KR20230100697A - Composition for improving neuroinflammatory disease or memory containing ginseng flower extract - Google Patents

Abstract

A ginseng flower extract according to the present invention (i) inhibits NO production in microglial cells, (ii) inhibits inflammatory cytokine expression, (iii) inhibits iNOS/COX-2 protein expression; (iv) and effectively inhibits neuroinflammation by inhibiting MAPK signal transduction. Therefore, the ginseng flower extract can be used as a pharmaceutical and food composition for the treatment, prevention, and alleviation of various diseases related to neuroinflammation and cognitive dysfunction.

Description

Composition for improving neuroinflammatory disease or memory containing ginseng flower extract

The present invention relates to a composition for inhibiting neuroinflammation containing a ginseng flower extract, and more particularly, the present invention relates to a composition for preventing or treating neuroinflammatory diseases comprising a ginseng flower hot water extract or a ginseng flower ethanol extract as an active ingredient. , Or provides a composition effective for preventing or treating memory or cognitive dysfunction.

The central nervous system consists of neurons and glial cells. Glial cells account for about 90% of all brain cells and account for about 50% of the entire brain in terms of volume. Glial cells can be further classified into three types: astrocytes, microglia, and oligodendrocytes. Among them, microglial cells are a type of specialized macrophages, and are widely distributed in the brain. Microglia not only act as phagocytes that engulf tissue debris and dead cells, but also play a role in biodefense activities in the brain.

Neuroinflammation is a kind of immune response of the nervous system, and is closely related to many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and multiple sclerosis, and is now considered a typical feature of neurodegenerative diseases. The neuroinflammatory response involves activation of innate immune cells (microglia), release of inflammatory mediators such as nitric oxide (NO), cytokines and chemokines, and macrophage infiltration, which leads to neuronal cell death. Activation of microglia inflammation is thought to be a pathological marker and an important mechanism in the progression of neurodegenerative diseases. Thus, tight regulation of microglia activity is essential for maintaining brain homeostasis and preventing infectious and inflammatory diseases.

Relatedly, cognitive impairment of the brain is clinically characterized by gradual impairment of memory, cognition, reasoning, executive functioning, planning, judgment, and emotional stability, and these disorders gradually lead to the deterioration of deep intelligence. A wide range of diseases can lead to cognitive impairment. Among them, Mild Cognitive Impairment (MCI) refers to a pre-stage of dementia in which cognitive function, especially memory, is reduced compared to the same age group, but the ability to perform daily life is maintained. People with mild cognitive impairment are pointed out as a high-risk group with a very high possibility of progressing to Alzheimer's disease.

On the other hand, ginseng (Panax ginseng C.A. Meyer) is a plant belonging to the genus Panax ginseng and has been used as a herbal medicine in Korea, China, and Japan for more than 2,000 years. It is known to be effective in alleviating disease, anti-cancer, enhancing immune function, anti-diabetes, and enhancing liver function. However, ginseng has mainly been researched on treatment methods for various diseases using the root, but the use of ginseng flowers has not been clearly studied yet.

Accordingly, the present inventors, while seeking ways to utilize ginseng flowers discarded as by-products after ginseng cultivation, confirmed that nerve inflammation can be effectively inhibited when ginseng flowers are extracted with hot water or ethanol, and the present invention has been completed.

Republic of Korea Patent Publication No. 10-2015-0113434

An object of the present invention is to provide a novel use of a ginseng flower extract.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating neuroinflammation comprising a ginseng flower extract as an active ingredient.

The present invention also provides a food composition for improving cognitive function, preventing or improving neuroinflammation, comprising a ginseng flower extract as an active ingredient.

In the present invention, the ginseng flower extract may be characterized in that it is a hot water extract or an ethanol extract.

In the present invention, the ginseng flower extract in nerve cells

(i) inhibition of NO production;

(ii) inhibition of inflammatory cytokine expression;

(iii) inhibition of iNOS/COX-2 protein expression; and/or

(iv) It can be characterized by improving cognitive function or suppressing neuroinflammation by inhibiting MAPK signal transduction.

The present invention also provides a health functional food composition containing the ginseng flower extract.

The food composition containing the ginseng flower extract of the present invention is effective not only for preventing and improving neuroinflammatory diseases and cognitive dysfunction, but also for improving (improving) memory.

In the present invention, the neuroinflammatory disease may be multiple sclerosis, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, post-traumatic stress disorder, depression, schizophrenia, amyotrophic lateral sclerosis or mild cognitive impairment.

The ginseng flower extract of the present invention may be included in the composition and ingested or administered to the subject at a concentration of 30 to 80 mg/kg.

