Comparative Effects of Ficus exasperata Aqueous Leaf Extract and
Furosemide on Urinary Excretion in DOCA-salt Hypertensive Rat
The prevalence of hypertension is increasing in many parts of the world. Management
of this disease requires the use of diuretics, ACE inhibitors and β-blockers.
In many pharmacopoeias, the leaves of Ficus exasperata are used to treat
hypertension. The hypotensive effects of leaves of this species have been shown.
The purpose of this study was to evaluate the diuretic effect of F. exasperata
aqueous leaf extract (FEFIX) comparing to those of furosemide in salt hypertensive
rats treated with deoxycorticosterone acetate (DOCA). Animals treated with DOCA
were divided into three groups which received intraperitoneally NaCl (9,
saline solution), FEFIX (100 mg kg-1 b.wt.) and furosemide (10 mg
kg-1 b.wt.), respectively. Urine output was collected for 24 h. At
the end of the experiment, the blood was collected and sampled. FEFIX and furosemide
increased urinary volume (EUV) to 153.32±6.89 and 105.71±9.37%,
respectively. F. exasperata aqueous leaf extract and furosemide increased
the urinary excretion of electrolytes (Na+, Cl- and Ca2+),
urea and creatinine. However, excretions induced by the plant extract were greater
than those induced by furosemide. FEFIX and furosemide decreased the rate of
plasma electrolytes with a decrease in serum sodium greater for this extract.
The studied extract decreased urea and plasma creatinine like furosemide. These
results showed that the diuretic effects of F. exasperata aqueous leaf
extract were similar to those of furosemide in hypertension due to salt overload.
However, the diuretic effects of this plant extract were superior to those of
to cite this article:
A.K. Amonkan, A.B. Konan, M.N. Bleyere, B.M.L. Ahui, L.K. Kouakou, G.M.K. Bouafou and S. Kati-Coulibaly, 2013. Comparative Effects of Ficus exasperata Aqueous Leaf Extract and
Furosemide on Urinary Excretion in DOCA-salt Hypertensive Rat. Journal of Medical Sciences, 13: 385-390.
Received: March 27, 2013;
Accepted: April 18, 2013;
Published: June 14, 2013
Hypertension is an elevation in blood pressure leading to a systolic blood
pressure greater than or equal to 140 mm Hg and a diastolic blood pressure greater
than or equal to 90 mm Hg (Erdine et al., 2006).
It is currently a public health problem worldwide due to its frequency and risk
of cardiovascular and renal diseases which are attached. Kearney
et al. (2005) indicate a prevalence of 1.6 billion hypertensive subjects
in 2025. Indeed, the prevalence of this disease increase in the world in general
and developing countries in particular (Whitworth et
al., 2003, Kearney et al., 2004). Generally,
the treatment of this disease requires the use of diuretics, ACE inhibitors
and beta-blockers (Armario and Waeber, 2013). So many
people in developing countries employ several herbals to treat this pathology
(Abrogoua et al., 2012; Anwar
et al., 2007). Ficus exasperata Vahl. 1805 (Moraceae) is one
of the plants used in this field. Its leaves are often found in many beverages
antihypertensive preparations. Previous studies showed that F. exasperata
aqueous leaf extract decreased the blood pressure in a dose-dependent manner
(Ayinde et al., 2007; Amonkan
et al., 2010; Adewole et al., 2011).
According to this disease etiology, the sodium intake via a diet is involved
in the onset of the disease. In addition, the control of sodium balance is necessary
in hypertensive subjects. Therefore reducing sodium intake and promote renal
excretion of sodium are necessary to reduce the blood pressure (Forman
et al., 2012; Karppanen and Mervaala, 2006).
Thus, the aim of this work was to evaluate the diuretic effect of F. exasperata
aqueous leaf extract comparing to those of furosemide in the case of hypertension
induced by salt overload.
MATERIALS AND METHODS
Ethics: Experimental procedures and protocols used in this study were
approved by Ethical Committee of Health Sciences, University Felix Houphouet-Boigny.
