KR100916025B1 - Pharmaceutical composition for treating or preventing a neurological brain disease comprising xanthorrhizol - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating or preventing cerebral neurological diseases containing xanthorrhizol, and more specifically, to treating or treating cerebral neurological diseases containing xanthorazole represented by the following formula (I) as an active ingredient: It relates to a prophylactic pharmaceutical composition. The pharmaceutical composition containing the xantholizole of the present invention as an active ingredient may be very useful for the treatment or prevention of cerebral neurological diseases.

[Formula I]

Xantolizole, Cranial Nerve Disease, Antioxidant  

Description

Pharmaceutical composition for treating or preventing a neurological brain disease comprising xanthorrhizol}

1 is a graph measuring the effect of the xantholizole of the present invention in HT-22 cells to inhibit the production of reactive oxygen species by glutamate.

Figure 2 is a graph measuring the effect of inhibiting the production of reactive oxygen species BSO-induced BSO by the present invention in cultured hippocampal cells.

3 is a graph measuring the inhibitory effect of the xantholizol of the present invention on lipid peroxidation of brain tissues induced by hydrogen peroxide at each concentration.

Figure 4 is a graph measuring the cytotoxic effect of xantolizole of the present invention.

FIG. 5 measures the inhibitory effect of the xantholizol of the present invention on apoptosis induced by glutamate. FIG.

   A: graph showing the inhibitory effect on apoptosis by concentration of xanthozol

   B: Photograph showing cell viability by Xanthozol 5 μM treatment

Figure 6 is a graph measuring the IL-6 inhibitory effect of the xantholizole of the present invention in microglia of cultured brain.

Figure 7 is a graph measuring the TNF-α inhibitory effect of the xantholizole of the present invention in microglia of cultured brain.

8 is a graph measuring the NO inhibitory effect of the xantholizole of the present invention in microglia of cultured brain.

9 is a measure of the inhibitory effect of the xantholizole of the present invention on the expression of iNOS and COX-2 induced by LPS in cultured microglia cells.

   A: Western blot analysis

   B: Graph quantifying the inhibitory effect of xantholizol on the expression of iNOS

   C: Graph quantifying the inhibitory effect of xantholizol on expression of COX-2

The present invention relates to a pharmaceutical composition for the treatment or prevention of cranial nerve disease containing xantolizole, and more particularly to the treatment or prevention of brain nerve disease containing xantolizole represented by the following formula (I) as an active ingredient To a red composition.

[Formula I]

As human life increases and aging society progresses, neurological diseases such as stroke, dementia and Parkinson's disease are increasing. The neurological diseases are characterized in that the death or degeneration of certain brain cells progresses temporarily or over a long period of time, and once dead brain cells are not regenerated, eventually leading to fatal brain function loss. In particular, brain dysfunction accompanied by progressive decline in cognitive function, sensory function, motor function, and systemic function eventually leads to changes in personality and behavior, leading to patients being unable to care for themselves. The main pathways for brain cell death include oxidative stress caused by oxidative stress, excitatory toxicity, and apoptosis, each of which induces cell death through a specific signaling process.

Specifically, oxidative damage of proteins, nucleic acids, and lipids has been suggested after accumulation of Reactive Oxygen Species as a major cause of brain cell death in patients with stroke, brain injury, Alzheimer's dementia, and Parkinson's disease. In particular, oxidative stress caused by free radicals has been reported as a major cause of cell death in tissues in the body, and has been suggested as one of the cycles of cell death in neurological diseases (Schapira, AH, Curr. Opin. Neurol ., 9 (4): 260-264, 1996). Evidence that free radicals are involved in the death of neurons in cerebral neurological diseases is evidenced by the increased production of reactive oxygen species after ischemia and the inhibitory effect of ischemic neurons by antioxidants (Flamm, ES et al., Stroke 9 (5)). : 445-447, 1978; Chan, PH, J. Neurotrauma 9 Suppl. 2: S417-423, 1992); Increased Fe ²⁺ in Huntington's striatum (Dexter, ET et al., Ann. Neutol ., 32 Suppl: S94-100, 1992), production of free radicals by beta amyloid in Alzheimer's disease (Richardson JS et al. , Ann.NY Acad. Sci ., 777: 362-367, 1996), point mutations in the Cu / Zn SOD-1 gene in amyotrophic lateral sclerosis (ALS) (Rosen, DR et al. , Nature , 362 (6415): 59-62, 1993).