The present invention relates to a composition for inhibiting neuroinflammation containing a ginseng flower extract, and the ginseng flower extract according to the present invention can inhibit (i) NO production in microglia; (ii) inhibition of inflammatory cytokine expression; (iii) inhibition of iNOS/COX-2 protein expression; (iv) Since neuroinflammation is effectively inhibited by inhibiting MAPK signal transmission, it can be used as a pharmaceutical and food composition for the treatment, prevention, and improvement of various diseases related to neuroinflammation.

1 shows the results of confirming the cytotoxicity of ginseng flower hot-water extract and 70% ethanol extract on BV2 microglia.
Figure 2 is the result of confirming the NO production inhibitory effect increased by LPS treatment in BV2 microglia of ginseng flower hot water extract and 70% ethanol extract.
Figure 3 is the result of confirming the effect of inhibiting the expression of iNOS and COX-2 proteins increased by LPS treatment in BV2 microglia of ginseng flower hot water extract.
Figure 4 confirms the expression changes of p38, ERK, and JNK, which are MAPK family members, increased by LPS treatment in BV2 microglia of ginseng flower hot water extract by Western blotting (A), p-ERK/ERK (B), This is the result of quantitatively confirming p-JNK/JNK (C) and p-p38/p38 (D).
5 is a result confirming the effect of ginseng flower hot water extract on the expression of IL-6 (A) and TNF-α (B), which are inflammatory cytokines, increased by LPS treatment in BV2 microglia.
6 shows the results of western blotting and immunofluorescence staining for the effect of ginseng flower extract on NF-κ activation by LPS treatment in BV2 microglia.
7 is a schematic diagram showing the timeline of the Morris Water Maze Test conducted to confirm the cognitive function improvement effect of a ginseng flower extract according to an embodiment of the present invention.
8 shows the y-maze results of the Morris water maze test conducted according to an embodiment of the present invention.
9 is a result confirming the expression changes of iNOS and COX-2 in mouse brain proteins after treatment with ginseng flower extract.
10 is a result of measuring the amount of change in p-IκBа in mouse brain protein after treatment with ginseng flower extract.
11 is a result of confirming the expression of BDNF and p-CREB in brain proteins after treatment with ginseng flower extract according to an embodiment of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.

In the present invention, ginseng flower extract inhibits NO production caused by neuroinflammatory stimulation without cytotoxicity to BV2 microglia, inhibits inflammatory cytokine expression, and also reduces inflammatory markers such as iNOS, COX-2, and MAPK It was confirmed that it exerted an inhibitory effect on neuroinflammation.

Accordingly, in one aspect, the present invention relates to a pharmaceutical composition for preventing or treating neuroinflammation comprising a ginseng flower extract as an active ingredient.

In another aspect, the present invention relates to a food composition for preventing or improving neuroinflammation comprising a ginseng flower extract as an active ingredient.

As used herein, the term "extract" refers to a liquid component obtained by immersing a desired substance in various solvents and then extracting at room temperature, low temperature or elevated temperature for a certain period of time, and removing the solvent from the liquid component. It means the result, such as the obtained solid content. In addition, in addition to the above results, it can be comprehensively interpreted as including all dilutions of the results, concentrates thereof, adjusted products, and purified products thereof.

The ginseng flower used for extraction in the present invention may be pulverized, minced, or properly dried.

The extraction solvent that can be used in the present invention is not particularly limited, and for example, water or an organic solvent may be used, and the organic solvent includes methanol, ethanol, propanol, and isopropanol. , alcohols having 1 to 4 carbon atoms, including butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, Various solvents such as hexane and cyclohexane may be used alone or in combination, but are not limited thereto.

In the present invention, the ginseng flower extract may be characterized in that it is preferably a hot water extract or an ethanol extract, but is not limited thereto.

In the present invention, as the extraction method, any one of methods such as hot water extraction, cold brew extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, and compression may be selected and used, preferably using hot water. It can be an extraction method. In addition, the desired extract may be additionally subjected to a conventional fractionation process or may be purified using a conventional purification method. There is no limitation on the preparation method of the extract according to the present invention, and any known method may be used.

In a preferred embodiment of the present invention, the ginseng flower extract may be a hot water extract of ginseng flower, and 3 to 20 times, preferably 5 to 15 times the weight of the medicinal material is added, and the temperature is 80 to 120 ° C., preferably 85 to 110 The extraction may be carried out for 30 minutes to 24 hours, preferably 1 to 6 hours, and more preferably about 2 hours at a temperature of 90 to 105 ° C more preferably than ° C, but the hot water extraction method is not limited thereto.

