KR101996487B1 - Medicinal composition and health functional food for preventing or treating inflammatory disease - Google Patents

Abstract

The present invention relates to a medicinal composition and a health functional food for preventing or treating an inflammatory disease, including a soybean curd extract. Specifically, the present invention relates to a composition for reducing or inhibiting the production of inflammatory cytokines such as TNF-a, IL- iNOS, and COX-2, inhibits the activity of inflammatory transcription factors, and can be preferably used as a composition for preventing or treating inflammatory diseases.

Description

TECHNICAL FIELD [0001] The present invention relates to a medicinal composition for preventing or treating an inflammatory disease,

The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating an inflammatory disease which exhibits an excellent inflammation-inhibiting effect.

Canavalia gladiata (sword bean) is a perennial vine plant belonging to the rosewood leguminosae. It is the largest among the various varieties of soybeans and is known to have excellent taste. Soybean is known to contain a variety of physiologically active substances such as hydrolytic enzymes urease, hemagglutinin, canavalin, linolenic acid, saponin, choline, lecithin, dietary fiber, and protein , Functional foods and pharmaceutical materials.

Soybean is called "Tozu" in the oriental medicine. It is effective for eliminating inflammation and enhances the immunity of the body, so it is effective for various boils and purulent diseases, warms the stomach, is effective for asthma, sputum and arthritis, It is said to cure arthritis, Shinhei low back pain, constipation, and obesity.

Inflammation is a defensive period that prevents human tissue damage from infection, and involves symptoms such as development, fever, and pain. If this inflammatory reaction is continuously repeated for a long time, it can cause various chronic diseases such as degenerative diseases and cancer. Macrophages are involved in and regulate the production of inflammatory mediators such as nitric oxide (NO), prostaglandin (PG) and pro-inflammatory cytokines in the human immune response. These inflammatory mediators induce an inflammatory response and, if the host immune response does not respond appropriately, induce inflammatory diseases. Nitric oxide synthase (NOS) includes neuronla NOS, endothelia NOS, and inducible NOS (iNOS), which are extremely abundant in NO production by iNOS and cause excessive tissue damage and gene mutation. Other cyclooxygenases (COX) are classified as COX-1, which acts on normal biological functions such as platelet formation, renal function maintenance, and gastric wall protection, and COX-2, which is involved in fever and pain. Thus, anti-inflammatory effects can be confirmed by confirming inhibition of the production of substances such as NO and PGE2 produced by iNOS and COX-2 in the inflammatory reaction.

In recent years, studies on natural anti-inflammatory substances that inhibit enzymes involved in inflammatory reaction and preventive and therapeutic effects on various chronic diseases using natural materials have been carried out, but they are still insufficient. Antioxidative effects of soybean and other antimicrobial activities related to soybean koji, antimicrobial activity extracts and their preparation methods, antimicrobial activity using soybean curd extracts, Development of antioxidant functional foods, and compositions for treating allergies and inflammations have been reported, but studies on anti-inflammation using soybean curd have been lacking.

Korean Patent No. 1680014

The present inventors have made efforts to develop a human-safe substance, particularly a plant-derived substance, capable of effectively preventing or treating an inflammatory disease. As a result, the present inventors have found that soybean extract inhibits the inflammatory response of macrophages with activated macrophages, The present invention has been completed.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition and a health functional food for the prophylactic or therapeutic treatment of inflammatory diseases including mature / immature soybean extract.

1. A pharmaceutical composition for the prophylaxis or treatment of an inflammatory disease comprising a soybean extract.

2. The pharmaceutical composition according to 1 above, wherein the soybean extract is soybean and pea extract.

3. The pharmaceutical composition according to item 1 above, wherein said soybean extract is a bean curd extract.

4. The pharmaceutical composition according to 1 above, wherein the soybean curd is a ripe soybean.

5. The pharmaceutical composition according to item 1 above, wherein the soybean curd extract is a mixed solvent extract of water and prtanol A.

6. The pharmaceutical composition according to 5 above, wherein the mixed solvent comprises water and prtanol A in a volume ratio of 1: 9 to 3: 7.

