KR102227261B1 - Composition for preventing, ameliorating or treating atopic dermatitis comprising enzyme treated Diospyros lotus as effective component - Google Patents

Abstract

The present invention relates to a composition for preventing, ameliorating or treating atopic dermatitis containing an enzyme-treated product as an active ingredient, wherein the enzyme hydrolyzate of the extract of Goyum leaves, which is an active ingredient of the present invention, inhibits the production of NO and is related to atopic dermatitis. It has the effect of inhibiting the secretion of factors (IL-4 and INF-γ). In addition, the enzyme hydrolyzate of the extract of Goyum leaves of the present invention is superior to the acid hydrolyzate of the extract of Goyum leaves or the ethanol extract of Goyum leaves, and has superior skin lesion improvement effect in an animal model caused by atopic dermatitis, and skin thickness, IL-4 in blood. , Since it has the effect of remarkably reducing the concentration of IgE and CCL17, the composition containing the enzymatic hydrolyzate of the extract of Koyotei of the present invention as an active ingredient is a quasi-drug for atopic dermatitis, a health functional food for atopic patients, and a material for atopic dermatitis treatment It can be usefully used.

Description

Composition for preventing, ameliorating or treating atopic dermatitis comprising enzyme treated Diospyros lotus as effective component

The present invention relates to a composition for the prevention, improvement or treatment of atopic dermatitis, which contains the enzyme treated product as an active ingredient.

Atopic dermatitis (atopic dermatitis) is caused by genetic, environmental, and immunological causes, and is an allergic disease that is more severe in dry climates as an abnormality in the stratum corneum that acts as a protective wall at the outermost part of the skin.

Atopic dermatitis has a prevalence rate of about 10 to 20% worldwide, and it mainly occurs in infants between 1 month and 1 year after birth, and is relieved to some extent at the age of 3, but may recur again due to living environment and individual differences.

The main symptoms of atopic dermatitis are severe itching, dry skin, rash, swelling, scab, scaly skin (phosphorus scales), etc., mainly accompanied by chronic skin inflammation. Itching, which arouses the feeling of wanting to scratch, is induced by an itching mediating substance such as histamine, and the symptoms are easily relieved by scratching in normal people, but the more atopic patients scratch, the more they want to scratch, the more severe the scratching is formed. Due to this scratching behavior of atopic patients, the skin barrier collapses and causes secondary infection, further exacerbating the inflammation. Atopic dermatitis may be temporarily relieved, but may recur and worsen due to irritants such as the environment and food, and the worsening and alleviation are repeated.

The cause of such atopic dermatitis has not been accurately identified yet, but it is mainly known to have many genetic factors and is related to the immune response, and in addition, dry skin, characteristics that easily feel itchy skin compared to normal people, bacteria, viruses, and fungi, etc. It is believed that infection, emotional and environmental factors work in combination with each other. Inflammation accompanying atopic dermatitis is caused by excessive immune cell action, leading to inflammatory cytokines and hydroxy acids such as TNF-α (tumor necrosis factor-α) and interleukin-1 (IL-1, interleukin-l). It causes damage to surrounding tissues because it mass-produces reactive oxygen species (ROS) such as radicals (·OH), superoxide radicals (·O ₂ -), and hydrogen peroxide (H ₂ O _{2 ).}

In addition, mast cells are known as key cells that cause acute and chronic inflammatory diseases in most allergic diseases including atopic dermatitis. When mast cells are activated, they not only produce inflammatory cytokines such as TNF-α, IL-1 and IL-6, but also release pruritogen such as histamine. In this way, mast cell-derived inflammatory cytokines further promote the inflammatory response and at the same time further promote the secretion of itching-inducing substances such as histamine, which causes the skin barrier to collapse. Therefore, if a substance that can suppress inflammatory cytokines and itching mediators without side effects is discovered, it will be effective in controlling various allergic diseases including atopic dermatitis.

