KR101861036B1 - A Composition comprising the extract of Oldenlandia diffusa Herba for preventing and treating cognitive dysfunction and improving memory - Google Patents

Abstract

The present invention relates to a composition for improving memory and treating and preventing cognitive dysfunction containing a Hedyotidis Diffusae Herba extract as an active ingredient. According to the present invention, the Hedyotidis Diffusae Herba extract has shown effectiveness in improving memory in an experiment for passive avoidance response and an underwater maze test using a mouse induced with memory impairment using scopolamine, and also increases expression of genes encoding brain-derived neurotrophic factor (BDNF) and p-CREB, thereby enabling production of health functional foods and pharmaceutical compositions effective in improving memory and cognitive dysfunction.

Description

[0001] The present invention relates to a composition for treating and preventing memory impairment and cognitive dysfunction, which comprises an extract of Baekhwa, as an effective ingredient, and a composition for treating and preventing cognitive dysfunction,

The present invention relates to a composition for treating and preventing memory impairment and cognitive dysfunction, which comprises an extract of Bacillus subtilis as an active ingredient.

Dementia is a complex clinical syndrome characterized by a marked impairment in human cognitive function, intellectual ability, emotional and behavioral changes. It causes cortical dysfunction such as memory, attention, language function, and spatio-temporal ability to lead daily life and social life (Kumar V et al., Robbins and Cotran Pathologic Basis of Disease. Elsevier Saunders., 7th, pp. 1386-1288, 2004). Dementia is defined as the presence of one or more of the other cognitive impairments, including memory, and not simply dementia when the memory is reduced (Dementia Society of Korea, Dementia, Clinical Approach. , 2006).

Alzheimer's Disease or Parkinson's Disease is the cause of dementia, which can be caused by cerebral hemorrhage, metabolic diseases such as hepatic encephalopathy, Wilson's Disease, infectious diseases such as neurosyphilis, acquired immunodeficiency, (2008), "A Study on the Prevalence of Alzheimer's Disease in the Elderly," Korean Journal of Health and Welfare, Vol. . Among them, dementia caused by Alzheimer's disease is the most common with 50 ~ 60%, followed by dementia caused by cerebrovascular disease.

It is estimated that the number of dementia patients among the elderly people aged 65 or older is about 460,000 in Korea, and that it will reach 700,000 in 2020 and 2 million in 2040 (Seoul National University Hospital). pp. 132, 2008). Dementia is more severe than other serious diseases such as cancer because of its long-term treatment and its symptomatic nature. It also has a great physical, psychological and economic burden on the care and protection of patients. With the rapid rate of increase in the elderly population and the prevalence of dementia, it is clear that dementia is a task that requires social and national solutions.

Memory cognitive impairment is the first and most common symptom of Alzheimer's disease. In the early stages of Alzheimer's disease, patients suffer from recent memory disorders that do not remember the details of recent conversations or work, due to the impaired ability of hippocampal neurons to damage recent memory . But at this time, the remote long-term memory of events in the distant past is relatively well preserved.

However, as the disease progresses, the memory of the past gradually becomes impaired as the cortex associated with the storage of long-term memory becomes impaired (Dementia Society of Korea, Dementia, Clinical Approach, Academeia, pp. 254, 2006).

These symptoms of Alzheimer's disease are related not only to cytotoxicity by β-amyloid deposition but also to synaptic dysfunction of the cholinergic system (PM et al., Interactions between the amyloid and cholinergic mechanisms in Alzheimer's disease. Neurochem Int., 53 (5), pp. 103-111, 2008). Cholinergic dysfunction is known to contribute to memory and cognitive dysfunction in Alzheimer's disease patients. Cholinergic neurons of the basal nucleus of Meynert are related to memory and cognitive function in association with temporal lobe, hippocampus and amygdala. In the brains of Alzheimer's patients, 78% in temporal lobe, 60% in hippocampus, (Dementia, Dementia, Clinical Approach, Acadamia, pp. 168, 2006, Korean Psychiatric Association, Neuropsychiatry, Central and Korean History, 2nd, pp. 507-509 , 2007). When brain cells are damaged by cytotoxicity, there is a barrier to the transmission of certain information, ie, the metabolism of neurotransmitters, which causes memory impairment. Many researchers have been steadily reporting the selective reduction of acetylcholine and its related enzymes (choline acetyltransferase) in Alzheimer's disease. Furthermore, in the brain of patients with Alzheimer's disease, as compared with the normal human brain function, not only the decrease of the nicotinic acetylcholine receptor, the muscarinic acetylcholine receptor but also the reabsorption of choline and the secretion of acetylcholine (Alzheimer's disease, Neurochem Int., 53 (5), pp. 103-111, 2008, Talesa VN, Acetylcholinesterase in Alzheimer's disease, Mechanisms of Aging and Development, 122, pp. 1961-1, 2001, Kasa P et al., The cholinergic system in Alzheimer's disease, Progress in neurobiology., 52, pp. 511-535, 1997).

