Biomedicine & Pharmacotherapy 84 (2016) 1524–1532

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Alteration in Oxidative/nitrosative imbalance, histochemical

expression of osteopontin and antiurolithiatic efficacy of Xanthium
strumarium (L.) in ethylene glycol induced urolithiasis
Padma Nibash Panigrahia,b,* , Sahadeb Deya , Monalisa Sahooc,
Shyam Sundar Choudharya , Sumit Mahajana
a
Division of Medicine, Indian Veterinary Research Institute, Izatnagar, Bareilly Uttar Pradesh-243122, India
b
Division of Medicine, Faculty of Veterinary and Animal Science, Banaras Hindu University, Varanasi, Uttar Pradesh-221005, India
c
Division of Pathology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh-243122, India

A R T I C L E I N F O A B S T R A C T

Article history:
Received 20 September 2016 Xanthium strumarium has traditionally been used in the treatment of urolitiasis especially by the rural
Received in revised form 24 October 2016 people in India, but its antiurolithiatic efficacy was not explored scientifically till now. Therefore, the
Accepted 8 November 2016 present study was designed to validate the ethnic practice scientifically, and explore the possible
antiurolithiatic effect to rationalize its medicinal use. Urolitiasis was induced in hyperoxaluric rat model
Keywords: by giving 0.75% ethylene glycol (EG) for 28 days along with 1% ammonium chloride (AC) for first 14 days.
Xanthium strumarium Antiurolithiatic effect of aqueous-ethanol extract of Xanthium strumarium bur (xanthium) was evaluated
Urolithiasis based on urine and serum biochemistry, oxidative/nitrosative stress indices, histopathology, kidney
Rat
calcium and calcium oxalate content and immunohistochemical expression of matrix glycoprotein,
Immunohistochemistry
osteopontin (OPN). Administration of EG and AC resulted in hyperoxaluria, crystalluria, hypocalciuria,
Osteopontin
Oxidative/Nitrosative stress polyurea, raised serum urea, creatinine, erythrocytic lipid peroxidise and nitric oxide, kidney calcium
content as well as crystal deposition in kidney section in lithiatic group rats. However, xanthium
treatment significantly restored the impairment in above kidney function test as that of standard
treatment, cystone. The up-regulation of OPN was also significantly decreased after xanthium treatment.
The present findings demonstrate the curative efficacy of xanthium in ethylene glycol induced
urolithiasis, possibly mediated through inhibition of various pathways involved in renal calcium oxalate
formation, antioxidant property and down regulation of matrix glycoprotein, OPN. Therefore, future
studies may be established to evaluate its efficacy and safety for clinical use.
ã 2016 Elsevier Masson SAS. All rights reserved.

1. Introduction epidemiological studies have shown that majority (70%) of stones

Urolithiasis is one of the important constraints in human and
animal health globally since last two decades, irrespective of
geographical, racial and cultural boundaries [1,2]. It is also
considered as the third most common problem of the urinary
tract with an estimated lifetime risk of around 1–5% in Asia, 8–15%
in America and Europe, and 20% in the Middle East countries [3,4].
The incidence of urolithiasis had been increases in India now a day
and two “stone belts” had also been scientifically identified in India
[3]. Oxalate, struvite, urate, brushite, cystine were the most
commonly reported urolith in man and animal. However,
* Corresponding author at: Division of Medicine, Faculty of Veterinary and
Animal Science, Banaras Hindu University, Varanasi, 221005, Uttar Pradesh, India.
E-mail address: pnpvetmed@gmail.com (P.N. Panigrahi).
commonly contain calcium oxalate (CaC2O4) [1,2]. In spite of
substantial progress in the study of physiology of urolithiasis, its
exact mechanism is still not clearly understood. The recent
proposed mechanism of stone formation involves urinary super-
saturation, crystal nucleation, precipitation, growth, aggregation of
crystals and their retention in renal tubular epithelial cells [1,5].
These processes are modulated by a variety of urinary macro-
molecules which become incorporated in the growing crystals and
eventually constitute organic component or matrix. Osteopontin
(OPN) is a negatively charged aspartic acid rich important matrix
glycoprotein usually associated with calcium oxalate stone
formation [6–8]. It is mainly present in the loop of Henle and
distal nephrons in normal kidneys in animal and humans, but its
expression is significantly up regulated after renal damage.
Although, OPN is a well known component of stone matrix, but

