Cardiovascular effects of the essential oil of Mentha x villosa in DOCA-salt-hypertensive rats.

Summary

The present study investigated the effects of chronic treatment with deoxycorticosterone-acetate (DOCA)-salt on cardiovascular responses to intravenous (i.v.) injection of the essential oil of Mentha x villosa (EOMV) in conscious rats.

In both DOCA-salt-hypertensive and uninephrectomized control, conscious rats, i.v. bolus injections of EOMV (1 to 20 mg/kg body wt.) decreased mean aortic pressure (MAP) and heart rate (HR) in a dose-dependent manner. Treatment with DOCA-salt significantly enhanced EOMV-induced decreases in MAP, without affecting bradycardia. Likewise, both maximal percent and absolute decreases in MAP elicited by i.v. injection of the ganglion blocker, hexamethonium (30 mg/kg body wt.), were significantly greater in DOCA-salt-hypertensive than in control rats. In DOCA-salt-hypertensive rats, i.v. pretreatment with hexamethonium (30 mg/kg body wt.) reduced the bradycardia elicited by EOMV (1 to 20 mg/kg body wt.) without affecting the enhancement of EOM V-induced hypotension.

These results show that i.v. treatment with EOMV decreases blood pressure in conscious DOCA-salt-hypertensive rats dose-dependently, and that this action is enhanced when compared with uninephrectomized controls. This enhancement could be related mainly to an increase in EOMV-induced vascular smooth muscle relaxation, rather than to enhanced sympathetic nervous system activity in this hypertensive model.

Key words: Mentha x villosa; Labiatae, essential oil, cardiovascular effects, DOCA-salt-hypertension, conscious rat

Introduction

Mentha x villosa Huds. (Labiatae) is an aromatic, creeping herb that is a hybrid of Men Mentha spicata L. and Mentha suaveolens Ehrh. and which is found commonly in small house gardens in northeastern Brazil for use as a medicinal plant (Matos, 2001). It is known popularly as hortela-rasteira (creeping mentha), hortela comun (regular mentha) or hortela-da-folha-miuda (small-leaved mentha) (Matos, 2001). Infusions or decoctions of leaves from Mentha x villosa are used commonly for their stomachic and anxiolytic effects, and also for treatment of menstrual colic and diarrhea with blood in the stools (Matos, 2001). Mentha x villosa has also been shown to exhibit antiparasitic actions in amebiasis, giardiasis and urogenital trichomoniasis and schistosomiasis (Borba et al. 1990; Melo et al. 1992; Santana et al. 1992; Hiruma, 1993).

Leaves of Mentha x villosa have an essential oil content of 0.2-0.5% of the plant's dry weight, comprised principally of mono- and sesquiterpenes (Matos et at. 1999). Essential oil of Mentha x villosa (EOMV), and its major constituent, piperitenone oxide (PO), have been reported to have intestinal myorelaxant and antispasmodic effects in laboratory animals (Sousa et al. 1997; Sousa, 1999), justifying the use of Mentha x villosa in folk medicine. Furthermore, they were shown to induce analgesia in either the acetic-acid-induced writhing or hot-plate tests, an effect that is unrelated to the opioid mechanism, as it was unaffected by naloxone (Sousa, 1999). Furthermore, it has been shown that intravenous (i.v.) treatment of anesthetized rats with EOMV induces dose-dependent decreases in mean aortic pressure (MAP) and heart rate (HR), which appear to be mostly attributable to the actions of the major constituent of EOMV, PO (Lahlou et al. 2001). These cardiovascular effects appear to be independent, as EOMV-induc ed bradycardia appears dependent upon the presence of an intact and functional sympathetic nerve drive to the heart, while EOMV-induced hypotension appears independent of the presence of an operational sympathetic nervous system, as it remained unaffected by i.v. hexamethonium administration (Lahlou et al. 2001). A previous study from our laboratory showed that i.v. treatment with the essential oil of Croton nepetaefolius (EOCN) also induces immediate and dose-dependent decreases in MAP and HR in either anesthetised or conscious normotensive rats (Lahlou et al. 1999). These cardiovascular responses also occurred independently, and in vivo and in vitro data suggested that the hypotensive response to EOCN results from its direct vasodilatory action upon vascular smooth muscle (Lahlou et al. 1999, 2000). It is possible that such a mechanism of action is not peculiar to EOCN alone, and that EOMV may also decrease blood pressure through an active vascular relaxation. The present investigation was undertaken to exa mine this possibility by assessing the influence of deoxycorticosterone-acetate (DOCA)-salt treatment, which increases basal sympathetic activity, on EOMV-induced hypotension and bradycardia.

