Chemical composition and vasorelaxant effect induced by the essential oil of Lippia alba (Mill.) N.E. Brown. (Verbenaceae) in rat mesenteric artery.

OBJECTIVES: To investigate the chemical composition and vasorelaxant effect of the essential oil of Lippia alba (EOLA) in rat mesenteric artery.
MATERIAL AND METHODS: Chemical composition of EOLA was investigated by gas chromatography-mass spectrometry (GC/MS). Vasorelaxant effect was evaluated in vitro in rat superior mesenteric artery rings.
RESULTS: GC/MS analysis revealed the presence of 19 compounds, with geranial (48.58%) and neral (35.42%) being the major constituents. In intact rings precontracted with phenylephrine (Phe: 1 μM), EOLA (100-1000 μg/mL) induced relaxation, where the maximal effect (Emax) was 110.8 ± 10.8%. This effect was not modified after endothelium removal (Emax = 134.8 ± 16.5%), after tetraethylammonium (TEA) (Emax = 117.2 ± 4.96%), or in rings precontracted with KCl (80 mM) (Emax = 112.6 ± 6.70%). In addition, EOLA was able to inhibit the contraction caused by CaCl(2) and produced a small but significant (P<0.05) additional effect (from 70.5 ± 3.4 to 105.3 ± 13.5%, n = 5) on the maximal relaxation of nifedipine (NIF: 10 μM).
CONCLUSIONS: The results demonstrated that EOLA induces endothelium-independent vasorelaxation, which appears to be caused, at least in part, by blocking Ca(2+) influx through voltage-operated Ca(2+) channels.

Introduction

The essential oils are a mixture of volatile substances commonly found in aromatic plants composed of terpenes and non-terpene components.[1] Studies in animals have demonstrated the beneficial properties of essential oils in the cardiovascular system as antithrombotic, antiplatelet, endothelial protective, vasorelaxant, and hypotensive activities.[12] Recent reports have shown that essential oils also produce CVS effects such as improvement in coronary flow, hypotension and bradycardia in humans.[34]

The genus Lippia comprises approximately 250 species, which produce characteristic aromatic essential oils.[5] In Brazil, the species Lippia alba (Mill.) N.E. Brown (Verbenaceae) is an aromatic medicinal plant popularly known as “Erva Cidreira Brasileira” or “Cidreira”. It has been one of the most commonly used aromatic herbs in Brazilian Folk Medicine for blood pressure control.[56]

In spite of the fact that the antihypertensive effect of Lippia alba has already been studied,[78] no data about the possible effect of the essential oil from this plant on vascular reactivity was found in the literature. Therefore, the objective of this work was to evaluate the chemical composition and vasorelaxant effect of the essential oil of L. alba (EOLA) in rat mesenteric artery and to investigate its possible mechanism of action.

Materials and Methods

Drugs and Solutions

The drugs used were: acetylcholine (ACh), L-phenylephrine (Phe), tetraethylammonium (TEA), cremophor (all from SIGMA), and nifedipine (NIF) (from RBI). The drugs were freely dissolved in distilled water. All stock solutions were maintained at 0°C and diluted to the desired concentration with Tyrode solution for experiments.

The composition of the normal Tyrode's solution was (mM): NaCl 158.3, KCl 4.0, CaCl22.0, NaHCO3 10.0, C6H12O6 5.6, MgCl2 1.05, and NaH2PO4 0.42. The K+-depolarizing solutions (KCl 80 and 60 mM) were made by replacing 80 or 60 mM KCl in the Tyrode's solution with equimolar NaCl. In nominally zero-Ca2+ solution, CaCl2 was omitted.

Animals

Male Wistar rats (200–300 g) were housed in controlled temperature (21±1° C) and exposed to a 12 hr light-dark cycle with free access to food (Purina-Brazil) and tap water. All procedures described in the present work are approved by the Animal Research Ethics Committee from the Federal University of Sergipe (CEPA n° 06/2009).

