KR101934794B1 - Composition for preventing, improving or treating atopic dermatitis comprising extract mixture of Diospyros lotus leaf and grape fruit stem as effective component - Google Patents

Abstract

The present invention relates to a composition for preventing, ameliorating or treating atopic dermatitis, which comprises a mixed extract of a gomule leaf and a grape clover, as an active ingredient. The present invention relates to a composition for preventing, improving or treating atopic dermatitis, Compared with the treatment with gomex leaf extract or grape clover extract, which is known to have an anti-atopic dermatitis effect, the thickness of skin induced by atopic dermatitis is remarkably reduced and the production of IgE and IL-4, (TNF-alpha) and IL-6 (interleukin 6) which are inflammatory cytokines, and inhibit itching. Accordingly, the present invention is effective for preventing, ameliorating or treating atopic dermatitis by inhibiting the progress of atopic dermatitis, so that it can be effectively used for the development of a therapeutic agent for atopic dermatitis, a health functional food for atopic patients or a cosmetic for atopic skin.

Description

[0001] The present invention relates to a composition for preventing, ameliorating or treating atopic dermatitis, which comprises as an active ingredient, a mixed extract of Gomex leaf and grape clover,

The present invention relates to a composition for preventing, ameliorating or treating atopic dermatitis, which comprises a mixed extract of gomoyobis and grape bunch branches as an active ingredient.

Atopic dermatitis is accompanied by severe itching (pruritus) with chronic skin inflammation. The itching that causes feelings of scratching is induced by an itch medication such as histamine. In the case of a normal person, the symptoms are easily alleviated by scratching. However, in the case of atopic patients, scratching is more likely to cause scratching and scratching. These scratching behaviors of atopic patients may cause skin barriers to collapse and secondary infections leading to further deterioration of inflammation. Atopic dermatitis can be temporarily alleviated, but it recurs and is aggravated by stimulants such as the environment and food, and deterioration and mitigation are repeated, and the cause is not clearly known yet. Thus, the inflammation associated with atopic dermatitis is caused by excessive immune cell action, resulting in inflammatory cytokines such as IgE, interleukin-4 (IL-4, interleukin-4), hydroxyl radicals (-OH), superoxide radicals ₂ ^- ), hydrogen peroxide (H ₂ O ₂ ), and the like. Therefore, in order to ameliorate skin diseases such as atopic dermatitis, materials that can relieve itching and effectively remove active oxygen species are needed.

In addition, mast cells are known to be a key participant in acute and chronic inflammatory diseases in most allergic diseases including atopic dermatitis. When mast cells are activated, they release inflammatory cytokines such as tumor necrosis factor (TNF) -α, interleukin (IL) -1 and IL-6, as well as pruritogens such as histamine. Thus, inflammatory cytokines derived from mast cells further promote the inflammatory reaction, while itching substances such as histamine further promote itching, thereby causing skin barrier disruption. Thus, identifying substances that can inhibit inflammatory cytokines and itch-mediated substances without side effects will be effective in controlling allergic diseases, including atopic dermatitis.

On the other hand, Goyom ( Diospyros lotus L.) is a deciduous plant which is naturally native in Asia including Korea and China. In the field of traditional medicines, Goyom, a mature fruit, has been used for calming, analgesic, convergence and constipation treatment. Goyom's biochemical components have been reported fatty acids, sugars, flavonoids and non-volatile substances. In recent years, there have been reports of anticoagulation, brain cell protection, antioxidant and anticancer effects of blood. Korean Patent Laid-Open Publication No. 2015-0122933 discloses a composition for preventing or ameliorating atopic dermatitis containing an ethyl acetate fraction of a methanol extract of Gomatopoeia as an active ingredient.

In addition, grapes ( Vitis vinifera L.) is known to exhibit various biological control functions due to its abundance of polyphenol components. Most of them are contained in seeds (60 ~ 70%) and peel (30%), The preference of processed foods such as juice and wine increased. The grapes cultivated in Korea are the American species ( Vitis labrusca L.), European species ( Vitis vinifera L.) and hybrids between these ( Vitis labruscana L.). Until now, grape clusters have not been utilized industrially, but recently research on biologically active substances by grape parts has been reported, and an opportunity to utilize grape byproducts industrially was established. For example, resveratrol, the main component of grapes in Campbell early varieties, contains higher amounts of grapevines than grapevines, rinds, and leaves, leaving the grapes that are thrown away by pruning Possibilities for use as a by-product have been reported. However, no case has been reported using grape clusters.

