Extract and chloroform fraction from Syzygium cumini leaves with vasorelaxant effect mediated by inhibition of calcium channels

1 Pharmacology Research and Post-Graduate Laboratory, Department of Physiological Sciences, Federal University of Maranhão, Av. Portugueses, 1966, 65085-580, São Luís, Maranhão, Brazil. 2 Laboratory of Biology, Department of Biology, Federal Institute of Education, Science and Technology of Maranhão (IFMA), BR-230, km-319, 65840-000, São Raimundo das Mangabeiras, Maranhão, Brazil. 3 Laboratory of Experimental Surgery, Department of Medicine, CEUMA University (UNICEUMA), Av. Josué Montello, 65075-120, São Luís, Maranhão, Brazil.


INTRODUCTION
Globally cardiovascular disease accounts for approximately 17 million deaths a year.Of these, complications of hypertension account for 9.4 million deaths worldwide every year.Hypertension is responsible for at least 45% of deaths due to ischemic heart disease and 51% of deaths due to stroke (WHO, 2013).
The objective of antihypertensive treatment is to achieve optimal blood pressure levels during therapy to reduce hypertension-related complications.The research literature indicates that secondary metabolites of herbs and spices exhibit antihypertensive effects contributing in reducing blood pressure levels and minimizing its complications (Al Disi et al., 2016).
Syzygium cumini (L.) Skeels, belongs to Myrtaceae family, is popularly known as jambolan, being is a medicinal plant known for its therapeutic properties for the treatment of different diseases such as inflammation, diabetes and hypertension (Morton, 1963;Pepato et al., 2005;Migliato et al., 2006;Dieckel et al., 2007;Abbas and Mushtaq, 2008).Its leaves are rich in phenolic compounds (Sanches et al., 2016) with great ability to act biologically in cardiometabolic disorders (Chagas et al., 2015).
According to Silva et al. (2012), hydroalcoholic extract of S. cumini leaves (HESc) exhibits no chronic toxicity.Additionally, the ability of HESc to reduce blood pressure in SHR suggesting antihypertensive property were demonstrated (Ribeiro et al., 2014).
In this context, this present study investigated the vasodilator effect of S. cumini leaves.This work assess the potential of HESc and and its active fraction in promoting vasorelaxation in resistance artery rings for the first time thereby, contributing to clarify the mechanism of its antihypertensive action.

Preparation of the hydroalcoholic extract of S. cumini
Leaves were dried at room temperature and pulverized.The crude extract was prepared by maceration of the leaf powder (300 g) in 70% ethanol (1:3 w/v), and concentration in a rotary evaporator under reduced pressure at a temperature below 60°C and lyophilized.The extract thus obtained was called the hydroalcoholic extract of S. cumini leaves (HESc) with a dry weight of 49.8 g and yield 16.6% (Ribeiro et al., 2014).
HESc aliquots were kept at 4ºC, protect from light, until further experimental use, when powdered HESc was resuspended in water at desired concentrations.HESc was partitioned by sequential extraction using hexane, chloroform (CF), ethyl acetate, and nbutanol.Based on previous results demonstrating that the CF was the most potent in inducing vascular relaxation in vitro (Ribeiro, 2007), we evaluated the effects of this fraction.The chloroform (CF) fraction were evaporated, with a yield 5.2% and tested to evaluate the vasorelaxant effect.In this study, phytochemical screening by CF revealed the presence of phenols.

Animals
Male 12-week-old spontaneously hypertensive rats (SHR) or normotensive (Wistar), Rattus norvegicus, weighing 250 to 300 g, obtained from the animal house of UFMA were used.The animals were housed under controlled conditions of temperature (21 ± 2°C) under a 12 h light-dark cycle, with ration and water available ad libitum.All The experimental protocols were reviewed and approved by the Animal Research Ethics Committee of UEMA, Brazil (Number 17/2012) and all the methods in this study were carried out in accordance with the approved guidelines.

Norepinephrine
hydrochloride, Acetylcholine and Tetraethylammonium chloride were purchased from Sigma Chemical Co.(St Louis, MO, USA).All other chemicals were of high analytical grade purity from Merck Darmstadt.

