KR20210030616A - Composition for preventing or treating dermatitis comprising mixed extracts of Astragalus membranaceus and Schizonepeta tenuifolia - Google Patents

Abstract

The present invention relates to a composition for preventing or treating dermatitis comprising a complex extract of Astragalus propinquus and Schizonepeta tenuifolia as an active ingredient. The composition has effects of: reducing the number of mast cells infiltrating the skin tissue; reducing the expression of a nerve growth factor (NGF) and tropomyosin receptor kinase A (TrkA); reducing phosphorylation of Raf, mitogen-activated protein kinase (MEK), and mitogen-activated protein kinase (ERK); reducing the expression of pro-inflammatory cytokines; increasing the expression of anti-inflammatory cytokines; and reducing the expression of Th2-specific cytokines, thereby being usefully used for preventing, ameliorating, and treating dermatitis.

Description

Composition for preventing or treating dermatitis comprising mixed extracts of Astragalus membranaceus and Schizonepeta tenuifolia}

The present invention relates to a composition for the prevention or treatment of dermatitis comprising a complex extract of Astragalus and Hyeonggae.

Atopic dermatitis is a chronic recurrent skin disease, and related symptoms include pruritus and erythematous papules, allergic diseases, and dryness. The causes of atopic dermatitis can be divided into environmental and genetic causes, and until now, a clear predisposition has not been identified, but it has been reported that immune-related factors are hypersensitive due to the action of external antigens. According to a recent study, the evidence for the symptom of abnormal surface barrier of the skin as the etiology of atopic dermatitis has been reported, and thus, studies on the immunological mechanisms of the stratum corneum constituents have been actively conducted.

It is known that the water retention of the skin epidermis is about 15% in the case of a normal person, and when the water retention falls below 10%, the concentration of ceramide in the stratum corneum decreases, causing dryness and pruritus. In addition, such damage is known to be involved in cell-mediated inflammation due to the infiltration of allergens and pathogens known as the cause of secondary skin diseases.

Atopic dermatitis is mainly observed in infancy and infancy, and can persist or develop into adulthood. The prevalence of atopic dermatitis is rapidly increasing in industrialized and developed countries, and it is reported as a refractory skin disease. In atopic dermatitis, immunopathological related factors interact to participate in skin inflammation, and typical related factors are T lymphocytes, Immunoglobulin E (IgE), and cytokines expressed in Th1 and Th2 cells ( cytokines).

Atopic dermatitis is divided into exogenous due to abnormal hyperactivity of serum IgE and endogenous of pathological symptoms similar to atopic dermatitis despite normal levels of IgE. IgE reacts with the corresponding antigen to promote the transfer of the antigen from Langerhans cells to T lymphocytes, and T lymphocytes induce lesions through a cell-mediated immune response and maintain the persistence of the immune response. Th1 and Th2 cell types are differentiated into Th1 and Th2 cell types by cytokines produced by T cells, and Th1 cells express cytokines such as TNF, IL-2, and IFN-γ to activate macrophages to induce subacute skin inflammation. Th2 cells express cytokines such as IL-4, IL-5, IL-6, and IL-10 to induce hypersensitivity reactions, induce differentiation of eosinophils and mast cells, and produce IgE. Tends to increase further. In general, the interaction between cells induces the balance of the immune response, but in atopic dermatitis, the inflammatory response of atopic dermatitis is induced and promoted due to an increase in cytokines related to the differentiation of Th2 cells. Interferon-gamma (IFN-γ), one of the cytokines produced in the Th1 cell type, induces a delayed immune response, and IL-4, one of the cytokines produced in the Th2 cell type, is IgE. By inducing the synthesis and differentiation of mast cells, inflammatory reactions and immunopathological symptoms are induced. Therefore, in the immune mechanism of atopic dermatitis, expression of Th1 cells is suppressed by relative promotion of Th2 cells. This humoral immune response affects the atopic inflammatory response of the skin. Consequently, in atopic dermatitis, changes in IgE concentration and the mechanism of expression of cytokines such as IL-4 can be seen as important indicators for treatment and alleviation.

