Cosmetic composition containing exosomes derived from iris bulbs as an active ingredient

Abstract

The present invention relates to a cosmetic composition containing exosomes derived from iris bulbs as an active ingredient, specifically, to a cosmetic composition that is highly stable because it contains iris bulb exosomes purified using ultra-high pressure pretreatment and an aqueous two-phase system as an active ingredient, and has excellent moisturizing, wrinkle improving and whitening effects on the skin.

Description

[Technical Field]

[0001] The present invention relates to a cosmetic composition containing exosomes derived from iris bulbs as an active ingredient. Specifically, the present invention relates to a cosmetic composition that is highly stable because it contains iris bulb exosomes purified using ultra-high pressure pretreatment and an aqueous two-phase system as an active ingredient, and that has excellent skin moisturizing, wrinkle improvement, and whitening effects. [Background technology]

[0002] In the present invention, "exosome" refers to small membrane-structured vesicles secreted by various cells and is defined as a type of extracellular vesicle (EVs). All cells secrete extracellular vesicles to exchange information with other cells or the external environment. Exosomes are approximately 50 to 200 nm in size and contain physiologically active substances such as proteins, lipids, and nucleic acids. Exosomes are present in various cells, including mammalian, bacterial, and plant cells, and reflect the state of their cell of origin, making them useful for diagnosis and treatment. Exosomes are double-phospholipid membrane structures that easily penetrate cells and perform various physiological and pathological functions, such as immune responses and signal transduction.

[0003] Recently, research has been conducted on the diverse effects of plant-derived exosomes, including their antioxidant and anti-inflammatory effects on the skin. Plant-derived exosomes contain physiologically active and signaling substances secreted by plant cells themselves, and are natural nanoparticles that contribute to intercellular transport and absorption. Exosomes purified from plants are known to have lower toxicity than exosomes derived from mammals.

[0004] In this regard, Korean Patent Registration No. 10-2125567 discloses a method for extracting highly purified plant exosomes from raw plant materials using centrifugation and tangential flow filtration (TFF), Korean Patent Publication No. 10-2020-0121062 discloses a method for purifying highly purified and high-quality extracellular vesicles using size exclusion chromatography, and Korean Patent Publication No. 10-2019-0050286 discloses a filler composition in which exosomes are incorporated into a hyaluronic acid-based filler composition, thereby increasing the stability of exosomes.

[0005] Due to their diverse benefits and activities, exosomes are a promising material for use in pharmaceuticals, cosmetics, food, and other fields. However, due to their structural characteristics consisting of a phospholipid bilayer, exosomes have low dispersibility and a tendency to aggregate. Furthermore, they are unstable at high temperatures and easily break down during the manufacturing process of cosmetic formulations. These properties can reduce the stability of exosomes in formulations and lead to precipitation. Therefore, to maintain their activity continuously, it is necessary to increase the solubility and dispersibility of exosomes in aqueous solutions and improve their stability in formulations.

[0006] The present inventors have completed the present invention as a result of their research into separating and purifying exosomes derived from various plants using an aqueous two-phase system and utilizing plant-derived exosomes with excellent skin effects as cosmetics. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to provide a cosmetic composition that contains exosomes derived from iris bulbs as an active ingredient and therefore has excellent stability, and that has excellent skin moisturizing, wrinkle improvement, and whitening effects. [Means for solving the problem]

[0008] In order to achieve the above object, the present invention provides a cosmetic composition containing iris bulb exosomes.

[0009] Preferably, the iris bulb exosomes are purified by a method comprising the steps of: (A) treating iris bulbs with ultra-high pressure; (B) squeezing the ultra-high pressure treated iris bulbs; (C) centrifuging the squeezed iris bulb juice at 1,000 xg to 10,000 xg to obtain a supernatant; (D) freeze-drying the supernatant containing exosomes; (E) forming an aqueous two-phase system with PEG (Polyethylene glycol) / dextran from the freeze-dried product; and (F) obtaining a lower layer of the aqueous two-phase system in which exosomes are concentrated.

[0010] The ultra-high pressure treatment is carried out at a temperature of 15 to 25° C. and a pressure of 200 to 500 MPa for 20 seconds to 2 minutes.

[0011] The iris bulb exosomes as an active ingredient are contained in an amount of 0.0001 to 30.0% (w / w) based on the total weight of the composition.

