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Endothelium-dependent hypotensive and vasorelaxant effects of the essential oil from aerial parts of Mentha x villosa in rats.

Abstract

Cardiovascular effects of an essential oil from the aerial parts of Mentha x villosa (OEMV) were tested in rats using a combined in vivo and in vitro approach. In non-anesthetized normotensive rats, OEMV (1, 5, 10, 20, 30 mg [kg.sup.-1] body wt., i.v.) induced a significant and dose-dependent hypotension (-3[+ or -]1.8%; -6[+ or -]0.7%; -40[+ or -]6.7%; -58[+ or -]3.8%; -57[+ or -]2.1%, respectively) associated with decreases in heart rate (-1[+ or -]0.3%; -9[+ or -]0.9%; -17[+ or -]3.2%; -72[+ or -]3.1%; -82[+ or -]1.4%, respectively). The hypotensive and bradycardic responses evoked by OEMV were attenuated and blocked by pre-treatment of the animals with atropine (2 mg [kg.sup.-1] body wt., i.v.). In isolated rat atrial preparations, OEMV (10, 100, 300, 500 [micro]g [ml.sup.-1]) produced concentration-related negative chronotropic and inotropic effects (I[C.sub.50] value -229[+ or -]17 and 120[+ or -]13 [micro]g [ml.sup.-1], respectively). In isolated rat aortic rings, increasing concentrations of OEMV (10, 100, 300, 500 [micro]g [ml.sup.-1]) were able to antagonize the effects of phenylephrine (1 [micro]M), prostaglandin [F.sub.2[alpha]] (10 [micro]M) and KCl (80 mM)-induced contractions (I[C.sub.50] value = 255[+ or -]9, 174[+ or -]4 and 165[+ or -]14 [micro]g [ml.sup.-1], respectively). The vasorelaxant activity induced by OEMV was attenuated significantly by either endothelium removal (I[C.sub.50] value = 304[+ or -]9 [micro]g [ml.sup.-1]), [N.sup.G]-nitro L-arginine methyl ester (L-NAME) 100 [micro]M (I[C.sub.50] value = 359[+ or -]18 [micro]g [ml.sup.-1]), L-NAME 300 [micro]M (I[C.sub.50] value = 488[+ or -]20 [micro]g [ml.sup.-1]) or indomethacin 10 [micro]M (I[C.sub.50] value = 334[+ or -]18 [micro]g [ml.sup.-1]). However, it was not affected by atropine 1 [micro]M (I[C.sub.50] value = 247[+ or -]12 [micro]g [ml.sup.-1]). Furthermore, the hypotensive response induced by OEMV was attenuated significantly after nitric oxide (NO) synthase blockade (L-NAME, 20 mg [kg.sup.-1] body wt., i.v.), while bradycardia was not altered. The results suggest that the hypotensive effect induced by OEMV is probably due to its direct cardiodepressant action and peripheral vasodilation, which can be attributed to both endothelium-dependent (via EDRFs, at least NO and prostacyclin) and endothelium-independent mechanisms (such as [Ca.sup.2+] channel blockade).

[c] 2004 Elsevier GmbH. All rights reserved.

Keywords: Mentha x. villosa; Essential oil; Cardiovascular effects; Isolated atria; Isolated rat aortic rings; Nitric oxide

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Introduction

The plant Mentha x villosa Hudson (Lamiaceae), known popularly as "hortela-da-folha-miuda," is a herb cultivated extensively in Brazil, where it is used for its beneficial effects such as anti-parasitic and tranquilizing action, relief of stomach disorders and of menstrual pain (Alencastro et al., 1965). The widespread use of this plant has stimulated pharmacological studies on its extracts and constituents. Studies have related some activity of its essential oil (oil from the aerial parts of Mentha x villosa--OEMV) and its major constituent rotundifolone (Piperitenone oxide) (Almeida et al., 1996; Lahlou et al., 2001). Almeida et al. (1996) showed that OEMV and rotundifolone exhibited central analgesic activity in mice and rats, and that the mechanism might involve the opioid system. In addition, Sousa et al. (1997) reported that rotundifolone exhibited a relaxant effect on intestinal smooth muscle, apparently mediated by an inhibition of [Ca.sup.2+]-release from intracellular stores.

