WO2017099413A1 - Composition comprising enzyme-treated rosa multiflora fruit extract as active ingredient for preventing or treating th2-mediated immunological disease - Google Patents

Abstract

The present invention relates to a composition comprising an enzyme-treated Rosa multiflora fruit extract as an active ingredient for preventing or treating a Th2-mediated immune disease. The composition according to the present invention can maximize the anti-allergic effect of the Rosa multiflora fruit extract by comprising an enzyme-treated Rosa multiflora fruit extract as an active ingredient. Specifically, the present inventors verified that, as a result of treating splenocytes of OVA-immunized mice with an enzyme-treated Rosa multiflora fruit extract, the enzyme-treated Rosa multiflora fruit extract remarkably promoted IL-4 production inhibitory activity compared with an enzyme-non-treated Rosa multiflora fruit extract and the in vivo oral administration of the enzyme-treated Rosa multiflora fruit extract remarkably reduced the blood IgE content in the OVA-immunized mice. In addition, the composition according to the present invention has advantages of being biocompatible and having excellent stability due to the use of Rosa multiflora fruit as a natural material, and thus, the composition is expected to be favorably used in the development of a safe therapeutic agent, a health functional food, a cosmetic product, and the like for the prevention, improvement, or treatment of a Th2-mediated immune disease.

Description

Composition for the prevention or treatment of TH2-mediated immune diseases, including enzyme-treated Youngsil extract as an active ingredient

The present invention relates to a composition for the prevention or treatment of Th2-mediated immune diseases, comprising an enzyme-treated Youngsil extract as an active ingredient.

The immune balance regulated by cytokines produced by Thl (type 1 helper T) and Th2 (type 2 helper T) cells is called Thl / Th2 balance, and Thl cells are important for the regulation of cellular immunity. Plays an important role in humoral regulation In steady state, the cytokine centered on IFN-γ, which is important for the differentiation of Thl, and the cytokine centered on IL-4, which is important for the differentiation of Th2, interfere with each other to maintain a constant Thl / Th2 balance. If the balance is off, a variety of immune diseases can occur. For example, when it is biased by Thl, cellular immunity is revived to increase infection resistance, and when it is biased by Th2, infection resistance is decreased, and conversely, allergy is enhanced.

Allergy is a malfunction of the immune system, a substance that does not affect the average person and causes hypersensitivity reactions such as hives, itching, runny nose, and cough only in certain people. Environmental pollution, westernization of diet and lifestyle, stress Environmental factors such as, heredity, and various allergens. 20-25% of the world's population is exposed to allergic diseases, and the prevalence continues to increase.

Allergic reactions are traditionally divided into four classes based on expression time and type, with type I-III being the humoral immune response involving antibodies (immediate type) and type IV with the cellular immune response directly involving cells. Delay type). Most allergic reactions include type I asthma, rhinitis, conjunctivitis, food and drug allergies, and atopic dermatitis. In severe cases, anaphylaxis may be induced.

Type I immediate hypersensitivity is divided into two stages, the first stage of which is a Th1 cell reaction that produces IL-12 and IFN-γ that inhibits the release of IgE and IgG1 and increases the secretion of IgG2a by the invasion of allergens. And the balance of Th2 (type2 helper T) cell responses that produce IL-4, IL-5, and IL-13 are inclined toward Th2, and IL-4, IL-13, etc. are secreted due to the excessive immune response of Th2, As a result, IgE-specific antibodies produced by B cells are attached to the surface of mast cells or basophils, and allergens are sensitized.

The second stage of allergic development is divided into early reaction and late reaction, and the initial reaction is reinvading allergens into the body to stimulate mast cells and induce degranulation reaction. Vasodilation due to released histamine, lipid metabolites and cytokines The back reaction occurs when neutrophils, eosinophils, macrophages, Th2 cells, basophils, etc. are activated by infiltration of the tissues, causing inflammation and causing atopic dermatitis, rhinitis, and asthma. Of these degranulated secretion substances, histamine is the most well known factor and is used as an important indicator of allergic symptoms related to immediate hypersensitivity.

