Cognitive Improving Effects by Highbush Blueberry (Vaccinium

Article pubs.acs.org/JAFC

Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Cognitive Improving Effects by Highbush Blueberry (Vaccinium crymbosum L.) Vinegar on Scopolamine-Induced Amnesia Mice Model Seong Min Hong,† Kyong Hee Soe,‡ Taek Hwan Lee,§,∥ In Sook Kim,§ Young Min Lee,‡ and Beong Ou Lim*,⊥,∇ †

BK21PLUS Glocal Education Program of Nutraceuticals Development, Konkuk University, Chungju-si, Chungcheongbuk-do 27478, Republic of Korea ‡ Department of Applied Life Science, College of Biomedical and Health Science, Konkuk University, Chungju-si, Chungcheongbuk-do 27478, Republic of Korea § Institute of Biomedical and Health Science, Kunkuk University, Chungju-si, Chungcheongbuk-do 27478, Republic of Korea ∥ Ahn-Gook Health., LTD., Seoul 07445, Republic of Korea ⊥ Research Institute of Inflammatory Diseases, Kunkuk University, Chungju-si, Chungcheongbuk-do 27478, Republic of Korea ∇ Department of Life Science, College of Biomedical and Health Science, Konkuk University, 268 Chungwondaero, Chungju-si, Chungcheongbuk-do 27478, Republic of Korea ABSTRACT: The present study aimed to evaluate the preventive effects of highbush blueberry (Vaccinium corymbosum L.) vinegar (BV) on cognitive functions in a scopolamine (Sco)-induced amnesia model in mice. In this study, Sco (1 mg/kg, intraperitoneal injection) was used to induce amnesia. ICR mice were orally administered donepezil (5 mg/kg), blueberry extract (120 mg/kg), and BV (120 mg/kg) for 7 days. After inducing cognitive impairment by Sco, a behavioral assessment using behavior tests (i.e., Y-maze and passive avoidance tests) was performed. The BV group showed significantly restored cognitive function in the behavioral tests. BV facilitated cholinergic activity by inhibiting acetylcholinesterase activity, and enhanced antioxidant enzyme activity. Furthermore, BV was found to be rehabilitated in the cornu ammonis 1 neurons of hippocampus. In our study, we demonstrated that the memory protection conferred by BV was linked to activation of brain-derived neurotrophic factor (BDNF)/cAMP response element binding protein (CREB)/serine−threonine kinase (AKT) signaling. KEYWORDS: blueberry vinegar, amnesia, scopolamine, behavior tests, AChE activity, BDNF/CREB/AKT pathway

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INTRODUCTION

It is well-known that the use of acetylcholinesterase (AChE) inhibitors such as tacrine, donepezil, and rivastigmine are beneficial for the treatment of AD symptoms.7 Unfortunately, they potentially tend to cause dangerous side effects including vasodilation, bradycardia, headache, and convulsions, and can be hepatotoxic.7 For these reasons, alternative antidementia therapies need to be developed. In response to the issues with the standard treatments, interest in treatments derived from natural sources, such as plants, has increased. Natural products, containing safer efficacy than synthetic chemicals, have played a vital role in improving health and are constantly suggested as new therapeutic options for AD.8 It was revealed that some components found in extracts obtained from natural sources may increase neuroprotection and cognition in animal models.9 Thus, the use of extracts derived from natural plants and their products might be able to help AD patients. Blueberries (Vaccinium corymbosum L.) have been mainly used in traditional healing to treat diabetes.10 Among the berry

Alzheimer’s disease (AD) is a chronic neurodegenerative disease that leads to cognitive impairment, memory loss, and progressive impairment of the ability to perform activities.1 This disease is thought to account for 60% of the dementia that occurs in individuals over age 65 worldwide.2 The AD pathological hallmarks involve the deposition of β-amyloid plaques, formation of intracellular and extracellular neurofibrillary tangles, neuroinflammation, and neuronal loss following traumatic injury in brain.3 Also, it has been mentioned that expression of related oxidative stress biomarkers (e.g., malondialdehyde (MDA) and reactive oxygen species (ROS)) in neurons is directly related to AD.4 Recently, more attention has been given to the development of therapeutic agents that prevent oxidative stress. Furthermore, several studies have reported that AD brains exhibit the relative loss of cholinergic neurons, and the levels and receptor numbers of reduced acetylcholine (ACh).5 It has also been shown that the impediment of ACh receptors leads to learning problems and memory loss.6 Therefore, the recuperation of cholinergic function in brain still remains a rational and challenging target for the development of therapeutic agents which focus on treating Alzheimer’s symptoms. © XXXX American Chemical Society

