Introduction

In recent years there has been a resurgence of interest in medicinal plants that are effective, safe and culturally acceptable as an alternative treatment for many human diseases [1]. About 25% of the drugs prescribed worldwide come from plants [2]. Phyllanthus niruri (P. niruri) is a plant belonging to the Euphorbiaceae family, which has a worldwide distribution. It is used in Brazilian folk medicine by patients with urolithiasis [3, 4]. Previous 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, as well as the normalization of calcium levels in hypercalciuric patients [5]. Experimental studies have shown that P. niruri reduced the uptake of calcium oxalate crystals by MDCK cells, without evidence of cytoxicity or biochemical alterations of the culture medium [6]. Moreover, it prevented the growth of calculi in a model of CaOx-induced urolithiasis in rats [7]. Finally it interfered with the CaOx crystallization process in vitro by reducing crystal growth and aggregation and favored the formation of a less adherent dihydrate CaOx crystalline structure [8]. Therefore, all these effects are related to the preventive potential of P. niruri. In the present study, we evaluated the effect of P. niruri treatment simultaneously or 30 days after the introduction of a CaOx seed into the bladder [9]. We also evaluated the mineral constitution and chemical composition of the stone formed with and without P. niruri treatment.

Materials and methods

An aqueous extract, as used in the popular medicine of P. niruri, was obtained from the whole plant, which was grown at the Experimental Center of the Estadual University of Campinas, São Paulo. Plant samples were cut and dried at 50°C for 2 months in a ventilated room. After drying, plants were ground in a mechanical mill and used for infusion preparation (5% w/v). The infusion was stirred for 30 min at 72°C and then vacuum filtered, concentrated, and lyophilized. P. niruri was administered (20 μg/g body weight/day) by gavage, diluted in 1 ml of distilled water.

CaOx seed

To prepare the CaOx seed, small disks of CaOx were obtained by a supersaturation reaction, as described previously [10]. Briefly, 100 ml of calcium chloride (0.4 mol/l) and 100 ml of potassium oxalate (0.4 mol/l) were mixed together by constant drop-wise addition to 300 ml of distilled water for 2 h with shaking at 75°C. The mixture was maintained under agitation at 75°C for an additional 5 h. Crystals were washed with distilled water to eliminate the excess of chloride and potassium, and the formed crystals were maintained in an oven at 37°C for 2 weeks to allow aggregation and to form a compact CaOx seed. The resultant material was transferred to a template containing cylinders of 4 mm diameter to obtain small disks of CaOx. Disks were weighed and sterilized before use.

Urolithiasis model

Urolithiasis was induced by introducing a CaOx disk (seed) into the bladders of adult male Wistar rats. Briefly, the bladder was exposed through a suprapubic incision under ether anesthesia and a CaOx seed was then introduced into the bladder. After suturing the bladder wall, muscle, and skin, the animals were maintained in individual cages for 24 h for observation. All animals were allowed free access to regular rat chow and tap water. The animals were then housed in cages (four animals/cage), in their respective groups (experimentals/control). The ethics committee of the São Paulo Federal University (no. 0957/02) approved the experimental protocol.

Experimental protocol

Animals were divided into six groups: C (n = 8): control animals, without treatment; CPn (n = 8): control animals treated with P. niruri (Pn); CaOx (n = 5): animals with CaOx seed with no treatment. Animals were sacrificed 50 days after the seed implantation: CaOxPn (n = 4): animals with CaOx seed receiving Pn treatment during 50 days starting on the day of surgery (early treatment); CaOxPn20 (n = 5): Pn treatment was initiated 30 days after surgery (late treatment), and lasted 20 days; CaOxPn40 (n = 5): Pn treatment was initiated 30 days after surgery and lasted 40 days. It was previously observed, in a pilot study, that a period of 30 days was sufficient for the development of calculi: thus, this period was adopted to start the P. niruri treatment. At the end of the protocol, animals were placed in metabolic cages (Nalgene, NalgeNunc Int., USA), to collect 24 h urine samples. After urine collection, animals were ether anesthetized for blood sampling (obtained from the aorta) and then killed by air injection. The bladder was exposed and the matrix calculus and satellites were removed, washed with distilled water, weighed, and stored. Biochemical determinations included urinary and plasma concentrations of sodium, potassium (photometry, Pegasus II, Tecnow, Brazil), calcium (atomic spectrophotometry), uric acid, magnesium, and creatinine (Labtest Diagnostics, Brazil).