In the present work, we attempted to characterize the cardiovascular effects induced by OEMV, using a combined in vivo and in vitro approach.

Material and methods

Plant material: Mentha x villosa leaves were collected in Joao Pessoa, Paraiba, Brazil, in September 1992, and authenticated by Dr. Harley of the Royal Botanic Gardens, Kew, England. A voucher specimen has been deposited in the Prisco Bezerra Herbarium of the Federal University of Ceara (No. 14996).

Preparation of the essential oil: Mentha x villosa fresh leaves (10 kg) were subjected to steam distillation for 8h. The oil obtained (0.1%) in the usual way (Craveiro and De Queiroz, 1993), was dried over [Na.sub.2]S[O.sub.4] and stored at 4[degrees]C.

Animals: Male Wistar rats (300-350 g each) were used for all experiments. Animals were housed under conditions of controlled temperature (21 [+ or -] 1[degrees]C) and exposed to a daily 12 h light-dark cycle.

Measurement of arterial blood pressure in conscious unrestrained rats

For measurement of arterial blood pressure, rats were anesthetized using sodium pentobarbital (60 mg [kg.sup.-1] body wt., i.p.), and a polyethylene (PE) catheter (heat-stretched PE-10 fused to a PE-50 extension) was inserted into the lower abdominal aorta via the left femoral artery. Stretching of the PE-10 intravascular part minimized the risk of ischemia in the catheterized leg, without altering the arterial pressure signal (Oliveira et al., 1996). Another catheter (PE-10 fused to PE-50) was inserted into the inferior vena cava via the left femoral vein for administration of drugs. Both catheters were filled with heparinized saline and led under the skin to exit between the scapulae.

After catheterization, rats were placed in large individual recording cages. Two days later, experiments were performed on conscious, freely moving rats accustomed to their environment and provided with food and water ad libitum. The arterial catheter was connected to a pre-calibrated pressure transducer (Statham P23 ID; Gould, Cleveland, OH, USA). The transducer was fed to an amplifier-recorder (Model TBM-4M, WPI, Sarasota, FL, USA) and to a personal computer (Pentium 166 MHz) equipped with an analog-to-digital converter board (CIO-DAS16/JR, Computer Boards, Inc., Mansfield, MA, USA). Using CVMS software (WPI, Sarasota, FL, USA), data were sampled every 500 Hz and stored on a CD-ROM.

Beat-to-beat time series were generated and processed off-line on another personal computer. For each cardiac cycle, the computer calculated mean arterial pressure (MAP), and pulse interval (referred to as heart rate). On the day of the experiments, after cardiovascular parameters had stabilized, sodium nitroprusside (10 [micro]g [kg.sup.-1] body wt.) was injected to check the patency of the venous catheter.

Studies using isolated rat aortic rings

Aortic rings (2-4 mm) were obtained free from connective tissue and fat and suspended by platinum hooks for isometric tension recording in a Krebs Henseleit solution maintained at 37[degrees]C and gassed with a mixture of 95% [O.sub.2] and 5% C[O.sub.2]. The rings were allowed to equilibrate for 1 h under a resting tension of 1 g. During this time, the bathing medium was changed every 15 min to protect against interfering metabolites (Altura and Altura, 1970). The isometric tension was measured and recorded using a force-displacement transducer (Ugo basile, Comerio, VA, Italy) coupled to a physiograph (Gemini 2, Ugo Basile, Comerio, VA, Italy).

Preparation of rat atrial muscle

Rat atria were isolated and perfused according to the technique described by Nasa et al. (1992). Briefly, rats were killed by cervical dislocation and the heart was rapidly excised. The whole left atrium and whole right atrium were cut perpendicular to the axis of the heart, and each atrium was suspended in an organ bath containing Krebs bicarbonate solution. The organ bath was maintained at 37[degrees]C and gassed with a mixture of 95% [O.sub.2] and 5% C[O.sub.2]. The resting tension of each atrium was adjusted to 500mg and the tissues were equilibrated for at least 60 min before the experiments. The left atrium was driven electrically through two parallel platinum electrodes by rectangular pulses with a frequency of 3 Hz, a duration of 3 ms and a voltage of 1.5-fold threshold. The isometric tension was measured and recorded using a force-displacement transducer (Ugo basile, Comerio, VA, Italy) coupled to a physiograph (Gemini 2, Ugo Basile, Comerio, VA, Italy).