Received: August 25, 2017 Revised: November 15, 2017 Accepted: November 15, 2017

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DOI: 10.1021/acs.jafc.7b03965 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Preparation of Blueberry (Vaccinium crymbosum L.) Vinegar. BV was prepared according to a modified method of Lee et al.20 The BV was prepared using a two-step fermentation process. Prior to the alcohol fermentation, blueberries were cut and crushed using a blender. Then, 500 mL of crushed blueberries and 300 mL of DW were mixed and fortified with 200 mL of sugar to 15°Brix. For the alcohol fermentation, Saccharomyces cerevisiae KCCM 34709 (10%, v/ v) was added to the mixed juice and then cultivated in the incubator at 30 °C for 3 days. At the end of alcoholic fermentation (one-step), the fermented blueberry wine was filtered by using Whatman No. 1 filter paper. At the process of acetic acid fermentation (two-step), Acetobacter sp. KCCM 40085 (10%, v/v) was added to the filtered blueberry wine and then incubated in a shaking incubator with at 30 °C and 200 rpm for 12 days. The obtained BV was centrifuged for 5 min at 13000g, after which the separated supernatants were collected. The total acidity and pH of the BV was 5.3% and 0.98, respectively. The BV was stored at 4 °C until further analysis. The yield value of BV was 60% HPLC Analysis of Polyphenols and Organic Acid in BE and BV. HPLC analysis for polyphenols in BE and BV was performed on a Waters system (Waters Corp., Milford, MA) on a Jshpere Supelco C18 column (Φ 250 mm × 4.6 mm, YMC Co. Ltd., Japan) at 40 °C. The mobile phase was consisted of 1% acetic acid (solvent A) and acetonitrile (solvent B) using a 35 min linear gradient from 0% to 100% of solvent B at a flow rate of 1 mL/min. For the HPLC analysis of organic acid (acetic acid) in each sample, the Jshpere ODS column (Φ 250 mm × 4.6 mm, YMC Co. Ltd., Japan) was used. The mobile phase was consisted of 1% acetic acid (solvent A) and methanol (solvent B) using a 45 min linear gradient from 0% to 100% of solvent B at a flow rate of 1 mL/min. An aliquot (20 μL) of the sample was injected into aforementioned HPLC system. Detection was performed with a UV−Vis spectrophotometer using two absorbance wavelengths of 280 and 520 nm. Animals and Sample Administration. Six-week-old ICR male mice (Central Laboratory Animal Inc., Seoul, Korea) were fed a AIN76A purified rodent diet (Central Laboratory Animal Inc., Seoul, Korea) and given water ad libitum. The animals were housed for 1 week at 23 ± 2 °C with 55 ± 5% relative humidity. A total of 80 mice were used for Y-maze and passive avoidance tests (40 mice per each test). In the behavioral tests, the mice were randomly divided into five groups (8 mice per each group): normal group (CON), donepezil group (positive control, PC), Sco group (negative control, NC), BE group, and BV group. The donepezil, BE, and BV were dissolved in DW and administered orally for a 1 week as follows: PC group, 5 mg/ kg body weights per day; BE group, 120 mg/kg body weights per day; BV group, 120 mg/kg body weights per day. The Sco (1 mg/kg) was mixed with saline solution and then injected intraperitoneally (i.p.) 30 min prior to training in the inhibitory Y-maze and passive avoidance tests. Behavioral tests were examined for 1 week, and then mice were sacrificed on day 8 for biochemical and immunochemical analysis. All animal experiments were carried out according to a protocol approved by the Institutional Animal Care and Use Committee of the Konkuk University (KU16015). Y-Maze Test. The Y-shaped maze was made up with three identical arms (40 cm × 3 cm × 12 cm) of dark gray polyvinyl plastic, with equal angles (120°) between each arm. The mice were carefully placed at the end of the start arm and then allowed to move throughout the other arm, during which the sequence and number of arm entries were manually measured and recorded for each mouse over 8 min. In the Ymaze test, a total of 40 mice were used (8 mice per each group). One hour prior to the beginning of the Y-maze test, the PC mice were given 1 mg/kg donepezil (per os; p.o.), and the other groups were given 120 mg/kg BE (p.o.), and 120 mg/kg of BV (p.o.). After 30 min, memory impairment was induced by using 1 mg/kg of Sco (i.p.). Passive Avoidance Test. This system (20 cm × 20 cm × 20 cm) consisted of dark and clear chambers separated by a guillotine door (5 cm × 5 cm). For the training trial test, each mouse was initially placed into the clear chamber. When mice entered the dark chamber, an electric foot shock (0.25 mA) 3s in duration was directly transferred through 2 mm stainless steel rods spaced 1 cm apart. A total of 40

