KR20190003415A - Composition for preventing or treating of Th2-mediated immune disease or inflammatory disease comprising enzyme-treated ellagic acid as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating, or treating Th2-mediated immune diseases or inflammatory diseases, which comprises enzyme-treated ellagic acid as an active ingredient. The composition according to the present invention, by including enzyme-treated ellagic acid as an active ingredient, may maximize antiallergenic effects and anti-inflammatory effects of ellagic acid. In particular, it was confirmed that treating splenocytes of OVA-immunized mice with enzyme-treated ellagic acid results in significantly enhanced inhibitory activities on IL-4 formation as compared to ellagic acid, and it was confirmed that treating macrophages with enzyme-treated ellagic acid results in enhanced inhibitory activities on MIP-2 and IL-6 formation as compared to ellagic acid. Accordingly, the composition according to the present invention may be beneficially used in the development of therapeutic agents, functional health foods, and cosmetics for preventing, alleviating, or treating Th2-mediated immune diseases or inflammatory diseases.

Description

[0001] The present invention relates to a composition for preventing or treating a Th2-mediated immune disease or an inflammatory disease, which comprises an enzyme-treated ellagic acid as an active ingredient.

The present invention relates to a composition for the prophylaxis or treatment of a Th2-mediated immune disease or inflammatory disease, which comprises an enzyme-treated elastase as an active ingredient.

The immune balance regulated by cytokines produced by Th1 (type 1 helper T) and Th2 (type 2 helper T) cells is called the Th1 / Th2 balance. Th1 cells are important for the regulation of cellular immunity, It plays an important role in humoral regulation. Th1 / Th2 balance is maintained constantly by interfering with cytokine mainly on IFN-γ and cytokine mainly on IL-4, which are important for Th1 differentiation, and Thl / Th2 If the balance is broken, a variety of immune diseases can be induced. For example, in the case of deflecting Thl, cellular immunity is resurfaced to enhance infection resistance, and when deflected to Th2, infection resistance is reduced, and conversely, allergy is enhanced.

Inflammation is a mechanism of recovery to regenerate damaged areas caused by chemical substances, bacterial infections, and physical effects that cause changes in organisms or tissues. When inflammation is manifested by harmful stimuli, infections and trauma, inflammation is induced by local release of vasoactive substances such as inflammatory components. If the inflammatory reaction is continued, mucous membrane damage is promoted, resulting in redness, fever, swelling, Inflammatory diseases such as rheumatoid arthritis, arteriosclerosis, gastritis, and asthma caused by pain, dysfunction, and the like.

Macrophages are involved in the maintenance of homeostasis in response to various host reactions such as acquired immunity as well as innate immunity, and play important roles in biological defense in the early stage of infection by producing nitric oxide (NO) and various cytokines during inflammatory reaction. Macrophage or monocytes such as RAW 264.7 have been shown to stimulate tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-1β by the stimulation of endotoxin lipopolysaccharide (LPS) 6 promotes the secretion of inflammatory mediators such as cytokines.

Allergy is a disease caused by a malfunction of the immune system that causes irritation such as urticaria, itching, runny nose, and cough in a specific person. It is a disease caused by environmental pollution, westernization of diet and lifestyle , Environmental factors such as stress, genetics, and various allergens. Twenty to 25 percent of the world's population is exposed to allergic diseases, and the prevalence of this disease continues to rise.

Allergic reactions are traditionally divided into four classes based on the time and type of expression: type I-type III is a humoral immune response (immediate type) involving antibodies, type IV is a cell-mediated immune response Delay type). Most allergic reactions are type I, and are typical diseases such as asthma, rhinitis, conjunctivitis, food and drug allergy, and atopic dermatitis. In severe cases, anaphylaxis may occur.

The type I immediate-type hypersensitivity reaction is divided into two stages. In the first step, the Th1 cell reaction which produces IL-12 and IFN-γ which suppresses the secretion of IgE and IgG1 by the invasion of the allergen and increases the secretion of IgG2a And Th2 (type 2 helper T) cell responses that produce IL-4, IL-5 and IL-13 are tilted toward Th2 and IL-4 and IL-13 are secreted by excessive immune response of Th2. It is said that IgE-specific antibodies produced by B cells are attached to the surface of mast cells or basophils, and allergens are sensitized to allergens.

