Evaluation of Antihypertensive Activity of Aqueous and Ethylic Alcohol Extracts of Stem Bark of Xylopia villosa Chipp (Annonaceae)

This study aims to evaluate Antihypertensive activity of aqueous and ethanolic extracts of Xylopia villosa stem bark. The induction of hypertension was made with adrenaline on albino rats of wistar strain. Treatments of hypertensive rats with aqueous and ethanolic extracts of Xylopia villosa at doses of 100 and 200 mg/kg bw and Atenolol (the reference molecule) at doses of 10 and 20 mg/kg bw have normalized cardiovascular parameters such as systolic blood pressure, diastolic blood pressure and heart rate. However, these doses all increased the blood concentration of Na of hypertensive rats. Only treatments with aqueous and ethanolic extracts at a dose of 200mg/kg bw were able to bring blood concentration in K of hypertensive rats to a concentration equivalent to that of normotensive rats.


Introduction
Hypertension is a chronic cardiovascular disease characterized by a rise in blood pressure above normal. It is a serious pathology, called by epidemiologists silent killer [1]. This disease rapidly and considerably alters the quality of life of the patient. It also reduces life expectancy if no effective therapy is undertaken in time. The severity of arterial hypertension is characterized by damage to organs such as the heart, brain, kidney and eye. Hypertension is now seen in most African countries as a real public health problem. Once the disease is declared, the cost of care is prohibitive for developing countries as it can represent up to 60% of all health care expenditures. With high costs of imported medicines, 80% of the population who use traditional medicine to meet their primary health care needs [2] find an emergency outlet. The work of Ake-Assi [3] identified 1421 species of medicinal plants in Côte d'Ivoire for the treatment of various pathologies including arterial hypertension.
Xylopia villosa Chipp, a species of this flora is used in traditional medicine in Africa to treat various pathologies including colds and headaches. Its crushed seeds are applied to leg ulcers and boils for healing [4]. Recently, Kouamé and al [5][6][7] carried out the study of triphytochemistry and acute toxicity, the study of the anti-inflammatory and antioxidant activities of Xylopia villosa Chipp. However, the antihypertensive activity of Xylopia villosa Chipp is not yet known. The objective of this study was to evaluate Xylopia villosa Chipp by evaluating its antihypertensive activity. To achieve this, the specific objectives were to induce hypertension with adrenaline and to determine the effect of extracts of Xylopia villosa Chipp On cardiovascular parameters and electrolyte balance by the determination of sodium and potassium.

Plant Material
Xylopia villosa stems bark were harvested in June 2014 at the National Floristic Center of Felix Houphouët Boigny University where can be found a sample recorded at the number 14712.

Preparation of Extracts
The stems bark of Xylopia villosa were dried for four weeks in the shade of the sun then made powder using an electric grinder IKAMAG RCT ® . Hundred (100) grams of powder of Xylopia villosa were macerated for 24 hours in 1

Determination of Trace Elements in Aqueous and Ethanolic Extracts of Xylopia villosa
The determination of the trace elements in aqueous and ethylic alcohol extracts of Xylopia villosa was carried out according to the method of Clément and Françoise [8].
The aqueous and ethylic alcohol extracts of Xylopia villosa were dried for 24 hours in a Memmert-Germany oven at 60°C. Then, they were kept in glass jars.

Mineralization by Calcination
For each of the extracts (aqueous and ethylic alcohol) dried in an oven at 60°C. 0.4 g was weighed using a Sartorius analytic (England) scale in a 30 ml porcelain crucible. This test portion was placed in the Naberthem-Germany muffle furnace set at 550°C for 5 hours. After cooling, 2 ml of 0.5 N chloridic acid were added to the ash obtained and then brought to total evaporation on a sand bath. The recovered final residue was filtered through a 100 mL volumetric flask and distilled water was added to reach the gauge mark. Five (5) mL of the filtrate were taken for the determination of the minerals Five (5) mL of the same filtrate was also removed for the determination of the minerals (Potassium, Iron, Zinc, Magnesium, Copper) by the atomic absorption spectrophotometer AAS 20 type VARIAN, Australia).

