Skin whitening and Anti-oxidating cosmetic composition containing kidney bean exosomes containing glutathione as active ingredient and functional cosmetics comprising same

Abstract

The present invention relates to a functional cosmetic composition comprising, as an active ingredient, plant exosomes derived from kidney beans, and a functional cosmetic containing same. Kidney bean exosomes have a lipid bilayer structure and are rich in glutathione, which has an effect on skin improvement and whitening. Glutathione, which is a powerful whitening ingredient, was proven to be absorbed in a large amount into skin cells by kidney bean exosomes and have inhibitory efficacy against melanin synthesis. Therefore, the glutathione-rich kidney bean exosomes presented in the present invention can be effectively used as a functional cosmetic composition for skin whitening and skin health improvement.

Description

Skin whitening and antioxidant cosmetic composition containing kidney bean-derived exosomes containing glutathione as an active ingredient and functional cosmetics containing the same

[0001] This application claims priority to Republic of Korea Application No. 10-2023-0049672, filed April 14, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to kidney bean-derived exosomes containing glutathione. More specifically, the present invention relates to cosmetic raw materials and product applications using kidney bean exosomes containing glutathione, which have skin whitening and antioxidant functions.

[0003] Plant cells, including animal cells, are known to secrete nano-sized vesicles called "exosomes." Exosomes are vesicles with a membrane structure identical to the cell membrane, and are known to play a role in delivering membrane components, proteins, RNA, etc. to surrounding cells and tissues. In particular, exosomes secreted by plants contain polyphenols, a powerful antioxidant derived from plants, and are known to regulate the oxidative environment caused by reactive oxygen species. The antioxidant properties of plant exosomes are known to have functions that improve skin health, such as whitening, anti-inflammation, and collagen regeneration. In particular, they are safe to use as a cosmetic ingredient as they are functional materials obtained from edible plants.

[0004] Exosomes are approximately 100–200 nm in size and consist of a double lipid membrane derived from cells. The membrane can contain hydrophilic proteins, genetic material, and polyphenols, while the double lipid membrane contains hydrophobic proteins, bioactive factors, and membrane proteins. They can contain a variety of bioactive factors and are known to be highly safe and stable in the body. In particular, exosomes carry out signaling and exchange between cells. As the mechanism by which therapeutic factors from parent cells are delivered to damaged cells and tissues via exosomes has been elucidated, research on using exosomes to regulate the behavior of tissues, including cells, is gaining attention.

[0005] Plant exosomes are rich in plant-derived antioxidants and are known to have maximized skin regeneration and health-enhancing properties. Research is currently underway to utilize plant exosomes as functional cosmetics ingredients. They are rich in polyphenols, a powerful antioxidant. Among these, glutathione, a type of amino acid complex, possesses potent antioxidant properties and plays a key role in reducing oxidative stress. Glutathione is abundant in plants such as kidney beans and broccoli, and plant exosomes derived from these plants are also rich in glutathione. In particular, kidney bean derivatives and kidney bean exosomes have high glutathione content, attracting significant attention in cosmetics and health functional foods due to their powerful whitening effects and various physiological benefits.

[0006] Plant exosomes contain a variety of proteins depending on their plant origin, but little is known about kidney bean exosomes. Most studies have identified plant exosomes based on their size and shape, and extracting plant exosomes requires a method that selects exosomes of an appropriate size and extracts only those exosomes.

[0007] In the case of conventional techniques, attempts have been made to extract plant exosomes through a density gradient ultracentrifugation method using a sucrose gradient, but this method is difficult to use in the production process for commercializing exosomes, and there is a problem that sucrose is mixed into the extract. In addition, the density gradient ultracentrifugation method using a sucrose gradient produces 10 per unit volume of 1 mL of extract. 7 It is impossible to obtain a highly pure exosome composition with a high concentration of exosomes in a content of more than 100. In addition, there is a method of filtering the separated extracellular vesicles to obtain a material smaller than the pores of the filter. However, when extracting exosomes from a large amount of plant extract, impurities or exosomes are adsorbed and accumulated in the pores of the filter, which drastically reduces the filtration efficiency and is very disadvantageous for long-term exosome extraction.

[0008] Numerous references and citations are provided throughout this specification. The disclosures of these references are incorporated herein by reference in their entirety to further clarify the state of the art and the scope of the present invention.

[0009] The purpose of the present invention is to provide a whitening cosmetic product using exosomes derived from kidney beans, and in particular, to provide a whitening cosmetic raw material that is non-cytotoxic and has an excellent whitening effect by using exosomes derived from kidney beans that are rich in glutathione.

