Ethanol Extract of Soybean Ameliorates Scopolamine-Induced Memory Impairment in Mice

Abstract

Soy (Glycine max, family Leguminosae) contains isoflavones and saponins as main constituents. In our preliminary study, soybean ethanol extract (SE) ameliorated scopolamine-induced memory impairment in mice in the passive avoidance task. Therefore, to confirm its ameliorating effect for memory impairments, we measured its effect in scopolamine-induced memory-impaired mice in Morris water maze task. SE significantly prevented scopolamine-induced memory impairment in the Morris water maze task. SE also increased the swimming time within quadrant section of the platform on the day after the final training session test. SE protected the reduction of brain-derived neurotrophic factor (BDNF) expression and cAMP response element-binding protein (CREB) phosphorylation in the hippocampi of scopolamine-treated mice. However, SE did not inhibit acetylcholinesterase. To understand the possible role of soysaponins in memory impairments, we prepared soyasaponins-rich (butanol) fraction of soybean (SRF) and investigated its protective effect against in the passive avoidance and Morris water maze tasks. SRF ameliorated scopolamine-induced memory impairment in mice. The memory impairment-ameliorating effect of SRF was more effective than that of SE. Based on these findings, soybean may improve memory impairment by regulating CREB phosphorylation and BDNF expression.

Introduction

Dementia is the most common neurodegenerative disorder characterized by the progressive deterioration of learning and memory as the brain becomes damaged. Of dementia, Alzheimer’s disease is irreversible, dislike thyroid disease. Alzheimer's disease (AD) caused by abnormal tau protein and β-amyloid deposition is progressively neurodegenerative (Whitehouse et al., 1981; Hardy and Seikoe, 2002; Goedert et al., 1991). Memory and cognitive functions in AD are impaired in cortical and hippocampal cholinergic systems areas (Araujo et al., 2005). Therefore, to develop the therapeutic agents for AD, many studies have been conducted on acetylcholinesterase (AChE) inhibitors donepezil and tacrine and cholinergic agonists carbachol using scopolamine-induced memory-impaired animals (Kotani et al., 2006; Tariot et al., 1996). Scopolamine, an anticholinergic drug, causes memory impairments, as well as the reduction of brainderived neurotrophic factor (BDNF), which regulates memory plasticity and formation.

Soy (Glycine max., family Leguminosae), which contains phytic acid, isoflavones and saponins as bioactive ingredients (Kitagawa et al., 1976 and 1988), has been reported to show anti-cancer, memory-enhancing, antilipidemic, and estrogen-like effects (File et al., 2001; Kim et al., 2006; Liu et al., 2007; Pan et al., 2010). Of soybean constituents, isoflavones genistin and daidzin have been reported to exhibit phytoestrogenic, antiinflammatory, and memory-enhancing effects. However, the memory-enhancing effects of soyasaponins, which are triterpenoid glycosides with one or two polysaccharide chains, have been studied.

In our preliminary study, soybean ethanol extract (SE) ameliorated scopolamine-induced memory impairment. Therefore, to confirm its ameliorating effect for memory impairments, we measured its effect in scopolamineinduced memory-impaired mice in passive avoidance, Ymaze and Morris water maze tasks.

 

Experimental

Materials − Tacrine, scopolamine hydrobromide, phosphatase inhibitor cocktail, radioimmuno-precipitation assay (RIPA) buffer, and a protease inhibitor cocktail were purchased from Sigma (U.S.A.). BDNF, cAMP response element-binding protein (CREB), p-CREB, β-actin and secondary antibodies were purchased from Santa Cruz Biotechnology (U.S.A.).

Preparation of SE and its soyasaponin-rich fraction (SRF) from soybeans − Dried soybeans (1 kg), which were purchased in KyungDong Market (Seoul, Korea), were ground to a fine powder, defatted with hexane (10 L), dried (0.8 kg), extracted with 80% ethanol (3 L) twice and concentrated in vacuo [127 g; soy ethanol extract (SE) contained 1.6% soyasaponin Bb, of which contents were analyzed by HPLC]. The concentrated extract was suspended in water, extracted with n-butanol (15 L), evaporated in vacuo [20 g; soyasaponin-rich fraction (SRF) containing 2.0% soyasaponin Ab and 7.1% soyasaponin Bb]

Animals − Male ICR mice (20 - 24 g, 6 weeks) were supplied from the Orient Animal Breeding Center (Korea). All animals were housed in wire cages at 20 - 22 ℃ and 50 ± 10% humidity, fed standard laboratory mouse chow (Samyang Co., Korea) and water ad libitum. All experiments were performed in accordance with the NIH and Kyung Hee University guidelines for Laboratory Animals Care and Use and approved by the Committee for the Care and Use of Laboratory Animals in the College of Pharmacy, Kyung Hee University.

