|Year : 2016 | Volume
| Issue : 1 | Page : 41-48
|Amelioration of cisplatin-induced nephrotoxicity by ethanolic extract of Bauhinia purpurea: An in vivo study in rats
Md Azmat Rana1, Rahat Ali Khan1, Mohammad Nasiruddin1, Aijaz Ahmed Khan2
1 Department of Pharmacology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
2 Department of Anatomy, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
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|Date of Web Publication||15-Jan-2016|
| Abstract|| |
Our objective is to study the nephroprotective activity and antioxidant potential of Bauhinia purpurea unripe pods and bark against cisplatin-induced nephrotoxicity. Healthy adult albino rats of either sex (150-200 g) were randomly divided into six groups of six animals each Group I (vehicle control) and Group II (negative control). Group III (BBE200) and Group IV (BBE400) were administered the ethanolic extract of Bauhinia purpurea bark in doses of 200 and 400 mg/kg/day p.o., respectively, and Group V (BPE200) and Group VI (BPE400) were administered the ethanolic extract of Bauhinia purpurea unripe pods at doses of 200 and 400 mg/kg/day p.o., respectively. All the treatments were given for nine days. Cisplatin in a single dose of 6 mg/kg i.p. was given on the 4 th day to all groups, except the vehicle control group. On the 10 th day, blood and urine were collected for biochemical tests and the rats were sacrificed. The kidney was removed for histology and lipid peroxidation-antioxidant test. Cisplatin caused nephrotoxicity as evidenced by elevated blood urea, serum creatinine and urine glucose, and there was decreased creatinine clearance in Group II as compared with Group I. Administration of BBE and BPE at doses of 200 and 400 mg/kg in Group III and Group VI caused a dose-dependant reduction in the rise of blood urea, serum creatinine and urine glucose, and there was a dosedependant increase in creatinine clearance compared with Group II. There was increased catalase and glutathione and decreased malondialdehyde levels in Group II, while BBE 400 (Group IV) and BPE 400 (Group VI) treatments significantly reversed the changes toward normal values. Histological examination of the kidney revealed protection in Group IV and Group VI compared with Group II. The ethanolic extract of Bauhinia purpurea unripe pods and bark has a nephroprotective activity against cisplatin-induced nephrotoxicity in rats.
|How to cite this article:|
Rana M, Khan RA, Nasiruddin M, Khan AA. Amelioration of cisplatin-induced nephrotoxicity by ethanolic extract of Bauhinia purpurea: An in vivo study in rats. Saudi J Kidney Dis Transpl 2016;27:41-8
|How to cite this URL:|
Rana M, Khan RA, Nasiruddin M, Khan AA. Amelioration of cisplatin-induced nephrotoxicity by ethanolic extract of Bauhinia purpurea: An in vivo study in rats. Saudi J Kidney Dis Transpl [serial online] 2016 [cited 2019 Nov 19];27:41-8. Available from: http://www.sjkdt.org/text.asp?2016/27/1/41/174068
| Introduction|| |
Cisplatin is one of the most widely used cancer chemotherapeutic agents. It is effective against many solid organ tumors, including those of the head, neck, lung, testis, ovary and breast. Its excellent anticancer activity is associated with numerous toxicities at therapeutic doses like ototoxicity, gastrotoxicity, myelosuppression and allergic reactions, but the main dose-limiting toxicity of cisplatin is nephrotoxicity. , Despite saline hydration and diuresis, the incidence of nephrotoxicity is in the range of 20-30%. 
Proximal tubules are the primary site of injury in cisplatin nephrotoxicity.  Recent studies indicate that oxidative stress plays an important role in cisplatin-induced proximal tubule injury, resulting in augmented oxidation of lipid, protein and nucleic acid and reducing the activity of enzymatic antioxidants such as superoxide dismutase (SOD), gluthathione peroxidase and catalase.  Reactive oxygen species (ROS) like superoxide radical and hydroxyl radical reduces renal blood flow and induces tubular damage associated with increased renal level of malondialdehyde (MDA), a biomarker of lipid peroxidation.  Apoptosis and necrosis of tubular epithelial cells are the key histological features in nephrotoxicity.  Therefore, several chemical and natural substances with antioxidant activity had been examined for their protective effects against cisplatin-induced nephrotoxicity. 
