Indian Journal of Experimental Biology Vol.51, August 2013, pp. 661-669

Antihypertensive and vasorelaxant effects of ethanol extract of stem barks from Zanthoxylum rhoifolium Lam. in rats

Edson Santos Ferreira-Filhoa, Daniel Dias Rufino Arcanjoa,*, Lucas Henrique Porfírio Mouraa, José Couras da Silva-Filhoa, Emanuel Tenório Paulinob, Êurica Adélia Nogueira Ribeirob, Mariana Helena Chavesc, Rita de Cássia Meneses Oliveiraa & Aldeídia Pereira de Oliveiraa aMedicinal Plants Research Center, Federal University of Piauí, Av. Nossa Senhora de Fátima s/n, SG-15, 64049-550, Teresina, PI, Brazil bCardiovascular Pharmacology Laboratory, Federal University of Alagoas, Brazil. cDepartment of Chemistry, Federal University of Piauí, Teresina, PI, Brazil.

Received 25 June 2012; revised 15 May 2013

Administration of ethanol extract of stem bark from Z. rhoifolium (EEtOH-ZR) induced hypotension associated with a dual effect in heart rate in normotensive rats. This response was highlighted in spontaneously hypertensive rats (SHR). In rat superior mesenteric artery rings, the cumulative addition of EEtOH-ZR (0.1–750 µg/mL) on a phenylephrine-induced pre-contraction (10-5 M) promoted a vasorelaxant effect by a concentration-dependent manner and independent of vascular endothelium. A similar effect was obtained on KCl-induced pre-contractions (80 mM). EEtOH-ZR attenuated contractions -6 -2 2+ induced by cumulative addition of CaCl2 (10 –3 × 10 M) in depolarizing medium without Ca only at 500 or 750 µg/mL. Likewise, on S-(–)-Bay K 8644-induced pre-contractions (10-7 M), the EEtOH-ZR-induced vasorelaxant effect was attenuated. EEtOH-ZR (27, 81, 243 or 500 µg/mL) inhibited contractions induced by cumulative addition of phenylephrine (10-9 - 10-5 M) in endothelium-denuded preparations or by a single concentration (10-5 M) in a Ca2+-free medium. The involvement of K+ channels was evaluated by tetraethylammonium (3 mM); the EEtOH-ZR-induced vasorelaxation was not attenuated. Thus, calcium influx blockade through voltage-operated calcium channels (CaVL) and inhibition of calcium release from intracellular stores are probably underlying EEtOH-ZR-induced cardiovascular effects.

Keywords: Anti-hypertensive, Cardiovascular, Hypertension, Vasorelaxation, Zanthoxylum rhoifolium Hypertension is a major public health problem several species are used in folk medicine to treat worldwide and, when untreated, predisposes to cardiovascular diseases5–8. Zanthoxylum rhoifolium cardiovascular morbidity and premature death1,2. Lam. (Rutaceae), popularly known as "mamica-de- Effective treatment of hypertension reduces cadela" or "mamica-de-porca"9, is a native tree of complications and improves life expectancy. Brazil, which grows up to 12 m found on the forest However, the difficulty of providing access to and savannah areas in almost all south-central antihypertensive medications in developing countries Brazil10. It has been popularly used in inflammatory, justifies the search for new antihypertensive agents of microbial, cancerous and malaria processes11. natural origin, especially in countries like Brazil, The stem bark is used in traditional medicine as teas where there is a large potential for the development of or infusions as antiinflammatory, antispasmodic, herbal preparations3. Further, natural products muscle relaxant, analgesic, sudorific, antifungal represent an extremely valuable source for production and diuretic12. of new chemical entities for the treatment of untreated From methanol extract of the bark of Z. rhoifolium, diseases, since they represent privileged structures three benzophenanthridine alkaloids have been isolated: selected by