Available online at www.sciencedirect.com Journal of Ethnopharmacology 114 (2007) 355–363 Antinociceptive action of ethanolic extract obtained from roots of Humirianthera ampla Miers Ana Paula Luiz a , Janaı́na D’Ávila Moura a , Flavia C. Meotti a , Giselle Guginski b , Cesar L.S. Guimarães c , Mariangela S. Azevedo c , Ana Lúcia S. Rodrigues d , Adair R.S. Santos a,∗ a Departamento de Ciências Fisiológicas, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, Florianópolis 88040-900, SC, Brazil b Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, Florianópolis 88040-900, SC, Brazil c Departamento de Quı́mica, Universidade Federal de Rondônia, Porto Velho, RO, Brazil d Departamento de Bioquı́mica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, Florianópolis 88040-900, SC, Brazil Received 2 March 2007; received in revised form 8 August 2007; accepted 12 August 2007 Available online 19 August 2007 Abstract Humirianthera ampla Miers is a member of the Icacinaceae family and presents great amounts of di and triterpenoids. These chemical constituents in roots of Humirianthera ampla sustain not only the ethnopharmacological use against snake venom, but also some anti-inflammatory and analgesic properties of the plant. In this study we investigated the antinociceptive action of the ethanolic extract (EE) from roots of the Humirianthera ampla in chemical and thermal models of pain in mice. The oral treatment with ethanolic extract dose-dependently inhibited glutamate-, capsaicin- and formalin-induced licking. However, it did not prevent the nociception caused by radiant heat on the tail-flick test. The ethanolic extract (30 mg/kg) caused marked inhibition of the nociceptive biting response induced by glutamate, (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (transACPD), N-methyl-d-aspartate (NMDA) and substance P. The antinociception caused by ethanolic extract was significantly attenuated by naloxone, l-arginine, WAY100635, ondansetron or ketanserin, but not by caffeine or naloxone methiodide. In conclusion, the ethanolic extract from roots of Humirianthera ampla produces antinociception against neurogenic and inflammatory models of nociception. The mechanisms of antinociception involve nitric oxide, opioid, serotonin and glutamate pathways. Therefore, our results support the ethnopharmacological use of the Humirianthera ampla against inflammatory and painful process caused by snake venom. © 2007 Published by Elsevier Ireland Ltd. Keywords: Humirianthera ampla; Icacinaceae; Antinociceptive action; Ethanolic extract 1. Introduction Humirianthera ampla Miers is a member of the Icacinaceae family that is popularly known as “surucucaı́na” or “surucuı́na”. Abbreviations: EE, ethanolic extract; l-NOARG, Nω -nitro-l-arginine; trans-ACPD, (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid; NMDA, N-methyl-d-aspartate; AMPA, ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; PBS, phosphate buffer saline; EAAs, excitatory amino acids; PGE2 , prostaglandin E2 ; TRPV, transient receptor potential vanilloid; NO, nitric oxide; MK-801, dizocilpine maleate; 5-HT, serotonin ∗ Corresponding author. Tel.: +55 48 3721 9352/9444; fax: +55 48 3721 9672. E-mail addresses: arssantos@ccb.ufsc.br, arssantos@ig.com.br (A.R.S. Santos). 0378-8741/$ – see front matter © 2007 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2007.08.016 The genus Humirianthera grows in a tropical clime and so, it is common in the South of America, more specifically in the Amazonia. The species Humirianthera ampla is greatly used in folk medicine against snake bite (Graebner et al., 2000). However, up to date there are no investigations supporting the pharmacological properties of this plant. Phytochemical studies carried out with plant of the genus Humirianthera revealed an abundant amount of diterpenoids (Kaplan et al., 1991), which is a feature of the plants belonging to the Icacinaceae family (Kaplan et al., 1991). The presence of chemical constituents of plants of genus Humirianthera sustains not only the antidote effect against snake venom (Mostafa et al., 2006), but also some anti-inflammatory properties (Hernandez- 356 A.P. Luiz et al. / Journal of Ethnopharmacology 114 (2007) 355–363 Perez et al., 1995; Liu and Lin, 2006). Indeed, diterpenoids and other terpenes have been intensively investigated because they counteract acute and chronic inflammation and pain (HernandezPerez et al., 1995; Fernandez et al., 2001; Yamashita et al., 2002; Spessoto et al., 2003; Liu and Lin, 2006). The roots of Humirianthera ampla are a rich source of terpenoids (Kaplan et al., 1991; Graebner