NeuroToxicology 45 (2014) 111–120

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NeuroToxicology

Protective effect of apple (Ralls) polyphenol extract against aluminum-induced cognitive impairment and oxidative damage in rat Dai Cheng a,b, Yu Xi a, Jiankang Cao a, Dongdong Cao c, Yuxia Ma d, Weibo Jiang a,* a

College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, People’s Republic of China Key Laboratory of Food Safety and Sanitation, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin, People’s Republic of China c Beijing Center for Diseases Prevention and Control, Beijing, People’s Republic of China d School of Public Health, Hebei Medical University, Shijiazhuang, People’s Republic of China b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 26 August 2014 Accepted 7 October 2014 Available online 17 October 2014

Aluminum (Al) has long been implicated in the pathogenesis of Alzheimer’s disease (AD). Dietary polyphenols have been strongly associated with reduced risk of AD and the other nervous diseases. We aimed to evaluate the preventive effect of the apple polyphenol extract (APE) on Al-induced biotoxicity, in order to provide a new focus on the design of strategies to prevent AD and the other human diseases related to Al overload. Control, Al-treated (171.8 mg Al kg1 day1 10 weeks), APE + Al (Al-treatment as previously plus 200 mg kg1 day1 10 weeks), and group of APE per se were used. Al intake caused memory impairment, significant decrease of acetylcholinesterase, CK, SOD, CAT activity and the rate of ATP synthesis, increase the Al content, the level of malondialdehyde and b-amyloid42. Administration of APE significantly improved memory retention, attenuated oxidative damage, acetylcholinesterase activity and Al level in Al treated rats. Furthermore, chlorogenic acid (ChA) was used for analyzing stability of polyphenols-Al3+ complex. Log K1 was 10.51, and the mole ratio of Al3+ to ligand was 1:1. We further found that the amounts of Al increased significantly in feces of the rats gavaged with AlCl3 plus ChA compared with AlCl3. Our finding has shown APE has neuroprotective effects against Al-induced biotoxicity. Chelating with Al and disturbing its absorption could account for the neuroprotective roles of dietary polyphenols against Al toxicity. ß 2014 Published by Elsevier Inc.

Keywords: Aluminum Apple polyphenols Neuroprotective Antioxidant Chelation

1. Introduction Aluminum (Al) is one of the most abundant elements in the earth crust and has been well known as a neurotoxicant (ElRahman, 2003). Al has been reported to alter the blood–brain barrier (BBB); as a result of which it gains an easy access to the central nervous system (CNS) under normal physiological conditions and accumulates in brain (Exley, 2001; Zatta et al., 2002). The administration of Al compounds can cause formation of intraneuronal neurofibrillary tangles (NFTs) (Wakayama et al., 1993), and consequently lead to neuronal loss in brain of model animals (Gupta et al., 2005). Study on the association, in elderly subjects, between exposure to Al in drinking water and Alzheimer’s disease (AD) showed that cognitive decline was greater in subjects with a higher daily Al intake, confirming that

* Corresponding author. Present address: Qinghua Donglu, No. 17, Beijing 100083, PR China. Tel.: +86 01062736565; fax: +86 01062736565. E-mail address: [email protected] (W. Jiang). http://dx.doi.org/10.1016/j.neuro.2014.10.006 0161-813X/ß 2014 Published by Elsevier Inc.

high consumption of Al may be a risk factor for AD (Walton, 2013). The full scale of mechanisms underlying Al neurotoxicity remains to be elucidated, and they are likely to involve multiple pathways. For instance, Al is a non-redox active metal which is capable of increasing the cellular oxidative milieu by potentiating the prooxidant properties of transition metals such as iron and copper (Bjertness et al., 1996). This may indirectly indicate its role in causing oxidative damage. Alternatively, considerable evidence has been provided for an interaction of Al with ATP synthesize via oxidative phosphorylation. It has been recently demonstrated that treatment of astrocytic cells with Al leads to dysfunctional mitochondria and a loss of energy synthesis (Lemire et al., 2009, 2011). This disruption in the ability of astrocytes subjected to Al to produce energy may limit the participation of the glial cells in their cognate neurophysiological tasks (Kumar et al., 2011). Moreover, it has been reported that Al was able to inhibit plasmin degradation of the Ab peptide (Korchazhkina et al., 2002). This may lead to the consideration that Al exposure altered conformation of Ab and enhanced its aggregation on the surface of cultured neurons the (Kawahara et al., 2001). Thus, cognitive deficits after

