KR20160081588A

KR20160081588A - COMPOSITION COMPRISING β-LAPACHONE OR PHARMACEUTICALLY ACCEPTABLE SALTS THEREOF AS AN ACTIVE INGREDIENT FOR PROTECTION OF BRAIN DAMAGE OR PREVENTION AND TREATMENT OF NEURODEGENERATIVE DISEASES

Info

Publication number: KR20160081588A
Application number: KR1020140195620A
Authority: KR
Inventors: 김희선; 박진선
Original assignee: 이화여자대학교 산학협력단
Priority date: 2014-12-31
Filing date: 2014-12-31
Publication date: 2016-07-08

Abstract

The present invention relates to a composition for preventing and treating brain damages or neurodegenerative diseases containing beta-lapachone or pharmaceutically acceptable salt thereof as an active component. The beta-lapachone or pharmaceutically acceptable salt can be usefully used as a composition for preventing and treating brain damages or neurodegenerative diseases by restraining active oxygen species generation in a cell when an oxidation stress is induced by using oxygenated water after pre-treating the beta-lapachone, increasing antioxidant enzyme expression functioning against the oxidation stress by activating an AMPK and an Akt singal transfer enzyme, and verifying an increase in a protection effect of an astrocyte on the oxidation stress thereby.

Description

The present invention relates to a composition for preventing or treating brain damage or degenerative brain diseases containing beta-raffone or a pharmaceutically acceptable salt thereof as an active ingredient. prevention and treatment of neurodegenerative diseases.

The present invention relates to a pharmaceutical composition for preventing or treating brain damage or degenerative brain diseases containing beta -lactone or a pharmaceutically acceptable salt thereof as an active ingredient.

The brain is rich in polyunsaturated fatty acids and metal ions that are highly saturated with oxygen and are a direct target for oxidative stress, while brain signaling substances (auto-oxidation) Which is very vulnerable to oxidative stress and has limited antioxidant and repair ability against oxidative stress.

Astrocyte is a cell in the brain that plays a role in supplying neurons with nutrients, which is closely related to the survival of neurons. Recently, however, the active role of astrocytes has been elucidated. Astrocytes have been shown to exert various functions ranging from neuronal synaptogenesis, synapse number, synapse function, and synaptic plasticity (Ullian et al., 2001; Hay, 2001; Horner and Palmer, 2003; Nedergaard et al., 2003). In addition, astrocytes play a crucial role in the differentiation of neural stem cells into neurons (Sevendsen, 2002; Song et al., 2002).

When injured in the brain, astrocytes undergo astrocytosis, swelling, and reactive astrocytes, such as astrogliosis (hypertrophy-hyperplasia) ). These reactive astrocytes are observed in AIDS dementia, brain damage, ischemic brain disease, Alzheimer's disease, and the like. In the early stages of brain injury, changes such as reactive astrocytic cells have a beneficial effect on nerve cells. However, persistent reactive astrocytes may inhibit neuronal regeneration or release toxic substances, leading to degenerative brain disease. Sustained reactive astrocytes induce apoptosis while inducing the death of surrounding astrocytes or neurons.

Oxidative stress is known to be associated with ischemic or degenerative brain disease. Oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS) and antioxidative capacity, and is caused by amyloid beta-peptide, cytokine, excitotoxic amino , nitric oxide (NO), and mitochondria (electron transport).

Degenerative brain disease refers to diseases that occur in the brain among degenerative diseases caused by age, and it can be classified according to the main symptom and the affected brain area. Downs syndrome, alzheimer's disease, Parkinson ' s disease, Huntington ' s disease, and the like. Degenerative brain diseases are known to cause neuronal cell death due to aggravation of neurodegeneration and genetic and environmental factors caused by aging. However, the precise cause of degenerative brain disease has not been clarified yet. .

The development of degenerative brain disease treatment is the most urgent task in Korea where aging is rapidly progressing. In 2026, it is predicted that over 20% will reach the aged society, and degenerative brain disease problem is expected to rise not only in Korea but also globally. The number of patients with dementia in Korea is estimated to be 750,000 in 2020 from 470,000 in 2010, and cerebrovascular disease remains the second leading cause of death in Korea over the past decade.

