Anti-urolithiatic Effect of Petroleum Ether Extract Stem Bark of Crataeva adansonii. in Rats

Abstract

Calcium oxalate nephrolithiasis in rats was induced by intraperitoneal injection of sodium oxalate (7 mg/100 g) daily for 7 days. A significant increase in the level of oxalate in urine and kidney with a decrease in urinary volume, pH, creatinine clearance, and lipid peroxide level was observed in animals receiving sodium oxalate as compared with saline-treated animals. Administration of petroleum ether extract (50 and 100 mg/kg, p.o.) of Crataeva adansonii. DC. along with sodium oxalate (prophylactically) and after 7 days of sodium oxalate injection (therapeutically) significantly decreased calcium oxalate content in urine and kidney and lipid peroxide level in liver and kidney compared with vehicle-treated group. Animals treated with petroleum ether extract (50 and 100 mg/kg, p.o.) of Crataeva adansonii. showed improved creatinine clearance as compared with vehicle group. Histological estimation of kidney treated with petroleum ether extract (50 and 100 mg/kg, p.o.) along with sodium oxalate strongly inhibited the growth of calculi and reduced the number of stones in kidney compared with group receiving vehicle. From this study, we conclude that both the prophylactic and therapeutic treatment with petroleum ether extract of bark of Crataeva adansonii. had an inhibitory effect on crystal growth, with improvement of kidney function as well as cytoprotective effect.

Keywords:

• Calcium oxalate
• Crataeva adansonii
• lipid peroxidation
• nephrolithiasis
• sodium oxalate
• urolithiasis

Introduction

Urolithiasis is the third most common disorder of the urinary tract. The wordwide incidence of urolithiasis is quite high, and in northern India more than 80% of urinary calculi are calcium oxalate stones alone or calcium oxalate mixed with calcium phosphate (Mitra et al., 1998). Hyperoxaluria is the main initiating factor of human idiopathic calcium oxalate (CaOx) stone disease. Oxalate is a powerful crystallization-driving factor present in the urine, retention of which enhances cell injury and causes early stages of lithogenesis (Kumar et al., 2002). In spite of tremendous advances in the field of medicine, there is no truly satisfactory drug for the treatment of renal calculi.

On the other hand, phytomedicines have offered an alternative source of therapy for many diseases and also have provided some additional information about the pathogenesis of diseases. Plants from the genus Crataeva were reported to have anti-urolithiatic activity (Anand et al., 1995). It was therefore thought worthwhile to investigate one of the species, Crataeva adansonii. DC., for its anti-urolithiatic activity in rats.

Crataeva adansonii. is a tropical tree that grows up to 5–10 m height. These trees are widely distributed all over India, Burma, and Ceylon. Traditionally, barks, leaves, flowers, and root barks are used for their medicinal properties.

In the Yunani system, the bark is mentioned to promote appetite, decrease the secretion of bile and phlegm, and remove disorders of the urinary organs. Traditionally in India, the bark is useful in some cases of urinary complaints and fever and in some mild forms of skin diseases. Although the bark of Crataeva adansonii. is reported for the treatment of urolithiasis in folklore literature (Nadkarni, 1982), there has been no published pharmacological data on Crataeva adansonii. for its potential anti-urolithiatic activity. The objective of the current study was to investigate the petroleum ether extract effect of stem bark of Crataeva adansonii. for anti-urolithiatic activity on experimentally induced urolithiasis in rats.

Materials and Methods

Plant material and extraction procedure

Stem bark of Crataeva adansonii. was purchased from a local market and authenticated at Agharkar Research Institute (Pune, India). Coarsely powdered shade-dried bark (350 g) was extracted with petroleum ether (60–80°) using a Soxhlet apparatus. The extraction process was repeated, and the extracts obtained were pooled and distilled to recover the solvents. The last traces of solvents from the extracts were removed under vaccum. The petroleum ether extract thus obtained was greenish in color, and the yield obtained was about 0.3%.

Chemicals and apparatus

Creatinine (Jaffe method) and uric acid (uricase method) estimation kit was purchased from Accurex Biomedical Pvt Ltd. (Mumbai, India). Zinc wire and sodium oxalate was purchased from S.D. Fine Chemicals Limited (Mumbai, India). Cystone (contains extracts shilapushpa 130 mg, pashanbheda 98 mg, manjishtha 32 mg, nagarmustha 32 mg, apamarga 32 mg, gojiha 32 mg, sahadevi 32 mg, and powders hajrul yahood bhasma 32 mg, shilajeet 26 mg) was purchased from Himalaya Herbal Healthcare (Banglore, India).