In another preferred embodiment of the present invention, the ginseng flower extract may be an ethanol extract, and 3 to 20 times the weight of the medicinal material, preferably 5 to 15 times the amount of ethanol added, and 1 hour to 24 hours at room temperature, preferably Can be extracted for 2 to 12 hours, but the ethanol extraction method is not limited thereto. In the present invention, the ethanol extract, preferably, after the first extraction, the extraction solvent is removed, and 3 to 20 times the weight of the medicinal material, preferably 5 to 15 times the amount of ethanol is added, and 1 hour to 24 times at room temperature. It may be subjected to an additional extraction step of extraction time, preferably 2 to 12 hours.

In the present invention, the ethanol extract may be characterized in that it is extracted using 60 to 80%, preferably 70% ethanol as a solvent, but is not limited thereto.

The extract according to the present invention may be subjected to additional processes such as filtration, concentration, and drying as necessary after the extraction. The method and conditions of the additional process are not particularly limited, and may be performed by a method known in the art or under commonly performed conditions.

The concentration of the ginseng flower extract in the composition may be 0.1 to 20000 ug/ml, preferably 10 to 500 ug/ml, and more preferably 50 to 300 ug/ml, but is not limited thereto.

In the present invention, the ginseng flower extract in nerve cells

(i) inhibition of NO production;

(ii) inhibition of inflammatory cytokine expression;

(iii) inhibition of iNOS/COX-2 protein expression; and/or

(iv) suppression of neuroinflammation by inhibiting MAPK signal transduction, but the mechanism of action of ginseng flower extract is not limited thereto.

In the present invention, the nerve cells may be characterized in that they are microglial cells, but are not limited thereto.

In the present invention, the pharmaceutical composition may include only an effective amount of ginseng flower extract or one or more pharmaceutically acceptable carriers, excipients, or diluents.

In the present invention, the term "effective amount (or effective amount)" means an amount that is very sufficient to deliver desired effects but is small enough to sufficiently prevent serious side effects within the scope of medical judgment. The amount of the composition administered to the body by the composition of the present invention may be appropriately adjusted in consideration of the route of administration and the subject of administration.

In addition, "pharmaceutically acceptable" as used herein refers to a composition that is physiologically acceptable and does not cause allergic reactions such as gastrointestinal disorders and dizziness or similar reactions when administered to humans.

The ginseng flower extract of the present invention is effective in improving cognitive function, improving, preventing, or treating cognitive dysfunction or neurodegenerative diseases.

As used herein, "cognitive impairment" includes loss of reasoning ability, forgetfulness, learning difficulties, concentration problems, decline in intelligence, and other mental function declines, including Alzheimer's disease, cerebrovascular disease, Dementia, Pick's disease and Creutzfeldt-Jakob's disease may be included, but are not limited thereto.

As used herein, "neuroinflammatory diseases" include Alzheimer's disease, Parkinson's disease, Niemann Pick's disease, amyotrophic sclerosis, multiple sclerosis, neuroblastoma, stroke, Lou Gehrig's disease, Huntington's disease, Creutzfeldt-Jakob disease, post-traumatic stress disorder, depression, schizophrenia and Spinal muscular atrophy may be included, but is not limited thereto.

As used herein, "neurodegenerative disease" includes multiple sclerosis, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, fronto-temporal dementia. temporal dementia), cortico basal degeneration, and progressive supranuclear palsy.

Carriers or media usable in the present invention include solvents, dispersants, coatings, absorption accelerators, controlled release agents (i.e. sustained release agents), and one or more inert excipients (starch, polyols, granules, microfine cellulose, microcrystalline cellulose (including, for example, cellspheres, cellphere beads, diluents, lubricants, binders, disintegrants, etc.), etc. Pharmaceutically acceptable carriers and pharmaceutical Excipients for use as acceptable inert carriers and examples of such additional ingredients include, but are not limited to, binders, fillers, disintegrants, lubricants, antimicrobial agents, and coating agents.

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

Liquid preparations for oral use include suspensions, solutions for oral use, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

The dosage form may be in the form of a powder, granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injectable solution, or sterile powder. In addition, the composition for preventing or treating diseases according to the present invention can be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient depends on the route of administration, the patient's age, sex, and body weight. And it can be appropriately selected according to various factors such as the severity of the patient, and can be administered in combination with a known compound having an effect of preventing, improving or treating symptoms of a disease.

The amount of the extract according to the present invention may vary depending on the patient's age, sex, and weight, but may be administered once or several times a day at 10 to 1000 mg/kg. In addition, the dosage may be increased or decreased depending on the route of administration, severity of disease, sex, weight, age, and the like. Therefore, the dosage is not intended to limit the scope of the present invention in any way.

