KR102197684B1 - Composition Comprising the 3,5-Dicaffeoylquinic Acid for strengthening skin barrier and preventing skin barrier damage - Google Patents

Abstract

The present invention relates to a composition for strengthening the skin barrier and preventing damage to the skin barrier, comprising 3,5-dicaffeoylquinic acid isolated from the extract of Aster glehni in a cosmetic composition and drug By introducing it as an active ingredient of the pharmaceutical composition, it is effective in preventing and improving atopic dermatitis by improving skin barrier damage and alleviating skin inflammation.

Description

Composition Comprising the 3,5-Dicaffeoylquinic Acid for strengthening skin barrier and preventing skin barrier damage, containing 3,5-dicafeoylquinic acid as an active ingredient

The present invention relates to a composition for strengthening the skin barrier and preventing damage to the skin barrier, and more particularly, to strengthen the skin barrier and damage the skin barrier containing 3,5-dicaffeoylquinic acid as an active ingredient. It relates to a preventive composition.

The skin, also known as the outer skin, consists of three main layers: the epidermis, the dermis and the subcutaneous tissue (subcutaneous). In particular, the epidermis is the outer area of the skin, and its structure is composed of four layers, and can be divided into the basal layer, the spinosum layer, the stratum granulosum and the stratum corneum (the outermost thin layer of the skin). Keratinocytes are the main cells that make up the epidermis, and through keratinization, they contribute to the construction of the body's defenses. In the spinosum layer, keratinocytes produce keratin1 and 10, and keratin10 is the main component of desmosome. In general, the skin barrier refers to the stratum corneum, and is composed of keratinocytes, stratum corneum, corneal epithelial cells, and lipids between keratinocytes. The stratum corneum is composed of transglutaminase, involucrin, loricrin, and filaggrin, which acts as an intact skin barrier by forming a multilayered structure of intercorneocyte lipids. Lipids between keratinocytes are ceramides, cholesterol, free fatty acids, and cholesterol. It contains sulphate and has the highest ceramide content. The main function of the skin barrier is to prevent loss of body fluids, attack of toxins, and invasion of pathogens (Department of Dermatology, Faculty of Medicine and Graduate School of Medicine Hokkaido University. Shimizu's Textbook of Dermatology. http://www.derm-hokudai .jp/shimizu-dermatology/ch01 (13.02.2017); HH Jang, SN Lee, Asian J Beauty Cosmetol, 14, 339, 2016). Thus, damage to the skin barrier can lead to serious immune reactions through the penetration of various pathogens, as well as simple mechanical destruction of the outer layer of the skin. Sodium dodecyl sulfate (SDS), an anionic surfactant, generally irritates the skin, and SDS, as a detergent, induces the polarity of the skin surface, the breakdown of the skin barrier, and the extraction of epidermal lipids. In addition, SDS increases percutaneous water loss from the stratum corneum and induces inflammation.

Atopic dermatitis (AD) occurs in early infancy but also occurs in adults. In general, AD is a chronic pruritic inflammatory skin disease characterized by increased immunoglobulin E (IgE) production, recurrence of eczema skin lesions, infiltration of inflammatory immune cells, and defects in the skin barrier. The underlying cause is hardly known, and the development of industrialization The incidence of AD is increasing. Therefore, finding an effective treatment for AD is very urgent and socioeconomic important. 2,4-Dinitrochlorobenzene (DNCB) has been known as a representative substance causing contact dermatitis and AD (Medscape. Atopic Dermatitis. http://emedicine.medscape.com/article) /1049085-overview#a4(15.02.2017); T. Bieber, N Engl J Med ., 358, 1483, 2008).

Aster glehni (AG) has been used in traditional Korean medicine to treat fever, pain, phlegm and cough. Other effects of AG have previously been reported. AG's ethyl acetate extract inhibited the protein expression of tyrosinase and tyrosinase-related protein 1, which are involved in melanocyte biosynthesis in melanocytes, and in another study, the ethyl acetate extract of AG was a nitric oxide synthase for inducing antioxidant effects and inflammation. The effect of inhibiting the protein expression of (iNOS) has been reported (Y. Fujii et al. , Skin Pharmacol Physiol ., 22 240, 2009).

On the other hand, it is not known that 3,5-dicaffeoylquinic acid isolated from the extract of Aster glehni has the effect of improving skin barrier damage or reducing skin inflammation.

Accordingly, the present inventors have made diligent efforts to find a composition having an effect of improving skin barrier damage and/or alleviating skin inflammation in order to prevent and ameliorate diseases such as atopy, and as a result, 3,5-D isolated from Aster glehni extract It was found that caffeoylquinic acid (3,5-dicaffeoylquinic acid) has excellent skin barrier damage improvement and/or skin inflammation alleviation effect, and the present invention was completed.

An object of the present invention is to provide a pharmaceutical composition for strengthening the skin barrier and preventing damage to the skin barrier, containing 3,5-dicaffeoylquinic acid as an active ingredient.

* Another object of the present invention is to contain 3,5-dicaffeoylquinic acid (3,5-dicaffeoylquinic acid) as an active ingredient, and liquid, ointment, cream, lotion, spray, patch, gel, or aerosol It is to provide a composition for external use in the first formulation.

In order to achieve the above object, the present invention provides a pharmaceutical composition for strengthening the skin barrier and preventing damage to the skin barrier containing 3,5-dicaffeoylquinic acid as an active ingredient.

The 3,5-dicapeoliquinic acid may be isolated from an extract of Aster glehni.

The present invention also contains 3,5-dicaffeoylquinic acid (3,5-dicaffeoylquinic acid) as an active ingredient, liquid, ointment, cream, lotion, spray, patch, gel, or aerosol formulation It provides a phosphorus external composition.

By introducing 3,5-dicaffeoylquinic acid of the present invention as an active ingredient of a cosmetic composition or pharmaceutical composition, there is an effect of strengthening the skin barrier and preventing damage to the skin barrier.

