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Antinociceptive effects of the essential oil of Alpinia zerumbet on mice
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Phytomedicine 12 (2005) 482–486 www.elsevier.de/phymed
Antinociceptive effects of the essential oil of Alpinia zerumbet on mice F.V.S. de Arau´jo Pinhoa, A.N. Coelho-de-Souzaa, S.M. Moraisb, C. Ferreira Santosa, J.H. Leal-Cardosoa, a
Departamento de Cieˆncias Fisiolo´gicas, Centro de Cieˆncias da Sau´de, Av. Paranjana 1700, Campus Itaperi, 60.740-000 Fortaleza, CE, Brasil b Departamento de Fı´sica e Quı´mica, Centro de Cieˆncias e Tecnologia, Universidade Estadual do Ceara´, Fortaleza, CE, Brasil Received 20 November 2003; accepted 30 April 2004
Abstract Alpinia zerumbet (Pers.) Burtt. et Smith is an aromatic plant that is distributed widely in the tropical and sub-tropical regions of the world. In Brazil, where A. zerumbet is called ‘‘colonia’’, it is used widely in folk medicine for the treatment of various diseases, including hypertension. In the present study, the antinociceptive effects of the orally administered essential oil of A. zerumbet (EOAz) were evaluated in male Swiss mice (20–25 g each). In the acetic acidinduced writhing test, EOAz (30, 100 and 300 mg/kg body wt.; n ¼ 10, n ¼ 13 and n ¼ 15, respectively) was effective at all doses. In the hot-plate test, EOAz significantly increased the latency at doses of 100 and 300 mg/kg body wt., but not at 30 mg/kg body wt., at all observation times up to the 180th min (n ¼ 10 for each dose). In the formalin test, EOAz significantly reduced paw licking time in the second phase of the test at 100 mg/kg body wt. (n ¼ 10), but decreased it in both phases at 300 mg/kg body wt. (n ¼ 10). At 30 mg/kg body wt., the effect of EOAz did not differ from control values in either phase of the formalin test (n ¼ 10). Pretreatment with naloxone (5 mg/kg body wt., i.p.) caused a significant reversal of the analgesic effect of 300 mg/kg body wt. EOAz (n ¼ 8) that was complete for the first phase, but only partial for the second phase of the formalin test. The data show that orally administered OEAz promotes a dosedependent antinociceptive effect, with a mechanism of action which probably involves the participation of opiate receptors. r 2005 Elsevier GmbH. All rights reserved. Keywords: Alpinia zerumbet; Essential oil; Antinociceptive effects; In vivo
Introduction Alpinia zerumbet is an aromatic plant found in tropical and sub-tropical regions of the world (Joly, 1966). Plants of the genus Alpinia are used in folk medicine all over the world (Itokawa et al., 1981a, b; Mendonc¸a et al., 1991; Prudente et al., 1993). A Corresponding author. Tel.: +85 299 2714.
E-mail address: [email protected] (J.H. Leal-Cardoso). 0944-7113/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2004.04.006
zerumbet, also named Alpinia speciosa, is known popularly as ‘‘colonia’’ in northeastern Brazil, where it receives widespread use in folk medicine, predominantly in the treatment of intestinal disorders and hypertension (Matos, 1987; Prudente et al., 1993; Leal-Cardoso and Fonteles, 1999). A. zerumbet medicinal users employ teas and infusates prepared from its leaves (Matos, 1987; Leal-Cardoso and Fonteles, 1999). The essential oil of A. zerumbet (EOAz), which is rich in terpinen-4-ol, 1,8-cineol and methyl-eugenol, is
ARTICLE IN PRESS F.V.S. de Arau´jo Pinho et al. / Phytomedicine 12 (2005) 482–486
present in relatively large quantities in the leaves of the plant (1% yield) (Craveiro et al., 1981). EOAz has shown efficacy as an antispasmodic agent in intestinal (Bezerra et al., 2000) and vascular smooth muscle and as an anti-hypertensive agent (Lahlou et al., 2002), effects consistent with its use in folk medicine. Despite the popularity of A. zerumbet as an herbal remedy and the documented efficacy of EOAz, A. zerumbet and its essential oil have received little scientific attention. Available studies have dealt only with the chemical composition of EOAz and its pharmacological effects on intestinal and vascular smooth muscle and on arterial pressure (Leal-Cardoso and Fonteles, 1999; Bezerra et al., 2000; Lahlou et al., 2002). Pharmacological investigations have shown EOAz to be a potential antispasmodic and anti-hypertensive agent, consistent with its use in folk medicine, and lend relevance to the question of whether EOAz has other biological activities. In the present study, we have attempted to answer that question by determining whether EOAz has antinociceptive activity. We found that EOAz induces antionociception via a mechanism that seems to partially involve opioid neurotransmission.
