KR101844890B1 - Composition for preventing or improving atopic dermatitis comprising ethyl acetate fraction of methanol extract from Diospyros lotus leaf as effective component - Google Patents

Abstract

The present invention relates to a health food composition, a pharmaceutical composition and a cosmetic composition for preventing or ameliorating atopic dermatitis, which comprises an ethyl acetate fraction of a methanol extract of a leaf of Diospyros lotus L. leaf as an active ingredient, wherein the ethyl acetate fraction The antioxidant and anti-itch effects of the present invention can be useful for improving skin diseases such as atopic dermatitis.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or improving atopic dermatitis, comprising ethyl acetate fraction of methanol extract of gomam tree leaf as an active ingredient.

The present invention relates to a composition for improving atopic dermatitis comprising an ethyl acetate fraction of a methanol extract of Gomatomivorus leaf as an active ingredient, and more particularly to a composition for improving atopic dermatitis comprising an ethyl acetate fraction of a methanol extract of a leaf of Dioscoros lotus L. leaf, To a health food composition, a pharmaceutical composition and a cosmetic composition for preventing or ameliorating atopic dermatitis.

Atopic dermatitis is accompanied by severe itching (pruritus) with chronic skin inflammation. The itching that causes feelings of scratching is induced by an itch medication such as histamine. In the case of a normal person, the symptoms are easily alleviated by scratching. However, in the case of atopic patients, scratching is more likely to cause scratching and scratching. These scratching behaviors of atopic patients may cause skin barriers to collapse and secondary infections leading to further deterioration of inflammation. Atopic dermatitis can be temporarily alleviated, but it recurs and is aggravated by stimulants such as the environment and food, and deterioration and mitigation are repeated, and the cause is not clearly known yet. The inflammation associated with atopic dermatitis is associated with inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1, interleukin-1) (ROS), such as hydroxyl radicals (OH), superoxide radicals (O ₂ ^- ), hydrogen peroxide (H ₂ O ₂ ) and the like. Therefore, in order to ameliorate skin diseases such as atopic dermatitis, materials that can relieve itching and effectively remove active oxygen species are needed.

Goomae ( Diospyros lotus L.) is a deciduous plant distributed in Korea including Korea and China, and can be directly consumed as mature fruit. Goyom has been used in traditional medicine as a sedative, astringent, anti-tumor, anti-diabetic, febrifuge and laxative. Recently, fatty acids, sugars, flavonoids and nonvolatile components have been known, and anticoagulation, brain cell protection, antioxidant and anticancer effects of blood have been reported.

Thus, the present inventors obtained water-soluble extracts and methanol extracts from Gomam tree leaves and obtained ethyl acetate (EA) fractions from methanol extracts. DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2 (3-ethylbenzthiazoline-6-sulfonic acid)) radical scavenging activity was investigated. In addition, TNF-α production and histamine release in rats peritoneal mast cells (RPMCs) activated with PMA (phorbol 12-myristate 13-acetate) and calcium ionophore A23187 in each extract and fraction , And the inhibitory effect of the extracts and fractions on the itchiness in the animal model induced by the compound 48/80 was very significant.

Korean Patent Laid-Open Publication No. 2013-0032099 discloses a caffeoyl alpha neoendolphin peptide derivative and its use as anti-itching and anti-atopic substances, and Korea Patent No. 1231590 discloses an antiviral, antioxidant and antimicrobial Has been disclosed, but there is no description of a composition for improving atopic dermatitis containing the ethyl acetate fraction of the methanol extract of Gomam leaf of the present invention as an active ingredient.

The present invention has been made in view of the above-mentioned needs. The present inventors have completed the present invention by confirming that the ethyl acetate fraction obtained from the methanol extract of Gomamine leaf has an antioxidant and anti-itch effect.

In order to solve the above problems, the present invention provides a health food composition for preventing or ameliorating atopic dermatitis, which comprises a fraction of Dioscorea lotus L. leaf methanol extract as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis, which comprises a fraction of methanol extract of Gomam leaf as an active ingredient.