http://dx.doi.org/10.1016/j.biopha.2016.11.029
0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
 P.N. Panigrahi et al. / Biomedicine & Pharmacotherapy 84 (2016) 1524–1532
whether it acts as a promoter or inhibitor of stone formation is
controversial till date [6,9,10].
Modern diagnostic and therapeutic aids like extracorporeal
shock wave lithotripsy, ureteroscopy and percutaneous nephro-
lithotomy had revolutionized the urological practices but cannot
altered the recurrence of stone formation and also have adverse
side effects. Pharmaceutical agents like thiazide and citrate
diuretics, alkali therapy, allopurinol etc have also limited efficacy
in addition to their less tolerability [11]. Indeed, urolithiasis is a
multifactorial disease and involves in alteration of several
biochemical pathways. Therefore, the present situation demands
a newer approach of therapy. Several recent studies have
highlighted the effectiveness of medicinal plants and natural
compounds for treatment and management of urolithiasis [2–
4,12–15]. Moreover, herbal remedies are known to contain
multiple constituents, acting through multiple pathways such as
antioxidant, analgesic, diuretic, pH neutralising etc. Xanthium
strumarium has traditionally been used as an herbal medicine in
India, China, Europe, Malaysia and America [16]. The whole plant
especially the root, bur and leaves had been reported to possess
antiulcerogenic [17], anti-cell proliferative [18,19], anti-inflamma-
tory and analgesic [20], antidiabetic and hypoglycaemic [16],
antiarthritic [21], diuretic and renoprotective [3,16], antimicrobial
[22], antitrypanosomal [23], antihelmintic [24] and anti-plasmo-
dial [25] properties. The bur extract had traditionally been used in
the treatment and management of urolithiasis and renal ailments
in some parts of India [3] but its antiurolithiatic efficacy was not
explored scientifically till now. Hence the study was designed to
validate the ethnic practice scientifically in ethylene glycol induced
lithiatic rat model, and to explore the possible mechanism of
antiurolithiatic effect of xanthium to rationalize its medicinal use.
2. Materials and methods
2.1. Animals
The animal care and handling was performed in accordance
with the internationally accepted standard guidelines for use of
animals and the protocol was approved by institutional animal
ethical committee, Indian Veterinary Research Institute, India
(vide letter no- F.26-1/2015-16/JDR). Thirty male albino Wistar rats
(weighing 150–200 g) were procured from the laboratory animal
research division and housed in clean polypropylene cages under
controlled temperature 25
2  C, humidity 45–55% and 12 h light-
dark cycle throughout the experimental period. Animals were
given standard diet and had free accesses to food and water ad
libitum throughout the study. However, food was withdrawn while
collecting 24 h urine samples inside metabolic cage.
2.2. Plant material and extraction
The plant Xanthium strumarium was collected from horticulture
section of the institute and was identified and authenticated from
Botanical Survey of India, Central National Herbarium, Howrah,
India. The bur of X. strumarium was separated from whole plant,
washed with distilled water, dried and uniformly powdered using
an electric grinder. Hydro-ethanol extract of the powdered X.
strumarium bur (xanthium) was extracted in room temperature

(37 C) for 24 h with occasional shaking using solvent system
(aqueous: ethanol 1:1) and filtered using filter paper (Whatman
no. 40). The solvent was evaporated using rotary evaporator under
pressure. The extract was dried in vacuo and stored refrigerated
until use [26]. The recovery percentage was 18% (w/w).
1525
2.3. Phytochemical screening
The preliminary phytochemical screening of xanthium was
carried out as per the standard procedures described by Harborne
[27]. For qualitative screening, Mayer’s test and Dragendroff test
were done to determine the presence of alkaloids; Benedict test,
Barfoed test and Fehling test for carbohydrate; Foam test for
saponin; Biuret test and Ninhydrin test for protein; Alkaline
reagent test for flavonoids; Sodium hydroxide test for coumarin;
Noller’s test for triterpinoids; and Gelatine test was done for
detection of tannins.
2.4. Study on animal model of urolithiasis
Antiurolithiatic activity of the test plant material was deter-
mined by hyperoxaluric rat model of calcium oxalate urolithiasis as
described previously [4,28]. Thirty male rats were divided with
matched body weights into five groups of six animals each, which
were then randomly selected to receive various treatments. Rats of
Group I served as healthy control without receiving any treatment.
Group II rats act as lithiatic control received stone-inducing
treatment comprised of 0.75% (w/v) ethylene glycol (EG) along
with 1% (w/v) ammonium chloride in drinking water for 14 days for
induction of crystal followed by EG alone for next 14 days. Group III
rats received stone inducing treatment for 14 days followed by
500 mg/kg hydo-ethanolic (1:1) extract of xanthium (EC50) orally
for next 14 days orally through gastric gavages (The EC50 dose was
calculated from earlier study, detailed not presented in this
manuscript, Panigrahi et al., 2016, PhD thesis submitted to Deemed
University- Indian Veterinary Research Institute). Group IV rats
received stone inducing treatment for 14 days followed by
standard drug-cystone at 100 mg/kg for next 14 days orally
through gastric gavages. Group V served as vehicle control and
received stone inducing treatment for 14 days followed by drinking
water for next 14 days without any treatment to check auto healing
process.
2.5. Sampling
Twenty four hour urine samples were collected at day 0, day 14
and day 28, housing rats individually in the metabolic cages.
Following volume and pH determination, part of each urine sample
was acidified to pH 2 with 5 M hydrochloric acid. Both acidified and
non-acidified urine sample were centrifuged at 1500g for 10 min to

