Article pubs.acs.org/JAFC
Soyasaponins Ab and Bb Prevent Scopolamine-Induced Memory Impairment in Mice without the Inhibition of Acetylcholinesterase Sung-Woon Hong, Dae-Hyung Yoo, Jae-Yeon Woo, Jin-Ju Jeong, Jeong-hwa Yang, and Dong-Hyun Kim* Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung Hee University, Hoegi, Dongdaemoon-gu, Seoul 130-701, Republic of South Korea ABSTRACT: Soy (Glycine max, family Leguminosae), which contains isoflavones and saponins as main constituents, is known to exhibit memory-enhancing effects. Therefore, to investigate the role of soyasaponins in memory impairments, we isolated soyasaponins Ab (SA) and Bb (SB) from soybean and measured their protective effects against scopolamine-induced memory impairment in mice. SA and SB significantly prevented scopolamine-induced memory impairment in passive avoidance and Ymaze tasks. Compared to SA, SB rescued memory impairment more potently. Treatment with SB (10 mg/kg, p.o.) protected memory impairment in passive avoidance and Y-maze tasks to 97% (F = 68.10, P < 0.05) and 78% (F = 35.57, P < 0.05) of untreated normal control level, respectively. SA and SB (10 mg/kg) also rescued scopolamine-induced memory impairment in Morris water maze task (F = 14.51, P < 0.05). In addition, soyasaponins preserved brain-derived neurotrophic factor (BNDF) expression (F = 33.69, P < 0.05) and cAMP response element-binding (CREB) protein phosphorylation (F = 91.62, P < 0.05) in the hippocampus of scopolamine-treated mice. However, SA and SB did not inhibit acetylcholinesterase in vitro and ex vivo. On the basis of these findings, we suggest that soybean, particularly soyasaponins, may protect memory impairment by increasing BDNF expression and CREB phosphorylation. KEYWORDS: Glycine max, soyasaponin, memory, brain-derived neurotrophic factor, cAMP response element-binding protein
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INTRODUCTION Dementia is characterized by loss of cognitive functions including memory, attention, language, and problem solving. This is primarily caused by Alzheimer’s disease (AD) or vascular dementia.1 AD is a progressive neurodegenerative disease caused by β-amyloid deposition and abnormal tau protein, leading to deterioration of memory and cognitive functions.2−4 Learning and memory can be affected by impairments in cortical and hippocampal cholinergic systems.5 Cholinergic impairment by scopolamine is prevented by acetylcholinesterase (AChE) inhibitors, such as, donepezil, and cholinergic agonists, such as, carbachol.6,7 Scopolamine not only causes memory impairments, but also reduces brainderived neurotrophic factor (BDNF) expression. BDNF, a representative neurotrophin, plays an important role in memory formation and plasticity7,8 and facilitates long-term potentiation (LTP).9,10 Therefore, scopolamine is frequently used to generate memory-impaired animal models.11,12 Soy (Glycine max, family Leguminosae), which contains isoflavones and saponins as bioactive ingredients, has been reported to exhibit anticarcinogenic, antilipidemic, and estrogen-like effects.13,14 Recently, the memory-enhancing effect of soy has been reported.14−17 Among its components, isoflavones have been reported to exhibit memory-enhancing, antiinflammatory, and phytoestrogenic effects. However, the memory-enhancing effects of soyasaponins have not been studied. To understand the possible role of soyasaponins in memory impairment, we isolated the primary soyasaponins Ab (SA) and Bb (SB) (Figure 1) from soybean and investigated their preventive effects in scopolamine-induced memory impairment in mice. © 2014 American Chemical Society
MATERIALS AND METHODS
Chemicals. Scopolamine hydrobromide, tacrine (9-amino-1,2,3,4tetrahydroacridine hydrochloride), acetylthiocholine (ATCh), AChE (electric eel type VI−S), radioimmuno-precipitation assay (RIPA) buffer, and a phosphatase inhibitor cocktail were purchased from Sigma (St. Louis, MO, U.S.). cAMP response element-binding protein (CREB) and p-CREB were purchased from Cell Signaling Technology (Beverly, MA, U.S.). BDNF, β-actin, and other secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.). Isolation of SA and SB from Soybeans. SA (purity, > 91%) and SB (purity, > 95%) were obtained as previously reported.18−20 Briefly, dried soybeans (3 kg) were pulverized using a coffee mill, defatted with hexane (10 L), allowed to dry, extracted with ethanol (10 L) twice and concentrated in vacuo (65 g; soybean ethanol extract [SE] contained 0.1% soyasaponin Ab [SA] and 1.6% soyasaponin Bb [SB], whose contents were analyzed by high-performance liquid chromatography [HPLC], Figure 1C). The extract was suspended in water, extracted with n-butanol (15 L), and evaporated to dryness under reduced pressure (40 g). The residue was then