Exosome secretion promoter, exosome uptake promoter, cosmetic composition for promoting exosome secretion, and method for promoting exosome secretion

Abstract

To provide novel beauty compositions having excellent beauty effects such as anti-aging effect, specifically, compositions capable of promoting exosome secretion from cells such as mesenchymal stem cells.SOLUTION: The present invention is an exosome secretagogue containing one or more selected from the group consisting of glycogen, Centella asiatica leaf extract, Vaccinium myrtillus L. leaf extract, Glycyrrhiza leaf extract, and Glycine max protein extract. The exosome secretagogue of the present invention preferably further contains a peptide represented by the amino acid sequence (Leu-Glu-His-Ala) of SEQ ID NO:1, or a salt thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an exosome secretion promoter, an exosome uptake promoter, a cosmetic composition for promoting exosome secretion, and a method for promoting exosome secretion. [Background technology]

[0002] Exosomes are extracellular vesicles covered with a lipid bilayer membrane secreted by most cell types, and are generally defined by a particle diameter of 50 to 150 nm and several membrane-expressed marker proteins (CD9, CD63, CD81, etc.). These vesicles contain cell membrane proteins, intracellular proteins, and nucleic acids such as DNA, mRNA, and miRNA, and are said to be involved in cell-cell interactions. In such cell-cell interactions, physiological and pathological functions have attracted attention, and new methods for diagnosing diseases using exosomes as an indicator and attempts to use exosomes themselves for disease treatment are also being considered.

[0003] Exosomes derived from stem cells such as bone marrow stem cells, umbilical cord blood stem cells, and adipose-derived stem cells are known to contain genes, proteins, growth factors, etc. related to cell proliferation, differentiation, and regeneration, and cosmetic compositions containing these as active ingredients for skin whitening, wrinkle improvement, or skin regeneration have been developed (see Patent Document 1).

[0004] In recent years, people's demands for beauty and health of the skin are increasing day by day, and new types of compositions, compositions with greater beauty effects are required. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2018-184446 A Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention has been made in view of such circumstances, and aims to provide a new type of cosmetic composition having excellent cosmetic effects such as anti-aging effects. Specifically, the present invention aims to provide a composition capable of promoting exosome secretion from cells such as mesenchymal stem cells. Since exosomes secreted from cells such as mesenchymal stem cells are known to have anti-aging effects, skin regeneration effects, and the like, it is believed that a composition capable of promoting the secretion of such exosomes will result in excellent cosmetic effects. [Means for solving the problem]

[0007] As a result of intensive research by the present inventors to solve the above problems, it was found that a composition containing one or more selected from the group consisting of glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract has an effect of promoting exosome secretion from cells, particularly from adipose tissue-derived stem cells. It was also revealed that the exosome secretion promoting effect of the composition can be further improved by containing a specific tetrapeptide in addition to these components. Furthermore, it was also found that the composition having excellent exosome secretion promoting effect exhibits excellent effects leading to beauty such as inhibition of cell aging in the skin, promotion of cell proliferation, promotion of collagen production, and promotion of elastin production, by which the secretion-promoted exosomes are taken into skin tissue, etc., as a result. Furthermore, it was also found that the composition has an effect of converting the M1 / M2 polarity of cells, that is, an effect of transforming inflammatory M1 macrophages into anti-inflammatory M2 macrophages. That is, the gist of the present invention is as follows.