On the other hand, Korean Patent Registration No. 0913436 discloses a composition for treating or preventing asthma, atopy or rhinitis, which contains grape extract as an active ingredient, as an example of prior art related to the anti-atopy of grape extract. However, the composition for preventing, ameliorating or treating atopic dermatitis containing the combined extract of goose's leaves and grape clusters of the present invention as an active ingredient has not yet been disclosed.

The present invention provides a composition for preventing, ameliorating or treating atopic dermatitis, which comprises as an active ingredient, a mixed extract of gomoyobis and grape clusters, Compared with single gomex leaf extract or grape gum extract, the mixed extracts of P. japonica significantly reduce the number of mast cells, which are major cells causing atopy and thickness of skin tissue induced by atopy, The amount of IgE and IL-4, which are the inducers of dermatitis, is markedly inhibited, thereby completing the present invention.

In order to achieve the above object, the present invention provides a health functional food composition for preventing or ameliorating atopic dermatitis comprising Diospyros lotus leaf and grape fruit stem mixed extract as an active ingredient.

The present invention also provides a cosmetic composition for preventing or ameliorating atopic dermatitis comprising Diospyros lotus leaf and grape fruit stem mixed extract as an active ingredient.

The present invention also provides a pharmaceutical composition for the prevention or treatment of atopic dermatitis comprising Diospyros lotus leaf and grape fruit stem mixed extract as an active ingredient.

The present invention relates to a composition for preventing, ameliorating or treating atopic dermatitis, which comprises a mixed extract of a gomule leaf and a grape clover, as an active ingredient. The present invention relates to a composition for preventing, improving or treating atopic dermatitis, The effect of alleviating the symptom of atopic dermatitis is more remarkable than the treatment of gomoma leaf extract or grape cloverleaf extract, which is known to have an anti-atopic dermatitis effect, so that it can be effectively used for the development of a health functional food, cosmetics or atopic dermatitis treatment for atopic patients Can be used.

FIG. 1 shows the results obtained by confirming the inhibitory effect of inflammatory cytokine production on the treatment of mixed extracts of Gomex leaf and grape seedlings of the present invention. (A) shows the degree of suppression of TNF-α production, while (B) shows the degree of suppression of IL-6 production. The DLE + MGFE was treated with atypia-induced group, and the MGFE-treated group was treated with the extract of grape seedlings at the atopy-induced group. And *** indicates that there is a statistically significant difference in the treatment of mixed extracts of goat's leaves and grape clusters compared to those treated with goat's leaf extract or grape clover extract alone, which means p <0.0001 do.
FIG. 2 shows the results of comparing the effects of the extracts of Gomex leaf, grape clover, and the extract of the present invention on the itching effect of the compound extract of 48/80, to be. *, ** means that the mixed extract (DLE + MGFE) of goose leaves and grape clusters of the present invention is statistically different from that of single goose's leaf extract (DLE) or grape cloverleaf extract (MGFE) * Is p < 0.05 and ** is p < 0.01.
FIG. 3 shows that when a mixed extract of goose leaves and grape clusters of the present invention was treated with a model mouse treated with DNCB (2,4-dinitrochlorobenzene) as an atopy inducer, The photograph (A) of the model mouse showing that the mitigation effect is excellent and the improvement effect of the skin lesion of the atopic dermatitis model mouse are shown by the clinical score (B).
FIG. 4 is a result of confirming the atopy mitigation effect after treating the ear skin tissue of a model mouse in which atopic dermatitis was induced, with the mixed extract of goose leaves and grape clusters of the present invention (DLE + MGFE). (A) shows the effect of reducing the infiltration of inflammatory cells in the ear skin tissue through H & E staining. (B) shows degranulation of mast cells inducing atopy through toluidine blue staining. (C) shows a decrease in the number of mast cells causing atopy, and (D) shows a decrease in ear skin tissue thickness. *, ** means that the mixed extract (DLE + MGFE) of goose leaves and grape clusters of the present invention is statistically different from that of single goose's leaf extract (DLE) or grape cloverleaf extract (MGFE) * Is p < 0.05 and ** is p < 0.01.
FIG. 5 shows the result of confirming the atopy mitigation effect after treating the dorsal skin tissue of a model mouse in which atopic dermatitis was induced with the mixed extract of goose leaves and grape clusters of the present invention (DLE + MGFE). (A) shows the effect of reducing the infiltration of inflammatory cells in the dorsal skin tissue through H & E staining. (B) shows the degranulation of mast cells inducing atopy through toluidine blue staining. (C) shows a decrease in the number of mast cells causing atopy, and (D) shows a decrease in dorsal skin tissue thickness. * And ** indicate that the treatment of the mixed extract of goose leaves and grape clusters of the present invention (DLE + MGFE) was statistically significantly different from the treatment of goose's leaf extract (DLE) or grape cloverleaf extract (MGFE) , P is &lt; 0.05, and ** is p &lt; 0.01.
FIG. 6 is a graph showing the changes in the levels of IgE (immunoglobulin E) (A) and IL-4 (interleukin-4) (B) in the atopic dermatitis model mice treated with the mixed extract of Dillon- Production inhibitory effect. * And ** indicate that the treatment of the mixed extract of goose leaves and grape clusters of the present invention (DLE + MGFE) was statistically significantly different from the treatment of goose's leaf extract (DLE) or grape cloverleaf extract (MGFE) , P is &lt; 0.05, and ** is p &lt; 0.01.