dissolved in methanol and adsorbed on silica gel (40 g). The adsorbed material was transferred to a silica gel column (550 g, 6 × 40 cm). The column was eluted with CHCl3−MeOH−H2O (65:35:10, v/v) to obtain 6 subfractions (FA1 ≈ FA6). Subfractions FA2 and FA3 were successively subjected to medium pressure liquid chromatography to obtain soyasaponins Bb (35 mg) and Ab (25 mg), respectively. Chromatographic separation was carried out on an Ultra-Pak C18 column (300 × 37 mm, Yamazen Co., Ltd., Tokyo, Japan), which was eluted with a gradient system of 5% acetonitrile and 100% acetonitrile for 2 h. The flow rate was 4 mL/ min. Isolated soyasaponins Ab and Bb were identified by comparing them with authentic standards using instrumental (FAB-MS, 1H NMR, Received: Revised: Accepted: Published: 2062
March 8, 2013 January 19, 2014 January 22, 2014 January 22, 2014 dx.doi.org/10.1021/jf4046528 | J. Agric. Food Chem. 2014, 62, 2062−2068
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(Sungnam, Korea). All animals were housed in wire cages under a 12 h light/dark cycle (light on 07:30−19:30) at 20−22 °C and 50 ± 10% humidity and fed standard laboratory chow and water ad libitum. Passive Avoidance Task. Acquisition and retention of the passive avoidance task was assessed using a two-compartment acrylic box wherein a lighted compartment (20 × 20 × 20 cm3) was connected to a dark compartment (20 × 20 × 20 cm3) by an entrance door (5 × 5 cm2), according to the method of Lee et al.22 Briefly, the mice underwent acquisition and retention trials. In the acquisition trial, a mouse was initially placed in the lighted chamber, and the entrance door was opened after 10 s. When the mouse entered the dark chamber, the door automatically closed, and a 0.3 mA electrical shock for 2 s was delivered through the grid floor. One hour before the retention trial, the mice were orally administered with soy ethanol extract (SE, 100 and 200 mg/kg), SA or SB (5, 10, 20, and 40 mg/kg), tacrine (10 mg/kg) as a positive control,23 and vehicle (2% Tween 80, the scopolamine group) as a negative control. Memory impairment was induced by intraperitoneal injection of scopolamine (0.9 mg/kg) 30 min after oral administrations of test agents. The passive avoidance maze task was carried out 30 min after scopolamine treatment. A retention trial was carried out 24 h after the acquisition trial. Thereafter, the mice were placed in the light compartment again and the latency time taken to enter the dark compartment was recorded for each mouse. If the mouse did not enter the dark compartment within 300 s, then we concluded that the mouse remembered the foot shock from the acquisition trial. Y-Maze Task. Y-maze was used to measure the immediate spatial working memory, which is a form of short-term memory, and was carried out as previously described.22 A Y-maze is a three-arm horizontal maze symmetrically separated by 120° angles from each other (length, 40 cm; width, 3 cm; and height, 12 cm). The floor and wall of the maze were constructed using dark polyvinyl plastic, as previously reported.22 During the maze task, the mice were placed inside one arm, and the sequence (i.e., ACCABC, etc.) of arm entries were manually recorded for each mouse for 8 min. An actual alternation was defined as consecutive entries into all three arms (i.e., ABC, CAB, or BAC, but not BAB). Memory impairment was induced by intraperitoneal injection of scopolamine (0.9 mg/kg). To remove mouse odors, maze arms were cleaned between two consecutive tasks. Test agents. i.e., SE (100 and 200 mg/kg), SA or SB (10 mg/kg, p.o.), tacrine (10 mg/kg, p.o.), or vehicle (p.o.) were orally administered 1 h before the maze task. The maze task was carried out 30 min after treatment with scopolamine. The alternation percentage was calculated: alternation percentage = [(number of alternations)/(total arm entries −2)] × 100. The number of arm entries was served as the indicator of locomotor activity. Morris Water Maze Task. Spatial memory and learning was studied with the Morris water maze task by using a circular pool (diameter, 90 cm; height, 45 cm), according to the method described by Lee et al.22 First, the pool was filled with water (20 ± 1 °C) up to 30 cm depth, and nonfat milk (500 mL) was mixed with water. A white platform (diameter, 6 cm; height, 29 cm) was centered in one of four quadrants of the milk-filled pool (southwest area) and hidden 1 cm below the water surface. On the first day of the maze task, the mice were given swimming training for 60 s in the platform-free pool. Starting from the second day, the mice were given four swimming training trial sessions per day for four consecutive days. Escape latencies for each mouse were measured and recorded. For each mouse, this parameter was averaged for each trial