KR101114506B1 - Herbal Composition Having Improving Ability for Atopy Dermatitis by TSLP Inhibiting Ability - Google Patents

Abstract

The present invention relates to a herbal composition having atopic dermatitis improving ability by TSLP inhibitory ability, and more particularly, to a cosmetic composition and a pharmaceutical composition containing betel hydrothermal extract and clove bark hydrothermal extract as an active ingredient.

The herbal composition having atopic dermatitis improving ability according to the present invention antagonizes the action of TSLP, which is a pathological immune mechanism of atopic dermatitis, thereby improving the immune response of atopic dermatitis, and using natural extracts. There is.

Atopy, TSLP, Betel Nut, Clove Bark, Hydrothermal Extraction, Ultrasonic Extraction, Supercritical Extraction

Description

Herbal Composition Having Improving Ability for Atopy Dermatitis by TSLP Inhibiting Ability

The present invention relates to a herbal composition having atopic dermatitis improving ability by TSLP inhibitory ability, and more particularly, to a cosmetic composition and a pharmaceutical composition containing betel hydrothermal extract and clove bark hydrothermal extract as an active ingredient.

Atopic dermatitis, also called civilization or advanced incurable disease, is a common disease that can occur at any age. 95% develop in infants under 5 years of age. Even in Korea, the problem of atopic dermatitis has reached a serious social and medical level. According to various reports, about 15% of all Koreans are atopic dermatitis patients, 18? 22 patients are known to have atopy.

The exact pathophysiology of atopic dermatitis is not yet fully understood, but it is thought that immunological and environmental factors may be involved along with genetic predisposition. Exogenous atopic dermatitis, which accounts for the majority of atopic dermatitis, is caused by an immune mechanism associated with IgE, and there are many reports that delayed immune responses caused by T-cell abnormalities are involved rather than immediate immune responses to certain allergens. It is still controversial to evaluate the direct correlation of atopic dermatitis with skin terminal test results, which is an immediate immune response test because is not an erythema and swelling on skin terminal test. It has been reported that cell-mediated immune dysfunction is associated with an increase in IgE in patients with atopic dermatitis, and is reported to be involved in delayed immune responses caused by T cell abnormalities rather than immediate immune responses to certain allergens. In patients with atopic dermatitis, up to 80% of cell-mediated immune disorders are known to occur, and their susceptibility to skin infections caused by viruses and dermatophytes is increased, and the sensitivity to contact allergens tends to decrease. Although there have been reports of T cell maturation deficiency and a decrease in the number of CD8 + inhibitory T cells, recently, the role of CD4 + T cells is known to be more important. That is, T cells that secrete IL-4, which induces IgE production from B cells, IL-5, which promotes IgE production from B cells, and IL-6, which amplify IgE production, are Th2 cells among CD4 + T cells. Antigen-specific T-cell clones isolated from peripheral blood and skin lesions of AD patients were found to be Th2, and IL-4 and IL-13 secreted from these cells promote IgE production and IL-5 is a basophil response. (Source: Immunopathology of Atopic Dermatitis, Lee Kwang-hoon). Recent reports suggest that the immune response varies according to the condition of atopic dermatitis. Th2 is involved in acute atopic dermatitis and Th1 is involved in chronic atopic dermatitis. Initially, IL 4 and IL 13 secreted from Th2 are important, and when itching starts to scratch, it moves to Th1 cells.

Immune imbalances, the underlying cause of atopic dermatitis, are the result of various biological signal transductions. The top level protein for this abnormal signaling is the thymic stromal lymphopoietin (TSLP) cytokine. When allergens penetrate into the body, substances such as TSLP stimulate dendritic cells to respond, causing symptoms of atopic dermatitis.

Recently, the increase in atopic dermatitis in children as well as in adults is becoming serious, and in severe cases, it causes serious social problems such as employment and marriage. In such cases, severe mental stress may cause extreme avoidance and depression. Although atopic dermatitis is a serious social and medical problem, there is no effective treatment to date.

 Existing atopic dermatitis treatments have been developed such as cyclosporin and FK506, which inhibits calcineurin and reduce cytokine expression.However, these drugs weaken the immune system during long-term use, reducing the resistance of secondary infections, and relieve skin itching and pruritus. The external treatment of the family also has problems such as reduced immunity and growth is inhibited when the child is used for a long time.

At present, many researches and products for improving atopic dermatitis are being researched and commercialized by many domestic school and bio company researchers, and most of the developed products disappear at the early stage of market entry. This is because hypoallergenic cosmetics are being developed as atopic dermatitis products to enhance the moisturizing effect of the skin vaguely than to develop a product corresponding to a pathological mechanism that causes and worsens atopic dermatitis.

