WO2018105926A1 - Anti-allergy composition containing lactobacillus-fermented composite medicinal herb extract as active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating allergic diseases, the composition containing lactobacillus-fermented extracts of Sophora root, Angelica gigas, Arctium lappa fruit, and Polygonum cuspidatum, and particularly, to a composition containing lactobacillus-fermented extracts of Sophora root, Angelica gigas, Arctium lappa fruit, and Polygonum cuspidatum, for preventing or treating atopic dermatitis, asthma, allergic rhinitis, or allergic conjunctivitis, and a method for preventing or treating allergic diseases comprising a step of administering the composition to an individual.

Description

Anti-allergic composition comprising lactic acid bacteria fermented complex herbal extract as an active ingredient

The present invention relates to a composition for the prophylaxis or treatment of allergic diseases, including a fermented extract of lactic acid bacteria of Sophora Root, Angelica gigas , Arctium lappa fruit, and Polygonum cuspidatum . Prophylaxis against atopic dermatitis, asthma, allergic rhinitis or allergic conjunctivitis, including lactobacillus fermented extracts of Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum cuspidatum Or a therapeutic composition, the method for preventing or treating an allergic disease comprising administering the composition to a subject.

Recently, allergic diseases are on the rise, and research and development of therapeutic agents are also in progress. An allergic disease refers to a disease caused by immune hypersensitivity reactions. An allergic reaction is a complex reaction involving various cells and mediators and also causes various diseases. Therefore, it is expected that the treatment of various diseases through the control of allergic reactions is ongoing. Allergic diseases include atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, and the like.

Atopic dermatitis is a worldwide disease that can occur at any age, with 70% to 95% occurring in infants under 5 years of age. In Korea, the social and medical problems caused by atopic dermatitis are already at a serious level. According to various reports, about 15% of Koreans are atopic dermatitis patients, and 18 to 22 of 100 infants aged 1 to 4 years are known as atopic patients. In recent years, the number of children with adult atopic dermatitis has also increased, and in severe cases, even social problems such as employment and marriage have become serious social problems. In these cases, severe mental stress can lead to extreme avoidance and depression. Although atopic dermatitis has become a serious social and medical problem, there is no effective treatment to date.

Allergic rhinitis is the most common allergic disease, and its incidence is gradually increasing, affecting the quality of life, and it has become a social and economic problem due to a decrease in production capacity and an increase in the cost of treatment. There is no situation. Currently used drug therapy is a basic treatment for allergic rhinitis, but recurrences are common and serious side effects when the drug is discontinued. Immunotherapeutic may be the only treatment that can restore the only modified immune system, but there are cost problems. .

Allergic conjunctivitis is an acute inflammatory disease of the conjunctiva (white egg) caused by certain allergens.Allergens such as pollen in the spring, dust in the air, and dandruff in animals come into contact with the conjunctiva of the eye, causing mast cells, eosinophils or basophils. When an allergic reaction through an immune cell such as a device is induced, various inflammatory substances such as histamine, inflammatory cytokines, and inflammatory lipid metabolites (Prostaglandins & Leukotrienes) are secreted to cause an inflammatory response of the conjunctiva. Such allergic conjunctivitis can be treated with glucocorticoid steroid eye drops.However, these anti-inflammatory drugs can cause sudden systemic weakness, fever, increased intraocular pressure due to acute adrenal insufficiency, etc. Side effects such as glaucoma and cataract may occur, and there is a need for an antiallergic agent without such problems.

The present inventors have made intensive efforts to develop a drug that is effective in the treatment of allergic diseases and ensures safety derived from natural products. It was confirmed that the antiallergic effect was remarkably superior to the herbal medicine alone, and thus had an excellent allergic disease prevention or treatment effect. Thus, it was confirmed that the lactic acid bacteria fermentation complex herbal extract can be effectively used for the prevention or treatment of allergic diseases, and completed the present invention.

One object of the present invention, the composition for the prevention or treatment of allergic diseases, including the extract of lactic acid bacteria fermentation of Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum cuspidatum To provide.

