Food and Chemical Toxicology 74 (2014) 156–163

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Food and Chemical Toxicology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f o o d c h e m t o x

The effects of Betula platyphylla bark on amyloid beta-induced learning and memory impairment in mice Namki Cho a, Hee Kyoung Lee b, Byung Ju Jeon b, Hyeon Woo Kim a, Hong Pyo Kim c, Jong- Hwan Lee d, Young Choong Kim a, Sang Hyun Sung a,* a

College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Seoul 151-742, Republic of Korea Institute for Life Science, Elcom Science Co. Ltd., Seoul, Republic of Korea c College of Pharmacy, Ajou University, Suwon 443-749, Republic of Korea d Department of Veterinary Anatomy, College of Veterinary Medicine, Konkuk University, Seoul, Republic of Korea b

A R T I C L E

I N F O

Article history: Received 21 April 2014 Accepted 29 September 2014 Available online 6 October 2014 Keywords: Betula platyphylla bark Alzheimer’s disease Amyloid beta Behavioral test CREB–BDNF pathway

A B S T R A C T

Alzheimer’s disease (AD) is closely associated with amyloid β (Aβ)-induced neurotoxicity and oxidative stress in the brain. Betula platyphylla, which has been used to treat various oxidative-stressed related diseases, has recently received attention for its preventive activity on age-related neurodegenerative diseases. In this study, we attempted to investigate the effects of B. platyphylla bark (BPB-316) on Aβ1–42-induced neurotoxicity and memory impairment. Oral treatment using BPB-316 significantly attenuated Aβinduced memory impairment which was evaluated by behavioral tests including the passive avoidance, Y-maze and Morris water maze test. BPB-316 also inhibited the elevation of β-secretase activity accompanying the reduced Aβ1–42 levels in the hippocampus of the brain. Furthermore, BPB-316 significantly decreased the acetylcholinesterase activity and increased the glutathione content in the hippocampus. In addition, we confirmed that the expression of both cAMP responsive element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) in the hippocampus of Aβ1–42-injected mice were markedly upregulated by the treatment of BPB-316. Our data suggest that the extracts of B. platyphylla bark might be a potential therapeutic agent against AD. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Alzheimer’s disease (AD) has been characterized as the progressive impairment of cognitive function in elderly people (Gibson and Zhang, 2001). This age-related neurodegenerative disease is highly implicated in extracellular deposits of amyloid beta (Aβ), which lead to the formation of neurofibrillary tangles and neuritic plaques in the brain (Selkoe, 2001). Aβ, a 40–42 amino acid peptide fragment, is the cleavage product of amyloid precursor protein (APP) through the combination of β-secretase (BACE1) and γ-secretase (Masters and Beyreuther, 2006). This amino acid peptide fragment can aggregate into dimers, oligomers and fibrils, and these aggregations can be toxic in the brain (Yankner et al., 1990). Synthetic Aβ in hippocampal neurons seem to be responsible for neurodegenerative processes through various biochemical pathways. Under the accumulation of abnormal Aβ deposits, the brain is particularly vulnerable to oxidative damage because of poor

* Corresponding author. College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Seoul 151-742, Republic of Korea. Tel.: +82 2 880 7859; fax: +82 2 877 7859. E-mail address: [email protected] (S.H. Sung). http://dx.doi.org/10.1016/j.fct.2014.09.019 0278-6915/© 2014 Elsevier Ltd. All rights reserved.

antioxidant systems. The intracellular accumulation of Aβ in neurons has been known to deplete intracellular GSH which is a major endogenous defense enzyme against oxidative stress in the body (Butterfield et al., 1994). Another marker for AD is degeneration and functional impairment within basal forebrain cholinergic neurons (Itoh et al., 1996). The level of acetylcholine within the hippocampus in the pathology of AD is low because of the hydrolysis of acetylcholine by AChE (acetylcholine esterase) around amyloid plaques (Giovannini et al., 1997). Thus, BACE1 and AchE inhibitors with antioxidant capacity might be therapeutic agents for AD by reducing the level of Aβ and increasing the contents of acetylcholine in the brain (Jeong et al., 2013). Phytochemical-rich medicinal plants could be an ideal source for multipotent agents related to the above mechanisms. Betula platyphylla (B. platyphylla) has been used as a natural drug in Korea, Japan and China. Various studies have reported that extract of B. platyphylla shows antioxidant, anti-inflammatory, anti-arthritis and anticancer activities (Huh et al., 2009). Especially, the bark of B. platyphylla has been used in traditional medicine to treat various oxidative-stressed related diseases such as arthritis, nephritis and dermatitis (Huh et al., 2009; Matsuda et al., 1998). In line with our previous study showing the cognitive enhancing activity of B. platyphylla in a scopolamine-induced model (Lee et al., 2012b),