In addition, the present invention provides a cosmetic composition for improving atopic dermatitis containing a fraction of methanol extract of Gomam leaf as an active ingredient.

In addition, the present invention provides a composition for antioxidant containing a fraction of methanol extract of Gomam leaf as an active ingredient.

According to the present invention, the ethylacetate fraction of Diospyros lotus L. leaf methanol extract is a plant-derived substance and thus has low cytotoxicity and shows radical scavenging activity, and inhibits the production of TNF-α, an inflammatory substance, Histamine release inhibitory effect and itching inhibitory effect and can be usefully used as a food for increasing antioxidant activity or for alleviating itching, medicines or cosmetics, and thus can be usefully used for improving skin diseases such as atopic dermatitis.

FIG. 1 shows the results of DPPH (A) and ABTS (B) radical scavenging activities of water soluble extract, methanol extract and ethyl acetate fraction (EA fraction) Each value represents the mean ± standard error of the results that were repeated three times.
FIG. 2 shows the effect of various concentrations of ethyl acetate fraction (EA) on rat peritoneal mast cells (RPMCs) as a result of examining the effect of water extract, methanol extract and ethylacetate fraction on the cell viability (B) is the result of analysis of cell viability of rat peritoneal mast cells by water soluble extract (WS), methanol extract (MeOH) and ethyl acetate fraction (EA) at the same concentration. The normal group was the untreated cell group such as PMA and A23187, the extract or fraction, and the control group was the group treated only with PMA and A23187 without treatment with the extract or fraction. #p <0.001 was the significance of the control group for the normal group, and * p <0.01 was the significance of the test group for the control group.
FIG. 3 shows the results of analysis of the effects of water-soluble extracts, methanol extracts and ethyl acetate fractions on the amount of histamine secreted from rat peritoneal mast cells by compound 48/80 treatment. (A) shows histamine secretion amount according to the treatment with various concentrations of ethyl acetate fraction (EA), (B) shows the amount of histamine secretion according to the treatment of water soluble extract (WS), methanol extract (MeOH) and ethyl acetate fraction . The normal group was the compound 48/80, the untreated cell group such as the extract or the fraction, and the control group was the compound 48/80 only group treated without the extract or fraction. * p <0.05, ** p <0.01 and *** p <0.001, respectively, were the significance of the control group for the control group.
FIG. 4 shows the results of analysis of the effects of water-soluble extracts, methanol extracts and ethylacetate fractions on the amount of TNF-α produced from rat peritoneal mast cells by PMA and A23187 treatment. (A) shows the production of TNF-α by treatment with various concentrations of ethyl acetate fraction (EA) and (B) shows the production of TNF-α by treatment with aqueous extracts (WS), methanol extracts (MeOH) and ethyl acetate fractions TNF-α production. The normal group was the untreated cell group such as PMA and A23187, the extract or fraction, and the control group was the group treated only with PMA and A23187 without treatment with the extract or fraction. * p <0.05, ** p <0.01 and *** p <0.001, respectively, were the significance of the control group for the control group.
FIG. 5 shows the anti-itching effect of water-soluble extracts, methanol extracts and ethyl acetate fractions of Gomam leaf on Balb / c mice induced by itching by compound 48/80. (A) shows the number of scratching of the mouse according to the treatment with various concentrations of ethyl acetate fraction (EA), and (B) shows the number of scratching of the mouse according to the treatment of water soluble extract (WS), methanol extract (MeOH) and ethyl acetate fraction It is the result of analyzing the number of scraping of the mouse. The normal group was the compound 48/80, the untreated group such as the extract or the fraction, the control group was the compound 48/80 only group treated without the extract or fraction, and the azelastine or methysergide was the positive control group . * p <0.05, ** p <0.01 and *** p <0.001, respectively, were the significance of the control group for the control group.

According to an aspect of the invention there is date-plum (Diospyros lotus The present invention provides a health food composition for preventing or ameliorating atopic dermatitis comprising a fraction of L. methanol extract as an active ingredient.

