KR102315090B1 - Composition for preventing, ameliorating or treating atopic dermatitis comprising extract of Diospyros lotus citric acid hydrolysate as effective component - Google Patents

Abstract

The present invention relates to a composition for the prevention, improvement or treatment of atopic dermatitis containing an extract of a hydrolyzate of goyme citric acid as an active ingredient, and the extract of a hydrolyzate of goyum citric acid, which is an active ingredient of the present invention, is prepared by mixing goyme and citric acid, Since the effect of alleviating the symptoms of atopic dermatitis is remarkable compared to the ethanol-extracted extract without hydrolysis, it can be usefully used as a health functional food, quasi-drug, or atopic dermatitis treatment for atopic patients.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing, improving or treating atopic dermatitis comprising an extract of a gourmet citric acid hydrolysate as an active ingredient.

The present invention relates to a composition for preventing, improving or treating atopic dermatitis comprising an extract of a hydrolyzate of goyme citric acid as an active ingredient.

Atopic dermatitis is accompanied by severe itching (pruritus) with chronic skin inflammation. The itch that evokes the feeling of wanting to scratch is induced by itching mediators such as histamine, and in the case of normal people, the symptoms are easily relieved by scratching, but in the case of atopic patients, the more they scratch, the more they want to scratch, and the more they scratch, the more they scratch. Due to this scratching behavior of atopic patients, the skin barrier is disrupted and secondary infection is caused, further exacerbating inflammation. Atopic dermatitis can be temporarily relieved, but it recurs and worsens due to irritants such as the environment and food, and the cause is not clearly known as a phenomenon of repeated exacerbation and remission. As described above, the inflammation accompanying atopic dermatitis is caused by excessive immune cell action, inflammatory cytokines such as IgE, interleukin-4 (IL-4, interleukin-4), hydroxy radicals (-OH·), superoxide radicals (-O ₂ ^- ), hydrogen peroxide (H ₂ O ₂ ), such as reactive oxygen species (ROS, reactive oxygen species), such as mass production causes damage to surrounding tissues. Therefore, in order to improve skin diseases such as atopic dermatitis, materials that can relieve itching and effectively remove active oxygen are needed.

In addition, mast cells are known to be key cells that cause acute and chronic inflammatory diseases in most allergic diseases, including atopic dermatitis. When mast cells are activated, inflammatory cytokines such as TNF-α (tumor necrosis factor-α), IL-1 (interleukin-1) and IL-6 are produced as well as pruritogens such as histamine. emits As such, mast cell-derived inflammatory cytokines further promote the inflammatory response, and at the same time, itch-inducing substances such as histamine further promote itching and cause the skin barrier to collapse. Therefore, if a substance that can suppress inflammatory cytokines and itch mediators without side effects is discovered, it will be effective in controlling various allergic diseases including atopic dermatitis.

On the other hand, the goyeum tree ( Diospyros lotus L.) is a deciduous plant that grows naturally in Asia including Korea and China, and in traditional medicine, the mature fruit, Goyom, has been used for soothing, analgesic, astringent and constipation treatment. Fatty acids, sugars, flavonoids and non-volatile substances have been reported as biochemical components of Goyam tree, and recently, it has been reported for blood anticoagulation, brain cell protection, antioxidant and anticancer effects. The physiologically active effect of the Goyam tree is mainly targeted to the fruit and seeds of the Goyam tree, and the evaluation of the physiologically active effect on the Goyam tree leaves is insufficient. In addition, the fruit of the Goyam tree is not only smaller than that of persimmon, but also has many seeds, and can be collected mainly in late autumn, so it is not easy to use industrially, and there is a limit to procurement of materials in large quantities. Therefore, it will be very easy to achieve industrialization by overcoming the limitations on the use of Goyam tree fruit and seeds if the physiological activity of Goyam leaves is identified and utilized.

On the other hand, Korea Patent Application Publication No. 2018-0041887 discloses a composition for the prevention, improvement or treatment of atopic dermatitis containing a mixed extract of bitter gourd leaves and grape cluster branches as active ingredients, but an extract of the hydrolyzate of bitter gourd citric acid of the present invention. It has not been disclosed with respect to a composition for preventing, improving or treating atopic dermatitis containing as an active ingredient.