As used herein, the term "treatment" relates to caring for a diseased subject in order to ameliorate, cure or reduce the symptoms of a disease or to reduce or halt the progression of a disease.

In the present invention, the term "prevention" relates to averting, delaying, impeding, or hindering the reduction of a disease.

In the present invention, the food in the composition for food may be a main ingredient of food, a supplementary ingredient, a food additive, a health functional food or a functional beverage, but is not limited thereto.

Food to which the food composition according to the present invention can be added includes, for example, various foods, beverages, chewing gum, tea, vitamin complexes, health functional foods, and other functional foods. In addition, in the present invention, foods include special nutritional foods (eg, formula milk, infant/young child food, etc.), processed meat products, fish meat products, tofu, jelly, noodles (eg, ramen, noodles, etc.), breads, health supplements, seasonings Foods (eg, soy sauce, soybean paste, gochujang, mixed paste, etc.), sauces, confectionery (eg, snacks), candies, chocolates, chewing gum, ice cream, dairy products (eg, fermented milk, cheese, etc.), other processed foods, kimchi, It includes, but is not limited to, pickled foods (various types of kimchi, pickled vegetables, etc.), beverages (eg, fruit drinks, vegetable drinks, soy milk, fermented beverages, etc.), and natural seasonings (eg, ramen soup, etc.). The food, beverage or food additive may be prepared by a conventional manufacturing method.

The health functional food refers to a food group or food composition that has added value so that the function of the food acts for a specific purpose by using physical, biochemical, or bioengineering methods, etc. It refers to food designed and processed to sufficiently express the body's regulatory function related to the living body. The functional food may include food additives that are acceptable in food science, and may further include one or more of appropriate carriers, additives, excipients, and diluents commonly used in the manufacture of functional foods.

The health functional food may be formulated as one selected from the group consisting of tablets, pills, powders, granules, powders, capsules and liquid formulations by further including one or more of carriers, diluents, excipients and additives. Examples of foods to which the extract of the present invention can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gum, tea, vitamin complexes, health functional foods, and the like.

Additives that may be further included in the present invention include natural carbohydrates, flavors, nutrients, vitamins, minerals (electrolytes), flavors (synthetic flavors, natural flavors, etc.), colorants, fillers (cheese, chocolate, etc.), One or more components selected from the group consisting of pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, antioxidants, glycerin, alcohols, carbonating agents and fruit flesh may be used. .

Examples of the aforementioned natural carbohydrates 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 dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents (thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

In addition to the above, the health functional food of the present invention is various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like. In addition, the composition according to the present invention may contain fruit flesh for preparing natural fruit juice and vegetable beverages. These components may be used independently or in combination.

Specific examples of the carrier, excipient, diluent, and additive include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium phosphate, calcium Silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate And at least one selected from the group consisting of mineral oil is preferably used.

When formulating the health functional food of the present invention, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

The content of the extract according to the present invention as an active ingredient in the above-described formulation can be appropriately adjusted according to the type and purpose of use, the patient's condition, the type and severity of symptoms, etc., and is 0.001 to 99.9% by weight based on the solid content weight, preferably. It may be 0.01 to 50% by weight, but is not limited thereto.

The dosage of the pharmaceutical composition and health functional food of the present invention may vary depending on the patient's age, weight, sex, dosage form, health condition, and degree of disease, and may be administered once a day at regular time intervals according to the judgment of a doctor or pharmacist. It may also be divided into several doses. For example, the daily dosage may be 10 to 1000 mg/kg (preferably, 30 to 80 mg/kg or 20 to 70 mg/kg) based on the active ingredient content. The above dosage is an example of an average case, and the dosage may be higher or lower depending on individual differences. If the daily dose of the health functional food of the present invention is less than the above dose, a significant effect may not be obtained, and if it exceeds more than that, it is uneconomical and may cause undesirable side effects because it is out of the range of the usual dose. there is.

In the present invention, the functional beverage refers to a general term for drinking to quench thirst or enjoy the taste, and there are no particular restrictions on other components other than including a composition for improving or preventing the disease symptoms as an essential component in the indicated ratio. As with conventional beverages, various flavoring agents or natural carbohydrates may be included as additional components.

Furthermore, in addition to the above, the food composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), natural carbohydrates, flavors such as synthetic flavors and natural flavors, coloring agents and fillers (cheese, chocolate, etc.) ), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages and fruit flesh, etc. can be used independently or in combination.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Experiment method

1-1. sample preparation

The ginseng flower used in the present invention was a product produced in Sinpyeong-myeon, Dangjin-si, Chungcheongnam-do, and was purchased and used at Jecheon Oriental Medicine Herb. Ginseng flowers were extracted with water (hot water extraction) and 70% ethanol to prepare two types of extracts.

