KR102267796B1 - Composition for Skin Soothing Comprising Exosomes Derived from Natural Extracts - Google Patents

Abstract

The present invention relates to a cosmetic composition for soothing the skin comprising natural product-based exosomes derived from antler as an active ingredient. The composition of the present invention can effectively soothe abnormal conditions of the skin (atopic dermatitis, inflammation, erythema, oxidation, cytotoxic substances in the skin, loss of moisture, freckles, itching, roughness, wrinkles, etc.) caused by various causes.

Description

A composition for skin soothing comprising exosomes derived from natural extracts as an active ingredient {Composition for Skin Soothing Comprising Exosomes Derived from Natural Extracts}

The present invention relates to a skin soothing composition comprising exosomes derived from natural extracts.

All cells secrete extracellular vesicles (EVs), an evolutionarily conserved phenomenon from bacteria to humans and plants. The extracellular endoplasmic reticulum enables the exchange of substances (proteins, lipids, genetic materials) between cells, and functions as a physiological/pathological mediator for signal transduction. The extracellular vesicles are largely classified into exosomes and microvesicles. The size of exosomes varies depending on the origin of the organism (human origin: 30-200 nm), and luminal vesicles are generated when the endosome membrane enters in the process of maturation of multi-vesicular endosomes. (intraluminal vesicles), which are secreted when multivesicular endosomes bind to the cell surface. Microvesicles are 50-1000 nm in size, and are vesicles from which the plasma membrane protrudes and separates and is secreted out of the cell. Each cell produces an extracellular ER differently according to the physiological state and secretes an extracellular ER with a specific lipid/protein/nucleic acid composition (Jaewook Lee (2019). A light on the cell biology of the extracellular ER. BRIC View 2019-R03 ).

Exosomes contain specific genetic material and bioactive factors according to the nature and state of the cell from which they are derived. That is, the exosomes contain various types of proteins, genetic materials (DNA, mRNA, miRNA), lipids, etc. derived from the cells. These exosomes have different characteristics from proteins and genetic materials present in biological fluids (blood, serum, lymph, etc.). Specifically, exosomes are composed of spherical particles surrounded by a lipid bilayer, and various cell-derived proteins exist on the surface of the exosomes, and cell-derived genetic materials exist inside. In particular, since there is an excess of RNA-degrading enzyme (RNase) in the serum, the genetic material secreted from the cell is easily degraded, whereas the genetic material present in the exosome is protected from RNA-degrading enzyme and is stably present and secreted. It may have an activity or therapeutic effect different from that of a cell or a culture of the cell.

Deer antler (鹿茸) refers to a medicinal material processed by collecting the unossified young antlers of various types of stags, such as flower deer and maroc, and is a weak child with slow growth or cartilage disease (軟骨).病), taking deer antlers not only promotes growth and strengthens the skeleton, but also develops intelligence and enhances the digestive and absorption capacity of the stomach. Deer antler contains a large amount of hormones and estrus hormones, so it is known that it has an excitatory effect on the decline of sexual function (Encyclopedia of National Culture of Korea, Central Research Institute of Korean Studies).

On the other hand, dermatitis (皮膚炎: dermatitis) refers to inflammation of the skin including eczema and is usually classified by type as follows (Doosan Encyclopedia):

(1) Atopic dermatitis: It is an eczema-shaped skin lesion that occurs in people with atopic constitution. It is also called intrinsic eczema or benier eczema. There is a genetic predisposition, but the cause is unknown. Unlike normal eczema or dermatitis, it shows specific symptoms and progress. 70-80% of childhood eczema is this eczema. Symptoms vary according to age, and are usually divided into three stages. ① Infancy (around 2 months to 3 years old): Redness, exudation, and snow fall on the face, especially the cheeks, and it is very itchy. When the symptoms worsen, the same changes and scabs appear on the head, and the skin all over the body becomes red and dripping. The entire skin becomes rough and becomes bluish-white. It occurs around 2-3 months after birth and heals well until 1 year of age, but there are cases where it repeats. In general, it tends to get worse in winter. ② Childhood (around 4-10 years old): From around 4-5 years old, papules and rashes appear on the extremities (especially in the flexion of the elbow and knee joints), and they fuse to form lichen. ③ Puberty (after 12 years old): In addition to the extremities, the face, chest, and nape of the neck are also lichenised. Children's asthma is often combined, and there are many cases of asthma or atopic dermatitis patients in the family. It is a disease that has a long course and does not heal well, so it is important to treat it with a relaxed mind and patiently. However, as the age increases, the disease becomes lighter. When the symptoms are severe, use an ointment (antihistamine ointment, vitamin A/vitamin D ointment), and use an ointment with anti-inflammatory drugs (止痒劑).