Figure 1 is the HPLC analysis chromatogram results for the ethyl acetate extract (1) 5-CQA: 5-caffeoylquinic acid (5-caffeoylquinic acid), 2) 3,4-DCQA: 3,4-D Caffeoylquinic acid (3,4-dicaffeoylquinic acid), 3) 3,5-epi-DCQA: 3,5-epi-dicaffeoylquinic acid (3,5-epi-dicaffeoylquinic acid), 4) 3,5 -DCQA: 3,5-dicaffeoylquinic acid, 5) 4,5-DCQA: 4,5-methyl 4,5-dicafeoylquinate, 6) 3,5- DCQA-Me: methyl 3,4-dicaffeoylquinic acid, 7) 4,5-DCQA-Me: methyl 4,5-dicafeoylquinic acid (methyl 4,5- dicaffeoylquinic acid)).
Figure 2 shows the results of Western blot analysis for PPARδ, AMPK and SPTLC2 in HaCaT cells treated with Aster glehni extract, SDS, DNCB and GSK0660 (Ctrl is untreated control, DNCB is DNCB treated group, D+AG is DNCB. + AG treatment group, D+A+GSK is DNCB + AG + GSK0660 treatment group, SDS is SDS treatment group, S+AG is SDS + AG treatment group, S+A+GSK is SDS + AG + GSK0660 treatment group).
Figure 3 is a result of performing an immunocytochemical test for keratin, involuclin, defensin and TNFα in HaCaT cells treated with Aster glehni extract, SDS, DNCB and GSK0660 (Ctrl is an untreated control group, DNCB is a DNCB-treated group , D+AG is DNCB + AG treatment group, D+A+GSK is DNCB + AG + GSK0660 treatment group, SDS is SDS treatment group, S+AG is SDS + AG treatment group, S+A+GSK is SDS + AG + GSK0660 treatment group).
4 is a result of immunohistochemical staining for keratin, involuclin, defensin, and TNFα in HaCaT cells treated with TRPV4 and AMPK antagonists.
5 is a result of Western blot for TRPV4, AMPK, PPARδ and SPTLC2 in HaCaT cells treated with 3,5-DCQA.
6 is a result of immunohistochemical staining for keratin, involuclin, defensing and TNFα in HaCaT cells treated with 3,5-DCQA.
Fig. 7 is a schematic diagram of the mechanism of action of the extract of Sumsukum chinensis in keratinocytes (arrows mean activation, horizontal lines mean inhibition, double vertical lines mean blocking).

In the present invention, cells treated with 3,5-dicaffeoylquinic acid (3,5-dicaffeoylquinic acid) compared to control cells treated with only SDS or DNCB, PPARδ, phosphorylated AMPK, SPTLC2, keratin, involuclin and defensin It was confirmed that the protein expression of was increased, and the increased TNFα expression in the cell group treated with only SDS or DNCB was decreased in the cell group to which 3,5-dicapeoliquinic acid isolated from the extract of S. From the results of the study using antagonist treatment, the order of the mechanisms of action related to the regulatory effect of the biomarker in the pathway of action of AG is TRPV4 → PPARδ → AMPK, and the 3,5-dicapeoliquinic acid isolated from the extract of sagebrush extract was TRPV4 → PPARδ. → It was confirmed that damage to keratinocytes caused by SDS or DNCB can be alleviated through sequential regulation of the AMPK pathway.

Therefore, in one aspect, the present invention strengthens the skin barrier and damages the skin barrier containing 3,5-dicaffeoylquinic acid isolated from the extract of Aster glehni as an active ingredient. It relates to a pharmaceutical composition for prevention.

The following Chemical Formula 1 is a structural formula of 3,5-dicapeoliquinic acid.

[Formula 1]

The term "skin barrier" as used in the present invention refers to a stratum corneum composed of keratinocytes as the upper layer of the epidermis, the outermost layer of the skin. It is the most important primary defense against toxic substances, microorganisms, mechanical irritation, and ultraviolet rays, and functions to provide an environment in which the skin can perform its normal functions by suppressing the loss of electrolyte or moisture through the skin.

The term "improving skin barrier damage" as used herein refers to treating and improving skin barrier damage by strengthening the barrier function of the stratum corneum located at the outermost part of the skin.

In the function of treating and improving skin barrier damage according to the present invention, the stratum corneum, which is the outermost layer of the epidermis, is mainly composed of non-nucleated flat corneocytes. A multi-lamella lipid layer formed of intercellular lipids such as ceramide, cholesterol, and fatty acids synthesized by keratinocytes of the skin barrier that is maintained through the process of division and differentiation of normal epidermal cells is a barrier to prevent moisture in the skin from evaporating. Plays a role. On the other hand, of these intercellular lipids, omega hydroxy ceramide is linked to involucrin, a protein in the outer layer of corneocytes, through a chemical covalent bond to form a corneocyte lipid envelope (CLE), thereby forming a multilayer lipid membrane. It plays the role of physically stabilizing the intercellular lipids in the form, thereby treating and improving barrier damage.

The composition of the present invention is delivered to the stratum corneum through skin application to promote the differentiation of keratinocytes, and has the effect of improving the thickness of the epidermal layer, as well as having an excellent effect of restoring damage to the skin barrier. It can be usefully used for the treatment and prevention of induced skin diseases. Skin diseases caused by the damage to the skin barrier include atopic dermatitis, xeroderma, psoriasis, ichthyosis, acne, and the like, but are not limited thereto.

In terms of the present invention, 　"for improving skin barrier damage" means 　skin 　 protection and 　 skin 　 condition 　 improvement, skin 　 protection and 　 alleviation of inflammatory reaction of the skin, immune disease 　 improvement ability, or 　 skin 　 barrier function 　 improvement, 　 skin irritation relief, 　 skin irritation relief It is a concept that includes all of the regeneration ability, antioxidant ability, and collagen synthesis enhancing ability.

In the present invention, alleviation of skin inflammation means improving and treating skin problems such as skin inflammation, itchiness, and the like.

