KR102248258B1 - Composition for skin improvement comprising green tea plant cell-derived exosomes as an active ingredient - Google Patents

Abstract

The present invention relates to a cosmetic composition for skin aging prevention, skin protection, and skin condition improvement comprising green tea plant cell-derived exosomes as an active ingredient. The composition of the present invention is safe for skin application, is effective in preventing skin aging and improving skin conditions, can protect the skin by strengthening the skin barrier, and has excellent wound healing effects, thereby being provided as functional cosmetics, pharmaceuticals, quasi-drugs, etc. derived from natural products.

Description

Composition for skin improvement comprising green tea plant cell-derived exosomes as an active ingredient}

The present invention relates to a cosmetic composition for suppressing skin aging and improving skin, including exosomes derived from green tea plant cells as an active ingredient.

The main causes of skin aging are increased age and factors such as ultraviolet rays, fine dust, and stress. Skin aging is divided into intrinsic, chronological aging according to time-dependent natural aging and extrinsic aging due to external factors, and the representative of exogenous aging is photoaging due to ultraviolet rays. As the cause of endogenous aging, an increase in age causes the action of fibroblasts and the number of cells to decrease, and as a result, the amount of synthesis of fiber components decreases and the structure becomes loose, resulting in a decrease in elasticity. In addition, loss of moisture in skin cells causes changes in the structure of the stratum corneum, which causes aging.

On the other hand, photoaging is caused by ultraviolet rays, and an increase in the generation of reactive oxygen species caused by ultraviolet rays induces the expression of MMPs (Matrix metalloproteinases) that promote collagen degradation and decreases the number of cells, thereby reducing skin elasticity and other aging effects. cause.

The skin consists of the epidermis, the dermis and the subcutaneous tissue. The epidermis, the outer layer of the skin, is composed of stratified squamous epithelium, and acts as a protective barrier for the body against the external environment. The epidermis is divided into stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale from the outside. The dermis is a layer between the epidermis and subcutaneous tissue, and is divided into a papillary dermis and a reticular dermis. Blood vessels, collagen, elastin fibers, pores, hair roots, sebaceous glands, sweat glands, and various other Sensory nerves, fibroblasts and macrophages are present and occupy the most part of the skin.

Collagen (Type I, II, III, IV, V), which is important for skin elasticity, is synthesized in the form of a precursor (pro-collagen), and is a major structural protein present in 80 to 90% of the dermal layer. Collagen is known to play the most important role in skin regeneration, skin moisture content, wound healing and wrinkle improvement, and it has the function of preventing skin damage from UV rays and containing a large amount of moisture, so it plays a role in supplying moisture to the dermis. It is an essential element in preventing wrinkles and maintaining elastic skin. In addition, when a wound occurs, the continuous collagen production of fibroblasts fills the wound area and acts as a wound healing.

On the other hand, exosomes are vesicles having a size of tens to hundreds of nanometers consisting of a double phospholipid membrane that is identical to the structure of the cell membrane. As a signaling medium secreted by cells, various cellular signals transmitted through it are activated, grown, and migrated to target cells. , Differentiation, dedifferentiation, apoptosis, and necrosis. In addition, effects such as wound healing and skin improvement of exosomes isolated from adult stem cells are known.

Exosomes contain specific genetic materials and bioactive factors depending on the nature and state of the derived cells. Exosomes isolated from stem cells of animals can exhibit effects other than the intended purpose, and exosomes There is a problem of stability in that stem cells can be included in the separation of the cells. However, the stability problem of exosomes derived from animal cells can be solved by using exosomes of plant cells. Accordingly, research on exosomes of plant cells is actively progressing, but it is still insufficient.

The present inventors completed the present invention by inducing green tea plant cells and separating exosomes therefrom to study various activities of green tea plant cells-derived exosomes.

KR 10-2018-0062854

The technical task of the present invention is to provide a composition for inhibiting skin aging, improving skin, strengthening skin barriers, improving scars, and treating wounds, including exosomes derived from green tea plant cells.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention provides a cosmetic composition for inhibiting or improving skin aging, comprising exosomes derived from green tea plant cells as an active ingredient.

As an embodiment of the present invention, the skin aging is endogenous aging and/or exogenous aging, and may include photoaging by ultraviolet rays.

In another embodiment of the present invention, the composition may suppress or improve photoaging by reducing apoptosis due to ultraviolet rays, and suppress or improve thermal aging by lowering the skin temperature.

In another embodiment of the present invention, the composition may increase cell survival and/or proliferation by enhancing telomerase reverse transcriptase (TERT) expression.

As another embodiment of the present invention, the composition may be to inhibit or improve skin aging due to environmental stress by enhancing the expression of a late embryogenesis abundant (LEA) protein that increases the resistance of cells to environmental stress, the Environmental stress can be ultraviolet light, dust, yellow dust, wind, and/or temperature (heat).

In addition, the present invention provides a cosmetic composition for improving skin, comprising an exosome derived from green tea plant cells as an active ingredient.

As an embodiment of the present invention, the skin improvement may be enhancing skin elasticity, moisturizing, whitening, soothing skin, reducing pores, reducing sebum secretion, and/or improving acne.

As another embodiment of the present invention, the composition may be one to reduce the activity of tyrosinase (tyrosinase) that contributes to the formation of melanin.

As another embodiment of the present invention, the composition may be to increase collagen synthesis in human fibroblasts.