[0008] [1] An exosome secretion promoter containing one or more selected from the group consisting of glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract. [2] The exosome secretion promoter according to [1], further comprising a tetrapeptide. [3] The exosome secretion promoter according to [2], wherein the tetrapeptide is a peptide represented by the amino acid sequence (Leu-Glu-His-Ala) of SEQ ID NO: 1 or a salt thereof. [4] An exosome secretion promoter according to any one of [1] to [3], which promotes exosome secretion in adipose-derived stem cells. [5] An agent for promoting exosome uptake into cells, comprising one or more components selected from the group consisting of glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract. [6] The agent for promoting exosome uptake into cells described in [5], further comprising a tetrapeptide. [7] The agent for promoting exosome uptake into cells according to [6], wherein the tetrapeptide is a peptide represented by the amino acid sequence (Leu-Glu-His-Ala) of SEQ ID NO: 1 or a salt thereof. [8] The agent for promoting exosome uptake into cells described in any one of [5] to [7], wherein the cells are skin fibroblasts. [9] The exosome secretion promoter according to any one of [1] to [4], which is used for promoting cell proliferation.

[10] The exosome secretion promoter according to any one of [1] to [4], which is used for M1 / M2 polarity conversion of cells.

[11] The exosome secretion promoter according to any one of [1] to [4], which is used for inhibiting cellular senescence.

[12] The exosome secretion promoter according to any one of [1] to [4], which is used for promoting collagen and / or elastin production.

[13] An exosome secretion promoter described in any of [1] to [4], which is used to improve skin firmness.

[14] An agent for promoting exosome uptake into cells, described in any of [5] to [8], which is used for promoting cell proliferation.

[15] An agent for promoting exosome uptake into cells, described in any of [5] to [8], which is used for M1 / M2 polarity conversion of cells.

[16] An agent for promoting exosome uptake into cells, described in any of [5] to [8], which is used for inhibiting cellular senescence.

[17] An agent for promoting exosome uptake into cells, described in any of [5] to [8], which is used for promoting collagen and / or elastin production.

[18] An exosome uptake promoter into cells, described in any of [5] to [8], which is used to improve skin firmness.

[19] A method for producing an exosome secretion promoter, comprising blending one or more selected from the group consisting of glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract.

[20] The method for producing an exosome secretion promoter according to

[19] , further comprising compounding a tetrapeptide.

[21] The method for producing an exosome secretion promoter according to

[20] , wherein the tetrapeptide is a peptide represented by the amino acid sequence (Leu-Glu-His-Ala) of SEQ ID NO: 1 or a salt thereof.

[22] A method for producing an agent for promoting exosome uptake into cells, comprising blending one or more components selected from the group consisting of glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract.

[23] A method for producing an exosome uptake promoter into cells, as described in

[22] , further comprising compounding a tetrapeptide.

[24] The method for producing an exosome uptake promoter into a cell described in

[23] , wherein the tetrapeptide is a peptide represented by the amino acid sequence (Leu-Glu-His-Ala) of SEQ ID NO: 1 or a salt thereof.

[25] A cosmetic composition for promoting exosome secretion, comprising any one of the exosome secretion promoters described in [1] to [4], or [9] to

[13] .

[26] A method for producing a cosmetic composition for promoting exosome secretion, comprising any one of the exosome secretion promoters described in [1] to [4], or [9] to

[13] .

[27] A cosmetic composition for promoting exosome uptake into cells, comprising an exosome uptake promoter according to any one of [5] to [8], or

[14] to

[18] .

[28] A method for producing a cosmetic composition for promoting exosome uptake into cells, comprising blending an exosome uptake promoter according to any one of [5] to [8], or

[14] to

[18] .

[29] A method for promoting exosome secretion using one or more selected from the group consisting of glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract.

[30] The method for promoting exosome secretion described in

[29] , further comprising using a tetrapeptide in combination.

[31] The method for promoting exosome secretion according to

[30] , wherein the tetrapeptide is a peptide represented by the amino acid sequence (Leu-Glu-His-Ala) of SEQ ID NO: 1 or a salt thereof.

[32] A method for promoting exosome uptake into cells, using one or more selected from the group consisting of glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract.

[33] A method for promoting exosome uptake into cells described in

[32] , further comprising the use of a tetrapeptide.