Parkinson's disease is a chronic disease that is caused by deficiency of neurotransmitters such as dopamine in the brain due to tremor, stiffness, slowed action and postural abnormality. Dopamine is produced in neurons called Substantia nigra pars compacta (SNpc), and the neurons in the black are complexly connected to the motor cortex and other parts of the brain so that the movement of the human body can be performed smoothly and in a harmonious and accurate manner. It is connected to the base called the basal gang. Parkinson's disease is caused by lack of dopamine, which is secreted to control basal ganglia function in the substantia nigra.

Symptoms of Parkinson's disease can be classified into primary symptoms and secondary symptoms. Primary symptoms include stiffness, tremors, slow or diminished body movements, and symptoms such as body imbalance and gait disorder, direct symptoms And secondary symptoms refer to symptoms that are caused by primary neurodegeneration or by other neurodegenerative conditions.

Currently, therapies used for the treatment of Parkinson's disease include drug therapy, surgical treatment and physical therapy. Drug therapy usually replenishes the deficient dopamine in the brain, and imbalances in neurotransmitters due to dopamine deficiency Drugs for the purpose of preventing or delaying the destruction of nerve cells and for controlling other symptoms such as depression have been used. For example, amantadine, anticholinergic drugs, el-dopa, cinemet, An agonist, an elderpyril, and an antidepressant. However, since these drugs can not revive dead neurons, they are not intended to cure but are intended to control symptoms.

Alzheimer's disease is a degenerative brain disease, in which mental function declines gradually as brain tissue loses function in the process of aging. The hallmark of this disease is that it causes serious obstacles to memory and emotion. In modern medicine, it is recognized as an 'incurable disease' with no clear treatment. It is one of the main causes of dementia which is mainly present in the elderly. Histopathological features include general atrophy of the brain, enlargement of the ventricles, multiple lesions of the nerve fibers and hyperchromatic spots. Clinical features include gradual decline of intellectual functions such as memory, judgment, and language ability, daily living skills, sight, and disability of behavior patterns. In addition, psychiatric symptoms such as depression, devastation of personality, and aggressive behavior are accompanied. These symptoms progress gradually and eventually lead to death. The period from the onset to the gradual death is about 6 to 8 years, but sometimes it is over 20 years depending on the person. In the United States, about 3 percent of the population aged 65 to 74, about 19 percent of the population aged 75 to 84, and 50 percent of the population aged 85 or older are suffering from the disease. Alzheimer's disease is caused by the damage of normal cells, acetylcholine (neurotransmitter) is reduced, memory, language function, judgment is lost, the personality is changed, and eventually the ability to take care of itself is lost.

Acetylcholine is an ester of choline and acetic acid. Numerous nervous systems or synapses and skeletal muscles transmit shock from the motor nerve endings. When nerve impulses reach the nerve endings, the acetylcholine stored in the synaptic pouch is released, and the permeability of the membrane is changed and the generator potential is generated by binding to the receptor in the membranes of post-synaptic cells or in myotubes. If the nerve impulse continues to reach, the effect accumulates. Since acetylcholine is degraded by enzymes, the lifetime of acetylcholine is extended by inhibitors of this enzyme.

As a result of efforts to develop a preventive and therapeutic agent for brain injury protection or degenerative brain disease, when oxidative stress was induced by using hydrogen peroxide after pretreatment of beta-lapachone, Production of AMPK and Akt signaling enzymes was activated to increase the expression of antioxidant enzymes that act against oxidative stress and thereby increase the protective effect of astrocytes against oxidative stress, Cone or a pharmaceutically acceptable salt thereof can be effectively used as an active ingredient of a composition for preventing or treating brain damage or for the prevention and treatment of degenerative brain diseases.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating brain damage protection or degenerative brain diseases containing beta -lactone or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for protecting brain damage containing beta -lactone or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health functional food for protecting brain damage containing beta-laicone or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a pharmaceutical composition for the prevention and treatment of degenerative brain diseases comprising beta-laicone or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health functional food for prevention and improvement of degenerative brain diseases comprising beta-raffone or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention relates to a composition for preventing or treating brain damage or degenerative brain diseases, which comprises beta -lactone or a pharmaceutically acceptable salt thereof as an active ingredient. The present invention relates to a composition for preventing and treating degenerative brain diseases, Induced oxidative stress induced the production of reactive oxygen species in the cell and increased the expression of antioxidant enzymes that act against AMPK and Akt signaling enzymes to act against oxidative stress, By confirming that the protective effect of astrocytes is increased, the beta-raffone or a pharmaceutically acceptable salt thereof can be effectively used as a composition for protecting brain damage or for the prevention and treatment of degenerative brain diseases.

