High-purity ginseng exosome composition and its use in functional cosmetics

Abstract

The present invention relates to a functional cosmetic composition containing ginseng-derived plant exosomes as an active ingredient and a functional cosmetic containing the same. Ginseng exosomes have a double lipid membrane structure and are rich in ginsenoside Rb1 and ginsenoside Rg1, which affect skin improvement and whitening. Such powerful functional components as ginsenoside Rb1 and ginsenoside Rg1 have been proven to be abundantly absorbed by skin cells by ginseng exosomes, improve collagen synthesis and hyaluronic acid synthesis in skin cells, and have a melanin synthesis inhibitory effect. Therefore, the ginseng exosomes rich in ginsenoside Rb1 and ginsenoside Rg1 presented in the present invention can be usefully used as a functional cosmetic composition for preventing skin aging, skin whitening, improving wrinkles, and skin cell regeneration.

Description

Technical Field

[0001] The present invention relates to a cosmetic composition containing ginseng exosomes as an active ingredient and a functional cosmetic containing the same.

[0002] This application claims priority based on Korean Patent Application No. 10-2022-0075171 filed on June 20, 2022, and all of the contents disclosed in the specification and drawings of the application are incorporated into this application.

Background Art

[0003] Exosomes are nano-sized microvesicles secreted from various cells, contain proteins, lipids, mRNA, and miRNA, and have been reported to act on the endocrine system and play an important role as mediators in cell-to-cell communication. Such a role has attracted attention as a new therapeutic agent that can overcome the problems of engraftment rate and immunogenicity of existing stem cell therapeutics.

[0004] According to recent research, exosomes isolated from edible plants are known to have very high in vivo safety and biocompatibility because they have no toxicity or immunogenicity compared to exosomes secreted from mammalian cells. In addition, exosomes isolated from mammalian cells contain about 20% cholesterol, but it has been reported that exosomes from edible plants have no cholesterol. In particular, raw material plants have the advantage of being obtained in large quantities compared to animal tissues or cell culture fluids.

[0005] Although exosomes contain various proteins inside depending on the source plant, little is known about ginseng exosomes yet. In many studies, the characteristics of plant exosomes have been confirmed as the size and morphology of exosomes, and in order to extract plant exosomes, a method of selecting an appropriate size and extracting only exosomes is required.

[0006] In the case of the prior art, there have been attempts to extract plant exosomes using an ultracentrifugation method with a sucrose gradient. However, such a method has difficulties in being applied to the production process for the commercialization of exosomes, and there is a problem that sucrose is mixed into the extract. Further, in the ultracentrifugation method using a sucrose gradient, it is not possible to obtain a high-purity exosome composition in which exosomes are highly concentrated at a content of 10 7 or more per unit volume of 1 mL of the extract. Also, there is a method of filtering the separated extracellular vesicles to obtain a substance smaller than the pores of the filter. However, when extracting exosomes from a large amount of plant juice, impurities or exosomes are adsorbed and accumulated in the pores of the filter, resulting in a sharp decrease in filtration efficiency, which is very disadvantageous for long-term exosome extraction.

[0007] Numerous documents are referred to throughout this specification and their citations are indicated. The disclosure content of the cited documents is incorporated herein by reference in its entirety to better explain the level in the technical field to which the present invention pertains and the content of the present invention.

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a high-purity ginseng exosome composition containing ginseng-derived exosomes as an active ingredient. More specifically, the present invention relates to the collagen synthesis, hyaluronic acid synthesis, and whitening efficacy of human skin tissue of plant exosomes containing ginsenoside Rg1 and ginsenoside Rb1 separated and purified from ginseng. Another object of the present invention is to provide a cosmetic composition containing ginseng exosomes having skin regeneration and whitening functions as an active ingredient.

[0009] Also, another object of the present invention is to provide functional cosmetics such as skin protection, wrinkle improvement, skin barrier improvement, and skin whitening, which are manufactured using the above composition as a cosmetic raw material.

[0010] Furthermore, another object of the present invention is to provide a skin whitening method including applying a cosmetic composition containing ginseng exosomes to a subject.

[0011] Furthermore, another object of the present invention is to provide the use of a cosmetic composition containing ginseng exosomes as a cosmetic for skin whitening.

[0012] Furthermore, another object of the present invention is to provide the use of a cosmetic composition containing ginseng exosomes as a cosmetic for improving wrinkles, skin regeneration, and improving skin barrier.

[0013] More specifically, an object of the present invention is to provide the following embodiments.