Tissue preparation
Preparations of the mesenteric artery were obtained as described by Borges et al. (1999), Abreu et al. (2003) and Amaechina and Omogbai (2007) and ring segments (3 to 5 mm) of the superior mesenteric artery were placed between stainless steel wires (50 m in diameter) and immersed in an organ bath chamber (5 mL) containing Krebs nutritive solution (118 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 15.5 mM NaHCO3, 2 mM CaCl2, and 11mM glucose, pH 7.4) at 37°C, equilibrated with 5% CO2/ 95% O2.The preparations were first equilibrated under a tension of 1.0 g and washed at intervals of 10 min, for 60 min.Changes in the isometric tension of the preparations were measured with an isometric force transducer (PowerLab, ADInstruments Pty. Ltd., Sydney, Australia).
Due to the endothelial dysfunction and the inefficient production of vasodilators by the epithelial cells present in hypertensive syndrome, we chose to perform all experiments on endotheliumdenuded mesenteric arteries, to demonstrate the vascular relaxing property of the extract and fractions without the interference of endothelium-derived factors.Vascular endothelium removal was confirmed by the absence of a relaxation response by Acetylcholine (10 M) to induce more than 70% inhibition of vessels precontract with norepinephrine (NE 10 M).

Effect of HESc on contraction induced by NE or KCl
After the stabilization period, endothelium-denuded mesenteric artery rings, obtained of SHRs, were pre-contracted with NE (10 M) or KCl (80 mM) and, on the tonic phase, different concentrations of HESc (0.1, 0.25, 0.5, 5 and 10 mg/ml) were added cumulatively to organ bath.The relaxant effect was expressed as the percentage of NE or KCl induced contraction.

CF on contraction induced by NE in presence or absences of K + -blocker
After the stabilization period, two successive contractions of similar magnitude were induced with NE (10 μM) in endotheliumdenuded rings, obtained of SHR.During the tonic phase of the third contract, different concentrations of CF (0.01, 0.05, 0.10, 0.50 and 1.0 mg/mL) were added cumulatively to the organ bath.To examine the role of K + channels in the HESc or CF induced relaxation, arteries rings pre-contract with NE (10 μM) were constructed in absence or presence of TEA (0.3 mM).The results were expressed as the percentage of NE induced contraction.

CF effect on arterial smooth muscle contraction induced by NE or Ca 2+
The preparations were first equilibrated under a tension of 1.0 g and washed at intervals of 10 min.After 60 min of successive washes, cumulative dose-response curves to NE (10 -9 to 10 -4 M) were constructed in the absence or presence of CF (0.25 and 0.5 mg/ml).To evaluate the antagonistic action of CF against Ca 2+ , vascular tissue was stabilized with normal Krebs solution.After 30 min, the fluid of the preparation was replaced with Ca 2+ -free Krebs solution (60 mM K + , nominally Ca 2+ free).
Also, after 30 min of successive washes, the basal tone was recovered, permitting to obtain cumulative concentration-response curves to CaCl2 (10 -6 to10 -2 M) in the absence or presence of CF (0.25 and 0.5 mg/ml).The concentration necessary to elicit 50% of the maximum response (EC50) was determined using nonlinear regression analysis.The negative logarithms of the EC50 values (pD2) were used for statistical analysis.In the experiments involving high extracellular K + , Krebs solution containing 60 mM KCl was prepared by replacing an equimolar concentration of NaCl with KCl.

Statistical analysis
Results were expressed as mean ± standard error of the mean (SEM).One-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls post-test was used for, multiple comparisons and Student t-test for comparison of unpaired data.A p-value < 0.05 was considered significant and the statistical analysis was performed using the GraphPad Prism ® 5.0 program.

