KR100861730B1 - Composition comprising an extract of nelumbinis semen for treating and preventing cognitive dysfunction - Google Patents

Abstract

An extract of Nelumbinis Semen is provided to show an effect of improving memory and learning ability and an effect of increasing brain cell proliferation during passive avoidance tests of a mouse, thereby being usefully used as a composition for preventing and treating cognitive impairment related diseases. A pharmaceutical composition for preventing and treating Alzheimer's disease, vascular dementia, Pick's disease, Creutzfeldt-jakob's disease, dementia caused by head injury or Parkinson's disease comprises 0.1-50 wt.% of 60-80% of methanol soluble extract of Nelumbinis Semen as an effective ingredient.

Description

Composition comprising an extract of Nelumbinis Semen for treating and preventing cognitive dysfunction

1 is a diagram showing the results of the passive avoidance experiment of the mice administered the control, lotus root extract and lotus root extract,

Figure 2 is a diagram showing the results of measuring the number of brain cells in the mice administered the control, lotus root extract and lotus root extract,

3 is a diagram showing the results of immunostaining using BrdU in the mice administered the control group, lotus root extract and lotus root extract,

4 is a diagram showing the results of immunostaining using Ki-67 in the control and mice administered the extract of lotus root meat,

5 is a diagram showing the results of immunostaining using DCX in the control group and mice administered the lotus leaf extract.

The present invention relates to a composition for the prevention and treatment of diseases related to cognitive impairment containing lotus leaf extract as an active ingredient.

As the elderly population increases in Korea as well as in the world, many degenerative diseases of the elderly are causing social and economic problems. Degenerative brain disease is a disease characterized by impaired cognitive function such as memory loss, and includes various diseases such as dementia, Parkinson's disease, stroke, and Huntington's disease. Also, in modern society, the increase of degenerative brain diseases such as senile dementia due to the rapid increase of the elderly population is a serious social problem, but until now, no effective preventive methods or treatments have been developed to prevent and treat the disease. It is true. Dementia, a typical degenerative brain disease, is a disorder of the general cognitive function. It is usually caused by chronic or progressive brain disease and impairs many senior cerebral functions such as memory, thinking, understanding, calculation, learning, and language judgment. The cause of dementia is not known, but the presumed causes include damage to choline neurons in the base of the cerebrum, reduction of neurotransmitters, accumulation of beta-amyloid proteins by inflammatory responses, and oxidative stress. (Davies et al., Lancet , 21 , p 1403, 1976; Rocher et al., J. Biol. Chem., 273 , p 29719, 1988; Coyle et al., Science, 262 , p689, 1993).

Memory degradation is closely related to neurotransmitters synthesized in neurons (Zarow, C et al., Arch. Neurol , 60 , pp337-341, 2003). Catecholamines synthesized from tyrosine in the sympathetic nerve ( Indolamines synthesized from catecholamines and tryptophan, along with their metabolites, are found in various parts of the body. The concentration of these substances in neurons is a biochemical function of sympathetic nervous system function. Can be used as an indicator. In particular, by tracking the amines and metabolites concentration in the brain, and the ability to act on the receptor as the aging progresses, it is possible to diagnose aging and related diseases of the brain. Measurement of glutamate or gamma aminobutyric acid (GABA) in the brain has been applied to the diagnosis of degenerative diseases and Alzheimer's in the brain. This is because excitatory amino acids (EAA) such as glutamate and aspartate act as major excitatory transporters in the central nervous system, leading to neuronal survival, synaptogenesis, learning and memory, and neuroplasticity. (Bowen, DM et al., Brain, 99 , pp 459-496, 1976). However, they accumulate neurotoxicity when they accumulate in the extracellular fluid, especially glutamate, such as hypoglycemia, status epilepticus, ischemia, hypoxia, head trauma, and hepatic brain disorders. It has been reported to cause neural cell necrosis in the back (Masotto, C et al., Phamacol . Res. Commun , 17 , pp 749-772, 1985). In addition, it has been reported that glutamate and GABA in the brain may cause neurological damage in relation to cerebral degenerative diseases and Alzheimer's (Choi, DW, J. Neurosci , 7 , p369, 1987).

