KR102343023B1 - Composition comprising Atractylenolide-Ⅰas an effective ingredient for preventing or treating of neurological disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of cranial nerve diseases and a functional health food composition for improvement containing atractylenolide-I as an active ingredient.

Description

A composition comprising Atractylenolide-I and an effective ingredient for preventing or treating of neurological disease, comprising atractylenolide-I and a salt thereof as an active ingredient

The present invention relates to a pharmaceutical composition for preventing or treating neuroinflammation comprising atactylnolide-I and a salt thereof as an active ingredient.

In the central nervous system, many studies have reported that a neuroinflammatory response to brain damage caused by Parkinson's disease, Alzheimer's disease, traumatic injury, stroke, and seizure is the cause (Block ML et al., Nat Rev Neurosci. 8(1)). , 57-69, 2007; Nelson, PT. et al., Ann Med, 34, 491-500, 2002; Griffin, WS et al., J Neuroinflammation, 3, 5, 2006). The inflammatory response at this time is a characteristic of acute brain injury. Neutrophils, monocytes, and phagocytic cells flow in due to activation of resting astrocytes in brain tissue, and pro-inflammatory cytokines and junction molecules and other Inflammatory mediators are active. Neuroinflammation occurs mainly due to the activation of glial cells, which make up about 20% of the brain.

Activation of microglia usually results from signal transduction of three pathways. First, the inflammatory enzymes inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), the second, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), well-known inflammatory cytokines, It has been reported to be induced by interleukin-6 (IL-6), and the transcription factor nuclear factor-κB (NF-κB) (Mc Geer et al., Neurology, 38, 1285-91, 1988; Minghetti, L. et al., Prog Neurobiol, 54, 99-125, 1998; Le, W. et al., J Neurosci, 21, 8447-55, 2001).

Induction of inflammation in glial cells is accompanied by the production of nitrite (NO) by LPS (lipopolysaccharide), and the increase in nitrite (NO) is a pathological mechanism that plays a very important role in cytotoxicity during brain damage. is known (Chen et al., J Biol Chem, 273, 19424-30, 1998).

Atractylenolide-I is the main active ingredient in Atractylodes macrocephala, and is used for indigestion (Zhang, Y. et al. Zhong Yao Cai 1999, 22, 636-640), antioxidant (Wang, CC, etc. Food Chem) Toxicol 2006, 44, 1308-1315) and anticancer (Huang, HL et al. J Ethnopharmacol 2005, 97, 21-29. ) are known to have efficacy. However, studies on the protection of nervous system diseases and inhibition of neuroinflammation are insufficient.

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cranial nerve diseases, etc. containing atractylenolide-I as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention provides a pharmaceutical composition for preventing or treating cranial nerve diseases containing atractylenolide-I as an active ingredient.

The composition can prevent the inflammatory response of glial cells (microglia) induced by LPS or MPTP.

The composition may inhibit the release of nitrite (NO) or reduce the expression of iNOS.

The composition may inhibit the expression of TNF-α, IL-1β or IL-6.

The composition may enhance antioxidant activity.

The composition may inhibit damage to the dopaminergic nervous system of the midbrain.

The cranial nerve disease may be one selected from the group consisting of dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, stroke, multiple sclerosis, learning disability, cognitive impairment, and memory impairment.

One embodiment of the present invention provides a functional health food composition for preventing or improving cranial nerve diseases containing atractylenolide-I as an active ingredient.

The present invention relates to a composition for the prevention or treatment of cranial nerve diseases comprising atactylenoride-I as an active ingredient, wherein the atactylenoride-I has the effect of inhibiting the activity of microglia, and thus A bar that can prevent or treat neuroinflammation, which can be used for prevention or treatment of cranial nerve diseases.

