KR101583755B1 - Composition for preventing and treating of nervous system disease containing beta-lapachone as an effective ingredient - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of neurological diseases, which comprises beta-rapacon as an active ingredient.
The beta-rapacon according to the present invention is useful for preventing neuronal cell proliferation and regulating the TLR4 signaling pathway activated by lipopolysaccharide to significantly inhibit neuronal apoptosis. Thus, Alzheimer's disease, Prion disease, Lou Gehrig's disease, Parkinson's disease, And can be useful as a preventive, ameliorating or therapeutic agent for neurological diseases such as stroke and multiple sclerosis.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition for preventing and treating neurological diseases comprising beta-rapacon as an active ingredient,

The present invention relates to a composition for the prevention and treatment of neurological diseases, which comprises beta-raffone as an active ingredient.

Inflammatory reactions are caused by various inflammatory mediators and immune cells in the local blood vessels and body fluids when they are infected with tissue (cell) damage or external infectious agents (bacteria, fungi, viruses, various kinds of allergens) It exhibits a series of complex physiological responses such as substance secretion, fluid infiltration, cell migration, and tissue destruction, and external symptoms such as erythema, edema, fever, and pain. In normal cases, the inflammatory reaction removes the external infectious agent and regenerates the damaged tissue to regenerate the organism's function. However, if the antigen is not removed or the internal substance causes the inflammatory reaction to occur excessively or continuously, Some lead to diseases such as cancer.

Recently, it has been found that inflammatory reaction is one of the main mechanism causing neurodegeneration. In other words, the immature cells of the central nervous system, the small glial ganglion cells can be activated by various exogenous and endogenous substances, and the activated small glial cells can be activated by the inflammatory cytokines TNF-α and IL-1β, And the like. The production of these substances induces an immune response in the short term, but excessive production or sustained production induces neuronal death of adjacent neurons, resulting in neural degeneration. In addition, since the substances released by the nerve cells that are killing lead to activation of the cervical ganglion cells, the neural degeneration falls into a constant vicious cycle. Actually, it has been reported that the activity of small glial cells is related to various degenerative neurological diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfelt-Jakob Disease (CJD) and multiple sclerosis.

Neurons exposed to endotoxin, such as lipopolysaccharide (LPS), express neurotoxicity mediated by the immune response by expressing iNOS (inducible nitric oxide synthase), which produces nitrogen monoxide (Cooper CL, Jeohn GH, Tobias P, Hong JS Serum-dependence of LPS-induced neurotoxicity in rat cortical neurons.Ann NY Acad Sci. 2002 May; 962: 306-17). Indolecarbazole compounds, known as PKC antagonists, are known to play a role in restoring damaged neurons by lipopolysaccharide and interferon-gamma (Molecular Brain Research, (2000) 79 (1-2) 3244). The neuroprotective function corresponds to the inhibition of the expression of iNOS and the expression of nitrogen monoxide.

In the case of lipopolysaccharide, it is an agonist of TLR4 which is one of Toll like receptors (TLRs) which is a pattern recognition receptor existing in neurons and nervous system. Toll-like receptors are widely expressed in the nervous system and nerve cells (Brain Res Rev. 2009. 59 (2): 278-292).

The TLRs signaling pathway in the immune cells of the nervous system is known to cause neurogenic diseases by causing degeneration of nerve cells through nitric oxide and inflammation and inflammatory cytokine expression (K. Takeda and S. Akira, " Toll -like receptors, " Current Protocols in Immunology, chapter 14, p. Unit 14.12, 2007, S. Lehnard, C. Lachance, S. Patrizi et al., " The Toll-like receptor TLR4 is necessary for lipopolysaccharide-induced oligodendrocyte injury in the CNS, "Journal of Neuroscience, vol. 22, no. 7, pp. 2478-2486, 2002, S. Lehnardt, L. Massillon, P. Follett et al.," Activation of innate immunity in the CNS triggers neurodegeneration 100, No. 14, pp. 8514-8519, 2003, O. Hoffmann, JS Braun, D, " Proceedings of the National Academy of Sciences, Becker et al., &Quot; TLR2 mediates neuroinflammation and neuronal damage, " Journal of Immunology, vol. 17 8, No. 10, pp. 6476-6481, 2007. S. Lehnardt, P. Henneke, E. Lien et al., &Quot; A mechanism for neurodegeneration induced by group B Streptococci through activation of the TLR2 / MyD88 pathway in microGlia, &Quot; Journal of Immunology, vol. 177, no. 1, pp. 583-592, 2006, A. Bal-Price and G. C. Brown, "Inflammatory neurodegeneration mediated by nitric oxide from activated Gliainhibiting neuronal respiration, causing glutamate release and excitotoxicity," Journal of Neuroscience, vol. 21, no. 17, pp. 6480-6491, 2001.)