Drugs used for atopic dermatitis mainly include immunosuppressants, topical steroids, and antihistamines. Immunosuppressants are toxic to the liver and kidneys, and may have adverse effects on the balance of cellular electrolytes. The topical steroid formulation is a short-term prescription drug formulation, and side effects such as skin atrophy and skin hardening have been reported. In particular, the use of topical steroids is limited due to the onset of skin diseases and the risk of chronic skin diseases. Antihistamines are drugs related to mast cells in the humoral immune response of atopic dermatitis, and their limitations in improving atopic dermatitis have been reported. Therefore, there is a need for a new drug formulation that has secured safety for the improvement of atopic dermatitis.

The Astragalus membranaceus BUNGE reaches 1m in height and has fine hairs in its entirety, and the leaf is a radix single-right biplane consisting of 6-11 pairs of lobules. Flowers bloom in July and August, are 15 ∼ 18 ㎜ long, light yellow, and form inflorescences with several flowers alternately on a long flower stalk. The roots are mainly used as medicinal materials, and in animal experiments, the excitatory and diuretic effects of the central nervous system were also remarkable, and when a large amount of powder was administered to rats, the occurrence of nephritis was suppressed, and the occurrence of proteinuria and cholesterolemia was also delayed. , Blood pressure lowering effect was also recognized. In addition, it is widely used for uterine drainage, gastric drainage, dehydration, and uterine bleeding, and it is reported to increase physical strength and increase the tension of the whole body muscles.

Hyeonggae ( Schizonepeta tenuifolia var. japonica) is an annual plant in the family Lamiaceae, and grows up to 60-100 cm tall, and its stem is greenish purple and has hairs. Leaves are opposite, split into 3~5 branches, and veins are not clear. Flowers bloom in August-September in purple, and hangs in layers of 5-25 cm long on the top of the stem. Fruits are 1.5 mm long and 0.8 mm in diameter, and ripen in dark brown color. Hyeonggae, which is used as a medicinal material, is the dried above-ground part of the hyunggae, and at the end of flowering when the flower stalks rise in the summer, the outpost is cut and dried in a well-ventilated shade. It is known that there are few stems or leaves and many stalks are good as medicinal materials. Hyunggae has an intense scent, very taste, and warm nature, and is known to act on menopause and liver disease.

Korean Patent Registration No. 10-1892865

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of dermatitis comprising a complex extract of Astragalus and Hyeonggae as active ingredients.

Another object of the present invention is to provide a health functional food for preventing or improving dermatitis comprising a complex extract of Astragalus membranaceus and Hyeonggae as an active ingredient.

Another object of the present invention is to provide a cosmetic composition for improving dermatitis comprising a complex extract of Astragalus and Hyeonggae as active ingredients.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of dermatitis comprising a complex extract of Astragalus and Hyeonggae as active ingredients.

In one embodiment of the present invention, the complex extract may be extracted with water, an organic solvent, or a mixture thereof, and the organic solvent is methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform , Ethyl acetate, methylene chloride, may be any one or more selected from the group consisting of hexane and cyclohexane.

In one embodiment of the present invention, the complex extract may be a mixture of the extracts of Astragalus and Hyeonggae in a weight ratio of 1:100 to 100:1, preferably 1:50 to 50:1 weight ratio. It may be, and more preferably, it may be mixed in a weight ratio of 1:10 to 10:1, but is not limited thereto.

In one embodiment of the present invention, the dermatitis is selected from the group consisting of atopic dermatitis, contact dermatitis, seborrhoic dermatitis, and allergic contact dermatitis. Can be.

In one embodiment of the present invention, the composition may be to reduce the number of mast cells penetrated into the skin tissue.