Material and methods Alpinia zerumbet (Pers.) Burt. et Smith leaves were collected in October 2000, at Pacoti county, Ceara´, Brazil. Its botanical identity was determined by Dr. Francisco J. Abreu Matos (Natural Products Laboratory of Federal University of Ceara´, UFC). A voucher specimen has been deposited in the Prisco Bezerra herbarium (UFC), under the number 10858. EOAz was extracted from freshly chopped plant leaves by steam distillation in a Clevenger-type apparatus (Craveiro et al., 1976) and analyzed by gas chromatography/mass spectroscopy. The identification of the constituents was performed by computer library search, retention indices and visual interpretation of the mass spectra (Adams, 1995; Alencar et al., 1984). The presence of the main constituents was confirmed 1H- and 13Cnuclear magnetic resonance spectroscopy. The main percentage composition of EOAz was 4-terpineol (28.09%), 1,8-cineol (15.05%) g-terpinene (13.71%), sabinene (7.09%), p-cymene (7.35%), a-tujeno (5.56%), b-pinene (5.35), a-terpinene (2.55%), limonene (2.42%), apinene (2.36%), trans-caryophyllene (2.33%), terpinolene (1.5%), a-terpineol (1.17%), and b-mircene (0.84%). Experiments were performed on male Swiss mice (20–25 g each) deprived of food, but with free access to drinking water for 12 h prior to the experiments. Animals were provided by the vivarium of UFC. All animals were cared for in compliance with the Guide for
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the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication 85–23, revised 1985). The EOAz antinociceptive activity was quantified using the writhing, the hot plate and formalin tests. The writhing test was performed according to the method of Koster et al. (1959). Briefly, 0.1 ml/10 g body wt. of an 0.8% (v/v) acetic acid solution in saline was administered by intraperitoneal injection. Abdominal contortions were counted during a 20-min period, starting 10 min after acetic acid injection. Antinocinociceptive effect was expressed as the reduction of the number of abdominal constrictions. To perform the hot-plate test, the method of Jacob et al. (1974) was used, as follows. A mouse was placed on a plate maintained at 50.071 1C and the latency of its response (number of seconds before it licked its hind paw or jumped) to this nociceptive stimulus was quantified, with an interruption time p45 s. Only mice which in a pretest showed a hot-plate reaction time of p20 s were used in this test. The latency of the reaction to nociception was measured at time 0 (60 min after pharmacological agent administration) and then at 30min intervals up to the 180th min. The formalin test was performed according to the method of Hunskaar et al. (1985). Briefly, 20 ml of a 1% (v/v) solution of formalin in saline was injected into the subplantar region of the right hind paw and the quantification of the time that the animal spent licking the paw during the first 5 min (early phase) and from 15 to 30 min (late phase) of post-injection time was performed. The test was performed at ambient temperature of 22–26 1C and care was taken to exclude environmental disturbances (high temperature, noise and excessive movement) that might interfere with the animal’s response (Tjolsen et al., 1992). In the writhing, hot-plate and formalin tests, EOAz solubilized in vehicle (a solution of 0.1% Tween 80 in sterile saline) or vehicle only (control) was administered orally with an orogastric cannula 60 min before initiation of noxious stimulation. Morphine was administered subcutaneously, while indomethacin and naloxone were administered by the intraperitoneal route. Tween 80, naloxone and indomethacin were from Sigma (St. Louis, MO, USA), morphine was obtained from Crista´lia (Rio de Janeiro, RJ, Brazil), and acetic acid and formalin were from Reagen (Rio de Janeiro, RJ, Brazil). EOAz solutions were prepared daily by vigorous manual shaking (3–5 min) or by vortexing the EOAz in vehicle. The final EOAz concentration was selected so as to inject a constant volume of 0.1 ml solution/10 g body wt. Results are reported as means7s.e.m. (n), with n indicating the number of animals. Data were analyzed using the Student t-test, ANOVA, or nonparametric tests as appropriate, and were considered significant at pp0:05.
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Results Writhing test Intraperitoneal injection of acetic acid induced 48.572.72 (n ¼ 18) contortions in control rats during the 20 min observation period. For the animals who received EOAz, dosed at 10, 30, 100 and 300 mg/ kg body wt., this number was 48.571.93 (n ¼ 5), 36.074.84 (n ¼ 9), 17.273.71 (n ¼ 13) and 10.872.22 (n ¼ 18), respectively (Fig. 1). For comparison, in a group of animals dosed with 25 mg/kg body wt. indomethacin (administered 30 min prior to noxious stimulation), this number was reduced to 8.071.93 (n ¼ 6). Only the alterations induced by EOAz dosed at 30, 100 and 300 mg/kg body wt. and by indomethacin were significant (pp0:05, ANOVA, Dunnett’s t-test).