(2) Contact dermatitis (接觸性皮膚炎): Inflammation of the skin caused by contact with external substances. It is characterized by so-called itching of the skin. It has the same symptoms as acute eczema, but it is different from eczema in that it occurs as a reaction to a specific substance acting from the outside. Substances susceptible to disease include lacquer tree, sword lacquer tree, fig tree, and ginkgo tree, as well as paints, synthetic resin products, leather products, rubber products, chromium plating, pharmaceuticals, cosmetics, and underwear made of synthetic fibers. Remove contact with the causative agent, stop makeup on the face, and do not scratch the area. When symptoms are mild, apply zinc borate ointment. For internal use, antihistamines, corticosteroids, vitamin B2, vitamin B6, etc. are used. If it doesn't get better after 2-3 days or if the symptoms are severe, see a dermatologist.

(3) Seborrhoic dermatitis (脂漏性皮膚炎: seborrhoic dermatitis): It is a dermatitis that occurs frequently in areas with a lot of sebum secretion, such as the head, forehead, and armpits. It is also called seborrheic eczema. It is a dermatitis mainly caused by erythema and fine scale (鱗屑: dandruff). It occurs in the 20s and 40s. Unlike normal eczema, it is caused by constitution or abnormal sebum secretion. The lightest type is dandruff and ringworm. It is sensitive to sunlight or heat, and it often worsens in spring and autumn, and even if it gets better, it is easy to recur. Avoid greasy food and avoid irritating the head by scratching with nails. In addition to treatment similar to eczema, vitamin B2, vitamin B6, nicotinic acid amide, etc. are taken orally.

In the treatment of the dermatitis, it is common to prescribe steroids (eg, topical corticosteroids) and antihistamines. However, when topical corticosteroids are used for a long time, there is a problem that various skin side effects such as skin atrophy, vasodilation, loss of pigment and generation of dilated striatum are caused, and in the case of antihistamines, tolerance and hypersensitivity reactions are a problem.

To date, there is no report that antler-derived exosomes have an action to normalize or soothe skin disorders including dermatitis, and development of such natural products-derived cosmetics for skin soothing is urgently required.

The matters described as the above background art are only for improving the understanding of the background of the present invention, and should not be taken as acknowledging that they correspond to the prior art already known to those of ordinary skill in the art.

The present inventors have tried to find a natural product-derived substance capable of soothing abnormally changed skin conditions due to various causes such as genetic, environmental or aging. As a result, the present invention was completed by confirming that exosomes derived from antlers are very effective in soothing the skin.

Accordingly, an object of the present invention is to provide a composition for soothing the skin comprising an antler-derived exosome as an active ingredient.

Another object of the present invention is to provide a method for preparing a composition for soothing the skin comprising an antler-derived exosome as an active ingredient.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a composition for soothing skin comprising an antler-derived exosome as an active ingredient.

In order to develop natural cosmetics and/or pharmaceuticals, the present inventors extracted ingredients derived from deer antlers using various extraction methods and compared and observed their effects for a long period of time. It was found that the clinical efficacy of the antler is significantly different depending on the extraction method, and surprisingly, the present invention was completed by confirming that the exosome derived from the antler is very effective in soothing the skin.

As used herein, the term “exosome” refers to a vesicle structure of a lipid bilayer naturally secreted from various cells, including specific molecules possessed by cells derived from proteins, nucleic acids (DNA, mRNA, miRNA, etc.), lipids, carbohydrates, etc. It refers to vesicle particles that are released into the environment outside the cells of the body, and is used in the same sense as extracellular vesicles.

The exosomes or extracellular vesicles have various diameters within the range of about 30-1,000 nm, depending on the type of animal or cell secreting the exosomes, but the antler-derived exosomes are preferably 100-800 nm, Most preferably, it has a diameter of 120 to 600 nm.

According to an embodiment of the present invention, the antler-derived exosomes or extracellular vesicles included in the present invention were analyzed to have a diameter of about 120 to 600 nm (Fig. 2a).

In the exosome or extracellular vesicle, specific proteins, nucleic acids, lipids, and carbohydrates are surrounded by a structure of a lipid bilayer, protecting them from the action of various enzymes such as RNA degrading enzymes (RNase) and proteolytic enzymes present in blood, plasma, or lymph can be received, and thereby may contain a composition completely different from the components of the blood or lymph or cells present therein or cultures of these cells.

