INTRODUCTION:

Despite the inception of new technologies and therapeutic advancements unfolding with every year passing by, no drug yet has proved utterly satisfactory, in managing and controlling the recurrent condition of Urolithiasis [1,2]. Urolithiasis, a condition refers to the presence of uroliths i.e. stones, crystals or calculi in the kidneys, bladder or anywhere else in the urinary tract [3, 4]. Based upon its location, the prevalent it can be broadly known as nephrolithiasis (formation of stone in the kidney), ureterolithiasis (formation of stone in the ureter) and cystolithiasis (formation of stone in the bladder).

Urinary stones are highly prevalent in India as it affects 15% of population which leads to loss of one or both kidneys. Northern part of India encounters 15% cases suffering from kidney stones [5,6]. Though the introduction of Extracorporeal Shock Wave Lithotripsy (ESWL) in 1980s was a noteworthy advancement, however, besides the traumatic effect of shockwaves, ESWL can cause acute renal injury, a decrease in renal function, an increase in stone recurrence, hypertension, hemorrhage etc. Moreover, it's the recrudescence of urolithiasis (70-81% in males and 47-60% in females) which poses a major challenge for pharmacologists [6, 7]. Hence, the latent potential for a safer and effective antiurolithiatic drug from natural sources has lately witnessed attention of many.

Terminalia arjuna is a deciduous and evergreen tree belonging to Combretaceae family. It contains an umpteen number of phytoconstituents like alkaloids, flavonoids, terpenoids, tannins, glycosides, polyphenols and saponin. It has been reported to possess wound healing [8], antidyslipidemic and antioxidant activities [9], cardio protective activity [10], anthelmintic activity [11], hypocholesterolaemic effects [12], analgesic and anti-inflammatory [13], antimicrobial [14] activity etc. Its bark extract has been found to be effective in protecting liver and kidney against CCl₄induced oxidative damage [15]. Moreover, it also inhibited the formation of calcium oxalate and calcium phosphate crystals in vitro [16]. However so far, no pharmacological experimentation has been performed to prove the speculations about its antiurolithiatic property in vivo.

Therefore, in the subsequent study and analysis, an effort has been made to establish the scientific validity for the antiurolithiatic property of Terminalia arjuna bark extract using ethylene glycol (EG) induced hyperoxaluria model in rats.

MATERIALS AND METHODS:

Procurement of plant material and preparation of extract:

The plant was collected from local region of Lovely Professional University, Phagwara, Punjab in the month of August and shade dried. The obtained sample of collected plant material was authenticated at Guru Nanak Dev University, Amritsar with a reference no. 1044. The dried barks were coarsely powdered and passed through sieve no. 40. Further the matter was packed in column for Soxhlet extraction with 95% (v/v) ethanol at 70–75 °C for 22 h [10]. The obtained extract (yield 47.22%, w/w) was evaporated at 45°C, then dried and stored in airtight container for further study.

Procurement of chemical and apparatus:

EG was procured from Loba Chemie Pvt. Ltd, Mumbai. Catalase, GSH and MDA were obtained from HiMedia Laboratories Pvt. Ltd., Mumbai. All other chemicals and reagents used were analytical grade and procured from approved chemical suppliers. Various kits used for biochemical estimations were purchased from Tulip Diagnostics (P) Ltd, Mumbai. Apparatus such as cold centrifuge (Remi Instruments, India), UV-spectrometer (Shimadzu Scientific Instruments), semi autoanalyzer (Piramal Healthcare) were used in the study.

Animals used:

Male Wistar albino rats weighing between 150–200g each were procured from National Institute of Pharmaceutical Education and Research (NIPER), Mohali, India used for present investigation. They were housed in polypropylene cages and maintained at 27± 2⁰C, relative humidity 65±10% under 12 h light/dark cycles. The animals were fed regularly with diet and water ad libitum. The protocol was approved by Institutional Animals Ethics Committee (954/ac/06/CPCSEA/11/18) constituted in accordance with the rules and guidelines of the CPCSEA (Committee for the purpose of Control and Supervision of Experiments on Animals), India.

