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Nutrition clinique et métabolisme xxx (2016) xxx–xxx

Original article

Protective effect of Raphanus sativus on D-galactosamine inducednephrotoxicity in rats

ffet néphroprotecteur de Raphanus sativus chez le rat après agression toxique à la D-galactosamine

Magesh S.B. a,∗, Rashmi Rajappa a, Dhana Rangesh Kumar V. b, Jayathi G. c

a Department of Water and Health, Faculty of Life Sciences, JSS University, SS Nagar, Mysore, Karnataka, Indiab Department of Biochemistry, NGP college of Arts and Science, Coimbatore, Tamil Nadu, India

c Department of Biochemistry, Women’s Arts and Science College, Krishnagiri, Tamil Nadu, India

Received 23 June 2015; received in revised form 30 November 2015; accepted 1st December 2015

bstract

The aim of the present study was to evaluate nephroprotective effect of Raphanus sativus ethanolic extract (RSEt) on tissue defense systemn galactosamine (GalN) induced renal damage in rats. GalN was administered intraperitoneally at a dose of 400 mg/kg/b.w for three alternateays and the renal toxicity was manifested by a significant (P < 0.05) increase in the levels of renal markers such as urea, creatinine and uric acid.his was found to be associated with decreased activities of renal antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT),lutathione-S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR) and depletion of renal reduced glutathione (GSH),itamin C and vitamin E. Administration of the RSEt (850 mg/kg/body weight, oral) for 15 days to rats reduced the levels of renal markers andignificantly increased the level of antioxidants. The activities of gamma glutamyl transpeptidase (GGT) and thiobarbituric acid reactive substancesTBARS) were also decreased in the kidney of RSEt treated group. Renal histology examination confirmed the damage to the kidney as it revealsevere necrosis of the proximal renal tubules with haemorrhage which was ameliorated by the treatment with RSEt. These results suggest that the. sativus has protective effects on GalN-mediated nephrotoxicity and this may be related to the action of the antioxidant content of the extract.

2015 Elsevier Masson SAS. All rights reserved.

eywords: Raphanus sativus; Galactosamine; Antioxidants; Free radicals; Kidney damage

ésumé

ntroduction. – Le but de l’étude était d’évaluer l’effet néphroprotecteur d’un extrait éthanolique de Raphanus sativus (RSEt ; radis) dans unodèle de lésions rénales induites par la galactosamine (GalN) chez le rat.éthodes. – Quatre groupes de rats étaient testés. Un groupe recevait per os du sérum physiologique (groupe I), un groupe recevait GalN par voie

ntra-péritonéale à une dose de 400 mg/kg de poids corporel pendant trois jours alternés (groupe II), un groupe recevait par la même voie GalN etar voie orale RSEt à raison de 850 mg/kg de poids corporel pendant 15 jours (groupe III), et un groupe recevait seulement RSEt (groupe IV) per

s durant 15 jours.ésultats. – Pour le groupe II, la toxicité rénale se manifestait par une augmentation significative (p < 0,05) des concentrations de marqueurs sériques

énaux tels que l’urée, la créatinine et l’acide urique. Ces variations étaient associées à une réduction des activités enzymatiques antioxydantes

Please cite this article in press as: S.B. M, et al. Protective effect of Raphanus sativus on D-galactosamine induced nephrotoxicity in rats. Nutrclin métab (2016), http://dx.doi.org/10.1016/j.nupar.2015.12.002

énales portant sur la superoxyde dismutase (SOD), la catalase (CAT), la glutathion-S-transférase (GST), la glutathion peroxydase (GPx) et lalutathion réductase (GR), ainsi qu’à une réduction au niveau rénal du glutathion réduit (GSH) et des vitamines C et E. L’administration aux rats dea RSEt (groupe III) réduisait les concentrations des marqueurs rénaux de toxicité et augmentait fortement le niveau des antioxydants. Les activitésénales de gamma glutamyl transférase (GGT) et le niveau des substances réactives à l’acide thiobarbiturique (TBARS) diminuaient égalementour ce groupe traité par RSEt. L’histologie révélait sous GalN l’induction d’une nécrose hémorragique grave des tubules rénaux proximaux,méliorée par le traitement par RSEt.

