KR20070116433A - Composition for preventing and improving allergic disease comprising extract of sophorae fructus - Google Patents

Abstract

A composition containing Sophora fruit extract is provided to be very useful for preventing and improving allergic disease by inhibiting the activity of Th2 cytokine and an eosinophil which cause allergy, infiltration of the eosinophil into the alveoli and airway hyperresponsiveness caused by methacholine. A composition for preventing and improving allergic disease contains Sophora fruit extract which is obtained by crushing Sophora fruit into a size of 20 to 40meshes, extracting it in water or C1-6 alcohol and treating with amylase or pectinase. The allergic disease is allergic dermatitis, allergic nasal inflammation, allergic asthma, allergic otitis media, anaphylactic shock and allergic conjunctivitis. The allergic dermatitis is atopic dermatitis, psoriasis, allergic contact dermatitis and urticaria. The recommended daily intake amount of the extract is orally 0.5mg/kg. A food composition for preventing and improving allergic disease contains functional food, nutritional supplement, health food and food additive.

Description

Composition for Preventing and Improving Allergic Disease Comprising Extract Of Sophorae Fructus}

Figure 1a is the result of analyzing the effect of the extract of the present invention on the proliferation of Y16 cell line by stimulation of IL-5.

#: Significant difference compared to the case without adding mouse IL-5 (p <0.01)

*: Significant difference compared to the case where no lump extract was added (p <0.01)

Figure 1b is the result of analyzing the effect of the extract of the present invention on the proliferation of BAF / B03 cell line by stimulation of IL-3.

#: Significant difference compared to the case without adding mouse IL-3 (p <0.01)

*: There was a significant difference compared with the case without the addition of the extract (p <0.01).

Figure 1c is the result of analyzing the effect of the extract of the present invention on the proliferation of transformed Y16 cells by stimulation of GM-CSF.

#: Significant difference compared with the case without adding mouse GM-CSF (p <0.01)

*: Significant difference compared to the case where no lump extract was added (p <0.01)

Figure 2 is a result of analyzing the effect of the extract of the present invention on the degree of LTC ₄ release of eosinophils by IL-5 stimulation.

#: Significant difference compared with no addition of human IL-5 (p <0.01)

*: Significant difference compared to the case where no lump extract was added (p <0.01)

Figure 3 is a photograph of microscopic observation of the degree of infiltration of eosinophils into bronchoalveolar alveolar in accordance with the treatment of the ingot extract of the present invention in egg yolk sensitizing allergic mouse model.

A: normal mouse

B: Egg yolk sensitizing allergy mouse model

C: Egg yolk sensitizing allergic mouse model treated with oyster extract

Figure 4 is the result of measuring the degree of airway resistance according to the treatment of the lump extract in egg yolk sensitizing allergic mouse model administered with methacholine.

*: Significant difference compared to the case where no lump extract was added (p <0.01)

The present invention relates to a composition for the prevention and improvement of allergic diseases containing a lump extract as an active ingredient. Specifically, the present invention relates to a food composition or cosmetic composition for the prevention and improvement of allergic diseases containing a lump extract as an active ingredient.

An allergic disease is a disease that occurs when an individual's immune mechanism is more sensitive than normal to an external substance (allergen). Such allergic diseases are immune diseases with high incidence due to climate change, climate, environmental pollution, occupation such as temperature and humidity, but fundamental therapeutics have not been developed yet.

Anti-allergic agents, which have been developed until now, have mostly inhibited the levels of chemical carriers produced by inflammatory cells that trigger seizures. That is, when the mast cells are degranulated by various allergens, chemical substances such as histamine, heparin, and proteolytic enzymes stored in granules in the mast cells are released, and an immediate allergic and inflammatory reaction occurs. As a therapeutic agent for alleviating the symptoms of allergic diseases, antihistamines that inhibit most of the above chemicals are used, and in severe cases, steroid agents are co-administered. However, such synthetic products can not be expected to be a complete treatment, and the long-term use has the disadvantage that the effect is poor and systemic side effects are severe.