The second stage of the allergic manifestation is divided into an initial reaction and a late reaction. The initial reaction re-enters the body to stimulate mast cells and triggers the degranulation reaction. At this time, the vasodilation by histamine, lipid metabolites, cytokines, And late response is activated by infiltration of neutrophils, eosinophils, macrophages, Th2 cells, basophils, and the like in the tissues, thereby causing inflammation and causing atopic dermatitis, rhinitis and asthma. Among these degranulation secretions, histamine is the most well-known factor and is also used as an important indicator of allergic symptoms in relation to immediate hypersensitivity reaction.

Allergy remedies have been the mainstream in the 21st century and have been actively researched. The global market of about 26 trillion won has been formed, and the health functional food market is expected to grow to about 10% by 6.5 trillion won. Approximately 50 million people in the US have diverse allergic diseases and about $ 5.2 billion in the drug market. In Korea, the market for anti-allergic drugs is 440 billion won, while the market for health functional food is steadily increasing to 145 billion won.

However, most of the drugs currently used for the treatment of allergy are focused on the suppression of the secretion of histamine secreted in the last stage of the allergy mechanism, or the treatment of the symptoms caused by allergy. Treatment of allergic diseases can be done for a long period of time. Most of the drugs with excellent therapeutic effect are steroids, which are widely used for atopic dermatitis, allergic rhinitis and asthma, but long term use can cause serious side effects And application. Ministry of Health and Welfare, 2010). Antihistamines also cause symptoms to feel alleviated, but long-term use causes side effects such as depression, concentration difficulties, lethargy, drowsiness, and hepatic disorders, and the problem of recurrence of allergic diseases is discontinued. Furthermore, since allergic diseases, especially atopic dermatitis, are mostly in children and adolescents, it is very important to develop a safe therapeutic agent free from toxicity and side effects, and researches to overcome these limitations are actively conducted. However, It is not easy. Accordingly, there is a growing need for the development of safe functional foods which can reduce the dose of these drugs and replace them to some extent, and have no side effects for long-term treatment.

Disclosure of the Invention The present invention has been conceived to solve the above-mentioned problems. The present inventors have made intensive studies to improve the preventive or therapeutic effect of a disease caused by a Th2 immune response such as an allergy or an inflammatory disease, acid has inhibitory activity against interleukin-4 (IL-4), interleukin-6 (IL-6) and macrophage inflammatory protein-2 (MIP-2) 4, IL-6, and MIP-2 in comparison with the untreated enzyme, the present inventors have completed the present invention based on this finding.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating a Th2-mediated immune disease or inflammatory disease, which comprises an enzyme-treated ellagic acid as an active ingredient.

Another object of the present invention is to provide a health functional food composition for preventing or ameliorating a Th2-mediated immunological disease or inflammatory disease, which comprises an enzyme-treated ellagic acid as an active ingredient.

Yet another object of the present invention is to provide a cosmetic composition for preventing or ameliorating a Th2-mediated immunological disease or inflammatory disease, which comprises an enzyme-treated ellagic acid as an active ingredient.

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

In order to achieve the above object,

There is provided a pharmaceutical composition for the prevention or treatment of a Th2-mediated immune disease or inflammatory disease, which comprises an enzyme-treated ellagic acid as an active ingredient.

In one embodiment of the present invention, the Th2-mediated immune disease may be an allergic disease.

In another embodiment of the present invention, the composition may inhibit the production of interleukin-4 (IL-4).

In another embodiment of the present invention, the composition may inhibit the production of macrophage inflammatory protein-2 (MIP-2) or interleukin-6 (IL-6).

In another embodiment of the present invention, the enzyme may be amylose sucrose (DGAS) derived from Deinococcus geothermalis.

The present invention also provides a health functional food composition for preventing or ameliorating a Th2-mediated immune disease or inflammatory disease, which comprises an enzyme-treated ellagic acid as an active ingredient.

Further, the present invention provides a cosmetic composition for preventing or ameliorating a Th2-mediated immunological disease or inflammatory disease, which comprises an enzyme-treated ellagic acid as an active ingredient.

The present invention also provides a method of preventing or treating a Th2-mediated immune disease or inflammatory disease, comprising the step of administering the composition to a subject.

The present invention also provides the use of the composition for the prevention or treatment of a Th2-mediated immune or inflammatory disease.

