KR102497941B1 - Compositions for anti-inflammation and anti-allergy using an extract of Artemisia stolonifera - Google Patents

Abstract

The present invention inhibits the production of IL-6 and IL-6, which are inflammatory cytokines, when HaCaT cells, a human keratinocyte cell line in which an inflammatory response is induced by TNF-α/IFN-γ treatment, are treated with the extract of artemisia broadleaf. Broadleaf Artemisia that inhibits degranulation when treated with RBL-2H3, a rat basophilic leukemia mast cell line activated with DNP-IgE (dinitrophenyl-immunoglobulin E) and DNP-BSA (anti-dinitrophenyl antibody) An anti-inflammatory composition and an anti-allergic composition using an extract are disclosed.

Description

Anti-inflammatory composition and anti-allergy composition using an extract of Artemisia stolonifera

The present invention discloses an anti-inflammatory composition and an anti-allergic composition using an extract of Artemisia stolonifera .

Skin exists in the outermost part of the human body and is in direct contact with the external environment, protecting the human body from external damage and preventing excessive evaporation of moisture and heat in the body. The skin is composed of three layers: epidermis, dermis, and subcutaneous tissue. Among them, the epidermis, which is the most superficial layer, is composed of keratinocytes, hair follicles, melanocytes, and Langerhans cells. Keratinocytes, which occupy most of the epidermal cells, not only form keratin, but also produce inflammatory factors such as IL-1β, TNF-α, IL-6, IL-8, and other cytokines depending on external environmental factors to stimulate the skin inflammatory response. (J Drugs Dermatol 7: 1038-1043, 2008; J Dermatol Sci 67: 101-110, 2012). The inflammatory response of these keratinocytes is closely related to skin diseases such as skin aging, atopic dermatitis, and psoriasis (Clin Exp Rheumatol 24: S1-6, 2006).

Allergic reactions are classified into five types: type I anaphylaxis type, type II cytolytic type, type III immune complex type, type IV cell-mediated type, and type V irritant hypersensitivity reaction type, most of which are type I. , and is generally used in the same sense as type I when it comes to allergic reactions (Roitt I, Brostoff J, Male D. Immunology 6th eds. U.S.A.. Mosby. 2001:323-383).

Representative allergic diseases include atopic dermatitis, bronchial asthma, allergic rhinitis, allergic keratitis, and cutaneous urticaria. In these allergic diseases, mast cells with abundant granules in the cytoplasm are known to be important effector cells. There is (Science, 282:2258-61, 1998; Science, 282:2261-2263, 1998).

Allergic response occurs when allergen binds to IgE bound to the IgE high-affinity receptor (FcεRI) on the surface of mast cells and mast cells are activated, followed by vacuolation of granules, fusion of granules, and fusion of granules and cell membranes, followed by degranulation. This occurs and is induced by the release of substances such as inflammatory cytokines IL-4 and TNF-α and histamine, which is an itch mediator (Journal of the Korean Medical Association 61:670-7, 2018). Therefore, substances that inhibit mast cell degranulation can be effective candidates for allergic diseases, and RBL-2H3, a rat basophil leukemia mast cell line, is widely used as a mast cell degranulation research model (The Biochemical Journal 323 (Pt 2):321-8, 1997)

Broad-leaved mugwort ( Artemisia stolonifera (Maxim.) Kom.) is a perennial herb that grows in mountainous areas. The leaves are wide, but it hangs on a single leaf, so it got the name broad-leaved mugwort. The stem is 30-120cm high, grows straight, branches at the top, and has hairs. Radical leaves wither when flowers bloom. Stem leaves are alternate, ovate or long elliptical, and the edge is feather-shaped, split shallowly or even to the middle. The front side of the leaf is green with some hairs, and the back side has dense white hairs. Flowers bloom in August-September, and head-shaped flowers with a diameter of 3-4mm hang on panicles. The gathered coriander is bell-shaped and covered with spider web-like hairs. The fruit is an achene. It grows wild throughout Korea and is distributed in Russia, Mongolia, Japan, and China. Young shoots are edible, and in the private sector, the outpost has been used as a diuretic, eye disease, and antipyretic medicine.

The present invention discloses the anti-inflammatory and anti-allergic activities of mugwort broadleaf.