In the health food composition for preventing or ameliorating atopic dermatitis according to an embodiment of the present invention, the gomam leaf methanol extract is preferably prepared by washing collected gomam leaf with water, steaming the gomam leaf for 4 to 10 minutes, After removing water and drying at 35 ~ 45 ℃ for 10 ~ 14 hours, 3 ~ 5,000ml methanol was added to 500 ~ 700g of dried gomam leaf and allowed to react for 5-10 days. The extract was filtered, Dried, and more preferably, the collected gomam leaf is washed with water, steamed for 5 minutes, removed at room temperature, dried in a dryer at 40 ° C for 12 hours, g, 4,000 ml of methanol, reacted for 7 days, and then the extract is filtered, reduced in pressure and lyophilized, but is not limited thereto.

In addition, in the composition according to an embodiment of the present invention, the fraction of the methanol extract of Gomam leaf may be an ethyl acetate fraction obtained by adding ethyl acetate to the methanol extract of Gomam leaf, Do not.

The ethyl acetate fraction of the methanol extract of Gomind tree leaf of the present invention is excellent in DPPH or ABTS radical scavenging activity, low in cytotoxicity, excellent in the inhibitory effect of histamine release which is involved in allergic reaction or inflammation, and proinflammatory cytokine the pro-inflammatory cytokine TNF-alpha is also excellently suppressed and the itching-relieving effect is also excellent. Thus, atopic dermatitis can be prevented or improved through antioxidant and / or anti-itching effects.

The health food is not particularly limited as long as it can be ingested to prevent or ameliorate atopic dermatitis.

When the fraction of the present invention is used as a food additive, the fraction may be added as it is, or may be used together with other food or food ingredients, and suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). Generally, the fraction of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material, in the production of food or beverage. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range .

There is no particular limitation on the kind of the food. Examples of foods to which the above substances can be added include dairy products including meats, sausages, breads, chocolates, candies, snacks, confectionery, pizza, ramen noodles, gums, ice cream, soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate may be generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the extract of the present invention, but is not limited thereto.

In addition to the above, the fraction of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the fractions of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis, which comprises a fraction of methanol extract of Gomam leaf as an active ingredient. In the pharmaceutical composition of the present invention, the fractions of methanol extract of Gomam leaf are as described in the above-mentioned health food composition.

The pharmaceutical composition of the present invention may be a composition for parenteral use such as a composition for external application for skin, an oral composition or an injection, and the formulation thereof is not particularly limited. In the case of composition for external application for skin, ointment, cream, paste, lotion, ), A liquid for external use, a tincture, an aerosol, and a plaster, and in the case of an oral composition, it may be formulated into a form such as a pill, a capsule, a single agent, a granule or a drink.

The pharmaceutical compositions comprising the fractions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition including the fraction include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium A variety of compounds or mixtures including silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, Or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical dosage forms of the fractions of the present invention may be used in the form of their pharmaceutically acceptable salts and may also be used alone or in combination with other pharmaceutically active compounds as well as in suitable aggregates.

The pharmaceutical composition of the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous, intraperitoneal, muscle, topical application, patch and iontophoresis, and local application and oral administration are preferred Do.

In addition, the dosage for preventing or treating atopic dermatitis in the composition according to the present invention may vary depending on the severity and formulation, and the number of application times may vary depending on the age, body weight, and constitution of the patient. For example, the dose of the composition for external application for skin of the present invention varies depending on age, sex, weight, symptom and administration method, but it is 1.0 to 3.0 ml per day, which is applied 1 to 5 times a day, It is good. In addition, for example, a typical daily dose of the oral composition of the present invention may range from 1 to 100 mg / kg body weight, preferably from 5 to 70 mg / kg body weight, divided once or several times Lt; / RTI &gt; However, the dose does not limit the scope of the present invention in any way.

In addition, the present invention provides a cosmetic composition for improving atopic dermatitis containing a fraction of methanol extract of Gomam leaf as an active ingredient. In the cosmetic composition of the present invention, the fraction of the methanol extract of Gomam tree leaves is as described in the above health food composition.

