Cosmetic composition containing exosomes derived from black yeast

Abstract

The present invention provides a cosmetic composition for improving skin, comprising black yeast-derived exosomes as an active ingredient, specifically, a cosmetic composition for improving skin elasticity, improving skin wrinkles, improving skin texture, improving skin tone, improving skin brightness, skin regeneration, skin moisturizing, and / or skin whitening. The cosmetic composition of the present invention has excellent effects of improving skin elasticity, improving skin wrinkles, improving skin texture, improving skin tone, improving skin brightness, skin regeneration, skin moisturizing, and / or skin whitening.

Description

[Technical Field]

[0001] The present invention relates to a cosmetic composition containing black yeast-derived exosomes as an active ingredient for improving skin elasticity, wrinkles, texture, tone, brightness, regeneration, moisturizing, and / or whitening. [Background technology]

[0002] As skin aging occurs, skin elasticity decreases and wrinkles increase. This is known to occur due to a decrease in collagen synthesis and an increase in the expression of matrix metalloproteinase (MMP), an enzyme that breaks down collagen.

[0003] Substances known to be effective in improving skin wrinkles include adenosine and retinoic acid, but adenosine has limited clinical efficacy, and retinoic acid cannot be used by pregnant women and has side effects such as erythema. As a result, functional cosmetics for skin anti-aging that utilize active ingredients from plants and natural products have recently been attracting attention. However, functional cosmetics containing plant extracts as active ingredients can cause a foreign body sensation as the solution evaporates after application to the skin, and the effects only last a short time.

[0004] Melanin, which determines human skin color, is produced by melanocytes. Specifically, melanin is a dark brown pigment formed by the polymerization and oxidation of tyrosine, an amino acid present in the body, via enzymes such as tyrosinase present in melanocytes. The melanin thus formed is transported to epidermal cells called keratinocytes via melanocyte dendrites. Once transported to keratinocytes, the melanin is removed from the skin as the keratinocytes leave the epidermis. However, since the body lacks the enzymes required to break down melanin, once formed, it cannot be broken down in the body. Therefore, inhibiting melanin production is crucial for skin lightening.

[0005] Research into skin-whitening agents for cosmetics has been gaining momentum as people in the East have increasingly favored paler skin and as darkening of the skin is increasingly recognized as a result of UV-induced skin aging. As a result, tyrosinase inhibitors such as ascorbic acid, hydroquinone, glutathione, and arbutin have been incorporated into cosmetics and pharmaceuticals. However, most of these have limited utility due to insufficient efficacy or unstable formulations. Compounds such as hydroquinone, which exhibit strong bleaching properties, are inherently skin sensitizers, potentially inducing skin allergies and altering normal skin functions, resulting in side effects such as vitiligo. Therefore, their use is restricted due to concerns about skin safety.

[0006] The skin is the human body's primary protective barrier, conserving moisture within the body, preventing excessive evaporation, and protecting the body from various external stimuli (chemicals, air pollutants, dry environments, ultraviolet rays, etc.). The skin is broadly composed of the epidermis, dermis, and subcutaneous fat. The epidermis is further divided into the stratum corneum, granular layer, spinous layer, and basal layer. The skin's protective function can be maintained only when the stratum corneum of the epidermis, which is composed of keratinocytes and lipid membranes between keratinocytes, is properly formed. The moisture retention function of keratinocytes and the lipid membranes between keratinocytes prevent moisture loss, allowing humans to maintain approximately 30% of the skin's moisture content.

[0007] Human keratinocytes express aquaporin 3 (AQP3), a transmembrane protein that transports water and glycerol into the cell. AQP3 regulates the movement of water and glycerol between cells, and in a mouse model in which AQP3 gene expression was suppressed, the skin barrier was damaged, resulting in a decrease in skin elasticity and moisture retention capacity.

[0008] Hyaluronic acid is also an important substrate for retaining moisture in the epidermis as a natural moisturizing factor for the skin. Hyaluronan synthase 2 (HAS2) and hyaluronan synthase 3 (HAS3), which are enzymes that synthesize hyaluronic acid, are expressed in the epidermis and are sometimes used as indicators for evaluating the skin's moisturizing ability.