FIG. 1 shows the inhibition of reactive oxygen species (ROS) production by β-lapachone:
β-LAP: Betalapcon;
H ₂ O ₂ : hydrogen peroxide; And
*: p < 0.05 (comparison value with hydrogen peroxide administration group).
FIG. 2 is a graph showing inhibition of cell death by beta-laakcon:
β-LAP: Betalapcon;
H ₂ O ₂ : hydrogen peroxide; And
*: p < 0.05 (comparison value with hydrogen peroxide administration group).
FIG. 3 shows the increase in antioxidant enzyme expression by beta-laicone:
β-LAP: Betalapcon.
Figure 4 shows the increase in glutathione expression by beta-laicone:
β-LAP: Betalapcon; And
*: p < 0.05 (comparison with control group).
Figure 5 shows the increase in migration of Nrf ₂ and c-Jun nuclei by beta-raffone cone:
β-LAP: Betalapcon.
FIG. 6 shows the increase in antioxidant activity and protein complex formation by beta-laakcon:
β-LAP: Betalapcon; And
ARE + protein complexes: antioxidant elements + protein complexes.
FIG. 7 is a graph showing an increase in gene transcription activity of antioxidant elements and antioxidant enzymes by beta-laakcon:
β-LAP: Betalapcon; And
*: p < 0.05 (comparison with control group).
Fig. 8 shows the increase in phosphorylation of signal transduction enzymes by beta-raffone:
β-LAP: Betalapcon.
Figure 9 is a graphical representation of the results of Figure 8:
β-LAP: Betalapcon; And
*: p < 0.05 (comparison with control group).
FIG. 10 shows inhibition of antioxidant enzyme expression by beta-raffone-induced inhibition by AMPK inhibitor (compound C) and Akt inhibitor (LY294002)
β-Lapachone: Betaparacon;
CC: compound C, an AMPK inhibitor; And
LY: Akt inhibitor LY294002.
FIG. 11 is a graph showing that the cell death-suppressing effect by beta-laakcon is canceled by the AMPK inhibitor:
compound C: AMPK inhibitor;
β-LAP: Betalapcon;
H ₂ O ₂ : hydrogen peroxide;
*: p < 0.05 (comparison with control); And
#: p < 0.05 (comparison with beta-rapacon treated group).

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for protecting brain damage containing beta -lactone or a pharmaceutically acceptable salt thereof as an active ingredient.

The beta-raffone is preferably a compound represented by the following formula (1).

It is preferable that the beta-raffone protects the glial cells against oxidative stress, and the glial cells are preferably astrocytes.

It is desirable that the brain injury protection protects brain damage induced by oxidative stress.

In a specific example of the present invention, the present inventors conducted pre-treatment of beta-raffaccine to induce oxidative stress using hydrogen peroxide to confirm the effect of intracellular reactive oxygen species on the astrocytes of beta-rapacon, It was confirmed that beta-raffone inhibited the intracellular reactive oxygen species produced in the astrocytes by hydrogen peroxide stimulation (see Fig. 1). As a result of confirming the effect of beta-raffone on the death of astrocytes, (Fig. 2). As a result, beta-raffone increased the expression of HO-1, NQO1, MnSOD, catalase and glutathione antioxidant enzyme (See Figs. 3 to 4).

In addition, the present inventors confirmed the migration of Nrf ₂ and c-Jun into the nucleus in association with the antioxidant reaction element, and found that the transfer of Nrf ₂ and c-Jun into the nucleus was increased by beta-raffone cone 5), antioxidant elements and protein complexes were increased by beta-raffone (see FIG. 6), antioxidant enzymes (HO-1 E1, HO-1 E2 and NQO1) Activity was increased (see Fig. 7).

In addition, the inventors of the present invention have confirmed that signal transduction factors involved in increasing the expression of antioxidant enzymes have been confirmed, and it has been confirmed that AMPK and Akt phosphorylation is increased by beta-raffone, thereby increasing AMPK and Akt activity (See FIGS. 8 to 9). As a result of examining the effect of AMPK and Akt activity on the antioxidant enzyme expression, it was confirmed that the antioxidant enzymes HO-1, NQO1, MnSOD and catalase were all reduced by AMPK and Akt inhibitor (See FIG. 10). As a result of confirming the effect of AMPK activity on the astrocyte apoptosis protective effect of beta-raffone, it was confirmed that the apoptosis-protecting effect of beta-raffone was counteracted in the treatment of AMPK activity inhibitor (see FIG. 11) .

