High-purity ginseng efflux composition and application thereof in functional cosmetics

Abstract

The present invention relates to a functional cosmetic composition comprising plant exosome derived from ginseng as an active ingredient thereof and a functional cosmetic comprising the same. Ginseng efflux has a dual lipid layer structure and is rich in ginsenosides Rb1 and Rg1 that affect skin improvement and whitening. The powerful functional substances ginsenosides Rb1 and Rg1 have been proved to be massively absorbed into skin cells through ginseng exosomes, enhance the synthesis of collagen and hyaluronic acid in the skin cells, and have the efficacy of inhibiting melanin synthesis. Therefore, the ginseng exosome rich in ginsenosides Rb1 and Rg1, as presented in the present invention, can be advantageously used in a functional cosmetic composition for skin aging prevention, skin whitening, wrinkle reduction, and skin cell regeneration.

Description

Technical Field

[0001] This application claims priority based on Korean Patent Application No. 10-2022-0075171 filed on June 20, 2022, and all contents disclosed in the specification and drawings of the corresponding application are incorporated into this application.

[0002] The present invention relates to a cosmetic composition comprising ginseng exosomes as an active ingredient and a functional cosmetic comprising the same. Background Art

[0003] Exosomes are reported to be nano-sized microvesicles secreted by various cells and contain proteins, lipids, mRNA and miRNA, and act on the endocrine system and play an important role as mediators of intercellular communication. Such an action is attracting attention as a new therapeutic agent that can overcome the engraftment rate and immunogenicity that are problems of conventional stem cell therapeutic agents.

[0004] According to recent studies, exosomes isolated from edible plants are known to have very high stability and biocompatibility in vivo because they are not toxic or immunogenic compared to exosomes secreted from mammalian cells. In addition, it is reported that exosomes secreted from mammalian cells contain about 20% cholesterol, but plant exosomes do not have cholesterol. In particular, raw materials plants have an advantage in that they can be obtained in large quantities compared to animal tissues or cell culture fluids.

[0005] Exosomes contain various proteins inside depending on the plant of origin, but little is known about ginseng exosomes until now. In most studies, plant exosomes are characterized by their size or shape, and a method is needed to extract plant exosomes by selecting the appropriate size to extract only the exosomes.

[0006] In the case of conventional technology, attempts have been made to extract plant exosomes by density gradient ultracentrifugation using a sucrose gradient, but such a method is difficult to use in the commercial production process of exosomes, and there is a problem of sucrose being mixed into the extract. In addition, with the density gradient ultracentrifugation using a sucrose gradient, it is impossible to obtain an extract in which the exosomes are present at a concentration of 10 mL per 1 mL unit volume of the extract. 7 In addition, there is a method of filtering the separated extracellular vesicles to obtain substances smaller than the pores of the filtration filter, and this is very disadvantageous for long-term exosome extraction because when the exosomes are extracted from a large amount of plant juice, impurities or exosomes are adsorbed and accumulated in the filter pores, and the filtration efficiency is significantly reduced.

[0007] Throughout this specification, a number of references are cited and their citations are indicated. The disclosures of the cited references are incorporated into this specification by reference in their entirety, and will more clearly describe the level of the technical field to which the present invention belongs and the content of the present invention. Summary of the invention

[0008] Technical issues

[0009] An object of the present invention is to provide a high-purity ginseng exosome composition comprising ginseng-derived exosomes as an active ingredient. More specifically, the present invention relates to the efficacy of plant exosomes comprising ginsenosides Rg1 and Rb1 isolated and purified from ginseng on collagen synthesis, hyaluronic acid synthesis, and whitening of human skin tissue, and an object of the present invention is to provide a cosmetic composition comprising ginseng exosomes having skin regeneration and whitening functions as an active ingredient.

[0010] Another object of the present invention is to provide functional cosmetics for skin protection, wrinkle improvement, skin barrier improvement, skin whitening, etc., prepared by using the composition as a cosmetic raw material.

[0011] Another object of the present invention is to provide a method for skin whitening comprising administering a cosmetic composition comprising ginseng exosomes to a subject.

[0012] Another object of the present invention is to provide a cosmetic use of a cosmetic composition comprising ginseng exosomes for skin whitening.

[0013] Another object of the present invention is to provide cosmetic uses of a cosmetic composition comprising ginseng exosomes for wrinkle improvement and skin regeneration as well as skin barrier improvement.

[0014] More specifically, an object of the present invention is to provide the following embodiments.

[0015] Embodiment 1. A high-purity ginseng exosome composition comprising ginseng-derived exosomes as an active ingredient.