[0009] Recently, due to factors such as changes in the environment and lifestyle, various stresses and environmental pollution arising from social life, and frequent face washing due to makeup habits, the moisture in the stratum corneum has decreased, causing the skin to become dry and rough, and the skin to lose its luster and appear dull. As a result, the issue of skin moisturization has become even more important.

[0010] Therefore, there is a need to develop new cosmetic materials that have excellent effects in improving skin conditions, such as wrinkle reduction, elasticity, texture, tone, brightness, regeneration, whitening, and / or moisturizing, and that are free of side effects and easy to use.

[0011] Meanwhile, recent research has shown that cell secretomes contain various bioactive factors that regulate cell behavior. In particular, cell secretomes contain exosomes, which have intercellular signaling functions, and active research is being conducted into their components and functions.

[0012] Cells release various membrane-type vesicles into the extracellular environment, and these released vesicles are commonly called extracellular vesicles (EVs). EVs are also known as plasma membrane-derived vesicles, ectosomes, shedding vesicles, microparticles, or exosomes, and are sometimes referred to separately from exosomes.

[0013] Exosomes are endoplasmic reticulum (ERs) measuring tens to hundreds of nanometers in size and composed of a double phospholipid membrane, similar to the structure of the cell membrane. They contain exosomal cargo, including proteins and nucleic acids (e.g., mRNA and miRNA). Exosome cargo contains a wide range of signaling factors, which are known to be cell-type specific and differentially regulated depending on the environment of the secreting cell. Exosomes are secreted by cells as intercellular signaling mediators, and the various cellular signals transmitted by them are known to regulate target cell behaviors, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis. Exosomes contain specific genetic material and bioactive factors depending on the nature and state of the cells from which they are derived. Exosomes derived from proliferating stem cells regulate cell behaviors, such as cell migration, proliferation, and differentiation, reflecting the stem cell properties related to tissue regeneration (Nature Review Immunology 2002(2) 569-579).

[0014] That is, exosomes, which are called avatars of cells, contain bioactive factors such as growth factors similar to cells, but act as carriers that load and transport bioactive factors between cells, i.e., as a means of communication between cells. Exosomes are known to be released not only from animal cells such as stem cells, immune cells, fibroblasts, and cancer cells, but also from cells of various organisms such as plants, bacteria, fungi, and algae.

[0015] Black yeast (Aureobasidium pullulans) is a fungus imperfecti found widely in nature. It produces melanin pigments and, depending on the culture conditions, produces large amounts of β-1,3-1,6-glucan extracellularly. Black yeast grows optimally at 30°C, but grows at temperatures between 10°C and 35°C. Pullulan, a polymeric polysaccharide produced by black yeast, is used in the food industry to impart physical properties to jellies and other foods. New foods have also been developed in which liquid foods are enclosed within pullulan films. Black yeast-derived beta-glucan is an exopolysaccharide biosynthesized in the culture medium, does not require purification, and is water-soluble, making it easy to process.

[0016] Therefore, black yeast is widely used in the food and cosmetics industries. However, research on the isolation, purification, and characterization of exosomes derived from black yeast remains insufficient. Therefore, more detailed characterization and functional studies on exosomes derived from black yeast are needed.

[0017] Cosmetics that use microbial fermentation products and culture broths have been introduced, but some fermentation products and culture broths can cause side effects such as skin troubles, redness, or dermatitis in people with sensitive skin.

[0018] On the other hand, it should be understood that the matters described above as background art are merely for the purpose of deepening understanding of the background of the present invention, and are not cited as an admission that they can be used as "prior art" for the present invention. [Prior art documents] [Non-patent literature]

[0019] [Non-Patent Document 1] Nature Review Immunology 2002(2) 569-579 Summary of the Invention [Problem to be solved by the invention]

[0020] In light of the above, the present inventors have conducted research to develop a cosmetic composition with improved effects compared to the fermentation product or culture solution of black yeast, and have found that exosomes derived from black yeast are excellent in improving skin elasticity, wrinkle reduction, skin texture, skin tone, skin brightness, skin regeneration, skin moisturizing, and skin whitening, thereby completing the present invention.

