KR20170049414A - Th2- Composition for preventing or treating of 2-mediated immune disease comprising fermented extracts from Rosa multiflora as an active ingredients - Google Patents

Abstract

The present invention relates to a composition for preventing or treating Th2-mediated immune diseases comprising a fermented extract of Rosa multiflora as an active ingredient. By fermenting Rosa multiflora, which is a natural ingredient with almost no side effects, the fermented extract of Rosa multiflora, with enhanced activities of Th2 immune reaction control, T cell differentiation control, and IgE production control, can be practically utilized for developing medicines, health functional foods, and cosmetics for preventing, alleviating, and treating Th2-mediated immune diseases.

Description

[0001] The present invention relates to a composition for prevention or treatment of a Th2-mediated immune disease,

The present invention relates to a composition for preventing, ameliorating, or treating a Th2-mediated immune disease, which comprises a fermented extract of Spirulina yezoensis as an active ingredient.

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. The prevalence rate is increasing worldwide, and 20 to 25% of all people suffer from allergic diseases such as rhinitis, asthma, atopic dermatitis, and food allergy.

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.

Type I immediate-type hypersensitivity is divided into two stages. In the first stage, IL-12, which suppresses the secretion of IgE and IgG1 by the invasion of allergen, increases the secretion of IgG2a, and Th1 (type 1 helper (IL-4), IL-13 (IL-4), IL-5, and IL-13 were induced by the excessive immune response of Th2 And IgE specific antibodies produced by B cells are adhered to the surface of mast cells or basophils to be sensitized to allergens.

The second stage of the allergic manifestation is divided into an initial reaction and a late reaction. The allergen is re-invaded into the body to stimulate mast cells and triggers a degranulation reaction. At this time, blood vessels caused by histamine, lipid metabolites, cytokines, And eosinophil, eosinophil, macrophage, Th2 cell, and basophil are infiltrated into the affected tissue to induce inflammation, resulting in atopic dermatitis, rhinitis, asthma, and the like. 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.

Currently, most medicines for the treatment of allergy are focused on suppressing the secretion of histamine secreted in the last stage of the allergy mechanism or treating the symptoms caused by allergy, and there are few therapeutic agents showing definite effects. The treatment of allergic diseases can be done for a long period of time. Most of the drugs with excellent therapeutic effect are widely used for atopic dermatitis, allergic rhinitis and asthma as long as they are steroid drugs. However, long term use can cause serious side effects. 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.

On the other hand, fermentation is a phenomenon in which microorganisms decompose or change organisms with their own enzymes and produce beneficial end products. They have been widely used as human food cooking methods, and one third of human food It is fermented food. Fermented food is considered to be the best health food with its natural taste and aroma. Yogurt, antioxidant and antioxidant wine, and miso and kimchi in Korea are representative. Furthermore, it is known that bacteria that are rich in fermented foods such as yogurt, cheese, miso, and cheese prevent allergic diseases. As well as taste, the beneficial effects of fermentation are widely known, and studies have been actively conducted to improve or treat diseases using fermented foods as well as fermented foods.

More specifically, many research results have been reported in order to improve natural functions or to obtain new effects by adding various nutritional ingredients in addition to original ingredients by fermenting natural products. For example, the fermented soybean fermented soybean fermented soybeans was recognized as a stabilizing raw material in the phytosanitary hospital after the postprandial glucose control function was suppressed by suppressing the absorption of sugar, and the toxicity reduction and immunity enhancement effect of the fermented extract of lactobacillus of mistletoe were also reported (KR10-1634670).

In addition to the development of safe natural material itself, there is no side effect for allergic response, and further research is needed to improve the efficacy or obtain new effects by using fermentation.

Accordingly, the present inventors have studied the effect of enhancing the antiallergic activity by preparing a fermented extract of Aspergillus sp.

The inventors of the present invention have conducted intensive studies to enhance the antiallergic activity of Youngsil and found that fermented extract is obtained by inoculating a microorganism in the preparation of Youngshil extract to thereby increase the inhibitory activity against Th2 immune response, T cell differentiation, and IgE production Thereby completing the present invention.

