EFFECT OF DIOSGENIN TREATMENT ON ETHYLENE GLYCOL …

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

*[email protected]

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

PAIDEUMA JOURNAL

Vol XII Issue XII 2019

Issn No : 0090-5674

http://www.paideumajournal.com487

mailto:*[email protected]

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

PAIDEUMA JOURNAL


Issn No : 0090-5674


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

PAIDEUMA JOURNAL


Issn No : 0090-5674


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

PAIDEUMA JOURNAL


Issn No : 0090-5674


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.

PAIDEUMA JOURNAL


Issn No : 0090-5674


-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)

PAIDEUMA JOURNAL


Issn No : 0090-5674


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)*: 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)

PAIDEUMA JOURNAL


Issn No : 0090-5674


Urine Urea level was expressed as mg/dl. Each group (n=6) represents mean ± SEM(b1 group



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



mean value was considered to be statistically significant p<0.01 and we have performed Tukey

test.

PAIDEUMA JOURNAL


Issn No : 0090-5674


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), *: 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.

PAIDEUMA JOURNAL


Issn No : 0090-5674


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




test.

PAIDEUMA JOURNAL


Issn No : 0090-5674


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




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)

PAIDEUMA JOURNAL


Issn No : 0090-5674


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



mean value was considered to bestatistically significant p<0.001 and we have performed Tukey

test.

PAIDEUMA JOURNAL


Issn No : 0090-5674


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




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)

PAIDEUMA JOURNAL


Issn No : 0090-5674


GSH level was expressed as µmol/mg. Each group (n=6) represents mean ± SEM(b1 group




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




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)

PAIDEUMA JOURNAL


Issn No : 0090-5674


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

PAIDEUMA JOURNAL


Issn No : 0090-5674


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

PAIDEUMA JOURNAL


Issn No : 0090-5674


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

PAIDEUMA JOURNAL


Issn No : 0090-5674


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

PAIDEUMA JOURNAL


Issn No : 0090-5674


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.

PAIDEUMA JOURNAL


Issn No : 0090-5674


https://en.wikipedia.org/wiki/Progesterone

REFERENCES

Adepu A, Narala S, Ganji A and Chilvalvar S. A Review on Natural Plant: Aervalanata.

International Journal of Pharma Sciences 3(6); 2013, 398-402.

Afroz R, Tanvir EM and Little JP. Honey-derived Flavonoids. Natural Products for the

Prevention Atherosclerosis and Cardiovascular Diseases.Journal of Clinical & Experimental

Pharmacology 6(3); 2016, 2161-1459.

Ahmed S, Montasheemul H M and Mohmood Z A. Journal of Pharmacognosy and

Phytochemistry 5(3); 2016, 28-35.

Alejandra A, Kedika B, Thotla K, Noole V and Chepyala K.Research Progress of Chrysin

derivatives with potential biological activities.Journal of Chemical and Pharmaceutical Research

8(8); 2016, 1210-1222.

Bramono AI, Rasyid N, Birowo P. Associations between BMI, serum uric acid, serum glucose,

and blood pressure with urinary tract stone opacity • Medicinal Journal of Indonesia 24;

2015,103-8.

Cherkaoui T. K., LachkarM,Wibo M and Morel N. Pharmacological studies on hypotensive,

diuretic and vasodilator activities of chrysin glucoside from Calycotomevillosa in rats.

Phytotherapy research Phytother. 22; 2008: 356–361.

Corbiere C, Liagre B, Terro F, et al. Induction of antiproliferative effect by diosgenin through

activation of p53, release of apoptosis-inducing factor (AIF) and modulation of caspase-3

activity in different human cancer cells [J]. Cell Res, 2004, 14(3): 188-196

Dash D K, Yeligar V C, Nayak S, Ghosh T, Rajalingam D, sengupta P, Maiti B C and Maity T

K. Evaluation of Hepatoprotective and Antioxidant Activity of IchnocarpusFrutescens (linn)

R.Br.on Paracetamol induced Hepatotoxicity in Rats. Tropical Journal of Pharmaceutical

Research 6(3); 2007, 755-765.

David A B, John R A, Marc D. G, Andrew P E., Walter R P, Kristen M A and Fredric L C.

Calcium oxalate stone formation in genetic hypercalciuric stone-forming rats. Kidney

International 61; (2002), 975–987.

David E. L, Ruslan K, Eric N. T, Jie T,| John R. A, David S. G, Mantu G and Gary C. Effect of

Vitamin D Repletion on Urinary Calcium Excretion among Kidney Stone Formers. Clinical

Journal of the American Society ofss Nephrology 7; 2012, 829–834.