Preparation of the Lanthanum Reagent (La 2 O 3 )
Under a hood, a mass of 58.65 g of La 2 O 3 was wetted with 50 mL of distilled water and 250 mL of concentrated hydrochloric acid was added thereto with slow stirring until the lanthanum was completely diluted. Before any reading, the atomic absorption spectrophotometer was calibrated. To do this, a standard solution of 100 ppm was prepared from a commercial solution called a multi-element of 1000 ppm. The preparation was carried out as follows: 2.5 mL of the stock solution (1000 ppm) was added to a 25 mL flask and supplemented with concentrated nitric acid to the gauge mark and this solution was used to prepare the standard ranges.

Preparation of Samples and Standards
Five (5) mL of each sample was taken from a 50 mL vial and 2 mL of 5% Lanthanum was added to it before completing with distilled water to the mark. For standards, dilutions from standard solutions of each mineral (100 mg/L) are performed by supplementing the initial volumes to 50 mL with distilled water, so as to obtain an accurate concentration range for each mineral. These calibration solutions are then used for the calibration of the flame atomic absorption spectrophotometer. To determine the different quantities of stock solution (mL) to be taken, the following procedure was adopted: With: C f : final concentration of the mineral solution (100 ppm) C i : initial concentration of the mineral solution (1 mg/mL) V i : initial volume taken (variable) (in mL) V f : final volume (mL) The wavelengths at which potassium, iron, zinc, magnesium and copper were read were 766.5 nm; 248.3 nm; 258 nm; 285.2 nm and 324.7 nm respectively. The results of the optical densities of each mineral made it possible to determine the quantities of minerals (ppm) contained in the aqueous and ethylic alcohol extracts. The mineral contents were determined as follows: T = [(C ess -C wh ) x V] / P ess With: C ess : concentration of the sample (mg/mL) C wh : white concentration in mg/mL P ess : test portion (Kg) V: recovery volume of test (mL) T: content of μg/g or mg/Kg

Reagents, Solvents and Drugs for Antidiabetic Activity
All products used in this study were provided by Sigma Aldrich Ltd (Paris, France).

Experimental Animals
In this experiment, 60 albino wistar strain rats aged fourteen (14) weeks, mean weight 187.03 ± 0.06 g were used. First, a control group of 6 non-hypertensive rats was formed. Secondly, 54 non-hypertensive rats received adrenaline intraperitoneally at a dose of 1 mL/kg body weight (bw) during 7 days and the evolution of systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate was followed during 7 days (day D 0 to D 7 ). After the 7th day, 42 hypertensive rats were selected and divided into 7 groups of 6 rats. Subsequently, a treatment by gavage took place during 6 days with the different drugs according to the following distribution:  Non-hypertensive control group → 1 mL of distilled water  Untreated hypertensive group → 1 mL of distilled water Before each recording, the caudal vein is dilated by passing the rats in a heating stem for 5 minutes. The rats are then placed in the containment cells and the pressure sensor inserted on the tail. The detected arterial pressure signal emits pressure vibrations that are displayed on the computer monitor as well as the corresponding pressure and heart rate values.

Blood Determination of Sodium and Potassium in Hypertensive Rats
Blood was taken from the retro orbital sinus in tubes containing sodium fluoride for the determination of electrolytes (sodium and potassium). Determination of sodium and potassium was carried out using a Hospitex Screen flame spectrophotometer (France).

Statistical Analysis
The values expressed as Mean ± SEM from 6 animals. The statistical analysis was carried out using one way analysis of variance (ANOVA) followed by Dunnett's test, P < 0.05 was considered as significant.

Trace Element Content
The trace element content (Potassium, Iron, Zinc, Magnesium, Copper) in aqueous and ethylic alcohol extracts of Xylopia villosa are summarized in Table 1.

Evolution of Systolic Blood Pressure and Diastolic Blood Pressure
The systolic blood pressure (SBP) and diastolic blood pressure (DBP) of normotensive rats and hypertensive rats are shown in Fig.1. The SBP of all rats prior to adrenaline injection was 121, 00 ± 1.42 mm Hg on day D0. After adrenaline injection into the test group, SBP was increased to 124.90 ± 2.79; 133.30 ± 3.03; 155.00 ± 2.17 and 176.30 ± 1.60 mmHg on days D2, D4, D6 and D7 respectively. In the case of DBP, before adrenaline injection, it was 88.53 ± 2.87 mm Hg on day D0 in all rats. After injection of adrenaline into the test group, the DBP was increased to 112.20 ± 3.00; 117.30 ± 0.75; 126.00 ± 1.97 and 155.90 ± 1.68 mmHg on days D2, D4, D6 and D7 respectively.

