QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.104.071779v1 313/1/310    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Otuki, M. F. Articles by Calixto, J. B. Search for Related Content PubMed PubMed Citation Articles by Otuki, M. F. Articles by Calixto, J. B.

NEUROPHARMACOLOGY

Antinociceptive Properties of Mixture of -Amyrin and -Amyrin Triterpenes: Evidence for Participation of Protein Kinase C and Protein Kinase A Pathways

Departamento de Farmacologia (M.F.O., J.F., J.B.C.), Ciências Fisiológicas (A.R.S.S.), e Química (R.A.Y.), Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil; Universidade do Vale do Itajaí, NIQFAR (C.M.-S., A.M., L.A.M., G.S.C.), Itajaí, Santa Catarina, Brazil; and Universidade do Sul de Santa Catarina (F.V.L.), Curso de Farmácia, Tubarão, Santa Catarina, Brazil

Received May 24, 2004; accepted December 1, 2004.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
The mixture of the two pentacyclic triterpenes -amyrin and -amyrin, isolated from the resin of Protium kleinii and given by intraperitoneal (i.p.) or oral (p.o.) routes, caused dose-related and significant antinociception against the visceral pain in mice produced by i.p. injection of acetic acid. Moreover, i.p., p.o., intracerebroventricular (i.c.v.), or intrathecal (i.t.) administration of ,-amyrin inhibited both neurogenic and inflammatory phases of the overt nociception caused by intraplantar (i.pl.) injection of formalin. Likewise, ,-amyrin given by i.p., p.o., i.t., or i.c.v. routes inhibits the neurogenic nociception induced by capsaicin. Moreover, i.p. treatment with ,-amyrin was able to reduce the nociception produced by 8-bromo-cAMP (8-Br-cAMP) and by 12-O-tetradecanoylphorbol-13-acetate (TPA) or the hyperalgesia caused by glutamate. On the other hand, in contrast to morphine, ,-amyrin failed to cause analgesia in thermal models of pain. The antinociception caused by the mixture of compounds seems to involve mechanisms independent of opioid, -adrenergic, serotoninergic, and nitrergic system mediation, since it was not affected by naloxone, prazosin, yohimbine, DL-p-chlorophenylalanine methyl ester, or L-arginine. Interestingly, the i.p. administration of ,-amyrin reduced the mechanical hyperalgesia produced by i.pl. injection of carrageenan, capsaicin, bradykinin, substance P, prostaglandin E₂, 8-Br-cAMP, and TPA in rats. However, the mixture of compounds failed to alter the binding sites of [³H]bradykinin, [³H]resiniferatoxin, or [³H]glutamate in vitro. It is concluded that the mixture of triterpene -amyrin and -amyrin produced consistent peripheral, spinal, and supraspinal antinociception in rodents, especially when assessed in inflammatory models of pain. The mechanisms involved in their action are not completely understood but seem to involve the inhibition of protein kinase A- and protein kinase C-sensitive pathways.

In early pharmacological studies, immunostimulant (Delaveau et al., 1980) and anti-inflammatory activities where demonstrated for the aqueous extract from resins of Burseraceae species (Duwiejua et al., 1993), along with anti-inflammatory activity of the essential oil, obtained from the leaves, and resin of some species of Protium (Siani et al., 1999).

Chemical analysis carried out with the resin from Protium kleinii reveled the presence of a mixture of two triterpenes belonging to the ursane (-amyrin) and oleanane (-amyrin) series. These triterpenes have been reported to exert anti-inflammatory effects when assessed in animal models of inflammation (Kweifio-Okai et al., 1994a,b; Recio et al., 1995; Akihisa et al., 1996). Furthermore, some in vitro studies have shown that -amyrin is a relatively specific inhibitor of the catalytic subunit of cyclic AMP-dependent protein kinase (Hasmeda et al., 1999). In addition, -amyrin and its palmitate and linoleate esters are effective in inhibiting collagenase (Kweifio-Okai et al., 1994a,b), whereas -amyrin palmitate inhibits ₁-adrenoceptors (Subarnas et al., 1993).

In our earlier study, we showed that the ether extract and the isolated pentacyclic triterpene brein obtained from P. kleinii caused pronounced and dose-related inhibition when tested in several models of nociception in mice (Otuki et al., 2001). The purpose of the present study was therefore to evaluate the peripheral, spinal, and supraspinal antinociceptive effects and some of the mechanisms underlying the action of the mixture of -amyrin and -amyrin (1:1) on chemical, mechanical, and thermal models of nociception in mice and rats.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Isolation and Chemical Identification of the Active Compound. Botanical material was collected in morro do Bau, state of Santa Catarina, Brazil, and was classified by Dr. Ademir Reis as being P. kleinii, a plant of the family Burseraceae. A voucher of this plant (excicata no. VC Filho 019) was deposited in the herbarium FLOR at the Federal University of Santa Catarina.

The resinous bark of P. kleinii (50 g) was powdered and extracted with diethylic ether in the proportion of 1:10 (w/v), being stirred and macerated at room temperature (21 ± 3°C) for approximately 2 weeks. The solvent was fully evaporated under reduced pressure, and the extract (33.42 g) was chromatographed (14.42 g) on a silica gel column eluted successively with hexane, hexane/ethyl acetate, ethyl acetate, ethyl acetate/methanol, methanol, and water, respectively. The fraction eluted with hexane/ethyl acetate (1:1) gave a crystalline solid (120 mg), which was identified as being the triterpene -amyrin and -amyrin (Fig. 1), the major natural compound present in this plant. This mixture was obtained in a 1:1 proportion by gas chromatography spectra. The chemical identification was obtained by using the ¹H and ¹³C NMR spectra.

View larger version (10K): [in this window] [in a new window]   Fig. 1. Molecular structure of -amyrin (A) and -amyrin (B).

Animals. Male Swiss mice (25–35 g) or male Wistar rats (200–300 g), housed at 22 ± 2°C under a 12-h light/12-h dark cycle and with access to food and water ad libitum, were used. Experiments were performed during the light phase of the cycle. The animals were allowed to adapt to the laboratory for at least 2 h before testing and were only used once. Experiments reported in this study were carried out in accordance with current guidelines for the ethical guidelines for investigation of experimental pain in conscious animals (Zimmermann, 1983).

