Cosmetic composition containing exosomes derived from ulleung chrysanthemum and ginkgo leaf as active ingredient

Abstract

The present invention relates to a cosmetic composition containing Ulleung chrysanthemum and ginkgo leaf exosomes as an active ingredient and, more specifically, to a cosmetic composition containing Ulleung chrysanthemum exosomes and / or ginkgo leaf exosomes as an active ingredient wherein the Ulleung chrysanthemum exosomes and / or ginkgo leaf exosomes are purified using freeze-thaw treatment, UV pretreatment, and an aqueous biphasic system, whereby the composition exhibits excellent wrinkle alleviation, cell regeneration, and skin barrier strengthening effects.

Description

Cosmetic composition containing exosomes derived from Ulleung chrysanthemum and ginkgo leaves as active ingredients

[0001] The present invention relates to a cosmetic composition containing exosomes derived from Ulleung chrysanthemum and ginkgo leaves as active ingredients, and more particularly, to a cosmetic composition having excellent effects of improving wrinkles, cell regeneration, and strengthening the skin barrier by containing Ulleung chrysanthemum exosomes and / or ginkgo leaf exosomes purified using freeze-thawing treatment and UV pretreatment and an aqueous two-phase system as active ingredients.

[0002] This invention is the result of research conducted as a result of the Science Belt Support Project supported by the Ministry of Science and ICT and the Korea Research and Development Special Zone Promotion Foundation (Project Number: 2023-SB-SB-0010-01).

[0003] The function of cosmetics has expanded beyond simple cleansing and makeup to include removing or suppressing the causes of skin aging, such as pollutants, stress, UV rays, free radicals, and peroxides. As the global population ages, the market for cosmetics designed to prevent skin aging continues to grow. With the desire for clean, healthy skin becoming a major concern for modern women, demand for ingredients with wrinkle-improving effects and cosmetics utilizing them continues to rise. Furthermore, skin aging is closely linked to hydration and maintenance. Dry skin results from a loss of moisture, which reduces the cohesion of keratinocytes, essentially a malfunction of the skin barrier.

[0004] Recently, there has been a surge in the development of cosmetics using safe, natural ingredients that offer excellent wrinkle-improving and skin barrier-strengthening effects. The inventors of this invention have researched and developed a raw material that promotes wrinkle improvement, cell regeneration, and skin barrier strengthening without causing skin irritation.

[0005] In the present invention, "exosome" refers to a small membrane-structured vesicle secreted from various cells, and is defined as a type of extracellular vesicle (EV). All cells secrete EVs 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 exist in various cells such as mammals, bacteria, and plants, and reflect the state of the cells from which they originate, so they can be used for diagnosis and treatment. Exosomes have a double phospholipid membrane structure, allowing them to easily penetrate cells and perform various physiological and pathological functions such as immune responses and signal transduction.

[0006] Recently, research has been conducted on the various benefits of plant-derived exosomes. Plant-derived exosomes are natural nanoparticles secreted by plant cells themselves, containing physiologically active and signaling substances that aid in intercellular movement and uptake. Furthermore, plant-purified exosomes are known to be less toxic than mammalian exosomes.

[0007] Due to their diverse advantages and activities, exosomes are potential materials for use in pharmaceuticals, cosmetics, and foods. However, their structural characteristics, consisting of a phospholipid bilayer, result in low dispersibility and a tendency to aggregate. Furthermore, they are unstable at high temperatures and can easily break during the manufacturing process of cosmetic formulations. These properties can reduce the stability of exosomes within formulations and lead to precipitation. Therefore, to maintain sustained activity, it is necessary to enhance the solubility and dispersibility of exosomes in aqueous solutions, thereby enhancing their stability within formulations.

[0008] The present inventors have attempted to produce plant-derived exosomes exhibiting excellent skin condition improvement activity by applying various methods and to use them as cosmetics. As a result, they have confirmed that Ulleung Chrysanthemum exosomes and / or Ginkgo biloba exosomes purified using freeze-thawing treatment and UV pretreatment under specific conditions and an aqueous two-phase system exhibit excellent skin improvement efficacy, thereby completing the present invention.

[0009] The purpose of the present invention is to provide a cosmetic composition containing Ulleung chrysanthemum exosomes, ginkgo leaf exosomes or mixed exosomes thereof as active ingredients, which has excellent stability and excellent effects in improving wrinkles, cell regeneration and strengthening the skin barrier.

[0010] In addition, another object of the present invention is to provide a method for purifying Ulleung chrysanthemum exosomes or ginkgo leaf exosomes.

[0011] In order to achieve the above purpose, according to the present invention, a cosmetic composition containing Ulleung chrysanthemum exosomes, ginkgo leaf exosomes or mixed exosomes thereof as an active ingredient is provided.

[0012] The above mixed exosomes are composed of Ulleung chrysanthemum exosomes and ginkgo leaf exosomes mixed in a weight ratio of 1 to 3:1 to 3, respectively.

[0013] As the above effective ingredients, Ulleung Chrysanthemum exosomes and Ginkgo leaf exosomes are

[0014] (A) a step of extracting Ulleung chrysanthemum or ginkgo leaves by adding a solvent and repeating freezing and thawing; (B) a step of treating Ulleung chrysanthemum or ginkgo leaves that have been pretreated by freezing and thawing with UV-A for 1 to 6 hours; (C) a step of extracting the UV-treated Ulleung chrysanthemum or ginkgo leaves; (D) a step of centrifuging the extracted juice at 1,000xg to 10,000xg to obtain a supernatant; (E) a step of lyophilizing the supernatant in which exosomes are present; (F) a step of forming an aqueous two-phase system using PEG (Polyethylene glycol) / Dextran in the lyophilized product; and (G) a step of obtaining a lower layer in which exosomes are concentrated in the aqueous two-phase system.

[0015] More preferably, the freeze-thaw pretreatment in step (A) is characterized by comprising the steps of adding water to Ulleung chrysanthemum or ginkgo leaves, freezing them at a temperature of -80 to -70°C for 15 to 20 hours, and then thawing them at a temperature of 40 to 50°C for 8 to 10 hours, which is repeated 2 to 5 times. Preferably, the UV treatment in step (B) is characterized by being performed for 4 to 6 hours with UV-A having a wavelength of 340 to 380 nm, and more preferably for 6 hours with UV-A having a wavelength of 365 nm.