[0014] Embodiment 1. A high-purity ginseng exosome composition containing ginseng exosomes separated from ginseng as an active ingredient.

[0015] Embodiment 2. A method for treating the skin of a subject in need thereof, including applying ginseng exosomes separated from ginseng or a ginseng exosome composition to the subject, wherein the separated ginseng exosomes or ginseng exosome composition provides anti-aging, antioxidant, collagen production, hyaluronic acid production, skin whitening, wrinkle improvement and / or skin cell regeneration effects on the skin.

[0016] Embodiment 3. The use of ginseng exosomes separated from ginseng or a ginseng exosome composition for providing (or producing) a cosmetic effect on the skin (or human skin), wherein the separated ginseng exosomes or ginseng exosome composition provides anti-aging, antioxidant, collagen production, hyaluronic acid production, skin whitening, wrinkle improvement and / or skin cell regeneration effects on the skin.

[0017] Embodiment 4. In any one of the foregoing embodiments, the ginseng exosomes separated from ginseng are contained at a content of 1×10 7 to 1×10 14 per unit volume of 1 mL of the composition.

[0018] Embodiment 5. In any one of the above-described embodiments, the composition is a cosmetic composition.

[0019] Embodiment 6. In any one of the above-described embodiments, the composition has any one or more skin beauty uses selected from the group consisting of anti-aging, antioxidant, collagen production, hyaluronic acid production, skin whitening, wrinkle improvement, and skin cell regeneration.

[0020] Embodiment 7. In any one of the above-described embodiments, the composition is in a dosage form selected from the group consisting of an aqueous solution, a suspension, an emulsion, a cream, a frozen solution, a spray-dried powder, and a freeze-dried powder.

[0021] Embodiment 8. In any one of the above-described embodiments, the ginseng exosomes isolated from the ginseng contain ginsenoside Rb1 and ginsenoside Rg1.

[0022] Embodiment 9. A cosmetic containing the ginseng exosomes or a ginseng exosome composition isolated from ginseng according to any one of the above-described embodiments.

[0023] Embodiment 10. In any one of the above-described embodiments, the cosmetic is in a dosage form selected from the group consisting of a mist, a serum, a nourishing lotion, a softening lotion, soft water, a milk lotion, a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisture lotion, a nourishing lotion, a massage cream, a nourishing cream, a moisture cream, a hand cream, a foundation, a powder, a makeup base, an essence, a nourishing essence, a pack, a soap, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion, a body cleanser, a facial wash, a treatment, a beauty liquid, a beauty pack, an ointment, a gel, a liniment, a liquid, a patch, a spray, a bath agent, a sunscreen, a sun oil, and a hair product.

[0024] Embodiment 11. In any one of the above-described embodiments, the cosmetic is a functional cosmetic having one or more functions selected from the group consisting of prevention of skin aging, skin whitening, wrinkle improvement, and skin cell regeneration.

[0025] Embodiment 12. In any one of the above-described embodiments, the cosmetic contains the ginseng exosome in a content of 1×10 6 to 1×10 12 per unit volume of 1 mL.

[0026] Embodiment 13. A method for producing a ginseng exosome composition according to any one of the above-described embodiments, the method including a centrifugation step and a tangential-flow filtration (TFF) step, wherein a high-purity ginseng exosome composition containing ginseng-derived exosomes in a content of 1×10 7 to 1×10 14 per unit volume of 1 mL of the composition is obtained.

[0027] Still other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings.

Means for Solving the Problem

[0028] One aspect of the present invention is to provide a high-purity ginseng exosome composition containing ginseng-derived exosomes as an active ingredient. The ginseng-derived exosomes may be contained in a content of 1×10 6 to 1×10 13 , preferably 1×10 7 to 1×10 14 , more preferably 1×10 11 to 1×10 14 , still more preferably 1×10 12 to 1×10 14 per unit volume of 1 mL of the composition.

[0029] The term "exosome" used in the present invention generically refers to exosomes having a size of about 50 to 300 nm, preferably 50 to 200 nm, extracted from ginseng, or extracellular vesicles containing exosomes. Ginseng exosomes form particles with a uniform size of 50 to 200 nm in the form of a double lipid membrane.

[0030] Although various existing plant extracts are known to contain anti-aging and antioxidant substances, in the form of water-soluble extracts, they are difficult to pass through the skin barrier, and the active ingredients are easily exposed to the extraction solvent and destroyed during the extraction process, so there is a problem that it is difficult to apply them as cosmetic raw materials. In contrast, exosomes are nanoparticles composed of a double lipid membrane, which are absorbed into skin cells by receptor-mediated endocytosis (RME) and membrane fusion, and the active ingredients contained inside are not directly exposed to the outside, so they are not easily deteriorated and can maintain biological activity for a long time. Therefore, it is very advantageous for commercial use in the cosmetics industry.

