Antiurolithiatic activity of selected plants extracts against calcium oxalate crystals

The present study was designed to evaluate the antiurolithiatic activity of selected plants extracts (Achyranthes aspera, Lawsonia inermis, Ficus benghalensis, Raphnus sativus and Macrotyloma uniflorum). The methanol extract of selected plants was analysed for in-vitro antiurolithiatic activity using nucleation, aggregation and growth assay of calcium oxalate (CaOX) monohydrate crystals. The nucleation, aggregation and growth of CaOx crystals were significantly inhibited by all selected plant extracts. The highest inhibition of CaOX nucleation was shown by R. sativus (55.21±1.9%) followed by M. uniflorum (53.91±1.1%) and the least by F. benghalensis (43.63±0.8%) at 1.0 mg/mL. The highest inhibition of calcium CaOX aggregation was shown by R. sativus (61.6±1.6%) which was very close to Cystone (63.28±2.5%). The growth of CaOX was highly inhibited by A. aspera (42.17±1.0%) followed by M. uniflorum (40.27±1.4%) with lowest inhibition by F. benghalensis (31.44±1.4%) as compared to Cystone (43.35 ±0.9%). The study showed that the selected plants showed the significant antiurolithiatic activity against CaOX crystals, which could be a potential source for the treatment of renal stone disease.


INTRODUCTION
Urolithiasis is the process of formation or deposition of calculi in the urinary tract, which is considered as the third most common disorder estimated to occur in around 12% of the global population worldwide (Sharma et al., 2016;Khan, 2013). The formation of calcified renal stone is a physiochemical event leading to crystal nucleation, aggregation and its growth assisted by many biological processes including urine volume, pH, increased calcium oxalate or sodium oxalate, and urates (Ratkalkar and Kleinman, 2011;Khan et al., 2016). At present, over 90% of upper urinary tract stones patients have been treated according to the size, type, and position of the stones with a success rate of 68 to 86% (Bahmani et al., 2016).
Increased dietary protein intake has been reported to elevate the rates of developing kidney stones and approximately 75% of all renal stones are composed of calcium oxalate and/or calcium phosphate (Stamatelou et al., 2003).
Technological progress has been remarkable in the treatment/management of kidney stones, but the associated complexities such as their tendency to increase recurrence, hemorrhage, hypertension, tubular nephrosis are a major limiting factor Patel and Shah).
Therefore, alternative or complementary medicine with minimum side effect might be useful. There is a growing public interest in herbal medicine for the management of urolithiasis in developed as well as developing country because of their wide biological and medicinal values, low toxicity and lesser costs (Sharma et al., 2016;Aggarwal et al., 2010). To date, most Nepalese people are unable to benefit from modern health care facilities for urinary stone treatment due to socio-economic factors (Lamichhane et al., 2017). Consequently, for the management of renal stone disease, they are relying on regionally available traditional herbal medicinal products (Gewali and Awale, 2008). The medicinal plants were used as principal ingredients in traditional medicine, available through tribal, home herbal remedy, and the Baidhya, Ayurveda and Amchi systems (Kunwar et al., 2010). Most of the plant-based medicines were used for the treatment of renal diseases (Bhattarai et al., 2010). Literature survey reveals that herbal formulation of plant-based medicine has been found to be successful in management of kidney stones in Nepal (Kumaran and Patki, 2011;Brardi et al., 2012).
Although these herbal medicines are popular in folk culture, the rationale behind their pharmacological and physiochemical antiurolithiatic efficacy has not been well elucidated. Only a few studies have suggested some evidence for the efficacy. In view of traditional and ethnomedicinal use, this study was designed to evaluate antiurolithiatic effects of selected plants, using methanol extracts in the in-vitro crystallization of calcium oxalate model.

