KR101816739B1 - Composition for preventing or treating inflammatory skin disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition, a cosmetic composition and a health functional food composition for preventing, treating or ameliorating an inflammatory skin diseases, which comprises an Ecklonia cava extract and a Sargassum Horneri extract. The composition blended in a specific ratio of the Ecklonia cava extract and Sargassum Horneri extract according to the present invention inhibits generation of inflammatory cytokines IL-1β, IL-4, and IFN-γ and also inhibits expression of inflammatory chemokines MDC and TARC, thereby having an anti-inflammatory effect regarding various inflammatory skin diseases. The Ecklonia cava extract and the Sargassum Horneri extract of the present invention are expected to be safely used in cosmetic, pharmaceutical and food compositions without cytotoxicity and skin side effects.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating inflammatory skin diseases,

The present invention relates to a composition for preventing or treating an inflammatory skin disease, which comprises a natural extract. More particularly, the present invention relates to a pharmaceutical composition for preventing, treating or ameliorating an inflammatory skin disease, And a health functional food composition.

Skin is the most extraterrestal organs of the human body. It protects the body's moisture and blocks invading factors from the outside, thus acting as an essential barrier to the human body. Factors that cause skin irritation include physicochemical stimuli, allergens, ultraviolet rays, oxidative stress, pathogen infections and secondary inflammation-related cytokines and chemokines produced thereby.

The common pathway of skin inflammation is the immune response to protect the body from external stimuli, and the control of inflammation through inhibition of inflammation has become a major target of therapy. In the lesion of skin inflammation, the initiation and maintenance of the inflammatory response involves cytokines or chemokines that are involved in the infiltration of various inflammatory cells such as T cells or eosinophils that express skin lymphocyte-associated antigens, Cells, keratinocytes, and fibroblasts.

Chemokines are a family of cytokines, now known as more than 50 species, which play a role in attracting appropriate immune cells to the site during the inflammatory response of the tissue. When the skin is stimulated by allergens or stimulants, the activated Langerhans cells migrate to the dermis, and T cells are activated by transferring the antigen to naive T cells that are not stimulated with the antigen. Memory T cells that have been exposed to an antigen once circulate along the blood vessels and migrate to the lesion area for quicker activation, leading to an effective immune response. T-cell mediated inflammatory diseases such as allergic contact dermatitis, psoriasis, and atopic dermatitis are representative skin diseases described by this mechanism.

Activated helper T cells, on the other hand, stimulate B cells and induce overproduction of immunoglobulin E. Immunoglobulin E causes an inflammatory reaction such as irritation and erythema itch and stimulates mast cells to activate another inflammatory pathway, COX (cyclooxygenase) signal. COX-1 is intrinsically constant in tissues, whereas COX-2 is induced by inflammatory responses and is rapidly expressed in a short period of time by various stimuli. Increased expression of COX-2, a typical inflammatory factor, is known to affect the production of nitric oxide.

Currently, steroid preparations, topical immunosuppressants, topical antibiotics, and antihistamines are currently used as therapeutic agents for inflammatory skin diseases. However, when the drug preparation is used for a long period of time, Side effects such as palpitation may occur. In particular, when steroids are applied to a wide area, systemic absorption of medicines through the skin may interfere with growth of children, and general side effects such as osteoporosis may occur in adults.

Therefore, it is necessary to develop a composition derived from a natural substance, which has few side effects and is safe for the human body and is excellent in an inflammatory skin disease-improving effect.

Accordingly, the present inventors have conducted intensive studies in order to find a substance having an inflammatory skin disease-improving effect and a low human side effect as described above. As a result, the present inventors have found that the combined extracts of Ganoderma lucidum and Rhododendron japonica have remarkably improved inflammatory skin disease The present invention has been completed.

Korean Patent Publication No. 10-2015-0139466 (Nov.

The present invention aims to provide a composition for preventing, treating or ameliorating an inflammatory skin disease, which comprises a gangrene extract and a hoe saengmabak extract.

1. A pharmaceutical composition for preventing or treating inflammatory skin diseases, which comprises a ghatti extract and a hornblende extract.

2. The pharmaceutical composition for preventing or treating inflammatory skin disease according to 1 above, wherein the ghatti extract and the horns extract are mixed at a weight ratio of 1: 0.5 to 1.5.

