Original Article

Effect of aqueous extract of on cisplatin‑induced nephrotoxicity in rat

Mousa-Al-Reza Hadjzadeh1,2, Ziba Rajaei3, Zakieh Keshavarzi1, Mohsen Ghasem Shirazi1, Vahedeh Toosi1

1Department of ABSTRACT 2 Physiology, Neuroscience Objective: The purpose of the present study was to examine whether Rheum ribes extract prevents Research Center, School of Medicine, cisplatin‑induced nephrotoxicity. Materials and Methods: The animals were divided into three groups: Group A Mashhad University considered as control group, group B were treated with cisplatin (3 mg/kg B.W. for 3 alternative days), and of Medical Sciences, group C further to cisplatin received the aqueous extract of Rheum ribes (150 mg/rat). Results: Blood urea Mashhad, 3Department nitrogen (BUN) level increased in group B on days 14 and 42 compared to day 0 (P < 0.001); it was also increased of Physiology, Isfahan in group B vs. group A on day 14 (P < 0.001). Rheum ribes extract decreased the serum BUN level on day 14 University of Medical compared to group B (P < 0.001). Serum creatinine level in group B had a similar profile as serum BUN level Sciences, Isfahan, but Rheum ribes had no effect on blood creatinine level. Serum cholesterol level was increased in group B on days 14 and 42 compared to day 0 (P < 0.001). Also, cholesterol level was significantly increased in group B Address for correspondence: Dr. Ziba Rajaei, when compared to group A on day 14 (P < 0.001). Rheum ribes decreased the blood cholesterol level on day E‑mail: [email protected] 42 in comparison to group B (P < 0.001). Serum glucose level was increased in group B on days 14 and 42 vs. day 0 (P < 0.001). Also, glucose level was significantly increased in group B when compared to group A on day 42 (P < 0.001). Rheum ribes increased the serum glucose level on days 14 and 42 compared to day 0 (P < 0.05). Histology of kidneys exposed to cisplatin showed renal injury, but Rheum ribes had no effect on the kidney architecture. Conclusion: Cisplatin‑induced nephrotoxicity was confirmed in our study. Although Rheum ribes had some effects on biochemical parameters; its effect on renal histology in injured kidney was insignificant. Received : 24‑12‑11 Review completed : 13‑05‑12 Accepted : 07‑08‑12 KEY WORDS: Blood urea nitrogen, cholesterol, cisplatin, creatinine, Rheum ribes

isplatin (cis‑diamminedichloroplatinum II; CDDP) is an temperate and subtropical regions of the world, chiefly in Cimportant chemotherapeutic agent that has been widely used Asian countries.[4] for its potent cytotoxic effects upon a variety of tumor types including testicular, ovarian, and cervical carcinoma.[1,2] However, Rheum ribes is a native of Iran which grows in several dose‑dependent and cumulative nephrotoxicity is the major side provinces including Khorasan and is locally known as “Rivas”.[5] effect of this compound, sometimes requiring a reduction in The thick leaf‑stalk of Rheum ribes is used as vegetable, jam or dose or dis‑continuation of treatment. Approximately 25‑35% of syrup by local people.[6] Leaf‑stalk powder and roots are used in patients develop evidence of nephrotoxicity following a single dose Iranian traditional medicine to relieve disorders such as: Gastric of cisplatin.[3] illnesses, constipation, headache, kidney and bladder pain, uterine pain and liver disorders; it also is used to increase appetite and Rheum ribes () from family, is a species bile secretion.[7] The roots of Rheum ribes are also used to treat of perennial and stout herbs that are distributed in the diabetes, , ulcer, diarrhea.[4] Hypocholesterolemic effects of both ethanolic and aqueous extracts of Rheum ribes in Access this article online rabbit have been also reported by Hadjzadeh, et al.[8] Quick Response Code: Website: www.jpbsonline.org The roots of Rheum ribes, collected from Bingöl, contain tannins (8%) and anthracene derivatives (0.025%). According to the results of the chemical study on the roots of the plant collected DOI: from Erzincan, chrysophanol, physcion, rhein, aloe‑emodin, 10.4103/0975-7406.120083 physcion‑8‑O‑glucoside, aloe‑emodin‑8‑O‑glucoside, sennoside A and rhaponticin have been isolated.[9] In a quantitative study from

How to cite this article: Hadjzadeh M, Rajaei Z, Keshavarzi Z, Shirazi MG, Toosi V. Effect of aqueous extract of Rheum ribes on cisplatin-induced nephrotoxicity in rat. J Pharm Bioall Sci 2013;5:309-13.