Abdominal Constriction Induced by Acetic Acid. The abdominal constriction induced by intraperitoneal injection of dilute acetic acid (0.6%) was carried according to the procedures described previously (Vaz et al., 1996). Animals were pretreated with ,-amyrin given i.p. (0.1–10 mg/kg) or p.o. (25–100 mg/kg) 30 and 60 min before testing, respectively. The control group received the same volume of 0.9% NaCl (10 ml/kg). After challenge, pairs of mice were placed in separate boxes, and the number of abdominal constrictions was cumulatively counted over a period of 20 min.

Formalin Test. The procedure used was essentially similar to that described previously (Vaz et al., 1996). Twenty microliters of 2.5% formalin solution (0.92% formaldehyde), made up in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, and 10 mM phosphate buffer), was injected intraplantarly under the surface of the right hindpaw. Animals were treated with ,-amyrin or vehicle (10 ml/kg) by i.p. (0.1–10 mg/kg) or p.o. (5–100 mg/kg) routes, 0.5 and 1 h before formalin injection, respectively. Other groups of animals were treated with ,-amyrin or with vehicle (5 µl/site) by i.c.v. (1–10 µg/site) or i.t. (1–30 µg/site) routes, as reported previously (Hylden and Wilcox, 1980; Vaz et al., 1996), 10 min before formalin injection. After intraplantar injection of formalin, the animals were immediately placed in a glass cylinder 20 cm in diameter, and the time spent licking the injected paw was timed with a chronometer and considered indicative of nociception.

Analysis of Possible Mechanism of Action of ,-Amyrin. To investigate the possible participation of the opioid system in the antinociceptive effect of ,-amyrin, animals were pretreated with naloxone (a nonselective antagonist of opioid receptors, 5 mg/kg i.p.), 15 min before the administration of ,-amyrin (10 mg/kg i.p.), morphine (5 mg/kg s.c.), or saline (0.9% NaCl solution, 10 ml/kg i.p.). The other groups of animals received only ,-amyrin, morphine, naloxone, or saline 30 min before the formalin injection (Vaz et al., 1996). To explore the possible contribution of serotonin to the antinociceptive effect of ,-amyrin, animals were pretreated with DL-p-chlorophenylalanine methyl ester (PCPA) (an inhibitor of serotonin synthesis, 100 mg/kg i.p.) once a day for 4 consecutive days, before administration of ,-amyrin (10 mg/kg i.p.) or saline (0.9% NaCl solution, 10 ml/kg i.p.).

To examine the possible contribution of ₁- and ₂-adrenoreceptors in the antinociceptive effect caused by ,-amyrin, animals were pretreated with prazosin (0.15 mg/kg i.p.) or with yohimbine (0.15 mg/kg i.p.), and after 15 min the animals received ,-amyrin (10 mg/kg i.p.), phenylephrine (10 mg/kg i.p.), clonidine (0.1 mg/kg i.p.), or saline (0.9% NaCl solution, 10 ml/kg i.p.). We further investigated the possible participation of nitric oxide-L-arginine pathway in the analgesic effect caused by ,-amyrin. Animals were pretreated with L-arginine (600 mg/kg), and after 15 min they received ,-amyrin (10 mg/kg i.p.), N^G-nitro-L-arginine (nitric-oxide synthase inhibitor, L-NOARG, 75 mg/kg i.p.), or saline (0.9% NaCl solution, 10 ml/kg i.p.).

Algogen-Induced Overt Nociception in Mice. The procedure used was similar to that described previously (Sakurada et al., 1992; Siebel et al., 2004). Twenty microliters of capsaicin (5.2 nmol/paw), 12-O-tetradecanoylphorbol-13-acetate (TPA) (50 pmol/paw), or 8-Br-cAMP (10 nmol/paw) was injected intraplantarly (i.pl.) under the surface of the right hindpaw. Before challenge, the animals were placed individually in transparent glass cylinders 20 cm in diameter, serving as observation chambers. Animals were observed individually for 5 min after capsaicin, 10 min after 8-Br-cAMP, or 15 to 45 min after TPA injection. The amount of time spent licking the injected paw timed with a chronometer was considered indicative of nociception. Animals were treated either with i.p. (0.3–30 mg/kg) or p.o. (5–100 mg/kg) injection of vehicle (10 ml/kg) or ,-amyrin, 0.5 and 1 h before algogen injection. Other groups of animals were treated with ,-amyrin or with vehicle (5 µl/site) by i.c.v. (0.3–3 µg/site) or i.t. (0.3–3 µg/site) routes 10 min before capsaicin injection.

Effect of ,-Amyrin on the Hyperalgesia in the Rat Paw. The procedures used were similar to those described previously (Randall and Selitto, 1957; Taiwo and Levine, 1991; Corrêa et al., 1996). The animals were pretreated i.p. with the ,-amyrin (30 mg/kg) 30 min before injection of 0.1 ml of bradykinin (3 nmol/paw), substance P (10 nmol/paw), capsaicin (20 nmol/paw), carrageenan (300 µg/paw), PGE₂ (10 nmol/paw), 8-Br-cAMP (1 nmol/paw), TPA (0.1 nmol/paw), or only with phosphate-buffer solution alone (control group), into the right hindpaw. The dose of ,-amyrin was defined in pilot experiments. The hyperalgesia was evaluated 0.5 h later, except for carrageenan, which was assessed at 3 h later. The nociception threshold (of squeak response or paw withdrawal) was assessed by applying increasing pressure to the dorsal site of inflamed or control paws, using a Basile analgesy meter (Ugo Basile, Milan, Italy) according to the method of Randall and Selitto (1957). The weight on the analgesy meter ranged from 0 to 750 g, and the threshold was expressed as load (grams) tolerated. When bradykinin was used, animals were pretreated with the angiotensin-converting enzyme inhibitor captopril (5 mg/kg s.c.), 1 h before experiments to prevent its degradation (Corrêa et al., 1996).