Allergy medicines are mainstream in the 21st century and are being actively researched, forming a global market of about 26 trillion won, and the functional food market is expected to grow to about 10% at 6.5 trillion won. In the United States, about 50 million people have a variety of allergic diseases and a $ 5.2 billion drug market. In Korea, the market for allergic diseases is 440 billion won, and the functional food market is 145 billion won.

However, most of the current drugs for the treatment of allergies are focused on suppressing the release of histamine released at the end of the allergic mechanism, or treating allergic symptoms, and few therapeutic agents show a definite effect. Allergic diseases can be treated for a long time. Most of the drugs showing excellent therapeutic effects are widely used as steroids such as atopic dermatitis, allergic rhinitis, asthma, but long-term use may cause serious side effects. Antihistamines also cause symptoms to be alleviated, but prolonged use causes side effects such as depression, concentration disorders, lethargy, drowsiness, and liver failure. In addition, since allergic diseases, especially atopic dermatitis, are mostly children and adolescent patients, it is very important to develop safety drugs without toxicity and side effects, and researches to overcome these limitations are actively conducted. It is not easy. Accordingly, the dose of these drugs can be reduced and replaced to some extent, and there is a need for developing a safe functional food without side effects even in long-term treatment.

On the other hand, Youngsil (Rosae multiflorae Fructus) is a fruit of the brier, which belongs to the family Rosaceae, and is commonly distributed in Korea and Japan. The appearance is spherical or oval, the outer surface is red or dark reddish brown, the pentagonal pistil remains at the end of the fruit, has a slightly sour smell and tastes a little sweet. Youngsil does not urinate well, or swelling, insomnia, decreased sexual function, boils, swelling, etc. It is effective in promoting blood circulation and removing poison. Pharmacological actions have been reported to coronary atherosclerosis, mouse life extension, lipid and protein metabolism improvement, and atherosclerosis formation inhibition. Recently, tannin-based compounds, which are phenolic compounds contained in Youngsil, have been reported to exhibit excellent antioxidant effects. Youngsil's protocatechuic acid, chlorogenic acid, and It has been reported that compounds such as quercetin have an antimicrobial effect against Helicobacter pylori (Helicobacter pylori) (Korean Patent Publication 2010-0043882). In addition, studies have been made to use Youngsil for the prevention or treatment of allergic diseases. However, since the anti-allergic effect of Youngsil extract extracted only by the general solvent extraction method has a limit, research that can improve the efficacy or obtain a new effect is necessary.

Thus, the present inventors studied the anti-allergic activity enhancing effect after the enzyme treatment in Youngsil extract, and completed the present invention based on this.

The present inventors have studied diligently to enhance the prophylactic or therapeutic effect of diseases caused by Th2 immune response such as allergy to Youngsil extract. As a result, enzyme-treated Youngsil extract is IL-4 as compared to extract without enzyme. And the present invention was completed by confirming that the IgE production inhibitory activity is significantly enhanced.

Accordingly, an object of the present invention is to provide a composition for preventing, ameliorating, or treating Th2-mediated immune disease, comprising an enzyme-treated Youngsil extract as an active ingredient.

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

In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition for preventing or treating Th2-mediated immune disease, comprising as an active ingredient the enzyme treatment Youngsil (Rosae multiflorae Fructus) extract.

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

In another embodiment of the invention, the enzyme may be beta-glucosidase (β-glucosidase).

In another embodiment of the present invention, the extract is a group consisting of water, C ₁ to C ₄ lower alcohol, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and a mixed solvent thereof It may be extracted with a solvent selected from.

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 immunoglobulin E (IgE).

In addition, the present invention provides a health functional food composition for improving Th2-mediated immune disease, comprising an enzyme-treated Youngsil (Rosae multiflorae Fructus) extract as an active ingredient.

In addition, the present invention provides a cosmetic composition for improving Th2-mediated immune disease, comprising an enzyme-treated Youngsil (Rosae multiflorae Fructus) extract as an active ingredient.