3. The method according to 1 or 2 above, wherein the inflammatory skin disease is at least one selected from the group consisting of atopic dermatitis, allergic dermatitis, psoriasis, seborrheic dermatitis, contact dermatitis, A pharmaceutical composition for preventing or treating skin diseases.

4. A cosmetic composition for the treatment of inflammatory skin diseases, which comprises a ghatti extract and a hornblende extract.

5. The cosmetic composition for the treatment of inflammatory skin diseases according to 4 above, wherein the ghatti extract and the hornblende extract are mixed at a weight ratio of 1: 0.5 to 1.5.

6. The method according to claim 4 or 5, wherein the inflammatory skin disease is at least one selected from the group consisting of atopic dermatitis, allergic dermatitis, psoriasis, seborrheic dermatitis, contact dermatitis, A cosmetic composition for improving skin diseases.

7. A health functional food composition for the treatment of inflammatory skin diseases, comprising a ghatti extract and a hornblende extract.

8. The health functional food composition for the treatment of inflammatory skin diseases according to 7 above, wherein the ghatti extract and the horn ginseng extract are mixed at a weight ratio of 1: 0.5 to 1.5.

9. The method according to 7 or 8 above, wherein said inflammatory skin disease is at least one selected from the group consisting of atopic dermatitis, allergic dermatitis, psoriasis, seborrheic dermatitis, contact dermatitis, A health functional food composition for improving skin diseases.

The present invention relates to a pharmaceutical composition, a cosmetic composition and a health functional food composition for preventing, treating or ameliorating an inflammatory skin disease, which comprises a ghatti extract and a horns extract, The composition blended in a specific ratio inhibits the production of inflammatory cytokines IL-1β, IL-4, and IFN-γ and also inhibits the expression of inflammatory chemokines MDC and TARC, . In addition, the phytotoxic extract of the present invention and the extract of Horns Cygnus sinensis extract are expected to be safely used in cosmetic, pharmaceutical and food compositions without cytotoxicity and skin side effects.

Figure 1 is an assessment of the effect of the combined extract (MES) of the present invention on cell viability and toxicity.
Fig. 2 shows the inhibitory effect of ECE, SHE, and the combined extract (MES) of the present invention on inflammatory cytokine and chemokine expression.
FIG. 3 is a graph showing the inhibitory effect of the compound extract of the present invention (5: 5 ratio) on the expression of TSLP, an initial stimulating factor for atopic dermatitis.
FIG. 4 is a graph showing inhibitory effects on the expression of inflammatory cytokines of the combined extract of the present invention (5: 5 ratio).
FIG. 5 shows the inhibitory effect on the expression of inflammatory chemokines of the combined extract of the present invention (5: 5 ratio).
FIG. 6 is a graph showing the inhibitory effect of the compound extract of the present invention (5: 5 ratio) on MAPK, a dermatitis-inducing signal transduction pathway.

The inventors of the present invention confirmed the cytotoxicity of the extracts of Ganoderma lucidum and Horns Cyprinus mellifera, and confirmed the inhibitory effect on the expression of inflammatory cytokines and chemokines of the extracts in the examples, and completed the present invention on the basis thereof.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for preventing, treating or ameliorating an inflammatory skin disease, which comprises a ghatti extract and a hoe saengmabak extract.

The composition of the present invention inhibits TSLP, an inflammatory cytokine, IL-1β, IL-4, and IFN-γ, and inhibits the expression of MDC and TARC, which are inflammatory chemokines, May be used for the prevention, treatment or amelioration of various inflammatory diseases.

In the present invention, inflammatory skin diseases are induced by a signal transduction system of the immune system, and specifically, there are atopic dermatitis, allergic dermatitis, psoriasis, seborrheic dermatitis, contact dermatitis, irritable lupus and globular urticaria , But is not limited thereto.

Ecklonia cava ) is a perennial brown algae belonging to kelp. It is widely used on the coast of Cheju Island, South Korea, and is used for edible purposes with sea tangle ( Laminaria japonica ) and undaria pinnatifida . In the conventional studies, there have been reported many useful physiological activities such as antioxidant, anti-cancer, anticoagulant, and MMP inhibitory activity.

