Vasorelaxant and antihypertensive effects of methanolic fraction of the essential oil of Alpinia zerumbet

Vascular Pharmacology 58 (2013) 337–345

Contents lists available at SciVerse ScienceDirect

Vascular Pharmacology journal homepage: www.elsevier.com/locate/vph

Vasorelaxant and antihypertensive effects of methanolic fraction of the essential oil of Alpinia zerumbet☆ Gilmara Holanda da Cunha a,⁎, Manoel Odorico de Moraes a, Francisco Vagnaldo Fechine a, Fernando Antônio Frota Bezerra a, Edilberto Rocha Silveira b, Kirley Marques Canuto c, Maria Elisabete Amaral de Moraes a a Clinical Pharmacology Unit, Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceará, Coronel Nunes de Melo 1127, 60430-270, Fortaleza, Ceará, Brazil b Department of Organic and Inorganic Chemistry, Federal University of Ceará, 12200, 60021-940, Fortaleza, Ceará, Brazil c Embrapa Tropical Agroindustry, Sara Mesquita 2270, 60511-110, Fortaleza, Ceará, Brazil

a r t i c l e

i n f o

Article history: Received 30 January 2013 Received in revised form 19 March 2013 Accepted 9 April 2013 Keywords: Alpinia Phytotherapy Vasodilatation Hypertension Calcium channel blocker

a b s t r a c t Alpinia zerumbet is used in folk medicine in Brazil to treat hypertension. However, several pathways involved in the mechanism of vasorelaxation are still unclear. This study was designed to verify the antihypertensive effect of the methanolic fraction of the essential oil of A. zerumbet (MFEOAz) and to characterize its mechanism of action. The thoracic aortic rings from the Wistar rats were perfused in the organ chambers filled with Kreb's solution, where the tension of each ring was measured. The antihypertensive effect of MFEOAz was assessed in rats submitted to chronic hypertension by inhibition of nitric oxide synthesis by indirect measurement of blood pressure with indirect tail cuff method. MFEOAz relaxed phenylephrine and KCl-induced contraction of either endothelium-intact or endothelium-denuded rat aortic rings in a concentration-dependent manner. Pre-incubation with MFEOAz (100 and 300 μg/mL) in Ca2+-free Krebs solution attenuated phenylephrine- or caffeine-induced contraction. Pre-incubation with L-NAME, ODQ, wortmannin, atropine, indomethacin, catalase, SOD, TEA, 4-aminopyridine, glibenclamide, apamin, charybdotoxin, or iberiotoxin did not affect MFEOAz-induced relaxation. The intragastric administration of MFEOAz induced an antihypertensive effect. MFEOAz it seems inhibited the calcium influx via voltage-operated calcium channels and receptor-operated calcium channels, as well as inhibition of calcium mobilization from intracellular stores. © 2013 Elsevier Inc. All rights reserved.

1. Introduction Alpinia zerumbet (Pers.) Burtt. et Smith (Zingiberaceae) is an aromatic plant, originating from West Asia, but it has a large distribution in South America (Santos et al., 2011). A. zerumbet is known popularly as “colonia” in Northeast Brazil, where it widely used in folk medicine, predominantly in the treatment of hypertension and anxiety (Lahlou et al., 2003; Santos et al., 2011). Abbreviations: MFEOAz, methanolic fraction of the essential oil of Alpinia zerumbet; GC-MS, gas chromatography coupled with mass spectrometry; GC-FID, flame ionization detector; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; TEA, tetraethylammonium; L-NAME, NG-nitro-L-arginine methyl ester; 1H-NMR, 1H-nuclear magnetic resonance spectroscopy; SOD, superoxide dismutase; EGTA, ethyleneglycol bis(β-aminoethylether)-N, N,N′,N′-tetraacetic acid; VOCC, voltage-operated calcium channels; ROCC, receptor-operated calcium channels; HR, heart rate; SAP, systolic arterial pressure; DAP, diastolic arterial pressure; MAP, mean arterial pressure. ☆ These authors contributed equally to this work. ⁎ Corresponding author at: Clinical Pharmacology Unit, Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceará, Coronel Nunes de Melo 1127, 60021-940, Fortaleza, Ceará, Brazil. Tel.: +55 85 3366 8346; fax: +55 85 3223 2903. E-mail address: [email protected] (G.H. Cunha). 1537-1891/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.vph.2013.04.001

Leaves of A. zerumbet possess an essential oil content of 0.2%–1% of plant dry weight, composed principally of mono- and sesquiterpenes (Pinto et al., 2009). Essential oils are natural complex mixtures that may have biological effects. Studies have shown that essential oil obtained from A. zerumbet leaves has vasorelaxant (Pinto et al., 2009) and antihypertensive effects (Lahlou et al., 2003), fungistatic activity (Lima et al., 1993), anxiolytic effect in mice (De Araújo et al., 2009), neuronal excitability blockade (Leal-Cardoso et al., 2004), relaxant effects on intestinal smooth muscle (Bezerra et al., 2000) and antioxidant activity (Elzaawely et al., 2007). Many studies have shown that essential oil of A. zerumbet has pharmacological effects; in particular, it lowers blood pressure by means of vascular smooth muscle relaxation (Pinto et al., 2009), but the mechanisms that govern this action are not yet completely elucidated. Considering that it is of great interest to explore the medicinal value of EOAz with regard to cardiovascular activities, its constituents were separated into three fractions, hexane, chloroform, and methanolic, to better understand their mechanisms of action. In fact, the effect of these fractions on vascular function has not been previously reported. In a study carried out in our laboratory, the vasodilator effect of these fractions was determined and MFEOAz was found to have the most

338

G.H. Cunha et al. / Vascular Pharmacology 58 (2013) 337–345

potent vasorelaxant effect in vitro (unpublished data). Thus, the purpose of this study was to verify the antihypertensive effect of MFEOAz in vivo and to characterize its mechanism of action in vitro.

MFEOAz on blood pressure measured by indirect tail cuff method in rats submitted to chronic hypertension by inhibition of nitric oxide. 2.3. In vitro experiments: studies on isolated rat thoracic aorta

