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Temporal variation of chemical composition and relaxant action of the essential oil of Ocimum gratissimum L. (Labiatae) on guinea-pig ileum.

Abstract

The medicinal plant Ocimum gratissimum L. (Labiatae) is widely encountered in the Northeast of Brasil where it is used to treat digestive problems. Its leaves have an essential oil (EOOG) content whose chemical composition varies according to the time of plant collection. We have compared the effects of the EOOG, collected at 08:00 a.m. (EOO[G.sub.8]) and at 12:00 a.m. (EOO[G.sub.12]), on the relaxation of guinea-pig isolated ileum. Both EOO[G.sub.8] and EOO[G.sub.12] (30-300 [micro]g/ml) reversibly relaxed the spontaneous tonus of the guinea-pig ileum in a concentration-dependent manner, with similar I[C.sub.50] values (49.3 and 23.8 [micro]g/ml, respectively). The magnitude of the decrease in resting tonus was similar to that of the recognised smooth muscle relaxant papaverine. EOO[G.sub.8] and EOO[G.sub.12] relaxed 60 mM KCl-precontracted preparations similarly (38.33 [+ or -] 9.91 [micro]g/ml and 35.53 [+ or -] 6.70), whereas a significantly more potent relaxant effect of EOO[G.sub.12] compared to EOO[G.sub.8] was observed when tissues were contracted using 10 [micro]M acetylcholine (I[C.sub.50] values of 69.55 [+ or -] 4.93 and 128.16 [+ or -] 15.70 [micro]g/ml, respectively; p < 0.05). The principal constituents of the essential oil, eugenol and cineole, also relaxed KCl-precontracted preparations, although they were less potent than EOOG, suggesting that they alone were not responsible for EOOG-induced relaxations. Our results show that the essential oil extracted from the leaves of O. gratissimum L., collected at different time periods, exerts significant relaxant effects on isolated guinea-pig ileum which may underlie the therapeutic action of the plant.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Ocimum gratissimum; Relaxation; Ileum; Eugenol; Chemical composition

Introduction

The plant Ocimum gratissimum L. (Labiatae) is widely encountered in the Northeast of Brasil, where it is commonly used for its medicinal properties and in cooking. One of the popular uses of this plant is for the treatment of intestinal disturbances, for which purpose it is usually prepared as a tea or infusate. We have recently reported that the essential oil of O. gratissimum L. (Labiatae) (EOOG) exerts a relaxant effect on isolated intestinal smooth muscle (Madeira et al., 2002), strengthening the argument that this fraction may constitute the active principal of the medicinal plant responsible for its therapeutic application in folk medicine.

Many types of O. gratissimum L. are encountered world-wide, each differing with respect to chemical composition of the essential oil. For example, the major constituent of O. gratissimum L. essential oil is thymol in the Rwandan variety (Ntezurubanza et al., 1987), whilst in Amazonia [rho]-cymene predominates (Maia et al., 1998). The variety indigenous to the Northeast of Brazil has eugenol and 1,8-cineol as the major components of its essential oil, although interestingly the exact chemical composition is dependent upon the hour of collection of the plant material (Silva et al., 1999).

In the present study, we were interested to evaluate whether the pharmacological properties would also vary according to the chemical composition, and to compare these effects with those of recognized smooth muscle relaxants papaverine and diltiazem. Thus, we have compared the effects of the EOOG, collected at 08:00 a.m. and at 12:00 a.m., on the relaxation of guinea-pig isolated ileum.

Materials and methods

The essential oil of the plant O. gratissimum L. (Labiatae), collected in Fortaleza (Brazil), registered in the Herbario Prisco Bezerra of the Universidade Federal do Ceara (EAC 14968), was extracted according to Craveiro et al. (1976) in the Departamento de Quimica Organica e Inorganica da Universidade Federal do Ceara. The composition of EOOG from leaves of O. gratissimum L. (Labiatae) was determined by gas chromatography and mass spectrometry from two different collection periods (08:00 and 12:00 a.m., designated as EOO[G.sub.8]: 1,8-cineol (54.94%), eugenol (20.99%), [beta]-ocimene (0.99%), [beta]-pinene (6.2%) and EOO[G.sub.12]: 1,8-cineole (38.37%), eugenol (34.9%), [beta]-ocimene (4.28%), [beta]-pinene (3.61%), respectively).