To prepare the hot water extract, 1L of distilled water was added per 100g of sample, boiled for 2 hours using an electric water heater (Daewoong Yaktang, Daewoong Bio Appliances, Korea), filtered using filter paper (Advantec, No.41), and reduced pressure. Concentrated and then lyophilized.

The manufacturing method of 70% ethanol extract was prepared by adding 1 L of solvent per 100 g of sample, sonicating for 1 hour, and then performing primary extraction overnight at room temperature. The first and second extracts were collected the next day, concentrated under reduced pressure after filtering, and freeze-dried to be used as samples.

1-2. cell culture

Experiments were conducted using BV2 cells, and cultured in an incubator while maintaining 5% CO2 and 37°C using DMEM medium (Gibco/Invitrogen; USA) containing 5% fetal bovine serum (FBS) and 1% penicillin streptomycin. did

1-3. Cytotoxicity assessment

The cytotoxicity evaluation of the ginseng flower extract was confirmed by the MTT assay method. BV2 cells were seeded in a 24-well plate at a density of 6Υ4, and the next day, ginseng flower extract (hot water extract, 70% ethanol extract) was treated, and 1 hour later, LPS (Sigma; USA) was treated at 200 ng/mL for another 24 hours. further cultured. After 24 hours, the medium was replaced with a medium containing MTT reagent (Sigma; USA), and further incubated at 37°C for 1 hour. The medium containing the MTT reagent was removed, and 400 μL of dimethyl sulfoxide (DMSO) was added to dissolve formazan produced in purple. After transferring the lysate to a 96 well plate by 150 μL, the absorbance was measured at 540 nm. The viability of the control group not treated with LPS was calculated as 100%, and the cell viability of the sample treated group was expressed as a percentage.

1-4. Nitric oxide (NO) production measurement

To measure NO production, BV2 cells were seeded in a 24-well plate at a density of 6X4, treated with ginseng flower extract (hot water extract, 70% ethanol extract) the next day, and treated with LPS one hour later and further cultured for another 20 hours. . After 20 hours, 100 μL of the cell culture medium was transferred to 96 wells, 100 μL of greiss reagent (Sigma; USA) was reacted, and the amount of NO production was measured at absorbance of 540 nm. The concentration of NO produced was calculated by preparing a standard curve using sodium nitrate (NaNO2) as a standard substance.

1-5. IL-6, TNF-α measurement using ELISA method

BV2 cells were divided into 35X10 ⁴ cells/well in a 6-well plate and cultured for about 24 hours. After pretreatment with ginseng flower hot water extract at concentrations of 50, 100, and 200 μg/mL for 1 hour, 200 ng/mL of LPS and further cultured for 6 hours or 20 hours. Thereafter, the culture medium was recovered, and IL-6 and TNF-α released in the culture medium were measured using an ELISA kit (BD Biosciences, NJ, USA).

1-6. Protein expression analysis (western blot)

BV2 cells were harvested, centrifuged, and the supernatant was discarded to recover the cell pellet. Protein was extracted using lysis buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS, containing fresh protease inhibitor cocktail), and the amount of extracted protein was determined by Pierce® protein assay kit (Rockford, IL, USA). ), and the same amount of protein was separated by sodium dodecyl sulfate-polyacrlyamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. Membrane was reacted with 5% skim milk for 1 hour to block reactivity to non-specific proteins, and incubated with primary antibody overnight at 4°C. The next day, they were incubated for 1 hour with a secondary antibody coupled to horseradish perox-idase (HRP). Between each reaction, it was washed three times for 10 minutes with TBST. Thereafter, the protein band corresponding to the antibody was confirmed using ECL kit (Amersham Pharmacia Biotech, UK) and ImageQuant LAS 500 (GE Healthcare Life Science, USA).

1-7. immunofluorescence staining

B항체(Santa Cruz Biotechnology; 미국)를 1시간 동안 처리하고 PBS로 5분 동안 세척하였다. 2차 형광 항체를 실온에서 1시간 동안 반응시키고 세척 후 DAPI 염색 후 슬라이드에 마운팅한 후 형광현미경 (Carl Zeiss Inc., Oberkochen, Germany)을 사용하여 관찰하였다. After washing BV2 cells with PBS, they were fixed with 4% paraformaldehyde. The fixed cells were permeabilized with 1% Triton X-100 dissolved in PBS for 10 minutes at room temperature, washed with PBS for 5 minutes, and blocked with 10% goat serum dissolved in PBS for 60 minutes. NF- at room temperature

Antibody B (Santa Cruz Biotechnology; USA) was treated for 1 hour and washed with PBS for 5 minutes. The secondary fluorescent antibody was reacted at room temperature for 1 hour, washed, stained with DAPI, mounted on a slide, and observed using a fluorescence microscope (Carl Zeiss Inc., Oberkochen, Germany).