A number of cholinergic drugs have been developed to reverse these cholinergic system disorders, but the most effective and widely used one is Acetylcholinesterase Inhibitor (AchEI). Currently marketed under FDA approval are donepezil, rivastigmine, and galanthamine, which inhibit the degradation of acetylcholine in synaptic clefts and increase the concentration, . These drugs show improvement in cognitive function and daily life in the early stages but they do not fundamentally prevent disease progression in that they return to the pre-administration state after 9 months to 1 year. The effect can be seen only if it is used relatively early, and it is not effective in the case of severe dementia. Common side effects include nausea, diarrhea, loss of appetite, dizziness, muscle cramps, and sleep disturbances due to increased acetylcholine. Serious side effects include bradycardia accompanied by syncope (Korean Dementia Society, Dementia, Clinical Approach. (2006), "The Korean Society for Research on Science and Technology," Dementia Therapeutics, pp. 41-44, 2002). In addition, there are Memantine (NMDA receptor blocker) to inhibit neuronal damage, Ginko biloba (Ginkgo biloba extract) to protect neurons, and Huperzine A (reversible AchEI) to extract Chinese herbal medicine. , Inhibition of deposition, antioxidant, antiinflammatory effect, and so on.

At present, in the case of Alzheimer's disease treatment in Korea, foreign affiliated companies are highly dependent on imports so that they occupy a share of 98%. In addition, one company accounts for 80% of the market, which is virtually monopolistic (Korea Institute of Science and Technology Information, Dementia Therapy, pp. 116, 2002). This shows that the growth of dementia treatment sector is urgent in terms of national competitiveness.

Therefore, the development of effective anti-cancer drugs and memory-improving agents with fewer side effects is urgent, but the research is still limited.

[Document 1] Kumar V, Abbas AK, Fausto N. Robbins and Cotran Pathologic Basis of Disease. 7th. Elsevier Saunders. 2004; 1386-1388. [2] The Korean Society of Alzheimer's Disease. Dementia, Clinical Approach. Academia. 2006; 62-63. [Literature 3] Seoul National University Hospital. Prevalence of senile dementia. Ministry of Health, Welfare and Family Affairs. 2008; 132.

Therefore, the inventors of the present invention have been studying a novel therapeutic agent capable of effectively preventing and treating memory and cognitive dysfunction. In addition to increasing the acetylcholine concentration and enhancing BDNF and p-CREB gene expression, And to increase the memory capacity.

Accordingly, an object of the present invention is to provide a novel diffusa Herba) extract as an active ingredient, and to provide a pharmaceutical composition for preventing and treating cognitive dysfunction.

In addition, another object of the present invention is to provide a method for producing diffusa Herba) extract as an active ingredient for the prevention and improvement of memory and cognitive dysfunction.

Another object of the present invention is to provide a method for increasing the expression of a memory-enhancing gene selected from BDNF or p-CREB gene by administering a pharmaceutically effective amount of a Bacillus subtilis extract to an individual.

According to an aspect of the present invention as described above, the present invention bleaching saseolcho (Oldenlandia diffusa Herba) extract as an active ingredient, and to provide a pharmaceutical composition for preventing and treating memory impairment and cognitive dysfunction.

In one embodiment of the present invention, the extract may be extracted by adding a 5 to 100% aqueous solution of ethanol to white freckles.

In one embodiment of the present invention, the extract may increase the concentration of acetylcholine and increase the expression of BDNF and p-CREB genes.

In one embodiment of the present invention, the cognitive dysfunction may be Alzheimer type dementia, cerebrovascular dementia, or dementia due to head injury.

In one embodiment of the present invention, the daily dose of the composition may be 0.5 to 300 mg / kg based on the content of the active ingredient.

The present invention also relates to a method for producing diffusa Herba) extract as an active ingredient to provide a health functional food for preventing and improving memory and cognitive dysfunction.

In one embodiment of the present invention, the health functional food may be in the form of powder, granule, tablet, capsule or beverage.

Further, the present invention provides a method for increasing the expression of a memory-enhancing gene selected from BDNF or p-CREB gene by administering a pharmaceutically effective amount of a Bacillus subtilis extract to an individual.