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Al exposure may be brought about by multiple actions of Al on nervous system. Growing evidence from in vitro, in vivo studies and clinical trials has shown that dietary polyphenols have been strongly associated with reduced risk of AD and the other nervous diseases (Scarmeas et al., 2006; Dai et al., 2006; Ebrahimi and Schluesener, 2012; Gao et al., 2012). The mechanisms involved in neuroprotection of polyphenols are mainly considered to act as free radical scavengers due to the antioxidant properties. Plant phenolics are being tried as chemoprotective agents in epidemiological and experimental studies to regulate the progression of Al-induced toxicity in brain (Kumar et al., 2009; Cheng et al., 2012; Margarity et al., 2013). According to Sanoner et al. (1999), apples have a unique polyphenol profile consisting of high levels of procyanidins, catechins, flavonols (such as quercetin) and chlorogenic acid. Experimental studies have shown that apple juice has the potential to improve cognitive performance, oxidative damage and synaptic signaling (Essa et al., 2012). Cell culture studies have shown that apple juice has the potential to reduce the levels of Ab and presenilin-1, and improve synaptic activity and acetylcholine level. The antioxidant activity of the phytonutrients in apple juice may be responsible for the observed neuroprotective effects (Remington et al., 2010). Moreover, mice supplemented with apple jucice concentrate prevented the overexpression of presenilin-1 (Chan and Shea, 2006). Apple polyphenol extract (APE) has been reported to have strong antioxidant properties and has been shown to exhibit wide variety of biological and pharmacological activities namely antioxidant, anti-inflammatory (D’Argenio et al., 2008), antiallergic (Zuercher et al., 2010) and anticarcinogenic activities (Daiki et al., 2007). Whether APE has a protective effect against brain related injury to aluminum exposure is unknown. Should this be the case, APE might represent a safe, risk-free, low-cost, natural way to prevent Al adverse effects in those people who are on long-term exposure to Al. Based on this background, present study was designed to investigate the neuroprotective effect of APE against aluminum-induced cognitive impairment and associated oxidative damage in rats. 2. Materials and methods 2.1. Preparation of apple polyphenol extract Fruits from the Ralls Genet (a cultivar of apple which originated in Virginia in the late 18th century, cv. Ralls) were harvested at the halfred stage of ripeness from orchards in Beijing, China. After harvest, the fruits were cleaned with tap water and carefully separated into peel and pulp using a stainless steel knife. Fresh apple pulp (100.0 g) was exhaustively extracted with 100 mL of 70% methanol (w/v) under ultrasound (SK8200H KuDos Ultrasonic Instrument Company, Shanghai, China) for 20 min in each extraction. The extraction process was repeated until the solvent became colorless. The solvent fractions of the extractions were filtered through Whatman No. 1 filter paper and concentrated in vacuo at 45 8C in a rotavapor (RE-52, YaRong Biochemistry Instrument Factory, Shanghai, China) until methanol was removed. The resulting concentrated solutions were extracted with 200 mL ethyl acetate and anhydrous ether (v:v = 1:1) until the solvent became colorless. The apple polyphenol extract (APE) was concentrated in vacuo at 30 8C in a rotavapor and dissolved with 10 mL distilled water, speed vacuumed to dryness, and stored at 80 8C until further use. The total phenolic content was determined by the method of Xue et al. (2009) and diluted to the concentration required before use. 2.2. HPLC analysis of phenolics As for phenolic acid analysis, high performance liquid chromatography equipped with DAD (diode array detector)

(LC-Prominence-20AT and SPD-M20A, Shimadzu Co., Japan) was employed. A analytical column C18 (Shim–pack VP-ODS 15 cm  4.6 mm ID, 5 mm, Shimadzu Co., Japan) was used and kept at 30 8C. A gradient event of mobile phase solvent A: water (acetic acid 1%, v/v) and B: methanol (acetic acid 1%, v/v) was as follows: 10–35% B (10 min), 35–42% B (15 min), 42–75% B (10 min), 75% B (5 min), 75–10% B (5 min), 10% B (5 min), and at a flow rate 1.0 mL/min. The injection volume was 10 mL. Identification of polyphenols was achieved by comparing retention times and UV spectra with those of standards. 2.3. Rat model for evaluating effect of APE against Al toxicity Seven-week-old male wistar rats, 160–180 g, clean grade. The experiments were performed according to Animal Management Rules of the Ministry of Health of the People’s Republic of China (documentation Number 55, 2001, Ministry of Health of PR China), with utilization permission from Animal Department of Academy of Military Medical Sciences, No. SCXK (Jun) 2007-004. All the rats were housed in a temperature-controlled room (25  2 8C) at relative humidity (60  5%) with a 12 h dark/light cycle and allowed free access to food and water for 7 days before the experiment and were randomly divided into four groups (six rats each group), then each as follows: Group 1 was served as untreated control; each of the rats received deionized water and normal chow diet. Group 2 was served as aluminum chloride control; each of the rats received deionized water and 171.8 mg Al kg1 day1 (1/5 LD50) in formulated diet (ElRahman, 2003). Group 3 received 200 mg APE kg1 day1 dissolved in deionized water and 171.8 mg Al kg1 day1 in formulated diet. Group 4 received 200 mg APE kg1 day1 dissolved in deionized water and normal chow diet. The doses of APE was selected based on the literature (Shoj et al., 2004) and our pre-experiments. The study was carried out for a period of 10 weeks. 2.4. Behavioral assessment 2.4.1. Step-down inhibitory avoidance task On the week 1, 3, 5, 7 and 9 following aluminum chloride treatment, We used the step-down inhibitory avoidance task since it has been widely used in the study of memory formation (Izquierdo and Medina, 1997; Prado-Alcala´ et al., 2003; Wyse et al., 2004). The experimental device is a 50 cm  30 cm  25 cm electronic avoidance-response chamber, made of Plexiglas on its three sides and hard black plastic on the other. The chamber has a bottom of parallel 0.1 cm stainless steel bars spaced 1 cm apart. A rubber platform (2.5 cm high, 7 cm in diameter of its top surface) was fixedly placed at a corner on the bottom of the chamber, providing rats a shelter from the electronic attack. Before normal test, rats were continually trained in a one-trial step-down inhibitory avoidance task for 4 times (one time/day, conducted between 14:00 and 17:00), and tested for their memory retention of the escape platform from electronic attack at the same time 24 h after training. Rats were placed on the platform, and their latency to step-down first, placing their four paws on the grids, was measured. In training sessions, immediately upon stepping down, the rats received a 0.3 mA, 2 s, scrambled foot shock. No foot shock was given in test sessions. Test session step-down latencies were taken as a measure of memory retention. 2.4.2. Assessment of cognitive performance 2.4.2.1. Spatial navigation task. The acquisition and retention of a spatial navigation task was evaluated by using Morris water maze (Frautschy et al., 2001). Animals were trained to swim to a visible platform in a circular pool (180 cm in diameter and 60 cm in