Korean Patent Registration No. 742,378 (composition for the treatment of atopic dermatitis containing herbal medicine), Korean Patent Registration No. 773,411 (NH herbal composition showing antioxidant, anti-cancer and antimicrobial effects) and Korean Patent Application No. 2008-0006837 (Atopic dermatitis A composition for preventing or alleviating and a method for preparing the same) disclose a mixed composition of various herbal ingredients that exhibit an antimicrobial effect on atopic dermatitis or skin bacteria using an extraction method using water or a lower alcohol. However, this extraction method has a problem that the extraction rate of the active ingredient of the herbal ingredients that are effective in dermatitis is somewhat reduced.

As a result of the present inventors' efforts to solve the above problems, betel hydrothermal extract and hydrothermal extract of clove bark among the extracts of various Chinese medicines and natural products extracted by using the hydrothermal extraction method, the action of TSLP which is a pathological immune mechanism of atopic dermatitis By antagonizing it was confirmed that the ability to improve atopic dermatitis is excellent, the present invention was completed.

It is an object of the present invention to provide a cosmetic composition having an atopic dermatitis improving ability by TSLP inhibitory activity, containing betel hydrothermal extract and clove bark hydrothermal extract as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for treating or preventing atopic dermatitis by TSLP inhibitory activity, containing betel hydrothermal extract and clove bark hydrothermal extract as an active ingredient.

In order to achieve the above object, the present invention provides a cosmetic composition having atopic dermatitis improving ability by TSLP inhibitory ability, containing betel hydrothermal extract and clove bark hydrothermal extract as an active ingredient.

The present invention also provides a pharmaceutical composition for the treatment or prevention of atopic dermatitis by TSLP inhibitory activity, containing betel hydrothermal extract and clove bark hydrothermal extract as an active ingredient.

The herbal composition having atopic dermatitis improving ability according to the present invention antagonizes the action of TSLP, which is a pathological immune mechanism of atopic dermatitis, thus improving the immune response of atopic dermatitis, and using natural extracts, thus having no side effects even when applied to human body for a long time. It works.

The present invention relates to a herbal composition having an atopic dermatitis improving ability by TSLP inhibiting ability, and specifically relates to a cosmetic composition and a pharmaceutical composition containing betel hydrothermal extract and clove bark hydrothermal extract as active ingredients.

In the present invention, "containing as an active ingredient" is meant to contain a range of doses that bring about the effect of improving, preventing, or treating atopic dermatitis, the dose range may vary depending on the severity and dosage form, and the number of application It may vary depending on the age, weight and constitution of the subject.

In the present invention, "atopic dermatitis" refers to a state in which an infection site of the skin is changed by atopic dermatitis, and this condition includes both a condition considered to be a skin disease and a condition not regarded as a skin disease.

“Treatment” in the present invention includes the cure of atopic dermatitis symptoms as well as partial cure, improvement and alleviation of atopic dermatitis symptoms as a result of applying the pharmaceutical composition of the present invention to the atopic dermatitis site.

In the present invention, "prevention" means to prevent or prevent atopic dermatitis symptoms in advance by applying the pharmaceutical composition of the present invention to the skin, in particular atopic dermatitis to inhibit or block the symptoms of atopic dermatitis on the skin.

In the present invention, one active ingredient "Binrang" is a kind of herbal medicine, has the effect of ganghwapa (降 氣 破 滯), passage conductor, hydration (습 水 化濕) in oriental medicine. Therefore, allergy to abdominal pain caused by alluvial, ulceration by dampness, hindsight, food carrier, thorax, and several species And mixed with various herbal medicines used for parasite control, glaucoma treatment, mouthwash, dysentery, skin disease and the like.
In the present invention, the betel nut means fruit of betel nut (Acaca catechu), that is, betel nut.

In the present invention, one active ingredient, "Caryophylli Cortex", warms the stomach and relieves pain in the stomach and pain in the scrotum with wags, dizziness, splitting and cold. It also improves sexual function, warms the waist and knees, relieves rubella, eliminates alcohol poisoning and wind poison, and heals various boils. It relieves the feeling and gives off various scents. In addition, the antioxidant effect and the removal of harmful oxygen is excellent.

TSLP, a cause of atopic dermatitis in the present invention, is a top-level protein that causes immune imbalance by abnormally transmitting various biological signals, and keratinocytes, fibroblasts, or mast cells constituting the skin upon contact with an external antigen or microorganism. Is a protein produced by). TSLP thus produced causes various immune mechanisms to bias the immune system to Th2. In particular, it activates dendritic cells (DC) in the premature stages to produce thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC), IL-8, eotaxin-2, and Th2-attracting chemokines. . It also produces IL-5, IL-13, GM-CSF, and IL-6 from mast cells. TSLP-activated dendritic cells express OX40L on the cell surface to differentiate allergen-specific naive CD4 + T cells into Th2, which produces IL-4, IL-5, IL-13, and TNF-alpha but does not produce IL-10 Let's do it. The inflammatory Th2 thus produced is transferred to the local inflammation site by the presence of TARC and MDC, and IL-4, IL-5, IL-13 and TNF-alpha produced by Th2 are inflammatory response mediators such as IgE, It promotes mucus production, eosinophilia, initiates over-inflammatory reactions and induces atopic dermatitis. TSLP is the most potent and superior derivative of atopic dermatitis among the various biological reasons that have been discovered so far.