Male guinea pigs (250-400 g) were used. Two centimeter pieces of ileum were mounted in a 10 ml chamber containing Tyrode solution (136.0 NaCl, 5.0 KCl, 0.98 Mg[Cl.sub.2], 2.0 Ca[Cl.sub.2], 0.36 Na[H.sub.2]P[O.sub.4], 11.9 NaHC[O.sub.3], and 5.5 glucose, in mM), pH 7.4, 37 [degrees]C and bubbled with air. An initial tension of 0.5 g was applied and tissue was allowed to equilibrate for 1 h. The mechanical responses in ileum were recorded with an isometric force transducer and computerised data aquisition system (DATAQ). EOOG (0.1-1000 [micro]g/ml) was applied in preparations pre-contracted with 60 mM [K.sup.+] solutions or acetylcholine (10 [micro]M). Papaverine and diltiazem were used as standard relaxant controls. Chemicals were purchased from Sigma Chemical Company (St. Louis, MO, USA). The results are presented as mean [+ or -] SEM of n observations. Values were analyzed using a Student's t-test or ANOVA, as appropriate, and were considered to differ significantly when p < 0.05. I[C.sub.50] values were calculated by interpolation from semi-logarithmic plots.

Results

Relaxant effects of EOO[G.sub.8], EOO[G.sub.12] and papaverine on basal tonus of the ileum

Both EOO[G.sub.8] and EOO[G.sub.12] (30-300 [micro]g/ml) significantly relaxed the spontaneous tonus of the guinea-pig ileum in a concentration-dependent manner, reaching a maximum effect at 300 [micro]g/ml (Fig. 1a). The respective I[C.sub.50] values for the EOO[G.sub.8]- and EOO[G.sub.12]-induced relaxations were 31.15 [+ or -] 4.06 [micro]g/ml (n = 6) and 21.68 [+ or -] 3.58 [micro]g/ml (n = 8), and were not significantly different. The relaxant effect of EOOG was fully reversible upon washout from the preparation in all cases. Papaverine (0.1-300 [micro]g/ml) also relaxed the basal tonus in a concentration-dependent manner to a similar extent, with an I[C.sub.50] value of 6.1 [micro]g/ml (n = 8, Fig. 1a).

[FIGURE 1 OMITTED]

Relaxant effects of EOO[G.sub.8] and EOO[G.sub.12] on Achinduced tonic contraction

Ach (10 [micro]M) elicited biphasic contractions of the isolated ileum, with a maintained plateau response of 0.44 [+ or -] 0.05 g (n = 12). EOO[G.sub.8] and EOO[G.sub.12] (10-300 [micro]g/ml) induced full concentration-dependent relaxations of this contraction with I[C.sub.50] values of 128.16 [+ or -] 15.70 [micro]g/ml (n = 6) and 69.55 [+ or -] 4.93 (n = 6), respectively (Fig. 1b). The concentration-response curve for EOO[G.sub.8] was slightly dislocated to the right of EOO[G.sub.12], with a small but significant difference observed between their relaxant I[C.sub.50] values.

Relaxant effects of EOO[G.sub.8], EOO[G.sub.12], eugenol, 1,8 cineole and diltiazem on KCl-induced tonic contraction

KCl (60 mM) induced reproducible tonic contractions of the ileum (0.37 [+ or -] 0.05 g, n = 15). EOO[G.sub.8] and EOO[G.sub.12] (3-300 [micro]g/ml) reversibly relaxed these contractions with I[C.sub.50] values of 38.33 [+ or -] 9.91 [micro]g/ml (n = 6) and 35.53 [+ or -] 6.70 [micro]g/ml (n = 6), respectively. Both EOO[G.sub.8] and EOO[G.sub.12] relaxed the KCl-precontracted more potently than Ach-precontracted preparations. Diltiazem ([10.sup.-8]-[10.sup.-6] M) also dose-dependently relaxed KCl-precontracted tissues to a similar magnitude to EOOG, although being significantly more potent than the oil with an I[C.sub.50] value of 0.084 [+ or -] 0.01 [micro]g/ml (Fig. 2a). Eugenol and 1,8-cineole also relaxed KCl-precontracted preparations in a concentration-dependent manner; however, they were less potent than the parent essential oil, with respective I[C.sub.50] values of 125.37 [+ or -] 25.40 [micro]g/ml (n = 6) and 409.27 [+ or -] 39.55 [micro]g/ml (n = 6) (Fig. 2b). Eugenol was significantly more potent than 1,8-cineole in relaxing the KCl-precontracted ileum.