In vitro antiurolithiatic activity:

The effect of extract on Calcium Oxalate (CaOx) crystallization was determined on artificial urine (AU) as described by Burns and Finlayson, 1980 [17]. One ml of AU was transferred into the cell further 0.5ml of distilled water was added to take blank reading. Then, 0.5 ml of 0.01M sodium oxalate was added and the absorbance measurement was recorded immediately started for a period of ten minutes. After that, the different concentrations of 0.5 ml of aqueous solution of extract (100, 200, 400, 800 and 1000μg/ml) were mixed with one ml of AU and a blank reading was taken. Six replicates were taken for samples of different concentrations and the percentage of inhibition were calculated [18].

In vivo Antiurolithiatic activity:

Ethylene glycol induced urolithiatic model-EG and ammonium chloride induced hyperoxaluria model was used to induce urolithiasis [19,20]. The animals were divided into eight groups (n=6). Group I (Normal) received regular food and distilled water ad libitum. Group II to VIII received renal calculi inducing treatment for 28 days, comprising of 0.75% v/v EG with 1% w/v ammonium chloride in distilled water ad libitum for 3 days to accelerate lithiasis followed by only 0.75% v/v EG for 25 days. Group II served as lithiatic control. Group III (preventive standard) received cystone (750 mg/kg body weight) from 1^st day to 28^th day of calculi induction. Group IV (preventive low dose) and Group V (preventive high dose) received oral EETA at 200 and 400 mg/kg body weight respectively in distilled water from 1^st day to 28^th day of calculi induction. Group VI (curative standard) received cystone (750 mg/kg body weight) in distilled water from 15^th day to 28^th day of calculi induction. Group VII (curative low dose) and Group VIII (curative high dose) received oral EETA at 200 and 400 mg/kg body weight respectively in distilled water from 15^th day to 28^th day of calculi induction. On 14th and 28th day of study, rats were weighed in order to analyze any significant change in their body weight.

Collection and analysis of urine:

Urine samples were collected on 0, 14^th and 28^th day of induction of urolithiasis and analyzed for urinary volume and calcium, magnesium, uric acid. Oxalate level was estimated using the method described by Hodgkinson, (1970) [21].

Serum analysis:

After the 28^th day of experimental period, rats were anaesthetized and blood was collected from the retro-orbital region without adding anticoagulant. Blood serum was collected after centrifugation for 10 min and analyzed for calcium, BUN, uric acid, and creatinine using kits.

Kidney homogenate analysis-

The rats were sacrificed by cervical dislocation; both kidneys were isolated and cleaned off extraneous tissue and rinsed in ice-cold physiological saline. The left kidney was weighed and then minced in a beaker and 20% homogenate was prepared in Tris-HCl buffer (0.02 mol/l, pH 7.4). The homogenate was used for assaying tissue calcium, with the help of kit using the method described by Khan et al. (2010) [22]. The kidney homogenate was also estimated for the malondialdehyde (MDA) content [23], reduced glutathione (GSH) [24] and Catalase level [23,25].

Histopathological Study of Kidney-

The right kidney was fixed in 10% neutral buffered formalin, processed in a series of graded alcohol and xylene, embedded in paraffin wax, sectioned at 5µm and stained with hematoxylin and eosin for histopathological examination [22].

Statistical analysis-

The obtained results has been expressed as mean ± SEM. The statistical significance was assessed using one-way analysis of variance (ANOVA) followed by Dunnett’s comparison test and level of significance set at p < 0.01.

RESULTS:

In vitro antiurolithiatic activity using AU:

There was a detectable increase in the turbidity of AU after induction of the crystallization with sodium oxalate. However, AU mixed with the extract showed a very less turbidity change (Figure 1). The percent inhibition in crystallization was found to be 51.56, 57.20, 62.15, 69.70 and 80.81% for 100, 200, 400, 800, and 1000µg/ml extract respectively.

In vivo Antiurolithiatic activity:

Table 1 represents the percent change in the body weights of all the animals on 14^th and 28^th day of treatment. The animals in lithiatic control group lost their body weights significantly as compared to normal animals. The animals in preventive groups showed significant increase in their body weight as compared to the lithiatic control group after two weeks of extract treatment. However, in the curative group of animals, there was significant gain in weight after four weeks of extract treatment.

Figure 1. The effect of different concentrations of EETA on CaOX crystal nucleation in AU.

Table 1. Percentage changes in body weight of the rat groups over 14 and 28 days of the experiment.

