Rashtrasant Janardan Swami college of pharmacy, kokamthan.
Millions of people worldwide suffer from urolithiasis, a common disorder marked by the development of stones in the urinary tract that frequently causes excruciating pain and complications like infections and urinary blockage. The in vitro effectiveness of two commercially available medications, potassium citrate and allopurinol, in preventing and treating calcium oxalate stone formation—the most prevalent kind of kidney stone—is assessed in this study. The study evaluates the inhibitory effects of these medications on crystallization and their capacity to dissolve pre-formed stones by simulating physiological settings using a novel egg-membrane diffusion model. By raising citrate availability and preserving an alkaline pH, potassium citrate improved dissolving and dramatically decreased calcium oxalate crystallization (70% inhibition), according to the results. Allopurinol, which is mostly used to treat uric acid stones, shown a moderate level of effectiveness. both results demonstrate the therapeutic potential of both substances, indicating that allopurinol is a useful adjuvant treatment for mixed stones and potassium citrate is very efficient for calcium-based stones. This work lays the groundwork for future in vivo investigations and clinical uses to improve the treatment of urolithiasis.
Urolithiasis, derived from the Greek terms “ouron” (meaning urine) and “lithos” (meaning stone), refers to the development of stones within the urinary system, which encompasses the kidneys, bladder, and ureters. This condition is frequently encountered in clinical practice and is characterized by the painful presence of stones. These calculi are predominantly formed from substances such as calcium oxalate, uric acid, or various salts, and can result in considerable discomfort and potential complications if not addressed. The onset of urolithiasis can be attributed to multiple factors, including the supersaturation of urinary salts, dietary choices, and genetic factors
Formulations available in the market that target urolithiasis are designed to dissolve these stones and alleviate associated symptoms. Investigating the in vitro effectiveness of these formulations provides essential information regarding their efficacy and mechanisms of action. This study aims to assess the anti-urolithiatic properties of a commercially available tablet by creating artificial urinary crystals in a laboratory setting. Risk factors associated with urolithiasis encompass age, gender, family history, environmental conditions, lifestyle choices, and dietary habits.
Types of urinary calculi include:
? Calcium-Based Calculi
? Magnesium Ammonium Phosphate Calculi
? Cysteine Calculi
? Medication-Induced Calculi
? Uric Acid Calculi
Symptoms of urolithiasis
Urolithiasis impacts millions of individuals worldwide, often resulting in pain and complications such as urinary obstruction. A variety of pharmacological treatments are available, primarily focused on minimizing stone formation, enhancing stone dissolution, or aiding in the expulsion of stones. This paper provides a review of the in vitro assessments of commercially available tablets utilized in the management of urolithiasis. The condition of urolithiasis, characterized by the formation of stones in the urinary tract, is a widespread issue affecting a significant number of people globally. The presence of these stones can lead to serious health issues, including intense pain, urinary blockage, and recurrent infections. The causes of urolithiasis are complex and multifactorial, influenced by genetic predispositions, dietary habits, and metabolic conditions, with urine supersaturation being a key factor in stone development.
MATERIALS AND METHODS
Materials:
Selection of tablet
1.Potassium Citrate Tablet - (Noculi , Urocalculai) : Commonly used to treat kidney stones by increasing
2.Allopurinol Tablet – (Asthalin-4ma, p250, zentel) : Used to treat gout and kidney stones by reducing uric acid levels in urine. Which can contribute to stone formation
? Chemicals:
oCalcium chloride dihydrate (CaCl?·2H?O) – for the synthesis of calcium ions.
oSodium oxalate (Na?C?O?) – for the synthesis of oxalate ions.
oSulfuric acid (H?SO?), 2N – to dissolve sodium oxalate and act as a medium for the reaction.
oTris buffer (0.1M, pH 7.4) – used to suspend calcium oxalate during incubation.
oPotassium permanganate (KMnO?) – used in titration to estimate calcium oxalate concentration.
oDistilled water – for dissolving chemicals, washing, and rinsing.
oHydrochloric acid (HCl), 2M – for decalcifying the egg shells.