In the present invention, the term'inflammation relief' refers to suppressing inflammation, and the inflammation is one of the defense responses of living tissues against a certain stimulus, and is a complex that causes tissue deterioration, circulatory disorders and effusion, and tissue proliferation. Refers to the lesion. More specifically, inflammation is part of innate immunity, and, as in other animals, innate immunity in humans recognizes patterns on the cell surface that are specific to pathogens. Phagocytes recognize cells with such surfaces as non-magnetic and attack pathogens. If pathogens break through the body's physical barriers, an inflammatory reaction occurs. The inflammatory reaction is a nonspecific defense action that creates a hostile environment for microorganisms invading the wound. In the inflammatory reaction, when a wound or an external infectious agent enters the body, white blood cells responsible for the early stage immune response gather and express cytokines. Therefore, the amount of expression of cytokines in cells becomes an indicator of activation of the inflammatory response. Examples of skin diseases related to inflammation include atopic dermatitis, psoriasis, erythematous disease triggered by radiation, chemicals, burns, etc., acid burns, bullous skin disease, lichen shape type disease, itching due to allergies, seborrheic eczema, rose acne, Pemphigus vulgaris, polymorphic exudative erythema, nodular erythema, balanitis, vulvitis, inflammatory hair loss such as alopecia areata, cutaneous T-cell lymphoma, and the like, but are not limited thereto.

As used herein, the term "prevention" refers to any action that suppresses atopic dermatitis or delays the onset of atopic dermatitis by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to any action in which symptoms of atopic dermatitis are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "improvement" means any action that at least reduces the severity of a parameter related to the condition being treated, for example, symptoms.

The term "extract" of the present invention refers to a material obtained by separating from the scallop.

In the present invention, the extract is characterized in that it is extracted with any one extraction solvent selected from an alcohol having 1 to 4 carbon atoms, an aqueous solution containing an alcohol having 1 to 4 carbon atoms, dichloromethane, acetone, and acetone aqueous solution. can do.

In the present invention, the extract of scallop serrata may be characterized in that it is a fractionated extract further fractionated by any one selected from the group consisting of ethyl acetate, hexane, chloroform and butanol.

The extraction may be performed by an extraction method known in the art, such as cold sedimentation, hot water extraction, ultrasonic extraction, reflux cooling extraction, or the like, but is not limited thereto. The extraction temperature may be adopted by a person skilled in the art in various temperature ranges suitable for the extraction method, and may be performed at, for example, 20° C. to 100° C., but is not limited thereto. In addition, the extraction time is different depending on the extraction method, and a person skilled in the art may adopt an appropriate extraction time, but is not limited thereto, but may be performed once or multiple times in the range of about 1 hour to 10 days. Preferably, the extraction may be performed by extracting with the above extraction solvent 2-3 times at room temperature for about 2 days each.

In the present invention, the composition is to increase the expression of TRPV (transient receptor potential cation channel subfamily V member 4), PPAR-δ (Peroxisome proliferator-activated receptor-delta) and AMPK (5' AMP-activated protein kinase). It can be characterized.

In the present invention, PPARδ and AMPK are involved in cell survival and anti-inflammatory response. Ceramide is an important and major lipid component in the skin barrier, and serine palmitoyltransferase is an enzyme that catalyzes the rate limiting step in ceramide biosynthesis. In addition, TRPV4 is known to be involved in the formation of intercellular junctions in keratinocytes. SPTLC2 is a long-chain base subunit of serine palmitoyltransferase.

In one aspect of the present invention, it was attempted to evaluate the protein expression levels of PPARδ, AMPK and SPTLC2 in HaCaT cells treated with SDS or DNCB together with AG. As a result, it was confirmed that the AG extract increased protein expression for PPARδ, P-AMPK, SPTLC2 and TRPV4 compared to the cell group treated with only DNCB and SDS (FIGS. 2A and 2B ).

In the present invention, the composition may be characterized by reducing the expression of TNF-α (Tumor necrosis factor-alpha).

In another aspect of the present invention, it was confirmed that the increased expression of TNFα in the cell group treated with only SDS or DNCB decreased in the cell group to which the AG extract was added (FIG. 2C ).

In the present invention, in order to clarify the effect and mechanism of AG on skin barrier protection, the expression level of biomarkers related to skin barrier maintenance in HaCaT cells treated with SDS or DNCB was measured. In particular, the expression of regulatory factors plays an important role in the skin protective mechanism of AG, which plays the role of peroxisome proliferator-activated receptor δ (PPARδ) and AMP-activated protein kinase (AMPK) and transient receptor potential cation channel subfamily V member 4 (TRPV4). We investigated whether it was doing.

As a result, it was confirmed that AG extract rich in caffeoylquinic compounds can protect the skin barrier through sequential regulation of the TRPV4-PPARδ-AMPK pathway in human keratinocytes HaCaT cells with SDS or DNCB. It was confirmed that the barrier was protected (FIG. 7).

In the present invention, Defensins are antimicrobial, antibacterial and antiviral small cationic peptides produced by various cell types, and include three subfamily of α, β and θ defensins. In keratinocytes, β-defensin is primarily a secreted subtype.

In the present invention, PPARδ as a nuclear receptor alleviates metabolic diseases such as obesity and atherosclerosis. PPARδ's protective effect on the skin barrier has been studied as follows: PPARδ ligand treatment stimulates stratum corneum formation and permeable barrier development in fetal rat explant culture model, and in PPARδ knockout mice, epidermal integrity is impaired and inflammation increases. do. In general, AMPK is involved in reducing inflammation as well as improving metabolic syndrome and is regulated by PPARδ. Activated AMPK inhibits the increase of matrix metalloprotenase1 (MMP1) induced by ultraviolet radiation in HaCaT cells, and is involved in autophagy regulation through inhibition of the mTOR signaling pathway by apigenin in human keratinocytes.

In the present invention, protein expression for keratin, involuclin and defensin, reduced by SDS or DNCB, was increased by AG extract. In addition, elevated expression of TNFα by SDS or DNCB was normalized by AG treatment. The expression of PPARδ and AMPK was increased by treatment with AG extract compared to administration of SDS or DNCB alone. In addition, since the effect of AG extract was offset by the PPARδ antagonist, the effect of AG on keratinocytes is thought to depend on PPARδ.

