KR102293111B1 - Composition for improving cognitive function, memory and activity comprising mixture of Schizandra chinensis extract and ascorbic acid as effective component - Google Patents

Abstract

The present invention relates to a composition for enhancing cognitive function, memory and activity containing a Schisandra extract and a mixture of ascorbic acid as active ingredients, wherein the group administered with the Schisandra extract and ascorbic acid mixture has enhanced memory compared to the group administered with the Schisandra extract or ascorbic acid alone, Since the total movement distance per unit time of the mouse increases, the expression of PSD95 protein in the brain hippocampus, which is involved in the regulation of synaptic plasticity important for memory improvement, is significantly increased, and the effect of increasing mitochondrial oxygen consumption in the hippocampus-derived cell line is excellent, The composition of the present invention containing a Schisandra extract and a mixture of ascorbic acid as an active ingredient can be usefully used as a health functional food for enhancing cognitive function, memory and activity or as a therapeutic agent for degenerative brain disease.

Description

Composition for improving cognitive function, memory and activity comprising mixture of Schizandra chinensis extract and ascorbic acid as effective component

The present invention relates to a composition for enhancing cognitive function, memory and activity containing a mixture of omija extract and ascorbic acid as active ingredients.

As the social environment is rapidly changing and diversified, modern people must engage in brain activities to quickly adapt by receiving a lot of information. After middle age, symptoms such as memory loss, forgetfulness, and increased anxiety due to central nervous system dysfunction appear. In addition, in the elderly, memory is deteriorated due to degenerative diseases such as dementia, and when the degree is severe, it not only makes social life impossible, but also it is a disease that requires enormous social costs in consideration of the effect on family and surrounding people. .

On the other hand, among the methods for improving memory or preventing memory deterioration, brain tissue-related methods may include surgical treatment, drug treatment, or taking a functional substance. Since surgical treatment is not easy, it is recommended to continuously consume drugs or functional substances that are effective in improving memory and lowering memory, focusing on prevention rather than treating diseases that deteriorate memory such as dementia after the onset. Therefore, there is a need for development of a method for improving memory by using a naturally-derived substance that has no side effects and can be safely and continuously ingested.

As a prior art related to cognitive function, memory improvement, and activity enhancement using natural products, Korea Patent No. 2023637 discloses a composition for food or feed for improving cognitive function or memory, including desalted tungtung nodule extract, and Korean Patent Publication No. No. 2018-0120604 discloses a composition for improving cognition or memory comprising an extract of sagebrush, but with respect to a composition for enhancing cognitive function, memory and activity containing the Schisandra extract and ascorbic acid mixture of the present invention as active ingredients has not been disclosed

The present invention was derived from the above needs, and provides a composition for improving cognitive function, memory and activity containing a Schisandra extract and a mixture of ascorbic acid as active ingredients, and the mice administered with the Schisandra extract and ascorbic acid mixture By confirming that the movement distance in the open field, the formation of fear memory and new object recognition are remarkable compared to the group administered with the single substance, the PSD95 protein expression in the hippocampus tissue is increased, and the mitochondrial oxygen consumption rate of the hippocampal-derived cell line is increased, the present invention completed.

In order to solve the above problems, the present invention provides a health functional food composition for enhancing cognitive function, memory and activity containing a Schisandra extract and a mixture of ascorbic acid as active ingredients.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of degenerative brain disease containing a Schisandra extract and a mixture of ascorbic acid as active ingredients.

The present invention relates to a composition for enhancing cognitive function, memory and activity containing a Schisandra extract and a mixture of ascorbic acid as active ingredients. The total movement distance per unit time of the mouse increases, the expression of PSD95 protein in the brain hippocampus, which is involved in the regulation of synaptic plasticity important for memory improvement, is significantly increased, and the mitochondrial oxygen consumption in the hippocampus-derived cell line has the effect of increasing.

