Preventive Potential of the Aqueous Extract of the Mixture of Bidens pilosa (Asteraceae) and Cymbopogon citratus (Poaceae) Aerial Parts on Hypertension Induced by a Chronic Salt and Alcohol Consumption on the Rats

High blood pressure (HBP) is currently one of the main risk factors for cardiovascular and kidney diseases. Nowadays, populations make extensive use of alternative medicine for their health problems. Bidens pilosa (B. pilosa) and Cymbopogon citratus (C. citratus) are used individually in the traditional treatment of cardiovascular disorders. This study assessed the effects of the mixture of these two plants aqueous extract on HBP in rats. Male rats (42) were divided into 7 groups of 6 rats each. Normotensive rats received only distilled water and formed group 1. The other animals received ethanol + salt preceded by distilled water (10 mL/kg; group 2) and spironolactone (10 mg/kg; group 3); the aqueous extracts of the mixture (100 and 200 mg/kg; groups 4 and 5) isolated plants B. pilosa (200 mg/kg; group 6) and C. citratus (200 mg/kg; group 7). Animals were treated for 7 weeks during which water consumption and urine volume were assessed; then, hemodynamic parameters were recorded, and rats were sacrificed. Serum and some organs (liver, kidney, heart, and aorta) were used to evaluate biochemical parameters. Ingestion of ethanol + salt leads to a significant increase in urinary volume and water intake that were significantly prevented by the extracts from the mixture and isolated plants. Ethanol + salt solution significantly increased the blood pressure, heart rate, triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-chol), very-low-density lipoprotein cholesterol (VLDL-chol), atherogenic indices, liver and kidney function parameters, and malondialdehyde (MDA) levels. However, the levels of high-density lipoprotein cholesterol (HDL-chol), albumin, reduced glutathione (GSH), catalase, and superoxide dismutase (SOD) activity were significantly reduced. The extracts of the mixture and isolated plants significantly prevented all these variations with a more pronounced action for the lowest dose of the mixture on the lipid profile, oxidative stress, and kidney function. These observations confirm the beneficial effects of B. pilosa and C. citratus to manage hypertension.


Introduction
Arterial hypertension (AHT) is a very common condition today, but for a long time, it was considered a rare and even nonexistent condition in Africa. Today, hypertension is a real public health problem. It refers to a permanent rise in blood pressure in the arteries, which is based on two points: the rise in blood pressure and its persistence [1].
According to the WHO, 1.28 billion people worldwide between the ages of 30 and 79 have hypertension, and most of them (two-thirds) live in low and middle-income countries [2]. e prevalence of hypertension varies across continents and country revenues. High-income countries have the lowest prevalence of hypertension compared to lower-income countries (40% versus 35% on average) [3]. Also, due to weak health systems in these countries, the number of undiagnosed and/or untreated people with hypertension is very high (46%) [2]. In the African continent, the prevalence of hypertension is the highest (27%) compared to the Americas where it is lower (18%) [2]. e prevalence of hypertension in Cameroon was estimated at 29.70% in 2015 [4]. According to a WHO analysis of ten countries on the African continent, cardiovascular disease, diabetes, and asthma are the comorbidities most associated with patients with COVID-19. South Africa, which recorded almost half of the cases and deaths on the continent, found that more than 60% of COVID-19 patients in hospitals had HBP and at least 50% had diabetes [5]. e increase in the prevalence of hypertension is attributable to population growth, ageing populations, and behavioural risk factors such as poor diet, alcohol and salt abuse, lack of physical activity, overweight, and constant exposure to stress [3]. Today, alcoholic beverages are consumed regularly by most societies in the world. However, its abuse is a public health problem [6]. Several studies have shown that chronic heavy alcohol consumption most often causes liver, gastrointestinal, nerve, and cardiovascular damages leading to physiological dysfunction [7,8]. It has been clearly established that chronic ethanol consumption leads to increased blood pressure and thus hypertension. Global salt consumption is far too high in both developed and developing countries [9]. Many epidemiological studies have established a causal relationship between high dietary sodium consumption and increased blood pressure and thus the risk of hypertension, which in the long term can lead to cardiovascular, liver, and kidney disorders [9]. Chronic consumption of both (alcohol and salt) could have adverse effects on the cardiovascular system, liver, and kidneys.
HBP is a chronic disease that requires lifelong treatment with dietary management and regular use of medications such as ACE inhibitors, calcium channel blockers, and diuretics. ese drugs have several limitations, namely, side effects, high cost, and inaccessibility. Despite a large number of modern medicines, people around the world are increasingly turning to herbal medicines for their primary health problems.
African flora in general and Cameroonian flora in particular abounds in diverse varieties of plant species. ese plants are most often used by populations for the treatment of several ailments. ey are used to treat sexual and fertility disorders, diabetes, obesity, and cardiovascular disorders [10]. Nowadays, the identification of new active ingredients and the discovery of new pharmacological properties, with minimal adverse effects, have contributed to promoting phytotherapy [11]. In view of the important place that these plants occupy in pharmacopoeia, the WHO has recommended that investigations be conducted on the effects of plants used in traditional medicine [12].
Despite the extensive use of herbs in traditional medicine, failures in monotherapy have most often been encountered in the treatment of various diseases. ese resistances encountered with monotherapy are leading healthcare personnel to advocate more and more for polytherapy. is renewed interest in the use of several molecules in combination with the treatment of diseases is related to both efficacy and the reduction of doses and possible side effects. erefore, since several plants have already proven their efficacy and limitations in monotherapy, there is an urgent need to develop new drugs combining several plants for the treatment of diseases such as hypertension.
B. pilosa is a perennial plant belonging to the Asteraceae family, globally distributed in tropical regions. Scientific studies, although not exhaustive, have shown that B. pilosa extracts are antitumor, anti-inflammatory, antioxidant, diuretic, antihypertensive, vasorelaxant, and antiulcerative [13][14][15][16]. In traditional medicine, the decoction of the whole plant is used against flu and fevers, while the leaves of the plant are used to treat ear infections, psoriasis, hepatitis, and hemorrhoids [15]. e plant is also considered to be antipoisonous [15]. C. citratus commonly known as lemongrass is an herb belonging to the Poaceae family. Scientific studies have shown that C. citratus has antispasmodic, antipyretic, sedative, diuretic, vasorelaxant, and hypotensive activities [17][18][19][20]. In traditional medicine, C. citratus is used for its many therapeutic properties. e decoction of the leaves is used to treat gastrointestinal pain, herpes, fever, headaches, and heart problems [19]. e two plants are diuretic, vasorelaxant, and antihypertensive, and so far, no scientific study has been conducted on the effects of the mixture. erefore, we propose to evaluate the preventive effects of the aqueous extract of the mixture of these two plants on a rat model of hypertension, using alcohol and salt.

