1. INTRODUCTION:

1.1 Background and Importance:

Urolithiasis, commonly known as kidney stone disease, is a prevalent urinary disorder characterized by the formation of mineral deposits in the urinary tract.¹ It affects millions worldwide, with an increasing incidence in both pediatric and geriatric populations.

Kidney stones primarily consist of calcium (64–92%)², mainly as calcium oxalate (32–46%) and calcium phosphate (3–5%) or both. Other components include cystine (1%), struvite (2–15%), uric acid (3–16%), carbapatite (15.6%), urate (12.4%), and brushite (1.7%).³

Their formation is influenced by various factors, including diet, metabolic disorders, dehydration, and genetic predisposition.^4,5 Clinically, urolithiasis manifests as severe pain (renal colic), hematuria, urinary obstruction, and recurrent infections, significantly impacting patients' quality of life.

The burden of urolithiasis has been rising globally, particularly in developing nations, due to changes in lifestyle and dietary habits.⁶ Current management strategies include pharmacological interventions, dietary modifications, extracorporeal shock wave lithotripsy (ESWL), and surgical procedures such as ureteroscopy and percutaneous nephrolithotomy.⁷ However, these approaches are often associated with high costs, recurrence rates, and potential side effects, underscoring the need for safer and more effective alternatives, particularly for vulnerable populations like children and the elderly.

In recent years, herbal-based therapies have gained considerable attention in urolithiasis management due to their multifaceted therapeutic effects, including diuretic, anti-inflammatory, and crystal-inhibiting properties. Traditional polyherbal formulations have been widely used in Ayurveda and other traditional medicine systems to prevent stone formation and facilitate stone dissolution.^8,9 Among commercially available herbal formulations, Cystone has demonstrated efficacy in inhibiting stone formation and promoting their elimination. However, it may cause gastrointestinal discomfort, allergic reactions, dizziness, headaches, and drug interactions, affecting efficacy and tolerability.¹⁰These limitations emphasize the need for safer, alternative polyherbal formulations.

This study aims to evaluate the in-vitro antiurolithiatic activity of selected polyherbal syrups and compare their efficacy with Cystone using the Egg Membrane Assay and the Simulation of the Sedimentary Crystal Formation Model. These models provide a reliable assessment of crystal dissolution and stone inhibition properties, thereby contributing to the development of alternative plant-based therapies for urolithiasis management.

1.2 LITERATURE REVIEW:

Several studies have highlighted the antiurolithiatic potential of medicinal plants rich in phenolics, flavonoids, and tannins, which contribute to stone inhibition, dissolution, and prevention of recurrence.¹¹ These bioactive compounds help by inhibiting crystal formation, dissolving stones, and modulating urinary factors. Phenolics (Phyllanthus niruri, Boerhavia diffusa) act as antioxidants and diuretics, reducing oxidative stress.¹² Flavonoids (Tribulus terrestris, Aerva lanata) provide anti-inflammatory and nephroprotective effects, helping to regulate urinary pH.¹³ Tannins (Crataeva nurvala, Terminalia arjuna) aid in mineral chelation and crystal dissolution.^14,15 These findings support the use of polyherbal formulations as effective, safer alternatives to conventional treatments for urolithiasis.

1.3 OBJECTIVES OF THE RESEARCH:

The primary objective of this study is to formulate a polyherbal syrup using leaf extracts of Trigonella foenum-graecum, Coleus amboinicus and Murraya koenigii with established antiurolithiatic properties. The formulated syrup was subjected to in vitro evaluation using the Egg Membrane Assay and Sedimentary Crystal Formation Model to assess its effectiveness in inhibiting stone formation and promoting dissolution. Furthermore, the study aims to compare the antiurolithiatic activity of the developed formulation with Cystone, a commercially available herbal preparation, to determine its relative efficacy as a potential alternative treatment (Figure 1).

Figure 1: Graphical Abstract of Research Work

2. MATERIALS AND METHODS:

2.1 Selection of Plant Materials and its Authentication:

Three medicinal plants-Trigonella foenum-graecum, Coleus amboinicus, and Murraya koenigii were selected for this study based on their traditional use in treating kidney-related ailments and their rich content of bioactive compounds such as phenolics, tannins, and flavonoids, which contribute to their antiurolithiatic properties. The leaves of Trigonella foenum-graecum and Murraya koenigii were procured from the APMC market, Sector-19, Vashi, Navi Mumbai, while Coleus amboinicus leaves were obtained from Hruturaj Green Nursery, Plot 125, Sector-26, Seawoods, Navi Mumbai, Maharashtra. The plant materials were authenticated by Dr. Rajendra D. Shinde, Principal and Head of the Department of Botany, and Director of the Blatter Herbarium, St. Xavier's College, Mumbai (400001). The authentication references provided were Shah-1600 (G. L. Shah), MRA-1582 (M. R. Almeida), and R-3594 (R. R. Fernandez). After collection, the leaves were dried, finely powdered, and stored in airtight containers for further phytochemical screening and formulation studies.

