Vol. 15, No. 3, 2009
159-166
African Journal of Urology
1110-5704
Original Article
Evaluation of Pyracantha crenulata Roem for
Antiurolithogenic Activity in Albino Rats
Y.M. Bahuguna1, M.S.M. Rawat2, V. Juyal3 and K. Gusain1
1
Division of Pharmaceutical Sciences, Shri Guru Ram Rai Institute of
Technology and Sciences, Patel Nagar, Dehradun-248 001, 2Department
of Chemistry, H.N.B. Garhwal University, Srinagar, Pauri Garhwal,
3
Department of Pharmacy, Kumaun University, Bhimtal Campus, Bhimtal,
Nainital, Uttarakhand, India
ABSTRACT
Objective: The aim of the present study was to investigate the effect of juice extract and alcohol
extract of fruit of Pyracantha crenulata (D.Don) Roem (Rosaceae) against ethylene glycolinduced urolithiasis in male albino rats.
Patients and Methods: Lithiasis was induced in rats by administering 0.75% ethylene glycol
in drinking water for 28 days and was manifested by hyperoxaluria as well as increased renal
excretion of calcium, phosphate and a low urinary magnesium content. Curative and preventive
treatment was then tried by supplementation with juice and alcohol extracts (250 mg/kg b.w., p.o.)
of P. crenulata fruit.
Results: The increased deposition of stone forming constituents in the kidneys of calculogenic rats
was significantly lowered by curative and preventive treatment using juice and alcohol extracts
(250 mg/kg b.w., p.o.) of P. crenulata fruit which showed a regulatory action on endogenous
oxalate synthesis.
Conclusion: From this study, we conclude that both prophylactic and therapeutic treatment with
juice and alcohol extract of fruit of P. crenulata may reduce precipitation of calcium oxalate, with
improvement of kidney function as well as cytoprotective effect
Keywords : Ethylene glycol, hyperoxaluria, nephrolithiasis, Pyracantha crenulata
Corresponding Author: Dr. Yogendr M. Bahuguna, Division of Pharmaceutical Sciences, SGRR
Institute of Technology and Sciences, Patel Nagar, Dehradun-248 001, Uttarakhand, India,
E-mail: yogendr.bahuguna@gmail.com
Article Info:
Date received : 14/4/2009
Date accepted (after revision): 14/11/2009
INTRODUCTION
Despite technological and conceptual developments in the practice of medicine, the
formation and growth of renal calculi continue to afflict mankind. The incidence of kidney stones has increased in western societies
in the last five decades, in association with
economic development. Most calculi in the
urinary system arise from a common compo-
nent of urine, calcium oxalate (CaOx), representing up to 80% of all analyzed stones1.
A large number of plant drugs have been
used in India since ancient times which claim
efficient cure of urinary stones2. Amongst
the medicinal plants used in the treatment of
urolithiasis are ‘patharphor’ (Didymocarpus
159
ANTIUROLITHOGENIC ACTIVITY OF P. CRENULATA
PATIENTS AND METHODS
Fresh Pyracantha crenulata fruit were
collected from local areas of Dehradun,
Uttarakhand, India during May 2008 and
authenticated at the Botanical Survey of India
(BSI), Dehradun, India. A voucher specimen
of the plant was deposited in the Botanical
Survey of India herbarium under the number
BSD 112215 for further reference.
The fresh and semi-ripe fruit were cut into
small pieces and fed through a juicer. The
juice was filtered and vacuum dried to obtain
P. crenulata fruit juice extract (PCJE, yield
17% w/w). In addition, the fruit were sliced
using a home slicer, then shade-dried, pulverized and passed through a 20-mesh sieve.
The dried, coarsely powdered plant material
was extracted with 70% (v/v) alcohol by the
hot continuous extraction method using a
soxhlet apparatus at a temperature of 6070ºC. The solvent was evaporated under vacuum yielding a semisolid mass (PCAE, yield
22% w/w with respect to the dried powder).
Both extracts were stored in tight containers
in a desiccator for further use16.
Fig. 1: Fruits and leaves of P. crenulata
pedicellata), several Bergenia species, three
species of Tribulus (T. systoides, T. terrestris
and T. alatus), ‘manjit’ (Rubia cordifolia and
Rubia tinctorum), ‘varuna’ (Crataeva nurvala) and ‘imli’ (Tamarindus indica)3, Costus
spiralis4, Raphanus sativus5, Moringa oleifera6, Crataeva adansonii7, Melia azedarach8
and Eleusine coracana9.