Sargassum Horneri ) is a brown algae belonging to the mating family, and lives in the coastal waters of the south of Korea and Japan. It is mainly used as feed. In the conventional studies, many useful physiological activities such as prevention of osteoporosis, prevention of blood clotting, herpes virus inhibitory effect, and the like have been reported in the hoe saengmyungbae.

The present invention has the advantage of being able to use safely even when taken for a long time without toxicity and side effects because it is a natural substance which can be used as a food grade and is very stable in the body.

The composition of the present invention includes the extracts obtained by extracting the natural materials such as Ganoderma lucidum and Rhododendron japonica, and the plant outbreaks, leaves, roots, stems, flowers and the like can be extracted by a conventional method. In addition, the extract may contain not only the extract extracted from the natural substance by a conventional method but also its dry powder or all the forms formulated using the same, and it is preferable that all the extracts, fractions , And purified water, their diluted solutions, concentrates or dried products.

The extract of the present invention can be extracted using water or an organic solvent, and the extracted liquid can be used in a liquid form or can be used by concentration and / or drying. The organic solvent may be selected from the group consisting of methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethylsulfoxide , 3-butylene glycol, propylene glycol, or a mixed solvent thereof. The extract may be extracted at room temperature or with heating under conditions where the active ingredient of the extract is not destroyed or minimized. Depending on the organic solvent to be extracted, the extraction degree and the degree of loss of the active ingredient may differ. Therefore, an appropriate organic solvent may be selected depending on the plant based on the basic knowledge in the field. The extraction method is not particularly limited, and for example, a cold extraction, an ultrasonic extraction, a reflux cooling extraction, or the like can be used.

Filtration is carried out according to a usual process. Filtration is a process of removing suspended solid particles from an extract. The filtration can be performed by filtering out particles using cotton, nylon, etc., or using ultrafiltration, freezing filtration, centrifugation, etc. But is not limited thereto.

In addition, it may further comprise a separation process by various chromatographies (size, charge, hydrophobicity or affinity chromatographic). In the step of drying the filtrate, methods such as freeze drying, vacuum drying, hot air drying, spray drying, vacuum drying, foam drying, high frequency drying and infrared drying can be used, but there is no particular limitation. In some cases, a step of pulverizing the final dried extract may be added.

The extracts of Ganoderma lucidum and P. vaginalis can be prepared by preparing a gentian extract and a ginseng extract, respectively, and mixing them at a predetermined ratio, or by preliminarily mixing gentian and horn ganoderma with a predetermined ratio and then extracting together.

The phytotoxic extracts and the hornblende extracts may be significantly improved in the anti-inflammatory effect when they are mixed at a specific weight ratio. Considering the unit price of seaweeds, when the phytotoxic extract of the present invention and the extract of Hornsby matsumabii are used together, they are more economical than those used alone.

The chewing gum extract and the horns lanceolate extract may be mixed in a weight ratio of 1: 0.1 to 4.5, preferably 1: 0.5 to 1.5, more preferably 1: 0.8 to 1.2, It is not.

The total weight of the gangrene extract and horn ginseng extract contained in the composition of the present invention is 0.001 to 20 wt%, preferably 0.01 to 10 wt%, based on the total weight of the composition. When the content of the extract is less than 0.001% by weight, it is difficult to obtain the anti-inflammatory effect. When the content of the extract is more than 20% by weight, the anti-inflammatory effect is not proportional to the content.

The composition of the present invention may be a pharmaceutical composition, a cosmetic composition or a food composition.

When the composition of the present invention is used as a pharmaceutical composition, it may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants other than the above-mentioned active ingredients, or may be administered to mammals. The adjuvant may be an excipient, a disintegrant, a sweetener, a binder, a coating agent, a swelling agent, a lubricant, a lubricant or a flavoring agent.

In addition, the pharmaceutical composition of the present invention may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the pharmaceutically effective amount of the above-described effective ingredients for administration.

A "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level will depend on the type of disease, severity, activity of the drug, The time of administration, the route of administration and the rate of excretion, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, body weight, the degree of absorption of the active ingredient in the body, the rate of inactivation and excretion, the type of disease, 0.001 to 150 mg, preferably 0.01 to 100 mg, per 1 kg of body weight may be administered daily or every other day, or one to three divided doses per day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

The term "pharmaceutically acceptable" as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to a human. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

The compositions of the present invention may also be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to a subject in need of treatment of an inflammatory skin disease, . Formulations may be powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

The pharmaceutical composition according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous or muscular, and the dose of the active ingredient may be appropriately determined depending on the administration route, age, sex, And can be appropriately selected according to various factors. In addition, it can be administered in combination with known compounds having an effect of preventing or ameliorating symptoms of inflammatory skin diseases.

