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Diuretic Effect of Compounds from Hibiscus

sabdariffa by Modulation of the Aldosterone
Activity

Article in Planta Medica November 2012

DOI: 10.1055/s-0032-1327864 Source: PubMed

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 Original Papers 1893

Diuretic Effect of Compounds from Hibiscus sabdariffa

by Modulation of the Aldosterone Activity

Authors Enrique Jimnez-Ferrer 1, Javier Alarcn-Alonso 1, Arturo Aguilar-Rojas 1, Alejandro Zamilpa 1, Itzia Jimnez-Ferrer C. 1, 2,
Jaime Tortoriello 1, Maribel Herrera-Ruiz 1

1
Affiliations Centro de Investigacin Biomdica del Sur, Instituto Mexicano del Seguro Social (IMSS), Xochitepec, Morelos, Mexico
2

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Depto. Medicina Molecular y Bioprocesos, Instituto de Biotecnologa (UNAM), Cuernavaca, Morelos, Mexico

Key words Abstract diminished until disappearance due to decrease

l
" Hibiscus sabdariffa Linn.
! of the polarity of the solvents used in the extrac-
l
" Malvaceae
Recent studies of Hibiscus sabdariffa Linn. have tion process, in contrast to the flavonoids and
l
" diuretic
demonstrated that it presents diuretic, natriuret- chlorogenic acid, which had their concentration
l
" aldosterone antagonism

received July 16, 2012
revised Sept. 24, 2012
accepted Sept. 25, 2012
Bibliography
DOI http://dx.doi.org/
10.1055/s-0032-1327864
Published online November 13,
2012
Planta Med 2012; 78:
18931898 Georg Thieme
Verlag KG Stuttgart New York
ISSN 00320943
Correspondence
Enrique Jimnez-Ferrer, PhD
Centro de Investigacin
Biomdica del Sur, CIBIS-IMSS
Argentina No. 1, Xochitepec
Morelos, 62790
Mxico
Phone: + 52 77 73 61 21 55
Phone: + 52 77 73 61 21 55
enriqueferrer_mx@yahoo.com
ic, and potassium sparing effects. However, the
mechanism that induces these effects has not yet
been elucidated. The aim of this study was to ex-
plore the possible mechanism of action for the di-
uretic effect of Hibiscus sabdariffa extract and its
fractions.
The aqueous extract from this plant and the frac-
tions obtained with solvents of different polar-
ities were administered to adrenalectomized rats,
and the diuretic effect was measured in the pres-
ence of deoxycorticosterone acetate (aldosterone
analog).
The effect on renal filtration was also evaluated in
an in situ kidney model, and finally, the effect of
diuretic active extracts on gene expression of the
alpha subunit from the transporter (ENaC) of re-
nal epithelial cell was quantified. The subsequent
results were obtained: The aqueous extract of Hi-
biscus sabdariffa presented the following chemi-
cal composition, 32.4 mg/g delphinidin-3-O-sam-
bubioside, 11.5 mg/g cyanidin-3-O-sambubio-
side, 11.5 mg/g quercetin, and chlorogenic acid
2.7 mg/g. The concentration of anthocyanins was
Introduction
! treatment [5]. In addition, these extracts can in-
Hibiscus sabdariffa Linn. (Malvaceae) is a crucial
resource used by Mexican traditional medicine in
the treatment of certain disorders included in the
metabolic syndrome such as dyslipidemia and
diabetes. H. sabdariffa extracts have been shown
to have different effects acting as hypocholestero-
lemic [1, 2], anti-hyperglucemic [2, 3], and espe-
cially as antihypertensive agents [4]. As to this last
characteristic, the H. sabdariffa extract is known
to possess specific effects on peripheral vascular
resistance and cardiac output, two therapeutic
increased. The diuretic effect caused by adre-
nalectomy in rats was reversed by deoxycortico-
sterone acetate activity. However, the effect of de-
oxycorticosterone acetate was antagonized by
spironolactone, the aqueous extract of Hibiscus
sabdariffa, and the acetonitrile : methanol 5 : 5
mixture extract, administered orally. A similar ef-
fect was observed on renal filtration obtained
from the isolated kidney model.
When the gene expression levels of ENaC was
measured in adrenalectomized rats, it was ob-
served that spironolactone, the aqueous extract
of Hibiscus sabdariffa, the acetonitrile : methanol
5 : 5 mixture, as well as the acetonitrile extract
significantly decreased the expression of this pro-
tein.
The conclusion of this work is that the diuretic,
natriuretic, and potassium sparing effects of Hi-
biscus sabdariffa are due in part to the modula-
tion of aldosterone activity by the presence in
the extract of this plant of compounds potentially
responsible for this modulation, as anthocyanins,
flavonoids, and chlorogenic acid.
targets of prime importance in hypertension
hibit the angiotensin-converting enzyme (ACE)
[6]. Regarding cardiac output, H. sabdariffa ex-
tracts have shown diuretic [710] and natriuretic
effects [1113], nonetheless, the mechanisms by
which they produce these effects on plasma vol-
ume and thereby in cardiac output have not been
described. The aim of the present work was to
demonstrate that H. sabdariffa calyx extracts pos-
sess diuretic, natriuretic, and potassium-sparing
effects with a mechanism of action by blocking
the expression of the epithelial sodium channel

