An in vivo study on the diuretic activity of Holarrhena antidysenterica
ABSTRACT Holarrhena antidysenterica is used as diuretic in traditional medicine. The aim of this study was to evaluate the crude extract of H. antidysentrica seeds (Ha.Cr) and its fractions, n-hexane (Ha.Hx), n-butanol (Ha.Bu) and aqueous (Ha.Aq), for their diuretic effect in Wistar rats and to investigate whether the activity is concentrated in any of the fractions. Wistar rats kept on fasting for 24 h with water ad labium, divided into normal, positive control and treated groups were orally given normal saline (20 ml/kg), hydrochlorothiazide (HCT; 10 mg/kg) and different doses of the plant material, respectively. Immediately after dosing, the rats were housed in the metabolic cages. The urine was collected at 2 h interval for 6 h and volume, pH and electrolytes levels were measured. Ha.Cr caused dose-dependent (30 and 100 mg/kg) increase in urine output, indicating the diuretic effect. In addition, Ha.Cr increased urine contents of Na+ and K+, suggesting that the diuretic effect is mediated through increased electrolyte excretion. Similarly, the reference drug, HCT (10 mg/kg), increased urine volume and Na+ and K+ excretion. None of the resultant fractions exhibited diuretic effect comparable to that of the parent crude extract. Ha.Hx was devoid of diuretic effect, Ha.Bu exhibited a mild diuretic effect at 30 mg/kg, whereas, Ha.Aq caused a significant increase in urine output only at 100 mg/kg, indicating that the diuretic activity is distributed among fractions but in an order of increasing polarity of the solvent. The enhanced diuretic effect in the crude extract as compared to any individual fraction is suggestive of the existence of additive and/or synergistic effect in the crude extract. This study shows the presence of diuretic activity in the H. antidysentrica possibly mediated through its saluretic effect, which rationalizes its medicinal use as diuretic.
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ABSTRACT: Holarrhena antidysenterica has a traditional use in the treatment of urolithiasis, therefore, its crude extract has been investigated for possible antiurolithic effect. The crude aqueous-methanolic extract of Holarrhena antidysenterica (Ha.Cr) was studied using the in vitro and in vivo methods. In the in vitro experiments, Ha.Cr demonstrated a concentration-dependent (0.25-4 mg/ml) inhibitory effect on the slope of aggregation. It decreased the size of crystals and transformed the calcium oxalate monohydrate (COM) to calcium oxalate dehydrate (COD) crystals, in calcium oxalate metastable solutions. It also showed concentration-dependent antioxidant effect against 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and lipid peroxidation induced in rat kidney tissue homogenate. Ha.Cr (0.3 mg/ml) reduced (p < 0.05) the cell toxicity and LDH release in renal epithelial cells (MDCK) exposed to oxalate (0.5 mM) and COM (66 μg/cm(2)) crystals. In male Wistar rats, receiving 0.75 % ethylene glycol (EG) for 21 days along with 1 % ammonium chloride (AC) in drinking water, Ha.Cr treatment (30-100 mg/kg) prevented the toxic changes caused by lithogenic agents; EG and AC, like loss of body weight, polyurea, oxaluria, raised serum urea and creatinine levels and crystal deposition in kidneys compared to their respective controls. These data indicate that Holarrhena antidysenterica possesses antiurolithic activity, possibly mediated through the inhibition of CaOx crystal aggregation, antioxidant and renal epithelial cell protective activities and may provide base for designing future studies to establish its efficacy and safety for clinical use.Urological Research 05/2012; · 1.59 Impact Factor
African Journal of Pharmacy and Pharmacology Vol. 6(7), pp. 454-458, 22 February, 2012
Available online at http://www.academicjournals.org/AJPP
ISSN 1996-0816 ©2012 Academic Journals
Full Length Research Paper
An in vivo study on the diuretic activity of Holarrhena
Aslam Khan1,2, Samra Bashir2 and Anwarul-Hassan Gilani2*
1Department of Pharmacology, Faculty of Pharmacy, University of Karachi, Karachi, Pakistan.
