Traditional extract of Pithecellobium dulce fruits protects mice against CCl 4 induced renal oxidative impairments and necrotic cell death

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1 Pathophysiology 19 (2012) Traditional extract of Pithecellobium dulce fruits protects mice against CCl 4 induced renal oxidative impairments and necrotic cell death Pabitra Bikash Pal, Sankhadeep Pal, Prasenjit Manna, Parames C. Sil Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata , West Bengal, India Received 4 April 2011; accepted 17 February 2012 Abstract The present study has been carried out to investigate the role of the aqueous extract of the fruits of Pithecellobium dulce (AEPD) against carbon tetrachloride (CCl 4 ) induced renal oxidative injury in mice. HPLC analysis shows that AEPD contains phenolics, flavonoids and saponins as the major active components. Creatinine and blood urea nitrogen (BUN) levels were assayed to determine renal protective action of AEPD in CCl 4 -induced renal pathophysiology. Its antioxidant activity was determined by measuring radical scavenging activity, antioxidant enzymes activities, GSH content, protein carbonylation and lipid peroxidation. In addition, FACS analysis, DNA fragmentation and histological studies were carried out to determine its effect in CCl 4 induced renal oxidative injury and cell death. CCl 4 exposure increased the intracellular reactive oxygen species production, decreased intracellular antioxidant defence, reduced mitochondrial membrane potential, attenuated the intracellular ATP content and caused renal cell death mainly via the necrotic pathway as revealed by DNA fragmentation analysis. Treatment with AEPD both prior and post to the toxin exposure protected the organ from CCl 4 induced oxidative insult. Histological studies also support our results. Combining, results suggest that the protective role of AEPD against CCl 4 induced renal oxidative impairments is probably due to the antioxidative properties present in its active constituents Elsevier Ireland Ltd. All rights reserved. Keywords: Carbon tetrachloride; Oxidative impairment and kidney dysfunction; Necrosis; Pithecellobium dulce fruit extract; Antioxidants; Renal-protection; Male mice 1. Introduction Carbon tetrachloride (CCl 4 ), an environmental toxin, is enormously used in chemical industry as an organic solvent. It is also used in medicine as a vermifuge in the treatment of hookworm disease. It is exposed to the whole body by Abbreviations: AEPD, aqueous extract of the fruits of Pithecellobium dulce; BSA, bovine serum albumin; BUN, blood urea nitrogen; CCl 4, carbon tetrachloride; CDNB, 1-chloro-2,4-dinitrobenzene; DPPH, 2,2-diphenyl-1-picrylhydrazyl; DTNB, 5,5 -dithiobis(2-nitrobenzoic acid) [(Ellman s reagent)]; EDTA, ethylene diamine tetraacetic acid; GSH, glutathione; GSSG, glutathione disulfide; H 2 O 2, hydrogen peroxide; MDA, malonaldehyde; NEM, N-ethylmaleimide; NADH, nicotinamide adenine dinucleotide reduced disodium salt; NBT, nitro blue tetrazolium chloride; PMT, phenazine methosulphate; ROS, reactive oxygen species; NaN 3, sodium azide; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substances; TCA, trichloroacetic acid. Corresponding author. Tel.: ; fax: addresses: parames@bosemain.boseinst.ac.in, parames 95@yahoo.co.in (P.C. Sil). inhalation, ingestion or absorption through the skin. Its exposure results poisoning in many cases although liver, brain, kidney, muscle, fat and blood are found to be seriously affected. Long time administration of CCl 4 caused fibrosis, cirrhosis and hepatic carcinoma. CCl 4 toxicity results from its bioactivation to the trichloromethyl free radical by cytochrome P450 isozymes [1]. The trichloromethyl radical reacts with oxygen to form the highly toxic reactive trichloromethyl peroxy radical, a reactive oxygen species (ROS). Free radical induced lipid peroxidation believed to be one of the major causes of cell membrane damage leading to a number of pathological situations. Recent reports show that in addition to hepatic disorders, CCl 4 also causes renal disorders by generating free radicals. Reports from Perez et al. [2], Ogeturk et al. [3] and Churchill et al. [4] suggest that acute and chronic renal injuries occur due to the exposure of this solvent. Many herbs and medicinal plants in India are rich in natural sources of antioxidants. These medicinal plants [e.g /$ see front matter 2012 Elsevier Ireland Ltd. All rights reserved. doi: /j.pathophys

2 102 P.B. Pal et al. / Pathophysiology 19 (2012) Andrographis paniculata [5,6], Phylanthus niruri [7 12], Cajanus indicus [13 18], Silybum marianum [19], Terminalia arjuna [20 23], etc.] are used for the treatment of renal, hepatic and other organ disorders. Pithecellobium dulce is a well known medicinal plant, generally used for fencing and tanning, fodder for feed and pods food. In this particular study, protective role of the aqueous extract of the fruits of P. dulce was evaluated against CCl 4 -induced renal pathophysiology using murine model. In our present study, free radical scavenging activity of the extract was determined from its 2,2-diphenyl-1- picrylhydrazyl (DPPH), hydroxyl (OH ) and superoxide (O 2 ) quenching ability. Renal oxidant antioxidant status was determined by measuring the levels of (a) lipid peroxidation and protein carbonylation; (b) activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione-s-transferase (GST), glutathione reductase (GR) and (c) the levels of cellular metabolites in the kidney tissue of the experimental mice. The mode of cell death in kidney tissue has been evaluated by DNA fragmentation analysis. The involvement of mitochondrial event and the intracellular ATP level has also been investigated in our study. In addition, histological studies were carried out to assess the ultrastructural changes in murine kidney. The results of the present study could clarify the role of AEPD in the protection of CCl 4 -induced nephrotoxicity and may shed light on a possible solution to the unavoidable ways of its exposures in humans. 