Hot plate test At 100 and 300 mg/kg body wt., EOAz, at the hotplate test, increased the latency time for nociception significantly above the control value (pp0:05, ANOVA, Dunnett test) throughout the period of observation (30–180 min; Fig. 2). At 30 mg/kg body wt., the significance of the EOAz effect did not surpass the 150-min observation. The latency increase induced by 100 and 300 mg/kg body wt. EOAz was smaller than that induced by a 10 mg/kg body wt. dose of morphine. However, at both doses, EOAz and morphine effect durations were similar.
Fig. 1. Effect of the essential oil of Alpinia zerumbet (EOAz) on the nociception induced by intraperitoneal injection of acetic acid. Ordinate, number of contortions. Mean7s.e.m.*, significantly different from control (po0:05, ANOVA, Dunnett’s t-test) at a given time.
Fig. 2. Time-course of the effects of the essential oil of Alpinia zerumbet (EOAz) on thermal nociception. Abscissa, time (min) after EOAz (oral), morphine (i.p.) or dipirone (i.p.) administration. Ordinate, latency time (s) for the response to thermal stimulation (5070.5 1C. Mean7s.e.m., n ¼ 12) for each EOAz dose.*, significantly different from control (po0:05, ANOVA, Dunnett’s t-test) at a given time.
Formalin test One hour after the administration of 300 mg/kg body wt. EOAz, there was a significant reduction (pp0:05, ANOVA, Bonferroni t-test) in the number of seconds the mice spent liking their paws in the first and second phase, which reached 22% and 93.99% (n ¼ 10), respectively, of controls (70.473.04 and 142.7715.09 s, n ¼ 11) (Fig. 3A). At the dose of 100 mg/kg body wt., EOAz (pp0:05, ANOVA, Bonferroni t-test, n ¼ 10) reduced (38.53%) only the second phase of the test significantly. At 30 mg/kg body wt., no significant reduction occurred. The analgesic effect of morphine (5 mg/kg body wt.) on both phases of the formalin test was reversed significantly by naloxone (5 mg/kg body wt.) (Fig. 2B). Naloxone also reversed the effect of 300 mg/kg body wt. EOAz totally in the first phase, as in the presence of naloxone and EOAz, the licking time of this phase returned to control. For the second phase, however, naloxone reversed only partially the effect of 300 mg/kg body wt. EOAz, as in the presence of naloxone and EOAz the licking time of this phase was significantly different from control and from the value measured in the presence of EOAz alone (Fig. 3B) (pp0:05, ANOVA, Dunnett’s t-test). Using a 300 mg/kg body wt. dose of EOAz, the time course of its effects in the formalin test was investigated. The results are shown in Fig. 4. The inhibition of second-phase response to formalin peaked at the 60th min after EOAz administration. Afterwards, this EOAz
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Fig. 4. Time-course of the effects of the essential oil of Alpinia zerumbet (EOAz) on the nociception induced by intraplantar injection of formaline (1%). EOAz, 300 mg/kg body wt., p.o. administration. Ordinate, time (s) that the mice spent licking the paw; h, hours. Mean7s.e.m., number on the top of columns, number of experiments.*, significantly different from control (po0:05, ANOVA, Dunnett’s t-test). h, hours.
Fig. 3. Effects of the essential oil of Alpinia zerumbet (EOAz) on the nociception induced by intraplantar injection of formaline (1%): (A) effect of EOAz (mg/kg body wt.) on the first and second phase of formaline test; (B) reversal, by naloxone (2 mg/kg body wt.), of the antinococeptive effects of morphine (Morph, 4 mg/kg body wt.); and EOAz (300 mg/ kg body wt.). Ordinate, time (s) that the mice spent licking the paw. Mean7s.e.m., number on the top of columns, number of experiments.*, significantly different from control; a, b, not different from control but significantly different from morphine and EOAz, alone, respectively; c, significantly different from control and from EOAz alone (po0:05, ANOVA, Dunnett’s t-test).
effect vanished progressively and full recovery was attained at the fourth hour. In the first phase of the test, a significant inhibition of the response was maintained from the end of the first to the third hour, but during this period of time the level of response depression remained approximately constant.