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height) located in a test room. In principle rats can escape from swimming by climbing onto the platform and over time the rats apparently learn the spatial location of the platform from any starting position at the circumference of the pool. The pool was filled with water (28  2 8C) to a height of 35 cm a movable circular platform (9 cm diameter), mounted on a column was placed in a pool 2 cm below the water level during the acquisition phase. The platform was removed in the pool for the maze retention phase. During acquisition phases the platform was placed in the center of one of the quadrants. Water was made cloudy by the addition of milk. Four equally spaced locations around the edge of the pool. 2.4.2.2. Maze acquisition phase (training). Animals received a training session consisting of four trials on day 35. The behavioral training procedure was modified from that of earlier studies (Morris et al., 1986). In all four trials, the starting position was different. A trial began by releasing the animal into the maze facing toward the wall of the pool. The latency to find the escape platform was recorded to a maximum of 90 s. If the rat did not escape onto the platform within this time it was guided to the platform and was allowed to remain there for 20 s. The time taken by rat to reach the platform was taken as the initial acquisition latency (IAL). At the end of the trial the rats were returned to their home cages and a 5 min gap was given between the subsequent trials. 2.4.2.3. Maze retention phase (testing for retention of the learned task). Following 24 h (day 36) and 34 days (day 70) after IAL, rat was released randomly at one of the edges facing the wall of the pool and tested for retention of response. In the test, the platform was removed, and the rat was allowed to swim freely in the pool for 90 s. The rats’ swimming time in the quadrant in which the platform had been placed in the training trials on day 36 and day 70 following start of aluminum chloride administration was recorded and termed as first retention latency (1st RL) and second retention latency (2nd RL), respectively was estimated 2.5. Biochemical assessment Biochemical tests were conducted 24 h after the last behavioral test. All rats were sacrificed by cervical decapitation. Brains were removed, rinsed with ice-cold isotonic saline, blotted with filter paper. One portion of brain was homogenized in ice-cold saline (1:10 w/v), and the homogenate was centrifuged (10,000  g at 4 8C for 30 min) and the supernatant so formed was used for the estimation of various biochemical estimations. The content of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD), catalase (CAT) and acetylcholinesterase (AChE) were estimated by reagent kits (NanJing JianCheng Bio Inst, Nanjing, China). Protein content was determined by the method of Lowery et al. (1951). 2.6. Creatine kinase (CK) activity assay CK activity was measured in brain homogenates pre-treated with 0.625 mM lauryl maltoside. The reaction mixture consisted of 60 mM Tris–HCl, pH 7.5, containing 7 mM phosphocreatine, 9 mM MgSO4 and approximately 0.4–1.2 mg protein in a final volume of 100 mL. After 15 min of pre-incubation at 37 8C, the reaction was started by the addition of 0.3 mmol of ADP plus 0.08 mmol of reduced glutathione. The reaction was stopped after 10 min by the addition of 1 mmol of p-hydroxymercuribenzoic acid. The creatine formed was estimated according to the colorimetric method of Hughes (1962). The color was developed by the addition of 100 mL 2% a-naphtol and 100 mL 0.05% diacetyl in a final volume of 1 mL