The composition according to the present invention has improved antiallergic and anti-inflammatory effects of elastase by containing enzyme-treated ellagic acid as an active ingredient. Specifically, the inventors of the present invention found that splenocytes treated with the enzyme-treated gelatin of mice immunized with OVA significantly enhanced the inhibitory activity of IL-4 production compared to the unlabeled elastase, Treated with the enzyme-treated ellagic acid showed that the activity of inhibiting MIP-2 and IL-6 production was enhanced as compared with that of eragacillus. Accordingly, the composition according to the present invention is expected to be useful for the development of therapeutic agents, health functional foods, and cosmetics for the prevention, improvement, or treatment of Th2-mediated immune diseases or inflammatory diseases.

FIG. 1 is a graph showing the results of measurement of the amount of IL-4 from the culture solution after ELISA treatment according to the present invention with mouse OVA-immunized mouse spleen cells for 72 hours and ELISA and cell proliferation ratio IL-4 / MTT).
2 shows the results of measuring the amount of MIP-2 and the cell proliferation ratio (MIP-2 / WST) after treating the enzyme-treated elastase of the present invention with the MH-S cell line and the RAW 264.7 cell line .
FIG. 3 shows the results of measuring the amount of IL-6 and the cell proliferation ratio (IL-6 / WST) after treating the enzyme-treated elastase of the present invention with the MH-S cell line.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating a Th2-mediated immune disease or inflammatory disease, which comprises an enzyme-treated ellagic acid as an active ingredient.

As used herein, the term " prophylactic " means any action that inhibits or delays the development of a Th2-mediated immune or inflammatory disease by administration of a pharmaceutical composition according to the present invention.

As used herein, the term " treatment " refers to any action that improves or alters the symptoms of a Th2-mediated immune disease or inflammatory disease by the administration of the pharmaceutical composition of the present invention.

The term enzyme as used in the present invention means a polymeric organic catalyst produced by a living body cell which becomes a catalyst for a chemical reaction in a living organism. There are various kinds of enzymes in the cells of all living things such as animals and plants, and they are involved in the chemical reaction of living things. Enzymes are very complex because their chemical structures are very complex. They are classified according to the kind of substrate and reaction type. Depending on the type of reaction, hydrolase, transferase, lyase and synthetase, It is classified as isomerase. Enzymes are used for a variety of purposes such as pharmaceuticals, large-scale use of starch saccharification by amylase, cheese production, juice purification, textile industry, synthesis of vitamins and amino acids. In the present invention, it is preferable to use amylose sucrose (DGAS) derived from Deinococcus geothermalis in order to enhance the activity of eragic acid. However, if it is a glycoprotein for promoting the activity of eragic acid Can be used without restrictions.

The term " T cell " used in the present invention refers to a cell that produces Th1 cells and IL-4, IL-5 and IL-13 that produce interleukin-2 (IL-2) and interferon- Th2 cells. The immune balance regulated by the cytokines produced by these Th1 and Th2 cells is called the Th1 / Th2 balance, and Th1 cells are important for the regulation of cellular immunity, and Th2 cells are known to play an important role in controlling humoral immune responses. In the normal state, the cytokine, which is important for the differentiation of Th1, is interfering with the cytokine mainly composed of IFN-γ and IL-4, which is important for differentiation of Th2, and the Thl / Th2 balance is kept constant. However, when the Th1 / Th2 balance is broken, various immune diseases can be induced. In the case of deflecting Thl, the cellular immunity is revived to increase the resistance to infection. When the Th2 is deflected, the infection resistance decreases. Conversely, do.

The term " Th2-mediated immune disease ", which is a disease targeted by the present invention, refers to a disease in which IgE and mast cells are involved in the production and activity of allergens, particularly Th2 cells. Examples of such diseases include allergies, Skin diseases including dermatitis, acute and chronic allergic rhinitis, asthma, food allergies, and the like.

"Inflammatory diseases" which are the object of the present invention include inflammatory bowel disease, inflammatory bowel disease, asthma, conjunctivitis, periodontitis, osteoarthritis, degenerative arthritis, rheumatoid arthritis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, Crohn's disease, And is characterized by chronic inflammatory bowel disease, chronic inflammatory bowel disease, diarrhea, type 2 diabetes, type 2 diabetes, reactive arthritis, psoriasis, scleroderma, osteoporosis, atherosclerosis, myocarditis, endocarditis, pericarditis, cystic fibrosis, But is not limited thereto.

