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ORIGINAL PAPER
Effects of an aqueous extract from
Phyllanthus niruri
on calcium oxalate crystallization in vitro
Received: 10 April 2002 / Accepted: 10 October 2002 / Published online: 21 January 2003
Springer-Verlag 2003
Abstract Phyllanthus niruri is a plant used in Brazilian
folk medicine for the treatment of urolithiasis. It was
previously observed that P. niruri shows no toxicity, po-
tentially increases calculus voiding by stone forming pa-
tients and inhibits the endocytosis of calcium oxalate
(CaOx) crystals by MDCK cells. In addition, in a rat
model of urolithiasis it reduced calculus growth. In the
present study, we evaluated the effect of an aqueous ex-
tract of P. niruri on CaOx crystallization in vitro. CaOx
precipitation was induced by the addition of 0.1 M sodi-
um oxalate to unfiltered urine samples from Wistar rats
(n=14) and normal humans (n=18) in the presence or
absence of P. niruri extract (0.25 mg/ml of urine). The
presence of CaOx crystals was evaluated immediately and
24 h later. In vitro crystallization of human urine pro-
duced typical mono- and dihydrated CaOx crystals, but
only a few typical CaOx crystals were found in rat urine.
The presence of P. niruri extract did not inhibit CaOx
precipitation and even more crystals were obtained, al-
though they were significantly smaller than those in the
control urine. Crystal aggregation observed 24 h after
crystallization was also inhibited by P. niruri extract. The
results showed an inhibitory effect of P. niruri extract on
CaOx crystal growth and aggregation in human urine,
suggesting that it may interfere with the early stages of
stone formation and may represent an alternative form of
treatment and/or prevention of urolithiasis
Keywords Calcium oxalate Æ In vitro crystallization Æ
Phyllanthus niruri Æ Renal stone Æ Urolithiasis Æ
Natural products
Introduction
Urinary stones affect 10–12% of the population in in-
dustrialized countries [27]. Their incidence has been in-
creasing over the last years while the age of onset is
decreasing [10]. In addition, the recurrence rate is high,
being more than 50% after 10 years [28, 29]. In spite of
substantial progress in the pathophysiology and treat-
ment of urolithiasis, there is no satisfactory drug to use
in clinical therapy. Thus a drug for the prevention of this
disease or its recurrence would be of grea t interest.
Phyllanthus niruri is a plant belonging to the Eu-
phorbiaceae family, which have a worldwide distribu-
tion. It is used in Brazilian folk medicine by patients
with urolithiasis [18, 21]. Many components of P. niruri
have been identified, including groups of active sub-
stances such as alkaloids, tannin, lignans, phenols, ste-
roids, flavanoids, triterpenes, as well as ricinoleic acid,
niruside, and phylitate [4]. However the components
involved in lithiasis prevention are not known. We have
been evaluating the potential effect of P. niruri in the
treatment of urolithiasis over the last few years. Exper-
imental and clinical studies have demonstrated that
P. niruri has no acute or chronic toxicity and preliminary
data suggest effects which promote stone elimination in
stone forming patients [25]. Moreover, oral administra-
tion of P. niruri aqueous extract to rats induced an in-
hibitory effect on vesical calcium oxalate (CaOx) crystal
growth, which was associated with a reduction in the
urinary excretion of glycosaminoglycans and with an
increase in the content of these macromolecules in the
calculi compared with untreated animals [11]. Also, P.
niruri significantly reduced the endocytosis of CaOx
crystals in MDCK cells in culture [5]. Despite the be n-
eficial effect of P. niruri observed in vivo in rats and
humans, its mechanism of action is not fully understood.
Most kidney stones contain calcium oxalate [9], and
the formation of urinary calculi involves a CaOx crys-
tallization process that includes nucleation, growth and
the aggregation of crystals [15, 16]. Thus, to better
Urol Res (2003) 30:374–379
DOI 10.1007/s00240-002-0285-y
M.E. Barros Æ N. Schor Æ M.A. Boim
M.E. Barros Æ N. Schor Æ M.A. Boim (&)
Nephrology Division, Escola Paulista de Medicina,
Universidade Federal de Sa
˜
o Paulo, Sa
˜
o Paulo, Brazil
E-mail: mirian@nefro.epm.br
Fax: +55-11-55739652
M.A. Boim
UNIFESP Renal Division,
Rua Botucatu 740, 04023-900, Sa
˜
o Paulo, SP, Brazil
understand the role of P. niruri in urinary stone for-
mation in the present study, we evaluated the effect of an
aqueous extract of this plant on CaOx crystallization
induced in vitro.