Discussion The main discovery of our study is that A. zerumbet essential oil possesses antinociceptive activity. This
conclusion is corroborated by three different evaluations, the writing, hot-plate and formalin tests. To the best of our knowledge this is the first report of such activity. This discovery enlarges the list of pharmacological effects already described for EOAz, which include antispasmodic (Bezerra et al., 2000) and antihypertensive (Lahlou et al., 2002) activities. At 300 mg/kg body wt., EOAz was effective during the first phase of the formalin test. Response during the first phase is attributed to a peripheral neural mechanism (Hunskaar and Hole, 1987). This suggests the participation of this type of mechanism in the antinociception induced by this dose of EOAz. It is important to note, however, that although EOAz affected the first phase, it did so only at the highest dose employed and with an efficacy much smaller than that of the same dose of EOAz in the second phase of this test, suggesting that activity through a peripheral neural mechanism is not the main route for EOAz antinociceptive activity. The effectiveness of EOAz in the hot-plate test demonstrated here indicates that this analgesic agent acts primarily in the spinal medulla and/or higher central nervous system levels or by an indirect mechanism (Hunskaar et al., 1985; Hunskaar and Hole, 1987; Yaksh and Rudy, 1977). This result implies therefore that EOAz activity probably includes a component with a central mechanism. EOAz showed antinociception in the second phase of the formalin test at 100 mg/ kg body wt., and noxious stimulation of this phase is attributed to inflammatory activity and/or alteration of central processing (Hunskaar and Hole, 1987; Shibata et al., 1989; Santos and Rao, 2000). Thus, the effectiveness during this phase of the formalin test is consistent with a central mechanism or with an indirect effect via anti-inflammatory action. Our data suggest that the participation in the central mechanism involves opiate receptors, because the naloxone (Jacob et al., 1974),
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which blocked the effect of morphine, reversed the EOAz-induced antinociception partially in the second and completely in the first phase of the formalin test. The major constituents of EOAz, 1,8-cineol, terpinen4-ol and g-terpinene, are likely to contribute to its overall effect. The 1,8-cineol is likely to participate in the induction of this effect, as this substance in the oral doses between 100 and 400 mg/kg body wt., has been reported to bear antinociceptive activity (Santos and Rao, 2000), and 15.05% of EOAz is 1,8-cineol. In connection with the mechanism of action of EOAz, 1,8cineol was found to act by an opioid mechanism (Santos and Rao, 2000). Terpineols (Moreira et al., 2001) have been reported as central and peripheral neural depressants, which raise the suspicion that they might act as antinociceptive agents. For the other constituents, we found no report in the literature of either antinociceptive or neural activity. A. zerumbet is used by the people of northeastern Brazil as an anti-hypertensive and diuretic agent. Our laboratory has confirmed by in vitro studies that EOAz has anti-hypertensive activity (Leal-Cardoso and Fonteles, 1999; Lahlou et al., 2002), which is consistent with the use of the plant in folk medicine. In the present study we detected analgesic activity as an additional pharmacological effect of EOAz. Unpublished experiments from this laboratory have shown that the LD50 value of EOAz (administered per os) is 42.33 g/kg body wt. Because the analgesic activity of EOAz was induced at doses far below the LD50 value, this effect is of potential therapeutical use, and this essential oil deserves further pharmacological investigation.
References Adams, R.P., 1995. Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Allured Publishing Corporation, Carol Stream, Illinois, USES. Alencar, J.W., Craveiro, A.A., Matos, F.J.A., 1984. Kovats indexes as a pre-s routine in mass spectra library search of volatiles. J. Nat. Prod. 47, 890–892. Bezerra, M.A.C., Leal-Cardoso, J.H., Coelho-de-Souza, A.N., Criddle, D.N., Fonteles, M.C., 2000. Myorelaxant and antispasmodic effects of the essential oil of Alpinia speciosa on rat ileum. Phytother. Res. 14 (7), 549–551. Craveiro, A.A., Matos, F.J.A., Alencar, J.W., 1976. A simple and inexpensive steam generator for essential oils extraction. J. Chem. Ed. 53, 652. Craveiro, A.A., Fernandes, A.G., Andrade, C.H.S., Matos, F.J., 1981. O`leos essenciais de plantas do Nordeste: 209. Edic¸o˜es UFC, Fortaleza, CE, Brasil.