Drug therapy such as steroids, antihistamines, antibiotics, etc. are mainly used to treat atopic dermatitis. Steroids (corticosteroids) are largely anti-inflammatory and immunosuppressive, and have excellent effects, but if applied for a long time, side effects such as skin weakness, systemic hormonal symptoms, and addiction may occur. The treatment direction of atopic dermatitis under recent research is the use of immunosuppressants and the use of new antihistamines. However, antihistamines alone do not completely block allergic reactions. This is because histamine is not the only chemical transporter that causes an allergic reaction. Therefore, there is a need to develop a composition for the treatment of atopic dermatitis, which is effective against atopic dermatitis and does not have side effects.

Meanwhile, Diospyros lotus L.) is a deciduous plant that grows naturally in Asia, including Korea and China. In the field of traditional medicine, Goyom, a mature fruit, has been used for soothing, pain relief, astringent and constipation treatment. Fatty acids, sugars, flavonoids, and non-volatile substances have been reported as biochemical components of Paleoanthus, and recently, anticoagulant, brain cell protective action, antioxidant and anticancer effects of blood have been reported. The physiologically active effect of the Goyum tree is mainly targeting the fruit and seeds of the Goyum tree, and the evaluation of the physiological activity effect on the leaves of the Goyum tree is insufficient. In addition, the fruit of the Goyom tree is not only small compared to persimmons, 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 procuring materials in large quantities. Therefore, if the physiological activity of the leaves of the Goyum tree was identified and utilized, it would be very easy to achieve industrialization by overcoming the limitations of the use of the Goyum tree fruit and seeds.

On the other hand, Korean Patent Application Publication No. 2018-0041887 discloses a composition for preventing, improving or treating atopic dermatitis containing a mixed extract of koyum leaves and grapefruit branches as an active ingredient, but the koyom enzyme treatment product of the present invention is effective. There is no disclosed composition for the prevention, improvement or treatment of atopic dermatitis containing as a component.

The present invention is derived from the above requirements, and provides a composition for the prevention, improvement or treatment of atopic dermatitis containing an enzyme hydrolyzate as an active ingredient, and wherein the enzyme hydrolyzate is an ethanol extract and The present invention was completed by confirming that the improvement effect of atopic dermatitis was remarkable compared to the acid hydrolyzate of Koyoteum.

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 enzyme hydrolyzate of an extract of Koyotei as an active ingredient.

In addition, the present invention provides a quasi-drug composition for the prevention or improvement of atopic dermatitis containing an enzyme hydrolyzate of a koyoteum leaf extract as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of atopic dermatitis containing the enzyme hydrolyzate of the extract of Koyotei as an active ingredient.

The present invention relates to a composition for the prevention, improvement, or treatment of atopic dermatitis containing a hydrolyzate as an active ingredient, and the enzyme hydrolyzate of the extract of Goyum leaves as an active ingredient of the present invention is an acid of the ethanol extract of Goyum and the extract of Goyum leaf. Compared to hydrolysates, since the effect of relieving symptoms of atopic dermatitis is remarkable, it can be effectively used in the development of health functional foods, quasi-drugs, or atopic dermatitis treatments for atopic patients.