As used herein, the term "skin soothing" refers to abnormal skin conditions (atopic dermatitis, inflammation, erythema, oxidation, cytotoxic substances in the skin (eg, nitric oxide (NO) Accumulation), loss of moisture, freckles, itching, roughness, wrinkles, etc.) and returning to normal skin condition.

According to one embodiment of the present invention, the antler-derived exosomes or extracellular vesicles contained in the present invention effectively inhibit the expression of TARC (thymus and activation-regulated chemokine), which is a specific factor for atopic dermatitis, and TNF-α and In contrast to significantly inhibiting the amount of MDC overexpressed by IFN-γ, such an effect was hardly observed in the general extract of deer antler, confirming that it is very useful as an immunosuppressant (eg, atopic dermatitis) ( FIGS. 4 and FIG. 4 and FIG. 5).

According to another embodiment of the present invention, the antler-derived exosomes or extracellular vesicles included in the present invention are cytotoxic in all treatment groups compared to the LPS-treated control group, unlike the general extract of antler, nitric oxide (NO). showed the inhibition rate of the production (FIG. 6).

In addition, according to another embodiment of the present invention, in the antler-derived exosomes or extracellular vesicles included in the present invention, a significant COX-2 gene expression inhibitory effect was observed compared to the negative control group and the antler extract treated only with LPS. , it was confirmed that it was used for the treatment and prevention of inflammatory diseases, or was effective in soothing the skin (FIG. 7).

Additionally, according to another embodiment of the present invention, the antler-derived exosomes or extracellular vesicles included in the present invention are MMP-1, which is a cause of wrinkles caused by environmental changes such as dry skin or ultraviolet rays and aging. It was confirmed that it can be effectively used for preventing and improving skin wrinkles by effectively inhibiting the production of protein (FIG. 8).

When the composition of the present invention is prepared as a cosmetic composition, the composition of the present invention includes not only the above-described antler-derived exosomes or extracellular vesicles, but also ingredients commonly used in cosmetic compositions, such as antioxidants, stabilizers, dissolving agents conventional adjuvants such as topical agents, vitamins, pigments and fragrances, and carriers. In addition, the composition of the present invention may be used by mixing with a skin soothing agent that has been conventionally used in addition to the antler-derived exosomes or extracellular vesicles described above.

As the carrier, purified water, monohydric alcohols (ethanol or propyl alcohol), polyhydric alcohols (glycerol, 1,3-butyrene glycol or propylene glycol), higher fatty acids (palmitylic acid or linolenic acid), oils and fats (wheat germ oil, Camellia oil, jojoba oil, olive oil, squalene, sunflower oil, macadamia peanut oil, avogard oil, soybean hydrogenated lecithin or fatty acid glyceride) may be used, but is not limited thereto. In addition, surfactants, disinfectants, antioxidants, ultraviolet absorbers, anti-inflammatory agents and refreshing agents may be added as needed.

Surfactants include polyoxyethylene, hydrogenated castor oil, polyoxyethylene, oleyl ether, polyoxyethylene monooleate, polyoxyethylene, glyceryl monostearate, sorbitan monostearate, polyoxyethylene monooleate, sorbitan, sucrose Fatty acid ester, monolauric acid hexaglycerin, polyoxyethylene reduced lanolin, POE, glyceryl pyroglutamic acid, isostearic acid, diester, N- acetyl glutamine and may be selectively included in the group consisting of isostearyl ester.

The fungicide may optionally include hinocthiol, tricroic acid, crohexidine gluconate, phenoxyethanol, resorcin, isopropylmethylphenol, azulene, salicylic acid and zinc pyritaone. .

As the antioxidant, any of butylhydroxyanisole, gallic acid, propyl gallate and erythorbic acid can be used.

UV absorbers include benzophenones such as dihydroxybenzophenone, melanin, ethyl para-aminobenzoate, para-dimethylaminobenzoic acid 2-ethylhexyl ester, synoxite, para-methoxycinnamic acid 2-ethylhexyl ester, 2-(2-hydroxyl) Any of hydroxy-5-methylphenyl) benzotriazole, urokanic acid and metal oxide fine particles can be used.

As an anti-inflammatory agent, dipotassium glitinate or allantoin may be used, and as a cooling agent, red pepper tincture or 1-menthol may be used.

