WO2014104681A1

WO2014104681A1 - Pharmaceutical composition for preventing or treating skin disease comprising eriodictyol compound or a pharmaceutically acceptable salt thereof as an active ingredient

Info

Publication number: WO2014104681A1
Application number: PCT/KR2013/012018
Authority: WO
Inventors: 김택중; 박세진
Original assignee: 연세대학교 원주산학협력단
Priority date: 2012-12-26
Filing date: 2013-12-23
Publication date: 2014-07-03

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating skin disease, and to a topical skin preparation, a skin-disease improving or alleviating cosmetic and health/functional food, comprising an eriodictyol compound, a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient, and, more specifically, the invention suppresses degranulation of mast cells and, in an allergic animal model, has been confirmed to be outstandingly effective in alleviating itching and with respect to inflammation, swelling, skin moisturisation and skin elasticity and hence can be used advantageously for the purpose of preventing, treating or improving various skin diseases.

Description

Pharmaceutical composition for preventing or treating skin diseases containing ERIODICTYOL compound or a pharmaceutically acceptable salt thereof as an active ingredient

The present invention relates to a composition for preventing or treating skin diseases containing an eriodictyol compound or a pharmaceutically acceptable salt thereof as an active ingredient.

In modern society, the incidence of skin diseases including allergic contact dermatitis has increased greatly due to the increase of environmental pollution and the production of synthetic substances due to industrial development. In addition, urban populations are increasingly exposed to allergic reactions caused by various antigens, such as mites, increased dust as the humidity decreases, the hair of dogs, pollen, new fruits and foods.

Mast cells and blood basophils are known as cells in the body that cause various allergic diseases, such as allergic rhinitis, allergic atopic dermatitis, allergic conjunctivitis, allergic asthma, food allergy and anaphylactic shock. .

Allergic skin diseases are caused by various allergens, which excessively stimulate the immune system, and in immune cells such as macrophages, TNF-α (tumor necrosis factor-α), IL-6 (interleukin-6), and IL-8 ( interleukin-8), prostaglandin, leucotriene and nitric oxide (NO) to induce excessive inflammatory substances, such as allergic purpura, atopic dermatitis, chronic asthma and immunity. Causes hypersensitivity symptoms.

As industrialization accelerates, modern people are exposed to various forms of allergens, which increases the likelihood of allergic and atopic skin diseases, and is a social problem that can alleviate allergic reactions from natural products with fewer side effects. The interest of many researchers has been focused on the research of the active research into a variety of raw materials with the prevention and treatment of these diseases, but the systematic research is insufficient.

Meanwhile, the eriodictyol compound is a type of flavanone of the plant, which is separated from the old maple ( Acer mono Max.) And Yerba santa ( Eriodictyon califonicum ). Known.

It has been reported that the erythridiol compound has a prophylactic effect in diseases such as atherosclerosis, bone disease and hypertension. However, there has been no report on the effect of the erythrothiol compound on the treatment of skin-related diseases.

The present inventors have completed the present invention by confirming that the eriodictyol compound, which is a kind of plant flavanone, is effective in alleviating the itch of dermatitis, treating inflammation, moisturizing the skin and enhancing skin elasticity.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating skin diseases containing an eriodictyol compound, a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides an external skin preparation for preventing or treating skin diseases containing an eriodictyol compound, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a cosmetic composition for improving or alleviating skin diseases containing an eriodictyol compound, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health functional food for improving or alleviating skin diseases containing an eriodictyol compound, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

By erythrothiol compound, a derivative thereof or a pharmaceutically acceptable salt thereof according to the present invention by inhibiting the degranulation of mast cells and by confirming the effect on relieving itching, inflammation, edema, skin moisturizing and skin elasticity excellent in allergic animal models, It is expected to be useful as a good medicine, cosmetics, external skin preparations or health functional food.

Figure 1 shows the chemical structure of riodic thiol.

2A and 2B show the results of experiments measuring the effect of erythrothiol on IgE / Ag-induced passive dermal anaphylaxis (PCA).

3A and 3B are graphs showing the results of experiments measuring the effect of erythrothiol on IgE / Ag-activated mast cell degranulation, 3a is the result of measuring the secretion amount of β-hexosaminidase, and 3b is cell survival Indicates the level measurement result.

Figure 4 shows the results of measuring the effect of the expression of TNF-α and IL-4 erythrothiol in IgE / Ag-sensitized mast cells.

Figure 5 shows the experimental results of measuring the effect of the cerioid thiol compound on ceramide kinase (CERK) in IgE / Ag-sensitized mast cells.

Figure 6 shows the experimental results of measuring the effect of the erythrothiol compound on ceramide levels in IgE / Ag-sensitized mast cells.

Figure 7 shows the results of a change measurement test of the scratching behavior of the dorsal epidermis of mice induced with allergic contact dermatitis according to the treatment of the erythrothiol compound.

8A and 8B show skin sensory evaluation photographs (A) and results (B) of the dorsal epidermis of mice induced with allergic contact dermatitis according to the treatment of the erythrothiol compound, respectively.