[0016] The above Ulleung Chrysanthemum exosome, Ginkgo leaf exosome or mixed exosome thereof as an effective ingredient is contained in an amount of 0.0001 to 30.0% (w / w) based on the total weight of the composition.

[0017] The above cosmetic composition is characterized by being used for improving wrinkles, regenerating cells, or strengthening the skin barrier.

[0018] According to the present invention, in order to achieve the above other objects,

[0019] (A) A step of adding a solvent to Ulleung chrysanthemum or ginkgo leaves and extracting them by repeating freezing and thawing;

[0020] (B) A step of treating Ulleung chrysanthemum or ginkgo leaves that have undergone freeze-thaw pretreatment with UV-A for 1 to 6 hours;

[0021] (C) Step of extracting UV-treated Ulleung chrysanthemum or ginkgo leaves;

[0022] (D) A step of centrifuging the above juice at 1,000xg to 10,000xg to obtain a supernatant;

[0023] (E) A step of freeze-drying the supernatant containing exosomes;

[0024] (F) a step of forming an aqueous two-phase system using PEG (Polyethylene glycol) / Dextran in the above lyophilized product; and

[0025] (G) A method for purifying exosomes from Ulleung chrysanthemum or ginkgo leaf is provided, including a step of obtaining a lower layer containing concentrated exosomes from the above two-phase aqueous solution.

[0026] More preferably, the freezing and thawing treatment in step (A) is characterized by repeating the process of freezing for 15 to 20 hours at a temperature condition of -80 to -70°C and then thawing for 8 to 10 hours at a temperature condition of 40 to 50°C 2 to 5 times, and the UV treatment in step (B) is characterized by performing the process with UV-A having a wavelength of 340 to 380 nm for 4 to 6 hours, more preferably with UV-A having a wavelength of 365 nm for 6 hours.

[0027] The Ulleung chrysanthemum exosomes, ginkgo leaf exosomes or mixed exosomes thereof of the present invention purified using freeze-thawing treatment and UV pretreatment and an aqueous two-phase system have excellent stability and exhibit excellent wrinkle improvement effects, cell regeneration effects and skin barrier strengthening effects, and thus can be usefully used as a cosmetic for skin improvement.

[0028] Figure 1 is a TEM image of an exosome derived from Ulleung chrysanthemum purified according to one embodiment of the present invention.

[0029] Figure 2 is a TEM image of exosomes derived from ginkgo leaves purified according to one embodiment of the present invention.

[0030] FIG. 3 is a graph showing the results of NTA analysis to confirm the size distribution and particle number of purified Ulleung chrysanthemum-derived exosome particles according to one embodiment of the present invention.

[0031] FIG. 4 is a graph showing the results of NTA analysis to confirm the size distribution and particle number of purified ginkgo leaf-derived exosome particles according to one embodiment of the present invention.

[0032] Figure 5 is a graph showing the results of evaluating the cytotoxicity of exosomes derived from Ulleung chrysanthemum and ginkgo leaves purified according to the present invention using an MTT assay.

[0033] Figure 6 is a graph showing the results of evaluating the wrinkle improvement efficacy of exosomes derived from Ulleung chrysanthemum and ginkgo leaves purified according to the present invention, based on COL1A1 expression.

[0034] Figure 7 is a graph showing the results of evaluating the wrinkle improvement efficacy of exosomes derived from Ulleung chrysanthemum and ginkgo leaves purified according to the present invention by suppressing MMP-1 expression.

[0035] Figure 8 is a graph showing the results of evaluating the skin barrier strengthening efficacy of exosomes derived from Ulleung chrysanthemum and ginkgo leaves purified according to the present invention, based on filaggrin expression.

[0036] Figure 9 is a graph showing the results of evaluating the skin barrier strengthening efficacy of exosomes derived from Ulleung chrysanthemum and ginkgo leaves purified according to the present invention, as measured by loricrin expression.

[0037] Figure 10 is an image showing the results of evaluating the cell regeneration efficacy of exosomes derived from Ulleung chrysanthemum and ginkgo leaves purified according to the present invention using a wound healing assay.

[0038] Figure 11 is a graph showing the results of evaluating the cell regeneration efficacy of exosomes derived from Ulleung chrysanthemum and ginkgo leaves purified according to the present invention as a wound area ratio.

[0039] Hereinafter, the present invention will be described in more detail.

[0040] 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. Due to these advantages, they are materials that can be utilized in fields such as pharmaceuticals, cosmetics, and foods. However, due to their structural characteristics of being composed of a phospholipid bilayer, they have low dispersibility and a tendency to aggregate, making it difficult to continuously maintain their activity within a formulation. The present invention is characterized by separating and purifying exosomes with excellent skin-improving activity from Ulleung chrysanthemum and ginkgo leaves with high purity through freeze-thawing treatment and UV pretreatment, and using them as cosmetics.

[0041] Ulleung Chrysanthemum (Dendranthema zawadskii var. lucidum (Nakai)) grows on the mountainous areas of Seonginbong Peak on Ulleung Island, Gyeongsangbuk-do Province. Its leaves are deeply and relatively finely lobed, with thick, glossy, lanceolate lobes and petioles. Similar to Chrysanthemum chinensis, but with more deeply lobed leaves, it is designated as a rare and endangered plant by the Korea Forest Service. It was first discovered by Nakai, a Japanese, and reported to a Japanese academic society. Subsequent research revealed it to be endemic to Ulleung Island, South Korea. Indiscriminate harvesting has led to the gradual disappearance of its native habitat. The area where it grows in the Nari Basin is designated a Natural Monument and is protected. It is effective for warming the body, improving circulation, and digestion, and has been used medicinally to treat menstrual irregularities, cold uterine symptoms, infertility, cold stomach, and indigestion. Ulleung chrysanthemum contains chlorogenic acid, luteoloside, cosmosiin, linarin, etc., and is rich in total flavonoids and polyphenols, so it has excellent antioxidant activity.

