KR20220041655A - Composition for improving cognition or memory abilities comprising genseong fine root concentrated extracts and the process for the preparation therof - Google Patents

Abstract

The present invention relates to a composition for improving cognitive ability and memory containing a concentrated extract of ginseng fine roots, and a method for preparing the same, in which the concentrated extract of ginseng fine roots is prepared by the method comprising the steps of: (S1) washing the ginseng fine roots and drying the same at 50 to 60 ℃ for 20 to 28 hours to obtain dried ginseng fine roots; (S2) obtaining a hot-water extract of the ginseng fine roots by extracting the dried ginseng fine roots at 120-130 ℃ in hot water by using distilled water; and (S3) obtaining a concentrated extract of the ginseng fine roots by filtering the hot water extract of the ginseng fine roots and concentrating to 1/8 to 1/12 by volume under reduced pressure. The composition containing the concentrated extract of ginseng fine roots prepared according to the present invention as an active ingredient increases signal transmission between synapses and restores memory and learning ability damaged by stress, and thus is more effective than whole raw ginseng. In addition, the present invention provides a food, a functional food, and a pharmaceutical composition for improving cognitive and memory abilities using the concentrated extract of ginseng fine roots, which has been considered a by-product of ginseng, because the use of the concentrated extract of ginseng fine roots has a more significant physiological activity than the use of whole raw ginseng as it is, and has price competitiveness and economic value, thereby having an effect of dramatically improving the overall added value of the ginseng industry.

Description

Composition for improving cognitive ability and memory by using concentrated extract of ginseng and manufacturing method thereof

The present invention relates to a composition for improving cognitive ability and memory comprising a concentrated extract of ginseng, and a method for preparing the same. It relates to a composition for improving cognitive dysfunction and memory loss due to stress, comprising as an active ingredient, and a method for preparing the same.

Modern people are suffering from various diseases due to stress caused by the westernization of diet and the physical environment such as fine dust, residential space, noise, environmental pollution, etc., unfair treatment in social life, relationships with others, rules, and the like.

Stress can be defined as physical and mental changes that occur when homeostasis and organ balance are disrupted. In particular, it is known that stress causes impairment of brain functions including memory, cognition, thinking, comprehension, calculation, and learning abilities.

Specific adaptive responses are activated to maintain homeostasis during stress, and when stress is applied, the brain adapts according to two basic axes of physiological processes. The first is by the autonomic (sympathetic) system, and the second is It is known as a physiological response of the corticotropin releasing hormone (CRH) system.

It has been reported that exposure to chronic stress and keeping the sympathetic nervous system active for a long time increases the risk of high blood pressure and coronary heart disease, and causes various diseases due to oxidative damage to the brain. It is known that stress causes a decline in memory and cognitive function, a decrease in choline absorption and acetylcholine synthesis in the hippocampus, and a decrease in nicotinic and muscarinic acetylcholine receptors.

Current dementia treatment drugs have the effect of improving memory and cognitive function by inhibiting acetylcholinesterase (AChE). There are reports that it has an effect on memory and cognitive function.

On the other hand, Korean ginseng is a perennial herb belonging to the genus Panax of the Araliaceae family. Korean ginseng is derived from Panax ginseng CA Meyer , which was registered as a scientific name at the World Botanical Society by Russian botanist Meyer in 1843. Panax is a compound word from the Greek words Pan, which means 'all', and ax, which means cure, from axon, which means 'to cure all diseases'. Meaning. Korean ginseng is distributed in the form of fresh ginseng, white ginseng, and red ginseng, and fine ginseng refers to the fine root part separated as a by-product obtained during ginseng processing.

Pharmaton Pharmaceuticals in Switzerland is known to generate billions of dollars in sales worldwide by developing a method to improve human cognitive ability using ginseng. Composition comprising guarana extract includes (i) an effective amount of an extract of ginseng (Panax ginseng); and (ii) an effective amount of an extract of guarana (Paullinia cupana).

On the other hand, Korean Patent Application Laid-Open No. 2002-0087787, which is a domestic prior patent application, "Ginseno-JK, a novel composition for enhancing memory using hair roots of ginseng and its manufacturing method," is a method for preparing ginseng hair roots obtained by tissue culture with 70% alcohol or ethanol. After extraction, the extract is adsorbed to benzeneethylene resin, the non-saponin component is eluted with 20% alcohol, and the desired component is eluted with 60 to 80% alcohol. The composition is composed of ginsenoside Rb₁ , Rb₂, Rc, Rd, Re, Rg₁, and the ginsenoside Rb 1 , Rb2 , Rc, Rd each content is less than 1/2 of Rg₁. A technology to provide a novel composition ginseno-JK, this ginseno- JK has the effect of enhancing memory, and there is a description that it can be used as a preventive and ameliorating agent for senile dementia, as well as as a raw material for beverages and food.