Recently, several research groups have discovered that cytokines produced by Th ₂ cells are the key to allergic development (O 'Garra, A. Immunity 8: 275, 1998; Glimcher LH et al., Genes Dev . 14: 1693, 2000; Murphy KM et al., Annul Rev. Immunol . 18: 451, 2000). Accordingly, studies are being actively conducted to develop drugs targeting the inhibition of the Th ₂ cytokine activity. For example, Schering-Plough is conducting a clinical trial to develop a single antibody of human IL-5 as an allergic agent, while Roche isothiazolone that inhibits IL-5. (isothiazolone) derivatives have been synthesized, but they are not suitable for drug development due to nonspecific reactions to other proteins.

Meanwhile, Sophorae Fructus refers to the fruit of a painting tree ( Sophora japonica Linne ). The painting tree is a deciduous tree belonging to the legumes, and is distributed in Korea, Japan, and China, and the crust, which is the fruit of the painting tree, is known to increase blood sugar and antimicrobial activity. It has been used for the treatment of keratinemia and anti-alveolar disease (Kim Chang-min et al., Medic.

Therefore, the present inventors can prevent and improve allergic diseases, and while searching for a new composition having no side effects, the extract of goose, which is a fig tree fruit, inhibits the activity of Th ₂ cytokines and eosinophils and prevents infiltration of eosinophils into bronchoalveolar alveoli. The present invention was completed by confirming that there is activity to prevent and improve allergic diseases by inhibiting and inhibiting airway hypersensitivity by methacholine.

Therefore, an object of the present invention is to provide a food composition for the prevention or improvement of allergic diseases containing the lump extract as an active ingredient.

Another object of the present invention is to provide a cosmetic composition for the prevention or improvement of allergic diseases containing a lump extract as an active ingredient.

In order to achieve the object of the present invention as described above, the present invention provides a food composition for the prevention or improvement of allergic diseases containing a lump extract as an active ingredient.

In addition, the present invention provides a cosmetic composition for the prevention or improvement of allergic diseases containing a lump extract as an active ingredient.

In the present invention, " Sophorae Fructus " refers to the fruit of the picture tree, which is a legume. Preferably, the ingot refers to the ripe fruit of the painting tree.

The ingot used for the preparation of the ingot extract of the present invention is a mature fruit, which has its own color and flavor, and is preferably free of odors.

The lump extract of the present invention can be prepared by a solvent extraction method known in the art and then by treating the extract with an enzyme. Examples of the extraction solvent include any one selected from alcohols having 1 to 6 carbon atoms such as water, ethanol and methanol, organic solvents such as acetone, ethyl acetate, n-nucleic acid, diethyl ether, acetone, and benzene, or a mixture thereof. Solvents may be used. Preferably, the extraction may be performed using water or an alcohol having 1 to 6 carbon atoms. By the solvent extraction method In preparing the extract, hot water extraction, ultrasonic extraction and reflux extraction methods can be used. Most preferably, the lump extract of the present invention may be prepared by a hydrothermal extraction method using water as a solvent.

The ratio of ingot and water during hot water extraction is not particularly limited, but water may be added to the ingot powder three to twenty times (by weight). Preferably, water is added to the ingot powder to increase the extraction efficiency. It can be added by 10 times (by weight).

The temperature during extraction is preferably performed at room temperature under atmospheric pressure and the extraction time depends on the extraction temperature, but is extracted from 1 hour to 6 hours, preferably 2 hours to 4 hours. In addition, the extraction efficiency can be further increased when stirring with a shaker (shaker) during extraction.

The lumps used for extraction can be harvested, washed and used as is or dried. As a drying method, both dry, shade, hot air drying and natural drying can be used. In addition, in order to increase the extraction efficiency, the ingot or the dry body may be used by grinding the mill.

Preferably, the lump extract of the present invention is pulverized the lump to the size of 20 to 40 mesh, and then the drinking water is added 3 to 20 times, preferably 5 to 10 times to the lump powder and 100 to 130 ℃, preferably Hot water is extracted for 1 to 3 hours at 120 to 125 ° C. By removing the precipitate by centrifugation of the hot water extract solution, the supernatant may be recovered to prepare a lump extract.

Enzymatic treatment of the lump extract obtained by the above method is preferably treated with 0.01-1% (v / v) of the enzyme in hot water extract for 4 to 24 hours and then concentrated and lyophilized. It can manufacture. As the enzyme, α- and β-amylase, pectinase may be used.

