Cosmetic composition for skin care containing milk exosomes and egg white exosomes stabilized by formation of lipid nanoparticles

Abstract

The present invention relates to a cosmetic composition for skin improvement containing milk exosomes and egg white exosomes stabilized by lipid nanoparticles as active ingredients. Specifically, the invention relates to a cosmetic composition containing milk exosomes and egg white exosomes stabilized by manufacturing them into nanoparticles using microfluidic chip technology as active ingredients, which exhibits excellent moisturizing, skin barrier improvement, skin wrinkle and elasticity improvement, and antioxidant effects.

Description

Technology Field

[0001] The present invention relates to a cosmetic composition for skin improvement containing milk exosomes and egg white exosomes stabilized by lipid nanoparticles as active ingredients. Specifically, the invention relates to a cosmetic composition containing milk exosomes and egg white exosomes stabilized by manufacturing them into nanoparticles using microfluidic chip technology as active ingredients, which exhibits excellent moisturizing, skin barrier improvement, skin wrinkle and elasticity improvement, and antioxidant effects. Background Technology

[0002] The functions of cosmetics have expanded beyond cleanliness and simple makeup to include removing or inhibiting the causes of skin aging, such as pollutants, stress, ultraviolet rays, free radicals, and peroxides. Skin aging is accelerating as the skin becomes rough, dry, and dull due to exposure to various environmental pollutants and stress, and this phenomenon is closely related to the supply and maintenance of moisture in the skin.

[0003] Recently, there has been active development of cosmetics with excellent wrinkle improvement and moisturizing effects using safe natural materials; however, there have been problems in that cosmetics containing various active ingredients are difficult to maintain formulation stability and have low skin absorption rates, making it difficult to expect sufficient skin improvement activity when used.

[0004] Among these active ingredients, milk and egg white extracts present a problem in that their activity decreases over time within standard formulations. Furthermore, as they deteriorate, their ability to penetrate cell membranes becomes very low, making absorption difficult. Consequently, there has been a demand to improve the stability and bioavailability of milk and egg white extracts.

[0005] The inventors conducted research on exosomes and confirmed that when milk exosomes and egg white exosomes are separated and purified by a specific method and manufactured together into nanoparticles using microfluidic chip technology, the activity stability within the formulation is improved and the skin absorption rate is enhanced, as well as exhibiting excellent skin improvement activity due to the synergistic effect, thereby completing the present invention. Prior art literature

[0006] (001) Republic of Korea Registered Patent No. 10-2265811 (June 16, 2021)(002) Republic of Korea Published Patent No. 10-2024-0139100 (September 23, 2024)(003) Republic of Korea Published Patent No. 10-2024-0173287 (December 11, 2024) The problem to be solved

[0007] The present invention aims to provide a cosmetic composition containing milk exosomes and egg white exosomes stabilized by lipid nanoparticles as active ingredients, which has high stability and exhibits excellent antioxidant, moisturizing, skin barrier improvement, and skin wrinkle and elasticity improvement effects. means of solving the problem

[0008] To achieve the above objective, according to the present invention, a cosmetic composition is provided containing milk exosomes and egg white exosomes stabilized with lipid nanoparticles as active ingredients.

[0009] Preferably, milk exosomes and egg white exosomes stabilized with lipid nanoparticles as the active ingredient are,

[0010] (A) a1) a step of inoculating sterilized milk with a starter, culturing at 22–43°C for 4–24 hours, and separating the whey by centrifugation; a2) a step of obtaining the supernatant by centrifuging the separated whey; a3) a step of freeze-drying the supernatant; a4) a step of forming an aqueous two-phase system using PEG (Polyethylene glycol) / Dextran on the freeze-dried product; a5) a step of preparing milk exosomes by obtaining a lower phase in which exosomes are concentrated; and a6) a step of preparing an aqueous phase by mixing 5–15% by weight of glutathione with 10–20% by weight of the milk exosomes and 65–75% by weight of purified water;

[0011] (B) b1) a step of centrifuging the whole egg to separate the yolk and egg white, obtaining the supernatant, and separating the egg white; b2) a step of mixing the egg white with purified water in a weight ratio of 1:5 to 1:10, stirring under cold water for 9 to 15 hours, and then removing the precipitate through filtration; b3) a step of centrifuging the filtrate at 1,000xg to 10,000xg, separating the supernatant containing egg white exosomes, and freeze-drying it; and b4) a step of preparing an oil phase by dissolving freeze-dried powder in dipropylene glycol and then mixing it with at least one emulsifying polymer selected from the group consisting of phosphatidyl choline, PCL (Poly-ε-caprolactone), PGA (Polyglycolic acid), PLLA (Poly-L-lactide), sphingomyelin, phosphatidyl serine, phosphatidyl inositol, phosphatidyl ethanolamine, and lecithin;

[0012] (C) c1) a step of homogenizing the above oil phase and water phase by passing them through a microfluidic chip using a weight ratio of 1:5 to 1:10, respectively; c2) a step of mixing and stirring a water-soluble solubilizing agent; and

[0013] (D) It is characterized by being manufactured by a method including the step of forming nanoparticles having a size of 50 to 400 nm under conditions of 300 to 800 bar using a high-pressure homogenizer (Microfluidizer).

[0014] At this time, preferably, the homogenization in step (C) is characterized by being performed by passing through a microfluidic chip having a flow channel of 1 μm to 5 μm repeatedly 1 to 3 times under conditions of 1 to 10 bar and 15 to 40°C.

[0015] Milk exosomes and egg white exosomes stabilized by the lipid nanoparticles as active ingredients are contained in an amount of 0.0001 to 30.0% (w / w) based on the total weight of the composition.

