Antioxidant Potential of Hesperidin Protects Gentamicin Induced Nephrotoxicity in Experimental Rats

Research Article

Austin J Pharmacol Ther. 2015; 3(2).1071.

Antioxidant Potential of Hesperidin Protects Gentamicin Induced Nephrotoxicity in Experimental Rats

Jain DP* and Somani RS

Department of Research, Suresh Gyan Vihar University, India

*Corresponding author: : Jain DP, Department of Research, Suresh Gyan Vihar University, Jaipur, 302 001, India

Received: April 07, 2015; Accepted: June 18, 2015; Published: June 23, 2015


Acute tubular necrosis is common with aminoglycoside therapy resulting in nephrotoxicity. The bioflavonoid, hesperidin is a specific flavonoid glycoside reported to act as a powerful antioxidant. Therefore, the present study investigates nephroprotective effect of hesperidin against gentamicin induced nephrotoxicity in rats. Thirty rats were randomly divided into five groups (n=6). Group I served as a control, Group II as a gentamicin control and was received vehicle for two days before and then treated with gentamicin (100 mg/kg/day) for eight days. Group III-V were received hesperidin orally at three doses (50, 100 and 200 mg/kg) two days before and eight days along with gentamicin (100 mg/kg/day). Hesperidin at doses of 100 and 200 mg/kg treatment significantly restored the body weight and kidney weight associated with the gentamicin. Moreover, significant changes in the biochemical parameters such as increased levels of BUN, serum creatinine and decreased urinary creatinine and creatinine clearance was observed in the hesperidin treated rats. Further increased MDA levels and decreased SOD and CAT activity as well as GSH levels were attenuated with hesperidin treatment (200 mg/kg). Necrosis and degenerative changes in glomeruli and tubules, observed in gentamicin treated rats were also been restored. Thus it suggests the antioxidant potential of hesperidin protect the gentamicin induced nephrotoxicity in rats.

Keywords: Hesperidin; Gentamicin; Urea nitrogen; Antioxidant; Nephrotoxicity


Acute tubular necrosis is a relatively common with aminoglycoside therapy resulting nephrotoxicity [1]. Gentamicin is an aminoglycoside antibiotic used to treat severe gram negative infections [2]. At physiologic pH, aminoglycoside molecules posses a cationic charge due to multiple amine groups, which bind to anionic charge on the phospholipids within the plasma membrane of the proximal tubule [3,4]. The accumulation of gentamicin in the proximal tubule interacts with intracellular metabolic processes, which depressed the renal function. Tubular cell injury due to gentamicin is characterized by cellular necrosis, mitochondrial structural alteration and suppression of free radical defense mechanism. The formations of free ions due to antibiotics results in production of hydrogen peroxide by the renal cortex and also inhibit the synthesis of phospholipase A2 and glutation [5]. Antioxidants have been observed the most consistent, safe and efficacious approaches used to ameliorate or protect gentamicin induced nephrotoxicity. Several reports suggest medicinal plant extracts with antioxidant properties protect nephrotoxicity induced by aminoglycosides [6,7].

Hesperidin is a flavanone glycoside derived from the word “hesperidium”, the kind of fruit produced by citrus trees as it is found abundantly in citrus fruits [8]. Hesperidin is mainly used as antioxidant, as it remarkably prevented indicators of oxidative stress, such as the ROS and lipid peroxidation levels in a dose-dependent manner [9]. Neves et al., proved that hesperidin is effective against sodium arsenite induced acute toxicity in mice, because it exhibits antioxidant activity [10]. It was also reported to protect and prevents embryos from oxidative stress, and may regenerate beta cells of endocrine pancreas in experimental diabetes pregnancy [11]. Moreover, it is reported as anti-inflammatory [12], anticancer [13], antihyperlipidemic [14], antihypertensive [15] and cardioprotective activity in ischemic heart disease in diabetic rats [16]. Therefore the present investigation was carried out to study the antioxidant potential of hesperidin protects gentamicin induced nephrotoxicity.