On the other hand, the lump extract of the present invention has the effect of preventing or treating allergic diseases. As used herein, the term "allergic disease" refers to a disease caused by hypersensitivity of the body's immune system to a substance that is sensitized to a substance of the human body, that is, a substance from outside. Examples of the allergic disease include, but are not limited to, allergic dermatitis, allergic rhinitis, allergic asthma, allergic otitis media, anaphylatic shock and allergic conjunctivitis. The allergic dermatitis may include atopic dermatitis, psoriasis, contact allergic dermatitis and urticaria.

The prevention or improvement effect of allergic disease of the lump extract according to the present invention was confirmed by the present inventors through in vitro and in vivo experiments.

That is, in one embodiment of the present invention, it was investigated whether the gangrene extract inhibits the activity of the Th ₂ cytokines IL-5, IL-3 and GM-CSF (see Example 3-1). As a result of the experiment, the lump extract according to the present invention was shown to dose-dependently inhibit the activity of IL-5, IL-3 and GM-CSF (see FIGS. 1A to 1C).

The IL-5, IL-3 and GM-CSF cause allergic diseases as cytokines released when Th ₂ cells are exposed to allergens. More specifically, the IL-5 promotes the differentiation of bone marrow cells into eosinophils, increases the adhesion between eosinophils and vascular endothelial cells, promotes the migration of eosinophils to target organs, inhibits apoptosis and activates eosinophils. Let's do it. Activated eosinophils release toxic proteins such as major basic protein (MBP) and eosinophil cationic protein (ECP), which damage epithelial cells in target organs. This causes allergic diseases such as bronchial asthma. IL-3 also induces allergic reactions by releasing histamine and leukotriene (LT) from mast cells. On the other hand, IL-5 must bind to a receptor consisting of alpha and beta chains in order to exhibit the cellular response as described above. IL-5 receptor beta-chains are identical to the receptor beta chains of IL-3 and GM-CSF. Therefore, the cytokines exhibit physiological activity through similar intracellular signal transduction and cause allergic diseases.

Thus goegak extract of the present invention can effectively suppress the allergic diseases caused by the activation of the Th ₂ cytokines by inhibiting the Th ₂ cytokine. For example, it has been reported in the atopic animal model that IL-5 monoantibodies bind IL-5 to water-soluble IL-5 receptors, thereby inhibiting infiltration and activation of eosinophils, resulting in anti-allergic effects (Devos , R. 1995). Therefore, even in the case of the extract of the present invention by inhibiting the activity of IL-5 can suppress allergic diseases such as atopic dermatitis.

Furthermore, the present inventors have found that it is goegak extract of the present invention inhibit the activity of the Th ₂ cytokine was investigated whether inhibition of eosinophil activation catalyzed by the Th ₂ cytokine. Eosinophils are enabled to emit LTC _4, LTC ₄ above, are known to cause respiratory allergic diseases such as asthma, as a material that exhibits an action to strongly contract the bronchi (Chari, S. et al., Am J. Clin Dermatol , 2: 1, 2001).

Thus, the present inventors have isolated eosinophils from asthma patients in one embodiment of the present invention and investigated whether the ingot extract according to the present invention inhibits LTC ₄ release of eosinophils by IL-5 stimulation (Example 3-2 Reference). As a result, the ingot extract according to the present invention was shown to dose-dependently inhibit the release of LTC ₄ from eosinophils (see FIG. 2).

Therefore, the present inventors were able to confirm that the ingot extract of the present invention inhibits the activity of eosinophils by inhibiting the activity of Th ₂ cytokines causing allergic diseases.

Furthermore, the present inventors confirmed that the ingot experiment of the present invention has an effect of preventing or improving allergic diseases through an in vivo experiment using an egg yolk sensitizing allergy animal model. In one embodiment of the present invention, a yolk sensitizing allergic mouse model was prepared and whether the ingot extract of the present invention inhibited the infiltration of eosinophils into the bronchial alveoli in the mouse model (see Example 4-1). As a result, in the control group, the number of eosinophils infiltrated into the bronchoalveolar alveolar was significantly increased compared to the non-sensitized mouse due to egg yolk sensitization, but in the experimental group to which the ingot extract according to the present invention was infiltrated into the bronchial alveolar The number of eosinophils was found to be drastically reduced (see Table 1 and FIG. 3).