Accordingly, it is an object of the present invention to provide a composition for preventing, ameliorating, or treating a Th2-mediated immune disease, which comprises a fermented extract of Aspergillus oryzae 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 object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating Th2-mediated immune diseases, comprising a fermented extract of Rosa multiflora as an active ingredient.

The present invention also provides a health functional food composition for the treatment of Th2-mediated immune diseases, comprising a fermented extract of Rosa multiflora as an active ingredient.

In addition, the present invention provides a cosmetic composition for improving Th2-mediated immune diseases, comprising a fermented extract of Rosa multiflora 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 fermented extract is obtained by inoculating a microorganism into a living room, culturing the microorganism for 4 to 10 days, adding water, a C ₁ to C ₄ lower alcohol, ethyl acetate, acetone, butyl acetate, And may be extracted with a solvent selected from the group consisting of ethylene glycol, propylene glycol, ethylene glycol, propylene glycol,

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

In another embodiment of the present invention, the composition can inhibit the differentiation of T cells.

In another embodiment of the present invention, the composition may inhibit the production of immunoglobulin E (IgE).

In addition, the present invention provides a method of preventing or treating a Th2-mediated immune disease, comprising the step of administering the pharmaceutical composition to a subject.

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

The present inventors are the IL-4 produced suppression and T cell inhibiting activity differentiation significantly increase compared to the results unleavened Youngsil extract treated Youngsil fermentation extract to splenocytes of immune mice with OVA, the Youngsil fermentation extract from in vivo It was confirmed that the IgE content in the blood of mice immunized with OVA by oral administration was significantly reduced. Therefore, the fermented extract of Ganoderma lucidum, in which Th2 immune response inhibitor, T cell differentiation and IgE production inhibitory activity are remarkably enhanced by fermenting a natural product having little side effect, is a therapeutic agent for prevention, improvement, or treatment of Th2- Health functional foods, cosmetics, and the like.

FIG. 1 shows the result of measuring the amount of IL-4 secreted by Th2 in the cell culture after treating the spleen cells isolated from mice immunized with OVA, that is, the control group, Youngsil Extract or Young's Silk Fermentation Extract, by ELISA (Young Sil 200: 200 ㎍ / ㎖ ginseng extract-treated group, 26 strains fermented 200: 200 ㎍ / ㎖ ginseng fermented extract treated group).
FIG. 2 shows the result of measuring the degree of T cell differentiation by performing MTT assay on the spleen cells after treating the spleen cells isolated from mice immunized with OVA, Mu] g / ml Young's Silk Extract treated group, 26 strains fermented 200: 200 [mu] g / ml Young's Silk Fermented Extract treated group).
FIG. 3 shows the results of ELISA assay of the serum IgE content of mouse after orally administered to mice immunized with OVA (Yeungsil: Youngsil extract group, 26 strains: Fermentation of Young silly fermented extract ).

The present invention relates to a composition for preventing, ameliorating, or treating a Th2-mediated immune disease, which comprises a fermented extract of Spirulina yezoensis as an active ingredient.

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 comprising Rosa multiflora fermented extract as an active ingredient.

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

As used herein, the term " treatment " means any action that improves or alters the symptoms of a Th2-mediated immune disorder by administering the pharmaceutical composition of the present invention.

"Youngsil", a natural material used in the present invention, is a fruit of a briar that often grows in Korea and Japan. It is spherical or elliptical. Outer surface is glossy with dark red or dark reddish brown color. to be. It has a slightly sour smell, a little taste, it does not see urine easily, it is used for edema, insomnia, sexual decline, boils, malt, promotes blood circulation and removes poison. In addition, pharmacological actions such as coronary artery dilation, prolonged mouse life, lipid, protein metabolism improving action, and atherosclerosis formation inhibitory action have been reported.

As used herein, the term " T cell " refers to a cell that expresses IL-4, IL-5, IL-6, , IL-10, IL-13, and the like. The immune balance regulated by the cytokines produced by these Th1 and Th2 cells is called the Th1 / Th2 balance. Th1 cells are important for the regulation of cellular immunity, and Th2 cells 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 to be treated in the present invention, refers to a disease in which IgE and mast cells are involved by the production and activation of allergens, particularly Th2 cells. Examples include allergies, Skin diseases including dermatitis, acute and chronic allergic rhinitis, asthma, food allergies, and the like.