Glutamate-Induced Thermal Hyperalgesia. To test the hypothesis whether or not the excitatory amino acid glutamate was involved in the ,-amyrin antinociception, we assessed the effect of ,-amyrin given by oral route on the hyperalgesic response caused by spinally administered glutamate (100 nmol/site i.t.) in mice. The analgesic effect was assessed in the hot-plate test as described previously (Ferreira et al., 1999) in control group (vehicle-treated) or in mice pretreated with ,-amyrin (0.03–5 mg/kg p.o., 60 min prior). The response to the thermal stimuli was measured on the hot-plate apparatus (model-DS 37; Ugo Basile) maintained at 50 ± 1°C as described for the hot-plate test. The maximal hyperalgesic response caused by i.t. injection of glutamate was observed at 5 min after the injection, and this time point was used in all future experiments. A cut-off of 30 s was used for the hot-plate test. The maximal possible effect (MPE) of glutamate-induced hyperalgesia was calculated as follows:

Hot-Plate Test. The hot-plate test was used to measure the response latencies according to the method described previously by Eddy and Leimbach (1953), with minor modifications. In these experiments, the hot-plate (model-DS 37; Ugo Basile) was maintained at 56 ± 1°C. Animals were placed into a glass cylinder with 24 cm diameter on the heated surface, and the time between placement and shaking or licking of the paws or jumping was recorded as the index of response latency. An automatic 30-s cut-off was used to prevent tissue damage. Each animal was tested before administration of drugs to obtain the baseline. Control animals (vehicle, 10 ml/kg i.p.) or mice pretreated with ,-amyrin (30 mg/kg i.p.) were injected 30 min earlier. Other groups of animals were treated with morphine (10 mg/kg s.c., 30 min prior).

Tail-Flick Test. A radiant heat tail-flick analgesimeter was used to measure response latencies according to the method described previously by D'Amour and Smith (1941), with minor modifications. Animals responded to a focused heat stimulus by flicking or removing their tail, exposing a photocell in the apparatus immediately below it. The reaction time was recorded for control mice (vehicle, 10 ml/kg i.p.) and for animals pretreated 30 min before with ,-amyrin (30 mg/kg i.p.) or with morphine (10 mg/kg s.c.). An automatic 20-s cut-off was used to minimize tissue damage. Animals were selected 24 h previously on the basis of their reactivity in the test. To determine the baseline, each animal was tested before administration of drugs.

Measurement of Motor Performance. To evaluate possible nonspecific muscle relaxant or sedative effects of ,-amyrin, mice were tested on the rotarod (Rosland et al., 1990). The apparatus consisted of a bar with a diameter of 2.5 cm, subdivided into six compartments by disks 25 cm in diameter (model 7600; Ugo Basile). The bar rotated at a constant speed of 22 revolutions per minute. The animals were selected 24 h previously by eliminating those mice that did not remain on the bar for two consecutive periods of 60 s. Animals were treated with ,-amyrin (30 mg/kg i.p.) or with the same volume of vehicle (10 ml/kg i.p.) 30 min before being tested. The results are expressed as the time (seconds) for which animals remained on the rotarod. The cut-off time used was 60 s.

[³H]Glutamate Binding Assay. Cerebral cortices obtained from rats (killed by decapitation) were dissected and homogenized in 20 volumes of ice-cold 0.32 M sucrose containing 10 mM Tris-HCl buffer, pH 7.4, and 1 mM MgCl₂. The homogenate was centrifuged at 1000g, and the pellet was rehomogenized and centrifuged again. The second pellet was discarded, and both supernatants were pooled and centrifuged at 27,000g for 15 min. The resulting pellet was dissolved in 1 mM Tris-HCl, pH 7.4, for 30 min. The pellet was washed three times in 10 mM Tris-HCl buffer, pH 7.4, at 27,000g for 15 min (Beirith et al., 1998). The final pellet was diluted in 10 mM Tris-HCl and frozen at -70°C until use. On the day of the experiment, the membranes were thawed and incubated with 0.04% Triton X-100 at 37°C for 30 min and were then washed three times in 10 mM Tris-HCl buffer, pH 7.4, at 27,000g for 15 min. The final pellet was resuspended in 10 mM Tris-HCl, and the suspensions were assayed for [³H]glutamate binding. Assays of [³H]glutamate binding were carried out in triplicate in a total volume of 0.5 ml containing 0.1 ml of membrane (0.2–0.3 mg of protein), 50 mM Tris-HCl, pH 7.4, and 40 nM radioactive ligand ([³H]glutamate, 53 Ci/mmol), in the presence or absence of ,-amyrin (concentration in the range 1–100 µg/ml). Nonspecific binding was assayed similarly, except that 40 M nonradioactive glutamate (displacer) was added to the incubation medium. Centrifugation at 12,000g for 25 min was used to separate [³H]glutamate not bound to membranes. The supernatant was discarded, and the walls of the Eppendorf tubes and the surfaces of the pellets were quickly and carefully rinsed with cold deionized water, followed by processing for radioactivity. Specific binding was calculated as the difference between binding values in the absence and the presence of the displacer. The results represent the means of three independent experiments.

[³H]Resiniferatoxin Binding Assay. Binding assays were carried out as described previously (Szallasi et al., 1998). To obtain membranes for the binding studies, spinal cord of rats were removed and disrupted with the aid of a tissue homogenizer in ice-cold buffer A, pH 7.4, which contained 5 mM KCl, 5.8 mM NaCl, 2 mM MgCl₂, 0.75 mM CaCl₂, 12 mM glucose, 137 mM sucrose, and 10 mM HEPES. The homogenate was first centrifuged for 10 min at 1000g in 4°C. The low-speed pellets were discarded, and the supernatants were further centrifuged for 30 min at 35,000g in 4°C. The resulting high-speed pellets, resuspended in buffer A, were stored at -70°C until assayed.

Binding assays were carried out in duplicate with a final volume of 500 µl, containing buffer A, supplemented with 0.25 mg/ml bovine serum albumin, membranes (100 µg/protein), and 50 pM [³H]RTX. For the measurement of the nonspecific binding, 100 nM nonradioactive RTX were included in some tubes.

Assay mixtures were set up on ice, and the binding reaction was then initiated by transferring the assay tubes to a 37°C water bath. After a 60-min incubation period, cooling the mixtures on ice terminated the binding reaction, and then 100 µg of bovine ₁-acid glyco-protein was added to each tube (to reduce nonspecific binding). Finally, the bound and free membranes [³H]RTX were separated by centrifuging for 15 min at 20,000g in 4°C. The pellet was quantified by scintillation counting. Specific binding was calculated as the difference of the total and nonspecific binding.