Hunskaar, S., Hole, K., 1987. The formalin test in mice: dissotiation between inflammatory and non-inflammatory pain. Pain 30, 103–114. Hunskaar, S., Fasmer, O.B., Hole, K., 1985. Formalin test in mice, a useful technique for evaluating mild analgesics. J. Neurosci. Meth. 14, 69–76. Itokawa, H., Aiyama, R., Ikuta, A.A., 1981a. Pungent diarylheptanoid from Alpinia oxyphylla. Phytochemistry 20 (4), 769–771. Itokawa, H., Morita, H., Mihashi, S., 1981b. Two new diarylheptanoid from Alpinia officinarum Hance. Chem. Pharm. Bull. 29 (8), 2383–2385. Jacob, J.J., Tremblay, E.C., Colombel, M.C., 1974. Enhancement of nociception reactions by naloxone in mice and rats. Psychopharmacology 37, 217–223. Joly, A.A., 1966. Botaˆnica: introduc¸a˜o a` taxonomia vegetal, Se´rie Cieˆncias Puras, 3. b.4: 5778–5783. Sa˜o Paulo, EDUSP. Koster, R., Anderson, M., Debeer, E.J., 1959. Acetic acid for analgesic screening. Fed. Proc. 18, 412–414. Lahlou, S., Galindo, C.A.B., Leal-Cardoso, J.H., et al., 2002. Cardiovascular effects of the essential oil of Alpinia zerumbet leaves and its main constituent, terpinen-4-ol, in rats: role of the autonomic nervous system. Planta Med. 68 (12), 1097–1102. Leal-Cardoso, J.H., Fonteles, M.C., 1999. Pharmacological effects of essential oils plants of the Northeast of Brazil. Anais da Academia Brasileira de Cieˆncias 71 (2), 207–213. Matos, F.J.A., 1987. O Formula´rio Fitotera´pico do Prof. Dias da Rocha, 1. 5. ed.: 11–18, 205. Colec¸a˜o ESAM, Ano 20, v. 18. Mendonc¸a, V.L.M., Oliveira, C.L.A., Craveiro, A.A., Rao, V.S., Fonteles, M.C., 1991. Pharmacological and toxicological evaluation of Alpinia speciosa. Mem. Inst. Oswaldo Cruz. 86, 93–97. Moreira, M.R., Cruz, G.M., Lopes, M.S., Albuquerque, A.A., Leal-Cardoso, J.H., 2001. Effects of terpineol on the compound action potential of the rat sciatic nerve. Braz. J. Med. Biol. Res. 34, 1337–1340. Prudente, D., Perineau, F., Bessiere, J.M., Michel, G., Bravo, R., 1993. Chemical analysis, bacteriostatic and fungistatic properties of the essential oil of the atouman from Martinique (Alpinia speciosa K. Schum). J. Essent. Oil Res. 5 (3), 255–264. Santos, F.A., Rao, V.S., 2000. Antiinflammatory and antinociceptive effects of 1,8-cineole, a terpenoid oxid present in many plant essential oil. Phytother. Res. 14, 240–244. Shibata, M., Ohkubo, T., Takahashi, H., Inoki, R., 1989. Modified formalin test: characteristic biphasic pain response. Pain 38, 347–352. Tjolsen, A., Berge, O.G., Hunskaar, S., Rosland, J.H., Hole, K., 1992. The formalin test: an evaluation of the method. Pain 51, 5–17. Yaksh, T.L., Rudy, T.A., 1977. Studies on the direct spinal action of narcotics in the prodution of analgesia in the rat. J. Pharmacol. Exp. Ther. 202 (2), 411–428.
Phytomedicine 12 (2005) 482–486 www.elsevier.de/phymed
Antinociceptive effects of the essential oil of Alpinia zerumbet on mice F.V.S. de Arau´jo Pinhoa, A.N. Coelho-de-Souzaa, S.M. Moraisb, C. Ferreira Santosa, J.H. Leal-Cardosoa, a
Departamento de Cieˆncias Fisiolo´gicas, Centro de Cieˆncias da Sau´de, Av. Paranjana 1700, Campus Itaperi, 60.740-000 Fortaleza, CE, Brasil b Departamento de Fı´sica e Quı´mica, Centro de Cieˆncias e Tecnologia, Universidade Estadual do Ceara´, Fortaleza, CE, Brasil Received 20 November 2003; accepted 30 April 2004
Abstract Alpinia zerumbet (Pers.) Burtt. et Smith is an aromatic plant that is distributed widely in the tropical and sub-tropical regions of the world. In Brazil, where A. zerumbet is called ‘‘colonia’’, it is used widely in folk medicine for the treatment of various diseases, including hypertension. In the present study, the antinociceptive effects of the orally administered essential oil of A. zerumbet (EOAz) were evaluated in male Swiss mice (20–25 g each). In the acetic acidinduced writhing test, EOAz (30, 100 and 300 mg/kg body wt.; n ¼ 10, n ¼ 13 and n ¼ 15, respectively) was effective at all doses. In the hot-plate test, EOAz significantly increased the latency at doses of 100 and 300 mg/kg body wt., but not at 30 mg/kg body wt., at all observation times up to the 180th min (n ¼ 10 for each dose). In the formalin test, EOAz significantly reduced paw licking time in the second phase of the test at 100 mg/kg body wt. (n ¼ 10), but decreased it in both phases at 300 mg/kg body wt. (n ¼ 10). At 30 mg/kg body wt., the effect of EOAz did not differ from control values in either phase of the formalin test (n ¼ 10). Pretreatment with naloxone (5 mg/kg body wt., i.p.) caused a significant reversal of the analgesic effect of 300 mg/kg body wt. EOAz (n ¼ 8) that was complete for the first phase, but only partial for the second phase of the formalin test. The data show that orally administered OEAz promotes a dosedependent antinociceptive effect, with a mechanism of action which probably involves the participation of opiate receptors. r 2005 Elsevier GmbH. All rights reserved. Keywords: Alpinia zerumbet; Essential oil; Antinociceptive effects; In vivo
Introduction Alpinia zerumbet is an aromatic plant found in tropical and sub-tropical regions of the world (Joly, 1966). Plants of the genus Alpinia are used in folk medicine all over the world (Itokawa et al., 1981a, b; Mendonc¸a et al., 1991; Prudente et al., 1993). A Corresponding author. Tel.: +85 299 2714.