Jimnez-Ferrer E et al. Diuretic Effect of Planta Med 2012; 78: 18931898

1894 Original Papers
(ENaC), which in turn is the target of aldosterone antagonists
[14].
Materials and Methods
! was excised together with the adjacent fat and the mesenteric
Plant material and extracts
This project was approved by IMSS (Mexican Social Security In-
stitute) on April 24, 2006 with the number FIS/IMSS/PROT/611
and by the National Council for Science and Technology (CONA-
CyT), Mexico, on August 18, 2008, with the number CB-2007-01
82 635.
The calyces of H. sabdariffa L. (Malvaceae) were obtained from a
controlled crop established in Xochitepec, Morelos, Mexico. A
specimen was prepared for reference, identified and deposited
at the IMSSM herbarium (Medicinal Plant Herbarium of the Mex-
ican Social Security Institute) with registration number 14 290.
The dried and ground plant material was extracted in water at
55 C for 2 h. The aqueous extract (HsAq) was concentrated in a
rotary evaporator and finally dried by lyophilization.
Extracts and fractions from H. sabdariffa
300 g of HsAq was suspended in 100 % acetronile for 24 h at room
temperature. The soluble fraction was filtered through Whatman
paper No. 4 (Whatman International Ltd.) and concentrated in a
rotary evaporator; the acetonitrile fraction (AN) was dried by lyo-
philization. The remaining precipitate was resuspended in a 9 : 1
acetonitrile : methanol mixture (9AN:1 M) and kept at room tem-
perature for 24 h. The 9AN:1 M extract was filtered, concentrated
and dried by lyophilization. The residual precipitate was resus-
pended in a 5 : 5 acetonitrile : methanol mixture (5AN:5 M), and
the same procedure was repeated until a dry extract was ob-
tained.
Chemical composition of H. sabdariffa
Compounds present in HsAq, AN, 9AN:1 M, 5AN:5 M, were deter-
mined and characterized by HPLC, equipped with diode array de-
tection and absorbance at 520 nm (Merck-Hitachi). The mobile
phase consisted of a 15 : 85 acetonitrile:1.1 % trifluoroacetic acid
system in water with a flow rate of 1 mL/min. The stationary
phase was a 0.45 20 cm RP-18 column (Merck). The H. sabdarif-
fa extracts were standardized by HPLC based on the concentra-
tion of delphinidin-3-O-sambubioside, cyanidin-3-O-sambubio-
side, quercitin, rutin, and chlorogenic acid.
Standard curves were built using commercial compounds (Sig-
ma-Aldrich). Anthocyanins were isolated and purified from plant
material following a previously reported procedure [6, 11].
Animals
!
Male albino SpragueDawley rats (280 g) (Harlan) were used for
all experiments. All animals were housed and maintained under
laboratory conditions at 25 C 3, with 12 h:12 h light/dark
schedule (lights on at 07 : 00 a. m.), with free access to water and
food (pellets from Harlan rodent lab diet). Animals were allowed
at least three weeks to adapt to the laboratory environment be-
fore experiments. All studies were carried out in accordance with
the official Mexican regulations NOM-062-ZOO-1999 (technical
specifications for production, care, and use of laboratory ani-
mals).
Jimnez-Ferrer E et al. Diuretic Effect of Planta Med 2012; 78: 18931898
Diuretic activity in adrenalectomized rats
Rats were adrenalectomized (ADX) under surgical anesthesia
with sodium pentobarbital (55 mg/kg). A 2-cm incision was made
at the costoventral angle, and the left and right adrenal glands