[0042] Unlike most gymnosperms, such as pine, juniper, spruce, and fir, which have needle-shaped leaves, the ginkgo leaf (Ginkgo biloba) is fan-shaped and splits down the middle. Ginkgo leaves grow alternately on long branches, while on short branches, they grow densely and alternately in groups of three to five, giving the appearance of growing from a single point. Furthermore, the leaves on long branches are deeply lobed, while those on short branches often have smooth margins. New leaves sprout in spring, and in fall, the leaves turn yellow before falling. Ginkgo leaves contain flavonoids, ginkgolides, bilobalides, and terpenoids as active ingredients, and have excellent antioxidant effects that remove harmful reactive oxygen species from the body. They are also known to improve blood circulation by activating nitric oxide (NO) and prostaglandin I2 (PGI2, prostacyclin), thereby inhibiting platelet aggregation and inducing vasodilation. It is also known to have memory benefits and improve cognitive abilities in dementia.

[0043] The exosomes derived from Ulleung chrysanthemum and ginkgo leaves as effective ingredients of the cosmetic composition of the present invention are purified by the following method.

[0044] (A) a step of extracting Ulleung chrysanthemum or ginkgo leaves by adding a solvent and repeating freezing and thawing; (B) a step of treating Ulleung chrysanthemum or ginkgo leaves that have been pretreated by freezing and thawing with UV-A for 1 to 6 hours; (C) a step of extracting the UV-treated Ulleung chrysanthemum or ginkgo leaves; (D) a step of centrifuging the extracted juice at 1,000xg to 10,000xg to obtain a supernatant; (E) a step of lyophilizing the supernatant in which exosomes are present; (F) a step of forming an aqueous two-phase system using PEG (Polyethylene glycol) / Dextran in the lyophilized product; and (G) a step of obtaining a lower layer in which exosomes are concentrated in the aqueous two-phase system.

[0045] In the present invention, in order to manufacture exosomes derived from Ulleung chrysanthemum and ginkgo leaves with excellent skin improvement activity, water is first added to the raw material, and freeze-thawing treatment and UV pretreatment are performed.

[0046] In plants, the secretion characteristics of exosomes can vary depending on environmental factors, and in particular, when the plant is stressed, the amount of exosomes secreted can be affected. Therefore, to determine the optimal conditions for exosome secretion in plants, various pretreatments were performed to confirm the yield and skin improvement activity. It was confirmed that freeze-thawing and UV pretreatment of Ulleung Chrysanthemum and Ginkgo leaves increased exosome secretion and skin improvement activity.

[0047] According to a preferred specific example of the present invention, the freeze-thaw treatment in step (A) is performed by repeating the process of freezing for 15 to 20 hours at a temperature of -80 to -70°C, then thawing for 8 to 10 hours at a temperature of 40 to 50°C, 2 to 5 times. Subsequently, irradiation treatment is performed with UV-A having a wavelength of 340 to 380 nm for 4 to 6 hours (step (B)).

[0048] UV treatment was performed after confirming that there was no change in the size and concentration of exosomes when plant exosomes were treated with UV.

[0049] It is preferable that the screw used in the juicing process in the above step (C) has a stirring speed of 20 to 50 rpm.

[0050] The centrifugation method used in the above step (D) refers to a method of separating particles in a solution using centrifugal force according to size, shape, density, viscosity, and rotor speed. It is necessary to sequentially adjust the rpm to remove large contaminants, and it is more preferable to perform the process at 10,000xg to obtain a final solution for forming an aqueous two-phase system.

[0051] The freeze-drying used in the above step (E) is a method in which the temperature of the container is rapidly lowered to freeze the material to be dried, and then the pressure inside the container is raised to near vacuum, thereby immediately sublimating the solidified solvent contained in the material into water vapor and drying it. Freezing is performed at -50 to -80°C for 15 to 24 hours, and drying is performed in a freeze-dryer under vacuum for 72 to 120 hours. The vacuum state here typically refers to the pressure state of the freeze-dryer.

[0052] In order to efficiently separate and purify exosomes, the present invention used the aqueous two-phase partition method, which is a separation method that utilizes the difference in affinity between two layers of two types of aqueous solutions that do not dissolve well in each other.

[0053] In general, PEG / salt (such as sulfate, phosphate, or citrate) can be used to form a two-phase aqueous system. However, to achieve the purpose of the present invention, PEG / Dextran is preferably used. Dextran is a natural polymer obtained through bacterial action and is used as a thickener, binder, and bulking agent in cosmetic formulations.

[0054] In the above step (F), in the formation of the aqueous two-phase system, PEG having a molecular weight of 10,000 to 35,000 is used in an amount of 1 to 15 wt%, preferably 2 to 5 wt%, and Dextran having a molecular weight of 300,000 to 650,000 is used in an amount of 1 to 8 wt%, preferably 1 to 3 wt%. When PEG and Dextran are used in a concentration ratio of 3.3 wt%: 1.7 wt%, the yield of exosomes is the highest and the stability is the best, which is more preferable.

[0055] To increase the purity of exosomes, a process of forming an aqueous two-phase system using an aqueous two-phase system solution of the same concentration and obtaining a lower layer solution may be additionally performed 2 to 3 times following the above step (G).

[0056] The Ulleung Chrysanthemum exosomes, Ginkgo biloba leaf exosomes and their mixed exosomes prepared by the above method showed excellent wrinkle improvement effects (Test Examples 7 and 8), cell regeneration effects (Test Example 11) and skin barrier strengthening effects (Test Examples 9 and 10) compared to general Ulleung Chrysanthemum extract and Ginkgo biloba leaf extract.

[0057] Therefore, the exosomes derived from Ulleung chrysanthemum and ginkgo leaf can be used in a cosmetic composition for improving wrinkles, regenerating cells, and strengthening the skin barrier. At this time, the exosomes derived from Ulleung chrysanthemum and ginkgo leaf or their mixed exosomes as the effective ingredients can be contained in an amount of 0.0001 to 30.0% (w / w) based on the total weight of the cosmetic composition. The mixed exosomes are formed by mixing Ulleung chrysanthemum exosomes and ginkgo leaf exosomes in a weight ratio of 1 to 3:1 to 3, respectively.