[0010] Another object of the present invention is to provide a skin whitening method comprising applying a cosmetic composition comprising kidney bean exosomes to a subject.

[0011] Another object of the present invention is to provide a cosmetic use of a cosmetic composition comprising kidney bean exosomes for skin whitening.

[0012] Another object of the present invention relates to a method for producing a whitening cosmetic raw material using exosomes derived from kidney beans, and provides a method for producing a cosmetic raw material and a cosmetic having a whitening effect using exosomes derived from kidney beans.

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

[0014] Embodiment 1. A high-purity kidney bean exosome composition comprising kidney bean-derived exosomes as an active ingredient; Use of kidney bean-derived exosomes or of a high-purity kidney bean exosome composition comprising kidney bean-derived exosomes as an active ingredient for providing a skin beauty effect and / or a skin cosmetic effect; Use of kidney bean-derived exosomes or of a high-purity kidney bean exosome composition comprising kidney bean-derived exosomes as an active ingredient for use in the manufacture of skin beauty products and / or cosmetics; A skin beauty and / or skin cosmetic method comprising applying kidney bean-derived exosomes or a high-purity kidney bean exosome composition comprising kidney bean-derived exosomes as an active ingredient to a subject; Or a method for preparing kidney bean-derived exosomes or of a high-purity kidney bean exosome composition comprising kidney bean-derived exosomes as an active ingredient, the method comprising the steps of: performing centrifugation; And a step of performing tangential flow filtration (TFF), wherein by the method, 1x10 per unit volume of 1 mL of the composition 7 Dog 1x10 14 A method characterized in that a high-purity kidney bean exosome composition comprising kidney bean-derived exosomes with a content of 100% is obtained.

[0015] Embodiment 2. In Embodiment 1, the soybean-derived exosomes are 1 x 10 per unit volume of 1 mL of the composition. 7 1 x 10 dog 14 A composition characterized by comprising a content of a dog; a use; or a method.

[0016] Embodiment 3. In Embodiment 1, a composition; use; or method according to any one of the preceding embodiments, characterized in that the composition is a cosmetic composition.

[0017] Embodiment 4. A composition; use; or method according to any one of the preceding embodiments, wherein the kidney bean-derived exosome or kidney bean exosome composition has at least one skin cosmetic use selected from the group consisting of antioxidation, skin whitening, skin cell regeneration, and skin health improvement.

[0018] Embodiment 5. A composition; use; or method according to any one of the preceding embodiments, wherein the kidney bean-derived exosome has a skin whitening use by exhibiting at least one effect selected from the group consisting of reducing melanin synthesis and reducing tyrosinase activity.

[0019] Embodiment 6. A composition; use; or method according to any one of the preceding embodiments, wherein the kidney bean exosome comprises glutathione.

[0020] Embodiment 7. A cosmetic comprising a kidney bean-derived exosome or kidney bean exosome composition according to any one of the preceding embodiments.

[0021] Embodiment 8. In any of the preceding embodiments, the cosmetic is a composition; use; method; or cosmetic in a formulation selected from the group consisting of an ampoule, a mist, a serum, a nourishing toner, an emollient toner, an emollient, an emulsion, a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing 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 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.

[0022] Embodiment 9. In any of the preceding embodiments, the cosmetic is a composition; use; method; or cosmetic characterized in that it is a functional cosmetic having at least one effect selected from the group consisting of skin whitening, wrinkle improvement, and skin cell regeneration.

[0023] Embodiment 10. In any of the preceding embodiments, the cosmetic comprises 1x10 of the kidney bean-derived exosomes per unit volume of 1 mL. 6 Dog 1x10 12 A composition characterized by comprising a content of a dog; a use; a method; or a cosmetic.

[0024] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0025] One aspect of the present invention is to provide a high-purity kidney bean exosome composition comprising kidney bean-derived exosomes as an active ingredient. The kidney bean-derived exosomes are present in an amount of 1 Х 10 per unit volume of 1 mL of the composition. 6 1 Х 10 14 Dog, preferably 1 Υ 10 6 1 Υ 10 13 dog or 1 Х 10 71 x 10 14 Dog, preferably 1 x 10 11 1 x 10 dog 14 dog or 1 x 10 11 1 x 10 dog 13 Dog, preferably 1 x 10 12 1 x 10 dog 14 dog or 1 x 10 12 1 x 10 dog 13 It may be included in the content of the dog.