session. Once the mouse located the platform, the mouse stayed on the platform for 10 s. If the mouse could not search the platform within 60 s, then the mouse was then placed on the platform for 10 s. During this period, a fixed platform was used. After each trial, the animals were taken to the cage and were dried under an infrared lamp. The trial time interval was 30 s. On the last day of training, the mice were underwent a probe trial session after the platform was removed, wherein they were allowed to swim for 60 s. The swimming time spent in platform quadrant section of the pool was recorded. SA or SB (10 mg/kg, p.o.), tacrine (10 mg/ kg, p.o.), or vehicle (p.o.) was administered 1 h before the maze task was conducted at every consecutive day. Scopolamine (0.9 mg/kg)
Figure 1. Structures of soyasaponins Ab (A) and Bb (B) Isolated from Glycine max Merr and HPLC chromatogram (C) of soybean ethanol extract (a) and isolated SA (b) and SB (c). C NMR) analysis, similar to as reported previously.18−21 The purities of the isolated soyasaponins were assayed by HPLC (Hewlett-Packard 1100 series, Ramsey, MN, U.S.) equipped with Zorbax Eclipse Plus C18 column (4.6 × 100 mm, 5 μm, Agilent Technologies, Santa Clara, CA, U.S.), a HP 1100 series binary pump, a HP 1100 series autosampler, and UV detector (203 nm). The HPLC was done by eluting with a linear gradient of 10% CH3CN to 60% CH3CN (flow rate, 1 mL/min) for 15 min. The retention times of soyasaponin Ab and Bb were 9.92 and 12.03 min, respectively. Soyasaponin Ab. White amorphous powder. FAB-MS: m/z 1436 [M − H]¯. Soyasaponin Bb. White amorphous powder. FAB-MS: m/z 941 [M + H]+. AChE Activity Assay. AChE activity was measured according to the method described by Lee et al.22 Briefly, a reaction mixture (225 μL) containing 15 mM ATCh (25 μL), 3 mM DTNB (125 μL), 50 mM Tris-HCl (50 μL, pH 8.0), and test agents (25 μL) in a microplate was preincubated for 10 min. AChE (0.226 U/mL, 25 μL) was then added in the mixture, and scanned for 10 min at 405 nm in a microplate reader (Model Biotek μQuant MQX200, Winooski, VT, U.S.). Enzyme activity was indicated as a percentage of the activity when treated with vehicle instead of inhibitors. Animals. All animal experiment protocols were performed according to the National Institute of Health (U.S.) and Kyung Hee University guidelines for Laboratory Animals Care and Usage and approved by the University Animal Care and Usage Committee of Laboratory Animals in the College of Pharmacy, Kyung Hee University. Male ICR mice (18−22 g, 5 weeks) were obtained from the Orient Animal Breeding Center, a branch of Charles River Laboratories 13
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was intraperitoneally administered 30 min after oral administrations of test agents. Immunoblotting. Mice were sacrificed 30 min after the acquisition trial of the passive avoidance task (2 h after scopolamine treatment). The hippocampi were promptly excised and homogenized in 200 μL of ice-cold RIPA buffer containing a protease inhibitor cocktail. The homogenates were centrifuged for 15 min at 13 000g. The resulting supernatants were aliquoted into Eppendorf tubes and stored at −80 °C until required. The expression levels of BDNF, CREB, p-CREB, and β-actin were measured by immunoblotting according to the method of Lee et al.22 The protein assay was performed using Bradford protein assay kit. Ex vivo AChE Activity Assay. The mice were randomly divided into 4 groups (n = 4) and were orally administered vehicle (2% tween), SA (10 mg/kg), SB (10 mg/kg), and tacrine (10 mg/kg). The mice were sacrificed 60 min after each administration, and their brains were removed. Their hippocampi were dissected and homogenized in cold RIPA buffer. The homogenates were centrifuged at 10 000g for 10 min at 4 °C, and the resulting supernatants were used as enzyme sources. Their AChE activities were measured as triplicates. The protein amount was determined using Bradford protein assay kits (Bio-Rad, Hercules, CA, U.S.). AChE activity per protein amount of the homogenate supernatant (mg) was calculated. Statistics. All experimental data are expressed as mean ± standard error of mean (SEM). Distributions in the passive avoidance and Y-maze tasks were depicted by box-plots. A box-plot consists of a box and tails for each group. The horizontal line through the box depicts the median, and the top and bottom of the box mark the upper and lower quartiles, respectively. For passive avoidance task, the significance among data was analyzed by a Kruskal−Wallis nonparametric one-way analysis of variance (ANOVA) test. For the Y-maze, and Morris water maze tasks, the significance was analyzed by ANOVA followed by Student− Newman−Keuls test for multiple comparisons. Statistical significance was set at p < 0.05.