[0058] The above cosmetic composition can be manufactured in any formulation that is commonly manufactured, and can be manufactured as, for example, a skin lotion, a skin toner, a pack, a nourishing cream, a moisturizing cream, an essence, a body cream, a body lotion, a body oil, a cleansing foam, a cleansing lotion, a soap, a patch, a foundation, a lipstick, a makeup base, a lipstick, etc.

[0059] [Example]

[0060] Hereinafter, the present invention will be described in more detail based on the following examples and test examples. However, the following examples are only for the purpose of illustrating the present invention, and the present invention is not limited to the following examples. It will be apparent to those skilled in the art to which the present invention pertains that the present invention may be changed into other equivalent examples and substitutions without departing from the technical spirit of the present invention.

[0061]

[0062] Example 1: Preparation of Ulleung Chrysanthemum exosomes

[0063] Ulleung Chrysanthemum Freezing and Thawing Pretreatment

[0064] Purified water was added to 100 g of Ulleung chrysanthemum, frozen at -80°C for 18 hours, and then thawed at 40°C for 8 hours. This process was repeated three times to create a condition conducive to exosome extraction.

[0065]

[0066] Ulleung Chrysanthemum UV-A treatment

[0067] Ulleung chrysanthemum, which had been pretreated by freeze-thawing, was treated with UV-A at a wavelength of 365 nm for 6 hours.

[0068]

[0069] Ulleung Chrysanthemum Juice

[0070] UV-treated Ulleung Chrysanthemum flowers were extracted using a conventional juicer with a low-speed screw of 30 rpm. The obtained Ulleung Chrysanthemum juice was filtered through a mesh screen to remove suspended matter. The recovered Ulleung Chrysanthemum juice was stored at -80℃ until purification.

[0071]

[0072] Supernatant recovery for exosome purification

[0073] Because the extract of Ulleung Chrysanthemum required the removal of large contaminants for exosome purification, centrifugation was performed at 10,000 × g for 10 min at 4°C. After centrifugation, the supernatant was collected to form an aqueous two-phase system.

[0074]

[0075] Freeze-drying of the supernatant

[0076] To reduce the volume of the supernatant for mass production of exosomes, lyophilization was performed. The solution was frozen at -80°C for 20 hours and then dried in a vacuum freeze dryer for 100 hours. The vacuum typically refers to the pressure of the freeze dryer, and the freezing and drying times may vary depending on the solution volume.

[0077]

[0078] Formation of a two-phase system on Mercury

[0079] Purified water was added to the freeze-dried 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 in an amount of 3.3 wt%, and Dextran (purchased from Sigma Aldrich) with a molecular weight of 300,000–650,000 was used in an amount of 1.7 wt% to form an aqueous two-phase system.

[0080]

[0081] Recovery of Ulleung Chrysanthemum exosomes

[0082] After mixing the supernatant and PEG / Dextran solution, centrifugation was performed at 1,000 × g for 10 minutes at 4°C. After centrifugation, the supernatant was removed to recover exosomes.

[0083]

[0084] Additional cleaning process

[0085] To increase purity, the recovered supernatant was subjected to an additional washing process using the same concentration of the aqueous two-phase solution. After three repeated treatments, the final supernatant, enriched in exosomes, was recovered.

[0086]

[0087] Example 2: Preparation of Ginkgo Leaf Exosomes

[0088] Exosomes derived from ginkgo leaves were purified in the same manner as in Example 1 above.

[0089]

[0090] Example 3: Preparation of mixed exosomes from Ulleung chrysanthemum and ginkgo leaves.

[0091] The Ulleung chrysanthemum exosomes and ginkgo leaf exosomes prepared in Examples 1 and 2 above were mixed in the same weight ratio to prepare mixed exosomes.

[0092]

[0093] Examples 4-9: Preparation of exosomes from Ulleung Chrysanthemum and Ginkgo leaves

[0094] In the case of plants, the secretion characteristics of exosomes can vary depending on environmental factors. Since the optimal conditions for exosome secretion for each plant are different, the yield of exosomes due to environmental stresses such as freeze-thawing treatment and UV treatment was compared to establish the optimal conditions. Exosomes were purified by varying the freeze-thawing treatment and UV treatment conditions of Ulleung chrysanthemum and ginkgo leaves. In order to confirm the optimal conditions, exosomes were purified in the same manner as in Example 1 except for the corresponding conditions, and the conditions are shown in Table 1 below. In Examples 4 and 7, Ulleung chrysanthemum and ginkgo leaves were purified by adding purified water and then extracting the juice without freeze-thawing treatment or UV pretreatment, and applying an aqueous two-phase system.

[0095] Whether the raw material was frozen and thawed or treated with UV treatment. Example 4 Ulleung Chrysanthemum XX. Example 5 Ulleung Chrysanthemum ○X. Example 6 Ulleung Chrysanthemum X○. Example 7 Ginkgo leaf XX. Example 8 Ginkgo leaf ○X. Example 9 Ginkgo leaf X○.

[0096]

[0097] Comparative Example 1: Preparation of Ulleung Chrysanthemum Extract

[0098] 10g of dried Ulleung Chrysanthemum was added to 100g of purified water and extracted at 80℃ for 3 hours. After extraction, vacuum filtration was performed to obtain Ulleung Chrysanthemum extract, which was then distilled using a rotary evaporator to obtain a powder sample.

[0099]

[0100] Comparative Example 2: Preparation of Ginkgo Leaf Extract

[0101] 10g of dried ginkgo leaves were added to 100g of purified water and extracted at 80℃ for 3 hours. After extraction, reduced pressure filtration was performed to obtain a ginkgo leaf extract, which was then distilled using a rotary evaporator to obtain a powder sample.

[0102]

[0103] Experimental Example 1: Characterization of exosomes derived from Ulleung Chrysanthemum: TEM analysis

[0104] To confirm the morphology of purified exosomes derived from Ulleung Chrysanthemum, transmission electron microscopy (TEM) analysis was performed. Figure 1 is a TEM image of exosomes derived from Ulleung Chrysanthemum, purified according to Example 1. The analysis results confirmed the presence of particles measuring approximately 150 nm with a spherical phospholipid bilayer structure.

