Background
Arterial hypertension is among the most frequent pathologies in elderly worldwide, with an incidence ranging from 40 % (about 65 years patients) to 90 % (patients older than 85) in developed countries [
1,
2]. This pathology raises more concerns as it constitutes a major risk of cardiovascular accident. In many of the developing countries, the use of plant drugs against arterial hypertension is increasing because modern life saving drugs are beyond the reach of their countries although they spend 40–50 % of their total wealth on drugs and healthcare [
3]. Many investigators demonstrated that studies of herbal plant used in traditional medicine as diuretics were in progressive elevation in the last decades, and might be a precious tool used in human pathology treatment [
4]. In Cameroon, different types of complementary and alternative treatments are believed to be effective for treating high blood pressure. Using a diuretic to prevent cardiovascular complications plays an important role in the management of arterial hypertension [
5]. These diuretics increase the drainage of water and salt (sodium) into the urine, causing a reduction of blood pressure, a decrease in blood volume, and thus lowering resistance to the flow of blood [
6]. Efforts of Cameroonians scientists and traditional therapists are growing in the direction of improvement and enhancement of the use of medicinal plants, to elucidate the pharmacological properties of these plants and possibly to extract active ingredients. The medicinal value of these plants lies in some chemical active substances that produce a definite physiological action on the human body. The most important of these chemically active (bioactive) constituents of plants are: saponin, glycosides and flavonoid compounds. Many of these indigenous medicinal plants are also used for medicinal purposes [
7].
Vepris heterophylla is a medicinal plant used empirically in the mountainous massifs in the northern part of Cameroon for the treatment of various illnesses such as malaria and cardio-vascular disorders [
8]. The vernacular names of this species are Kounikoutchoum (Guiziga, Mofou), Hohoum (Zoulgo), Gougouvetche (Mafa), Kotokolhi (fulfulde) which testifies its importance in this region [
9]. The medicinal virtues of this Rutaceae on high blood pressure have already been pointed out by several authors: [
10,
11]. The efficacy of
V. heterophylla leaf extract in bringing relieve to patients with cardiovascular diseases was previously demonstrated. The vascular effects of
V. heterophylla methanol leaf extract in experimental rat thoracic aortic strips, with a view of providing a pharmacological justification (or otherwise) to the ethno-medical uses of the plant leaf in the management, control and/or treatment of hypertension and certain cardiac dysfunctions was demonstrated [
12]. This study indicated that, the vasorelaxation induced by the methanol leaf extract plant was endothelium dependent effect, likely via the NO–cGMP pathway or independent associated mediators such as prostacyclin [
13]. A flavonoid (6,8-dihydroxy-4′- methoxyflavone) isolated from stem bark was found to induced nitric oxide-dependent vasodilation in rat aorta [
14]. In the same order a triterpen (3β-16β, 23, 29-tetrahydroxyoleane-12-ene) isolated from stem bark was found to increase intracellular Ca
2+ in rat aorta endothelial cells [
15]. To our knowledge, there have been no reports on the diuretic and antioxidant activities of
V. heterophylla leaves. The present work aim was to measure the diuretic and antioxidant activities of the aqueous extract of
V. heterophylla leaves in rats.
Discussion
Preliminary phytochemical studies showed that aqueous extract of the leaves of
V. heterophylla contains several chemical compounds that could be partially or fully responsible for the increase of diuresis and moderate natriuretic activity. Saponin is used as a mild detergent and in intracellular histochemistry staining to allow antibody access to intracellular proteins. In medicine, it is used in hypercholesterolaemia, hyperglycaemia, diuresis, antioxidant, anti-cancer, anti-inflammatory and weight loss etc. It is also known to have anti-fugal properties [
19]. Tannin are reported to exhibit antiviral, antibacterial, anti-tumor activities. It was also reported that certain tannin are able to inhibit HIV replication selectivity and is also used as diuretic [
20]. Cardiac glycosides are known to work by inhibiting the Na
+/K
+ pump. This causes an increase in the level of sodium ions in the myocytes, which then lead to a rise in the level of calcium ions. This inhibition increases the amount of Ca
2+ ions available for contraction of the heart muscle, which improves cardiac output and reduces distention of the heart; thus, they are used in the treatment of congestive heart failure and cardiac arrhythmia. They are also, used to strengthen a weakened heart and allow it to function more efficiently, though the dosage must be controlled carefully, since the therapeutic dose is close to the toxic dose [
21]. Plant steroids are known to be important for their cardiotonic activities, they possess insecticidal and anti-microbial properties. They are also used innutrition, herbal medicine and cosmetics, they are routinely used in medicine because of their profound biological activities [
21]. Flavonoid have been referred to as nature’s biological response modifiers because of strong experimental evidence of their inherent ability to modify the body’s reaction to allergies, virus and carcinogens. They also show diuresis, anti-allergic, anti-inflammatory, anti-microbial and anti-cancer activity [
19].