Journal of Pharmacy and Bioallied Sciences October-December 2013 Vol 5 Issue 4 309  Hadjzadeh, et al.: Effect of Rheum ribes on cisplatin‑induced nephrotoxicity

400 g of and roots of Rheum ribes, these compounds were determined according to Olympus Kits in Olympus AU600 were found: chrysophanol (7 mg), physcion (14 mg), rhein (9 mg), auto‑analyzer (Olympus Corp., Tokyo, Japan). aleo‑emodine (29 mg), physcion‑8‑O‑glucoside (12 mg), aleo‑emodin‑8‑o‑glycoside (35 mg), sennoside‑A (4 mg) and Histological examination rhaponticin (9 mg).[9] In another study, the aerial parts of Rheum ribes, collected from Hakkari, have also been studied, and chrysophanol, physcion, emodin, quercetin, 5‑desoxyquercetin, At the end of the experiment (day 42), animals were decapitated quercetin 3‑O‑rhamnoside, quercetin, 3‑O‑galactoside and and kidneys were excised, trimmed of connective tissues, rinsed quercetin 3‑O‑rutinoside have been isolated.[10] with saline to eliminate blood contamination and preserved in 10% formalin solution for histological examination. After In view of the fact that there is no systematic study on the effect of preparation and staining with hematoxylin and eosin (H and E), aqueous extract of Rheum ribes on cisplatin‑induced nephrotoxicity specimens were examined by a pathologist who was unaware of in rat, the aim of the present study was to examine the protective details of animal groups with light microscopy. effect of Rheum ribes on cisplatin‑induced toxicity in rats. Statistical analysis Materials and Methods The values were expressed as mean ± SEM. The statistical analysis Plant material and preparation of extract of data was realized by one‑way analysis of variance (ANOVA), followed by Tukey multiple comparison tests using 5% level of The aerial parts of Rheum ribes, i.e., thick leaf‑stalk with newly significance. The statistical package used was statistical package growing leaves were obtained from heals of Zoshk (southwest for the social sciences (SPSS 15). of Mashhad, Iran) in June and identified by botanists in a of Ferdowsi University. The plant was cleaned, Results chapped into small pieces and dried while protected from light and wetness. The dried powder (120 g) was mixed with distilled Serum BUN level water in a balloon; the balloon was shaken for two days. The preparation was then filtered off a Gauze mesh and the solvent As shown in Figure 1, serum BUN level was increased in group B was dried by evaporation under reduced pressure at 40°C. The on days 14 and 42 when compared to day 0 (P < 0.001). Also, final product yielded an 8% w/w dried extract. BUN level was significantly increased in group B compared to group A on day 14 (P < 0.001). Treatment with Rheum ribes Animals extract decreased the blood BUN level on day 14 compared to group B (P < 0.001). Albino male rats (weighing 270‑320 g) were purchased from animal house of Mashhad University of Medical Sciences, Serum creatinine level Iran. The animals were acclimatized under room temperature and humidity with a regular light/dark cycle. All experiments in this study were performed in accordance with the guidelines Serum creatinine level was increased in group B on days 14 and for the care and use of laboratory animals and the study was 42 when compared to day 0 (P < 0.001). Also, creatinine level was approved by Mashhad University of Medical Sciences. significantly increased in group B when compared to group A on

Experimental groups

The animals were divided into three experimental groups and underwent following treatment: Group A (n = 7): Control group, received distilled water (0.75ml/rat) for three alternate days. Group B (n = 7): Received cisplatin (3 mg/kg, i.p.) for three alternate days. Group C (n = 8): Received cisplatin (3 mg/kg, i.p.) for three alternate days; then two weeks after the first injection of cisplatin (day 0), the aqueous extract of Rheum ribes (150 mg/rat) was added daily in drinking water throughout the experiment.

