Studies on the in vitro and in vivo antiurolithic activity of Holarrhena antidysenterica.

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.

Full text

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ORIGINAL PAPER
Studies on the in vitro and in vivo antiurolithic activity
of Holarrhena antidysenterica
Aslam Khan•Saeed R. Khan•Anwar H. Gilani
Received: 3 February 2012/Accepted: 2 May 2012
? Springer-Verlag 2012
Abstract
inthetreatmentofurolithiasis,therefore,itscrudeextracthas
been investigated for possible antiurolithic effect.The crude
aqueous-methanolic extract of Holarrhena antidysenterica
(Ha.Cr)wasstudiedusingtheinvitroandinvivomethods.In
the in vitro experiments, Ha.Cr demonstrated a concentra-
tion-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 oxa-
late metastable solutions. It also showed concentration-
dependent antioxidant effect against 2,2-diphenyl-1-picryl-
hydrazyl (DPPH) free radicals and lipid peroxidation
induced inrat kidneytissue homogenate.Ha.Cr(0.3 mg/ml)
reduced(p\0.05)thecelltoxicityandLDHreleaseinrenal
epithelial cells (MDCK) exposed to oxalate (0.5 mM) and
COM (66 lg/cm2) crystals. In male Wistar rats, receiving
0.75 % ethylene glycol (EG) for 21 days along with 1 %
ammoniumchloride(AC)indrinkingwater,Ha.Crtreatment
(30–100 mg/kg) prevented the toxic changes caused by
lithogenic agents; EG and AC, like loss of body weight,
Holarrhenaantidysentericahasa traditional use
polyurea, oxaluria, raised serum urea and creatinine levels
and crystal deposition in kidneys compared to their respec-
tive controls. These data indicate that Holarrhena antidy-
senterica possesses antiurolithic activity, possibly mediated
through the inhibition of CaOx crystal aggregation, antiox-
idant and renal epithelial cell protective activities and may
provide base for designing future studies to establish its
efficacy and safety for clinical use.
Keywords
In vitro ? In vivo ? MDCK cell line ? Rats
Urolithiasis ? Holarrhena antidysenterica ?
Introduction
Urolithiasis, urinary stone formation, is the third most
common problem of the urinary tract, sparing no geo-
graphical, racial and cultural boundaries and is associated
with high rate of recurrence [1, 2]. In Pakistan its preva-
lence is 10–15 %, while its complication can lead to acute
or chronic renal failure, pyelonephritis, pyonephritis and
perinephric abscess, which can present with life threatening
situations and even death [3].
Despitethefactthatthecurrentlyavailablestoneremoval
techniques like extracorporeal shock wave lithotripsy
(ESWL), ureteroscopy (URS), and percutaneous nephroli-
thotomy (PNL), are considered to be effective, but they are
costly, making them an option of choice for limited number
of patients and a compelling data suggest that these tech-
niqueshavesomeserioussideeffects[4].Moreover,thehigh
rate of recurrence in stone formation, which is around 50 %
ata5-yearfollow-up[3],makesitachronicconditionwhich
underscores the importance of preventive therapy [5]. In
spiteofsubstantialprogressinthestudyofthebiologicaland
physical manifestation of urolithiasis, its mechanism is still
A. Khan
Department of Pharmacology, Faculty of Pharmacy,
University of Karachi, Karachi, Pakistan
e-mail: aslamkhan_mkd@yahoo.co.uk
A. Khan ? A. H. Gilani (&)
Natural Product Research Division, Department of Biological
and Biomedical Sciences, Aga Khan University Medical
College, Karachi 74800, Pakistan
e-mail: anwar.gilani@aku.edu
S. R. Khan
Centre for the Study of Lithiasis, Department of Pathology,
College of Medicine, University of Florida, Gainesville, USA
e-mail: khan@pathology.ulf.edu
123
Urol Res
DOI 10.1007/s00240-012-0483-1

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not clearly understood [6] and there is no satisfactory drug
available for the treatment of urolithiasis, especially for the
prevention of recurrence of the stones [7]. The agents used
clinically for prophylactic therapy are primarily aimed to
correct the underlying metabolic disorders, but the evidence
for their effectiveness is still not convincing in addition to
their side effects and tolerability [8, 9]. One reason for a
limited success of chemical drugs in urolithiasis is that
multiple factors are involved in its pathogenesis [10] and
thus treatment demands multiple targets, such as antispas-
modic, antioxidant, anti-inflammatory activities [11]. The
medicinalplantsthatareknowntocontainmultiplechemical
constituents, which could offer a synergistic and/or side-
effect neutralizing combinations [12], are likely to offer
more effective and safer remedy particularly in urolithiasis.
