KR20130057878A - Pharmaceutical composition for preventing or treating neurodegenerative diseases comprising compounds isolated from daphne genkwa extract - Google Patents

Abstract

The present invention is a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases comprising genkwanine N or yuanhuacine, or a pharmaceutically acceptable salt thereof, which is a compound isolated from the extract of Azalea, as an active ingredient It is about. More specifically, the present invention provides a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, including Genkwanine N or Yuanhuacine, or a pharmaceutically acceptable salt thereof, isolated from the organic solvent extract of Sorbus almond, Nurr1 dysfunction. It relates to a pharmaceutical composition for the prevention or treatment of diseases caused by, Nurr1 activation composition and functional food additives for the prevention or improvement of neurodegenerative diseases comprising the Genkwanine N or Yuanhuacine as an active ingredient.
Compounds isolated from the red bean extract of the present invention are natural compounds derived from Parkinson's disease caused by inhibition of Nurr1 protein by exhibiting an excellent effect in restoring the inhibition of Nurr1 protein activity by neuronal damage without showing any side effects. And other neurodegenerative diseases can be prevented or treated.

Description

Pharmaceutical composition for preventing or treating neurodegenerative diseases comprising compounds isolated from Daphne genkwa extract}

The present invention is a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases comprising genkwanine N or yuanhuacine, or a pharmaceutically acceptable salt thereof, which is a compound isolated from the extract of Azalea, as an active ingredient It is about. More specifically, the present invention provides a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, including Genkwanine N or Yuanhuacine, or a pharmaceutically acceptable salt thereof, isolated from the organic solvent extract of Sorbus almond, Nurr1 dysfunction. It relates to a pharmaceutical composition for the prevention or treatment of diseases caused by, Nurr1 activation composition and functional food additives for the prevention or improvement of neurodegenerative diseases comprising the Genkwanine N or Yuanhuacine as an active ingredient.

Neurodeegenerative diseases are associated with symptoms when neurons degenerate, lose function, and often die. Because they are primarily progressive, the consequences of neurodegenerative diseases are often very devastating. Patients with neurodegenerative diseases may experience extreme degeneration in cognitive or motor ability. As a result, their quality of life and their expectations for life can be significantly reduced. In humans, these diseases include Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and frontal lobe dementia (Fronto-Temporal). Dementia, Cortico Basal Degeneration, progressive supranuclear palsy (PSP), and other diseases.

On the other hand, many neurodegenerative diseases are known to be related to the Nuclear Receptor Related 1 (Nurr1) protein. The Nurr1 refers to a nuclear receptor-related protein, also known as NR4A2 (nuclear receptor subfamily 4, group A, member 2), which is known to be encoded by the human NR4A2 gene. Although the Nurr1 protein is an orphan nuclear receptor and a ligand for the protein has not yet been identified, the Nurr1 is a protein belonging to the nuclear receptor family of intracellular transcription factors. It has been found to play a key role in maintaining the dopaminergic system. When the Nurr1 or NR4A2 gene abnormality occurs, it is known that the function of the dopaminergic system is impaired to induce Parkinson's disease, and it causes a wide range of inflammatory and neurological diseases such as rheumatoid arthritis, schizophrenia and bipolar disorder.

Parkinson's disease, which is a representative neurodegenerative disorder caused by dysfunction of Nurr1, is a chronic progressive neurological disease that affects the nerve cells that control muscle movement, and makes dopamine, a neurotransmitter in the substantia nigra area of the midbrain. Inflammation occurs when cells are suddenly damaged, degenerated, or significantly reduced in number. Dopamine is an important chemical for transmitting signals between cells to facilitate the body's movement. Motor disease symptoms such as tremor, rigidity, bradykinesia, postural instability and impaired speech are caused. The cause of damage to dopamine-producing cells is not clear. However, metabolic and traumatic encephalitis sequelae, such as atherosclerosis of the brain, carbon monoxide poisoning, drugs and hypothyroidism, have been studied.