In one embodiment of the present invention, the composition may be to reduce the expression of NGF (nerve growth factor) and TrkA (tropomyosin receptor kinase A).

In one embodiment of the present invention, the composition may be to reduce phosphorylation of Raf, mitogen-activated protein kinase kinase (MEK), and mitogen-activated protein kinase (ERK).

In one embodiment of the present invention, the composition may reduce the expression of pro-inflammatory cytokines, increase the expression of anti-inflammatory cytokines, and decrease the expression of Th2-specific cytokines.

In addition, the present invention provides a health functional food for the prevention or improvement of dermatitis comprising a complex extract of Astragalus and Hyeonggae as active ingredients.

In addition, the present invention provides a cosmetic composition for improving dermatitis comprising a complex extract of Astragalus and Hyeonggae as active ingredients.

The composition according to the present invention reduces the number of mast cells infiltrating the skin tissue, reduces the expression of nerve growth factor (NGF) and tropomyosin receptor kinase A (TrkA), and reduces the expression of Raf, mitogen-activated protein kinase kinase (MEK). And reducing the phosphorylation of mitogen-activated protein kinase (ERK), and the composition reduces the expression of pro-inflammatory cytokines, increases the expression of anti-inflammatory cytokines, and reduces the expression of Th2-specific cytokines. It is effective, and can be usefully used in the prevention, improvement or treatment of dermatitis.

1 is a mouse (atopic dermatitis animal model) with a lesion of allergic contact dermatitis induced by DNCB, dexamethasone (DEX, positive control), astragalus (AM), hyeonggae (ST), or a complex (AM+) ST) is a result of measuring the dermatitis score after treatment (data are expressed as mean ± SEM (n=5); ^### p <0.001: compared with normal control (NOR); and ^* p <0.05, ^** p <0.01 and ^*** p <0.001: compared to disease control (DNCB)).
Figure 2 is a mouse with a lesion of allergic contact dermatitis induced by DNCB (atopic dermatitis animal model), dexamethasone (DEX, positive control), astragalus (AM), hyeonggae (ST) or complex (AM+) ST) is the result of measuring the thickness of the epidermis and dermis by staining the skin tissue with H&E after treatment (x100; ^### p <0.001: compared with normal control (NOR); And ^* p <0.05 and ^*** p <0.001: compared to disease control (DNCB)).
Figure 3 is a mouse with a lesion of allergic contact dermatitis induced by DNCB (atopic dermatitis animal model), dexamethasone (DEX, positive control), astragalus (AM), hyeonggae (ST), or complex (AM+) ST) is the result of measuring the number of mast cells by staining the skin tissue with Toluidine blue after treatment (x200, scale bar: 100 μm; ^### p <0.001: compared with normal control (NOR); And ^*** p <0.001: when compared to disease control (DNCB)).
Figure 4 shows dexamethasone (DEX, positive control), Astragalus (AM), Hyeonggae (ST), or complex (AM+) in mice with lesions of allergic contact dermatitis induced by DNCB (atopic dermatitis animal model). ST) is the result of measuring the expression level of NGF (nerve growth factor) in the dorsal of rats through Western blot ( ^### p <0.001: compared with normal control (NOR); And ^** p <0.01 and ^*** p <0.001: compared to disease control (DNCB)).
Figure 5 shows dexamethasone (DEX, positive control), Astragalus (AM), Hyeonggae (ST), or complex (AM+) in mice with lesions of allergic contact dermatitis induced by DNCB (atopic dermatitis animal model). ST) is the result of measuring the TrKA expression level, Raf, MEK, and ERK phosphorylation level in the dorsal of mice through Western blot after treatment ( ^### p <0.001: compared with normal control (NOR). Case; and ^** p <0.01 and ^*** p <0.001: compared to disease control (DNCB)).
Figure 6 is a mouse with a lesion (lesion) of allergic contact dermatitis induced by DNCB (atopic dermatitis animal model), dexamethasone (DEX, positive control), Astragalus (AM), hyeonggae (ST), or complex (AM+) ST), then pro-inflammatory cytokines IL-6 and TNF-α (A); Anti-inflammatory cytokines IL-10 and TGF-β (B); And the expression levels of Th2-specific cytokines IL-4, IL-13 and IL-31 (C) were measured through Western blot ( ^### p <0.001: compared with normal control (NOR) ; And ^* p <0.05, ^** p <0.01 and ^*** p <0.001: when compared to disease control (DNCB)).

