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812 Protein & Peptide Letters, 2012, 19, 812-819
A Novel Antilithiatic Protein from Tribulus terrestris Having Cytoprotec-
tive Potency
Anshu Aggarwala, Simran Tandona, Surinder Kumar Singlab and Chanderdeep Tandona,*
aDepartment of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan -
173234, Himachal Pradesh, India, bDepartment of Biochemistry, Panjab University, Chandigarh. India
Abstract: Adhesion of calcium oxalate (CaOx) crystals to kidney cells is a key event in kidney stones associated with
marked hyperoxaluria. As the propensity of stone recurrence and persistent side effects are not altered by surgical tech-
niques available, phytotherapeutic agents could be useful as an adjuvant therapy. The present study is aimed at examining
the antilithiatic potency of the protein biomolecules of Tribulus terrestris, a plant which is a common constituent of herbal
marketed preparations to treat urolithiasis. Various biochemical methods with mass spectrometry were used to purify and
characterize the purified protein. The protective potency of the protein was tested on the oxalate induced injury on renal
epithelial cell lines (NRK 52E). An antilithiatic protein having molecular weight of ~ 60kDa was purified. This purified
protein showed similarities with Carotenoid cleavage dioxygenase 7 (CCD7) of Arabidopsis thaliana after matching pep-
tide mass fingerprints in MASCOT search engine. An EF hand domain was identified in CCD7 by SCAN PROSITE.
Presence of an EF hand domain, a characteristic feature of calcium binding proteins and a role in the synthesis of retinol
which is transported by retinol binding protein, a protein found in kidney stone matrix; of CCD7 support the role of TTP
as an antilithiatic protein. The protective potency of TTP on NRK 52E was quite comparable to the aqueous extract of
cystone. Our findings suggest that this purified protein biomolecule from Tribulus terrestris could open new vista in
medical management of urolithiasis.
Keywords: Calcium oxalate, carotenoid cleavage dioxygenase 7, NRK 52E, Tribulus terrestris, urolithiasis
INTRODUCTION
Nephrolithiasis is quite common, affecting 10–12% of
the population in industrialized countries [1]. Calcareous
stones are the most commonly occurring ones to an extent of
60-80% followed by magnesium ammonium phosphate
(Struvite) to an extent of 10-15%, uric acid 5-10% and cys-
tine 0.5-1% [2]. The genesis of a calcium oxalate stone is a
complex process which would ensue from an imbalance be-
tween promoters and inhibitors of urinary crystallisation.
The natural progression of the urine chemistry leading to
stone development is urine saturation, urine supersaturation,
crystal nucleation, aggregation, the retention of crystals by
the urothelium, and the continued growth of the stone on the
retained crystals [3]. However, incompletely understood bio-
logical processes can anchor these crystals to the urothelium.
A widely held theory is that of Randall’s plaques, which
proposes that subepithelial interstitial calcium-based deposits
act as nuclei for stone formation [4].
Experiments with cultured renal cells indicate that ox-
alate can activate pathways that either trigger cell growth or
produce a cellular injury that can ultimately result in cell
death. Increase in oxalate concentration leads to the activa-
tion of lipid signalling pathways that produce arachidonic
acid, lysophospholipids, and ceramide. These lipids disrupt
mitochondrial function by increasing reactive oxygen species
*Address correspondence to this author at the Biotechnology and Bioinfor-
matics, Jaypee University of Information Technology, Waknaghat- 173234,
Solan, India; Emails: chanderdeep.tandon@juit.ac.in
(ROS), decreasing mitochondrial membrane potential, and
increasing mitochondrial permeability. The net response is
cytochrome C release, activation of caspases, and apoptosis
or necrosis [5].
Various surgical procedures introduced for the manage-
ment of renal stones like extracorporeal shock wave
lithotripsy (ESWL), ureteroscopy (URS), or percutaneous
nephrolithotomy (PNL) have not proved to be quite effective
and the propensity for stone recurrence is still high. In addi-
tion, ESWL might show some significant side effects such as
renal damage, ESWL induced hypertension or renal impair-
ment [6]. Literature suggests that phytotherapeutic agents
could be useful as either an alternative or a complementary
therapy in the management of urolithiasis. Plant medicines
are in great demand because of their wide biological and
medicinal activities, higher safety margin and lesser costs
[7].
