d Department of Pharmacology, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences (AJUMS), Ahvaz, Iran e Department of Physiology, School of Medicine, Physiology Research Center, Ahvaz Jundishapur University of Medical sciences (AJUMS), Ahvaz, Iran b

art ic l e i nf o

a b s t r a c t

Article history: Received 19 November 2012 Received in revised form 6 March 2013 Accepted 7 March 2013 Available online 23 March 2013

The present study was conducted to evaluate the analgesic effects of p.o., i.p., or i.c.v. administration of ellagic acid (EA), and investigate the possible mechanisms underlying the systemic antinociceptive activities in different animal models of pain. Using radiant heat tail-flick test, EA (100–1000 μmol/kg, p. o.) only resulted in antinociception at 1000 μmol/kg. Also, EA (10–660 μmol/kg, i.p.) produced the antinociceptive effect in a dose-dependent manner with an ED50 of 122 μmol/kg. In addition, the i.c.v. administration of EA (0.1–2 μmol/rat) resulted in dose-dependent antinociception with an ED50 of 0.33 μmol/rat. EA induced antinociception (330 μmol/kg. i.p.) was reversed by naloxone (1 mg/kg, i.p.). Likewise, EA (1–33 μmol/kg, i.p.) produced significant dose-dependent antinociception when assessed using acetic acid-induced abdominal writhing test with an ED50 of 3.5 μmol/kg. It was also demonstrated that pre-treatment with L-arginine (100 mg/kg, i.p.), a nitric oxide (NO) precursor, and methylene blue (20 mg/kg, i.p.), a guanylate cyclase (GC) inhibitor, significantly enhanced antinociception produced by EA suggesting the involvement of L-arginine–NO–cGMP pathway. Additionally, administration of glibenclamide (10 mg/kg, i.p.), an ATP-sensitive K+ channel blocker, significantly reversed antinociceptive activity induced by EA. Moreover, EA treatment had no effect on the motor activity of rats when tested in rota-rod task. The present results indicate that the dose-related antinociceptive action of EA has both peripheral and central components which involve mediation by opioidergic system and L-arginine–NO– cGMP–ATP sensitive K+ channels pathway. & 2013 Elsevier B.V. All rights reserved.

Keywords: Ellagic acid Antinociception Nitric oxide Potassium channel Opioid receptor Tail-flick Writhing test

1. Introduction Phenolic compounds are secondary metabolites widely found in fruits, mostly represented by flavonoids and phenolic acids. They are usually referred to a diverse group of naturally occurring compounds with phenolic structural features (Tsao, 2010). They have synthetic, medicinal and industrial value. Naturally occurring polyphenols and or flavonoids are known to have numerous biological activities. They are found to be potential candidates for use as drugs, for example, in diseases like AIDS, heart ailments, ulcer formation, bacterial infection, mutagenesis and neural disorders. In contrast, little is known about the effects of polyphenols on the modulation of pain transmission (Handique and Baruah, 2002).

n Corresponding author at: Department of Pharmacology, School of Medicine, Atherosclerosis Research Center, Ahvaz Jundishapur University of Medical Sciences (AJUMS), Ahvaz, Iran. Tel.: +98 913 3178795; fax: +98 611 3332036. E-mail addresses: [email protected], [email protected], [email protected] (M. Taghi Mansouri).

0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.03.031

Ellagic acid (EA, 2,3,7,8-tetrahydroxybenzopyrano[5,4,3-cde]benzopyran-5-10-dione) is an excretion product of many plant species of economic importance, particularly fruits and nuts (Clifford and Scalbert, 2000; Wada and Ou, 2002). It is found in strawberries, cranberries, walnuts, pecans, and red raspberry seeds (Whitley et al., 2003). Also, this compound is a major component of pomegranate juice, an increasingly popular dietary supplement used by the American adult population (Corbett et al., 2010). It has been reported to show different pharmacological effects including chemoprevention (Townsend and Tew, 2003), inhibition of tumorigenesis (Buniatian, 2003), anti-inflammation and antioxidant (Festa et al., 2001; Lei et al., 2003; Solon et al., 2000), neuroprotection against diabetic neuropathy (Liu et al., 2011), inhibition of anaphylactic reaction in vivo and in vitro (Choi and Yan, 2009), and also inhibition of lipopolysaccharide-induced prostaglandin E2 synthesis in human monocytes (Karlsson et al., 2010). Few studies have investigated the antinociceptive effects of EA in vivo. Rogerio et al. (2006) examined the anti-inflammatory and antinociceptive effects of EA in animal models. Their findings