1 is a graph showing (a) nitrite inhibitory efficacy and (b) cytotoxicity test results of atactylenoride-I in BV-2 glial cells stimulated with LPS.
FIG. 2 is a graph showing the enzyme iNOS gene and the MCP-1 and MMP-9 genes of the proinflammatory mediators TNF-α, IL-6, IL1-β and IL-6 genes in LPS-stimulated BV-2 glial cells. It shows the inhibitory efficacy of atactylenolide-I for
3 shows the inhibitory effect of atactylenolide-I on the enzyme iNOS protein involved in the production of inflammation in LPS-stimulated BV-2 glial cells.
4 shows the phosphorylation inhibitory effect of a single compound, atactylenoride-I, on MAPK in BV-2 glial cells stimulated with LPS.
5 shows the concentration-dependent results of the concentration-dependent NF-κB nuclear transcription factor activity inhibitory efficacy and the IκB-α degradation inhibition and phosphorylation inhibition efficacy results of a single compound, atractylenoride-I, in BV-2 glial cells stimulated with LPS. will be.
Figure 6 is an enzyme iNOS gene involved in the production of inflammation in a neurodegenerative animal disease model induced by MPTP, TNF-?, a pro-inflammatory mediator, GFAP, an inflammatory marker of microglial cells, Mac-1 gene, and HO-1 gene, an antioxidant enzyme; Shows the inhibitory efficacy of atactylenolide-I for
7 shows the inhibitory efficacy of atactylenolide-I on GFAP and Mac-1 protein, which are inflammatory markers of microglia in a model of an animal neurodegenerative disease induced by MPTP.
Figure 8 shows the neuroprotective efficacy of atactylenoride-I in a neurodegenerative animal disease model induced by MPTP.
9 is a graph showing the antioxidant power of atactylenoride-I and the increase in the activity of HO-1, an antioxidant enzyme, in a neurodegenerative animal disease model induced by MPTP.

The present inventors performed an analysis of the efficacy of atactylenolide-I through a mechanism study related to the expression of products, genes and proteins of inflammation-related factors induced by lipopolysaccharide (LPS), and MPTP-induced The behavioral power and protection of nerve cells were confirmed in a neurodegenerative animal model, and the present invention was completed by confirming the neuroprotective effect of atactylenoride-I.

Hereinafter, the present invention will be described in detail.

Pharmaceutical composition for prevention or treatment of cranial nerve disease

The present invention provides a pharmaceutical composition for preventing or treating cranial nerve diseases containing atractylenolide-I as an active ingredient. Specifically, the atractylenolide-I may be represented by the following formula (1), and as a major active ingredient of baekchul (Atractylodes macrocephala), may be extracted or purified from baekchul, but is not limited thereto.

[Formula 1]

The composition according to the present invention prevents the inflammatory response of glial cells in LPS or MPTP-induced glial cells (microglia), inhibits the release of nitrite (NO) or reduces the expression of iNOS, TNF-α, IL-1β Alternatively, by suppressing the expression of inflammatory cytokines such as IL-6, it is possible to prevent or treat cranial nerve diseases.

In addition, the composition prevents the degradation of IkB-a, inhibits the activity of NF-κB, inhibits it through the ERK pathway, which is one of MAPKs, which is a representative pathway by LPS, and inhibits the dopaminergic nervous system of the midbrain by MPTP. By inhibiting the damage, it is possible to prevent or treat cranial nerve diseases.

The cranial nerve disease that can be prevented or treated by the composition of the present invention is selected from the group consisting of dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, stroke, multiple sclerosis, learning disability, cognitive impairment and memory impairment It may be, but it is preferable that it is Parkinson's disease, but is not limited thereto.

The dose of atractylenoride-I that may be contained in the composition of the present invention is preferably in a concentration of 0.1 mM to 100 mM, but is not limited thereto. At this time, when the concentration of atactylenolide-I is less than the above range, there is a problem in that it is difficult to prevent apoptosis and thus it is difficult to exert the effect of preventing or treating cranial nerve disease, and when the concentration is exceeded, cytotoxicity There may be toxicity concerns, including

The pharmaceutical composition for preventing or treating cranial nerve disease according to the present invention can be prepared according to a conventional method for oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions, respectively. It may be formulated and used in the form, and may include an appropriate carrier, excipient or diluent commonly used in the preparation of a pharmaceutical composition for formulation.

The carrier or excipient or diluent may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, unknown. various compounds or mixtures including vaginal cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, it can be prepared using commonly used diluents or excipients such as fillers, weight agents, binders, wetting agents, disintegrants, and surfactants.

A solid formulation for oral administration may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. with the atactylenolide-I. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. may be included. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous agents, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol gelatin and the like can be used.

The preferred dosage of the pharmaceutical composition for preventing or treating cranial nerve disease according to the present invention varies depending on the patient's condition, body weight, degree of disease, drug form, administration route and period, but may be appropriately selected by those skilled in the art. However, for a desirable effect, it may be administered at 0.0001 to 2,000 mg/kg per day, preferably 0.001 to 2,000 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. However, the scope of the present invention is not limited by the dosage.