As drugs for the treatment of Parkinson's disease, there are known l-dopa drugs, dopamine receptor agonists, anticholinergic drugs, and Eldepryl = depreyl. Most of these drugs are not causal treatment, And therefore requires continuous and continuous drug use. However, long-term administration of these drugs causes problems with drug side effects. For example, anticholinergic drugs may show autonomic nervous system abnormalities or mental dysfunctions, and thus are limited to continuous administration to older patients. In addition, in the case of the L-dopa formulation, the effect gradually decreases according to the long-term administration, and the side effects such as twisting the body and abnormal movements of the hands or feet are generated.

In order to prevent such side effects, efforts to develop a therapeutic agent for Parkinson's disease derived from natural products have been actively conducted. For example, Japanese Patent Application Laid-Open No. 2001-0081188 discloses a composition for preventing and treating neurological diseases such as Parkinson's disease, which includes the extract of Scutellariae Radix having a neuroprotective effect as an active ingredient, and brain diseases, including senile dementia. Patent Publication No. 2004-0012396 discloses a composition for treating degenerative brain diseases such as Parkinson's disease and stroke, which includes extracts of Beauveria Bassiana 101A, and Patent Publication No. 2004-0029072. A pharmaceutical composition for treating Parkinson's disease comprising an extract obtained from the guangguo (wenguanguo) peel is disclosed, and Patent Publication No. 2010-0060123 discloses a pharmaceutical for the prevention or treatment of Parkinson's disease, including ginger extract or shogaol The composition is disclosed, and Patent Publication No. 2010-0060949 discloses the prevention or prevention of diseases such as Parkinson's disease comprising peach leaf extract as an active ingredient And a composition for protecting nerve cells used in the treatment is disclosed, in Patent Publication No. 2011-0013466 discloses is a pharmaceutical composition for treating Parkinson's Disease disclosed comprising at least one compound derived from grape seed extract or an active ingredient therefrom. However, the components derived from these natural products have a disadvantage in that the treatment efficiency of Parkinson's disease is low, without side effects.

Thus, the present inventors, by extracting from the stem and root of the Daphne genkwa (from Daphne genkwa) is derived from natural products without any side effects, by activating Nurr1, thereby promoting the differentiation, growth and maintenance of dopaminergic neurons, and as a result It was confirmed that the therapeutic effect of various neurodegenerative diseases including Parkinson's disease caused by Nurr1 dysfunction was excellent. Furthermore, by analyzing the active ingredient of the sorbus extract, it was confirmed that Genkwanine N and Yuanhuacine compounds in the extract have an excellent effect on the Nurr1 protein activation, and completed the present invention.

One object of the present invention to provide a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases (neurodegenerative diseases) comprising Genkwanine N or Yuanhuacine, or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of diseases caused by Nurr1 dysfunction, including Genkwanine N or Yuanhuacine, or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention to provide a functional food additive for the prevention or improvement of neurodegenerative diseases comprising Genkwanine N or Yuanhuacine as an active ingredient.

Still another object of the present invention is to provide a Nurr1 activating composition comprising Genkwanine N or Yuanhuacine as an active ingredient.

As one aspect for achieving the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases (neurodegenerative diseases) comprising Genkwanine N or Yuanhuacine, or a pharmaceutically acceptable salt thereof as an active ingredient do.

Genkwanine N of the present invention is a terpenoid, specifically a diterpene ester compound represented by the following Formula 1, and may be prepared according to a method known to those skilled in the art. For example, Genkwanine N can be isolated and purified from plants known in the art using polar or nonpolar solvents. Alternatively, commercially available compounds may be purchased or synthesized. Preferably it can be extracted and separated from the red bean.

Yuanhuacine of the present invention is a terpenoid represented by Formula 2, specifically, a diterpene ester compound, and may be prepared according to a method known to those skilled in the art. For example, Yuanhuacine can be isolated and purified from plants known in the art using polar or nonpolar solvents. Alternatively, commercially available compounds may be purchased or synthesized. Preferably it can be extracted and separated from the red bean.

The term "Daphne genkwa" of the present invention refers to a deciduous shrub of the dicotyledon plant plated cedar. It is also known as early flowering tree and red bean tree, and it grows mainly near the beach. In Oriental medicine, it is used to treat symptoms such as diuresis, species, and nephritis.