Animal Material.
Male Wistar rats aged 6-8 weeks and weighing 150-160 g were randomly selected from the colony of Animal Biology and Physiology Animal House of Yaoundé I University. ey were reared in Plexiglas cages. Every effort was made to minimise animal suffering and to reduce the number of animals used. e rats were housed 3 per cage and exposed to a daily 12-hour light/dark cycle. ey were maintained at room temperature (25 ± 3°C) with free access to a standard diet (composed of 40% maize, 20% wheat bran, 24% fish meal, 4% palm kernel meal, 8% peanut meal, 2% cottonseed meal, 2% bone meal, and a 10 g vitamin complex) and tap water. All procedures and protocols involving animals and their care were conducted in accordance with institutional guidelines and approved by the National Ethics Committee of Cameroon (Reg. No. FWA-IRB00001954).

Plants Extraction.
e collected aerial parts of C. citratus and B. pilosa were washed, shade-dried, and crushed to obtain the powder of each plant. 200 g of each plant was separately introduced into 2 L of boiling distilled water. e infusion was done until it cooled down. e solutions obtained were filtered with Whatman No. 3 paper, and the filtrates were oven-dried at 45°C to give 10.04 g and 15.96 g of aqueous extract of B. pilosa and C. citratus, respectively, for a yield of 5.02% and 7.98%. e doses of the mixture of the two plants were chosen on the basis of screening for direct hypotensive effects.
is screening was carried out using combinations of proportions 25-75, 50-50, and 75-25 of the mixture of aqueous extracts of B. pilosa and C. citratus. e best combination (50-50) of the mixture was chosen and represented the dose of 100 mg/kg. is dose was multiplied by two to obtain the dose of 200 mg/kg. Knowing the best combination, the doses of 125 g for B. pilosa and 200 g for C. citratus were obtained by setting the mass of extract obtained after extraction to 50 g for each plant and also by using the extraction yields of each separate plant. us, the amount of crude plant of each plant per 50 g of extract was determined by calculation. 125 g of B. pilosa and 200 g of C. citratus were introduced into 3.25 L of boiling distilled water. is gave 33 g of aqueous extract of the mixture of B. pilosa and C. citratus, a yield of 10.15%.

Screening for Diuretic
Activity. All extracts were tested for diuretic activity using the method described by Kau et al. [21]. After checking the urinary excretion, the animals with the best urine volume were selected and divided into 6 groups of 6 animals each. e first group (TN) was treated with distilled water (10 mL/kg) and the second and third (BC 100 and BC 200) with the aqueous extract of the mixture of B. pilosa and C. citratus at doses of 100 and 200 mg/kg, respectively. e fourth and fifth groups (B 100 and C 200) were treated with aqueous extracts of B. pilosa (200 mg/kg) and C. citratus (200 mg/kg), respectively, while the sixth group (FURO 10) was treated with furosemide (10 mg/kg). e animals of the different groups were placed in standard metabolic cages and food and water were removed 12 hours before the experiment. All extracts were dissolved in distilled water to the required concentrations and administered orally by gavage. Cumulative urine excretion was measured at 3, 6, 9, 12, and 24 hours in all groups. e ratio of urine excretion in the test group to urine excretion in the control group is used as a value of the diuretic action for a given dose of a drug. As diuretic action is subjected to variability, diuretic activity was calculated [21]. To obtain the diuretic activity, the diuretic action of the test substance was compared to that of a standard drug in the test group. e diuretic activity was considered as "no," "low," "moderate," and "good" if the values were, respectively, <0.72, 0.72-1.00, 1.00-1.5, and > 1.5 [21].
Urinary excretion (UE) � total urine output ×100/total fluid administered; diuretic action � urinary excretion of the test group/urinary excretion of the control group; diuretic activity � diuretic action of the test group/diuretic action of a standard drug.

Animal Grouping, Treatment, and Hypertension
Induction.
e preventive effects of the mixture of B. pilosa and C. citratus were evaluated in normotensive male Wistar rats. e plants extracts and the hypertension inducer made up of 40°alcohol (5 g/kg) + salt (2.46 g/kg) for 49 days (7 weeks) were given concomitantly to the animals starting with the extracts. Forty-two (42) male rats were divided into seven groups (1-7) of 6 animals each. ese seven groups were treated daily by gavage for 49 days as follows: groups 1 and 2 were receiving distilled water (10 mL/kg), group 3 spironolactone (10 mg/kg), groups 4 and 5 the aqueous extract of the mixture of B. pilosa and C. citratus at doses of 100 and 200 mg/kg, respectively, and groups 6 and 7 the aqueous extracts of B. pilosa (200 mg/kg) and C. citratus (200 mg/kg), respectively. irty minutes after, the inducer (alcohol + salt solution) was administrated to rats in groups 2-7 at a rate of 1.66 mL/100 g bodyweight; while, group 1 was considered as the normotensive control group. Water intake and urinary excretion volume were assessed at the beginning and the end of the experiment using metabolic cages.

Drugs and Chemicals.
All the drugs and chemicals used in this study were purchased from Sigma Chemical Company (St. Louis, MO, USA).

