KR20180066941A - Anti-allergic Composition Comprising Herbal Extract fermented by Lactic acid Bacteria as an Active Ingredient - Google Patents

Abstract

The present invention relates to a method for producing Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum The present invention relates to a composition for preventing or treating an allergic disease, which comprises a fermented extract of a lactic acid bacterium of the order Sophora Root, Angelica gigas , Arctium a composition for preventing or treating atopic dermatitis, asthma, allergic rhinitis or allergic conjunctivitis, comprising a fermented extract of a lactic acid bacterium of Lappa fruit and Polygonum cuspidatum , And a method of preventing or treating a sexual disorder.

Description

(Anti-allergic Composition Comprising Herbal Extract Fermented by Lactic Acid Bacteria as an Active Ingredient)

The present invention relates to a method for producing Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum The present invention relates to a composition for preventing or treating an allergic disease, which comprises a fermented extract of a lactic acid bacterium of the order Sophora Root, Angelica gigas , Arctium a composition for preventing or treating atopic dermatitis, asthma, allergic rhinitis or allergic conjunctivitis, comprising a fermented extract of a lactic acid bacterium of Lappa fruit and Polygonum cuspidatum , And a method of preventing or treating a sexual disorder.

Recently, allergic diseases are on the rise, and research and development of therapeutic agents are under way. Allergic disease means a disease caused by immune hypersensitivity reaction. Allergic reaction is a complicated reaction involving various cells and mediators, and acts as a cause of various diseases. Therefore, it is expected that various diseases can be treated through the control of allergic reactions. Allergic diseases include atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis and the like.

Atopic dermatitis is a common worldwide disease that can occur in any age group, with 70% to 95% occurring at infancy under 5 years of age. In Korea, social and medical problems caused by atopic dermatitis have already reached serious level. According to various reports, about 15% of the total population in Korea are atopic dermatitis patients, of which 18-22 per 100 children aged 1 to 4 years are known to be atopic. In recent years, the number of patients with atopic dermatitis has increased, as well as children, and serious cases of social problems such as employment and marriage are causing serious social problems. In such cases, severe psychological stress can cause severe depression and avoidance of interpersonal abuse. Despite the fact that atopic dermatitis is a serious social and medical problem, there is no effective treatment until now.

Allergic rhinitis is the most common allergic disease, and its incidence is gradually increasing, affecting the quality of life, reducing the production capacity and increasing the burden of the treatment, which is a social and economic problem. However, It is absent. The currently used pharmacotherapy is a basic treatment for allergic rhinitis, but recurrence is common and can cause serious side effects, and immunotherapy can be the primary treatment to restore the originally modified immune system, but it is costly .

Allergic conjunctivitis is an allergic disease that is caused by an allergic reaction caused by a specific allergen causing conjunctival (white) inflammatory disease. Allergic conjunctivitis is caused by allergens such as pollen of spring, airborne dust, animal dander, When an allergic reaction occurs through contact with immune cells such as mast cells, eosinophils or basophils, various inflammatory substances such as histamine, inflammatory cytokines and inflammatory lipid metabolites (Prostaglandins & Leukotrienes) are secreted, Inflammatory response of the immune system. These allergic conjunctivitis can be treated with glucocorticoid based steroid eye drops. However, these antiinflammatory drugs can cause sudden adrenal insufficiency, fever, fever, elevated intraocular pressure, Glaucoma, cataracts, etc., and thus an antiallergic agent having no such problems is required.

Under these circumstances, the present inventors have made intensive efforts to develop drugs capable of securing safety derived from natural products, while effectively treating allergic diseases. As a result, in case of lactic acid fermented extract of Gosam, Angelicae, It was confirmed that the antiallergic effect is remarkably superior to that of the herbal medicine alone and that it has an excellent allergic disease prevention or therapeutic effect. Thus, it has been confirmed that the herbal extract of fermented lactic acid bacteria can be effectively used for the prevention or treatment of allergic diseases, and the present invention has been completed.

One object of the present invention, Sophora (Sophora Root), Angelica (Angelica gigas), woobangja (Arctium lappa fruit and Polygonum The present invention also provides a composition for preventing or treating an allergic disease, comprising a fermented extract of a lactic acid bacterium of the genus Cuspidatum .

It is another object of the present invention to provide a method for producing Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum cuspidatum , which comprises administering to a subject other than a human, a composition comprising a fermented extract of a lactic acid bacterium of the present invention, a method for preventing or treating an allergic disease.

