http://informahealthcare.com/ijf ISSN: 0963-7486 (print), 1465-3478 (electronic) Int J Food Sci Nutr, Early Online: 1–7 ! 2015 Informa UK Ltd. DOI: 10.3109/09637486.2015.1064865
RESEARCH ARTICLE
Lactobacillus pentosus var. plantarum C29 increases the protective effect of soybean against scopolamine-induced memory impairment in mice Dae-Hyoung Yoo and Dong-Hyun Kim
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Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung Hee University, Seoul, Korea
Abstract
Keywords
Biological activities of soybean saponins are dependent on their metabolism by gut microbiota, which generate absorbable bioactive metabolites. Therefore, to enhance the pharmacological effect of soybean, we fermented defatted soybean powder (SP) with Lactobacillus pentosus var. plantarum C29 and measured its protective effect against scopolamine-induced memory impairment in mice using the passive avoidance, Y-maze and Morris water maze tasks. Fermentation increased soyasapogenol B, genistein and daidzein content of soybean and enhanced the protective effect of soybean against scopolamine-induced memory impairment. Additionally, compared with the exthanol extract of soybean, fermented SP (FSP) increased the expression of brain-derived neurotrophic factor (BDNF) in the hippocampi of scopolaminetreated mice. Furthermore, FSP inhibited acetylcholinesterase (AChE) activity in vitro and ex vivo. These findings suggest that C29 fermentation might increase the ameliorating effect of soybean against memory impairments by inhibiting AChE activity and increasing BDNF expression.
Fermentation, Lactobacillus pentosus var. plantarum C29, memory, soybean
Introduction Dementia is a common neurodegenerative disorder characterized by the progressive impairment of memory and cognition functions. In Alzheimer’s disease, which is a common type of dementia, learning and memory functions are irreversibly impaired due to the progressive deterioration of cortical and hippocampal cholinergic systems (Araujo et al., 2005; Tariot et al., 1996; Whitehouse et al., 1981). Therefore, acetylcholinesterase (AChE) inhibitors and cholinergic agonists have been investigated as therapeutic agents for Alzheimer’s disease (Araujo et al., 2005; Bejar et al., 1999; Hardy & Selkoe, 2002). Orally administered functional foods such as ginseng and soybean are inevitably metabolized by the microbiota in the gastrointestinal tract and constituents are transformed into absorbable metabolites by the intestinal microflora before absorption from the gastrointestinal tract to the blood (Akao et al., 1998a; Kim, 2012). Numerous metabolites exhibit stronger biological effects than their molecules. For example, when ginsenoside Rb1 is administered to humans or rats, it is transformed to 20-O-b-D-glucopyranosyl-20(S)-protopanaxadiol (compound K) by the gastrointestinal microbiota, and is absorbed into the blood (Akao et al., 1998a, 1998b). Compound K exhibits more potent anti-tumor and anti-inflammatory effects than ginsenoside Rb1 (Joh et al., 2011; Wakabayashi et al., 1998). Soybean (Glycine max., family Leguminosae), which contains bioactive ingredients such as isoflavones and saponins (Kitagawa et al., 1976, 1988), has been reported to show antilipidemic Correspondence: Prof. Dr. Dong-Hyun Kim, College of Pharmacy, Kyung Hee University, 1, Hoegi, Dongdaemun-Gu, Seoul 130-701, Korea. Tel: +82 2 961 0374. Fax: +82 2 957 5030. E-mail: [email protected]
History Received 20 November 2014 Revised 2 June 2015 Accepted 3 June 2015 Published online 14 July 2015
(Liu et al., 2007), phytoestrogenic (Pan et al., 2010) and memoryenhancing effects (File et al., 2001). Similarly, its constituents, isoflavones and saponins, exhibit phytoestrogenic (Pan et al., 2010), anti-inflammatory (Choi et al., 2011; Lee et al., 2010) and memory-enhancing effects (Ding et al., 2011; Hong et al., 2013, 2014; Pan et al., 2010; Xi et al., 2013). These constituents (daidzin, genistin, soyasaponins I and Ab) are metabolized to aglycones by gut microbiota (Chang et al., 2009, 2010; Park et al., 2006), and aglycones exhibit more potent phytoestrogenic effects than their parental molecules (Chang et al., 2009; Park et al., 2006). In a preliminary study, we found that soyasaponin I ameliorated scopolamine-induced memory impairment in a gut microbiota-dependent manner in mice. This result suggests that the protective effect of soybean might be dependent on the metabolism of its constituents, such as soyasaponins, by the gut microbiota. Therefore, to enhance the pharmacological effect of soybean, we fermented defatted soybean powder (SP) with Lactobacillus pentosus var. plantarum C29, and measured the protective effect of fermented SP (FSP) against the scopolamine-induced memory impairment in mice using the passive avoidance, Y-maze and Morris water maze tasks.
Materials and methods Materials Tacrine, scopolamine hydrobromide, phosphatase inhibitor cocktail, radioimmuno-precipitation assay (RIPA) buffer, acetylthiocholine (ATCh), AChE (electric eel type VI-S) and ampicillin were purchased from Sigma (St Louis, MO). Tryptic soy broth
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was purchased from BD (Franklin Lakes, NJ). Soyasaponin I was isolated according to the previously reported method of Lee et al. (2010). Bacterial cells Lactobacillus pentosus var. plantarum C29 (KCCM10885, Korea Culture Center of Microorganisms, Seoul, Korea) was cultured at a tryptic soy broth for 20 h at 37 C, collected and used in in vivo study, as previously described (Jung et al., 2013).