In the cosmetic composition according to one embodiment of the present invention, the cosmetic composition for improving atopic dermatitis may be at least one selected from the group consisting of ointment for external use, cream, softener, nutritional lotion, pack, essence, hair tonic, shampoo, rinse, hair conditioner, hair treatment, Skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, eye cream, moisturizing cream, hand cream, foundation, nutrition essence, sunscreen, soap, cleansing But are not limited to, a formulation selected from the group consisting of a foam, a cleansing lotion, a cleansing cream, a body lotion and a body cleanser. The composition of each of these formulations may contain various kinds of bases and additives necessary for formulation of the formulation, and the kinds and amounts of these ingredients can be easily selected by those skilled in the art.

When the formulation of the present invention is a paste, cream or gel, animal fibers, plant fibers, wax, paraffin, starch, tracant, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide are used as carrier components .

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.

In the case of the solution or emulsion of the present invention, a solvent, a solvent or an emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

When the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component is selected from aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolenic derivatives or ethoxylated glycerol fatty acid esters.

In the cosmetic composition according to one embodiment of the present invention, the cosmetic composition for improving atopic dermatitis may contain 0.0001% to 10% by weight of the ethyl acetate fraction of the methanol extract of Gomam tree leaf, based on the total weight of the composition, Preferably 0.005 to 1%, but is not limited thereto, including an effective amount that can provide a desired improvement effect of atopic dermatitis.

The invention also goyom tree (Diospyros lotus L.) leaf methanol extract as an active ingredient. In the antioxidant composition of the present invention, the fraction may be, but is not limited to, the ethyl acetate fraction. Since the fractions have excellent DPPH and ABTS radical scavenging activity, they can be used as an excellent antioxidant composition.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

Reagent material

An ELISA kit for the measurement of TNF-α and histamine was purchased from R & D Systems (Minneapolis, Minn., USA), and the percoll, compound 48/80, phorbol 12-myristate 13- acetate, calcium ionophore A23187, penicillin / streptomycin, methysergide maleate, butylated hydroxytoluene, DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS 2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid), potassium persulfate, sodium bicarbonate, azelastine, lipopolysaccharide, methysergide, , Trolox ((±) -6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) and other reagents were purchased from Sigma-Aldrich (Louis, MO, USA) as a reagent grade.

Guillaume Leaf Extract

Diospyros lotus L.) leaves were collected on July 10, 2013 in Shigi Village, Suwang-ri, Buyeon-myeon, Jinan-myeon, Jeollabuk-do, and received sympathy from Professor Kim Hong-joon of Woosuk University School of Oriental Medicine. The goat leaf specimen (# 2013-07-10) is kept in the Health Management Laboratory of Chonju University Medical School. The collected gomilla leaf was immediately washed with distilled water, steamed for 5 minutes, dried using a fan at room temperature, and finally dried at 40 ° C for 12 hours in a dryer. The dried goat leaves were quantified as 500 g, 3,000 ml of distilled water was added and boiled for 10 minutes. The extract was filtered using a 0.45 ㎛ filter and dried in a freeze dryer (EYELA FDU-2100, Japan) to obtain 45.3 g. The dried goat leaves were weighed to 600 g, and 4,000 ml of methanol was added. After 7 days, the extracts were filtered using a 0.45 ㎛ filter, and the organic solvent was removed by reduced pressure. Finally, the filtrate was lyophilized to obtain 70.3 g 50 g of ethyl acetate (EA) was sufficiently injected (500 ml) to obtain about 650 mg of the compound, which was stored at -20 ° C.

