Cosmetic composition containing iris bulb-derived exosomes as an active ingredient

Abstract

The present invention relates to a cosmetic composition containing iris bulb-derived exosomes as an effective ingredient, and more particularly to a cosmetic composition containing iris bulb-derived exosomes purified by ultrahigh pressure pretreatment and a two-aqueous phase system as an effective ingredient, which has excellent stability and excellent skin moisturizing, skin wrinkle improving and skin whitening effects.

Description

Technical Field

[0001] The present invention relates to a cosmetic composition containing iris bulb-derived exosomes as an effective ingredient, and more particularly to a cosmetic composition containing iris bulb-derived exosomes purified by ultrahigh pressure pretreatment and a two-aqueous phase system as an effective ingredient, which has excellent stability and has excellent skin moisturizing, skin wrinkle improvement and skin whitening effects. Background Art

[0002] In the present invention, "exosome" refers to a small membrane-structured vesicle secreted from a variety of cells, which is defined as a type of extracellular vesicle (EVs). All cells exchange information with other cells or the external environment, and secrete extracellular vesicles for this purpose. Exosomes have a size of about 50 to 200 nm and contain physiologically active substances such as proteins, lipids, and nucleic acids. Exosomes exist in a variety of cells such as mammals, bacteria, and plants and reflect the state of the origin cells, so they can be used for diagnosis and treatment. As a biphospholipid membrane structure, exosomes can easily penetrate into cells and perform a variety of physiological and pathological functions such as immune response and signal transduction.

[0003] In recent years, research on the various effects of plant-derived exosomes has been ongoing, and research has also begun on their antioxidant, anti-inflammatory and other skin effects. It is well known that plant-derived exosomes are natural nanoparticles that contain physiologically active substances and signal transduction substances secreted by plant cells themselves and facilitate cell-to-cell transport and absorption. Compared with mammalian-derived exosomes, exosomes purified from plants are non-toxic.

[0004] In this regard, Korean Patent No. 10-2125567 discloses a method for extracting high-purity plant exosomes from raw plants using centrifugal separation and tangential flow filtration (TFF), Korean Patent No. 10-2020-0121062 discloses a method for purifying high-purity, high-quality extracellular vesicles using size exclusion chromatography, and Korean Patent No. 10-2019-0050286 discloses a filler composition that contains exosomes in a hyaluronic acid-based filler composition to increase the stability of the exosomes.

[0005] Such exosomes are materials that can be used in the fields of medicine, cosmetics, food, etc. due to their various advantages and activities. However, due to their structural characteristics consisting of a biphospholipid membrane, they have low dispersibility and the property of agglomeration. In addition, they are unstable at high temperatures and may be easily broken during the preparation of cosmetic dosage forms. This property may reduce the stability of exosomes in the dosage form and produce precipitation. Therefore, in order to maintain continuous activity, it is necessary to improve the solubility and dispersibility of exosomes in aqueous solutions, thereby improving the stability in the dosage form.

[0006] The inventors of the present invention have completed the present invention as a result of conducting research aimed at separating and purifying various plant-derived exosomes using an aqueous two-phase system and using plant-derived exosomes with excellent skin efficacy as cosmetics. Summary of the invention

[0007] Technical issues

[0008] An object of the present invention is to provide a cosmetic composition containing iris bulb-derived exosomes as an active ingredient, which has excellent stability and has excellent skin moisturizing, skin wrinkle improving and skin whitening effects.

[0009] Technical Solution

[0010] In order to achieve the above object, according to the present invention, a cosmetic composition containing iris bulb exosomes is provided.

[0011] Preferably, the iris bulb exosomes are purified by a method comprising the following steps: (A) subjecting the iris bulbs to ultrahigh pressure treatment; (B) extracting juice from the iris bulbs that have been subjected to ultrahigh pressure treatment; (C) centrifuging the iris bulb juice at 1,000×g to 10,000×g to obtain an upper layer liquid; (D) freeze-drying the upper layer liquid containing exosomes; (E) forming a two-phase aqueous system using polyethylene glycol (PEG) / dextran on the freeze-dried product after the freeze-drying; and (F) obtaining a lower layer liquid concentrated with exosomes in the two-phase aqueous system.

[0012] The cosmetic composition is characterized in that the ultrahigh pressure treatment is performed at a temperature of 15 to 25° C. and a pressure of 200 to 500 MPa for 20 seconds to 2 minutes.