fruits, highbush blueberries are known to contain the polyphenolic compounds and have strong antioxidant properties.11 Indeed, this species contains various phytochemical compounds including several anthocyanin pigments (e.g., cyanidin and malvidin glycosides), which are related to antioxidant effects.12 In addition to their antioxidant activity, we previously demonstrated that the polyphenols found in highbush blueberries showed the anti-inflammatory properties in animal models.13 Highbush blueberries also have a rich source of dietary fibers, which have potential metabolic effects in the gastrointestinal tract.14 Especially, anthocyanins having antiinflammatory effects are predominantly included in blueberries.15 Anthocyanins have displayed the prevention of neurotoxicity caused by reperfusion damage in the cerebral ischemia model.16,17 Moreover, previous studies reported that flavonoids modulated memory and learning, and cognitive function in amnesia animal model.18 This evidence implied that blueberries containing polyphenols and flavonoids could be potentially beneficial in AD patients of both Parkinson’s and AD. However, few studies have shown the effects of fermented blueberries on memory impairment using animal models. Fermentation techniques have been shown to enhance the efficiency of bioactivity in natural products. The production of more effective compounds from natural sources may be possible using various species of yeast.19 Fermented products, such as vinegar, might act to preserve the phenolic compounds that are easily oxidized during food processing and that are impacted by factors such as maturity, storage, and processing. We have successfully developed optimum fermentation conditions for the blueberry, and found that fermentation increases its biological activity.20 This suggests that blueberry vinegar (BV) may be a good source of extract that possesses antiamnesic effects. According to the aforementioned information and studies, we investigated the protective effects of blueberry extract (BE) and BV on the performance of Sco-induced animals in Y-maze and passive avoidance tests, and evaluated their underlying mechanisms of action.

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MATERIALS AND METHODS

Materials and Chemicals. Highbush blueberries were obtained from Blueberry Suite company (Chae-Hyang-Won, Gangwon-do, Korea). Sco was obtained from Sigma-Aldrich Inc. (St. Louis, MO). AChE and ACh were purchased from the Nanjing Jiancheng Institute (Nanjing, China). Antibodies for brain-derived neurotrophic factor (BDNF), phosphorylated-CREB (pCREB), cAMP response element binding protein (CREB), phosphorylated-AKT (pAKT), serine− threonine kinase (AKT), and β-actin were obtained from Santa Cruz Biotech Inc. (Santa Cruz, CA). Saccharomyces cerevisiae KCCM 34709 and Acetobacter sp. KCCM 40085 were purchased from the Korea Culture Center of Microorganisms (Seoul, Korea). In this study, all other chemicals and reagents were reagent-grade and were used without further purification. Preparation of Blueberry (Vaccinium crymbosum L.) Extract. Dried blueberries were ground to powder with a grinding machine. Approximately 100 g of the powder was extracted three times with 700 mL of distilled water (DW). This experiment for extraction was done each time during 3 h in the water bath at 90 °C, and then their residue was filtered through Whatman No. 1 filter paper (Whatman Ltd., England). The extracts were evaporated using a rotary evaporator under vacuum condition. The residual crude extracts were freeze-dried at −80 °C. The BE were stored at −20 °C until further use. The yield value of BE was 12% B


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Western Blot Analysis. After removing the hippocampus, the tissues (Con, PC, BE, and BV group) were sliced into 400 μm-thick coronal sections. Each tissue section was homogenized with lysis buffer (containing 50 mM Tris-Cl, pH 8.0, 0.1% SDS, 150 mM NaCl, 1% NP-40, 0.02% sodium azide, 0.5% sodium deoxycholate, 100 pg/mL PMSF and 1 pg/mL), and then centrifuged at 13,000 × g for 20 min at 4 °C. After centrifugation, the separated supernatant was collected and measured by using Bradford reagent (Bio-Rad, Hercules, CA, USA) with a standard of bovine serum albumin. Equal amounts (20 μg) were separated on a 10% SDS-PAGE gel and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Each membrane was blocked using TBST solution (containing 5% nonfat dry milk in Trisbuffered saline with 0.1% Tween 20) for 1 h at room temperature. The membranes were incubated with the following primary rabbit antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA): antiBDNF (1:1000), anti-CREB (1:1000), anti-pCREB (1:1000), antiAKT (1:1000), anti-pAKT (1:1000) and monoclonal β-actin (1:1000) at 4 °C overnight. The membranes were then washed with TBTS solution and incubated with secondary rabbit antibodies (1:2000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at room temperature. Finally, the expressed protein levels were detected using a chemiluminescence kit (ECL, SurModics, MN, USA). The amounts of expressed protein were calculated by using ImageJ 1.46 software (NIH, Bethesda, MD, USA). Protein levels were normalized to β-actin. Statistical Analyses. The results of behavioral tests, and the determination of AChE activity and ACh levels are expressed as mean ± standard deviation (S.D.). Data from the behavioral tests, and the determination of ACh levels and AChE activity, as well as SOD, catalase, and MDA were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test using GraphPad Prism 5 (GraphPad software, Inc., La Jolla, CA). Statistical significance was set at P < 0.05.