1 is a result of confirming the cytotoxicity (A) and the amount of NO (Nitric Oxide) production (B) by the hydrolyzate treatment of the extract of Koolai leaf in RAW 264.7 cells. LPS (Lipopolysaccharide) is an inducer of NO production. Control is a sample and LPS-treated group, and prednisolone is a positive control group treated with LPS and prednisolone. DLE is LPS and ethanol extract treatment group, HDLE is LPS and acid hydrolyzate treatment group, and EDLE is LPS and enzyme hydrolyzate treatment group. a~f means that there is a statistically significant difference from each other, and p<0.05.
Figure 2 is a result of confirming the change in the secretion amount of atopic-related factors (IL-4 and INF-γ) according to the treatment of the enzyme hydrolyzate (EDLE) of the present invention in mouse splenocytes through ELISA. ConA is Concanavalin A, a substance that stimulates splenocytes. Control is a sample and a group that is not treated with ConA, and prednisolone is a positive control group that is treated with ConA and prednisolone. DLE is a group treated with ConA and Ethanol extract of Koolai leaf, HDLE is a group treated with acid hydrolyzate of ConA and Koyotei leaf extract, and EDLE is a group treated with enzymatic hydrolyzate of ConA and Koyotei leaf extract. a~f means that there is a statistically significant difference from each other, and p<0.05.
FIG. 3 is a photograph confirming the effect of improving atopic dermatitis by administration of an enzyme hydrolyzate of Atopic dermatitis in an atopic dermatitis animal model that caused atopic dermatitis by applying DNFB (2,4-dinitrofluorobenzene) and house dust mite antigen. The normal group was the control group that did not induce atopic dermatitis, AD is the atopic dermatitis-inducing group, prednisolone is a positive control group administered with prednisolone to the atopic dermatitis-causing animal model, and DLE is the black leaf ethanol in the atopic dermatitis-induced animal model. The extract was administered, and HDLE was the group administered with the acid hydrolyzate of the Atopic dermatitis-inducing animal model, and EDLE was the group administered the enzyme hydrolyzate of the edible oleander extract to the atopic dermatitis-induced animal model.
FIG. 4 is a clinical score (A) and an effect of improving atopic dermatitis by administering an enzyme hydrolyzate of Atopic dermatitis in an animal model of atopic dermatitis inducing atopic dermatitis by applying DNFB (2,4-dinitrofluorobenzene) and house dust mite antigen. This is the result of checking through the change in the thickness of the back skin tissue (B). The normal group was the control group that did not induce atopic dermatitis, AD is the atopic dermatitis-inducing group, prednisolone is a positive control group administered with prednisolone to the atopic dermatitis-causing animal model, and DLE is the black leaf ethanol in the atopic dermatitis-induced animal model. The extract was administered, and HDLE was the group administered with the acid hydrolyzate of the Atopic dermatitis-inducing animal model, and EDLE was the group administered the enzyme hydrolyzate of the edible oleander extract to the atopic dermatitis-induced animal model. A to d mean that there is a statistically significant difference from each other, and p<0.05.
FIG. 5 shows the effect of improving atopic dermatitis by administration of an enzyme hydrolyzate of Kool leaf extract in an animal model of atopic dermatitis inducing atopic dermatitis by applying DNFB (2,4-dinitrofluorobenzene) and house dust mite antigen. ), IgE(B) and CCL17(C). The normal group was the control group that did not induce atopic dermatitis, AD is the atopic dermatitis-inducing group, prednisolone is a positive control group administered with prednisolone to the atopic dermatitis-causing animal model, and DLE is the black leaf ethanol in the atopic dermatitis-induced animal model. The extract was administered, and HDLE was the group administered with the acid hydrolyzate of the Atopic dermatitis-inducing animal model, and EDLE was the group administered the enzyme hydrolyzate of the edible oleander extract to the atopic dermatitis-induced animal model. A to d 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 the prevention or improvement of atopic dermatitis containing the enzyme hydrolyzate of the extract of Koyotei as an active ingredient.

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

The enzyme may be a sugar removal enzyme, preferably an enzyme that hydrolyzes a rhamnose residue, more preferably a rhamnosidase, but is not limited thereto.

The enzymatic hydrolyzate of the extract of Koyotei of the present invention

1) extracting by adding ethanol to the dried koyotei leaves, filtering, and then concentrating under reduced pressure;

2) adjusting the pH of the vacuum concentrate in step 1) to a pH of 5.5 to 7.5, followed by enzymatic hydrolysis by adding a rhamnosidase enzyme; And

3) inactivating the enzyme of the enzyme hydrolyzate of step 2) and then lyophilizing it;

Specifically

1) adding 150 to 250 mL of 50% (v/v) ethanol to 10 g of dried yellow leaves, extracting for 12 to 36 hours, filtering, and then concentrating under reduced pressure;

2) adjusting the pH of the vacuum concentrate in step 1) to pH 5.5 to 7.5, adding a rhamnosidase enzyme to enzymatic hydrolysis in a 40 to 60°C constant temperature water bath for 12 to 36 hours; And