In another embodiment of the present baldeo, the composition may be one to increase the expression of Aquaporin-3 and/or filaggrin.

In addition, the present invention provides a composition for enhancing skin barrier or improving scars, comprising exosomes derived from green tea plant cells as an active ingredient.

As an embodiment of the present invention, the composition may strengthen the skin barrier by increasing the expression of filaggrin.

In addition, the present invention provides a pharmaceutical composition for treating wounds comprising exosomes derived from green tea plant cells as an active ingredient.

In addition, the present invention provides a method for treating wounds or scars comprising administering to an individual an exosome derived from green tea plant cells.

In addition, the present invention provides the use of green tea plant cell-derived exosomes for the manufacture of a drug for treating wounds or scars.

The present invention contains exosomes derived from green tea plant cells as an active ingredient, is safe for skin application, is effective in preventing skin aging and improving skin conditions, and has excellent skin barrier reinforcement and wound healing effects, such as natural product-derived functional cosmetics, pharmaceuticals, quasi-drugs, etc. Can be provided.

1 is a diagram showing the results of a wound healing assay for confirming the wound healing effect of exosomes derived from green tea plant cells.

The present inventors solved the safety problem of exosomes derived from animal cells and searched for a substance that is more effective in improving skin condition.As a result, using exosomes derived from green tea plant cells, preclinical skin-related effects such as skin aging inhibition and skin condition improvement The present invention was completed by performing evaluation and confirming the effect.

Specifically, the present inventors induce green tea plant cells by wounding and culturing green tea leaves, subculture in a solid state, and then liquid culture to prepare a green tea plant cell culture solution, and extract exosomes by centrifuging the culture solution, The effect of the green tea plant cell-derived exosomes on the skin was confirmed (see Examples 1 and 2).

First, in order to confirm the stability of the green tea plant cell-derived exosomes, the present inventors treated the green tea plant cell-derived exosomes by concentration in keratinocytes and performed an MTT assay. As a result, it was confirmed that the exosomes derived from green tea plant cells did not show toxicity at all concentrations. This means that the green tea plant cell-derived exosomes have a very low possibility of causing skin irritation and are safe for application to cosmetic compositions (see Experimental Example 1).

Next, the present inventors tried to confirm the skin protective effect of the green tea plant cell-derived exosomes. Ultraviolet light activates MPPs and induces collagen degradation as well as apoptosis. Thus, after irradiating keratinocytes with ultraviolet rays, which cause skin aging, and treating exosomes derived from green tea plant cells by concentration, cell viability was confirmed. As a result, it was confirmed that the cell viability was increased depending on the concentration of the green tea plant cell-derived exosome treatment. From this, it means that the exosomes derived from green tea plant cells can reduce apoptosis due to ultraviolet rays by protecting cells from ultraviolet rays (see Experimental Example 2).

Meanwhile, melanin is a pigment that causes spots and freckles, and tyrosinase contributes to the formation of melanin pigments. In order to confirm the whitening effect of exosomes derived from green tea plant cells, the present inventors confirmed the inhibitory effect of tyrosinase activity of exosomes derived from green tea plant cells. As a result, it was found that the plant cell-derived exosomes have a whitening effect by effectively inhibiting the activity of tyrosinase in a concentration-dependent manner (see Experimental Example 3).

In addition, the present inventors confirmed that the green tea plant cell-derived exosomes induce the biosynthesis of collagen, the main structural protein of the dermal layer, and found that the green tea plant cell-derived exosomes increased the amount of collagen synthesis in a concentration-dependent manner (Experimental See example 4). From this, it can be seen that exosomes derived from green tea plant cells can contribute to enhancing skin elasticity and improving wrinkles. On the other hand, the increase in the production of collagen in the wound area is helpful in wound healing and scar improvement.

On the other hand, aquaporin (AQP) is a membrane protein that actively transports water molecules into cells, and among them, aquaporin-3 is a protein that selectively passes water and glycerol, and plays an important role in moisturizing the skin and healing wounds. And, telomerase reverse transcriptase (TERT) is involved in maintaining the length of telomeres and is associated with cellular senescence. In addition, filaggrin is a keratin-binding protein of the epidermis that is involved in the binding of membrane proteins of keratinocytes, contributing to the maintenance and strengthening of the skin barrier, and performing a moisturizing function.

The present inventors confirmed the effect of green tea plant cell-derived exosomes on the expression of AQP, TERT, and filaggrin through specific experiments, and confirmed that green tea plant cell-derived exosomes increased the expression of the three genes in a concentration-dependent manner. From this, it can be seen that exosomes derived from green tea plant cells have skin moisturizing, skin aging prevention, and skin barrier strengthening effects (see Experimental Example 5).

The late embryogenesis abundant (LEA) protein protects cells from external environmental stress. As the expression of the LEA protein increases, the cells are more resistant to environmental stress. Through specific experiments, the present inventors were able to confirm that the concentration-dependent treatment of green tea plant cells increased the expression level of the LEA protein, thereby enhancing the resistance of cells to environmental stress (see Experimental Example 6).

In addition, the present inventors performed a wound healing assay to confirm the effect of green tea plant cell-derived exosomes on wound healing in addition to enhancing collagen production. As a result, it was found that the exosomes derived from green tea plant cells were effective in wound healing by promoting cell proliferation and migration (see Experimental Example 7).