2. Materials and methods 2.1. Plant P. dulce (PD), one of the important traditional medicinal plants in India, belongs to the family of Fabaceae. In this study, the fruits of PD were collected in the summer session of the current year from the local market, Kolkata, India. After collection, one voucher specimen of this fruit was deposited in the Central National Herbarium (CNH), Botanical Survey of India (BSI), Howrah, West Bengal, India Experimental animals All experiments in this study were performed on Swiss albino mice (male, body weight 20 ± 2 g, purchased from M/S Gosh Enterprises, Kolkata, India). The animals were acclimatized under laboratory conditions for two weeks prior to the experiments. They were maintained under standard conditions of temperature (23 ± 2 C) and humidity (50 ± 10%) with an alternating 12 h light/dark cycles. The animals had free access to tap water and fed standard pellet diet (Agro Corporation Private Ltd., Bangalore, India). All the experiments with animals were carried out according to the Guidelines of the Institutional Animal Ethical Committee Chemicals and other reagents 2,2-Diphenyl-1-picryl hydrazyl (DPPH), Bradford reagent, bovine serum albumin (BSA), and protein estimation kit were purchased from Sigma Aldrich Chemical Company (St. Louis, MO, USA). 1-Chloro-2,4-dinitrobenzene (CDNB), carbontetrachloride (CCl 4 ), disodium hydrogen phosphate (Na 2 HPO 4 ), sodium dihydrogen phosphate (NaH 2 PO 4 ), 5,5 -dithiobis(2-nitrobenzoic acid) [DTNB, (Ellman s reagent)], glacial acetic acid, hydrogen peroxide (H 2 O 2 ), ethylenediaminetetraacetic acid (EDTA), phenazine methosulphate (PMT), nitro blue tetrazolium (NBT), sodium pyrophosphate, potassium dihydrogen phosphate (KH 2 PO 4 ), trichloro acetic acid (TCA), thiobarbituric acid (TBA), reduced glutathione (GSH) were bought from Sisco Research Laboratory, India Preparation of fruit extract of P. dulce After collection, the fruits were cut into small pieces and they were homogenized in 50 mm phosphate buffer, ph 7.2 at 4 C. The homogenized mixture was centrifuged at 12,000 g for 30 min to get rid of unwanted debris and lyophilized. The freeze-dried material was weighed, dissolved in the same phosphate buffer and used in different experiments needed for this study Qualitative analysis of AEPD Phytochemical analysis of AEPD was carried out to investigate the presence of active ingredients such as phenolics, flavonoids, and saponins (Figs. 1 and 2). About 0.5 g of crude powder was heated with 5 ml of ethyl alcohol over a steam bath for 3 min and then few drops of neutral ferric chloride solution were added. Appearance of violet colouration confirmed the presence of phenolic compounds in the extract [24]. A portion of crude powder was heated with 10 ml of ethyl acetate over a steam bath for 3 min. The mixture was filtered and 4 ml of the filtrate was shaken with 1 ml of dilute ammonia solution. Appearance of yellow colouration confirmed the presence of flavonoids in the extract [25]. To find out the presence of saponins, small amount (0.5 g) of crude powder was shaken with water in a test tube and it was warmed in a water bath. Persistent froth formation indicated the presence of saponins in the extract [26] Quantitative analysis of AEPD Total phenolic compounds estimation The content of total phenolic compounds of the extract was determined according to McDonald s method using Folin Ciocalteau reagent (gallic acid as a standard) [27].

3 P.B. Pal et al. / Pathophysiology 19 (2012) whole solution was filtered through Whatman filter paper no. 42 (125 m). The filtrate was transferred into a crucible, evaporated into dryness and weighed to a constant weight [28] Determination of saponin content Crude powder (20 g) was put into a conical flask and 100 ml of 20% aqueous ethanol was added. The samples were heated over a hot water bath for 4 h with continuous stirring at about 55 C. The mixture was filtered and the residue was extracted with another 200 ml of 20% ethanol. The extract was reduced to 40 ml over water bath at about 90 C. The concentrate was transferred into 250 ml separating funnel, 20 ml of diethyl ether was added and the mixture was shaken vigorously. The aqueous layer was recovered while the ether layer was discarded. The purification process was repeated. 60 ml of n-butanol was then added. The combined n-butanol extracts were washed twice with 10 ml of 5% aqueous sodium chloride. The remaining solution was heated in a water bath. After evaporation, the samples were dried in the oven to constant weight and the saponin content was calculated [29] HPLC analysis of AEPD Fig. 1. Upper panel: reverse phase HPLC analysis of AEPD. Lower panel: reverse phase HPLC analysis of a mixture of pure compounds gallic acid (peak I), quercetin (peak II) and digitonin (peak III) Flavonoid content estimation Crude powder (10 g) was extracted repeatedly with 100 ml of 80% aqueous methanol at room temperature. The HPLC analysis of AEPD was carried out using a C 18 column (8 nm 10 cm). The column was eluted with a mobile phase 70:30 methanol/water, flow rate 1 ml/min. UV detection was done at 254 nm. A standard curve was prepared using the mixture of standard compounds, such as gallic acid (standard for phenolic compounds), quercetinn (standard for flavonoid compounds) and digitonin (standard for saponin compounds). Fig. 2. (A) DPPH radical scavenging activity of AEPD in cell free system. Each measurement was made six times. Data represent the averages ± SD of 6 separate experiments. * the optimum dose of AEPD at which it shows its maximum DPPH radical scavenging activity. (B) Hydroxyl (black square) and superoxide radical (red circle) scavenging activities of AEPD in cell free system. Each measurement was made six times. Data represent the averages ± SD of 6 separate experiments. * the optimum dose of AEPD at which it shows its maximum hydroxyl and superoxide radicals scavenging activities. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