evolutionary mechanisms over a period of 4 dihydronitidine, oxynitidine and zanthoxyline, together millions of years . with the known compounds, dihydronitidine, The genus Zanthoxylum (Rutaceae) comprises 6-oxynitidine and skimmianine13. Also, analyses of more than 200 species distributed worldwide and the essential oil from Z. rhoifolium leaves showed that —————— among its numerous constituents, there are some *Correspondent author components described as cytotoxic substances against Telephone: +55-86-3215 5872 Fax: +55-86-3215 5872 tumoral cells, such as β-caryophyllene, β-elemene, 11 E-mail: [email protected] δ-elemene, α-humulene . 662 INDIAN J EXP BIOL, AUGUST 2013

The phytochemical screening of ethanol extract of were maintained on normal laboratory chow and stem barks from Z. rhoifolium by thin-layer silica gel water ad libitum. All experiments were approved by chromatography, using specific spray reagents, Committee of Ethics for Animal Research of Federal suggested the presence of isoprene nature substances University of Piaui, Brazil (CEEA No. 029/09). (triterpenes and steroids), as well as flavonoids and The protocols agree with the guidelines of the alkaloids. The presence of triterpenes was confirmed National Institute of Health (NIH Publication 85-23, by the isolation and identification of lupeol, a 1985) for laboratory animal care and use. pentacyclic triterpenoid obtained from hexane fraction Drugs—The following drugs were used: of partition and determined by 1H and 13C NMR L-phenylephrine hydrochloride (PHE), acetylcholine spectroscopic analyses. Besides, the ethanol extract hydrochloride (ACh), tetraethylammonium (TEA), elicit antinociceptive and gastroprotective effects in S-(–)-Bay K 8644 (Sigma Chemical Co., Saint Louis, models of chemical nociception and gastric ulcers, MO, USA), heparin sodium salt (Roche Brazil, São respectively, in rodents14,15. Paulo, Brazil) and sodium thiopental (Cristalia, The effects of Z. rhoifolium on cardiovascular Butantã, Brazil). All the stock solutions were prepared system, especially in hemodynamic parameters in vivo in distilled water. S-(–)-Bay K 8644 was previously and its mechanisms of vasorelaxation still remain dissolved in ethanol. unknown. Therefore, the aim of this study is to Solutions—The composition of the Tyrode´s characterize the vasorelaxant and the antihypertensive solution used was (in mM): NaCl, 158.3; KCl, 4.0; effects elicited by the ethanolic extract from stem CaCl2·2H2O, 2.0; MgCl2·6H2O, 1.05; NaHCO3, 10.0; barks of Z. rhoifolium Lam. (EEtOH-ZR) and also NaH2PO4·H2O, 0.42; and glucose, 5.6, all from Sigma contribute towards the pharmacological knowledge Chemical Co., Saint Louis, MO, USA16. Depolarizing about this plant. Tyrode solutions with KCl 20, 60 or 80 mM were prepared by an equimolar replacement of Na+ for K+ Materials and Methods 17. In nominally without Ca2+ depolarizing solution, Plant material identification and extraction—Stem 2+ CaCl2 was not added, and in Ca -free solution, CaCl2 barks of Z. rhoifolium were collected in 2005 from was omitted and 1 mM ethylene-diamine-tetraacetic Pedro II city, Piauí state, Brazil. The voucher specimen acid (EDTA), was added18. (TEPB 13870) was identified by a botanist and Effects of EEtOH-ZR on cardiovascular deposited at Graziela Barroso Herbarium of Federal parameters in non-anaesthetized rats—The University of Piauí (UFPI). EEtOH-ZR was obtained 14 procedures for arterial pressure (AP) and heart rate according to Pereira et al . Dried and powdered barks (HR) recordings were done as previously described19. of Z. rhoifolium (1.0 kg) were exhaustively extracted at Briefly, under sodium thiopental