Figures 9a and 9b is taken after the hematoxylin and eosin staining process after the tissue of the dorsal epidermis of the mouse induced allergic contact dermatitis in accordance with the treatment of the erythrodiol thiol compound of the present invention was taken under a microscope (A) and a graph showing the experimental results numerically. (It is about microscope picture which enlarged 40 times (a, b, c), 100 times (d, e, f).)

Figure 10 shows the results of the measurement of the change in serum IgE following the treatment with the eriodithiol compound.

Figure 11 is a graph showing the results of measuring the skin moisturizing effect of the cream-type cosmetics containing erythridiol with MY-808S Moisture Checker (Scalar, Japan).

Figure 12 is a graph showing the results of measuring the skin moisturizing effect of the serum type cosmetics containing erythridiol with MY-808S Moisture Checker (Scalar, Japan).

The present invention relates to a pharmaceutical composition for preventing or treating skin diseases containing an eriodictyol compound, a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also relates to an external preparation for skin disease prevention or treatment containing an eriodictyol compound, a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also relates to a cosmetic composition for improving or alleviating skin diseases containing an eriodictyol compound, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also relates to a health functional food for improving or alleviating skin diseases containing an eriodictyol compound, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention is characterized by providing a pharmaceutical composition for preventing or treating skin diseases containing an eriodictyol compound represented by Formula 1, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. :

[Formula 1]

.

The invention Erie ohdik thiol (eriodictyol) compounds IUPAC name as a kind of flavanone (flavanone) are (2 S) -2- and (3,4-dihydroxyphenyl) -5,7-dihydroxy -4-chromanone, a The molecular formula of the material is C ₁₅ H ₁₂ O ₆ , with a molecular weight of 288.25.

The eriodithiol compound according to the present invention may be used in the form of a salt, preferably a pharmaceutically acceptable salt, wherein the salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid. The free acid may be an organic acid or an inorganic acid. The organic acid is not limited thereto, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Glutamic acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.

In addition, the eriodithiol compound of the present invention can be isolated from natural products or prepared and used by chemical synthesis methods known in the art, and commercially available riodicthiol compounds can be obtained commercially.

In addition, the pharmaceutical composition of the present invention is hardly toxic and harmless to the human body since the active ingredient is a component isolated from a natural product extract or a derivative thereof.

The pharmaceutical dosage forms of erythridiol according to the present invention may be used in the form of their pharmaceutically acceptable salts, and may also be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

In one embodiment according to the invention, the pharmaceutical composition may exhibit an itching relief, inflammatory response inhibition, edema inhibition, skin moisturizing and skin elasticity effect, the composition is characterized in that for the prevention or treatment of contact dermatitis Can be.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. Mix is prepared. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like can also be used.

Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories. It can have one formulation.

In one embodiment according to the invention, the eriodictyol compound, derivative thereof or pharmaceutically acceptable salt thereof in the pharmaceutical composition is 0.0001 to 50% by weight of the total pharmaceutical composition, more specifically 0.005 to 25% by weight, more specifically may be included in an amount of 0.001 to 10% by weight.

If it is less than 0.0001% by weight of the eriodictyol compound, derivative thereof, or pharmaceutically acceptable salt thereof, the eriodicthiol compound, derivative thereof, or pharmaceutically acceptable salt thereof does not affect the skin disease and will be effective. It may not be possible. In addition, when it exceeds 50% by weight, it does not show an effect similar to or higher than that of 50% by weight.

In another embodiment of the present invention, a skin external preparation for preventing or treating skin diseases containing an eriodictyol compound represented by Formula 1 below, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient is provided. It is characterized by:

[Formula 1]

.

In one embodiment of the present invention, the external skin preparation has an effect selected from the group consisting of alleviation of itching, inhibition of inflammatory response, edema inhibition, skin moisturization and skin elasticity, wherein the external skin preparation is for the prevention or treatment of contact dermatitis It is characterized by.

In one embodiment of the present invention, the external skin preparation is formulated into any one selected from the group consisting of ointments, creams, lotions, solutions, dressings, patches, blisters, tapes, aerosols, external preparations and sprays. Can be.

In one embodiment according to the present invention, the eriodictyol compound, a derivative thereof or a pharmaceutically acceptable salt thereof in the external preparation for skin is 0.00001 to 25% by weight of the total external skin composition, more specifically 0.0005 To 10% by weight, more specifically, may be included in an amount of 0.0001 to 5% by weight.

In addition to the active ingredient of the formulation of the present invention, any ingredient generally used in external preparations for skin may be used. For ointments, creams, gels and lotions, white and petrolatum (waceline), yellow and petrolatum, lanolin, refined beeswax, cetanol, stearyl alcohol, stearic acid, hydrogenated oil, hydrocarbon gel, polyethylene glycol, liquid paraffin and squalane Base such as; Oleic acid, isopropyl myristate, glycerol triisooctanoate, crotamiton, diethyl sebacate, diisopropyl sebacate, diisopropyladipate, hexylulaate, fatty acids, fatty acid esters, vegetable oils, fatty alcohols and Solvents or stabilizers such as alcohols; Antioxidants such as tocopherol derivatives, L-ascorbic acid, dibutylhydroxytoluene and butylhydroxyanisol; preservatives such as p-hydroxybenzoate; Curb agents such as glycerin, propylene glycol and sodium hyaluronate; Surfactants such as polyoxyethylene derivatives, glycerol fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters and lecithin; Thickeners such as carboxyvinyl polymers, xan gum, carboxymethyl cellulose and sodium carboxymethyl cellulose, hydroxypropyl cellulose and hydroxypropylmethyl cellulose; Stabilizer; Preservatives; Adsorption promoters; And other suitable fillers may be added.