[0012] The cosmetic composition is characterized in that it further contains at least one selected from the group consisting of polyethylene glycol, polypropylene glycol, a copolymer of polyethylene glycol and polypropylene glycol, or a derivative thereof, butylene glycol, propylene glycol, and glycerin, in order to enhance the stability of the properties of iris bulb exosomes.

[0013] The cosmetic composition is characterized by being for moisturizing skin, improving skin wrinkles, or whitening skin. [Effects of the Invention]

[0014] The iris bulb-derived exosomes of the present invention, purified using ultra-high pressure pretreatment and an aqueous two-phase system, are highly stable and exhibit excellent moisturizing effects on the skin, as well as wrinkle improvement and whitening effects, making them useful as anti-aging cosmetics. [Brief explanation of the drawings]

[0015] [Figure 1] TEM image of exosomes derived from iris bulbs purified by one embodiment of the present invention. [Figure 2] Graph showing the results of NTA analysis to confirm the size distribution and particle number of exosome particles derived from iris bulbs purified according to one embodiment of the present invention. [Figure 3] Graph showing the results of NTA analysis to confirm the size distribution and particle number of exosome particles derived from iris bulbs purified without ultra-high pressure pretreatment. [Figure 4] Graph showing the results of evaluating the cytotoxicity of exosomes derived from iris bulbs purified according to the present invention using an MTT assay. [Figure 5] Graph showing the results of evaluating the moisturizing effect of exosomes derived from iris bulbs purified according to the present invention based on AQP3 expression. [Figure 6] Graph showing the results of evaluating the wrinkle-improving effect of iris bulb-derived exosomes purified according to the present invention on skin based on MMP-1 expression. [Figure 7] Graph showing the results of evaluating the wrinkle-improving effect of exosomes derived from iris bulbs purified according to the present invention on skin based on COL1A1 expression. [Figure 8] Graph showing the results of evaluating the whitening effect of exosomes derived from iris bulbs purified according to the present invention in terms of the tyrosinase activity inhibition rate. [Figure 9] Graph showing the results of evaluating the whitening effect of exosomes derived from iris bulbs purified according to the present invention in terms of melanin production inhibition rate. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention will be described in more detail below.

[0017] Plant-derived exosomes contain physiologically active and signaling substances secreted by plant cells themselves and are known to be less toxic than mammalian-derived exosomes. These advantages make them suitable for use in pharmaceuticals, cosmetics, food, and other fields. However, due to their structural characteristics, consisting of a phospholipid bilayer membrane, they have low dispersibility and a tendency to aggregate, making it difficult to maintain their activity in formulations. The technical feature of the present invention is the isolation and purification of highly purified exosomes from iris bulbs and their use in cosmetics.

[0018] Iris is a perennial plant belonging to the Iridaceae family. Iris bulbs have digestive and anti-inflammatory properties, making them a popular treatment for indigestion, hemorrhoids, bruises, skin conditions, sore throats, and tonsillitis. Iris bulbs contain high amounts of fatty acids, including linoleic acid, myristic acid, and lauric acid. Iris germanica, a species of iris, is a perennial plant native to Europe. It is a monocotyledonous perennial plant of the Iridaceae family in the order Liliales, grown in dry locations. It is highly cold-tolerant and can survive winter outdoors. Its largest flowers, among the irises, bloom abundantly at the end of flower stalks. Its thick rhizomes also make it a bulbous plant. Iris germanica bulbs contain isoflavones, such as irisolone and irigenin.

[0019] The iris bulb exosomes of the present invention are purified by the following method.

[0020] (A) subjecting iris bulbs to ultra-high pressure treatment; (B) squeezing the ultra-high pressure treated iris bulbs; (C) centrifuging the juice of the iris bulbs at 1,000 xg to 10,000 xg to obtain a supernatant; (D) freeze-drying the supernatant containing exosomes; (E) forming an aqueous two-phase system using PEG (Polyethylene glycol) / dextran on the freeze-dried product; and (F) obtaining a lower layer of the aqueous two-phase system in which exosomes are concentrated.

[0021] In step (A), either live or dried iris bulbs can be used, and the ultra-high pressure treatment is carried out at a temperature of 15 to 25°C and a pressure of 200 MPa or higher, preferably 200 to 500 MPa, for 20 seconds to 2 minutes. Exosomes are physiologically active substances, and the treatment temperature is preferably 15 to 25°C.