The present invention has been derived from the above needs, and provides a composition for the prevention, improvement or treatment of atopic dermatitis containing an extract of a hydrolyzate of goyme citric acid as an active ingredient, and an ethanol extract of the hydrolyzate of goyum and After mixing citric acid, the present invention was completed by confirming that the improvement effect of atopic dermatitis was remarkable compared to the extract obtained by ethanol extraction without hydrolysis.

In order to solve the above problems, the present invention provides a health functional food composition for the prevention or improvement of atopic dermatitis containing an extract of a hydrolyzate of citric acid from goyum as an active ingredient.

In addition, the present invention provides a quasi-drug composition for preventing or improving atopic dermatitis containing an extract of a hydrolyzate of citric acid from Goyum as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis containing an extract of a hydrolyzate of citric acid from Goyum as an active ingredient.

The present invention relates to a composition for the prevention, improvement or treatment of atopic dermatitis, comprising an extract of the hydrolyzate of goyme citric acid as an active ingredient, and the ethanol extract of the hydrolyzate of goyeum citric acid, which is the active ingredient of the present invention, is prepared by mixing goyme and citric acid. Since the effect of alleviating the symptoms of atopic dermatitis is remarkable compared to the ethanol-extracted extract without hydrolysis, it can be used as a material for health functional food, quasi-drugs, or atopic dermatitis treatment for atopic patients.

1 is a photograph (A) and skin lesion evaluation results confirming the improvement effect of atopic dermatitis of an extract of citric acid hydrolyzate in atopic dermatitis induced atopic dermatitis by applying DNFB (2,4-dinitrofluorobenzene) and house dust mite antigen. (B). Control is a negative control group that does not induce atopic dermatitis, AD model is a group that induces atopic dermatitis, and EDLE is a group administered with an ethanol-extracted extract without hydrolysis after mixing goyme and citric acid in atopic dermatitis-induced animal model, CDLE is the group administered with an ethanol extract of citric acid hydrolyzate to atopic dermatitis-induced animal model, CA is the group administered with citric acid to atopic dermatitis-induced animal model, and BEO is borage oil and evening cream for atopic dermatitis-induced animal model. A positive control group administered with a rose seed oil mixture. For the evaluation of skin lesions, dry condition, scaling, erosion, abrasions, and bleeding of the skin were checked, and 0 points for no lesion and 3 points for severe condition were evaluated as the sum of the total scores. Different characters in the drawing mean that there is a statistically significant difference from each other, and p<0.05.
Figure 2 shows the changes in skin tissue when treated with an extract of citric acid hydrolyzate in an atopic dermatitis animal model induced by atopic dermatitis by applying DNFB (2,4-dinitrofluorobenzene) and house dust mite antigen by H&E (hematoxylin & Eosin staining) staining. These are the results confirmed by a photograph (A), a photograph taken by staining with toluidine blue (B), and a speculum (C) of the infiltrated mast cells. Control is a negative control group that does not induce atopic dermatitis, AD model is a group that induces atopic dermatitis, and EDLE is a group administered with an ethanol-extracted extract without hydrolysis after mixing goyme and citric acid in atopic dermatitis-induced animal model, CDLE is the group administered with an ethanol extract of citric acid hydrolyzate to atopic dermatitis-induced animal model, CA is the group administered with citric acid to atopic dermatitis-induced animal model, and BEO is borage oil and evening cream for atopic dermatitis-induced animal model. A positive control group administered with a rose seed oil mixture. Red arrows point to infiltrated leukocytes. Different characters in the drawing mean that there is a statistically significant difference from each other, and p<0.05.
3 is a result confirming the change in the secretion amount of atopy-related factors (TSLP, CCL17, IL-6 and IgE) according to the treatment of the extract (CDLE) of the hydrolyzate of citric acid of the present invention through ELISA. Control is a negative control group that does not induce atopic dermatitis, AD model is a group that induces atopic dermatitis, and EDLE is a group administered with an ethanol-extracted extract without hydrolysis after mixing goyme and citric acid in atopic dermatitis-induced animal model, CDLE is the group administered with an ethanol extract of citric acid hydrolyzate to atopic dermatitis-induced animal model, CA is the group administered with citric acid to atopic dermatitis-induced animal model, and BEO is borage oil and evening cream for atopic dermatitis-induced animal model. A positive control group administered with a rose seed oil mixture. Different characters in the drawing mean that there is a statistically significant difference from each other, and p<0.05.
_{4 is a result of confirming the NO production amount (A) and the PGE 2} content (B) of the extract of the hydrolyzate of citric acid in RAW 264.7 cells. An inflammatory response was induced by treatment with LPS. DLE is a group treated with high-yum ethanol extract, HDLE is a group treated with hydrochloric acid (HCl) hydrolyzate of high-yum ethanol extract, and CDLE is a group treated with ethanol extract of hydrolyzate from high-yum citric acid. Different characters in the drawing mean that there is a statistically significant difference from each other, and p<0.05.
5 is a photograph of the degree of improvement of atopic dermatitis according to the lapse of time after applying the cream containing the ethanol extract of the hydrolyzate goyme citric acid of the present invention.