As used herein, the term "Adzuki bean extract" refers to an extract obtained from adzuki bean, preferably an extract obtained from a stem or root of adzuki bean, and more preferably, a stem or root of adzuki bean. Means an extract obtained by extraction, specifically It means an extract obtained by extracting the stem or root of the red bean flower with C1 to C5 lower alkyl alcohol. As is well known, the sorbus extract is largely composed of five kinds of compounds of coumarins, flavonoids, lignans, terpenoids and other compounds. The number of compounds can reach several hundred [Xu, W.-C. meat al . , Chem . Biodiversity , 2011, 8: 1215-1233.

According to one embodiment of the present invention, the stem and / or root pulverized stalk of the red bean biloba is about 2 to 20 times the dry weight, preferably about 3 to 5 times the volume of water, methanol, ethanol, butanol and the like. Organic solvents such as C1 to C5 lower alkyl alcohols are used as a solvent, soaked for 3 to 5 hours, filtered to separate solids and primary liquid components, and the solids are immersed and filtered in the same manner to form secondary liquid components. Obtained and mixed. Thereafter, the mixture was concentrated under reduced pressure, and the residue was lyophilized to obtain an adzuki bean extract. The sorbus extract may include the Genkwanine N or Yuanhuacine compound.

According to another embodiment of the present invention, the red bean flower extract obtained is obtained by separating the fractionation layer of each solvent using a mixed solvent of an organic solvent such as hexane, ethyl acetate, butanol, or distilled water, The active ingredient can be separated and purified in high purity from each fraction using separation methods known in the art. The hexane fraction of the sorbus extract may include the Genkwanine N or Yuanhuacine compound of the present invention.

The inventors of the present invention are directed to extracts of various forms obtained from various natural products, in order to identify components derived from natural products which are excellent in preventing or treating various diseases including Parkinson's disease caused by Nurr1 dysfunction without any side effects. As a result of the study, it was confirmed that the organic solvent extracts of Daphne genkwa can increase Nurr1 activity (FIG. 1).

In addition, the present inventors confirmed that the red bean extract extract that increases the Nurr1 activity has an effect of preventing nerve cell damage caused by compounds known to induce neuronal cell death (eg, 6-OHDA) (FIG. 2). In addition, as a result of feeding a mixture of adzuki bean extract to an animal model that killed dopaminergic neurons in the rat brain, it was confirmed that the nerve function impaired by the killed dopaminergic neurons gradually improved (FIG. 3). ).

As such, the present inventors have found that a variety of diseases caused by damage to dopaminergic neurons that are directly affected by the activity of Nurr1 protein, such as Parkinson's and Parkinson's disease, by increasing the activity of Nurr1 protein by Azalea extract In addition, it was confirmed that the effect on the prevention or treatment of various diseases caused by dysfunction of Nurr1.

The present inventors analyzed the active fractions and active ingredients for activating the Nurr1 glycoprotein in the extract of Adzuki bean, and found that the hexane or ethyl acetate fraction of the Adzuki bean has an effect on the Nurr1 protein activation (Fig. 4), among which Genkwanine It was confirmed that the N and Yuanhuacine compounds showed an excellent effect on Nurr1 protein activation (FIG. 5). Nurr1 protein is involved in the establishment and maintenance of dopaminergic phenotypes in certain neuronal populations of the central nervous system, and it has been shown that decreased expression of Nurr1 is involved in neurodegenerative diseases including Parkinson's disease [Chu, Y. et. al ., J. Comp . Neurol ., 2006, 494 (3): 495-514], the hexane or ethyl acetate fractions of the zinnia and Genkwanine N or Yuanhuacine increase the Nurr1 protein activity, thereby directly affecting dopaminergic activity by Nurr1 protein activity. It can be seen that it is effective in preventing or treating various diseases caused by nerve cell damage, for example, various diseases caused by dysfunction of Nurr1 other than Parkinson's disease and Parkinson's disease.