The extract of Bacillus leptolepis according to the present invention exhibits memory enhancing effect in the underwater maze test and passive avoidance test using the scopolamine-induced mouse memory impairment and has an activity to increase the expression of BDNF and p-CREB gene , A pharmaceutical composition effective for improving memory and cognitive dysfunction, and a health functional food.

Figure 1 shows the results of weight and brain weights of six experimental treatment groups.
Fig. 2 shows the result of underwater labyrinth experiment with six experimental treatment groups.
FIG. 3 shows the results of the passive avoidance test on six experimental treatment groups.
FIG. 4 shows the results of measurement of acetylcholine concentration and acetylcholinesterase activity in 6 experimental treatment groups.
FIG. 5 shows the results of measurement of expression amounts of BDNF and p-CREB genes in 6 experimental treatment groups.
FIG. 6 shows the results of immunohistochemical staining of the amount of BDNF protein expression in the brain in six experimental treatment groups.
FIG. 7 shows the results of immunohistochemical staining of p-CREB protein expression in brain in six experimental treatment groups.

The present invention relates to a method for producing diffusa Herba) extract as an active ingredient, and to provide a pharmaceutical composition for preventing and treating memory impairment and cognitive dysfunction.

The present invention also relates to a method for producing diffusa Herba) extract as an active ingredient to provide a health functional food for preventing and improving memory and cognitive dysfunction.

In the present invention, the cognitive dysfunction may include Alzheimer type dementia, cerebrovascular dementia, and dementia caused by head injury.

The therapeutic or preventive mechanism for the memory enhancement and cognitive dysfunction is characterized by enhancing the expression of acetylcholine synthetase and receptor and exerting a mechanism of inhibiting acetylcholinesterase.

The extracts defined herein may be water containing purified water, C1 to C4 lower alcohols such as methanol, ethanol, butanol, or mixtures thereof, but may be, but not limited to, 100% ethanol.

The present invention relates to a method for measuring memory performance using an experimental animal that induces memory damage by scopolamine, such as Morris water-maze, Passive Avoidance test (ACE) and Brain-derived neurotrophic factor (BDNF) in order to elucidate the mechanisms of action of these compounds and their effects on the expression of these compounds. And corticosteroid-induced short-term memory impairment, and the therapeutic or preventive mechanism for the memory enhancement and cognitive dysfunction was shown to enhance acetylcholine synthetase and receptor expression And that the extract was exerted through a mechanism of inhibiting acetylcholinesterase, It is useful for the prevention and treatment of memory enhancement and cognitive dysfunction.

The composition comprising the extract of the present invention may further comprise an appropriate carrier, excipient or diluent according to a conventional method.

Examples of carriers, excipients and diluents that can be included in the extract of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The composition containing the extract of the present invention can be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories or sterilized injection solutions, Can be used.

More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The extract according to the present invention may vary depending on the patient's age, sex and body weight, but is generally administered in an amount of 0.01 to 500 mg / kg, preferably 0.5 to 300 mg / kg, . The dose of the extract may also be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Accordingly, the dosage is not limited in any way to the scope of the present invention.

The extract of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

Since the extract of the present invention has little toxicity and side effects, it can be safely used for prolonged use for preventive purposes.

The present invention also provides a health functional food for preventing and ameliorating memory impairment and cognitive dysfunction, which contains the above-described Baekhwasa reed extract as an effective ingredient.

Accordingly, the present invention also provides a food and food additive containing the above-described Baekhwa-ri, reed extract as an active ingredient, which has an effect of preventing and improving memory and cognitive dysfunction.

The composition containing the extract of the present invention can be used variously for medicine, food and beverage for the treatment and prevention of memory improvement and cognitive dysfunction.

Examples of the foods to which the extract of the present invention can be added include various foods such as beverages, gums, tea, vitamin complexes and health supplements. Examples of such foods include pills, powders, granules, infusions, tablets, Can be used.

At this time, the amount of the extract in the food or beverage may generally be 0.01 to 15% by weight of the total food weight of the health food composition of the present invention, 0.02 to 10 g, Preferably in a proportion of 0.3 to 1 g

The health beverage composition of the present invention has no particular limitation on the liquid ingredient other than the above-mentioned extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; Polysaccharides such as dextrin, cyclodextrins; And sugar alcohols such as xylitol, sorbitol, and erythritol. As natural flavors other than those described above, natural flavors (such as tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin)) and synthetic flavors (saccharin, aspartame, etc.) have. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract of the present invention can also be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and aging agents (cheese, chocolate, etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like.

In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that the following examples are merely illustrative of the present invention and that the scope of the present invention is not limited to these examples.

