effect of diosgenin treatment on ethylene glycol …
TRANSCRIPT
EFFECT OF DIOSGENIN TREATMENT ON ETHYLENE
GLYCOL INDUCED UROLITHIASIS IN RATS.
Surbhi Guptaa, Reema Mitraa, Dr. M. Arockia Babua
aChandigarh College of Pharmacy, Landran, Mohali, Punjab- 140307
ABSTRACT
The anti-urolithiatic effect of Diosgenin was determined on ethylene glycol induced urolithiasis
in male wistar rats. The urolithiasis was induced in rats by administration of ethylene glycol in
drinking water (0.75%v/v) for 28 days. Suspension of Diosgenin(50mg/kg and 100mg/kg) was
administered orally from 1st day for preventive treatment and from 15th day for curative regimen.
Ethylene glycol administration elevated the calcium, oxalate, urea and uric acid in urine.
Treatment with Diosgenin significantly reduced these biochemical parameters in urine. Also
treatment with Diosgenin significantly elevated the urinary concentration of citrate. The elevated
serum creatinine and blood urea nitrogen levels of urolithiatic rats were reduced by prophylactic
and curative treatment with Diosgenin. A considerable reduction in oxidative stress and
histopathological parameters were also noted. In vitro and ex vivo antiurolithiatic activity of
diosgenin were carried out using aggregation assay in synthetic urine and in rat plasma. The
study revealed that diosgenin in plasma has significant inhibitory potential.
Key words: Urolithiatic, Ethylene glycol, Diosgenin, Antiurolithiatic
INTRODUCTION
Urolithiasis (UL) is the formation of stone in the urinary system, when it occurs in kidneys it is
called as nephrolithiasis, when stone occurs in ureter it is called as ureterolithiasis and when it
occurs in urinary bladder and urethra it is called as Cystolithiasis.(Mohammadet.al.2010).
Kidney stone formation or urolithiasis is a complex process that is a consequence of an
imbalance between promoters and inhibitors in the kidneys. Various kinds of stone have been
identified, calcium stones are the most common in human as well as in rats (Onaran M
et.al.2016). Urolithiasis is three times higher in men than women because of enhancing capacity
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of testosterone and inhibitory capacity of oestrogen in stone formation (Joy et.al.2012). Stone
formation is considered as a medical challenge, as the etiology and pathophysiology is still
widely unknown and governed by a number of factors. An imbalance between promoters and
inhibitors leads to precipitation and formation of stones (Vijaya et.al.21013). It has also been
found that the formation of renal stones and also the composition of stones depend on age and
gender of the patients. It is more commonly found to occur in older patients. The incidences of
urolithiasis in children are rare. In present days increase in obesity among children may pose a
risk factor for stone formation as in adult (Mohammadet.al.2010).
Studies have indicated that plants containing steroidal saponins have good antiurolithiatic
potential due to anti-crystallisation and estrogenic activity. Saponins are a group of naturally
occurring plant glycosides, characterized by their strong foam-forming properties in aqueous
solution. The cardiac glycosides also possess this property but are classified separately because
of their specific biological activity. Unlike the cardiac glycosides, saponins generally do not
affect the heart. These are classified as steroidal triterpenoid saponins. Depending on the nature
of the aglycone pharmacological activities of different plant extracts have shown that plants
containing saponin have good antiurolithiatic activity. Diosgenin is a saponin with several
pharmacological properties that include anticancer, cardiovascular protective, anti-diabetes,
neuroprotective, immunomodulatory, estrogenic, and skin protective effects, mainly by inducing
apoptosis, suppressing malignant transformation, decreasing oxidative stress, preventing
inflammatory events (Yanchen et.al.2015). Plants containing diosgenin as saponin e.g. Tribulus
terrestris has been tested for their action against kidney stone and the results are promising
(Ranandet.al.2013). The present study therefore intends to find out the efficacy of diosgenin
which is saponin in treatment of stone (Mafalda et.al.2016)
The present study was planned to investigate the effect of diosgenin in ethylene glycol induced
urolithiasis in rats.