[0021] Therefore, an object of the present invention is to provide a cosmetic composition for improving skin conditions, which contains exosomes derived from black yeast as an active ingredient. [Means for solving the problem]

[0022] To achieve the above object, the present invention provides a cosmetic composition for improving skin conditions, which contains exosomes derived from black yeast (Aureobasidium pullulans) as an active ingredient.

[0023] The term "black yeast (Aureobasidium pullulans)" used in this specification refers to a fungus belonging to the genus Aureobasidium, which is a unicellular organism without hyphae, and which is neither motile nor photosynthetic. Black yeast is a fungus imperfecti found widely in nature, which produces melanin pigment and, depending on the culture conditions, produces large amounts of β-1,3-1,6-glucan outside the fungal body.

[0024] The term "exosomes" used herein refers to nano-sized vesicles having a membrane structure secreted or released from black yeast into the extracellular space, and is also defined as exosome-like vesicles or exosome-like particles.

[0025] The term "exosomes derived from black yeast" as used herein is intended to encompass all exosomes, for example, exosomes isolated from black yeast culture medium, black yeast fermentation product, or equivalent black yeast biological solutions, or exosomes secreted and / or released from black yeast itself. Various black yeast strains that are currently used in the art or that may be used in the future can be used to produce black yeast-derived exosomes, and the black yeast strain used in the examples should be understood as an example of a black yeast strain that may be used in the present invention, and the present invention is not limited thereto.

[0026] The term "improving skin condition" as used herein refers to reducing the severity of symptoms associated with aggravated skin condition, and the skin condition may be parameters associated with skin elasticity, skin wrinkles, skin texture, skin tone, skin brightness, skin regeneration, skin whitening, and skin moisturization.

[0027] Specifically, "improving skin elasticity" means maintaining or increasing skin elasticity by promoting collagen synthesis and increasing the volume of fat tissue in the skin, thereby contracting sagging skin tissue.

[0028] Furthermore, "skin wrinkles" refer to fine lines that appear due to skin aging, which can be caused by genetic factors, a decrease in collagen and elastin present in the dermis of the skin, external environments, etc. Therefore, the term "skin wrinkle improvement" used herein means suppressing or inhibiting the formation of wrinkles on the skin or alleviating wrinkles that have already formed.

[0029] Meanwhile, the term "skin tone" as used herein refers to the degree of dark or light skin color, and "skin tone improvement" refers to making inconsistent skin tone uniform. "Skin whitening" and "skin brightness improvement" include increasing the brightness of skin that has been reduced due to an excess of pigments such as melanin, or maintaining the brightness of skin at a constant level.

[0030] "Skin regeneration" refers to the restoration of skin tissue damaged by external and internal causes. Examples of external causes include ultraviolet rays, external pollutants, wounds, trauma, etc., while examples of internal causes include illness, stress, etc. "Skin moisturization" refers to inhibiting or suppressing the loss of skin moisture or increasing the moisture content of the skin, thereby smoothing the skin surface and providing a radiant glow.

[0031] As can be seen from the following examples, the black yeast-derived exosomes are delivered to skin fibroblasts (Figure 2) and increase collagen synthesis (Figure 3). In melanoma cells, they suppress melanin production (Figure 5) and promote hyaluronic acid synthase or aquaporin synthesis.

[0032] Therefore, a cosmetic composition containing exosomes derived from black yeast can exhibit one or more effects selected from the group consisting of improved skin elasticity, improved skin wrinkles, improved skin texture, improved skin tone, improved skin brightness, skin regeneration, skin whitening, and skin moisturizing effects.

[0033] Meanwhile, the cosmetic composition for improving skin condition of the present invention may be prepared in at least one form selected from the group consisting of lotions, liquids, creams, deposition masks, gels, aerosols, and powders.

[0034] The lotion is a formulation prepared in a viscous liquid state by mixing the active ingredient of the cosmetic composition with an emulsifier and the like to homogenize the oily and aqueous components, the cream is a formulation prepared in a semi-solid state by mixing the active ingredient of the cosmetic composition with an emulsifier and the like to homogenize the oily and aqueous components, the liquid is a formulation prepared in a liquid state by dissolving the active ingredient of the cosmetic composition and other ingredients in a solvent, and the gel is a semi-solid state made of organic molecules with a large molecular weight that are permeated with a liquid.