7. The pharmaceutical composition according to 1 above, wherein said soybean extract is an ethyl acetate fraction.

8. The method of claim 1 wherein the inflammatory disease is selected from the group consisting of atopic dermatitis, edema, dermatitis, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, Diseases selected from the group consisting of rheumatoid spondylitis, rheumatoid arthritis, osteoarthritis, rheumatoid arthritis, shoulder inflammation, nephritis, nephritis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome and multiple sclerosis / RTI >

9. The pharmaceutical composition according to 1 above, wherein said inflammatory disease is Crohn's disease or ulcerative colitis.

10. A health functional food for preventing or improving an inflammatory disease including soybean extract.

11. The composition of claim 10, wherein the soybean curd is a ripe soybean.

12. The composition of claim 10, wherein said soybean extract is a mixed solvent extract of water and prtanol A.

13. The health functional food according to the above 10, wherein the soybean extract is an ethyl acetate fraction.

14. The method of claim 10, wherein the inflammatory disease is selected from the group consisting of atopic dermatitis, edema, dermatitis, allergies, asthma, conjunctivitis, periodontitis, rhinitis, otitis, rhinitis, tonsillitis, pneumonia, gastric ulcer, gastritis, A syndrome consisting of sjogren's syndrome and multiple sclerosis, including but not limited to rheumatoid arthritis, rheumatoid spondylitis, rheumatoid arthritis, osteoarthritis, rheumatoid arthritis, rheumatoid arthritis, shoulder pain, nephritis, ≪ / RTI >

15. The health functional food according to item 10 above, wherein said inflammatory disease is Crohn's disease or ulcerative colitis.

The extracts of soybean curd according to the present invention inhibit the production and suppression of inflammatory cytokines such as TNF-a, IL-6 and the like against macrophages, suppress the expression of iNOS and COX-2, and inhibit the activity of inflammatory transcription factors. Therefore, it can be preferably used as a composition for preventing or treating inflammatory diseases.

Fig. 1 is a diagram showing the appearance of whole pods of ripe and immature seeds.
Fig. 2 shows the effect of RAW264.7 cells (A) and mouse embryonic stem cell (B) survival rate on the concentration of mature / immature soybean extract.
FIG. 3 is a graph showing the degree of inhibition of the production of inflammatory mediators by LPS by mature / immature soybean extract. (A) NO, (B) PGE2, (C) iNOS protein, (D) COX-2 protein
FIG. 4 is a graph showing the degree of inhibition of inflammatory cytokine activity by (C, D) LPS in RAW264.7 cells (A, B) and mouse peritoneal macrophages of mature / immature soybean extracts. (A, C: IL-6, B, D: TNF-α)
FIG. 5 is a graph showing the inhibition of the activity of an electron regulatory factor (NF-KB) induced by PMA and LPS by mature / immature soybean extract. (A: NF-κB report assay, B: NF-κB immunofluorescence analysis, C: cytoplasmic and nuclear IB protein and NF-κB)
FIG. 6 is a graph showing protein inhibition involved in the inflammation mechanism by mature / immature soybean extract. FIG.
FIG. 7 is a graph showing changes in body weight and length after administration of ripe soybean extract according to each experimental group in the DSS-induced inflammatory bowel disease model.
8 is a graph showing the degree of suppression of inflammatory mediator protein after administration of ripe soybean extract according to each experimental group in the DSS-induced inflammatory bowel disease model.
FIG. 9 is a graph showing the degree of suppression of inflammatory cytokine activity after administration of ripe soybean extract of each experimental group in the DSS-induced inflammatory bowel disease model.
FIG. 10 shows the results of searching for the amount of gallic acid in soybean curd extract using HPLC analysis system.
Fig. 11 shows the cytotoxicity test results of the soybean extract and its fractions.
Fig. 12 is a test result showing the NO inhibitory effect of soybean curd extract and its fractions.
Fig. 13 is a test result showing the effect of inhibiting the secretion of inflammatory cytokines from the extracts of soybean curd and its fractions.

Hereinafter, the present invention will be described in detail.

The pharmaceutical composition for preventing or treating the inflammatory disease of the present invention comprises soybean curd extract.

