Effect of homotaurine in experimental analgesia tests.

1. The possible antinociceptive action of GABA A receptor agonist homotaurine, has been studied through a battery of chemical (acetic acid) and thermal (hot plate, tail flick and tail immersion) tests in rats and mice. 2. The aminoacid was used at the following doses 22.25; 55.62 and 111.24 mg/kg i.p. and 50-100 micrograms i.c.v.; and measurements were made at the time of and at 5, 15 and 30 min after drug administration. 3. Homotaurine exhibited a significantly antinociceptive effect in all the above mentioned test except hot plate and when administered i.c.v. in the tail flick test. 4. The antinociceptive effect in the chemical test was dose and time dependent. 5. In the tail immersion test, latency time for withdrawal of the tail was significantly increased with the dose of 55.62 mg/kg at 15 min and 111.24 mg/kg at 30 min. 6. In the tail flick test the antinociceptive effect was dose dependent at 15 and 30 min. 7. From the above results the implication of peripheral and spinal mechanisms in the antinociceptive effect of homotaurine may be concluded.     

Combined use of tertiary amine parasympathomimetics with a quaternary amine parasympatholitic--a new perspective to use parasympathomimetic drugs for systemic analgesia

The interactions on antinociception between a muscarinic agonist arecoline (arec), an anticholinesterase physostigmine (physo) which both cross CNS, and a peripherally acting antimuscarinic hyoscine-N-butyl bromide (hyo), were assessed by tail flick test in mice. All drugs were administered intraperitoneally (i.p.). While hyoscine-N-butyl bromide (0.15 and 4.00 mg/kg, i.p.) did not produce antinociception, physostigmine salicylate (0.3 mg/kg, i.p.) and arecoline hydrobromide (8.00 mg/kg, i.p.) exerted significant antinociceptive effect. In combined applications, physo + hyo (0.075 + 0.15; 0.15 + 0.30; 0.30 + 0.60 mg/kg) and arec + hyo (1.00 + 0.50; 2.00 + 1.00; 4.00 + 2.00; 8.00 + 4.00 mg/kg), respectively, produced significant antinociception and the tail flick latencies produced by physo 0.30 + hyo 0.60 mg/kg and arec 8.00 + hyo 4.00 mg/kg were not significantly different from those of physo 0.30 mg/kg and arec 8.00 mg/kg, respectively, showing that hyo did not antagonise the antinociceptive effects of physo and arec. We believe that combining an centrally acting cholinergic drug applied systemically with a peripherally acting (quaternary amine) antimuscarinic compound might be used as an effective analgesic in clinical practice.     

Dopamine D(2) receptor antagonists prevent delta(9)-tetrahydrocannabinol-induced antinociception in rats

Delta(9)-Tetrahydrocannabinol (1 and 5 mg/kg, i.p.) produced, dose-dependently, antinociceptive effects using hot plate and tail flick tests in rats. These effects were suppressed not only by the cannabinoid CB(1) receptor antagonist SR 141716A (0.5 mg/kg; i.p.) but also by the dopamine D(2) receptor antagonists S(-)-sulpiride (5 and 10 mg/kg; i.p.) and S(-)-raclopride (1.5 and 3 mg/kg; i.p.). Conversely, Delta(9)-tetrahydrocannabinol antinociceptive effects were potentiated by the dopamine D(2) receptor agonists (-)-quinpirole (0.025 mg/kg, s.c.) and (+)-bromocriptine (0.5 and 1 mg/kg; i.p.). Our results indicate that the antinociceptive effects of Delta(9)-tetrahydrocannabinol are mediated by the concomitant activation of cannabinoid CB(1) and dopamine D(2) receptors and that dopamine D(2) receptor agonists may be useful in improving the analgesic effects of cannabinoids.     