The formulation of the composition is any formulation capable of blending antler-derived exosomes or extracellular vesicles as an active ingredient, and in the form of cosmetics, powder, gel, cream, essence, lotion, sol-gel, emulsion, oil , wax, spray, mist, etc. may be prepared in various forms, but is not limited thereto. In addition, it may be prepared in the form of a mask pack containing an antler-derived exosome or extracellular vesicle or a composition comprising the same.

According to another aspect of the present invention, the present invention provides a food composition or a pharmaceutical composition comprising the antler-derived exosome.

When the composition of the present invention is prepared as a food composition, it includes not only the antler-derived exosomes or extracellular vesicles as an active ingredient, but also ingredients commonly added during food preparation, for example, proteins, carbohydrates, fats, Nutrients, seasonings and flavoring agents. Examples of the above-mentioned carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents (taumatine, stevia extract [eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

For example, when the food composition of the present invention is prepared as a drink, in addition to the antler-derived exosomes or extracellular vesicles of the present invention, citric acid, high fructose, sugar, glucose, acetic acid, malic acid, fruit juice, Euclidean extract, jujube extract, licorice extract, etc. may be additionally included.

The food composition of the present invention has a very good action in soothing the skin. Moreover, the composition of the present invention is very safe for the human body because it uses a natural product that has already been proven in biosafety as an active ingredient.

According to a preferred embodiment of the present invention, the pharmaceutical composition of the present invention is used for preventing or treating skin inflammatory diseases, atopic dermatitis, or skin wrinkles.

When the present invention is used in the pharmaceutical composition, the pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is commonly used in formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, Gum Acacia, Calcium Phosphate, Alginates, Gelatin, Calcium Silicate, Microcrystalline Cellulose, Polyvinylpyrrolidone, Cellulose, Water, Syrup, Methyl Cellulose, Methylhydroxybenzoate, Propylhydroxybenzoate, Talc, Stearic Acid Magnesium and mineral oil, and the like, but are not limited thereto. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by nasal administration, eye drop administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, and the like.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration mode, age, weight, sex, morbidity, food, administration time, administration route, excretion rate and response sensitivity of the patient, An ordinarily skilled physician can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.001-100 mg/kg.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

The pharmaceutical composition of the present invention may be prepared in the form of external preparations for skin, aerosols, sprays, eye drops, oral preparations, medicinal needles and injections.

When the pharmaceutical composition of the present invention is prepared as an injection, the mixing ratio of the antler-derived exosomes or extracellular vesicles of the present invention can also be appropriately selected according to the type and the type, amount, form, etc. of other components to be mixed, In general, with respect to the total amount of the injection, 0.01 to 99% by weight, preferably 0.1 to 30% by weight of the antler-derived exosome or extracellular vesicles of the present invention may be included.

In the injection of the present invention, other various other ingredients may be blended within a range that does not impair the desired effect of the present invention. As other components, for example, water for injection, NaCl, NaOH, glucose, other active ingredients other than the antler-derived exosomes or extracellular vesicles of the present invention, etc. can be appropriately selected and formulated by selecting one or two or more types. .

As used herein, the terms “cosmetic composition”, “food composition” and “pharmaceutical composition” are used in the meaning of including applications for animals including livestock and pets as well as for humans.

According to another aspect of the present invention, the present invention provides a method for preparing a skin soothing composition comprising an antler-derived exosome as an active ingredient, comprising the following steps:

a) washing and grinding the antler to obtain a powder;

b) preparing a solution by dissolving the powder in an exosome extraction buffer; and

c) separating the antler-derived exosomes from the solution.

The exosome extraction buffer available in the present invention means a buffer capable of preparing a solution by dissolving the exosome powder, EXO-BB, ExoQuick®-ULTRA, ExoQuick®-TC, Capturem™ Exosome Isolation Kit, Various exosome isolation kits existing in the art, such as Total Exosome Isolation Kit, ExoTrap™ Exosome Isolation Spin Column Kit, and Exo2D™, may be used, but are not limited thereto.

As used herein, the term “isolation” refers to selectively obtaining a desired substance (eg, exosome) in a biological sample (eg, a solution in which antler powder is dissolved in an exosome extraction buffer). It includes both the process of selectively removing impurities other than the target material (negative isolation) as well as the process (positive isolation). Therefore, the term “separation” may be used synonymously with “obtain”, “extract”, and “purify”. In the process of isolating exosomes in the present invention, any method commonly used in the art may be used without limitation, for example, utilizing the commercialized exosome separation kit, or using the specific gravity difference between components in the solution. separation (e.g., centrifugation), separation by size (e.g., ultrafiltration or vacuum filter), separation based on affinity for a particular substrate (e.g., affinity chromatography) Any method commonly used in the art may be used without limitation as a separation method based on the intrinsic properties of the target material in a non-homogeneous sample.