This will be described in detail as follows. On the other hand, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present invention. Further, the scope of the present invention is not limited by the detailed description described below.

As one aspect for achieving the above object, the present invention provides a method for producing a sophora root, Angelica gigas , Arctium the present invention provides a composition for preventing or treating an allergic disease, comprising a fermented extract of Lactobacillus of Lappa fruit and Polygonum cuspidatum .

The term " Sophora Root" in the present invention is a perennial plant of a perennial plant of rosemary roots, which is also called a spermatozoa of a cane, a pupa, a snake, and it is known to be prescribed for dyspepsia, neuralgia, hepatitis, jaundice, have.

In the present invention, the term " Angelica gigas "is a perennial herbaceous herbaceous plant belonging to the genus Liliaceae. It is known to have a blood-generating, anti-cancer effect and blood pressure lowering effect.

The term " Arctium " lappa fruit ") is a dried mature seed of burdock belonging to Asteraceae, burdock is about 1.5 m in height, branches are split from the stems, and roots are known to grow thick and long. , Eczema, swelling and so on.

The term " Polygonum " cuspidatum ") is a perennial plant of Oriental herbaceous plant, and it is known to use roots as a diuretic, sedative and the like in herbal medicine.

The ginseng root, angelica gigas, angelica gigas, angelica gigas, and angiosaccharides used in the present invention may be collected from nature or commercially available, but are not limited thereto.

According to one embodiment of the present invention, the combined fermented extracts of Gosam, Angelica gigas and Wisteria fermentation extracts, Gosam, Angelica gigas, Worcester, It was confirmed that the combined fermented extract of Gosam, Angelica gigas, Worsted ginger and Root canola showed more excellent antiallergic activity and could be used as a composition for preventing or treating allergic diseases (FIGS. 3 and 5).

The solvent used in the preparation of the extract may be water, a C ₁ to C ₄ alcohol, preferably methanol, ethanol or butanol, or a mixed solvent thereof, but is not limited thereto . Examples of the extraction method include, but are not limited to, solvent extraction, hot water extraction, cold extraction, reflux extraction, ultrasonic extraction, or steam extraction.

In the lactic acid fermentation extract of the present invention, isolated strains or various commercially available lactic acid bacteria can be used without limitation. Specifically, the lactic acid bacteria fermentation can be carried out by using lactobacillus rhamnosus, lactobacillus ashdophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus fermentum, lactobacillus bulgaricus, lactobacillus delbrueckerium lactis, Gasseri and bifidobacterium breve. More specifically, the lactic acid bacteria fermentation may be made of Lactobacillus rhamnosus . However, the lactic acid bacteria that can be used are not limited to the above-mentioned lactic acid bacteria, and the lactic acid bacteria culture medium may also be selected from MRS (Man-Rogosa-Sharpe), Lactose, M17 and APT medium (Asparagine Enrichment Broth) But is not limited thereto.

The term "allergic disease" in the present invention means a disease caused by an immunological hypersensitivity reaction. The allergic reaction is a complex reaction involving various cells and mediators, and acts as a cause of various diseases. Specifically, the allergic disease may be, but is not limited to, atopic dermatitis, asthma, allergic rhinitis or allergic conjunctivitis.

Asthma is a chronic disease that causes respiratory distress symptoms because of the chronic inflammation of the airways causing airway hyperreactivity and intermittent airway constriction. Asthma is caused by allergens (allergens) such as house dust mites and pollen which are present in the external environment, resulting in excessive inflammation of the airway mucosal tissues due to excessive immune reaction (allergic reaction), resulting in cough, dyspnea (Lemanske RF Jr., JAMA 1997, 278, 1855-1873). Recently, despite the development of immunology and molecular genetics, understanding the pathophysiology of asthma and developing new therapies, the prevalence of asthma is increasing. In particular, steroids or bronchodilators, which are used as conventional therapeutic agents, must be used for a long time as medicaments for the control and prevention of symptoms, not the fundamental healing of diseases, resulting in a great economic burden on individuals and society.

In one embodiment of the present invention, the treatment of the allergic reaction-induced mast cells with the fermented extracts of Gosam, Angelica gigas, Wolverine, and Fagopyrum japonica resulted in the secretion of β-Hexosaminidase (degranulation biomarker) In addition, it was found that the inhibition of the secretion of allergen-inducing substances was inhibited more than the existing herbal fermentation extracts in a dose-dependent manner. Thus, it is possible to prevent allergic diseases such as atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis or rheumatoid arthritis (Fig. 4 to Fig. 8).

