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Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:http://www.elsevier.com/copyright Author's personal copy Naringin alleviates cognitive impairment, mitochondrial dysfunction andoxidative stress induced by D -galactose in mice Anil Kumar * , Atish Prakash, Samrita Dogra Pharmacology Division, University Institute of Pharmaceutical Sciences, UGC Centre for Advance Studies, Panjab University, Chandigarh 160014, India a r t i c l e i n f o Article history: Received 12 August 2009Accepted 19 November 2009 Keywords: SenescenceMitochondriaAlzheimer’s diseaseOxidative stressNeuroprotection a b s t r a c t Role of mitochondrial dysfunction and oxidative stress has been well documented in aging and relateddisorders such as Alzheimer’s disease. Bioflavonoids have been reported to have a therapeutic potentialagainst several age related processes. Bioflavonoids are being used as a neuroprotectants in the treatmentof various neurological disorders including aging. Therefore, present study has been conducted in order toexplore the possible role of naringin against D -galactose induced cognitive dysfunction, oxidative damageand mitochondrial dysfunction in mice. Chronic administration of D -galactose (100 mg/kg) for 6 weekssignificantly impaired cognitive performance (both in Morris water maze and elevated plus maze), loco-motor activity, oxidative defense and mitochondrial complex (I, II and III) enzymes activities as comparedto sham group. Six weeks naringin (40 and 80 mg/kg) treatment significantly improved cognitive perfor-mance, oxidative defense and restored mitochondria complex enzyme activities as compared to control( D -galactose). Naringin treatment significantly attenuated acetylcholine esterase activity in D -galactosetreated mice. In conclusion, present study highlights the potential role of naringin against D -galactoseinduced cognitive impairment, biochemical and mitochondrial dysfunction in mice.Crown Copyright 2009 Published by Elsevier Ltd. All rights reserved. 1. Introduction Aging is a gradual slow process that alters physiological, cellularprocess with time and leads to several age related diseases such asAlzheimer’s disease (AD), Parkinson’s disease (PD) and Hunting-ton’s disease (HD). Cognitive deterioration is a well-known factwhich accelerates with age (Barnes, 1979; Rosenzweig and Barnes,2003). Several theories have been put forward to explain the pro-cess of aging (Miquel et al., 1980). Oxidative damage has been re-ported to play a key role in aging process (Floyd and Hensley,2002). In addition, mitochondrial free radical theory also explainsthe mechanistic basis of aging (Miquel et al., 1980). This theorypostulates that aging and related diseases are the consequence of free radical mediated damage to cellular macromolecules and theirinability to counterbalance endogenous anti-oxidant defensesmechanism ( Jang and Remmen, 2009).Further, evidence suggests that mitochondria are both produc-ers as well as targets of reactive oxygen species which increasesoxidative damage (Turrens, 2003). As a consequence, damagedmitochondria progressively become less efficient, losing theirfunctional integrity and release more reactive oxygen molecules(Reddy, 2007). Increasing oxidative burden deteriorates functionalmitochondria during aging. Mitochondria are the major source of energy or adenosine triphosphate (ATP) for the normal functioningof eukaryotic cells. Dysfunction of mitochondria is well known togenerate reactive oxygen species (ROS), reduce mitochondrialATP production, increased mitochondrial deoxyribonucleic acid(DNA) mutations, increase in abnormal mitochondrial criste struc-tures and impairs intracellular calcium level (Reddy and Beal,2005). Increased ROS generation with compromised mitochondrialfunction ultimately affects neurons and accelerates neurodegener-ative process (Zeevalk et al., 2005). D -Galactose is a physiological nutrient obtains from lactose inmilk. The hydrolysis of lactose in the intestine results monosac-charide glucose and galactose. In animals, galactose is normallymetabolized by D -galactokinase and