Therefore, Genkwanine N and Yuanhuacine compounds of the present invention having an effect on the activation of Nurr1 can be usefully used for the prevention or treatment of neurodegenerative diseases including Parkinson's disease. As used herein, the term "neurodegenerative disease" refers to a disease associated with symptoms that occur when neurons degenerate, lose function, and die. It is mainly progressive and the consequences are very destructive and patients with the disease may experience severe degeneration in cognitive or motor skills. The neurodegenerative diseases are not particularly limited thereto, but Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and temporal lobe dementia (Fronto-Temporal Dementia), Cortico Basal Degeneration, and progressive supranuclear palsy (PSP).

The pharmaceutical composition of the present invention may be used in the form of Genkwanine N of Formula 1 or Yuanhuacine of Formula 2, or a pharmaceutically acceptable salt thereof. The term "pharmaceutically acceptable salts" of the present invention means all salts that retain the desired biological and / or physiological activity of the compound and exhibit undesired toxicological effects to a minimum. As salts are acid addition salts formed with pharmaceutically acceptable free acids. Acid addition salts are prepared by conventional methods, for example by dissolving a 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. Equal molar amounts of the compound and acid or alcohol (eg, glycol monomethyl ether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered. In this case, inorganic acids and organic acids may be used as the free acid, and hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, tartaric acid, and the like may be used as the inorganic acid, and methane sulfonic acid, p -toluene sulfonic acid, acetic acid, and triacid may be used as the organic acid. Fluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid (glycollic) acid), gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, etc. Can be, and not limited to these.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble compound salt, and evaporating and drying the filtrate. At this time, as the metal salt, it is preferable to produce sodium, potassium or calcium salt particularly, but not limited thereto. The corresponding silver salt can also be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

Pharmaceutically acceptable salts of Genkwanine N of Formula 1 or Yuanhuacine compound of Formula 2 include salts of acidic or basic groups which may be present in Genkwanine N or Yuanhuacine compound, unless otherwise indicated. For example, pharmaceutically acceptable salts may include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, Hydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p -toluenesulfonate (tosylate) salts; and the like through the methods for preparing salts known in the art. Can be prepared.

According to another embodiment of the present invention, the present invention comprises the Genkwanine N or Yuanhuacine, or a pharmaceutically acceptable salt thereof as an active ingredient for the prevention or treatment of neurodegenerative diseases comprising a pharmaceutically acceptable carrier To provide a composition.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate an organism and does not inhibit the biological activity and properties of the administered compound. Acceptable pharmaceutical carriers in compositions formulated as liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, bacteriostatic agents, etc. may be added as necessary.

According to another embodiment of the present invention, the present invention provides a method for preventing or treating a neurodegenerative disease, comprising administering the pharmaceutical composition to an individual in need thereof.

The term "individual" of the present invention refers to all animals including humans having or may develop a neurodegenerative disease, and can effectively prevent or treat the neurodegenerative disease by administering the pharmaceutical composition of the present invention to an individual. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent, or in combination with an existing neurodegenerative disease therapeutic agent, and may be administered sequentially or simultaneously with the conventional therapeutic agent.

The term "administration" of the present invention means introducing a predetermined substance into a patient in an appropriate manner and the route of administration of the composition can be administered via any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which solid preparations, in addition to the composition, include at least one excipient such as starch, calcium carbonate, sucrose, or lactose, Prepare by mixing gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups.In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives are available. May be included. However, upon oral administration, since the active ingredient of the sorbus extract derived from natural products may be lost due to digestion, it is preferable that the oral composition is formulated to coat or protect the active agent from degradation in the stomach. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, utopsol, macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In addition, the pharmaceutical composition of the present invention may be administered by any device capable of moving the active substance to the target cell. The preferred modes of administration and formulations are intravenous, subcutaneous, intradermal, intramuscular, and drip injections. Injections include non-aqueous solvents such as aqueous solvents such as physiological saline solution and ring gel solution, vegetable oils, higher fatty acid esters (e.g., oleic acid, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Stabilizers (e.g. ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, to prevent microbial growth Preservatives (eg, mercury nitrate, chimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) may be included.