The composite according to the present invention may be obtained by extracting and separating from nature using a method for extracting and separating known in the art, and "composite extract" or "formula" as defined in the present invention is an appropriate It is an extract extracted from Astragalus (Astragalus membranaceus BUNGE) and Hyeonggae (Schizonepeta tenuifolia var.japonica ) using a solvent, and includes, for example, a crude extract of Astragalus and Hyeonggae, a polar solvent-soluble extract, or a non-polar solvent-soluble extract.

As an appropriate solvent for extracting the complex of Astragalus membranaceus and Hyeonggae, any organic solvent may be used as long as it is a pharmaceutically acceptable organic solvent, and water or an organic solvent may be used, but is not limited thereto, for example, purified water, Alcohols having 1 to 4 carbon atoms, including methanol, ethanol, propanol, isopropanol, butanol, etc., acetone, ether, benzene, Various solvents such as chloroform, ethyl acetate, methylene chloride, hexane, and cyclohexane may be used alone or in combination.

As the extraction method, any one of methods such as hot water extraction, cold precipitation extraction, reflux cooling extraction, solvent extraction, steam distillation method, ultrasonic extraction method, elution method, and compression method may be used. In addition, the desired extract may be further subjected to a conventional fractionation process, or may be purified using a conventional purification method. There is no limitation on the method for producing the composite of the present invention, and any known method may be used.

For example, the composite material included in the composition of the present invention may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze drying or spray drying of the primary extract extracted by the hot water extraction or solvent extraction method described above. In addition, the primary extract was further purified using various chromatography such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, etc. You can also get

Therefore, in the present invention, the complex is a concept including all extracts, fractions, and purified products obtained in each step of extraction, fractionation, or purification, and their dilutions, concentrates, or dried products.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. In the present invention, the term "pharmaceutically acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition. The carrier may be used without limitation as long as it is known in the art such as a buffering agent, a preservative, a painless agent, a solubilizing agent, an isotonic agent, a stabilizer, a base agent, an excipient, and a lubricant.

In addition, the pharmaceutical composition of the present invention is formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. I can. Further, it may be used in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel or gel for external skin. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, such as starch, calcium carbonate, in the Cycotria rubra extract, It is prepared by mixing sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administration" of the present invention means introducing a predetermined substance to an individual by an appropriate method, and the route of administration of the composition may be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration may be administered, but are not limited thereto.

The term "individual" refers to all animals including humans, such as rats, mice, and domestic animals. Preferably, it may be a mammal including a human.

The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not cause side effects, and the effective dose level is the sex, age, and Weight, health condition, type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, and rate of excretion, duration of treatment, factors including drugs used in combination or concurrently, and other fields of medicine. It can be readily determined by a person skilled in the art according to well-known factors. Administration may be administered once a day at the recommended dosage, or may be divided several times.

The food composition of the present invention may include all foods in a conventional sense, and may be mixed with terms known in the art, such as functional foods and health functional foods.

The term "functional food" of the present invention refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body according to the Health Functional Food Act No.6727, and the term "functional" It refers to ingestion for the purpose of obtaining useful effects for health purposes such as controlling nutrients for structure and function or for physiological effects.