On the other hand, when the temperature of the skin rises, moisture evaporates due to heat, thereby reducing collagen, and increasing MMPs that decompose collagen, resulting in thermal aging, which decreases the elasticity of the skin. The present inventors produced a lotion by including exosomes derived from green tea plant cells at different concentrations, and applied them to the skin to confirm the cooling effect.As a result, they showed a skin soothing effect in a concentration-dependent manner of exosomes derived from green tea plant cells. It was found that moth has an effect of preventing heat aging (see Experimental Example 9).

In addition, the present inventors tried to confirm the pore shrinking effect through the degree of hemoglobin precipitation by mixing green tea plant cell-derived exosomes and hemoglobin. As a result, it was found that the precipitation of hemoglobin was increased depending on the concentration of the green tea plant cell-derived exosomes, and through this, it was found that the green tea plant cell-derived exosomes had an excellent pore-constricting effect (see Experimental Example 10).

In addition, the present inventors confirmed that the results of the panel test using the lotion of the thermal aging prevention effect confirmation experiment showed that the exosomes derived from green tea plant cells had the effect of suppressing sebum and improving acne (see Experimental Examples 11 and 12).

From the above, the present inventors can provide a composition comprising exosomes derived from green tea plant cells as an active ingredient for use in inhibiting or improving skin aging, improving skin conditions, strengthening skin barriers, improving scars, and treating wounds. .

In the present invention, skin aging includes endogenous aging due to time-dependent natural aging and exogenous aging due to ultraviolet rays, wind, temperature, pollution (yellow sand, fine dust, etc.), and ultraviolet rays, and endogenous aging decreases skin elasticity and reduces wrinkles caused by it. Includes.

In addition, the improvement of skin condition in the present invention includes improving skin elasticity, whitening, moisturizing, soothing skin by cooling effect, reducing pores, reducing sebum secretion, and improving acne.

The exosomes derived from green tea plant cells of the present invention can protect cells from ultraviolet rays, increase the expression of LEA protein, and increase the cell's own resistance to environmental stress, and increase collagen production and increase the expression of TERT in the skin. It can inhibit or ameliorate endogenous and exogenous aging.

In the present specification, suppression of aging refers to all actions that delay aging, and improvement of aging refers to all actions of reducing the severity of symptoms of aging.

In the present specification, an exosome is a microvesicle originating from an endosome, and is a membrane-structured vesicle secreted from green tea plant cells.

The cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions in addition to exosomes derived from green tea plant cells, and non-limiting examples thereof include antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, It may contain conventional adjuvants and carriers.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , Oil, powder foundation, emulsion foundation, wax foundation, and may be formulated as a spray, but is not limited thereto. In more detail, it may be prepared in the form of a flexible lotion, astringent lotion, a nutrition lotion, a nutrition cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder.

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

When the formulation of the cosmetic composition of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Propellants such as carbon, propane/butane or dimethyl ether.

When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene Glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

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

When the formulation of the cosmetic composition of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate , Fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester, and the like may be used.

Meanwhile, the exosomes derived from green tea plant cells of the present invention are effective in wound healing by increasing the proliferation and migration of cells in a wound healing assay, and thus can be provided in the form of a pharmaceutical composition for the purpose of wound healing. Silver may further contain suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. In addition, it can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, and sterile injectable solutions according to a conventional method.

In addition, the pharmaceutical composition of the present invention may be formulated and used in the form of external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injectable solutions, respectively, according to a conventional method. May have a formulation of cream, gel, patch, spray, ointment, warning agent, lotion, liniment, pasta or cataplasma.

Carriers, excipients, and diluents that may be included in the composition containing piperonylic acid include lactose, dextrose, sucrose, oligosaccharide, 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 and mineral oil. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient in the extract, such as starch, calcium carbonate, and sucrose. ) Or lactose (lactose), gelatin, etc. are mixed to prepare. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "a pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, drug activity, Sensitivity to drugs, 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 can be determined. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may 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 this can be easily determined by a person skilled in the art to which the present invention pertains.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be expected and can be administered by, for example, subcutaneous, intravenous, intramuscular, intrauterine dura mater or intracerebrovascular injection. The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient, along with various related factors such as the disease to be treated, the route of administration, the age, sex, weight, and severity of the disease of the patient.

In the present invention, various transformations can be applied and various embodiments may be applied. Hereinafter, specific embodiments will be illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to a specific embodiment, it should be understood to include all conversions, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, when it is determined that a detailed description of a related known technology may obscure the subject matter of the present invention, a detailed description thereof will be omitted.

[Example]

Example 1: Induction of green tea plant cells

The green tea leaves used in the present invention (purchased from the Jeju Resources Plant Research Institute) were immersed in 70% ethanol for 30 seconds, washed with sterile water, and then sterilized again with a disinfectant solution (30% Lax + Tween 20) for 20 minutes, and then used with sterile water. After washing three times, cut green tea leaves with a sharp knife at once, and basic MS medium containing 1 mg/L 2,4-D, 3% Sucrose, and 8g/L agar (Murashige and Skoog 1962, Duchefa Co., Ltd., Cat No. M0221) was cultured at 25°C and under the conditions of a humidity of 70%. Early green tea plant cells were induced. The medium was adjusted to pH 5.8 using 1N sodium hydroxide (NaOH). Subculture in solid state was carried out for 2 weeks, and air supply to 20 L bioreactor (from: Samsung Science Co., Ltd.) is 0.1 under the condition of temperature condition 23±2 ℃ and 70% humidity in culture room for liquid mass culture. Prepare a green tea plant cell culture solution by incubating for 5 weeks at about vvm.