Figure 2. The protecting effect of SE against scopolamine-induced memory impairment in mice. (A) Effect of SE in passive avoidance task. (B) Effect of SE in Y-maze task. SE (100 or 200 mg/kg, p.o.), tacrine (TC, 10 mg/kg, p.o.) or vehicle (the same volume of 2% Tween 80) was administered to mice 1 h before treatment with scopolamine. Memory impairment was induced by scopolamine treatment (0.9 mg/kg i.p.). Normal control mice were treated with vehicle instead of scopolamine. Six different animals were used in each treatment group. Acquisition trials were carried out 30 min after a single scopolamine treatment. Test agents (NOR, none; SCO, scopolamine alone; SE100, 100 mg/kg SE with scopolamine; SE200, 200 mg/kg of SE with scopolamine; closed triangle, 10 mg/kg tacrine with scopolamine) was orally treated 1 h before maze task. BoxplotsTo depict a distribution, a box-plot consists of a box and tails for each group. The horizontal line through the box depicts the median, and the top and bottom of the box mark the upper and lower quartiles. #Significantly different from normal mice (P < 0.05). *Significantly different from scopolamine-treated control (P < 0.05).
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RESULTS SE Protected Scopolamine-Induced Memory Impairment. Preliminary experiment indicated that SE potently prevented scopolamine-induced memory impairment in passive avoidance and Y-maze tasks. The performance of mice treated with SE (200 mg/kg, p.o.) and scopolamine in passive avoidance and Y-maze tasks was 43% (F = 11.97, P < 0.05) and 98% (F = 4.84, P < 0.05), respectively, of vehicle-treated normal control mice (Figure 2). Therefore, to understand the possible role of soyasaponins in memory impairment, we isolated the two main soyasaponins SA and SB and identified them by FAB-MS and 1H- and 13C NMR. Isolated SA and SB exhibited soyasponin-specific seven angular methyl singlet signals [δ 0.70 (25-CH3), 0.86 (26CH3), 0.96 (29-CH3), 0.99 (28-CH3), 1.23 (27-CH3), 1.30 (30CH3), 1.45 (23-CH3)] and [δ 0.67 (25-CH3), 0.90 (26-CH3), 1.23 (29-CH3), 1.26 (28-CH3), 1.33 (27-CH3), 1.35 (30-CH3), and 1.41 (23-CH3)], respectively, in 1H NMR and m/z 1436 [M − H]− and m/z 941 [M + H]+, respectively, in FAB-MS. SA and SB Prevented Scopolamine-Induced Memory Impairment. We investigated the preventive effects of SA and SB against scopolamine-induced memory impairment in mice in the passive avoidance task (Figure 3A,B). Both SA and SB significantly prevented scopolamine-induced memory impairment. SB protected memory impairment; however, the effect was more with SB than with SA. SA and SB at a dose of 10 mg/ kg (p.o.) restored memory impairment to 86% (F = 85.33, P < 0.05) and 97% (F = 68.10, P < 0.05) of untreated normal control mice, respectively. These effects are comparable to that of tacrine (10 mg/kg), a positive control, which restored the
step-through latency to 91% of untreated normal control mice. However, no significant differences among the groups treated with SA (10 mg/kg), SB (10 mg/kg), and tacrine (10 mg/kg) were observed. During the acquisition trial, no latency differences were observed among test groups (Figure 3A,B). On the Y-maze task, SA and SB (10 mg/kg) restored spontaneous alteration, which was lowered by scopolamine (Figure 3C). Compared to SA, SB restored memory impairment more potently. SB (10 mg/kg, p.o.) significantly restored the lowered spontaneous alteration induced by scopolamine to 78% of that of untreated normal control mice (F = 35.57, P < 0.05). However, no significant differences between those of SA and SB were observed. 2064
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We also investigated the effect of SA and SB (10 mg/kg, p.o.) on escape latencies in the Morris water maze task (Figure 4A). Escape latencies were prolonged by scopolamine treatment. SA significantly shortened the escape latencies on the fifth day (F = 14.51, P < 0.05). However, SB-treated mice showed
Figure 4. Effects of soyasaponins Ab (SA) and Bb (SB) on scopolamine-induced memory impairment in morris water maze tasks. (A) Effects in escape latencies during training trial sessions. (B) Effects in swimming times spent in target quadrant. (C) Effects in swimming speed. Soyasaponins (10 mg/kg, p.o.) or tacrine (TC, 10 mg/kg, p.o.) was given 1 h before the first trial session at every consecutive day. Memory impairment was induced with scopolamine (0.9 mg/kg, i.p.) at 30 min after treatment with each test agent. Normal group was treated with vehicle instead of scopolamine and test agents. The training trials for 5 days and the probe trial sessions in the last day were performed as described in the Materials and Methods. Open square, group treated with scopolamine; closed square, normal control group; open circle, group treated with scopolamine in the presence of SA; closed circle, group treated with scopolamine in the presence of SB; closed triangle, group treated with scopolamine in the presence of tacrine. Values are expressed as mean ± SEM (n = 6). # Significantly different from normal mice (P < 0.05). *Significantly different from scopolamine-treated control (P < 0.05).
