KR20210079528A - Composition for preventing, ameliorating or treating atopic dermatitis comprising Apium graveolens citric acid hydrolysate as effective component - Google Patents

Abstract

The present invention relates to a composition for the prevention, alleviation or treatment of atopic dermatitis containing Apium graveolens citric acid hydrolysates as an active ingredient. The Apium graveolens citric acid hydrolysate, which is the active ingredient of the present invention, after mixing the Apium graveolens ethanol extract and citric acid, has a remarkable effect of alleviating the symptoms of atopic dermatitis compared to the non-hydrolyzed product that is not hydrolyzed, thereby being usefully used as a health functional food, quasi-drug, or atopic dermatitis treatment agent for atopic patients.

Description

A composition for preventing, improving or treating atopic dermatitis comprising a hydrolyzate of celery citrate as an active ingredient TECHNICAL FIELD

The present invention relates to a composition for preventing, improving, or treating atopic dermatitis, comprising a hydrolyzate of celery citrate 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 is recurred and aggravated by irritants such as the environment and food, and the cause of atopic dermatitis is not clearly known yet as the aggravation and remission are repeated. 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 ) Because it mass-produces reactive oxygen species (ROS), such as, it 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 required.

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, celery ( Apium graveolens ) is an annual or biennial herb of the dicotyledonous umbel family. It is native to Southern Europe, North Africa, and Western Asia. Wild celery has a strong bitter taste, so it has been improved by Italians since the 17th century and has reached the present day. Celery is grown in the field, and the height is 60-90 cm. Leaves and stems are green, hairless, and ridged. The root leaf has a long petiole, egg-shaped or long oval, and is divided into several from above. Stem leaves are alternate phyllotaxis, and the lower part becomes a sheath, and the upper part is divided into a feather shape. Flowers bloom in June-September, are white, hang in umbel, and fruits are flat round.

Since celery is fragrant throughout, its tender leaves and stems are edible, and it is an essential ingredient in Western cuisine. Therefore, after softening the petiole by covering it with soil or paper, it is eaten. Celery likes a cool climate and grows worse when it is 23~24℃ or higher. Varieties include dark green leaves, pale yellow leaves, and intermediate varieties. Green varieties are easy to grow.

Celery is rich in vitamins A, B ₁ , B ₂ , C and contains various minerals such as calcium, iron, potassium and magnesium. do. Abundant fiber aids in bowel movements and lowers cholesterol levels to prevent aging, relieve constipation, and prevent cancer. It also enhances stamina and purifies blood, so it is effective in enhancing stamina and beauty. In addition, it is effective for nerve stabilization, food poisoning prevention, menopausal disorders, diabetes, and neuralgia. Magnesium and iron help blood formation.

On the other hand, Korea Patent Publication No. 2017-0025356 discloses a composition for improving atopic dermatitis comprising a celery extract as an active ingredient, but for preventing, improving or treating atopic dermatitis containing the hydrolyzate of celery citric acid of the present invention as an active ingredient No composition has been disclosed.

The present invention has been derived from the above needs, and provides a composition for preventing, improving or treating atopic dermatitis containing hydrolyzate of celery citrate as an active ingredient, wherein the hydrolyzate of celery citrate is mixed with an ethanol extract of celery and citric acid Then, the present invention was completed by confirming that the improvement effect of atopic dermatitis was remarkable compared to the non-hydrolyzed product that was not hydrolyzed.

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

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

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

The present invention relates to a composition for preventing, improving or treating atopic dermatitis containing hydrolyzate of celery citrate as an active ingredient, wherein the hydrolyzate of citric acid of celery extract, which is the active ingredient of the present invention, is prepared by mixing celery ethanol extract and citric acid, Since the effect of alleviating the symptoms of atopic dermatitis is remarkable compared to the non-hydrolyzed product that is not hydrolyzed, it can be used as a material for health functional food, quasi-drugs or atopic dermatitis treatment for atopic patients.