In another embodiment of the present invention, the degree of airway hypersensitivity was measured after sequentially inhaling methacholine in the yolk sensitizing allergic mouse model according to the concentration. As a result of the experiment, the worm extract according to the present invention was shown to significantly reduce the degree of airway resistance induced by methacholine in egg yolk sensitized allergic mice. From the results of the experiment, it was confirmed that the lump extract according to the present invention has an antiallergic activity.

Therefore, the present invention may be provided in the form of a food composition for the purpose of preventing or improving allergic diseases containing the lump extract as an active ingredient. The food composition of the present invention includes all forms such as functional foods, nutritional supplements, health foods and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, as a health food, the ingot extract itself of the present invention may be prepared in the form of tea, juice and drink for drinking, or granulated, encapsulated and powdered. In addition, it can be prepared in the form of a composition by mixing with the gangbang extract of the present invention and known active ingredients known to have the effect of improving and preventing menopausal diseases.

In addition, functional foods include beverages (including alcoholic beverages), fruits and processed foods (e.g. canned fruit, canned foods, jams, marmalade, etc.), fish, meat and processed foods (e.g. ham, sausage corned beef) Breads and noodles (e.g. udon, soba noodles, ramen, spagate, macaroni, etc.), fruit juices, various drinks, cookies, syrups, dairy products (e.g. butter, cheese), edible vegetable oils, margarine, vegetable Protein, retort foods, frozen foods, various seasonings (eg, miso, soy sauce, sauce, etc.) can be prepared by adding the extract of the present invention.

In addition, in order to use the lump extract of the present invention in the form of food additives can be prepared and used in the form of powder or concentrate.

The preferred content of the lump extract of the present invention in the food composition of the present invention may include about 0.1 to 50% by weight relative to the total weight of the food. Preferably, when the intake of the lump extract of the present invention in the amount of 0.5mg / kg / day to 5mg / kg / day may have the effect of preventing and improving allergic diseases.

In addition, the present invention provides a cosmetic composition for the prevention or treatment of allergic diseases containing the gangseo extract according to the present invention as an active ingredient. The cosmetic composition according to the present invention can be prepared in any form of formulation suitable for topical application according to methods known in the art by additionally containing cosmetically and / or dermatologically acceptable excipients in addition to the ingot extract as an active ingredient. For example, but not limited to, the cosmetic composition according to the invention may be a solution, gel, solid, emulsion, suspension, microemulsion, microcapsules, microgranules, ionic and / or nonionic vesicle dispersants, creams, skins, It may be prepared in the form of a lotion, powder, ointment, spray or stick.

Such cosmetically and / or dermatologically acceptable excipients may include emollients, emulsifiers, thickening agents and solvents.

The emollients are used to soften, sooth, coat, smooth or moisturize the skin. In the present invention, the emollient may use all kinds known in the art and may be, for example, petroleum based, fatty acid esters, alkyl ethoxylates, fatty acid ester ethoxylates, fatty alcohols, polysiloxanes or mixtures thereof. In addition to propylene glycol, butylene glycol, glycerin, triethylene glycol, meridians, waxes, fatty acids, fatty alcohol ethers, glycerides, acetoglycerides, ethoxylated glycerides, other fatty acid esters of polyhydroxy alcohols, lanolin and derivatives thereof Conventional emollients such as;

The emulsifier serves to mix the water phase and the oil phase of the cosmetic composition. In the present invention, the emulsifier may optionally contain one or more, and may include both nonionic, anionic or cationic. The type of the emulsifier may be determined depending on whether the emulsion is in water-in-oil dispersion or oil-in-water dispersion. Examples of suitable emulsifiers include, but are not limited to, sorbitan trioleate, sorbitan tristearate, glycerol monooleate, glycerol monostearate, glycerol monolaurate, sorbitan sesquioleate, sorbitan monooleate, Sorbitan monostearate, polyoxyethylene stearyl ether, polyoxyethylene sorbitol beeswax derivative, PEG 200 dilaurate, PEG 200 monostearate, PEG 400 dioleate, sorbican monopalmitate, polyoxyethylene monostearate and Polyoxyethylene sorbitan monostearate and the like.

Thickeners include crosslinked carboxypolymethylene polymers, ethyl cellulose, polyethylene glycol, tracant gum, karaya gum, xanthan gum, bentonite, hydroxyethyl cellulose and hydroxypropyl cellulose.

As the solvent, purified water, alcohol or a mixture of purified water and alcohol may be used.