The present invention relates to a health functional food composition for preventing or ameliorating atopic dermatitis comprising Diospyros lotus leaf and a grape fruit stem mixed extract as an active ingredient. The solvent of the mixed extract is preferably water, a C ₁ -C ₄ lower alcohol, or a mixture thereof, but is not limited thereto. The combined extracts of Gomex leaf and Grapeseed mulberry leaves are prepared by 1) cutting gyoomum and grape seeds and drying at 35 ~ 45 ° C for 12 ~ 20 hours; 2) adding 1.0 to 3.5 liters and 0.4 to 2 liters of ethanol to the 50 to 150 g of the goat leaves and the 20 to 100 g of the grape seedlings, respectively, dried in the step 1), and extracting them for 2 to 5 days; And 3) mixing each of the extracts obtained in the step 2). Preferably, the extract is obtained by 1) cutting the gomule leaf and the grape seedlings, Drying; 2) To 2 g of 50% (v / v) ethanol was added to 100 g of goat leaves dried in step 1), and 1 liter of 80% (v / v) ethanol was added to 50 g of dried grape clusters. ; And 3) a step of mixing the extract of Guillaume Leaf Leaf and the grape seedling extract obtained in the step 2), but the present invention is not limited thereto. The mixed extracts of guanyum leaf and grape clover are preferably mixed at a weight ratio of 1: 0.1 to 10, more preferably 2: 6: 0.5 to 3.0, and still more preferably, Eggplant extract in a weight ratio of 4: 1 or 2: 1, but the present invention is not limited thereto.

The health functional food composition for preventing or ameliorating atopic dermatitis of the present invention may be prepared in any form of powder, granule, ring, tablet, capsule, candy, syrup and beverage, but is not limited thereto. The mixed extract may include any one of an extract obtained by the extraction treatment, a diluted or concentrated liquid of the extracted liquid, a dried product obtained by drying the extracted liquid, an adjusted product, or a purified product. The health functional food composition is not particularly limited as long as it can be ingested to prevent or ameliorate atopic dermatitis. When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is, or may be used together with other food or food ingredients, and suitably used according to a conventional method. The active ingredient may be suitably used depending on its intended use (prevention or improvement). Generally, in the production of food or beverage, it is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the health functional food composition of the present invention. However, in the case of long-term intake for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range. There is no particular limitation on the kind of the food. Examples of the foods to which the health functional food composition can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, soups, Drinks, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense. In addition, the health functional food composition of the present invention can be produced as a food, particularly a functional food. The functional food of the present invention includes components that are ordinarily added in food production, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, in the case of a drink, a natural carbohydrate or a flavoring agent may be included as an additional ingredient in addition to the active ingredient. The natural carbohydrate may be selected from the group consisting of monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, , Xylitol, sorbitol, erythritol, etc.). The flavoring agent may be a natural flavoring agent (e.g., tau Martin, stevia extract, etc.) and a synthetic flavoring agent (e.g., saccharin, aspartame, etc.). In addition to the above-mentioned health functional food composition, it is possible to use various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, A carbonating agent used in beverages, and the like.

