Antinociceptive effect of leaf essential oil from Croton sonderianus in mice

Life Sciences 77 (2005) 2953 – 2963 www.elsevier.com/locate/lifescie

Antinociceptive effect of leaf essential oil from Croton sonderianus in mice F.A. Santos a, F.A. Jeferson a, C.C. Santos b, E.R. Silveira b, V.S.N. Rao a,T a

Departments of Physiology and Pharmacology, Federal University of Ceara´, C.P. 3157, 60430-270 Fortaleza, CE, Brazil Department of Organic and Inorganic Chemistry, Federal University of Ceara´, C.P. 3157, 60430-270 Fortaleza, CE, Brazil

b

Received 3 August 2004; accepted 2 May 2005

Abstract The leaf essential oil from Croton sonderianus (EOCS) was evaluated for antinociceptive activity in mice using chemical and thermal models of nociception. Given orally, the essential oil at doses of 50, 100 and 200 mg/kg produced significant inhibitions on chemical nociception induced by intraperitoneal acetic acid and subplantar formalin or capsaicin injections. However, it evidenced no efficacy against thermal nociception in hot-plate test. More prominent inhibition of acetic acid-induced writhing and capsaicin-induced hind-paw licking responses was observed at 100 and 200 mg/kg of EOCS. At similar doses, the paw licking behavior in formalin test was more potently suppressed during the late phase (20–25 min, inflammatory) than in early phase (0–5 min, neurogenic). The EOCS-induced antinociception in both capsaicin and formalin tests was insensitive to naloxone (1 mg/kg, s.c.), but was significantly antagonized by glibenclamide (2 mg/kg, i.p.). In mice, the essential oil (100 and 200 mg/kg) neither significantly enhanced the pentobarbital-sleeping time nor impaired the motor performance in rota-rod test, indicating that the observed antinociception is unlikely due to sedation or motor abnormality. These results suggest that EOCS produces antinociception possibly involving glibenclamidesensitive KATP+ channels, which merit further studies on its efficacy in more specific models of hyperalgesia and neuropathic pain. D 2005 Elsevier Inc. All rights reserved. Keywords: Croton sonderianus; Antinociception; Essential oil; Chemical nociception; Mouse

T Corresponding author. Tel.: +55 85 288 8341; fax: +55 85 288 8333. E-mail address: [email protected] (V.S.N. Rao). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.05.032

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Introduction Croton sonderianus Muell. Arg. (Euphorbiaceae), popularly known as bmarmeleiro pretoQ, is a widespread shrub largely grown in northeastern parts of Brazil. Species of this type provide wood to make fences; construct lobster traps or for other uses requiring a wood of great durability due to their high decay resistance. It is also used as fire wood due to the high content of essential oil that may vary from 0.5% to 1.5%, the leaves and barks are used as an infusion or simply chewed as a folk medicine for the treatment of gastrointestinal disturbances, rheumatism and headache (Mattos, 1999; Chaves and Reinhard, 2003). This plant is rich in clerodane and cleisthantane type diterpenes with diverse biological activity (McChesney et al., 1991). From the root extracts of C. sonderianus, two diterpene compounds, hardwickic and 3,4-secotrachylobanoic acids have been isolated and described to possess antibacterial and antifungal properties (McChesney et al., 1991). Essential oils in general exhibit antimicrobial, anti-inflammatory and analgesic properties (Pattnaick et al., 1997; Siani et al., 1999; Santos and Rao, 2000; Medeiros et al., 2003). Besides, they have an ecological role and they are found to possess attractant, repellent, feeding deteriorate, and ovipositional stimulant activities against various insect species (Enan, 2001). Essential oil and its constituents play a prominent role as flavoring agents in the food industry as fragrances for the perfume industry and in drug formulations by the pharmaceutical industry (Levison et al., 1994; De Vincenzi et al., 1996; Bruneton, 1999). Past studies have described the antinociceptive effect of essential oils obtained from some related species such as Croton cajucara Benth and Croton nepetaefolius Baill (marmeleiro vermelho) in mice (Bighetti et al., 1999; Abdon et al., 2002). Despite the popular use of leaf extracts/infusions of C. sonderianus in pain relief, there have been no published reports and therefore the present study aims to examine its leaf essential oil for possible analgesic activity in experimental models of nociception induced by chemical and thermal stimuli.