[0016] Embodiment 2. A method for treating the skin of a subject in need thereof, comprising administering ginseng exosomes isolated from ginseng or a ginseng exosome composition to the subject, wherein the isolated ginseng exosomes or the ginseng exosome composition provides anti-aging, anti-oxidation, collagen production, hyaluronic acid production, skin whitening, wrinkle improvement and / or skin cell regeneration effects on the skin.

[0017] Embodiment 3. Use of ginseng exosomes isolated from ginseng or a ginseng exosome composition for providing (or producing) a cosmetic effect on the skin (or human skin), wherein the isolated ginseng exosomes or the ginseng exosome composition provides anti-aging, anti-oxidation, collagen production, hyaluronic acid production, skin whitening, wrinkle improvement and / or skin cell regeneration effects on the skin.

[0018] Embodiment 4. According to any one of the preceding embodiments, the ginseng exosomes isolated from ginseng are present in an amount of 1×10 7 Up to 1×10 14 The content of each is included.

[0019] Embodiment 5. According to any one of the preceding embodiments, the composition is a cosmetic composition.

[0020] Embodiment 6. According to any one of the preceding embodiments, the composition has at least one skin cosmetic use selected from anti-aging, anti-oxidation, collagen production, hyaluronic acid production, skin whitening, wrinkle improvement and / or skin cell regeneration.

[0021] Embodiment 7. According to any one of the preceding embodiments, the composition is a formulation selected from an aqueous solution, a suspension, an emulsion, a cream, a frozen solution, a spray-dried powder, and a freeze-dried powder.

[0022] Embodiment 8. According to any one of the preceding embodiments, the ginseng exosomes isolated from ginseng comprise ginsenoside Rb1 and ginsenoside Rg1.

[0023] Embodiment 9. According to any one of the preceding embodiments, a cosmetic comprising ginseng exosomes isolated from ginseng or a ginseng exosome composition.

[0024] Embodiment 10. According to any one of the preceding embodiments, the cosmetic is a preparation selected from the following: spray, serum, nourishing lotion, soft lotion, soft water, emulsion, skin lotion, skin softener, firming agent, astringent, lotion, emulsion lotion, moisturizing lotion, nourishing lotion, massage cream, nourishing cream, moisturizing cream, hand cream, foundation, powder, cosmetic base, essence, nourishing essence, peel-off mask (pack), soap, cleansing foam, cleansing lotion, cleansing cream, body wash, body cleanser, facial cleanser, treatment, cosmetic solution, cosmetic mask, ointment, gel, liniment, liquid, patch, spray, bath preparation, sunscreen, sunscreen oil and hair care product.

[0025] Embodiment 11. According to any one of the preceding embodiments, the cosmetic is a functional cosmetic having at least one effect selected from the group consisting of skin aging prevention, skin whitening, wrinkle improvement, and skin cell regeneration.

[0026] Embodiment 12. According to any one of the preceding embodiments, the cosmetic has a volume of 1×10 6 Up to 1×10 12 The content of each contains the ginseng exosomes.

[0027] Embodiment 13. A method for preparing the ginseng exosome composition according to any one of the preceding embodiments, the method comprising centrifugation; and tangential-flow filtration (TFF), wherein the method obtains 1×10 7 Up to 1×10 14 A high-purity ginseng exosome composition containing ginseng-derived exosomes.

[0028] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, the claims and the accompanying drawings.

[0029] Technical Solution

[0030] One aspect of the present invention is to provide a high-purity ginseng exosome composition comprising ginseng-derived exosomes as an active ingredient. The ginseng-derived exosomes can be present in an amount of 1×10 6 Up to 1×10 13 , preferably 1×10 7 Up to 1×10 14 , more preferably 1×10 11 Up to 1×10 14 More preferably, 1×10 12 Up to 1×10 14 The content of each is included.

[0031] The term "exosome" used in the present invention refers to an exosome extracted from ginseng with a size of about 50nm to 300nm, preferably 50nm to 200nm, or an extracellular vesicle including an exosome. Ginseng exosomes form particles with a uniform size of 50nm to 200nm in the form of a double lipid membrane.

[0032] Conventional various plant extracts are known to contain anti-aging and antioxidant substances, but there are problems in that it is difficult to apply them to cosmetic raw materials because it is difficult to pass through the skin barrier in the form of a water-soluble extract, and further, the active ingredients are exposed to the extraction solvent during the extraction process and are easily destroyed. On the other hand, exosomes are nanoparticles composed of a double lipid membrane, and are absorbed into skin cells through receptor-mediated endocytosis and membrane fusion, and further, the active ingredients contained inside are not directly exposed, so it is not easy to deteriorate and can maintain biological activity for a long period of time, so it is very advantageous for its industrial use in the cosmetic industry.

