In-vitro Evaluation of Anti-Urolithiatic Activity of Hydroalcoholic Extract of Centella asiatica Leaves

Dr.Sundhararajan R, Mrs.K. Gayathri*, S. Madhan, Mohamed Sameer Akthar M,

Mohamed Absar A, Jamil Rahman. J

Mohamed Sathak A.J. Collage of Pharmacy, Chennai, Tamil Nadu.

*Correspondence: vanigayu1905@gmail.com

DOI: https://doi.org/10.71431/IJRPAS.2026.5902

INTRODUCTION

Urolithiasis, commonly known as urinary stone disease, is one of the most prevalent disorders of the urinary tract, characterized by the formation of solid crystalline masses within the kidneys, ureters, bladder, or urethra [1]. It may cause renal colic, haematuria, dysuria, urinary tract obstruction, infection and, in severe or recurrent cases, impairment of renal function. Reported prevalence ranges from approximately 1% to 20% depending on geography, climate, diet and genetic susceptibility, and nearly half of affected individuals experience recurrence [2,3]. Calcium oxalate is the most common stone constituent, and its formation follows a multistep process of urinary supersaturation, nucleation, crystal growth, aggregation and retention within the renal tubular epithelium [3,4].

Current management includes conservative therapy, medical expulsive therapy, pharmacological prevention, and interventional procedures such as extracorporeal shock wave lithotripsy, ureteroscopy and percutaneous nephrolithotomy. While effective for stone removal, these approaches are limited by cost, invasiveness, and high recurrence rates, and do not address the underlying tendency toward stone formation [4]. This has renewed interest in medicinal plants as sources of diuretic, litholytic, antioxidant, anti-inflammatory and anti-crystallization agents capable of interfering with nucleation, crystal growth, aggregation and adhesion [8,9]. Since oxidative stress and inflammatory injury to renal epithelium are believed to promote crystal retention, plants with antioxidant and anti-inflammatory activity are of particular interest [7].

Centella asiatica (L.) Urb. (Apiaceae), commonly known as Gotu kola or Indian pennywort, is a traditionally used medicinal herb whose leaves are rich in triterpenoid saponins (asiaticoside, madecassoside, asiatic acid, madecassic acid), flavonoids and phenolic compounds, and which possess documented antioxidant, anti-inflammatory, antimicrobial and wound-healing activity [15-22]. Despite this pharmacological profile, systematic evaluation of C. asiatica for anti-urolithiatic activity remains limited. The present study was therefore undertaken to evaluate the in-vitro anti-urolithiatic activity of a hydroalcoholic extract of C. asiatica leaves by assessing its effect on calcium oxalate nucleation, aggregation and crystal dissolution, with the following objectives: (i) authentication and extraction of C. asiatica leaves; (ii) preliminary phytochemical screening of the extract; and (iii) evaluation of the extract's calcium oxalate nucleation-inhibitory, aggregation-inhibitory and crystal-dissolution activity in comparison with a standard agent.

MATERIALS AND METHODS

Collection and extraction of plant material

Authenticated leaves of Centella asiatica were collected, washed to remove adhering dirt, and shade-dried at room temperature. The dried leaves were pulverized using a mechanical grinder and stored in an airtight container. A known quantity of the powdered material was extracted with a hydroalcoholic (ethanol) solvent system using a Soxhlet apparatus until the siphoning solvent became nearly colourless. The extract was concentrated under reduced pressure using a rotary Centella asiatica Percentage yield was calculated as: Percentage Yield = (Weight of extract obtained / Weight of plant material taken) × 100.

Preliminary phytochemical screening

The hydroalcoholic extract was screened for major phytoconstituents using standard qualitative chemical tests: Dragendorff's and Mayer's tests for alkaloids; Molisch's and Fehling's tests for carbohydrates; foam and Liebermann-Burchard tests for saponins; Biuret and Million's tests for proteins; Ninhydrin test for amino acids; Salkowski and Liebermann-Burchard tests for steroids/triterpenoids; Keller-Killiani test for glycosides; Shinoda and lead-acetate tests for flavonoids; ferric chloride and lead-acetate tests for tannins; and ferric chloride test for phenolic compounds [57].

Calcium oxalate nucleation assay

Calcium chloride and sodium oxalate solutions were mixed in the presence of different concentrations of the extract (25-400 µg/mL) under controlled conditions, with a control (no extract) and a standard drug (100 µg/mL) run in parallel. Crystal formation was monitored spectrophotometrically, and percentage inhibition was calculated as % Inhibition = [(Ac - As) / Ac] × 100, where Ac and As are the absorbance of the control and sample, respectively [58].