4 104 P.B. Pal et al. / Pathophysiology 19 (2012) Determination of free radical scavenging activity of AEPD in cell free system Determination of DPPH radical quenching activity The free radical scavenging activity of AEPD was measured by the DPPH radical quenching method [30]. Various concentrations of extract were added to DPPH in methanol (125 M, 2 ml) solution and the final volume was by water to 4 ml. The solution was shaken and incubated at 37 C for 30 min in dark. The decrease in absorbance of DPPH was measured at 517 nm. Percent inhibition was calculated by comparing the absorbance values of control and the extract. Percent inhibition was calculated by comparing the absorbance values of control and the sample. Percentage inhibition = A 1 A A 1 A 1 is the absorbance of the blank and A 2 is the absorbance in the presence of AEPD Determination of hydroxyl radical scavenging activity The hydroxyl radical scavenging activity of the extract has been measured following the method of Nash [31]. In vitro hydroxyl radicals were generated by Fe 3+ /ascorbic acid system. The detection of hydroxyl radicals was carried out by measuring the amount of formaldehyde produced from the oxidation of dimethyl sulfoxide (DMSO). The production of formaldehyde was detected spectrophotometrically at 412 nm Determination of superoxide radical scavenging activity The superoxide radical scavenging activity of the extract was measured following the method of Siddhuraju and Becker [32]. Compositions of reaction mixture are 0.1 M phosphate buffer, ph 7.4, 150 M nitroblue tetrazolium (NBT), 60 M phenazine methosulphate (PMT), 468 M NADH and various concentrations of the AEPD. Then the mixture was incubated in the dark for 10 min at 25 C and the absorbance was measured at 560 nm. Results were expressed as percentage inhibition of the superoxide radicals Determination of dose and time dependent effect of AEPD To find out the dose of AEPD necessary for optimum protection against CCl 4 induced renal-toxicity mice were divided into ten groups each consisting of six mice in each. First two groups were served as normal control (received only water as vehicle) and toxin control (received CCl 4 at a dose of 1 ml/kg body weight for 2 days, orally) respectively. Other eight groups of animals were treated with AEPD orally at a dose of 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg and 300 mg/kg body weight for 7 days prior to CCl 4 treatment (at a dose of 1 ml/kg body weight for 2 days, orally) respectively. To determine the time dependent effects of AEPD, mice were divided into nine groups each consisting of six animals. First two groups were served as normal control (received only water as vehicle) and toxin control (received CCl 4 at a dose of 1 ml/kg body weight for 2 days, orally) respectively. Other seven groups of animals were treated with AEPD orally at a dose of 200 mg/kg body weight, once daily for 1, 3, 5, 7, 10, 12 and 14 days prior to CCl 4 intoxication (at a dose of 1 ml/kg body weight for 2 days, orally). After 24 h to the administration of CCl 4 all mice were sacrificed. The CAT activities were measured in the kidney tissue homogenates of all the experimental mice Experimental mice groups and treatment The animals were divided into seven groups each consisted of six mice and they were treated as follows. Group 1 normal control (received only water as vehicle). Group 2 animals were administered with AEPD at a dose of 200 mg/kg body weight for 7 days, orally. Group 3 toxin control; animals were administered with CCl 4 orally at a dose of 1 ml/kg body weight in liquid paraffin (1:1, v/v) for 2 days. Group 4 animals were administered with AEPD at a dose of 200 mg/kg body weight for 7 days, orally prior to CCl 4 administration (at a dose of 1 ml/kg body weight for 2 days). Group 5 animals were administered with AEPD at a dose of 200 mg/kg body weight for 7 days, orally after CCl 4 administration (at a dose of 1 ml/kg body weight for 2 days). Group 6 animals were administered with vitamin C at a dose of 100 mg/kg body weight for 7 days, orally, prior to CCl 4 administration (at a dose of 1 ml/kg body weight for 2 days). Group 7 animals were administered with CCl 4 at a dose of 1 ml/kg body weight for 2 days and received normal diet for next 7 days. This group was included just to check the natural healing of the animals after toxin treatment. The in vivo experimental protocol has been summarized in Fig Kidney tissue homogenate preparation About 150 mg of kidney tissue was homogenized in 10 volume of 100 mm KH 2 PO 4 buffer containing 1 mm EDTA, ph 7.4 and centrifuged at 12,000 g for 30 min at 4 C from each mouse of separated groups. The supernatant was collected and used for following experiments as described below. Protein concentration of the supernatant was measured according to the method of Bradford [33] using crystalline BSA as standard.

