Antihypertensive and Vasorelaxant effects of the ethanolic extract from the stem bark of Aspidosperma tomentosum Mart

Article history: Received on: 23/05/2017 Accepted on: 23/09/2017 Available online: 30/10/2017 Aspidosperma species have been traditionally used for the treatment of cardiovascular disease. These species are important sources of indole alkaloids, which are responsible for different cardiovascular effects. Based on this premise, and as there are still no studies related to the cardiovascular effects of the ethanolic extract from the stem bark of Aspidosperma tomentosum (EEAT). The goal of the survey was to characterize the mechanism of the vasorelaxant action of EEAT in isolated rat mesenteric rings and to establish its antihypertensive activity. The mean arterial pressure (MAP) and Heart Rate (HR) were measured in hypertensive rats through a catheter implanted in abdominal aorta via the femoral artery. The vasodilator effect of EEAT in isolated pre-contracted rat mesenteric rings was examined. The bioactive extract was investigated via mass spectrometry fingerprinting. EEAT induced relaxation in mesenteric rings pre-contracted with Phe independent of the endothelium. In denuded rings, when incubation with KCl 20 mM or TEA did not change the relaxation. EEAT inhibited the concentration-response curves induced by CaCl2 or Phe and inhibited 80 mM KCl-induced contraction. In precontracted preparations with BayK 8644, the EEAT induced relaxation. The transient contraction induced by Phe was inhibited. The oral administration of EEAT reduced MAP and did not alter the HR. Phenolic acids, flavonoids, and indole alkaloids were identified in EEAT. In conclusion, the EEAT induced vasorelaxation through a blockade of Ca 2+ channels and inhibition of the mobilization of Ca 2+ of the IP3-sensitive Ca 2+ stores and caused an antihypertensive effect.


INTRODUCTION
Hypertension is considerable public health challenge w w ff 26.4% f h w ' p p (Kearney et al., 2005).Complications of hypertension are held for 9.4 million deaths worldwide every year (Lim et al., 2012).
Nevertheless, less than 25% of treated hypertensive patients achieve target mean arterial pressure (MAP) (Evans et al., 2005) and, furthermore, 20% to 30% of hypertensive individuals may be resistant to antihypertensive treatment (Hajjar et al., 2003).Thus, the search for new, safer and more efficient antihypertensive drugs with fewer side effects is extremely needed (Cogolludo et al., 2005).
The use of medicinal plants is common in patients with cardiovascular disease, including hypertension (Tirapelli et al., 2010).In this context, medicinal plants can be considered as a potential source for new antihypertensive drugs.

The
Aspidosperma genus, which comprises approximately 43 species, is found from Mexico to Argentina (Jacomé et al., 2004).Several Aspidosperma species have been traditionally used for the treatment of cardiovascular diseases, including diabetes, hypercholesterolemia, erectile dysfunction, anti-hyperlipidemic, and also used as a vasodilator (Campos et al., 2006;Tresvenzol et al., 2006;Oliveira et al., 2009).These species are important sources of indole alkaloids, which are responsible for different cardiovascular effects, such as diuretic, hypotensive α-adrenergic receptors blocker (Lyon et al., 1973;Deutsch et al., 1994;Craveiro et al., 1983).
In the literature, there are reports on the cardiovascular effects of Aspidosperma species.For example, in spontaneously hypertensive rats, the ethanolic extract of the wood of Aspidosperma pyrifolium produced hypotensive effects (Herculano et al., 2012).The ethanolic extract of the leaves of Aspidosperma macrocarpum elicited hypotensive, antihypertensive and vasorelaxant effects (Oliveira et al., 2012).The ethanol extract of Aspidosperma subincanum induced hypotension associated with bradycardia and vascular relaxation (Bernardes et al., 2013).
Aspidosperma tomentosum Mart. is a tree commonly recognized in Brazil as "peroba-do-campo".The majority of Aspidosperma species have already been chemically characterized, in which several indole alkaloids were isolated and identified, nevertheless, the pharmacological properties have not yet been largely studied.Based on this premise, and as there are still no studies related to the cardiovascular effects of the ethanolic extract from the stem bark of Aspidosperma tomentosum (EEAT), we aimed to assess the mechanism(s) involved in the vasorelaxant effect of EEAT in rat mesenteric rings, and to establish its antihypertensive activity in vivo.