In one embodiment of the present invention, the following experiment was conducted to confirm the improvement or therapeutic effect of the enzyme-treated ellagic acid on a Th2-mediated immunological disease or an inflammatory disease.

In one embodiment of the present invention, splenocytes isolated from OVA-immunized mice were treated with various concentrations of OVA, enzyme-treated ellagic acid, and then the cells were cultured. As a result, the enzyme- ellagic acid) significantly enhanced the IL-4 inhibitory activity compared to the untreated eragic acid (see Example 1).

In another embodiment of the present invention, the enzyme-treated elastase was treated at various concentrations in the macrophage (MH-S cell line, RAW 264.7 cell line), and the MIP-2 and IL- It was confirmed that the enzyme was significantly enhanced compared with the untreated enzyme (see Example 2).

Thus, it was specifically confirmed that the composition of the present invention can inhibit the production of IL-4, MIP-2, and IL-6.

The term " macrophage " as used in the present invention refers to a cell that is distributed in all tissues of an animal body and is responsible for immunity. It predisposes and digests foreign substances, bacteria, viruses, reticulocytes and the like, To the lymphocytes. Macrophages play an important role in innate immune and inflammatory responses as well as in acquired immune responses, and its typical function is antigen presentation to T cells. Macrophages not only present antigens to T cells but also secrete cytokines such as IL-1 and promote T cell activation. In addition, macrophages also function as effector cells that exhibit immune responses in cell-mediated immune responses and humoral immune responses in addition to their ability to present antigens necessary for the initiation of an immune response. Macrophages activated by T cells function to remove antigens from delayed type hypersensitivity. Macrophages have the function of eliminating antigens bound by antibodies with phagocytosis.

The term " MIP (macrophage inflammatory protein) " used in the present invention is a heparin-binding inflammatory protein produced by macrophages and is a kind of chemokine. MIP family includes MIP-1α, β belonging to CC chemokine, and MIP- There are two.

The pharmaceutical composition according to the present invention comprises an enzyme-treated ellagic acid as an active ingredient, and may further comprise a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are those conventionally used in the field of application and include, but are not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, And may further contain other conventional additives such as antioxidants and buffers as needed. In addition, it can be formulated into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like by additionally adding diluents, dispersants, surfactants, binders, lubricants and the like. Suitable pharmaceutically acceptable carriers and formulations can be suitably formulated according to the respective ingredients using the methods disclosed in Remington's reference. The pharmaceutical composition of the present invention is not particularly limited to a formulation, but may be formulated into injections, inhalants, external skin preparations, and the like.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may vary depending on the condition and the weight of the patient, The mode of administration, the route of administration, and the time, but may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, a pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. The effective dose level is determined depending on the type of disease, severity, drug activity, , The time of administration, the route of administration and rate of excretion, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, body weight, the degree of absorption of the active ingredient in the body, the rate of inactivation and the excretion rate, the type of disease, 0.001 to 150 mg, preferably 0.01 to 100 mg per kg of body weight, may be administered daily or every other day, or one to three divided doses per day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

As another aspect of the present invention, the present invention provides a health functional food composition for preventing or ameliorating a Th2-mediated immune disease or inflammatory disease, which comprises an enzyme-treated ellagic acid as an active ingredient.

As used herein, the term "improvement" means all actions that at least reduce the degree of symptom associated with the condition being treated. Herein, the health functional food composition may be used simultaneously with or separately from the medicament for treatment before or after the onset of the disease for the improvement of the Th2-mediated immunological disease or inflammatory disease.

The term health-functional food composition as used in the present invention includes at least one of a carrier, a diluent, an excipient, and an additive, and is formulated into one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, . Examples of foods that can be added to the extract of the present invention include various foods, powders, granules, tablets, capsules, syrups, drinks, gums, tea, vitamin complexes, and health functional foods. Examples of the additive that 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 alginic acid and its salts, 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 such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As the above-mentioned flavors, natural flavors (tautatin, stevia extract (for example, rebaudioside A and glycyrrhizin) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used. The composition according to the present invention can be used in various forms such as flavorings such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies, factic acid and its salts, alginic acid and its salts, , pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, etc. Other compositions according to the present invention may contain flesh for the production of natural fruit juices and vegetable drinks . These components may be used independently or in combination. Specific examples of the carrier, excipient, diluent, and additive include, but are not limited to, lactose, dextrose But are not limited to, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylquilolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water , At least one selected from the group consisting of sugar syrup, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil is preferably used.

