Comparative study of the antihypertensive effects of hexane, chloroform and methanol fractions of essential oil of Alpinia zerumbet in rats Wistar

The aim of this study was to characterize components of the EOAz and its hexane (HFEOAz), chloroform (CFEOAz) and methanol (MFEOAz) fractions, and its antihypertensive effect. EOAz was extracted from leaves by hydrodistillation. Aliquot was subjected to selective desorption with silica gel column and eluted with hexane, chloroform and methanol. The components of the EOAz and fractions were analyzed by gas chromatography coupled with mass spectrometry and nuclear magnetic resonance spectroscopy of hydrogen. Experiments of vascular reactivity were performed with isolated aortic rings of male Wistar rats. Antihypertensive effect was evaluated in hypertensive rats submitted to the inhibition of synthesis of nitric oxide. Blood pressure was measured indirectly by tail plethysmography. MFEOAz showed the lowest EC50 (150.45 μg/mL), 1,8-cineole (27.81%) and terpinen-4-ol (57.35%) as main components. Single administration by nasogastric tube of EOAz, fractions and captopril significantly reduced the blood pressure of hypertensive rats, when compared to animals of the negative control group with distilled water. In conclusion, the potency of the MFEOAz was higher than that of EOAz and other fractions. The antihypertensive effect of EOAz and fractions was similar, higher than the negative control and lower than that of captopril.


INTRODUCTION
Hypertension is a multifactorial systemic chronic disorder through macrovascular and microvascular alterations at the functional and structural levels (Yannoutsos et al., 2014). High blood pressure may cause serious diseases such as heart failure, kidney failure and stroke (Sharifi et al., 2013). Several classes of drugs are indicated to hypertension. However, there has been an increase in use of herbal medicines in the last decades, which has encouraged studies of herbal products (Palhares et al., 2015).
Considering that it is of great interest to explore the medicinal value of EOAz with regard to cardiovascular activities, and that this study can also serve as a support for the realization of future clinical trials, its constituents were separated into three fractions, hexane (HFEOAz), chloroform (CFEOAz), and methanol (MFEOAz) fractions, to better understand their pharmacological effects. In fact, the effect of these fractions on vascular function has not yet been reported. Thus, the purpose of this study was to characterize the components of the EOAz and its fractions and to determine their vasorelaxant and antihypertensive effects.

MATERIALS AND METHODS P l a n t m a t e r i a l , e x t r a c t i o n a n d chromatographic analysis
A. zerumbet leaves were collected in November 2009, in Maranguape County, Ceará, Brazil. Botanical identification was determined in Prisco Bezerra Herbarium of School of Agronomy, Federal University of Ceará, where voucher specimen has been deposited under No. 50312. EOAz was extracted by hydrodistillation in Clevenger-type glass apparatus. Aliquot of oil was chromatographed in silica gel column and eluted with hexane, followed by chloroform and methanol. Crude oil and fractions were analyzed by gas chromatography coupled with mass spectrometry (GC-MS) and flame ionization detection (GC-FID) according to method described (Cavalcanti et al., 2012). GC-MS analysis was carried out on a Shimadzu QP5050 instrument equipped with non-polar OV-5 fused silica capillary column, while GC-FID analysis was carried out on a Shimadzu GC 2010 Plus instrument provided with non-polar CP-Sil-8 fused silica capillary column. Main constituents were confirmed by 1H-nuclear magnetic resonance spectroscopy (1H-NMR).

Animals and experiments
Male Wistar rats weighing 200-250g (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. Procedures were performed in accordance with the Animal Ethics Committee of Federal University of Ceará, Brazil, under registration No. 18/2011. There were two experimental phases: in vitro study to determine the vasorelaxant effect of EOAz and fractions in isolated aortic rings, and in vivo study to determine the effect of fractions on blood pressure measured by indirect tail cuff method in rats subjected to chronic hypertension by inhibition of nitric oxide.

In vitro experiments: studies on isolated rat thoracic aorta
Rats were sacrificed by cervical dislocation followed by exsanguination. Thoracic aorta was removed, cleaned of adherent connective tissue and cut into rings (3-4 mm in length). Two stainless-steel 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. Rings were placed in a 10 mL organ chamber containing Krebs solution, gassed with 95% O 2 and 5% CO 2 and maintained at 37°C and pH 7.4. Composition of Krebs solution was as follows (mmol/L): NaCl, 118.0; KCl, 4.7; KH 2 PO 4 , 1.2; MgSO 4 .7H 2 O, 1.2; NaHCO 3 , 15.0; CaCl 2 , 2.5 and glucose, 5.5 (Hipólito et al., 2011). Aortic rings were stretched until they reached a resting tension of 10 millinewtons (mN). They were then allowed to equilibrate for 60 minutes, during which time, bath solution was changed every 15-20 min. Endothelial integrity was assessed by degree of relaxation caused by acetylcholine (10 mmol/L) in presence of contractile tone induced by phenylephrine (1 mmol/L). After 60 minutes equilibration period, aortic rings were exposed to KCl (80 mmol/L) (Estrada-Soto et al., 2010). Steady tension was evoked by phenylephrine (1 mmol/L) (Chen et al., 2009), and EOAz and fractions were added cumulatively (0.1-3,000 mg/mL).