anaesthesia room temperature with ethanol. The solvent was (45 mg/kg, ip), rats were fitted with polyethylene removed by evaporation under reduced pressure using catheters inserted into the lower abdominal aorta and a Hedolph Rotary Evaporator to yield the ethanolic inferior vena cava through left femoral artery and extract (85.0 g, 8.5%). Prior to the experiments, vein, respectively. Both catheters were filled with EEtOH-ZR was dissolved in a mixture of distilled heparinized saline, tunnelled subcutaneously, water and 3% cremophor solution and kept at -4 ºC. exteriorized and sutured at the dorsal surface of the The stock solution (10 mg/mL) was diluted to desired neck. After 24 h of surgery, experiments were concentrations with distilled water just before use. The performed in conscious rats. The arterial catheter was final concentration of cremophor in the bath never connected to a precalibrated pressure transducer, exceeded 0.01% and had no effect on tension which was connected to an amplifier–recorder (AVS development when tested in control preparations (data Projetos, São Paulo, Brazil). Beat-to-beat time not shown). waveforms were generated and processed offline. For Animals—Male Wistar rats (Rattus norvegicus, each cardiac cycle, the software calculated mean 8–10 weeks old, 250–350 g) were used. For the arterial pressure (MAP) and pulse interval (PI), used evaluation of the antihypertensive effect, male Wistar to derive HR. rats and Spontaneously Hypertensive Rats (SHR) were used. The animals were kept in well-ventilated Evaluation of EEtOH-ZR-induced vasorelaxant effects cages at controlled temperature (24±2 ºC) and Preparation of superior mesenteric artery rings— under controlled light cycles (12:12 h L:D). They Male Wistar rats were euthanized by cervical FERREIRA-FILHO et al.: CARDIOVASCULAR EFFECTS OF ZANTHOXYLUM RHOIFOLIUM STEM BARK 663

dislocation, dissected and the superior mesenteric Effect of EEtOH-ZR on S-(–)-Bay K 8644-induced arteries were quickly removed, cleaned of adherent pre-contractions—After endothelium removal, aorta connective tissues and cut into rings (3–4 mm length), ring preparations were pre-incubated with a according to Oliveira et al.20. The rings were depolarizing Tyrode solution of KCl 20 mM for 20 suspended by cotton threads in organ baths containing min, a procedure performed to obtain a better 6 mL Tyrode’s solution, maintained at 37 ºC and response for the contractile agent. Then, S-(–)-Bay K -7 gassed with carbogenic mixture (95% O2 and 5% 8644 (10 M), a CaVL channels activator was added CO2). Rings were stabilized with a resting tension of to induce a sustained contraction, and EEtOH-ZR 0.75 gf for at least 60 min. The Tyrode’s solution was (0.1, 0.3, 1, 3, 9, 27, 81, 243, 500 and 750 g/mL) replaced every 15 min to prevent the accumulation of was cumulative added on this contractile tonus23. The 21 metabolites . The force of contraction was results were expressed as EC50 values and compared isometrically recorded using a force transducer with KCl-induced pre-contractions (80 mM) in coupled to an amplifier–recorder (AECAD 1604, endothelium-denuded preparations. AQCAD 2.0.3, AVS Projetos, São Paulo, Brazil). Effect of EEtOH-ZR on cumulative PHE-induced When necessary the endothelium was removed by contractions—Cumulative-response curves for PHE gently rubbing the intimal layer with moistened (10-9–10-5 M) were also designed. Curves were cotton strings. The endothelium integrity was verified obtained in endothelium-denuded rings by a stepwise by relaxation to ACh (10-5 M) in rings pre-contracted increase in the concentration of PHE, in the absence by PHE (10-5 M), according to