TLRs are closely related to the development of various neurodegenerative diseases such as Alzheimer's disease, prion disease, Amyotrophic Lateral Sclerosis, Parkinson's disease, ischemic stroke, multiple sclerosis Brain Res Rev. 2009. 59 (2): 278-292 / Dorit Trudler, Dorit Farfara, and Dan Frenkel, Toll-Like Receptors Expression and Signaling in Glia Cells in Neuro-Amyloidogenic Diseases: Towards Future Therapeutic Application. Mediators of InflammationVolume 2010 (2010) Article ID 497987, 12 pages)

Thus, a variety of materials have been studied for the treatment and prevention of neurological diseases associated with the TLRs signaling pathway. For example, Korean Patent Laid-Open Publication No. 2012-0051621 effectively inhibits the activity of small glial cells associated with neuroinflammation by reducing gene expression in the TLR4 signaling pathway, which is a representative signal transduction pathway activated by lipopolysaccharide (LPS) The present invention provides pyridylimidazole derivatives useful as preventive or therapeutic agents for neuroinflammatory diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt Jakob disease, multiple sclerosis and the like.

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On the other hand, beta-lapachol is a natural quinone substance obtained from lapacho tree (Tabebuia avellandedae), which is extracted from various plants.

Previous studies have shown that beta-raffonin has anticancer effects [Terai, Kaoru et al ., Cisplatin enhances the anticancer effect of beta-lapachone by upregulating NQO1. , Anticancer Drugs , Nov, 01; 20 (10): 901-9 (2009); United States Patent Nos. 7,074,824, 6,962,944, 6,664,288, 7,361,691, WO 2003/11224], antitumor effect [HR Shah et al ., Beta - lapachone inhibits proliferation and induces apoptosis in retinoblastoma cell linesBeta - lapachone in retinoblastoma cell lines , Eye 22, 454-460 (March 2008); U.S. Patent Application Publication Nos. 2009/0226395 and 7,070,797], anti-inflammatory effects [Dong-Oh Moon et al ., Anti-inflammatory effects of β- lapachone in lipopolysaccharide - stimulated BV2 microglia , International Immunopharmacology , Vol 7, Issue 4, 506-514, (April 2007)], antifungal effect [Shing-Hwa Liu et al ., Inhibition of inducible nitric oxide synthase by β- lapachone in rat alveolar macrophages and aorta , Br J Pharmacol . 126 (3): 746-50. (February 1999)].

Korean Patent Publication No. 2012-0051621 U.S. Patent No. 7,074,824 U.S. Patent No. 6,962,944 U.S. Patent No. 6,664,288 U.S. Patent No. 7,361,691 International Patent Publication No. WO 2003/11224

Cooper CL, Jeohn GH, Tobias P, Hong JS Serum-dependence of LPS-induced neurotoxicity in rat cortical neurons. Ann N Y Acad Sci. 2002 May; 962: 306-17 Brain Res Rev. 2009. 59 (2): 278-292 Dorit Trudler, Dorit Farfara, and Dan Frenkel, Toll-Like Receptors Expression and Signaling in Glia Cells in Neuro-Amyloidogenic Diseases Towards Future Therapeutic Application. Mediators of Inflammation Volume 2010 (2010) Article ID 497987, 12 pages

The inventors of the present invention discovered that beta-lapachone enhances cell proliferation of neuronal cells and has a restoration effect on endotoxin-induced apoptosis, and completed the present invention.