[0026] The term "exosome" used in the present invention refers to exosomes or extracellular vesicles containing exosomes, extracted from kidney beans, having a size of about 50 to 300 nm, preferably 50 to 200 nm. Kidney bean exosomes form uniformly sized particles of 50 to 200 nm in the form of a double lipid membrane.

[0027] Although various existing plant extracts are known to contain anti-aging and antioxidant substances, they have difficulties in penetrating the skin barrier in the form of water-soluble extracts, and the active ingredients are easily destroyed by exposure to the extraction solvent during the extraction process, making them difficult to apply as cosmetic raw materials. 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. Furthermore, the active ingredients contained within are not directly exposed to the outside, so they are not easily deteriorated and can maintain biological activity for a long time, making them extremely advantageous for commercial use in the cosmetics industry.

[0028] The raw material plant of the present invention, kidney bean, belongs to Phaseolus vulgaris L., a species of the genus Kidney Bean. Kidney bean fruit can be used as the raw material plant part.

[0029] In a preferred embodiment, the kidney bean exosome composition of the present invention is a cosmetic composition, wherein the composition may have one or more skin cosmetic uses selected from the group consisting of anti-oxidation, skin whitening, wrinkle improvement, skin cell regeneration, and skin health improvement.

[0030] The kidney bean exosome presented in the present invention contains an excessive amount of glutathione, and has the function of alleviating intracellular oxidative stress and inhibiting melanin synthesis.

[0031] In the present invention, kidney bean exosomes possess skin regeneration and whitening properties due to functional substances, including glutathione, derived from the raw kidney bean. Specifically, kidney bean exosomes, which are rich in glutathione and polyphenols, known to possess powerful antioxidant properties, suppress active oxygen radicals accumulated in skin cells, thereby exhibiting skin regeneration and whitening effects.

[0032] In addition, exosomes derived from kidney beans can have skin whitening properties by exhibiting at least one effect selected from the group consisting of reducing melanin synthesis and reducing tyrosinase activity.

[0033] Kidney bean exosomes have anti-aging and antioxidant functions due to the polyphenol components they contain, which promote the decomposition of active oxygen existing outside the cell and active oxygen accumulated inside the cell.

[0034] In particular, kidney bean exosomes with antioxidant function are absorbed into skin cells through receptor-mediated endocytosis and membrane fusion, and the absorbed kidney bean exosomes stimulate antioxidant signaling mechanisms within skin cells, increasing gene expression such as SOD and catalases (CAT), and exhibiting anti-aging and antioxidant effects.

[0035] The kidney bean exosomes developed in this invention contain high amounts of glutathione, a crystalline peptide composed of three amino acids: glutamic acid, cysteine, and glycine. Glutathione has been clinically proven to have skin-enhancing properties, including whitening and antioxidant properties, as well as boosting immunity, alleviating fatigue, detoxifying the liver, and treating Parkinson's disease.

[0036] In addition, the kidney bean exosome of the present invention contains thioredoxin-dependent peroxiredoxin, superoxide dismutase, lipoxygenase, glutathione transferase, GST N-terminal domain-containing protein, elongation factor 1-gamma, glutathione S-transferase, glutathione reductase, dihydrolipoyl dehydrogenase, glutathione-disulfide reductase, and metallo-beta-lactamase domain-containing protein inside the exosome. It may comprise one or more proteins selected from the group consisting of a domain-containing protein.

[0037] In one embodiment, a cosmetic composition comprising the kidney bean exosome of the present invention as an active ingredient, more specifically, a cosmetic composition for anti-oxidation, skin whitening, skin cell regeneration and / or skin health improvement, may be provided in a formulation 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.

[0038] Another aspect of the present invention is to provide a cosmetic composition comprising kidney bean exosomes as an active ingredient, more specifically, a cosmetic composition comprising a cosmetic composition for antioxidation, skin whitening, skin cell regeneration and / or skin health improvement.

[0039] The above cosmetics can be formulated with the necessary amount of common ingredients used in the manufacture of general skin cosmetics, such as oil, water, surfactants, moisturizers, low-grade alcohols, thickeners, chelating agents, pigments, preservatives, fragrances, etc.

[0040] In addition, the above cosmetics may be manufactured in a formulation selected from the group consisting of mist, serum, nourishing toner, emollient toner, emollient, milk lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, powder, makeup base, essence, nourishing essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, facial cleanser, treatment, beauty liquid, beauty pack, ointment, gel, liniment, liquid, patch, spray, bath agent, sunscreen, sun oil, and hair product, and may include various conventional carriers and additives suitable for each of these formulations and well known in the art.