The present invention also relates to a cosmetic composition for preventing or improving atopic dermatitis comprising Diospyros lotus leaf and grape fruit stem mixed extract as an active ingredient. In the cosmetic composition for improving atopic dermatitis according to the present invention, the cosmetic composition for improving atopic dermatitis may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing oil, powder foundation A cream, a softening agent, a nutrient lotion, a pack, an essence, a hair tonic, a shampoo, a rinse, a hair conditioner, a hair conditioner, a hair conditioner, an emulsion, a foundation, a wax foundation and a spray. , Hair treatment, gel, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutritional cream, eye cream, moisturizing cream, hand cream, foundation, nutrition essence, line Screen, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser Any one of the formulations may be used, but is not limited thereto. The composition of each of these formulations may contain various kinds of bases and additives necessary for formulation of the formulation, and the kinds and amounts of these ingredients can be easily selected by those skilled in the art. When the formulation of the present invention is a paste, cream or gel, animal fibers, plant fibers, wax, paraffin, starch, tracant, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide are used as carrier components . 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 as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether. In the case of the solution or emulsion of the present invention, a solvent, a solvent or an emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters. When the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used. When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component is selected from aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolenic derivatives or ethoxylated glycerol fatty acid esters.

The present invention also relates to a pharmaceutical composition for preventing or treating atopic dermatitis comprising Diospyros lotus leaf and grape fruit stem mixed extract as an active ingredient. The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions. The pharmaceutical dosage forms of the compositions according to the invention may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set. The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and injections according to conventional methods . Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition containing the mixed extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. have. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

Materials and methods

1. Goose leaves and Grape bunch  Manufacture of Egg Mixture Extracts

Gomel leaf was collected from Shingi Village, Suwang - ri, Buyeon - myeon, Jinan - gun, Jeollabuk - do, and was commissioned by Professor Kim Hong - joon of the School of Oriental Medicine, Woosuk University, and the identified specimen was stored in the specimen storage room of Ato Q & A Institute of Venture Business of Jeonju University. The prepared goose leaves were finely cut and then dried at 40 DEG C for 16 hours. The dried goose leaves (100 g) were ground and added to 50% (v / v) ethanol (2,000 ml) for 3 days, filtered using a 0.45 μm filter, lyophilized and recovered and stored at -20 ° C., An appropriate amount of the sample was dissolved in distilled water to give a concentration of 5 μg / ml and used in the following examples.

Muscat Bailey A grape fruit stem was purchased from Baek San-ri Hope Farm in Shintae-eup, Jeongeup, Jeonbuk Province and cut into small pieces and dried at 40 ℃ for 16 hours. Dried grape clusters (50 g) were pulverized and added to 80% (v / v) ethanol (1,000 ml), and the mixture was extracted for 3 days, filtered through a 0.45 μm filter, lyophilized, collected and stored at -20 ° C. , And an appropriate amount of the sample was dissolved in distilled water to give a concentration of 1.25 μg / ml and used in the following examples.

The final concentration of the mixed extracts was 2.5 mu g / ml of goat's leaf extract and 0.625 mu g / ml of grape clover extract, respectively. The extract and 1.25 占 퐂 / ml of grape seedling extract were mixed in the same volume and used in the following examples.