Analgesic Activity of Cistus laurifolius in Mice

The possible analgesic activity of Cistus laurifolius extracts has been evaluated by using tail flick and acetic acid-induced writhing tests in mice. The chloroform extract (500?mg/kg, i.p.) and the precipitated fraction (10, 30, 100?mg/kg, i.p.) obtained from C. laurifolius leaves showed significant analgesic activity on tail flick assay, while aqueous, ethanol and butanol extracts of the plant had no activity on the same test. Chloroform extract (500?mg/kg, i.p.) and precipitate fraction (30?mg/kg, i.p.) also inhibited number of writhings induced by acetic acid. These observations suggest that C. laurifolius leaves possess antinociceptive compound(s) which act through a central mechanism.     

Do Diuretics have Antinociceptive Actions: Studies of Spironolactone,Eplerenone, Furosemide and Chlorothiazide,Individually and with Oxycodone and Morphine

Spironolactone, eplerenone, chlorothiazide and furosemide are diuretics that have been suggested to have antinociceptive properties, for example via mineralocorticoid receptor antagonism. In co‐administration, diuretics might enhance the antinociceptive effect of opioids via pharmacodynamic and pharmacokinetic mechanisms. Effects of spironolactone (100 mg/kg, i.p.), eplerenone (100 mg/kg, i.p.), chlorothiazide (50 mg/kg, i.p.) and furosemide (100 mg/kg, i.p.) were studied on acute oxycodone (0.75 mg/kg, s.c.)‐ and morphine (3 mg/kg, s.c.)‐induced antinociception using tail‐flick and hot plate tests in male Sprague Dawley rats. The diuretics were administered 30 min. before the opioids, and behavioural tests were performed 30 and 90 min. after the opioids. Concentrations of oxycodone, morphine and their major metabolites in plasma and brain were quantified by mass spectrometry. In the hot plate test at 30 and 90 min., spironolactone significantly enhanced the antinociceptive effect (% of maximum possible effect) of oxycodone from 10% to 78% and from 0% to 50%, respectively, and that of morphine from 12% to 73% and from 4% to 83%, respectively. The brain oxycodone and morphine concentrations were significantly increased at 30 min. (oxycodone, 46%) and at 90 min. (morphine, 190%). We did not detect any independent antinociceptive effects with the diuretics. Eplerenone and chlorothiazide did not enhance the antinociceptive effect of either opioid. The results suggest that spironolactone enhances the antinociceptive effect of both oxycodone and morphine by increasing their concentrations in the central nervous system.     

An antinociceptive profile of kojic amine: an analogue of gamma-aminobutyric acid (GABA)

Kojic amine [2-(aminomethyl)-5-hydroxy-4H-pyran-4-one], an analogue of gamma-aminobutyric acid (GABA), produced dose-related, but short-lived, antinociceptive activity in the 48 degrees C [ED50 = 9.2 (8.2-10.3) mg/kg i.p.] and 55 degrees C [ED50 = 13.8 (12.2-15.7) mg/kg i.p.] hot-plate tests in the mouse. The antinociceptive activity of kojic amine at 48 degrees C was found to be insensitive to bicuculline (1.0 mg/kg i.p.) and picrotoxin (0.5 mg/kg i.p.). At this temperature, antinociception was distinctly separate from the impairment of motor function (measured by a rotorod assay) and was not significantly affected by prior treatment with the cholinergic antagonist, atropine sulfate (10.0 mg/kg i.p.). However, at 55 degrees C, the antinociceptive effect of a large dose (20 mg/kg i.p.) of kojic amine was significantly attenuated by similar pretreatment with atropine sulfate, but not by the peripheral cholinergic antagonist, atropine methylnitrate (10.0 mg/kg i.p.). Kojic amine exhibited no significant interaction with haloperidol (0.5 mg/kg i.p.) at this temperature. In animals made tolerant to morphine, THIP or baclofen, there was analgesic cross-tolerance between kojic amine, morphine and baclofen but not between kojic amine and THIP. It is suggested that kojic amine-induced antinociception is similar to that produced by both THIP and baclofen. Thus, kojic amine represents a unique tool with which to study GABA-ergic antinociceptive processes.     