The object of the present invention is to provide a composition for helping the proliferation of neurons and inhibiting lipopolysaccharide-induced neuronal cell death and for improving, preventing, and treating neurological diseases having neuronal cell protection effect.

To this end, the present invention provides a pharmaceutical composition for the prevention and treatment of neurological diseases, which comprises, as an active ingredient, beta-raffone or a pharmaceutically acceptable salt thereof represented by the following general formula (1).

[Chemical Formula 1]

The present invention also provides a health food composition for preventing or ameliorating a neurological disease, which comprises the beta-rapacon represented by the above formula (1) as an active ingredient.

The beta-rapacon according to the present invention is useful for preventing neuronal cell proliferation and regulating the TLR4 signaling pathway activated by lipopolysaccharide to significantly inhibit neuronal apoptosis. Thus, Alzheimer's disease, Prion disease, Lou Gehrig's disease, Parkinson's disease, And can be useful as a preventive, ameliorating or therapeutic agent for neurological diseases such as stroke and multiple sclerosis.

FIG. 1 is a graph showing the results of measuring the survival rate of neuroblast cells according to beta-rapacon treatment concentration in order to confirm neuronal proliferative activity of beta-raffone.
FIG. 2 is a graph showing the results of measuring the survival rate of neuroblast cells according to beta-raffonin treatment concentration in order to confirm the inhibitory effect of beta-rapacon on lipopolysaccharide-induced neuronal cell death.

The beta-raffone conferred by the present invention is 3,4-dihydro-2,2-dimethyl-2H-naphtho (1,2-b) pyran-5,6- 4-Dihydro-2,2-dimethyl-2H-naphtho (1,2-b) pyran-5,6-

[Chemical Formula 1]

The beta-rapacon is a known substance having a CAS No. 1707-32-8, which can be extracted from natural plants or purchased commercially.

Beta extracted from natural plant-Rapa cone bignoniaceae (Bignoniaceae), verbenaceae (Verbenaceae), proteaceae (Proteaceae), leguminous (Lequminosae), sandpaper tagwa (Sapotaceae), Scrophulariaceae (Scrophulariaceae), Malvaceae (Malvaceae), etc. Of the plant.

For example, it can be produced by a conventional method known in the art, that is, a conventional method such as hot water extraction, room temperature extraction, warming extraction, ultrasonic extraction, supercritical extraction and the like using a conventional solvent.

Is preferably extracted with a solvent selected from the group consisting of water, lower glycols having 1 to 6 carbon atoms, lower alcohols having 1 to 4 carbon atoms, and mixtures thereof. Most preferably, 1,3-butylene glycol is used as an extraction solvent and extracted with a sonicator at room temperature for 1 to 12 hours. The time of the ultrasonic extraction may be varied depending on the amount of the sample to be extracted, and the result of the extraction may be filtered or purified.

In the case of commercially available beta-rapacon, it is used after refining. The purification method used at this time can be a method commonly used in this field. Liquid chromatography using various organic solvents, column chromatography using various synthetic resins such as silica gel and activated alumina, and high performance liquid chromatography (HPLC), and the method is not limited to the above purification method .

Experimental Example 1 according to the present invention was conducted to examine the effect of beta-raffonin on neuronal cell proliferation, and it was confirmed that neuronal cell protection effect was enhanced by nerve cell proliferation.

Also, in Experimental Example 2 according to the present invention, beta-raffonne increased neuronal cell survival by about 10 to 20% in neuronal cell death induced by lipopolysaccharide treatment with neurotoxicity, Respectively.

These results suggest that the beta-raffone conferred by the present invention can be used for the treatment of neuronal diseases such as Alzheimer's, prion diseases, Lou Gehrig's disease, Parkinson's disease, ischemic stroke and multiple sclerosis associated with regulation of TLR4 signaling pathway activated by lipopolysaccharide Can be used for the improvement, treatment and prevention of diseases.

The beta-raffone according to the present invention can be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Propyl sulphonate, naphthalene-1-yne, xylenesulfonate, phenylsulfate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, Sulfonate, naphthalene-2-sulfonate or mandelate.