et al., 2000). Studies of chemical constituents of this plant revealed the presence of the di and triterpenoids: annonalide, humirianthol, acrenol and lupeol. In addition, substances as ␤-sitosterol and glycosylated sitosterol were also isolated from the ethanolic extract (EE) from the roots of Humirianthera ampla (Graebner et al., 2000, 2002). The presence of these compounds might explain the exceptional effect of the Humirianthera ampla against inflammation and pain caused by snake bite and consequently validates the large use of this plant by the Amazonia native people in this condition. Considering the chemical constituents present in the roots of Humirianthera ampla as well as the ethnopharmacological employment of this plant, the present study examined the antinociceptive effects of the EE from roots of Humirianthera ampla in chemical and thermal models of nociception in mice. The nociception induced by formalin was chosen because it permits us to evaluate the effects of EE in neurogenic and inflammatory nociception. Additional studies investigated the effects of EE on glutamatergic system in the peripheral and spinal sites. The involvement of opioid, adenosinergic, serotonergic and nitric oxide–l-arginine pathways was also investigated. Finally, the effects of EE on learning and memory were studied regarding the effects of EE on the glutamatergic system. Brazil). A voucher specimen of Humirianthera ampla was deposited in the Herbarium of the Institute National of Research from Amazon (INPA) under the number 214579. The dried roots were triturated and extracted with ethanol, being stirred and macerated at room temperature (28 ± 2 ◦ C). The solvent was evaporated under reduced pressure, and the extract (yield 0.2–0.3%) was concentrated and stored in a freezer at −20 ◦ C until use. 2. Material and methods 2.4. Assessment of antinociceptive effect of EE from Humirianthera ampla 2.3. Drugs The following substances were used: formalin and morphine hydrochloride (Merck, Darmstadt, Germany); capsaicin, l-glutamic acid, naloxone hydrochloride, naloxone methiodide, l-arginine, d-arginine, N␻ -nitro-l-arginine (l-NOARG), caffeine and WAY100635 (Sigma Chemical Co., St. Louis, MO, USA); (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), N-methyl-d-aspartate (NMDA), ␣-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainic acid (kainate), substance P and ketanserin (Tocris Cookson, Bristol, UK); ondansetron hydrochloride (Cristália, São Paulo, Brazil). The ethanolic extract was obtained from the roots of Humirianthera ampla at the Departamento de Quı́mica, Universidade Federal de Rondônia, Brazil, as described above. Drugs were dissolved in 0.9% of NaCl solution. Glutamate, NMDA, trans-ACPD, AMPA, kainic acid, substance P and formalin were dissolved in phosphate buffer saline (pH 7.4). Capsaicin was dissolved in 5% ethanol:95% phosphate buffer saline (PBS). EE from Humirianthera ampla was dissolved in saline. 2.1. Animals Adult Swiss mice of either sex (25–35 g) and female Wistar rats (2-month-old, 250–300 g) were housed in single-sex cages under a 12-h light:12-h dark cycle (lights on at 6:00 h) in a controlled temperature room (22 ± 2 ◦ C). They had free access to food and water. The male and female mice were homogeneously distributed among groups. Mice and rats were acclimatized to the laboratory for at least 1 h before testing that was carried out during light cycle. The experiments were performed after the approval of the protocol by the local Institutional Ethics Committee. All experiments were carried out in accordance with the current guidelines for the care of laboratory animals and the ethical guidelines for investigations of experimental pain in conscious animals (Zimmermann, 1983). The number of animals and intensities of noxious stimuli used were the minimum necessary to demonstrate the consistent effects of the drug treatments. 2.2. Preparation of the ethanolic extract (EE) of Humirianthera ampla Miers The plants were collected in the Amazon (Parque Natural city of Porto Velho, Rondônia, Brazil) in the point S08◦ 41′ 09.9153′′ ; W063◦ 52′ 10.02′′ . Botanical material was classified by Mr. José Ferreira Ramos (Departamento de Botânica, INPA, Manaus, 2.4.1. Glutamate-induced nociception The procedure used was similar to that described previously (Beirith et al., 2002). A volume of 20 ␮l of glutamate (10 ␮mol/paw prepared in PBS solution) was injected intraplantary (i.pl.) in the ventral surface of the right hindpaw. Animals were observed individually for 15 min following glutamate injection. The amount of time they spent licking the