remove debris and supernatants and were stored at 20 C until
analysed. Blood was collected through cardiac puncture from
individual animals under ether anaesthesia at day 0, day 14 and day
28 for harvesting serum required for kidney function test. Animals
were sacrificed after end of study and both the kidneys were
excised for histopathology, estimation of tissue calcium content
and Immunohistochemistry.
2.6. Biochemical analysis of urine and serum
In acidified urine sample calcium, phosphorus, sodium and
potassium were determined using commercial kits. Oxalate
content of acidified urine was estimated using quantitative ELISA
kit (Quibiotech, China). Microscopic examination of urine was
performed in non acidified first 3 h morning samples. Urine urea
nitrogen (UUN) and urine creatinine was estimated in non acidified
urine sample. Blood urea nitrogen (BUN), creatinine, total protein
and albumin were estimated in serum using commercial kits.
1526 P.N. Panigrahi et al. / Biomedicine & Pharmacotherapy 84 (2016) 1524–1532

2.7. Estimation of oxidative/nitrosative stress indices 2.9. Immunohistochemistry for OPN

Lipid peroxidases (LPO) level in 10% RBC haemolysate was
estimated spectrophotometrically following the method of Placer
[29]. Lipid peroxidation was calculated using 1.56  105 as
extinction coefficient to express the value in nanomole of
Malonaldehyde (MDA) per millilitre of haemolysate [30]. Super-
oxide dismutase (SOD) was measured in the supernatant of 10%
RBC haemolysate following the method of Marklund [31] with
certain modifications suggested by Menami and Yoshikawa [32].
Each unit of SOD activity is defined as the quantity of enzyme that
inhibits auto oxidation of pyrogallol by 50% under suitable
experimental conditions. Catalase (CAT) activity in 10% RBC
haemolysate was estimated spectrophotometrically at wave length
of 240 nm after appropriate dilution following the method of
Cohen [33] and the values were expressed in units per milligram of
haemoglobin. Glutathione (GSH) was estimated in packed RBC
following DTNB (di-thiobis2-nitro benzoic acid) method [34].
Nitric oxide (NO) level of blood plasma was measured by nitrate
reduction on copper cadmium alloy (Cu–Cd alloy) followed by
colour development with Griess reagent (0.1% naphthalene
diamine dihydrochloride in 3 N hydrochloric acid and 1%
sulphanilamide 1:1) as described by Sastry [35].
2.8. Histopathology and calcium oxalate crystal deposition study
The kidneys were rinsed in ice cold physiological saline and
weighed. The left kidney was fixed in 10% neutral buffered
formalin, sectioned at 5 mm thickness and stained with Hematox-
ylin and Eosin (H and E) for histopathological examination and also
stained with Pizzolato’s method for in situ demonstration of
calcium oxalate crystal deposition [36]. The right kidney was taken
for estimation of tissue calcium content by atomic absorption
spectrophotometer.
Calcium oxalate crystal deposition within the kidneys was
determined using the semiquantitative scoring methods described
previously [14]. Briefly, ten randomly selected microscopic fields
were examined under 10X magnification and crystal deposits were
graded according to the scale: 0 = <1 crystal, 1 = 1-10, 2 = 11–30,
3 = 31–50, 4 =  51–75 and 5 = >75 crystals [4].
The left kidneys were fixed in 10% neutral buffered formalin,
sectioned at 5 mm thicknesses, deparaffinised by immersion in
xylene and dehydrated in a graded series of ethanol. Then, the
kidney sections were boiled in 10 mM sodium citrate buffer (pH
6.0) for 10 min for antigen retrieval and cooled at room
temperature. Endogenous peroxidase activity was inhibited by
incubation with 0.3% H2O2 for 10 min to prevent non-specific
bindings sections were blocked by incubation in 3% normal goat
serum for 60 min. The sections were stained with primary antibody
for OPN (mouse monoclonal to osteopontin, 1: 50 dilutions, Santa

