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Research Article
Pharmaceutical Sciences
ANTIUROLITHIATIC ACTIVITY
OF THE PLANT EXTRACTS OF SOLANUM VIRGINIANUM
ON ETHYLENE GLYCOL INDUCED UROLITHIASIS IN RATS
Krishna Mohan Chinnala*, Srinivas Shanigarm, Madhan Mohan Elsani
Department of Pharmacology, St. John College of Pharmacy, Yellapur,
Hasanparthy, Warangal- 506 371. Andhra Pradesh, INDIA.
*Corresponding Author Email: drchinnala@gmail.com

ABSTRACT
Objective: To study the antiurolithiatic activity of Solanum virginianum (Solanaceae) in ethylene glycol induced
urolithiasis in male Spargue Dawley rats.
Methods: The urolithiasis was induced in rats by oral feeding of ethylene glycolated water (0.75%v/v) for 28 days.
Ethanolic extract of Solanum virginianm (200 mg/kg, 400 mg/kg) was administered orally from 1st day for
preventive regimen and from 15th day for curative regimen. Results: It was observed that the inducing agent
ethylene glycol elevated the ionic parameters, calcium and phosphate levels in urine, blood urea nitrogen (BUN),
serum creatinine and serum uric acid levels. Treatment with ethonolic extract of Solanum virginianm significantly
(P<0.001) reduced the elevated levels of ions in urine as well as BUN, serum creatinine and seum uric acid levels.
Conclusion: The elevated calcium and phosphate levels in urine, serum creatinine, blood urea nitrogen (BUN) and
uric acid levels of urolithiasis induced rats were reduced with preventive and curative regimens of plant extract
treatment. The histological findings also showed improvement in kidney architecture after treatment with the
plant extract. These observations enable us to conclude that the Solanum virginianum has curative and preventive
properties for ethylene glycol induced urolithiasis in rats.
KEY WORDS
Urolithiasis, Ethylene glycol, Solanum virginianum, Hyperoxaluria, Micro crystals.

INTRODUCTION major problem as patients who have formed one

Urolithiasis is a complex process which is a stone are more likely to form another.
consequence of an imbalance between promoters The synthetic drugs used to prevent urolithiasis are
1
and inhibitors in the kidneys . Even after the not effective in all patients, and many of them have
technological developments in the present medical adverse effects that compromise their long term use.
practice, the formation and growth of renal calculi In the present day management of urolithiasis with
continues to afflict humankind. Though various kinds open renal surgery is an unusual and rarely used one
of stones have been identified, calcium stones are the since the introduction of Extracorporeal Shock Wave
most common in human as well as in rats2. Lithotripsy (ESWL) which has almost become the
Urolithiasis is a common disorder estimated to occur standard procedure for eliminating kidney stones4.
in approximately 12% of the world population, with a However, in addition to the traumatic effect of
recurrence rate of 70‐81% in males and 47‐60% in shockwaves, persistent residue stone fragments and
328

3
females . The recurrence of urolithiasis represents a the possibility of infection suggests that ESWL may
cause acute renal injury, a decrease in renal function
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and an increase in stone recurrence . Hence, the 177/99 CPCSEA), Hyderabad, Andhra Pradesh. And
search for effective antilithiatic drugs without or they were housed at CPCSEA (Rag, no.
minimum side effects from natural sources has gained 1278/ac/09/cpcsea) approved animal house of St.
a great potential. John College of Pharmacy, Warangal. The animals
Solanum virginianm is a very prickly perennial herb were kept in polypropylene cages (6 in each cage)
with woody base plant from the family Solanaceae under standard laboratory condition (12 hr light and
which is common throughout India, Sri Lanka, Burma, 12 hr dark cycle) and had free access to commercial
Malaysia and Nepal. The plant is used in rheumatism, pellet diet (Hindustan lever L.td, Bombay, India) with
asthma, also as diuretic, anthelmentic and anti- water ad labium. The animal house temperature was
inflammatory substance. The aerial parts and the maintained at 25 ± 20C with relative humidity at (50
roots are used in herbal preparations. S. virginianum ±15%). Ethical norms were strictly followed during all
is endowed with various chemical components such experiments and the study was approved by the
as alkaloids, flavonoids, phytosterols, mucilage and Institutional Animal Ethical Committee of St. John
fixed oil etc., which possibly contribute to its vast uses College of Pharmacy (Reference No:
5
in folklore medicine . The present study was planned 001/IAEC/StJCOP/2011).
to evaluate the antiurolithiatic activity of ethanolic Acute Toxicity Study
extract of Solanum virginianm whole plant on The acute toxicity of ethanolic extract of Solanum
ethylene glycol induced calcium oxalate urolithiasis in virginianm was determined as per the OECD guideline
male rats. no.423 (Acute Toxic Class Method). It was observed
that the test extract was mortal even at 2000mg/kg
MATERIALS AND METHODS dose. Hence, 1/10th (200mg/kg) and 1/5th (400mg/kg)
Plant material of this dose were taken as a therapeutic dose for
The plants of Solanum virginianum were collected determination of antiurolithiatic effect7.
from surroundings of Warangal, Andhra Pradesh, Pharmacological screening for anti urolithiatic
India and the plant was authenticated by plant activity
taxonomist Prof. D.V.S.Raju., Department of Botany Animals were divided into seven groups, each
and Plant anatomy research center, Kakatiya containing six animals. Group I animals served as
University, Warangal, Andhra Pradesh. The voucher normal control and maintained on regular laboratory
specimen of the plant was deposited at the college diet and water ad labium. Group II to VII animals were
for the future reference. fed with 0.75% ethylene glycol (EG) in water to induce
Preparation of ethanolic extract renal calculi till 28th day8. Group III animals received
The whole plants of Solanum virginianum were standard antiurolithiatic drug cystone® (750 mg/kg
cleaned and chopped into small pieces and dried body weight) from 15th to 28th day9-10. Group IV & V
under shade. The coarse powder was obtained by animals served as curative regimen and received
mechanical grinding. The powdered material (100 g) ethanolic extract of the plant of Solanum virginianm
was subjected to continue hot extraction in soxhlet at a dose of 200 mg/kg and 400 mg/kg body weight
0
apparatus at a temperature of (60- 70 C) by using from 15th day to 28th day. Group VI & VII animals were
ethanol (95% v/v) as solvent6. After complete served as preventive regimen and received ethanolic
extraction, the extract was dried. The yield was about extract of the plant of Solanum virginianum at a dose
st
5% w/w and it was stored at 4oC in desiccator. The of 200 mg/kg and 400 mg/kg body weight from day 1
th
extract was suspended in distilled water using 1% day to 28 day. Both the extracts were administered
acasia as suspending agent for oral administration to once daily by oral route.
animals.
Experimental animals ASSESSMENT OF ANTIUROLITHIATIC ACTIVITY
329