On the other hand, senile dementia, which is a gradual loss of cognitive ability, is also associated with the activity of cholinergic neurons in the central nervous system, which is mainly caused by a marked decrease in acetylcholine and choline acetyltransferase activity in the brain. Known as More than 60 neurotransmitters have been discovered, and acetylcholine, catecholamines, glutamate, and GABA play important roles in various aspects of learning and memory (Cummings JL et al., Neurology , 44 , pp 2308-14, 1994).

Lotus meat is the fruit and seed of lotus flower, which contains a large amount of starch and raffinose, protein 16.6%, fat 2%, carbohydrate 62%, calcium 0.089%, phosphorus 0.285%, iron 0.0064%. Jarae contains nuciferine, n-nornuciferine, n-nornuciferine, oxoushinsunine (lixiodenine), and n-norarmepavine (n-norarmepavine). Oxo-synsinin has a function of suppressing non-human diseases. Conscience (養 心), 신 xin (보), Bobby (補脾), the effect of the eleven. Yam-daam (유 多 夢), Yujeong (遺精), Im Tak (淋 濁), grilled (久 痢), vulgarity (虛 瀉), Mrs. Vung daedae (婦人 崩 漏 帶下) is treated. In addition, tonic, governor, jigal, soothing, Jingu, health medicinal herbs, Efficacy and commonly used to treat geumgui (禁 口 痢) (Bo Bo-seop and Shin Mingyo, Hyangjedae, Yeonglimsa, p514, 1998)

It is very well known that memory is closely related to neurotransmitters. Therefore, it is important to evaluate the dynamics and effects of neurotransmitters in the development of brain function improving substances. In addition, considering the long-term use of drugs applicable to memory loss, the study on high efficacy and safe herbal medicines will have very beneficial results.

Therefore, the present inventors completed the present invention by confirming that the composition containing the extract of lotus root meat has a significant memory enhancement and learning improvement effect in the passive avoidance experiment and the brain cell proliferation effect is excellent in order to develop cognitive improvement and the treatment of brain disease. It was.

An object of the present invention is to provide a composition for the prevention and treatment of cognitive impairment-related diseases containing the extract of lotus root meat as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of diseases related to cognitive impairment containing the extract of lotus root meat as an active ingredient.

Extracts as defined herein include extracts soluble in water, including purified water, lower alcohols having 1 to 4 carbon atoms or mixed solvents thereof, preferably in water and methanol mixed solvents, more preferably 60-80% methanol. Include.

The cognitive dysfunction related diseases include Alzheimer's dementia, cerebrovascular dementia, pick name, Creutzfeldt-jakob disease, dementia due to head injury and Parkinson's disease, preferably Is characterized by Alzheimer's dementia or cerebrovascular dementia.

The lotus meat (Nelumbinis Semen) is characterized in that the seed or fruit of the lotus.

Hereinafter, the method for obtaining the extract of the present invention will be described in detail.

The extract of the present invention is water containing about 1 to 50 times, preferably about 5 to 30 times the volume of soft meat, lower alcohols having 1 to 4 carbon atoms or a solvent selected from a mixed solvent thereof, preferably water and methanol Hot water extraction, reflux cooling with a mixed solvent, more preferably 60 to 80% methanol at an extraction temperature of 20 to 100 ° C., preferably 80 to 100 ° C. for about 1 to 5 hours, preferably 1 to 3 hours Extraction method, such as extraction, ultrasonic extraction, preferably by extraction method of hot water extraction 1 to 5 times, preferably 2 to 3 times to obtain the extract, and then filtered under reduced pressure and freeze-dried to soften the meat of the present invention Extracts can be obtained.

The present invention provides a pharmaceutical composition for the prevention and treatment of cognitive impairment disorders containing as an active ingredient the extract of lotus root meat obtained by the above method.

The composition for preventing and treating diseases related to cognitive dysfunction of the present invention comprises 0.1 to 50% by weight of the extract of lotus root meat based on the total weight of the composition. However, the composition as described above is not necessarily limited thereto, and may vary according to the condition of the patient and the type and extent of the disease.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical compositions of the present invention, each can be used in the form of oral dosage forms, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injectable solutions according to conventional methods, Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered at 1 to 1000 mg / kg, preferably 50 to 500 mg / kg per day based on healthy adults. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention provides a dietary supplement for the prevention and improvement of cognitive impairment-related diseases containing the extract of lotus root meat as an active ingredient.