In addition, Korean Patent Laid-Open No. 10-2017-0054157 "Composition for improving cognitive function comprising ginseng extract as an active ingredient and manufacturing method thereof" is effective in a mixture of an alcohol extract of ginseng and a water extract of ginseng filtrate remaining after extraction. It describes a composition for improving cognitive function, a health functional food, a pharmaceutical composition, a food additive, and a manufacturing method thereof, including as a component.

In general, studies on the efficacy of ginseng have been centered on whole Won ginseng so far, so fine ginseng has been treated as a by-product. is becoming

Accordingly, the present inventors used the hippocampus induced with scopolamine, which causes memory and cognitive impairment, to increase the usability of fine ginseng, which was considered a by-product, to observe the cognitive function and memory improvement effects of various herbal ingredients. The present invention was completed by discovering that the unripe ginseng extract, in particular the hot water extract of ginseng extract, showed an excellent effect in preventing cognitive dysfunction and memory loss due to stress rather than (original root).

1. Republic of Korea Patent Publication No. 2002-0087787 "Ginseno-JK, a novel composition for enhancing memory using hair roots of ginseng and its manufacturing method" 2. Republic of Korea Patent Publication No. 10-2017-0054157 "Composition for improving cognitive function comprising ginseng extract as an active ingredient and manufacturing method thereof" 3. Korean Patent Publication No. 10-2005-0047130 Composition comprising ginseng and guarana extract

The technical problem to be solved by the present invention is to provide a composition for improving cognitive ability and memory containing the hot water extract of ginseng while solving the above problems.

The technical problem to be solved by the present invention is to provide a method for preparing a composition for improving cognitive ability and memory containing the hot water extract of ginseng.

In order to solve the above technical problem, the present invention provides a composition for improving cognitive ability and memory, characterized in that it contains a concentrated extract of ginseng as an active ingredient.

In order to solve the above other technical problems, the present invention provides a method for manufacturing a composition for improving cognitive ability and memory containing a concentrated extract of ginseng, characterized in that it comprises the following steps:

(S1) washing the raw ginseng and then drying it at 50 to 60 ° C. for 20 to 28 hours to obtain a dried ginseng;

(S2) hot water extraction of the dried ginseng product using distilled water at 120 ~ 130 ° C. to obtain a hot water extract of unginseng; and

(S3) After filtering the hot water extract of ginseng, it is reduced pressure and concentrated to 1/8 to 1/12 volume to obtain a concentrated extract of ginseng.

According to one embodiment of the present invention, the concentrated extract of ginseng may be in the form of a dry powder of hot water extract of ginseng obtained by further freeze-drying and then pulverizing it.

As used herein, the term "extract" refers to a preparation obtained by extracting a crude drug with a solvent such as water or squeezing it with an appropriate leachate and evaporating the leachate, but is not limited thereto. It may be a dried product obtained by drying a diluted or concentrated solution or an extract, a crude product or a purified product thereof.

In the present invention, "concentrated extract" is used in the form of finely pulverized powder by lyophilizing and lyophilizing the concentrated extract obtained by heating for 1 to 5 hours after extracting or juicing the crude drug with a solvent such as water.

On the other hand, the present invention provides a pharmaceutical composition for improving cognitive ability and memory comprising the above-mentioned concentrated extract of ginseng.

In addition, the pharmaceutical composition for improving cognitive ability and memory is tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, non-aqueous solutions, suspensions and suppositories Any one formulation selected from the group consisting of It provides a composition characterized in that it has.

The pharmaceutical composition according to the present invention may be prepared in a form in which the active ingredient is incorporated into a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

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

In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient in the active ingredient, for example, starch, calcium carbonate, sucrose, lactose, gelatin. It can be prepared by mixing and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral administration include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. can Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As the base of the suppository, witepsol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The pharmaceutical composition according to the present invention may be administered to an individual by various routes. Any mode of administration can be envisaged, for example, by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the individual's age, weight, sex, physical condition, and the like. It is self-evident that the concentration of the peptide contained in the pharmaceutical composition can be variously selected depending on the subject, and is preferably contained in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 μg/ml, pharmaceutical activity may not appear, and if it exceeds 5,000 μg/ml, it may be toxic to the human body.

In addition, the present invention provides a food composition for improving cognitive ability and memory comprising the concentrated extract of ginseng.

The food composition of the present invention can be used in various ways, such as food and beverages for improving cognitive ability and memory. Foods that can contain the concentrated extract of ginseng of the present invention include various foods, for example, beverages, gum, tea, vitamin complexes, health supplements, etc., and include pills, powders, granules, needles, tablets, capsules, or It can be used in beverage form.

At this time, the amount of the concentrated extract of ginseng in the food or beverage may be added as 0.01 to 15% by weight of the total food weight in the case of the health food composition of the present invention, and 0.02 to 10g based on 100ml in the case of the health drink composition. , preferably in a ratio of 0.3 to 1 g.