[0016] The above cosmetic composition is characterized by being for skin moisturization, skin barrier improvement, skin wrinkle and elasticity improvement, or antioxidant use. Effects of the invention

[0017] Stabilized milk exosomes and egg white exosomes as active ingredients, which are purified using a pretreatment under specific conditions and an aqueous two-phase system and then formed into nanoparticles using microfluidic chip technology, have high activity stability within the formulation and exhibit excellent antioxidant, skin moisturizing, skin barrier improvement, and skin wrinkle and elasticity improvement effects, so they can be usefully used as cosmetic ingredients for skin improvement. Brief explanation of the drawing

[0018] Figure 1 is a graph showing the results of NTA analysis to confirm the size distribution and number of milk exosome particles according to one embodiment of the present invention. Figure 2 is a graph showing the results of NTA analysis to confirm the size distribution and number of egg white exosome particles according to one embodiment of the present invention. Figure 3 is a graph showing the results of NTA analysis to confirm the size distribution and number of milk exosomes and egg white exosomes stabilized with lipid nanoparticles according to one embodiment of the present invention. Figure 4 is a graph showing the results of evaluating the cytotoxicity of milk exosomes and egg white exosomes according to one embodiment of the present invention using an MTT assay. Figure 5 is a graph showing the results of evaluating the skin moisturizing efficacy of milk exosomes and egg white exosomes according to one embodiment of the invention based on the Aquaporin3 mRNA expression rate. Figure 6 is a graph showing the results of evaluating the skin barrier improvement efficacy of milk exosomes and egg white exosomes according to one embodiment of the present invention based on the Filaggrin expression rate. Figure 7 is a graph showing the results of evaluating the wrinkle-improving efficacy of milk exosomes and egg white exosomes according to one embodiment of the present invention based on the COLA1A1 mRNA expression rate. Figure 8 is a graph showing the results of evaluating the wrinkle-improving efficacy of milk exosomes and egg white exosomes according to one embodiment of the present invention based on the MMP-1 mRNA expression rate. Figure 9 is a graph showing the results of evaluating the skin elasticity efficacy of milk exosomes and egg white exosomes according to one embodiment of the present invention based on the Elastin mRNA expression rate. Figure 10 is a graph showing the results of evaluating the antioxidant efficacy of milk exosomes and egg white exosomes according to one embodiment of the present invention as SOD-2 mRNA expression rate. Specific details for implementing the invention

[0019] The present invention will be described in more detail below.

[0020] According to the present invention, a cosmetic composition is provided that contains milk exosomes and egg white exosomes stabilized with lipid nanoparticles as active ingredients.

[0021] Milk is one of nature's complete foods, containing a large amount of essential nutrients, and has been used for skin care since ancient times. The lipid components and proteins in milk are effective for moisturizing the skin, while whey protein and casein aid in skin regeneration and strengthening the skin barrier function. Milk has the particular advantage of being suitable even for sensitive skin due to its soft and mild texture.

[0022] Egg white is the transparent liquid portion of a whole egg excluding the yolk, composed primarily of protein and water. It contains almost no fat or cholesterol, making it a high-quality source of protein. Furthermore, the lysozyme component in egg white provides antibacterial effects, helping to strengthen the immune system. Additionally, the moisturizing properties of the proteins in egg white help improve skin elasticity and pore health.

[0023] In the present invention, exosomes are purified from milk and egg white, then nano-sized using microfluidic chip technology and used as a cosmetic.

[0024] Preferably, milk exosomes and egg white exosomes stabilized with lipid nanoparticles as the active ingredient are,

[0025] (A) a1) a step of inoculating sterilized milk with a starter, culturing at 22–43°C for 4–24 hours, and separating the whey by centrifugation; a2) a step of obtaining the supernatant by centrifuging the separated whey; a3) a step of freeze-drying the supernatant; a4) a step of forming an aqueous two-phase system using PEG (Polyethylene glycol) / Dextran on the freeze-dried product; a5) a step of preparing milk exosomes by obtaining a lower phase in which exosomes are concentrated; and a6) a step of preparing an aqueous phase by mixing 5–15% by weight of glutathione with 10–20% by weight of the milk exosomes and 65–75% by weight of purified water;

[0026] (B) b1) a step of centrifuging the whole egg to separate the yolk and egg white, obtaining the supernatant, and separating the egg white; b2) a step of mixing the egg white with purified water in a weight ratio of 1:5 to 1:10, stirring under cold water for 9 to 15 hours, and then removing the precipitate through filtration; b3) a step of centrifuging the filtrate at 1,000xg to 10,000xg, separating the supernatant containing egg white exosomes, and freeze-drying it; and b4) a step of preparing an oil phase by dissolving freeze-dried powder in dipropylene glycol and then mixing it with at least one emulsifying polymer selected from the group consisting of phosphatidyl choline, PCL (Poly-ε-caprolactone), PGA (Polyglycolic acid), PLLA (Poly-L-lactide), sphingomyelin, phosphatidyl serine, phosphatidyl inositol, phosphatidyl ethanolamine, and lecithin;

[0027] (C) c1) a step of homogenizing the above oil phase and water phase by passing them through a microfluidic chip using a weight ratio of 1:5 to 1:10, respectively; c2) a step of mixing and stirring a water-soluble solubilizing agent; and

[0028] (D) It is manufactured by a method comprising the step of forming nanoparticles having a size of 50 to 400 nm under conditions of 300 to 800 bar using a microfluidizer.

[0029] First, exosomes are purified from milk.

[0030] According to one embodiment of the present invention, a starter is inoculated into sterilized milk, cultured at 22 to 43°C for 4 to 24 hours, and the whey is separated by centrifugation.

[0031] A thermophilic starter or a mesophilic starter may be used as the above starter. When using a thermophilic starter, the mixture is inoculated uniformly and cultured at 42–43°C for 4–8 hours, and when using a mesophilic starter, the mixture is cultured at 22–25°C for 12–24 hours.

[0032] The obtained culture is centrifuged at 3–5°C and about 4,000xg for 15–20 minutes, and the supernatant is obtained to separate the whey from the curd. At this time, to remove suspended proteins, the whey may be additionally centrifuged at 3–5°C and about 10,000xg for 10–15 minutes.