Furchgott (control) or after a 30 min incubation period with and Zawadzki22. In endothelium-intact studies, EEtOH-ZR (27, 81, 243 and 500 g/mL). The results preparations were considered when ACh-induced were expressed as percentages of the maximal relaxation was higher than 80%. Endothelium- response for only PHE-induced response, and curves denuded preparations were considered when were statistically compared17. relaxation was lower than 10%. Effect of EEtOH-ZR on PHE-sensitive calcium Effect of EEtOH-ZR on PHE- or KCl-induced intracellular stores–The effect of EEtOH-ZR on contractions—After equilibration, a PHE (10-5 M)- PHE-sensitive calcium intracellular stores was induced pre-contraction was elicited in endothelium- assessed. The transient contractions were obtained in intact and endothelium-denuded rings to promote endothelium-denuded rings by PHE (10-5 M) in Ca2+- similar magnitude contractions. Then, EEtOH-ZR was free Tyrode solution before and after incubation of added cumulatively (0.1, 0.3, 1, 3, 9, 27, 81, 243, EEtOH-ZR (27, 81, 243 and 500 g/mL) for 3 min. 500 and 750 g/mL), after response to PHE had The results were expressed as percentages of the stabilized, approximately 30 minutes later. Then, a response induced by PHE alone24. concentration–response curve was obtained. In Effect of K+ channels involvement in EEtOH-ZR- another set of experiments, the ability of EEtOH-ZR induced vasorelaxant response—To investigate the to attenuate KCl-induced tonic contraction (80 mM) possible involvement of potassium channels in in the rings was also examined. The results were EEtOH-ZR-induced vasorelaxation, endothelium- expressed as EC50 values and compared with PHE- denuded rings were pre-incubated with TEA (3 mM), or KCl-induced pre-contractions in endothelium- a non-specific K+ channel blocker, for 30 min25. After denuded preparations. stabilization of PHE-induced tonic vasoconstriction, Effect of EEtOH-ZR on CaCl2-induced cumulative concentrations of EEtOH-ZR were added contractions—After the stabilization period, the effect of to the organ bath. The results were expressed as EC50 EEtOH-ZR on CaCl2-induced contractions in values and compared with PHE-induced pre- endothelium-denuded rings was assessed. Cumulative contractions in endothelium-denuded preparations17. -6 -2 concentration-response curves for CaCl2 (10 –3×10 M) Statistical analysis—All values were expressed were obtained in endothelium-denuded rings exposed to as mean ± SE. Curves and EC50 values were nominally without Ca2+ Tyrode solution with 60 mM obtained by nonlinear regression. Student’s t-test KCl before and after pre-incubation, separately with and ANOVA-One-way or Two-way followed by EEtOH-ZR (81, 243, 500 or 750 µg/mL) for Bonferroni post-test were used in the data analysis 30 min. The results were expressed as percentages of the and results were considered significant when P<0.05. maximal response for only CaCl2-induced response, and All analysis was performed using GraphPad Prism™ curves were statistically compared20. 5.0 (GraphPad Software, San Diego, CA, USA). 664 INDIAN J EXP BIOL, AUGUST 2013