[0033] Ginseng, which is a raw material plant of the present invention, belongs to Panax ginseng, which is a species of the genus Panax. As the raw material plant part, the whole ginseng or at least a part including flower, stem, bud and fruit etc. may be mixed and used.

[0034] In a preferred embodiment, the ginseng exosome composition of the present invention is a cosmetic composition, and the composition may have at least one skin cosmetic use selected from anti-aging, anti-oxidation, collagen production, hyaluronic acid production, skin whitening, wrinkle improvement and skin cell regeneration.

[0035] The ginseng exosomes proposed in the present invention contain excessive amounts of ginsenosides Rg1 and Rb1, and have the functions of improving collagen production and hyaluronic acid production of human skin and inhibiting melanin synthesis.

[0036] In the present invention, ginseng exosomes have skin regeneration and whitening functions by containing functional substances of ginsenosides derived from ginseng as a raw material plant. Specifically, ginseng exosomes containing a large amount of ginsenoside Rg1 and ginsenoside Rb1, polyphenols, etc., which are known to have antioxidant functions, exhibit skin regeneration and whitening effects by inhibiting active oxygen accumulated in skin cells.

[0037] Ginseng exosomes have anti-aging and anti-oxidation functions by polyphenol components contained by themselves, and they promote degradation of active oxygen present outside cells and active oxygen accumulated inside cells.

[0038] In particular, ginseng exosomes having antioxidant functions are absorbed into skin cells through receptor-mediated endocytosis and membrane fusion, and the absorbed ginseng exosomes stimulate the antioxidant signaling mechanism inside skin cells to increase the expression of genes such as SOD, catalase (CAT), etc. and exhibit anti-aging and antioxidant effects.

[0039] The ginseng exosome developed in the present invention contains a large amount of ginsenosides or natural steroid glycosides, which are a type of triterpene saponins. Among them, ginsenosides Rg1 and Rb1 have been shown to have an antioxidant mechanism of action and not only have skin improvements such as skin whitening and collagen regeneration, but also have clinical effects such as immune enhancement, fatigue improvement, blood flow improvement, memory improvement, etc.

[0040] In one embodiment, the skin regenerating and whitening cosmetic composition comprising the ginseng exosomes of the present invention as an active ingredient may be provided in a form selected from aqueous solution, suspension, emulsion, cream, frozen solution, spray-dried powder and freeze-dried powder.

[0041] Another aspect of the present invention is to provide a cosmetic comprising a skin regenerating and whitening cosmetic composition containing ginseng exosomes as an active ingredient.

[0042] The cosmetic may apply and mix general components for preparing general skin cosmetics, such as oil, water, surfactant, moisturizer, lower alcohol, thickener, chelating agent, pigment, preservative, flavoring agent, etc., in required amounts.

[0043] In addition, the cosmetic can be prepared in a formulation selected from sprays, serums, nourishing lotions, soft lotions, soft water, emulsions, skin lotions, skin softeners, skin tighteners, astringents, lotions, emulsion lotions, moisturizing lotions, nourishing lotions, massage creams, nourishing creams, moisturizing creams, hand creams, foundations, powders, cosmetic bases, essences, nourishing essences, peel-off masks, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, facial cleansers, care agents, cosmetic solutions, cosmetic masks, ointments, gels, liniments, liquids, patches, sprays, bath preparations, sunscreens, sunscreen oils, and hair care products, and can contain various common carriers and additives suitable for each formulation thereof and known in the art.

[0044] Such cosmetics of the present invention may be functional cosmetics having at least one effect selected from the group consisting of collagen production, hyaluronic acid production, skin whitening, wrinkle improvement, skin barrier improvement, and skin cell regeneration.

[0045] In the present invention, the ginseng exosomes extracted from ginseng can be present in an amount 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 The content of the number of particles or more is included, and preferably, it can be 1×10 6 Up to 1×1012 , more preferably 1×10 6 Up to 1×10 12 , and more preferably 1×10 7 Up to 1×10 12 The content of each particle is included.

[0046] Another aspect of the present invention is to provide a method for preparing a high-purity ginseng composition comprising ginseng exosomes as an active ingredient, the method comprising performing centrifugation; and performing tangential flow filtration (TFF).

[0047] More specifically, the method for preparing of the present invention may include: crushing ginseng; performing microfiltration and centrifugation; and performing tangential flow filtration (TFF).

