KR20140148168A - Pharmaceutical Composition and Functional Food for Preventing or Treating Neurodegenerative Disease by Cnidium officinale Extracts, and Extracting Method of Cnidium officinale Extracts - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and health food containing a Cnidium officinale extract for preventing or treating neurodegenerative diseases, and to a method for preparing a Cnidium officinale extract. The present invention relates to a pharmaceutical composition and health food for preventing or treating neurodegenerative diseases, in which a Cnidium officinale extract suppresses proinflammatory mediator of activated microglia in neuroinflammation caused by neuroglia cells, thereby having an effect of supprssing neuroinflammation; and to a method for preparing a Cnidium officinale extract.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases and a method for extracting cnidium officinale extracts from cnidium officinale extracts,

The present invention relates to a composition for preventing or treating neurodegenerative diseases containing Cnidium officinale extracts (CO), a health functional food, and a method for extracting such a cnidium extract. More particularly, To a composition for preventing or treating neurodegenerative diseases, a health functional food, and a method for extracting cucurbitaceae extract, which contain a cucurbitaceae extract exhibiting nerve inflammation inhibitory effect by inhibiting an inflammatory mediator.

In the past, there have been many studies that neuronal inflammation acts as a cause of various neurodegenerative diseases.

Activated glial cells are continuously exposed to nitrite (NO), reactive oxygen species (ROS), inflammatory enzymes Inducible nitric oxide synthase (iNOS), Cyclooxygenase-2 (COX-2), and proinflammatory mediators IL- 6, and TNF-a have been reported to cause various central nervous system diseases such as multiple sclerosis, Parkinson's disease, Alzheimer's disease and Huntington's disease (Gao et al., 2002; Nelson et al., 2002; Eikelenboom and Van Gool, 2004).

At this time, the reduction of proinflammatory mediators in the glial cells can suppress the expression of these diseases (Matsumoto et al., 1992; Liu and Hong, 2003).

In the cell-based model, various proinflammatory factors and neurotoxins induced by LPS in LPS-induced neuronal damage in inflammatory processes are well known in the mechanism studies (Kim et al., 2004a, b).

It has also been reported that LPS-activated glial cells increase neurotoxicity by proinflammatory mediators and cytotoxins (Dawson et al., 1994).

Given the importance of inflammatory responses in neuroinflammation, inhibiting the production of these inflammatory-related factors in activated glial cells will be the goal of treating various neuroinflammatory diseases.

Cnidium officinale Makino has long been recognized as one of the most important medicinal plants in China and Korea and has been recognized as an antioxidant (Jeong et al., 2009), anti-angiogenesis (Kwak et al., 2002) al., 1995; Kim et al., 2003).

However, there has been no research to utilize the extract of Astragalus membranaceus as a composition for preventing or treating neurodegenerative diseases, paying attention to the effect of preventing and treating neurodegenerative diseases of astrocytes.

For example, Korean Patent Registration No. 10-0375921 entitled " Tungkang Jelly and Its Production Method " describes a technique for manufacturing a celestial gland as a jelly in order to utilize the effect of the celestial gland. However, It was not a remarkable technique.

The object of the present invention is to provide a composition and health functional food for preventing and treating neurodegenerative diseases, which contains astragalus extract exhibiting nerve inflammation inhibitory effect by inhibiting the proinflammatory mediators of activated glial cells, .

The present invention provides a composition for preventing or treating a neurodegenerative disease, wherein the ethanol solvent comprises 1 to 20 parts by weight of water or 1 to 20 parts by weight of an alcohol having 1 to 4 carbon atoms per 100 parts by weight of ethanol And is a submixed mixed solvent.

The present invention also provides a formulation formulated by adding a pharmaceutically acceptable excipient in the form of tablets, capsules, solutions, suspensions, syrups or beverages to such compositions, and provides a herbal medicine or a health functional food.

The present invention also provides a method for extracting ethanol extract of celadon shell, comprising the steps of: (A) collecting and drying a piece of celadon; (B) mixing the dried piece of astragalus with a polar solvent containing ethanol and pulverizing using an ultrasonic grinder; And (C) a step of extracting the celadon root extract from the mixture of the crushed celadon shell and the polar solvent, followed by filtration, followed by vacuum concentration and lyophilization.