Herbal composition of the betel nut hot water extract and clove bark hot water extract according to the present invention can improve or treat atopic dermatitis by inhibiting the activity of TSLP which is the cause of atopic dermatitis.

The present invention relates to a cosmetic composition having an atopic dermatitis improving ability by TSLP inhibitory activity, containing betel hydrothermal extract and clove bark hydrothermal extract.

In the present invention, the content of the betel hydrothermal extract and clove bark hydrothermal extract is characterized in that 10 to 30 parts by weight with respect to 100 parts by weight of the total composition, if the extract content is less than 10 parts by weight does not show a significant improvement effect in a short time There is a problem, and if it exceeds 30 parts by weight, the physical safety of the entire cosmetic composition is impaired or there is a problem such as skin irritation during long-term use.

In the present invention, the clove bark hot water extract may be characterized in that the weight ratio of 0.3 to 3 times with respect to betel nut hot water extract, out of the ratio, there is no marked improvement in a short period of time, or of the total cosmetic composition There are problems such as inhibition of physical safety and skin irritation during long-term use.

In the present invention, the cosmetic composition is a lotion, cream, massage cream, nutrition cream, moisturizing cream, hand cream, lotion, moisturizing lotion, milk lotion, nutrition lotion, skin lotion, skin softener, skin toner, ice cream, pack , Foundation, essence, nutritional essence, skin gel, skin oil, skin toner, face lotion, converging water and deodorant.

The cosmetic composition of the present invention may be for cleaning the skin, the cosmetic composition for cleaning the skin is a cream, lotion, lotion, cleanser, foam, cleansing foam, peeling gel, body wash, scrub, soap, oil, emulsion, hair soap, hair It may have a formulation selected from the group consisting of rinse, body cleanser, shampoo, emulsion, solid detergent and liquid detergent.

The cosmetic composition of the present invention is a fatty substance, an organic solvent, a dissolving agent, a thickening agent and a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant, purified water, an ionic or nonionic type. Emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, anti-inflammatory agents, colorants, water or other ingredients commonly used in cosmetics, etc. And may be present in amounts of about 98.0 to 99.1 weight percent of the total composition.

Surfactants used in the cosmetic compositions of the present invention include both anionic, cationic and amphoteric, and the amount used is typically at least 30% by weight, preferably at least 70% by weight. Selecting and determining the type and amount of surfactant can be easily made by those skilled in the art.

Preferred stabilizers that can be used in the cosmetic compositions of the invention include, but are not limited to, glycol stearate. When stabilizers are used, they are generally about 0.1 to 5 weight percent of the composition.

Preferred preservatives that can be used in the cosmetic compositions of the invention include, but are not limited to, tetrasodium ethylenediamine tetraacetic acid (EDTA), 1- (3-chloroallyl) -3,5,7-traaza-1 Adamantane, paraben, methylparaben, a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one, phenoxyethanol, benzyl alcohol , Benzophenone-4, methylchloroisothiazolinone and methylisothiazolinone or mixtures thereof. If a preservative is used, its amount is typically about 0.01 to 6% by weight of the composition, the preferred amount is about 0.05 to 4% by weight, more preferably about 0.1 to 2% by weight.

Preferred wetting agents that can be used in the cosmetic compositions of the invention include wheat protein (e.g., laudimonium hydroxypropyl hydrolyzed wheat protein), hair keratin amino acids, sodium peroxylincarbolic acid, panthenol, tocopherol (vitamin E) And dimethicone or mixtures thereof. Sodium chloride may also be present, especially when hair keratin amino acids are included as wetting agents. Wetting agents are typically used from about 0.01 to 10% by weight, preferably from about 0.05 to 1.5% by weight, more preferably from about 0.01 to 1% by weight of the composition.

Preferred colorants that can be used in the cosmetic compositions of the invention include FD & C Green No. 3, Ext. D & C Violet 2, FD & C Yellow 5 and FD & C Red 40, or mixtures thereof. Colorants, if used, are typically from about 0.001 to 0.1% by weight of the composition, preferably from about 0.005 to 0.05% by weight.

Preferred anti-inflammatory agents that can be used in the cosmetic compositions of the invention include any pharmaceutically acceptable compound suitable for topical administration, preferably allantoin. Anti-inflammatory agents, when used, are used in an amount sufficient to inhibit or relieve inflammation and are typically from about 0.1 to 2% by weight, preferably from about 0.3 to 1.5% by weight, more preferably from about 0.1 to 1% by weight of the composition.