Example 2: Isolation of exosomes derived from green tea plant cell culture solution

The green tea plant cell culture solution separated as in Example 1 was sequentially centrifuged at 1000 g for 10 minutes, 3000 g for 20 minutes, and 10,0000 g for 40 minutes, and then only the supernatant was taken and centrifuged again at 150,000 g for 90 minutes. After centrifugation, the supernatant was discarded and a pellet was taken to separate exosomes. And the pellet (exosome) was prepared by dissolving in 1 ml of PBS.

[Experimental Example]

Experimental Example 1: Cytotoxicity check (MTT assay)

In order to find out whether the composition of the present invention causes toxicity in skin cells, thereby causing skin irritation, an MTT assay was performed. HaCaT cells (human keratinocytes, keratinocytes) were dispensed in a 96-well plate with DMEM (Dubelcco's Modified Eagle's Medium) medium containing 10% FBS (Fetal Bovine Serum), and 5% CO ₂ , in a thermostat under conditions of 37°C. Incubated for 24 hours. Subsequently, the control group to which purified water was added and the composition of the present invention were treated to the cells to a final concentration of 0.5, 5.0, 10, and 20% (v/v), and further cultured for 24 hours. Subsequently, 4 μL of 5 mg/mL MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide) solution was added to each well and incubated for 4 hours. After removing the culture medium, 100 μL of a DMSO (dimethyl sulfoxide) solution was added to dissolve the cells, and the absorbance was measured at 540 nm using a spectrophotometer, and the results are summarized in Table 1 below.

sample Treatment concentration (%) Cytotoxicity rate (%)
(Cytotoxicity) Control 99 Green tea plant cell-derived exosomes 0.5% 101 5.0% 103 10.0% 106 20.0% 103

As shown in Table 1, it was confirmed that the green tea plant cell-derived exosomes did not show toxicity in skin cells at a concentration of 0.1 to 20%, so that there was no irritation.

Experimental Example 2: Confirmation of cell protection effect from ultraviolet rays

As a light source to be used for UV irradiation, an ultraviolet crosslinker CL-1000 (Ultra-Violet Products, CA) that emits ultraviolet rays with a wavelength of 302 nm was used. HaCaT cells were placed ^{in a 24-well plate at a concentration of 1×10 5} cells/ml and incubated for 24 hours in an incubator, and then the medium was removed and washed with phosphate buffer. After 500 μL of phosphate buffer was added, ultraviolet rays were irradiated at 10 mJ/cm², and then replaced with fresh cell culture medium containing no FBS, and the composition according to the present invention was treated by concentration, followed by additional culture for 24 hours. Here, 5 mg/ml of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma) was added and then incubated for 3 hours in an incubator, and the formed fomazan was dissolved in DMSO, After transferring to a 96 well plate, the absorbance was measured with an ELISA reader at 595 nm, and the results of comparing the cell viability with the control group that did not treat the sample after UV irradiation are summarized in Table 2 below.

division UV irradiation Treatment concentration (%) Cell protection effect by UV irradiation
(% of Control) Control - - 100 Control + - 52 Green tea plant cell-derived exosomes 0.5 68 5.0 75 10.0 89 20.0 94

In order to evaluate the keratinocyte protective effect of the composition according to the present invention, human keratinocytes (HaCaT) are exposed to UV irradiation 20 mJ/cm² and cultured for an additional 24 hours, and then the cell viability can be confirmed. As a result of performing, it was confirmed that as the treatment concentration of the green tea plant cell-derived exosomes increased, cell vialability increased compared to the control group.

Experimental Example 3: Confirmation of whitening effect by inhibition of tyrosinase activity (in vitro)

In order to confirm the whitening effect by the composition of the present invention, the effect on the activity of tyrosinase, which plays an important role in melanin formation, was tested.

Each 50 μl of phosphate buffer was dispensed into a 96 well plate, and 10 μl of an exosome sample derived from green tea plant cells according to the concentration was added. Then, mushroom tyrosinase was added to 50 μg/ml in purified water, and 20 μl each was dispensed. After that, 2.5mM L-DOPA was added to each well by 20 μl, mixed, reacted at 37° C., and absorbance was measured at 475 nm for about 1 hour at 10 minute intervals, and the results are summarized in Table 3 below. As a positive control, 250 μM kojic acid was added.

division Treatment concentration (%) Tyrosinase activity (%) Control - 100 Positive control (kojic acid) 250 μM 65 Green tea plant cell-derived exosomes 0.5 92 5.0 83 10.0 76 20.0 74

As confirmed by the above table, a significant decrease in Tyrosinase activity was confirmed by exosomes derived from green tea plant cells. From the decrease in tyrosinase activity, it can be seen that the exosomes derived from green tea plant cells have a whitening effect by inducing melanin formation inhibition.