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we focused on the cognitive enhancing effects of B. platyphylla bark (BPB-316) in an Aβ-induced AD mice model in this study. After oral treatment using BPB-316 followed by injection of Aβ1–42 (2 mg/ mouse, i.c.v.), the restored degree of impairment was measured using the passive avoidance, Y-maze and Morris water maze tests. Furthermore, the effects of BPB-316 on the activity of BACE1 and AchE, and the levels of Aβ and GSH, related to the accumulation of Aβ in the hippocampus, were evaluated. Because the enhancement of short-term or long-term memory has been known to be controlled at molecular levels in neurons, we further examined whether BPB-316 modulates the cAMP-response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) expression which belongs to the neurotrophin family of growth factors, and affects the survival of neurons in the brain (Williams et al., 2008).

2. Materials and methods 2.1. Sample preparation The dried bark of B. platyphylla was collected at the afforested land of SK E&C (Korea) and ground into powder (Lee et al., 2012b). The powdered B. platyphylla bark (1.2 kg) was weighed accurately and extracted with 80 % ethanol for 48 h at room temperature. This extract (90.2 g) was filtered and evaporated in vacuum at 55 °C and 70 cmHg. A voucher specimen (SNU-0316) was deposited at the College of Pharmacy, Seoul National University, Korea. For the simultaneous determination of three major components of B. platyphylla bark, chromatographic analyses were performed using a Thermo Dionex HPLC system with an autosampler, and a photodiode array detector (Supplementary Fig. S1). The wavelength for detection was set at 200 nm, where the three components showed the maximum absorption. Determination of major compounds was carried out with INNO C18 (5 μm, 4.6 × 150 mm) in the mobile phase composed of ACN–water at a flow rate of 1.0 ml/min in the mobile phase composed of acetonitrile (a) and water (b) with gradient elution as follows: 0 min, 85% b; 20 min, 0% b; 30 min, 85% b; 40 min, 85% b (v/v). The presence of platyphylloside, aceroside VIII and betulin in this herb was verified by comparing each retention time (platyphylloside: 13.2 min; aceroside VIII: 17.5 min; betulin: 26.3 min) and UV spectrum (Supplementary Fig. S1).

2.2. Animal experimental design Male ICR (6 weeks old) mice (KOATEC. Co. Ltd., Gyunggi, Korea), weighing approximately 27 ± 2 g, were used after a 5-day adaptation period (20–23 °C; 12 h light cycle from 09:00 to 21:00; food, Agribrand Purina Korea, and water ad libitum). ICR mice were randomly assigned to seven groups: saline-CMC-treated normal control, Aβ1–42-CMC-treated group, Aβ1–42-BPB-316-treated group (25, 50, 100 and 200 mg/ kg) and Aβ1–42-donepezil-treated group (1 mg/kg). All experiments and the method used for euthanasia were according to the guidelines of the Committee on the Care and Use of Laboratory Animals of Seoul National University. The Aβ1–42 (Sigma, St. Louis, MO, USA) was dissolved and diluted in distilled water to a final concentration of 1 mg/2.5 ml. The dissolved Aβ1–42 was incubated at 37 °C for 4 days to induce the fibrillized form. Sterile saline (0.9% NaCl) containing aggregated Aβ1–42 was injected directly into the mice in the third ventricle 0.25 mm posterior to the bregma with a 10 μl Hamilton syringe fitted with a 28-gauge needle, which was adjusted to be inserted to a depth of 3.0 mm (anteroposterior, −2.5 mm; mediolateral, 0 mm; dorsoventral, −3.0 mm relative to the bregma). The selection of the third ventricle was based on its vicinity to brain regions most affected in AD. The dissolved Aβ1–42 (2 μg/mouse) was injected using stereotaxic instruments and the injection rate was approximately 1 μl/s and the volume was 5 μl. Ten mice were used per treatment. BPB-316 (25–200 mg/kg, p.o.) or donepezil (1 mg/kg, p.o.) were dissolved in 0.5%CMC at the concentration of 10 mg/ml and administered orally to mice once a day for 18 consecutive days. The administration of BPB-316 or donepezil began 5 days before Aβ1–42 i.c.v. infusion (day 0) and continued for 13 days after Aβ1–42 i.c.v. infusion. On day 4, the behavioral tests were started followed by resting for 3 days (days 0–3), and a study of their possible mechanisms was performed in separate brain regions including the hippocampus.