In addition, glutamate, an excitatory neurotransmitter, acts as a neurotransmitter in the normal state, but when excessively secreted by various causes, neuronal cell death occurs. Transient activity of glutamate receptors, NMDA, AMPA and kainate receptors, is also known to be a major cause of neuronal cell death by stroke and brain injury (Choi DW Neuron, 1: 623-634, 1988). Neurotoxicity by glutamate has been shown to be involved in neuronal cell death in ALS, evidence of glutamate synthase abnormalities, glutamate transporter proteins, and glutamate receptor protein increases in ALS patients (Rothstein, JD Clin). Neurosci ., 3 (6): 348-359, 1995; Shaw, PJ et al., J. Neurol ., 244: Suppl 2 S3-14, 1997).

Apoptosis has also been reported as another cause of brain cell death, which is a major form of apoptosis in ischemia, brain injury, spinal injury, Alzheimer's dementia, and Parkinson's disease (Smale et al., Exp. Neurol). , 133: 225-230, 1995; Crow et al., Nat. Med ., 3: 73-76, 1997).

These various reports show that oxidative toxicity, excitatory toxicity and apoptosis kill brain cells as a major cause of various neurological diseases. Inhibition of and / or suppression of apoptosis of brain cells has become a major target.

Accordingly, the present inventors searched for a long time to find a natural-derived compound that can be used as a therapeutic agent for cerebral neurological diseases. As a result, xanthorrhizol isolated and purified from curcuma xanthorrhiza extract was used for the treatment / prevention of cerebral neurological diseases. The present invention has been completed by elucidating that it has an excellent effect.

Accordingly, an object of the present invention is to provide a novel use of xantolizol separated and purified from curcuma xantoriza extract.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the treatment or prevention of cerebral neurological diseases containing xantolizol or a pharmaceutically acceptable salt thereof represented by the following formula (I) as an active ingredient.

[Formula I]

Hereinafter, the present invention will be described in detail.

The present invention is characterized by providing a novel use of xanthozol separated and purified from Curcuma xanthorrhiza.

Curcuma zantorija is a traditional medicinal plant known as temu lawak or Javanese turmeric, which is known as the Zingiberaceae plant. , terpenoid compounds such as β-turmerone and xanthorrhizol, 7-30% essential oil, 30-40% carbohydrate, and curcuminoid, 0.02-2.0% aromatic pigment (Lin SC et al., Am J. Chin.Med., 23: 243-254, 1995).

Xantorisol is a type of sesquiterpene that is a representative constituent found in Curcuma xantoriza. Xanthozol has antimicrobial activity against oral microorganisms (Hwang, JK et al., Fitoterapia , 71: 321-323; Hwang, JK et al., Planta Medica , 66: 196-197, 2000) Inhibitory activity (Kim, SH et al., Toxicology and Applied Pharmacology , 196: 346-355, 2004); Kim, SH et al., Food and Chemical Toxicology , 43: 117-122, 2005), inhibiting cancer metastasis (Choi, MA et al., Biochem. Biophys. Res. Commun., 326, 210-217, 2005) Etc. are known. However, there has been no report on the use of xantorizole as a therapeutic agent for cranial nerve disease of the present invention.

As an extraction method of Curcuma xanthorrhiza, organic solvent extraction method, supercritical fluid extraction method, microwave extraction method and ultrasonic extraction method as disclosed in Korean Patent Application Publication No. 2000-0000342 and PCT Patent No. WO88 / 05304 And the like can be used. Separation and purification of xanthozol from the Curcuma xantoriza extract has been disclosed in Korean Patent Laid-Open Publication No. 2000-0073295.

Xantorazole according to the present invention is represented by the following formula (I).

[Formula I]

Xantoriazoles of the present invention can be obtained from plants or parts of plants using methods of extracting and separating conventional materials. Stems, roots or leaves are properly dehydrated and macerated or only dehydrated to obtain the desired extracts, and the desired extracts are purified using purification methods known to those skilled in the art to which this invention pertains. In addition, the synthetic compounds or derivatives thereof corresponding to the xantholizol represented by Chemical Formula I are generally commercially available materials, or may be chemically prepared using known synthetic methods.