2. Materials and methods 2.1. Plant material, extraction and chromatographical analysis A. zerumbet leaves were collected in November 2009, in Maranguape County, State of Ceará, Brazil. Its botanical identification was determined in the Prisco Bezerra Herbarium of the School of Agronomy, Federal University of Ceará, where a voucher specimen has been deposited under No. 50312. A. zerumbet leaves were extracted by hydrodistillation in a Clevenger-type glass apparatus, affording a yellowish oil, which was chromatographed on silica gel column and eluted with hexane, followed by chloroform and methanol. The three fractions obtained were analyzed by gas chromatography coupled with mass spectrometry (GC-MS) and flame ionization detector (GC-FID) according to the method described previously (Cavalcanti et al., 2012). The GC-MS analysis was carried out on a Shimadzu QP5050 instrument equipped with a non-polar OV-5 fused silica capillary column, while the GC-FID analysis was accomplished on a Shimadzu GC 2010 Plus instrument provided with a non-polar CP-Sil-8 fused silica capillary column. The GC-MS and GC-FID analysis of the MFEOAz obtained by the chromatographic separation permitted the identification of 15 constituents. All compounds identified were already reported in previous works for essential oil of this species (Lahlou et al., 2003; Elzaawely et al., 2007). The MFEOAz presented 1,8-cineol (27.81%) and terpinen-4-ol (57.35%) as the major components. The presence of the main constituents was confirmed by 1H-nuclear magnetic resonance spectroscopy (1H-NMR). In this study, we characterized only MFEOAz, which had the chemical composition shown in Table 1. 2.2. Animals and experiments Male Wistar rats weighing 200–250 g (50–60 days old) were used for in vitro experiments, and those weighing 250–330 g were used for in vivo experiments. Animals were kept under a 12 h–12 h light/dark cycle and allowed free access to food and water. All procedures were performed in accordance with the Animal Ethics Committee of the Federal University of Ceará, Brazil, under registration no. 18/2011. The experiments were performed in two phases: an initial in vitro study to determine the relaxant effect of MFEOAz in isolated aortic rings and application of specific protocols to elucidate the possible mechanism of action, and an in vivo study to determine the effect of Table 1 Chemical composition, kovats retention indices and relative area of the constituents of MFEOAz. Constituents

KIa

Relative area (%)b

%

1,8-Cineol 4-Thujanol Linalool cis-β-dihydro-terpineol cis-p-Menth-2-en-1-ol trans-p-Menth-2-en-1-ol trans-Dihydro-α-terpineol Borneol Terpinen-4-ol p-Cymen-8-ol α-Terpineol p-Menth-1-en-3-ol Bornyl acetate Caryophyllene oxide β-eudesmol

1046 1082 1106 1110 1135 1153 1180 1182 1190 1197 1204 1219 1291 1588 1662

27.81 1.64 1.48 2.14 1.77 1.47 0.29 0.31 57.35 0.08 3.82 0.38 0.05 1.00 0.40

27.81 1.64 1.48 2.14 1.77 1.47 0.29 0.31 57.35 0.08 3.82 0.38 0.05 1.00 0.4

a Kovats retention indices calculated from a homologous series of n-alkanes (C7–C30) analyzed on a CP-Sil-8 column. b Relative area percentage determined by GC-FID.

Rats were sacrificed by cervical dislocation followed by exsanguination. The thoracic aorta was quickly removed, cleaned of adherent connective tissue and cut into rings (3-4 mm in length). Two stainlesssteel stirrups were passed through the lumen of each ring. One stirrup was connected to an isometric force transducer (Force Transducer, MLT0201, Panlab, Spain) to measure tension in the vessels. The rings were placed in a 10-mL organ chamber containing Krebs solution, gassed with 95% O2 and 5% CO2, maintained at 37 °C and pH 7.4. The composition of Krebs solution was as follows (mmol/L): NaCl, 118.0; KCl, 4.7; KH2PO4, 1.2; MgSO4∙7H2O, 1.2; NaHCO3, 15.0; CaCl2, 2.5 and glucose, 5.5 (Hipólito et al., 2011). The aortic rings were stretched until they reached a resting tension of 10 millinewtons (mN), which was determined by length–tension relationship experiments and were then allowed to equilibrate for 60 min; during this time, the bath fluid was changed every 15–20 min. Endothelial integrity was assessed qualitatively by the degree of relaxation caused by acetylcholine (10 μmol/L) in the presence of contractile tone induced by phenylephrine (1 μmol/L). For studies of endothelium-intact vessels, a ring was discarded if relaxation with acetylcholine was not 80% or greater. In some experiments, the endothelium of the aortic rings was mechanically removed by gently rolling the lumen vessel on a thin wire. For studies of endothelium-denuded vessels, the ring was discarded if there was any degree of relaxation by acetylcholine. After a 60-min equilibration period, all aortic rings were initially exposed to KCl (80 mmol/L). 2.3.1. Effect of MFEOAz on rat aortic rings pre-contracted with phenylephrine or KCl Steady tension was evoked by phenylephrine (1 μmol/L) (Chen et al., 2009) or KCl (80 mmol/L) (Estrada-Soto et al., 2010) for endothelium-intact and endothelium-denuded rings (n = 6), and MFEOAz was added cumulatively (0.1–3000 μg/mL). 2.3.2. Role of nitric oxide (NO), guanylyl cyclase and phosphatidylinositol 3-kinase (PI3K) in MFEOAz-induced vascular response To determine if NO, guanylyl cyclase soluble and PI3K were involved in the relaxant effect of MFEOAz, endothelium-intact rings (n = 6) were incubated with NG-nitro-L-arginine methyl ester (L-NAME) (a NO synthase inhibitor, 100 μmol/L) (Magalhães et al., 2008; Pinto et al., 2009), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (a guanylyl cyclase inhibitor, 10 μmol/L) (Yeh et al., 2005) or wortmannin (a PI3K inhibitor, 0.5 μmol/L) (Hipólito et al., 2011) for 30 min prior to precontraction with phenylephrine (1 μmol/L). The cumulative concentration–response curves of MFEOAz were then constructed and compared with those obtained with untreated rings. 2.3.3. Role of muscarinic receptors in MFEOAz-induced vascular response To assess whether MFEOAz produced vasodilatation through the activation of muscarinic receptors, endothelium-intact aortic rings (n = 6) were incubated with atropine (a muscarinic receptor antagonist, 1 μmol/L)(Assreuy et al., 2011) for 30 min prior to pre-contraction with phenylephrine(1 μmol/L). 2.3.4. Role of prostanoids in MFEOAz-induced vascular response To determine if prostanoids were involved in the relaxant effect of MFEOAz, endothelium-intact rings (n = 6) were incubated with indomethacin (a non-selective COX inhibitor, 10 μmol/L) (Kamadyaapa et al., 2009) for 30 min prior to pre-contraction with phenylephrine (1 μmol/L).

G.H. Cunha et al. / Vascular Pharmacology 58 (2013) 337–345

339

Fig. 1. Vasorelaxant response induced by MFEOAz on rat aortic rings pre-contracted with phenylephrine (1 μmol/L) or KCl (80 mmol/L). Vasodilator effect was evaluated on endothelium-intact and endothelium-denuded preparations. Data are expressed as means ± SEM of 6 experiments performed on preparations obtained from different animals. *P = 0.0343 and ***P b 0.0001 denote a significant difference between the pEC50 values (unpaired t test).