The health drink composition of the present invention has no particular limitation on the liquid component except that it contains the above-mentioned concentrated extract of ginseng as an essential component in the indicated ratio, and it can contain various flavoring agents or natural carbohydrates as additional components like a conventional beverage. there is. 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 dextrins, cyclodextrins; and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion 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 composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof , organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the compositions of the present invention may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The proportion of these additives is not 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. There is no particular limitation on the other ingredients except for the composition having an effect on improving cognitive ability and memory, which includes the hot water extract or pharmaceutically acceptable salt thereof as an essential ingredient that can be included in the functional food composition of the present invention It may contain various herbal extracts, food supplement additives or natural carbohydrates as additional ingredients.

In addition, as mentioned above, food supplement additives may be additionally added. The food supplement additives include food supplement additives conventional in the art, for example, flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like. . Examples of the natural carbohydrate include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. .

In addition to the above, the functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, it may contain the pulp for the production of natural fruit juice and fruit juice beverages and vegetable beverages. These components may be used independently or in combination.

The composition comprising the concentrated extract of ginseng prepared according to the present invention as an active ingredient increases signal transduction between synapses, restores memory and learning ability damaged by stress, and is more effective than that of tongwon ginseng.

In addition, the present invention is a food for improving cognitive ability and memory using raw ginseng, which was considered a by-product because it has a significantly more physiologically active effect and has price competitiveness and economic utility value to use raw ginseng concentrate than to use whole-won ginseng as it is. , has the effect of dramatically improving the overall added value of the ginseng industry by providing functional foods and pharmaceutical compositions.

1 is a process for preparing a concentrate and freeze-dried powder of a hot water extract of ginseng according to an embodiment of the present invention.
Figure 2 is OHSCs and MEA probe mounting and process according to an embodiment of the present invention.
3 is a long-term potentiation (LTP) induction and experimental method of the brain hippocampus slice tissue according to an embodiment of the present invention.
Figure 4 is a drug treatment in the brain hippocampus tissue for Western Blot analysis according to an embodiment of the present invention, after incubation at 36 ℃, 5% CO₂ for 24 hours, BDNF, CREB, TrkB, measuring the protein expression amount of AChE A photograph showing the experimental method.
5 is a diagram showing a comparative analysis of fEPSP of Ginseng Body and Ginseng Root according to an embodiment of the present invention.
6 is a diagram showing a comparative analysis of fEPSP when scopolamine of Ginseng Body is treated according to an embodiment of the present invention.
7 is a diagram showing a comparative analysis of fEPSP when scopolamine of raw ginseng (Ginseng Body) is treated according to an embodiment of the present invention.
8 is a view showing a comparative analysis of fEPSP when scopolamine of whole won ginseng (Ginseng Body) and ginseng root (Ginseng Root) is treated according to an embodiment of the present invention.
9 is a diagram showing BDNF, CREB, TrkB expression measurement values of raw ginseng and tongwon ginseng according to an embodiment of the present invention.
10 is a diagram showing a comparison of fEPSP between hot water-extracted Ginseng Fine Root and ethanol (EtOH)-extracted raw ginseng extract according to an embodiment of the present invention.

Hereinafter, examples and the like will be described in detail to help the understanding of the present invention. However, the embodiments according to the present invention may be modified in various other forms, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

Research materials and methods

Tongwon ginseng used in the experiment was obtained from Punggi-eup, Yeongju-si, Gyeongsangbuk-do, and was purchased from Gyeongpoong Ginseng Company (Youngju, Korea) and used in the experiment. The fine ginseng is made from the fine root part of this whole Won ginseng. Products of Sigma-Aldrich (St. Louis, MO, USA) were used for HEPES (H4034), L-glutamine (G-8540), D-glucose (G-7528), and Scopolamine (S-0929). Minimum essential medium (MEM, LM 007-01), Hank's balanced salt solution (HBSS, LB 003-01), and Horse serum (S104-01) were purchased from Welgene (Seoul, Korea). Penicillin-streptomycin was purchased from Gibco BRL (LS 202-02, Rockville, MD, USA). The animals used in this experiment were 7-day-old rats (Sprague-Dawley rats, SD rats) purchased from Saeron Bio (Gyeonggi, Korea). All procedures were carried out in accordance with the policies of the Laboratory Animal Ethics Committee of Kyunghee University and regulations related to animal testing (IRB approval number: KHGASP-19-337).

<Example 1>

After separating raw ginseng from raw ginseng, washing and drying at 55°C for 24 hours, add 2.25L of tertiary distilled water to 225g of raw ginseng, and extract with hot water twice for 30 minutes at 121°C and 15psi to obtain an extract.

Then, it was filtered and concentrated under reduced pressure to 400 ml using a rotary evaporator (Eyela Co., Tokyo, Japan). Thereafter, freeze-drying was performed for 48 hours using a freeze-dryer and pulverized to obtain 38.15 g of dry powder.

1 is a process for preparing a hot water extract of fresh ginseng according to an embodiment of the present invention. A process of concentration under reduced pressure using (D) a concentrate obtained after concentration under reduced pressure. (E) The process of freeze-drying the concentrate of raw ginseng, (F) the freeze-dried powder finally obtained from the raw ginseng.