In the case of a foaming agent, adhesives, such as a polyacrylic acid and a polyacrylic acid copolymer; Crosslinking agents such as aluminum sulfate, aluminum potassium sulfate, aluminum chloride, magnesium aluminosilicate and dihydroxyaluminum aminoacetate; Thickeners such as sodium polyacrylate, polyvinyl alcohol, polyvinylpyrrolidone, gelatin, sodium alginate, carboxymethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose and hydroxypropylmethyl cellulose; Polyhydric alcohols such as glycerin, polyethylene glycol (macrogol), polyethylene glycol and 1,3-butanediol; Surfactants such as polyoxyethylene derivatives; flavorings such as l-menthol; preservatives such as p-hydroxybenzoate; Purified water; And other suitable fillers may be added.

In the case of a tape agent, adhesives, such as a styrene-isoprene- styrene block copolymer and an acrylate resin; Adhesive resins such as acrylic saturated hydrocarbon resins, hydrogenated rosine resins and terpene resins; Softeners such as liquid gums and liquid paraffins; Antioxidants such as dibutylhydroxytoluene; Polyhydric alcohols such as polyethylene glycol; Adsorption promoters such as oleic acid; Surfactants such as polyoxyethylene derivatives; And other suitable fillers may be added. In addition, water-absorbing polymers such as sodium polyacrylate and polyvinyl alcohol and a small amount of purified water may be added to the preparation of the tape agent containing water.

In the case of aerosols, bases such as white and petrolatum (waceline), yellow and petrolatum, lanolin, purified beeswax, cetanol, stearyl alcohol, stearic acid, hydrogenated oil, hydrocarbon gel, polyethylene glycol, liquid paraffin and squalane; Oleic acid, isopropyl myristate, glycerol triisooctanoate, crotamiton, diethyl sebacate, diisopropyl sebacate, isopropyl adipate, hexyl laurate, fatty acids, fatty acid esters, vegetable oils, aliphatic alcohols and alcohols Solvents or stabilizers such as; Antioxidants such as tocopherol derivatives, L-ascorbic acid, dibutylhydroxytoluene and butylhydroxyanisol; preservatives such as p-hydroxybenzoate; Curb agents such as glycerin, propylene glycol and sodium hyaluronate; Carboxyvinyl polymers, such as those used in surfactant ointments, creams, gels or lotions, xanthan gum, carboxymethyl, such as polyoxyethylene derivatives, glycerol fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters and lecithin Thickeners such as cellulose and sodium carboxymethyl cellulose, hydroxypropyl cellulose and hydroxypropylmethyl cellulose; Stabilizer; Buffers; Sweetener suspending agents; Emulsifiers; Condiment; antiseptic; Adsorption promoters and other suitable fillers may be added.

For external acids, potato starch, rice starch, corn starch, fillers such as talc and zinc oxide, and other suitable additives may be added.

The external skin preparation of the present invention may be prepared by well mixing each component with a suitable base as necessary according to a well-known method for preparing the external skin preparation, and the preparation thus prepared is applied to the lesion as necessary.

In another embodiment of the present invention, to provide a cosmetic composition for improving or alleviating skin diseases containing an eriodictyol compound, a derivative thereof or a pharmaceutically acceptable salt thereof represented by the following formula (1) as an active ingredient It is characterized by:

[Formula 1]

.

In one embodiment of the present invention, the cosmetic composition for improving or alleviating skin diseases exhibits an effect selected from the group consisting of alleviating itching, inhibiting inflammatory reactions, suppressing edema, skin moisturizing and skin elasticity, and improving or alleviating the skin disease. Cosmetic composition for is characterized in that for the prevention or treatment of contact dermatitis.

In one embodiment according to the invention, the eriodictyol compound, a derivative thereof or a pharmaceutically acceptable salt thereof in the cosmetic composition is 0.0000001 to 10% by weight of the total cosmetic composition weight, more specifically from 0.0000005 to 5% by weight, more specifically, it may be included in an amount of 0.000001 to 1% by weight.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, skin cosmetic ointment, essence, whitening cream, lotion, emulsion, pack, general cosmetics, skin milk, cream, It can be prepared in various forms, such as serum, cosmetic soap, soft cosmetics, medicinal cosmetics, hair tonic, systemic cleanser and oil gel.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide are used as carrier components. Can be.

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

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

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Sung cellulose, aluminum metahydroxy, bentonite, agar or tragacanth and the like can be used.

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

In another embodiment of the present invention, a health functional food for improving or alleviating skin diseases containing an eriodictyol compound, a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient Its features include:

[Formula 1]

.

