KR20240020098A - Composition for preventing or treating neurodegenerative diseases comprising diterpenoid compound - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating neurodegenerative diseases containing a diterpene-based compound or a pharmaceutically acceptable salt thereof.
Specifically, the diterpene-based compound of the present invention can prevent or treat neurodegenerative diseases caused by inhibition of the activity of Nurr1 protein by activating Nurr1 protein.

Description

Composition for preventing or treating neurodegenerative diseases comprising diterpenoid compound}

The present invention relates to a pharmaceutical composition for preventing or treating neurodegenerative diseases containing a diterpene-based compound or a pharmaceutically acceptable salt thereof.

In addition, the present invention relates to a health functional food for preventing or improving neurodegenerative diseases containing diterpene-based compounds or pharmaceutically acceptable salts thereof.

As the various bioregulatory functions of natural bioactive substances isolated from natural products become known, research in fields such as new drug development and health functional foods using natural bioactive substances is actively conducted. However, natural products contain a wide variety of bioactive substances, and each substance has different physical and chemical properties, so even when extracted from the same natural product, the types of bioactive substances separated are different depending on the type of extraction solvent. . In addition, it has been reported that even if bioactive substances are isolated from the same natural product, their activity and effects have different characteristics depending on the type. Therefore, recently, research has been actively conducted to isolate new, previously unknown bioactive substances from the same natural product using different extraction solvents.

Neurodegenerative diseases involve symptoms when nerve cells degenerate, lose function, and often die. Patients with neurodegenerative diseases can experience extreme decline in cognitive or motor abilities, and because these diseases are often progressive, their quality of life and expectations of life can be significantly reduced as a result. .

These diseases include Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), frontotemporal dementia, and cortico-basal ganglia. Includes corticobasal degeneration, progressive supranuclear palsy (PSP) and other diseases.

Meanwhile, a significant number of neurodegenerative diseases are significantly related to Nurr1 protein. Specifically, U.S. Patent Publication No. 2009-0226401 discloses that Parkinson's disease, a type of neurodegenerative disease, is a disease related to dopamine neurons, and that activation of Nurr1 exhibits a therapeutic effect for Parkinson's disease. In addition, International Patent Publication No. WO2010-04221 states that Nurr1 plays an essential role in activating dopamine, and discloses a method of treating Parkinson's disease by activating Nurr1 and regulating neurotransmission that activates dopamine.

A representative neurodegenerative disease caused by dysfunction of Nurr1 is Parkinson's disease. Parkinson's disease is one of the important diseases in modern aging society whose main symptoms are tremor, stiffness, bradykinesia, and gait abnormality. It is caused by a neurotransmission called dopamine in the substantia nigra and corpus striatum of the brain. It is a chronic disease caused by a lack of substances.

Drugs for the treatment of Parkinson's disease include L-dopa drugs, dopamine receptor agonists, anticholinergic drugs, and Eldepryl. Most of these drugs treat symptoms rather than treating the cause. Since it plays a regulating role, continuous medication is required. To date, many drugs have been created and commercialized to treat Parkinson's disease, but an essential treatment to completely treat Parkinson's disease has not yet been developed.

Accordingly, the present inventor confirmed the effect of compounds obtained from Daphne genkwa flower buds using various extraction and fractionation solvents on activating Nurr1 and suppressing inflammatory responses, and found that Parkinson's disease caused by Nurr1 dysfunction was confirmed. The present invention was completed after confirming that it has excellent therapeutic effects on various neurodegenerative diseases, including diseases.

US Patent Publication No. 2009-0226401 International Patent Publication No. WO2010-04221

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating neurodegenerative diseases containing a diterpene-based compound or a pharmaceutically acceptable salt thereof.

Additionally, the present invention aims to provide a health functional food for preventing or improving neurodegenerative diseases containing a diterpene-based compound or a pharmaceutically acceptable salt thereof.

The present invention provides a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising a diterpene-based compound or a pharmaceutically acceptable salt thereof as an active ingredient.

Specifically, to provide a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases comprising as an active ingredient any one selected from the group consisting of compounds represented by Formulas 1 to 4 or pharmaceutically acceptable salts thereof, wherein the formula The compounds represented by numbers 1 to 4 are yuanhuagine (Formula 1), yuanhuahine (Formula 2), isoyuanhuacine (Formula 3), or yuanhuajine (Formula 4).

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

In another aspect, the present invention provides a method for producing a diterpene-based compound or a pharmaceutically acceptable salt thereof.