Sample collection and biochemical assays Figure 1: Comparison of serum BUN levels (mg/dl) in different groups of rats. Data are expressed as mean±SEM. (Group A) Control group The serum samples were collected on the 0, 14 and 42nd day after (n=7), (Group B) cisplatin-treated group (3 mg/kg, n=7), (Group C) the first injection of cysplatin. Blood samples were collected for cisplatin+Rheum ribes-treated group (150 mg/rat, n=8). aP<0.001 as determination of serum creatinine, blood urea nitrogen (BUN), compared to day 0. bP<0.001 as compared to control group. cP<0.001 as cholesterol and glucose levels. The levels of these parameters compared to day 14 in group B

 310 Journal of Pharmacy and Bioallied Sciences October-December 2013 Vol 5 Issue 4 Hadjzadeh, et al.: Effect of Rheum ribes on cisplatin‑induced nephrotoxicity day 14 (P < 0.001). Treatment of rats with Rheum ribes extract group B when compared to control group on day 42 (P < 0.001). had no effect on the blood creatinine level [Figure 2]. Rheum ribes extract treatment increased the serum glucose level on days 14 and 42 compared to day 0 (P < 0.05). Serum cholesterol level Histopathologic parameters Serum cholesterol level was increased in group B on days 14 and 42 in comparison with day 0 (P < 0.001); it was also The results of histopathologic examination of kidneys are shown significantly increased in group B when compared to group A in Figure 5. In control (normal) group, renal tissue sections had on day 14 (P < 0.001). Treatment with Rheum ribes extract a normal morphology [Figure 5a]. Histological examination of decreased the blood cholesterol level on day 42 when compared kidneys exposed to cisplatin showed the distinctive pattern, which included widespread degeneration of tubular architecture, to group B (P < 0.001) [Figure 3]. sloughing tubular epithelial cells from the basement membrane, tubular cell necrosis and intra‑tubular cast formation especially Serum glucose level in the outer medulla [Figure 5b]. Renal sections obtained from rats treated with Rheum ribes extract demonstrated features As shown in Figure 4, serum glucose level was increased very similar to cisplatin‑treated group; degeneration of tubular in group B on both days 14 and 42 in comparison to day cells, basement membrane impairment and intra‑tubular cast P 0 ( < 0.001). Also, glucose level was significantly increased in formation were found in this group.

Discussion

As shown in the present study, histopathologic and biochemical data demonstrated that cisplatin induced renal failure lasting until the end of the experiment (day 42). Much attention

Figure 2: Comparison of serum creatinine levels (mg/dl) in different groups of rats. Data are expressed as mean±SEM. (Group A) Control group (n=7), (Group B) cisplatin-treated group (3 mg/kg, n=7), (Group C) cisplatin+Rheum ribes-treated group (150 mg/rat, n=8). aP<0.001 as compared to day 0. bP<0.001 in comparison to control group

Figure 3: Comparison of serum cholesterol levels (mg/dl) in different groups of rats. Data are expressd as mean±SEM. (Group A) Control group (n=7), (Group B) cisplatin-treated group (3 mg/kg, n=7), (Group C) cisplatin+Rheum ribes-treated group (150 mg/rat, n=8). aP<0.001 as compared to day 0. bP<0.001 as compared to day 14 in control group. cP<0.05 as compared to day 42 in cisplatin-treated group

Figure 4: Comparison of serum glucose levels (mg/dl) in different groups of rats. Data are expressed as mean±SEM. (Group A) Control a group (n=7), (Group B) cisplatin-treated group (3 mg/kg, n=7), (Group b C) cisplatin+Rheum ribes-treated group (150 mg/rat, n=8). aP<0.001 as Figure 5: Light photomicrographs of the kidney sections (H and E, compared to day 0. bP<0.001 as compared to control group. cP<0.05 ×200). (a) Kidney sections from control group with normal renal as compared to day 0 in the same group morphology. (b) The group B shows the distinctive pattern of renal injury