The health functional food of the present invention includes the form of tablets, capsules, pills, or liquids, and as a food to which the composition of the present invention can be added, for example, various foods, beverages, gums, teas, vitamin complexes, etc. And health functional foods.

It may also be added to food or beverages for the purpose of preventing and improving diseases related to cognitive impairment. At this time, the amount of the composition in the food or beverage may be added at 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 10 g, preferably 0.3 to 1 g based on 100ml. have.

In addition to containing the extract as an essential ingredient in the indicated proportions, the health beverage composition of the present invention has no particular limitation on the liquid component, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose, for example polysaccharides such as maltose and sucrose, and conventional sugars such as dextrin and cyclodextrin. And sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from 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 detail by Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

Comparative example  1. Preparation of Lotus Root Extract

300 g of lotus root (Dayeon, Jeonnam Muan) was placed in a 10 L round flask, and 6 L of 70% methanol was added thereto, followed by stirring for 2 hours by connecting a chiller. The whole solution was filtered on a filter paper, concentrated in a vacuum concentrator, and freeze-dried with a lyophilizer to obtain 45 g (yield: 15%) of lyophilized extract of lotus root extract.

Example  One. Lotus root  Preparation of Extract

300 g of lotus root meat (Wonkwang Pharmaceutical Co., Ltd., Korea) was placed in a 10 L round flask, and 6 L of 70% methanol was added thereto, followed by stirring for 2 hours by connecting a cooling device. The lyophilized solution was filtered through a filter paper, concentrated under a reduced pressure concentrator, and lyophilized with a lyophilizer to obtain lyophilized extract of 36 g (yield: 12%) of lotus leaf extract.

Reference Example  1. Experimental Animal

1-1. Preparation of Laboratory Animals

Five-week-old male rats (180-200 g) of Wistar system supplied from the central laboratory animals were used in the experiment of the present invention, and water and feed (Purina Inc., Korea) were freely eaten, and the temperature was 18 The experiment was carried out after adapting for 10 days in a nursery environment with a temperature of ～ 24 ℃ and a humidity of 40 to 60%. Day and night cycles were 12 hours each and night time was from 7 am to 7 pm. The laboratory environment was the same as the breeding room and the same environment was maintained for the duration of the experiment.

1-2. Sample Administration of Laboratory Animals

The experimental animals of Reference Example 1-1, which were adapted to the laboratory environment for 10 days, were divided into a control group, lotus root administration group, and lotus root administration group, and each group was 10 animals. The animals weighed 250-300 g at the time of the experiment. While supplying enough water and feed, the control group dissolved primary distilled water, and the experimental group dissolved the samples of Example 1 and Comparative Example 1 in the primary distilled water at a concentration of 1 g / 10 ml for 21 consecutive administrations once daily for 21 days (1.0 g / kg / day, po).

Reference Example  2. Statistical Processing

The results of the behavior test were expressed as Mean ± SD, and the significance evaluation was performed using the Mann-Whitney U-test using SPSS 11.0 for Window (SPSS Inc., USA). .

The results of the tissue experiments were expressed as Mean ± S.D., And the significance test was conducted using Student's t-test using SPSS 11.0 (SPSS 11.0 for Window (SPSS Inc., U.S.A.)).

Experimental Example  Passive avoidance test

To investigate the effects of lotus roots and lotus roots on the learning and memory abilities of rats, Lee's method (Lee, Jin-woo and Goji-ja) was used to evaluate the memory and forgetfulness of white papers. effect of速度), Journal of physiology and Pathology, 15 (1), pp84 ~ 9, 2001), method of Manuel (Manuel Sanchez-Alavez et al. , Cortistatin modulates memory processes in rats, Brain Research, 858, pp78 ~ 83 , 2000) and Schtle using methods such as Park et al. (Cheol Hyoung Park et al., Glutamate and aspartate impair memory retention and damage hypothalamic neurons in adult mice, Toxicology Letters , 115 , pp117-125, 2000). A passive avoidance test was conducted using a shuttle box.

One hour before the start of each experiment, the animals were transferred to the behavioral laboratory and stabilized, and the experiment was conducted in a dark state where the illumination of the behavioral laboratory was turned off.