Groups (n=6)	Relative Body Weight (%)
Groups (n=6)	On 14^th day	On 28^th day
I	22.72 ± 2.83	34.88 ± 2.36
II	-12.83 ± 2.39^a***	-28.77 ± 1.98^a***
III	13.37 ± 1.10^a,b*	30.89 ± 1.98^b***
IV	5.49 ± 0.93^a*,b*	17.25 ± 0.52^a*,b*
V	11.13 ± 1.52^a*,b*	2.48 ± 2.70^b***
VI	-13.83 ± 1.43^a***	15.48 ± 3.21^a*,b*
VII	-12.90 ± 1.87^a***	0.29 ± 3.53^a*,b*
VIII	-14.41 ± 1.69^a***	2.47 ± 2.70^a*,b*

Values are expressed as mean ± SEM (n=6), ^***p < 0.001, ^**p < 0.01, ^acompared with normal,^bcompared with lithiatic control.

In spite of the EG administration, the urine output was significantly increased in all the preventive groups as compared to the lithiatic group (Table 2). In the curative groups, urine output was almost same to the lithiatic group on the 14^th day of the treatment but was markedly increased on the 28^th day.

Table 2. Effect of EETA on the urinary output in urolithiasis induced model.

Groups	Urinary output (mL/24h)
Groups	0 DAY	14 DAY	28 DAY
I	5.25 ± 0.30	5.53 ± 0.29	5.25 ± 0.21
II	5.25 ± 0.31	2.2 ± 0.061^a***	1.19 ± 0.04^a***
III	4.91 ± 0.55	4.82 ± 0.32^b***	4.91 ± 0.22^b***
IV	5.41 ± 0.41	3.13 ± 0.06^{a*, b}	3.28 ± 0.05^{a*, b*}
V	4.91 ± 0.49	3.85 ± 0.23^{a*, b*}	3.91 ± 0.09^{a*, b*}
VI	5.08 ± 0.45	2.75 ± 0.08^a***	2.78 ± 0.03^{a*, b*}
VII	4.81 ± 0.45	1.83 ± 0.14^a***	2.11 ± 0.06^{a*, b*}
VIII	5.03 ± 0.36	2.36 ± 0.08^a***	2.52 ± 0.05^{a*, b*}

Values are expressed as mean ± SEM (n=6), ^***p < 0.001, ^**p < 0.01, ^acompared with normal,^bcompared with lithiatic control.

The administration of 0.75% v/v EG along with 1% ammonium chloride resulted in hyperoxaluria in rats as represented in Table 3. The preventive groups remain protected due to continuous administration of cystone and EETA throughout the experimental period. However, the curative groups showed significant protection after four weeks of treatment.

Table 3. Effect of EETA on urinary oxalate, uric acid, calcium and magnesium levels in urolithiasis induced rats.

Days	Groups
Days	I	II	III	IV	V	VI	VII	VIII
Oxalate (mg/24 h)
0	0.34±0.01	0.33±0.01	0.33±0.02	0.33±0.01	0.034±0.01	0.34±0.01	0.33±0.01	0.34±0.01
14	0.30±0.01	3.25±0.26^a***	1.04±0.02^b***	2.1±0.16^a*,b	2.06±0.27^a*,b	3.17±0.34^a***	3.09±0.21^a***	3.32±0.26^a***
28	0.33±0.01	3.78±0.34^a***	1.11±0.15^b***	1.91±0.05^a*,b*	1.71±0.05^a*,b*	1.95±0.15^a*,b*	2.66±0.34^a*,b	2.45±0.27^a*,b*
Uric acid (mg/24 h)
0	1.33±0.02	1.31±0.02	1.31±0.01	1.31±0.02	1.31±0.01	1.32±0.02	1.30±0.01	1.3±0.02
14	1.29±0.02	1.91±0.05^a***	1.36±0.07^b***	1.63±0.04^a*,b	1.56±0.03^a,b*	1.73±0.07^a***	1.81±0.07^a***	1.76±0.05^a***
28	1.28±0.02	1.98±0.04^a***	1.32±0.03^b***	1.61±0.07^a*,b*	1.57±0.06^a,b*	1.63±0.07^a*,b*	1.65±0.05^a*,b*	1.61±0.05^a*,b*
Calcium (mg/24 h)
0	1.20±0.00	1.20±0.01	1.20±0.00	1.21±0.00	1.20±0.01	1.20±0.00	1.20±0.01	1.21±0.01
14	1.21±0.01	3.23±0.15^a***	1.83±0.22^b***	2.34±0.09^a*,b	2.12±0.03^a,b*	3.38±0.25^a***	3.42±0.25^a***	3.16±0.22^a***
28	1.22±0.01	4.29±0.33^a***	1.70±0.27^b***	2.39±0.11^a,b*	2.35±0.13^a,b*	2.41±0.13^a,b*	3.16±0.32^a*,b	2.99±0.21^a*,b*
Magnesium (mg/24 h)
0	2.63±0.07	2.69±0.07	2.69±0.06	2.71±0.05	2.73±0.04	2.69±0.06	2.73±0.04	2.71±0.05
14	2.74±0.04	1.55±0.07^a***	2.64±0.11^b***	1.94±0.07^a*,b*	2.06±0.09^a*,b*	1.92±0.01^a*,b	1.89±0.02^a*,b	1.91±0.02^a*,b
28	2.72±0.04	1.41±0.02^a***	2.71±0.02^b***	2.15±0.04^a*,b*	2.27±0.07^a*,b*	2.12±0.02^a*,b*	1.88±0.15^a*,b*	2.08±0.02^a*,b*