Bauhinia purpurea is an ornamental plant of subtropical regions like India, Nepal and North and South America. In India, the habitat is the sub-Himalayan forest, northern India, Assam and Khasi hills. It belongs to the family Caesalpiniaceae and is commonly known as Raktakanchan and Khairwaal in folk and purple orchid tree in English.  The Bauhinia purpurea plant has been extensively used in Indian traditional and folklore medicine to cure various human ailments such as dropsy, pain, rheumatism, convulsions, wound healing, delirium and septicemia. The bark of the plant is used as an astringent and its decoctions are recommended for ulcers as a useful wash solution. Various pharmacological activities of Bauhinia purpurea have been reported, like analgesic and anti-inflammatory,  antimalarial, antimycobacterial, antifungal and cytotoxic,  cardiotonic,  hypo-lipidemic,  antioxidant,  hepatoprotective,  antidiabetic  and nephroprotective activities.  The nephroprotective activity has been studied using pods of Bauhinia purpurea against gentamicin-induced nephrotoxicity.  In this study, we have evaluated the nephroprotective potential of Bauhinia purpurea and for its antioxidant activity against cisplatin-induced nephrotoxicity.
| Materials and Methods|| |
The unripe pods and bark of Bauhinia purpurea were collected in the months of April- May from the botanical garden of the Aligarh Muslim University, Aligarh and were identified and authenticated by Dr. Athar Ahmed, Assistant Professor, Department of Botany of our university and a voucher specimen with voucher no. DWS/VS/01 was submitted to the department. These plant materials were separately washed with tap water, shade-dried and pulverized to a coarse powder using a mechanical grinder. The powder was then separately subjected to soxhlet extraction with ethanol (95%). Extraction was carried out for 36-48 h. Extracts thus obtained were dried by slow heat treatment. Thus, the yield obtained was 47.37% (w/w) and 4.88% (w/w) from the bark and unripe pods of Bauhinia purpurea, respectively. The dried ethanolic extract were suspended in 0.5% w/v of sodium carboxymethyl cellulose (CMC) in distilled water to prepare the test doses (200 and 400 mg/kg/mL).
Injection cisplatin (Neon Laboratories Ltd, Mumbai, India.) and diagnostic kits for serum creatinine, blood urea, urine sugar and urine creatinine (Span Diagnostic Ltd. Hyderabad, India) were employed in the study.
Healthy adult albino rats weighing between 150 and 200 g of either sex were procured from the central animal house of the Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh (Vide Registration no. 401/CPCSEA). The animals were housed in maintained laboratory conditions according to the Committee for Supervision on Experiments on Animals guidelines. They were supplied with drinking water ad libitum and a pellet diet (Ashirwad Industries, Chandigarh, India). Rats were acclimatized for one week prior to experimentation in the laboratory conditions. Ethical clearance for the study was obtained from the Institutional Animal Ethics Committee of the Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh.
The rats were randomly divided into six groups, each group containing six rats. Group I served as the vehicle control group and received normal saline intraperitoneally (1 mL/ kg) and 0.5% CMC per orum daily (1 mL/kg). Group II served as the negative control group and received 0.5% CMC per orum daily (1 mL/kg). Group III (BBE 200) and Group IV (BBE400) were the test groups that received the ethanolic extract of the Bauhinia purpurea bark at doses of 200 and 400 mg/kg per orum daily. Similarly, Group V (BPE 200) and Group VI (BPE 400) were the test groups to which ethanolic extracts of the Bauhinia purpurea unripe pod was given at a doses of 200 and 400 mg/kg per orum daily. All the groups were given treatment over a period of nine consecutive days. Cisplatin in a single dose of 6 mg/kg  i.p. was administered on the 4 th day to all groups except the vehicle control group. Extracts were given 1 h prior to the administration of cisplatin in the treatment groups. Following the last dose of treatment, the animals were housed individually in separate metabolic cages to collect the 24-h urine. Twenty-four hours after the last dosing, i.e. on the 10 th day, the rats were anaesthetized with ketamine and sacrificed. Blood samples were collected by cardiac puncture. Serum was separated by centrifuging the blood at 2500 rpm (1500 g) for 10 min and stored. Kidneys were dissected out and weighed. One kidney was preserved in 10% formalin for histological examination and the other kidney was cut into small pieces and homogenized. Homogenization was performed at 5000 rpm (3000 g) in potassium-phosphate buffer (pH 7.36, 0.1 M) and the supernatant was taken out for biochemical analysis for assessment of oxidative stress and antioxidant activity. For the estimation of reduced glutathione (GSH), 1 mL of homogenate of each rat as such was stored separately. All the samples were labeled properly and stored at -20°C for further analysis.
Serum urea, serum creatinine, urine creatinine and urine glucose levels were determined using diagnostic kits from Span Diagnostics, Hyderabad, India. Creatinine clearance (CrCl) was calculated using the following formula:
For estimation of oxidative stress (MDA) and antioxidant enzymes, the standard protocol was followed. Malonyldialdehyde (MDA) level was determined by condensation of MDA with two equivalents of thiobarbituric acid to give a fluorescent red derivative that was assayed spectrophotometrically at 532 nm.  The level of catalase, an antioxidant enzyme, was estimated by breakdown of hydrogen peroxide.  Reduced GSH level estimation was performed according to the method of Ellman. 