Results denuded rings. The concentration-response curve Effect of EEtOH-ZR on mean arterial pressure was significantly rightward shifted with inhibition of (MAP) and heart rate (HR) in non-anaesthetized rats— maximal effect (Fig. 6). Systemic haemodynamic changes induced by EEtOH- Effects of EEtOH-ZR on PHE-sensitive calcium ZR were investigated in conscious normotensive and intracellular stores—In Ca2+-free depolarizing Tyrode spontaneously hypertensive rats (SHR). In normotensive solution, EEtOH-ZR promoted a concentration- rats, the baseline values of MAP and HR were 122±5 dependent inhibition of PHE-induced contractions on mmHg and 300±10 bpm, respectively. Administration rat mesenteric artery rings (Fig. 7; Emax values: of EEtOH-ZR at doses 0.5, 1.0, 5.0 and 10.0 mg/kg 27 µg/mL=78.48±7.71; 81 µg/mL=54.46±8.22; (iv) induced hypotension by 17.4±3.5, 15.8±2.3 and 243 µg/mL=25.51±4.06 and 500 µg/mL=11.58±4.50%). 11.3±1.3%, respectively (expressed as percentage of Participation of K+ channels in EEtOH-ZR- baseline values) that was associated with a dual effect induced vasorelaxant response—In rat endothelium- in HR. Higher doses (20.0 and 30.0 mg/kg, iv) were denuded isolated superior mesenteric rings, able to elicit hypertension and tachycardia. pretreatment by TEA (3 mM) did not attenuated the In SHR, the baseline values of MAP and HR were EEtOH-ZR-induced vasorelaxation, and the 175±10 mmHg and 400±10 bpm, respectively. concentration-response curve was leftward shifted Administration of EEtOH-ZR at doses 0.5, 1.0 and (Fig. 8), indicating the non-participation of the K+ 5.0 mg/kg (iv) induced hypotension by 15.0±4.7%, channels in this response. 25.3±6.3% and 22.1±2.1%, respectively that was also associated with a dual effect in HR. Higher doses (10.0, 20.0 and 30.0 mg/kg, iv) also elicited hypertension and tachycardia (Fig. 1). Effect of EEtOH-ZR on PHE- or KCl-induced contractions—In rat endothelium-intact isolated superior mesenteric rings, EEtOH-ZR induced a vasorelaxant response on PHE-induced pre-contraction (Fig. 2; EC50=240.0±19.91 µg/mL), which was attenuated neither after removal of the vascular endothelium (EC50=301.82±38.46 µg/mL) nor on KCl- induced pre-contractions in endothelium-denuded preparations (Fig. 3; EC50=262.68±6.28 µg/mL). Effects of EEtOH-ZR on cumulative CaCl2-induced contractions—In a nominally without Ca2+ depolarizing Tyrode solution, EEtOH-ZR only reduced the CaCl2-induced maximal contractile response (Emax) at 500 or 750 µg/mL (Fig. 4) Effects of EEtOH-ZR on S-(–)-Bay K 8644-induced pre-contractions—In endothelium-denuded rat superior mesenteric rings, increasing concentrations of EEtOH-ZR (0.1–750 g/mL) induced a vasorelaxant response on S-(–)-Bay K 8644 (10-7 M)- induced pre-contraction. However, the concentration- response curve obtained was rightward shifted compared with EEtOH-ZR vasorelaxant effect on

KCl-induced pre-contraction (Fig. 5) The, EEtOH- ZR-induced vasorelaxant effect was then attenuated. Fig. 1—Changes in (A) mean arterial pressure (MAP) and (B) Effects of EEtOH-ZR on cumulative PHE-induced heart rate (HR) induced by the acute administration of different contractions—The pre-incubation of EEtOH-ZR for doses of EEtOH-ZR to non-anesthetized normotensive and spontaneously hypertensive rats. Data are mean ± SE from 30 min was able to inhibit in a concentration- 3 experiments. P values: a<0.001 vs normotensive rats 0.5 mg/kg; dependent manner the maximal contractile response b<0.001 vs SHR 0.5 mg/kg (one-way ANOVA following -9 -5 (Emax) induced by PHE (10 – 10 M) on endothelium- Bonferroni’s post-test) FERREIRA-FILHO et al.: CARDIOVASCULAR EFFECTS OF ZANTHOXYLUM RHOIFOLIUM STEM BARK 665

Fig. 4—Concentration-response curves to EEtOH-ZR (81, 243, 500 -6 -2 or 750 µg/mL) on CaCl2-induced contractions (10 –3×10 M) in Fig. 2—Vasorelaxant response induced by EEtOH-ZR endothelium-denuded rat mesenteric artery rings in nominally (0.1–750 µg/mL) on PHE-induced pre-contractions in without Ca2+ depolarizing Tyrode solution. Values are mean±SE endothelium-intact or -denuded rat superior mesenteric from 5 experiments. P values: ** <0.01 and *** <0.001 vs. artery isolated rings. Values are mean±SE from 5 experiments. control.