In another aspect of the present invention, the present invention provides a cosmetic composition for preventing or ameliorating a Th2-mediated immune disease or inflammatory disease, comprising an enzyme-treated ellagic acid (ellagic aicd) as an active ingredient.

The cosmetic composition of the present invention may contain not only enzyme-treated ellagic acid but also components commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, A conventional adjuvant, and a carrier.

In addition, the composition of the present invention may be used in combination with enzyme-treated ellagic acid (ellagic aicd) in combination with enzyme-treated ellagic acid (ellagic aicd) have. Examples of the organic UV blocking agent include glyceryl paraben, drometrizol trisiloxane, drometrizol, dipaloyyl triolate, disodium phenyldibenzimidazole tetrasulfonate, diethylhexylbutamidotriazone, diethylamino Hydroxybenzoylhexyl benzoate, di-methoxycinnamate, a mixture of Rawson and dihydroxyacetone, methylene bis-benzotriazolyltetramethylbutylphenol, 4-methylbenzylidene camphor, menthyl anthranylate, benzophenone (Benzophenone-4), benzophenone-8 (dioxyphenylbenzone), butylmethoxydibenzoylmethane, bisethylhexyloxyphenol methoxyphenyltriazine, synoxate, ethyl dihydroxypropylparaben, Ethylhexyldimethylpivalate, ethylhexylmethoxycinnamate, ethylhexylsalicylate, ethylhexyltriazone, isoamyl-p-methoxy cinnamate, polysilicon-15 (dimethicodimethylbenzalmate, At least one selected from the group consisting of terephthalylidene dicam peresophilic acid and its salts, thiai-salicylate and aminobenzoic acid (parabe) can be used.

Examples of products to which the cosmetic composition of the present invention can be added include cosmetics such as astringent lotion, softening longevity lotion, nutrition lotion, various creams, essences, packs, foundation and the like, cleansing, cleanser, soap, . Specific formulations of the cosmetic composition of the present invention include skin lotions, skin softeners, skin toners, astringents, lotions, milk lotions, moisturizing lotions, nutritional lotions, massage creams, nutritional creams, moisturizing creams, hand creams, essences, It includes formulations such as soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, latex, lipstick, makeup base, foundation, press powder, loose powder, eye shadow and the like.

According to a preferred embodiment of the present invention, the content of the enzyme-treated ellagic acid (ellagic aicd) of the present invention is 0.00001-30% by weight, preferably 0.5-20%, more preferably 1.0- 10% by weight. If the content of the enzyme-treated ellagic acid (ellagic acid) is less than 0.00001% by weight, the effect of absorbing ultraviolet light is greatly reduced. If the content of the enzyme-treated ellagic acid is more than 30% by weight, skin irritation may be caused.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Confirmation of antiallergic effect in mouse splenocytes treated with enzyme-treated eragucan

1-1. Enokokusu Geothermal Malis  ( Deinococcus geothermal ) Origin Amylose sucrase  ( DGAS ) Of enzyme treatment

The ellagic acid was purchased from Sigma-Aldrich used in the present invention, the enzyme used enoic Lactococcus Geo sseoreu Marlies (Deinococcus be a geothermalis) derived from amyl Klein dehydratase (DGAS) is a geo-diethoxy no Lactococcus sseoreu was produced and purified from Marlies (Deinococcus geothermalis) sucrase Kinase (recombinant Escherichia coli (E. coli transformants of the amylosucrase gene conversion)) as derived from amyl.

Specifically, a recombinant vector pHC-DGAS containing an amylose sucrase gene derived from Deinococcus geothermalis was added to a pHCEXHD expression vector (His tag-containing vector), and E. coli transformed with pHC-DGAS ( E. coli ) MC1061. Thereafter, the transformed Escherichia coli was inoculated in 5 ml of LB liquid medium containing ampicillin, cultured at 37 캜 for 12 hours, and centrifuged to obtain cells.