Experimental design

Animals

Male Wistar rats (200–250 g) were purchased from National Toxicology Centre (Pune, India). They were maintained at a temperature of 25 ± 1°C and relative humidity of 45% to 55% under 12-h light and 12-h dark cycle. The animals had free access to food pellets (Chakan Oil Mills, Pune, India) and water. The experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC) of Poona College of Pharmacy (Pune, India) and were in accordance with the guidelines of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA). Experiments were carried out between 0900 and 1600 h.

Anti-urolithiatic activity

Sodium oxalate–induced urolithiatic model in rat was used to assess the effect of petroleum ether extract Crataeva adansonii.. The study was designed to find out the effect of petroleum ether extract of Crataeva adansonii. on prophylactic and therapeutic usage against sodium oxalate–induced urolithiasis. The groups are divided as follows:

Prophylactic groups (drug treatment for 7 days along with sodium oxalate)

• Group I Saline (1 ml/kg)

• Group II Sodium oxalate (7 mg/100 g, i.p.)

• Group III Sodium oxalate (7 mg/100 g, i.p.) + Tween 80 (1% v/v)

• Group IV Sodium oxalate (7 mg/100 g, i.p.) + petroleum ether extract (50 mg/kg, p.o.)

• Group V Sodium oxalate (7 mg/100 g, i.p.) + petroleum ether extract (100 mg/kg, p.o.)

• Group VI Sodium oxalate (7 mg/100 g, i.p.) + cystone (500 mg/kg, p.o.)

Therapeutic groups (drug treatment for 8–14 days after sodium oxalate treatment)

• Group VII Saline (1 ml/kg)

• Group VIII Sodium oxalate (7 mg/100 g, i.p.)

• Group IX Sodium oxalate (7 mg/100 g, i.p.) + Tween 80 (1% v/v)

• Group X Sodium oxalate (7 mg/100 g, i.p.) + petroleum ether extract (50 mg/kg, p.o.)

• Group XI Sodium oxalate (7 mg/100 g, i.p.) + petroleum ether extract (100 mg/kg, p.o.)

• Group XII Sodium oxalate (7 mg/100 g, i.p.) + Cystone (500 mg/kg, p.o.)

All rats were housed in metabolic cages individually for the entire duration of the experiment. The urine of each rat was collected on the 7th and 14th days after 6 h of sodium oxlalate injection with thymol as a preservative. Prophylactic groups and therapeutic groups were sacrificed on the 7th and 14th days, respectively. The right kidney was examined for the presence of calcium oxalate crystals and stone formation by histological techniques. The left kidneys of three animals were homogenized with Tris buffer (0.605 g Tris in 30 ml H₂O + 0.33 ml of conc. HCl diluted to 100 ml with water), and the homogenate after centrifugation at 4000 rpm was used to determine the concentration of calcium oxalate in the kidney. The left kidneys of the remaining three animals and the livers of all six animals of the respective group were perfused with KCl and homogenated with KCl (0.15 M) solution. The homogenate obtained after centrifugation at 7000 rpm was used to study the lipid peroxide effect.

Qualitative crystal deposition was graded as grade 0, no deposition of crystals; grade 1, mild deposition of crystals; grade 2, moderate deposition of crystals; and grade 3, higher amount of calcium oxalate crystals in kidney.

Parameters

Serum and urine electrolytes (sodium, potassium, chloride) were determined by electrolyte analyzer (Biolyte 2000, Taiwan), and serum/urine creatinine and uric acid were determined with the help of an autoanalyzer (Secomam). Urine and kidney oxalate concentration was determined by the method of Hodgkins and Williams (1972). Liver and kidney lipid peroxide level was determined by the method of Sreejayan et al. (1994) and Ohkawa (1979). The pH of the urine was determined with a pH meter (model EQ-614).

Statistical analysis

The values are represented as mean ±SEM. Statistical significance between means was analyzed using one-way analysis of variance (ANOVA) followed by Tukey test. p < 0.05 was considered statistically significant.