[0033] The above supernatant containing whey is freeze-dried. Freeze-drying is a method of drying in which the material to be dried is frozen by rapidly lowering the temperature of the container, and then the pressure inside the container is brought close to a vacuum to immediately sublimate the solidified solvent contained in the material into water vapor. The material is frozen at -50 to -80°C for 15 to 25 hours and dried in a freeze-dryer under a vacuum for 72 to 120 hours. At this time, the vacuum state typically refers to the input state of the freeze-dryer.

[0034] In order to efficiently separate and purify exosomes, the present invention used an aqueous two-phase partition method, which is a separation method utilizing the difference in affinity to each layer by creating two layers with two types of aqueous solutions that do not dissolve well in each other.

[0035] Generally, PEG / salt (such as sulfate, phosphate, citrate, etc. as salts) can be used to form an aqueous two-phase system, but to achieve the objective of the present invention, it is preferable to use PEG / Dextran. Dextran is a natural polymer obtained by bacterial action and is used as a thickening agent, binder, and bulking agent in cosmetic formulations.

[0036] Purified water is added to the above freeze-dried product, and an aqueous two-phase system is formed using PEG (Polyethylene glycol) / Dextran.

[0037] In the formation of an aqueous two-phase system, PEG having a molecular weight of 10,000 to 35,000 is used in an amount of 1 to 15% by weight, preferably 2 to 5% by weight, and Dextran having a molecular weight of 300,000 to 650,000 is used in an amount of 1 to 8% by weight, preferably 1 to 3% by weight. It is more preferable that the PEG and Dextran are used in a concentration ratio of 3.3% by weight:1.7% by weight, as this results in the highest exosome yield and the best stability.

[0038] Milk exosomes are prepared by forming an aqueous two-phase system and obtaining the lower layer in which exosomes are concentrated. To increase purity, an additional washing process can be repeated 2 to 3 times by adding the above aqueous two-phase solution of the same concentration to the recovered lower layer.

[0039] According to one embodiment of the present invention, the egg white exosome is purified by the following method.

[0040] To separate the egg white from the whole egg, the whole egg is centrifuged to obtain the supernatant. According to one embodiment, the supernatant is obtained by centrifuging at 3,000 to 4,000 xg for 10 to 20 minutes at 3 to 5°C.

[0041] The above egg white is mixed with purified water in a weight ratio of 1:5 to 1:10 and stirred under refrigeration for 9 to 15 hours, then filtered to remove precipitates. The filtrate is centrifuged at 1,000xg to 10,000xg, and the supernatant containing egg white exosomes is separated to produce egg white exosomes.

[0042] To stabilize the milk exosomes and egg white exosomes produced in this way, they are manufactured into nanoparticles using microfluidic chip technology.

[0043] A microfluidic chip is a chip configured to perform various experiments and diagnoses by flowing fluid through microchannels based on microfluidics technology. Microfluidic chip technology is also being applied to emulsion manufacturing. Korean Published Patent No. 10-2015-0126561 discloses a microfluidic chip and a method for manufacturing liposomes using the same, wherein a micro thin plate is interposed between stacked upper and lower plates, two types of solutions are injected, and the flow rate ratio of each solution is adjusted to generate liposomes with controlled uniformity and particle size.

[0044] To this end, an aqueous phase is prepared by mixing 5 to 15 weight percent of glutathione with 10 to 20 weight percent of the milk exosomes prepared above and 65 to 75 weight percent of purified water.

[0045] In addition, the egg white exosomes are freeze-dried and dissolved in a solvent, then mixed with an emulsifying polymer to prepare an oil phase. According to a preferred embodiment of the present invention, egg white exosome freeze-dried powder, prepared by freezing at -80°C for 20 hours and drying in a freeze-dryer under vacuum for 100 hours, is dissolved in dipropylene glycol and then mixed with at least one emulsifying polymer selected from the group consisting of phosphatidyl choline, PCL (Poly-ε-caprolactone), PGA (Polyglycolic acid), PLLA (Poly-L-lactide), sphingomyelin, phosphatidyl serine, phosphatidyl inositol, phosphatidyl ethanolamine, and lecithin to prepare an oil phase.

[0046] The above oil phase and water phase are homogenized by passing them through a microfluidic chip in a weight ratio of 1:5 to 1:10, respectively. Preferably, the homogenization is performed by passing the mixture through a microfluidic chip having flow channels of 1 μm to 5 μm repeatedly 1 to 3 times under conditions of 1 to 10 bar and 15 to 40°C. A water-soluble solubilizing agent is mixed and stirred. According to one embodiment of the present invention, Polyglyceryl-10 Laurate is used as the water-soluble solubilizing agent.

[0047] The above mixture is stabilized by forming nanoparticles with a size of 50 to 400 nm using a microfluidizer under conditions of 300 to 800 bar.

[0048] Milk exosomes and egg white exosomes stabilized with lipid nanoparticles produced by this method exhibited superior antioxidant, skin moisturizing, skin barrier improvement, and skin wrinkle and elasticity improvement effects due to their synergistic effects.

[0049] At this time, milk exosomes and egg white exosomes stabilized by the lipid nanoparticles as active ingredients may be contained in an amount of 0.0001 to 30.0% (w / w) based on the total weight of the composition.

[0050] The above cosmetic composition can be manufactured in any formulation that is commonly produced, for example, skin lotion, skin toner, pack, nourishing cream, moisturizing cream, essence, body cream, body lotion, body oil, shampoo, rinse, hair conditioner, hair gel, cleansing foam, cleansing lotion, soap, patch, foundation, lipstick, makeup base, etc.

[0051] [Example]

[0052] The present invention will be explained in more detail below based on the following examples and test examples. However, the following examples are merely for illustrating the present invention and are not limited by the following examples; it will be obvious to those skilled in the art that the present invention can be modified by substitutions and equivalent alternative examples within the scope of the technical concept of the present invention.

[0054] Example 1: Preparation of milk exosomes

[0055] Milk was sterilized at low temperature for 30 minutes at 62–68°C. Mesophilic Starter was uniformly inoculated into the sterilized milk, and the mixture was cultured at 22–25°C for 12 hours to induce whey and curd formation. To separate the solids and whey from the milk, centrifugation was performed at 4,000xg for 15 minutes at 4°C to obtain the supernatant.