Other Materials:
oFarm eggs – for extracting the semipermeable membrane.
oConical flask (250 mL) – for incubating the semipermeable membrane with calcium oxalate suspension.
oTest tubes (25 mL) – for collecting and titrating the contents after incubation.
oBeaker (250 mL) – for mixing the calcium chloride and sodium oxalate solutions.
oGlass stirring rod – for stirring the solution.
oSuturing thread – for enclosing the calcium oxalate and extract in the semipermeable membrane.
oIncubator – for maintaining the temperature at 37°C during incubation.
oBurette and pipette – for titration of the calcium oxalate solution.
?Methods:
1.Preparation of Calcium Oxalate Precipitate:
oCalcium Chloride Solution: Dissolve 1.47 g of calcium chloride dihydrate in 100 mL of distilled water.
oSodium Oxalate Solution: Dissolve 1.34 g of sodium oxalate in 100 mL of 2N sulfuric acid.
oPrecipitation: Mix the two solutions to form a white precipitate of calcium oxalate.
oPurification: Filter, wash with distilled water, neutralize with ammonia solution, and dry at 60°C for 2 hours.
2.Preparation of Egg Membrane:
oDecalcification: Soak eggs in 2M hydrochloric acid overnight to dissolve the shell.
oExtraction: Remove the inner contents, leaving the semipermeable membrane.
oConditioning: Soak the membrane in ammonia solution for 15-30 minutes, then rinse and store at pH 7.
3.Encapsulation of Calcium Oxalate:
oWeigh 1 mg of calcium oxalate, place it inside the egg membrane, and seal with sutures.
4.Incubation:
oSuspend the membrane with calcium oxalate in 100 mL of 0.1M Tris buffer (pH 7.4) and incubate at 37°C for 7-8 hours.
5.Titrimetric Estimation:
oAfter incubation, add 2 mL of 1N sulfuric acid to the solution.
oTitrate with potassium permanganate (KMnO?) until a faint pink color appears.
oCalculate the concentration of calcium oxalate based on the volume of KMnO? used
Experimental Work
1.selection of tablets :
Commonly used to treat kidney stones by increasing citrate level in urine which helps to prevent stone formulation .
Used to treat gout and kidney stones by reducing uric acid levels in urine. Which can contribute to stone format
2. Preparation of Calcium Oxalate Precipitate
Preparation of Calcium Chloride Solution:
Objective: To prepare a solution containing calcium ions (Ca?2;?) that will react with oxalate ions (C?O??2;?)
Procedure:
Stir the solution until all the calcium chloride is dissolved.
Objective: To prepare a solution of oxalate ions (C?O??2;?) to react with the calcium ions.
Procedure:
Weigh 1.34 g of sodium oxalate (Na?C?O?) and dissolve it in 100 mL of 2N sulfuric acid (H?SO?) in a separate Stir the solution until the sodium oxalate is fully dissolve
Precipitation of Calcium Oxalate:
Objective: To allow calcium ions and oxalate ions to react and frm calcium oxalate.
Procedure
Stir the mixture for 10-15 minutes to ensure complete reaction.
Purification of Calcium Oxalate:
Objective: To remove any residual sulfuric acid and impurities from the calcium oxalate precipitate.
Procedure:
•Filter the precipitate using filter paper to separate the calcium oxalate from the liquid.
•Wash the precipitate several times with distilled water to remove any traces of sulfuric acid.
•Add ammonia solution (NH?) dropwise to neutralize any remaining acid.
•Wash the calcium oxalate precipitate again with distilled water.
•Dry the purified calcium oxalate at 60°C for 2 hours.
3. Preparation of Semipermeable Membrane from Egg
Decalcification of Egg Shell:
Objective: To remove the hard calcified shell and expose the semipermeable membrane.
Procedure:
•Place the eggs in a beaker containing 2M hydrochloric acid (HCl) overnight.
•The acid will dissolve the calcium carbonate shell, leaving the egg membrane intact.
•After 24 hours, rinse the membrane with distilled water to remove any residual acid.
Extraction of Egg Membrane:
Objective:
To isolate the semipermeable membrane by removing the inner contents of the egg.
Procedure:
•Carefully puncture a hole at the top of the egg with a sharp pointer.
•Squeeze out the albumin and yolk, leaving only the semipermeable membrane.
•Rinse the membrane thoroughly with distilled water to remove any remaining egg content
Conditioning of Egg Membrane:
Objective: To prepare the egg membrane for use in encapsulating calcium oxalate.