As another aspect of the present invention, the present invention provides a method for producing a compound of the present invention, which comprises the steps of: Sophora Root, Angelica gigas , Arctium the present invention provides a method for preventing or treating an allergic disease comprising administering to a subject other than a human a composition comprising a lactic acid fermented extract of Lappa fruit and Polygonum cuspidatum .

As for the method for the prevention or treatment of the allergic diseases, the ginseng root, Angelica gigas, Wisteria root, Root muscle, lactic acid fermentation, or allergic diseases are as described above.

In the present invention, "prevention" means any action that inhibits or slows the onset of inflammation by the administration of the composition, and "treatment" Means action.

The term "administering" in the present invention means introducing the pharmaceutical composition of the present invention into a subject by any appropriate method, and the administration route of the composition may be administered through various routes of oral or parenteral administration, And can be administered in a conventional manner via oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, intradermal or intradermal routes.

In one embodiment of the present invention, the fermented extract of Gosam, Angelicae, Wisteria, and Rootgut showed almost no cytotoxicity (Fig. 9), and the signaling proteins Syk, Fyn (PLCγ1 / 2-PKCδ pathway) (Fig. 12), inhibited the phosphorylation of Syk and Lyn in Lyn (Fig. 11) (JNK, ERK1 / 2, p38, and Akt phosphorylation) (Fig. 13), and thus it can be effectively utilized for prevention or treatment of allergic diseases.

In addition, in one embodiment of the present invention, the above-mentioned fermented extract was treated with a passive skin hypersensitive model mouse to show that the allergic reaction was inhibited compared with the negative control, and the antiallergic activity was superior to that of the positive control, dexamethasone , And can be useful for prevention or treatment of allergic diseases (Fig. 14).

The composition of the present invention may contain, for administration, a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-described effective ingredients. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The composition of the present invention may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like oral preparation, external preparation, suppository or sterilized injection solution according to a conventional method. In detail, when formulating, it can be prepared by using diluents or excipients such as fillers, weighing agents, binders, humectants, disintegrants, surfactants and the like which are generally used. Solid formulations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin and the like in the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration, liquid paraffin, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

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

In another aspect, the present invention provides a cosmetic composition for preventing or ameliorating an allergic disease, which comprises a fermented extract of Lactobacillus acidus in a mixture of Gossam, Angelica gigantea, Wisteria, and Rhizoma root.

The formulation of the cosmetic composition of the present invention is not particularly limited, but it is preferably a formulation of an external preparation for skin, and a preferred form of the composition for external application of the skin includes a softening agent, a nutritional lotion, a massage cream, A cosmetic composition having a formulation of an adhesive type cosmetic composition, and another preferred form is a transdermal dosage form such as lotion, ointment, gel, cream, patch or spray. Further, in the external preparation for each formulation, components other than the above-mentioned essential components can be mixed and selected without difficulty by those skilled in the art depending on the formulation or purpose of use of the other external preparation.

In another aspect, the present invention provides a health functional food for preventing or ameliorating an allergic disease, which comprises a fermented extract of Lactobacillus acidus of a mixture of Gossam, Angelica, Wisteria, and Hojang.

The fermented extract of the present invention can be added to a health food for the purpose of improving allergic diseases and can be used as it is or can be used together with other food or food ingredients and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the herbal extract or herbal medicine fermentation product is added in an amount of not more than 30% by weight, preferably not more than 10% by weight based on the raw material when the food or beverage is produced. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range .

There is no particular limitation on the kind of the food. Examples of foods to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

Sophora Root, Angelica gigas , Arctium lappa fruit , and Polygonum cuspidatum may be more effectively used as a composition for the prevention or treatment of allergic diseases because it can inhibit the production of allergen-inducing substances more effectively than the known antiallergic extracts.