Acquisition and extraction of herbal materials
The stem bark of Aspidosperma tomentosum Mart.was collected in Planaltina (Goiás/Brazil).The plant species was identified by Botanist Prof. José Elias de Paula, of the Vegetal Anatomy Laboratory (Institute of Biology), University of Brasilia (UnB).A voucher specimen (no.UB-3732) was deposited in the Herbarium of UnB.
Dried and powdered plant material (3.64 kg) was extracted with ethanol/water (1:9, v/v, 20 L) for 96 h at room temperature.The solvent was removed under reduced pressure to give the crude extract (EEAT, 316.55 g).

Evaluation of chemical composition by ESI-MS analysis
In this study, to characterize and identify the compounds present in the extract, we employ the method by electrospray ionization mass spectrometry (ESI-MS) in the positive and negative ion mode.Capillary and cone voltages were kept at 3.0 kV and 30.0 V, using a source temperature of 100° C and desolvation temperature of 350° C. The equipment used was the Micromass-Waters Q-TOF mass spectrometer (Waters, Manchester, England).The data were processed and stored over 60 s.The spectral scan was performed in an interval between m/z 100 and 1000.ESI-MS was conducted by direct infusion.The flow rate was standardized in 10 μLmin −1 .The equipment used was a syringe pump (Harvard Apparatus, MA, USA).The identification of compounds was made by comparing the ESI-MS/MS fragmentation spectra with fragmentation spectra of the pattern samples and with literature results.

Chemicals and drugs
Acetylcholine chloride (ACh), BAYK-(-) 8644, caffeine, L-phenylephrine chloride (Phe), tetraethylammonium (TEA) were purchased from Sigma (Sigma Chemical Co., St. Louis, MO, USA).The extract was solubilized in cremofor® (3%) and diluted with water to obtain the concentration 10 mg/mL.The final concentration of cremophor ® did not exceed 0.01%.This concentration did not induce any effects on the mesenteric artery rings (data not shown).The other compounds were dissolved in distilled water.

Animals
Male spontaneously hypertensive rats (SHR) and Wistar rats with 12 weeks old were used in all the experimental protocols.
The animals came from central bioterium of the UFPB.The SHR and Wistar rats were kept in plastic cages (5 rats per cage), under conditions of temperature control and they were subjected to 12h-12h light-dark cycle, without restriction of feed and tap water.All experimental protocols and techniques performed in this study were accepted by the Institutional Animal Care and Use Committee of the UFPB (Number of the certification 0106/08).

Pharmacological Evaluation Isolated mesenteric ring studies
The animal was anesthetized and euthanized.The abdominal incision was then performed, and the superior mesenteric artery was removed.The connective and adipose tissues were carefully dissected of the artery.After this procedure, the mesenteric artery was cut into rings of 1-2 mm.The rings were suspended on cotton threads in isolated organ baths filled with 10 mL of Tyrode's solution (in mM): sodium chloride (NaCl 158.3); potassium chloride (KCl 4.0); calcium chloride (CaCl 2 2.0); magnesium chloride(MgCl 2 1.05); sodium bicarbonate (NaHCO 3 10.0); sodium phosphate(NaH 2 PO 4 0.42) and Glucose 5.6, at 37 °C and bubbled with carbogenic mixture (95% O 2 and 5% CO 2 ).The mesenteric rings were subjected to a constant tension of 0.75 g for a period of 60 min.
For this period, the preparations were washed with Tyrode's solution every 15 min to prevent interference of metabolites.The isometric tension was determined by use ofa force transducer (FORT-10, WPI, Sarasota, FL, USA) connected to an amplifier (Miobath-4, WPI, Sarasota, FL, USA).
The vascular endothelium was removed by rubbing the intimal surface of the artery with stainless steel rod.The functionality of the endothelium was verified by the relaxation y y h (ACh 10 μM) p -contracted rings w h h (10 μM).R x q h 90% induced by ACh was considered that the mesenteric rings had functional endothelium, while the mesenteric rings with the relaxation of less than 10% were considered without vascular endothelium.

Effect of EEAT on the contraction induced by phenylephrine or KCl
In the first set of experiments, after stabilization for 1 hour, contractions were induced by Phe 10 µM in rings with and without vascular endothelium.Then, the extract was added y ( f 0.03 300 μ / L) concentration-response curve.The capability of EEAT to reduce the 80 mM KCl induced sustained contraction was also verified in rings without endothelium.

Investigation of the role of K + channels in the EEAT-induced vasorelaxant response
In another set of experiments, rings without endothelium were obtained with 20 mM KCl, an inhibitor of K + efflux, plus Phe or TEA (3 mM), a nonselective blocker of K + channels.Thus, concentration-response curves to EEAT were acquired.The TEA w f 30 f h w h h .

