KR20170054314A - Composition comprising extract of Eleutherococcus sessiliflorus fruits for prevention and treatment of allergic diseases or inflammatory diseases - Google Patents

Abstract

The present invention relates to a composition for prevention and treatment of allergic diseases or inflammatory diseases, comprising an extract of Eleutherococcus sessiliflorus fruits as effective ingredients, and more specifically, to a pharmaceutical composition and health functional food for prevention and treatment of allergic diseases or inflammatory diseases, comprising an extract of Eleutherococcus sessiliflorus fruits as effective ingredients. The extract of Eleutherococcus sessiliflorus fruits inhibits release of beta-hexosaminidase, inflammatory cytokines and IgE, thereby being effective in treatment of inflammatory or allergic diseases, and being effective due to low cytotoxicity even at a high concentration.

Description

[0001] The present invention relates to a composition for preventing and treating allergic diseases or inflammatory diseases,

The present invention relates to a composition for the prevention and treatment of allergic diseases or inflammatory diseases comprising an oregano extract as an active ingredient, and more particularly to a composition for preventing or treating allergic diseases or inflammatory diseases containing an oregano extract as an active ingredient Compositions and health functional foods.

When foreign substances come into the body from the outside of the body, an immune reaction that protects our body from these bodies occurs, which is called a normal immune response. However, when the immune response is excessive, an allergic reaction is caused by hypersensitivity reaction that causes physical abnormality, and when a specific mechanism of hypersensitivity causes an abnormality in our body.

The cause of the allergic reaction is caused by the immune system of the human body reacting to external substances acting as allergen, allergen. Allergens are presented on the surface of cells by antigen presenting cells, which are recognized by B lymphocytes and converted to plasma cells, releasing antibodies (IgE) that specifically recognize the antigen.

Allergic diseases are caused by repeated immune reactions to allergens in memory cells, and IgE binding to antigen binds to receptors of mast cells (FcεRI) of tissues, leading to degranulation of mast cells . The degranulation of mast cells secretes histamine, heparin, leukotrienes, prostaglandine and other anaphylatic agents. These secreted immune activation factors activate the immune system and cause inflammation and allergic reactions.

Hypersensitivity reactions can be classified into four types. Type 1 hypersensitivity reactions, commonly referred to as allergic reactions, are reactions that occur to specific antigens and are induced by binding IgE antibodies specific for that antigen to mast cells or basophils.

When allergen-specific IgE antibodies are produced, these antibodies bind to FcεRI receptors on the surface of mast cells or basophils, and allergens bind to these antibodies to activate these cells, secrete various physiologically active substances, Expansion, increased penetration of blood vessels, smooth muscle contraction, and inflammatory response.

These reactions may occur locally or systemically depending on the type and amount of mediators produced by these cells. Specific allergic diseases include atopic dermatitis, rhinitis, and asthma.

Mast cells are very important in various allergic diseases such as atopic dermatitis, asthma, allergic rhinitis and rheumatoid arthritis. In contrast to other cells, mast cells contain large and coarsely stained granules. When IgE is stimulated by an antigen in the state of being bound to FcεRI (high-affinity receptor for IgE) of mast cells, It is involved in the inflammatory reaction.

Thus, mast cells may be involved in a variety of physiological, immunological, and pathological processes that occur during allergic reactions, such as wound healing, angiogenesis, host response to parasites and neoplasms, and chronic inflammatory and IgE mediated immediate allergic responses .

The mediators secreted during mast cell activation in allergic reactions include histamine and 5-HT (serotonin), biogenic amines that cause vasodilatation and pain, and IL-1, 4, 6, 10 , 13, MIF, and TNF to induce inflammation and leukocyte migration and proliferation.

In addition, by secretion of the phospholipid metabolites leukotrien B4, LTB4, leukotrien C4, LTC4, platelet activating factor (PAF), prostaglandin D2 (prostaglandin D2, PGD2) Leukocyte chemotaxis, airway constriction and pain.

In addition, there is a growing interest in the use of enzymes such as chymase and tryptase to induce tissue damage, pain, angiotensin II synthesis, corticotropin-releasing hormone (CRH), endorphin endothelin, kinins, somatostatin substance P, SP, vasoactive intestinal peptide (VIP), urocortin, vascular endothelial growth factor (VEGF) vascular endothelial growth factor (VEGF), and the like, to induce a series of allergic reactions such as inflammation, inflammation, pain, vasodilation, and airway constriction.

The allergic disease drugs developed so far are predominantly antagonistic to histamine and leukotriene receptors secreted by allergens in mast cells. However, since these drugs show tolerance within a short period of time after administration, most of the patients are symptomatic to improve their symptoms, not only to prevent the cause of the disease but also to have side effects due to medicines (Rabe KF, et al ., Eur Respir J Suppl., 34: 34s-40s, 2001).

On the other hand, wood senticosus (Eleutherococcus sessiliflorus (Rupr. & Maxim.) SYHu or Acanthopanax sessiliflorus (Rupr. & Maxim. ) Seem.) is a deciduous broad-leaved shrub with senticosus hawthorn (Eleutherococcus) plant belonging to Araliaceae (Araliaceae), the key is 34cm and the kind of near the roots Leaves are alternate phyllotaxis, umbilical leaflets, and lobules (oval) are ovate with 35 eggs.

It is reported that the phytopathogenic fungi are excellent in pharmacological efficacy in the classical medicinal herbs ranging from Oriental herb medicine to Oriental medicinal herbs, medicinal herbs, medicinal herbs, neurotoxins, and diabetes. Recently, it has been scientifically proved that it has blood pressure lowering action, liver function protecting action, hematopoietic promotion and immune function increasing action, blood cholesterol lowering action, antioxidant action, antiallergic action and anti- There is a growing trend of interest and use in many areas, including.

