Dillenia indica fruit prevents cisplatin-induced kidney injury in experimental rats through modulation of oxidative stress, marker enzyme, and biochemical changes

Dillenia indica fruit prevents cisplatin-induced kidney injury in experimental rats through... Background: Dillenia indica fruit is known for its numerous health benefits in folk medicine including its use to cure kidney diseases. The present study was designed to investigate the protective effect of D. indica fruit extracts on cisplatin-induced nephrotoxicity. Methods: A nephrotoxic dose of cisplatin (3 mg/kg b.w./day, i.p) was administered every fifth day to the animals receiving vehicle or fruit extracts (methanol, ethyl acetate, and petroleum ether) daily for 25 days. Rats were sacrificed on the 25th day, and the effect of extracts was assessed by determining the alterations in various serum and urine parameters, membrane-bound enzyme, and antioxidant defense system in kidney tissue. Results: Increase in serum urea, uric acid, creatinine, blood urea nitrogen, phospholipid, and cholesterol and a decrease in urine urea, uric acid, creatinine, and creatinine clearance rate were reported in the cisplatin control group. Cisplatin alters electrolyte balance, brush border membrane marker enzyme (i.e., alkaline phosphatase, γ- glutamyl transferase, leucine aminopeptidase) activity, and redox balance significantly. Methanol and ethyl acetate extracts of D. indica fruit produced beneficial effect and ameliorated serum and urine parameter to normal. Extract + + administration increases Na /K -ATPase activity and different enzymatic and non-enzymatic antioxidants positively, whereas lipid peroxidation reduced significantly. Extracts exhibited a potent in vitro antioxidant activity. Conclusion: Taking into account these results, it can be assumed that D. indica fruit could be the future key candidate which may maximize the clinical use of cisplatin in the treatment of different cancer without nephrotoxicity. Keywords: Dillenia indica, Fruit, Antioxidant, Cisplatin, Kidney function test Background due to frequent reversible and irreversible side effects The incidence of drug-induced nephrotoxicity has been like nephrotoxicity, neurotoxicity, bone marrow toxicity, increasing at a frightening rate with the increasing uses and gastrointestinal toxicity and ototoxicity [2]. of antibiotics and anticancer drugs [1]. Oxidative stress Dillenia indica Linn. (Family: Dilleniaceae) is a wildly is considered as a key factor for drug-induced nephro- available medicinal plant found in Northeast India. The toxicity [2]. Cisplatin [cis-diamminedichloroplatinum common name of the plant is outenga in Assamese and (II)] is the most common and potent anticancer drug chalita in Bengali. The plant grows in abundance in this used against a diverse spectrum of malignancies. How- part of India, but most of the fruits of this plant are ever, the use of cisplatin in combating cancer is limited wasted due to the lack of technical knowledge. Fruits are edible; the fleshy calyx of the fruit can be consumed directly and also be prepared with vegetable and pickle. * Correspondence: saikat.pharm@rediffmail.com The fruits and other parts of the plant are used Department of Pharmacy, Assam Down Town University, Guwahati, Assam 781026, India traditionally to cure a number of ailments like stomach CES College of Pharmacy, Kurnool, Andhra Pradesh 518218, India © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sen et al. Nutrire (2018) 43:15 Page 2 of 9 ● ● problem, fevers, and cough and to combat hair fall and acid and rutin were used as standard in DPPH ,NO dandruff [3]. The juice of the fruit was used by the folk scavenging, and lipid peroxidation inhibition assay, medicinal practitioners of Bangladesh to cure kidney dis- respectively. eases [4]. The previous investigation found that D. indica fruits contain ascorbic acid, tocopherol, carotene, Acute toxicity study and phenolic compounds [5]. The fruits of D. indica In this study, we adopted acute oral toxicity-acute toxic found to possess several beneficial functions such as class method as per OECD guideline 423 (Annexure 4d). anticancer and antidiarrheal [6], antioxidant [5], CNS This method is considered reproducible, required a very depressant, and anti-inflammatory activity [7]. limited number of animals, and is able to categorized Food with antioxidant potential or antioxidant mole- substances in a parallel mode to the other acute toxicity cules such as phenolic acids, flavonoids, and tannins par- study methods like OECD guidelines 420 and 425. The ticularly from plant sources have proved their potential extract (2000 mg/kg) is administered orally to three role as a prophylactic or curative agent against various healthy nulliparous and non-pregnant female albino oxidative stress-related diseases [8]. The present study mice (20–25 g). Mice were observed individually at least was undertaken to evaluate the protective activity of D. once in half an hour during the first 24 h, with special indica fruits against cisplatin-induced nephrotoxicity. notice given during the initial 4 h, and daily thereafter, for a total of 14 days [11, 12]. Methods Plant materials Induction of nephrotoxicity and treatment schedule Fruits of Dillenia indica Linn. were collected from the Healthy animals were acclimatized to the standard labora- state of Tripura, India, and authenticated by Dr. BK Datta, tory facility for a week. Rats were fed with standard rat Department of Botany, Tripura University, Tripura, India chow and water ad libitum under controlled conditions. (voucher specimen no. TU/BOT/HEB/RC25092011a). Treatment schedule and induction of nephrotoxicity using cisplatin was performed as per the procedure described by Extraction of plant material Khan et al. [2]. At the end of acclimatization, period ani- The fruits were cleaned to remove unwanted material mals were divided into major four groups (control, DIME, and cut into small pieces. The fruits (without seed) were DIEE, DIPE) each containing 12 animals. Animals in dif- dried under the shed as required to grind into coarse ferent major groups were treated with water (control), powder. The powder of D. indica fruit was extracted DIME (300 mg/kg/day, p.o.), DIEE (300 mg/kg/day, p.o.), with methanol, ethyl acetate, and petroleum ether separ- and DIPE (300 mg/kg/day, p.o.) for 5 days. Animals of ately using Soxhlet apparatus. The filtrate was concen- each major group were divided into two subgroups each trated, and the solvent was evaporated under reduced (six animals/subgroup) and continued to receive their ex- pressure to obtain methanol extract (DIME), ethyl tract treatment daily at a dose of 300 mg/kg orally. Based acetate extract (DIEE), and petroleum ether extract on preliminary study, 300 mg/kg dose was selected. Cis- (DIPE) of D. indica fruit. platin (CP, 3 mg/kg b.w./day, i.p) in 0.9% saline is injected every fifth day (four injections in total) for 25 days to in- Experimental animals duce nephrotoxicity to one of the subgroups designated as Healthy albino female mice were used for acute toxicity CP-control, CP-DIME, CP-DIEE, and CP-DIPE. Normal study, and male Wistar rats were used for the nephropro- saline in equivalent volume was given to the animals of tective activity. Animals were maintained under standard the other subgroup from each group for the same period. environmental conditions. The animal experiments were The rats were sacrificed on the 25th day, 5 days after the carried out according to the guidelines of Committee for last injection of cisplatin under light ether anesthesia Purpose of Control and Supervision of Experiments on (Fig. 1, grouping of animal and treatment schedule). Animals (CPCSEA), Ministry of Environment and Forests, Government of India. The study was approved by the Estimation of biochemical parameters and in vivo Institutional Animal Ethical Committee (Reg. No. 1305/ antioxidant assay ac/09/CPCSEA). On the 25th day, urine samples (24 h) were collected using metabolic cages. Blood samples were collected by Antioxidant activity of extracts cardiac puncture under light ether anesthesia, and serum The free radical scavenging activity of extracts of D. sample was used for estimation of different biochemical indica fruits was determined by 2,2-diphenyl-1-picrylhy- parameters. After euthanasia, one kidney of each rat was drazyl radical (DPPH ) scavenging assay method [8], ni- rapidly removed and washed thoroughly with ice-cold tric oxide radical (NO ) scavenging assay method [9], normal saline, and homogenates (10% w/v) were pre- and lipid peroxidation inhibition assay [10]. Ascorbic pared 0.1 M Tris-HCl buffer, pH 7.5. The homogenate Sen et al. Nutrire (2018) 43:15 Page 3 of 9 Fig. 1 Experimental design (grouping of animal and treatment schedule) was centrifuged at 3000×g for 15 min to remove the cell adopted by Asokkumar et al. [17] in which decompos- debris. The supernatant was used for the determination ition of hydrogen peroxide (H O ) in the presence of 2 2 of different biochemical parameters. CAT was measured at 254 nm. Glutathione reductase (GR) activity was determined by the method involving Urea, uric acid, creatinine, and creatinine clearance oxidation of NADPH into NADP in the presence of ox- Levels of urea, uric acid, and creatinine (Cr) in serum idized glutathione. Glutathione peroxidase (GPx) activity and urine were determined spectrophotometrically using was estimated by monitoring the oxidation of reduced commercially available kits (Agapee Diagnostics, India). NADPH at 340 nm. The level of reduced glutathione Creatinine clearance rate was determined using the (GSH) was determined using the method of Asokkumar following equation [13], et al. [17]. Total malondialdehyde (MDA) was deter- mined as an index of the extent of lipid peroxidation in Creatinine clearanceðÞ ml=kg body weight= min kidney tissue using standard method [17]. ¼½ Urinary CrðÞ mg=dl urine volumeðÞ ml  1000 =½ serum CrðÞ mg=dl 1440ðÞ min Statistical analysis The results are expressed as mean ± S.E.M (n = 6 for in vivo test and n = 3 for in vitro study) Statistical differ- Blood urea nitrogen, cholesterol, and phospholipid ence was tested by using