The step (A) may include: (A1) drying the alabaster fragments collected from the alabaster fragments at a temperature of 15 ° C to 20 ° C for 3 days to 5 days; And (A2) pulverizing the dried celestial pieces to have a particle size of 0.1 mm to 5 mm.

Also, the polar solvent in step (B) is a mixed solvent comprising 1 to 20 parts by weight of water or 1 to 20 parts by weight of an alcohol having 1 to 4 carbon atoms per 100 parts by weight of ethanol.

The step (C) may include: (C1) extracting the ethanol extract of the moxibustion extract using an ultrasonic mill while maintaining the extraction temperature at 15 to 25 ° C; (C2) Vacuum concentrating and extracting the extract of the citronelles ethanol at 50 to 60 ° C and lyophilization; And (C3) a lyophilized ethanol extract, and concentrating the mixture at 40 to 50 ° C under reduced pressure.

According to the present invention, there is provided a pharmaceutical composition and a health functional food composition for preventing or treating neurodegenerative diseases containing astragalusae which exhibit neuroinflammatory activity by inhibiting the proinflammatory mediators of activated neuroglial cells, There is an effect to provide.

FIGS. 1A and 1B are graphs showing the cytotoxicity and nitrite inhibition activity of the extract of T. kansen according to the present invention.
FIG. 2A and FIG. 2B are graphs showing the effect of inhibiting the active oxygen of the extract of Angelica keiskei according to the present invention.
FIG. 3A and FIG. 3B are graphs showing the inhibitory effect of genes and proteins on iNOS, an inflammation-inducing enzyme involved in the extract of Astragalus membranes according to the present invention.
FIGS. 4A and 4B are graphs showing the inhibitory effect of gene and protein on COX-2, an inflammation-inducing enzyme involved in the extract of Astragalus membranes according to the present invention.
FIGS. 5A to 5D are graphs showing inhibitory effects on the proinflammatory mediators TNF-.alpha., IL-1.beta., And IL-6 gene of astragalus extract according to the present invention.
6A to 6C are graphs showing the inhibitory effects of the extract of T. gentianus according to the present invention on NF-κB activity.
FIGS. 7A and 7B are graphs showing the inhibitory effects of the extracts of ciliopsis thaliana according to the present invention on the p38 phosphorylation activity.

Before describing the specific details for the practice of the invention, terms and words used in the specification and claims should be construed to enable the inventor to properly define the concept of a term in order to best describe its invention It should be interpreted as meaning and concept consistent with the technical idea of the present invention.

It is to be noted that the detailed description of known functions and constructions related to the present invention is omitted when it is determined that the gist of the present invention may be unnecessarily blurred.

The present inventors have conducted a mechanism research on the expression of products, genes and proteins of inflammatory factors induced by Lipopolysaccharide (LPS). Based on molecular biochemical experimental analysis, the efficacy of Cnidium officinale Makino (CO) extract Respectively.

In particular, the production of abnormal proinflammatory cytokines has been reported to cause inflammatory damage (Feldmann et al., 1998; Dinarello, 1999; De Nardin, 2001) (Boka et al., 1994; Hunot et al., 1999), the efficacy of inhibiting the production of cytokines is a major analytical efficacy for inhibiting neuroinflammation.

Accordingly, the present invention provides a composition containing an extract of Angelica keiskei koidz. Exhibiting an excellent neurodegenerative disease preventing or treating effect, and a method of extracting Angelica keiskei koidz.

The present invention also relates to a composition formulated by adding a pharmaceutically acceptable excipient in the form of tablets, capsules, solutions, suspensions, syrups or edible beverages to astragalus extracts, a herbal medicine preparation containing astragalus extract, Or drinks.

Hereinafter, a method for extracting citrine ethanol extract according to a preferred embodiment of the present invention will be described in detail.

First, a dry celestial sample is added to liquid ethanol to produce a celestial ethanol mixture (A).

(A) comprises the steps of: (A1) drying a piece of astragm harvested by taking a piece of astragmatum from 3 to 5 days while maintaining a temperature condition of 15 ° C to 20 ° C; And (A2) pulverizing the dried celestial pieces to have a particle size of 0.1 mm to 5 mm.

The ethanol of step (A) can be used for mixed polarity daily. Such a polar solvent may be used in an amount of 1 to 20 parts by weight or 1 to 20 parts by weight of water, have.