[0041] The cosmetic of the present invention may be a functional cosmetic having one or more effects selected from the group consisting of skin whitening, wrinkle improvement, skin barrier improvement, and skin cell regeneration.

[0042] In the present invention, the kidney bean exosomes extracted from kidney beans are present in an amount of 1 Х 10 or more, 1 Х 10 per unit volume of 1 mL of the cosmetic. 2 Above, 1 Х 10 3 Above, 1 Х 10 4 Above, 1 Х 10 5 Ideal or 1 Х 10 6 It may be included in the content of the above particle number, preferably 1 Х 106 1 Х 10 12 Dog, more preferably 1 Х 10 6 1 Х 10 12 Dog, more preferably 1 Х 10 7 1 Х 10 12 It may be included in the content of dog particles.

[0043] Another aspect of the present invention provides a method for producing a high-purity kidney bean composition comprising the kidney bean exosome as an active ingredient, the method comprising the steps of performing centrifugation; and performing tangential-flow filtration (TFF).

[0044] More specifically, the manufacturing method of the present invention may include a step of crushing kidney beans; a step of performing microfiltration and centrifugation; and a step of performing tangential-flow filtration (TFF).

[0045] The process of crushing the above raw plant material may include a process of mechanically crushing a mixture of the raw plant material and the buffer solution in a weight ratio of 1:1 to 1:10, and this may be performed by mechanically crushing the plant mixture by a blade rotating at a speed of 10 to 1,000 rpm.

[0046] The above microfiltration method is performed by filtering a plant mixture through a filter membrane having pores of 10 μm to 200 nm in size, and preferably, the filtering can be performed from a combination of one or more types of filter membranes having different pores.

[0047] The centrifugation method may be selected from low-speed centrifugation, high-speed centrifugation, ultracentrifugation, and combinations thereof, and preferably includes 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 easily precipitated, such as cells or nuclei, high-speed centrifugation has a maximum speed of about 20,000-25,000 rpm (60,000 xg), and ultracentrifugation refers to a centrifugation method having a maximum speed of about 40,000-80,000 rpm (600,000 xg).

[0048] The above tangential flow filtration (TFF) is a filtration method in which a solution flows in a direction perpendicular to a filtration membrane, filtering out small-sized impurities present in the solution and separating large-sized exosomes. Compared to conventional filtration methods, it can minimize adsorption of exosomes to the pores of the filtration filter or clogging of the membrane pores, and is thus easy to apply to process scale-up and cGMP (cosmetic good manufacturing procedure) processes. The above tangential flow filtration may be at least one selected from the group consisting of hollow fiber TFF and membrane TFF capable of performing ultrafiltration, and preferably, a TFF filter having a molecular weight cutoff (MWCO) of 100,000 Da to 500,000 Da may be used.

[0049] In this way, the kidney bean exosomes according to the present invention can be separated from kidney beans through mechanical grinding, stepwise ultracentrifugation, and tangential flow filtration (TFF), and these exosomes contain a large amount of glutathione and polyphenols, which have skin regeneration, whitening, and antioxidant functions.

[0050] All ingredients described in the present invention preferably do not exceed the maximum usage levels stipulated in relevant laws and regulations of Korea, China, the United States, Europe, Japan, etc. (e.g., Regulations on Cosmetic Safety Standards (Korea), Cosmetic Safety Technical Standards (China)). That is, preferably, the cosmetic composition and cosmetics according to the present invention contain the ingredients according to the present invention within the content limits permitted by the relevant laws and regulations of each country.

[0051] Kidney bean exosomes are absorbed into skin cells as a double lipid membrane structure, and glutathione and polyphenols contained within the absorbed kidney bean exosomes exhibit antioxidant effects by stimulating antioxidant signaling mechanisms within skin cells and increasing the expression of various antioxidant genes. Kidney bean exosomes have various functional cosmetic applications, such as whitening, anti-aging, and skin cell activation, derived from the antioxidant effects of glutathione and polyphenols contained in kidney bean exosomes. In addition, the excellent stability of kidney bean exosomes allows them to be utilized in functional cosmetics in various forms, such as aqueous solutions, frozen solutions, and freeze-dried powders.

[0052] Figure 1 is a process schematic diagram of a plant exosome extraction method for isolating plant exosomes from plant raw materials with high purity and high yield according to one embodiment.

[0053] Figure 2 shows the results of analyzing the characteristics of kidney bean exosomes manufactured according to one embodiment using nanoparticle tracking analysis (NTA).

[0054] Figure 3 shows the results of evaluating the total protein and polyphenol content in kidney bean exosomes manufactured according to one embodiment.

