Protective effect of Cassia fistula fruit extract on bromobenzene-induced nephrotoxicity in mice

Introduction

Use of herbal remedies is one of the oldest forms of medicine that played an integral role in the development of civilization. Today, pharmaceutical companies also focus their efforts on creating and improving plant-based pharmaceuticals. In recent years, there has been considerable pursuit to identify antioxidant plant products, as free radicals are considered to play significant role in several chronic debilitating diseases and aging. Extracts made from Cassia fistula (Golden shower tree) may be a candidate, since it is reported to possess various therapeutic uses, for example as a laxative or purgative, an antipyretic and an analgesic drug.^1,2 Moreover, Cassia fistula was found to be efficacious against hyperglycaemia, hypercholesterolaemia and gastric ulcer,^3–5 furthermore it was shown to have antitumor, antibacterial, antifungal and contraceptive properties as well.^6–9 A hepatoprotective effect exerted by Cassia fistula leaf^10,11 and fruit ¹² has also been documented.

However, to the best of our knowledge, the effect of Cassia fistula on toxic renal damage has not been investigated. Therefore, the present study was designed to evaluate the effect of a crude hydroalcoholic extract of Cassia fistula fruit against bromobenzene-induced toxicity in the mouse kidney.

Bromobenzene (monobromobenzene, bromobenzol) is an industrial compound used to manufacture a number of chemicals and drugs. Primary metabolites of bromobenzene, generated during hepatic phase I reactions, are hepatotoxic.^13–15 In turn, secondary metabolites, formed in hepatic phase II reactions via conjugation, show nephrotoxicity, such as bromophenol isomers,^16,17 4-bromocatechol, ¹⁶ 2-bromohydroquinone ¹⁸ and benzoquinone. ¹⁵

Methods

The investigation conforms to the European Community guidelines for the use of experimental animals. The protocols have been approved by the institutional ethics committee (Committee of Animal Research, University of Debrecen, DE MÁB 35/2007).

Adult male albino mice, aged 6 to 8 weeks, were used. Mice were maintained in a conventional animal house at ambient temperature of 22°C, and were supplied with commercial mice food pellets and tap water ad libitum.

Bromobenzene was purchased from Sigma (St. Louis, Missouri, USA). Chemicals (methanol, ethanol, formalin, paraffin and salts) were purchased from the local market. Before the administration to the mice, bromobenzene was dissolved in liquid paraffin (1/2 v/v, respectively).

Cassia fistula fruit was obtained from the United Arab Emirates and was authenticated by the Department of Pharmacognosy of Ahwaz Jundishapur University of Medical Sciences. The Cassia fistula fruit extract was prepared according to the method of Santosh et al. ¹⁹ Three hundred grams of Cassia fistula fruit was comminuted, crushed, saturated with 90% ethanol for 3 days, and the extracted material was filtered. The residue was re-extracted with 90% ethanol three times. The pooled filtrate was concentrated under vacuum to dryness using vacuum evaporator (Adolphe, Model 462, Germany). The dried extract was suspended in normal saline before administration to the mice.

Mice were divided into six groups, each containing ten animals. All mice were treated daily by oral gavage for 10 days. Group 1 was treated with 0.2 mL normal saline (negative control); group 2 received 460 mg/kg of bromobenzene (positive control); groups 3 to 6 were treated with Cassia fistula fruit extract in doses of 200 mg/kg, 400 mg/kg, 600 mg/kg, and 800 mg/kg, respectively, followed by oral gavage of 460 mg/kg bromobenzene 2 hours later. This 2-hour delay was performed to improve the protective effect of the Cassia fistula extract. Animals were sacrificed on the 11th day.

To measure enzyme activities, blood was drawn from the jugular vein of mice. Blood urea nitrogen (BUN) and creatinine were assayed according to methods of Bretaudiere et al. ²⁰ and Spencer, respectively. ²¹ For light microscopy, kidneys were removed, conserved in 10% neutral phosphate-buffered formalin, processed by routine paraffin sectioning (6 μm), and stained with hematoxylin and eosin (H&E).

All data are presented as the mean ± standard error of the mean (SEM). Statistical analysis was carried out with GraphPad Prism version 4.03 for Windows, performing one-way ANOVA followed by Tukey’s post-testing. Probability values <0.05 were considered to be statistically significant.

Results

We found that a 10-day-long administration of 460 mg/kg bromobenzene alone (positive control; group 2) produced a significant elevation in levels of both BUN and creatinine when compared to the normal saline-treated mice (negative control; group 1). In addition, the ratio of BUN to creatinine showed a moderate decrease in response to bromobenzene (Table 1 ). However, the administration of Cassia fistula fruit extract in addition to bromobenzene (groups 3-6) dose-dependently reduced the BUN and serum creatinine levels, which approached the negative control values, when the Cassia fistula fruit extract was administered in the highest dose (800 mg/kg). Consistently, BUN and creatinine levels of mice treated with 800 mg/kg Cassia fistula fruit extract differed significantly from the positive control values (Table 1). Interestingly, the BUN-to-creatinine ratio remained low at 200, 400 and 600 mg/kg, but it recovered at 800 mg/kg Cassia fistula fruit extract dose (Table 1).

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Table 1.