1 is a result confirming the effect of improving memory by administering a mixture of Schisandra extract and ascorbic acid through a fear memory formation test. CN is a control group administered by mixing NMP (N-Methyl-2-pyrrolidone) used as a solvent and physiological saline, SCE+AA is a group administered with a Schisandra extract and ascorbic acid mixture, SCE is a group administered with a Schisandra extract alone, and AA is ascorbic acid alone. *** means that the stiffness of the SCE+AA administration group was statistically significantly increased compared to the CN, SCE and AA administration groups, and p<0.001.
2 is a result of confirming the cognitive function to distinguish new objects by administration of a mixture of Schisandra extract and ascorbic acid. Old is a familiar object, and Novel is a new object. CN is a control group administered by mixing NMP (N-Methyl-2-pyrrolidone) used as a solvent and physiological saline, SCE+AA is a group administered with a Schisandra extract and ascorbic acid mixture, SCE is a group administered with a Schisandra extract alone, and AA is ascorbic acid alone. * indicates that the search time for new objects in the SCE+AA administration group increased significantly compared to the CN, SCE and AA administration groups, and p<0.05.
3 is a result confirming the activity enhancing effect of the mouse after administration of a mixture of Schisandra extract and ascorbic acid. A is the result of tracing the movement of the mouse with a red solid line, and B is the result of quantifying the red solid line. CN is a control group administered by mixing NMP (N-Methyl-2-pyrrolidone) used as a solvent and physiological saline, SCE+AA is a group administered with a Schisandra extract and ascorbic acid mixture, SCE is a group administered with a Schisandra extract alone, and AA is ascorbic acid alone. *, ** means that the movement of the SCE+AA administration group increased statistically significantly compared to the CN, SCE and AA administration groups, * is p<0.05, ** is p<0.01.
4 is a Western blot confirming the change in PSD95 protein expression in the hippocampal tissue of mice administered with Schisandra extract and ascorbic acid mixture (A), and quantifying it (B). CN is a control group administered by mixing NMP (N-Methyl-2-pyrrolidone) used as a solvent and physiological saline, SCE+AA is a group administered with a Schisandra extract and ascorbic acid mixture, SCE is a group administered with a Schisandra extract alone, and AA is ascorbic acid alone. ** means that the PSD95 protein content of the SCE+AA administration group increased statistically significantly compared to the CN, SCE and AA administration groups, and ** is p<0.01.
5 is a result of measuring mitochondrial oxygen consumption rate (OCR) by administering a mixture of Schisandra extract and ascorbic acid in a hippocampal-derived cell line.
A and B are experimental results of administration of Schisandra extract, ascorbic acid, and a mixture thereof, CN is a control group treated by mixing NMP (N-Methyl-2-pyrrolidone) and physiological saline used as a solvent, SCE + AA is a group treated with Schisandra extract and ascorbic acid mixture, SCE is a group treated with Schisandra extract alone, and AA is a group treated with ascorbic acid alone. * means that the mitochondrial oxygen consumption of the SCE + AA treatment group was statistically significantly increased compared to the CN treatment group, and * is p<0.05.
C and D are experimental results of administering a mixture according to the mixing ratio of Schisandra extract and ascorbic acid, and Schisandra extract:ascorbic acid was mixed in a weight ratio of 4:1, 2:3 or 1:4. * and ** indicate a statistically significant increase in mitochondrial oxygen consumption in the group treated with the Schisandra extract mixed in the 4: 1 weight ratio: ascorbic acid mixture compared to the 2:3 or 1:4 weight ratio mixture: ascorbic acid mixture means, * is p<0.05, ** is p<0.01.

The present invention relates to a health functional food composition for enhancing cognitive function, memory and activity containing a mixture of omija extract and ascorbic acid as active ingredients.

The solvent of the Schisandra extract may be water, a C ₁ to C ₄ lower alcohol or a mixture thereof, preferably ethanol, but is not limited thereto.

The Schisandra extract and ascorbic acid mixture is preferably mixed Schisandra extract:ascorbic acid in a weight ratio of 3 to 5:1, and more preferably, Schisandra extract:ascorbic acid is mixed in a weight ratio of 4:1, but is not limited thereto.

When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient may be appropriately used depending on the purpose of its use (prevention or improvement). In general, in the production of food or beverage, the health functional food composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the total raw materials. However, in the case of long-term intake for health purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.

There is no particular limitation on the type of the health functional food. Examples of foods to which the health functional food composition can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea There are drinks, alcoholic beverages, vitamin complexes, and the like, and includes all health foods in the ordinary sense.

In addition, the health functional food composition of the present invention may be prepared as a food, particularly a functional food. The functional food of the present invention includes ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when it is prepared as a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), oligosaccharides, polysaccharides (eg, dextrin, cyclodextrin, etc.) or sugar alcohols ( For example, xylitol, sorbitol, erythritol, etc.) is preferable. As the flavoring agent, natural flavoring agents (eg, taumatine, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) may be used.

In addition to the health functional food composition, various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid It may further contain a carbonation agent and the like used in beverages. The ratio of these added ingredients is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition of the present invention.

In addition, the present invention relates to a pharmaceutical composition for the prevention or treatment of degenerative brain disease containing a mixture of Schisandra extract and ascorbic acid as active ingredients.

The degenerative brain disease may be Alzheimer's disease, Parkinson's disease, mild cognitive impairment, meningitis, stroke, dementia, Huntington's disease or Creutzfeldt-Jakob disease, preferably dementia or Parkinson's disease, but is not limited thereto.

The pharmaceutical composition according to the present invention may be formulated in the form of oral dosage forms such as capsules, powders, granules, tablets, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. can

The pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent.

Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate , cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, mineral oil, and the like.