The composition may be administered in single or multiple amounts in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to prevent or treat a disease at a reasonable benefit / risk ratio applicable to medical prophylaxis or treatment, and an effective dose level refers to the severity of the disease, the activity of the drug, Including the age, body weight, health, sex of the patient, sensitivity to the drug of the patient, time of administration of the composition of the invention used, route of administration and rate of release, duration of treatment, drugs used in combination or concurrently with the composition of the invention used Factors, and factors well known in the medical arts.

As another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of diseases caused by Nurr1 dysfunction, including Genkwanine N or Yuanhuacine, or a pharmaceutically acceptable salt thereof as an active ingredient.

"Nurr1 (nuclear receptor related 1)" in the present invention means a nuclear receptor related 1 protein, also known as nuclear receptor subfamily 4, group A, member 2 (NR4A2), which is known to be encoded by the human NR4A2 gene. . It is also known to be involved in many neurodegenerative diseases. Although the Nurr1 protein is an orphan nuclear receptor and a ligand for the protein has not yet been identified, the Nurr1 is a protein belonging to the nuclear receptor family of intracellular transcription factors. It has been found to play a key role in maintaining the dopaminergic system. Diseases caused by the Nurr1 dysfunction are not limited thereto, but Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), Rheumatoid arthritis, schizophrenia, and mood swings caused by dopamine dysfunction, including neurodegenerative disorders such as Fronto-Temporal Dementia, Cortico Basal Degeneration, and progressive supranuclear palsy (PSP) And a wide variety of inflammatory diseases.

As another aspect, the present invention provides a functional food additive exhibiting a prophylactic or ameliorating effect of neurodegenerative diseases comprising Genkwanine N or Yuanhuacine as an active ingredient.

Since the composition of the present invention has been confirmed that the stability as a compound isolated from a natural extract of the red bean flower, the common sense for a long time according to the usual eating habits, as well as preventing various neurodegenerative diseases including Parkinson's disease, the disease caused It may have an effect of improving symptoms.

In the present invention, the term "prevention" refers to any action of inhibiting or delaying the onset of neurodegenerative diseases by activating the Nurr1 protein using a composition comprising Genkwanine N of Formula 1 or Yuanhuacine compound of Formula 2 as an active ingredient.

In the present invention, the term "improvement" is to activate the Nurr1 protein using a composition comprising the Genkwanine N of Formula 1 or Yuanhuacine compound of Formula 2 as an active ingredient to improve or benefit the symptoms of suspected and onset of neurodegenerative diseases Say all the acts.

When the Genkwanine N or Yuanhuacine compound of the present invention is used as a food additive, the compound may be added to the food or beverage as it is or in combination with other food additives. When the food additive of the present invention is added at the time of the production of a food or beverage, the addition amount thereof is not particularly limited, but may be in the range of 1 to 5% by weight, preferably 1 to 3% by weight, Can be added. However, in the case of long-term intake for the purpose of health and hygiene or for health control, the added amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

In addition, the kind of the food or drink is not particularly limited, but includes all food or drink in a general sense, preferably meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, etc. Dairy products including noodles, gums, ice creams, various soups, beverages, teas, drinks, alcoholic beverages, vitamin complexes and the like.

At this time, when adding a food additive containing a compound isolated from the red bean extract of the present invention to food, various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids , Protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, may be added together with auxiliary components, such as alcohol, the content of the auxiliary component is not particularly limited, but 0.01 to 0.1 by weight of the final food It is preferably in weight percent.

In addition, when adding a food additive containing a compound isolated from the sorbus extract of the present invention to the beverage, it may include various sweeteners or natural carbohydrates, and the like as an additional component like a conventional beverage. At this time, the sweetener is not particularly limited, but natural sweeteners such as tautin, stevia extract, synthetic sweeteners such as saccharin, aspartame, and the like may be used, and the natural carbohydrate is not particularly limited thereto. For example, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), polysaccharides (eg, dextrin, cyclodextrin, etc.), or sugar alcohols (eg, xylitol, sorbitol, erythritol, etc.) ), And the content of the natural carbohydrate is not particularly limited, but is preferably included in a ratio of about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the final beverage product.