The present inventors have found that the extract of Canavalia gladiata (sword bean) is excellent in the inhibitory effect on the NO production of cells and the suppression of intracellular inflammation-related protein expression, and that the extract of the present invention has an excellent recovery effect in the inflammatory disease model The present invention has been devised.

The soybean extract of the present invention is an extract of soybean curd, which may be an extract of soybean curd alone, an extract of soybean curd alone, or a mixed extract of soybean curd and bean curd.

The soybean extract of the present invention may be an immature soybean extract, a ripe soybean extract or a mixed extract thereof. Preferably a ripe soybean extract.

In the present specification, the immature soybean is a green-colored period with fruit pods (the bottom of Fig. 1, for example, sown in March to April and harvested in June to July), ripe pods are brown (The upper part of Fig. 1, for example, harvested from March to April and harvested from August to October). The sowing time, harvesting time, etc. may vary depending on cultivation conditions.

The soybean extract of the present invention may be one extracted by a conventional method with a conventional solvent.

The extraction solvent may be, for example, water, an alcohol having 1 to 4 carbon atoms, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane, hexane, prtanol A or a mixed solvent thereof.

Specifically, a mixed solvent of water and prtanol A can be used. The mixing ratio may be, for example, water and prtanol A in a volume ratio of 1: 9 to 3: 7, but is not limited thereto.

The extraction temperature may be 20 to 100 占 폚, and the extraction period may be about 1 to 4 days. The extraction can be performed using extraction methods such as hot water extraction, cold extraction, reflux extraction, or ultrasonic extraction.

In addition, filtration, concentration, drying and the like may be performed under conditions well known in the art.

In addition, the soybean extract of the present invention may be a specific solvent fraction of the solvent extract. For example, it may be a fraction such as nucleic acid (Hexane), chloroform (CHCl3), ethyl acetate (EA), butanol (n-BuOH) and the like. Among these solvents, ethyl acetate fraction may be preferable.

As used herein, inflammatory disease refers to any disease that is mediated by inflammation. Specifically, there is provided a method for treating atopic dermatitis, edema, dermatitis, allergies, asthma, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn disease, colitis, hemorrhoids, but are not limited to, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, shoulder periarmitis, tendonitis, nephritis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome and multiple sclerosis. More specifically Crohn's disease, ulcerative colitis.

The medicinal composition of the present invention is excellent in inhibiting the production of inflammatory mediators (NO, TNF-α, IL-6) in macrophages related to congenital immunity, and thus exhibits excellent pharmaceutical effects against various inflammatory diseases.

The pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method.

Examples of carriers, excipients and diluents that may be contained in the composition containing the extract of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, , Alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil have. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose , Gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The dosage of the medicinal composition of the present invention may vary depending on the age, sex and body weight of the patient, but may be 0.1 to 100 mg / kg, preferably 0.1 to 10 mg / kg, once to several times per day. The dosage may also be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Accordingly, the dosage is not limited in any way to the scope of the present invention.

In addition, the present invention provides a health functional food for preventing or ameliorating an inflammatory disease comprising a soybean extract.

The contents of soybean extract, inflammatory diseases are as described above.

The health functional food of the present invention is a food prepared and processed including the soybean curd extract having the above excellent functionality, and includes all foods regardless of the formulation.

The health functional food of the present invention can be used in a variety of foods including beverages including alcoholic beverages including soybean extract, fruits and processed foods thereof (e.g., canned fruit, jar, jam, maarelade, etc.), fish, (Eg udon, buckwheat noodles, ramen noodles, spaghetti, macaroni, etc.), juice, various drinks, cookies, candy, dairy products such as butter, cheese, Margarine, vegetable protein, retort food, frozen food, various seasonings (eg, miso, soy sauce, sauces, etc.).

In addition, the health functional food of the present invention may be formulated into tablets, pills, powders, granules, powders, capsules, liquid preparations and the like. These may be formulated further comprising one or more of carriers, diluents, excipients and additives.

Examples of the additive which can be further included in the present invention include natural carbohydrates, flavors, nutrients, vitamins, minerals (electrolytes), flavors (synthetic flavors, natural flavors and the like), colorants, fillers At least one component selected from the group consisting of acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, antioxidants, glycerin, alcohols, carbonating agents and fats can be used.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tautatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharine, aspartame, etc.) can be advantageously used as the flavor.