mice (8 mice per each group) were used in this test. In addition, this test underwent two separate trials as follows: 1) a training trial and 2) a test trial 24 h later. Prior to each training trial test, the PC mice were given 5 mg/kg of donepezil (p.o.), and the other mice were given 120 mg/kg of BE (p.o.) or 120 mg/kg of BV (p.o.). After 30 min, memory impairment was induced by using 1 mg/kg of Sco (i.p.). At 24 h after the training trial, each mouse was again placed into bright chamber. The time taken for each mouse to enter the dark chamber after the door opening was defined as latency for both the training and the test trials. In this experiment, each sample was administered 1 h before the training trial without Sco treatment for the avoidance of a ceiling effect in unimpaired animals. The latency time to move the dark chamber was manually recorded up to 300 s. Also, the intensity of electric foot shock was set at 0.25 mA for 3 s. The level of low intensity shock contributed to investigate the potentially improving effects of donepezil, BE, and BV. Determination of ACh and AChE Activity in the Hippocampus and Cortex. To measure the activity of ACh and AChE in the hippocampus and cerebral cortex, an enzyme-linked immunosorbent (ELISA) assay (The Amplex Red ACh/AChE assay kit, Molecular probes, Grand Island, NY) was used. A working mixture solution containing 400 μM Amplex Red reagent, 0.2 U/mL choline oxidase, and 2 U/mL horseradish peroxidase were prepared, along with 100 μM ACh. The working mixture solution (100 μL) and 100 μM ACh were mixed and then reacted in microplate wells containing the samples. The fluorescence released by the samples was measured by using a fluorescence microplate reader (Molecular Devices, Sunnyvale, CA) at an excitation wavelength of 571 nm and an emission wavelength of 590 nm. Measurement of Superoxide Dismutase (SOD), Catalase, and MDA in the Cerebral Cortex. Following behavioral testing, all mice were anesthetized using ether. The brain was immediately removed after the sacrifice of 16-fasted mice, and then washed with ice-cold saline. The brain was homogenized with 400 mM NaCl and 12.5 mM sodium phosphate buffer (pH 7.0). The homogenized brain was centrifuged at 3000g for 10 min at 4 °C and the separated supernatant was used for the SOD, catalase, and MDA tests. The level of SOD and catalase in the cerebral cortex were detected by using analysis kits of antioxidant enzyme (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). MDA content in the brain was analyzed by using the thiobarbituric acid-reactive substances (TBARS) assay according to a modified method of Mihara and Uchiyama.21 Tissue Preparation and Cresyl Violet Staining. The brains from each group were removed and postfixed for 6 h. Each brain tissue was cryoprotected by soaking them in 30% sucrose during 12 h. Thereafter, each tissue was frozen and sliced into 30 μm thick coronal sections by using CM1100 Leica cryomicrotome (Leica, Wetzlar, Germany). The tissue sections were collected in the 6-well plates with phosphate-buffered saline (PBS, pH 7.4). The neuronal damaged cells in the cornu ammonis (CA) 1 area of the hippocampus was observed using cresyl violet staining. Each tissue section that were analyzed came from the mice that performed the behavioral tests. For the cresyl violet staining, hippocampus tissues were immersed in a 0.5% cresyl violet solution for 3 min. The tissue sections were imaged using the image analysis system Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD). In addition, the number of viable neuronal cells in a 50 × 50 μm2 area of CA1 regions of the hippocampus was counted using image analysis system. Immunohistochemistry Detection of Expressed pCREB. After behavioral testing, CON, NC, and BV group were anaesthetized with ether, and then the brains were removed and fixed in 5% paraformaldehyde for 6 h. Each brain tissue was cryoprotected by soaking in 30% sucrose during 12 h, and then the tissues were sliced into 5 μm thick coronal sections by CM1100 Leica cryomicrotome (Leica, Wetzlar). Each tissue section was incubated in 5% normal coat serum and 0.3% Triton X-100 and for 1 h, followed by incubation with a pCREB antibody. The sections were incubated with the secondary rabbit antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and then detected using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD).

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RESULTS HPLC Analysis of BE and BV. The chemical composition of BE and BV were analyzed using a HPLC system (Table 1, Table 1. Contents (mg/mL) of Polyphenol Compounds in the Blueberry Extract and Vinegara content (mg/mL) compds L-ascorbic acid ellagic acid gallic acid D-catechin vanillic acid caffeic acid cyanidin chloride epicatechin chlorogenic acid myricetin quinic acid naringin kaempferol

BEb 1.73 0.66 0.21 0.41 2.25 2.02 26.34 20.24 2.43 2.68 1.35 1.15 1.35

± ± ± ± ± ± ± ± ± ± ± ± ±

BVc 0.03 0.04 0.01 0.04 0.15 0.42 0.54 0.66 0.48 0.35 0.31 0.43 0.31

0.34 0.56 0.25 1.74 0.31 5.54 28.54 22.24 8.68 5.35 6.98 6.25 6.62

± ± ± ± ± ± ± ± ± ± ± ± ±

0.03 0.04 0.01 0.04 0.04 0.52 0.54 0.56 0.35 0.31 0.34 0.43 0.38

Data represent means ± SD (n = 3). bBE, blueberry extract. cBV, blueberry vinegar.