[0055] Figure 4 shows the results of evaluating the surface charge and size distribution (DLS) of kidney bean exosomes manufactured according to one embodiment.

[0056] Figure 5 shows the results of detecting glutathione in kidney bean exosomes manufactured according to one embodiment.

[0057] Figure 6 shows the results of a cytotoxicity test of kidney bean exosomes manufactured according to one embodiment.

[0058] Figure 7 shows the results of confirming the extracellular and intracellular melanin formation inhibition efficacy of kidney bean exosomes manufactured according to one embodiment.

[0059] Figure 8 shows the results of confirming the antioxidant efficacy of kidney bean exosomes manufactured according to one embodiment.

[0060] Figure 9 shows the results of confirming Tyrosinase activity inhibition in each experimental group.

[0061] Figure 10 shows the results of classifying the protein profiling contained in kidney bean exosomes manufactured according to one embodiment into molecular function categories.

[0062] Figure 11 shows the results of classifying the protein profiling contained in kidney bean exosomes manufactured according to one embodiment into biological response categories.

[0063] Figure 12 shows the results of classifying the protein profiling contained in kidney bean exosomes manufactured according to one embodiment into cell component categories.

[0064] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0065] Example

[0066] <Example 1> Isolation of exosomes from various plant raw materials

[0067] By performing ultracentrifugation and tangential-flow filtration (TFF), plant exosomes can be isolated with high purity and yield from various plant raw materials, with vacuoles completely removed (Fig. 1). In this example, a high-purity plant exosome composition was prepared using aloe, garlic, seaweed, and kidney beans.

[0068] (1) Preparation of aloe-derived exosome composition

[0069] Aloe exosomes were isolated from the raw material plant, aloe, 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 (w / w) and then ground. The supernatant was centrifuged at 1,000 xg for 10 minutes, 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. The supernatant was then ultracentrifuged at 100,000 xg for 70 minutes at 4°C to remove vacuoles. Finally, the supernatant was discarded, and the remaining pellet was suspended and exosomes were isolated using a TFF system. Specifically, the solution containing exosomes was filtered using a filter having a molecular cut-off of 100 to 500 kDa within a TFF system, removing other impurity particles smaller than the pores of the filter and concentrating the solution.

[0070] As a result of extracting exosomes derived from aloe peel by performing ultrafiltration (TFF), it was confirmed that the extracted exosomes derived from aloe peel had a spherical microstructure of less than 200 nm, and nanoparticle tracking analysis (NTA) showed that the number of exosomes per unit volume of 1 mL was 3.7 x 10 10 It was confirmed that a high-purity exosome composition was obtained, in which aloe exosomes were concentrated at a concentration of 100 μg / ml (Fig. 2a).

[0071] (2) Preparation of garlic-derived exosome composition

[0072] Garlic-derived exosomes were isolated from the raw garlic plant using ultracentrifugation and tangential flow filtration (TFF). Specifically, peeled garlic was placed in a blender with phosphate-buffered saline (PBS) at a weight ratio of 1:2 (w / w) and ground. The supernatant was centrifuged at 1,000 xg for 10 min, and the supernatant was sequentially centrifuged at 2,000 xg for 20 min, 3,000 xg for 30 min, and 10,000 xg for 60 min. The supernatant was then ultracentrifuged at 100,000 xg for 70 min at 4°C to remove vacuoles. Finally, the supernatant was discarded, and the remaining pellet was suspended and exosomes were isolated using a TFF system. Specifically, the solution was filtered using a filter having a molecular cut-off of 100 to 500 kDa within a TFF system, removing other impurity particles smaller than the pores of the filter and concentrating the solution containing garlic-derived exosomes.

[0073] As a result of extracting garlic-derived exosomes by performing ultrafiltration (TFF), it was confirmed that garlic-derived exosomes have a spherical microstructure of less than 200 nm, and nanoparticle tracking analysis (NTA) showed that there were 3.2 x 10 per unit volume of 1 mL. 11 It was confirmed that a high-purity exosome composition was obtained, in which garlic exosomes were concentrated at a concentration of 100 μg / ml (Fig. 2b).