1 is a photo (A) and skin lesion evaluation results confirming the improvement effect of atopic dermatitis of a citric acid hydrolyzate of celery extract in atopic dermatitis animal model induced 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 ECE is a group administered with a non-hydrolyzate that is not hydrolyzed after mixing celery extract and citric acid in an atopic dermatitis-induced animal model. , CCE is the group administered with citric acid hydrolyzate of celery extract 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 were checked, and 0 points for no lesion and 3 points for severe condition were evaluated as a total. Different characters in the drawing mean that there is a statistically significant difference from each other, and p<0.05.
Figure 2 shows the improvement effect of citric acid hydrolyzate of celery extract in atopic dermatitis animal model in which atopic dermatitis was induced by applying DNFB (2,4-dinitrofluorobenzene) and house dust mite antigen to skin tissue by H&E (hematoxylin & Eosin staining) A photograph taken by staining (A), a photograph taken by staining with toluidine blue (B), and a microscopic result (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 ECE is a group administered with a non-hydrolyzate that is not hydrolyzed after mixing celery extract and citric acid in an atopic dermatitis-induced animal model. , CCE is the group administered with citric acid hydrolyzate of celery extract 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 of confirming the secretion amount of atopy-related factors (TSLP, CCL17, IL-6 and IgE) according to the citric acid hydrolyzate (CCE) treatment of the celery extract 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 ECE is a group administered with a non-hydrolyzate that is not hydrolyzed after mixing celery extract and citric acid in an atopic dermatitis-induced animal model. , CCE is the group administered with citric acid hydrolyzate of celery extract 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} expression level (B) of the citric acid hydrolyzate of the celery extract in RAW 264.7 cells. An inflammatory response was induced by treatment with LPS. CE is a group treated with an ethanol extract of celery, HCE is a group treated with hydrochloric acid (HCl) hydrolyzate of celery extract, and CCE is a group treated with citric acid hydrolyzate of celery extract. Different characters in the drawing mean that there is a statistically significant difference from each other, and p<0.05.

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

The celery extract may be used at any part of celery, and is preferably extracted using a celery leaf part, but is not limited thereto.

The solvent of the celery 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 citric acid hydrolyzate of the celery extract of the present invention is

1) adding ethanol to the ground celery for extraction, followed by filtration and concentration under reduced pressure;

2) freeze-drying the concentrate under reduced pressure of step 1);

3) adding citric acid to the freeze-dried product of step 2) and hydrolyzing at 80-100° C. for 3-5 hours, followed by cooling; and

4) freeze-drying the cooled hydrolyzate of step 3); can be prepared by a manufacturing method comprising;

Specifically

1) After adding 200~400mL of 50mM PBS (pH 7) to 15g of crushed celery leaf, stirring at room temperature (20±5℃) for 1~3 hours, 95% (v/v) ethanol was added to the stirring reaction. After addition, the final ethanol concentration is 50 to 70% (v/v), and then stirred at 40 to 60° C. for 10 to 18 hours for extraction, centrifugation to recover the supernatant, filtration of the supernatant, and then concentration under reduced pressure to do;

2) freeze-drying the concentrate under reduced pressure of step 1);

3) adding 50-70 mL of 1M citric acid to 6 g of the freeze-dried product of step 2), hydrolyzing at 85-95° C. for 3-5 hours, and cooling; and

4) freeze-drying the cooled hydrolyzate of step 3); can be prepared by a manufacturing method comprising;

more specifically

1) After adding 300 mL of 50 mM PBS (pH 7) to 15 g of crushed celery leaf and stirring at room temperature for 2 hours, 95% (v/v) ethanol was added to the stirred reaction mixture to obtain a final ethanol concentration of 60% (v /v) and then stirred for 14 hours at 50° C. for extraction, centrifugation to recover the supernatant, filtration of the supernatant, and concentration under reduced pressure;

2) freeze-drying the concentrate under reduced pressure of step 1);

3) adding 60 mL of 1M citric acid to 6 g of the freeze-dried product of step 2), hydrolyzing at 90° C. for 4 hours, and cooling; and

4) freeze-drying the cooled hydrolyzate of step 3); may be prepared by a manufacturing method comprising, but is not limited thereto.