injected paw was recorded with a chronometer and was considered as indicative of nociception. Animals were treated with EE of Humirianthera ampla (3–300 mg/kg) by oral route (p.o.) 1 h before glutamate injection. Control animals received a similar volume of vehicle (10 ml/kg). In separate series of experiments, we investigated the timecourse of the antinociceptive effect of EE from Humirianthera ampla (30 mg/kg) given by oral route at the time points 1, 2, 4, 6, 8, 10 and 12 h before glutamate administration. Control animals received a similar volume of vehicle by p.o. (10 ml/kg) route and were observed in the same intervals of time. 2.4.2. Formalin-induced nociception The procedure used was essentially the same as that described previously (Hunskaar and Hole, 1987; Tjølsen et al., 1992). Animals received 20 ␮l of a 2.5% formalin solution (0.92% formaldehyde, in saline) in the ventral surface of the right hind- A.P. Luiz et al. / Journal of Ethnopharmacology 114 (2007) 355–363 paw (i.pl.). Animals were observed from 0 to 5 min (neurogenic phase) and from 15 to 30 min (inflammatory phase) and the time that they spent licking the injected paw was recorded and considered as indicative of nociception. Animals received EE from Humirianthera ampla (30–500 mg/kg, p.o.) 1 h beforehand. Control animals received vehicle (10 ml/kg, p.o.). 2.4.3. Capsaicin-induced nociception To provide direct evidence concerning the antinociceptive effect of the Humirianthera ampla on neurogenic nociception, the EE of Humirianthera ampla was tested against capsaicininduced licking in the mouse paw. The procedure used was similar to that described previously (Sakurada et al., 1992; Santos and Calixto, 1997). After an adaptation period (1 h), 20 ␮l of capsaicin (1.6 ␮g/paw prepared in saline) was injected in the ventral surface of the right hindpaw. Animals were observed individually for 5 min following capsaicin injection. The amount of time they spent licking the injected paw was recorded and considered as indicative of nociception. Animals were treated with EE from Humirianthera ampla (3–300 mg/kg, p.o.) 1 h before capsaicin injection. Control animals received vehicle (10 ml/kg). 2.4.4. Intrathecal injection of excitatory amino acids (EAAs) and substance P induced pain behavior in mice The intrathecal (i.t.) injection of excitatory amino acids and substance P was carried out as described previously (Hylden and Wilcox, 1980; Scheidt et al., 2002; Gadotti et al., 2006) The EE from Humirianthera ampla (30 mg/kg) was given by oral route 1 h beforehand. Injections were given to fully conscious mice using the method described by Hylden and Wilcox (1980). The mice were restrained manually, and a 30-gauge needle attached to a 25-␮l microsyringe, was inserted through the skin and between the vertebras into the subdural space of the L5–L6 spinal segments. A volume of 5 ␮l of the EAAs, substance P or vehicle was given over a period of 5 s and the nociceptive response was elicited by glutamate (an excitatory amino acid, 175 nmol/site), AMPA (a selective agonist of AMPA-subtype of glutamatergic ionotropic receptors, 135 pmol/site), NMDA (a selective agonist of NMDA-subtype of glutamatergic ionotropic receptors, 450 pmol/site), kainate (a selective agonist of kainatesubtype of glutamatergic ionotropic receptors, 110 pmol/site), trans-ACPD (an agonist of metabotropic glutamate receptors, 50 nmol/site), substance P (0.1 nmol/site) or saline. The amount of time that animal spent biting was timed and considered as indicative of nociception. In this regard, a bite was defined as a single head movement directed at the flanks or hind limbs, resulting in contact of the animal’s snout with the target organ. The behavioral nociception was evaluated immediately following local post-injections of each agonist: glutamate, 3 min; AMPA, 1 min; NMDA, 5 min; kainite, 4 min; trans-ACPD, 15 min; and SP, 6 min as described previously (Scheidt et al., 2002). 2.4.5. Tail-flick test On the tail-flick test, a radiant heat analgesiometer was used to measure response latencies according to the method described previously by D’Amour and Smith (1941), with 357 minor modifications. Mice responded to a focused heat stimulus (90 W) by flicking or removing their tail, exposing a photocell in the apparatus immediately under it. An automatic cut-off of 30 s was used to minimize tissue damage. The latency for removing the tail was quantified by an average of three measurements. The mice were tested before administration of drugs in order to obtain the baseline latency. Animals were treated with EE (10–100 mg/kg, p.o.) or with vehicle (10 ml/kg, p.o.) 1 h beforehand or morphine (10 mg/kg) subcutaneously (s.c.) 30 min beforehand. 