[0031] The raw material plant of the present invention, ginseng, belongs to Panax ginseng, a species of the genus Panax. As the part of the raw material plant, one or more parts including the whole ginseng or flowers, stems, young buds, fruits, etc. can be mixed and used.

[0032] In a preferred embodiment, the ginseng exosome composition of the present invention is a cosmetic composition, and the composition may have one or more skin beautifying uses selected from the group consisting of anti-aging, antioxidant, collagen production, hyaluronic acid production, skin whitening, wrinkle improvement, and skin cell regeneration.

[0033] The ginseng exosomes presented by the present invention contain an excessive amount of ginsenoside Rg1 and ginsenoside Rb1, and have the function of promoting collagen production and hyaluronic acid production in human skin and suppressing melanin synthesis.

[0034] In the present invention, ginseng exosomes have skin regeneration and whitening functions due to functional substances containing ginsenosides derived from the raw material plant, ginseng. Specifically, ginseng exosomes containing a large amount of ginsenoside Rg1 and ginsenoside Rb1, which are known to have antioxidant functions, and polyphenols suppress reactive oxygen species accumulated in skin cells and exhibit effects such as skin regeneration and whitening.

[0035] Ginseng exosomes have anti-aging and antioxidant functions due to the polyphenol components they contain, which promote the decomposition of reactive oxygen species existing outside cells and those accumulated inside cells.

[0036] In particular, ginseng exosomes with antioxidant functions are absorbed into skin cells through receptor-mediated endocytosis (RME) and membrane fusion, and the absorbed ginseng exosomes stimulate the antioxidant signaling mechanism in skin cells to increase the gene expression of superoxide dismutase (SOD), catalase (CAT), etc., showing anti-aging and antioxidant effects.

[0037] The ginseng exosomes developed in the present invention contain an excessive amount of ginsenoside, which is a kind of natural steroid glycoside or triterpene saponin. Among them, ginsenoside Rg1 and ginsenoside Rb1 have the mechanism of antioxidant action, and it has been proven that they have not only skin improvement effects such as skin whitening and collagen regeneration, but also clinical effects such as enhancing immunity, improving fatigue, improving blood circulation, and improving memory.

[0038] In one embodiment, a skin regeneration and whitening cosmetic composition containing the ginseng exosomes of the present invention as an active ingredient can be provided in a dosage form selected from the group consisting of an aqueous solution, a suspension, an emulsion, a cream, a frozen solution, a spray-dried powder, and a freeze-dried powder.

[0039] According to another aspect of the present invention, there is provided a cosmetic containing a cosmetic composition for skin regeneration and whitening containing ginseng exosomes as an active ingredient.

[0040] The cosmetic may be formulated with appropriate amounts of ordinary ingredients used in the production of ordinary skin cosmetics, such as oils, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments, preservatives, fragrances, etc., as needed.

[0041] Also, the cosmetic can be manufactured into dosage forms selected from the group consisting of mists, serums, nutritive lotions, emollient lotions, softeners, emulsions, skin lotions, skin softeners, skin toners, astringents, lotions, milk lotions, moisture lotions, nutritive lotions, massage creams, nutritive creams, moisture creams, hand creams, foundations, powders, makeup bases, essences, nutritive essences, packs, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, facial cleansers, treatments, beauty liquids, beauty packs, ointments, gels, liniments, liquids, patches, sprays, bath salts, sunscreen agents, sun oils, and hair products. It is suitable for each of these dosage forms and may contain various ordinary carriers and additives well-known in the industry.

[0042] Such a cosmetic of the present invention can be a functional cosmetic having one or more effects selected from the group consisting of collagen production, hyaluronic acid production, skin whitening, wrinkle improvement, skin barrier improvement, and skin cell regeneration.

[0043] In the present invention, the ginseng exosomes extracted from ginseng may be contained at a content of 1×10 or more, 1×10 2 or more, 1×10 3 or more, 1×10 4 or more, 1×10 5 or more, or 1×10 6 or more particles per unit volume of 1 mL of the cosmetic. Desirably, it may be contained at a content of 1×10 6 to 1×10 12 particles, more desirably at a content of 1×10 6 to 1×10 12 particles, and even more desirably at a content of 1×10 7 to 1×10 12 particles.