In addition, when the composition of the present invention is used as a cosmetic composition, conventional auxiliary agents such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, May be further added.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, the present invention relates to a cream, essence, cosmetic lotion, spray, gel, pack, sunscreen, make-up such as lotion such as lotion, facial lotion and body lotion such as soft lotion or nutrition lotion, nutrition cream, Such as a foundation, a liquid, a solid or spray type, a powder, a cleansing cream, a cleansing lotion, a makeup remover such as a cleansing oil, a cleansing foam, a soap, a body wash and the like. In this case, the cosmetic composition may contain a surfactant, an emulsifier, a soap acid, a solvent, a coloring agent, a preservative, an antioxidant, a defoamer, an antibacterial agent, an anticorrosion agent, an enzyme, a plant or mineral oil, a fat, a fluorescent substance, a fungicide, Preservatives, proteins, silicones, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, waxes, and the like.

When the formulation of the present invention is a paste, a cream or a gel, an animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide .

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

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

In the case where 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 tragacanth.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include 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, lanolin derivatives or ethoxylated glycerol fatty acid esters.

In addition, when the composition of the present invention is used as a food composition, it can be easily utilized in the production of foodstuffs, additives, food additives, functional foods or beverages which are effective for prevention and improvement of inflammatory skin diseases .

The term "food" as used herein means a natural product or a processed product containing one or more nutrients, preferably a state of being able to be directly eaten through a certain degree of processing, , Food additives, functional foods and beverages.

Foods to which the composition according to the present invention can be added include, for example, various foods, beverages, gums, tea, vitamin complexes, and functional foods. In addition, the above foods may include special nutritional foods (eg, crude oil, spirit, baby food, etc.), meat products, fish products, tofu, jelly, noodles (eg, ramen, noodles, etc.) (Such as soy sauce, soybean paste, hot pepper paste, mixed sauce, etc.), sauces, confectionery (eg snacks), candy, chocolate, gums, ice cream, milk products (E.g., various kinds of kimchi, pickles, etc.), beverages (e.g. fruit drinks, vegetable beverages, beverages, fermented beverages, etc.) The additive can be produced by a conventional production method.

The above-mentioned "functional food" refers to a food group which is imparted with added value to function and express the function of the food by physical, biochemical or biotechnological techniques, or to control the bio-defense rhythm of the food composition, Refers to a food prepared by processing a body so as to sufficiently express the body's control function with respect to the body, and preferably, it may be a health functional food. The functional food may include a food-acceptable food-aid additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of functional foods.

In the present invention, the term "beverage " means a general term for drinking or enjoying a taste, and includes a functional beverage. The beverage includes, as an essential ingredient, a composition comprising a topical extract for preventing or ameliorating inflammatory symptoms, and there is no particular limitation on other ingredients, and various flavors or natural carbohydrates such as ordinary beverages are added as an additional ingredient can do.

In addition to the above, the food containing the composition of the present invention may be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and fillers (cheese, chocolate, etc.) An organic acid, a protective colloid thickener, a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, a carbonating agent used in a carbonated beverage, and the like, Can be used in combination.

In the food containing the food composition according to the present invention, the amount of the composition comprising the topical extract as an active ingredient may be 0.001% by weight to 90% by weight, preferably 0.1% by weight or less, And may be contained in an amount of 0.001 g to 2 g, preferably 0.01 g to 0.1 g, based on 100 ml of the beverage. In the case of long-term intake, the content may be less than the above range, Since the active ingredient has no problem in terms of safety, it can be used in an amount of more than the above range, so it is not limited to the above range.

Therefore, the present invention can provide a health functional food composition for improving inflammatory skin diseases, which comprises a complex extract of Ganoderma lucidum and Rhododendron japonica as an active ingredient, wherein the form of the food is not limited thereto, but may be a powder, a granule, Capsule or beverage form.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

Example

In this example, the cytotoxicity of the extracts of Ganoderma lucidum and Huaxiai ganoderma lucidum were examined, and the extracts of the present invention were tested for their inhibitory effects on the expression of inflammatory cytokines and chemokines.