Effect of EEAT on the concentration-response curve for CaCl 2
After removal of endothelium, the mesenteric artery rings were washed for 15 min in nominally Ca 2+ -free Tyrode's solution.After this time, the preparations were exposed to the solution of 60 mM KCl nominally without calcium for 15 min.Then, cumulative concentration-response curves for CaCl 2 were induced.Different concentrations of the EEAT (10, 30, 50, 100 or 300 µg/mL) were pre-incubated for 15 min.Soon after, new cumulative concentration-response curves for CaCl 2 were determined.

Effect of EEAT on Ca 2+ release from intracellular stores sensitive to phenylephrine and caffeine
The mesenteric artery rings without endothelium were pre-contracted with 60 mM KCl.After this procedure, the preparations were washed with Ca 2+ -free Tyrode's solution containing ethylene glycol-bis (2-aminoethyl ether)-N,N,N′,′tetraacetic acid (EGTA 1 mM) for 2-3 min.After this time ph y ph (10 μM) ff (CA 20 M) was then added to the organ baths to contract the rings.The contractions induced by phenylephrine or caffeine were obtained in the absence (control) or after incubation with isolated concentrations of the EEAT (10, 30, 100 or 300 µg/mL).

Antihypertensive activity
The measurements of the MAP and HR were performed in SHR rats.The SHR rats were randomly divided into two groups (5 animals per cage), which were denominated of control rats (Saline group) and treated rats (Treated group).The first group received saline and second group a single intragastric dose of 200 mg of EEAT/kg body weight.For measurements of the MAP and HR, the SHR rats were anesthetized with sodium thiopental (45 mg/kg, i.p.) and polyethylene (PE) catheter was implanted into the lower abdominal aorta via the left femoral artery.Then, the catheter was fixed, filled with a heparin solution, and tunneled subcutaneously to exit between the scapulae.The rats were placed in individual cages and 24 h after the surgical procedure the MAP and HR were measured.The measurements of the MAP and HR were performed are described above.They were acquired before and after the treatment with EEAT at 0, 1, 2, 4, and 6 h.The animals were kept in acclimation period of at least 30 min for stabilization of hemodynamic parameters.Percent decrease in MAP and HR were calculated.

Statistical analysis
Two pharmacological parameters were used to analyze the data, the E max (maximum effect elicited by the agonist) and EC 50 .All values are displayed as the mean ± standard error of the mean (SEM, n = number of experiments).The drug concentration that is capable of inhibiting the contractile response by 50% (EC 50 ) was obtained by non-linear regression.Student´s t-test or two-way analysis of variance (ANOVA) were used to analyze the results statistically.The differences between the mean were considered significant when the value obtained for "p" was lower than 0.05 (p <0.05).All statistical analysis and plotting of the curves were performed by GraphPad Prism TM software, version 3.0 (GraphPad Software, Inc., San Diego, CA, USA).

Analysis of the chemical constituents by ESI-MS
Using direct infusion electrospray ionization mass spectrometry (ESI-MS/MS) data in the negative and positive ion mode, the ethanol:water (1:9, v/v) extract from the stem bark of A. tomentosum were characterized.These analyses demonstrated that the detected constituents in EEAT were equivalent to the mass of the phenolic acids, flavonoids and indole alkaloids (Table 1, Figure 1).Thus, 10 compounds were tentatively identified (caffeic acid, quinic acid, kaempferol, quercetin, isorhamnetin, dimethyluleine, uleine, aparicine, and quebrachamine) based on CID experiments of the observed precursors and make a comparison of the ESI-MS/MS fragmentation spectra with the spectra of the standard samples, and with literature results.ESI-MS is a technique simple to execute.The sample preparation and the process of acquisition of spectra were performed for 5 min, and it provides valuable information about the constituents present in the extract

Effect of EEAT on the contraction induced by phenylephrine or KCl
A h w T 2 EEAT (0.03 300 μ / L) induced relaxation in rat superior mesenteric artery rings in a concentration-dependent manner of Phe-induced tonus.The concentration-response curve to EEAT was not significantly shifted to right without a reduction in the maximal response after endothelium removal.In rings without endothelium, precontracted with a depolarizing agent KCl 80 mM, the cumulative addition of EEAT (0.03-300 μ / L) -dependent vasorelaxant effect with [EC 50 = 26.4 ± 3.1; E max = (94.4± 4.5 %)] (Table 2).