In vivo experiments: effects of the fractions and EOAz on arterial pressure
Hypertension was induced in rats by administration of L-NAME (30 mg/kg) dissolved in drinking water for 60 days. First 30 days represented the induction phase of hypertension and was called pre-treatment. On the 31 st day, the rats were already hypertensive, and treatments studied were started and maintained for 30 days, corresponding to the treatment phase. Animals were randomly allocated into seven groups: negative control (0.5 mL distilled water; n=9), positive control (30 mg/kg captopril; n=11), EOAz (n=8), HFEOAz (n=9), CFEOAz (n=8), MFEOAz (n=8), all at 100 mg/kg, diluted in 0.5 mL distilled water.
EOAz and fractions dosages were determined from pre-clinical toxicology studies (Cunha, 2012). The dose of EOAz used in this protocol was assessed from acute and chronic toxicity studies in mice, where the dose at which adverse events were not observed was 200 mg/ kg. For conversion of the dose of drug used in mice to the equivalent dose in rats the following formula was applied: Equivalent dose in rats = Drug dose at which adverse events were not observed in the animal (mg/kg) × (K m factor mouse) ⁄ (K m factor rat). In the formula, K m factor mouse is 3, K m factor rat is 6, and the dose calculations were 100 mg/kg for OEAz and fractions (HFEOAz, CFEOAz, MFEOAz) (Reagan-Shaw et al., 2008).
Tr e a t m e n t s w e r e b y i n t r a g a s t r i c administration, at the same time (8:00 AM), for 30 days until 60 th day. Measurements (blood pressure and heart rate) were recorded at pre-treatment every 6 days and in treatment phase every 3 days by tail cuff plethysmography method (IITC Life Science, model 229, Woodland Hills, CA).
Before measurements, conscious rats were restrained for 5-10 minutes 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 (MAP) pressure were measured, the mean of three measurements was recorded. To evaluate overall antihypertensive effect of treatments used in this study, area under the curve (AUC) of SAP versus time was calculated by the trapezoidal method. Were determined the rate of systolic arterial pressure decline (mmHg/day) during treatment phase, which was obtained by following formula: Rate of SAP decline=[SAP(day 30) -SAP(day 60)]/30 days.

Solutions and drugs
Following drugs and reagents were used in the in vitro studies: phenylephrine hydrochloride, acetylcholine hydrochloride, potassium chloride; diluents included Tween-80 and Krebs solution (NaCl, KCl, KH 2 PO 4 , MgSO 4 .7H 2 O, NaHCO 3 , CaCl 2 , glucose). All substances were from Sigma-Aldrich, St. Louis, MO, USA. EOAz and fractions were prepared as stock solutions in Tween 80 and sonicated; bath concentration of Tween 80 did not exceed 0.01%. Concentration of Tween 80 did not exceed 0.5%, which was shown to have no effect per se on the basal tonus of preparations or on the agonist-mediated contraction or relaxation (Hipólito et al., 2011;Lima-Accioly et al., 2006). For in vivo experiments, EOAz, fractions and captopril were diluted in distilled water and sonicated. Solutions were prepared fresh on the day of experiments.

Data and statistical analysis
In vascular reactivity studies, vasorelaxant response of EOAz and fractions was expressed as percentage relaxation of phenylephrine contraction. Concentration-response curves were constructed using non-linear regression according to the following sigmoidal equation: where y is the response (relaxation or contraction), x is the logarithm of concentration, EC 50 is the concentration required to achieve a half-maximal response, a and b correspond to the minimum and maximum response values, respectively. Two pharmacological parameters were determined from the concentrationresponse curves: EC 50 and E max , the maximal effect of test substance. Data were expressed as mean and standard error of mean (SEM) of at least 8 experiments performed on preparations obtained from different animals. Comparisons between five groups, concerning the EC 50 and E max values, were carried out using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test to compare all pairs of groups.
For evaluating the antihypertensive effect of MFEOAz, variables were analyzed by Kolmogorov-Smirnov test for normal distribution of data. As such criteria were met in all analyses, mean and standard deviation (SD) were calculated for descriptive statistics, and parametric tests for analytical statistics. Comparisons between groups, concerning the temporal progression of SAP, DAP, MAP, HR, rate of SAP decline and AUC of SAP, were performed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Significance level was set at 0.05 (5%) so that values of P<0.05 were considered significant. GraphPad Prism® version 5.00 for Windows® was used to perform statistical procedures and graphs.