galactose-1-phosphate uri-dyltransferase but over-supply of D -galactose results its abnormalmetabolism (Kaplan and Pesce, 1996). D -Galactose converts into 0278-6915/$ - see front matter Crown Copyright 2009 Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.fct.2009.11.043 Abbreviations: ATP, adenosine triphosphate; ROS, reactive oxygen species; DNA,deoxyribonucleic acid; AGE, advanced glycation end products; RAGE, receptor foradvanced glycation end products; IAL, initial acquisition latency; RL, retentionlatency; EGTA, ethylene glycol tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; BSA, bovine serum albumin; SDH, succinatedehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-H-tetrazoliumbromide; MDA, malondialdehyde; DTNB,5,5 0 -dithio-bis (2-nitrobenzoic acid); NBT,nitro blue tetrazolium; NOS, nitric oxide synthase; SOD, super oxide dismutase;TBARS, thiobarbituric acid reactive substances; 6-OHDA, 6-hydroxydopamine;AChE, acetyl cholinesterase. * Corresponding author. Tel.: +91 172 2534106; fax: +91 172 2541142. E-mail address:
[email protected] (A. Kumar).Food and Chemical Toxicology 48 (2010) 626–632 Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox Author's personal copy galactitol, which is not metabolize by above enzymes butaccumulate in the cell, that leads to osmotic stress and ROS pro-duction (Hsieh et al., 2009). D -Galactose is a reducing sugar thatreacts with free amines of amino acids in proteins and peptidesto from advanced glycation end products (AGE), which in turncauses activation of receptor for advanced glycation end products.These sequences of events cause oxidative stress and cellulardamage (Song et al., 1999; Baynes, 2001). AGE increases withage and has been linked pathologically in many age relatedpathologies such as diabetes, arteriosclerosis, nephropathy andAlzheimer’s disease.Recently, several dietary supplements such as spinach, straw-berries or citrus fruits extracts have been suggested to producebeneficial effects in age related neurological deficits (Gemmaet al., 2002; Wang et al., 2005). Naringin (4 0 ,5,7-trihydroxyflava-none 7-rhamnoglucoside) is a well-known flavanone glycoside of grape fruits, e.g., Citrus paradise, Citrus sinensis, Citrus unshiu , and Artemisia selengensis (Swiader and Lamer-Zarawaska, 1996), rootsof Cudrania cochinchinensis and fruits of Pon cirus . Naringin hasbeen reported to possess potent anti-oxidant, superoxide scaveng-ing, anti-apoptotic, anti-atherogenic and metal chelating activity( Jung et al., 1983; Jeon et al., 2004; Jagetia and Reddy, 2005). Orallyadministered naringin is metabolized to naringenin (4 0 ,5,7-trihydr-oxyflavanone) (Fuhr and Kummert, 1995) which crosses the bloodbrain barrier (Zbarsky et al., 2005). Despite, many studies on thebeneficial effects of naringin, its therapeutic potential as a neuro-protectant against mitochondrial dysfunction and free radicalmediated toxicity have not been well understood.Therefore, present study has been designed to explore the pos-sible role of naringin against D -galactose induced cognitive dys-function, oxidative damage and mitochondrial dysfunction in mice. 2. Materials and methods 2.1. Drugs and treatment schedule D -Galactose (CDH, India) solution and naringin suspensions were made fresheach day during the experiment. D -Galactose was dissolved in distilled water forsubcutaneous (s.c.) administration. Naringin was suspended in 0.25% w/v sodiumcarboxy-methyl-cellulose and administered orally in a dose of 1 ml/100 g bodyweight. Animals were randomized into five groups, consists of 12 animals in each. Group I: naïve group received 0.25% w/v sodium carboxy-methyl-cellulose. Group II: received D -galactose (100 mg/kg) subcutaneously. Group III: naringin (40 mg/kg, p.o.) + D -galactose (100 mg/kg). Group IV: naringin (80 mg/kg, p.o.) + D -galactose (100 mg/kg). Group V: naringin (80 mg/kg, p.o.) per se .The study was carried out for a period of 42 days (6 weeks). 