Calcium oxalate aggregation assay

Preformed calcium oxalate crystals were incubated with different concentrations of the extract (25-400 µg/mL), alongside a control and a standard drug (100 µg/mL). Aggregation was monitored spectrophotometrically at fixed intervals, and percentage inhibition was calculated using the same formula as above [59].

Calcium oxalate crystal dissolution assay

A known quantity of preformed calcium oxalate crystals was incubated with different concentrations of the extract (25-400 µg/mL), a control, and a standard drug (100 µg/mL). Residual crystal mass was estimated after incubation, and percentage dissolution was calculated as: % Dissolution = [(Initial amount - Final amount) / Initial amount] × 100 [60].

RESULT AND DISCUSSION

Extraction yield

Soxhlet extraction of C. asiatica leaves with a hydroalcoholic ethanol system yielded a dark green, semisolid extract with a percentage yield of 7% w/w (Table 1), indicating efficient extraction of polar and moderately polar constituents, including phenolics, flavonoids, glycosides and triterpenoids, and confirming the extract's suitability for further phytochemical and in-vitro anti-urolithiatic evaluation.

Table 1. Extraction yield of hydroalcoholic extract of C. asiatica leaves

Preliminary phytochemical screening

Phytochemical screening (Table 2) revealed the presence of alkaloids, flavonoids, tannins, phenolic compounds, saponins, triterpenoids, steroids, glycosides, carbohydrates, proteins and amino acids in the hydroalcoholic extract. Flavonoids, phenolic compounds, saponins and triterpenoids are of particular interest given their reported antioxidant and anti-inflammatory activity, and are plausible contributors to the anti-urolithiatic effects described below; however, this screening is qualitative, and further isolation studies are required to identify the constituent(s) responsible for the observed activity.

Table 2. Preliminary phytochemical screening of hydroalcoholic extract of C. asiatica leaves

Calcium oxalate nucleation inhibitory activity

The extract produced concentration-dependent inhibition of calcium oxalate nucleation, from 7.93±1.21% at 25 µg/mL to 57.20±0.91% at 400 µg/mL, compared with 61.22±0.73% for the standard at 100 µg/mL (Table 3). Since nucleation represents the initial, rate-limiting step of stone formation, this progressive inhibition suggests that the extract's phytoconstituents interfere with the earliest stage of calcium oxalate crystal formation, an effect plausibly linked to its flavonoid, phenolic, saponin and triterpenoid content.

Table 3. Effect of C. asiatica hydroalcoholic extract on calcium oxalate nucleation

Calcium oxalate crystal aggregation inhibitory activity

Inhibition of crystal aggregation increased in a concentration-dependent manner from 7.37±1.05% at 25 µg/mL to 57.11±0.82% at 400 µg/mL, compared with 60.79±0.65% for the standard (Table 4). Since aggregation of individual crystals into larger particles increases the likelihood of retention within the urinary tract, this activity indicates that the extract may reduce the formation of larger, retention-prone calcium oxalate aggregates.

Table 4. Effect of C. asiatica hydroalcoholic extract on calcium oxalate crystal aggregation

Calcium oxalate crystal dissolution activity

The extract showed concentration-dependent dissolution of preformed calcium oxalate crystals, increasing from 8.00% at 25 µg/mL to 38.00% at 400 µg/mL, compared with 41.00% for the standard at 100 µg/mL (Table 5). Although slightly lower than the standard, this appreciable dissolution activity suggests that extract constituents can also destabilize preformed crystals, complementing its anti-nucleation and anti-aggregation effects.

Table 5. Effect of C. asiatica hydroalcoholic extract on calcium oxalate crystal dissolution

CONCLUSION

The hydroalcoholic extract of Centella asiatica leaves demonstrated concentration-dependent in-vitro anti-urolithiatic activity, producing 57.20±0.91% inhibition of calcium oxalate nucleation, 57.11±0.82% inhibition of aggregation, and 38.00% dissolution of preformed crystals at 400 µg/mL, approaching but not exceeding standard activity across all three assays. Preliminary phytochemical screening indicated the presence of alkaloids, flavonoids, tannins, phenolic compounds, saponins, triterpenoids, steroids, glycosides, carbohydrates, proteins and amino acids, of which flavonoids, phenolic compounds, saponins and triterpenoids are considered the most plausible contributors to the observed activity, potentially via antioxidant and crystal-modifying mechanisms. These findings support the traditional and pharmacological interest in C. asiatica and provide a preliminary scientific basis for its evaluation as an anti-urolithiatic agent. However, the results are limited to in-vitro models and should not be interpreted as evidence of clinical efficacy; further studies involving isolation and characterization of active constituents, mechanistic investigation, toxicity evaluation, and appropriate in-vivo models are warranted.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest associated with this manuscript.

ACKNOWLEDGEMENT

The authors are thankful to [Institution Name] for providing the necessary facilities to carry out this research work.

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