[0105]

[0106] Experimental Example 2: Characterization of exosomes derived from ginkgo leaves: TEM analysis

[0107] To confirm the morphology of purified Ginkgo leaf-derived exosomes, transmission electron microscopy (TEM) analysis was performed. Figure 2 shows a TEM image of Ginkgo leaf-derived exosomes purified according to Example 2. The analysis results confirmed the presence of approximately 150 nm particles with a spherical phospholipid bilayer structure.

[0108]

[0109] Experimental Example 3: Characterization of Ulleung Chrysanthemum-derived exosomes: NTA analysis

[0110] To confirm the particle size distribution and number of particles per unit volume of purified Ulleung Chrysanthemum-derived exosomes, nanoparticle tracking analysis (NTA) was used.

[0111] Figure 3 is a graph showing the results of NTA analysis of exosomes derived from Ulleung chrysanthemum purified according to Example 1. As a result of the analysis, the average particle size was 150.1 nm and the concentration was 3.3E+10 per unit volume of 1 mL.

[0112]

[0113] Experimental Example 4: Characterization of Ginkgo Leaf-Derived Exosomes: NTA Analysis

[0114] To confirm the particle size distribution and number of particles per unit volume of purified ginkgo leaf-derived exosomes, nanoparticle tracking analysis (NTA) was used.

[0115] Figure 4 is a graph showing the results of NTA analysis of exosomes derived from ginkgo leaves purified according to the above Example 2. As a result of the analysis, the average particle size was 178.5 nm and the concentration was 5.0E+10 per unit volume of 1 mL.

[0116]

[0117] Experimental Example 5: Comparison of the yields of exosomes derived from Ulleung Chrysanthemum and Ginkgo leaves.

[0118] To determine the optimal conditions for freeze-thaw treatment and UV treatment, the yields of exosomes derived from Ulleung chrysanthemum and ginkgo leaves purified according to Examples 1, 2, and 4-9 were compared. Table 2 below shows the yield comparison based on nanoparticle tracking analysis (NTA) results.

[0119] NumberParticle Number(Particles / ml)Particle Size(nm)Example 13.3E+10150.1Example 25.0E+10178.5Example 46.2E+08148.7Example 53.1E+09143.2Example 62.7E+09147.3Example 78.0E+08176.3Example 85.2E+09170.6Example 96.4E+09173.7

[0120] As confirmed in Table 2 above, the yield of exosomes was the highest in Examples 1 and 2, which were freeze-thaw treatment and UV pretreatment conditions. Looking at the results of Examples 4 and 7, the yield of exosomes that were not freeze-thawed or UV pretreatment was the lowest. This indicates that the pretreatment conditions have a great influence on the yield of exosomes, and it was confirmed that the most optimal conditions were freeze-thaw treatment and UV pretreatment.

[0121]

[0122] Test Example 6: Cytotoxicity Evaluation

[0123] To confirm the cytotoxicity of Ulleung Chrysanthemum exosomes (Example 1), Ginkgo leaf exosomes (Example 2), and their mixed exosomes (Example 3), Ulleung Chrysanthemum extract (Comparative Example 1), and Ginkgo leaf extract (Comparative Example 2), an MTT assay was performed. Human keratinocyte (HaCaT) cells were seeded in a 96-well plate at a density of 1 × 10 5 After inoculation at a concentration of 10 cells / mL, the cells were cultured at 37°C for 18 hours under 5% CO2. After incubation, the medium was removed, washed with PBS buffer, and then Ulleung Chrysanthemum exosomes, Ginkgo biloba exosomes, their mixed exosomes, Ulleung Chrysanthemum extract, and Ginkgo biloba leaf extract were added to new medium at different concentrations, and cultured for another 24 hours. To measure the cell viability, MTT solution (5 mg / mL) was added, and the formazan formed over 4 hours was dissolved with Dimethyl sulfoxide (DMSO), and the absorbance was measured at 570 nm using an ELISA reader.

[0124] Figure 5 is a graph showing the results of the MTT assay for evaluating the cytotoxicity of the above samples. The test results showed that no cytotoxicity was observed at any concentration when treated with Ulleung Chrysanthemum exosomes, Ginkgo leaf exosomes, mixed Ulleung Chrysanthemum and Ginkgo leaf exosomes, and Ulleung Chrysanthemum extracts and Ginkgo leaf extracts.

[0125]

[0126] Test Example 7: Evaluation of the Wrinkle-Improving Efficacy of Ulleung Chrysanthemum and Ginkgo Leaf Exosomes (COL1A1 Expression)

[0127] To confirm the anti-wrinkle efficacy of exosomes derived from Ulleung chrysanthemum and Ginkgo biloba leaves, their effects on COL1A1 expression were evaluated compared to the extracts. After inoculation of human dermal fibroblast (HDFa) cell lines, they were cultured in Fibroblast Basal Medium (Medium 106) culture medium supplemented with 100 IU / mL penicillin and 100 μg / mL streptomycin at 37°C for 24 h under 5% CO2. The medium in each well was removed and replaced with fresh serum-free medium. Ulleung chrysanthemum exosomes, Ginkgo biloba leaf exosomes, Ulleung chrysanthemum extract, and Ginkgo biloba leaf extract were administered to each well at different concentrations and pretreated for 4 h. Each well plate was exposed to 20 mJ / cm UVB irradiation using a UVB irradiation device (vilber loumet, France). 2UVB was irradiated. After treating with serum-free medium in which the sample was diluted, the cells were further cultured for 24 hours. At this time, PBS was treated as a negative control and 10 nM TGF-β1 was treated as a positive control. After completing cell culture, cells were lysed using Ribo Ex ™ Total RNA Isolation Solution (GeneAll Biotechnology, Korea) and a scraper, 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 volume of isopropanol (Merck-Millipore, Germany) was added to the supernatant, inverted, and centrifuged (12,000 rpm, 4°C, 30 min). RNA was precipitated, the supernatant was discarded, and the remaining precipitate was washed with 70% ethanol (Merck-Millipore, Germany) and centrifuged (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 the A260 / A280 wavelength using a Micro-volume Spectrophotometer (MN-913, Maestrogen, USA), and the ratio of 260 nm to 280 nm was confirmed to be in the range of 2.0-2.2. cDNA was synthesized by preparing 1 μg RNA, Oligo dT (Bionics, Korea), dNTP (Takara, Korea), and nuclease-free water in a total volume of 13 μL in a PCR tube, incubating at 65°C for 5 minutes, and then incubating at 37°C for 50 minutes with M-MLV Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA). qRT-PCR was performed to quantitatively analyze the gene expression pattern occurring in each cell by the sample. qRT-PCR was performed by mixing primers, cDNA, 2X SYBR green PCR Master Mix (Applied Biosystems, USA), and HPLC (J. T baker, USA) in a PCR tube to create a reaction solution (total 20 μL), and using the StepOnePlus Real-Time PCR System (Applied Biosystems, USA).