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Fermentation and ethanol extraction of defatted SP Defatted SP (60 g, Sejong K&C, Seoul, Korea) was suspended in 3 l distilled water, divided into two groups (30 g each), and sterilized. Then, the sterilized defatted soy powder was incubated with or without C29 (1 108 CFU/g SP) at 37 C for 48 h. The SP (30 g) treated with (FSP) or without C29 (SP) was freeze-dried. Yields of SP and FSP were 101% and 98%, respectively. Half of the FSP and SP was extracted with 70% ethanol (Yoo et al., 2013) twice, and was concentrated in vacuum. The ethanol extract of FSP and SP were named FSE and SE, respectively. Yields of SE and FSE were 19.5% and 19.9%, respectively. Genistin, genistein, daidzin, daidzein, soyasaponin I and soyasapogenol B in the SE and FSE were analyzed using HPLC system, which consisted of the Hewlett Packard series 1050, a UV detector (Ramsey, MN) and an Eclipse plus C18 column (4.6 mm 100 mm i.d., 5.0 mm, Agilent, Santa Clara, CA). For the analysis of soyasaponins, a gradient with a mixture of two solvents was applied (A, 0.05% trifluoroacetic acid; B, acetonitrile; 015 min, 90:10–30:70; 1520 min 30:70). Flow rate of the mixture was 1 ml/min, the detection wavelength was 203 nm. Retention times of soyasaponin Ab and soyasaponin Ab were 7.540 and 10.817 min, respectively. For analysis of soyasapogenols, a mixture of two solvents was applied (A, 0.05% trifluoroacetic acid; B, acetonitrile; 020 min; 40:60). Flow rate pf the mixture was 1 ml/min and the detection wavelength was 203 nm. Retention times of soyasapogenol A and soyasapogenol B were 4.391 and 11.239 min, respectively. For analysis of isoflavones, a gradient with a mixture of two solvents was applied (A, 0.05% trifluoroacetic acid; B, acetonitrile; 025 min, 85:15–65:35). Flow rate of the mixture was 1 ml/min and the detection wavelength was 254 nm. Retention times of genistin, genistein, daidzin and daidzein were 6.796, 18.964, 3.899 and 12.804 min, respectively. Animals Male ICR mice (24–28 g, 6 weeks) were supplied from the Orient Animal Breeding Center (Seoul, Korea). All the animals were housed in wire cages at 20–22 C and 50 ± 10% humidity, fed commercial standard laboratory mouse chow and water ad libitum. Ampicillin (100 mg/kg) was orally administered once a day for 2 days and soyasaponin I was treated 72 h after treatment with ampicillin. All the 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. Animal study Acquisition and retention of the passive avoidance task was conducted using a two-compartment acrylic box wherein a lighted compartment (20 cm 20 cm 20 cm) was connected to a dark compartment (20 cm 20 cm 20 cm) by an entrance door (5 cm 5 cm), like previously reported (Jung et al., 2013). Memory impairment was induced by the intraperitoneal injection
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of scopolamine (1 mg/kg) 30 min after the oral administrations of test agents. The passive avoidance maze task was performed 30 min after scopolamine treatment. Test agents [SE (100 mg/kg), FSE (100 mg/kg), SP (500 mg/kg), FSP (500 mg/kg), tacrine (10 mg/kg) and vehicle (2% Tween-80, the scopolamine group)] were orally administered into mice 1 h before the retention trial. Y-maze was used to evaluate the immediate spatial working memory, as previously described (Jung et al., 2013). Memory impairment was induced by the intraperitoneal injection of scopolamine (1 mg/kg). The maze task was performed 30 min after treatment with scopolamine. Test agents [SE (100 mg/kg), FSE (100 mg/kg), SP (500 mg/kg), FSP (500 mg), tacrine (10 mg/ kg) and vehicle (2% Tween-80, the scopolamine group)] were administered orally into mice 1 h before the task. The alternation percentage was calculated: alternation percentage ¼ [(number of alternations)/(total arm entries 2)] 100. The number of arm entries was served as the indicator of locomotor activity. The Morris water maze task was performed, like the previously reported (Jung et al., 2013). Test agents [SE (100 mg/kg), FSE (100 mg/kg), tacrine (10 mg/kg) and vehicle (2% Tween-80, the scopolamine group)] were administered orally into mice 1 h before test was performed at every consecutive day. Scopolamine (1 mg/kg, i.p.) was administered 30 min after the administration of test agents. Immunoblotting After stimulation with scopolamine, hippocampus lysates were prepared with ice-cold lysis RIPA buffer containing 50 mM Tris–HCl (pH 8.0), 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1% phosphatase inhibitor cocktail and a protease inhibitor cocktail. Hippocampus lysates were electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to an Immobilon-P nylon membrane. Brain-derived neurotrophic factor (BDNF) and b-actin were analyzed using the corresponding antibodies as previously reported (Hong et al., 2014). Immunodetection was carried out using an ECL detection kit. Ex vivo assay of AChE activity For the ex vivo AChE activity assay, the mice were randomly divided into four groups (n ¼ 4) and were orally administered vehicle (2% Tween), SP (500 mg/kg), FSP (500 mg/kg) and tacrine (10 mg/kg) (Hong et al., 2014). The mice were sacrificed after 60 min after each administration and their brains were removed. Their hippocampus or cerebral cortices were dissected and homogenized in cold 50 mM Tris–HCl buffer. The homogenates were centrifuged at 10 000 g for 10 min at 4 C, and the resulting supernatants were used enzyme sources. Their AChE activities were measured as triplicates. AChE activity per protein amount of the homogenate supernatant (mg) was calculated as a percentage compared to buffer without any inhibitor. In vitro AChE activity assay AChE activity was measured using Ellman’s coupled enzyme assay. The reaction mixture consisted of 125 ml of 3 mM dithiobis(2-nitrobenzoic acid), 25 ml of 15 mM ATCh, 50 ml of 50 mM Tris–HCl (pH ¼ 8.0) and 25 ml of test agents in a microplate. The mixture was pre-incubated for 10 min, and then 25 ml AChE was added before scanning at 405 nm for 10 min in a microplate reader, Model Biotek mQuant MQX200 (Winooski, VT). AChE was prepared from the brain cerebral cortices according to the previously reported method of Konrath et al. (2012). The protein amount was determined using Bradford
DOI: 10.3109/09637486.2015.1064865
Lactobacillus C29 increases memory-enhancing effect of soy
protein assay kits (Bio-Rad, Hercules, CA). Enzyme activity was calculated as a percentage compared to buffer without any inhibitor.
To evaluate the role of gastrointestinal microbiota on the memory impairment alleviating effect of soyasaponin I, we administered soyasaponin I orally to mice previously treated or not treated with antibiotics, and measured the memory impairment using the passive avoidance and Y-maze tasks (Figure 1). In mice not treated with antibiotics, soyasaponin I potently ameliorated the scopolamine-induced memory impairment. However, in mice pretreated with antibiotics, soyasaponin I did not improve the memory impairment significantly. Next, we fermented defatted SP with C29, a lactic acid bacterium, extracted the solution with 70% ethanol and measured memory impairment-alleviating effect of the soybean extracts SP
and FSP using the passive avoidance task in mice subjected to scopolamine-induced memory impairment (Figure 2A). Treatment with scopolamine alone significantly impaired the memory. Treatment with SP or FSP ameliorated the scopolamineinduced memory impairment. FSP (500 mg/kg, p.o.) reversed memory impairment to 27.8% of normal control mice, and it was more potent than SP. During the acquisition trial, no differences were observed in the latency among the test groups. Similarly, we measured the effects of SP and FSP using Y-maze task in mice subjected to scopolamine-induced memory impairment (Figure 2B). While scopolamine decreased the number of spontaneous alterations, SP and FSP mitigated this effect. FSP (500 mg/kg) significantly restored the number of spontaneous alterations to 84.2% of that in scopolamine-untreated mice, and the effect of FSP was more potent than of SP. To clarify the protective role of SP and FSP against memory impairment, we extracted SP and FSP with 70% ethanol and measured their memory-enhancing effects using the passive avoidance task in mice with scopolamine-induced memory impairment (Figure 3A). FSE significantly reversed the reduced latency time induced by scopolamine (p50.05), but SE (100 mg/kg) did not significantly enhance memory. However, FSE (100 mg/kg, p.o.) administered to scopolamine-treated mice significantly memory to 41.4% of control mice. During the
Figure 1. Effect of soyasaponin I on scopolamine-induced memory impairment in mice treated or non-treated with in the passive avoidance (A) and Y-maze tasks (B). Memory impairment was induced with scopolamine (SCO, 0.9 mg/kg, i.p.) in mice treated or not treated with antibiotics (ab, 300 mg/kg ampicillin). Test agents were administered to mice 1 h before treatment with scopolamine: NOR, vehicle alone; SCO + ab, scopolamine with antibiotics; SI, 10 mg/kg soyasaponin I with scopolamine and SI + ab, 10 mg/kg soyasaponin I with scopolamine and antibiotics. All the values are expressed as mean ± SD (n ¼ 6). #Significantly different from vehicle-treated mice (p50.05). *Significantly different from scopolamine-treated control (p50.05).