Among them, Ogaki (fruit) It is known that the fruit of Ogaki is the effect of protecting the liver, improving the learning power, improving the immunity, anticancer activity, lowering of cholesterol, and treatment of gastric ulcer. However, the inhibitory effect of β-hexosaminidase on the extract of fruit juice has not been reported.

Accordingly, the present inventors confirmed the atopy treatment effect, the skin inflammation inhibiting effect and the anti-allergic effect of the extract of Orgama, and completed the present invention.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of an allergic disease or inflammatory disease comprising an oregano extract as an active ingredient.

It is still another object of the present invention to provide a health functional food for preventing or ameliorating allergic diseases or inflammatory diseases comprising an extract of Orgama as an active ingredient.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing or treating an allergic disease or an inflammatory disease comprising an oregano extract as an active ingredient.

According to another preferred embodiment of the present invention, the extract is selected from the group consisting of water, C ₁ -C ₄ alcohol, hexane, ethyl acetate, butylene glycol, propylene glycol, glycerin, acetic acid ethyl ether, chloroform, And the like can be extracted with any one of the extraction solvents selected from the group consisting of

According to another preferred embodiment of the present invention, the extract may be obtained by immersing the dried oregano in an extraction solvent at room temperature or extracting it using Accelerated Solvent Extraction (ASE).

According to another preferred embodiment of the present invention, the extract may be contained in the composition at a concentration of 0.1 to 50 mg / ml.

According to another preferred embodiment of the present invention, the above-mentioned allergic diseases include allergic rhinitis, allergic conjunctivitis, allergic asthma, anaphylactic shock, food allergies, hay fever, drug allergies, plant allergies, urticaria, eczema and allergic dermatitis Lt; / RTI >

According to another preferred embodiment of the present invention, the inflammatory disease may be a skin inflammatory disease, and more preferably, it is atopic dermatitis.

According to another preferred embodiment of the present invention, the Ogaja extract has an activity of inhibiting the secretion of beta -hexosaminidase and inhibiting inflammatory cytokines and immunoglobulin E Lt; / RTI > Preferably, the inflammatory cytokine is IL-4 (interleukin 4) or TSLP (thymic stromal lymphopoietin).

The present invention also relates to a pharmaceutical composition containing an oregano extract as an active ingredient And health functional foods for the prevention and improvement of allergic diseases or inflammatory diseases.

According to another preferred embodiment of the present invention, the above-mentioned allergic diseases include allergic rhinitis, allergic conjunctivitis, allergic asthma, anaphylactic shock, food allergies, hay fever, drug allergies, plant allergies, urticaria, eczema and allergic dermatitis Lt; / RTI >

According to another preferred embodiment of the present invention, the inflammatory disease is skin inflammatory disease or atopic dermatitis. Or an inflammatory disease.

Accordingly, the present invention provides a composition for prevention and treatment of allergic diseases or inflammatory diseases, which comprises an oregano extract as an active ingredient, and a health functional food. The present extract of the present invention is effective in inhibiting beta-hexosaminidase release, inhibiting inflammatory cytokines and IgE, and being effective in the treatment of inflammatory diseases or allergic diseases, and low in cytotoxicity even at high concentrations.

FIG. 1 is a graph showing inhibition of cell proliferation at a concentration of 2, 10 and 50 μg / ml of Ogata extract.
FIG. 2 is a graph showing the inhibition of beta-hexosaminidase secretion at the concentration of 2, 10 and 50 μg / ml of Ogata extract.
FIG. 3 is a graph showing the inhibitory effect of IL-4 on the secretion of Ogata extract as an OD value in comparison with the positive control substance R406.
Fig. 4 is a graph showing the inhibitory effect of IL-4 on the secretion of Ogawa extract as a percentage compared with the positive control substance R406.
FIG. 5 is a graph showing the cytotoxicity test of the extract of Orgama on HaCaT cells, which are keratinocytes.
FIG. 6 is a graph showing the cytotoxicity of Jurcat T cells derived from an acute lymphocyte-derived peripheral blood-derived T cell line.
FIG. 7 is a graph showing changes in the level of mRNA expression of TNF-.alpha. In HaCaT cells stimulated with allergic stimulants according to the extract of Orgama.
FIG. 8 is a graph showing changes in mRNA expression level of IL-2 according to the extract of Orgaca in Jurcat T cells stimulated with allergic stimulants.
FIG. 9 is a graph showing the number of scratching actions of a mouse when applied to an atopy-induced mouse.
FIG. 10 is a graph showing the amount of inflammatory cytokine IL-4 released from the skin tissue of mice when the extract was applied to atopy-induced mice.
FIG. 11 is a graph showing IgE in serum of mice when they are applied to atopy-induced mice.
Fig. Fig. 2 is an electrophoresis photograph and graph showing the effect of TSLP reduction of atopic skin tissue of the extract.

Hereinafter, the present invention will be described in more detail.

As described above, the inhibitory effect of Beta-hexosaminidase release on the extract of Ogaki nuts (Ogaja) has not been reported so far.

The present extract of the present invention is effective in inhibiting beta-hexosaminidase release, inhibiting inflammatory cytokines and IgE, and being effective in the treatment of inflammatory diseases or allergic diseases, and low in cytotoxicity even at high concentrations.