2.2 Preparation of Hydroalcoholic Leaves Extract:

Dried, coarsely powdered leaves (70g) of each plant were extracted using a Soxhlet apparatus with 500mL of 70% ethanol and 30% water for three days. The process was repeated twice to ensure maximum extraction. The extract was then concentrated by evaporation at 70°C for 8hours. The extract was dried and the percentage yield was calculated. The final extract was stored at room temperature for further phytochemical screening.^16,17

2.3 Phytochemical Screening:

Phytochemical screening was carried out to detect the presence of phenolic compounds, tannins, flavonoids, and other bioactive constituents in the plant extracts using standard qualitative tests.¹⁸

2.4 Preparation of Polyherbal Syrup:

Two polyherbal syrup formulations, Formulation 1(F-1) and Formulation 2(F-2), were prepared using a standardized method with simple syrup as the base, according to the composition given in Table 1.

Table 1: Polyherbal Syrup Formulations

Ingredients	Function	Formulation
Ingredients	Function	F₁	F₂
Trigonella foenum-graecum	Active Drug	500mg	1000mg
Coleus amboinicus	Active Drug	500mg	1000mg
Murraya koenigii	Active Drug	500mg	1000mg
Propylene Glycol	Solubilizer	6.0ml	6.0ml
Methyl Paraben	Preservative	0.025gm	0.025gm
Propyl Paraben	Preservative	0.0025gm	0.0025 gm
Glycerine	Thickening agent	0.5ml	0.5ml
Simple Syrup	As a Base	Up to 100 ml	Up to 100ml

Method:

The required quantity of each ingredient was accurately weighed. The extract was first mixed with a small amount of water using a mortar and pestle. Preservatives (methyl paraben, propyl paraben) and solubilizer (propylene glycol) were then incorporated. Simple syrup was gradually added, followed by glycerine as a thickening agent. The final volume was adjusted to 100 mL with distilled water, ensuring a homogeneous mixture. The solubility was assessed visually by checking the clarity of the solution. The prepared syrup was transferred into a bottle, labeled, and stored for further use.^19,20

2.5 Evaluation of Polyherbal Syrup Formulations:

2.5.1 Organoleptic Properties:

The color, taste, appearance and odour of the polyherbal syrup were immediately determined using sensory and visual examination.

2.5.2 pH Determination:

The pH of polyherbal syrup was determined by using digital pen-style pH meter and pH paper.

2.5.3 Determination of Density of Syrup:

The empty bottle of known volume was weighed (W₁) and then filled with syrup and weighed again (W₂). The difference between the weight of the empty bottle and the bottle containing the syrup was recorded (W₃) and density was calculated using the given formula:

Weight of Syrup

Density of Syrup = -------------------------------

Volume of Bottle

2.5.4 Determination of Specific Gravity of Syrup:

The pycnometer was used to determine specific gravity (g/mL) of the syrup formulations. The weight of the empty pycnometer and its weight when filled with water were recorded as W₁ and W₂ respectively. The weight of the syrup in the pycnometer was recorded as W₃. Mass of syrup was calculated by subtracting the weight of the empty pycnometer from the weight of the syrup in the pycnometer. Mass of water was calculated by subtracting the weight of the empty pycnometer from the weight of pycnometer filled with distilled water. Specific gravity was computed using the formula below;

Mass of Syrup

Specific Gravity of Syrup = -------------------------------

Mass of Distilled Water

2.5.5 Viscosity:

Using an Ostwald viscometer, the viscosity of polyherbal syrup formulations was measured. The viscometer was mounted vertical position on a suitable stand. Syrup was filled into the viscometer up to mark A. The time was counted for syrup to flow from A to mark B. Viscosity was measured in triplicate.²¹

2.5.6 Determination of Crystal Growth:

The crystal growth was determined after 24hours.