An object of the present invention is to provide an anti-inflammatory composition and an anti-allergic composition using an extract of mugwort broadleaf.

Other objects or specific objects of the present invention will be presented below.

As confirmed in the following Examples and Experimental Examples, the present invention is an inflammatory cytokine when HaCaT cells, a human keratinocyte cell line in which an inflammatory response is induced by TNF-α/IFN-γ treatment, are treated with mugwort extract. RBL-, a rat basophilic leukemia mast cell line that inhibits the production of IL-6 and IL-6 and is activated with DNP-IgE (dinitrophenyl-immunoglobulin E) and DNP-BSA (anti-dinitrophenyl antibody) It was completed by confirming that degranulation was inhibited when treated with 2H3.

Considering the foregoing, the present invention can be identified as an anti-inflammatory composition comprising an extract of mugwort leaf as an active ingredient in one aspect, and an anti-allergic composition comprising an extract of mugwort leaf as an active ingredient in another aspect, or It can be identified as an antihistamine composition. In particular, the anti-inflammatory composition of the present invention can be useful for application of dermatitis such as atopic dermatitis for the purpose of inhibiting the skin inflammatory reaction, especially in that it is based on the result of suppressing the anti-inflammatory reaction in human keratinocytes.

In the present specification, "extract of broad leaf mugwort" means the stem, leaf, fruit, flower, root, underground part, aerial part, outpost or a mixture thereof of mugwort broadleaf, which is an extraction target, in water, lower alcohol having 1 to 4 carbon atoms (methanol, ethanol) , butanol, etc.), methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, It refers to an extract obtained by leaching using propylene glycol or a mixture thereof, an extract obtained using a supercritical extraction solvent such as carbon dioxide or pentane, or a fraction obtained by fractionating the extract. , considering the degree of preservation, any method such as cooling, reflux, heating, ultrasonic radiation, and supercritical extraction can be applied. In the case of the fractionated extract, after suspending the extract in a specific solvent, the fraction obtained by mixing and standing with a solvent of different polarity, and the crude extract are adsorbed on a column filled with silica gel, etc., and then a hydrophobic solvent, a hydrophilic solvent, or a mixed solvent thereof It is meant to include fractions obtained as a mobile phase. In addition, the meaning of the extract includes a concentrated liquid extract or solid extract from which the extraction solvent is removed by lyophilization, vacuum drying, hot air drying, spray drying, or the like. Preferably, it refers to an extract obtained by using water, ethanol or a mixed solvent thereof as an extraction solvent, and more preferably an extract obtained by using a mixed solvent of water and ethanol as an extraction solvent.

In addition, in the present specification, "active ingredient" means a component that exhibits the desired activity alone or can exhibit activity in combination with a carrier having no activity itself.

Also, as used herein, "anti-inflammatory" is meant to include improvement (relief of symptoms), treatment, suppression or delay of onset of an inflammatory disease defined below.

Also, in the present specification, "anti-allergy" is meant to include improvement (relief of symptoms), treatment, and prevention (inhibition or delay of onset) of allergic diseases defined below.

In addition, in the present specification, the "inflammatory disease" refers to pathological symptoms caused by an inflammatory reaction mediated by inflammatory cytokines, and includes bacterial infection, fungal infection, conjunctivitis, iritis, scleritis, uveitis, and dermatitis (including atopic dermatitis). ), skin eczema, psoriasis, etc.

In addition, in the present specification, "allergic disease" refers to pathological symptoms caused by allergic reactions mediated by mast cell activation, such as mast cell degranulation, and such allergic diseases include atopic dermatitis, asthma , allergic rhinitis, allergic conjunctivitis, allergic dermatitis, allergic contact dermatitis, and allergic keratitis.

The anti-inflammatory composition, anti-allergic composition, and anti-histamine composition (hereinafter referred to as "the composition of the present invention") of the present invention exhibits an activity to improve inflammatory diseases and allergic diseases, etc. It can be included in any amount (effective amount) as long as possible, but a typical effective amount will be determined within the range of 0.001% by weight to 15% by weight based on the total weight of the composition. Here, "effective amount" means that when the composition of the present invention is administered to a mammal, preferably a human, to which it is applied, during the administration period according to the advice of medical experts, etc., the intended medical and pharmacological effects such as improvement of inflammatory diseases and allergic diseases It refers to the amount of the active ingredient included in the composition of the present invention that can exhibit an effect. Such an effective amount can be determined empirically within the ordinary skill of the skilled artisan.