RESULTS
In endothelium-denuded rings HESc (0.1 to 10 mg/ml) inhibited the sustained tonic contraction induced by NE 10 µM (Figure 1) in a concentration-dependent manner (E max values= 97.20 ± 2.90%; EC 50 values= 2.82 mg/ml).Additionally, HESc also promoved a prominent vasorelaxant effect in arteries rings contracted with KCl (Figure 1), with a maximum relaxation of 100.0 ± 0.0% (EC 50 1.11mg/ml).The relaxant effect of HESc was reversible as the tissue regained its spontaneous activity at least within one hour after repeated washout.
In Figure 2A was observed CF (0.1 to 1 mg/ml) also exhibited vasorelaxant activity in preparations endothelium-denuded pre-contracted with NE (10µM), in a concentration-dependent manner (Table 1).The relaxant effect of CF also was reversible after repeated washout.The vasorelaxant effect induced by HESc or CF was available in preparations with TEA (1mM).The Figure 2B showed that 30 min of TEA-pretreatment in  endothelium-denuded rings did not change the vasorelaxant response of HESc (E max values= 99.75 ± 0.25%; EC 50 values= 2.24 mg/ml).In addition, in relation to vasorelaxation promoted by CF, TEA reduced relaxative effect only in concentration of 0.1 mg/mL (Figure 2A and Table 1).It was also observed that in a lower concentration CF demonstrated a potential higher vasorelaxant HESc (Figure 3).CF at concentrations of 0.25 and 0.5 mg/ml reduced the E max value for NE in endothelium-denuded mesenteric artery (Figure 4 and Table 2).In addition, contractions induced by CaCl 2 in endothelium-deprived mesenteric arterial rings were reduced in a concentration-dependent manner after incubation with 0.25 and 0.5 mg/ml CF, (Figure 5 and Table 2).There was a displacement of the CaCl 2 curves to the right, changing pD2.These effects were reversed after washing with Krebs solution.

DISCUSSION
This study is the first to demonstrate the vasorelaxant activity of S. cumini leaves in mesenteric artery of SHR and possibly acting involving intracellular Ca 2+ stores.These results support the elucidation of possible mechanisms involved in the antihypertensive effect of this plant species, a pharmacological property recently demonstrated by our group.
Structural and functional alterations occur in arterial hypertension and are important in the mechanisms that determine blood pressure and target of antihypertensive therapy (Oh et al., 2007;Oh et al., 2008).The sympathetic nervous system also participates in the development and maintenance of various forms of hypertension (Piascik et al., 1996).The magnitude of sympathetic over activity has been closely related to  hypertension-related end organ damage and predicts mortality and cardiovascular outcomes (Hering and Narkiewicz, 2013).The contractile response of vascular smooth muscle cells (VSMCs) by NE is dependent on the Ca 2+ influx from extracellular space through receptor-operated Ca 2+ channels (ROCCs) (Karaki and Weiss, 1988;Nelson et al., 1990;Qayyum et al., 2016)  The values indicate the mean ± SEM of the pD2 and the Emax obtained from 5 experiments.*Significantlyvs Control; a Significantly vs CF 0.25 mg/ml.
In VSMCs, K + channels play important roles in the regulation of vascular tone (Tanaka et al., 2004).Largeconductance Ca 2+ -activated K + channel (BK Ca channels), which have been extensively studied in VSMCs, contribute to the control of vascular tone (Brenner et al., 2000) and have been therapeutic targets in the treatment of cardiovascular disease (Saponara et al., 2006).
To determine if the vasorelaxant effect induced by