[0013] 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.

[0014] The cosmetic composition is characterized in that, in order to improve the stability of the properties of iris bulb exosomes, it can also contain 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 glycerol.

[0015] The cosmetic composition is characterized in that it is a cosmetic composition for skin moisturizing, for skin wrinkle improvement or for skin whitening.

[0016] Technical Effects

[0017] The iris bulb-derived exosomes of the present invention purified by ultrahigh pressure pretreatment and a two-phase aqueous system have excellent stability and exhibit excellent skin moisturizing effects, skin wrinkle improvement, and whitening effects, and can therefore be effectively used as anti-aging cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a TEM image of exosomes purified from iris bulbs according to an embodiment of the present invention.

[0019] Figure 2 : is a graph showing the NTA analysis results for confirming the size distribution and the number of exosome particles derived from iris bulbs purified according to one embodiment of the present invention.

[0020] Figure 3 The graph shows the results of NTA analysis for confirming the size distribution and number of exosome particles derived from iris bulbs that were not purified by ultrahigh pressure pretreatment.

[0021] Figure 4 This is a graph showing the results of evaluating the cytotoxicity of the iris bulb-derived exosomes purified according to the present invention by a cell proliferation assay (MTT assay).

[0022] Figure 5 This is a graph showing the results of evaluating the moisturizing effect of the exosomes derived from iris bulbs purified according to the present invention by AQP3 expression.

[0023] Figure 6 This is a graph showing the results of evaluating the skin wrinkle improving effect of the iris bulb-derived exosomes purified according to the present invention by the expression of MMP-1.

[0024] Figure 7 This graph shows the results of evaluating the skin wrinkle-improving effect of the iris bulb-derived exosomes purified according to the present invention based on COL1A1 expression.

[0025] Figure 8This is a graph showing the results of evaluating the skin-whitening effect of the iris bulb-derived exosomes purified according to the present invention by the tyrosinase activity inhibition rate.

[0026] Fig. 9 This is a graph showing the results of evaluating the whitening effect of the iris bulb-derived exosomes purified according to the present invention by the melanin production inhibition rate. DETAILED DESCRIPTION

[0027] Hereinafter, the present invention will be described in more detail.

[0028] As is known to all, plant-derived exosomes contain physiologically active substances and signal transduction substances secreted by plant cells themselves, and are non-toxic, compared to mammal-derived exosomes. Due to this advantage, it is a material that can be used in the fields of medicine, cosmetics, food, etc. However, due to its structural characteristics consisting of a biphospholipid membrane, it has low dispersion and the property of agglomeration, so it is difficult to maintain its activity continuously in the dosage form. The technical feature of the present invention is that exosomes are separated and purified from iris bulbs with high purity and used as cosmetics.

[0029] Iris is a perennial herb belonging to the genus Iris of the family Iridaceae. The bulb of iris has the effects of digestion and detumescence, and is therefore used as a therapeutic medicinal material for indigestion, hemorrhoids, contusions, skin diseases, pharyngitis, tonsillitis, etc. The bulb of iris contains a large amount of fatty acids such as linoleic acid, myristic acid, and lauric acid. As one of them, German iris (Iris germanica) is a perennial herb native to Europe. It is a perennial herb of the monocotyledonous plant Liliales Iridaceae plant cultivated in dry places. Because it is very cold-resistant, it can survive the winter in the open field, and the largest flower in the iris class blooms several flowers at the end of the flower stem. Because it has a thick underground rhizome, it is also regarded as a bulbous plant. The bulb of German iris (Iris germanica) contains isoflavones such as irisolone and irigenin.

[0030] The iris bulb exosomes of the present invention can be purified by the method described below.

[0031] Purification is performed using a method comprising the following steps: (A) subjecting iris bulbs to ultrahigh pressure treatment; (B) extracting juice from the iris bulbs that have been subjected to ultrahigh pressure treatment; (C) centrifuging the iris bulb juice at 1,000×g to 10,000×g to obtain an upper layer liquid; (D) freeze-drying the upper layer liquid containing exosomes; (E) using polyethylene glycol (PEG) / dextran to form a two-phase aqueous system with the freeze-dried product after the freeze-drying; and (F) obtaining a lower layer liquid in which exosomes are concentrated in the two-phase aqueous system.