were removed without exposing the kidney. The complete gland
junction [15]. The rats were placed on a normal diet and 1 % sa-
line solution. On the 4th day after surgery, 7 groups (n = 6) were
formed, and each group started on the following 5-day oral treat-
ment: group 1 or basal group), 300 L of isotonic saline solution;
group 2) 30 mg/kg of deoxycorticosterone acetate (DOCA; Sigma-
Aldrich); group 3) 9 mg/kg spironolactone; group 4) 1000 mg/kg
aqueous extract of H. sabdariffa (HsAq); group 5) the combined
DOCA/spironolactone treatment; group 6) the combined HsAq/
DOCA treatment; group 7) the combined spironolactone/HsAq
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treatment. On the day of diuresis determination, each rat was
orally administered 3 mL of water, and urine elimination/24 h
was measured in metabolic cages. Sodium and potassium levels
were determined in the excreted urine.
Thereafter, the fractions obtained (HsAq, AN, 9AN:1 M, 5AN:5 M)
were used to perform the diuresis assay, applying the selected
treatment to group 6 which received the combination of H. sab-
dariffa fractions and DOCA, all else followed the described exper-
imental method.
In situ kidney diuresis model in adrenalectomized rats
Groups of adrenalectomized rats (n = 6) were formed. On day 4
after surgery, rats were subjected to a 10-day treatment with ex-
tracts HsAq, AN, 9AN:1 M, and 5AN:5 M administered orally at a
dose of 1000 mg/kg/day. The positive control group received oral
spiralactone at 9 mg/kg/day. After the treatment period, rats
were anesthetized (urethane 1.5 g/kg, ip) to prepare the in situ
kidney model. The animals were placed in the ventral position
on a surgery platform. In order to expose the kidney, a left lateral
surgical incision was done. The renal artery was tied at the aortic
bifurcation. The renal artery and vein, as well as the ureter were
channeled, and a catheter was inserted into the ureter to collect
samples. The channeled renal artery was used to perfuse the kid-
ney with Ringer-Krebs buffer (KRB) (123.3 mM NaCl; 6.17 mM
KCl; 3.29 mM CaCl2 7H2O; 0.78 mM MgSO4 7H2O; 32.14 mM
NaHCO3; 1.54 mM KH2PO4; 6.4 mM sodium pyruvate; 6.4 mM so-
dium glutamate; 7 mM sodium fumarate, and 11.1 mM glucose)
at a rate of 5 mL/min for 10 min, then the rate was decreased at
0.7 mL/min for 30 min, which corresponded to the test period.
During this period, the kidney was perfused, and samples were
collected. Afterwards, the kidney was removed, weighed and ho-
mogenized to determine protein content by the Bradford method
(Sigma-Aldrich). The glomerular filtration rate (FR) of each kid-
ney was calculated with the following formula:
FR = Excreted volume in 30 min/protein (mg)
RNA purification and amplification of the ENaC
receptor by RT PCR
Adrenalectomized rats that on day 4 after surgery began receiv-
ing 9 mg/kg/day oral spiranolactone were used. On days 0, 2, 5,
and 10 of this treatment, animals were sacrificed by overexpo-
sure to anesthesia, kidneys removed, and the -subunit from ep-
ithelial Na channels (ENaC) was determined. Kidneys from one
group of adrenalectomized rats without any treatment were
used to compare ENaC expression.
Having established the period of treatment needed to reveal the
effect of spironolactone on ENaC expression was up to 10 days,