As used herein, the term " fermentation " means that an organic material is decomposed and changed by microbial action to produce a useful substance. The fermentation may be various depending on the environment. Typically, there are fermentation of alcohol by yeast, fermentation of glycerol, fermentation of lactic acid bacteria with lactic acid, fermentation with lactic acid, methane fermentation with methane bacteria, and mixed organic acid fermentation with Escherichia coli. Oxidative fermentation utilizes the accumulation of intermediate metabolites by incomplete oxidation of the substrate, such as fermentation of acetic acid with acetic acid bacteria, fermentation with gluconic acid, fermentation with sorbose or organic acids such as citric acid, gluconic acid, fumaric acid and oxalic acid. In the present invention, fermentation can be carried out by adding water to the living room at a weight ratio of 5 to 50, then inoculating the microorganism and culturing for 4 to 10 days, preferably 6 to 8 days, more preferably 7 Lt; / RTI > for one day.

The microorganism which can be used for the fermentation in the present invention is not limited as long as it is a microorganism capable of promoting the activity of the spirit chamber through fermentation. Preferably, it may be a strain of Bacillus amyloliquefaciens , May be the Bacillus amyloliquefaciens strain 26N (Accession No. KCCM 11287P).

The fermentation extract can be extracted using a conventional solvent known in the art for extracting the extract from a natural product, that is, under ordinary temperature and pressure conditions. For example, in the present invention, the fermentation extract of Youngsil is fermented with a mixture of water, alcohols having 1 to 4 carbon atoms, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, The solvent may be extracted using one or more solvents selected from the group consisting of ethanol and ethanol, preferably ethanol. In addition, the method for extracting the fermentation extract of Young Root can be carried out by various methods such as room temperature extraction, hot water extraction, cold extraction, reflux extraction, microwave extraction and ultrasonic extraction. Preferably, But is not limited to this.

The extract thus prepared may be filtered, concentrated or dried to remove the solvent, and may be subjected to both filtration, concentration and drying. For example, the filtration can be performed using a filter paper or a vacuum filter, the concentration can be carried out using a vacuum concentrator, and the lyophilization can be carried out, but the present invention is not limited thereto.

Further, 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 캜 to 120 캜, but is not limited thereto.

In the examples of the present invention, the following experiment was conducted to confirm the improvement or therapeutic effect of Th2-mediated immune disease on the fermented extract of Spirulina japonica.

In one embodiment of the present invention, when OVA and non-fermented spirulina cells isolated from OVA-immunized mice were treated with fermented extract of Youngsil or Youngsil, the fermented extract of Youngju was treated It was confirmed that the degree of decrease in the amount of IL-4 production in the culture solution was remarkably increased (see Example 2).

In another embodiment of the present invention, MTT assay was performed on spleen cells treated with each of the above extracts, and it was found that when the fermented extract of Youngsil was fermented, the activity of inhibiting the differentiation of T cells was enhanced as compared with that of the Ganoderma lucidum extract-treated group (See Example 3).

In another embodiment of the present invention, the IgE content of the mouse immunized with OVA was orally administered to the mice immunized with OVA, and as a result, the IgE content in the group treated with the Ganoderma lucidum extract was lower than that of the Ganoderma lucidum extract group (See Example 4).

Therefore, the composition of the present invention can inhibit the production of IL-4.

The composition of the present invention can inhibit the differentiation of T cells.

The composition of the present invention can inhibit IgE production.

The pharmaceutical composition according to the present invention may further comprise a pharmacologically acceptable carrier, wherein the pharmaceutical composition comprises a fermented Young's milk extract as an active ingredient. 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. It may also be formulated into injectable formulations, pills, capsules, granules or tablets, such as aqueous solutions, suspensions, emulsions and the like, with the addition of diluents, dispersants, surfactants, binders and lubricants. 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, " 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, The time of administration, the route of administration and the 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.