[0056] To remove suspended proteins, centrifugation was additionally performed at 10,000 xg for 10 minutes at 4°C. Freeze-drying was performed to reduce volume for the mass production of exosomes. The samples were frozen at -80°C for 20 hours and dried under vacuum in a freeze-dryer for 100 hours.

[0057] Purified water was added to the freeze-dried material, and an aqueous two-phase system was formed using PEG (Polyethylene glycol) / Dextran. An aqueous two-phase system was formed using 3.3 wt% of PEG (purchased from Sigma Aldrich) with a molecular weight of 10,000–35,000 and 1.7 wt% of Dextran (purchased from Sigma Aldrich) with a molecular weight of 300,000–650,000. Centrifugation was performed at 1,000 xg for 10 minutes at 4°C, and the supernatant was removed to recover exosomes.

[0058] To increase purity, an additional washing process was performed by adding the above aqueous two-phase solution of the same concentration to the recovered lower layer. After repeating the treatment three times, the lower layer containing the final exosomes was recovered.

[0060] Example 2: Preparation of Egg White Exosomes

[0061] To separate the yolk and egg white from the whole egg, the whole egg was centrifuged at 4,000 xg for 15 minutes at 4°C to obtain the supernatant (egg white). The obtained egg white was mixed with purified water at a weight ratio of 1:10 and stirred under refrigeration for 15 hours, after which a filtration step was performed to remove the precipitate.

[0062] After centrifuging the filtrate at 10,000xg, the supernatant containing egg white exosomes was separated to prepare egg white exosomes.

[0064] Example 3: Preparation of Stabilized Milk Exosomes and Egg White Exosomes

[0065] An aqueous phase was prepared by mixing 10 g of glutathione with 20 g of milk exosomes prepared in Example 1 above and 70 g of purified water.

[0066] The egg white exosome prepared in Example 2 above was frozen at -80°C for 20 hours and dried under vacuum in a freeze-dryer for 100 hours. 4g of the powder obtained after freeze-drying was dissolved in 10g of dipropylene glycol heated to 40~50°C, and then mixed with 3g of phosphatidyl choline to prepare an oil phase.

[0067] The oil phase prepared above was homogenized by passing it through a microfluidic chip. Homogenization was achieved by passing it through a microfluidic chip with 5㎛ flow channels at 3 bar at room temperature. This process was repeated a total of three times to increase the degree of homogenization through self-assembly. After homogenization, Polyglyceryl-10 Laurate, a water-soluble solubilizer, was added and mixed and stirred.

[0068] Here, nanoparticle capsules were prepared using a microfluidizer under conditions of 800 bar.

[0070] Comparative Example 1: Preparation of milk extract

[0071] 10g of whey protein powder was added to 100g of purified water and extracted for 1 hour at 50℃ with low-speed stirring. After extraction, centrifugation was performed at 10,000xg at 4℃ for 15 minutes to remove insoluble precipitates. The supernatant was filtered under reduced pressure to obtain a whey extract, which was then freeze-dried in a freeze-dryer to obtain a sample in powder form.

[0073] Comparative Example 2: Preparation of Egg White Extract

[0074] To separate the yolk and egg white from the whole egg, the whole egg was centrifuged at 4,000xg for 15 minutes at 4°C to obtain the supernatant. The obtained egg white was freeze-dried. 10g of the freeze-dried egg white powder and 100g of purified water were added, and extraction was performed at 50°C for 1 hour with low-speed stirring. After extraction, insoluble precipitates were removed by centrifugation at 10,000xg for 15 minutes at 4°C. The supernatant was subjected to vacuum filtration to obtain the egg white extract, which was then freeze-dried in a freeze-dryer to obtain a sample in powder form.

[0076] Test Example 1: Characterization of Milk-Derived Exosomes: NTA Analysis

[0077] Nanoparticle Tracking Analysis (NTA) was performed to determine the particle size distribution and the number of particles per unit volume of purified milk-derived exosomes. Figure 1 shows the NTA analysis results of the milk-derived exosomes purified according to Example 1 above. The average particle size was 147.5 nm and the concentration was 1.6E+11 particles per unit volume of 1 mL.

[0079] Test Example 2: Characterization of Egg White-Derived Exosomes: NTA Analysis

[0080] Nanoparticle Tracking Analysis (NTA) was performed to determine the particle size distribution and the number of particles per unit volume of the purified egg white-derived exosomes. Figure 2 shows the NTA analysis results of the egg white-derived exosomes purified according to Example 2 above. The average particle size was 149.1 nm and the concentration was 4.1E+10 particles per unit volume of 1 mL.

[0082] Test Example 3: Characterization of the final manufactured liposomes: NTA analysis

[0083] Nanoparticle Tracking Analysis (NTA) was performed to determine the particle size distribution and the number of particles per unit volume of liposomes prepared with an aqueous phase containing milk exosomes and an oil phase containing egg white exosomes. Figure 3 shows the NTA analysis results of the liposomes prepared according to Example 3 above. The average particle size was 148.5 nm and the concentration was 2.8E+11 particles per unit volume of 1 mL.

[0085] Test Example 4: Cytotoxicity Evaluation

[0086] An MTT assay was performed to confirm cytotoxicity. Human keratinocytes (HaCaT) were placed in a 96-well plate at a ratio of 1 x 10⁻⁶ 5 After inoculation at a concentration of cells / mL, the cells were cultured at 37°C for 18 hours under 5% CO2. After culture, the medium was removed and washed with PBS buffer. Then, fresh medium was added along with milk exosomes (Example 1), egg white exosomes (Example 2), and milk exosomes and egg white exosomes stabilized with lipid nanoparticles (Example 3) at various concentrations, and the cells were cultured again for 24 hours. To measure cell viability, formazan formed after 4 hours of adding MTT solution (5 mg / mL) was dissolved in dimethyl sulfoxide (DMSO), and the absorbance was measured at 570 nm using an ELISA reader.