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and read spectrophotometrically after 20 min at 540 nm. Results were expressed as units/min mg protein. 2.7. Isolation of mitochondria Rat brain mitochondria were isolated by the method of Berman and Hastings (1999). The brain regions were homogenized in isolation buffer with EGTA (215 mM Mannitol, 75 mM sucrose, 0.1% BSA, 1 mM EGTA, pH-7.2). Homogenatewas centrifuged at 1300  g for 5 min at 4 8C. Pellet was resuspended in isolation buffer with EGTA and spun again at 13,000  g for 5 min. The resulting supernatant was transferred to new tubes and topped off with isolation buffer with EGTA and again spun at 13,000  g for 10 min. Pellet containing mitochondrial rich fraction was resuspended in isolation buffer without EGTA. 2.8. ATP synthesis ATP synthesis was measured using a glucose/hexokinase trap system as described by Griffiths et al. (1977). Phosphorylation was determined in terms of disappearance of inorganic phosphorus. The assay mixture (1.1 mL) contained 0.25 M sucrose, 10 mM Tris– HCl, 22 mM glucose, 5 mM KH2PO4, 2 mM MgCl2, 0.5 mM EDTA, 20 U of hexokinase (EC units, 1 mmol substrate/, min), 2 mM ADP (pH 7.4), 20 mM succinate and appropriate amount of mitochondrial protein. The reaction was terminated by addition of 10% trichloroacetic (TCA) acid after 20 min of incubation at 30 8C. The supernatant was assayed for inorganic phosphorus. 2.9. Mitochondrial ATPase (ATP hydrolysis) ATPase was assayed using the method of Griffiths and Houghton (1974). The method involves measurement of inorganic phosphorus liberated following catalytic hydrolysis of ATP to ADP. The reaction mixture (1.0 mL) containing 5 mM ATP, 2 mM MgCl2, 50 mM Tris–HCl (pH8.5) and appropriate amount of mitochondrial protein were incubated for 5 min at 30 8C. The reaction was terminated by the addition of 10% TCA and the supernatant assayed for inorganic phosphorus. 2.10. Inorganic phosphorus determination The phosphorus content was determined according to the method of Fiske and Subbarow, 1925. 2.11. Determination of Ab42 levels by Western blot Hemi-forebrains were homogenized in a buffer containing 8 M urea, 0.5% SDS, 2% b-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor cocktail. Brain homogenates of all group were respectively loaded in each lane of 10% SDS polyacrylamide gel (10% separating gel and 4%stacking gel) and transferred onto nitrocellulose filter membrane. Blots were probed with a polyclonal antibody directed against Ab42 (ab10148, 1:1500; Abcam) in 1 M Tris–HCl, pH 8.0, 5 M NaCl, 5% skim milk, and 0.1% Tween 20. Blots were visualized using anti-rabbit secondary antibody tagged with horse-radish peroxidase (1:10,000; Boisynthesis) and enhanced chemiluminescence (EarthOx) and quantification was done by determining density of the bands using the ImageJ software (http://rsb.info.nih.gov/ij/) (Rivest et al., 2009). 2.12. Histological study Brain samples were cut down and fixed in 10% formalin solution. The specimens embedded with paraffin were cut into 5 mm thick sections and stained with hematoxylin–eosin. The

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sections were examined by an experienced observer who was blind to the treatment under light microscope and then photomicrographs were taken. 2.13. Estimation of Al content in the brain Weighed brain tissue samples (0.1–0.3 g) were digested with nitric and perchloric acids as described by Puchades et al. (1989). Residues were taken into 1% v/v nitric acid and the aluminum concentrations determined by inductively coupled plasma optical emission spectrometry (ICP-OES) (OPTIMA7000, PerkinElmer Corporation, USA). 2.14. Potentiometric study of chlorogenic acid-Al (III) complexes According to the Bjerrum (1957) method, two titrations were carried out: (1) chlorogenic acid titration and (2) Al(III)/chlorogenic acid titration. A JinMai (Shanghai) ZD-2A automatic tirator was used to measure pH. To determine the protonation constants, the solutions of chlorogenic acid was titrated potentiometrically using 9.916  102 M NaOH, at constant ionic strength (0.1 M) and temperature (25 8C). Potentiometric titration curves 1 (Fig. 6A) of chlorogenic acid system were used to calculate the average values nA. The equation used for the calculation is nA ¼

  C1  V 1 ½OH  ½H 3 þ TL V 0  TL

(1)

where 3 is the number of protons which can be released by chlorogenic acid; V0 is the initial volume; C1 is the molarity of NaOH; V1 is the volumes of alkali added to reach the pH reading in both titrations; TL is the initial concentration of ligand; [OH] is the molarity of OH; [H] is the molarity of H+. The protonation constants were determined from the graph of nA vs pH, named the formation curve. The pH values at nA = 0.5, nA = 1.5 and nA = 2.5 designate the lg KH1, lg KH2 and lg KH3 respectively. To determine the stability constants of the complexes, the NaCl + chlorogenic acid + Al(III) mixture was titrated potentiometrically using 9.916  102 M NaOH at constant ionic strength (0.1 M) and temperature (25 8C) (curve 2 in Fig. 6A). nL and pL values were calculated using the nA, KH1, KH2 and KH3 values and the equation given below: nL ¼

CL ¼

T L  C L ð1 þ K H1 ½H þ K H1 K H2 ½H2 þ K H1 K H2 K H3 ½H3 TM nA  T L K H1 ½H þ 2K H1 K H2 ½H2 þ 3K H1 K H2 K H3 ½H3

pL ¼ lg

K H1 ½H þ 2K H1 K H2 ½H þ 3K H1 K H2 K H3 ½H3 nA  T L

(2)