Discussion

Recently, it has been reported that the chemical composition of the essential oil of the medicinal plant O. gratissimum L., found abundantly in the Northeast of Brazil, varies according to the time that the plant is collected (Silva et al., 1999). Our current study has confirmed such a temporal variation, and has additionally shown that this change in composition does not substantially alter the basic pharmacological activity of EOOG. Thus both EOO[G.sub.8] and EOO[G.sub.12] manifested strong relaxant properties on isolated intestinal smooth muscle, coherent with the therapeutic application of this plant in local folk medicine. The depression of basal tonus was of a similar magnitude to that of the established relaxant papaverine, delineating the importance of the present findings. Since the plant is usually prepared as teas or infusates from the leaves and it is likely that quite high local concentrations of the essential oil will be present in the gut following medication, which would exert a calmant action on gastrointestinal motility.

[FIGURE 2 OMITTED]

EOOG concentration-dependently relaxed contractions induced both by high potassium and by acetylcholine. The relaxation induced by EOOG in potassium-precontracted tissues was of a similar magnitude to that produced by the voltage-dependent calcium channel blocker diltiazem, although EOOG was significantly less potent. There was no difference observed between the effects of EOO[G.sub.8] and EOO[G.sub.12] on depression of basal tone and reversal of high potassium-induced tone; however, a slight but clear shift in the concentration-response curve was observed between the two essential oils when acetylcholine was employed as the contractile agent.

The most obvious change between EOO[G.sub.8] and EOO[G.sub.12] is the increase in eugenol content from approximately 21% to 35%. Our functional evaluation has shown that eugenol exerts a concentration-dependent relaxation of the guinea-pig ileum, in agreement with previous studies in both visceral and vascular smooth muscle preparations (Reiter and Brandt, 1985; Nishijima et al., 1999). Currently, the mechanism of action of eugenol in smooth muscle remains undefined, but evidence in vascular tissue has suggested that modulation of the calcium sensitivity of the contractile proteins may be involved (Nishijima et al., 1999; Criddle et al., 2003). It thus appears that eugenol should contribute to the effects of EOOG observed in the present study. However, the pure substance alone was less potent than the parent essential oil in its relaxant action arguing against this compound being the sole active principle. Similarly 1,8 cineole, the major constituent of EOOG, was even less potent than eugenol. It is therefore possible that another minor component of EOOG may exert a more potent relaxant action in the guinea-pig ileum than the substances tested so far. Alternatively, since essential oils are complex mixtures of biologically active compounds, there may be an interplay of synergistic and/or antagonistic influences contributing to the final observed pharmacological effect.

Interestingly, EOO[G.sub.8] and EOO[G.sub.12] were significantly less potent in relaxing Ach--than high potassium-induced tone, which may reflect the transduction mechanisms utilized by the respective spasmogens. In guinea-pig ileum the contraction elicited by high potassium is due to influx of extracellular calcium into the muscle cell via voltage-dependent calcium channels (Bolton, 1979), and was completely inhibited by diltiazem, whereas ACh-induced responses may instead involve activation of non-selective cation channels (Chen et al., 1993). Since inhibitory effects of eugenol on calcium currents have been reported in cardiac myocytes (Sensch et al., 2000), this may explain a preferential inhibition of high potassium-induced spasms in guinea-pig ileum.

In conclusion, our results show that the time of collection of the leaves of O. gratissimum L. is important in determining the chemical composition of its essential oil; however, the potency of its relaxant effect on intestinal smooth muscle is not altered greatly. Thus if the therapeutic action of O. gratissimum L. observed in folk medicine is mediated via its essential oil, the time of collection of the plant appears unlikely to significantly influence its beneficial effects.