The calculi were photographed by stereomicroscopy (Carl Zeiss, model Stemi SV11), and further analyzed by scanning electron microscopy (SEM). The compositions of calculi were obtained by X-ray diffraction (XRD) using a Siemens/Brucker model D5000 diffractometer (Germany) with Cu-Kα radiation (λ = 0.15437 nm) at 40 kW and 40 μA (step size: 0.05°θ: time per step: 1 s), and analyzed by computer program (Evaluation Diffract Plus, 2001, France).

Data are expressed as means ± standard error. Statistical analysis was carried out using ANOVA (SigmaStat for Windows version 2.0), followed by the Tukey or Dunn’s tests when necessary. Parametric Student t-test was used when appropriate. Differences between the data were considered significant at P < 0.05.

Results

Biochemical analysis of blood and urine did not reveal significant alterations among groups (data not shown), thus the presence of calculus or P. niruri treatment did not induce detectable alterations in the animal metabolism or urinary excretions.

Figure 1 shows representative photographs of the calculi removed from the different groups. For comparison, panel A shows a CaOx seed before implantation into the bladder. The calculi removed from the untreated CaOx group consisted of a main calculus and many satellites, which were rigid and had spicule-shaped aspects (Fig. 1b). In contrast, the material (calculi) obtained from a CaOx animal treated early with P. niruri consisted of the CaOx seed covered by a soft material, probably an organic matrix (Fig. 1c). The animals of group CaOxPn20 (Fig. 1d) had a larger number of larger sized satellites. However, there was a clear difference in the shape of the calculi, they had a more regular surface compared with those in the untreated CaOx group.

Fig. 1
figure 1

Optical microscopy of the calculi. a CaOx seed. b Main calculus and satellites obtained from untreated CaOx animals. c Main calculus coated with an organic material from a P. niruri treated CaOx animal. d Main calculus and satellites from a CaOx animal treated with P. niruri in the presence of preformed calculi. The difference in calculus shape between untreated CaOx and P. niruri treated animals is clear

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Table 1 summarizes the data concerning number of calculi and mass. The mean mass of CaOx disks introduced into the bladder was similar among groups. As previously reported for this experimental model, the CaOx seed served as a nidus where organic and inorganic material precipitated, allowing the growth of a main calculus, and satellites. Fifty days after the introduction of CaOx seeds, the resulting calculi were, on average, 25-fold larger in mass than the original seed (0.3283 ± 0.1350 vs. 0.0131 ± 0.0004 g, P < 0.05). CaOx animals treated early with P. niruri exhibited a significant reduction in both calculus mass and satellite number compared with untreated CaOx animals. However, P. niruri treatment initiated after growth of calculi induced a further increase both in calculus mass (Fig. 2) and in the number of satellites.

Table 1 Analyses of calculi
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Fig. 2
figure 2

Weight of calculi. Values represent the sum of the main calculus and the satellites. Horizontal bars indicate the median. P < 0.05: ⊗versus CaOx: ∇versus CaOxPn

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We were surprised to find that P. niruri treatment initiated in the presence of preformed calculi did not minimize the growth of calculi. However, P. niruri clearly induced critical changes in calculus shape. These changes varied from a flattening of the edges of spicules to a complete smoothening of calculi surfaces. To observe better this effect, we next analyzed the calculi by stereomicroscopy and SEM, which allowed us to evaluate the calculi in more detail. Figure 3a shows a typical calculus and satellites grown in CaOx animals without treatment, all of them exhibited an irregular surface and a spicule shape. Group CaOxPn (Fig. 3b) exhibited almost no satellites, when present, however, they had an appearance of a predominantly organic, non-crystalline material. The calculi, when found in this group, were smaller, and had a less irregular surface compared to those found in the CaOx group with no treatment. P. niruri treatment initiated in the presence of a preformed calculus (groups CaOxPn20 and CaOxPn40, Fig. 3c, d, respectively) induced a clear difference in calculus surface observed with more detail under SEM (Fig. 4b, c) compared to the CaOx group (Fig. 4a). The spicule flattening or the smoother surface of calculi observed after P. niruri treatment on the preformed calculi can be clearly observed in Fig. 4c and d.