[0048] The process of crushing the raw material plant may include a process of mechanically crushing a mixture of the raw material plant and the buffer solution mixed in a range of 1:1 to 1:10 as a weight ratio, and this may be performed by mechanically crushing the plant mixture with a rotating blade at a rate of 10 rpm to 1,000 rpm.

[0049] The microfiltration method may be performed by filtering the plant mixture through a filter membrane having pores with a size of 10 μm to 200 nm, and preferably, may be filtered through at least one combination of filter membranes having pores different from each other.

[0050] The centrifugation method may be selected from low-speed centrifugation, high-speed centrifugation, ultracentrifugation and combinations thereof, and preferably, it may include ultracentrifugation. Low-speed centrifugation may achieve a speed of 6,000 rpm (6,000 x g) or less, and is mainly used for centrifugation of samples that are easily precipitated, such as cells or nuclei, etc., and the maximum speed of high-speed centrifugation is about 20,000 rpm to 25,000 rpm (60,000 x g), and ultracentrifugation refers to a centrifugation method with a maximum speed of about 40,000 rpm to 80,000 rpm (600,000 x g).

[0051] Tangential flow filtration (TFF) is a filtration method in which a solution flows in a direction perpendicular to a filter membrane, and impurities with small sizes present in the solution are filtered, and exosomes with large sizes are separated. Compared with conventional filtration methods, it can minimize the adsorption of exosomes to the holes of the filter filter or the clogging of the membrane pores, and it is easily applied to process amplification and cGMP (cosmetic good manufacturing procedure, cosmetics manufacturing procedure) processes. Tangential flow filtration can be selected from at least one of hollow fiber TFF and membrane TFF that can perform ultrafiltration, and preferably, it can use a TFF filter with a molecular weight cutoff (MWCO) of 100,000Da to 500,000Da.

[0052] Therefore, ginseng exosomes according to the present invention can be separated from ginseng by mechanical crushing, stepwise ultracentrifugation and tangential flow filtration (TFF) methods, and these exosomes contain a large amount of polyphenols and ginsenosides, etc., which have skin regeneration, whitening and antioxidant functions.

[0053] Beneficial Effects

[0054] Ginseng exosomes have a double lipid membrane structure and are absorbed into skin cells, and ginsenosides Rg1 and Rb1, polyphenols, etc. contained in the absorbed ginseng exosomes exhibit antioxidant efficacy by stimulating the antioxidant signaling mechanism in skin cells to increase the expression of various antioxidant genes. It has various functional cosmetic uses derived from the antioxidant efficacy of ginsenosides Rg1 and Rb1 and polyphenols contained in ginseng exosomes, such as collagen production, hyaluronic acid production, whitening and anti-aging, skin cell activation, etc. In addition, the excellent stability of ginseng exosomes can be used for functional cosmetics in various forms such as aqueous solutions, frozen solutions, and freeze-dried powders. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Schematic diagram of the process of plant exosome extraction for isolating plant exosomes from plant materials with high purity and high yield.

[0056] Figure 2a The results are the results of analyzing the characteristics of Aloe exosomes by nanoparticle tracking analysis (NTA).

[0057] Figure 2b The results of NTA analysis of the characteristics of garlic exosomes.

[0058] Figure 2c The results of analyzing the characteristics of the exosomes of Sea mustard by NTA.

[0059] Figure 2d Shown are the results of determining the amount of ginseng exosomes by NTA (left) and the results of measuring the protein content in the exosomes (right).

[0060] Figure 3 The results of evaluating the total polyphenol content in ginseng exosomes are shown.

[0061] Figure 4 Shown are the results of evaluating the morphological stability of ginseng exosomes using cryo-TEM after storing the ginseng exosomes at frozen (-70°C) and room temperature (25°C) for 3 months.

[0062] Figure 5a Ginsenoside Rb1 detection reference is shown.

[0063] Figure 5b Ginsenoside Rg1 detection reference is shown.

[0064] Figure 5c The results of detecting ginsenoside Rg1 and ginsenoside Rb1 in ginseng exosomes are shown.

[0065] Figure 5d The results of detecting total polyphenolic compounds in ginseng exosomes are shown.

[0066] Figure 6 Results determining the effectiveness of ginseng exosomes in increasing intracellular collagen production are shown.

[0067] Figure 7 Shown are results determining the effectiveness of ginseng exosomes in increasing intracellular hyaluronic acid production.

[0068] Figure 8 Shown are the results of determining the potency of ginseng exosomes in inhibiting extracellular and intracellular melanin formation. DETAILED DESCRIPTION

[0069] Hereinafter, the present invention will be described in more detail by way of examples. These examples are only intended to describe the present invention in more detail, and it will be apparent to those skilled in the art to which the present invention pertains that the scope of the present invention is not limited by these examples.