The composition (mM [l.sup.-1]) of the physiological salt solution was as follows:

Krebs Henseleit solution: NaCl 118.0; KCl 4.7; NaHC[O.sub.3] 25.00; Ca[Cl.sub.2] * 2[H.sub.2]O 2.5; glucose 11.1; K[H.sub.2]P[O.sub.4]. 1.2 and MgS[O.sub.4] * 7[H.sub.2]O 1.2.

Krebs bicarbonate solution (KBS): NaCl 118.4; KCl 4.7; NaHC[O.sub.3] 25.00; Ca[Cl.sub.2] * 2[H.sub.2]O 2.5; glucose 10.0; Na[H.sub.2]P[O.sub.4] * [H.sub.2]O 2.5 and MgS[O.sub.4] * [H.sub.2]O 1.2.

Drugs: The drugs used were heparin sodium salt (Roche); sodium nitroprusside, [N.sup.G]-nitro L-arginine methyl ester (L-NAME), atropine, phenylephrine, prostaglandin [F.sub.2[alpha]], and acetylcholine hydrochloride (all from Sigma); and sodium pentobarbital (Jansen). The stock solutions were prepared in distilled water and kept at 4[degrees]C.

Experimental protocols

Systemic hemodynamic effects of OEMV in conscious rats

After hemodynamic parameters had stabilized, sodium nitroprusside (10 [micro]g [kg.sup.-1] body wt., i.v.) was injected to check the patency of the venous catheter. Ten to 15 min later, different doses of OEMV (1, 5, 10, 20 and 30 mg [kg.sup.-1] body wt., i.v.) were administered randomly and the responses were recorded. Successive injections were separated by a time sufficient to allow full recovery of arterial pressure, usually 15-20 min.

Effects of cardiac muscarinic blockade on the responses induced by OEMV in conscious freely moving rats

After stabilization of haemodynamic parameters. OEMV (5, 10, 20 and 30 mg [kg.sup.-1] body wt., i.v.) was administered and the effects were recorded. After a 30-min period, atropine (2 mg [kg.sup.-1] body wt., i.v.) was administered and 10 min later, OEMV was repeated. Changes in MAP and HR induced by OEMV were compared before (baseline conditions) and after atropine treatment.

Effects of nitric oxide synthase blockade on the responses induced by OEMV in conscious freely moving rats

Pilot experiments showed that doses of OEMV (5, 10, 20 and 30 mg [kg.sup.-1] body wt., i.v.) present responses of similar magnitude when administered 3-4 times repeatedly. After stabilization of hemodynamic parameters, OEMV (5, 10, 20 and 30 mg [kg.sup.-1] body wt., i.v.) was administered and the effects recorded. After a 30-min period, L-NAME (20 mg [kg.sup.-1] body wt., i.v.) was administered and 30 min later OEMV was repeated. Changes in MAP and HR induced by OEMV were compared before (baseline conditions) and after L-NAME treatment.

Effects of OEMV on isolated rat aortic rings

Different concentrations of OEMV (10, 100, 300 and 500 [micro]g [ml.sup.-1]) were added cumulatively to isolated aortic preparations pre-contracted with either phenylephrine (1 [micro]M). prostaglandin [F.sub.2[alpha]] (10 [micro]M) or KCl (80 mM). Some experiments were also conducted in which phenylephrine, prostaglandin [F.sub.2[alpha]] or KCl was added to the tissue and left for at least 2 h to observe whether the tension was maintained during the period. The relaxation was measured by comparing the developed tension before and after addition of OEMV. I[C.sub.50] values were calculated from linear regression fitted to individual curves using GraphPad Prism[TM] software.

Influence of muscarinic blockade on the relaxing response of OEMV against phenylephrine-induced contractions

Two tonic responses to 1 [micro]M phenylephrine, which stabilized in 15 min, were registered. Aortic rings were then incubated in the presence of atropine (1 [micro]M) for 30 min. A third response was obtained and OEMV (10, 100, 300 and 500 [micro]g [ml.sup.-1]) was added to preparations. The effectiveness of the muscarinic blockade was verified by the loss of relaxing response of the preparations to 1 [micro]M acetylcholine in the presence of 1 [micro]M atropine.