The composition (cosmetic composition, food composition, pharmaceutical composition) prepared by the method of the present invention, unlike the general extract of antler, is overexpressed by TARC expression, COX-2 gene expression, and TNF-α and IFN-γ. Effectively suppresses inflammation including atopic dermatitis by significantly suppressing the amount of MDC produced, and effectively soothes abnormal skin conditions changed due to genetic, environmental or aging reasons by inhibiting the production of cytotoxic nitric oxide can do it

The features and advantages of the present invention are summarized as follows:

(i) The present invention provides a cosmetic composition for soothing the skin comprising an antler-derived exosome, which is a natural product based, as an active ingredient.

(ii) The composition of the present invention can effectively calm abnormal conditions of the skin caused by various causes (atopic dermatitis, inflammation, erythema, oxidation, cytotoxic substances in the skin, loss of moisture, spots, itchiness, roughness, wrinkles, etc.) have.

1 is a result of confirming the antler-derived exosomes included in the present invention by analyzing the content of CD9 (FIG. 1a), CD63 (FIG. 1b), and CD81 (FIG. 1c) proteins.
2 is a result of analyzing the diameter of exosomes derived from antlers (FIG. 2a) and ear plate (FIG. 2b) using a transmission electron microscope and DLS.
3 is a result confirming the effect on the cytotoxicity of the antler-derived exosomes.
4 is a result confirming the anti-atopic efficacy (inhibition of TARC production overexpressed by TNF-α and IFN-γ) of the antler-derived exosomes.
5 is a result confirming the anti-atopic efficacy (inhibition of MDC production amount overexpressed by TTNF-α and IFN-γ) of the antler-derived exosomes.
6 is a result confirming the nitric oxide production inhibitory effect of antler-derived exosomes.
7 is a result confirming the anti-inflammatory effect of the antler-derived exosomes.
8 is a result confirming the anti-wrinkle effect of antler-derived exosomes.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

1. Preparation of antler-derived exosomes (Experimental Example 1)

10 g of deer antler (Hans Pharmaceutical Co., Ltd., Korea) was washed with purified water, mixed and dried to prepare a mixed sample. The prepared mixed sample was pulverized and refined to obtain a powder. After adding a 10-fold volume of EXO-BB buffer (system biosciences, Palo Alto, CA, USA) to the dry powder, it was dissolved by vortexing for 5 minutes. The dissolved solution was placed in a 2 mL collection tube, loaded in a filter cartridge, and centrifuged for 2 minutes at 18,000 G conditions. The down layer was transferred to a microfuge tube with a capacity of 1.5 mL, taking care not to separate the pellet from the centrifuged tube. The exosome separation solution was added to the microtube in double volume, and incubated at 4° C. for 48 hours to increase the exosome yield, followed by centrifugation at 10,000 G for 60 minutes. The supernatant was removed from the centrifuged tube to separate exosome components derived from antlers in the form of pellets. The exosome pellet was re-dissolved in EXO-BB buffer using a sonicator and stored at 4° C. for subsequent experiments.

2. Preparation of deer antler extract (Experimental Example 2)

An antler extract was prepared as a comparison group for comparing the activity with the antler-derived exosomes. 100 g of deer antler (Hans Pharmaceutical Co., Ltd., Korea) was washed with purified water, mixed and dried to prepare a mixed sample. The prepared mixed sample was pulverized and refined to obtain a powder. 1 L of purified water was used as a solvent in the dry powder, followed by heating and reflux extraction for 12 hours, followed by cooling, followed by filtration with filter paper having a permeation size of 1.2 μm to obtain a filtrate. The obtained filtrate was concentrated under reduced pressure and dried under reduced pressure to prepare a dry powder of deer antler extract.