[0022] According to one embodiment of the present invention, the ultra-high pressure treatment is carried out by placing dried iris bulbs together with distilled water in a vinyl bag, sealing it tightly to prevent air from getting in, and then using an ultra-high pressure machine to treat them at a temperature of 15 to 25°C and a pressure of 200 MPa or more for 20 seconds to 2 minutes. When using living iris bulbs, they can be treated by placing them in a vinyl bag without using distilled water.

[0023] In step (B), the screw used in the process of squeezing the iris bulbs preferably has an agitation speed of 20 to 50 rpm. Because the ultra-high pressure treatment causes a change in the permeability of the plant cell wall, which makes it easier for the juice to escape, it is preferable to use a screw with an agitation speed below a certain speed.

[0024] The centrifugation method used in step (C) refers to a method of separating particles in a solution using centrifugal force depending on the size, shape, density, viscosity, and rotor speed. The rpm needs to be adjusted to remove large contaminants, and it is more preferable to perform the centrifugation at 10,000 x g to obtain a solution that will ultimately form an aqueous two-phase system.

[0025] The freeze-drying used in step (D) involves rapidly lowering the temperature of a container to freeze the substance to be dried, and then lowering the pressure inside the container to a vacuum, causing the solidified solvent contained in the material to immediately sublimate into water vapor and dry. The material is frozen at -50 to -80°C for 15 to 24 hours, and then dried in a freeze dryer under vacuum for 72 to 120 hours. In this case, the vacuum state usually refers to the pressure state of the freeze dryer.

[0026] In order to efficiently separate and purify exosomes, the present invention uses the aqueous two-phase partition method, which is a separation method that creates two layers from two types of aqueous solution that are poorly soluble in each other and utilizes the difference in affinity for each layer.

[0027] Although PEG / salt (such as sulfate, phosphate, or citrate) can be commonly used to form an aqueous two-phase system, the use of PEG / dextran is preferred for achieving the objectives of the present invention. Dextran is a natural polymer obtained by bacterial action and is used as a thickener, binder, and bulking agent in cosmetic formulations.

[0028] In step (E), when forming the aqueous two-phase system, PEG with a molecular weight of 10,000 to 35,000 is used at 1 to 15 wt%, preferably 2 to 5 wt%, and dextran with a molecular weight of 300,000 to 650,000 is used at 1 to 8 wt%, preferably 1 to 3 wt%. A concentration ratio of PEG to dextran of 3.3 wt%:1.7 wt% is more preferred, as it provides the highest exosome yield and the best stability.

[0029] To increase the purity of exosomes, after step (F), an additional step of forming an aqueous two-phase system using an aqueous two-phase solution of the same concentration and obtaining a lower layer solution can be performed 2 to 3 times.

[0030] Iris bulb-derived exosomes produced by this method exhibited superior moisturizing effects (Test Example 4), wrinkle improvement effects (Test Examples 5, 6, and 9), and whitening effects (Test Examples 7 and 8) compared to iris bulb extracts. Furthermore, the iris bulb-derived exosomes exhibited excellent stability in formulations (Test Examples 9 and 10).

[0031] Therefore, the iris bulb-derived exosomes can be used in cosmetic compositions for moisturizing, wrinkle reduction, or whitening. In this case, the iris bulb-derived exosomes as the active ingredient may be contained in an amount of 0.0001 to 30.0% (w / w) based on the total weight of the cosmetic composition.

[0032] Preferably, the cosmetic composition further contains at least one selected from the group consisting of polyethylene glycol, polypropylene glycol, a copolymer of polyethylene glycol and polypropylene glycol or a derivative thereof, butylene glycol, propylene glycol, and glycerin, more preferably at least one selected from the group consisting of glycerin, butylene glycol, and 1,3-propylene glycol, and most preferably butylene glycol and 1,3-propylene glycol, in order to enhance the stability of the properties of iris bulb exosomes.

[0033] In general, exosomes have a structural characteristic of being composed of a phospholipid bilayer, which means that they have low dispersibility and tend to aggregate, making it difficult to continuously maintain their activity within the formulation.The stability of iris bulb exosomes was further improved when they were used in combination with a compound containing an alcohol group selected from the group consisting of polyethylene glycol, polypropylene glycol, copolymers of polyethylene glycol and polypropylene glycol or their derivatives, butylene glycol, propylene glycol, and glycerin.

