KR101545495B1 - Pharmaceutical Composition and Functional Food for Preventing or Treating Neurodegenerative Disease by Saururus Chinesis Extracts and Quercetin as Effective Component, and Extracting Method of Saururus Chinesis Extracts - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating neurodegenerative diseases containing quercetin, an effective ingredient of Saururus chinensis extract, and a method for extracting Saururus chinensis extract. A pharmaceutical composition and a health functional food for preventing or treating neurodegenerative diseases which enable quercetin, which is an effective ingredient thereof, to have an effect on nerve inflammation and inhibition of neuronal cell death by inhibiting the proinflammatory mediators of activated neuritic cells, And an extraction method for extracting the extract.

Description

FIELD OF THE INVENTION The present invention relates to a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases comprising quercetin, an effective ingredient of Saururus chinensis extract, a health functional food, and a method for extracting Saururus chinensis extract by Saururus chinensis extracts and quercetin as effective Component, and Extracting Method of Saururus Chinesis Extracts}

The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating neurodegenerative diseases which contain Saururus chinensis extract and quercetin as an active ingredient thereof, and a method for extracting Saururus chinensis extract. In the neuronal inflammation caused by glial cells, And quercetin as an active ingredient thereof are effective for inhibiting neuronal inflammation and neuronal cell death by inhibiting proinflammatory mediators of activated neuritic cells, and a pharmaceutical composition and a health functional food for preventing or treating neurodegenerative diseases, And an extraction method for extracting Saururus chinensis extract.

The glial cells are located in the central nervous system (CNS) and are involved in the primary protection and recovery of neurons. However, many studies have reported that activated glial cells induce neuroinflammatory responses, and that neuroinflammation plays a role in various neurodegenerative diseases. Continuous neuroinflammation due to activation of the glial cells is a prominent pathological feature in various neurodegenerative diseases (Kim and Joh, 2006; Koning et al., 2007; Krause and Muller, 2010;

Neuroglial cells activated by brain injury or neurotoxin have been shown to inhibit nitrite (NO), reactive oxygen species (ROS), inflammatory enzymes Inducible Nitric Oxide Synthase (iNOS) and Cyclooxygenase-2 (COX-2), and proinflammatory cytokines (Graeber and Streit, 2010). These substances are involved in the pathogenesis of degenerative neurodegenerative diseases such as multiple sclerosis, Parkinson's disease, Alzheimer's disease and Huntington's disease (Kim and Joh, 2006; Koning et al., 2007; Krause and Muller, 2010; Moller, 2010) have been reported. Therefore, it can be inferred that the regulation of neuroinflammation by inhibition of these glial cell activation plays an important role in the prevention of degenerative neurodegenerative diseases.

Meanwhile, Saururus chinensis (Lour.) Baill; SC] has not yet been studied in its intrinsic mechanism of neuroinflammation efficacy. Korean Patent No. 10-0358384, for example, discloses a health supplement containing raw saururus as a raw material and a method for producing the same, but it is not a technique relating to the effect of inhibiting neuronal inflammation .

The object of the present invention is to provide a pharmaceutical composition and health functional food for preventing or treating neurodegenerative diseases containing Saururus chinensis extract and quercetin as an active ingredient thereof, and a method for extracting Saururus chinensis extract.

The present invention provides a composition for preventing or treating a neurodegenerative disease, which comprises a saururus ethanol solvent extract, 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.

In addition, the composition provided by the present invention is characterized by containing quercetin (QCT) as its active ingredient.

The present invention also provides a method for extracting Saururus chinensis ethanol extract, comprising the steps of: (A) extracting Saururus chinensis, drying and pulverizing; (B) mixing the crushed 3-hundred-millisecond piece with a polar solvent comprising ethanol; And (C) extracting the Saururus chinensis ethanol extract from the mixture of the milled Saururus chinensis and the polar solvent, followed by filtration, vacuum concentration and lyophilization.