The pharmaceutical composition for preventing or treating cranial nerve disease according to the present invention may be administered to mammals such as rats, mice, livestock, and humans by various routes. All modes of administration can be administered by, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

As described above, atractylenoride-I according to the present invention inhibits the expression of iNOS gene and protein and the expression of inflammatory cytokines in a concentration-dependent manner, and prevents the degradation of IkB-a, thereby enhancing the activity of NF-κB. By inhibiting the ERK signaling pathway and damage to the dopaminergic nervous tissue of the midbrain, it has the effect of inhibiting the activity of microglia, so it is possible to prevent or treat microglia-mediated neuroinflammation. It can be used for prevention or treatment.

Health functional food for prevention or improvement of cranial nerve disease

The present invention provides a health functional food for the prevention or improvement of cranial nerve diseases containing atractylenolide-I as an active ingredient. Specific details of the atlactylenolide-I are the same as described above.

In the health functional food for preventing or improving cranial nerves according to the present invention, when the atactylenolide-I is used as an additive of a health functional food, it can be added as it is or used with other foods or food ingredients, and the conventional It can be used appropriately depending on the method. The mixing amount of the active ingredient may be appropriately determined according to each purpose of use, such as prevention, health or treatment.

The formulation of health functional food may be in the form of powders, granules, pills, tablets, and capsules, as well as in the form of general food or beverages.

The type of the food is not particularly limited, and examples of food to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and dairy products including ice cream. , various soups, beverages, teas, drinks, alcoholic beverages, vitamin complexes, etc., and may include all foods in a conventional sense.

In general, in the production of food or beverage, the atractylenolide-I may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on 100 parts by weight of the raw material. However, in the case of long-term intake for health and hygiene or health control, the amount may be less than or equal to the above range, and in the present invention, there is no problem in terms of safety in terms of using a fraction from a natural product. It can also be used in larger amounts.

Beverages among health functional foods according to the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional beverages. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumartin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the beverage according to the present invention.

In addition to the above, the health functional food for the prevention or improvement of cranial nerve diseases according to the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents. , stabilizers, preservatives, glycerin, alcohols, and carbonates used in carbonated beverages. In addition, the sleep improvement composition of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The proportion of these additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight relative to 100 parts by weight of the health functional food of the present invention.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[ preparation example ]

LPS (E. coli 0111:B4), Tween-20, bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), p-nitrophenyl phosphate, 3-(4, 5-dimethylthiazol-2-yl)-2,5- iphenyltetrazolium bromide (MTT), pyrrolidine dithiocarbamate (PDTC) (Sigma Chemical Co, USA). 6-well, 12-well, 96-well tissue culture plate 100 mm culture plate. (NUNC Inc, IL, USA)

DMEM was used as a cell culture medium, and 5% FBS and 100 units/ml penicillin/streptomycin as an antibiotic were used (Gibco/Invitrogen, USA). Products of Roche (USA) were used as protease inhibitors and phosphatase inhibitors, and the antibodies used in the experiment were iNOS, ERK, p-ERK, Akt, p-Akt, PI3K, p-PI3K, inhibitor of kappa. B-alpha (IκB-α), p-IκB-α and β-actin (Cell signaling, Co,USA), HO-1, manganese superoxide dismutase (MnSOD) (Enzo Life Sciences, USA), macrophage-1 antigen ( Mac-1) (AbD Serotec, USA), glial fibrillary acidic protein (GFAP) (Abcam, USA), and tyrosine hydroxylase (TH) (Calbiochem, USA) were used.

[Test method]

One. BV -2 cell culture

The BV-2 immortalized murine microglial cell line was provided by Dr. Kyungho Seok of Kyungpook National University. BV-2 cells were grown and maintained in DMEM supplemented with 5% heat-inactivated FBS. Penicillin/streptomycin (100 U/mL) was added to the DMEM medium, and the cells were maintained at _{37° C., 5% CO 2 in a humidified incubator.} ART-I was dissolved in DMSO, and the control/vehicle group was treated with DMSO only.

2. Animals and MPTP administration

Male C57BL6/J mice (25-28 g) (Samtako Bio Korea) aged 8-9 weeks were acclimatized for 14 days before drug treatment. Animal experiments were ethically performed in accordance with the National Institute of Laboratory Animal Care and Use (NIH publication No. 85-23, revised in 1985), and the experimental procedures were approved by the Institutional Animal Care and Use Committee of Konkuk University. Experimental mice were allowed to freely ingest food and water in a controlled environment (temperature 23°C ± 1°C and humidity 50% ± 5%). Indoor lighting was supplied between 8 am and 8 pm. 126 mice were randomized into 7 groups of 18 mice: vehicle group, MPTP group, ATR-I (30 mg/kg/10 ml) group, Selegiline (10 mg/kg/10 ml)+MPTP group, ATR -I (3mg/kg/10ml)+MPTP group, ATR-I (10mg/kg/10ml)+MPTP group, and ATR-I (30mg/kg/10ml)+MPTP group.