In the case of formulation, it is prepared using diluents or excipients, such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the pharmaceutical composition, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. is prepared by mixing In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents, may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, and the like can be used.

A suitable dosage of the pharmaceutical composition of the present invention may be prescribed variously depending on factors such as formulation method, administration mode, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity of the patient. can

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by topical application to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, and the like.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.

Experimental materials and methods

1. Schisandra extract and ascorbic acid

Schisandra extract was provided by the Korea Plant Extracts Bank, Korea Research Institute of Bioscience and Biotechnology. The extract was specifically pulverized and powdered by the dried plants, and then 1000 ml of 70% (v/v) ethanol was added to 100 g of the powder and extracted at room temperature (20±5° C.) for 72 hours. The extract was obtained from Whatman No. After filtration with 2 filters, the mixture was concentrated using a rotary concentrator at a temperature of 40-50° C., and dried under reduced pressure. The dried Schisandra extract was dissolved in NMP (N-Methyl-2-pyrrolidone, Sigma-Aldrich, MO, USA) solution and administered to mice.

Ascorbic acid was purchased from Sigma-Aldfich (Sigma-Aldfich, MO, USA) and used.

2. Mouse Behavior Analysis

Animal experiments were conducted with the approval of the Laboratory Animal Ethics Committee of Chungnam National University (approval number: 201903A-CNU-46).

C57BL/6 mice were acclimatized by adjusting the illuminance so that day and night were repeated at 12 hour intervals under a temperature condition of 22 ° C in an aseptic facility, and the Schisandra extract of the present invention, ascorbic acid (hereinafter, AA) alone, or Schisandra The extract and the AA mixture (4:1 weight ratio) were each injected into the abdominal cavity of an 8-week-old male mouse three times at an interval of 24 hours at 10 mg/kg. As a control group, NMP (N-Methyl-2-pyrrolidone, Sigma-Aldrich, MO, USA) used as a solvent and physiological saline were mixed and injected in the same volume as the experimental group. After 3 injections, the following behavioral analysis was performed, respectively.

(1) open field experiment

After 3 injections, each mouse was placed in a 40x40x40cm square box, and the movement for 1 hour was recorded by video. Then, the moving distance was analyzed using EthoVision XT 11.5 software, and the total moving distance was measured and statistically processed.

(2) Fear memory formation experiment

After 3 injections, a mouse was placed in a fear chamber capable of giving electrical stimulation, and allowed to freely explore the space for 5 minutes. After 5 minutes, 3 kHz, 80 dB of sound was heard for 20 seconds, and an electric shock (0.4 mA) for 1 second was repeated 3 times at 1 minute intervals. After 24 hours, the mouse was placed in the same fear chamber and allowed to move freely without any stimulation for 5 minutes. During the formation of the first fear memory, the same sound was heard and the movement of the mouse was recorded on video. The image was analyzed with freeze frame 4 software, and the percentage of the time that the mouse was immobile without moving was quantified as a percentage.

(3) New object recognition experiment

After 3 injections, the mouse was placed in a cylinder with a diameter of 30 cm and a height of 40 cm installed in the center of two cylinders, and allowed to freely explore objects for 10 minutes. After that, one of the two cylinders was replaced with a square iron pillar, and the movement of the mouse was recorded on video for 10 minutes so that the time of the mouse turning and looking at, smelling, or touching an object was measured between a familiar object and a new one. All measurements were made on the object and quantitatively compared.

3. Protein extraction and Western blot

After 3 injections, the hippocampal tissue of the mouse was isolated from the brain and a 10% (w/v) protease inhibitor cocktail (Roche, Basel, Switzerland) containing RIPA buffer [50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% (w/v) SDS, 0.5% (v/v) deoxycholate and 1% (v/v) Nonidet P-40] and crushed. Thereafter, the supernatant was obtained through centrifugation, the protein was quantified, and 12 μg of the protein was mixed with 5X sample buffer, boiled, and then loaded onto an SDS-PAGE gel for electrophoresis. After electrophoresis, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA), the membrane was blocked with 5% (w/v) skim milk, and then the primary antibody [anti -PSD95 (Thermo Scientific, MA, USA), α-Tubulin (Sigma-Aldrich, MO, USA) antibody] was added to diluted skim milk and reacted at 4° C. for 16 hours. Thereafter, the secondary antibody [IgG horseradish peroxidase (HRP) antibody (Pierce Biotechnology, MA, USA)] corresponding to the host of the primary antibody was reacted at room temperature for 1 hour, and then the protein band was reacted with an ECL system (Thermo Scientific). , MA, USA) was used.

4. Cell Culture and Measurement of Oxygen Consumption Rate

mHippoE-14 cells, a mouse hippocampal-derived cell line, were treated with DMEM (Dulbecco's) containing 10% (v/v) FBS (Hyclone, MA, USA), 1% (v/v) penicillin and streptomycin (Hyclone, MA, USA). In Modified Eagle's medium, Sigma-Aldrich, MO, USA) medium, 37° C., 5% CO ₂ and 21% O ₂ Conditions were maintained and cultured.