Formalin-preserved kidney tissues were embedded in paraffin and 5-6 μm sections were cut using a rotary microtome and stained with hematoxylin and eosin (H&E).  All sections were examined with a light microscope and graded for glomerular congestion, mononuclear cell infiltration, tubular necrosis and tubular hyaline casts.
| Statistical Analysis|| |
All the data of the study have been expressed as mean ± standard error of mean (SEM). The Statistically significant differences were determined by one-way analysis of variance (ANOVA) followed by the post hoc Dunnets test for multiple comparisons. Probability values (P) <0.05 were considered to be statistically significant.
| Results|| |
The physical parameters studied after nine days of oral treatment were percentage change in body weight, kidney weight per 100 g of body weight, kidney volume and urine volume. Administration of a single injection of cisplatin 6 mg/kg body weight, i.p., in the negative control group caused a decrease in body weight and an increase in kidney weight, kidney volume and urine volume as shown in [Table 1]. The changes were statistically significant (P <0.001) as compared with the normal control group. Administration of the ethanolic extract of Bauhinia purpurea unripe pods and bark at doses of 200 and 400 mg/kg for nine days caused dose-dependant mild protection in cisplatin-induced nephrotoxicity. There was a lower reduction in body weight as compared with the negative control group. The increase in kidney weight, kidney volume and urine volume in Bauhinia purpurea unripe pods and bark-treated groups was also lower compared with the negative control group. However, the increase in kidney volume in Bauhinia Purpurea unripe pods (BPE 400) and bark (BBE 400)-treated groups was significantly (P <0.05) less as compared with the negative control group.
|Table 1: Effect of ethanolic extracts of bark and unripe pod of Bauhinia purpurea on physical parameters in cisplatin-induced nephrotoxicity.|
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Blood urea, serum creatinine, urine glucose and creatinine clearance were taken as markers of kidney function. In the negative control group, animals treated with a single injection of cisplatin 6 mg/kg, i.p., the levels of blood urea, serum creatinine and urine glucose were significantly elevated (P <0.001) while creatinine clearance was significantly decreased (P <0.001). Data are shown in [Table 2]. Administration of ethanolic extracts of Bauhinia Purpurea unripe pods and bark at doses of 200 and 400 mg/kg caused dose-dependant protection against cisplatin-induced nephrotoxicity. The rise in blood urea, serum creatinine and urine glucose was less compared with that in the negative control group. There was improvement in creatinine clearance, i.e. the reduction in creatinine clearance was lesser in the test groups compared with the negative control group. Changes in the above parameters in the Bauhinia purpurea unripe pods (BPE 400) and bark (BBE 400)-treated groups were statistically significant (P <0.01) compared with the negative control group.
|Table 2: Effect of ethanolic extracts of bark and unripe pod of Bauhinia purpurea on biochemical parameters of cisplatin-induced nephrotoxicity.|
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Lipid peroxidation markers malonyldialdehyde (MDA) test and antioxidant test, viz. catalase and reduced GSH, were performed on homogenized kidney tissue taken on the 10 th day. Cisplatin administration significantly elevated the MDA (P <0.001) level and decreased the catalase (P <0.001) and GSH (P <0.001) levels, as shown in [Table 3]. Administration of ethanolic extracts of unripe pods (BPE 400) and bark (BBE 400) of Bauhinia purpurea 400 mg/kg caused a significant change in the levels of MDA (P <0.01), catalase (P <0.01) and GSH (P <0.01) in the test groups compared with the negative control group.
|Table 3: Effect of ethanolic extracts of bark and unripe pod of Bauhinia purpurea on cisplatin-induced oxidative stress and antioxidant defence system.|
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Histopathology of the kidney section of the normal control group [Figure 1]A showed normal glomerulus and tubules with normal architecture. The kidney sections of the cisplatinonly treated negative control group [Figure 1]B, showing extensive degeneration, desquamation of tubular epithelium, hyaline casts, interstitial edema and inflammatory cell infiltration with loss of normal architecture. These features suggested extensive necrosis of the kidney, while the BBE 400 and BPE 400treated groups (Group IV, [Figure 1]C and Group VI, [Figure 1]D) showed lesser damage as compared with the negative control with respect to necrotic tubules, infiltration, interstitial cell edema, congestion and hyaline casts, as shown in [Table 4]. These observations substantiate the biochemical findings in the above groups.