Materials and methods
An aqueous extract of P. niruri was obtained from the whole plant,
as occurs in popular medicine. The plant was grown at the Ex-
perimental Center of the Universidade Estadual de Campinas, Sa
˜
o
Paulo. Plant samples were dried at 50C for 2 months in a venti-
lated room. After drying, samples were ground in a mechanical mill
and used for tea preparation (5% w/v). The infusion was stirred for
30 min at 72C and then vacuum filtered, concentrated and
lyophilized.
CaOx crystallization was induced in the urine obtained from
normal humans and rats. Isolated human urine samples were ob-
tained from six healthy subjects, three males and three females,
with no personal or family history of kidney stone disease. Urine
was collected on three different occasions from each individual,
with an interval of at least 15 days between each sampling time
(n=18). Urine samples collected over 24 h were obtained from
normal, adult Wistar rats (n=14) in a metabolic cage. Rats were
maintained on standard chow and tap water during the collection
period. Human and rat urine samples were centrifuged at
5,000 rpm (4,815 g) for 8 min, the supernatant was then transferred
to a clean tube, and the pH was adjusted to 6.0.
Experimental protocol
CaOx precipitation was induced by adding 40 ll of 0.1 M sodium
oxalate per ml of urine (corresponding to 0.536 mg), every 30 min
(0, 30, 60 and 90 min) under shaking at 37C, resulting in a final
concentration of 2.14 mg/ml of urine. Each urine sample was di-
vided into two aliquots, one of which was used as a control
(crystallization without P. niruri extract) while in the other CaOx
precipitation was induced in the presence of P. niruri extract, which
was added to the sample 30 min before the crystallization process.
Lyophilized P. niruri extract was resuspended in distilled water
(25 mg/ml), filtered through a 0.22 lm filter, and used at a final
concentration of 0.25 mg/ml urine, based on a dose-response curve.
The present protocol was approved by the Ethics Committee of
the Universidade Federal de Sa
˜
o Paulo.
Analysis of crystals
The crystals obtained were analyzed immediately and 24 h after the
crystallization process. The semiquantitative analysis of crystals
was estimated by turbidity [26]. After crystallization, 100 ll
aliquots were loaded onto a 96 well microplate and the absorbance
was measured with a plate reader (Original Multiskan EX,
Labsystens, Finland), at 590 nm (OD
590
). The absorbance of each
sample was measured in quadruplicate and the mean was used to
calculate the turbidity index TI=(DOt·DOb)/DOb, where DOt is
the mean sample absorbance after CaOX precipitation and DOb is
the mean sample absorbance before precipitation. The number and
size of the crystals were determined using an automated particle
counter [22] (Coulter counter, model Z1, Coulter Electronics, En-
gland), using a 50 lm filter calibrated with latex particles measur-
ing 10 lm in diameter. The number of particles was counted
according to size, which was classified from 5.0 to 30.0 lm. The
number of crystals was expressed on a percentage scale. Crystals
were also analyzed by light microscopy. After crystallization,
samples were centrifuged at 3,000 rpm (720 g) for 5 min and the
supernatant was partially discarded, with approximately 10% of
the initial volume being left and rehomogenized. One drop
was transferred to a Neubauer chamber and the crystals were
qualitatively analyzed in terms of size and shape. Images were re-
corded with a digitalized video-camera (Model SSC-DC54A, Sony
Exwave HAD, Japan), transferred to a computer and analyzed
using Imagelab 2000 (Brazil) software.
Statistical analysis
All results are reported as means±SEM. Results for control and
experimental samples were compared by the paired Student’s t-test.
Differences between human and rat urine were compared by the
unpaired Student’s t-test. P values of less than 0.05 were considered
significant.
Results
A dose-response curve was constructed using human
urine, based on the effect of different doses of P. niruri
extract on the turbidity of the solution. Concentrations
of P. niruri extr act varied from 0.00 to 1.00 mg/ml of
urine. Figure 1 shows that the absorbance of the solu-
tion increased with increasing doses of P. niruri extract.