Antioxidants that can be used in the cosmetic composition of the present invention can be used both enzymatic and non-enzymatic antioxidant. Examples of natural enzymatic antioxidants include peroxide dismutase (SOD), catalase and glutathione peroxidase. Suitable non-enzymatic antioxidants include vitamin E (e.g. tocopherol), vitamin C (ascorbic acid), carotenoids, echinacoside, caffeoyl derivatives, oligomeric proanthocyanidins, proantanol (e.g. grapes). Seed extract), silymarin (eg, milk thistle extract, silyboom marianium), Ginkgo biloba and green tea polyphenols or mixtures thereof. Carotenoids are potent antioxidants and examples include beta-carotene, canthaxanthin, zeaxanthin, lycopene, lutein, crocetin, and capxanthin. Preferred antioxidants are vitamin E, vitamin C or carotenoids. Antioxidants, if used, are used in an amount sufficient to inhibit or mitigate the effects of free-radicals on the skin. Typical amounts of antioxidants are from about 0.001 to 1% by weight of the composition, with a preferred amount of about 0.01 to 0.5% by weight.

Cosmetic compositions of the present invention may contain other adjuvants commonly used in the art. Examples of such adjuvants include perfumes, dyes (including dyes that simultaneously dye or tint hair), solvents, opacifiers, pearlescent agents (eg esters of fatty acids and polyols, magnesium salts of fatty acids and zinc) Salts), copolymer-based dispersions, thickening agents (e.g. sodium chloride, potassium chloride, ammonium chloride and sodium sulfate), fatty acid alkylolamides, cellulose derivatives, natural gums, plant extracts, albumin derivatives (e.g. gelatin), collagen valences Degradation products, natural or synthetic polypeptides, egg yolk, lecithin, lanolin or lanolin derivatives, fats, oils, fatty alcohols, silicones, diorrents, antibacterial substances, anti-seborrheic substances, keratinants (e.g. sulfur, salicylic acid, benzoyl) Peroxides, selenium sulfides, PG, FA, enzymes) and keratant (eg tars, sulfur, salicylic acid, selenium sulfides, antibacterial agents, benzoyl peroxides, L'hexyl City of Aberdeen, iodine, triclosan, an antifungal agent and an enzyme), or a mixture thereof.

In one embodiment, in the following examples, vegetable hardened oil, petrolatum, glycerin, monostearic acid polyoxyethylene sorbitan, tocopheryl acetate, butyleneglycol, citric acid, paraoxybenzoic acid ester, methylbaraben, perfume, purified water An example of preparing a cosmetic composition by mixing in a ratio is provided, but is not limited thereto.

In another aspect, the present invention relates to a pharmaceutical composition for treating or preventing atopic dermatitis by TSLP inhibitory activity, containing betel hydrothermal extract and clove bark hydrothermal extract.

In the following examples, by confirming that the composition containing the betel hydrothermal extract and clove bark extract has a TSLP inhibitory ability, not only atopic dermatitis treatment, but also includes a function that can be prevented in advance.

In the present invention, the content of the betel hydrothermal extract and clove bark hydrothermal extract is characterized in that 10 to 30 parts by weight with respect to 100 parts by weight of the total composition, if the extract content is less than 10 parts by weight does not show a clear improvement effect in a short time There is a problem, and if it exceeds 30 parts by weight, the physical safety of the entire pharmaceutical composition is impaired or there is a problem such as skin irritation during long-term use. Within this range the concentrations of specific agents are determined by the intended use of them, and certain formulations, such as concentrated formulations, which must be diluted prior to use, may contain higher concentrations. All of the above formulations according to the invention are formulated in the appropriate desired form using techniques conventional in the art. Various preparations containing the betel hydrothermal extract and clove bark hydrothermal extract prepared by the present invention can be used in a conventional manner, preferably applied to atopic dermatitis or massaged.

In the present invention, the clove bark hydrothermal extract may be characterized in that the weight ratio of 0.3 to 3 times with respect to the betel hydrothermal extract, outside the ratio, there is no marked improvement in a short period of time, or the physical of the entire pharmaceutical composition There is a problem such as inhibition of safety and skin irritation during long-term use.

The pharmaceutical composition of the present invention may be prepared including various pharmaceutically acceptable carriers, excipients or diluents, etc., which are known at the time of the present application, and according to known methods, such as dispersing agents, suspending agents, emulsifiers, solutions and ointments It may be prepared in the form.

Specifically, the pharmaceutical composition may contain a thickening agent for imparting a viscosity to the formulation in the range of about 4,000 to about 9,000 cps at room temperature. When the thickener is added, the formulation can be maintained at various temperature ranges, a stable viscosity can be obtained, and at the same time, the usability of the skin can be improved. Such thickeners include Carbopol 1342, a copolymer of sucrose allyl ether and C _10-30 alkyl acrylate, acrylic acid and / or methacrylic acid. Other thickeners that may be used in addition include cellulose derivatives such as hydroxypropyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose and the like. Small additions of salts (NaCl) also serve to control the viscosity of the final formulation. Usually the amount of salt added is about 0.25 to 0.6% by weight. The pharmaceutical composition contains acid, base and buffer if necessary to maintain the pH at 4-10, preferably 4.5-8, more preferably 4.5-7. The nature and manner of use of such pH adjusting materials are well known in the art.