Effect of the EOAz and fractions on rat aortic rings pre-contracted with phenylephrine
Were obtained concentration-response curves of relaxation induced by different concentrations (0.01 to 3,000 mg/mL) of EOAz, fractions and control (Tween 80). Were used preparations of aortic rings with intact endothelium pre-contracted with phenylephrine (1 mmol/l). EOAz and fractions caused concentration-dependent relaxation, and at the highest concentration (3,000 mg/ml), the fractions caused full relaxation of aortic rings. Contractile response was more markedly induced by HFEOAz, primarily at doses of 10, 30 and 100 mg/mL. This effect also occurred to a lesser extent with EOAz, CFEOAz and MFEOAz ( Figure 1). E max values of EOAz and fractions were significantly higher (***P<0.001) than control (Figure  (Table 2).
In vivo experiments: effects of EOAz and fractions on arterial pressure SAP, DAP, MAP and HR were measured throughout the experiment. Figure 2 shows the induction phase of hypertension by chronic administration of L-NAME. Progressive increase in blood pressure was observed in the six groups. HR varied little in induction phase (Figure 2).
From the 31st day, which marked the start of treatments, control group maintained upward pressure values. In the captopril group, antihypertensive action has already started on day 3 of treatment (study day 33) and remained stable until the end of the study (day 60). However, in the HFEOAz and MFEOAz groups, antihypertensive effect was seen from day 6 (36 days of study) and day 9 (39 days of study) in the EOAz and CFEOAz groups. During treatment phase, antihypertensive effect of EOAz and fractions was lower than the captopril, except at the end of study, mostly on the 60th day, at which time the antihypertensive activity of both compounds was similar. In relation to the temporal behavior of HR in the treatment groups, similar temporal pattern was observed in the six groups characterized by specific alterations, demonstrating that there was little interference of treatment with HR (Figures 3, 4, 5, 6). FIGURE 1. Vasorelaxant response induced by EOAz, HFEOAz, CFEOAz and MFEOAz in rat aortic rings precontracted with phenylephrine (1 mmol/l). Vasodilator effect was evaluated on endothelium-intact preparations. Data expressed as mean ± SEM of 8 experiments performed on preparations obtained from different animals. EC 50 of MFEOAz was significantly lower than the EOAz ( +++ P<0.001), CFEOAz ( +++ P<0.001) and HFEOAz ( ++ P<0.01), and EC 50 of CFEOAz was significantly lower ( + P<0.05) than the FHOEAz. E max values of EOAz, HFEOAz, CFEOAz and MFEOAz were significantly higher (***P<0.001) than the control.       , mean (C) arterial pressure, and heart rate (D) in groups EOAz, HFEOAz, CFEOAz and MFEOAz. Hypertension was induced and sustained by chronic administration of L-NAME for 60 days. The first 30 days corresponded to hypertension induction phase, and the last 30 days to treatment phase. At each time point, data represent mean and standard deviation. Were no statistically significant differences between groups at any time point (ANOVA followed by Tukey's multiple comparison test).
Comparing the antihypertensive effects of EOAz and fractions in the treatment phase, there was similar temporal pattern, so that there was no statistically significant difference between them at any of the times evaluated. This fact was also observed in relation to HR (Figure 7).
Overall antihypertensive effect was evaluated in six treatment groups by determining of area under the curve of time versus SBP ( Figure  8). Reduction rate of PAS, in turn, reflects the speed at which PAS decreased over the treatment phase. Overall antihypertensive effect provided by EOAz and fractions was higher than the observed in negative control and less than the observed in group treated with captopril (A and B). EOAz, fractions and captopril induced a significant increase in rate of decay of SBP compared to the negative control (C). FIGURE 8. Global evaluation of antihypertensive effect verified in the groups control (n=9), captopril (n=11), EOAz (n=8), HFEOAz (n=9), CFEOAz (n=8) and MFEOAz (n=8). Area under the curve (A) of systolic arterial pressure (SAP) versus time denotes the overall antihypertensive effect of treatments. It was calculated according to the trapezoidal method (B). Reduction rate of SAP (C) denotes velocity of SAP decrease over in treatment phase. Data represent mean (A) and standard deviation (B, C). ***P<0.001, **P<0.01 compared to control group; +++ P<0.001, ++ P<0.01, + P<0.05 compared to captopril group (ANOVA followed by Tukey's multiple comparison test).