Measurement of DPPH radical scavenging activity

DPPH radical scavenging activity was measured by the method of Blois ( Nature , 1958, 181: 1199-1200). The sample was dissolved in methanol to give a final concentration of 3.9-1,000 .mu.g / ml. 100 .mu.l of each sample was injected into a 96-well plate, and 100 .mu.l of 0.3 mM DPPH was added to make a total amount of 200 .mu.l. After incubation at room temperature for 10 minutes, the absorbance was measured at 540 nm with an ELISA reader (Tecan Group Ltd., Mannedorf, Swizerland). The DPPH radical scavenging activity was expressed as a percentage of absorbance difference between the addition group and the no addition group of the sample solution.

DPPH radical scavenging activity (%) = {1- (absorbance of sample addition group / absorbance of sample addition group)} x 100

Measurement of ABTS radical scavenging activity

The ABTS radical scavenging activity was measured by the method of Re et al. ( Free Radical Biol. Med. 1999, 26: 1231-1237). 7.4 mM ABTS and 2.6 mM potassium persulfate were mixed and allowed to stand at room temperature for 24 hours to form radicals. The ABTS solution was diluted with methanol to an absorbance of 0.70 ± 0.03 at 732 nm immediately before the experiment . The final concentration of each extract and Trolox was determined to be 3.9-1,000 ㎍ / ㎖. After adding 950 ㎕ of ABTS solution to 50 ㎕, the reaction was allowed to proceed in the dark for 30 minutes, and the absorbance was measured at 732 nm. The scavenging activity of ABTS was expressed as a percentage of absorbance difference between the addition group and the no addition group of the sample solution.

ABTS radical scavenging activity (%) = {1- (absorbance of sample addition group / absorbance of sample addition group)} x 100

Experimental animal

Seven weeks old male Balb / c mice and 10-week old Sprague-Dawley rats raised in sterile environment were purchased from Central Laboratory Animals (Seoul City, Korea), purified and fed for a week with sufficient feed and water, Lt; / RTI &gt; The incubation environment consisted of day and night cycles for 12 hours, and the temperature (20-22 ℃) and humidity (50-60%) were kept constant and tested according to the provisions of the Laboratory Animal Committee.

Isolation of RPMCs and treatment of guillofolate leaf fractions

Separation of rat peritoneal mast cells (RPMCs) was performed according to the method of Martynova et al. ( Cell Res . 2005, 15: 811-816). Briefly, ether rats were anesthetized and then anesthetized with 10 ml of calcium free HEPES-Tyrode buffer (10 mM HEPES, 113 mM NaCl, 4.7 mM KCl, 2.13 mM MgCl ₂ , 0.6 mM NaH ₂ PO ₄ , 10 mM glucose, pH 7.4) Was infused into the abdominal cavity and gently massaged the abdominal cavity for about 90 seconds. The abdominal cavity was carefully opened, and a cell suspension was obtained using a pastel pipette to obtain RPMCs by a percoll concentration gradient method. For the purpose of this experiment, RPMCs obtained from 5 to 10 rats were mixed and used to obtain sufficient cells. Ethyl acetate (EA) fractions (5-20 μg / ml) were pretreated with RPMCs ( ⁵ × 10 ⁵ ) for 10 min or 2 h and then stimulated with compound 48/80 or PMA and A23187 to induce histamine and proinflammatory cytokines -inflammatory cyotokine).

Cell viability measurement

Cell viability of RPMCs was measured by MTT assay. In summary, RPMCs (5x10 ⁵ / ㎖) inoculated with the cells in 24-well culture plate, and then treated with ethyl acetate fraction in various concentrations as described above and stimulated with PMA and A23187, after 12 hours MTT solution (5 ㎎ / Ml) was added, and the mixture was allowed to stand at 37 ° C for 4 hours. The supernatant was removed, and the formazan product was dissolved in DMSO, transferred to a 96-well plate, and absorbance was measured at 540 nm.