FIG. 1 is a graph showing the inhibitory effect of F-PASA extract on the secretion of? -Hexosaminidase from mast cells. (** P <0.01 compared to DNP-IgE / DNP-HSA group)
FIG. 2 is a graph illustrating the inhibitory effect of F-PASA extract and KIOM-MA128 on the secretion inhibition of β-Hexosaminidase from mast cells. (## P <0.01 compared to each concentration group of KIOM-MA128)
FIG. 3 is a graph comparing the inhibitory effects of β-Hexosaminidase from mast cells on the F-PASA extract, Wisteria fermented extract, Angelica giganteus fermentation extract, and Gossam fungus extract. (** P <0.01 compared to DNP-IgE / DNP-HSA group, ## P <0.01 compared to F-PASA group)
Figure 4 is a graphical representation of the inhibitory effect of F-PASA extract on the production of TNF-a from mast cells. (** P <0.01 compared to DNP-IgE / DNP-HSA group)
FIG. 5 is a graph illustrating the inhibitory effect of F-PASA extract, Wisteria fermentation extract, Angelica gigantea fermentation extract, and Gossam fermentation extract on the production of TNF-α from mast cells. (** P <0.01 compared to DNP-IgE / DNP-HSA group, ## P <0.01 compared to F-PASA group)
FIG. 6 is a graph showing the inhibitory effect of F-PASA extract on IL-4 (Interleukin 4) production. (** P <0.01 compared to DNP-IgE / DNP-HSA group)
FIG. 7 is a graph illustrating the inhibitory effect of F-PASA extract on the production of PGD ₂ (Prostaglandin D ₂ ). (** P <0.01 compared to DNP-IgE / DNP-HSA group)
FIG. 8 is a graph illustrating the inhibitory effect of F-PASA extract on LTC ₄ (Leukotriene C ₄ ) production. (** P <0.01 compared to DNP-IgE / DNP-HSA group)
FIG. 9 is a graph showing the degree of cytotoxicity of the F-PASA extract. FIG.
Figure _{10, cPLA 2 (Cytosolic phospholipase A 2} ) and 5-LO (5-Lipoxygenase) phosphorylation, COX-2 (Cyclooxygenase-2 ), PGDS (Prostaglandin-D synthase) and _{LTC 4 S (Leukotriene C 4 synthase} ) of The results of Western blot electrophoresis show the degree of inhibition of phosphorylation and expression of F-PASA extract.
FIG. 11 is an electrophoresis image showing Western blot results showing the degree of inhibition of F-PASA extract activity on the phosphorylation of Syk, Fyn, and Lyn, signaling proteins of the FcεRI receptor signaling system.
FIG. 12 is an electrophoresis photograph of the Western blot showing the degree of inhibition of the activation of the degranulation-related mechanism (PLCγ1 / 2-PKCδ pathway) of the F-PASA extract.
Fig. 13 is an electrophoresis image showing the Western blot results showing the degree of inhibition of the activation of the cytokine production-related mechanism (JNK, ERK1 / 2, p38, Akt phosphorylation) of the F-PASA extract.
14 is a graphical representation of the results of measuring the absorbance of Evans blue for the antiallergic effect of F-PASA extract on mice with passive skin hypersensitivity. (* P <0.05, ** P <0.01 compared to DNP-IgE / DNP-HSA group)

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: F- PASA  Extract preparation

In order to prepare the F-PASA extract of the present invention (fermented product of Angelica gigasso, Ganoderma lucidum, Alzheimer's radix, and Root mixture), 1 kg of PASA (1: 1: 1: 1 w / w / w / w) was placed in 10 liters of distilled water and heated to a temperature of 115 DEG C for about 3 hours. The resulting solution was sieved through a sieve having a diameter of 150 to 500 μm and then sterilized by heating for about 5 minutes. Lactobacillus rhamnosus (KFRI 128, FCTC 2182) was inoculated with 1N sodium hydroxide (NaOH) to adjust the pH of the sterilized extract to 7.0 and fermented at 37 ° C for two days. Thereafter, the fermented product was filtered through a nylon mesh having a diameter of 60 mu m and allowed to stand for one day. The supernatant was taken and lyophilized to obtain F-PASA extract (dried). The dried material was stored at a temperature of -20 ° C and dissolved in a 10% DMSO solution in all experiments.

Comparative Example  1: Lactic acid bacteria extract of herbal medicine Fermentation product  Produce

In order to compare the antiallergic effect with the F-PASA extract prepared according to Example 1, the same method as described in Korean Patent Laid-Open No. 10-2010-0093901 of the present inventor, the fermented product of lactic acid bacteria (KIOM- MA128).