The step (A) comprises: (A1) drying the saururus pieces collected from the saururus pieces for 3 days to 5 days while maintaining the temperature condition at 15 ° C to 20 ° C; And (A2) pulverizing the dried three hundred milligram 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 Saururus chinensis ethanol extract using an ultrasonic grinder while maintaining the extraction temperature at 15 ° C to 25 ° C; (C2) concentrating the extracted Saururus chinensis ethanol extract at 50 to 60 캜 in vacuo and freeze-drying; And (C3) lyophilized Saururus chinensis ethanol extract into a container, followed by concentration under reduced pressure at 40 ° C to 50 ° C.

According to the present invention, there is provided an effect of providing a pharmaceutical composition, a health functional food, and a method for extracting Saururus chinensis extract, which contains Saururus chinensis extract and quercetin as an active ingredient thereof, for the prevention or treatment of neurodegenerative diseases.

FIGS. 1A and 1B are graphs showing cytotoxicity and nitrite inhibition activity of Saururus chinensis extract according to the present invention. FIG.
FIG. 2A and FIG. 2B are graphs showing the activity of inhibiting active oxygen of Saururus chinensis extract according to the present invention.
FIGS. 3A to 3D are graphs showing the inhibitory effects of genes and proteins on inflammation-inducing enzymes of Saururus chinensis extract according to the present invention.
FIGS. 4A and 4B are graphs showing the inhibitory effects on TNF-.alpha. And IL-6 gene, pro-inflammatory mediators of Saururus chinensis extract according to the present invention.
5A to 5C are graphs showing the inhibitory effects of the Saururus chinensis extract according to the present invention on NF-κB activity.
6A to 6E are graphs showing the anti-inflammatory activity of quercetin, an active ingredient of Saururus chinensis extract according to the present invention.

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 carried out a mechanism research on the expression of products, genes and proteins of inflammatory factors induced by Lipopolysaccharide (LPS). Based on molecular biochemical assay, Saururus chinensis Baill; SC ) extracts.

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 and the activity of glial cells is a major therapeutic target for inhibition of neuroinflammation do.

The present inventors confirmed the nitrite inhibitory effect of Quercetin (QCT), which is a representative active ingredient of Saururus chinensis extract, by using various inflammation mediators according to the degree of inhibition of gene and protein stage. Thus, Saururus chinensis extract has anti- And that it is quercetin.

Accordingly, the present invention provides a composition comprising an extract of Saururus chinensis and an effective ingredient thereof, quercetin, which exhibits an excellent neurodegenerative disease preventing or treating effect, and a method for extracting Saururus chinensis extract.

The present invention also relates to a composition comprising the Saururus chinensis extract and a formulation formulated by adding a pharmaceutically acceptable excipient in the form of a tablet, a capsule, a solution, a suspension, a syrup or an edible beverage, a herb medicine preparation containing a Saururus chinensis extract, Or drinks.

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

First, the pieces were sowed from Saururus chinensis.

Specifically, three hundred milligram pieces were randomly picked from the stem, root, and leaf of Saururus chinensis, and more than 100 pieces of Saururus chinensis were collected from more than 5 kilograms of Saururus chinensis.

Then, the sampled hundreds of millions of pieces were dried for 3 to 5 days while maintaining the temperature condition of 15 to 20 캜.

Next, the dried three hundred milligram pieces were crushed.

The dried Saururus chinensis was crushed to have even particles and the particle size was distributed between 0.1 mm and 5 mm.

Next, the pulverized saururus was mixed with a polar solvent containing ethanol.

At this time, the polar solvent to be mixed may be 100% ethanol, or may be mixed daily for 1 to 20 parts by weight of water or 1 to 20 parts by weight of alcohol having 1 to 4 carbon atoms per 100 parts by weight of ethanol.

The weight of the polar solvent to be mixed is preferably 0.2 to 2 times the weight of the pulverized saururus. Specifically, 1 L of mixed polar solvent consisting of 95% of ethanol and 5% of water is mixed with 1 kg of pulverized saururus.

Next, the Saururus chinensis ethanol extract is extracted from the Saururus chinensis mixture, filtered and concentrated in vacuo and lyophilized to obtain the Saururus chinensis ethanol extract according to the present invention.