Fig. 5—Vasorelaxant response induced by EEtOH-ZR (0.1–750 Fig. 3—Vasorelaxant response induced by EEtOH-ZR µg/mL) on KCl (80 mM) or S-(–)-Bay K 8644 (10-7 M) induced (0.1–750 µg/mL) on PHE (10-5 M) or KCl (80 mM) induced pre-contractions in endothelium-denuded rat mesenteric artery pre-contractions in endothelium-denuded rat mesenteric artery rings. Values are mean±SE from 5 experiments. P value: **<0.01 rings. Values are mean±SE from 5 experiments. vs. KCl 80 mM. 666 INDIAN J EXP BIOL, AUGUST 2013

Fig. 8—Vasorelaxant response induced by EEtOH-ZR (0.1–750 Fig. 6—Concentration-response curves to EEtOH-ZR (27, 81, µg/mL) in endothelium-denuded rat mesenteric artery rings after 243 or 500 µg/mL) on PHE-induced contractions (10-9–10-5 M) TEA 3 mM pretreatment. Values are mean±SE from 5 in endothelium-denuded rat mesenteric artery rings. Values are experiments. P value: *<0.05 vs. control E. mean±SE from 5 experiments. P values: *<0.05 and ***<0.001 vs. control. of inducing both hypotensive and antihypertensive effects, which come with dual response on heart rate: the tachycardia observed for doses of 1.0 mg/kg (normotensive) and 5.0 mg/kg (SHR) may probably due to baroreflex response after the direct hypotensive effects of EEtOH-ZR26. Otherwise, the bradycardia observed for 5.0 mg/kg (normotensive) and 10.0 mg/kg (SHR) doses may probably due to decreased cardiac inotropism, which is shown by the majority of the most common antihypertensive and vasodilator drugs27. Regarding the highest doses tested, it is possible that the increase in mean arterial pressure values is due to a cardiac action of the extract, implying a positive inotropic effect which eventually led to an increase of cardiac output and subsequently overcame the effect on the peripheral vascular Fig. 7—Inhibitory effect of EEtOH-ZR (27, 81, 243 or 500 resistance. A particularly interesting aspect was that µg/mL) on PHE 10-5 M-induced transient contractions in 2+ similar responses were observed for both endothelium-denuded rat mesenteric artery in Ca -free depolarizing Tyrode solution. Values are mean±SE from 5 normotensive and spontaneously hypertensive rats, experiments.P values: **<0.01, ***<0.001 vs. control. with different potencies: the behaviour of cardiovascular parameters of a dose of EEtOH-ZR in Discussion normotensive animals can be predicted by observing The present study shows that EEtOH-ZR the effects of the immediately lower dose on these intravenously administrated in conscious parameters in SHR, which can be explained by normotensive and SHR rats evoked biphasic changes different functioning of the physiology of these on mean arterial pressure and dual effects on heart animals when it comes to expression of receptors, ion rate (Fig. 1). At lower doses, EEtOH-ZR was capable channels and contractile proteins28. FERREIRA-FILHO et al.: CARDIOVASCULAR EFFECTS OF ZANTHOXYLUM RHOIFOLIUM STEM BARK 667

These results are consistent with the cardiovascular Vascular smooth muscle cells (VSMCs) use effect reported for several species from genus calcium as a molecular signal for multiple functions. Zanthoxylum. In isolated rabbit aortic rings, In the majority of VSMCs, excitation-contraction the crude extract of Z. armatum exhibited coupling is maintained by L-type Ca2+ channels cardiodepressant and vasodilator effect against (CaVL), regulating the calcium influx leading to phenylephrine and K+-induced contractions6. vasoconstriction32,33. The main voltage-operated Likewise, Z. schinifolium elicits an increase in calcium channel found in vascular smooth muscle is atrial dynamics, cAMP efflux, and decrease in ANP the CaV1.2, a CaVL subtype present in various smooth secretion, suggesting a cardioprotective effect by muscle cells, including VSMCs34. Thus, 2+ activation of β1-adrenoceptor and cAMP-PKA-Ca concentration-response curves to EEtOH-ZR were signaling pathway in