Water or C1-C6 organic solvent may be used as the extracted organic solvent. Preferably purified water, methanol (ethanol), ethanol (propanol), isopropanol (isopropanol), butanol (acetone), acetone (ether), benzene (benzene), chloroform (chloroform), Various solvents such as ethyl acetate, methylene chloride, hexane, cyclohexane and petroleum ether may be used alone or in combination. More preferably methanol or hexane can be used. Separation and purification of the xantholizole of the present invention from the Curcuma xantoriza extract is carried out by column chromatography and high performance liquid chromatography (HPLC) filled with various synthetic resins such as silica gel or activated alumina. ) May be used alone or in combination. However, the extraction and separation and purification method of the active ingredient is not necessarily limited to the above method.

As such, the xantholizole of the present invention may be used in the form of a purely separated and purified compound, and may also be used in the form of an extract containing the same. The extract can be obtained by extracting the Curcuma zantorija with water or an organic solvent of C1-C6 as described above.

Xantoriazoles according to the invention may be used in the form of salts, preferably pharmaceutically acceptable salts. As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is preferable. Organic acids and inorganic acids may be used as the free acid. The organic acid is not limited thereto, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Glutamic acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.

Reactive oxygen species are substances that cause oxidative toxicity in the body, causing the peroxidation of lipids, which are components of the cell membrane, and destroying the bioprotection and signaling system of the cell membrane, oxidative damage of DNA, and destruction of red blood cells. Phenomenon reduces the function of various enzymes in the body. Through this, reactive oxygen species are known to cause various diseases such as aging, cancer, brain diseases such as stroke, Parkinson's disease, heart disease, ischemia, arteriosclerosis, skin disease, digestive diseases, inflammation, rheumatism, autoimmune diseases, etc. . Reactive oxygen species have been raised as a major cause of Alzheimer's disease in particular (Maccioni et al., Arch. Med. Res ., 32: 367-281, 2001). Therefore, in one embodiment of the present invention it was investigated whether the xantholizole of the present invention inhibits the generation of reactive oxygen species. As a result, it was confirmed that xantholizole of the present invention inhibits the production of reactive oxygen species induced by glutamate in concentration-dependently in cultured hippocampal neurons as well as HT22 cells, which are hippocampal-derived cell lines (see FIGS. 1 and 2). .

Lipid peroxidation is an indicator of brain damage following oxidative stress (Sewerynek et al., Neuroscience Letter , 195: 203-205, 1995). Hydrogen peroxide breaks down into water and oxygen, producing hydroxyl free radicals. The free radicals cause DNA damage, protein carbonylation, lipid peroxidation. Therefore, in another embodiment of the present invention it was investigated whether the xantholizol of the present invention inhibits lipid peroxidation by hydrogen peroxide. As a result, it was confirmed that xantolizole of the present invention inhibits lipid peroxidation in a concentration-dependent manner (see FIG. 3).

In another embodiment of the present invention, it was investigated whether the xantholizol of the present invention exhibits cytotoxicity. As a result, it was confirmed that the xantholizole of the present invention did not show cytotoxicity even at 5 μM (see FIG. 4).

Other causes of brain cell death include excitatory neurotransmitters glutamate and cell death through its receptors (Olney, JW, Int Rev. Neurobiol ., 27: 337-362, 1985). Therefore, in another embodiment of the present invention it was investigated whether the xantholizole of the present invention inhibits the death of brain cells induced by glutamate. As a result, it was confirmed that the xantholizole of the present invention inhibits the death of brain cells induced by glutamate in a concentration-dependent manner (see FIG. 5).

In addition, epidemiologic evidence has been reported that nonsteroidal anti-inflammatory drugs, such as ibuprofen, delay the progression of Alzheimer's disease (McGeer and McGeer, Exp. Gerontol ., 33: 371-378, 1998). In particular, administration of ibuprofen in mice mimicking Alzheimer's disease slowed disease progression (Lim et al, J. Neurosci ., 20: 5709-5714, 2000). Therefore, compounds having anti-inflammatory activity as well as antioxidant activity have high efficacy as a treatment / prophylactic agent for cranial nerve disease. Therefore, in another embodiment of the present invention, the anti-inflammatory activity of the xantholizol of the present invention was investigated by treating LPS (lipopolysaccharide) to microglia, which are brain immune cells. As a result, it was confirmed that the xantholizole of the present invention significantly reduced the expression and production of various immune mediators (IL-6, TNF-α, NO, iNOS and COX-2) induced by LPS. (See FIGS. 6-9).