E-mail address: [email protected] (J.H. Leal-Cardoso). 0944-7113/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2004.04.006
zerumbet, also named Alpinia speciosa, is known popularly as ‘‘colonia’’ in northeastern Brazil, where it receives widespread use in folk medicine, predominantly in the treatment of intestinal disorders and hypertension (Matos, 1987; Prudente et al., 1993; Leal-Cardoso and Fonteles, 1999). A. zerumbet medicinal users employ teas and infusates prepared from its leaves (Matos, 1987; Leal-Cardoso and Fonteles, 1999). The essential oil of A. zerumbet (EOAz), which is rich in terpinen-4-ol, 1,8-cineol and methyl-eugenol, is
ARTICLE IN PRESS F.V.S. de Arau´jo Pinho et al. / Phytomedicine 12 (2005) 482–486
present in relatively large quantities in the leaves of the plant (1% yield) (Craveiro et al., 1981). EOAz has shown efficacy as an antispasmodic agent in intestinal (Bezerra et al., 2000) and vascular smooth muscle and as an anti-hypertensive agent (Lahlou et al., 2002), effects consistent with its use in folk medicine. Despite the popularity of A. zerumbet as an herbal remedy and the documented efficacy of EOAz, A. zerumbet and its essential oil have received little scientific attention. Available studies have dealt only with the chemical composition of EOAz and its pharmacological effects on intestinal and vascular smooth muscle and on arterial pressure (Leal-Cardoso and Fonteles, 1999; Bezerra et al., 2000; Lahlou et al., 2002). Pharmacological investigations have shown EOAz to be a potential antispasmodic and anti-hypertensive agent, consistent with its use in folk medicine, and lend relevance to the question of whether EOAz has other biological activities. In the present study, we have attempted to answer that question by determining whether EOAz has antinociceptive activity. We found that EOAz induces antionociception via a mechanism that seems to partially involve opioid neurotransmission.
Material and methods Alpinia zerumbet (Pers.) Burt. et Smith leaves were collected in October 2000, at Pacoti county, Ceara´, Brazil. Its botanical identity was determined by Dr. Francisco J. Abreu Matos (Natural Products Laboratory of Federal University of Ceara´, UFC). A voucher specimen has been deposited in the Prisco Bezerra herbarium (UFC), under the number 10858. EOAz was extracted from freshly chopped plant leaves by steam distillation in a Clevenger-type apparatus (Craveiro et al., 1976) and analyzed by gas chromatography/mass spectroscopy. The identification of the constituents was performed by computer library search, retention indices and visual interpretation of the mass spectra (Adams, 1995; Alencar et al., 1984). The presence of the main constituents was confirmed 1H- and 13Cnuclear magnetic resonance spectroscopy. The main percentage composition of EOAz was 4-terpineol (28.09%), 1,8-cineol (15.05%) g-terpinene (13.71%), sabinene (7.09%), p-cymene (7.35%), a-tujeno (5.56%), b-pinene (5.35), a-terpinene (2.55%), limonene (2.42%), apinene (2.36%), trans-caryophyllene (2.33%), terpinolene (1.5%), a-terpineol (1.17%), and b-mircene (0.84%). Experiments were performed on male Swiss mice (20–25 g each) deprived of food, but with free access to drinking water for 12 h prior to the experiments. Animals were provided by the vivarium of UFC. All animals were cared for in compliance with the Guide for
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the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication 85–23, revised 1985). The EOAz antinociceptive activity was quantified using the writhing, the hot plate and formalin tests. The writhing test was performed according to the method of Koster et al. (1959). Briefly, 0.1 ml/10 g body wt. of an 0.8% (v/v) acetic acid solution in saline was administered by intraperitoneal injection. Abdominal contortions were counted during a 20-min period, starting 10 min after acetic acid injection. Antinocinociceptive effect was expressed as the reduction of the number of abdominal constrictions. To perform the hot-plate test, the method of Jacob et al. (1974) was used, as follows. A mouse was placed on a plate maintained at 50.071 1C and the latency of its response (number of seconds before it licked its hind paw or jumped) to this nociceptive stimulus was quantified, with an interruption time p45 s. Only mice which in a pretest showed a hot-plate reaction time of p20 s were used in this test. The latency of the reaction to nociception was measured at time 0 (60 min after pharmacological agent administration) and then at 30min intervals up to the 180th min. The formalin test was performed according to the method of Hunskaar et al. (1985). Briefly, 20 ml of a 1% (v/v) solution of formalin in saline was injected into the subplantar region of the right hind paw and the quantification of the time that the animal spent licking the paw during the first 5 min (early phase) and from 15 to 30 min (late phase) of post-injection time was performed. The test was performed at ambient temperature of 22–26 1C and care was taken to exclude environmental disturbances (high temperature, noise and excessive movement) that might interfere with the animal’s response (Tjolsen et al., 1992). In the writhing, hot-plate and formalin tests, EOAz solubilized in vehicle (a solution of 0.1% Tween 80 in sterile saline) or vehicle only (control) was administered orally with an orogastric cannula 60 min before initiation of noxious stimulation. Morphine was administered subcutaneously, while indomethacin and naloxone were administered by the intraperitoneal route. Tween 80, naloxone and indomethacin were from Sigma (St. Louis, MO, USA), morphine was obtained from Crista´lia (Rio de Janeiro, RJ, Brazil), and acetic acid and formalin were from Reagen (Rio de Janeiro, RJ, Brazil). EOAz solutions were prepared daily by vigorous manual shaking (3–5 min) or by vortexing the EOAz in vehicle. The final EOAz concentration was selected so as to inject a constant volume of 0.1 ml solution/10 g body wt. Results are reported as means7s.e.m. (n), with n indicating the number of animals. Data were analyzed using the Student t-test, ANOVA, or nonparametric tests as appropriate, and were considered significant at pp0:05.