100 mg/kg/10 ml MPTP was intraperitoneally administered to all groups except for the vehicle group and the ATR-I (30 mg/kg/10 ml) group, 4 times at 1 hour intervals. 12 hours after the 4th MPTP injection, ATR-I (i.p) was administered 3 times a day for 3 days and 7 days. Selegiline (i.p) was also administered once a day for 3 and 7 days after the 4th injection of MPTP. Experimental mice were sacrificed 3 and 7 days after the last administration of ATR-I. MPTP was prepared by washing with saline before use. Before administration of ART-I, it was suspended in a 0.5% methyl cellulose solution containing 1% Tween 80. The control/vehicle group was administered with a normal methyl cellulose solution.

3. Pole test

In order to measure the degree of motility, which is a typical symptom of Parkinson's disease, the pole test of the modified procedure was performed, and it was performed three times consecutively for each mouse. The experimenter was kept in the dark during the experiment for all groups.

4. Nitrite measurement assay and cell viability assay

The release of nitric oxide (NO) in the cell supernatant was measured using the Griess reaction. BV-2 cells (2.5×10 ⁴ cell/mL) were inoculated into a 96-well plate with 200 μL of a culture medium pretreated with ATR-I at concentrations of 25 μM, 50 μM, and 100 μM for 1 hour, and then for 24 hours. Stimulation was carried out in the presence or absence of LPS (100 ng/mL). Additional procedures were performed according to previously published data. Results are representative of three independent experiments.

Cytotoxicity of ART-I was assessed using MTT-based colorimetry, and MTT reduction was measured with formazan. BV-2 cells were pretreated with various doses of ART-I for 1 hour regardless of exposure to LPS (100 ng/mL) for 24 hours. Cell viability measurements were performed in a conventional manner and expressed as a percentage of control (untreated cells).

5. Immunohistochemistry

Mac-1 (1:500; 131 AbD Serotec, Oxford, UK) and GFAP (1:2000) in free floating brain sections of mouse striatum (STR) and SNpc sacrificed 3 days after MPTP injection ; Abcam) were tagged with primary antibodies. The fragment labeled Mac-1 was incubated with a biotinylated anti-rat (1:200) (Vector Laboratories, Burlingame, CA, USA) antibody for 2 hours. Thereafter, the avidin biotin-peroxidase complex of the Vectastain Elite ABC Kit (Vector Laboratories) was incubated for 60 minutes at room temperature according to the supplier's recommendations. Finally, each section was reacted for 80 seconds with Vector DAB substrate kit (Vector Laboratories) to develop color, and the stained section was observed using a bright field microscope (Carl Zeiss Inc., Oberkochen, Germany). . GFAP fragments were incubated for 1 hour using rabbit antigoat GFAP antibody (1:200) (Alexa Flour, Invitrogen, Carlsbad, CA, USA). After the sections were dehydrated with ethanol (70%, 80% and 90%), they were treated with 100% xylene to prevent slipping, and the stained sections were subjected to a fluorescent microscope (Carl Zeiss Inc., Oberkochen, Germany). was observed using STR and SNpc sections of mice sacrificed on day 7 were labeled with primary TH antibody (1:1500; Calbiochem, Billerica, MA, USA) and stored at 4°C for one day. In addition, the sections were treated with biotinylated anti-rabbit (1:500) (Vector Laboratories, Burlingame, CA, USA) antibody and incubated for 2 hours. Thereafter, the avidin biotin-peroxidase complex of the Vectastain Elite ABC Kit (Vector Laboratories) was incubated for 60 minutes at room temperature according to the supplier's recommendations. Finally, each section was treated with Vector DAB substrate kit (Vector Laboratories) for 80 seconds to develop color. The sections were dehydrated with ethanol (70%, 80% and 90%) and then mounted and covered with 100% xylene treatment for 60 seconds. The stained sections were observed using a bright field microscope (Carl Zeiss Inc., Oberkochen, Germany). Effect quantification in brain-tissue sections was performed by measuring the number of TH-immunopositive neurons in the SNpc using Image J software (Bethesda, MD, USA) and measuring the optical density of TH-positive fibers in the STR. Data are expressed as a percentage of the measured values of the control group.