(3)

(4)

where TL is the initial concentration of ligand; [OH] is the molarity of OH; [H] is the molarity of H+; The stability constants were determined from the nL vs pL curve, where the pL values at nL = 0.5 designate the log K1. All titrations were carried out at constant values of both temperature and ionic strength. 2.15. Experimental rat model for evaluating effect of chlorogenic acid on Al excretion The doses of chlorogenic acid (ChA) and AlCl3 were selected based on those reported in literature (El-Rahman, 2003; Shi et al., 2009). The rats were randomly divided into three groups (six rats each group): Group 1 was served as untreated control; each of the rats was gavaged with 1.5 mL deionized water. Group 2 was served as Al-control; each of the rats was gavaged with AlCl3 (about 1.5 mL), equal to 171.8 mg Al kg1 bw day1. Group 3, each of the rats was gavaged with AlCl3 as Group 2; 30 min later, each of the rats gavaged with ChA (about 1.5 mL), equal to 60 mg Al kg1 bw day1. The urine and feces were collected, respectively for each of the groups in the period of 0–24 h after the intragastric administration. All the samples of urine or feces were dried to dried to constant weight at 100 8C, then digested with nitric acid and submitted for measuring Al content by ICP-OES (see above). 2.16. Statistical analysis The results were expressed as the means  standard deviation (SD) of triplicate using SPSS 17.0 for windows. The Statistical significances of data were determined using one-way analysis of variance (ANOVA) followed by Dunnett’s contrast, P value < 0.05 was regarded as significant.

3. Results The main components in the apple polyphenol extract (APE) determined by HPLC were chlorogenic acid (3-O-Caffeoylquinic acid, 45.1%), epicatechin (32.8%), catechin (5.5%) and caffeic acid (3.5%). The APE was used to evaluate the effects of dietary polyphenols against Al-neurotoxicity with rats as an experimental model. Since Alzheimer’s disease is associated with declines in navigational skills, the Morris water maze (MWM) is commonly used to assess absolute navigation in rodents, by measuring the initial acquisition latency (IAL) and retention latency (RL) in MWM. Compared with control rats, the Al-exposed rats spent much more time to learn reaching the platform, namely the IAL was significantly prolonged. Meanwhile, the rat RL (a parameter for indicating the memory retention) was significantly shortened by the Al-exposure (Fig. 1). These neural impaired effects of aluminum could remarkably prevented by the administration with APE. As shown in Fig. 1, the IAL, 1st RL or 2nd RL of the Al

Fig. 1. Effect of APE on spatial navigation task in the Al-treated rats. The initial acquisition latencies (IAL) on day 35 and retention latencies on days 36 (1 st RL) and 70 (2nd RL) following Al-treatment were observed in Morris water maze. Note: Data were expressed as mean  SD (n = 6), *: only the Al treatment was compared against the control treatment (P < 0.05); #: only the APE-Al treatment was compared against the Al treatment (P < 0.05).

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Fig. 2. Effect of APE on step-down inhibitory avoidance task of rats testing on week 1, 3, 5, 7 and 9 following Al-treatment. The step-down latency time (in s) was quantified. Note: Data were expressed as mean  SD (n = 6), *: only the Al treatment was compared against the control treatment (P < 0.05); #: only the APE-Al treatment was compared against the Al treatment (P < 0.05).

exposed rats plus the APE-diet was 70.2%, 156.1% or 181.3% that of Control, respectively. In a step-down inhibitory avoidance task, the latency time gradually decreased in the Al exposed rats, and was 56.4%, 29.4%, or, 7.5% of that in control rat after 5, 7, or 9 weeks of the administration, respectively (Fig. 2). There was no significant difference in the latency between the APE-diet and control during the experiment. However, feeding the rat with APE significantly prevented declining in the latency induced by Al exposure.

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As shown in Fig. 3A, in brain tissue of the rats with Al diet, acetylcholinesterase (AChE) activity showed a significant decrease as compared to the control. However, APE treatment in Al-exposed significantly enhanced AChE activity in the brain. Creatine kinase (CK) catalyzing the reversible transfer of the Nphosphoryl group from phosphocreatine to ADP to regenerate ATP, is a major enzyme of higher eukaryotes that deal with high and fluctuating energy demands to maintain cellular energy homeostasis and to guarantee stable, locally buffered ATP/ADP ratios. In the present work, we evaluated CK activity to assess energy metabolism after treated with Al and APE (Fig. 3B). The activity of CK declined significantly in cerebral cortex of the rats exposed to Al. The administration of APE remarkably enhanced activities of CK in the Al-exposed rats. However, activities of CK were not affected remarkably by the administration with APE alone. We also studied the process of oxidative phosphorylation in terms of ATP synthesis and ATP hydrolysis in order to see how brain mitochondria react to APE and Al treated (Fig. 3C, D). We observed significant difference in ATP synthesis in the cerebral cortex of aluminum treated rats as compared to controls. The ATP synthesis in cerebral cortex of rats with Al diet was 61.5% lower than that in the control rats after 10-week of the feedings. As observed in the present study, the ATP hydrolysis rate increased significantly in cerebral cortex showing 128.9% of control. The administration of APE remarkably enhanced the rate of ATP synthesis and reduced the rate of ATP hydrolysis in the Al-exposed rats. However, the rate of ATP synthesis and hydrolysis was not affected remarkably by the administration with APE alone. This result might suggest that APE can reduce