2.3.5. Role of reactive oxygen species in MFEOAz-induced vascular response To determine if reactive oxygen species were involved in the relaxant effect of MFEOAz, endothelium-intact rings (n = 6) were incubated with catalase (catalyzes the decomposition of hydrogen peroxide to water and oxygen, 500 U/mL) (Sato et al., 2003) or superoxide dismutase (SOD) (catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide) (300 U/mL)(Campana et al., 2009) for 30 min prior to pre-contraction with phenylephrine (1 μmol/L). +

2.3.6. Role of K channels in MFEOAz-induced vascular response The involvement of K+ channels in MFEOAz-induced relaxation was assessed by incubating endothelium-intact rings (n = 6) for 30 min prior to pre-contraction with phenylephrine (1 μmol/L) with several K+ channel inhibitors: tetraethylammonium (TEA) (a non-selective K+ channel inhibitor, 10 mmol/L) (Zhang et al., 2010), 4-aminopyridine (a voltage-dependent K+ channel blocker, 1 mmol/L) (Chen et al.,

Table 2 Values of pEC50 and Emax (%) related to the vasorelaxant response of MFOEAz on endothelium-intact and endothelium-denuded rat aortic rings pre-contracted with phenylephrine (1 μmol/L) or KCl (80 mmol/L). Contractile agent

Endothelium-intact

Endothelium-denuded

pEC50

Emax (%)

pEC50

Emax (%)

KCl Phenylephrine

−1.57 ± 0.05 −2.13 ± 0.08†

105.74 ± 2.32 109.72 ± 5.11

−1.72 ± 0.04⁎ −2.12 ± 0.04‡

104.78 ± 1.79 109.20 ± 2.36

Data are expressed as means ± SEM of 6 experiments performed on preparations obtained from different animals. ⁎ P = 0.0343 compared to endothelium-intact aortic rings pre-contracted with KCl. † P b 0.0001 compared to endothelium-intact aortic rings pre-contracted with KCl. ‡ P b 0.0001 compared to endothelium-denuded aortic rings pre-contracted with KCl.

2009), glibenclamide (a non-specific ATP-sensitive K + channel blocker, 10 μmol/L) (Xue et al., 2011; Shen et al., 2013), apamin (1 μmol/L), charybdotoxin (15 nmol/L), and iberiotoxin (30 nmol/L) (selective small, intermediate and large conductance Ca2+-activated K + channel blockers, respectively) (Senejoux et al., 2011; Seok et al., 2011).

Table 3 Effect of L-NAME, ODQ, wortmannin, atropine, indomethacin, catalase, SOD, TEA, 4-aminopyridine, glibenclamide, apamin, charybdotoxin and iberiotoxin on MFEOAz-induced relaxant responses of endothelium-intact rat aortic rings pre-contracted with phenylephrine. Groups

pEC50

b

Control (MFEOAz) L-NAME (100 μmol/L) ODQ (10 μmol/L) Wortmannin (0.5 μmol/L) Atropine (1 μmol/L) Indomethacin (10 μmol/L) Catalase (500 U/mL) SOD (300 U/mL) TEA (10 mmol/L) 4-Aminopyridine (1 mmol/L) Glibenclamide (10 μmol/L) Apamin (1 μmol/L) Charybdotoxin (15 nmol/L) Iberiotoxin (30 nmol/L)

Emax (%)

Mean

SEM†

Mean

SEMa

−2.04 −1.99 −1.91 −1.78 −1.93 −2.28 −1.91 −2.00 −1.97 −2.33 −2.08 −2.20 −2.24 −2.34

0.12 0.07 0.06 0.10 0.06 0.17 0.13 0.09 0.08 0.06 0.10 0.10 0.09 0.08

107.59 109.14 108.65 101.23 109.41 115.79 109.98 110.53 109.80 114.38 110.98 113.51 112.46 116.62

6.53 3.67 3.13 4.26 3.24 13.00 7.78 5.75 4.54 4.01 6.02 6.53 5.95 6.43

In each group, data correspond to measurements obtained from 6 experiments performed on preparations from different animals. No statistically significant differences were found between treated groups and control group, by ANOVA followed by Dunnett's multiple comparison test. a SEM: standard error of the mean. b Control corresponds to MFEOAz-induced relaxant responses in the absence inhibitors.

340

G.H. Cunha et al. / Vascular Pharmacology 58 (2013) 337–345

containing EGTA (1 mmol/L) for 15 min is placed and removed. The rings were then rinsed in Ca 2 +-free Krebs solution (without EGTA), followed by the addition of KCl (60 mmol/L) or phenylephrine (1 μmol/L) (Hipólito et al., 2011). The cumulative concentration– response curves for CaCl2 (0.01–3 mmol/L) were obtained in the absence of MFEOAz (control group) or after 30 min incubation in the presence of MFEOAz (300 μg/mL). 2.3.9. Effect of MFEOAz pre-treatment on Ca 2+ release from intracellular stores To investigate whether MFEOAz could interfere with Ca 2 + release from intracellular stores, the inhibitory effects of MFEOAz on phenylephrine- or caffeine-induced contractions in the absence of extracellular Ca 2 + were determined in endothelium-intact (n = 6) and endothelium-denuded aortic rings (n = 6). Normal Krebs solution was replaced with Ca 2+-free solution containing EGTA (1 mmol/L) for 15 min and then washed with Ca 2 +-free solution. The rings were stimulated with phenylephrine (1 μmol/L) or caffeine (30 mmol/L) (Hipólito et al., 2011). The contractions induced by both agonists were obtained in the absence of MFEOAz (control group) or after 30 min incubation in the presence of MFEOAz (30, 100 or 300 μg/mL), using endothelium-intact and endothelium-denuded rings. 2.4. In vivo experiments: effects of MFEOAz on arterial pressure

Fig. 2. Effect of MFEOAz on CaCl2-induced contractile response in endothelium-intact and -denuded rat aortic rings. Concentration–response curves were determined in Ca2+-free solution after the depletion of extracellular calcium, where the CaCl2-contractile effect was dependent on Ca2+ influx through VOCC activated by KCl (60 mmol/L). The curves were constructed in the absence of added substance (control) or after 30 min incubation in the presence of nifedipine (100 μmol/L) or MFEOAz (30, 100 and 300 μg/mL) prior to the cumulative addition of CaCl2. Data correspond to the means ± SEM of 6 experiments performed on preparations obtained from different animals. *, + and # denote that Emax values of 300 μg/mL MFEOAz, nifedipine and 100 μg/mL MFEOAz, respectively, are significantly less than that of control and 30 μg/mL MFEOAz (P b 0.001). § denotes a significant difference between Emax values of 30 μg/mL MFEOAz and control (P b 0.001). Symbols † and ‡ denote that Emax values of 300 μg/mL MFEOAz and nifedipine, respectively, are significantly less than that of control and 30 μg/mL and 100 μg/mL MFEOAz (P b 0.001).