After dispensing the cultured mHippoE-14 cells to 2x10 ⁴ cells per well, Schisandra extract, AA, and a mixture thereof (4:1, w/w) were added to the medium to a concentration of 10 μg/ml each. , further incubated for 24 hours. Thereafter, the basal oxygen consumption rate (OCR) was measured using an XF24 analyzer (Seahorse, MA, USA), and the ATPase inhibitor oligomycin A (oligomycin A, 20 μg/ml, Sigma-Aldrich, MO, USA), an oxidative phosphorylation inhibitor as an uncoupler (carbonyl cyanide 3-chlorophenylhydrazone, 50 μM, Sigma-Aldrich, MO, USA) and mitochondrial respiratory complex I inhibitor rotenone (20 μM, Sigma-Aldrich) , MO, USA) were sequentially injected and the OCR change was analyzed at 37°C.

Example 1. Fear memory formation experiment

As a result of the fear memory formation experiment, as shown in FIG. 1 , the movement stop time of the Schisandra extract and the AA mixture group was significantly increased compared to the Schisandra extract alone and the AA alone group. This means that the memory of the mice was improved by the administration of the Schisandra extract and the AA mixture, and the time at which they were still without movement due to the memory of the previous electrical stimulation was increased compared to the group treated alone.

Example 2. New object recognition experiment

As a result of the new object recognition experiment, as shown in FIG. 2 , the detection ratio of a new object (Novel) compared to a familiar object (Old) in the group administered with the Schisandra extract and AA mixture was significantly increased compared to the group administered with the Schisandra extract alone and AA alone. This means that when the Schisandra extract and AA mixture were administered, the cognitive function and memory were improved, and the ability to distinguish new objects was improved.

Example 3. Open Field Experiment

After administration of the Schisandra extract of the present invention, AA, and a mixture thereof, as a result of an open field experiment, as shown in FIG. 3 , it was confirmed that the movement of the group administered with the Schisandra extract and AA mixture was significantly improved compared to the group administered with the single substance. Through this, it was found that the Schisandra extract and AA mixture enhance the activity.

Example 4. Changes in protein expression in the hippocampus

PSD95 is a synaptic plasticity regulatory protein important for memory enhancement in hippocampal tissues. As a result of confirming the change in the expression of PSD95 protein in the hippocampus when the Schisandra extract, AA, and a mixture thereof were administered, it was found that the expression of PSD95 protein significantly increased when the Schisandra extract and the AA mixture were administered compared to the Schisandra extract or AA alone, as shown in FIG. 4 . Confirmed.

Through this, it was confirmed that the Schisandra extract and AA mixture helped to strengthen memory by increasing the expression of PSD95 protein in the hippocampus.

Example 5. Measurement of mitochondrial oxygen consumption rate

Increased mitochondrial activity in the brain is known to help improve memory. Therefore, mitochondrial activity was confirmed by measuring the oxygen consumption rate of mitochondria in mHippoE-14 cells, a cell line derived from the hippocampus. increased significantly.

In addition, as shown in FIGS. 5C and 5D , as a result of treatment with cells by adjusting the mixing ratio of the Schisandra extract and the AA mixture, the oxygen consumption rate by the Schisandra extract and the AA mixture mixed in a 4:1 weight ratio of the present invention was 2:3 or It was found to be higher than that of Schisandra extract and AA mixture mixed at a weight ratio of 1:4. Through this, it was confirmed that the Schisandra extract and the AA mixture mixed at a 4:1 weight ratio of the present invention were superior in enhancing mitochondrial activity compared to the Schisandra extract and the AA mixture mixed in other ratios.

Claims (5)

A health functional food composition for enhancing cognitive function, memory and activity, comprising as an active ingredient a mixture of Schisandra extract and ascorbic acid in which ascorbic acid is mixed in a dry matter of Schisandra extract: ascorbic acid in a weight ratio of 3 to 5:1. The health functional food composition for enhancing cognitive function, memory and activity according to claim 1, wherein the solvent of the Schisandra extract is water, C ₁ ~ C _{4 lower alcohol, or a mixture thereof.} delete Schisandra extract: A pharmaceutical composition for the prevention or treatment of degenerative brain disease, comprising the Schisandra extract and ascorbic acid mixture in which ascorbic acid is mixed in a weight ratio of 3 to 5:1 as active ingredients. The pharmaceutical composition for preventing or treating degenerative brain disease according to claim 4, wherein the degenerative brain disease is Alzheimer's disease, Parkinson's disease, mild cognitive impairment, meningitis, stroke, dementia, Huntington's disease or Creutzfeldt-Jakob disease.

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