[0032] In the above step (A), the iris bulbs can be used in their entirety or in a dried form, and the ultrahigh pressure treatment is characterized by being carried out at a temperature of 15 to 25°C and a pressure of 200 MPa or more, 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.

[0033] According to a specific example of the present invention, the ultra-high pressure treatment is performed as follows: the dried iris bulbs are placed in a plastic bag together with distilled water and sealed to prevent air from entering, and then ultra-high pressure treatment is performed at a temperature of 15 to 25° C. and a pressure of 200 MPa or more for 20 seconds to 2 minutes using an ultra-high pressure machine. In the case of using biological iris bulbs, the treatment can be performed by placing the bulbs in a plastic bag without distilled water.

[0034] In the above step (B), preferably, the propeller used in the process of extracting juice from iris bulbs uses a propeller with a stirring speed of 20 to 50 rpm. Since the change in the permeability of the plant cell wall caused by ultrahigh pressure treatment can easily cause the juice to escape, it is preferred to use a propeller with a stirring speed below a predetermined speed.

[0035] The centrifugal separation method used in the above step (C) is a method of separating particles in a solution by centrifugal force according to size, shape, density, viscosity, and rotor speed. It is necessary to remove larger contaminants by adjusting the rotation speed (rpm) in sequence, and in order to obtain a final solution for forming a two-phase aqueous system, it is more preferably carried out at 10,000 x g.

[0036] The freeze drying used in the above-mentioned step (D) is a method of drying in the following manner, that is, the material to be dried is frozen by rapidly lowering the temperature of the container, and then the pressure inside the container is made close to vacuum so that the solid solvent contained in the material is directly sublimated into water vapor, thereby drying. Freeze at -50 to -80°C for 15 to 24 hours, and dry in a freeze dryer in a vacuum state for 72 to 120 hours. At this time, the vacuum state refers to the pressure state of a normal freeze dryer.

[0037] In order to effectively separate and purify exosomes, the present invention uses an aqueous two-phase partition method, which is a separation method that uses two aqueous solutions that are not easily soluble in each other to form two layers and uses the difference in affinity for each layer.

[0038] In the method of forming a two-phase aqueous system, PEG / salt (salts include sulfate, phosphate, citrate, etc.) can also be generally used, but in order to achieve the purpose of the present invention, PEG / dextran is preferably used. Dextran is a natural polymer obtained by the action of bacteria and is used as a thickener, binder, and filler in cosmetic dosage forms.

[0039] In the above step (E), the formation of the aqueous two-phase system is characterized in that PEG uses 1 to 15% by weight of PEG with a molecular weight of 10,000 to 35,000, preferably 2 to 5% by weight of PEG, and dextran uses 1 to 8% by weight of dextran with a molecular weight of 300,000 to 650,000, preferably 1 to 3% by weight of dextran. When the above PEG and dextran are used at a concentration ratio of 3.3% by weight: 1.7% by weight, the yield of exosomes is the highest and the stability is the best, so it is more preferred.

[0040] In order to improve the purity of exosomes, after the above step (F), a process of forming an aqueous two-phase system by using an aqueous two-phase system solution of the same concentration and performing a process of obtaining a lower layer solution 2 to 3 times may be performed.

[0041] Compared with the iris bulb extract, the iris bulb-derived exosomes prepared by the above method showed excellent moisturizing effect (Test Example 4), wrinkle improvement effect (Test Example 5, Test Example 6, Test Example 9), and whitening effect (Test Example 7, Test Example 8). In addition, the iris bulb-derived exosomes have excellent stability in the dosage form (Test Examples 9 and 10).

[0042] Therefore, the iris bulb-derived exosomes can be used in a cosmetic composition for moisturizing, wrinkle improvement, or whitening. In this case, the iris bulb-derived exosomes as the active ingredient may contain 0.0001 to 30.0% (w / w) relative to the total weight of the cosmetic composition.

[0043] Preferably, in order to improve the stability of the properties of iris bulb exosomes, the cosmetic composition is characterized in that it further comprises at least one selected from the group consisting of polyethylene glycol, polypropylene glycol, copolymers of polyethylene glycol and polypropylene glycol or derivatives thereof, butylene glycol, propylene glycol and glycerol, more preferably, it may further comprise at least one selected from the group consisting of glycerol, butylene glycol and 1,3-propylene glycol, and most preferably, it further comprises butylene glycol and 1,3-propylene glycol.