rabbit atrium7. performed after precontraction with S-(–)-Bay K 23 Moreover, EEtOH-ZR induced a vasorelaxation 8644, a CaV1.2 activator . EEtOH-ZR promoted on pre-contracted rat mesenteric artery rings in vasorelaxation of endothelium-denuded mesenteric a concentration-dependent, which may partially rings pre-contracted with S-(–)-Bay K 8644 only at justify its effect on blood pressure and heart rate. 243, 500 or 750 µg/mL, attenuating the response by It is well known that the endothelium is an rightward shift of the concentration-response curve important regulator of the vascular tone by releasing compared with preparations pre-contracted by KCl endothelium-derived relaxing factors29. In order to 80 mM. These results suggest that EEtOH-ZR investigate its participation in the vasorelaxant probably may acts on vascular smooth muscle by a effect induced by EEtOH-ZR, experiments were decrease of calcium influx through CaVL channels performed in absence of functional endothelium. only at higher concentrations, as observed on CaCl2- This vasorelaxant effect was not decreased in induced contractions. endothelium-denuded preparations, suggesting an Otherwise, EEtOH-ZR inhibited the endothelium-independent effect and a direct action vasoconstriction induced by cumulative addition of on vascular smooth muscle. The choice of this phenylephrine by a concentration-dependent manner. artery is due to its substantial contribution to the Phenylephrine promotes increased vascular tone by regulation of systemic circulation and also reflects stimulation of α1-adrenergic receptor, leading to an the variation of vascular resistance in circulation30. increase of two second messengers: inositol 1,4,5- For an endothelium-independent vasodilator effect, trisphosphate (IP3), which promotes the release of several mechanisms may be involved, including: calcium from intracellular IP3-sensitive calcium blockade of extracellular Ca2+ influx through stores, and diacylglycerol (DAG), which promotes the transmembrane Ca2+ channels; inhibition of agonist- activation of contractile proteins and increases 2+ calcium influx through voltage-dependent calcium mediated release of Ca from intracellular stores; 35 opening of K+ channels; inhibition of the contractile channel . These evidences might explain that apparatus; among others31. Since EEtOH-ZR also EEtOH-ZR possess vasorelaxant effects on voltage- produced relaxation of isolated arteries pre-contracted operated calcium channels only at higher by depolarization with KCl, which elicits contraction concentrations, probably an indirect effect of by allowing the influx of extracellular Ca2+ through activation of phenylephrine signaling pathway. voltage-dependent Ca2+ channels, the hypothesis that Whereas the inhibition of calcium transmembrane EEtOH-ZR can inhibit vasoconstriction induced by influx probably acts only at highest concentrations, extracellular Ca2+ influx should be previously the participation of EEtOH-ZR in lower considered. To confirm this observation, CaCl2- concentrations on mobilization of calcium induced concentration-response curves before and intracellular stores was evaluated. After after EEtOH-ZR addition were obtained in a phenylephrine binding to a α1-receptor, inositol-1,4,5- nominally without Ca2+ depolarizing Tyrode solution triphosphate (IP3) is produced, which binds and (KCl 60 mM). However, EEtOH-ZR induced an activates a IP3-specific receptor (IP3R) on the inhibition of CaCl2-induced contractions only at sarcoplasmic reticulum (SR) membrane, inducing a 500 or 750 µg/mL, indicating a possible effect on Ca2+ internal release and promoting a transient voltage-operated calcium channels only in the higher vasoconstriction31,36. Thus, in a Ca2+-free Tyrode concentrations. solution, EEtOH-ZR inhibited phenylephrine-induced 668 INDIAN J EXP BIOL, AUGUST 2013