2.3. Passive avoidance test The step-through passive avoidance test was carried out as previously described (Jeong et al., 2009). The mice were placed in a light chamber, and the door between compartments was opened 10 s later. When mice entered the dark compartment, the door automatically closed and an electrical foot shock (0.1 mA/10 g body weight) for a time period of 2 s was delivered through the stainless steel rods (one trial training). Twenty-four hours after the training trial, the mice were again

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placed in the light compartment. The escape latency to enter the dark compartment was measured. If the mice did not enter the dark compartment within 300 s, the experiment was stopped.

2.4. Y maze test The Y-maze test was performed as previously described (Mansouri et al., 2013). The Y-maze was a three-arm maze with all arms at equal angles, 20 cm in length and 5 cm in width with walls 12 cm high. Mice were initially placed within one arm, and allowed to explore over an 8-min period. The sequence and total number of arms entered were recorded. Arm entry was considered to be complete when the hind paws of the mouse were completely within the arm. The percentage of alternation was calculated with the following equation: % alternation = [(number of alternations)/ (total arm entries − 2)] × 100.

2.5. Morris water maze test The Morris water maze test was performed as previously described (Kim et al., 1999). The white pool was circular (100 cm in diameter and 45 cm in height) filled with water in which 500 ml was milk (20 ± 1 °C). The pool was divided into four quadrants of equal area, and a white platform (6 cm in diameter and 29 cm in height) was centered in one of the four quadrants of the pool. The location of each swimming mouse was monitored by a video tracking system (Smart 2.5). Each mouse was dedicated to swim for training and in the following days, the mice were given two trial sessions each day for 4 consecutive days. During each trial, the time taken to swim to the platform (escape latency) was recorded. Once the mouse located the platform, it was permitted to remain on it for 10 s. If the mouse did not locate the platform within 120 s, it was placed on the platform for 10 s and then removed from the pool by the experimenter (trial 1). The mouse was given a second trial (trial 2) with an inter-trial interval of 20 min. The point of entry into the pool and the location of the platform for escape remained unchanged between trial 1 and trail 2 but was changed each day thereafter. This parameter was averaged for each session of the trials and for each mouse. The decreased latency from day to day in trial 1 represents the long-term or reference memory, while that from trial 1 to trial 2 represents the short-term or working memory.

2.6. Brain collection and preservation After the behavioral test (water maze test), the mice were euthanized, and the brains were removed. The brains were immediately collected and separated into the cortical and hippocampal regions. The hippocampal region was immediately stored at −80 °C and used for measuring various biological assays (Jeong et al., 2013).

2.7. Determination of the Aβ1–42 level Aβ1–42 levels in the mouse hippocampus were determined using specific ELISAs (IBL, Immuno-Biological Co., Ltd., Japan) as previously described (Flood et al., 1991). One hundred microliters of sample were applied to a precoated plate and was incubated overnight at 4 °C. After each well of the precoated plate was washed with washing buffer, 100 μl of labeled antibody solution was added and the mixture was incubated for 1 h at 4 °C in the dark. After another wash, chromogen was added and the mixture was incubated for 30 min at room temperature in the dark. Finally, the resulting color was assayed at 450 nm using a microplate absorbance reader (Sunrise, Tecan, Switzerland) after the addition of a stop solution (Kim et al., 2009).