Histamine release measurement

RPMCs ( ⁵ × 10 ⁵ / ml) were pretreated with ethylacetate fractions of various concentrations (50-200 μg / ml) at 37 ° C. for 10 minutes, stimulated with 0.5 μg / ml of compound 48/80 and left for 30 minutes To stop the reaction, the culture tube was placed in ice water and sufficiently cooled. After centrifugation at 1,200 rpm for 15 minutes, supernatant was obtained and histamine was measured. To summarize, an anti-histamine antibody is used to react with the supernatant obtained as described above, and a secondary antibody with a substrate enzyme specifically acting on the anti-histamine antibody is injected thereinto, followed by color development. The concentration of histamine was determined by histamine treatment and the standard curve was determined. The histamine concentration was measured by ELISA reader (Molecular Devices, USA).

TNF-a measurement

Cytokines from RPMCs were pretreated with an ethyl acetate (EA) fraction (2.5-10 μg / ml) for 2 hours, stimulated simultaneously with 20 nM PMA and 1 μM A23187, and then 12 hours later, TNF -α was measured. TNF-α was measured and quantified using an ELISA kit using the anti-mouse TNF-α antibody according to the method provided by the manufacturer. In summary, 100 μl of the supernatant or 5-fold diluted serum was injected into the plate coated with antibody, reacted, washed well, injected with secondary antibody with HRP (horseradish peroxidase), reacted, And the reaction was measured with an ELISA reader. The quantification of each substance was performed by treating each substance with a concentration to determine a standard curve, and the amount of substance contained in the supernatant or serum was calculated.

Measures itching and scratching behavior

Five weekly Balb / c mice were inoculated into a transparent acrylic cage (20 cm) each of five mice per experimental group and left in the same experimental environment for 30 minutes for stability. The control group was then administered orally with physiological saline and the experimental group was treated with 10 mg / kg of the ethyl acetate (EA) fraction (2.5-10 mg / kg) and the positive control methysergide or azelastine After 60 minutes of oral administration, 100 μl of compound 48/80 (50 μg / site) or histamine (100 μg / site) was subcutaneously injected at the level between both shoulders such as mice. Mice injected with the itch inducer were immediately recorded for 60 minutes using a micro camera (ONCCTV, Seoul, Korea) according to the method of Mihara ( Br. J. Dermatol . 2004, 151: 335-345) The number of times scratching the injected area was assessed by counting with a double blind method. Experiments on each inducer were conducted on different days and the mice used in each experiment were used once.

Statistical processing

Statistical analysis was performed with ANOVA and Student's t-test. The significance level was set at p <0.05.

Example 1 DPPH and ABTS Radical Scavenging Activity of Ethyl Acetate Fraction Derived from Goat Leaf Leaf

To investigate the DPPH and ABTS radical scavenging activities of ethyl acetate fraction (EA) (hereinafter referred to as ethyl acetate fraction or ethylacetate fraction derived from gomule leaf) of methanol extract of Goat Leaf, the ethyl acetate fraction, Methanol extracts, and radical antioxidants BHT (butylated hydroxytoluene) and Trolox. As a result, the ethyl acetate fraction derived from goat leaf showed better DPPH radical scavenging activity than BHT, as well as water soluble extract and methanol extract at all concentrations (FIG. 1A). In the case of water - soluble extracts and methanol extracts, DPPH radical scavenging activity was similar to that of BHT at a low concentration of less than 31.3 ㎍ / ㎖, but DPPH radical scavenging activity was better than BHT at higher concentrations. In addition, the IC ₅₀ for the DPPH radical scavenging activity of the ethyl acetate fractions was 5.3 ㎍ / ㎖, about 5-7 (w / w) more soluble than water soluble exports (37.9 ㎍ / ㎖), methanol extract (41.2 ㎍ / It was confirmed that the concentration was low. Also, in the case of ABTS radical scavenging activity, the ethyl acetate fraction derived from gomoma leaf showed better ABTS radical scavenging activity than Trolox as well as water soluble extract and methanol extract at all concentrations. Water soluble extracts and methanol extracts showed lower ABTS radical scavenging activity at each concentration than Trolox but increased activity as concentration increased. The IC ₅₀ for the ABTS radical scavenging activity of the ethyl acetate fractions was 53.8 ug / ml, about 3-7 fold higher than the aqueous extract (311.2 / / ml), methanol extract (402.5 / / ml) and Trolox (178.6 / / And showed its activity at low concentrations (FIG. 1B).