Plant Material, Preparation and Phytochemical Analyses of the Essential Oil

Leaves of L. alba were collected from the medicinal plants garden at the Federal University of Sergipe (Brazil) (latitude 11°00' S and longitude 37°12’ W). A voucher specimen was identified and deposited at the Herbarium of the Federal University of Sergipe under code ASE 13495. EOLA was obtained from the dried leaves by hydrodistillation in a clevenger apparatus for 160 min and stored at 4° C. When required, EOLA was dissolved with Tyrode's solution plus cremophor (0.1% v/v) for experiments at the desired concentrations.

EOLA was analyzed by gas chromatography coupled with mass spectrometry (GC/MS) according to these experimental conditions: capillary column DB-5MS (30 × 0.25 × 0.25 mm, i.d.), electron impact 70 eV, helium (99.999%) was used as carrier gas at a constant flow of 1.2 mL min-1 and an injection volume of 0.5 μL (dilution in ethyl acetate), injector temperature 250°C, and detector temperature 280°C. The oven temperature was programmed from 50° C (isothermal for 2 min), with an increase of 4°C/min, to 200° C, then 10° C/min to 300° C, ending with a 10-min isothermal at 300° C. Mass spectra were taken at 70 eV, a scan interval of 0.5 s, and fragments from 40 to 550 Da. Quantitative analysis of the chemical constituents was performed by flame ionization gas chromatography (FID) under the same conditions as GC-MS. Identification of individual components of the essential oil was performed by computerized matching of the acquired mass spectra with those stored in the NIST21 and NIST107 mass spectral library of the GC-MS data system. Retention indices (RI) for all compounds were determined according to literature for each constituent as previously described.[910]

Pharmacological experiments

Tissue preparation

Tissue preparation was performed as described by Menezes et al.[11] After animal sacrifice, superior mesenteric artery was removed, cleaned from connective and fat tissues, and sectioned in rings (1–2 mm). These rings were suspended by fine stainless steel hooks connected to a force transducer (Letica, Model TRI210, Italy) with cotton threads in organ baths containing 10 mL of Tyrode's solution. This solution was continually gassed with carbogen at 37° C and the rings maintained under a resting tension of 0.75 g for 60 min (stabilization period). The isometric tension was recorded through the force transducer coupled to an amplifier-recorder (AVS, SP, Brazil). When necessary, endothelium was removed by gently rubbing the intimal surface of the vessels with a fine stainless steel wire, and its functionality was assessed by the ability of ACh (1 μM) to induce more than 75% relaxation of Phe (1 μM) tonus. The absence of the relaxation to ACh was taken as evidence that the rings were functionally denuded of endothelium.

EOLA Effect on Phe (1 μM) Tonus in Rings with or without Endothelium

After a stabilization period, contractions were induced with 1 μM of Phe in intact rings (with endothelium) or after endothelium removal (without endothelium). During the tonic phase of the contraction, vehicle (Tyrode's solution + cremophor) or different concentrations of EOLA (1, 3, 10, 30, 100, 300 and 1000 μg/mL, cumulatively) were added to the organ bath. The relaxations were measured in function of the developed tension before and after the addition of EOLA and expressed as percentage of relaxation from induced tonus. EOLA-induced vasorelaxation was statistically compared with the vehicle effect.

EOLA Effect on Phe (1 μM) or KCl (80 mM) Tonus in Rings Without Endothelium, or after TEA Incubation

After a stabilization period, contractions were induced either with 1 μM of Phe or with a high concentration of K+ (KCl 80 mM) in rings without endothelium. The vasorelaxation was obtained and measured as previously described. Furthermore, vasorelaxation for EOLA was also obtained after preincubation (30 min) with 0.1 mM of TEA, a non-selective K+ channel blocker.[12]

EOLA Effect on Concentration-Response Curves for CaCl2 in Rings Without Endothelium

After a stabilization period, rings without endothelium were incubated with nominally zero-Ca2+ solution for 15 min and then exposed to nominally zero-Ca2+ solution with KCl at 60 mM for next 15 min. Then, a first cumulative concentration-response curve for CaCl2(10-6, 3×10-6, 10-5, 3×10-5, 10-4, 3×10-4, 10-3, 3×10-3, 10-2, and 3×10-2 M) was obtained. In these same preparations, after washout and exposed to nominally zero-Ca2+ solution with KCl at 60 mM again, concentrations of EOLA (10, 30 and 300 μg/mL) were individually preincubated for 15 min and a second cumulative concentration-response curve for CaCl2 was obtained. The results were expressed as percentages of the maximal response for CaCl2 alone, and the curves were statistically compared.