Also, in step (A), the concentration of ethanol is preferably 90% to 99%, and the amount of ethanol is preferably 1000 parts by weight to 1500 parts by weight with respect to 100 parts by weight of the Angelite sample. In the present invention, Was added to 10 L of 95% ethanol.

Next, the celestial ethanol mixture is pulverized at room temperature using an ultrasonic pulverizer (B).

In the present invention, a product of BRANSON Ultrason's Corporation, USA is used as the ultrasonic mill in step (B), and the pulverization in step (B) is performed 10 to 20 times for 10 minutes to 20 minutes at intervals of 1 hour to 3 hours .

Next, a crushed celestan ethanol mixture is obtained, concentrated, and lyophilized to obtain celadon ethanol extract (C).

The ethanol mixture obtained in step (C) is preferably carried out using a 0.22 μM film filter, and the concentration is preferably carried out using a rotary evaporator.

The step (C) may further comprise: (C1) extracting the ethanol extract of ciliaceae using an ultrasonic mill while maintaining the temperature at 15 to 25 ° C; (C2) Vacuum concentrating and extracting the extract of the citronelles ethanol at 50 to 60 ° C and lyophilization; And (C3) lyophilized astragalus ethanol extract into a container, followed by concentration under reduced pressure at 40 ° C to 50 ° C.

In step (C), 43 g of ethanol extract of Angelica keiskei koidz. Were obtained in step (A) after 1 kg of Angelica koreana sample was added to 10 L of 95% ethanol.

The ethanol extract according to the preferred embodiment of the present invention extracted as described above inhibits neuroglial cells to reduce neuronal inflammation.

Specifically, the ethanol extract according to a preferred embodiment of the present invention is characterized in that it contains a nitrite, an iNOS and an inflammation-related enzyme iNOS and COX-2, a protein stage, a proinflammatory cytokine such as TNF-a, IL- , Which is related to the entry of the nuclear protein p65 into the nucleus and to the phosphorylation of p38.

In the case of LPS treatment, IκB-α is rapidly denatured and phosphorylation is increased, but treatment with cynomolgus monkey extract inhibits phosphorylation of IκB-α in a concentration-dependent manner and prevents the entry of nuclear protein into the nucleus. Therefore, cecum extract inhibits NF-κB signaling pathways in LPS-activated glial cells, thereby controlling proinflammatory mediators.

Activated glial cells transmit signals through three pathways of MAPKs (ERK, p38, JNK) (Ock and Suk, 2010; Park et al., 2010) Thus inhibiting nerve inflammation.

Murine BV-2 microglial cells were maintained in DMEM supplemented with 5% FBS and 100 units / ml penicillin / streptomycin in an inactivated condition. The cells were maintained in a humidified atmosphere of 5% CO ₂ , 95% O ₂ incubator.

The number of cells of Murine BV-2 microglial cells was 5 × 10 ⁵ cells / ㎖. The ethanol extract of celadon was diluted in the medium and treated with 1 ㎍ / ㎖, 10 ㎍ / ㎖, 100 ㎍ / , And LPS was diluted in medium to 100 ng / ml.

In order to confirm the reproducibility of the experimental results, the experimental results in the following experimental examples were expressed as mean value ± standard deviation, and each experiment was carried out three times. In addition, two-way ANOVA was performed using the Bonferroni method using Sigmaplot 11.1 software for the statistical analysis of the experimental results.

Experimental Example 1 - Cell culture  And cytotoxicity test ( MTT assay )

The MTT assay can be used to perform cytotoxicity tests on ethanol extracts of the ciliates.

Specifically, 30 μl of MTT at a concentration of 0.5 mg / ml was added to the cells on the microplate, reacted for 2 hours in an incubator, and dissolved in formazan crystals using 100 μl of DMSO. Then, using a Vmax microplate reader with a wavelength of 540 nm Absorbance was measured.

Experimental Example 2 - Assay of NO

Assay of NO is a microplate (96 well) assay method for quantitatively measuring NO (nitric oxide) production.

After 5 × 10 ⁴ cells / well of microcells were added to the microplate, LPS and celecoxib were added so that the concentration was different. The reaction was carried out in a 24 h incubator, Was reacted with Griess reagent (1% sulfanilamide / 0.1% N- (1-naphthyl) -ethylenediamine dihydrochloride / 2.5% H ₃ PO ₄ ) and the value was measured using a Vmax 96-well microplate spectrophotometer with a wavelength of 550 nm .