Specifically, preparing a Daphne genkwa extract; and separating and identifying the active ingredient from the extract. A method for producing a compound selected from the group consisting of compounds represented by Formulas 1 to 4 is provided.

The diterpene-based compounds can be prepared by methods known to those skilled in the art, can be prepared by purchasing or synthesizing commercially available compounds, and can be separated and purified from plants known in the art using a polar or non-polar solvent. there is. Specifically, the compound can be extracted and isolated from Red Bean Blossom Tree. More specifically, the compound can be isolated from the extract of red bean flower buds, but is not limited thereto.

The term “Daphne genkwa” used in the present invention refers to a deciduous shrub of the dicotyledonous plant Myrtle family. It is also called early flowering tree or red bean tree, and has the characteristic of growing mainly near the seaside.

The red bean flower tree extract of the present invention refers to an extract obtained from the red bean flower tree. Specifically, the extract may be an extract obtained by extracting the red bean flower tree with water or an organic solvent, and more specifically, it may be an extract obtained by extracting it with water, a C1 to C5 lower alkyl alcohol, or a mixed solvent thereof. The alkyl alcohol may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% alkyl alcohol.

The Red Bean Blossom extract according to an embodiment of the present invention is an extract extracted with 60% to 80(v/v)% ethanol, preferably an extract extracted with 70(v/v)% ethanol, but is not limited thereto.

The Red Bean Blossom extract may be a fraction of the extract, and the fraction refers to an active fraction obtained by fractionating the compound of the present invention from the Red Bean Blossom extract using a specific solvent.

According to one embodiment of the present invention, the obtained Red Bean Flower extract is obtained by separating the fraction layer of each solvent using an organic solvent such as hexane, chloroform, ethyl acetate, butanol, or distilled water, or a mixed solvent thereof, and chromatography Fractions can be prepared by separating and purifying the compounds of the present invention to high purity using separation methods known in the art, such as graphy.

The pharmaceutical composition of the present invention may be used in the form of any one compound selected from the group including compounds represented by Formulas 1 to 4, or a pharmaceutically acceptable salt thereof.

As used herein, the term “pharmaceutically acceptable salt” refers to any salt that retains the desired biological and/or physiological activity of the compound and exhibits minimal undesirable toxicological effects. As a salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts are prepared by conventional methods, for example, by dissolving the compound in an excess of aqueous acid solution and precipitating the salt using a water-miscible organic solvent, such as methanol, ethanol, acetone or acetonitrile. Equimolar amounts of the compound and an acid or alcohol (e.g., glycol monomethyl ether) in water can be heated, and the mixture can then be evaporated to dryness, or the precipitated salt can be filtered off with suction. At this time, inorganic acids and organic acids can be used as free acids. Hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, tartaric acid, etc. can be used as inorganic acids, and methane sulfonic acid, p-toluene sulfonic acid, acetic acid, and trihydrocytic acid can be used as organic acids. Fluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid. acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, etc. possible, and is not limited to these.

Additionally, a pharmaceutically acceptable metal salt can be prepared using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. At this time, it is particularly pharmaceutically suitable to prepare sodium, potassium or calcium salts as metal salts, but is not limited to these. Additionally, the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with an appropriate silver salt (eg, silver nitrate).

Pharmaceutically acceptable salts of the compounds of formulas 1 to 4 include almost all salts of acidic or basic groups that may be present in the compounds, unless otherwise indicated. For example, pharmaceutically acceptable salts may include sodium, calcium and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, etc. There are salts such as hydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate), and p-toluenesulfonate (tosylate), and salts can be prepared through methods known in the art. can be manufactured.

The pharmaceutical composition containing the diterpene-based compound of the present invention, or a pharmaceutically acceptable salt thereof, may be provided for the prevention or treatment of neurodegenerative diseases. Specifically, the neurodegenerative diseases include Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and frontotemporal dementia (Fronto- It may be any one selected from the group consisting of Temporal Dementia, Cortico Basal Degeneration, and progressive supranuclear palsy (PSP).

According to one embodiment of the present invention, it was confirmed that the compounds of Formulas 1 to 4 increase the activity of Nurr1 protein (FIGS. 1 to 4).

Therefore, a pharmaceutical composition containing any one compound selected from the group containing compounds of formulas 1 to 4 of the present invention or a pharmaceutically acceptable salt thereof can be usefully used for the prevention or treatment of neurodegenerative diseases.