∗ Corresponding author.E-mail address: kumar.magesh06@gmail.com (M. S.B.).

http://dx.doi.org/10.1016/j.nupar.2015.12.002985-0562/© 2015 Elsevier Masson SAS. All rights reserved.

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onclusion. – Ces résultats suggèrent que R. sativus a des effets protecteurs sur la néphrotoxicité médiée par GalN ; ceci peut être lié à l’actione la teneur en antioxydants de l’extrait.

2015 Elsevier Masson SAS. Tous droits réservés.

ots clés : Raphanus sativus ; Galactosamine ; Antioxidants ; Radicaux libres ; Lésions rénales

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. Introduction

Nephrotoxicity is a serious health problem among all popula-ions which may be due to hypertension, inflammatory disease,iabetes, obesity, and toxic nephropathies from environmentalnd occupational hazards that include heavy metals, drugs, andhemicals [1,2]. Recent studies have documented that oxida-ive stress is highly prevalent in patients with renal disease [3].mino sugar, galactosamine (GalN) with its unique properties

auses many metabolic and morphological abnormalities in theiver and advanced renal failure in experimental animals [4].he toxic effect of GalN has been reported to associate with

he depletion of UTP nucleotides followed by the formationf UDP hexosamines resulted in the inhibition of transcriptionnd consequently the translation processes [5,6]. GalN intoxica-ion increased reactive oxygen species (ROS), reactive nitrogenpecies (RNS) and tumor necrosis factor-� (TNF-�) productionnd disturbed the antioxidant machineries in the kidney tissue7]. Moreover GalN damages the enzymes responsible for mito-hondrial substrate transportation leading to decrease in renallood flow and vasoconstriction [8]. The detailed mechanismy which GalN induces nephrotoxicity is unclear. Neverthe-ess, a role has been postulated for free radical induced lipideroxidation [9].

Many numbers of plants are extensively used in indige-ous system of medicine and also have been reported for itsephroprotective effect [10,11]. The leaves of Aegle marmelos,eratonia silique, Ocimum sanctum; roots of Cassia auricu-

ata, Withania somnifera; seeds of Carica papaya, Cucurbitaepo; fruits of Crataeva nurvula, Morinda citrifolia, Gymnemaontanum and many other plants are proved to have protective

ffect against nephrotoxicity [12,13]. It has been reported thatephroprotective activities of the compounds may be well corre-ated with its antioxidant and free radical scavenging properties14,15].

Raphanus sativus Linn (family - Cruciferae) is commonlyalled “radish”, is an aromatic annual herb, which is used in tra-itional medicine to treat various diseases. The roots and leavesf this plant have been reported to possess various pharmaco-ogical values like hepatoprotective [16–18], cardioprotective19], antioxidant [20], antiurolithiatic effects [21] and gastro-ynic pains [22]. So far, limited information exists concerninghe beneficial effects of R. sativus against the oxidative injuries

Please cite this article in press as: S.B. M, et al. Protective effect of Raphaclin métab (2016), http://dx.doi.org/10.1016/j.nupar.2015.12.002

n kidney. Hence, the present study aimed to evaluate nephropro-ective effect of RSEt on tissue defense system in GalN inducedenal damage in rats.

012

. Materials and methods

.1. Chemicals

D-galactosamine was purchased from Sigma Aldrich LimitedSt Louis, MO, USA). All other chemicals used were of highesturity and analytical grade.

.2. Extraction of R. sativus

R. sativus roots were collected from JSS Institute of Natur-pathy and Yogic Sciences, Ooty, Tamil Nadu. The roots wereried at ambient temperature and stored in dry place prior to use.he roots were then cleaned and washed with 10% saline, shaderied and powdered. About 500 g of powder was subjected tothanolic extraction using soxlet apparatus. The organic fractionas collected and concentrated using a rotary evaporator under

educed pressure and lyophilized to get powder and used fornalysis. The final yield (w/w) of the extract was 7.30%.