EOLA Effect after Inhibition of the Dihydropyridine-Sensitive Voltage-Operated Calcium Channels (Cavs) by NIF (10 μM) in rings without endothelium

The involvement of dihydropyridine-sensitive Cavs in the effect induced by EOLA was assessed by testing the oil (300 μg/mL) in rings without endothelium precontracted with Phe (1 μM) in the absence or presence of NIF (10 μM), a selective blocker of the dihydropyridine-sensitive Cavs.[1314]

Statistical analysis

Values are expressed as mean ± standard error mean (SEM). When appropriate, one- or two-way ANOVA followed by the Bonferroni post-test were conducted in order to evaluate the differences between means. All statistical analyses were done by using Graph Pad Prism™ software.

Results

Phytochemical Screening

The chemical composition of EOLA revealed presence of 19 compounds, with geranial (48.58%) and neral (35.42%) being the major constituents [Table 1].

Table 1

Chemical composition of the essential oil of L. alba leaves

Pharmacological Results

Rings with intact endothelium (control), EOLA produced relaxations of tonus induced by Phe. This effect was not different from those obtained in endothelium-denuded rings [Figure 1].

Figure 1

Vasorelaxant effect of vehicle in the control condition (with endothelium) or of EOLA (1 - 1000 μg/mL, cumulatively) in the control condition (with endothelium) and after removal of endothelium in rings of rat superior mesenteric artery precontracted with Phe (1 μM). Values are mean ± SEM of six experiments. The data were analyzed with one-way ANOVA followed by the Bonferroni post-test

In endothelium-denuded rings precontracted with K+-depolarizing solution (KCl 80 mM), EOLA-induced vasorelaxation was similar to that obtained in endothelium-denuded rings precontracted with Phe or in the presence of TEA [Figure 2].

Figure 2

Vasorelaxant effect of EOLA (1 - 1000 μg/mL, cumulatively) in rings of rat superior mesenteric artery without endothelium precontracted with Phe (1 μM), precontracted with KCl 80 mM, or precontracted with Phe (1 μM) after incubation with TEA (100 μM, 30 min). Values are mean ± SEM of six experiments. The data were analyzed with one-way ANOVA followed by the Bonferroni post-test

EOLA significantly (P<0.001) antagonized CaCl2-induced contraction at 10 mg/ml and significantly (P<0.001) abolished at 30 and 300 μg/ml [Figure 3].

Figure 3

Concentration-response curves for CaCl2 (10-6 to 3.10-3 M) before (Control) and after the incubation of preparations with EOLA (10, 30 and 300 μg/mL) in rings of rat mesenteric artery without endothelium. Values are expressed as mean ± SEM of six experiments. The data were analyzed with repeated-measures two-way ANOVA followed by the Bonferroni post-test. *P < 0.05, **P < 0.01 and ***P < 0.001 versus Control

Preparations without endothelium precontracted with Phe, both EOLA (300 μg/mL) and NIF (10 μM), separately, were able to induce vasorelaxations. In rings preincubated with NIF, EOLA (300 μg/mL) induced a small but significant (P<0.05) additional effect [Figure 4].

Figure 4

Vasorelaxant effect of nifedipine (NIF: 10 μM), EOLA (300 μg/mL) and EOLA (300 μg/mL) after the maximum relaxation of NIF (10 μM) in rings of rat mesenteric artery without endothelium precontracted with Phe (1 μM). Values are expressed as mean ± SEM of 6 experiments. The data were analyzed with one-way ANOVA followed by the Bonferroni post-test.

Discussion

This study demonstrated the possible benefits of EOLA on the cardiovascular system by producing vasorelaxation that appeared to have a calcium-blocking property similar to other drugs used in the treatment of hypertension, such as nifedipine and verapamil.[1314]

The phytochemical analysis of EOLA demonstrated the presence of several compounds, among them geranial and neral, which were the major constituents of the oil.