2.5.7 After Taste:

The taste is strong and remains unchanged over the week except for the ambient temperature sample. Healthy male and female candidates, were selected to participate in the evaluation of taste. About 1 ml of polyherbal syrup formulations was given to selected candidates and ask for the taste.

2.5.8 Stability Studies:

The aim of stability studies was to guarantee that the polyherbal syrup formulations can be used and can continue to have the same properties over time. The prepared polyherbal syrup formulations were stored at room temperature, in the refrigerator and at accelerated temperature (40°C) for 30 days and assessed visually for presence/absence of growth or crystals.

2.6 In vitro Antiurolithiatic Activity Testing:

2.6.1 Egg Membrane Assay:

2.6.1.1 Preparation of Synthetic Kidney Stones: Calcium oxalate (CaOx) experimental kidney stones were synthesized using homogeneous precipitation. Calcium chloride dihydrate (1.47g) was dissolved in 100mL of distilled water, while sodium oxalate (1.34g) was dissolved in 100mL of 1 M H₂SO₄. The solutions were mixed in a beaker with continuous stirring, leading to CaOx precipitation. Ammonia solution was added to neutralize excess H₂SO₄, and the precipitate was thoroughly washed with distilled water. The crystals were then dried at 60°C for four hours. The prepared stones were further used for studies involving artificial urine.²²

2.6.1.2 Preparation of Semi-permeable Membrane: The semipermeable membrane was prepared from egg (Figure 2). A glass rod was used to puncture the apex of the eggs and the entire contents were squeezed out. Empty egg shells were washed thoroughly in distilled water, after that the shells were placed in a beaker containing 4.0mL concentrated HCl in 200mL distilled water. The semi-permeable membrane was completely decalcified after being kept for an overnight period. The next day the semi permeable membranes were carefully removed from the egg shell and washed thoroughly with distilled water. The trace of acid present in the membrane is neutralized by placing the shell membranes in an ammonia solution, and rinsed with distilled water. It was kept in a moistened state in the refrigerator at a pH of 7–7.4.²³

Figure 2: Preparation of Semi-permeable Membrane from Eggs

2.6.1.3 Preparation of Standard Drug: The color coating on a 500mg Cystone tablet was removed with absolute ethanol, yielding 400mg. Cystone tablets were crushed into powder and mixed with 100mL distilled water before being filtered. Cystone filtrate was used as a positive control for anti-urolithiatic activity in vitro.²⁴

2.6.1.4 Estimation of Calcium Oxalate by Using Dissolution Model:

Totally 07 semi permeable egg membranes were collected. Each egg semi permeable membrane was packed separately with the different drugs or extracts as given in the experimental setup table 2.

These were allowed to suspend in a conical flask containing 100ml of 0.1M Tris buffer by tying the membrane with the thread. In order to make the shell hanging a stick is placed in the mouth of a conical flask and covered with aluminum foil at the other end of the thread (Figure 3). All the conical flasks were kept in an incubator to 37°C for 2hours. Remove the contents of semi-permeable membranes from each group into separate test tubes, add 2ml of 1N sulfuric acid to each test tube and titrated with 0.9494 N KMnO₄ till a light pink color (endpoint) obtained (Figure 4). Consequently, 1ml of 0.9494 N KMnO4 was equivalent to 0.1898 mg of calcium. All the experiments were performed in replicate. Finally, the undissolved Calcium oxalate was subtracted from the total quantity used in the experiment, in the beginning, to know the total quantity of dissolved Calcium oxalate by various dosage.^25,26,27

The following formulas were used to calculate dissolved Calcium oxalate and Percentage dissolution respectively.

Dissolved Calcium ocalate

= Total quantity used in the experiment in the beginning

-undissolved Calcium oxalate

% Dissolution = Dissolved Calcium oxalate/Wt. of CaOx in the beginning x 100

Figure 3: Experimental Setup for Egg Membrane Assay

Figure 4: Percentage Dissolution of CaOx by Titrimetric Method

2.2.11.3 Sedimentary Crystal Formation Model:

Hennequin et al., 1993, described a method for crystal formation that was used with slight modifications. To avoid sedimentation, the mixture was kept agitated. At various time intervals, light microscopy was used to track the crystal size development. At 10μg/ml concentration, both formulations of polyherbal syrup, hydroalcoholic extract of Trigonella foenum-graecum, Coleus amboinicus, and Murraya koenigii and cystone were taken. At a one-hour interval, crystal drops were examined by placing the sample on a clear grease-free slide and covering it with a cover slip, and the crystals were identified using a microscope with a 10X magnifying lens.^28,29

3. RESULT AND DISCUSSION:

The % yield of hydroalcoholic leaves extract of Trigonella foenum-graecum, Coleus amboinicus and Murraya koenigii was found to be 10.99%, 18.5% and 17.32% respectively. The preliminary phytochemical screening of hydroalcoholic leaves extract of Trigonella foenum-graecum, Coleus amboinicus and Murraya koenigii revealed the presence of phenolic compounds, tannins, flavonoids, alkaloids, phytosterols and terpenoids.