Examples of other optional cosmetic additives that may optionally be used include, but are not limited to, preservatives such as para-hydroxybenzoate esters, butyl hydroxy toluene, ascorbic acid and derivatives thereof, and antioxidants such as tocopherols and derivatives thereof, glyce Wetting agents such as rolls, sorbitol, 2-pyrrolidone-5-carboxylate, dibutylphthalate, gelatin, polyethylene glycol, pH buffers such as acetates, phosphates, citrate, triethanolamine and carbonates, beeswax, paraffins and The same waxes, thickeners, activity enhancers, colorants and flavorings can be used.

Preferred content of the lump extract of the present invention in the cosmetic composition of the present invention may include about 0.1 to 50% by weight relative to the total weight of the cosmetic. Preferably in the amount of 0.1mg ~ 10mg of the ingot extract of the present invention on the skin surface Treatment may have the effect of preventing and ameliorating allergic diseases.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

<Example 1>

Preparation of Oyster Extract

20 kg of lumps (Jeseong Pharmaceutical Co., Ltd., Gyeongdong Market) were ground to a size of 30 mesh using a dry mill. Drinking water was added to the pulverized product and diluted 10 times (grind product: drinking water = 9: 1), followed by heating at 100 ° C. for 4 hours. Thereafter, the mixture was cooled to 50 ° C., filtered using a 100 mesh filter cloth, and filtered again using a 200 mesh filter cloth to remove precipitates, thereby obtaining a filtrate. The supernatant was concentrated to a volume of 1/2 using a concentrator to prepare an extract. To the concentration of 0.5% (v / v) was added to the concentrate of the shell was subjected to the enzyme reaction for 16 hours at 50 ℃. The reaction solution was centrifuged to obtain a precipitate, which was extracted with ethanol for 1 hour. The extract was filtered through a 200mesh filter cloth to remove precipitates, and the obtained filtrate was concentrated to a volume of 1/5 by using a concentrator, thereby preparing an enzyme-degradable extract of lumps. The enzyme decomposition extract was powdered by spray drying using a spray dryer.

<Example 2>

In vitro ( in vitro Investigation of Prevention and Treatment of Allergic Diseases of Necrosis Extracts through Experiments

<2-1> Th of lump extract ₂  Inhibitory Effect of Cytokines

We investigated whether the enzyme-decomposed extract of the ganglia according to the present invention has an effect of inhibiting the activity of Th ₂ cytokines that play an important role in allergic reactions. Th ₂ cytokines were targeted to IL-5, IL-3 and GM-CSF, which are known to be the main causes of allergic diseases.

The inhibitory effect of IL-5, IL-3 and GM-CSF on the enzyme enzyme extract of the gangrene of Example 1 is characterized in that Y16 cell line proliferates depending on mouse IL-5, BAF / BO3 cell line mouse IL-3 The Y16 cell line transformed with cDNA encoding GM-CSF receptor α-chain, depending on the mouse GM-CSF, and propagated depending on the mouse GM-CSF were used for the enzymatic extract of the ganglia. It was confirmed by measuring whether or not to inhibit the proliferation of.

That is, 1 × 10 ⁵ Y16 cell lines (provided by Professor Kiyoshi Takatsu, the Institute of Medical Science, the University of Tokyo University of Japan) were dispensed 1 × 10 ⁵ per well into a 96-well plate, and 3U / ml of mouse IL-5 (Sigma-Aldrich) and the above example. Enzymatic digestion extracts of 1 gangle were added together at concentrations of 1, 3, 10 and 30 µg / ml, respectively, and cultured for 48 hours at 37 ° C. and 5% CO ₂ . Then, WST-1 coloring reagent was added thereto, and the proliferation of Y16 cell line was analyzed by measuring absorbance at 450 nm. At this time, the enzyme was not added to the enzyme control enzyme extract. The inhibition rate of cell proliferation was expressed as a percentage of the decrease in cell proliferation of the group treated with the enzyme hydrolyzate extract of IL-5 and the ganglia relative to the cell proliferation of the group treated with IL-5 only.