Material and methods Plant material C. sonderianus Muell. Arg. (Euphorbiaceae) leaves were collected during March 2003 from the municipal area of Caucaia, Ceara´ State of Brazil. A voucher specimen (#31423) has been deposited at the Herbarium Prisco Bezerra of the Federal University of Ceara´. Essential oil extraction and its major constituents The fresh leaves of C. sonderianus (290 g) were subjected to hydro-distillation for 2 h using a modified cleavenger type essential oil assay apparatus. The deep blue colored oil (yield, 3 ml) was separated and dried over anhydrous sodium sulphate and stored under refrigeration. The volatile components were analysed in a Hewlett-Packard 5971 by gas liquid chromatography (GLC), and GLC coupled to mass spectrometry (GLC/MS) in the following conditions: DB-5 column (30  0.25 mm); helium (1 ml/min; programmed temperature 35–180 8C (4 8C/min); 180–280 8C (20 8C/ min); injector temperature (250 8C) and detector temperature (200 8C). Identifications were made by comparison with MS literature data and by comparison of their Kovats indices.

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Animals Male Swiss mice (20–25 g) were used. Experimental groups consisted of 8 animals per group. They were housed at 22 F 2 8C under a 12 h light/12 h dark cycle and had free access to standard pellet diet (Purina chow) and tap water. The animals were deprived of food for 15 h before experimentation, but had free access to drinking water. Each animal was used only once for experimentation. The experimental protocols were approved by the Animal Care and Use Committee of our Institute and complied with the recommendations of International Association for the study of pain (Zimmermann, 1983). Drugs and chemicals The following drugs were used: morphine hydrochloride (Cristalia, RJ, Brazil), diazepam (Novaquimica, Brazil) pentobarbital sodium, capsaicin, acetyl-salicylic acid, chlorpromazine, naloxone, glibenclamide and acetic acid (Sigma Chemical Co. MO, USA). Morphine hydrochloride, chlorpromazine, acetyl salicylic acid, glibenclamide, pentobarbital sodium and naloxone were dissolved in physiological saline (0.9% NaCl), capsaicin was dissolved in 1% ethanol and 1% Tween 80 in saline (1:1:8). The vehicles used alone had no effects per se on the nociceptive responses in mice. Antinociceptive activity The antinociceptive activity of the essential oil of C. sonderianus (EOCS) was evaluated on chemical nociception in the test models of acetic acid-induced writhing (Koster et al., 1959), capsaicin- and formalin-induced hind paw licking (Santos and Calixto, 1997; Hunskaar and Hole, 1987) and on thermal nociception in hot-plate test (Eddy and Leimbach, 1953). In all the nocifensive tests, conscious (unanesthetized) mice were used. EOCS (50, 100 and 200 mg/kg) was administered orally, using an orogastric polyethylene cannula, in a volume of 10 ml/kg. Control groups were treated with a similar volume of vehicle that has been used to dilute EOCS. The dose selection for EOCS was based on our pilot experiments and doses b 50 mg/kg and N 200 mg/kg did not manifest significant antinociception. Also, the doses employed in this study were considered non-toxic since EOCS in doses up to 3.0 g/kg do not cause any behavioral impairment or overt toxicity in mice (unpublished observations). In acetic acid-induced nociception, groups of overnight fasted mice (n = 8) were treated orally with EOCS (50, 100 and 200 mg/kg), vehicle (2% Tween 80, 10 ml/kg) or acetylsalicylic acid (250 mg/ kg), 1 h before the administration of acetic acid (0.6%, 10 ml/kg, i.p.). The number of abdominal constrictions (writhings) was counted for each animal, starting 10 min after acetic acid injection over a period of 20 min. In formalin test, groups of mice were treated as above with EOCS (50, 100 and 200 mg/kg, p.o.) or vehicle (10 ml/kg, p.o.) and 60 min later, each mouse was given 20 Al of 1% formalin (in 0.9% saline, subplantar) into the right hindpaw. The duration of paw licking (s) as an index of painful response was determined at 0–5 min (early phase, neurogenic) and 20–25 min (late phase, inflammatory) after formalin injection. Morphine (7.5 mg/kg, s.c., 30 min before the test) pre-treated animals were included in the study as a positive control. In order to verify the possible mechanism of EOCS