3) inactivating the enzyme of the enzyme hydrolyzate of step 2) and then lyophilizing it;

More specifically

1) adding 200 mL of 50% (v/v) ethanol to 10 g of dried yellow leaves, extracting for 24 hours, filtering, and then concentrating under reduced pressure;

2) adjusting the pH of the vacuum concentrate in step 1) to pH 6.5, adding a rhamnosidase enzyme to enzymatic hydrolysis in a 50°C constant temperature water bath for 24 hours; And

3) Inactivating the enzyme of the enzymatic hydrolyzate of step 2), followed by lyophilization, can be used to prepare a rhamnosidase enzyme hydrolyzate of the extract of Koolai oleracea, but is not limited thereto. .

The health functional food composition for preventing or improving atopic dermatitis of the present invention may be prepared in any one formulation selected from powders, granules, pills, tablets, capsules, candy, syrup, and beverages, but is not limited thereto. In addition, the mixed extract shall contain any one of an extract obtained by an extraction treatment, a dilute or concentrated solution of the extract, a dried product obtained by drying the extract, a crude product, or a purified product. The health functional food composition is not particularly limited as long as it can be ingested to prevent or improve atopic dermatitis.

When using the health functional food composition of the present invention as a food additive, the health functional food composition may be added as it is or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method.

The active ingredient can be appropriately used depending on the purpose of use (prevention or improvement). In general, when preparing food or beverage, 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. However, in the case of long-term intake for the purpose of health control, the amount may be less than the above range, and there is no problem in terms of safety, so 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, and vitamin complexes, and all health foods in the usual sense are included.

In addition, the health functional food composition of the present invention may be prepared as a food, particularly a functional food. Functional foods of the present invention include ingredients that are commonly added during food production, and include, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when prepared as a drink, natural carbohydrates or flavoring agents may be included as an additional ingredient in addition to the active ingredient. The natural carbohydrates are monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrins, cyclodextrins, etc.) or sugar alcohols (e.g. , Xylitol, sorbitol, erythritol, etc.). As the flavoring agent, natural flavoring agents (eg, taumatin, 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, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonic acid It may further contain a carbonation agent used in beverages.

In addition, the present invention relates to a quasi-drug composition for the prevention or improvement of atopic dermatitis containing an enzyme hydrolyzate of a koyoteum leaf extract as an active ingredient.

The term "quasi-drug" as used in the present invention refers to items that are less effective than medicines among items used for the purpose of diagnosing, treating, improving, alleviating, treating, or preventing diseases of humans or animals. According to this, quasi-drugs exclude items used for pharmaceutical purposes, and include products used for the treatment or prevention of diseases of humans and animals, and products that have 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 is not particularly limited in its formulation, and may be, for example, a formulation for transdermal administration such as a lotion, ointment, gel, cream, patch or spray. .

In addition, in each formulation, the quasi-drug composition may be arbitrarily selected and blended 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 (inhibition or mitigation). For example, thickeners, stabilizers, solubilizers, conventional adjuvants such as vitamins, pigments and flavors, and carriers may be included.

In addition, the present invention relates to a pharmaceutical composition for the prevention or treatment of atopic dermatitis, containing the enzyme hydrolyzate of the extract of Koyotei 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 pharmaceutical compositions. 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 a suitable set.

The pharmaceutical composition according to the present invention may be formulated and used in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and injections according to a conventional method. .

Carriers, excipients, and diluents that may be included in the pharmaceutical composition containing the mixed extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate. , Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. have.

In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. in the mixed extract. It is prepared by mixing. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, and other excipients, such as water and liquid paraffin, which are commonly used simple diluents, such as humectants, sweeteners, fragrances, and preservatives. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like may be used. As a base for suppositories, Witepsol, Macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.