In addition to the above, the health functional food of the present invention may contain flavorings such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, And salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health functional food of the present invention may contain flesh for the production of natural fruit juice and vegetable beverages. These components may be used independently or in combination.

Specific examples of the carrier, excipient, diluent and additives include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium phosphate, calcium Silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate And a mineral oil are preferably used.

The content of the health functional food of the present invention as the above-mentioned functional ingredient in the formulation can be appropriately adjusted depending on the mode and purpose of use, the condition of the user, the type of symptoms and the severity, and is 0.001 to 99.9% by weight, May be 0.01 to 50% by weight, but is not limited thereto.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to examples.

Example

Example . Preparation of soybean extract

2 L extraction solvent (400 mL of distilled water, 1600 mL of Prednol A (alcohol)) was added to 2 kg of full ripened soybeans (Fig. 1), and each extract was obtained by refluxing at 70 캜 for 3 hours. The obtained extract was filtered through a filter paper, and the filtrate was concentrated under reduced pressure using a rotary vacuum concentrator. Then, each extract obtained by lyophilization was dissolved in DPBS at a concentration of 200 mg / mL, and then syringe filter (0.22 mu m) was used. This was used as an experimental sample (soybean koji, RSBE, Mi-Sook; GSBE).

Experimental Example

Cell culture

Mouse macrophages RAW264.7 and 293T cells were obtained from ATCC (American Type Culture Collection) and passaged in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin. Cells were cultured in a 37 ° C, 5% CO ₂ incubator.

Isolation and culture of mouse embryonic cell

Approximately 10 mL of RPMI1640 medium was injected into the abdominal cavity of the mouse, the abdominal cavity was gently massaged, and the abdominal cavity was taken out to collect the peritoneal macrophages. The cells were washed twice with the medium and suspended in RPMI 1640 medium supplemented with 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin. After incubation at 37 ° C. in a 5% CO ₂ incubator for 2 hours, .

Cytotoxicity test

Cytotoxicity tests were performed by MTS assay. RAW264.7 cells (1 × 10 ⁴ cells / well) were cultured overnight, and the samples were treated for 24 hours at a concentration of 1 × 10 ⁵ cells / well The samples were treated by concentration for 48 hours. After incubation, 20 uL of MTS reagent was added and the absorbance was measured at 490 nm using a microplate spectrophotometer (BioTek Instruments, Inc., VT, USA) after 1 to 4 hours. This is shown in FIG. Cytotoxicity test results showed that RSBE and GSBE did not inhibit cell growth, that is, they were not cytotoxic.

NO and PGE ₂  Production amount measurement

The nitrite concentration in the cell culture solution was measured by using a grease reagent. RAW264.7 cells were cultured in a 48-well plate at a density of 1 × 105 cells / well for 24 hours. Thereafter, the sample was treated with RSBE and GSBE for 1 hour before induction of NO production with LPS (1 ug / mL) for 24 hours. Then, the culture supernatant and the Griess reagent were mixed at a ratio of 1: 1 and analyzed using a microplate spectrophotometer The amount of production was measured. Sodium nitrite (NaNO ₂ ) was used to prepare a standard curve and used for NO determination. The amount of PGE ₂ produced in the culture supernatant was assayed using an ELISA kit (BD Bioscience, MN, USA) according to the manufacturer's protocol. The results are shown in FIG.

As a result of confirming the anti - inflammatory effect on NO production inhibition, the NO production of LPS - treated cells was increased compared to the control without LPS treatment. RSBE and GSBE samples dose-dependently inhibited NO production by LPS. As shown in FIG. 3 (A), the NO inhibiting activity of GSBE sample was larger than that of RSBE at 2 mg / mL concentration.