[0044] Generally, exosomes have low dispersibility and tend to aggregate due to their structural characteristics of being composed of a biphospholipid membrane, and thus have a problem of difficulty in maintaining their activity continuously in a dosage form. When a compound containing an alcohol group 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 glycerol is used together, the stability of iris bulb exosomes is further improved.

[0045] The above-mentioned cosmetic composition can be prepared into any commonly prepared dosage form, for example, it can be prepared into a moisturizing lotion, a softening milk, a toner, a skin care water, a skin care lotion, a moisturizing milk, a nutrient water, a massage cream, a nutrient cream, a moisturizing cream, a hand cream, an essence, a facial mask, a soap, a shampoo, a cleansing foam, a cleansing milk, a cleansing cream, a body lotion, a shower gel, an emulsion, a pressed powder, a loose powder, etc.

[0046] In addition, when preparing the above-mentioned cosmetic composition, those skilled in the art can appropriately select and formulate ingredients commonly used in cosmetics without difficulty as needed within the scope of not impairing the effects of the present invention, for example, common auxiliary agents such as moisturizers, antioxidants, surfactants, alcohols, thickeners, aqueous ingredients, water, emulsifiers, vitamins, pigments and fragrances.

[0047] [Example]

[0048] Hereinafter, the present invention will be described in more detail according to the following examples and test examples. However, the following examples are only used to illustrate the present invention, and the present invention is not limited by the following examples. It is obvious to those with common knowledge in the technical field to which the present invention belongs that other equivalent embodiments can be replaced and changed within the scope of the technical idea of ​​the present invention.

[0049] Example 1: Preparation of exosomes from iris bulbs

[0050] Ultra-high pressure treatment of iris bulbs

[0051] 100 g of dried iris ( Iris germanica ) bulbs were placed in a plastic bag together with distilled water and sealed to prevent air from entering. The bag was then ultrahigh pressure treated at 25° C. and 200 MPa for 30 seconds using an ultrahigh pressure machine.

[0052] Iris bulb juice

[0053] The iris (Iris germanica) bulbs treated with ultrahigh pressure were squeezed with a common juicer at a low speed of 30 rpm, and the obtained iris bulb juice was filtered with a mesh to remove suspended matter. The recovered iris bulb juice was stored at -80°C until purification.

[0054] Recovery of supernatant for purification of exosomes

[0055] In order to purify exosomes, the iris bulb extract needed to remove large contaminants, so it was centrifuged at 10,000 x g at 4° C. for 10 minutes. After centrifugation, the supernatant was recovered to form a two-phase aqueous system.

[0056] Freeze drying of supernatant

[0057] In order to achieve mass production of exosomes, freeze drying was performed to reduce the volume of the supernatant. The solution was frozen at -80°C for 20 hours and dried in a freeze dryer in a vacuum state for 100 hours. At this time, the vacuum state refers to the pressure state of a normal freeze dryer, and the freezing time and drying time may vary depending on the volume of the solution.

[0058] Aqueous two-phase system formation

[0059] Purified water was added to the freeze-dried upper liquid and polyethylene glycol (PEG: Polyethylene glycol) / dextran was used to form a two-phase aqueous system. PEG (purchased from Sigma Aldrich) used 3.3% by weight of PEG with a molecular weight of 10,000 to 35,000, and dextran (purchased from Sigma Aldrich) used 1.7% by weight of dextran with a molecular weight of 300,000 to 650,000, thereby forming a two-phase aqueous system.

[0060] Recovery of exosomes from iris bulbs

[0061] The supernatant and the 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 to recover exosomes.

[0062] Additional washing process

[0063] In order to improve the purity, the same concentration of the aqueous two-phase system solution was added to the recovered lower layer liquid to perform an additional washing process. After repeating the process three times, the lower layer liquid finally concentrated with exosomes was recovered.

[0064] Comparative Example 1: Purification of exosomes from iris bulbs without ultrahigh pressure treatment

[0065] The iris bulb-derived exosomes were purified in the same manner as in the above example, except that the ultrahigh pressure treatment was not performed.

[0066] Comparative Example 2: Preparation of hot water extract of iris bulb

[0067] 20 g of dried iris (Iris germanica) bulbs were added to 800 g of purified water and extracted at 80° C. for 3 hours. After extraction, the iris bulb hot water extract was obtained by filtration under reduced pressure, and then distilled using a rotary evaporator to obtain a powdered sample.