Fredric L.C.Uric acid and calcium oxalate nephrolithiasis.Kidney International 24 ;1983, 392-

403.

Griffith D P. Struvite stones. Kidney International 13; 1978, 372 —382.

Gong G, Qin Y, Huang W, et al. Protective effects of diosgenin in the hyperlipidemic rat model

and in human vascular endothelial cells against hydrogen peroxide-induced apoptosis [J]. Chem-

Biol Interact, 2010, 184(3): 366-375.

PAIDEUMA JOURNAL


Issn No : 0090-5674


Holmes RP, Goodman HD, Assimos DG. The distribution of urinary calcium excretion in

individuals on control diets. Journal of Urology 1995; 153:350. Abstract, 468.

Hodgkinson A and Ann WillIams M.R.C Mineral Metabolism Unit, The General Infirmary,

Leeds LSI3EX . (U.K.) (Received July 22, 1971).

He Z, Tian Y, Zhang X, et al. Anti-tumour and immunomodulating activities of diosgenin, a

naturally occurring steroidal saponin [J]. Nat Prod Res, 2012, 26(23): 2243-2246.

Hirai S, Uemura T, Mizoguchi N, et al. Diosgenin attenuates inflammatory changes in the

interaction between adipocytes and macrophages [J]. Mol Nutr Food Res, 2010, 54(6): 797-804.

Inmaculada C V, Milagros G, Rocı´o V, Francisco O’V, Marı´Fr G S, Antonio Z, Juan Duarte.

Effects of the Dietary Flavonoid Chrysin in Isolated Rat Mesenteric Vascular Bed. Jouranal of

vascular research 4; 2004, 509-516.

Ebrahimi H, Badalzadeh R, Mohammadi M, et al. Diosgenin attenuates inflammatory response

induced by myocardial reperfusion injury: role of mitochondrial ATP-sensitive potassium

channels [J]. J PhysiolBiochem, 2014, 70(2): 435-432.

Jamshid G, Yahya E, Ramin S. Effect of Garlic (Allium sativum) Aqueous Extract on serum

values of Urea, Uric-Acid and Creatinine compared with Chromium Chloride in Male Rats

Scholars Research Library Annals of Biological Research3 (9); 2012, 4485-4490.

Jebouri M and Madish AS. The role of sex hormone in the formation of renal stones with

reference to urinary tract infection of Iraqi patients. World Journal Of Pharmacy and

Pharmaceutical Sciences 3( 4); 2014, 352-365.

Joy M J, Prathyusha S, Monanalakshmi S, Kumar AVS Praveen, Kumar CK Ashok. Innovative

Drug discovery. Potent herbal wealth with litholytic activity : A review 2(2); 2012, 66-75.

Joy M Jyoti, Prathyusha S, Monanalakshmi S, Kumar AVS Praveen, Kumar CK Ashok.

Innovative Drug discovery. Potent herbal wealth with litholytic activity : A review, 2(2); 2012,

66-75.

Kambadakone A R , Eisner H B, Catalano O A and Sahani V D.New and Evolving Con cepts in

the Imaging and Management of Urolithiasis: Urologists’ Perspective.

www.rsna.org.education/rg_cme.html 30; 2010, 603–623.

Kelly G, Cathy L, Dauglas P, John M. Calcium oxalate urolithiasis. Compendium Education for

veterinarians 2009 (compendium vet.com.)

Kelly G, Cathy L. calcium oxalate urolithiasis. Compendium education for veterinarins 2009,

496-498.

Khashayar S., Beveley A. H., Orson W. M, Charles Y.C. Pak.Pathophysiologic basis for

normouricosuric uric acid nephrolithiasis.Kidney International, Vol. 62 ; 2002, 971–979.

PAIDEUMA JOURNAL


Issn No : 0090-5674


Khoo Y B, Chua S S and Balaram P. Apoptotic Effects of Chrysin in Human Cancer Cell

Lines.International Journal of Molecular Sciences11; 2010, 2188-2199.

Kitamura T , Homma Y ,Takahashi S, ArugaS,Yamamoto Y, Matsumoto T.Urinary Oxalate

Excretion Decreased in Androgen Receptor-Knockout Mice by Suppressing Oxalate Synthesis in

the Liver.Open Journal of Urology 5; 2015, 123-132.

Kitamura T , Homma Y ,Takahashi S, ArugaS,Yamamoto Y, Matsumoto T. Urinary Oxalate

Excretion Decreased in Androgen Receptor-Knockout Mice by Suppressing OxalateSynthesis in

the Liver. Open Journal of Urology 5; 2015, 123-132.

Knoll T. Epidemiology, Pathogenesis and Pathophysiology of Urolithiasis. European Uroloy

Supplements 9; 2010, 802-806.