[0034] The cosmetic composition may be any commonly produced formulation, and can be produced, for example, into a skin lotion, skin softener, skin toner, lotion, milk lotion, moisturizing lotion, nourishing lotion, massage cream, nourishing cream, moisturizing cream, hand cream, essence, pack, soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, emulsion, pressed powder, loose powder, etc.

[0035] Meanwhile, when producing the cosmetic composition, those skilled in the art can easily select and blend, as needed, ingredients commonly used in cosmetics, such as moisturizers, antioxidants, surfactants, alcohols, thickeners, aqueous components, water, emulsifiers, vitamins, pigments, and common adjuvants such as fragrances, within the scope that does not impair the effects of the present invention. [Example]

[0036] The present invention will be described in more detail with reference to the following examples and test examples, however, the following examples are for illustrative purposes only and it will be apparent to those skilled in the art that the present invention is not limited to the following examples and can be substituted or modified into other equivalent examples within the scope of the technical concept of the present invention.

[0037] Example 1: Production of exosomes derived from iris bulbs Ultra-high pressure treatment of iris bulbs 100 g of dried iris (Iris germanica) bulbs were placed in a vinyl bag together with distilled water, tightly sealed to prevent air from entering, and then subjected to ultra-high pressure treatment at 200 MPa pressure at 25°C for 30 seconds using an ultra-high pressure machine.

[0038] Iris bulb juice Ultra-high pressure-treated iris (Iris germanica) bulbs were squeezed using a standard juicer with a low-speed screw at 30 rpm, and the resulting juice was filtered through a mesh screen to remove suspended matter. The collected juice was stored at -80°C until purification began.

[0039] Collection of supernatant for exosome purification To purify exosomes, the juice of iris bulbs was centrifuged at 10,000 x g for 10 minutes at 4°C to remove large contaminants. After centrifugation, the supernatant was collected to form an aqueous two-phase system.

[0040] Freeze-drying of the supernatant For mass production of exosomes, the supernatant was freeze-dried to reduce its volume. It was frozen at -80°C for 20 hours and then dried in a freeze-dryer under vacuum for 100 hours. Here, "vacuum" usually refers to the pressure of the freeze-dryer, and the freezing and drying times vary depending on the volume of the solution.

[0041] Formation of an aqueous two-phase system Purified water was added to the lyophilized supernatant, and an aqueous two-phase system was formed using PEG (polyethylene glycol) / dextran. PEG (purchased from Sigma-Aldrich) with a molecular weight of 10,000–35,000 was used at 3.3 wt% and dextran (purchased from Sigma-Aldrich) with a molecular weight of 300,000–650,000 was used at 1.7 wt%.

[0042] Extraction of exosomes from iris bulbs The supernatant and PEG / dextran solution were mixed and then centrifuged at 1,000 x g for 10 minutes at 4°C. After centrifugation, the supernatant was removed and the exosomes were collected.

[0043] Additional cleaning steps To increase the purity, the collected sublayer was washed with the same concentration of the aqueous two-phase solution. After repeating this process three times, the final sublayer was enriched in exosomes.

[0044] Comparative Example 1: Purification of exosomes from iris bulbs that have not been subjected to ultra-high pressure treatment Exosomes derived from iris bulbs were purified in the same manner as in Example 1 above, except that ultra-high pressure treatment was not performed.

[0045] Comparative Example 2: Preparation of Iris Bulb Hot Water Extract 20 g of dried iris (Iris germanica) bulbs were placed in 800 g of purified water and extracted for 3 hours at 80°C. After extraction, the extract was filtered under reduced pressure to obtain a hot water extract of the iris bulbs, which was then distilled using a rotary evaporator to obtain a powdered sample.

[0046] Comparative Example 3: Preparation of methanol extract of iris bulbs 20 g of dried iris (Iris germanica) bulbs were placed in 800 g of methanol and extracted for 3 hours at 60°C. After extraction, the extract was filtered under reduced pressure to obtain a methanol extract of the iris bulbs, which was then distilled using a rotary evaporator to obtain a powdered sample.

[0047] Comparative Example 4: Preparation of ethanol extract of iris bulbs 20 g of dried iris (Iris germanica) bulbs were placed in 800 g of ethanol and extracted for 3 hours at 60°C. After extraction, the ethanol extract of the iris bulbs was obtained by filtration under reduced pressure, and then distilled using a rotary evaporator to obtain a powdered sample.