As the extraction method, a cold extract extraction method, a hot extraction extraction method, a heat extraction method, or the like can be used, and a conventional extraction device, an ultrasonic pulverizer or a fractionator can be used. Specifically, an ultrasonic pulverizer (BRANSON Ultrasonic Corporation, Lt; RTI ID = 0.0 > 15 C < / RTI > to < RTI ID = 0.0 > 25 C. < / RTI >

Specifically, filtration was carried out using a 0.22 μM film filter. Vacuum concentration was carried out using a rotary evaporator at 50 ° C to 60 ° C, and 14.4g of Saururus chinensis ethanol extract in powder form was obtained through lyophilization.

Then, the Saururus chinensis ethanol extract obtained for use as an experimental sample was put in a tube, concentrated under reduced pressure at 40 ° C to 50 ° C and stored at low temperature, and 15 or more tubes having a capacity of 20 mg ± 0.5 mg were obtained. And the average value was adopted as the experimental result.

Meanwhile, quercetin (QCT) (Sigma Chemical Co, USA) was purchased and confirmed by HPLC (Waters, Ontario, Canada) analysis to confirm that quercetin was contained in the Saururus chinensis ethanol extract.

Experimental Example

reagent

Saururus Chinensis (SC), Lipopolysaccharide (LPS), Tween-20, Bovine Serum Albumin (BSA), 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT). (Sigma Chemical Co, USA) 6-well, 12 well, 96 wel tissue culture plate 100 mm culture plate. (NUNC Inc, IL, USA)

The reagents used for the experiments were DMEM medium for cell culture, penicillin / streptomycin (Gibco, BRL) with 5% FBS and 100 units / ml of antibiotics were used. We also used antibodies NF-κB p65, COX-2 (Santa Cruz, CA), iNOS, IκB-α, phosphor (p) -IκB-α and β-actin (Cell signaling, Co, USA).

Cell culture and cytotoxicity test ( MTT assay )

Was Murine BV-2 microglial cells are obtained from Gyeongsangbuk University pharmacological class, that the humidity is maintained at a 5% FBS for deactivation state with the addition of 100 units / ㎖ penicillin / streptomycin DMEM 5% CO 2, the 95% O ₂ incubator Lt; / RTI >

In all experiments, the number of cells was 5 × 10 ⁵ cells / ㎖. Saururus chinensis extract was diluted in the medium and treated with 1 μg / ml, 5 μg / ml, 10 μg / 100 ng / ml was used as a dilution in the medium.

The cytotoxicity of SC was confirmed by MTT assay. After MTT assay, 30 μl of 0.5 mg / ml MTT was added to the cells in the microplate and reacted for 2 hours in an incubator. The absorbance was measured using a Vmax microplate reader with a wavelength of 540 nm.

Assay of NO

Assay of NO is a microplate (96 well) assay method for quantitatively measuring NO (nitric oxide) production. The cells were seeded at 5 × 10 ⁴ cells / well in microplate, and the LPS and Saururus chinensis extracts were added at different concentrations. The cells were reacted in a 24 h incubator. The supernatants were washed with Griess reagent (1% sulfanilamide / 0.1% - (1-naphthyl) -ethylenediamine dihydrochloride / 2.5% H3PO4), and then measured using a Vmax 96-well microplate spectrophotometer with a wavelength of 550 nm.

Isolation of total RNA and RT - PCR

All RNAs in the Microglia BV-2 cell line were isolated using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. Add 1 ml of TRizol reagent to the microglia BV-2 cell line, add 200 μl of chloroform, stir, react at room temperature for 5 minutes, and centrifuge (4 ° C, 10,000 rpm, 10 minutes).

Transfer 500 μl of the supernatant to a new tube. Add 500 μl of the same amount of isopropanol, react at room temperature for 10 minutes, and centrifuge (4 ° C, 14,000 rpm, 15 minutes). Remove the supernatant, wash twice with 75% EtOH, dry the pellet and dissolve using DEPC-water.

