Urolithiasis is a multifaceted urologic condition that requires frequent emergency department visits and immediate urological intervention.1 Technological advancement witnessed for the removal of stones has been outstanding, but the associated complexities like, their tendency to enhance stone recurrence is a major limiting factor.2
Development of plant-based medicine as alternative or complementary to the conventional system of medicine have drawn tremendous attention and serves as an immense source of new drug entities.3 Based on these grounds, carrots (Daucus carota L., family Apiaceae) were selected for the present study. Carrots are considered functional food and are widely cultivated all around the globe as well as are extensively exploited commercially. Carrots are of high nutritional value and are a rich source of phenolics, polyacetylenes and carotenoids.4 Despite their established nephroprotective role5 as well as traditional claims for their antilithic potential,6 no screening has been carried out so far to establish the antiurolithiatic efficacy of D. carota. Therefore, current investigation was carried out for the screening of the antilithic potential of D. carota against calcium oxalate crystallization in an in vitro setting.
MATERIALS AND METHODS
Daucus carota L. roots (carrots) were procured from a grocery store of Bhimtal, Uttarakhand and were identified and authenticated from Botanical Survey of India (BSI), Dehradun. A voucher specimen of D. carota (accession number 116593) was also deposited in the herbarium of BSI.
Processing and extraction
D. carota roots were cut into thin slices, shade dried, and finely powdered. Cold maceration method was employed for the extraction of powdered carrot roots with 70% v/v ethanol. D. carota root extract (DCRE) thus obtained was dried in a rotary evaporator under reduced temperature and pressure.7
Preliminary phytochemical screening
Preliminary phytochemical evaluation of DCRE was carried out for qualitative estimation of phytoconstituents.8
Effect of DCRE on calcium oxalate (CaOx) crystal formation was determined by means of nucleation assay. Calcium chloride (CaCl2) (5 mmol/l) and sodium oxalate (Na2C2O4) solution (7.5 mmol/l) were prepared in Tris-HCl (0.05 mol/l) and NaCl (0.15 mol/l) buffer (pH 6.5). Dilutions of DCRE ranging from 100-1000 µg/ml were prepared in distilled water. One milliliter of each DCRE concentration was mixed with 3 ml CaCl2 solution followed by the addition of 3 ml Na2C2O4 solution. Final mixtures were incubated for 30 min at 37ºC. The optical density (OD) of the mixtures was then measured at 620 nm wavelength. Percent inhibition of nucleation by DCRE was calculated using the under mentioned formula and compared to that calculated for the standard polyherbal drug, Cystone.9
Number, size and morphology of CaOx crystals formed in absence or presence of DCRE were determined using a Leica DM 2500 LED microscope at 1000× magnification.
Effect of DCRE on CaOx crystal aggregation was determined by means of aggregation assay. CaCl2 and Na2C2O4 solutions (50 mmol/l each) were mixed together, heated to 60 ºC in a water bath for 1 h and then incubated overnight at 37ºC to prepare seed CaOx crystals. After drying, CaOx crystal solution (0.8 mg/ml) was prepared in a 0.05 mol/l Tris-HCl and 0.15 mol/l NaCl buffer (pH 6.5). One milliliter of aliquots (100-1000 µg/ml) of DCRE were added to 3 ml CaOx solution, vortexed and then incubated at 37ºC for 30 min. OD of the final mixtures was then read at 620 nm wavelength and percent inhibition of aggregation was then calculated as described for nucleation assay.9
Oxalate depletion assay
Effect of DCRE on the growth of CaOx crystals was determined by oxalate depletion assay. Varying concentrations of DCRE (100 µg/ml, 500 µg/ml and 1000 µg/ml) were prepared in distilled water. CaOx crystal slurry at a concentration of 1.5 mg/ml was prepared in a 50 mM sodium acetate buffer (pH 5.7). 4 mM CaCl2 solution and 4 mM Na2C2O4 solution (1 ml each) were added to 1.5 ml Tris-HCl (10 mM) and NaCl (90 mM) buffer (pH 7.4). To this was added 30 µl of CaOx crystal slurry. The growth of CaOx crystals was then determined by measuring the rate of oxalate depletion from the solution at 214 nm wavelength for 600 s. Effect of each concentration of DCRE on crystal growth was then determined by addition of 1 ml of DCRE (100 µg/ml, 500 µg/ml and 1000 µg/ml) to the reaction mixture and change in OD was again recorded. Percent inhibition of crystal growth was then calculated as described for nucleation assay.10
CaOx crystal characterization by FT-IR
FT-IR analysis was used to confirm the CaOx crystals prepared in vitro using attenuated total reflectance (ATR) technique.
