Effect of Unex on ethylene glycol-induced urolithiasis in rats.

This study was aimed to evaluate the effectiveness of the Unex capsule on albino rats as a preventive agent against the development of kidney stones. The Unex capsule is a marketed product of Unijules Life Sciences, Nagpur, containing the extracts of Boerhaavia diffusa and Tribulus terrestris. Activity of Unex was studied using the ethylene glycol-induced urolithiasis model. Standard drug used was Cystone. Several parameters were used including urinary volume, urine pH, urine analysis, and serum analysis to assess the activity. The results indicated that the administration of Unex to rats with ethylene glycol-induced lithiasis significantly reduced and prevented the growth of urinary stones (P < 0.01). Also, the treatment of lithiasis-induced rats by Unex restored all the elevated biochemical parameters (creatinine, uric acid, and blood urea nitrogen), restored the urine pH to normal, and increased the urine volume significantly (P < 0.01) when compared to the model control drug. This study supports the usage of Unex in urolithiasis and the utility could further be confirmed in other animal models.

Introduction

Urinary stone disease has affected humankind since antiquity and can persist, with serious medical consequences, throughout the patient's lifetime. In addition, the incidence of kidney stones has been increased in western societies in the last five decades, in association with economic development. Most calculi in the urinary system arise from a common component of urine, calcium oxalate (CaO), representing up to 80% of analyzed stones.[1-3] This may cause obstruction, hydronephrosis, infection, and hemorrhage in the urinary tract system. Surgical operation, lithotripsy, and local calculus disruption using high-power laser are widely used to remove the calculi.[4] Many remedies have been employed since ages to treat renal stones and most of them were from plants and proved to be useful.[5-7] The present day medical management of nephrolithiasis is either costly or not without side effects.[8] The current study was aimed to evaluate the effectiveness of the Unex capsule on albino rats as a preventive agent against the development of kidney stones. The Unex capsule (Herbajules Tricare in Malaysia) is a marketed product of Unijules Life Sciences, Nagpur, containing the extracts of Boerhaavia diffusa and Tribulus terrestris.

Materials and Methods

Procurement of Materials

The product Unex containing the extracts of B. diffusa and T. terrestris was provided by the Unijules Life science Ltd. and Associated Companies, Nagpur. Cystone containing the extracts of Didymocarpus pedicellata, Saxifraga ligulata, Rubia cordifolia, Cyperus scariosus, Achyranthes aspera, Onosma bracteatum, Vernonia cinerea, Shilajeet (purified) and hajrul yahood bhasma was procured from Himalaya herbal health care, Bangalore. The urea kit, creatinine mono reagent test kit, and triglycerides test kit were procured from Central Store, BRNCOP.

Animals

Healthy adult male Wistar albino rats weighing between 150-200 g were selected for the antiurolithiatic activity. The animals were acclimatized to standard laboratory conditions (temperature: 25±2°C) and maintained on 12-h light:12-h dark cycle. They were provided with regular rat chow and drinking water ad libitum. The animal care and experimental protocols were approved by Institutional Animal Ethical Committee (IAEC) Reg no. (918/ac/05/CPCSEA).

Ethylene Glycol Induced Urolithiasis Model

Ethylene glycol-induced hyperoxaluria model was used to assess the antilithiatic activity in albino rats. Animals were divided into five groups containing six animals in each. Group I served as control and received regular rat food and drinking water ad libitum. Ethylene glycol (0.75%) in drinking water was fed to Groups II–V for induction of renal calculi for 28 days. Group III received standard antiurolithiatic drug, cystone (750 mg/kg body weight) from 15th till 28th day. Group IV and V received Unex at high dose (400 mg/kg) and low dose (200 mg/kg) (po) from 15th till 28th day.[19]

Collection and analysis of urine

All the animals were kept in individual metabolic cages and urine samples of 24 h were collected on the 28th day. Animals had free access to drinking water during the urine collection period. A drop of concentrated hydrochloric acid was added to the urine before being stored at 4°C. Urine was analyzed for calcium, phosphate, and oxalate content using the method of Bahuguna et al.[8]

