Krishnaveni Janapareddi et al./ Vol 3 / Issue 2 / 2013 / 46-52.

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International Journal of

Pharmacological Screening Methods

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ANTIUROLITHIATIC ACTIVITY OF CUCUMIS SATIVUS

Krishnaveni Janapareddi1*, Rajkiran Ellandala2, Manjula Pulluru2, Sudheer K Dundigalla2
1

University College of Pharm.Sciences, Kakatiya University, Warangal, Andhra Pradesh-506009, India.

Care College of Pharmach, Oglapur (V), Atmakur (M), Warangal Dist. 506 006, Andhra Pradesh, India.

ABSTRACT
Ayurveda offers vast scope for the successful treatment of urolithiasis. Although most remedies were herbal and
proved useful, a systematic scientific evaluation has been reported for only few remedies. Cucumis sativus is claimed to be
useful in the treatment of urinary stones in ayurvedic literature. Hence a systematic study was planned and conducted to see the
influence of the extract of the fruits of Cucumis sativus when used as preventive and curative regimens for treatment in
urolithiasis induced by administration of ethylene glycol in albino rats. Various biochemical estimations in serum ,urine ,
kidney homogenates and histological examination of the kidneys showed that the test extract has beneficial action in urolithiasis
when given in Preventive and curative regimens. However if the active constituents can be further isolated and concentrated
and then their pharmacological evaluation is done, a product with greater efficacy becomes possible.
key words: Urolithiasis, Kidney stones, Cucumis sativus, Allopurinol, Calcium Oxalate.
INTRODUCTION
Urolithiasis is the third most common affliction of
urinary tract. The major causative factor for formation of
stones is supersaturation of precipitating salts in the urine.
The treatment of urolithiasis costly and in most cases is
invasive and with high recurrence rate. Hence it is
worthwhile to look for an alternative to the conventional
therapy in the treatment of urolithiasis and one approach is
use of herbal drugs.
Ayurveda offers vast scope for the successful
treatment of urolithiasis [1] and many remedies have been
employed during the ages to treat urolithiasis. Although
most remedies were herbal and proved useful, a systematic
scientific evaluation has been reported for only few
remedies [2]. In view of this, it is interesting to know that
Cucumis sativus is claimed to be useful in the treatment of
urinary stones in ayurvedic literature and a systematic
search and survey of the literature showed that there is no
record of systematic pharmacological study on the plant to

verify the claim. Hence, the present study is taken up to

investigate the potential of the alcoholic extract of Cucumis
sativus as an antiurolithiatic agent.
MATERIALS AND METHODS
Experimental Animals
Rats and mice were procured from Sainath animal
agencies, Hyderabad. Male wistar albino rats weighing
between 150- 200gm were selected for the study. Female
albino mice weighing between 25- 30gm were selected for
the acute oral toxicity studies. The animals were maintained
on 12hr/12hr light and dark cycle at ambient room
temperature and at relative humidity (50%). They were kept
in polypropylene cages in a well-ventilated room under
hygienic conditions throughout the study. The animals were
fed with commercial rat feed pellets and were given water
ad libitum. Maintenance of animals was as per CPCSEA
guidelines. All animal studies were carried out only after
approval of IAEC.

Corresponding Author:- Krishnaveni Janapareddi Email:- krishnavenij153@gmail.com

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Chemicals
Ethylene glycol was purchased from Merck India.
Allopurinol was procured from Unicure remedies Pvt Ltd
as a gift sample. Ethanol, Tween 80 and Formaldehyde
were purchased from S.S Pharma, Warangal.
Plant material
Ripe mature fruits of Cucumis sativus were
procured from local vegetable markets of Warangal,
chopped into small pieces, shade dried and coarsely
powdered. The powdered plant material was subjected to
soxhelation with 60% v/v ethanol at 60oC for 18 hours and
the extract obtained was evaporated to dryness and used for
various evaluations.
Acute toxicity test
Acute toxicity test was conducted as per the limit
test specification of OECD revised guidelines (423) in adult
female mice. Five animals were treated after fasting for 3-4
hours with an oral dose of 2000 mg/ kg body weight with
the ethanolic extract of the whole fruit of Cucumis sativus,
suspended with 5% Tween 80 in water. The animals were
observed for 2-3 h for general behavioral, neurological,
autonomic profiles and mortality after 24 h.
Induction of Renal Calculi
Renal calculi was induced by administration of
ethylene glycol (0.75%v/v), in drinking water ad libitum
over a period of 28 days [3,4].
Study design
Male wistar albino rats were divided into five
groups of six animals each. All the groups except control
were treated with ethylene glycol 0.75% in water orally for
a period of 28 days. The extract of Cucumis sativus was
given at 500mg/kg dose for 28 days to group II as
preventive regimen and from 15th day to 28 day as curative
regimen to group III.
Collection and analysis of Urine
Urine samples were collected on 28th day for 24
hrs using metabolic cages. Samples were stored at 4C after
adding a drop of conc. HCl. The stored samples were
analyzed for Calcium, Phosphate and Oxalate content.
Urine samples were centrifuged to pool the crystals and
observed under light microscope. Size and shape of crystals
were observed and reported.
Collection and analysis of Serum
After the 28days experimental period, blood was
collected from the retro-orbital plexusof the rats and
subjected to centrifugation at10,000 rpm for 10min.The
serum was analyzed for serum creatinine, uric acid and
blood urea nitrogen.