[³H]Bradykinin Binding Assay. The specific binding of [³H]bradykinin (a high-affinity bradykinin B₂ receptor ligand) was assayed according to the method described previously (Manning et al., 1986) with minor modifications. The ilea from guinea pigs were removed and homogenized in ice-cold buffer (50 mM TES and 1 mM 1,10-phenanthroline, pH 6.8) with a Polytron. The homogenate was centrifuged to remove cellular debris (1000g, 20 min, 4°C), and the supernatant was centrifuged (100,000g, 60 min, 4°C). Then, the pellet was resuspended in ice-cold assay buffer (50 mM TES, 1 mM 1,10-phenanthroline, 140 g/ml bacitracin, 1 mM dithiothreitol, 1 M captopril, and 0.1% bovine serum albumin, pH 6.8) and used.

In the binding assay, membranes were incubated with [³H]bradykinin (final concentration 0.5 nM), in the absence or presence of ,-amyrin (100 µg/ml) at room temperature for 90 min. For the measurement of the nonspecific binding, 1 µM nonradioactive bradykinin was included in some tubes. Receptor-bound [³H]bradykinin was harvested by filtration through Whatman GF/B glass fiber filters under reduced pressure, and the filter was washed four times with 2 ml of ice-cold buffer (50 mM Tris-HCl). The radioactivity retained on the washed filter was measured with a liquid scintillation counter. The experiments were performed three times in duplicate.

Drugs. Formalin, morphine hydrochloride, and acetic acid were from Merck (Darmstadt, Germany); naloxone hydrochloride was from DuPont (Garden City, NY); capsaicin was from Calbiochem (San Diego, CA); and PCPA, glutamate, L-arginine, L-NOARG, yohimbine, clonidine, L-phenylephrine, prazosin, TPA, 8-Br-cAMP, PGE₂, substance P, and carrageenan were from Sigma-Aldrich (St. Louis, MO). [³H]BK and [³H]glutamate were from Amersham Biosciences, Inc. (Piscataway, NJ), and [³H]RTX was from PerkinElmer Life and Analytical Sciences (Boston, MA). Morphine and naloxone were dissolved in 0.9% NaCl solution just before use, whereas capsaicin and ,-amyrin were dissolved in ethanol and Tween 80 plus 0.9% NaCl solution, respectively. The final concentration of Tween 80 or ethanol did not exceed 5% and did not cause any effect per se.

Statistical Analysis. The results are presented as means ± S.E.M., except the ID₅₀ values (i.e., the dose of - and -amyrin reducing the pain responses by 50% relative to the control value), which are reported as geometric means accompanied by their respective 95% confidence limits. Data were analyzed by analysis of variance or t test and complemented by Dunnett's or Newman-Keuls post hoc test. P values less than 0.05 (P < 0.05) were considered as indicative of significance. The ID₅₀ values were determined by linear regression analysis from individual experiments using GraphPad software (GraphPad Software, Inc., San Diego, CA), and are reported as geometric means accompanied by their respective 95% confidence limits. In the glutamate-induced hyperalgesia, the ID₅₀ value was calculated on the peak (5 min) of glutamate response

	Results

Top Abstract Materials and Methods Results Discussion References

 
Abdominal Constriction Induced by Acetic Acid. Results of Fig. 2 show that ,-amyrin, given by i.p. (0.1–10 mg/kg) or by p.o. (25–100 mg/kg) route, dose-dependently inhibited acetic acid-induced abdominal constrictions. The calculated mean ID₅₀ values and the inhibitions obtained were 0.79 (0.63–1.01) mg/kg and 41.0 (26.10–66.01) mg/kg and 84 ± 3 and 83 ± 7%, respectively.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Effects of ,-amyrin given intraperitoneally (), and orally () against the acetic acid-induced abdominal constriction. The total time (mean ± S.E.M.). Each point represents the mean ± S.E.M. for 6 to 10 animals. The asterisks denote the significance levels compared with control groups. Significantly different from controls, **, P < 0.01. In some cases, the S.E.M.s are hidden within the symbols.

Formalin Test. The results of Fig. 3, A and C, show that ,-amyrin, given i.p. (0.1–10 mg/kg) caused a significant and dose-related inhibition of the neurogenic (0–5 min) and the inflammatory phase (15–30 min) of the formalin-induced licking. The calculated mean ID₅₀ values for these effects were >10 and 0.16 (0.07–0.37) mg/kg, respectively. Intraperitoneal administration with ,-amyrin was significantly more active to inhibit the inflammatory phase than the neurogenic phase of the formalin response. The inhibitions obtained were 45 ± 6 and 99 ± 1%, respectively.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Effects of ,-amyrin given intraperitoneally (, 0.1–10.0 mg/kg), orally (, 5.0–100 mg/kg), intracerebroventricularly (, 1.0–10.0 µg/site), and intrathecally (, 1.0–30.0 µg/site) against the early (A and B) and the second phase (C and D) of formalin-induced licking in mice. The total time (mean ± S.E.M.) spent licking the hindpaw was measured in the first phase (0–5 min) and the second phase (15–30 min) after subplantar injection of formalin into the hindpaw. Each point represents the mean ± S.E.M. for 6 to 10 animals. The asterisks denote the significance levels compared with control groups. Significantly different from controls, **, P < 0.01. In some cases, the S.E.M.s are hidden within the symbols.

When administered by p.o. route, ,-amyrin (5.0–100 mg/kg) produced dose-related inhibition of both the neurogenic and the inflammatory phase of the formalin test, being more potent (about 3-fold) and efficacious against the second phase (Fig. 3, A and C). The calculated mean ID₅₀ values and the inhibitions observed for the early and the late phase were 70.0 (63.0–78.0) and 19.7 (8.7–44.0) mg/kg with inhibitions of 56 ± 4 and 99 ± 1%, respectively.

Given i.c.v. (1.0–10 µg/site) ,-amyrin produced dose-dependent inhibition of both phases of the formalin-induced licking (Fig. 3, B and D). The calculated mean ID₅₀ values for these effects (micrograms per site) were 4.96 (2.55–9.62) and 0.72 (0.14–3.6), against the early and the late phase of the formalin response, respectively. The inhibitions observed for the early and the late phase were 67 ± 7 and 84 ± 4%, respectively.