Antinociceptive effects of tricyclic antidepressants and their noradrenergic metabolites.

This study evaluates the antinociceptive effect of several tricyclic antidepressants in four nociceptive tests which employ either thermal (hot plate and tail flick tests) or chemical (formalin and acetic acid tests) stimuli. Forced swimming test was also performed as a model of depression and an activity test was also performed. Mixed antidepressants in current clinical use: amitriptyline, imipramine and clorimipramine and their respective main secondary metabolites which preferentially inhibit noradrenaline reuptake: nortriptyline, desipramine and desmethylclorimipramine, were tested (2.5-20 mg/kg, i.p.) in mice. The results show a stronger antinociceptive effect in chemical tests induced by all the drugs, compared with thermal tests. The doses needed to produce antinociception were lower than those inducing an antidepressive effect, both effects being mutually independent. The overall results show that preferentially noradrenergic tricyclics induced an antinociceptive effect comparable with that of mixed tricyclics, indicating that noradrenaline reuptake plays an important role in tricyclic-induced antinociception.     

Analgesic Effect Of Dioclenol And Dioflorin Isolated From Dioclea Grandiflora

The analgesic activity of dioflorin and dioclenol, two constituents of Dioclea grandiflora Mart., has been studied in mice. It was found that dioclenol (10 mg/kg, i.p.) and dioclenol (10 mg/kg, i.p.) were effective in the acetic acid-induced writhing and tail immersion tests. These observations suggest that dioflorin and dio-clenol possesses central antinociceptive activity.     

Pharmacological characterization of AM1710, a putative cannabinoid CB2 agonist from the cannabilactone class: antinociception without central nervous system side-effects

Cannabinoid CB₂ agonists produce antinociception without central nervous system (CNS) side-effects. This study was designed to characterize the pharmacological and antinociceptive profile of AM1710, a CB₂ agonist from the cannabilactone class of cannabinoids. AM1710 did not exhibit off-target activity at 63 sites evaluated. AM1710 also exhibited limited blood brain barrier penetration. AM1710 was evaluated in tests of antinociception and CNS activity. CNS side-effects were evaluated in a modified tetrad (tail flick, rectal temperature, locomotor activity and rota-rod). Pharmacological specificity was established using CB₁ (SR141716) and CB₂ (SR144528) antagonists. AM1710 (0.1-10 mg/kg i.p.) produced antinociception to thermal but not mechanical stimulation of the hindpaw. AM1710 (5 mg/kg i.p.) produced a longer duration of antinociceptive action than the aminoalkylindole CB₂ agonist (R,S)-AM1241 (1 mg/kg i.p.) at maximally antinociceptive doses. Antinociception produced by the low (0.1 mg/kg i.p.) dose of AM1710 was blocked selectively by the CB₂ antagonist SR144528 (6 mg/kg i.p.), whereas antinociception produced by the high dose of AM1710 (5 mg/kg i.p.) was blocked by either SR144528 (6 mg/kg i.p.) or SR141716 (6 mg/kg i.p.). AM1710 did not produce hypoactivity, hypothermia, tail flick antinociception, or motor ataxia when evaluated in the tetrad at any dose. In conclusion, AM1710, a CB₂-preferring cannabilactone, produced antinociception in the absence of CNS side-effects. Thus, any CB₁-mediated antinociceptive effects of this compound may be attributable to peripheral CB₁ activity. The observed pattern of pharmacological specificity produced by AM1710 is consistent with limited blood brain barrier penetration of this compound and absence of CNS side-effects.     