Isolation and analysis of Kidney homogenate

Animals were sacrificed by cervical dislocation
under anesthesia. The abdomen was cut open to remove the
two kidneys from each animal. The isolated kidneys were
cleaned off the extraneous tissue and one kidney was dried
at 80C in a hot air oven. A sample of 100mg of the dried
kidney was boiled in 10ml of 1 N HCl for 30 min,
homogenized and the homogenate obtained was subjected
to centrifugation at 2000 rpm for 10 min. The supernatant is
separated and analyzed for Calcium, Phosphate and Oxalate
content.
Histopathology of Kidney
The other isolated kidney was preserved in 10%
neutral formalin. The specimens were dehydrated in
descending grades of ethanol, cleared in xylene and
embedded in paraffin wax. Sections of 4-5m thickness
were cut and stained with haematoxylin and eosin and
examined under microscope.
RESULTS
Preliminary phytochemical Screening
The preliminary phytochemical analysis of
ethanolic extract of whole fruit of Cucumis sativus
indicated the presence of various chemical constituents like
steroids, carbohydrates, glycosides, tannins, saponins,
triterpenoids, flavonoids and alkaloids. Literature review
conducted on Cucumis sativus indicated the presence of the
above mentioned active constituents. As the plant extract
was not soluble in water, it was dispersed in 5% Tween 80
and the suspension was used for the study.
Anti Urolithiatic activity
Serum analysis
The serum uric acid and BUN and serum
creatinine were remarkably increased in calculi-induced
animals (Table 1). In the preventive and curative treatment
groups, the elevated serum levels of creatinine, uric acid,
and BUN were significantly (P<0.001) reduced; However,
the results were significantly (P<0.001) comparable to
those of Allopurinol treated animals (Table 1, Fig 1-3)
Urine analysis
Biochemical examination of Urine
After 28 days study period, on the 29th day urine
was collected for 24hrs into the bottom of the cages where
animals were put on metallic mesh 4cm above the bottom
of the cage and the collected urine sample was analyzed for
Calcium, Oxalate and Phosphate content. Increased
excretion of Calcium, Oxalate and Phosphate was seen in
Group II animals as compared to Group I animals. (Table
2).A significant (p<0.001) lowering of elevated levels of
Urinary Calcium, Oxalate and Phosphate was found in
curative and prophylactic (PR) treatment of ethanolic fruit
extract of C.sativus. These results obtained were significant

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(p<0.001) and comparable to Group III animals. (Table 2,

Fig 4-6).
Microscopic Examination of Urine
The microscopic examination of the urine samples
was done under 10X magnification and images are as
shown in Fig: 7 (A-E).The urine of Group I (normal)
animals was found to be devoid of crystals or crystal like
structures. Group II (calculi induced) animals showed
abundant and large Calcium oxalate crystals with
characteristic rectangular shape (Fig 7B). Group III
(Allopurinol treated) animals showed very few and small
(almost dissolved) crystals, when compared to that of the
group II(Fig 7C). Extract treated Group IV (Preventive
treatment) animals showed fewer crystals in the urine when
compared to the positive control group but the number of
crystals were more when compared to the standard group.