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showed that EA significantly decreased paw edema, as measured by calipers after an injection of 1% carrageenan, and the number of acid-induced writhing periods in mice. They suggested that the reduction in writhing periods may be via cyclooxygenase inhibition or another antinociceptive pathway. In 2008, Beltz et al. examined the antinociceptive effects of EA in the rat hot-plate model. Fischer et al. (2008) evaluated the antinociceptive potential of two derivatives of EA, 3,4,3′-trimethoxyflavellagic acid and 3,4,3′-trimethoxy flavellagic acid 4′-O-glucoside, in the formalin model of pain. They showed that these compounds inhibited both phases of formalin induced nociception. Additionally, Corbett et al. (2010) showed that EA also decreases paw edema induced by 3% carrageenan and may interact with known cyclooxygenase inhibitor, ketorolac. However, research studies have not clearly demonstrated the underlying mechanisms involved in the antinociceptive effects of EA. So, the primary aim of this study is to investigate the peripheral and central antinociceptive effects of EA using tailflick and writhing tests. The second one is to evaluate whether there are interactions among EA, the opioid receptors and also Larginine–NO–cGMP–K+ channel pathway in the EA-induced antinociception.

2. Materials and methods 2.1. Animals All animal care and experimental procedures were in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. We followed the ethical guidelines for investigations of experimental pain in conscious animals (Zimmermann, 1983), as well as our institutional guidelines for experiments with animals, designed to avoid suffering and limit the number of animals. The number of animals and intensities of noxious stimuli used were the minimum necessary to demonstrate consistent effects of drug treatments. Experiments were conducted using adult male Wistar rats (220 720 g) and also Swiss mice (25–30 g) purchased from the central animal house of the Jundishapur University of Medical Sciences (Ahvaz-Iran). They were housed at 2272 1C and 12 h light/dark cycles (light from 7:00 to 19:00 h) with free access to food and water ad libitum. All animals were randomly divided into groups of 8 in each, acclimatized and habituated to the laboratory environment for at least 1 week prior to the experiments and were used only once throughout the experiments. In all experiments, data were collected by a blinded, randomized and controlled design. 2.2. Drugs and chemicals The following drugs were used: Ellagic acid (EA), Nω-nitro-Larginine methyl ester hydrochloride (L-NAME), L-arginine hydrochloride (L-arginine), methylene blue, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Glibenclamide donated by Poursina Pharmaceutical Co. (Tehran, Iran). Naloxone hydrochloride and morphine sulfate (5H2O) were donated by Temad Pharmaceutical Co. (Tehran, Iran). All drugs were dissolved in normal saline (0.9% NaCl) and buffered to a pH of 7, while EA was dissolved in 10% DMSO/normal saline. Respective controls received only solvent vehicle. Drug concentrations were freshly prepared in such a way that the necessary dose could be injected in a volume of 5 ml/kg by both p.o. and i.p. routes unless otherwise stated in the method. The vehicle alone had no effects per se on the nociceptive responses. Doses and drug administration schedules were selected based on previous reports