5 P.B. Pal et al. / Pathophysiology 19 (2012) Fig. 3. Schematic diagram of in vivo experimental protocol Assessment of the levels of blood urea nitrogen (BUN) and creatinine Blood samples collected from puncturing rat heart were kept overnight to clot and then centrifuged at 3000 g for 10 min. Creatinine and BUN levels were estimated by using standard kits (Span diagnostic Ltd., India) Lipid peroxidation and protein carbonylation estimation The extent of lipid peroxidation in terms of malondialdehyde (MDA) formation was measured according to the method of Esterbauer and Cheeseman [34]. Sample containing 1 mg protein was mixed with 1 ml TCA (20%), 2 ml TBA (0.67%) and heated for 1 h at 100 C. After cooling, the precipitate was removed by centrifugation. The absorbance of the sample was measured at 535 nm using a blank containing all the reagents except the sample. MDA content of the sample was calculated using the extinction co-efficient of MDA, which is M 1 cm 1. Protein carbonyl contents were determined according to the methods of Uchida and Stadtman [35]. The sample was treated with an equal volume of 0.1% (w/v) 2,4-DNPH in 2 N HCl and incubated for 1 h at room temperature and then treated with 20% TCA. After centrifugation, the precipitate was extracted three times with EtOH/EtOAc and dissolved in 8 M guanidine hydrochloride in 133 mm Tris solution containing 13 mm EDTA. The absorbance was recorded at 365 nm. The results were expressed as nmol of DNPH incorporated/mg protein based on the molar extinction coefficient of 22,000 M 1 cm 1 for aliphatic hydrazones Determination of the activities of antioxidant enzymes The activities of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione-s-transferase (GST) and glutathione reductase (GR) were measured in all experimental sets following the method of Sinha et al. [36]. Briefly, in case of SOD assay tissue homogenates were centrifuged at 600 g for 8 min at 4 C. Supernatants were decanted and re-centrifuged at 5500 g for 15 min to form mitochondrial pellets. The supernatant was kept as cytosolic fraction. Five micrograms protein of the cytosolic fraction was mixed with sodium pyrophosphate buffer, PMT and NBT. The reaction was started by the addition of NADH. Then the reaction mixture was incubated at 30 C for 90 s and stopped by the addition of 1 ml of glacial acetic acid. The absorbance of the chromogen formed was measured at 560 nm. One unit of SOD activity is defined as the enzyme concentration required to inhibit chromogen production by 50% in 1 min under the assay condition. CAT activity was determined by following the decomposition of H 2 O 2 (7.5 mm) at 240 nm for 10 min and it was monitored spectrophotometrically. One unit of CAT activity

6 106 P.B. Pal et al. / Pathophysiology 19 (2012) is defined as the amount of enzyme, which reduces 1 mol of H 2 O 2 per minute. GST activity was assayed based on the conjugation reaction with glutathione in the first step of mercapturic acid synthesis. The reaction mixture contains supernatant 25 g protein sample, KH 2 PO 4 buffer, EDTA, CDNB and GSH. The reaction was carried out at 37 C and monitored spectrophotometrically at 340 nm for 5 min. One unit of GST activity is 1 mol product formation per minute. GR activity was determined spectrophotometrically by monitoring the absorbance at 412 nm for 3 min at 24 C. Reaction mixture contained 1 mm EDTA, 0.3 mm DTNB, 2 mm NADPH and 20 mm GSSG. The enzyme activity was calculated using molar extinction coefficient of 13,600 M 1 cm 1. One unit of enzyme activity is defined as the amount of enzyme, which catalyzes the oxidation of 1 mol NADPH per minute Estimation of the levels of cellular metabolites Determination of GSH content Intracellular GSH level was measured according to the method of Ellman [37]. 720 L of sample was double diluted and 5% TCA was added to precipitate the protein content. After centrifugation (at 10,000 g for 5 min) the supernatant was taken, DTNB solution (Ellman s reagent) was added to it and the absorbance was measured at 412 nm. A standard curve was drawn using different known concentrations of GSH solution. With the help of this standard curve, GSH contents were calculated Determination of GSSG content Glutathione disulfide (GSSG) was assessed by measuring its level using a kit from Calbiochem, USA following the method of Finck et al. [38]. Briefly, kidney tissue samples were homogenized in ice cold m-phosphoric acid (100 g/l, Fluka). This treatment resulted in the precipitation of the proteins. After allowing the mixture to stand for 5 min at room temperature, homogenates were centrifuged (10,000 g; 10 min at 4 C). Supernatants were frozen at 80 C until further analysis. Prior to the estimation of GSSG, neutralization was achieved by adding 5 L of triethanolamine (4 mmol/l, Sigma Chemicals) to a 100 L supernatant. For GSSG assay, the thiol scavenging reagent 1-methyl-2-vinylpyridinium trifluoromethanesulfonate (M2VP) was used to mask GSH very rapidly. 10 L of M2VP was added to 100 L of neutralized supernatant. Then 100 L of the recycling reagent, which contained NADPH (0.30 mm), DTNB (0.225 mm), and GR (1.6 units/ml) in a 100 mm phosphate/1 mm EDTA buffer (ph 7.4), was added. The change of absorbance was monitored at 412 nm for 3 min with a spectrophotometer. Standards and test samples were run in triplicate for each assay and the measurements were repeated three times. The reaction rate and calibration curves were used to calculate concentrations of GSSG Determination of total thiols content The total thiols (total sulfhydryl groups) content was measured according to the method of Sedlak and Lindsay [39] with some modifications. About 50 L of sample was mixed with 0.6 ml of Tris EDTA buffer, 40 L of 10 mm DTNB in methanol. The final volume was made up to 1 ml by adding methanol. The reaction mixture was incubated at room temperature for 20 min and the absorbance was measured at 417 nm. The content of total thiols was calculated using molar extinction coefficient of 13,600 M 1 cm Measurement of ROS level ROS level was estimated by using 2,7-dichlorofluorescein diacetate (DCFDA) as a probe following the method of LeBel and Bondy [40] as modified by Kim et al. DCF-DA diffuses through the cell membrane where it is enzymatically deacetylated by intracellular esterases to the more hydrophilic nonfluorescent reduced dye dichlorofluorescin. In the presence of reactive oxygen metabolites, nonfluorescent DCFH rapidly oxidized to highly fluorescent product DCF. For in vivo study, 100 L of tissue homogenates were incubated with the assay media (20 mm tris HCl, 130 mm KCl, 5 mm MgCl 2, 20 mm NaH 2 PO 4, 30 mm glucose and 5 M DCFDA) at 37 C for 15 min. The formation of DCF was measured at the excitation wavelength of 488 nm and emission wavelength of 610 nm for 10 min by using fluorescence spectrometer (HITACHI, Model No F4500) equipped with a FITC filter DNA fragmentation analysis The extent of DNA fragmentation (DNA ladder) has been assayed by the procedure as described by Sellins and Cohen [41]. DNA samples were isolated from the kidney tissue of the normal as well as experimental mice and they were analyzed by electrophoresing the DNA samples on agarose/ethydium bromide gel Assessment of mitochondrial membrane potential and integrity Reduction in mitochondrial membrane potential ( ) and hence the mitochondrial integrity plays an important role in CCl 4 induced cell death. For the detection of mitochondrial, mitochondria were isolated from the renal tissue of experimental mice. Briefly, renal tissue was minced, washed with ice-cold isolation buffer (10 ml Tris MOPS [0.1 M; ph 7.4], 20 ml sucrose [1 M], and 1 ml EGTA Tris buffer [0.1 M; ph 7.4]) and the buffer was discarded. 5 ml of fresh isolation buffer was then added, and the mixture was homogenized. The homogenates were centrifuged at 600 g at 4 C for 10 min, and then the supernatants were centrifuged at 7000 g at 4 C for 10 min, after which the supernatants were discarded. The pellets were washed once with isolation buffer, and the centrifugation steps were repeated twice.