one-way analysis of variance Blood urea nitrogen (BUN) and cholesterol level in followed by Tukey tests. A level of p < 0.05 was used as serum were determined by commercially available kits. the criterion for statistical significance. Phospholipid content in serum was estimated using a standard method [14]. + + Na ,K , and brush border membrane marker enzymes Results Potassium and sodium level in plasma was estimated Acute toxicity study + + using flame photometer. Level of Na /K -ATPase and In principle, the acute toxicity study method is not brush border membrane marker enzymes like alkaline intended to permit precise LD calculation but used to phosphatase (ALP), γ-glutamyl transferase (GGTase), find exposure ranges to check lethality since the death of and leucine aminopeptidase (LAP) was assayed by previ- a proportion of experimental animals is still the main ously described method [15, 16]. endpoint of the test. In acute toxicity study of samples, mortality was not observed at the dose of 2000 mg/kg; Enzymatic and non-enzymatic antioxidant activity thus, further lower dose is not administered. Consider- Superoxide dismutase (SOD) activity was estimated by ing OECD guideline, 2000 mg/kg dose was categorized the inhibition of formation of autocatalyzed adreno- under Globally Harmonized Classification System (GHS) chrome in the presence of tissue homogenate at 480 nm. category 5 (safe dose), as per OECD guideline 423 Catalase (CAT) activity was determined by the method (Annexure 2d). Sen et al. Nutrire (2018) 43:15 Page 4 of 9 In vitro antioxidant activity of D. indica fruits extracts phospholipid, and cholesterol level compared to the In vitro antioxidant activity of different extracts of D. nephrotoxic group (Table 3). indica fruit was investigated. Our present findings There was only a small insignificant decrease in serum showed that DIEE and DIME exhibited strong free radical sodium level (− 5.2%) and a significant decrease in serum scavenging activity. The IC values of DIEE (15.8 ± potassium level (− 40.2%) in the cisplatin-treated group 0.02 μg/ml) and DIME (29.3 ± 0.03 μg/ml) against DPPH in comparison to the normal control group. DIME and were observed while IC values of DIME (28.22 ± DIEE administered at a dose of 300 mg/kg along with cis- 0.34 μg/ml) and DIEE (34.0 ± 0.55 μg/ml) against NO and platin have shown significant (p < 0.01) decrease in the the IC values of DIME (61.5 ± 0.60 μg/ml) and DIEE serum potassium level in comparison to the cisplatin con- (70.7 ± 0.54 μg/ml) against lipid peroxidation inhibition trol group. Both urinary excretion of sodium (+ 118.7%) assay (Table 1). and potassium (+ 62.7%) was increased significantly after cisplatin administration. Administration of DIME in nephrotoxic animals caused 39.6 and 35.9% reduction in Effect of extracts on urea, uric acid, creatinine, and urinary sodium and potassium excretion, respectively. creatinine clearance DIEE reduced excretion of urinary sodium and potassium Effects of D. indica fruit extracts on serum and urine significantly, though DIPE had an insignificant effect on level of urea, uric acid, and creatinine were tabulated in administered in the nephrotoxic group (Table 3). Table 2. Treatment with cisplatin causes a significant rise in serum urea, serum uric acid, and serum creatin- ine level, whereas the level of urea, uric acid, and cre- Effect of extracts marker enzymes atinine in urine significantly reduced in the cisplatin A significant reduction in the activities of Na / control group. Treatment with DIME in the nephrotoxic K -ATPase, ALP, GGTase, and LAP was observed after group (CP-DIME) cause significant reduction in serum cisplatin treatment. DIME and DIEA treatment in ani- urea (− 32.2%), serum uric acid (− 46.0%), and serum mals that also received cisplatin increased Na / creatinine (− 37.1%) level and an increase in urine urea K -ATPase by 50 and 37%, ALP by 44 and 28%, GGTase (+ 14.8%), urine uric acid (+ 37.4%), and urine creatinine by 59 and 49%, and LAP by 45 and 34%, respectively, level (+ 39.6%). Results showed that DIEE also produced compared to the CP-control group. Result reviled that a significant nephroprotective effect on cisplatin-induced marker enzyme activity was restored after the adminis- nephrotoxicity. None of the extracts produces any sig- tration of DIME and DIEA, which further indicated to- nificant variation in serum and urine parameters ward the protective effect of D. indica fruit (Fig. 2). when administered in healthy animals. Cisplatin also induces 18.5% reduction in creatinine clearance rate Effect of D. indica fruits on enzymatic and non-enzymatic when compared with the control. DIME and DIEE antioxidants treatment significantly elevate creatinine clearance The effect of DIME, DIEE, and DIPE was determined on rate in nephrotoxic animals. the level of various enzymatic and non-enzymatic anti- oxidants when administered in nephrotoxic and healthy Effect of D. indica fruit on BUN, phospholipid, cholesterol, rats (Table 4). Level of SOD, CAT, GPx, GR, and GSH and sodium and potassium level significantly reduced after cisplatin induction, whereas The administration of cisplatin to healthy rats caused a the level of MDA increases. Cisplatin-induced reduction significant increase in BUN (+ 89.6%), serum phospholipid in antioxidant activity was inhibited significantly after (+ 25.8%), and cholesterol (+ 34.2%) level. The extracts the administration of DIME and DIEE. After receiving had no effect on these parameters when administered on the respective drug treatment in nephrotoxic animals, healthy animals. Although, DIME and DIEE (300 mg/kg) there is a significant change in the level of MDA (− 63.3% when administered in rats that received cisplatin signifi- by DIME, − 54.1% by DIEE, − 24.7% by DIPE) compared cantly prevent such alteration and reduce BUN, serum to the CP-control group. Table 1 In vitro antioxidant effect of D. indica fruit extracts Screening method IC value (μg/ml) DIME DIEE DIPE Standard DPPH scavenging assay 29.3 ± 0.03 15.8 ± 0.02 79.9 ± 0.07 2.6 ± 0.02 (ascorbic acid) NO scavenging assay 28.22 ± 0.34 34.0 ± 0.55 81.0 ± 0.81 24.1 ± 0.24 (ascorbic acid) Lipid peroxidation inhibition assay 61.5 ± 0.60 70.7 ± 0.54 92.6 ± 0.44 59.9 ± 0.20 (rutin) Values are expressed as mean ± SEM Sen et al. Nutrire (2018) 43:15 Page 5 of 9 Table 2 Effect of D. indica fruit extracts on serum and urine parameters in an animal with/without cisplatin treatment Group Serum urea Serum uric acid Serum creatinine Urine urea Urine uric acid Urine creatinine Creatinine clearance rate (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) (ml/min/kg body weight) Control 38.32 ± 2.01 1.24 ± 0.21 0.85 ± 0.01 140.66 ± 6.06 6.92 ± 1.06 32.05 ± 0.36 0.068 c c b b b c b CP-control 64.09 ± 3.99 2.65 ± 0.33 1.43 ± 0.10 109.80 ± 5.86 4.38 ± 0.86 21.40 ± 0.22 0.053 [↓18.5] [↑67.2] [↑113.7] [↑68.2] [↓21.9] [↓36.7] [↓33.2] DIME 37.00 ± 2.12 1.21 ± 0.13 0.89 ± 0.07 135.82 ± 8.33 6.70 ± 0.98 31.99 ± 0.19 0.067 b b b a b b a CP-DIME 43.45 ± 3.11 1.43 ± 0.28 0.90 ± 0.09 126.00 ± 6.98 6.02 ± 1.01 29.88 ± 0.29 0.071 [↑34.0] [↓32.2] [↓46.0] [↓37.1] [↑14.8] [↑37.4] [↑39.6] DIEA 38.97 ± 2.77 1.19 ± 0.22 0.87 ± 0.05 141.35 ± 8.01 6.63 ± 0.88 32.13 ± 0.34 0.067 b b b a a b a CP-DIEA 48.32 ± 2.89 1.66 ± 0.19 1.01 ± 0.04 130.33 ± 7.23 5.59 ± 1.02 26.81 ± 0.37 0.070 [↑32.0] [↓24.6] [↓37.4] [↓29.4] [↑18.7] [↑27.6] [↑25.3] DIPE 37.79 ± 2.32 1.11 ± 0.28 0.82 ± 0.07 138.68 ± 6.87 6.82 ± 1.26 30.82 ± 0.38 0.069 CP-DIPE 59.33 ± 4.02 2.32 ± 0.31 1.39 ± 0.11 114.38 ± 6.01 4.45 ± 0.77 22.47 ± 0.28 0.058 [↑9.4] [↓7.4] [↓12.5] [↓2.8] [↑4.2] [↑1.6] [↑6.4] Values are expressed as mean ± SEM for six mice in each group. CP cisplatin, DIME methanol extract of D. indica (300 mg/kg), DIEA ethanol extract of D. indica (300 mg/kg), DIPE petroleum ether extract of D. indica (300 mg/kg) Statistics: ANOVA followed by the Tukey test a b c p <0.05, p <0.01, p < 0.001—when the extract-treated groups (CP-DIME, CP-DIEA, CP-DIPE) compared with the diseases control (CP-control) group and the CP-control compared with the healthy control group Values in parentheses represent percent change from control/cisplatin-control group Discussion used as a vegetable, and an ethnomedicinal survey con- Cisplatin is a most widely used drug for the treatment of ducted in Bangladesh showed that D. indica fruit juice different cancers and solid tumors. However, nephrotox- was used by the patients with kidney problems [4]. icity caused by the drug is a main limiting factor of its Oxidative stress is an emergent factor for the caus- widespread clinical use for the long-term treatment [2]. ation of many diseases, and nephrotoxicity is one of A number of investigations have been carried out to them. Antioxidant molecules present in the plant/food find/monitor the protective effect of different natural can act synergistically along with exogenous antioxidants substances like grape seed extract, fish oil [18], green tea to avert oxidative stress-induced damage [8]. In vitro [2], Momordica dioica fruit [19], Ganoderma lucidum,a study reveals that D. indica extracts particularly metha- mushroom [20], etc. when administered along with cis- nol and ethyl acetate extract possess strong in vitro anti- platin; however, their use in clinical practice could not oxidant activity tested through lipid peroxidation be achieved till now. Fruits of D. indica are commonly inhibition assay, DPPH radical, and nitric oxide radical Table 3 Effect of D. indica fruit extracts on BUN, phospholipid, cholesterol, and sodium and potassium level + + + + Group BUN (mg/dl) Phospholipid Cholesterol Serum Na Serum K Urinary Na excretion Urinary K excretion (mg/dl) (mg/dl) (mmol/l) (mmol/l) (μmol/24 h) (μmol/24 h) Control 17.9 ± 1.11 101.32 ± 7.06 119.3 ± 8.17 137.22 ± 2.10 6.44 ± 0.21 101.50 ± 7.22 289.06 ± 14.11 c a a b c c CP-control 33.93 ± 2.66 127.53 ± 13.02 160.05 ± 9.29 130.11 ± 2.21 3.85 ± 0.33 222.03 ± 9.12 470.18 ± 15.60 [↑89.6] [↑25.8] [↑34.2] [↓5.2] [↓40.2] [↑118.7] [↑62.7] DIME 17.28 ± 1.43 99.72 ± 9.11 117.39 ± 7.10 136.12 ± 1.99 6.29 ± 0.30 110.23 ± 8.72 280.35 ± 13.99 c a a b a a CP-DIME 19.29 ± 1.51 107.31 ± 13.96 116.67 ± 8.66 138.50 ± 2.31 6.03 ± 0.20 134.11 ± 8.22 [↓39.6] 301.33 ± 14.07 [↓43.1] [↓15.9] [↓27.1] [↑6.4] [↑56.6] [↓35.9] DIEA 18.20 ± 0.99 103.73 ± 9.15 120.39 ± 8.03 138.07 ± 1.77 6.39 ± 0.32 108.66 ± 7.29 293.55 ± 14.66 b a a b b a a CP-DIEA 21.57 ± 1.20 108.34 ± 11.11 121.93 ± 9.21 136.55 ± 2.33 5.86 ± 0.19 145.01 ± 7.94 [↓34.7] 330.20 ± 15.33 [↓36.4] [↓15.0] [↓23.8] [↑4.9] [↑52.2] [↓29.8] DIPE 17.65 ± 1.18 98.92 ± 7.97 120.63 ± 6.99 140.01 ± 2.03 6.45 ± 0.35 115.33 ± 9.02 300.05 ± 14.05 CP-DIPE 29.71 ± 1.87 118.63 ± 10.79 127.42 ± 9.04 132.93 ± 2.62 4.72 ± 0.30 179.42 ± 0.11 [↓19.2] 428.44 ± 16.44 [↓12.4] [↓7.0] [↓20.3] [↑2.2] [↑22.6] [↓8.9] Values are expressed as mean ± SEM for six mice in each group. CP cisplatin, DIME methanol extract of D. indica (300 mg/kg), DIEA ethanol extract of D. indica (300 mg/kg), DIPE petroleum ether extract of D. indica (300 mg/kg) Statistics: ANOVA followed by the Tukey test a b c p <0.05, p <0.01, p < 0.001—when the extract-treated groups (CP-DIME, CP-DIEA, CP-DIPE) compared with the diseases control (CP-control) group and the CP-control compared with the healthy control group Values in parentheses represent percent change from control/cisplatin-control group Sen et al. Nutrire (2018) 43:15 Page 6 of 9 Fig. 2 Effects of D. indica fruit extracts on different marker enzyme activity. Values are expressed as mean ± SEM (n = 6). DIME, methanol extract of D. indica fruits; DIEA, ethyl acetate extract of D. indica fruits; DIPE, petroleum ether extract of D. indica fruits; ALP, alkaline phosphatase; GGTase, g-glutamyl transferase; LAP, leucine aminopeptidase scavenging activity. These results are similar to that of in serum is linked with renal damage and considered as previous investigations carried out by different the indicator of nephrotoxicity. Increased level of urea researchers [5, 21, 22]. Inhibition of lipid peroxidation concentrations in serum may increase after parenchymal by extracts further expands their role as a potent injury. Hyperuricemia is considered as a renal prognostic antioxidant. factor, which may indicate the physical response to an Repeated injection of cisplatin was found to induce amplified generation of endogenous oxygen species as uric marked renal dysfunction as evidenced by increased acid scavenges peroxynitrite [23, 26]. Serum creatinine serum urea, uric acid, and creatinine diagnostic indicators concentration is considered as a potent indicator of the of nephrotoxicity. Urea is the key nitrogen-containing first phase of any kidney disease than the urea and uric metabolic product produced during protein metabolism acid levels [23]. The most penetrable organ in a living sys- [23]. Uric acid is considered as the last substance pro- tem is the kidney through which the toxic substances are duced from an exogenous pool of purines and endogenous eliminated from the living system. Urinalysis is a major purine metabolism [24]. Creatinine is a breakdown prod- pathway to define whether kidney is functioning properly uct of creatine phosphate in muscle, is generally formed at [27]. Cisplatin causes decrease in the level of urine cre- a fairly constant rate based on muscle mass, and consid- atinine, urea, uric acid, and creatinine clearance. These ered as a measure of kidney function [25]. The serum changes may occur due to the reduction in the glomerular urea, creatinine, and uric acid may induce the alteration of filtration rate (GFR) or may be secondary due to the oxi- the glomerular filtration rate, and increase in their levels dative stress, which can cause contraction of mesangial Table 4 Effect of D. indica fruit extracts on enzymatic and non-enzymatic antioxidant level Group SOD CAT GPx GR GSH MDA (μmol/min/mg protein) (μmol/min/mg protein) (μmol/min/mg protein) (μmol/min/mg protein) (μM GSH/gm tissue) (nM/min/mg protein) Control 4.62 ± 0.99 34.33 ± 3.91 22.68 ± 1.72 1.09 ± 0.18 12.08 ± 1.83 0.97 ± 0.13 c c c c c c CP-control 2.88 ± 0.70 [↓37.7] 20.33 ± 3.70 [↓40.8] 12.11 ± 1.05 [↓46.6] 0.68 ± 0.07 [↓37.6] 6.91 ± 1.04 [↓42.8] 3.68 ± 0.37 [↑279.4] DIME 5.05 ± 0.90 35.40 ± 2.87 23.30 ± 2.08 1.11 ± 0.23 13.01 ± 2.97 1.02 ± 0.29 c c c c c c CP-DIME 4.20 ± 0.80 [↑45.8] 34.36 ± 3.01 [↑69.0] 23.01 ± 1.88 [↑90.0] 1.05 ± 0.18 [↑54.4] 11.44 ± 2.33 [↑65.6] 1.35 ± 0.34 [↓63.3] DIEA 4.82 ± 0.76 37.01 ± 3.27 22.93 ± 1.28 1.12 ± 0.27 11.96 ± 1.78 0.95 ± 0.20 c c c c c c CP-DIEA 3.97 ± 0.73 [↑37.8] 34.92 ± 2.79 [↑71.8] 22.13 ± 2.01 [↑80.9] 0.99 ± 0.11 [↑45.6] 10.73 ± 2.06 [↑55.3] 1.69 ± 0.36 [↓54.1] DIPE 4.70 ± 0.82 35.03 ± 3.44 22.70 ± 1.66 1.08 ± 0.09 12.07 ± 2.96 1.06 ± 0.19 a a b CP-DIPE 3.02 ± 0.77 [↑4.9] 24.32 ± 2.58 [↑19.6] 15.32 ± 1.04 [↑26.5] 0.73 ± 0.20 [↑7.4] 7.02 ± 1.84 [↑1.6] 2.77 ± 0.38 [↓24.7] Values are expressed as mean ± SEM for six mice in each group. CP cisplatin, DIME methanol extract of D. indica (300 mg/kg), DIEA ethanol extract of D. indica (300 mg/kg), DIPE petroleum ether extract of D. indica (300 mg/kg) Statistics: ANOVA followed by the Tukey test a b c p < 0.05, p < 0.01, p < 0.001—when the extract-treated groups (CP-DIME, CP-DIEA, CP-DIPE) compared with the diseases control (CP-control) group and the CP-control compared with the healthy control group. Values in parentheses represent percent change from control/cisplatin-control group Sen et al. Nutrire (2018) 43:15 Page 7 of 9 cells, alteration of filtration surface area, and modification enzymes [27]. ATPases are considered as integral mem- of the ultrafiltration coefficient factors that thereby brane proteins which require phospholipids and thiol reduces GFR [19]. In the present study, administration of groups to preserve their function and structure [28]. Cis- DIME and DIEE positively ameliorated the serum and platin induces toxicity by causing the disturbances in the urine level of urea, uric acid, and creatinine levels in all electrolytes homeostasis which ultimately reduced the + + the rats treated with cisplatin. Creatinine clearance rate activity of Na /K -ATPase and leading to cell death [29]. was also increased after the extract administration. The Cisplatin is a water-soluble molecule which crosses the results indicated the protective effect of DIME and DIEE plasma membrane and penetrates into the cell. After ac- against cisplatin-induced nephrotoxicity. tivation inside the cell, cisplatin causes direct interaction The BUN is considered as an indicator of renal func- with the isolated C45 loop that is a major part of Na / tion. Cisplatin induces the destruction of proximal and K -ATPase [30]. A significant reduction in the activities distal tubules preceded the renal hemodynamics, re- of other marker enzymes (ALP, GGTase, and LAP) indi- duced the reabsorption, enhanced vascular resistance, cated that the kidney was adversely affected by cisplatin and caused an elevation in the level of BUN [18]. Cis- treatment. This alteration due to cisplatin enzymes could platin administration increases BUN level significantly, be related to the oxidative modification of enzymes which was back to normal after DIME and DIEE admin- through the generation free radicals [27]. Result reviled istration. There was a significant (p < 0.001) increase in that marker enzyme activity was restored after adminis- the levels of serum phospholipids of nephrotoxic animals tration of methanol and ethyl acetate extracts. compared to healthy animals, which is in line with earl- Cisplatin has been found to augment oxidative stress ier reported studies [2]. The cellular injury is linked with by increasing levels of superoxide anion, hydroxyl radical the alterations in lipid composition of tissue [28]. Hence, and H O [31]. In our study, cisplatin induction causes a 2 2 the elevation in the level of serum phospholipids in the significant decrease in the level of SOD, CAT, GPx, GR, CP-control group may due to the damage of membrane and GSH level, whereas the level of MDA increases in phospholipids caused by cisplatin. Serum cholesterol the nephrotoxic animal. Maintenance of redox balance is profoundly enhanced upon cisplatin treatment. The important for the maintenance of the integrity of the phospholipids are vital membrane components, and a cell. Antioxidant enzymes such as SOD, CAT, GPx, and significant decrease in the serum phospholipid and re- GR protect the cells against oxidative stress-induced cell duction in serum cholesterol level by DIME and DIEE in damage by converting the reactive species to a non-toxic animals of the CP-DIME and CP-DIEE group may link or less reactive substance [17, 32]. Reduced glutathione with the beneficial effect of extracts during cisplatin (GSH) is a non-enzymatic component that plays an im- treatment. portant role in maintaining the cell integrity and consid- Toxic chemicals, certain drugs, infectious agents, etc. ered to play a central role in the antioxidant network. It can induce damage to the kidney that ultimately leads to also detoxifies certain endogenous toxins, including cis- the imbalance of electrolyte [26]. In this study, serum platin, through the production of GSH adducts [33]. sodium level did not alter significantly (only 5% de- Cisplatin-induced nephrotoxicity causes inhibition of crease) in the cisplatin-induced nephrotoxic group when protein synthesis and depletion of intracellular GSH and compared to the normal group. Serum level of potas- protein-SH level [33]. MDA is an excellent indicator of sium decreased significantly (p < 0.01) in nephrotoxic the degree of lipid peroxidation. Increase in the level of re- animal compared to the normal group. Hypokalemia is a active species or depletion of enzymatic and non-enzymatic common electrolyte abnormality found during cisplatin antioxidant can cause oxidative stress, which in turn induce treatment due to enhanced renal reabsorption capacity cell injury [32]. Significant depletion of GSH and increase observed in response to reduced intestinal absorption of in MDA level were observed in the cisplatin-induced + + potassium [26]. Urinary excretion of Na and K in- nephrotoxic group. The present study showed that the ad- creased significantly after treatment with cisplatin, which ministration DIME and DIEE in animals that receive also indicated the abnormality in kidney function. The ad- cisplatin significantly increase the activity of