As used herein, the term "treatment" refers to clinical intervention to alter the natural processes of the individual or cell being treated, which may be performed during the course of the clinical pathology or to prevent it. . The desired therapeutic effect includes preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing all direct or indirect pathological consequences of the disease, reducing the rate of disease progression, alleviating or temporarily alleviating the disease state, and This includes remission or improving prognosis. Preferably, the present invention includes all actions to improve the course of neurodegenerative diseases by administering a composition containing a diterpene compound or a pharmaceutically acceptable salt thereof. In addition, “prevention” refers to any act of suppressing or delaying the onset of the neurodegenerative disease by administering a composition containing a diterpene-based compound or a pharmaceutically acceptable salt thereof according to the present invention.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier in addition to containing the diterpene-based compound or a pharmaceutically acceptable salt thereof as an active ingredient.

Specifically, a pharmaceutical for the prevention or treatment of neurodegenerative diseases comprising a therapeutically effective amount of a compound represented by Formulas 1 to 4 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. A composition is provided.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

The type of carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art can be used. Non-limiting examples of the carrier include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, etc. You can. These may be used individually or in combination of two or more types.

In addition, the pharmaceutical composition of the present invention can be used by adding other pharmaceutically acceptable additives, such as excipients, diluents, antioxidants, buffers or bacteriostatic agents, if necessary, as well as fillers, extenders, wetting agents, disintegrants, dispersants, and surfactants. , it can be used by additionally adding a binder or lubricant.

In the pharmaceutical composition of the present invention, the diterpene-based compound, or a pharmaceutically acceptable salt thereof, may be included in an amount of 001% to 9000% by weight, preferably 001% by weight, based on the total weight of the pharmaceutical composition. It may be included in % to 9000% by weight, more preferably 01% to 70% by weight, and even more preferably 01% to 50% by weight, but is not limited thereto and depends on the condition of the administration subject, the type of specific disease, and the degree of progression. It may change in various ways depending on the situation. If necessary, it may also be included in the total content of the pharmaceutical composition.

The pharmaceutical composition of the present invention can be formulated and used in various suitable dosage forms for oral or parenteral administration.

Non-limiting examples of preparations for oral administration using the pharmaceutical composition of the present invention include troches, lozenges, tablets, aqueous suspensions, oily suspensions, preparation powders, granules, emulsions, hard capsules, and soft capsules. Capsules, syrups, or elixirs may be used.

In order to formulate the pharmaceutical composition of the present invention for oral administration, binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax can be used, and sweeteners, fragrances, syrups, etc. can also be used. Furthermore, in the case of capsules, in addition to the above-mentioned substances, a liquid carrier such as fatty oil can be additionally used.

Non-limiting examples of parenteral preparations using the pharmaceutical composition of the present invention include injections, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for application, oils, creams, etc.

To prepare the pharmaceutical composition of the present invention for parenteral administration, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, topical preparations, etc. can be used. Examples of the non-aqueous solvents and suspensions include propylene glycol and polyethylene. Glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, etc. may be used.

When the pharmaceutical composition of the present invention is formulated as an injection solution, the pharmaceutical composition of the present invention is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is then administered in ampoules or vials for unit administration. It can be formulated.

When the pharmaceutical composition of the present invention is formulated as an aerosol, a propellant or the like can be mixed with additives to disperse the water-dispersed concentrate or wet powder.

When the pharmaceutical composition of the present invention is formulated into ointments, creams, powders for application, oils, external skin preparations, etc., animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivatives, polyethylene glycol, silicone, bentonite , silica, talc, zinc oxide, etc. can be used as carriers.

The pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time, and/or administration route of the pharmaceutical composition, and the type of response to be achieved by administration of the pharmaceutical composition. and severity, type of subject to be administered, age, weight, general health condition, symptoms or severity of disease, gender, diet, excretion, drugs used simultaneously or simultaneously with the subject, other composition ingredients, etc. It may vary depending on factors and similar factors well known in the pharmaceutical field, and a person skilled in the art can easily determine and prescribe an effective dosage for the desired treatment.

The pharmaceutical composition of the present invention may be administered once a day, or may be administered in several divided doses. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Considering all of the above factors, it can be administered in an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

“Pharmaceutically effective amount” for treatment means an amount sufficient to suppress or alleviate the disease at a reasonable rate applicable to medical use, and the effective dose level is determined by the type and severity of the subject, age, gender, activity of the drug, drug It can be determined based on factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple times.

Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and can be easily determined by a person skilled in the art. For example, the pharmaceutically effective amount is 001 mg/day/kg to 100 mg/day/kg of body weight, specifically 01 mg/day/kg to 01 mg/day/kg of body weight for the diterpene compound or a pharmaceutically acceptable salt thereof. It is 10 mg/day/kg of body weight.

The administration route and administration method of the pharmaceutical composition of the present invention may be independent, and any administration route and administration method may be used without particular limitation as long as the pharmaceutical composition can reach the desired area. The pharmaceutical composition can be administered orally or parenterally.

Parenteral administration methods of the pharmaceutical composition of the present invention include intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, or subcutaneous administration. Methods include applying the composition to the diseased area, spraying it, or inhaling it. It can also be used, but is not limited to this.

The pharmaceutical composition of the present invention can exhibit excellent effects when used alone, but can be used in combination with various cancer treatment methods such as radiation therapy and chemotherapy to increase treatment efficiency.

In the present invention, “Nurr1 (nuclear receptor related 1)” refers to nuclear receptor related 1 protein, also known as NR4A2 (nuclear receptor subfamily 4, group A, member 2), which is encoded by the human NR4A2 gene. It is known that It is also known to be involved in a significant number of neurodegenerative diseases. Although the Nurr1 protein is an orphan nuclear receptor, the ligand for this protein has not yet been identified. However, Nurr1 is a protein belonging to the nuclear receptor family of intracellular transcription factors and is involved in the dopamine system in the brain. It has been found that it plays a key role in maintaining the dopaminergic system. Diseases caused by Nurr1 dysfunction include, but are not limited to, Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), Neurodegenerative diseases such as fronto-temporal dementia, cortico-basal degeneration, progressive supranuclear palsy (PSP), and rheumatoid arthritis, schizophrenia, and bipolar disorder caused by dopamine dysfunction. Includes a wide range of inflammatory diseases such as

In another aspect of the present invention, a health functional food for preventing or improving neurodegenerative diseases containing as an active ingredient any one selected from the group consisting of compounds represented by formulas 1 to 4 or foodologically acceptable salts thereof. provides.

The above-mentioned health functional food is a food that emphasizes the bioregulatory function of food and is a food that has added value to act and express for a specific purpose using physical, biochemical, and biotechnological methods. The ingredients of these health functional foods are designed and processed to fully exert the body's regulatory functions related to biological defense, regulation of body rhythm, prevention and recovery of disease, and contain food auxiliary additives, sweeteners or functional ingredients that are acceptable as food. May contain raw materials.

When using the diterpene-based compound of the present invention, or a pharmaceutically acceptable salt thereof, as a health functional food (or health functional beverage additive), the compound is added as is or used together with other foods or food ingredients, using the usual method. It can be used appropriately. The amount of the compound mixed can be appropriately determined depending on the purpose of its use (prevention, health or improvement, therapeutic treatment).

The health functional foods include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, organic acids, and protective colloid thickeners. It may contain agents, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Additionally, the health functional food of the present invention may contain pulp for the production of fruit and vegetable drinks. These components can be used alone or in combination, and the ratio of these additives is generally selected in the range of 0001 to 50 parts by weight per total weight of the composition.

There are no particular restrictions on the types of health functional foods. Foods to which the above compounds can be added include sausages, meat, bread, chocolates, snacks, candies, confectionery, ramen, pizza, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, and alcohol. There are beverages and vitamin complexes. When formulated into a beverage, the liquid ingredients added in addition to the novel lactic acid bacteria are not limited to this, but may contain various flavoring agents or natural carbohydrates as additional ingredients like regular beverages. The above-mentioned natural carbohydrates include monosaccharides (e.g. glucose, fructose, etc.), disaccharides (e.g. maltose, sucrose, etc.) and polysaccharides (e.g. common sugars such as dextrins, cyclodextrins, etc.), and xylitol, sorbitol. It may be a sugar alcohol such as erythritol.

The present invention provides a health functional food for preventing or improving neurodegenerative diseases comprising an effective amount of any one selected from the group consisting of a compound represented by Formulas 1 to 4 or a foodologically acceptable salt thereof and a foodologically acceptable carrier. to provide.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

The present invention provides a method for treating neurodegenerative diseases, comprising administering to a subject a pharmaceutical composition containing as an active ingredient any one selected from the group consisting of compounds represented by Formulas 1 to 4 or pharmaceutically acceptable salts thereof. to provide.