Fruits of Tribulus terrestris (Zygophyllaceae) locally
named as “gokhru” in India are commonly used in folklore
to treat urolithiasis. So far, its diuretic properties have been
documented in literature [8, 9] and it is actively used in vari-
ous drug formulations of kidney stone treatments like
Cystone and Uriflow. The present study is aimed at identifi-
cation of the most potent biomolecules(s) responsible for the
antilithiatic property of Tribulus terrestris and examining the
efficacy of the same on oxalate induced injury in NRK 52E
(rat renal tubular epithelial) cells.
-/12 $58.00+.00 © 2012 Bentham Science Publishers
Cytoprotective Effect of Tribulus terrestris Protein Protein & Peptide Letters, 2012, Vol. 19, No. 8 813
MATERIALS AND METHODS
Materials
The dried and matured fruits of Tribulus terrestris were
obtained from “Natural Remedies Pvt. Ltd.” at Bangalore in
India. A collection of voucher specimen is available with the
company. Other materials required were Macro Prep® 25 Q
Strong anion exchanger (Bio-Rad laboratories), Bio gel® P-
100 gel (Medium, 90-180 m), Molecular sieve support
(Bio-Rad laboratories), bovine serum albumin, Trypsin pro-
file IGD kit (Sigma, St. Lois, USA), Solvents used were of
HPLC grade. All other chemicals were of analytical grade.
Crystal Growth Inhibition
Inhibitory activity against CaOx crystal growth was
measured using the seeded, solution-depletion assay [10].
Briefly, CaOx crystal seed (from FTIR identified clinical
kidney stones) slurry, 1.5mg/ml were added to a solution
containing 1mM calcium chloride (CaCl2) and 1mM sodium
oxalate (Na2C2O4). The reaction between CaCl2 and Na2C2O4
would lead to deposition of CaOx on the crystal surface lead-
ing to the depletion in free oxalate that is detectable spectro-
photometry at 214 nm. When a protein is added into this
solution, the rate of depletion of free oxalate will decrease if
the protein inhibits CaOx crystal growth. Rate of reduction
of free oxalate was calculated using the baseline value and
the value after incubation with or without protein. The rela-
tive inhibitory activity was calculated as follows: % Relative
inhibitory activity = [(C–S)/C] 100, where C is the rate of
reduction of free oxalate without any test protein and S is the
rate of reduction of free oxalate with a test protein.
Protein Purification
The active inhibitory protein was isolated as described by
Kaur et al [11]. Powdered Tribulus terrestris was extracted
with 50mM Tris-Cl buffer (pH 7.4), containing 0.25M NaCl,
1mM PMSF and 0.01% sodium azide for 24 h with gentle
stirring at 4oC. The slurry was centrifuged at 10,000g for 20
min at 4oC to recover the supernatant. Protein in the super-
natant was estimated using Lowry et al. The protein was
precipitated with ammonium sulphate (40-60% saturation) to
recover the pellet with CaOx inhibitory activity. The pellet
was dissolved in 50mM Tris-Cl buffer containing 50mM
NaCl (pH 7.4) and dialyzed against the same buffer before
fractionation on strong anion exchanger Macro Prep® 25 Q
column (20 X 1.5cm) pre-equilibrated with 20mM Tris
buffer with 0.1mM NaCl (pH 7.4) using Biologic LP system
(Bio-Rad). The elution was carried out with same buffer us-
ing a linear gradient of 0.1-1.0M NaCl at flow rate of
1.5ml/min. The fractions having anticalcifying activity were
pooled, dialyzed against 50mM Tris-Cl containing 50mM
NaCl and further fractionated on a Bio gel® P-100 gel mo-
lecular sieve column (70 X 2.5cm) equilibrated and eluted
with the 20mM Tris buffer (pH 7.4) at a flow rate of
0.1ml/min. The homogeneity of the active protein was
evaluated by electrophoresis. The purified protein was stored
at -80 0C for further analysis.
Electrophoresis
SDS/PAGE was carried out using 10% polyacrylamide
gels and stained with silver nitrate.
Peptide Mass Fingerprinting by MALDI-TOF MS
The protein band was excised and subjected to in-gel
tryptic digestion as using Trypsin profile IGD kit (Sigma).
The resulting peptide mixtures were, eluted on the sample
plate with the matrix solution (10 mg/ml of -cyano-
carboxycinnamic acid in 50% acetonitrile/0.1% trifluoroace-
tic acid) and then analyzed on Bruker Ultraflex MALDI–
TOF/TOF mass spectrometer. Peptide matching was per-
formed using the MASCOT search engine
(http://www.matrixscience.com) assuming that peptides were
monoisotopic, carbamidomethylated at cysteine residues, and
oxidized at methionine residues. A mass tolerance was 1.2
Da, and only 1 maximal cleavage was allowed for peptide
matching. Proteins with probability based MOWSE scores
exceeding their threshold (P<0.05) were considered to be
positively identified [12].