Experimental Example 3 - Isolation of total RNA and RT - PCR

The total RNA of the Microglia BV-2 cell line was isolated using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer's instructions.

1 μl of TRIZol reagent was added to the microglia BV-2 cell line, 200 μl of chloroform was added, and the mixture was stirred at room temperature for 5 minutes and centrifuged (4 ° C., 10,000 rpm, 10 minutes) Lt; / RTI > was transferred to a new tube.

500 ㎕ of the same amount of isopropanol was added and reacted at room temperature for 10 minutes, followed by centrifugation (4 캜, 14000 rpm, 15 minutes). The supernatant was removed, washed twice with 75% EtOH, And dissolved using DEPC-water.

DNase (Roche AC, USA) was diluted at a ratio of 1: 10 and 2 μl was added. After reacting at 25 ° C for 15 minutes, 78 μl of DEPC-water was added to 22 μl of sample to prepare 100 μl of sample Respectively.

After adding 100 μl of TRIZol reagent and stirring, 25 μl of chloroform was added and stirred. The mixture was reacted at room temperature for 5 minutes and centrifuged (4 ° C., 14000 rpm, 15 minutes).

Approximately 140 μl of the supernatant was transferred to a new tube, reacted with isopropanol for 10 minutes, and centrifuged (4 ° C., 14000 rpm, 15 minutes). The supernatant was removed again and washed twice with 75% EtOH The pellet was then dried and dissolved in DEPC-water.

The cDNA was synthesized according to the cDNA synthesis method of SuperScript III (Invitrogen CA, USA) at a concentration of 1 μg / μl for RT (reverse transcription) PCR, and 1 μl of cDNA (0.5 μg / μl) ≪ / RTI >

1 μl of template, 2 μl of dyemix, 1 μl of primer-F, 1 μl of primer-R, and 7 μl of distilled water were incubated at 95 ° C for 2 minutes, 95 ° C 20 sec at a reaction temperature of 30 sec - 58 캜 for 1 min - 72 캜 for 1 min 25 times, 72 캜 for 5 min and 4 캜 storage.

The primers used in this experiment, its nucleotide sequence, genebank accession number and experimental conditions are as shown in Table 1 below.

Abbreviations: iNOS, inducible nitric oxide synthase; COX-1, cyclooxygenase-1; COX-2, cyclooxygenase-2; IL-1 [beta], interleukin-1 [beta]; IL-6, interleukin-6; TNF-alpha, tissue necrosis factor-alpha; GAPDH, glyceraldehydes-3-phosphate dehydrogenase.

Experimental Example 4 - Western blot 분석

For identification of proteins in Microglia BV-2 cells, cells were washed twice with 1 × PBS and lysis buffer [1% Triton X-100, 1% deoxycholate, 0.1% NaN3] was used.

For the quantification of proteins, the bradford method of DC protein assay (bio-rad CA) was used. The same amount of protein was electrophoresed on 10% SDS-PAGE and then gelated using 0.45 μm polyvinylidene fluoride (PVDF: Millipore) To the membrane.

As for the antibody, anti-β-actin (1: 2000; Cell Signaling), anti-iNOS, anti-COX-1, anti-COX-2, anti-IκB- (1: 1000; Santa Cruz), anti-p65 (1: 1000; Santa Cruz) and incubated at 4 ° C (Horseradish peroxidase (HRP) as a secondary antibody) was reacted with ECL kit (Pierce, CA) for 1 hour at room temperature and reacted with LAS-3000 (Fuji Co, Japan) was used to confirm the results.

Experimental Example 5 - Immunofluorescence assay

In order to measure the migration of NF-κB p65 in Microglia BV-2 cells (5 × 10 ⁴ cells / well in 24-well plate), the cells were incubated on cover slips for 24 h.

After the culture medium was pretreated for 1 hour, 250 μl of methanol was added to the supernatant after 30 minutes of LPS treatment, and the plate was fixed at -20 ° C. for 20 minutes, washed with PBS (in 0.02% Tween) And the cells were treated with 1% Triton for 10 minutes to penetrate the cell membrane.

The primary antibody was treated with 1 mg / ml of monoclonal mouse anti-human NF-κB (p65) (Santa Cruz Biotechnology Inc, Santa Cruz, CA) for 60 min at room temperature.

