KR20220077610A - Pharmaceutical composition for preventing or treating Alzheimer’s disease comprising of ginsenosides as active ingredients - Google Patents

Abstract

The present invention confirms the neuroprotective effect of five ginsenosides (G75, Rk2, C-Mx, PPD, and Rg5) and provides them for the use of Alzheimer's improvement, prevention or treatment, and the composition of the present invention is a natural product As it is derived from it, it has few or no side effects, so it is expected that it can be used in various fields such as medicine, quasi-drug, functional bio material, and functional food material for Alzheimer's prevention, treatment, and improvement.

Description

A pharmaceutical composition for preventing or treating Alzheimer's disease comprising ginsenoside as an active ingredient {Pharmaceutical composition for preventing or treating Alzheimer's disease comprising of ginsenosides as active ingredients}

The present invention confirms the microglia protective effect of five types of ginsenosides (G75, Rk2, C-Mx, PPD, and Rg5) and a synergistic effect according to a mixture thereof, and provides it for the use of Alzheimer's improvement, prevention or treatment will be.

Dementia is one of the three major diseases of the 21st century set by the World Health Organization, and is a representative chronic progressive degenerative brain disease. In senile dementia, mainly the hippocampus, forebrain, and temporal lobe are sequentially damaged, and about 30-40% of nerves are lost in this process. Typical neuropathological features of senile dementia include the accumulation of senile plaques in the extraneuronal and the formation of neurofibrillary tangles in the intraneuronal. At this time, the main component of senile plaque is beta-amyloid (Aβ) peptide, and the toxicity of beta-amyloid to nerve cells is considered to be the main etiology of Alzheimer's disease.

Neurotoxicity caused by beta-amyloid can be divided into direct or indirect mechanisms depending on whether glial cells are mediated. In a pathological state, microglia activated by beta-amyloid generate and release proinflammatory cytokines, nitric oxide (NO), reactive oxygen species (ROS), etc. mediates neurotoxicity. On the other hand, microglia in the normal state are the main immune cells of the central nervous system and play a beneficial role in removing accumulated beta-amyloid. Close interaction with nerve cells, glial cells, and microglia is essential to suppress the degeneration of nerve cells due to aging and keep nerve cells healthy. . Therefore, discovering a neuroprotective substance that improves the cell viability of microglia, which is reduced in the process of beta-amyloid-induced neuronal death, and elucidating the mechanism is a treatment for preventing and controlling dementia. It can be said to be a very important issue in technology development.

An object of the present invention is to provide a composition for improving, preventing, or treating Alzheimer's disease comprising ginsenoside as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating Alzheimer's disease comprising ginsenoside as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving Alzheimer's disease comprising ginsenoside as an active ingredient.

In one embodiment of the present invention, the ginsenoside may include G75, Rk2, C-Mx, PPD, and Rg5.

In another embodiment of the present invention, the ginsenoside may protect microglia from beta-amyloid, and the ginsenoside may protect nerve cells from oxidative stress caused by beta-amyloid.

In addition, the present invention provides a method for preventing or treating Alzheimer's disease, comprising administering ginsenoside to a subject.

In addition, the present invention provides a use of ginsenoside for the preparation of a medicament for preventing or treating Alzheimer's disease.

It was confirmed that the five types of ginsenosides (G75, Rk2, C-Mx, PPD, and Rg5) of the present invention are excellent in protecting microglia by beta-amyloid, each of which is pointed as the etiology of dementia, and by combining the A synergistic effect on cell protection was confirmed. Accordingly, the present invention proposes a novel use of the five types of ginsenoside mixture for preventing, treating, and improving Alzheimer's disease. The five kinds of ginsenosides of the present invention are derived from natural products and have little or no side effects, so they are used for preventing, treating, and improving Alzheimer's disease. is expected to be available.

1 is a view confirming the cytotoxicity according to the concentration of gypenoside LXXV and the cytotoxicity reduction effect by Aβ.
2 is a view confirming the cytotoxicity according to the concentration of Rk2 and the cytotoxicity reduction effect by Aβ.
3 is a view confirming the cytotoxicity according to the concentration of C-Mx and the cytotoxicity reduction effect by Aβ.
4 is a view confirming the cytotoxicity according to the concentration of PPD and the cytotoxicity reduction effect by Aβ.
5 is a view confirming the cytotoxicity according to the concentration of Rg5 and the cytotoxicity reduction effect by Aβ.
6 is a view comparing and confirming the cytotoxicity reduction effect by Aβ of each of G75, Rk2, C-Mx, PPD, and Rg5 ginsenosides and their complexes.
7 is a view confirming the change in the expression level of NLRP3 inflammasome according to the treatment of G75, Rk2, C-Mx, PPD, or Rg5 ginsenoside.