Doses above 0.25 mg/ml were not able to induce a fur-
ther change in absorbance and therefore this dose was
used in the subsequent experiments. The increase in
absorbance indicates a higher density of crystals, an
unexpected result for P. niruri extract. However, the
observation of crystals by light microscopy showed that
increasing doses of P. niruri extract actually produced a
higher crystal density but of smaller crystals (Fig. 2),
explaining why P. niruri induced an increase rather than
a decrease in absorbance.
The number of crystals related to size was estimated
by automatic counting. As shown in Fig. 3, in the
presence of 0.25 mg/ml of P. niruri extract there was an
increase in the number of smaller crystals between 5.0–
7.5 lm and a decrease in the number of larger crystals
(10–30 lm) compared with urine without P. niruri ext-
raxt. Figure 4 illustrates the effect of P. niruri on the
crystal form of CaOx analyzed by micros copy in human
urine. In control urine, crystals were identified as a
Fig. 1 The induction of CaOx crystallization measured by optical
density (OD
590
), in normal human urine by adding sodium oxalate
in the presence of increasing amounts of P. niruri extract
375
mixture of mono- and dihydrate (COM and COD)
CaOx crystals (Fig. 4A). The presence of P. niruri ex-
tract was associated with a partial shift from the COM
form to the COD form (Fig 4B). A semiquantitative
analysis of the COM and COD fractions estimated by
visual examination under the microscope showed that in
the control urine 15% of the crystals were COD and this
fraction increased significantly to 30% in the presence of
P. niruri extract.
An intense aggregation of CaOx crystals was
observed 24 h after crystallization in control urine
(Fig 5A), a phenomenon that was substantially inhibited
in the presence of P. niruri extract (Fig. 5B).
In contrast to human urine, typical CaOx crystals
were almost absent in rat urine after the induction of
crystallization. Small particles, but no typical CaOx
crystals were observed in these samples, either immedi-
ately or 24 h after the crystallization process. P. niruri
extract had no effect on rat urine.
Discussion
Increasing evidence has pointed to the beneficial effects
of P. niruri in the treatment of uroli thiasis [18, 21].
In vivo studies have shown that P. niruri may be
effective in promoting calculus voiding by stone forming
patients [25] and significantly reduced calculus growth
in a model of vesical calculi in rats [19, 25]. In addition,
no toxic effect was observed in individuals ingesting P.
niruri tea over a period of 3 months [19, 25]. In spite of
these exciting results obtained in vivo, little is known
about the mechanism of action of P. niruri. Many steps
and a favorable environment are necessary for the de-
velopment of a calculus. The supersaturation of urine is
a prerequisite for precipitation to occur but not suffi-
cient to produce a stone [15, 16]. Thereafter, nucleation,
growth and crystal aggregation take place as a result of
a favorable environment, including the adhesion and
internalization of the crystal into the tubular epithelial
cells [12]. Recently, Campos and Schor [5] showed that
an aqueous extract of P. niruri significantly reduced the
Fig. 2 Light microscopy of CaOx crystals induced in human urine
by adding sodium oxalate solution in the absence and presence
of P. niruri extract at different concentrations. 100· and 400·
magnification
Fig. 3 Number of particles analyzed by automatic counting
classified by size in the absence and presence of P. niruri extract
in human urine
376
endocytosis of CaOx crystals by MD CK cells . More-
over, the presence of defined substances such as
magnesium and citrate or macromolecules such as
glycosaminoglycans, osteopotin, nephrocalcin, etc, in
urine may act as protectors [7, 8, 23, 31] inhibiting
calculus development by interfering with many phases
of calculus formation. Studies conducted by Fr eitas et
al. [11] using a model of urolithiasis in rats demon-
strated that P. niruri did not interfere with the urinary
excretion of Mg
++
, citrate or glycosaminoglycans, but
instead promoted adsorption of the latter substances
into the calcu li, making them softer and smaller. In
order to provide further evidence on the potential role
of this plant as an inhibitor of stone formation, and to
better understand its mechanism of action, we evaluated
here the effect of P. niruri extract on the CaOx
crystallization process induced in vitro in human and
rat urine.
In vitro CaOx crystallization in human urine under
the conditions employed in the present study produced a
mixture of typical mono- and dihydrate CaOx crystals.