The term "health functional food" of the present invention refers to a food manufactured and processed by extracting, concentrating, refining, mixing, or extracting, concentrating, refining, and mixing specific ingredients contained in food ingredients or using specific ingredients for the purpose of health supplementation. It refers to foods designed and processed to sufficiently exert biological control functions such as biological defense, biological rhythm control, disease prevention and recovery, etc., by ingredients, and the health food composition is used to prevent diseases and recover diseases. Can perform functions related to etc.

There is no limitation on the kind of food in which the composition of the present invention can be used. In addition, the composition of the present invention can be prepared by mixing suitable other auxiliary ingredients that may be included in food and known additives according to the choice of a person skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and There are vitamin complexes and the like, and can be prepared by adding the extract according to the present invention and a fraction thereof as a main component, such as juice, tea, jelly, and juice.

In addition, foods that can be applied to the present invention include, for example, special nutritional foods (e.g., formulas, infant foods, etc.), processed meat products, fish meat products, tofu, rice cakes, noodles (e.g., ramen, noodles, etc.), health supplement foods. , Seasoned foods (e.g. soy sauce, miso, red pepper paste, mixed sauce, etc.), sauces, sweets (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.) ), beverages (eg, fruit, vegetable beverages, soy milk, fermented beverages, etc.), natural seasonings (eg, ramen soup, etc.).

When the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweetening agents, flavoring agents, natural carbohydrates, and the like as an additional component, as in ordinary beverages. In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin. , Alcohol, carbonated beverages, etc. may contain. In addition, it may contain flesh for the manufacture of natural fruit juice, fruit juice beverage and vegetable beverage.

The cosmetic composition of the present invention may include commonly used ingredients, and may include conventional adjuvants and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , Oil, powder foundation, emulsion foundation, wax foundation, and may be formulated as a spray, but is not limited thereto. In more detail, it may be prepared in the form of a flexible lotion, nutritional lotion, nutritional cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component. I can.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally chlorofluorohydrocarbon, propane / May contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and crystallites Sex cellulose, aluminum metahydroxide, bentonite, agar or tracanth, and the like can be used.

When the formulation of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters may be used.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited to these examples.

Example 1. Experimental materials and methods

1.1. Preparation of the formula

The Hwanggi and Hyeonggae used in the present invention were purchased from Jeongdo Pharmacy (Seoul, Korea). The hwanggi and hyeonggae were each extracted with 10-fold distilled water, and the solution was filtered and concentrated, and then lyophilized and used. Astragalus and Hyeonggae complex extract (hereinafter, a compound (formula)) was prepared by mixing Astragalus extract and Hyeonggae extract in a weight ratio of 3:2. Freeze-dried specimens (Voucher specimen: #AM-W100-2018, #ST-W100-2018, #Derma-H-2018) of the obtained Astragalus, Hyeonggae, and composites were stored at -20°C.

1.2. Atopic dermatitis animal model and dermatitis score measurement method

In order to prepare an atopic dermatitis animal model, the present inventors purchased a 5-week-old male mouse C57BL/6J with a weight of 19 to 21 g, and the day and night of a 12-hour cycle are alternated with a room temperature of 22 ± 2°C and 50 It was reared in an environment of ± 5% humidity. Food and water were allowed to be eaten freely, raised for one week, and after a 7-day adaptation period, the back area was depilated with an epilator, and left for 24 hours so that microscopic wounds could heal spontaneously. In order to induce atopic dermatitis in the mouse, 200 μL of a 1% DNCB (2,4-dinitrochlorobenzene) solution was sensitized on the same back area once a day for 3 days, and the vehicle (acetone: olive) was used in the normal control group. oil = 4: 1) was treated. After the incubation period for 4 days, 200 μL of 0.5% DNCB solution was applied to all groups except for the normal control group to induce a second sensitization. After treating with 4% SDS solution to facilitate skin absorption, 30 mg/mL Astragalus extract (AM), 20 mg/mL Hyeonggae extract (ST), or 50 mg/mL complex (AM+ST) were applied at 200 μL each I did. The positive control dexamethasone was applied at a concentration of 10 μM. The 0.5% DNCB solution was treated 3 hours after the extract treatment. Sample treatment and secondary sensitization were treated once a day for 11 days, and the experimental process was carried out for a total of 18 days. On the 19th day, after 12 hours fasting, mice were sacrificed according to the purpose of the experiment.