Figure 2. Effect of SP and FSP in mice against scopolamine-induced memory impairment in the passive avoidance (A) and Y-maze tasks (B). Memory impairment was induced by treatment with scopolamine (0.9 mg/kg, i.p.). Test agents were administered to mice 1 h before treatment with scopolamine: NOR, vehicle alone; SCO, scopolamine alone; SP, 500 mg/kg SP with scopolamine; FSP, 500 mg/kg of FSP with scopolamine and TC, 10 mg/kg tacrine with scopolamine. All the values are expressed as mean ± SD (n ¼ 6). #Significantly different from vehicletreated mice (p50.05). *Significantly different from scopolamine-treated control (p50.05).
Statistical analysis All the values are expressed as means ± SD. For the passive avoidance task, data were analyzed by a Kruskal–Wallis nonparametric ANOVA test. Statistical significance was set at p50.05.
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Results
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Figure 3. Effect of SE and FSE in mice against scopolamine-induced memory impairment in the passive avoidance (A) and Y-maze tasks (B). Memory impairment was induced by treatment with scopolamine (0.9 mg/kg, i.p.). Test agents were administered to mice 1 h before treatment with scopolamine: NOR, vehicle alone; SCO, scopolamine alone; SE, 100 mg/kg SE with scopolamine; FSE, 100 mg/kg of SE with scopolamine and TC, 10 mg/kg tacrine with scopolamine. All the values are expressed as mean ± SD (n ¼ 6). #Significantly different from normal mice (p50.05). *Significantly different from scopolamine-treated control (p50.05).
acquisition trial, no differences were observed in the latency time among the test groups. Similarly, we measured the memoryenhancing effects of SE and FSE using the Y-maze task in mice with scopolamine-induced memory impairment (Figure 3B). SE and FSE restored the scopolamine-induced reduction of spontaneous alterations. FSE (100 mg/kg) alone significantly restored the number of spontaneous alterations to 83.0% of that in scopolamine-untreated mice. Next, we measured the effect of SE (100 mg/kg) and FSE (100 mg/kg, p.o.) on the escape latency in the Morris water maze task (Figure 4). Scopolamine increased the escape latencies. However, SE and FSE decreased the escape latency on the fourth day. FSE alone significantly reduced the escape latency between from the third day to the fourth day. In scopolamine-treated mice, FSE significantly increased the swimming time within the platform quadrant 24 h after the last training session. The effect of FSE (100 mg/kg) was comparable to that of tacrine (10 mg/kg). We found no significant differences in the swimming speed within the target quadrant among the groups treated with vehicle alone, scopolamine, or the test agents.
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Figure 4. Effect of SE and FSE on scopolamine-induced memory impairment in Morris water maze tasks. (A) Effects in escape latencies during training trial sessions. (B) Effects on swimming times spent in target quadrant. (C) Effects on swimming speed. Test agents were administered to mice 1 h before treatment with scopolamine: open circle, vehicle alone; closed, scopolamine alone; open square, 100 mg/kg SE with scopolamine; closed square, 100 mg/kg of FSE with scopolamine and closed triangle, 10 mg/kg tacrine with scopolamine. Normal group (open circle) was treated with vehicle instead of scopolamine and test agents. All the values are expressed as mean ± SD (n ¼ 6). #Significantly different from normal mice (p50.05). *Significantly different from scopolamine-treated control (p50.05).
Next, we measured the inhibitory effects of SE and FSE on AChE activity in vitro (Figure 5A). AChE activity was inhibited by FSE more potently than SE. Hale maximal inhibition concentrations of SE, FSE and tacrine were 410, 4.1 and 0.04 mg/ml, respectively. Furthermore, we measured the AChE-inhibitory activities of SE and FSE ex vivo (Figure 5B). SE and FSE significantly inhibited the AChE activity in the cortex and hippocampus of mice. The inhibitory potency of FSE was more prominent than that of SE. We measured the effect of SE and FSE on BDNF expression in the hippocampi mice treated with scopolamine (Figure 5C). Scopolamine significantly inhibited BDNF expression, but upon SE or FSE treatment, the
DOI: 10.3109/09637486.2015.1064865
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Table 1. The contents of main constituents in soybeans fermented with or without C29. Content (mg/g) Constituent Genistin Genistein Daidzin Daidzein Soyasaponin Ab Soyasaponin I Soyasapogenol A Soyasapogenol B
Defatted soybean
Fermented defatted soybean
203.6 ± 27.8a 8.6 ± 0.8 185.1 ± 19.1 3.5 ± 2.7 918.5 ± 106.4 1037.2 ± 55.2 –b –
6.6 ± 2.0 45.9 ± 15.4 18.6 ± 3.3 34 ± 10.3 190.6 ± 48.4 767.5 ± 37.9 160.9 ± 67.1 91.2 ± 41.6
a
Mean ± SD. Not detected.
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b
Figure 5. Effects of SP and FSP on acetylcholinesterase activity and BDNF expression in the hippocampi of scopolamine-treated mice. (A) Effect on acetylcholinesterase activity in vitro. Test agents were as follows: closed circle, tacrine; closed triangle, SP and closed sequare, FSP. (B) Effect on acetylcholinesterase activity ex vivo in the hippocampus (closed bar) and cortex (open bar) of brain. (C) Effect on BDNF expression. SP (500 mg/kg, p.o.), FSP (500 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 and b-actin were measured by immunoblotting. Values are expressed as mean ± SD (n ¼ 4). #Significantly different from normal mice (p50.05). *Significantly different from scopolamine-treated control (p50.05).
scopolamine-induced reduction of BDNF expression was significantly suppressed. We found no significant difference in b-actin expression. Additionally, we measured genistin, genistein, daidizin, daidzein, soyasaponin I and soyasapogenol B content of SP and FSP. The amounts of genistein, daidzein and soyasapogenol B were higher in C29-fermented SP (FSH) than those in non-FSH, but the amounts of genistin, daidzin and soyasaponin I were lower in C29-fermented soybean than those in non-FSH (Table 1).