[0048] Comparative Example 5: Preparation of hexane extract of iris bulbs 20 g of dried iris (Iris germanica) bulbs were placed in 800 g of hexane and extracted for 3 hours at 60°C. After extraction, the hexane extract was obtained by filtration under reduced pressure, and then distilled using a rotary evaporator to obtain a powdered sample. Table 1 below shows the extraction yields for each solvent.

[0049] [Table 1]

[0050] Test Example 1: Analysis of the characteristics of exosomes derived from iris bulbs: TEM analysis To confirm the shape of the purified iris (Iris germanica) bulb-derived exosomes, they were analyzed using a transmission electron microscope (TEM). Figure 1 shows a TEM analysis image of the iris bulb-derived exosomes purified in Example 1 above. The analysis confirmed the presence of spherical particles of approximately 160.7 nm consisting of a phospholipid bilayer membrane structure.

[0051] Test Example 2: Analysis of the characteristics of exosomes derived from iris bulbs: NTA analysis The purified exosomes derived from iris (Iris germanica) bulbs were analyzed by nanoparticle tracking analysis (NTA) to confirm the particle size distribution and particle number per unit volume. To compare the particle number of exosomes with and without ultra-high pressure treatment, NTA analysis of exosomes derived from iris bulbs purified according to Comparative Example 1 was also performed. Figure 2 is a graph showing the results of NTA analysis of exosomes derived from iris (Iris germanica) bulbs purified according to Example 1. The analysis results showed that the average particle size was 160.7 nm and the number of particles was 9.90 × 10 per mL of unit volume. 9 Figure 3 is a graph showing the results of NTA analysis of the exosomes derived from iris bulbs purified by the above Comparative Example 1. The average particle size was 172.1 nm and the concentration was 1.61 × 10 per mL of unit volume. 9 The same amount of exosomes was used in Example 1 and Comparative Example 1, and the yields were compared. It was confirmed that a larger amount of exosomes could be extracted when ultra-high pressure treatment was performed (Example 1).

[0052] Test Example 3: Evaluation of the cytotoxicity of exosomes derived from iris bulbs To confirm the cytotoxicity of exosomes derived from iris (Iris germanica) bulbs (Example 1) and the solvent-based iris (Iris germanica) bulb extracts (Comparative Examples 2, 3, and 4), an MTT assay was performed. Human dermal fibroblast (HDFa) cells were cultured at 1 × 10 in a 96-well plate. 5 After inoculation at a concentration of 1000 cells / mL, the cells were cultured at 37°C for 18 hours under 5% CO2. After the culture, the medium was removed, washed with PBS buffer, and then fresh medium and iris bulb-derived exosomes (stock concentration 9.90 × 10 9 The cells were then cultured for 48 hours. After adding MTT solution (5 mg / mL), the formazan formed within 4 hours was dissolved in dimethyl sulfoxide (DMSO) and the absorbance was measured at 570 nm using an ELISA reader. Figure 4 is a graph showing the results of an MTT assay evaluating the cytotoxicity of exosomes derived from iris bulbs purified in Example 1. The test results showed that 9.90 x 10 exosomes derived from iris bulbs were cultured at 100 x 10 9 When the stock particles / mL concentration was treated at 0.1% or less, no cytotoxicity was observed, and when treated at 1% or more, cell viability was confirmed to be approximately 90%.

[0053] Test Example 4: Evaluation of the moisturizing effect of exosomes derived from iris bulbs (AQP3 expression) To confirm the moisturizing effect of iris bulb-derived exosomes, we compared their effect on AQP3 expression with that of solvent-based iris (Iris germanica) bulb extracts. Human epidermal keratinocytes (HEKa) were inoculated and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 100 IU / mL penicillin and 100 μg / mL streptomycin at 37°C under 5% CO2 for 24 hours. After incubation, the medium was discarded and iris bulb-derived exosomes (stock concentration 9.90 × 10) were added. 9 The cells were treated with iris bulb extract (stock concentration 100 mg / mL) and each solvent (100 mg / mL) and cultured for 48 hours. RNA was extracted from the cultured cells using TransZol reagent, followed by RT-PCR (real-time polymerase chain reaction) to measure changes in mRNA levels of AQP3 (Aquaporin 3), a protein that plays an important role in skin moisturization as a moisturizing factor involved in cellular water transport. PCR products were electrophoresed on a 1% agarose gel and analyzed using a gel documentation system. PBS was used as the negative control, and 0.01% hyaluronic acid was used as the positive control. Figure 5 is a graph showing the results of evaluating the moisturizing effect of iris bulb-derived exosomes purified in Example 1 based on AQP3 expression. The test results showed that treatment with iris bulb-derived exosomes and iris bulb extract increased AQP3 mRNA expression in a concentration-dependent manner, and that treatment with iris bulb-derived exosomes further increased expression. In this case, when treated with 0.01% hyaluronic acid, the positive control group, the expression rate was 155.69%.