3. Identification of antler-derived exosomes

1) Identification using exosome markers

The antler-derived exosome component isolated in Experimental Example 1 was analyzed using an enzyme-linked immunosorbent assay (ELISA, Enzyme-Linked Immunosorbent Assay) kit (system biosciences, Palo Alto, CA, USA) present in the antler-derived exosome. The content of CD9, CD63, and CD81 proteins was analyzed. The CD9, CD63, and CD81 proteins are representative exosome markers. For a comparative experiment, gupan extract (sample 1) prepared by extracting and processing in the same manner as in Experimental Example 1, using animal-derived herbal medicines gupan (Daon Co., Ltd., Korea) and musk (Seosa-hyang Co., Ltd., Korea) as raw materials. , musk extract (sample 2) was used as a control. As a result, it was confirmed that the antler-derived exosomes prepared in Experimental Example 1 contained significantly more exosome markers compared to the gupan and musk extracts (samples 1 to 2), and through this, it was confirmed that Compared to that, it was confirmed that the antler, a raw material of the present invention, contained significantly more exosome components (Fig. 1a: CD9, Fig. 1b: CD63, and Fig. 1c: CD81).

2) Confirmation using a transmission electron microscope

The antler-derived exosome of Experimental Example 1 and the spherical plate-derived exosome component of Sample 1 were analyzed using a transmission electron microscope and dynamic light scattering (DLS). As a result of analyzing the shape and size of exosomes derived from antler and ear plate, they are generally spherical and have a diameter of about 60 to 600 nm It was confirmed that the size was between (Fig. 2a: antler-derived exosome; Fig. 2b: ear plate-derived exosome) .

4. Confirmation of effect on cytotoxicity of antler-derived exosomes

The effect of the antler-derived exosome prepared in Experimental Example 1 and the antler extract prepared in Experimental Example 2 on the growth of human keratinocytes (HaCaT, Human immortalized keratinocyte, ATCC) was measured using MTT colorimetric assay. to confirm. First, using DMEM (Dulbeccos modified Eagles medium, GIBCO) medium containing 10% FBS (fetal bovine serum, Cambrex), human keratinocytes were inoculated at a concentration of ^{1×10 5 in a 24-well cell culture dish.} After that, it was cultured at 37° C., 5% CO _{2 wet conditions for 24 hours.} Thereafter, the medium was removed and the lactic acid bacteria fermentation thyme extract prepared in Example 1 diluted with serum-free DMEM medium was treated at different concentrations (10 and 100 μg/ml) and cultured for 24 hours. After 24 hours, 1 mg/ml of MTT reagent was treated, and 2 hours later, formazan generated in cells due to MTT treatment was dissolved in DMSO and absorbance was measured at 570 nm. As a positive control, tacrolimus (1 and 10 μg/ml) was treated. As a result, it was confirmed that the antler extract and the antler-derived exosome did not significantly affect the cell viability of human keratinocytes, and it was confirmed that the antler extract and the antler-derived exosome did not show toxicity (Fig. 3).

5. Confirmation of anti-atopic efficacy of antler-derived exosomes

After culturing a skin keratinocyte line (HaCaT, 5×10 ⁵ /well) in FBS DMEM medium for 24 hours, the antler-derived exosomes prepared in Experimental Example 1 and the antler extract prepared in Experimental Example 2 were added to DMEM without FBS. % and 10% (v/v) concentrations were diluted with TNF-α and IFN-γ (10 ng/ml), respectively, and co-treated, and further cultured. After 24 hours, the supernatant of each well was quantified using a human TARC ELISA kit (R&D system) and a human MDC ELISA kit (R&D system). The group treated with the same amount of distilled water was used as a negative control group, and the group treated with tacrolimus was used as a positive control group. The tacrolimus is known to inhibit phosphatase activity inherent in calcineurin as an inhibitor of calcineurin (Sieber, M., Karanik, M., Brandt, C., Blex, C., et al., 2007, Inhibition of calcineurin NFAT signaling by the pyrazolopyrimidine compound NCI3., European Journal of Immunology , 37, 2617-2626), recently, administration of tacrolimus to atopic patients resistant to corticosteroid treatment Studies have shown that it is effective (Russell, JJ, 2002, Topical tacrolimus: a new therapy for atopic dermatitis. American Family Physician , 66, 1899-1902). Referring to FIG. 4 , it was confirmed that the TARC production amount overexpressed by TNF-α and IFN-γ was significantly inhibited by treatment with antler-derived exosomes, and the test group treated with 10% (v/v) concentration. It was confirmed that there was a significantly improved effect compared to the tacrolimus-treated positive control group. From the above results, it can be confirmed that the exosome component derived from antler of the present invention can prevent and effectively improve atopic dermatitis by inhibiting the expression of TARC (thymus and activation-regulated chemokine), which is a specific factor for atopic dermatitis. could In addition, referring to FIG. 5 , it could be confirmed that the amount of MDC overexpressed by TNF-α and IFN-γ was significantly inhibited by treatment with antler-derived exosomes, and treated with 10% (v/v) concentration. In the test group, it was confirmed that there was a significantly improved effect compared to the tacrolimus treatment positive control group. It was confirmed that the antler-derived exosome of the present invention has an excellent effect compared to the conventional immunosuppressant tacrolimus compound used for atopy, and thus can be used as a natural medicine to replace it.