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(Beltz et al., 2008; Rogerio et al., 2006) and on pilot experiments in our laboratory. For i.c.v. injections, a permanent guide cannula was implanted in the right ventricle of the brain as described by Stoppa et al. (2008). Briefly, each rat was anesthetized with a mixture of ketamine hydrochloride (50 mg/kg, i.p.) and xylazine (5 mg/kg, i. p.) and a 23-gauge, 12-mm stainless steel guide cannula was stereotaxically placed (Stoelting Stereotaxic Apparatus, Wood Lane, IL, USA) in the right lateral ventricle of the brain. The stereotaxic coordinates, according to Paxinos and Watson (2006), were: 0.8 mm posterior to the bregma, 2 mm lateral to the midline and 4 mm below the top of the skull. The guide cannula was anchored with jewelers' screws and dental acrylic cement. A 12.5 mm stylet was inserted into the guide cannula to keep it patent prior to injection. All animals were allowed 7 days to recover from surgery. Drugs or saline were delivered in a volume of 5 ml/rat. To ascertain the exact site of i.c.v. injection, some rats were injected i.c.v. with 5 ml of 1:10 diluted Indian ink and their brains were examined macroscopically after sectioning. The i.c.v. injections of the vehicle and EA (0.1–2 μM/rat) were performed using a 10-ml Hamilton syringe over a period of 30 s with a constant volume of 1 ml. After completion of each i.c.v. injection, the needle was left in place for further 30 s to facilitate infusion of the drug solution. The i.c.v. injections of the drugs were performed 10 min before the nociceptive test. 2.3. Antinociceptive analysis 2.3.1. Central nociceptive model induced by radiant heat stimulation in rats The antinociceptive effects of EA and the reference drug (morphine), represented by the time required for rat tail-flick after exposure to a source of radiant heat, were evaluated according to the description of D’Amour and Smith (1941). Briefly, each animal was placed in a Plexiglas box that allowed its tail to be free, then the box was placed on the tail stimulator analgesia meter (IITC Inc., USA) with the tail occluding a slit over a photocell for radiant heat stimulation generated by a power lamp mounted in a reflector. The tail-flick response was elicited by applying radiant heat to the point 1/3 of length away from the tip of the tail. When the rat felt pain and flicked its tail, the light of the lamp fell on the photocell such that the timer was automatically stopped. The intensity of the heat stimulus in the tail-flick test was adjusted so that the rat flicked its tail within 3–6 s. A 10 s cut-off time was set in order to prevent tissue damage. Before experiment, the heat stimulation latency of all animals were tested, and those with response time of <2 or >6 s to heat stimulation were excluded. The tail-flick responses were measured before, and 15, 45, 90, 150 and 210 min after the administration of EA (100–1000 μmol/kg, p.o.; 10–660 μmol/kg, i.p.; and 0.1–2 μmol/rat, i.c.v.), morphine (13.2 μmol/kg, i.p.), or respective vehicles. Tail-flick latencies were converted to % MPE (maximal possible effect) as follows: % MPE ¼100x(post drug latency-predrug latency)/(cut-off timepredrug latency). Each animal was used as its own control. 2.3.2. Visceral nociceptive model induced by acetic acid stimulation in mice All animals were acclimatized to laboratory environment for at least 2 h before testing. The abdominal writhing test induced by acetic acid stimulation in mice as originally described by Siegmund et al. (1957). Briefly, EA (1–33 μmol/kg) or the vehicle were intraperitoneally administrated 30 min prior to acetic acid injection. Immediately after intraperitoneal injection of 0.1 ml/10 g acetic acid (0.6% v/v) in normal saline (0.9% w/v NaCl), animals were isolated for observation. The numbers of abdominal writhing