.3. Qualitative phytochemical estimation

The ethanolic extract of R. sativus was examined by standardethods to determine the presence of phytochemicals such as

lkaloids, proteins, flavonoids, phenolics, tannins, saponins andarbohydrates.

.4. Quantitative phytochemical estimation

.4.1. Estimation of total phenolic contentThe total phenolic content in RSEt was determined by

olin-ciocalteu method [23] spectrometrically. A 0.2-mL ofest extract solution was mixed with 1 mL of folin-ciocalteueagent and then 1 mL of sodium carbonate was added fol-owed by distilled water. The reaction mixture is allowed totand for 2 hours at room temperature and the absorbance waseasured at 760 nm. The gallic acid is used as a standard. The

otal phenolic content was expressed in the terms of gallic acidquivalents.

.4.2. Estimation of total flavanoid contentTotal flavonoid content in RSEt was determined colorimetri-

ally according to the method described by Chang et al. [24]. A

nus sativus on D-galactosamine induced nephrotoxicity in rats. Nutr

.5 mL of extract was mixed with 1.5 mL of methanol, 0.1 mL of0% aluminum chloride, 0.1 mL of 1 M potassium acetate and.8 mL of distilled water. The reaction mixture was incubated

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M. S.B. et al. / Nutrition cliniqu

or 30 min at room temperature and the absorbance was mea-ured at 415 nm. The total flavonoid content was expressed ing quercetin equivalent flavones per mg extract.

.5. Animals

Female albino wistar rats weighing around 150–200 g wererocured from Kerala Agriculture University, Mannuthy, Ker-la, India. The study protocol was approved by the Institutionalnimal Ethics Committee and the animals were maintained in

ccordance with the guidelines of the National Institute of Nutri-ion, Indian council of Medical Research, Hyderabad, India. Thenimals were maintained in the controlled environment withtandard temperature of (25 ± 2 ◦C) and humidity with an alter-ating 12 h light/12 h dark cycle. The animals were fed with aalanced commercial diet and water ad libitium.

.6. Experimental protocol

All animals were randomly divided into four groups. Group: control animals given physiological saline solution, orally.roup II: animals were given intraperitoneal injection of GalN

400 mg/kg body weight on alternative days for 3 days). GroupII: animals injected with GalN (400 mg/kg body weight on alter-ative days for 3 days) and treated with R. sativus ethanol extract850 mg/kg body weight) orally for 15 days. Group IV: controlnimals administered with R. sativus ethanol extract (850 mg/kgody weight) orally for 15 days. At the end of the experi-ental period, the animals were deprived of food overnight

nd then sacrificed by decapitation. Serum was prepared fromhole blood for biochemical analysis such as urea, creatinine

nd uric acid. The kidney tissues were dissected, washed andomogenized to 10% (w/v) with 0.1 M Tris- HCl buffer, pH 7.5.he homogenates were centrifuged at 10,000 rpm for 15 min at◦ C and the clear supernatants were used for the estimationf enzymatic antioxidants (SOD, CAT, GPX, GST, and GR)nd non-enzymatic antioxidants such as GSH, vitamin C, anditamin E.

.7. Preparation of brush border membrane

Brush border membrane vesicles (BBMV) were preparedrom whole cortex using the MgCl2 precipitation method asescribed previously [25]. Briefly, freshly minced cortical slicesere homogenized in 50 mM Mannitol and 5 mM Tris-HEPESuffer, pH 7.0 for 10 minutes. MgCl2 was added to final concen-ration of homogenate and the mixture stirred for 20 minutes once. The homogenate was centrifuged at 2000 × g for 10 minnd the supernatant was then recentrifuged at 35,000 × g for0 min. The pellet was resuspended in 300 mM Mannitol and

mM Tris-HEPES, pH 7.4 and and centrifuged at 35,000 × gor 20 minutes. The outer white fluffy pellet of BBM was resus-

Please cite this article in press as: S.B. M, et al. Protective effect of Raphaclin métab (2016), http://dx.doi.org/10.1016/j.nupar.2015.12.002

ended in small volume of buffered 300 mM Mannitol. Freshlyrepared BBM vesicles were used for the renal cortex markers,amma GT and lipid peroxidation levels (TBARS).