<Example 2>

As in Example 1, dried raw ginseng was separated from raw ginseng, washed and dried at 55 ° C. for 24 hours. Then, 2.25 L of tertiary distilled water was added to 225 g of dried ginseng, and hot water was extracted twice for 30 minutes at 121 ° C. and 15 psi. After filtering the extract, it was concentrated under reduced pressure to 400 ml using a rotary evaporator (Eyela Co., Tokyo, Japan).

<Comparative Example 1>

Using the same hot water extraction method as in Example 1, 40.35 g and 38.15 g of dry extract were obtained from 225 g of dried ginseng and 225 g of hot water extract, respectively. After measuring the dry weight of each extract, it was calculated as a percentage of the dry matter of the raw material used to prepare the extract.

- Whole Won Ginseng Extraction Yield (%)= (40.35g/225g) x 100 = 17.93 (%)

- Unginseng Extraction Yield (%)= (38.15g/225g) x 100 = 16.95 (%)

division dry weight dry extract transference number tongwon ginseng 225g 40.35g 17.93 (%) ginseng 225g 38.15g 16.95 (%)

<Test Example 1> Cognitive function and memory improvement efficacy experiment of ginseng extract

(1) Organotypic hippocampal slice cultures (OHSCs)

Organotypic hippocampal slice cultures (OHSCs) using slices and thin films of the brain hippocampus, which have been commonly used before, were used (Stoppini et al., 1991). Specifically, 7-day-old SD rat brain hippocampus was extracted, sliced to a thickness of 350 μm using a tissue slicer (Mickle Laboratory Engineering Co., Surrey, UK), and then 5-6 tissue sections were cut into numerous 0.4 μm-sized sections. Place the thin film insert (Millicell-CM; Millipore, MA) with a hole and place the thin film insert into a 6-well plate. Plates were cultured in an incubator maintained at 36°C and 5% CO₂, and culture medium (50% MEM, 20mM HEPES, 25% Hank'balanced salt solution, 6g/L D-glucose, 1mM L-glutamine, 25 % Horse serum, 1% penicillin-streptomycin, pH = 7.1) was replaced, and the total incubation time was 12 to 14 days before use in the experiment.

(2) MEA system preparation of cultured brain and hippocampal section tissue

After separating the cultured brain and hippocampus tissue from the thin film of the insert using a soft brush, an 8 8 microelectrode array with a diameter of 10 μm and an interval of 100 μm coated with 0.01% Polyethylenimine (MEA; Multi-Channel Systems, Germany) put on The MEA system consists of a stimulator (STG1004), an amplifier (MEA1060), a thermostat, and software for data analysis (Egert et al., 1998). The tissue sections were prepared in sterile artificial spinal solution (aCSF; 114 mM NaCl, 26 mM NaHCO₃, 10 mM glucose, 3 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 20 mM HEPES, pH=7.4) for 30 minutes. It was stabilized while maintaining 33℃ while injecting 5% CO₂ and 95% O₂ mixed gas. After stabilization, the array on which the tissue of the brain and hippocampus was mounted was mounted on the MEA1060 amplifier. The artificial spinal cord solution in the MEA was grounded using Ag/AgCl. During the experiment, the artificial spinal cord solution was continuously flowed at a rate of 3ml/min. After the experiment was completed, the array was washed with 1% Tergazyme solution, rinsed with distilled water, and stored at room temperature with distilled water.

(3) MEA system preparation of cultured brain and hippocampal section tissue

After separating the cultured brain and hippocampus tissue from the thin film of the insert using a soft brush, an 8 8 microelectrode array with a diameter of 10 μm and an interval of 100 μm coated with 0.01% Polyethylenimine (MEA; Multi-Channel Systems, Germany) put on The MEA system consists of a stimulator (STG1004), an amplifier (MEA1060), a thermostat, and software for data analysis (Egert et al., 1998). The tissue sections were prepared in sterile artificial spinal solution (aCSF; 114 mM NaCl, 26 mM NaHCO₃, 10 mM glucose, 3 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 20 mM HEPES, pH=7.4) for 30 minutes. It was stabilized while maintaining 33℃ while injecting 5% CO₂ and 95% O₂ mixed gas. After stabilization, the array on which the tissue of the brain and hippocampus was mounted was mounted on the MEA1060 amplifier. The artificial spinal cord solution in the MEA was grounded using Ag/AgCl. During the experiment, the artificial spinal cord solution was continuously flowed at a rate of 3ml/min. After the experiment was completed, the array was washed with 1% Tergazyme solution, rinsed with distilled water, and stored at room temperature with distilled water.