2.8. β-Secretase assay The inhibitory activities of BPB-316 on β-secretase in the mouse hippocampus were measured using a specific β-secretase ELISA kit (Biovision Co. Ltd., USA) according to the manufacturer’s instructions. This formation of fluorescence was read using a fluorescence plate reader with excitation at 335–355 nm and emission at 495–510 nm. The enzyme activity of β-secretase was expressed as the relative fluorescence units/μg protein (Jeong et al., 2013).

2.9. Acetylcholinesterase (AChE) assay AChE activity was measured as previously described (Ellman et al., 1961). The hippocampus was rapidly homogenized with sodium phosphate buffer (0.1 mM, pH 7.4). Each homogenate was preincubated for 5 min at 37 °C with 0.1 mM tetraisopropyl pyrophosphoramide (TPPA) which is a selective inhibitor of butyryl cholinesterase. And then, a reaction mixture that contained 470 μl sodium phosphate (0.1 mM, pH 8.0), 167 μl of 4% 5,5′-dithio-bis (2-nitro benzoic acid) and 33 μl of homogenate was incubated at 37 °C for 5 min. For the determination of the AChE activity, 280 μl of 1 mM acetylcholine iodide solution was added to the reaction mixture. After incubation for 3 min at 37 °C, the reaction was terminated by adding 50 μl of 2 mM neostigmine. The absorption was read using an ELISA reader at 412 nm.

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The AChE activity was calculated as the optical density (OD) value per mg protein (Jeong et al., 2009).

2.10. Evaluation of the reduced glutathione content The hippocampus was homogenated in 0.1 M phosphate buffer (pH 7.4). The homogenates were centrifuged for 30 min at 3000 g at 4 °C and the supernatant was collected to assess the GSH content. Total GSH was determined spectrophotometrically with the enzymatic cycling method (Tietz, 1969).

2.11. Congo red staining Mice were anesthetized, and the brains were dissected and fixed with 4% paraformaldehyde (PFA) after transcardial wash-out with heparinized 0.5% sodium nitrite saline. Serial coronal sections of brain were cut with a freezing microtome and stained with cresyl violet (Lee et al., 2012a). Congo red staining was also used to detect Aβ deposits in the mouse hippocampus (Rofina et al., 2004). The removed brain tissues were fixed in 10% neural formalin, dehydrated, and embedded in paraffin. The brain slices were deparaffinized with xylene and rehydrated by a gradient ethanol series. After deparaffinization, slices were incubated in Congo red working solution for 20 min, and then, slices were quickly differentiated with alkaline alcohol solution. Slices were dehydrated through a graded ethanol series, cleared in xylene, and cover-slipped.

2.12. Tissue preparation and western blot analysis The hippocampus was promptly excised and homogenized in 200 μl of lysis buffer (50 mM Tris–HCl, pH 7.4, 1% Nonidet P-40, 0.25% Na-deoxycholate, 150 mM NaCl, 2 mM EDTA, 1 mM Na3VO4, and 1 mM NaF) containing protease inhibitors (2 mMPMSF, 100 lg/ml leupeptin, 10 lg/ml pepstatin, and 1 lg/ml aprotinin). After centrifugation for 15 min at 13,000 × g, supernatants were divided into eppendorf tubes and stored at −70 °C, and then, a protein assay was performed using the Bradford protein assay kit (Bio-Rad, USA). For western blot analysis, a mixture of sample loading buffer (Biosaesang Co., Korea) and 30 μg of tissue protein was boiled at 100 °C for 10 min. Denatured proteins were separated by 14% polyacrylamide gel electrophoresis for 2–3 h at 100 V and transferred to a 0.2 μm nitrocellulose membranes for 1.5 h at 100 V. Membranes were then washed three times for 10 min in 0.1% Tween-20 PBS between each of the following steps: 1 h block in 5% milk and over-night incubation at 4 °C with primary antibodies (BDNF, p-CREB, CREB and β-actin). The immunoreactive bands were visualized by using secondary antibodies and an ECL chemiluminescence detection kit (Amersham Biosciences, USA). 2.13. Statistical analysis All data were expressed as the mean ± SEM. The results of the behavioral tests and assays were analyzed by Graph Pad Prism version 5.00 for Windows (Graph Pad Software, San Diego, CA, USA). The data were considered to be statistically significant if the probability had a value of 0.05 or less.