[0128] The PCR primer sequence of the COL1A1 gene is shown in Table 3 below, and Fig. 6 is a graph showing the results of evaluating the wrinkle improvement efficacy of exosomes derived from Ulleung chrysanthemum and Ginkgo biloba leaves purified according to the above example, based on COL1A1 mRNA expression. As a result of the test, when Ulleung chrysanthemum, exosomes, and Ginkgo biloba leaf exosomes and Ulleung chrysanthemum extract and Ginkgo biloba leaf extract were treated, COLA1A mRNA, a collagen production factor, increased in a concentration-dependent manner, and it was confirmed that the expression rate increased much more in the range of exosome treatment concentrations compared to the extract. At this time, when TGF-β1 10 nM, which is a positive control, was treated, the fold change value was confirmed to be 2.998.

[0129] GeneForward primer (5'→3')Reverse primer (5'→3')COL1A15'-GAG AGC ATG ACC GAT GGA TT-3'5'-CCT TCT TGA GGT TGC CAG TC-3'GAPDH5'-GTC TCC TCT GAC TTC AAC AGC G-3'5'-ACC ACC CTG TTG CTG TAG CCA A-3'

[0130]

[0131] Test Example 8: Evaluation of the wrinkle-improving efficacy of exosomes from Ulleung Chrysanthemum and Ginkgo leaves (inhibition of MMP-1 expression)

[0132] In order to confirm the wrinkle improvement efficacy of exosomes derived from Ulleung chrysanthemum and Ginkgo biloba leaves, the effect on MMP-1 expression was confirmed by comparing them with the extracts. The experimental method is the same as Test Example 7 above, and the PCR primer sequences of the MMP-1 gene are shown in Table 4 below. Figure 7 is a graph showing the results of evaluating the wrinkle improvement efficacy of Ulleung chrysanthemum and Ginkgo biloba leaves-derived exosomes purified according to the above example by suppression of MMP-1 mRNA expression. As a result of the test, when Ulleung chrysanthemum exosomes, Ginkgo biloba leaf exosomes, Ulleung chrysanthemum extract, and Ginkgo biloba leaf extract were treated, the collagenase factor MMP-1 mRNA decreased in a concentration-dependent manner, and it was confirmed that the inhibition rate was much higher in the range of exosome treatment concentrations compared to the extracts. At this time, when treated with 10 nM of TGF-β1, which is a positive control, the fold change value was confirmed to be 2.680.

[0133] GeneForward primer (5'→3')Reverse primer (5'→3')MMP-15'-GGG CTT GAA GCT GCT TAC GA-3'5'-ACA GCC CAG TAC TTA TTC CCT TTG-3'GAPDH5'-GTC TCC TCT GAC TTC AAC AGC G-3'5'-ACC ACC CTG TTG CTG TAG CCA A-3'

[0134]

[0135] Test Example 9: Evaluation of the Skin Barrier Strengthening Efficacy of Ulleung Chrysanthemum and Ginkgo Leaf Exosomes (Filaggrin Expression)

[0136] To confirm the skin barrier strengthening efficacy of exosomes derived from Ulleung Chrysanthemum and Ginkgo biloba leaves, their effects on filaggrin expression were evaluated compared to the extracts. After inoculation of human keratinocyte (HaCaT) cell lines, they were cultured in Fibroblast Basal Medium (Medium 106) culture medium supplemented with 100 IU / mL penicillin and 100 μg / mL streptomycin at 37°C for 24 h under 5% CO2. The medium in each well was removed and replaced with fresh serum-free medium. Ulleung Chrysanthemum exosomes, Ginkgo biloba leaf exosomes, Ulleung Chrysanthemum extract, and Ginkgo biloba leaf extract were administered to each well at different concentrations and pretreated for 4 h. Each well plate was exposed to 20 mJ / cm UVB irradiation using a UVB irradiation device (vilber loumet, France). 2UVB was irradiated. After treating with serum-free medium in which the sample was diluted, the cells were further cultured for 24 hours. At this time, PBS was treated as a negative control and 100 nM CaCl2 was treated as a positive control. After completing cell culture, the cells 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 volume of isopropanol (Merck-Millipore, Germany) was added to the supernatant, inverted, and centrifuged (12,000 rpm, 4°C, 30 min). RNA was precipitated, the supernatant was discarded, and the remaining precipitate was washed with 70% ethanol (Merck-Millipore, Germany) and centrifuged (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 the A260 / A280 wavelength using a Micro-volume Spectrophotometer (MN-913, Maestrogen, USA), and the ratio of 260 nm to 280 nm was confirmed to be in the range of 2.0-2.2. cDNA was synthesized by preparing 1 μg RNA, Oligo dT (Bionics, Korea), dNTP (Takara, Korea), and nuclease-free water in a total volume of 13 μL in a PCR tube, incubating at 65°C for 5 minutes, and then incubating at 37°C for 50 minutes with M-MLV Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA). qRT-PCR was performed to quantitatively analyze the gene expression pattern occurring in each cell by the sample. qRT-PCR was performed by mixing primers, cDNA, 2X SYBR green PCR Master Mix (Applied Biosystems, USA), and HPLC (J. T baker, USA) in a PCR tube to create a reaction solution (total 20 μL), and using the StepOnePlus Real-Time PCR System (Applied Biosystems, USA).