Cruz Biotechnologies, USA) and incubated for overnight at 4 C.
After three successive washing with PBS for 10 min, the sections
were incubated with goat-anti mouse immunoglobulin-G horse-
radish peroxidase-conjugated secondary antibody (1: 200 dilu-
tions, Santa Cruz Biotechnologies, USA) for 1 h. Thereafter, sections
were washed 3 times with PBS and stained with 3.30 -diamino-
benzidine (Abcam Biotechnologies, India) and counterstained with
Mayer’s hematoxylin solution (Sigma Aldrich biochemical, USA).
Finally, the sections were mounted with CC/mount (Sigma Aldrich
biochemical, USA) and photographed under light microscope.
2.10. Statistical analysis
The data were expressed as mean
standard error of mean
(SEM) and median effective concentration (EC50 value) with 95%
confidence interval (CI). All statistical comparisons between the
groups were made by t-test (comparison between two groups) or
ANOVA with post hoc Dunnett’s test using SPSS. P value <0.05 is
considered as significant.
3. Results
3.1. Phytochemical screening
The preliminary phytochemical screening of xanthium revealed
positive for alkaloids, flavonoids, triterpenoids, tannins, saponins
and proteins, and negative for carbohydrate and coumarin.

Table 1
Effect of Xanthium strumarium and standard drug on 24 h urinary biochemical parameters.

Parameters Day UC Lithiatic Xanthium Cystone Vehicle

Volume (ml) 14 6.533
1.091 11.333
1.453** 11.333
0.882** 10.700
0.814* 10.900
1.320*
28 6.500
0.764 14.867
0.926** 8.633
0.895## 7.533
0.712## 11.600
0.757**
pH 14 7.100
0.115 6.567
0.088 6.433
0.338 6.367
0.384 6.700
0.355
28 6.600
0.305 5.467
0.437* 6.300
0.264# 6.600
0.312# 5.817
0.361*
Oxalate (mg) 14 0.727
0.044 1.700
0.156** 2.123
0.072** 2.050
0.201** 1.883
0.034**
28 0 0.697
0.045 2.703
0.086** 0.903
0.075## 0.740
0.064## 2.007
0.118**##
Calcium (mg) 14 6.935
0.452 3.837
0.238** 4.753
0.320** 4.287
0.424** 4.006
0.471**
28 7.239
0.704 3.374
0.492** 5.750
0.308## 6.084
0.206## 3.670
0.329**
Phosphorus (mg) 14 4.963
0.422 6.102
0.629 6.042
0.251 5.262
0.746 5.683
0.361
28 5.014
0.191 7.411
0.329 5.633
0.477 5.077
0.310 6.501
0.290
UUN (mg) 14 8.526
0.346 12.483
0.800* 14.219
1.671** 14.567
0.652** 12.721
1.193*
28 7.335
0.736 20.677
1.753** 11.894
0.904*## 7.803
0.460## 18.262
2.678**
Creatinine (mg) 14 0.344
0.052 0.681
0.088** 0.774
0.026** 0.814
0.089** 0.799
0.034**
28 0.362
0.014 1.389
0.026** 0.697
0.055**## 0.544
0.037*## 1.268
0.064**
Potassium (mg) 14 10.219
0.147 9.483
0.219 10.192
0.108 10.399
0.395 9.840
0.254
28 13.057
0.116 12.267
0.333 12.733
0.316 12.556
0.378 11.482
0.376
Sodium (mg) 14 81.10
9.71 95.40
7.32 98.77
13.15 89.59
5.71 88.93
5.96
28 74.78
12.50 101.91
9.09 89.65
8.45 89.04
1.80 84.38
0.90

UC = untreated healthy control, Lithiatic = lithogenic control group, Xanthium = 500 mg/kg Xanthium strumarium, Cystone = 100 mg/kg standard drug cystone, Vehicle =
vehicle control group. *p < 0.05 and **p < 0.01 vs Healthy control (group A), #p < 0.05 and # #p < 0.01 vs. Lithogenic group (group B). UUN = urinary urea nitrogen.
 P.N. Panigrahi et al. / Biomedicine & Pharmacotherapy 84 (2016) 1524–1532 1527

Table 2
Effect of Xanthium strumarium and standard drug on serum biochemical parameters.

Parameters Day UC Lithiatic Xanthium Cystone Vehicle

BUN (mg/dl) 14 16.194
0.531 45.456
1.564** 41.615
3.247** 48.893
2.141** 41.298
1.957**
28 21.795
1.873 53.655
3.459** 28.856
3.314*## 23.776
1.596## 36.581
1.815**##
Creatinine (mg/dl) 14 0.324
0.032 0.466
0.023* 0.614
0.014** 0.562
0.050** 0.541
0.046**
28 0.282
0.047 1.445
0.075** 0.407
0.018*## 0.354
0.052## 0.801
0.043**##
Total Protein (g/dl) 14 4.239
0.212 5.949
0.244** 5.747
0.125** 5.379
0.114** 5.131
0.195**
28 5.220
0.210 6.742
0.177** 5.636
0.105## 5.626
0.168## 6.334
0.237**
Albumin (g/dl) 14 2.735
0.056 3.061
0.136 3.123
0.089 3.048
0.218 3.123
0.125
28 3.152
0.169 3.518
0.167 3.053
0.150 3.127
0.071 3.361
0.095

UC = untreated control, Lithiatic = lithogenic control group, Xanthium = 500 mg/kg Xanthium strumarium, Cystone = 100 mg/kg standard drug cystone, Vehicle = vehicle control
group. *p < 0.05 and **p < 0.01 vs Healthy control (group A), #p < 0.05 and ##p < 0.01 vs. Lithogenic group (group B).