In the present invention, an individual refers to any animal, including humans, that possesses or develops a neurodegenerative disease, and may be an individual other than a human. By administering the pharmaceutical composition of the present invention to a subject, it shows excellent effects in the treatment of neurodegenerative diseases.

The present invention provides the use of a composition containing a compound represented by Formulas 1 to 4 or a pharmaceutically acceptable salt thereof in the manufacture of a drug for the treatment of neurodegenerative diseases.

The present invention provides a composition comprising a compound represented by Formulas 1 to 4 or a pharmaceutically acceptable salt thereof for use in the treatment of neurodegenerative diseases.

The diterpene compound of the present invention improves Nurr1 protein activity and has the effect of suppressing inflammatory responses in nerve cells, thereby preventing or treating neurodegenerative diseases, including Parkinson's disease, caused by inhibiting the activity of Nurr1 protein. It can be used efficiently.

Figure 1 is a graph confirming the Nurr1 activation effect of yuanhuagine of the present invention.
Figure 2 is a graph confirming the Nurr1 activation effect of yuanhuahine of the present invention.
Figure 3 is a graph confirming the Nurr1 activation effect of isoyuanhuacine of the present invention.
Figure 4 is a graph confirming the Nurr1 activation effect of yuanhuajine of the present invention.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Preparation of Red Bean Flower Extract

1.3 kg of dried flower buds of Daphne genkwa were immersed in 26 L 70(v/v)% ethanol for 72 hours and filtered to obtain a liquid component. The obtained liquid component was concentrated under reduced pressure, and then 376 g of Red Bean Flower bud extract was prepared.

Example 2: Separation of active ingredients from Red Bean Blossom extract using various solvents

The red bean flower bud extract obtained in Example 1 was fractionated with distilled water and dichloromethane to produce two fractions (distilled water and dichloromethane). For the dichloromethane fraction (45.7 g), column chromatography using Silica gel 70-230 mesh resin (Φ 6.7 × 52.5 cm, 100% n-hexane → n-hexane/ethyl acetate/methanol = 10/0/0 → 6 /3.6/0.4 → 100% ethyl acetate → 100% methanol) and divided into 9 small fractions (Fr.1 ~ Fr.9).

Column chromatography (Φ 4.5 × 62.3 cm, dichloromethane/methanol = 5/5 → 0/10) was performed on the subfraction Fr.6 (6.6 g) using Sephadex LH-20 resin and divided into four subfractions. (Fr.6-1 ~ Fr.6-4). Among these, the small fraction Fr.6-2 (3.6 g) was subjected to column chromatography using Silica gel 230-400 mesh resin [Φ 4.0 × 25.5 cm, mobile phase: 100% n-hexane → n-hexane/ethyl acetate/ methanol = 8/1.8/0.2 → 5/4.5/0.5 → 0/0/10], and through this, 6 small fractions were obtained (Fr.6-2-1 ~ Fr.6-2-6).

Using flash column chromatography (RP C18, 130 g) [mobile phase: acetonitrile/distilled water = 6/4 → 8.5/1.5], nine subfractions ( Fr.6-2-3-1 ~ Fr.6-2-3-9) were obtained, and the small fraction Fr.6-2-3-5 (157.2 mg) was obtained by preparative HPLC (Gemini 5 μm NX-C18). , 250 minutes), flow rate 7.0 ml/min] to obtain the compound of Formula 2 (2.0 mg) and the compound of Formula 4 (2.1 mg).

Using flash column chromatography (RP C18 130 g) [mobile phase: acetonitrile/distilled water = 6.5/3.5 → 8.5/1.5], the small fraction Fr.6-2-4 (783.5 mg) was used to obtain yuanhuadine (348.3 mg). Five small fractions (Fr.6-2-4-1 to Fr.6-2-4-5) were obtained. Small fraction Fr.6-2-4-5 (139.1 mg) was repeatedly preparative HPLC (Gemini 5 μm NX-C18, 250 ), composition ratio: 80-90% B (90 minutes) → 90-100% B (5 minutes) → 100% B (10 minutes), flow rate 7.0 ml/min] to obtain the compound of formula 3 (1.3 mg), Yuanhuacine (19.1 mg) and compound of formula 4 (1.2 mg) were obtained. The small fraction Fr.6-2-5 (967.8 mg) was purified using flash column chromatography (80 g of RP C18) [mobile phase: acetonitrile/distilled water = 5.5/4.5 → 7.5/2.5] to obtain the compound of Formula 1 (212.3 mg) Six subfractions (Fr.6-2-5-1 to Fr.6-2-5-6) containing Genkwadaphnin (198.2 mg) were obtained.

