Pharmacological Investigation of Traditional Medicinal Plants for the Management of Urolithiasis

Urolithiasis (kidney stone disease) is a common and recurrent disorder of the urinary system characterized by the formation of calculi due to the supersaturation of urine with minerals such as calcium, oxalate, and phosphate. It is associated with severe pain, urinary obstruction, and a high rate of recurrence, making its management a significant clinical challenge. Although modern medical and surgical interventions such as lithotripsy and drug therapy are effective in removing stones, they are often associated with high cost, potential side effects, and limited ability to prevent recurrence. This has led to increasing interest in alternative and complementary therapies, particularly those derived from medicinal plants.[1]

Traditional medicinal systems have long utilized plant-based remedies for the treatment of kidney stones. Among these, Bryophyllum pinnatum (commonly known as Patharchatta or life plant) and Phagnalon rupestre have gained attention for their potential antiurolithiatic properties. Bryophyllum pinnatum is widely used in Ayurveda and folk medicine for its diuretic, anti-inflammatory, and lithotriptic (stone-breaking) activities.[2] It is rich in bioactive compounds such as flavonoids, alkaloids, and glycosides, which are believed to inhibit crystal formation, reduce urinary supersaturation, and promote the dissolution and expulsion of renal calculi. Similarly, Phagnalon rupestre, though less extensively studied, has been traditionally used in certain regions for urinary disorders, including kidney stones. It is reported to possess antioxidant and anti-inflammatory properties, which may play a role in preventing renal tissue damage and inhibiting stone formation. The phytoconstituents present in this plant are thought to interfere with the processes of nucleation, aggregation, and growth of urinary crystals.[3]

The pharmacological investigation of these plants is essential to scientifically validate their traditional use and to identify their mechanisms of action in the management of urolithiasis.[4] Therefore, the present study focuses on evaluating the antiurolithiatic potential of Bryophyllum pinnatum and Phagnalon rupestre using appropriate experimental models. This research aims to contribute to the development of effective, safe, and affordable herbal alternatives for the prevention and treatment of kidney stone disease.[5]

MATERIALS AND METHODS

Plant Material

The plant materials used in the present study were procured from an authorized commercial supplier and authenticated by experts at the Department of Botany, Shri Shivaji Science and Arts College, Chikhli, District Buldana, Maharashtra, India.

Table : Plant Used

Preparation of Hydroalcoholic Extract[6-9]

The collected plant materials were washed with distilled water and shade-dried at 25–30°C for 10–15 days. The dried materials were powdered using a mechanical grinder and passed through sieve No. 40.Approximately 200 g of powdered material was defatted using petroleum ether (60–80°C) in a Soxhlet apparatus for 6–8 hours. The defatted marc was then extracted with 70% ethanol (ethanol: water, 70:30 v/v) for 24–48 hours. The extract was filtered (Whatman No. 1), concentrated using a rotary evaporator at 40–45°C, and further dried to obtain a semisolid mass. The final extract was stored in airtight amber-colored containers at 4°C.

 Preliminary Phytochemical Screening[10]

The hydroalcoholic extracts were subjected to qualitative phytochemical analysis using standard procedures to detect:

Table: Preliminary Phytochemical Screening Tests

Pharmacological Screening for Antiurolithiatic Activity[11-12]

Experimental Animals

• Swiss albino mice (toxicity study)
• Wistar rats (antiurolithiatic study)
• Weight: 150–200 g
• Standard laboratory conditions maintained

Ethical Approval

The study protocol was approved by the Institutional Animal Ethics Committee (IAEC) under CPCSEA guidelines.

Acute Oral Toxicity Study: Conducted as per OECD Guideline 425 (Up-and-Down method):

• Initial dose: 175 mg/kg
• Dose progression factor: 3.2
• Observations: behavioral changes, mortality, toxicity signs
• Duration: 24 hours to 14 days

Induction of Urolithiasis[13]

Urolithiasis was induced using:

• Ethylene glycol (0.75% v/v) in drinking water
• Duration: 28 days
• Mechanism: conversion to oxalic acid → calcium oxalate crystal formation

Experimental Design

Evaluation Parameters (Tests)[14-20]

Urine Analysis

• Urine volume (diuretic effect)
• pH (crystal formation)
• Oxalate (KMnO? titration)
• Calcium (OCPC method)
• Magnesium (Calmagite method)

Serum Biochemical Tests

• Uric acid (Uricase method)
• Creatinine (Jaffe’s method)
• Urea (DAM method)

Kidney Homogenate Analysis

• ACP, ALP
• AST, ALT
• LDH

Physical Parameters

• Body weight
• Kidney weight
• Relative organ weight

Histopathological Studies

• Calcium oxalate crystal deposition
• Tubular dilation
• Epithelial degeneration
• Inflammation
• Tissue necrosis

Polyherbal Formulation

A combination (1:1) of:

• Bryophyllum pinnatum
• Phagnalon rupestre

Prepared using 1% CMC as a suspending agent and administered orally.

Statistical Analysis

Results were expressed as mean ± SEM (n = 6) and analyzed using ANOVA followed by post hoc tests. A p value < 0.05 was considered statistically significant.