2. Inflammatory cytokines in HMC-1 cells ( TNF -α and IL-6) measurements

HMC-1 cells were seeded in a 6-well plate to a final concentration of 5 × 10 ⁵ cells / ml and cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours. Then, 5 μg / ml of goat's leaf extract, 1.25 μg / (The final concentration of each extract in the mixture is 2.5 mu g / ml of goat's leaf extract and 0.625 mu g / ml of grape bunch branch extract), and then the inflammatory reaction inducer PMA (Minneapolis, USA) using an ELISA kit for TNF- [alpha] and IL-6 from the supernatant after 16 hours of stimulation with A23187 (1 [mu] M) .

3. Experimental animals

Seven week old male hairless mice and four week old male ICR mice raised in sterile environment were purchased from Central Laboratory Animals Co., Ltd. (Seoul, Korea) and purified for one week while feeding with sufficient feed and water. Respectively. The incubation environment consisted of day and night cycles for 12 hours, and the temperature (20 ~ 22 ℃) and humidity (50 ~ 60%) were kept constant and tested according to the regulations of the Laboratory Animals Committee.

4. Itching inhibition effect

In order to confirm the inhibitory effect of the mixed extract of goat's leaves and grape clusters of the present invention on ICR mice, 5 ICR mice were put into each of the transparent acrylic cages (20 x 26 x 13 cm) for each test group and left in the same environment for 30 minutes . In the control group, oral administration of physiological saline was carried out. In the experimental group, 40 mg / kg of goose's leaf extract (DLE), 20 mg / kg of grape cloverleaf extract (MGFE) + MGFE 10 mg / kg) was orally administered to the mice at a dose of 0.1 ml each. After 60 minutes, the compound 48/80 (compound 48/80), which is an inducer of itching, was injected at a concentration of 50 μg / (Sc injection) to induce itching. Mice injected with the itch inducing substance were administered with a micro-camera (ONCCTV, Seoul, Korea) according to the method of Mihara et al., 2004, Br J Dermatol, 151 (2), 335-345) And the number of scratching of the area where the itching inducer was injected into the hind paw was counted by a double blind test.

5. DNFB (2,4- dinitrofluorobenzene ) And House  Induction of atopic dermatitis by application of mite antigen

To induce chemoattractant-induced atopic dermatitis model animals, 0.15% DNFB (2,4-dinitrofluorobenzene) was prepared in a solvent prepared from acetone and olive oil at a ratio of 3: 1. Mu] l). From the second sensitization day 4, the control group was orally administered the physiological saline, and a mixture of 40 mg / kg of goatee leaf extract (DLE), 20 mg / kg of grape cloverleaf extract (MGFE) and 30 mg / kg of goat The extracts (DLE 20 mg / kg + MGFE 10 mg / kg) were orally administered by 0.1 ml each day once a day until the end of the experiment. On the 7th, 10th, and 13th days after the first sensitization, 0.2% DNFB was applied at 3-day intervals and completely dried. After applying Biostir, Hyogo, Janpan 100mg of house dust mite antigen derived from house dust mite antigen three times Challenge.

6. Serum and tissue thickness measurements

Five hours after the last challenge, blood was collected from the liver portal vein of the mouse to obtain serum, and the thickness of the back and ear skin tissues of the mice was measured using a digital caliper (Mitutoyo, Japan).

7. Evaluation of skin lesions

The skin condition was examined by taking pictures after the experiment. The skin was checked for dryness, scaling, erosion, excoriation, bleeding, and the lesion was classified as 0 (none), mild as 1 point (mild) (Moderate) and severe (severe), respectively, and the total score was assigned to each step.

8. Skin tissue H & E staining and toluidine blue  dyeing

For histopathological analysis, H & E staining (Hematoxylin & Eosin staining) was performed. It was found that the hairless mouse model which induced the itching-induced ICR mouse model and atopic dermatitis and the skin model of about 5x5 mm were sacrificed by oral administration of the gomoma leaf extract, the grape clover extract and the mixed extract of goose leaf and grape clover, Respectively. After washing with saline, water was removed and fixed with 4% paraformaldehyde (pH 7.4). Paraffin blocks were prepared through a series of procedures. The tissue sections cut to a thickness of 5 μm were deparaffinized and functionalized and stained with H & E and observed under a microscope (× 100, Olympus, Tokyo, Japan). The cells were stained with toluidine blue and photographed at 100 × (H & E) and 400 × (toluidine blue) microscope fields at low magnification (40 ×) to observe leukocyte infiltration and mast cells.