Another object of the present invention, comprising administering to a subject a composition comprising a fermented extract of lactic acid bacteria of Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum cuspidatum To provide a method for preventing or treating allergic diseases.

Another object of the present invention is to ferment lactic acid bacteria of Sophora Root, Angelica gigas , Arctium lappa fruit , and Polygonum cuspidatum for the manufacture of a medicament for preventing or treating allergic diseases. It is to provide a use of the extract.

Another object of the present invention, to prepare a health functional food or cosmetics to improve the allergic diseases, of Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum cuspidatum It is to provide a use of lactic acid bacteria fermented extract.

Still another object of the present invention is to prevent, ameliorate or treat allergic diseases, including extracts of fermented lactic acid bacteria of Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum cuspidatum . It is to provide a use.

The composition comprising the lactobacillus fermentation extracts of Sophora Root, Angelica gigas , Arctium lappa fruit, and Polygonum cuspidatum of the present invention is more effective in producing allergens than conventionally known antiallergic extracts. Since it can be effectively suppressed, it can be usefully used as a composition for preventing or treating allergic diseases.

1 is a graph depicting the inhibitory effect of F-PASA extract on the secretion of β-hexosaminidase from mast cells. (** P <0.01 compared to DNP-IgE / DNP-HSA group)

Fig. 2 is a graph comparing the inhibitory effect of β-hexosaminidase from mast cells on F-PASA extract and KIOM-MA128. (## P <0.01 for each concentration group of KIOM-MA128)

Figure 3 is a graph plotting the inhibitory effect of β-hexosaminidase from mast cells of F-PASA extract, woofer fermentation extract, Angelica fermentation extract, and Ginseng fermentation extract. (** P <0.01 for DNP-IgE / DNP-HSA group, ## P <0.01 for F-PASA group)

4 is a graph depicting the inhibitory effect of F-PASA extract on the production of TNF-α from mast cells. (** P <0.01 compared to DNP-IgE / DNP-HSA group)

Fig. 5 is a graph illustrating the comparison of the inhibitory effect of TNF-α production from mast cells of F-PASA extract, woofer fermentation extract, Angelica fermentation extract, and Ginseng fermentation extract. (** P <0.01 for DNP-IgE / DNP-HSA group, ## P <0.01 for F-PASA group)

6 is a graph illustrating the inhibitory effect of IL-4 (Interleukin 4) production of F-PASA extract. (** P <0.01 compared to DNP-IgE / DNP-HSA group)

7 is a graph illustrating the inhibitory effect of PGD ₂ (Prostaglandin D ₂ ) production of F-PASA extract. (** P <0.01 compared to DNP-IgE / DNP-HSA group)

8 is a graph illustrating the inhibitory efficacy of LTC ₄ (Leukotriene C ₄ ) production of F-PASA extract. (** P <0.01 compared to DNP-IgE / DNP-HSA group)

9 is a graph showing the degree of cytotoxicity induced by F-PASA extract.

10 shows phosphorylation of cPLA ₂ (Cytosolic phospholipase A ₂ ) and 5-LO (5-Lipoxygenase), COX-2 (Cyclooxygenase-2), PGDS (Prostaglandin-D synthase) and LTC ₄ S (Leukotriene C ₄ synthase) Electrophoresis picture of Western blot results showing the degree of phosphorylation and inhibition of expression of F-PASA extract for. (* P <0.05 and ** P <0.01 compared to DNP-IgE / DNP-HSA group)

FIG. 11 is an electrophoretic photograph of the Western blot showing the degree of inhibition of the activity of the F-PASA extract on phosphorylation of the signaling proteins Syk, Fyn, and Lyn of the FcεRI receptor signaling system. (** P <0.01 compared to DNP-IgE / DNP-HSA group)

12 is an electrophoresis photograph of the Western blot result showing the degree of inhibition of activation of the degranulation-related mechanism (PLCγ1 / 2-PKCδ pathway) of the F-PASA extract. (* P <0.05 and ** P <0.01 compared to DNP-IgE / DNP-HSA group)

FIG. 13 is an electrophoretic photograph of Western blot results showing the degree of inhibition of the activation of cytokine production-related mechanisms (phosphorylation of JNK, ERK1 / 2, p38, Akt) of F-PASA extract. (* P <0.05 and ** P <0.01 compared to DNP-IgE / DNP-HSA group)

14 is a graph illustrating the results obtained by measuring Evans blue absorbance for verifying the anti-allergic effect of F-PASA extract on passive skin hypersensitivity model mice. (* P <0.05, ** P <0.01 compared to DNP-IgE / DNP-HSA group)

This will be described in detail as follows. In addition, each description and embodiment disclosed in this invention is applicable to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention is not to be limited by the specific description described below.