The formulation of the pharmaceutical composition is not particularly limited, but may be characterized in that it is for transdermal administration, and the composition for transdermal administration may include ointments, creams, pastes, lotions, liniments, pastes. It may have a formulation selected from the group consisting of external preparations, tinctures, glycerogelatins, external preparations, aerosols and plasters. These formulations are described in the literature, which is a prescription generally known in all pharmaceutical chemistry (Blaug et al ., Remington's Pharmaceutical Science , 15th Edition, 1975).

A preferred embodiment of the formulation is an ointment. The ointment is petrolatum, white petrolatum, yellow ointment, hydrocarbon base such as mineral oil, hydrophilic petrolatum, absorbent base such as anhydrous lanolin, lanolin, cold cream, washable base such as hydrophilic ointment, polyethylene glycol Any water-soluble base such as an ointment can be prepared. Their selection and preparation take into account factors such as the rate of release of the drug from the base, the expected increase in percutaneous absorption by the base, whether the skin is sealed by the base, the stability of the drug in the base, and the effect of the drug on the base. But belong to those of ordinary skill in the art.

A preferred embodiment of the formulation is a cream. The creams include W / O types, such as cold creams and emollient creams, and O / W types, such as shaving creams, varnishing creams, hand creams, and lining creams. More preferred creams are typically varnishing creams containing water and stearic acid. Usually patients or doctors prefer creams to ointments because o / w creams are easier to wash away than ointments.

A preferred embodiment of the formulation is a lotion. The lotions are prepared by methods such as suspending agents, emulsions, solutions and the like, which are also common to those skilled in the art of pharmaceutical formulation. More preferred in the present invention is a white lotion and this formulation also belongs to conventional techniques for those skilled in the formulation arts.

Another preferred embodiment of the formulation is a wax. The friction agent may be prepared as either an oily agent or an ethanol agent. Preferred are oily waxes with low irritation to the skin. In the case of oil-based lubricants, there is a combination of nonvolatile oils such as almond oil, peanut oil, cottonseed oil, volatile oils such as wintergreen and turpentine, or nonvolatile oils.

In addition, the range of prophylactic or therapeutic doses for atopic dermatitis may vary with severity and formulation. In addition, the frequency of application may also vary depending on the age, weight and constitution of the patient.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples. In addition, in the following experimental example of the present invention, the experiment to confirm the improvement ability of atopic dermatitis by preparing a cosmetic composition is illustrated, but in the case of the pharmaceutical composition because it contains betel hydrothermal extract and clove bark hydrothermal extract as an active ingredient, a known method It will be apparent to those skilled in the art that even in the case of the pharmaceutical composition prepared by the same, the same atopic dermatitis has a therapeutic or prophylactic effect.

Example 1 Preparation of Natural Product Sample for Atopic Dermatitis

In order to search for atopic improvement natural products through TSLP inhibitory activity used in the present invention, 400 kinds of herbal or natural products described in Table 1 were extracted with hot water.

First, the medicinal herbs of Table 1 are sufficiently washed and dried in purified water, and then crushed. Then, 2L of purified water is added to 100 g of the dried and crushed natural products, followed by hot water extraction at 100 ° C. for 2 hours. After heating, the mixture was heated to 80 ° C. for 1 hour to obtain a hydrothermal extract. The obtained natural product hydrothermal extracts were sufficiently cooled at room temperature after removing solids using three-ply gauze. Thereafter, after freezing at -70 ℃ for more than 4 hours, water was evaporated in a freeze dryer to obtain lyophilized natural product hot water extracts. In order to confirm the TSLP inhibitory activity of the extracts so obtained, it was dissolved uniformly in pure water at a concentration of 2 mg / ml, and then sterilized with a syringe filler of 0.22 μm and used for further experiments.

Table 1 Library for Searching for TSLP Inhibitory Natural Products

Example 2: TSLP Inhibition Test of Herbal Extracts Using Real-time PCR

2-1. Human skin keratinocyte line HaCaT cell culture

In order to perform a screening test of a natural extract that can reduce the production of TSLP produced in the skin, a constituent cell line of human skin, a keratinocyte HaCaT cell line (CLC Cell line serveice, Germany) was used. HaCaT cell lines were cultured using 10% fetal bovine serum (FBS; Gibco BRL, USA) and DMEM medium containing antibiotics (Gibco BRL, USA). The culture vessel was a 75T-flask and 6 well plate and incubated in a 37 ℃ incubator supplied with 5% CO2. The cultures were exchanged every 3 to 4 days and subcultured when cells proliferated excessively. Treatment test of natural extracts was treated with 0.3 ml of the extract obtained in Example 1, 10% of 3 ml of culture medium when the appropriate passage number of HaCaT cells proliferated more than 90% of the culture vessel area in 6 well plates. After 48 hours of incubation, total RNA was isolated and cDNA synthesis was performed.