Alexa Fluor 568-labeled goat anti-mouse antibody (Invitrogen, CA) diluted 1: 100 was used as the secondary antibody for 60 min at room temperature.

The cells were fixed on a cover glass with a mounting solution for the stuffing, and the fluorescence microscopic observation was performed in the dark room to observe the fluorescence stained cells after the stuffing.

Experiment result

One. BV -2 microglia cells in CO  And / or LPS Of cytotoxicity and nitrite release

The effect of LPS and corticosteroid extract (CO) on cell viability in BV-2 microglia cells stimulated with LPS was confirmed by cytotoxicity assay using MTT assay.

As shown in FIG. 1A, the MTT assay showed that the cell survival rate did not change in all experimental groups treated with LPS alone or in combination with the astragalus extract, compared with the control group. It does not affect survival.

In order to analyze the anti-inflammatory effects of the extracts of astragalus, it was confirmed that the concentration-dependent inhibitory effect of nitrite (NO) produced by mouse BV-2 microglia cells stimulated with the same neuroinflammatory factor LPS (100 ng /

To determine nitrites, NO assay using Griess reagent was used. The amount of nitrite (NO) released by LPS was 100% in BV-2 cells. As shown in FIG. 1B, it was confirmed that the amount of nitrite (NO) released was decreased in a concentration-dependent manner compared with the group treated with LPS alone.

FIGS. 1A and 1B show the effects of the extract of T. koreensis on cell viability and nitrite inhibition activity in LPS-stimulated mouse BV-2 microglia cells. BV-2 microglia cells were plated on a 96-well plate at 5 × 10 ⁴ cells / well and cultured for 24 hours. Cells were stimulated with LPS (100 ng / ml). After 24 hours, the supernatant was removed and the amount of nitrite released was measured by Griess assay. The remaining supernatant was removed, MTT reagent was added, and surviving cells were measured and the results were expressed as a percentage. The displayed experimental results are the average of three repeated experiments.

2. ESR  Free radicals of cynomolgus sinensis extracts using spectroscopic instrument Scatters  Verification Result

The free radical scavenging activity was measured using an ESR spectrometer in order to confirm the ability of CO to remove the active oxygen.

As shown in Fig. 2, the removal efficiencies of DPPH radicals were 44.00%, 71.68%, 83.00%, and 86.13%, respectively, at various concentrations (0.25, 0.51 and 2 mg / The value of ₅₀ was 0.3042 mg / mL.

Figs. 2A and 2B show the effect of suppressing the active oxygen of the extract of cynomolgus sinensis. FIG. 2 shows the results obtained by mixing the DPPH reagent and the celestial concentration sample at a ratio of 1: 1, reacting for 2 minutes, introducing the reaction reagent into the capillary tube, and measuring using the ESR instrument.

3. LPS Stimulated by mouse BV -2 microglia  The concentration-dependent iNOS  Inhibition of gene and protein expression

In order to analyze the anti-inflammatory effect of the extract of Cynomolgus sinensis, it was confirmed whether iNOS produced by mouse BV-2 microglia stimulated with LPS (100 ng / ml), which is the same neuroinflammatory factor, had a concentration-dependent inhibitory effect.

Referring to FIG. 3, it can be confirmed that the extract of Angelica keiskei koidz. Inhibits expression in a concentration-dependent manner in the gene and protein level of iNOS, which is an enzyme inducing nitrite (NO) synthesis.

In order to analyze the expression pattern of iNOS gene, BV-2 glioma cells were treated with LPS and cynomolgus extracts at different concentrations and cultured for 6 hours. RNA was identified and RT-PCR was performed to quantitatively express the gene expression pattern Respectively.

In order to confirm the results shown in FIG. 3, the extracts were treated with LPS (100 ng / ml) for 1 hour before treatment with the extracts of cucurbitaceae at concentrations of 1, 10 and 50 占 퐂 / Respectively. Cells were cultured in a 6-well plate at 1 × 10 ⁶ cells / well. After 6 hours, the supernatant was removed, and the RNA was isolated from the cells. The iNOS gene was analyzed by RT-PCR. Cells were cultured in 1 × 10 ⁶ cells / well in 6 well plates. Cells were incubated with iNOS (polyclonal antibody) for 18 h.