[34] The method for promoting exosome uptake into cells described in

[33] , wherein the tetrapeptide is a peptide represented by the amino acid sequence (Leu-Glu-His-Ala) of SEQ ID NO: 1 or a salt thereof. Effect of the Invention

[0009] According to the present invention, a new type of cosmetic composition having excellent cosmetic effects such as anti-aging effects can be provided. Specifically, the exosome secretion promoter of the present invention promotes exosome secretion from cells, particularly adipose tissue-derived stem cells, and the secretion-promoted exosomes are taken up into skin tissues, etc., resulting in cosmetic effects such as inhibition of skin cell aging, promotion of cell proliferation, promotion of collagen production, and promotion of elastin production. Furthermore, it can also have an effect of converting M1 / M2 polarity of cells, that is, changing the phenotype of inflammatory M1 macrophages to anti-inflammatory M2 macrophages. Therefore, the exosome secretion promoter of the present invention is suitable for use in products aimed at anti-aging, skin regeneration, and improvement of moisturizing properties of the skin. More specifically, it is expected to improve dullness of the skin, restore firmness of the skin, improve firmness of the skin, and improve the visibility of pores, and is also effective in improving aging symptoms such as dryness, improving and enhancing skin tone, and whitening the skin. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows the state in which exosomes secreted from adipose tissue-derived mesenchymal stem cells cultured in the composition of Example 2 were taken up by human fibroblasts. [Diagram 2] FIG. 1 shows the cellular aging inhibitory effect on skin fibroblasts of exosomes secreted from adipose tissue-derived mesenchymal stem cells cultured in the composition of Example 2, in comparison with the effect when a comparative composition was used (β-galactosidase staining). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention will now be described in detail. In addition, the terms used in this specification are to be interpreted as having the meanings usually used in the technical field unless otherwise specified.

[0012] <Exosome secretion promoter> The exosome secretion promoter of the present invention is characterized by containing one or more selected from the group consisting of glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract as an active ingredient. The exosome secretion promoter of the present invention can improve the exosome secretion promoting effect by further containing a specific tetrapeptide. The exosome secretion promoter of the present invention contains the above-mentioned specific ingredient as an active ingredient, thereby promoting exosome secretion from cells, particularly adipose tissue-derived stem cells, and the secretion-promoted exosomes are taken into skin tissue, etc., and as a result, it can exhibit effects that lead to beauty such as inhibition of cell aging in the skin, promotion of cell proliferation, promotion of collagen production, and promotion of elastin production. Furthermore, it can also exhibit the effect of converting the M1 / M2 polarity of cells, that is, the effect of transforming inflammatory M1 macrophages into anti-inflammatory M2 macrophages. Below, each essential ingredient, optional ingredient, etc. contained in the exosome secretion promoter of the present invention will be specifically explained.

[0013] In the present invention, glycogen refers to a polymer in which α-D-glucose (grape sugar) is a structural unit and is linked only by α-1,4-glucosidic bonds and α-1,6-glucosidic bonds. Examples of such glycogen include vegetable glycogen derived from corn seeds, barley, rice, potatoes, etc.; and animal glycogen derived from the liver and muscle of animals such as cows and pigs, and shellfish such as scallops, abalone, oysters, mussels, pearl oysters, and corbiculas. As glycogen in the present invention, glycogen contained in natural products prepared by a conventional method may be used as it is, or glycogen synthesized using an enzyme may be used. Examples of synthesis methods using the above enzymes include a method in which sucrose phosphorylase (EC2.4.1.7), α-glucan phosphorylase (EC2.4.1.1) and branching enzyme (EC2.4.1.18) are allowed to act on sucrose and primer molecules (malto-oligosaccharides, etc., or dextrin), and a method in which branching enzyme (EC2.4.1.18) is allowed to act on short-chain amylose. Glycogens synthesized by these methods are known to have similar chemical and physical structures to natural glycogens, and these enzyme-synthesized glycogens can also be used. The glycogen of the present invention may be not only the above-mentioned natural or synthetic glycogens, but also their derivatives or equivalents.