DISCUSSION
In this research, we did not use isolated components, since our main interest was to use the knowledge gained to further studies in humans using phytotherapeutics and not isolated substances. Evaluation of herbal purification to isolate or even identify chemical constituents is not necessary. Pharmacologically active ingredients can be analyzed by focusing on one or more ingredients purportedly responsible for the activity of plant, by studying a compound makes up a considerable part of total composition or by chromatographic profile of total ingredients (Brasil, 2008).
The investigation of naturally occurring agents acting on the cardiovascular system, especially those that reduce blood pressure is a growing area, with potential for the discovery of molecules that can be used to develop new antihypertensive drugs (Mahmood et al., 2010, Tirapelli et al., 2010. All major components of EOAz and fractions were monoterpenes. Monoterpenes are secondary metabolites of plants. They have various pharmacological properties including antifungal, antibacterial, antioxidant, anticancer, anti-spasmodic, hypotensive, and vasorelaxant (Santos et al., 2011b). Compounds tested must act by different mechanisms of action. In fact, monoterpene alcohols, aldehydes, ethers, and hydrocarbons show effects on the cardiovascular system via vasorelaxation, decreased heart rate or hypotension, among others (Lima et al., 2012).
In the initial in vitro evaluation of EOAz and fractions, it was found that these substances cause relaxation of rat aortic preparations with intact endothelium pre-contracted with phenylephrine in a dose-dependent manner, so that at the higher concentration (3,000 µg/ml) there was complete relaxation of aortic rings with the tested substances.
Existence of a biphasic effect, equivalent to a slight vasoconstriction, driven by HFEOAz at doses of 10, 30 and 100 µg/mL was observed. At high doses, there was no biphasic effect, but significant vasodilation in aortic preparations with intact endothelium pre-contracted with phenylephrine. No previous studies with EOAz have reported occurrence of biphasic effect. In relation to fractions, no studies have examined this aspect, and thus, this is the first study we know of to use hexane, chloroform and methanol fractions of EOAz. Biphasic effect has been identified in other studies with herbal products that evaluated vasodilator activity of natural substances extracted from plants. Regarding this effect, the authors mentioned that it was due to a transient increase in Ca 2+ influx from the extracellular medium (Chiwororo & Ojewole, 2008;Zhang et al., 2010).
Considering the findings in the literature and because MFEOAz had the lowest EC 50 and showed the terpinen-4-ol and 1,8-cineolas the main constituents, we infer that its vasodilator effect was probably due to the presence of these constituents, particularly for terpinen-4-ol, since CFEOAz had the second lowest EC 50 and had more 1,8-cineol than terpinen-4-ol.
In this study, we used tail plethysmography to determine indirect blood pressure, a method that measures SAP in rodents. The advantages of this method is that it can be easily used in many animals, allows for monitoring blood pressure for long periods of time, and is a validated and widely accepted method for experiments of this type (Irvine et al., 1997;Fritz & Rinaldi, 2008). The more precisely parameter analyzed is SAP, but DAP, MAP and HR were also determined. Other studies with substances from plants have also analyzed all parameters obtained by this method (Fritz & Rinaldi, 2008;Vergara-Galicia, 2008).
Animals were subjected to chronic hypertension by inhibiting nitric oxide synthesis, which was performed by administration of L-NAME. This model of hypertension is widely accepted and validated (Baylis et al., 1992;Fazan et al., 2001;Ribeiro & Muscará, 2001;Rang et al., 2012). Were found that the group treated with L-NAME had a statistically significant increase in PAS when compared to baseline time (day 0) and the control group, treated withdrinking water. There were no major changes in heart rate and body mass of the animals treated with L-NAME.
Results showed that a single administration by gavage of EOAz, HFEOAz, CFEOAz and MFEOAz as well as the positive control with captopril was able to significantly reduce blood pressure in rats subjected to chronic hypertension model by inhibition of nitric oxide when compared to the control treated with distilled water. Overall, the experiments performed here showed that hypotensive effect was also observed in other pre-clinical studies that evaluated the essential oil of A. zerumbet (Barcelos et al., 2010;Santos et al., 2011a). It has been found that hypotension in rats occurs independently of the operational presence of the sympathetic nervous system, suggesting that EOAz is a direct vasodilator (Lahlou et al., 2002a;Lahlou et al., 2002b).
In conclusion, the potency of the MFEOAz was higher than that of EOAz and other fractions. The antihypertensive effect of EOAz and fractions was similar, higher than the negative control and lower than that of captopril. We emphasize that more studies are needed to identify the mechanism of action of EOAz and its fractions.