In addition to PPARδ and AMPK, TRPV4 has also been announced to play an important role in the maintenance or protection of the skin barrier. TRPV4 is commonly known as a calcium ion (Ca ⁺⁺ )-permeable cation transporter and responds to mechanical stresses such as edema. In the area of dermatology, TRPV4 protects the skin barrier through strengthening of tight junctions and increases keratin synthesis in HaCaT cells treated with baicalein. The fact that TRPV4 acts as a Ca ⁺⁺ messenger suggests that it is involved in calcium signaling in keratinocytes (keratinocytes). Ca ⁺⁺ generally acts as a signaling molecule in cells and stimulates the expression of biomarkers such as keratin1/10, involucrin, loricrin, and transglutaminase 1 involved in keratinocyte differentiation. In addition, Ca ⁺⁺ promotes the conversion of profiraggrin to filaggrin. When the skin barrier is damaged, the Ca ⁺⁺ gradient disappears and the expression of loricrin, filaggrin and involucrin is lowered.

In the present invention, the skin inflammation may be characterized in that atopic dermatitis.

In another aspect, the present invention relates to a composition for external application comprising the composition, and is a liquid, ointment, cream, lotion, spray, patch, gel, or aerosol formulation.

The composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment in medical treatment, and the effective dose level refers to the type and severity of the individual, and age. , Sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and can be easily determined by a person skilled in the art.

The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier, and oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, respectively, according to conventional methods. And it may be formulated in the form of a sterile injectable solution.

The pharmaceutically acceptable carriers are those commonly used in the art, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. In addition, the pharmaceutical composition of the present invention includes diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and other pharmaceutically acceptable additives.

When the pharmaceutical composition of the present invention is formulated as an oral solid preparation, it includes tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient, such as starch, calcium carbonate , Sucrose, lactose, gelatin, and the like, and include, but are not limited to, lubricants such as magnesium stearate and talc.

When the pharmaceutical composition of the present invention is formulated in a liquid form for oral use, it includes a suspension, an inner solution, an emulsion, and a syrup, and includes, but is not limited to, diluents such as water and liquid paraffin, wetting agents, sweetening agents, fragrances, preservatives, etc. .

When the pharmaceutical composition of the present invention is formulated for parenteral use, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories are included, and non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, Vegetable oils such as olive oil, injectable esters such as ethyloleate, etc. are included, but are not limited thereto. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used, but are not limited thereto.

The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intravenous, subcutaneous, intraperitoneal or topical application) according to a desired method, and the dosage is It depends on the degree, drug form, administration route and time, but may be appropriately selected by those skilled in the art.

The dosage of 3,5-dicaffeoylquinic acid isolated from the extract of Aster glehni contained in the pharmaceutical composition of the present invention is the patient's condition and weight, age, and disease. It depends on the degree of, drug form, administration route and duration, but can be appropriately selected by those skilled in the art. For example, the 3,5-dicapeoliquinic acid isolated from the extract of sagebrush extract can be administered at a dose of 1 to 2000 mg/kg per day, preferably 10 to 2000 mg/kg, and the administration is performed per day. It may be administered once or several times.

In another aspect, the present invention strengthens the skin barrier and prevents damage to the skin barrier containing 3,5-dicaffeoylquinic acid isolated from the extract of Aster glehni as an active ingredient. It relates to a cosmetic composition for.

In the present invention, the extract is characterized in that it is extracted with any one extraction solvent selected from an alcohol having 1 to 4 carbon atoms, an aqueous solution containing an alcohol having 1 to 4 carbon atoms, dichloromethane, acetone, and acetone aqueous solution. can do.

In the present invention, the skin inflammation may be characterized in that atopic dermatitis.

In the present invention, the composition is to increase the expression of TRPV (transient receptor potential cation channel subfamily V member 4), PPAR-δ (Peroxisome proliferator-activated receptor-delta) and AMPK (5' AMP-activated protein kinase). It can be characterized.

In the present invention, the composition may be characterized by reducing the expression of TNF-α (Tumor necrosis factor-alpha).

In another aspect, the present invention strengthens the skin barrier and prevents damage to the skin barrier containing 3,5-dicaffeoylquinic acid isolated from the extract of Aster glehni as an active ingredient. It relates to a health functional food composition for.

In the present invention, the extract is characterized in that it is extracted with any one extraction solvent selected from an alcohol having 1 to 4 carbon atoms, an aqueous solution containing an alcohol having 1 to 4 carbon atoms, dichloromethane, acetone, and acetone aqueous solution. can do.

In the present invention, the skin inflammation may be characterized in that atopic dermatitis.

In the present invention, the composition is to increase the expression of TRPV (transient receptor potential cation channel subfamily V member 4), PPAR-δ (Peroxisome proliferator-activated receptor-delta) and AMPK (5' AMP-activated protein kinase). It can be characterized.

In the present invention, the composition may be characterized by reducing the expression of TNF-α (Tumor necrosis factor-alpha).

In the present invention, the term "health functional food" refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body according to the Health Functional Food Act No. 6727, and the term "functional" means It means ingestion for the purpose of obtaining useful effects for health purposes such as controlling nutrients or physiological effects on the structure and function of the human body.

The food composition of the present invention may contain conventional food additives, and the suitability as the "food additive" is, unless otherwise specified, according to the general rules and general test methods of food additives approved by the Ministry of Food and Drug Safety. It is determined according to the standards and standards for the item.

* Items listed in the "Food Additives Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as reduced pigment, licorice extract, crystalline cellulose, high color pigment, guar gum, etc. , L-sodium glutamate, an alkali additive for noodles, a preservative, and a tar colorant.

The food composition of the present invention is a 3,5-dicaffeoylquinic acid isolated from Aster glehni extract based on the total weight of the composition for the purpose of improving skin barrier damage and/or alleviating skin inflammation. acid) from 0.01 to 95% by weight, preferably from 1 to 80% by weight.

In addition, the food composition of the present invention is manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of improving skin barrier damage and/or alleviating skin inflammation, in particular, preventing and/or improving atopic dermatitis. can do.