antioxidant en- ministration of DIME and DIEE significantly increased zymes. Cisplatin-induced depletion of GSH and enhance- the serum potassium concentration and reduced urinary ment of MDAproductioninrenal tissue also averted by + + excretion of Na and K toward normal values in the D. indica fruit extracts. Thus, the antioxidant activity of cisplatin-treated animals, which indicates the potentiality the extracts may contribute to the protective effect of D. of D. indica fruit to overcome electrolyte imbalance. indica fruit on cisplatin-induced nephrotoxicity. A significant reduction in the activities of Na / In light of biochemical results and antioxidant activity, K -ATPase, ALP, GGTase, and LAP was indicated that it was observed that D. indica fruit extracts particularly the kidney was adversely affected by cisplatin treatment, methanol and ethyl acetate extracts exert a protective ef- which may be linked with the oxidative modification of fect on the cisplatin-induced renal injury, at least in part, Sen et al. Nutrire (2018) 43:15 Page 8 of 9 can be due to antioxidant and free radical scavenging ac- Received: 8 April 2018 Accepted: 24 May 2018 tivity exerted by the extracts. A number of chemical constituents are isolated from the plant. Previous inves- tigations found the presence of betulinaldehyde, betuli- References 1. Singh NP, Ganguli A, Prakash A. Drug-induced kidney diseases. J Assoc Phy nic acid, lupeol, and dillenetin in D. indica bark, fruit, Ind. 2003;51:970–9. leaf, and stem. Myricetin and isorhamnetin found to be 2. Khan SA, Priyamvada S, Khan W, Khan S, Farooq N, Yusufi ANK. Studies on present in fruit and stem. Leaves, fruit, and bark contain the protective effect of green tea against cisplatin induced nephrotoxicity. Pharmacol Res. 2009;60:382–91. cycloartenone and n-hentriacontanol. The fruits also 3. Gogoi BJ, Tsering J, Goswami B. Antioxidant activity and phytochemical contain rosmarinic acid. The fruits of the plant also consid- analysis of Dillenia indica L. fruit of Sonitpur, Assam, India. Int J Pharm Sci ered as arichsourceofphenoliccompounds [3, 21, 22]. Res. 2012;3(12):4909–12. 4. Rahmatullah M, Hasan ME, Islam MA, Islam MT, Jahan FI, Seraj S, et al. A survey on medicinal plants used by the folk medicinal practitioners in three villages of Panchagarh and Thakurgaon District, Bangladesh. Ame-Eur J Sust Conclusion Agr. 2010;4(3):291–301. In conclusion, in the light of various biochemical results and 5. Abdille MH, Singh RP, Jayaprakasha GK, Jena BS. Antioxidant activity of the antioxidant investigations, the present data confirmed that extracts from Dillenia indica fruits. Food Chem. 2005;90:891–6. 6. Talukdar A, Talukdar N, Deka S, Sahariah BJ. A review: Dillenia indica (outenga) methanol and ethyl acetate extract of Dillenia indica fruit as anti-diabetic herb found in Assam. Int J Pharm Sci Res. 2012;3:2482–6. confers a protective effect against cisplatin-induced nephro- 7. Singh AP, Brindavanam BN, Kimothi PG, Aerim V. Evaluation of in vivo anti- toxicity and other damaging effects. Treatment with metha- inflammatory and analgesic activity of Dillenia indica f. elongata (Miq.) Miq. And Shorea robusta stem bark extracts. Asian Pac J Trop Dis. 2016;6(1):75–81. nol extract to a larger extent prevented cisplatin-induced 8. Sen S, De B, Devanna N, Chakraborty R. Total phenolic, total flavonoids nephrotoxicity by protecting the kidney from damaging and content, and antioxidant capacity of the leaves of Meyna spinosa Roxb., an by strengthening antioxidant defense mechanism. Protective Indian medicinal plant. Chin J Nat Med. 2013;2:149–57. effect of D. indica fruit, at least in part, can be due to their 9. Yen G, Lai H, Chou H. Nitric oxide-scavenging and antioxidant effects of Uraria crinita root. Food Chem. 2001;74:471–8. antioxidant and free radical scavenging activity. Further 10. Tai Z, Cai L, Dai L, Dong L, Wang M, Yang Y, Cao Q, Ding Z. Antioxidant study is required to find the molecular mechanism and to activity and chemical constituents of edible flower of Sophora viciifolia. isolate bioactive molecule responsible for the nephroprotec- Food Chem. 2011;126:1648–54. 11. Chanda S, Deb L, Tiwari RK, Singh K, Ahmed S. Gastroprotective mechanism tive activity of D. indica fruit. of Paederia foetida Linn. (Rubiaceae) - a popular edible plant used by the tribal community of North-East India. BMC Comp Alt Med. 2015;15(304):1–9. Abbreviations 12. Organisation for Economic Co-operation and Development (OECD). OECD ALP: Alkaline phosphatase; CAT: Catalase; Cr: Creatinine; DPPH: 2,2-Diphenyl- guideline for testing of chemicals (test no. 423: acute toxicity—acute toxic 1-picrylhydrazyl; GGTase: γ-Glutamyl transferase; GPx: Glutathione peroxidase; class method), 2001. Available in: https://www.oecd-ilibrary.org/environment/ GR: Glutathione reductase; GSH: Reduced glutathione; LAP: Leucine test-no-423-acute-oral-toxicity-acute-toxic-class-method_9789264071001-en. aminopeptidase; MDA: Total malondialdehyde; NO: Nitric oxide; 13. Ogundipe DJ, Akomolafe RO, Sanusi AA, Imafidon CE, Olukiran OS, Oladele SOD: Superoxide dismutase AA. Effects of two weeks administration of Ocimum gratissimum leaf on feeding pattern and markers of renal function in rats treated with gentamicin. Egypt J Bas Applied Sci. 2016;3:219–31. Acknowledgements 14. Fiske CH, Subbarow Y. The colorimetric determination of phosphorus. J Bio The authors are thankful to CES College of Pharmacy, Kurnool, and Assam Chem. 1925;66:375–400. Down Town University, Guwahati and to all the friends, lab colleagues, and 15. Jung K, An JM, Eom D, Kang KS, Kim S. Preventive effect of fermented black the fellows who directly or indirectly helped us during this experiment. No ginseng against cisplatin-induced nephrotoxicity in rats. J Gins Res. 2017; financial support was received from any sources for this work. 41(2):188–94. 16. Farooq N, Yusufi ANK, Mahmood R. Effect of fasting on enzymes of carbohydrate metabolism and brush border membrane in rat intestine. Nut Availability of data and materials Res. 2004;24:407–16. All the generated and analyzed data are included in this published article. 17. Asokkumar K, Sen S, Umamaheswari M, Sivashanmugam AT, Subhadradevi V. Synergistic effect of the combination of gallic acid and famotidine in protection of rat gastric mucosa. Pharmacol Rep. 2014;66:594–9. Authors’ contributions 18. Hassan I, Chibber S, Naseem I. Ameliorative effect of riboflavin on the SS and RC are involved in designing the experiment and the collection of cisplatin induced nephrotoxicity and hepatotoxicity under the sample. SS, RC, and PK performed the experimental work and statistical photoillumination. Food Chem Toxicol. 2010;48:2052–8. analysis. SS, RC, and PK managed the literature and wrote the first and final 19. Jain A, Singhai AK. Nephroprotective activity of Momordica dioica roxb.in draft. All the authors read and approved the final draft. cisplatin-induced nephrotoxicity. Nat Prod Res. 2010;24(9):846–54. 20. Sheena N, Ajith TA, Janardhanan KK. Prevention of nephrotoxicity induced Ethics approval and consent to participate by the anticancer drug cisplatin, using Ganoderma lucidum, a medicinal Not applicable as no study on human was performed. mushroom occurring in South India. Curr Sci. 2003;85:478–82. 21. Das M, Sarma BP, Ahmed G, Nirmala CB, Choudhury MK. In-vitro anti oxidant activity and total phenolic content of Dillenia indica and Garcinia Competing interests penducalata, commonly used fruits in Assamese cuisine. Free Radic The authors declare that they have no competing interests. Antioxid. 2012;2:30–6. 22. Singh DR, Singh S, Salim KM, Srivastava RC. Estimation of phytochemicals and antioxidant activity of underutilized fruits of Andaman Islands (India). Publisher’sNote Int J Food Sci Nutr. 2012;63(4):446–52. Springer Nature remains neutral with regard to jurisdictional claims in 23. Renugadevi J, Prabu SM. Naringenin protects against cadmium-induced published maps and institutional affiliations. oxidative renal dysfunction in rats. Toxicology. 2009;256:128–34. Sen et al. Nutrire (2018) 43:15 Page 9 of 9 24. Maiuolo J, Oppedisano F, Gratteri S, Muscoli C, Mollace V. Regulation of uric acid metabolism and excretion. Int J Cardiol. 2016;213:8–14. 25. Gowda S, Desai PB, Kulkarni SS, Hull VV, Math AAK, Vernekar SN. Markers of renal function tests. North Ame J Med Sci. 2010;2(4):170–3. 26. Rajakrishnan R, Lekshmi R, Benil PB, Thomas J, AlFarhan AH, Rakesh V, Khalaf S. Phytochemical evaluation of roots of Plumbago zeylanica L. and assessment of its potential as a nephroprotective age. Saudi J Bio Sci. 2017;24(4):760–6. 27. Farooquia Z, Ahmeda F, Rizwana S, Shahida F, Khanb AA, Khana F. Protective effect of Nigella sativa oil on cisplatin induced nephrotoxicity and oxidative damage in rat kidney. Biomed & Pharmaco. 2017;85:7–15. 28. Shiny KS. Biochemical studies on the protective effect of taurine on experimentally induced myocardial infarction in rats, Thesis (PhD). Kerala: Cochin University; 2007. 29. Noori S, Mahboob T. Role of electrolytes disturbances and Na+-K+-ATPase in cisplatin-induced renal toxicity and effects of ethanolic extract of Cichorium intybus. Pak J Pharm Sci. 2012;25:857–62. 30. Kubala M, Geleticova J, Huliciak M, Zatloukalova M, Vacek J, Sebela M. Na+/K +-ATPase inhibition by cisplatin and consequences for cisplatin nephrotoxicity. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2014;158(2):194–200. 31. Deavall DG, Martin EA, Horner JM, Roberts R. Drug-induced oxidative stress and toxicity. J Toxicol. 2012; https://doi.org/10.1155/2012/645460. Available from: https://www.hindawi.com/journals/jt/2012/645460/cta/. [Accessed 21 May 2015]. 32. Sen S, Chakraborty R. The role of antioxidants in human health. In: Silvana A, Hepel M, editors. Oxidative stress: diagnostics, prevention and therapy. Washington D: American Chemical Society; 2011. p. 1–37. 33. Ognjanovic BI, Djordjevic NZ, Matic MM, Obradovic JM, Mladenovic JM, Stajn AS, Saicic ZS. Lipid peroxidative damage on cisplatin exposure and alterations in antioxidant defense system in rat kidneys: a possible protective effect of selenium. Int J Mol Sci. 2012;13:1790–803. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nutrire Springer Journals