Results

Urine output and excretion constituents

Administration of sodium oxalate (7 mg/100 g, i.p.) did not significantly alter the output of urine; however, it showed a slight decrease in urine output as compared with untreated group. Administration of petroleum ether extract of Crataeva adansonii. (50 and 100 mg/kg) and cystone (500 mg/kg) caused a non-significant increase in urine output in both prophylactic and therapeutic groups on 7th day and 14th day of treatment. Administration of sodium oxalate (7 mg/100 g, i.p.) significantly (p < 0.001) decreased urine pH as compared with untreated group. Administration of Crataeva adansonii. (50 and 100 mg/kg) and cystone (500 mg/kg) caused significant increase in urine pH in both prophylactic and therapeutic group on 7th day and 14th day of treatment compared with vehicle-treated group (Table 1).

Table 1 Effect of petroleum ether extract of Crataeva adansonii. on urine parameters of rats intoxicated with sodium oxalate.

					Pet. ether extract
Urine parameter	Time in days	Saline (1 ml/kg)	NaOx (7 mg/100 g)	NaOx + Tween 80 (7 mg/100 g + 1%)	(50 mg/kg)	(100 mg/kg)	Cystone (500 mg/kg)
Urine output	Day 7	2.82 ± 0.29	2.09 ± 0.26	1.70 ± 0.24	1.43 ± 0.17	1.38 ± 0.21	3.49 ± 0.38
	Day 14	2.83 ± 0.18	1.90 ± 0.23	1.78 ± 0.25	2.57 ± 0.44	2.85 ± 0.39	4.02 ± 0.38
pH	Day 7	7.74 ± 0.17	6.42 ± 0.22***	6.52 ± 0.14***	7.35 ± 0.11^##	6.97 ± 0.08	7.83 ± 0.14^###
	Day 14	7.77 ± 0.16	6.70 ± 0.16***	6.78 ± 0.15***	7.53 ± 0.13^##	7.73 ± 0.10^###	8.03 ± 0.13^###
Sodium	Day 7	148.85 ± 5.45	98.37 ± 3.91***	102.31 ± 3.14	113.33 ± 6.94	135.35 ± 4.73#	184.28 ± 7.38^###
	Day 14	147.21 ± 5.20	96.34 ± 2.93***	102.60 ± 2.21	100.37 ± 5.68	130.47 ± 3.09^##	259.58 ± 6.97^###
Potassium	Day 7	213.37 ± 6.46	250.40 ± 7.37*	241.30 ± 5.27	258.48 ± 9.88	144.67 ± 13.04^###	236.87 ± 6.53
	Day 14	221.02 ± 5.03	241.50 ± 4.34	254.30 ± 4.73	271.03 ± 4.24	247.45 ± 9.87	298.35 ± 6.67^###
Chloride	Day 7	22.65 ± 8.10	177.50 ± 11.12***	189.20 ± 6.98	204.52 ± 7.56	238.67 ± 7.60#	382.23 ± 16.06^###
	Day 14	241.60 ± 3.84	156.34 ± 4.82***	169.83 ± 6.46	187.83 ± 7.42	238.67 ± 7.60^###	419.98 ± 7.14^###
Calcium oxalate	Day 7	19.63 ± 1.64	170.99 ± 6.00***	154.97 ± 5.37	124.80 ± 3.66^###	119.56 ± 3.88^###	79.13 ± 3.23^###
	Day 14	20.04 ± 1.58	152.35 ± 6.50***	145.20 ± 4.10	115.35 ± 2.78^###	93.49 ± 3.58^###	73.92 ± 3.48^###
Creatinine clearance	Day 7	0.46 ± 0.07	0.09 ± 0.01***	0.08 ± 0.01	0.24 ± 0.02	0.16 ± 0.04	0.44 ± 0.06^###
	Day 14	0.47 ± 0.04	0.09 ± 0.09***	0.09 ± 0.01	0.45 ± 0.06^###	0.54 ± 0.07^###	0.64 ± 0.07^###

Values are expressed as mean ± SEM, n = 6.

*p < 0.05, **p < 0.01, ***p < 0.001 vs. saline treated rats.

^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. NaOx + Tween 80, one-way ANOVA followed by Tukey test.