Kidney homogenate analysis

Hyperoxaluric condition and deposition of calcium
and phosphate was observed in kidney homogenates of
Group II animals(Table 3) as compared to negative control
group. In the standard , preventive and curative groups a
significant reduction in the levels of calcium phosphate
and oxalate in the kidney homogenate was seen but the
reduction was greatest in the standard group followed by
preventive and then curative group.
A significant (p<0.001) reduction in the deposition
of kidney stone forming constituents (Calcium, Oxalate and
Phosphate) were found in the kidneys of preventive (Group
IV) and curative (Group V) animals. The results obtained
were significant (p<0.001) when compared to Group III
(Allopurinol treated standard group) animals. (Table 3, Fig
8)

Table 1. Changes in serum parameters in control and experimental animals

Serum parameters (mean SEM)
S.No.
Group
Uric acid (mg/dl)
Creatinine (mg%)
BUN (mg%)
36.500.42
0.520.01
16.80.20
1.
Negative control
52.670.47c
1.030.02
24.420.22c
2.
Positive control
42.420.15c,3
0.570.005a,3,
19.70.13c,3
3.
Standard
c,3,A
c,3,C
42.920.20
0.610.007
20.20.14c,3,A
4.
Preventive regimen (Extract)
c,3,C
c,3,C
44.500.18
0.880.005
20.820.07c,3,C
5.
Curative regimen (Extract)
All values are expressed as Mean SEM (n=6)
Compared to Normal: a= P<0.05, b= P<0.01, c= P<0.001.
Compared to Positive control: 1= P<0.05, 2=P<0.01, 3=P<0.001
Compared to Standard treatment: A=P<0.05, B=P<0.01, C=P<0.001 by using (ANOVA) with Dunnetts t-test.

Table 2. Changes in Urine excretion parameters in control and experimental animals

Serum parameters (mean SEM)
S.No.
Group
Dose (mg/kg)
Uric acid (mg/dl) Creatinine (mg%)
BUN (mg%)
Vehicle
36.500.42
0.520.01
16.80.20
1.
Negative control
Vehicle
52.670.47c
1.030.02
24.420.22c
2.
Positive control
50
42.420.15c,3
0.570.005a,3,
19.70.13c,3
3.
Standard
Preventive regimen [PR]
500
42.920.20c,3,A
0.610.007c,3,C
20.20.14c,3,A
4.
(AlcEC.sativus fruits)
Curative regimen [CR]
500
44.500.18c,3,C
0.880.005c,3,C
20.820.07c,3,C
5.
(AlcEC.sativus fruits)
Values are expressed as Mean SEM n=6
Compared to Normal: a= P<0.05, b= P<0.01, c= P<0.001.
Compared to Positive control: 1= P<0.05, 2=P<0.01, 3=P<0.001
Compared to Standard treatment: A=P<0.05, B=P<0.01, C=P<0.001 by using (ANOVA) with Dunnetts t-test.

Table 3. Changes in Kidney deposition parameters in control and experimental animals

Kidney deposition parameters (mean SEM)
S.No.
Group
Calcium (mg/gm tissue)
Oxalate (mg/gm tissue)
Phosphate (mg/gm tissue)
2.520.04
1.790.03
2.460.09
1.
Negative control
3.740.09c
3.730.02c
3.700.05c
2.
Positive control
2.860.01c,3
2.150.07c,3
2.580.01c,3
3.
Standard
c,3,C
c,3,C
2.930.07
2.200.07
2.710.01c,3,C
4.
Preventive regimen
c,3,C
c,3,C
3.140.08
2.310.6
2.920.01c,3,C
5.
Curative regimen
Values are expressed as Mean SEM n=6
Compared to Normal: a= P<0.05, b= P<0.01, c= P<0.001.
Compared to Positive control: 1= P<0.05, 2=P<0.01, 3=P<0.001
Compared to Standard treatment: A=P<0.05, B=P<0.01, C=P<0.001 by using (ANOVA) with Dunnetts t-test.

