Therapeutic Implications of Punica Granatum Seeds on Doxorubicin-Induced Nephrotoxicity in Wistar Rats

Research Article

Austin J Pharmacol Ther. 2014; 2 (8).1043

Therapeutic Implications of Punica Granatum Seeds on Doxorubicin-Induced Nephrotoxicity in Wistar Rats

Nirwane AM1*, Gupta PV2 and Patil RA1

1Department of Pharmacology, Bombay College of pharmacy, India

2Department of Pharmacology, Bombay College of Pharmacy, Mumbai, India

*Corresponding author: : Nirwane AM, Department of Pharmacology, Bombay Collage of Pharmacy, India

Received: May 24, 2014; Accepted: September 24, 2014; Published: September 24, 2014


Punica granatum Linn (Lythraceae) popularly known as ‘Pomegranate’ is a rich source of flavonoids. Flavonoids have been implicated in nephroprotective activity.

Objectives: To evaluate effect of Punica granatum seeds and its flavonoids on doxorubicin-induced nephrotoxicity in Wistar rats.

Materials and Methods: The anthocyanidins of P. granatum (A-PJ) (100 and 300 mg/kg, p.o., 21 days) and P. granatum juice (PJ) extract (300 mg/kg, p.o., 21 days) was gavaged daily to the rats along with Doxorubicin (DXR) (60 mg/kg, i.p., once 48 h before sacrifice). The levels of blood urea nitrogen (BUN), uric acid, creatinine and Superoxide Dismutase (SOD), catalase (CAT) and ATPase levels in kidney were measured. Morphological and histopathological indices in the kidney of healthy and DXR treated rats were also measured.

Result: In the present study, DXR exhibited increase in the renal biomarkers (BUN, uric acid and creatinine) which were significantly decreased after pretreatment with A-PJ (100, 300 mg/kg) and PJ (300 mg/kg). DXR significantly decreased levels of SOD and CAT which were restored with A-PJ and PJ pre-treatment. The results were similar to that of Limarin (25 mg/kg, p.o., 21 days) which served as a reference standard. Histopathological studies were also in agreement of above.

Conclusion: The study concludes that P. granatum possess potential nephroprotective activity which may be attributive to its flavonoids (anthocyanidins), having preventive potential in treatment of kidney disorders.

Keywords: Blood urea nitrogen; Anthocyanidins; Flavonoids, Pomegranate


The introduction of Doxorubicin (DXR) for the treatment of cancer in 1969, this chemical has demonstrated high antitumor efficacy. However, its use in chemotherapy has been limited largely due to its diverse toxicities, including cardiac, renal, hematological and testicular toxicity [1,2], alterations of DNA and the production of free radicals [3-5] mitochondrial damage [6] and iron-dependent oxidative damage to biological macromolecules [7]. DXR is an anthracyclic antibiotic with broad spectrum of anti-neoplastic activity. Oxidative stress has been demonstrated in DXR mediated myocardial, renal [8] and liver [9] damages. DXR injection substantially increased the peroxidative damage in the myocardium, hepatic and renal tissues and markedly increased the serum CK-MB, LDH and the other biochemical variables such as, creatinine, BUN, ALT and AST [10]. DXR treatment also induced peroxidative alterations in various tissues [11,12] which was evident by significant decrease in SOD and CAT. Flavonoids possess a variety of biochemical and pharmacological activities such as antioxidant, antiviral, anticarcinogenic, and anti-inflammatory effects believed to be beneficial for human health [13]. Polyphenols are the major class of pomegranate fruit phytochemicals, including flavonoids (anthocyanins), condensed tannins, (proanthocyanidins) and hydrolysable tannins (ellagitannins and gallotannins) [14-16]. Pomegranate phytochemicals also show potential in chemoprevention of various types of cancers, by exerting antiproliferative effects on tumor cells [17]. The presence of anthocyanins is responsible for the appealing bright red color of juice and other products of pomegranate fruit [18]. So, in the light of above literature, the present study was planned to study the effect of Punica granatum seeds on DXR-induced nephrotoxicity in Wistar rats.

Materials and Methods


Albino rats (Wistar strain) of either sex weighing between 200- 250 g, were obtained from Bharat Serum and Vaccines Ltd., Thane, India. Animals were housed into groups of five under standard laboratory conditions of temperature 25 ± 1°C with free access to food and water. The experiments were performed during the light portion. The experiments were carried out according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India, and approved by the Institutional Animal Ethical Committee of MGV’s Pharmacy College, Nashik, India.