In addition to the active ingredients, the composition of the present invention may further include any compounds or natural extracts whose safety has already been verified and are known to have anti-inflammatory and anti-allergic activities in order to increase or enhance anti-inflammatory and anti-allergic activities. can

These compounds or extracts include compounds or extracts, pharmaceuticals listed in compendials such as pharmacopoeia of each country ("Korean Pharmacopoeia" in Korea) and health functional food codes of each country (in Korea, it is "standards and specifications for health functional foods", a notice of the Ministry of Food and Drug Safety). In accordance with the laws of each country regulating the manufacture and sale of products (in Korea, it is the "Pharmaceutical Affairs Act"), the laws of each country regulating the manufacture and sale of compounds or extracts and health functional foods (in Korea, the "Act on Health Functional Foods") "), compounds or extracts whose functionality has been individually recognized according to For example, MSM (dimethylsulfonylmethane) with 'arthritis improvement' function of the Korea Health Functional Food Code, N-acetylglucosamine with 'arthritis improvement' function and 'skin moisturizing' function, etc. Enterococcus faecalis heat-treated dry powder, which has been individually recognized for its functionality as 'relieving hypersensitivity immune response', complexes such as guava leaf extract, complexes such as chinensis extract, leaf extract, picao preto powder, etc., PLAG (1-palmitoyl-2-linoleoyl- 3-acetyl-rac-glycerol), etc., L. sakei Probio 65, which has been individually recognized for its functionality as 'improvement of hypersensitive skin conditions', oil containing gamma-linolenic acid, L.plantarum CJLP133, a lactic acid bacterium derived from fruits and vegetables, and probiotics ATP μ, are these compounds or will correspond to the extract.

One or more of these compounds or natural extracts may be included in the anti-allergic composition of the present invention together with the active ingredient.

In a specific aspect, the composition of the present invention can be regarded as a food composition. When the composition of the present invention is understood as a food composition, its use may be understood as suppressing an inflammatory reaction or an allergic reaction.

The food composition of the present invention can be prepared in any form, for example, beverages such as tea, juice, carbonated beverages, and ionic beverages, processed oils such as milk and yogurt, chewing gum, rice cakes, traditional Korean snacks, bread, snacks, noodles, etc. It can be manufactured into health functional food preparations such as foods, tablets, capsules, pills, granules, liquid, powder, flakes, pastes, syrups, gels, jellies, and bars.

In addition, the food composition of the present invention may take any product classification as long as it conforms to the enforcement regulations at the time of manufacture and distribution in legal and functional classification. For example, health functional food according to the "Health Functional Food Act", or snacks, legumes, teas, and beverages according to each food type according to the Food Code of the Korean "Food Sanitation Act" (Ministry of Food and Drug Safety notification "Standards and Specifications for Food") , special purpose foods, etc.

The food composition of the present invention may include food additives in addition to the active ingredient. Food additives can generally be understood as substances that are added to, mixed with, or infiltrated into food in manufacturing, processing, or preserving food. Since food is consumed daily and for a long period of time, its safety must be guaranteed. According to the Food Additives Code under the laws of each country governing the manufacture and distribution of food (“Food Sanitation Act” in Korea), safety-guaranteed food additives are limitedly regulated in terms of ingredients or functions. In the Korean Food Additives Codex (Ministry of Food and Drug Safety Notice "Standards and Specifications for Food Additives"), food additives are classified into chemical synthetic products, natural additives, and mixed formulations in terms of ingredients, and these food additives are classified as sweeteners and flavors in terms of functions It is divided into additives, preservatives, emulsifiers, acidulants, and thickeners.