2.3.7. Effect of different concentrations of MFEOAz on calcium-induced contraction-dependent on extracellular Ca 2+ To investigate the inhibitory effects of MFEOAz (30, 100 and 300 μg/mL) on Ca 2+ influx through voltage-operated calcium channels (VOCC), endothelium-intact (n = 6) and endothelium-denuded aortic rings (n = 6) were exposed to a Ca2+-free Krebs solution in the presence of K + (60 mmol/L). Ca 2+-free Krebs solution had the same composition as normal Krebs solution except that CaCl2 was omitted. In addition, EGTA (1 mmol/L) was added to ensure total elimination of extracellular Ca2+ for 15 min and afterwards removed. Cumulative concentration–response curves for Ca2+ (0.01–3 mmol/L) were obtained. Three different concentrations of MFEOAz, vehicle or positive control (nifedipine, 100 μmol/L) were added to the bath and allowed to act for 30 min before recording the cumulative concentration–response curve for Ca 2+. Each preparation was exposed to only one concentration of MFEOAz. 2.3.8. Effect of MFEOAz on Ca 2+ influx through voltage-operated calcium channels (VOCC) and receptor-operated calcium channels (ROCC) To investigate the inhibitory effects of MFEOAz on Ca 2+ influx through VOCC and ROCC, after equilibration, the endothelium-intact (n = 6) and endothelium-denuded aortic rings (n = 6) were washed with Ca2+-free Krebs solution. Afterwards, phenylephrine (1 μmol/L) was added to induce transient vasoconstriction and depletion of intracellular Ca2+ stores in Ca2+-free Krebs solution (approximately 45 min) containing EGTA (1 mmol/L). Then again Ca 2 +-free Krebs solution

Hypertension was induced in rats by the administration of L-NAME (30 mg/kg) dissolved in drinking water for 60 days. The first 30 days represented the induction phase of hypertension and was called pretreatment. On the 31st day, the rats were already hypertensive, and the therapies studied were started and maintained for 30 days, corresponding to the treatment phase. Animals were randomly allocated into three groups: control (0.5 mL distilled water; n = 9), MFEOAz (100 mg/kg, diluted in 0.5 mL distilled water; n = 8), and nifedipine (10 mg/kg, 0.5 mL distilled water; n = 10). The dose of MFEOAz was established in previous toxicity studies (unpublished data). Treatments were for intragastric administration, at the same time (8:00 AM), for 30 days until the 60th day. Measurements (blood pressure and heart rate) were recorded at pre-treatment every 6 days and in the treatment phase every 3 days by a tail cuff plethysmography method (IITC Life Science, model 229, Woodland Hills, CA). Before the measurements, conscious rats were restrained for 5–10 min in a warm chamber in a quiet room and conditioned to numerous cuff inflation–deflation cycles by a trained operator. Heart rate (HR), systolic (SAP), diastolic (DAP) and mean arterial pressure (MAP) were measured, and the mean of three measurements was recorded. To evaluate the overall antihypertensive effect of the treatments used in this study, the area under the curve (AUC) was calculated by the trapezoidal method. In addition, we determined the rate of systolic arterial pressure decline (mm Hg/day) during the treatment phase, which was obtained by the following formula:

Rate of SAP decline ¼

SAP ðday 30Þ−SAP ðday 60Þ 30 days

2.5. Solutions and drugs The following drugs and reagents were used in the in vitro studies: phenylephrine hydrochloride, acetylcholine hydrochloride, potassium chloride, L-NAME, ODQ, wortmannin, indomethacin, atropine, catalase, SOD, tetraethylammonium, 4-aminopyridine, apamin, charybdotoxin, iberiotoxin, glibenclamide, ethyleneglycol bis(β-aminoethylether)-N, N,N′,N′-tetraacetic acid (EGTA), calcium chloride, nifedipine and caffeine; the diluents included Tween-80, dimethyl sulfoxide (DMSO), and Krebs solution (NaCl, KCl, KH2PO4, MgSO4∙7H2O, NaHCO3, CaCl2 and glucose). All these substances were from Sigma-Aldrich, St. Louis,

G.H. Cunha et al. / Vascular Pharmacology 58 (2013) 337–345

341

Fig. 3. Effect of MFEOAz on CaCl2-induced contractile response in endothelium-intact and -denuded rat aortic rings. Concentration–response curves were determined in Ca2+-free solution after the depletion of intra- and extracellular calcium, so that the CaCl2-contractile effect was dependent on Ca2+ influx via ROCC and VOCC induced by phenylephrine (1 μmol/L) and KCl (60 mmol/L), respectively. The curves were constructed in the absence of MFEOAz (control) or after 30 min incubation with MFEOAz (300 μg/mL) prior to the cumulative addition of CaCl2. Data correspond to the means ± SEM of 6 experiments performed on preparations obtained from different animals. ***P b 0.0001 denotes a significant difference between Emax values (unpaired t test).

MO, USA. MFEOAz was prepared as stock solutions in Tween 80 and sonicated; the bath concentration of Tween 80 did not exceed 0.01%. Glibenclamide, nifedipine and wortmannin were prepared as stock solutions in DMSO. Bath concentration of Tween 80 and DMSO did not exceed 0.5%, which was shown to have no effect per se on the basal tonus of the preparations or on the agonist-mediated contraction or relaxation (Lima-Accioly et al., 2006; Hipólito et al., 2011). Indomethacin was dissolved at pH 8.4. The other drugs were dissolved in distilled water. For the in vivo experiments, MFEOAz and capsules of nifedipine (Bayer Schering Pharma AG, Germany) were diluted in distilled water and sonicated. The solutions were prepared fresh on the day of experiments. Table 4 Effect of MFEOAz on contraction induced by CaCl2 on endothelium-intact and -denuded rat aortic rings evaluated in Ca2+-free solution containing phenylephrine (1 μmol/L) or KCl (60 mmol/L). MFEOAz [μg/mL]

Stimulant: phenylephrine 0 (control) 300 Stimulant: KCl 0 (control) 300

Endothelium-intact

Endothelium-denuded

pEC50

Emax (%)

pEC50

Emax (%)

0.67 ± 0.07 −0.97 ± 1.71

15.39 ± 0.54 0.87 ± 2.33⁎

0.61 ± 0.11 0.46 ± 0.25

14.51 ± 0.80 0.58 ± 0.07†

0.78 ± 0.14 0.93 ± 0.17

10.93 ± 0.59 0.17 ± 0.01†

0.66 ± 0.11 0.75 ± 0.11

9.73 ± 0.47 0.22 ± 0.01†

Data are expressed as means ± SEM of 6 experiments performed on preparations obtained from different animals. ⁎ P = 0.0001 compared to control. † P b 0.0001 compared to control.

2.6. Data and statistical analysis In vascular reactivity studies, the vasorelaxant response of MFEOAz was expressed as percentage relaxation of the phenylephrine or KCl contraction. The vasocontractile actions of CaCl2, phenylephrine and caffeine were measured in millinewtons (mN) and the attenuating effect of MFEOAz on the agonist contraction was calculated as the difference in relation to baseline. The concentration–response curves were constructed using non-linear regression according to the following sigmoidal equation: y¼aþ

b−a 1 þ 10ð logEC50 −xÞ

where y is the response (relaxation or contraction), x is the logarithm of the concentration, EC50 is the concentration required to achieve a half-maximal response, and a and b correspond to the minimum and maximum response values, respectively. Two pharmacological parameters were determined from the concentration–response curves: pEC50, the negative logarithm of EC50, and Emax, the maximal effect of the test substance. Data were expressed as mean and standard error of the mean (SEM) of at least 6 experiments performed on preparations obtained from different animals. The unpaired t test was applied to compare the pEC50 and Emax values of two groups. Comparisons between three or more groups, concerning the pEC50 and Emax values, were carried out using one-way analysis of variance (ANOVA) followed by one of two post-hoc tests: Tukey's multiple comparison test was employed to compare all pairs of groups, and Dunnett's multiple comparison test was used to