[0054] Test Example 5: Evaluation of the wrinkle improvement effect of exosomes derived from iris bulbs (MMP-1 expression) To confirm the anti-aging effect of exosomes derived from iris (Iris germanica) bulbs, we compared their effects on MMP-1 expression with those of solvent-based iris (Iris germanica) bulb extracts. 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 for 24 hours under 5% CO2. After incubation, the medium was discarded and iris bulb-derived exosomes (stock concentration 9.90 × 10) were added. 9 The cells were treated with iris bulb extract (stock concentration 100 mg / mL) and the solvent (particles / mL) and cultured for 48 hours. To induce cell damage, the remaining cultured cells, except for the non-UVB-irradiated group, were exposed to UVB at 20 mJ / cm after removing the medium. 2 The cells were irradiated, replaced with fresh medium, and cultured for 24 hours. After 24 hours, RNA was extracted using TransZol reagent, and real-time polymerase chain reaction (RT-PCR) was performed to measure changes in MMP-1 mRNA, which is associated with skin aging and wrinkle formation. The PCR products were electrophoresed on a 1% agarose gel and analyzed using a gel image analysis system. PBS was used as the negative control, and 50 μM retinyl palmitate was used as the positive control. Figure 6 is a graph showing the results of evaluating the anti-aging effect of iris bulb-derived exosomes purified in Example 1 above based on MMP-1 expression. The test results showed that treatment with iris bulb-derived exosomes and iris bulb extracts in each solvent reduced MMP-1 expression in a concentration-dependent manner, with a significantly greater reduction observed with iris bulb-derived exosomes. In this case, treatment with 50 μM retinyl palmitate, the positive control, showed a 72.81% expression rate.

[0055] Test Example 6: Evaluation of the wrinkle improvement effect of exosomes derived from iris bulbs (COL1A1 expression) To confirm the anti-aging effect of exosomes derived from iris (Iris germanica) bulbs, we compared their effects on COL1A1 expression with those of other iris (Iris germanica) bulb extracts. 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 for 24 hours under 5% CO2. After incubation, the medium was discarded and iris bulb-derived exosomes (stock concentration 9.90 × 10) were added. 9 The cells were treated with iris bulb extract (stock concentration 100 mg / mL) and each solvent (particles / mL) and cultured for 48 hours. After culture, RNA was extracted using TransZol reagent, followed by RT-PCR (real-time polymerase chain reaction) to measure changes in mRNA levels of collagen, which maintains skin connective tissue. The PCR products were electrophoresed on a 1% agarose gel and analyzed using a gel image analysis system. PBS was used as the negative control, and 50 μM retinyl palmitate was used as the positive control. Figure 7 is a graph showing the results of evaluating the anti-aging effect of iris bulb-derived exosomes purified in Example 1 based on COL1A1 expression. The test results confirmed that treatment with iris bulb-derived exosomes and iris bulb extract increased COL1A1 expression in a concentration-dependent manner, and that treatment with iris bulb-derived exosomes further increased expression. In this case, treatment with 50 μM retinyl palmitate, the positive control, showed an expression rate of 173.93%.

[0056] Test Example 7: Evaluation of the whitening effect of exosomes derived from iris bulbs (inhibition of tyrosinase activity) To confirm the skin whitening effect of exosomes derived from iris (Iris germanica) bulbs, a tyrosinase activity inhibition test was conducted in comparison with the solvent-based iris (Iris germanica) bulb extract. Mushroom-derived tyrosinase and tyrosine were purchased from Sigma Chemical. Tyrosinase activity was measured by incubating iris bulb-derived exosomes (stock concentration 9.90 × 10) with 150 μl of 0.1 M phosphate buffer (pH 6.5), 8 μl of mushroom tyrosinase (2100 units / ml, 0.05 M phosphate buffer, pH 6.5), and 36 μl of 1.5 mM L-tyrosine. 9 The samples were treated with various concentrations of iris bulb extract (stock concentration: 100 mg / mL) and various solvents. The tyrosinase inhibitory activity was confirmed by measuring the absorbance at 490 nm after incubating the samples at 37°C for 15 minutes. PBS was used as a negative control, and arbutin, a synthetic substance known as a skin-whitening agent, was used as a standard. The results were converted to percentages. Figure 8 is a graph showing the results of evaluating the whitening effect of iris bulb-derived exosomes purified in Example 1 above, as measured by the tyrosinase activity inhibition rate. The test results showed that the tyrosinase activity inhibition rate increased concentration-dependently when treated with iris bulb-derived exosomes and iris bulb extract, and an even higher inhibition rate was observed when treated with iris bulb-derived exosomes.