As another aspect, the present invention provides a Nurr1 activating composition comprising Genkwanine N or Yuanhuacine, or a pharmaceutically acceptable salt thereof as an active ingredient.

Nurr1 protein of the present invention is an orphan nuclear receptor (orphan nuclear receptor), the ligand for the protein has not yet been identified, but Nurr1 is a protein belonging to the nuclear receptor family of intracellular transcription factors, the dopamine system ( It has been found to play a key role in maintaining the dopaminergic system. In addition, it has been known that abnormalities of the Nurr1 protein may cause disorders of the dopamine system and cause neurodegenerative diseases. Nurr1 has been studied as a neurodegenerative protein target. Therefore, the Genkwanine N or Yuanhuacine compound of the present invention can be used for Nurr1 function research and the like, but is not limited thereto.

Compounds isolated from the red bean extract of the present invention are natural compounds derived from Parkinson's disease caused by inhibition of Nurr1 protein by exhibiting an excellent effect in restoring the inhibition of Nurr1 protein activity by neuronal damage without showing any side effects. And other neurodegenerative diseases can be prevented or treated.

Figure 1 is a graph showing the change of Nurr1 protein activity according to the concentration of methanol extract of Azalea through Luciferase analysis.
Figure 2 is a graph showing the inhibitory effect of the concentration of sorbus extract on neuronal cell death induced by 6-OHDA (6-hydroxydopamine).
Figure 3 is a graph showing the therapeutic effect of the bean extract methanol extract in Parkinson's animal model.
Figure 4 is a graph showing the Nurr1 protein activity change by the various solvent extracts of the red bean flower.
Figure 5 is a graph showing the increase in Nurr1 protein activity according to the concentration of Genkwanine N and Yuanhuacine compound isolated from the extract of the Red Beanstalk.

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

Example  One: Red bean flower  Preparation of extract

1,260.93 g of the stem and 1,487.17 g of the stem of the Daphne genkwa were cut into small pieces, immersed in 13 L methanol for 4 hours, and filtered to separate the solids and the first liquid component. The separated solid was again immersed in 13 L methanol for 4 hours and filtered to obtain a secondary liquid component. The obtained primary liquid component and the secondary liquid component were mixed, the mixture was concentrated under reduced pressure, and the residue was lyophilized to prepare 172.71 g of red bean extract.

Example  2: Red bean flower  Isolation of Active Ingredients from Various Extracts by Various Solvents

Methanol extract and the extract of the red bean root and stem obtained in Example 1 was concentrated under reduced pressure to add a variety of solvents in order to confirm the effect on the Nurr1 protein activity of the fraction for each solvent. At this time, the solvent used is hexane, ethyl acetate (EtO Ac), distilled water (dH ₂ O), and butanol (BuOH).

First, the dried red bean flower extract was dissolved in 1: 1 mixed solvent of distilled water and hexane and fractionated to obtain a hexane layer. 2 L of distilled water and a 1: 1 mixed solvent of ethyl acetate were added to the remaining distilled water layer to obtain an ethyl acetate layer, and a butanol layer and a distilled water layer were obtained in the same manner. Nurr1 activity experiments were performed on the fractions of the solvent layers obtained by the separation, and it was confirmed that the hexane and ethyl acetate fractions had high activity. Thus, in order to proceed with mass separation of the active fraction, similarly to the method of Example 1, 2.75 kg of red bean root and stem dry samples were repeatedly cooled and concentrated twice in 13 L of methanol solvent to obtain 172.71 g of methanol extract. Obtained. 30.1 g of this was suspended in distilled water and fractionated in the order of hexane and ethyl acetate. The hexane fraction (3 g) identified as the higher activity of the two fractions was a gradient mixed solvent of hexane and ethyl acetate using silica gel column chromatography (10: 1, 5: 1, 2: 1, 1). : 1, 1: 2, 1: 5, 1:10) to give a total of 50 fractions. Among them, Nurr1 activity was observed in 32 to 38 fractions (268 mg) eluted at 1: 2 solvent condition of hexane: ethyl acetate. Therefore, the 32 to 38 fractions were collected, concentrated under reduced pressure, and subjected to reverse phase silica gel prep TLC under 85% acetonitrile to obtain a total of 11 bands. Among these, Nurr1 activation was observed in band 6 (Rf = 0.44) and band 8 (Rf = 0.33). The active material was extracted from the two bands with methanol and concentrated under reduced pressure to obtain two compounds, B6 and B8, respectively. Finally, ODS HPLC was performed using 80% acetonitrile (0.025% trifluoroacetic acid; TFA) as an eluent, and two active ingredients, genkwanine N and Yuanhuacine was successfully isolated as a single substance.