The amount of other inflammatory substance PGE2 produced is shown in Fig. 3 (B). When the samples were treated with RSBE and GSBE, the production was inhibited in a concentration dependent manner, and the inhibition of PGE ₂ formation at RSBE 2 mg / mL concentration was high.

iNOS  And COX-2 expression inhibition (Western-blotting)

Western blot analysis was used to measure the expression pattern of inflammatory proteins in the cells. The amount of iNOS and COX-2 protein was measured to investigate the cellular inflammation inhibitory effect of RSBE and GSBE. RAW264.7 cells were plated at a density of 1 × 10 ⁶ cells / well in a 6-well plate and cultured for 4 hours. The samples (RSBE, GSBE: 2 mg / mL) and control drug (celecoxib: 20 μM) , And treated with LPS (1 ug / mL) for 24 hours. After incubation, the cells were washed twice with DPBS and then lysed with a phosphatase inhibitor cocktail buffer (25 mM Tris pH 7.5 with H ₃ PO ₄ , 2 mM CDTA, 1% Triton X-100, 2 mM DTT, The cell lysate was prepared and centrifuged at 13,000 rpm for 10 minutes at 4 ° C. The supernatant was used as total protein extract. Protein was quantitated by Bradford assay (Bio-Rad Laboratories, CA, USA). 30 μg of the protein was boiled at 100 ° C. for 10 minutes and then electrophoresed at 100 V on 10% SDS-PAGE. Protein was attached to PVDF and blocked with 5% skim milk. After iNOS and COX-2 were reacted with primary antibody, secondary antibody was treated with HRP-conjugated anti-rabbit antibody. To identify the target protein, ECL (enhanced chemiluminescence, Advansta Inc., CA, USA) was used as a substrate, and the band was developed using a C300 chemiluminescence imager (Azure Biosystems, Inc., CA, USA).

The inhibitory activity of iNOS and COX-2 protein induced by LPS of RSBE and GSBE was confirmed as shown in Fig. 3 (C, D). The expression of iNOS and COX-2 protein is increased by LPS. RSBE and GSBE at a concentration of 2 mg / mL were found to simultaneously inhibit the two proteins of iNOS and COX-2, and they were found to have a strong inhibitory activity as much as the control drug celecoxib of the COX-2 protein of RSBE .

Measure inflammatory cytokine secretion

RAW264.7 cells (1 × 10 ⁵ cells / well) were cultured in a 48-well plate for 24 hours, and mouse embryonic cells (4 × 10 ⁵ cells / well) -well plate. The cells were pre-treated for 1 hour for each concentration, treated with 1 μg / mL of LPS and re-cultured for 24 hours. The amount of cytokine secreted by IL-6 (Biolegend, CA, USA) and TNF-α (BD Bioscience, MN, USA) in the culture was assayed by ELISA kit according to the protocol of the manufacturer. Absorption at 490 nm using a microplate spectrophotometer Were measured.

As a result, the amount of each cytokine secreted by the sample RSBE and GSBE concentration showed a concentration-dependent decrease (FIG. 4). In the RAW264.7 cell and mouse embryonic cell-treated RSBE 2 mg / mL samples, the IL-6 and TNF-α cytokine secretion levels were significantly inhibited compared to the LPS-treated control group.

Inflammatory transcription factor NF -B reporter assay

100 ng of pNF-κB-SEAP report plasmid was treated with HilyMax (Dojindo molecular technologies, Inc., MD, USA) in each well of a 96-well plate containing 293T cells (1 × 10 ⁴ cells / well) Lt; / RTI > The samples were then pretreated for 1 hour and stimulated with PMA (3 ng / mL) for 24 hours. After incubation with QUANTI-Blue (Invitrogen, MA, USA) for 4 hours, the activity of NF-B was measured by measuring the absorbance at 630 nm using a microplate spectrophotometer.

The activity of NF-B was significantly increased by PMA stimulation, indicating that NF-B activated by PMA stimulation increased the expression of SEAP with NF-b promoter. The inhibitory effect of RSBE and GSBE was significantly decreased by concentration. In the case of RSBE 2 mg / mL, the result of inhibition by 48% or more as compared with the PMA control was also shown in FIG. 5 (A).