Plant material and extraction

Fruits of P. granatum (PG) (1Kg) were brought from Malegaon and authenticated by Mr. P. G. Devakar, Botanical Survey of India, Pune, India. Voucher specimen has been retained there (PUGAN12). The seeds were separated and ground to obtain juice. The juice subjected to dryness and concentrated under reduced pressure to obtain juice extract (PJ) (yield: 4% w/w). Anthocyanidins, (A-PJ) (yield: 1.6% w/w), were isolated from PG by slight modification of method described earlier [19]. dissolved in 2-4 drops of methanolic HCl and was subjected to thin layer chromatography using TLC silica gel 60 F254 plates (Merck) and BAW [n-butanol: acetic acid: water (4:1:5)], forestal [Concentrated HCl: acetic acid: water (3:30:10)] and formic acid [Concentrated HCl: formic acid: water (2:5:3)] as mobile phases. Iodine vapors were used for visualizing the spots on the plates and Rf values were calculated (Data not shown). The phytochemical testing of extract was also carried out.

Drugs and Treatment schedule

Doxorubicin (ADRIM 50 mg) was purchased from Dabur Pharma Ltd., India. Limarin was purchased from Serum institute, India). BUN kit (Reckon Diagnostics Pvt. Ltd., India), URIC ACID kit (Crest Biosystems, Goa, India), CREATININE kit (Vital Diagnostics (P) Ltd. India) were used for the study. All drug solutions were freshly prepared in saline before each experiment. PJ and A-PJ was dissolved in distilled water and administered orally. In DXR treated groups, DXR was given once, 48 h before sacrifice. The animals were divided in following experimental groups, 5 animals in each.

Group I: Vehicle (saline) (1 ml/kg, p.o., 21 days), Group II: DXR (60 mg/kg, i.p.), Group III: A-PJ (100 mg/kg, p.o., 21 days), Group IV: PJ (300 mg/kg, p.o., 21 days) + DXR (60 mg/kg, i.p.), Group V: A-PJ (100 mg/kg, p.o., 21 days) + DXR (60 mg/kg, i.p.), Group VI: A-PJ (300 mg/kg, p.o., 21days) + DXR (60 mg/kg, i.p.), Group VII: Limarin (25 mg/kg, p.o., 21 days) + DXR (60 mg/kg, i.p.).

After DXR treatment, animal was sacrificed by cervical dislocation and blood samples were collected by heart puncture method for determination of various biochemical parameters. The both Kidney tissues were immediately dissected out, washed with ice-cold 0.9% saline and weighed. They were then subjected to antioxidant and histopathology studies.

Estimation of body and kidney weight

In each group, body weight of rats was taken before and after DXR treatment. Isolated kidney was weighed after keeping them in ice-cold saline and squeeze out the blood.

Biochemical estimation

Serum was separated from collected blood using centrifuge at 3000 g for 15 min and used for estimation of BUN [20], URIC ACID [21], and CREATININE [22]. The one of the excised kidney was then weighed and homogenized in chilled Tris buffer (10 mM, pH 7.4) at a concentration of 10% w/v. The homogenates were centrifuged at 10,000 g for 20 min. The clear supernatants were used for the assays of Superoxide Dismutase (SOD) [23], catalase (CAT) [24]. The sediment after centrifugation of kidney tissue homogenate were resuspended in ice cold Tris buffer (10 mM, pH 7.4) to get a final concentration of 10% and were used for the estimation of different membrane bound enzymes such as Na+ K+ ATPase [25], Ca2+ ATPase [26], and Mg 2+ ATPase [27], Total proteins [28].

Histopathological studies

The remaining kidney was fixed in 10% formalin. The specimens were then processed for standard procedure and were embedded in paraffin wax. The blocks were then stained according to hematoxylin and eosin method. Five-micrometer thick histological sections were obtained from the paraffin blocks. The sections were examined under the light microscope and photographs were taken under 10X using Motic camera.


The mean ± SD values were calculated for each group. One-way ANOVA followed by Dunnett’s multiple comparison tests were used for statistical analysis. Values of p<0.05 were considered statistically significant.


The changes in weights of rats among the experimental group after 21 days were found to be significant. Significant reduction (p<0.05) were observed in the body weight and isolated kidney weight of DXR alone treated group compared to vehicle treated group. This failure to thrive in animals pretreated with PJ (300 mg/kg), A-PJ (100 mg/kg, 300 mg/kg), and Limarin (25 mg/kg) and also treated with DXR and was probably due to DXR. However, pretreatment with PJ, A-PJ and Limarin exhibited significant (p<0.01) elevation in the body weight and isolated kidney weight in these groups compared to DXR treated group (Table 1).