3A, the expression level of the iNOS gene in the control group not treated with LPS was found to be almost zero. In the test group treated with LPS, the expression level of the iNOS gene was significantly increased, Which can be confirmed by the expression pattern. GAPDH was used as a control for gene quantitative analysis.

3B, in order to analyze the expression pattern of iNOS protein, LPS and cynomolgus koreanum extracts were treated at the same concentration as that of the iNOS gene. After the same incubation time, proteins were identified and Western blot was performed, The expression changes were quantitatively analyzed.

4. LPS Stimulated by BV -2 in neuron cells COX -2 Inhibition of gene and protein expression

In order to examine the expression pattern of genes and proteins of COX-2 (cyclooxygenase type 2), an enzyme involved in inflammation production, BV-2 cells were treated with LPS and cynomolgus koreanum extracts at different concentrations and cultured over time.

The expression of COX-2 gene was confirmed by quantitative analysis of gene expression by RT-PCR after identification of RNA after 6 hours.

As shown in FIG. 4A, the expression level of the LPS-treated group was increased compared with that of the control group, whereas the expression level of the LPS-treated extracts (1, 10, and 50 μg / The expression level of the COX-2 gene is decreased in a concentration-dependent manner.

As shown in FIG. 4A, the cells were cultured in a 6-well plate at 1 × 10 ⁶ cells / well. After 6 hours, the supernatant was removed, and RNA was identified from the cells. The COX-2 gene Were analyzed by RT-PCR.

After 18 hours, the expression of COX-2 protein was confirmed by Western blot analysis.

As shown in FIG. 4B, the expression level of LPS-treated group was increased compared with that of the control group, and the expression level of LPS-treated extracts (1, 10, 50 ㎍ / The expression of COX-2 protein is decreased in a concentration-dependent manner.

4B, as described above, the cells were cultured in a 6-well plate at 1 × 10 ⁶ cells / well, and the proteins were identified in the cells after 18 hours, and the cells were treated with western blotting using a polyclonal antibody, COX-2 This is the result of analysis through blot.

5. LPS Stimulated by mouse BV -2 microglia  Inflammatory mediators in cells cytokine Concentration-dependent inhibitory effect of cynomolgus sinensis extract

To determine the expression pattern of the inflammatory cytokines TNF-α, IL-1β, and IL-6 gene, BV-2 cells were treated with LPS and cynomolgus extracts at different concentrations and cultured for 6 hours. The expression patterns of the genes were analyzed quantitatively by PCR to reveal common expression patterns in the two genes.

As shown in FIG. 5, the expression levels of TNF-α and IL-1β genes were almost absent in the LPS-untreated control group. In the experimental group treated with LPS (100 ng / -α and IL-1β genes were significantly increased, and the expression pattern of IL-1β was decreased in a dose-dependent manner. GAPDH was used as a control for gene quantitative analysis.

In order to confirm the results shown in FIG. 5, BV-2 glioma cells were cultured in a 6-well plate at 5 × 10 ⁵ cells / ml, cultured for 24 hours, and treated with astragalus extract for 1 hour . Then, the cells were stimulated with LPS (100 ng / ml) and after 6 hours, the supernatant was removed and RNA was identified in the cells, and TNF-α, IL-1β and IL-6 gene were analyzed by RT-PCR.

6. LPS Stimulated by BV -2 in neuron cells NF Concentration-dependent efficacy mediated by inhibition of -KB

In order to confirm the efficacy of the extracts, the activation of NF-κB, a transcription factor that expresses proinflammatory cytokines, was induced by the induction of inflammation by LPS in the nucleus of p65 constituting NF-κB, Expression of -IκBα was confirmed by Western blot.

6A, BV-2 cells were treated with LPS (100 ng / ml) and cortex extract at different concentrations and cultured for 30 minutes. Immunofluorescence analysis revealed that the p65 protein constituting NF- The degree of influx can be confirmed.

It can be confirmed that the protein p65 is intrinsic to the nucleolus at the time of the LPS treatment and that the protein p65 is introduced into the nucleus at the time of the LPS treatment and that the protein p65 is prevented from entering into the nucleus when the extract is treated have.

In order to confirm the results shown in FIG. 6A, BV-2 glioma cells were cultured in a 12-well plate at 5 × 10 ⁴ cells / well for 24 hours, . The cells were stimulated with LPS (100 ng / ml) and after 30 minutes, the supernatant was removed and observed by immunofluorescence.