2.2. Animals Male Laca mice (25–30 g), 2–3 months old (Central Animal House, Panjab Uni-versity, Chandigarh) were used. Animals were acclimatized to the laboratory condi-tions at room temperature prior to the experiment. Animals were kept understandard condition of 12 h light/dark cycle with food and water ad libitum in plasticcages with soft bedding. All the behavioral observations were started in the morn-ing between 9.00 and 10.00 AM. Animals were scarified immediately after behav-ioral experiment between 15.00 and 16.00 h. Sixty animals were used in thepresent study. The protocol was approved by the Institutional Animal Ethics Com-mittee and carried out in accordance with the Indian National Science AcademyGuidelines for the use and care of animals. 2.3. Behavioral assessments 2.3.1. Assessment of cognitive performance 2.3.1.1. Morris water maze task. The acquisition and retention of memory was eval-uated by using Morris water maze (Kumar and Dogra, 2009). Morris water mazeconsisted of large circular pool (150 cm in diameter, 45 cm in height, filled to adepth of 30 cm with water at 28 ± 1 C). Pool was divided into four equal quadrantswith help of two threads, fixed at right angle to each other. The pool was placed inilluminated light room among several colored clues. These external clues are re-mained unchanged through out the experimental period and used as referencememory. A circular platform (4.5 cm diameter) was placed in one quadrant of thepool, 1 cm above the water level during the acquisition phase. A similar platformwas placed 1 cm below the water level for retention phase. The position of the plat-form was not changed in any quadrant during assessment of both the phases. Eachanimal was subjected to four consecutive trials with gap of 5 min. The mouse wasgently placed in the water pool between quadrants, facing the wall of pool and al-lowed 120 s to locate the platform. Then, it was allowed to stay on the platform fornext 20 s. If animal failed to reach the platform within 120 s, same was guided toreach the platform and allowed to stay there for next 20 s. 2.3.1.1.1. Maze acquisition phase (training) . Animals received a training session,consist of four trials on day 20. Starting position was different in all the four trials.The time taken by the mouse to reach the visual platform was taken as the initialacquisition latency (IAL). At the end of each trial, mice were returned to theirrespective home cages. 2.3.1.1.2. Maze retention phase(testing for retention of the learned task) . Following24 h (day 21) and 21 days (day 42) after IAL, mouse was released randomly atone of the edges facing the wall of the pool to assess for memory retention. Time taken bymice to find the hidden platform on day 21 and 42 following start of D -galactoseadministration was recorded, termed as first retention latency (first RL) and secondretention latency (second RL), respectively. 2.3.1.2. Elevated plus maze paradigm. The elevated plus maze consists of two oppo-site white open arms (16 5 cm), crossed with two closed walls (16 5 cm) with12 cm high walls. The arms were connected with a central square of dimensions5 5 cm. The entire maze was placed 25 cm high above the ground. Acquisitionof memory was tested on day 20. A mouse was placed individually at one end of the open arm facing away from the central square. The time taken by the animalto move from the open arm to the closed arm was recorded as the initial transferlatency (ITL). Animal was allowed to explore the maze for next 10 s after recordingITL. If animal did not enter the closed arm within 90 s, same was guided to theclosed arm and ITL was recorded as 90 s. Similarly, retention of memory was as-sessed by placing mouse in an open arm on day 21 and day 42 of the ITL, termedas the first retention transfer latency (first RTL) and second retention transfer la-tency (second RTL), respectively (Sharma and Kulkarni, 1992). 2.3.2. Assessment of gross behavioral activity Gross behavioral activity was observed at weekly interval. Each animal wasplaced in a square (30 cm) closed arena equipped with infra-red light sensitive pho-tocells using digital actophotometer. The animal was observed for a period of 5 minand expressed as counts/5 min. The apparatus was placed in a darkened, light andsound attenuated and ventilated test room (Reddy and Kulkarni, 1998). 