The celery citrate 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 intake 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 greater than 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 a hydrolyzate of citric acid of celery extract 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 containing a hydrolyzate of citric acid of celery extract 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, aerosols, external preparations, suppositories, and injections, respectively, according to conventional methods. . Carriers, excipients and diluents that may be included in the pharmaceutical composition containing the citric acid hydrolyzate of the celery extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, 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 in the citric acid hydrolyzate of the celery extract, for example, starch, calcium carbonate, sucrose or lactose. , 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 patient's condition and weight, the degree of disease, drug form, administration route and period, 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 Celery Ethanol Extract (CE)

Celery ( Apium graveolens ) 300 mL of 50 mM PBS (pH 7) was added to 15 g of leaf powder and stirred at room temperature for 2 hours. 95% (v/v) ethanol was added to the stirred reaction mixture so that the final ethanol concentration became 60% (v/v), and the mixture was stirred and extracted at 50° C. for 14 hours. The extract was centrifuged at 10,000×g, 4° C. for 10 minutes to recover the supernatant, and the recovered supernatant was filtered with filter paper using a vacuum filtration device, and then ethanol was removed using a rotary vacuum concentrator and concentrated. The concentrate was freeze-dried to recover the celery extract in powder form, and the powder of the celery extract was stored at -80°C and used as a sample for the experiment.

2. Preparation of citric acid hydrolyzate (CCE) of celery extract

60 mL of 1M citric acid was added to 6 g of the freeze-dried product (powder) of the celery extract, followed by reaction at 90° C. for 4 hours, followed by cooling.

As a control, the same amount of citric acid was not reacted (ECE) and citric acid alone (CA) was used.

After the reaction, the sample to be used for the animal experiment was cooled and freeze-dried to recover the extract powder and stored at -80°C for use in the experiment.

3. Preparation of Hydrochloric Acid Hydrolyzate (HCE) of Celery Extract

Celery leaves (100 g) were crushed, added to 80% (v/v) ethanol (2,000 ml), extracted for 2 days, filtered using a 0.45 μm 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, and then 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, and the experiments were conducted according to the regulations of the Jeonju National University Experimental Animals Committee.

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

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 mixed 200 mg/kg of celery ethanol extract and citric acid, followed by non-hydrolyzed non-hydrolyzate (ECE) and citric acid hydrolyzate (CCE) of celery ethanol extract. ), 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. Biostir AD ointment derived from house dust mite antigen at 100 mg per animal (Biostir, Hyogo, Janpan) 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 given 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. A hairless mouse model induced by atopic dermatitis and a mouse model in which ECE, CCE, 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 ECE, CCE, CA and BEO was 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, and then cultured at 37° C. and 5% CO _{2 in an} incubator for 24 hours, and then treated with CE, HCE, and CCE at 200 μg/mL. 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 ) was treated in the same way as above, and then stimulated with LPS and incubated for 16 hours. Then, the supernatant was used for PGE ₂ by using an anti-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 citric acid hydrolyzate (CCE) of celery extract in atopic dermatitis animal model

In the animal model in which atopic dermatitis was induced by application of DNFB and house dust mite antigen, celery ethanol extract and citric acid were mixed, and then non-hydrolyzed non-hydrolyzate (ECE), citric acid hydrolyzate (CCE), citric acid ( CA) and borage oil + evening primrose seed oil (BEO) were confirmed to improve atopic dermatitis, and in particular, the atopic improvement effect was remarkable when CCE was administered (FIG. 1A).

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 reduced in all administration groups, and in particular, the clinical score of the CCE administration group was significant compared to the ECE and CA administration groups (FIG. 1B).