Fig. 1. The cognitive-enhancing activity of BPB-316 was investigated by the passive avoidance, Y-maze and water maze tests. The experiments were designed as in Supplementary Fig. S2. (A) Effect of BPB-316 on Aβ-induced learning and memory deficits in passive avoidance. The latency to enter the dark compartment was measured. (B) Effect of BPB-316 on Aβ-induced memory deficits in the Y-maze test. The sequence and total number of arms entered were recorded. (C) Effect of BPB-316 during the probe trial of the Morris water maze. Probe trial sessions were carried out for 60 s. Data represent the means ± S.E.M. (###P < 0.001 and #P < 0.05 compared to the control group. *P < 0.05 and **P < 0.01 compared to Aβ-treated groups).

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3. Results 3.1. Memory enhancing activity of BPB-316 on Aβ-induced amnesic mice We determined the cognitive enhancing effects of BPB-316 on Aβ-induced amnesic mice by using the passive avoidance, Y-maze and Morris water maze tests. The effect of BPB-316 on long-term memory was evaluated in the step-through passive avoidance test. The step through latency was shortened in the Aβ-injected mice (9.3 ± 2.6 s, protection rate = 8.7%) compared to that of the normal control mice (107.3 ± 17.8 s, protection rate = 100.0%) (Fig. 1A). The administration of BPB-316 (50 mg/kg body weight, p.o.) slightly increased the latency to a level of 66.4% of the normal control mice. At the highest effective dose of BPB-316 (200 mg/kg body weight, p.o.), the memory impairment recovered up to 90.3% (97.0 ± 18.6 s). In addition, the cognitive enhancing effects of BPB-316 at a dose of 100 mg/kg (106.2 ± 23.7 s, protection rate = 98.9%) were even more potent than those of the donepezil (1 mg/kg body weight, p.o.)treated group (104.6 ± 22.6 s, protection rate = 97.5%). We used the Y-maze test to observe the effects of BPB-316 on short-term or working memory (Mansouri et al., 2013). In the spontaneous alternation behavior in the Y-maze test, the Aβ-injected group (38.3 ± 8.4 s) showed a significant reduction compared to the control group (62.8 ± 9.4 s) (Fig. 1B). However, treatment with BPB316 at 50–200 mg/kg inhibited the decrease in spontaneous alternation induced by Aβ. These results indicate that BPB-316 improved short-term or working memory. We further evaluated whether BPB-316 affected spatial memory by using the Morris water maze test. The control mice rapidly learned the location of the platform from day 1 to day 4, whereas the Aβtreated group had delayed latencies compared to the controls showing memory impairment (Fig. 2). This memory impairment was significantly prevented by treatment with BPB-316 at concentrations ranging from 25 to 200 mg/kg. In addition, BPB-316 treatment tended to decrease the swimming time and swimming distance

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(Supplementary Fig. S3) over the 4 days compared to the mice given Aβ only. In the amnesic mice treated with BPB-316 at a dose of 100 mg/kg, the mice exhibited a shorter escape latency by the training. Particularly, the mice group treated with BPB-316 at a dose of 100 mg/kg exhibited a marked decrease in escape latencies from trial 1 and trial 2 suggesting that the prolonged treatment of amnesic mice with BPB-316 (100 mg/kg bodyweight, p.o.) significantly improved the deficits in working and reference memories. The probe trials followed after the acquisition tests. BPB-316 dramatically increased the time spent in the training quadrant. The amnesic mice treated with BPB-316 at a dose of 100 mg/kg significantly remained longer in the target quadrant compared to the Aβ-treated mice. 3.2. Inhibitory effects of BPB-316 on AChE and β-secretase activity In the mice group treated with Aβ, the activity of AChE and β-secretase within the hippocampus was significantly increased compared to the normal control group. Oral administration with a highdose of BPB-316 (100 mg/kg, 200 mg/kg body weight) significantly inhibited the increased AChE (Fig. 3C) and β-secretase activity (Fig. 3B) induced by Aβ. Although the change in β-secretase activity was not statistically significant after a low-dose of BPB-316, it decreased to some extent in the 25 mg/kg- and 50 mg/kg-treated group (Fig. 3B). 3.3. Effect of BPB-316 on Aβ accumulation in the mouse hippocampus In line with the inhibitory effects of BPB-316 on the β-secretase activity, we further estimated the levels of Aβ in the mouse hippocampus. The accumulation of Aβ was observed by Congo red staining (Fig. 4). The area of brown colored Aβ deposition was enlarged in the CA1, CA3 and CA4 hippocampus regions through the cerebrospinal fluid (CSF) of mice injected with Aβ. However, BPB316 (100 mg/kg) treatment markedly decreased the appearance of

