Effect of soybean supplementation on the memory of alprazolam-induced amnesic mice.

Soybean, Glycine max (L.) Merr. (Leguminoseae), is known as golden bean. It contains vegetable protein, oligosaccharide, dietary fiber, vitamins, isoflavones and minerals. Earlier studies have demonstrated a cholesterol lowering, skin protective, antitumour, antidiabetic and antioxidative potential of soybean. Soy isoflavones are also utilized as estrogen replacement therapy in postmenopausal women. The present study was undertaken to investigate the effect of soybean on memory of mice when consumed along with diet. Soybean was administered chronically for 60 consecutive days as three soybean diets viz. Soy2, Soy5, Soy10. These diet contains soybean in normal diet at concentration of 2%, 5%, 10% w/w respectively. Passive avoidance paradigm and elevated plus maze served as exteroceptive behavioral models for testing memory. Alprazolam (0.5 mg/kg; i.p.) induced amnesia served as interoceptive behavioral model. The administration of soybean significantly reversed alprazolam-induced amnesia in a dose-dependent manner as indicated by the increased step down latency of mice using passive avoidance paradigm and increased transfer latency using elevated plus maze. Theses results suggest that consumption of soybean in diet may not only improve memory but also reverse the memory deficits, owing to its multifarious activities. It would be worthwhile to explore the potential of this nutrient in the management of Alzheimer's disease.

Dementia is a syndrome of the gradual onset and continuing decline of higher cognitive functioning. It is a common disorder in older persons and becomes more prevalent in each decade of life.[1] Approximately 10% of the adults, 65 years or older, and 50% of the adults older than 90 years have dementia.[2] The most common cause of dementia is Alzheimer's disease, which is a progressive neurodegenerative disorder, associated with loss of neurons in distinct brain areas.[3] Currently, the allopathic system of medicine principally relies on nootropic agents and anticholinesterases.[4] In addition to medicines, dietary supplementation with useful nutrients may be advocated to minimize the number of patients with dementia. Soybean may be one such type of nutrient.

Soybean, Glycine max (L.) Merr., (Leguminoseae), is a singular food because of its rich nutrient content.[5] Soybean contains vegetable protein, oligosaccharides, dietary fiber, vitamins, and minerals.[6] It is consumed in different forms like soybean flour, soy milk, tofu, and temphe.[6] Soybean intake is also advocated in patients with diabetes mellitus,[7] osteoporosis,[8] skin disorders,[9] and cancer.[1011] Soy protein has been seen to be useful in lowering blood cholesterol, improving coronary artery function, and also preventing the development of heart disease.[12] Dietary intake of soybean fiber has a positive effect on diarrhea as well as constipation and can be useful as a therapy for irritable bowel disease.[13] Soy isoflavones have also been reported to be effective anti-inflammatory[14] and antioxidative agents.[15] Soy isoflavones are utilized as estrogen replacement therapy in postmenopausal women.[16] Soy isoflavones include glycone forms of genistein, daidzein, and glycetin.[17] Soy isoflavones and their metabolites can act as agonists or partial agonists at estrogen receptors.[18] Reports in the literature suggest that soy isoflavones may improve verbal and non-verbal memory in men and women.[19] However, the reports available in literature are not sufficient to reflect the neuroprotective properties of soybean, when consumed along with the diet.

In the light of the above, the current study was undertaken to investigate the effect of soybean supplementation on the memory of alprazolam-induced amnesia in mice.

Materials and Methods

Animals

Young (3 – 4 months, 20 g) Swiss albino mice of either sex, were employed in this study. The animals were procured from the disease-free small animal house of CCS Haryana Agriculture University, Hisar, Haryana, India. The animals had free access to food and water. They were housed in a natural (12 hour each) light–dark cycle. The animals were acclimatized for at least five days to the laboratory conditions, prior to behavioral experiments. The experiments were carried out between 0900 hours and 1800 hours. The experimental protocol was approved by the Institutional Animal Ethics Committee and the laboratory animals were taken care of as per the guidance of CPCSEA, Ministry of Forests and Environment, Government of India.

Drugs

The drugs were obtained from the following drug houses. Alprazolam (Ind-Swift, Baddi, India) and piracetam (NootropilR, UCB India Ltd, India). Alprazolam was suspended in 1% carboxymethylcellulose sodium before administration.