Specifically, the herbal medicine extract is a oriental herbal medicine prepared by extracting ginseng, licorice, gonghwaseong, angelica, cheongwoo, hanbok, windbreak, sanjoin, 80 g of ginseng, 80 g of licorice, 80 g of ginseng, 80 g of ginseng, 80 g of virgin, 80 g of wheat germ, 80 g of wind gusts, 80 g of sanjiton 80 g, 160 g, muscle wax 80 g, fat 80 g, ginseng 80 g, ginseng 160 g, and goryanggang 80 g were prepared and added to 18.4 L distilled water, which was 10 times the amount of 1,840 g of the crude drug mixture, to prepare 1 , And then filtered using a high-speed vacuum low-temperature extractor (cosmos-600, Korea) and a standard testing sieve (Aperture 500 ㎛ and 150 ㎛) at 115 ° C for 3 hours to prepare herbal extracts .

Lactobacillus acidophilus (KFRI # 128), Lactobacillus casei (KFRI # 127), Lactobacillus plantarum (KFRI # 144, KFRI # 402), Lactobacillus fermentum KFRI &amp;num; 164), Lactobacillus bulgaricus (KFRI # 344), Lactobacillus delbrueckiliaci lactis (KFRI # 442), Lactobacillus gasseri (KFRI # 658, KCTC 3163), and Bifidobacterium breve # 744). The specific fermentation process is as described in Korean Patent Publication No. 10-2010-0093901.

Comparative Example  2: Fermentation Warder  Extract, fermented Angelica gigantea extract, and fermentation GoSam  Preparation of extract

Fermented Angelicae extract (F-Angelica gigantea), fermented Angelica gigantosa extract (F-Angelica) and fermented ginseng extract (F-gossam) were prepared for comparison of antiallergic efficacy with the F-PASA extract prepared in Example 1, A specific fermentation extract was prepared according to Example 1, and each strain for fermentation was prepared according to Korean Patent Publication No. 10-2010-0054272; Seo, Min-joon et al. Journal of the Korean Society of Herbals 28 (5): 39-44, 2013; And Korean Patent Publication No. 10-2014-0008618 were used.

Experimental Example  1: F- PASA  Measurement of antiallergic activity of extract

1-1. Induce allergic reaction of mast cell

Allergic reactions were induced in mast cells to compare the antiallergic activity of the F-PASA extract prepared in Example 1 and the fermented extract prepared in Comparative Examples 1 and 2.

The RBL-2H3 cells in 24-well plates 1 × 10 ⁵ cells / well and dispensed to a concentration, 100 units / mL penicillin, 100 ㎍ / mL streptomycin (streptomycin) and 5% (v / v) fetal calf serum is Ml &lt; / RTI &gt; medium at 37 &lt; 0 &gt; C, 5% CO ₂ . After completion of the incubation, the cells were washed with 1 x PBS buffer and the cells were sensitized by culturing with DNP-IgE 50 ng / mL (Sigma, Co., Ltd) for 24 hours.

The F-PASA extract prepared in Example 1 was added to the sensitized cells; Fermented product of the herbal medicine extract prepared in Comparative Example 1; And fermented guinea ginseng extract and fermented ginseng extract prepared in Comparative Example 2 were added for 1 hour. Then, 0.1 μg / mL of DNP-HSA (Dinitrophenyl-human serum albumin) was added to induce hypersensitive reaction (Sigma, Co., Ltd.) was added and incubated again for 4 hours. The supernatant was centrifuged at 17,000 g for 10 minutes at 4 ° C and the supernatant was stored at -80 ° C and thawed for analysis.

1-2. F- PASA  Inhibitory Effect of Extracts on the Secretion of Allergic Matter to Mast Cells

The inhibitory effect of β-Hexosaminidase, a marker of degranulation, on the secretion of allergen-inducing substances from mast cells was assayed.

To measure the activity of beta -hexosaminidase secreted from the cells into the medium, 25 [mu] l of the supernatant of 1-1 and 0.1 [mu] M sodium citrate buffer (pH 4.5) 50 μl of 10 mM p-NAG (4-Nitropheyl N- acetyl-β- D- glucosaminide) was added to a 96-well plate and cultured at 37 ° C. After incubation 1 hours 0.1M Na ₂ CO ₃ buffer (pH 10.0) to terminate the reaction and the absorbance at 405 nm as a p - is β- hexyl sosami by measuring the amount of the nitrophenol (p -Nitrophenol) kinase (β-Hexosaminidase ) Was determined. Each result was expressed as the average value of three experiments.