7 P.B. Pal et al. / Pathophysiology 19 (2012) After the three-step centrifugation, the supernatants were discarded, and the pellets were suspended in 1 ml of isolation buffer and used for further analysis [42]. Analytic flow cytometric measurements for the membrane potential ( ψ m )of isolated mitochondria were performed using a FACScan flow cytometer with an argon laser excitation at 488 nm. Mitochondrial membrane potential ( ψ m ) was estimated to the exclusion of rhodamine 123 from intact mitochondria. ROS stimulated changes of mitochondrial membrane potential will promote the uptake of rhodamine Assay of ATP level For the intracellular ATP assay, renal homogenate was mixed with TCA (6.5%, w/v) and left on ice for 10 min. The TCA extract was removed and used immediately for analysis of ATP as described by Kalbheim and Koch [43] Histological studies Kidneys from the normal and experimental mice were fixed in 10% buffered formalin and were processed for paraffin sectioning. Sections of about 5 m thickness were stained with hematoxylin and eosin to evaluate under light microscope Statistical analysis All the values are expressed as means ± SD (n = 6). Significant differences between the groups were determined with SPSS 10.0 software (SPSS Inc., Chicago, IL, USA) for Windows using one-way analysis of variance (ANOVA) and the group means were compared by Student Newman Keuls post hoc tests. A difference was considered significant at the p < 0.05 level. 3. Results 3.1. Phytochemical screening of AEPD for active components Qualitative analysis of AEPD (Table 1A) revealed the presence of phenolics, flavonoids, and saponins as the major constituents in the extract. Quantitative estimation of the chemical constituents in the studied medicinal plant is summarized in Table 1B. HPLC profile of AEPD extract (Fig. 1) also shows the presence phenolics (peak I), flavonoids (peak Table 1A Qualitative extract analysis of AEPD. Serial no. Component Abundance 1 Phenolics + 2 Flavonoids + 3 Saponins + + sign indicates the presence of the compounds in the AEPD. Table 1B Quantitative extract analysis of AEPD. Serial no. Component Concentration 1 Phenol content (mg/g dry weight material) ± Flavonoid content (mg/g dry weight material) ± Saponin content (mg/g dry weight material) ± 2.35 II) and saponins (peak III). The presence of these compounds in the extract has been characterized by comparing the HPLC profiles of the respective pure standard compounds Free radical scavenging activity of AEPD To begin with the evaluation of the antioxidant nature of AEPD, we looked for an effective radical scavenging cell free assay. So, we determined its radical scavenging power using the DPPH radical. The experimental results on the radical scavenging effect of AEPD have been shown in Fig. 2A. The plot shows that with increase in concentration of AEPD the color of the DPPH radical vanishes rapidly at 517 nm. It has been observed that AEPD showed maximum inhibition when it was incubated at a concentration of 20 mg/ml with DPPH solution. Hence, it can be anticipated that the active principle(s) present in AEPD possesses potent free radical scavenging activity. Hydroxyl (OH ) and superoxide (O 2 ) radicals scavenging activity of AEPD has been investigated in cell free system and Fig. 2B represents those results. It has been observed that at a dose of 20 mg/ml, AEPD can effectively scavenge OH and O 2 radicals Dose and time dependent protective action of AEPD In the present study, we used CAT assay to determine the optimum dose and time necessary for AEPD to protect mouse kidney against CCl 4 -induced renal oxidative damages. The experimental results have been represented in Fig. 4A and B. CCl 4 exposure decreased the CAT activity. AEPD administration increased the CAT activity linearly up to a dose of 200 mg/kg body weight, when applied for 7 days prior to CCl 4 intoxication. This dose and time of AEPD treatment were used for the subsequent experiments Kidney body weight ratio CCl 4 intoxication usually reduces the weight of different organs in pathophysiological situations. We, therefore, checked whether the kidney weight of the experimental animals was affected by CCl 4 exposure and if that happened whether AEPD could protect it. Table 2A shows that CCl 4 toxicity lowers kidney weight to body weight ratio and that could be prevented by the treatment with AEPD prior to toxin exposure. Post treatment with AEPD also showed similar result as the pre treated groups. It is evident from the data

8 108 P.B. Pal et al. / Pathophysiology 19 (2012) Fig. 4. (A) Dose dependent effect of AEPD on CAT activity against CCl 4 induced renal-toxicity. Cont: CAT activity in normal mice, CCl 4 : CAT activity in CCl 4 treated mice, AEPD-25, AEPD-50, AEPD-75, AEPD-100, AEPD-150, AEPD-200, AEPD-250, AEPD-300: CAT activity in AEPD treated mice for 7 days at a dose of 25, 50, 75, 100, 150, 200, 250 and 300 mg/kg body weight prior to CCl 4 intoxication. Data are mean ± SD, for 6 animals per group and were analyzed by one-way ANOVA, with Student Newman Keuls post hoc tests. Differences were attributed at p < 0.05, and homogeneous subgroups share common superscripted letters. (B) Time dependent effect of AEPD on CAT activity against CCl 4 induced renal-toxicity. Cont: CAT activity in normal mice, CCl 4 : CAT activity in CCl 4 treated mice, AEPD-1, AEPD-3, AEPD-5, AEPD-7, AEPD-10, AEPD-12 and AEPD-14: CAT activity in AEPD treated mice at a dose of 200 mg/kg body weight prior to CCl 4 intoxication for 1, 3, 5, 7, 10, 12 and 14 days respectively. Data are mean ± SD, for 6 animals per group and were analyzed by one-way ANOVA, with Student Newman Keuls post hoc tests. Differences were attributed at p < 0.05, and homogeneous subgroups