RESULT

Table :  Preliminary Phytochemical Screening of Extract

Acute Oral Toxicity Study of Extracts (OECD Guideline 425)

Table : Acute Toxicity Study of Bryophyllum pinnatum

Table :Acute Toxicity Study of Phagnalon rupestre

Acute toxicity studies of Bryophyllum pinnatum and Phagnalon rupestre extracts showed no mortality up to 2000 mg/kg. Only mild, temporary lethargy was observed at higher doses, with no severe toxic effects. The LD?? is >2000 mg/kg, indicating low toxicity and good safety for further pharmacological studies.

Urine Analysis of Bryophyllum pinnatum and Phagnalon rupestre

Figure: Urine Analysis of Bryophyllum pinnatum and Phagnalon rupestre

Table : Urine Analysis of Bryophyllum pinnatum and Phagnalon rupestre

Disease induction promoted stone formation (low urine volume, acidic pH, high calcium/oxalate, low magnesium). Treatment with Bryophyllum pinnatum and Phagnalon rupestre normalized these, showing strong antiurolithiatic and renal protective effects.

Table: Serum Biochemical Analysis of Bryophyllum pinnatum and Phagnalon rupestre

Figure:  Serum Biochemical Analysis of Bryophyllum pinnatum and Phagnalon rupestre

Serum analysis showed increased uric acid, creatinine, and urea in the disease group, indicating renal damage. Treatment with Bryophyllum pinnatum and Phagnalon rupestre significantly reduced these levels, showing improved kidney function and strong nephroprotective and antiurolithiatic effects.

Table: Effect of Extracts on Urine Volume and Urinary pH

Figure : Serum Biochemical Analysis of Bryophyllum pinnatum and Phagnalon rupestre

Kidney homogenate analysis showed elevated ACP, ALP, AST, ALT, and LDH in the disease group, indicating renal damage. Treatment with Bryophyllum pinnatum and Phagnalon rupestre significantly reduced these levels, suggesting membrane stabilization and strong nephroprotective activity.

Table: Effect of Extracts on Physical Parameters

Data (mean ± SEM, n = 6) analyzed by one-way ANOVA with Dunnett’s test showed decreased body weight and increased kidney weight in the disease group. Treatment with Bryophyllum pinnatum and Phagnalon rupestresignificantly (***p < 0.001) restored these, indicating renal protection and improved overall condition.

Table: Effect of Polyherbal Extract on Urine Volume and Urine pH.

Ethylene glycol significantly reduced urine volume and pH, promoting stone formation. Treatment with the polyherbal formulation restored these parameters toward normal, indicating a potent diuretic and antiurolithiatic effect.

Figure:  Effect on Urine Volume

Bar graph representing the effect of treatments on urine volume in ethylene glycol-induced urolithiasis in Wistar rats. Values are expressed as Mean ± SEM (n = 6). ***p < 0.001 compared with the disease control group.

Effect on Urine pH

Bar graph showing the effect of treatments on urinary pH. Values are expressed as Mean ± SEM (n = 6). ***p < 0.001 compared with the disease control group.

Effect on Urine pH

Bar graph showing the effect of treatments on urinary pH. Values are expressed as Mean ± SEM (n = 6). ***p < 0.001 compared with the disease control group.

Table: Effect on Urinary Biochemical Parameters

The disease control group exhibited hypercalciuria and hyperoxaluria with decreased magnesium levels. The polyherbal formulation significantly reduced calcium and oxalate excretion while restoring magnesium levels.

Figure  : Effect on Urinary Biochemical Parameters

Biochemical analysis showed increased calcium and oxalate with decreased magnesium in the disease group, confirming urolithiasis. Treatment significantly (***p < 0.001) reduced calcium and oxalate and restored magnesium, with Group III showing slightly better effect, indicating strong antiurolithiatic activity.

Table : Effect on Serum Biochemical Parameters

Elevated serum markers in the disease group indicate renal impairment. Treatment with the polyherbal formulation significantly improved kidney function.

Figure: Effect on Serum Biochemical Parameters

Renal parameters showed increased urea, creatinine, and uric acid in the disease group, indicating kidney damage. Treatment significantly (***p < 0.001) reduced these levels toward normal, with Group III showing slightly better improvement, indicating strong nephroprotective activity.

Table : Effect on Kidney Homogenate Enzymes

The extracts significantly reduced enzyme levels, indicating protection against renal tissue damage.

Figure Effect on Kidney Homogenate Enzymes

Enzymatic parameters showed elevated ACP, ALP, AST, ALT, and LDH in the disease group, indicating tissue damage. Treatment significantly (***p < 0.001) reduced these levels toward normal, with Group III showing slightly better recovery, indicating strong tissue protective activity.

Table  Effect on Kidney Weight and Body Weight

The polyherbal treatment prevented kidney enlargement and restored normal body weight.

Figure  Effect on Kidney Weight and Body Weight

Disease induction decreased body weight and increased kidney weight, indicating toxicity and renal damage. Treatment significantly (***p < 0.001) restored body weight and reduced kidney weight, showing strong nephroprotective and overall protective effects. Histopathological Observations

Figure  Histopathological Observations

Histopathology showed normal kidney structure in control, while the disease group had calcium oxalate crystals, tubular damage, and inflammation. Standard (Cystone) and test (Bryophyllum pinnatum + Phagnalon rupestre) groups showed reduced crystal deposition and near-normal renal architecture, indicating strong antiurolithiatic and nephroprotective effects.

Table  Histopathological Findings