Therefore, there is a need to look for an alternative therapy,
especially herbal remedies, for the management and treat-
ment of urolithiasis, which have the potential to be effective
in urolithiasis [13]. In addition, herbal medicines are con-
sidered to be relatively safer, economical and accessible to
large population of the developing countries like Pakistan
[12]. Therefore, we evaluated the seeds of Holarrhena an-
tidysenterica Wall (Apocynaceae) for its antiurolithic
activity because of its traditional use as a lithotriptic, along
with other uses like colic, diarrhea, dysentery, constipation,
flatulence, carminative, antispasmodic, astringent, diuretic
and antihypertensive [14, 15].
Holarrhena antidysenterica is known to contain cones-
sine, ergosterol, holarrhenine, kurchicine, resin and tannin
[16, 17] and has been reported to possess antimutagenic
[18], antibacterial [19], immuno-modulatory [20], anti-
spasmodic [21] and diuretic [22] properties.
This study has been undertaken to investigate the anti-
urolithic activity of the seeds of Holarrhena antidysenter-
ica using Ethylene glycol-induced rat model of CaOx
urolithiasis and the in vitro assays to investigate its calcium
oxalate crystallization inhibitory, antioxidant, renal cell
protective and antispasmodic activities.
Materials and methods
Chemicals and reagents
The following chemicals were obtained from the Merck,
Darmstadt, Germany, BDH Laboratory supplies, Poole,
EnglandandSigmaChemicalCompany,St.Louis,MO,USA.
Kits used in this study for the determination of calcium,
magnesium,andbloodureanitrogen(BUN)weresuppliedby
RandoxLaboratoriesLtd.Ardmore,DiamondRoad,Crumlin,
Co.Antrim,UK.Oxalateestimationwasdonebythekitfrom
Trinity Biotech Plc, IDA business park, Bray, Co. Wicklow.
All the chemicals used were of analytical grade available.
Animals
Experiments were performed in compliance with the rul-
ings of the Institute of Laboratory Animal Resources,
Commission on Life Sciences, National Research Council
(1996) [23] and approved by the Ethical Committee for
Research on Animals (ECRA) of the Aga Khan University,
Karachi, Pakistan.
Plant material and extraction
Dried seeds of Holarrhena antidysenterica were taken
from local herbal store, identified by a taxonomist, Prof.
Dr. Jhandar Shah, University of Malakand, Chakdara,
Khyber Pakhtunkhwa, Pakistan 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 clean seeds were
extracted with 70 % aqueous-ethanol and kept for 3 days
with occasional shaking. It was filtered through a muslin
cloth and then through a Whatman qualitative grade 1 filter
paper. This procedure was repeated twice and the com-
bined filtrate was evaporated on rotary evaporator under
reduced pressure (-60mmHg) to a thick, semi-solid mass
of brown color, i.e. the crude extract of Holarrhena anti-
dysenterica seeds (Ha.Cr), yielding *18 % w/w [24].
In vitro experiments
Determination of CaOx crystallization
The effect of the Ha.Cr on kinetics of calcium oxalate
(CaOx) crystallization was determined by the time course
measurement of turbidity changes due to the crystal
nucleation and aggregation after mixing metastable solu-
tions of calcium (Ca??) and oxalate (Ox). While the effect
on the number, size and morphology of the crystals was
determined by incubating different concentrations of Ha.Cr
with the metastable solutions of Ca??and Ox as described
previously [25].