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Results Writhing test Intraperitoneal injection of acetic acid induced 48.572.72 (n ¼ 18) contortions in control rats during the 20 min observation period. For the animals who received EOAz, dosed at 10, 30, 100 and 300 mg/ kg body wt., this number was 48.571.93 (n ¼ 5), 36.074.84 (n ¼ 9), 17.273.71 (n ¼ 13) and 10.872.22 (n ¼ 18), respectively (Fig. 1). For comparison, in a group of animals dosed with 25 mg/kg body wt. indomethacin (administered 30 min prior to noxious stimulation), this number was reduced to 8.071.93 (n ¼ 6). Only the alterations induced by EOAz dosed at 30, 100 and 300 mg/kg body wt. and by indomethacin were significant (pp0:05, ANOVA, Dunnett’s t-test).
Hot plate test At 100 and 300 mg/kg body wt., EOAz, at the hotplate test, increased the latency time for nociception significantly above the control value (pp0:05, ANOVA, Dunnett test) throughout the period of observation (30–180 min; Fig. 2). At 30 mg/kg body wt., the significance of the EOAz effect did not surpass the 150-min observation. The latency increase induced by 100 and 300 mg/kg body wt. EOAz was smaller than that induced by a 10 mg/kg body wt. dose of morphine. However, at both doses, EOAz and morphine effect durations were similar.
Fig. 1. Effect of the essential oil of Alpinia zerumbet (EOAz) on the nociception induced by intraperitoneal injection of acetic acid. Ordinate, number of contortions. Mean7s.e.m.*, significantly different from control (po0:05, ANOVA, Dunnett’s t-test) at a given time.
Fig. 2. Time-course of the effects of the essential oil of Alpinia zerumbet (EOAz) on thermal nociception. Abscissa, time (min) after EOAz (oral), morphine (i.p.) or dipirone (i.p.) administration. Ordinate, latency time (s) for the response to thermal stimulation (5070.5 1C. Mean7s.e.m., n ¼ 12) for each EOAz dose.*, significantly different from control (po0:05, ANOVA, Dunnett’s t-test) at a given time.
Formalin test One hour after the administration of 300 mg/kg body wt. EOAz, there was a significant reduction (pp0:05, ANOVA, Bonferroni t-test) in the number of seconds the mice spent liking their paws in the first and second phase, which reached 22% and 93.99% (n ¼ 10), respectively, of controls (70.473.04 and 142.7715.09 s, n ¼ 11) (Fig. 3A). At the dose of 100 mg/kg body wt., EOAz (pp0:05, ANOVA, Bonferroni t-test, n ¼ 10) reduced (38.53%) only the second phase of the test significantly. At 30 mg/kg body wt., no significant reduction occurred. The analgesic effect of morphine (5 mg/kg body wt.) on both phases of the formalin test was reversed significantly by naloxone (5 mg/kg body wt.) (Fig. 2B). Naloxone also reversed the effect of 300 mg/kg body wt. EOAz totally in the first phase, as in the presence of naloxone and EOAz, the licking time of this phase returned to control. For the second phase, however, naloxone reversed only partially the effect of 300 mg/kg body wt. EOAz, as in the presence of naloxone and EOAz the licking time of this phase was significantly different from control and from the value measured in the presence of EOAz alone (Fig. 3B) (pp0:05, ANOVA, Dunnett’s t-test). Using a 300 mg/kg body wt. dose of EOAz, the time course of its effects in the formalin test was investigated. The results are shown in Fig. 4. The inhibition of second-phase response to formalin peaked at the 60th min after EOAz administration. Afterwards, this EOAz
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Fig. 4. Time-course of the effects of the essential oil of Alpinia zerumbet (EOAz) on the nociception induced by intraplantar injection of formaline (1%). EOAz, 300 mg/kg body wt., p.o. administration. Ordinate, time (s) that the mice spent licking the paw; h, hours. Mean7s.e.m., number on the top of columns, number of experiments.*, significantly different from control (po0:05, ANOVA, Dunnett’s t-test). h, hours.