MATERIALS AND METHODS
IN VIVO STUDY-
Preparation of suspension of Diosgenin
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The powdered drug of diosgenin (50 and 100mg/kg) was weighed and macerated using 1%
Tween 80 and then the volume was made up by using distilled water with occasional shaking as
required. The prepared suspension of diosgenin was poured into individual beakers and stored
under normal temperature conditions of refrigerator and to be taken out while dosing only.
Experimental animals
Male wistar rats weighing between 180-220g were used, the animals were fed with commercial
rat feed pellets and were given water ad libitum. Animals were housed in plastic cages with filter
tops under controlled conditions of 12:12 light dark cycle, 50% humidity and 28°C. All animal
experiments and maintenance were carried out according to the ethical guidelines suggested by
the Institutional Animal Ethical Committee (IAEC).
Pharmacological screening for anti-urolithiatic activity
Animals were divided into five groups containing six animals in each group. Group I served as
normal control and received regular food and drinking water ad libitum. Ethylene glycol
(0.75%v/v) in drinking water was fed to Group II-V for induction of urolithiasis till 28th day.
Group II served as urolithiatic (disease) control group. Group III received standard anti-
urolithiatic drug cystone (750 mg/kg) from 15th day till 28th day. Group IV received suspension
of diosgenin (50 mg/kg p.o.) from 15th day till 28th day and served as curative regimen. Group V
received suspension of diosgenin (100 mg/kg p.o.) from 1st day till 28th day and served as
preventive regimen. All drugs were given once daily by oral route ( p.o.) using oral cannula
(Mohemidet.al. 2014).
Assessment of antiurolithiatic activity
On day 28 animals of all the groups were analyzed for the following parameters:
Collection and analysis of urine:
All animals were kept in individual metabolic cages and urine samples of 24h were collected on
28th day and analyzed for urine output volume, urea, uric acid, citrate, calcium and oxalate by
using standard methods (Taylor et.al.2008).
Serum analysis:
After the experimental period, blood was collected from the cardiac-puncture under anesthetic
conditions and animals were sacrificed by cervical decapitation. Serum was separated by
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centrifugation at 1000rpm for 10min and analyzed for serum creatinine and blood urea nitrogen
by using standard methods (Husdanet.al.1968, Faweettet.al.1960).
Kidney homogenate analysis:
The abdomen was cut open to remove right kidneys from each animal. Isolated kidneys were
cleaned off extraneous tissue and preserved in buffer (8.2g sodium chloride + 2.5g sodium
phosphate monobasic + 2.5g sodium phosphate dibasic). The homogenate was centrifuged at
2000rpm for 10min and supernatant was separated. The levels of thiobarbituric acid reactive
species (TBARS), reduced glutathione (GSH) and superoxide dismutase (SOD) content in
kidney homogenate were determined by using standard methods (Niehiuset.al.1968).
Histopathological study:
To confirm the incidence of urolithiasis, the animals were sacrificed and their left kidneys were
isolated and subjected to histopathological studies. The kidneys were washed, weighed and fixed
rapidly with 10 % neutralized formalin (pH 7.4) and soaked in paraffin, cut at 5µm intervals and
the slides were stained with hematoxylin and eosin. Tissue slides were photographed using
optical microscopy and observed the pathological changes at 10X and then images were taken at
40X. (Thangarathinam et.al.2016)
Statistical analysis:
The results were expressed as mean ± SEM by using graph pad prism software. Statistical
analysis was performed by using one way ANOVA followed by Tukey's multiple range test for
multiple comparisons and p<0.05 was considered as significant.
IN VITRO STUDY-
1) In Vitro Aggregation Assay:
Inhibition assay of oxalate crystals aggregation was carried out for diosgenin extract. Solutions
of calcium chloride and sodium oxalate were prepared at concentrations of 6.0 mmol/L and 6.5
mmol/L respectively in a buffer containing Tris 0.05 mol/L and sodium chloride 0.15 mol/L at
pH 6.5. The calcium chloride solution (950 µL) was mixed with 100 µL of diosgenin at the
different concentrations (100-1000 µg/mL). Crystallization was started by adding 950 µL of
sodium oxalate solution. In the case of control experiment, 100µL of buffer was added to
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calcium chloride solution. The temperature was maintained at 37°C throughout for 1 h of the
incubation period. The optical density (OD) of the crystallized suspension was monitored at 620
nm. The percentage aggregation inhibition was then calculated by comparing the turbidity in the
presence of sample with that obtained in the control using the following formula:
Percentage of inhibition = [ 1 - ( Turbidity of sample / Turbidity of control ) ] × 100.