These guidelines were in accordance with the internationally accepted principles
for laboratory animals use and care (NRC, 1996; Mosihuzzaman
and Choudhary, 2008).
F. exasperata Aqueous leaf extract: Fresh leaves of F. exasperata
Vahl. 1805 (Moraceae) were collected in a forest of the Southern region of Côte
dIvoire (Region des Lagunes). This plant was authenticated by a Botany
expert, Prof. Ake-Assi Laurent of the Centre National de Floristique,
UFR-Biosciences, Felix Houphouët-Boigny University, Abidjan, Côte
dIvoire. Ficus exasperata aqueous leaf extract (FEFIX) preparation
was previously described (Amonkan et al., 2010
and 2013). Fresh leaves of F. exasperata were
washed and dried in an oven at a temperature of 40±2°C. They were
pulverized to obtain a fine powder which was left to macerate in n-hexane at
a rate of 10 g of powder in 100 mL of n-hexane for 24 h. After filtration, the
residue was collected and dried to be subjected to further maceration in distilled
water at a rate of 5 g per 100 mL of solvent. The filtrate was then collected
and dried using a rotavapor (Buchi, France). A powder of F. exasperata
aqueous leaf extract (FEFIX) was obtained with a yield of 14.27±3.26
%. FEFIX was stored at 4°C until experiments.
Animals: Male Wistar rats weighing 200-250 g were used for these experiments.
From Pasteur Institute, Abidjan (Côte d'Ivoire), the animals were acclimatized
in plexiglass cages for 14 days before experimentation. They were maintained
at a temperature of 25±2°C with dark and light cycle (12/12 h). They
have free access to standard dry pellet diet and water ad libitum. Animals
were treated with deoxycorticosterone acetate (DOCA) for 4 weeks. They received
DOCA subcutaneously twice a week (25 mg kg-1 b.wt.). They were normally
fed with a drink of NaCl 1% and KCl 0.2% ad libitum (Fournie-Zaluski
et al., 2004; Bodineau et al., 2008).
After 4 weeks of treatment, the animals become hypertensive and exhibit hemodynamic
parameters and kidney similar to those observed in the spontaneously hypertensive
rat (Johnson et al., 2004). The day before the
experiment, all animals were fasted overnight. At the end of the experiment,
the animals were anesthetized with ether and blood rats were sampled from the
inferior vena cava.
Evaluation of the diuretic: The day of the experiment, the animals were
divided into three groups of six rats and placed individually in metabolic cages.
Fluid overload was conducted at 50 mL kg-1 and the animals received
immediately following substances according to the group: saline (NaCl 9,
control) FEFIX (100 mg kg-1 b.wt.) and furosemide (10 mg kg-1
b.wt.). The urine were collected separately every two hours for 24 h and sampled.
They were stored at -20°C prior to determination of the levels of electrolytes,
creatinine and urea. Excreted urine volume (EUV) was determined from the ratio
of the volume of urine excreted and the volume of fluid overload.
Determination of plasma and urinary electrolytes: Automatic analyzer
(Hitachi 902, Roche) was used to determine plasma and urinary electrolytes,
creatinine and urea. The determination of sodium and potassium in urine and
plasma was performed by the technique of photometry. Levels of calcium, chlorine
and creatinine were performed by the technique of colorimetry. The levels of
urea were determined by the principle of kinetics.
Chemicals used: The following reference drugs were used: Furosemide
(Lasilix®, Sanofi-Aventis, France), Deoxycorticosterone acetate
(DOCA, Sigma ). FEFIX and Furosemide were dissolved and diluted in saline solution
(NaCl 9). DOCA was dissolved in sesame oil on each day of our experiments.
Statistical analysis: The experimental results were expressed as the
mean with standard error of mean (m±sem). Data were assessed by the method
of analysis of ANOVA followed by Tukey-Kramer test with GraphPad Instat software
(Microsoft, San Diego, California, USA). Graphical representations of data were
performed by GraphPad Prism 5 software (Microsoft, San Diego, California, USA).