Materials and Experiments

One. Moth  And Horns  Ethanol extract preparation

The dried samples of Ganoderma lucidum and P. vaginalis were extracted by shaking for 24 hours in 70% ethanol (substrate: solvent = 1: 10-100) and repeated three times to obtain a crude, The extract was obtained.

The extracts of ethanol extracts of horseradish peroxidase and horseradish peroxidase were 60 ± 20% and 38 ± 20%, respectively, and the extracts were obtained. (ECE) and hoe saxiflora (SHE) extracts at a ratio of 0:10, 2: 8, 4: 6, 5: 5, 6: 4, 8: 2 and 10: 0, respectively, (MES) were prepared.

2. Cytotoxicity assays ( MTT  analysis)

HaCaT cells were cultured in DMEM medium containing 10% FBS and 1% antibiotics in order to measure the effect of the skin keratinocyte survival morphology, hornfish moth, Suspended in a 5% CO ₂ incubator at 37 ° C. The cultured cells were equally divided into 1 × 10 ⁴ cells per well in a 96-well plate, and cultured for 24 hours. After the cells were attached, the cells were treated with an ethanol extract (ECE), a herbal extract (SHE) (MES) were each treated for 24 hours, and then 15 μl of MTT (5 mg / ml) was added to each well. After addition, the cells were incubated for another 4 hours. The supernatant was removed and 100 μl of DMSO was added to dissolve the formed formazan precipitate. The absorbance was measured at 540 nm using a microplate reader.

3. Cytotoxicity assays ( LDH  analysis)

HaCaT cells were suspended in DMEM medium containing 10% FBS and 1% antibiotics in order to measure the toxicity of HaCaT cells as a keratinocyte, toxicity of Haemophilus influenzae, And cultured in a 5% CO ₂ incubator at 37 ° C. The cultured cells were seeded on a 96-well plate at the same rate of 1 × 10 ⁴ cells per well and cultured for 24 hours. After the cells were attached, the cell extracts were extracted with ethanol (ECE), ethanol extract of hornblende (SHE) And 50 μl of the cell culture supernatant and 50 μl of the LDH cytotoxicity detection kit were incubated for 30 min. After stopping the reaction with 50 μl of Stop solution, Absorbance was measured at 490 nm.

4. RT- PCR  (reverse transcription-polymerase chain reaction) analysis

Evaluation of expression of inflammatory factor was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). More specifically, HaCaT cells were first seeded in 6-wells and then treated with ECE, SHE, and MES at a concentration of 62.5 μg / ml or 125 μg / ml for 2 hours. TNF-α and IFN- 10 ng / ml each. After 2 hours, the supernatant was removed, triazol reagent was added to the cells, chloroform was added, vortexing was performed for 10 seconds, centrifugation was carried out at 12,000 rpm for 15 minutes, the supernatant was taken, and isopropanol was mixed and shaken. After centrifugation at 12000 rpm for 15 minutes, the supernatant was removed and pellets were mixed and used for RT-PCR. CDNA was synthesized using a cDNA synthesis kit, and denaturation was performed at 45 ° C for 30 minutes, at 94 ° C for 30 seconds, and annealed at 55 ° C for 30 seconds using an RT-PCR kit The next 30 sec extraction cycle at 72 < 0 > C was repeated 32 times and the last extension was performed at 72 < 0 > C for 5 min. Each PCR product was loaded on 1.5% agarose gel and analyzed by electrophoresis at 100 V for 30 minutes.

5. Western Blot  (western blot) analysis

Expression of proteins involved in inflammation was confirmed by western blotting. More specifically, HaCaT cells were cultured on a 6-cm culture dish and then treated with MES (5: 5) for 2 hours. TNF-α and IFN-γ were treated with 10 ng / ml. After 30 min, the supernatant was removed, and lysis buffer was added to the cells. Vortexing was performed 6 times for 10 min each. Centrifugation was carried out at 4 ° C and 14000 rpm for 10 min. Using the BCA kit To quantify the protein. Proteins of the same concentration were mixed with a sample buffer and boiled at 100 ° C for 3 minutes to induce protein denaturation. Proteins were separated by 10% SDS-PAGE and transferred to PVDF membrane. Nonspecific protein binding sites were blocked by reaction in Tris-buffered saline (TBS) containing 0.1% Tween 20 and blocking buffer for 2 hours. Subsequently, the solutions containing the primary antibodies p38 and phospho-p38 were mixed for 12 hours and washed three times with TBST for 10 minutes. Then, the membrane was reacted with a solution containing goat anti-rabbit IgG, which is a secondary antibody, at room temperature for 1 hour and 30 minutes, and then washed with TBST for 10 minutes for 3 times. The ECL- The expression of the target protein was measured.