Influence of endothelium on the relaxing response of OEMV against phenylephrine-induced contractions

In this experiment, phenylephrine-induced sustained contractions were obtained in endothelium-free rings or in preparations under the blockade of NO-synthase by 100 [micro]M L-NAME or blockade of cyclooxigenase by 10 [micro]M indomethacin. L-NAME or indomethacin was added 30 min before the administration of phenylephrine. After a third phenylephrine contraction, OEMV (10, 100, 300 and 500 [micro]g [ml.sup.-1]) was added to preparations. The effectiveness of endothelium removal and NO-synthase blockade was verified by the loss of relaxing response of the preparations to 1 [micro]M acetylcholine. The results obtained were compared with those verified after addition of OEMV (10, 100, 300 and 500 [micro]g [ml.sup.-1]) in the course of the concentration-response experiment.

Effects of OEMV on isolated perfused rat atria

Cumulative concentration response curves to OEMV (10, 100, 300 and 500 [micro]g [ml.sup.-1]) were constructed by stepwise addition of essential oil. In some experiments, the rate of espontaneous beating of right atrium was measured, being defined as atrial rate, in order to assess the chronotropic efftects of OEMV. The inotropic effect of OEMV was studied in the electrically stimulated left atrium. Both the chronotropic and inotropic effects were observed 5 min after addition of OEMV.

Data analysis

Values are expressed as mean [+ or -] s.e.m. Statistical analysis was performed by means of one-way analysis of variance for repeated measures. Paired and unpaired students t-tests were also used when appropriate. Linear regressions were performed using the least-squares method, using GraphPad Prism[TM] software, version 2.0 (GraphPad Software, Inc.).

Results

Systemic hemodynamic effects of OEMV in conscious rats

In conscious, unrestrained rats, OEMV (1, 5, 10, 20 and 30 mg [kg.sup.-1] body wt., i.v., randomly) produced a significant and dose-related hypotension and bradycardia. At these doses, OEMV decreased MAP by: -2.5 [+ or -] 1.8%; -6.3 [+ or -] 0.7%; -39.6 [+ or -] 6.7%; -58.4 [+ or -] 3.8% and -57.4 [+ or -] 2.1%, respectively. OEMV also induced a significant (p < 0.05) decrease in heart rate: -0.9 [+ or -] 0.3%; -9.1 [+ or -] 0.9%; -16.6 [+ or -] 3.2%; -71.5 [+ or -] 3.1% and -81.9 [+ or -] 1.4%, respectively.

Influence of the cardiac muscarinic blockade on the effects induced by OEMV in conscious freely moving rats

During cardiac muscarinic blockade with atropine (2 mg [kg.sup.-1] body wt., i.v.), The hypotensive effect induced by OEMV (5, 10, 20 and 30 mg [kg.sup.-1] body wt. i.v., randomly) was attenuated significantly as compared to the response evoked by the essential oil given alone (Fig. 1). Under these conditions, OEMV-induced bradycardia was abolished by atropine treatment (Fig. 1).

Effect of NO-synthase inhibition on hypotensive response to OEMV in conscious freely moving rats

In conscious, unrestrained rats, L-NAME (20 mg [kg.sup.-1] body wt., i.v.) induced a sustained increase in MAP (136 [+ or -] 4 from 118 [+ or -] 3 mmHg, p < 0.01) accompanied by bradycardia (340 [+ or -] 10 from 360 [+ or -] 8 bpm, p < 0.005). After NO-synthase inhibition, the hypotensive response induced by OEMV was attenuated significantly as compared to control; however, the bradycardic effect of OEMV was not changed (Fig. 1).