Materials and Methods

Plant material

Mentha x villosa was collected in January, 1997, at the Experimental Farm of the Federal University of Ceara, Ceara, Brazil. Its identification was confirmed by Dr. F. J. Abreu Matos (Laboratory of Natural Products, Federal University of Ceara) and by Dr. R. Harley (Herbarium, Royal Botanic Gardens, Kew, UK). A voucher specimen (No 27286) is deposited in the herbarium of Prisco Viana at the University of Ceara.

Extraction and chemical analysis

EOMV was kindly provided by the Laboratory of Natural Products of the Federal University of Ceara. It was prepared from fresh chopped leaves by steam distillation and analyzed chemically as described previously (Craveiro et al. 1976; Matos et al. 1999). Briefly, analytical conditions were as follows. EOMV analysis was performed on a Hewlett-Packard 6971 gas chromatography and mass spectrometry (GC/MS) instrument. Column: dimethylpolysiloxane DB-1 fused silica capillary column (20 m x 0.25 mm; 0.1 [micro]m); carrier gas: helium (1 ml/min); injector temperature: 250 [degrees]C; detector temperature: 280 [degrees]C; column temperature: 50-180 [degrees]C at 4 [degrees]C/min, then 180-250 [degrees]C at 20 [degrees]C/min; mass spectra: electronic impact 70 eV. The composition of EOMV used in the present study was (%): piperitenone oxide, 95.87; piperitenone, 2.24 and 1.8-cineole, 1.89. These compounds were identified using a mass spectral library search and (13) C-NMR spectroscopy. Their retention indices were 1337 , 1269 and 1009, respectively.

Solutions and drugs

EOMV was dissolved in Tween 80 solution (2%), brought to the chosen volume with sterile isotonic saline and sonicated just before use. Control preliminary experiments showed that this vehicle had no effects on either baseline MAP or HR during a period of 20 min. Sodium pentobarbitone (Sanofi, Libourne, France) and heparin (Laboratoires Leo S. A., Montigny-le-Bretonneux, France) were used as commercially available injectable solutions. Hexamethonium bromide and acetylcholine chloride were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and dissolved in saline just before use. Hexamethonium bromide was administered at a volume of 1 ml/kg body wt., while acetylcholine chloride was given at a volume of 100 [micro]1.

DOCA-salt treatment

Male Wistar rats, weighing 200-220 g each, were kept under conditions of constant temperature (23 [+ or -] 2 [degrees]C) with a standard light/dark cycle (12 h light/12 h dark) and free access to food and water. All animals were cared for in compliance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication 85-23, revised 1996). Under ether anaesthesia, all rats were subjected to unilateral nephrectomy. After a one-week recovery period, they were divided into two groups: rats of group 1 (DOCA-salt-hypertensive group) were treated weekly with subcutaneous injections of a suspension of DOCA (25 mg/kg body wt.) dissolved in olive oil (vehicle), and salt was administered by substitution of 1% NaCl solution for drinking water ad libitum. Rats of group 2 (uninephrectomized control group), allowed to drink tap water ad libitum, received subcutaneous injections of an equal volume of vehicle once a week. Circulatory experiments were performed four w eeks after the initiation of DOCA-salt or vehicle treatment.

Catheterization procedure

On the lfirst day of treatment, rats of both groups were anesthetized with intraperitoneal sodium pentobarbitone (50 mg/kg body wt.) and catheters (PE-10 fused to PE-50) were implanted in the left femoral artery and femoral vein for the recording of arterial blood pressure and drug administration, respectively. These catheters, filled with heparin-saline solution (125 IU/ml), were exteriorized at the dorsal neck level. Postoperatively, rats were housed individually in plastic cages and allowed to recover for 24 h before any circulatory experiments.