As one embodiment for achieving the above object, the present invention includes allergic disease, including lactic acid bacteria fermented extract of Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum cuspidatum It provides a composition for the prevention or treatment of.

In another embodiment, the present invention provides a pharmaceutical composition of Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum cuspidatum for the manufacture of a medicament for preventing or treating allergic diseases. It provides the use of lactic acid bacteria fermented extract.

As another aspect, the present invention includes the lactic acid bacteria fermented extracts of Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum cuspidatum , preventing, improving allergic diseases Or for therapeutic use.

In the present invention, the term " Sophora Root" is a perennial plant of the dicotyledonous rosewood legume, also called the cane of the thief, the ginseng, and the sperm of the snake. have.

In the present invention, the term "angelica gigas " is a perennial aromatic herb belonging to the Apiaceae, which is known to have a blood-producing action, an anti-cancer effect, and a blood pressure-lowering effect to produce blood when blood is scarce.

In the present invention, the term " Arctium lappa fruit " is a dry seed of burdock belonging to the chrysanthemum, the burdock is about 1.5 m in height, the stem is known to grow branches thick and long roots. It is also called a chamber, bookstore, etc., and is known to be effective in urination disorders, eczema, and swelling.

In the present invention, the term " Polygonum cuspidatum " is a perennial plant of the family Mardiaceae, and it is known that the root is used as a diuretic, a sedative in herbal medicine.

The ginseng, Angelica, alligator, and knotweed used in the present invention may be used in nature or purchased commercially, but is not limited thereto.

In one embodiment of the present invention, the result of comparing the efficacy of the fermented extracts of red ginseng, tangui and allies fermentation extracts, and ginseng, tangui, allies and hojanggeun as a result, compared to the case of using the herbal raw materials alone, When the combined fermentation extracts of red ginseng, Angelica, alligator and Escherichia coli were used, they showed more excellent anti-allergic activity, confirming that they can be used as a composition for preventing or treating allergic diseases (FIGS. 3 and 5).

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

In the lactic acid bacteria fermented extract of the present invention, as the lactic acid bacteria, various strains or commercially available lactic acid bacteria can be used without limitation. Specifically, the lactobacillus fermentation is Lactobacillus rhamnosus, Lactobacillus ashdophyllus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus vulgaris, Lactobacillus delbruecchi subspecies lactis, Lactobacillus It may be composed of one or more strains selected from the group consisting of gasseri and Bifidobacterium breve, and more specifically, the lactic acid bacteria fermentation may be made of Lactobacillus rhamnosus . However, lactic acid bacteria that can be used is not limited to the lactic acid bacteria exemplified above, lactic acid bacteria culture medium is also to be selected from MRS (Man-Rogosa-Sharpe), lactose, M17 and APT medium (Asparagine Enrichment Broth) for each lactic acid bacteria But it is not limited thereto.

In the present invention, the term "allergic disease" refers to a disease caused by an immune hypersensitivity reaction, and an allergic reaction is a complex reaction involving various cells and mediators and at the same time causes various diseases. Specifically, the allergic disease may be atopic dermatitis, asthma, allergic rhinitis or allergic conjunctivitis, but is not limited thereto.