(4) LTP induction method in the brain and hippocampus tissue

While the signal amplifier was mounted in a Faraday cage, bipolar electrical stimulation was applied to the stratum radiatum of the CA2 site of the Schaffer collateral and commissural pathways. The intensity per phase of the bipolar test pulse is 80 μ and the time interval is 120 μ. This is an optimized value of 40~65% of the value to which the tissue responds best. Theta-Burst stimulation (TBS) for LTP induction consisted of 100 Hz high-frequency stimulation (HFS) for 1 second, followed by 3 stimulations at 5-minute intervals. The experimental group was treated with Control (aCSF only), Scop (Scopolamine 300μM), whole Won ginseng (100 μg/ml whole Won ginseng extract), Unginseng (Unginseng extract 100 μg/ml), Scop + Won ginseng (scopolamine 300 μM and Won Won ginseng extract 100 μg/ml) simultaneously ), Scop + unginseng (treated with scopolamine 300μM and unginseng extract 100μg/ml simultaneously), divided into 6 groups, and treated with aCSF from 10 minutes after the start of the experiment. For all experiments, the baseline values of field excitatory postsynaptic potentials (fEPSPs) were measured by stimulation once every minute for the first 30 minutes, followed by TBS for 10 minutes (3 HFS at 5-minute intervals), and then post-TBS fEPSP values for 50 minutes. was recorded. For quantitative comparison, the average of the fEPSP values for 30-40 minutes after post-TBS was compared with the average of the baseline (fEPSP values for the first 10 minutes). During the experiment, the artificial spinal cord solution was continuously flowed at a rate of 3ml/min. Unfiltered data were recorded and stored using Recorder-Rack software (Multi Channel Systems) at a sampling rate of 25 KHz from 60 probes.

(5) processing of electrophysiological data

Using MC Rack (v.3.2.1.0, Multi-Channel Systems), the experimental result data consisting of analog MEA signals was converted into digital form, EPSP values of 40 μ or more were selected using trigger mode, and MATLAB (v.7.0.1, Mathworks, Inc.) was used to remove unnecessary abnormal signals generated when stimulation was applied, and the area of the previously used signal trajectory was calculated.

(A) Activity is measured as the area under the field potential trajectory during the activation period. Areas included in the measurement were expressed with a black circle, and stimulus artifact points were omitted from the calculation. (B) Tetha Burst Stimulation (3 trains of 100Hz for 1s, train interval 5min, total 300 pulses) is injected to induce LTP in brain hippocampal slices. (C) Treatment schedule for each group.

2 to 5 show an experiment to determine the cognitive function and memory improvement efficacy using scopolamine-induced hippocampus, which causes memory and cognitive impairment, of hot water extracts of raw ginseng and comparator tongwon ginseng.

Figure 2 is a process for mounting OHSCs and MEA probes according to an embodiment of the present invention (A) the process of extracting and culturing the hippocampus part from the brain of a 7-day-old mouse, (B) the MEA system test process for the experiment, (C) MEA The data analysis process from the system is shown, and FIG. 3 shows the long-term potentiation (LTP) induction and experimental method of the hippocampal section of the brain according to an embodiment of the present invention, and FIG. 4 is a Western Blot analysis according to an embodiment of the present invention. This is a photograph showing the experimental method for measuring the protein expression levels of BDNF, CREB, TrkB, and AChE after drug treatment on brain hippocampal tissue and incubation at 36℃, 5% CO₂ for 24 hours.

(6) Protein expression measurement (Western blot)

Control, Scopolamine, Scopolamine + Wonwon ginseng, and Scopolamine + Misam were divided into 4 groups to measure protein expression in the tissue of the brain hippocampus. Thin-film inserts containing 8 brain and hippocampal tissue sections cultured for 12 to 14 days were randomly selected, and the control group contained only 1ml of culture medium, and the other group included 1ml of culture medium with 300μM Scopolamine, 300μM Scopolamine+100μg/ml After treatment with Wonwon ginseng extract and 300μM Scopolamine+100μg/ml unginseng extract, it was maintained at 36℃, 5% CO₂ incubator for 24 hours. The treated brain and hippocampal sections were collected and homogenized with cold cell lysis buffer containing phosphatase inhibitors and protease inhibitor cocktail. After homogenization for 1 hour, the supernatant was collected by centrifugation at 4° C. and 14,000 rpm for 10 minutes and used in the experiment. After quantifying the protein content by the Bradford protein assay (Bradford, 1976) method, electrophoresis was performed on a 10% gel, and the developed gel was transferred to PVDF membranes. The membranes were inhibited from non-specific binding with 5% dry skim milk-TBST containing 0.1% Tween 20. The primary antibody was diluted in 5% dry skim milk and reacted overnight at 4°C. Primary antibodies are polyclonal antibody against brain-derived neurotrophic factor (BDNF, sc-33904, Santa Crus Biotechnology), cAMP response element-binding protein (CREB, ab32512, abcam), tropomyosin-related kinase receptor B (TrkB, ab18987, abcam) ), acetylcholinesterase (AChE, ab183591, abcam), and GAPDH (ab8245, abcam). After washing the membranes to which the primary antibody is attached with TBST 3 times for 15 minutes, the secondary antibody Anti-rabbit IgG HRP-linked Antibody (#7074, cell signaling technology), Goat anti mouse IgG HRP-linked Antibody (ab205719, abcam ) was reacted at room temperature for 1 hour. Finally, after washing with TBST three times, it was reacted with an enhanced chemoluminescence (ECL) solution, and band images were taken using the ECL Western Blotting Detection System (ATTO System) equipment. The captured images were digitized using the image J program. This experiment was repeated at least 3 times with other tissue samples to obtain the results.