In one embodiment according to the present invention, the eriodictyol compound, a derivative thereof or a pharmaceutically acceptable salt thereof in the dietary supplement is 0.0001 to 10% by weight of the total dietary supplement, more specifically 0.0005 to 5% by weight, more specifically may be included in an amount of 0.001 to 1% by weight.

Hereinafter, the present invention will be described in more detail with reference to Experimental Examples and Preparation Examples. However, this is to help the understanding of the present invention, not intended to limit the scope of the present invention to this.

Experimental Example

(1) Eriodithiol was purchased from Elcomb Science (Seoul, South Korea), and mouse monoclonal anti-DNP IgE antibody, dinitriphenol-conjugated human serum albumin (DNP-HSA), and Evans Blue were sigma -N -Oleoyl-D-erythro-spingosin (C ₁₇ base) was purchased from Aldrich (St. Louis, Mo.) and p -nitrophenyl- N -acetyl-- from Avantiola Lipid (Alabaster, AL). D-glucosaminid was purchased from MP Biomedical (Solon, OH).

(2) cell culture

Rat mast cell line RBL-2H3 was purchased from the American Type Culture Collection (ATCC, Rockville, MD). Cells were cultured in Eagle minimal minimal medium (WelGENE, Daegu, Korea) containing 10% (v / v) fetal bovine serum and 100 units / mL penicillin-streptomycin (Lonza Walkersville, Walkersville, MD). Incubated at 5% CO ₂ . Cells were aliquoted in trypsin-EDTA solution and resuspended in fresh medium for use in the experiment.

(3) experimental animals

Male BALB / C mice (7 weeks old) were purchased from Orient Bio (Gangneung, South Korea) and bred at 40-50% relative humidity at 20-22. Animals were given free access to diet and water purchased from Samyang (Wonju, South Korea). The animal testing protocol has been approved by the Yonsei Institutional Animal Care and Use Committee. This experiment was confirmed by the NIH guidelines for the use and care of laboratory animals (NIH publication no. 85-23, revised 1996).

Experimental Example 1 Passive cutaneous anaphylaxis analysis

The animals were largely divided into 5 groups: normal control group (vehicle, n = 5), control group (antigen stimulated group, n = 5), and a group containing a small amount of erythrodiol (10 mg / kg, n = 5), the group containing medium amounts of eriodicthiol (30 mg / kg, n = 5) and the group containing a large amount of erythoxythiol (50 mg / kg, n = 5). . One ear of each mouse was injected into the skin with an anti-DNP-specific IgE antibody (0.5 μg). After 24 hours, mice were injected intravenously with 200 μL of DNP-HSA (200 μg; antigen) dissolved in PBS 3% Evans Blue. After one hour, eriodithiol was orally administered in an amount of 10-50 mg / kg. Two hours after antigen challenge, the mice were euthanized and the treated ears were removed to determine the amount of die to determine the amount of antigen response. The extracted ears were immersed in 500 μL of formamide and left overnight at 63 ° C. to extract the die, and quantified by measuring die absorbance at 620 nm.

As a result, in the passive dermal anaphylaxis (PCA) reaction, erythridiol showed a concentration-dependent antiallergic effect in the allergic reaction (see FIG. 2).

Experimental Example 2 Analysis of β-hexosaminidase Secretion Effect

As markers of degranulation, secretion of β-hexosaminidase was measured. RBL-2H3 cells were aliquoted into 24-well plates in 2 × 10 ⁵ cells / well and sensitized with anti-DNP-specific IgE 200 ng / mL overnight. IgE-sensitized cells were washed twice with PIPES buffer (25 mM PIPES, pH 7.2, 110 mM NaCl, 4 mM KCl, 0.4 mM MgCl, 40 mM HCl, 5.6 mM glucose, 1 mM CaCl ₂ , and 0.1% BSA). In addition, the treatment was performed by the solution of erythridiol solution dissolved in PIPES buffer for each concentration, the temperature was set to 37 ℃, was treated for 30 minutes. It was then activated with DNP-HSA (25 ng / mL) at 37 ° C. for 15 minutes and cooled with ice to stop activation. Supernatants from antigen-activated cells and P -hexosaminidase substrate ( p -nitrophenyl- N -acetyl-D-glucosaminid) in PIPES buffer were measured to determine β-hexosaminidase release. Well plates were mixed in 0.1 M citrate buffer (pH 4.5) and incubated at 37 ° C. for 1 hour. 0.1 M carbonate buffer (pH 10.5) was added to terminate the reaction, and the absorbance was measured at 405 nm using a microplate reader.

As a result, eriodicthiol was shown to inhibit IgE / Ag-induced β-hexosaminidase release in a concentration-dependent manner, and the effect was remarkable (see FIG. 3A).

Experimental Example 3 Cell Viability Assay

Cytotoxicity measurement of eriodic thiol was measured using the Ez-Cytox Enhanced Cell Viability Kit (Daily Lab Service, Seoul, Korea), and the protocol was followed. RBL-2H3 cells were dispensed into 96 × well plates at 2 × 10 ⁴ cells / well, inoculated with 200-ng / mL anti-DNP-specific IgE, and left overnight. After incubation, the medium was replaced with serum-free medium and treated for 23 hours with various concentrations of eriodicthiol and 20 ng / mL DNP-HSA. Ez-Cytox kit reaction was added to the medium and cells were incubated at 37 ° C. for 1 hour. Cell survival levels were determined by measuring absorbance at 450 nm using a microplate reader.