Given i.t. (1.0–30 µg/site), ,-amyrin produced dose-dependent inhibition of both phases of the formalin-induced licking (Fig. 3, B and D). The calculated mean ID₅₀ values for these effects (micrograms per site) were 4.71 (1.28–17.33) and 7.84 (4.74–12.95), against the early and the late phase of the formalin response, respectively, with inhibitions of 66 ± 3 and 67 ± 3%, respectively.

Analysis of Possible Mechanism of Action of ,-Amyrin. The pretreatment of animals with naloxone given 15 min before injection of morphine largely reversed the antinociception caused by morphine when analyzed for both phases of the formalin-induced licking, leaving the antinociceptive effect of ,-amyrin unaffected (Table 1). The pre-treatment of animals with L-arginine, given 15 min prior, completely reversed the antinociception effect caused by N^G-nitro-L-arginine, but it did not reverse the antinociception caused by ,-amyrin against either phases of the formalin test (Table 1). Treatment of the animals with prazosin or yohimbine, 10 min prior, significantly reversed the antinociception caused by phenylephrine and clonidine, respectively, but it failed to interfere significantly with the antinociception caused by ,-amyrin against both phases of formalin-induced nociception (Table 1). The pretreatment of animals with PCPA (once a day for 4 days) did not significantly modify the antinociceptive effect caused by ,-amyrin against both phases of formalin-induced nociception (Table 1).

View this table: [in this window] [in a new window]   TABLE 1 Summary of the effects of the various drugs on the antinociception caused by ,-amyrin assessed in the formalin test Each group represents means ± S.E.M. for 6 to10 animals.

Algogen-Induced Overt Nociception. The results of Fig. 4, A and B, show that ,-amyrin, given orally (5–100 mg/kg), by i.p. (0.3–30 mg/kg), by i.t. (0.3–3.0 µg/site), or by i.c.v. (0.3–3.0 µg/site) route, dose-dependently inhibited capsaicin-induced licking. The calculated mean ID₅₀ values and the inhibitions observed were 8.04 (1.98–32.68) mg/kg, 1.5 (0.7–3.0) mg/kg, 0.3 (0.07–1.32) µg/site, and 0.89 (0.69–1.15) µg/site and 89 ± 2, 82 ± 4, 84 ± 2, and 88 ± 2%, respectively. Moreover, i.p. treatment with ,-amyrin is able to reduce the overt nociception produced by TPA or 8-Br-cAMP (Fig. 4, C and D). The calculated inhibitions observed were 77 ± 4 and 59 ± 3% for 8-Br-cAMP and TPA, respectively.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Effects of ,-amyrin given orally (, 5–100 mg/kg), intraperitoneally (, 0.3–30 µmol/paw) (A), intrathecally (, 0.3–3.0 µg/site), and intracerebroventricularly (, 0.3–3.0 µg/site) (B) against capsaicin-induced licking in mice. The total time spent licking the hindpaw was measured in the 0 to 5 min after subplantar injection of capsaicin into the hindpaw. Effects of ,-amyrin given intraperitoneally (10.0 mg/kg). The total time spent licking the hindpaw was measured after subplantar injection of TPA (C) and 8-Br-cAMP (D) into the hindpaw. Each point represents the mean ± S.E.M. for 8 to 10 animals. The asterisks denote the significance levels compared with control groups. Significantly different from controls, *, P < 0.05 and **, P < 0.01.

Hyperalgesia in the Rat Paw. When assessed in the Randall-Selitto model of nociception, ,-amyrin (30 mg/kg i.p.) partially, but significantly, reversed the hyperalgesia caused by intraplantar injection of bradykinin (3 nmol/paw), substance P (10 nmol/paw), capsaicin (20 nmol/paw), carrageenan (300 µg/paw), PGE₂ (10 nmol/paw), 8-Br-cAMP (1 nmol/paw), or TPA (0.1 nmol/paw). The inhibitions observed were 85 ± 26, 29 ± 5, 47 ± 5, 52 ± 17, 75 ± 9, 93 ± 8, and 83 ± 7%, respectively (Fig. 5).

View larger version (25K): [in this window] [in a new window]   Fig. 5. Effects of i.p. injection of the ,-amyrin (30 mg/kg) on bradykinin- (A), capsaicin- (B), carrageenan- (C), substance P- (D), 8-Br-cAMP- (E), and TPA (F)-induced hyperalgesia in the rat paw. The closed column indicates the control values (C, phosphate-buffered solution, injection paws), and the diagonally hatched column indicates the BK-, capsaicin- (CAP), carrageenan- (CAR), substance P- (SP), 8-Br-cAMP- (E), or TPA-injected paws, in the absence of the ,-amyrin. Each column represents the mean ± S.E.M. for 8 to 10 animals. The asterisks denote the significance levels compared with control groups. Significantly different from controls, *, P < 0.05 and **, P < 0.01.

Glutamate-Induced Hyperalgesia. Results of Fig. 6 show that ,-amyrin, given orally (0.03–5.01 mg/kg), dose-dependently inhibited glutamate-induced hyperalgesia. The calculated mean ID₅₀ value (estimated at 5 min) was 1.14 (0.82–1.6) mg/kg, and the maximal inhibition was 100%. Given alone, ,-amyrin, over the same range of doses where it was effective in inhibiting glutamate-induced hyperalgesia, had no effect in the hot-plate assay (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 6. Effects of ,-amyrin given orally against glutamate-induced hyperalgesia in mice. The latency (mean ± S.E.M.) in the hot-plate was measured at 5 min after intrathecal injection of glutamate. Each point represents the mean ± S.E.M. for six to eight animals. The asterisks denote the significance levels compared with control groups. Significantly different from controls, **, P < 0.01. In some cases, the S.E.M.s are hidden within the symbols.

Hot-Plate and Tail-Flick Tests. ,-Amyrin (30 mg/kg i.p.), given 30 min prior, did not cause any significant increase change in the latency response in either hot-plate or the tail-flick assays. Under similar conditions, morphine (10 mg/kg s.c.), used as a reference drug and given 30 min before, caused significant and marked analgesic effect in both models (Table 2).

View this table: [in this window] [in a new window]   TABLE 2 Analgesic effect of morphine (s.c.) and ,-amyrin (i.p.) in the hot-plate and tail-flick tests in mice Each group represents means ± S.E.M. for 6 to 10 animals.