Effect of bromobenzene and Cassia fistul a fruit extract on BUN and serum creatinine levels in micea

Groups	BUN	Cr	BUN/Cr
1 (negative control)	18.86 ± 2.10	0.26 ± 0.03	73.3
2 (positive control)	28.17 ± 1.78c	0.43 ± 0.04c	66.3
3 (200 mg/kg)	23.50 ± 2.41 ns, ns	0.35 ± 0.03 ns, ns	67.8
4 (400 mg/kg)	22.00 ± 1.05 ns, ns	0.33 ± 0.02 ns, ns	66.7
5 (600 mg/kg)	21.00 ± 2.20 ns, ns	0.31 ± 0.03 ns, ns	67.7
6 (800 mg/kg)	20.00 ± 0.82 ns,b	0.26 ± 0.04 ns,c	76.9

BUN: blood urea nitrogen, Cr: creatinine. ^aAnimals in group 1 were treated with physiological saline solution, while animals in groups 2-6 were given 460 mg/kg of bromobenzene by oral gavage for 10 days. In addition, mice in groups 3-6 were treated daily with Cassia fistula fruit extract (200, 400, 600, 800 mg/kg, respectively). Results are expressed as mean ± SEM. BUN and Cr are presented in mg/dL. Levels of statistical significance (ns: p > 0.05; ^b: p < 0.05; ^c: p < 0.01; are indicated in the following arrangement: at the group 2: group 2 versus group 1; at the groups 3-6: the given group versus group 1, and then the given group versus group 2.

The protective effect of Cassia fistula fruit extract was confirmed by comparing the histological status of kidneys obtained from the different groups. Histological profile of renal samples of mice treated with bromobenzene alone revealed necrotic lesions (Figure 1B versus Figure 1A). Administration of the herbal extract in four doses exhibited a significant improvement in the architecture of the kidneys indicated by the reduction of necrosis (Figure 1C-F versus Figure 1B).

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Figure 1.

Kidney sections (stained with H&E) from mice given normal saline (A), 460 mg/kg of bromobenzene alone (B), 460 mg/kg of bromobenzene with 200 (C), 400 (D), 600 (E) and 800 (F) mg/kg of Cassia fistula fruit extract.

Discussion

To the best of our knowledge, this is the first study to show the protective effect of Cassia fistula extract in nephrotoxicity. We found that the hydroalcoholic crude extract made from Cassia fistula fruit reduces both the functional and the structural damages induced by bromobenzene in the mouse kidney, although a significant protection can only be expected from a high dose of this extract.

In murine models, the orally administered bromobenzene was usually administered once in bolus.^{15,16,22–25} In the case of this protocol, bromobenzene doses smaller than 800-900 mg/kg did not significantly elevate BUN levels,^22,23 while 1495 mg/kg and 2355.15 mg/kg of bromobenzene were found to produce about 2.8- and 2.6-fold increases in the BUN value, respectively.^24,25 In a chronic toxicity study, 50−600 mg/kg bromobenzene added by gavage 5 days/week for 90 days failed to exert significant nephrotoxic effect. ²⁶ The previous investigations also established that intensity of the bromobenzene-induced nephrotoxicity strongly depends on the species and even on the strain of the animal. ²⁷ In the present study, 10-day-long oral bromobenzene treatment (alone) induced renal injury that was evidenced by the elevation of BUN and serum creatinine levels, the two most important laboratory parameters of renal function. ²⁸ It should be noted, however, that the approximately 1.5-fold increase in the BUN level (as well as the about 1.7-fold rise in the serum creatinine) elicited by bromobenzene (Table 1) might be labeled mild in a study that focuses on the toxic effects per se. Nevertheless, this deterioration in the renal function proved to be sufficient to detect the effect of the Cassia fistula fruit extract on the action of bromobenzene.

In addition, the bromobenzene treatment decreased the BUN-to-creatinine ratio (Table 1). Upon elevated BUN and serum creatinine levels, their ratio may denote the cause of renal failure. An increased ratio may indicate an extrarenal origin (e.g. inadequate renal perfusion), while decrease of the ratio suggests an intrarenal cause (i.e. renal parenchymal lesion) that has led to tubular dysfunction. ²⁸ Thus, the decreased BUN-to-creatinine ratio observed in this study corroborates the bromobenzene-induced nephrotoxicity. In accordance with these changes, renal parenchymal necrosis was detected histologically (Figure 1).

Cassia fistula has been used extensively as an ayurvedic medicament. ¹ Also, growing body of scientific evidence emerged concerning its numerous beneficial properties supporting its use in the amelioration of various disorders.^1–8 Former studies have reported the hepatoprotective effect of Cassia fistula extract, which was made from the leaf^10,11 or from the fruit of the plant. ¹²

In the current study, we were able to provide evidence for the efficacy of Cassia fistula fruit to prevent the bromobenzene-mediated nephrotoxicity. Co-treatment of mice with Cassia fistula fruit extract besides bromobenzene reduced levels of both BUN and creatinine in the blood, which effect was especially obvious at 800 mg/kg, the highest herbal extract dose used in the present study (Table 1). Consistent with this, the BUN-to-creatinine ratio was restored to normal in response to the 800 mg/kg Cassia fistula fruit extract dose. Interestingly, lower doses of Cassia fistula were unable to affect the BUN-to-creatinine ratio, although BUN and serum creatinine levels per se showed a dose-dependent improvement in response to the Cassia fistula treatment (Table 1). Thus, it may be hypothesized that renal tubular function is more sensitive to the bromobenzene-induced toxicity than the glomerular filtration. Beneficial effects of Cassia fistula fruit extract against the bromobenzene-evoked nephrotoxicity could be verified by histological examination (Figure 1). Since Cassia fistula has been reported to possess effective antioxidant properties,^29,30 it may be supposed that phytochemicals in Cassia fistula fruit scavenged the toxic metabolite of bromobenzene.

In summary, it may be proposed that Cassia fistula fruit extract can contribute to the preservation of functional and morphological integrity of the kidney against a nephrotoxic agent.