[0087] Figure 4 is a graph showing the results of evaluating the cytotoxicity of the samples of the above examples using an MTT assay. As a result of the test, no cytotoxicity was observed in any of the samples at any concentration.

[0089] Test Example 5: Evaluation of moisturizing efficacy (AQP3 expression)

[0090] To confirm the moisturizing efficacy, the effect on AQP3 expression was evaluated. Human dermal fibroblast (HDFa) cells were inoculated and cultured in Fibroblast Basal Medium (Medium 106) supplemented with 100 IU / mL penicillin and 100 μg / mL streptomycin at 37°C under 5% CO2 for 24 hours. The medium was removed from each well and replaced with fresh serum-free medium. Samples corresponding to the above examples were administered to each well at various concentrations, and a 4-hour pretreatment was performed. Each well plate was irradiated with 15 mJ / cm² using a UVB irradiator (Vilber Loumet, France). 2 UVB irradiation was performed. The samples were treated with serum-free medium diluted with the sample and cultured for an additional 24 hours. During this time, PBS was used as a negative control, and 0.005% hyaluronic acid was used as a positive control. Cells that had finished cell culture were lysed using Ribo Ex™ Total RNA Isolation Solution (GeneAll Biotechnology, Korea) and a scraper, and then 0.2 mL of chloroform (Sigma-Aldrich, USA) was added and centrifuged (12,000 rpm, 4°C, 30 min). The supernatant containing RNA was separated, and an equal amount of isopropanol (Merck-Millipore, Germany) was added to the supernatant; after inversion, the mixture was centrifuged (12,000 rpm, 4°C, 30 min). RNA was precipitated, and the supernatant was discarded after removing the precipitate. The remaining precipitate was washed by adding 70% ethanol (Merck-Millipore, Germany) and centrifuging (12,000 rpm, 4°C, 10 min). After removing the ethanol and drying at room temperature, total RNA was extracted by dissolving in nuclease-free water (Affymetrix, USA). MaestroNano ®The purity and concentration of RNA were measured at A260 / A280 wavelengths using a micro-volume spectrophotometer (MN-913, Maestrogen, USA), and it was confirmed that the ratio of 260 nm to 280 nm fell within the range of 2.0–2.2. cDNA was synthesized by preparing a PCR tube containing 1 μg RNA, Oligo dT (Bionics, Korea), dNTP (Takara, Korea), and nuclease-free water, mixing for a total of 13 μL, reacting at 65°C for 5 minutes, and then reacting with M-MLV Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA) at 37°C for 50 minutes. qRT-PCR was performed to quantitatively analyze the gene expression patterns occurring within each cell based on the samples. For qRT-PCR, a reaction mixture was prepared by mixing primer, cDNA, 2X SYBR green PCR Master Mix (Applied Biosystems, USA), and HPLC (J. T. Baker, USA) in a PCR tube to a total of 20 μL, and PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems, USA).

[0091] The PCR primer sequences of the AQP3 gene are shown in Table 1 below, and Figure 5 is a graph showing the results of evaluating the moisturizing efficacy of the above samples based on the AQP3 mRNA expression rate.

[0092] As a result of the test, when treated with Example 3, AQP3 mRNA, a moisturizing factor involved in cell water transport and a protein that plays an important role in skin moisturization, increased in a concentration-dependent manner, and it was confirmed that the expression rate increased significantly within the treatment concentration range compared to the samples corresponding to Examples 1 and 2. At this time, when treated with the positive control, Hyaluronic acid 0.005%, an expression rate of 159.63% was observed.

[0093] gene Forward primer (5'→3') Reverse primer (5'→3') AQP3 5'-CCT TTG GCT TTG CTG CAC TC-3' 5'-ACG GGG TTG TTG TAA GGG TCA-3' GAPDH 5'-GTC TCC TCT GAC TTC AAC AGC G-3' 5'-ACC ACC CTG TTG CTG TAG CCA A-3'

[0095] Test Example 6: Evaluation of Skin Barrier Improvement Efficacy

[0096] To confirm the skin barrier-strengthening efficacy of exosomes derived from milk and egg white, their effect on Filaggrin expression was evaluated. Human Keratinocyte (HaCaT) cell lines were inoculated and cultured in Fibroblast Basal Medium (Medium 106) supplemented with 100 IU / mL penicillin and 100 μg / mL streptomycin at 37°C under 5% CO2 for 24 hours. The medium was removed from each well and replaced with fresh serum-free medium. The exosome samples from the above example were administered to each well at various concentrations, and a 4-hour pretreatment was performed. Each well plate was irradiated with 20 mJ / cm² using a UVB irradiation device (Vilber Loumet, France). 2UVB irradiation was performed. The samples were treated with serum-free medium diluted with the sample and cultured for an additional 24 hours. During this time, PBS was used as a negative control, and 100 nM CaCl2 was used as a positive control. Cells that had finished cell culture were lysed using Ribo Ex™ Total RNA Isolation Solution (GeneAll Biotechnology, Korea) and a scraper, and then 0.2 mL of chloroform (Sigma-Aldrich, USA) was added and centrifuged (12,000 rpm, 4°C, 30 min). The supernatant containing RNA was separated, and an equal amount of isopropanol (Merck-Millipore, Germany) was added to the supernatant; after inversion, the mixture was centrifuged (12,000 rpm, 4°C, 30 min). RNA was precipitated, and the supernatant was discarded after removing the precipitate. The remaining precipitate was then washed by adding 70% ethanol (Merck-Millipore, Germany) and centrifuging (12,000 rpm, 4°C, 10 min). After removing the ethanol and drying at room temperature, total RNA was extracted by dissolving in nuclease-free water (Affymetrix, USA). MaestroNano ®The purity and concentration of RNA were measured at A260 / A280 wavelengths using a micro-volume spectrophotometer (MN-913, Maestrogen, USA), and it was confirmed that the ratio of 260 nm to 280 nm fell within the range of 2.0–2.2. cDNA was synthesized by preparing a PCR tube containing 1 μg RNA, Oligo dT (Bionics, Korea), dNTP (Takara, Korea), and nuclease-free water, mixing the mixture in a total of 13 μL, reacting at 65°C for 5 minutes, and then reacting with M-MLV Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA) at 37°C for 50 minutes. qRT-PCR was performed to quantitatively analyze the gene expression patterns occurring within each cell based on the samples. For qRT-PCR, a reaction mixture was prepared by mixing primer, cDNA, 2X SYBR green PCR Master Mix (Applied Biosystems, USA), and HPLC (J. T. Baker, USA) in a PCR tube to a total of 20 μL, and PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems, USA).