mui

étabolisme xxx (2016) xxx–xxx 3

.8. Biochemical assays

Estimation of renal functional markers was determined byrea, uric acid and creatinine by spectrophotometrically. Serumrea was estimated according to the method of Fawcett and Scott26], serum uric acid was estimated based on the enzymaticethod described by Buchanan et al. [27] and serum creatinineas determined by Peake and Whiting [28] color reaction. The

ctivity of SOD was assayed by the method of Kakkar et al. [29],atalase by Sinha [30] and Rotruck et al. [31] method for GPx.he GST activity was determined spectrophotometrically by theethod of Habig [32] and GR activity is measured according to

he method of Smith et al. [33].GSH content of kidney tissues was measured according to the

ethod of Ellman [34]. Vitamin C concentration was determinedpectrophotometrically by dinitrophenyl-hydrazine method at30 nm [35]. Vitamin E was measured by the method of Desai36]. The serum GGT was assayed according to the method ofosalki and Rau [37]. Lipid peroxidation in kidney tissue wasssessed by analysing the formation of TBARS spectrophoto-etrically by the method of Fraga et a1. [38] and the protein

ontent was estimated by the method of Lowry et al. [39].

.9. Histopathological assessment

The collected kidney tissues were fixed in 10% neutraluffered formalin solutions. The tissues were embedded inaraffin. The paraffin blocks were cut to 5 �m thick slices andtained with hematoxylin and eosin (H & E stain). Photomicro-raphs of the stained slides were taken using a light microscopeLeica, Germany).

.10. Statistical analysis

The results were expressed as mean ± S.D. and the resultsere evaluated using statistical analysis at significance level

< 0.05 using SPSS package version 10.0. One way Anovaollowed by post hoc analysis of “least significant difference”LSD) was performed.

. Results

.1. Qualitative and quantitative phytochemical estimation

The preliminary phytochemical screening of RSEt revealedhe presence of alkaloids, proteins, flavonoids, phenolics, tan-ins, saponins (Table 1). Moreover the total phenolic andavonoid content of ethanolic extract of R. sativus. was esti-ated as 26.99 ± 1.4 mg/g and 45.63 ± 1.32 mg/g, respectively.

.2. R. sativus prevents biochemical changes which areaused by GalN

nus sativus on D-galactosamine induced nephrotoxicity in rats. Nutr

Serum urea, uric acid and creatinine are reported as renalarkers and elevated during the damage to the kidney. Serum

rea, uric acid and creatinine levels were found to be signif-cantly higher in GalN group compared with control group

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Table 1Qualitative phytochemical tests of ethanolic extract of R. sativus.

Phytochemicals Ethanolic extract of R. sativus

Alkaloids +Protein and amino acid +Flavonoids +Phenolics +Tannins +Saponins +Carbohydrates −+

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Table 2). These levels were reduced by RSEt treatment in GalN-dministered group and appeared to near normal levels. TheSEt administered control rats showed no remarkable changes

n the renal markers.The kidney SOD, CAT, GPx, GST and GR activities were

easured as an index of tissue antioxidant status (Table 3). Allhese antioxidant enzymes were remarkably declined in ratsdministered GalN when compared to the other experimen-al groups. In response to RSEt treatment, the activities wererought back to near normal levels.

The non-enzymatic antioxidant levels of the control andxperimental groups were depicted (Table 4). The tissue antiox-dant levels were found to be decreased in GalN-treated grouphen compared to control groups. On the other hand, the RSEt

dministration to GalN-administered rats markedly restored theoncentrations of antioxidants in the kidney tissue.