Rotarod Test. ,-Amyrin (30 mg/kg), given i.p. 30 min prior, did not significantly affect the motor response of the animals. The control response in the rotarod test was 60 s versus 60 s in the presence of ,-amyrin (n = 8) (data not shown).

[³H]BK, [³H]RTX, and [³H]Glutamate Binding Studies. High concentrations of ,-amyrin (up to 100 µg/ml) were not able to alter either [³H]bradykinin, [³H]RTX, [³H]glutamate specific binding to guinea pig ilium, rat spinal cord membranes, or cerebral cortex in vitro (data not shown). In the same conditions, Hoe140 (100 nM), capsaicin (30 µM), or nonradioactive glutamate (30 µM) blocked the specific binding of [³H]bradykinin, [³H]RTX, and [³H]glutamate to membranes (inhibition of 100%).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The results presented in the current study show that the mixture of ,-amyrin triterpenes administered systemically, spinally, and supraspinally to mice produces pronounced and dose-related antinociception. Furthermore, this effect seems to be related with its ability to interfere with PKC- and PKA-sensitive pathways.

The antinociception elicited by ,-amyrin also seems to be independent of the activation of important endogenous analgesic systems, namely, opioidergic, serotoninergic, and nor-adrenergic. In fact, the treatment of animals with PCPA, at a dose known to inhibit serotonin synthesis (Vaz et al., 1996; Beirith et al., 1998), fails to interfere with ,-amyrin-induced antinociception when assessed in the formalin model of pain. Furthermore, the 1- and 2-adrenoceptors seem unlikely to be involved in the antinociceptive action of ,-amyrin, evidenced by the fact that selective antagonists of these receptors fail to alter the antinociception caused by ,-amyrin, in conditions where they produce significant inhibition of the antinociception provoked by the selective agonists. Finally, the mechanism underlying the antinociceptive action of ,-amyrin seems to be unrelated to activation of the opioid system. The antinociceptive action of ,-amyrin, in contrast to that reported for morphine, was not reversed by naloxone, a nonselective opioid antagonist.

,-Amyrin was devoid of analgesic action when assessed in thermal models of nociception, the tail-flick and hot-plate tests, under conditions where morphine had a marked antinociceptive effect. The hot-plate and tail-flick tests are commonly used to assess narcotic analgesic or other centrally acting drugs, including sedatives and psychomimetics (Vaz et al., 1996; Beirith et al., 1998). However, these thermal tests are not sensitive to the analgesic action of some drugs that act in the central nervous system, including weak agonists of opioid receptors. Apart from a its lack of action in these thermal models of pain, the antinociceptive effect of ,-amyrin may possess a central component, since i.t. and i.c.v. injection of amyrin was just as efficacious as systemically administered ,-amyrin in producing antinociception.

Also relevant are our findings showing that ,-amyrin was able to produce dose-dependent systemic inhibition of the hyperalgesia induced by i.t. injection of glutamate in mice. However, ,-amyrin was not able to alter the [³H]glutamate binding sites to cerebral cortex membranes. These results suggest that the antinociceptive effect of ,-amyrin is unrelated with a direct interaction with glutamate receptors. Some nociceptive actions produced by glutamate are mediated by nitric oxide (NO)-cGMP pathway activation (Ferreira et al., 1999; Beirith et al., 2002). NO is a modulator of the nociceptive processes, being able to produce hyperalgesia or antinociceptive effects, depending on the experimental model and the dose or site of administration tested (Aley et al., 1998). However, the systemic treatment with NO-synthase inhibitors usually produces an antinociceptive effect in the formalin model (Beirith et al., 1998). Our results show that the antinociception caused by ,-amyrin is unlikely to involve any interaction with nitric oxide, since the treatment of animals with L-arginine, a precursor of nitric oxide, in conditions where it consistently reversed the antinociception caused by N^G-nitro-L-arginine (a nitric-oxide synthase inhibitor) (Vaz et al., 1996; Beirith et al., 1998), failed to interfere with ,-amyrin-induced antinociception.

In addition, ,-amyrin also failed to directly interact with vanilloid or with kinin B₂ receptor binding sites, apart from its inhibitory activity in capsaicin-, bradykinin-, or carrageenan-induced mechanical hyperalgesia. Several inflammatory mediators produce nociception by peripheral and spinal sensory fiber sensitization through protein kinase activation, including PKC, PKA, and mitogen-activated kinases (Scholz and Woolf, 2002). Evidence now suggests that the mechanical hyperalgesia produced by PGE₂ could be mediated by peripheral PKA stimulation (Malmberg et al., 1997; Aley and Levine, 1999). Moreover, bradykinin-induced overt nociception and mechanical hyperalgesia is mediated by peripheral activation of PKC and vanilloid receptor (Ferreira et al., 2004). On the other hand, intraplantar capsaicin seems to induce nociceptive action via direct activation of peripheral vanilloid receptors (Ferreira et al., 2004). Also, it has been demonstrated that both PKA and PKC stimulation in spinal cord are involved in capsaicin-induced hyperalgesia (Sluka and Willis, 1997). On top of this, the nociception caused by carrageenan, formalin, and acetic acid is sensitive to PKA or PKC inhibitors or gene deletion (Malmberg et al., 1997; Khasar et al., 1999). Interestingly, systemic treatment with ,-amyrin almost abolishes the mechanical hyperalgesia or the overt nociception produced by intraplantar injection of direct activators of PKA (8-Br-cAMP) or direct activators of PKC (TPA) into the rat or mouse paw. Of note, in vitro studies have shown that ,-amyrin is capable of blocking the activity of both PKC, and, in a special manner, PKA (Hasmeda et al., 1999). Therefore, the ability of ,-amyrin to interact with kinase pathways might explain its ability to inhibit PGE₂-, capsaicin-, and bradykinin-induced nociception without directly interfering with their respective receptor binding sites. Apart from its nociceptive-producing effects, PKC pathway seems also to be involved in the tolerance to the analgesic effect of morphine (Granados-Soto et al., 2000). However, studies must be carried out to verify whether ,-amyrin treatment also could be effective in reducing morphine tolerance.