Knoll Thomas Epidemiology, Pathogenesis and Pathophysiology of Urolithiasis. European

Urology Supplements 9; 2010, 802-806.

Kshetrimayum B S, and Saitluangpuii S. Understanding epidemiology and etiologic factors of

urolithiasis: an overview. www.sciencevision.org 13(4); 2013, (print) 0975-6175 (online) 2229-

6026.

Krishnaveni J, Rajkiran E, Manjula P, Sudheer K. Antiurolithiatic activity of Cucumis

sativus.International Journal of Pharmacological Screening Methods 3 ( 2 ); 2013, 46-52.

Lee J, Jung K, Kim YS, et al. Diosgenin inhibits melanogenesis through the activation of

phosphatidylinositol- 3-kinase pathway (PI3K) signaling [J]. Life Sci, 2007, 81(3): 249-254.

Matlaga B R, Ojas D S and Assimos D G. Drug-Induced Urinary Calculi. Review in urology

5(4); 2003, 227-231.

Medigović I, Ristić N, Živanović J, et al. Diosgenin does not express e activity: a uterotrophic

assay [J]. Can JPhysiol Pharm, 2014, 92(4): 292-298.

Millmam S, Strauss A L, Parks J S and Fredric LC. Pathogenesis and clinical course of mixed

calcium oxalate and uric acid nephrolithiasis. Kidney International 22; 1982, 366-370.

Mohan Harsh. Textbook of pathology. Introduction of urolithiasis 5; 2005, 714-716

Mohemid MA and Salih AM. The role of sex hormones in the formation of renal stones with

references to urinary tract infections of Iraqi patients. World journal of pharmacy and

pharmaceutical sciences 3(4); 2014, 352-365.

Mshelbwala PM, Ameh EA, and Mbibu HN. Urinary stones in children: Review article. Niger J

Surg Res 79(3–4); 2005, 238–243.

Naim MM, Alicia HS, Brian JW, Barbara VH, Barbara BC, Khashayar S, and John A R.

Postmenopausal Hormone Use and the Risk of Nephrolithiasis :Results From the Women’s

Health Initiative Hormone Therapy Trials. American Medical Association 170(18); 2010, 1678–

1685.

PAIDEUMA JOURNAL


Issn No : 0090-5674


http://www.sciencevision.org/

Naim MM, Alicia HS,Brian JW, Barbara VH, Barbara BC, Khashayar S, and John A R.

Postmenopausal Hormone Use and the Risk of Nephrolithiasis :Results From the Women’s

Health Initiative Hormone Therapy Trials.American Medical association 170(18) : 2010, 1678–

1685.

Naryana Raj K, Reddy M. Sripal, Rhaluvadi M.R., KrishanaD.R.. Bioflavonoids classification,

pharmacological, biochemical effect and therapeutic potential. Indian Journal of Pharmacology

33; 2001, (2-16).

Nélida N, Cristina Q, Felipe J, Cristina T, Gabriela E, Beatriz L, Elba L and Guillermo S.

Antibacterial Activity, Antioxidant Effect and Chemical Composition of Propolis from the

Región del Maule Central Chile www.mdpi.com/journal/molecules 20; 2015, 18144-18167.

Omar M, Monga M, Calle J C. Relationship between Patient’s Demographics and Stone

Composition in Men and Women. Journal of nephrology and therapeutics. 5(2); 2015.

Onaran M, Orhan N, Farahvash A, Ekin NH, Kocabiyik M, Gonul II. Sucessfully treatment of

sodium oxalate induced urolithiasis with Heli chrysum flowers. Journal of Ethnophamacology

186; 2016, 322-328.

Onaran M, Orhan N, Farahvash A, Ekin NH, Kocabiyik M, Gonul II. Sucessfully treatment of

sodium oxalate induced urolithiasis with Heli chrysum flowers. Journal of Ethnophamacology

186; 2016, 322-328.

Panda A K. Simple synthetic processes for Chrysin norwogonin and their derivatives.

International Journal of Research in Ayurveda & Pharmacy 1( 1); 2010, 225-233.

Philip M, Mshelbwala Hyacinth N, Mbibu. Chapter 97 urolithiasis.

Premlatha M and Parameswari.Renoprotective effect of chrysin (5,7 dihydroxy flavone) in

streptozotocin induced diabetic nephropathy in rats. International Journal of Pharmacy and

Pharmaceutical Sciences 4(3); 2012, 0975.

Pullaiah C P, Kumar G V, Madhuri P V, Dhanunjaya S, Kumari B P, Rangana Y D.