[0014] As the glycogen in the present invention, fractions having appropriate molecular weights may be used that have been separated by a method well known to those skilled in the art. Examples of the above separation method include chromatographic separation methods such as gel filtration chromatography and HPLC, and membrane separation methods. In addition, precipitation methods using solvents such as methanol and ethanol, used alone or in combination, may also be used. Furthermore, methods for derivatizing natural or synthetic glycogen (etherification, esterification, cross-linking, and grafting) include methods commonly used for starch modification well known to those skilled in the art. For phosphorylation of glycogen, a method of reacting glycogen with phosphorus oxychloride in dimethylformamide can be used. The average molecular weight of glycogen in the present invention is 300,000 to 1,500,000, preferably 400,000 to 1,200,000, and more preferably 600,000 to 900,000, from the viewpoint of promoting exosome secretion.

[0015] As the glycogen in the present invention, commercially available products can be used, such as BIOSACCHARIDES LS / HG (manufactured by LABORATORIES SEROBIOLOGIQUES) derived from Mytilus edulis, Bioglycogen (Glico Nutrition Foods Co., Ltd.), and the like.

[0016] The glycogen content in the exosome secretion promoter of the present invention may be 0.001 to 10% by mass, preferably 0.005 to 5% by mass, more preferably 0.01 to 3% by mass, and even more preferably 0.05 to 2.0% by mass. By setting the glycogen content in the exosome secretion promoter of the present invention within the above numerical range, an excellent exosome secretion promoting effect can be obtained.

[0017] As the Centella asiatica leaf extract, bilberry leaf extract, and licorice leaf extract of the present invention, extracts extracted from each plant using techniques conventionally known to those skilled in the art can be used. The extraction method and extraction solvent for each plant extract are not particularly limited. In addition, each plant extract may be decomposed, and the decomposition method is not particularly limited, and known techniques can be used. As the Centella asiatica leaf extract, bilberry leaf extract, and licorice leaf extract of the present invention, commercially available ones can also be used. The content of the Centella asiatica leaf extract, bilberry leaf extract, and licorice leaf extract in the exosome secretion promoter of the present invention is not particularly limited as long as it is within a range that has an exosome secretion promoting effect on cells, and is appropriately set according to the type and content of other blended components, the formulation form of the exosome secretion promoter, etc. From the viewpoint of more significantly exerting the exosome secretion promoting effect of the present invention, for example, the concentration is preferably 0.00001 to 5% by mass, more preferably 0.0001 to 4% by mass, even more preferably 0.0005 to 3% by mass, even more preferably 0.001 to 2% by mass, and particularly preferably 0.002 to 1% by mass. The dosage to humans is preferably 0.001 to 200 mg per day, more preferably 0.01 to 150 mg, even more preferably 0.1 to 100 mg, and particularly preferably 1 to 80 mg.

[0018] The soy protein extract in the present invention is not particularly limited as long as it is derived from soybeans of the legume family, and one or more types of soybean plants may be selected and used. The plant parts used for the soy protein extract may be any parts, and one or more types of parts may be selected and used. In addition, the extraction method and extraction solvent for the soy protein extract are not particularly limited, and known techniques can be used. The soy protein extract in the exosome secretion promoter of the present invention may be a commercially available product. The content of the soy protein extract in the exosome secretion promoter of the present invention is not particularly limited as long as it is within a range that has an exosome secretion promoting effect, and is appropriately set depending on the type and content of other blended ingredients, the formulation form of the exosome secretion promoter, etc. From the viewpoint of more significantly exerting the exosome secretion promoting effect of the present invention, for example, it is preferably 0.00001 to 5% by mass, more preferably 0.0001 to 3% by mass, even more preferably 0.0005 to 2% by mass, and particularly preferably 0.001 to 1% by mass. The dosage to humans is preferably 0.001 to 100 mg per day, more preferably 0.01 to 80 mg, even more preferably 0.1 to 60 mg, and particularly preferably 1 to 50 mg.