[0035] Deposited masks are formulations made by depositing lotions, creams, liquids, gels, etc. onto a support such as nonwoven fabric, and can be made into patches, mask packs, mask sheets, etc. Aerosols are formulations that are sprayed in mist or foam form using the pressure of a propellant (liquefied gas, compressed gas, etc.) filled in the same container or a separate container as the concentrate. Powders are formulations made into homogeneous powder or fine particles, and excipients can be used.

[0036] For example, the cosmetic composition for improving skin condition of the present invention can be made into a lotion or cream, or can be applied or deposited on at least one surface of a patch, mask pack, or mask sheet.

[0037] Meanwhile, the cosmetic composition for improving skin condition of the present invention is used for the purposes of improving skin elasticity, wrinkle reduction, skin texture, skin tone, skin brightness, skin regeneration, skin moisturizing, and / or skin whitening, and may be prepared in any form commonly used in the art, such as, but not limited to, a patch, a mask pack, a mask sheet, a softening lotion, a nutritious lotion, an astringent lotion, a nutritious cream, a massage cream, an eye cream, a cleansing cream, an essence, an eye essence, a cleansing lotion, a cleansing foam, a cleansing water, a sunscreen, a lipstick, a soap, a shampoo, a surfactant-containing cleanser, a bath additive, a body lotion, a body cream, a body oil, a body essence, a body wash, a hair dye, or a hair tonic.

[0038] Meanwhile, the cosmetic composition for improving skin condition according to one embodiment of the present invention may further contain, as needed, ingredients commonly used in cosmetic compositions, such as moisturizers, antioxidants, oily ingredients, UV absorbers, emulsifiers, surfactants, thickeners, alcohols, powder ingredients, colorants, aqueous ingredients, water, various skin nutrients, etc., within a range that does not impair the effects of the present invention.

[0039] Furthermore, the cosmetic composition for improving skin conditions according to one embodiment of the present invention can be used in combination with conventional skin improving agents, antioxidants, and / or moisturizing agents, in addition to the black yeast-derived exosomes, to the extent that their functions (such as improving skin elasticity, reducing skin wrinkles, improving skin texture, skin regeneration, skin moisturizing, improving skin tone and brightness, skin whitening, and skin beauty) are not impaired. For example, the black yeast-derived exosomes of the present invention may be supported by or mixed with at least one of hydrogel, hyaluronic acid, hyaluronate salts (e.g., sodium hyaluronate), and hyaluronic acid gel. The type of hydrogel used in the cosmetic composition according to one embodiment of the present invention is not limited, but may preferably be a hydrogel obtained by dispersing a gelling polymer in a polyhydric alcohol. The gelling polymer may be at least one selected from the group consisting of pluronic, purified agar, agarose, gellan gum, alginic acid, carrageenan, cassia gum, xanthan gum, galactomannan, glucomannan, pectin, cellulose, guar gum, and locust bean gum, or may be at least one selected from the group consisting of polyhydric alcohols, ethylene glycol, propylene glycol, 1,3-butylene glycol, isobutylene glycol, dipropylene glycol, sorbitol, xylitol, and glycerin.

[0040] In addition, the cosmetic composition for improving skin condition of the present invention may additionally contain one or more cosmetically acceptable carriers that are blended into general skin cosmetics, and may be appropriately blended with common ingredients such as oils, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments, preservatives, fragrances, etc., but is not limited thereto.

[0041] When the dosage form of the present invention is a powder or an aerosol, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or a mixture thereof may be used as a carrier component, and particularly when it is an aerosol, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether may be additionally contained.

[0042] When the dosage form of the present invention is a cream or gel, animal fibers, plant fibers, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. may be used as the carrier component.

[0043] When the dosage form of the present invention is a solution or emulsion among liquid preparations, a solvent, solvating agent or emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan.

[0044] When the dosage form of the present invention is a suspension among liquid preparations, the carrier component may be a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agarose or tragacanth.