Values are expressed as mean ± SEM (n=6), ^***p < 0.001, ^**p < 0.01, ^acompared with normal,^bcompared with lithiatic control.

Table 4. Effect of EETA on serum in experimental hyperoxaluria.

Groups	BUN (mg/dl)	Creatinine (mg/dl)	Calcium (mg/dl)	Uric acid (mg/dl)
I	33.04±0.63	0.65± 0.01	5.05± 0.30	1.34±0.01
II	51.87±0.93^a***	3.33±0.33^a***	14.82±0.32^a***	2.45±0.03^a***
III	37.04±1.25^b***	1.33±0.22^b***	6.91±0.45^b***	1.40±0.03^b***
IV	43.19±1.59^a*,b*	2.22±0.23^a*,b	8.91±0.70^a*,b*	1.59±0.02^{a*, b*}
V	41.04±1.98^a*,b*	1.84±0.16^a,b*	7.74±0.45^a,b*	1.53±0.03 ^{a*, b*}
VI	43.42±1.49^a*,b*	2.24±0.22^{a*, b}	9.58±0.89^a*,b*	1.61±0.01^{a*, b*}
VII	45.26± 0.80^a*,b	2.31±0.10^a*,b	11.23± 0.35^a*,b*	1.81±0.02^{a*, b*}
VIII	44.09±1.22^a*,b*	2.29±0.24^a*,b	10.75±0.49^a*,b*	1.77±0.03^{a*, b*}

Values are expressed as mean ± SEM (n=6), ^***p < 0.001, ^**p < 0.01, ^acompared with normal,^bcompared with lithiatic control.

Table 5. Effect of EETA on CAT, GSH, and MDA in EG induced urolithiasis on tissue homogenate

Groups	Calcium (mg/g)	Oxalate (mg/g)	CAT (μmol H₂O₂/min/100 mg protein)	GSH (μmol/100mg)	MDA (nmol/mg)
I	1.45±0.04	1.48±0.04	41.89 ± 0.94	152.83±2.72	15.01±0.64
II	5.90±0.21^a***	6.01±0.23^a***	11.95 ± 0.76^a***	39.79±2.31^a***	96.86±2.99^a***
III	2.64±0.11^a,b*	2.94±0.30^a*,b*	35.68±0.66^a*,b*	138.09±2.59^a,b**	25.04±2.63^a,b*
IV	3.01±0.26^a*,b*	3.27±0.25^a*,b*	27.04±0.48^a*,b*	109.29±2.21^a*,b*	49.28±2.63^a*,b*
V	2.94±0.30^a*,b*	3.10±0.29^a*,b*	31.11±0.42^a*,b*	122.33±2.79^a*,b*	33.28±1.31^a*,b*
VI	3.03±0.26^a*,b*	3.22±0.25^a*,b*	24.33±0.57^a*,b*	91.83±2.16^a*,b*	56.89±0.67^a*,b*
VII	3.28±0.32^a*,b*	3.55±0.13^a*,b*	15.72±0.61^a*,b	49.54±5.12^a***	80.22±0.49^a*,b*
VIII	3.11±0.29^a*,b*	3.39±0.26^a*,b*	21.51±0.76^a*,b*	73.46±4.30^a*,b*	65.07±1.88^a*,b*

Values are expressed as mean ± SEM (n=6), ^***p < 0.001, ^**p < 0.01, ^*p < 0.05, ^acompared with normal,^bcompared with lithiatic control.