As a result of intensive research on natural products for inhibiting the pathogenesis of Alzheimer's by inhibiting microglia death by beta-amyloid peptide, the present inventors confirmed the cytoprotective effect from beta-amyloid in 5 types of ginsenosides, The present invention was completed by confirming a synergistic effect on cell protection by mixing senosides.

Ginsenoside is a pharmacologically active ingredient of ginseng, mainly known for its anticancer activity and immune enhancing effect, but is not known about its effect on inhibiting neuronal cell death by beta-amyloid.

Ginsenosides can be divided into protopanaxadiol (PPD) and protopanaxatriol (PPT) types according to their chemical structural formula, and they are named Ginsenoside Rx according to the polarity sequence by column chromatography.

The present inventors searched for compounds derived from natural products that protect nerve cells from beta-amyloid and screened about 40 types of ginsenosides, especially 5 types of ginsenosides (G75, Rk2, C-Mx, PPD, and Rg5). The microglia protective effect was confirmed.

In addition, the five ginsenosides of the present invention effectively inhibited the activation response of NLRP3 inflammasome among inflammasomes in immune response-inducing cells, and, in particular, the level of decreased expression of NLRP3 in PPD and Rk2 was remarkable.

Accordingly, the present invention provides a composition for improving, preventing, or treating Alzheimer's disease, comprising a G75, Rk2, C-Mx, PPD, and Rg5 ginsenoside mixture as an active ingredient.

As used herein, the term "prevention" refers to any action that inhibits or delays the occurrence, spread or recurrence of Alzheimer's disease by administration of the composition of the present invention, and "treatment" means that the symptoms of the disease are reduced by administration of the composition of the present invention. It means any action that is improved or is changed to a beneficial effect, and “improvement” means any action that reduces a parameter related to Alzheimer's disease, for example, the degree of symptom by administration of the composition according to the present invention.

In the present invention, the term "pharmaceutical composition" means one prepared for the purpose of preventing or treating a disease, and each may be formulated in various forms according to a conventional method and used. For example, it can be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and the like.

In addition, according to each dosage form, a pharmaceutically acceptable carrier, such as a buffer, preservative, analgesic agent, solubilizer, isotonic agent, stabilizer, base, excipient, lubricant, etc., may be prepared by further including a carrier known in the art. can

'On the other hand, the pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects, and the effective dose level depends on the patient's health condition, Severity, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, treatment period, factors including drugs used in combination or concomitantly, and other factors well known in the medical field.

Accordingly, Alzheimer's can be prevented or treated by administering the pharmaceutical composition of the present invention to an individual.

As used herein, the term “individual” may be a mammal, such as a rat, livestock, mouse, or human, preferably a human.

The pharmaceutical composition of the present invention may be formulated in various forms for administration to an individual, and a representative formulation for parenteral administration is an injection formulation, preferably an isotonic aqueous solution or suspension. Formulations for injection may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component can be dissolved in saline or buffer to be formulated for injection. In addition, formulations for oral administration include, for example, ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers. , sucrose, mannitol, sorbitol, cellulose and/or glycine) and glidants (eg silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol). The tablet may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and optionally starch, agar, alginic acid or its It may further contain disintegrating agents such as sodium salts, absorbents, coloring agents, flavoring agents and/or sweetening agents. The formulation may be prepared by conventional mixing, granulating or coating methods.

In addition, the pharmaceutical composition of the present invention may further contain adjuvants such as preservatives, wettable powders, emulsification accelerators, salts or buffers for osmotic pressure control, and other therapeutically useful substances, and may be formulated according to a conventional method. .

The pharmaceutical composition according to the present invention may be administered through several routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient may vary depending on the route of administration, age, sex, weight, and severity of the patient. It may be appropriately selected according to several factors. In addition, the composition of the present invention may be administered in parallel with a known compound capable of enhancing the desired effect.

The pharmaceutical composition according to the present invention may be administered orally or parenterally, such as intravenously, subcutaneously, intranasally or intraperitoneally, to humans and animals. Oral administration also includes sublingual application. Parenteral administration includes injection methods such as subcutaneous injection, intramuscular injection and intravenous injection and drip method.