two groups of adrenalectomized rats (n = 8) were formed and
orally treated with the same dose of 1000 mg/kg/day HsAq,
HsMeCN, 9AN:1 M, and 5AN:5 M. The positive control group re-
ceived spironolactone. All groups were treated for 10 days, after
which rats were sacrificed and both kidneys removed. They were
placed in 0.5 mL of TRIzol (Invitrogen) and frozen in liquid ni-
trogen at 80 C until total mRNA was extracted.
Kidneys were homogenized in TRIzol and subsequently centri-
fuged at 14 000 rpm for 10 min at 4 C. The supernatant was re-
covered with 200 L chloroform (Merck) per mL of TRIzol,
vigorously shaken (15 sec), set aside and left to rest (3 min) at
room temperature. Thereafter, the sample was centrifuged
(14 000 rpm/15 min/4 C). The inorganic phase was recovered
from the interphase and added with a volume of isopropanol
(70%, Merck). The RNA purification was performed using a con-
ventional methodology which can be summarized as follows:
The total volume was passed through a nucleic acid extraction
column (Qiagen). DNAse (Roche) nucleic acid digestion was al-
lowed to take place within the column (15 min) at room temper-
ature. The column was then washed with 700 L of buffer 1 (Qia-
gen) and centrifuged for 15 s, followed by two washings with
500 L of buffer 2 and centrifugation for 15 s. The column was
then centrifuged at 14 000 rpm for 2 min. RNA was eluted from
the centrifugation column with 60 L of water containing diethyl
pirocarbonate (DEPC) (Sigma-Aldrich, Cat. 40718). Samples were
kept at 20 C until used. RNA was quantified by spectrophotom-
etry.
For cDNA synthesis, 1 g of total RNA was added with 0.5 L of
50 M oligo (dT) 18 (Roche) and DEPC to a total volume of 6.5 L.
The reaction mixture was placed in a thermocycler (BIORAD) for
10 min at 65 C. Two L of buffer (250 mM Tris-HCl, 150 mM KCl,
40 mM MgCl, pH 8.5) were then added, 0.25 L of RNAse inhibitor
(40 U/L), 1 L of each of the four deoxyribonucleotides (10 mM),
and 0.25 L of inverse transcriptase (20 U/L). The mixture was
incubated for 10 min at 25 C and then for 60 min at 50 C.
Thereafter, cDNA was amplified by PCR. First, 2 L of cDNA were
added with 2.5 L of buffer (200 mM Tris-HCl, pH 8.4, 500 mM
KCl), 0.75 L of 50 mM magnesium chloride, 0.5 L of deoxynu-
cleotide mixture (dNTP, each at 10 mM), 0.5 L of rat oligo 1
scnn1a (48 nM) forward (Fw) (5TGCACCCTTAATCCTTACAGA-
TAC3), 0.5 L of rat oligo 2 scnn1a (39 nM) reverse (Rv) 3CAG
TGTCAGGGACAAACCATT5, and 0.5 L of Taq DNA polymerase
(platinum, 5 U/L; Invitrogen) and diluted to 25 L final volume
with milli-Q water. The reaction was amplified with a thermocy-
cler under the following conditions: 5 min at 94 C incubation fol-
lowed by 35 cycles of 1 min at 94 C for denaturalization, 1 min at
47 C for alignment, 1 min at 72 C for extension, and final incuba-
tion of 10 min at 72 C. In parallel, the same procedure was fol-
lowed using -actin oligos: 0.5 L (38.8 nM) (Fw) TCT ATG AGG
GTT ACG CGC TC, 0.5 L, and (44.6 nM) (Rv) CTT CTG CAT CCT
GTC AGC AA, which was used as an internal control. Oligonucleo-
tides used for ENaC were designed following the sequence re-
ported in the GenBank. The corresponding fragments are from
nucleotide 840863 for Fw and from nucleotide 14451425 for
Rv, which amplified a fragment of 606 bp corresponding to sub-
unit EnaC. The actin control was amplified for a fragment of 432
bp, corresponding to Fw nucleotides 575594 and Rv nucleotides
10071026. The DNA works software was used to determine the
mean alignment temperature (Tm) of each oligonucleotide.
Segments amplified by PCR were analyzed by 1.5 % gel agarose
electrophoresis using a 100 bp DNA marker (Invitrogen). The gel
was run (30 min/90 volts) and revealed with ethidium bromide,