Discussion Numerous hydrophilic constituents, such as glycosides and polysaccharides, of orally administered functional foods are
metabolized into bioactive compounds by the gastrointestinal microbiota in the alimentary tract of humans and animals (Akao et al., 1998a; Kim, 2012). The microbiota of different individuals is a unique collection of 4500 different bacterial species (De Filippo et al., 2010; Simon & Gorbach, 1986). Composition of the gastrointestinal microbiota is relatively stable throughout adulthood in the absence of disease or antimicrobial therapy. Indigenous metabolic enzymec activities can be attributed to the unique intestinal microbiota of each individual (Mykkanen et al., 1998; Reddy et al., 1980). Therefore, biological activities of functional foods containing hydrophilic constituents are dependent on the metabolic activities of the intestinal microbiota. In the present study, we found that soyasaponin I ameliorated the scopolamine-induced memory impairment in mice, but this effect was reduced in pseudo-germ-free mice, which were treated with ampicillin, as previously reported (Yoo et al., 2014). These results are consistent with previous report suggesting that soyasaponin I is metabolized to hydrophobic and absorbable compounds by the gut microbiota (Chang et al., 2009). Therefore, to elicit the protective effects of soybean against memory impairment in a gut microbiota-independent manner, we fermented soybean with C29 and measured the effect of FSP in mice with scopolamine-induced memory impairment. FSP was more protective against memory impairments than SP. Similarly, the ethanol extract of FSP (FSE, 100 mg/kg) significantly improved the memory impairment, and it had more prominent effects than SP. Furthermore, fermentation of soybean by the C29 strain increased the concentrations of isoflavone and soyasaponin aglycones genistein, daidzein and soyasapogenols A and B. These results suggest that C29 b-glycosidase(s) might biotransform soybean isoflavone and soyasaponin glycosides to aglycones and these metabolites might elicit more potent protective effects against memory impairment than their molecules. Both SE and FSE suppressed the reduction of BDNF expression induced by scopolamine. This is consistent with previous works reporting that SE and soyasaponins Ab and I significantly inhibit the scopolamine-induced reduction of BDNF expression (Hong et al., 2014; Yoo et al., 2013). Importantly, FSE alleviated this reduction more potently than SE. BDNF influences neuronal synaptic plasticity by depolarizing neurons (Kafitz et al., 1999), enhancing glutamatergic synaptic transmission (Figurov et al., 1996), increasing the phosphorylation of N-methyl-D-aspartate receptor subunit and facilitating hippocampal long-term potentiation (Connor et al., 1997). Furthermore, BDNF expression is lower in the hippocampus of patients with Alzheimer’s disease than that of healthy people (Bourtchuladze et al., 1994; Hock et al., 2000). In the present study, FSE restored the scopolamine-induced reduction of BDNF
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expression. Furthermore, SE and FSE inhibited AChE activity in vitro and ex vivo, unlike soyasaponins and their metabolites soyasapogenol A and B (File et al., 2001). These results suggest that AChE-inhibitory activities of certain SE constituents, such as isoflavones but not soyasaponins, might be activated by the gut microbiota or C29 fermentation. This is supported by a previous study of Orhan et al. (2007), which showed that genistein, a soy isoflavone that is transformed by the gut microbiota or lactic acid bacteria, can inhibit AChE activity in vitro. Therefore, the potent protective effect of the ethanol extract C-29-fermented soybean against memory impairment might be due to the biotransformation of soybean constituents into easily absorbable bioactive compounds by C29 rather than by the gut microbiota.
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Conclusions Fermentation of SP by C29 increased the amount of soyasapogenol B, genstein and daidzein. FSP and its ethanol extract improved the scopolamine-induced memory impairment more potently than SP and its ethanol extract in the passive avoidance, Y-maze and Morris water maze tasks. Additionally, in the hippocampi of mice restored the scopolamine-induced reduction of BDNF expression more potently than SE. Furthermore, FSP inhibited AChE activity in vitro and ex vivo. Based on these findings, C29-fermentation might increase the protective effects of soybean against memory impairment by inhibiting AChE activity and inducing BDNF expression.
Declaration of interest The authors declare that there is no conflict of interest. This study was supported by grants from the Bio & Medical Technology Development Program (2013M3A9B6076413) of the National Research Foundation (NRF) funded by the Korean government (MSIP).
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Ding BJ, Ma WW, He LL, Zhou X, Yuan LH, Yu HL, Feng JF, Xiao R. 2011. Soybean isoflavone alleviates b-amyloid 1-42 induced inflammatory response to improve ability by down regulation of Toll-like receptor 4 expression and nuclear factor-kB activity in rats. Int J Dev Neurosci 29:537–542. Figurov A, Pozzo-Miller LD, Olafsson P, Wang T, Lu B. 1996. Regulation of synaptic responses to high-frequency stimulation and LTP by neurotrophins in the hippocampus. Nature 381:706–709. File SE, Jarrett N, Fluck E, Duffy R, Casey K, Wiseman H. 2001. Eating soya improves human memory. Psychopharmacology 157:430–436. Hardy J, Selkoe DJ. 2002. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297:353–356. Hock C, Heese K, Hulette C, Rosenberg C, Otten U. 2000. Regionspecific 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 57:846–851. Hong SW, Heo H, Yang JH, Han M, Kim DH, Kwon YK. 2013. Soyasaponin I improved neuroprotection and regeneration in memory deficient model rats. PLoS One 8:e81556. Hong SW, Yoo DH, Woo JY, Jeong JJ, Yang JH, Kim DH. 2014. Soyasaponins Ab and Bb prevent scopolamine-induced memory impairment in mice without the inhibition of acetylcholinesterase. J Agric Food Chem 62:2062–2068. Joh EH, Lee IA, Jung IH, Kim DH. 2011. Ginsenoside Rb1 and its metabolite compound K inhibit IRAK-1 activation – the key step of inflammation. Biochem Pharmacol 82:278–286. Jung IH, Jung MA, Kim EJ, Han MJ, Kim DH. 2013. Lactobacillus pentosus var. plantarum C29 protects scopolamine-induced memory deficit in mice. J Appl Microbiol 113:1498–506. Kafitz KW, Rose CR, Thoenen H, Konnerth A. 1999. Neurotrophinevoked rapid excitation through TrkB receptors. Nature 401:918–921. Kim DH. 2012. Chemical diversity of Panax ginseng, Panax quinquifolium, and Panax notoginseng. J Ginseng Res 36:1–15. Kitagawa I, Taniyama T, Nagahama Y, Okubo K, Yamauchi F, Yoshikawa M. 1988. 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 36:2819–2828. Kitagawa I, Yoshikawa M, Yosioka I. 1976. Saponin and sapogenol. XIII. structures of three soybean saponins: soyasaponin I, soyasaponin II, and soyasaponin III. Chem Pharm Bull 24:121–129. Konrath EL, Neves BM, Lunardi PS, Passos Cdos S, Simo˜es-Pires A, Ortega MG, Gonc¸alves CA, et al. 2012. Investigation of the in vitro and ex vivo acetylcholinesterase and antioxidant activities of traditionally used Lycopodium species from South America on alkaloid extracts. J Ethnopharmacol 139:58–67. Lee IA, Park YJ, Yeo HK, Han MJ, Kim DH. 2010. Soyasaponin I attenuates TNBS-induced colitis in mice by inhibiting NF-kB pathway. J Agric Food Chem 58:10929–10934. Liu YQ, Xin TR, Lu¨ XY, Ji Q, Jin Y, Yang HD. 2007. Memory performance of hypercholesterolemic mice in response to treatment with soy isoflavones. Neurosci Res 57:544–549. Mykkanen H, Laiho K, Salminen S. 1998. Variations in faecal bacterial enzyme activities and associations with bowel function and diet in elderly subjects. J Appl Microbiol 85:37–41. Orhan I, Kartal M, Tosun F, Sener B. 2007. Screening of various phenolic acids and flavonoid derivatives for their anticholinesterase potential. Z Naturforsch 62:829–832. Pan M, Li Z, Yeung V, Xu RJ. 2010. 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 7:75. Park EK, Shin J, Bae EA, Lee YC, Kim DH. 2006. Intestinal bacteria activate estrogenic effect of main constituents puerarin and daidzin of Pueraria thunbergiana. Biol Pharm Bull 29:2432–2435. Reddy BS, Hanson D, Mangat S, Mathews L, Sbaschnig M, Sharma C, Simi B. 1980. Effect of high-fat, high-beef diet and of mode of cooking of beef in the diet on fecal bacterial enzymes and fecal bile acids and neutral sterols. J Nutr 110:1880–1887. Simon GL, Gorbach SL. 1986. The human intestinal microflora. Dig Dis Sci 31:147S–162S. Tariot PN, Patel SV, Cox C, Henderson RE. 1996. Age-related decline in central cholinergic function demonstrated with scopolamine. Psychopharmacology 125:50–56.