Putative Function of Protein and Domain Identification
The amino acid sequences of protein were subjected to
BLASTp analysis to determine putative function and family
of protein at the National Center for Biotechnology Informa-
tion (www.ncbi.nlm.nih.gov/BLAST) using all non-
redundant GenBank CDS. Further to reveal presence of ac-
tive domains in the protein, SCAN PROSITE
(http://expasy.org/tools/scanprosite) was employed.
Cell Culture
Normal rat renal tubular (NRK 52E) epithelial cells were
obtained from National Centre of Cell Sciences (NCCS,
Pune). The cells were maintained as monolayers in Dul-
becco’s Modified Eagle’s Medium (DMEM) with 2.0 mM L-
glutamine adjusted to contain 3.7 g/L sodium bicarbonate,
4.5 g/L glucose. Media was supplemented with 1% Penicillin
(10,000 units/ml)-Streptomycin (10,000 g/ml) and 10%
fetal bovine serum. Cells were cultured in 25 cm2 tissue-
culture treated flasks at 37oC and 5% CO2 in humidified
chambers.
Preparation of the Tribulus terrestris Extract
For cell culture studies, the purified protein was dialysed
through amicon tube (3kDa) and was reconstituted in 0.22
filtered distilled water. This was treated as a stock solution of
the Tribulus terrestris protein.
Oxalate-induced Cell Injury
NRK 52E cells were incubated in DMEM containing 1
mM sodium oxalate in the presence of different concentra-
tions of cystone as the positive control (1g/ml, 2g/ml and
4g/ml) and that of the plant purified protein (1g/ml,
2g/ml and 4g/ml) for 72 h [13, 14]. Cell injury was as-
sessed by measuring the cell viability through trypan blue
and monitoring the lactate dehydrogenase (LDH) leakage
into the medium.
814 Protein & Peptide Letters, 2012, Vol. 19, No. 8 Aggarwal et al.
CYTOTOXICITY
Trypan Blue Assay
The cytotoxicity of the purified protein of T. terrestris
was assessed by cell viability using trypan blue exclusion
method. For the determination of cell viability, cells were
plated at the density of 4 104 cells/well and cultured for 72
h. The medium was replaced with serum-free medium and
the cells were treated with various concentrations of cystone
(1g/ml, 2g/ml and 4g/ml) and the purified protein of the
plant (1g/ml, 2g/ml and 4g/ml) for a further 72 h. The
percentage viability for the cells was calculated as (live
cells/total cells) x100.
Lactate Dehydrogenase (LDH) Leakage Assay
Lactate dehydrogenase (LDH) leakage assay was per-
formed by the method described by Wagner et al. [15].
Briefly, 6.6 mM NADH and 30 mM sodium pyruvate were
prepared in Tris (0.2M, pH 7.3). Reaction was initiated with
the addition of 50 l of the test sample and the disappearance
of NADH was monitored at 340nm, for 5 min at an interval
of 1 min. The percentage of LDH release was calculated by
dividing the activity of LDH in the supernatant by the total
LDH activity measured after complete cell lysis achieved by
sonication.
Statistical Analysis
Data were expressed as mean values of three independent
experiments (each in triplicate) and analyzed by the analysis
of variance (p<0.05) to estimate the differences between
values of extracts tested.
RESULTS
Purification of Potential Inhibitor (Antilithiatic Protein)
of CaOx Crystal Growth
The maximum inhibitory activity was recovered in the
fraction precipitating at 40-60% ammonium sulphate satura-
tion. The pellet so obtained was desalted by dialysis and
loaded on to anion exchanger. The fractions under the peak
were pooled and the protein content was determined. The
fractions which were obtained between conductivity 71.13
mS/cm and 85.99 mS/cm (Fig. 1), had protein concentration
of 100g/ml and showed the maximum inhibitory activity
(51.6%) towards CaOx growth. The pooled fractions were
analyzed by SDS PAGE, which revealed two bands signify-
ing it as a mixture of proteins (figure not shown). Further the
pooled fraction of anion exchange chromatography was par-
titioned by molecular sieve chromatography on Bio gel (P-
100 gel) column. The fractions under the peak (Fig. 2) which
was eluted in a time span of 890.23 min to 998.70 min show-
ing inhibitory potency (78.3%) was pooled and stored at -
80oC till further use. The SDS profile of this fraction re-
vealed a single band of molecular mass of 60kDa (approxi-
mate) giving an idea that the protein isolated was purified
(Fig. 3). Table 1 shows the protein concentration and the
inhibitory activity of the isolated fraction after each step of
purification.