3.2. Urine and serum biochemistry
All the day 0 parameters of urine and serum biochemistry
recorded just before the start of experiment were statistically
similar among all the groups; hence the values were not given in
the table. Tables 1 and 2 summarise the change in urine and serum
biochemistry among different groups at different interval of study,
respectively. The volume of urine, urinary oxalate concentration,
UUN and urine creatinine concentration increased significantly
(p < 0.01) on day 14 in all four grouped rats received stone inducing
treatment as compared to healthy untreated control rats (Table 1).
The values were again increased on day 28 in lithiatic control
(group B) and vehicle control (group E) rats but treatment with
xanthium significantly reduced the above parameters as that of
standard treatment (group D). On contrary, the pH of urine and Ca
concentration decreased significantly (p < 0.01) after stone induc-
ing treatment, but the above parameters were increased signifi-
cantly after treatment with xanthium and the values were
statistically similar to standard treatment. The inorganic phos-
phate, sodium and potassium concentration differ non-signifi-
cantly before and after treatment in all groups and the values were
statistically similar (Table 1). EG treatment cause significant
impairment of renal functions as evident from significantly raised
BUN, creatinine and TP concentration in all four grouped rats
except healthy control on day 14; and lithiatic group on day 28,
which were prevented after treatment with xanthium and cystone
(Table 2). On contrary, only drinking water (group E) could not
resolve the problem as observed from kidney function test.
3.3. Alteration of oxidative/nitrosative indices
The mean LPO and NO activity increased significantly (p < 0.01)
in all the rats received stone inducing treatment after induction of
urolithiasis (Table 3). However, xanthium treatment significantly
reduced the above oxidative/nitrosative stress and the mean values
were statistically similar to rats treated with standard drug,
cystone. On contrary, the antioxidant enzymes GSH, SOD and CAT
decreased significantly (p < 0.01) on day 14 in all the rats received
stone inducing treatment and further decreased (p < 0.01) on day
28 in lithiatic control rats as compared to untreated rats. But, they
were increased significantly after treatment with xanthium and
the values were statistically similar to standard treatment and
untreated control (Table 3).
3.4. Histopathology and calcium oxalate crystal deposition study
The microscopy of histological section of kidney revealed
presence of calcium oxalate crystals mostly in renal tubules in
cortex region and less commonly in medulla both in Pizzolato’s
(Fig. 1) and H&E (Fig. 3) method of staining in lithiatic rats. The
healthy control rats did not show any calcium oxalate crystals in
kidney section whereas, a high score of crystal deposits were
recorded in lithiatic and vehicle group by Pizzolato staining (Fig. 2).
However, xanthium treatment significantly (p < 0.01) reduce the
calcium oxalate crystal deposits as compared to lithiatic group and
the crystal deposits score is statistically similar with standard
treatment (Fig. 2). Lithiatic treatment affected proximal nephrons,
distal nephrons, collecting ducts and CaC2O4 crystals induced
injury to the renal epithelium resulting in leakage of cellular
content into the lumen which was observed from the histology
examination of the kidney tissue section stained with H & E stain
(Fig. 3). There was severe mononuclear cell infiltration, necrosis,
tubular degeneration, dilatation of tubules casts, and fibrin
deposits in kidney of lithiatic control (Fig. 3B) and vehicle control
rats (Fig. 3E). The renal parenchyma was almost intact, tubules
appeared normal and significant reduction in cellular infiltration
and haemorrhages were observed in rats treated with xanthium
(Fig. 3C) and standard treatment (Fig. 3D). While there was
complete absence of crystals observed in the healthy control rats
(Fig. 3A).
The tissue Ca concentration was significantly increased in
lithiatic rats and vehicle control (p < 0.01) as compared to healthy
control (Fig. 4). However, treatment with xanthium significantly
reduces the tissue Ca content and the value is statistically similar to
healthy control as that of standard treatment.

Table 3
Effect of Xanthium strumarium and standard drug on oxidative/nitrosative indices.