International Bioscience. Supplementation of Ethanolic extract of Abutilon Indicum (L)

Urolithiasis in Experimental Rats 1(1); 2016, 07-13.

Ramos M, Santana L, Rasvickas C, Teixeira V, Schor N. Effect of vitamin D3 overdose and

calcium supplementation in experimental nephrolithiasis model. Department of Nephrology -

UNIFESP/ EPM (Paulista School of Medicine . 55 (11); 2013, 5573-9652.

Ranjitha J, Vijiyalakshmi S, Anand M and Bhagiyalakshmi M. Biological Assay of In Vitro

Antioxidant and Antibacterial Activity of the Whole Plant Material Cleome

gynandraLinn.Research Journal of Pharmaceutical, Biological and Chemical Sciences 4(3) ;

2013, 0975-8585.

Rathod N, Chitme HR, Chandra R. In vivo - In vitro models for Evaluating Anti-urolithiatic

activity of Herbal Drugs. International Journal of Pharmaceutical Research and Science 3(5);

2014, 309-329.

PAIDEUMA JOURNAL


Issn No : 0090-5674


http://www.mdpi.com/journal/molecules%2020

Robert P, Kathryn S, Graeme M, Mary K, Thomas L, Ruth T, Jennifer B, John N, Gladys M,

Alison M, Kirsty S, Katie G, Kenneth A, Charles B, James N D, Neil B, Terry C, Sarah C and

Samuel M. Use of drug therapy in the management of symptomatic ureteric stones in

hospitalised adults: a multicentre, placebo-controlled, randomised controlled trial and cost-

effectiveness analysis of a calcium channel blocker (nifedipine) and an alpha-blocker

(tamsulosin) (the SUSPEND trial). Health technology assessment 19(63); 2015, 1366-5278.

Salimeh A, Mohammadi M, Rashidi B. Preconditioning with diosgenin and treadmill exercise

preserves the cardiac toxicity of isoproterenol in rats [J]. J PhysiolBiochem, 2013, 69(2): 255-

265.

Sarmistha S and Ramtej J.Antinephrolithiatic and antioxidative efficacy of Dolichosbiflorus

seeds in a lithiasic rat model.Department of Zoology, University School of Sciences, Gujarat

University, Ahmedabad, Gujarat, Indiahttp://informahealthcare.com/phb ; 2014, ISSN 1388-

0209 print/ISSN 1744-5116 online Editor-in-Chief.

Singh K B and Sailo S.Understanding epidemiology and etiologic factors of urolithiasis: an

overview.Science Vision © 2013 MAS. All rights reserved 13(4); 2013,2229-6026.

Taylor N E and Curhan C G. Journal list clin J Am SOC Nephrol. Determinant of 24 hour

urinary oxalate excretion 3(5); 2008, 1453-1460.

Thangarathinam N, Jayshree N, Me tha A.V and Ramanathan L. Effect of polyherbal

formulation on ethylene glycol induced urolithiasis. International Journal of Pharmacy and

Pharmaceutical Sciences 5(3); 2013, 0975-1491. Türk C, Knoll T, Petrik A S, Seitz C and Straub

M. Guidelines on Urolithiasis March 2011.

Tsujihata M, Miyaka O, Yoshimura K, Kakimoto K.-I, Takahara S and Okuyama A,

``Fibronectin as a potent inhibitor of calcium oxalate urolithiasis,``Journal of Urology, vol. 164,

no.5, pp. 1718-1723,2000.

Umekaw T, Uemura H, Khan SR. Super oxide from NADPH oxidase as second messenger

forexpression of osteopontin and monocyte chemoattractant-1 in renal epithelial cells exposed to

calcium oxalate crystals BJU International 104(1); 2009 ,115–120.

Vijaya T, Kumar M Sathish, Ramarav N.V, Babu A Naredra, Ramarao N. Urolithiasis and its

cause- short review. The journal of phytopharmacology 2(3); 2013, 1-6.

Wang YJ, Liu YC, Chang HD, et al. Diosgenin, a plant-derived sapogenin, stimulates Ca2+-

activated K+ current in human cortical HCN-1A neuronal cells [J]. Planta Med, 2006, 72(5):

430-436.

Yoshioka I, Tsujihata M, Momohara C, Akanae W, Nonomura N, and Okuyamaz A. Effect of

Sex Hormones on Crystal Formation in a Stone-forming Rat Model.Urology 75 ;2010, 907–913.

PAIDEUMA JOURNAL


Issn No : 0090-5674


http://informahealthcare.com/phb

Date post:	03-Oct-2021
Category:	Documents
Upload:	others
View:	11 times
Download:	0 times

EFFECT OF DIOSGENIN TREATMENT ON ETHYLENE GLYCOL …

Documents