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

The present invention also provides a use of the pharmaceutical composition for the prophylaxis or treatment of Th2-mediated immune diseases.

The composition according to the present invention can maximize the anti-allergic effect of the enzyme treatment Youngsil extract as an active ingredient, Youngsil extract. Specifically, the inventors of the present invention treated the enzyme-treated Youngsil extract on splenocytes of mice immunized with OVA, which significantly enhanced IL-4 production inhibitory activity compared to the enzyme-treated Youngsil extract, and the enzyme-treated Youngsil in vivo . It was confirmed that blood IgE content of mice immunized with OVA was significantly decreased by oral administration of the extract. In addition, since the use of the natural product Youngsil has the advantages of bio-friendly and excellent stability, the composition according to the present invention is a safe treatment for the prevention, improvement, or treatment of Th2-mediated immune diseases, health functional foods, and the development of cosmetics, etc. It is expected to be usefully used.

1 is treated with mouse spleen cells immunized with OVA at different concentrations of each of the Youngsil extracts (Youngsil water extract, Youngsil room temperature alcohol extract, and Youngsil alcohol extract) with or without beta-glucosidase, After culturing the cells for 72 hours, the amount of IL-4 from the culture was measured by ELISA.

Figure 2 is a result of oral administration of the control of the Youngsil extract or enzyme-treated Youngsil extract as a control to the mice with OVA, the blood IgE content of the mice was measured by ELISA (Youngsil: Youngsil extract administration group, β-glucosidase treatment: enzyme treatment Youngsil extract administration group).

The present invention relates to Youngsil extracts whose activity is enhanced through enzyme treatment and their use.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating Th2-mediated immune disease, comprising an enzyme-treated Youngsil (Rosae multiflorae Fructus) extract as an active ingredient.

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

As used herein, the term "treatment" refers to any action in which symptoms for Th2-mediated immune disease improve or benefit altered by administration of the pharmaceutical composition according to the present invention.

“Youngsil”, a natural material used in the present invention, belongs to the family Rosaceae and is a fruit of the brier commonly grown in Korea and Japan. It is the appearance that remains. It has a slightly sour smell and tastes a little, and it is not used to urinate well, or is used for swelling, insomnia, decreased sexual function, boil, swelling, and promotes blood circulation and removes poison. In addition, pharmacological actions such as coronary artery dilatation, mouse life extension, lipid, protein metabolism improvement, and atherosclerosis suppression are reported. In the present invention, Youngsil may be used as it is without extracting the fruit as a raw material, it may be used in powder form.

The disease targeted by the present invention, "Th2-mediated immune disease", refers to a disease involving allergens, particularly IgE and mast cells by the production and activation of Th2 cells, including allergy, and more specifically, atopic dermatitis. Skin diseases including dermatitis, acute and chronic allergic rhinitis, asthma, food and drug allergies, and anaphylaxis, and the like, but are not limited thereto.

As used herein, the term “enzyme” refers to a protein-based high molecular organic catalyst produced by living cells as a catalyst for chemical reactions in living organisms. Various kinds of enzymes exist in the cells of all living things such as animals and plants, and they are involved in the chemical reactions of living things. Since enzymes are very complex in their chemical structure, they are classified according to the type of substrate or the type of reaction, and according to the type of reaction, hydrolase, transferase, lyase and synthetase, Are classified as isomerases. Enzymes are used in a wide variety of applications, including pharmaceuticals, large-scale utilization of starch saccharification by amylase, cheese production, fruit juice purification, textile industry, and the synthesis of vitamins and amino acids. In the present invention, beta-glucosidase was used to enhance activity of Youngsil. The beta-glucosidase is an enzyme that acts on a β1-> 4 bond connecting two glucose or glucose-substituted molecules and is a cellulose or an associated polysaccharide. Cellulase involved in the degradation of (cellulase), more specifically exocellulase (exocellulase) having a variety of beta-D-glucoside substrate specificity.