Figure 3. Effects of soyasaponins Ab (SA) and Bb (SB) on scopolamine-induced memory impairment in mice in the passive avoidance and Y-maze tasks. (A) Effect of SA in passive avoidance task. (B) Effect of SB in passive avoidance task. (C) Effects of SA and SB in Y-maze task. Soyasaponins (5, 10, 20, or 40 mg/kg, p.o.), tacrine (TC, 10 mg/kg, p.o.) or vehicle (the same volume of 2% Tween 80) was administered to mice 1 h before treatment with scopolamine. Memory impairment was induced by scopolamine treatment (0.9 mg/kg i.p.). Normal control mice were treated with vehicle instead of scopolamine. Six different animals were used in each treatment group. Acquisition trials were carried out 30 min after a single scopolamine treatment. The retention trials were carried out for 5 min, 24 h after the acquisition trials. Box plot details see Figure 2. #Significantly different from normal mice (P < 0.05). *Significantly different from scopolamine-treated control (P < 0.05). 2065
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Figure 5. Effects of soyasaponins Ab (SA) and Bb (SB) on aceytlcholinesterase (AChE) activity in vitro and on BDNF expression and CREB phosphorylation in the hippocampus of scopolamine-treated mice. (A) Effect on AChE activity. The inhibitory potencies of SA (closed circle), SB (closed triangle), and tacrine (closed square) against AChE activity were measured in vitro by using electric eel type VI−S AChE. (B) Effect in hippocampal acetylcholinesterase activity. SA (10 mg/kg, p.o.), SB (10 mg/kg, p.o.) or tacrine (TC, 10 mg/kg, p.o.) was orally administered to mice only once and sacrificed 60 min after treatment with test agents and measured hippocampal AChE activity (n = 4). Each sample was measured as triplicates. (C) Effect on BDNF expression. SA (10 mg/kg, p.o.), SB (10 mg/kg, p.o.) or tacrine (TC, 10 mg/kg, p.o.) was orally administered to mice for once. Control group received an equal volume of vehicle for the same period. Memory impairment was induced by scopolamine treatment (0.9 mg/kg, i.p.) 1 h after the administration of each test agent. The normal control group was treated with vehicle instead of scopolamine and test agents. The expression levels of BDNF, CREB, and p-CREB were measured by immunoblotting. (D) Intensity graph showing the quantification of BDNF/β-actin and p-CREB/CREB expression levels measured by densitometry. β-actin was used as an internal control. Values are expressed as mean ± SEM (n = 3). #Significantly different from normal mice (P < 0.05). *Significantly different from scopolamine-treated control (P < 0.05).
increased, as previously reported.7,12 No significant difference in β-actin level was observed.