Evaluation of the role of K + channels in the EEAT-produced vasorelaxant effect
In endothelium-denuded rings pre-incubated with 20 mM KCl w h h (10 μM) EEAT vasorelaxant effect with no change as compared with control with Phe.The incubation with TEA (3 mM), the concentration-response curves of EEAT not was significantly affected(Table 2).

Effect of EEAT on Ca 2+ release from intracellular stores sensitive to phenylephrine and caffeine
As illustrated in Figure 4, in the Ca 2+ -free solution, the EEAT (30 100 300 μ / L) w h concentration-dependent manner the transient contractions induced y h (10 μM) h w f h ff induced contraction (CAF 20 mM).

Antihypertensive activity of EEAT in SHR
In this battery of experiments, baseline MAP and HR values were registered at 0 h and assuming as 100% of activity.After 1 and 2 h of extract administration, the MAP was significantly decreased without changed HR when compared the saline group.Nevertheless, after this time, pressure values returned to baseline (Figure 5).

DISCUSSION
The main finding in this paper is that the extract induced a vasorelaxant activity in mesenteric rings and the oral administration caused a reduction in the mean arterial pressure in non-anesthetized SHR.
To investigate the direct effect of EEAT on the vasculature, we performed experiments in rat isolated superior mesenteric arteries.This artery was used because it has greater resistance to blood flow.Therefore, it is widely involved in regulating blood pressure (Mulvany and Aalkjaer, 1990).We found that EEAT concentration-dependently induced relaxation in mesenteric rings with or without functional endothelium precontracted with phenylephrine.The E max and EC 50 values of EEAT were not statistically different.Thus, it suggests that the vasorelaxant effect of EEAT is independent of endothelium and this effect is probably due to a direct action of the extract in vascular smooth muscle.The K + channels contribute to the regulation of vascular tone.The activation of these channels induces vasorelaxation by membrane hyperpolarization, due to inactivation of voltage-dependent Ca 2+ channels and consequently decrease in intracellular Ca 2+ levels.In contrast, inhibition of K + channels function leads to membrane depolarization and vasoconstriction.The potassium channels present in the vascular smooth muscle were four types: voltage-dependent K + (K v ) channels, Ca 2+ -activated K + (BK Ca ) channels, ATP-sensitive K + (K ATP ) channels, and inward rectifier K + (Kir) (Nelson and Quayle, 1995).To verify the participation of the K + channels in the relaxant response induced by the extract, we executed experiments in the presence partial inhibitor of K + efflux, as the solution 20 mM KCl.According to the literature, the drugs that induce relaxation by opening K + channels are less efficient in the presence of high external concentration of K + (Campbell et al., 1996).In denuded rings, the incubation with KCl 20 mM did not change the relaxation when compared with those obtained in denuded rings, suggesting no involvement of K + channels in this response.To confirm this hypothesis, the endothelium-denuded rings were pretreated with TEA (3 mM), a non-selective blocker of K + channels.The concentration-response curves of EEAT were not significantly affected, indicating the lack of participation of K + channels in the vasorelaxant action induced by this extract.
h y ph α 1 -adrenergic receptor agonist.The contractions induced by adrenergic agonists in the vascular smooth muscle are initiated by the release of calcium from intracellular IP 3 receptor-sensitive stores.The increase in intracellular calcium concentration causes the activation of Ca 2+ -activated channels and consequently depolarisation (Criddle et al., 1996).On the other hand, the contractions induced by 80 mM KCl are caused almost completely through the opening of Ca v (Karaki et al., 1997).
Therefore, it was evaluated the effect of the extract in rat mesenteric rings precontracted by depolarizing solution of 80 mM KCl.Operating in this conditions, EEAT promoted concentrationdependent relaxation in mesenteric artery rings.It is important to mention that EEAT was able to inhibit both KCl-induced and phenylephrine-induced contractions similarly.These results suggest that the EEAT probably acts by inhibiting Ca 2+ influx via Ca v .According to the literature, two types of Ca 2+ channels are expressed in smooth muscle cells: voltage-dependent Ca 2+ channels (high KCl induced contraction is due to membrane depolarization, consequently increasing the Ca 2+ influx through these channels) and receptor operated Ca 2+ channels (contraction induced by Phe or CAF is due to intracellular Ca 2+ release, through sarcoplasmic reticulum Ca 2+ channels activated by IP 3 or ryanodine) (Clapham, 