For example, the health functional food in the form of a tablet is a mixture of 3,5-dicapeoliquinic acid isolated from the extract of Ssumbus serrata or a mixture of excipients, binders, disintegrants, and other additives by a conventional method. Next, compression molding may be performed by putting a lubricant or the like, or the mixture may be directly compression molded. In addition, the health functional food in the form of a tablet may contain a mating agent or the like, if necessary, and may be coated with a suitable coating agent if necessary.

Among capsule-type health functional foods, hard capsules are filled with a mixture of additives such as 3,5-dicafeoliquinic acid and excipients separated from the Ssugwort extract into ordinary hard capsules, or granular or coated granules thereof. The soft capsules can be prepared by filling a mixture of 3,5-dicapeoliquinic acid separated from the Ssumbus squirrel extract and additives such as excipients into a capsule base such as gelatin. The soft capsules may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, and the like, if necessary.

For health functional foods in the form of rings, a mixture of 3,5-dicaffeoylquinic acid, excipients, binders, disintegrants, etc. isolated from the extract of Aster glehni is molded in an appropriate way. If necessary, the skin can be coated with a white sugar or other suitable skin, or with starch, talc, or a suitable substance.

For health functional foods in the form of granules, a mixture of 3,5-dicaffeoylquinic acid, excipients, binders, disintegrants, etc. separated from the extract of Aster glehni is granulated in an appropriate manner. It can be prepared by, and if necessary, may contain a flavoring agent, a flavoring agent, and the like. For health functional foods in the form of granules, use No. 12 (1680 μm), No. 14 (1410 μm), and No. 45 (350 μm) sieves. It is less than 5.0% and passing through sieve 45 may be less than 15.0% of the total amount.

The definitions of terms for the excipients, binders, disintegrants, lubricants, flavoring agents, flavoring agents, etc. are described in documents known in the art, and include those having the same or similar functions (the Korean Pharmacopoeia commentary, Moonseongsa, Korea College of Pharmacy Council, 5th edition, p.33-48, 1989).

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Example 1: Preparation of extract of sageum wormwood

In November 2012, peeled and dried Aster glehni (AG) was purchased from Ulleungdo Island (N37° 30', E130° 52') and confirmed by Emeritus Professor Kwak Chang-soo (Pharmacology Department, Kyunghee University, Seoul). , The voucher specimen (971-12A-P) was stored in the herbarium of the Korea Institute of Science and Technology.

To obtain a methanol-soluble extract, 12Kg of chopped AG was extracted three times using 70 L of methanol at room temperature. 2.6 kg of the dry extraction residue was suspended in water and then partitioned sequentially with ethyl acetate. The ethyl acetate fraction was evaporated under reduced pressure to give 41.0 g of a residue. AG's ethyl acetate extract was analyzed by reverse phase high performance liquid chromatography (Waters 1500 Series System) using a 2996 PDA detector (254 nm, Waters, Worcester, MA, USA).

For separation, 10 μL of a sample was injected at 30° C. into a Luna C18 column (5 μm, 250×4.6 mm, Phenomenex, Torrance, CA, USA). The mobile phase used a gradient of acetonitrile and 1% phosphoric acid. The gradient system is 20% acetonitrile (0 min), 20% acetonitrile (0-10 min), 30% acetonitrile (10-20 min), 40% acetonitrile (20-30 min), 80 % Acetonitrile (30-40 minutes) and 100% acetonitrile (40-50 minutes). The flow rate of the mobile phase was 1.0 ml/min. The organic solvent used for the extraction process and HPLC analysis was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.).

As a result, the ethyl acetate fraction extracted from AG was mainly 5-caffeoylquinic acid, 3,4-dicaffeoylquinic acid, and 3,5-epi-dicafe. Oilquinic acid (3,5-epi-dicaffeoylquinic acid), 3,5-dicaffeoylquinic acid (3,5-DCQA)), 4,5-dicafeoylquinic acid (4, 5-dicaffeoylquinic acid), methyl 3,5-dicaffeoylquinic acid, and methyl 4,5-dicaffeoylquinic acid (methyl 4,5-dicaffeoylquinic acid) were included (Fig. One). It was confirmed that 3,5-DCQA was most abundant in the seven caffeoylquinic compounds of the AG ethyl acetate fraction.

Example 2: Culture of skin keratinocytes (HaCaT)

HaCaT cells (human keratinocytes) were cultured in Dulbecco's modified Eagle's medium (DMEM) medium containing 10% fetal bovine serum (FBS) and 1% antibiotic-antibacterial solution in a 37°C, 5% CO ₂ incubator. I did. The cell culture medium was replaced with fresh DMEM medium every 48-72 hours. HaCaT cells from passages 5 to 17 per well in 8-well chamber slides at a density of 1 x 10 ⁴ cells per well, or in 6 well culture plates in DMEM containing 10% fetal bovine serum and 1% antibiotics-antibacterial Plated at 1 x 10 ⁶ cell density. The cells were cultured in an incubator at 37° C. and 5% CO ₂ for 24 to 48 hours, and then the medium was replaced with DMEM containing 1% fetal bovine serum. Thereafter, cells with DNCB, 5 micromoles (Sigma-Aldrich, Louis, MO, USA) or SDS, 30 micromoles (Sigma-Aldrich) containing AG extract (50 micrograms) and PPARδ antagonist GSK0660 (Sigma-Aldrich) Were treated for 24 hours. In addition, 3,5-dicaffeoylquinic acid (3,5-DCQA) was treated with 1 uM. HaCaT cells were donated by Dr. Sun-Wook Sun. All reagents for cell culture were purchased from WELGENE Inc. (Daegu, Korea). The concentrations of all reagents were final.

Example 3: Observation of protein expression of PPARδ, AMPK, SPTLC2, and TRPV4 in HaCaT cells treated with DNCB or SDS upon treatment with S.

In order to evaluate the protein expression levels of PPARδ, AMPK, and SPTLC2 in HaCaT cells treated with SDS or DNCB together with AG, after treatment with the treatment conditions of Example 2, Western blot was performed.