Experimental design

In the current investigation, the mice were divided into 12 different groups. Each group comprised of a minimum of six animals. Soybean (Soy) was administered chronically for 60 consecutive days as three soybean diets namely, Soy2, Soy5, and Soy10. These diets contained soybean in the normal diet at concentrations of 2, 5, and 10% w/w, respectively. The doses of soybean were determined on the basis of a pilot study and literature reports.[1720] Soybean administration was continued during the maze studies. The animals were given food ad libitum. During the experiment period, the body weight was measured once each week and the extent of food consumption was measured daily by weighing the remaining (uneaten) diet in the cage. Behavioral studies were carried out using passive avoidance and elevated plus maze. Amnesia was induced in mice by the administration of alprazolam (0.5 mg/kg; i.p.).[21]

Group I (Control group) — A standard diet was administered to the mice. The mice were exposed to training sessions using the passive avoidance paradigm. Retention (memory) was recorded after 24 hours.

Group II (Alprazolam group) — Alprazolam (0.5 mg/Kg; i.p.) was administered to the mice of this group. The mice were exposed to the training session using the passive avoidance paradigm after 45 minutes of injecting alprazolam. Retention (memory) was recorded after 24 hours.

Groups III, IV, and V (Soybean + Alprazolam groups) — Soy (2, 5, and 10% w/w, respectively) was administered for 60 successive days. Alprazolam (0.5 mg/kg; i.p.) was injected after 90 minutes of the last dose of soybean administration (on the sixtieth day). The mice were exposed to the training session using the passive avoidance paradigm after 45 minutes of injecting alprazolam. Retention (memory) was recorded after 24 hours (on the sixty-first day).

Group VI (Piracetam + Alprazolam) — Piracetam is an established memory enhancer available in the market,[22] it serves as a positive control for comparing the memory enhancing activity of soybean in both exteroceptive behavioral models. Piracetam (400 mg/kg; i.p.) was administered for seven successive days to the mice. Alprazolam (0.5 mg/kg; i.p.) was injected after 90 minutes of the last dose of piracetam (on the seventh day). The mice were exposed to the training session using the passive avoidance paradigm after 45 minutes of the injection. Retention was recorded again after 24 hours (on the eighth day).

The elevated plus maze study (Groups VII to XII) was employed along similar lines.

Passive avoidance paradigm

Passive avoidance behavior based on negative reinforcement is used to examine long-term memory.[2324] The apparatus consisted of a box (27 cm × 27 cm × 27 cm) having three walls of wood and one wall of Plexiglas, featuring a grid floor (made up of 3 mm stainless steel rods set 8 mm apart), with a wooden platform (10 cm × 7 cm × 1.7 cm), in the center of the grid floor. The box was illuminated with a 15W bulb during the experimental period. Electric shock (20V, A.C.) was delivered to the grid floor. Training (on the sixtieth day of soy administration) was carried out in two similar sessions. Each mouse was gently placed on the wooden platform set in the center of the grid floor. When the mouse stepped-down, placing all its paws on the grid floor, shocks were delivered for 15 seconds and the step-down latency (SDL), which was defined as the time (in seconds) taken by the mouse to step down from the wooden platform to the grid floor, with all its paws on the grid floor, was recorded. Animals showing SDL in the range of 2 – 15 seconds during the first test were used for the second session and the retention test. The second-session was carried out 90 minutes after the first test. During the second session, if the animals stepped down before 60 seconds, electric shocks were delivered once again for 15 seconds. During the second test, the animals were removed from the shock-free zone if they did not step down for a period of 60 seconds and were subjected to the retention test. Retention (memory) was tested after 24 hours (i.e., the sixty-first day) in a similar manner, except that the electric shocks were not applied to the grid floor, observing an upper cutoff time of 300 seconds. A significant increase in SDL value indicated improvement in memory.

Elevated plus maze

Elevated plus-maze (EPM) served as the exteroceptive behavioral model to evaluate the memory in mice. The procedure, technique, and end point for testing memory was followed as per the parameters described earlier.[2325] Briefly, the elevated plus maze for mice consisted of two open arms (16 cm Χ 5 cm) and two covered arms (16 cm × 5 cm × 12 cm) extending from a central platform (5 cm × 5 cm), and the maze was elevated to a height of 25 cm from the floor. On the first day (i.e., the sixtieth day of soy administration), each mouse was placed at the end of an open arm, facing away from the central platform. Transfer latency (TL) was defined as the time (in seconds) taken by the animal to move from the open arm into one of the covered arms with all its four legs. The mouse was allowed to explore the maze for another two minutes and then returned to its home cage. Retention of this learned-task (memory) was examined 24 hours after the last trial (on the sixty-first day). A significant reduction in TL value indicated improvement in memory.

Measurement of locomotor activity

The spontaneous locomotor activities of control and test animals were recorded for a period of 10 minutes using Medicraft photoactometer (INCO, Ambala, India).

Statistical analysis

All the results were expressed as mean ± SEM. Data were analyzed using one way ANOVA followed by post hoc Tukey's multiple range test. P < 0.05 was considered to be statistically significant.