[0068] Comparative Example 3: Preparation of methanol extract of iris bulb

[0069] 20 g of dried iris (Iris germanica) bulbs were added to 800 g of methanol and extracted at 60° C. for 3 hours. After extraction, the iris bulb methanol extract was obtained by filtration under reduced pressure, and then distilled using a rotary evaporator to obtain a sample in powder form.

[0070] Comparative Example 4: Preparation of Iris bulb ethanol extract

[0071] 20 g of dried iris (Iris germanica) bulbs were added to 800 g of ethanol and extracted at 60° C. for 3 hours. After extraction, the iris bulb ethanol extract was obtained by filtration under reduced pressure, and then distilled using a rotary evaporator to obtain a sample in powder form.

[0072] Comparative Example 5: Preparation of Hexane Extract from Iris Bulbs

[0073] 20 g of dried iris (Iris germanica) bulbs were added to 800 g of hexane and extracted at 60°C for 3 hours. After extraction, the iris bulb hexane extract was obtained by filtration under reduced pressure, and then distilled using a rotary evaporator to obtain a powdered sample. The following Table 1 shows the extraction yields according to different solvents.

[0074] [Table 1]

[0075] Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Extraction solvent Purified water Methanol Ethanol Hexane Powder weight (g) 3.32 2.95 2.41 2.44 Yield (%) 16.60 14.75 12.05 12.20

[0076] Experimental Example 1: Characteristic Analysis of Exosomes from Iris Bulbs: TEM Analysis

[0077] In order to confirm the shape of the purified Iris germanica bulb-derived exosomes, analysis was performed using a transmission electron microscope (TEM). Figure 1 This is a TEM analysis image of the exosomes derived from iris bulbs purified according to the above-mentioned Example. According to the analysis results, the presence of spherical phospholipid bilayer structured particles of about 160.7 nm was confirmed.

[0078] Experimental Example 2: Characteristic Analysis of Exosomes from Iris Bulbs: NTA Analysis

[0079] In order to confirm the particle size distribution and the number of particles per unit volume of the purified exosomes from Iris germanica bulbs, analysis was performed by nanoparticle tracking analysis (NTA). In order to compare the number of particles of exosomes that were treated with ultrahigh pressure or not, NTA analysis of exosomes from Iris germanica 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 germanica bulbs purified according to the above example. According to the analysis results, it was confirmed that the average size of the particles was 160.7 nm and the concentration per unit volume of 1 mL was 9.90×10 9 indivual. Figure 3 is a graph showing the results of NTA analysis of exosomes derived from iris bulbs purified according to Comparative Example 1, confirming that the average particle size was 172.1 nm and the concentration per unit volume of 1 mL was 1.61×10 9 The same amount was used in the experiment in Example and Comparative Example 1, and the yield was compared to confirm that a larger amount of exosomes could be extracted when ultrahigh pressure treatment was performed (Example).

[0080] Experimental Example 3: Evaluation of cytotoxicity of exosomes derived from iris bulbs

[0081] In order to confirm the cytotoxicity of exosomes derived from iris germanica bulbs (Example 1) and iris germanica bulb extracts in different solvents (Comparative Examples 2, 3, and 4), a cell proliferation test (MTT assay) was performed. Human dermal fibroblast cell line (HDFa) was cultured at 1×10 5The cells were seeded in a 96-well plate at a concentration of 10 cells / mL and then incubated at 37°C in 5% CO 2 After culturing, the culture medium was removed, the cells were washed with phosphate buffer (PBS buffer), and then new culture medium and iris bulb-derived exosomes (stock concentration was 9.90×10 9 The cells were incubated for 48 hours with 100 mg / mL of iris bulb extract in different solvents. To determine the cell survival rate, formazan was dissolved in dimethyl sulfoxide (DMSO) and added with MTT solution (5 mg / mL) for 4 hours, and the absorbance was measured at 570 nm using an ELISA reader. Figure 4 : is a graph showing the results of evaluating the cytotoxicity of the iris bulb-derived exosomes purified according to the above example by a cell proliferation assay (MTT assay). 9 When the concentration of iris bulb-derived exosome stock was less than 0.1% in particles / mL, no cytotoxicity was observed. When the concentration was more than 1%, the cell survival rate was about 90%.