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

Hereinafter, the present invention will be described in more detail using examples. These examples are for illustrative purposes only, and it is obvious 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 sample

-Enzyme of Koyotei leaf extract Hydrolyzate

For the preparation of the enzyme hydrolyzate of the ethanol extract of Koyotei, 50% (v/v) ethanol (200ml) was added to the dried koyotei leaf (10g), followed by extraction at room temperature (20~25℃) for 24 hours, and then 0.45 Filtration was performed using a µm filter, and the filtrate was concentrated under reduced pressure to remove ethanol. Thereafter, the pH of the vacuum concentrate was adjusted to 6.5, and 100 µl of ramnosidase (α-L-RHAMNOSIDASE (Lot 110501c) derived from prokaryote; Megazyme, Chicago, IL, USA) was added, and then in a 50°C constant temperature water bath for 24 hours. After the reaction during, the enzyme was inactivated and lyophilized. After that, the rhamnosidase enzyme hydrolyzate of the freeze-dried Koyotei leaf extract was stored at -80°C and used in the experiment.

-Ethanol extract of Koyotei leaf

For the preparation of ethanol extract of Koyotei leaf, 50% (v/v) ethanol (200ml) was added to dried koyotei leaf (10g), and extracted for 24 hours at room temperature (20~25℃), and then a 0.45㎛ filter was used. Then, it was filtered, and the filtrate was concentrated under reduced pressure to remove ethanol. After that, the concentrated product under reduced pressure was lyophilized, and the powder was stored at -80°C and used in the experiment.

-Goyomnip Mountain Hydrolyzate

For the preparation of the enzyme hydrolyzate of the ethanol extract of Koyotei, 50% (v/v) ethanol (200ml) was added to the dried koyotei leaf (10g), followed by extraction at room temperature (20~25℃) for 24 hours, and then 0.45 Filtration was performed using a µm filter, and the filtrate was concentrated under reduced pressure to remove ethanol. After injecting an appropriate amount of 1N hydrochloric acid solution to a pH of 2.7 to the concentrated under reduced pressure, it was hydrolyzed by reacting in a constant temperature water bath at 80°C for 2 hours, neutralized by injecting a 1N sodium hydroxide solution, lyophilized to powder, and then Recovered and stored at -80 ℃ was used in the experiment.

2. DPPH Radical Erasing ability

DPPH radical scavenging activity was measured by the method of Blois (1958). The sample was dissolved in methanol and quantified so that the final concentration was 20, 40, 60, 80, and 100 μg/mL, and 100 μl of each sample was injected into a 96-well plate, and 100 μl of 0.3 mM DPPH was simultaneously added to the total amount of 200 μl. Μl. Then, after reacting at room temperature for 10 minutes, absorbance was measured at a wavelength of 540 nm with an ELISA reader (Tecan Group Ltd., Mannedorf, Swizerland). The DPPH radical scavenging ability was expressed as a percentage of the difference in absorbance between the added group and the non-added group using the following equation 1, and the concentration having 50% scavenging activity was confirmed.

[Equation 1]

DPPH radical scavenging ability (%) = 1-(absorbance in added group/absorbance in no added group) × 100

3. RAW 264.7 Cell culture and kohlrabi enzyme Hydrolyzate Cytotoxicity assay

The cells used in the present invention were RAW264.7 cells, a mouse-derived macrophage cell line, and were purchased from the American Type Culture Collection (ATCC). DMEM (Dulbecco's modified eagle medium) medium (GIBCO-BRL, Invitrogen, Carlsbad, CA, USA) added with 10% (v/v) FBS (fetal bovine serum), 100 units/mL penicillin, and 100 μg/mL streptomycin was added. And cultured in an incubator at 37° C. and 5% CO _2. After treating the cultured cells with samples (12.5, 25, 50, 100 and 200㎍/mL) and incubating again for 24 hours, cells using EZ-Cytox Cell Viability Analysis Kit (Daeil Biotech Co., Ltd., Korea) Toxicity was measured.

