In vitro Anti-Urolithiatic Evaluation of a Polyherbal Formulation against Calcium Oxalate-Induced Urolithiasis in Rats

Kidney stones are one of the most common nephrological disorders (Rodriguez Cuellaret al., 2020). Kidney stones recur due to urinary tract formation, causing pain, hematuria, and hydronephrosis. Prevalence (16% globally) varies by diet, lifestyle, location, sex, and genetics, with India having the highest incidence (Madsen 2013; Guhaet al., 2019). About 70-80% of kidney stones consist of calcium phosphates and oxalates, with 1-5% prevalence in Asia. They form via crystal nucleation and aggregation of calcium, magnesium, phosphates, cystine, and uric acid, affecting both genders equally (Öneret al., 2015; Teoet al., 2021; Edvardsson et al., 2022). In modern times, kidney stone treatment often involves complex, sophisticated, and expensive surgical procedures. However, herbal remedies provide a cost-effective and widely accepted alternative (Papatsoriset al., 2024). Traditional medicinal plants, particularly in Ayurveda, have long been used to prevent crystallization, nucleation, and aggregation of urinary calculi, offering therapeutic benefits in the management of urolithiasis along with other pharmacological advantages (Rukariet al., 2024). The lotus, native to Asia, is revered as a sacred plant in traditional cultures. In tropical regions, it features a whip-shaped rhizome that retains its green coloration and continues to flower throughout its lifecycle (Caoet al., 2021). The lotus contains proteins, carbohydrates, and sugars (arabinose, galactose, mannose, xylose, rhamnose, glucuronic acid). It is rich in magnesium, calcium salts of ghattic acid, and phytochemicals like alkaloids, flavonoids, phenols, terpenoids, sterols, saponins, tannins, coumarins, quinones, and ellagic acid (Khanet al., 2024). Extensive phytochemical screening has successfully identified and extracted key bioactive compounds, particularly saponins, in significant proportions (Dalmarco,et al., 2010). The lotus contains vitamins, lipids, glycosides, and essential oils. Leucas aspera (Lamiaceae) derives its name from the Greek “Leukos” (white) and is widespread in India, especially the Himalayas, and Sri Lanka. The lotus is among the largest flowering plants. (Abdel-Alimet al., 2023). Another notable species, Anogeissus latifolia, commonly known as Gum Ghatti, Axle-wood, or the Indian Gum Tree, belongs to the Combretaceae family (Singhet al., 2023). It is widely distributed across Asian countries such as India, Sri Lanka, Myanmar, and Nepal, thriving in tropical and subtropical regions (Mahawaret al., 2024). This study aims to evaluate the in vitro and in vivo anti-urolithiatic activity of NNE, ALE, LAE, and PHF, focusing on the extraction and pharmacological significance of saponins.

N. nucifera, A. latifolia, and L. aspera were collected from Sri Venkateswara University, Tirupati, and authenticated by Dr. K. Madhava Chetty (IDs: 1078, 1080, 1088). Plants were air-dried for seven days in the shade.

Calcium Oxalate-AR, (Merck, India), and Cystone (Himalaya Company Limited, India) are selected for the practical studies. If needed selection of the required chemicals and reagents were included with analytical grades. Auto analyzer Semi followed by UV- spectrophotometer UV-1700 Pharma-spec, Shimadzu.

Male Wistar rats (150-200 g) were procured and acclimatized to laboratory conditions before the study. They were housed in a controlled environment with regulated temperature, relative humidity, and hygienic conditions. Standard feed and water were provided ad libitum in accordance with quarantine protocols. Animal experiments followed CPCSEA guidelines and were approved by IAEC, Jeeva Life Sciences (CPCSEA/IAEC/ JLS/18/07/22/029), adhering to ethical regulations.

Toxicity studies were conducted according to Organization for Economic Cooperation and Development rules and regulations (Sewellet al., 2024).

To induce urolithiasis in rats, all groups (except the control group) were fed a commercially available pelleted diet with drinking water containing calcium oxalate (70 mg/kg, intraperitoneally) for 28 days (Table 1). Renal and serum biochemical parameters, along with histopathological studies, were subsequently performed (Ćorić-Martinovićet al., 2024).

Histopathological evaluation was carried out using a microtome. The kidneys were isolated, fixed in formalin (10%), and embedded in paraffin for 5 mm thick sections. Sections measuring 5 mm thick were stained with hematoxylin and eosin. The specimen was examined under a microscope (Li et al., 2023).

All the results were scrutinized by analysis of variance (Dunnett’s comparison test) using Prism (v8.2) and articulated as mean and SD. The significance (p<0.05) was considered.

Preliminary phytochemical evaluation revealed the presence of supportive active phyto-constituents such as alkaloids, flavonoids, tannins, phenols, saponins, phytosterol, terpenoids, and glycosides (Table 2).

In acute oral toxicity evaluated on selected rats (both sexes) for 14 days, no signs and symptoms of toxicity were noticed at the limit-test dose of 2000 mg/kg administered as per the OECD guidelines.

Methanolic extracts of NNE, ALE, LAE, and PHF improved creatinine, calcium, phosphorus, uric acid, and ALP in CaOx-induced rats. Ethylene glycol raised calcium, magnesium, and phosphate in the disease group, while extracts reduced these levels. PHF showed superior efficacy, also increasing oxalate levels more effectively than high-dose extracts (Tables 3, 4 and Figure 1).