[0097] The PCR primer sequences for the Filaggrin gene are shown in Table 2 below, and Figure 6 is a graph showing the results of evaluating the skin barrier strengthening efficacy of milk and egg white-derived exosomes according to the above examples based on Filaggrin mRNA expression. As a result of the test, it was confirmed that when Example 3, a sample in which milk exosomes and egg white exosomes were stabilized together as nanoparticles, was treated, the increase rate of Filaggrin mRNA, a skin barrier protein, was greater than that of Examples 1 and 2. At this time, when treated with the positive control CaCl2 100 nM, an expression rate of 152.8% was observed.

[0098] gene Forward primer (5'→3') Reverse primer (5'→3') Filaggrin 5'-GCT GAA GGA ACT TCT GGA AAA GG-3' 5'-GTT GTG GTC TAT ATC CAA GTG ATC-3' GAPDH 5'-GTC TCC TCT GAC TTC AAC AGC G-3' 5'-ACC ACC CTG TTG CTG TAG CCA A-3'

[0100] Test Example 7: Evaluation of Anti-aging Efficacy (COL1A1 Expression)

[0101] To confirm the anti-aging (wrinkle improvement) efficacy, the effect on COL1A1 expression was evaluated. Human dermal fibroblast (HDFa) cells were inoculated and cultured at 37°C for 24 hours under 5% CO2 in Fibroblast Basal Medium (Medium 106) supplemented with 100 IU / mL penicillin and 100 μg / mL streptomycin. The medium was removed from each well and replaced with fresh serum-free medium. Samples corresponding to Example 3 and Examples 1–2 were administered to each well at various concentrations, and a 4-hour pretreatment was performed. Each well plate was irradiated with 15 mJ / cm² using a UVB irradiator (Vilber Loumet, France). 2UVB irradiation was performed. The samples were treated with serum-free medium diluted with the sample and cultured for an additional 24 hours. During this time, PBS was used as a negative control, and TGF-β 5 ng / mL was used as a positive control. Cells that had finished cell culture were lysed using Ribo Ex™ Total RNA Isolation Solution (GeneAll Biotechnology, Korea) and a scraper, and then 0.2 mL of chloroform (Sigma-Aldrich, USA) was added and centrifuged (12,000 rpm, 4°C, 30 min). The supernatant containing RNA was separated, and an equal amount of isopropanol (Merck-Millipore, Germany) was added to the supernatant; the mixture was inverted and centrifuged (12,000 rpm, 4°C, 30 min). RNA was precipitated, and the supernatant was discarded after removing the precipitate. The remaining precipitate was washed by adding 70% ethanol (Merck-Millipore, Germany) and centrifuging (12,000 rpm, 4°C, 10 min). After removing the ethanol and drying at room temperature, total RNA was extracted by dissolving in nuclease-free water (Affymetrix, USA). MaestroNano ®The purity and concentration of RNA were measured at A260 / A280 wavelengths using a micro-volume spectrophotometer (MN-913, Maestrogen, USA), and it was confirmed that the ratio of 260 nm to 280 nm fell within the range of 2.0–2.2. cDNA was synthesized by preparing a PCR tube containing 1 μg RNA, Oligo dT (Bionics, Korea), dNTP (Takara, Korea), and nuclease-free water, mixing for a total of 13 μL, reacting at 65°C for 5 minutes, and then reacting with M-MLV Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA) at 37°C for 50 minutes. qRT-PCR was performed to quantitatively analyze the gene expression patterns occurring within each cell based on the samples. For qRT-PCR, a reaction mixture was prepared by mixing primer, cDNA, 2X SYBR green PCR Master Mix (Applied Biosystems, USA), and HPLC (J. T. Baker, USA) in a PCR tube to a total of 20 μL, and PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems, USA).

[0102] The PCR primer sequences for the COL1A1 gene are shown in Table 3 below, and Figure 7 is a graph showing the results of evaluating the anti-aging efficacy of the samples based on COL1A1 mRNA expression rates. The test results confirmed that when the sample of Example 3 was treated, the collagen-producing factor COLA1A mRNA increased in a concentration-dependent manner, and the expression rate increased significantly within the treatment concentration range compared to the samples corresponding to Examples 1 and 2. At this time, when treated with the positive control TGF-β 5 ng / mL, an expression rate of 156.4% was observed.

[0103] gene Forward primer (5'→3') Reverse primer (5'→3') COL1A1 5'-GAG AGC ATG ACC GAT GGA TT-3' 5'-CCT TCT TGA GGT TGC CAG TC-3' GAPDH 5'-GTC TCC TCT GAC TTC AAC AGC G-3' 5'-ACC ACC CTG TTG CTG TAG CCA A-3'

[0105] Test Example 8: Evaluation of anti-aging efficacy (inhibition of MMP-1 expression)

[0106] To confirm the anti-aging (wrinkle improvement) efficacy, the effect on MMP-1 expression was examined. The experimental method was the same as in the above test example, and the PCR primer sequences of the MMP-1 gene are shown in Table 4 below. Figure 8 is a graph showing the results of evaluating the wrinkle improvement efficacy of the above samples based on the MMP-1 mRNA expression rate.