Determination of antioxidant activity
Antioxidant potential of the Ha.Cr was estimated by its
effect on free radical scavenging activity [26] and its
inhibitory effect against lipid peroxidation induced in rat
kidney homogenate with ferrous-ascorbate system, as
described previously [27].
Cell lines (MDCK) experiment
Madin Darby canine kidney (MDCK) cells, obtained from
ATCC (Cat.# CRL-34; Manassa, VA, U.S.A), were
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maintained as sub-confluent monolayers at 37 ?C in 5 %
CO2. The culture was grown and maintained in 75 cm2
Falcon tissue culture flasks as discussed previously.
Determination of cell viability and LDH release
After incubating the cells for 24 h with Ox (0.5 mM) or
COM crystals (67 lg/cm2), renal cells toxicity was deter-
mined by XTT cell viability Assay Kit (Biotium, Inc.,
Haward, Ca, USA Cat # 30007), while LDH release was
estimated by CytoTox 96 Non-Radioactive Cytotoxicity
assay kit (Fisher Scientific, Norcross, GA, Promega Cat #
PR-G1780).
In vivo experiments
Study on animal model of urolithiasis
Antiurolithic activity of the test material was determined
usinghyperoxaluricratmodelofCaOxurolithiasisdescribed
previously [11, 28, 29]. Male Wistar rats (weighing
180–220 g) were divided with matched body weights into
groups of 6–8 animals each, which were then randomly
selectedtoreceivevarioustreatments.Inthisstudy,ratswere
divided into five groups. Group 1 serving as normal control,
received intraperitoneal (i.p.) injections of normal saline
(2.5 ml/kg), once in 24 h. Group II serving as the Ha.Cr
control received only the Ha.Cr (100 mg/kg), Group III
serving as the lithogenic control received stone inducing
treatment, while group IV and V received the lithogenic
treatment along with the Ha.Cr 30 and 100 mg/kg, respec-
tively. Lithogenic treatment was given for 21 days and
comprised of 0.75 % (w/v) ethylene glycol (EG) with 1 %
(w/v)ammoniumchloride(AC)indrinkingwaterfor5 days,
following this the water supply was switched to 0.75 % EG
alone in water [28, 30], along with saline treatment. Animal
weight and activity were regularly monitored to assess their
overall health. 24 h urine samples were collected immedi-
ately before the onset and at the end of total 21 days of
treatment, for which animals were housed individually in
metabolic and diuretic cages. The 3 h morning urine for
crystalluria study was also collected at the end of 21 days of
treatments. The number of the crystals/mm3was counted
under light microscope using hemocytometer.
Following volume and pH determination, part of each
24-h urine sample was acidified to pH 2 with 5 M HCl.
Both acidified and non-acidified urine samples were then
centrifuged at 1,500g for 10 min to remove debris and
supernatants were stored at -20 ?C until analyzed. Blood
was collected through cardiac puncture from animals under
ether anesthesia for serum separation to assess serum cre-
atinine and BUN. Animals were sacrificed and both the
kidneys were excised, rinsed in ice cold physiological
saline and weighed. The right kidney was fixed in 10 %
neutral buffered formalin, processed, embedded in paraffin
wax, sectioned at 5 lm and stained with hematoxylin and
eosin (H & E) and by Pizzolato’s method, for calcium
oxalate crystals [31], for microscopic examination.
Calcium oxalate crystal deposition in kidney
Crystaldistributionwithinthekidneyswasdeterminedusing
thesemiquantitative scoringbythemethods usedpreviously
[11, 32]. Briefly, the crystal deposits in stained sections with
visible in a field of 109 magnification were counted and
severity grades were assigned
1 = B1–10, 2 = B11–30, 3 = B31–50, 4 = B51–75 and
5 =[75 crystals. Most of the crystals were located in the
outer modularly and cortical region of the kidney.
as0 =\1crystals,
Biochemical analysis of urine and serum
In acidified urine samples, Ox, Ca??and Mg??contents
were determined using commercially available kits. Inor-
ganic phosphate (PO4
molybdenum blue reaction [33]. In non-acidified urine
samples, citrate, creatinine and uric acid (UA), while in
serum, creatinine and BUN were estimated with the help of
kit-based methods. Total protein in non-acidified urine was
estimated by lowery method [34] based on the develop-
ment of blue color as a result of a combination of two
reaction involving the interaction Cu2?in the presence of
base with peptide bonds and Folin–Ciocalteu phenol
reagent with tyrosine and tryptophan residues.