Preparation of methanol extracts
Five fully mature plants (Table 1) were collected from the Rupandehi, Nepal and further authenticated by Professor Subodh Khanal, a Botanist at the Institute of Agriculture and Animal Sciences, Tribhuvan University. The voucher specimens of collected plants were deposited in the Pharmacognosy Laboratory, Department of Pharmacy, Universal College of Medical Sciences, Tribhuvan University, Nepal. The collected plants were dehydrated and pulverized. The grounded coarse powders (70 g) were immersed in methanol (350 mL) for 7 days at room temperature with frequent agitation. The extracts were filtered using a Buckner funnel and Whatman No. 1 filter paper. Each filtrate was concentrated to dryness in a rotary evaporator (Büchi Labortechnik, Germany) under reduced pressure and controlled temperature (40-50°C) to give final extracts, which was stored at 4°C in an airtight container until further use.

Experimental procedure
The effect of extract on CaOx crystallization was determined by the time course measurement of turbidity changes due to the crystallization in artificial urine on addition of 0.01 M sodium oxalate solution. The precipitation of calcium oxalate at 37°C and pH 6.8 was studied by the measurement of absorbance at 620 nm using UV/Visible spectrophotometer (Sharma et al., 2016).

Preparation of artificial urine
The artificial urine (AU) was prepared as per previously described method (Sharma et al., 2016) using the following composition: Calcium chloride dehydrates solution (10 mM) and sodium oxalate solution (1.0 mM), sodium chloride (200 mM) and sodium acetate trihydrate (10 mM). The AU was prepared fresh each time and pH adjusted to 5.7.

Nucleation and aggregation assay of CaOX crystals
The spectrophotometric assay method was used to determine the inhibitory activity of the extracts in the nucleation and aggregation of CaOx crystals (Sharma et al., 2016;Hess et al., 2000). Briefly, the calcium chloride dehydrate aqueous solution (10 mM) and sodium oxalate solution (1.0 mM), was diluted by a buffer of sodium chloride (200 mM) and sodium acetate trihydrate (10 mM) at pH 5.7 maintaining temperature at 37°C with circulating water bath. For Where Tsc = turbidity slope of the control and Tsi = turbidity slope in the presence of the inhibitor. All the experiments were performed in triplicate.

Growth assay
The inhibitory activity of the extracts on the CaOx crystals growth was determined by a spectrophotometric assay (Sharma et al., 2016;Aggarwal et al., 2010;Chaudhary et al., 2010). Briefly, calcium chloride solution (4 mM, 20 ml) and 4 mM sodium oxalate (4 mM, 20 ) mL were added to a buffer solution (30 mL) of sodium chloride (90 mM) and Tris HCl (10 mM) at pH 7.2 followed by the addition of calcium oxalate monohydrate (COM) crystal slurry (600 μl) prepared in acetate buffer (1.5 mg/mL). Consumption of oxalate commences immediately after the addition of the COM slurry, which was monitored at 214 nm for 600 s absorbance disappearance. Further, cystone or extract test solution (1 mL) at 0.5 or 1.0 mg/mL in water was added separately into the above solution and depletion of free oxalate ions was calculated by spectrophotometer as inhibition in calcium oxalate crystals growth using the following.
Inhibitory of crystal growth (%) = (C -S) /C) × 100 Where C = reduction of free oxalate without any extract; and S = reduction of free oxalate with inhibitor. All the experiments were performed in triplicate.

Statistical analysis
The data were reported as mean ± standard deviation. Statistical significance of the observations from nucleation aggregation and growth assay was calculated using one way analysis of variance test (ANOVA), followed by Dunnett's t-test, using GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA, USA). P<0.05 is considered as statistically significant level.

Nucleation and aggregation assay
The changes in the turbidity of different solutions (control, cystone and all five selected extracts at 0.5 and 1 mg/mL), were plotted at different time intervals. All the plants extracts at tested concentration showed the significant reduction (P<0.01) in nucleation and aggregation of CaOx crystals when compared with cystone. The methanol extract of R. sativus (55.21±1.9%) showed highest inhibition compared to cystone (57.7±1.1%) and F. benghalensis (43.63±0.8%) showed lowest inhibition of at 1 mg/mL (Figure 1). In aggregation assay, the extract of R. sativus (61.6±1.6%) showed highest inhibition compared to cystone (63.28±2.5%) and the extract of F. benghalensis (45.18±1.05%) showed lowest inhibition at 1 mg/mL (Figure 2). It was found that increasing the concentration of plant extracts resulted in the increase in percentage inhibition of calcium oxalate crystallization.