2007).The extract inhibited contractions induced by phenylephrine or KCl in endothelium-denuded mesenteric rings.Analyzing the results obtained with EEAT, we can suggest that the vasorelaxant mechanism may involve blocking both voltage-dependent and receptor operated Ca 2+ channels.Furthermore, in endothelium-denuded rings incubating with depolarizing and nominally without Ca 2+ solution, CaCl 2 (1 µM -30 mM) was able to induce contractions that were significantly inhibited by EEAT, supporting the idea that EEAT possesses a Ca 2+ influx blocking activity.In order to evaluate the Ca v subtype involved in the inhibition of Ca 2+ influx promoted by the EEAT, we perform experiments with S-(-)-Bay K8644, an Ltype Ca v agonist (Spedding and Paoletti, 1992).EEAT induced relaxation in rat superior mesenteric artery rings in a concentration-dependent manner of S-(-) BayK 8644-induced tonus.Our results suggest that the EEAT probably decreases the calcium influx via L-type channel.Further studies involving voltage-dependent Ca 2+ currents in the smooth muscle cell are needed to confirm the participation of L-type voltage-gated calcium channels (VGCC) in EEAT-induced relaxation.However, in the Ca 2+ -free solution, EEAT significantly inhibited the Phe (10 μM)-induced contraction in a concentration-dependent manner.EEAT (300 µg/mL) did not inhibit transient contractions induced by caffeine (20 mM) in a Ca 2+ -free solution containing EGTA.This result suggests that EEAT may also inhibit Ca 2+ mobilization from intracellular stores by a possible IP 3 signaling blockade.The different effects mentioned in this study were unlikely caused by toxicity of EEAT on vascular cells.We can make this affirmation because the relaxant effect induced by extract was reproduced, and the contraction induced by Phe was completely restored following a 60 min of EEAT.
Literature data confirm that the essential hypertension is in part determined by genetic factors.The SHR are models of animals that develop hypertension of genetic origin.Based on this premise, they were used to analyze the antihypertensive effect of the extract.The intragastric administration of EEAT in nonanesthetized SHR induced reduction significant in MAP (first and second hours after oral administration of the extract) and did not change the heart rate.Nevertheless, after this time, pressure values returned to baseline.The effects induced by EEAT on the cardiovascular system in rats are in agreement with literature data, related with bioactivity of Apocynaceae species and with bioactive chemical compounds isolated from species of the Aspidosperma, which are rich alkaloids, including aspidospermine, quebrachamine, and yohimbine (Lyon et al., 1973;Craveiro et al., 1983;Sperling et al., 2002).Also, other natural products have been documented, such as the occurrence of triterpenes (Barbosa et al., 2010), flavonol glycosides, quercetin, kaempferol and isorhamnetin derivatives (Pelotto and Del Pero, 1995) in genus Aspidosperma.The EEAT was analyzed by ESI-MS in the positive and negative ion mode.These analyses showed that the detected constituents in EEAT coincided with the mass of the phenolic acids, flavonoids, and indole alkaloids.Thus, 10 compounds were tentatively identified (caffeic acid, quinic acid, kaempferol, quercetin, isorhamnetin, dimethyl-uleine, uleine, aparicine, and quebrachamine), based on CID experiments of the observed precursors and comparison of their ESI-MS/MS spectra with authentic standard spectra, and with literature data.The experiment performed by ESI-MS provided relevant information about the active principles present in the extract.It is reported that flavonoids such as rutin, quercetin, and apigenin promote vasorelaxation by inhibiting the influx of Ca 2+ , and flavonoids like flavones and flavonols, inhibit the release of Ca 2+ from intracellular stores (Chan et al., 2000).The caffeic acid phenethyl ester inhibited contractile responses to phenylephrine or KCl in thoracic aortic rings, and also inhibited the contractile response to phenylephrine obtained in a Ca 2+ -free medium (Cicala et al., 2003).Using the ESI-MS technique, it was possible to detect the presence of some phenolic acids, flavonoids and, alkaloids in EEAT.The presence of these bioactive components could explain, in part, the antihypertensive and vasorelaxant action of the extract.

CONCLUSIONS
Our results suggest that EEAT induces vasorelaxation through a blockade of Ca 2+ channels and inhibition of the mobilization of Ca 2+ of the IP 3 -sensitive Ca 2+ stores, and also causes an antihypertensive effect.Our findings have highlighted the therapeutic potential of A. tomentosum for the treatment of cardiovascular diseases.

Table 2 :
Emax and EC50 values of experiments in the presence of EEAT.