[0074] (3) Preparation of exosome composition derived from seaweed

[0075] Exosomes derived from seaweed were isolated from the raw plant, seaweed, using ultracentrifugation and tangential flow filtration (TFF). Specifically, the dried seaweed was mixed with phosphate-buffered saline (PBS) at a weight ratio of 1:2 (w / w) and ground in a blender. The supernatant was centrifuged at 1,000 xg for 10 min, and the supernatant was sequentially centrifuged at 2,000 xg for 20 min, 3,000 xg for 30 min, and 10,000 xg for 60 min. The supernatant was then ultracentrifuged at 100,000 xg for 70 min at 4°C to remove vacuoles. Finally, the supernatant was discarded, and the remaining pellet was suspended and exosomes were isolated using a TFF system. Specifically, the solution containing exosomes was filtered using a filter having a molecular cut-off of 100 to 500 kDa within a TFF system, removing other impurity particles smaller than the pores of the filter and concentrating the solution.

[0076] As a result of extracting exosomes derived from seaweed by performing ultrafiltration (TFF), it was confirmed that the exosomes derived from seaweed had a spherical microstructure of less than 200 nm, and nanoparticle tracking analysis (NTA) showed that the number of exosomes per unit volume of 1 mL was 5.17 x 10 10 It was confirmed that a high-purity exosome composition was obtained, in which seaweed exosomes were concentrated at a concentration of 100 μg / ml (Fig. 2c).

[0077] (4) Exosomes derived from kidney beans

[0078] Kidney bean exosomes were isolated from the raw material, kidney bean, using ultracentrifugation and tangential flow filtration (TFF) methods. Specifically, kidney beans were placed in a blender with distilled water at a weight ratio of 1:4 (w / w) and ground. The supernatant was collected by centrifugation at 1,000 Xg for 10 min, and the supernatant was sequentially centrifuged at 2,000 Xg for 20 min, 3,000 Xg for 30 min, 10,000 Xg for 60 min, and 20,000 Xg for 30 min. The supernatant was then filtered through a 40 μm cell strainer and ultracentrifuged at 100,000 Xg for 70 min at 4°C, removing vacuoles. Finally, the supernatant was discarded, and the remaining pellet was suspended, filtered under reduced pressure through a 0.22 μm filter, and then exosomes were isolated using a TFF system. Specifically, filtration was performed using a filter with a molecular cutoff of 100 to 500 kDa within the TFF system, removing other impurity particles smaller than the pores of the filter and concentrating the solution containing exosomes.

[0079] As a result of extracting exosomes derived from kidney beans by performing ultrafiltration (TFF), it was confirmed that the exosomes derived from kidney beans had a size of 50 to 300 nm, and nanoparticle tracking analysis (NTA) showed that there were 1Х10 exosomes per 1 mL of unit volume. 12 It was confirmed that a high-purity exosome composition was obtained in which kidney bean exosomes were concentrated to a concentration of more than 10 (Fig. 2d).

[0080] As described above, the combination of ultracentrifugation and TFF methods has been shown to achieve a highly pure exosome composition enriched with exosomes at a remarkably high particle count per unit volume when applied to kidney beans. Therefore, further studies were conducted using kidney bean exosomes, which were found to provide the highest purity exosome composition.

[0081] <Example 2> Characterization of kidney bean exosomes

[0082] The kidney bean exosomes isolated in Example 1 above were subjected to exosome characteristic evaluation using the following method. The size of the microparticles was analyzed using nanoparticle tracking analysis (NTA), and as described above, it was confirmed that the kidney bean exosomes had a size of 50 to 300 nm, and the kidney bean exosomes produced once were approximately 1Х10 per lot through nanoparticle tracking analysis (NTA). 13 It was confirmed that the yield was more than 100 (Fig. 2d). In addition, the protein content in exosomes was measured using Micro BCA from Thermo Scientific. TM The protein content was measured using a Protein Assay Kit and confirmed to be 30 to 3,000 μg / mL per 1 mL unit volume (Fig. 3). The total polyphenol content in kidney bean exosomes was 10 12 It was confirmed that 100 to 500 μg of kidney bean exosomes were contained per unit cell number (Fig. 3).

[0083] Kidney bean exosomes were confirmed to have a zeta surface potential of less than -20 mV, indicating that they maintain a stable colloidal form in aqueous solutions (Fig. 4). DLS analysis confirmed that kidney bean exosomes have a size distribution ranging from 30 to 300 nm, with an average size of approximately 100 nm (Fig. 4).

[0084] <Example 3> Evaluation of total glutathione content in kidney bean exosomes

[0085] The kidney bean exosomes isolated in Example 1 were evaluated for their total glutathione content in the exosomes using the following method. The total glutathione content in the kidney bean exosomes isolated in Example 1 was evaluated using the Glutathione assay kit from NWLLS. The glutathione content was evaluated before and after exosome disruption using a mixture of 0.5% Triton X-100 and 0.05% SDS as a lysis buffer, and a glutathione content of 4.0 to 5.0 μM per 1 mL of unit volume was confirmed (Fig. 5).