Crystalluria is a common event observed even in
Fig. 4 CaOx crystals observed
in human urine by light
microscopy in the presence (A)
and absence (B)ofP. niruri
extract. 10·, 100· and 400·
magnification
Fig. 5 CaOx crystals observed
in human urine 24 h after
crystallization by light
microscopy in the absence (A)
and presence (B)ofP. niruri
extract. 10·, 100· and 400·
magnification
377
non-stone forming ind ividuals, and these crystals are
predominantly of the COD form [9]. In the present
study, the major crystalline form found after CaOx
precipitation was COM (85%), prevailing over the COD
(15%) form. This discrepancy probably reflects the dif-
ferences between spontaneous CaOx nucleation in vivo
and induced precipitation in vitro. In spite of this dif-
ference, P. niruri extract induced an increase in the COD
fraction from 15% to 30%. It has been suggested that
COM has a stronger affinity for cell membranes than
COD [17, 30], and thus COM crystals may constitute a
form of higher potential risk for stone formation.
Moreover, the most common form of CaOx crystals
found in kidney ston es is COM [10], although many
stones contain both crystal forms. Thus, the presence of
P. niruri extract induced alterations in CaOx crystal
morphology, favoring the formation of the CaOx dihy-
drate (COD) form, which is less likely to bind to renal
cells [30].
Our results also showed that P. niruri extract did not
inhibit CaOx nucleation, but inhibited crystal growth,
since the size of the particles was significantly smaller
than that of the particles found in control samples. Also,
the aggregation of crystals was reduced in the samples
containing P. niruri extract. These properties may con-
stitute an important advantage in the prevention of
lithiasis, inhibiting calculus growth and keeping the
crystals dispersed in the urine, with their subsequent
easier elimination through the urine. The aqueous ex-
tract of P. niruri must contain substances that interfere
with these processes. Many active compounds have been
described in P. niruri [3, 13, 20, 24]; however, the isolated
effect of these compounds on CaOx crystallization have
not been tested. Recently, Atmani and Khan [1] showed
similar results of CaOx crystallization in vitro obtained
with Herniaria hirsuta, a plant from Morocco, but the
specific compound(s) involved in this protector mecha-
nism is not known.
Finally it is interesting to note that CaOx crystalliza-
tion did not occur in rat urine and thus no effect of P.
niruri extract was observed. In fact, spontaneous pre-
cipitation of calcium salts in vivo is a rare event in rats [6,
14] and even in induced urolithiasis in these animals,
apatite but not oxalate stones are observed [2, 14]. The
relatively alkaline rat urine (pH7.0) compared to
human urine (pH5.5) may have a role in the failure to
induce CaOx precipitation [6]. However even at an acidic
pH, spontaneous CaOx crystallization in rats may be a
sporadic event, even when oxalate is in excess in the urine,
because the rat urine has a high level of oxalate relative to
that of calcium [14] and thus a further increase in oxalate
concentration does not increase the level of supersatu-
ration by very much. In contrast, normal human urine
has a calcium concentration about 15 ti mes that of oxa-
late. Moreover, in the present study the pH was adjusted
to 6.0 in all rat and human urine samples, with the sub-
sequent elimination of pH as an interfering factor. Thus,
the presence of unknown, specific inhibitor molecules in
rat urine should be investigated.
In summary, we showed that P. niruri extract inter-
fered with the CaOx crystallization process by reduci ng
CaOx crystal growth and aggregation and that this ex-
tract favored the formation of a less adherent dihydrate
CaOx crystalline structure. These results contribute to
the accumulating evidence obtained over the last
10 years pointing to the beneficial effects of P. niruri on
many stages of stone formation and/or elimination [25],
including crystallization, aggregation, cellular adherence
[5] and adsorption of macromolecules into the calculi
[11]. Thus, P. niruri can potentially interfere with the
pathogenesis of urolithiasis and may represent an at-
tractive alternative for the prevention of lithiasis of the
urinary tract.
Acknowledgements We are indebted to Dr. A.J. Lapa, Pharma-
cology Department, Universidade Federal de Sa
˜
o Paulo, for pre-
paring the lyophilized form of Phyllanthus niruri. This study was
supported by Fundac¸ a
˜
o de Amparo a
`
Pesquisa (FAPESP), Coor-
denac¸ a
˜
o de Aperfeic¸ oamento de Nı
´
vel Superior (CAPES), Conse-
lho Nacional Cientı
´
fico Tecnolo
´
gico (CNPq) and Fundac¸ a
˜
o
Oswaldo Ramos.
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