Asthma is a chronic disease that causes respiratory distress due to irritability of the airways and intermittent airway contractions due to chronic inflammation of the airways. Asthma is caused by allergens (antigens), such as house dust mites and pollen, present in the external environment, resulting in excessive immune reactions (allergic reactions), resulting in inflammation of airway mucosal tissues. Causes asthma symptoms (Lemanske RF Jr. JAMA 1997, 278, 1855-1873). With the recent advances in immunology and molecular genetics, the prevalence of asthma is increasing despite the understanding of the pathophysiology of asthma and the development of new therapeutic agents. In particular, steroids or bronchodilators, which are used as conventional therapeutics, should be used for a lifetime as a medicament for the control and prevention of symptoms rather than the fundamental cure of a disease, which causes a significant economic burden on individuals and society.

In one embodiment of the present invention, as a result of treating the allergic reaction-induced mast cells fermented extracts of Ginseng, Angelica, alligator, and Escherichia coli, secretion of β-hexosaminidase (degranulated biomarker) from mast cells In addition to suppressing concentration-dependently, it was confirmed that the suppression of allergen-induced secretion is more concentration-dependent than conventional herbal fermented extracts, and allergies such as atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis or rheumatoid arthritis. It was confirmed that it can be used as a composition for preventing or treating sexual diseases (FIGS. 1 to 8).

In another aspect of the present invention, the present invention comprises the steps of administering to a subject a composition comprising a fermented extract of lactic acid bacteria of Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum cuspidatum It includes, it provides a method of preventing or treating allergic diseases.

Regarding the method for preventing or treating the allergic disease, red ginseng, Angelica, alligator, Escherichia coli, fermentation of lactic acid bacteria or allergic disease are as described above.

In the present invention, "prevention" means any action that suppresses or delays the onset of inflammation by the administration of the composition, and "treatment" means any action that improves or advantageously changes the symptoms of inflammation by administration of the composition. Means.

As used herein, the term "administration" refers to introducing the pharmaceutical composition of the present invention to an individual in any suitable manner, and the route of administration of the composition is through various routes, oral or parenteral, as long as the target tissue can be reached. It may be administered, and specifically, in a conventional manner via the oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal, inhaled or intradermal routes.

In one embodiment of the present invention, fermented extracts of Ginseng, Angelica, alligator, and K. koji rarely induced cytotoxicity (FIG. 9), and Syk, Fyn, which are signaling proteins of the rate-limiting step of the FcεRI receptor signal transduction system. , Lyn inhibited the phosphorylation of Syk, Lyn (Fig. 11), by inhibiting the activation of Lyn inhibits the degranulation-related mechanism (PLCγ1 / 2-PKCδ pathway), a downstream signaling system (Fig. 12), cytokine production It was confirmed that the activation of the relevant mechanism (JNK, ERK1 / 2, p38, Akt phosphorylation) can be inhibited (Fig. 13), it was confirmed that it can be usefully used for the prevention or treatment of allergic diseases.

In addition, in one embodiment of the present invention, as a result of treatment of the fermented extract to the passive skin hypersensitivity model mouse, skin hypersensitivity reaction (allergic reaction) was inhibited compared to the negative control, in particular, more excellent anti-allergic activity than the positive control dexamethasone By confirming that, it was confirmed that it can be usefully used for the prevention or treatment of allergic diseases (Fig. 14).

The composition of the present invention may include a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-described active ingredient for administration. The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The compositions of the present invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, or the like, oral preparations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. Specifically, when formulated, it may be prepared using diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used. Solid form preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such solid preparations may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin and the like in the composition. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. It may be prepared by adding various excipients such as humectants, sweeteners, fragrances, preservatives and the like in addition to liquid oral liquids or liquid paraffin for oral use. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, utopsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The composition of the present invention may be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the condition and weight of the patient, the degree of disease, Depending on the drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.

As another aspect, the present invention provides a cosmetic composition for preventing or ameliorating allergic diseases, including a lactobacillus fermented extract of a mixture of red ginseng, Angelica, allium and ephedra.

As another aspect, the present invention is a fermented extract of lactic acid bacteria of Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum cuspidatum for preparing cosmetics for improving allergic diseases Serves the purpose of.