Inflammatory transcription factor NF Of -B In immunofluorescence analysis  Activity analysis by

RAW264.7 cells were plated at 2 × 10 ⁴ cells / well in an 8-well chamber slide (Lab-Tek, MD, USA). Cells were pretreated with RSBE, GSBE at a concentration of 2 mg / mL and control drug bay11-7082 at a concentration of 30 uM, and treated with 1 ㎍ / mL of LPS for 2 hours. Cells were fixed with 4% paraformaldehyde for 15 min and blocked with 1% BSA / PBS containing 0.1% Triton X-100. After washing the cells, the NF-κB antibody was reacted with O / N at 4 ° C. After washing the cells, Alexa Fluor 488-labeled secondary antibody was reacted and the nuclei were stained with DAPI. Cells were observed with confocal microscopy (Carl Zeiss, Jena, Germany).

NF-κB / p65 (green) was mostly distributed around the DAPI-stained nucleus (blue) in the absence of any stimulation, but most of the green NF-κB / , Indicating that NF-κB was activated and migrated to the nucleus. When these cells were treated with 30 uM of the control drug (bay 11-3082), NF-κB / p65 was observed to be distributed in the cytoplasm around the nucleus. When NF-κB / p65 was pre-treated at 2 mg / mL of each of RSBE and GSBE, the expression of NF-κB / p65 was observed in the cytoplasm of nuclear periplasm, and the activation of NF-κB was remarkably inhibited in RSBE compared to GSBE (B).

Cytoplasmic and nuclear Inflammatory transcription factor NF Confirmation of inhibition of -B

In order to analyze the degree of activation of NF-κB, cytoplasmic and nuclear protein extracts were separately prepared from RAW264.7 cells treated with RSBE, GSBE and LPS, respectively. Specifically, the treated cells (1 × 10 ⁶ cells / dish) were washed with PBS and recovered. Then, 180 μL of hypotonic buffer [10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl ₂ , 0.02% NaN ₃ , 0.5 mM DTT, 1 mM PMSF, pH 7.4] was added, and 20 μl of 5% nonidet NP-40 was added thereto, followed by reaction for 5 minutes. After centrifugation (1,800 g, 4 ° C, 5 minutes), the supernatant was used as a cytoplasmic extract. The precipitate was washed once with hypotonic buffer and added with hypertonic buffer (20 mM HEPES, 25% glycerol, 40 mM NaCl, 1.5 mM MgCl ₂ , 0.2 mM EDTA, 0.02% NaN ₃ , 0.5 mM DTT, 1 mM PMSF, (13,000 g, 4 ° C, 10 minutes), and the supernatant was recovered and used as the nuclear protein extract. The degree of activation of NF-κB and IB was analyzed by Western blot.

The translocation of NF-κB into the nucleus is due to the phosphorylation of IB, a protein that inhibits it. As shown in Fig. 5 (C), the amount of IB in the cytoplasm was decreased by LPS stimulation, and the increase in the amount of NF-κB caused decomposition by increase of IB phosphorylation and NF-κB activation was observed. The amount of IB increased by RSBE and GSBE treatment resulted in a decrease in the level of NF-κB in the nucleus.

By LPS  Induced MARKs  Inhibitory effect on activation

MARKs (ERK, JNK, p38) phosphorylation was analyzed by Western blot to determine the signal proteins involved in the NF-B signal pathway. As shown in FIG. 6, JNK and ERK phosphorylation was reduced by RSBE and GSBE treatment in RAW264.7 cells induced by LPS but did not affect phosphorylation of p38. These results indicate that they have anti-inflammatory activity by regulating phosphorylation of IB as well as NF-b and phosphorylation of higher protein (Fig. 6).

Experimental animal

(24 ± 1 ℃, humidity 55 ± 5%, 12-hour light cycle) were obtained for two days while feeding a diet of BALB / C 16-18g and 6-week old mice from Daehan Science , And then started the experiment.

The experimental group was divided into three groups of 5 mice per group: control (control), inflammatory bowel disease induced group (DSS), inflammatory bowel disease, and RSBE prepared in the above example for 4 days (DSS + RSBE) and then fed for 8 days. The body weight changes of the experimental animals were examined during the experimental period, and the results are shown in FIG.