Sweeteners are used to impart appropriate sweetness to foods, and both natural and synthetic sweeteners can be used in the composition of the present invention. Preferably, a natural sweetener is used, and examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavors may be used to improve taste or aroma, and both natural and synthetic flavors may be used. Preferably, it is the case of using a natural one. In case of using natural ones, in addition to flavor, the purpose of enhancing nutrition can also be combined. As a natural flavoring agent, it may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or obtained from green tea leaves, roundworms, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo, etc. can be used. Natural flavors can be liquid concentrates or solid extracts. In some cases, synthetic flavors may be used, and synthetic flavors may include esters, alcohols, aldehydes, terpenes, and the like.

As preservatives, sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. may be used, and as emulsifiers, acacia gum, carboxymethylcellulose, xanthan gum, pectin and the like, and examples of the acidulant include acid salt, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like. Acidulants may be added to the food composition to have an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to improving taste.

As the thickening agent, a suspending agent, a sedimentation agent, a gel forming agent, a swelling agent, and the like may be used.

In addition to the food additives described above, the food composition of the present invention may include physiologically active substances or minerals known in the art and whose stability is guaranteed as food additives for the purpose of supplementing or reinforcing functionality and nutrition.

Examples of such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoylthiamine, and the like. Examples of minerals include calcium preparations such as calcium citrate and magnesium stearate. magnesium preparations such as the like, iron preparations such as iron citrate, chromium chloride, potassium iodine, selenium, germanium, vanadium, zinc, and the like.

The food composition of the present invention may include the above-mentioned food additives in an appropriate amount to achieve the purpose of addition according to the type of product.

Regarding other food additives that may be included in the food composition of the present invention, reference may be made to national food codes or food additive codes.

The composition of the present invention can be understood as a pharmaceutical composition in another specific aspect.

The pharmaceutical composition of the present invention may be formulated into an oral formulation or a parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. The route of administration herein may be any appropriate route including topical route, oral route, intravenous route, intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination. An example of a combination of two or more routes is a case in which two or more formulations of drugs are combined according to the route of administration, for example, one drug is firstly administered intravenously and the other drug is secondly administered through a topical route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or dosage form, and specifically, reference may be made to the pharmacopoeia of each country including the "Korean Pharmacopoeia".

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, powder, granule, tablet, pill, dragee, capsule, liquid, gel, syrup, suspension, wafer according to a method known in the art together with a suitable carrier. It can be prepared in formulations such as Examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, and wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Celluloses such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, glycerol etc. can be mentioned. When formulated, appropriate binders, lubricants, disintegrants, coloring agents, diluents, etc. may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, and the like, and sodium oleate as a lubricant , sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts and calcium salts thereof, polyethylene glycol, etc. as disintegrants, starch, methyl cellulose, agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt, and the like. Examples of the diluent include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine.

When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it may be formulated in the form of an injection, transdermal administration, nasal inhalation, and suppository along with a suitable carrier according to a method known in the art. When formulated as an injection, an aqueous isotonic solution or suspension may be used as a suitable carrier, and specifically, an isotonic solution such as phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, or 5% dextrose may be used. . When formulated as a transdermal formulation, it may be formulated in the form of ointments, creams, lotions, gels, external solutions, pastas, liniments, air rolls, and the like. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc., and when formulated as a suppository, the carrier is Witepsol ( witepsol), tween 61, polyethylene glycols, cacao fat, laurin fat, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, and the like can be used.

Regarding the specific formulation of the pharmaceutical composition, it is known in the art, and for example, reference may be made to Remington's Pharmaceutical Sciences (19th ed., 1995) and the like. These documents are considered as part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, sex, age, severity of the patient, and route of administration. /kg can be in the range. Administration can be done once a day or divided into several times. These dosages should not be construed as limiting the scope of the present invention in any respect.

In another specific aspect, the composition of the present invention can be identified as a cosmetic composition. When the composition of the present invention is understood as a cosmetic composition, its use can be understood as the use of suppressing inflammatory skin trouble, relieving inflammatory skin irritation, suppressing allergic skin trouble, relieving allergic skin irritation, and the like.

Even when the composition of the present invention is identified as a cosmetic composition, the cosmetic composition may be classified as an arbitrary product in terms of its use and law, and specifically, functional cosmetics and non-functional cosmetics with uses such as improving skin troubles and atopic dermatitis. It may be a general cosmetic or the like. In product form, it may take any product form, specifically, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax It may take the form of a product such as foundation or spray. In a specific product form, it may be a softening lotion, nutrient lotion, nutrient cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, or powder formulation.