The present invention relates to a health functional food composition for preventing or improving atopic dermatitis, comprising an extract of a hydrolyzate of goyme citric acid as an active ingredient.

The Goyam may be used in any part of the Goyam tree, and preferably, the leaves of the Goyam tree are used, but the present invention is not limited thereto.

The solvent of the extract of the present invention may be water, a C ₁ ~ C ₄ lower alcohol or a mixture thereof, and preferably ethanol is used as the solvent, but is not limited thereto.

The extract of the hydrolyzate goyme citric acid of the present invention is

1) after adding citric acid to the high-grade pulverized product, hydrolyzing to obtain a high-grade citric acid hydrolyzate; and

2) extracting by adding ethanol to the hydrolyzate of step 1), filtering, and then concentrating under reduced pressure;

preferably

1) 15 to 25 times (v/w) of 100 mM citric acid is added to 10 g of the pulverized goyme leaf, and then reacted at 80 to 100° C. for 3 to 7 hours to hydrolyze to obtain a high goyme citric acid hydrolyzate ; and

2) 95% (v/v) ethanol was added to the hydrolyzate of step 1) to adjust the final concentration to be 50 to 70% (v/v), and then extracted at 15 to 25° C. for 10 to 14 hours. It may be prepared by a manufacturing method comprising a; then filtration and then concentration under reduced pressure,

more preferably

1) 20 times (v/w) of 100 mM citric acid was added to 10 g of the pulverized goyme leaf, followed by reaction at 90° C. for 5 hours to hydrolyze to obtain a hydrolyzate of goyme citric acid; and

2) 95% (v/v) ethanol was added to the hydrolyzate of step 1) to adjust the final concentration to 60% (v/v), extracted at 20° C. for 12 hours, filtered, and then concentrated under reduced pressure It may be prepared by a manufacturing method comprising a; but is not limited thereto.

The ethanol extract of the citric acid hydrolyzate of the present invention has an effect of reducing the secretion of one or more factors selected from TSLP, CCL17, IL-6 and IgE.

The health functional food composition for preventing or improving atopic dermatitis of the present invention may be prepared in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup and beverage, but is not limited thereto.

The health functional food composition is not particularly limited as long as it can be ingested to prevent or improve 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 used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The active ingredient may be used appropriately depending on the purpose of its use (prevention or improvement). In general, it is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the health functional food composition of the present invention when preparing food or beverage. However, in the case of long-term ingestion for the purpose of health control, 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 above the above range.

There is no particular limitation on the type of the food. Examples of foods to which the health functional food composition 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 There are drinks, alcoholic beverages, vitamin complexes, and the like, and includes all health foods in the ordinary sense. In addition, the health functional food composition of the present invention may be prepared as a food, particularly a functional food. The functional food of the present invention includes ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when manufactured as a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), oligosaccharides, polysaccharides (eg, dextrin, cyclodextrin, etc.) or sugar alcohols (eg, , xylitol, sorbitol, erythritol, etc.). As the flavoring agent, natural flavoring agents (eg, taumatine, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) may be used. In addition to the health functional food composition, various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid It may further contain a carbonation agent and the like used in beverages.