Hemodynamic Parameters Recording.
At the end of the respective treatments, arterial blood pressure and heart rate of all rats were measured as previously described [22]. Briefly, the rat was anaesthetised by an intraperitoneal injection of urethane (1.5 g/kg). e trachea was exposed and connected to a pipe to allow breathing. e arterial blood pressure was measured from a carotid artery through an arterial pipe connected to a pressure transducer coupled with a hemodynamic recorder Biopac Student Lab. (MP35) and computer.

Blood and Organs
Collection. Immediately after hemodynamic parameters measurement, blood samples were collected from the abdominal artery and centrifuged at 3000 rpm for 15 min. e plasma obtained was kept at −20°C for biochemical analysis. ereafter, the heart, the kidney, Evidence-Based Complementary and Alternative Medicine 3 the liver, and the thoracic aorta were dissected out, washed in saline, and weighed; parts of all these organs were kept for oxidative stress markers. For histological analysis, other parts of the kidney, the liver, and the aorta were fixed in 10% formalin.

Histological Analysis.
For microscopic evaluation, the investigated organs were dehydrated and paraffin-embedded for microscopic examination in accordance with routine laboratory procedures. Paraffin sections of 4 µm were prepared and stained with haematoxylin and eosin (H&E) for histological examination. Morphometric measurements of the thickness of arteries were performed using Image J 1.3.

Statistical Analysis.
Results were expressed as the mean ± SEM. e difference between the groups was compared using one-way analysis of variance (ANOVA) followed by Turkey's post hoc test using GraphPad Prism software version 8.01. A value of p < 0.05 was considered statistically significant.  Figure 1. 24 hours after administration of the different substances, the aqueous extract of the mixture resulted in a significant (p < 0.05) increase in the urine volume at the dose of 200 mg/kg compared to rats given distilled water. C. citratus aqueous extract (200 mg/kg) caused a significant (p < 0.05) increase in urinary volume excreted at 6 and 24 hours compared to rats given distilled water.

Effects on Diuretic
Activity. Details of the urinary excretion rate, diuretic action, and diuretic activity are given in Table 3. e results showed that furosemide and extracts increased urine output at 24 h compared to control rats. Like furosemide and separate extracts, the aqueous extract of the mixture of B. pilosa and C. citratus showed diuretic activity at all doses. e diuretic activity of a drug is considered to be nil if it is less than 0.72, low if it is between 0.72 and 1.00, moderate if it is between 1.00 and 1.50, and good if it is greater than 1.50. With respect to this, the aqueous extract of the mixture of B. pilosa and C. citratus shows a good diuretic activity much more at the dose of 200 mg/kg.

Effects on Water Intake and Urinary Excretion Volume.
Administration of distilled water (10 mL/kg) concomitantly with the inducer (ethanol + salt solution, hypertensive rats) induced a significant (p < 0.001) increase in water intake and urinary excretion in rats compared to rats given distilled water alone (normotensive rats, Figure 2). e increase in is increase induced by alcohol + salt solution was also significantly (p < 0.01) prevented in animals treated concomitantly with C. citratus (200 mg/kg) and the inducer by 18.47%. At the end of the experiment, a significant (p < 0.001) decrease in the volume of urinary excretion was observed in rats treated concomitantly with inducer and B. pilosa aqueous extract (200 mg/kg) by 35.71% as compared to hypertensive rats.

Effects of the Aqueous Extract of the Mixture of B. pilosa and C. citratus on Blood Pressure and Heart Rate.
Administration of the inducer (ethanol + salt solution) and distilled water (10 mL/kg) to normotensive rats for 7 weeks resulted in a significant (p < 0.001) increase in systolic (SBP), diastolic (DBP), and mean (MBP) blood pressure and heart rate ( Figure 3). is increase was 34.09%, 51.37%, 37.42%, and 15.41%, respectively, compared to normotensive rats. e increase in SBP, DBP, MBP, and heart rate observed in the group treated with distilled water and the inducer (hypertensive rats) was significantly (p < 0.001) prevented in the group treated concomitantly with aqueous extract of B. pilosa and C. citratus mixture (100 mg/kg) and the inducer by 30.51%, 30.16%, 32.30%, and 14.12%, respectively. Treatment concomitantly with the aqueous extract of B. pilosa and C. citratus mixture (200 mg/kg) and the inducer significantly prevented the increase in SBP, DBP, MBP, and heart rate. e values decreased by 28.89% (p < 0.001), 33.13% (p < 0.001), 32.11% (p < 0.001), and 11.25% (p < 0.01), respectively, compared to hypertensive rats. In animals treated concomitantly with spironolactone and the inducer, there was a significant decrease in SBP, DBP, MBP, and heart rate of 23.67% (p < 0.001), 29.06% (p < 0.01), 25.45% (p < 0.001), and 8.59% (p < 0.05), respectively, as compared to hypertensive rats. In animals treated concomitantly with aqueous extract of B. pilosa (200 mg/kg) or C. citratus (200 mg/kg) and the inducer, there was a significant decrease in SBP (p < 0.001), DBP (p < 0.01; p < 0.005, respectively), MBP (p < 0.001; p < 0.005, respectively), and heart rate (p < 0.001; p < 0.05, respectively) compared to hypertensive rats.    (Table 4) as compared to normal rats receiving only distilled water (normotensive rats). e increase in TG and VLDL-chol induced by ethanol + salt solution was significantly (p < 0.001) prevented by the aqueous extract of the mixture of B. pilosa and C. citratus (100 mg/kg) by 62.31% and 62.33%, respectively, as compared with hypertensive rats. e mixture's aqueous extract (200 mg/kg) significantly (p < 0.01) prevented the increase induced by the alcohol + salt solution in TG and VLDL-chol by 33.48% and 33.53%, respectively, as compared to hypertensive rats. e mixture's aqueous extract at doses of 100 mg/kg (p < 0.001) and 200 mg/kg (p < 0.05) significantly prevented the increase in TC induced by the alcohol + salt solution as compared with the hypertensive rats. e aqueous extract of the mixture of B. pilosa and C. citratus (200 mg/kg) significantly (p < 0.01) prevented the decrease induced by the alcohol + salt solution in HDL-chol by 33.21% as compared with the hypertensive rats. In animals given alcohol + salt and spironolactone (10 mg/kg), there was a significant (p < 0.01) prevention of the increase induced by the alcohol + salt solution in TC levels by 12.34%, in TG (p < 0.001) by 53.38%, in LDL-chol (p < 0.05) by 41.42%, and in VLDL-chol (p < 0.001) by 62.33% as compared to hypertensive rats. B. pilosa aqueous extract (200 mg/kg) significantly prevented the increase induced by the alcohol + salt solution in TG levels (p < 0.001) by 60.79% and in VLDL-chol (p < 0.001) by 60.76% as compared to hypertensive rats. e aqueous extract of C. citratus (200 mg/ kg) significantly prevented the increase induced by the alcohol + salt solution in TG (p < 0.001) by 53.81%, in LDLchol (p < 0.01) by 55.67%, and in VLDL-chol (p < 0.001) by 53.81% and the decrease in HDL-chol by 50.43% (p < 0.001) as compared with hypertensive rats.