Example 2. Changes in skin tissue of citric acid hydrolyzate (CCE) of celery extract 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 contrast, after mixing the celery ethanol extract and citric acid, In the non-hydrolyzed non-hydrolyzate (ECE), citric acid hydrolyzate (CCE) of celery ethanol extract, citric acid (CA), and borage oil + evening primrose seed oil (BEO) administered group, atopy-induced inflammatory cell infiltration and mast cells and skin thickness were reduced. In particular, in the case of the CCE-administered 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 citric acid hydrolyzate (CCE) of celery extract in atopic dermatitis animal model

In an atopic dermatitis animal model, the atopic dermatitis improvement effect of the citric acid hydrolyzate of celery extract was confirmed 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 in serum increased by atopic dermatitis upon administration of non-hydrolyzed non-hydrolyzate (ECE), citric acid hydrolyzate (CCE) of celery ethanol extract, citric acid (CA) and borage oil + evening primrose seed oil (BEO), The concentrations of CCL17, IL-6 and IgE were decreased. In particular, the reducing effect of the CCE-administered group was significant compared to the ECE, CA and BEO-administered groups (FIG. 3).

Example 4. NO Production and PGE of Citric Acid Hydrolyzate (CCE) of Celery Extract 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 were treated with CE, HCE and CCE, it was confirmed to generate changes of NO and PGE _{2. As a result, NO and PGE 2} increased by LPS treatment were decreased after treatment with celery ethanol extract (CE), hydrolyzate hydrolyzate of celery ethanol extract (HCE), and citric acid hydrolyzate (CCE) of celery ethanol extract, especially CCE treatment The time reduction effect was remarkable.

Example 5. Clinical trial

Atopic dermatitis improvement effect by preparing a cream with Preparation Example 1 containing citric acid hydrolyzate (CCE) of celery extract, which has a marked improvement effect on atopic dermatitis in the above Example, as an active ingredient, and Comparative Example 1 without it as shown in Table 1 below. and usability evaluation were performed.

Cosmetic (cream) ingredients and content ingredient Preparation Example 1 (wt%) Comparative Example 1 (wt%) water base Citric Acid Hydrolyzate (CCE) of Celery Extract 10 - 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

Twenty patients with atopic dermatitis under the age of 25 with atopic lesions were classified into 2 groups and 10 people per group were applied. The value divided by 10 was made into an evaluation value. As a result, as shown in Table 2 below, it was confirmed that the cream of Preparation Example 1 was excellent in suppressing and alleviating itching and symptoms of atopic skin without irritating the atopic skin.

Evaluation items Preparation Example 1 Comparative Example 1 skin non-irritation 5.0 4.7 itching suppression 4.6 4.0 Inhibits erythema and inflammation 4.6 3.9 moisturizing effect 4.8 4.1 Total atopy suppression 4.7 3.8 <Evaluation criteria> 5 points: Very good / 4: Good / 3: Average

Claims (5)

A health functional food composition for preventing or improving atopic dermatitis comprising hydrolyzate of citric acid of celery extract as an active ingredient. According to claim 1, wherein the solvent of the celery extract is water, C ₁ ~ C ₄ Health functional food composition for the prevention or improvement of atopic dermatitis, characterized in that the lower alcohol or a mixture thereof. The method of claim 1, wherein the citric acid hydrolyzate of the celery extract is
1) adding ethanol to the ground celery for extraction, followed by filtration and concentration under reduced pressure;
2) freeze-drying the concentrate under reduced pressure of step 1);
3) adding citric acid to the freeze-dried product of step 2) and hydrolyzing at 80-100° C. for 3-5 hours, followed by cooling; and
4) freeze-drying the cooled hydrolyzate of step 3); a health functional food composition for preventing or improving atopic dermatitis, characterized in that it is prepared by a manufacturing method comprising a. A quasi-drug composition for preventing or improving atopic dermatitis comprising hydrolyzate of citric acid of celery extract as an active ingredient. A pharmaceutical composition for the prevention or treatment of atopic dermatitis comprising a hydrolyzate of citric acid of celery extract as an active ingredient.

Images