Recording of MAP and HR

At the time of experiment, the arterial catheter was connected to a blood-pressure transducer (Statham P23 ID) coupled to a polygraph recorder; HR was obtained from a cardiotachometer triggered by the pressure pulses. Both signals were recorded on a Gilson model 5/6H (Medical Electronics Inc., Middletown, WI, USA). Mean aortic pressure was calculated as diastolic + [(systolic -- diastolic)/3].

Experimental protocol

Before each experiment, blood pressure and HR were allowed to stabilize for at least 45 min and were recorded during a period of 10 to 15 min (according to the duration of effects) after i.v. treatment with EOMV. When subsequent doses of EOMV were administered, MAP and HR were first allowed to return to their baseline levels, obtained before the first injection of the compound. All of the following experiments were performed on conscious, freely moving rats.

* Series 1: This series of experiments was carried out to establish a dose-effect relationship. Each DOCA-salt-hypertensive (n = 8) or uninephrectomized control (n = 6) rat received a series of increasing bolus (100 [micro]1) doses of EOMV (1, 5, 10 and 20 mg/kg body wt.) (Lahlou et al. 2001) via the i.v. catheter, and time course of the changes in MAP and HR was recorded. Acetylcholine (5 [micro]g/kg body wt.) was used as a positive control (Degouville and Cavero, 1991) and induced a decrease in MAP of 59.20 [+ or -] 2.10% (mean [+ or -] SEM, n = 3).

* Series 2: Maximal decreases in MAP elicited by i.v. hexamethonium bromide (30 mg/kg body wt.) were assessed in both DOCA-salt-hypertensive (n = 5) and uninephrectomized control (n = 5) rats. At this dose, hexamethonium elicits complete ganglionic blockade (Sapru et al. 1982).

* Series 3: The time course of the changes in MAP and HR elicited by i.v. injections of EOMV was also determined for DOCA-salt-hypertensive rats which had been pretreated intravenously 10 min earlier with saline (1 ml/kg body wt., n = 8) or hexamethonium (30 mg/kg body wt., n = 5).

Statistical analysis

All results are expressed as mean [+ or -] s.e.m. Maximal changes (expressed as a percentage of baseline values) in MAP and HR after each dose of the EOMV, were used to construct a dose-response curve. Significance (p <0.05) of the results was assessed using of unpaired or paired Student's t-tests, Mann-Whitney U-test and one-way (doses) or two-way (treatment x doses) analysis of variance (ANOVA).

Results

Baseline MAP in rats treated with DOCA and given 1% saline to drink was 177 [+ or -] 4 mmHg (pooled data from 16 rats), and was significantly (p < 0.001, unpaired Student's t-test) higher than that measured in uninephrectomized control rats (110 [+ or -] 4 mmHg, pooled data from 11 rats). However, baseline HR in DOCA-salt-hypertensive rats (372 [+ or -] 12 bpm) was not different statistically (p > 0.05, unpaired Student's t-test) from that in normotensive rats (361 [+ or -] 14 bpm).

In both DOCA-salt-hypertensive and normotensive control rats, one-way ANOVA showed that baseline MAP and HR before injection of each dose of EOMV remained essentially invariant (p > 0.05). Intravenous injections of EOMV (1 to 20 mg/kg body wt.) induced immediate and dose-dependent decreases in MAP and HR (Fig. 1; p <0.001, one-way ANOVA), the magnitude of which was maximal within the first 20-30 sec after EOMV treatment. Both the hypotensive and bradycardic effects of EOMV became significant at 1 mg/kg body wt. (Fig. 1; p <0.01, paired Student's t-test). At all doses tested, pre-dose values of MAP were fully recovered within the first mm following EOMV treatment. However, HR remained significantly reduced 1-5 min following administration of 5, 10 and 20 mg/kg body wt. of EOMV (p <0.05, paired Student's t-test). Two-way analysis of variance showed that maximal percent decreases in MAP elicited by EOMV were significantly greater than those in uninephrectomized control rats (Fig. 1A; p <0.001). By contrast, EOMV -induced bradycardia in DOCA-salt-hypertensive rats did not differ significantly from that in normotensive rats (Fig. IB; p> 0.05, two-way ANOVA).