The DPPH radical scavenging activity (IC ₅₀ : 5.3 / / ㎖) of the ethyl acetate fraction derived from goat leaf was higher than that of D. lotus fruit (72.6 / / ㎖) (Loizzo et al., 2009, Plant Foods Hum Nutr . 64: 264-270), and Gooseum-derived quercetin (5.8 [mu] g / ml) (Moghaddam et al., 2012, Act Po Phar 69: 687-692), Solanum belonging to the group reported by Zadra et al. ( Molecules , 2012, 17: 12560-12574) guaraniticum (9.11 占 퐂 / ml). Therefore, ethyl acetate fraction derived from goat 's leaf is considered to be able to exert a strong antioxidative effect.

Example  2. Ethyl acetate derived from goat leaf The fraction  Effect on cell survival rate

To investigate the cell survival rate according to the concentration of ethyl acetate fraction derived from goat leaf, before treatment of compound 48/80 (0.5 / / ml) used to activate rat peritoneal mast cells (RPMCs), ethyl acetate Fractions (25-100 ㎍ / ㎖), aqueous extracts or methanol extracts were treated for 2 hours to compare cell viability with MTT assays. As a result, as shown in FIG. 2, the compound 48/80-treated control group was more cytotoxic than the normal control group. However, the cell survival rate was similar to that of the normal control group at all concentration levels of the ethyl acetate fraction, Similar results were obtained in the methanol extract treated group. Therefore, it was confirmed that all ethyl acetate fractions, water-soluble extracts and methanol extracts used in the present invention had no cytotoxicity.

Example 3 Inhibitory Effect of Ethyl Acetate Fraction Derived from Goat Leaf Leaf on Histamine Release

(RPMCs) were inoculated and ethylacetate fractions (25-100 ㎍ / ㎖) were injected and incubated at 37 ℃ for 30 min at 37 ℃. The inhibitory effect of ethyl acetate fraction on the inhibition of histamine release, After pretreatment for 10 minutes, histamine release was measured by stimulation with compound 48/80 (0.5 / / ml). As a result, when the RPMCs were stimulated with compound 48/80, the histamine release was 63.49 ± 5.67 ng / ml, which was significantly higher than that of the normal control (10.17 ± 1.40 ng / ml) ( p <0.001). However, when the ethyl acetate fraction was treated, it was confirmed that the release of histamine was inhibited in a concentration-dependent manner. Especially, 100 ㎍ / ㎖ treated group had histamine release of 31.57 ± 1.40 ng / ㎖, which was about 47.6% lower than that of the control group ( p <0.001). In order to compare histamine release inhibition effects of the ethyl acetate fraction, the water soluble extract of methanol extract and methanol extract, the fraction and each extract were fixed at a concentration of 100 μg / ml. As a result, as shown in FIG. 3B, The extracts and methanol extracts showed 48.97 ± 3.70 ng / ㎖ and 46.87 ± 3.54 ng / ㎖ inhibition of histamine release (p <0.05), respectively. The ethyl acetate fraction showed higher histamine release inhibitory effect Respectively.

Compound 48/80 is known to activate mast cells, and activated mast cells secrete itching mediators such as histamine, serotonin or substance P. Itching caused by substances secreted from these mast cells leads to symptoms similar to itching, which is common in allergic skin diseases such as atopic dermatitis. Therefore, the ethyl acetate fraction of the present invention was confirmed to be an excellent material that can be applied to allergic skin diseases, which has an effect of inhibiting histamine release.