2.4. Mitochondrial complex estimation 2.4.1. Isolation of mice brain mitochondria The whole brain (excluding cerebellum) was used for mitochondrial isolation.Mice brain mitochondria were isolated by differential centrifugation (Berman andHastings, 1997). The mice brain was homogenized in 10 ml of homogenizing buffercontaining 225 mM mannitol, 75 mM sucrose, 5 mM HEPES, 1 mM EGTA, 1 mg/mlBSA, pH 7.4. The homogenate was brought to 30 ml with the same buffer and cen-trifuged at 2000 g for 3 min at 4 C. The pellet was discarded and the supernatantwas divided into two tubes and centrifuged at 12,000 g for 10 min. The pellet con-taining the mixture of synaptosomes and mitochondria was suspended in 10 mlof homogenization buffer containing 0.02% digitonin to lyse the synaptosomes fol-lowed by centrifugation at 12,000 g for 10 min to pellet down both extra synaptoso-mal and intra synaptosomal mitochondria. The mitochondrial pellet is washedtwice in the same buffer without EGTA, BSA and digitonin. 2.4.1.1. Complex-I (NADHdehydrogenase activity). Complex-I was measured spectro-photometrically by the method of King and Howard (1967). The method involvescatalytic oxidation of NADH to NAD+ with subsequent reduction of cytochrome c .The reaction mixture contained 0.2 M glycyl glycine buffer pH 8.5, 6 mM NADHin 2 mM glycyl glycine buffer and 10.5 mM cytochrome c . The reaction was initiatedby addition of requisite amount of solubilized mitochondrial sample and followedabsorbance change at 550 nm for 2 min. 2.4.1.2. Complex-II (succinate dehydrogenase (SDH) activity). SDH was measuredspectrophotometrically according to King (1967). The method involves oxidationof succinate by an artificial electron acceptor, potassium ferricyanide. The reactionmixture contained 0.2 M phosphate buffer pH 7.8, 1% BSA, 0.6 M succinic acid, and0.03 M potassium ferricyanide. The reaction was initiated by the addition of mito-chondrial sample and absorbance change was followed at 420 nm for 2 min. 2.4.1.3. Complex-III (MTT ability). The MTT assay was based on the reduction of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-H-tetrazolium bromide (MTT) by hydroge-nase activity in functionally intact mitochondria. The MTT reduction rate was usedto assess the activity of the mitochondrial respiratory chain in isolated mitochon- A. Kumar et al./Food and Chemical Toxicology 48 (2010) 626–632 627 Author's personal copy dria by the method of Liu et al. (1997). Briefly, 100 l l mitochondrial samples wereincubated with 10 l l MTT for 3 h at 37 C. The blue formazan crystals were solubi-lized with dimethylsulfoxide and measured by an ELISA reader at 580 nm filter. 2.5. Biochemical assessments Biochemical tests were conducted 24 h after last behavioral test. The animalswere sacrificed by decapitation. Brains were removed and rinsed with ice-cold iso-tonic saline. Brains were then homogenized with ice-cold 0.1 mmol/l phosphatebuffer (pH 7.4). The homogenates (10% w/v) were then centrifuged at 10,000 g for15 min and the supernatant so formed was used for the biochemical estimations. 2.5.1. Measurement of lipid peroxidation The extent of lipid peroxidation in the brain was determined quantitatively byperforming the method as described by Wills (1966). The amount of malondialde-hyde (MDA) was measured by reaction with thiobarbituric acid at 532 nm usingPerkin Elmer Lambda 20 spectrophotometer. The values were calculated usingthe molar extinction co-efficient of chromophore (1.56 105 (mol/l) 1 cm 1 ). 2.5.2. Estimation of nitrite The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide was determined by a colorimetric assay with Greiss reagent (0.1% N-(1-napththyl) ethylene diamine dihydrochloride, 1% sulphanilamide and 5% phospho-ric acid) (Green et al., 1982). Equal volumes of the supernatant and Greiss reagentwere mixed and incubated for 10 min at room temperature in the dark. The absor-bance was measured at 540 nm using Perkin Elmer Lambda 20 spectrophotometer.The concentration of nitrite in the supernatant was determined from sodium nitritestandard curve. 