Figure 1:
Effect of extracts on in vitro antioxidant activity. *p<0.05, **p<0.01, ***p<0.01. Values are mean±SEM, n=6, when compared with control by using one-way ANOVA followed by Tukey’s multiple comparison test. ANOVA: Analysis of variance, SEM: Standard error of the mean, NNE, ALE, LAE, and PHF: Nelumbo nucifera, Anogeissus latifolia, Leucas aspera, and PHF extract.

Titrimetric analysis measured calcium oxalate stone dissolution. Nelumbo nucifera and Leucas aspera showed 80% dissolution, while Anogeissus latifolia achieved 74%, compared to the blank and positive control (Neeri) (Figure 2). This demonstrates that selected plants showed significant anti-urolithiasis activity in vitro. These plants can be used effectively to treat urolithiasis.

Figure 2:
Effect of extracts on in vitro anti-urolithiasis activity. *p<0.05, **p<0.01, ***p<0.01. Values are mean±SEM, n=6, when compared with control by using one-way ANOVA followed by Tukey’s multiple comparison test. ANOVA: Analysis of variance, SEM: Standard error of the mean, NNE, ALE, LAE, and PHF: Nelumbo nucifera, Anogeissus latifolia, Leucas aspera, and PHF extract.

Calcium oxalate-induced renal stones caused crystal deposits, tubular atrophy, cystic changes, and inflammation. Cystone (70 mg/kg) and plant extracts (250-500 mg/kg, 28 days) significantly reduced these abnormalities (Figure 3). CaOx treatment significantly (p<0.01) reduced urine volume without altering pH. Cystone and plant extracts (250-500 mg/kg) increased urine volume (p<0.05-0.01) compared to controls, with no pH change. Similarly, rats treated with N. nucifera (NNE), A. latifolia (ALE), L. aspera (LAE), and PHF at doses of 250, 500, and 375 mg/kg body weight showed a significant (p<0.05 to 0.01) increase in urine volume, without affecting urine pH (Figure 4).

Figure 3:
Effect of selected plant extracts on Calcium oxalate-induced Urolithiasis Rats on kidney tissue histology.

Figure 4:
Hydroxyl (OH·) radical scavenging assay. The graph depicts the percentage inhibition of hydroxyl radicals at varying concentrations (1000-250 μg/mL) for AA (Ascorbic Acid), NNE (sample), ALE (sample), and LAE (sample). Data points represent mean values, showing a dose-dependent inhibition trend.

CaOx (70 mg/kg, i.p.) induced hyperoxaluria, altering urinary markers and causing tubular damage. Cystone (500-750 mg/ kg) and plant extracts (250-500 mg/kg) restored levels, reduced oxidative stress, and prevented lithiasis. PHF was most effective. Histological analysis showed reduced tubular damage and crystal deposits with treatment.

In the current study, the calcium oxalate-induced model in Wistar rats was used as a representation of the human urinary system (Wanget al., 2025). Previous research has shown that renal crystal deposition is more pronounced in male rats compared to females (Peeterset al., 2022; Shi,et al., 2025). Calcium Oxalate (CaOx) renal stone formation begins with the deposition of calcium phosphate plaques, also known as Randall’s plaques. These plaques attract cations, which, in turn, form insoluble calcium oxalate salts that deposit on the renal interstitium. The oxalate crystals then attach to renal cells, damaging cell walls, promoting crystal aggregation, and facilitating further crystal growth, ultimately leading to retention in the renal tubules (Kauret al., 2025; Chung H.J. 2014; Kostovet al., 2025). NNE, ALE, LAE, and PHF reduced urinary oxalate levels and LDH activity, lowering oxalate accumulation. CaOx crystals increased LPO and oxidative stress, leading to kidney damage. Treated groups showed reduced urine output and filtration rates, causing nitrogenous waste buildup (Ranet al., 2025; Kanlayaet al., 2024). Treatments with PHF reduced the deposition of calcium oxalate crystals and lowered the levels of creatinine, phosphorus, and uric acid. The extracts of NNE, ALE, and LAE also decreased calcium oxalate deposition, minimizing damage to the urinary system (Simhadri and Leslie, 2024).

The deposition of CaOx crystals causes renal and histological damage in various parts of the renal system. A microscopic study of kidney sections from lithiatic control rats showed CaOx crystals in tubular and interstitial spaces, accompanied by glomerular congestion and tubular necrosis. However, treatment with NNE, ALE, LAE, and PHF extracts resulted in a reduction of CaOx crystal deposition. High concentrations of CaOx can lead to renal tubular obstruction, glomerular impairment, and tubular necrosis (Kaleet al., 2024; Houet al., 2025; Rajet al., 2024). The plant extracts showed stone-dissolution properties, likely due to their saponin content. NNE, ALE, and LAE contained saponins, flavonoids, and phytosterols, contributing to anti-lithiatic and antioxidant effects. These phytochemicals may prevent CaOx supersaturation, reducing crystal deposition. The antioxidant activity improved renal markers by inhibiting oxidative stress. The study confirms their inhibitory and preventive effects on urolithiasis (Mohamedet al., 2024).

These studies conclude that the administration of NNE, ALE, LAE, and particularly PHF decreases urinary stone formation. Therefore, in the early stages of stone formation, Nelumbo nucifera, Anogeissus latifolia, and Leucas aspera plant extracts are recommended in the recurrent pattern of renal stone disease.

The mechanism behind this effect may be due to antioxidant nephroprotective properties as well as a reduction in urinary stone-forming constituent concentrations. However, further research is needed to determine the precise mechanism of this behavior.