Enzymatically treated Youngsil extract of the present invention comprises the steps of obtaining Youngsil extract; And it can be prepared by a method comprising the step of reacting by adding an enzyme to the Youngsil extract. Specifically, the step of adding and reacting the enzyme may be performed at 37 ° C. for 15 hours to 40 hours, more preferably 20 to 30 hours.

The Youngsil extract can be extracted according to a conventional method known in the art for extracting the extract from natural products, that is, using a conventional solvent under the conditions of conventional temperature, pressure. For example, the Youngsil extract in the present invention is at least one selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and a mixed solvent thereof The extraction can be carried out using a solvent, preferably extraction with water or ethanol. In addition, the extraction method of Youngsil extract may be extracted through various methods such as room temperature extraction, hot water extraction, cold extraction, reflux extraction, microwave extraction, and ultrasonic extraction, preferably using a room temperature extraction or reflux extraction method May be, but is not limited to this.

The prepared extract can then be filtered or concentrated or dried to remove the solvent, it can be carried out both filtration, concentration and drying. For example, the filtration may use a filter paper or a vacuum filter, the concentration may be a vacuum concentrator, the drying may be a freeze-drying method, but is not limited thereto.

In addition, the extract extracted with the solvent may be further fractionated with a solvent selected from the group consisting of butanol, hexane, methylene chloride, acetone, ethyl acetate, ethyl ether, chloroform, water, and a mixed solvent thereof. The fractionation temperature may be 4 ℃ to 120 ℃, but is not limited thereto.

In one embodiment of the present invention, the following experiment was conducted to confirm the improvement or treatment effect of the Th2-mediated immune disease against the enzyme-treated Youngsil extract.

In one embodiment of the present invention, after treatment with various concentrations of OVA, non-enzymatically treated Youngsil extract or enzyme-treated Youngsil extract in splenocytes isolated and cultured from mice immunized with OVA at various concentrations, the enzyme treatment. It was confirmed that the IL-4 inhibitory activity of Youngsil extract was significantly enhanced (see Example 2).

In another embodiment of the present invention, after the oral administration of the enzyme-treated Youngsil extract or enzyme-treated Youngsil extract in mice immunized with OVA and compared the blood IgE content, the enzyme-treated Youngsil extract compared to the Youngsil extract administration group It was confirmed that the degree of IgE content reduction was enhanced in the administration group (see Example 3).

Therefore, it was confirmed that the composition of the present invention can inhibit the production of IL-4 and IgE.

The pharmaceutical composition according to the present invention includes the enzyme-treated Youngsil extract as an active ingredient, and may further include a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are conventionally used in the preparation, and include, but are not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and the like. If necessary, other conventional additives such as antioxidants and buffers may be further included. In addition, diluents, dispersants, surfactants, binders, lubricants and the like may be additionally added to formulate injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Suitable pharmaceutically acceptable carriers and formulations can be preferably formulated according to the individual components using methods disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection, inhalant, or external skin preparation.

The pharmaceutical compositions of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is determined by the condition and weight of the patient, Depending on the extent, drug form, route of administration, and time, it 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, “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level refers to a disease type, severity, drug activity, drug, and drug of a patient. Sensitivity to, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The pharmaceutical compositions according to the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum 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, weight of the patient, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the type of disease, the drug used in general 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 divided into 1 to 3 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.

In another aspect of the present invention, the present invention provides a health functional food composition for improving Th2-mediated immune disease, comprising an enzyme-treated Youngsil (Rosae multiflorae Fructus) extract as an active ingredient.

As used herein, the term “improvement” means any action that at least reduces the parameters associated with the condition being treated, for example, the extent of symptoms. At this time, the health functional food composition may be used simultaneously or separately with the agent for treatment before or after the onset of the disease in order to improve the Th2-mediated immune disease.