Therefore, when oxidative stress was induced by using hydrogen peroxide after pretreatment of the beta-rapaicone of the present invention, production of active oxygen species in the cell was inhibited and activation of AMPK and Akt signal transduction enzymes to act against oxidative stress The present invention provides a pharmaceutical composition for protecting brain damage, which comprises an effective amount of the beta-raffone or a pharmaceutically acceptable salt thereof as an active ingredient of a pharmaceutical composition for protecting brain damage by confirming that the protective effect of astrocytes on oxidative stress is increased by increasing the expression of antioxidant enzymes Lt; / RTI >

The betalapacon of the present invention can be used in the form of a pharmaceutically acceptable salt, and the acid addition salt formed by a pharmaceutically acceptable free acid is useful as a salt. 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 Hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfo, naphthalene-1-sulfonate Naphthalene-2-sulfonate, or mandelate.

The addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving betalaparcone in a water-miscible organic solvent such as acetone, methanol, ethanol, or acetonitrile, adding an excessive amount of an organic acid, Followed by precipitation or crystallization with an aqueous acid solution. Subsequently, in this mixture, a solvent or an excess acid is evaporated and dried to obtain an additional salt, or the precipitated salt may be produced by suction filtration.

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 of the present invention includes not only pharmaceutically acceptable salts, but also all salts, hydrates and solvates which can be prepared by conventional methods.

When the composition of the present invention is used as a medicine, a pharmaceutical composition containing beta-raffone or a pharmaceutically acceptable salt thereof as an active ingredient may be formulated into various oral or parenteral dosage forms at the time of clinical administration and administered But is not limited thereto.

Examples of the formulations for oral administration include tablets, pills, light / soft capsules, liquids, suspensions, emulsions, syrups, granules and elixirs. These formulations may contain, in addition to the active ingredient, a diluent (e.g., lactose, dextrose, (E.g., silica, talc, stearic acid and magnesium or calcium salts thereof and / or polyethylene glycols), such as, for example, water, rosin, sucrose, mannitol, sorbitol, cellulose and / or glycine. The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine and may optionally contain additives such as starch, agar, alginic acid or its sodium salt A disintegrating or boiling mixture and / or an absorbent, a colorant, a flavoring agent, and a sweetening agent.

The pharmaceutical composition containing the beta-raffone or its pharmaceutically acceptable salt as an active ingredient of the present invention can be administered parenterally, and parenteral administration can be carried out by subcutaneous injection, intravenous injection, intramuscular injection, And the like. In this case, in order to formulate the formulation for parenteral administration, the pharmaceutical composition containing the beta-raffone or its pharmaceutically acceptable salt as an active ingredient is mixed with water together with a stabilizer or a buffer to prepare a solution or suspension, Or vial unit dosage forms. The compositions may contain sterilized and / or preservatives, stabilizers, wettable or emulsifying accelerators, adjuvants such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, Or may be formulated according to the coating method.

The dose of the composition of the present invention to the human body may be varied depending on the age, body weight, sex, dosage form, health condition, and disease severity of the patient. When the patient is 60 kg body weight, And may be 0.01 to 500 mg / day, preferably 0.01 to 500 mg / day, and may be administered once or several times a day at regular intervals according to the judgment of a doctor or pharmacist.

The beta-raffone is preferably a compound represented by the formula (1).

When oxidative stress was induced by using hydrogen peroxide after pretreatment with the beta-raffone of the present invention, production of active oxygen species in the cell was inhibited, and antioxidant enzymes which act against AMPK and Akt signaling enzymes to act against oxidative stress , And that the protective effect of the astrocytic cells against oxidative stress is increased. Thus, the beta-raffone or its pharmaceutically acceptable salt thereof is useful as an active ingredient of a health functional food for protecting brain damage .

In order for beta-rapacon or its pharmaceutically acceptable salt of the present invention to be used for protecting brain damage as described above, it may be prepared by various methods known in the field of food science or pharmacology, And may be prepared in any food form that can be ingested orally by mixing with a carrier, excipient, diluent or the like. Preferably in the form of beverage, ring, granule, tablet or capsule.