transient contractions (10-5 M) in a concentration- 4 Vergara-Galicia J, Ortiz-Andrade R, Rivera-Leyva J, dependent manner, suggesting its possible interference Castillo-España P, Villalobos-Molina R, Ibarra-Barajas M, Gallardo-Ortiz I & Estrada-Soto S, Vasorelaxant and in the calcium mobilization from IP3-sensitive antihypertensive effects of methanolic extract from roots of intracellular stores. Laelia anceps are mediated by calcium-channel antagonism, Membrane potential is a key variable that regulates Fitoterapia, 81 (2010) 350. Ca2+ influx through voltage-gated Ca2+ channels. 5 Facundo V A, Silveira A S P, Braz Filho R, Pinto A C & Rezende C M, Constituintes químicos de Zanthoxylum Further, potassium channels activity importantly ekmanii (URB.) Alain, Quim Nova, 28 (2005) 224. contributes to determination and regulation of 6 Gilani S N, Khan A U & Gilani A H, Pharmacological basis membrane potential and vascular tone37. Actually, for the medicinal use of Zanthoxylum armatum in gut, potassium channels opening in cell membranes of airways and cardiovascular disorders, Phytother Res, 24 smooth muscle increases K+ efflux, causing (2010) 553. 38 7 Cui H Z, Choi H R, Choi D H, Cho K W, Kang D G & Lee membrane hyperpolarization , which constitutes an H S, Aqueous extract of Zanthoxylum schinifolium elicits alternative mechanism of vasodilation of natural contractile and secretory responses via β1-adrenoceptor products. In order to investigate K+ channels activation in beating rabbit atria, J Ethnopharmacol, 126 participation in the vasorelaxant effect induced by (2009) 300. EEtOH-ZR, experiments were performed in presence 8 Li X, Kim H Y, Cui H Z, Cho K W, Kang D G & Lee H S, Water extract of Zanthoxylum piperitum induces vascular of tetraethylammonium 3 mM (TEA), a non-selective relaxation via endothelium-dependent NO-cGMP signaling, J potassium channel blocker. In the present study, TEA Ethnopharmacol, 129 (2010) 197. did not attenuated the vasorelaxant effect of EEtOH- 9 Moreira F, Plantas que curam: Cuide da sua saúde através ZR in mesenteric artery rings, suggesting that K+ da natureza, 5th ed. (Hermus: São Paulo) 1996. channels opening may not be involved in the 10 Silva I A, Valenti M W & Silva-Matos D M, Fire effects on vasorelaxation of EEtOH-ZR. the population structure of Zanthoxylum rhoifolium Lam (Rutaceae) in a Brazilian savanna, Braz J Biol, 69 (2009) In conclusion, the present study demonstrates that 813. the ethanol extract from stem barks of Z. rhoifolium 11 Silva S L, Figueiredo P M & Yano T, Chemotherapeutic Lam. is capable of modifying the hemodynamic potential of the volatile oils from Zanthoxylum rhoifolium parameters in normotensive and hypertensive rats, Lam leaves, Eur J Pharmacol, 576 (2007) 180. 12 Silva C V, Detoni C B, Velozo E S & Guedes M L S, promoting decrease of arterial blood pressure Alcalóides e outros metabólitos do caule e frutos de probably modulating peripheral vascular resistance by Zanthoxylum tingoassuiba A. St. Hil., Quim Nova, 31 (2008) acting on mobilization of calcium intracellular stores. 2071. 13 Moura N F, Ribeiro H B, Machado E C S, Ethur E M, Acknowledgment Zanatta N & Morel A F, Benzophenanthridine alkaloids from Zanthoxylum rhoifolium, Phytochemistry, 46 (1997) 1443. This work was supported by UFPI (Federal 14 Pereira S S, Lopes L S, Marques R B, Figueiredo K A, Costa University of Piauí, Brazil), FAPEPI (Fundação de D A, Chaves M H & Almeida F R, Antinociceptive effect of Amparo à Pesquisa do Estado do Piauí, Brazil) and Zanthoxylum rhoifolium Lam. (Rutaceae) in models of acute CNPq (Conselho Nacional de Desenvolvimento pain in rodents, J Ethnopharmacol, 129 (2010) 227. Científico e Tecnológico). There is no conflict of 15 Freitas F F B P, Fernandes H B, Piauilino C A, Pereira S S, Carvalho K I M, Chaves M H, Soares P M G, Miura L M C interest associated with the study. 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