The dermatitis score is (1) erythema/bleeding, (2) dryness/scarring, (3) swelling, and (4) erosion/exhaustion: 0 (normal), 1 (mild), 2 (moderate), or 3 It was evaluated as (Severe). The dermatitis index was measured by 3 researchers without providing information for the group. The sum of the separate scores was described as the dermatitis score.

1.3. Histological measurement method of skin tissue

The skin tissue sample was fixed in 10% formalin for 24 hours, and the fixative solution penetrating the tissue was sufficiently removed through a sufficient washing process. Thereafter, after dehydration in the order of alcohol concentration of 70%, 90%, 95%, and 100% to remove moisture from the tissue, a paraffin block was prepared using xylene as a transparent agent. The finished paraffin block was cut at 4 μm intervals to make sections, followed by deparaffinization and hydration, and then stained with H&E (hematoxylin & eosin) solution. The stained slides were observed at 400 magnification with an optical microscope, and the Image J program was used to analyze the thickness of the epidermis and the dermis.

1.4. Method for measuring penetration of mast cells

The prepared skin tissue section was stained with toluidine blue to observe mast cell infiltration in the dermis, and the number of mast cells was counted. Five randomly selected measurement images were observed at 100 magnification in an optical microscope.

1.5. Western blot analysis

From skin tissue using RIPA assay buffer (50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM EDTA) containing protease inhibitors cocktail. Protein was extracted. Skin tissues such as mice (n=5/group) were crushed with buffer, and the extracted protein was quantified in the same amount of 30 μg each using a Dc Protein assay kit and used in the experiment. Protein was isolated through electrophoresis. The separated protein was transferred to a Nitrocellulose membrane, blocked with 5% BSA (in TBS-T), and washed three times for 15 minutes with TBS-T. The primary antibodies Anti-β-actin, -NGF, -TrKA, -Raf, -MEK, -ERK (in TBS-T, 1:1000) were treated and bound overnight at 4°C. Wash three times with TBS-T for 15 minutes, and then add goat-anti-rabbit or goat-anti-mouse secondary antibody with HRP (horseradish-peroxidase) attached to TBS-T at 1:2000, and add at room temperature for 1 hour. After treatment, the ECL kit was used. The density of each band was measured using the LAS Image Gauge program.

Example 2. Dermatitis index relief effect

The present inventors performed an experiment to measure the dermatitis score after administering the complex to the skin tissues of mice in which dermatitis was induced in order to confirm the therapeutic effect of the complex on dermatitis.

As a result, the dermatitis index was 6.07 ± 2.35 in mice induced dermatitis by DNCB, 4.93 ± 1.39 when treated with Astragalus in mice with dermatitis induced by DCNB, 4.53 ± 1.09 when treated with Hyeonggae It was found to be 4.40 ± 0.80 when treated (Fig. 1). That is, it was confirmed that the treatment of the complex has the effect of alleviating the dermatitis induced by DCNB compared to the single extract. However, in the case of treatment with dexamethasone (DEX), which is a positive control, it was 2.67 ± 0.78, and the efficacy of the complex was not greater than that of the positive control.

Example 3. Inflammation Relief Effect in Skin Tissue

Dermatitis is an inflammation of the skin tissue, and has a characteristic that the thickness of the epidermis and the dermis forming the skin tissue becomes thick due to an inflammatory reaction. Accordingly, the present inventors observed the histological characteristics of the skin tissue by staining the skin tissue of the mouse caused by dermatitis with H&E.