Discussion
Concerning the antihypertensive activity of aqueous and ethanolic extracts of the stem bark of Xylopia villosa, it was investigated on systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) after induction of arterial hypertension by adrenaline. The results of the study showed that extracts of Xylopia villosa resulted in a significant reduction in adrenaline-induced hypertension by normalizing the cardiovascular parameters (SBP, DBP and HR) of hypertensive rats. This suggests that the aqueous and ethanolic extracts of the stem bark of Xylopia villosa contain antihypertensive compounds. These results are in agreement with those of N'Go [10], Tiékpa et al [11] which showed that the treatment of hypertensive rats with the aqueous extract of Terminalia superba Englers & Diels (Combretaceae) and Wakouba Salt extracted from Elaeis guineensis normalized the cardiovascular parameters of said rats. The antihypertensive effects of Xylopia villosa extracts also resemble those of the extracts of Stephania tetranda (Menispermaceae) Yu et al [12] and Berberis vulgaris (Berberidaceae) Fatehi-Hassanabab et al [13].
The determination of the trace elements revealed the presence of magnesium and potassium in the aqueous and ethanolic extracts of Xylopia villosa. This decrease in blood pressure after administration of extracts of Xylopia villosa could be explained by the presence of these two trace elements. Indeed, studies by Kass et al [14] showed that magnesium supplementation in hypertensive patients led to a modest decrease in blood pressure. Thus, Whelton et al [15], Tunstall-Pedoe [16] and Geleijnse [17] showed that a potassium-rich diet lowered systolic and diastolic pressures and led to decreased cardiovascular events and even mortality. This hypotensive effect could militate for the use of Xylopia villosa in the treatment of hypertension. Concerning the Na + and K + electrolytes of hypertensive rats, there was a significant increase in Na + concentration in untreated hypertensive rats and those treated with extracts of Xylopia villosa and Atenolol. Nevertheless, the concentration of K + ion has had three phases. A first phase in which it increased in untreated hypertensive rats and those treated with Atenolol (10 mg/kg bw) and aqueous and ethanolic extracts at a dose of 100 mg/kg bw. A second phase in which Atenolol (20 mg/kg bw) resulted in a decrease in K + ion concentration compared to normotensive control rats. Finally, a third phase in which aqueous and ethanolic extracts at a dose of 200 mg/kg bw reduced the concentration of K + ion similar to that of normotensive control rats. Indeed, the ability of the kidneys to excrete or conserve sodium is a key factor in the regulation of blood pressure. Most studies show that reduced salt intake reduces blood pressure. On the other hand, the increase in potassium intake makes it possible to lower the blood pressure, a phenomenon which could be explained by the potassium's ability to increase the excretion of sodium and by its vasoactive effects on the blood vessels [18]. The Na + and K + ion concentrations obtained are consistent with the distribution of sodium and potassium in the body. Indeed, the distribution of sodium and potassium in the body is one of the conditions of cell life, especially for nerve and muscle function. There is about 15 times more sodium in the blood than in the cell and there are about 28 times more potassium in the cell than in the blood [19]. To bring nutrients to different cells, a mechanism, using sodium, among others, allows cells to let in what they need to function. Once in the cell, sodium has done its job and must come out because if the cell remained swollen in sodium and water, it could no longer function. A mechanism then allows the sodium to leave the cell, but it requires the presence of potassium to replace it thanks to an ingenious system of "pumps". Sodium and potassium form a necessary tandem for the correct regulation of blood pressure [19].

Conclusions
The results of the studies showed that aqueous and ethanolic extract of Xylopia villosa resulted in a significant reduction in adrenaline-induce hypertension by normalizing the cardiovascular parameters (SBP, DBP and HR) of hypertensive rats. This suggests that aqueous and ethanolic extracts of stem bark of Xylopia villosa contain antihypertensive compounds. Treatment of hypertensive rats with different drugs resulted in an increase in the concentration of Na + ions compared to normotensive rats. However, the aqueous and ethanolic extracts of Xylopia villosa at a dose of 200 mg/kg bw resulted in a concentration of K + ion similar to normotensive rats.

Ethical Approval
The experimental procedures and protocols used in this study were approved by the Ethical Committee of Health Sciences, University Félix Houphouet-Boigny. These guidelines were in accordance with the European Council Legislation 87/607/EEC for the protection of experimental animals. All efforts were made to minimize animal suffering and reduce the number of animals used.