The Ogaja of the present invention refers to the fruit of Ogaki, and the Ogphi has the tongue such as Ogphi, Ogalli, Seoul Ogphi, and Seoul Ogphi. The term " orza " used in the present invention refers to all of the fruits of plants belonging to the ocarob.

Preferably, the ogphi of the present invention is any one selected from the group consisting of a hairy eucalyptus, a thorny oak, a seagrass, an oak tree, an oak tree, a jiric acid oak, a short oak tree, Or more.

Eleutherococcus divaricatus , and Acanthopanax senticosus Eleutherococcus sessiliflorus ( Rupr. et Maxim.) SY Hu or Acanthopanax sessiliflorus (Rupr. et Maxim.) Seem., Seoul ogphi is represented by Eleutherococcus seoulensis or Acanthopanax seoulense Nakai, and the eucalyptus tree is Eleutherococcus gracilistylus (WWSm.) SYHu or Acanthopanax can be expressed as koreanum Nakai. In addition, visibility is Eleutherococcus senticosus Senticosus (Rupr. & Maxim.) Max ., Acanthopanax senticosus ( Rupr. & Maxim.) Harms. Or Acanthopanax senticosus for. inermis., and the Jirisan aquapi can be represented by Eleutherococcus There is divaricatus . chiisanensis (Nakai) CHKim & BYSun or Acanthopanax can be represented by chiisanensis Nakai, minor senticosus trees can be represented as Acanthopanax sessilitlorus, per tree is Acanthopanax senticosus siebololianum , and the royal palm tree is Acanthopanax I have senticosus . koreanus. , And the hair coat can be represented as Acanthopanax rufinerve Nakai.

More preferably, the ogulf of the present invention may be an islet oak tree, an oak tree, or an oak tree.

The present invention provides a pharmaceutical composition for preventing or treating an allergic disease or an inflammatory disease, which comprises an oregano extract as an active ingredient.

The inventive extract of the present invention is preferably but not limited to be produced by a manufacturing method comprising the following steps:

1) extracting the extract by adding an extraction solvent;

2) filtering the extract of step 1); And

3) Concentrating the filtered extract of step 2) under reduced pressure and drying.

The present invention relates to the above-mentioned extract, wherein the extract is selected from the group consisting of water, a C ₁ -C ₄ alcohol, hexane, ethyl acetate, butylene glycol, propylene glycol, glycerin, acetic acid ethyl ether, chloroform, And is extracted with one extraction solvent. Preferably, water, a lower alcohol of a C ₁ to C ₄ alcohol, or a mixed solvent thereof may be used, and more preferably, any one selected from the group consisting of water, methanol, ethanol, It can be extracted with an extraction solvent.

In the above method, the extraction method of the organs extract may be a conventional method in the art such as filtration, hot water extraction, immersion extraction, room temperature extraction, accelerated solvent extraction, reflux cooling extraction and ultrasonic extraction, Extraction, room temperature extraction or accelerated solvent extraction. The accelerated solvent extraction method is a method of extracting the accelerated solvent at a high rate at a high temperature and a high pressure by adding an organic solvent and an extraction solvent to the accelerated solvent extraction apparatus. The yield of the organic solvent is increased because the structural change of the useful active ingredient in the organic solvent is minimized, Can be increased.

The extraction solvent is preferably extracted by adding 2 to 20 times the amount of the dried oregano. The extraction temperature is preferably 20 to 80 ° C, but is not limited thereto. More preferably, when the extract is prepared by the filtration method, the immersion extraction method and the room temperature extraction method, the extraction temperature is preferably 20 to 40 ° C. When the extract is prepared by the accelerated solvent extraction method, the extraction temperature is 45 to 60 ° C .

When the extract is prepared by the above-described immersion extraction and room temperature extraction method, the extraction time is preferably 10 to 100 hours, more preferably 24 to 96 hours, most preferably 24 to 36 hours But is not limited thereto.

In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.

In addition, the extract of the present invention has no toxicity and side effects. In one embodiment of the present invention, the cell proliferation of Ogaja extract was confirmed, and it was found that the cell proliferative activity was 80% or more at a concentration of 50 μg / ml and showed little or no toxicity and side effects.

Thus, the above extract may preferably be contained in the composition at a concentration of 0.1 to 50 占 퐂 / ml.

The allergic diseases of the present invention may be selected from the group consisting of allergic rhinitis, allergic conjunctivitis, allergic asthma, anaphylactic shock, food allergy, hay fever, drug allergy, plant allergy, urticaria, eczema, have.

The inflammatory disease may be a skin inflammatory disease, and more preferably, it is atopic dermatitis.

The above extract of the present invention may be one having an activity of inhibiting secretion of beta-hexosaminidase and inhibiting inflammatory cytokine and immunoglobulin E. Wherein the inflammatory cytokine may be IL-4 or TSLP.

According to FIG. 2 or Example <2-2> of the present invention, it can be confirmed that the secretion of beta-hexosaminidase is suppressed when the extract is treated with the extract, and most preferably, the extract of Orgama extract has a concentration of 50 μg / Concentration, it was confirmed that it was suppressed to the highest level.

3 and 4 or Example 2-3 of the present invention, it was confirmed that the extract of Orgama was superior to the positive control group in inhibiting IL-4 secretion.

According to Figs. 5 and 6 or Example < 3-1 > of the present invention, the Ogaya extract prepared under various extraction conditions exhibited a high cell proliferation rate on dermal keratinocytes and T-cell derived from pseudo- .