Acknowledgements

This study was supported by FUNCAP (Brazil).

References

Bolton, T.B., 1979. Mechanisms of action of transmitters and other substances on vascular smooth muscle. Physiol. Rev. 59, 606-718.

Chen, S., Inoue, R., Ito, Y., 1993. Pharmacological characterization of muscarinic receptor-activated cation channels in guinea-pig ileum. Br. J. Pharmacol. 109, 793-801.

Craveiro, A.A., Alencar, J.W., Matos, F.J.A., 1976. Estudo de oleos essenciais de plantas do nordeste brasileiro. Ciencia e Cultura 28 (Suppl.), 180.

Criddle, D.N., Madeira, S.V., Soares de Moura, R., 2003. Endothelium-dependent and -independent vasodilator effects of eugenol in the rat mesenteric vascular bed. J. Pharm. Pharmacol. 55 (3), 359-365.

Madeira, S.V.F., Matos, J.F.A., Cardoso, J.H.L., Criddle, D.N., 2002. Relaxant effects of the essential oil of Ocimum gratissimum on isolated ileum of the guinea-pig. J. Ethnopharmacol. 81 (1), 1-4.

Maia, J.G.S., Ramos, L.S., Luz, A.I.R., Silva, M.L., Zoghbi, M.G.B., 1998. Uncommon Brazilian essential oils of the Labiatae and composital. In: Laurence, B.M., Mookherjee, B.D., Willis, B.J. (Eds.), Flavors and Fragrances: A World Perspective, Proceedings of the 10th International Congress of Essential Oils. Fragrances and Flavors, Washington, DC, pp. 177-187.

Nishijima, H., Uchida, R., Kameyama, K., Ohkubo, T., Kitamura, K., 1999. Mechanisms mediating vasorelaxing action of eugenol, a pungent oil, on rabbit arterial tissue. Jpn. J. Pharmacol. 79 (3), 327-334.

Ntezurubanza, L., Scheffer, J.J.C., Svendsen, A.B., 1987. Composition of the essential oil of Ocimum gratissimum grown in Rwanda. Planta Med. 123, 421-423.

Reiter, M., Brandt, W., 1985. Eugenol relaxant effects on tracheal and ileal smooth muscles of the guinea pig. Arzneim.-Forsch 35 (1A), 408-414.

Sensch, O., Vierling, W., Brandt, W., Reiter, M., 2000. Effects of inhibition of calcium and potassium currents in guineapig cardiac contraction: comparison of [beta]-caryophyllene oxide, eugenol, and nifedipine. Br. J. Pharmacol. 131, 1089-1096.

Silva, M.G.V., Craveiro, A.A., Matos, F.J.A., Machado, M.I.L., Alencar, J.W., 1999. Chemical variation during daytime of constituents of the essential oil Ocimum gratissimum leaves. Fitoterapia 70, 32-34.

S.V.F. Madeira (a), M. Rabelo (a), P.M.G. Soares (a), E.P. Souza (a), A.V.P. Meireles (a), C. Montenegro (a), R.F. Lima (a), A.M.S. Assreuy (a), D.N. Criddle (a,b,*)

(a) Laboratorio de Farmacologia dos Canais Ionicos--LAFACI, Mestrado Academico em Ciencias Fisiologicas, CCS, Universidade Estadual do Ceara, Av. Paranjana 1700, Fortaleza CE 60740-000, Brazil

(b) MRC Secretory Control Research Group, The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, UK

Received 8 July 2003; accepted 11 November 2003

*Corresponding author. MRC Secretory Control Research Group, The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, UK. Tel.: +44 55(0)151 794 5355; fax: +44 55(0)151 794 5327.

E-mail address: criddle@liv.ac.uk (D.N. Criddle).
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Title Annotation:SHORT COMMUNICATION
Author:Madeira, S.V.F.; Rabelo, M.; Soares, P.M.G.; Souza, E.P.; Meireles, A.V.P.; Montenegro, C.; Lima, R.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:3BRAZ
Date:Jun 1, 2005
Words:2532
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