[0070] Example

[0071] <Example 1> Isolation of exosomes from various plant materials

[0072] By performing ultracentrifugation and tangential flow filtration (TFF), plant exosomes can be isolated from various plant materials with high purity and yield. Figure 1 In this example, a high-purity plant exosome composition was prepared using aloe vera, garlic, sea mustard and ginseng.

[0073] (1) Preparation of Aloe-derived Exosome Composition

[0074] Aloe exosomes were isolated from the raw material plant aloe by using ultracentrifugation and tangential flow filtration (TFF). Specifically, aloe peel was placed in a blender with phosphate buffered saline (PBS) at a weight ratio of 1:2 (weight / weight) and then crushed. The supernatant was collected by centrifugation at 1,000×g for 10 minutes, and the supernatant was sequentially centrifuged at 2000×g for 20 minutes, 3,000×g for 30 minutes, and 10,000×g for 60 minutes. After that, the supernatant was ultracentrifuged at 100,000×g for 70 minutes at 4°C, and the vacuoles were removed by the process. Finally, the supernatant was discarded, and the remaining pellet was suspended, and then the exosomes were separated by the TFF system. Specifically, they were filtered using a filter with a molecular cutoff of 100kDa to 500kDa in the TFF system, and other impurity particles smaller than the pores of the filter were removed and the solution containing the exosomes was concentrated.

[0075] As a result of extracting aloe peel-derived exosomes by ultrafiltration and TFF, it was determined that the extracted aloe peel-derived exosomes had a spherical microstructure of less than 200 nm, and that aloe peel-derived exosomes were obtained at a concentration of 1×10 9～11 The concentration of the high-purity exosome composition ( Figure 2a ).

[0076] (2) Preparation of garlic-derived exosome composition

[0077] Garlic-derived exosomes were isolated from the raw material plant garlic by using ultracentrifugation and tangential flow filtration (TFF) methods. Specifically, peeled garlic was placed in a blender with phosphate buffered saline (PBS) at a weight ratio of 1:2 (weight / weight) and then crushed. The supernatant was collected by centrifugation at 1,000×g for 10 minutes, and the supernatant was sequentially centrifuged at 2000×g for 20 minutes, 3,000×g for 30 minutes, and 10,000×g for 60 minutes. After that, the supernatant was ultracentrifuged at 100,000×g for 70 minutes at 4°C, and the vacuoles were removed by the process. Finally, the supernatant was discarded, and the remaining precipitate was suspended, and then the exosomes were separated by the TFF system. Specifically, they were filtered using a filter with a molecular cutoff of 100kDa to 500kDa in the TFF system, and other impurity particles smaller than the pores of the filter were removed and the solution containing garlic-derived exosomes was concentrated.

[0078] As a result of extracting garlic-derived exosomes by ultrafiltration and TFF, it was determined that the garlic-derived exosomes had a spherical microstructure of less than 200 nm, and that the garlic exosomes were obtained at a concentration of 1×10 9～11 The concentration of the high-purity exosome composition ( Figure 2b ).

[0079] (3) Preparation of exosome composition derived from sea mustard

[0080] Exosomes derived from sea mustard are separated from the raw material plant sea mustard by using ultracentrifugation and tangential flow filtration (TFF). Specifically, the sea mustard from which water is removed is removed and placed in a blender with phosphate buffered saline (PBS) at a weight ratio of 1:2 (weight / weight), and then crushed. The supernatant is collected by centrifugation at 1,000×g for 10 minutes, and the supernatant is sequentially centrifuged at 2000×g for 20 minutes, at 3,000×g for 30 minutes, and at 10,000×g for 60 minutes. After this, the supernatant is ultracentrifuged at 100,000×g for 70 minutes at 4°C, and the vacuoles are removed by this process. Finally, the supernatant is discarded, and the remaining precipitate is suspended, and then the exosomes are separated by a TFF system. Specifically, they are filtered using a filter with a molecular cutoff of 100kDa to 500kDa in the TFF system, and other impurity particles smaller than the pores of the filter are removed and the solution containing the exosomes is concentrated.

[0081] As a result of extracting the sea mustard-derived exosomes by ultrafiltration and TFF, it was confirmed that the sea mustard-derived exosomes had a spherical microstructure of less than 200 nm, and that the sea mustard exosomes were obtained at a concentration of 1×10 per 1 mL unit volume by nanoparticle tracking analysis (NTA). 9～11 The concentration of the high-purity exosome composition ( Figure 2c ).