Hyperalgesia is often observed during painful pathological processes. This sensitization results from activation of different intracellular kinase cascades leading to the phosphorylation of key membrane receptors and channels (Ji and Woolf, 2001). Thus, the interaction of kinases also could explain why ,-amyrin is capable of reducing the thermal hyperalgesia produced by glutamate, which is dependent on retrograde sensitization of primary afferents (Ferreira and Lorenzetti, 1994), without changing the response to acute thermal stimulation. This seems to not be associated with the transmission of acute thermal stimuli (Malmberg et al., 1997; Khasar et al., 1999).

In addition, the antinociceptive effects of ,-amyrin herein reported extend the antiarthritic activity of ,-amyrin triterpenes (Kweifio-Okai et al., 1994a,b) and the antiedematogenic of ,-amyrin effect in carrageenan-induced mouse paw edema (Recio et al., 1995). They also confirm and extend the antinociceptive activity of the triterpene mixture (-amyrin, -amyrin, and baurenol) in the writhes induced by acetic acid (Villasenor et al., 2004). Moreover, these results demonstrate that, at least in part, ,-amyrin is responsible for the antinociceptive effect of P. kleinii.

In summary, we have demonstrated that the mixture of -amyrin and -amyrin triterpenes, isolated from resins of P. kleinii, exhibit dose-related antinociception when assessed in chemical, but not thermal, models of nociception in mice, as well as producing antihyperalgesic effects in models of painful mechanical hypersensitivity in rats. Currently, the precise mechanism involved in its action is not completely understood, but the inhibition of both PKA and PKC pathways stimulated by different algogen mediators seems to account for ,-amyrin's antinociceptive effect.

	Acknowledgements

 
We are indebted to Dr. Ademir Reis for botanical classification of P. kleinii.

	Footnotes

 
This study was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico, Financiadora de Estudos e Projetos, and Programa de Apoio aos Núcleos de Excelência, Brazil. M.F.O. and J.F. are Ph.D. students in pharmacology, respectively, and they thank Conselho Nacional de Desenvolvimento Científico e Tecnológico and Companha de Aperfeiçoamento de Pessoal de Nível Superior for fellowship support.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.104.071779.

ABBREVIATIONS: PCPA, DL-p-chlorophenylalanine methyl ester; L-NOARG, N^G-nitro-L-arginine; BK, bradykinin; TPA, 12-O-tetraradecanoylphorbol-13-acetate; 8-Br-cAMP, 8-bromo-cAMP; i.pl., intraplantar; PGE₂, prostaglandin E₂; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; RTX, resiniferatoxin; PKC, protein kinase C; PKA, protein kinase A; NO, nitric oxide.

Address correspondence to: Dr. João B. Calixto, Department of Pharmacology, Centre of Biological Sciences, Federal University of Santa Catarina, Campus Universitario-Trindade, Bloco D-CCB, Cx. Postal: 476, CEP 88049-900, Florianópolis, SC, Brazil. E-mail: calixto{at}farmaco.ufsc.br or calixto3{at}terra.com.br

	References

Top Abstract Materials and Methods Results Discussion References

 

Akihisa T, Yasukawa K, Oinuma H, Kasahara Y, Yamanouchi S, Takido M, Kumaki K, and Tamura T (1996) Triterpene alcohols from the flowers of Compositae and their anti-inflammatory effects. Phytochemistry 43: 1255-1260.[CrossRef][Medline]

Aley KO and Levine JD (1999) Role of protein kinase A in the maintenance of inflammatory pain. J Neurosci 19: 2181-2186.[Abstract/Free Full Text]

Aley KO, McCarter G, and Levine JD (1998) Nitric oxide signaling in pain and nociceptor sensitization in the rat. J Neurosci 18: 7008-7014.[Abstract/Free Full Text]

Beirith A, Santos AR, and Calixto JB (2002) Mechanisms underlying the nociception and paw oedema caused by injection of glutamate into the mouse paw. Brain Res 924: 219-228.[CrossRef][Medline]

Beirith A, Santos ARS, Rodrigues ALS, Creczynski-Pasa TB, and Calixto JB (1998) Spinal and supraspinal antinociceptive action of dipyrone on formalin, capsaicin and glutamate test. Study of the mechanism of action. Eur J Pharmacol 345: 233-245.[CrossRef][Medline]

Calixto JB, Beirith A, Ferreira J, Santos AR, Filho VC, and Yunes RA (2000) Naturally occurring antinociceptive substances from plants. Phytother Res 14: 401-418.[CrossRef][Medline]

Corrêa CR, Kyle DJ, Chakravarty S, and Calixto JB (1996) Antinociceptive profile of the pseudopeptide B2 bradykinin receptor antagonist NPC 18688 in mice. Br J Pharmacol 117: 552-558.[Medline]

Corrêa P (1984) Dicionário de plantas úteis do Brasil e das Exóticas cultivadas. Imprensa Nacional, vol 1, p 82, Ministério da Agricultura, Rio de Janeiro, Brazil.

D'Amour FE and Smith DL (1941) A method for determining loss of pain sensation. J Pharmacol Exp Ther 72: 74-79.[Abstract/Free Full Text]

Delaveau P, Lallouette P, and Tessier AM (1980) Drogues végétales stimulant l'activitè phagocytaire du système réticulo-endothélial. Planta Med 40: 49-54.[Medline]

Duwiejua M, Zeitlin IJ, Waterman PG, Chapman J, Mhango GJ, and Provan GJ (1993) Anti-inflammatory activity of resins from some species of the plant family Burseraceae. Planta Med 59: 12-16.[Medline]

Eddy NB and Leimbach D (1953) Synthetic analgesics II. Dithienylbutenyl and dithienylbutylamines. J Pharmacol Exp Ther 107: 385-393.[Free Full Text]

Ferreira J, Santos ARS, and Calixto JB (1999) The role of systemic, spinal and supraspinal L-arginine-nitric oxide-cGMP pathway in thermal hyperalgesia caused by intrathecal injection of glutamate in mice. Neuropharmacology 38: 835-842.[CrossRef][Medline]

Ferreira J, Silva GL, and Calixto JB (2004) Contribution of vanilloid receptors to the overt nociception induced by B2 kinin receptor activation in mice. Br J Pharmacol 141: 787-794.[CrossRef][Medline]

Ferreira SH and Lorenzetti BB (1994). Glutamate spinal retrograde sensitization of primary sensory neurons associated with nociception. Neuropharmacology 33: 1479-1485.[CrossRef][Medline]