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episodes, which consisted of contraction of the abdominal area with extension of hind legs, were recorded for 30 min, starting from 5 min after injection of acetic acid in each animal. Antinociceptive activity (reduction in writhes) was expressed as percent maximum possible effect (% MPE) that was calculated by the following equation: %MPE¼ [100  (mean of writhes in control group-mean of writhes in drug(s)-treated group)]/mean of writhes in control group (Jain et al., 2001). 2.4. Analysis of the possible mechanism of EA action 2.4.1. Involvement of L-arginine/NO pathway To investigate the role of nitric oxide-L-arginine pathway in the antinociception caused by EA in the acetic acid test, the mice were pre-treated with L-arginine (100 mg/kg, i.p.) and after 20 min., EA (10 μmol/kg, i.p.), L-NAME (30 mg/ kg, i.p.) or vehicle (10 ml/kg, i. p.) were administered as described previously with slight modifications (Abacioglu et al., 2000; Perimal et al., 2011). Nociceptive responses against acetic acid injection were observed for 30 min. The numbers of abdominal writhing were counted as indication of pain behavior. 2.4.2. Involvement of cyclic guanosine monophosphate (cGMP) pathway To verify the possible involvement of cGMP in the antinociceptive action caused by EA in the acetic acid test, the mice were pretreated with methylene blue (20 mg/kg) 15 min before administration of EA (10 μmol/kg, i.p.), as described previously with slight modification (Abacioglu et al., 2000; Perimal et al., 2011). Nociceptive responses against acetic acid injection were observed for 30 min. The numbers of abdominal writhing were counted as indication of pain behavior. 2.4.3. Involvement of ATP-sensitive K+ channel pathway Possible contribution of K+ channel in the antinociceptive effect of EA was evaluated using the method previously described (Mohamad et al., 2011; Perimal et al., 2011). The mice were pretreated with glibenclamide (10 mg/kg, i.p.) 15 min before injection of either vehicle (10 ml/kg, i.p.) or EA (10 μmol/kg, i.p.). The mice were challenged with i.p. injection of 0.6% v/v acetic acid, 30 min post-treatment. Following the injection of acetic acid, the animals were immediately placed in a Plexiglas chamber and the number of writhing was recorded for 30 min, starting from 5 min post injection. 2.4.4. Involvement of opioid receptors To evaluate the involvement of opioid receptors in the antinociceptive activity of EA, animals were pre-treated with naloxone (1 mg/kg, i.p.) as a non-selective opioid antagonist, 15 min before administration of EA (330 μmol/kg, i.p. and or 0.33 μmol/rat, i.c.v.) in rats using tail-flick test. The tail-flick responses were measured before, and 15, 45, 90, 150 and 210 min after the drug administration (Pini et al., 1997). 2.5. Rota-rod test The integrity of motor coordination was assessed on the basis of endurance time of the animals on the rotating rod according to Dunham and Miya (1957) accelerating from 0 to 40 revolutions/ 5 min, three times on the day prior to the experiment (Ugo Basile, Italy). On the day of the test, the performance time was measured before and 15, 30, 60 and 90 min after the drug or vehicle administration.

2.6. Statistical analysis The results were presented as mean 7S.E.M., except the effective dose 50 (ED50) values (i.e., the dose of EA reducing the nociceptive response by 50% relative to the control value), which were reported as geometric means accompanied by the irrespective 95% confidence limits. The ED50 value was determined by nonlinear regression from individual experiments using Graph Pad software (Graph Pad Prism 5, San Diego, CA, USA). The statistical analyses were performed by one-way ANOVA followed Tukey's post hoc test unless otherwise stated. A two-way ANOVA followed by Bonferroni's test was carried out for the time-course effect of EA. The difference was considered significant at 5% level.

3. Results 3.1. Tail-flick test 3.1.1. Antinociceptive effects of p.o. EA The mean baselines of tail-flick latency in the four groups of rats (8 each) were 3.12 70.18, 3.057 0.12, 2.88 70.17 and 2.727 0.17 s, respectively. There was no significant difference (P>0.05) between the mean baselines of tail-flick latency in the vehicle-treated and EA-treated rats. As shown in Fig. 1a, EA did not produce any dose-dependent antinociception following p.o. administration. Only at dose of 1000 μmol/kg, EA treatment showed significant antinociceptive activity as compared to the vehicle group (P<0.001), while the rest showed no significant difference. In the group with dose of 1000 μmol/kg, the effective antinociception started 15 min after EA administration, reached a peak after 45 min and lasted more than 100 min. 3.1.2. Antinociceptive effects of i.p. EA The mean baselines of tail-flick latency in the six groups of rats (8 each) were 3.22 70.18, 3.45 70.21, 2.91 70.19, 2.74 70.21, 3.217 0.22, and 2.79 70.19 s, respectively. There was no significant difference (P>0.05) between the mean baselines of tail-flick latency in the vehicle-treated and EA-treated rats. As shown in Fig. 1b, EA produced dose-dependent antinociception following i.p. administration. In the groups with larger doses 330 and 660 μmol/ kg, the effective antinociception started 15 min after EA administration, reached a peak after 45 min and lasted more than 100 min. The dose–response line for the i.p. administration of EA at the time of peak effects, 45 min, is presented in Fig. 2a, with ED50 (the dose of 50% increase of tail-flick latency) calculated to be 122 μmol/kg (95% C.L. 66.2–227 μmol/kg). Furthermore, morphine (13.2 μmol/ kg, i.p.) had significant antinociceptive effect as compared to EA (330 μmol/kg) showing more efficacy (more than 4-fold) than EA (Fig. 3). 3.1.3. Antinociceptive effects of i.c.v. EA EA was administered via the i.c.v. route at doses up to 2 μM per rat. The mean baselines of tail-flick latency in the six groups of rat (8 each) were 3.16 70.15, 3.057 0.18, 2.95 7 0.15, 3.35 70.28, 3.237 0.18 and 3.277 0.22 s, respectively. There was no significant difference (P>0.05) between the mean baselines of tail-flick latency in the vehicle-treated and EA-treated rats. The i.c.v. administration of EA resulted in dose-dependent antinociceptive effects according to Fig. 1c. Only 0.1 μmol/rat EA treatment showed no significant difference compared with the vehicle group, while the rest showed significant antinociceptive activity. In the groups with the doses of 0.33–2 μmol/rat, the antinociception started 15 min after EA administration, reached a peak after 45 min and lasted more than 200 min. The dose-response line for the i.c.v. administration of EA was plotted at the time of peak effects,