The health functional food of the present invention may further comprise ingredients that are conventionally added at the time of food production and which are pharmaceutically acceptable. For example, in the case of beverage preparation, one or more components may be further contained in citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, etc., in addition to the beta-raffone or its pharmaceutically acceptable salts of the present invention.

The amount that can be included as an active ingredient of the health functional food according to the present invention can be appropriately selected according to the age, sex, weight, condition, and symptom of a person who desires to protect the brain damage, preferably 0.01 g / To 10.0 g, and the health functional food having such a content can be taken into consideration to provide a protective effect for brain damage.

The degenerative brain disease may be selected from the group consisting of Alzheimer's disease, Parkinson's disease, dementia, Huntington's disease, Creutzfeldt-Jakob disease, Pick's disease, Lewy body disease, amyotrophic lateral sclerosis, multiple sclerosis, and ischemic brain disease.

When oxidative stress was induced by using hydrogen peroxide after pretreatment with the beta-raffone of the present invention, production of active oxygen species in the cell was inhibited, and antioxidant enzymes which act against AMPK and Akt signaling enzymes to act against oxidative stress Of the present invention, thereby confirming that the protective effect of astrocytes on oxidative stress is increased. Thus, the beta-raffone or a pharmaceutically acceptable salt thereof can be used as an active ingredient of a pharmaceutical composition for the prevention and treatment of degenerative brain diseases . &Lt; / RTI >

It is preferable that the degenerative brain disease is any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, dementia, Huntington's disease, Creutzfeldt-Jakob disease, Peak disease, Louicher's disease, amyotrophic lsarosclerosis, multiple sclerosis and ischemic brain disease.

When oxidative stress was induced by using hydrogen peroxide after pretreatment with the beta-raffone of the present invention, production of active oxygen species in the cell was inhibited, and antioxidant enzymes which act against AMPK and Akt signaling enzymes to act against oxidative stress Of the active ingredient of the health functional food for preventing and ameliorating degenerative brain diseases, and confirming that the protective effect of the astrocytic cells against the oxidative stress is increased, . &Lt; / RTI >

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

< Example 1> astrocytes ( astrocyte ) Culture

The cerebral cortex of the day-old SD rats was changed and replaced with 10% fetal bovine serum (Welgene, Korea), penicillin / streptomycin (Welgene), 2 mM L-glutamine, Using a minimum essential medium (Welgene) containing 5% CO ₂ at 37 ° C for one week. In cerebral cortical cultured cells, astrocytes were separated and cultured using a rotary shaker.

< Example 2> Activation of oxygen and apoptosis of astrocytes

The primary cultured astrocytes prepared by the method of Example 1 were divided into 48-well plates at a concentration of 1.0 x 10 ⁵ cells / ml and then added to wells of the experimental group with β-lapachone ) (Sigma-Aldrich) was pretreated for 1 hour, treated with 60 uM hydrogen peroxide (H ₂ O ₂ ), and incubated for 1 hour. In addition, in order to examine the effect of beta-rapacon on apoptosis, beta-rapacon was pretreated for 1 hour and hydrogen peroxide was treated for 24 hours.

< Example 3> Cell extract from astrocytes ( cell extracts Separation and Quantification

Cell extracts were isolated from astrocytes treated with beta-lapachone over time (Woo et al., Mol . Brain Res . , 113, pp 86-96, 2003). Cells (1.0 × 10 ⁵ cell number) in 1 ㎖ lysis buffer (lysis buffer; 10 mM Tris- HCl (pH 7.4), 30 mM NaCl, 1% Triton x-100, 0.1% SDS, 0.1% sodium deoxycholate, 1 mM EDTA) was added, and the mixture was allowed to stand on ice for 15 minutes and then centrifuged at 13200 rpm for 15 minutes to obtain a cell extract. The protein content of the cell extract was measured using a protein quantification kit from Bio-Rad (USA).

< Experimental Example 1> Beta Rafacon Active oxygen species in astrocytes ( reactive oxygen species, ROS ) Production inhibitory effect

The effect of beta-raffone of the present invention on intracellular reactive oxygen species of astrocytes, which plays an important role in the development of neurodegenerative diseases, was confirmed.

Specifically, the stromal cells cultured and obtained in the same manner as in <Example 1> were pretreated with beta-raffaccine (0, 0.5, 1, 2 uM) in the same manner as in <Example 2> Cells treated with hydrogen peroxide were treated with 50 uM H ₂ DCF-DA (Sigma Aldrich, USA), and incubated at 37 ° C for 1 hour in the absence of light. The degree of DCF fluorescence was measured using a fluorescence photometer.