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antinociception (100 mg/kg) animal groups pretreated with naloxone (1 mg/kg, s.c.) or glibenclamide (2 mg/kg, i.p.) were used. Naloxone and glibenclamide were administered 15 min before the EOCS or morphine. In capsaicin test, mice were pretreated with EOCS (50, 100 and 200 mg/kg, p.o.) or vehicle (10 ml/kg, p.o.) 60 min before the subplantar injection of capsaicin (1.6 Ag, 20 Al) into the right hind paw (Santos and Calixto, 1997). Morphine (7.5 mg/kg, s.c.)-treated animal group was included as a positive control. The amount of time each mouse spent (s) in licking the injected paw was recorded over the first 5-min period. In order to verify the possible involvement of endogenous opioids, and or the KATP+ channels in the antinociceptive activity, the essential oil was examined in groups of mice pretreated with naloxone (1 mg/kg, s.c.) or glibenclamide (2 mg/kg, i.p.). Naloxone and glibenclamide were administered 15 min before the EOCS or morphine. In thermal nociceptive test, the reaction time (time in seconds elapsed between placement and the animal starts to lick its hind paw or jumping as an index of painful response) in a hotplate (Ugo Basile, model-DS 37) maintained at 51 F 0.5 8C was measured before and after 30, 60, and 90 min of drug administration. Mice with baseline latencies of more than 15 s were eliminated from the study. Animal groups were treated with the vehicle (10 ml/kg, p.o.), EOCS (50, 100 and 200 mg/kg, p.o.) or morphine (7.5 mg/kg, s.c.) 60 min or 30 min (in case of morphine), before the hot-plate test. The cut-off time was set at 45 s. Pentobarbitone-induced sleeping time The sleeping time in mice was studied by a previously described method (Dandiya and Collumbine, 1959). Groups of mice (n = 8) were treated orally with EOCS (100 and 200 mg/kg), and vehicle (10 ml/kg) or chlorpromazine (10 mg/kg, i.m.), 60 min before the injection of sodium pentobarbitone (40 mg/kg, i.p.). The loss and regain of righting reflex was considered as the duration of sleep time in seconds. Rota-rod test To verify a possible effect on motor coordination, EOCS (100 and 200 mg/kg, p.o.) was examined on rota-rod apparatus (Rosland et al., 1990). The apparatus consisted of a horizontal bar with a diameter of 5 cm, subdivided into four compartments (INSIGHT, RT-2002, Brazil). The mice were placed on the bar rotating at a speed of 4 rpm and mice that were able to remain on the rod longer than 120 s were selected 24 h before the test. They were divided into four groups (n = 8 per group) and were treated as above with the vehicle or the mixture of a- and h-amyrin (100 and 200 mg/kg, p.o.), or the reference compound diazepam (1.0 mg/kg, i.p.). One hour following the treatments, each animal was tested on the rota-rod and the time of permanence (s) on the bar during a 2-min period was registered before (0 h), 1 and 2 h after drug administration. Statistical analysis The results were expressed as mean F SEM. Statistical significance was analysed by the one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. Values were considered statistically significant at P b 0.05.

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Results Essential oil constituents The results of GC-MS analysis on EOCS showed predominance of mono and sesquiterpenes. Sixteen constituents were identified, representing 81.33% of the whole oil. The levels of the 11 major components identified (73.72%) were the monoterpenes, h-phellandrene (6.16%) and 1,8-cineole (4.17%); and the sesquiterpenes, bicyclogermacrene (10.22%), (E)-caryophyllene (6.90%), E-calaminene (3.41%), (Z)-calaminene (10.86%), h-elemene (4.96%), germacrene d (4.77%), guaiazulene (8.31%), a-guaiene (6.67%), and spathulenol (7.29%). Among the remaining constituents (7.61%) germacrene A, germacrene B, a-humulene, a-pinene and sativene were detected in percentages ranging from 1.09% to 2.39%. Effect on nociception EOCS evidenced significant antinociception relative to the control group in all the test models of nociception induced by chemical agents. In acetic acid-induced writhing test, EOCS produced a doserelated inhibition on the mean number of writhes, when compared to vehicle-treated control group (Fig. 1). These were in the order of 48.38 F 5.92, 20.22 F 2.98, 20.25 F 3.00, and 14.12 F 1.10 s, respectively, for the controls and EOCS at the tested doses 50, 100 and 200 mg/kg. The positive control group treated with acetylsalicylic acid (250 mg/kg) also manifested significantly diminished number of writhes (18.49 F 3.30 s). In formalin test, vehicle-treated animals showed the mean licking times (s) of 69.50 F 5.28 in the first phase and 29.50 F 5.49 in the second phase (Fig. 2A). Pretreatment with the EOCS caused significant diminutions of both first phase (neurogenic) (49.87 F 6.06, 40.47 F 4.42, and 40.55 F 6.17 s) and second phase (inflammatory) (37.00 F 9.90, 2.15 F 1.27, and 3.22 F 1.73 s) pain responses, at the tested doses of 50, 100 and 200 mg/kg, respectively. Morphine (7.5 mg/kg), the reference standard also significantly suppressed the formalin-response at both phases (first phase, 24.75 F 6.65 and second phase, 1.71 F1.71 s). When used alone, naloxone (1 mg/kg, s.c.) and glibenclamide (2 mg/kg, i.p.), the respective opioid-