2.5.3. Estimation of reduced glutathione Reduced glutathione was estimated according to the method as described byEllman (1959). A 1 ml supernatant was precipitated with 1 ml of 4% sulphosalicylicacid and cold digested for 1 h at 4 C. The samples were then centrifuged at 1200 g for 15 min at 4 C. To 1 ml of the supernatant obtained, 2.7 ml of phosphate buffer(0.1 mmol/l, pH 8) and 0.2 ml of 5,5 0 -dithio-bis (2-nitrobenzoic acid) (DTNB) wasadded. The yellow color developed was measured at 412 nm using Perkin ElmerLambda 20 spectrophotometer. Results were calculated using molar extinctionco-efficient of the chromophore (1.36 10 4 (mol/l) 1 cm 1 ). 2.5.4. Superoxide dismutase activity Superoxide dismutase (SOD) activity was assayed by the method of Kono(1978). The assay system consists of EDTA 0.1 mM, sodium carbonate 50 and96 mM of nitro blue tetrazolium (NBT). In the cuvette, 2 ml of the above mixture,0.05 ml of hydroxylamine and 0.05 ml of the supernatant was added and auto-oxi-dation of hydroxylamine was measured for 2 min at 30 s interval by measuringabsorbance at 560 nm using Perkin Elmer Lambda 20 spectrophotometer. 2.5.5. Catalase activity Catalase activity was assessed by the method of Luck (1971), wherein thebreakdown of H 2 O 2 is measured. Briefly, assay mixture consists of 3 ml of H 2 O 2 phosphate buffer and 0.05 ml of the supernatant of the tissue homogenate. Thechange in absorbance was recorded for 2 min at 30 s interval at 240 nm using Per-kin Elmer Lambda 20 spectrophotometer. The results were expressed as micro-moles of hydrogen peroxide decomposed/min/mg of protein. 2.5.6. Glutathione-S-transferase activity The activity of glutathione-S-transferase was assayed by the method of Habigand Jakoby (1981). Briefly, the assay mixture consisted of 2.7 ml of phosphate buf-fer, 0.1 ml of reduced glutathione, 0.1 ml of 1-chloro-2,4-dinitrobenzene (CDNB) assubstrate and 0.1 ml of supernatant. The increase in the absorbance was recorded at340 nm for 5 min at 1 min interval using Perkin Elmer Lambda 20 spectrophotom-eter. The results were expressed as nmoles of CDNB conjugated/min/mg of protein. 2.5.7. Estimation of acetyl cholinesterase (AChE) activity AChE is a marker of loss of cholinergic neurons in the forebrain. The AChEactivity was assessed by Ellman method (Ellman et al., 1961). The assay mixturecontained 0.05 ml of supernatant, 3 ml of sodium phosphate buffer (pH 8), 0.1 mlof acetylthiocholine iodide and 0.1 ml of DTNB (Ellman reagent). The change inabsorbance was measured for 2 min at 30 s interval at 412 nm using Perkin ElmerLambda 20 spectrophotometer. Results were expressed as micromoles of acetylthi-ocholine iodide hydrolyzed/min/mg of protein. 2.5.8. Protein estimation The protein content was estimated by Biuret method (Gornall et al., 1949) usingbovine serum albumin as a standard. 2.6. Statistical analysis Values are expressed as mean ± SEM. The behavioral assessment data wasanalyzed by a repeated measures two-way analysis of variance (ANOVA) withdrug-treated groups as between and sessions as the within-subjects factors. Thebiochemical estimations were analyzed by one-way ANOVA. Post hoc comparisonsbetween groups were made using Tukey’s test. P < 0.05 was considered significant. 3. Results 3.1. Effect of naringin on memory performance in Morris water mazetask in D -galactose treated mice In the Morris water maze task, naïve and naringin (80 mg/kg) per se group of animals quickly learned to swim directly to theplatform in the Morris water maze on day 20. D -Galactose treatedmice showed an initial increase in escape latency, which declinedwith continuing training during acquisition trial on day 20. Therewas a significant difference in the mean IAL of D -galactose treatedgroup as compared to naïve group on day 20 indicating thatchronic administration of D -galactose impaired acquisition of memory ( P < 0.05). In contrast, concomitant administration of naringin (40 and 80 mg/kg) with D -galactose significantly attenu-ated D -galactose induced memory impairment (IAL to reach theplatform) as compared to D -galactose treated mice on day 20(Table 1).Following training, the mean retention latencies (first and sec-ond RL) to escape onto the hidden