As used herein, the term “health functional food composition” is selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, including one or more of carriers, diluents, excipients, and additives. Characterized in one. Examples of the food that can be added to the extract of the present invention include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods. As additives which may be further included in the present invention, natural carbohydrates, flavoring agents, nutrients, vitamins, minerals (electrolytes), flavoring agents (synthetic flavoring agents, natural flavoring agents, etc.), colorants, fillers, pactic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, antioxidants, glycerin, alcohols, carbonation agents and one or more components selected from the group consisting of pulp 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 conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. Compositions according to the invention include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, colorants and neutralizers, fact acids and their salts, alginic acids and their salts, organic acids, protective colloidal thickeners , pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, etc. In addition, other compositions of the present invention may contain flesh for the production of natural fruit juices 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, dex , Sucrose, sorbitol, mannitol, erythritol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water It is preferable to use at least one selected from the group consisting of sugar syrup, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil.

As another aspect of the present invention, the present invention provides a cosmetic composition for improving Th2-mediated immune disease, comprising an enzyme-treated Youngsil (Rosae multiflorae Fructus) extract as an active ingredient.

The cosmetic composition of the present invention may include not only enzyme-treated Youngsil extract, but also components commonly used in cosmetic compositions, such as conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and perfumes, And a carrier.

In addition, the composition of the present invention may be used in addition to the enzyme-treated Youngsil extract, a mixture of organic sunscreens that have been conventionally used insofar as it does not impair the skin protection effect by reacting with the enzyme-treated Youngsil extract. Examples of the organic sunscreen include glyceryl pava, drometrizole trisiloxane, drometrizole, digaloyltrioleate, disodium phenyldibenzimidazole tetrasulfonate, diethylhexyl butamidotriazone, diethylamino Hydroxybenzoylhexylbenzoate, die-methoxycinnamate, a mixture of lowson and dihydroxyacetone, methylenebis-benzotriazolyltetramethylbutylphenol, 4-methylbenzylidene camphor, menthyl anthranilate, benzophenone -3 (oxybenzone), benzophenone-4, benzophenone-8 (dioxyphenbenzone), butylmethoxydibenzoylmethane, bisethylhexyloxyphenol methoxyphenyltriazine, synoxate, ethyldihydroxypropylpava, Octocrylene, ethylhexyldimethylpava, ethylhexylmethoxycinnamate, ethylhexylsalicylate, ethylhexyltrizone, isoamyl-p-methoxycinnamate, polysilicon-15 (dimethicodiethylbenzalmal Ronate), terephthalylidene dicamphor sulfonic acid and salts thereof, thy-salicylate and aminobenzoic acid (Pava) can be used.

Examples of products to which the cosmetic composition of the present invention may be added include, for example, cosmetics such as astringent cosmetics, soft cosmetics, nourishing cosmetics, various creams, essences, packs, foundations, and the like, cleansing agents, soaps, treatments, and essences. Etc. Specific formulations of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, Formulations such as soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, lipsticks, makeup bases, foundations, press powders, loose powders, eye shadows and the like.

According to a preferred embodiment of the present invention, the content of the enzyme-treated Youngsil extract of the present invention is 0.00001-30% by weight, preferably 0.5-20%, more preferably 1.0-10% by weight based on the total weight of the composition. . When the content of the enzyme-treated Youngsil extract is less than 0.00001% by weight, the ultraviolet absorbing effect is greatly reduced, and when it exceeds 30% by weight, skin irritation may occur, and formulation problems may occur.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

Example 1 Experiment Preparation and Methods

1-1. Youngsil Extract Manufacturer

Youngsil was purchased from Tojong Village Co., Ltd. in Buyeosan, Chungcheongnam-do and used for experiment. Youngsil extract was prepared by three different types of extracts, namely, Youngsil water extract, Youngsil room temperature spirit extract, and Youngsil spirit water extract.

Specifically, Youngsil water extract was obtained by mixing Youngsil and water in a weight ratio of 1:10, and then reflux extraction at 85 ℃ for 3 hours. Youngsil room temperature ethanol extract was obtained by mixing Youngsil and ethanol (95% ethanol) in a weight ratio of 1:10, immersing in a shaking water bath, shaking at a speed of 45 rpm for 15 hours at 25 ℃. . Finally, the water extract after the Youngsil alcohol was obtained by recovering all the spirits after obtaining the Youngsil room temperature alcohol extract according to the above method, by adding reflux of 10 times the weight of the Youngsil residue to reflux extraction at 85 ℃ for 3 hours. Thereafter, the extracts were filtered, concentrated, and lyophilized using filter paper.