DNase I (Roche AC, USA) diluted 1:10, and 2 μl of the mixture is reacted at 25 ° C for 15 minutes. 78 μl of DEPC-water was added to 22 μl of the sample to prepare a total of 100 μl. Add 100 μl of TRizol reagent and stir. Add 25 μl of chloroform and stir.

The reaction is carried out at room temperature for 5 minutes and centrifuged (4 ° C, 14000 rpm, 15 minutes). About 140 μl of the supernatant is taken out in a new tube, reacted with isopropanol for 10 minutes, and centrifuged (4 ° C, 14,000 rpm, 15 minutes). Remove the supernatant and wash twice with 75% EtOH, dry the pellet, and dissolve using DEPC-water.

RT (reverse transcription) PCR was performed to synthesize cDNA according to the cDNA synthesis method of SuperScript III (Invitrogen CA, USA) with a sample concentration of 1 μg / μl and 1 μl of cDNA (0.5 μg / μl) Lt; / RTI >

1 μl of dyemix, 2 μl of dyemix, 1 μl of primer-F, 1 μl of primer-R and 7 μl of distilled water were reacted at 95 ° C for 2 minutes, 95 ° C for 2 minutes, 20 ℃ for 30 seconds, 58 ℃ for 1 minute, 72 ℃ for 1 minute, 25 ℃, 72 ℃ for 5 minutes and 4 ℃. The primers used, their nucleotide sequence, genebank accession number and experimental conditions are listed in [Table 1] below.

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.

Protein quantitation was performed using the bradford method of the DC protein assay (bio-rad CA). The same amount of protein was electrophoresed on 10% SDS-PAGE and the membrane was electrophoresed with 0.45 μm polyvinylidene fluoride (PVDF: Millipore) .

The use of anti-β-actin (1: 2000; Cell Signaling), anti-iNOS, anti-IκB-α, anti-phospho-IκB- The cells were diluted with p65 (1: 1000; Santa Cruz), anti-COX-2 (1: 1000; Santa Cruz) and incubated overnight at 4 ° C with Horseradish peroxidase (HRP) The antibody was reacted at room temperature for 1 hour and then reacted with ECL kit (pierce, CA), and the results were confirmed using LAS-3000 (Fuji Co, Japan).

Double - immunofluorescence labeling assay

In order to measure the migration of NF-κB p65 in Microglia BV-2 cells (1 × 10 ⁵ cells / well in 12-well plate), the cells were incubated on cover slip for 24 hours. After the material was treated for 1 hour, the supernatant was removed after 30 minutes of LPS treatment, and 250 μl of methanol was added and fixed at -20 ° C for 20 minutes.

After washing with PBS (0.02% Tween), the nuclei were stained with 1 μM Hoechst. The cells were treated with 1% Triton for 10 minutes to penetrate the anti-bodies.

Monoclonal mouse anti-human NF-κB (p65) (Santa Cruz Biotechnology Inc, Santa Cruz, CA) diluted 1: 200 with primary antibody was applied for 60 minutes at room temperature. Alexa Fluor 568-labeled goat anti-mouse antibody (Invitrogen, CA) diluted 1: 100 was used as the secondary antibody for 60 minutes at room temperature.

Fixed to cover glass with mounting solution for stuffing. After staining, fluorescence microscopy was used to observe the fluorescently stained cells and the results were confirmed.

Statistical 분석

In order to confirm the reproducibility of the experimental results, the following experimental results were expressed as mean ± standard deviation, and each experiment was carried out three times. Statistical analysis of the experimental results was performed using Graphpad Prism V5.01. One-way ANOVA was performed using Dunnett's method using software.

Key abbreviation explanation

iNOS: inducible nitric oxide synthase

COX-2: cyclooxygenase-2

IL-6: interleukin-6

TNF-α: tumor necrosis factor-alpha

GAPDH: glyceraldehydes-3-phosphate dehydrogenase.

Experiment result

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

The effect of LPS and Saururus chinensis extract (SC) on cell viability was examined in BV-2 microglia cells stimulated with LPS.