Fig. 2. The enhancing effect of BPB-316 (100 mg/kg body weight) on spatial memory impairment induced by amyloid beta in mice. Mice were given two sessions of trials each day for 4 consecutive days. The swimming time required for the mouse to escape to the platform was recorded in the saline-treated group (A) or Aβ-treated group or BPB-316 [25 (C), 50 (D), 100 (E) and 200 (F) mg/kg]-treated group. The values shown are the mean escape latency ± S.E.M. Results significantly differ from the values in trial 1: ***P < 0.001 and *P < 0.05.

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Fig. 3. After behavioral tests, the mice were euthanized and the brains were removed. (A) Inhibitory effect of BPB-316 on the Aβ level induced by Aβ in the hippocampus. Aβ levels were determined using specific ELISAs. (B) The effect of BPB-316 on β-secretase activity within the hippocampus of Aβ-injected mice. The activities of β-secretase in the hippocampus were measured as relative fluorescence units (RFU)/mg protein. (C) The effect of BPB-316 on AchE activity within the hippocampus of Aβ-injected mice. The activity of AchE in the hippocampus was calculated as the optical density (OD) value per mg protein. (D) Effect of BPB-316 on the reduced glutathione level in the hippocampus of Aβ-injected mice. Results significantly differ from the values of the normal control group: #P < 0.05, ##P < 0.01 and ###P < 0.001 compared to the control group. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to the Aβ-treated groups.

Aβ. In addition, dying neurons, which had a shrunken morphology in the Aβ-treated group, were found in the hippocampal slices stained with cresyl violet (Fig. 4); whereas, BPB-316 (100 mg/kg) treatment increased the viable neurons which had a round and intact morphology. Furthermore, the elevated level of Aβ in the hippocampus was reduced by the administration of BPB-316 (100 mg/ kg) (Fig. 3A). 3.4. Effect of BPB-316 on GSH content To assess whether Aβ injection causes any change in the antioxidant status within the brain, we further evaluated the effects of BPB-316 on the GSH contents in the hippocampus. Under our experimental conditions, i.c.v. injection of Aβ depleted the GSH contents by 25% (Fig. 3D). The GSH contents reduced by Aβ insult were restored by BPB-316 at concentrations ranging from 25 to 200 mg/kg. 3.5. Effects of BPB-316 on pCREB and BDNF expression CREB phosphorylation and BDNF expression in the hippocampus were determined using western blotting for the mechanistic

study related to short-term or long-term memory. In our experiments, the Aβ-treated mice significantly inhibited the CREB phosphorylation and subsequent BDNF expression in the hippocampus (Fig. 5). The treatment of amnesic mice with BPB-316 significantly increased the CREB phosphorylation and subsequent BDNF expression in the hippocampus. Donepezil, used as positive control, also increased the CREB phosphorylation and BDNF expression; whereas, there were no significant alterations in the total levels of CREB and β-actin.