Effects of the Aqueous Extract of the Mixture of B. pilosa and C. citratus on Atherogenic Indices.
e effects of the aqueous extract of the mixture of B. pilosa and C. citratus on the levels of the atherogenic indices are given in Table 5. Administration of the inducer (alcohol + salt solution) and distilled water (10 mL/kg) resulted in a significant (p < 0.001) increase in CRR, AC, and AIP compared to normal rats given distilled water alone (normotensive rats). e aqueous extract of the mixture of B. pilosa and C. citratus (100 mg/kg) significantly prevented the increase induced by the alcohol + salt solution in CRR (p < 0.001), AC (p < 0.01), and AIP (p < 0.001) as compared with hypertensive rats. e  aqueous extract of the plant mixture (200 mg/kg) significantly (p < 0.001) prevented the increase induced by the alcohol + salt solution in CRR, AC, and AIP as compared with hypertensive rats. Treatment concomitantly with alcohol + salt and C. citratus aqueous extract (200 mg/kg) significantly (p < 0.001) prevented increases induced by the alcohol + salt solution in CRR, AC, and AIP as compared with hypertensive rats. e aqueous extract of B. pilosa aqueous extract (200 mg/kg) significantly (p < 0.001) prevented the increase induced by the alcohol + salt solution in AIP as compared with hypertensive rats. Treatment concomitantly with alcohol + salt and spironolactone (10 mg/kg) significantly prevented the increase of CRR (p < 0.001), AC (p < 0.05), and AIP (p < 0.001) as compared with hypertensive rats.

Effects of the Aqueous
Heart rate (Beats/min) Figure 3: Effects of the aqueous extract of the mixture of B. pilosa and C. citratus on blood pressure (a) and heart rate (b). Each bar represents the mean ± SEM (n � 6). SBP, systolic blood pressure; DBP, diastolic blood pressure; MBP, mean blood pressure; DW, normal rats receiving only distilled water (10 mL/kg); DW + Al-salt, rats receiving distilled water (10 mL/kg) and the inducer (alcohol + salt solution) concomitantly; BC 100+Al-salt and BC 200+Al-salt, rats receiving the aqueous extract of B. pilosa and C. citratus mixture at 100 and 200 mg/ kg, respectively, and the inducer concomitantly; SPIRO + Al-salt, rats receiving spironolactone (10 mg/kg) and the inducer simultaneously; B 200+Al-salt, rats receiving B. pilosa aqueous extract (200 mg/kg) and the inducer simultaneously; C 200+Al-salt, rats receiving C. citratus extract (200 mg/kg) and the inducer; 3 p < 0.001, significant difference compared to normotensive rats receiving only distilled water; a p < 0.05; b p < 0.05; c p < 0.001, significant difference compared to rats given distilled water and the inducer.

Urinary Level Effects.
At the urinary level, the alcohol + salt solution resulted in a significant (p < 0.001) decrease in creatinine, urea, and uric acid levels of 50.88%, 79.67%, and 55.72%, respectively, as well as a significant increase (p < 0.001) in magnesium, sodium, and potassium levels of 158.15%, 33.07%, and 181.78%, respectively, as compared to normotensive rats (Table 8).
e aqueous extract of the mixture of the plants (100 and 200 mg/kg) significantly prevented the decrease induced by inducer in creatinine (p < 0.001; p < 0.01, respectively) and urea (p < 0.001; p < 0.05, respectively) levels as well as the increase (p < 0.001) of sodium and potassium levels as compared with hypertensive rats. Treatment concomitantly with alcohol-+ salt solution and B. pilosa aqueous extract (200 mg/kg) significantly (p < 0.01) prevented the decrease induced by inducer in urea levels and the increase (p < 0.001) in sodium and potassium levels compared with hypertensive rats. e aqueous extract of C. citratus (200 mg/kg) significantly (p < 0.05) prevented the decrease induced by inducer in urea and uric acid levels and the increase (p < 0.001) in sodium and potassium levels as compared with hypertensive rats. Spironolactone significantly (p < 0.001) prevented the decrease in uric acid levels and the increase (p< 0.001) in sodium and potassium levels as compared with hypertensive rats.