Meanwhile, in order to purify the His-tag (His-tag) -binding recombinant protein, the transformed E. coli MC1061 was cultured at 37 ° C for 15 hours. After the incubation, the cells were recovered by performing centrifugation at 4 ° C and 4,000 rpm for 30 minutes. Thereafter, the mixture was suspended in a lysis buffer (50 mM NaH ₂ PO ₄ , pH 7.0, 300 mM NaCl, 10 mM imidazole, pH 7.0) and sonicated.

Thereafter, the pulverized liquid pulverized by using the ultrasonic wave was subjected to centrifugation for 10 minutes at 4 ° C and 12,000 rpm, and a supernatant was obtained. The supernatant was injected into Ni-NTA affinity chromatography to purify the recombinant protein.

 300 mg sucrose and DGAS (1 U / L) prepared above were added to 10 mg of eragacillus. The volume was adjusted to 100 mL with 50 mM Tris-HCl pH 8.0 buffer solution and reacted at 40 ° C for 48 hours, respectively. After the reaction, the solution was boiled for 5 minutes and then filtered using a Whatman filter paper.

1-2. Culture of mouse splenocytes immunized with OVA

In order to induce allergy to female BALB / c mice at 5 weeks of age, 10 μg of ovalbumin (OVA) and 1 mg of alum were mixed for 30 minutes, and 100 μl of each mouse was injected every 2 weeks (N = 5) twice a week. One week after the immunization, the mice were sacrificed by cervical disassembly, and the spleen was removed from the aseptic state. Then, the spleen was transferred to a small petri dish containing 1 ml of the basic medium, i.e., 2-mercaptoethanol and RPMI1640 medium supplemented with antibiotics And stored on ice. The spleen was then single-celled using a mesh, washed twice with 5 ml of basal medium, and then centrifuged at 1500 rpm for 5 minutes. After removing the supernatant, 1 ml of RBC (red blood cell) lysing buffer (SIGMA R7757) was added and incubated for 3 minutes on ice. 10 ml of basal medium was further added, ≪ / RTI > centrifugation twice.

On the other hand, the 96-well plate was treated with OVA as an antigen at a concentration of 100 μg / ml, and each of the DGAS-treated and DGAS-untreated eragic acid prepared in Example 1-1 was diluted 2.5, And 5.0 [mu] g / ml, and the spleen cells of the mouse cultured by the above method were treated at 5 x 10 < ⁶ > cells / well. Thereafter, the cells were cultured in a CO ₂ incubator at 37 ° C for 72 hours, and the cell culture medium was recovered.

1-3. Cytokine analysis of spleen cell cultures using ELISA

In order to measure the amount of IL-4 cytokine from the culture medium of the mouse spleen cells recovered according to the method of Example 1-2, an experiment using an IL-4 BD OptEIATM MOUSE ELISA kit (enzyme-linked immunosorbent assay) Respectively.

Specifically, a capture antibody was added to a 96-well plate and reacted overnight at 4 ° C. The plate was coated with an antibody, washed with a washing buffer (phosphate buffered saline with Tween 20; PBST), and diluted with assay diluent (10% FBS in PBS) was added to each well and reacted at room temperature for 1 hour to perform a blocking process. Next, 100 μl of the standard solution and the culture solution of mouse spleen cells recovered through the method of Example 1-2 were dispensed into the wells, and reacted at room temperature for 2 hours. After the reaction, 100 쨉 l of the secondary antibody conjugated with the IL-4 specific primary antibody capable of detecting IL-4 and streptavidin-Horseradish peroxidase (HRP) was dispensed into each well The reaction was allowed to proceed at room temperature for 1 hour. Then, 100 μl of a substrate solution (0.01% TMB in phosphate-citrate buffer) was added and developed at room temperature for 30 minutes. Then, 50 μl of 2 M H ₂ SO ₄ was added to each well to stop the color reaction. The absorbance was measured with a microplate reader at.

In addition, MTT assay was performed to measure cell proliferation rate. Specifically, after recovering the culture solution according to the method of Example 1, the cells were treated with 3- (4,5-diethyl thiazol-2-yl) -2,5-diphenyl 20 μl of tetrazolium bromide (MTT) reagent was treated. After 4 hours, 100 μl of 10% SDS solution was added and allowed to stand overnight. Absorbance was measured at 595 nm using a microplate reader.