According to Fig. 7 and Fig. 8 or Example < 3-2 > of the present invention, the extract of Ogaya produced under various extraction conditions showed that TNF-alpha and IL-2, which are cytokines inducing inflammation in cells stimulated with allergic stimulants Allergic effects of the compounds of the present invention.

According to FIG. 9 of the present invention, it was confirmed that the number of scratches was significantly reduced when the extract was applied to an experimental animal.

According to Fig. 10 and Fig. 12 of the present invention, IL-4 inflammatory cytokine and TSLP cytokine were observed to be low in the skin tissue of the atopy-induced mouse coated with the Ogata extract. According to Fig. 11 of the present invention, It was confirmed that IgE was low in plasma of one mouse.

The pharmaceutical composition containing the extract according to the present invention can be administered orally in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions And can be used as formulations. Examples of carriers, excipients and diluents that can be included in the composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

The preferred dosage of the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The extract of the present invention can be administered to mammals such as rats, mice, livestock, humans and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention provides a health functional food for preventing or ameliorating an allergic disease or an inflammatory disease comprising an oregano extract as an active ingredient.

The allergic diseases of the present invention may be selected from the group consisting of allergic rhinitis, allergic conjunctivitis, allergic asthma, anaphylactic shock, food allergy, hay fever, drug allergy, plant allergy, urticaria, eczema, And can be preferably allergic dermatitis or atopic dermatitis.

The inflammatory disease may be a skin inflammatory disease, and more preferably, it is atopic dermatitis.

The above extract of the present invention may be one having an activity of inhibiting secretion of beta-hexosaminidase and inhibiting inflammatory cytokine and immunoglobulin E. Wherein the inflammatory cytokine may be IL-4 or TSLP.

Examples of foods to which the extract can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, tea, vitamin complexes and health functional foods. It can also be added to foods or beverages for the purpose of preventing hypertension.

At this time, the amount of the extract in the food or drink may be 0.01 to 15% by weight of the total food, and the health beverage composition may be added in a proportion of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 ml .

The health functional beverage composition of the present invention has no particular limitation on the other ingredients other than the above-mentioned extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract of the present invention can also be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and aging agents (cheese, chocolate, etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the extracts of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Preparation of experimental material

&Lt; 1-1 > Preparation of Extract of Ogata Extract at Room Temperature

Eleutherococcus used in the experiment sessiliflorus (Rupr. & Maxim.) SYHu or Acanthopanax sessiliflorus (Rupr. & Maxim.) Seem. fruits were purchased from the market in 2012. The dried ores were crushed and immersed in water, ethanol or ethanol aqueous solution of 10 times the above amount according to the conditions described in [Table 1] below, and after three times of extraction for 24 hours, the solvent was removed using a freeze dryer, The extract was obtained. The amount of extract (g) and the extraction yield (%) were confirmed to be as shown in Table 1 below.

The extraction conditions and extract yields of the Ogaya extract prepared in the present invention Sample number Extraction condition Sample weight (g) Amount of extract (g) yield(%) Collection place One 100% ethanol extraction at room temperature 200 18.10 9.1 Jecheon 2 70% Ethanol Extraction at room temperature 200 41.29 20.6 Jecheon 3 50% Ethanol Extraction at room temperature 200 60.04 30.0 Jecheon 4 30% ethanol extract at room temperature 200 55.96 28.0 Jecheon 5 Room temperature and water extraction 200 51.05 25.5 Jecheon 6 100% ethanol ASE extraction 179.67 12.82 7.1 Jecheon 7 70% ethanol ASE extraction 209.82 44.47 21.2 Jecheon 8 30% Ethanol ASE Extraction 72.47 22.92 31.6 Jecheon 9 50% Ethanol ASE Extraction 414.75 103.11 24.9 Jecheon 10 100% ASE extraction 200 51.05 25.5 Jecheon

<1-2> Preparation of Accelerated Solvent Extraction Ogata Extract

Eleutherococcus sessiliflorus (Rupr. & Maxim.) SYHu or Acanthopanax sessiliflorus (Rupr. & Maxim.) Seem. Fruits) were purchased from the market in 2012. The dried orega was pulverized and then added to an accelerated solvent extraction apparatus (ASE) under normal pressure and room temperature conditions. Then, water, ethanol or ethanol aqueous solution of 10 times the amount of the above organism was subjected to ASE , Extracted at 50 DEG C / high pressure, and then the solvent was removed using a freeze dryer to obtain an extract at room temperature or ASE extract. The amount of extract (g) and the extraction yield (%) were confirmed to be as shown in Table 1 above.

<1-3> Culture of cells

The RBL-2H3 cell line used in the experiment was purchased from Korean Cell Line Bank (KCLB), and the cells were cultured in RPMI-1640 supplemented with 15% fetal bovine serum (FBS), 100 mM sodium pyruvate, Well plate at 2.5 ⁵ cells / well in a 5% CO ₂ incubator at 37 ° C.

HaCa T cells and Jurkat T cells (both purchased from Korean Cell Line Bank), which are keratinocytes, were cultured in RPMI640 medium containing 10% fetal bovine serum, 100 IU / ml penicillin G, 100 ug / ml streptomycin and 2 mM L- Lt; / RTI &gt; Each cell line was cultured in 12-well, 6-well or 60-mm petri dishes in accordance with each experiment, corresponding to the log-phase.