Original Papers 1895
and photographed with light from an ultraviolet transilluminator
(FOTO/Analyst Apprentice Systems Fotodyne, Inc.). The same
procedure was followed for the actin oligo internal control. Varia-
tions in sample expression were evaluated by the Fotodyne pro-
gram (FOTO/Analyst Apprentice Systems Fotodyne, Inc.). An in-
dex of expression was prepared by contrasting the actin control
band with the experimental group band, assigning the values of
1 to maximum expression and 0 to minimum expression.
Statistical analysis
For all models, analysis of variance was applied with the Tukey
post hoc test to find statistically significant differences consider-
ing p < 0.05. SPSS 11.0 program was used.
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Results
!
The chemical characterization of H. sabdariffa extracts showed
that when the extract HsAq was fractionated and the polarity di-
minished, then the concentration of anthocyanins also fell until it
disappeared: delphinidin-3-sambubioside (Rt = 8.22 min) and cy-
anidin-3-sambubioside (Rt = 8.52 min) (l " Fig. 1 a). In the HsAq
extract, the two anthocyanins respectively showed concentra-
tions of 32.4 mg/g and 19.9 mg/g; and in the 5AN:5 M, concentra-
tions were 30.5 mg/g and 20.3 mg/g.
Other identified compounds included 3-caffeoyl quinic acid
(Rt = 7.35 min), 5-caffeoyl quinic acid (Rt = 8.14 min), 4-caffeoyl
quinic acid (Rt = 8.59 min), rutin (Rt = 10.02 min), quercitin
(Rt = 11.40 min). The chlorogenic acids (caffeoyl quinic acid com-
pounds) showed a general tendency to increase in concentration
as the polarity of the dissolving systems diminished (l " Fig. 1 b).
Total concentration of chlorogenic acids increased from 11.5 mg/
g in the HsAq (highest polarity) to 16.9 mg/g in the AN extract
(lowest polarity). From the whole content of chlorogenic acid
compounds, 55 to 59 % corresponded to 5-caffeoyl quinic acid
(Rt = 8.14 min). l " Fig. 1 also shows that flavonoid-type com-
pounds displayed a tendency to increase in concentration with
decreasing polarity; thus, rutin was increased from undetectable
levels until 0.12 mg/g and quercitin from 2.71 mg/g to 19.69 mg/
g. The lower concentration values of polyphenol compounds
were found in the extremely polar HsAq extract and the higher
values in the AN extract of the lowest polarity.
The results for diuretic activity of H. sabdariffa extracts in ADX
rats are shown in l " Table 1, where the ADX group significantly
increased diuresis (92.8 %, p < 0.05) compared with this parame-
ter for the rats in the basal group. The increments of sodium and
potassium elimination measured as saluretic index (S. i.) behaved
similarly, although these increments were higher when com-
pared to the diuretic effect (2.25 Na+ and 1.55 K+). DOCA (an aldo-
sterone analog) administration restores the effects on diuresis,
natriuresis, and kaliuresis provoked by ADX. Spironolactone and
HsAq extract induced an increment of diuresis, and this effect
was similar with both treatments. However, spironolactone pre-
sented a Na+/K+ ratio with higher natriuresis than kaliuresis lev-
els, although there is no significant difference between them. Si-
multaneous administration of DOCA and spironolactone as well
as DOCA and HsAq showed an antagonistic effect; in contrast,
the coadministration of spironolactone with the HsAq extract
showed a subadditive effect (l " Table 1).
Results from HsAq, 5AN:5 M, and 9AN:1 M extracts treatments in
ADX rats with DOCA showed that these treatments induced a sig-
nificant increase in diuresis in comparison to the basal condition
Jimnez-Ferrer E et al. Diuretic Effect of Planta Med 2012; 78: 18931898
1896 Original Papers