a

and Figure 1). The chromatogram of each sample was obtained at 520 nm and successfully separated into 13 polyphenol compounds. As shown in Table 1, the polyphenol contents of BV containing cyanidin chloride (28.45 ± 0.54 mg/mL), epicatechin (22.24 ± 0.56 mg/mL), and chlorogenic acid (8.68 ± 0.35 mg/mL) were slightly higher than those of BE. Also, BV was identified that the content of acetic acid (42.31 ± 1.53 mg/ mL) was increased by a two-stage fermentation (Figure 1). C


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Figure 1. HPLC chromatogram of compounds in water extract and vinegar of highbush blueberry (Vaccinium crymbosum L). (A) HPLC chromatogram of blueberry extract (BE). (B) HPLC chromatogram of blueberry vinegar (BV). Data represent means ± SD (n = 3).

Figure 2. Effects of blueberry extract and vinegar on average spontaneous alternation (A) and total arm entry (B) during the Y-maze test. Donepezil as positive control (PC; 5 mg/kg, p.o.), blueberry extract (BE, 120 mg/kg, p.o.), or blueberry vinegar (BV, 120 mg/kg, p.o.) was administered to mice 1 h prior to the Y-maze tests. After 30 min, the mice were injected with scopolamine (Sco; 1 mg/kg, i.p.) and tested in the Y-maze. Data represent means ± SD (n = 8). ###P < 0.001 compared with the control (CON) group; **P < 0.01 and ***P < 0.001 compared with the negative control (NC) group.

Effects of BE and BV on Spontaneous Alternation Behavior in the Y-Maze Test. The effects of BE and BV on short-term memory were investigated in the spontaneous alternation behavior Y-maze test (Figure 2). Only NC group exhibited a significant decrease in the spontaneous alternation behavior compared with the CON group (Figure 2A, ###P < 0.001). The behavioral decline of NC group was significantly reversed by BE (Figure 2A, **P < 0.01) and BV (Figure 2A, ***P < 0.001) groups. Interestingly, BV group alone (***P < 0.001) dramatically enhanced the spontaneous alternation behavior as similar to the results from the PC group, but no significant difference in the number of arm entries among all the groups (Figure 2B). Effects of BE and BV on the Passive Avoidance Test. We also examined the effects of BE and BV on short-term memory using the passive avoidance test as shown in Figure 3. During the training trials test of passive avoidance, there was no significant difference in latency between any groups, but a significant group effect was clearly observed in latency from the test trials. From the results, the NC group displayed significantly reduced the step-through latency time in the test trials (###P < 0.001). Whereas, the PC (***P < 0.001), BE (*P < 0.05), and BV (**P < 0.01) groups did not experience a negative effect. Furthermore, the mice treated with BV

Figure 3. Effects of blueberry extract and vinegar on the average latency time during the passive avoidance test. Donepezil as positive control (PC; 5 mg/kg, p.o.), blueberry extract (BE, 120 mg/kg, p.o.), or blueberry vinegar (BV, 120 mg/kg, p.o.) was administered to mice 1 h prior to the passive avoidance test. After 30 min, the mice were injected with scopolamine (Sco; 1 mg/kg, i.p.) and tested for passive avoidance. Data represent means ± SD (n = 8). ###P < 0.001 compared with the control (CON) group; *P < 0.05 and ***P < 0.001 compared with the negative control (NC) group. D


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Figure 4. Effects of blueberry extract and vinegar on acetylcholine (ACh) levels and acetylcholinesterase (AChE) activity. Mice were decapitated after training in the Y-maze test. The cerebral cortex (A) and hippocampus (B) of each group were dissected and measured using an ACh/AChE assay kit. Data represent means ± SD (n = 3). ###P < 0.001 compared with the control (CON) group; *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the negative control (NC) group. CON, Control; NC, negative control; PC, positive control; BE, blueberry extract; BV, blueberry vinegar.

Figure 5. Effects of blueberry extract and vinegar on superoxide dismutase (SOD; A), catalase (B) activity, and malondialdehyde level (MDA; C). The cerebral cortex of each mouse was dissected and measured using a thiobarbituric acid-reactive substances (TBARS) assay. Data represent means ± SD (n = 3). ###P < 0.001 compared with the control (CON) group; *P < 0.05 and **P < 0.01 compared with the negative control (NC) group. CON, Control; NC, negative control; PC, positive control; BE, blueberry extract; BV, blueberry vinegar.