[0082] (4) Preparation of Ginseng-derived Exosome Composition

[0083] Ginseng exosomes were isolated from the raw material plant ginseng by using ultracentrifugation and tangential flow filtration (TFF) methods. Specifically, ginseng was removed, placed in a blender with phosphate buffered saline (PBS) at a weight ratio of 1:2 (weight / weight), and then crushed. The supernatant was collected by centrifugation at 1,000Xg for 10 minutes, and the supernatant was sequentially centrifuged at 2000Xg for 20 minutes, 3,000Xg for 30 minutes, and 10,000Xg for 60 minutes. After this, the supernatant was ultracentrifuged at 100,000Xg for 70 minutes at 4°C, and the vacuoles were removed by this process. Finally, the supernatant was discarded, and the remaining precipitate was suspended, and then the exosomes were separated by a TFF system. Specifically, they were filtered using a filter with a molecular cutoff of 100kDa to 500kDa in the TFF system, and other impurity particles smaller than the pores of the filter were removed and the solution containing the exosomes was concentrated.

[0084] As a result of extracting ginseng-derived exosomes by ultrafiltration and TFF as described above, it was confirmed that the ginseng-derived exosomes had a spherical microstructure of 50 nm to 300 nm, and that ginseng exosomes were obtained at a concentration of 1×10 13 or more concentrated high-purity exosome compositions ( Figure 2d left image).

[0085] When a combination of ultracentrifugation and TFF methods were applied to ginseng, in particular among various plant materials such as above, it was determined that a high purity exosome composition with a significantly high particle count per unit volume could be achieved. Thus, it was determined that further studies using ginseng exosomes provided the following exosome composition with the highest purity.

[0086] <Example 2> Evaluation of Characteristics of Ginseng Exosomes

[0087] For the ginseng exosomes separated in Example 1 above, the characteristics of the exosomes were evaluated by the following method. The size of the microparticles was analyzed by nanoparticle tracking analysis (NTA), and as described above, the size of the ginseng exosomes was determined to be 50 nm to 300 nm, and the yield of the ginseng exosomes produced once was determined to be about 1×10 per batch by nanoparticle tracking analysis (NTA). 13 or more ( Figure 2d In addition, the MicroBCA TM The protein assay kit measures the protein content in the exosomes and determines the protein content of 10 μg / mL to 1,000 μg / mL per 1 mL unit volume ( Figure 2d (right image).

[0088] <Example 3> Evaluation of the total polyphenol content of ginseng exosomes

[0089] For the ginseng exosomes separated in Example 1 above, the total polyphenol content in the exosomes was evaluated by the following method: Figure 3 ). The ginseng exosomes separated in Example 1 were extracted in water at 1 mg / mL with an ultrasonic oscillator, and the ginseng exosomes were used as a sample. 20 mg of ginseng exosomes were ultrasonically extracted with 5 mL of purified water at room temperature for 10 minutes, and filtered with a 0.45 μm PVDF membrane filter and used as 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 each of 2N-Folin-ciocalteu reagent was added to the test tube, and 1 mL each of 23% sodium carbonate solution was added, and they were reacted in a dark place at room temperature for 10 minutes. As a standard substance, 1 mg of gallic acid was ultrasonically extracted with 1 mL of purified water at room temperature for 10 minutes, filtered with a 0.45 μm PVDF membrane filter and used as a standard undiluted solution (250 ppm, 125 ppm, 62.5 ppm, 31.25 ppm, 15.625 ppm, 7.8125 ppm, 3.90625 ppm), and the standard solution was added to a test tube by concentration at 1 mL each, and 0.5 mL of 2N-Folin-ciocalteu reagent was added to the test tube, and 1 mL of 23% sodium carbonate solution was added, and they were reacted in a dark place at room temperature for 1 hour. The absorbance was measured under the condition of a wavelength of 760 nm using an Epoch microplate spectrophotometer, and the total polyphenol content in the ginseng exosome was quantified. As an experimental result, the total polyphenol content in the ginseng exosome composition was determined to be 20 mg / g to 30 mg / g. On the other hand, the total polyphenol content in the aloe exosome composition was determined to be 38 mg / g to 41 mg / g.

[0090] <Example 4> Evaluation of the shape and storage stability of ginseng exosomes

[0091] The shape stability of the ginseng exosomes isolated in Example 1 was evaluated under the conditions of ultra-freezing and room temperature storage. 8 p / mL to 10 10 p / mL, and then prepared a cosmetic raw material formulation to which trehalose and 1,3 propanediol and 1,2 hexanediol were added. After preparation, it was stored at a frozen state (-70°C) and at room temperature (25°C) for 3 months, and then the shape stability was evaluated using cryo-TEM. It was determined that both the ginseng exosome and the raw material formulation maintained the shape of the exosome even after storage for 3 months or longer ( Figure 4 ).