As a result, it was confirmed that the amount of IL-4, which is a cytokine secreted by Th2 cells, was significantly increased in order to induce an allergic immune response when OVA alone was administered to spleen cells. In the case of treatment with the enzyme- Although the amount of IL-4 was decreased, it was confirmed that the amount of IL-4 was significantly reduced in the case of the DGAS enzyme-treated elagic acid than in the case of treatment with the enzyme-untreated elagic acid. The results are shown in Figure 1 as the IL-4 / MTT ratio value.

Example  2. Enzyme treatment ElaGrass  Identification of anti-inflammatory effects in treated macrophages

2-1. Enzyme treatment and cell viability measurement

After removing all of the media in Dish, the cells were washed twice with 5 ml of DPBS (Dulbecco's phosphate-buffered saline), and DPBS was removed. 10 ml of the MH-S cell line and RAW 264.7 cell line medium were dispensed into the dish, The cells were scraped off using a scrapper. This was transferred to a 15 ml tube, centrifuged at 1000 rpm at 24 ° C for 10 minutes, the supernatant was removed from the tube, and 1 ml of the medium was dispensed. Then cell count was performed using cell counter ADAM.

MH-S cell line and RAW 264.7 cell line were diluted to 2 × 10 6 / ml, and 250 μl of the cells were added to a 96-well culture plate (SPL, cat.

Thereafter, the cells were diluted with an MH-S cell line and RAW 264.7 cell line medium at a concentration of 100 ng / ml of LPS, and the DGAS-treated elastase and DGAS prepared according to the method of Example 1-1 Eulagic acid was diluted to a concentration of 2.5 and 5.0 μg / ml, and 250 μl of each was diluted into appropriate wells and cultured for 24 hours.

After culturing for 24 hours, the supernatant in 96 wells was separately removed for cytokine determination, and the MH-S cell line medium, RAW 264.7 cell line medium and cellvia solution were mixed at a ratio of 10: 1 Respectively.

After 20 minutes, absorbance was measured at 450 nm to calculate cell viability.

2-2. MIP-2 measurement using macrophage supernatant

On the day before the experiment, the capture antibody was diluted to the appropriate concentration in PBS, and 100 ㎕ per well was dispensed per well in a 96-well immune plate (SPL). After washing three times with 350 ㎕ of each well using a washing machine, Subsequently, 300 μl of the reagent diluent was treated per well and cultured at room temperature for 1 hour. After 1 hour, two wash steps were carried out. Elaginic acid treated with DGAS and unlabeled elagic acid prepared according to the method of Example 1-1 were diluted to an appropriate concentration using a reagent diluent, 100 ㎕ per well was added and incubated at room temperature for 2 hours. After 2 hours, two washing steps were carried out. The detection antibody was diluted to an appropriate concentration using a reagent diluent, treated with 100 μl per well, and incubated at room temperature for 2 hours. After 2 hours, two wash steps were carried out. Streptavidin-HRP was diluted to an appropriate concentration using a reagent diluent, treated with 100 μl per well, and incubated at room temperature for 20 minutes. Two hours later, two wash steps were carried out. The substrate solution was treated with 100 μl per well, followed by incubation at room temperature for 20 minutes without exposure to light. After 20 minutes, the stop solution was treated with 50 μl per well. The plate was patched gently so that the solution was well mixed, and the absorbance was measured at 450 nm using a microplate reader.

As a result, it was confirmed that the amount of MIP-2 was significantly increased when LPS alone was administered to the MH-S cell line. When 2.5 μg / ml of the enzyme-untreated elagantic acid was treated and when the DGAS enzyme- Ug / ml, the amount of MIP-2 was decreased, and the amount of MIP-2 was slightly decreased in the case of elagic acid treated with DGAS enzyme than in the case of treatment with no enzyme-treated elagic acid .

In the RAW 264.7 cell line, the amount of MIP2 was not decreased in the case of treatment with 5 g / ml of the enzyme-untreated elagantic acid, but the amount of 5 g / ml of DGAS enzyme-treated elagant was treated The amount of MIP-2 was decreased.

The results for the MH-S cell line and the RAW 264.7 cell line are shown in FIG. 2 and are shown as MIP-2 / WST ratio values.