4. RAW 264.7 in cell line NO ( Nitric Oxide ) Measure

After dispensing RAW264.7 cells in a 96 well plate so that the final concentration is 2×10 ⁵ cells/mL, incubated for 24 hours in a 37°C and 5% CO ₂ incubator, 50 μg/mL sample or 5 μg/mL After prednisolone treatment, 1 hour later, LPS (1 μg/mL) was treated and incubated for 16 hours. After incubation, 100 µl of cell culture solution and 100 µl of grease reagent were mixed and reacted for 15 minutes at room temperature, and the absorbance was measured at 540 nm using a microplate reader, and a standard curve was prepared with sodium nitrate. The amount of NO (nitric oxide) produced was calculated.

5. ELISA ( Enzyme - linked immunosorbent assay ) Using mouse In splenocytes Analysis of atopic related factors

To obtain splenocytes from mice, 4-week-old male BALB/c mice bred in a sterile environment were purchased from Orient Bio Co., Ltd. (Gwangju Metropolitan City, Korea), and after being purified for 1 week while supplying enough feed and water, they were used for the experiment. I did. In the breeding environment, the cycle of day and night was performed for 12 hours each, and the temperature (20~22℃) and humidity (50~60%) were kept constant, and the experiment was conducted in accordance with the regulations of the Experimental Animal Committee of Jeonju University.

The spleen was aseptically removed from the purified mouse, washed in cold PBS, ground finely with a sterilized glass slide, passed through a 40 μm cell strainer, and hemolyzed red blood cells using an RBC lysis buffer. After being centrifuged, splenocytes were obtained.

The obtained splenocytes were 6 wells using RPMI 1640 medium (GIBCO-BRL, Invitrogen, Carlsbad, CA, USA) added with 10% (v/v) FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. After dispensing the plate to a final concentration of 5×10 ⁶ cells/mL, it was incubated in an incubator at 37°C and 5% CO _{2 for 24 hours.} Then, the cultured cells were pretreated with a sample (100 μg/mL) or 5 μg/mL of prednisolone for 1 hour, and then concanavalin A (Con A, 2 μg/mL) was treated and cultured again for 48 hours. After the supernatant was recovered, the concentrations of IL-4 (interleukin-4) and IFN-γ (interferon-γ) were measured. IL-4 and IFN-γ ELISA kits were measured according to the manufacturer's protocol using a kit from R&D Systems (Minneapolis, MN, USA).

6. In the animal model DNFB And House dust Induction of atopic dermatitis by application of tick antigen

A 7-week-old male hairless mouse raised in a sterile environment was purchased from Orient Bio Co., Ltd. (Gwangju, Korea), and was used in the experiment after being purified for one week while supplying enough feed and water. In the breeding environment, the cycle of day and night was performed for 12 hours each, and the temperature (20~22℃) and humidity (50~60%) were kept constant, and the experiment was conducted in accordance with the regulations of the Experimental Animal Committee of Jeonju University.

0.15% (w/v) DNFB (2,4-dinitrofluorobenzene) prepared using a solvent prepared in a volume ratio of 3:1 to acetone and olive oil in order to induce chemical antigen-induced atopic dermatitis in animals separated from groups after purification. ) Were sensitized on the skin of the dorsal side (100µl) on the 1st and 4th days after group separation, respectively. From the first sensitization day, 0.1 ㎖ of physiological saline was orally administered to the negative control group (AD), and 0.1 ㎖ of 200 mg/kg sample was orally administered to the experimental group, and 0.1 ㎖ of 10 mg/kg prednisolone was orally administered as a positive control group. Oral administration was performed once a day until the end of the experiment.

In addition, after applying 0.2% (w/v) DNFB at 3 days intervals on the 7th, 10th and 13th from the first sensitization, 100mg of house dust mite antigen-derived Biostir AD ointment (Biostir, Hyogo, Janpan) was applied 3 times to perform a challenge.

7. Tissue thickness measurement

5 hours after the last challenge, the thickness of the back and ear skin tissue of the mouse was measured using a digital caliper (Mitutoyo, Japan).

8. Evaluation of skin lesions (clinical score)

The skin condition was investigated by taking a picture after the end of the experiment. Dryness, scaling, erosion, abrasion, bleeding, etc. of the skin are checked. 2 points (moderate) and 3 points (severe) were given for severe conditions, and a total score was assigned for each step to evaluate.