Example  3: Nurr1  For protein activity Red bean flower  Effect of extract

After combining the gene with which the GLA4 gene can bind (5'-CTCGGAGGACAGTACTCCG-3, SEQ ID NO: 1) to the reporter gene luciferase, a vector was synthesized, and DNA and beta containing Nurr1-LBD were beta. Six hours after transfection of three kinds of plasmid DNA such as DNA having -galactosidase (β-galactosidase) into BE (2) C cells, the sorbus extract was treated at a concentration of 1 to 200 ppm. . Cells thus treated were incubated in a 5% carbon dioxide incubator at 37 ° C. for 20 hours, followed by luciferase analysis (FIG. 1).

Figure 1 is a graph showing the change of Nurr1 protein activity according to the concentration of methanol extract of Azalea through Luciferase analysis. 0.1% DMSO was used as a control, and the activity was increased in multiples of the activity at this time. When the sorbus extract was treated with a concentration of 20 ppm, the maximum activity was shown, and the value reached about three times that of the control group. As shown in Figure 1, it was confirmed that the activity of Nurr1 protein by the sorbus extract in BE (2) C cells, which are human-derived neuroblasts (neuroblast).

Example  4: SH - SY5Y  For cells Red bean flower  Effect of extract

Human-derived neuroblastoma SH-SY5Y cells were dispensed in 24-well plates at a cell concentration of 5 × 10 ⁵ cells / well and incubated in a carbon dioxide incubator for 24 hours. The cultured cells were treated with 6-OHDA (6-hydroxydopamine) (50 μM) which induces apoptosis alone or with different concentrations (5-20 ppm) of S. aureus extracts, and again at 37 ° C. for 24 hours. , 5% carbon dioxide incubator. After 24 hours, MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) solution was treated in each well at a concentration of 1 mg / ml, followed by a 5% carbon dioxide incubator for 1 hour. After storage in MTT extraction solution (extraction solution) was added. Then stored at 37 ℃ for 24 hours, the absorbance was measured at 570 nm (Fig. 2).

Figure 2 is a graph showing the effect of the concentration of sorbus extract on neuronal cell death induced by 6-OHDA. As shown in Figure 2, 6-OHDA-induced neuronal cell death was inhibited by the sorbus extract so that the cell viability of the sorbus extract treatment group recovered to 70% or more of the control group.

Example  5: damaging nerve cells In rats  About Red bean flower  Effect of extract

In order to specifically kill dopaminergic neurons in the melanoma of the midbrain of 6-week-old SD rats (Cortech Co., Ltd.), AP (-4.3), ML ( -1.8), 4 μg of 6-OHDA (1 μg / μl) at DV (-8.2), AP (-5.0), ML (-1.8) and DV (-8.2) at 1 μl / min. Direct injection At this time, by administering desipramine at a dose of 25 mg / kg 30 minutes before the 6-OHDA administration, cell death other than dopaminergic neurons was inhibited.

Thereafter, the rats were fed with the feed mixed with the extract for 2 weeks so that the daily dose of the red bean flower extract became 500 mg / kg. At this time, as a control group, rats to which the extract had not been mixed were used.

A stepping test was performed, which is a non-pharmacological test method to reinforce the error of the pharmacological test. Specifically, while controlling the three legs of the rat by hand on the table 90 cm in length for 15 seconds while moving on the table surface, the number of uncontrolled legs hit the table surface was repeated three times for each leg. The measurement time point of the step test was 3 days before 6-OHDA injection, 2 weeks, 4 weeks and 6 weeks after the 6-OHDA injection (Fig. 3).