MTT assay was used for the cytotoxicity test. As shown in FIG. 1A, MTT measurement showed that cell survival rate did not change in all the experimental groups treated with LPS and SC alone or in the same manner with LPS and SC Respectively. These results indicate that the neuroinflammatory inducers LPS and Saururus chinensis extract do not affect cell survival.

In order to analyze the anti-inflammatory effect of Saururus chinensis extract, it was confirmed that the concentration-dependent inhibitory effect on nitrite (NO) produced in mouse BV-2 microglia cells stimulated with LPS (100 ng / ml) .

To determine nitrites, NO assay using Griess reagent was used. The amount of nitrite (NO) released by LPS in BV-2 cells was 100% Mu] g / ml), as shown in Fig. 1B, the amount of nitrite (NO) released was decreased in a concentration-dependent manner compared to the group treated with LPS only.

1A and 1B show the effect of Saururus chinense extract 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, cultured for 24 hours, and treated with Saururus chinensis extract for 1 hour before concentration. 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  Active oxygen of Saururus chinensis extract using spectroscopic instrument Scatters  Verification Result

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

As shown in FIG. 2A, the SC extracts of various concentrations (0.031, 0.062, 0.125, 0.25, 0.5 1 and 2 mg / mL) were 48.9% ± 0.6%, 53.2% ± 2.4% , 63.0% ± 3.1%, 77.8% ± 1.6%, 86.8% ± 2.2%, 88.9% ± 1.3% and 90.8% ± 0.9%, respectively, and the IC ₅₀ value was 0.039 ± 0.004 mg / mL.

In addition, the alkyl radicals of APPH are degraded to an alkyl radical, which induces lipid peroxidation by the reaction of an alkyl radical with oxygen (O ₂ ). When APPH is incubated at 37 ° C for 30 minutes, an alkyl radical spin product of 4-PBON / free radical is generated.

As shown in FIG. 2B, SC extracts of various concentrations (0.25, 0.5 and 1 mg / mL) exhibited a scavenging ability of 41.0% ± 1.3%, 56.0% ± 0.5%, and 62.1% ± 5.6% ₅₀ value was 0.35 ± 0.07 mg / mL.

Figs. 2A and 2B show the effect of inhibiting the active oxygen of Saururus chinensis extract. FIG. 2A shows the results obtained by mixing a DPPH reagent and a Samples of Saururus concentration at a ratio of 1: 1, reacting for 2 minutes, introducing a reaction reagent into a capillary tube, and measuring using an ESR instrument. FIG. 2B is a graph showing the results obtained by mixing sterilized distilled water and Saururus chinensis AAPH and POBN at a ratio of 1: 1: 1: 1, reacting the mixture in a water bath at 37 ° C for 30 minutes, introducing the reaction reagent into a capillary tube, .

3. LPS Stimulated by BV -2 in glial cells SC Concentration-dependent iNOS , COX -2 as a result of inhibiting the expression of genes and proteins

To investigate the expression patterns of genes and proteins of iNOS and COX-2 (cyclooxygenase type 2), which are involved in inflammation, BV-2 cells were treated with LPS and Saururus chinensis extract Lt; / RTI >

The expression of iNOS and COX-2 gene was confirmed by quantitative analysis of the expression pattern of the gene after RT-PCR.

As shown in FIG. 3A and FIG. 3C, it was confirmed that the expression level in the group treated with LPS alone was increased compared with that in the control group, and the expression level of Saururus chinense extract treated with concentrations (1, 5, 10 / / Showed that the expression level of iNOS and COX-2 gene was decreased in a concentration-dependent manner compared to the group treated with LPS alone.

After 18 hours, the protein was identified and Western blot was performed to confirm the expression pattern of iNOS and COX-2 protein.

As shown in FIG. 3B and FIG. 3D, it was confirmed that the expression level in the group treated with LPS alone was increased compared with that in the control group, and the expression level of Saururus chinensis extract treated with concentrations (1, 5, 10 / / Showed that the expression of iNOS and COX-2 protein was decreased in a concentration-dependent manner compared to the group treated with LPS alone. β-actin was used as a control for protein expression analysis.