[0045] Another aspect of the present invention provides a cosmetic method for improving the skin of a mammal, excluding medical treatment, comprising the step of applying the cosmetic composition for improving skin conditions to the skin of the mammal.

[0046] The mammal may be, but is not limited to, a human, dog, cat, rodent, horse, cow, monkey, or pig.

[0047] In the cosmetic method of the present invention, improving skin condition means a visually and / or tactilely perceptible positive change in the appearance and feel of skin tissue. For example, improving skin condition may be improving skin elasticity, reducing skin wrinkles, improving skin texture, improving skin tone, improving skin brightness, skin regeneration, skin moisturizing, and / or skin whitening.

[0048] In the present invention, the step of applying the cosmetic composition for improving skin condition to the skin of a mammal may be (a) a step of directly applying the cosmetic composition for improving skin condition to the skin of a mammal, or (b) a step of contacting or attaching a patch, mask pack, or mask sheet onto which the cosmetic composition for improving skin condition has been applied or deposited to the skin of a mammal, or a step of sequentially performing (a) and (b). In step (a), a cosmetic composition for improving skin condition in the form of a lotion or cream may be used.

[0049] Furthermore, the cosmetic method of the present invention may further include (c) removing the patch, mask pack, or mask sheet from the mammalian skin after step (b) and applying the cosmetic composition for improving skin condition to the mammalian skin. In step (c), the cosmetic composition for improving skin condition may be in the form of a lotion or cream.

[0050] Meanwhile, all of the components described in the present invention may be preferably contained in the composition of the present invention within a range not exceeding the maximum use value specified in regulations on cosmetic safety standards and the Chinese "Cosmetic Safety Technical Standards." [Effects of the Invention]

[0051] The cosmetic composition of the present invention has excellent effects of improving skin elasticity, wrinkles, texture, tone and brightness, skin regeneration, moisturizing, and / or whitening, and can therefore be usefully used for improving skin conditions. [Brief explanation of the drawings]

[0052] [Figure 1] FIG. 1 is a graph showing the particle size distribution and particle number obtained by performing nanoparticle tracking analysis (NTA) on the exosomes derived from black yeast of the present invention. [Figure 2]Figure 2 shows a cell fluorescence microscope image showing the delivery of fluorescently stained exosomes derived from black yeast to human skin fibroblasts (green: exosomes delivered into cells, blue: cell nuclei). [Figure 3] FIG. 3 is a graph showing the relative collagen content after treating human skin fibroblasts with low and high concentrations of black yeast-derived exosomes. [Figure 4] Figure 4 shows a cell fluorescence microscope image showing the delivery of fluorescently stained exosomes derived from black yeast to melanoma cells (green: exosomes delivered into cells, blue: cell nuclei). [Figure 5] FIG. 5 is a graph showing that melanin production was reduced after treating melanoma cells with low and high concentrations of yeast-derived exosomes. DETAILED DESCRIPTION OF THE INVENTION

[0053] The present invention will be described in more detail with reference to the following examples. However, the following examples are merely illustrative of the contents of the present invention and are not intended to limit or restrict the scope of the present invention. Anything that can be easily inferred by a person skilled in the art from the detailed description and examples of the present invention is considered to be within the scope of the present invention. The references cited in this specification are incorporated herein by reference.

[0054] Throughout the specification, when a part is said to "comprise" certain elements, this does not mean that it excludes other elements, but that it may further include other elements, unless specifically stated to the contrary. [Example]

[0055] Example 1: Production of exosomes derived from black yeast

[0056] A black yeast strain, Aureobasidium Pullulans OFY11-1 strain (obtained from KCTC; accession number 14158BP), was cultured with shaking, and the culture medium was centrifuged to pellet the black yeast cells to remove them.

[0057] Black yeast-derived exosomes were isolated from the culture medium from which the black yeast cells had been removed using a tangential flow filtration (TFF) device.

[0058] The size and concentration of the isolated exosomes derived from black yeast were confirmed by nanoparticle tracking analysis (NTA) using NS300 (purchased from Malvern Panalytical) (Figure 1).