DISCUSSION:

Urolithiasis or kidney stone formation is the consequence of an imbalance between promoters and inhibitors in the kidney. Its recurrence is the serious concern as the patients who have formed one stone are more likely to form another. Human kidney stones are usually composed of CaOx [26,27].

Male rats were selected to induce urolithiasis because the urinary system of male rats resembles that of humans and earlier studies shown that the amount of stone deposition in female rats was significantly less [19,20]. The most common agent used for the development and progression of urolithiasis in animals is EG [4]. EG undergoes metabolism in the liver to glycoaldehyde acid, glycolic acid, glyoxylic acid and oxalic acid. The formed oxalic acid is excreted as oxalate in urine, where it is precipitated by calcium ions to form CaOx crystals [28]. The EG induced lithiasis can be accelerated by administration of ammonium chloride [29]. Therefore, this model was selected to evaluate the effect of plant extract against urolithiasis.

The basic mechanism behind EG induced calculi is hypercalciuria and hyperoxaluria leading to CaOx crystal formation. In addition to development of oxalate crystal, it is also associated with severe oxidative stress to renal tissue. In urolithiasis, the glomerular filtration rate (GFR) decreases due to stones in the urinary system obstructing urine outflow. This leads to the accumulation of waste products in the blood, particularly nitrogenous substances such as urea, creatinine and uric acid [30]. In addition, increased lipid peroxidation and decreased levels of antioxidant potential have been reported in the kidneys of rats supplemented with a calculi-producing diet. Oxalate has been reported to induce lipid peroxidation and causes renal tissue damage by reacting with polyunsaturated fatty acids in cell membranes and by generation of reactive oxygen species like hydroxyl and superoxide ions. Due to renal papillary hypertrophy and crystal depositions, kidney weight increases in EG treated rats. It increases blood urea and creatinine levels significantly due to impairment in renal function [19,20, 26,28,31-33]

There was a remarkable loss in the relative body weights of lithiatic control animals which might be due to disturbances in metabolism of carbohydrates, proteins or fat [31]. When compared to lithiatic control group, all the treatment groups showed a significant gain in the relative body weights which may be due to the fact that plant extract stimulates the absorption or metabolism of nutrients.

The extract has increased the urine volume in preventive as well as curative groups indicating its diuretic activity. Chemistry of urine is one of the important factors in determining the type of crystal formed and the nature of macromolecules included on the surface of the crystals. Hence, urinary chemistry related to the calculi forming minerals was studied in order to provide a good indication of the extent of stone formation.

The increased urinary calcium is a factor favoring the nucleation and precipitation of calcium oxalate from urine and subsequently crystal growth. As consistent with the previous reports, EG was found to produce renal calculi, composed of mainly calcium oxalate, along with an increase in the level of calcium, phosphate and oxalate in the urine of lithiatic control rats. Treatment with the plant extract reduced the urinary excretion of calcium and oxalate decreasing the super-saturation of urine. Similarly, administration of EG also led to an increase in the excretion of uric acid which has been previously found to promote stone formation by altering calcium oxalate solubility. Treatment with extract and standard drug lowered the risk of stone formation by reducing the uric acid excretion.

Magnesium is one of the well-known inhibitors of crystallization found in normal urine. It complexes with oxalate reducing the growth and nucleation rate of calcium oxalate crystals [20]. In the lithiatic control group, the level of magnesium was found to be very low. However, it was significantly increased in standard drug and extract treated groups.

Furthermore, the induction of nephrotoxicity led to marked elevation of serum BUN, calcium, uric acid and creatinine which are markers of glomerular and tubular damage. A remarkable decrease in BUN, calcium, uric acid and creatinine was observed in the groups treated with extract and standard drug. Also, there was an increased deposition of the crystalline components (calcium and oxalate) in the renal tissue of stone forming animals (Group II). The EETA treatment significantly reduced the stone forming constituents in renal tissue in both the regimens. Since urolithiasis is the third most common disorder of the urinary tract [34] and here reduction in oxalate and calcium level shows the efficiency of the plant extract in prevention of stone formation. In addition to the development of oxalate and calcium crystals, EG is also associated with severe oxidative stress in the renal tissue that leads to renal dysfunction [28].