[0057] Test Example 8: Evaluation of the whitening effect of exosomes derived from iris bulbs (inhibition of melanin production) To confirm the skin whitening effect of exosomes derived from iris (Iris germanica) bulbs, a melanin production inhibition test was conducted in comparison with each solvent-based iris (Iris germanica) bulb extract. Mouse-derived B16F10 cell line (melanin-secreting cells) was inoculated and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS at 37°C for 24 hours under 5% CO2. After the culture, the medium was discarded and replaced with new medium. Then, exosomes derived from iris bulbs (stock concentration 9.90 x 10 9The cells were treated with iris bulb extract (stock concentration: 100 mg / mL) and each solvent (particles / mL) and cultured for 72 hours. After culture, the cells were treated with trypsin-EDTA and collected by centrifugation. The collected cells were washed, then treated with 500 μL of 1N NaOH and incubated at 100°C for 10 minutes to dissolve the melanin. The amount of melanin was determined by measuring absorbance at 405 nm. PBS was used as a negative control, and arbutin, a synthetic substance known as a skin-whitening agent, was used as a standard sample. The results were converted to percentages. Figure 9 is a graph showing the results of evaluating the whitening effect of iris bulb-derived exosomes purified in Example 1 above, as measured by the melanin production inhibition rate. The test results showed that the melanin production inhibition rate increased concentration-dependently when treated with iris bulb-derived exosomes and iris bulb extract, and an even higher inhibition rate was observed when treated with iris bulb-derived exosomes.

[0058] Formulation Example 1: Manufacture of aqueous formulation An aqueous formulation containing exosomes derived from iris bulbs purified in Example 1 above was prepared according to the composition shown in Table 2 below. To further enhance the stability of the exosomes purified in the aqueous two-phase system in the formulation, the formulation was prepared by adding one or more compounds selected from the group consisting of compounds containing an alcohol group, such as polyethylene glycol, polypropylene glycol, their copolymers and derivatives, butylene glycol, propylene glycol, and glycerin. A formulation not containing such compounds is designated Comparative Formulation Example 1.

[0059] [Table 2]

[0060] Test Example 9: Temperature-dependent stability analysis of exosomes derived from iris bulbs in aqueous formulations A stability test was conducted depending on temperature for the aqueous formulations prepared according to Formulation Example 1 and Comparative Formulation Example 1. Table 3 below shows the particle size and concentration measured by nanoparticle tracking analysis (NTA) after Formulation Example 1 and Comparative Formulation Example 1 were stored at 4°C, 25°C, and 45°C for 12 weeks.

[0061] [Table 3]

[0062] Analysis of temperature-dependent stability over 12 weeks confirmed that the size and concentration of exosomes derived from iris bulbs corresponding to Formulation Example 1, which contains the polyol of Formulation Example 1, were stable at all temperatures of 4°C, 25°C, and 45°C. On the other hand, in the case of Comparative Formulation Example 1, it was confirmed that the exosome concentration decreased at all temperatures of 4°C, 25°C, and 45°C.

[0063] Formulation Example 2: Cream preparation A cream containing exosomes derived from iris bulbs purified in Example 1 above was prepared by a conventional method using the composition shown in Table 4 below. The cream containing the iris bulb hot water extract of Comparative Example 2, which showed good effects when comparing the effects of iris bulb extracts for each solvent, was designated Comparative Formulation Example 2.

[0064] [Table 4]

[0065] Test Example 10: Analysis of stability of formulations of exosomes derived from iris bulbs A stability test was conducted on the cream prepared according to Formulation Example 2. The prepared cream was placed in a cycling chamber for 12 weeks, and the temperature was changed from 4°C to 45°C every 24 hours to observe the changes in its properties. The results are shown in Table 5 below.