As a result, the thickness of the epidermis in the skin tissue was 155.2 ± 22.3 μm in the rats caused by DNCB dermatitis, and 77.48 ± 19.2 when treated with Astragalus, hyeonggae, or complex in the rats caused by DNCB dermatitis, respectively, and It was found to be 70.34 ± 13.5, 69.5 ± 11.6 μm (Fig. 2 lower left panel). In other words, it was confirmed that there is an effect of recovering the epidermis thickened by DNCB again when the composite is treated. This had a recovery effect similar to that of treatment with the positive control dexamethasone (55.27 ± 7.1 μm).

In addition, the thickness of the dermis was 384.2 ± 57.3 μm in DCNB-treated mice, which was about twice as thick as that of the normal control mice, and decreased to 272.1 ± 56.4 μm when treated with the positive control group. When Astragalus, Hyeonggae, or the complex was treated, the thickness of the dermis was 322.8 ± 73.1, 296.4 ± 88.8, and 274.0 ± 53.9 μm, respectively. Bottom panel).

Accordingly, the present inventors have confirmed that when the complex is administered to skin tissue in which dermatitis is induced, the thickness of the skin tissue is restored to a normal level compared to a single extract.

Example 4. Mast cell penetration reduction effect

The present inventors performed an experiment to determine whether mast cells penetrated into the skin tissues of mice in which dermatitis was induced, and whether the complex had an effect of reducing the number of penetrated mast cells.

As a result, the number of mast cells in the skin tissues of mice induced dermatitis by DNCB increased 8.7 times compared to the normal control, but the number of mast cells was treated with Astragalus, Hyeonggae, or complex in mice induced by DNCB. It was confirmed that the effect was reduced to 38.4%, 43.0%, and 49.1%, respectively (FIG. 3).

Therefore, the present inventors have confirmed that when the complex is administered to skin tissue in which dermatitis is induced, it has an effect of reducing the number of mast cells infiltrating the skin tissue compared to a single extract.

Example 5. Effect on the expression level of NGF (nerve growth factor)

In dermatitis, it has been reported that NGF (nerve growth factor) is overexpressed. Accordingly, the present inventors performed a Western blot to confirm whether the complex has a change in the expression level of NGF.

As a result, it was confirmed that the expression level of NGF in the skin tissues of rats caused by dermatitis by DNCB increased by about 2 times compared to the normal control group. It was confirmed that the expression levels of NFG were reduced to 11.8%, 22.1%, and 32.5%, respectively (FIG. 4). In particular, in the case of treatment with the complex, there was an effect of reducing the expression level of NFG to a degree similar to that of treatment with the positive control dexamethasone (41.9% reduction).

Therefore, the present inventors have confirmed that when the complex is administered to skin tissues in which dermatitis is induced, it has an effect of reducing the expression level of NGF, which was increased in dermatitis, compared to a single extract.

Example 6. Effect on expression level or phosphorylation of dermatitis-related protein

The present inventors performed a Western blot to confirm the level of phosphorylation of dermatitis-related TrkA (tropomyosin receptor kinase A) protein expression, Raf, mitogen-activated protein kinase kinase (MEK), and ERK (mitogen-activated protein kinase) protein. .

As a result, TrkA expression, Raf, MEK and ERK phosphorylation were significantly increased in DNCB-induced dermatitis-induced rats, and TrkA expression when DNCB-induced dermatitis-induced rats were treated with Astragalus, H. In the case of treatment with the complex, there was an effect of reducing the expression level to a level similar to that of the normal control or positive control (FIG. 5). For Raf and MEK, there was an effect of reducing phosphorylation to a level similar to that of the normal control or positive control when astragalus, hyeonggae, or complex was treated (FIG. 5). In addition, for ERK, when Astragalus was treated, phosphorylation was slightly reduced, and when the complex was treated, phosphorylation was reduced to a level similar to that of the positive control group (FIG. 5).