Although the formulation of the cosmetic composition of the present invention is not particularly limited, it is preferably a formulation of an external preparation for skin, and one preferred form of the external preparation for skin is a softening cosmetic, nourishing cosmetic, massage cream, nourishing cream, pack, gel or skin Cosmetic compositions having formulations of adhesive cosmetics, and other preferred forms include transdermal dosage forms such as lotions, ointments, gels, creams, patches or sprays. In addition, in the external preparation composition of each formulation, other components than the essential components described above may be appropriately selected and blended by those skilled in the art without difficulty according to the formulation or purpose of use of the other external preparation.

As another aspect, the present invention provides a dietary supplement for preventing or ameliorating allergic diseases, including fermented extract of lactic acid bacteria of a mixture of red ginseng, Angelica, alligator and Escherichia coli.

In another aspect, the present invention is a lactic acid bacteria of Sophora Root, Angelica gigas , Arctium lappa fruit , and Polygonum cuspidatum Provides the use of fermented extracts.

Fermented extract of the present invention may be added to health food for the purpose of improving allergic diseases, it may be added as it is or used with other food or food ingredients, and may be appropriately used according to conventional methods. The mixed amount of the active ingredient can be determined suitably according to the purpose of use (prevention, health or therapeutic treatment). Generally, in the manufacture of foods or beverages, herbal extracts or herbal fermenters are added in amounts of up to 30% by weight, preferably up to 10% by weight relative to the raw materials. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .

There is no particular limitation on the kind of food. Examples of the food to which the substance may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, dairy products including other noodles, gum, ice cream, various soups, beverages, teas, drinks, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional sense.

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: Preparation of F-PASA Extract

To prepare the F-PASA extract of the present invention (fermented product of Angelica ginseng, ginseng, allium, e. / w / w / w)) in 10 liters of distilled water and heated to a temperature of 115 ℃ for about 3 hours. The resulting solution was filtered through a strainer having a diameter of 150-500 μm and then sterilized by heating for about 5 minutes. After calibrating the pH of the sterilized extract with 1N sodium hydroxide (NaOH) to 7.0, Lactobacillus rhamnosus (KFRI 128, FCTC 2182) was inoculated and fermented at 37 ° C for two days. The fermentation was then filtered through a nylon net having a diameter of 60 μm and allowed to stand for one day. The supernatant was taken and lyophilized to obtain an F-PASA extract (dried). The dry matter was stored at a temperature of -20 ° C and used in all experiments dissolved in 10% DMSO solution.

Comparative Example 1: Preparation of Lactic Acid Bacteria Fermented Herbal Extract

For comparison of antiallergic efficacy with F-PASA extract prepared according to Example 1, lactic acid bacterium fermentation product of the herbal extract in the same manner as the method described in Korean Patent Laid-Open Publication No. 10-2010-0093901 of the present inventors (KIOM- MA128) was prepared.

Specifically, the herbal extract is a herbal preparation prepared by extracting red ginseng, licorice, gilt, angui, venom, mokyang, windproof, sanjoin, eoseongcho, yeonkyo, woobangja, yinyanggak, ginseng, japonica, fat milk, cheongung, hyeonsam and jangjanggeun , 160 g of red ginseng, 80 g of licorice, 80 g of gold and silver coins, donkey 80 g, poisonous g 80 g, tree root 80 g, windproof 80 g, Sanjoin 80 g, Eoseongcho 160 g, kyokyo 80 g, alligator 160 g, Yin Yang 80 g, ginseng A herbal mixture was prepared at a ratio of 160 g, 80 g root, 80 g fat, 80 g cheongung, 160 g of Hyunsam and 80 g of rye root, and then put it in 18.4 L of distilled water, 10 times the 1,840 g of the herbal mixture 1 After soaking for 3 hours at 115 ℃ ultrafast vacuum low temperature extractor (Korea, Gyeongseo extract cosmos-600), filtered using a standard test sieve (standard testing sieve, Aperture 500 ㎛ and 150 ㎛) to prepare a herbal extract .

Lactobacillus ashdophyllus (KFRI # 128), Lactobacillus casei (KFRI # 127), Lactobacillus plantarum (KFRI # 144, KFRI # 402), Lactobacillus fermentum KFRI # 164), Lactobacillus bulgaricus (KFRI # 344), Lactobacillus delbrueki subspecies Lactis (KFRI # 442), Lactobacillus gasseri (KFRI # 658, KCTC 3163) and Bifidobacterium breve (KFRI # 744), and the specific fermentation process is as described in Korean Patent Publication No. 10-2010-0093901.