Peptide Mass Fingerprinting
MALDI TOF analysis spectrum of Tribulus terrestris
protein is shown in Figure 4. MASCOT search engine
showed highest similarity (score: 41) of this protein with
carotenoid cleavage dioxygenase 7 (CCD7) of Arabidopsis
thaliana (CCD7_ARATH) having 17% sequence coverage.
Figure 1. Purification of active protein from Tribulus terrestris. Elution profile of the protein sample loaded on anion exchange chromatog-
raphy column after ammonium sulphate precipitation. The eluting proteins were detectable at 280nm.
Cytoprotective Effect of Tribulus terrestris Protein Protein & Peptide Letters, 2012, Vol. 19, No. 8 815
Figure 3. SDS PAGE of Tribulus terrestris protein showing a sin-
gle band of molecular mass of 60kDa.
Putative Function and Domain Prediction
The BLASTp analysis of CCD7 of Arabidopsis thaliana
showed its similarity with NP_182026.4 (9-cis-epoxycaro-
tenoid dioxygenase [Arabidopsis thaliana]), ACY01408.1
(Carotenoid cleavage dioxygenase 7 [Petunia hybrid]),
NP_001183928.1 (Carotenoid cleavage dioxygenase [Zea
mays] showing that the purified protein belong to carotenoid
cleavage dioxygenase family. Figure 5 shows domains in the
purified protein of Tribulus terrestris by SCAN PROSITE.
This protein has EF hand calcium binding domain from 228-
240.
Reduction of Oxalate-induced Renal Tubular Epithelial
cell injury by Purified Protein of Tribulus terrestris
Figure 6 depicts the protective effect of the purified pro-
tein towards the renal tubular epithelial cells, NRK 52E. The
oxalate induced a significant injury to the cells as it reduced
the viability from 100% in the untreated cells (control) to
75.1%. The purified protein proved to be protective towards
oxalate induced injury as it increased the viability signifi-
cantly in a concentration dependent manner from 1g/ml to
4g/ml. The percentage viability obtained with the purified
Table 1. Summary of Purification of Inhibitory Protein from the Fruits of Tribulus terrestris. %Age Inhibition of CaOx Represents
Results as Mean of Three Independent Experiments.
Purification Steps Amount of Protein Yield (%) %Age Inhibition of CaOx Growth
Tris-Cl Buffer Extract 286 mg 100 37.56
40-60% Ammonium sulphate precipitate (dial-
ysed) 118.5 mg 41.4 38.61
Anion Exchange Chromatography 1.5 mg 0.5 51.61
Molecular Sieve Chromatography 0.74 mg 0.3 78.26
Figure 2. Purification of active protein from Tribulus terrestris. Elution profile of the protein sample loaded on molecular sieve chromatog-
raphy column after anion exchange chromatography column. The eluting proteins were detectable at 280nm.
816 Protein & Peptide Letters, 2012, Vol. 19, No. 8 Aggarwal et al.
protein was comparable to the cystone (positive control). The
percentage viability with 1μg/mL, 2μg/mL and 4μg/mL of
the protein was 77.1, 80.9 and 85.9 respectively.
The release of lactate dehydrogenase (LDH), a stable
cytosolic enzyme, was estimated to check the cell injury
(Fig. 7). In the cells injured with the exposure of oxalate,
LDH release was significantly high (217.9%). The LDH re-
lease was significantly reduced in the cells which had the
exposure of different concentrations of the purified protein
with oxalate in a concentration dependent manner to 115.9%
Figure 4. The peptide mass fingerprinting by MALDI TOF MS obtained from trypsinised Tribulus terrestris protein.
Figure 5. Domain identified in Carotenoid cleavage Dioxygenase 7(CCD7) of Arabidopsis thaliana by SCAN PROSITE. The sequence of
the EF hand domain is represented.
Figure 6. Effect of Tribulus terrestris on the % viability. Data are mean ± SEM of three independent observations. * p < 0.05 versus un-
treated control, ** p < 0.05 versus oxalate control.