Inflammatory Bowel Disease Induced by DSS

Intestinal disease models using dextran sodium sulfate (MW: 36,000-50,000 Da, MP Biomedicals, CA, USA) were performed according to the same method (Chemically induced mouse models of intestinal inflammation, Wirt et al., Nature Protocols 2 541 -546 (2007)).

Specifically, 6-week-old mice were fed DSS with 3.5% free water. After 4 days, the RSBE prepared in the above example was administered intraperitoneally for 4 days at 2 g / kg.

After the experimental breeding, the abdomen was excised by cervical dislocation and the large intestine was excised. FIG. 7 shows the mouse body weight and the colon length according to the RSBE administration of the present invention. In the case of RSBE administration of the present invention, the intestinal length was significantly lower in the DSS-administered group, and the RSBE-administered group recovered the colon length to a level not induced by the colonic inflammation. In the case of mouse body weight, about 10% of body weight was lost in DSS - treated group, and after 3 days of administration of RSBE, body weight was recovered to almost the same level as normal body weight.

Inflammatory Parameter  Determination of cytokine secretion and identification of inflammatory indicator proteins

The amount of cytokine secreted by inflammatory markers and the inflammatory indicator protein in the proteins isolated from mouse colon tissues were determined.

Colonic tissues were homogenized by adding protein extraction solution (Biovision, CA, USA) in an ice-cold state. The supernatant was used for the experiment by centrifugation (13,000 rpm, 10 min, 4 ° C). This was electrophoresed at 100 V on 10% SDS-PAGE. Protein was attached to PVDF and blocked with 5% skim milk. After reacting with iNOS, COX-2, NF-κB and IκB-α as primary antibodies, HRP-conjugated anti-rabbit antibody Respectively. To identify the target protein, ECL (enhanced chemiluminescence, Advansta Inc., CA, USA) was used as a substrate, and the band was developed using a C300 chemiluminescence imager (Azure Biosystems, Inc., CA, USA).

The results of confirming the inhibitory activity of iNOS and COX-2 protein induced by LPS of RSBE are shown in Fig. The expression of iNOS and COX-2 protein is increased by DSS. Inhibition activity of iNOS and COX-2 proteins in RSBE-treated group was concurrently observed.

The secretion levels of IL-6, IFN-γ (Biolegend, CA, USA) and TNF-α, IL-1β (BD Bioscience, MN, USA) cytokines in the supernatants obtained from the tissues were measured by ELISA kit The absorbance was measured at 490 nm using a microplate spectrophotometer.

As shown in Fig. 9, all cytokines were increased in the DSS-administered group, but the amount of cytokines in the RSBE-administered group was significantly lower than that in the DSS-administered group.

HPLC  Quantification by analysis

HPLC analysis was performed to quantify gallic acid for RSBE and GSBE. The column used was LUNA C18 column (5 μm, 4.6 × 250 mm, CA, USA) and the mobile phase was water: acetonitrile (95: 5) 10 minutes. The injection amount of the sample was 20 μL, the flow rate of the mobile phase was 1.0 mL / min, and the wavelength of the UV detector was 271 nm.

The results of searching for the amount of gallic acid in the sample using the HPLC analysis system are shown in FIG. As shown in Fig. 10, the gallic acid standard product was detected at the time of 6.2 min. The amount of gallic acid in RSBE and GSBE was 0.00147% (w / w) and 0.00019% (w / w), respectively. The amount of gallic acid in RSBE was about 10 times higher than that of GSBE.

Example . Ripe soybean extract Fraction  Produce

Ripe soybean extract prepared from the above extract; 10 g of RSBE was suspended in 1 L of distilled water and extracted twice with the same amount of nucleic acid (hexane), chloroform (CHCl ₃ ), ethyl acetate (EA), and butanol (n-BuOH) , And concentrated under reduced pressure using a rotary vacuum concentrator to prepare a ripe soybean extract fraction. Then, each fraction obtained by lyophilization was dried in a water layer (1.065 g in the nucleic acid layer, 0.503 g in the chloroform layer, 0.362 g in the ethyl acetate layer, 1.115 g in the butanol layer, . In the following experiment, it is referred to as Hex fraction, CH fraction, EA fraction, Bu fraction and water fraction.