(Tsujihata. Met.al.2000).
2) Ex Vivo Turbidity Assay:
The oxalate crystal inhibition potential of diosgenin extract was carried out in rat plasma to
provide the biological environment. The plasma sample was diluted with equal volume of
calcium chloride and sodium oxalate (12 mmol/L each), separately. The 950 µL of plasma
containing sodium oxalate (6.0 mmol/L) was mixed with 100 µL of diosgenin extract at the
different concentrations (100-1000 µg/mL). Crystallization was carried out by adding 950 µL of
plasma containing calcium chloride (6.0 mmol/L). The mixture solution was incubated for 1 h
and the temperature was maintained at 37°C. The OD of the crystallized suspension was
measured at 620 nm and the inhibition potential was estimated by comparing with control. The
percentage of aggregation inhibition was then calculated by comparing the turbidity in the
presence of the extract with that obtained in the control using following formula:
Percentage of inhibition = [ 1 - ( Turbidity of sample / Turbidity of control ) ] × 100.
(Tsujihata. Met.al.2000).
RESULTS
Assessment of urolithiasis was done by measuring different biochemical parameters (urine
volume, calcium, oxalate, creatinine, urea, uric acid, citrate, BUN and body weight level were
measured by using specific diagnostic kits (Taylor et.al.2008). Homogenate analysis was done
for TBARS, GSH and SOD using chemical methods (Chitikelaet.al.2016).
On the basis of results obtained in the present study following finding may be summarized
-Ethylene glycol administration in a dose of 0.75%v/v in experimental animals induced
significant changes in biochemical parameter, histopathology, and homogenate analysis.
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-Treatment with Diosgeninp.o. (50mg/kg, 100mg/kg) resulted in significant preventive and
curative against ethylene glycol induced urolithiasis. The urinary output was decreased
significantly in disease control group. The urinary output of standard, test1(low dose &
test2(high dose) is increased significantly(p<0.001). In treated groups, the urine output was
higher than the disease group.
In the present study, there was an increased uric acid, oxalate, urea, calcium, BUN in disease
group. In treatment groups these parameters were found to be significantly decreased.
Levels of creatinine & TBARS were elevated in disease group & animal receiving treatment had
decreased levels.
Urinary citrate, SOD, GSH ( Reduced Glutathione) was decreased gradually by renal stone
induction. However, in standard and treatment there was a significant increase in these
parameters showing an inhibitory action on stone formation.
The following are the results from the analysis of body weight, biochemical parameters in
urine and serum and the results for homogenate and histopathology analysis and the
results for invitro study.
Results of Body weight analysis:
Graph 1 Effect of Diosgenin treatment on body weight (g)
Norm
al
Dis
ease
contr
ol
Sta
ndard
Low d
ose
Hig
h dose
0
200
400
600
800
1000
a
b1
b2
b3
body weight (g)
Group(s)
Bo
dy w
eig
ht(
g)
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Body Weight was expressed in gms. Each group (n=6) represents mean ± SEM(b1 group
compared with disease control group, b2 group compared with disease control group, b3 group
compared with disease control group), *: p< 0.05, **: p< 0.01, ***: p< 0.001. A difference in the
mean value was considered to be statistically significant at p<0.01 and we have performed Tukey
test.
Results of Urine analysis:
Graph 2 Effect of Diosgenin treatment , curative(a) and preventive (b)on urine volume (ml)
urine volume day28
0
5
10
15
N D S T1 T2
uri
ne v
olu
me(m
l)
Urine volume level was expressed as ml. Each group (n=6) represents mean ± SEM(b1 group
compared with disease control group, b2 group compared with disease control group, b3 group
compared with disease control group)*: p< 0.05, **: p< 0.01, ***: p< 0.001. A difference in the
mean value was considered to be statistically significant at p<0.001 and we have performed
Tukey test.