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DISCUSSION
The urinary system of rats resemble that of
humans and urine also is similar to human urine in
characteristics believed to be significant in stone formation.
This forms the basis for selection of rats to induce urinary
stones in present study [5]. Most animal models for calcium
oxalate stone disease employed ethylene glycol
administered in drinking water, oxalate infusion or feeding
and pyridoxine depletion [6]. Hyperoxaluria certainly
causes crystalluria; unless crystal formation is massive
these crystals are not retained within the kidney for
sufficient time to form stones [7]. The main limitations of
the above mentioned models are high incidence of
nephrotoxicity, metabolic acidosis and occurrence of calculi
in renal cortex that is a situation opposite to as found in
human urolithiasis.
Treatment with 0.75% (v/v) of ethylene glycol was
reported to induce stone formation by causing kidney
damage. Chronic administration of ethylene glycol
increases calcium and oxalate super saturation, renal
tubular injury and produce conditions conducive to the
formation and growth of calcium oxalate stone [3,4].
Administration of ethanolic whole fruit extract of Cucumis
sativus significantly reduced both calcium and oxalate
deposition in the kidneys when compared to the positive
control group.
Evidences in previous studies indicated that, in
response to 14 days period of ethylene glycol (0.75 % v/v)
administration, young albino rats form renal calculi
composed mainly of Calcium oxalate). Hyperoxaluria plays
almost a secondary role in the genesis of calcium oxalate
stone formation. The increased deposition of calcium and
oxalate in renal tissues is known to lead to papillary
calcification and eventual calculi formation [8]. In the
present animal model, hyperoxaluria induces not only
calcium oxalate crystallization but also papillary damage
and incrustations.
In the present study, a significant increase in
urinary excretion of calcium, oxalate and phosphate was
observed along with the formation of calcium oxalate type
of stones, which is similar to the reports of Prasad et al.,
1997 [2]. Treatment with ethanolic extract of Cucumis
sativus markedly reduced the excretion of calcium, oxalate
and phosphate in both preventive and curative groups when
compared to the positive control group. It lowered the
levels of oxalate and calcium in urine and even their
retention in kidney. This indicates the ability of C. sativus
in both preventing formation of and dissolving the calcium
oxalate type of stones.
Urinary phosphate levels were increased in the
positive control group and this increased level of phosphate
along with oxalate stress provides an appropriate
environment for stone formation by forming calcium
phosphate crystals which induces calcium oxalate
deposition. Ethanolic whole fruit extract reduced the risk of
stone formation by restoring urinary phosphate levels.

In urolithiatic condition, obstruction to the outflow

of urine by stones in urinary system takes place and as a
result nitrogenous substances such as urea, creatinine and
uric acid get accumulated in blood due to reduced excretion
by the kidneys. The elevated serum levels of creatinine,
uric acid and BUN indicate marked renal damage in Calculi
induced animals. Another evidence of increased lipid
peroxidation and decreased levels of antioxidant potential
have been reported in the kidneys of rats supplemented
with calculi producing diet.
Enzymatic and non-enzymatic systems preserve
the antioxidant/oxidant status, these defense systems
become overwhelmed during oxidative stress, a metabolic
derangement due to an imbalance caused by excessive
generation of ROS or a diminished antioxidant capacity.
Oxalate and free radicals generated during the metabolism
of oxalate, impose oxidative stress on hyperoxaluric
animals. The damaged epithelium of the kidney, due to
increased oxidative stress and with reduced antiadherent
glycosoaminoglycan layer, might act as a nidus for stone
formation.
Lipid peroxidation (LPO) is a degenerative
pathway for membrane components mediated through free
radicals produced in the cells. Oxalate has pro-oxidant
nature and the increased LPO formation by oxalate is
probably associated with generation of free radicals. The
evidence of involvement of oxalate in free radical mediated
LPO reaction for the membrane injury is further
strengthened by the subsequent observations made in
several studies [9,10].
From the results of our studies on the whole fruit
extract of C.sativus it has been seen that the extract
hastened the process of dissolving the preformed stones in
curative regimen and reduced stone formation when used as
prophylactic treatment. The histopathological study
supported the results obtained. Elevated levels of serum
parameters such as BUN, uric acid and creatinine in
untreated group animals were indicative of prominent
necrosis of renal epithelia and the depositions of calcium
oxalate crystals were observed in untreated group. Elevated
levels of oxalate in urine and even its retention in kidney
may be one of the causative factors for the peroxidative
degeneration of renal epithelia. However, the preventive
and curative treatment with ethanolic whole fruit extract of
C. sativus prevents oxalate induced lipid peroxidation and
causes regeneration of renal epithelium.
CONCLUSION
Renal calculi were induced in the rats by 0.75%
V/V ethylene glycol treatment. Both preventive and
curative regimens were found to be effective in reducing
urolithiasis. Further studies are required to exploit the
antiurolithiatic potential of C. sativus and develop it into a
herbal formulation.

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