[0137] The PCR primer sequences of the filaggrin gene are shown in Table 5 below, and Fig. 8 is a graph showing the results of evaluating the skin barrier strengthening efficacy of exosomes derived from Ulleung chrysanthemum and Ginkgo biloba leaves purified according to the above examples, based on Filaggrin mRNA expression. As a result of the test, when Ulleung chrysanthemum exosomes, Ginkgo biloba leaf exosomes, Ulleung chrysanthemum extract, and Ginkgo biloba leaf extract were treated, Filaggrin mRNA, a skin barrier protein, increased in a concentration-dependent manner, and it was confirmed that the expression increased significantly more in the range of exosome treatment concentrations compared to the extract. At this time, when CaCl2100 nM, which was the positive control group, was treated, the fold change value was confirmed to be 2.620.

[0138] GeneForward primer (5'→3')Reverse primer (5'→3')Filaggrin5'-GCT GAA GGA ACT TCT GGA AAA GG-3'5'-GTT GTG GTC TAT ATC CAA GTG ATC-3'GAPDH5'-GTC TCC TCT GAC TTC AAC AGC G-3'5'-ACC ACC CTG TTG CTG TAG CCA A-3'

[0139]

[0140] Test Example 10: Evaluation of the Skin Barrier Strengthening Efficacy of Ulleung Chrysanthemum and Ginkgo Leaf Exosomes (Loricrin Expression)

[0141] In order to confirm the skin barrier strengthening efficacy of exosomes derived from Ulleung chrysanthemum and Ginkgo biloba leaves, the effect on Loricrin expression was confirmed by comparing them with the extracts. The experimental method was the same as Test Example 9 above, and the PCR primer sequences of the Loricrin gene are shown in Table 6 below. Figure 9 is a graph showing the results of evaluating the skin barrier strengthening efficacy of Ulleung chrysanthemum and Ginkgo biloba leaves-derived exosomes purified according to the above example, as measured by Loricrin mRNA expression. As a result of the test, when Ulleung chrysanthemum exosomes, Ginkgo biloba leaf exosomes, Ulleung chrysanthemum extract, and Ginkgo biloba leaf extract were treated, Loricrin mRNA, a skin barrier protein, increased in a concentration-dependent manner, and it was confirmed that the expression increased significantly more in the range of exosome treatment concentrations compared to the extracts. At this time, when CaCl2100 nM, which was the positive control, was treated, the fold change value was confirmed to be 2.600.

[0142] GeneForward primer (5'→3')Reverse primer (5'→3')Loricrin5'- GTC TGC GGA GGT GGT TCC TCT-3'5'- TGC TGG GTC TGG TGG CAG ATC-3'GAPDH5'-GTC TCC TCT GAC TTC AAC AGC G-3'5'-ACC ACC CTG TTG CTG TAG CCA A-3'

[0143]

[0144] Test Example 11: Evaluation of the Cell Regeneration Efficacy of Exosomes Derived from Cnidium officinalis and Peony Root (Wound Healing Assay)

[0145] To investigate the cell regeneration efficacy of exosomes derived from Ulleung Chrysanthemum and Ginkgo biloba leaves, a wound healing assay was conducted. When cells are damaged by chemical and physical stimuli, they secrete various chemokines and cytokines, and simultaneously migrate to the damaged skin site to induce cell proliferation. Therefore, the cell regeneration-inducing effect of the samples was confirmed through a wound healing assay that tests skin cell growth and migration. Keratinocytes (HaCaT) were seeded in 6-well plates and cultured at 37°C for 24 hours under 5% CO2. After incubation, cells were scraped using a 200 μL pipette tip. The detached cells were repeatedly washed with PBS buffer and replaced with DMEM medium containing 1.5% bovine serum. The cells were then treated with Ulleung Chrysanthemum exosomes, Ginkgo biloba leaf exosomes, Ulleung Chrysanthemum extract, and Ginkgo biloba leaf extract. As a positive control, 10 ng / mL of TGF-β1 was added. After culturing for 18 hours, the same location at 0 and 18 hours after the wound was formed was photographed under a microscope, and the area reduced for 18 hours was measured with Image J (National Institutes of Health, USA) and the average value was calculated. Fig. 10 is an image showing the results of evaluating the skin regeneration efficacy of the purified Ulleung chrysanthemum and ginkgo leaf-derived exosomes according to the above example using a wound healing assay, and shows the results when 1.0E+08 particles / mL of Ulleung chrysanthemum and ginkgo leaf exosomes were treated and 20% of Ulleung chrysanthemum and ginkgo leaf extracts were treated. Fig. 11 shows the graph results for the wound area ratio. As a result of the test, the wound area decreased when Ulleung chrysanthemum-derived exosomes and ginkgo leaf-derived exosomes were treated, and a higher area reduction rate was shown when Ulleung chrysanthemum and ginkgo leaf mixed exosomes were treated. It was confirmed that this increased cell growth and migration, which was effective in cell regeneration.At this time, when the positive control group, TGF-β1 10ng / mL, was treated, the area ratio was 37.05%.

[0146]

[0147] Formulation Examples 1-3: Preparation of Cream

[0148] A cream containing purified Ulleung chrysanthemum exosomes and ginkgo biloba exosomes according to the above examples was prepared by a conventional method with the compositions shown in Table 7 below. The cream containing the Ulleung chrysanthemum extract of Comparative Example 1 was designated as Comparative Formulation Example 1, and the cream containing the ginkgo biloba extract of Comparative Example 2 was designated as Comparative Formulation Example 2.