platform was significantly de-creased in naïve group on days 21 and 42, respectively, ascompared to IAL on day 20. On the contrary, the performance inthe D -galactose treated mice had changed after initial trainingin the water maze on days 21 and 42, with significant increase inmean retention latencies as compared to IAL on day 20. The resultssuggest that D -galactose caused significant cognitive impairment.However, there was a significant decrease in first and second RL on days 21 and 42, respectively, when naringin (40 and 80 mg/kg) were co-administered chronically to D -galactose treated miceas compared to D -galactose group (Table 1). 3.2. Effect of naringin on memory performance in elevated plus maze paradigm in D -galactose treated mice In the elevated plus maze task, mean ITL on day 20 for eachmouse was relatively stable and showed no significant variation.All the mice entered the closed arm within 60 s. Following training,naïve and or naringin (80 mg/kg)-treated mice entered closed armquickly and mean retention transfer latencies (first and secondRTL) to enter closed arm on days 21 and 42 were shorter as Table 1 Effect of naringin (NAR; 40 and 80 mg/kg, p.o.) on memory performance in Morriswater maze in D -galactose treated mice. Treatment (mg/kg) Day 20 (IAL) Day 21 (1st RL) Day 42 (2nd RL)Naïve 48.1 ± 2.9 17.3 ± 3.2 12.3 ± 1.4 D -Gal (100) 49.8 ± 3.0 58.0 ± 2.2 a 55.1 ± 1.7 a NAR (80) 48.5 ± 2.4 14.0 ± 2.7 11.6 ± 2.4NAR (40) + D -gal (100) 47.3 ± 2.6 41.5 ± 2.1 b,NS 38.0 ± 2.2 b,NS NAR (80) + D -gal (100) 47.1 ± 2.6 26.6 ± 2.4 b,c,NS 23.8 ± 1.9 b,c,NS The initial acquisition latencies (IAL) on day 20 and retention latencies on days 21(first RL) and 42 (second RL) following D -gal concurrent treatment were observed.Values are mean ± SEM. NS P < 0.05 as compared to na group (repeated measures two-way ANOVA followedby Tukey’s test for multiple comparisons) [ D -gal, D -galactose; NAR, naringin; NS, notsignificant]. a P < 0.05 as compared to na group. b P < 0.05 as compared to D -gal treated group. c P < 0.05 as compared to NAR (40) + D -gal group.628 A. Kumar et al./Food and Chemical Toxicology 48 (2010) 626–632 Author's personal copy compared to ITL on day 20 of each group, respectively. In contrast, D -galactose treated (control) mice performed poorly throughoutthe experiment as compared to naïve mice and did not show anysignificant change in the mean retention transfer latencies on days21 and 42 as compared to pre-training latency on day 20, demon-strating that chronic D -galactose administration induced markedmemory impairment. Chronic administration of naringin (40 and80 mg/kg) following D -galactose administration significantly de-creased the mean retention latencies on days 21 and 42 ( P < 0.05vs D -galactose treated group) (Table 2). The mean transfer latenciesof naringin (40 and 80 mg/kg) with D -galactose treated groupswere significantly different as compared to D -galactose (control)treated mice on days 21 and 42 ( P < 0.05) (Table 2). 3.3. Effect of naringin on locomotor activity in D -galactose treated mice Chronic naringin (40 and 80 mg/kg) per se treatment for6 weeks did not produce any significant effect on the locomotoractivity as compared to naïve mice. Further, naringin (40 and80 mg/kg) treatment did not cause any significant alteration inthe locomotor activity as compared to control ( D -galactose) (Fig. 1). 3.4. Effect of naringin on brain lipid peroxidation and nitrite in D - galactose treated mice Chronic D -galactose treatment for 6 weeks significantly raisedbrain MDA and nitrite concentration as compared to naïve mice( P < 0.05). Chronic naringin (40 and 80 mg/kg) treatment signifi-cantly attenuated the increase in MDA and nitrite concentrationas compared to control ( D -galactose treated) group. However,naringin (80 mg/kg) per se treatment did not produce any signifi-cant effect as compared to naïve mice (Table 3). 