and hippocampus. The BE and BV groups not only had significantly decreased amounts of AChE activity in the cerebral cortex of 16.09% (*P < 0.05) and 24.86% (**P < 0.001), respectively, compared with the NC group, but also showed the amounts of AChE activity in the hippocampus that were decreased by 21.95% (**P < 0.01) and 34.17% (***P < 0.001), respectively. Furthermore, the levels of ACh in the BV group were significantly increased by 21.76% (***P < 0.001) and 11.24% (*P < 0.05), respectively, similar to the PC group. Effects of BE and BV on SOD, Catalase, and MDA. The biomarkers of antioxidant enzymes (SOD and catalase) and

exhibited a longer latency than the donepezil treated group (PC group). Effects of BE and BV on ACh Levels and AChE Activity. The dysfunction of the cholinergic system plays a crucial role in the memory impairment in AD patients, and the changes of AChE activity in brain directly connect to the decline in cognitive function.5 Thus, we evaluated the effects of BE and BV on ACh levels and AChE activity in the cerebral cortex and hippocampus. The NC group showed significantly decreased ACh levels (Figure 4A, ###P < 0.001) and increased AChE activity (Figure 4B, ###P < 0.001) in both the cerebral cortex E


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Journal of Agricultural and Food Chemistry lipid peroxidation (MDA) in the cerebral cortex were measured as shown in Figure 5. The NC group had SOD and catalase activity that was decreased by 27.64% (###P < 0.001) and 29.93% (###P < 0.001), respectively (Figure 5A, B). Whereas, MDA content in the NC group was 6.82 ± 0.20 nmol/mg protein (Figure 5C, ###P < 0.001), which was significantly higher than that in the CON group (2.34 ± 0.02 nmol/mg protein). The BE and BV groups were found to have an increase in SOD activity (9.58% and 16.27% for BE and BV, respectively) and catalase (2.43% and 9.10% for BE and BV, respectively) compared to the NC group. Meanwhile, MDA was decreased (BE: 5.22 ± 0.20 nmol/mg protein, BV: 4.79 ± 0.91 nmol/mg protein) compared to the NC group. Neuronal Protective Effects of BE and BV in the Hippocampus. The protective effects by treatment with BE and BV on damaged neuronal cells (pyramidal cells) from the hippocampal CA1 region were measured. In the NC group (Figure 6B), the stained CA1 pyramidal cells in the stratum

Figure 7. Immunohistochemistry detection of expressed phosphorylated c-AMP-responsive element binding protein (pCREB) in the scopolamine-induced mice hippocampus. The hippocampus of the control (CON), negative control (NC), and blueberry vinegar (BV) groups were dissected and incubated with pCREB antibody.

Effects of BE and BV on the Expression of Proteins (BDNF, CREB, and AKT) in the Hippocampus. Previous studies have reported that BDNF, which is related to neural cell proliferations (i.e., growth, survival, protection, and repair etc.), showed the high-level expression in the hippocampus region.22 Thus, we evaluated the expression level of BDNF and downstream signaling pathways of CREB and AKT by Western blot assay. The level of BDNF expression was significantly increased in the BE (**P < 0.01) and BV groups (*P < 0.05); this was absent in the NC group (Figure 8A). Moreover, the profound reductions of pCREB and pAKT that are found following oxidative brain damage by Sco were attenuated by treatment with BV (***P < 0.001 and **P < 0.01, respectively) in comparison to the PC group (Figure 8B, C).

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DISCUSSION The study aimed to assess the neuronal protective effects of BV on Sco-induced memory impairment. Our data shows the consolidation of memory impairment induced by Sco in the trained animal model via the two behavioral assessment tasks. Intriguingly, we found that administrated BV (at a dose of 120 mg/kg for a 1 week) prevented the Sco-induced amnesia model. It is well-known that Sco is an antagonist of muscarinic acetylcholine receptors. It has been studied in animal models of AD, particularly for the processes of short-term memory and learning acquisition.23 Indeed, the increment of brain oxidative level which arise from administration of Sco proves the useful value of Sco-induced memory deficits in the animal model in which to evaluate for functional foods or drugs as therapeutic effects for dementia. ACh, which is a neurotransmitter, mainly acts to regulate cognitive functions.24 In one clinical study it was found that the reduction of ACh level in the brain of AD patients was a critical element in dementia.25 Furthermore, the ACh activity is mainly terminated by hydrolysis reaction of AChE, which is responsible for the formation of the degraded ACh products (i.g. acetate and choline) in the cortex and hippocampus region.26 Thus, one of the purposes of the present study was to evaluate the effects of BV on Sco-induced ACh levels and AChE activity in the cortex and hippocampus (Figure 4). It was displayed that Sco extremely increased AChE activity in the brains as shown in NC group (Figure 4A, B). Meanwhile, we found that BE and BV groups significantly prevented the AChE activity in the cortex and hippocampus (Figure 4B). In particular, BV greatly inhibited ACh degradation (Figure 4B), suggesting that it may inhibit Scoinduced memory impairments by mediating the inhibition of AChE activity. In the Y-maze test, spontaneous alternation behavior is directly indicative of short-term memory loss.27 The BE, BV, and PC groups had significantly increased spontaneous