In addition, 2 × 10 ⁵ BAF / BO3 cell lines (provided by Professor Toshio Hirano, Osaka Medical University, Japan) were dispensed in 96-well plates at 2 × 10 ⁵ per well, 10U / ml of mouse IL-3 (Sigma-Aldrich) and the above example. Enzymatic digestion extracts of 1 gangle were added together at concentrations of 1, 3, 10 and 30 µg / ml, respectively, and cultured for 48 hours at 37 ° C. and 5% CO ₂ . Then, the absorbance was measured in the same manner as above. At this time, the enzyme decomposition extract of the gangrene was not added to the negative control. The percentage of cell proliferation inhibition was expressed as a percentage of the decrease in cell proliferation of the group treated with the enzyme hydrolyzed extract of IL-3 and the ganglia relative to the cell proliferation of the group treated with IL-3 only.

Y16 cell line transformed with cDNA encoding the GM-CSF receptor α-chain (provided by Professor Kiyoshi Takatsu, the Institute of Medical Science, Tokyo University of Japan) was dispensed 5 × 10 ⁵ per well into a 96-well plate and mouse GM-CSF 2.5 U / ml and the enzymatic digestion extract of the ingots of Example 1 were added together at concentrations of 1, 3, 10 and 30 µg / ml, respectively, and incubated for 48 hours at 37 ° C. and 5% CO ₂ . Then, the absorbance was measured in the same manner as above. At this time, the enzyme decomposition extract of the gangrene was not added to the negative control. Inhibition rate of the cell proliferation was expressed as a percentage of the cell proliferation of the GM-CSF-treated and enzyme-extracted extracts of the shells as a percentage of the cell proliferation of the GM-CSF-only group.

The experiment was repeated five times independently, and the measured results were expressed as mean ± standard deviation, and the significant difference was verified by Dunnett's test method.

As a result, the proliferation of Y16 cell line was increased by the treatment of IL-5, and the proliferation of this Y16 cell line was dose-dependently inhibited by the enzymatic extract of the ganglia. In other words, when 1 μg / ml of the enzyme was extracted, the percentage of cell proliferation was 21%, 53% when 3 μg / ml was added, and 72%, 30 when 10 μg / ml was added. In the case of adding μg / ml, the proliferation inhibition rate was 87%. The concentration (ED ₅₀ ) showing a 50% growth inhibitory effect was 2.8 μg / ml (FIG. 1A).

In addition, the proliferation of BAF / B03 cell lines was increased by the treatment of IL-3, and the proliferation of these BAF / BO3 cell lines was dose dependently inhibited by the enzymatic extract of the ganglia. In other words, the cell growth inhibition rate was 15% when 1 μg / ml of enzyme extract of the ganglia was added, and 46% when 3 μg / ml was added and 74% when 10 μg / ml was added. , 85% was added when 30 μg / ml was added. ED ₅₀ value was 4.1 μg / ml (FIG. 1B).

In addition, the proliferation of cell lines transformed with cDNA encoding GM-CSF receptor α-chain by GM-CSF was increased and the proliferation of such transformed cell lines was dose dependently inhibited by the enzymatic extract of the ganglia. In other words, when 1 μg / ml concentration of the enzyme was extracted, the cell proliferation inhibition rate was 16%. When 3 μg / ml was added, 32% and 57 μg was added. % And 76 ㎍ / ml was 76%. ED ₅₀ value was found to be 8.3 μg / ml (FIG. 1C).

Therefore, it was found that the extract of the present invention inhibits the activity of Th ₂ cytokines.

<2-2> Eosinophil Activity Inhibitory Effect of Extracts

It was investigated whether the enzyme-decomposed extract of the ganglia according to the present invention has an effect of inhibiting the activity of eosinophils isolated from patients suffering from allergic diseases. The degree of inhibitory activity of eosinophils was analyzed by measuring the decrease in the amount of LTC ₄ released by IL-5 stimulation. LTC ₄ is a kind of leukotriene secreted by activated eosinophils and is a mediator of an inflammatory response.

To this end, eosinophils were first isolated from the blood of patients diagnosed with asthma by negative immunoprecipitation using CD16 antibody. Dissociate eosinophils isolated from asthma patients in 6-well plates 1 × 10 ⁶ per well, and 10U / ml of human IL-5 (Sigma Aldrich) and enzyme extracts of the ingots of Example 1, respectively. , 10, 30 ㎍ / ml was added together and incubated for 2 hours. After the incubation was completed, the supernatant was obtained by centrifugation. The amount of LTC ₄ present in the supernatant was measured by ELISA. Release inhibition of LTC ₄ exhibited a reduction in IL-5 and goegak LTC ₄ bangchulyang of the group treated with the enzyme extract in the ratio of the bangchulyang of the LTC ₄ group treated with IL-5 only as a percentage.