[0044] According to still another aspect of the present invention, provided is a method for manufacturing a high-purity ginseng composition containing the ginseng exosomes as an active ingredient, the method including a centrifugation step and a tangential flow filtration (TFF) step.

[0045] More specifically, the manufacturing method of the present invention may include a step of crushing ginseng, a step of performing microfiltration and centrifugation, and a step of performing tangential flow filtration (TFF).

[0046] The process of crushing the raw material plant may include a process of mechanically crushing a mixture of the raw material plant and a buffer solution in a weight ratio of 1:1 to 1:10, which can be performed by mechanically crushing the plant mixture with a blade rotating at a speed of 10 to 1,000 rpm.

[0047] The microfiltration method is performed by filtering the plant mixture through a filtration membrane having voids with a size of 10 μm to 200 nm, and desirably, one or more combinations of filtration membranes having different voids can be used for filtration.

[0048] The centrifugation method can be selected from low-speed centrifugation, high-speed centrifugation, ultracentrifugation, and combinations thereof, and desirably, it can be ultracentrifugation. Low-speed centrifugation can achieve a speed of 6,000 rpm (6,000 xg) or less and is mainly used for centrifugation of samples that are likely to precipitate, such as cells and nuclei. High-speed centrifugation has a maximum speed of about 20,000 to 25,000 rpm (60,000 xg), and ultracentrifugation means a centrifugation method with a maximum speed of about 40,000 to 80,000 rpm (600,000 xg).

[0049] The aforementioned tangential-flow filtration (TFF) is a filtration method in which a solution flows in a direction perpendicular to a filtration membrane, filtering small-sized impurities present in the solution and separating large-sized exosomes. It can minimize the adsorption of exosomes to the pores of the filtration filter or the blockage of the pores compared to conventional filtration methods, facilitating the scale-up of the process and the application of the process according to cosmetic good manufacturing procedure (cGMP). The tangential-flow filtration is one or more selected from the group consisting of hollow fiber TFF and membrane TFF capable of performing ultrafiltration, and preferably, it may use a TFF filter with a molecular weight cutoff (MWCO) of 100,000 Da to 500,000 Da.

[0050] Thus, the ginseng exosomes according to the present invention can be separated from ginseng by mechanical grinding, stepwise ultracentrifugation, and tangential-flow filtration (TFF) methods, and these exosomes contain a large amount of polyphenols, ginsenosides, etc. having skin regeneration, whitening, and antioxidant functions.

Effects of the Invention

[0051] Ginseng exosomes have a double lipid membrane structure and are absorbed by skin cells. Ginsenoside Rg1, ginsenoside Rb1, polyphenols, etc. contained in the absorbed ginseng exosomes exhibit antioxidant efficacy by stimulating the antioxidant signaling mechanism in skin cells and increasing the expression of various antioxidant genes. Ginsenoside Rg1, ginsenoside Rb1, and polyphenols contained in ginseng exosomes have applications in various functional cosmetics such as collagen production, hyaluronic acid production, whitening, anti-aging, and skin cell activation derived from their antioxidant efficacy. In addition, the excellent safety of ginseng exosomes can be applied to functional cosmetics in various forms such as aqueous solutions, frozen liquids, and freeze-dried powders.

Brief Description of the Drawings

[0052]

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Modes for Carrying Out the Invention

[0053] Hereinafter, the present invention will be described in more detail by way of examples. These examples are for more specifically explaining the present invention, and it is self-evident to those with ordinary knowledge in the technical field to which the present invention pertains that the scope of the present invention is not limited by these examples.

[0054] Examples <Example 1> Separation of Exosomes from Various Plant Raw Materials By performing ultracentrifugation and tangential-flow filtration (TFF), plant exosomes can be separated from various plant raw materials with high purity and high yield (Figure 1). In this example, aloe, garlic, wakame, and ginseng were used to produce a high-purity plant exosome composition.

[0055] (1) Production of Aloe-Derived Exosome Composition Aloe exosomes were separated from the raw material plant aloe using ultracentrifugation and tangential-flow filtration (TFF). Specifically, the skin of aloe was put into a blender with phosphate-buffered saline (PBS) at a weight ratio of 1:2 (w / w) and pulverized. Centrifuged at 1,000 Xg for 10 minutes to remove the supernatant, and the supernatant was sequentially centrifuged at 2,000 Xg for 20 minutes, 3,000 Xg for 30 minutes, and 10,000 Xg for 60 minutes. Then, the supernatant was ultracentrifuged at 100,000 Xg for 70 minutes at 4°C to remove vacuoles during this process. Finally, the supernatant was discarded, and the remaining pellet was suspended and then exosomes were separated by the TFF system. Specifically, filtered using a filter with a molecular cutoff of 100 - 500 kDa in the TFF system to remove other impurity particles smaller than the pores of the filter and concentrate the solution containing exosomes.