Experiments using the Ez-Cytox kit in the presence or absence of Ag (25 ng / mL) showed no induction of IgE / Ag reactions (Ag, 25 ng / mL) and obesity in the various concentrations of riodicthiol treated groups. There was no cytotoxicity against the cells (see FIG. 3B). In other words, the survival rate of the cells with erythrothiol was not less than 95%, indicating no toxicity to the cells.

Experimental Example 4 RNA Extraction and Reverse Transcription Polymerase Chain Reaction (RT-PCR)

RBL-2H3 cells were seeded in 6-well plates at 8 × 10 ⁵ cells / well and incubated overnight in medium containing 200 ng / mL anti-DNP-specific IgE. After washing twice, they were resuspended in PIPES buffer and activated for 1 hour with DNP-HSA (25 ng / mL) in the absence of erythrothiol and in the presence of erythoxythiol. After incubation, the cells were washed twice with ice-cold PBS. Total RNA was isolated using TRI reaction solution (Sigma-Aldrich, St. Louis, MO), but followed protocol. The concentration of total RNA was measured using a spectrophotometer. Total RNA (1 μg) was used as a template for cDNA synthesis and PCR using AccuPower RT / PCR premix kit (Bionia, Daejeon, Korea). The following primers were used:

rat IL-4 sense 5'-ACCTTGCTGTCACCCTGTTC-3 ';

rat IL-4 antisense 5'-TTGTGAGCGTHHACTCATTC-3 ';

rat TNF-α sense 5'-CAAGGAGGAGAAGTTCCCAA-3 ';

TNF-α antisense 5'-CGGACTCCGTGATGTCTAAG-3 ';

rat CERK sense 5'-AGGAGACTTTATACGAGATCA-3 '

rat CERK antisense 5'-GACTCGATAAACTTCAACGAA-3 ';

beta-actin sense 5'- ATGCCATCCTGCGTCTGGACCTGGC-3 '; And

beta-actin antisense 5'-AGCATTTGCGGTGCACGATGGAGGG-3 '.

Denaturation, annealing, extension conditions and number of cycles are as follows:

For IL-4, 1 minute at 94 ° C., 45 seconds at 50 ° C., 45 seconds at 72 ° C., 35 cycles,

For TNF-α, 1 minute at 94 ° C., 45 seconds at 49 ° C., 45 seconds at 72 ° C., 35 cycles,

For CERK, 30 cycles at 94 ° C., 30 seconds at 52 ° C., 1 minute at 72 ° C., 35 cycles.

PCR products were electrophoresed on 2% agarose gels and visualized by addition of ethidium bromide. Gels were tested using a transilluminator (Vilber Lourmat, France).

Inflammatory cytokines IL-4 and TNF-α are cytokines used as experimental indicators in allergic reactions. IL-4 induces IgE production in B cells to promote naïve T cells into Th2 cells. TNF-α is an inflammatory mediator secreted from mast cells via IgE challenge. As shown in FIG. 4, IgE / Ag-induced expression of IL-4 mRNA was shown to be significantly inhibited, and the effect was concentration dependent. The experimental results show that erythrodiol inhibits IgE production in B cells by down-regulating Il-4 expression, and also inhibits IgE / Ag-mediated degranulation.

Ca ²⁺ -dependent trigger in mast cell degranulation appears to be present in the enzyme CERK, which phosphorylates ceramide to C1P. Formation of C1P induces arachidonic acid release, synthesis of inflammatory metabolites, and Ca ²⁺ -dependently activates MAPK. When mast cells were activated for 1 hour with IgE / Ag, CERK mRNA was found to be increased and measured by RT-PCR. However, in the cells treated with erythridiol, CERK mRNA levels were significantly lower than those in cells activated only with IgE / Ag (see FIG. 5). It can be seen that it inhibits the ^{2 +} -dependent process.

Experimental Example 5 Measurement of Ceramide

RBL-2H3 cells were washed twice and harvested. Cell pellets were lysed with 0.2 N NaOH. Total lipids were extracted with ethanol and incubated at 37 ° C. for 1 hour with C ₁₇ ceramide ( N -oleoyl-D-erythro-sphingosine) as internal standard. The extract was centrifuged at 12,000 rpm for 10 minutes. The supernatant was dried in a vacuum centrifuge. The dried residue was dissolved in methanol, spotted on a high performance thin layer chromatography silica gel plate (Merck, Darmstadt, Germany), and isopropylether / methanol / 29% NH ₄ OH (40/10/1, v / v / v). The plate was immersed in 10% H ₂ SO ₄ and dried at 180 ° C. The region of the sample lane corresponding to the ceramide standard band was then cut out and the ceramide was eluted with methanol. The eluate was incubated for 1 hour and dried in a vacuum centrifuge. The ceramide residue was mixed with reaction buffer (pH 7.5) containing 25 mM Tris-HCl, 1% sodium cholate and 15% fatty acid-free BSA. The mixture was incubated at 37 ° C. overnight with 500 μU of sphingolipid ceramide N-deacylase (SCDase; Takara, Ohtsu, Japan) and dried in a vacuum centrifuge. The resulting extract was dissolved in methanol with an OPA reaction solution (50 mg OPA, 1 mL ethanol, 200 μL β-mercaptoethanol and 50 mL 3% boric acid, pH 10.5) and 30 minutes while derivatizing Liver cultures. The isocratic mobile phase for HPLC was then composed of methanol / water / triethylamine (85: 15: 0.1, v / v / v) and the flow rate was 1 mL / min. Intelligent fluorescence detectors were set to 340 nm (excitation wavelength) and 455 nm (emission wavelength).