Fig. 3
figure 3

Stereomicroscopy of the calculi taken from two representative animals (upper and lower panels) of each group. a Calculi with a homogeneous spicular shape from untreated CaOx animals. b Calculi from CaOx animals treated with P. niruri from the first day after CaOx seed implantation showing smaller calculi and very few satellites, mostly composed of an amorphous material (arrows). CaOx animals treated with P. niruri after growth of calculi for 20 (c) or 40 days (d). The variability in the response to P. niruri treatment is clear in (c). The upper panel shows calculi with a smooth surface and the lower panel shows multiple fragments of a probably more fragile calculus

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Fig. 4
figure 4

Scanning electronic microscopy (SEM) of the calculi taken from CaOx untreated and late treated animals during 20 days (CaOXPn20) or 40 days (CaOxPn40). a Pictures show a general visualization of the calculi. P. niruri treated animals exhibited calculi with more regular shapes. b Images show a panoramic view of the calculus surface. c Presents the lateral view of the calculus surface in greater detail. It can be seen that the spicules are more flattened and have a smoother surface in groups treated with P. niruri. d Shows a frontal view of the calculus surface. The differential crystal deposition in calculi from P. niruri treated compared to untreated animals is clear

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A summary of our XRD results is presented in Table 2. Main calculi exhibited the presence of both COM (nidus) and struvite (precipitated material) and the satellites presented only struvite.

Table 2 Inorganic composition of the calculi analyzed by XRD
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Figure 5 shows a main calculus from an untreated CaOx animal, which was fragmented. A different material (struvite) was deposited on the CaOx seed, which acted as a central nidus.

Fig. 5
figure 5

SEM showing the central nidus consisting of the CaOx seed and the surrounding material formed by struvite

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Discussion

The experimental model of urolithiasis used in the present study was induced by a vesical foreign body (CaOx seed) implantation method that was obtained with no significant metabolic or systemic alterations. The vesical CaOx seed acts as a supporting surface, allowing organic, and inorganic material to precipitate over the central nidus, thereby mimicking a spontaneous calculus growth. Furthermore, the functional parameters of renal function, urinary pH, and urinary solute excretions were not significantly modified in this model.

The present urolithiasis model revealed that a main calculus grew over the CaOx seed together with a variable number of satellites. Since this model utilized rats, the deposited inorganic material consisted of magnesium ammonium phosphate, resulting in struvite calculi. However, the central nidus consisted of pure CaOx crystals. This result was expected since the alkaline nature of rat urine propitiates the precipitation of struvite and not CaOx crystals [11].

Results obtained with X-ray diffraction analysis confirmed the presence of both CaOX and struvite in the main calculi, and only struvite in the satellites, this suggests that the satellites probably grew on small fragments released from the main stone and acted as struvite seeds. Moreover, struvite was the main crystalline chemical composition found in all groups, both treated and untreated with P. niruri, indicating that the effects of P. niruri on the growth of calculi were not related to alterations in the inorganic elements of the calculi.