9 . The serum IgE ( immunoglobulin E) and IL-4 ( interleukin- 4) cytokine assay

IL-4 and IgE were measured in atopic dermatitis model mice by administering a single extract of goose leaves and grape clusters and a mixed extract thereof. After 5 hours, serum was taken and IgE and IL-4 were measured from the serum. IL-4, and IgE were assayed by a method provided by R & D Systems and Shibayagi (Hyogo, Japan) using an ELISA kit that specifically works with each of anti-mouse IL-4 and anti- Were measured and quantified. In summary, 100 μl of cell supernatant or 5-fold diluted serum was injected into each antibody-coated plate, reacted, washed well, injected with secondary antibody with horseradish peroxidase, (Molecular Devices, USA). The quantification of each substance was performed by treating each substance with a specific concentration to prepare a standard curve, and the substance contained in the cell supernatant or serum Was calculated.

10. Statistical processing

Statistical analysis was performed with ANOVA and Student's t-test. Statistical significance limits were expressed as *, ** and ***, * means p <0.05, and * * Means p < 0.01 and *** means p < 0.001.

Example  1. In the HMC-1 cell line, Grape bunch  Inhibition of Inflammatory Cytokine Formation by Mixed Extract of Eggplant

TNF-α is a typical cytokine that causes inflammatory responses in vivo, and is mainly produced in macrophages, which are mainly activated. However, lymphoid cells such as lymphoid cells, mast cells, endothelial cells and the like It is also produced in various cells present in the body. Therefore, in Example 1, the effect of the mixed extract of goose leaves and grape clover on the inhibition of the production of TNF-α and IL-6, which are inflammatory cytokines, , The amount of TNF-α and IL-6, which are inflammatory cytokines, was measured in HMC-1 cell culture medium. As a result, as shown in FIG. 1, when the mixed extract of goose leaves and grape seedlings of the present invention was treated with a single extract of gourd leaf and grape clover, which is known to have an anti-atopic dermatitis effect, TNF- And IL-6 production inhibitory effect were statistically significantly superior.

Example  2. The goat leaf of the present invention and Grape bunch  Confirmation of itching inhibition effect by treatment of mixed extract of eggplant

The skin tissue of the mouse injected subcutaneously with the compound 48/80, which is an inducer of itching in ICR mice, a hypodermic injection of the above compound 48/80, a gomoma leaf extract, a grape clover extract, and a mixed extract of gourd leaf and grape clover Were orally administered to the mice.

As a result, as shown in Figs. 2A, 2B, and 2C, it was confirmed that infiltration of inflammatory cells and mast cell and skin thickness were increased in the experimental group treated with Compound 48/80 to induce atopy In contrast, inflammatory cell infiltration and mast cell and skin thickness were reduced in the atopy - induced group treated with the combined extract of gomup leaf extract, grape clover extract, and gourd leaf and grape clover. In particular, the oral administration of the mixed extract of goose leaves and grape seedlings of the present invention orally reduces the infiltration of inflammatory cells and the mast cell and skin thickness more effectively than the oral administration of the extracts of gomate leaf and grape clover, .

In addition, the compound 48/80 (compound 48/80) was treated, and the extracts of the goats' leaves and grape clusters alone and the mixed extract thereof were individually treated to confirm the inhibitory effect of itching. As a result, as shown in FIG. 2D, it was confirmed that the extract of Guillaume leaf and grape cluster of the present invention had an effect of more effectively inhibiting itch than the extract of Guillaume leaf and grape clover, It was found that the combined extract had a synergistic effect.

Example 3. Skin tissue analysis of mouse model of atopic dermatitis according to the treatment of mixed extract of goose leaves and grape seeds of the present invention

The combination of extracts of gomoma leaf, grape cloverleaf, and gourd leaf and grape clover were administered orally to model mice of atopic dermatitis induced with DNFB (2,4-dinitrochlorobenzene) and house dust mite antigen, The change was confirmed.