2-3. Measurement of IL-6 using macrophage supernatant

On the day before the experiment, dilute the capture antibody to the appropriate concentration in PBS, dispense 100 μl per well in a 96-well immune plate (SPL), overnight at 4 ° C, wash 3 times 300 μl per well using a washing machine, , The assay diluent was treated with 300 μl per well and cultured at room temperature for 1 hour. Then, the remaining solution was washed 5 times with 300 μl per well using a washing machine, and the remaining solution was bled off. Using the assay diluent, the DGAS-treated elastase and DGAS prepared according to the method of Example 1-1 were not treated Elagic acid was diluted to an appropriate concentration, treated with 100 μl per well, and cultured at room temperature for 2 hours. After washing with 300 μL / well of the wash machine, the remaining solution was blotted off. The working detector was diluted to an appropriate concentration using an assay diluent, treated with 100 μL per well, incubated at room temperature for 1 hour Respectively. Subsequently, the substrate solution was treated with 100 μl per well, followed by incubation at room temperature for 30 minutes without exposing to light. After 30 minutes, the stop solution was treated with 50 μl per well, and the plate was gently patted so that the solution was well mixed. The absorbance was then measured at 450 nm using a microplate reader.

As a result, it was confirmed that the amount of IL-6 was significantly increased when LPS alone was administered to the MH-S cell line. When the enzyme-treated unlabeled elagance 2.5 and 5 μg / ml were treated and the DGAS enzyme- The amount of IL-6 was decreased in all of the treatment groups of 2.5 and 5 μg / ml, but the amount of IL-6 was significantly decreased in the case of the DGAS enzyme-treated elagic acid than in the untreated enzyme-treated elagic acid .

The results in the MH-S cell line are shown in FIG. 3 and are shown as the IL-6 / WST ratio values.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the embodiments described above are illustrative in all aspects and not restrictive.

Claims (15)

A pharmaceutical composition for the prophylaxis or treatment of a Th2-mediated immune disease or inflammatory disease, comprising an enzyme-treated ellagic acid as an active ingredient.
The pharmaceutical composition according to claim 1, wherein the Th2-mediated immune disease is an allergic disease.
The pharmaceutical composition according to claim 1, wherein said composition inhibits the production of interleukin-4 (IL-4).
The method according to claim 1, wherein the composition inhibits the production of macrophage inflammatory protein-2 (MIP-2) or interleukin-6 (IL-6) A pharmaceutical composition for preventing or treating diseases.
The method of claim 1, wherein the enzyme is selected from the group consisting of Deinococcus A medicinal composition for the prevention or treatment of a Th2-mediated immune disease or inflammatory disease, characterized in that it is an amylose sucrose derived from geothermal origin.
A health-functional food composition for preventing or ameliorating a Th2-mediated immunological disease or inflammatory disease, which comprises an enzyme-treated ellagic acid as an active ingredient.
The health functional food composition according to claim 6, wherein the Th2-mediated immune disease is an allergic disease.
The health functional food composition according to claim 6, wherein the composition inhibits the production of interleukin-4 (IL-4).
7. The method according to claim 6, wherein the composition inhibits the production of macrophage inflammatory protein-2 (MIP-2) or interleukin-6 (IL-6) A health functional food composition for preventing or ameliorating a disease.
The method of claim 6, wherein the enzyme is selected from the group consisting of Deinococcus A medicament for preventing or ameliorating a Th2-mediated immune disease or inflammatory disease, characterized in that it is an amylose sucrose derived from geothermalis .
A cosmetic composition for preventing or ameliorating a Th2-mediated immunological disease or inflammatory disease, comprising an enzyme-treated ellagic acid as an active ingredient.
12. The cosmetic composition according to claim 11, wherein the Th2-mediated immune disease is an allergic disease.
12. The cosmetic composition according to claim 11, wherein the composition inhibits the production of interleukin-4 (IL-4).
12. The method of claim 11, wherein the composition inhibits the production of macrophage inflammatory protein-2 (MIP-2) or interleukin-6 (IL-6) A cosmetic composition for preventing or improving disease.
12. The method of claim 11, wherein the enzyme is selected from the group consisting of Deinococcus A medicinal composition for preventing or ameliorating a Th2-mediated immunological disease or an inflammatory disease, characterized in that it is amylosucrase derived from geothermalis .

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