In another aspect of the present invention, there is provided a health functional food composition for improving a Th2-mediated immune disease comprising Rosa multiflora fermented extract 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 or separately with the medicament for treatment before or after the onset of the disease for the prevention or improvement of the Th2-mediated immune disease.

The term " health functional food composition " as used in the present invention means a food composition comprising at least one of a carrier, diluent, excipient, and additives, selected from the group consisting of tablets, pills, powders, granules, powders, capsules, Which is characterized in that it is formulated into one. 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 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.

As another embodiment of the present invention, the present invention provides a cosmetic composition for the treatment of Th2-mediated immune disorders, comprising a fermented extract of Rosa multiflora as an active ingredient.

The cosmetic composition of the present invention may contain components commonly used in cosmetic compositions as well as fermentation extracts of real-time fermentation, such as conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and perfumes, and Carrier.

In addition, the composition of the present invention may be used in combination with a conventional ultraviolet screening agent as long as it does not deteriorate the skin protecting effect by reacting with the fermentation extract of Spirulina et al. 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 gamma-fermented 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. If the content of the fermentation extract is less than 0.00001% by weight, the effect of absorbing ultraviolet light is greatly reduced. If the content 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. Experimental preparation and method

1-1. Preparation of Young-Sil fermented extract

Youngsil was purchased from Jongno Village, Gyeongsangnam - do, and used for the experiment. Bacillus amyloliquefaciens strain 26N (Accession No. KCCM 11287P) used for fermentation of Youngsil was separated and cultured from conventionally prepared meju using a tryptic case soybean agar (Difco Co., USA) medium .

To prepare a fermented extract of Youngsil, the Young's chamber and water were mixed at a ratio of 1:10, and the Bacillus amyloliquefaciens strain 26N was inoculated, followed by shaking fermentation at 170 rpm for 7 days at 170 rpm. After that, the fermented product was added to the fermented product so that the ethanol content was 50%, and then microwave extraction was carried out at 90 W for 5 minutes using Prolabo's Soxwave 100 (Fontenay-Sous-Bois, France). The extract was filtered through a filter paper, concentrated under reduced pressure, and lyophilized to use in the experiment. At this time, as a control group of the fermentation extract of Youngsil, fermented yeast extract was used which was prepared by fermenting the strain but not fermenting the same.

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 then 100 μl of each mouse was weighed every week 2 times (n = 5). One week after the immunization, the mice were sacrificed by cervical dislocation, the spleens were removed from the aseptic state, and then transferred to a small petri dish containing RPMI 1640 medium supplemented with 1 ml of the basic medium, i.e., 2-mercaptoethanol and antibiotics. 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, OVA, an antigen, was treated at a concentration of 100 占 퐂 / ml on a 96-well plate, and a Young's chamber fermentation extract prepared according to the method of Example 1-1 and a control group Youngsil extract were added at a concentration of 200 占 퐂 / Splenocytes from the cultured mice were treated at 5 x ¹⁰ cells / well. After incubation for 72 hours in a CO ₂ incubator at 37 ° C, the cell culture medium was recovered.

Example 2 Th1 / Th2 Cytokine Balance Regulatory Activity Test

The immune balance regulated by the cytokines produced by Th1 and Th2 cells is called the Thl / Th2 balance. Th1 cells are important for the regulation of cellular immunity, and Th2 cells play an important role in the regulation of humoral immune. Th1 / Th2 balance is maintained constantly by interfering with cytokine mainly on IFN-γ and cytokine mainly on IL-4, which are important for differentiation of Th2, If broken, various 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.

In order to evaluate the inhibitory activity of Th2 immunoreactivity induced by the fermentation of Youngju, the amount of IL-4 cytokine was measured using an IL-4 BD OptEIATM MOUSE ELISA kit and TGF-β kit (R & D Systems) Were measured.

To this end, a capture antibody was added to a 96-well plate and incubated at 4 ° C overnight. The plate was coated with antibody, washed with a washing buffer (phosphate buffered saline with Tween 20; PBST) 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 substrate solution (0.01% TMB in phosphate-citrate buffer) was added and developed at room temperature for 30 min. Then, 50 μl of 2 M H ₂ SO ₄ was added to stop the color reaction. Absorbance was measured with a plate reader.