Central components in the antinociceptive activity of dipyrone in mice

Dipyrone is classified as a nonsteroidal anti-inflammatory drug. It has analgesic, antipyretic and anti-inflammatory properties and exerts its analgesic effect via both peripheral and central action. Dipyrone at the dose of 250 and 500 mg/kg showed dose-dependent antinociceptive activity in the hot plate, tail flick tests to radiant heat and tail clip test and the writhing test induced by acetic acid in mice. The antinociceptive effects of dipyrone (500 mg/kg) were antagonized by naloxone (1, 2, 5 mg/kg) in the tail flick test to radiant heat and tail clip test and hot plate tests but not in the writhing test. Cyproheptadine (100 g/kg) decreased the antinociceptive effect of dipyrone. There was an increase in the antinociceptive effects of dipyrone (500 mg/kg) when combined with buspiron (0.5 mg/kg) in the tail flick test to radiant heat and tail clip test. The results provide evidence for a central antinociceptive effect of dipyrone antagonized by naloxone which suggests that its activity may also involve the serotoninergic system.     

Dopaminergic involvement in adenosine A1 receptor-mediated antinociception in the tail flick latency model in mice

The interaction between the adenosinergic and dopaminergic systems in nociception was assessed in the tail flick latency (TFL) test in mice. Adenosine exhibited qualitatively different responses depending on the dose: Adenosine 10 and 100 mg/kg i.p. caused antinociception as evidenced by an increase in TFL while the middle dose of 30 mg/kg decreased TFL. On the other hand, the specific adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) at doses of 0.05, 0.1, 0.25 and 0.5 mg/kg (i.p.) resulted in dose-dependent antinociception. The antinociceptive effect of CPA was reversed by classical albeit nonspecific adenosine receptor antagonist theophylline (5 mg/kg) at a dose which had no effect on TFL per se. A low dose (1 mg/kg i.p.) of the dopaminergic agonist apomorphine caused an early mild hyperalgesic response while the high dose (10 mg/kg i.p.) had no significant effect on TFL. The antinociceptive effect of CPA was attenuated by pretreatment with low dose apomorphine while pretreatment with the high dose caused mild but insignificant potentiation. Theophylline, when administered prior to apomorphine failed to modify the nociceptive response. The results suggest that an interaction between adenosine and dopamine may be involved in nociception.     

Gender and test dependence of a type of kappa mediated stress induced analgesia in mice

1. In male mice, 80 inescapable footshocks (S-80) induce analgesic responses measured by the tail flick test that are blocked by naloxone and the kappa opioid antagonist, nor-binaltorphimine. We now study the nociceptive responses, induced after this particular stress, measured by the writhing test, the tail immersion test and a high intensity tail immersion test both in male and female mice.2. In stressed males, analgesic responses are seen in all the nociceptive tests. Naloxone (10 mg/kg) does not prevent them.3. In stressed females, in contrast with males, no analgesia is produced in the tail flick test. The writhing test and the tail immersion test registered analgesic responses that were not prevented by naloxone (10 mg/kg).4. We conclude that only the antinociceptive kappa opioid mediated component of the stress we study is strongly dependent on gender, in contrast to other types of analgesia triggered by the same stress.     

Antinociceptive activity of <Emphasis Type="Italic">Hypericum grandifolium</Emphasis> Choisy in mice

The present study investigated the antinociceptive activity of different extracts prepared from the aerial parts of blossom of Hypericum grandifolium Choisy—a species native to the Macaronesian Region—using the acetic acid-induced writhing, formalin and tail flick test in mice. Oral administration of methanol extract (500 and 1,000 mg/kg p.o.), the aqueous, butanol and chloroform fractions (500 mg/kg p.o.) as well as subfractions F2 and F3 (45 mg/kg p.o.) from the chloroform fraction significantly inhibited acetic acid-induced writhing, with values ranging from 28 to 50% of inhibition. The methanol extract (1,000 mg/kg p.o.) and chloroform fraction (500 mg/kg p.o.) significantly reduced both phases of formalin-induced pain (with inhibition values ranging from 18 to 53%), whereas subfraction F2 (45 mg/kg p.o.) significantly inhibited the late phase (30%). In the tail flick assay, only the chloroform fraction (500 mg/kg p.o.) significantly prolonged the tail flick response. Different constituents, such as flavonoids and benzophenone derivatives, could account for the effects observed. Taking together, the results indicate that Hypericum grandifolium Choisy possesses both peripheral and central antinociceptive activities in mice, suggesting an interesting therapeutic potential for this species in pain diseases.     