Materials and Methods

Drugs and chemicals

Gentamicin was purchased from local market of Pune (Genticyn, Piramal Healthcare, India), Hesperidin was obtained as gift sample (NANS Product, Mumbai), malondialdehyde (MDA), tetrabutyl ammonium and superoxide dismutase (Sigma-Aldrich, St. Louis), Catalase (Hi Media Laboratories Pvt. Ltd., Mumbai) and all other reagents and chemical were of analytical grade and purchased from local suppliers of Pune.


Sprague Dawley (SD) rats (180- 220 gm) were procured from National Institute of Biosciences, Pune. Rats were placed separately in polypropylene cages with paddy husk as bedding. The animals were maintained under standard laboratory conditions at temperature 23 ± 2°C with relative humidity 55 ± 10 % in a 12 h light and 12 h dark cycle throughout the experiment. Animals had free access to water and standard laboratory feed ad libitum (Nutrivet Lab, India). All the experimental procedures and protocols used in this study were reviewed and approved (IAEC/2011-12/33) by the Institutional Animal Ethics Committee (IAEC). Ethical guidelines were strictly followed during all the experimental procedures.

Experimental design

Thirty rats were randomly divided into five equal groups (n=6). Group 1: rats in this group were administered a vehicle 2% (W/V) gum acacia orally and served as a control. Group 2: rats in this group were administered a vehicle two days before and then injected with gentamicin (100 mg/kg/day) intraperitoneally for eight day and served as gentamicin control. Group 3-5: rats in these groups were treated orally with hesperidin at three doses (50, 100 and 200 mg/ kg) two days before and eight days concomitantly with gentamicin intraperitoneally (100 mg/kg). The hesperidin was suspended in 2% (W/V) gum acacia [17]. At the end of the treatment each rat was individually placed in metabolic cage for 24 h urine was collected and centrifuged at 1000 rpm for 10 min to remove cells and debris. Blood was collected from retrorbital plexus under light anesthesia and serum was separated by centrifugation at 3000 rpm for 15 min. The rats were sacrificed by cervical dislocation under ether anesthesia. The abdominal cavity was immediately opened; kidneys were removed and processed for antioxidant as well as histopathological studies.

Body weight and kidney weight change

Body weight of all animals was measured using digital electronic balance. After sacrificing, a kidney was dissected, rinsed in chilled saline, decapsulated blotted on filter paper and quickly weighed.

Relative kidney weight (%) = [Absolute kidney weight/Body weight at sacrifice] × 100

Biochemical estimations

A serum blood urea nitrogen levels was estimated using urea enzymatic colorimetric kit [18]. Serum and urinary levels of creatinine were estimated according to the method of Bartels et al. [19]. Creatinine clearance was calculated as per the following formula;

Ccr (mL/min/kg) = [urinary Cr (mg/dL) × urinary volume (mL)/ serum Cr (mg/dL)] × [1000/body weight (g)] × [1/1440 (min)]

Oxidative stress

Kidney was homogenized in chilled 50mM phosphate buffer saline (pH 7.4) in volume of nine times of its weight to yield 10% (w/v) tissue homogenate. The homogenates were centrifuged at 10500 rpm for 15 min at 4°C. The homogenate was used for the determination of malondialdehyde levels (MDA) [20], reduced glutathione (GSH) [21], and activities of SOD [22] and catalase (CAT) [23]. Protein concentrations of homogenates were determined according to Lowry et al. [24].

Histopathological studies

Kidney of individual rat stored in 10% formalin solution were embedded with paraffin and stained with Hematoxyline-Eosin (HE). HE stained sample was observed under light microscope (100 x).

Statistical analysis

All the data were expressed as the mean ± S.E.M (n=6). Data were subjected to one-way analysis of variance (ANOVA) followed by the Tukey’s multiple comparison test. Whereas, P<0.05 was set minimum levels of significance.


Body weight, kidney weight and relative kidney weight

Intraperitoneal injection of gentamicin produced significant decrease in the body weight and increase in kidney weight compared to control (P<0.01). Whereas, hesperidin (200 mg/kg) produced most significant effect on the body weight and kidney weight which amounted to 8.09% (P<0.05) and 23.96 % (P<0.01) compared to gentamicin control rats. However, hesperidin at doses of 50 and 100 mg/kg body weight could not produce significant changes in the body weight and kidney weight compared to gentamicin control rats (Table 1).