1-1. Training trial; first day (Day 1st))

The light in the left room of the passive avoidance box was turned on and the animals were placed in the left room for 10 seconds with the left and right radiators closed. When the left and right radiators are opened, the experiment animal instinctively moves to the dark right room. At this time, the left and right radiators are closed and the right room is sufficiently searched for 1 minute. Then, the animals are taken out to the cage. Moved. The above method was repeated so that the time from the left room to the right room was less than 20 seconds.

1-2. Acquisition trial; Second day (Day 2nd))

The light in the left room of the passive avoidance box was turned on and the animals were placed in the left room for 10 seconds with the left and right radiators closed. When the left and right radiators opened, the experimental animals moved to the dark right room. The left and right radiators were closed and 2.0 mA electric stimulation was applied to the soles of the animals for 5 seconds. After the electrical stimulation, the left and right radiator doors were opened, and the animals avoided immediately to the left room, where they stayed for 30 seconds before being transferred to the cage.

1-3. Retention test; third day (Day 3rd))

After 24 hours of electrical stimulation, the left room of the passive avoidance box was turned on and the animals were placed in the left room for 30 seconds with the left and right doors closed. The left and right radiators were opened and the time taken for the animals to enter the right chamber was measured. All four feet of the test animals were measured to enter the right chamber. If 300 seconds did not enter, the retention time was recorded. After that, the memory capacity was measured after 24 hours of learning.

1-4. Experiment result

The retention time of the electrical stimulation resulted in 179.1 ± 96.4 seconds (Mean ± SD, n = 9) in the soft-fed group compared to the control group 66.9 ± 78.0 seconds (Mean ± SD, n = 9). 0.01), the lotus root administration group was 124.8 ± 115.1 seconds (Mean ± SD, n = 9) did not appear significant (see Figure 1).

Experimental Example  2. Immunohistostaining method ( Immunohistochemistry )

Park's method (Chan Park et al., Inhibition of neuronal nitric oxide synthase enhances cell proliferation in the dentate gyrus of the adrenalecctomized rat, to investigate the effect of cerebral hippocampus on brain cell proliferation) Immunohistochemical staining (Immunohistochemistry) using BrdU, Ki-67 and DCX was performed with reference to Neuroscience Letters , 309 , pp9 ~ 12, 2001).

2-1. BrdU Immunohistostaining using

2-1-1. BrdU  Injection

As a result of the retention test of Experimental Examples 1-3, 8 animals were selected for each group by excluding one animal having the fastest retention latency and one animal having the latest retention time in the control group and the experimental group, respectively, and BrdU (B5002). , Sigma Chemical, USA). The solution of BrdU dissolved in normal saline was administered three times (50 mg / kg, i.p.) two or four hours prior to cardiac perfusion of the experimental animals in the evening and the next morning of the retention test.

2-1-2. Tissue preparation

Experimental animals were anesthetized with pentobarbital sodium (50 mg / kg, ip) and perfused with 4% paraformaldehyde (PFA) in 0.1M phosphate buffer. Brains were removed and soaked in 4% PFA for overnight postfixation. Transfer was performed with 30% sucrose (sucrose in 0.05M Phosphate buffered saline (PBS)) for 5 days. The brain tissue was 40 μ m thick coronal sections in (coronal section), and place it in a 30% ethylene glycol (ethylene glycol), 30% glycerol (glycerin), 0.05M phosphate buffer (phosphate buffer) Storage solution.

2-1-3. Immunocytochemical Test ( Immunocytochemical  detection)