Anti-allergic effects of Ogaja extract in mast cells

<2-1> Evaluation of cytotoxicity using MTT method

After the mast cells prepared in Example <1-3> were cultured, the water extracts prepared at room temperature or ASE prepared in Example <1-1> were treated with mast cells for 24 hours, and as a negative control, dimethyl sulfoxide (DMSO) was added. After incubation for 3 days at 37 ° C in a 5% CO 2 incubator for 4 h, the cells were cultured in a 5% CO 2 incubator at 37 ° C for one day. To measure cytotoxicity, 200 μg / ml of MTT (USB, Cleveland, Lt; / RTI &gt; After removing the MTT solution, 100 μl of DMSO was added and the absorbance was measured at 550 nm using a Biotek Synergy HT plate reader (Biotek, Winooski, VT, USA).

As a result, as shown in FIG. 1, the Ogaya extract tended to decrease cell proliferation in proportion to the treatment concentration, but showed a relatively high cell proliferation of about 120% even at the treatment concentration of 50 ㎍ / ㎖. Based on these results, it was confirmed that Ogaja extract showed low cytotoxicity at a relatively high concentration.

<2-2> Measurement of release of beta-hexosaminidase related to degranulation of mast cells

Allergic reactions are a type of inflammatory disease that occurs when mast cells are degranulated, releasing histamine. It is known that histamine is synthesized and stored in mast cells and has a great influence on acute inflammatory reaction. Beta-hexosaminidase is an enzyme which coexists in histamine and mast cell and is known to be proportional to the amount of histamine released by degranulation And is used as an index of the anti-allergy effect confirmation. Thus, in order to confirm the antiallergic reaction of the Ogaya extract, the release amount of beta-hexosaminidase was measured.

After mast cells were cultured, the cells were replaced with Tyrode's buffer and 200 ng / μl of DNP-IgE (Sigma-Aldrich, St. Louis, Mo., USA) was added and cultured at 37 ° C for 24 hours. Then, 50 ng / DN of DNP-BSA (Sigma-Aldrich, St. Louis, Mo., USA) was added to the aqueous extract prepared in Example <1-1> And cultured at 37 ° C. Then the degranulation reaction was stopped on ice. 25 μl of the supernatant and 100 μl of 2.4 mM 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (Sigma-Aldrich, St. Louis, Mo., USA) were mixed on a 96-well plate and incubated for 1 hour at 37 ° C. Then, 175 μl of 0.1 M glycine-carbonate buffer (pH 10.0) was added to stop the reaction and fluorescence was measured (ex: 360 nm em: 450 nm) using a Biotek Synergy HT plate reader (Biotek, Winooski, Respectively.

As a result, as shown in FIG. 2, the inhibitory effect of Beta-hexosaminidase was shown to be 8% in the test group treated with 2 ㎍ / ㎖ of Ogaya extract and in the test group treated with 10 ㎍ / %. On the other hand, the experimental group treated with 50 ㎍ / ㎖ of Ogaya extract showed about 20% inhibition of beta - hexosaminidase release and exhibited excellent beta - hexosaminidase inhibitory effect.

<2-3> Measurement of the amount of IL-4 cytokine production associated with allergic response

Overproduction of cytokine IL-4 is associated with allergic disease. Thus, in Example 4, changes in the amount of IL-4 cytokine produced by the Ogata extract treatment were measured.

To each of the mast cells prepared in Example <1-3>, 100 ㎕ of blank control, each standard substance (positive control R406) and 100 쨉 l of the room temperature water extract prepared in Example <1-1> 50 μl of 1 × anti-IL-4 was added to all wells, followed by incubation at room temperature (18 to 25 ° C.) for 4 hours. After removing the cover and washing the plates several times, 100 μl of 1 × solution was added, re-covered at room temperature for 30 min, and incubated and washed. 100 [mu] l of chromogen TMB substrate solution was added to each well, and the plate in the aluminum foil was wrapped and incubated at room temperature for 10 to 20 minutes in a dark place. 100 μl of stop reagent was added to each well and the absorbance was immediately measured at 450 nm using a Biotek Synergy HT plate reader (Biotek, Winooski, VT, USA).

As a result, as shown in FIG. 3 and FIG. 4, it was confirmed that the extract of Orgama was superior to the positive control substance R406 in inhibiting IL-4 secretion.

Extract of Ogawa from keratinocytes Anti-allergy  Check the effect

<3-1> Evaluation of cytotoxicity using MTT method

Specifically, HaCaT cells or Jurkat T cells were inoculated into a 48-well plate in DMEM medium containing 10% FBS and 1% antibiotic (ampicillin, streptomycin) at a concentration of 5 × 10 ⁵ cells / , And cultured in a 5% CO2 incubator at 37 ° C for 24 hours. After 24 hours, the extracts obtained by extracting at room temperature or accelerated solvent prepared in Examples <1-1> and <1-2> were treated at a concentration of 20 μg / ml and further cultured for 24 hours. After incubation, 100 쨉 l of 0.1% MTT (Sigma M2128) solution was added to each well, followed by incubation for 3 hours at 37 째 C in a 5% carbon dioxide incubator. After removing the culture solution and MTT solution, 100 μl of DMSO was added and the absorbance was measured at 540 nm using a Biotek Synergy HT plate reader (Biotek, Winooski, VT, USA).

As a result, as shown in FIG. 5 and FIG. 6, it was confirmed that the extract of Ogaya produced with various solvents and extraction conditions exhibited a growth rate of 90% or more on keratinocytes. In the case of HaCaT cells, it was confirmed that all the extracts showed high cell proliferation rate of 91% or higher. Especially, when extracting with water as a solvent in the accelerated solvent extraction apparatus (ASE), the highest proliferation effect of 105.6% (Fig. 5). Jurkat T cells, a T cell line derived from acute lymphocyte-derived peripheral blood, showed a high cell proliferation rate of 94% or higher in all groups treated with Ogana extract. Especially, the cells were extracted with 70% ethanol aqueous solution at room temperature And the extract showed a cell proliferation rate of 99.6% (Fig. 6).