Quantitative results of all the experiments performed in triplicates were expressed as mean ± S.E.M. (Standard Error of Mean). Statistical computations were performed on GraphPad Prism 6 software using one-way analysis of variance (ANOVA) followed by Tukey Kramer’s multiple comparison test. P values less than 0.05 were considered statistically significant.
Preliminary phytochemical screening
Percent yield of DCRE was 23.73%. Qualitative phytoconstituent determination in DCRE showed the presence of carbohydrates, saponins, flavonoids, tannins and phenols.
Addition of Na2C2O4 solution to the reaction mixture consisting of CaCl2 resulted in the formation of numerous CaOx crystals. Presence of DCRE (1000 µg/ml) in the reaction mixture produced a percent reduction in nucleation of 56.1±1.55% which was significantly higher (P<0.01) than that produced by Cystone (41.67±1.03%) (Figure 1).
Microscopy of CaOx crystals
Numerous large CaOx monohydrate (COM) crystals of either rectangular habit or dendrites with sharp edges were predominant in the control group. DCRE at higher concentrations (Figure 2) and Cystone from lower concentrations itself (Figure 3) favored the formation of tetrahedral shaped calcium oxalate dihydrate (COD) crystals with smoother morphology. DCRE and Cystone also reduced the size and number of CaOx crystals. Percent reduction in size of CaOx crystals produced by DCRE (62.34%) was comparable to that produced by Cystone (70.10%) (Figure 4). Number of CaOx crystals was reduced to a far greater extent by DCRE (84.71%) than Cystone (58.51%) (Figure 4).
DCRE produced a significant reduction (P<0.0001) in aggregation of preformed CaOx crystals. Percent reduction in aggregation produced by DCRE was found to be 49.94±2.07% comparable to that of Cystone (57.15±2.53%) at 1000 µg/ml concentration (Figure 1).
Oxalate depletion Assay
Percent reduction in growth in the presence of DCRE was found to be 48.58±0.88% while, 57.44±3.7% with Cystone at 1000 µg/ml. CaOx crystal growth inhibitory effect of DCRE was significantly lower than Cystone (P<0.001) at lower concentrations but was comparable to Cystone at highest concentration (Figure 1).
FT-IR characterization of CaOx crystal
FT-IR analysis of CaOx crystals produced seven bands (Figure 5). Spectrum showed a broad O─H stretching band at 3330.42 cm-1; a strong band at 1614.47 cm-1 representing C═O; another band with high absorbance at 1316.74 cm-1 representing C─O stretch; C─H bending at 778.65 cm-1; out-of-plane O─H bending at 651.68 cm-1; and O─C═O in-plane bending at 511.79 cm-1 as has also been reported in previous studies.11-12
CaOx urolithiasis is the most prevalent type of all urinary stone diseases. Key events involved in its pathological biomineralization include crystal nucleation, growth and aggregation.13 Present study was designed to address these key events involved in CaOx stone formation as a means to investigate the efficacy of D. carota roots as an antiurolithiatic.