As a result, as shown in Fig. 1, production of IL-4 was reduced in both of the fermented extract of Yeongsil (26 strains fermentation 200) and the extract of Youngsil (Youngsil 200), compared with the case of OVA alone. However, when the fermented extract was treated with Youngju, it showed a remarkable reduction of IL-4 production by about 3 times as compared with the control.

From the above results, it can be seen that the Th2 immune response inhibitory activity of the Ganoderma lucidum extract is remarkably enhanced by the fermentation process of the Ganoderma lucidum.

Example 3 Test for inhibiting T cell differentiation

To evaluate the T cell differentiation inhibitory activity by the fermentation extract of Young silly, the culture solution was recovered according to the method of Example 1-2, and then the spleen cells were diluted with PBS to a concentration of 2 mg / -diethyl thiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) reagent. After incubating the cells for 4 hours, 100 μl of 10% SDS (sodium dodecyl sulfate) solution was added and left overnight. Absorbance was measured at 595 nm using a microplate reader.

As a result, as shown in FIG. 2, the Young Seil extract (Youngsil 200) showed a slight decrease in the degree of T cell differentiation in proportion to the treatment concentration, compared with the case of OVA treatment alone, Fermentation 200), it was confirmed that the degree of differentiation of T cells was significantly decreased compared with the same concentration of Ganoderma lucidum extract.

From the above results, it was found that the activity of inhibiting the differentiation of T cell of T. ganoderma extract was remarkably enhanced by the fermentation process of Ganoderma lucidum.

Example 4. In vivo Of Antiallergic Effect by Fermented Extract of Young Sil

Based on the results of Examples 2 and 3 above, the antiallergic activity of the fermented extract of Youngsil flour in an in vivo allergen-induced mouse model was evaluated. To this end, 10 μg of ovalbumin (OVA) and 1 mg of alum were mixed in a 6-week-old BALB / c mouse for 30 minutes, and then 100 μl per mouse was injected at 7 days and 21 days And secondary immunization. In the physiological saline from the 14th to 27th days from the start of the experiment, the yeast extract and the control group Youngju extract prepared in Example 1-1 were prepared and administered orally at a dose of 200 mg / kg per day for 14 days. Mice were sacrificed and blood and spleen cells were collected.

In order to analyze the content of IgE in the blood collected from the mouse administered with the fermentation extract of Youngju or Youngsun extract according to the above method, the amount of total IgE was measured using a mouse IgE ELISA kit of BD Company.

As a result, as shown in FIG. 3, the IgE content was slightly decreased compared with the positive control sham even when the extract of Youngsil was administered. However, when the Youngju extract (26 strains fermented) was administered, And the IgE content was significantly decreased compared with the case of the administration of the < RTI ID = 0.0 >

From the above results, it was found that the activity of inhibiting the secretion of IgE from persimmon extract was remarkably enhanced by the fermentation process of Ganoderma lucidum.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. There will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (8)

A pharmaceutical composition for the prevention or treatment of a Th2-mediated immune disorder, comprising an extract of Rosa multiflora fermented as an active ingredient.
The method according to claim 1,
Wherein said Th2-mediated immune disease is an allergic disease.
The method according to claim 1,
The fermentation extract is prepared by inoculating microorganisms into a living room and culturing the microorganism for 4 to 10 days. Thereafter, water, C ₁ to C ₄ lower alcohols, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, Of the total weight of the composition.
The method according to claim 1,
Wherein said composition inhibits the production of IL-4.
The method according to claim 1,
Wherein said composition inhibits T cell differentiation.
The method according to claim 1,
Wherein said composition inhibits the production of immunoglobulin E (IgE).
A health-functional food composition for improving a Th2-mediated immunological disease, comprising Rosa multiflora fermented extract as an active ingredient.
A cosmetic composition for improving a Th2-mediated immune disorder, comprising a fermented extract of Rosa multiflora as an active ingredient.

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