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Wakabayashi C, Murakami K, Hasegawa H, Murata J, Saiki I. 1998. An intestinal bacterial metabolite of ginseng protopanaxadiol saponins has the ability to induce apoptosis in tumor cells. Biochem Biophys Res Commun 246:725–730. Whitehouse PJ, Price DL, Clark AW, Coyle JT, DeLong MR. 1981. Alzheimer disease: evidence for selective loss of cholinergic neurons in the nucleus basalis. Ann Neurol 10:122–126. Xi YD, Li XY, Ding J, Yu HL, Ma WW, Yuan LH, Wu J, Xiao R. 2013. Soy isoflavone alleviates Ab1-42-induced impairment of
learning and memory ability through the regulation of RAGE/ LRP-1 in neuronal and vascular tissue. Curr Neurovasc Res 10: 144–156. Yoo DH, Kim IS, Le TK, Jung IH, Yoo HH, Kim DH. 2014. Gut microbiota-mediated drug interactions between lovastatin and antibiotics. Drug Metab Dispos 42:1508–1513. Yoo DH, Woo JY, Kim DH. 2013. Ethanol extract of soybean ameliorates scopolamine-induced memory impairment in mice. Nat Prod Sci 19: 324–328.
RESEARCH ARTICLE
Lactobacillus pentosus var. plantarum C29 increases the protective effect of soybean against scopolamine-induced memory impairment in mice Dae-Hyoung Yoo and Dong-Hyun Kim
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Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung Hee University, Seoul, Korea
Abstract
Keywords
Biological activities of soybean saponins are dependent on their metabolism by gut microbiota, which generate absorbable bioactive metabolites. Therefore, to enhance the pharmacological effect of soybean, we fermented defatted soybean powder (SP) with Lactobacillus pentosus var. plantarum C29 and measured its protective effect against scopolamine-induced memory impairment in mice using the passive avoidance, Y-maze and Morris water maze tasks. Fermentation increased soyasapogenol B, genistein and daidzein content of soybean and enhanced the protective effect of soybean against scopolamine-induced memory impairment. Additionally, compared with the exthanol extract of soybean, fermented SP (FSP) increased the expression of brain-derived neurotrophic factor (BDNF) in the hippocampi of scopolaminetreated mice. Furthermore, FSP inhibited acetylcholinesterase (AChE) activity in vitro and ex vivo. These findings suggest that C29 fermentation might increase the ameliorating effect of soybean against memory impairments by inhibiting AChE activity and increasing BDNF expression.
Fermentation, Lactobacillus pentosus var. plantarum C29, memory, soybean
Introduction Dementia is a common neurodegenerative disorder characterized by the progressive impairment of memory and cognition functions. In Alzheimer’s disease, which is a common type of dementia, learning and memory functions are irreversibly impaired due to the progressive deterioration of cortical and hippocampal cholinergic systems (Araujo et al., 2005; Tariot et al., 1996; Whitehouse et al., 1981). Therefore, acetylcholinesterase (AChE) inhibitors and cholinergic agonists have been investigated as therapeutic agents for Alzheimer’s disease (Araujo et al., 2005; Bejar et al., 1999; Hardy & Selkoe, 2002). Orally administered functional foods such as ginseng and soybean are inevitably metabolized by the microbiota in the gastrointestinal tract and constituents are transformed into absorbable metabolites by the intestinal microflora before absorption from the gastrointestinal tract to the blood (Akao et al., 1998a; Kim, 2012). Numerous metabolites exhibit stronger biological effects than their molecules. For example, when ginsenoside Rb1 is administered to humans or rats, it is transformed to 20-O-b-D-glucopyranosyl-20(S)-protopanaxadiol (compound K) by the gastrointestinal microbiota, and is absorbed into the blood (Akao et al., 1998a, 1998b). Compound K exhibits more potent anti-tumor and anti-inflammatory effects than ginsenoside Rb1 (Joh et al., 2011; Wakabayashi et al., 1998). Soybean (Glycine max., family Leguminosae), which contains bioactive ingredients such as isoflavones and saponins (Kitagawa et al., 1976, 1988), has been reported to show antilipidemic Correspondence: Prof. Dr. Dong-Hyun Kim, College of Pharmacy, Kyung Hee University, 1, Hoegi, Dongdaemun-Gu, Seoul 130-701, Korea. Tel: +82 2 961 0374. Fax: +82 2 957 5030. E-mail: [email protected]
History Received 20 November 2014 Revised 2 June 2015 Accepted 3 June 2015 Published online 14 July 2015
(Liu et al., 2007), phytoestrogenic (Pan et al., 2010) and memoryenhancing effects (File et al., 2001). Similarly, its constituents, isoflavones and saponins, exhibit phytoestrogenic (Pan et al., 2010), anti-inflammatory (Choi et al., 2011; Lee et al., 2010) and memory-enhancing effects (Ding et al., 2011; Hong et al., 2013, 2014; Pan et al., 2010; Xi et al., 2013). These constituents (daidzin, genistin, soyasaponins I and Ab) are metabolized to aglycones by gut microbiota (Chang et al., 2009, 2010; Park et al., 2006), and aglycones exhibit more potent phytoestrogenic effects than their parental molecules (Chang et al., 2009; Park et al., 2006). In a preliminary study, we found that soyasaponin I ameliorated scopolamine-induced memory impairment in a gut microbiota-dependent manner in mice. This result suggests that the protective effect of soybean might be dependent on the metabolism of its constituents, such as soyasaponins, by the gut microbiota. Therefore, to enhance the pharmacological effect of soybean, we fermented defatted soybean powder (SP) with Lactobacillus pentosus var. plantarum C29, and measured the protective effect of fermented SP (FSP) against the scopolamine-induced memory impairment in mice using the passive avoidance, Y-maze and Morris water maze tasks.