5 is a comparative analysis of fEPSP between Ginseng Body and Ginseng Root according to an embodiment of the present invention. (A) Control, CA1 of hippocampus treated with 100 μg/ml of control, 100 μg/ml of raw ginseng and 100 μg/ml of whole-won ginseng Changes in fEPSP according to time of the site, (B) A diagram showing the average of fEPSP values between 30 and 40 minutes after TBS. In 300ml of aCSF, 100μg/ml of raw ginseng and 100μg/ml of raw ginseng were treated, and LTP was measured and recorded in the CA1 part. As a result, when only visiting ginseng was treated, the average fEPSP value was significantly increased compared to the control group without any treatment (n=3, p<0.05), and the fEPSP value after TBS was applied between 30 and 40 minutes was also increased in the control group. was found to increase to 151% and 218% in the three outpatient group (n=3, p<0.01; Fig. 5A). In addition, the average fEPSP value of the group treated with raw ginseng increased compared to the control group (n=3, p<0.05), and the post-TBS fEPSP value of the raw ginseng group also increased by 240% compared to the control group (n= 3, p<0.001; Fig. 5). When the treatments of raw ginseng and whole-won ginseng were compared, no statistical significance was found in raw ginseng compared to whole-won ginseng, but the mean fEPSP and post-TBS fEPSP values were higher.

6 is a comparative analysis of fEPSP when scopolamine of Ginseng Body is treated according to an embodiment of the present invention. Changes in fEPSP according to time in the CA1 site, (B) is a diagram showing the average of fEPSP values for 10 minutes between 30 and 40 minutes after TBS.

Looking at the fEPSP of the CA1 part of the brain hippocampus of raw ginseng and whole-won ginseng injected with scopolamine, it was investigated through LTP whether the treatment of raw ginseng and whole-won ginseng could restore cognitive and memory abilities after scopolamine was injected. Control, the group treated with scopolamine 300 μM only, the group treated only with raw ginseng, the group treated with only raw ginseng, the group treated with scopolamine and raw ginseng 100 μg/ml, and the group treated with scopolamine and raw ginseng 100 μg/ml were compared with each other. . The group treated with both scopolamine and whole Won ginseng was 156%, and the group treated only with scopolamine was 121%. 0.05; Fig. 6B), showed similar values to the control.

Moreover, the group treated with scopolamine and raw ginseng showed a significantly higher fEPSP value than the group treated with scopolamine and whole-won ginseng (n=3, p<0.01; Fig. 8B).

7 is a comparative analysis of fEPSP when Scopolamine of raw ginseng (Ginseng Body) is treated according to an embodiment of the present invention (A) Scopolamine 300 μM, 100 μg/ml of raw ginseng and Scopolamine + time of the CA1 site of the groups treated together Changes in fEPSP according to (B) is a diagram showing the average of the fEPSP values for 10 minutes between 30 and 40 minutes after giving TBS, and FIG. Ginseng Root) scopolamine-treated fEPSP comparison analysis. (A) Changes in fEPSP over time in the CA1 region of groups treated with scopolamine 300μM, 100㎍/ml ginseng and scopolamine + ginseng. (B) A diagram showing the average of fEPSP values for 10 minutes between 30 and 40 minutes after giving TBS.

Control, scopolamine 300μM, scopolamine 300μM + raw ginseng 100㎍/ml, scopolamine 300μM + raw ginseng 100㎍/ml, fEPSP values after 30-40 minutes post-TBS can increase LTP, prevent LTP damage caused by scopolamine treatment, and the effect is statistically significantly superior to that of raw ginseng. (Table 2 below)

Looking at the effects of ginseng and whole-won ginseng on the expression of neurotrophic factors and activation of upstream transcription factors, scopolamin The expression levels of BDNF, CREB, and TrkB were higher in the group treated with 300 μM of scopolamin and 100 μg/ml of whole won ginseng than the group treated with only 300 μM (n=3, p<0.05, Fig. 10). The expression levels of BDNF, CREB, and TrkB in the group treated with scopolamin 300 μM and raw ginseng 100 μg/ml were also higher than those of the scopolamin group (n=3, p<0.01, Fig. 10). In particular, the group treated with scopolamine and raw ginseng showed a higher expression level than the group treated with whole-won ginseng, indicating that the recovery effect on memory and learning ability impairment was greater when treated with raw ginseng than whole-won ginseng.