6. Isolation of total RNA and reverse transcription-polymerase chain reaction (RT- PCR )

25 μM, 50 μM, and 100 μM concentrations of ART-I were ^{pre-incubated with BV-2 cells (5.0×10 5} cell/mL) for 1 hour, followed by LPS (100 ng/mL) presence. or in the absence of treatment for 6 hours each. Animal tissues were washed with 0.1M cold PBS (pH 7.4), homogenized using a tuberculin syringe, and stored at -80°C until RNA was extracted. Total RNA was isolated by extraction with TRIzol (Invitrogen). For RT-PCR, using the First-Strand cDNA Synthesis Kit (Invitrogen), 2.5 μg of total RNA of each group of Example 2 was reverse transcribed. Then, cDNA was amplified using the specific primers shown in Table 1. PCR products were analyzed on 1% agarose gel stained with ethidium bromide and the bands were visualized by UV light.

target gene Primer sequences from BV-2 microglia and C57BL6/J mice SEQ ID NO: iNOS F-5'-CTTGCAAGTCCAAGTCTTGC-3'' One R-5'-GTATGTGTCTGCAGATGTGCTG-3'' 2 TNF-α F-5'-TTCGAGTGACAAGCCTGTAGC-3'' 3 R-5'-AGATTGACCTCAGCGCTGAGT-3'' 4 IL-6 F-5'-GGAGGCTTAATTACACATGTT-3'' 5 R-5'-TGATTTCAAAGATGAATTGGAT-3' 6 IL-1β F-5'-CATATGAGCTGAAAGCTCTCCA-3'' 7 R-5'-GACACAGATTCCATGGTGAAGTC-3'' 8 MCP-1 F-5'-AGATGCAGTTAACGCCCCAC-3'' 9 R-5'-GACCCATTCCTTCTTGGGT-3'' 10 MMP-9 F-5'-CGCTCATGTACCCGCTGTAT-3'' 11 R-5'-TGTCTGCCGGACTCAAAGAC-3'' 12 HO-1 F-5'-GGATTTGGGGCTGCTGGTTTTC-3'' 13 R-5'-GCAGTGGCAAAGTGGAGATTG-3'' 14 Mae-1 F-5'-TCAAGCGGCAGTACAAGGAC-3'' 15 R-5'-GCCACACACAGAGCTTGCTT -3'' 16 GFAP R-5'-CCCTTCAGGACTGCCTTAGT -3'' 17 R-5'-CCCTTCAGGACTGCCTTAGT -3'' 18 GAPDH F-5'-GCAGTGGCAAAGTGGAGATTG-3'' 19 R-5'-TGCAGGATGCATTGCTGACA-3'' 20

7. SDS-PAGE and western blot Western blot analysis

BV-2 cells (5.0×10 ⁵ cell/mL) were cultured in a 6-well plate, pretreated with ART-I at concentrations of 25 μM, 50 μM, and 100 μM for 1 hour, followed by LPS (100 ng/mL). Each was exposed for 18 hours. Preparation of cell lysates (whole cells and nuclei), electrophoresis and immunoblotting procedures were performed by known methods. For animal experiments, STR and ventral midbrain (VM) tissues were processed according to a known method. PVDF membrane with anti-iNOS(1:1000), anti-IκB-α(1:1000), anti-p-IκB-α(1:1000), anti-ERK(1:1000), anti-p-ERK (1:1000), anti-Akt(1:1000), anti-p-Akt(1:1000), anti-PI3K(1:1000), anti-p-PI3K(1:1000), anti-HO- 1 (1:1000), anti-β-actin (1:2000), anti-MnSOD (1:1000), anti-GFAP (1:50,000), anti-Mac-1 (1:500), anti-TH (1:1000), anti-NF-κB/p65 (1:500), and anti-nucleolin (1:500) were treated and incubated for one day. Thereafter, horseradish peroxidase-conjugated secondary antibodies (1:1000-5000) (Cell Signaling Technology and Santa Cruz biotechnology) were treated and incubated for 1 hour. The optical density of the antibody-specific band was analyzed with a Luminescent Image Analyzer, LAS-3000 (Fuji, Tokyo, Japan).