342

G.H. Cunha et al. / Vascular Pharmacology 58 (2013) 337–345

Table 5 Effect of MFEOAz on contraction (mN) induced by phenylephrine (1 μmol/L) and caffeine (30 mmol/L) on endothelium-intact and -denuded rat aortic rings evaluated in Ca2+-free solution. MFEOAz [μg/mL]

0 (control) 30 100 300

Phenylephrine

Caffeine

Endothelium-intact

Endothelium-denuded

Endothelium-intact

Endothelium-denuded

11.55 ± 0.85 7.97 ± 0.41⁎ 3.67 ± 0.26⁎,† 0.60 ± 0.09⁎,†,‡

10.58 ± 0.76 7.25 ± 0.14⁎ 2.95 ± 0.10⁎,† 0.47 ± 0.02⁎,†,‡

1.85 1.30 0.77 0.22

1.35 0.88 0.57 0.18

± ± ± ±

0.22 0.24 0.11§ 0.03⁎,

± ± ± ±

0.17 0.19 0.15§ 0.03⁎,¶

Data are expressed as means ± SEM of 6 experiments performed on preparations obtained from different animals. The preparations incubated with vehicle (control) or MFEOAz (30, 100 and 300 μg/mL) for 30 min prior to the addition of phenylephrine (1 μmol/L). ⁎ P b 0.001 compared to control. † P b 0.001 compared to 30 μg/mL MFEOAz. ‡ P b 0.01 compared to 100 μg/mL MFEOAz. § P b 0.01 compared to control. P b 0.01 compared to 30 μg/mL MFEOAz. ¶ P b 0.05 compared to 30 μg/mL MFEOAz.

compare all groups with the control. In the studies for evaluating the antihypertensive effect of MFEOAz, the variables were first analyzed by the Kolmogorov–Smirnov test for normal distribution of the data. As such criteria were met in all analyses, the mean and standard deviation (SD) were calculated for descriptive statistics, and parametric tests were applied for analytical statistics. Thus, comparisons between the control, nifedipine and MFEOAz groups concerning the temporal progression of SAP, DAP, MAP and HR, as well as the rate of SAP decline and AUC of SAP, were performed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. In all analyses, the significance level was set at 0.05 (5%) so that values of P b 0.05 were considered significant. GraphPad Prism® version 5.00 for Windows® (GraphPad Software, San Diego, California, USA, 2007) was used to perform all statistical procedures and to plot the graphs as well.

3.2. Effect of several inhibitors on MFEOAz-induced relaxation in phenylephrine pre-contracted rings

3. Results

Pre-treatment with MFEOAz at 30, 100 and 300 μg/mL for 30 min attenuated the CaCl2-induced contraction of endothelium-intact aortic rings exposed to Ca 2 +-free medium containing high K + in a concentration-dependent manner. The Emax values of MFEOAz at 100 and 300 μg/mL and nifedipine were significantly less than that of control and 30 μg/mL MFEOAz (P b 0.001). Furthermore, the Emax value of 30 μg/mL MFEOAz was less than the corresponding value of control (P b 0.001). However, in endothelium-denuded rings, only MFEOAz at 300 μg/mL significantly reduced the CaCl2-induced contraction. Indeed, the Emax values of 300 μg/mL MFEOAz and nifedipine were significantly less than that of control and 30 and 100 μg/mL MFEOAz (P b 0.001). In both endothelium-intact and endothelium-denuded aortic rings, the magnitude of Emax in preparations pre-treated with 300 μg/mL of MFEOAz or nifedipine was similar. Pre-incubation of the rings with MFEOAz at 30, 100 and 300 μg/mL did not significantly influence the pEC50 values in both endothelium-intact and -denuded aortic rings (Fig. 2).

3.1. Effect of MFEOAz on rat aortic rings pre-contracted with phenylephrine or KCl MFEOAz, at concentrations ranging from 0.1 to 3000 μg/mL, significantly reduced the sustained contractions induced by phenylephrine (1 μmol/L) and KCl (80 mmol/L) in a concentration-dependent manner (Fig. 1, Table 2). The Emax values for the relaxant effect of MFEOAz in endotheliumintact and endothelium-denuded rings, pre-contracted with KCl, were not significantly different (105.74 ± 2.32% and 104.78 ± 1.79%, respectively). Conversely, a significant difference was found between the pEC50 values for the vasorelaxant response induced by MFEOAz in endothelium-intact and -denuded rings (− 1.57 ± 0.05 and − 1.72 ± 0.04, respectively; P = 0.0343). In the aortic rings pre-contracted with phenylephrine, the Emax values for the relaxant effect of MFEOAz in endothelium-intact and endothelium-denuded preparations were not significantly different (109.72 ± 5.11 and 109.20 ± 2.36, respectively). Similarly, no significant difference was found in the pEC50 values for MFEOAz in endothelium-intact and -denuded rings (−2.13 ± 0.08 and −2.12 ± 0.04, respectively). Comparisons were also made between the relaxant effect of MFEOAz in phenylephrine- and KCl-pre-contracted rings considering preparations with intact and denuded endothelium. It was found that in both endothelium preparations, the pEC50 value for MFEOAz in the rings pre-contracted with KCl was significantly different from that found in phenylephrine-pre-contracted rings (P b 0.0001). However, a significant difference was not observed with the Emax values for MFEOAz in preparations pre-contracted with KCl and phenylephrine using endothelium-intact or endothelium-denuded rings.

The effect of pre-treatment with L-NAME, ODQ, wortmannin, atropine, indomethacin, catalase, SOD, TEA, 4-aminopyridine, glibenclamide, apamin, charybdotoxin and iberiotoxin on MFEOAz relaxant response was determined in endothelium-intact rings. The inhibitors did not have any significant effect on MFEOAz-induced relaxation in endothelium-intact aortic rings (Table 3). The magnitude of contraction induced by phenylephrine was similar in the presence of the different inhibitors. 3.3. Effect of different concentrations of MFEOAz on calcium-induced contraction dependent on extracellular Ca 2+

3.4. Effect of MFEOAz on Ca 2+ influx through VOCC and ROCC Pre-treatment with MFEOAz at 300 μg/mL inhibited the contraction induced by phenylephrine (P = 0.0001) and KCl (P b 0.0001) in endothelium-intact and endothelium-denuded aortic rings (Fig. 3, Table 4). Pre-treatment with MFEOAz at 300 μg/mL completely inhibited the contraction induced by CaCl2 in endothelium-intact and endothelium-denuded aortic rings exposed to Ca 2+-free medium containing phenylephrine or KCl (Fig. 3, Table 4). When Ca2+ influx through of ROCC was stimulated by phenylephrine, the Emax values of 300 μg/mL MFEOAz were significantly less than that of both control and 30 μg/mL MFEOAz both in endothelium-intact (P b 0.0001) and endothelium-denuded (P b 0.0001) aortic rings. Similarly, in experiments where Ca 2 + influx through VOCC was stimulated by