FIGS. 3A-3D show the inhibitory effects of iNOS and COX-2 on the gene and protein levels of Saururus chinense extract in BV-2 glial cells stimulated by LPS. FIGS. 3A and 3C show the results of RT-PCR of iNOS and COX-2 gene by identifying the RNA in the cells, removing the cell supernatant after 6 hours, culturing the cells in a 6-well plate at 1 × 10 ⁶ cells / . FIG. 3B and FIG. 3D were obtained by incubating cells in a ^6- well plate at 1 × 10 ⁶ cells / well. After 18 hours, the proteins were identified in cells and analyzed by Western blot using polyclonal antibodies iNOS and COX-2 Results. The experimental results are obtained from three repeated experiments.

4. LPS Stimulated by mouse BV -2 microglia  The inhibitory effects of Saururus chinensis extract on the production of inflammatory mediators

In order to elucidate the expression pattern of TNF-α and IL-6 gene, inflammation-mediated cytokines, BV-2 cells were treated with LPS and Saururus chinensis extracts for various concentrations and cultured for 6 hours. The expression pattern of the gene was analyzed quantitatively by PCR to confirm common expression patterns of the two genes.

As shown in FIGS. 4A and 4B, in the control group not treated with LPS, the amount of expression of TNF-α and IL-6 gene was almost not found. In the group treated with LPS (100 ng / α and IL-6 gene were significantly increased, and the change in expression pattern was observed in a dose-dependent manner when Saururus chinense extract was treated. GAPDH was used as a control for gene quantitative analysis.

FIGS. 4A and 4B show the concentration-dependent inhibitory effect of Saururus chinense extract on TNF-a and IL-6 genes, proinflammatory mediators, in BV-2 glial cells stimulated by LPS. BV-2 glial cells were plated at 5 × 10 ⁵ cells / ml on a 6-well plate, cultured for 24 hours, and treated with SC for 1 hour before concentration. Cells were stimulated with LPS (100 ng / ml). After 6 hours, the supernatant was removed, RNA was identified from the cells, and the TNF-a and IL-6 genes were analyzed by RT-PCR. The experimental results are obtained from three repeated experiments.

5. LPS Stimulated by BV -2 in the glial cells NF - κB inhibition mediated concentration-dependent efficacy results

In order to analyze the efficacy of Saururus chinensis extract, the activation of NF-κB, a transcription factor that expresses proinflammatory cytokines in the induction of inflammation by LPS, was confirmed. In the nucleus of p65 constituting NF-κB, , And the expression of p-IκBα was confirmed by Western blot analysis by decomposition of IκBα.

As shown in FIG. 5A, the BV-2 cell was treated with LPS (100 ng / ml) and Saururus chinensis extract at different concentrations and cultured for 30 minutes. Then, the p65 protein constituting NF- And the degree of influx was confirmed. In the absence of any treatment, it was confirmed that the protein p65 was contained in the nucleolus, and that the protein p65 was introduced into the nucleus by the LPS treatment, and that treatment with the Saururus chinensis extract inhibited the inflow of the protein p65 into the nucleus .

As shown in Figs. 5B and 5C, IκBα was decomposed to increase the amount of p-IκBα during LPS treatment, and it was confirmed that the amount of protein p-IκBα decreased in a concentration dependent manner of SC. β-actin is a control protein for quantitative confirmation.

Figures 5A-5C show the concentration-dependent inhibitory effect of SC on NF-kB activity in LPS-stimulated BV-2 glial cells. FIG. 5A is a graph showing the results of stimulation of cells with LPS (100 ng / ml). BV-2 glioma cells were plated on a 12-well plate at a concentration of 1 × 10 ⁵ cells / well and cultured for 24 hours. After 30 minutes, the supernatant was removed and the result was observed by immunofluorescence. FIG. 5B shows the results of stimulating cells with LPS (100 ng / ml), treating BV-2 microglia cells with 1 x 10 ⁶ cells / ml on a ^6- well plate, culturing them for 24 hours, After 30 minutes, the supernatant was removed and the protein was identified in the cytoplasm. The result was analyzed by Western blot using monoclonal antibody NF-κB p65 and polyclonal antibody IκBα, p-IκBα. FIG. 5C is a result of quantitative analysis of the experimental results shown in FIG. 5B. The experimental results are obtained from three repeated experiments.