Baclofen induces catatonia in rats

Baclofen (10 and 20 mg/kg, i.p.) induced catatonia in rats within 10 min of its administration and the effect lasted for 3 hr. Muscimol (100 ng i.c.v. or 1 mg/kg, i.p.) as well as GABA (5 micrograms i.c.v.) potentiated the effect without producing any effect per se. Bicuculline, bromocriptine and scopolamine failed to modify the catatonia induced by baclofen, thereby ruling out the involvement of GABAA receptors, dopaminergic and cholinergic mechanisms. However, GABAB receptor antagonists, such as homotaurine and delta-amino-n-valeric acid, reversed the catatonia induced by baclofen in rats. Since baclofen is known to bind to a subpopulation of GABA receptors (bicuculline-insensitive) and baclofen-induced catatonia was susceptible to reversal by homotaurine and delta-amino-n-valeric acid, it is suggested that this effect could be mediated through GABAB receptors.     

The involvement of central cholinergic system in (+)-matrine-induced antinociception in mice

The antinociceptive effect of (+)-matrine was examined in mice by writhing, tail-pressure and hot-plate tests. (+)-Matrine (5, 10 and 20 mg/kg s.c.) produced antinociception in a dose-dependent manner. In hot-plate test, the antinociception produced by (+)-matrine (10 mg/kg s.c.) was attenuated by muscarinic receptor antagonists atropine (5 mg/kg i.p.) and pirenzepine (0.1 mug/mouse i.c.v.) and acetylcholine depletor hemicholinium-3 (HC-3) (1 mug/mouse i.c.v.), but not by opioid receptor antagonist naloxone (2 mg/kg i.p.), dopamine D(2) receptor agonist (-)-quinpirole (0.1 mg/kg i.p.) or catecholamine depletor reserpine (2.5 mg/kg i.p.). Radioligand binding assay demonstrated that (+)-matrine had no affinity for mu-, kappa- or delta-opioid receptors in a wide concentration range (1 x 10(-11)-1 x 10(-3) M). The results suggest that (+)-matrine exerts its antinociceptive effect through multiple mechanism(s) such as increasing cholinergic activation in the CNS rather than acting on opioid receptors directly.     

Mechanisms of L-NG-nitro arginine methyl ester-induced antinociception in mice: a role for serotonergic and adrenergic neurons.

1. The mechanisms involved in the antinociceptive action of L-NG-nitro arginine methyl ester (L-NAME) were investigated in mice. 2. Intraperitoneal administration of L-NAME produced a dose-dependent antinociception in the tail-flick, hot-plate and phenyl-p-quinone-induced writhing tests. 3. Pretreatment with the catecholamine depletors 6-hydroxydopamine (5 micrograms i.c.v.) or reserpine (5 mg/kg i.p.) or the serotonin synthesis inhibitor, p-chlorophenylalanine methyl ester (200 mg/kg i.p. on 2 consecutive days) resulted in a significant decrease in the antinociceptive effect of L-NAME. 4. Similarly, pretreatment with the selective alpha 1-adrenoceptor antagonist prazonin (2.5 mg/kg, i.p.), or the non-selective alpha-adrenoceptor blocker, phentolamine (5 mg/kg, i.p.) antagonized the antinociceptive effect of L-NAME. 5. However, the administration of the selective alpha 2-adrenoceptor antagonist, idazoxan (2.5 mg/kg i.p.) was without effect. 6. Likewise, pretreatment with the serotonin 5-HT2 receptor blocker, ketanserin (1 mg/kg, i.p.), the D2 dopamine receptor antagonist (+/-) sulpiride (30 mg/kg, i.p.) or the opioid antagonist naloxone (5 mg/kg, i.p.) did not inhibit the antinociceptive effect of L-NAME. 7. These results suggest that L-NAME produces antinociception in the mouse probably by an action on adrenergic and serotonergic synapses.     