|Figure 1: Photomicrograph of rat kidney tissue section. (A) Normal histology of the kidney tissue in the control group (Group I) showing normal architecture of the glomerulus and tubules (H&E × 400). (B) Cisplatin-only treated negative control group (Group II) showing mononuclear cell infiltration, glomerular congestion, tubular necrosis and hyaline casts (H&E × 400). (C) Cisplatin plus BBE400-treated group (Group IV) showing mild mononuclear cell infiltration with mild glomerular congestion (H&E × 400). (D) Cisplatin plus BPE400-treated group (Group VI) showing improvement in the architecture resembling the control group (H&E × 400).|
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|Table 4: Scoring of the histological features of the rat kidney tissue sections in cisplatin-induced nephrotoxicity.|
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| Discussion|| |
Cisplatin, which is the most widely used anti-cancer agent, is most effective against many solid organ tumors. However, its major side-effect of nephrotoxicity is the doselimiting factor for its clinical use as dose escalation therapy.  Since its discovery, various strategies have been tried to prevent its side-effect of nephrotoxicity. One of the most commonly used strategies is saline hydration and forced diuresis. Despite saline hydration and forced diuresis, nephrotoxicity is in the range of 20-30%. 
In our study, cisplatin administration in rats produced a significant decrease in body weight and increase in kidney weight, kidney volume and urine volume [Table 1]. The decreased body weight observed may have resulted from the increased catabolism and decreased food intake. The increase in normalized kidney weight and kidney volume with respect to the body weight of rats of the cisplatin-treated negative control group resulted from the edema due to druginduced tubular necrosis. The significant increase in urine volume per day in the cisplatin-treated negative control group compared with the normal control group confirms the cisplatin-induced non-oliguric acute renal failure. Biochemical markers of serum and urine confirmed that cisplatin in a single dose of 6 mg/kg produced significant nephrotoxicity as indicated by a significant increase (P <0.001) in blood urea, serum creatinine and urine glucose following a single dose of cisplatin treatment. Further, it was characterized by significant reduction in creatinine clearance (P <0.001) compared with the normal control group.
Bauhinia purpurea unripe pods and bark treatment produced dose-dependant improvement in loss in body weight compared with the negative control group. But, the improvement was lesser than in the normal control group, suggesting that supplementary energy must be supplied through diet. Similarly, extract treatment failed to completely prevent the edema caused by cisplatin administration. Although ethanolic extracts of unripe pod and bark of Bauhinia purpurea at a dose of 400 mg/kg significantly (P <0.001) decreased the edema, it still did not return to normal. This was probably because of the shorter duration of the study.
Nine days' treatment of ethanolic extract of Bauhinia purpurea pod and bark at doses of 200 and 400 mg/kg produced dose-dependant protection in the alteration of markers of nephrotoxicity compared with the negative control group, although the protection was insignificant at a dose of 200 mg/kg for both pods and bark extracts. But, the Bauhinia purpurea extracts' treatment at a dose of 400 mg/kg for both unripe pod and bark produced significant protection compared with the negative control group. Bauhinia purpurea unripe pod extract at 400 mg/kg reduced the blood urea, serum creatinine and urine glucose by 29.22%, 26.32% and 19.90%, respectively, and increased the creatinine clearance by 12.74%. The Bauhinia purpurea bark extract at 400 mg/kg reduced the blood urea, serum creatinine and urine glucose by 28.29%, 24.21% and 19.56%, respectively, and increased creatinine clearance by 12.42% [Table 2]. Several possible mechanisms have been proposed to explain the cisplatin-induced nephrotoxicity. 
In earlier studies, it is reported that cisplatin preferentially accumulates in the epithelial cells of the proximal tubule and covalently binds to the protein and DNA. , This can affect their antioxidant enzymes, which protect the cell from oxidative damage. This may be the primary reason behind the significant increase in lipid peroxidation indicated by elevated MDA level and reduction in GSH and catalase activity after the treatment of cisplatin in the negative control group. These observations were also reported earlier. , Administration of the ethanolic extract of the unripe pod and bark of Bauhinia purpurea in Group IV and Group VI produced protection against lipid peroxidation and increased the activity of catalase and the level of GSH against cisplatin-induced oxidative stress [Table 3].
The results of our study showed that treatment with ethanolic extracts of Bauhinia Purpurea unripe pod and bark produced significant protection against nephrotoxicity induced by cisplatin treatment. The beneficial effect of Bauhinia purpurea as evidenced by biochemical findings and supported by histological findings in cisplatin toxicity might be due to the scavenging effect of the extract. These findings indicate that the Bauhinia purpurea ethanolic extract supplementation may reduce cisplatin-induced nephrotoxicty. Therefore we suggest that the Bauhinia purpurea extract modulates oxidative stress in the kidney.
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Md Azmat Rana
Department of Pharmacology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh - 202 002, Uttar Pradesh
[Table 1], [Table 2], [Table 3], [Table 4]
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