Fig. 1 Chromatograms of H. sabdariffa extracts:

HsAq extract line (1); 5AN:5 M extract line (2); ex-
tract 9AN:1 M line (3); AN extract line (4).
Panel a shows the detection at 520 nm of delphini-
din-3-sambubioside (Rt = 8.22 min) and cyanidin-3-
sambubioside (Rt = 8.52 min). Panel b shows the
detection at 350 nm of chlorogenic acid (Rt =
8.14 min), rutin (Rt = 10.02 min), and quercetin
(Rt = 11.40 min).

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Table 1 Effect produced by different treatments on adrenalectomized rat diuresis and urinary electrolyte excretion. ADX: adrenalectomized rats; DOCA: des-
oxicorticosterone acetate 30 mg/kg; Spr: spironolactone 9 mg/kg; HsAq: aqueous extract of H. sabdariffa 1 g/kg. Each value is a mean SD (n = 8); (S. i.) saluretic
index = mEq excreted problem group/mEq excreted control group. ANOVA and post hoc Tukey test. * = p < 0.05 compared to basal conditions; = p < 0.05 com-
pared to ADX.

Treatment Diuresis Na+ K+ S. i.

(mL) SD (mEq/100 g/5 h) SD (mEq/100 g/5 h) SD Na+ K+ Na+/K+
Basal 8.4 1.5 0.57 0.16 0.29 0.05 1.96
ADX 16.2 2.2* 1.29 0.37* 0.45 0.08 2.25 1.55 2.85
ADX/DOCA 10.0 1.5 0.45 0.19 0.28 0.08 0.80 0.96 1.60
ADX/Spr 29.6 2.1* 3.39 0.39* 0.85 0.07* 5.94 2.93 3.99
ADX/HsAq 24.3 7.6* 2.73 0.64* 0.79 0.17* 4.78 2.72 3.45
ADX/DOCA + Spr 39.8 5.3* 2.28 0.28* 1.02 0.17* 3.99 3.52 2.23
ADX/DOCA + HsAq 19.5 4.0* 2.04 0.18* 0.95 0.14* 3.57 3.27 2.14
ADX/HsAq + Spr 27.5 7.3* 1.64 0.35* 0.87 0.23* 2.56 3.00 1.88

(* p < 0.05), antagonizing the effect of DOCA, but no significant
difference was found when compared to the ADX group
(p > 0.05, l" Fig. 2).
The results from the in situ kidney diuresis model in ADX rats are
shown in l " Fig. 3. Mean filtration rate was compared by the fil-
tered volume (L) in respect to the concentration of protein (mg/
at day 10 of oral spironolactone treatment (9 mg/kg) at a level ap-
proximately half the basal expression value.
Based on the results obtained in the assay for the ENaC subunit
expression reduction, it was determined that the optimum peri-
od of treatment was 10 days of spironolactone or H. sabdariffa
extract administration. l " Fig. 5 shows the agarose gel which al-