-2) excretion was determined by the
Data analysis
The data expressed are mean ± standard error of mean
(SEM) and the median effective concentration (EC50
value) with 95 % confidence intervals (CI). All statistical
comparisons between the groups are made by means of
t test (comparison between two groups) or one way anal-
ysis of variance (ANOVA) with post hoc Dunnett’s test
using GraphPad Prism (GraphPad Software, San Diego,
CA, USA). p value\0.05 is regarded as significant.
Results
In vitro experiment
Effect on in vitro crystallization
EffectofHa.CronvariousphasesofCaOxcrystallizationwas
determined by time course measurement of turbidity under
standard conditions (4.25 mM Ca??and 0.75 mM Ox).
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Typical tracings of the experiment in the presence of Ha.Cr
andpotassiumcitrateare showninFig. 1a,b.Ha.Crinhibited
the SAwith an IC50of 0.27 mg/ml (95 % CI 0 23–0.33),
similar to potassium citrate, which caused dose-dependent
inhibitionwithIC50valueof0.31 mM(95 %CI0.30–0.52)as
shown in Fig. 1c. Ha.Cr did not significantly decrease the
slopes of nucleation (1–4 mg/ml), while potassium citrate
caused 31 ± 2.31, 42 ± 4.0, 65 ± 4.2 and 85 ± 5.2 %
inhibition at a concentration of 0.5, 1, 2 and 4 mM, respec-
tively (Fig. 1d). In the incubation study, mixing the metasta-
ble solutions of Ca??and Ox resulted in the formation of
CaOxcrystalspredominatelyofCOM(Fig. 2a).Ha.Crdidnot
influence the number of crystal formation, but significantly
decreasedthe sizeandmorphologyofthe crystals fromCOM
to COD (Fig. 2b), whereas potassium citrate reduced the
numberaswellasthesizeandmorphologyofthecrystalsfrom
COM to COD (Fig. 2c).
Antioxidant effect
Ha.Cr caused inhibition of DPPH free radical with IC50
value of 14.4 lg/ml (95 % CI 11.21–18.35), while the
control drug BHT inhibited DPPH with IC50 value of
3.42 lg/ml (95 % CI 3.166–3.692) as shown in Fig. 3a.
Ha.Cr inhibited lipid peroxidation, induced in rat kidney
homogenate by 13.40 ± 3.40 and 42.40 ± 3.10, while
BHT caused 30.85 ± 2.60 and 71.60 ± 3.84 % inhibition
of lipid peroxidation at 50 and 150 lg/ml, respectively
(Fig. 3b).
Effect on kidney epithelial cell lines (MDCK)
Ha.Cr had no toxic effect on MDCK cells up to 0.3 mg/kg.
However, it reduced significantly the cell viability at a
concentration of 1 mg/ml or more (Fig. 4a). The cell via-
bility was decreased significantly (p\0.001) in the
untreatedcontrolafterexposureto0.5 mMOxor66 lg/cm2
of COM. However, the treatment of Ha.Cr significantly
(p\0.05) increased the cell viability at a concentration of
0.3 mg/ml compared to untreated control (Fig. 4b, c). LDH
releasewassignificantlyincreasedafterexposureto0.5 mM
oxalate or 66 lg/cm2COM versus untreated control. How-
ever, after pretreatment with Ha.Cr, the LDH decreased was
significantly at the concentration of 0.3 mg/ml (Fig. 4d, e).