6. Confirmation of the inhibitory efficacy of antler-derived exosomes on nitric oxide production

In order to confirm the anti-inflammatory effect of the antler-derived exosome of the present invention, the inhibition rate of the production of nitric oxide (NO), which is a representative cytotoxic substance involved in induction of inflammation, was measured. First, RAW 264.7 cells, a macrophage cell line, were purchased from the Korea Cell Line Bank (KTCC, Seoul, Korea) and used. Specifically, a medium in which 10% FBS, 100 μg/ml penicillin and 100 μg/ml streptomycin was added to DMEM (Dulbecco's modified Eagle's medium) containing 10% Fetal bovine serum (FBS) was used, and the cells were inoculated. and incubated at 37° C., 5% CO _{2 conditions.} Specifically, the RAW 264.7 cells ^{were inoculated into a 96-well plate at a concentration of 1×10 6} cells/mL (in DMEM) and cultured for 24 hours, in the exosomes derived from deer antlers prepared in Experimental Example 1 and Experimental Example 2 The prepared antler extract was treated with a sample diluted to 1% and 10% (v/v) concentrations and a fresh medium containing LPS (1 μg/ml) known as an endotoxin, and cultured for 24 hours. Then, 100 μl of the cell culture supernatant and 100 μl of Griess reagent [1% (w/v) sulfanilamide, 0.1% (w/v) naphtylethylenediamine in 2.5% (v/v) phosphoric acid] were mixed, After reacting for a minute, the amount of nitric oxide produced was measured by measuring absorbance at 540 nm using an ELISA reader. The concentration of the produced nitrite (nitrite) was calculated using a standard curve in which sodium nitrite (sodium nitrite) was dissolved in DMEM medium. Based on the difference in the amount of nitrite produced in the control group treated with LPS (Cont) and the control group not treated with LPS (Nor), the nitric oxide production inhibitory activity of each sample was confirmed. As a result, the antler-derived exosomes of the present invention were observed to exhibit excellent anti-inflammatory effects by inhibiting nitric oxide production in all treated groups compared to the LPS-treated control group (Cont), unlike the antler extract (FIG. 6).

7. Confirmation of anti-inflammatory efficacy of antler-derived exosomes

RAW 264.7 cells, a macrophage cell line, were purchased from the Korea Cell Line Bank (KTCC, Seoul, Korea) and used in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS (Fetal bovine serum), 10% FBS, 100 μg/ml The cells were inoculated in a medium supplemented with penicillin and 100 μg/ml streptomycin, and cultured at _{37° C., 5% CO 2 condition.} Specifically, the RAW 264.7 cells ^{were inoculated in a 6-well plate at a concentration (in DMEM) of 1×10 6} cells/ml and cultured for 24 hours, in the exosomes derived from deer antlers prepared in Experimental Example 1 and Experimental Example 2 The prepared deer antler extract was treated with a sample diluted to 1% and 10% (v/v) concentrations and a fresh medium containing LPS (1 μg/ml) known as an endotoxin, and cultured for 24 hours. The experiment confirmed the mRNA expression level of the COX-2 gene using the RT-PCR method. The primers used were as follows:

COX-2: Forward 5' -AAG ACT TGC CAG GCT GAA CT-3', Reverse 5'-CTT CTG CAG TCC AGG TTC AA-3'

GAPDH: Forward 5'-TGA AGG TCG GTG TGA ACG GAT TTT GGC-3', Reverse 5'-TGG TTC ACA CCC ATC ACA AAC ATG G-3'.

RT-PCR was performed using the primers as described above, and the experimental results are summarized in FIG. 7 . As a result, in the antler-derived exosome-treated group of the present invention, a significant COX-2 gene expression inhibitory effect was observed compared to the negative control group and the antler extract treated only with LPS, and used for treatment and prevention of inflammatory diseases, or skin It was confirmed that it had a sedative effect.