G.H. Cunha et al. / Vascular Pharmacology 58 (2013) 337–345

343

3.5. Effect of MFEOAz pre-treatment on Ca 2+ release from intracellular stores Pre-incubation with MFEOAz at 100 and 300 μg/mL for 30 min in Ca 2 +-free Krebs solution significantly attenuated phenylephrine- or caffeine-induced contraction, whereas 30 μg/mL MFEOAz reduced only phenylephrine-evoked vasoconstriction. These results suggest that MFEOAz inhibited the reduction of sarcoplasmic reticulum calcium release (Table 5). 3.6. In vivo experiments: effects of MFEOAz on arterial pressure SAP, DAP, MAP and HR (Fig. 4) were measured at least 1 h after the administration of a single dose of MFEOAz (100 mg/kg). The antihypertensive effect of nifedipine occurred early on day 33 and persisted until day 60. The antihypertensive action induced by MFEOAz was observed from day 36 until day 60 and intensified with time. Although the MFEOAz antihypertensive activity was lower than that of nifedipine over almost the entire treatment period, this difference was reversed at the end of the study. Heart rate showed a similar temporal pattern for the three groups. The overall antihypertensive effect in the MFEOAz-treated group was greater than in the negative control and lower than in the group treated with nifedipine, according to the analysis of the AUC for SAP (Fig. 5A and B). Similarly, nifedipine and MFEOAz significantly decreased the rate of SAP decline, but there was no significant difference between them (Fig. 5C). 4. Discussion

Fig. 4. Temporal progression of systolic, diastolic and mean arterial pressure and heart rate in the control, nifedipine and MFEOAz groups. Hypertension was induced and sustained by chronic administration of L-NAME for 60 days. The first 30 days corresponds to the hypertension induction phase and the last 30 days corresponds to the treatment phase. The arrow indicates the beginning of the treatments. At each time point, data represent the mean and standard deviation of the measurements performed in 9, 10 and 8 rats of the control, nifedipine and MFEOAz groups, respectively. ***P b 0.001, **P b 0.01, *P b 0.05 compared to control group; +++P b 0.001, ++P b 0.01, + P b 0.05 compared to nifedipine group (ANOVA followed by Tukey's multiple comparison test).

KCl, 300 μg/mL MFEOAz also significantly reduced the Emax values for CaCl2 in endothelium-intact (P b 0.0001) and endothelium-denuded (P b 0.0001) aortic rings.

Although previous studies have shown the antihypertensive effects of the essential oil of A. zerumbet (Lahlou et al., 2003; Pinto et al., 2009), their fractions, especially the methanolic one, had not yet been studied with regard to their cardiovascular activities. This study demonstrated, for the first time, the vasorelaxant and antihypertensive effects of MFEOAz and the mechanisms of action involved. The chemical characterization of MFEOAz by gas chromatography and mass spectrometry showed that the major constituents were terpinen-4-ol (57.35%) and 1,8-cineole (27.81%). This result confirmed previous data from other groups (Lahlou et al., 2003; Padalia et al., 2010), where these components have been identified as being responsible for the vasodilator and hypotensive effect of the essential oil of A. zerumbet (EOAz). Different pathways were evaluated to elucidate the mechanism of action of MFEOAz. Vascular endothelium plays a crucial role in controlling vascular tone. A previous study showed that the NO pathway is involved in the vasorelaxant effect of EOAz (Pinto et al., 2009). Therefore, we also investigated if the NO–cGMP pathway was involved in the vasorelaxant effect of MFEOAz. It was found that pretreatment of endothelium-intact aortic rings with L-NAME, ODQ or wortmannin did not influence MFEOAz-induced vasorelaxation. Pre-incubation with atropine, indomethacin, catalase and SOD also did not interfere with the vasodilator effect of MFEOAz. It is well known that K+ channels play an important role in the regulation of muscle contractility and vascular tone (Jackson, 2005). Direct activation of K+ channels in arterial smooth muscle cells normally hyperpolarizes the cell membrane, inhibits Ca2+ influx through VOCC, and suppresses smooth muscle contraction (Eichhorn and Dobrev, 2007). Thus, we evaluated the influence of different types of K+ channels inhibitors on vasorelaxant response induced by MFEOAz. However, in this study, a non-selective K+ channel inhibitor (TEA), a voltage-dependent K+ channel blocker (4-aminopyridine), a non-specific ATP-sensitive K+ channel blocker (glibenclamide) and selective small, intermediate and large conductance Ca2+-activated K + channel blockers (apamin, charybdotoxin and iberiotoxin, respectively) did not influence the relaxant effect of MFEOAz.

344

G.H. Cunha et al. / Vascular Pharmacology 58 (2013) 337–345

Fig. 5. Global evaluation of anti-hypertensive effect in the control, nifedipine and MFEOAz groups. The area under the curve (A) of systolic arterial pressure (SAP) denotes the overall antihypertensive effect of the treatments. It was calculated according to the trapezoidal method (B). The rate of SAP decline (C) during the treatment phase was determined. Data represent the mean (A) and standard deviation (B, C) of the measurements performed in 9, 10 and 8 rats of the control, nifedipine and MFEOAz groups, respectively. ***P b 0.001 compared to control group; +++P b 0.001 compared to nifedipine group (ANOVA followed by Tukey's multiple comparison test).

Two types of stimulants are widely used in vascular smooth muscle to increase the cytosolic Ca2+ level: high-K+-induced membrane depolarization and contractile agonists such as phenylephrine. The influx of extracellular Ca 2 + is mainly through two kinds of transmembrane Ca2+ channels: receptor-operated Ca 2+ channels (ROCC) and voltageoperated Ca2+ channels (VOCC) (Jones et al., 2003). An increase in cytosolic Ca2+ concentration is the major trigger for smooth muscle contraction, because it leads to the formation of the Ca2+–calmodulin complex, inducing signaling that culminates in muscle contraction (Somlyo and Somlyo, 1994). Phenylephrine-induced contraction is mediated by an increase in Ca2+ influx through receptor-operated channels and voltagesensitive channels (Lee et al., 2001), whereas KCl-induced contraction in smooth muscle is mediated by cell membrane depolarization and an increase in Ca 2+ influx through voltage-operated Ca2+ channels (Somlyo and Somlyo, 1994). MFEOAz at 300 μg/mL attenuated the CaCl2-induced contraction of endothelium-intact and endothelium-denuded aortic rings exposed to Ca2+-free medium containing high K+. In addition, the magnitude of the inhibitory effect of MFEOAz and nifedipine, an L-type calcium channel blocker, was similar. The effect of MFEOAz on Ca2+ influx through ROCC and VOCC was evaluated in Ca2+-free solution. MFEOAz inhibited the contraction induced by CaCl2 in a Ca2+-free solution containing KCl or phenylephrine in both endothelium-intact and -denuded preparations. Thus, MFEOAz blocks Ca2+ influx through interference with both ROCC and VOCC. Moreover, it was observed that MFEOAz induced endothelium-independent relaxation in aortic rings pre-contracted with phenylephrine and KCl and that it was more potent in relaxing preparations pre-contracted with KCl than phenylephrine. These findings suggest that FMEOAz is more selective at inhibiting Ca2+ influx through VOCC. MFEOAz was found to inhibit Ca 2+ release from phenylephrineand caffeine-sensitive intracellular stores. Phenylephrine induces the production of inositol triphosphate (IP3), which activates IP3 receptors