Graph 3 Effect of Diosgenintreatment (curative and preventive) on urine urea level(mg/dl)
urine urea level(mg/dl)
Norm
al
Dis
ease
contr
ol
Standar
d
Low d
ose
Hig
h dose
0
50
100
150
200
a
b1 b2 b3
Group(s)
uri
ne u
rea c
on
c.(
mg
/dl)
urine urea level
0
20
40
60
80
N D S T1 T2
ure
a c
on
c.(
mg
/dl)
urine volume(ml)
Normal
Disease c
ontrol
Standard
Low d
ose
High d
ose
0
10
20
30
Group(s)
a
b1b2
b3
uri
ne
volu
me(
ml)
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Urine Urea level was expressed as mg/dl. Each group (n=6) represents mean ± SEM(b1 group
compared with disease control group, b2 group compared with disease control group, b3 group
compared with disease control group), *: p< 0.05, **: p< 0.01, ***: p< 0.001. A difference in the
mean value was considered to be statistically significant at p<0.001 and we have performed
Tukey test.
Graph 4 Effect of Diosgenin treatment (curative and preventive) on urine uric acid
level(mg/dl)
urine uric acid level(mg/dl)
Nor
mal
Disea
se cont
rol
Sta
ndar
d
Low
dos
e
Hig
h dose
0
5
10
15
20a
b1
b2
b3
Group(s)
urin
e u
ric
acid
co
nc.(
mg
/dl)
0
2
4
6
8
N D S T1 T2
uric
acid
co
nc.(
mg
/dl)
Urine uric acid level was expressed as mg/dl. Each group (n=6) represents mean ± SEM(b1 group
compared with disease control group, b2 group compared with disease control group, b3 group
compared with disease control group), *: p< 0.05, **: p< 0.01, ***: p< 0.001. A difference in the
mean value was considered to be statistically significant p<0.01 and we have performed Tukey
test.
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Graph 5 Effect of Diosgenin treatment (curative and preventive) on urine citrate level
(mmol/l)
urine citrate level(mmol/l)
Nor
mal
Disea
se con
trol
Sta
ndar
d
Low d
ose
Hig
h do
se
0
20
40
60
80
100
a
b1
b2
b3
Group(s)
urin
e c
itrate
co
nc.(
mm
ol/l)
0
10
20
30
40
N D S T1 T2
cit
rate
co
nc.(
mm
ol/l)
Urine citrate level was expressed as mmol/L. Each group (n=6) represents mean ± SEM(b1 group
compared with disease control group, b2 group compared with disease control group, b3 group
compared with disease control group), *: p< 0.05, **: p< 0.01, ***: p< 0.001. A
difference in the mean value was considered to be statistically significant p<0.05 and we have
performed Tukey test.
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Graph 6 Effect of Diosgenin treatment (curative and preventive) on urine calcium level
(mg/dl)
urine calcium level (mg/dl)
Nor
mal
Disea
se cont
rol
Sta
ndar
d
Low
dos
e
Hig
h dose
0
10
20
30
40
50
a
b1b2 b3
Group(s)
urin
e c
alc
ium
co
nc.(
mg
/dl)
0
5
10
15
20
N D S T1 T2
uri
ne c
al.(m
g/d
l)
Urine
Calcium level was expressed as mg/dl. Each group (n=6) represents mean ± SEM(b1 group
compared with disease control group, b2 group compared with disease control group, b3 group
compared with disease control group), *: p< 0.05, **: p< 0.01, ***: p< 0.001. A difference in the
mean value was considered to be statistically significant p<0.001 and we have performed Tukey
test.
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Graph 7 Effect of Diosgenin treatment on urine oxalate level (mmol/l)
urine oxalate level(mmol/l)
Normal
Disease c
ontrol
Standard
Low d
ose
High d
ose
0
50
100
150
200
a
b1
b2
b3
Group(s)
urin
e ox
alat
e co
nc.(
mm
ol/l)
Urine oxalate level was expressed as mmol/l. Each group (n=6) represents mean ± SEM(b1 group
compared with disease control group, b2 group compared with disease control group, b3 group
compared with disease control group), *: p< 0.05, **: p< 0.01, ***: p< 0.001. A difference in the
mean value was considered to be statistically significant p<0.05 and we have performed Tukey
test.