2Natural Product Research Division, Department of Biological and Biomedical Sciences,
the Aga Khan University Medical College, Karachi-74800, Pakistan.
Accepted 28 November, 2011
Holarrhena antidysenterica is used as diuretic in traditional medicine. The aim of this study was to
evaluate the crude extract of H. antidysentrica seeds (Ha.Cr) and its fractions, n-hexane (Ha.Hx), n-
butanol (Ha.Bu) and aqueous (Ha.Aq), for their diuretic effect in Wistar rats and to investigate whether
the activity is concentrated in any of the fractions. Wistar rats kept on fasting for 24 h with water ad
labium, divided into normal, positive control and treated groups were orally given normal saline (20
ml/kg), hydrochlorothiazide (HCT; 10 mg/kg) and different doses of the plant material, respectively.
Immediately after dosing, the rats were housed in the metabolic cages. The urine was collected at 2 h
interval for 6 h and volume, pH and electrolytes levels were measured. Ha.Cr caused dose-dependent
(30 and 100 mg/kg) increase in urine output, indicating the diuretic effect. In addition, Ha.Cr increased
urine contents of Na+ and K+, suggesting that the diuretic effect is mediated through increased
electrolyte excretion. Similarly, the reference drug, HCT (10 mg/kg), increased urine volume and Na+ and
K+ excretion. None of the resultant fractions exhibited diuretic effect comparable to that of the parent
crude extract. Ha.Hx was devoid of diuretic effect, Ha.Bu exhibited a mild diuretic effect at 30 mg/kg,
whereas, Ha.Aq caused a significant increase in urine output only at 100 mg/kg, indicating that the
diuretic activity is distributed among fractions but in an order of increasing polarity of the solvent. The
enhanced diuretic effect in the crude extract as compared to any individual fraction is suggestive of the
existence of additive and/or synergistic effect in the crude extract. This study shows the presence of
diuretic activity in the H. antidysentrica possibly mediated through its saluretic effect, which
rationalizes its medicinal use as diuretic.
Key words: Holarrhena antidysenterica, diuretic, rats, Na+, K+.
Diuretics are drugs that promote the rate of urine flow
and sodium excretion. Diuretics alone or in combination
with other drugs are used in a variety of clinical situations
like hypertension, heart and renal failure, nephritic
syndrome and cirrhosis (Jackson, 2006). Along with this,
*Corresponding author. E-mail: email@example.com. Tel:
(+92) 21-3486 4571. Fax: (+92) 21-3493 4294, 3494 2095.
diuretics can also be helpful in diluting the ion contents of
the urine, leading to reduction in the supersaturation of
stone forming ions and also help in expulsion of crystals,
thus preventing recurrent renal stones (Ulmann et al.,
1984). The two commonly used diuretics, that is,
thiazides and furosemide, have been associated with
many side effects, like disturbances of electrolytes, acid-
base and water balance, changes in uric-acid,
carbohydrate and lipid metabolism and drug interactions
(Losse et al., 1983; Ide and Sunagawa, 2007). Therefore,
there is a need to look for safe diuretics. Herbal
medicines are considered to be more safe and econo-
mical sources of drugs and also contain synergistic
and/or side effects neutralizing potential (Gilani and Atta-
ur-Rahman, 2005). Therefore, herbal diuretics can be
considered as better therapeutic option, because of their
relatively safer and milder actions as compared to
diuretics used nowadays which produce several adverse
effects due to their strong saluretic effects. As most of the
plants contain potassium, along with other nutritional
elements like Na+, Mg2+, Ca2+, Zn2+, etc (Jan, 2011), it
can be assumed that they would not lead to potassium
depletion (Meléndez, 2004; Gasparotto et al., 2009).