3.4 Preparation of Polyherbal Syrup Formulations (F-1 and F-2):

Two formulations of polyherbal syrup: Formulation 1 (F-1) and Formulation 2 (F-2) were prepared (Figure 5).

Figure 5: Polyherbal Syrup Formulations

3.5 Evaluation of Polyherbal Syrup Formulations:

Both prepared formulations (F-1 and F-2) of polyherbal syrup were subjected for evaluation parameters. The results of evaluation parameters are presented in table 3.

Table 3: Evaluation Parameters of Both Polyherbal Syrup Formulations

S. No.	Parameters	F-1	F-2
1	Color	Dark Brown	Dark Brown
2	Odour	Aromatic	Aromatic
3	Taste	Mildely sweet	Mildely sweet in both columns
4	Appearance	Clear	Clear
5	pH (By Digital pen style pH meter)	6.6	6.4
6	pH (By pH Paper)	6.7	6.7
7	Density	1.77	1.63
8	Specific Gravity	1.240	1.253
9	Viscosity	16.75 cps	13.10 cps
10	Crystal Growth	No Growth	No Growth
11	After Taste	Same	Same
Stability Studies
12	Change in color	No Change	No Change
	Odour	No Change	No Change
	Physical Separation	No Change	No Change

SCREENING OF POLYHERBAL SYRUP FORMULATIONS FOR ANTIUROLITHIATIC ACTIVITY:

3.8.1 Egg Membrane Assay:

Table 4 shows, the standard cystone solution showed the mean weight of calcium reduction 1.93mg in standard group, 2.10mg in F1-Formulation Test group, 2.83mg in F2-Formulation Test group, 1.40mg in Trigonella foenum-graecum Extract test group, 1.05mg in Coleus amboinicus Extract test group and 1.02mg in Murraya koenigii Extract test group.

The dissolution percentage of the standard cystone solution and F-1 formulation showed 38.6% and 42.0% respectively. The F-2 formulation of polyherbal syrup showed the percentage dissolution 56.6%. Trigonella foenum-graecum and Coleus amboinicus leaves extract showed 28.0% and 21.0% respectively. Murraya koenigii leaves extract showed the least dissolution percentage i.e. 20.4% hence it can be concluded that the F-2 formulation of polyherbal syrup showed the higher dissolution percentage than the Standard solution and other test samples (Figure 6).

Figure 6: % Dissolution of CaOx

3.8.2 Sedimentary Crystal Formation Model:

In sedimentary crystal formation model, inhibition of stone formation was examined using a light microscope. Microscopic observations revealed a marked decrease in both the size and quantity of crystals. Among all groups, the F-2 formulation demonstrated the most pronounced crystal reduction, suggesting its strong inhibitory effect on the nucleation, growth, and aggregation of calcium oxalate crystals (Figure 7).

Normal Control Group Standard (Cystone)

F-1 Formulation

F-2 Formulation Trigonella foenum-graecum Extract

Coleus amboinicus Extract

Murraya koenigii Extract

Figure 7: Inhibition of Stone Formation in Different Formulations

DISCUSSION:

The present study focused on the in vitro evaluation of two polyherbal syrup formulations (F-1 and F-2) for their antiurolithiatic activity, with a comparative analysis against the commercially available standard, Cystone. The chosen plant extracts Trigonella foenum-graecum, Coleus amboinicus, and Murraya koenigii have long been used in traditional medicine systems for renal health, and their inclusion in the polyherbal syrups was supported by phytochemical analysis indicating the presence of phenolic compounds, flavonoids, tannins, alkaloids, and phytosterols. These phytoconstituents are known to contribute to antiurolithiatic activity through antioxidant, anti-inflammatory, and crystal-inhibiting mechanisms.