In addition, the present invention relates to a quasi-drug composition for preventing or improving atopic dermatitis containing an extract of a hydrolyzate of citric acid from Goyum as an active ingredient.

The term "quasi-drug" as used in the present invention refers to items with a milder action than pharmaceuticals among items used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing diseases of humans or animals, for example, in the Pharmaceutical Affairs Act. According to the report, quasi-drugs exclude products used for pharmaceutical purposes, and include products used for the treatment or prevention of diseases in humans and animals, and products with minor or no direct action on the human body. The quasi-drug composition of the present invention is used for the purpose of improving atopic dermatitis, and the formulation is not particularly limited. For example, it may be a formulation for transdermal administration such as a lotion, ointment, gel, cream, patch or spray. .

In addition, in each formulation, the quasi-drug composition can be formulated by selecting other components arbitrarily according to the formulation or purpose of use of other quasi-drugs. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (suppression or alleviation). For example, it may contain conventional adjuvants such as thickeners, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers and the like.

In addition, the present invention relates to a pharmaceutical composition for preventing or treating atopic dermatitis comprising an extract of a hydrolyzate of goyme citric acid as an active ingredient.

The pharmaceutical composition of the present invention may further include an appropriate carrier, excipient or diluent commonly used in the preparation of the pharmaceutical composition. The pharmaceutical dosage form of the composition according to the present invention may be used alone or in combination with other pharmaceutically active compounds, as well as in any suitable group. The pharmaceutical composition according to the present invention may be formulated in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, and injections, respectively, according to conventional methods. . Carriers, excipients and diluents that may be included in the pharmaceutical composition comprising the extract of the hydrolyzate of high citric acid include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, various compounds including gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil; mixtures. In the case of formulation, it is prepared using diluents or excipients, such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, for example, starch, calcium carbonate, sucrose or lactose in the extract of the citric acid hydrolyzate. , gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, 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 sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin, glycerogelatin, etc. may be used.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the patient, the degree of disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. The pharmaceutical composition of the present invention may be administered by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

Hereinafter, the present invention will be described in more detail using Preparation Examples and Examples. These preparations and examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.

Materials and Methods

1. Preparation of ethanol extract (CDLE) of citric acid hydrolyzate

10 g of dry powdery mildew leaves were quantified, and a citric acid solution prepared in 100 mM was added 20 times (v/w) and mixed. After the mixture was reacted in a constant temperature water bath at 90° C. for 5 hours, 95% (v/v) ethanol was added to adjust the final concentration of ethanol to 60% (v/v), and then stirred extraction (20° C., 12 time). Then, after centrifugation to obtain a supernatant, the obtained supernatant was filtered with filter paper using a reduced pressure filtration device, ethanol was removed using a reduced pressure concentrator, and then freeze-dried to ethanol The extract powder was recovered and stored at -80°C for use in the experiment.

As a control, citric acid alone (CA) and 10 g of dry gourd leaf powder were quantified, and the same amount of citric acid prepared at 100 mM was added. v/v), followed by stirring extraction (20 ° C, 12 hours), centrifugation to obtain a supernatant, and then filtering the obtained supernatant with paper filter paper using a reduced pressure filtration device, and using a reduced pressure concentrator After removing the ethanol, lyophilized extract powder (EDLE) was used, and it was used in the experiment while being stored at -80°C.

2. Preparation of Goyom Ethanol Extract (DLE)

After quantifying 10 g of dry powdery mildew leaves, 95% (v/v) ethanol was added to adjust the final concentration of ethanol to 60% (v/v), followed by extraction with stirring (20°C, 12 hours). Thereafter, after centrifugation to obtain a supernatant, the obtained supernatant was filtered with filter paper using a reduced pressure filtration device, ethanol was removed using a reduced pressure concentrator, and lyophilized to recover the extract powder. It was used in the experiment while being stored at 80°C.