Treatment with eriodicthiol showed ceramide concentrations comparable to that of unactivated mast cells and significantly lower ceramide concentrations compared to IgE / Ag-activated mast cells. (Refer to FIG. 6) It can be seen from the experimental results that erythrothiol shows an effect of inhibiting mast cell degranulation.

Laboratory Animals and Treatment

Eriodicthiol was purchased from Sigma-Aldrich (St. Louis, Mo. U.S.A.). Male ICR mice (6 weeks old) were purchased from Orient Bio (Gangneung, South Korea) and were bred at 40-50% relative humidity at 20-22 ° C. Animals were given free access to diet and water purchased from Samyang (Wonju, South Korea). The animal testing protocol has been approved by the Yonsei Institutional Animal Care and Use Committee.

Experimental Example 6 Induction of Allergic Contact Dermatitis (Preparation of Experimental Animal)

To induce allergic contact dermatitis, dissolve DNCB (2,4-dinitrochlorobenzene) as 1w / v% in a mixed solution of acetone and olive oil (mixed volume ratio 3: 1) for 7 weeks. Primary immunization was performed by applying 300 μL to 2 cm ² of the dorsal region of male ICR mice. After 4 days of primary immunization, 250 μL of 0.5% DNCB was applied to the dorsal area and secondary immunization was performed by continuously treating the same concentration and the same amount for about 2 weeks. In the group to which riodic thiol was applied, 200 μL was applied to the dorsal area at the concentration of 0.2 mg / mL once every other day from the second DNCB administration.

Experimental Example 7 Change in Scraping Behavior

Scraping behavior verification experiments were conducted to measure the number of scratching behaviors caused by contact dermatitis through video recording at 24 hour intervals after the second challenge. After secondary application of DNCB, scratching of the dorsal area of the subject continued to increase after a lack of dorsal epidermis. In the group that was administered with riodic thiol, it was observed that after the third administration, the scratching gradually decreased and recovered (see FIG. 7).

Experimental Example 8 Skin Sensory Evaluation

The skin condition is graded as erythema, itching and dry skin, edema and hematoma, soreness, thyroiditis, no symptoms 0 points, symptom weakness 1 point, normal 2 points, severe 3 points for these 5 items The total score was given a score between a minimum of 0 (no symptoms) and a maximum of 15 (all symptoms severe). In the skin sensory evaluation, the score was gradually increased in the group in which contact dermatitis was induced by the application of DNCB and reached 13 or more. However, in the experimental group in which riodic thiol was applied, the score was significantly lowered than that of the normal group. (See FIGS. 8A and 8B).

Experimental Example 9 Change in the Thickness of the Dorsal Epidermis (Identification of Dorsal Epidermal Edema)

The thickness of the dorsal epidermis was confirmed by tissue extraction of the dorsal epidermis and staining with hematoxylin and eosin. Compared to the normal group, the thickness of the dorsal epidermis was increased continuously after the second application of DNCB. In the group of riodic thiol administered, the edema was significantly alleviated from the third dose, and it can be seen that there is an anti-inflammatory effect on riodic thiol due to the result (see FIGS. 9A and 9B).

Experimental Example 10 Changes in Serum IgE

Blood IgE levels were measured at 450 nm using the IgE ELISA kit for mice. Compared to the normal group, the concentration of IgE increased more than three times in the group of DNCB-induced contact dermatitis, but IgE was lowered in the group administered with both DNCB and erythrothiol. As a result, it could be confirmed that eriodic thiol causes an allergic reaction due to an increase in immunoglobulin (see FIG. 10).

Experimental Example 11 Skin Moisturizing Effect Test of Eriodithiol

Skin moisturizing effect experiment was performed by making a cream and serum containing 0.0006% of pure eriodictyol powder. The device used for the skin moisturizing effect was MY-808S Moisture Checker (Scalar, Japan), and clinical trials were performed using the inside of the forearm of a person.

In the moisturizing power measurement method, the skin moisturizing power before application was checked including the control group, and each sample 0.25 g / cm ² was applied, and the skin moisturizing power after 1 hour, 2 hours, 3 hours, and 4 hours was measured.

Skin moisturizing power is very sensitive to temperature and humidity, so it was carried out in a constant temperature and humidity room controlled at a temperature of 20 ° C. and a humidity of 60%. The test subjects were 5 males and 20s.