Parameters Day UC Lithiatic Xanthium Cystone Vehicle

LPO (nmol MDA/mg Hb) 14 2.368
0.275 3.887
0.193** 3.661
0.207** 4.095
0.171** 4.063
0.189**
28 3.155
0.175 5.783
0.209** 3.688
0.276## 3.181
0.242## 4.362
0.216**##
GSH (mmol/mg Hb) 14 0.304
0.034 0.162
0.032** 0.191
0.028 ** 0.206
0.048* 0.182
0.011**
28 0.310
0.027 0.139
0.021** 0.213
0.020 * 0.214
0.022 * 0.150
0.010 **
CAT (U/mg Hb) 14 1.252
0.034 0.773
0.042** 0.839
0.037** 0.722
0.026** 0.834
0.036**
28 1.205
0.118 0.534
0.041** 1.022
0.114## 1.137
0.068## 0.654
0.044**
SOD (U/mg Hb) 14 0.864
0.032 0.553
0.035** 0.587
0.027** 0.684
0.030** 0.524
0.023**
28 0.970
0.073 0.374
0.021** 0.720
0.031*## 0.838
0.048## 0.425
0.026**
NO (mmol/ml) 14 3.288
0.039 6.155
0.197** 6.384
0.320** 6.438
0.225** 6.448
0.190**
28 2.876
0.038 10.285
0.519** 3.791
0.148## 3.047
0.216## 7.320
0.404**#

UC = untreated control, Lithiatic = lithogenic control group, Xanthium = 500 mg/kg Xanthium strumarium, Cystone = 100 mg/kg standard drug cystone, Vehicle = vehicle control
group. *p < 0.05 and **p < 0.01 vs Healthy control (group A), #p < 0.05 and ##p < 0.01 vs. Lithogenic group (group B).
1528 P.N. Panigrahi et al. / Biomedicine & Pharmacotherapy 84 (2016) 1524–1532

Fig. 1. Histology of representative kidney section stained with Pizzolato’s stain (X100).
Black colour particles showing calcium oxalate crystal. A = Untreated healthy control, B = Lithiatic control, C = 500 mg/kg Xanthium strumarium, D = 100 mg/kg Cystone,
E = Vehicle control

3.5. Immunohistochemistry and expression of OPN 4. Discussion

Immunohistochemical staining for detection of OPN revealed
expression of OPN in all groups of rats (Fig. 5). However, it was
barely detectable in the healthy control group only mild expression
in the distal limb of Henle in the outer medulla (Fig. 5A). On
contrary, its expression was intensely increased throughout the
renal tubules, proximal and distal convoluted tubules, collecting
ducts in lithiatic group (group B) (Fig. 5B). Treatment with
xanthium significantly reduced the expression of OPN (Fig. 5C)
which was comparable to the standard treatment (Fig. 5D). The
quantitative analysis of immunohistochemical expression of OPN
in kidney tissue also revealed significant (p < 0.01) reduction in
expression of OPN after treatment with xanthium in rats (Fig. 6).
6 was not explored scientifically till now. Hence, the study was
CaOx crystal deposition Score
India has been known to be rich repository of medicinal plants
since ancient civilization. Xanthium strumarium is a commonly
available weed in India and has traditionally been used as a
medicinal herb in most parts of India, China, Europe and America
[16]. The whole plant especially the root, bur and leaves had been
reported to possess antiulcerogenic [17], anti-cell proliferative
[18,19], anti-inflammatory and analgesic [20], antidiabetic and
hypoglycaemic [16], antiarthritic [21], diuretic and renoprotective
[3,16], antimicrobial [22], antitrypanosomal [23], antihelmintic
[24] and anti-plasmodial [25] properties. The bur extract contains
rich source of bioactive compounds [37] and has traditionally been
used in the treatment and management of urolithiasis and renal
ailments in some parts of India [3] but its antiurolithiatic efficacy
designed to evaluate the antiurolithiatic and reno-protective

efficacy of xanthium in ethylene glycol induced rat model.

5
Calcium oxalate stone formation is a multi-factorial process
4.67 involving various etiological factors. Hyperoxaluric rat model is the
4
## 4.17 most potent experimental model for preclinical evaluation of
## antiurolithiatic efficacy of medicinal herbs because the physiolog-
3
ical process mimics the etiology of kidney stone formation in
2.53 human and animal [38]. The hepatic enzymes metabolize EG to
2 2.27 oxalic acid by glyoxalate mechanism, which combine with calcium
ion in the renal tubular epithelium to form calcium oxalate crystals
1
[39]. The lithogenesis can be induced either by ethylene glycol
0 alone [38], or in combination with ammonium chloride [4,14,15].
0 Administration of EG and AC resulted in increased calcium oxalate
A B C D E
crystalluria, hypocalciuria and polyuria in the present study in
Fig. 2. Calcium oxalate crystal deposition score of kidney section stained with lithiatic group as compared to healthy control, which might be due
Pizzolato’s stain. to impaired renal function [4]. The impairment of renal function
Severity grade were assigned as 0 = <1 crystal, 1 = 1-10, 2 = 11-30, 3 = 31-50, was also clearly indicated by elevation of serum creatinine, BUN, TP
4 =  51–75 and 5 = >75 crystals; data are expressed as mean
SEM. #p < 0.05,
in lithiatic group as compared to untreated healthy control, which
##p < 0.01 vs Lithiatic control (group B). A = Untreated healthy control, B = Lithiatic
group, C = 500 mg/kg Xanthium strumarium, D = 100 mg/kg cystone, E = Vehicle has been restored by xanthium treatment and the values were
control. statistically similar to standard cystone treatment. Similar finding
 P.N. Panigrahi et al. / Biomedicine & Pharmacotherapy 84 (2016) 1524–1532 1529