[0019] The exosome secretion promoter of the present invention preferably contains one or more selected from the group consisting of glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract, and further contains a specific tetrapeptide.

[0020] The tetrapeptide of the present invention is preferably a peptide represented by the amino acid sequence (Leu-Glu-His-Ala) of SEQ ID NO: 1 or a salt thereof. The peptide or salt thereof also includes the amino acid sequence represented by SEQ ID NO: 1, which has one or more amino acid substitutions and / or additions, and which is characterized in that the exosome secretion promoter of the present invention has excellent exosome secretion promoting ability. When the total number of amino acids constituting the peptide exceeds 4 due to the addition of amino acids, the name "tetrapeptide" is generally not suitable, but in the present invention, it is referred to as the tetrapeptide of the present invention for convenience. The above amino acid substitution is not particularly limited, but is preferably a conservative amino acid substitution, i.e., a conservative amino acid substitution. Conservative amino acid substitutions are shown in Table 1 below.

[0021] [Table 1]

[0022] In the present invention, "the exosome secretion promoter has excellent exosome secretion promoting ability" means that when the exosome secretion promoter containing the above-mentioned tetrapeptide or a salt thereof is allowed to act on cells, the amount of exosomes secreted from the cells is increased compared to when the exosome secretion promoter is not allowed to act on the cells.

[0023] In the present invention, the salt of a peptide refers to any pharmacologically acceptable salt of a peptide (including inorganic salts and organic salts), and examples thereof include sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, hydrochloride salt, sulfate salt, nitrate salt, organic acid salt (acetate salt, citrate salt, maleate salt, malate salt, oxalate salt, lactate salt, succinate salt, fumarate salt, propionate salt, formate salt, benzoate salt, picrate salt, benzenesulfonate salt, etc.) of a peptide, and the like. Ammonium salt, hydrochloride salt, sulfate salt, and acetate salt are preferred, and ammonium salt and acetate salt are more preferred.

[0024] In the present invention, the peptides can be prepared by methods known in the art. For example, the peptides in the present invention can be synthesized by chemical synthesis methods (e.g., solid-phase methods (e.g., Fmoc methods), liquid-phase methods, etc.), or can be prepared by methods such as recombinant expression. The amino acids constituting the peptides of the present invention may be either L- or D-forms, but are preferably L-forms.

[0025] The peptide of the present invention can also be prepared by excising a peptide consisting of a target amino acid sequence from the amino acid sequence of a protein containing the target amino acid sequence by known means such as protease treatment.

[0026] The peptide salts of the present invention can be prepared by any method known in the art.

[0027] The content of the tetrapeptide or a salt thereof in the exosome secretion promoter of the present invention is not particularly limited as long as it is within a range that has an exosome secretion promoting effect, and is appropriately set depending on the type and content of other blended ingredients, the formulation form of the exosome secretion promoter, etc. From the viewpoint of more significantly exhibiting the exosome secretion promoting effect according to the present invention, for example, the content of the tetrapeptide or a salt thereof in the exosome secretion promoter of the present invention is 0.000001% by mass to 7% by mass, preferably 0.00001% by mass to 5% by mass, more preferably 0.0001% by mass to 2% by mass, and even more preferably 0.001% by mass to 1% by mass.

[0028] In addition to the above-mentioned essential components, the exosome secretion promoter of the present invention may contain other optional components within the scope of the present invention. Such optional components may include excipients, thickeners, binders, disintegrants, dispersants, surfactants, suspending agents, emulsifiers, stabilizers, gelling agents, sweeteners, sour agents, umami agents, salty agents, bitter agents, colorants, UV inhibitors, antioxidants, preservatives, antibacterial agents, pH adjusters, buffers, chelating agents, vitamins, flavorings, additives such as minerals, amino acids or their salts, sugars, moisturizing agents, organic acids or their salts, dietary fiber, biological extracts (excluding Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract), etc. These may be appropriately selected from known ones and produced using conventional methods.