[0086] <Example 4> Evaluation of cytotoxicity of kidney bean exosomes

[0087] The kidney bean exosomes isolated in Example 1 were evaluated for their toxicity to human cells using the following method. 10,000 HaCaT cells were seeded in each well of a 96-well plate and cultured for 24 hours. After culture, the kidney bean exosomes isolated in Example 1 were added to 10 7 10 inland 10 After treatment at a concentration of 10 particles / mL, the cells were cultured for 24 and 72 hours. After culture, the cell count was performed using the MTT assay, and from this result, 10 10 No toxicity to human keratinocytes was observed even with high doses of kidney bean exosomes (particles / mL) (Fig. 6).

[0088] <Example 5> Evaluation of the inhibition of intracellular melanin synthesis by kidney bean exosomes

[0089] In order to confirm the cosmetic efficacy of the kidney bean exosomes isolated in Example 1, skin melanoma cells (B16F10) were treated with the exosomes to determine whether they inhibited melanin production. When measuring the amount of melanin production, cell culture medium containing no test substance was used as a solvent control, and 200 μM arbutin was used as a positive control. The total melanin production of cells was measured by seeding 5 × 10 B16F10 cells in a 12-well plate. 4 The cells were seeded at a concentration of 10 cells / well and cultured for 24 hours. After 24 hours, the medium from each well was removed, the cells were washed with PBS, and then 10 9 10 inland 10 Fresh culture medium containing p / mL of kidney bean exosomes and 100 ng / mL α-MSH was added and cultured for 48 hours. The cultured cells were used to measure the amount of intracellular melanin. For the measurement of the amount of intracellular melanin, the cultured B16F10 cells were washed with PBS, lysed with lysis buffer, and centrifuged at 12,000 rpm for 5 minutes. The supernatant was then removed, and lysed with 1 N NaOH (10% DMSO) at 60°C. The cell lysate was measured for absorbance at 490 nm, and the amount of melanin was determined from a calibration curve using synthetic melanin, and the amount of melanin was converted to the amount of melanin per protein (Fig. 7).

[0090] Kidney bean exosomes are 10 9 At a concentration of 100 particles / mL, it was confirmed to be effective in inhibiting extracellular and intracellular melanin formation (Fig. 7). It was confirmed that kidney bean exosomes effectively inhibit intracellular melanin formation. In this example, the efficacy of kidney bean exosomes is not limited to whitening efficacy, but includes all functional cosmetic efficacy related to antioxidant activity, such as whitening and antioxidant effects.

[0091] <Example 6> Evaluation of the antioxidant efficacy of kidney bean exosomes

[0092] To confirm the antioxidant efficacy of the kidney bean exosomes isolated in Example 1, SOD and DPPH activity were evaluated. 10 10 SOD activity is observed at particles above 10 11 In particles above, high SOD and DPPH activation of more than 50% was observed (Fig. 8). It was confirmed that kidney bean exosomes effectively alleviate oxidative stress. In this example, the efficacy of kidney bean exosomes is not limited to antioxidant efficacy, but includes all functional cosmetic effects related to antioxidant activity, such as whitening and antioxidant effects.

[0093] <Example 7> Evaluation of the inhibition of intracellular tyrosinase activity by kidney bean exosomes

[0094] In order to confirm the cosmetic efficacy of the kidney bean exosomes isolated in Example 1, skin melanoma cells (B16F10) were treated with the exosomes to confirm whether they inhibited tyrosinase activity. When measuring the amount of melanin production, cell culture medium containing no test substance was used as a solvent control, and 600 μM arbutin was used as a positive control. The measurement of cell tyrosinase activity was performed by seeding 1.5 × 10 B16F10 cells in a 6-well plate. 5 The cells were seeded at a concentration of 10 cells / well and cultured for 24 hours. After 24 hours, the medium from each well was removed, the cells were washed with PBS, and then 10 10 10 inland 11 Fresh culture medium containing p / mL of kidney bean exosomes and 100 ng / mL α-MSH was added and cultured for 24 hours.