As a result, as shown in Fig. 1, it was confirmed that beta-raffone inhibited intracellular reactive oxygen species produced in astrocytes by hydrogen peroxide stimulation, and in particular, at various concentrations of beta- And inhibited the production of oxygen species (Fig. 1).

< Experimental Example 2> Beta Rafacon Confirming the effect of inhibiting the death of astrocytes by hydrogen peroxide stimulation

The effect of the beta-raffone of the present invention on the death of astrocytes, which plays an important role in the protection of nerve cells, was confirmed.

Specifically, beta-raffaccine was pretreated with concentrations of (0, 0.5, 1, 2 uM) in the same manner as in Example 2, treated with hydrogen peroxide, and cultured for 24 hours in a cell culture medium at a concentration of 5 mg / (20 μL) of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) solution prepared in Example 1 was treated at 37 ° C. for 1 hour. The product, formazan, was dissolved in dimethyl sulfoxide (DMSO) and measured using a spectrophotometer.

As a result, it was confirmed that beta-raffone inhibited the death of astrocytes caused by hydrogen peroxide stimulation as shown in Fig. 2, and it was confirmed that treatment with beta-rafficon alone did not affect cell death (Fig. 2) .

< Experimental Example 3> Beta Rafacon Increased expression of antioxidant enzymes in astrocytes

In the cell, antioxidant enzyme expression is increased by antioxidant stress, which reduces oxidative stress. In order to confirm the effect of beta-raffone of the present invention on the expression of antioxidant enzymes in astrocytes, antioxidant enzymes hemeoxygenase-1 (HO-1), NADPH quinone oxidoreductase (NAD (P The expression of manganese superoxide dismutase (MnSOD) and catalase was confirmed by western blotting.

Specifically, 50 μg of the cell extract was electrophoresed using 12% SDS (sodium dodecyl sulfate) -polyacrylamide gel (PAGE) with the cell extract obtained in the same manner as in Example 3, And transferred to a nitrocellulose membrane. The membranes were separated by size and blocked with 5% skim milk in TBST buffer (10 mM Tris-HCl, 150 mM NaCl, 0.5% Tween-20) to prevent nonspecific binding with antibodies (Santa Cruz, USA), which recognizes the proteins of HO-1, NQO1, MnSOD and catalase, was diluted with 3% BSA (bovine serum albumin) at a ratio of 1: 1000 . The HRP-conjugated secondary antibody (Bio-rad), which recognizes the primary antibody, was diluted at a ratio of 1: 1000 in skim milk dissolved in TBST buffer to 5%, reacted, and then subjected to an ECL (Enhanced Chemiluminescence) . &Lt; / RTI >

In addition, glutathione assay kit (Cayman, USA) was used to confirm the expression of another antioxidant enzyme, glutathione.

As a result, as shown in FIG. 3 to FIG. 4, it was confirmed that beta-raffone increased the expression of HO-1, NQO1, MnSOD, catalase and glutathione antioxidant enzyme (FIGS.

These results suggest that beta - arachidonic acid increases the antioxidant enzymes and increases the defense mechanism against oxidative stress.

< Experimental Example 4> Beta Rafacon Identification of antioxidant and transcription factors in astrocytes

<4-1> Nrf2 ( nuclear factor erythroid 2- related factor 2) and c- Jun Confirmation of movement into nucleus

The transfer of Nrf2 and c-Jun into the nucleus, known to bind to the antioxidant elements in astrocytes of the present invention, was confirmed by Western blotting.

Specifically, the nuclear extracts were isolated from astrocytes after beta-raffone con- tent treatment for astrocytes cultured in the same manner as in Example 1 (Woo et al., Mol . Brain Res . , 113, pp 86-96, 2003). 1 mM lysis buffer (10 mM Tris-HCl (pH 7.9), 10 mM NaCl, 3 mM MgCl ₂ , 1% NP-40) was added to the cells (1.0 × 10 ⁵ cells) 20 mM HEPES, 20% Glycerol, 1.5 mM MgCl ₂ , 0.2 mM EDTA, 300 mM NaCl, 1 mM DTT, 1 mM) in an extraction buffer (extraction buffer; PMSF) was added, and the mixture was left on ice for 30 minutes and centrifuged at 13200 rpm for 20 minutes to obtain a nuclear extract. The protein content of the nuclear extract was measured using a protein quantification kit from Bio-Rad. To examine the nuclear translocation of the protein, the protein of the nuclear extract obtained was confirmed by using the primary antibody (Santa Cruz) of Nrf2 and c-Jun in the same manner as in <Experiment 3>.