Effects of OEMV on isolated aortic rings

As illustrated in Fig. 2, OEMV (10, 100, 300 and 500 [micro]g [ml.sup.-1]) antagonized in a significant (p < 0.05) and concentration-dependent manner the phenylephrine-(1 [micro]M), prostaglandin [F.sub.2[alpha]]- (10 [micro]M) and KCl (80 mM)-induced contractions (I[C.sub.50] value = 255[+ or -]9, 174[+ or -]4 and 165[+ or -]14 [micro]g [ml.sup.-1], respectively). The maximal relaxing response was obtained with 500 [micro]g [ml.sup.-1], which was not significantly different from that induced by 1000 [micro]g [ml.sup.-1]. Experiments also demonstrated that part of the vasorelaxant response to OEMV was endothelium-dependent, because after endothelium-removal, exposure to L-NAME (100 or 300 [micro]M) or indomethacin (10 [micro]M), the responses induced by OEMV were attenuated significantly (I[C.sub.50] value = 304[+ or -]9, 359[+ or -]18, 488[+ or -]20 and 334[+ or -]18 [micro]g [ml.sup.-1], respectively). Nevertheless, the vasorelaxant effect of OEMV was not altered significantly after pre-incubation of the preparations with 1 [micro]M atropine (I[C.sub.50] value = 247[+ or -]12 [micro]g [ml.sup.-1]) (Table 1).

[FIGURE 1 OMITTED]

Effects of OEMV on isolated perfused rat atria

As shown in Fig. 3, OEMV (10, 100, 300 and 500 [micro]g [ml.sup.-1]) produced dose-dependent negative chronotropic and inotropic effects (I[C.sub.50] value = 229[+ or -]17 and 120[+ or -]14 [micro]g [ml.sup.-1], respectively). Both effects reached a maximum within 5 min after addition of OEMV and remained unchanged for at least 10 min.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Discussion

The major finding of this work is that OEMV decreases MAP due to cardiac depressant and vasorelaxant actions significantly.

To minimize the influence of anesthesia and stress on hemodynamic parameters (Smith and Hutchins, 1980; Fluckiger et al., 1985), studies were performed in conscious, freely moving Wistar rats. Furthermore, we used isolated perfused preparations which are more suitable to study specific effects.

Because OEMV induced hypotension and bradycardia in conscious rats, in this work, the effects of atropine on the above effects were evaluated. It is well established that the parasympathetic nervous system plays a significant role in the control of cardiac activity and arterial blood pressure (Higgins et al., 1973). Our data showed that hypotension was attenuated significantly and bradycardia was abolished completely by atropine, indicating a strong participation of muscarinic receptors in these responses.

To investigate a direct cardiac action of OEMV, we designed experiments on isolated atrial preparations. In these preparations, OEMV had negative inotropic and chronotropic effects, which were concentration-dependent. Although mammalian heart is thought to possess predominantly [M.sub.2] muscarinic receptors, as confirmed by the localization of [M.sub.2] mRNA in rat heart by in situ hybridization, expression of [M.sub.1], [M.sub.3] and [M.sub.4] muscarinic receptor genes have been reported (Caulfield, 1993; Peralta et al., 1987; Hulme et al., 1990; Brodde and Michel, 1999), as confirmed by the localization of M2 mRNA in rat heart by in situ hybridization (Hoover et al., 1994). The role of these gene products and/or their presence has not yet been associated with any functional responses in atria (Stengel et al., 2000). Because [M.sub.2]-muscarinic receptors are known to be expressed in the heart, and the cholinergic stimulation of this organ decreases the rate of the spontaneous diastolic depolarization, as well as increasing the repolarizing current at the SA node (Di Francesco, 1993), we suggest that OEMV could decrease heart rate by stimulation of cardiac [M.sub.2]-muscarinic receptors. This cardiac muscarinic receptor stimulation could in turn reduce cardiac output momentarily and, consequently, decrease MAP. Because Lahlou et al. (2001) showed that the essential oil of Mentha x villosa was able to induce hypotension in anesthetized rats even after a cervical bilateral vagotomy, we rule out a possible effect of OEMV due to stimulation of the parasympathetic efferent pathways (vagal) with subsequent acetylcholine release and stimulation of muscarinic receptors (Thoren, 1979). Taken together with results obtained in conscious rats, where atropine completely abolished bradycardia induced by OEMV, we suggest that the essential oil could stimulate muscarinic receptors in the heart to decrease heart rate and contractility.

Because OEMV-induced hypotension was significantly attenuated in conscious unrestrained normotensive rats, but was not completely abolished after atropine administration, we hypothesized an additional relaxant activity of the essential oil on the vascular smooth muscle, which was verified in isolated rat aortic rings. In these preparations, OEMV antagonized the phenylephrine-, KCl- and prostaglandin [F.sub.2[alpha]]-induced-contractions in a concentration-dependent manner.