significantly shorter latencies from the third day (F = 6.70, P < 0.05) to the fifth day (F = 14.51, P < 0.05). On the day after the last training session, the mean swimming time within the platform quadrant was significantly lower in mice treated with scopolamine alone than in those untreated normal control mice. Soyasaponins increased the swimming time within the platform quadrant (F = 21.52, P < 0.05) (Figure 4B). SB increased the target quadrant swim time more than SA. The effect of SB is comparable to that of tacrine in Morris water maze task. No significant differences in swimming speed within the target quadrant were observed between the groups treated with vehicle alone, scopolamine, and/or test agents (Figure 4C). This demonstrates that scopolamine and soyasaponins have no effect on general locomotor behavior. Next, we measured the inhibitory effects of these soyasaponins on AChE activity in vitro. These soyasaponins did not inhibit AChE activity (IC50, > 0.2 mM), however, tacrine potently inhibited it (IC50 = 0.12 μM) (Figure 5A). We also measured AChE activity in hippocampi of mice orally treated with SA, SB, or tacrine (10 mg/kg). Treatment with SA or SB did not inhibit hippocampal AChE activity, whereas treatment with tacrine inhibited it by 31% (Figure 5B). Next, we investigated the effect of SB on BDNF expression and CREB phosphorylation in the hippocampi of scopolaminetreated memory-impaired mice (Figure 5C,D). Scopolamine significantly reduced BDNF expressions and CREB phosphorylation. However, when SA or SB was administered in these mice, BDNF expression (F = 33.69; P < 0.05) and CREB phosphorylation (F = 91.62; P < 0.05) were significantly
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DISCUSSION A decrease in cholinergic function, particularly within the basal forebrain, can decline memory and cognitive function with age.5 A commercial anticholinergic drug, scopolamine, causes memory impairment in healthy young people, which is the equivalent of memory impairment observed in nondemented drug-free elderly people. Furthermore, several studies have reported that scopolamine is increasingly disruptive with age and declines cognitive function.7 This is consistent with the previously reported hypothesis that the function of cholinergic neurons decreases with increasing age and dementia.5,7 In the present study, SE and its constituents SA and SB significantly prevented scopolamine-induced memory impairment in mice in the passive avoidance, Y-maze and Morris water maze tasks. SB rescued memory impairment more potently than SA. However, both SA and SB did not inhibit AChE activity in vitro and ex vivo. Orally administered SA and SB are metabolized to their aglycones by intestinal microbiota of mouse and human feces.19,24 Therefore, to elucidate the ability of SA, SB, and their metabolites soyasapogenols A and B, their AChEinhibitory activities were measured. Soyasaponins and their metabolites did not inhibit AChE activity. These results suggest that the amelioration of memory impairment by SA and SB may not be dependent on AChE inhibition. When soyasaponins were orally administered to mice, these compounds might be metabolized to their aglycones soyasapogenols A and B, which are possibly the active 2066
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Cholinergic Neurons in the Nucleus Basalis. Ann. Neurol. 1981, 10, 122−126. (3) Hardy, J.; Selkoe, D. J. The Amyloid Hypothesis of Alzheimer’S Disease: Progress and Problems on the Road to Therapeutics. Science 2002, 297, 353−356. (4) Goedert, M.; Spillantini, M. G.; Crowther, R. A. Tau Proteins and Neurofibrillary Degeneration. Brain Pathol. 1991, 1, 279−286. (5) Araujo, J. A.; Studzinski, C. M.; Milgram, N. W. Further Evidence for the Cholinergic Hypothesis of Aging and Dementia from the Canine Model of Aging. Prog. Neuropsychopharmacol. Biol. Psych. 2005, 29, 411−422. (6) Kotani, S.; Yamauchi, T.; Teramoto, T.; Ogura, H. Pharmacological Evidence of Cholinergic Involvement in Adult Hippocampal Neurogenesis in Rats. Neuroscience 2006, 142, 505−514. (7) Tariot, P. N.; Patel, S. V.; Cox, C.; Henderson, R. E. Age-Related Decline in Central Cholinergic Function Demonstrated with Scopolamine. Psychopharmacology 1996, 125, 50−56. (8) Hock, C.; Heese, K.; Hulette, C.; Rosenberg, C.; Otten, U. Region-Specific Neurotrophin Imbalances in Alzheimer Disease: Decreased Levels of Brain-Derived Neurotrophic Factor and Increased Levels of Nerve Growth Factor in Hippocampus and Cortical Areas. Arch. Neurol. 