Experimental Example 4: Confirmation of the effect of promoting collagen synthesis

DMEM (Dulbecco's) containing 10% FBS, Penicillin (50 U/ml) and Streptomycin (50/ml) in a cell culture device that maintains constant humidity in a 37°C, 5% CO _{2 incubator for human skin fibroblasts.} Modified Eagle's Medium, Gibco, USA), and incubated for 24 hours after dispensing 500 µl each into a 48 well plate at 1 ^{×10 5 cells/ml.} A control (DMEM medium) and a medium containing exosomes derived from green tea plant cells at a concentration of 0.5 to 20% were added and cultured in a 37°C, 5% CO ₂ incubator for 48 hours. After 48 hours, the amount of newly synthesized collagen was measured by taking 20 µl of each of the supernatant of the medium and measuring it using a Procollagen Type I C-Peptide EIA Kit (PICP, Takara, Cat No. MK101). The increase rate of collagen biosynthesis was calculated according to Equation 1 below, and the results are summarized in Table 4 below.

[Equation 1]

Treatment concentration (%) Procollagen biosynthesis (% of control) Control 100 Green tea plant cell-derived exosomes 0.5% 102 5.0% 110 10.0% 119 20.0% 132

As shown in Table 4, it can be seen that the amount of collagen biosynthesis increases depending on the treatment concentration of the exosomes derived from green tea plant cells.

Experimental Example 5: Confirmation of the effect on the expression of AQP, TERT, and Filaggrin genes

Aquaporin (AQP) is a membrane protein that actively transports water molecules into cells, and among them, aquaporin-3 is a protein that selectively passes water and glycerol, and plays an important role in skin moisturization and wound healing. It is known to be.

Telomerase reverse transcriptase (TERT) is involved in maintaining the length of telomeres and is associated with cellular senescence.

In addition, filaggrin is a keratin-binding protein of the epidermis that is involved in the binding of membrane proteins of keratinocytes, thereby contributing to the maintenance and strengthening of the skin barrier.

In the following examples, the present inventors observed changes in the expression levels of AQP, TERT, and filaggrin genes in keratinocytes treated with exosomes derived from green tea plant cells to moisturize the skin of exosomes derived from green tea plant cells, prevent skin aging, and skin barriers. It was intended to confirm the reinforcing effect.

5-1. Cell culture

Human keratinocytes (HaCaT) were mixed with Dulbecco's Modified Eagle's Medium (DMEM), 10% Fatal bovine serum (FBS), and 1% Antibiotic-Antimycotic (GIBCO, Cat.# 15240-062) in a 100 mm/60.1 cm² culture dish. Incubate under conditions of 37°C and 5% CO _2. When the human keratinocytes are more than 80% confluence, dispense 1×10⁴ cells/well into a 96 well plate and incubate until the confluence reaches 80% or more under cell culture conditions. If the confluence is more than 80%, the medium is DMEM-free. After exchange with medium (without FBS), the composition according to the present invention is treated. After treatment with the composition according to the present invention, it is further cultured for 24 hours to see if there is any effect on the expression of each gene by the composition according to the present invention. The test method performs the Real-time PCR method, and the test sequence is as follows.

5-2. real-time PCR

For RNA isolation, use FastLane Cell one-step buffer set (QIAGEN Cat.# 216213). Cells from which the medium was removed were washed with FCW (cell wash buffer), and 50ul cell processing mixture (a mixture of buffer FCPW added with Buffer FCPL and gDAN wipeout buffer added) was added and incubated for 5 minutes at room temperature, and then added to a new E-tube. After transfer, the reaction was performed at 75°C for 5 minutes.

To analyze this, the RNA extracted above was used as a template and proceeded with a Rotor Gene Q Real-time PCR Machine (Qiagen) according to the manual of the 2X QuantiTect SYBR Green RT-PCR Master mix. The primers used in the experiment were normalized with Qiagen's QuantiTect ^® primerassays (Aquaporin3 Cat.#QT00212996, Telomerase reverse transcriptase (TERT) Cat.#QT00073409, Filaggrin Cat.#QT01192646) and GAPDH (Cat.# QT01192646).

Real-time PCR conditions are shown in Table 5 below, and the expression level of the gene according to the exosome treatment was confirmed by comparing with the control group as shown in Equation 2 below.

<Real-time cycler conditions> Step Time Temperature Reverse transcription 30 min 50 ℃ PCR initial activation step 15 min 95 ℃ 3-step cycling Denaturation 15 s 94 ℃ Annealing 30 s 60 ℃ Extension 30 s 72 ℃ Number of cycles 45

[Equation 2]

Expression rate (fold) = (expression rate of sample treatment group)/(expression rate of control group)

5-3. Confirmation of moisturizing effect by increasing AQP3 expression

It was found that the biosynthetic amount of the AQP-3 (Aquaporin-3) gene related to the moisturizing effect increased as the amount of exosomes derived from the treated green tea plant cells increased.

Treatment concentration (%) AQP-3 expression level (% of control) Control 100 Green tea plant cell-derived exosomes 0.5% 104 5.0% 114 10.0% 123 20.0% 136

5-4. Checking the effect of preventing skin aging by increasing the expression of TERT

As a result of confirming the evaluation of the efficacy of extending cell life through the expression of TERT, an anti-aging-related gene by exosomes derived from green tea plant cells, exosomes derived from green tea plant cells were similar to 1mM Shikimic acid (SA) used as a positive control. It was confirmed that it has excellent TERT expression ability.