The Spargue dawley rats, weighing 150-200g, were Collection and analysis of urine
procured from Teen baobabs Pvt. Ltd. (Rag, no. Urine samples were collected on 28th day for 24 h by
keeping the animals in individual propylene metabolic
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cages. Animals had free access to drinking water calcium and phosphate. Whereas the cystone-treated
during the urine collection period. The collected urine group III animals were shown significant reduction in
was analyzed for calcium and phosphate using calcium (P<0.0001) and phosphate (P<0.001) levels.
standard methods. The volume of urine collected Similarly treatment with Solanum virginianm
from all groups was recorded. Further, microscopy of significantly lowered the elevated levels of calcium
the urine was performed to identify the presence of (P<0.0001) and phosphate (P<0.0001) in urine
crystals. curative regimens and preventive regimen as
Serum analysis compared to EG induced group II animals (Table 1).
After the experimental period, the blood was Serum analysis
collected from the retro orbital puncture of rat eye The blood urea nitrogen (BUN), serum creatinine and
under ether anesthesia. Serum was separated by serum uric acid levels were significantly (P<0.0001)
centrifugation of the blood samples at 10,000 RPM increased in calculi-induced group II animals. While
for 10 minutes and analyzed for creatinine, uric acid the BUN, serum creatinine and serum uric acid levels
and BUN using standard methods. were significantly (P<0.001) decreased in cystone-
Effect of S. virginianum plant extracts on the kidney treated group III animals. However, the BUN and
weight serum creatinine levels were significantly (P<0.0001)
The weights of the kidney of normal, induced, decreased in both preventive and curative regimen
standard drug treated and extract treated group rats groups. It is also found that there is no significant
were weighed. The Group II animals gained the least reduction in serum uric acid levels in curative
body weight as compared to the normal control and regimens (Group IV and Group V), but the preventive
extract treated groups. In addition, the wet weight of regimens shows a significant (P<0.0001) reduction in
kidneys were taken and compared between the elevated serum uric acid levels (Table 1).
groups. Kidney Weights and Urinary Volume
Histopathology The kidney weight was significantly (P<0.001)
To confirm the incidence of urolithiasis the animals increased in ethylene glycol induced group –II animals
were sacrificed and their kidneys were isolated and when compare to the control group animals. Whereas
subjected to histopathological studies. The kidneys the standard treated group animals was shown
were cleaned off from extraneous tissue and significant (P<0.001) reduction in the kidney weight
transferred to 10% neutralized formalin solution (pH when compared to the EG induced group II animals.
7.4). Sections of kidney was fixed in paraffin, stained Similarly a significant (P<0.0001) decrease in the
with hematoxylin and eosin and observed for kidney weight is also identified in Solanum virginianm
histopathological studies11. treated preventive and curative regimens (Table 2).
Statistical analysis The urinary volume was significantly (P<0.001)
Statistical analysis was carried out using Graphpad decreased in ethylene glycol induced group-II animals
Pism (version 5) software. Results were expressed as when compared to the control group animals.
mean ± SEM. Groups of the data were compared Whereas the standard treated group animals was
applying one-way ANOVA followed by Dunnett’s shown significant (P<0.01) increase in urine output
Multiple Comparison test. Differences between the when compared to the EG induced group II animals.
data were considered significant at P<0.05. Similarly a significantly increase the urinary volume is
also identified in Solanum virginianm treated
RESULTS preventive regimen (P<0.0001) and curative regimen
Antiurolithiatic study (P<0.05) animals (Table 2).
Urinary excretion of calcium and phosphorous Histopathological studies
In the present study, chronic induction of EG (0.75% Histopathological studies of kidneys clearly revealed
330