A B
0.50 1.00
[mg/ml]
2.004.00
0
15
30
45
60
75
90
D
K. Citrate
Ha.Cr
*
*
**
SN(% inhibition)
0
25
50
75
100
Ha.Cr
K.Citrate
21
0.25
0.090.03
C
[mg/ml]
SA (% of Control)
SN
SA
Ha.Cr
Fig. 1 Calcium oxalate
crystallization study. Effect of
Holarrhena antidysenterica
(Ha.Cr) and Potassium Citrate
(k-Cit) on calcium oxalate
crystallization. a, b Typical
tracing of the control in the
presence of Ha.Cr and
potassium citrate. c The
concentration response curves
of Holarrhena antidysenterica
and potassium citrate on SAof
the turbidity curves, d the %
inhibition on the SN. Symbols
shown are mean ± SEM
(n = 3). SNand SArepresent
slope of nucleation and slop of
aggregation, respectively
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In vivo experiments
In the in vivo study, all the parameters, like body weights,
24 h water intake, urine volume, urinary pH and compo-
sition, recorded before the treatment were not significantly
different among the groups. The parameters recorded at
day 0 and at the end of 3 weeks of treatment period are
listed in the Table 1.
Ha.Cr had no significant effect on the CaOx crystalluria,
however, the crystals (mostly CaOx dehydrate) found in
the treated groups were quite smaller than those found in
the untreated lithogenic group (Fig. 5). The body weight
was significantly (p\0.01 vs. normal) reduced in stone
forming group as compared to the normal saline group. The
co-administration of Ha.Cr (30–100 mg/ml) prevented
(p\0.05 vs. stone forming group) the loss in body weight.
The 24-h urine volume and water intake were higher
(p\0.01) in the stone forming group compared to that in
the normal saline animals. Urine pH was slightly reduced
in the stone forming group, though not to a significant
extent. A co-treatment with Ha.Cr significantly reduced
(p\0.05) polyurea and water intake compared to stone
forming group. Similarly, oxalate excretion was signifi-
cantly increased (p\0.01) in stone forming animals,
whereas Ca??excretion was decreased (p\0.05). Urine
Fig. 2 Calcium oxalate incubation study. Representative photo-
graphs, under inverted microscope (2009), of CaOx crystals devel-
oped in the metastable solutions in the absence (a) and in the presence
of Holarrhena antidysenterica (Ha.Cr) 2 mg/ml (b) and 2 mM
K-citrate (c)
0
15
30
45
60
75
50150
Ha.Cr (µg/ml)
BHT (µg/ml)
B
*
*
% inhibition
0.1 110100
0
25
50
75
100
BHT (µg/ml)
Ha.Cr (µg/ml)
A
% Inhibition
Fig. 3 Antioxidant activity. Concentration response curves of the
free radical scavenging activity of the butylated hydroxytoluene
(BHT) and Holarrhena antidysenterica (Ha.Cr), while bar chart
(b) representing lipid peroxidation inhibitory activity of two different
concentrations of Ha.Cr and BHT. Inhibition is measure as % of the
respective control experiments. The values shown are mean ± SEM
(n = 3)
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contents of citrate, phosphate, uric acid (UA), Mg2?, Na?
and K?were not altered to a significant level (p[0.05).
Co-administration of Ha.Cr (30–100 mg/kg) to EG treat-
ment group significantly (p\0.05) decreased oxalate
excretion, whereas unlike control group, Ca2?excretion
remained unchanged in the treated groups as compared
to the normal group. EG treatment caused impairment
of renal functions of the untreated rats as evident from
total protein loss and raised BUN and serum creatinine
(p\0.05), which were prevented in the animals treated
with Ha.Cr (Table 1).
In the histological preparations of kidneys, the normal
saline groupdid notshow any crystalline deposits.Whereas,
a high score of crystal deposits were observed under polar-
ized light in all regions of kidneys in lithogenic group.
However, in Ha.Cr treated groups, significantly less number
of CaOx crystal deposits (p\0.5) were observed as com-
pared to untreated EG (lithogenic) group (Figs. 6, 7).