(Zhu et al., 2007), while caffeine stimulates ryanodine receptors (Xu et al., 1998). Both receptors mediate the release of Ca 2+ from the sarcoplasmic reticulum, an essential step in smooth muscle contraction. However, in addition to the production of IP3, stimulation of α1 receptors by phenylephrine results in the regulation of multiple effector systems such as the production of diacylglycerol, which in turn activates protein kinase C. The latter phosphorylates the light chain of myosin, which is associated with the development of tension (Stull et al., 1990). The activation of protein kinase C by phenylephrine possibly induces a contractile response greater than that produced by caffeine. MFEOAz significantly inhibited the contraction induced by both phenylephrine and caffeine. These results indicate that MFEOAz blocks Ca2+ mobilization from intracellular stores. In addition, a previous study evaluating the action of EOAz on cardiac contractility indicated that it diminishes contractile force and heart rate possibly due to a reduction in Ca2+ entry through voltage-dependent L-type Ca2+ channels (Santos et al., 2011). To determine whether the in vitro vasorelaxation properties of MFEOAz also occurred in vivo, the effect intragastric administration of MFEOAz was investigated in conscious hypertensive rats through the temporal monitoring of the cardiovascular parameters SAP, DAP, MAP and HR. A. zerumbet is a plant commonly used by certain populations to treat hypertension, but there are no clinical studies proving its efficacy. Previously published preclinical studies have evaluated the effect of EOAz on anesthetized normotensive rats, finding that hypotension results independent of the presence of an operating sympathetic nervous system, suggesting that EOAz may be a direct vasorelaxant agent (Lahlou et al., 2002a, 2002b). In conscious DOCA-salt hypertensive rats, it was shown that EOAz decreases MAP in a dose-dependent fashion (Lahlou et al., 2003). Similarly, EOAz was also found to reduce MAP in anesthetized spontaneously hypertensive rats Barcelos et al. (2010). However, in all these studies, the cardiovascular parameters were not monitored over time.

G.H. Cunha et al. / Vascular Pharmacology 58 (2013) 337–345

During the treatment phase, MFEOAz evoked a significant reduction in SAP, DAP and MAP, although its antihypertensive activity was less than that of nifedipine. However, this difference disappeared at the end of treatment because MFEOAz action exhibited a time-dependent pattern. Thus, the overall antihypertensive effect of MFEOAz was lower compared to the negative control and higher relative to nifedipine, according to the AUC of SAP, but the rate of SAP decrease with MFEOAz and nifedipine was not different. On the other hand, the temporal change in heart rate was similar between the treatments. Additional studies are needed to confirm the present findings, and these would include voltage clamp, patch clamp technique or measurement of cytosolic calcium concentration by confocal microscopy using the fluorescent probe Fluo-3AM. 5. Conclusions In conclusion, our results suggest that MFEOAz induces relaxation in rat aortic rings through an endothelium-independent pathway and that such effect is the result of inhibition of Ca 2+ influx via ROCC and VOCC, as well as inhibition of Ca 2+ mobilization from intracellular stores. Furthermore, intragastric administration of MFEOAz induces an antihypertensive response in a time-dependent manner. This antihypertensive activity is certainly the consequence of a Ca 2+ antagonist effect of MFEOAz. References Assreuy, A.M., Pinto, N.V., Lima Mota, M.R., Passos Meireles, A.V., Cajazeiras, J.B., Nobre, C.B., Soares, P.M., Cavada, B.S., 2011. Vascular smooth muscle relaxation by a lectin from Pisum arvense: evidences of endothelial NOS pathway. Protein Pept. Lett. 18, 1107–1111. Barcelos, F.F., Oliveira, M.L., Giovaninni, N.P.B., Lins, T.P., Filomeno, C.A., Schneider, S.Z., Pinto, V.D., Endringer, D.C., Andrade, T.U., 2010. Phytochemistry and cardiovascular biological activity of the essential oil from leaves of Alpinia zerumbet (Pers.) B.L. Burtt & R.M.Sm. in rats. Rev. Bras. Plant. Med. 12, 48–56. Bezerra, M.A., 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, 549–551. Campana, P.R., Braga, F.C., Cortes, S.F., 2009. Endothelium-dependent vasorelaxation in rat thoracic aorta by Mansoa hirsuta D.C. Phytomedicine 16, 456–461. Cavalcanti, B.C., Ferreira, J.R., Cabral, I.O., Magalhães, H.I., de Oliveira, C.C., Rodrigues, F.A., Rocha, D.D., Barros, F.W., da Silva, C.R., Júnior, H.V., Canuto, K.M., Silveira, E.R., Pessoa, C., Moraes, M.O., 2012. Genetic toxicology evaluation of essential oil of Alpinia zerumbet and its chemoprotective effects against H2O2-induced DNA damage in cultured human leukocytes. Food Chem. Toxicol. 50, 4051–4061. Chen, G., Ye, Y., Li, L., Yang, Y., Qian, A., Hu, S., 2009. Endothelium-independent vasorelaxant effect of sodium ferulate on rat thoracic aorta. Life Sci. 84, 81–88. De Araújo, F.Y., Silva, M.I., Moura, B.A., de Oliveira, G.V., Leal, L.K., Vasconcelos, S.M., Viana, G.S., de Moraes, M.O., de Souza, F.C., Macêdo, D.S., 2009. Central nervous system effects of the essential oil of the leaves of Alpinia zerumbet in mice. J. Pharm. Pharmacol. 61, 1521–1527. Eichhorn, B., Dobrev, D., 2007. Vascular large conductance calcium-activated potassium channels: functional role and therapeutic potential. Naunyn Schmiedeberg's Arch. Pharmacol. 376, 145–155. Elzaawely, A.A., Xuan, T.D., Tawata, S., 2007. Essential oils, kava pyrones and phenolic compounds from leaves and rhizomes of Alpinia zerumbet (Pers.) B.L.Burtt. & R.M. Sm. and their antioxidant activity. Food Chem. 103, 486–494. Estrada-Soto, S., Rivera-Leyva, J., Ramírez-Espinosa, J.J., Castillo-España, P., Aguirre-Crespo, F., Hernández-Abreu, O., 2010. Vasorelaxant effect of Valeriana edulis ssp. procera (Valerianaceae) and its mode of action as calcium channel blocker. J. Pharm. Pharmacol. 62, 1167–1174. Hipólito, U.V., Rocha, J.T., Palazzin, N.B., Rodrigues, G.J., Crestani, C.C., Corrêa, F.M., Bonaventura, D., Ambrosio, S.R., Bendhack, L.M., Resstel, L.B., Tirapelli, C.R., 2011. The semi-synthetic kaurane ent-16α-methoxykauran-19-oic acid induces vascular relaxation and hypotension in rats. Eur. J. Pharmacol. 660, 402–410. Jackson, W.F., 2005. Potassium channels in the peripheral microcirculation. Microcirculation 12, 113–127.