As described above, the xantholizole of the present invention is very excellent in the antioxidant effect of inhibiting lipid peroxidation and generation of reactive oxygen species, the brain cell protection effect of inhibiting atoptoses of brain cells by glutamate, and the anti-inflammatory effect. Therefore, the present invention provides a pharmaceutical composition for the treatment or prevention of cerebral neurological diseases, which comprises xantolizol or a pharmaceutically acceptable salt thereof represented by Chemical Formula I as an active ingredient. In another aspect, the present invention provides a pharmaceutical composition for the treatment or prevention of cerebral neurological diseases containing Curcuma zantoriza extract as an active ingredient. Preparation of the Curcuma Xantoriza extract is as described above.

The composition of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of a general pharmaceutical formulation. When formulated in the form of a general medicine, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which solid preparations comprise at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin The mixture is prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, ointments, creams. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.

In addition, the composition of the present invention can be administered parenterally, parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method. To formulate into a parenteral formulation, xantolizol of Formula I is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is formulated in unit dosage forms of ampoules or vials. Dosage units may contain, for example, one, two, three or four times, or 1/2, 1/3 or 1/4 times the individual dosage. The individual dosage preferably contains an amount in which the effective drug is administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dose.

The effective dose of xantholizol represented by formula (I) of the present invention is a single dose of 0.1-50 mg / kg, preferably 1-10 mg / kg, and may be administered 1-3 times a day. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, severity of the disease, and the like.

As a result of the toxicity experiment when oral administration of the xantholizole of the present invention to rats, the 50% lethal dose (LD ₅₀ ) by oral toxicity test was found to be more than 2,000 mg / kg (results not shown).

Neurological diseases to which the pharmaceutical composition of the present invention can be applied include oxidative stress caused by lipid peroxidation, reactive oxygen species and / or free radicals; Excitatory toxicity by glutamate; And / or diseases caused by death or degeneration of brain cells caused by apoptosis. The cranial nerve disease includes dementia, mild cognitive impairment, degenerative cranial nerve disease such as Parkinson's disease, Huntington's disease, and ischemic cranial nerve disease such as stroke and convulsive cranial nerve disease such as epilepsy. Specifically, the neurological disease is not limited thereto, but is not limited to dementia, Parkinson's disease, stroke, Huntington's disease, Creutzfeldt-Jay Jacob disease, Pick's disease, amyotrophic lateral sclerosis (ALS), Parkin-ALS-dementia complex Symptoms, Wilson's disease, progressive supranuclear palsy, mild cognitive impairment and epilepsy. The dementia includes senile dementia, Alzheimer's dementia, vascular dementia, alcoholic dementia, and thalamic dementia.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1>

Inhibitory effect on generation of reactive oxygen species

HT-22 (obtained from Dr. David Schubert), a hippocampal cell line responsible for memory of the brain, simultaneously treated with 5 mM glutamate and the xantolizole (2 μM) of the present invention for 8 hours It was. The control group was not treated with the xantholizole of the present invention. Thereafter, CM-H ₂ DCFDA (chloromethyl derivative of dichlorodihydrofluorescein diacetate, Molecular Probes) was stained to investigate the production of reactive oxygen species. As a result, as shown in Fig. 1 , the xantholizole of the present invention inhibited the generation of reactive oxygen species induced by glutamate. Xantolizole of the present invention also significantly inhibited the generation of natural reactive oxygen species in cells in the control group not treated with glutamate.