[0056] As a result of performing TFF, which is ultrafiltration, up to this point to extract aloe vera skin-derived exosomes, it was confirmed that the extracted aloe vera skin-derived exosomes have a spherical microstructure of 200 nm or less, and by nanoparticle tracking analysis (NTA), 1 × 10 9~11 It was confirmed that a high-purity exosome composition in which aloe exosomes were concentrated to a concentration of particles per unit volume of 1 mL was obtained (Fig. 2a).

[0057] (2) Production of garlic-derived exosome composition Garlic-derived exosomes were separated from the raw material plant garlic using ultracentrifugation and tangential flow filtration (TFF). Specifically, garlic with the skin removed was put into a blender at a weight ratio of 1:2 (w / w) with phosphate buffered saline (PBS) and crushed. It was centrifuged at 1,000 Xg for 10 minutes to remove the supernatant, and the supernatant was sequentially centrifuged at 2,000 Xg for 20 minutes, 3,000 Xg for 30 minutes, and 10,000 Xg for 60 minutes. Then, the supernatant was ultracentrifuged at 100,000 Xg for 70 minutes at 4°C to remove vacuoles in this process. Finally, the supernatant was discarded, and after suspending the remaining precipitate, exosomes were separated by a TFF system. Specifically, filtration was performed using a filter having a molecular cut-off of 100 to 500 kDa in the TFF system to remove other impurity particles smaller than the pores of the filter and concentrate the solution containing garlic-derived exosomes.

[0058] As a result of performing TFF, which is ultrafiltration, up to this point to extract garlic-derived exosomes, it was confirmed that the garlic-derived exosomes have a spherical microstructure of 200 nm or less, and by nanoparticle tracking analysis (NTA), 1 × 10 9~11 It was confirmed that a high-purity exosome composition in which garlic exosomes were concentrated at a concentration of particles per unit volume of 1 mL was obtained (Fig. 2b).

[0059] (3) Production of kelp-derived exosome composition The wakame-derived exosomes were separated from the raw material plant, wakame, using ultracentrifugation and tangential flow filtration (TFF). Specifically, the wakame with water removed was put into a blender at a weight ratio of 1:2 (w / w) with phosphate-buffered saline (PBS) and pulverized. It was centrifuged at 1,000 Xg for 10 minutes to remove the supernatant, and the supernatant was successively centrifuged at 2,000 Xg for 20 minutes, 3,000 Xg for 30 minutes, and 10,000 Xg for 60 minutes. Then, the supernatant was ultracentrifuged at 100,000 Xg for 70 minutes at 4°C to remove the vacuoles in this process. Finally, the supernatant was discarded, and after suspending the remaining precipitate, the exosomes were separated by the TFF system. Specifically, it was filtered using a filter with a molecular cut-off of 100 - 500 kDa in the TFF system to remove other impurity particles smaller than the pores of the filter and concentrate the solution containing the exosomes.

[0060] As a result of extracting the wakame-derived exosomes by performing up to TFF, which is ultrafiltration, it was confirmed that the wakame-derived exosomes have a spherical microstructure with a size of 200 nm or less, and by nanoparticle tracking analysis (NTA), it was confirmed that a high-purity exosome composition with the concentration of wakame exosomes concentrated to 1×10 9~11 particles per unit volume of 1 mL was obtained (Figure 2c).

[0061] (4) Production of ginseng-derived exosomes Ginseng exosomes were separated from the raw material plant, ginseng, using ultracentrifugation and tangential flow filtration (TFF). Specifically, ginseng was placed in a blender at a weight ratio of 1:2 (w / w) with phosphate buffered saline (PBS) and then pulverized. It was centrifuged at 1,000 Xg for 10 minutes to remove the supernatant, and the supernatant was sequentially centrifuged at 2,000 Xg for 20 minutes, 3,000 Xg for 30 minutes, and 10,000 Xg for 60 minutes. Subsequently, the supernatant was ultracentrifuged at 100,000 Xg for 70 minutes at 4 °C to remove vacuoles in this process. Finally, the supernatant was discarded, and the remaining precipitate was suspended, and then exosomes were separated by the TFF system. Specifically, filtration was performed using a filter with a molecular cut-off of 100 - 500 kDa in the TFF system to remove other impurity particles smaller than the pores of the filter and concentrate the solution containing exosomes.