Fig. 3. Effects of the essential oil of Alpinia zerumbet (EOAz) on the nociception induced by intraplantar injection of formaline (1%): (A) effect of EOAz (mg/kg body wt.) on the first and second phase of formaline test; (B) reversal, by naloxone (2 mg/kg body wt.), of the antinococeptive effects of morphine (Morph, 4 mg/kg body wt.); and EOAz (300 mg/ kg body wt.). Ordinate, time (s) that the mice spent licking the paw. Mean7s.e.m., number on the top of columns, number of experiments.*, significantly different from control; a, b, not different from control but significantly different from morphine and EOAz, alone, respectively; c, significantly different from control and from EOAz alone (po0:05, ANOVA, Dunnett’s t-test).
effect vanished progressively and full recovery was attained at the fourth hour. In the first phase of the test, a significant inhibition of the response was maintained from the end of the first to the third hour, but during this period of time the level of response depression remained approximately constant.
Discussion The main discovery of our study is that A. zerumbet essential oil possesses antinociceptive activity. This
conclusion is corroborated by three different evaluations, the writing, hot-plate and formalin tests. To the best of our knowledge this is the first report of such activity. This discovery enlarges the list of pharmacological effects already described for EOAz, which include antispasmodic (Bezerra et al., 2000) and antihypertensive (Lahlou et al., 2002) activities. At 300 mg/kg body wt., EOAz was effective during the first phase of the formalin test. Response during the first phase is attributed to a peripheral neural mechanism (Hunskaar and Hole, 1987). This suggests the participation of this type of mechanism in the antinociception induced by this dose of EOAz. It is important to note, however, that although EOAz affected the first phase, it did so only at the highest dose employed and with an efficacy much smaller than that of the same dose of EOAz in the second phase of this test, suggesting that activity through a peripheral neural mechanism is not the main route for EOAz antinociceptive activity. The effectiveness of EOAz in the hot-plate test demonstrated here indicates that this analgesic agent acts primarily in the spinal medulla and/or higher central nervous system levels or by an indirect mechanism (Hunskaar et al., 1985; Hunskaar and Hole, 1987; Yaksh and Rudy, 1977). This result implies therefore that EOAz activity probably includes a component with a central mechanism. EOAz showed antinociception in the second phase of the formalin test at 100 mg/ kg body wt., and noxious stimulation of this phase is attributed to inflammatory activity and/or alteration of central processing (Hunskaar and Hole, 1987; Shibata et al., 1989; Santos and Rao, 2000). Thus, the effectiveness during this phase of the formalin test is consistent with a central mechanism or with an indirect effect via anti-inflammatory action. Our data suggest that the participation in the central mechanism involves opiate receptors, because the naloxone (Jacob et al., 1974),
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which blocked the effect of morphine, reversed the EOAz-induced antinociception partially in the second and completely in the first phase of the formalin test. The major constituents of EOAz, 1,8-cineol, terpinen4-ol and g-terpinene, are likely to contribute to its overall effect. The 1,8-cineol is likely to participate in the induction of this effect, as this substance in the oral doses between 100 and 400 mg/kg body wt., has been reported to bear antinociceptive activity (Santos and Rao, 2000), and 15.05% of EOAz is 1,8-cineol. In connection with the mechanism of action of EOAz, 1,8cineol was found to act by an opioid mechanism (Santos and Rao, 2000). Terpineols (Moreira et al., 2001) have been reported as central and peripheral neural depressants, which raise the suspicion that they might act as antinociceptive agents. For the other constituents, we found no report in the literature of either antinociceptive or neural activity. A. zerumbet is used by the people of northeastern Brazil as an anti-hypertensive and diuretic agent. Our laboratory has confirmed by in vitro studies that EOAz has anti-hypertensive activity (Leal-Cardoso and Fonteles, 1999; Lahlou et al., 2002), which is consistent with the use of the plant in folk medicine. In the present study we detected analgesic activity as an additional pharmacological effect of EOAz. Unpublished experiments from this laboratory have shown that the LD50 value of EOAz (administered per os) is 42.33 g/kg body wt. Because the analgesic activity of EOAz was induced at doses far below the LD50 value, this effect is of potential therapeutical use, and this essential oil deserves further pharmacological investigation.
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