3. Preparation of hydrochloric acid hydrolyzate (HDLE) of Goyam extract

50% (v/v) ethanol (200ml) was added to dried bitter gourd leaves (10g) for the preparation of an enzymatic hydrolyzate of the ethanol extract of Goyam leaves, and then extracted at room temperature (20~25℃) for 24 hours. It was filtered using a 0.45㎛ filter, and the filtrate was concentrated under reduced pressure to remove ethanol. After that, an appropriate amount of 1N hydrochloric acid solution was injected into the concentrated under reduced pressure so as to have a pH of 2.7, followed by reaction in a constant temperature water bath at 80° C. for 2 hours to hydrolyze, neutralized by injecting 1N sodium hydroxide solution, freeze-dried to powder It was recovered and stored at -80°C for use in the experiment.

4. Experimental animals

7-week-old male hairless mice bred in a sterile environment were purchased from Orient Bio (Gwangju Metropolitan City, Republic of Korea), acclimatized for 1 week while sufficiently supplied with feed and water, and then used in the experiment. The breeding environment had a day and night cycle of 12 hours each, and the temperature (20~22℃) and humidity (50~60%) were kept constant.

5. Induction of atopic dermatitis by application of DNFB (2,4-dinitrofluorobenzene) and house dust mite antigen

In order to induce chemical antigen-induced atopic dermatitis model animals, 0.15% (w/v) DNFB (2,4-dinitrofluorobenzene) was prepared in a solvent mixed with acetone and olive oil at a ratio of 3:1 (v/v) for 1 day and On the 4th day, 100 μl of each was applied to the skin on the back for sensitization. From the first day of sensitization, the control group was orally administered with physiological saline, and the experimental group was mixed with 200 mg/kg of goyme and citric acid and then extracted with ethanol without hydrolysis (EDLE), and ethanol extract (CDLE) of the high-yum citric acid hydrolyzate (CDLE). , citric acid (CA) and borage oil + evening primrose seed oil (1:1 volume ratio, BEO) were orally administered by 0.1 ml each once a day until the end of the experiment. On the 7th, 10th and 13th days from the first sensitization day, 0.2% (w/v) DNFB was applied at 3-day intervals and completely dried. was applied three times to induce atopic dermatitis.

6. Serum

Five hours after the last induction of atopic dermatitis, blood was collected from the liver portal vein of mice to obtain serum.

7. Assessment of skin lesions

The skin condition was investigated by taking pictures after the end of the experiment. By checking the dryness, scaling, erosion, excoriation, and bleeding of the skin, a condition without lesion is 0 (none), a mild condition is 1 point (mild), and a moderate condition is checked. was given 2 points (moderate) and severe condition as 3 points (severe), and a total score was assigned to each stage for evaluation.

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

For histopathological analysis, H&E staining (Hematoxylin & Eosin staining) was performed. The hairless mouse model induced atopic dermatitis and the mouse model in which EDLE, CDLE, CA and BEO were orally administered were sacrificed and skin tissues of about 5 × 5 mm were excised, respectively. Thereafter, after washing with saline, water was removed, fixed with 4% (v/v) paraformaldehyde (pH 7.4), and a paraffin block was prepared through a conventional block manufacturing process. The tissue sections cut to a thickness of 5 μm were subjected to deparaffin and hydrous processes, stained with H&E, and observed under a microscope (×100, Olympus, Tokyo, Japan). In addition, it was stained with toluidine blue, observed at low magnification (40×), and magnified while taking pictures in a microscope field of 100× (H&E) and 400× (toluidine blue) to examine leukocyte infiltration and mast cells.

9. Measurement of TSLP, CCL17, IgE (immunoglobulin E) and IL-6 (interleukin-6) cytokines in serum

200 mg/kg of EDLE, CDLE, CA and BEO were administered to atopic dermatitis model mice, and TSLP, CCL17, IL-6 and IgE were measured from the serum collected 5 hours later. TSLP, CCL17, IL-6 and IgE were analyzed by R&D Systems using an ELISA kit that specifically acts on each using anti-mouse TSLP, anti-mouse CCL17, anti-mouse IL-6 and anti-mouse IgE antibodies. and Shibayagi (Hyogo, Japan) were measured and quantified according to the method provided.