[0062] As a result of performing TFF, which is ultrafiltration, up to this point to extract ginseng-derived exosomes, it was confirmed that ginseng-derived exosomes have a size of 50 - 300 nm, and by nanoparticle tracking analysis (NTA), the concentration of ginseng exosomes was 13 1×10 or more per unit volume of 1 mL. It was confirmed that a high-purity exosome composition was obtained (upper graph in Figure 2d).

[0063] As described above, it was confirmed that the combination of ultracentrifugation and TFF methods can achieve a high-purity exosome composition in which exosomes are concentrated to a significantly high number of particles per unit volume, especially when applied to ginseng among many plant raw materials. Then, additional studies were conducted using ginseng exosomes, for which it was confirmed that the highest-purity exosome composition could be obtained.

[0064] <Example 2> Characterization of Ginseng Exosomes The ginseng exosomes isolated in Example 1 were characterized by the following method. The size of fine particles was analyzed by nanoparticle tracking analysis (NTA), and it was confirmed that the ginseng exosomes had a size of 50 to 300 nm. The ginseng exosomes produced at one time were approximately 1 x 10 per lot by nanoparticle tracking analysis (NTA). 13 It was confirmed that the yield of exosomes was more than 10 ... TM Protein Assay Kit (Micro BCA TM The results were measured using a protein assay kit and confirmed to contain 10 to 1,000 μg / mL of protein per 1 mL unit volume (lower graph in Figure 2d).

[0065] Example 3: Evaluation of total polyphenol content in ginseng exosomes The ginseng exosomes isolated in Example 1 were evaluated for the total content of polyphenols in the exosomes by the following method (Figure 3). The ginseng exosomes isolated in Example 1 were extracted with an ultrasonic crusher at 1 mg / mL in water and then used as samples. 20 mg of ginseng exosomes were added to 5 mL of purified water and ultrasonically extracted at room temperature for 10 minutes, and then filtered through a 0.45 μm PVDF membrane filter to obtain a test solution. 1 mL of the test solution and 7.5 mL of purified water were placed in a test tube, and 0.5 mL of 2N Folin's phenol reagent was added to the test tube one by one, and 1 mL of 23% sodium carbonate aqueous solution was added one by one, and the reaction was carried out in the dark at room temperature for 1 hour. As a standard substance, 1 mg of gallic acid was ultrasonically extracted in 1 mL of purified water at room temperature for 10 minutes and filtered through a 0.45 μm PVDF membrane filter to obtain a standard stock solution (250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625 ppm). 1 mL of the standard solution was added to the test tube according to the concentration, 0.5 mL of 2N Folin's phenol reagent was added to the test tube one by one, and 1 mL of 23% sodium carbonate aqueous solution was added one by one, and the reaction was carried out in the dark at room temperature for 1 hour. The total content of polyphenols in the ginseng exosomes was quantified by measuring the absorbance at a wavelength of 760 nm using an Epoch microplate spectrophotometer. As a result of the experiment, it was confirmed that the total content of polyphenols in the ginseng exosome composition was 20 - 30 mg / g. On the other hand, it was confirmed that the total content of polyphenols in the aloe exosome composition was 38 - 41 mg / g.

[0066] <Example 4> Evaluation of the morphology and storage safety of ginseng exosomes The morphological safety of the ginseng exosomes isolated in Example 1 was evaluated under ultra-low temperature freezing and room temperature storage conditions. The ginseng exosomes were at 10 8 p / mL~10 10After diluting to a concentration of p / mL, a dosage form of a cosmetic raw material was prepared by adding trehalose, 1,3-propanediol, and 1,2-hexanediol. After production, it was stored at -70°C (frozen) and 25°C (room temperature) for 3 months, and then the morphological safety was evaluated using cryo-TEM. It was confirmed that the exosome morphology of both ginseng exosomes and the raw material dosage form was maintained even after storage for 3 months or more under normal temperature and ultra-low temperature freezing conditions (Figure 4).