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Fig. 1. Effects of orally administered essential oil from Croton sonderianus (EOCS) and acetyl salicylic acid (ASA) on acetic acid-induced writhings in mice. The vehicle (Control, 10 ml/kg), EOCS (50, 100, and 200 mg/kg) or ACS (250 mg/kg) were administered orally, 1 h before the intraperitoneal administration of acetic acid (0.6%, 10 ml/kg) and the number of writhes were counted over a period of 20 min. Each column represents the mean F S.E.M. (n = 8). Asterisks indicate significant difference from control. ***P b 0.001 (ANOVA followed by Dunnett’s test).

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Fig. 2. The upper panel (A) shows the effects of essential oil from Croton sonderianus (EOCS) and morphine (Morph) on formalin-induced nociception in mice. The total time spent (s) in licking the injected hind-paw was measured in the early phase (0– 5 min, open column) and the late phase (20–25 min, closed column). The vehicle (Control, 10 ml/kg) or EOCS (50, 100, and 200 mg/kg) was administered orally and Morphine (7.5 mg/kg) subcutaneously. EOCS was administered 1 h before and morphine 30 min before the test. The effects of naloxone and glibenclamide on EOCS and morphine antinociception are shown in lower panel (B). Naloxone (Nalox, 1 mg/kg s.c.) or glibenclamide (Glib, 2 mg/kg, i.p.) were administered 15 min before EOCS or morphine. Each column represents the mean F S.E.M. (n = 8). Asterisks indicate significant difference from control. *P b 0.05; ***P b 0.01; ***P b 0.001; aP b 0.001 vs. control; bP b 0.001 vs. Morph; cP b 0.001 vs. EOCS (ANOVA followed by Dunnett’s test).

receptor and KATP+ channel antagonists failed to modify the formalin-induced nociceptive responses in a significant manner (Fig. 2B) (naloxone: first phase, 72.71 F 9.30 and second phase, 26.00 F 3.56; glibenclamide: first phase, 77.23 F 11.23 and second phase, 37.99 F 7.03). In combination studies, naloxone significantly antagonized only the morphine antinociception but not of EOCS (naloxone + morphine: first phase, 60.62 F 11.35 and second phase, 33.50 F 12.04; naloxone + EOCS (100 mg/kg): first phase, 50.75 F 6.29 and second phase, 5.62 F 2.26). In contrast, glibenclamide manifested significant antagonism only to EOCS but not to morphine (glibenclamide + EOCS: first phase, 64.97 F 7.10 and second phase, 34.51 F 3.44; glibenclamide + morphine: first phase, 37.08 F 13.04 and second phase, 3.79 F 1.69). Fig. 3A shows the antinociceptive effects of EOCS and morphine against capsaicin-induced nociception in mice. When compared to vehicle-treated controls (56.37 F 6.83 s), a dose-dependent decrease in the duration of paw licking was observed in mice pretreated with EOCS (18.00 F 4.88, 15.87 F 6.81, and 9.00 F 3.61 s, respectively for the doses of 50, 100 and 200 mg/kg). Morphine, the positive control used in the study also caused significant antinociception (2.43 F 1.63 s). The effects of naloxone (1 mg/kg, s.c.) and glibenclamide (2 mg/kg, i.p.) on the antinociceptive effect of morphine (7.5 mg/kg, s.c.) and EOCS (100 mg/kg) are shown in Fig. 3B. At the doses employed, naloxone and