[0107] As a result of the test, it was confirmed that when the sample of Example 3 was treated, the mRNA of the collagenase factor MMP-1 decreased in a concentration-dependent manner, and the inhibition rate was significantly increased within the treatment concentration range compared to the samples corresponding to Examples 1 and 2. At this time, when treated with the positive control TGF-β 5 ng / mL, an expression rate of 44.75% was observed.

[0108] gene Forward primer (5'→3') Reverse primer (5'→3') MMP-1 5'-GGG CTT GAA GCT GCT TAC GA-3' ACA GCC CAG TAC TTA TTC CCT TTG-3' GAPDH 5'-GTC TCC TCT GAC TTC AAC AGC G-3' 5'-ACC ACC CTG TTG CTG TAG CCA A-3'

[0110] Test Example 9: Evaluation of Skin Elasticity Efficacy (Elastin Expression)

[0111] To confirm the efficacy on skin elasticity, the effect on Elastin expression was examined. HDF cells were cultured in Fibroblast Basal Medium (Medium 106, GIBCO, Cat No. M-106-500) supplemented with 100 IU / mL penicillin (GIBCO, Cat No. 15140122), 100 μg / mL streptomycin (GIBCO, Cat No. 15140122), and Low Serum Growth Supplement (LSGS, GIBCO, Cat No. S00310) at 37°C under 5% CO2 for 18 hours. After culture, the medium was removed, and the cells were treated with the samples at various concentrations and cultured for 48 hours. After culture, RNA was extracted using an RNA extraction kit (Bioneer, Cat No. K-3140), and qRT-PCR was performed using a qPCR master mix (Bioneer, Cat No. K6251). The Real-Time PCR System was performed using Applied Biosystems, USA.

[0112] The PCR primer sequences of the Elastin gene are shown in Table 5 below. Figure 9 is a graph showing the results of evaluating the skin elasticity efficacy of the above samples based on the Elastin mRNA expression rate.

[0113] As a result of the test, it was confirmed that Elastin mRNA increased in a concentration-dependent manner when the sample of Example 3 was treated, and that the expression rate increased significantly within the treatment concentration range compared to the samples corresponding to Examples 1 and 2. At this time, when treated with the positive control TGF-β 10 ng / mL, an expression rate of 158.25% was observed.

[0114] gene Forward primer (5'→3') Reverse primer (5'→3') Elastin 5'-GGCCATTCCTGGTGGAGTTCC-3' 5-AACTGG CTTAAGAGGTTTGCC-3' GAPDH 5'-GTC TCC TCT GAC TTC AAC AGC G-3' 5'-ACC ACC CTG TTG CTG TAG CCA A-3'

[0116] Test Example 10: Evaluation of Antioxidant Efficacy

[0117] To confirm the antioxidant efficacy, the effect on SOD-2 expression was evaluated. Human dermal fibroblast (HDFa) cells were inoculated and cultured at 37°C for 18 hours under 5% CO2 in Fibroblast Basal Medium (Medium 106) supplemented with 100 IU / mL penicillin, 100 μg / mL streptomycin, and Low Serum Growth Supplement (LSGS, GIBCO, Cat No. S00310). The medium was removed from each well, and the samples were treated at different concentrations in fresh serum-free medium and cultured for 48 hours. Vitamin C 0.005% was used as a positive control. After incubation, total RNA was extracted using an RNA extraction kit (Bioneer, Cat No. K-3140). RNA purity and concentration were measured at A260 / A280 wavelengths using a SpextraDrop Micro-Volume Microplate (MOLECULAR DEVICES), and it was confirmed that the ratio of 260 nm to 280 nm fell within the range of 2.0–2.2. cDNA was synthesized using Reverse Transcription 5X Premix (ELPis, Cat No. EBT-1515) to prepare a total volume of 20 μL. qRT-PCR was performed to quantitatively analyze the gene expression patterns occurring within each cell of the sample. For qRT-PCR, primers, cDNA, and AccuPower were placed in the PCR tube. ® 2X GreenStar TMA reaction mixture was prepared by mixing a total of 20 μL of qPCR Master Mix (Bioneer, Cat No. K6251), and PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems, USA). The PCR primer sequences for the SOD-2 gene are shown in Table 6 below, and Figure 10 is a graph showing the results of evaluating the antioxidant efficacy of the above samples based on the SOD-2 mRNA expression rate.

[0118] As a result of the test, when the sample prepared according to Example 3 and the samples corresponding to Examples 1 and 2 were treated, SOD-2 expression increased in a concentration-dependent manner, and the sample of Example 3 showed a higher rate of expression increase. At this time, when treated with the positive control, Vitamin C 0.005%, an expression rate of 168.1% was observed.

[0119] gene Forward primer (5'→3') Reverse primer (5'→3') SOD-2 5'-CGTCACCGAGGAGAAGTACC -3' 5'-CTGATTTGGACAAGCAGCAA-3' GAPDH 5'-GTC TCC TCT GAC TTC AAC AGC G-3' 5'-ACC ACC CTG TTG CTG TAG CCA A-3'

[0121] Formulation Example 1: Preparation of a cream

[0122] A cream containing the stabilized milk exosomes and egg white exosomes of Example 3 was prepared by a conventional method according to the composition of Table 7 below. A cream containing the exosomes of Examples 1 and 2, the milk extract of Comparative Example 1, and the egg white extract of Comparative Example 2 was used as a comparative formulation example.