Specifically, oxalate has been reported to induce lipid peroxidation and cause tissue damage by reacting with polyunsaturated fatty acids in cell membranes. In the present study, a significant increase in LPO and decrease in GSH and catalase were observed in the lithiatic control group. Treatment with the plant extract significantly increased the levels of catalase and GSH along with a decrease in LPO as compared to lithiatic control group. Microscopic examination of kidney sections derived from EG induced urolithic rats showed polymorphic irregular crystal deposits inside the tubules which cause dilation of the proximal tubules along with interstitial inflammation that might be attributed to oxalate. Co-treatment with the extract decreased the calcium oxalate deposits in different parts of the renal tubules and also prevented damages to the tubules.

CONCLUSION:

As a conclusion, the presented data indicated that administration of EETA to rats with EG induced lithiasis reduced and prevented the growth of urinary stones. The plant extract is more effective in preventing the formation of kidney stones rather than treating the same. The mechanism underlying this effect is still unknown, but is apparently related to lowering the concentrations of stone forming constituents in the urine. This investigation could be regarded as preliminary probes, requiring extensive studies to establish the molecular mechanism for its urolithiatic activity. Prospective studies should include among other investigations.

ACKNOWLEDGMENT:

Authors are thankful to second International Conference of Pharmacy, held by School of Pharmaceutical Sciences, Lovely Professional University on September 13-14, 2019 to fund the publication of this manuscript.

REFERENCES:

1. S. Mitra, S. Gopumadhavan, M. Venkataranganna, and R. Sundaram, “Effect of cystone, a herbal formulation, on glycolic acid‐induced urolithiasis in rats,” Phytotherapy Research: An International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives, vol. 12, no. 5, pp. 372-374. 1998.

2. A. Makasana, V. Ranpariya, D. Desai, J. Mendpara, and V. Parekh, “Evaluation for the anti-urolithiatic activity of Launaea procumbens against ethylene glycol-induced renal calculi in rats,” Toxicology reports, vol. 1, pp. 46-52. 2014.

3. A. P. Evan, “Physiopathology and etiology of stone formation in the kidney and the urinary tract,” Pediatric Nephrology, vol. 25, no. 5, pp. 831-841. 2010.

4. Y. Bayir, Z. Halici, M. S. Keles, S. Colak, A. Cakir, Y. Kaya, and F. Akçay, “Helichrysum plicatum DC. subsp. plicatum extract as a preventive agent in experimentally induced urolithiasis model,” Journal of ethnopharmacology, vol. 138, no. 2, pp. 408-414. 2011.

5. S. K. Gupta, M. S. Baghel, C. Bhuyan, B. Ravishankar, B. Ashok, and P. D. Patil, “Evaluation of anti-urolithiatic activity of Pashanabhedadi Ghrita against experimentally induced renal calculi in rats,” Ayu, vol. 33, no. 3, pp. 429. 2012.

6. R. Ganesamoni, and S. K. Singh, "Epidemiology of stone disease in Northern India," Urolithiasis, pp. 39-46: Springer, 2012.

7. S. Jafar, L. Mehri, B. Hadi, and M. Jamshid, “The antiurolithiasic and hepatocurative activities of aqueous extracts of Petroselinum sativum on ethylene glycol-induced kidney calculi in rats,” Scientific Research and Essays, vol. 7, no. 15, pp. 1577-1583. 2012.

8. M. M. Rane, and S. A. Mengi, “Comparative effect of oral administration and topical application of alcoholic extract of Terminalia arjuna bark on incision and excision wounds in rats,” Fitoterapia, vol. 74, no. 6, pp. 553-558. 2003.

9. R. Chander, K. Singh, A. Khanna, S. Kaul, A. Puri, R. Saxena, G. Bhatia, F. Rizvi, and A. K. Rastogi, “Antidyslipidemic and antioxidant activities of different fractions ofTerminalia arjuna stem bark,” Indian Journal of Clinical Biochemistry, vol. 19, no. 2, pp. 141. 2004.