[0067] <Example 5> Evaluation of Ginsenoside Content in Ginseng Exosomes The ginseng exosomes separated in Example 1 were evaluated for the content of ginsenosides in the exosomes by the following method. The ginseng exosomes separated in Example 1 were lyophilized to prepare a sample. After dissolving the lyophilized exosomes in methanol at a concentration of 117.9 mg / mL, they were dissolved with an ultrasonic crusher for 20 minutes and then filtered through a 0.45 μm PVDF membrane filter to obtain a test solution. Ginsenoside Rg1 and ginsenoside Rb1 were each dissolved in 1 mg of methanol to a concentration of 1 mg / mL and then diluted (312.5, 195.313, 76.294, 47.684, 29.803 ppm) to prepare a standard stock solution. After dissolving with an ultrasonic crusher for 20 minutes, it was filtered through a 0.45 μm PVDF membrane filter to obtain a standard solution. HPLC was used to measure the contents of ginsenoside Rg1 and ginsenoside Rb1 in ginseng exosomes, and the mobile phase was purified under gradient solution conditions of purified water and acetonitrile. Ginsenoside Rg1 and ginsenoside Rb1 in ginseng exosomes were detected, and it was confirmed that 0.378 - 0.386 mg of ginsenoside Rg1 and 0.214 - 0.224 mg of ginsenoside Rb1 were contained in 1 g of lyophilized ginseng exosomes (Figure 5).

[0068] <Example 6> Evaluation of In Vitro Collagen Production Efficacy of Ginseng Exosomes To confirm the cosmetic efficacy evaluation of the ginseng exosomes isolated in Example 1, exosomes were treated with human dermal fibroblasts (HDFs) to confirm the amount of collagen produced (PIP) in the cells. Before measuring the amount of collagen produced, it was confirmed by cell viability analysis that there was no cytotoxicity at a concentration of 6.3×10 8 p / mL or less. When measuring the amount of collagen produced in the cells, a cell culture medium without the test substance was used as a solvent control group, and TGF-β1 10 ng / mL was used as a positive control group. To confirm the amount of collagen produced, a cell culture solution containing ginseng exosomes was treated with 5×10 4 HDFs cells. After culturing for 24 hours, each cultured supernatant was centrifuged at 10,000 rpm for 10 minutes, and the amount of procollagen in the supernatant from which debris had been removed was measured at 450 nm using a procollagen type I C-terminal peptide (PIP) kit by measuring the absorbance.

[0069] It was confirmed that the amount of collagen produced in the cells increased at a concentration of 1.6×10 8 ~6.3×10 8 p / mL (Figure 6). Desirably, the amount of collagen produced in the cells was increased at a concentration of 2×10 8 ~6×10 8 p / mL, which has a similar efficacy to TGF-β1 (10 ng / mL), which is the positive control group. In this example, the efficacy of ginseng exosomes is not limited to the collagen production efficacy, but includes all the efficacy of functional cosmetics such as antioxidant, whitening, anti-aging, and collagen production.

[0070] <Example 7> In vitro evaluation of the hyaluronic acid production efficacy of ginseng exosomes To confirm the cosmetic efficacy evaluation of the ginseng exosomes isolated in Example 1, exosomes were treated with human dermal fibroblasts (HDFs), which are primary cells isolated from adult skin, to measure the amount of hyaluronic acid produced intracellularly. Before measuring the amount of hyaluronic acid produced, it was confirmed by cell viability analysis that there was no cytotoxicity at a concentration of 6.3×10 8 p / mL or less. When measuring the amount of intracellular hyaluronic acid produced, a cell culture medium without the test substance was used as the solvent control group, and 3 μg / mL of retinoic acid was used as the positive control group. In the measurement of the amount of intracellular hyaluronic acid produced, HDFs were inoculated at 5×10 4 cells / well in a 24-well plate and cultured for 24 hours, and then replaced with a medium without FBS containing ginseng exosomes diluted by concentration and cultured for 24 hours. After 24 hours, each culture supernatant was centrifuged at 10,000 rpm for 10 minutes, and the absorbance of the hyaluronic acid contained in the medium was measured at 450 nm using a human hyaluronic acid ELISA kit (Human Hyaluronic Acid (HA) ELISA Kit) with the debris-removed supernatant.

[0071] Ginseng exosomes were confirmed to increase the amount of intracellular hyaluronic acid produced at a concentration of 1.6×10 8 ~6.3×10 8 p / mL (Figure 7). Desirably, the intracellular production of hyaluronic acid was effectively increased at a concentration of 2×10 8 ~4×10 8 p / mL, which has a similar efficacy to retinoic acid, the positive control group. In this example, the efficacy of ginseng exosomes is not limited to the efficacy of hyaluronic acid production, but includes all the efficacy of functional cosmetics such as antioxidant, whitening, anti-aging, and collagen production.