share common superscripted letters. that CCl 4 -induced renal pathophysiology could be protected by AEPD Effect of AEPD on BUN and creatinine levels In our study we found that CCl 4 exposure did not alter the levels of serum markers; BUN and creatinine related to renal dysfunction (Table 2A) and treatment with AEPD also did not show any alteration Lipid peroxidation and protein carbonylation inhibition In oxidative stress related organ pathophysiology, lipid peroxidation and protein carbonylation are considered to be the two important parameters. In our study, lipid peroxidation has been measured by estimating the concentration of MDA (lipid peroxidation end product). Table 2B represents the changes in MDA and protein carbonylation in the kidney tissue of the normal and experimental animals. CCl 4 intoxication increased the levels of MDA and protein carbonylation. However, both pre and post treatment with AEPD decreased the levels of lipid peroxidation as well as protein carbonylation suggesting the anti-oxidant nature of AEPD in CCl 4 -induced renal dysfunction Protective role of AEPD against CCl 4 induced ROS production ROS plays a major role in CCl 4 induced kidney dysfunction and cellular death of kidney. In order to assess Table 2A Kidney body weight ratios and the activities of the serum markers related to renal dysfunction in male mice. Animal groups Ratio of the kidney weight to the body weight (%) Activities of the serum markers BUN a Creatinine a Normal 1.11 ± 0.04a ± 3.24a 0.56 ± 0.02a AEPD 1.05 ± 0.05a ± 2.62a 0.58 ± 0.02a CCl ± 0.06b ± 3.27a 0.62 ± 0.03a AEPD + CCl ± 0.03a ± 4.52a 0.56 ± 0.02a CCl 4 + AEPD 0.99 ± 0.04a ± 5.52a 0.60 ± 0.04a VitC + CCl ± 0.05a ± 4.82a 0.57 ± 0.06a Recovery 0.65 ± 0.06b ± 10.57a 0.63 ± 0.04a Data are means ± SD, for 6 mice per group and were analyzed by one-way ANOVA, with Student Newman Keuls post hoc tests. Differences were attributed at p < 0.05, and homogeneous subgroups share common letters (a, b). a mg/dl.

9 P.B. Pal et al. / Pathophysiology 19 (2012) Table 2B Levels of the parameters as the index of lipid peroxidation (MDA) and protein carbonylation (PC) in the kidney tissue of the control and experimental mice. Animal groups MDA a PC a Normal 4.72 ± 0.25a 11.5 ± 0.57a AEPD 5.23 ± 0.27a ± 0.53a CCl ± 0.52b ± 1.43b AEPD + CCl ± 0.36c ± 0.64a CCl 4 + AEPD 7.85 ± 0.41c ± 0.71a VitC + CCl ± 0.34c ± 0.66a Recovery 9.34 ± 0.45b ± 1.28b Data are means ± SD, for 6 animals per group and were analyzed by oneway ANOVA, with Student Newman Keuls post hoc tests. Differences were attributed at p < 0.05, and homogeneous subgroups share common letters (a, b). a nmol/mg protein. the changes in the production of intracellular ROS under oxidative stress, kidneys were isolated from the experimental animals and studied the ROS production assay using DCFDA by fluorescence spectrophotometer. Fig. 5 shows the effect of CCl 4 on the intracellular ROS production and its reversal by the treatment with AEPD. Here, we observed that CCl 4 administration caused increased production of intracellular ROS. Treatment with AEPD both prior and post to toxin administration decreased the intracellular ROS level compared to toxin control suggesting the intracellular ROS scavenging nature of AEPD. Fig. 6. DNA fragmentation on agarose/ethydium bromide gel. DNA isolated from experimental kidney tissues was loaded onto 1% (w/v) agarose gels. Lane 1: Marker (1 kb DNA ladder); Lane 2: DNA isolated from normal kidney tissue; Lane 3: DNA isolated from AEPD treated kidney samples; Lane 4: DNA isolated from CCl 4 intoxicated kidney; Lane 5: DNA isolated from AEPD pre-treated kidney samples; Lane 6: DNA isolated from kidney sample of the AEPD post-treated mouse and Lane 7: DNA isolated from kidney tissue of the mouse of recovery group Antioxidant enzyme activities Antioxidant enzymes are considered to be the first line of cellular defence that prevents cellular ingredients from oxidative damage. Among them SOD and CAT act as important enzymes in the elimination of ROS. In order to remove excess free radicals from the system, GST utilizes GSH during their course of reactions. Due to decrease in GSH content for toxicity simultaneously decreased the activities of GST with a concomitant decrease in the activity of GSH regenerating enzyme, GR. We, therefore, determined the activities of these enzymes and presented the data in Table 2C. A significant reduction in the activities of all antioxidant enzymes have been observed in the kidney tissue of the CCl 4 intoxicated experimental animals. Results suggest that both pre as well as post treatment with AEPD could protect the first line of renal dysfunction in oxidative damage induced by CCl 4. Fig. 5. The intracellular ROS production was detected by DCF-DA method. Cont: ROS level in normal animals, AEPD: ROS level in the animals treated with AEPD only; CCl 4 : ROS level in the CCl 4 intoxicated animals; AEPD + CCl 4 : ROS level in the animals treated with AEPD prior to CCl 4 intoxication; CCl 4 + AEPD: ROS level in the animals treated with AEPD post to CCl 4 intoxication and Recovery: ROS level in the animals of recovery group. Data are means ± SD, for 6 animals per group and were analyzed by one-way ANOVA, with Student Newman Keuls post hoc tests. Differences were attributed at p < 0.05, and homogeneous subgroups share common superscripted letters Effect on cellular metabolites Table 2D depicts the effects of AEPD and CCl 4 on the levels of the cellular metabolites in the kidney tissue of the experimental animals. CCl 4 decreased the levels of total thiols as well as GSH and increased the level of GSSG. Administration of AEPD could ameliorate the alteration in the thiol parameters in the CCl 4 intoxicated animals.