8. Confirmation of anti-wrinkle effect of antler-derived exosomes

Healthy young male volunteers were selected and skin biopsied to obtain normal human dermal fibroblasts (NHDFs). Cells were plated in 100 mm tissue culture plates and cultured in DMEM medium supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin in a humid environment containing 5% CO2 at 37°C. All experiments were performed using only cells between passages 6 and 10. The normal human dermal fibroblasts were seeded in 40 mm tissue culture plates (1.2×10 ⁵ cells). Normal human dermal fibroblasts were cultured until 80% confluence was reached, washed twice with phosphate buffered saline (PBS), and 1980 μl of fresh serum-free medium and 20 μl of sample were added to each well. , and cultured for 3 days. Thereafter, the culture medium was harvested, centrifuged at 4°C, 7,500 rpm for 5 minutes, and MMP-1 (Human Total MMP-1 kit, R&D Systems, Inc., Minneapolis, MN, USA) using an ELISA method. ) of the protein expression level was confirmed. The expression of MMP-1 protein showed a marked decrease by the treatment with exosomes derived from deer antler of Experimental Example 1. Specifically, the antler-derived exosome-treated group exhibited 41%, 45%, and 47% improved MMP-1 protein production inhibitory effects at 0.01%, 0.1%, and 1% doses, respectively, compared to the untreated group (Fig. 8). ). Through these results, it was confirmed that the exosome derived from antler of the present invention can be effectively used for preventing and improving skin wrinkles by effectively inhibiting the production of MMP-1 protein, which is the cause of wrinkles.

manufacturing example

Preparation Example 1: Preparation of cosmetics

Deer antler-derived exosome: 2 ml, 1,3-butylene glycol: 7.0 mg, glycerin: 1.0 mg, polysorbate 60: 1.5 mg, lipophilic glyceryl stearate: 2.0 mg, mineral oil: 4.0 mg, cetea Reel alcohol: 3.0 mg, a small amount of preservative, an appropriate amount of combined fragrance, and purified water (added to make a total of 100 mg) according to the composition of the cosmetic was prepared in a conventional manner.

Preparation Example 2: Preparation of pharmaceuticals

Additives such as NaCl and NaOH and 900 ml of water for injection to 2 ml of the antler-derived exosomes were used to prepare a pH 7.0, 0.9% isotonic solution, followed by filtering 3 times with a PES 0.1 filter. The required vial bottle, rubber stopper and aluminum cap were sterilized, and 20 ml each was divided into vial bottles, capped with rubber stoppers and aluminum caps, and then put into an autoclave and autoclaved. After performing a foreign material test through a foreign material tester, microbiological test and endotoxin test, they were labeled and stored in a refrigerator.

Preparation Example 3: Preparation of granules

To 2 ml of the antler-derived exosome, an appropriate amount of a vitamin mixture, 10 μg of biotin, 1.7 mg of nicotinic acid amide, 50 μg of folic acid, 0.5 mg of calcium pantothenate, an appropriate amount of a mineral mixture, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate , 15 mg of potassium phosphate monobasic, 55 mg of dibasic calcium phosphate, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride were mixed according to a conventional health functional food manufacturing method to prepare a health functional food.

Preparation Example 4: Preparation of a mask pack

0.05% by weight of the antler-derived exosome, 5.0% by weight of glycerin, 4.0% by weight of propylene glycol, 15.0% by weight of polyvinyl alcohol, 8.0% by weight of ethanol, 1.0% by weight of polyoxyethylene oleyl ethyl, 0.2% by weight of methyl paraoxybenzoate A mask pack was prepared by mixing the appropriate amount of , dye, and fragrance according to a conventional mask pack manufacturing method.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (5)

A cosmetic composition for soothing the skin comprising an antler-derived exosome as an active ingredient, wherein the diameter of the exosome is 120 to 600 nm, and the skin soothing is inflammation and wrinkle improvement.
The composition according to claim 1, wherein the formulation of the composition is in the form of a gel, cream, essence, lotion, sol-gel, emulsion, oil, wax, spray or mist.
A mask pack comprising the cosmetic composition of claim 1.
A food composition for skin soothing comprising antler-derived exosomes as an active ingredient, wherein the diameter of the exosomes is 120 to 600 nm, and the skin soothing is inflammation and wrinkle improvement.
A pharmaceutical composition for skin soothing comprising an antler-derived exosome as an active ingredient, wherein the exosome has a diameter of 120 to 600 nm, and the skin soothing is inflammation and wrinkle improvement.

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