In the pharmaceutical composition of the present invention, the total effective amount of ginsenosides according to the present invention may be administered to a patient as a single dose, and a divided treatment method in which multiple doses are administered for a long period of time ( fractionated treatment protocol). The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease, but may be administered repeatedly several times a day at an effective dose of 100 μg to 3,000 mg when administered once for adults. . However, the concentration of the ginsenoside can be determined by considering various factors such as the age, weight, health status, sex, severity of disease, diet and excretion rate of the patient, as well as the route of administration and number of treatments, and the effective dosage for the patient can be determined. have. Therefore, in consideration of these points, a person of ordinary skill in the art can determine an appropriate effective dosage according to the specific use of the ginsenoside as an agent for treating or preventing Alzheimer's disease, and the pharmaceutical composition according to the present invention is the It is not particularly limited to the dosage form, administration route and administration method as long as the effect is shown.

In addition, the pharmaceutical composition of the present invention may further include a known agent for preventing or treating Alzheimer's disease in addition to ginsenoside as an active ingredient, and may be used in combination with other known treatments for the treatment of these diseases.

As another aspect of the present invention, there is provided a health functional food for preventing or improving Alzheimer's disease comprising the ginsenoside of the present invention as an active ingredient.

The composition comprising the ginsenoside of the present invention as an active ingredient can be used in various ways, such as drugs, food and beverages effective for preventing or improving Alzheimer's disease. Foods to which the composition containing the ginsenoside of the present invention can be added as an active ingredient include, for example, various foods, beverages, gum, tea, vitamin complexes, health supplements, and the like, and powders, granules, tablets , can be used in the form of capsules or beverages.

In addition, the composition comprising the ginsenoside of the present invention as an active ingredient may be added to food or beverage for the purpose of preventing and improving Alzheimer's disease. At this time, the amount of the compound in the food or beverage may be generally added in 0.01 to 15% by weight of the total food weight, and the health beverage composition is 0.02 to 10 g, preferably 0.3 to 1 g based on 100 g. can apply

The health functional food of the present invention may contain, in addition to containing the above compound as an essential ingredient in the indicated ratio, a food pharmaceutically acceptable food supplement additive, such as various flavoring agents or natural carbohydrates, as an additional ingredient. Examples of the above-described natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and conventional sugars such as polysaccharides 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. . The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, colorants 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, carbonates used in carbonated beverages, and the like. In addition, the health functional food of the present invention may contain natural fruit juice, fruit juice for the production of fruit juice drinks, and vegetable drinks. 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.

The present invention can apply various transformations and can have various embodiments. Hereinafter, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

[Experiment method]

1. Cell Culture

BV2 cells, a mouse microglia cell line, were cultured in DMEM/High glucose (HyClone) containing 10% FBS (HyClone) and 1% penicillin-streptomycin (Sigma-aldrich) at 5% CO ₂ and maintained at 37°C. did.

2. Ginsenoside treatment

Cultured BV2 cells were seeded in a 96-well plate (SPL) at a density of 3X10 ³ cells/well, and after 24 hours, 0.5 μM Aβ (Alpha diagnosis international) in 0.1% DMSO (Tocris) was treated. After 2 hours, ginsenoside (G75, Rk2, C-Mx, PPD, or Rg5 in 0.1% DMSO) was treated with various concentrations (0-10uM), respectively, and the medium and cells were collected after 24 hours. 3 hours before cell collection, 100ng/mL LPS was treated to activate microglia. At this time, 1% Triton X-100 was treated to be used as a positive control to measure LDH enzyme activity. Each material was treated in the medium in consideration of the final treatment concentration of the vehicle.

3. Cell viability assay

Aβ peptide has cytotoxicity, and the viability of cells was analyzed to determine whether the five types of ginsenosides and a mixture thereof exhibit a cytoprotective effect from Aβ. Specifically, after taking the medium so that the cells do not fall off the bottom when collecting the medium, centrifugation at 13,200 rpm for 5 minutes was performed to sink the cells. To check the cytotoxicity, the LDH cytotoxicity kit (Roche) was used. After reacting the kit mixture with 30 μL of the collected medium and the same amount to the immunoplate, absorbance at 492 nm using a microplate reader (BioTek) was confirmed. Toxicity was analyzed as a percentage (%) relative to the positive control group treated with Triton X-100.