0
10
20
30
40
50
60
***
D
#
% of Control (LDH)
67 µg/cm2 0.100.30
0
10
20
30
40
50
60
70
***
E
##
% of Control (LDH)
0.0 0.003 0.010.03 0.1 0.31.0 3.0
0
25
50
75
100
125
*
**
A
Ha.Cr [mg/mL]
% Cell viability
0
25
50
75
100
***
B
#
Control
Ox
Ox. + Ha.Cr (mg/ml)
0.5 mM0.100.30
% Cell viability
0
25
50
75
100
***
C
#
Control
COM
COM + Ha.Cr (mg/ml)
66 µg/cm2
0.10 0.30
% Cell viability
0.5 mM 0.100.30
Fig. 4 Effect on MDCK cells.
Effects of various
concentrations of Ha.Cr on
MDCK cell survival in
acclimatization media (a). b,
c The protective effect of Ha.Cr
after exposure to 0.5 mM
oxalate or 66 mg/cm2COM,
respectively. d, e The percent
increase in LDH release against
control by MDCK cells exposed
Ox. (0.5 mM) and COM
(66 lg/cm2) for 24 h. Data
shown are mean ± SEM of two
separate experiments with three
independent replicates.
*p\0.05, **p\0.05 and
***p\0.001
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Discussion
To investigate the medicinal use of Holarrhena antidy-
senterica in urolithiasis, we evaluated crude extract for its
antiurolithic activity using different in vitro assays and in
vivo rat model of urolithiasis.
The effect of Ha.Cr on CaOx crystallization kinetics was
studied by the time course measurement of turbidity using
aggregometer, as already reported in previous studies [25,
35]. The initial positive slope of the turbidity curve, called
the slope of nucleation (SN), is mainly due to an increase in
the particle number resulting from crystal nucleation. After
a plateau is achieved, a gradual decrease of absorbance
(negative slope) despite continuous stirring reflects the
decrease in the particle number, due to crystal aggregation
[25, 35]. In this experiment, unlike potassium citrate, Ha.Cr
had no effect on the slope of nucleation but inhibited the
CaOx crystals aggregation in a concentration-dependent
manner, similar to potassium citrate, a well-known inhib-
itor of CaOx crystallization and clinically used for the
management of urolithiasis [36]. Similarly, in the incuba-
tion study, Ha.Cr caused a decrease in crystal size and
transformed COM to COD crystals, which are less likely to
attach with the kidney epithelial cells than COM crystals
[38, 39] like that of citrate and Mg2?[37]. Crystal poly-
morphism plays an important role in calcium oxalates
nephrolithiasis. COM crystals are thermodynamically more
stable and more frequent in kidney stones as compared to
COD. Macromolecules isolated from urine of normal
healthy individuals inhibit COM crystals and favors the
formation of COD, which are less likely to adhere to renal
epithelial cells. This suggests that COD formation protects
against stone disease because of its reduced capacity to
form stable aggregates and strong adhesion contacts to
renal epithelial cells and thus potentially inhibiting a crit-
ical step in the formation of kidney stones [38, 39].
Calcification is a multifactorial phenomenon [40],
developing as a result of a cascade of events initiated
by supersaturation, including crystal nucleation, growth,
aggregation and retention [41]. Various crystal inhibitors
like potassium-sodium citrate and magnesium oxide have
been shown to decrease the saturation of CaOx and inhibit
crystal nucleation, growth and aggregation, while reduced
crystallization in urine of stone forming patients [42].