1-2. Treatment of β-glucosidase

In order to treat beta-glucosidase in the Youngsil extract, each Youngsil extract prepared by the method of 1-1 was dissolved in 50% DMSO (dimethyl sulfoxide) at a concentration of 100 mg / ml. , 0.05 M sodium acetate buffer (pH 5.0) was diluted to 10 mg / ㎖ concentration. Each dilute Youngsil extract was treated with β-glucosidase at 10 units / ml, incubated at 37 ° C. for 24 hours, and each extract was used for the experiment.

1-3. Culture of Mouse Splenocytes Immunized with OVA

To induce allergy in 5-week-old female BALB / c mice, 100 μl per mouse was mixed with a mixture of 10 μg of ovalbumin (OVA) and 1 mg of alum for 30 minutes. Intraperitoneal administration was performed twice (n = 5). After 1 week of immunization, mice were sacrificed by cervical distal bone and spleens were removed in aseptic state and transferred to a small petri dish containing 1 ml of basic medium, RPMI1640 medium containing 2-mercaptoethanol and antibiotics. Stored on ice. The spleen was then unicellularized using a mesh, washed twice with 5 ml of basal medium, and centrifuged at 1500 rpm for 5 minutes. After removal of the supernatant, 1 ml of red blood cell (RBC) lysing buffer (SIGMA R7757) was added and suspended. The mixture was allowed to react on ice for 3 minutes. Then, 10 ml of basic medium was added and resuspended. Then, 5 minutes at 1500 rpm. Centrifugation was performed twice.

On the other hand, the 96-well plate was treated with an antigen of OVA at a concentration of 100 μg / ml, and each of the three types of Youngsil extracts prepared according to the method of Example 1-1 was 100, 200, or 400 μg / ml. Each of the three types of Youngsil extract treated with β-glucosidase prepared according to the method of 1-2 was added at a concentration of 10, 50, 200 ㎍ / ml, and the splenocytes of the mice cultured by the above method were added. Treated with 5 × 10 ⁶ cells / well. After incubation for 72 hours in a CO ₂ incubator at 37 ℃ cell culture was recovered.

1-4. Cytokine Analysis of Splenocyte Cultures Using ELISA

In order to measure the amount of IL-4 cytokines from the culture of mouse splenocytes recovered according to the method of Example 1-3, IL-4 BD OptEIATM MOUSE enzyme-linked immunosorbent assay (ELISA) kit and TGF-β kit The experiment was performed using (R & D Systems).

Specifically, a capture antibody was added to a 96-well plate and reacted overnight at 4 ° C. to coat the plate with an antibody, washed with washing buffer (phosphate buffered saline with Tween20; PBST), and then 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 culture solution of mouse splenocytes recovered through the standard solution and the method of Example 1-3 were dispensed into the wells, followed by reaction at room temperature for 2 hours. After the reaction, 100 μl of an IL-4 specific primary antibody capable of detecting IL-4 and a secondary antibody conjugated with streptavidin-horseradish peroxidase (streptoavidin-HRP) were injected into each well. The reaction was performed at room temperature for 1 hour. Then, 100 μl of substrate solution (0.01% TMB in phosphate-citrate buffer) was added and developed for 30 minutes at room temperature. Then, 50 μl of 2 M H ₂ SO ₄ was added to each well to stop the color reaction. Absorbance was measured with a micro plate reader at nm.

Example 2 Th1 / Th2 Cytokine Balanced Activity Test

In order to evaluate the inhibitory activity of Th2 immune response by β-glucosidase-treated Youngsil extract, the amount of IL-4 cytokines was measured from the culture medium of mouse splenocytes according to the methods of Examples 1-3 and 1-4. It was.