[0066] [Table 5]

[0067] (-: no change, +: slight color change, ++: color change or slight precipitation, +++: color change and precipitation)

[0068] The stability of a cream formulation containing exosomes derived from iris bulbs (Example 1) purified in an aqueous two-phase system was observed, and it was confirmed that the stability of the properties was maintained.

[0069] Test Example 11: Skin wrinkle improvement effect of exosomes derived from iris bulbs Thirty adult women in their 30s to 50s were divided into two groups and applied a cream containing iris bulb-derived exosomes purified according to Example 1 (Formulation Example 2) and a cream containing the iris bulb hot water extract of Comparative Example 2 (Comparative Formulation Example 2) to both sides of the face. After irradiating the samples with 633 nm red light from an LED light source device for a total of 24 hours over a six-week period, the wrinkle improvement effect was evaluated. Table 6 below shows the results of this evaluation.

[0070] [Table 6]

[0071] As can be seen from the results in Table 6 above, the cream containing exosomes derived from iris bulbs of the present invention showed excellent wrinkle improvement effects. <Additional Notes> The present invention includes the following aspects. <Section 1> A cosmetic composition containing iris bulb exosomes. <Section 2> The iris bulb exosomes are (A) subjecting iris bulbs to ultra-high pressure treatment; (B) squeezing the ultra-high pressure treated iris bulbs; (C) centrifuging the juice of the iris bulbs at 1,000 x g to 10,000 x g to obtain a supernatant; (D) freeze-drying the supernatant containing exosomes; (E) forming an aqueous two-phase system using PEG (Polyethylene glycol) / Dextran in the lyophilized product; (F) obtaining a lower layer solution from the aqueous two-phase system in which exosomes are concentrated. <Section 3> The cosmetic composition according to item 2, wherein the ultra-high pressure treatment is carried out at a temperature of 15 to 25°C and a pressure of 200 to 500 MPa for 20 seconds to 2 minutes. <Section 4> The cosmetic composition according to <Item 1>, wherein the iris bulb exosomes are contained in an amount of 0.0001 to 30.0% (w / w) based on the total weight of the composition. <Section 5> The cosmetic composition according to <Item 1>, further comprising at least one selected from the group consisting of polyethylene glycol, polypropylene glycol, a copolymer of polyethylene glycol and polypropylene glycol or a derivative thereof, butylene glycol, propylene glycol, and glycerin, in order to enhance the stability of the properties of iris bulb exosomes. <Section 6> The cosmetic composition according to <Item 1>, characterized in that the cosmetic composition is for moisturizing the skin. <Section 7> The cosmetic composition according to <Item 1>, characterized in that the cosmetic composition is used to improve skin wrinkles. <Section 8> The cosmetic composition according to <Item 1>, characterized in that the cosmetic composition is for skin whitening.

Claims

1. The present invention comprises iris bulb exosomes and at least one selected from the group consisting of polyethylene glycol, polypropylene glycol, a copolymer of polyethylene glycol and polypropylene glycol or a derivative thereof, butylene glycol, propylene glycol, and glycerin, The iris bulb exosomes are (A) subjecting iris bulbs to ultra-high pressure treatment; (B) squeezing the ultra-high pressure treated iris bulbs; (C) centrifuging the juice of the iris bulbs at 1,000 x g to 10,000 x g to obtain a supernatant; (D) freeze-drying the supernatant containing exosomes; (E) forming an aqueous two-phase system using PEG (Polyethylene glycol) / Dextran on the lyophilized product; (F) obtaining a lower layer solution from the aqueous two-phase system in which exosomes are concentrated.

2. The cosmetic composition according to claim 1, wherein the ultra-high pressure treatment is carried out at a temperature of 15 to 25°C and a pressure of 200 to 500 MPa for 20 seconds to 2 minutes.

3. The cosmetic composition according to claim 1, wherein the iris bulb exosomes are contained in an amount of 0.0001 to 30.0% (w / w) relative to the total weight of the composition.

4. The cosmetic composition according to claim 1, further comprising at least one selected from the group consisting of polyethylene glycol, polypropylene glycol, a copolymer of polyethylene glycol and polypropylene glycol or a derivative thereof, butylene glycol, propylene glycol, and glycerin, in order to enhance the stability of the properties of iris bulb exosomes.

5. The cosmetic composition according to claim 1, which is for moisturizing skin.

6. The cosmetic composition according to claim 1, wherein the cosmetic composition is for improving skin wrinkles.

7. The cosmetic composition according to claim 1, which is for skin whitening.

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