Figure 3 is a graph showing the results of confirming the effect of the Red Bean Extract on the rats in which dopaminergic neurons were killed through a step test. As shown in FIG. 3, the rats to which the sorbus extract was administered were found to increase the number of steps compared to the rats not administered.

Example  6: by various solvent Red bean flower  Effect of extract

The effect on the Nurr1 protein activity of the fraction of the red bean flower extract obtained using the various solvents in Example 2 was confirmed according to a method similar to that described in Example 3. Hexane (hexane), ethyl acetate (EtOAc), distilled water (dH ₂ O), and butanol (BuOH) were used as the solvent.

As shown in FIG. 4, a strong Nurr1 protein activation effect was observed in the hexane fraction, and the 1 ppm hexane fraction increased the activity of the Nurr1 protein by 380%.

Example  7: Effects of Genkwanine N and Yuanhuacine Active Ingredients

In a similar manner to Example 3, the concentration dependence of Genkwanine N and Yuanhuaicine effects on the activity of Nurr1 protein was tested and the results are shown in FIG. 5.

As shown in FIG. 5, Genkwanine N and Yuanhuaicine showed more than 300% activity at both compounds at 0.3 ppm, especially at low nanomolar levels of 16 nM and 15 nM for 0.01 ppm, respectively. The activity was improved by 60 and 70%, respectively.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Pharmaceutical composition for preventing or treating          neurodegenerative diseases comprising compounds isolated from          Daphne genkwa extract <130> PA110885 / KR <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GAL4 gene <400> 1 ctcggaggac agtactccg 19

Claims (12)

Prevention of neurodegenerative diseases comprising a terpenoid compound selected from the group consisting of Genkwanine N and Yuanhuacine, or a pharmaceutically acceptable salt thereof as an active ingredient Or therapeutic pharmaceutical compositions.
The method of claim 1,
The neurodegenerative diseases include Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and frontal lobe dementia (Fronto-Temporal Dementia). Pharmaceutical composition, which is selected from the group consisting of Cortico Basal Degeneration, and progressive supranuclear palsy (PSP).
The method of claim 1,
The neurodegenerative disease is Parkinson's disease (Parkinson's disease; PD) pharmaceutical composition.
The method of claim 1,
The genkwanine N (Genkwanine N) or yuanhuacine (Yuanhuacine) is a pharmaceutical composition that is a compound isolated from the extract of the bean.
The method of claim 1,
A pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
Of diseases caused by Nurr1 dysfunction comprising a terpenoid compound selected from the group consisting of Genkwanine N and Yuanhuacine, or a pharmaceutically acceptable salt thereof as an active ingredient Prophylactic or therapeutic pharmaceutical compositions.
The method according to claim 6,
The genkwanine N (Genkwanine N) or yuanhuacine (Yuanhuacine) is a pharmaceutical composition that is a compound isolated from the extract of the bean.
The method according to claim 6,
Diseases caused by Nurr1 dysfunction include Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and temporal lobe dementia ( Any one selected from the group consisting of Fronto-Temporal Dementia, Cortico Basal Degeneration, progressive supranuclear palsy (PSP), rheumatoid arthritis, schizophrenia and manic depression Pharmaceutical composition.
Functional food additive for the prevention or improvement of neurodegenerative diseases comprising a terpenoid compound selected from the group consisting of Genkwanine N and Yuanhuacine as an active ingredient.
10. The method of claim 9,
The genkwanine N (Genkwanine N) or yuanhuacine (Yuanhuacine) is a functional food additive that is a compound isolated from the extract of the bean.
10. The method of claim 9,
The neurodegenerative diseases include Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and frontal lobe dementia (Fronto-Temporal Dementia). A functional food additive, which is any one selected from the group consisting of Cortico Basal Degeneration, and Progressive supranuclear palsy (PSP).
Nurr1 activating composition comprising a terpenoid compound selected from the group consisting of Genkwanine N and Yuanhuacine as an active ingredient.

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