“The plant Pyracantha crenulata Roem,
syn. Crataegus crenulata Roxb (Rosaceae),
locally known as Ghingaaru, is found in the
Himalayas from Sutlaj to Bhutan, at altitudes
of 800-2500 m. In Ayurvedic medicine it
is reported to be useful in the treatment of
a number of ailments, including hepatic,
stomach and skin diseases due to its diuretic,
depurative, tonic, antirheumatic, cardiotonic,
hypoglycemic, hypotensive, anti-inflammatory and lithontripic properties10-14. However,
there are no records of systematic pharmacological studies that support its antiurolithogenic effect. Due to the wide distribution in
Uttarakhand, India, and in continuation of our
research work on P. crenulata15, the present
investigation was carried out to determinate
the antiurolithogenic property of the alcohol
and juice extracts of the fruit of P. crenulata
using an ethylene glycol induced hyperoxaluria model in albino rats and to confirm the
traditional medicinal use of the plant.
For acute toxicity studies, albino rats of
either sex weighing 150-200 g were selected,
and healthy adult male albino rats weighing
150-200 g were selected for assessing the
antiurolithogenic activity. The animals
were acclimatized to standard laboratory
conditions (temperature: 25 ± 2ºC) and
maintained on a 12-h light: 12-h dark cycle.
They were housed in polypropylene cages
and provided with regular rat chow (Lipton
India Ltd., Mumbai, India) and drinking water
ad libitum. The animal care and experimental
protocols were approved by the Institutional
Animal Ethical Committee (IAEC).
The acute oral toxicity study17 was
carried out as per the guidelines set by the
Organization for Economic Co-Operation
and Development (OECD) received from
the Committee for the Purpose of Control
and Supervision of Experiments on Animals
160
ANTIUROLITHOGENIC ACTIVITY OF P. CRENULATA
Table 1: Urinary excretion of stone forming constituents in control and experimental animals
Urine parameters (mg/dl)
Groups
Dose (mg/kg)
Oxalate
Calcium
Phosphate
Normal (N)
Vehicle
0.28 ± 0.01
1.18 ± 0.03
2.96 ± 0.04
Calculi induced (PR)
Vehicle
2.61 ± 0.17*
3.85 ± 0.18*
6.82 ± 0.13*
Cystone treated (CR)
750
0.46 ± 0.10***
1.34 ± 0.10***
3.36 ± 0.03***
Alcohol extract (CR I)
250
0.86 ± 0.01***
2.03 ± 0.24***
3.40 ± 0.26***
Juice extract (CR II)
250
2.25 ± 0.24**
3.52 ± 0.31**
5.98 ± 0.28**
Alcohol extract (PR I)
250
1.28 ± 0.13***
2.27 ± 0.13***
4.52 ± 0.22***
Juice extract (PR II)
250
2.36 ± 0.16**
3.41 ± 0.23**
6.48 ± 0.32**
Values are expressed as mean ± SEM of 6 observations; Statistical comparisons are made between Group N vs Group PR and CR
(*P<0.001); Group PR vs PR I and PR II (**P<0.01); Group CR vs Group CR I and CR II (***P<0.01)
(CPCSEA). One tenth of the median lethal
dose (LD50) was taken as an effective dose18.
From the acute toxicity study, the LD50
cut-off dose was found to be 2500 mg/kg
body weight for both extracts. Hence, the
therapeutic dose was taken as 250 mg/kg
body weight for both extracts.
From day 1 - 28, group PR I received
alcohol extract (250 mg/kg body weight)
and group PR II juice extract (250 mg/kg
body weight). Group PR did not receive any
extract.
From day 15 – 28, group CR received a
standard antiurolithogenic drug (cystone; 750
mg/kg body weight20-23), while groups CR I
and CR II received alcohol extract (250 mg/
kg body weight) and juice extract (250 mg/kg
body weight), respectively.
An
ethylene
glycol
induced
hyperoxaluria model19 was used to assess
the antiurolithogenic activity in albino rats.
The animals were divided into seven groups
containing six animals each.
•
Group N served as a normal control
group and received regular rat food
and drinking water ad libitum.
•
Groups PR, PR I and PR II for the
preventive regimen
•
Groups CR, CR I and CR II for the
curative regimen
All extracts were given orally once daily.
All animals were kept in individual metabolic
cages, and 24-h urine samples were collected
on day 28. The animals had free access to
drinking water during the urine collection
period. A drop of concentrated hydrochloric
acid was added to the urine before it was
stored at 4ºC. The urine was analyzed for
calcium24, phosphate25 and oxalate26.
Starting from day 1, ethylene glycol
(0.75% v/v) in the drinking water was given
to all groups except the control group for the
induction of renal calculi.