2.5. Measurement of motor performance and locomotor activity To evaluate some non-specific muscle-relaxant or sedative effects of EE from Humirianthera ampla, mice were submitted to the rota-rod task (Santos et al., 1999) and open-field test (Rodrigues et al., 1996). The rota-rod apparatus (Ugo Basile, Model 7600) consisted of a bar with a diameter of 2.5 cm, subdivided into four compartments by disks 25 cm in diameter. The bar rotated at a constant speed of 17 revolutions per min. The animals were selected 24 h previously by eliminating those mice which did not remain on the bar for three consecutive periods of 60 s. Animals were treated with EE from Humirianthera ampla (30 or 500 mg/kg, p.o.) or vehicle (10 ml/kg, p.o.), 1 h before the test. The results are expressed as the time in sets for which animals remained on the rota-rod. The cut-off time used was 60 s. The ambulatory behavior was assessed in the open-field test as described previously (Rodrigues et al., 1996). The apparatus consisted of a wooden box measuring 40 cm × 60 cm × 50 cm. The floor of the arena was divided into 12 equal squares, and the number of squares crossed with all paws (crossings) was counted in a 6-min session. Mice were treated with EE from Humirianthera ampla (30 or 500 mg/kg, p.o.) or vehicle (10 ml/kg, p.o.) 1 h beforehand. 2.6. Step-down inhibitory avoidance Rats were trained in an inhibitory avoidance paradigm, a hippocampal-dependent learning task in which stepping-down from a platform present in a given context is associated with a foot shock resulting in an increase in step-down latency (Bernabeu et al., 1997; Cammarota et al., 2004). The step-down apparatus consists of a 50 cm × 25 cm × 25 cm Plexiglas box with a platform on the left end of a series of bronze bars that make the floor of the box. In the training session (day 1), animals were gently placed on the platform facing the left rear corner of the training box. When they stepped down and placed their four paws on the grid, the animals received a 0.4 mA 1 s scrambled foot shock and were immediately withdrawn from the training box. The animals were treated with EE of Humirianthera ampla (30 mg/kg, p.o.) or saline (10 ml/kg, p.o.) soon training (retention memory) or 1 h before training (acquisition memory). Short-term memory (STM) and long-term memory (LTM) were evaluated in step-down inhibitory avoidance test 358 A.P. Luiz et al. / Journal of Ethnopharmacology 114 (2007) 355–363 sessions carried out 1.5 and 24 h after training, respectively. At test, trained animals were put back on the training box platform until they eventually stepped down to the grid. The latency to step-down was taken as an indicator of memory retention. A 180 s ceiling was imposed on step-down latency during test sessions. ampla, mice were pretreated with caffeine (3 mg/kg, i.p., a nonselective adenosine receptor antagonist). After 20 min the mice received EE from Humirianthera ampla (30 mg/kg, p.o.) or vehicle (10 ml/kg, p.o.). The nociceptive response to the glutamate intraplantar injection was recorded 1 h after administration of EE or vehicle. 2.7. Analysis of possible mechanism of action of EE 2.8. Statistical analyses The mechanisms by which EE from Humirianthera ampla causes antinociception were evaluated employing the model of nociception induced by glutamate. The doses of the antagonists, agonists and other drugs were selected with basis on the literature (Santos et al., 1999, 2005; Kaster et al., 2005; Meotti et al., 2006; Pietrovski et al., 2006) or in previous results from our laboratory. The results are presented as mean ± S.E.M., except the ID50 values (i.e., the dose of EE reducing the nociceptive response by 50% relative to the control value), which are reported as geometric means accompanied by their respective 95% confidence limits. The ID50 value was determined by linear regression from individual experiments using linear regression GraphPad software (GraphPad software, San Diego, CA, USA). The statistical analyses were performed by one-way ANOVA followed by Newman–Keuls’ test or ttest when appropriated. A one-way ANOVA with repeated measures was carried out for the time-course effect of the Humirianthera ampla. P-values less than 0.05 (P < 0.05) were considered as indicative of significance. The step-down inhibitory avoidance is presented as median ± interquartile range and the data were analyzed by Kruskal–Wallis non-parametric test followed by Dunn’s post hoc comparisons. 