8. Detection of intracellular ROS generation using a flow cytometer

Intracellular reactive oxygen species (ROS) production was measured using the _{non-fluorescent compound H 2 DCFDA, as described above.} H ₂ DCFDA is a stable, non-polar compound that readily diffuses into cells to form DCFH. In the presence of peroxidase, intracellular ROS transforms DCFH into a highly fluorescent compound, DCF. Thus, the amount of ROS is proportional to the fluorescence sensitivity exhibited by the cells. Briefly, BV-2 cells (5.0×10 ⁵ cells/6-well plate) stimulated with LPS for 4 hours were treated with DMSO or ART-I (25 μM, 50 μM and 100 μM) and incubated at 37° C. for 1 hour. did In addition, cells were _{treated with 10 μM of H 2} DCFDA (dissolved in DMSO) at 37° C. for 45 min. Thereafter, the cells were treated with trypsin, and the fluorescence intensity was measured using a FACS Calibur flow-cytometry system.

9. Immunofluorescence ( Immunofluorescence assay)

BV-2 cells ( ⁵ ×10 5 cells/well) were cultured on sterile coverslips of 24-well plates, and treated with ATR-I (100 μM) for 1 hour. Then, in order to detect the p65 sub-unit in NF-κB cells, LPS (100 ng/mL) was treated for 0.5 hours. Thereafter, further procedures were performed for immunofluorescence analysis according to known methods.

[ Example ]

Example One. microglia BV in -2 cells Atractylenolide -I toxicity test

In order to determine the effect of LPS and a single compound, atlactylenolide-I, on cell survival in LPS-stimulated Microglia BV-2 cells, MTT assay was performed.

As a result, as shown in FIG. 1B, as a result of MTT measurement, it was confirmed that cell viability did not change in all experimental groups treated with LPS and a single compound, atlactylenoride-I, alone or together, compared to the control group. It can be seen that the neuroinflammation inducing substance LPS and the single compound atractylenoride-I do not affect cell survival.

Example 2. by LPS stimulated microglia BV A single compound in -2 cells Atractylenolide -I concentration-dependent NO release, iNOS Inhibition of gene and protein expression

2-1) NO release inhibitory action

In order to analyze the anti-inflammatory efficacy of a single compound, atractylenoride-I, the concentration-dependent inhibitory effect of nitrite (NO) produced in Microglia BV-2 cells stimulated with LPS (100 ng/ml), the same neuroinflammation factor was confirmed to be visible. For the measurement of nitrite (NO), NO assay using Griess reagent was performed.

As a result, nitrite (NO) induced by LPS in BV-2 cells showed an increase of about 40 times compared to the control group, and a single compound, atactylenolide-I, was added to the experimental group by concentration (25 μM, 50 μM, and 100 μM ), as shown in FIG. 1A , it was confirmed that the amount of nitrite (NO) released was decreased in a concentration-dependent manner with atactylenolide-I.

2-2) iNOS gene expression inhibition

The expression pattern of the iNOS gene was treated with LPS and a single compound, atactylenolide-I, at different concentrations (25 μM, 50 μM, and 100 μM) in BV-2 and cultured for 6 hours, then RNA was identified and RT-PCR was performed to analyze the gene expression pattern through a quantitative method.

As a result, as shown in FIGS. 2 and 3 , it can be confirmed that the concentration-dependent inhibition of the expression of atactylenolide-I, a single compound at the gene and protein levels of iNOS, an enzyme inducing the synthesis of nitrite (NO), in a concentration-dependent manner. there was. That is, as shown in FIG. 2 , it can be seen that the expression level of the iNOS gene was almost absent in the control group not treated with LPS, whereas the expression level of the iNOS gene was significantly increased in the experimental group treated with LPS, and the single compound atractyleno It was confirmed that the expression change pattern of Ride-I decreased in a concentration-dependent manner.

2-2) iNOS protein expression inhibition

In order to analyze the expression pattern of iNOS protein, LPS and a single compound, atractylenolide-I, were treated at the same concentration as in the analysis process of iNOS gene. Expression changes were quantitatively analyzed.

As a result, as shown in FIG. 3 , it was confirmed that the iNOS protein expression level was almost absent in the control group not treated with LPS. In the LPS-treated experimental group, the expression level of iNOS protein was significantly increased, and in the case of a single compound, atractylenoride-I, it was confirmed that the expression change in a concentration-dependent manner decreased.

Example 3. by LPS stimulated microglia BV -2 In the production of inflammatory-mediated cytokines in cells, a single compound Atractylenolide I concentration-dependent inhibitory efficacy of

In order to derive a change in the expression pattern of the inflammatory-mediated cytokine TNF-α, IL-1β, and IL-6 genes, the expression pattern of the inflammation-mediated cytokine gene was analyzed in BV-2 cells by LPS and a single compound, atractylenolide- I was treated at different concentrations (25 μM, 50 μM, and 100 μM) and incubated for 6 hours, RNA was identified and RT-PCR was performed, and the expression pattern of the gene was analyzed through a quantitative method in three genes. Common expression changes were identified. GAPDH was used as a control for gene quantitative analysis.