10 110 P.B. Pal et al. / Pathophysiology 19 (2012) Table 2C Effect of CCl 4 and AEPD on the activities of the antioxidant enzymes in kidneys of the normal and experimental mice. Animal groups SOD a CAT b GST b GR c Normal ± 3.45a ± 2.44a 3.47 ± 0.21a ± 0.95a AEPD ± 2.81a ± 2.12a 2.12 ± 0.17a ± 0.81a CCl ± 2.62b ± 0.85b 0.85 ± 0.09b 5.25 ± 0.35b AEPD + CCl ± 3.08a ± 1.92a 1.95 ± 0.15a ± 0.77a CCl 4 + AEPD ± 3.62a ± 1.75a 1.75 ± 0.12a ± 0.69a VitC + CCl ± 3.05a ± 2.15a 2.05 ± 0.14a ± 0.81a Recovery ± 2.67b ± 1.01b 0.91 ± 0.11b 6.89 ± 0.42b Data are means ± SD, for 6 mice per group and were analyzed by one-way ANOVA, with Student Newman Keuls post hoc tests. Differences were attributed at p < 0.05, and homogeneous subgroups share common letters (a, b). a Unit/mg protein. b mol/min/mg protein. c nmol/min/mg protein Effect on DNA damage Fig. 6 represents the results carried out to evaluate whether AEPD protects CCl 4 induced renal DNA damage. DNA fragmentation was examined by agarose gel electrophoresis. A smear on agarose gel was observed in CCl 4 treated group, indicating random DNA fragmentation which is a hallmark of cellular necrosis. AEPD treatment was found to be effective in preventing the toxin induced smear formation suggesting its protective power for the prevention of kidney cells from CCl 4 -induced DNA damage and necrotic death Effect on mitochondrial membrane potential and ATP level Oxidative stress-induced cell death is directly related to mitochondrial dysfunction. Disruption of mitochondrial membrane potential and loss of ATP production cause cell death. Therefore, we assessed the mitochondrial membrane potential (Fig. 7) and the ATP level (Fig. 8). It has been observed that CCl 4 exposure reduced the mitochondrial membrane potential as well as intracellular ATP level. AEPD administration however found to be effective in preventing this event. Table 2D Status of the thiol based antioxidants in the kidney tissue of the CCl 4 and AEPD treated mice. Animal groups GSH a GSSG a Total thiols a Normal 7.89 ± 0.38a 0.27 ± 0.014a ± 6.26a AEPD 7.87 ± 0.39a 0.26 ± 0.016a ± 5.77a CCl ± 0.142b 0.87 ± 0.042b ± 2.99b AEPD + CCl ± 0.33a 0.59 ± 0.028a ± 5.21a CCl 4 + AEPD 6.55 ± 0.31a 0.51 ± 0.024a ± 4.87a VitC + CCl ± 0.33a 0.57 ± 0.027a ± 5.19a Recovery 2.89 ± 0.143b 0.86 ± 0.042b ± 2.98b Data are means ± SD, for 6 mice per group and were analyzed by oneway ANOVA, with Student Newman Keuls post hoc tests. Differences were attributed at p < 0.05, and homogeneous subgroups share common letters (a, b). a nmol/mg protein. Fig. 7. Mitochondrial membrane potentials ( ) were measured in the kidney tissue of normal and experimental animals. Upper panel represents the histogram analysis and lower panel represents the corresponding levels as percentage over control. Cont: in normal animals, AEPD: in the animals treated with AEPD only; CCl 4 : in the CCl 4 intoxicated animals; AEPD + CCl 4 : in the animals treated with AEPD prior to CCl 4 intoxication; CCl 4 + AEPD: in the animals treated with AEPD post to CCl 4 intoxication and Recovery: in the animals of recovery group. Data are mean ± SD, for 6 animals per group and were analyzed by one-way ANOVA, with Student Newman Keuls post hoc tests. Differences were attributed at p < 0.05, and homogeneous subgroups share common superscripted letters.

11 P.B. Pal et al. / Pathophysiology 19 (2012) in Fig. 9. CCl 4 exposed animals showed extensive tubular damage by swollen and necrotic epithelial cells. Treatment with AEPD both prior to and after the toxin exposure showed a considerable improvement in kidney morphology. Results of the histological assessment support the outcome of the earlier studies by exhibiting CCl 4 -induced necrosis in the kidney tissue and its protection by AEPD. 4. Discussion Fig. 8. ATP levels were measured in the kidney tissue of normal and experimental animals. Cont: level in normal animals, AEPD: level in the animals treated with AEPD only; CCl 4 : level in the CCl 4 intoxicated animals; AEPD + CCl 4 : level in the animals treated with AEPD prior to CCl 4 intoxication; CCl 4 + AEPD: level in the animals treated with AEPD post to CCl 4 intoxication and Recovery: level in the animals of recovery group. Data are mean ± SD, for 6 animals per group and were analyzed by one-way ANOVA, with Student Newman Keuls post hoc tests. Differences were attributed at p < 0.05, and homogeneous subgroups share common superscripted letters Histological assessment Histological assessments of different kidney segments of the normal and experimental animals have been presented We observed that CCl 4 administration caused murine renal dysfunction and cell death by disturbing the status of antioxidant enzyme activities, lipid peroxidation, protein carbonylation followed by the reduction in mitochondrial membrane potential, loss in, unprogrammed cell death, necrosis. This impairment of renal function and cell death could, however, be protected by the treatment of AEPD prior and post to CCl 4 intoxication. As the first step of defining a substance to be antioxidant, we usually determine its radical scavenging activity. DPPH radical scavenging assay is considered to be a convenient tool in cell free system for this purpose. We found that incubation of AEPD with increased concentrations and DPPH radical at a fixed concentration diminished the absorbance at 517 nm (Fig. 2A). This experiment shows the ability of the extract to scavenge DPPH radicals. In addition, the hydroxyl as well as superoxide radicals scavenging activity of AEPD were also investigated in cell free systems and found that it could effectively scavenge these radicals (Fig. 2B). We Fig. 9. Haematoxylin and eosin stained kidney section of experimental mice. Cont: kidney section from normal mice ( 100); AEPD: kidney section of the mice treated with AEPD only ( 100); CCl 4 : mice administered with CCl 4 ( 100); AEPD + CCl 4 : kidney section from the AEPD pre treated mice ( 100); CCl 4 + AEPD: kidney section from the AEPD post treated mice ( 100) and Recovery: kidney section from the mice of recovery group ( 100).