After the experimental period, blood was
collected from the retro-orbital area under
161
ANTIUROLITHOGENIC ACTIVITY OF P. CRENULATA
Table 2: Kidney retention of stone forming constituents in control and experimental animals
Kidney parameters (mg/g)
Groups
Dose (mg/kg)
Oxalate
Calcium
Phosphate
Normal (N)
Vehicle
1.36 ± 0.02
3.02 ± 0.03
2.58 ± 0.04
Calculi induced (PR)
Vehicle
4.85 ± 0.11*
4.94 ± 0.21*
3.88 ± 0.11*
Cystone treated (CR)
750
2.15 ± 0.03***
1.82 ± 0.27***
1.62 ± 0.03***
Alcohol extract (CR I)
250
2.18 ± 0.03***
2.82 ± 0.21***
1.80 ± 0.06***
Juice extract (CR II)
250
3.42 ± 0.06**
4.26 ± 0.34**
3.47 ± 0.16**
Alcohol extract (PR I)
250
4.28 ± 0.36***
3.46 ± 0.22***
3.02 ± 0.07***
Juice extract (PR II)
250
4.11 ± 0.23**
4.49 ± 0.32**
3.50 ± 0.16**
Values are expressed as mean ± SEM of 6 observations; Statistical comparisons are made between Group N vs Group PR and CR
(*P<0.001); Group PR vs PR I and PR II (**P<0.01); Group CR vs Group CR I and CR II (***P<0.01)
RESULTS
anesthetic conditions, and the animals were
sacrificed by cervical decapitation. The serum
was separated by centrifugation at 10,000
rpm for 10 min and analyzed for creatinine,
urea nitrogen27 and uric acid28.
In the
present
study, chronic
administration of 0.75% (v/v) ethylene
glycol aqueous solution to male albino rats
resulted in hyperoxaluria. Oxalate, calcium
and phosphate excretion were grossly
increased in calculi-induced animals (Table
1). However, supplementation with juice
and alcohol extracts of P. crenulata fruit
significantly (p<0.001) lowered the elevated
levels of oxalate, calcium and phosphate in
urine and kidney in the animals of groups CR
I and CR II (curative regimen) and those of
groups PR I and PR II (preventive regimen)
as compared to group PR (calculi-induced)
(Table 1).
The abdomen was cut open to remove
both kidneys, which were dissected from
extraneous tissue and preserved in 10%
neutral formalin. They were then dried
at 80ºC in a hot-air oven. A sample of 100
mg of the dried kidney was boiled in 10
ml of 1N hydrochloric acid for 30 min
and homogenized. The homogenate was
centrifuged at 2,000 rpm for 10 min and the
supernatant was separated29. The calcium24,
phosphate25 and oxalate26 contents in the
kidney homogenate were determined.
The deposition of crystalline components
in the renal tissues, namely oxalate, phosphate
and calcium, was increased in the stone-forming rats (Table 2). Treatment with juice and
alcohol extracts of P. crenulata fruit significantly (p<0.001) reduced the renal content of
these stone-forming constituents in both regimens (Table 2, CR I, CR II, PR I, PR II).
The results were expressed as mean ± SD.
Differences among the data were statistically
analyzed using one-way ANOVA followed
by Student Newman Keul’s test (GraphPad
Prism software for Windows, Version
5.01.2007). Statistical significance was set
accordingly30.
162
ANTIUROLITHOGENIC ACTIVITY OF P. CRENULATA
Table 3: Serum parameters in control and experimental animals
Serum parameters (mg/dl)
Groups
Dose (mg/kg)
BUN
Creatinine
Uric acid
Normal (N)
Vehicle
38.21 ± 0.16
0.56 ± 0.03
1.26 ± 0.04
Calculi induced (PR)
Vehicle
52.67 ± 0.46*
0.98 ± 0.08*
3.48 ± 0.11*
Cystone treated (CR)
750
42.60 ± 0.38***
0.58 ± 0.01***
1.88 ± 0.03***
Alcohol extract (CR I)
250
40.31 ± 0.22***
0.60 ± 0.03***
2.06 ± 0.04***
Juice extract (CR II)
250
52.90 ± 0.42**
0.78 ± 0.07**
3.18 ± 0.08**
Alcohol extract (PR I)
250
46.23 ± 0.23***
0.61 ± 0.06***
2.46 ± 0.06***
Juice extract (PR II)
250
51.87 ± 0.36**
0.84 ± 0.10**
3.12 ± 0.16**
Values are expressed as mean ± SEM of 6 observations; Statistical comparisons are made between Group N vs Group PR and CR
(*P<0.001); Group PR vs PR I and PR II (**P<0.01); Group CR vs Group CR I and CR II (***P<0.01)
Although the extent of reduction was insignificant on inter-regimen comparison
(curative versus preventive regimens), the
differences were significant (p<0.001) when
compared with the cystone-treated animals
(Table 2).
amount of stone deposition in female rats was
significantly less31,32.