As a result of comparing IL-4 production inhibitory activity of each extract, water, alcohol, and alcoholic water extracts were 26%, 14%, and 400% at the concentration of 400 ㎍ / ml for β-glucosidase-treated Youngsil extracts. 65% showed IL-4 inhibitory activity. On the other hand, in the Youngsil extract prepared by β-glucosidase treatment, water, alcohol, and alcoholic water extracts showed 89%, 92%, and 90% IL-4 production inhibitory activity at the concentration of 200 ㎍ / ml, respectively. It was. Also, at the same 200 ㎍ / ㎖ concentration of β-glucosidase-treated Youngsil extract showed little IL-4 inhibitory activity.

In conclusion, it was confirmed that IL-4 inhibitory activity of Young-sil extract was significantly enhanced by the treatment of β-glucosidase.

Example 3. In vivo Antiallergic Effect by Enzyme-treated Youngsil Extract in

Based on the results of Example 2, it was intended to evaluate the anti-allergic activity by enzyme-treated Youngsil extract in an in vivo allergy-induced mouse model. To this end, 10 μg of ovalbumin (OVA) and 1 mg of alum were mixed in 6-week-old BALB / c mice for 30 minutes, and then 100 μl per mouse 1 7 days and the first day of the experiment. And secondary immunization. In addition, from 14 to 27 days from the beginning of the experiment to prepare the Youngsil water extract prepared in Example 1-1 and the Youngsil extract treated with β-glucosidase prepared in Example 1-2 in saline solution 200 mg / kg per horse Oral administration was performed daily for 14 days, and blood and splenocytes were collected at the sacrifice of mice on the 28th day of the experiment.

In order to analyze the content of IgE in the blood collected from the mice administered the Youngsil extract or enzyme-treated Youngsil extract according to the above method, the total IgE amount was measured using BD mouse IgE ELISA kit.

As a result, as shown in Figure 2, IgE content was slightly reduced compared to the positive control group (Sham) even when administration of Youngsil extract (youngsil), β-glucosidase treated enzyme-treated Youngsil extract (β-glucosidase treatment) ), The IgE content was significantly reduced compared to the administration of Youngsil extract.

The results showed that the IgE production inhibitory activity of Youngsil extract was significantly enhanced by the treatment of β-glucosidase.

The description of the present invention set forth above is for illustrative purposes, and one of ordinary skill in the art may understand that the present invention may be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

The composition for preventing, ameliorating, or treating a Th2-mediated immune disease according to the present invention is excellent in inhibiting Th2 immune response and inhibiting IgE production, thereby preventing, improving, or treating a Th2-mediated immune disease, preferably an allergic disease. It may be usefully used in industrial fields for the development of medicines, dietary supplements, and cosmetics.

Claims (10)

1. Enzyme-treated Youngsil (Rosae multiflorae Fructus) comprising a extract as an active ingredient, Th2-mediated immune disease preventing or treating pharmaceutical composition.
2. The method of claim 1,

   The Th2-mediated immune disease is characterized in that allergic disease, composition.
3. The method of claim 1,

   The enzyme is characterized in that beta-glucosidase (β-glucosidase), composition.
4. The method of claim 1,

   The extract is characterized in that extracted with a solvent selected from the group consisting of water, C ₁ to C ₄ lower alcohols, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and a mixed solvent thereof Composition.
5. The method of claim 1,

   Wherein the composition inhibits the production of interleukin-4 (IL-4).
6. The method of claim 1,

   Wherein said composition is characterized by inhibiting immunoglobulin E (IgE) production.
7. Enzyme-treated Youngsil (Rosae multiflorae Fructus) comprising a extract as an active ingredient, Th2-mediated immune disease improvement health functional food composition.
8. Enzymatic treatment Youngsil (Rosae multiflorae Fructus) comprising a extract as an active ingredient, a cosmetic composition for improving Th2-mediated immune disease.
9. Enzymatic treatment comprising administering a pharmaceutical composition comprising Rosae multiflorae Fructus extract as an active ingredient to an individual, Th2-mediated immune disease prevention or treatment method.
10. Use of Rosae multiflorae Fructus extract for the prevention or treatment of Th2-mediated immune diseases.

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