In Vitro Evaluation of Ethyl Acetate Extract of Tridax Procumbens On Calcium Oxalate Crystal Dissolution: A Potential Anti?Urolithiatic Agent

Urolithiasis, characterized by the formation of calculi within the urinary tract, is a significant global health problem due to its high prevalence, painful clinical presentation, and tendency to recur. Epidemiological data indicate that calcium-containing stones, especially calcium oxalate (CaOx), represent the majority of urinary calculi worldwide. The pathogenesis is multifactorial and involves urinary supersaturation, nucleation, crystal growth, aggregation, and retention within renal tubules. While modern management includes hydration, medical expulsive therapy, and surgical options such as shock wave lithotripsy, pharmacologic dissolution of established calcium oxalate monohydrate (COM) stones remains limited. Herbal medicines have long been explored for their litholytic and antiurolithiatic potential. Phytochemicals like flavonoids, saponins, and polyphenols have been implicated in modulation of crystal formation through mechanisms such as chelation of divalent cations (reducing free Ca2+), adsorption onto crystal faces (altering growth habit), antioxidant protection of renal epithelium (reducing sites for crystal attachment), and surfactant effects that impede aggregation. Tridax procumbens, a pantropical weed used in traditional medicine, is rich in flavonoids and saponins and has reported antioxidant and anti?inflammatory properties that could be relevant to urolithiasis prevention. Although there are a few scattered reports on the in vitro antiurolithiatic activity of T. procumbens and other ethnobotanical remedies, systematic in vitro evaluation with standardized controls remains sparse. This study aims to investigate the ethyl acetate fraction of T. procumbens—expected to be enriched in flavonoids and moderately polar saponins—for its ability to inhibit nucleation and aggregation of COM crystals in vitro, using Cystone® as a positive herbal control.

MATERIALS AND METHODS

Plant material and authentication: Fresh aerial parts of Tridax procumbens were collected from the campus vicinity of PRMSS Anuradha College of Pharmacy, Chikhli. Specimens were authenticated by the departmental botanist and a voucher specimen (TP-2025-AP) was deposited in the institutional herbarium.

Preparation of ethyl acetate extract (EAE): Dried aerial parts were powdered (500 g) and macerated with ethyl acetate (2.5 L) for 72 h at room temperature, with intermittent shaking. The combined filtrate was concentrated under reduced pressure to yield the ethyl acetate extract (EAE). The extract was dried, weighed, and stored at 4 °C until use. Percentage yield was calculated relative to dried plant material.

Phytochemical screening and standardization: Qualitative tests for flavonoids, saponins, tannins, terpenoids, and phenolics were performed using standard chemical tests. Total flavonoid content (TFC) was estimated by the aluminium chloride colorimetric assay and expressed as quercetin equivalents (QE) per gram of extract.

Synthesis of calcium oxalate monohydrate (COM): COM crystals were synthesized by mixing equimolar solutions of CaCl2 and Na2C2O4 under controlled stirring at 25 °C. The precipitate was washed repeatedly with distilled water and dried at 40 °C for 24 h. The crystalline phase was confirmed by visual microscopy and previous standard protocols were followed.

Preparation of test solutions: EAE was dissolved in minimal DMSO and diluted with Tris–HCl buffer (pH 6.5) to obtain final concentrations of 100, 200, 300, 400, and 500 µg/mL. Final DMSO concentration in assays was <0.5% (v/v), which did not affect crystallization. Cystone® (Himalaya Drug Company) was prepared as a 250 µg/mL solution for comparative testing.

Nucleation assay (turbidity method): The nucleation assay followed established spectrophotometric protocols. Equal volumes of 4 mM CaCl2 and 0.5 mM Na2C2O4 in Tris–HCl buffer (pH 6.5) were mixed at 37 °C in a quartz cuvette containing test extract or control. Absorbance at 620 nm was measured at t = 0 and then every 5 minutes for 30 minutes. Increase in turbidity reflects nucleation; percent inhibition was calculated as: [(A_control − A_test)/A_control] × 100 at 30 min. Each experiment was performed in triplicate.

Aggregation assay: Aggregation of preformed seed COM microcrystals was monitored by measuring optical density (OD) changes at 620 nm. Seed crystals (0.5 mg/mL) were suspended in buffer and incubated with test extract or control at 37°C. OD was recorded at 0, 10, 20, and 30 minutes. Aggregation inhibition was calculated using a similar formula to nucleation and presented as percent inhibition at 30 minutes.

Mechanistic assays: Calcium chelation was inferred by incubating extract with known Ca2+ concentration and measuring residual Ca2+ colorimetrically (o-cresolphthalein complexone method). Surface tension of extract solutions was measured using a tensiometer to evaluate surfactant-like behavior suggestive of saponins.

Statistical analysis: Data are presented as mean ± SD (n=3). One-way ANOVA followed by Tukey’s post-hoc test was used to assess significance. A p-value < 0.05 was considered statistically significant.

RESULTS

The ethyl acetate extract (EAE) of Tridax procumbens demonstrated concentration-dependent inhibition of both nucleation and aggregation of calcium oxalate monohydrate crystals. Results (mean ± SD, n=3) are summarized below.

Table 1: Nucleation assay results

Figure 1: Concentration-dependent nucleation inhibition by EAE of Tridax procumbens. Dashed line denotes Cystone® (250 µg/mL) control.

Table 2: Aggregation assay results

Figure 2: Concentration-dependent aggregation inhibition by EAE of Tridax procumbens. Dashed line denotes Cystone® (250 µg/mL) control.