As a result, the F-PASA extract of the present invention was able to inhibit the secretion of β-Hexosaminidase from mast cells in a concentration-dependent manner, and the IC ₅₀ value was 510.9 ㎍ / mL. In particular, the extract of F-PASA inhibited the secretion of allergen-inducing substances by more than 75% at a concentration of 2 mg / mL (FIG. 1).

In addition, it can be seen that the F-PASA extract inhibits the secretion of allergen-inducing substances from mast cells in a concentration-dependent manner, compared to the conventional herbal fermentation extract (KIOM-MA128) of Comparative Example 1. The maximum concentration of 2 mg / mL (Fig. 2). Fig. In addition, when the efficacy of the extract of Comparative Example 2 and the extract of F-PASA on the prior art samples was compared with the effect of inhibiting the secretion of β-hexosaminidase from mast cells at the same concentration (0.5 mg / mL), the extract of Comparative Example 2 (Fig. 3), while F-PASA was able to inhibit about 50%.

1-3. F- PASA  Inhibitory Effect of Extracts on the Production of Cytokines in Mast Cells

The inhibitory effect of F-PASA extract on the production of tumor necrosis factor-α (TNF-α), well known as a cytokine secreted from mast cells associated with allergy, was observed by the following method.

More specifically, the concentration of TNF-? In the supernatant of the allergic reaction-induced mast cells of Experimental Example 1-1 was measured according to the manufacturer's manual using an ELISA kit (e-Bioscience, Inc.) 4 and Fig. 5, respectively.

As a result, when the F-PASA extract of the present invention was treated, the production of TNF-α was significantly inhibited in a concentration-dependent manner, and the production of TNF-α was inhibited by 80% or more at a concentration of 1 mg / (Fig. 4).

In addition, it was confirmed that the F-PASA extract of the present invention showed more excellent inhibitory effect on TNF-a production of mast cells compared to the extract of Comparative Example 2 relating to the fermentation product of a single substance (FIG. 5).

1-4. F- PASA  Extracts of prostaglandins and mast cells against mast cells Leukotriene  Generation inhibition effect assay

The inhibitory effects of prostaglandins and leukotrienes, which are known as inflammatory and allergic disease mediators, as inflammatory lipid mediators, in mast cells were observed by the following methods.

The concentration of IL-4 (Interleukin 4), PGD ₂ (Prostaglandin D ₂ ) and LTC ₄ (Leukotriene C ₄ ) in the supernatant of allergen-induced mast cells of Experimental Example 1-1 was measured using an EIA kit (Cayman Chemical, Inc.). ) Were measured according to the manufacturer's manual, and the results are shown in FIGS. 6 to 8, respectively.

As a result, it was confirmed that the F-PASA extract of the present invention significantly inhibited the production of IL-4 of FIG. 6, PGD ₂ of FIG. 7 and LTC ₄ of FIG. In particular, the F-PASA extract of the present invention inhibited the production of IL-4 by about 100% at a concentration of 0.2 mg / mL and inhibited the production of PGD ₂ by about 98% at a concentration of 0.5 mg / mL.

Experimental Example  2: F- PASA  Cytotoxicity measurement of extract

Cell respiration was measured by the mitochondria-dependent reduction of WST-1 to a water-soluble tetrazolium salt as an indicator of cell viability by the following method.

More specifically, RBL-2H3 cells were divided into 96-well plates at a concentration of 1 × 10 ⁴ cells / well and then cultured in MEM-α medium containing 5% (v / v) fetal bovine serum at 37 ° C. &Lt; / RTI &gt; for 12 hours. Cells cultured the next day were washed with 1 x PBS buffer and incubated with DNP-IgE (Dinitrophenyl-immunoglobulin E) 50 ng / mL (Sigma, Co., Ltd.) for 24 hours to sensitize the cells.

F-PASA (0 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL) prepared in Example 1 was added to the sensitized cells, 10 μl of WST-1 reagent (Daeil Lab Service Co., Ltd.) and 0.1 μg / mL of DNP-HSA (Dinitrophenyl-human serum albumin) (Sigma, Co., Ltd.) Lt; / RTI &gt; The absorbance at 450 nm was measured using a micro-plate reader (Emax, Molecular Devices Inc., Sunnyvale, Calif., USA) to confirm cell viability, and the results are shown in FIG. As a result, it was confirmed that the F-PASA extract of the present invention did not cause any cytotoxicity to a concentration of 2 mg / mL (FIG. 9).