Contraction
HESc involves BK Ca -channel activation, it was incubated in mesenteric artery without endothelium preparations with a selective inhibitor of this channel TEA (1mM) (Garcia and Kaczorowski, 1992;Campbell, 1993;Jackson, 2005;Eichhorn and Dobrev, 2007).Similar study was realized by Matsumoto et al. (2010) when assessing the vasodilator alkaloid isolated from Cassia siamea in mesenteric artery without endothelium preparations of rats.The results in Figure 2B showed that vasorelaxant effect of S. cumini leaves does not occur by direct activation of BK Ca channels.
A particular feature of phytomedicines is their complex composition, that is, the "phytocomplex", with different specific effects, however a wider array of effects and healing properties are guaranteed only by the phytocomplex (Medeiros et al., 2008).In order to further characterize this effect, HESc was submitted to liquid partition, as described under material and methods.To continue the study, we decided to work with CF, whose previous results suggest potential vasorelaxant effect in mesentric arteries of normotensive rats (Ribeiro, 2007).
In Figure 2A and 3 it was observed that, CF in the lower concentration exhibited remarkable vasorelaxant ability in a concentration-dependent manner, when compared to HESc at the same concentration, suggesting that CF contains the active compounds present in phytocomplex and the vasorelaxant response probably implicate an endothelium-independent pathway or inhibits Ca 2+ influx or by hyperpolarization produced by K + -channels (Karaki and Weiss, 1988;Eichhorn and Dobrev, 2007).
Figure 2B and Table 2 suggest that the presence of TEA did not alter the vasorelaxant response pattern of CF at higher concentrations.Additionally, reduced inhibitory effect of the CF at 0.1 mg/mL by TEA showed implication of BK Ca -channel activation in the induced vasorelaxation at lower concentrations of fraction (Table 1).
Studies have shown that a number of plant products including polyphenols, flavonoids, and various plant extract exert antihypertensive effects that might be owing to vasorelaxant action (Curin and Andriantsitohaina, 2005;Wang et al., 2014;Van Rymenant et al., 2017).Chemical studies of S. cumini leaves were performed by Ruan et al. (2008) and showed that CF contains phenolic acids, the other complex phenolic compounds.The results of phytochemical screening showed that CF is rich in these compounds, which may be responsible for the vasorelaxant property of the plant.
Previously, we have shown that the maximal response induced by α 1 -adrenoceptor agonist NE in SHRs, suggest that HESc contains components that interfere with the reactive response of the vascular musculature.In the present study, the incubation of CF (0.25 and 0.5 mg/ml) reduced in a concentration-dependent way the E max induced by NE in mesenteric artery, however, did not altered the pD 2 of the NE, suggesting that CF contains components that interfere with the reactive response of the vascular musculature, possibly interfering with the mechanisms of Ca 2+ homeostasis in VSMCs.Contractions of smooth muscles induced by high K + have been widely used in understanding Ca 2+ roles in biological systems.To check the effect of the CF in [Ca  2+ ] i , a concentration-response curve to Ca 2+ (10 -6 and 10 -2 M) in presence of K + -depolarizing solution (KCl 60 mM) was constructed, before and after incubation with CF 0.25 and 0.5 mg/ml, that induced concentrationdependent manner, maximal effect inhibition and also led to a significant rightward shift in the concentrationresponse curve for Ca 2+ in endothelium-denuded rings (Figure 5 and Table 2).These findings support the notion that, the CF can block Ca-influx from the extracellular space and acts as a non-competitive Ca 2+ antagonist (Figure 5).Clinically, Ca 2+ antagonist are potentially used to treat hypertension (Tep-Areenan and Sawasdee, 2011).

Conclusion
The results demonstrate that S. cumini L. (Skeels) causes vasorelaxant effect and interfere with the responsiveness of vascular smooth muscle cell, probably as a result of the blockade of Ca 2+ channels, as demonstrated in this study.These effects can be attributed to the presence of phenolic compounds detected by phytochemical screening.
The S. cumini leaves showed an excellent potential as a vasodilator agent for the treatment of hypertension.The findings may provide a possible candidate drug for clinical medical use to treat cardiovascular diseases in the future.However, further experiments are necessary to clearly elucidate this assumption.

Figure 3 .
Figure 3. Comparative Emax (% values in terms of vasorelaxant effects) of HESc and CF at 1 mg/ml.Values are expressed as mean ± SEM of (n = 5) experiments.*** p < 0.00001 in comparison to HESc as reference.
. In addition, potassium causes VSMCs contraction through activation of voltage dependent

Table 1 .
Parameters of concentration-relaxation curves for chloroform fraction (CF) from Syzygium cumini leaves in presence or absence of TEA.

Table 2 .
Parameters of concentration-response curves for the effects of chloroform fraction (CF) from Syzygium cumini leaves on contraction induced by NE or Ca 2+ in mesenteric arteries.