2.7.1. Involvement of opioid system To assess the participation of the opioid system, mice were pretreated intraperitoneally (i.p.) with naloxone (1 mg/kg, a nonselective opioid receptor antagonist) or naloxone methiodide (1 mg/kg, s.c., a non-selective opioid receptor antagonist not permeable into the blood-brain barrier). After 20 min the animals received EE (30 mg/kg, p.o.), morphine (2.5 mg/kg, s.c.) or vehicle (10 ml/kg, p.o.). The nociceptive response to the glutamate intraplantar injection was recorded 1 h after administration of EE or vehicle and 30 min after administration of morphine. Another group of mice was pretreated with vehicle and after 20 min, received EE, morphine or vehicle, 1, 0.5 and 1 h before glutamate injection, respectively. 3. Results 3.1. Glutamate-induced nociception 2.7.2. Involvement of l-arginine–nitric oxide pathway To investigate the role played by the l-arginine–nitric oxide pathway in the antinociception caused by EE from Humirianthera ampla, mice were pretreated with l-arginine (40 mg/kg, i.p., a nitric oxide precursor) or d-arginine (40 mg/kg, i.p., an inactive isomer of l-arginine). After 20 min the mice received the EE from Humirianthera ampla (30 mg/kg, p.o.), N␻ -nitrol-arginine (25 mg/kg, i.p., a nitric oxide inhibitor) or vehicle (10 ml/kg, p.o.). The nociceptive response to the glutamate intraplantar injection was recorded 1 h after administration of EE or vehicle and 30 min after administration of l-NOARG. The oral administration of EE from Humirianthera ampla produced marked and dose-dependent attenuation of the glutamate-induced nociception. The ID50 value (and its respective 95% confidence limits) was 19.9 (15.6–25.2) mg/kg. The peak of inhibition was 71 ± 5% at 300 mg/kg (Fig. 1A). A timecourse analysis of the antinociceptive effect of EE given by p.o. route is shown in Fig. 1B. EE produced marked antinociception as early as 1 h after p.o. administration, an action that remained significant up to 10 h after the administration. Thus, the time point of 1 h was chosen for all further studies with independent groups of animals. 2.7.3. Involvement of serotonergic system The participation of the serotonergic system in the antinociceptive action of EE of Humirianthera ampla was investigated using WAY100635 (0.1 mg/kg, i.p., a selective 5-HT1A receptor antagonist), ketanserin (1 mg/kg, i.p., a selective 5-HT2A receptor antagonist), ondansetron (0.5 mg/kg, i.p., a 5-HT3 receptor antagonist) or vehicle (10 ml/kg, i.p.). After 20 min the mice received EE (30 mg/kg, p.o.) or saline, 1 h before glutamate intraplantar injection. 3.2. Formalin-induced nociception 2.7.4. Involvement of adenosinergic system To investigate the role played by the adenosinergic systems in the antinociception caused by EE of Humirianthera The results depicted in Fig. 2A and B show that the EE from Humirianthera ampla (30–500 mg/kg, p.o.) caused a significant inhibition of both neurogenic (0–5 min) and inflammatory (15–30 min) phases of formalin-induced licking. However, its antinociceptive effects were significantly more pronounced against the second phase of this model of pain. The calculated mean ID50 value (and its respective 95% confidence limits) for these effects were: >500 and 144.5 (119.3–175.1) mg/kg and the inhibitions observed were 31 ± 11 and 97 ± 2% at a dose of 500 mg/kg, for first and second phase, respectively. A.P. Luiz et al. / Journal of Ethnopharmacology 114 (2007) 355–363 359 Fig. 1. (A) Dose–response and (B) time-course of the antinociceptive effect of the oral treatment with ethanolic extract from Humirianthera ampla against glutamate (10 ␮mol/paw)-induced nociception in mice. Each column represents the mean of 6–8 animals and vertical lines indicate the S.E.M. The asterisks denote the significance levels, when compared with control groups C, * p < 0.05 and *** p < 0.001 by one-way ANOVA followed by Student–Newman–Keuls test. Fig. 2. Effects of the ethanolic extract from Humirianthera ampla administered orally against formalin-induced licking (early phase, panel A, and late phase, panel B) in mice. Each column represents the mean of 6–8 animals and vertical lines indicate the S.E.M. The asterisks denote the significance levels, when compared with control groups C, * p < 0.05 and *** p < 0.001 one-way ANOVA followed by Student–Newman–Keuls test. 3.3. Capsaicin-induced nociception The results depicted in Fig. 3 show that EE from Humirianthera ampla produced marked and dose-related inhibition of the capsaicin-induced neurogenic pain in mice. The ID50 value (and its respective 95% confidence limits) was 20.0 (17.4–23.0) mg/kg. The maximal inhibition was 80 ± 4% at a dose of 300 mg/kg. Fig. 3. Effects of the ethanolic extract from Humirianthera ampla administered orally against capsaicin (1.6 ␮g/paw)-induced nociception in mice. Each column represents the mean of 6–8 animals and vertical lines indicate the S.E.M. The asterisks denote the significance levels, when compared with control groups C, *** p < 0.001 by one-way ANOVA followed by Student–Newman–Keuls test. 3.4. Effect of EE on spinal excitatory amino acids- and substance P-induced biting response The results depicted in Fig. 4 show that EE of Humirianthera ampla (30 mg/kg) inhibited the nociceptive responses induced by spinal injections of glutamate, trans-ACPD, NMDA and substance P in mice. The inhibition values were 84 ± 2, 41 ± 10, 55 ± 8 and 53 ± 9%, respectively. In contrast, EE had no effect against AMPA- and kainate-mediated biting responses (Fig. 4). Fig. 4. Effect of the ethanolic extract from Humirianthera ampla administered orally on biting response caused by intrathecal injection of glutamate (175 nmol/site), trans-ACPD (50 nmol/site), NMDA (450 pmol/site), AMPA (135 pmol/site), kainate (110 pmol/site) and substance P (SP, 100 pmol/site) in mice. Each column represents the mean of 6–8 animals and vertical lines indicate the S.E.M. The asterisks denote the significance levels, when compared with untreated groups, * p < 0.05 and *** p < 0.001 by paired t-test. 360 A.P. Luiz et al. / Journal of Ethnopharmacology 114 (2007) 355–363 Fig. 5. Effect of pretreatment of animals with naloxone (1 mg/kg, panel A) or naloxone methiodide (1 mg/kg, panel B) on the antinociceptive profiles of ethanolic extract from Humirianthera ampla (30 mg/kg, p.o.) and morphine (2.5 mg/kg, s.c.) against the glutamate-induced licking in mice. Each column represents the mean of 6–8 animals and vertical lines indicate the S.E.M. The symbols denote the significance levels ** p < 0.01 and *** p < 0.001 when compared with control groups; # p < 0.001 when compared with morphine or ethanolic extract treated group by one-way ANOVA followed by Student–Newman–Keuls test. 3.5. Tail-flick test The EE from Humirianthera ampla (10–100 mg/kg) did not alter the latency response to the tail-flick test. In contrast, morphine (10 mg/kg, s.c.) caused a significant increase in the latency response (results not shown). 3.6. Evaluation of motor performance and locomotor activity The EE from Humirianthera ampla (30 and 500 mg/kg) did not affect the motor performance on the rota-rod task and locomotor activity in the open-field test when compared with animals that received vehicle (control group). The means ± S.E.M. on the rota-rod task were 60.0 ± 0.00; 60.0 ± 0.00 and 56.67 ± 3.33 s for the control, 30 and 500 mg/kg group, respectively. In the locomotor activity the means ± S.E.M. of crossings number were 169.5 ± 11.69; 190.5 ± 16.53 and 171.5 ± 13.51 for the control, 30 and 500 mg/kg group, respectively. tion of EE against glutamate-induced pain (Fig. 5B). However, the antinociception produced by morphine was significantly reversed (Fig. 5B). The systemic pretreatment of mice with the nitric oxide precursor l-arginine (40 mg/kg, i.p.), but not with d-arginine (40 mg/kg, i.p., an inactive isomer of l-arginine), completely reversed the antinociception caused by l-NOARG (25 mg/kg, i.p., a nitric oxide inhibitor) when analyzed against glutamateinduced nociception (Fig. 6). Under the same conditions, l-arginine significantly reversed the antinociception caused by EE from Humirianthera ampla (Fig. 6). The results depicted in Fig. 7 show that the previous treatment of mice with WAY100635 (0.1 mg/kg, i.p., a selective 5-HT1A receptor antagonist), ketanserin (1 mg/kg, i.p., a selective 5-HT2A receptor antagonist) or ondansetron (0.5 mg/kg, i.p., a selective 5-HT3 receptor antagonist), significantly reversed 3.7. Inhibitory avoidance training The EE from Humirianthera ampla (30 mg/kg, p.o.) did not affect the acquisition or retention of the memory in rats submitted to the step-down inhibitory avoidance task. Furthermore, EE at the same dose did not alter the short-term memory and long-term memory (data not shown). 