Cytoprotective Effect of Tribulus terrestris Protein Protein & Peptide Letters, 2012, Vol. 19, No. 8 817
with 4g/ml protein sample. The LDH release in the pres-
ence of test protein was quite comparable with that of the
positive control, cystone.
DISCUSSION
Urolithiasis can be traced to the earliest antiquity of hu-
man history though the mechanism governing the induction
of various physicochemical events leading to stone formation
still remain speculative. The medical management therapies
available are quite costly and in most cases are invasive and
with serious side effects. Therefore, it is worthwhile to look
for an alternative to these conventional methods by using
medicinal plants or phytotherapy [1]. Till date, many plants
have been studied by various groups to reduce the incidence
of calcium stone deposition both in vitro and in vivo [7] but
the identification of naturally occurring CaOx inhibitory
biomolecules from plants was hampered in the past by the
limitation of the purification methods.
Many plants are also known to produce CaOx as crystal-
line deposits, having an organic matrix constituting of differ-
ent proteins [16, 17]. Recently, it was observed that water
soluble protein matrix associated with calcium oxalate crys-
tals from bean seed coat (Phaseolus vulgaris) contains many
polypeptides out of which two proteins were isolated and
they showed strong inhibition towards nucleation of CaOx in
a concentration dependent manner [18]. Calsequestrinlike
calcium binding protein was isolated from calcium accumu-
lating cells of Pistia stratiotes [19]. Recently our group has
also purified two anticalcifying proteins each from Dolichos
biflorus [20] and Trachyspermum ammi [11] with high con-
tent of acidic amino acids. The purified protein from Trachy-
spermum ammi was also found to be effective against in-
duced kidney stones in vivo [21]. Aspartic acid and glutamic
acid are quoted to play a vital role in the antilithiatic potency
of Tribulus terrestris by disintegrating the stone outer shell,
according to the datasheet of an herbal composition “Uri-
flow” marketed by Bioneutrix. So, it is worthwhile to look
for a CaOx inhibitory protein from the fruits of Tribulus ter-
restris since it showed strong anticalcifying properties in
vitro [22].
In the present study, an antilithiatic protein was isolated
from the fruits of Tribulus terrestris inhibiting calcium ox-
alate crystallisation. The protein was purified through am-
monium sulphate precipitation, anionic adsorption and fi-
nally molecular size separation. This isolated purified protein
had a molecular weight of around 60kDa. The diuretic po-
tency of Tribulus terrestris has been explored by various
groups in vitro and in vivo but the purification of active bio-
molecules is still not done [8, 9]. The inhibitory potency of
the purified protein was found to be as high as 78.3% and the
presence of an EF hand domain in a homologous protein
indicates that this protein probably imparts its inhibitory ef-
fect by binding to calcium ions and thus minimizing the
availability of calcium for the formation of CaOx crystals.
The isolated protein was subjected to MALDI-TOF fol-
lowed by peptide mass fingerprinting analysis and MASCOT
search engine, which showed the maximum similarity (17%
sequence coverage) with carotenoid cleavage dioxygenase 7
(CCD7) of Arabidopsis thaliana with a molecular weight of
65kDa and pI 6.01. BLASTp analysis of CCD7 showed
similar hits, further verifying the hit obtained from MAS-
COT.
CCD7 belong to a family of dioxygenases which possess
characteristic five conserved histidines spread throughout
their primary protein sequence, secondly, they require Fe2+
ions thought to be coordinated by the five histidine residues
and they contain a conserved polypeptide segment at their
carboxy terminus that minimally constitutes a signature se-
quence for the family [23]. Histidine is said to play a role in
urolithiasis as histidine coordinated manganese is critical for
Figure 7. Effect of Tribulus terrestris on the %LDH release. Data are mean ± SEM of three independent observations. * p < 0.05 versus
untreated control, ** p < 0.05 versus oxalate control.
818 Protein & Peptide Letters, 2012, Vol. 19, No. 8 Aggarwal et al.
substrate recognition and is directly involved in the catalysis
action of oxalate decarboxylase, enzyme involved is conver-
sion of oxalate to formic acid and CO2 [24].
CCD7 cleaves its substrates specifically at the 9,10 dou-
ble bond asymmetrically [25]. With -carotene as a sub-
strate, which is also called as provitamin A, CCD7 produces
one -ionone product and the C27 product, 10’-apo--
carotenal that is required for the normal inhibition of shoot
growth from axillary meristems. Another significant member
of this family is carotene 15, 15’ monooxygenase (BCO),
formerly known as -carotene 15,15’-dioxygenase, based on
biochemical and amino acid sequence data [26, 27]. This
enzyme shares 58% homology with CCD7 of Arabidopsis
thaliana as established through BLASTp. BCO catalyzes the
first step in the synthesis of retinol from dietary carotenoids.