<3-2> Identification of anti-allergic activity of Ozai extract by confirmation of TNF-α and IL-2 mRNA expression levels

In order to examine whether or not the extract of the present invention can exhibit an anti-allergic effect on dermal keratinocytes that have undergone an allergic stimulation reaction in addition to mast cells mediating induction of allergic responses, the expression of TNF-a and IL- 2 &lt; / RTI &gt;

Specifically, HaCaT cells or Jurkat T cells, which are keratinocytes, were each divided into 6-well plates at a concentration of 2 × 10 ⁶ cells / well, and then allergic stimulants were added to each cell culture medium. As stimulants for stimulating HaCaT cells, TNF-α and INF-γ (both R & D systems, USA) were added. As stimulants for stimulating Jurlat T cells, 12-myristate 13-acetate (PMA) and A23187 Sigma, USA) was added to the medium. After culturing the cells for 24 hours, the extracts obtained by extracting at room temperature or accelerated solvent prepared in Examples <1-1> and <1-2> were respectively treated at a concentration of 20 μg / ml and further cultured for 4 hours .

After incubation, the cells were harvested, chloroform added, vortexed for 20-30 seconds and centrifuged at 15,000 rpm for 15 minutes at 4 &lt; 0 &gt; C. After centrifugation, the uppermost layer of the three-layered sample was transferred to a new tube, and an equal amount of isopropanol was added. The mixture was allowed to stand at room temperature for 10 minutes and then centrifuged at 15,000 rpm for 20 minutes at 4 ° C. The separated supernatant was discarded and only the precipitate was obtained, washed with 75% aqueous ethanol solution containing nucleic acid hydrolase inhibitor, and then centrifuged at 13,000 rpm for 5 minutes at 4 ° C. The separated supernatant was again removed, and the obtained precipitate was dissolved in distilled water containing a nucleic acid hydrolase inhibitor (DEPC) to extract mRNA from the cells. Extracted mRNA was stored at -80 ℃ and total RNA concentration was determined by dissolving in 0.1% DEPC aqueous solution treated with RNase and measuring absorbance at 260 ㎚ / 280 ㎚ by UV spectrophotometer. 1 μg of mRNA was extracted and mixed with 100 pmole oligonucleotide dT primer and Accupower ^ⓡ RT PreMix (Bioneer Co., Korea), and the reaction volume was adjusted to 20 증 with distilled water. Min, 42 ° C for 1 hour, and 80 ° C for 15 minutes to reverse-transcribe the cDNA.

A ^{1 ㎍ cDNA, Accupower ⓡ RT PreMix} (Bioneer Co, Korea) , and the forward / reverse primer 10 pmol mixing TNF-α or IL-4 mRNA sequences to prepare a PCR reaction and the PCR reaction respectively with the synthesized cDNA as a template, Respectively. The amplified PCR products were quantitatively analyzed by ImageJ 1.44d program using the thickness and brightness of the band by electrophoresis on 1% agarose gel.

In addition, real-time PCR was performed for real-time quantitative analysis of mRNA expression levels. The 2 × PCR master mix and forward / reverse primer of DyNAmo SYBR Green qPCR kit (TaKaRa Bio Inc., Japan) were mixed using the reverse transcription-synthesized cDNA as a template, and DNA Engine Opticon (MJ Research, USA) PCR amplification was performed. The levels of TNF-α and IL-2 expression were corrected based on the expression level of GAPDH in each sample group for correction for relative DNA quantitation. The expression levels of TNF-α and IL-2 in the extract-treated group were compared with those of the solvent-treated group, and the levels of relative gene expression were compared with those of the solvent control group Respectively. As a negative control group (NC), allergic stimulants were treated and the extracts were not treated.

As a result, as shown in FIG. 7 and FIG. 8, it was confirmed that the extract of Ogaya produced with various solvents and extraction conditions significantly inhibited the expression levels of TNF-α and IL-2 in keratinocytes. In the case of HaCaT cells, it was confirmed that all the extracts of Ogaya inhibited TNF-α mRNA expression in the range of -2.9% to 94.9%. Especially, in the case of the Ogaya extract prepared by using 30% ethanol aqueous solution at room temperature, And the expression of TNF-α was 94.4%. When compared with the solvent control (CON), the solvent control showed inhibition of TNF-α expression at a level of 93.9% as compared with the negative control. Thus, the extract of the present invention showed TNF- alpha expression level (Fig. 7). In addition, when extracted with 100% ethanol as a solvent, the expression level of TNF-α was 51.7% as compared with the negative control, and when extracted with 100% ethanol at room temperature, the expression level of TNF-α was 24.7% , Indicating that it exhibits a relatively excellent TNF-a expression inhibiting effect (Fig. 7).

In addition, in the case of Jurkat T cells, it was confirmed that the extract of all organs suppressed the expression of IL-2 mRNA in the range of 3.5 to 34.8%, and that of the extract of water extracted with water at room temperature was 34.8% 2 expression in the cells. In addition, when extracted with 50% ethanol aqueous solution as the solvent, the expression level of IL-2 was 33.6% in ASE, 33.3% in the extract of 30% ethanol aqueous solution and 33.3% % Ethanol aqueous solution was inhibited at 30.8% level in the Ogaja extract, which showed a relatively excellent inhibitory effect on IL-2 expression (FIG. 8).