Two important effects of P. niruri were found in the present study. First was its ability to prevent the growth of calculi when the treatment was initiated soon after CaOx seed implantation. Second was the drastic modification in the shape and texture of the preformed calculus and after 20 or 40 days of P. niruri treatment the resultant stones exhibited different features. The early P. niruri treatment, initiated on the first day after seed implantation, caused a significant inhibition in the growth of calculi, indicating a potential prophylactic effect of P. niruri. This result confirmed previous data from our laboratory using the same animal model and experimental protocol [7] and suggests that P. niruri may have a potential inhibitory effect on the development of urinary calculi, probably by hindering the deposition of crystalline material on the CaOx seed. In contrast, P. niruri treatment initiated 30 days after CaOx seed implantation (late treatment), and thus in the presence of a preformed calculus, did not prevent the growth of further calculi, but rather induced substantial variations in the spatial arrangement of crystals, resulting in stones with a distinct shape and texture compared to untreated CaOx animals. These differences were analyzed in greater detail by SEM, the results of which revealed that an irregular precipitation of crystals was responsible for the spicule-shaped surface of the calculi taken from untreated CaOX animals. The calculi raised in P. niruri treated animals exhibited an anomalous crystal deposition with a filling of the spaces between the spicules, resulting in a more homogeneous and compact surface. We have previously observed that P. niruri interfered in the CaOx crystallization process induced in isolated human urine by reducing both crystal size and aggregation, and also by favoring the formation of a less adherent dihydrate CaOx crystalline structure [8]. Taken together, these results suggest that P. niruri probably interferes in the biomineralization process by promoting a different interaction between the crystal and the macromolecules of the organic matrix. It is well accepted that this interaction greatly controls crystal nucleation, size, morphology, structure, and growth rate [12–14].

The interaction between matrix proteins and crystals ensures that only crystals of a particular composition are formed, and guarantees that their various faces will grow only in certain directions to produce a structure of defined shape [15, 16]. On the other hand, the mechanisms involved in the pathological mineralization, as observed in urinary stone formation, are not well understood. However, it has become clear in recent years that the interaction between the matrix macromolecules and the nascent crystal may determine the crystal growth rate and morphology [17]. It has been demonstrated, for example, that the different isoforms of nephrocalcin induce different effects on CaOx mineralization [17]. The A and B isoforms of nephrocalcin appear to coat CaOx crystals, leaving their hydrophobic faces exposed, thereby inhibiting further mineralization. In contrast, while the C and D isoforms also coat the crystals, they promote aggregation of multiple crystals by exposing their hydrophilic face [17]. We did not analyze the organic matrix composition in the present study, however, we found in a previous study that used the same model of urolithiasis that calculi taken from CaOx animals treated with P. niruri from the first day after the seed implantation showed smaller and softer calculi with a higher incorporation of the matrix macromolecules glycosaminoglycans (GAGs) in the calculus structure [7]. It has been suggested that P. niruri-induced retardation of calculus growth was related to an increased incorporation of GAGs into the calculus, which acted to reduce the rate of calculus growth [7]. It is not presently clear if the more regular and smooth surface of the stones in animals with preexisting calculi was also the result of different matrix deposition induced by P. niruri.

We found some variability in both the shape and texture of calculi even among animals of the same group receiving the late treatment for 30 or 40 days. Some animals formed stones with a smooth surface (three animals), whereas others formed a less smooth surface, but flattened spicule-shaped stones and a more fragile appearance (two animals). The heterogeneity of the stones raised in this group indicates variability in the biological response to P. niruri treatment. Although the alterations induced by P. niruri were visible in all animals with preexisting calculi, further analysis of larger populations of calculi will be required to further explore this effect.

It is not clear at this time which substance(s) present in P. niruri would be responsible for this effect. More than 50 compounds were identified in P. niruri, including alkaloids, flavanoids, lignans, and triterpenes [18]. Among these substances, the triterpenes have been found to inhibit the cytotoxicity induced by calcium oxalate [19], they are also known to reduce excretion of stone forming constituents [20] and the markers of crystal deposition in the kidneys [21]. These findings point to an antilithiasic activity of these compounds, however, their effect on the growth and shape of crystals warrants further investigation.

The present study corroborates previous data suggesting the potential prophylactic effect of P. niruri [6–8] on the growth of calculi when the plant extract was administered before calculus development. The novel data shown here is related to the potential therapeutic effect of P. niruri on previously formed vesical calculi. Indeed it was clear that P. niruri did not prevent the growth of preexisting calculi, however, it certainly interfered with crystal deposition and substantially modified stone shape and texture. This finding raises the possibility for an alternative use of P. niruri, namely, to induce changes in calculi that might aid in elimination and/or dissolution of calculi.

Although the effect of P. niruri must be further evaluated in lithiasic patients, the results obtained in the present study suggest that P. niruri may have useful therapeutic applications in patients who already have stones, while it might have a prophylactic role in persons who are at high risk but have not yet developed stones.