9. Serum IgE , IL -4 and CCL17 Cytokine measurement

IL-4, IgE and CCL17 (chemokine ligand 17) of atopic dermatitis model mice were measured by collecting blood from the portal vein of the mouse after evaluating skin lesions of the atopic dermatitis model mice, and then obtaining serum.

Serum IL-4, IgE and CCL17 were identified using anti-mouse IL-4, anti-mouse IgE and anti-mouse CCL17 antibodies, and using ELISA kits that specifically act on each of R&D Systems Co. and Shibayagi It was measured and quantified according to the method provided by (Hyogo, Japan).

Specifically, 100 µl of serum diluted 5 times was injected into the plate coated with each antibody, washed well, and then a secondary antibody to which horseradish peroxidase was attached was injected and reacted. The chromogenic substrate was injected and reacted and measured with an ELISA reader (Molecular Devices, USA).For quantification of each substance, each substance was treated by concentration and reacted to obtain a standard curve, and then the amount of substance contained in the serum was measured. Calculated.

10. Statistical Analysis

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

Example 1. Enzyme of Koyotei Leaf Extract Hydrolyzate Antioxidative effect

As a result of confirming the DPPH radical scavenging activity of the Ethanol Extract (DLE), the acid hydrolyzate (HDLE) of the Ethanol Extract and the Enzyme Hydrolyzate (EDLE) of the Ethanol Extract from the Ethanol Leaf of the present invention, as disclosed in Table 1 below. It was confirmed that the IC ₅₀ value of EDLE was significantly lower than that of DLE and HDLE by more than half.

sample DPPH radical scavenging activity
(IC ₅₀ , μg/mL) DLE (ethanol extract) 55.00±0.85 ^a HDLE (acid hydrolyzate) 54.45±0.16 ^a EDLE (Enzyme Hydrolyzate) 24.76±0.28 ^b

Example 2. Enzyme of Koyotei Leaf Extract Hydrolyzate NO Production inhibitory effect

It was confirmed that there was no cytotoxicity up to 200 μg/mL by treating RAW 264.7 cells with different preparation methods by concentration (Fig. 1A), and after inducing the production of NO by treating LPS, the preparation method was changed. In the case of treatment with a concentration of 50 μg/mL, it was confirmed that the production of NO increased by the LPS treatment was decreased by the treatment of the koyotei leaf sample, and the ethanol extract (DLE) and the ethanol extract of koyomib were Compared to the acid hydrolyzate (HDLE) treatment group, the enzymatic hydrolyzate (EDLE) treatment group of the ethanol extract of Koyotei leaves had a remarkable effect of inhibiting NO production (FIG. 1B).

Example 3. Enzyme of Koyotei Leaf Extract Hydrolyzate Changes in the amount of secretion of atopic related factors (IL-4, INF-γ) by treatment

It was confirmed by ELISA how to change the secretion of atopy-related factors increased by ConA when splenocytes were treated with ConA with a sample of oleander leaves with different preparation methods. As a result, when ConA was treated alone, the increased secretion of IL-4 and INF-γ was decreased when treated with Koyotei leaf samples. In particular, IL- The effect of reducing the secretion amount of 4 and INF-γ was remarkable compared to the acid hydrolyzate (HDLE)-treated group of the ethanol extract (DLE) and the ethanol extract of koyoteum leaf (FIG. 2).

Example 4. Enzymes of Koyotei Leaf Extract in Animal Model of Atopic Dermatitis Hydrolyzate Atopic dermatitis improvement effect

In the animal model in which atopic dermatitis was induced by the application of DNFB and house dust mite antigen, it could be confirmed that atopic dermatitis was improved when the administration of the kool leaf sample or prednisolone. The atopy improvement effect was remarkable compared to the group administered with the extract (DLE) and the acid hydrolyzate (HDLE) of the ethanol extract of Koyotei (Fig. 3).