Effects of the Aqueous Extract of the Mixture of B. pilosa and C. citratus on Some Oxidative Stress Parameters.
Administration of the inducer (ethanol + salt solution) and distilled water (10 mL/kg) to normotensive rats resulted in a significant (p < 0.001) decrease in the concentration of GSH in the aorta by 66.06%, the heart by 40.84%, the kidney by 30.62%, and the liver by 59.62% as compared to normotensive rats (Figure 4(a)). e aqueous extract of the mixture of B. pilosa and C. citratus (100 mg/kg) prevented this decrease in GSH significantly (p < 0.001) in the heart, kidney, and liver. e values were 55.90%, 92.08%, and 142.16%, respectively, higher in these organs as compared to Each value represents the mean ± SEM (n � 6). DW, normal rats receiving only distilled water (10 mL/kg); DW + Al-salt, rats receiving distilled water (10 mL/ kg) and the inducer (alcohol + salt solution) simultaneously; BC 100 + Al-salt and BC 200 + Al-salt, rats receiving the aqueous extract of the mixture of B. pilosa and C. citratus at doses of 100 and 200 mg/kg, respectively, and the inducer; SPIRO + Al-salt, rats receiving spironolactone (10 mg/kg) and the inducer simultaneously; B 200 + Al-salt, rats receiving B. pilosa aqueous extract (200 mg/kg) and the inducer; C 200 + Al-salt, rats receiving the aqueous extract of C. citratus (200 mg/kg) and the inducer; 1 p < 0.05; 2 p < 0.01; 3 p < 0.001, significant difference compared to normal rats receiving only distilled water; a p < 0.05; b p < 0.01; c p < 0.001, significant difference compared to rats receiving distilled water and the inducer. Catalase activity in the investigated organs of rats concomitantly administered distilled water (10 mg/kg) and the inducer (ethanol + salt solution) was significantly (p ˂ 0.001) decreased in the aorta by 24.14%, the heart by 42.58%, the kidney by 31.94%, and the liver by 46.49%, compared to normal rats administered distilled water alone (normotensive rats, Figure 4(b)). is decrease was significantly prevented in the liver by the aqueous extracts of the mixture of B. pilosa and C. citratus (100 mg/kg; p ˂0.05), B. pilosa (200 mg/kg; p ˂ 0.001), or C. citratus (200 mg/kg; p ˂ 0.001). e values were 47.66%, 76.99%, and 77.67%, respectively, higher as compared to hypertensive rats. e different aqueous extracts and spironolactone significantly (p ˂ 0.001) prevented the decrease in catalase activity at the cardiac level as compared to hypertensive rats. At the aortic level, there was a significant (p ˂ 0.05) prevention of the decrease in catalase activity induced by alcohol + salt solution in rats, with B. pilosa extract (200 mg/kg) by 13.23% as compared to hypertensive rats. Figure 4(c) shows the effects of the aqueous extract of the mixture of B. pilosa and C. citratus on MDA levels. It can be seen that the concentration of MDA was significantly increased (p < 0.001) in the heart by 263.41%, in the aorta by 241.71%, in the liver by 42.88%, and in the kidneys by 94.48% in rats receiving distilled water and the inducer (hypertensive rats) concomitantly, compared to normotensive rats. e aqueous extract of the mixture of B. pilosa and C. citratus significantly prevented the increase (p < 0.001) in MDA levels induced by alcohol + salt solution in the aorta, heart, and kidney by 55.95%, by 64.89%, and by 48.04% at 100 mg/kg and by 40.30%, by 22.60%, and by 54.23% at 200 mg/kg, respectively, lower as compared to hypertensive rats. In the liver, the aqueous extract of the mixture of B. pilosa and C. citratus (100 and 200 mg/kg) prevented the significant increase in MDA level by 9.68% (p < 0.05) and 25.76% (p < 0.001), respectively, as compared to hypertensive rats. Treatment concomitantly with alcohol + salt and B. pilosa aqueous extract (200 mg/kg) significantly (p < 0.001) prevented the increase in MDA levels by 56.92% in the heart, 42.17% in the aorta, 50.83% in the kidney, and 21.99% in the liver compared to rats treated with distilled water and inducer. C. citratus extract (200 mg/kg) produced the same effects in the heart by 34.63%, in the aorta by 46.72%, and in the kidney by 45.12% as compared to hypertensive rats. Spironolactone (10 mg/kg) significantly prevented the increase in MDA levels induced by alcohol + salt solution in the heart by 20.30% (p < 0.01), in the liver by 25.75% (p < 0.001), in the aorta by 60.75% (p < 0.001), and in the kidney by 26.49% (p < 0.001) as compared to hypertensive rats.
SOD activity was significantly (p < 0.001) decreased in the liver by 63.22%, in the heart by 59.56%, and in the aorta by 91.66% of hypertensive rats as compared to normotensive rats (Figure 4(d)). e aqueous extract of the mixture of B. pilosa and C. citratus (100 mg/kg) significantly (p < 0.001) prevented the decrease in SOD activity induced by alcohol + salt solution in the liver, heart, and aorta. Values were 175.00%, 154.58%, and 140.38%, respectively, greater in these organs compared to hypertensive rats. e aqueous extract of the mixture of B. pilosa and C. citratus (200 mg/kg) also significantly (p < 0.001) prevented the decrease in SOD activity induced by alcohol + salt solution in the liver and the heart. e values were 134.38% and 227.30%, respectively, higher in these organs compared to hypertensive rats. At the same dose of the mixture's aqueous extract, SOD activity was significantly (p < 0.01) higher in the heart by 32.35% as compared to normotensive rats. Spironolactone significantly prevented the decrease in SOD activity induced by alcohol + salt solution in the liver (p < 0.001), heart (p < 0.001), and aorta (p < 0.05). e values were 110.02%, 200.02%, and  in the liver by 70.68%, heart by 61.66%, aorta by 86.69%, and kidney by 17.77% (p < 0.05) as compared to normotensive rats (Figure 4(e)). e aqueous extract of the B. pilosa and C. citratus mixture significantly (p < 0.001) prevented the decrease in nitrite levels induced by alcohol + salt solution in the aorta, liver, kidney, and heart. e values were, respectively, by 215.00%, 220.00%, 111.11%, and 106.25% at 100 mg/kg and 355.00%, 200.00%, and 127.77% at 200 mg/ kg, higher as compared to hypertensive rats. In the spironolactone (10 mg/kg) treated group, there was a significant (p < 0.001; p < 0.05 for aorta) prevention of nitrite decline in the heart, liver, kidney, and aorta. Values were 78.91%, 150.00%, 50.00%, and 115.00%, respectively, greater in these organs as compared to hypertensive rats. B. pilosa aqueous extract (200 mg/kg) significantly (p < 0.001) prevented the decrease in nitrite levels in the kidney, liver, and aorta induced by alcohol + salt solution. e values were 114.78%, 266.10%, and 576.78%, respectively, higher in these organs as compared to hypertensive rats. In concomitant treatment with alcohol + salt solution, C. citratus aqueous extract (200 mg/kg) also significantly (p < 0.001) prevented the decrease in nitrite levels in the kidney, liver, and aorta. e values were 74.30%, 265.19%, and 696.48%, respectively, higher in these organs as compared to hypertensive rats. Figure 5 shows the effects of the aqueous extract of the mixture of B. pilosa and C. citratus on tissue protein levels. It can be seen that protein levels were significantly (p < 0.001) decreased in the aorta, heart, liver, and kidney by 90.85%, 76.02%, 41.66%, and 34.86%, respectively, in hypertensive rats as compared to normotensive rats. e aqueous extract of the plant mixture (100 mg/kg) significantly (p < 0.001) prevented the decrease induced by alcohol + salt solution in protein levels in the liver by 64.28% and in the kidney by 66.98%, respectively, as compared to hypertensive rats. e aqueous extract of the mixture of B. pilosa and C. citratus (200 mg/kg) significantly prevented the decrease induced by alcohol + salt solution in tissue protein levels in the kidney, liver, and aorta as compared to hypertensive rats. e values were 85.66% (p < 0.001), 71.42% (p < 0.001), and 712.5% (p < 0.01), respectively, higher in these organs as compared to hypertensive rats. Spironolactone (10 mg/kg) administered under the same conditions as the aqueous extract of the plant mixture significantly prevented the decrease in protein levels in the kidney, liver, and aorta. Values were 61.88% (p < 0.001), 37.36% (p < 0.001), and 691.66% (p < 0.01), respectively, greater in these organs as compared to hypertensive rats. e aqueous extracts of B. pilosa (200 mg/kg) and C. citratus (200 mg/kg) significantly (p < 0.001) prevented the decrease induced by alcohol + salt solution in protein levels in the kidney, liver, and aorta compared to hypertensive rats.