2000, 57, 846−851. (9) Lu, B.; Pang, P. T.; Woo, N. H. The Yin and Yang of Neurotrophin Action. Nat. Rev. Neurosci. 2005, 6, 603−614. (10) Lu, Y.; Christian, K.; Lu, B. BDNF: a Key Regulator for Protein Synthesis-Dependent LTP and Long-Term Memory? Neurobiol. Learn. Mem. 2008, 89, 312−323. (11) Kim, D. H.; Jeon, S. J.; Son, K. H.; Jung, J. W.; Lee, S.; Yoon, B. H.; Lee, J. J.; Cho, Y. W.; Cheong, J. H.; Ko, K. H.; Ryu, J. H. The Ameliorating Effect of Oroxylin a on Scopolamine-Induced Memory Impairment in Mice. Neurobiol. Learn. Mem. 2007, 87, 536−546. (12) Joh, E. H.; Yang, J. W.; Kim, D. H. Gypenoside LXXIV Ameliorates Scopolamine-Induced Learning Impairment in Mice. Planta Med. 2010, 76, 793−795. (13) Kim, S. L.; Berhow, M. A.; Kim, J. T.; Chi, H. Y.; Lee, S. J.; Chung, I. M. Evaluation of Soyasaponin, Isoflavone, Protein, Lipid, And Free Sugar Accumulation in Developing Soybean Seeds. J. Agric. Food Chem. 2006, 54, 10003−10010. (14) Liu, Y. Q.; Xin, T. R.; Lü, X. Y.; Ji, Q.; Jin, Y.; Yang, H. D. Memory Performance of Hypercholesterolemic Mice in Response to Treatment with Soy Isoflavones. Neurosci. Res. 2007, 57, 544−549. (15) Kato-Kataoka, A.; Sakai, M.; Ebina, R.; Nonaka, C.; Asano, T.; Miyamori, T. Soybean-Derived Phosphatidylserine Improves Memory Function of the Elderly Japanese Subjects with Memory Complaints. J. Clin. Biochem. Nutr. 2010, 47, 246−255. (16) File, S. E.; Jarrett, N.; Fluck, E.; Duffy, R.; Casey, K.; Wiseman, H. Eating Soya Improves Human Memory. Psychopharmacology 2001, 157, 430−436. (17) Pan, M.; Li, Z.; Yeung, V.; Xu, R. J. Dietary Supplementation of Soy Germ Phytoestrogens or Estradiol Improves Spatial Memory Performance and Increases Gene Expression of BDNF, TrkB Receptor and Synaptic Factors in Ovariectomized Rats. Nutr. Metab. 2010, 7, 75. (18) Lee, I. A.; Park, Y. J.; Yeo, H. K.; Han, M. J.; Kim, D. H. Soyasaponin I Attenuates TNBS-Induced Colitis in Mice by Inhibiting NF-kappaB Pathway. J. Agric. Food Chem. 2011, 58, 10929−10934. (19) Chang, S. Y.; Han, M. J.; Joh, E. H.; Kim, D. H. Liquid Chromatography/Mass Spectrometry-Based Structural Analysis of Soyasaponin Ab Metabolites by Human Fecal Microflora. J. Pharm. Biomed. Anal. 2010, 52, 752−756. (20) Kitagawa, I.; Taniyama, T.; Nagahama, Y.; Okubo, K.; Yamauchi, F.; Yoshikawa, M. Structure of Acetyl-Soyasaponin A1, A2 and A3, Astringent Partially Acetylated Bisdesmoside of Soyasapogenol a Form American Soybean, The Seed of Glycine Max MERRILL. Chem. Pharm. Bull. 1988, 36, 2819−2828. (21) Kitagawa, I.; Yoshikawa, M.; Yosioka, I. Saponin and Sapogenol. XIII. Structures of Three Soybean Saponins: Soyasaponin I, Soyasaponin II, and Soyasaponin III. Chem. Pharm. Bull. 1976, 24, 121−129.
candidates responsible for preventing memory impairment. However, these metabolisms may not be quickly catalyzed by intestinal microbiota. These results suggest that the effect of SA and SB may be due to parental compounds rather than their metabolites by intestinal microbiota. Nevertheless, both SA and SB prevented scopolamine-induced memory impairment. The memory impairment-ameliorating effect of SB was more potent than that of SA in passive avoidance (5 mg/kg, p.o.) and Ymaze tasks (10 mg/kg, p.o.). However, the effects of SA and SB were not significantly different. Although the AChE-inhibitory effects of SA and SB may be different between in vitro and in vivo studies, these soyasaponins may ameliorate scopolamineinduced memory impairment in mice without the inhibiton of acetylcholinesterase. Furthermore, in the mouse hippocampus, SA and SB potently restored scopolamine-mediated reduced expression of BDNF. BDNF influences neuronal synaptic plasticity by depolarizing neurons as rapidly as glutamate does by activating tyrosine kinase receptors,25 enhancing glutamatergic synaptic transmission,26 increasing the N-methyl-Daspartate receptor subunit phosphorylation in the hippocampus, and facilitating hippocampal LTP.27 Notably, the expression of BDNF in the entorhinal cortex and hippocampus is lower in patients with AD than in healthy male.8,28 CREB may be the most important transcription factor regulating BDNF expression.29 Being a cellular transcription factor, CREB regulates the expression of BDNF, tyrosine hydroxylase, and many neuropeptides.29−31 Several studies have