4. Immunoblot analysis

In order to confirm the mechanism by which the five types of ginsenosides and their mixture induce a decrease in the cytotoxicity of the Aβ peptide, the NLRP3 expression level of cells treated with the Aβ peptide and the cells treated with the 5 types of ginsenosides was examined. comparison was confirmed. Specifically, SDS-PAGE was performed to prepare cell lysates and immunoblot each with an antibody, and protein signals were detected and quantified using GAPDH as a control.

[Experiment result]

1. Cytotoxicity Assessment

In order to confirm the effect of Gypenoside LXXV (G75) on cells, as a result of processing a range of concentrations, as can be seen in FIG. 1 to left). In the case of IC ₅₀ , it was estimated to be 8.295 μM. Based on this, treatment with 0, 0.1, 1, or 2 μM of Gypenoside LXXV and confirming the cytotoxicity mitigating effect of Aβ treatment in BV2 cells was confirmed. (Right in Figure 1).

Similarly, as a result of confirming the concentration of Rk2 without cytotoxicity and the concentration showing the cytotoxic effect induced by Aβ in BV2 cells within the concentration range, Rk2 showed low cytotoxicity in the range of 0 to 10 μM, and in the case of IC ₅₀ It was estimated to be 163.7 μM (left in FIG. 2). And Rk2 significantly reduced the cytotoxicity caused by Aβ within the above concentration range, and it was confirmed that, in particular, it significantly reduced the cytotoxicity at 10nM (right in FIG. 2).

Subsequently, in the case of C-Mx, it exhibited low cytotoxicity in the concentration range of 0-1 μM, and in the case of IC ₅₀ , it was estimated to be 10.89 μM (left in FIG. 3). In addition, it was confirmed that C-Mx significantly reduced the cytotoxicity caused by Aβ within the above concentration range, and in particular, significantly reduced the cytotoxicity at 100nM (right in FIG. 3).

In addition, in the case of PPD, it showed little cytotoxicity in the range of 0-1 μM, and in the case of IC ₅₀ , it was estimated to be 12.47 μM (left in FIG. 4). And, it was confirmed that PPD significantly reduced the cytotoxicity caused by Aβ at a concentration of 1 μM (right in FIG. 4).

And, in the case of Rg5, it was confirmed that low cytotoxicity was confirmed in the concentration range of 0 to 130 μM (left in Fig. 5), and it was confirmed that both Aβ-induced cytotoxicity was significantly reduced in the concentration range of 0.1 to 10 μM (FIG. 5) uh).

On the other hand, in order to confirm the synergistic effect by mixing 5 types of ginsenosides, the optimal conditions (G75, Rk2, C-Mx, PPD, and Rg5) of each ginsenoside individually were sequentially 2 μM, 10 nM, respectively. , 100 nM, 1 μM, and 1 μM) of cells treated with 20% and the same amount of 5 ginsenoside complexes (80 nM G75, 0.4 nM Rk2, 4 nM C-Mx, 40 nM PPD, and 40 nM Rg5) As a result of confirming the survival rate of , the individual treatment of ginsenoside did not show a significant difference in cell viability, but the treatment of the complex showed a significantly significant cytoprotective effect ( FIG. 6 ).

2. Confirmation of NLRP3 inflammasome expression level

Activation of NLRP3 inflammasome is known to induce apoptosis along with autophagy. In order to confirm the cytoprotective mechanism of the five types of ginsenosides, the expression level of NLRP3 inflammasome was confirmed in the cells treated with the optimal conditions of the ginsenosides. As a result, treatment with Aβ peptide increased the expression of NLRP3, and in particular, it was confirmed that the expression of NLRP3 was significantly reduced in cells treated with Rk2 and PPD.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, 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 (5)

As a pharmaceutical composition for preventing or treating Alzheimer's disease comprising five types of ginsenoside complex as an active ingredient,
The five ginsenosides are G75, Rk2, C-Mx, PPD, and Rg5, a pharmaceutical composition.
The method of claim 1,
The complex is characterized in that inhibiting the death of nerve cells, a pharmaceutical composition.
The method of claim 1,
The complex is characterized in that it inhibits the death of nerve cells from toxicity caused by beta-amyloid, a pharmaceutical composition.
A health functional food composition for preventing and treating or improving Alzheimer's disease, comprising five kinds of ginsenoside complex as an active ingredient,
The five kinds of ginsenosides are G75, Rk2, C-Mx, PPD, and Rg5, a health functional food composition.
5. The method of claim 4,
The complex is to protect nerve cells and microglia from beta-amyloid, health functional food composition.

Images