Journal of Pharmacy and Bioallied Sciences October-December 2013 Vol 5 Issue 4 311  Hadjzadeh, et al.: Effect of Rheum ribes on cisplatin‑induced nephrotoxicity has been focused on the direct toxic effects of cisplatin on cholesterol and low‑density lipoprotein cholesterol (LDL‑C) renal tubular cells in vitro.[11] In this setting, cisplatin induces were decreased in diabetic rats treated with Rhuem ribes. They DNA damages,[12,13] mitochondrial dysfunction,[13] formation concluded that Rhuem ribes exerts a beneficial effect on the of reactive oxygen species,[14] caspase activation,[15] and either experimental diabetic nephropathy.[32] In a randomized trial, necrotic or apoptotic cell death, depending on the level of Chinese rhubarb extract was found to lower total cholesterol cisplatin.[16,17] Inflammatory mediators appear to play an and LDL‑C levels and increase serum high‑density lipoprotein important role in pathogenesis of acute renal failure (ARF).[18‑20] cholesterol (HDL‑C) and albumin levels in patients with CRF Faubel, et al., demonstrated that cisplatin‑induced acute renal compared to controls.[33] Another study in rats with diabetic failure was associated with increased in IL‑1, IL‑18, and IL‑6 nephropathy found that rhubarb extract speeded nitrogen levels and neutrophil infiltration in kidney. However, inhibition excretion and alleviated hyperlipidemia compared to control of IL‑1, IL‑18, and IL‑6 or neutrophil infiltration in the kidney rats.[25] Hypocholesterolemic effects of both ethanolic and was not sufficient to prevent cisplatin‑induced ARF.[21] aqueous extracts of Rheum ribes in hypercholesterolemic rabbit have been also reported by Hadjzadeh, et al.[8] As shown in Figures 1 and 2, cisplatin increased the serum BUN and creatinine levels and Rheum ribes was not able to decrease Rheum ribes has been used as a traditional Turkish therapeutic the creatinine levels but decreased the BUN levels on day 14. agent for diabetes mellitus in Eastern . ÖzbekIn H, Maliakel, et al., demonstrated that administration of cisplatin et al., reported that the decoction extract of Rheum ribes significantly increased the serum creatinine and urea levels roots possesses significant blood‑sugar lowering activity in compared to normal group.[22] In agreement with our study, alloxan‑induced diabetic mice. However, this extract did not Hanzlicek reported that administration of Rheum officinalis show hypoglycemic action in healthy mice. This study has shown orally as 75 mg tablet every 12 hrs in cats with chronic kidney that Rheum ribes roots decoction extract is more potent than disease had no effect on body weight, serum creatinine, or glibenclamide during the first hour following the application. [23] systemic blood pressure. In other study, Rheum ribes considered as potential candidates for amelioration/management of type 2 diabetes.[34] It has been reported that glomerulosclerosis was reduced after administration of aqueous extract of Chinese rhubarb to rats In conclusion, cisplatin‑induced nephrotoxicity was confirmed which underwent subtotal nephrectomy compared to those by our study, but daily administration of 150 mg/kg aqueous [24] given only plain water. Rhubarb has long held an esteemed extract of Rheum ribes had a little effect on biochemical factors place in traditional Chinese herbalism as part of treatment in this dose. This may partially be due to its time‑course action protocols for patients with chronic renal failure (CRF). Chinese on biochemical parameters. Thus, further investigation is rhubarb has been investigated in preliminary clinical trials in needed to determine the proper dose and mechanism(s) of China and shown to have beneficial effects on clinical symptoms action of Rheum ribes. and on serum BUN and creatinine levels in CRF patients. In a similar trial, a combined herbal protocol with Chinese rhubarb Acknowledgment was shown to reduce BUN and clinical symptoms in patients with CRF underwent hemodialysis compared to controls treated The results presented in this work have been taken from the student’s [25] with hemodialysis alone. Chinese rhubarb extracts have thesis. This study was supported by a grant from the Council of significant potential to improve treatment protocols for patients Research, Mashhad University of Medical Sciences. with CRF. In one perspective clinical and experimental study, the progression rate of renal failure was reduced markedly in References both Rhubarb and Rhubarb plus captopril group.[26] 1. 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