The cosmetic composition of the present invention may include, in addition to the active ingredient, components commonly used in cosmetic compositions, for example, stabilizers, solubilizers, surfactants, vitamins, conventional adjuvants such as pigments and fragrances, and carriers.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. can

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane / May contain a propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, specifically water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol, fatty acid esters of sorbitan, and the like may be used.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester, microcrystals Star cellulose, aluminum metahydroxide, bentonite, agar, and the like can be used.

When the formulation of the present invention is surfactant-containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The cosmetic composition of the present invention may be prepared according to a method for preparing a cosmetic composition conventionally performed in the art, except for including an active ingredient exhibiting anti-inflammatory activity and anti-allergic activity.

As described above, according to the present invention, it is possible to provide an anti-inflammatory and anti-allergic composition using the extract of mugwort leaf.

The composition of the present invention can be commercialized as food, cosmetics, drugs, etc. for inflammatory diseases, relief of inflammatory skin irritation, allergic diseases, and relief of allergic skin irritation.

Figure 1 shows the cytotoxicity results of the broad leaf mugwort extract on HaCaT cells, a human keratinocyte cell line.
2 and 3 are results of evaluation of the anti-inflammatory activity of the broadleaf mugwort extract in human keratinocyte HaCaT cells.
Figure 4 is an evaluation result of the anti-allergic activity of the broadleaf mugwort extract in the rat basophilic leukemia mast cell line RBL-2H3.
Figure 5 is a result of cytotoxicity of the broadleaf mugwort extract on rat basophilic leukemia mast cell line RBL-2H3.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these Examples and Experimental Examples.

<Example> Preparation of broad leaf mugwort extract

To 100 g of dried broad leaf mugwort leaf powder, 10 times the weight of 70% ethanol was added, immersed for 24 hours, extracted twice, filtered, and the filtrate was concentrated under reduced pressure and freeze-dried to solid broad leaf mugwort leaf. An extract (yield 12.41%) was prepared.

<Experimental Example> Evaluation of anti-inflammatory activity and anti-allergic activity of mugwort extract

<Experimental Example 1> Evaluation of the anti-inflammatory activity of the broad leaf mugwort extract

HaCaT cells, a human keratinocyte cell line, were distributed from the Korea Cell Line Bank (KCLB, Seoul, Korea), and DMEM medium (hereafter HaCaT culture) supplemented with 10% fetal bovine serum (FBS) and 1% penicilin/streptomycin (P/C) was added. Medium) was used, and subcultured once every 2-3 days in an incubator maintained at 37 ° C. and 5% CO ₂ conditions, and used for experiments.

HaCaT cells were plated in a 96-well plate at 2×10 ⁴ cells/mL and cultured in an incubator at 37° C., 5% CO ₂ to stabilize the cells. After 24 hours, the supernatant was removed and new HaCaT cells were added. -Inflammation? IFN-γ (interferon-gamma) and TNF-α (tumor necrosis factor-alpha) were added at 10 ng/mL each as an inducing substance, and cultured for 24 hours. As a positive control, dexamethasone (20 μg/mL) was used.

The toxicity of HaCaT cells to the sample was derived by adding a 0.5 mg/mL MTT solution to the attached cells from which the culture supernatant was removed, reacting for 30 minutes, and measuring the color developed at a wavelength of 550 nm. It was expressed as cell viability (%) for .

On the other hand, only the culture supernatant was recovered, and the contents of IL-6 and IL-8, which were inflammatory cytokines released into the supernatant, were calculated through a standard curve prepared using a recombinant human protein provided by the manufacturer (pg/mL). was calculated as

Statistical analysis of the experimental results was calculated by Student's t-test using the SPSS statistical program (Statistical Package for the Social Science, Ver. 12.0, SPSS Inc., Chicago, IL, USA), p<0.05, p<0.01 and p<0.01, respectively. Significance was tested at the p<0.001 level.

Results are shown in Figures 1 to 3. Referring to Figure 1, the broad leaf mugwort leaf extract did not show cytotoxicity at all treatment concentrations of 20 to 50 μg / ml. In addition, referring to Figures 2 and 3, the broad leaf mugwort leaf extract significantly inhibited the production of inflammatory cytokines IL-6 and IL-8 at all treatment concentrations.