[0072] <Example 8> Evaluation of the in vitro melanin production inhibitory efficacy of ginseng exosomes To confirm the cosmetic efficacy evaluation of the ginseng exosomes isolated in Example 1, it was confirmed whether the treatment of exosomes on skin melanoma cells (B16F10) inhibited the production of melanin. Before measuring the amount of melanin production, it was confirmed that there was no cytotoxicity at a concentration of 4×10 7 p / mL or less. When measuring the amount of melanin production, a cell culture medium without the test substance was used as the solvent control group, and 100 mg / mL of arbutin was used as the positive control group. The measurement of the total melanin production amount of the cells was carried out by inoculating B16F10 cells into a 6-well plate at a concentration of 1×10 5 cells / well and culturing for 24 hours. After 24 hours, the medium in each well was removed, and the cells were washed with PBS. Then, a new culture medium containing no phenol red and containing 4×10 7 p / mL of ginseng exosomes and 100 nM of α-MSH was replaced and cultured for 72 hours. The cultured cells were used for measuring the amount of intracellular melanin, and the cell culture solution was used for measuring the amount of extracellular melanin. The total melanin production amount of the cells was calculated by adding the amounts of melanin in the intracellular and extracellular compartments. The B16F10 cells cultured for measuring the amount of intracellular melanin were washed with PBS, and 1 M of NaOH was added to each well and dissolved at 60°C. After measuring the absorbance of the cell lysate at 400 nm, the amount of melanin was determined from the calibration curve using synthetic melanin, and the amount of melanin was converted to the amount of melanin per protein. After centrifuging the cell culture solution for measuring the amount of extracellular melanin, the supernatant was transferred to a 96-well plate and the absorbance was measured at 400 nm. The amount of melanin was determined from the calibration curve using synthetic melanin, and the amount of melanin was converted to the amount of melanin per protein.

[0073] Ginseng exosomes were at 4×10 7It was confirmed that it was effective in suppressing extracellular and intracellular melanogenesis at a concentration of p / mL (Figure 8). It was confirmed that ginseng exosomes effectively inhibited extracellular and intracellular melanogenesis. In this example, the efficacy of ginseng exosomes is not limited to the whitening efficacy, but includes all the efficacy of functional cosmetics, such as whitening related to antioxidant, anti-aging, and collagen production.

Claims

1. A high-purity ginseng exosome composition containing ginseng-derived exosomes as an active ingredient.

2. The ginseng-derived exosomes are contained in an amount of 1×10 ７ to 1×10 １４ per unit volume of 1 mL of the composition, and the ginseng exosome composition according to claim 1 is characterized by this.

3. The ginseng exosome composition according to claim 1, wherein the composition is a cosmetic composition.

4. The ginseng exosome composition according to claim 1, wherein the composition has any one or more skin beautifying uses selected from the group consisting of anti-aging, antioxidant, collagen production, hyaluronic acid production, skin whitening, wrinkle improvement, and skin cell regeneration.

5. The ginseng exosome composition according to claim 1, wherein the composition is in a dosage form selected from the group consisting of an aqueous solution, a suspension, an emulsion, a cream, a frozen solution, a spray-dried powder, and a freeze-dried powder.

6. The ginseng exosome composition according to claim 1, wherein the ginseng-derived exosomes contain ginsenoside Rb1 and ginsenoside Rg1.

7. A cosmetic containing the ginseng exosome composition according to any one of claims 1 to 6.

8. The cosmetic according to claim 7, wherein the cosmetic is in a dosage form selected from the group consisting of a mist, a serum, a nourishing lotion, a softening lotion, a softening water, a milk lotion, a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisture lotion, a nourishing lotion, a massage cream, a nourishing cream, a moisture cream, a hand cream, a foundation, a powder, a makeup base, an essence, a nourishing essence, a pack, a soap, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion, a body cleanser, a facial cleanser, a treatment, a beauty liquid, a beauty pack, an ointment, a gel, a liniment, a liquid, a patch, a spray, a bath agent, a sunscreen, a sun oil, and a hair product.

9. The cosmetic according to claim 7, wherein the cosmetic is a functional cosmetic having one or more effects selected from the group consisting of preventing skin aging, skin whitening, wrinkle improvement, and skin cell regeneration.

10. The cosmetic contains the ginseng-derived exosomes in a content of 1×10 ６ to 1×10 １２ per unit volume of 1 mL. The cosmetic according to claim 7, characterized in that it contains the exosomes in such a content.

11. A method for manufacturing the ginseng exosome composition according to any one of claims 1 to 6, comprising: a step of performing centrifugation; a step of performing tangential flow filtration (TFF). By the above method, a high-purity ginseng exosome composition containing ginseng-derived exosomes at a content of 1×10 ７ to 1×10 １４ per mL of the composition is obtained. A method characterized by this.

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