2-2. Total RNA Isolation and cDNA Synthesis

The cDNA to be used for real-time PCR was synthesized by extracting total RNA 48 hours after treatment with the natural hot water extract. Specifically, total RNA was extracted using 1 ml of Trizol (Invitrogen, Carlsbad, CA) per 6 well plate.

In order to perform cDNA synthesis using Total RNA, 20 μl of the total reaction solution was composed of the following compositions. 4 μl of 5X PrimeScript Buffer (Kit code RR037A, Lot A1201-1) and 1 μl of PrimeScript RT Enzyme Mix (Kit code RR037A, Lot A1201-1) with Oligo dT Primer (50 μM) (Kit code RR037A, Lot A1201-1) ) 1µl and 4µl of Random 6mers (100µM) (Kit code RR037A, Lot A1201-1) were mixed and placed in a tube and stored on ice. 10 μl of total RNA was added to the tube to make the total reaction solution 20 μl. The mixture was mixed three times with mild vortexing and stored on ice. The reaction was carried out at 37 ° C. for 15 minutes using a PCR machine, followed by 5 seconds reaction at 85 ° C., and 4 ° C. treatment. Treated cDNA was used for real-time PCR analysis in the following manner.

2-3. TSLP Inhibitory Activity of Herbal Extracts

In order to confirm the reduction effect of the natural hydrothermal extract of Example 1 on TSLP expression of human keratinocytes HaCaT, the Taqman method using the cDNA real-time PCR was performed. The reaction was carried out using Taqman Primer & Probe (HS00263639, AB Applied Biosystems, USA) and Taqman Gene Expression Master Mix specific to TSLP for 1 μL of cDNA template at 1 μg / μL concentration. Final reaction samples were prepared and measured using an Applied Biosystems ABI 7500 Real-time PCR system (AB Applied Biosystems, USA).

In more detail, the reaction solution pre-mix for GAPDH and human beta defensin 3 analysis was performed to perform real-time PCR. The reaction solution pre-mix for GAPDH analysis consisted of 1 μl of GAPDH (Hs99999905, Lot 682967), 5 μl of DW, and 10 μl of Master Mix (2X) (TaqMan Gene Expression Master Mix L / N 0810037, P / N 4369016) and cDNA 4 Made by mixing well. The reaction solution pre-mix for analysis of human beta defensin 3 was 1 μl of DEF3 (Hs00218678, Lot 660126), 5 μl of DW and Master Mix (2X) (10 μl of TaqMan Gene Expression Master Mix L / N 0810037, P / N 4369016). 4 μl of cDNA was mixed well. The prepared reaction solution pre-mixes were added to 16 μl each in a real-time PCR reaction plate (optical 96-well Reaction Plate with Barcode, part No.4306737). To the same plate, add 2µl of HaCaT cDNA and Easy Dilution (Kit code RR037A, Lot A1201-1) and 2µl of cDNA diluted X10, X100. 4 μl of the prepared cDNA was added to the reaction solution of 16 μl in the plate and reacted with real-time PCR Cycles. The real-time PCR reaction first reacted the samples at 94 ° C. for 10 minutes for initial denaturation and then amplified the entire 60 cycles. The time and temperature for each cycle consisted of 10 seconds at 94 ° C, 10 seconds at 60 ° C, and 30 seconds at 25 ° C. Expression of the target genes was compared with relative values through the corrected quantification of the internal control gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) by the threshold cycle (Ct) method.

As a result, twelve of the 400 natural hydrothermal extracts showed a TSLP reduction of 25% or more compared to the control using water, among which 80% or more in the hydrothermal extracts of Arecaemen and Caryophylli Cortex. It showed a TSLP reduction effect (FIGS. 1-2). The reduction effect of the total 12 natural extracts showing a reduction effect of more than 25% compared to the water control is shown in Table 2 and FIG.

[Table 2] TSLP Reduction Effect of 12 Hot Water Extracts

number designation TSLP Reduction Rate (%) 23 Gugija (North) 26 ± 6 29 Ear woo 31 ± 7 48 Doin 29 ± 12 91 Betel nut 82 ± 6 116 Mokhyang (sec) 26 ± 12 129 Ejection (sec) 35 ± 11 168 Fellowship 28 ± 6 224 Sine (autumn) 33 ± 9 250 Clove Bark 87 ± 8 287 Gardenia (sec) 39 ± 12 305 Fellow ruler 28 ± 2 389 Golden (Sat) 26 ± 8

Example 3: Comparison of TSLP Inhibitory Activity According to Various Extraction Methods

In order to test whether the Arecaemen and Caryophylli Cortex showing high TSLP expression inhibitory effects of 82 ± 6% and 87 ± 8%, respectively, in Example 2, the same TSLP inhibition effect was obtained under various extraction conditions. General extraction with% ethanol, 50% methanol, 50% hexane, ultrasonic extraction, and supercritical extraction were performed (Table 3).