Materials and methods Materials Tacrine, scopolamine hydrobromide, phosphatase inhibitor cocktail, radioimmuno-precipitation assay (RIPA) buffer, acetylthiocholine (ATCh), AChE (electric eel type VI-S) and ampicillin were purchased from Sigma (St Louis, MO). Tryptic soy broth
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was purchased from BD (Franklin Lakes, NJ). Soyasaponin I was isolated according to the previously reported method of Lee et al. (2010). Bacterial cells Lactobacillus pentosus var. plantarum C29 (KCCM10885, Korea Culture Center of Microorganisms, Seoul, Korea) was cultured at a tryptic soy broth for 20 h at 37 C, collected and used in in vivo study, as previously described (Jung et al., 2013).
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Fermentation and ethanol extraction of defatted SP Defatted SP (60 g, Sejong K&C, Seoul, Korea) was suspended in 3 l distilled water, divided into two groups (30 g each), and sterilized. Then, the sterilized defatted soy powder was incubated with or without C29 (1 108 CFU/g SP) at 37 C for 48 h. The SP (30 g) treated with (FSP) or without C29 (SP) was freeze-dried. Yields of SP and FSP were 101% and 98%, respectively. Half of the FSP and SP was extracted with 70% ethanol (Yoo et al., 2013) twice, and was concentrated in vacuum. The ethanol extract of FSP and SP were named FSE and SE, respectively. Yields of SE and FSE were 19.5% and 19.9%, respectively. Genistin, genistein, daidzin, daidzein, soyasaponin I and soyasapogenol B in the SE and FSE were analyzed using HPLC system, which consisted of the Hewlett Packard series 1050, a UV detector (Ramsey, MN) and an Eclipse plus C18 column (4.6 mm 100 mm i.d., 5.0 mm, Agilent, Santa Clara, CA). For the analysis of soyasaponins, a gradient with a mixture of two solvents was applied (A, 0.05% trifluoroacetic acid; B, acetonitrile; 015 min, 90:10–30:70; 1520 min 30:70). Flow rate of the mixture was 1 ml/min, the detection wavelength was 203 nm. Retention times of soyasaponin Ab and soyasaponin Ab were 7.540 and 10.817 min, respectively. For analysis of soyasapogenols, a mixture of two solvents was applied (A, 0.05% trifluoroacetic acid; B, acetonitrile; 020 min; 40:60). Flow rate pf the mixture was 1 ml/min and the detection wavelength was 203 nm. Retention times of soyasapogenol A and soyasapogenol B were 4.391 and 11.239 min, respectively. For analysis of isoflavones, a gradient with a mixture of two solvents was applied (A, 0.05% trifluoroacetic acid; B, acetonitrile; 025 min, 85:15–65:35). Flow rate of the mixture was 1 ml/min and the detection wavelength was 254 nm. Retention times of genistin, genistein, daidzin and daidzein were 6.796, 18.964, 3.899 and 12.804 min, respectively. Animals Male ICR mice (24–28 g, 6 weeks) were supplied from the Orient Animal Breeding Center (Seoul, Korea). All the animals were housed in wire cages at 20–22 C and 50 ± 10% humidity, fed commercial standard laboratory mouse chow and water ad libitum. Ampicillin (100 mg/kg) was orally administered once a day for 2 days and soyasaponin I was treated 72 h after treatment with ampicillin. All the 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. Animal study Acquisition and retention of the passive avoidance task was conducted using a two-compartment acrylic box wherein a lighted compartment (20 cm 20 cm 20 cm) was connected to a dark compartment (20 cm 20 cm 20 cm) by an entrance door (5 cm 5 cm), like previously reported (Jung et al., 2013). Memory impairment was induced by the intraperitoneal injection
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of scopolamine (1 mg/kg) 30 min after the oral administrations of test agents. The passive avoidance maze task was performed 30 min after scopolamine treatment. Test agents [SE (100 mg/kg), FSE (100 mg/kg), SP (500 mg/kg), FSP (500 mg/kg), tacrine (10 mg/kg) and vehicle (2% Tween-80, the scopolamine group)] were orally administered into mice 1 h before the retention trial. Y-maze was used to evaluate the immediate spatial working memory, as previously described (Jung et al., 2013). Memory impairment was induced by the intraperitoneal injection of scopolamine (1 mg/kg). The maze task was performed 30 min after treatment with scopolamine. Test agents [SE (100 mg/kg), FSE (100 mg/kg), SP (500 mg/kg), FSP (500 mg), tacrine (10 mg/ kg) and vehicle (2% Tween-80, the scopolamine group)] were administered orally into mice 1 h before the task. The alternation percentage was calculated: alternation percentage ¼ [(number of alternations)/(total arm entries 2)] 100. The number of arm entries was served as the indicator of locomotor activity. The Morris water maze task was performed, like the previously reported (Jung et al., 2013). Test agents [SE (100 mg/kg), FSE (100 mg/kg), tacrine (10 mg/kg) and vehicle (2% Tween-80, the scopolamine group)] were administered orally into mice 1 h before test was performed at every consecutive day. Scopolamine (1 mg/kg, i.p.) was administered 30 min after the administration of test agents. Immunoblotting After stimulation with scopolamine, hippocampus lysates were prepared with ice-cold lysis RIPA buffer containing 50 mM Tris–HCl (pH 8.0), 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1% phosphatase inhibitor cocktail and a protease inhibitor cocktail. Hippocampus lysates were electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to an Immobilon-P nylon membrane. Brain-derived neurotrophic factor (BDNF) and b-actin were analyzed using the corresponding antibodies as previously reported (Hong et al., 2014). Immunodetection was carried out using an ECL detection kit. Ex vivo assay of AChE activity For the ex vivo AChE activity assay, the mice were randomly divided into four groups (n ¼ 4) and were orally administered vehicle (2% Tween), SP (500 mg/kg), FSP (500 mg/kg) and tacrine (10 mg/kg) (Hong et al., 2014). The mice were sacrificed after 60 min after each administration and their brains were removed. Their hippocampus or cerebral cortices were dissected and homogenized in cold 50 mM Tris–HCl buffer. The homogenates were centrifuged at 10 000 g for 10 min at 4 C, and the resulting supernatants were used enzyme sources. Their AChE activities were measured as triplicates. AChE activity per protein amount of the homogenate supernatant (mg) was calculated as a percentage compared to buffer without any inhibitor. In vitro AChE activity assay AChE activity was measured using Ellman’s coupled enzyme assay. The reaction mixture consisted of 125 ml of 3 mM dithiobis(2-nitrobenzoic acid), 25 ml of 15 mM ATCh, 50 ml of 50 mM Tris–HCl (pH ¼ 8.0) and 25 ml of test agents in a microplate. The mixture was pre-incubated for 10 min, and then 25 ml AChE was added before scanning at 405 nm for 10 min in a microplate reader, Model Biotek mQuant MQX200 (Winooski, VT). AChE was prepared from the brain cerebral cortices according to the previously reported method of Konrath et al. (2012). The protein amount was determined using Bradford
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Lactobacillus C29 increases memory-enhancing effect of soy
protein assay kits (Bio-Rad, Hercules, CA). Enzyme activity was calculated as a percentage compared to buffer without any inhibitor.