Immunocytochemical detection was carried out by selecting four tissue sections from Bregma-2.56 mm to 4.16 mm per animal among the stored tissue sections. Immunocytochemical testing of BrdU-labeled nuclei requires DNA denaturation. Prior to incubation in anti-BrdU primary antibodies, free-floating brain sections were treated with 50% formamide-2X saline sodium citrate buffer (SSC; 1X SSC = 0.15M sodium chloride, 0.015M sodium citrate, pH 7.0)) and then incubated in 2N HCl for 30 minutes at 37 ℃. The tissues were then rinsed in 0.1M boric acid (pH 8.5) at 25 ° C. for 10 minutes. Wash three times with 5 minutes each time with 0.05M phosphate buffered saline (Phosphate buffered saline). Tissues were incubated with primary antibody overnight at 25 ° C. The primary antibody is then diluted with phosphate buffer containing 0.3% Triton X-100, 0.5mg / ml bovine serum albumin, and 1.5% normal goat serum. I was. Tissues were incubated with a horse anti-mouse secondary antibody at room temperature for 90 minutes, followed by incubation with an avidin-biotin-peroxidase complex at room temperature for 1 hour. The tissues were reacted with 0.02% 3,3-diaminobenzidine tetrahydrochloride, 0.01% hydrogen peroxide, 0.04% NiCl2 for 3 minutes. Tissues were washed with 0.05M PBS three times for five minutes each time at each incubation. The tissue was mounted on a gelatin coated slide, dried sufficiently, counterstained with cresyl violet, and then mounted with polymount. After the slides were dried, the position of the dentate gyrus of the hippocampus was observed at 100 times magnification by an optical microscope, and then BrdU stained cells at the granule cell layer at 400 times magnification. Counted. At this time, tissue sections damaged during staining were excluded and the cells were counted.

2-1-4. Experiment result

As a result of immunostaining using BrdU, the number of proliferated brain cells was 4.9 ± 1.0 (Mean ± SD, n = 9) in the control group and 8.6 ± 1.5 in the lotus root-administered group (Yan ± SD, n = 9). 10.9 ± 2.9 dogs (Mean ± SD, n = 9) the administration group showed an excellent increase effect (see Fig. 2 and 3).

2-2. Ki Immunohistostaining using -67

To stain Ki-67 antigens of brain cells, a monoclonal antibody (MIB-1) (Immunotech, SA MAC, Inc. Germany) was used. Thinly sliced brain tissue embedded in paraffin is deparaffinized with xylene and sequentially passed through 100, 90 and 70% ethanol to absorb moisture, and then 1% PBS. (phosphate-buffered solution, pH 7.4). Sections were heated three times, five minutes each, at maximum power in a 600 W microwave. The heated sections were left at room temperature for 40 minutes and then blocked with nonspecific protein binding by addition of 2% normal horse serum. After blocking, the sections were incubated with a MIB-1 antibody diluted 1:50 for 24 hours in a chamber kept at 4 ° C in humidity. Avidin DH: biotinylated horseradish peroxidase H complex with 3,3-diaminobenzidine (Polysciences, Inc.) bound to 3,3-diaminobenzidine to identify MIB-1 antibodies bound to brain tissue sections. , USA) and Vectastain Elite ABC reagents (Vector Laboratories, Inc., USA) and Mayer's hematoxylin (Fisher Scientific, USA) were used as staining reagents.

2-2-1. Experiment result

Based on the experimental results of Experimental Example 2-1, Ki-67 immunostaining was performed on the soft-skinned administration group, which showed the effect on the proliferation of brain cells. See FIG. 4).

2-3. DCX  ( Doublecortin Immunohistostaining using

DCX is a protein expressed in the brain throughout the process of nerve migration and differentiation to form cortex, and is involved in nerve migration and development. Cells that are immune to DCX in the growing mammalian brain include specific granule neurons that appear in the pharyngeal region of the granule layer of the dental gyrus.

2-3-1. Immunocytochemical Test ( Immunocytochemical  detection)

For immunohistochemical staining of DCX, one of the tissue sections stored in the storage solution was selected as a visual observation of hippocampus and washed on a vibrating pad three times for 5 minutes with PBS. To inactivate endogenous peroxidase, react for 15 minutes in a 1% hydrogen peroxide solution diluted in PBS, wash three times with PBS three times each, and then goat anti-doublecortin (Goma Biotech). (Korea)) diluted 1: 4,000 to a primary antibody solution containing 0.05% bovine serum albumin, 1.5% goat serum and 0.3% Triton X-100. The plate was placed in a vibrating pad at 4 ° C. and overnight. After the reaction in the primary antibody solution, the tissues were washed three times with PBS for 5 minutes each, followed by dilution of the biotinylated anti-goat IgG of the Vectastain-Elite kit to 1: 200 (0.3% Triton X-100). After reacting at room temperature for 1 hour, washed three times with PBS again three times for 5 minutes and then ABC solution (Avidin-biotin-peroxidase complex solution / Vectastatin-Elite kit A solution 1: 100, B solution 1: 100, 0.3% Triton X-100) at room temperature for 1 hour. As a colorant, DAB solution (0.02% 3,30 diaminobenzidine tetrahydrochloride (Sigma, USA), 0.003% H ₂ O ₂ ) in 0.05M Tris buffer at pH 7.6 was used. The color reaction was performed at room temperature for 3-5 minutes, and after the reaction, the tissues were washed three times with PBS for 5 minutes each. The colored tissue was placed on a gelatin-coated slide, dried at room temperature for 2 hours, then dehydrated in xylene through alcohol dehydration, and encapsulated in polymount.