[0029] The dosage form of the exosome secretion promoter of the present invention can be used without being limited to a specific product or property, and can be appropriately prepared by a known method in any dosage form such as liquid, gel, cream, paste, solid, aerosol or patch. Examples of liquid forms include lotion, emulsion, beauty essence, oral liquid, syrup, etc. Examples of solid forms include stick, tablet, chewable, granule, powder, capsule, ointment, etc. In addition, although the dosage form may be either an external preparation or an internal preparation, from the viewpoint of exerting the effects of the present invention, it is preferable that the agent is an external preparation. The external preparation may be a pharmaceutical or a cosmetic, and is not limited. These can be manufactured using conventional methods.

[0030] The cells that can obtain an excellent exosome secretion promoting effect by the exosome secretion promoter of the present invention are not particularly limited as long as they can obtain the above-mentioned effect, and examples thereof include adipose-derived stem cells, bone marrow-derived stem cells, and umbilical cord-derived stem cells. Adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and umbilical cord-derived mesenchymal stem cells are preferable, adipose-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells are more preferable, and adipose-derived mesenchymal stem cells are even more preferable.

[0031] The exosome secretion promoter of the present invention promotes exosome secretion from cells, particularly adipose tissue-derived stem cells, by the action of one or more selected from the group consisting of glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract, and the tetrapeptide or a salt thereof used in combination, and the secretion-promoted exosomes are taken up into skin tissue, etc., resulting in cosmetic effects such as inhibition of skin cellular aging, promotion of cell proliferation, promotion of collagen production, and promotion of elastin production. Furthermore, it can also exert an effect of converting M1 / M2 polarity of cells, that is, an effect of transforming inflammatory M1 macrophages into anti-inflammatory M2 macrophages. Therefore, the exosome secretion promoter of the present invention is used for promoting cell proliferation, for converting M1 / M2 polarity of cells, for inhibiting cellular aging, and for promoting collagen and / or elastin production.

[0032] Therefore, the exosome secretion promoter of the present invention may be used in medicines, quasi-drugs, cosmetics, and foods in which the above-mentioned effects are expected, and can be suitably used in cosmetic compositions for the purposes of skin anti-aging, skin regeneration, improving moisturizing properties, etc., as described below.

[0033] [Method of producing exosome secretion promoter] The present invention also includes a method for producing the exosome secretion promoter described above. The method for producing the exosome secretion promoter of the present invention is characterized by blending one or more selected from the group consisting of glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract. It is also preferable that the above-mentioned tetrapeptide or a salt thereof is further blended. The above-mentioned exosome secretion promoter can be produced by blending one or more selected from the group consisting of glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract, preferably the above-mentioned tetrapeptide or a salt thereof, and appropriately selecting, blending, and mixing other optional components (base or carrier, additive, etc.) as necessary, according to a conventional method.

[0034] <Beauty composition for promoting exosome secretion> The present invention also includes a cosmetic composition for promoting exosome secretion, which contains the exosome secretion promoter of the present invention. The cosmetic composition for promoting exosome secretion of the present invention can be said to be characterized in that it contains one or more selected from the group consisting of glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract, and preferably further contains the above-mentioned specific tetrapeptide or a salt thereof. Note that the explanation of the exosome secretion promoter, glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, soy protein extract, and tetrapeptide can be directly applied to the explanation in the section on exosome secretion promoter.

[0035] The cosmetic composition for promoting exosome secretion of the present invention may contain, in addition to the essential components and optional components described in the section on the exosome secretion promoter above, other optional components that are preferably used in cosmetic compositions, as long as the effects of the present invention are not impaired. The other optional components are described below.