Treatment concentration (%) TERT expression amount
(% of control) Negative control 100 Positive control (1mM SA) 580 Green tea plant cell-derived exosomes 0.5% 104 5.0% 140 10.0% 230 20.0% 360

5-5. Confirmation of skin barrier strengthening and moisturizing effect according to the increase in filaggrin expression

It shows that the skin barrier function can be improved by promoting the normal differentiation of keratinocytes by the exosomes derived from green tea plant cells according to the present invention, and promoting intradermal expression of filaggrin, which is converted into a natural moisturizing factor in the skin. That is, as a result of measuring the expression of the Filaggrin gene in the exosomes derived from green tea plant cells, it was confirmed that the gene expression rate significantly increased the amount of gene expression.

division Treatment concentration (%) Filaggrin gene expression rate
(Relative Expression of Filaggrin) Control - 1.00 Green tea plant cell-derived exosomes 0.5 1.14 5.0 1.59 10.0 2.55 20.0 3.76

Experimental Example 6: Confirmation of anti-stress effect according to increase in LEA expression

It is known that the higher the expression level of LEA (Late embryogenesis abundant), the more resistance to stress increases, thereby protecting it from cell damage. In the following experiments, in order to confirm whether the exosomes derived from green tea plant cells exhibit the effect of enhancing stress tolerance of cells, changes in the expression of the LEA protein according to the treatment of exosomes derived from green tea plant cells were observed.

Specifically, after dispensing HaCaT cells into a 6-well plate, they were cultured for 24 hours under cell culture conditions. Thereafter, the control and the composition of the test substance to which purified water was added were treated to the cells to a final concentration of 0.5, 5, 10, 20%, and then the cells were further cultured for 24 hours, and In- Cell ELISA Colorimetric Detection kit was used.

After the culture was completed, the medium of the cells was removed, and the cells were fixed by incubating at room temperature for 15 minutes with 100 μL fixing solution. After washing with PBS, 100 μL of Permeabilization solution was added and reacted at room temperature for 15 minutes. After washing with PBS, 100 μL Quenching solution was added and reacted at room temperature for 20 minutes. After washing with PBS, 100 μL Blocking solution was added and reacted at room temperature for 30 minutes. The solution was removed and 50 μL of LEA primary antibody was added, followed by reaction at 4° C. for 24 hours. After removing the antibody, it was washed with PBS, and 50 μL of HRP conjugated secondary antibody was added and reacted for 1 hour. After removing the antibody and washing with PBS, 100 μL of TMB substrate solution was added and reacted at room temperature for 15 minutes in a light-shielded state. Then, 100 μL stop solution (1 NH ₂ SO ₄ ) was added, and absorbance at 450 nm was measured.

division Treatment concentration (%) LEA Protein content (%) Control - 100 Green tea plant cell-derived exosomes 0.5 101 5.0 108 10.0 115 20.0 123

As a result, it was confirmed that the green tea plant cell-derived exosomes protected cell damage by increasing the expression level of the LEA protein at 0.5 to 20% treatment concentration.

Experimental Example 7: Checking the anti-aging effect of wound oil using Wound healing assay

The human keratinocyte line HaCaT (human keratinocyte) was mixed with Dulbecco's Modified Eagle's Medium (DMEM), 10% Fatal bovine serum (FBS), 1% Antibiotic-Antimycotic (GIBCO) in a 100 mm/60.1 cm2 culture dish at 37°C, 5%. Incubated under the condition of CO2. When HaCaT is confluenced by about 90%, dispense 1.5×10 ^{5 cells/12 well plate in 100 mm/60.1 cm 2} dish, incubate for 24 hours, replace with medium that does not contain FBS, and use 200P tip to # Scratched into shape and treated the composition according to the present invention. After the material treatment, the cell image was taken with a microscopic photograph at 0 hours and cultured under conditions of ^{37°C and 5% CO 2.} After material treatment, the culture medium was removed for 18-20 hours, added with a fixing solution (4% paraformaldehyde), incubated at room temperature for 15 minutes, and washed 3 times with PBS. Fixed cells could be stored at 4℃ for one month. The degree of wound healing was calculated using a program called Image J by taking a picture with a microscope. As a positive control, 300ng/ml EGF was used.

We tried to evaluate the anti-aging efficacy by observing whether the test substance promotes cell proliferation and migration by using Wound healing assay. 100 ng/ml EGF was used as a positive control.

As a result of the experiment by treating the green tea plant cell exosomes, as shown in FIG. 1, it was confirmed that the healing area increased as the skin cells grew attached to the floor compared to the control group.

Experimental Example 8: Confirmation of the energy enhancing effect of cells

In order to examine the effect of enhancing cellular energy of the composition of the present invention, the degree of activity of ATP released by cells during metabolic processes was measured.

For this purpose, Detroit cells were dispensed into a 12-well plate and cultured for 24 hours under cell culture conditions. Thereafter, the control and the composition as a test substance to which purified water was added were treated to the cells to a final concentration of 0.5, 5, 10 and 20%, and the cells were further cultured for 24 hours thereafter. Take 50 μL of a portion of the lysate of the cells cultured through the above process, and add 50 μL of the ATP standard material in the ATP assay kit (Abcam, Cat.ab83355) into a 96-well plate, respectively, and 50 μL of ATP reaction mix solution Was also added to each well and allowed to react at room temperature for 30 minutes in a light-shielded state. After measuring the absorbance at 570 nm, the ATP standard concentration curve was created to calculate the amount of ATP in the sample.

division Treatment concentration (%) ATP Content (%) Control - 100 Green tea plant cell-derived exosomes 0.5 98 5.0 105 10.0 113 20.0 120

As a result, it was confirmed that the composition of the present invention has an energy activity enhancing effect by increasing the amount of ATP at 0.5 to 20% treatment concentration.