Nucleation is a prerequisite in the pathogenesis of CaOx urolithiasis. Nucleation basically marks a thermodynamically driven event of phase change wherein dissolved substances in a supersaturated solution spontaneously crystallize.13-14 Similar phase change and formation of CaOx crystals was witnessed while carrying out nucleation assay. Significant inhibition in the nucleation of CaOx crystals was observed in the presence of DCRE which was even better than in the presence of Cystone. This suggests the anticrystallization activity of DCRE against CaOx crystallization. One possible mechanism of anticrystallization activity of DCRE could be its ability to complex with free calcium and oxalate ions, thus preventing the formation of CaOx complexes, as has also been suggested for Sarghassum wightti.15
CaOx polymorphism is a common phenomenon and of utmost significance in urolithiasis. COM and COD crystals are commonly found in CaOx uroliths.16 Of the two polymorphs, COM is thermodynamically more stable with more aggregatory and adhesive tendency. Hence, COM tends to form large crystal aggregates and adheres strongly to renal epithelial tissue, injuring the same. Therefore, of the two polymorphs, COM significantly promotes crystal retention and eventual stone formation.17 Therefore, a transformation from COM to COD is advocated as a crucial step in inhibition of calculi formation.16 In present study, DCRE also promoted transformation of pointy edged dendritic COM crystals to smoother edged COD crystals of extremely reduced size and number. Reduction in size of CaOx crystals is critical as smaller crystals tend to spontaneously pass out in urine.18
Growth of CaOx crystals marks the event of deposition of crystal forming ions present in the supersaturated solution on preformed CaOx crystal lattice.13,19 This event of growth of CaOx crystals was also tracked in the present study. DCRE exhibited growth inhibitory activity as was also confirmed from the crystals of reduced size produced in the presence of DCRE.
Aggregation of crystals marks the process wherein numerous crystals in the solution come together and adhere forming large crystal agglomerates. Aggregation is a key determinant of crystal retention as large crystal agglomerates are the ones that produce renal tubular obstruction thereby promoting stone formation.13 DCRE showed significant inhibitory effect on CaOx crystal aggregation.
Qualitative phytochemical estimation of DCRE revealed the presence of flavonoids, phenolic compounds, saponins and tannins. These phytoconstituents are of utmost significance for inhibiting urinary stone formation. Saponins possess antilithic properties9 and are known to disintegrate mucoproteins that are crucial components of stone matrix.20 Tannins and polyphenols inhibit CaOx crystal formation as well as dissolve the preformed CaOx crystals by aiding calcium complexation.21 Flavonoids also possess CaOx crystal dissolution potency. Phenolics and flavonoids exhibit antioxidant activity.20 Therefore, the anti-crystallization, anti-aggregatory and crystal growth defying activity of DCRE would have been an outcome of these phytoconstituents present in DCRE.
As COM crystals are the most predominant form of all the polymorphs of CaOx found in kidney stones, formation of COM crystals was a mandatory requirement to test the efficacy of DCRE against COM crystallization. In order to confirm the formation of COM crystals to be utilized for aggregation and growth assays, CaOx crystal characterization was carried out by FT-IR spectrophotometry. Formation of COM crystals was confirmed from the sharp peak at 778.65 cm-1 and a comparatively broader peak at 651.68 cm-1 which are characteristic of COM crystals. Whereas, a sharp peak at about 910 cm-1 is known to be specific to COD crystals.22 In addition, peaks at 3330.42, 1614.47, 778.65 and 511.79 cm-1 that were prominent in the FT-IR spectrum of CaOx crystals closely correlate to the typical COM identification bands.23
It is true that in vitro data cannot be simply extrapolated to infer results of more complex in vivo systems, but in vitro studies give an insight into the activity related efficacy of tested compounds or extracts. Present study also demonstrated prominent inhibitory activity of DCRE against CaOx crystallization.
Findings of the present study clearly demonstrate antiurolithiatic potential of DCRE against CaOx urolithiasis in vitro. DCRE showed prominent inhibition of all the phases of CaOx stone formation viz. nucleation, growth and aggregation, and favored the formation of more amenable COD crystals. Although further in vivo and clinical explorations are required to confirm the efficacy of D. carota as an antiurolithiatic, still, considering the vast consumption and availability of D. carota all around the globe together with its antilithic potential, D. carota could serve as an easily accessible and beneficial alternative or adjunctive treatment for CaOx urolithiasis.