As a result, as shown in Fig. 5, it was confirmed that beta-raffone increased migration into Nrf2 and c-Jun nuclei (Fig. 5).

<4-2> Antioxidant response element ( Antioxidant Response Element , ARE )

The electrophoretic mobility shift assay (EMSA) confirmed the binding changes of antioxidant elements and proteins, which plays an important role in regulating the expression of antioxidant enzymes in astrocyte of the present invention.

Specifically, 15 μg of the nuclear extract obtained in the same manner as in Experimental Example <4-1> was incubated with an antioxidant reaction oligonucleotide probe labeled with γ- ³² P-ATP (5'-TGG GGA ACC TGT GCT GAG TCA CTG GAG-3 'SEQ ID NO: 1) and left on ice for 30 minutes to produce a DNA-protein complex. The DNA-protein complexes were identified by electrophoresis using 5% acrylamide gel followed by autoradiography.

As a result, as shown in Fig. 6, it was confirmed that the antioxidant factor and protein complex formation were increased by beta-raffone (Fig. 6).

<4-3> Transcriptional Activity of Antioxidant Enzymes and Antioxidant Elements transcriptional activity

The transactivation activities of antioxidant enzymes (HO-1 E1, E2 and NQO1) and antioxidant elements in beta-laakcon astrocytes of the present invention were confirmed by gene transfection technology.

Specifically, a cloning vector containing a reporter gene in which luciferase is expressed to examine the transcriptional activity of the gene is referred to as HO-1 E1, HO-1 E2 , NQO1 and an antioxidant reaction element promoter were transiently transfected into astrocytes using Convoy injection reagent (ACTGene, USA). Approximately 24 hours later, beta-rapacon was treated and cellular extracts were isolated from astrocytes. (Lysis buffer (pH 7.8); 25 mM glycylglycin, 15 mM MgSO ₄ , 1% Triton X-100, 1 mM DTT) was added to the cells (1.0 x 10 ⁵ cells) For 10 minutes and centrifuged at 13200 rpm for 15 minutes to obtain a cell extract. The obtained cell extract was mixed with lx luciferin in an assay buffer (25 mM glycylglycin, 15 mM MgSO ₄ , 20 mM KPO ₄ , 2.5 mM ATP, 1 mM DTT) Respectively.

As a result, as shown in Fig. 7, it was confirmed that the gene transcription activity of HO-1 E1, HO-1 E2, NQO1 and antioxidant factor promoter was increased by beta-raffone (Fig. 7).

As a result of <Example 4>, beta-raffone increased the nuclear activity of Nrf2 and c-Jun to increase the binding activity with the antioxidant reaction element (ARE) and enhanced the transcription activity of the antioxidant enzyme promoter gene, Lt; / RTI >

< Experimental Example 5> Beta Rafacon Identification of activation effect of signaling enzyme in astrocytes

It is known that mitogen-activated protein kinases (MAP kinases), Akt, and AMP-activated protein kinase (EMAK) act on the expression of antioxidant enzymes. The signaling involved in increasing the expression of antioxidant enzymes of beta- In order to identify the factors, signaling enzymes were identified by Western blotting.

Specifically, a primary antibody (Cell) recognizing each phosphorylated protein (p-AMPK, p-Akt, p-p38, p-ERK, p-JNK, Signaling Technology, USA) was used for Western blotting.

In addition, visualization results by Western blotting are shown in a graph.

As a result, as shown in Fig. 8 to Fig. 9, it was confirmed that beta-raffone increased phosphorylation of AMPK and Akt among various signal transduction enzymes, thereby confirming that the activity of AMPK and Akt was increased 9).

< Experimental Example 6> Beta Rafacon Increased expression of antioxidant enzymes by activation of signaling enzymes in astrocytes

To investigate the effect of AMPK and Akt on the signaling activity of the antagonistic activity of AMPK and Akt in the expression of antioxidant enzyme, beta-raffone of the present invention was screened by western blotting for antioxidant enzymes Respectively.