Serum analysis

After the experimental period, blood was collected from the retro-orbital under anesthetic condition and animals were sacrificed by cervical decapitation. Serum was separated by centrifugation at 10 000 × g for 10 min and analyzed for creatinine, uricacid, and urea nitrogen using the method of Atef and Attar.[10]

Urine volume

Animals were placed in separate metabolic cages for 24 h and total urinary volume was measured using the measuring cylinder and reported in ml.[8]

Urine pH

Uric acid crystals were found to deposit most frequently in the concentrated acid urine. Thus, the acidity of the urine was tested using the pH meter.[8]

Statistical Analysis

Statistical evaluation was done using one-way analysis of variance (ANOVA) followed by the Bonferroni test. Statistical significant was set at P < 0.05. Results are presented as mean ± standard error of mean (SEM).

Results

In this study, the administration of 0.75% (v/v) ethylene glycol aqueous solution to male Wistar albino rats produced hyperoxaluria. Oxalate, calcium, and phosphate excretion were grossly increased in the calculi-induced animals However, supplementation with Unex significantly lowered the elevated level of oxalate, calcium, and phosphate in urine when compared to the model control group [Table 1].

Table 1

Effect of Unex on urinary salts of experimental animals

The level of serum uric acid, blood urea nitrogen (BUN), and creatinine were found to increase in the calculi-induced animal [Table 2]. In the case of Unex-treated groups, the treatment significantly (P < 0.001) lowered the elevated level of creatinine, uric acid, and BUN. Urine volumes were increased by Unex and the standard drug cystone compared to the model control group. Urinary pH was significantly increased in the animals treated with the 0.75% of ethylene glycol. Unex and the standard drug cystone significantly decreased the pH [Table 3]. No significant differences were observed in the activity of Unex between low dose and high dose.

Table 2

Effect of Unex on serum parameters of experimental animals

Table 3

Effect of Unex on urine volume of experimental animals

Discussion

Urinary supersaturation with respect to stone forming constituents is generally considered to be one of the causative factors in calculogenesis. Evidence in previous studies indicated that after 14 days period of ethylene glycol (0.75%, v/v) administration, renal calculi was formed in the young male albino rat composed mainly of calcium oxalate.[11] Stone formation in ethylene glycol-fed rats is caused by hyperoxaluria, which cause increased renal retention and excessive excretion of oxalate in urine.[8] In this study, oxalate and calcium excretion were increased in calculi-induced animals (Group II). An increase in urinary phosphate is also observed in calculi induced rats (Group II). Increased urinary phosphate excretion along with oxalate stress seems to provide an environment appropriate for stone formation by forming calcium phosphate crystals, which induce calcium oxalate deposition. Treatment with the Unex restored the phosphate level, thus reducing the risk of stone formation.[12] In urolithiasis, the glomerular filtration rate (GFR) decreases due to the obstruction to the outflow of urine by stones in the urinary system. Due to this, the waste products, particularly nitrogenous substances such as urea, creatinine, and uric acid get accumulated in blood. Also, increased lipid peroxidation and decreased levels of antioxidant potential have been reported in the kidneys of rats supplemented with a calculi producing diet. In this context, oxalate has been reported to induce lipid peroxidation and to cause renal tissue damage by reacting with polyunsaturated fatty acids in the cell membrane. In calculi-induced rats (Group II), marked renal damage was seen by the elevated serum levels of creatinine and uric acid, and BUN.[10] However, the curative treatment with product unex and cystone caused diuresis and hastened the process of dissolving the preformed stones and prevention of new stone formation in the urinary system. The diuretic effect of Unex was evident from urine volumes collected when compared to the model control group.

Conclusion

In conclusion, the results indicated that the administration of Unex to rats with ethylene glycol-induced lithiasis reduced the growth of the urinary stone. The dose requirement of Unex to produce the activity is less than the standard drug used.