10. K. Gauthaman, T. Mohamed Saleem, V. Ravi, P. Sita, and D. Niranjali, “Alcoholic Extract of Terminalia Arjuna Protects Rabbit Heart against Ischemic-Reperfusion Injury, Role of Antioxidant Enzymes and Heat Shock Protein,” World Acad Sci Eng Technol, vol. 18, pp. 488-98. 2008.

11. H. A. Bachaya, Z. Iqbal, M. N. Khan, A. Jabbar, A. H. Gilani, and I.-u. Din, “In vitro and in vivo anthelmintic activity of Terminalia arjuna bark,” International Journal of Agriculture & Biology, vol. 11, pp. 273. 2009.

12. A. Ram, P. Lauria, R. Gupta, P. Kumar, and V. Sharma, “Hypocholesterolaemic effects of Terminalia arjuna tree bark,” Journal of Ethnopharmacology, vol. 55, no. 3, pp. 165-169. 1997.

13. M. Biswas, K. Biswas, T. K. Karan, S. Bhattacharya, A. K. Ghosh, and P. K. Haldar, “Evaluation of analgesic and anti-inflammatory activities of Terminalia arjuna leaf,” Journal of Phytology. 2011.

14. M. A. Morshed, A. Uddin, A. Rahman, T. Hasan, S. Roy, A. Amin, R. Ahsan, and R. Islam, “In vitro antimicrobial and cytotoxicity screening of Terminalia arjuna ethanol extract,” International Journal of Biosciences, vol. 1, no. 2, pp. 31-38. 2011.

15. P. Manna, M. Sinha, and P. C. Sil, “Aqueous extract of Terminalia arjuna prevents carbon tetrachloride induced hepatic and renal disorders,” BMC complementary and alternative medicine, vol. 6, no. 1, pp. 33. 2006.

16. A. Chaudhary, S. Singla, and C. Tandon, “In vitro evaluation of Terminalia arjuna on calcium phosphate and calcium oxalate crystallization,” Indian journal of pharmaceutical sciences, vol. 72, no. 3, pp. 340. 2010.

17. J. Burns, and B. Finlayson, “A proposal for a standard reference artificial urine in in vitro urolithiasis experiments,” Investigative urology, vol. 18, no. 2, pp. 167-169. 1980.

18. G. P. Kumar, M. Arun, and K. Rishi, “Evaluation of Tinospora cordifolia for antiurolithiatic potential,” Journal of Pharmaceutical and Biomedical Sciences, vol. 9, no. 14, pp. 1-5. 2011.

19. R. V. Karadi, N. B. Gadge, K. Alagawadi, and R. V. Savadi, “Effect of Moringa oleifera Lam. root-wood on ethylene glycol induced urolithiasis in rats,” Journal of ethnopharmacology, vol. 105, no. 1-2, pp. 306-311. 2006.

20. K. Divakar, A. Pawar, S. Chandrasekhar, S. Dighe, and G. Divakar, “Protective effect of the hydro-alcoholic extract of Rubia cordifolia roots against ethylene glycol induced urolithiasis in rats,” Food and chemical toxicology, vol. 48, no. 4, pp. 1013-1018. 2010.

21. A. Hodgkinson, “Determination of oxalic acid in biological material,” Clinical Chemistry, vol. 16, no. 7, pp. 547-557. 1970.

22. N. Khan, J. Shinge, and N. Naikwade, “Antilithiatic effect of Helianthus annuus Linn. Leaf extract in ethylene glycol and ammonium chloride induced nephrolithiasis,” Int J Pharm Pharm Sci, vol. 2, no. 2010, pp. 181. 2010.

23. S. Kaur, A. Rana, S. Gangwani, and R. Sharma, “Punica granatum attenuates sciatic nerve ligation induced-neuropathic pain,” International Journal of Pharmaceutical Sciences and Research, vol. 3, no. 2, pp. 509. 2012.

24. G. Ellman, “Archs: Tissue sulfhydril groups,” Biochem Biophys, vol. 82, pp. 70-77. 1959.

25. A. Ray, S. R. Chaudhuri, B. Majumdar, and S. K. Bandyopadhyay, “Antioxidant activity of ethanol extract of rhizome of Picrorhiza kurroa on indomethacin induced gastric ulcer during healing,” Indian Journal of Clinical Biochemistry, vol. 17, no. 2, pp. 44-51. 2002.