The difference between the averages is considered statistically significant
Volume of urine excreted: Urinary excretion induced by furosemide after
2 h was greater than that induced by F. exasperata aqueous leaf extract
(FEFIX). The values obtained were respectively 32.04±6.87 and 20.04±3.84%.
From the fourth hour, FEFIX achieved relatively high urinary volumes and higher
than those induced by furosemide administration. After 24 hours, FEFIX caused
urinary excretion of 153.32±6.89% while that measured with furosemide
was 105.71±9.37% (Fig. 1).
Urinary excretion of electrolytes: After 24 h, FEFIX caused a urinary
sodium excretion of 15.77±0.62 mEq (Fig. 2). Furosemide
induced urinary excretion of sodium, which amounted to 12.78±0.69 mEq.
Urinary potassium levels were 1.54±0.12 and 1.19±0.09 mEq, respectively
for FEFIX and furosemide. The rate of chlorine and calcium excreted after 24
h under the action of FEFIX were relatively higher than those induced by furosemide.
Concerning chlorine, excretions recorded were 12.35±0.62 and 08.27±0.59
mEq, respectively following FEFIX and furosemide treatments. Calcium excretions
were 4.84±0.48 mEq (FEFIX) and 4.32±0.42 mEq (furosemide).
Plasma electrolytes: After 24 h, FEFIX and furosemide caused a decrease
in plasma electrolytes (Fig. 3). The serum sodium measured
were 115.17±6.06 and 127.50±6.59 mEq L-1, respectively
for FEFIX and furosemide.
||Evolution of urinary excretion volume measured for three groups
of DOCA salt hypertensive rats: Saline solution (NaCl 9 , Control),
FEFIX (100 mg kg-1 b.wt.) and furosemide (10 mg kg-1
b.wt.). Urine output was measured every two hours for 24 h. FEFIX: F.
exasperata aqueous leaf extract, DOCA: deoxycorticosterone acetate,
n = 6, M±SEM
||Rate of electrolyte urinary excretion in rats at 24 hours
in three groups of DOCA salt hypertensive rats treated with Saline solution
(NaCl 9 , Control), FEFIX (100 mg kg-1 b.wt.) and furosemide
(10 mg kg-1 b.wt.) respectively. Electrolytes were measured in
all urine sampled for 24 h after treatment in each group of rats. FEFIX:
F. exasperata aqueous leaf extract, DOCA: deoxycorticosterone acetate,
n = 6, M±SEM, ***: p<0.001 , **: p<0.01, *p<0.05
The plasma chlorine obtained were 75.00±4.74 mEq L-1 (FEFIX)
and 76.32±6.51 mEq L-1 (furosemide).
|| Effects of FEFIX and furosemide on creatinine and urea in
urine output and plasma in DOCA salt hypertensive rat
|Saline solution (NaCl 9 , Control), FEFIX (F. exasperata
aqueous leaf extract, 100 mg kg-1 b.wt.): furosemide (10 mg kg-1
b.wt.). DOCA: deoxycorticosterone acetate, u: urine, p: plasma, n = 6, m±sem;
***p<0.001, **p<0.01, *p<0.05
||Plasma levels of electrolytes at 24 h after administration
of Saline solution (NaCl 9, Control), FEFIX (100 mg kg-1
b.wt.) and furosemide (10 mg kg-1 b.wt.) in three groups of DOCA
salt hypertensive rats respectively. The rate of electrolyte was measured
on blood samples. FEFIX: F. exasperata aqueous leaf extract, DOCA:
deoxycorticosterone acetate, n = 6, M±SEM, ***: p<0.001 , **:
The serum calcium obtained were 16.50±3.64 mEq L-1 (FEFIX)
and 17.42±3.14 mEq L-1 (furosemide). However, changes in serum
potassium induced by furosemide and FEFIX showed no significant difference to
that obtained with the control (p>0.05).
Urea and creatinine in urine and plasma: FEFIX and furosemide induced
significant urinary excretion of creatinine and urea (Table 1).