Maximal percent and absolute decreases in MAP following i.v. administration of hexamethonium (30 mg/kg body wt.) were 49 [+ or -] 3% and 83.50 [+ or -] 5.40 mmHg, respectively, in DOCA-salt-hypertensive rats, versus 34 [+ or -] 3% and 38 [+ or -] 2 mmHg in uninephrectomized control rats. They were significantly greater in DOCA-salt-hypertensive rats than in controls (Fig. 2; p <0.01, Mann-Whitney U-test). In DOCA-salt-hypertensive rats, pretreatment with hexamethonium (30 mg/kg body wt., i.v.) did not significantly affect maximal percent decreases in MAP elicited by EOMV (1 to 20 mg/kg body wt.) (Fig. 3; p>0.05, two-way ANOVA), the magnitude of which remained statistically greater than in uninephrectomized control rats (Fig. 3; p <0.001, two-way ANOVA). However, such pretreatment reduced partially, but significantly, the magnitude of maximal EOMV-induced bradycardia (Fig. 3; p <0.01, two-way ANOVA). Such a reduction became significant at the dose of 5 mg/kg body wt. (p <0.05, Mann-Whitney U-test). Residual ma ximal decreases in HR after i.v. hex-amethonium were still significant with respect to pre-injection values (Fig. 3;p < 0.001, paired Student's t-test) and remained related to the dose of EOMV (Fig. 3; p < 0.00 1, one-way ANOVA).

* Discussion

Baseline MAP and HR values of conscious, uninephrectomized normotensive, rats were of the same order of magnitude as those reported previously for the same preparation (Lahlou and Pinto Duarte, 1998; Lahlou, 1999; Lahlou et al. 2000) or in conscious, normotensive intact rats (Lahlou et al. 1993). Furthermore, maximal percent decreases in MAP and HR elicited by EOMV in uninephrectomized control rats were of similar magnitude to those reported in normotensive intact rats (Lahlou et al. 2001). Finally, only baseline MAP was increased significantly after 4 weeks of DOCA-salt treatment, as reported previously (Lahlou and Pinto Duarte, 1998; Lahlou, 1999; Lahlou et al. 2000).

The current study shows that i.v. treatment with EOMV induces dose-dependent decreases in MAP and HR in both DOCA-salt-hypertensive and uninephrectomized control rats. This agrees with a previous report regarding cardiovascular responses to i.v. EOMV in anesthetized, normotensive rats (Lahlou et al. 2001).

The observed cardiovascular effects of EOMV in DOCA-salt-hypertensive rats may add antihypertensive activity to the list of therapeutic uses of Mentha x villosa in folk medicine. The magnitude of EOMV-induced hypotension is significantly greater in DOCA-salt-hypertensive rats, as compared with uninephrectomized controls. It has been reported that the basal level of norepinephrine, an index of sympathetic activity (Kopin et al. 1978), is greater in DOCA-salt-hypertensive rats than in controls (Nagahama et al. 1985; Lahlou et al. 2000). Furthermore, the observed increases in both maximal percent and absolute decreases in MAP, in response to hexamethonium bromide after DOCA-salt treatment, further support the increased basal sympathetic activity in this model of hypertension. Therefore, enhancement of EOMV-induced hypotension after DOCA-salt treatment may be related to increased sympathetic activity in this hypertensive model. Two lines of evidence do not, however, support this explanation. First, the magnitude of EOMV-induced hypotension in intact rats remained unaltered even when the central sympathetic nerve drive, contributing to the maintenance of blood pressure, was eliminated by ganglionic blockade with hexamethonium (Lahlou et al. 2001). This indicates that EOMV does not require the presence of an operational central autonomic drive to the cardiovascular system in order to reduce blood pressure, because the latter effect occurs irrespective of whether vessels are constricted by the sympathetic neural drive. Second, the enhancement of EOMV-induced hypotension after DOCA-salt treatment appears to be in dependent of the degree of vascular tone, as it was unaffected by blockade of ganglionic transmission with hexamethonium.