In addition, the position and morphology of neurons that showed a positive immune response to DCX in the dentate gyrus of the hippocampus were observed by light microscopy. (grayscale). AOD (Average optical density) was set to 0 for white and 255 for black, and the stained tissue had a value therebetween. In this case, since the value of AOD was changed according to the brightness of the light source, the measurement was performed after fixing the light source. The measured brightness of the light source was measured by the image analyzer to measure the stained and non-tissue areas according to the brightness of the light source, and then obtained the correlation standard curve between the light source and the AOD. It was set as the measurement light source. In this case, at least 15 regions of each experimental group received images from a 200x optical microscope through a CCD camera and measured with an image analyzer.

The dentate gyrus of the hippocampus was measured using an optical microscope to determine the number of neurons that showed a positive immune response to DCX. The number of neurons observed in one field in a 100-fold field of view was visually measured through a microscope. The location and name of the detailed structure at each site were classified based on bregma described in paxinos and Watson brain map.

2-3-2. Experiment result

As a result of observing the dentate gyrus of the hippocampus, DCX-positive cells in the soft-skin-treated group were significantly increased compared to the control group (see FIG. 5).

Having described an example of the formulation of the pharmaceutical composition comprising the composition of the present invention, the present invention is intended to explain in detail only, not intended to limit it.

Formulation example  One. Powder  Produce

200 mg of lotus root extract of Example 1

Lactose 1000 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation example  2. Preparation of Tablets

200 mg of lotus root extract of Example 1

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation example  3. Manufacture of capsule

50 mg of lotus root extract of Example 1

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation example  4. Preparation of Injectables

50 mg of lotus root extract of Example 1

Appropriate sterile distilled water for injection

pH adjustment agent

According to the conventional method for preparing an injection, the amount of the above ingredient per 1 ampoule (2 ml)

Joe.

Formulation example  5. Liquid  Produce

100 mg of lotus root extract of Example 1

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve it, the lemon flavor is added appropriately, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then filled in a brown bottle. The solution is prepared by sterilization.

Formulation example  6. Manufacture of healthy food

1000 mg of lotus root extract of Example 1

Vitamin mixture proper amount

70 ㎍ Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients may be mixed according to a conventional health food manufacturing method. Next, the granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation example  7. Manufacture of health drinks

1000 mg of lotus root extract of Example 1

Citric acid 1000 mg

100 g oligosaccharides

Vitamin C 500 mg

10 mg caramel

Add 900 ml of purified water

After mixing the above components according to the conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilized and then refrigerated and stored Used to prepare the healthy beverage composition of the invention. Although the composition ratio is mixed with a component suitable for a favorite beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

As described above, the composition containing the lotus leaf meat of the present invention as an active ingredient showed an excellent memory and learning ability improvement effect and an excellent increase effect on the proliferation of brain cells in the manual avoidance test of the mouse, preventing and It can be used as a therapeutic composition.

Claims (6)

Alzheimer's dementia, cerebrovascular dementia, pick disease, Creutzfeldt-jakob disease, dementia due to head injury, or Parkinson's, containing soft extract of 60-80% methanol soluble extract as an active ingredient Pharmaceutical composition for the prevention and treatment of diseases. delete delete The pharmaceutical composition of claim 1, wherein the extract comprises 0.1 to 50% by weight, based on the total weight of the composition. Alzheimer's dementia, cerebrovascular dementia, pick disease, Creutzfeldt-jakob disease, dementia due to head injury, or Parkinson's, containing soft extract of 60-80% methanol soluble extract as an active ingredient Health functional food for the prevention and improvement of disease. delete

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