Figure 6. Effects of blueberry extract and vinegar in cornu ammonis (CA) 1 neurons in the hippocampus. The hippocampus of the control (CON; A), negative control (NC; B), positive control (PC; C), blueberry extract (BE; D), and blueberry vinegar (BV; E) groups were dissected and immediately stained with cresyl violet. The graph (F) showed the quantitative results of the CA1 neuronal cell counts. Data represent means ± SD (n = 3). ##P < 0.01 compared with the control (CON) group; *P < 0.05 compared with the negative control (NC) group.

pyramidale (SP) were damaged in comparison to the CON group (Figure 6A). Meanwhile, the BE (Figure 6D) and BV (Figure 6E) groups were recovered the same areas as similar to the PC group (Figure 6C). Indeed, the number of restored pyramidal cells (Figure 6F) by treatment with BE (75%) and BV (85%, *P < 0.05) increased in comparison to NC group (56%, ##P < 0.01), and was similar to PC group (86%, *P < 0.05). Moreover, it was found that the expression of pCREB was restored at a protein level after administration of BV in the hippocampal CA1 pyramidal cells in comparison to NC group (Figure 7). F


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Figure 8. Effects of blueberry extract and vinegar on expression of brain-derived neurotrophic factor (BDNF), cAMP response element binding protein (CREB), phosphorylated CREB (pCREB), serine−threonine kinase (AKT), and phosphorylated AKT (pAKT) in the hippocampus. (A) BDNF levels in the hippocampus measured by Western blot assay and quantified BDNF/β-actin intensity. (B) pCREB and CREB levels in the hippocampus measured by Western blot assay and quantified pCREB/CREB intensity. (C) pAKT and AKT levels in the hippocampus measured by Western blot assay and quantified pAKT/AKT intensity. Data represent means ± SD (n = 3). ###P < 0.001 compared with the control (CON) group; *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the negative control (NC) group. CON, Control; NC, negative control; PC, positive control; BE, blueberry extract; BV, blueberry vinegar.

finding that Sco strongly increases the value of MDA content in the cortex and hippocampus33 supports our present findings. We also found that BE and BV significantly inhibited MDA levels and rescued the activities of ROS and catalase in the cortex (Figure 5), suggesting that the neuroprotective effects of BE and BV might be able to directly remove free radicals or inhibit oxidative stress in the brain. The chemical composition of BV was analyzed using HPLC (Table 1). The chromatogram of BV was obtained at 520 nm and successfully separated into 13 compounds. The results showed that the BV involving cyanidin chloride, epicatechin, and chlorogenic acid may participate in the regulation of oxidative stress as antioxidant agents and decrease lipid peroxidation activity in brain. These findings are in accord with those reported by Diaconeasa et al.,34 who found that the predominant phenolic acids in blueberries are delphinidin, cyanidin, and malvidin. In addition, it was already revealed that polyphenol-rich blueberry extract showed higher antioxidant properties and inhibition of AChE activity,9 suggesting that administration of BV involving various polyphenol contents could be possibly have neuroprotective effect. Also, the increased content of acetic acid in BV (Figure 1B) could be responsible for suppressed the MDA content and increased activity of antioxidant enzymes. It was previously reported that acetic acid bacteria produce precursors of alkali-stable lipids known as sphingolipids, and that consumption of these products such as vinegar expected improved memory and cognitive ability.35 This suggests that the administration of BV, which contained phenolic and organic acids, in our study suppressed Sco-induced memory impairment via the improvement of antioxidative functions in the brain. These results demonstrated that BV restores the memory dysfunction of cholinergic system in brain tissue sections by preventing Sco-induced neuronal damage. The present results were further supported by the morphological evaluation that showed that the damaged neuron cells induced by Sco in hippocampal CA1 regions were restored by treatment with BV (Figure 6). It has been shown that a blueberry-rich diet can reduce neuronal loss in the same region.36 These findings also strongly suggest that BV could be a useful functional material or food to provide the neuroprotective action against Sco-induced oxidative damage in hippocampal tissue.

alternation behavior (Figure 2A), which prevented the Scoinduced reduction in the behavior. The BV alone significantly ameliorated the spontaneous alternation behavior, implying that BV could be improved the short-term memory and protected the memory loss via the declined the AChE activity in the acetylcholine system. However, the total mean numbers of the arm entries were not different across all experimental groups, which revealed that all samples did not affected the locomotor activity (Figure 2B). Also, the passive avoidance test widely used for the investigation of memory impairment.22 It was revealed that administration of BE and BV inhibited the reduction in latency times of test trials induced by Sco, but did not different affect the latency during the training trials (Figure 3). This result implied that BE and BV improved the Scoinduced memory impairment without influence of locomotor and exploratory activity of the mice in the passive avoidance test. Additionally, BV alone strongly improved the latency time in the passive avoidance test, similar to that of the PC group (Figure 3), suggesting that BV prevented the Sco-induced short-term cognitive impairments via rescuing effect of acetylcholine system involving improvement of ACh activity and blockade of AChE hydrolysis (see Figure 4). Several studies have shown that the Sco-induced cognitive impairment in animal models was related to oxidative stress causing neuronal injury or cell death (e.g., cell apoptosis) in the brain, which illuminates the role of disturbed antioxidant activities (e.g., SOD, catalase, glutathione, etc.).28,29 Oxidative stress is known to be of cytotoxic consequence due to its production of oxyradicals and oxidants that react with cellular constituents.30 ROS are byproducts of the oxidative process and cause deterioration of normal metabolism and neuronal activity.31 Antioxidant enzyme and lipid peroxidation activity represent important biomarkers for the investigation of cognitive ability. In the preclinical and clinical study, there is already strong experimental evidence that oxidative stress was contributed to subsequent apoptosis in AD patients and directly associated in the AD pathogenesis.31,32 In order to investigate the regulating mechanism of BV activity on the memory impairment amnesia model in mice, we additionally measured the effects of BV on the activity of antioxidant enzyme (i.e., ROS and catalase) and level of lipid peroxide (i.e., MDA) in the mouse cerebral cortex (Figure 5). The previous G