It is well-established that NO is a major endothelium-derived relaxing factor, both in vivo and in vitro (Moncada et al., 1991), and that the release of NO from endothelial cells leads to relaxation of vascular smooth muscle cells and plays a critical role in the maintenance of vascular tone (Moncada et al., 1991; Moncada and Higgs, 1993). In order to determine whether part of the relaxant effect produced by OEMV in isolated aortic rings could be due to NO release, we performed experiments in intact aortic preparations (pre-contracted with 1 [micro]M phenylephrine) incubated with increasing concentrations (100 and 300 [micro]M) of L-NAME, a competitive inhibitor of NO-synthase (Moncada and Higgs, 1993). Under these conditions, OEMV-induced smooth muscle relaxation was attenuated significantly, but not abolished completely. Furthermore, in these preparations in which the endothelium was mechanically removed, OEMV-induced vasorelaxant action was attenuated significantly. These data indicate that, at least in part, the vasorelaxant effect of OEMV is endothelium-dependent.

In most vascular beds, the stimulation of muscarinic ([M.sub.3] subtype) receptors produces an intense dilation, despite the lack of vascular cholinergic innervation (Brunning et al., 1994). The muscarinic receptors responsible for relaxation are located on the endothelial cells and their stimulation leads to the release of the endothelium-derived relaxing factors (Moncada et al., 1991), mainly NO, which diffuse to adjacent smooth muscle cells and cause them to relax (Furchgott, 1983; Vanhoutte et al., 1995). To determine whether OEMV-induced NO release could be secondary to the stimulation of the endothelial [M.sub.3] receptors (Sawada et al., 1994), we performed experiments in intact aortic preparations that were pre-incubated with atropine. In these preparations, the vasorelaxant effect of OEMV was unaffected, which allowed us to rule out the participation of a direct vascular muscarinic receptor activation. In addition, the involvement of the vasodilator cyclo-oxygenase products was also investigated in intact isolated aortic preparations that were pre-incubated with indomethacin. The inhibition of phenylephrine-induced contractions produced by OEMV was also attenuated significantly by indomethacin. Taken together, these results indicated clearly that relaxing factors released by the endothelium, mostly NO and prostacyclin, play an important role in the vasorelaxant response induced by OEMV.

It is well known that the maintenance of smooth muscle contraction depends upon [Ca.sup.2+] entry from extracellular space through voltage- and/or receptor-operated calcium channels (VOCCs and/or ROCCs, respectively) (Nelson et al., 1990; Karaki and Weiss, 1988). It is well known that KCl induces smooth muscle contraction through activation of VOCCs and subsequent release of calcium from the sarcoplasmic reticulum (Gurney, 1994; Nasa et al., 1992), without changing other signal transduction systems including phosphatidylinositol turnover and calcium sensitization (Karaki et al., 1997). OEMV seems to block VOCCs as the contractions induced by potassium were inhibited in a concentration-dependent manner by the essential oil of Mentha x villosa. The activation of phosphoinositide turnover by G-protein coupling of phospholipase C induced by the activation of [alpha]-adrenergic and/or prostanoid receptors is crucial for the cytoplasmic calcium increase involved in the contraction induced by activation of these receptors, and involves calcium from intracellular stores (Clapham, 1995; Bootman and Berridge, 1995). OEMV inhibited phenylephrine, and PG[F.sub.2[alpha]]-induced contractions with similar potencies (Table 1).

Because the major resulting effect after KCl, phenylephrine and PG[F.sub.2[alpha]] stimulation is an increase in intracellular calcium concentration through calcium entry, we suggest that the residual vasorelaxant effect observed after NO-synthase inhibition, endothelium removal and cyclo-oxygenase inhibition, is due to an endothelium-independent mechanism, possibly linked to L-type calcium blocking activity.

Endothelium-derived NO plays a major role in the regulation of blood pressure and vascular tone (Moncada et al., 1991; Ress et al., 1989). In non-anesthesized normotensive rats, OEMV-induced hypotension was significantly attenuated, but not abolished, after L-NAME administration. Nevertheless, in these conditions OEMV-induced bradycardia remained unchanged after NO-synthase inhibition. These results confirm that OEMV decreases MAP due to a decrease in peripheral vascular resistance, mainly but not exclusively, through the activation of NO release by vascular endothelium.