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 an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

In addition, the beta-rapacon according to the present invention may include not only pharmaceutically acceptable salts, but also possible solvates, hydrates and the like which can be prepared therefrom.

The pharmaceutical composition of the present invention can be formulated by a known method in the pharmaceutical field and can be mixed with a pharmaceutically acceptable carrier, excipient or the like to prepare a conventional pharmaceutical preparation such as a solution, a syrup, a capsule, a granule, , Ointments, emulsions, gels, creams, and the like, and they can be administered by various routes.

The pharmaceutical composition of the present invention contains 0.00001 to 10.0% by weight of the above-mentioned beta-rapacon in the entire pharmaceutical composition. If the content of the compound is less than the above range, a significant effect can not be expected. On the other hand, if the content exceeds the above range, the effect is not increased and an amount of cytotoxicity may be slightly increased. The pharmaceutical composition thus prepared is used at an effective dose of 0.00001 mg / kg to 400 mg / kg per dose, and the actual dose of the active ingredient is determined according to the severity of symptoms, the route of administration selected, the age, sex, Should be determined in light of the various relevant factors of the disease. Accordingly, the above dose is not intended to limit the scope of the present invention in any way. The administration can be administered once to several times a day.

In addition, the present invention provides a health food composition for preventing and ameliorating a neurological disorder containing beta-rapacon as an active ingredient.

 Preferably, the neurological disease is any one selected from the group consisting of Alzheimer's disease, Prion disease, Lou Gehrig's disease, Parkinson's disease, ischemic stroke, and multiple sclerosis.

Foods to which the beta-rapacon of the present invention can be added include, for example, various foods, beverages, gums, tea, vitamin complexes, and health functional foods.

It can also be added to foods or beverages for the purpose of preventing neuronal cell protection and degenerative brain diseases. At this time, the amount of the compound in the food or drink may be 0.01 to 15% by weight of the total food, and the health beverage composition may be added in a proportion of 0.02 to 5 g, preferably 0.3 to 1 g, have.

The health functional beverage composition of the present invention is not particularly limited to other ingredients other than the above-mentioned beta-rapacon as an essential ingredient in the indicated ratios and may contain various flavors or natural carbohydrates as an additional ingredient . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

Hereinafter, preferred embodiments of the present invention will be described. The following examples are provided for the purpose of more clearly illustrating the present invention and the present invention is not limited to the following examples.

Example  1: Beta- Rafacon  Identification of neuronal proliferation activity

The beta-rapacon was a known substance having a CAS No. 1707-32-8, which was extracted from natural plants or purchased commercially.

In order to confirm the neuronal cell protection activity of beta-rapacon, an in vitro assay system using neuroblastoma cell line SH-SY5Y cells was established and cell viability was measured.

The SH-SY5Y cell line was added to MEM medium supplemented with 10% fetal calf serum, 100 U / ml penicillin and 100 쨉 g / ml streptomycin, and incubated at 37 ° C in 5% CO ₂ Lt; / RTI > The SH-SY5Y cells cultured as described above were inoculated in a 96-well dish at ⁵ × 10 ⁴ cells / well, and samples containing 0 ug / ml, 0.0625 ug / ml, 0.1 ug / ml and 0.25 ug / ml of beta- Lt; / RTI > To measure formazan formation, 20ul of 5 mg / ml 3- (4,5-dimethylthiazoyl-2-yl) -2,5-diphenyltetrazolium bromide (MTT) solution was added and reacted for 4 minutes. Respectively. The resulting formazan was dissolved in DMSO to measure A ₅₄₀ , and the survival rate was measured. The results are shown in FIG.

Referring to FIG. 1, it was confirmed that when 0.1 ug / mml and 0.25 ug / ml beta- raffoncon were applied, they exhibited the highest neuronal cell protection activity as compared to the beta-rafficon untreated group.

Example  2: SH - SY5Y In neuronal cell lines  beta- Rafacon LPS  Treatment-induced inhibition of cell death

In order to confirm neuronal cell protection activity of beta-rapacon, SH-SY5Y cell line, neuroblastoma cell line, was used to induce apoptosis using LPS. To confirm the effect of beta-rapacon on apoptosis inhibition, Respectively.