As a result, as shown in FIG. 3A, the atopic dermatitis of the model mouse was more relaxed than the single extract of the goat's leaves and the grape clusters when the mixed extract of goose leaves and grape clusters of the present invention was treated. As described above, by confirming that the skin lesion of the combined extract of goose leaves and grape seedlings of the present invention is lower in clinical score than the single extract, the skin lesion state is more effective than the single extract extract, And the mixed extract of grape clusters showed better skin lesions.

Example  4. The goat leaf of the present invention and Grape bunch  Analysis of skin tissue of atopic dermatitis model mice treated with mixed extract of eggplant

Hairy mice were induced with atopic dermatitis using DNFB (2,4-dinitrochlorobenzene) and house dust mite antigens. To the model mice, a mixture of goat's leaf extract, grape clover extract, Were orally administered, respectively.

As a result, as shown in FIG. 4 and FIG. 5, when the atopic dermatitis was induced, it was confirmed that the infiltration of inflammatory cells and the skin thickness were increased compared to the normal control group. In contrast, It was confirmed that infiltration of inflammatory cells and skin thickness were alleviated in the extracts and the mixed extracts of Gomel leaf and grape clover. Particularly, it was confirmed that the oral administration of the mixed extract of goose leaves and grape seedlings of the present invention alleviated the infiltration of inflammatory cells and the skin thickness, as compared with the case of oral administration of the extracts of gomate leaf and grape clover , It was found that the mixed extract of the present invention had a synergistic effect.

Example  5. The goat leaf of the present invention and Grape bunch  In the atopic dermatitis model mice treated with the mixed extract of eggplant, IgE  And IL-4 production inhibitory effect

In hairless mice, atopic dermatitis was induced by using DNFB (2,4-dinitrochlorobenzene) and house dust mite antigen, and the extracts of the gomoyle leaf and grape cloverleaf and the mixed extracts thereof were treated respectively and the atopic markers IgE and The production ability of IL-4 was measured in the serum of the model mouse.

As a result, as shown in FIG. 6, in the case of the mixed extract of goose leaves and grape clusters of the present invention, the production of IgE and IL-4 in serum was more effectively inhibited than that of the extract of goose leaves and grape clusters alone, It was confirmed that there was an effect.

Claims (9)

1. A health functional food composition for preventing or ameliorating atopic dermatitis comprising as an active ingredient, a mixed extract of Diospyros lotus leaf and grape fruit stem mixed at a ratio of 0.1 to 10 by weight. The health functional food composition according to claim 1, wherein the solvent of the mixed extract is water, a C ₁ -C ₄ lower alcohol, or a mixture thereof. [Claim 2] The method according to claim 1,
1) cutting gyom leaf and grape clusters and drying at 35-45 ° C for 12-20 hours;
2) adding 1.0 to 3.5 liters and 0.4 to 2.0 liters of ethanol to the 50 to 150 g of the gomule leaf and the 20 to 100 g of the grape seedlings which have been dried in the step 1), and extracting them for 2 to 5 days; And
3) mixing the gourdocyte leaf extract obtained in step 2) with grape cloverleaf extract in a weight ratio of 1: 0.1 ~ 10; and extracting the extract with the extracting method comprising at least one of: a health functional food for preventing or improving atopic dermatitis Composition. delete 1. A cosmetic composition for preventing or ameliorating atopic dermatitis comprising as an active ingredient, a mixed extract of Diospyros lotus leaf and grape fruit stem mixed at a ratio of 0.1 to 10 by weight. 6. The composition of claim 5, wherein the composition is selected from a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, The cosmetic composition for prevention or improvement of atopic dermatitis according to any one of the preceding claims, 1. A pharmaceutical composition for preventing or treating atopic dermatitis, comprising as an active ingredient, a mixed extract of Diospyros lotus leaf and grape fruit stem mixed at a ratio of 0.1 to 10 by weight. The pharmaceutical composition for preventing or treating atopic dermatitis according to claim 7, further comprising a carrier, an excipient or a diluent, in addition to the active ingredient. [8] The composition according to claim 7, wherein the composition is prepared by any one of formulations selected from powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and injections. &Lt; / RTI &gt;

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