Experimental Example 9: Skin soothing effect evaluation (skin temperature reduction rate)

The effect of preventing heat aging by the composition of the present invention was evaluated. That is, the composition according to the present invention was formulated to contain a lotion by concentration, and then applied to the skin to confirm the effect of preventing heat aging. It was conducted on the forearm of 25 healthy subjects in their 20s to 30s, and the subject's test site (forearm) was exposed to direct sunlight for 20 minutes before the test. The test sample and the control sample were applied in an amount of 50 μL each using a micro pipette to the selected test area (2 cm * 2 cm) of the forearm, and the temperature before, immediately after, 5 minutes after application, and 20 minutes after application Was measured, and the results are summarized in Table 11. Water was used as a negative control for the comparison of heat aging prevention efficacy, and the temperature reduction rate of each sample after measurement was derived according to Equation 3 below.

Heat reduction rate (%) Composition of the present invention (%) Immediately after application 5 minutes later 20 minutes later 0 6.1 2.1 0 0.5 7.2 5.5 3.5 5 9.3 7.4 5.8 10 11.8 9.8 7.9 20 15.9 13.4 11.1

[Equation 3]

Heat reduction rate (%) = (1-temperature of the sample application area/ temperature of the sample non-application area) * 100

As a result, in the case of the control group, the rate of decrease in temperature rapidly decreased with the passage of time, but it was confirmed that the sample containing the composition of the present invention maintained a continuous rate of decrease in heat even after 20 minutes in a concentration-dependent manner even after 20 minutes.

Experimental Example 10: Confirmation of pore shrinking effect (In vitro Test)

In order to measure the pore shrinking effect of the composition of the present invention, the composition prepared in the above example was tested for the pore shrinking effect using hemoglobin as a protein to replace the skin.

A hemoglobin solution (0.05g/50ml) was prepared with 0.9% Phosphate Buffer Saline (0.1mM, pH 7.4), and 2 ml of the exosomes derived from green tea plant cells obtained in Example 2 were added to 2 ml of the hemoglobin solution, followed by 30 seconds. After shaking and mixing, centrifugation at 3,500 rpm for 10 minutes, 2 ml of purified water was added to 1 ml of the supernatant, and measured by UV-Visible spectrum at 407 nm.

As a control, 2 ml of PBS (Phosphate Buffer Saline) was added.

Concentration (%) Hemoglobin precipitation (%) PBS (negative control) 0 0.1 Tannic acid (positive control) One 36 Green tea plant cell-derived exosomes 0.5 6 5.0 11 10.0 21 20.0 25

The shrinkage effect of the green tea plant cell-derived exosome was measured by measuring the amount of hemoglobin precipitated in the composition, and it was confirmed that the higher the precipitation (%) of hemoglobin, the better the pore shrinking effect.

Experimental Example 11: Sebum inhibition effect confirmation

In order to confirm the sebum inhibitory effect of the composition according to the present invention, the amount of sebum secretion of the skin was measured using a skin oil meter (SM810) after 4 weeks of 10 test subjects. The experiment was conducted at 22±2°C and 40±5% humidity. For oily skin, the measured value is 220 µg/cm ² or more, and for normal skin, it is 100-220 µg/cm ² . Purified water was used as a control, and the experimental results are shown in Table 9.

sample Sebum inhibitory effect 0 days (before use) 28 days (after use) One Control 230 223 2 10% green tea plant cell-derived exosomes 230 173 3 20% green tea plant cell-derived exosomes 232 151

Compared with the control group, exosomes derived from green tea plant cells showed excellent sebum secretion inhibitory effect.

Experimental Example 12: Acne improvement effect confirmation

For 10 men and women with acne, the lotion was evenly applied to the entire face in the morning and evening for 4 weeks, and the lotion containing the 10% concentration of green tea plant cell-derived exosomes prepared in Example 1 was evenly applied. To determine the acne improvement effect, the pre-test condition was evaluated with 1-3 points based on the following criteria according to the degree of feeling of the person participating in the experiment, and then the acne healing effect was evaluated, and the results are shown in Table 14.

<Evaluation of the state before the test>

1 = Slight acne 2 = Slight acne 3 = Severe acne

<Acne effect judgment>

-: Little effect, +: slight effect, ++: much relief, +++: completely healed

Subject State before test Effect after 2 weeks Effect after 4 weeks Subject 1 2 + + Subject 2 One + ++ Subject 3 One - + Subject 4 2 - - Subject 5 2 + ++ Subject 6 2 + + Subject 7 3 ++ +++ Subject 8 2 - ++ Subject 9 3 + + Subject 10 2 + ++

After using the flexible lotion containing exosomes derived from green tea plant cells according to the present invention for 4 weeks, the effect of improving acne was confirmed in most subjects.

[Production Example]

Preparation of composition for external application for skin

As cosmetics containing exosomes derived from green tea plant cells of the present invention as an active ingredient, cosmetics in emulsified formulations such as nutrient lotion, cream, essence, and solubilized cosmetics such as softening lotion were prepared.

Preparation Example 1: Lotion

It was prepared according to a conventional lotion preparation method according to the following prescription.