[0149] Ingredient Formulation Example 1 Formulation Example 2 Formulation Example 3 Comparative Formulation Example 1 Comparative Formulation Example 2 Content (weight %) Ulleung Chrysanthemum exosome (Example 1) 5----Ginkgo leaf exosome (Example 2) - 5---Ulleung Chrysanthemum + Ginkgo leaf exosome (Example 3) - 5---Ulleung Chrysanthemum extract (Comparative Example 1) - 5-Ginkgo leaf extract (Comparative Example 2)----5 Glycerin 10 10 10 10 10 Butylene Glycol 5 5 5 5 Glyceryl Oleate 1.8 1.8 1.8 1.8 1.8 Cetearyl Olivate 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Sorbitan Olivate 0.5 0.5 0.5 0.5 0.5 Caprylic / Capric Triglyceride 5.0 5 0.5 5 0.5 5 0.0 Cetyl Ethylhexanoate 1.0 1 0.0 1 0.0 1 0 Beeswax 0.5 0.5 0.50.50.5 Squalane 0.20.20.20.20.21,2-Hexanediol 0.20.20.20.20.2 Cholesteryl / Behenyl / Octyldodecyl Lauroyl Glutamate 1.01.01.01.01.0 Dimethicone 0.50.50.50.50.5 Cyclopentasilon / Cyclohexasiloxane 2.02.02.02.02.0 Cetearyl Alcohol 1.01.01.01.01.0 Mineral Oil 2.5 2.5 2.5 2.5 2.5 Disodium EDTA 0.020 020 020 020 020 02 BHT 0.05 0.05 0.05 0.05 0.05 Tocopheryl Acetate 0.30 30 30 30 30 30 3 Panthenol 0.20 20 20 20 20 20 20 20 20 2 Ethylhexyl Methoxycinnamate 0.20 ...

[0150]

[0151] Test Example 12: Evaluation of Wrinkle Improvement Effects of Exosomes from Ulleung Chrysanthemum and Ginkgo Leaves

[0152] In order to confirm the wrinkle improvement efficacy of Ulleung Chrysanthemum exosomes (Example 1), Ginkgo leaf exosomes (Example 2), and their mixed exosomes (Example 3), 20 adult women in their 30s to 50s were tested. The cream prepared in the above formulation example was applied to both sides of the face, and irradiated with red light of 633 nm using an LED light source for a cumulative 24 hours for 6 weeks. The wrinkle improvement effect was then evaluated through an actual use test.

[0153] Table 8 shows the results of an actual use test evaluating the wrinkle improvement effect of exosomes derived from Ulleung chrysanthemum and ginkgo leaves purified according to the above examples.

[0154] Wrinkle improvement effect Excellent Slightly None Formulation example 16104 Formulation example 2884 Formulation example 31271 Comparative formulation example 12414 Comparative formulation example 23413

[0155] As can be seen from the results in Table 8 above, the cream containing the Ulleung chrysanthemum exosomes, the ginkgo leaf exosomes, and the mixed exosomes of Ulleung chrysanthemum and ginkgo leaves of the present invention exhibited an excellent skin wrinkle improvement effect.

Claims

1. A cosmetic composition containing Ulleung chrysanthemum exosomes, ginkgo leaf exosomes, or mixed exosomes thereof as an effective ingredient.

2. In the first paragraph, the Ulleung Chrysanthemum exosome and the Ginkgo leaf exosome as the effective ingredients are, A cosmetic composition characterized in that it is purified by a method including: (A) a step of extracting Ulleung chrysanthemum or ginkgo leaf by adding a solvent and repeating freezing and thawing; (B) a step of treating Ulleung chrysanthemum or ginkgo leaf that has been subjected to freeze-thawing pretreatment with UV-A for 1 to 6 hours; (C) a step of extracting the UV-treated Ulleung chrysanthemum or ginkgo leaf; (D) a step of centrifuging the extracted juice at 1,000 xg to 10,000 xg to obtain a supernatant; (E) a step of lyophilizing the supernatant in which exosomes are present; (F) a step of forming an aqueous two-phase system using PEG (Polyethylene glycol) / Dextran in the lyophilized product; and (G) a step of obtaining a lower layer in which exosomes are concentrated among the aqueous two-phase system.

3. A cosmetic composition characterized in that in the second paragraph, the freeze-thaw treatment in step (A) is a process of freezing at a temperature of -80 to -70°C for 15 to 20 hours and then thawing at a temperature of 40 to 50°C for 8 to 10 hours, repeated 2 to 5 times.

4. A cosmetic composition characterized in that in the second paragraph, the UV treatment in step (B) is performed for 4 to 6 hours with UV-A having a wavelength of 340 to 380 nm.

5. A cosmetic composition characterized in that, in paragraph 1, the Ulleung chrysanthemum exosome, the ginkgo leaf exosome, or the mixed exosome thereof is contained in an amount of 0.0001 to 30.0% (w / w) based on the total weight of the composition.

6. A cosmetic composition according to claim 1, characterized in that the cosmetic composition is for improving wrinkles.

7. A cosmetic composition according to claim 1, characterized in that the cosmetic composition is for cell regeneration.

8. A cosmetic composition according to claim 1, characterized in that the cosmetic composition is for strengthening the skin barrier. 9.(A) A step of extracting by adding a solvent to Ulleung chrysanthemum or ginkgo leaves and repeating freezing and thawing; (B) Step of treating Ulleung chrysanthemum or ginkgo leaves that have undergone freeze-thaw pretreatment with UV-A for 1 to 6 hours; (C) Step of juicing UV-treated Ulleung chrysanthemum or ginkgo leaves; (D) a step of centrifuging the juice at 1,000xg to 10,000xg to obtain a supernatant; (E) A step of freeze-drying the supernatant containing exosomes; (F) a step of forming an aqueous two-phase system using PEG (Polyethylene glycol) / Dextran in the above freeze-dried product; and (G) A method for purifying exosomes from Ulleung chrysanthemum or ginkgo leaf, comprising the step of obtaining a lower layer containing concentrated exosomes from the above two-phase aqueous solution.

10. In the 9th paragraph, the freeze-thaw treatment in step (A) is characterized in that the process of freezing at a temperature of -80 to -70°C for 15 to 20 hours and then thawing at a temperature of 40 to 50°C for 8 to 10 hours is repeated 2 to 5 times, and the UV treatment in step (B) is performed with UV-A having a wavelength of 340 to 380 nm for 4 to 6 hours. A method for purifying exosomes of Ulleung chrysanthemum or ginkgo leaf.

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