v/v) to male Spargue dawley rats resulted in that the tissue section of Group I rats showing normal
significant (P<0.001) increase in urinary excretion of size tubules with single epithelial lining along the
margin. Whereas the Group II rats showed dilated
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tubules and degeneration of epithelial lining with standard and extract treated groups showed
presence of crystals. But the kidney specimen from characters similar normal control group (Figure 1).

Table: 1 Effect of Solanum virginianum extracts on urinary and serum parameters in control and experimental
urolithiasis

Parameters Normal Inducer Standard Curative regimen Preventive regimen

Group-I Group-II Group III
Group-IV Group-V Group-VI Group-VII
(200 mg/k.g) (400 mg/kg) (200 mg/kg) (400 mg/kg)
Urine (mg/dl)

*** ** ** *** **
Calicium 9.18±0.18 21.0±20.36 11.77±0.36 16.52 ±0.13 15.12±0.12 14.12±0.13 12.58±0.17

** ** ** ** ***
Phosphorous 2.005±0.02 3.938±0.05 2.520±0.02 3.477±0.01 3.323±0.05 2.825±0.01 2.723±0.01

Serum

Blood urea nitrogen 18.95±0.805 39.47±1.702 22.68±0.419* 33.55±2.325*** 27.01±1.24*** 24.01±0.42*** 2.76±0.365***

Serum creatinine 2.283±0.08 4.433±0.16 2.033±0.13** 4.033±0.20ns 2.333±0.09*** 2.800±0.23*** 1.800±0.14***

Uric acid 4.175±0.17 5.812±0.19 4.797±0.13** 5.613±0.14ns 5.823±0.09ns 4.700±0.12** 3.132±0.18**

The values are expressed as Mean ± SEM (n=6).

Comparisons were made between: Control Vs Group-II & Group-II Vs Group-III, IV, V, VI, and VII.
Statistical significant test for comparisons was done by ANOVA, followed by Dunnet’- ‘t’test
p-values ***p<0.001, **p<0.01 and *p<0.05. Ns – Non significant

Table 2: Effect of Solanum virginianum Plant extracts on the Urinary volume and kidney weight
Parameters Normal Inducer Standard Curative regimen Preventive regimen
Group-I Group-II Group III
Group-IV Group-V (400 Group-VI Group-VII
(200 mg/k.g) mg/kg) (200 mg/k.g) (400 mg/kg)

Urinary Volume 31.94±1.22 15.08±0.81 25.37±1.05*** 24.34±1.35*** 25.67±0.92*** 21.56±0.95** 19.86±1.62*

*** *** *** *** ***

Kidney Weight 32.73±0.05 46.51±0.10 39.00±0.16 44.08±0.04 43.16±0.01 42.03±0.08 40.62±0.01

The values are expressed as Mean ± SEM (n=6).

Comparisons were made between: Control vs Group-II & Group-II vs Group-III,IV,V,VI,VII.
Statistical significant test for comparisons was done by ANOVA, followed by Dunnet’-‘t’test
p-values ***p<0.001, **p<0.01 and *p<0.05. Ns – Non significant
331
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Figure 1: Histopathological studies

DISCUSSION precipitates as a calcium oxalate crystals in kidney

Urolithiasis can be produced in rats by induction of since the oxalate metabolism is considered almost
acute or chronic hyperoxaluria by using a variety of identical between rats and humans.
agents such as ethylene glycol, sodium oxalate, Calcium and phosphate play a vital role in renal
ammonium oxalate, hydroxyl-L-proline and glycolic calculogenesis. In EG induced rats, the urinary
12
acid . Kidney being the principal target for EG excretion of calcium, phosphate was significantly
induced toxicity. EG is broken down in-vivo into four increased. The increase in calcium and phosphate
organic acids viz., glycolaldehyde, glycolic acid, excretion could be due to defective tubular
glycooxalic acid and oxalic acid leading to reabsorption in the kidneys14. While treatment with
hyperoxaluria which is the main initiative factor for standard, curative and preventive regimens of S.
lithiasis13. Therefore in the present study, EG was virginianum markedly reduced the levels of these
preferred to induce lithiasis. Administration EG to the ions, suggested protective effect of S. virginianum
experimental animals for 28 days resulted in against urolithiasis.
332

substantial elevation of oxalate and deposition of In urolithiasis, the calculi formed in the renal tissue
microcrystal’s in kidney. In addition, oxalate leads to obstruction in the urinary system that
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*Corresponding Author:
Dr. Krishna Mohan Chinnala
Professor & Principal
St. John College of Pharmacy,
Yellapur, Hasanparthy, Warangal-506371,
Andhra Pradesh, INDIA.
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