Finally, assuming a total blood volume of about 20 ml in a 300 g rat (Ise et al., 1998), a peak blood OEMV concentration of 300 [micro]g [ml.sup.-1] can be expected at the 20 mg [kg.sup.-1] body wt. dose, which is in the range of the I[C.sub.50] value for the vasorelaxant action of OEMV in aortic rings.

Nitric oxide (NO) can be synthesized and released in the endocardium (Schulz et al., 1991), cardiac myocytes (Schulz et al., 1991; Brady et al., 1993), cardiac nerve fibers and neurons (Hassall et al., 1993; Klimaschewski et al., 1992), and can also induce negative effects on the contractile force of the myocardium (Finkel et al., 1992) and isolated myocytes (Brady et al., 1993). Based on the fact that vascular effect in isolated aortic rings is possibly mediated by endothelium-derived relaxing factors, we hypothesize that NO may also mediate local cardiac effects of OEMV. Further studies are, however, required to investigate the relationship between the actions of OEMV and NO in the heart.

In conclusion, the present study, using a combined approach (in vivo and in vitro experiments), demonstrated that OEMV lowers arterial pressure and heart rate markedly in non-anesthetized rats. Whatever the underlying additional mechanisms, the results shown here suggest that the hypotensive action of the essential oil of Mentha x villosa can be a consequence of decrease in both heart rate/heart contractility and peripheral vascular resistances. The vasorelaxant action of OEMV can be attributed to both endothelium-dependent mechanisms (via EDRFs, at least NO and prostacyclin) and other endothelium-independent mechanisms such as [Ca.sup.2+] channel blockade.

In this context it is important to note that menthol, the major constituent of Mentha piperita essential oil, which has been reported to exert [Ca.sup.2+]-channel blocking activity (Hawthorn et al., 1988; Schafer et al., 1986; Neuhaus-Carlisle et al., 1997), could not be detected in the essential oil of Mentha x villosa. Instead of menthol, the major monoterpenoid of Mentha x villosa oil is piperitenone oxide (55.4%). Therefore, it can be suggested that also piperitenone oxide possess Ca-antagonistic activity and is at least in part responsible for the hypotensive and vasorelaxant activity of the Mentha x villosa oil.
Table 1. Comparison of I[C.sub.50] values of OEMV against tonic
contractions induced by phenylephrine (1 [micro]M) in isolated rat
aortic rings

OEMV (condition) n I[C.sub.50] value
 ([micro]g [ml.sup.-1])

Endothelium intact 6 255[+ or -]9
Endothelium denuded 6 304[+ or -]9*
L-NAME (100 [micro]M) 6 359[+ or -]18*
L-NAME (300 [micro]M) 6 488[+ or -]20*
Atropine (1 [micro]M) 6 247[+ or -]12
Indomethacin (10 [micro]M) 6 334[+ or -]18*

OEMV (condition) [r.sup.2]

Endothelium intact 0.9993[+ or -]0.01
Endothelium denuded 0.9891[+ or -]0.08
L-NAME (100 [micro]M) 0.9732[+ or -]0.01
L-NAME (300 [micro]M) 0.9172[+ or -]0.09
Atropine (1 [micro]M) 0.9230[+ or -]0.021
Indomethacin (10 [micro]M) 0.9822[+ or -]0.011

Results are means [+ or -] s.e.m.
*p<0.05 versus endothelium intact; n, number of experiments; [r.sup.2],
correlation coefficient.


Acknowledgements

The authors wish to express their sincere thanks to Jose Crispim Duarte and Raimundo Nonato da Silva for technical assistance. Financial support from CAPES-Brazil is also acknowledged gratefully.

Received 25 July 2001; accepted 25 February 2003

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D. Nunes Guedes, D.F. Silva, J.M. Barbosa-Filho, I. Almeida de Medeiros*

Laboratorio de Tecnologia Farmaceutica, Universidade Federal da Paraiba, Caixa Postal 5009, 58.051-970 Joao Pessoa, PB. Brazil

*Corresponding author.

E-mail address: isacmed@uol.com.br (I. Almeida de Medeiros).
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Author:Guedes, D. Nunes; Silva, D.F.; Barbosa-Filho, J.M.; de Medeiros, I. Almeida
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:Sep 1, 2004
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