1-8. statistical processing

The experiment performed in the present invention was repeated more than three times in total, and the statistical significance of the data was verified by the Student t-test and ANOVA method using the GraphPad Prism 5.0 program, and the result value is expressed as mean ± standard deviation (SD), A p value of less than 0.05 was considered statistically significant.

Extraction yield of ginseng flower extract

The yields of the hot water extract and the 70% ethanol extract were 25.70% and 33.22%, respectively, and the yields when extracted with 70% ethanol were higher.

Confirmation of cytotoxicity of ginseng flower extract

To confirm the cytotoxicity of hot water extract and 70% ethanol extract of ginseng flower, cell viability was measured by MTT assay using BV2 microglia. After culturing the cells in a 24-well plate 6 X 10 ⁴ cells/well for about 24 hours, the cells were treated with ginseng flower hot water extract and 70% ethanol extract at concentrations of 12.5, 25, 50, 100, and 200 μg/mL for 24 hours. The survival rate was confirmed.

As a result, both the ginseng flower hot water extract and the ethanol extract showed no cytotoxicity, with cell viability exceeding 95% up to the highest concentration of 200 μg/mL (FIG. 1).

Effects of Ginseng Flower Extract on NO Production

In order to investigate the neuroinflammation inhibitory effect of ginseng flower extract, BV2 cells were pretreated with ginseng flower extract and then treated with LPS (200 ng/mL) to induce a neuroinflammatory response and the production of NO, a representative indicator of inflammatory response, was measured by Griess A NO assay kit (Abcam) using a reagent was used, and the measurement method was performed according to the instruction manual of the kit.

As a result, NO production increased in the experimental group treated with LPS alone compared to the control group not treated with LPS in BV2 cells. In the experimental group, it was confirmed that NO secretion was significantly reduced depending on the concentration of ginseng flower extract. In particular, it was confirmed that the hot water extract reduced NO production more effectively than the ethanol extract (FIG. 2). These results indicate that the hot-water extract of ginseng flower has the potential to be used as a neuroinflammatory inhibitor that can reduce the inflammatory response of BV2 cells induced by LPS.

Effects of ginseng flower extract on iNOS and COX-2 protein expression

In order to confirm whether the hot-water extract of ginseng flower affects iNOS and COX-2 protein expression in BV2 cells stimulated with LPS, iNOS (Cell Signaling Technology; USA) and COX-2 (Santa Cruz Biotechnology; USA) antibodies were used. Protein expression was analyzed by Western blot.

As a result, the expression of iNOS and COX-2 proteins, which were increased by LPS treatment, was inhibited by hot-water extract of ginseng flower (FIG. 3). In particular, referring to FIG. 4 , it can be seen that treatment at a concentration of 50 mg/ml is more effective in inhibiting iNOS and COX-2 than treatment with a ginseng flower extract at a concentration of 100 mg/kg. Therefore, the ginseng flower extract of the present invention is 20mg/kg to 80mg/kg (preferably, 25mg/kg to 75mg/kg, 30mg/kg to 70mg/kg) in order to derive the effect of improving cognitive function (memory improvement). It is suitable to use it at a concentration (amount) of (preferably on a daily basis). These results show that ginseng flower hot-water extract can control the neuroinflammatory response by inhibiting the expression of iNOS and COX-2 proteins, which are early inflammatory response regulators, and that ginseng flower hot-water extract has functional properties for the treatment of various neurological diseases based on the inflammatory response. This means that there is potential for use as a material.

Effect of ginseng flower extract on MPAK protein expression

Mitogen activated protein kinases (MAPK) are known to play an important role in regulating cell proliferation and differentiation and various immune responses, and in particular in inducing the production of inflammatory mediators by being activated by external infection. Therefore, LPS-stimulated BV2 cells were treated with ginseng flower extract, and changes in the expression of p38, ERK, and JNK, which are MAPK family members involved in the suppression of inflammatory responses, were measured.