[0082] Test Example 4: Evaluation of the moisturizing effect of exosomes derived from iris bulbs (AQP3 expression)

[0083] In order to confirm the moisturizing effect of iris bulb exosomes, the effect on AQP3 expression was confirmed by comparing with Iris germanica bulb extracts in different solvents. After inoculation of human epidermal keratinocytes (HEKa), the cells were incubated in Dulbecco Modified Eagle Medium (DMEM) supplemented with 100 IU / mL penicillin and 100 μg / mL streptomycin at 37°C under 5% CO. 2 After culturing, the culture medium was discarded and exosomes from iris bulbs (stock concentration was 9.90×10 9The cells were treated with iris bulb extract (stock concentration was 100 mg / mL) and iris bulb extract in different solvents (stock concentration was 100 mg / mL) and cultured for 48 hours. Then, RNA was extracted from the cultured cells using TransZol reagent, and RT-PCR (real-time polymerase chain reaction) was performed to measure the mRNA changes of aquaporin 3 (AQP3: Aquaporin 3), a protein that is a moisturizing factor involved in the water migration of cells and plays an important role in skin moisturizing. The products generated by PCR were electrophoresed in 1% agarose gel and confirmed using a gel documentation system. At this time, PBS was used as a negative control group, and 0.01% hyaluronic acid was used as a positive control group. Figure 5 This is a graph showing the results of evaluating the moisturizing effect of the iris bulb-derived exosomes purified according to the above example by AQP3 expression. According to the test results, it was confirmed that the mRNA expression of AQP3 increased in a concentration-dependent manner when treated with iris bulb-derived exosomes and iris bulb extract, and the expression was further increased when using iris bulb-derived exosomes. At this time, the expression rate of 155.69% was shown when treated with 0.01% hyaluronic acid as a positive control group.

[0084] Test Example 5: Evaluation of wrinkle improvement efficacy of exosomes derived from iris bulbs (MMP-1 expression)

[0085] In order to confirm the anti-aging effect of exosomes derived from Iris germanica bulbs, the effect on MMP-1 expression was confirmed by comparing with Iris germanica bulb extracts in different solvents. After inoculation of human dermal fibroblast cell line (HDFa), 100 IU / mL penicillin and 100 μg / mL streptomycin were added to Fibroblast Basal Medium (106 culture medium) at 37°C under 5% CO 2 After culturing, the culture medium was discarded and exosomes from iris bulbs (stock concentration was 9.90×10 9The cells were treated with 20 mJ / cm2 of UVB-irradiated cells and 100 mg / mL of iris bulb extract in different solvents and cultured for 48 hours. 2 The cells were irradiated with UVB, the culture medium was replaced, and then cultured for 24 hours. After 24 hours, RNA was extracted using TransZol reagent, and RT-PCR (real-time gene polymerase chain reaction) was performed to measure the mRNA changes of MMP-1 related to skin aging and wrinkle formation. The products generated by PCR were electrophoresed in 1% agarose gel and confirmed using a gel documentation system. At this time, PBS was used as a negative control group, and 50μM retinyl palmitate was used as a positive control group. Figure 6 This is a graph showing the results of evaluating the anti-aging effect of the iris bulb-derived exosomes purified according to the above example by MMP-1 expression. According to the test results, it was confirmed that when treated with iris bulb-derived exosomes and iris bulb extracts in different solvents, MMP-1 expression decreased in a concentration-dependent manner, and the decrease rate was significantly higher when treated with iris bulb-derived exosomes. At this time, when treated with 50μM retinyl palmitate as a positive control group, an expression rate of 72.81% was shown.

[0086] Test Example 6: Evaluation of wrinkle improvement efficacy of exosomes derived from iris bulbs (COL1A1 expression)