[0123] ingredient Formulation Example 1 Comparative formulation example 1 2 3 4 Content (weight %) Example 3 5 - - - - Example 1 - 5 - - - Example 2 - - 5 - - Comparative Example 1 - - - 5 - Comparative Example 2 - - - - 5 glycerin 10 10 10 10 10 Butylene glycol 5 5 5 5 5 Glyceryl oleate 1.8 1.8 1.8 1.8 1.8 Cetearyl Oliveate 0.5 0.5 0.5 0.5 0.5 Sorbitan Oliveate 0.5 0.5 0.5 0.5 0.5 Caprylic / Capric Triglyceride 5.0 5.0 5.0 5.0 5.0 Cetylethylhexanoate 1.0 1.0 1.0 1.0 1.0 Beeswax 0.5 0.5 0.5 0.5 0.5 Squalan 0.2 0.2 0.2 0.2 0.2 1,2-hexanediol 0.2 0.2 0.2 0.2 0.2 Cholesteril / Behenyl / Octyldodecyl Lauroyl Glutamate 1.0 1.0 1.0 1.0 1.0 Dimethicone 0.5 0.5 0.5 0.5 0.5 Cyclopentasilonsaccharide / Cyclohexasiloxane 2.0 2.0 2.0 2.0 2.0 Cetearyl alcohol 1.0 1.0 1.0 1.0 1.0 mineral oil 2.5 2.5 2.5 2.5 2.5 Disodium EDTA 0.02 0.02 0.02 0.02 0.02 BHT 0.05 0.05 0.05 0.05 0.05 Tocopheryl acetate 0.3 0.3 0.3 0.3 0.3 Panthenol 0.2 0.2 0.2 0.2 0.2 Ethylhexyl methoxycinnamate 0.2 0.2 0.2 0.2 0.2 incense 0.01 0.01 0.01 0.01 0.01 purified water Remaining amount Remaining amount Remaining amount Remaining amount Remaining amount

[0125] Test Example 11: Effect of inhibiting transdermal water evaporation

[0126] A turbometer (Tewameter, Courage & Khazaka) was used to confirm the effect of inhibiting transepidermal water evaporation (moisturizing activity). Twenty adult men and women aged 30 to 40 with dry skin were tested. The inner lower part of the left arm was divided into sections, and transepidermal water loss was measured three times per section. Subsequently, Formulation Example 1 and Comparative Formulation Examples 1 to 4 were applied twice daily for five days, and transepidermal water loss (TEWL) was measured. Measurements were conducted in a constant temperature and humidity room at an indoor temperature of 24±2℃ and a relative humidity of 40±2%. The average value obtained from three measurements per condition was used. The results represent the amount of water evaporation after 3 and 5 days of application, based on the transepidermal water loss before application as 100%. Table 8 shows the results of evaluating the effect of inhibiting transepidermal water evaporation by the above samples.

[0127] Moisture evaporation (%) beginning 3 days after application 5 days after application Formulation Example 1 100 52 41 Comparative Formulation Example 1 100 70 59 Comparative Formulation Example 2 100 65 56 Comparative Formulation Example 3 100 60 49 Comparative Formulation Example 4 100 59 48

[0128] As can be seen from the results in Table 8 above, it was confirmed that the cosmetic composition of Formulation Example 1 containing milk exosomes and egg white exosomes stabilized with lipid nanoparticles of Example 3 of the present invention has a superior transdermal water evaporation inhibition effect compared to the cosmetic composition of Comparative Formulation Example containing milk exosomes, egg white exosomes, milk extract, and egg white extract.

Claims

Claim 1 A cosmetic composition containing milk exosomes and egg white exosomes stabilized by lipid nanoparticles as active ingredients, wherein the milk exosomes and egg white exosomes stabilized by lipid nanoparticles as active ingredients comprises: (A) a1) a step of inoculating sterilized milk with a starter, culturing it at 22–43°C for 4–24 hours, and separating the whey by centrifugation; a2) a step of obtaining a supernatant by centrifuging the separated whey; a3) a step of freeze-drying the supernatant; a4) a step of forming an aqueous two-phase system using PEG (Polyethylene glycol) / Dextran on the freeze-dried product; a5) a milk exosome preparation step of obtaining a lower layer in which exosomes are concentrated; and a6) a step of preparing an aqueous phase by mixing 5-15% by weight of glutathione with 10-20% by weight of the milk exosomes and 65-75% by weight of purified water; (B) b1) a step of separating the egg yolk and egg white by centrifuging the whole egg to obtain the supernatant and separating the egg white; b2) a filtration step of mixing the egg white with purified water at a weight ratio of 1:5 to 1:10, stirring under refrigeration for 9 to 15 hours, and then removing the precipitate; b3) a step of separating the supernatant containing egg white exosomes by centrifuging the filtrate at 1,000xg to 10,000xg and freeze-drying it; and b4) a step of preparing an oil phase by dissolving freeze-dried powder in dipropylene glycol and then mixing it with at least one emulsifying polymer selected from the group consisting of phosphatidyl choline, PCL (Poly-ε-caprolactone), PGA (Polyglycolic acid), PLLA (Poly-L-lactide), sphingomyelin, phosphatidyl serine, phosphatidyl inositol, phosphatidyl ethanolamine, and lecithin;(C) A cosmetic composition characterized by being prepared by a method comprising: c1) a step of homogenizing the oil phase and the aqueous phase by passing them through a microfluidic chip using a weight ratio of 1:5 to 1:10, respectively; c2) a step of mixing and stirring a water-soluble solubilizing agent; and (D) a step of forming nanoparticles having a size of 50 to 400 nm under conditions of 300 to 800 bar using a high-pressure homogenizer (Microfluidizer).; Claim 2 delete Claim 3 A cosmetic composition according to claim 1, characterized in that, in step (C), homogenization is performed by repeatedly passing through a microfluidic chip having a flow channel of 1 μm to 5 μm at conditions of 1 to 10 bar and 15 to 40°C 1 to 3 times. Claim 4 A cosmetic composition according to claim 1, characterized in that the milk exosomes and egg white exosomes stabilized by the lipid nanoparticles as active ingredients are contained in an amount of 0.0001 to 30.0% (w / w) based on the total weight of the composition. Claim 5 A cosmetic composition according to claim 1, characterized in that the cosmetic composition is for moisturizing the skin. Claim 6 A cosmetic composition according to claim 1, characterized in that the cosmetic composition is for improving the skin barrier. Claim 7 A cosmetic composition according to claim 1, characterized in that the cosmetic composition is for improving skin wrinkles and elasticity. Claim 8 A cosmetic composition according to claim 1, characterized in that the cosmetic composition is for antioxidant use.

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