345

Jones, R.D., Pugh, P.J., Jones, T.H., Channer, K.S., 2003. The vasodilatory action of testosterone: a potassium-channel opening or a calcium antagonist action? Br. J. Pharmacol. 138, 733–744. Kamadyaapa, D.R., Gondwe, M.M., Moodley, K., Ojewole, J.A., Musabayane, C.T., 2009. Cardiovascular effects of Ekebergia capensis Sparrm (Meliaceae) ethanolic leaf extract in experimental animal paradigms. Cardiovasc. J. Afr. 20, 162–167. Lahlou, S., Figueiredo, A.F., Magalhães, P.J., Leal-Cardoso, J.H., 2002a. Cardiovascular effects of 1,8-cineole, a terpenoide oxide present in many plant essential oils, in normotensive rats. Can. J. Physiol. Pharmacol. 80, 1125–1131. Lahlou, M.S., Galindo, C.A., Leal-Cardoso, J.H., Fonteles, M.C., Duarte, G.P., 2002b. 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, 1097–1102. Lahlou, S., Interaminense, L.F., Leal-Cardoso, J.H., Duarte, G.P., 2003. Antihypertensive effects of the essential oil of Alpinia zerumbet and its main constituent terpinen-4-ol, in DOCA-salt hypertensive conscious rats. Fundam. Clin. Pharmacol. 17, 323–330. Leal-Cardoso, J.H., Moreira, M.R., da Cruz, G.M., de Morais, S.M., Lahlou, M.S., Coelhode-Souza, A.N., 2004. Effects of essential oil of Alpinia zerumbet on the compound action potential of the rat sciatic nerve. Phytomedicine 11, 549–553. Lee, C.H., Poburko, D., Sahota, P., Sandhu, J., Ruehlmann, D.O., van Breemen, C., 2001. The mechanism of phenylephrine-mediated [Ca(2+)](i) oscillations underlying tonic contraction in the rabbit inferior vena cava. J. Physiol. 534, 641–650. Lima, E.O., Gompertz, O.F., Giesbrecht, A.M., Paulo, M.Q., 1993. In vitro antifungal activity of essential oils obtained from officinal plants against dermatophytes. Mycoses 36, 333–336. Lima-Accioly, P.M., Lavor-Porto, P.R., Cavalcante, F.S., Magalhães, P.J., Lahlou, S., Morais, S.M., Leal-Cardoso, J.H., 2006. Essential oil of Croton nepetaefolius and its main constituent, 1,8-cineole, block excitability of rat sciatic nerve in vitro. Clin. Exp. Pharmacol. Physiol. 33, 1158–1163. Magalhães, P.J.C., Lahlou, S., Lucá, D.M., Coelho-De-Souza, L.N., Frota, P.T.T., Costa, A.M.G., Leal-Cardoso, J.H., 2008. Vasorelaxation induced by the essential oil of Croton nepetaefolius and its constituents in rat aorta are partially mediated by the endothelium. Fundam. Clin. Pharmacol. 22, 169–177. Padalia, R.C., Chanotiya, C.S., Sundaresan, V., 2010. Compositional variability in essential oil from different parts of Alpinia speciosa from India. Nat. Prod. Commun. 5, 279–282. Pinto, N.V., Assreuy, A.M., Coelho-de-Souza, A.N., Ceccatto, V.M., Magalhães, P.J., Lahlou, S., Leal-Cardoso, J.H., 2009. Endothelium-dependent vasorelaxant effects of the essential oil from aerial parts of Alpinia zerumbet and its main constituent 1,8-cineole in rats. Phytomedicine 16, 1151–1155. Santos, B.A., Roman-Campos, D., Carvalho, M.S., Miranda, F.M.F., Carneiro, D.C., Cavalcante, P.H., Cândido, E.A.F., Xavier, Filho L., Cruz, J.S., Gondim, A.N.S., 2011. Cardiodepressive effect elicited by the essential oil of Alpinia speciosa is related to L-type Ca2+ current blockade. Phytomedicine 18, 539–543. Sato, A., Sakuma, I., Gutterman, D.D., 2003. Mechanism of dilation to reactive oxygen species in human coronary arterioles. Am. J. Physiol. Heart Circ. Physiol. 285, 2345–2354. Senejoux, F., Girard, C., Aisa, H.A., Bakri, M., Kerram, P., Berthelot, A., Bévalot, F., Demougeot, C., 2011. Vasorelaxant and hypotensive effects of a hydroalcoholic extract from the fruits of Nitraria sibirica Pall. (Nitrariaceae). J. Ethnopharmacol. 141, 629–634. Seok, Y.M., Choi, Y.W., Kim, G.D., Kim, H.Y., Takuwa, Y., Kim, I.K., 2011. Effects of gomisin A on vascular contraction in rat aortic rings. Naunyn Schmiedebergs Arch. Pharmacol. 383, 45–56. Shen, M., Zhao, L., Wu, R., Yue, S.Q., Pei, J.M., 2013. The vasorelaxing effects of resveratrol on abdominal aorta from rats and its underlying mechanism. Vasc. Pharmacol. 58, 64–70. Somlyo, A.P., Somlyo, A.V., 1994. Signal transduction and regulation in smooth muscle. Nature 372, 231–236. Stull, J.T., Bowman, B.F., Gallagher, P.J., Herring, B.P., Hsu, L.C., Kamm, K.E., Kubota, Y., Leachman, S.A., Sweeney, H.L., Tansey, M.G., 1990. Myosin phosphorylation in smooth and skeletal muscles: regulation and function. Prog. Clin. Biol. Res. 327, 107–126. Xu, L., Tripathy, A., Pasek, D., Meissner, G., 1998. Potential for pharmacology of ryanodine receptor/calcium release channels. Ann. N. Y. Acad. Sci. 16, 130–148. Xue, Y., Shi, H., Murad, F., Bian, K., 2011. Vasodilatory effects of cinnamaldehyde and its mechanism of action in the rat aorta. Vasc. Health Risk Manag. 7, 273–280. Yeh, J., Liou, S., Liang, J., Lee, C., Chiu, C., Lin, Y., Chen, I., 2005. Labedipinedilol-C: a thirdgeneration dihydropyridine-type calcium channel antagonist displaying K+ channel opening, NO-dependent and adrenergic antagonist activities. J. Cardiovasc. Pharmacol. 46, 130–140. Zhang, N., Zou, H., Jin, L., Wang, J., Zhong, M.F., Huang, P., Gu, B.Q., Mao, S.L., Zhang, C., Chen, H., 2010. Biphasic effects of sodium danshensu on vessel function in isolated rat aorta. Acta Pharmacol. Sin. 31, 421–428. Zhu, X.M., Fang, L.H., Li, Y.J., Du, G.H., 2007. Endothelium-dependent and -independent relaxation induced by pinocembrin in rat aortic rings. Vasc. Pharmacol. 46, 160–165.