Interference with crystal growth and aggregation, there-
fore, seems a possible therapeutic strategy for the preven-
tion of recurrent stone disease. The effect on CaOx crystal
aggregation might be due to the presence of saponins,
present in the crude extract, which is evident from the
phytochemical screening in our previous study [21], as
saponins have been reported to have anti-crystallization
property by disaggregating the suspension of mucopro-
teins, which are promoters of crystallization [43]. This
shows that the Ha.Cr may contain substances that inhibit
CaOx crystal aggregations and thus preventing a critical
step in urinary stone formation, as larger particles are less
Table 1 Parameters recorded from urine and blood of groups of rats after 21 days of in vivo study
ParametersBaseline N. SalineHa.Cr 100 EG (0.75 %)EG ? Ha.Cr 30EG ? Ha.Cr 100
Change in BW 16.1 ± 1.418 ± 3.4-3.3 ± 2.3** 8.33 ± 4.1##
11.7 ± 1.7#
12.4 ± 1.0#
10.74 ± 2.8##
8.5 ± 0.6#
10 ± 0.6#
Urine vol (ml/24 h)6.5 ± 0.87.6 ± 1.212.4 ± 1.6 15± 1.5**
Urine vol (ml/24 h)8.2 ± 0.78.5 ± 1.110.8 ± 1.3 14.4 ± 0.9**
pH6.6 ± 0.08 6.5 ± 0.21 6.6 ± 0.066.4 ± 0.126.9 ± 0.2 6.6 ± 0.29
Urinary (mg/24 h)
Oxalate 0.80 ± 0.200.72 ± 0.190.82 ± 0.102.34 ± 0.32*1.39 ± 0.271.37 ± 0.15#
Citrate12.21 ± 2.113.59 ± 1.3 5.5 ± 0.71 4.47 ± 0.59 4.02 ± 0.544.39 ± 0.54
Phosphate4.01 ± 0.71 4.41 ± 0.525.5 ± 0.71 4.47 ± 0.594.02 ± 0.544.39 ± 0.54
Uric acid0.62 ± 0.06 0.7 ± 0.070.72 ± 0.060.63 ± 0.05 0.60 ± 0.080.61 ± 0.10
Calcium
Mg??
K?
4.06 ± 0.48 5.01 ± 0.52 5.75 ± 0.712.67* ± 0.41 3.15 ± 0.36 3.28 ± 0.34
4.1 ± 0.685.05 ± 0.534.21 ± 0.53 5.11 ± 0.904.03 ± 0.614.38 ± 0.87
0.76 ± 0.08 0.77 ± 0.070.71 ± 0.100.72 ± 0.06 0.78 ± 0.08
7.48 ± 0.93#
0.72 ± 0.09
9.09 ± 0.57#
Total protein 3.5 ± 1.00 4.6 ± 1.205.5 ± 0.7012.4 ± 1.07**
Serum
Creatinine (mg/dl) 0.73 ± 0.11 0.45 ± 0.061.4 ± 0.15** 0.81 ± 0.08*0.81 ± 0.08#
21.58 ± 3.47#
BUN (mg/ml)22.9 ± 3.925.14 ± 3.940.19 ± 3* 26.28 ± 3.9
Values are expressed as Mean ± SEM
* p\0.05, ** p\0.05 and *** p\0.001 versus normal
#
p\0.05,##p\0.01 and###p\0.001 versus lithogenic group
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likely to pass spontaneously in urinary tract. If the extract
keeps CaOx particles dispersed in solution then they can be
easily eliminated [44].
Animal and cellular studies have revealed that oxalate
ions, or COM and hydroxy apatite (HA) crystals cause
injury to the kidney cells [45, 46], most probably by the
production of reactive oxygen species (ROS). Epithelial
injury is considered to be a risk factor for the crystallization
and crystals deposition in the kidney, as it promotes crystal
nucleation, aggregation, retention and stone development
[45, 47]. Antioxidants, such as vitamin E, catechin and
selenium, have been shown to provide protection against
oxidative injury by oxalate and crystal deposition [48, 49].
When tested for its antioxidant activity, Ha.Cr caused
scavenging of DPPH free radical and inhibited ferrous-
ascorbate-induced lipid peroxidation
homogenate similar to BHT, a standard antioxidant [50].
These results are in line with earlier study [51] which
showed the presence of antioxidant activity in Holarrhena
antidysenterica. As hyperoxaluria and COM crystals are
known to cause oxidative stress resulting in toxicity of
renal epithelial cells [49, 52], the protective effect of Ha.Cr
on renal epithelial cell might be due to its antioxidant
activity, as pretreatment with the Ha.Cr significantly
increased the survival rate and reduced the LDH release, a
marker of cell membrane damage, in MDCK cells, when
exposed to oxalate and COM crystals.