[0087] In order to confirm the anti-aging effect of exosomes derived from Iris germanica bulbs, the effect on COL1A1 expression was confirmed by comparing with Iris germanica bulb extracts in different solvents. After inoculation of human dermal fibroblast cell line (HDFa), 100 IU / mL penicillin and 100 μg / mL streptomycin were added to Fibroblast Basal Medium (106 medium) at 37°C under 5% CO 2 After culturing, the culture medium was discarded and exosomes from iris bulbs (stock concentration was 9.90×10 9The cells were treated with iris bulb extract (stock concentration was 100 mg / mL) and iris bulb extract in different solvents (stock concentration was 100 mg / mL) and cultured for 48 hours. After culture, RNA was extracted using TransZol reagent and RT-PCR (real-time polymerase chain reaction) was performed to measure the mRNA changes of collagen, which maintains the connective tissue of the skin. The products generated by PCR were electrophoresed in 1% agarose gel and confirmed using a gel documentation system. At this time, PBS was used as a negative control group, and 50 μM retinyl palmitate was used as a positive control group. Figure 7 This is a graph showing the results of evaluating the anti-aging effect of the iris bulb-derived exosomes purified according to the above example by COL1A1 expression. According to the test results, it was confirmed that the expression of COL1A1 increased in a concentration-dependent manner when treated with iris bulb-derived exosomes and iris bulb extract, and the expression further increased when treated with iris bulb-derived exosomes. At this time, when 50μM retinylpalmitate was used as a positive control group, an expression rate of 173.93% was shown.

[0088] Test Example 7: Evaluation of the whitening effect of exosomes derived from iris bulbs (inhibition of tyrosinase activity)

[0089] In order to confirm the whitening effect of exosomes derived from iris germanica bulbs, a tyrosinase activity inhibition test was performed by comparing with iris germanica bulb extracts in different solvents. Mushroom-derived tyrosinase and tyrosine were purchased from Sigma Chemical and used. Tyrosinase activity was measured by mixing 150 μL of 0.1 M phosphate buffer (pH 6.5) with 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 with iris bulb-derived exosomes (stock concentration 9.90 × 10 9 The samples were treated with 100 mg / mL of iris bulb extract (stock concentration: 100 mg / mL) and 100 mg / mL of iris bulb extract in different solvents. The tyrosinase inhibitory activity was confirmed by reacting the samples at 37°C for 15 minutes and measuring the absorbance at 490 nm. At this time, PBS was used as a negative control group, and arbutin, a synthetic substance known as a whitening agent, was used as a standard sample, and the results were converted into %. Figure 8This is a graph showing the results of evaluating the whitening effect of the iris bulb-derived exosomes purified according to the above example by the tyrosinase activity inhibition rate. According to the test results, it was confirmed that the tyrosinase activity inhibition rate increased in a concentration-dependent manner when treated with iris bulb-derived exosomes and iris bulb extract, and the inhibition rate was higher when treated with iris bulb-derived exosomes.

[0090] Test Example 8: Evaluation of the whitening effect of exosomes derived from iris bulbs (inhibition of melanin production)

[0091] In order to confirm the whitening effect of exosomes derived from iris germanica bulbs, a melanin production inhibition test was conducted by comparing with iris germanica bulb extracts in different solvents. The mouse-derived B16F10 cell line (melanin-secreting cells) was inoculated and then incubated in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C under 5% CO 2 After culturing, the culture medium was discarded and replaced with new culture medium. Then, exosomes from iris bulbs (stock concentration was 9.90×10 9 The cells were treated with 100 mg / mL of iris bulb extract (stock concentration was 100 mg / mL) and cultured for 72 hours. After culture, the cells were treated with trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA) and recovered by centrifugation. After washing the recovered cells, they were treated with 500 μL of 1N NaOH and reacted at 100°C for 10 minutes to dissolve the melanin. The amount of melanin was confirmed by measuring the absorbance at 405 nm. At this time, as a negative control group, PBS was used for treatment, and arbutin, a synthetic substance known as a whitening agent, was used as a standard sample and the results were converted to %. Fig. 9 This is a graph showing the results of evaluating the whitening effect of the iris bulb-derived exosomes purified according to the above example by the melanin production inhibition rate. According to the test results, it was confirmed that the melanin production inhibition rate increased in a concentration-dependent manner when treated with iris bulb-derived exosomes and iris bulb extract, and the inhibition rate was higher when treated with iris bulb-derived exosomes.

[0092] Dosage Form Example 1: Preparation of Several Types of Dosage Forms

[0093] Several types of dosage forms containing exosomes derived from iris bulbs purified according to the above examples were prepared using the composition of Table 2 below. In order to further improve the stability of the exosomes purified by the two-phase aqueous system, one or more of the group consisting of compounds containing alcohol groups such as polyethylene glycol, polypropylene glycol and copolymers and derivatives thereof, butanediol, propylene glycol and glycerol were included. The dosage form not containing the above-mentioned compound was used as comparative dosage form example 1.