The level of expression of the renal cortex marker enzyme,

Please cite this article in press as: S.B. M, et al. Protective effect of Raphaclin métab (2016), http://dx.doi.org/10.1016/j.nupar.2015.12.002

GT was shown (Table 5). The GGT expression was increasedn the GalN induced animal when compared to the control groupsndicating the renal tubular damage. RSEt treatment showed a l

able 2ffect of R. sativus on renal profile in serum of control and experimental rats.

arameters (mg/dL) Group I Group II

ric acid 11.36 ± 0.30a 13.49 ±

rea 34.24 ± 9.25a 98.05 ±

reatinine 0.91 ± 0.07c 3.65 ±

roup I: control rats; group II: rats injected with GalN (400 mg/kg body weight on athanolic extract of R. sativus (850 mg/kg body weight) for 15 days; group IV: rats adean ± SD for six rats in each group. Statistical evaluation was done by Anova follow

t P < 0.05.

able 3ffect of R. sativus on enzymatic antioxidants in kidney of control and experimental

arameters Group I Group II

ODA 10.09 ± 0.88a 3.73 ± 0.2atalaseR 52.36 ± 3.52a 26.31 ± 2.4Pxc 27.95 ± 2.80a 13.07 ± 2.0STD 1.54 ± 0.21a 0.55 ± 0.0RE 11.37 ± 0.51a 10.53 ± 0.6

: units/min/mg protein; B: �mol of H202 decomposed/min/mg protein; C: �mol of N: �mol of NADPH oxidized/min/mg protein. Group I: control rats; group II: rats inje

II: rats injected with GaIN and treated with ethanolic extract of R. sativus (850 mg/kf R. sativus for 15 days. Values are given as mean ± SD for six rats in each group. Sommon superscript letter differ significantly at P < 0.05.

étabolisme xxx (2016) xxx–xxx

onsiderable reduction in the expression levels of renal cortexarker enzyme. There was a notable difference in the level ofBARS in the control and experimental animals (Table 5). InalN group, a significant increase in kidney TBARS levels wasbserved. The groups treated with the RSEt showed a signifi-ant reduction of the TBARS which shows the nephroprotectiveffect of the extract.

.3. R. sativus prevents histopathological changes causedy GaIN

The kidney tissues of the control rats showed normal his-ological appearance and no difference was observed betweenhe kidney tissues of control animals administered physi-logical saline solution (group I) and those treated withSEt alone (group IV). In the experimental animals admin-

stered GalN (group II), in glomeruli, the enlargement ofapsular space and the decrease of glomerular mass wasbserved.

There was a significant decrease in the degenerative changesn tubules of the kidney tissues of group III animals adminis-ered with GalN and treated with RSEt. Mild tubular changesere observed when compared to the kidney tissues of group

I (Fig. 1). These results obviously indicate that nephroprotec-ive effect of RSEt to the kidney tissues of rats subjected toalN intoxication. These results are well correlated with theiochemical findings of our study.

nus sativus on D-galactosamine induced nephrotoxicity in rats. Nutr

Cumulative data suggest a role for reactive oxygen metabo-ites as one of the postulated mechanisms in the pathogenesis

Group III Group IV

0.22b 12.21 ± 0.47c 12.03 + 0.05c

8.24b 52.47 ± 7.69c 35.16 ± 4.21a

0.39b 2.47 ± 0.31c 1.55 ± 0.21d

lternative days for 3 days); group III: rats injected with GalN and treated withministered with ethanolic extract of R. sativus for 15 days. Values are given ased by LSD. Values not sharing a common superscript letter differ significantly

rats.

Group III Group IV

3b 8.91 ± 0.32c 9.97 ± 0.77a

5b 50.21 ± 2.07a 52.87 ± 2.19a

6b 29.33 ± 1.58a 29.46 ± 2.71a

7b 1.62 + 0.20a 1.90 ± 0.01c

8b 11.32 ± 0.18a 11.64 ± 0.10a

APDH oxidized/min/mg protein; D: �mol of CDNB utilized/min/mg protein;cted with GaIN (400 mg/kg body weight on alternative days for 3 days); groupg body weight) for 15 days; group IV: rats administered with ethanolic extract

tatistical evaluation was done by Anova followed by LSD. Values not sharing a

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Table 4Effect of R. sativus on non-enzymatic antioxidants in kidney of control and experimental rats.