Passive avoidance task − The passive avoidance task was performed as previously described (Joh et al., 2012; Lee et al., 2009). SE (100 or 200 mg/kg, p.o.), SRF (50 or 100 mg/kg, p.o.), tacrine (10 mg/kg, p.o.), or vehicle (p.o.) were administered 1 h before the test was conducted at every consecutive day. Scopolamine (1 mg/kg, i.p.) was administered 30 min after these oral administrations.

Morris water maze task − The Morris water maze task was performed as previously described (Lee et al., 2009; Joh et al., 2012). SE (100 or 200 mg/kg, p.o.), SRF (50 or 100 mg/kg, p.o.), tacrine (10 mg/kg, p.o.), or vehicle (p.o.) were administered 1 h before the test was conducted at every consecutive day. Scopolamine (1 mg/kg, i.p.) was administered 30 min after these oral administrations.

Immunoblotting − Mice were sacrificed by cervical dislocation 30 minutes after the acquisition trial of the passive avoidance task. The hippocampi were then removed and homogenized in 200 μl of protease inhibitor cocktail-contained ice-cold RIPA buffer. After centrifugation for 15 min at 10,000 × g, supernatants were divided into Eppendorf tubes and stored at −80 ℃ until required for protein assay, which was performed using Bradford protein assay kits (Bio-Rad, USA). The expression levels of CREB, p-CREB, BDNF, and β-actin were measured by immunoblotting according to the previous method of Lee et al. (2009).

Statistics − All values are expressed as means ± SD. For the passive avoidance task, data was analyzed by a Kruskal-Wallis non-parametric ANOVA test. Statistical significance was set at p < 0.05.

 

Results and Discussion

When SE orally administered to mice memory-impaired by scopolamine, its ameliorating effect was evaluated in the passive avoidance task. SE reversed the lowered spontaneous alteration induced by scopolamine in mice (Fig. 1A). SE (200 mg/kg, p.o.) significantly reversed it to 85.3 % of normal control mice. During the acquisition trial, latencies between among tested groups were not significantly different. Furthermore, SE significantly shortened the escape latencies prolonged by scopolamine treatment in mice in the Morris water maze task (Fig. 2A). On the day after the last training session test, mean swimming time within quadrant section of the platform in mice treated with scopolamine alone was significantly lower than in normal mice. SE increased the swimming time within the platform quadrant (Fig. 2 B). Next, we measured the inhibitory effect of SE against AChE in vitro. However, SE did not inhibit AChE (IC50, > 0.2 mM), although tacrine potently inhibit it (IC50 = 0.12 μM). We also measured the effect of SE on BDNF expression and CREB phosphorylation in hippocampi of memoryimpaired mice induced with scopolamine (Fig. 3). Scopolamine significantly inhibited BDNF expression and CREB phosphorylation, but when scopolamine with SE was given, BDNF expression and CREB phosphorylation were significantly increased. No significant difference in β-actin expression was found.

Fig. 1.Effect of SE in Mice against Scopolamine-induced Memory Impairment in Passive Avoidance Task. Memory impairment was induced by treatment with scopolamine (1 mg/kg i.p.). Normal control mice were treated with vehicle instead of scopolamine. Acquisition trials were carried out 30min 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; TC10, 10 mg/kg tacrine with scopolamine) were administered to mice 1 h before treatment with scopolamine. All values are expressed as mean ± S.D (n = 6). #Significantly different from normal mice (p < 0.05). *Significantly different from scopolamine-treated control (p < 0.05).