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Fig. 2. Dose-response curve of peak % MPE after (a) i.p. and (b) i.c.v. administration of EA in the rat tail-flick test. Ordinate shows percentage changes of tail-flick latency 45 min after EA administration. All points represent the mean 7S.E.M (n ¼8). The ED50 were 122 μmol/kg and 0.33 μmol/rat for i.p. and i.c.v. routes, respectively.

Fig. 1. Antinociceptive effects of EA following (a) p.o.; (b) i.p.; and (c) i.c.v. administration in the rat tail-flick test. Antinociception was determined by tailflick latency. Ordinate shows percentage of maximal possible effect (% MPE) from baseline level. All points represent the mean7 S.E.M. (n)P<0.05; (n)(n)P<0.01; (n)(n) (n ) P<0.001 (doses of EA vs. vehicle-treated group, n¼ 8).

45 min, and is shown in Fig. 2b, with ED50 calculated to be 0.33 μmol/rat (95% C.L. 0.28–0.40 μmol/rat).

3.1.4. Effects of i.p. naloxone on the antinociceptive effect induced by i.p. EA The mean baselines for tail-flick latency in the two groups (8 each) were 3.14 70.21 and 3.697 0.21 s, without a significant

Fig. 3. Comparison of antinociceptive effects of i.p. EA (330 μmol/kg) and morphine (MPH, 13.2 μmol/kg) in rats. Antinociception was determined by tail-flick latency in the tail-flick test. Ordinate shows percentage of maximal possible effect (% MPE) from baseline level. All points represent the mean 7S.E.M (n¼ 8). (n)(n)(n)P<0.001 vs. vehicle-treated group.

difference (P>0.05). As shown in Fig. 4a, the antinociceptive effect of EA was antagonized significantly by naloxone, starting 15 min after naloxone administration and lasting more than 80 min. The increase in tail-flick latency 45 min after the i.p. administration of EA were 14.8 72.4% and 4.1 70.8% in the saline-treated and the naloxone-treated groups, respectively. Moreover, it was found that naloxone (1 mg/kg, i.p.) itself did not alter tail-flick latency significantly over the 200 min observation period (data not shown).

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Fig. 5. Dose–response curve of % MPE after i.p. administration of EA on the inhibition of writhing response. All points represent the mean7 S.E.M. from 12 mice. The ED50 was 3.5 μmol/kg.

3.2.2. Involvement of L-arginine/NO pathway The involvement of L-arginine/NO pathway (Fig. 6a) was analyzed with the pre-treatment of nitric oxide precursor, Larginine (100 mg/kg, given 15 min prior to test). L-arginine which was administered at a dose that was not notably different from the control group, was able to significantly reverse the antinociceptive activity exhibited by NO synthase (NOS) inhibitor, L-NAME (30 mg/ kg, i.p.), and EA (10 μmol/kg, i.p.) in acetic acid-induced abdominal writhing test.