[0059] <Example 2: Confirmation of the ability of black yeast-derived exosomes to be delivered to skin fibroblasts>

[0060] Whether or not black yeast-derived exosomes can be delivered into human dermal fibroblasts (HDFs, purchased from ATCC) was confirmed as follows.

[0061] To fluorescently stain the membranes of the black yeast-derived exosomes prepared in Example 1, they were reacted with PKH67 (Sigma-Aldrich) fluorescent dye. After the reaction, the reaction solution was fractionated using an MW3000 (Thermo Fisher) column to remove free PKH67 fluorescent dye that had not stained the exosome membrane. As a negative control, PKH67 fluorescent dye was reacted with a buffer solution and then fractionated using the MW3000. The PKH67-stained exosomes were then cultured with pre-cultured human dermal fibroblasts, and the intracellular delivery of exosomes over time was observed using a fluorescence microscope. Hoechst fluorescent dye (Thermo Fisher) was used to stain cell nuclei, and CellMask Orange fluorescent dye (Thermo Fisher) was used to stain cell membranes.

[0062] When the delivery of black yeast-derived exosomes into cells was confirmed using a fluorescence microscope, it was confirmed that green fluorescence accumulated inside the cells over time, indicating that the black yeast-derived exosomes were delivered into the cells (Figure 2).

[0063] Example 3: Confirmation of the collagen production promoting effect of black yeast-derived exosomes

[0064] Human dermal fibroblasts dispersed in DMEM medium containing fetal bovine serum (FBS) were plated in a 24-well plate at 5x10 cells per well. 4 The cultures were divided into different concentrations and cultured for 24 hours.

[0065] Then, the black yeast culture solution or black yeast-derived exosomes prepared in Example 1 were diluted in serum-free medium and applied to human dermal fibroblasts, which were then cultured for 24 hours. To confirm the efficacy of collagen production using human dermal fibroblasts, the experimental groups were divided into the following groups:

[0066] (1) Negative control group (denoted as “control group” in Figure 3 ): the experimental group treated with serum-free medium only;

[0067] (2) Black yeast culture solution treatment group (shown as "CM" in FIG. 3): An experimental group in which the black yeast culture solution prepared in Example 1 was diluted in a serum-free medium and treated (particle number-based treatment concentration: 3.0×10 9 particles / mL);

[0068] (3) Low-concentration treatment group of black yeast-derived exosomes (shown as "low-concentration exosomes" in Figure 3): An experimental group in which the black yeast-derived exosomes prepared in Example 1 were diluted at a low concentration in a serum-free medium and treated (particle number-based treatment concentration: 3.0x10 9 particles / mL);

[0069] (4) High-concentration treatment group of black yeast-derived exosomes (shown as "high-concentration exosomes" in Figure 3): An experimental group in which the black yeast-derived exosomes prepared in Example 1 were diluted at a high concentration in a serum-free medium and treated (particle number-based treatment concentration: 3.0x10 10 particles / mL).

[0070] After culturing human dermal fibroblasts for 24 hours, the culture medium was collected and centrifuged to prepare the centrifuged culture medium. The amount of collagen synthesized by human dermal fibroblasts and accumulated in the culture medium was measured using an EIA kit for procollagen type IC-peptide (PIP) (purchased from Takara). The measured collagen amount was divided by the total cell number measured using an MTT assay kit (purchased from Sigma-Aldrich) and normalized to determine the relative collagen amount.

[0071] As a result, it was confirmed that black yeast-derived exosomes significantly increased collagen synthesis in human skin fibroblasts compared to the negative control group, and in particular, collagen synthesis increased further as the treatment concentration increased (Figure 3). In other words, it was confirmed that the black yeast-derived exosomes of the present invention increased collagen synthesis in human skin fibroblasts in a concentration-dependent manner.

[0072] In addition, the same concentration (particle number standard treatment concentration: 3.0X10 9 It was found that the amount of collagen produced by human skin fibroblasts in the group treated with low concentrations of black yeast-derived exosomes was greater than that in the group treated with black yeast culture medium (particles / mL) (Figure 3).