Fig. 3. Effect of APE on the activities of AchE (A), CK (B), Al level (E), Ab42 levels (F) the rate of ATP synthesis (C) and ATP hydrolysis (D) in the brain of rats treated with AlCl3 for 10-week. Note: Data were expressed as mean  SD (n = 6), *: only the Al treatment was compared against the control treatment (P < 0.05); #: only the APE-Al treatment was compared against the Al treatment (P < 0.05).

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the toxicity of Al on the impairment of mitochondrial energy metabolism in brain. After 10-week feeding, significantly increased concentration of Al was noticed in the cerebral cortex of rats fed with Al diet comparing to the control, and this increasing of Al accumulation in the cerebral cortex was significantly prevented by the administration with APE (Fig. 3E) We determined Ab42 levels in forebrain from treated rats using a specific anti-Ab42 antibody (Fig. 3F). Ab42 levels were increased by 27.7% in the forebrain of Al-treated animals compared with controls. APE treatment in Al-exposed group significantly decreased the level of Ab42 in brain. These analyses of protein levels show that APE treatment is effective at preventing Ab42 accumulation in the brain induced by Al. The MDA level in brain of rats exposed to Al increased substantially to about 50.7% of that in control (Fig. 4A). The APE diet effectively prevented the increase of MDA level in brain of the rats. The activities of superoxide dismutase (SOD) and catalase (CAT) declined significantly in brain of the rats exposed to Al. The administration of APE remarkably enhanced activities of SOD and CAT in the Al-exposed rats (Fig. 4B, C). We observed that APE was no effect on the MDA level and the activities of SOD, CAT in brain by itself alone. Fig. 5 shows the control, aluminum intoxicated and effect of feeding with diet of the APE on the histology of brain tissue in aluminum intoxicated rat, respectively. Control group (Fig. 5A) and APE per se group (Fig. 5D) showed normal appearance. The cerebral cortex showed variable neurons varying in size and shape. The intercellular area is occupied by nerve fibers and neuroglial cells. The brain tissue from rats fed with Al diet exhibited severe histopathological changes, such as degenerative changes of nerve fibers with

Fig. 4. Effects of apple polyphenols on activities of MDA (A), SOD (B) and CAT (C) in brain of the Al-exposed rat. Note: Data were expressed as mean  SD (n = 6), *: only the Al treatment was compared against the control treatment (P < 0.05); #: only the APE-Al treatment was compared against the Al treatment (P < 0.05).

congestion of blood vessels, disruption of nucleus and vacuolization around the neuron (Fig. 5B). However, in the Al plus APE treated rat (Fig. 5C), the cerebral cortex specimens exhibited marked improvement with almost normal morphological appearance of mild degenerative changes of nerve fibers, reduced congestion in blood vessels and edema. Stability constant for complex of polyphenols and Al3+ is broadly extrapolated as the strength of polyphenols interaction with Al3+, therefore, which has been used as a parameter of capacity reducing aluminum toxicity. In our present study, ChA (typical phenol in APE) was used as a model chemical for analyzing stability of polyphenols-Al3+ complex. Every titration carried out is exhibited in Fig. 6A, which was further used to calculate the complex stability constant of ChA plus Al3+ according to IrvingRossotti method (1954). As shown in Fig. 6C, the pL values at nL = 0.5 designate log K1 of ChA was 10.51, and the mole ratio of Al3+ to ligand was kept at 1:1 for ChA to reach the maximum coordination of the ligand. We further found that the amounts of Al increased by 52.4% in feces of the rats gavaged with AlCl3 plus ChA compared with AlCl3 alone in 24 h after the intragastric administration (Fig. 7). This result suggests that the dietary phenolic compounds, such as ChA, indeed can reduce aluminum absorption. 4. Discussion Aluminum (Al) is a well-known neurotoxic agent, which has been involved in neuro-disorders such as Alzheimer’s disease (AD) and other serious neurodegenerative diseases (Esparza et al., 2011). Aluminum exacerbates brain oxidative damage (Nehru et al., 2007), causes inflammation and induces Ab deposition. The present study investigates the neuroprotective potential of APE (200 mg kg1 day1) dissolved in deionized water to adult rat that were exposed to Al intake (171.8 mg Al/kg/day) through their formulated diet for a 10-week period. In present study, chronic exposure of aluminum increased aluminum concentration in cerebral cortex as compared to the control animals. It has been observed that high aluminum level in brain is associated with decline in visual memory and attention concentration in hemodialysis patients (Bolla et al., 1992). Our findings showed that longterm Al intake results in progressive deterioration of spatial memory in both step-down inhibitory avoidance task and Morris water maze. Impaired performance of rabbits on step-down inhibitory avoidance task has been shown by Petit et al. (1980) after made an infusion of 5 mM aluminum into the lateral ventricles. Also, it has been shown that administration of AlCl3 (50 mg kg1 day1) in drinking water for 6 months causes learning deficits in Morris-water maze test in rats (Sethi et al., 2008). Now, the low aluminum level in cerebral cortex and the improved behavioral performance could be observed in the Al-exposed rat which was received drinking water with the apple polyphenol extract (APE) added. The results suggest the potential role of APE as a neuroprotectant against aluminum-induced neurotoxicity. Animal cells derive more than 90% of the required energy from oxidative phosphorylation associated with inner mitochondrial membrane. As energy is intimately linked to functional neurophysiology, diminished ATP production in astrocytes will have a major impact within the brain (Verderio and Matteoli, 2011). It has been demonstrated that oxidative metabolism is heavily reliant on Fe to execute the combustion of citric acid (Hentze et al., 2004). Appanna and Lemire (2011) stands to reason that if Fe homeostasis was to be affected under Al-insult, oxidative ATP production would be severely compromised. On the previous experimental results, the chronic aluminum exposure would result in the reduced rate of ATP synthesis and enhanced rate of ATP hydrolysis in rat brain (Dua and Gill, 2004; Gill et al., 2010). Meanwhile, creatine kinase