Caffeine induces central cholinergic analgesia

The antinociceptive effect of caffeine was examined by using the hot-plate, abdominal constriction tests in mice and the tail flick and paw-pressure tests in rats. Caffeine (1–5 mg kg^–1 s.c. in mice; 2.5–5 mg kg^–1 i.p. in rats) produced significant antinociception in both species which was prevented by atropine (5 mg kg^–1 i.p.), pirenzepine (0.1 μg per mouse i.c.v.), hemicholinium-3 (1 μg/ mouse i.c.v.) and N⁶-cyclopentyladenosine (5 μg/mouse i.c.v.), but not by naloxone (1 mg kg^–1 i.p.), CGP 35348 (100 mg kg^–1 i.p.), α-methyl p-tyrosine (100 mg kg^–1 i.p.) and reserpine (2 mg kg^–1 i.p.). Intracerebroventricular injection of caffeine in mice at doses (2.5–5 μg per mouse) which were largely ineffective by parenteral routes, induces an antinociception whose intensity equalled that obtainable s.c. or i.p. In the antinociceptive dose-range, caffeine did not produce any behavioural impairment as revealed by the rotarod and Irwing tests. On the basis of the above data, it can be postulated that caffeine exerts an antinociceptive effect mediated by central amplification of cholinergic transmission.     

The synthetic TRH analogue taltirelin exerts modality-specific antinociceptive effects via distinct descending monoaminergic systems

BACKGROUND AND PURPOSE: Exogenously administered thyrotropin-releasing hormone (TRH) is known to exert potent but short-acting centrally-mediated antinociceptive effects. We sought to investigate the mechanisms underlying these effects using the synthetic TRH analogue taltirelin, focusing on the descending monoaminergic systems in mice. EXPERIMENTAL APPROACH: The mice received systemic or local injections of taltirelin combined with either central noradrenaline (NA) or 5-hydroxytryptamine (5-HT) depletion by 6-hydroxydopamine (6-OHDA) or DL-p-chlorophenylalanine (PCPA), respectively, or blockade of their receptors. The degree of antinociception was determined using the tail flick and tail pressure tests. KEY RESULTS: Subcutaneously (s.c.) administered taltirelin exhibited dose-dependent antinociceptive effects in the tail flick and tail pressure tests. These effects appeared to be primarily supraspinally mediated, since intracerebroventricularly (i.c.v.) but not intrathecally (i.t.) injected taltirelin generated similar effects. Depletion of central NA abolished only the analgesic effect of taltirelin (s.c. and i.c.v.) on mechanical nociception. By contrast, depletion of central 5-HT abolished only its analgesic effect on thermal nociception. Intraperitoneal (i.p.) and i.t. injection of the alpha2-adrenoceptor antagonist yohimbine respectively reduced the analgesic effect of taltirelin (s.c. and i.c.v.) on mechanical nociception. By contrast, the 5-HT1A receptor antagonist WAY-100635 (i.p. and i.t.) reduced the effect of taltirelin (s.c. and i.c.v.) on thermal nociception. Neither the 5-HT2 receptor antagonist ketanserin nor the opioid receptor antagonist naloxone altered the antinociceptive effect of taltirelin. CONCLUSIONS AND IMPLICATIONS: These findings suggest that taltirelin activates the descending noradrenergic and serotonergic pain inhibitory systems, respectively, to exert its analgesic effects on mechanical and thermal nociception.     