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Fig. 3. The upper panel (A) shows the effects of essential oil from Croton sonderianus (EOCS) and morphine on Capsaicininduced paw licking response in mice. The vehicle (Control, 10 ml/kg) or EOCS (50, 100, and 200 mg/kg) was administered orally and Morphine (7.5 mg/kg) subcutaneously. EOCS was administered 1 h before and morphine 30 min before the subplantar injection of capsaicin (1.6 Ag, 20 Al) into the hind paw and the time in seconds (s) the animal licks the injected paw was noted over a period of 5 min. The lower panel (B) shows the pretreatment effects of naloxone (Nalox, 1 mg/kg, s.c.), Glibenclamide (Glib, 2 mg/ kg, i.p.) on EOCS and morphine antinociception. Glibenclamide and naloxone were administered 15 before EOCS or morphine administrations.Each column represents the mean F S.E.M. (n = 8). Asterisks indicate significant difference from control. ***P b 0.001; aP b 0.001 vs. control; bP b 0.001 vs. Morph; cP b 0.001 vs. EOCS (ANOVA followed by Dunnett’s test).

glibenclamide produced no per se effects on capsaicin-induced paw licking response (naloxone: 49.57 F 7.07 s; glibenclamide: 45.31 F 6.75 s). In combination studies, while naloxone pretreatment selectively antagonized the antinociceptive effect of morphine (morphine + naloxone: 44.06 F 7.27 s; EOCS + naloxone: 10.41 F 3.74 s), glibenclamide pretreatment abolished only the EOCS antinociception (morphine + glibenclamide: 5.86 F 2.82 s; EOCS + glibenclamide: 48.00 F 6.76 s). In the hot-plate test, the EOCS (50, 100 and 200 mg/kg) pretreatments showed no significant analgesia whereas the opioid agonist, morphine (7.5 mg/kg, s.c.) demonstrated significant antinociception at the time points of 30 and 60 min (Fig. 4). Effect on pentobarbital sleeping time The effects of EOCS and chlorpromazine on pentobarbitone sodium-induced sleeping times were as follows. Vehicle-treated controls: 31.00 F 1.92 s; EOCS at 100 and 200 mg/kg: 33.73 F 7.75 and

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Fig. 4. Effect of Croton sonderianus essential oil (EOCS) in the hot-plate test. The reaction time was mesured in seconds (s) before (0 min) and 30, 60 and 90 min after drug treatment. Horizontal axis shows time intervals (min) and the bars represent reaction time (s) in each animal group treated with vehicle (control) , EOCS 50 mg/kg , EOCS 100 mg/kg , EOCS 200 mg/kg or Morphine 7.5 mg/kg . Each column represents the mean F S.E.M. (n=8). Asterisks indicate significant difference from control. ***P b 0.001 (ANOVA followed by Dunnett’s test).

42.25 F 7.41 s, respectively; Chlorpromazine: 172.21 F16.34 s. Only chlorpromazine but not EOCS prolonged the sleeping time significantly produced ( P b 0.001). Effect in rota-rod test EOCS (100 and 200 mg/kg) did not affect the motor coordination in mice. The mean permanence time (s) of animals on rota-rod, obtained in EOCS-treated groups, were not statistically different from vehicle-treated control group at time points of 0, 1 and 2 h. Only diazepam (1 mg/kg, i.p.) but not either dose of EOCS-treatments significantly ( P b 0.01) affected the rota-rod performance (data not shown).

Discussion EOCS is a complex mixture of several terpenes and hydrocarbons, the principal components being the sesquiterpenes, which comprises a greater part of the oil. Among sesquiterpenes, hcaryophyllene, guaiazulene and h-elemene are largely present. In the present experiments, the essential oil demonstrated antinociceptive activity against chemical nociception induced in mice by intraperitoneal acetic acid, subplantar capsaicin or formalin. EOCS lacked efficacy against thermal nociception. A previous study reported the antinociceptive activity of an essential oil from a related species, C. nepetaefolius (EOCN) in acetic acid, and formalin tests of nociception (Abdon et al., 2002). However, EOCN but not EOCS demonstrated suppression of thermal nociception in mice, which may be due to variation in essential oil composition between the two species. EOCN presented large amounts of methyleugenol (10.3%) and 1,8-cineole (31.5%) that could explain its potential to suppress thermal nociception (Dallmeier and Carlini, 1981; Santos and Rao, 2000). Methyleugenol was absent in EOCS and further it showed only a very small amount of 1,8-cineole (4.17%).