In vivo extract of Ogawa Anti-allergy  Verify Active

<4-1> Preparation of experimental animals and manufacture of atopy model group

NC / Nga mouse was used as an experimental animal for atopic experiment, and it was adapted to the experimental environment for more than one week prior to the experiment. The incubation room maintained a light cycle of 12 hours and a room temperature of 23 ° C. The feed was fed sterilized solid feed purchased from Korea BioLink and water sterilized by ultraviolet rays was supplied.

Atopic induction was induced by using 4% SDS (sodium dodecyl sulfate) to induce skin barrier disruption 24 hours after epilating the neck and back and 3 hours after using house dust mite. The water extract prepared at the room temperature prepared in Example <1-1> was dissolved in 70% ethanol at a concentration of 1% and 5%, and applied to the skin every morning after preparation.

Experiments with the Ogaja extracts on the atopy - induced animal models were divided into two groups. First, as the first experiment condition, atopy induction was induced by applying 50 ㎎ of 15 times for 55 days twice a week. The extract was applied to the skin 6 times a day for 22 days from 29 days after the onset of atopy was confirmed Respectively. As a second experimental condition, atopy induction was induced by applying 50 ㎎ of a total of 4 times for 14 days twice a week. Ogaja extract was applied to the skin for 13 days (6 times / week) every morning in parallel with atopy induction.

<4-2> Measurement of number of scratches of experimental animals

The number of scratches was measured in order to confirm the improvement effect of Ogaya extract on animal behavior in mice coated with Ogaja extract for 22 days while inducing atopy for 55 days. Animal specific behavior measurement system (METRIS, LABORAS, The Netherlands) was used as the measurement equipment.

As a result, as shown in Fig. 9, the number of scratches was two times higher in a mouse (NC) causing atopy than a normal mouse. On the other hand, the number of scratches was significantly decreased in the mice coated with the extract of Ogaja than in the negative control group (NC) induced with atopy. The number of scratches was significantly decreased when the extract was applied at a concentration of 5% than when the extract was applied at a concentration of 1%. On the other hand, it was confirmed that the Ogaja extract at a concentration of 5% showed better effects than the tacrolimus ointment used as a positive control, and thus it was confirmed that the extract of Ogaja of the present invention is effective for the treatment of atopy.

<4-3> Reduction of inflammatory cytokine IL-4 in mouse skin tissue induced by atopy of Ogaja extract

The atopy-induced mouse prepared in Example <4-1> was coated with the aqueous extract prepared at the room temperature prepared in Example <1-1> according to the second experimental condition. The skin tissue was then removed to prepare a homogenate of the dermal tissue. 300 mg of depilated mouse skin tissue was suspended in 1.6 ml of Tissue homogenate buffer (10 mM HEPES, pH 7.8, 10 mM KCl, 2 mM MgCl 2, 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 0.1 mM PMSF, 1 mM p-aminobenzamidine). After 15 minutes, 125 μl of 10% Nonidet p40 was added, mixed for 20 seconds, and centrifuged at 12,000 rpm for 5 minutes to obtain a supernatant. Next, the tissue homogenate containing 40 μg of protein for Western blotting was mixed with gel loading buffer (0.125 M Tris, pH 6.8, 4% SDS, 10% 2-mercaptoethanol, 0.2% bromphenol blue) And heated for 5 minutes. Each sample was electrophoresed at 110 V for 2 hours in a 12% SDS-polyamide mini-gel. The membrane was transferred to polyvinylidene difluoride (PVDF) membrane using Towbin buffer (25 mM Tris, 192 mM glycine, 20% methanol) and the membrane was incubated in wash buffer containing 5% nonfat milk After stirring, it was washed and reacted with the primary antibody at 4 ° C for 16 hours. Washed with wash buffer and reacted with horseradish-peroxidase conjugated secondary antibody for 30 minutes at room temperature. After washing, ECL detection reagent was added and the fluorescence was photographed with an image analyzer to confirm the signal.

As a result, as shown in FIG. 10, IL-4 reduction was not as effective as the tacrolimus ointment, which is a positive control substance, in the skin tissues of mice that had been exposed to the extract for 13 days while inducing atopy for 14 days. However, -4.

<4-4> Reduction of IgE in mouse sera induced by atopy of Ogaya extract

The serum of the mice prepared in the second method of Example <4-1> was taken from the abdominal aorta, refrigerated for 15 minutes and centrifuged, and each standard solution was added to each well, 100 μl of water extract was added and the mixture was shaken at room temperature for 2.5 hours. After discarding the solution, 100 μl of 1X Biotinylated IgE Detection Antibody was added, incubated at room temperature for 1 hour with shaking, and again the solution was removed and washed. 100 μl of 1X HRP-Streptavidin solution was added, incubated at room temperature for 45 min with shaking, and solution removal and washing were repeated. Then, 100 μl of TMB One-Step Substrate Reagent was added to each well and incubated at room temperature for 30 minutes. 50 μl of Stop Solution was added to each well and immediately measured at 450 nm.

As a result, as shown in FIG. 11, it was confirmed that the mice that had been exposed to Ogata extract for 13 days while inducing atopy for 14 days had increased IgE concentration compared with the normal group. On the other hand, compared with the negative control group, the test group treated with OGa extract had an IgE reduction effect, which was superior to the positive control substance.

<4-5> Analysis of TSLP (thymic stromal lymphopoietin) content in skin tissue

In skin tissue, TSLP is a cytokine with a structure similar to that of IL-7, and is expressed in keratinocytes of skin, pheosome, smooth muscle cells and fibroblasts of lung. TSLP activates dendritic cells, differentiates helper T cells into Th2 cells, and differentiation of Th2 cells plays an important role in inducing symptoms of atopic dermatitis. Therefore, the TSLP inhibitory effect of Ogaya extract was measured.