The present findings give strong support to our previous hypothesis that hypotensive response to EOMV results from its vasodilatory action directly upon vascular smooth muscle. Such a hypothesis has been corroborated recently by in vitro experiments showing that, in isolated thoracic aorta preparations, EOMV was able to induce a concentration-dependent reduction of potassium-induced contraction (unpublished data). The effects of EOMV on isolated aorta are not peculiar to that tissue alone, as EOMV and OP were reported to induce a concentration-dependent relaxation of basal tone of guinea pig isolated ileum segments, and to inhibit potassium-induced contracture of the same preparation (Sousa et al. 1997; Sousa, 1999). Such a myorelaxant activity justifies the use of Mentha x villosa in folk medicine as an antispasmodic. Thus, it could be postulated that enhancement of EOMV-induced hypotension in DOCA-salt-hypertensive rats is related to an increased vascular smooth muscle relaxation induced by EOMV, as was dem onstrated previously for EOCN-induced hypotension in the same hypertensive model (Lahlou et al. 2000). Further investigations are presently underway in our laboratory to test this hypothesis. As was reported for normotensive rats (Lahlou et al. 2001), bradycardic effects of EOMV in DOCA-salt-hypertensive rats appear dependent upon an intact and operational autonomic nerve drive to the heart, as they were reduced by i.v. hexamethonium administration. Finally, the findings that DOCA-salt treatment enhanced EOMV-induced hypotension without affecting brady-cardia, and that i.v. hexamethonium reduced the magnitude of bradycardia elicited by EOMV without affecting hypotension, lend further support to the concept of independent mechanisms for EOMV-induced hypotension and bradycardia (Lahlou et al. 2001).

In conclusion, the current study shows that: (1) hypotensive responses to i.v. EOMV and hexamethonium bromide are greater in conscious DOCA-salt-hypertensive rats than in uninephrectomized controls, and (2) enhancement of EOMV-induced hypotension remained unaffected by i.v. pretreatment with the ganglion blocker hexamethonium. Thus, our previous hypothesis that the hypotensive response to EOMV is due to an active vascular relaxation rather than to withdrawal of sympathetic tone, is corroborated by the present results. The enhancement of EOMV-induced hypotension in DOCA-salt-hypertensive rats could he related mainly to increased vascular responsiveness to EOMV, rather than to enhanced sympathetic nervous system activity in this hypertensive model.

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Acknowledgements

We would like to thank Dr. Sergio Horta Mattos, Dr. Francisco Jose de Abreu Matos, Dr. Jose Wilson Alencar, Dr. M. Iracema Lacerda Machado and Dr. Afranio Aragao Craveiro from the Department of Organic and Inorganic Chemistry of the Federal University of Ceara, for kindly providing us with EOMV, and Leylliane F.L. Interaminense for technical assistance. This work was supported by the Fundacao de Amparo a Ciencia e Tecnologia, Governo de Estado de Pernambuco (FACEPE). R.F.L. Carneiro-Leao and L.F.L. Interaminense are undergraduate students (PIBIC/CNPq Fellows).

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* Address

S. Lahlou, Departamento de Fisiologia e Farmacologia, Centro de Ciencias Biologicas, Universidade Federal de Pernambuco, 50670-901, Recife, PE, Brasil. Tel.: ++55-81-3271-8531; Fax: ++55-81-3271-8976; e-mail: [e-mail]

S. Lahlou (1), R. Ferreira Lima Carneiro-Leao (1) and J. H. Leal-Cardoso (2)

(1.) Departamento de Fisiologia e Farmacologia, Universidade Federal de Pernambuco, Recife-PE, Brasil

(2.) Departmento de Ciencias Fisiologicas, Universidade Estadual do Ceara, Fortaleza-CE, Brasil