Fig. 3. Histology of representative kidney section stained with H and E stain (X 100).
A. Kidney section of untreated healthy control rat showing normal architecture; B. Kidney section of Lithiatic control rat showing crystals, haemorrhages, tubular
degeneration and dilatation; C. Kidney section of rat treated with Xanthium @ 500 mg/kg showing almost normal architecture with mild tubular dilatations; D. Kidney section
of rat treated with Cystone @ 100 mg/kg showing almost normal architecture with mild tubular dilatations and mono nuclear infiltration; E. Kidney section of vehicle control
rat showing crystal, tubular dilatation and infiltration of cells.

were also reported by taking various herbs such as Origanum
vulgare [4], Astilbin containing rhizome of Smilax china [12],
Holarrhena antidysenterica [14], Gokshuradi polyherbal ayurvedic
formulation [28], Punica granatum [40] and Adiantum capillus
veneris [41]. However, withdrawal of lithogenic treatment after
14 days in vehicle control group (group E) caused mild recovery in
kidney function test was observed but the values were significantly
different from healthy control as well as xanthium and cystone
treatment group. Khan [4] also reported similar findings while
evaluate the curative effect of Origanum vulgare (Linn.) against
urolithiatic rats.
Hyperoxaluria and hypercalciuria are considered as the major
risk factor in pathogenesis of kidney stone formation. Continuing
hypercalciuria promotes the nucleation and subsequent precipita-
tion of calcium oxalate crystals from the urine. Some studies have
suggested that disorders of renal tubular calcium reabsorption are
Renal ssue Calcium
2
**
1.8
1.6
1.4
##
1.2 ##
mg/g
the major cause of hypercalciuria. In the present study, hyper-
calciuria was not observed in the EG group as previously described
in some of the study [42,43]. A possible explanation for this result
is that the formation of CaC2O4 crystal consumes free calcium (Ca)
and normalizes Ca levels in the urine. However, oxalate is a more
important risk factor in the process of urinary calculi formation
than hypercalciuria [28,44,45]. Because, hyperoxaluria is associat-
ed with production of free radical resulting in oxidative damage of
renal epithelium thereby providing a nidus for crystal attachment
and ultimately causes crystal aggregation and retention in renal
tubules [46–48]. Therefore, decrease in oxalate concentration in
urine may explain its decrease in oxidative stress and renal crystal
deposition which was observed after xanthium treatment.
Moreover, the antioxidant property of xanthium was also recently
reported by some researchers due to presence of various bioactive
compounds [16,37].
It has been reported that reactive oxygen species (ROS) and
reactive nitrogen species (RNS) play an important role in renal
stone formation as a signalling molecule, as well as agent of injury
and inflammation [49,50]. ROS are produced through the involve-
ment of both mitochondria [51,52] and NADPH oxidase, but NADPH
**
oxidase is a major source of ROS in the kidney [45,50].
Experimental studies suggest that renal cellular exposure to high
oxalate, calcium oxalate or Calcium phosphate crystals results in
increased gene expression and production of molecules involved in

1 tissue remodelling, inflammation and biomineralization [50].

0.8 Hyperoxaluria and CaC2O4 crystal deposition induce rennin up-
0.6 regulation and generation of angiotensin II which activate the non
0.4 phagocytic NADPH oxidase [53] leading to the production of ROS/
0.2 RNS. ROS induced damage to the cells leads to cell death and the
0 formation of membrane bound vesicles which support crystal
A B C D E nucleation [50]. As a result crystallization inhibitors became
Fig. 4. Renal tissue calcium content. defective or damaged by exposure to excess free radicals and thus
A = Untreated healthy control, B = Lithiatic group, C = 500 mg/kg Xanthium strumar- not able to provide adequate protection resulting in crystal growth
ium, D = 100 mg/kg cystone, E = Vehicle control. *p < 0.05 and **p < 0.01 vs healthy and aggregation. Cell death also leads to the formation of new cells
control group (group A), #p < 0.05, ##p < 0.01 vs Lithiatic control (group B)
1530 P.N. Panigrahi et al. / Biomedicine & Pharmacotherapy 84 (2016) 1524–1532

Fig. 5. Immunohistichemical expression of OPN in kidney section (X100).