4. Discussion Diarylheptanoids and triterpenoids are the major constituents of B. platyphylla bark with various pharmacological effects (Matsuda et al., 1998). With respect to neuroprotection, our previous studies confirmed that B. platyphylla bark and its major diarylheptanoids, aceroside VIII and platyphylloside significantly suppressed neurotoxicity induced by glutamate in HT22 cells and scopolamine in mice (Lee et al., 2012b). Moreover, in our laboratory, betulin showed a significant anti-amnesic activity on memory deficits induced by scopolamine in mice (unpublished data). Scopolamine is a muscarinic antagonist that induces central cholinergic blockade and

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Fig. 4. The effect of BPB-316 on Aβ level in the CA1, CA3 and CA4 hippocampal regions. The level of extracellular Aβ was observed by Cresyl violet and Congo red staining. The accumulation of Aβ is shown in the representative photomicrographs of the saline-treated group (A,B,C,a,b,c), Aβ-treated group (D,E,F,d,e,f) or BPB-316-treated group (G,H,I,g,h,i) (magnification, ×100).

produces a reversible cognitive impairment. It has been known that memory impairment in a scopolamine-induced animal model is associated with the altered status of brain oxidative stress (Jeong et al., 2009). However, the pathophysiology of AD is highly implicated in not only indirect causes such as cholinergic blockade and oxidative stress but also direct damages such as Aβ deposition and formation of neuritic plaques in the brain (Selkoe, 2001). Single intracerebroventricular (icv) injections of Aβ peptides into mice impair learning and memory as tested with passive avoidance tests

as well as with the water maze test or Y-maze tests (McDonald et al., 1994). It was also shown that icv injection of the aggregated form of Aβ significantly induced amyloid deposits and neuronal loss in the mouse brain (Kim et al., 2009; Maurice et al., 1998). In line with these previous studies, our aim was to study the cognitive enhancing effects of BPB-316 in a mouse model of AD induced by Aβ. We applied donepezil, which has been well known as a potent and selective inhibitor of brain cholinesterase and Aβ-induced impairment (Yamada et al., 2006). To verify the memory enhancing

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Fig. 5. The effects of BPB-316 on CREB–BDNF signaling by Western blot analysis (A). The relative expression levels of pCREB and BDNF in the hippocampus were determined by densitometry and normalized by CREB and β-actin (B). Three animals were used per treatment group. Data represent the means ± SD (#P < 0.05 vs. control group; *P < 0.05, **P < 0.01 vs amyloid beta-treated group).

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potential of B. platyphylla bark in an Aβ-induced AD model, we prepared the bioactive fraction of 80% ethanol extracts of B. platyphylla bark. In our experimental comparison of the chemical composition between the inner bark extracts and outer bark extracts of B. platyphylla, we found that the outer bark extracts included more contents of betulin compared to the inner bark extracts. Consistent with the difference in the chemical composition of the barks, the antioxidant effects of the outer bark extracts were much more potent compared with those of the inner bark extracts (data not shown). Therefore, in this study, we extracted the outer bark more than inner bark of B. platyphylla, which was denoted as BPB-316. The cognitive-enhancing activities of BPB-316 were estimated by the passive avoidance, Y-maze and water maze tests. Aβtreated groups decreased the step through latency to a level of 8.7% compared to the control group. Prolonged treatment with BPB316 (50–200 mg/kg body weight) exhibited mitigation of memory deficit in mice suggesting that BPB-316 has cognitive enhancing effects on long-term memory impairment induced by Aβ treatment. Although the change in step-through latency was not statistically significant, it increased to some extent with a lowdose of BPB-316 (50 mg/kg body weight). The memory enhancing effects of BPB-316 were shown to be most potent in the BPB-316treated group at a dose of 100 mg/kg. It is well known that spontaneous alternation behavior in the Y-maze is an indication of short-term and working memory (Mansouri et al., 2013). In our experiments, we observed that Aβ caused a decrease in spontaneous alternation in the Y-maze test. Oral administration of BPB316 at dosages of 50–200 mg/kg significantly inhibited the decreased spontaneous alternation induced by Aβ. This outcome was consistent with the results of the passive avoidance test. In the Morris water maze test, it was shown that Aβ interferes with memory and cognitive function, and subsequently causes impairment of the working (=short-term) and reference (=long-term) memories. In our experiments, the normal control group exhibited that both working and reference memories were being well formed whereas the Aβ-treated group showed a memory deficit. In the low-dose BPB-316 treated group (25 and 50 mg/kg), there were little cognitive enhancing effects showing a similar pattern to that of the Aβ group. However, the mice given BPB-316 at a dose of 200 mg/kg improved the amnesic deficits in the reference memory but not in the working memory. The prolonged treatment of amnesic mice with BPB-316 (100 mg/kg bodyweight, p.o.) significantly improved the deficits in the working and reference memories. The BPB-316 (100 mg/kg body weight p.o.)-treated group reduced the deficits in the working memory on day 1 and gradually improved the reference memory over the 4 testing days showing a similar pattern to that of the control group. The disruption of Aβ homeostasis is one of the major factors of AD. The changes in Aβ metabolism increase the levels of Aβ, and the accumulated amyloid plaques in the brain are a characteristic feature of AD. Thus, there have been many efforts to develop therapeutics controlling β-secretase activity to reduce Aβ levels in the brain (Jeong et al., 2013). In our studies, the activities of BACE1 were markedly reduced by BPB-316. Since BACE1 is the Aβ converting enzyme that cleaves APP into Aβ, in accordance with the reduction in BACE1 activity by BPB-316, the Aβ level in the hippocampus was significantly reduced by BPB-316. In addition, a marked reduction in Aβ aggregation in the hippocampal CA1, CA3, and CA4 regions were observed in the BPB-316-treated group compared to the Aβ-treated group. Many clinical studies have reported that impairments in learning and memory in AD patients are caused by changes within the cholinergic system (Jeong et al., 2009). Cholinergic transmission is terminated mainly by acetylcholinesterase which is the acetylcholine hydrolysis enzyme. Consequently, this enzyme could be a