Injection of sodium oxalate (7 mg/100 g, i.p., once a day for 7 days) in rats increased the urinary level of oxalate significantly compared with the saline (1 ml/kg, i.p.) treated group indicating formation of sodium oxalate–induced lithiasis. There was no significant change in urinary oxalate level in the urine of vehicle-treated group compared with sodium oxalate (7 mg/100 g, i.p.) treated group. The petroleum ether extract (50 and 100 mg/kg) and cystone (500 mg/kg) showed a significant (p < 0.001) decrease in urine calcium oxalate content in prophylactic and therapeutic group on the 7th day and 14th day of treatment compared with vehicle group (Table 1). Administration of sodium oxalate caused a significant decrease in sodium and chloride concentration in urine, whereas the potassium content was not changed compared with the untreated group. Significant increase in sodium and chloride was observed with extract at 100 mg/kg dose with both the prophylactic and therapeutic group. Significant decrease in potassium was observed with prophylactic group of 100 mg/kg of petroleum ether extract (Table 1). Creatinine clearance was found to be significantly (p < 0.001) decreased in the sodium oxalate–treated group compared with untreated group. After treatment with petroleum ether extract (50 and 100 mg/kg), a significant (p < 0.001)increase in creatinine clearance was observed in the therapeutic group but not in the prophylactic group. Cystone-treated group showed significant (p < 0.001) increase in creatinine clearance in both the prophylactic and therapeutic group compared with vehicle-treated group (Table 1).

Effect on lipid peroxide level

After treatment with sodium oxalate (7 mg/100 g, i.p.), the lipid peroxide level was found to be higher when compared with untreated group in kidney and liver. Administration of extract (50 and 100 mg/kg) and cystone (500 mg/kg) showed significant inhibition of lipid peroxide level in rat kidney in both the prophylactic and therapeutic group. In the liver, extract at 50 mg/kg showed significant (p < 0.05) inhibition of lipid peroxide level in prophylactic group on 7th day while 100 mg/kg showed significant (p < 0.001) inhibition in therapeutic-treated group on 14th day compared with vehicle-treated group. No significant inhibition of lipid peroxide was found with cystone (500 mg/kg) in rat liver compared with vehicle-treated group (Table 2).

Table 2 Effect of petroleum ether extract of Crataeva adansonii. on lipid peroxide level in liver and kidney of rat treated with sodium oxalate.

		Treatment
		Liver		Kidney
Group	Dose	Day 7	Day 14	Day 7	Day 14
NaOx + Tween 80	7 mg/100 g + 1%	13.74 ± 1.08	20.20 ± 3.03	11.27 ± 3.38	12.71 ± 3.04
Petroleum ether extract	50 mg/kg	59.89 ± 15.83^#	53.54 ± 10.91	63.79 ± 8.54^##	64.71 ± 6.73^##
	100 mg/kg	53.85 ± 10.31^#	65.15 ± 10.89^###	57.79 ± 11.57^#	58.12 ± 10.19^##
Cystone	500 mg/kg	36.54 ± 5.18	50.25 ± 8.04	67.15 ± 4.97^##	64.41 ± 3.60^##

Values are expressed as mean ± SEM, n = 6.

^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. NaOx + Tween 80, one-way ANOVA followed by Tukey test.

A dose-dependent inhibition of lipid peroxide level was observed in in vitro. study, which was maximum at 500 µg of petroleum ether extract of Crataeva adansonii. in both liver and kidney homogenate (unpublished data).

Calcium oxalate estimation

After administration of sodium oxalate (7 mg/100 g, i.p.), the calcium oxalate level was significantly (p < 0.001) higher in kidney on 7th day and 14th day as compared with the untreated group. A significant (p < 0.001) decrease in calcium oxalate level in kidney was observed in both the prophylactic and therapeutic group of extract (50 and 100 mg/kg) and cystone (500 mg/kg). Maximum decrease in calcium oxalate content was observed in therapeutic group of 50 mg/kg (Table 3).

Table 3 Effect of petroleum ether extract on calcium oxalate content and deposition in rat kidney.

	Treatment		Crystal deposit in kidney
Group	7 Days	14 Days	7 Days	14 Days
Saline	50.82 ± 3.85	57.25 ± 3.66	ND	ND
NaOx	246.16 ± 10.02 ***	223.01 ± 8.00***	2.67 ± 0.21	2.50 ± 0.22
NaOx + Tween 80	215.11 ± 7.17	238.30 ± 5.34	2.83 ± 0.17	2.67 ± 0.33
50 mg/kg	128.54 ± 6.13^###	109.73 ± 2.93^###	1.33 ± 0.33*	1 ± 0.00**
100 mg/kg	120.01 ± 5.12^###	112.82 ± 4.47^###	1.33 ± 0.21	1.50 ± 0.22
Cystone (500 mg/kg)	65.44 ± 5.82^###	78.44 ± 6.71^###	1.33 ± 0.21	1.17 ± 0.17*

Values are expressed as mean ± SEM, n = 6.