To evaluate the role of gastrointestinal microbiota on the memory impairment alleviating effect of soyasaponin I, we administered soyasaponin I orally to mice previously treated or not treated with antibiotics, and measured the memory impairment using the passive avoidance and Y-maze tasks (Figure 1). In mice not treated with antibiotics, soyasaponin I potently ameliorated the scopolamine-induced memory impairment. However, in mice pretreated with antibiotics, soyasaponin I did not improve the memory impairment significantly. Next, we fermented defatted SP with C29, a lactic acid bacterium, extracted the solution with 70% ethanol and measured memory impairment-alleviating effect of the soybean extracts SP
and FSP using the passive avoidance task in mice subjected to scopolamine-induced memory impairment (Figure 2A). Treatment with scopolamine alone significantly impaired the memory. Treatment with SP or FSP ameliorated the scopolamineinduced memory impairment. FSP (500 mg/kg, p.o.) reversed memory impairment to 27.8% of normal control mice, and it was more potent than SP. During the acquisition trial, no differences were observed in the latency among the test groups. Similarly, we measured the effects of SP and FSP using Y-maze task in mice subjected to scopolamine-induced memory impairment (Figure 2B). While scopolamine decreased the number of spontaneous alterations, SP and FSP mitigated this effect. FSP (500 mg/kg) significantly restored the number of spontaneous alterations to 84.2% of that in scopolamine-untreated mice, and the effect of FSP was more potent than of SP. To clarify the protective role of SP and FSP against memory impairment, we extracted SP and FSP with 70% ethanol and measured their memory-enhancing effects using the passive avoidance task in mice with scopolamine-induced memory impairment (Figure 3A). FSE significantly reversed the reduced latency time induced by scopolamine (p50.05), but SE (100 mg/kg) did not significantly enhance memory. However, FSE (100 mg/kg, p.o.) administered to scopolamine-treated mice significantly memory to 41.4% of control mice. During the
Figure 1. Effect of soyasaponin I on scopolamine-induced memory impairment in mice treated or non-treated with in the passive avoidance (A) and Y-maze tasks (B). Memory impairment was induced with scopolamine (SCO, 0.9 mg/kg, i.p.) in mice treated or not treated with antibiotics (ab, 300 mg/kg ampicillin). Test agents were administered to mice 1 h before treatment with scopolamine: NOR, vehicle alone; SCO + ab, scopolamine with antibiotics; SI, 10 mg/kg soyasaponin I with scopolamine and SI + ab, 10 mg/kg soyasaponin I with scopolamine and antibiotics. All the values are expressed as mean ± SD (n ¼ 6). #Significantly different from vehicle-treated mice (p50.05). *Significantly different from scopolamine-treated control (p50.05).
Figure 2. Effect of SP and FSP in mice against scopolamine-induced memory impairment in the passive avoidance (A) and Y-maze tasks (B). Memory impairment was induced by treatment with scopolamine (0.9 mg/kg, i.p.). Test agents were administered to mice 1 h before treatment with scopolamine: NOR, vehicle alone; SCO, scopolamine alone; SP, 500 mg/kg SP with scopolamine; FSP, 500 mg/kg of FSP with scopolamine and TC, 10 mg/kg tacrine with scopolamine. All the values are expressed as mean ± SD (n ¼ 6). #Significantly different from vehicletreated mice (p50.05). *Significantly different from scopolamine-treated control (p50.05).
Statistical analysis All the values are expressed as means ± SD. For the passive avoidance task, data were analyzed by a Kruskal–Wallis nonparametric ANOVA test. Statistical significance was set at p50.05.
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Results
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Figure 3. Effect of SE and FSE in mice against scopolamine-induced memory impairment in the passive avoidance (A) and Y-maze tasks (B). Memory impairment was induced by treatment with scopolamine (0.9 mg/kg, i.p.). Test agents were administered to mice 1 h before treatment with scopolamine: NOR, vehicle alone; SCO, scopolamine alone; SE, 100 mg/kg SE with scopolamine; FSE, 100 mg/kg of SE with scopolamine and TC, 10 mg/kg tacrine with scopolamine. All the values are expressed as mean ± SD (n ¼ 6). #Significantly different from normal mice (p50.05). *Significantly different from scopolamine-treated control (p50.05).
acquisition trial, no differences were observed in the latency time among the test groups. Similarly, we measured the memoryenhancing effects of SE and FSE using the Y-maze task in mice with scopolamine-induced memory impairment (Figure 3B). SE and FSE restored the scopolamine-induced reduction of spontaneous alterations. FSE (100 mg/kg) alone significantly restored the number of spontaneous alterations to 83.0% of that in scopolamine-untreated mice. Next, we measured the effect of SE (100 mg/kg) and FSE (100 mg/kg, p.o.) on the escape latency in the Morris water maze task (Figure 4). Scopolamine increased the escape latencies. However, SE and FSE decreased the escape latency on the fourth day. FSE alone significantly reduced the escape latency between from the third day to the fourth day. In scopolamine-treated mice, FSE significantly increased the swimming time within the platform quadrant 24 h after the last training session. The effect of FSE (100 mg/kg) was comparable to that of tacrine (10 mg/kg). We found no significant differences in the swimming speed within the target quadrant among the groups treated with vehicle alone, scopolamine, or the test agents.
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Figure 4. Effect of SE and FSE on scopolamine-induced memory impairment in Morris water maze tasks. (A) Effects in escape latencies during training trial sessions. (B) Effects on swimming times spent in target quadrant. (C) Effects on swimming speed. Test agents were administered to mice 1 h before treatment with scopolamine: open circle, vehicle alone; closed, scopolamine alone; open square, 100 mg/kg SE with scopolamine; closed square, 100 mg/kg of FSE with scopolamine and closed triangle, 10 mg/kg tacrine with scopolamine. Normal group (open circle) was treated with vehicle instead of scopolamine and test agents. All the values are expressed as mean ± SD (n ¼ 6). #Significantly different from normal mice (p50.05). *Significantly different from scopolamine-treated control (p50.05).