10. Nitric Oxide (NO) and PGE in RAW 264.7 cell line ₂ measurement of

The cells used in the present invention were RAW264.7 cells, a mouse-derived macrophage cell line, and were purchased from ATCC (American Type Culture Collection). DMEM (Dulbecco's modified eagle medium) medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin (GIBCO-BRL, Invitrogen, Carlsbad, CA, USA) was used and cultured in an incubator at 37° C. and 5% CO _{2 .}

RAW264.7 cells were aliquoted to a final concentration of 2×10 ⁵ cells/mL in a 96-well plate, incubated at 37° C. and 5% CO _{2 in an} incubator for 24 hours, and then treated with 200 μg/mL of DLE, HDLE and CDLE. Then, after 1 hour, it was treated with LPS (1 μg/mL) and incubated for 16 hours. After incubation, 100 μl of cell culture solution and 100 μl of grease reagent were mixed and reacted at room temperature for 15 minutes, absorbance was measured at 540 nm using a microplate reader, and a standard curve was prepared with sodium nitrate. NO (nitric oxide) production was calculated.

In addition, PGE ₂ (prostaglandin E ₂₎ is treated with a sample in the same manner as described above, and then, after stimulation with LPS 16 h of incubation and then further on PGE ₂ targeting the supernatant-utilizing mouse PGE ₂ ELISA kit It was measured and quantified by the method provided by R&D Systems (Minneapolis, USA). In summary, 100 μl of the cell supernatant was injected into each antibody-coated plate, reacted, washed well, and then a secondary antibody attached with horseradish peroxidase was injected and reacted, followed by reaction with a chromogenic substrate. Injection and reaction were measured with an ELISA reader (Molecular Devices, USA). For the quantification of each substance, a standard curve was determined by treating each substance by concentration and reacting, and the amount of substance contained in the cell supernatant was calculated.

11. Statistical analysis

All experimental values were expressed as mean ± standard error, and statistical analysis was processed by ANOVA and Duncan analysis. When p<0.05, statistical significance was analyzed.

Example 1. Atopic dermatitis improvement effect of ethanol extract (CDLE) of citric acid hydrolyzate in atopic dermatitis animal model

In animal models in which atopic dermatitis was induced by application of DNFB and house dust mite antigen, ethanol extract (EDLE), ethanol extract (CDLE) and citric acid (CA) extracted from ethanol without hydrolysis after mixing goyme and citric acid ) and borage oil + evening primrose seed oil (BEO) were confirmed to improve atopic dermatitis.

In addition, as a result of confirming the dermatitis improvement effect as a clinical score, the clinical score for the severity of skin lesions was decreased in all the sample administration groups, and in particular, the clinical score of the CDLE administration group was significant compared to the EDLE and CA administration groups (FIG. 1B) .

Example 2. Changes in skin tissue of ethanol extract (CDLE) of citric acid hydrolyzate in atopic dermatitis animal model

After the sample was orally administered to the atopic dermatitis animal model, changes in the skin tissue were confirmed. In the case of the atopic dermatitis-induced group (AD model), it was confirmed that the infiltration of inflammatory cells, the increase of mast cells, and the skin thickness were increased compared to the normal control group. In the group administered with ethanol-extracted extract (EDLE), ethanol extract (CDLE) of citric acid hydrolyzate, citric acid (CA) and borage oil + evening primrose seed oil (BEO), the infiltration of inflammatory cells induced by atopy and It was confirmed that mast cells and skin thickness were reduced. In particular, in the case of the CDLE administration group, the most remarkable alleviation effect was shown ( FIG. 2 ).