Growth assay
In calcium oxalate growth assay, all the plants extracts at tested concentration showed the significantly (P<0.01) inhibited calcium oxalate monohydrate (COM) growth when compared with cystone. For checking inhibitory activity on COM crystal growth, it was observed that inhibition increases with increase in time; probably the secondary metabolites present in extract interfere with the growth of calcium and oxalate onto the slurry and thus inhibits the growth process. Out of the five methanolic extracts, the extract of A. aspera (42.17±1.03%) showed highest inhibition compared to cystone (43.35±0.95%) and the extract of F. benghalensis (36.44±1.36%) showed lowest inhibition at 1 mg/mL (Figure 3). The increasing the concentration of plant extracts resulted in the increase in percentage inhibition of COM growth.

DISCUSSION
With effective ethnopharmacological value, Achyranthes aspera, Lawsonia inermis, Ficus benghalensis, Raphnus sativus and Macrotyloma uniflorum are widely used by different tribes as an effective herbal medicine (Acharya, 2012;Tamang et al., 2017;Malla et al., 2014). The aim of this study was to evaluate and compare the antiurolithiatic activity of selected plants extracts, to ascertain scientific rationales behind its ethnomedicinal practice. CaOx nucleation is a phase-change thermodynamic driven process in which dissolved substances spontaneously crystallize in a supersaturated solution. A comparable phase change and formation of CaOx crystals was observed during the nucleation assay, which can imitate the biological process. The nucleation of CaOx crystals was significantly inhibited in the presence of selected plants extracts, which was highly comparable to Cystone. This indicates the anti-crystallization property of selected plant extracts, which could be due to their ability to complex with free calcium and oxalate ions and prevent CaOx crystallization, as suggested for Daucus carota (Bawari et al., 2018).
COM, a CaOx polymorph, is most commonly found in urolithiasis, which is more stable with a more aggregatory and adhesive ability (Sheng et al., 2005). Thereby, COM generates large crystal aggregates which deposits heavily into the renal epithelial tissue, causing injuries. In this study, all the plant extracts inhibited the aggregation of COM, which could be due to presence of flavonoids, phenolic compounds, saponins and tannins, which was reported to inhibit urinary stone formation studies (Vargas et al., 1999;Asif et al., 2014). Moreover, CaOx polymorph growth is a marker of deposition event for crystal forming ions in the supersaturated solution. This incidence of CaOx crystals growth was also monitored in the current study. All the selected plant extracts showed growth inhibitory activity. Natural extracts shows a varied range of phytochemicals of which tannins, polyphenols, flavonoids alkaloids and saponins are most abundance. There are reports that flavonoids inhibit calcium oxalate crystallization in human urine as well as in animal models (Zhong et al., 2012) as well as crystal deposition (Noorafshan et al., 2013). Saponins showed anticrystallization properties by disaggregating the suspension of mucoproteins, which are the promoters of crystallization (Gürocak and Küpeli, 2006). In this study, maximum antiurolithiatic activity may be apparent due to flavonoids, saponins, alkaloids, polyphenols that may be present in higher quantity and also by CaOx crystal inhibition, diuretic, hypermagneseuric and antioxidant effects which are reported by previous studies (Vargas et al., 1999;Asif et al., 2014). Many literatures report reveals that the physical (agitation, temperature, and supersaturation) and chemical (concentration of acidic or basic additives) conditions of urine are responsible for precipitation of calcium oxalates (Šter et al., 2018). The overall condition could be modified by phytochemicals from selected plant extracts to inhibit crystallization.

Conclusion
Out of selected plants, R. sativus possessed comparable antiurolithiatic activity to cystone in nucleation and aggregation and A. aspera in growth assay. The result of the study showed that the selected plants possess antiurolithiaic activity, which was directly proportional to the concentration of the extracts. These findings substantiate the traditional use of the plants in the treatment of urinary stones and kidney problems.