As a result of the skin moisturizing effect, the results of testing with MY-808S Moisture Checker are shown in FIG. 11. As shown in FIG. 11, a cream containing 0.0006% pure eriodictyol single powder had a skin moisturizing effect of 29.52% before application and a 32.88% increase in skin moisturizing effect after application. As a result, it was confirmed that after 4 hours, the skin moisturizing effect was maintained to almost the same level as the right skin.

In addition, as shown in FIG. 12, a serum containing 0.0006% of pure eriodictyol single powder had a skin moisturizing effect of 33.54% after application on 30.2% of skin moisturizing power before application. After 4 hours, the skin moisturizing effect was maintained to the same degree as the right skin.

Table 1

Preparation Example 1 Preparation of Powder

Eriodichol	0.001% by weight
Sucrose
	100 mg
Talc
	10 mg

The powders are mixed and mixed, followed by filling into an airtight fabric to prepare a powder.

TABLE 2

Preparation Example 2 Preparation of Tablet

Eriodichol	0.001% by weight
Starch
	100 mg
Sucrose
	100 mg
Magnesium Stearate
	2 mg

Tablets are prepared by mixing the above components according to a conventional method for preparing tablets and then compressing them.

TABLE 3

Preparation Example 3 Preparation of Capsule

Eriodichol	0.001% by weight
Crystalline cellulose
	3 mg
Lactose	15 mg
Magnesium Stearate
	1 mg

The capsules are prepared by mixing the above components and filling the gelatin capsules according to a conventional capsule preparation method.

Table 4

Preparation Example 4 Preparation of Granules

Eriodichol	0.001% by weight
Soybean Extract
	50 mg
glucose
	200 mg
Starch
	500 mg

After mixing the above components, 100 mL of 30% ethanol was added, dried at 60 ° C. to form granules, and then filled into a cloth to prepare granules.

Table 5

Preparation Example 5 Preparation of Pill

Eriodichol	0.001% by weight
Lactose	1,500 mg
glycerin	1,500 mg
Starch	980 mg

After mixing the above components it is prepared according to the conventional manufacturing method of pills.

Table 6

Preparation Example 6 Preparation of Injection

Eriodichol	0.001% by weight
Mannitol	180 mg
Sterile Distilled Water for Injection	2,970 mg
Na _₂ HPO ₄ · 12H ₂ O	30 mg

Prepared by mixing the above components to make (2 mL) per ampoules according to a conventional injection method.

TABLE 7

Preparation Example 7 Preparation of Liquid

Eriodichol	0.001% by weight
Iseong Hwadang	10,000 mg
Mannitol	5,000 mg
Purified water	Quantity

It is prepared by dissolving the above components in purified water according to a conventional liquid preparation method, adding an appropriate flavor and then filling the bottle with sterilization.

Table 8

Preparation Example 8 Cream

A. Paid
Eriodichol	0.001% by weight
Stearic acid
	10
Stearyl alcohol	4
Butyl stearate	8
Stearic Acid Monoglycerin Ester	2
Vitamin E Acetate	0.5
Vitamin A Palmitate	0.1
Octylmethoxycinnamate	2
Macadamia Nuts You	One
Tea tree oil	One
Spices	0.4
antiseptic	Quantity

B. Award
Eriodichol	0.001% by weight
glycerin
	4
1,2-pentadiol	3
Sodium hyaluronate	One
Potassium hydroxide	2
Ascorbic Acid Magnesium Phosphate	0.1
L-arginine hydrochloride	0.01
Trisodium Edetate	0.05
Purified water	residual

quite

The oil phase part of A and the water phase part of B are respectively heated to 70 ° C. and completely dissolved. Phase A is added to phase B and emulsified with an emulsifier. The emulsion was cooled using a heat exchanger to obtain a cream.

Table 9

Preparation Example 9 Latex

A. Paid
Eriodichol	0.001% by weight
Squalane
	5
Oleyl oleate
	3
Vaseline	2
Solbitan sesquioleic acid ester	0.8
Polyoxyethylene oleyl ether	1.2
Evening Primrose Oil	0.5
Spices	0.3
antiseptic	Quantity

B. Award
Eriodichol	0.001% by weight
1,3-butylene glycol	4.5
ethanol	3
Carboxy Vinyl Polymer	0.2
Potassium hydroxide	0.1
L-asparaginate	0.01
Edetate	0.05
Purified water	residual

quite

The oil phase of A is added to the water phase of B to obtain an emulsion.

Table 10

Preparation Example 10

A. Alcohol Award
ethanol	5.0 wt%
POE oleyl alcohol ether	2
Spices	0.05

B. Award
Eriodichol	0.001% by weight
1,3-butylene glycol	9.5
Pyrrolidone Sodium Carbonate	0.5
Nicotinic acid amide	0.3
glycerin	5
Dimorpholino pyridadione	0.1
Purified water	Remaining amount

quite

The alcohol phase of A was added to the aqueous phase of B and solubilized to obtain a lotion.