Results of serum anlysis:
Graph 8 Effect of Diosgenin treatment (curative and preventive) on serum creatinine level
(mg/dl)
serum creatinine level(mg/dl)
Norm
al
Dis
ease
contr
ol
Standar
d
Low d
ose
Hig
h dose
0
2
4
6
8
a
b1
b2b3
Group(s)
seru
m c
reati
nin
e(m
g/d
l)
0
1
2
3
4
N D S T1 T2
cre
ati
nin
e (
mg
/dl)
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Serum Creatinine level was expressed as mg/dl. Each group (n=6) represents mean ± SEM(b1
group compared with disease control group, b2 group compared with disease control group, b3
group compared with disease control group), *: p< 0.05, **: p< 0.01, ***: p< 0.001. A difference
in the mean value was considered to be statistically significant p<0.01 and we have performed
Tukey test.
Graph 9 Effect of Diosgenin treatment (curative and preventive) on Serum Blood Urea
nitrogen (BUN) level(mg/dl)
serum BUN level (mg/dl)
Normal
Disease c
ontrol
Standard
Low d
ose
High d
ose
0
20
40
60
80
100
a
b1
b2
b3
Group(s)
seru
m B
UN
co
nc.
(mg
/dl)
0
10
20
30
40
N D S T1 T2
BU
N c
on
c.(
mg
/dl)
Serum BUN level was expressed as mg/dl. Each group (n=6) represents mean ± SEM(b1 group
compared with disease control group, b2 group compared with disease control group, b3 group
compared with disease control group), *: p< 0.05, **: p< 0.01, ***: p< 0.001. A difference in the
mean value was considered to bestatistically significant p<0.001 and we have performed Tukey
test.
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KIDNEY HOMOGENATE RESULT ANALYSIS:
Graph 10 Effect of Diosgenin treatment (curative and preventive) on Thiobarbituric acid
reactive substances (TBARS) level (nm/mg)
TBARS level(nm/mg)
Norm
al
Dis
ease
contr
ol
Standar
d
Low d
ose
Hig
h dose
0
10
20
30
40
50a
b1
b2
b3
Group(s)
TB
AR
S(n
m/m
g)
0
5
10
15
20
N D S T1 T2
TB
AR
S(n
m/m
g)
TBARS level was expressed as nm/mg. Each group (n=6) represents mean ± SEM(b1 group
compared with disease control group, b2 group compared with disease control group, b3 group
compared with disease control group), *: p< 0.05, **: p< 0.01, ***: p< 0.001. A difference in the
mean value was considered to be statistically significant p<0.05 and we have performed Tukey
test.
Graph 11 Effect of Diosgenintreatment (curative and preventive) on Glutathione
(GSH)level (µM/mg)
GSH level (µM/mg)
Nor
mal
Disea
se con
trol
Sta
ndar
d
Low d
ose
Hig
h do
se
0
20
40
60
a
b1
b2
b3
Group(s)
GS
H(µ
M/m
g)
0
5
10
15
20
25
N D S T1 T2
GS
H(u
M/m
g)
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GSH level was expressed as µmol/mg. Each group (n=6) represents mean ± SEM(b1 group
compared with disease control group, b2 group compared with disease control group, b3 group
compared with disease control group), *: p< 0.05, **: p< 0.01, ***: p< 0.001. A difference in the
mean value was considered to be statistically significant p<0.001 and we have performed Tukey
test.
Graph 12 Effect of Diosgenin treatment on Superoxide dismutase(SOD) level (Units/ml)
SOD level Units/ml
Norm
al
Dis
ease
control
Standar
d
Low d
ose
Hig
h dose
0
200
400
600
800
1000
a
b1
b2
b3
Group(s)
SO
D c
on
. U
nit
s/m
l
SOD level was expressed as units/ml. Each group (n=6) represents mean ± SEM(b1 group
compared with disease control group, b2 group compared with disease control group, b3 group
compared with disease control group), *: p< 0.05, **: p< 0.01, ***: p< 0.001. A difference in the
mean value was considered to be statistically significant p<0.001 and we have performed Tukey
test.