Granados-Soto V, Kalcheva I, Hua XY, Newton A, and Yaksh TL (2000) Spinal PKC activity and expression: role in tolerance produced by continuous spinal morphine infusion. Pain 85: 395-404.[CrossRef][Medline]

Hasmeda M, Kweifio-Okai G, Macrides T, and Polya GM (1999) Selective inhibition of eukaryote protein kinases by anti-inflammatory triterpenoids. Planta Med 65: 14-18.[CrossRef][Medline]

Hylden JKL and Wilcox GL (1980) Intrathecal morphine in mice: a new technique. Eur J Pharmacol 67: 313-316.[CrossRef][Medline]

Ji RR and Woolf CJ (2001) Neuronal plasticity and signal transduction in nociceptive neurons: implications for the initiation and maintenance of pathological pain. Neurobiol Dis 8: 1-10.[CrossRef][Medline]

Khasar SG, Lin YH, Martin A, Dadgar J, McMahon T, Wang D, Hundle B, Aley KO, Isenberg W, McCarter G, et al. (1999) A novel nociceptor signaling pathway revealed in protein kinase C epsilon mutant mice. Neuron 24: 253-260.[CrossRef][Medline]

Kweifio-Okai G, Bird D, Eu P, Carroll AR, Ambrose R, and Field B (1994a) Effect of alpha-amyrin palmitate on adjuvant arthritis. Drugs Exp Clin Res 20: 1-5.[Medline]

Kweifio-Okai G, De Munk F, Rumble BA, Macrides TA, and Cropley M (1994b) Antiarthritic mechanisms of amyrin triterpenes. Res Commun Chem Pathol Pharmacol 85: 45-55.

Malmberg AB, Brandon EP, Idzerda RL, LI H, Mcknight GS, and Basbaum AI (1997) Diminished inflammation and nociceptive pain with preservation of neuropathic pain in mice with a targeted mutation of the type I regulatory subunit of cAMP-dependent protein kinase. J Neurosci 17: 7462-7470.[Abstract/Free Full Text]

Manning DC, Vavrek R, Stewart JM, and Snyder SH (1986) Two bradykinin binding sites with picomolar affinities. J Pharmacol Exp Ther 237: 504-512[Abstract/Free Full Text]

Otuki MF, Lima FV, Malheiros A, Cechinel-Filho V, Delle Monache F, Yunes RA, and Calixto JB (2001) Evaluation of the antinociceptive action caused by ether fraction and a triterpene isolated from resin of Protium kleinii. Life Sci 69: 2225-2236.[CrossRef][Medline]

Randall LO and Selitto JJ (1957) A method for measurement of analgesic activity on inflamed tissue. Arch Int Pharmacodyn Ther 111: 409-419.[Medline]

Recio MC, Giner RM, Máñez S, and Ríos JL (1995) Structural requirements for the anti-inflammatory activity of natural triterpenoids. Planta Med 61: 182-185.[Medline]

Rosland JH, Hunskaar S, and Hole K (1990) Diazepam attenuates morphine antinociception test-dependently in mice. Pharmacol Toxicol 66: 382-386.[Medline]

Sakurada T, Katsumata K, Tan-no K, Sakurada S, and Kisara K (1992) The capsaicin test in mice for evaluating tachynin antagonist in the spinal cord. Neuropharmacology 31: 1279-1285.[CrossRef][Medline]

Scholz J and Woolf CJ (2002) Can we conquer pain? Nat Neurosci 5: 1062-1067.

Siani AC, Ramos MFS, Menezes-de-Lima O Jr, Ribeiro-dos-Santos R, Fernandez-Ferreira E, Soares ROA, Rosas EC, Susunaga GS, Guimarães AC, Zoghbi MGB, et al. (1999) Evaluation of anti-inflammatory-related activity of essentials oils from the leaves and resin of species of Protium. J Ethnopharmacol 66: 57-69.[CrossRef][Medline]

Siebel JS, Beirith A, and Calixto JB (2004) Evidence for the involvement of metabotropic glutamatergic, neurokinin 1 receptor pathways and protein kinase C in the antinociceptive effect of dipyrone in mice. Brain Res 1003: 61-67.[CrossRef][Medline]

Siqueira JBG, Zoghbi MGB, Cabral JA, and Filho WW (1995) Lignans from Protium tenuifolium. J Nat Prod 58: 730-732.[CrossRef]

Sluka KA and Willis WD (1997) The effects of G-protein and protein kinase inhibitors on the behavioural responses of rats to intradermal injection of capsaicin. Pain 71: 165-178.[CrossRef][Medline]

Subarnas A, Tadano T, Kisara K, and Ohizumi Y (1993). An alpha-adrenoceptor-mediated mechanism of hypoactivity induced by -amyrin palmitate. J Pharm Pharmacol 45: 1006-1008.[Medline]

Szallasi A, Biro T, Modarres S, Garlaschelli L, Petersen M, Klusch A, Vidari G, Jonassohn M, De Rosa S, Sterner O, et al. (1998) Dialdehyde sesquiterpenes and other terpenoids as vanilloids. Eur J Pharmacol 356: 81-89.[CrossRef][Medline]

Taiwo YO and Levine JD (1991) Further confirmation of the role of adenyl cyclase and of cAMP-dependent protein kinase in primary afferent hyperalgesia. Neuroscience 44: 131-135.[CrossRef][Medline]

Vaz ZR, Cechinel Filho V, Yunes RA, and Calixto JB (1996) Antinociceptive action of 2-(4-bromobenzoyl)-3-methyl-4,6-dimethoxy benzofuran, a novel xanthoxyline derivative on chemical and thermal models of nociception in mice. J Pharmacol Exp Ther 278: 304-312.[Abstract/Free Full Text]

Villasenor IM, Canlas AP, Faustino KM, and Plana KG (2004) Evaluation of the bioactivity of triterpene mixture isolated from Carmona retusa (Vahl.) masam leaves. J Ethnopharmacol 92: 53-56.[CrossRef][Medline]

Zimmermann M (1983) Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16: 109-110.[CrossRef][Medline]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.104.071779v1 313/1/310    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Otuki, M. F. Articles by Calixto, J. B. Search for Related Content PubMed PubMed Citation Articles by Otuki, M. F. Articles by Calixto, J. B.