Effects of the Aqueous Extract of the Mixture of B. pilosa and C. citratus on Histomorphometry of the Aorta and the
Microarchitecture of the Kidney and Liver. Figure 6 shows the effects of preventive treatment of the aqueous extract of the mixture of B. pilosa and C. citratus on histomorphometry of the aorta (A) and the microarchitecture of the kidney and aorta (B). e administration of the inducer (alcohol + salt solution) in rats resulted in a significant increase in media thickness by 73.84% (p < 0.001) as compared to normotensive rats. e aqueous extract of the mixture of B. pilosa and C. citratus (100 and 200 mg/kg) significantly (p < 0.001) prevented the increase induced by alcohol + salt solution in the thickness of this tunic as compared to hypertensive rats. e values were, respectively, 25.55% and 22.24% lower as compared to hypertensive rats. e aqueous extracts of B. pilosa (200 mg/kg) and C. citratus (200 mg/kg) significantly prevented the increase induced by alcohol + salt solution in media size as compared to hypertensive rats. e values were 14.70% (p < 0.01), 27.42% (p < 0.001), and 16.17% (p < 0.01), respectively, lower as compared to hypertensive rats. Treatment with alcohol + salt solution resulted in renal leukocyte infiltration and renal mesenchymal expansion as compared to normotensive rats. e aqueous extract of the mixture of B. pilosa and C. citratus as well as the separate extracts of these two plants prevented the damages induced by alcohol + salt solution as compared to hypertensive rats.