Holarrhena antidysenterica Wall, commonly known as
bitter oleander and locally as “Inderjo Tulkh” or “Kurchi”
belongs to the family of Apocynaceae. It is a small
deciduous tree found at Himalayan and sub-Himalayan
tracts (Baquar, 1989). Traditionally, H. antidysentrica is
used in the treatment of various disorders like colic,
diarrhea, dysentery and fever (Bajrai, 2010). It is also
used as carminative, astringent, lithontriptic, tonic, aphro-
disiac, cardio suppressant, diuretic and antihypertensive
(Baquar, 1989; Usmanghani et al., 1997; Duke, 2002).
In our previous study (Gilani et al., 2010), the
phytochemical screening of H. antidysentrica has shown
the presence of alkaloids, coumarins, flavonoids,
saponins and tannins, whereas, conessine, ergostenol,
holarrhenine, kurchicine, resin and tannin have been
reported among the plant constituents (Kapoor, 1990;
H. antidysentrica has been studied for antimutagenic
(Atal et al., 1986), antibacterial (Ahmad et al., 1998; Aqil
and Ahmad, 2007), immuno-modulatory (Aqil et al.,
2008), antioxidant (Kalim et al., 2010), antihyperglycemic
(Ali et al., 2011), anti-malarial (Verma et al., 2011),
spasmolytic and spasmogenic properties (Gilani et al.,
2010). Since the scientific data on the traditional use of
H. antidysentrica as diuretic is sparse, this study was
undertaken to investigate the diuretic activity of its crude
extract and the resultant fractions using rats as
MATERIALS AND METHODS
Plant, preparation of crude extract and fractions
Dried seeds of H. antidysenterica were purchased from local herbal
store, identified by a taxonomist, Professor Dr. Jhandar Shah,
University of Malakand, Chakdara, Khyber Pakhtunkhwa and
voucher specimen (HA-SE-01-08-71) was submitted to the
herbarium of the Department of Biological and Biomedical
Sciences, the Aga Khan University, Karachi. The detail of the 70%
aqueous-ethanol crude extract and its fraction preparation is given
in our earlier publication of this plant (Gilani et al., 2010). Yield of
the crude extract was approximately 18% (w/w), while n-hexane, n-
Butanol and aqueous fractions yielded 10, 25 and 65%,
Khan et al. 455
Chemicals and drugs
The solvents, methanol, n-butanol and n-hexane, for the extraction
and fractionation were obtained from Mark, Darmstadt, Germany.
Hydrochlorothiazide (HCT); reference diuretic drug, was obtained
from the Sigma Chemicals Co, St Louise, MO, USA. All drugs and
chemical used were of analytical grade.
Wistar rats (180 to 220 g) used for this study were sourced locally
and housed at the animal house of the Aga Khan University, kept in
plastic cages (47 × 34 × 18 cm3) with saw dust (renewed after every
48 h), under a controlled temperature of 23 to 25°C and 12 h light-
dark cycle. Animals were given standard diet consisting of flour (5
kg), bran (5 kg), molasses (150 g), salt (75 g), nutrivet L (33 g),
potassium meta bisulphate (15 g), oil (500 g), fish meal (2.25 kg)
and powdered milk (2 kg) for a total of 13 kg of the food material.
Animals had access to food and water ad-libitum throughout the
study except 24 h before and during 6 h of diuretic study.
Experiments performed complied with the rulings of the Institute of
Laboratory Animal Resources, Commission on Life Sciences,
National Research Council (1996).
Determination of diuretic activity
Diuretic activity of the crude extract (Ha.Cr) and its fractions, n-
hexane (Ha.Hx), n-butanol (Ha.Bu) and aqueous (Ha.Aq), was
studied on Wistar rats (180 to 220 g), as described previously
(Consolini et al., 1999). Animals were divided with matched body
weight and sex into groups of 6 animals each. Normal saline and
positive control groups received oral doses of saline (20 ml/kg) and
standard diuretic drug: HCT, 10 mg/kg body weight, respectively.