In order to perform the ultrasonic extraction, 10 g of the powder of dry crushed natural products were extracted using an ultrasonic extraction system by adding 100 ml of extractant water and 100 ml to a 500 ml beaker. In order to apply the same extraction method, 100 100 KHz ultrasonic energy (300 Watt ± 2) was applied, and the extraction temperature was fixed at 25 ° C. by circulating water in the reactor through a chiller. Ultrasonic extraction time was applied to 60 min. Since the extract contains a large amount of impurities, the solvent is extracted under reduced pressure with a filter (pore size: 5 μm), and separated from the sample residue. The sample is separated from the membrane filter (FH-0.2 μm, Waters, Milford, MA, USA). ) Was used as a TSLP inhibition test solution.

To perform supercritical extraction, ethanol (95% edible alcohol, v / v) was added to 10 g of the powder sample of the natural product as a co-solvent in an amount equivalent to 29.4 ml / kg CO ₂ with respect to carbon dioxide, the main solvent, and mixed immersion. I was. The ethanol immersed natural product was then placed in a 100 ml supercritical extraction container, extraction pressure 300 bar, extraction temperature 70 ℃, CO ₂ flow rate 8.5 g / min (this corresponds to 119.5 ml CO ₂ / min per liter of total void volume in the extraction vessel), Supercritical fluid extraction was performed for 40 minutes under conditions of 3.5 minutes. The powder was recovered and dried in a dry oven at 105 ° C. for 12 hours to remove moisture and ethanol components, and then tested for TSLP inhibition using the obtained supercritical extract.

As a result, even when the test method was calibrated as a control, it showed a TSLP inhibitory effect of 80% or more of the hot water extract effect, but both betel and clove bark showed the best activity in the hot water extract.

Table 3 Comparison of TSLP Inhibitory Activity According to Various Extraction Solvents and Methods

Extraction Method Of Arecae Semen Extract
Inhibitory activity (%) Inhibitory Activity of Caryophylli Cortex Extract (%) Hydrothermal extraction 100 ± 16 100 ± 6 50% Ethanol Extract 89 ± 9 95 ± 12 50% methanol extraction 87 ± 10 92 ± 17 50% Hexane Extraction 95 ± 11 86 ± 9 Ultrasonic extraction 96 ± 12 94 ± 4 Supercritical Extraction 92 ± 22 92 ± 6

Example 4 Determination of Skin Irritability of Natural Extracts According to the Present Invention

Patch test was performed on 11 subjects in order to measure the degree of skin irritation in the Arecaemen and Caryophylli Cortex showing high TSLP expression inhibition effect in Example 2. The clean skin of the arm of 11 healthy volunteers was treated with 10% SLS (Sodium Lauryl Sulfate) solution as a positive control, pure water as a negative control, and the same amount of betel and clove bark hot water extracts with a concentration of 2 mg / ml were included. Patch was attached. After 48 hours, the patch was removed, washed with running water, and after 30 minutes the degree of irritation was determined.

Skin irritation was evaluated by dividing the skin irritation levels into five stages. The skin irritation of the TSLP inhibitory natural extracts was 4.5 in the negative control group and 70.5 in the positive control group, whereas it was 9.1 and 9 in the TSLP inhibitory natural extracts, It was confirmed by 6.8 that there is no skin irritation (Table 4 and Figure 4).

[Table 4] Skin irritation test results of TSLP inhibitory betel nut and clove bark

division Stimulation value (persons) Skin irritation
Average degree (%) pepper
Judgment 4 3 2 One 0 Negative Control (Water) 0 0 0 2 9 4.5 One Positive control group (10% SLS) One 7 3 0 0 70.5 4 Betel Extract 0 0 0 4 7 9.1 One Clove Bark Extract 0 0 0 3 8 6.8 One

* Stimulation values: 0 (no stimulation), 1 (very weak stimulation), 2 (weak stimulation), 3 (severe stimulation), 4 (very severe stimulation)

* Average skin irritation (%) =

* Irritation determination: 1 (less than 10, minimum stimulation), 2 (10-20, mild stimulation) 3 (20-30, normal stimulation), 4 (more than 30, severe stimulation)

Example 5 Atopic Dermatitis Improvement Effect

5-1. Confirmation of Atopic Dermatitis Improvement in Extracts of Betel Nuts and Clove Bark

In order to confirm the effect of atopic dermatitis, 13 of 17 adults and men with atopic dermatitis were applied with a mixed extract of betel nut and clove bark hot water extract in a 1: 1 ratio, and the other 4 were water as a control, 1 Twice a day, 1 ml or 1 g was applied to the affected area by moistening a cotton pad was tested for 8 days. After 8 days of application, the improvement effect was quantified in four stages: very effective, slightly effective, no effect, and worsened.