For the in vivo antiurolithic effect of Ha.Cr, 0.75 %
ethylene glycol (EG) and 1 % ammonium chloride (AC)-
induced hyperoxaluric rat model of urolithiasis was used.
Since the stone inducing treatment, Ethylene glycol (EG),
was given orally, therefore, the extract was given i.p. to
prevent any potential interaction of EG with plant con-
stituents inside gut, interfering with absorption of either of
the two. We have used intra-peritoneal (i.p) route, which is
also commonly used in previous studies [11, 25, 53, 54].
Administration of EG and AC resulted in the increased
CaOx crystalluria, with larger crystals due to hyperoxalu-
ria, increase in water intake and urine output, which might
be due to the renal impairment [29], as evident by increase
in serum creatinine, BUN and total urinary protein loss in
lithiatic group as compared to normal. Consistent with
some previous reports, stone induction by hyperoxaluria
caused an increase in oxalate and decrease in Ca2?
excretion in the untreated group [52, 55]. Hyperoxaluria is
one of the major risk factor in the pathogenesis of kidney
stone formation [56], as it causes oxidative stress and
damages the renal epithelial cells thereby providing a nidus
for crystals attachment and ultimately causes crystal
aggregation retention and deposition in the kidney [45, 47].
Therefore, decrease in oxalate may explain its decrease in
oxidative stress and renal crystal deposition. It is not
uncommon that plant extracts may interfere with oxalate
metabolism in animal model of urolithiasis, e.g. extract of
Aerva lanata decreases the oxalate excretion, in ethylene
glycol fed rats, by decreasing the formation of oxalate
synthesizing enzymes like glycolic acid oxidase (GAO) in
liver and lactate dehydrogenase (LDH) in liver and kidney
[57]. Similar results were found in the extract of Tribulus
terrestris [58]. This can be speculated that decrease in
ofrat kidney
Fig. 5 Images of crystalluria. Images of calcium oxalate crystals in
3 h morning urine collected from normal control (a), lithogenic
control (b) and treated with Holarrhena antidysenterica (Ha.Cr) (c),
under light microscope at 4009 magnification
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Fig. 6 Microscopic images of kidney sections in the in vivo study.
Representative microscopic images of the H and E stain of the kidney
sections from normal (a), lithogenic group (b) and treated (c) with
Ha.Cr. A1, B1 and C1 show the Pizzolato’s staining of the respective
kidney sections
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oxalate might be useful in hyperoxaluric kidney stone
formers.
There was hypertrophy and extensive CaOx crystal
deposition in kidneys of untreated rats. The renal tubules
were markedly dilated, which might be due to the
obstruction in distal renal tubular flow by large crystals
[29]. Several in vivo and in vitro studies have demonstrated
that hyperoxaluria, a major risk factor for calcium oxalate
nephrolithiasis, results in greater production of superoxide
and hydroxyl free radicals, leading to antioxidant imbal-
ance, cell membrane rapture and cell death [49, 59] which
leads to CaOx crystal adherence and retention in renal
tubules [49, 60]. Thus, it can be speculated that the
inhibitory effect of the plant extract on CaOx crystal
deposition in renal tubules is possibly caused by its anti-
oxidant activity. The plant is considered relatively safer, as
it has been used in different herbal preparations and sup-
plements, such as Kutaj (Holarrhena antidysenterica), a
herbal supplement, is used in humans for ailment of diar-
rhea and urinary disorders, which has also undergone
clinical trials for several studies, with no reported side
effect, for ulcerative colitis and bleeding piles [61–63].
Thus, these data suggest that the preventive effect of
Holarrhena antidysenterica in urolithiasis is mediated
through multiple pathways including inhibition of the
CaOx crystal aggregation, antioxidant and epithelial cell
protective effects, which provide a step forward for
designing further studies on Holarrhena antidysenterica to
establish its safety and efficacy for clinical use.
Acknowledgments
cation Commission (HEC) of Pakistan as (1) indigenous PhD and (2)
This study was supported by the Higher Edu-
International Research Support Initiative Program (IRSIP) scholar-
ships awarded to Aslam Khan.
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1
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Lithogenic Group Normal Saline
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***
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