6. LPS Stimulated by BV -2 The activity of quercetin, the active ingredient of Saururus chinensis extract in glial cells Quercetin, QCT) NO  Production and various inflammation related bio Marker  Results of inhibition of expression

The constituents of Saururus chinensis have been reported to consist of several phenolic compounds (Chen et al., 2010). Accordingly, the present inventors confirmed whether or not quercetin is contained in the Saururus chinensis extract obtained by selecting quercetin (QCT) which has not been reported in the glial cells of known compounds, using HPLC analysis. The results are shown in FIG. 6A .

It has already been reported that quercetin exhibits potent antioxidant activity (Choi et al., 2002) and anti-inflammatory efficacy (Kao et al., 2010). The present inventors confirmed the absence of toxicity by conducting a cytotoxicity test on BV-2 glial cells using a quercetin compound (Fig. 6B), and found that concentration of quitetin at various concentrations (1, 5, 10 μM) (Fig. 6D). In addition, in order to confirm the inhibitory effect of quercetin on neuroinflammation, various inflammation-related biomarkers were used to confirm that it inhibited concentration-dependently (Fig. 6E).

6A to 6E show the anti-inflammatory effect of quercetin, the active ingredient of Saururus chinense extract in BV-2 glial cells stimulated with LPS. FIG. 6A shows HPLC analysis of QCT, an extract of Saururus chinensis and an indicator substance. BV-2 glial cells were plated at 5 × 10 ⁴ cells / well on a 96-well plate and cultured for 24 hours. Quercetin, an active ingredient of Saururus chinensis extract, was treated with LPS (100 ng / Lt; / RTI > FIG. 6B is a result of removing the supernatant after 24 hours and conducting a cytotoxicity test using MTT reagent. FIG. 6D shows the result of measuring the amount of nitrite released using the Griess assay using the supernatant removed. Figure 6C shows the chemical structure of quercetin. FIG. 6E shows the results of culturing the cells in a ^6- well plate at 1 × 10 ⁶ cells / well for one day to identify nitrite (NO) synthesis-inducing enzyme iNOS, inflammatory mediator protein COX-2 and proinflammatory cytokine IL- , QCT was pretreated for 1 hour by concentration, and stimulated with LPS (100 ng / ml). After 6 hours or 18 hours in each experiment, genes and proteins were identified in the cells and analyzed by RT-PCR and western blot. Experimental results are based on 3 repeated experiments and show similar experimental results.

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)

(IL-1b), IL-6, and TNF-α were inhibited by inhibiting the activity of neuroglial cells. A composition for preventing or treating a neurodegenerative disease, comprising a Saururus chinensis extract prepared by the following production method containing quercetin (QCT) as an active ingredient having an inhibitory effect.
(1) drying three to five seconds at a temperature of 15 to 20 DEG C and
(2) pulverizing the dried Saururus chinensis
(3) mixing 100 parts by weight of ethanol with 1 to 20 parts by weight of water or 1 to 20 parts by weight of an alcohol having 1 to 4 carbon atoms to prepare a solvent; and
(4) mixing the pulverized saururus prepared in the above step (2) with the solvent prepared in step (3) of 0.2 to 2.0 times the weight of the saururus pulverized to prepare a mixture; and
(5) preparing the Saururus chinensis extract by using an ultrasonic disintegrator while maintaining the extraction temperature of 15 to 25 캜 in the mixture prepared in the step (4); and
(6) a step of vacuum-concentrating and freeze-drying the Saururus chinensis extract prepared at step (5) at 50 to 60 ° C and
(7) The step of injecting the extract lyophilized by the above step (6) into a container, followed by concentration under reduced pressure at 40 to 50 캜.
delete delete A herbal medicine preparation comprising the composition of claim 1.
A health functional food comprising the composition of claim 1.
delete delete delete delete

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