Experimental Example  3: F- PASA  Extract FcεRI  Measurement of effect on receptor signaling system

The total protein was extracted by dissolving allergen-induced cells prepared in Experimental Example 1-1, and the protein of the FcεRI receptor signaling system was quantitated by Western blot. Antibodies used in Western blot were Cell signaling Technology, Inc. Were purchased and used.

An electrophoresis photograph of the result of the Western blot is shown in Figs. 10 to 13. Fig. Here, β-actin was used as a loading control.

As a result, the F-PASA extract of the present invention was found to inhibit the expression of cPLA ₂ (cytosolic phospholipase A ₂ ) and 5-LO (5- Lipoxygenase) and the expression of COX-2 (cyclooxygenase-2). In addition, the F-PASA extract inhibited the expression of PGDS (Prostaglandin-D synthase) and LTC ₄ S (Leukotriene C ₄ synthase) (FIG. 10). Here, cPLA ₂ is an enzyme participating in the rate-determining step of the arachidonic acid chain reaction, 5-LO is an enzyme involved in the rate-determining step of leukotriene production, and COX-2 is an enzyme involved in the rate-determining step of prostaglandin production. PGDS is an enzyme involved in PGD ₂ production, and LTC ₄ S is an enzyme involved in LTC ₄ production.

In addition, the F-PASA extract of the present invention inhibited the phosphorylation of Syk, Lyn among Syk, Fyn, and Lyn, which are signaling proteins of the rate-limiting step of FcεRI receptor signal transduction system (FIG. 11) (PLCγ1 / 2 - PKCδ pathway), which is a delivery system, (Fig. 12). Further, it was confirmed that the F-PASA extract of the present invention can inhibit the activation of the cytokine production-related mechanism (JNK, ERK1 / 2, p38, Akt phosphorylation) (FIG. 13) .

Experimental Example  4: F- PASA  Anti-allergic efficacy of extracts on passive skin hypersensitivity model mice

To prepare passive skin hypersensitive model mice, the ear of ICR mice was injected intradermally with DNP-IgE (100 ng). After one day, F-PASA at 0, 12.5, 25, 50 and 100 mg / kg were respectively administered orally and after 1 hour, 0.5% Evans blue aqueous solution containing 100 μg of DNP- Were injected intravenously. Dexamethasone, commonly used as an antiallergic drug, was orally administered as a positive control. After 30 minutes, the mice were euthanized, and the ears were cut and placed in 1 ml of Formamide, and the extract was extracted at 80 DEG C for 2 hours. The extract was centrifuged and the supernatant was taken and the absorbance at 620 nm was measured and shown in Fig.

As a result, in the negative control group to which F-PASA or the positive control was not administered, high absorbance was shown by the allergic reaction caused by DNP-HSA and DNP-IgE. However, when the F-PASA extract of the present invention was administered, It was confirmed that the allergic reaction was suppressed (Fig. 14). Particularly, when the F-PASA extract of 100 mg / kg was administered, the anti-allergic activity was superior to that of the positive control, dexamethasone. Thus, the fermented extract of the present invention is a composition for preventing or treating antiallergic diseases Can be used.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (8)

Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum cuspidatum &lt; / RTI &gt; of the present invention.
The method according to claim 1, wherein the lactic acid bacteria fermentation is carried out by using lactobacillus laminosus, lactobacillus ashidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus fermentum, lactobacillus bulgaricus, lactobacillus delbrueckerium lactis , Lactobacillus gasseri and Bifidobacterium breve. &Lt; Desc / Clms Page number 24 &gt;
The composition of claim 1, wherein the lactic acid fermentation comprises Lactobacillus rhamnosus .
The composition according to claim 1, wherein the allergic disease is atopic dermatitis, asthma, allergic rhinitis or allergic conjunctivitis.
Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum comprising administering to a subject other than a human a composition comprising a fermented extract of a lactic acid bacterium of the genus Cuspidatum .
6. The method of claim 5, wherein the allergic disease is atopic dermatitis, asthma, allergic rhinitis or allergic conjunctivitis.
Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum cuspidatum ) of fermented extract of Lactobacillus.
Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum cuspidatum ), comprising the fermented extract of Lactobacillus, for the prevention or amelioration of an allergic disease.

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