suggested that BDNF and CREB may possess therapeutic potential in patients with AD.22,32,33 In the present study, we demonstrated that scopolamine inhibited CREB phosphorylation, as previously reported.22,33 Orally administered SA and SB potently restored scopolamine-mediated reduction of CREB phosphorylation. Overall, compared to SA, SB prevented scopolamine-induced memory impairment more potently. These studies suggest that soyasaponins restore the reduction of BDNF expression and CREB phosphorylation caused by scopolamine and reverse neuronal synaptic plasticity mediated by BDNF, which binds cell surface receptors such as TrkB and low-affinity nerve growth factor receptor. On the basis of these findings, we suggest that soybean, particularly soyasaponins, ameliorates scopolamine-induced memory impairment by increasing BDNF expression and CREB activation.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +82-2-961-0374; fax: +82-2-957-5030; e-mail: dhkim@ khu.ac.kr. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED AChE,acetylcholinesterase; AD,Alzheimer’s disease; ATCh,acetylthiocholine; BDNF,brain-derived neurotrophic factor; CREB,cAMP response element-binding protein; LTP,longterm potentiation; RIPA,radioimmuno-precipitation assay; SA,soyasaponin Ab; SB,soyasaponin Bb; SE,soy ethanol extract
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REFERENCES
(1) Everitt, B. J.; Robbins, T. W. Central Cholinergic Systems and Cognition. Annu. Rev. Psychol. 1997, 48, 649−684. (2) Whitehouse, P. J.; Price, D. L.; Clark, A. W.; Coyle, J. T.; DeLong, M. R. Alzheimer Disease: Evidence for Selective Loss of 2067
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(22) Lee, B.; Jung, K.; Kim, D. H. Timosaponin AIII, a Saponin Isolated from Anemarrhena asphodeloides, Ameliorates Learning and Memory Impairments in Mice. Pharmacol., Biochem. Behav. 2009, 93, 121−127. (23) Nielsen, J. A.; Mena, E. E.; Williams, I. H.; Nocerini, M. R.; Liston, D. Correlation of Brain Levels of 9-Amino-1,2,3,4-tetrahydroacridine (THA) with Neurochemical and Behavioral Changes. Eur. J. Pharmacol. 1989, 173, 53−64. (24) Chang, S. Y.; Han, M. J.; Joh, E. H.; Kim, D. H. Metabolism of Soyasaponin I by Human Intestinal Microflora and Its Estrogenic and Cytotoxic Effects. Biomol. Therap. 2009, 17, 430−437. (25) Kafitz, K. W.; Rose, C. R.; Thoenen, H.; Konnerth, A. Neurotrophin-Evoked Rapid Excitation through TrkB Receptors. Nature 1999, 401, 918−921. (26) Figurov, A.; Pozzo-Miller, L. D.; Olafsson, P.; Wang, T.; Lu, B. Regulation of Synaptic Responses to High-Frequency Stimulation and LTP by Neurotrophins in the Hippocampus. Nature 1996, 381, 706− 709. (27) Connor, B.; Young, D.; Yan, Q.; Faull, R. L.; Synek, B.; Dragunow, M. Brain-Derived Neurotrophic Factor Is Reduced in Alzheimer’S Disease. Brain Res. Mol. Brain Res. 1997, 49, 71−81. (28) Levine, E. S.; Dreyfus, C. F.; Black, I. B.; Plummer, M. R. BrainDerived Neurotrophic Factor Rapidly Enhances Synaptic Transmission in Hippocampal Neurons via Postsynaptic Tyrosine Kinase Receptors. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 8074−8077. (29) Bourtchuladze, R.; Frenguelli, B.; Blendy, J.; Cioffi, D.; Schutz, G.; Silva, A. J. Deficient Long-Term Memory in Mice with a Targeted Mutation of the cAMP-Responsive Element-Binding Protein. Cell 1994, 79, 59−68. (30) Pugazhenthi, S.; Wang, M.; Pham, S.; Sze, C. I.; Eckman, C. B. Downregulation of CREB Expression in Alzheimer’S Brain and in AβTreated Rat Hippocampal Neurons. Mol. Neurodegener. 2011, 6, 60. (31) Jeon, S. J.; Rhee, S. Y.; Seo, J. E.; Bak, H. R.; Lee, S. H.; Ryu, J. H.; Cheong, J. H.; Shin, C. Y.; Kim, G. H.; Lee, Y. S.; Ko, K. H. Oroxylin A Increases BDNF Production by Activation of MAPKCREB Pathway in Rat Primary Cortical Neuronal Culture. Neurosci. Res. 2011, 69, 214−222. (32) Nagahara, A. H.; Merrill, D. A.; Coppola, G.; Tsukada, S.; Schroeder, B. E.; Shaked, G. M.; Wang, L.; Blesch, A.; Kim, A.; Conner, J. M.; Rockenstein, E.; Chao, M. V.; Koo, E. H.; Geschwind, D.; Masliah, E.; Chiba, A. A.; Tuszynski, M. H. Neuroprotective Effects of Brain-Derived Neurotrophic Factor in Rodent and Primate Models of Alzheimer’S Disease. Nat. Med. 2000, 15, 331−337. (33) Bejar, C.; Wang, R. H.; Weinstock, M. Effect of Rivastigmine on Scopolamine-Induced Memory Impairment in Rats. Eur. J. Pharmacol. 1999, 383, 231−240.
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