<Experimental Example 2> Evaluation of anti-allergic activity of leaf extract of mugwort broadleaf

The anti-allergic activity of the leaf extract of artemisia broadleaf was evaluated by the degranulation inhibitory activity of mast cell lines using β-hexosaminidase assay.

RBL-2H3 cells, a rat basophilic leukemia mast cell line for evaluation of anti-allergic activity, were purchased from Korea Cell Line Bank (KCLB, Seoul, Korea) and used in the experiments. For the cells, DMEM medium (hereafter referred to as RBL-culture medium) supplemented with 10% FBS and 1% P/S was used, and subcultured once every 2 to 3 days in an incubator maintained at 37°C and 5% CO2 conditions. used

After adjusting the BL-2H3 cells to 5×10 ⁵ cells/mL, 450 μL of each was applied to a 24-well plate, and 50 μL of DNP-IgE (dinitrophenyl-immunoglobulin E) at a concentration of 5 μg/mL was added as an allergen. Incubated for 24 hours.

Cells were cultured in Siraganian buffer (119 mM NaCl, 5 mM KCl, 5.6 mM glucose, 0.4 mM MgCl ₂ , 25 mM PIPES, 40 mM NaOH, 1 mM CaCl ₂ , 0.1% BSA; pH 7.2). After washing, 400 μL of Syraganian buffer and 50 μL of the sample diluted to an appropriate concentration were added and re-incubated for 1 hour. Then, 50 μL of 10 μg/mL DNP-BSA (anti-dinitrophenyl antibody) was added, followed by 1 hour. After reacting, the reaction was stopped by leaving it on an ice bath for 10 minutes.

Transfer 50 μL of the reaction supernatant to a 96-well plate, add 50 μL of substrate buffer (2 mM 4-p-nitrophenyl-N-acetyl-β-D-glucosaminide/0.0 5M citrate buffer; pH 4.5), and react for 2 hours. After that, 50 μL of 0.1M sodium carbonate buffer (pH 10.0) was added to terminate the reaction, and absorbance was measured at 405 nm using a microplate reader to detect β-hexosaminidase (β). -hexosaminidase) secretion inhibitory activity is shown in Figure 4 as a percentage (%) compared to the sample untreated group. As a positive control, dexamethasone (20 μg/mL) was used.

As mentioned above, the cytotoxicity of the sample was measured by the MTT method and is shown in FIG. 5 as cell viability (%) relative to the untreated sample.

Statistical analysis of the experimental results was tested for significance in the same way as above.

Referring to Figures 4 and 5, the broadleaf mugwort leaf extract inhibited the secretion of β-hexosaminidase in a concentration-dependent manner without showing cytotoxicity.

Claims (12)

As an anti-inflammatory pharmaceutical composition comprising a water and ethanol mixture solvent extract of leaves of artemisia broadleaf as an active ingredient,
The anti-inflammatory is a symptom improvement, prevention, or treatment of an inflammatory disease,
The inflammatory disease is a composition, characterized in that atopic dermatitis or skin eczema.
An anti-inflammatory food composition comprising extract of water and ethanol mixed solvent of leaves of artemisia broadleaf as an active ingredient,
The anti-inflammatory is a symptom improvement, prevention, or treatment of an inflammatory disease,
The inflammatory disease is a composition, characterized in that atopic dermatitis or skin eczema.
An anti-inflammatory cosmetic composition comprising a water and ethanol mixture solvent extract of leaves of artemisia broadleaf as an active ingredient,
The anti-inflammatory is a symptom improvement, prevention, or treatment of an inflammatory disease,
The inflammatory disease is a composition, characterized in that atopic dermatitis or skin eczema.
delete delete delete An anti-allergic composition comprising a mixture of water and ethanol extract from the leaves of artemisia broadleaf as an active ingredient.
delete delete According to claim 7,
The composition, characterized in that the composition is a pharmaceutical composition.
According to claim 7,
The composition is an anti-allergic composition, characterized in that the food composition.
According to claim 7,
The composition is an anti-allergic composition, characterized in that the cosmetic composition.

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