Comparative Example 2: Preparation of Fermented Allium Extract, Fermented Angelica Extract, and Fermented Ginseng Extract

In order to compare the anti-allergic efficacy with the F-PASA extract prepared according to Example 1, fermented alligator extract (F-allergen), fermented Angelica extract (F-Dangri), and fermented red ginseng extract (F-Ginseng) were prepared, respectively. A method for preparing a specific fermented extract was prepared according to Example 1, and the strain for each fermentation was disclosed in Korean Patent Publication No. 10-2010-0054272; Seo, Min-Jun et al. Journal of the Korean Herb Society, vol. 28, no. 39-44, 2013; And the strain described in Korean Patent Publication No. 10-2014-0008618.

Experimental Example 1: Determination of anti-allergic activity of F-PASA extract

1-1. Induction of allergic reactions of mast cells

In order to compare the anti-allergic activity of the F-PASA extract prepared in Example 1, the fermented extracts prepared in Comparative Examples 1 and 2, allergic reactions were induced in mast cells.

RBL-2H3 cells were dispensed in 24-well plates at a concentration of 1 × 10 ⁵ cells / well, 100 units / mL penicillin, 100 μg / mL streptomycin and 5% (v / v) fetal bovine serum. Incubated overnight at 37 ° C., 5% CO ₂ , humidified conditions in MEM-α medium. After incubation, cells were washed with 1 × PBS buffer and incubated with DNP-IgE 50 ng / mL for 24 hours with Sigma, Co., Ltd to sensitize the cells.

F-PASA extract prepared in Example 1 on sensitized cells; Lactic acid bacteria fermented product of herbal extract prepared in Comparative Example 1; And fermented alligator extract, fermented Angelica extract and fermented red ginseng extract prepared in Comparative Example 2, respectively, incubated for 1 hour, and then cultured for 1 hour, in order to induce hypersensitivity reaction, DNP-HSA (Dinitrophenyl-human serum albumin) 0.1 μg / mL (Sigma, Co., Ltd.) was added and incubated for another 4 hours. The supernatant was taken and centrifuged at 17,000 g at 4 ° C. for 10 minutes, and the supernatant was stored at −80 ° C. and thawed and used for analysis.

1-2. Inhibitory Effect of F-PASA Extract on Allergen-induced Secretion of Mast Cells

Inhibition of secretion of allergens from mast cells was determined by measuring the activity of β-hexosaminidase, a marker of degranulation.

In order to measure the activity of β-hexosaminidase secreted from the cells into the medium, 10 mM p-NAG prepared with 25 μL of the supernatant of 1-1 and 0.1M sodium citrate buffer (pH 4.5). 50 μL of (4-Nitropheyl N- acetyl-β- D- glucosaminide) was placed in a 96-well plate and incubated at 37 ° C. For by measuring the amount of the nitrophenol (p -Nitrophenol) is β- hexyl sosami azepin-one hours after culturing 0.1M sodium carbonate (Na ₂ CO ₃₎ buffer to terminate the reaction with (pH 10.0) and 405 nm to the absorbance at p Activity was determined. Each result is shown as the average of three experiments.

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

In addition, compared to the conventional herbal fermented extract (KIOM-MA128) of Comparative Example 1, it can be seen that the F-PASA extract further suppresses the secretion of allergens from mast cells in a concentration-dependent manner, and the maximum concentration is 2 mg / mL. Was found to be more effectively suppressed by about 4 to 5 times (FIG. 2). In addition, the extract of Comparative Example 2 when the extract of Comparative Example 2 and the F-PASA extract related to prior art samples were compared at the same concentration (0.5 mg / mL) to inhibit the secretion of β-hexosaminidase from mast cells. Did not inhibit degranulation while F-PASA was able to inhibit about 50% (FIG. 3).