*p < 0.05, **p < 0.01, ***p < 0.001 vs. control, ^###p < 0.001 vs. NaOx + Tween 80. Concentration of calcium oxalate in kidney was analyzed using one-way ANOVA followed by Tukey test, and crystal deposition in kidney was analyzed using Kruskal-Wallis test followed by Dunn test.

Serum clinical chemistry parameters

No significant change was observed in serum sodium, chloride, and uric acid level. The petroleum ether extract (50 and 100 mg/kg) and cystone (500 mg/kg) showed significant (p < 0.001) increase in potassium level on day 14 of treatment. The extract (50 and 100 mg/kg) and cystone (500 mg/kg) showed significant (p < 0.001) decrease in the serum creatinine level compared with vehicle-treated group.

Histopathological evaluation

The histopathology of rats treated with NaOx (7 mg/kg) showed moderate crystals (++) in 2 of 6 animals, and maximum crystals deposition (+++) in 4 of 6 animals with focal acute tubular necrosis (ATN), dilated collecting tubules, and focal tubular atrophy in tubules. These groups also showed mild infiltration of interstitum mononuclear cells (+). The blood vessels and glomeruli were found to be normal.

The histopathology of rats treated with NaOx (7 mg/kg, i.p.) + Tween 80 (1%) showed moderate crystals (++) in 1 of 6 animals, and maximum crystals deposition (+++) in 5 of 6 of animals with focal ATN, dilated collecting tubules, and focal tubular atrophy in tubules. This group also showed mild infiltration of interstitum mononuclear cells (MNC) (+). The blood vessels and glomeruli were found to be normal.

The histopathology of rats treated with petroleum ether extract of Crataeva adansonii. at the dose of 50 mg/kg (once a day for 7 days) showed mild crystals deposition with fragmentation in 5 of 6 animals and maximum crystal deposition (+++) in 1 of 6 animals with dilated collecting tubules. This group also showed MNC infiltration with normal blood vessels and glomeruli.

The histopathology of rats treated with petroleum ether extract of Crataeva adansonii. at the dose of 50 mg/kg (once a day from 8 to 14 days) showed mild deposition with fragmentation in 6 of 6 animals, and MNC infiltration was maximum.

The histopathology of rats treated with petroleum ether extract of Crataeva adansonii. at the dose of 100 mg/kg (once a day from 8 to 14 days) showed moderate crystals deposition (++) in 3 of 6 animals and mild crystal deposition (+) in 3 of 6 animals with dilated collecting no effect on damages on tubules with mild MNC infiltration.

The dose of 100 mg/kg (once daily for 7 days) showed moderate deposition (++) in 2 of 6 animals and mild crystal deposition (+) in 4 of 6 animals with mild to maximum MNC infiltration. The blood vessels and glomeruli showed normal.

The histopathology of cystone (500 mg/kg) once a day for 7 days showed mild crystals deposition (+) with fragmentation and partially dissolved, in 4 of 6 animals and moderate crystal deposition (++) in 2 of 6 animals with no effect on damage on tubules with mild to maximum MNC infiltration.

The histopathology of cystone (500 mg/kg) once a day from 8 to 14 days showed mild crystals deposition (+) in 5 of 6 animals and moderate crystal deposition (++) in 1 of 6 animals with focal tubular atrophy. Maximum MNC infiltration was found with normal blood vessels and glomeruli.

Discussion

A number of models have been used for the study of nephrocalcinosis and nephrolithiasis. Rats have been a suitable species for study of anti-urolithiatic activity because of its urinary system resembling closely that of the human (Anand et al., 1995). Various experimental procedures result in basically two types of hyperoxaluria: (1) acute, when the rat is challenged by a single, large dose of lithogen, (2) chronic, when the rat is continuously challenged with generally small doses of lithogen for a period of time. Hyperoxaluria is measured by determining urinary oxalate, and crystal deposition in the kidney is confirmed by examining paraffin sections of kidney (Khan et al., 1991).