< Example  1>

The chemicals and reagents used in the present invention

Tacrine and ursolic acid were purchased from Sigma Aldrich (St. Louis, Mo., USA). (200 g) were extracted twice in 100% ethanol (EtOH, 1 L × 2) for 3 days each. The extracted solution was filtered and evaporated to give an EtOH extract (9.4 g, yield = 4.7%). BDNF (Santa Cruz, CA, USA), p-CREB (Ser133), total-CREB (Santa Cruz, CA, USA) and GAPDH (Cell signaling, Beverly, MA, USA)

< Example  2>

Preparation of experimental animals

ICR mice (Male Institute of Cancer Research, 5 weeks old) were purchased from Samtako, BIOKOREA (Osan, South Korea). Animals were acclimated for 7 days before the experiment. The animals were maintained at a controlled temperature (22 ± 3 ° C) and humidity (50 ± 10%) at 12 hours of light period (07:00 to 19:00). All animal experiments were approved by the Institutional Animal Care and Use Committee (SEMI 15-07) and were performed in accordance with the National Institutes of Health (NIH) Laboratory Animal Care and Use Guide.

< Example  3>

Experimental Design

After 1 week of adaptation, mice were randomly divided into 10 groups of 6 groups.

Six treatment groups were normal mouse control, control, 2 mg / kg tacrine, 2 mg / kg uronic acid, 100 mg / kg bloom extract and 200 mg / kg bloom extract See Table 1).

In the present invention, the tacrine administration group was used as a positive control group, and the uronic acid was one of the main compounds of ODH. In the case of the normal and control groups, physiological saline was orally administered for 28 days. In other treatments, oral gavage is administered daily using intragastric gavage for 28 days. After 28 days of dieting, scopolamine (5 mg / kg), which causes transient dementia, was injected intraperitoneally in all groups and mice were sacrificed 24 hours later.

< Example  4>

Mice weighing and brain weighing results

The results of weight and brain weights of the six treatment groups of the present invention are shown in FIG.

The whiteness extract of the present invention ( Oldenlandia diffusa Herba; ODH) were administered orally for 28 days by the above concentrations. There was no significant difference in the weight of rats between the groups. In addition, when the brain was extracted and weighed after 28 weeks of experimentation, there was no significant difference between the groups, and thus it was found that the white whitener extract extract of the present invention had less toxicity and side effects.

< Example  5>

Evaluation of memory ability

<5-1> Underwater maze  Experiment (Morris Water Maze Test)

For the experiment of the present invention, a circular pool (diameter 100 cm, height 35 cm) was filled with water (21-32 ° C) of 15 cm depth. The mouse has been trained to discover hidden platforms in the water maze for five consecutive days. I first gave 90 seconds to find the hidden platform, and if I could not find the platform for 90 seconds, I left the mouse for 15 seconds. After 30 seconds, the time to wait for the escape (finding the dive platform) was recorded, and when all the mice found the platform within 90 seconds, the experiment was completed. The experiment was carried out using the same test method for 3 days and the escape latency was recorded.

Morris water maze test was performed on the experimental animals. The time required for platform search, which was increased by scopolamine, was significantly reduced in the present invention of the whitening reeds treated group compared to the positive control and ursolic acid groups (See FIG. 2).

This means that the Baekwajeopsaek is protected by the spatial memory ability of the scopolamine.

<5-2> Passive Avoidance Test

A shuttle box (50 × 15 × 40 cm, electric grid floor, Ugo, Italy) consisting of one light (20 W lamp) and one dark room (25 × 15 cm each) and a guillotine connection for guillotine (10 x 10 cm) were prepared. The experiment was performed in a quiet environment (<60 dB). Mice were placed in an illuminated room (1500 Lux) in five consecutive tests. When the mice were transferred to the dark room, an electric shock (3 mA) was given through the stainless steel grid for 3 seconds. Twenty-four hours later, with the door of the connected guillotine open, the mouse was placed in a lighted box. The waiting time to enter the dark room was recorded for 5 minutes.

Passive evacuation tests on experimental animals showed that delayed avoidance response time by scopolamine was significantly reduced in the Bacillus subtilis treated groups (see FIG. 3).

This is because the white freckle of the present invention suppresses the decline of memory caused by scopolamine.

<5-3> Biochemical analysis

<5-3-1> Preparation of tissue for biochemical analysis

All rats were anesthetized with CO2 for 12 hours starvation, the brains were removed and weighed. The brain was then homogenized with homogenization buffer (12.5 mM sodium phosphate buffer, pH 7.0, 400 mM sodium chloride). The tissue homogenate was centrifuged at 1,000 xg for 10 min at 4 ° C, and the supernatant was used for biochemical analysis.