potential target for the treatment of AD. In our experiments, the administration of BPB-316 significantly inhibited the acetylcholinesterase activity in the hippocampus. In this regard, the memoryenhancing action of BPB-316 in Aβ-induced amnesia could be explained, in part, by the changes in AchE in the brain. Much evidence has suggested that memory impairment by Aβ is associated with an altered status in brain oxidative stress. Generally, the mammalian brain has an antioxidant defense system, which includes GSH. Under oxidative stress, GSH is reversibly oxidized to glutathione disulfide (GSSG) and decreases in the GSH/ GSSG ratio (Cho et al., 2013). In our experimental conditions, exposing mice to Aβ1–42 reduced the GSH content by 25%. After BPB-316 treatment, it increased to some extent at concentrations ranging from 25 to 200 mg/kg. Under pathological conditions of memory impairment, various signaling pathways including calcium-calmodulin kinases (CaMK) and mitogen-activated protein kinases (MAPKs) are related with memory-enhancing pathways. These pathways eventually converge to the CREB, which is a transcription factor leading to changes in neurogenesis. The phosphorylation of CREB is also responsible for transcriptional activation causing gene expression such as BDNF, which is the second neurotrophic factor acting on certain neurons in the brain. These signals are active in the hippocampus related to memory, learning and cognition (Cho et al., 2013). We evaluated the effects of BPB-316 on CREB phosphorylation and BDNF expression ex vivo by western blot analysis. Treating mice with Aβ markedly reduced the CREB phosphorylation and subsequent BDNF expression in the hippocampus of amnesic mice. However, treating amnesic mice with BPB-316 significantly increased the CREB phosphorylation and subsequent BDNF expression in the hippocampus. The cognitive-enhancing effects of BPB-316 in vivo were well correlated with the up-regulation of the CREB–BDNF pathway which plays a crucial role in inducing synaptic plasticity and cognition. In summary, BPB-316 improves learning and memory deficits in Aβ1–42-treated mouse models and this is partly mediated by an activation of CREB–BDNF signaling pathway. These cognitiveenhancing effects of BPB-316 might result from inhibiting the accumulation of Aβ and the activities of BACE1. In addition, BPB316 significantly suppressed the AChE activity and recovered the content of GSH in the hippocampus. Based on these findings, BPB316 containing aceroside VIII, platyphylloside and betulin could be a potential therapeutic agent against Alzheimer’s disease through multiple cognitive-enhancing mechanisms. Conflict 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. Acknowledgements This work was carried out with the support of ‘Forest Science and Technology Projects (S121214L120100)’ provided by Korea Forest Service. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.fct.2014.09.019.

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