Figure 9 is the BDNF, CREB, TrkB expression measurement values of raw ginseng and raw ginseng according to an embodiment of the present invention. Control, scopolamine 300 μM, scopolamine + whole won ginseng 100 μg/ml and scopolamine 300 μM + raw ginseng 100 μg/ml were treated in the hippocampus. (A) BDNF, (B) CREB, (C) TrkB expression levels divided by GAPDH (upper figure for each) and representative immunoblotting photo (lower figure for each). Looking at the effect on the expression of choline metabolizing enzymes, as a result of western blot measurement of the efficacy of raw ginseng and tongwon ginseng on the protein expression of acetylcholine esterase (AChE), 140.3% of the group treated with scopolamine 300μM was 140.3%, scopolamine 300μM and Wonwon ginseng. In the group treated with 100 μg/ml together, the expression of AChE was significantly reduced in the group treated with whole Won ginseng compared to the scopolamine group (n=3, p<0.01) to 76.79%. In the group treated together with scopolamine 300 μM and ginseng 100 μg/ml, the expression of AChE was reduced to 114.6% compared to the scopolamine group (n=3, p<0.05). As a result of the comparison between whole won ginseng and unginseng, there was a statistically significant difference between whole won ginseng and unstained ginseng (n=3, p<0.01).3.4. Looking at the effects of ginseng and whole-won ginseng on the expression of neurotrophic factors and activation of upstream transcription factors, in the present invention, the expression of neurotrophic factor BDNF, its upstream transcription factor CREB, and BDNF receptor TrkB protein was analyzed through Western blot. As a result, the expression levels of BDNF, CREB, and TrkB were higher in the group treated with scopolamin 300 μM and 100 μg/ml whole Wonjin ginseng than the group treated with only 300 μM of scopolamin (n=3, p<0.05). The expression levels of BDNF, CREB, and TrkB in the group treated with scopolamin 300μM and 100 μg/ml raw ginseng were also higher than those of the scopolamin group (n=3, p<0.01). In particular, the group treated with scopolamine and fine ginseng showed a higher expression level than the group treated with whole-won ginseng, indicating that the recovery effect on memory and learning ability impairment was greater when treated with raw ginseng than that of whole-won ginseng.

FIG. 10 is a comparison (n=3) of fEPSP of hot water extracted Ginseng Fine Root, ground ginseng extract, and ethanol (EtOH) extracted ginseng extract according to an embodiment of the present invention.

(A) Control, changes in fEPSP according to time in CA1 region of hippocampus treated with 100㎍/ml hot water extract, 100㎍/ml ground ginseng extract, and 100㎍/ml ethanol-extracted raw ginseng extract, (B) TBS It is a diagram showing the average of the fEPSP values between 30 and 40 minutes after.

In the above experiment, 100 μg/mL of raw ginseng (Ginseng Fine Root HW-Ex) extracted with hot water, 100 μg/mL of raw ginseng extracted through grind (Ginseng Fine Root Grind) 100 μg/mL, and ethanol-extracted fine ginseng (Ginseng Fine Root EtOH) were treated with CA1 part of the brain and hippocampus. was measured and recorded. As a result, the average fEPSP value between 30 and 40 minutes after TBS was added was 242.90 ± 9.18 % for hot water extracted fine ginseng and 214.67 ± 10.83 % for fine ginseng extracted through grind, both in the control (151.31 ± 6.01 %) group. was significantly increased compared to that ( ^** p<0.01; Fig. 1B). However, when hot-water-extracted fine ginseng was compared with fine ginseng extracted through grind, post-TBS fEPSP of hot-water-extracted fine ginseng was slightly higher, but there was no statistical significance (p=0.59). The fEPSP value of ethanol-extracted raw ginseng was 67.17 ± 2.53 %, which was significantly decreased compared to that of hot-water extracted raw ginseng ( ^## p<0.001; 1B). Also, when compared with the control group, the value of ethanol-extracted ginseng was significantly reduced ( ^*** p<0.001;1B).

Mean (%) S.E.M. Control 151.31 6.01 Ginseng Fine root (HW-Ex) 242.90 9.18 Ginseng Fine root (Grind) 214.67 10.83 Ginseng Fine root (EtOH) 67.17 2.53

Therefore, it was confirmed that the hot water extract of ginseng was superior to the ethanol extract in improving cognitive ability and memory.

In the present invention, as a method of examining whether raw ginseng has an effect of improvement in the scopolamine-treated experimental model that causes memory impairment due to stress, it is treated with raw ginseng and visiting ginseng, which are thought to be effective in the impairment of memory and learning ability due to stress. and the learning function improvement effect were compared. For this purpose, it was investigated whether the treatment of scopolamine-induced learning and memory impairments with fine ginseng and tongwon ginseng relieved the symptoms through electrophysiological and molecular biological methods.

LTP has been widely used as an experimental model to test the synaptic action of memory and learning in neurophysiology, and is also frequently used to study neuropathy related to memory and learning due to stress.

In this study, when TBS stimulation was applied to a section of brain hippocampal tissue cultured with the OHSCs method to induce LTP, a significant increase effect was seen in the group treated with raw ginseng and whole-won ginseng. In particular, fine ginseng was more effective than tongwon ginseng.