[0036] Optional components contained in the cosmetic composition for promoting exosome secretion of the present invention include, for example, the essential components and optional components described in the section on exosome secretion promoters above, other than whitening components, turnover promoters, antiglycation components, antioxidant components, antiaging components, anti-inflammatory agents, refreshing agents, bactericides, vitamins, organic acids, moisturizing components, polyhydric alcohols, emulsifiers, scrubbing agents, UV absorbing components, UV scattering components, astringent components, peptides or derivatives thereof (excluding the tetrapeptide of the present invention), amino acids or derivatives thereof, cleansing components, keratin softening components, cell activating components, blood circulation promoting components, penetrating agents, etc. In addition, in the cosmetic composition for promoting exosome secretion of the present invention, these components may be used alone or in combination of two or more types.

[0037] [Method of manufacturing a cosmetic composition for promoting exosome secretion] The method for producing the cosmetic composition for promoting exosome secretion of the present invention is not particularly limited, and is characterized by including the essential component of the exosome secretion promoter of the present invention (a preparation characterized by including at least one selected from the group consisting of glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract, and further including the above-mentioned tetrapeptide or a salt thereof). The above-mentioned exosome secretion promoter (one or more selected from the group consisting of glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract, preferably further including the above-mentioned tetrapeptide or a salt thereof), and other optional components (base or carrier, additive, etc.) as necessary are appropriately selected, mixed, and produced by a conventional method.

[0038] The cosmetic composition for promoting exosome secretion of the present invention is a new type of cosmetic composition that has excellent cosmetic effects such as anti-aging effects. Specifically, the cosmetic composition for promoting exosome secretion of the present invention promotes exosome secretion from cells, particularly adipose tissue-derived stem cells, and the secretion-promoted exosomes are taken up into skin tissue, etc., resulting in cosmetic effects such as inhibition of skin cellular aging, promotion of cell proliferation, promotion of collagen production, and promotion of elastin production. Furthermore, it can also have an effect of converting M1 / M2 polarity of cells, that is, changing the phenotype of inflammatory M1 macrophages to anti-inflammatory M2 macrophages. Therefore, the cosmetic composition for promoting exosome secretion of the present invention is suitable for use in products aimed at anti-aging, skin regeneration, and improvement of moisturizing properties of the skin. More specifically, it is expected to improve dull skin, restore skin firmness, improve skin firmness, and improve the visibility of pores, and is also effective in improving aging symptoms such as dryness, improving and enhancing skin tone, and whitening the skin. In this way, the cosmetic composition for promoting exosome secretion of the present invention can be suitably used as an external composition such as a beauty serum, lotion, emulsion, cream, gel cream, beauty mask (face mask), makeup base, foundation, sunscreen, cleansing, detergent, etc., and more preferably can be used as a skin care product such as a beauty serum, lotion, emulsion, cream, gel cream, etc.

[0039] In addition, the cosmetic composition for promoting exosome secretion of the present invention can be a food composition. This food composition can be suitably used as a health food or a nutritional supplement (including balanced nutritional food, supplements, etc.). It is also suitable for a health functional food (specified health food (including disease risk reduction labeling, standard type)).

[0040] [Preparation (external composition)] When the cosmetic composition for promoting exosome secretion of the present invention is a composition for external use, the essential components and other optional components described above can be mixed with a base or carrier normally used in cosmetics, medicines, and quasi-drugs, and, if necessary, known additives added to cosmetics, medicines, and quasi-drugs, such as surfactants, antioxidants, colorants, pearl luster imparting agents, chelating agents, pH adjusters, preservatives, thickeners, etc., according to a conventional method, and emulsified or solubilized as necessary to form a cosmetic composition in various formulations.