6. Statistical Analysis

Statistical significance was assessed using the PASW Statistics 19.0 software (SPSS, Chicago, IL, USA). The significance of the mean values among the experimental groups was compared using the Duncan's test at P <0.05.

result

One. ECE , SHE and its combination extract ( MES ) Toxicity assessment

MTT assay was performed to assess the effect of the combined extract (MES) of the present invention on cell viability.

As a result, when the complex extract (MES) prepared at various ratios was treated at a concentration of 62.5 and 125 μg / ml, as shown in FIG. 1A, treatment was carried out at a ratio of 4: 6, 5: 5, and 6: 4, respectively MES showed cell survival similar to that of the untreated control group or the group treated with TNF-α and IFN-γ and the ratio of the remaining 0:10, 2: 8, 8: 2, and 10: 0 The MES treated with MES was slightly toxic compared to the above-mentioned groups.

Next, the effect of the combined extract (MES) of the present invention on the amount of LDH produced was evaluated. The amount of lactate dehydrogenase (LDH) produced from the cells passes through the cell membrane and catalyzes the dehydrogenation of lactic acid to produce pyruvate and NADH. NADH is converted to tetrazolium salt (INT) by diaphorase To form a red formazan pigment, which is then measured for cytotoxicity.

As a result, as shown in Fig. 1B, in the case of MES treated at the ratios of 4: 6, 5: 5 and 10: 0, respectively, the control group or TNF-α and IFN- Showed similar LDH production and the rest 0:10, 2: 8. MES treated at the ratio of 6: 4 and 8: 2 showed slightly increased LDH production.

As a result of synthesis of the above contents, when the HaCaT cells were induced to induce inflammation with TNF-α and IFN-γ, MES treated with the complex extract of the present invention, particularly 4: 6 and 5: 5, And it was confirmed that it does not affect the toxicity.

2. Inflammatory cytokines and Chemokine Complex extracts for expression  ( MES )

HaCaT cells are essential for the formation of the stratum corneum, and act as a primary derivative and target of the immune response that occurs in the skin. In other words, keratinocytes secrete various cytokines such as IL-4, TNF-α, and IFN-γ by several stimuli and have receptors of TGF-β and keratinocyte growth factor (KGF) It is also affected. TNF-α, an inflammatory cytokine, acts on the production of various cytokines, cell growth and differentiation, and necrosis, and affects dermatitis. TGF-β is involved in cell proliferation and differentiation, immune response, wound healing . In addition, there are high concentrations of TSLP and MDC in keratinocytes in dermatitis patients, and TSLP stimulates CD11c + dendritic cells to increase TARC and MDC production. This concentration of chemokine is closely related to the symptoms of dermatitis.

Based on the above, the effect of the combined extract (MES) of the present invention on the inhibition of inflammatory cytokine production in HaCaT cells was confirmed by RT-PCR.

As a result, as shown in Fig. 2, the expression of the inflammatory cytokines IL-1β, IL-4, and IFN-γ was markedly increased in the inflammatory stimulus group treated with TNF-α and IFN- The expression of IL-1β, IL-4, and IFN-γ, which are inflammatory cytokines, decreased in the MES-treated experimental group compared to the control group. In particular, MES, in which ECE and SHE were mixed at a ratio of 5: 5, showed a marked decrease in IL-1β, IL-4, and IFN-γ expression compared to the other sample-treated groups.

In addition, the inhibitory effect of the combined extract (MES) of the present invention on inflammatory chemokine production in HaCaT cells was confirmed by RT-PCR.