Then, it was confirmed whether xantolizole of the present invention exhibited the same antioxidant effect in tissue cultured hippocampal neurons. For this purpose, the hippocampus of fetuses extracted from 18-day-old rats were cultured for 10 days in neurobasal media (Gibco BRL) supplemented with B-27 supplement and 2 mM L-glutamine. Tissue cultured hippocampal neurons were treated with 1 mM BTH (buthionine sulfoxide) for 8 hours to induce oxidative stress. At this time, the xantholizole of the present invention was treated together by concentration (0.1, 0.5 and 1 μM) to investigate the effect of reducing the active oxygen by the compound. The control group was not treated with BSO. Thereafter, the amount of active oxygen was measured according to methods known in the art (Jung, YS, Biochem Biophys Res Commun ., 320 (3): 789-94, 2004) using 10 μM Molecular probes (DCFDA). As a result, as shown in Figure 2 , xantholizole of the present invention inhibited the generation of reactive oxygen species in tissue culture hippocampal neurons to a level similar to the control.

<Example 2>

Lipid Peroxidation Inhibitory Effect

After anesthesia, the rat tissue was refluxed with 0.9% saline containing EDTA. The brains were then extracted and washed with ice-cold 20 mM Tris-HCl, pH 7.4. After draining, the brain weight was measured. Brain-cold 20 mM Tris-HCl (pH 7.4) was mixed at a dose of 0.1 g / ml and homogenized. Centrifugation gave supernatant. Lipid peroxidation was induced by adding 40 mM hydrogen peroxide to 40 μl of the supernatant. At the same time, xantholizol isolated in Example 1 was added by concentration (0.5, 1, 5 and 10 μM), respectively. After incubation for 30-60 minutes in water at 37 ° C., 162.5 μl of R1 solution (lipid peroxidation Assay Kit, Cat. No. 437634, Calbiochem) and 37.5 μl of R2 solution (lipid peroxidation Assay Kit, Cat. No. 437634) , Calbiochem) was added and further incubated at 45 ° C. for 40 minutes. The absorbance of the culture was then measured at 586 nm to quantify the degree of lipid peroxidation.

As a result, as shown in FIG. 3 , it was confirmed that lipid peroxidation caused by hydrogen peroxide was inhibited in a concentration-dependent manner by the xantholizole of the present invention. In particular, when 10 μM of the xantolizole of the present invention was administered, lipid peroxidation was inhibited to a level almost similar to that of the control group (when the lipid peroxidation was not induced by hydrogen peroxide).

<Example 3>

Cytotoxic effect

In order to measure the cytotoxic effect of the xantholizol itself of the present invention, the xantholizol was treated with HT-22, a hippocampal-derived cell line at each concentration (0.5, 1 and 5 μM) for 24 hours. As a result, as shown in Figure 4 , the xantholizole of the present invention did not induce cytotoxicity even at 5 μM.

<Example 4>

Apoptosis Inhibitory Effect of Brain Cells

Hippocampus-derived cell line HT-22 was treated with 5 mM glutamate for 24 hours to induce apoptosis. The control group was not treated with glutamate. Thereafter, xantolizole of the present invention was treated by concentration (1, 2, 5 and 10 μM) for 24 hours. Apoptosis was observed using WST-1 (Roche). As a result, as shown in FIG. 5 , it was confirmed that the xantholizole of the present invention suppressed apoptosis of brain cells induced by glutamate in a concentration-dependent manner.

<Example 5>

Anti-inflammatory effect

<6-1> Inhibitory effect of proinflammatory cytokines

Microglia are the only cells of the mesoderm originating from the central nervous system and are greatly increased when there is an inflammatory response in brain tissue (Streit, WJ Prog. Neurobiol ., 57: 563-581, 1999). When microglia are activated by LPS, they synthesize and secrete various proinflammatory cytokines such as IL-1, IL-6 and TNF-α (Chen, S. Neurobiol. Aging, 17: 781-787, 1996). . Therefore, in order to confirm the anti-inflammatory effect of the xantholizole of the present invention, the effect of the xantholizole of the present invention on the production of IL-6 and TNF-α in activated microglia cells was investigated.