As a result, as shown in Table 5, 15 out of 17 people using betel and clove bark hot water extract showed a very effective and slightly improved effect, the ratio accounted for 88.2%, which is significant for atopic dermatitis It was confirmed that there is an effect. Most of the control regimens used were found to be ineffective, with a proportion of 60.0%.

[Table 5] Atopic Dermatitis Improvement Effects of Betel and Clove Bark Hot Water Extracts

Betel nut and clove bark hot water extract prescription Very effective 6 people 35.3% Slightly effect 9 people 52.9% no effect 2 people 11.8% worse 0 people 0.0% Control (water) prescription Very effective 0 people 0.0% Slightly effect 1 person 20.0% no effect 3 people 60.0% worse 1 person 20.0%

5-2. Confirmation of Atopic Dermatitis Improvement of Cosmetic Composition Containing Betel Nut and Clove Bark Hot Water Extract

In order to confirm the effect of atopic dermatitis, 13 of 17 adults and men with atopic dermatitis were coated with a cosmetic extract containing a mixed extract of betel nut hot water extract and clove bark hot water extract in a 1: 1 ratio, and the other 4 extracts The cosmetic composition containing no was used as a control, and was tested for 8 days by a method of applying it to the affected part by soaking in a cotton pad 1 ml or 1 g twice a day. After 8 days of application, the improvement effect was quantified in four stages: very effective, slightly effective, no effect, and worsened.

Table 6 shows the component ratios of the cosmetic compositions used in this example.

[Table 6] Cosmetic composition ratio for atopic dermatitis improvement test

Raw material name Example 5-2 Control Betel and Clove Bark Mixed Hot Water Extract 20.00 - Vegetable Curing Oil 10.00 10.00 Vaseline 10.00 10.00 glycerin 10.00 10.00 Monostearic acid polyoxyethylene sorbitan 0.10 0.10 Tocopheryl acetate 3.00 3.00 Butylene Glycol 1.00 1.00 Citric acid 0.10 0.10 Paraoxybenzoic acid ester, methyl paraben Quantity Quantity Spices Quantity Quantity Purified water Balance Balance

[Table 7] Atopic Dermatitis Improvement Effect of Cosmetic Composition Containing Betel Nuts and Clove Bark Hot Water Extract

Prescription cosmetic containing betel nut and clove bark hot water extract Very effective 4 people 23.5% Slightly effect 12 people 70.6% no effect 1 person 5.9% worse 0 people 0.0%
Comparative Cosmetic Prescription Very effective 0 people 0.0% Slightly effect 1 person 20.0% no effect 3 people 60.0% worse 1 person 20.0%

As a result, as shown in Table 7, 4 out of 17 people and 12 out of 17 people were found to be very effective, with 94.1% of the total improvement. In the subjects who showed a significant improvement in effectiveness, it was confirmed that atopic dermatitis with itching was generally calmed on the 4th day of application of TSLP-inhibited betel nuts and clove bark hydrothermal extract, and most lesions disappeared on the 8th day.

In the above, the cosmetic composition was confirmed to improve atopic dermatitis, but as shown in 5-1, because the betel hot water extract and the clove bark hot water extract have an excellent effect on atopic dermatitis, a pharmaceutical composition containing the same as an active ingredient is known. It will be apparent to those skilled in the art that the results prepared by containing the components of the pharmaceutical composition generally contained according to the method also have the same treatment or prophylactic effect of atopic dermatitis.

Having described the specific part of the present invention in detail, to those skilled in the art, such a specific description is only a preferred embodiment, whereby the scope of the present invention is not limited, it will be apparent. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 is a graph showing the TSLP expression reduction effect of the natural product betel nut hot water extract (No. 91).

2 is a graph showing the TSLP expression reduction effect of the natural clove bark hot water extract (No. 250).

Figure 3 shows a graph of 12 natural hot water extracts having a TSLP reducing effect.

Figure 4 is a photograph showing the skin irritant of betel nut and clove bark extract.

Claims (6)

Cosmetic composition having the ability to improve atopic dermatitis by TSLP (Thymic Stromal Lymphopoientin) inhibitory ability, containing betel nut hot water extract and clove bark hot water extract. According to claim 1, wherein the content of the betel nut hot water extract and clove bark hot water extract is a cosmetic composition, characterized in that 10 to 30 parts by weight based on 100 parts by weight of the total composition. The cosmetic composition according to claim 1, wherein the clove bark hydrothermal extract has a weight ratio of 0.3 to 3 times the betel nut hydrothermal extract. Pharmaceutical composition for the treatment or prevention of atopic dermatitis by TSLP (Thymic Stromal Lymphopoientin) inhibitory ability, containing betel nut hot water extract and clove bark hot water extract. The pharmaceutical composition according to claim 4, wherein the content of the betel nut hydrothermal extract and the clove bark hydrothermal extract is 10 to 30 parts by weight based on 100 parts by weight of the total composition. The pharmaceutical composition according to claim 4, wherein the clove bark hydrothermal extract has a weight ratio of 0.3 to 3 times the betel nut hydrothermal extract.

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