Four types of diterpenoid compounds (compounds of formulas 1 to 4) were isolated from extracts of red bean flower buds using repetitive chromatography, and their chemical structures were determined through analysis of spectroscopic data (1H-NMR, DEPT-NMR, etc.) They were identified as Yuanhuagine, isoYuanhuacine, Yuanhuahine, and Yuanhuajine, respectively.

[Formula 1] Yuanhuagine

[Formula 2] Yuanhuahine

[Formula 3] isoyuanhuacine

[Formula 4] Yuanhuajine

Example 3: Effect of compounds on Nurr1 protein activity

As in Example 2, the Nurr1 protein activity according to the concentration of diterpene-based compounds isolated from the red bean flower bud extract was confirmed through luciferase analysis (see Korean Patent No. 10-1964889).

Specifically, after synthesizing a vector in which a gene with a nucleotide sequence (5'-CTCGGAGGACAGTACTCCG-3') to which the GLA4 gene can bind repeated 8 times was linked to the reporter gene luciferase, DNA containing Nurr1-LBD and beta -Three types of plasmid DNA, including DNA containing galactosidase (β-galactosidase), were injected (transfected) into BE(2)C cells. After 6 hours, compounds 1 to 10 isolated in Example 2 were treated according to the concentrations shown in Table 1 below. The cells thus treated were cultured in a 5% carbon dioxide incubator at 37°C for 20 hours, and then luciferase analysis was performed.

As a result of the analysis, as shown in Figures 1 to 4, all compounds of formulas 1 to 4 activated Nurr1. Specifically, it was confirmed that all compounds of Formulas 1 to 4 had an excellent effect of activating Nurr1 in a concentration-dependent manner at a concentration of 0.01 μM or higher. Through this, it was found that compounds isolated from red bean flower extract activate Nurr1 and that their activities may vary depending on the structure of the compound.

The contents of this specification can be fully recognized and inferred by those of ordinary skill in the technical field of the present invention, and various modifications can be made without changing the structure. Accordingly, the present invention may be practiced in ways other than those specifically described and exemplified in this specification, which can be understood by those skilled in the art.

Claims (12)

A pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, comprising as an active ingredient any one selected from the group consisting of compounds represented by Formulas 1 to 4 or pharmaceutically acceptable salts thereof.
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]
The method of claim 1, wherein the neurodegenerative disease is Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and frontotemporal dementia. (Fronto-Temporal Dementia), cortico-basal degeneration (Cortico Basal Degeneration), and progressive supranuclear palsy (PSP), any one selected from the group consisting of a pharmaceutical composition for the prevention or treatment of neurodegenerative disease . The pharmaceutical composition for preventing or treating a neurodegenerative disease according to claim 1, wherein the neurodegenerative disease is Parkinson's disease (PD). A health functional food for preventing or improving neurodegenerative diseases, comprising as an active ingredient any one selected from the group consisting of compounds represented by Formulas 1 to 4 or foodologically acceptable salts thereof.
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]
The method of claim 1, wherein the neurodegenerative disease is Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and frontotemporal dementia. (Fronto-Temporal Dementia), cortico-basal degeneration (Cortico Basal Degeneration), and progressive supranuclear palsy (PSP), any one selected from the group consisting of a health functional food for preventing or improving neurodegenerative diseases . The health functional food for preventing or improving neurodegenerative diseases according to claim 4, wherein the neurodegenerative disease is Parkinson's disease (PD). A pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, comprising a therapeutically effective amount of a compound represented by Formulas 1 to 4 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]
A health functional food for preventing or improving neurodegenerative diseases, comprising an effective amount of any one selected from the group consisting of a compound represented by Formulas 1 to 4 or a foodologically acceptable salt thereof, and a foodologically acceptable carrier.
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]
Preparing Daphne genkwa extract; and
A method for producing a compound selected from the group consisting of compounds represented by Formulas 1 to 4, comprising the step of separating and identifying the active ingredient from the extract.
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]
The method of claim 9, wherein the Red Bean Blossom extract is extracted from flower buds. The method of claim 9, wherein the Red Bean Flower extract is any one compound selected from the group consisting of compounds represented by Formulas 1 to 4, which are extracted with water, lower alcohols having 1 to 4 carbon atoms, or a mixture thereof. Manufacturing method. The method of claim 9, wherein the Red Bean Blossom extract is extracted with 60 to 80 (v/v)% ethanol.