12 112 P.B. Pal et al. / Pathophysiology 19 (2012) Fig. 10. Schematic diagram of the antioxidative role AEPD and its protective role against CCl 4 induced renal pathophysiology. then determined its effect on intracellular ROS production by using 2,7-dichlorofluorescein diacetate (DCFDA) as a probe. Results showed that CCl 4 intoxication increased the rate of DCF formation, an indicator of intracellular ROS production, in the renal tissue of the experimental animals. Treatment with AEPD, both pre and post to the toxin administration, could, however, prevent the increased production of ROS and maintain the intracellular antioxidant power close to normal. Results of these studies clearly established AEPD as a potent radical scavenger. It is, therefore, likely to believe that AEPD might alter the toxic effects of any toxicant-induced excessive free radicals by quenching those harmful species. It has been well established that exposure to various organic compounds including a number of environmental pollutants and drugs can cause cellular damages through metabolic activation of those compounds to highly reactive substances such as reactive oxygen species (ROS). CCl 4 is one of such extensively studied environmental toxicant. Reports from our laboratory and other investigators have already established that in addition to hepatic problems, the industrial solvent CCl 4 also causes disorders in kidneys, lungs, testis and brain as well as in blood by generating free radicals [44]. The reactive metabolite trichloromethyl radical ( CCl 3 ) has been formed from the metabolic conversion of CCl 4 by cytochrome P-450 [45]. AsO 2 tension rises, a greater fraction of CCl 3 present in the system reacts very rapidly with O 2 and many orders of magnitude more reactive free radical, CCl 3 OO is generated from CCl 3 [46]. Although various enzymatic and non-enzymatic systems in the cell try to cope up with the ROS and other free radicals, a condition of oxidative stress establishes when the defense capacities inside the cells against ROS become insufficient [47]. In the present study, it has been observed that CCl 4 caused a significant decrease in antioxidant enzyme activities, depleted the GSH content and enhanced lipid peroxidation as well as protein carbonylation in kidney tissue although levels of the markers related to renal damages practically remained unaltered. The exact reason for this in CCl 4 induced renal pathophysiology is not clearly known. One possible explanation is that, although the time of CCl 4 exposure to the animals was sufficient to induce renal oxidative stress but not enough to cause sufficient damage for its dysfunction. Similar results have been reported by other investigators in CCl 4 induced renal damage in rats [48]. AEPD, on the other hand, was found to be highly effective in this organ pathophysiology as its administration both prior and post to CCl 4 intoxication could not only prevent the CCl 4 induced increased levels of lipid peroxidation and protein carbonylation, but also protected the antioxidant machineries of the kidney as revealed from the enhanced levels of SOD, CAT GST and GR activities, increased level of GSH. Oxidative stress induced by oxygen-derived species can produce a multiplicity of modifications in DNA including base and sugar lesions, strand breaks, DNA-protein

13 P.B. Pal et al. / Pathophysiology 19 (2012) cross-links and base-free sites. If left un-repaired, oxidative DNA damage can lead to detrimental biological consequences in organisms, including cell death, mutations and transformation of cells to malignant cells. So, in addition to excessive oxidative stress, DNA fragmentation acts as a mediator of cell death [49]. In the present study it has been observed that CCl 4 intoxication caused random fragmentation of genomic DNA leading to the formation of a DNA smear on agarose gel (Fig. 6), suggesting that CCl 4 induced renal cell damage and death occur via necrotic pathway. CCl 4 induced increased production of ROS caused reduction in mitochondrial membrane potential and loss in intracellular ATP content. AEPD treatment could, however, prevent CCl 4 -induced over production of ROS, degree of DNA fragmentation, reduction in mitochondrial membrane potential (Fig. 7) and loss in ATP content (Fig. 8). These results clearly showed the antioxidative and antinecrotic nature of AEPD in CCl 4 -induced renal pathophysiology and dysfunction. The fruits of P. dulce contain about, 2.5% protein, 0.4% fat, 18% carbohydrate, 77.8% water and 1.3% fiber as reported by the Indian Council of Scientific and Industrial Research (CSIR) [50]. The report also mentioned that the fruits are enriched with vital vitamins such as thiamine, riboflavin, niacin and ascorbic acid and contain some essential minerals such as Ca, P, Fe, Na and K. Few essential amino acids like valine, lysine, phenylalanine, and tryptophan have also been found in this fruit. Alcoholic extraction yielded saponin, sterol glucoside, flavone and lecithin. In the present study, we also verified the presence of phenolics, flavonoids and saponins in the plant extract. The observed effects of the extract could be related to chemically defined compounds. Flavonoids show their antioxidative action through scavenging or chelating process. Phenolic content is also important because of the presence of hydroxyl groups possessing scavenging ability. It can, therefore, be speculated that the observed antioxidant effects of AEPD could be due to the presence of flavonoids and phenolic contents. Histopathological examination clearly demonstrated that CCl 4 administration caused a significant abnormality in the renal morphology showing marked tubular damages. Complete loss of brush borders, extensive tubular casts and debris as well as tubular dilatations was also observed in the renal tissue of the experimental animal. Treatment of AEPD prevented any such CCl 4 -induced alterations and kept the kidney histologically close to normal (Fig. 9). In conclusion, aqueous extract of the fruits of P. dulce protects kidneys against CCl 4 induced oxidative damages in mice model and could be used as an effective protector in studies pathophysiology (Fig. 10). 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