As a result, phosphorylation of p38, ERK and JNK was induced in BV2 cells treated with LPS, but phosphorylation of ERK, JNK and p38 was significantly inhibited in the group pretreated with ginseng flower extract (FIG. 4). From these results, it was confirmed that the anti-inflammatory effect of ginseng flower extract can be exerted through inhibition of MAPK signal transduction.

Effects of Ginseng Flower Extract on Inflammatory Cytokine Expression

To confirm the inhibitory effect of ginseng flower extract on the expression of inflammatory cytokines, BV2 cells were pretreated with hot water extract of ginseng flower for 1 hour, and then inflammatory responses were induced with LPS, and changes in the expression of inflammatory cytokines were observed.

IL-6 and TNF-α ELISA kit was used to confirm changes in the expression of IL-6 and TNF-α, which are inflammatory cytokines. , Although the expression level of TNF-α was significantly increased, it was confirmed that the amount of inflammatory cytokines was decreased in a concentration-dependent manner in the group pretreated with the hot water extract of ginseng flower (FIG. 5). This means that the ginseng flower extract is effective in inhibiting the expression of inflammatory mediators.

Effect of ginseng flower extract on NF-κ activation

The expression of nuclear transcription factor-kappa-B (NF-κB), an important transcription factor involved in mediating the inflammatory response, and IκBα, known as a negative regulator of NF-κB, were evaluated. To this end, IκBα expression (Santa Cruz Biotechnology, USA) was measured at the protein level by Western blot method, and intracellular distribution of NF-κB protein was confirmed using immunofluorescence staining method.

As a result, the expression of phosphorylated IκBα protein was increased by LPS treatment in BV2 cells, but the expression was suppressed in a concentration-dependent manner when treated with ginseng flower extract (FIG. 6A). Through these results, it was found that when BV2 cells were treated with LPS, NF-κB was activated and migrated to the nucleus, but when ginseng flower extract was also treated, NF-κB migration into the nucleus could be inhibited. In addition, as a result of observing NF-κB migration to the nucleus using immunofluorescence staining, most of the BV2 cells cultured under normal conditions not stimulated with LPS expressed NF-κB in the cytoplasm, but BV2 cells stimulated with LPS , NF-κB was expressed in the nucleus. On the other hand, when the ginseng flower extract was also treated, it was confirmed that the movement of NF-κB into the nucleus by LPS treatment was significantly inhibited (FIG. 6B). These results indicate that the ginseng flower extract blocks the NF-κB pathway, an inflammatory signaling pathway induced by LPS, by inhibiting the nuclear migration of NF-κB protein by LPS.

Confirmation of cognitive function improvement effect of ginseng flower extract (Morris Water Maze Test)

Ginseng flower extract (50mg/kg, 100mg/kg) and donepezil (3mg/kg) were orally administered to C57BL/6 mice for one week. For each training, each mouse was given 2 minutes for the 1st trial and 1 minute for the 2nd trial, and the goal position was recognized at the beginning and end of each trial.), and scopolamine (2mg/kg) was administered on the day of the experiment. ) was injected to induce short-term memory loss 30 minutes later, the Morris Water Maze Test was performed to measure the time it took for mice to reach their destination (goal) (FIG. 7).

8 shows the cognitive function improvement effect of the ginseng flower extract according to the present Example in a scopolamine model. According to FIG. 7, ginseng flower extract (50mg/kg) and donepezil (3mg/kg) were orally administered to mice for a week, and then scopolamine (2mg/kg) was injected to induce short-term memory loss. Morris after 30 minutes The water maze test was conducted to measure the time it took for the mouse to reach the goal. Referring to FIG. 8, the experimental result showed that the time to reach the goal, which was increased due to memory impairment caused by scopolamine, was determined by ginseng flower extract. It can be seen that this reduces That is, according to this experiment, it can be seen that the ginseng flower extract of the present invention is effective in improving, preventing or treating cognitive dysfunction.

Expression of iNOS, COX-2 and p-I***B according to the treatment of ginseng flower extract

Ginseng flower extract (50mg/kg, 100mg/kg) and donepezil (3mg/kg) were orally administered to C57BL/6 mice for one week, respectively, and donepezil was used as a positive control. On the day of the experiment, scopolamine (2mg/kg) was injected to induce short-term memory loss 30 minutes after the mouse was necropsied, and the brain was obtained and used for the experiment (see FIG. 7).