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promising functional material or food for the protective agents of amnesia-related cognitive impairment.

We furthermore elucidated the antiamnesic effects of BV administration by studying several protein levels, including BDNF, pBDNF, CREB, pCREB, and AKT in the hippocampus. The memory improvement is regulated at the molecular level in neurons and by major signaling pathways such as hippocampal cAMP/cAMP-dependent protein kinase/pCREB signaling pathways. These pathways converge to activate the CREB in which binding of the promoter regions of various genes related to memory and synaptic plasticity.37 Several studies have already shown that the activation of CREB is specifically linked with the memory formation and affects members of the neurotrophin family involving BDNF, which is responsible for neuronal growth and cellular differentiation and proliferation in the central nervous system.38 The expression of BDNF is known to enhance neurogenesis via pCREB signaling, and it is directly related to the neuroprotection potency of growth factors involving proliferation, differentiation, and neurodegeneration.39 It reported that BDNF partially leads to control the neuronal function and memory through the AKT signaling pathway.40 Furthermore, it has been shown previously that AKT signaling is involved in promoting neuronal survival and is regulated by BDNF signaling.40 Our study demonstrated that administration of BV increased BDNF protein levels in hippocampal tissue (Figure 8A). In addition, the pCREB within the cells was markedly decreased by Sco, and increased by administration of BE (Figure 8B). Previous studies have demonstrated that increased level of CREB showed the neuroprotective effect against from oxidative stress, ROS-induced cell toxicity, and that its activity ameliorates cognitive impairment via the cholinergic system.41,42 This indicates that BV upregulates the expression of CREB, which is correlated to memory improvement in the Scoinduced mouse model. Indeed, we demonstrated through immunohistochemistry, which has high sensitivity to detect and locate targeted proteins in tissues, that pCREB was restored in the hippocampus CA1 region by treatment with BV in comparison to the NC group (Figure 7). These findings also support the present study. Moreover, we performed Western blotting for AKT and pAKT on hippocampus from Sco-induced mice model. The AKT family provides neuron cells with a survival signal that allows them to inhibit apoptosis in the brain.40 Several studies reported that phosphoinositide 3-kinease/AKT pathway has an antiapoptotic activity in a wide range of neuron cells.43 As shown in Figure 8C, BV was increased the levels of pAKT signaling in comparison to NC, suggesting that BV was improved the effect of neuronal protective via pAKT in our designed model. These findings are accordance with previous data from literature implying that AKT is an important key for researching the therapeutic agents of AD.44 In conclusion, we successfully developed BV by using a fermentation technique and investigated its effects on cognitive function using several behavioral tasks. The designed model of Sco-mediated Alzheimer’s-type is mainly related with cognitive impairments. BV showed the improvement of ACh level via inhibition of AChE activity, suggesting that BV could be exerted the memory-enhancing effect. Moreover, the BV group was visually observed the preventive effect of neuron dysfunction in the CA1 neurons of the hippocampus. Also, we demonstrated that fermented blueberry has potential antiamnesic effects that might be regulated via hippocampal BDNF/CREB/AKT signaling pathways. BV may be a

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +82 43-840-3570. Fax: +82 43-856-3572. E-mail: [email protected]. ORCID

Beong Ou Lim: 0000-0002-9618-5956 Funding

This paper was supported by Konkuk University in 2016. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED ACh, acetylcholine; AChE, acetylcholinesterase; AD, Alzheimer’s disease; AKT, serine−threonine kinase; BE, blueberry extract; BDNF, brain-derived neurotrophic factor;; BV, blueberry vinegar; CA, cornu ammonis; CON, control; CREB, cAMP response element binding protein; D.W., distilled water; MDA, malondialdehyde; NC, negative control; pAKT, phosphorylated-AKT; PC, positive control; pCREB, phosphorylated-CREB; ROS, reactive oxygen species; Sco, scopolamine; SOD, superoxide dismutase

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REFERENCES

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Cognitive Improving Effects by Highbush Blueberry (Vaccinium

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