Parameters (mg/g tissue) Group I Group II Group III Group IV

GSH 2.31 ± 0.70a 1.19 ± 0.16b 1.89 ± 0.10c 2.28 ± 0.42a

Vitamin E 1.03 ± 0.02a 0.90 ± 0.08b 0.99 ± 0.02a 1.06 ± 0.03a

Vitamin C 1.52 ± 0.02a 0.90 ± 0.04b 1.42 ± 0.03a 1.56 ± 0.03a

Group I: control rats; group II: rats injected with GalN (400 mg/kg body weight on alternative days for 3 days); group III: rats injected with GalN and treated withethanolic extract of R. sativus (850 mg/kg body weight) for 15 days; group IV: rats administered with ethanolic extract of R. sativus for 15 days. Values are given asmean ± SD for six rats in each group. Statistical evaluation was done by Anova followed by LSD. Values not sharing a common superscript letter differ significantlyat P < 0.05.

Table 5Effect of R. sativus on renal cortex marker enzymes of control and experimental rats.

Parameters Group I Group II Group III Group IV

Gamma GTA 37.35 ± 7.38a 46.06 ± 17.58b 32.56 ± 3.82c 39.76 ± 6.22d

LPOB 0.43 ± 0.03a 0.33 ± 0.01b 0.16 ± 0.01c 0.01a

A: units/min/mg protein; B: �mol of MDA/gm tissue. Group I: control rats; group II: rats injected with GalN (400 mg/kg body weight on alternative days for 3 days);group III: rats injected with GalN and treated with ethanolic extract of R. sativus (850 mg/kg body weight) for 15 days; group IV: rats administered with ethanolicextract of R. sativus for 15 days. Values are given as mean ± SD for six rats in each group. Statistical evaluation was done by Anova followed by LSD. Values notsharing a common superscript letter differ significantly at P < 0.05.

Fig. 1. Histopathological changes in kidney of control and experimental rats. A. Control showing normal architecture. B. GalN-treated at the dose of 400 mg/kgb ose of imal

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ody weight – showing enlargement of capsular space. C. GalN-treated at the dor 15 days – showing significant decrease in the tubular change. D. Control anormal architecture.

f galactosamine induced nephrotoxicity [4]. The induction ofhe cell death was primarily due to oxidative and nitrosativetress and alteration in multiple gene regulation which lead to

Please cite this article in press as: S.B. M, et al. Protective effect of Raphaclin métab (2016), http://dx.doi.org/10.1016/j.nupar.2015.12.002

ell death [9] and instability of cellular membrane due to lipideroxidation [40]. In addition, galactosamine induces plasmaembrane injury initiated by the RNA and protein synthesis

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f 400 mg/kg body weight and treated with R. sativus (850 mg/kg body weight)administered with R. sativus (850 mg/kg body weight) for 15 days – showing

nhibition caused by the deficiency of uridine phosphate andDP-hexoses [41]. Increase in the calcium concentration in turn

ncreases phospholipases leads to advanced membrane injury

nus sativus on D-galactosamine induced nephrotoxicity in rats. Nutr

hich leads to abnormalities in the kidneys, like decreasedenal blood flow due to vasoconstriction and also shows reducedlomerular filtration [42].

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M. S.B. et al. / Nutrition cliniqu

Results of this study confirmed that GalN at a dose of00 mg/kg produces significant nephrotoxicity as evidenced byncrease in blood urea nitrogen, serum creatinine, urea and uriccid, and renal tubular necrosis which corroborated with pre-ious reports [43]. The generation of free radicals leads to themplication of renal dysfunction which in turn disorders the per-

eability of the system leads to the loss of enzyme activitiesesulted increase in the levels of urea, uric acid and creati-ine. The treatment with the RSEt provided marked functionalnd histological protection against acute renal damage in ratsnduced by GalN.