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Acetic acid-induced writhing assay is used for detecting both central and peripheral analgesia whereas hot-plate assay is more sensitive to centrally acting analgesics (Fukawa et al., 1980). In formalin test, EOCS showed more potent inhibition on the second phase inflammatory response rather than on the first phase neurogenic response. Several reports suggest that both capsaicin and formalin-induced licking responses are mediated by release of the excitatory amino acid glutamate and by sensory neuropeptides like substance P released from sensory neurons at the spinal cord (Santos and Calixto, 1997; Otuki et al., 2001). Our results indicate that the EOCS is active in suppressing neurogenic as well as the inflammatory nociception. The antinociception caused by EOCS seems to be unrelated to motor impairment or sedation since mice tested in rota-rod and barbiturate-sleeping time tests showed no significant effects on these behaviors. Some of the active components monoterpenes and sesquiterpenes present in EOCS particularly, 1,8-cineole, guaiazulene, and h-caryophyllene possess anti-inflammatory, analgesic, and anti-oxidant properties (Santos and Rao, 2000; Tambe et al., 1996; Kourounakis et al., 1997; Trentin et al., 1999; Andre et al., 2004). Since guaiazulene and h-caryophyllene are the principal sesquiterpenes present in EOCS, we assume that these are responsible for the observed nociception in chemical tests. In a most recent study, Andre et al. (2004) have described the attenuating effect of some other sesquiterpenes like polygodiol and drimanial from Drymis winteri against formalin and capsaicin induced nociception. In the present study, due to complex nature of the essential oil, it would be difficult to attribute the observed activity to any single component present in it. This may equally be true for not obtaining a clear dose–response curve for the antinociceptive effect in tests of acetic acid and formalin. To verify the possible mechanisms by which EOCH produces antinociception, we have examined the effect of naloxone, an opioid antagonist and glibenclamide, a blocker of KATP+ channels. Interestingly, the data obtained in both formalin and capsaicin tests show that the antinociception produced by EOCS is not naloxone-sensitive but sensitive to glibenclamide, suggesting the involvement of KATP+ channels. In contrast, antinociception produced by morphine was found to be naloxone-sensitive and glibenclamideinsensitive. It is well known that the KATP+ channel openers such as levcromakalim and morphine induce cell hyperpolarization, decrease the intracellular Ca (2+) level and neurotransmitter release and thereby the antinociception (Ocana et al., 1990; Lohmann and Welch, 1999). Since naloxone failed to reverse the EOCS antinociception in formalin and capsaicin tests, mechanisms other than the opioid might be involved. Intraplantar injections of formalin as well as capsaicin evoke increases in primary afferent activity (Peterson et al., 1997; Ren et al., 2005). In primary afferent neurons, capsaicin activates vanilloid receptor (TRPV1) and causes accumulation of intracellular Ca2+, which is necessary for capsaicin-evoked transmitter release (Sakurada et al., 2003). EOCH antinociception may thus involve opening of glibenclamide-sensitive K+ channels, hyperpolarization of capsaicin-sensitive sensory afferent neurons and decreased release of neurotransmitters. Future studies using neonatal capsaicin-treated animals and immunohistochemical studies that show Fos-like immunoreactivity in spinal cord may help in elucidating the mechanism of EOCH antinociception. In the future, compounds that open K+ channels by direct activation may gain importance as effective pain relievers since these were shown to be very effective in models of acute and chronic pain (Ocana et al., 2004). In conclusion, the study demonstrates the antinociceptive activity of essential oil from C. sonderianus in the test models of chemical nociception induced by acetic acid, capsaicin and formalin, and further suggests that its antinociceptive action is unrelated to classical opioid receptor stimulation, but may be related to activation of ATP-gated K+ channels, which merits further studies regarding the precise site and mechanism of action.

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Acknowledgements The authors are grateful to CNPq, Brazil for financial support and fellowship awards.

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