DISCUSSION
Urinary supersaturation with stoneforming constituents is generally considered
one of the causative factors in calculogenesis.
Previous studies indicate that in response to
the administration of ethylene glycol (0.75%,
v/v) over a 14-day period, young male albino
rats form renal calculi composed mainly
of calcium oxalate18,33,34. The biochemical
mechanisms of this process are related to
an increase in the urinary concentration of
oxalate. Stone formation in ethylene glycolfed animals is caused by hyperoxaluria,
which causes increased urinary retention and
excretion of oxalate33. Similar results have
been obtained when rats were treated with
ethylene glycol and ammonium oxalate35,36.
In the present study, male rats were
selected to induce urolithiasis because the
urinary system of male rats resembles that of
humans and earlier studies showed that the
In the present study, oxalate and calcium
excretion progressively increased in stoneforming animals (PR). Since it is accepted
that hyperoxaluria is a more significant risk
Serum uric acid and blood urea nitrogen
(BUN) were remarkably increased in the
stone-forming animals (Table 3), while serum creatinine was only slightly elevated in
group PR indicating marked renal damage.
However, P. crenulata extracts significantly
(p<0.001) lowered the elevated serum levels of creatinine, uric acid and BUN in the
groups of curative and preventive regimen
(Table 3).
163
ANTIUROLITHOGENIC ACTIVITY OF P. CRENULATA
factor in the pathogenesis of renal stones
than hypercalciuria, the changes in urinary
oxalate levels are relatively more important
than those of calcium37,38. Increased urinary
calcium is a factor favoring the nucleation
and precipitation of calcium oxalate or
apatite (calcium phosphate) from urine
and subsequent crystal growth39. However,
juice and alcohol extracts of P. crenulata
fruit lowered the levels of oxalate as well as
calcium excretion.
In conclusion, the presented data indicate
that administration of juice and alcohol
extracts of P. crenulata fruit to rats with
ethylene glycol-induced lithiasis reduced
and prevented the growth of urinary
stones, thus supporting folk information
regarding the antiurolithogenic activity of
the plant. The mechanism underlying this
effect is still unknown, but is apparently
related to increased diuresis and lowering
of urinary concentrations of stone forming
constituents. These effects could explain the
antiurolithogenic property of P. crenulata.
An increase in urinary phosphate is
observed in stone-forming rats (PR).
Increased urinary phosphate excretion along
with oxalate stress seems to provide an
environment appropriate for stone formation
by forming calcium phosphate crystals,
which epitaxially induces calcium oxalate
deposition40. Treatment with P. crenulata
fruit extract restored the phosphate level, thus
reducing the risk of stone formation.
Acknowledgements
The authors express their thanks to
Botanical Survey of India Dehradun,
Uttarakhand, India for authentification of
the plant material. The authors also express
their gratitude to Shri Mahant Devendra
Dass Ji Maharaj, Chairman, Shri Guru Ram
Rai Institute of Technology and Sciences,
Dehradun, Uttarakhand, India for providing
the facilities necessary to carry out the
research work.
In urolithiasis, the glomerular filtration
rate (GFR) decreases due to obstruction of
urine outflow by stones in the urinary system.
Due to this, the waste products, particularly
nitrogenous substances such as urea,
creatinine, and uric acid accumulate in the
blood41. Also, increased lipid peroxidation
and decreased levels of antioxidant potential
have been reported in the kidneys of rats
supplemented with a calculi-producing
diet42,43. In this context, oxalate has been
reported to induce lipid peroxidation and
to cause renal tissue damage by reacting
with polyunsaturated fatty acids in the cell
membrane44. In the stone-forming rats (PR)
marked renal damage was seen by the elevated
serum levels of creatinine, uric acid and
BUN. However, curative and prophylactic
treatment with juice and alcohol extracts of P.
crenulata fruit caused diuresis15 and hastened
the process of dissolving the preformed
stones and preventing new stone formation in
the urinary system.
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EDITORIAL COMMENT
This study is interesting and should be published, as based on the investigations and results presented by the
authors Pyracantha crenulata may gain clinical importance for the inhibition of urinary calculi. However,
clinical studies on patients with urinary calculi are still necessary to confirm the said effect in humans
Dietmar Bach
Prof. emerit. Rheinische Friedrich Wilhelm Universität Bonn, Germany
166