<5-3-2> Acetylcholine concentration measurement

For acetylcholine concentration, 50 μl of the brain homogenate was mixed with 50 μl of 1% hydroxylamine and 500 μl of FeCl ₃ (10% in 0.1 N HCl). Absorbance (490 nm) was evaluated using a spectrophotometer.

<5-3-3> Measurement of acetylcholinesterase activity Acetylcholinesterase  Activity)

2.6 ml of 0.1 M phosphate buffer solution (pH 8.0) was added to measure the homogenization solution of the brain. 100 μl of 10 mM Ellman solution (10 mM 5,5'-dithio-bis-2-nitrobenzoic acid, 15 mM sodium bicarbonate) Changes in absorbance were observed using 0.2 mL of zymogen at 405 nm for 2 minutes using a spectrophotometer.

In dementia, the concentration of acetylcholine, a neurotransmitter, decreases and the activity of acetylcholinesterase, which degrades acetylcholine, is increased. Therefore, the concentration and activity of these acetylcholine were measured in the brain of an experimental animal as described above.

As a result, the concentration of acetylcholine reduced due to scopolamine was significantly increased in the group of Paired Leaves of the present invention, and the acetylcholinesterase activity increased due to scopolamine, (See Fig. 4).

<5-3-4> Western blotting  analysis

The Western blotting analysis method used in the present invention is as follows.

PBS buffer (300)) was added to the frozen brain tissue, homogenized with a homogenizer, and centrifuged at 800 rpm for 10 seconds. The supernatant was dissolved in 50 μl of ice buffer for 30 minutes and centrifuged at 4 ° C and 14,000 rpm for 20 minutes.

Protein concentrations were calculated using the Bradford assay. The same amount of protein was separated by electrophoresis on an SDS-PAGE gel and electroporated into a NC (nitrocellulose) membrane using a semi-dry transfer system (Bio-Rad, USA). After blocking with blocking buffer (0.5% skim milk, 1 × PBST buffer) for 1 hour and washing with 1 × PBST buffer for 10 minutes (3 times), the membrane was incubated with primary antibody of BDNF (Santa Cruz, CA, USA) (Santa Cruz, CA, USA), p-CREB (Ser133), total-CREB (Santa Cruz, CA, USA) and GAPDH (Cell signaling, Beverly, MA, USA).

After 2 hours, the membranes were incubated with horseradish peroxidase (HRP) -binding secondary antibody or rabbit antibody (AbD serotec, kidlington, UK). Protein bands were visualized with Chemi-Doc using Western Blot Detection Kit (Biorad, Hercules, USA).

Brain-derived neurotrophic factor (BDNF) plays an important role in learning and memory, and the activity of cAMP response element-binding protein (CREB, Ser133) is required for the expression of BDNF in hippocampal neurons. BDNF and p-CREB expression is reduced in the hippocampus of patients with dementia.

In the present invention, the hippocampus was extracted from the brain of each group of experimental animals, and the amount of each hippocampus was measured by Western blot analysis. As a result, it was found that the expression amount of BDNF and p-CREB decreased due to scopolamine, because of the administration of Bacillus specialis (see FIG. 5).

<5-3-5> Immunohistochemical staining analysis

The dissected brain was washed and fixed with Bouin solution for 4 hours. Brains were frozen in 30% sucrose, cut into tissue-freezing medium with liquid nitrogen, and coronal frozen sections (4 μm) were cut.

The 10 μm tissue sections were paraffin-free using heated Xylol and then rehydrated with dehydrated EtOH (100%, 80%, 50%, H ₂ O) and then blocked with blocking buffer (5% normal (Santa Cruz, CA, USA) and p-CREB (ser133) (Santa Cruz, CA, USA) diluted 1: 100 in PBS and 0.3% Triton X- Lt; RTI ID = 0.0 &gt; 4 C. &lt; / RTI &gt;

Protein expression was visualized using 3.3'-diaminobenzidine tetrahydrochloride (DAB). All immune responses were observed using an Axio vision 4.0 fluorescence microscope (Carl Zeiss, Germany).

The expression of BDNF and p-CREB in the hippocampal region of each group was analyzed by immunohistochemical staining as described above. As a result of western blot analysis, decreased expression of scopolamine was recovered in the group treated with Bacillus subtilis (See Figs. 6 and 7).

< Example  5>

Statistical analysis

All data are expressed as mean ± standard deviation. Statistical significance of behavioral tests was determined by one-way analysis of variance (ANOVA) using the Statview program.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (8)

delete delete delete delete delete delete delete A method of increasing the expression of a memory-enhancing gene selected from the BDNF or p-CREB gene by administering an extract of Bacillus subtilis in a sample.

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