1-3. Inhibitory Effect of F-PASA Extract on Cytokine Production on Mast Cells

The inhibitory effect of F-PASA extract on the production of tumor necrosis factor-α (TNF-α), a well-known cytokine secreted from allergic mast cells, was observed in the following manner.

More specifically, the concentration of TNF-α 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.) and the results are shown. 4 and 5, respectively.

As a result, when the F-PASA extract of the present invention was treated, it significantly inhibited the production of TNF-α in a concentration-dependent manner, in particular, it was possible to inhibit the production of TNF-α by 80% or more at a concentration of 1 mg / mL. It was confirmed (FIG. 4).

In addition, it was confirmed that the F-PASA extract of the present invention exhibited a superior ability to inhibit TNF-α production of mast cells compared to the extract of Comparative Example 2 regarding a fermentation product of a single substance (FIG. 5).

1-4. Inhibitory Effect of F-PASA Extract on Prostaglandin and Leucoterien Production on Mast Cells

The inhibitory effects of prostaglandins and leukotrienes on mast cells, known as key mediators of inflammation and allergic diseases, were observed in the following ways.

The concentrations of IL-4 (Interleukin 4), PGD ₂ (Prostaglandin D ₂ ), and LTC ₄ (Leukotriene C ₄ ) in the supernatant of allergic reaction-induced mast cells of Experimental Example 1-1 were measured using EIA kit (Cayman Chemical, Inc. ) Are 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 inhibits the production of IL-4 of FIG. 6, PGD ₂ of FIG. 7, and LTC ₄ of FIG. 8. In particular, the F-PASA extract of the present invention inhibited the production of IL-4 close to 100% at a concentration of 0.2 mg / mL, and confirmed that about 98% of PGD ₂ production at a concentration of 0.5 mg / mL.

Experimental Example 2: Measurement of Cytotoxicity of F-PASA Extract

Cell respiration was measured by mitochondrial dependent reduction of WST-1 to water-soluble tetrazolium salt as an indicator of cell viability.

More specifically, RBL-2H3 cells were dispensed in 96-well plates at a concentration of 1 × 10 ⁴ cells / well, followed by 37 ° C. in MEM-α medium containing 5% (v / v) fetal bovine serum. Incubate overnight at temperature. The cells that were cultured the next day were washed with 1 × PBS buffer and incubated with 50 ng / mL of DNP-IgE (Dinitrophenyl-immunoglobulin E; 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 and further 1 hour After incubation, 10 μL of WST-1 reagent (Daeil Lab Service. Co., Ltd.) and 0.1 μg / mL of DNP-HSA (Dinitrophenyl-human serum albumin) were added at the same time. Incubated for hours. In order to confirm cell viability, absorbance at 450 nm was measured using a micro-plate reader (Emax, Molecular Devices Inc. Sunnyvale, California, USA), and the results are shown in FIG. 9. As a result, it was confirmed that the F-PASA extract of the present invention did not cause any cytotoxicity up to a concentration of 2 mg / mL (Fig. 9).

Experimental Example 3: Effect of F-PASA Extract on FcεRI Receptor Signaling System

Allergic reaction-induced cells prepared in Experimental Example 1-1 were lysed to extract total protein, and then proteins of the FcεRI receptor signaling system were quantified by Western blot. Antibodies used in Western blot were Cell signaling Technology, Inc. It was purchased and used.

Electrophoresis pictures of the Western blot results are shown in FIGS. 10 to 13. Here, β-actin was used as a loading control.

As a result, the F-PASA extract of the present invention is a cPLA ₂ (Cytosolic phospholipase A ₂ ) and 5-LO (5- (5), which are related enzymes of Arachdonate cascade, a subsignal of Syk (Spleen Tyrosine Kinase). Lipoxygenase) phosphorylation and COX-2 (Cyclooxygenase-2) can be confirmed to inhibit the expression. F-PASA extract also inhibited the expression of PGDS (Prostaglandin-D synthase) and LTC ₄ S (Leukotriene C ₄ synthase) (FIG. 10). Here, cPLA ₂ is an enzyme involved in the rate step of arachidonic acid chain reaction, 5-LO is an enzyme involved in the rate step of leukotriene production, and COX-2 is an enzyme involved in the rate step of prostaglandin production. PGDS is a PGD ₂ production related enzyme and LTC ₄ S is an LTC ₄ production related enzyme.