The protein concentration of the sample was evaluated by the Bradford method. 10 μg of the extracted protein was loaded onto a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel, and protein blotting was performed on a nitrocellulose membrane for 90 minutes. The membrane was blocked with 5% skim milk overnight and washed 3 times for 10 minutes with TBS-T. The primary antibody was bound to the membrane for 2 hours at room temperature. The primary antibody of PPARδ was purchased from Abcam. Primary antibodies to total and phosphorylated forms of AMPK were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). The primary antibody against serine palmitoyltransferase 2 (SPTLC 2) was purchased from Novus, and the primary antibody of β-actin was purchased from Santa Cruz Biotechnology, Inc. The dilution conditions for the primary antibody are as follows. PPARδ was 1:500, AMPK, P-AMPK (at Thr172) and SPTLC 2 were 1:1000, and β-actin was 1:800. After washing three times with TBS-T for 10 minutes, a secondary antibody (Santa Cruz Biotechnology, Inc.) was bound to the membrane for 1 hour at room temperature. The dilution conditions for the secondary antibody are as follows. The IgG antibody against anti-rabbit PPARδ, AMPK, p-AMPK and SPTLC 2 was 1:5000 and the anti-mouse IgG antibody against β-actin was 1:5000. After washing three times with TBS-T for 10 minutes, TBS washing was performed once again for 10 minutes, and a chemiluminescent substrate and enhancer solution (Bio-Rad, Hercules, CA, USA) were applied to the membrane to determine the protein expression state. Measured. Images were manually processed using Kodak GBX developer and fixative reagent (CARESTREAM HEALTH, INC., Rochester, NY, USA) and analyzed using ImageJ program. β-actin was used as a normal control to normalize the loaded protein.

As a result, the AG extract increased protein expression for PPARδ, P-AMPK, SPTLC2 and TRPV4 compared to the cell group treated with only DNCB and SDS. However, these protein expression increasing effects of the AG extract were almost eliminated by GSK0660, a PPARδ antagonist, except for TRPV4 (FIGS. 2(A) and 2(B)).

Example 4: Observation of keratin, involuclin, β-defensin, and TNFα expression of DNCB or SDS-treated HaCaT cells upon treatment with S.

In order to evaluate the expression of β-defensin1, one of the representative β-defensins, along with skin barrier components such as keratin and involuclin, and the inflammatory cytokine TNFα, after treatment with the treatment conditions of Example 2, immunocytochemistry (ICC)) was carried out.

The cells on the chamber slide were fixed with ice-cold methanol for 15 minutes. The fixed cells were reacted with a 0.3% hydrogen peroxide (H2O2) solution containing 0.3% normal serum for 5 minutes to remove peroxidase activity from the cells. After washing PBS for 5 minutes, the cells were blocked with non-specific antigens using normal serum diluted for 20 minutes, and then reacted with the diluted primary antibody for 1 hour. Primary antibodies against involuclin and TNFα were purchased from Novus (Littleton, CO 80120, U.S.A.). The primary antibody against β-defensin1 was obtained from Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA). The primary antibody against pan-keratin was purchased from Abcam (Cambridge, UK). After washing with PBS, the secondary antibody was reacted to the cells for 30 minutes. After washing with PBS, the premixed VECTASTATIN ABC reagent solution was reacted to the cells for 30 minutes. Cells were washed with PBS and reacted with DAB substrate solution until an appropriate color change appeared. After washing for 3 minutes with tap water, the cells were counter-stained with hematoxylin. The cells were washed with tap water, dried in the air, and the last was mounted. Immunocytochemistry kit (including secondary antibody) was purchased from Vector laboratories (Burlingame, CA, USA).

Protein extracts were electrophoresed on a 10% polyacrylamide gel and blotted onto a nitrocellulose membrane. After the nitrocellulose membrane was sequentially bound with the primary and secondary antibodies, hemiluminescence was exposed to the X-ray film. The band density of the Xray film was analyzed with the Image J program. HaCaT cells in the slide chamber were fixed and stained immunocytochemically with keratin, involuclin, defensin, and TNFα antibodies. Images were taken at 200 magnification. The density of the image was analyzed with the Image J program. Results were expressed as mean ㅁ SEM. Values were statistically analyzed by unpaired t-test. All experiments were repeated three times.

As a result, AG extract increased the expression of proteins of keratin, involuclin and β-defensin1, which were reduced by DNCB and SDS. However, increased protein expression was offset by the PPARδ antagonist GSK0660. TNFα expression increased by DNCB and SDS was reduced by AG extract, and the improvement effect of AG extract was eliminated by GSK0660 (FIG. 3).

Example 5: Observation of keratin, involuclin, β-defensin and TNFα expression in HaCaT cells

To investigate the effect of TRPV4 and AMPK on the expression of skin barrier components, biomarkers related to defense and inflammation, protein expression against keratin, involuclin, β-defensin and TNFα in HaCaT cells treated with antagonists against TRPV4 and AMPK Was investigated by immunocytochemical staining.

Western blots for TRPV4, AMPK and PPARδ were performed in HaCaT cells treated with antagonists against TRPV4 and AMPK and treated with antagonists against TRPV4, PPARδ and AMPK. HaCaT cells in the slide chamber were fixed and stained immunocytochemically with antibodies of keratin, involuclin, defensin, and TNFα. Images were taken at 200 magnification. The density of the images was analyzed with the Image J program. The protein extract was electrophoresed on a 10% polyacrylamide gel and adsorbed onto a nitrocellulose membrane. The nitrocellulose membrane was sequentially bound with the primary and secondary antibodies, and chemiluminescence was exposed to the Xray film. The band density of the X-ray film was analyzed by the Image J program. Results were expressed as mean ㅁ SEM. Values were statistically analyzed by unpaired t-test. All experiments were repeated three times.

As a result, TNFα protein expression was increased by TRPV4 antagonist or AMPK antagonist compared to the control in HaCaT cells. However, under the same conditions, the protein expression of keratin, involuclin and β-defensin1 was significantly reduced compared to the control group (FIG. 4).

Example 6: Observation of correlation between protein expression of TRPV4, PPARδ and AMPK and antagonists for TRPV4, PPARδ and AMPK in HaCaT cells

In order to find out the order of action of major regulatory factors such as TRPV4, PPARδ and AMPK, the protein expression levels of TRPV4, PPARδ and AMPK were measured by Western blot in HaCaT cells treated with TRPV4, PPARδ or AMPK antagonists.