The experiment was repeated three times independently, and the measured results were expressed as mean ± standard deviation, and the significant difference was verified by Dunnett's method.

As a result, the enzyme-decomposed extract of the ganglia according to the present invention was shown to dose-dependently inhibit the release of LTC ₄ of eosinophils isolated from asthma patients by IL-5 stimulation. In other words, when the enzyme-degraded extract of the ganglia was added at a concentration of 1 µg / ml, the release inhibition rate of LTC ₄ was 15%, and when 3 µg / ml was added, 32% and 10 µg / ml were added. When 74% and 30 µg / ml were added, the inhibition rate was 93%. ED ₅₀ value was 5.9 μg / ml (FIG. 2).

Therefore, it was confirmed that the kerchief extract of the present invention inhibits the activity of eosinophils.

<Example 3>

In vivo ( in vivo Investigation of Prevention and Treatment of Allergic Diseases of Necrosis Extracts through Experiments

<3-1> Antiallergic Activity of Extracts from Shells in Animal Models of Egg Yolk Sensitization

In order to confirm whether the extract according to the present invention has the effect of preventing or treating allergic diseases, an animal model of egg yolk sensitization allergy was prepared and the antiallergic activity of the extract was investigated. Antiallergic activity of the ganglia extract was determined by measuring the degree of infiltration of eosinophils into bronchial alveoli in the animal model.

First, an egg yolk sensitizing allergic animal model was prepared by the following method, and after administration of the enzyme-degrading extract of the ganglia, the bronchoalveolar lavage fluid was collected, and the number of eosinophils infiltrated with alveoli was measured by an optical microscope. 6 to 8 weeks old male Balb / c mice (weight 25 to 30 g) were used as experimental animals. 20 μg of egg yolk was dissolved in 100 μl of physiological saline, and then the same volume of Imject Alum complex was added thereto. 200 μl of the yolk solution was injected intraperitoneally of the Balb / c mice. Intraperitoneal injections were performed twice, once each on the first and 14th days of the experiment. 2% egg yolk were sensitized for 2 days on a 28-day and 29-day trial using a nebulizer (neubilizer, Pari-Inhaler Boy) for 30 minutes each day. Then, seven mice were divided into a control group and an experimental group, and on the 30th day of the experiment, 100 mg / kg of the enzyme-degraded extract of the ingot prepared by dissolving the enzyme-dissolved extract of the ingot in Example 1 in physiological saline containing 10% DMSO. After intravenous injection, 2% egg yolk was inhaled for 30 minutes using a nebulizer. The control group was injected with physiological saline containing 10% DMSO used for dissolution of the enzyme decomposition extract of the gangrene.

After the experiment was completed, the thoracic sections of the control and experimental mice were incised, the airways were separated, and the incisions were made to intubate the polyethylene tubes. The bronchial alveolar lavage fluid was collected by gently injecting 1 ml of saline into the airway through the tube and gently massaging the lungs. Then 0.8 ml of saline was injected in the same manner as above and the bronchoalveolar lavage fluid was collected. A total of 1.8 ml of the bronchial alveolar lavage fluid collected above was centrifuged to separate cells, and a slide was prepared using cytospin, an automatic cell smearing device. After staining with Bright-Giemsa, the number of infiltrating eosinophils was calculated as a percentage by measuring the number of cells in a × 1000 magnification field with an optical microscope. In addition, neutrophils and macrophages were also examined by an optical microscope to measure the number of cells.

Experimental results showed that the number of eosinophils infiltrated with bronchoalveolar alveolar was significantly increased in the control group compared to non-sensitized mice. In the experimental group to which the ingot extract according to the present invention was administered, the number of eosinophils infiltrated with bronchoalveolar algae was rapidly reduced (FIG. 3). On the other hand, in the experimental group to which the ganglia extract was administered, the number of macrophages infiltrated into the bronchoalveolar algae was significantly increased compared to the control group (Table 1). These experimental results may play an important role in the study of molecular targets to elucidate the mechanism of action of allergic gangrene extracts.