Example 4. Inhibitory Effect of Ethyl Acetate Fraction Derived from Goat Leaf Leaf on TNF-α Production

In order to investigate the effect of acetoacetate fraction derived from goat's leaf on the inhibition of the production of TNF-α, a proinflammatory cytokine of activated rat peritoneal mast cells (RPMCs), RPMCs were inoculated and fractionated with ethyl acetate (25-100 ㎍ / Was pretreated for 2 hours, and PMA (30 ng / ml) and A23187 (10 μM) were simultaneously treated. After 12 hours, the amount of TNF-α accumulated in the culture solution was measured. As shown in FIG. 4A, the production of TNF-α was significantly increased in the control group treated with PMA and A23187 (594.51 ± 28.99 pg / ㎖) ( p <0.001) TNF-α production was inhibited, and significant inhibition was observed at all concentrations (p <0.05, p <0.001). In addition, in order to compare the effect of the ethyl acetate fraction on the inhibition of TNF-α production of the water extract of Guoyom leaf and the methanol extract, it was fixed at a concentration of 100 μg / ml. As a result, as shown in FIG. 4B, (P <0.05), whereas the ethyl acetate fraction inhibited TNF-α production more than watery extracts and methanol extracts of gom- yle leaves (p <0.05) The effect was excellent.

TNF-α is a typical cytokine that causes inflammatory responses in vivo, and is mainly produced in macrophages, which are mainly activated. However, lymphoid cells such as lymphoid cells, mast cells, endothelial cells and the like It is also produced in various cells present in the body. In particular, mast cells are known to be produced in large quantities when TNF-α is stimulated simultaneously with PMA and A23187. Therefore, in order to alleviate the inflammatory reaction, a substance capable of effectively controlling TNF-a is required. Thus, the ethyl acetate fraction derived from the gomoma leaf of the present invention is considered to be an effective substance capable of controlling the inflammatory reaction in this respect.

Example 5. Inhibitory effect of ethyl acetate fraction derived from goat's leaf on itching

(2.5-10 mg / kg), aqueous extracts and methanol extracts (10 mg / kg, respectively) were orally administered to Balb / c mice in order to investigate the effect of ethyl acetate fraction derived from goat leaf on skin itching inhibition After 1 hour, the itch inducer compound 48/80 was injected subcutaneously at the level between the shoulders of the back of the mouse. As a result, mice injected with compound 48/80 subcutaneously showed a greater number of scratches than those of the normal control group (32.3 ± 4.9 times / 60 minutes) with 255.3 ± 39.5 times / 60 minutes of scratching (FIG. 5A, p <0.001 ). However, the experimental group treated with ethyl acetate fraction showed inhibitory effects on the concentration - dependent manner. Especially, in the group treated with 10 mg / kg ethyl acetate fraction derived from goat leaf, the number of scratching was 120.7 ± 24.2 times / 60 minutes, which was about 53% (p <0.001). The anti-itching effect of the ethyl acetate fraction was found to be lower than that of azelastine, but was found to be higher than that of methicillinide at concentrations such as azelastine and methysergide, which are known to be excellent anti-itching drugs 5B).

Azelastine is an anti-itching drug known as an antagonist to the H1 histamine receptor and is known to effectively alleviate itching induced by itch inducers such as compound 48/80 as well as histamine and substance P. Metisergide is also well known as a drug that can relieve compound 48/80 induced itching. Although it may be necessary to elucidate the molecular mechanism of the inhibition of itching in the later ethyl acetate fraction, based on the above results, the excellent anti-itching effect of the ethyl acetate fraction derived from the goat leaf of the present invention is effective for skin itching such as atopic skin disease It is considered to be a very good material that can be used.

Claims (8)

A health food composition for preventing or ameliorating atopic dermatitis containing ethyl acetate fraction of methanol extract of Gomam ( Diospyros lotus L.) leaf as an active ingredient. delete delete A pharmaceutical composition for the prevention or treatment of atopic dermatitis comprising an ethyl acetate fraction of a methanol extract of Gomam ( Diospyros lotus L.) leaf as an active ingredient. delete A cosmetic composition for improving atopic dermatitis comprising ethyl acetate fraction of methanol extract of Gomam ( Diospyros lotus L.) leaf as an active ingredient. delete An antioxidative composition comprising as an active ingredient an ethyl acetate fraction of methanol extract of Gomam ( Diospyros lotus L.) leaf.

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