Dillenia indica fruit prevents cisplatin-induced kidney injury in experimental rats through modulation of oxidative stress, marker enzyme, and biochemical changes

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Abstract

Background: Dillenia indica fruit is known for its numerous health benefits in folk medicine including its use to cure kidney diseases. The present study was designed to investigate the protective effect of D. indica fruit extracts on cisplatin-induced nephrotoxicity. Methods: A nephrotoxic dose of cisplatin (3 mg/kg b.w./day, i.p) was administered every fifth day to the animals receiving vehicle or fruit extracts (methanol, ethyl acetate, and petroleum ether) daily for 25 days. Rats were sacrificed on the 25th day, and the effect of extracts was assessed by determining the alterations in various serum and urine parameters, membrane-bound enzyme, and antioxidant defense system in kidney tissue. Results: Increase in serum urea, uric acid, creatinine, blood urea nitrogen, phospholipid, and cholesterol and a decrease in urine urea, uric acid, creatinine, and creatinine clearance rate were reported in the cisplatin control group. Cisplatin alters electrolyte balance, brush border membrane marker enzyme (i.e., alkaline phosphatase, γ- glutamyl transferase, leucine aminopeptidase) activity, and redox balance significantly. Methanol and ethyl acetate extracts of D. indica fruit produced beneficial effect and ameliorated serum and urine parameter to normal. Extract + + administration increases Na /K -ATPase activity and different enzymatic and non-enzymatic antioxidants positively, whereas lipid peroxidation reduced significantly. Extracts exhibited a potent in vitro antioxidant activity. Conclusion: Taking into account these results, it can be assumed that D. indica fruit could be the future key candidate which may maximize the clinical use of cisplatin in the treatment of different cancer without nephrotoxicity. Keywords: Dillenia indica, Fruit, Antioxidant, Cisplatin, Kidney function test Background due to frequent reversible and irreversible side effects The incidence of drug-induced nephrotoxicity has been like nephrotoxicity, neurotoxicity, bone marrow toxicity, increasing at a frightening rate with the increasing uses and gastrointestinal toxicity and ototoxicity [2]. of antibiotics and anticancer drugs [1]. Oxidative stress Dillenia indica Linn. (Family: Dilleniaceae) is a wildly is considered as a key factor for drug-induced nephro- available medicinal plant found in Northeast India. The toxicity [2]. Cisplatin [cis-diamminedichloroplatinum common name of the plant is outenga in Assamese and (II)] is the most common and potent anticancer drug chalita in Bengali. The plant grows in abundance in this used against a diverse spectrum of malignancies. How- part of India, but most of the fruits of this plant are ever, the use of cisplatin in combating cancer is limited wasted due to the lack of technical knowledge. Fruits are edible; the fleshy calyx of the fruit can be consumed directly and also be prepared with vegetable and pickle. * Correspondence: saikat.pharm@rediffmail.com The fruits and other parts of the plant are used Department of Pharmacy, Assam Down Town University, Guwahati, Assam 781026, India traditionally to cure a number of ailments like stomach CES College of Pharmacy, Kurnool, Andhra Pradesh 518218, India © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sen et al. Nutrire (2018) 43:15 Page 2 of 9 ● ● problem, fevers, and cough and to combat hair fall and acid and rutin were used as standard in DPPH ,NO dandruff [3]. The juice of the fruit was used by the folk scavenging, and lipid peroxidation inhibition assay, medicinal practitioners of Bangladesh to cure kidney dis- respectively. eases [4]. The previous investigation found that D. indica fruits contain ascorbic acid, tocopherol, carotene, Acute toxicity study and phenolic compounds [5]. The fruits of D. indica In this study, we adopted acute oral toxicity-acute toxic found to possess several beneficial functions such as class method as per OECD guideline 423 (Annexure 4d). anticancer and antidiarrheal [6], antioxidant [5], CNS This method is considered reproducible, required a very depressant, and anti-inflammatory activity [7]. limited number of animals, and is able to categorized Food with antioxidant potential or antioxidant mole- substances in a parallel mode to the other acute toxicity cules such as phenolic acids, flavonoids, and tannins par- study methods like OECD guidelines 420 and 425. The ticularly from plant sources have proved their potential extract (2000 mg/kg) is administered orally to three role as a prophylactic or curative agent against various healthy nulliparous and non-pregnant female albino oxidative stress-related diseases [8]. The present study mice (20–25 g). Mice were observed individually at least was undertaken to evaluate the protective activity of D. once in half an hour during the first 24 h, with special indica fruits against cisplatin-induced nephrotoxicity. notice given during the initial 4 h, and daily thereafter, for a total of 14 days [11, 12]. Methods Plant materials Induction of nephrotoxicity and treatment schedule Fruits of Dillenia indica Linn. were collected from the Healthy animals were acclimatized to the standard labora- state of Tripura, India, and authenticated by Dr. BK Datta, tory facility for a week. Rats were fed with standard rat Department of Botany, Tripura University, Tripura, India chow and water ad libitum under controlled conditions. (voucher specimen no. TU/BOT/HEB/RC25092011a). Treatment schedule and induction of nephrotoxicity using cisplatin was performed as per the procedure described by Extraction of plant material Khan et al. [2]. At the end of acclimatization, period ani- The fruits were cleaned to remove unwanted material mals were divided into major four groups (control, DIME, and cut into small pieces. The fruits (without seed) were DIEE, DIPE) each containing 12 animals. Animals in dif- dried under the shed as required to grind into coarse ferent major groups were treated with water (control), powder. The powder of D. indica fruit was extracted DIME (300 mg/kg/day, p.o.), DIEE (300 mg/kg/day, p.o.), with methanol, ethyl acetate, and petroleum ether separ- and DIPE (300 mg/kg/day, p.o.) for 5 days. Animals of ately using Soxhlet apparatus. The filtrate was concen- each major group were divided into two subgroups each trated, and the solvent was evaporated under reduced (six animals/subgroup) and continued to receive their ex- pressure to obtain methanol extract (DIME), ethyl tract treatment daily at a dose of 300 mg/kg orally. Based acetate extract (DIEE), and petroleum ether extract on preliminary study, 300 mg/kg dose was selected. Cis- (DIPE) of D. indica fruit. platin (CP, 3 mg/kg b.w./day, i.p) in 0.9% saline is injected every fifth day (four injections in total) for 25 days to in- Experimental animals duce nephrotoxicity to one of the subgroups designated as Healthy albino female mice were used for acute toxicity CP-control, CP-DIME, CP-DIEE, and CP-DIPE. Normal study, and male Wistar rats were used for the nephropro- saline in equivalent volume was given to the animals of tective activity. Animals were maintained under standard the other subgroup from each group for the same period. environmental conditions. The animal experiments were The rats were sacrificed on the 25th day, 5 days after the carried out according to the guidelines of Committee for last injection of cisplatin under light ether anesthesia Purpose of Control and Supervision of Experiments on (Fig. 1, grouping of animal and treatment schedule). Animals (CPCSEA), Ministry of Environment and Forests, Government of India. The study was approved by the Estimation of biochemical parameters and in vivo Institutional Animal Ethical Committee (Reg. No. 1305/ antioxidant assay ac/09/CPCSEA). On the 25th day, urine samples (24 h) were collected using metabolic cages. Blood samples were collected by Antioxidant activity of extracts cardiac puncture under light ether anesthesia, and serum The free radical scavenging activity of extracts of D. sample was used for estimation of different biochemical indica fruits was determined by 2,2-diphenyl-1-picrylhy- parameters. After euthanasia, one kidney of each rat was drazyl radical (DPPH ) scavenging assay method [8], ni- rapidly removed and washed thoroughly with ice-cold tric oxide radical (NO ) scavenging assay method [9], normal saline, and homogenates (10% w/v) were pre- and lipid peroxidation inhibition assay [10]. Ascorbic pared 0.1 M Tris-HCl buffer, pH 7.5. The homogenate Sen et al. Nutrire (2018) 43:15 Page 3 of 9 Fig. 1 Experimental design (grouping of animal and treatment schedule) was centrifuged at 3000×g for 15 min to remove the cell adopted by Asokkumar et al. [17] in which decompos- debris. The supernatant was used for the determination ition of hydrogen peroxide (H O ) in the presence of 2 2 of different biochemical parameters. CAT was measured at 254 nm. Glutathione reductase (GR) activity was determined by the method involving Urea, uric acid, creatinine, and creatinine clearance oxidation of NADPH into NADP in the presence of ox- Levels of urea, uric acid, and creatinine (Cr) in serum idized glutathione. Glutathione peroxidase (GPx) activity and urine were determined spectrophotometrically using was estimated by monitoring the oxidation of reduced commercially available kits (Agapee Diagnostics, India). NADPH at 340 nm. The level of reduced glutathione Creatinine clearance rate was determined using the (GSH) was determined using the method of Asokkumar following equation [13], et al. [17]. Total malondialdehyde (MDA) was deter- mined as an index of the extent of lipid peroxidation in Creatinine clearanceðÞ ml=kg body weight= min kidney tissue using standard method [17]. ¼½ Urinary CrðÞ mg=dl urine volumeðÞ ml  1000 =½ serum CrðÞ mg=dl 1440ðÞ min Statistical analysis The results are expressed as mean ± S.E.M (n = 6 for in vivo test and n = 3 for in vitro study) Statistical differ- Blood urea nitrogen, cholesterol, and phospholipid ence was tested by using one-way