In our current investigation, we used NaOx (7 mg/kg, i.p. for 7 days) induced hyperoxaluria model for rapid screening. Experimental induction of hyperoxaluria results in rapid formation of calculi oxalate crystals in renal tubules of experimental animals. The major reason for this instantaneous crystal formation after a sodium oxalate challenge is the rapid increase in urinary excretion of oxalate. The speed with which crystals appears in renal tubules is indicative of efficiency of transport to kidneys of this added oxalate and its eventual excretion in the urine. After administration of NaOx, the crystalline deposition first reaches in the cortex, then in medulla and then renal tubules (Khan et al., 1982). Results of oxalate administration in this study confirm the histological findings of a relationship between the amounts of calcium oxalate deposition.

A semi-quantitative assay (microscopic scoring system) and a quantitative assay (chemical assay for calcium oxalate determination in kidney) of the crystal deposits were used in this study to compare severity of kidney calcium oxalate crystal deposition among various study groups. NaOx administration in rats decrease in urine volume and pH shows the supersaturation of urine and developed significant calcium oxalate crystal deposition in the kidneys. The severity of microscopic kidney crystal deposition correlated well with calculi oxalate concentration in kidney (Fan et al., 1999).

After treatment with petroleum ether extract (50 mg/kg and 100 mg/kg) and cystone (500 mg/kg), increases in urine volume, sodium, chloride excretion, and pH proved its beneficial effect in preventing calculi formation due to supersaturation of these lithogenic substances.

The petroleum ether extract of Crataeva adansonii. and cystone showed significant decrease in calcium oxalate concentration in kidney. Oxalate determination in the kidney sections (histology) confirmed the relationship between the amount of calcium oxalate crystal deposition and the sodium oxalate administration.

The appearance of calcium oxalate crystal in renal tubules after NaOx injection is associated with necrosis of tubular cells, which results in exposure of tubular basal lamina and formation of luminal cellular debris. The calcium oxalate crystals do cause cytolysis of polymorphonuclear leukocytes after phagocytosis. The crystal aggregates may be destructive to renal epithelium because they are large and irregular and mechanically disrupt the epithelium (Khan et al.,1982).

The extract treatment significantly (p < 0.001), lowered oxalate level in urine and kidney. Extract-treated group showing a significant increase in the creatinine clearance is indicative of improvement in kidney function.

NaOx and vehicle treated group exhibited an increased level of lipid peroxides measured as thiobarbituric acid (TBA) reactive substance. Oxalate itself has been reported to incite tissue damage; the antioxidant protective mechanisms of cells and tissues are disturbed when oxalate-induced oxidative stress evades or overwhelm the cellular balance of pro- and antioxidants. Hence, under such conditions, control over the urinary concentration of oxalate and further crystallization process seems to be the only way out.

Preliminary HPTLC studies in our laboratory confirm the presence of lupeol in petroleum ether extract of stem bark of Crataeva adansonii.. The triterpenes lupeol and betulin are capable of achieving this object by way of increasing the urine volume, the mechanism of which is still to be explored (Malini et al., 1999). Treatment of stone-forming animals with the petroleum ether extracts of Crataeva adansonii. restricts the sodium oxalate–induced peroxidative changes in the renal tissue to a greater extent. Lupeol to reduce the level of oxalate supersaturation in the tissue by way of their diuretic activity or cytoprotective effect by exhibiting protection against peroxidative changes by imparting cellular membrane stability (Anand et al., 1995; Malini et al., 1999). The mechanism of antilithiatic activity of petroleum ether extract may involve the inhibition of oxalate-induced toxic manifestations and free radical production along with enhancement of the body defense system. Drug-treated group showing cytoprotection due to its effect on prevention of deposition or aggregation of calcium oxalate in tubules so the mechanical disruption of epithelium is less or protection against free radicals rearrangements (Malini et al.,1999).

In the current investigation, histopathological evaluation showed the maximum prevention of crystal deposition at the dose of 50 mg/kg compared with 100 mg/kg, which may be due to the active compound that is present in petroleum ether extract showing activity at lower dose. The active ingredients at higher dose may become inactive or a less active metabolite is formed, which reduced its activity.

Acknowledgments

The authors would like to thank Dr. Avinash Pradhan, Pradhan Laboratories, Pune, India, for histopathological estimation of kidney samples. The authors are also thankful to Dr. S.S. Kadam, principal, Poona College of Pharmacy, Pune, for providing all the necessary facilities for the research work.