As a result, as shown in FIG. 2 , the expression levels of TNF-α, IL-1β, and IL-6 genes were almost absent in the control group not treated with LPS, and the LPS (100ng/ml) treated experimental group compared to the control group. Upon treatment with the compound, atractylenoride-I, it was confirmed that the expression change pattern decreased in a concentration-dependent manner.

Example 4. by LPS stimulated BV -2 in glial cells Atractylenolide - of I MAPKs pathway ERK Inhibition of phosphorylation and inflammation-related protein PI3K / Akt Concentration-dependent inhibitory efficacy results of phosphorylation

It has been previously known that the inflammatory response through LPS treatment typically proceeds through the MAPKs (mitogen-activated protein kinase) pathway. It was confirmed whether atractylenoride-I inhibits the inflammatory response through the MAPK pathway.

As a result, as shown in FIG. 4 , it was confirmed that the phosphorylation amount in the ERK pathway in ERK, one of the components of MAPKs, increased compared to the control group when LPS was treated. However, it was confirmed that the degree of phosphorylation decreased in a concentration-dependent manner of atactylenolide-I when atactylenolide-I was treated. It was confirmed that I was inhibited in a concentration-dependent manner.

Example 5. by LPS stimulated BV -2 In glial cells, a single compound Atractylenora Efficacy of Id-I in inhibiting the activity of NF-κB nuclear transcription factor

In order to analyze the efficacy of atractylenolide-I, the effect on the activation of NF-κB, a transcription factor that expresses pro-inflammatory cytokines during induction of inflammation by LPS, was confirmed. The influx of p65 constituting NF-κB into the nucleus from the nucleolus was confirmed through immunofluorescence staining, the nucleoprotein was identified, and the expression of NF-κB p65 in the nucleus was confirmed by Western blot.

BV-2 cells were treated with LPS (100ng/ml) and atactylenoride-I at a concentration of 100 μM and cultured for 30 minutes. The degree was confirmed (FIG. 5B). When nothing was treated, it was confirmed that a lot of protein p65 was embedded in the nucleus, and it was confirmed that a lot of protein p65 was introduced into the nucleus during LPS treatment, and when treated with atactylenolide I, it was confirmed that protein p65 was inserted into the nucleus of protein p65. was confirmed to prevent the inflow of

5A is the result of quantitatively confirming the expression pattern of the nuclear protein through Western blot by identifying the nuclear protein in order to more accurately confirm the fact confirmed by the immunofluorescence method. In the control group not treated with LPS, there was almost no expression of NF-κB p65, but in the experimental group treated with LPS, the expression of NF-κB p65 was increased. Confirmed. Nucleolin is a control protein for quantitative confirmation of nucleoprotein.

5A and 5B show the concentration-dependent inhibitory efficacy of atlactylenoride I according to NF-κB activity in LPS-stimulated BV-2 glial cells.

Example 6. MPTP -Inflammatory response improvement effect on treated neurodegenerative mouse model

MPTP is widely known as a neurotoxin capable of inducing neuroinflammation and dopaminergic neurodegeneration by inducing the activation of glial cells in the central nervous system (CNS). The neuroinflammation and neuroprotective effects of atactylenoride-I on MPTP-induced neuroinflammation in a neurodegenerative mouse model were analyzed.

As a result, as shown in FIG. 6, at the gene level of iNOS, an enzyme inducing nitrite (NO) synthesis, and TNF-α, an inflammation-mediated cytokine in the inflammatory response of glial cells induced by MPTP, at the gene level, atractylenolide- It was confirmed that iNOS and TNF-α gene expression was inhibited in a concentration-dependent manner of I.

In addition, as shown in FIGS. 6 and 7 , it was confirmed that the expression of atactylenolide-I was inhibited in a concentration-dependent manner at the gene level and protein level of GFAP and Mac-1, which are activation markers of glial cells. As a result of confirming this using the tissue immunostaining method, it was confirmed that the gene level of Mac-1 and the expression of the protein were inhibited in a concentration-dependent manner of atactylenoride-I.

Example 7. MPTP -treated neurodegenerative mice Neuroprotective effect on the model

The loss of dopaminergic neurons by MPTP in SNpc (substantia nigra pars compacta) was measured through TH (tyrosine hydroxylase)-positive neurons.