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Fig. 5. Photomicrograph of cerebral cortex from each experimental group. A, control group (deionized water and normal chow diet), showing intact neurons, without any spongiosis; B, aluminum group (171.8 mg Al/kg/day add in the formulated diet for 10 weeks), showing degenerative changes of nerve fibers with congestion of blood vessels, disruption of nucleus and vacuolization around the neuron; C, APE (200 mg kg1 day1 in the drinking water for 10 weeks) + aluminum group (171.8 mg Al/kg/day add in the formulated diet for 10 weeks), showing marked improvement with almost normal morphological appearance of mild degenerative changes of nerve fibers, reduced congestion in blood vessels and edema; D, APE (200 mg kg1 day1 in the drinking water for 10 weeks) group cerebral cortex is similar to control (magnification = 100). 1: congestion of blood vessels, 2: vacuolization around the neuron, 3: disruption of nucleus.

(CK) is a key enzyme in energy metabolism and CK reaction has a much higher maximal rate of ATP synthesis than oxidative phosphorylation (Walliman et al., 1992). It has been reported in literature that Alzheimer’s disease (AD) brain creatine kinase CK is modified such that the nucleotide binding site of CK is blocked and CK activity was 86% decreased in AD brain homogenates in comparison to age-matched controls (Haley et al., 1998). However, long-term Al intake decreases CK activity in rat brain has not been reported. Diminished ATP production and decreased CK activity have also been found in brain tissue of the rats with Al diet compared to control in our study (Fig. 2). Adding APE into drinking water in the Al-exposed rat was able to reverse the mitochondrial dysfunction and this effective energy production may result in improved performance in the behavioral experiment. On the other hand, Al has been demonstrated to induce reactive oxygen species (ROS) (Mailloux et al., 2011) and the impairment of mitochondrial functions may attribute to the formation of excessive ROS in cell. A mechanism has been elucidated to explain the full gamut of the oxidative potential of aluminum and it implicates the binding of Al3+ by the superoxide radical anion to form an aluminum superoxide semi-reduced radical ion (Exley, 2004). The prooxidative potential of aluminum has been considered a serious protagonist in ROS-mediated damage in neurodegenerative diseases. When (ROS) begin to accumulate, astrocytic cells exhibit a defensive mechanism by using various antioxidant enzymes. The main bio-detoxifying systems for peroxides are superoxide dismutase (SOD) and catalase (Meister, 1983). In the current study, aluminum increased the levels of MDA in the brain, which is an end product of lipid peroxidation and indicator of oxidative damage in vivo (Fig. 4A). The level of MDA in the brain of Al-exposed rat was significantly reduced when the rats were received drinking water

with the APE added. APE treatment was also able to restore the activity of SOD and catalase in aluminum chloride treated rats. It has been reported in literature that APE reduce the MDA concentration and increases the levels of SOD and catalase in rat gastric mucosa with oxidative damage (Graziani et al., 2005). The chlorogenic acid (ChA), which is 45.1% of the total phenol content in APE has been shown to have multiple biological effects, including antioxidant (Feng et al., 2005), neuroprotective (Lee et al., 2008; Kwon et al., 2010) and neurotrophic activity (Ito et al., 2008). In H2O2-induced oxidative neuronal death, ChA up-regulated the antioxidant enzyme and the anti-apoptotic proteins, which probably exerted a neuroprotective effect on this population of neurons (Kim et al., 2012). In Verzelloni’ (2011) study, low molecular-weight colonic catabolites of dietary polyphenols, that pass through the circulatory system before being excreted in urine, were shown to protect cultured neuroblastoma cells against mild oxidative stress. The catabolites linked to coffee-derived ChA intake were the most effective, showing a 16% protection with respect to untreated cells. Aluminum treatment confers pleiotropic effect on different biochemical parameters undertaken in this study. Our data showed the dysfunction of acetylcholinesterase (AChE) and an overexpression of Ab42 immunoreactivity induced by Al exposure. Merging with previous studies, Al triggers the intracellular accumulation of proteins and peptides, including Ab peptide (Korchazhkina et al., 2002). The accumulation of Ab peptides might cause the formation of the neuritic plaques, and the neurotoxicity (Sultana et al., 2004). Among membrane-associated proteins, AChE activity was observed to be reduced in Alintoxicated in mouse brain (Zatta et al., 2002). Our results were in accordance with the recent reports. APE treatment was able to prevent the accumulation of Ab42 and restore the activity of AChE.