[0073] Therefore, a cosmetic composition containing the black yeast-derived exosomes of the present invention as an active ingredient has the activity of increasing collagen synthesis, and the black yeast-derived exosomes can be usefully utilized as an active ingredient in functional cosmetic compositions for improving skin wrinkles, improving skin elasticity, and / or skin regeneration.

[0074] <Example 4: Confirmation of the ability of black yeast-derived exosomes to be delivered to mouse melanoma cells>

[0075] Whether or not exosomes derived from black yeast can be delivered into mouse melanoma (B16F10; purchased from ATCC) was confirmed as follows.

[0076] The membranes of the black yeast-derived exosomes prepared in Example 1 were reacted with PKH67 fluorescent dye to fluorescently stain them. After the reaction, the reaction solution was fractionated using an MW3000 column to remove free PKH67 fluorescent dye that had not stained the exosome membrane. As a negative control, PKH67 fluorescent dye was reacted with a buffer solution and then fractionated using the MW3000. The PKH67-stained exosomes were cultured with pre-cultured B16F10 cells, and the intracellular delivery of exosomes over time was observed using a fluorescence microscope. Cell nuclei were stained using Hoechst fluorescent dye, and cell membranes were stained using CellMask Orange fluorescent dye.

[0077] When the delivery of black yeast-derived exosomes into cells was confirmed using a fluorescence microscope, it was confirmed that green fluorescence accumulated inside the cells over time, indicating that black yeast-derived exosomes were delivered into melanoma cells (Figure 4).

[0078] Example 5: Inhibitory effect of melanin production

[0079] The whitening effect of black yeast-derived exosomes was confirmed through the degree of inhibition of melanin production in melanoma cells. The melanoma cells were derived from mouse melanoma and secrete the black pigment called melanin. Melanoma cells were plated in a 48-well plate at 8 x 10 cells per well. 3 After dividing the cells at a concentration of 1000 kJ / ml, the cells were cultured for 24 hours in phenol red-free DMEM medium containing 10% fetal bovine serum.

[0080] Then, the black yeast-derived exosomes prepared in Example 1 were diluted in a culture medium containing α-melanocyte stimulating hormone (α-MSH), a melanin synthesis stimulator, and then applied to melanoma cells, which were then cultured for 48 hours. The experimental groups were divided as follows:

[0081] (1) Negative control group (shown as "negative control group" in Figure 5): an experimental group treated with culture medium containing a-MSH, a melanin synthesis stimulator;

[0082] (2) Positive control group (shown as "positive control group" in Figure 5): an experimental group treated with a culture medium containing a mixture of melanin synthesis stimulators a-MSH and arbutin (final concentration: 1 mM);

[0083] (3) Low-concentration treatment group of black yeast-derived exosomes (shown as "low-concentration exosomes" in Figure 5): An experimental group treated with a culture medium containing a mixture of a-MSH, a melanin synthesis stimulator, and a low concentration of black yeast-derived exosomes (particle number-based treatment concentration: 3.0 × 10 9 particles / mL);

[0084] (4) High-concentration treatment group of black yeast-derived exosomes (shown as "high-concentration exosomes" in Figure 5): An experimental group treated with a culture medium containing a mixture of a-MSH, a melanin synthesis stimulator, and a high concentration of black yeast-derived exosomes (particle number-based treatment concentration: 3.0 × 10 10 particles / mL).

[0085] After culturing the melanoma cells for 48 hours, the culture medium was collected and the melanoma cells were washed with a washing solution (PBS; purchased from Thermo Fisher).

[0086] The collected culture medium was mixed with CCK-8 assay reagent (purchased from Dojindo) and incubated for 2 hours at 37°C under 5% CO2. The supernatant was then transferred to a 96-well plate and the absorbance measured at 450 nm. The washed melanoma cells were treated with 1N NaOH (purchased from Merck-Millipore) containing 10% DMSO. The plate was then sealed and heated at 85°C for 20 minutes to extract melanin from the cells. The absorbance of the extracted melanin was measured at 405 nm to calculate the amount of melanin.

[0087] As a result, it was confirmed that black yeast-derived exosomes reduced melanin synthesis in melanoma cells in a concentration-dependent manner (Figure 5).