As a result, all protein expressions for TRPV4, PPARδ and p-AMPK were significantly reduced in HaCaT cells treated with TRPV4 antagonists (FIG. 4 ). PPARδ antagonist treatment lowered protein expression for PPARδ and p-AMPK, but did not change TRPV4 protein expression (FIG. 4 ). Compound C, an AMPK antagonist, reduced protein expression only for p-AMPK, but did not affect protein expression for TRPV4 and PPARδ (FIG. 4).

Example 7: Upon treatment with 3,5-dicaffeoylquinic acid (3,5-DCQA), observation of protein expression for TRPV4, PPARδ, AMPK and SPTLC2 in HaCaT cells

In order to observe the protein expression of TRPV4, PPARδ, AMPK, and SPTLC2 in HaCaT cells upon treatment with 3,5-dicapeoliquinic acid, Western blot was performed after treatment under the treatment conditions of Example 2.

As a result, 3,5-DCQA increased protein expression for PPARδ, AMPK, and SPTLC2 in HaCaT cells like AG ethyl acetate extract (FIG. 5).

Example 8: When 3,5-dicaffeoylquinic acid (3,5-DCQA) was treated, keratin, involuclin, β-defensin and TNFα expression were observed in HaCaT cells.

In order to observe the protein expression of keratin, involuclin, β-defensin and TNFα in HaCaT cells during treatment with 3,5-dicapeoliquinic acid, after treatment with the treatment conditions of Example 2, immunohistochemical staining was performed. .

Protein extracts were electrophoresed on a 10% polyacrylamide gel and blotted onto a nitrocellulose membrane. The nitrocellulose membrane was bound to the primary and secondary antibodies, irradiated sequentially, and then exposed to the X-ray film with chemiluminescence. The band density of the X-ray film was analyzed by the Image J program. HaCaT cells in the slide chamber were fixed and stained immunocytochemically with keratin, bulcurin, defense and TNFα antibodies. Images were taken at 200x magnification. The density of the image was analyzed with the Image J program. Results were expressed as mean ㅁ SEM. Values were statistically analyzed by unpaired t-test. All experiments were repeated three times.

As a result, 3,5-DCQA treatment in HaCaT cells increased the protein expression of keratin, involuclin and β-defensin1 involved in the integrity of the skin barrier, but decreased TNFα expression related to skin inflammation (FIG. 6 ).

Therefore, the TRPV4 protein level was increased by AG extract compared to the DNCB or SDS treatment group, and since the TRPV4 antagonist counteracted the effect of AG in keratinocytes, it can be seen that TRPV4 is involved in the maintenance of the skin barrier.

Experimental results obtained from HaCaT cells treated with antagonists against PPARδ, AMPK and TRPV4 showed that AG can protect keratinocytes through sequential regulation of the TRPV4 → PPARδ → AMPK pathway. In addition, 3,5-dicaffeoylquinic acid, which is the most abundant of the 7 caffeoylquinic acids of AG extract, showed the same effect as ethyl acetate extract on the integrity of the skin barrier of HaCaT cells and the expression of biomarkers related to inflammation. It has been shown to be due to quinic compounds.

AG extract rich in caffeoylquinic compounds is TRPV4 ??in keratinocytes HaCaT cells with SDS or DNCB? It was confirmed that the skin barrier can be protected through sequential regulation of the PPARδ-AMPK pathway (FIG. 7).

statistics

Data are presented as mean ㅁ SE (measure of standard error). Statistically significant differences between the two groups were calculated by unpaired t-test with GraphPad Prism software. A value of p <0.05 was considered significant.

Preparation Example 1: Pharmaceutical composition and health functional food composition containing 3,5-dicapeoliquinic acid as an active ingredient

<Production Example 1> Preparation of pharmaceutical formulation

<1-1> Preparation of powder

3,5-dicafeoliquinic acid 2 g

1 g lactose

The above ingredients were mixed and filled in an airtight cloth to prepare a powder.

<1-2> Preparation of tablets

3,5-dicafeoliquinic acid 100 mg

Corn starch 100 mg

100 mg lactose

Stearic acid magnet 2mg

After mixing the above ingredients, tablets were prepared by tableting according to a conventional tablet preparation method.

<1-3> Preparation of capsules

3,5-dicafeoliquinic acid 100 mg

Corn starch 100 mg

100 mg lactose

2 mg of magnesium stearate

After mixing the above ingredients, a gelatin capsule was filled according to a conventional capsule preparation method to prepare a capsule.

<1-4> Preparation of ring

1 g of 3,5-dicafeoliquinic acid

1.5 g lactose

1 g glycerin

0.5 g xylitol

After mixing the above components, it was prepared so as to be 4 g per ring according to a conventional method.

<1-5> Preparation of granules

150 mg of 3,5-dicafeoliquinic acid

Soybean extract 50 mg

200 mg of glucose

Starch 600 mg

After mixing the above ingredients, 100 mg of 30% ethanol was added and dried at 60°C to form granules, and then filled into a cloth.

<Production Example 2> Preparation of food

<2-1> Preparation of flour food

0.5 to 5.0 parts by weight of 3,5-dicafeoliquinic acid of the present invention was added to the flour based on 100 parts by weight of flour, and bread, cakes, cookies, crackers and noodles were prepared using this mixture.

<2-2> Preparation of soup or gravies

To 100 parts by weight of soup or gravy, 0.1 to 5.0 parts by weight of 3,5-dicafeoliquinic acid of the present invention were added to the soup or juice to prepare a health-promoting meat product, a soup of noodles, or a gravy.

<2-3> Preparation of ground beef

Ground beef for health promotion was prepared by adding 10 parts by weight of 3,5-dicapeoliquinic acid of the present invention to ground beef based on 100 parts by weight of ground beef.

<2-4> Manufacture of dairy products

5 to 10 parts by weight of 3,5-dicafeoliquinic acid of the present invention were added to milk based on 100 parts by weight of 100 parts by weight of milk ground beef, and various dairy products such as butter and ice cream were prepared using the milk. .