FIG. 6B is a result of quantitatively confirming the expression pattern of the nuclear protein by identifying the nuclear protein and Western blotting for more accurate confirmation.

In the LPS-untreated control group, expression of NF-κB p65 was scarcely observed. However, the expression of NF-κB p65 was increased in the LPS-treated experimental group, and the expression level of the extract was decreased when the extract was treated. Nucleolin is a control protein for quantification of nuclear proteins.

In order to confirm the results shown in FIG. 6B, the BV-2 glial cells were cultured for 24 hours in a 6-well plate at a concentration of 1 × 10 ⁶ cells / ml. . The cells were stimulated with LPS (100 ng / ml), and after 30 minutes, the supernatant was removed and only nuclear proteins were identified in the cells. And analyzed by Western blot using mouse monoclonal antibody, NF-κB p65.

Referring to FIG. 6C, it can be confirmed that IκBα is decomposed to increase the amount of p-IκBα during LPS treatment, and that the amount of protein p-IκBα decreases in a concentration-dependent manner. β-actin is a control protein for quantitative confirmation.

In order to confirm the results shown in FIG. 6C, BV-2 microglia cells were cultured in a 6-well plate at 1 × 10 ⁶ cells / ml for 24 hours, . The cells were stimulated with LPS (100 ng / ml), and after 30 minutes, the supernatant was removed and the protein was identified in the cytoplasm. And analyzed by Western blot using monoclonal antibody NF-κB p65 and polyclonal antibodies IκBα and p-IκBα.

7. LPS Stimulated by BV -2 in neuron cells MAPKs Concentration-dependent efficacy mediated by inhibition of

The fact that the inflammatory response through the LPS treatment is expressed through the mitogen-activated protein kinase (MAPK) pathway has been reported in a number of articles. The present inventors have found that the extracts of the astrocytes suppress the inflammatory response through the MAPK pathway Respectively.

Referring to FIG. 7A, it can be seen that the phosphorylation amount of p38 in the p38 pathway is increased in the p38, which is one of the constituents of MAPKs, compared to the control group. On the contrary, the phosphorylation degree of the citrus extract Dependent manner.

For confirmation of the results shown in FIG. 7A, BV-2 glioma cells were cultured in a 6-well plate at a concentration of 1 × 10 ⁶ cells / ml for 4 hours. The cells were stimulated with LPS (100 ng / ml), and after 30 minutes, the supernatant was removed to identify the protein. And analyzed using Western blot using LPS polyclonal antibodies p38 and p-p38.

While the present invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. It will be appreciated by those skilled in the art that numerous changes and modifications can be made without departing from the invention. Accordingly, all such modifications and variations are intended to be included within the scope of the present invention.

Claims (9)

A composition for preventing or treating a neurodegenerative disease, which comprises ethanol extract of Corynebuide.
The method according to claim 1,
Wherein the ethanol solvent is a mixed solvent comprising 1 to 20 parts by weight of water or 1 to 20 parts by weight of an alcohol having 1 to 4 carbon atoms per 100 parts by weight of ethanol.
A composition formulated by adding to the composition of claims 1 or 2 a pharmaceutically acceptable excipient in the form of tablets, capsules, solutions, suspensions, syrups or edible beverages.
A herbal medicine or a health functional food comprising the composition of claim 1 or 2.
3. The method according to claim 1 or 2,
Wherein the composition comprises a transcriptional inhibitory function of the proinflammatory cytokines TNF-a, IL-l [beta] and IL-6.
In the method for extracting ethanol extract from Chennung,
(A) collecting and drying a piece of astragalus;
(B) mixing the dried piece of astragalus with a polar solvent containing ethanol and pulverizing using an ultrasonic grinder; And
(C) Extracting the ethanol extract of celadon from the mixture of crushed celadon shell and the polar solvent, followed by filtration, vacuum concentration and lyophilization.
The method according to claim 6,
Wherein the concentration of ethanol into which the herbarium samples are added in step (B) is 90% to 99%.
The method according to claim 6,
Wherein the amount of ethanol to be mixed in step (B) is 1000 parts by weight to 1500 parts by weight with respect to 100 parts by weight of the Angelite sample.
The method according to claim 6,
Wherein the pulverization of step (B) is performed 10 to 20 times for 10 minutes to 20 minutes at intervals of 1 hour to 3 hours using an ultrasonic grinder.

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