First, only the neocortex was isolated from the brains of mice 1 day old. The microglia-star glial cell mixtures were then prepared according to methods known in the art (Kim, HY et al ., J. Immunol ., 171: 6072-6079, 2003). The microglia-star glial cell mixture was incubated for 2 weeks in a 75 cm ² flask at a ratio of 4 flasks per head in MEM medium containing 10% FBS. Only microglia were removed from the cultured cells and cultured in MEM medium containing 5% FBS for 24 hours. The cultured microglia were washed twice with serum-free medium and then treated with LPS (1 μg / ml) and the xantholizole (2.5 and 10 μM) of the present invention together for 24 hours. Thereafter, the amounts of IL-6 and TNF-α secreted into the cell culture were measured using a solid-phase ELISA system (RPN2742 for IL-6, RPN2744 for TNF-α, Amersham Bioscience). To this end, first, culture supernatants of microglia and standard of each material (pure isolated and quantified IL-6 or TNF-a) were prepared in 96-well plates coated with murine IL-6 and TNF-α specific antibodies. 50 μl each was added. After 2 hours of reaction at room temperature, each well was washed three times with washing buffer (Amersham Bioscience). Biotin-treated IL-6 or TNF-α specific antibodies were added at 100 μM and reacted at room temperature for 1 hour. Each well was washed three times again with a wash buffer, and then 100 μl of HRP-bound streptapidine solution (Amersham Bioscience) was added and reacted at room temperature for 30 minutes. Each well was washed three times with a wash buffer, and then 100 μl of TMB-substrate solution (Amersham Bioscience) was added and allowed to react for 30 minutes at room temperature under dark conditions. Thereafter, 100 µl of a stop solution (Amersham Bioscience) was added to terminate the reaction, and then the absorbance was measured at 450 nm using a microreader. As a result, as shown in FIG . 6 and FIG. 7 , the production of proinflammatory cytokines (IL-6 and TNF-α) induced by LPS was inhibited concentration-dependently by the xantholizole of the present invention.

<6-2> NO Production Inhibition Effect in Microglial Cells Activated by LPS

Activation of microglia leads to the expression of NO, a mediator of neurotransmission and immune responses (Liu, B. et al., Ann. NY Acad. Sci., 962: 318-331, 2002) . Therefore, the effect of the xantholizole of the present invention on the production of NO in microblasts was examined.

First, only the neocortex was isolated from the brains of mice 1 day old. The microglia-star glial cell mixtures were then prepared according to methods known in the art (Kim, HY et al ., J. Immunol ., 171: 6072-6079, 2003). The microglia-star glial cell mixture was incubated for 2 weeks in a 75 cm ² flask at a ratio of 4 flasks per head in MEM medium containing 10% FBS. Only microglia were removed from the cultured cells and cultured in MEM medium containing 5% FBS for 24 hours. Tissue cultured microglial cells were inoculated in 5% FBS-containing MEM medium at a concentration of 1.5 × 10 ⁴ cells / well and cultured in 96-well plates. After 1 day, LPS (1 μg / ml, Sigma) was treated to induce activation of microglia. At this time, the xantholizole (2.5 and 10 μM) of the present invention was treated with LPS, and then reacted for 16 hours or 24 hours. Thereafter, cell culture was taken to measure NO production. NO production was confirmed by measuring the amount of nitrite, a stable metabolite of NO, in cell culture using a Gries's reagent kit (Molecular Probe). The measurement method was as follows: 150 µl of the cell culture was taken and mixed with 20 µl of Gries reagent and 130 µl of water, and then allowed to react at room temperature in a microplate for 30 minutes. Then, absorbance at 548 nm was measured using a microplate reader.

As a result, as shown in Figure 8 , it was confirmed that the production of NO induced by LPS in tissue-cultured microglia cells is concentration-dependently inhibited by the xantholizole of the present invention. In particular, the xantholizol of the present invention was treated with 10 μM showed an inhibition rate of about 97%.

<6-3> Inhibitory Effect of iNOS and COX-2

Through Example <6-2> it was confirmed that the production of NO induced by LPS in the microglia cells of tissue culture mice is inhibited by the xantholizole of the present invention. NO was produced by the iNOS enzyme, which induces expression when microglial cells are activated. Therefore, we investigated whether the reduction of NO production is associated with the inhibition of iNOS expression. We also observed changes in COX-2 protein involved in the production of other inflammatory mediators.