Fig. 4. Effect of i.p. naloxone (NLX, 1 mg/kg) on the antinociceptive effect induced by (a) i.p. and (b) i.c.v. EA (330 μmol/kg and 0.33 μmol/rat, respectively). NLX or NS (normal saline) was given i.p. 15 min before EA administration. Antinociception was determined by tail-flick latency in the tail-flick test. Ordinates show percentage of maximal possible effect (% MPE) from baseline level. All points represent the mean 7S.E.M (n¼ 8). (n)P<0.05; (n)(n)P<0.01; (n)(n)(n)P<0.001 vs. EA+NS group.

3.1.5. Effects of i.p. naloxone on the antinociceptive effect induced by i.c.v. EA The mean baselines for tail-flick latency in the two groups (8 each) were 3.24 70.18 and 3.29 70.22 s, without a significant difference (P>0.05). As shown in Fig. 4b, the antinociceptive effect of EA was antagonized significantly by naloxone, starting 15 min after naloxone administration and lasting more than 80 min. The increase in tail-flick latency 45 min after the i.p. administration of EA were 7.67 1.1% and 2.9 70.36% in the saline-treated and the naloxone-treated groups, respectively. Moreover, it was found that naloxone (1 mg/kg, i.p.) itself did not alter tail-flick latency significantly over the 200 min observation period (data not shown).

3.2.3. Involvement of cyclic guanosine monophosphate (cGMP) pathway EA (10 μmol/kg, i.p.) and methylene blue (20 mg/kg, i.p.) when administered alone, both significantly inhibited acetic acidinduced abdominal writhing. Given together, methylene blue significantly potentiated EA-induced antinociception (Fig. 6b). 3.2.4. Involvement of ATP-sensitive K+ channel pathway It was also found that glibenclamide (10 mg/kg, i.p.) administration alone did not alter abdominal writhing counts when assessed through the injection of 0.6% (v/v) acetic acid (Fig. 6c). Given together, antinociceptive activity of EA (10 μmol/kg, i.p.) was significantly reversed by glibenclamide. 3.3. Motor coordination The neuromuscular coordination was not significantly affected by i.p. administration of EA at doses of 10–100 μmol/kg, while at dose of 330 μmol/kg (i.p.) EA significantly decreased the endurance time on the rotating rod at 60 and 90 min after administration. Also, morphine (13.2 μmol/kg, i.p.) significantly decreased the endurance time on the rotating rod (Fig. 7).

4. Discussion 3.2. Acetic acid-induced abdominal writhing test 3.2.1. Antinociceptive effects of i.p. EA The results in Fig. 5 showed the effect of systemic administration of EA (1–33 μmol/kg, i.p.) in acetic acid-induced abdominal writhing test. EA exhibited significant dose-dependent inhibition on abdominal writhing when administered intraperitoneally with percentage of inhibition of 18.9, 41.1, 92.4 and 100%. The calculated mean ED50 for i.p. administration of EA was 3.5 μmol/kg (C.L. 95%, 2.88–4 μmol/kg).

The present study was undertaken to provide further pharmacological information about the site of action and the mechanisms involved in the antinociceptive effects of EA. The analgesic activity was assessed in rats and mice using two well-accepted pain models, namely the tail-flick and writhing tests. The tail-flick test has been reported to be useful to investigate central antinociceptive activity and refers predominantly to a spinal reflex with modest control by supraspinal structures. Additionally, this test is a validated model for opioid-derived analgesic compounds. Intraperitoneal injection of algogenic chemical agents (acetic acid)

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Fig. 6. Effects of pre-treatment with (a) L-arginine (100 mg/kg) on antinociceptive profiles of EA (10 μmol/kg) and L-NAME (30 mg/kg); (b) methylene blue (20 mg/kg); and (c) glibenclamide (10 mg/kg) in acetic acid-induced abdominal writhing test in mice. Each column represents the mean 7 S.E.M (n¼ 12–14). (n)(n)(n)P<0.001 when compared to control group; ###P<0.001 when compared to L-NAME or #P<0.05 and ##P<0.01 when compared to EA-treated group (one-way ANOVA followed by Tukey's post hoc test).