[0088] Therefore, a cosmetic composition containing the black yeast-derived exosomes of the present invention as an active ingredient has a whitening effect, and the black yeast-derived exosomes can be usefully utilized as an active ingredient in functional cosmetic compositions for improving skin tone and brightness and / or skin whitening.

[0089] <Example 6: Confirmation of the promoting effect of hyaluronic acid synthase or aquaporin synthesis>

[0090] Human keratinocytes (HaCaT) were cultured in a 6-well cell culture plate at 2x10 per well. 5 The cells were cultured in DMEM medium containing 10% fetal bovine serum for 24 hours. After that, the medium was changed to 10% FBS and calcium (Ca 2+After the medium was replaced with DMEM medium without ATP, black yeast exosomes were diluted in the medium and incubated for 24 hours. After 24 hours, the cells were harvested and washed with chilled phosphate-buffered saline (PBS). Total RNA was extracted using an RNeasy mini kit (Qiagen, Germany). Using the extracted RNA as a template, reverse transcription was performed to synthesize cDNA using a cDNA Synthesis Kit (PhileKorea, Korea). The synthesized cDNA was quantified and diluted with water, and the same amount was used for all reactions. TaqMan® Universal Master Mix II and TaqMan® Gene Expression Assays (Thermo Fisher, USA) were used, along with HAS2 primer (ID: Hs00193435_m1) and AQP3 primer (ID: HS01105469_G1). Quantitative real-time PCR was performed using a StepOnePlus® Real-Time PCR System (Applied Biosystems, USA).

[0091] The experimental results were calculated using the ΔΔCt method with the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the standard. The mRNA expression level of the negative control group, which was not treated with black yeast-derived exosomes, was set at approximately 1.0, and the expression levels of the HAS2 (hyaluronan synthase 2) and AQP3 (aquaporin 3) genes in the experimental groups were quantified using this as the standard (Tables 1 and 2).

[0092] [Table 1]

[0093] [Table 2]

[0094] The expression level of the HAS2 gene, a hyaluronic acid synthase, was measured following treatment with black yeast-derived exosomes. As can be seen in Table 1, when treated with black yeast-derived exosomes, the expression of the HAS2 gene increased in a concentration-dependent manner, up to approximately three-fold, compared to the untreated group.

[0095] In addition, the expression level of the transmembrane protein aquaporin 3 (AQP3) gene was measured after treatment with black yeast-derived exosomes. As can be seen from Table 2, when treated with black yeast-derived exosomes, the expression of the AQP3 gene increased in a concentration-dependent manner compared to the untreated group, with the expression of the AQP3 gene increasing by up to approximately 4.5 times.

[0096] Therefore, a cosmetic composition containing the black yeast-derived exosomes of the present invention as an active ingredient has a skin moisturizing effect through an increase in hyaluronic acid or aquaporin synthesis, and can be usefully utilized as an active ingredient in a cosmetic composition for moisturizing skin.

[0097] Although the present invention has been described above with reference to the above-mentioned embodiments, the present invention is not limited thereto. Those skilled in the art will recognize that modifications and variations can be made without departing from the spirit and scope of the present invention, and that such modifications and variations are also included in the present invention.

Claims

1. A cosmetic composition for improving skin condition, comprising isolated exosomes derived from black yeast (Aureobasidium pullulans) as an active ingredient.

2. 2. The cosmetic composition for improving skin condition according to claim 1, wherein the improvement of skin condition is improvement of skin elasticity, improvement of skin wrinkles, improvement of skin texture, improvement of skin tone, improvement of skin brightness, skin regeneration, skin moisturizing, or skin whitening.

3. 3. The cosmetic composition for improving skin conditions according to claim 1 or 2, wherein the black yeast-derived exosomes are isolated from a black yeast culture solution, a black yeast fermentation product, or a biological solution of black yeast.

4. 3. The cosmetic composition for improving skin condition according to claim 1 or claim 2, which is in at least one dosage form selected from the group consisting of lotions, liquids, creams, deposition masks, gels, aerosols, and powders.

5. 3. The cosmetic composition for improving skin conditions according to claim 1, which is applied to or deposited on at least one surface of a patch, a mask pack, or a mask sheet.

Images