[0095] Cultured cells were used to measure intracellular tyrosinase activity. For the measurement of intracellular tyrosinase activity, cultured B16F10 cells were washed with PBS and lysed with 50 mM phosphate buffer (1% Triton X-100, 1% protease inhibitor cocktail) at 4℃ for 10 minutes, and centrifuged at 10,000 rpm for 20 minutes. The supernatant was then collected for protein quantification. Then, the supernatants of each control and experimental group were adjusted to the same volume and protein concentration and added to a 96-well plate at an amount of 50 μL / well. After that, 5 mM L-DOPA was added to the wells with the supernatant at an amount of 50 μL / well, and the reaction was performed in a dark room at 37℃ for 2 hours. Afterwards, the mixture was measured for absorbance at 490 nm, and the absorbance for the control and experimental groups was divided by the absorbance for the solvent control group to convert it into a percentage to measure the tyrosinase activity value. Based on these results, the kidney bean exosome was 10 10 10 inland 11 It was confirmed that the exosome was effective in inhibiting intracellular tyrosinase activity at a concentration of 100 p / mL (Fig. 9). In this example, the efficacy of kidney bean exosomes is not limited to whitening efficacy, but includes all functional cosmetic efficacy related to antioxidant activity, such as whitening and antioxidant efficacy.

[0096] <Example 8> Protein profiling evaluation of kidney bean exosomes

[0097] In order to identify the protein components of the kidney bean exosomes isolated in Example 1, the protein components of the exosomes were evaluated using LC-MS / MS analysis. The kidney bean exosomes in solution were concentrated using a 3K cut-off filter, and the supernatant was dissolved by adding 8 M urea, 100 mM Tris-HCl, and protease inhibitor, and then protein quantification was performed. Afterwards, the in-solution digestion process was performed according to the “KBIOHealth-SOP-NDTM-038 Procedure” for exosome analysis. Afterwards, the dried sample was dissolved in a 0.1% formic acid aqueous solution to become 0.5 μg / μL, and 2 μL (1 μg) of the peptide sample was injected into the analytical column using a NanoLC system, and the ratio of the organic solvent (Sol B) was separated for 45 minutes with a gradual concentration gradient (5-50%). The flow rate was 300 nL / min, and the total analysis time was 60 minutes, and the analysis was repeated three times. The resulting data were then analyzed for Phaseolus vulgaris using Proteome Discoverer 2.4 software, and the results were extracted and organized. The profiled proteins were classified into three categories: molecular function, cellular component, and biological response, and the proteins with the highest abundance were shown in the diagram (Figs. 10 to 12). Among the entire protein list, protein components involved in antioxidant function were separately organized and shown in Table 1 below.

[0098]

[0099] The above results confirmed that kidney bean exosomes contain protein components involved in antioxidant function. In this example, the efficacy of kidney bean exosomes is not limited to antioxidant effect, but includes all functional cosmetic effects related to antioxidant activity, such as whitening and antioxidant effect.

Claims

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

2. In the first paragraph, the soybean-derived exosomes are 1 x 10 per unit volume of 1 mL of the composition. 7 1 x 10 dog 14 A kidney bean exosome composition characterized by comprising a content of:

3. A kidney bean exosome composition according to claim 1, characterized in that the composition is a cosmetic composition.

4. A kidney bean exosome composition according to claim 1, characterized in that the composition has at least one skin cosmetic use selected from the group consisting of antioxidant, skin whitening, skin cell regeneration, and skin health improvement.

5. In the fourth paragraph, a kidney bean exosome composition characterized in that the kidney bean exosome has a skin whitening use by exhibiting at least one effect selected from the group consisting of reducing melanin synthesis and reducing tyrosinase activity.

6. A kidney bean exosome composition according to claim 1, characterized in that the kidney bean exosome contains glutathione.

7. A cosmetic comprising a kidney bean exosome composition according to any one of claims 1 to 6.

8. In paragraph 1, the cosmetic is a cosmetic having a formulation selected from the group consisting of an ampoule, a mist, a serum, a nourishing toner, an emollient toner, an emollient, an emulsion, a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing 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. A cosmetic according to claim 8, characterized in that the cosmetic is a functional cosmetic having at least one effect selected from the group consisting of skin whitening, wrinkle improvement, and skin cell regeneration.

10. In the 8th paragraph, the cosmetic product contains 1 x 10 of the kidney bean-derived exosomes per unit volume of 1 mL. 6 1 x 10 dog 12 A cosmetic product characterized by containing a content of dog.

11. A method for producing a kidney bean exosome composition according to any one of claims 1 to 6, a step of performing centrifugation; and Comprising a step of performing tangential flow filtration (TFF), 1 x 10 per unit volume of 1 mL of the composition by the above method 7 1 x 10 dog 14 A method characterized in that a high-purity kidney bean exosome composition comprising kidney bean-derived exosomes with a content of 100% is obtained.

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