26. M. Touhami, A. Laroubi, K. Elhabazi, F. Loubna, I. Zrara, Y. Eljahiri, A. Oussama, F. Grases, and A. Chait, “Lemon juice has protective activity in a rat urolithiasis model,” BMC urology, vol. 7, no. 1, pp. 18. 2007.

27. A. Khajavi Rad, M.-A.-R. Hajzadeh, Z. Rajaei, M.-H. Sadeghian, N. Hashemi, and Z. Keshavarzi, “Preventive effect of Cynodon dactylon against ethylene glycol-induced nephrolithiasis in male rats,” Avicenna Journal of Phytomedicine, vol. 1, no. 1, pp. 14-23. 2011.

28. A. Shukla, D. Mandavia, M. Barvaliya, S. Baxi, and C. Tripathi, “Anti-urolithiatic effect of cow urine Ark on ethylene glycol-induced Renal Calculi,” International braz j urol, vol. 39, no. 4, pp. 565-571. 2013.

29. J. Fan, M. A. Glass, and P. S. Chandhoke, “Impact of ammonium chloride administration on a rat ethylene glycol urolithiasis model,” Scanning Microsc, vol. 13, no. 2-3, pp. 299-306. 1999.

30. D. A. K. Chaitanya, M. Kumar, A. Reddy, N. Mukherjee, M. Sumanth, and A. Ramesh, “Anti urolithiatic activity of Macrotyloma uniflorum seed extract on ethylene glycol induced urolithiasis in albino rats,” J Innov Trends Pharm Sci, vol. 1, pp. 216-26. 2010.

31. S. Bouanani, C. Henchiri, E. Migianu-Griffoni, N. Aouf, and M. Lecouvey, “Pharmacological and toxicological effects of Paronychia argentea in experimental calcium oxalate nephrolithiasis in rats,” Journal of ethnopharmacology, vol. 129, no. 1, pp. 38-45. 2010.

32. A. Aggarwal, S. K. Singla, M. Gandhi, and C. Tandon, “Preventive and curative effects of Achyranthes aspera Linn. extract in experimentally induced nephrolithiasis.” 2012.

33. P. Ashok, B. C. Koti, and A. Vishwanathswamy, “Antiurolithiatic and antioxidant activity of Mimusops elengi on ethylene glycol-induced urolithiasis in rats,” Indian journal of pharmacology, vol. 42, no. 6, pp. 380. 2010.

34. S. B. SAYANA, V. R. CHAVAN, and R. MUDIUM, “Antilithiatic effect of Cissampelospareiraleaves in ammonium chloride and ethylene glycol induced urolithiasis in rats,” Asian J. Pharm. Clin. Res, vol. 7, pp. 103-106. 2014.

Received on 19.11.2019 Modified on 26.02.2020

Research J. Pharm. and Tech. 2020; 13(12):6132-6139.

DOI: 10.5958/0974-360X.2020.01070.7

Groups	Calcium (mg/g)	Oxalate (mg/g)	CAT (μmol H₂O₂/min/100 mg protein)	GSH (μmol/100mg)	MDA (nmol/mg)
I	1.45±0.04	1.48±0.04	41.89 ± 0.94	152.83±2.72	15.01±0.64
II	5.90±0.21^a***	6.01±0.23^a***	11.95 ± 0.76^a***	39.79±2.31^a***	96.86±2.99^a***
III	2.64±0.11^a,b*	2.94±0.30^a*,b*	35.68±0.66^a*,b*	138.09±2.59^a,b**	25.04±2.63^a,b*
IV	3.01±0.26^a*,b*	3.27±0.25^a*,b*	27.04±0.48^a*,b*	109.29±2.21^a*,b*	49.28±2.63^a*,b*
V	2.94±0.30^a*,b*	3.10±0.29^a*,b*	31.11±0.42^a*,b*	122.33±2.79^a*,b*	33.28±1.31^a*,b*
VI	3.03±0.26^a*,b*	3.22±0.25^a*,b*	24.33±0.57^a*,b*	91.83±2.16^a*,b*	56.89±0.67^a*,b*
VII	3.28±0.32^a*,b*	3.55±0.13^a*,b*	15.72±0.61^a*,b	49.54±5.12^a***	80.22±0.49^a*,b*
VIII	3.11±0.29^a*,b*	3.39±0.26^a*,b*	21.51±0.76^a*,b*	73.46±4.30^a*,b*	65.07±1.88^a*,b*