Retinol, also referred to as vitamin A, is a fat-soluble poly-
isoprenoid essential for tissue development, growth, and
vision. Retinol can be either ingested or synthesized within
the body from dietary carotenoids. The major substrate for
the in vivo synthesis of retinol is the plant carotenoid -
carotene. Of the more than 600 different carotenoids isolated
from nature, almost 50 possess biological activity; hence,
these compounds are termed provitamin A carotenoids [28].
Kidney is said to play a vital role in retinol metabolism
by managing the levels of Retinol Binding Protein (RBP4).
The serum retinol level in the lithiatic patients was found to
be low [29]. The mechanism by which retinol (vitamin A)
deficiency causes calculus formation is still unclear. The
squamous metaplasia of the urinary tract can result in keratin
debris which promotes calculus formation. Histological
analysis showed squamous metaplasia that was confined to
urinary tract [30]. Vitamin A deficiency caused important
changes in urine composition and there was a decrease in the
concentration of urinary glycoaminoglycans and zinc. Le-
sions of cuboidal epithelia covering the papillae in rats
treated with vitamin A deficient diet were severe. It was
studied that in urolithiatic humans too, there is an increase in
Vit E/Vit A ratio. These results could be related to the possi-
ble deficit of vitamin A in kidney of stone formers, this be-
ing one factor for urolith development. Moreover, deficit of
important urinary crystallization inhibitors in stone formers
(pyrophosphate and phytate) can also be related to presence
of low levels of renal vitamin A which prevents enzymatic
degradation of such inhibitors [31].
Since, CCD7 belong to the same family as BCO [27] and
retinol happens to be a product of BCO which as cited in
literature is known to possess antilithiatic potency [30, 31],
we hypothesize that in the light of above findings, even the
product of CCD7 i.e. 10’ apo -carotenal might be having a
key role to play in preventing kidney stone formation. This
study has to be further undertaken.
CCD7 homologous to the purified protein from Tribulus
terrestris is also shown to contain an EF hand domain (228 –
240) which is a characteristic of Ca2+ binding proteins. It is
common calcium binding helix-loop-helix motif. Most of the
kidney stone inhibitory proteins like nephrocalcin, os-
teonectin and calgranulin have EF hand domain [32].
We tested the protective potency of the purified protein
towards oxalate induced cell injury on NRK 52E. In the pre-
sent study, oxalate exposure resulted in a significant increase
of LDH release and decreased the cell viability. The cellular
injury potentiates calcium oxalate crystal formation and re-
tention [5]. In the presence of the plant protein, the cell vi-
ability and LDH release were significantly normalised. The
percentage viability and the LDH release with 4g/ml pro-
tein in the presence of 1mM oxalate were quite comparable
with the 50g/ml crude aqueous extract as reported earlier
[22]. This data further validates the high potency of the puri-
fied protein in playing a crucial role in protecting the renal
epithelia cells against oxalate damage.
CONCLUSION
In conclusion, a plant protein, anionic in nature, from the
fruits of Tribulus terrestris was shown to attain the ability of
inhibiting CaOx crystallization in vitro. This protein had
similarity with CCD7 of Arabidopsis thaliana which belongs
to the family of proteins known to form retinol that plays a
significant role in preventing calculi formation. The presence
of EF hand domain in this protein signifies its calcium bind-
ing properties which is a feature of most of the kidney stone
inhibitors. Activity of this plant protein from T. terrestris
adds a new vista to study plant proteins for their therapeutic
use to treat kidney stones.
ACKNOWLEDGEMENT
The authors would like to thank Department of Biotech-
nology (DBT), Government of India and Jaypee University
of Information Technology, Solan, India for funding this
research work.
CONFLICT OF INTEREST
None declared.
ABBREVIATIONS
NRK 52E = Normal rat kidney epithelial cell line
CaOx = Calcium Oxalate
MALDI TOF MS = Matrix-assisted laser desorp-
tion/ionization time of flight mass
spectroscopy
BLAST = Basic local alignment search tool
LDH = Lactate dehydrogenase
CCD7 = Carotenoid Cleavage Dioxygenase 7
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Received: September 26, 20 11 Rev ised: February 6, 2012 Accepted: February 22 , 2012