Results and Discussion

The administration of soybean for sixty consecutive days had not significantly affected the diet intake and weight of experimental animals. The administration of alprazolam (0.5 mg/kg; i.p.) to the mice before the training trials showed a significant (p < 0.05) decrease in SDL 101.7 ± 2.6 vs. 168.1 ± 3.6; Figure 1), when using the passive avoidance paradigm, and an increase in TL 50.2 ± 4.7 vs. 23.5 ± 3.4; Figure 2), when using the elevated plus maze, as compared to the control group mice, indicating induction of amnesia. However, administration of all Soy2, Soy5, and Soy10 successfully (p < 0.05) reversed the alprazolam-induced memory deficit in a dose-dependent manner as indicated by the increased SDL 119.2 ± 3.9, 128.2 ± 3.5, 139.3 ± 4.1, vs. 101.7 ± 2.6; Figure 1) and decreased TL 43.3 ± 4.3, 32.2 ± 1.8, 28.2 ± 2.2, vs. 50.2 ± 4.7; Figure 2). Piracetam (400 mg/kg; i.p.) served as a positive control and pretreatment with piracetam reversed the alprazolam-induced amnesia as expected. Soybean did not show any significant change in the locomotor activity of mice as compared to control animals.

Figure 1

Effect of soybean on the step down latency (SDL) of mice using the passive avoidance paradigm. Values are mean ± SEM. a - denotes P < 0.05 as compared to the control group; b - denotes P < 0.05 as compared to the alprazolam group

Figure 2

Effect of soybean on the transfer latency (TL) of mice using the elevated plus maze. Values are mean ± SEM. a - denotes P < 0.05 as compared to the control group; b - denotes P < 0.05 as compared to the alprazolam group

In the present study, administration of alprazolam successfully induced amnesia in the experimental animals. It acted on the benzodiazepine site of the GABA / benzodiazepine receptor complex and impaired both prospective and retrospective memory.[2627] The benzodiazepine binding sites are present on the GABA receptor, in the hippocampal neurons.[28] It has been suggested that the blocking of long-term potentiation in the slices of hippocampus and piriform cortex by benzodiazepines has occurred by the increased action on the GABA-mediated inhibitory potential, mediating the amnesic effects of benzodiazepines.[29] These findings are further confirmed by the studies where flumazenil, a benzodiazepine antagonist, has been shown to block alprazolam-induced amnesia.[30] Alprazolam is therefore, a useful candidate in the evaluation of antiamnesic drugs. In the present study, the passive avoidance paradigm and elevated plus maze studies revealed that soybean administration reversed alprazolam-induced amnesia. It is possible that soybean reverses the inhibition of long-term potentiation in the hippocampus in exerting antiamnesic effects.

Soybean contains isoflavones (genistein, daidzein, and glycetin) in abundant quantity;[31] it is quite possible that the observed anti-amnesic effects are due to these isoflavones. Soy isoflavones have an estrogenic effect, and it has been reported that soy isoflavones may improve cognitive functions by mimicking the effects of estrogen (especially through estrogen receptor β) in the brain.[18] Various studies have shown that estrogen can significantly enhance the basal forebrain cholinergic function by increasing the uptake of choline, and stimulating acetylcholine release.[3233] Acetylcholine is considered as the most important neurotransmitter involved in the regulation of cognitive functions.[4] There is extensive evidence linking the central cholinergic system to the memory.[3435] Cognitive dysfunction has been shown to be associated with impaired cholinergic function and the facilitation of central cholinergic activity, with improved memory.[36] Soy isoflavones are reported to increase cholinergic transmission due to indirect facilitation of acetylcholine in the brain. This increased cholinergic transmission may be exerted through activation of choline acetyltransferase[37] or inhibition of acetylcholinesterase activity.[38] These suggestions are also supported by a study in which the aqueous extract of soybean has shown a contractile action on the guinea pig ileum via activation of muscarinic acetylcholine receptors, and this response was inhibited by atropine.[39]

In addition, soy isoflavones have been found to promote the activity of neurotrophic factors such as, the brain derived neurotrophic factor (BDNF)[40] and nerve growth factor (NGF)[37] in the brain. The agents having cholesterol lowering and antioxidant properties play a potential role in the treatment of Alzheimer's disease.[41] Soy isoflavones have shown cholesterol lowering and antioxidant activities in animals as well as humans.[313842] These properties of soy isoflavones might contribute to the anti-amnesic activity of soybean.

Conclusion

In light of the earlier discussion, it may be concluded that consumption of soybean in the diet reversed alprazolam-induced amnesia, owing to its multifarious activities. However, further investigations are necessary to explore the potential of this nutrient in the management of Alzheimer's disease.