[0092] <Example 5> Evaluation of ginsenoside content of ginseng exosomes

[0093] For the ginseng exosomes separated in Example 1 above, the ginsenoside content in the exosomes was evaluated by the following method. The ginseng exosomes separated in Example 1 were freeze-dried and a sample was prepared. The freeze-dried exosomes were dissolved in methanol at a concentration of 117.9 mg / mL, then melted with an ultrasonic oscillator for 20 minutes, then filtered with a 0.45 μm PVDF membrane filter and used as a test solution. 1 mg of each ginsenoside Rg1 and Rb1 was dissolved in methanol and melted to become 1 mg / mL, then diluted (312.5 ppm, 195.313 ppm, 76.294 ppm, 47.684 ppm, 29.803 ppm) and used as a standard undiluted solution, and melted with an ultrasonic oscillator for 20 minutes, then filtered with a 0.45 μm PVDF membrane filter and used as a standard solution. The content of ginsenoside Rg1 and ginsenoside Rb1 in ginseng exosomes was measured using HPLC, and the mobile phase was purified under a gradient solution condition of purified water and acetonitrile. Ginsenoside Rg1 and ginsenoside Rb1 were detected in ginseng exosomes, and it was determined that 0.378 mg to 0.386 mg of ginsenoside Rg1 and 0.214 mg to 0.224 mg of ginsenoside Rb1 were contained inside 1 g of freeze-dried ginseng exosomes ( FIG. 5 ).

[0094] <Example 6> Evaluation of the in vitro collagen production efficacy of ginseng exosomes

[0095] In order to evaluate the cosmetic efficacy of the ginseng exosomes isolated in Example 1, the exosomes were treated to human dermal fibroblasts (HDF) to determine intracellular collagen production (PIP). Before measuring collagen production, the PIP was determined by cell viability analysis at 6.3×10 8 There was no cytotoxicity at concentrations of p / mL or less. When measuring intracellular collagen production, cell culture medium without the test substance was used as a solvent control, and as a positive control, 10 ng / mL TGF-β1 was used. To determine collagen production, samples containing ginseng exosomes were treated to 5×10 4 HDF cells were cultured, and after culturing for 24 hours, each culture supernatant was centrifuged at 10,000 rpm for 10 minutes, and in the case of the supernatant from which the debris was removed, for the amount of procollagen, the absorbance was measured at 450 nm using a type I procollagen C-peptide (PIP) kit.

[0096] In the ginseng exosome, it was determined that 8 p / mL to 6.3×10 8p / mL concentration, the intracellular collagen production increased ( Figure 6 ). Preferably, at 2×10 8 p / mL to 6×10 8 p / mL concentration, intracellular collagen production increased, and they have a similar efficacy to the positive control TGF-β1 (10ng / mL). In this embodiment, the efficacy of ginseng exosomes is not limited to collagen production efficacy, and includes all functional cosmetics efficacy such as anti-oxidation, whitening, aging prevention, collagen production, etc.

[0097] <Example 7> Evaluation of the in vitro hyaluronic acid production efficacy of ginseng exosomes

[0098] To determine the cosmetic efficacy evaluation of the ginseng exosomes isolated in Example 1 above, the exosomes were treated to human dermal fibroblasts (HDF), which are primary cells isolated from adult skin, to measure intracellular hyaluronic acid production. Prior to measuring hyaluronic acid production, the cell viability assay determined that the hyaluronic acid production at 6.3×10 8 There was no cytotoxicity at a concentration of p / mL or less. When measuring intracellular hyaluronic acid production, a cell culture medium without the test substance was used as a solvent control, and as a positive control, 3 μg / mL retinoic acid was used. In the measurement of intracellular hyaluronic acid production, HDFs were plated at 5×10 4 Cells / well were seeded in a 24-well plate and incubated for 24 hours, and then replaced with a FBS-free medium containing ginseng exosomes diluted according to the concentration, and then incubated for 24 hours. After 24 hours, each culture supernatant was centrifuged at 10,000 rpm for 10 minutes, and in the case of the supernatant in which the debris was removed, for the production of hyaluronic acid contained in the culture medium, the absorbance was measured at 450 nm using a human hyaluronic acid (HA) ELISA kit.