g). The tendency was similar to that observed in the diuresis as-
say (l" Fig. 2) which determined the diuretic capacity of the dif-
ferent extracts. Firstly, the adrenalectomy provoked a signifi-
cantly increase of the renal filtration rate, which was reverted
by the administration of DOCA. The clearest effects on the renal
filtration rate were obtained using the more polar extracts (HsAq
and 5AN:5 M). However, the most conspicuous difference with
respect to the diuresis assay in adrenalectomized rats was that
the treatment with the 5AN:5 M extract showed significant var-
iation (p < 0.05) compared with basal conditions in controls and
adrenalectomized rats. Extracts with a lower polarity (AN and
9AN:1 M) showed an evident increase in the filtration rate, which
was significant compared with basal conditions.
The modulation of gene expression of ENaC after treatment
with H. sabdariffa extracts is shown in l " Fig. 4: the index of E-
NaC/-actin expression was measured by the band expression
density (approx. 600 bp) obtained in agarose gel was revealed
with ethydium bromide and is shown in the inset of l " Fig. 4. Sig-
nificant differences (p < 0.05) in basal expression appear from
day 5 (l " Fig. 4). This decrease in expression apparently stabilizes
lowed evidencing for the amplified segment that was measured
by densitometry, as well as the index of the density value of the
ENaC segment band induced in relation to -actin expression,
which is constitutive and is not affected by treatment adminis-
tration. Treatments with spironolactone, HsAq, 5AN:5 M, and AN
significantly reduced ENaC (* p < 0.05) compared with vehicle
administration. Although the differences among treatments were
not quantified, spironolactone administration was apparently
the most effective in reducing ENaC expression. Treatment with
HsAq followed in efficacy, and 5AN:5 M and AN showed similar
results and were the least effective.
Discussion
!
Recent studies established the diuretic, natriuretic, and potassi-
um-sparing effects, produced by acute administration of H. sab-
dariffa aqueous extract and measured them in an in situ kidney
model [13].