In addition, the F-PASA extract of the present invention inhibited the phosphorylation of Syk, Lyn among Syk, Fyn, and Lyn, the signaling proteins of the rate-limiting step of the FcεRI receptor signal transduction system (FIG. 11). It was confirmed to inhibit the mechanism of degranulation related to the delivery system (PLCγ1 / 2-PKCδ pathway) (FIG. 12). Furthermore, it was confirmed that the F-PASA extract of the present invention can inhibit the activation of cytokine production-related mechanisms (JNK, ERK1 / 2, p38, Akt phosphorylation) (Fig. 13), usefully used as an antiallergic substance It can be confirmed that.

Experimental Example 4: Validation of anti-allergic effect of F-PASA extract on passive skin hypersensitivity model mice

To prepare passive skin hypersensitivity model mice, DNP-IgE (100 ng) was injected intradermal into the ears of ICR mice. After 1 day, 0.5% Evans blue aqueous solution containing 0, 12.5, 25, 50 and 100 mg / kg of F-PASA was administered orally and 100 μg of DNP-HSA after 1 hour. Was injected into the vein. As a positive control, dexamethasone, which is commonly used as an antiallergic drug, was orally administered. After 30 minutes, the mice were euthanized, their ears were cut out, placed in 1 mL of formamide, and the extracts were extracted for 2 hours at a temperature of 80 ° C. The extract was centrifuged and then the supernatant was taken and measured for absorbance at 620 nm, as shown in FIG. 14.

As a result, in the negative control group to which the F-PASA or the positive control group was not administered, allergic reaction by DNP-HSA and DNP-IgE resulted in high absorbance. However, when the F-PASA extract of the present invention was administered, the absorbance was lowered. It was confirmed that the skin hypersensitivity reaction is suppressed (FIG. 14). In particular, when the 100 mg / kg F-PASA extract is administered, by confirming that the anti-allergic activity is superior to the positive control dexamethasone, the fermented extract of the present invention as a composition for the prevention or treatment of anti-allergic diseases It was confirmed that it can be used.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (11)

A composition for the prophylaxis or treatment of allergic diseases, comprising a fermented extract of lactic acid bacteria of Sophora Root, Angelica gigas , Arctium lappa fruit , and Polygonum cuspidatum . The method of claim 1, wherein the lactic acid bacteria fermentation is Lactobacillus rhamnosus, Lactobacillus ashdophyllus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus vulgaris, Lactobacillus delbruechi subspecies lactis , Lactobacillus gaseri and Bifidobacterium breve consisting of one or more strains selected from the group consisting of. The composition of claim 1, wherein the lactic acid bacteria fermentation consists of Lactobacillus rhamnosus . The composition of claim 1, wherein the allergic disease is atopic dermatitis, asthma, allergic rhinitis or allergic conjunctivitis. Preventing or treating allergic diseases, comprising administering to a subject a composition comprising a lactobacillus fermentation extract of Sophora Root, Angelica gigas , Arctium lappa fruit , and Polygonum cuspidatum Way. The method of claim 5, wherein the allergic disease is atopic dermatitis, asthma, allergic rhinitis or allergic conjunctivitis. A cosmetic composition for preventing or ameliorating allergic diseases, comprising a fermented extract of lactic acid bacteria of Sophora Root, Angelica gigas , Arctium lappa fruit , and Polygonum cuspidatum . A health functional food for preventing or ameliorating allergic diseases, including extract of lactic acid bacteria fermentation of Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum cuspidatum . The use of lactic acid bacteria fermented extracts of Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum cuspidatum for the manufacture of a medicament for preventing or treating allergic diseases. Use of Lactic Acid Bacteria Fermented Extracts of Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum cuspidatum for the manufacture of dietary supplements or cosmetics that ameliorate allergic diseases. Use for the prevention, amelioration or treatment of allergic diseases, including lactobacillus fermented extracts of Sophora Root, Angelica gigas , Arctium lappa fruit and Polygonum cuspidatum .

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