[0099] It was determined that at 1.6×10 8 p / mL to 6.3×10 8 p / mL, ginseng exosomes increased intracellular hyaluronic acid production ( Figure 7 ). Preferably, at 2×10 8 p / mL to 4×10 8 p / mL concentration, they effectively increased intracellular hyaluronic acid production, and they had similar efficacy to the positive control retinoic acid. In this embodiment, the efficacy of ginseng exosomes is not limited to hyaluronic acid production efficacy, but includes all functional cosmetic efficacy such as anti-oxidation, whitening, aging prevention, collagen production, etc.

[0100] <Example 8> Evaluation of the Inhibitory Effect of Ginseng Exosomes on Melanin Production in Vitro

[0101] To determine the cosmetic efficacy evaluation of the ginseng exosomes isolated in Example 1 above, the exosomes were treated to skin melanoma cells (B16F10) to determine whether they inhibit melanin production. Prior to measuring melanin production, it was determined that the exosomes were 4×10 7 There was no cytotoxicity at concentrations of p / mL or less. When measuring melanin production, cell culture medium containing no test substance was used as a solvent control, and 100 mg / mL arbutin was used as a positive control. To measure total melanin production by cells, B16F10 cells were cultured at 1×10 5 The cells were seeded at a concentration of 10 cells / well in a 6-well plate and incubated for 24 hours. After 24 hours, the medium in each well was removed, the cells were washed with PBS, and then 4×10 7 p / mL of ginseng exosomes and 100nM α-MSH, and a new culture medium without phenol red, and incubated for 72 hours. The incubated cells are used to measure the amount of intracellular melanin, and the culture medium is used to measure the amount of extracellular melanin. The total melanin production of the cell is calculated by adding each of the intracellular and extracellular melanin amounts. In order to measure the amount of intracellular melanin, the cultured B16F10 cells are washed with PBS, and 1M NaOH is added to each well and dissolved at 60°C. For cell lysates, the absorbance is measured at 400nm, and the amount of melanin is then calculated by a calibration curve using synthetic melanin, and the amount of melanin is converted to the amount of melanin per protein. In order to measure the amount of extracellular melanin, the cell culture medium is centrifuged, and the supernatant is then transferred to a 96-well plate, and the absorbance is measured at 400nm. The amount of melanin is calculated by a calibration curve using synthetic melanin, and the amount of melanin is converted to the amount of melanin per protein.

[0102] It was determined that the ginseng exosomes were 4×10 7 p / mL concentration has the effect of inhibiting the formation of extracellular and intracellular melanin ( Figure 8 ). It was determined that ginseng exosomes effectively inhibit extracellular and intracellular melanin formation. In this embodiment, the efficacy of ginseng exosomes is not limited to whitening efficacy, but includes all functional cosmetic efficacy related to anti-oxidation such as whitening, aging prevention, collagen production, etc.

Claims

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

2. The ginseng exosome composition according to claim 1, wherein the ginseng exosomes are present in an amount of 1×10 7 Up to 1×10 14 The content of each is included.

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 at least one skin cosmetic use selected from anti-aging, anti-oxidation, 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 a preparation 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. The ginseng exosome composition according to claim 1 , wherein the ginseng-derived exosomes comprise ginsenoside Rb1 and ginsenoside Rg1.

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

8. The cosmetic according to claim 7, wherein the cosmetic is a preparation selected from the group consisting of a spray, a serum, a nourishing lotion, a soft lotion, a soft water, an emulsion, a skin lotion, a skin softener, a firming agent, an astringent, a lotion, a milky lotion, a moisturizing lotion, a nourishing lotion, a massage cream, a nourishing cream, a moisturizing cream, a hand cream, a foundation, a powder, a makeup base, an essence, a nourishing essence, a peel-off mask, a soap, a cleansing foam, a cleansing lotion, a cleansing cream, a body wash, a body cleanser, a facial cleanser, a care agent, a cosmetic solution, a cosmetic mask, an ointment, a gel, a liniment, a liquid, a patch, a spray, a bath preparation, a sunscreen, a sunscreen oil, and a hair care product.

9. The cosmetic according to claim 7, wherein the cosmetic is a functional cosmetic having at least one effect selected from the group consisting of skin aging prevention, skin whitening, wrinkle improvement, and skin cell regeneration.

10. The cosmetic according to claim 7, wherein the cosmetic has a volume of 1×10 6 Up to 1×10 12 The content of each comprises the ginseng-derived exosomes.

11. A method for preparing the ginseng exosome composition according to any one of claims 1 to 6, comprising: Perform centrifugation; as well as Perform tangential flow filtration (TFF), The method is used to obtain 1×10 7 Up to 1×10 14 A high-purity ginseng exosome composition containing ginseng-derived exosomes.

Images