As confirmed by Western blot and tissue immunostaining, the injection of MPTP decreased the expression of TH and the number of TH-bearing neurons. However, it was confirmed that the MPTP-induced loss of TH expression was reduced in the group administered with atractylenoride-I ( FIG. 8 ).

Therefore, it can be seen that atractylenoride-I protects neurons from MPTP-induced neuroinflammation.

Example 8. MPTP -Antioxidant activity against treated neurodegenerative mouse model

In the MPTP-treated neurodegenerative mouse model, it was confirmed whether ROS increased in the brain through flow cytometry and decreased expression of antioxidant enzymes HO-1 and Mn-SOD.

As a result, as shown in FIG. 9A , there was almost no increase in ROS in the control group not treated with MPTP, and the expression level of ROS in the experimental group treated with MPTP in a concentration-dependent manner when treated with atractylenolide-I compared to the control group It can be seen that this decreases.

In addition, as shown in FIGS. 9B and C, it was confirmed that the expression of antioxidant enzymes HO-1 and Mn-SOD was increased in a concentration-dependent manner when atactylenoride-I was treated.

Example 9. Statistical Analysis

Statistical analysis was performed using GraphPad software version 5 (GraphPad, La Jolla, 199 CA, USA). Data mean mean ± standard error (SEM) of at least 3 independent experiments. Significant differences between groups were determined through one-way analysis (ANOVA) and Tukey's post-hoc analysis. P <0.05 is considered statistically significant.

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> Konkuk University Glocal Industry-Academic Collaboration Foundation <120> Composition comprising Atractylenolide-1 as an effective ingredient for preventing or treating of neurological disease <130> 1062241 <160> 20 <170> KopatentIn 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer of iNOS <400> 1 cttgcaagtc caagtcttgc 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of iNOS <400> 2 gtatgtgtct gcagatgtgc tg 22 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer of TNF-alpha <400> 3 ttcgagtgac aagcctgtag c 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of TNF-alpha <400> 4 agattgacct cagcgctgag t 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer of IL-6 <400> 5 ggaggcttaa ttacacatgt t 21 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of IL-6 <400> 6 tgatttcaaa gatgaattgg at 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer of IL-6 beta <400> 7 catatgagct gaaagctctc ca 22 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of IL-6 beta <400> 8 gacacagatt ccatggtgaa gtc 23 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer of MCP-1 <400> 9 agatgcagtt aacgccccac 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of MCP-1 <400> 10 gacccattcc ttcttggggt 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer of MMP-9 <400> 11 cgctcatgta cccgctgtat 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of MMP-9 <400> 12 tgtctgccgg actcaaagac 20 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer of HO-1 <400> 13 ggatttgggg ctgctggttt c 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of HO-1 <400> 14 gcagtggcaa agtggagatt g 21 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer of Mae-1 <400> 15 tcaagcggca gtacaaggac 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of Mae-1 <400> 16 gccacacaca gagcttgctt 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GFAP <400> 17 cccttcagga ctgccttagt 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GFAP <400> 18 cccttcagga ctgccttagt 20 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GAPDH <400> 19 gcagtggcaa agtggagatt g 21 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GAPDH <400> 20 tgcaggatgc attgctgaca 20

Claims (8)

A pharmaceutical composition for preventing or treating Parkinson's disease caused by damage to dopaminergic nervous tissue in the midbrain, containing atractylenolide-I as an active ingredient.
According to claim 1,
The composition is characterized in that it prevents the inflammatory reaction of glial cells (microglia) induced by LPS (Lipopolysaccharide) or MPTP (1-methy-4-phenyl-1,2,3,6-tetrahydropyridine), midbrain dopamine A pharmaceutical composition for preventing or treating Parkinson's disease caused by damage to sexual nervous tissue.
According to claim 1,
The composition inhibits the release of nitrite (NO) or reduces the expression of iNOS, a pharmaceutical composition for preventing or treating Parkinson's disease caused by damage to the midbrain dopaminergic nervous tissue.
According to claim 1,
The composition is a pharmaceutical composition for preventing or treating Parkinson's disease caused by damage to the midbrain dopaminergic nervous tissue, characterized in that it inhibits the expression of TNF-α, IL-1β or IL-6.
According to claim 1,
The composition is a pharmaceutical composition for preventing or treating Parkinson's disease caused by damage to the dopaminergic nervous system of the midbrain, characterized in that it enhances antioxidant activity.
delete delete A functional health food composition for preventing or improving Parkinson's disease caused by damage to dopaminergic nervous tissue in the midbrain, containing atractylenolide-I as an active ingredient.

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