Discussion
e present study investigated the preventive effects of the aqueous extract of the mixture of the aerial parts of B. pilosa and C. citratus on a Wistar rat model of hypertension induced by oral administration of alcohol + salt solution at doses of 5 g/kg and 2.46 g/kg, respectively, for 49 days.
Alcohol and salt are the most important causes for HBP and increased cardiovascular risk worldwide [30]. Chronic alcohol and salt abuse is the cause of cardiovascular, renal, and liver dysfunction as well as oxidative stress and dyslipidemias associated with atherosclerosis that contributes to the pathogenesis of hypertension. In the present study, seven weeks of alcohol + salt solution (inducer) consumption resulted in an important increase in blood pressure and heart rate. e increase in those hemodynamic parameters by the inducer was significantly prevented by the substances given concomitantly with the inducer. Dyslipidemias and atherosclerosis, which are risk factors for cardiovascular disease [31], were observed in this study by decreased HDLchol levels and increased TC, TG, VLDL-chol, and LDL-chol levels as well as atherogenic indices (CRR, AC, and AIP).
is damage was confirmed by the histology and histomorphometry of the aorta characterised by thickening of the media. is might be the result of the effects of ethanol and/ or salt in rats treated with the inducing solution. Indeed, alcohol could act by increasing the bioavailability of free fatty acids (glycerophosphates), reducing the activity of the enzymes such as lipoprotein lipase and triglyceride lipase, and decreasing the oxidation of fats, thus causing the accumulation of lipids in the serum [32]. ese results are in agreement with the work of Olaleye et al. [33] who showed that a high-salt diet (8%) for six weeks caused a significant increase in serum TG, LDL-chol, and TC concentrations and a reduction in serum HDL-chol levels. According to these authors, dyslipidemias observed could promote the development of atheromatous plaques in the arteries and consequently HBP. According to Ikewuchi [28], atherogenic indices are strong predictors of heart disease risk, and the risk of developing cardiovascular diseases increases with increasing values of these indices and vice versa. In the pathogenesis of atherosclerosis described by Badimon and Vilahur [31], the increase in plasma LDL-chol leads to an increase in LDL-chol in the arterial wall. Once in arteries, they promote vascular resistance which leads to AHT. Furthermore, an increase in HDL-chol and a reduction in LDL-chol and TC levels could be considered beneficial in the long-term prognosis of cardiovascular disease [31]. Treatment of rats with spironolactone, the aqueous extract of the mixture of B. pilosa and C. citratus, and separate extracts of both plants prevented the onset of dyslipidemia and atherosclerosis induced by alcohol + salt solution. ese protective effects were more marked with the mixture of both plant extracts, especially at the lowest dose (100 mg/kg). is confirms a lipid-lowering synergism between the two plants. ese results are in agreement with those of Ogbonnia et al. [34] who showed that the hydroethanolic extract of the mixture of Treculia africana and Bryophyllum pinnatum had better hypoglycaemic and hypolipidemic effects than the extracts of isolate T. africana or B. pinnatum on streptozotocin-induced diabetic rats. erefore, the lipid-lowering effects of this mixture could be attributed to the synergistic effects of each of the bioactive compounds present in each plant. ese results confirm the beneficial effects of plant mixtures as hypolipidemic agents and consequently as antihypertensive. ese lipid-lowering effects could therefore be one of the mechanisms by which the aqueous extract of the mixture exerts its antihypertensive effect.
In addition to dyslipidemias as risk factors for AHT, alcohol and salt caused cardiovascular dysfunctions characterised by an abnormal increase in the heart rate and blood pressure and a decrease in vasodilators such as nitric oxide. According to Wake (2021), alcohol or its metabolic derivatives act on the cardiovascular system or on the blood pressure regulation systems.
is is achieved through a decrease in nitric oxide in the vascular endothelium due to inhibition of endothelial nitric oxide synthase [30]. is leads to inflammatory/oxidative damage to the vascular endothelium and therefore to vasoconstriction which increases vascular resistance and leads to HBP [30]. Alcohol may have a direct effect on smooth muscle cells by increasing both vascular sensitivity to vasomotor amines and the entry of calcium into muscle cells, which leads to hypertension via increased peripheral vascular resistance [30]. Alcohol also induces the activation of the sympathetic nervous system via increased hypothalamic corticoliberin, resulting in hypersecretion of catecholamines, cortisol, angiotensin, and aldosterone responsible for HBP [30]. In animal models of salt-sensitive hypertension, the increase in blood pressure after a salt load is characterised by a decrease in nitric oxide production [35]. Chronic salt consumption induces the production of superoxide anions via increased xanthine oxidase, which causes vasoconstriction through the decrease in nitric oxide [36]. In addition, a high-salt diet leads to K + depletion, with a negative impact on blood pressure regulation, promoting cardiovascular diseases. e concomitant treatment with alcohol + salt and the aqueous extract of the mixture of B. pilosa and C. citratus or extracts of isolated plants significantly prevented the increase in blood pressure and heart rate and decrease in nitrite levels.
e results obtained with the two extracts of the isolated plants confirm the antihypertensive effects of B. pilosa [16] and C. citratus [20] already demonstrated. e prevention of AHT was more marked by the concomitant treatment with alcohol + salt and the aqueous extract of the mixture of B. pilosa and C. citratus, especially at the dose of 100 mg/kg. is confirms a synergistic effect between the two plants in preventing HBP. Moreover, these results obtained with the aqueous extract of the mixture of B. pilosa and C. citratus corroborate those of Dzeufiet et al. [37] who demonstrated the benefits of the aqueous extract of a mixture of plants to prevent ethanol and fructose-induced hypertension in rats.
is phenomenon of beneficial interaction with plants is known as a synergy of pharmacodynamic effects [38]. is suggests that in the aqueous extract of the mixture of B. pilosa and C. citratus, bioactive compounds present such as the phytochemicals mainly flavonoids and phenolic compounds act synergistically to cause a reduction in cardiac output and/or total peripheral resistance and consequently a decrease in blood pressure [39]. ese effects would therefore be one of the mechanisms by which the aqueous extract of the mixture exerts its antihypertensive effect.
High alcohol and salt consumption is associated with oxidative stress and increased free radicals, which play an important role in oxidative alterations of proteins and the pathogenesis of hypertension [40]. Oxidative stress in the present study is characterised by a decreased level of GSH, SOD, and catalase activity with an increase in the MDA level. Glutathione plays an excellent role in protecting cells from free radical damages by directly scavenging reactive oxygen species [41]. It reduces hydrogen peroxide (H 2 O 2 ) production and therefore prevents any deleterious effects such as lipid peroxidation, which is characterised by the production of MDA [42]. SOD and catalase are important antioxidant enzymes that contribute to getting rid of superoxide anion (O 2 -) and H 2 O 2 , respectively [43]. Alcohol and salt are thought to act synergistically to create oxidative imbalance. Indeed, chronic alcohol consumption could result in the depletion of antioxidant enzymes due to the excessive generation of O 2 and H 2 O 2 [44]. Alcohol leads to the production of free radicals which by lipid peroxidation causes cellular damages accompanied by a significant increase in MDA levels [45]. Excessive salt consumption is known to be associated with increased tissue production of reactive oxygen species [36]. According to Bopda et al. [35], long-term stress due to chronic salt intake induces a massive and toxic amount of reactive oxygen species, leading to a decrease in GSH and SOD levels. In fact, salt consumption enhances O 2 and H 2 O 2 generation accompanied by increased NADPH oxidase expression and activity with decreased SOD in the vessels and kidney, contributing to the pathogenesis of hypertension [36]. Treatment of rats with the aqueous extract of the mixture of B. pilosa and C. citratus as well as the extracts from isolated plants significantly prevented the installation of oxidative stress observed in hypertensive rats. e lower dose (100 mg/kg) was more effective in preventing oxidative imbalance.