Table 11

Preparation Example 11 Latex

Eriodichol	0.001% by weight
Cetyl alcohol ether	One
Silicon KF96 (20cs)	2
Liquid paraffin	3
Propylene glycol	5
glycerin	2
Ethyl alcohol	5
Carboxy Vinyl Polymer	0.3
Hydroxypropyl cellulose	0.1
2-aminomethylpropanol	0.1
Vitamin A Acid	0.05
Alkylene oxide derivatives	2
Hydroxyproline	0.1
Trehalose	One
antiseptic	Quantity
Spices	Quantity
Distilled water	Remaining amount

quite

Latex was prepared by the conventional method by the said prescription.

Table 12

Preparation Example 12 Converging Cosmetics

Eriodichol	0.0001% by weight
Dipropylene glycol
	2
Citric acid	0.03
Citric acid	0.05
Alkylene oxide derivatives	0.1
Ethyl alcohol	5
Hydroxyproline	0.03
Trehalose	0.5
antiseptic	Quantity
Spices	Quantity
Distilled water	Remaining amount

quite

The lotion was manufactured by the conventional method by the said prescription.

Table 13

Preparation Example 13 Cream

A. Paid
ethanol	2.0 wt%
Vaseline
	2
Squalane	20
Glycerol Monofatty Acid Ester	2
Tween60	3
Isopropyl Millistate	6
Glycylyte sterate
1	0.5
Glycerin Mono-2-ethylhexanoyl-diparamethoxycinnamate	0.05
4-methoxy-4'-t-butyldibenzoylmethane	0.05
BHT	0.01
antiseptic	0.3
Spices	0.2

B. Award
Eriodichol	0.0001% by weight
glycerin
	10
Propylene glycol	5
Alkylene oxide derivatives	0.1
Hydroxyproline	0.05
Trehalose	5
Trisodium Edetate	0.1
Potassium hydroxide	One
Distilled water	Remaining amount

quite

The cream was manufactured by the conventional method by the said prescription.

The prevalence of patients with dermatitis diseases is gradually increasing, and currently, antihistamines or steroids are used as symptom relievers, and there are few products that can cure skin diseases fundamentally. According to the pharmaceutical composition for preventing and treating skin diseases and the external preparation for skin, cosmetic composition for improving or alleviating skin disease and health functional food, the demand is expected to be large.

Claims

A pharmaceutical composition for preventing or treating skin diseases containing an eriodictyol compound represented by Formula 1, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

.
The method of claim 1,

The pharmaceutical composition is a pharmaceutical composition for preventing or treating skin diseases, characterized in that it exhibits an effect selected from the group consisting of relief of itching, inhibition of inflammatory response, edema inhibition, skin moisturizing and skin elasticity.
The method of claim 1,

The pharmaceutical composition is a pharmaceutical composition for preventing or treating skin diseases, characterized in that for the prevention or treatment of contact dermatitis.
The method of claim 1,

A pharmaceutical composition for preventing or treating skin diseases, wherein the eriodictyol compound, a derivative thereof, or a pharmaceutically acceptable salt thereof is 0.001 to 10% by weight of the total weight of the pharmaceutical composition.
Skin external preparation for preventing or treating skin diseases containing an eriodictyol compound represented by Formula 1, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

.
The method of claim 5,

The external skin preparation for external skin prevention or treatment of skin diseases, characterized in that it exhibits an effect selected from the group consisting of relief of itching, inhibition of inflammatory response, edema inhibition, skin moisturizing and skin elasticity.
The method of claim 5,

The skin external preparation for skin disease prevention or treatment, characterized in that for the prevention or treatment of contact dermatitis.
The method of claim 5,

The skin external preparation is any one selected from the group consisting of ointments, creams, lotions, solutions, dressings, patches, blisters, tapes, aerosols, external acids and sprays. Skin external preparations.
The method of claim 5,

The skin external preparation for contact dermatitis prevention or treatment, characterized in that the eriodictyol compound, a derivative thereof or a pharmaceutically acceptable salt thereof is 0.0001 to 5% by weight of the total weight of the external skin preparation.
A cosmetic composition for improving or alleviating skin diseases containing an eriodictyol compound represented by Formula 1, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

.
The method of claim 10,

The cosmetic composition is a cosmetic composition for improving or alleviating skin diseases, characterized in that it exhibits an effect selected from the group consisting of itching, inflammatory response inhibition, edema inhibition, skin moisturizing and skin elasticity enhancement.
The method of claim 10,

The cosmetic composition is a cosmetic composition for improving or alleviating skin diseases, characterized in that for the prevention or treatment of contact dermatitis.
The method of claim 10,

The cosmetic composition for improving or alleviating skin diseases, characterized in that the eriodictyol compound, a derivative thereof or a pharmaceutically acceptable salt thereof in the cosmetic composition is 0.000001 to 1% by weight of the total cosmetic composition weight.
A health functional food for improving or alleviating skin diseases containing an eriodictyol compound represented by Formula 1, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

.
The method of claim 14,

Health functional food for improving or alleviating skin diseases, characterized in that the eriodictyol compound, a derivative thereof or a pharmaceutically acceptable salt thereof is 0.001 to 1% by weight of the total health functional food.
Use of preventing, ameliorating, alleviating or treating skin diseases of an eriodictyol compound represented by Formula 1, a derivative thereof or a pharmaceutically acceptable salt thereof:

[Formula 1]

.