The extract was applied to the atopy-induced mice prepared in Example <4-1> according to the second experimental conditions. The skin tissue was then removed to prepare a homogenate of the dermal tissue. 300 mg of depilated mouse skin tissue was suspended in 1.6 ml of Tissue homogenate buffer (10 mM HEPES, pH 7.8, 10 mM KCL, 2 mM MgCl 2, 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 0.1 mM PMSF, 1 μM pepstatin, mM p-aminobenzamidine). After 15 minutes, 125 μl of 10% Nonidet p40 was added, mixed for 20 seconds, and centrifuged at 12,000 rpm for 5 minutes to obtain a supernatant. Next, the tissue homogenate containing 40 μg of protein for Western blotting was mixed with gel loading buffer (0.125 M Tris, pH 6.8, 4% SDS, 10% 2-mercaptoethanol, 0.2% bromphenol blue) And heated for 5 minutes. Each sample was electrophoresed at 110 V for 2 hours in a 12% SDS-polyamide mini-gel. The membrane was transferred to polyvinylidene difluoride (PVDF) membrane using Towbin buffer (25 mM Tris, 192 mM glycine, 20% methanol) and the membrane was incubated in wash buffer containing 5% nonfat milk After stirring, it was washed and reacted with the primary antibody at 4 ° C for 16 hours. Washed with wash buffer and reacted with horseradish-peroxidase conjugated secondary antibody for 30 minutes at room temperature. After washing, ECL detection reagent was added and the fluorescence was photographed with an image analyzer to confirm the signal.

As a result, as shown in Fig. 12, it was confirmed that the TSLP of the skin tissue was slightly reduced in the mouse treated with the extract of Ogana extract for 22 days while inducing atopy for 55 days.

The preparation examples of the pharmaceutical composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically explained.

[Formulation Example 1] Preparation of powders

The extract of Example 1 20 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

[Formulation Example 2] Preparation of tablet

The extract of Example 1  10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate   2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

[Formulation Example 3] Preparation of capsules

The extract of Example 1 10 mg

Crystalline cellulose  3 mg

Lactose 14.8 mg

Magnesium stearate  0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

[Formulation Example 4] Preparation of injections

The extract of Example 1 10 mg

Mannitol 180 mg

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ , 12H ₂ O 26 mg

(2 ml) per ampoule in accordance with the usual injection method.

[Formulation Example 5] Preparation of liquid agent

The extract of Example 1 20 mg

Isomer 10 g

Mannitol  5 g

Purified water Suitable amount

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

[Formulation Example 6] Preparation of health drink

The extract of Example 1  100 mg

Vitamin C   15 g

Vitamin E (powder)  100 g

Iron lactate 19.75 g

Zinc oxide  3.5 g

Nicotinic acid amide  3.5 g

Vitamin A  0.2 g

Vitamin B1  0.25 g

Vitamin B2  0.3 g

water    dose

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 ° C for about 1 hour. The resulting solution was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, It is used in the production of the health beverage composition of the invention.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Claims (11)

A pharmaceutical composition for preventing or treating an allergic disease or an inflammatory disease comprising an extract as an active ingredient.
The method of claim 1, wherein the extract is selected from the group consisting of water, a C ₁ to C ₄ alcohol, hexane, ethyl acetate, butylene glycol, propylene glycol, glycerin, acetic acid, ether, chloroform, Or a pharmaceutically acceptable salt thereof. The pharmaceutical composition for preventing or treating allergic diseases or inflammatory diseases according to claim 1,
[2] The method according to claim 1, wherein the extract is obtained by immersing the dried oregano in an extraction solvent at room temperature or extracting it using Accelerated Solvent Extraction (ASE) &Lt; / RTI &gt;
The pharmaceutical composition according to claim 1, wherein the extract is contained in the composition at a concentration of 0.1 to 50 占 퐂 / ml.
The method according to claim 1, wherein the allergic disease is selected from the group consisting of allergic rhinitis, allergic conjunctivitis, allergic asthma, anaphylactic shock, food allergy, hay fever, drug allergy, plant allergies, urticaria, eczema and allergic dermatitis Characterized by Allergy disease Or a pharmaceutical composition for the prevention or treatment of an inflammatory disease.
The use according to claim 1, wherein the inflammatory disease is skin inflammatory disease or atopic dermatitis Or a pharmaceutical composition for the prevention or treatment of an inflammatory disease.
The method according to claim 1, wherein the extract has an activity of inhibiting the secretion of beta-hexoxaminidase and inhibiting inflammatory cytokines and immunoglobulin E Or a pharmaceutical composition for the prevention or treatment of an inflammatory disease.
The pharmaceutical composition according to claim 7, wherein the inflammatory cytokine is IL-4 (interleukin 4) or TSLP (thymic stromal lymphopoietin).
A health-functional food for preventing or ameliorating an allergic disease or an inflammatory disease containing an extract as an active ingredient.
10. The method of claim 9, wherein the allergic disease is selected from the group consisting of allergic rhinitis, allergic conjunctivitis, allergic asthma, anaphylactic shock, food allergies, hay fever, drug allergies, plant allergies, urticaria, eczema, and allergic dermatitis Characterized by Allergy disease Or health functional food for the prevention and improvement of inflammatory diseases.
10. The health functional food according to claim 9, wherein the inflammatory disease is skin inflammatory disease or atopic dermatitis.

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