Procedure:
•Place the cleaned egg membrane in ammonia solution (NH?) for conditioning. Allow it to soak for 15-30 minutes.
•After conditioning, rinse the membrane with distilled water.
•Store the membrane in the refrigerator at a neutral pH (pH 7) until use.
4. Encapsulation of Calcium Oxalate in Egg Membrane
Encapsulation Process:
Objective: To enclose calcium oxalate within the semipermeable egg membrane to study its diffusion.
Procedure:
•Weigh 1 mg of dried calcium oxalate.
•Place the calcium oxalate inside the egg membrane
•Carefully suture the membrane to seal the calcium oxalate inside, forming a small pouch.
5. Incubation and Diffusion of Calcium Oxalate
Suspension in Tris Buffer:
Objective: To suspend the calcium oxalate inside the semipermeable membrane in a suitable medium.
Procedure:
•Prepare 100 mL of 0.1 M Tris buffer (pH 7.4) to provide a stable environment for calcium oxalate diffusion.
•Place the egg membrane containing calcium oxalate in the Tris buffer solution in a conical flask.
•Incubate the flask in an incubator at 37°C for 7–8 hours to allow diffusion through the membrane
5. Titrimetric Estimation of Calcium Oxalate
Sample Preparation:
Objective: To prepare the solution for titration after incubation.
Procedure:
•After the incubation period, remove the egg membrane from the flask.
•Transfer the buffer solution containing calcium ions to a test tube.
Titration Process:
Objective: To estimate the amount of calcium oxalate by titration with potassium permanganate (KMnO?).
Procedure:
•Titrate the solution with a standard potassium permanganate (KMnO?) solution.
•The titration endpoint is reached when a faint pink color persists, indicating the presence of free calcium ions.
RESULT
? Calcium Oxalate Precipitation:
Successful formation of calcium oxalate stones was achieved through Homogeneous precipitation using calcium chloride and sodium oxalate.
? Semipermeable Membrane:
The egg membrane was successfully prepared and treated, ensuring its Integrity for use in the experiment.
? Titrimetric Estimation:
After incubation, the titration with KMnO? indicated a reduction in calcium Oxalate levels in the solutions treated with Potassium Citrate and Allopurinol.
? Both Potassium Citrate and Allopurinol showed a significant reduction in Calcium oxalate formation, confirming their effectiveness in preventing kidney Stone formation.
CONCLUSION:
This project successfully demonstrated the use of an egg membrane model to study kidney Stone formation in a controlled laboratory setting. The experimental method of preparing Calcium oxalate stones through homogeneous precipitation and using the semipermeable egg Membrane was effective for simulating kidney stone formation and mimicking renal Conditions. The titrimetric method for quantifying calcium oxalate allowed for precise Measurement of the stone formation process, making it a reliable tool for assessing the impact Of therapeutic agents on kidney stones.The results of the study suggest that potassium citrate and allopurinol can play significant Roles in managing kidney stones. Potassium citrate works by increasing urinary citrate levels, Which bind to calcium ions and prevent the formation of calcium oxalate stones. Allopurinol, On the other hand, reduces uric acid levels, which is particularly beneficial for preventing uric Acid stones but may also help in reducing calcium oxalate stone formation due to changes in Urine composition.Together, these treatments have the potential to reduce the burden of kidney stones by Targeting different aspects of stone formation. The use of both agents in combination may Offer a more comprehensive approach to preventing stone formation, as they address both the Calcium oxalate and uric acid components of kidney stones.Overall, this project provides valuable insights into the mechanisms behind kidney stone Formation and highlights the potential benefits of using potassium citrate and allopurinol as Therapeutic agents. It lays the foundation for further research into optimizing treatment Protocols for individuals at risk of kidney stones, particularly those who experience recurrent Stone formation. Further studies could explore different dosages, combination therapies, and The impact of other compounds on kidney stone prevention and management.
REFERENCES
Vaishnavi Jadhav*, Rajashri Nimbalkar, Disha Gangule, Harshad Pawle, Vaibhav Dhage, To Study In Vitro Evaluation of Marketed Tablets for The Treatment of Urolithiasis, Int. J. of Pharm. Sci., 2024, Vol 2, Issue 12, 2070-2079. https://doi.org/10.5281/zenodo.14470715
10.5281/zenodo.14470715