3.8. Analysis of mechanism of action of EE The results presented in Fig. 5A show that the pretreatment of mice with naloxone (1 mg/kg, i.p., a non-selective opioid receptor antagonist), given 20 min beforehand, completely reversed the antinociception of EE from Humirianthera ampla (30 mg/kg, p.o.) and morphine (2.5 mg/kg, s.c.) (Fig. 5A). The systemic treatment of mice with naloxone methiodide (1 mg/kg, s.c., a non-selective opioid receptor antagonist that is not permeable into the blood brain barrier) did not reverse the antinocicep- Fig. 6. Effect of pretreatment of animals with l-arginine (40 mg/kg i.p.) or darginine (40 mg/kg i.p.) on the antinociceptive profiles of ethanolic extract of Humirianthera ampla (30 mg/kg, p.o.) and l-NOARG (25 mg/kg, i.p.) against the glutamate-induced licking in mice. Each column represents the mean of 6–8 animals and vertical lines indicate the S.E.M. The symbols denote the significance levels ** p < 0.01 when compared with control groups; # p < 0.001 when compared with l-NOARG or ethanolic extract treated group by one-way ANOVA followed by Student–Newman–Keuls test. A.P. Luiz et al. / Journal of Ethnopharmacology 114 (2007) 355–363 Fig. 7. Effect of pretreatment of animals with WAY100635 (0.1 mg/kg, i.p.), ketanserin (1 mg/kg, i.p.) or ondansetron (0.5 mg/kg, i.p.) on the antinociceptive profiles of ethanolic extract of Humirianthera ampla (30 mg/kg, p.o.) against the glutamate-induced licking in mice. Each column represents the mean of 6–8 animals and vertical lines indicate the S.E.M. The symbols denote the significance levels *** p < 0.001 when compared with control groups; # p < 0.001 and ## p < 0.001 when compared with ethanolic extract treated group by one-way ANOVA followed by Student–Newman–Keuls test. the antinociception caused by EE (30 mg/kg, p.o.) against glutamate-induced nociception. The systemic pretreatment of mice with caffeine (3 mg/kg, i.p., a non-selective adenosine receptor antagonist) did not significantly reverse the antinociception caused by EE from Humirianthera ampla (30 mg/kg, p.o.) against glutamateinduced nociception (results not shown). 4. Discussion In the present study, we demonstrated that the oral treatment of mice with EE from roots of the Humirianthera ampla caused potent antinociception against the chemical agents of nociception: formalin, glutamate and capsaicin. Hence, the chemical models of nociception here employed gave us an approach about the pathways involved on the antinociception of the Humirianthera ampla. It is worth to mention that Humirianthera ampla produced antinociception against both neurogenic and inflammatory phases of formalin. However, the effect was more pronounced against the inflammatory phase. The formalin-induced nociception is a well-described model of nociception and can be consistently inhibited by typical analgesic and anti-inflammatory drugs, including morphine, indomethacin and dexamethasone (Hunskaar and Hole, 1987). Considering the inhibitory property of Humirianthera ampla on the second phase of formalin, we might suggest an antiinflammatory action of the plant extract. In fact, the chemical constituents present in the roots of the Humirianthera ampla: diterpenoids, triterpenoids and sterols have been reported as potent anti-inflammatory agents (Hernandez-Perez et al., 1995; Fernandez et al., 2001; Yamashita et al., 2002; Spessoto et al., 2003; Liu and Lin, 2006). 361 The EE from Humirianthera ampla strongly inhibited the nociception induced by capsaicin and glutamate. Doses from 10 and 30 mg/kg significantly inhibited the capsaicin and glutamate-induced licking, whereas doses of 100 and 500 mg/kg were necessary to inhibit the inflammatory and neurogenic phases of formalin. This suggests a straight action of Humirianthera ampla on pathways dependent on the glutamatergic and vanilloid systems. Hence, an effect of the plant extract directly on the receptors or second messengers related to these transmitters could avoid the nociceptive response. Interestingly, the Humirianthera ampla antinociception was extended up to 10 h after the treatment, an effect that is hardly reached for analgesic clinically used. The licking response induced by formalin, capsaicin and glutamate results from a combination of peripheral input and spinal cord sensitization (Tjølsen et al., 1992; Santos and Calixto, 1997; Beirith et al., 2002; Sakurada et al., 2003). The intraplantar injection of formalin, capsaicin or glutamate releases EAAs, PGE2 , NO and kinins in the spinal cord (Tjølsen et al., 1992; Santos and Calixto, 1997; Beirith et al., 2002; Sakurada et al., 2003). Taking this into account, the antinociception of Humirianthera ampla could be dependent on either peripheral or central sites of action. Indeed, the systemic treatment of mice with EE from Humirianthera ampla consistently inhibited the nociception caused by spinal administration of glutamate and neurokinin receptor agonists. Interestingly, Humirianthera ampla inhibited specifically the nociception induced by NMDA, trans-ACPD and substance P receptor agonists. Therefore, it is plausible