Free radical production, which increases with clinical pro-ression of diseases, involves increased lipid peroxidation, as aesult of which there are cellular membrane degeneration andNA damage. Lipid peroxidation and free radical generation

ncreases in the renal tubular cells and depletion in enzymaticnd non-enzymatic antioxidant systems were reported [44].xtent of lipid peroxidation could be determined by estimationf the final lipid peroxidation products- malondialdehyde andhe byproduct TBARS, a marker for enhanced lipid peroxidation45]. A relationship between oxidative stress and nephrotoxi-ity has been well-demonstrated in many experimental animalodels [46]. In the present study, we determined the oxidative

tatus of kidney tissues after the administration of the RSEt inats. The administration of RSEt showed decrease in the levelsf TBARS in kidney tissue, implicated reduction in oxidativetress. These findings suggest that the RSEt may exert antioxi-ant activities and protect the tissues from lipid peroxidation.

Evidence suggests that various enzymatic and non-enzymaticystems have been generated by the cells to deal with ROS andther free radicals [47]. However, when a condition of oxidativetress establishes, the defense capacities against ROS becomensufficient [48]. From the present study, it has been observedhat GalN induced significant decrease in SOD, CAT, GSH-Px,ST and GR activities, depleted the GSH content and enhanced

ipid peroxidation in kidney. It has been reported that SOD,AT, GSH-Px and GST constitute a team of defense againstOS [49]. The decreased activities of SOD, CAT and GPx inidney in GalN-treated rats were possibly due to the enhancedipid peroxidation or inactivation of the antioxidative enzymes.his would cause an increased accumulation of superoxide radi-als, which could further stimulate lipid peroxidation. DecreasedST activity during GalN-treated groups might be due to theecreased availability of GSH resulted during the enhanced lipideroxidation. Administration of the RSEt may have stimulatedhe antioxidant machineries of the kidney, as revealed fromnhanced levels of the antioxidant enzymes, increased GSHontent and decreased lipid peroxidation.

Histopathological alterations common to GalN-treated ratsere moderate necrosis and tubule interstitial alterations. It iselieved that the capacity for tubular absorption may have beenltered, thus bringing about functional overload of nephrons withubsequent renal dysfunctions. On the other hand, the RSEt

Please cite this article in press as: S.B. M, et al. Protective effect of Raphaclin métab (2016), http://dx.doi.org/10.1016/j.nupar.2015.12.002

rotects kidney tissue against oxidative damages induced byalN.In clinical practice, an increased serum GGT activity is

onventionally interpreted as a marker of liver disease [50].

étabolisme xxx (2016) xxx–xxx

owever, numerous reports have indicated that serum GGT levelay be an early predictor for the development of chronic kid-

ey disease [51,52]. The serum GGT has been proposed as aensitive and reliable marker of oxidative stress, having a directroportionality, thus giving a strong association of serum GGTith the incidence of chronic kidney disease [53]. The GalN

dministrated rats showed increase in GGT level indicated byhe proximal tubular lesions [54]. The increased activity of GGTbserved in the present study may be attributed to the adminis-ration of GalN, which is capable of altering the redox balancef the cell by drastic reduction of GSH in the tissues. The RSEtas able to significantly reverse this condition of renal damage.In the present study, RSEt was initially subjected to a prelim-

nary phytochemical study and the results revealed the presencef alkaloids, phenols and flavonoids which are responsible forifferent pharmacological activities. Moreover, Beevi et al. [20]eported the presence of array of polyphenols in the extractsf R. sativus such as catechin, protocatechuic acid, vanilliccid, syringic acid, ferulic acid, sinapic acid, O-coumaric acid,yricetin and quercetin, and demonstrated that the antioxidant

roperty of the extract is due to the presence of these polyphe-ols. Moreover ferulic acid [55], vanillic acid [56] and quercetin15] are well reported for its nephroprotective activity. Ethanolicxtract of R. sativus showed nephroprotective activity on galac-osamine induced kidney damage and it can be concluded thatossible mechanism of nephroprotective activity of R. sativuserb may be due to its composition of phenolic compounds. Fur-hermore, more mechanistic knowledge is essential to elucidatehe exact mechanism of this protective effect.

isclosure of interest

The authors declare that they have no competing interest.

cknowledgement

We thank Kongunadu Arts & Science College, Coimbatore,amil Nadu for providing lab facilities.

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