analysis of variance Blood urea nitrogen (BUN) and cholesterol level in followed by Tukey tests. A level of p < 0.05 was used as serum were determined by commercially available kits. the criterion for statistical significance. Phospholipid content in serum was estimated using a standard method [14]. + + Na ,K , and brush border membrane marker enzymes Results Potassium and sodium level in plasma was estimated Acute toxicity study + + using flame photometer. Level of Na /K -ATPase and In principle, the acute toxicity study method is not brush border membrane marker enzymes like alkaline intended to permit precise LD calculation but used to phosphatase (ALP), γ-glutamyl transferase (GGTase), find exposure ranges to check lethality since the death of and leucine aminopeptidase (LAP) was assayed by previ- a proportion of experimental animals is still the main ously described method [15, 16]. endpoint of the test. In acute toxicity study of samples, mortality was not observed at the dose of 2000 mg/kg; Enzymatic and non-enzymatic antioxidant activity thus, further lower dose is not administered. Consider- Superoxide dismutase (SOD) activity was estimated by ing OECD guideline, 2000 mg/kg dose was categorized the inhibition of formation of autocatalyzed adreno- under Globally Harmonized Classification System (GHS) chrome in the presence of tissue homogenate at 480 nm. category 5 (safe dose), as per OECD guideline 423 Catalase (CAT) activity was determined by the method (Annexure 2d). Sen et al. Nutrire (2018) 43:15 Page 4 of 9 In vitro antioxidant activity of D. indica fruits extracts phospholipid, and cholesterol level compared to the In vitro antioxidant activity of different extracts of D. nephrotoxic group (Table 3). indica fruit was investigated. Our present findings There was only a small insignificant decrease in serum showed that DIEE and DIME exhibited strong free radical sodium level (− 5.2%) and a significant decrease in serum scavenging activity. The IC values of DIEE (15.8 ± potassium level (− 40.2%) in the cisplatin-treated group 0.02 μg/ml) and DIME (29.3 ± 0.03 μg/ml) against DPPH in comparison to the normal control group. DIME and were observed while IC values of DIME (28.22 ± DIEE administered at a dose of 300 mg/kg along with cis- 0.34 μg/ml) and DIEE (34.0 ± 0.55 μg/ml) against NO and platin have shown significant (p < 0.01) decrease in the the IC values of DIME (61.5 ± 0.60 μg/ml) and DIEE serum potassium level in comparison to the cisplatin con- (70.7 ± 0.54 μg/ml) against lipid peroxidation inhibition trol group. Both urinary excretion of sodium (+ 118.7%) assay (Table 1). and potassium (+ 62.7%) was increased significantly after cisplatin administration. Administration of DIME in nephrotoxic animals caused 39.6 and 35.9% reduction in Effect of extracts on urea, uric acid, creatinine, and urinary sodium and potassium excretion, respectively. creatinine clearance DIEE reduced excretion of urinary sodium and potassium Effects of D. indica fruit extracts on serum and urine significantly, though DIPE had an insignificant effect on level of urea, uric acid, and creatinine were tabulated in administered in the nephrotoxic group (Table 3). Table 2. Treatment with cisplatin causes a significant rise in serum urea, serum uric acid, and serum creatin- ine level, whereas the level of urea, uric acid, and cre- Effect of extracts marker enzymes atinine in urine significantly reduced in the cisplatin A significant reduction in the activities of Na / control group. Treatment with DIME in the nephrotoxic K -ATPase, ALP, GGTase, and LAP was observed after group (CP-DIME) cause significant reduction in serum cisplatin treatment. DIME and DIEA treatment in ani- urea (− 32.2%), serum uric acid (− 46.0%), and serum mals that also received cisplatin increased Na / creatinine (− 37.1%) level and an increase in urine urea K -ATPase by 50 and 37%, ALP by 44 and 28%, GGTase (+ 14.8%), urine uric acid (+ 37.4%), and urine creatinine by 59 and 49%, and LAP by 45 and 34%, respectively, level (+ 39.6%). Results showed that DIEE also produced compared to the CP-control group. Result reviled that a significant nephroprotective effect on cisplatin-induced marker enzyme activity was restored after the adminis- nephrotoxicity. None of the extracts produces any sig- tration of DIME and DIEA, which further indicated to- nificant variation in serum and urine parameters ward the protective effect of D. indica fruit (Fig. 2). when administered in healthy animals. Cisplatin also induces 18.5% reduction in creatinine clearance rate Effect of D. indica fruits on enzymatic and non-enzymatic when compared with the control. DIME and DIEE antioxidants treatment significantly elevate creatinine clearance The effect of DIME, DIEE, and DIPE was determined on rate in nephrotoxic animals. the level of various enzymatic and non-enzymatic anti- oxidants when administered in nephrotoxic and healthy Effect of D. indica fruit on BUN, phospholipid, cholesterol, rats (Table 4). Level of SOD, CAT, GPx, GR, and GSH and sodium and potassium level significantly reduced after cisplatin induction, whereas The administration of cisplatin to healthy rats caused a the level of MDA increases. Cisplatin-induced reduction significant increase in BUN (+ 89.6%), serum phospholipid in antioxidant activity was inhibited significantly after (+ 25.8%), and cholesterol (+ 34.2%) level. The extracts the administration of DIME and DIEE. After receiving had no effect on these parameters when administered on the respective drug treatment in nephrotoxic animals, healthy animals. Although, DIME and DIEE (300 mg/kg) there is a significant change in the level of MDA (− 63.3% when administered in rats that received cisplatin signifi- by DIME, − 54.1% by DIEE, − 24.7% by DIPE) compared cantly prevent such alteration and reduce BUN, serum to the CP-control group. Table 1 In vitro antioxidant effect of D. indica fruit extracts Screening method IC value (μg/ml) DIME DIEE DIPE Standard DPPH scavenging assay 29.3 ± 0.03 15.8 ± 0.02 79.9 ± 0.07 2.6 ± 0.02 (ascorbic acid) NO scavenging assay 28.22 ± 0.34 34.0 ± 0.55 81.0 ± 0.81 24.1 ± 0.24 (ascorbic acid) Lipid peroxidation inhibition assay 61.5 ± 0.60 70.7 ± 0.54 92.6 ± 0.44 59.9 ± 0.20 (rutin) Values are expressed as mean ± SEM Sen et al. Nutrire (2018) 43:15 Page 5 of 9 Table 2 Effect of D. indica fruit extracts on serum and urine parameters in an animal with/without cisplatin treatment Group Serum urea Serum uric acid Serum creatinine Urine urea Urine uric acid Urine creatinine Creatinine clearance rate (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) (ml/min/kg body weight) Control 38.32 ± 2.01 1.24 ± 0.21 0.85 ± 0.01 140.66 ± 6.06 6.92 ± 1.06 32.05 ± 0.36 0.068 c c b b b c b CP-control 64.09 ± 3.99 2.65 ± 0.33 1.43 ± 0.10 109.80 ± 5.86 4.38 ± 0.86 21.40 ± 0.22 0.053 [↓18.5] [↑67.2] [↑113.7] [↑68.2] [↓21.9] [↓36.7] [↓33.2] DIME 37.00 ± 2.12 1.21 ± 0.13 0.89 ± 0.07 135.82 ± 8.33 6.70 ± 0.98 31.99 ± 0.19 0.067 b b b a b b a CP-DIME 43.45 ± 3.11 1.43 ± 0.28 0.90 ± 0.09 126.00 ± 6.98 6.02 ± 1.01 29.88 ± 0.29 0.071 [↑34.0] [↓32.2] [↓46.0] [↓37.1] [↑14.8] [↑37.4] [↑39.6] DIEA 38.97 ± 2.77 1.19 ± 0.22 0.87 ± 0.05 141.35 ± 8.01 6.63 ± 0.88 32.13 ± 0.34 0.067 b b b a a b a CP-DIEA 48.32 ± 2.89 1.66 ± 0.19 1.01 ± 0.04 130.33 ± 7.23 5.59 ± 1.02 26.81 ± 0.37 0.070 [↑32.0] [↓24.6] [↓37.4] [↓29.4] [↑18.7] [↑27.6] [↑25.3] DIPE 37.79 ± 2.32 1.11 ± 0.28 0.82 ± 0.07 138.68 ± 6.87 6.82 ± 1.26 30.82 ± 0.38 0.069 CP-DIPE 59.33 ± 4.02 2.32 ± 0.31 1.39 ± 0.11 114.38 ± 6.01 4.45 ± 0.77 22.47 ± 0.28 0.058 [↑9.4] [↓7.4] [↓12.5] [↓2.8] [↑4.2] [↑1.6] [↑6.4] Values are expressed as mean ± SEM for six mice in each group. CP cisplatin, DIME methanol extract of D. indica (300 mg/kg), DIEA ethanol extract of D. indica (300 mg/kg), DIPE petroleum ether extract of D. indica (300 mg/kg) Statistics: ANOVA followed by the Tukey test a b c p <0.05, p <0.01, p < 0.001—when the extract-treated groups (CP-DIME, CP-DIEA, CP-DIPE) compared with the diseases control (CP-control) group and the CP-control compared with the healthy control group Values in parentheses represent percent change from control/cisplatin-control group Discussion used as a vegetable, and an ethnomedicinal survey con- Cisplatin is a most widely used drug for the treatment of ducted in Bangladesh showed that D. indica fruit juice different cancers and solid tumors. However, nephrotox- was used by the patients with kidney problems [4]. icity caused by the drug is a main limiting factor of its Oxidative stress is an emergent factor for the caus- widespread clinical use for the long-term treatment [2]. ation of many diseases, and nephrotoxicity is one of A number of investigations have been carried out to them. Antioxidant molecules present in the plant/food find/monitor the protective effect of different natural can act synergistically along with exogenous antioxidants substances like grape seed extract, fish oil [18], green tea to avert oxidative stress-induced damage [8]. In vitro [2], Momordica dioica fruit [19], Ganoderma lucidum,a study reveals that D. indica extracts particularly metha- mushroom [20], etc. when administered along with cis- nol and ethyl acetate extract possess strong in vitro anti- platin; however, their use in clinical practice could not oxidant activity tested through lipid peroxidation be achieved till now. Fruits of D. indica are commonly inhibition assay, DPPH radical, and nitric oxide radical Table 3 Effect of D. indica fruit extracts on BUN, phospholipid, cholesterol, and sodium and potassium level + + + + Group BUN (mg/dl) Phospholipid Cholesterol Serum Na Serum K Urinary Na excretion Urinary K excretion (mg/dl) (mg/dl) (mmol/l) (mmol/l) (μmol/24 h) (μmol/24 h) Control 17.9 ± 1.11 101.32 ± 7.06 119.3 ± 8.17 137.22 ± 2.10 6.44 ± 0.21 101.50 ± 7.22 289.06 ± 14.11 c a a b c c CP-control 33.93 ± 2.66 127.53 ± 13.02 160.05 ± 9.29 130.11 ± 2.21 3.85 ± 0.33 222.03 ± 9.12 470.18 ± 15.60 [↑89.6] [↑25.8] [↑34.2] [↓5.2] [↓40.2] [↑118.7] [↑62.7] DIME 17.28 ± 1.43 99.72 ± 9.11 117.39 ± 7.10 136.12 ± 1.99 6.29 ± 0.30 110.23 ± 8.72 280.35 ± 13.99 c a a b a a CP-DIME 19.29 ± 1.51 107.31 ± 13.96 116.67 ± 8.66 138.50 ± 2.31 6.03 ± 0.20 134.11 ± 8.22 [↓39.6] 301.33 ± 14.07 [↓43.1] [↓15.9] [↓27.1] [↑6.4] [↑56.6] [↓35.9] DIEA 18.20 ± 0.99 103.73 ± 9.15 120.39 ± 8.03 138.07 ± 1.77 6.39 ± 0.32 108.66 ± 7.29 293.55 ± 14.66 b a a b b a a CP-DIEA 21.57 ± 1.20 108.34 ± 11.11 121.93 ± 9.21 136.55 ± 2.33 5.86 ± 0.19 145.01 ± 7.94 [↓34.7] 330.20 ± 15.33 [↓36.4] [↓15.0] [↓23.8] [↑4.9] [↑52.2] [↓29.8] DIPE 17.65 ± 1.18 98.92 ± 7.97 120.63 ± 6.99 140.01 ± 2.03 6.45 ± 0.35 115.33 ± 9.02 300.05 ± 14.05 CP-DIPE 29.71 ± 1.87 118.63 ± 10.79 127.42 ± 9.04 132.93 ± 2.62 4.72 ± 0.30 179.42 ± 0.11 [↓19.2] 428.44 ± 16.44 [↓12.4] [↓7.0] [↓20.3] [↑2.2] [↑22.6] [↓8.9] Values are expressed as mean ± SEM for six mice in each group. CP cisplatin, DIME methanol extract of D. indica (300 mg/kg), DIEA ethanol extract of D. indica (300 mg/kg), DIPE petroleum ether extract of D. indica (300 mg/kg) Statistics: ANOVA followed by the Tukey test a b c p <0.05, p <0.01, p < 0.001—when the extract-treated groups (CP-DIME, CP-DIEA, CP-DIPE) compared with the diseases control (CP-control) group and the CP-control compared with the healthy control group Values in parentheses represent percent change from control/cisplatin-control group Sen et al. Nutrire (2018) 43:15 Page 6 of 9 Fig. 2 Effects of D. indica fruit extracts on different marker enzyme activity. Values are expressed as mean ± SEM (n = 6). DIME, methanol extract of D. indica fruits; DIEA, ethyl acetate extract of D. indica fruits; DIPE, petroleum ether extract of D. indica fruits; ALP, alkaline phosphatase; GGTase, g-glutamyl transferase; LAP, leucine aminopeptidase scavenging activity. These results are similar to that of in serum is linked with renal damage and considered as previous investigations carried out by different the indicator of nephrotoxicity. Increased level of urea researchers [5, 21, 22]. Inhibition of lipid peroxidation concentrations in serum may increase after parenchymal by extracts further expands their role as a potent injury. Hyperuricemia is considered as a renal prognostic antioxidant. factor, which may indicate the physical response to an Repeated injection of cisplatin was found to induce amplified generation of endogenous oxygen species as uric marked renal dysfunction as evidenced by increased acid scavenges peroxynitrite [23, 26]. Serum creatinine serum urea, uric acid, and creatinine diagnostic indicators concentration is considered as a potent indicator of the of nephrotoxicity. Urea is the key nitrogen-containing first phase of any kidney disease than the urea and uric metabolic product produced during protein metabolism acid levels [23]. The most penetrable organ in a living sys- [23]. Uric acid is considered as the last substance pro- tem is the kidney through which the toxic substances are duced from an exogenous pool of purines and endogenous eliminated from the living system. Urinalysis is a major purine metabolism [24]. Creatinine is a breakdown prod- pathway to define whether kidney is functioning properly uct of creatine phosphate in muscle, is generally formed at [27]. Cisplatin causes decrease in the level of urine cre- a fairly constant rate based on muscle mass, and consid- atinine, urea, uric acid, and creatinine clearance. These ered as a measure of kidney function [25]. The serum changes may occur due to the reduction in the glomerular urea, creatinine, and uric acid may induce the alteration of filtration rate (GFR) or may be secondary due to the oxi- the glomerular filtration rate, and increase in their levels dative stress, which can cause contraction of mesangial Table 4 Effect of D. indica fruit extracts on enzymatic and non-enzymatic antioxidant level Group SOD CAT GPx GR GSH MDA (μmol/min/mg protein) (μmol/min/mg protein) (μmol/min/mg protein) (μmol/min/mg protein) (μM GSH/gm tissue) (nM/min/mg protein) Control 4.62 ± 0.99 34.33 ± 3.91 22.68 ± 1.72 1.09 ± 0.18 12.08 ± 1.83 0.97 ± 0.13 c c c c c c CP-control 2.88 ± 0.70 [↓37.7] 20.33 ± 3.70 [↓40.8] 12.11 ± 1.05 [↓46.6] 0.68 ± 0.07 [↓37.6] 6.91 ± 1.04 [↓42.8] 3.68 ± 0.37 [↑279.4] DIME 5.05 ± 0.90 35.40 ± 2.87 23.30 ± 2.08 1.11 ± 0.23 13.01 ± 2.97 1.02 ± 0.29 c c c c c c CP-DIME 4.20 ± 0.80 [↑45.8] 34.36 ± 3.01 [↑69.0] 23.01 ± 1.88 [↑90.0] 1.05 ± 0.18 [↑54.4] 11.44 ± 2.33 [↑65.6] 1.35 ± 0.34 [↓63.3] DIEA 4.82 ± 0.76 37.01 ± 3.27 22.93 ± 1.28 1.12 ± 0.27 11.96 ± 1.78 0.95 ± 0.20 c c c c c c CP-DIEA 3.97 ± 0.73 [↑37.8] 34.92 ± 2.79 [↑71.8] 22.13 ± 2.01 [↑80.9] 0.99 ± 0.11 [↑45.6] 10.73 ± 2.06 [↑55.3] 1.69 ± 0.36 [↓54.1] DIPE 4.70 ± 0.82 35.03 ± 3.44 22.70 ± 1.66 1.08 ± 0.09 12.07 ± 2.96 1.06 ± 0.19 a a b CP-DIPE 3.02 ± 0.77 [↑4.9] 24.32 ± 2.58 [↑19.6] 15.32 ± 1.04 [↑26.5] 0.73 ± 0.20 [↑7.4] 7.02 ± 1.84 [↑1.6] 2.77 ± 0.38 [↓24.7] Values are expressed as mean ± SEM for six mice in each group. CP cisplatin, DIME methanol extract of D. indica (300 mg/kg), DIEA ethanol extract of D. indica (300 mg/kg), DIPE petroleum ether extract of D. indica (300 mg/kg) Statistics: ANOVA followed by the Tukey test a b c p < 0.05, p < 0.01, p < 0.001—when the extract-treated groups (CP-DIME, CP-DIEA, CP-DIPE) compared with the diseases control (CP-control) group and the CP-control compared with the healthy control group. Values in parentheses represent percent change from control/cisplatin-control group Sen et al. Nutrire (2018) 43:15 Page 7 of 9 cells, alteration of filtration surface area, and modification enzymes [27]. ATPases are considered as integral mem- of the ultrafiltration coefficient factors that thereby brane proteins which require phospholipids and thiol reduces GFR [19]. In the present study, administration of groups to preserve their function and structure [28]. Cis- DIME and DIEE positively ameliorated the serum and platin induces toxicity by causing the disturbances in the urine level of urea, uric acid, and creatinine levels in all electrolytes homeostasis which ultimately reduced the + + the rats treated with cisplatin. Creatinine clearance rate activity of Na /K -ATPase and leading to cell death [29]. was also increased after the extract administration. The Cisplatin is a water-soluble molecule which crosses the results indicated the protective effect of DIME and DIEE plasma membrane and penetrates into the cell. After ac- against cisplatin-induced nephrotoxicity. tivation inside the cell, cisplatin causes direct interaction The BUN is considered as an indicator of renal func- with the isolated C45 loop that is a major part of Na / tion. Cisplatin induces the destruction of proximal and K -ATPase [30]. A significant reduction in the activities distal tubules preceded the renal hemodynamics, re- of other marker enzymes (ALP, GGTase, and LAP) indi- duced the reabsorption, enhanced vascular resistance, cated that the kidney was adversely affected by cisplatin and caused an elevation in the level of BUN [18]. Cis- treatment. This alteration due to cisplatin enzymes could platin administration increases BUN level significantly, be related to the oxidative modification of enzymes which was back to normal after DIME and DIEE admin- through the generation free radicals [27]. Result reviled istration. There was a significant (p < 0.001) increase in that marker enzyme activity was restored after adminis- the levels of serum phospholipids of nephrotoxic animals tration of methanol and ethyl acetate extracts. compared to healthy animals, which is in line with earl- Cisplatin has been found to augment oxidative stress ier reported studies [2]. The cellular injury is linked with by increasing levels of superoxide anion, hydroxyl radical the alterations in lipid composition of tissue [28]. Hence, and H O [31]. In our study, cisplatin induction causes a 2 2 the elevation in the level of serum phospholipids in the significant decrease in the level of SOD, CAT, GPx, GR, CP-control group may due to the damage of membrane and GSH level, whereas the level of MDA increases in phospholipids caused by cisplatin. Serum cholesterol the nephrotoxic animal. Maintenance of redox balance is profoundly enhanced upon cisplatin treatment. The important for the maintenance of the integrity of the phospholipids are vital membrane components, and a cell. Antioxidant enzymes such as SOD, CAT, GPx, and significant decrease in the serum phospholipid and re- GR protect the cells against oxidative stress-induced cell duction in serum cholesterol level by DIME and DIEE in damage by converting the reactive species to a non-toxic animals of the CP-DIME and CP-DIEE group may link or less reactive substance [17, 32]. Reduced glutathione with the beneficial effect of extracts during cisplatin (GSH) is a non-enzymatic component that plays an im- treatment. portant role in maintaining the cell integrity and consid- Toxic chemicals, certain drugs, infectious agents, etc. ered to play a central role in the antioxidant network. It can induce damage to the kidney that ultimately leads to also detoxifies certain endogenous toxins, including cis- the imbalance of electrolyte [26]. In this study, serum platin, through the production of GSH adducts [33]. sodium level did not alter significantly (only 5% de- Cisplatin-induced nephrotoxicity causes inhibition of crease) in the cisplatin-induced nephrotoxic group when protein synthesis and depletion of intracellular GSH and compared to the normal group. Serum level of potas- protein-SH level [33]. MDA is an excellent indicator of sium decreased significantly (p < 0.01) in nephrotoxic the degree of lipid peroxidation. Increase in the level of re- animal compared to the normal group. Hypokalemia is a active species or depletion of enzymatic and non-enzymatic common electrolyte abnormality found during cisplatin antioxidant can cause oxidative stress, which in turn induce treatment due to enhanced renal reabsorption capacity cell injury [32]. Significant depletion of GSH and increase observed in response to reduced intestinal absorption of in MDA level were observed in the cisplatin-induced + + potassium [26]. Urinary excretion of Na and K in- nephrotoxic group. The present study showed that the ad- creased significantly after treatment with cisplatin, which ministration DIME and DIEE in animals that receive also indicated the abnormality in kidney function. The ad- cisplatin significantly increase the activity of