that some constituents of Humirianthera ampla may achieve the central nervous system and interact with the pathways dependent on activation of neurokinin-1, NMDA and metabotropic glutamate receptor, without interacting with kainate and AMPA receptors. The NO production is greatly dependent on the NMDA receptor activation. Hence, NMDA and substance P act synergistically to promote neuronal excitability in dorsal horn through mechanisms that involve NO (Sakurada et al., 1996; Liu et al., 1997; Ji and Strichartz, 2004; Caruso et al., 2005). As a result, our and other findings showed that the block of NO production efficiently decreased nociceptive transmission (Beirith et al., 2002). Here, we clearly demonstrated that the synthesis of NO from larginine prevented the antinociceptive effect of Humirianthera ampla. Hence, the inhibition of NO production/release is an important step for the antinociceptive action of Humirianthera ampla and may contribute for the plant effects against substance P, glutamate, capsaicin and formalin-induced nociception. Concerning the central effects of Humirianthera ampla on the glutamatergic system, non-specific effects, such as ataxia, motor disability, memory impairment and depression of the central nervous system could also be displayed by the plant extract (Coderre and Van Empel, 1994; Izquierdo and Medina, 1997; Vinadé et al., 2004). Here, we observed that EE from Humirianthera ampla did alter motor performance, locomotor activity and memory in the doses that caused significant antinociception. The involvement of opioid system on the antinociceptive action of Humirianthera ampla was demonstrated through the pretreatment of animals with the non-selective opioid receptor 362 A.P. Luiz et al. / Journal of Ethnopharmacology 114 (2007) 355–363 antagonist naloxone. Interestingly, the effects of Humirianthera ampla on the opioid system were restricted to the central nervous system, since the antagonism of the peripheral opioid system by naloxone methiodide did not counteract the antinociception of Humirianthera ampla. Although Humirianthera ampla has antinociceptive properties that are, at least in part, dependent on opiod system, the plant extract did not prevent the thermal nociception in the tail-flick test. The absence of effect against nociceptive transmission on the tail-flick test could be explained because the response caused by this stimulus corresponds to a reflex action from the spinal medulla and does not involve supraspinal centers (Chapman et al., 1985; Le Bars et al., 2001). Another remarkable finding of this study resides on the involvement of the serotonergic system on the antinociceptive action of Humirianthera ampla. Serotonergic neurons have a crucial role in the control of pain (Fields et al., 1991; Alhaider et al., 1991; Millan, 2002). The diversity of subtype receptors for serotonin, with different patterns of coupling to the intracellular transduction mechanisms, makes this system able to exert either facilitatory or inhibitory function (Bardin et al., 2000). It is well postulated that the activation of spinal serotonergic subtype receptors 5-HT1A , 5-HT2 and 5-HT3 produces antinociception (Bardin et al., 2000; Millan, 2002). Corroborating with these previous studies, we found here that the antagonism of these receptors by WAY100635, ketanserin and ondansetron counteracted the antinociception of Humirianthera ampla. The antinociception caused by serotonin is partially due to a release of adenosine in the spinal cord (Sawynok and Reid, 1996). However, the antagonism of adenosine receptors did not prevent the antinociception of Humirianthera ampla. Regarding this finding, the antinociception caused by Humirianthera ampla is likely not related with a modulation of the adenosinergic system. 5. Conclusion In summary, the present results provide convincing evidence that EE from Humirianthera ampla exerts a long-lasting and pronounced systemic antinociception against nociceptive response caused by formalin, glutamate and capsaicin in mice. The decrease on nociception response was greatly associated with an effect of the plant extract in the central nervous system. Our results indicate that glutamatergic, neurokinin, NO, opioid and serotonergic pathways are closely involved with the antinociception of the Humirianthera ampla. These effects are probably due to the presence of different constituents in the plant extract. Hence, additional studies are being carried out to identify the compounds of Humirianthera ampla responsible by its antinociceptive action. 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