Therefore, the present inventors confirmed that when the complex is administered to skin tissues in which dermatitis is induced, the expression level of TrkA, Raf, MEK, and ERK phosphorylation, which have been increased in the signaling pathway, are reduced.

Example 7. Effect on the expression level of cytokines

The present inventors performed an experiment to measure the expression level of cytokines through western blot in order to confirm the effect of the complex on inflammatory cytokines.

As a result, the expression levels of IL (interleukin)-6 and TNF-α, known as pro-inflammatory cytokines, were remarkably increased in the mice induced by DNCB compared to the normal control, and in mice induced by DNCB dermatitis. It was confirmed that IL-6 and TNF-α were significantly reduced when treated with Astragalus, Hyeonggae or the complex (FIG. 6A). In particular, when the complex was treated, it was decreased compared to the case where dexamethasone, which was a positive control, was treated, and it was found to be at a level similar to that of the normal control.

In addition, in mice induced dermatitis by DNCB, the expression levels of IL-10 and TGF-β, known as anti-inflammatory cytokines, were significantly reduced compared to the normal control group. When the complex was treated, there was an effect of increasing the expression levels of IL-10 and TGF-β (FIG. 6B).

In addition, the expression levels of Th2-specific cytokines IL-4, IL-13, and IL-31 were significantly increased in DNCB-induced dermatitis-induced rats compared to the normal control, and NCB-induced dermatitis-induced rats Astragalus , It was confirmed that there is an effect of reducing the expression levels of IL-4, IL-13 and IL-31 when treated with a mold or complex (Fig. 6C). In particular, for IL-31, when the complex was treated, there was an effect of remarkably reducing the expression level of IL-31 compared to the single extract of Astragalus or Hyeonggae, which is a more remarkable reduction effect than the positive control, and similar to the normal control. It was confirmed to be the level.

Therefore, the present inventors consider dermatitis by remarkably reducing pro-inflammatory cytokines, remarkably increasing anti-inflammatory cytokines, and remarkably reducing Th2-specific cytokines when the complex is administered to dermatitis-induced skin tissue. It was confirmed that there is a therapeutic effect on.

Claims (11)

A pharmaceutical composition for the prevention or treatment of dermatitis comprising a complex extract of Astragalus membranaceus BUNGE) and Hyeonggae (Schizonepeta tenuifolia var. japonica) as an active ingredient. The method of claim 1,
The composite extract is a composition, characterized in that extracted with water, an organic solvent, or a mixture thereof. The method of claim 2,
The organic solvent is a composition characterized in that at least one selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane. The method of claim 1,
The composite extract is a composition characterized in that the mixture of the extracts of Astragalus and Hyeonggae 1:100 to 100:1 by weight. The method of claim 1,
The dermatitis is atopic dermatitis (atopic dermatitis), contact dermatitis (contact dermatitis), seborrheic dermatitis (seborrhoic dermatitis) and allergic contact dermatitis (allergic contact dermatitis) composition characterized in that selected from the group consisting of. The method of claim 1,
The composition is a composition, characterized in that reducing the number of mast cells infiltrating the skin tissue. The method of claim 1,
The composition is a composition characterized in that reducing the expression of NGF (nerve growth factor) and TrkA (tropomyosin receptor kinase A). The method of claim 1,
The composition is a composition characterized in that reducing the phosphorylation of Raf, mitogen-activated protein kinase (MEK) and ERK (mitogen-activated protein kinase). The method of claim 1,
The composition is a composition characterized in that it reduces the expression of pro-inflammatory cytokines, increases the expression of anti-inflammatory cytokines, and decreases the expression of Th2 specific cytokines. Health functional food for the prevention or improvement of dermatitis containing a complex extract of Astragalus and Hyeonggae as active ingredients. A cosmetic composition for improving dermatitis comprising a complex extract of Astragalus and Hyeonggae as an active ingredient.

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