Fig. 2.Effect of SE on Scopolamine-induced Memory Impairment in Morris water maze Tasks. (A) Effect in escape latencies during training trial sessions. (B) Effect in swimming times spent in target quadrant. Acquisition trials were carried out 30 min after a single scopolamine treatment. Test agents (open circle, none; closed, scopolamine alone; open square, 100 mg/kg SE with scopolamine; closed square, 200 mg/kg of SE with scopolamine; closed triangle, 10 mg/kg tacrine with scopolamine) were administered to mice 1 h before treatment with scopolamine. Normal group was treated with vehicle instead of scopolamine and test agents. The training trial and the probe trial sessions were performed as described in MATERIALS AND METHODS. Values are expressed as mean ± S.D (n = 6). #Significantly different from normal mice (p < 0.05). *Significantly different from scopolamine-treated control (p < 0.05).

Fig. 3.Effect of SE on BDNF Expression and CREB Phosphorylation in the Hippocampi of Scopolamine-treated Mice. SE (100 or 200 mg/kg, p.o.) or tacrine (TC, 10 mg/kg, p.o.) was orally administered to mice at 1 h before treatment with scopolamine. Normal control group was treated with vehicle instead of scopolamine and test agents. BDNF, CREB, p-CREB and β-Actin were measured by immunoblotting.

Of the constituents of soybeans, isoflavones alleviate β-amyloid 1-42 induced learning and memory impairment by down regulation of Toll-like receptor 4 expression (Ding et al., 2011) and nuclear factor-κB activity in rats and lecithin also improve memory impairment in aged rats (Su et al., 2010; Suzuki et al., 2001). However, the ameliorating effect of its saponins has not been reported.

Therefore, to understand the possible role of soysaponins in memory impairments, we prepared SRF and measured its protective effect against in the passive avoidance and Morris water maze tasks (Fig. 4). SRF significantly protected scopolamine-induced memory deficit in the passive avoidance task (Fig. 4 A). During the acquisition trial test, no latency differences were observed among tested groups. SRF (50 and 100 mg/kg, p.o.) also significantly shortened the escape latencies prolonged by scopolamine treatment in mice in the Morris water maze task (Fig. 4 B). The memory impairment-ameliorating effect of SRF was more effective than that of SE.

Fig. 4.Effect of SRF on Scopolamine-induced Memory Impairment in Mice in the Passive Avoidance and Morris Water Maze Tasks. (A) Effect in passive avoidance task. (B) Effect in Morris water maze task. Test agents (NOR [open circle], none; SCO [closed circle], scopolamine alone; SRF50 [open square], 50 mg/kg SRF with scopolamine; SRF100 [closed square], 100 mg/kg of SE with scopolamine; TC10 [closed triangle], 10 mg/kg tacrine with scopolamine) were administered to mice 1 h before treatment with scopolamine. Passive avoidance and Morris water maze tasks were performed as described in MATERIALS AND METHODS. Values are expressed means ± S.D (n = 6). #Significantly different from normal mice (p < 0.05). *Significantly different from scopolamine-treated control (p < 0.05).

SE and SRF significantly ameliorated scopolamineinduced memory deficit in mice in passive avoidance and Morris water maze tests. Of them, SRF reversed memory deficit more potently than SE. Although SE did not inhibit AChE, SE potently inhibited the reduction of CREB phosphorylation and BDNF expression by scopolamine. BDNF influences neuronal synaptic plasticity by rapidly depolarizing neurons, as glutamate stimulates neuronal synaptic plasticity by activating tyrosine kinase receptors, enhancing glutamatergic synaptic transmission, increasing phosphorylation of the subunits of N-methyl-D-aspartate receptors in the hippocampus, facilitating hippocampal LTP (Figurov et al., 1996; Hock et al., 2000). Particularly, BDNF expression in the hippocampus and entorhinal cortex in AD is lower than in healthy mans. Of transcription factors for BDNF expression, CREB regulates the expression of tyrosine hydroxylase, BDNF, and many neuropeptides as a cellular transcription factor (Bourtchuladze et al., 1994; Pugazhenthi et al., 2011; Levine et al., 1995). Therefore, in many studies, it was suggested that the increase of BDNF expression and CREB phosphorylation may possess therapeutic potential for patients that have AD (Nagahar et al., 2000; Bejar et al., 1999). Orally administered SE, particularly SRF, may protect memory impairment as well as the reduction of BDNF expression and CREB phosphorylation by scopolamine. At least, memory impairment-ameliorating effect of soybean may be dependent on isoflavone, lecithin and soyasaponins.

Based on these results, ethanol extract of soybean ameliorates scopolamine-induced memory deficit by increasing BDNF expression and CREB phosphorylation.