The creatinine level obtained under FEFIX treatment after 24 h was 0.40±0.05
mmol. That induced by furosemide was 0.36±0.03 mmol. The urea measured
in urine were 290.21±17.48 mmol for FEFIX and 249.42±18.15 mmol
for furosemide. In the plasma, FEFIX and furosemide also caused significant
changes in creatinine and urea. The creatinine levels measured after 24 h were
0.41±0.05 and 0.44±0.08 mM, respectively for FEFIX and furosemide.
Uremia obtained in presence of FEFIX (2.08±0.33 mM) was less than that
obtained when furosemide (2.54±0.25 mM) was administrated to rats.
Ficus exasperata aqueous leaf extract (FEFIX) and furosemide induced
urinary excretion volume (EUV) relatively large. The two substances had similar
urinary excretion kinetics. However, the urinary excretion obtained with FEFIX
was greater than that obtained with furosemide. The increase in urinary excretion
induced by FEFIX could result from stimulation of renal excretory function.
Similar results were reported in previous works concerning several plants used
in Thai Pharmacopoeia (Sripanidkulchai et al., 2001).
Urtica dioica aqueous leaf extract increased urinary excretion according
to the dose. For infusions of 4-24 mg/kg/h, this extract increased urine volume
by 11 to 84% (Tahri et al., 2000). In addition,
crude ethanolic extract of leaves Melothria maderaspatana protects the
kidney in salt hypertensive rats. This protection is manifested by the reduction
in histological damage associated with hypertension (Veeramani
et al., 2012).
Urinary excretion induced by FEFIX and furosemide were associated with significant
loss of sodium, chloride and calcium. Electrolytes Urinary excretion caused
by FEFIX were relatively higher than those obtained under furosemide treatment.
As furosemide, F. exasperata aqueous leaf extract inhibited renal reabsorption
of electrolytes. Indeed, Tribulus terrestris aqueous extract increased
the excreted urinary volume. Diuresis induced by this extract was relatively
higher than that induced by furosemide (Al-Ali et al.,
2003). In addition, the infusion of Salvia scutellarioides caused
high urinary excretion of electrolytes which increased dose-dependent manner
(Ramirez et al., 2006).
Diuretic effects of FEFIX and furosemide altered the plasma electrolytes. Both
substances decreased serum sodium, chloride and serum calcium without affecting
significantly the plasma potassium. The decrease in plasma electrolytes could
result from their significant urinary excretion. Indeed, previous studies had
shown that administration of saponins from Herniara glabra decreased
arterial blood pressure by reducing the reabsorption of sodium and water in
the renal tubules. Saponins of H. glabra, thereby increasing urine flow
and excretion of sodium and potassium (Rhiouani et al.,
1999). Furosemide increased urine volume and urinary excretion of sodium.
This increase of diuresis and natriuresis resulted from the inhibition of electrolytes
cotransporter along the ascending limb of the nephron (Carmosino
et al., 2001; Haque et al., 2011).
In addition, the ethanolic extract of Tropaeolum majus and his purified
fraction containing isoquercitrin increased diuresis with potassium-sparing.
This effect observed in spontaneously hypertensive rats results from inhibition
of angiotensin converting enzyme and activity of Na+/K+-ATPase
(Gasparotto et al., 2012). In addition, the
Flavangenol extracted from pine bark marine attenuated significantly renal lesions
in salt hypertensive rats. This protective effect could be attributed to its
antioxidant property which protects against endothelial dysfunction (Kwak
et al., 2009; Ohkita et al., 2011).
In salt hypertension, F. exasperata aqueous leaf extract induced a diuretic
effect relatively large and greater than that induced by furosemide. This diuresis
was associated with a significant loss of electrolytes in the urine. These urine
outputs of electrolytes, creatinine and urea decreased their levels in plasma.
We are grateful to Pr YAO Datte Jacques and Pr OFFOUMOU Atte Michel for their
critical suggestions and encouragement.
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