Jimnez-Ferrer E et al. Diuretic Effect of Planta Med 2012; 78: 18931898


Fig. 2 Diuretic effect produced by oral administration of the aqueous ex-
tract of H. sabdariffa (HsAq) and its fractions in adrenalectomized rats
(ADX). Each bar on the graph represents the average of six observations.
* = p < 0.05 on ANOVA test compared with basal conditions; = p < 0.05 on
ANOVA test compared with the ADX group.
The present study could establish the composition of the active
extracts, indicating that the compounds of higher polarity were
responsible for the pharmacological effect. This was confirmed
with an adrenalectomized rat kidney model, subchronically ad-
ministered with the different extracts, demonstrating again that
the more polar the compounds, the most effective they are as di-
uretics. Moreover, it was possible to establish that these same ex-
tracts decreased the expression of the ENaC subunit in rat kid-
neys exposed to the same treatment.
Initially, it was proposed that the mechanism of action resulting
in the diuretic effect could be explained by the relaxing effect on
smooth vascular muscle provoked by calcium antagonism or by
ACE inhibition, which could cause the increase in the glomerular
filtration rate and so partially justify the diuretic effect [13].
Based on this, the present work aimed to demonstrate that the
potassium-sparing diuretic effect of H. sabdariffa was due in part
to the modulation of the aldosterone action.
The adrenalectomized rat model caused an increase in diuresis
with Na+ and K+ elimination. The Na+/K+ ratio revealed that the
natriuretic effect was higher. Subchronic administration of DOCA
antagonized the aldosterone stimulus suppression effect associ-
ated to adrenalectomy; this became evident by the values
Original Papers 1897
Fig. 3 Renal filtration in the in situ kidney model. The basal conditions
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were perfusion with Ringer solution. The treatments were the aqueous ex-
tract of H. sabdariffa and fractions, n = 6, ANOVA and post hoc Tukey test.
* = p < 0.05 compared to basal conditions; = p < 0.05 compared to treat-
ment without the H. sabdariffa extract.
Fig. 4 Effect on gene expression of the ENaC
subunit in a daily dosage of spironolactone (9 mg/
kg v. o.) in the rat. The index for gene expression of
the transporter subunit on the expression of a con-
stitutive gene (-actin) is reported. The inset shows
the agarose gel of the amplified products by
RTPCR, revealed with ethidium bromide and read
under UV light. At the top, days of treatment are
shown and in the left margin, the size of the ampli-
fied segments. The ENaC subunit resulted in 600
bp and the actin segment in 450 bp. (*) ANOVA
p < 0.05 and post hoc Tukey test.
reached in diuresis, as well as Na+ and K+ saluretic indexes, but
mainly because of the Na+/K+ ratio. In this model, it was demon-
strated that H. sabdariffa induced a diuretic action, and it was de-
termined that the aqueous extract (HsAq) and the fraction of
highest polarity (5AN:5 M) were responsible for these effects. Ad-
ministration of HsAq had a diuretic effect, but the Na+ loss was
notably higher than K+ elimination since the value of the Na+/K+
ratio was 3.45. HsAq and 5AN:5 M administration to adre-
nalectomized rats treated with DOCA revealed a modulator effect
of the two treatments.
This was confirmed by the in situ kidney assay, in which no differ-
ence was found in the renal filtration rate when treated with
HsAq or 5AN:5 M. These treatments from H. sabdariffa contained
mainly anthocyanins: delphinidin-3-sambubioside and cyanidin-
3-sambubioside; phenyl propanoids such as chlorogenic acid
(mostly 5-caffeofyl quinic acid) and, to a lesser extent, flavonoids
such as quercitin and rutin. Also, these kinds of compounds were
capable of inhibiting the genomic effect of aldosterone on the ge-
netic expression of ENaC.
The main compounds found in the HsAq and the 5AN:5 M ex-
tracts, delphinidin-3-sambubioside and cyanidin-3-sambubio-
side [6, 11], and the flavonoids quercitin and rutin [16, 17], also
Jimnez-Ferrer E et al. Diuretic Effect of Planta Med 2012; 78: 18931898
1898 Original Papers
Fig. 5 Effect of daily oral dosage for 10 days of the aqueous extract and
fractions of Hibiscus sabdariffa (100 mg/kg). Values represent the gene ex-
pression index of ENaC regarding the expression of -actin. The agarose
gel revealed with ethidium bromide is shown at the bottom of the figure.
Each lane corresponds to the bars of the graphs. The amplified segment of
the ENaC subunit had a length of 600 bp. (*) ANOVA p < 0.05 and post
hoc Tukey test.
present in the tested extracts, have been described as efficient
ACE inhibitors which provoke a decrement of blood pressure by
diminishing the AGII levels. Another explanation for the diuretic
effect of H. sabdariffa as a modulator of aldosterone action is
through the presence of flavonoids. For example, it was demon-
strated that the application of quercitin in an in vitro epithelial
cell culture model, significantly diminished ENaC expression. It
is known that transepithelial Na+ hypotonicity in kidney cells
stimulates Na+ reabsorption by increasing mRNA expression of
the ENaC subunit and leads to a decrease in intracellular Cl con-
centration. Then, the activation of the Na+-K+ 2 Cl cotrans-
porter by quercitin increases the cytosolic concentration of Cl
and suppresses mRNA expression of the ENaC subunit [18].
In consequence, the decrease in AGII levels by ACE inhibition pro-
duced by H. sabdariffa administration diminishes the genetic ex-
pression of ENaC as shown in the assays presented herein. Fur-
thermore, the presence of quercitin may specifically decrease E-
NaC expression. The compounds contained in H. sabdariffa have
great therapeutic potential since they could control alterations
associated to overstimulation of RAAS, which have grave delete-
rious consequences as vascular damage secondary to endothelial
dysfunction. To conclude, H. sabdariffa has diuretic, natriuretic,
and potassium-sparing effects, at least in part by downregulation
of aldosterone, and compounds such as anthocyanins and flavo-
noids, present in this plant, are potentially responsible for the
modulation of aldosterone activity.
Acknowledgements
! pharmacol 2011; 134: 363372
This work was partially supported by CONACyT (project refer-
ence CB-2007-0182635) and the Mexican Institute of Social Se-
curity (project reference FIS/IMSS/PROT/611). We wish to thank
Abigail Aguilar for the identification of the H. sabdariffa speci-
men. Isabel Perez Montfort and Galia Lombardo Earl translated
the manuscript into English.
Jimnez-Ferrer E et al. Diuretic Effect of Planta Med 2012; 78: 18931898
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Conflict of Interest
!
All authors contributed to and have approved the final manu-
script. We declare not to have any conflict of interest regarding
the publication of this manuscript.
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