The rest of the groups were given 2 to 3 different doses of the test
material dissolved in saline. Subsequently, the animals were placed
individually in metabolic and diuretic cages (Techniplast, 21020
Buguggiate-Va-Italy). The urine was collected in graduated
cylinders for 6 h at 2 h intervals. Total urine excreted out was
collected and the volume was determined. pH of the pooled urine
from each animal was determined by using pH meter (Orion* 320
PerpHecT* Thermo Scientific) and Na+ and K+ concentrations was
determined on flame photometer (Flame Photometer 410, Corning).
The data are expressed as mean ± standard error of mean (SEM).
All statistical comparisons between the groups are made by mean
of one-way analysis of variance (ANOVA) with post hoc Dunnett’s
test, using GraphPad Prism (GraphPad Software, San Diego, CA,
USA). P value less than 0.05 is regarded as significant.
RESULTS AND DISCUSSION
In this study, the diuretic activity of the crude extract of H.
antidysentrica and its fractions were investigated to
rationalize its medicinal use as a diuretic agent, which
might also be contributing to its antihypertensive and
antiurolithic activities. Urine output/100 g body weight/6 h
in saline treated group was 0.98 ± 0.12 ml (mean ± SEM),
while HCT (10 mg/kg) increased it to 2.68 ± 0.23 ml (P <
456 Afr. J. Pharm. Pharmacol.
K+ (mmol/100 g body weight/6 h)
Na+ (mmol/100 g body weight/6 h)
Urine volume/100 g body weight/6 h
Figure 1. Effect of the crude extract of H. antidysentrica and hydrochlorothiazide (HCT) on
urine volume (A), pH (B) K+ (C) and Na+ contents (D), collected after 6 h of their administration
(values shown are means ± SEM, n = 6). *P < 0.05, **P < 0.01 versus saline treated group.
0.001), as shown in Figure 1A. Ha.Cr at the doses of 30,
100 and 300 mg/kg increased the urine volume to 1.68 ±
0.119 ml (P < 0.05), 2.070 ± 0.18 ml (P < 0.01) and 1.76
± 0.22 ml (P < 0.05), respectively, indicating its diuretic
effect (Figure 1A). At the highest dose (300 mg/kg) used
in this study, there was blunting of the response by
approximately 20% as compared to 100 mg/kg. The
maximum diuretic efficacy of Ha.Cr was about 20% less
than that of HCT in terms of cumulative urine volume.
The decrease in the diuretic activity on higher doses
could be due to the co-existence of antidiuretic compo-
nent(s) in Ha.Cr, as plant extract may exhibit multiple
therapeutic activities probably on account of having a
mixture of phytochemical. The presence of synergistic
and/or side effect neutralizing effect in plants is known to
exist, which is probably meant by nature not to allow the
pharmacological effect go beyond its therapeutic limit,
above which it could have caused toxic effects (Gilani
and Atta-ur-Rahman, 2005). In addition to its effect on
urine volume, Ha.Cr also increased urine excretion of Na+
and K+ (Figure 1C and D). Total K+ and Na+ contents
(mmol/100 g body weight) in 6 h pooled urine samples
were 0.21 ± 0.02 (P < 0.05) and 0.16 ± 0.01, 0.23 ± 0.02
(P < 0.01) and 0.23 ± 0.03 (P < 0.05), and 0.21 ± 0.02 (P
< 0.05) and 0.16 ± 0.03 (P < 0.01), respectively, at 30,
100 and 300 mg/kg, as opposed to, 0.13 ± 0.02 and 0.11
± 0.03, with saline group. The reference drug HCT also
increased the K+ and Na+ excretion to 0.22 ± 0.01 (P <
0.01) and 0.31 ± 0.04 (P < 0.01) mmol/100 g body
weight/6 h, respectively (Figure 1 C and D), whereas, the
urine pH did not change significantly in any group (Figure
1B). The mechanism of diuretic actions of Ha.Cr is
possibly due to the increase of electrolytes, like K+ and
Na+ excretion by inhibiting their tubular reabsorption, a
Khan et al. 457
Urine volume/100 g body weight/6 h
K+ (mmol/100 g body weight/6 h)
Na+ (mmol/100 g body weight/6 h)
Figure 2. Effect of the n-hexane, n-butanol and aqueous fractions of H. antidysentrica extract on
urine volume (A), pH (B), K+ (C) and Na+ (D) contents, collected after 6 h of their administration.