HISTOPATHOLOGICAL RESULT
In histopathological observations gross examination of rats kidney from normal control group
showed a normal cortical structure of the kidney including no stone depositions with normal
glomeruli, distended tubules, proximal and distal convoluted tubules without any inflammatory
changes (fig-a) (Claydenet.al.1971)
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Light microscopic architecture and calcification in the kidney section of all group Paraffin
section of kidney, haematoxylin and eosin (H&E).
Standard (Cystone750 mg/kg) recovered distended tubules and increased cellularity between
tubules (Fig-c). No stone deposition or other abnormalities in the segment of high dose(Fig-d2)
treatment group. On the other hand, in disease group (Fig-b) many deposits inside the tubules
and dilations of the tubules along with interstitial inflammations were observed in the tissue of
urolithiatic rats.
a b c
d 1
c
d 2
a b
d 1 d 2
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Results of Invitro Study:
Graph 13 Comparison between In Vitro Aggregation Assay and Ex Vivo Turbidity Assay.
In Vitro Aggregation Assay
0 100 200 300 400 500 600 700 800 90010000
5
10
15
20
25
Diosgenin concentration (µg/ml)
Perc
en
tag
e o
f in
hib
itio
n
Ex Vivo Turbidity Assay
0 100 200 300 400 500 600 700 800 90010000
20
40
60
80
Diosgenin concentration (µg/ml)
Percen
tag
e o
f in
hib
itio
n
The study revealed that the inhibitory potency of diosgenin to inhibit formation of stones by ex
vivo turbidity assay was more significant and potent than in vitro aggregation assay.
DISCUSSION
The development of urolithiasis is well reported. Development of urolithiasis requires a total
period of 28 days. The dose of ethylene glycol was selected on the basis of previously reported
studies i.e 0.75% v/v in drinking water (Nizamiet.al.2014), (Makasanaet.al.2010). Diosgenin was
given from 1st day and 15th day of ethylene glycol administration at a low dose of 50mg/kg and
high dose of 100 mg/kg p.o once a day (Ahmed LA et.al.2014).
In the present study, the urolithiasis developed after 28 days and was tested by estimation of
different parameters after 28th day. The results showed that there was a considerable difference in
the groups administered ethylene glycol as compared to the others. This proved that there was
development of urolithiasis in all the 4 groups other than the normal control. The experiment was
continued as per the protocol and parameters were evaluated after the 28th day.
Pharmacological activities of different plant extracts have shown that plants containing saponins
have good antiurolithiatic activity (RajashekharV et.al.2012) Plants containing diosgenin (a
steroidal saponin) have also been found to have diuretic activity. According to some studies
diosgenin exhibits several pharmacological properties that include antioxidant, anti-inflammator
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and anti- apoptotic activity. There is inflammation and also oxidative stress during kidney
stones so diosgenin might be beneficial in kidney stones.
The body weight of animals was significantly reduced in disease control group when compared
with standard and treatment group due to formation of stones and marked renal damage.
Urine Volume of animals for all the four groups were observed for the change in volume (ml)
after 28th day. After 28th day it was observed that disease control group showed highly decreased
level of urine volume due to the urinary supersaturation with respect to stone formation. The
urine volume in standard control group showed an improvement which was statistically
significant. In the low dose treatment group there was significant difference as compared to
disease control group. In the high dose treatment group the improvement in urine volume was
more as compared to disease control group.
Urea is synthesized in the body of many organisms as part of the urea cycle, either from the
oxidation of amino acids or from ammonia. In this cycle, amino groups donated by ammonia and
L-aspartate are converted to urea, while L-ornithine, citrulline, L-argininosuccinate, and L-
arginine act as intermediates. Urea production occurs in the liver and is regulated by N-
acetylglutamate. Urea is found dissolved in blood (in the reference range of 2.5 to 6.7 mmol/L)
and is excreted by the kidney as a component of urine (Sakami W et.al.1963). The urine
chemistry showed that urea levels in urine were increased significantly in the disease control
group due to the decrease in the glomerular filtration rate due to the obstruction to the outflow of
urine by stones. There was a significant decrease in standard control group as compared to
disease control. There was also a marked decrease in two treatment groups with the decrease
being more with the higher dose because diosgenin causes diuresis and hastens the process of
dissolving the performed stones and prevention of new stone formation.