For Western blot analysis, tissue cultured microglia were cultured at a concentration of 7.5 × 10 ⁵ cells in a 60 mm cell culture dish. After 1 day, LPS (1 μg / ml, Sigma) was treated to induce activation of microglia. At this time, the xantholizole (2.5 and 10 μM) of the present invention was treated with LPS, and then reacted for 16 hours or 24 hours. After culturing the cells twice with cold PBS, using cold lysis buffer (1% SDS, 1 mM Na ₃ VO ₄ , 10 mM NaF, 10 mM Tris-Cl, pH 7.4 containing 1 X protease inhibitors cocktail) Cells were lysed. Cell lysates were centrifuged at 12,000 g for 10 minutes at 4 ° C to take supernatant. Thereafter, the protein amount was quantified by the BCA method. The same amount of protein was separated via SDS-PAGE and then transferred to the PVDF membrane. Each membrane was blocked with 3% BSA solution and then washed three times with TBS-T solution (10 mM Tris-Cl, pH 7.5, 150% NaCl containing 0.1% Tween 20). Subsequently, a primary antibody specific for iNOS (rabbit polyclonal Ab, Upstate, 06-573) and COX-2 (rabbit polyclonal Ab, Santa Cruz Biotechnology, sc-7951) was added to the membrane for 1 hour at room temperature. I was. The membrane was washed three times with TBS-T solution, and then reacted for 1 hour at room temperature by adding a specific secondary antibody bound to HRP. Again the membrane was washed three times with TBS-T solution, and then each band was identified using an ECL system (Sigma).

As a result, as shown in Figure 9 , it was confirmed that the expression of iNOS and COX-2 induced by LPS in tissue culture microglia reduced concentration-dependently by the xantholizole of the present invention.

<Manufacture example 1>

Preparation of a medicament containing the pharmaceutical composition for the treatment or prevention of cerebral neurological diseases according to the present invention

<1-1> Preparation of Tablet

25 mg of xantolizole of the present invention was prepared with 26 mg of lactose for excipients and 3.5 mg of avicel (microcrystalline cellulose), 1.5 mg of sodium starch glyconate as a disintegration aid, and 8 mg of low-hydrosyprophylcellulose (L-HPC) for binders. The U-type mixer was put together and mixed for 20 minutes. After mixing was completed, 1 mg of magnesium stearate was further added as a lubricant and mixed for 3 minutes. Tablets were prepared by tableting and film coating after a quantitative test and a humidity test.

<1-2> Preparation of Syrup

A syrup containing the xantholizole of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient of 2% (W / V) was prepared by the following method: 2g of acid addition salt of xantolizole of the present invention, saccharin 0.8 g and 25.4 g of sugar were dissolved in 80 g of warm water. After cooling the solution, 8.0 g of glycerin, 0.04 g of flavor, 4.0 g of ethanol, 0.4 g of sorbic acid, and an appropriate amount of distilled water were mixed thereto. Water was added to the mixture to make 100 mL.

<1-3> Preparation of Capsule

A capsule was prepared by mixing 50 mg of xantolizol, 50 mg of lactose, 46.5 mg of starch, 1 mg of talc, and an appropriate amount of magnesium stearate and filling it into a hard gelatin capsule.

<1-4> Preparation of Injection Solution

Injectable solutions containing 10 mg of the active ingredient were prepared in the following manner: 100 ml were prepared by dissolving 1 g of hydrochloride, 0.6 g of sodium chloride, and 0.1 g of ascorbic acid of the xantholizole of the present invention in distilled water. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

<Application example 1>

Parkinson's disease

Parkinson's disease is a central nervous system degenerative brain disease that occurs in older people. It causes symptoms such as limb tremor, muscle stiffness and difficulty in starting exercise, and mental depression is accompanied. Parkinson's disease is caused by the death of mesenchymal melanoma dopaminergic neurons (Fahn S., Parkinsons disease in: Diseases of the nervous system, (ED) by A. Asbury, G. Mckhann, pp. 1217-). 1238, Saunders, 1986. Oxidative stress, abnormal energy metabolism, mitochondrial gene mutations, and excitatory amino acid toxicity have been reported as causes of neuronal death associated with Parkinson's disease, but oxidative stress has been reported to be the major cause (Fahn S. and Cohen). , G., Ann. Neurol . 32 (6): 804-812, 1992; Foley P. and Riederer P., J. Neurol ., 247 [Sppl. 2] II / 82-II / 94, 2000). Therefore, the pharmaceutical composition of the present invention which has the activity of protecting brain cells against oxidative stress and inhibiting the death of brain cells by apoptosis can be very useful for treating or preventing Parkinson's disease.