When the ginseng flower extract was treated, it was confirmed that the inflammation-related proteins iNOS and COX-2 were suppressed compared to the scopolamine-administered group (FIG. 9). In addition, in the group administered with the ginseng flower extract, the increased phosphorylation of IκB-α was suppressed by scopolamine treatment, and through this, it was judged to suppress neuroinflammation by inhibiting the NF-KB pathway (FIG. 10). In particular, when treated at a concentration (amount) of 50 mg/kg, the levels of iNOS, COX-2, and p-IκBα were higher than that of the donepezil-treated group. It was confirmed that protein expression was inhibited

Expression of BDNF and p-CREB according to the treatment of ginseng flower extract

Ginseng flower extract (50mg/kg, 100mg/kg) and donepezil (3mg/kg) were orally administered to C57BL/6 mice for one week, respectively, and donepezil was used as a positive control. On the day of the experiment, scopolamine (2mg/kg) was injected to induce short-term memory loss 30 minutes after the mouse was necropsied, and the brain was obtained and used for the experiment (see FIG. 7).

The brains of the mice were disrupted to extract brain proteins. The expression of BNDF and p-CREB in the disrupted brain proteins was confirmed by Western blot.

BDNF (brain-derived neurotrophic factor) plays an important role in memory formation in relation to nerve cell survival, long-term memory enhancement, and synaptic plasticity. It is known. CREB is a transcription factor that plays an important role in neuronal survival and protection and synaptic plasticity in relation to degenerative brain diseases, and has been found to induce transcription of related genes through an important signal transduction mechanism related to memory formation and reinforcement. In particular, BDNF increased by CREB plays a critical role in neuronal differentiation and survival, and also increases synaptic plasticity and neuronal activity (Tao et al., 1998; Gordon, 2009; Bekinschtein et al., 2008).

11 shows the results of confirming the expression changes of BDNF protein and p-CREB protein when the ginseng flower extract was treated. As shown in FIG. 11, the expression of BDNF protein and p-CREB protein in the experimental group treated with ginseng flower extract was significantly increased compared to the non-treated group and the control group (p<0.01).

That is, it can be seen that the ginseng flower extract according to the present invention increases the expression of BDNF protein and p-CREB protein, is effective for nerve cell survival and long-term memory enhancement, and helps improve cognitive function (memory formation).

As described above, specific parts of the present invention have been described in detail, to those skilled in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (13)

A pharmaceutical composition for preventing or treating neuroinflammation comprising a ginseng flower extract as an active ingredient.
According to claim 1,
The ginseng flower extract is a hot water extract or an ethanol extract, a pharmaceutical composition.
According to claim 1,
The ginseng flower extract in nerve cells
(i) inhibition of NO production;
(ii) inhibition of inflammatory cytokine expression;
(iii) inhibition of iNOS/COX-2 protein expression; or
(iv) A pharmaceutical composition that suppresses neuroinflammation by inhibiting MAPK signal transduction.
According to claim 1,
Wherein the ginseng flower extract is administered to a subject at a concentration of 30 to 80 mg/kg, a pharmaceutical composition.
According to claim 1,
The composition is effective for preventing, treating or improving neuroinflammatory diseases,
The neuroinflammatory disease is at least one selected from the group consisting of multiple sclerosis, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, post-traumatic stress disorder, depression, schizophrenia, amyotrophic lateral sclerosis, or mild cognitive impairment, pharmaceutical composition.
A health functional food composition for preventing or improving neuroinflammation, comprising ginseng flower extract as an active ingredient.
According to claim 6,
The ginseng flower extract is a hot water extract or ethanol extract, health functional food composition.
According to claim 6,
The ginseng flower extract in nerve cells
(i) inhibition of NO production;
(ii) inhibition of inflammatory cytokine expression;
(iii) inhibition of iNOS/COX-2 protein expression; or
(iv) A functional health food composition that suppresses neuroinflammation by inhibiting MAPK signal transduction.
According to claim 6,
The composition is effective for preventing or improving neuroinflammatory diseases,
The neuroinflammatory disease is at least one selected from the group consisting of multiple sclerosis, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, post-traumatic stress disorder, depression, schizophrenia, amyotrophic lateral sclerosis and mild cognitive impairment, Health functional food composition.
A health functional food composition for improving memory or preventing or improving cognitive dysfunction, comprising ginseng flower extract as an active ingredient.
According to claim 10,
The ginseng flower extract is characterized in that the hot water extract or ethanol extract, health functional food composition.
According to claim 10,
The ginseng flower extract is to be ingested at a concentration of 30 to 80 mg / kg to the subject, health functional food composition.
According to claim 10,
The cognitive dysfunctions include loss of reasoning ability, forgetfulness, learning difficulties, concentration problems, decline in intelligence, decline in mental function, Alzheimer's disease, cerebrovascular dementia, Pick's disease and Creutzfeldt-Jacob ( Creutzfeldt-jakob) disease, at least one symptom selected from the group consisting of, health functional food composition.

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