The increase in Urine oxalate level was in accordance with the previous studies with ethylene
glycol in various animal species (Hodgkinson et.al.1971). The increase in urine oxalate level
could be due to the fact that ethylene glycol leads to the hyperoxaluria, which causes increased
renal retention and excretion of oxalate (Selvam et.al.2001) so because of this the oxalate level
was found to be increased in disease control group and decreased in standard group and two of
the
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treatment groups respectively becausediosgenin lower the levels of oxalate excretion. However
the decrease was more significant in the high dose treatment group.
An increased urinary level of calcium favours the nucleation and precipitation of Calcium
oxalate crystal attachment and more centers for nucleation of new crystals (Lemann et.al. 1991).
The Urine calcium level showed increase in disease control group while, Standard group showed
decreased calcium level. The calcium level was found to be decreased more in high dose
treatment group.
The Creatinine level, was found increased in disease control group because in this the glomerular
filtration rate decreases due to the obstruction to the outflow of urine by stones as creatinine get
accumulated in blood leads to marked renal damage and creatinine level was found decreased in
standard and low dose treatment group. The decrease was more in the high dose treatment group
as diosgenin causes diuresis and hastens the process of dissolving the performed stones and
prevention of new stone formation at higher dose.
In Urolithiatic condition, obstruction to the outflow of urine by stones in urinary system takes
place and as a result nitrogenous substances such as BUN get accumulated in blood due to
reduced excretion by the kidneys. The elevated serum levels of creatinine and BUN indicate
marked renal damage in calculi induced animals (KrishnaveniJanapareddiet.al.2013). In serum
BUN was increased in disease control group and there was significant decrease in standard
control group as compared to disease control group. The BUN levels also decreased in low and
high dose treatment group with decrease being more with high dose.
Citrate level was lower in rats treated with EG than intact control animals. Low level of citrate is
an important risk factor for the stone formation in kidney (Zuckerman and Assimos,2009).
Citrate complexes with calcium can increase its solubility and reduce free calcium content in the
urine.
The uric acid level was also significantly increased in disease control group because the GFR
decreases due to the obstruction to the outflow of urine by stones in urinary system which get
accumulated in blood leading to marked renal damage. There was a significant decrease in uric
acid level in standard control group. While, the higher dose treatment group showed a
considerable improvement as compared to low dose treatment group because the treatment with
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diosgenin causes diuresis and hastens the process of dissolving the performed stones and
prevention of new stone formation in urinary system at higher dose.Furthermore, levels of
antioxidative enzymes such as glutathione (GSH), superoxide dismutase in the renal cortex were
found to be significantly decreased in disease group while TBARS was found increased in
disease group. While in TBARS, it was observed that there was increased MDA level in disease
control group and simultaneously decreased in standard control group and two of the treatment
groups. So it can be concluded that, biochemical estimations also showed promising results.
There was also considerable improvement in oxidative stress with administration of diosgenin as
seen with improvement in the assay of various parameters like, glutathione level (GSH).
The histopathological findings showed that administration of diosgenin decreased the renal
damage to glomeruli as compared to the disease control group. Histopathological picture of high
dose treatment group resembled that of normal control group.
CONCLUSION
The current study was able to show the effect of diosgenin in ethylene glycol induced renal
calculi model. Also, these results indicated administration of Diosgenin cure and prevent the
growth of urinary stones. Earlier study reported plant containing saponin is useful in renal
disorder (Rajasekhar V et.al.2012).
Diosgenin also showed good antioxidant activity as oxidative stress is common in kidney stones.
Diosgenin also showed good diuretic action and improvement in biochemical parameters so from
the results obtained.
The mechanism underlying this effect is possibly mediated through antioxidant nephroprotective
properties and also lowering the concentration of urinary stone forming constituents.
Testosterone is a promoter in kidney stone formation on the other hand estrogen and
progesterone is inhibitor in kidney stone formation. Diosgenin is a steroidal saponin & precursor
for progesterone therefore it shows inhibitory effect in the formation of kidney stone. However,
it requires more investigation to clarify the exact mechanism of this action. It can be concluded
that Diosgeninhas a curative and preventive effect against the formation of kidney stone.
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