Fig. 7. Effect of i.p. EA on rotarod performance. EA (10–330 μmol/kg), morphine (MPH, 13.2 μmol/kg) and vehicle (Veh) were administered i.p. 30 min before recording the fall latency in rotarod performance. Ordinate shows percentage change of falling latency from baseline level. All points represent the mean 7 S.E.M. from eight rats. (n)P<0.05; (n)(n)P<0.01; (n)(n)(n)P<0.001 vs. baseline level.

usually produces a longer duration or tonic stimulus as compared with phasic pain tests, in which thermal thresholds are determined. This tonic pain test is very sensitive method to evaluate new molecules whose pharmacodynamic properties are unknown and also valuable to detect analgesic drugs acting on the peripheral nervous system (Collier et al., 1968; Chiang and Zhuo, 1989; LeBars et al., 2001). The results revealed that EA exhibited dose-dependent analgesic activity in rats in the radiant heat tail-flick test when administered via i.p. and i.c.v. routes. In contrast, orally administration of

EA inhibited the nociceptive response only at dose of 1000 μmol/ kg and this is due to low bioavailability of EA via oral route (Larrosa et al., 2010). In addition, it was also demonstrated that at the ED50 level, administration of EA via i.c.v. route was approximately 82-fold more potent than that of i.p. Also, the antinociceptive effect of EA was comparable to morphine (the positive control), since the latency time was significantly increased by morphine and EA. This effect is in agreement with the Beltz’s et al. (2008) report describing the antinociceptive effect of EA in the hot-plate test. Moreover, we found that while the i.p. injection of naloxone itself had no significant influence on the pain threshold, which is consistent with the data reported by Woolf (1980), it significantly counteracted the antinociceptive effect induced by i.p. and also i.c.v. EA. These results suggest that the central nervous system may be the primary site for EA to exert its analgesic effect and also the activation of opioid receptors and/or an increment of endogenous opioids might be involved in the antinociceptive effect of EA in the tail-flick model (Akil et al., 1984). In the writhing model, EA produced marked and also dosedependent antinociceptive effect. This was in agreement with Rogerio et al. (2006) study which reported the antinociceptive effect of EA in the acetic acid-induced abdominal writhing test. This is a good model of visceral pain and the algesic effects of acetic acid is due to liberation and increase the levels of several mediators such as histamine, serotonin, bradykinin, cytokines and eicosanoids in the peritoneal fluid. These mediators are able to increase vascular permeability, reduce the threshold of the nociception and stimulate nociceptive fibers (Deraedt et al., 1980; Ikeda et al., 2001). Additionally, involvements of different mechanisms such as noradrenergic and cholinergic systems as well as NO/ cGMP/PKG/ATP pathway have been suggested in the acetic acid nociceptive response. A growing amount of evidences point out the L-arginine–NO– cGMP–ATP-sensitive K+ channel pathway as a relevant factor for antinociceptive effect of many drugs. It seems that NO is involved