A. Mild expression of OPN in outer medulla in healthy untreated control rat; B. Increased expression of OPN throughout renal tubules of cortex and medulla in Lithiatic control
rat; C. Mild expression of OPN in rat treated with xanthium @ 500 mg/kg; D. Mild expression of OPN in rat treated with Cystone @ 100 mg/kg; E. Increased expression of OPN in
vehicle control rat. DAB counterstained with Mayer’s Hematoxylene

to repopulate the epithelium. The surfaces of the new cells as well
as the exposed basement membrane are conducive to crystal
attachment and retention [54]. Crystals retained in the terminal
collecting ducts produce Randall’ plugs which will act as stone
nidus when exposed to the pelvic urine.
In the present study, there was significantly increased activity
of LPO and NO; and significantly decreased activity of SOD, CAT and
GSH in lithiatic rats as compared to healthy control which might be
due to production of ROS and RNS. However, treatment with
xanthium significantly reduced the activity of LPO and NO, and
increased the mean activity of SOD, CAT and GSH, and the values
were statistically similar to standard treatment demonstrating the
optimal antioxidant effect of both the plant extract. Shirfule [28], Li
80
**
70
% of OPN positive cells
[45], and Jagannath [42] also reported the increased activity of LPO
in both the renal tissue and urine in rats with hyperoxaluria and
CaC2O4 nephrolithiasis. Similar renoprotective and antioxidant
effect was also reported in other medicinal herbs namely rhizome
of Smilax china containing Astilbin [12], Artemisia arborescens [55]
and green tea extract [56] against oxidative related renal injury.
Additionally, Selvam [57] and Sumitra [58] reported that treatment
with antioxidant like vitamin E improved the tissue levels of
antioxidant enzymes like SOD, CAT and GSH, reduced injury and
totally eliminated CaC2O4 crystal deposition in the kidneys.
The microscopic examination of renal histology showed intra-
tubular and interstitial CaC2O4 crystal deposits, mononuclear cell
infiltration, dilated renal tubule in untreated rats, consistent with
finding of other researchers [7,14]. The presence of polycrystalline,
rosette like arranged CaC2O4 crystals are evidence of adhesion and
retention of particles within the renal tubules. In contrast,
treatment with xanthium significantly reduced the CaC2O4 crystal
deposit evident by Pizzolato’s method of staining and renal

** histology was also nearly same as healthy control.

60
Osteopontin is an important component of stone matrix and
50 thought to be involved in many pathogenic and physiologic
*## *## processes, including cell migration, adhesion, inflammation, renal
40
injury and cancer [6,9,10]. OPN is mainly expressed in specific sites
30 like the loop of Henle and distal nephron in normal kidneys in
animal and humans. However, OPN expression may be significant-
20
ly up-regulated in all tubular segments after renal injury especially
10 in nephrolithiasis [6,15,45]. OPN is always associated with renal
stone formation, but whether it acts as a promoter or inhibitor of
0
stone formation is controversial. It was thought that, OPN may act
A B C D E
as a modulator of CaC2O4 crystallization, involved in nucleation,
Fig. 6. Quantitative analysis of immunohistochemical expression OPN in kidney growth and aggregation of crystal in the kidney [6,8]. In the present
tissue of rats. study, there was up-regulation of OPN in urolithiatic rats without
The number of stained (OPN positive) cells/100cells was counted across 10 fields/
any treatment. However, xanthium significantly decreased the
section. A = Untreated healthy control, B = Lithiatic group, C = 500 mg/kg Xanthium
strumarium, D = 100 mg/kg cystone, E = Vehicle control. Data represented mean
OPN expression and associated renal injury induced by EG
SD. * p < 0.05, ** p < 0.01 vs healthy control (group A); #p < 0.05, ##p < 0.01 vs suggesting a potent anti-inflammatory and anti-urolithiatic effect.
Lithiatic control (group B).
 P.N. Panigrahi et al. / Biomedicine & Pharmacotherapy 84 (2016) 1524–1532
5. Conclusion
The present findings demonstrate the curative efficacy of
Xanthium strumarium bur in ethylene glycol induced urolithiasis,
which might be mediated through inhibiting various pathways
involved in renal calcium oxalate formation, antioxidant effect, and
potential to inhibit biochemical parameters involving in im-
pairment of renal function and down regulation of matrix
glycoprotein, OPN. Therefore, future studies may be established
to quantify the active components in the extracts and also to
evaluate its efficacy and safety for clinical use.
Acknowledgement
Financial assistance given by the Director, Indian Veterinary
Research Institute to undertake this investigation is thankfully
acknowledged.
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