As a result, as shown in FIG. 2, the expression of MDC and TARC in the experimental group treated with TNF-α and IFN-γ was significantly increased compared to the control group, whereas in the experimental group treated with MES mixed at various ratios MDC and TARC expression was decreased. Interestingly, MES mixed with ECE and SHE at a ratio of 5: 5 showed MDC expression similar to that of MES at 0:10 or 10: 0, but showed significantly higher inhibitory potency than the other groups and expression of TARC The highest inhibitory effect was observed. Subsequent experiments were performed on MES in which ECE and SHE were mixed at a ratio of 5: 5.

3. TSLP  For expression MES  (5: 5) of  Confirmation of inhibition

RT-PCR was performed in order to confirm the inhibitory effect of the complex extract of the present invention (especially, 5: 5 ratio) on the expression of TSLP, a dermatitis-inducing cytokine known as an initial stimulating factor, when atopic dermatitis was induced .

As a result, as shown in Fig. 3, in the experimental group treated with TNF-α and IFN-γ, the expression of TSLP was markedly increased as compared with the control group. On the other hand, in the case of ECE and SHE mixed at a ratio of 5: 5, Showed that TSLP significantly suppressed mRNA expression in a concentration-dependent manner.

4. Inflammatory cytokines and Chemokine  For expression MES  (5: 5) of  Confirmation of inhibition

RT-PCR was performed in order to confirm the inhibitory effect of the complex extract of the present invention (especially, 5: 5 ratio) on mRNA expression of inflammatory cytokines and chemokines which are increased when atopic dermatitis is induced.

As a result, as shown in Fig. 4, inflammatory cytokines such as IL-1β, IL-4, IL-6, IFN-γ and TNF-α MES, which is a mixture of ECE and SHE at a ratio of 5: 5, was significantly higher than that of cells stimulated with TNF-α and IFN-γ. IL-1β, IL-4 and IL-6 , IFN-y, and TNF-alpha in a dose-dependent manner. Furthermore, as shown in Fig. 5, gene expression of MDC, RANTES and TARC significantly increased by the stimulation of TNF-α and IFN-γ was inhibited by treatment of MES in which ECE and SHE were mixed at a ratio of 5: 5 .

In general, about 80% of patients with atopic dermatitis among dermatitis have an increase in serum IgE. Since IL-4, a cytokine produced by lymphocytes, is known to induce the production of IgE, inhibition of the expression of these cytokines may lead to a decrease in dermatitis symptoms. Furthermore, by inhibiting the expression of TSLP, inflammatory cytokine, and chemokine, which are early factors of atopic dermatitis, it is possible to exhibit the preventive, ameliorative or therapeutic effect of dermatitis.

Therefore, it is anticipated that the extract of the present invention and the extract of Horns monocotyledonous complex (especially, 5: 5 ratio) inhibit the expression of the inflammatory cytokine and the chemokine, thereby exhibiting the effect of preventing, improving or treating dermatitis.

5. For dermatitis-induced associative signaling pathways MES  (5: 5) of  Confirmation of inhibition

The MAPKs signaling pathway plays an important regulatory role in both innate and acquired immunity, and responds extensively to changes in the cell interior and exterior, interacting with other signaling pathways and contributing to intracellular gene expression and cellular function regulation. In particular, there are three types of MAPK pathways in mammals: ERK, JNK, and p38. Thus, the inhibitory effect of the complex extract of the present invention (particularly, 5: 5 ratio) on the expression of MAPKs protein, which is a basic factor expressed in the dermatitis expression process, was confirmed.

As a result, as shown in Fig. 6, the expression of p-p38 was increased by phosphorylation of p38 in the group treated with TNF-α and IFN-γ, but the MES in which ECE and SHE were mixed at a ratio of 5: 5 In one experimental group, the expression of p-p38 was significantly reduced in a concentration-dependent manner compared to the untreated control.

Claims (9)

A method for preventing or treating atopic dermatitis, comprising the steps of: (a) providing an aqueous extract of hornblende ethanol and a hornblende ethanol extract in a weight ratio of 1: 0.8 to 1.2;
delete delete Wherein the composition comprises at least one of ethanol extracts of horseradish peroxidase and horseradish peroxidase at a weight ratio of 1: 0.8 to 1.2.
delete delete Wherein the composition comprises at least one selected from the group consisting of ethanol extracts and ethanol extracts of hornblende at a weight ratio of 1: 0.8 to 1.2. delete delete

Images