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in all three levels of pain pathway, which are the peripheral nerve fibers, dorsal horn of spinal cord and cerebral cortex where perception of pain is processed (Anbar and Gratt, 1997). NO is able to increase the concentration of cGMP which will lead to activation of potassium channels. The opening of these channels induces the membrane hyperpolarization and reduces the depolarization and action potential transmission abilities of neurons, thereby inducing analgesia (Nguelefack et al., 2010). In the same sense, Sachs et al. (2004) and Vale et al. (2007) have described that the NO/cGMP/ATP-sensitive K+ channel pathway is an additional mechanism for the action of some peripheral analgesics and this events' sequence would be considered as an antagonistic mechanism to the hyperalgesic state that occurs in inflammed tissues. Then, to further understand the mechanism of EA activity and determine the possible participation of this pathway in the analgesia induced by EA, its activity was evaluated in presence of L-arginine (a nitric oxide synthase substrate or a NO precursor), LNAME (a non-selective NOS inhibitor), methylene blue (a guanylyl cyclase inhibitor) and glibenclamide (an ATP-sensitive K+ channels blocker) in the writhing model. This design was based on the previous data showing the activation of the L-arginine–NO–cGMP– ATP sensitive K+ channel pathway in the antinociceptive effect of several compounds including some natural products (DecigaCampos et al., 2006; Lazaro-Ibanez et al., 2001; Taylor et al., 1998). The present study found that pre-treatment with L-arginine, at a dose that did not notably change acetic acid-induced nociception compared to the control, was able to significantly reverse the antinociceptive activity exhibited by L-NAME (30 mg/kg, i.p.) and EA (10 μmol/kg, i.p.) in acetic acid-induced abdominal writhing test. Furthermore, treatment with L-NAME produced significant inhibition of the effect observed following L-arginine pretreatment but was significantly higher as compared to L-NAME alone. These findings may possibly suggest the involvement of the L-arginine/NO pathway in the antinociceptive activity of EA. Previous studies have reported that NOS inhibitors reduced nociception caused by acetic acid (Larson et al., 2000; Meotti et al., 2006; Moore et al., 1993). Therefore, the inhibitory effect of L-NAME observed in the present study are in accordance with the notion that NO is one of the mediators of acetic acid induced nociception, justifying the use of this model. In addition, some previous studies reported that EA has potent inhibitory effect on NO production (Soliman and Mazzio, 1998; Umesalma and Sudhandiran, 2010). In addition to the above findings, NO also increases the level of cGMP through the activation of soluble guanylyl cyclase, which influences a wide range of physiological functions including pain and analgesia (Abacioglu et al., 2000). cGMP acts on the ion channels directly or through the activation of protein kinases and phosphodiesterases (Yu Xu et al., 1995). In order to further prove the involvement/inhibition of cGMP in EA-induced antinociception, methylene blue as a guanylyl cyclase inhibitor was administered prior to injection of acetic acid. It was demonstrated that methylene blue pre-treatment significantly enhanced the antinociceptive effect of EA. It has been suggested that methylene blue promotes antinociception by sequentially inhibiting peripheral NOS and GC, resulting in NO interference (Abacioglu et al., 2000). The activation or deactivation of nociresponsive neurons is dependent on the availability of cGMP. Intracellular cGMP concentration is regulated by the action of GCs and also the rate of degradation by cGMP-specific phosphodiesterases (Jain et al., 2001). Therefore, cGMP is very important for the function of nociceptors. NO activates the soluble GC, which in turn catalyzes the formation of cGMP from guanosine triphosphate, whereas cyclic GMP-specific phosphodiesterase catalyzes the hydrolysis of cGMP to GMP, thus consequently ending the signal transduction (Pyne et al., 1996). Our findings showed that the ability of EA to

inhibit the L-arginine-induced NO and cGMP increase, may partially explain the significant increase in the antinociceptive effect of EA (Desoky and Fouad, 2005). The present study also showed the involvement of ATPsensitive K+ channel activation in EA-induced antinociception. Administration of ATP-sensitive K+ channel antagonist, glibenclamide, significantly reversed the antinociceptive activity evoked by EA. There are many reports on the fact that glibenclamide specifically blockes ATP-sensitive K+ channels, with no effects on other types of K+ channels such as Ca2+ activated and voltagegated potassium channels (Alves and Duarte, 2002; Jesse et al., 2007). Therefore, the present findings might suggest that EA exerted its antinociceptive activity through the opening of ATPsensitive K+ channel that allows the efflux of K+ ion, thus leading to membrane repolarization and/or hyperpolarization state which reduces the membrane excitability (Lawson, 1996). Altogether, the present results provide strong evidence on the participation of Larginine/NO/cGMP/ATP-sensitive K+ channel pathway in EAinduced antinociception.

5. Conclusion As a conclusion, EA at doses that did produce any toxic and sedative effects, provides conclusive evidence on the mediation of opioid receptors and L-arginine/NO/cGMP/ATP-sensitive K+ channel pathway in EA-induced antinociceptive activity, apart from its ability to produce peripheral and central effects. Further studies indicating the mechanism(s) of EA-induced antinociception, are underway in our laboratory to fully comprehend the role of other receptors possibly involved. Taken together, EA might represent potential therapeutic options for the treatment of pain-related diseases.

Conflict of interest The authors declare that there is no conflict of interest.

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