ese results corroborate those of Eshrat and Hussain [46] who obtained better antioxidant effects with the mixture of Curcuma longa and Abroma augusta as compared to the two plants used separately in streptozotocin-induced diabetes. us, it can be suggested that the synergistic interaction of the bioactive compounds contained in each mixture of plants confers better antioxidant properties to the aqueous extract of the mixture of B. pilosa and C. citratus.
ese free radicalscavenging properties of the aqueous extract of the mixture could therefore be one of the mechanisms by which it would exert its antihypertensive activity. e development of hypertension in the present study has had a negative impact on the functioning of vital organs such as the kidneys. Oxidative stress is the main pathological pathway involved in renal dysfunction [47]. In the present study, oxidative stress-mediated by chronic alcohol and/or salt consumption contributed to the development of nephrotoxicity through increased water intake, urinary excretion volume, serum creatinine, uric acid, and urea concentrations, as well as decreased creatinine clearance.
is renal injury was confirmed in this study by histological analyses characterised by leucocytic infiltration and mesangial expansion. Indeed, alcohol acts by decreasing the number of vasopressin neurons in the hypothalamus, leading to a decrease in plasma antidiuretic hormone levels [48]. is could explain the increase in urinary excretion volume in rats treated concomitantly with water and inducing solution. e increase in water intake is thought to be due to the stimulation of the renin-angiotensin system by salt leading to the production of angiotensin II. e latter will act at the level of the hypothalamus inducing a sensation of thirst which increases water consumption, blood volume, and therefore blood pressure [49]. Urea, creatinine, and uric acid are the catabolites of ammonia, creatine, and purine nucleotides released into blood and eliminated by the kidney, respectively. Due to higher sensitivity of the glomerular region to alcohol and/or salt-induced oxidative damage, the result of the inducing solution intake will be a decrease of the filtration rate and clearance of these substances in the rats [29]. Studies have reported that increased plasma urea and creatinine levels are a consequence of impaired renal function leading to acute glomerulonephritis, nephrosclerosis, and tubular necrosis [50]. In the present study, the concentrations of biochemically and physiologically important electrolytes and minerals were measured. Sodium, potassium, calcium, and magnesium levels were decreased in plasma and increased in the urine of hypertensive rats as compared to normotensive controls. Plasma electrolytes maintain intravascular homeostasis with interstitial and intracellular space. Alcohol consumption induces polyuria and severe loss of electrolytes and minerals which would result in plasma hyponatremia, hypokalemia, hypomagnesaemia, and hypocalcemia [51]. Adewale and Ifudu studies reported that alcohol-generated free radicals cause severe membrane damage, resulting in decreased mineral reabsorption at the distal tubules and ascending branches of the loops of Henle [51]. Salt induces oxidative stress at the renal level through the expression of NADPH oxidase which stimulates the production of O 2 in mesangial cells and ascending limbic cells [30]. Treatment of rats with inducing solution and the aqueous extract of the mixture of B. pilosa and C. citratus and separate extracts of these two plants significantly prevented kidney damages in rats and maintained plasma electrolytes and minerals at normal levels. e nephroprotective effects were the best with the aqueous extract of the mixture of B. pilosa and C. citratus especially at the lowest dose (100 mg/kg). ese results are in agreement with the work who Manzoor et al. [52] showing nephroprotective effects of different doses of aqueous extracts of Foeniculum vulgare seeds, Solanum nigrum fruits, and their mixture on gentamicin-induced nephrotoxicity in albino rabbits. According to these authors, the aqueous extract of the mixture of F. vulgare and S. nigrum had the best effects as compared to the extracts of each individual plant. ese therapeutic effects may be due to the synergistic effects of the phytocompounds (polyphenols and flavonoids) present in each of the plants, which are thought to modify membrane fatty acid composition, fluidity, permeability, and electrolyte homeostasis by reducing alcohol and salt-induced oxidative stress through free radical-scavenging activity. e aqueous extract of the mixture would therefore partly exert its antihypertensive effect due to its nephroprotective properties, knowing the important role played by the kidney in blood pressure regulation. e effects of the mixture were not dosedependent on lowering blood lipid and nephroprotection; this could be due to antagonism. In fact, the extract of the mixture contained many molecules, some of which could be antagonistic. erefore, at low doses, the concentration of these antagonistic molecules was low, thus offering no hindrance to the hypolipidemic and nephroprotective causative substances. Similar results were reported by Dzeufiet et al. [53] when working on the effect of Ceiba pentandra on type 2 diabetes.
In addition to the imbalance of the prooxidant/antioxidant equilibrium in favor of prooxidants, alcohol and salt are known to produce free radicals, which affect the cellular permeability of hepatocytes, subsequently leading to elevated levels of serum biochemical parameters such as ALT and AST [54]. is marks an impairment of the structural and functional integrity of liver cell membranes, as these cytosolic enzymes are mainly released into circulation after liver cell injury [55]. An increase in ALT enzyme activity is almost always due to hepatocellular injury and is usually accompanied by an increase in AST [55]. In the present study, hypertensive rats showed an increase in serum ALT and AST activities and bilirubin concentration indicating liver damages. ese liver damages were prevented by the aqueous extract of the mixture of B. pilosa and C. citratus as well as the extracts of the plants taken individually. e present results suggest that the aqueous extract of the mixture of B. pilosa and C. citratus (100 mg/kg) is more effective as compared to the aqueous extracts of isolated B. pilosa or C. citratus. ese results obtained with the mixture's aqueous extract are in agreement with the work of Tripathi and Trilochana [56] who showed that the ethanolic Evidence-Based Complementary and Alternative Medicine extract of the mixture of the leaves and flowers of Bassia latifolia had better hepatoprotective effects than the separate ethanolic extracts of the leaves and flowers in paracetamolinduced hepatotoxic rats.
is hepatoprotective activity could be one of the mechanisms whereby the mixture's aqueous extract exerts its antihypertensive effect.

Conclusion
e results of the present study showed that the aqueous extract of the mixture of B. pilosa and C. citratus (100 and 200 mg/kg) significantly prevented the development of hypertension in rats treated with the alcohol + salt solution. According to the biochemical results, which were supported by histopathological evidences, the aqueous extract of the B. pilosa and C. citratus mixture, especially at the lower dose, synergistically protected the cardiovascular system as well as the kidney and liver against adverse effects of the alcohol-+ salt solution compared to the plants taken separately. is study also suggests that the possible antihypertensive mechanisms of this herbal mixture could be attributed to the vasorelaxant, antioxidant, lipid-lowering, hepato, and nephroprotection activities of its phenolic and flavonoid components.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.