3.5. Effect of naringin on reduced glutathione, superoxide dismutase, glutathione-S-transferase activity and catalase levels in D -galactosetreated mice Chronic D -galactose treatment for 6 weeks significantly de-creased reduced glutathione, superoxide dismutase level, glutathi-one-S-transferase and catalase activity in brain as compared tonaïve mice ( P < 0.05). However chronic naringin (40 and 80 mg/kg) treatment significantly restored reduced glutathione, superox-ide dismutase levels, glutathione-S-transferase and catalase activ-ity as compared to control ( D -gal treated mice). Further, naringin(80 mg/kg) per se treatment did not produce any significant effecton these parameters as compared to naïve mice (Table 3). 3.6. Effect of naringin on acetylcholinesterase activity in D -galactosetreated mice Chronic D -galactose treatment for 6 weeks significant increasedacetyl cholinesterase activity in brain on as compared to naïvemice ( P < 0.05). However, chronic naringin (40 and 80 mg/kg)treatment significantly decreased acetyl cholinesterase activity ascompared to control ( D -galactose treated mice). Further, naringin(80 mg/kg) per se treatment did not produce any significant effecton acetyl cholinesterase activity as compared to naïve mice (Fig. 2). 3.7. Effect of naringin on NADH dehydrogenase activity, succinatedehydrogenase activity and MTT ability in D -galactose treated mice Chronic administration of D -galactose for 6 weeks caused signif-icant alterations in mitochondrial enzyme complex activity. D -gal-actose treatment significantly decreased NADH dehydrogenase,succinate dehydrogenase and MTT ability as compared to naïvemice ( P < 0.05). However, naringin (40 and 80 mg/kg) treatmentfor 6 weeks significantly restored these mitochondrial enzymecomplex activity which was significant as compared to control( D -galactose treated) (Fig. 3). Further, naringin (40 and 80 mg/kg) per se treatment did not produce significant effect on altered NADHdehydrogenase, succinate dehydrogenase and MTT abilities ascompared to naïve mice. 4. Discussion Cognitive dysfunction is a very common problem in aged peo-ple. However, several theories have been proposed to explain thisage related problem. Oxidative stress theory gained more impor-tance from last few decades. A recent study also demonstrated areduced mitochondrialfunctional activity and increased mitochon-drial DNA damage with more reactive oxygen generation duringaging (Figueiredo et al., 2008). D -Galactose induced toxicity is anexperimental model for studying aging and to develop suitabledrug strategy against such problem. Study showed that D -galactoseinduces behavioral impairment in C57 mice (Wei et al., 2005).Although, the exact mechanism underlying D -galactose-inducedaging has not been understood so far. Existing data indicate thatoxidative stress could be one of the possible reason (Li et al.,2007). It has also been hypothesized that accumulated D -galactosemay react with proteins and peptides to form advanced glycationend products (AGEs) in vivo and accelerate aging process. Besides,abnormal accumulation of galactitol from excess D -galactose by al-dose reductase in cell, leading to osmotic stress and generatingROS (Song et al., 1999).In the present study, chronic D -galactose treatment results animpairment of cognitive tasks both in morris water maze and Table 2 Effect of naringin (NAR; 40 and 80 mg/kg, p.o.) on memory performance in elevated plus maze paradigm in D -galactose treated mice. Treatment (mg/kg) Day 20 (ITL) Day 21 (first RTL) Day 42 (second RTL)Na 77.5 ± 2.4 15.0 ± 2.7 10.0 ± 2.5 D -Gal (100) 77.6 ± 2.3 87.8 ± 2.6 a 84.3 ± 2.3 a NAR (80) 78.0 ± 1.6 13.6 ± 2.5 11.0 ± 2.3NAR (40) + D -gal (100) 78.3 ± 1.9 50.0 ± 1.8 b,NS 48.6 ± 2.5 b,NS NAR (80) + D -gal (100) 77.5 ± 2.4 32.1 ± 1.4 b,c,NS 31.5 ± 2.2 b,c,NS The initial acquisition latencies (IAL) on day 20 and retention latencies on days 21 (first RL) and 42 (second RL) following D -gal concurrent treatment were observed. Valuesare mean ± SEM. NS P < 0.05 as compared to na group (repeated measures two-way ANOVA followed by Tukey’s test for multiple comparisons) [ D -gal, D -galactose; NAR, naringin; NS, notsignificant]. a P < 0.05 as compared to na group. b P < 0.05 as compared to D -gal treated group. c P < 0.05 as compared to NAR (40) + D -gal group. A. Kumar et al./Food and Chemical Toxicology 48 (2010) 626–632 629