Next, we measured the inhibitory effects of SE and FSE on AChE activity in vitro (Figure 5A). AChE activity was inhibited by FSE more potently than SE. Hale maximal inhibition concentrations of SE, FSE and tacrine were 410, 4.1 and 0.04 mg/ml, respectively. Furthermore, we measured the AChE-inhibitory activities of SE and FSE ex vivo (Figure 5B). SE and FSE significantly inhibited the AChE activity in the cortex and hippocampus of mice. The inhibitory potency of FSE was more prominent than that of SE. We measured the effect of SE and FSE on BDNF expression in the hippocampi mice treated with scopolamine (Figure 5C). Scopolamine significantly inhibited BDNF expression, but upon SE or FSE treatment, the
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Table 1. The contents of main constituents in soybeans fermented with or without C29. Content (mg/g) Constituent Genistin Genistein Daidzin Daidzein Soyasaponin Ab Soyasaponin I Soyasapogenol A Soyasapogenol B
Defatted soybean
Fermented defatted soybean
203.6 ± 27.8a 8.6 ± 0.8 185.1 ± 19.1 3.5 ± 2.7 918.5 ± 106.4 1037.2 ± 55.2 –b –
6.6 ± 2.0 45.9 ± 15.4 18.6 ± 3.3 34 ± 10.3 190.6 ± 48.4 767.5 ± 37.9 160.9 ± 67.1 91.2 ± 41.6
a
Mean ± SD. Not detected.
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b
Figure 5. Effects of SP and FSP on acetylcholinesterase activity and BDNF expression in the hippocampi of scopolamine-treated mice. (A) Effect on acetylcholinesterase activity in vitro. Test agents were as follows: closed circle, tacrine; closed triangle, SP and closed sequare, FSP. (B) Effect on acetylcholinesterase activity ex vivo in the hippocampus (closed bar) and cortex (open bar) of brain. (C) Effect on BDNF expression. SP (500 mg/kg, p.o.), FSP (500 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 and b-actin were measured by immunoblotting. Values are expressed as mean ± SD (n ¼ 4). #Significantly different from normal mice (p50.05). *Significantly different from scopolamine-treated control (p50.05).
scopolamine-induced reduction of BDNF expression was significantly suppressed. We found no significant difference in b-actin expression. Additionally, we measured genistin, genistein, daidizin, daidzein, soyasaponin I and soyasapogenol B content of SP and FSP. The amounts of genistein, daidzein and soyasapogenol B were higher in C29-fermented SP (FSH) than those in non-FSH, but the amounts of genistin, daidzin and soyasaponin I were lower in C29-fermented soybean than those in non-FSH (Table 1).
Discussion Numerous hydrophilic constituents, such as glycosides and polysaccharides, of orally administered functional foods are
metabolized into bioactive compounds by the gastrointestinal microbiota in the alimentary tract of humans and animals (Akao et al., 1998a; Kim, 2012). The microbiota of different individuals is a unique collection of 4500 different bacterial species (De Filippo et al., 2010; Simon & Gorbach, 1986). Composition of the gastrointestinal microbiota is relatively stable throughout adulthood in the absence of disease or antimicrobial therapy. Indigenous metabolic enzymec activities can be attributed to the unique intestinal microbiota of each individual (Mykkanen et al., 1998; Reddy et al., 1980). Therefore, biological activities of functional foods containing hydrophilic constituents are dependent on the metabolic activities of the intestinal microbiota. In the present study, we found that soyasaponin I ameliorated the scopolamine-induced memory impairment in mice, but this effect was reduced in pseudo-germ-free mice, which were treated with ampicillin, as previously reported (Yoo et al., 2014). These results are consistent with previous report suggesting that soyasaponin I is metabolized to hydrophobic and absorbable compounds by the gut microbiota (Chang et al., 2009). Therefore, to elicit the protective effects of soybean against memory impairment in a gut microbiota-independent manner, we fermented soybean with C29 and measured the effect of FSP in mice with scopolamine-induced memory impairment. FSP was more protective against memory impairments than SP. Similarly, the ethanol extract of FSP (FSE, 100 mg/kg) significantly improved the memory impairment, and it had more prominent effects than SP. Furthermore, fermentation of soybean by the C29 strain increased the concentrations of isoflavone and soyasaponin aglycones genistein, daidzein and soyasapogenols A and B. These results suggest that C29 b-glycosidase(s) might biotransform soybean isoflavone and soyasaponin glycosides to aglycones and these metabolites might elicit more potent protective effects against memory impairment than their molecules. Both SE and FSE suppressed the reduction of BDNF expression induced by scopolamine. This is consistent with previous works reporting that SE and soyasaponins Ab and I significantly inhibit the scopolamine-induced reduction of BDNF expression (Hong et al., 2014; Yoo et al., 2013). Importantly, FSE alleviated this reduction more potently than SE. BDNF influences neuronal synaptic plasticity by depolarizing neurons (Kafitz et al., 1999), enhancing glutamatergic synaptic transmission (Figurov et al., 1996), increasing the phosphorylation of N-methyl-D-aspartate receptor subunit and facilitating hippocampal long-term potentiation (Connor et al., 1997). Furthermore, BDNF expression is lower in the hippocampus of patients with Alzheimer’s disease than that of healthy people (Bourtchuladze et al., 1994; Hock et al., 2000). In the present study, FSE restored the scopolamine-induced reduction of BDNF
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expression. Furthermore, SE and FSE inhibited AChE activity in vitro and ex vivo, unlike soyasaponins and their metabolites soyasapogenol A and B (File et al., 2001). These results suggest that AChE-inhibitory activities of certain SE constituents, such as isoflavones but not soyasaponins, might be activated by the gut microbiota or C29 fermentation. This is supported by a previous study of Orhan et al. (2007), which showed that genistein, a soy isoflavone that is transformed by the gut microbiota or lactic acid bacteria, can inhibit AChE activity in vitro. Therefore, the potent protective effect of the ethanol extract C-29-fermented soybean against memory impairment might be due to the biotransformation of soybean constituents into easily absorbable bioactive compounds by C29 rather than by the gut microbiota.
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Conclusions Fermentation of SP by C29 increased the amount of soyasapogenol B, genstein and daidzein. FSP and its ethanol extract improved the scopolamine-induced memory impairment more potently than SP and its ethanol extract in the passive avoidance, Y-maze and Morris water maze tasks. Additionally, in the hippocampi of mice restored the scopolamine-induced reduction of BDNF expression more potently than SE. Furthermore, FSP inhibited AChE activity in vitro and ex vivo. Based on these findings, C29-fermentation might increase the protective effects of soybean against memory impairment by inhibiting AChE activity and inducing BDNF expression.
Declaration of interest The authors declare that there is no conflict of interest. This study was supported by grants from the Bio & Medical Technology Development Program (2013M3A9B6076413) of the National Research Foundation (NRF) funded by the Korean government (MSIP).
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DOI: 10.3109/09637486.2015.1064865
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