Anti-allergic Activity According to the Administration of Oyster Extract in Egg Yolk Sensitized Mouse Model Group / Immune Cell Type Eosinophils Neutral sphere Macrophage Etc Control 82% 2% 8% 8% Experimental group 8% 18% 56% 18%

<3-2> Antiallergic Activity of Extracts from Shells by Methacholine Bronchial Induction Test

A methacholine bronchial challenge test was performed to determine whether the ganglia extract inhibited airway hypersensitivity, a hallmark of bronchial asthma. The airway hyperresponsiveness was investigated by measuring the degree of airway resistance caused by airway obstruction.

Sensitized with egg yolk in Example <3-1> Metacholine was divided into 5 levels of 2.5, 6.25, 12.5, 25, and 50mg / ml in the mouse and inhaled for 3 minutes, respectively.The degree of airway resistance was measured using OCP-2000 (one chamber plethylsmography) for 3 minutes. Measured. The pen reflects the degree of airway resistance as an index automatically calculated from the expiration time Ti, the relaxation time RT, the maximum exhalation flow rate PEF, and the maximum intake flow rate PIF, which are automatically measured by the OCP-2000.

The experiment was repeated two times independently, and the measured results were expressed as the mean standard deviation and the significant difference was verified by Dunnett's test method.

As a result, the control mice showed that the degree of airway resistance increased depending on the dose of inhaled methacholine. On the other hand, in the experimental group administered with the extract according to the present invention it was shown that the degree of airway resistance induced by methacholine is significantly reduced compared to the control group. In particular, in the experimental group inhaled 12.5 to 50 mg / ml of methacholine, the gangrene extract of the present invention was shown to suppress airway resistance by 65% compared to the control (Fig. 4).

Therefore, it was confirmed that the extract of the present invention is an antiallergic activity.

<Manufacture example 1>

Manufacture of beverages

According to the conventional beverage preparation method was mixed with the ingredients as shown in Table 2 and then heated with stirring at 85 ℃ for about 1 hour and then prepared by filtering the sterilized solution to prepare a beverage containing a lump extract.

Beverage composition containing oyster extract ingredient content Oyster Extract of Example 1 1000mg (0.1%) Citric acid 1000mg (0.1%) oligosaccharide 100g (10%) Taurine 1000mg (0.1%) Purified water Add up to 1000 ml total volume

<Manufacture example 2>

Manufacture of soap

A solid soap composition was prepared by mixing 99.5% by weight of a soap base (including water) and 0.5% (w / w) of the ingot extract prepared in Example 1 with a mixer and then extruding, cutting, and moldting in a soap maker.

<Manufacture example 3>

Manufacture of cream

The cream base 40g containing 1.5 g of stearic acid, 2.2 g of stearyl alcohol, 0.5 g of stearate, 0.5 g of propylene glycol, 0.5 g of propylene glycol, 2.0 g of monostearate, 0.3 g of potassium hydroxide, an aqueous component, a surfactant, etc. The lump extract prepared in Example 1 was mixed at a concentration of 0.5% (w / w) to emulsify well in a mixer, degassed, filtered and cooled to prepare a cream composition. A chelating agent, flavoring agent and pigment were added to the composition as an additive and the formulation was prepared in oil / water so that a small amount of oily ingredient was present.

As described above, the ingot extract according to the present invention inhibits the activity of Th ₂ cytokines and eosinophils causing allergy, inhibits infiltration of eosinophils into bronchoalveolar alveoli and inhibits airway hypersensitivity reactions by methacholine. It is very useful for preventing or improving allergic diseases.

Claims (5)

      Food composition for the prevention or improvement of allergic diseases containing the enzyme-enzyme extract of the gangster obtained by extracting the gangbang with water or alcohol having 1 to 6 carbon atoms and then treating amylase or pectinase. The food composition according to claim 1, wherein the water extract of the lump is manufactured by hot water extraction. The food composition of claim 1, wherein the allergic disease is selected from the group consisting of allergic dermatitis, allergic rhinitis, allergic asthma, allergic otitis media, anaphylatic shock, and allergic conjunctivitis. The food composition according to claim 3, wherein the allergic dermatitis is atopic dermatitis, psoriasis, contact allergic dermatitis and urticaria. A cosmetic composition for the prevention or improvement of allergic diseases, comprising as an active ingredient the enzyme decomposition extract of the shell obtained by extracting the shell with water or alcohol having 1 to 6 carbon atoms and then treating with amylase or pectinase.

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