The SH-SY5Y cell line was added to MEM medium supplemented with 10% fetal calf serum, 100 U / ml penicillin and 100 쨉 g / ml streptomycin, and incubated at 37 ° C in 5% CO ₂ Lt; / RTI > The SH-SY5Y cells cultured in the above manner were inoculated to a 96-well dish at ⁵ × 10 ⁴ cells / well, treated with 1 μg / ml of 1 μg / ml of lipopolysaccharide (LPS) and then treated with 0 μg / ml, 0.0625 ug / ml, 0.1 ug / ml and 0.25 ug / ml were treated for 48 hours. To measure formazan formation, 20 μl of 5 mg / ml 3- (4,5-dimethylthiazoyl-2-yl) -2,5-diphenyltetrazolium bromide (MTT) solution was added and reacted for 4 minutes. . The resulting formazan was dissolved in DMSO to measure A ₅₄₀ , and the survival rate was measured. The results are shown in FIG.

As shown in Fig. 2, when LPS alone was treated, the cell viability of SH-SY5Y was found to be 65% lower than that of the untreated group. When beta-rapacon was treated after pretreatment with LPS, it was increased by about 82% to about 17%.

The preparation examples for the composition of the present invention are illustrated below.

Manufacturing example  1: Preparation of pharmaceutical preparations

1-1. Manufacture of Powder

After mixing the components shown in Table 1, tablets were prepared by tableting according to a conventional method for producing tablets.

Beta-Lapcon  2 g Lactose 1 g

 1-2. Manufacture of tablets

The ingredients in Table 2 below were mixed and filled in airtight bags to prepare powders.

Beta-Lapcon 100 mg Corn starch 100 mg Lactose 100 mg Magnesite stearate 2 mg

1-3. Preparation of capsules

The components shown in Table 3 were mixed and filled in gelatin capsules according to the conventional preparation method of capsules to prepare capsules.

Beta-Lapcon 100 mg Corn starch 100 mg Lactose 100 mg Magnesium stearate 2 mg

1-4. Injection preparation

According to the conventional method for preparing injections, injectable preparations were prepared by incorporating the ingredients shown in Table 4 below in the prescribed amounts.

Beta-Lapcon 100 mg Mannitol 180 mg Na2 _H and PO ₄ 2H ₂ O 26 mg Distilled water 2974 mg

Production Example 2: Production of Health Functional Foods

2-1. Manufacturing of beverages

The beverage was prepared using the conventional method according to the composition and content shown in Table 5 below.

honey 522 mg Nicotinic acid amide 10 mg Sodium riboflavin hydrochloride 10 mg Pyridoxine hydrochloride 2 mg Inositol 30 mg Ortho acid 50 mg Beta-Lapcon 0.48-1.28 mg water 200 ml

2-2. Manufacture of chewing gum

Chewing gum was prepared using the conventional method according to the composition and content of the following Table 6 (content: wt%).

Gum base One% Sugar 76.36-76.76% Beta-Lapcon 0.24-0.64% Fruits One% water 2%

2-3. Manufacture of candy

The candies were prepared using the conventional method according to the composition and content of the following Table 7 (content:% by weight).

Sugar 50-60% corn syrup 39.26-49.66% Beta-Lapcon 0.24-0.64% Orange incense 0.1%

Claims (7)

A pharmaceutical composition for the prevention and treatment of Lou Gehrig's disease comprising, as an active ingredient, beta-raffone or a pharmaceutically acceptable salt thereof represented by the following formula (1)
[Chemical Formula 1]

The composition of claim 1, wherein the composition is beta-rapaconic
In a content of 0.00001 to 10.0% by weight. delete delete A health food composition for preventing and ameliorating Lou Gehrig's disease comprising beta-rapacon represented by the following formula (1) as an active ingredient:
[Chemical Formula 1]

6. The composition of claim 5, wherein the composition comprises beta-
0.00001 to 10.0% by weight, based on the total weight of the composition. delete

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