Example 3. Changes in the content of atopic-related factors (TSLP, CCL17, IL-6, and IgE) in serum by administration of an ethanol extract (CDLE) of citric acid hydrolyzate in atopic dermatitis animal model

In the atopic dermatitis animal model, the ethanol extract of the hydrolyzate of citric acid was confirmed to improve the atopic dermatitis by the concentrations of TSLP, CCL17, IL-6 and IgE in the serum. As a result, it was confirmed that the concentrations of TSLP, CCL17, IL-6 and IgE in the serum of animal models induced by atopic dermatitis were increased by application of DNFB and house dust mite antigen. TSLP, CCL17 in serum increased by atopic dermatitis when administered without ethanol extract (EDLE), ethanol extract (CDLE) of citric acid hydrolyzate, citric acid (CA), and borage oil + evening primrose seed oil (BEO) , the concentrations of IL-6 and IgE were decreased. In particular, the reduction effect of the CDLE-administered group was significant compared to the EDLE, CA and BEO-administered groups (FIG. 3).

Example 4. NO Production and PGE of Ethanol Extract (CDLE) of Goyme Citric Acid Hydrolyzate ₂ Check the production inhibitory effect

After inducing the production of PGE ₂ in the NO generation and treated with LPS in RAW 264.7 cells treated with DLE, and HDLE CDLE was then confirmed that the content change of NO and PGE _{2. As a result, NO and PGE 2} increased by the LPS treatment were decreased after treatment with Goyom ethanol extract (DLE), hydrolyzate of Goyum ethanol extract (HDLE), and ethanol extract (CDLE) of Goyum citric acid hydrolyzate (CDLE), especially CDLE The reduction effect upon treatment was remarkable.

Example 5. Clinical trial

Atopic dermatitis was prepared by preparing the cream of Preparation Example 1 and Comparative Example 1 without containing the ethanol extract (CDLE) of the hydrolyzate citric acid (CDLE), which had a significant improvement effect on atopic dermatitis in the above example, as an active ingredient, as shown in Table 1 below. Improvement effect and feeling of use were evaluated.

Cosmetic (cream) ingredients and content ingredient Preparation Example 1 (wt%) Comparative Example 1 (wt%) water base Ethanol extract (CDLE) of citric acid hydrolyzate 5 - Butylene Glycol 4 4 xanthan gum 0.1 0.1 Sodium Hyaluronate 0.05 0.05 Purified water remaining amount remaining amount oil base Hydrogenated Polydecene 8.4 8.4 squalane 4 4 C12-20 Alkylglucoside 2 2 C14-22 alcohol 2 2 cetyl alcohol 2 2 ceramide 0.05 0.05

In order to evaluate the improvement effect of the cream on atopic dermatitis, 20 skin disease patients under the age of 25 with atopic lesions were divided into 2 groups and 10 people per group were applied. It was used continuously for 2 weeks, and the evaluation score was divided by 10 as the evaluation value.

As a result, it was confirmed that the cream of Preparation Example 1 was excellent in suppressing and alleviating itching and symptoms of atopic skin without irritation to the atopic skin as shown in Table 2 and FIG. 5 below.

Evaluation items Preparation Example 1 Comparative Example 1 skin non-irritation 5.0 4.6 itching suppression 4.8 3.9 Inhibits erythema and inflammation 4.7 4.0 moisturizing effect 4.9 4.2 Total atopy suppression 4.8 3.9 <Evaluation criteria> 5 points: Very good / 4: Good / 3: Average

Claims (5)

A health functional food composition for the prevention or improvement of atopic dermatitis, comprising an ethanol extract of a hydrolyzate of high citric acid as an active ingredient. delete The method according to claim 1, wherein the extract of the citric acid hydrolyzate is
1) after adding citric acid to the pulverized goyum, hydrolyzing to obtain a hydrolyzate of goyum citric acid; and
2) A health functional food composition for preventing or improving atopic dermatitis, characterized in that it is prepared by a manufacturing method comprising; 2) adding ethanol to the hydrolyzate of step 1) for extraction, followed by filtration and concentration under reduced pressure. A quasi-drug composition for the prevention or improvement of atopic dermatitis, comprising an ethanol extract of a hydrolyzate of goyme citric acid as an active ingredient. A pharmaceutical composition for the prevention or treatment of atopic dermatitis, comprising an ethanol extract of a hydrolyzate of citric acid from Goyum as an active ingredient.

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