[0094] [Table 2]

[0095]

[0096]

[0097] Experimental Example 9: Stability analysis of several types of dosage forms of exosomes derived from iris bulbs at different temperatures

[0098] Several types of dosage forms prepared according to the above-mentioned dosage form example 1 and comparative dosage form example 1 were subjected to stability tests at different temperatures. The following Table 3 is a table showing the size and concentration of particles after the above-mentioned dosage form example 1 and comparative dosage form example 1 were stored at 4°C, 25°C, and 45°C for 12 weeks by nanoparticle tracking analysis (NTA).

[0099] [Table 3]

[0100]

[0101]

[0102] As a result of analyzing the 12-week stability at different temperatures, it was confirmed that the size and concentration changes of the exosomes derived from the iris bulb of Formulation Example 1, which is equivalent to Formulation Example 1 formulated with a polyol, were stable at 4°C, 25°C, and 45°C. In addition, in the case of Comparative Formulation Example 1, the concentration of the exosomes decreased at 4°C, 25°C, and 45°C.

[0103] Dosage Form Example 2: Preparation of Cream

[0104] A cream containing exosomes derived from iris bulbs purified according to the above example was prepared by a conventional method using the composition shown in Table 4 below. The cream containing the iris bulbs hot water extract of Comparative Example 2 was used as Comparative Formulation Example 2. The iris bulbs hot water extract of Comparative Example 2 had a better efficacy when the efficacy of the iris bulbs extract was evaluated by different solvents.

[0105] [Table 4]

[0106]

[0107] Test Example 10: Analysis of the Stability of Dosage Forms of Exosomes from Iris Bulbs

[0108] The cream prepared according to the above-mentioned Formulation Example 2 was subjected to a property stability test. The prepared cream was administered at intervals of 24 hours in a cycling chamber. The temperature changes were observed for 12 weeks. The results are shown in Table 5 below.

[0109] [Table 5]

[0110] Week Dosage Form Example 2 Early stage - Week 2 - Week 4 - Week 8 - Week 12 +

[0111] (-; no change, +; slight color change, ++; color change or slight precipitation, +++; color change and precipitation)

[0112] The stability of a cream formulation containing exosomes derived from iris bulbs purified by an aqueous two-phase system (Example) was observed, and it was confirmed that the stability of the properties was maintained.

[0113] Test Example 11: Skin wrinkle improvement effect of exosomes derived from iris bulbs

[0114] Thirty adult women aged 30 to 50 were divided into two groups, and the cream containing the exosomes derived from the iris bulbs purified according to the above Example (Formulation Example 2) and the cream containing the hot water extract of the iris bulbs of Comparative Example 2 (Comparative Formulation Example 2) were applied to both sides of the face, and the wrinkle improvement effect was evaluated by cumulative irradiation of 633 nm red light for 24 hours over 6 weeks using an LED light source device. The following Table 6 is a table showing the results of the wrinkle improvement effect based on the evaluation.

[0115] [Table 6]

[0116]

[0117] As can be seen from the results in Table 6, the cream containing the exosomes derived from iris bulbs of the present invention exhibits an excellent skin wrinkle improvement effect.

Claims

1. A cosmetic composition, It is characterized in that The cosmetic composition contains iris bulb exosomes and at least one selected from the group consisting of butanediol and 1,3-propylene glycol for improving the property stability of the iris bulb exosomes. Wherein, the iris bulb exosomes are purified by a method comprising the following steps: (A) subjecting iris bulbs to ultrahigh pressure treatment at a temperature of 15 to 25°C and a pressure of 200 to 500 MPa for 20 seconds to 2 minutes; (B) extracting juice from the iris bulbs treated with ultra-high pressure; (C) centrifuging the iris bulb juice at 1,000 x g to 10,000 x g to obtain a supernatant; (D) freeze-drying the upper layer containing exosomes; (E) using PEG / dextran in the lyophilized product to form an aqueous two-phase system; and (F) obtaining a lower layer liquid containing concentrated exosomes in the aqueous two-phase system, The iris bulb exosomes contain 0.0001 to 10.0% (w / w) based on the total weight of the composition.

2. The cosmetic composition according to claim 1, It is characterized in that The cosmetic composition is a cosmetic composition for skin moisturizing.

3. The cosmetic composition according to claim 1, It is characterized in that The cosmetic composition is a cosmetic composition for improving skin wrinkles.

4. The cosmetic composition according to claim 1, It is characterized in that The cosmetic composition is a cosmetic composition for skin whitening.

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