Evaluation of Antiurolithiatic activity of Celosia argentea seeds by UV Spectroscopic method

Urolithiasis, commonly known as kidney stone disease. It is a significant global health problem characterized by the formation of stones in the urinary tract due to the supersaturation of urine with stone-forming constituents such as calcium, oxalate, phosphate and uric acid. The pathophysiology of kidney stone formation involves nucleation, crystal growth, aggregation and retention within the renal tubules. Factors such as urinary pH imbalance, reduced citrate levels, oxidative stress, increase calcium levels and renal tubular injury contribute to stone formation. Additionally, metabolic disorders like hypercalciuria, hyperoxaluria and hypocitraturia play a crucial role in urolithiasis.

Several treatment are available for managing kidney stones, ranging from traditional approaches to minimally invasive and surgical interventions. Extracorporeal Shock Wave Lithotripsy (ESWL) is a non-invasive technique that uses shock waves to break into fragment stones, facilitating their natural removal through urine. However, ESWL may not be effective for harder or larger stones and can sometimes lead to renal injury. Ureteroscopy (URS) involves the insertion of a thin scope through the urethra to directly visualize and remove or break stones using laser lithotripsy. For larger renal stones, Percutaneous Nephrolithotomy (PNL) is often chosen, where a small cut is made in the back to access and remove stones using specialized instruments. In severe cases or when other methods fail, open surgery may be required, although it is less commonly performed due to advancements in minimally invasive procedures. Despite these interventions, reappearance rates remain high and complications such as infections, bleeding and renal damage require the examination of alternative and safer treatment options. (1) (2) Medicinal plants have gained increasing attention for their potential in preventing and managing urolithiasis due to their diuretic, anti-inflammatory and stone-dissolving properties. Celosia argentea, a plant belonging to the Amaranthaceae family, has been traditionally used for renal disorders. The seeds of Celosia argentea contain bioactive constituents such as flavonoids, saponins and alkaloids, which are reported to have antiurolithiatic properties. (3) (4)  This study aims to evaluate the antiurolithiatic activity of Celosia argentea seeds using a UV spectroscopic method. UV spectroscopy is a reliable analytical tool for quantifying the dissolution of kidney stone-forming constituents under in vitro conditions. By assessing the extract’s ability to inhibit or dissolve calcium oxalate crystals, this study seeks to provide scientific evidence supporting the traditional claims of Celosia argentea as a natural remedy for kidney stone.

MATERIALS AND METHODS:

Plant material collection and extraction:

The fresh plant seeds were collected from Maharastra state by gently uprooting the plant. Authenticated by­ Vilas M. Suroshe Msc., Phd (Ethnobotanist). Seeds collected are allow for shade drying process later grinded into fine powder. Extracted by reflux extraction technique by using 70% ethanol as solvent.

Apparatus used:

Reflux condenser, Mortar-pestle, UV Spectrophotometer A1900i Shimadzu

METHOD:

Preparation of Extracts:

The seeds of Celosia argentea were collected from Thane district of Maharastra during the month of October 2024. Seeds of celosia argentea were shade dried and grinded to obtain fine powder which then stored in closed container in dark place. The 20 gm of powdered were subjected to reflux extraction in 200 mL of 70% ethanol for 2-4 hours. The obtained extract were filtered and filtrate were evaporated to viscous mass, % yield was calculated and used for further study.

Nucleation assay

The nucleation assay was performed to evaluate the inhibitory effect of Celosia argentea seed extract on calcium oxalate crystal formation. This assay mimics the early stages of kidney stone formation, where calcium and oxalate ions supersaturate and nucleate into crystals.

Materials and Reagents

Calcium chloride (CaCl?) – 5 mmol/L

Sodium oxalate (Na?C?O?) – 7.5 mmol/L

Tris-HCl buffer (0.05 mol/L, pH 6.5)

NaCl solution (0.15 mol/L)

Test extract solutions of Celosia argentea (100, 300, 500, 700, 1000 µg/ml)

Preparation of Solutions:

5 mmol/L CaCl? and 7.5 mmol/L Na?C?O? solutions were prepared in 0.05 mol/L Tris-HCl buffer (pH 6.5) containing 0.15 mol/L NaCl to maintain ionic strength.

Reaction Setup:

Equal volumes of CaCl? and Na?C?O? (950mL) solutions were mixed in a reaction medium to initiate calcium oxalate crystallization.

The reaction was carried out in the absence (control) and presence of different concentrations of Celosia argentea seed extract (100mL).

Spectrophotometric Measurement:

The rate of nucleation was monitored by measuring the absorbance at 621 nm using a UV-visible spectrophotometer.

A decrease in absorbance compared to the control indicated inhibition of crystal nucleation by the extract.

Calculation of turbidity: The turbidity of calcium oxalate in nucleation was calculated using the formula:

Turbidity = 2.3 X Abs / L

Aggregation Assay

The aggregation assay was conducted to evaluate the ability of Celosia argentea seed extract to inhibit the growth and clustering of preformed calcium oxalate monohydrate (COM) crystals. Crystal aggregation plays a crucial role in kidney stone formation, as larger aggregates increase the risk of urinary obstruction and stone retention in the renal system.

Materials and Reagents

Calcium chloride (CaCl?) – 50 mmol/L

Sodium oxalate (Na?C?O?) – 50 mmol/L

Tris-HCl buffer (0.05 mol/L, pH 6.5)

NaCl solution (0.15 mol/L)

Preformed calcium oxalate monohydrate (COM) crystals

Test extract solutions of Celosia argentea (100, 300, 500, 700, 1000 µg/ml)

Preparation of Calcium Oxalate Monohydrate (COM) Crystals:

Equimolar solutions of 50 mmol/L CaCl? and 50 mmol/L Na?C?O? were mixed in Tris-HCl buffer (pH 6.5) containing 0.15 mol/L NaCl to induce calcium oxalate crystal formation.

The formed crystals were filtered and collected to prepare 0.8 mg/ml solution.

Reaction Setup:

The crystal solution was incubated with different concentrations of Celosia argentea seed extract.

A control sample was prepared without the extract for comparison.

Spectrophotometric Measurement:

The degree of crystal aggregation was monitored by measuring absorbance at 621 nm using a UV-visible spectrophotometer against extract concentrations.

A decrease in absorbance indicated inhibition of crystal aggregation by the extract.

Calculation of Aggregation Inhibition Percentage:

The percentage inhibition of crystal aggregation was calculated using the formula:

I %= A control – A test/Abs control X 100

Where, A_control is the absorbance of the control (without extract), and A_test is the absorbance in the presence of the extract. (5)

RESULT AND DISCUSSION:

Determination of λ max:

Take the plant seed extract and dilute to 100 µg/mL, 300 µg/mL, 500 µg/mL. Scanned 3 samples by UV and λ max was found at 621 nm.

Turbidity calculation by Nucleation assay:

The formula for turbidity as per Lambered and Beers law:

Turbidity = (2.3 X absorbance) / L

Table 1: Table for Nucleation assay

Fig 1: Effect of different concentration of seed extract in nucleation assay.

Aggregation assay:

The calcium oxalate monohydrate crystals prepared are taken in concentration on 0.8mg/ml as control. In control there is no herb extract was present.

Table 2: CaOx crystallization of control

Determination of Rate of Inhibition (IR):

Table 3: Effect of concentration of C. argentea extract on CaOx crystallization inhibition rate:

Fig 2: Effect of different concentration of seed extract in nucleation assay