(Values shown are means ± SEM, n = 6). *P < 0.05, **P < 0.01 versus saline treated group.
a mechanism attributed to the standard drug used in this
study (hydrochlorothiazide). The presence of flavonoids
and saponins in the plant extract (Gilani et al., 2010)
could be contributing in its diuretic effect, as these
phytochemicals are known to possess diuretic properties
(Maghrani et al., 2005), however, additional mecha-
nism(s) may not be ruled out.
In order to check whether the diuretic effect is
concentrated in any of the fractions, we carried out the
activity-guided fractionation and evaluated their diuretic
effect. The doses of the fractions were selected in
proportion to the percentage yield of the fractions,
obtained from crude extract. Figure 2A shows the effect
of different fractions on the urine output. Ha.Hx was
devoid of any diuretic affect, the Ha.Bu showed a mild
increase in the urine volume (1.75 ± 0.11, P > 0.05) at the
dose of 30 mg/kg, while the aqueous fraction showed
significant increase (1.92 ± 0.24, P < 0.05) only at the
dose of 100 mg/kg, as compared to normal saline (1.07 ±
0.16) (Figure 2A). Like crude extract, there was no
change observed in pH and Na+ excretion of all the
groups (Figure 2B and D), whereas, urinary excretion of
K+ was slightly increased (0.12 ± 0.012, P > 0.05) by
Ha.Bu at the dose of 30 mg/kg and significantly by Ha.Aq
(0.14 ± 0.01, P < 0.05) at the dose of 100 mg/kg, as
compared to the normal saline group (0.08 ± 0.008), as
shown in Figure 2C. Whereas, HCT very significantly
increase urinary excretion of both K+ (0.16 ± 0.01, P <
0.01) and Na+ (0.25 ± 0.02, P < 0.01) at 10 mg/kg (Figure
2C and D). Loss of efficacy with fractionation suggests
the existence of multiple constituents exhibiting additive
and/or synergistic effect in the crude extract that is
458 Afr. J. Pharm. Pharmacol.
segregated among the fractions, thereby, reducing
intensity of the diuretic action.
Diuretics alone or in combination with other antihy-
pertensive drugs are considered to be more effective
than the calcium channel blockers and angiotensin
converting enzymes inhibitors as the first line treatment of
hypertension. It also helps in the prevention of one or
more forms of cardiovascular diseases in high-risk
patients with hypertension (Boger-Megiddo et al., 2010).
The seventh report guidelines issued in the United States
by the Joint National Committee on prevention,
evaluation, and treatment of high blood pressure, and
England and Wales, the National Institute for Health and
Clinical Excellence guidelines recommend the use of low
dose diuretics as first line pharmacological treatment for
high blood pressure (Boger-Megiddo et al., 2010). The
diuretic therapy is also useful in the treatment of edema,
hypercalcemia, hepercalceuria, diabetes insipidus and
acute renal failure (Krumlovsky and del Greco, 1976).
The presence of diuretic activity in H. antidysentrica may
explain the medicinal use of the plant in hypertension and
urolithiasis, though additional mechanism(s) may not be
In this study, we found the presence of diuretic effect in
H. antidysentrica, possibly mediated through its saluritic
potential. All the fractionations were found less
efficacious than the parent crude extract suggesting the
existence of additive and/or synergistic effect in the crude
extract. These results provide rational for its medicinal
use as a diuretic agent.
This study was supported by the Higher Education
Commission (HEC) of Pakistan as indigenous M.Phil/PhD
scholarship awarded to Aslam Khan.
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