<Application example 2>

Alzheimer's Dementia

Alzheimer's disease is a degenerative neurological disorder with severe memory and mental disorders, with an incidence of 10-15% / year. Autopsy findings in Alzheimer's patients show Senile Plaque and Neurofibrillary tangles. Oxidative damage is involved in the senile plaque induction, and the senile plaque itself causes an inflammatory response. Epidemiologic evidence has been reported that anti-inflammatory agents, such as ibuprofen, delay the progression of Alzheimer's disease (McGeer and McGeer, Exp. Gerontol ., 33: 371-378, 1998). Ibuprofen treatment delayed disease progression in mice mimicking Alzheimer's disease (Lim et al , J. Neurosci ., 20: 5709-5714, 2000). Therefore, the pharmaceutical composition of the present invention that protects brain cells against oxidative stress and has an anti-inflammatory effect can be very useful for treating or preventing Alzheimer's dementia.

<Application Example 3>

Stroke

Stroke is a neurological condition that results from the blockage or bursting of blood vessels that supply blood to the brain. The brain performs a number of functions, and the damaged part of the brain fails to function, resulting in motor and memory problems. Strokes occur mainly in older people, but they can also occur in their 20s or 30s, and the rate of strokes has not declined in the past decade. The cause of the stroke is known to be due to hypersecretion of glutamate inducing cell death by overexciting NMDA (N-methyl-D-aspartate) receptors. The activity of microglia contributes to NMDA toxicity (Tikka and Koistinaho, J immunol. , 166 (12): 7527-33, 2001). Oxidative stress is also a cause of stroke. Therefore, the pharmaceutical composition of the present invention that protects brain cells against oxidative stress and has an anti-inflammatory effect can be very useful for treating or preventing stroke.

<Application example 4>

Mild cognitive impairment

Many older people have some memory problems but do not have serious difficulties in normal daily life. This stage is called mild cognitive impairment (MCI), and older people diagnosed with mild cognitive impairment are more likely to develop degenerative neurological disorders such as Alzheimer's disease in a few years (10-15% / year). As such, mild cognitive impairment is the transitional stage between normal aging and early Alzheimer's disease and is a predromal degenerative neurodegenerative disease. Autopsy findings in patients with mild cognitive impairment revealed multiple pathologies of senile plaques and nerve fibers. Oxidative damage is involved in the senile plaque induction, and the senile plaque itself causes an inflammatory response. Therefore, the pharmaceutical composition of the present invention that protects brain cells against oxidative stress and has an anti-inflammatory effect can be very useful for treating or preventing mild cognitive impairment.

As described above, the xantholizole of the present invention has an effect of inhibiting various mediators and their activities that cause death of brain cells. In particular, the antioxidant effect of inhibiting the production of lipid peroxidation and reactive oxygen species, the brain cell protective effect of inhibiting atoptoses of brain cells and the anti-inflammatory effect is very excellent. Therefore, the pharmaceutical composition containing the xantholizole of the present invention as an active ingredient can be very useful for the treatment or prevention of cerebral neurological diseases.

Claims (3)

Senile dementia, vascular dementia, alcoholic dementia, thalamic dementia, Parkinson's disease, stroke, Huntington's disease, Creutzfeldt-Jajacob disease, containing xantolizol or a pharmaceutically acceptable salt thereof as an active ingredient, Any one or more diseases selected from the group consisting of pick disease, amyotrophic lateral sclerosis, Parkins-ALS-dementia complications, Wilson's disease, progressive supranuclear palsy, mild cognitive impairment and epilepsy Pharmaceutical compositions for the treatment or prophylaxis. [Formula I]

Senile dementia, vascular dementia, alcoholic dementia, thalamic dementia, Parkinson's disease, stroke, Huntington's disease, Crutzfeldt's disease containing curcuma xanthorrhiza extracted with water or C1-C6 organic solvent as an active ingredient Any one selected from the group consisting of Jay Jakob disease, pick disease, amyotrophic lateral sclerosis, Parkins-ALS-dementia complications, Wilson's disease, progressive supranuclear palsy, mild cognitive impairment and epilepsy Pharmaceutical compositions for the treatment or prevention of one or more diseases. delete

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