In the Egg Membrane Assay, the F-2 formulation demonstrated the highest percentage dissolution of calcium oxalate crystals (56.6%), surpassing the marketed product Cystone (38.6%) and F-1 formulation (42.0%). This suggests a potential synergistic effect of higher concentrations of active plant extracts in F-2, making it more effective in dissolving preformed crystals. Among individual extracts, Trigonella foenum-graecum exhibited the greatest dissolution capacity (28.0%), followed by Coleus amboinicus (21.0%) and Murraya koenigii (20.4%), highlighting the enhanced efficacy of combined formulations over single-plant extracts.

The Sedimentary Crystal Formation Model further validated the inhibitory potential of the polyherbal syrups. Microscopic analysis showed a significant reduction in crystal size and number in the F-2 group, suggesting that the formulation effectively hinders the nucleation, growth, and aggregation of calcium oxalate crystals. The presence of phenolics and flavonoids likely contributes to this inhibition by chelating calcium ions and modulating urinary pH, thereby reducing supersaturation and crystal formation.

Physicochemical evaluations confirmed that both formulations maintained desirable syrup characteristics such as pH, viscosity, and stability, making them suitable for oral administration, especially in pediatric and geriatric populations. The taste, odour, and clarity remained stable over 30 days under various storage conditions, indicating good shelf-life potential.

A notable advantage of the polyherbal syrups is their liquid dosage form, which ensures better compliance among individuals who face difficulty swallowing tablets. Furthermore, by formulating using known edible herbs, the syrups may present a reduced risk of adverse effects compared to Cystone, which, despite its efficacy, has been associated with gastrointestinal discomfort and potential allergic reactions.

However, it is important to acknowledge that this study was limited to in vitro evaluations. While the results are promising, they may not fully translate to in vivo scenarios where factors like bioavailability, metabolism, and systemic interactions play critical roles. Therefore, further in vivo studies and clinical trials are required to validate the therapeutic potential and safety profile of the developed polyherbal syrups.

CONCLUSION:

The findings of this study strongly support the antiurolithiatic potential of the developed polyherbal syrup formulations, particularly F-2, which exhibited superior calcium oxalate dissolution and inhibition of crystal formation compared to the marketed preparation Cystone. The efficacy of the formulations can be attributed to the presence of phytoconstituents such as flavonoids, phenolic compounds, and tannins. These results suggest that polyherbal syrups, especially those with higher concentrations of active plant extracts, offer a promising, natural, and patient-friendly alternative for the prevention and management of urolithiasis.

Given the growing interest in herbal therapeutics and the limitations of current pharmacological treatments, the formulation of polyherbal syrups could represent a significant step forward in renal care, particularly for populations requiring non-invasive, safe, and effective alternatives. Future in vivo studies and clinical evaluations are warranted to confirm these findings and to explore their long-term safety, efficacy, and mechanism of action in human subjects.

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Received on 24.06.2025 Revised on 22.11.2025

Accepted on 28.01.2026 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):3143-3150.

DOI: 10.52711/0974-360X.2026.00446

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Group	Group Name	Test Parameters	Acronym
Group I	Negative Control	5 mg of Calcium oxalate + 2 ml of distilled water	NC
Group II	Standard (Positive Control)	5 mg of Calcium oxalate + 2 ml of Cystone solution (10 mg/ml)	PC
Group III	Test Group 1	5 mg of Calcium oxalate + 2 ml of polyherbal syrup formulation (F-1)	TG1
Group IV	Test Group 2	5 mg of Calcium oxalate + 2 ml of polyherbal syrup formulation (F-2)	TG2
Group V	Test Group 3	5 mg of Calcium oxalate + 2 ml of hydroalcoholic extract of Trigonella foenum-graecum leaves (10 mg/ml).	TG3
Group VI	Test Group 4	5 mg of Calcium oxalate + 2 ml of hydroalcoholic extract of Coleus amboinicus leaves (10 mg/ml).	TG4
Group VII	Test Group 5	5 mg of Calcium oxalate + 2 ml of hydroalcoholic extract of Murraya koenigii leaves (10 mg/ml).	TG5

Groups	Vol. of KMnO₄ (ml)	Weight of Calcium oxalate Estimated (mg)	Mean Weight of Calcium Reduced (mg)	Percentage Dissolution
Negative Control	27.4	5	0	0%
Standard (Cystone)	18.34	5	1.93	38.6%
F1- Formulation	17.83	5	2.10	42.0%
F2- Formulation	16.55	5	2.83	56.6%
Trigonella foenum-graecum Extract	22.54	5	1.40	28.0%
Coleus amboinicus Extract	23.12	5	1.05	21.0%
Murraya koenigii Extract	23.45	5	1.02	20.4%