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Fig. 6. Potentiometric study of chlorogenic acid Al complexes. A, Potentiometric titration curves of chlorogenic acid + Al in water at 25 8C and 0.1 M NaCl ionic strength. B, Formation curves of chlorogenic acid in water at 25 8C and 0.1 M NaCl ionic strength. C, nL vs pL plots of: chlorogenic acid + Al in water at 25 8C and 0.1 M NaCl ionic strength.

This might be one mechanistic pathways for the neuroprotective effect of APE in cognitive dysfunction of aluminum treated rats. Our histological observations indicated that Al-treated groups exhibited disorganized nerve fibers with congestion of blood vessels, disruption of nucleus and vacuolization around the neuron. Similar histological changes have also been reported by others researchers upon Al treatment. It was demonstrated that the toxic effects of Al on mice brain, confirmed a damage in the hippocampus and cortex, including neurofibrillary degeneration, due to the accumulation of Al in these regions (Rebai and Djebli, 2008). In the present study, it revealed a partial restoration of induced damage, mild degenerative changes of nerve fibers, reduced congestion in blood vessels and edema after treatment with APE. It indicate that the APE could attenuate the brain injury in rat induced by Al. Accumulation of Al has been determined in different experimental animals (Erasmus et al., 1993). Accumulation of Al is the

net consequence of uptake, biotransformation and elimination processes within an individual. Successful chelation therapy for metal poisoning lies in the mobilization of the metal and its excretion from the body by the chelating agents used. This reduces the body burden of the metal and reduces the metal’s toxic effects. It has been indicated that dietary phenols, such as catecholates, salicylates, curcumin and epigallocatechin, can chelate with Al3+, therefore, be capable to mobilize Al and to reduce its body burden (Exley et al., 2006; Crisponi et al., 2012). Results of the present study suggested that most of the above parameters responded positively to therapy with APE. Main structural classes of APE include phenol carboxylic acid derivatives, catechins and di-, tri-, and oligomeric procyanidins, dihydrochalcones and flavonoid glycosides (Henriette et al., 2008), which possesses sufficient phenolic hydroxyl groups. Al3+ can coordinate via the carboxylate group and one phenolate group (with the other phenolic group remaining protonated). In our study, we anlysis the stability constants for Al (III) complexes with the chlorogenic acid (ChA), which is 45.1% of the total phenol content in APE added to rat diet. The stability constant for the chlorogenic acid-Al (III) complex (log K1 = 10.51) is a high value considering its 1:1 stoichiometry. To further investigate how dietary phenolic compounds may affect the bioavailability of Al, we carried an experiment with rats as an animal model of testing effect of ChA on Al excretion. We found that the amounts of Al increased significantly in feces of the rats gavaged with AlCl3 plus ChA compared with AlCl3 alone in 24 h after the intragastric administration as compared to the control group. Our result suggests that the dietary phenolic, such as ChA, indeed can reduce aluminum absorption. Our previous studies suggest the phenols extracted from jujube peel and pulp to be effective not only in reducing some of the biochemical variables indicative of oxidative stress but also in restoring activity of the various antioxidant enzymes in the aluminum-treated (intraperitoneally) rats (Cheng et al., 2012). Although we find that APE-mediated protection against chronic Al-toxicity is connected with APE chelation properties, we should not exclude the contribution of APE antioxidant effects against Al-toxicity. We hypothesize that the neuroprotective role of APE against Al-toxicity involve chelation and antioxidant effects. These two mechanisms’ respective contribution to APE mediates protection should be analyzed in subsequent research. In summary, although the neurotoxicity of Al is likely to be the result of a combination of several mechanisms, including oxidative brain injury, induced amyloid deposits, ineffective energy production, and reduced neurotransmitter biosynthesis, it was clearly demonstrates that APE has a neuroprotective effect against Al induced behavioral and biochemical changes. Further study should focus on molecular studies to elucidate the mechanisms underlying the protective effects of APE. Conflicts of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgments

Fig. 7. Effect of ChA diet on Al excretion of rats gavaged with AlCl3. Data were expressed as mean  SD (n = 6).

This work was partly supported by National Natural Science Foundation of China and National Basic Research Program of China (973 Program No: 2013CB127106).

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Protective effect of apple (Ralls) polyphenol extract against aluminum-induced cognitive impairment and oxidative damage in rat.

Aluminum (Al) has long been implicated in the pathogenesis of Alzheimer's disease (AD). Dietary polyphenols have been strongly associated with reduced...
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