<2-5> Manufacturing of Seonsik

Brown rice, barley, glutinous rice, and adlay were gelatinized and dried by a known method, and then roasted, and then prepared into a powder having a particle size of 60 mesh with a grinder.

Black soybeans, black sesame seeds, and perilla seeds were also steamed and dried by a known method, and then roasted, and then prepared into a powder having a particle size of 60 mesh with a grinder.

The 3,5-dicafeoliquinic acid of the present invention was concentrated under reduced pressure in a vacuum concentrator, sprayed and dried with a hot air dryer, and the resulting dried product was pulverized with a grinder to a particle size of 60 mesh to obtain a dry powder.

It was prepared by blending the grains, seeds, and 3,5-dicafeoliquinic acid prepared above in the following ratio.

Grains (40% by weight of brown rice, 15% by weight of barley, 20% by weight of barley),

*Seeds (perilla 7% by weight, black beans 8% by weight, black sesame 7% by weight),

3,5-dicafeoliquinic acid (3% by weight),

Ganoderma lucidum (0.5% by weight),

Rehmannia (0.5% by weight)

<Production Example 3> Preparation of beverage

<3-1> Manufacturing of health drinks

Subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%), water (75%) and 5 g of 3,5-dicapeoliquinic acid of the present invention are homogeneous After mixing and sterilizing instantaneously, it was prepared by packaging it in a small container such as a glass bottle or a plastic bottle.

<3-2> Preparation of vegetable juice

Vegetable juice was prepared by adding 5 g of 3,5-dicapeoliquinic acid of the present invention to 1,000 ml of tomato or carrot juice.

<3-3> Preparation of fruit juice

Fruit juice was prepared by adding 1 g of 3,5-dicapeoliquinic acid of the present invention to 1,000 ml of apple or grape juice.

Preparation Example 2: Skin external composition and cosmetic composition containing 3,5-dicapeoliquinic acid as an active ingredient

Examples of the formulation of the invention include skin ointment, soft lotion, astringent lotion, nutritional lotion, massage cream, essence, and pack, but the formulation of the cosmetic composition of the present invention should not be construed as being limited thereto, and within the scope of the invention. Conventional variations of those skilled in the art are possible.

Formulation Example 1: Ointment for external use for skin

As shown in Table 1 below, an ointment for external use for skin containing 3,5-dicapeoliquinic acid was prepared according to a conventional method.

Formulation Example 2: Softening lotion

Formulation Example 3: Converging lotion

Formulation Example 4: Nutrient Cosmetic Water

Formulation Example 5: Essence

Formulation Example 6: Pack

Example 9: Checking the effect of improving the skin barrier function of the cosmetic composition according to the present invention

* It was confirmed that the 3,5-dicafeoliquinic acid of the present invention enhances the lipid barrier recovery after skin barrier damage.

As subjects, 10 volunteers with normal skin with good health and no dermatological disorders were selected. Their skin barrier was damaged until the amount of transdermal moisture loss was around 30g/m2/h by tape stripping the forearm according to the following procedure. Thereafter, TEWL (Transepidermal water loss, transdermal moisture loss) was evaluated, and the external ointment of Preparation Example 2 at a concentration of 20 µl for 30 minutes twice a day was applied to each of 9 cm 2 area on the arms of the volunteers. TEWL was measured every day until 7 days after application, and the degree of decrease in TEWL was measured to evaluate the degree of recovery of skin barrier function. The recovery rate used in the efficacy evaluation was measured according to the following formula 1.

[Equation 1]

Br(damage recovery rate)=(1-(Bt=i-Bt=0)/(Bt=d-Bt=0)×100

Bt = i = TEWL measurement after skin barrier damage at each time

Bt = 0 = TEWL measurement before skin barrier damage

Bt = d = TEWL measurement after skin barrier injury

As a result, as shown in Table 7, the skin barrier recovery rate of the skin to which the cream containing 3,5-dicaffeoliquinic acid of the present invention (Formulation Example) was applied is a cream containing no 3,5-dicaffeoliquinic acid. Compared to the (Comparative Formulation Example), it was 19.6% higher after 1 day and 17.7% higher after 3 days. Through this, it was confirmed that the 3,5-dicafeoliquinic acid of the present invention is excellent in the recovery effect of the damaged skin barrier.

As described above, specific parts of the present invention have been described in detail, and it will be apparent to those of ordinary skill in the art that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (7)

A pharmaceutical composition for strengthening the skin barrier and preventing damage to the skin barrier, containing 3,5-dicaffeoylquinic acid as an active ingredient.
The pharmaceutical composition according to claim 1, wherein the 3,5-dicapeoliquinic acid is isolated from the extract of Aster glehni, skin barrier strengthening and skin barrier damage prevention.
The method of claim 2, wherein the extract is extracted with any one extraction solvent selected from an alcohol having 1 to 4 carbon atoms, an aqueous solution containing an alcohol having 1 to 4 carbon atoms, dichloromethane, acetone, and an aqueous acetone solution. A pharmaceutical composition for strengthening the skin barrier and preventing skin barrier damage.
The pharmaceutical for strengthening skin barrier and preventing skin barrier damage according to claim 3, wherein the extract is a fractionated extract further fractionated by any one selected from the group consisting of ethyl acetate, hexane, chloroform, and butanol. Ever composition.
The method of claim 1, wherein the composition increases the expression of TRPV (transient receptor potential cation channel subfamily V member 4), PPAR-δ (Peroxisome proliferator-activated receptor-delta), and AMPK (5' AMP-activated protein kinase). A pharmaceutical composition for strengthening the skin barrier and preventing damage to the skin barrier, characterized in that.
The pharmaceutical composition for strengthening skin barrier and preventing skin barrier damage according to claim 1, wherein the composition reduces the expression of TNF-α (Tumor necrosis factor-alpha).
A pharmaceutical composition comprising the composition of any one of claims 1 to 6, and which is an external preparation in the form of a liquid, ointment, cream, lotion, spray, patch, gel, or aerosol.

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