In addition, as a result of confirming the clinical score of the dermatitis improvement effect and the change in the thickness of the back skin tissue, the clinical score of the severity of the skin lesion decreased when administering the Kool leaf sample and prednisolone, and the thickness of the back skin tissue was not administered. It was confirmed that it decreased compared to the control group. In particular, the effect of reducing the thickness of skin tissues such as the clinical score of the enzyme hydrolyzate (EDLE) administration group of the Ethanol leaf ethanol extract was remarkable compared to the acid hydrolyzate (HDLE) administration group of the Ethanol leaf ethanol extract (DLE) and the ethanol extract of Ethanol leaf. (Fig. 4).

Example 5. Enzyme of Koyotei Leaf Extract Hydrolyzate Changes in the concentration of IL-4, IgE and CCL17 in serum of atopic dermatitis animal model by administration

In an animal model of atopic dermatitis, the atopic dermatitis improvement effect of the enzyme hydrolyzate of the extract of oleander leaves was confirmed by the concentrations of IL-4, IgE, and CCL17 in serum. As a result, it was confirmed that the concentration of IL-4 and IgE in the serum of the animal model in which atopic dermatitis was induced by the application of DNFB and house dust mite antigen increased. The concentration decreased.

In addition, it was confirmed that the concentration of CCL17 in the serum of the animal model caused by atopic dermatitis was increased by the application of DNFB and house dust mite antigen. It was confirmed that the increased concentration of CCL17 decreased in the group administered with the extract (DLE) and the enzyme hydrolyzate (EDLE) of the ethanol extract of Koyotei.

In particular, the effect of reducing the concentration of IL-4, IgE, and CCL17 in the serum of the enzyme hydrolyzate (EDLE)-administered group of Ethanol leaf ethanol extract compared to the acid hydrolyzate (HDLE)-administered group of Ethanol extract (DLE) and Ethanol extract. It was remarkable (Fig. 5).

Claims (3)

1) adding 200 mL of 50% (v/v) ethanol to 10 g of dried yellow leaves, extracting for 24 hours, filtering, and then concentrating under reduced pressure;
2) adjusting the pH of the vacuum concentrate in step 1) to 6.5, adding 100 µl of a rhamnosidase enzyme, and performing enzymatic hydrolysis in a 50°C constant temperature water bath for 24 hours; And
3) Inactivating the enzyme of the rhamnosidase enzyme hydrolyzate of step 2) and then freeze-drying it; containing the rhamnosidase enzyme hydrolyzate of the koala leaf extract prepared by a manufacturing method comprising as an active ingredient Health functional food composition for preventing or improving atopic dermatitis. 1) adding 200 mL of 50% (v/v) ethanol to 10 g of dried yellow leaves, extracting for 24 hours, filtering, and then concentrating under reduced pressure;
2) adjusting the pH of the vacuum concentrate in step 1) to 6.5, adding 100 µl of a rhamnosidase enzyme, and performing enzymatic hydrolysis in a 50°C constant temperature water bath for 24 hours; And
3) Inactivating the enzyme of the rhamnosidase enzyme hydrolyzate of step 2) and then freeze-drying it; containing the rhamnosidase enzyme hydrolyzate of the koala leaf extract prepared by a manufacturing method comprising as an active ingredient A quasi-drug composition for preventing or improving atopic dermatitis. 1) adding 200 mL of 50% (v/v) ethanol to 10 g of dried yellow leaves, extracting for 24 hours, filtering, and then concentrating under reduced pressure;
2) adjusting the pH of the vacuum concentrate in step 1) to 6.5, adding 100 µl of rhamnosidase enzyme, and enzymatic hydrolysis in a 50°C constant temperature water bath for 24 hours; And
3) Inactivating the enzyme of the rhamnosidase enzyme hydrolyzate of step 2) and then freeze-drying it; containing the rhamnosidase enzyme hydrolyzate of the koala leaf extract prepared by a manufacturing method comprising as an active ingredient A pharmaceutical composition for the prevention or treatment of atopic dermatitis.

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