Raw material name Weight% (w/w) glycerin 5.0 Dipropylene glycol 3.0 Hyaluronic acid 0.5 Polyoxyethylene hydrogenated castor oil 0.1 Polyethylene oleylethyl 0.1 ethanol 5.0 antiseptic 0.15 Perfume Appropriate amount flash Appropriate amount Green tea plant cell-derived exosomes 10.0 Purified water to 100

Preparation Example 2: Essence

It was prepared according to a conventional essence manufacturing method according to the following prescription.

Raw material name Weight% (w/w) Cetostearyl alcohol 1.0 Self-emulsifying monostearic acid 1.0 Beeswax 0.5 Squalane 5.0 Isocetyloctanoate 3.0 Dimethylsiloxane 0.3 Sorbitan monostearate 0.5 Polyethylene glycol monostearate 8.0 glycerin 4.0 Propylene glycol 0.2 Carboxypolymer 0.22 Triethanolamine 0.25 antiseptic Appropriate amount Spices Appropriate amount flash Appropriate amount Green tea plant cell-derived exosomes 10.0 Purified water to 100

Preparation Example 3: Lotion

It was prepared according to a conventional lotion manufacturing method according to the following prescription.

Raw material name Weight% (w/w) Cetostearyl alcohol 0.8 Self-emulsifying monostearic acid 1.0 Beeswax 0.5 Stearic acid 0.5 Floating paraffin 7.0 Squalane 5.0 Macadamia Oil 3.0 Isocetyloctanoate 2.0 Dimethylsiloxane 0.3 Sorbitan monostearate 0.5 Polyethylene glycol monostearate 1.2 glycerin 4.0 Propylene glycol 4.0 Betaine 4.0 Carboxypolymer 0.12 Triethanolamine 0.15 antiseptic 0.25 Spices Appropriate amount flash Appropriate amount Green tea plant cell-derived exosomes 10.0 Purified water to 100

Preparation Example 4: Cream

It was prepared according to a conventional cream preparation method according to the following prescription.

Raw material name Weight% (w/w) Cetostearyl alcohol 3.0 Self-emulsifying monostearic acid 1.5 Lipophilic monostearic acid 1.5 Beeswax 0.5 Floating paraffin 8.0 Squalane 7.0 Isocetyloctanoate 4.0 Refined Jojoba Oil 4.0 Dimethylsiloxane 0.3 Sorbitan monostearate 1.0 Polyethylene glycol monostearate 1.2 glycerin 6.0 Propylene glycol 4.0 Betaine 4.0 Xanthan gum 0.06 Triethanolamine 0.10 antiseptic 0.25 Spices Appropriate amount flash Appropriate amount Green tea plant cell-derived exosomes 10.0 Purified water to 100

Preparation Example 5: Gel

It was prepared according to a conventional gel preparation method according to the following prescription.

Raw material name Weight% (w/w) glycerin 4.0 Propylene glycol 4.0 ethanol 10 Polyoxyethylene hardened castor oil 0.1 Carboxypolymer 0.30 Triethanolamine 0.30 antiseptic Appropriate amount Spices Appropriate amount flash Appropriate amount Green tea plant cell-derived exosomes 10.0 Purified water to 100

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is obvious that this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (13)

A cosmetic composition for inhibiting or improving skin aging without cytotoxicity, comprising an exosome derived from green tea plant cells as an active ingredient.
The method of claim 1,
The skin aging is endogenous aging and / or exogenous aging, cosmetic composition. The method of claim 1,
The composition is to reduce apoptosis due to ultraviolet rays, a cosmetic composition. The method of claim 1,
The composition is to enhance the expression of telomerase reverse transcriptase (TERT), a cosmetic composition. The method of claim 1,
The composition inhibits skin aging due to environmental stress by enhancing the expression of a late embryogenesis abundant (LEA) protein,
The environmental stress is characterized in that ultraviolet, pollution, wind, or temperature, cosmetic composition. As a cosmetic composition for improving skin condition without cytotoxicity, comprising exosomes derived from green tea plant cells as an active ingredient,
The improvement of the skin condition is a cosmetic composition that is improving skin elasticity, moisturizing, whitening, soothing skin, reducing pores, reducing sebum secretion, and/or improving acne.
The method of claim 6,
The composition is to promote collagen synthesis, cosmetic composition. The method of claim 6,
The composition is to enhance the expression of Aquaporin-3 (Aquaporin-3) and filaggrin (filaggrin), a cosmetic composition. The method of claim 6,
The composition is to inhibit the activity of tyrosinase (tyrosinase), a cosmetic composition. A composition for reinforcing skin barriers or improving scars without cytotoxicity, comprising exosomes derived from green tea plant cells as an active ingredient.
A pharmaceutical composition for treating wounds without cytotoxicity, comprising exosomes derived from green tea plant cells as an active ingredient.
The method of any one of claims 1, 6, 10, and 11,
The exosome is a composition, characterized in that obtained by sequentially centrifuging the green tea leaves after dark culture.
(a) inducing green tea plant cells by wounding green tea leaves and culturing them with cancer;
(b) subculturing the green tea plant cells in a solid state;
(c) after step (b), mass culturing in a liquid state; And
(d) comprising the step of separating exosomes by sequentially centrifuging the green tea plant cell culture solution and then centrifuging again at high RPM,
As a method for producing exosomes derived from green tea plant cells,
A composition comprising the exosomes as an active ingredient is a method for producing exosomes for inhibiting or improving skin aging, improving skin conditions, strengthening skin barriers, improving scars, and/or treating wounds without cytotoxicity.

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