Based on these results, it is believed that M. ginseng and Toongwon ginseng further increased LTP-induced synaptic signal transduction and restored memory and learning ability damaged by scopolamine. As a result of a comparative effect experiment of treatment with scopolamine of raw ginseng and whole-won ginseng, it was found that the raw ginseng group (240%) showed a statistically significant recovery (218%) compared to the whole-won ginseng group.

The cholinergic system in the brain activates phosphorylation of cAMP response elemnent binding protein (CREB) by regulating BDNF expression. Activated CREB is known to increase the survival and proliferation of neurons (Luo et al., 2017). In this study, the expression of BDNF, CREB, and TrkB was increased by scopolamine in the extracts of ginseng and ginseng tongwon. From this, it can be inferred that the ginseng extract improved the BDNF-CREB-TrkB signaling system and restored the survival and activity of nerve cells.

Therefore, in the results of comparative treatment of extracts of raw ginseng and whole-won ginseng in this study, it was found that raw ginseng increased the expression of all factors of the BDNF-CREB-TrkB pathway more statistically and significantly more than that of whole-won ginseng, thus reducing LTP due to stress. It was confirmed that fine ginseng can recover more effectively than whole-won ginseng.

Impairment of memory and learning functions is common in patients with neurodegenerative diseases and is closely related to the acetylcholine system. Therefore, in order to elucidate the molecular mechanism for the effect of ginseng on memory and learning ability, an experiment was conducted to verify the expression of an enzyme that degrades acetylcholine in the brain and hippocampus. Scopolamine is a muscarinic receptor antagonist in the cholinergic nervous system. By binding to the site of action, acetylcholine temporarily blocks receptor binding in the synaptic cleft, leading to deterioration of memory and learning functions. AChE is an enzyme that degrades ligands that bind to acetylcholine receptors. An increase in the expression of acetylcholine esterase means a decrease in the amount of acetylcholine, which impairs memory and learning ability.

Accordingly, in this study, hot water extracts of raw ginseng and raw ginseng decreased the amount of acetylcholine esterase expression in the brain hippocampus where scopolamine-induced memory and learning abilities were impaired, but compared to raw ginseng, raw ginseng was statistically significantly more decreased. made it On the other hand, these results show that, when looking at the physiological activity of LTP damage caused by scopolamine, Misam is more statistically significantly restored than that of outpatient ginseng. It seems that the better effect on hemp is not due to the expression of acetylcholine esterase.

When comparing the results of treatment with raw ginseng and raw ginseng, it was found that LTP physiological activity had a statistically significant effect, and had an effect on improving memory and learning ability decline caused by inhibition of nerve action. This effect was more effective than that of raw ginseng compared to tongwon ginseng.

In addition, until now, fine ginseng had no commercial value because it was recognized that the value of the cosmetic product was lower than that of whole-won ginseng, and thus the physiological activity effect was also lowered. However, due to the results of this study, the commercial value of fine ginseng will increase because it has a physiologically active effect and has price competitiveness and economic utility value than whole-won ginseng.

As described above, preferred embodiments of the present invention have been disclosed in the present specification and drawings, and although specific terms are used, these are only used in a general sense to easily explain the technical content of the present invention and to help the understanding of the present invention. , it is not intended to limit the scope of the present invention. It will be apparent to those of ordinary skill in the art to which the present invention pertains that other modifications based on the technical spirit of the present invention can be implemented in addition to the embodiments disclosed herein.

Claims (7)

A composition for improving cognitive ability and memory comprising a concentrated extract of ginseng. The method of claim 1,
A composition for improving cognitive ability and memory, characterized in that the ginseng concentrate extract is prepared according to a method comprising the following steps:
(S1) washing the raw ginseng and then drying it at 50 to 60 ° C. for 20 to 28 hours to obtain a dried ginseng;
(S2) hot water extraction of the dried ginseng product using distilled water at 120 ~ 130 ° C. to obtain a hot water extract of unginseng; and
(S3) After filtering the hot water extract of ginseng, it is reduced pressure and concentrated to 1/8 to 1/12 volume to obtain a concentrated extract of ginseng. The method of claim 1,
The composition for improving cognitive ability and memory, characterized in that the concentrated extract of raw ginseng is in the form of hot water extract of raw ginseng obtained by further freeze-drying and then pulverizing. Method for preparing a composition for improving cognitive ability and memory comprising a concentrated extract of ginseng, characterized in that it was prepared according to a method comprising the following steps:
(S1) washing the raw ginseng and then drying it at 50 to 60 ° C. for 20 to 28 hours to obtain a dried ginseng;
(S2) hot water extraction of the dried ginseng product using distilled water at 120 ~ 130 ° C. to obtain a hot water extract of unginseng; and
(S3) After filtering the hot water extract of ginseng, it is reduced pressure and concentrated to 1/8 to 1/12 volume to obtain a concentrated extract of ginseng. 5. The method of claim 4,
After the step (S3), freeze-drying and then pulverizing the concentrated extract of ginseng. A pharmaceutical composition comprising a concentrated extract of ginseng as an active ingredient. A food composition comprising a concentrated extract of ginseng as an active ingredient.

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