The possible mechanisms of protocatechuic acid-induced central analgesia

It is aimed to investigate the central antinociceptive effect of protocatechuic acid and the involvement of stimulation of opioidergic, serotonin 5-HT_2A/2C, α2-adrenergic and muscarinic receptors in protocatechuic acid-induced central analgesia in mice. Time-dependent antinociceptive effects of protocatechuic acid at the oral doses of 75, 150 and 300?mg/kg were tested in hot-plate (integrated supraspinal response) and tail-immersion (spinal reflex) tests in mice. To investigate the mechanisms of action; the mice administered 300?mg/kg protocatechuic acid (p.o.) were pre-treated with non-specific opioid antagonist naloxone (5?mg/kg, i.p.), serotonin 5-HT_2A/2C receptor antagonist ketanserin (1?mg/kg, i.p.), α2-adrenoceptor antagonist yohimbine (1?mg/kg, i.p.) and non-specific muscarinic antagonist atropine (5?mg/kg, i.p.), respectively. The antinociceptive effect of protocatechuic acid was observed at the doses of 75, 150 and 300?mg/kg in tail-immersion test, at the doses of 150 and 300?mg/kg in hot-plate test at different time interval. The enhancement in the latency of protocatechuic acid-induced response to thermal stimuli was antagonized by yohimbine, naloxone and atropine in tail-immersion test, while it was antagonized only by yohimbine and naloxone pretreatments in hot-plate test. These results indicated that protocatechuic acid has the central antinociceptive action that is probably organized by spinal mediated cholinergic and opiodiergic, also spinal and supraspinal mediated noradrenergic modulation. However, further studies are required to understand how protocatechuic acid organizes the interactions of these modulatory systems. As a whole, these findings reinforce that protocatechuic acid is a potential agent that might be used for pain relief. Additionally, the clarification of the effect and mechanisms of action of protocatechuic acid will contribute to new therapeutic approaches and provide guidance for new drug development studies.     

Mechanisms involved in the antinociception caused by ethanolic extract obtained from the leaves of Melissa officinalis (lemon balm) in mice

The present study examined the antinociceptive effect of the ethanolic extract from Melissa officinalis L. and of the rosmarinic acid in chemical behavioral models of nociception and investigates some of the mechanisms underlying this effect. The extract (3-1000 mg/kg), given orally (p.o.) 1 h prior to testing, produced dose-dependent inhibition of acetic acid-induced visceral pain, with ID50 value of 241.9 mg/kg. In the formalin test, the extract (30-1000 mg/kg, p.o.) also caused significant inhibition of both, the early (neurogenic pain) and the late (inflammatory pain), phases of formalin-induced licking. The extract (10-1000 mg/kg, p.o.) also caused significant and dose-dependent inhibition of glutamate-induced pain, with ID50 value of 198.5 mg/kg. Furthermore, the rosmarinic acid (0.3-3 mg/kg), given p.o. 1 h prior, produced dose-related inhibition of glutamate-induced pain, with ID50 value of 2.64 mg/kg. The antinociception caused by the extract (100 mg/kg, p.o.) in the glutamate test was significantly attenuated by intraperitoneal (i.p.) treatment of mice with atropine (1 mg/kg), mecamylamine (2 mg/kg) or l-arginine (40 mg/kg). In contrast, the extract (100 mg/kg, p.o.) antinociception was not affected by i.p. treatment with naloxone (1 mg/kg) or d-arginine (40 mg/kg). It was also not associated with non-specific effects, such as muscle relaxation or sedation. Collectively, the present results suggest that the extract produced dose-related antinociception in several models of chemical pain through mechanisms that involved cholinergic systems (i.e. through muscarinic and nicotinic acetylcholine receptors) and the l-arginine-nitric oxide pathway. In addition, the rosmarinic acid contained in this plant appears to contribute for the antinociceptive property of the extract. Moreover, the antinociceptive action demonstrated in the present study supports, at least partly, the ethnomedical uses of this plant.