antioxidant en- ministration of DIME and DIEE significantly increased zymes. Cisplatin-induced depletion of GSH and enhance- the serum potassium concentration and reduced urinary ment of MDAproductioninrenal tissue also averted by + + excretion of Na and K toward normal values in the D. indica fruit extracts. Thus, the antioxidant activity of cisplatin-treated animals, which indicates the potentiality the extracts may contribute to the protective effect of D. of D. indica fruit to overcome electrolyte imbalance. indica fruit on cisplatin-induced nephrotoxicity. A significant reduction in the activities of Na / In light of biochemical results and antioxidant activity, K -ATPase, ALP, GGTase, and LAP was indicated that it was observed that D. indica fruit extracts particularly the kidney was adversely affected by cisplatin treatment, methanol and ethyl acetate extracts exert a protective ef- which may be linked with the oxidative modification of fect on the cisplatin-induced renal injury, at least in part, Sen et al. Nutrire (2018) 43:15 Page 8 of 9 can be due to antioxidant and free radical scavenging ac- Received: 8 April 2018 Accepted: 24 May 2018 tivity exerted by the extracts. A number of chemical constituents are isolated from the plant. Previous inves- tigations found the presence of betulinaldehyde, betuli- References 1. Singh NP, Ganguli A, Prakash A. Drug-induced kidney diseases. J Assoc Phy nic acid, lupeol, and dillenetin in D. indica bark, fruit, Ind. 2003;51:970–9. leaf, and stem. Myricetin and isorhamnetin found to be 2. Khan SA, Priyamvada S, Khan W, Khan S, Farooq N, Yusufi ANK. Studies on present in fruit and stem. Leaves, fruit, and bark contain the protective effect of green tea against cisplatin induced nephrotoxicity. Pharmacol Res. 2009;60:382–91. cycloartenone and n-hentriacontanol. The fruits also 3. Gogoi BJ, Tsering J, Goswami B. Antioxidant activity and phytochemical contain rosmarinic acid. The fruits of the plant also consid- analysis of Dillenia indica L. fruit of Sonitpur, Assam, India. Int J Pharm Sci ered as arichsourceofphenoliccompounds [3, 21, 22]. Res. 2012;3(12):4909–12. 4. Rahmatullah M, Hasan ME, Islam MA, Islam MT, Jahan FI, Seraj S, et al. A survey on medicinal plants used by the folk medicinal practitioners in three villages of Panchagarh and Thakurgaon District, Bangladesh. Ame-Eur J Sust Conclusion Agr. 2010;4(3):291–301. In conclusion, in the light of various biochemical results and 5. Abdille MH, Singh RP, Jayaprakasha GK, Jena BS. Antioxidant activity of the antioxidant investigations, the present data confirmed that extracts from Dillenia indica fruits. Food Chem. 2005;90:891–6. 6. Talukdar A, Talukdar N, Deka S, Sahariah BJ. A review: Dillenia indica (outenga) methanol and ethyl acetate extract of Dillenia indica fruit as anti-diabetic herb found in Assam. Int J Pharm Sci Res. 2012;3:2482–6. confers a protective effect against cisplatin-induced nephro- 7. Singh AP, Brindavanam BN, Kimothi PG, Aerim V. Evaluation of in vivo anti- toxicity and other damaging effects. Treatment with metha- inflammatory and analgesic activity of Dillenia indica f. elongata (Miq.) Miq. And Shorea robusta stem bark extracts. Asian Pac J Trop Dis. 2016;6(1):75–81. nol extract to a larger extent prevented cisplatin-induced 8. Sen S, De B, Devanna N, Chakraborty R. Total phenolic, total flavonoids nephrotoxicity by protecting the kidney from damaging and content, and antioxidant capacity of the leaves of Meyna spinosa Roxb., an by strengthening antioxidant defense mechanism. Protective Indian medicinal plant. Chin J Nat Med. 2013;2:149–57. effect of D. indica fruit, at least in part, can be due to their 9. Yen G, Lai H, Chou H. Nitric oxide-scavenging and antioxidant effects of Uraria crinita root. Food Chem. 2001;74:471–8. antioxidant and free radical scavenging activity. Further 10. Tai Z, Cai L, Dai L, Dong L, Wang M, Yang Y, Cao Q, Ding Z. Antioxidant study is required to find the molecular mechanism and to activity and chemical constituents of edible flower of Sophora viciifolia. isolate bioactive molecule responsible for the nephroprotec- Food Chem. 2011;126:1648–54. 11. Chanda S, Deb L, Tiwari RK, Singh K, Ahmed S. Gastroprotective mechanism tive activity of D. indica fruit. of Paederia foetida Linn. (Rubiaceae) - a popular edible plant used by the tribal community of North-East India. BMC Comp Alt Med. 2015;15(304):1–9. Abbreviations 12. Organisation for Economic Co-operation and Development (OECD). OECD ALP: Alkaline phosphatase; CAT: Catalase; Cr: Creatinine; DPPH: 2,2-Diphenyl- guideline for testing of chemicals (test no. 423: acute toxicity—acute toxic 1-picrylhydrazyl; GGTase: γ-Glutamyl transferase; GPx: Glutathione peroxidase; class method), 2001. Available in: https://www.oecd-ilibrary.org/environment/ GR: Glutathione reductase; GSH: Reduced glutathione; LAP: Leucine test-no-423-acute-oral-toxicity-acute-toxic-class-method_9789264071001-en. aminopeptidase; MDA: Total malondialdehyde; NO: Nitric oxide; 13. Ogundipe DJ, Akomolafe RO, Sanusi AA, Imafidon CE, Olukiran OS, Oladele SOD: Superoxide dismutase AA. Effects of two weeks administration of Ocimum gratissimum leaf on feeding pattern and markers of renal function in rats treated with gentamicin. Egypt J Bas Applied Sci. 2016;3:219–31. Acknowledgements 14. Fiske CH, Subbarow Y. The colorimetric determination of phosphorus. J Bio The authors are thankful to CES College of Pharmacy, Kurnool, and Assam Chem. 1925;66:375–400. Down Town University, Guwahati and to all the friends, lab colleagues, and 15. Jung K, An JM, Eom D, Kang KS, Kim S. Preventive effect of fermented black the fellows who directly or indirectly helped us during this experiment. No ginseng against cisplatin-induced nephrotoxicity in rats. J Gins Res. 2017; financial support was received from any sources for this work. 41(2):188–94. 16. Farooq N, Yusufi ANK, Mahmood R. Effect of fasting on enzymes of carbohydrate metabolism and brush border membrane in rat intestine. Nut Availability of data and materials Res. 2004;24:407–16. All the generated and analyzed data are included in this published article. 17. Asokkumar K, Sen S, Umamaheswari M, Sivashanmugam AT, Subhadradevi V. Synergistic effect of the combination of gallic acid and famotidine in protection of rat gastric mucosa. Pharmacol Rep. 2014;66:594–9. Authors’ contributions 18. Hassan I, Chibber S, Naseem I. Ameliorative effect of riboflavin on the SS and RC are involved in designing the experiment and the collection of cisplatin induced nephrotoxicity and hepatotoxicity under the sample. SS, RC, and PK performed the experimental work and statistical photoillumination. Food Chem Toxicol. 2010;48:2052–8. analysis. SS, RC, and PK managed the literature and wrote the first and final 19. Jain A, Singhai AK. Nephroprotective activity of Momordica dioica roxb.in draft. All the authors read and approved the final draft. cisplatin-induced nephrotoxicity. Nat Prod Res. 2010;24(9):846–54. 20. Sheena N, Ajith TA, Janardhanan KK. Prevention of nephrotoxicity induced Ethics approval and consent to participate by the anticancer drug cisplatin, using Ganoderma lucidum, a medicinal Not applicable as no study on human was performed. mushroom occurring in South India. Curr Sci. 2003;85:478–82. 21. Das M, Sarma BP, Ahmed G, Nirmala CB, Choudhury MK. In-vitro anti oxidant activity and total phenolic content of Dillenia indica and Garcinia Competing interests penducalata, commonly used fruits in Assamese cuisine. Free Radic The authors declare that they have no competing interests. Antioxid. 2012;2:30–6. 22. Singh DR, Singh S, Salim KM, Srivastava RC. Estimation of phytochemicals and antioxidant activity of underutilized fruits of Andaman Islands (India). Publisher’sNote Int J Food Sci Nutr. 2012;63(4):446–52. Springer Nature remains neutral with regard to jurisdictional claims in 23. Renugadevi J, Prabu SM. Naringenin protects against cadmium-induced published maps and institutional affiliations. oxidative renal dysfunction in rats. Toxicology. 2009;256:128–34. Sen et al. Nutrire (2018) 43:15 Page 9 of 9 24. Maiuolo J, Oppedisano F, Gratteri S, Muscoli C, Mollace V. Regulation of uric acid metabolism and excretion. Int J Cardiol. 2016;213:8–14. 25. Gowda S, Desai PB, Kulkarni SS, Hull VV, Math AAK, Vernekar SN. Markers of renal function tests. North Ame J Med Sci. 2010;2(4):170–3. 26. Rajakrishnan R, Lekshmi R, Benil PB, Thomas J, AlFarhan AH, Rakesh V, Khalaf S. Phytochemical evaluation of roots of Plumbago zeylanica L. and assessment of its potential as a nephroprotective age. Saudi J Bio Sci. 2017;24(4):760–6. 27. Farooquia Z, Ahmeda F, Rizwana S, Shahida F, Khanb AA, Khana F. Protective effect of Nigella sativa oil on cisplatin induced nephrotoxicity and oxidative damage in rat kidney. Biomed & Pharmaco. 2017;85:7–15. 28. Shiny KS. Biochemical studies on the protective effect of taurine on experimentally induced myocardial infarction in rats, Thesis (PhD). Kerala: Cochin University; 2007. 29. Noori S, Mahboob T. Role of electrolytes disturbances and Na+-K+-ATPase in cisplatin-induced renal toxicity and effects of ethanolic extract of Cichorium intybus. Pak J Pharm Sci. 2012;25:857–62. 30. Kubala M, Geleticova J, Huliciak M, Zatloukalova M, Vacek J, Sebela M. Na+/K +-ATPase inhibition by cisplatin and consequences for cisplatin nephrotoxicity. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2014;158(2):194–200. 31. Deavall DG, Martin EA, Horner JM, Roberts R. Drug-induced oxidative stress and toxicity. J Toxicol. 2012; https://doi.org/10.1155/2012/645460. Available from: https://www.hindawi.com/journals/jt/2012/645460/cta/. [Accessed 21 May 2015]. 32. Sen S, Chakraborty R. The role of antioxidants in human health. In: Silvana A, Hepel M, editors. Oxidative stress: diagnostics, prevention and therapy. Washington D: American Chemical Society; 2011. p. 1–37. 33. Ognjanovic BI, Djordjevic NZ, Matic MM, Obradovic JM, Mladenovic JM, Stajn AS, Saicic ZS. Lipid peroxidative damage on cisplatin exposure and alterations in antioxidant defense system in rat kidneys: a possible protective effect of selenium. Int J Mol Sci. 2012;13:1790–803.

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