Experimental Example

Cytotoxicity test

Cytotoxicity test was performed by MTS assay. RAW264.7 cells (1 × 10 ⁴ cells / well) were cultured overnight and the samples were treated for 24 hours by concentration. The absorbance of the culture was measured at 490 nm using a microplate spectrophotometer (BioTek Instruments, Inc., VT, USA) after 1 hour of addition of 20 uL of MTS reagent. This is shown in FIG. Hex fraction inhibited cell growth by about 20% at 1 mg / mL concentration, and CH fraction inhibited cell growth and showed toxicity at all concentrations.

NO production measurement

The nitrite concentration in the cell culture solution was measured by using a grease reagent. RAW264.7 cells were seeded on a 48-well plate at a density of 1 × 10 ⁵ cells / well and incubated for 24 hours. Then, each fraction of the ripe soybean extract and RSBE were treated 1 hour before, and the NO production was induced with LPS (1 ug / mL) for 24 hours. Then, the culture supernatant and Griess reagent were mixed 1: 1 and microplate spectrophotometer was used to measure 570 nm was measured. Sodium nitrite (NaNO ₂ ) was used to prepare a standard curve and used for NO determination. The results are shown in Fig.

The NO production of LPS - treated cells was increased when compared with the control without LPS treatment. RSBE significantly inhibited NO production by LPS. Hex fraction increased NO production at the concentration of 1 mg / mL with cytotoxicity, but inhibited NO production at a concentration below 0.3 mg / mL in a concentration dependent manner. The CH fraction also inhibited NO production at the concentration of 1, 0.3 mg / mL, which was cytotoxic, but inhibited NO production at the concentration of 0.1 mg / mL. EA fraction significantly inhibited NO production in a concentration dependent manner, and Bu fraction inhibited NO production only at the concentration of 1, 0.3 mg / mL. Although not shown in the data, the water fraction showed NO inhibition at a high concentration of 1 mg / mL, but showed very slight inhibition.

Measure inflammatory cytokine secretion

RAW264.7 cells (1 × 10 ⁵ cells / well) were cultured on a 48-well plate for 24 hours to examine the effect of inhibiting inflammatory cytokine secretion. The cells were pre-treated for 1 hour by concentration, and then treated with 1 μg / mL of LPS and re-cultured for 24 hours. The amount of cytokine secreted by IL-6 (Biolegend, CA, USA) and TNF-α (BD Bioscience, MN, USA) in the culture was assayed by ELISA kit according to the protocol of the manufacturer. Absorption at 490 nm using a microplate spectrophotometer Were measured.

As a result, cytokine production of LPS-treated cells was increased when compared to the control without LPS treatment (Fig. 13). The CH fraction inhibited the cytokine production at the concentration of 0.1 mg / mL, and the EA fraction significantly inhibited cytokine production in a concentration dependent manner. This fraction inhibited cytokine at 1 mg / mL concentration.

Claims (15)

A pharmaceutical composition for the prophylaxis or treatment of inflammatory bowel disease, comprising soybean curd extract.
The pharmaceutical composition according to claim 1, wherein the soybean extract is soybean and pea extract.
The pharmaceutical composition according to claim 1, wherein the soybean curd extract is a bean curd extract.
The pharmaceutical composition according to claim 1, wherein the soybean curd is a ripe soybean.
The pharmaceutical composition according to claim 1, wherein the soybean curd extract is a mixed solvent extract of water and prtanol A.
[Claim 7] The pharmaceutical composition according to claim 5, wherein the mixed solvent comprises water and prtanol A in a volume ratio of 1: 9 to 3: 7.
The pharmaceutical composition according to claim 1, wherein the soybean extract is an ethyl acetate fraction.
delete The pharmaceutical composition according to claim 1, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.
A health functional food for preventing or ameliorating inflammatory bowel disease including soybean extract.
The health functional food according to claim 10, wherein the soybean koji is a ripe soybean.
[Claim 10] The health food according to claim 10, wherein the soybean extract is a mixed solvent extract of water and prtanol A.
The health functional food according to claim 10, wherein the soybean extract is an ethyl acetate fraction.
delete [Claim 11] The health food according to claim 10, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

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