Findings of the study also indicated that
V. heterophylla may have antioxidant effects. Comparable observations were reported in a number of other plants with diuretic properties [
22]. In addition, the extract also decreased hydroperoxide levels in homogenates, malondialdehyde levels in plasma and the activity of catalase in homogenates and hemolysates, which are markers of oxidative stress [
23]. The present study indicated that the aqueous extract of
V. heterophylla at doses ranging from 150 to 250 mg/kg caused a significant and dose-dependent increase of urinary water and electrolytes excretion in normal rats. It was noted that
V. heterophylla treatment caused increase in both water and electrolytes excretion qualitatively similar to furosemide which is known by its potential saluretic and diuretic effects [
24]. Determination of urinary carbonic anhydrase inhibition revealed that carbonic anhydrase inhibition was not significantly increased. This shows that diuretic activity of aqueous extract of
V. heterophylla does not make use of carbonic anhydrase inhibition as its mechanism of action [
25]. The increase in the ratio of concentration of excreted sodium and potassium ion indicates that the extract increases sodium ion excretion to a greater extent than potassium which is a very essential quality of a good diuretic with lesser hyperkalemia side effect [
26].
This study suggest that the aqueous extract leaves of
V. heterophylla administrated
per os had stronger diuretic effects at the doses used in acute studies. The urinary output of rats after oral administration of aqueous extract of
V. heterophylla showed a statistical difference observed between the test groups given the extract and the control groups during the 1st h and after 24 h. There is biological significance in urine volume of positive control in relation to the negative control during the 1st h and at the 24th h. Similar observations were reported in studies assessing the other plants with diuretic activity such as
Retama raetam [
27], and
Ficus glumosa [
17]. The acute administration of the extract at the dose with the more marked response (250 mg/kg) induced an increase of 82.58 % in urinary excretion (compared to negative control group), against 91.20 % and 13.87 % with furosemide and HCTZ, respectively. Diuretics modulate the volume and composition of body fluids in variety of clinical conditions like hypertension. The extract also accelerated the elimination of fluid overload and decreased the latency of the first urination 30.54 % overload was eliminated in the negative control group, whereas HCTZ eliminated 85.34 % and furosemide 90.66 %. The response of the extract was dose-dependent, and eliminated 80.62 % of overload at 150 mg/kg, 92.44 % at 200 mg/kg and 127.38 % at 250 mg/kg against negative control group. and the diuretic index of groups treated with the extract was higher (1.72 at 250 mg/kg) than HCTZ -treated (1.13) but lower than furosemide-treated (1.91). Such rapid diuretic activity may be due to very high concentration of active molecules of the saponin, Cardiac glycosides and flavonoïd families [
28], which presence in extracts of
V. heterophylla was detected by phytochemical analysis in our study and previously reported [
13]. The increase natriuresis in response to acute treatment of aqueous extract of leaves of
V. heterophylla may partly explain the increase in diuresis [
29,
30].
V. heterophylla also caused the acidification of urine. There was a significant reduction in the osmolarity of urine in rats treated with the extract.
V. heterophylla may impair the basal secretion of ADH and reduce the responsiveness of uriniferous tubules to the action of ADH. Inhibition of ADH causes polyurea with low osmolarity [
31]. Aldosterone hormone measured by radioimmunoassay was slightly increased in animals treated with aqueous extract, and the lack of correlation between plasma aldosterone and sodium concentration in the blood as well as in urine seem to imply that aldosterone is not involved in the natriuresis observed and suggested that stimulation of diuresis by the aqueous extract of the leaves of
V. heterophylla could be similar to that of furosemide. furosemide increases urinary excretion of sodium by inhibiting Na
+/K
+/2Cl
− symporter (co-transporter system) in the thick ascending limb of the Henley loop [
32], while HCTZ inhibits the Na
+/Cl
− symporter (co-transporter system) in the distal convoluted tubule, by competing for the Cl
− binding site, and increasing the excretion of Na
+ and Cl
− [
33]. Whether the extract induces the suppression of renal tubular reabsorption of water and electrolytes by one of these processes or by another mechanism is still to be determined. The increase of the Na
+ excretion tend to reduced GFR by increasing the Na
+ load available for Na
+/K
+ exchange, stimulating further such exchange by hyperaldosteronism, which causes a reduction in blood volume [
34].
Glucose and albumin were not present in treated rats’ urine, and no significant change was observed in the urinary creatinine levels. Instead, a marked reduction was observed in the concentration of urea in the urine compared to negative control group, the K
+ plasmatic concentration was increased, and Na
+ and Cl
− concentrations in the plasma were significantly decreased. Glomerular filtration measured by creatinine clearance does not vary according to treatment compared to controls, which suggest that the increase in diuresis would rather have a tubular origin as seems to show the clearance of free water [
16].
Taken together, these results indicate that V. heterophylla may act as a loop diuretic which inhibit the Na+/K+/Cl− co-transporter system in the thick ascending loop of the nephron, thus increasing natriuresis and kaliuresis.
Acknowledgments
We are profoundly grateful to the University of Yaounde I (Cameroon), the Institute of Medical Research and Medicinal Plants Studies (Cameroon), The authors thank Mr ALLARAMADJI Ndohortongar of N’Djaména hospital Le Bon Samaritain, for their assistance in this project. The authors also thank the Laboratory of the Medicinal Plants, Health and Galenic Formulation of the Department of Biological Sciences.