Specifically, the primary cultured astrocytes cultured and cultured in the same manner as in <Example 1> were dispensed into a 6-well plate at a concentration of 1.0 x 10 <^5> cells / ml, and then the AMPK inhibitor Compound C and Akt inhibitor LY294002 were pretreated and treated with beta-laicone for 1 hour and cultured for 6 hours. In order to confirm the activity of the antioxidant enzyme protein in the cells treated with the signal transduction enzyme inhibitor and beta-laicone, the cell extracts obtained in the same manner as in Example 3 were tested in the same manner as in Experimental Example 3 Western blotting was performed using a primary antibody (Santa Cruz) that recognizes proteins (HO-1, NQO1, MnSOD and catalase).

As a result, as shown in FIG. 10, the antioxidative enzymes HO-1, NQO1, MnSOD and catalase were all reduced by AMPK inhibitor and Akt inhibitor (FIG. 10).

As a result, it was confirmed that beta - rafaconon stimulates AMPK and Akt signal transduction enzymes in astrocytes to increase the expression of antioxidant enzymes against oxidative stress.

< Experimental Example 7> Beta Rafacon AMPK Confirmation of astrocyte protective effect by activation

Based on the results of suppressing the death of astrocytes by oxidative stress and the inhibition of antioxidant enzyme expression by the AMPK inhibitor, when the signal transduction enzyme AMPK was inhibited in the state of oxidative stress, the beta- The effect of MTT on the protective effect of astrocytes was confirmed by MTT assay.

Specifically, the primary cultured astrocytes cultured and cultured in the same manner as in <Example 1> were dispensed into a 6-well plate at a concentration of 1.0 x 10 <^5> cells / ml, and then the AMPK inhibitor Compound C was pretreated, treated with hydrogen peroxide in the same manner as in <Example 2> for 30 minutes, and cultured for 24 hours. Then, MTT assay was performed by the same method as that of <Experimental Example 2>, and the result was measured with a spectrophotometer.

As a result, as shown in Fig. 11, beta-raffone conferred a protective effect against cell death by hydrogen peroxide. It was confirmed that the apoptosis-protecting effect of beta- rapacon was canceled by treating AMPK inhibitor (Fig. 11).

As a result, it was confirmed that beta - raffone increased the signal transduction activity of AMPK in astrocytes and increased antioxidant enzyme expression, thereby increasing the protective effect of astrocyte against oxidative stress.

<110> Ewha University - Industry Collaboration Foundation <120> Composition comprising beta-lapachone or medicament acceptable salts thereof as an active ingredient for protection of brain damage or prevention and treatment of neurodegenerative diseases <130> 2014P-11-032 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> 32P-ATP-probe <400> 1 tggggaacct gtgctgagtc actggag 27

Claims

A pharmaceutical composition for protecting brain damage containing beta -lactone or a pharmaceutically acceptable salt thereof as an active ingredient.

The pharmaceutical composition for protecting brain damage according to claim 1, wherein the beta-raffone is a compound represented by the following formula (1).
[Chemical Formula 1]

The pharmaceutical composition for protecting brain damage according to claim 1, wherein the beta-rapacon protects the glial cells against oxidative stress.

[Claim 5] The pharmaceutical composition for protecting brain damage according to claim 3, wherein the glial cells are astrocytes.

A health functional food for protecting brain damage containing beta-raffone or a pharmaceutically acceptable salt thereof as an active ingredient.

A pharmaceutical composition for the prevention and treatment of degenerative brain diseases comprising beta-raffone or a pharmaceutically acceptable salt thereof as an active ingredient.

7. The method of claim 6, wherein the degenerative brain disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, dementia, Huntington's disease, Creutzfeldt-Jakob disease, Wherein the disease is any one selected from the group consisting of Lewy body disease, amyotrophic lateral sclerosis, multiple sclerosis and ischemic brain disease. A pharmaceutical composition for preventing and treating brain diseases.

A health functional food for preventing and improving degenerative brain diseases containing beta-raffone or its pharmaceutically acceptable salt as an active ingredient.

10. The method according to claim 8, wherein the degenerative brain disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, dementia, Huntington's disease, Creutzfeldt-Jakob disease, Peak disease, Lewy body disease, amyotrophic lsarosclerosis, multiple sclerosis and ischemic brain disease Wherein the functional food for preventing and improving degenerative brain diseases is one.