EOLA produced relaxations of Phe tonus in rings with intact endothelium. According to literature, vascular tone is modulated by endothelium-derived relaxant factors (EDRFs), including nitric oxide (NO) and prostacyclin (PGI2).[15] To verify the role of the endothelium in EOLA-induced vasorelaxation, experiments were performed on endothelium-denuded rings. As observed, there was no difference between intact (control) and endothelium-denuded ring relaxations, suggesting that this effect is not mediated by vascular endothelium.

It is well known that the maintenance of smooth muscle contraction depends on Ca2+ influx through mainly Cavs.[16] It is also reported that the increase of external K+ concentration induces smooth muscle contraction through Cavs activation and subsequent calcium release from the sarcoplasmic reticulum.[16] This contraction is inhibited by Ca2+ channel blockers or by removal of external Ca2+ and is, therefore, entirely dependent on Ca2+ influx.[16] Based on this assumption, in another set of experiments, EOLA was tested on endothelium-denuded rings precontracted with K+-depolarizing solution. It was observed that EOLA-induced vasorelaxation was similar to that obtained in rings precontracted with Phe, which suggests that EOLA appears to be acting through a similar pathway between both contractile agents, i.e., the inhibition of the Ca+2 influx.

In order to emphasize the hypothesis above, we obtained concentration-response curves for CaCl2 before and after incubation with EOLA. In this condition, EOLA was able to inhibit the contractions induced by CaCl2. As reported by Chan et al.,[17] NIF, a dihydropyridine-sensitive Cavs selective blocker, also inhibited the concentration-response curve for CaCl2, which strongly supports that EOLA could possibly be acting as a calcium channel blocker.

Furthermore, in preparations without endothelium precontracted with Phe, EOLA was able to induce a small but significant additional vasorelaxant effect on the maximal relaxation of NIF, demonstrating that EOLA can be acting mainly through a similar pathway to NIF, i.e., blocking dihydropyridine-sensitive Cavs.

Although the results show an important participation of the Ca+2 channels in EOLA effects, the possible involvement of K+ channels can not be discarded. According to the literature, potassium channels are the dominant ion conductive pathways in vascular muscle cells. The electrochemical gradient for K+ ions is such that the opening of K+ channels results in diffusion of this cation out of the cells and membrane hyperpolarization. This effect closes Ca2+ channels and leads to vasodilatation.[18]

Thus, the participation of the K+ channels in EOLA-induced vasorelaxation was investigated by using rings without functional endothelium precontracted with Phe in the absence or presence of TEA, a non-selective blocker of these channels.[12] In this condition, EOLA was able to induce relaxation that was not significantly different from the control. This result suggests that K+ channels do not seem to be involved in the vasorelaxant effect induced by EOLA.

A previous study by Guerrero et al.,[7] using the ethanol extract from Lippia alba, has demonstrated vasorelaxant effect in rat-isolated aorta rings. These results are in agreement with our findings. On the other hand, unlike our results, Guerrero et al.[7] have also demonstrated that this extract does not produce a hypotensive effect in anesthetized rats. The observed difference can be due to the type of extract or dose, or anesthetic agent used in the animal.

The literature has also demonstrated that essential oil produces vasorelaxation by several mechanisms of action, among them inhibition of calcium influx or through endothelial mediators. The results of the present work are analogous to that described by Lahlou et al., Menezes et al., and Moreira et al.,[31119] who have demonstrated that the essential oils of Aniba canelilla Bark, Cymbopongon winterianus, and Cymbopongon citratus, respectively, medicinal plants used in folk medicine for hypertension treatment, produce vasorelaxation through an inhibition of calcium inward current. On the other hand, Guedes et al.[20] showed that the essential oil of Mentha x villosa in rats induces vasorelaxation by a pathway involving endothelial mediators.

In conclusion, these results demonstrate that EOLA induces vasorelaxation in rat mesenteric artery possibly due to an inhibition of the Ca2+ influx through dihydropyridine-sensitive Cavs. This plant seems to present a potential clinical use for hypertension treatment; however, further studies are necessary to evaluate its safety and therapeutic margin before human use.