[0041] When the cosmetic composition for promoting exosome secretion of the present invention is a topical composition, the formulation form is not particularly limited, and examples thereof include liquid preparations (lotion, lotion, beauty essence, etc.), cream preparations (milky lotion, cream, gel cream, balm, etc.), gel preparations, jelly preparations, sherbet preparations, aerosol preparations, foam preparations, spray preparations, patches, stick preparations, other ointments, solid preparations, etc. These preparations can be manufactured according to conventional methods, for example, methods described in the General Provisions of Preparations in the 17th Revised Japanese Pharmacopoeia, etc. In addition, dosage forms such as emulsions and solubilizations can be appropriately selected in consideration of the desired stability, usability, etc. In addition, the patch may be, for example, a nonwoven fabric sheet, gel sheet, etc. impregnated with a beauty essence composition, a beauty essence composition, an emulsion composition, etc.

[0042] [Preparation (food composition)] When the cosmetic composition for promoting exosome secretion of the present invention is a food composition, the essential components and the other optional components described above may be mixed with a base or carrier commonly used in foods, and, if necessary, food additives such as oils and fats, emulsifiers, dispersants, suspending agents, stabilizers, thickeners, sweeteners, colorants, flavors, preservatives, antioxidants, and organic acids to prepare a formulation.

[0043] [pH] The pH of the cosmetic composition of the present invention is usually pH 3.0 to 8.0, and preferably pH 3.5 to 7.5. The pH can be adjusted, for example, by using a pH adjuster. However, this does not apply to formulations in which pH measurement is impossible or difficult.

[0044] Examples of the administration method or application method of the exosome secretion promoter or the cosmetic composition for promoting exosome secretion of the present invention include transdermal administration and oral administration, and a suitable method may be appropriately selected depending on the type of disease or symptom and the purpose of use.

[0045] <Method for promoting exosome secretion> The present invention also includes a method for promoting exosome secretion, characterized by using one or more selected from the group consisting of glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract. In the method for promoting exosome secretion of the present invention, it is preferable to further use the specific tetrapeptide in combination. Here, the above-mentioned use may be any method such as external use or internal use. According to the present invention, excellent cosmetic effects such as anti-aging effects can be obtained. Specifically, according to the method of the present invention, exosome secretion from cells, particularly adipose tissue-derived stem cells is promoted, and the secretion-promoted exosomes are taken into skin tissue, etc., resulting in cosmetic effects such as inhibition of cell aging in the skin, promotion of cell proliferation, promotion of collagen production, and promotion of elastin production. Furthermore, it can also have an effect of converting the M1 / M2 polarity of cells, that is, the effect of transforming inflammatory M1 macrophages into anti-inflammatory M2 macrophages. Therefore, the method of the present invention can be used for the purpose of anti-aging of the skin, regeneration of the skin, improving moisturizing properties, etc. For specific explanations of the various components used in the method for promoting exosome secretion, the explanations in the section on exosome secretion promoters can be applied as is.

[0046] <Exosome uptake promoter> The exosome uptake promoter of the present invention is a composition that promotes the uptake of exosomes into cells. The exosome uptake promoter of the present invention is characterized by containing one or more selected from the group consisting of glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract as an active ingredient. In addition, the exosome uptake promoter of the present invention can promote the uptake of exosomes into cells by further containing a specific tetrapeptide. The exosome uptake promoter of the present invention promotes exosome secretion from cells, particularly adipose tissue-derived stem cells, by containing the above-mentioned specific ingredient as an active ingredient, and the secretion-promoted exosomes are taken up in more cells such as skin tissue, resulting in effects that lead to beauty such as inhibition of cell aging in the skin, promotion of cell proliferation, promotion of collagen production, and promotion of elastin production. In addition, it can also exhibit the effect of converting the M1 / M2 polarity of cells, that is, the effect of transforming inflammatory M1 macrophages into anti-inflammatory M2 macrophages. The explanations of the essential components, optional components, dosage forms, uses, etc. contained in the exosome uptake promoter of the present invention can be applied as they are by replacing "exosome secretion promoter" with "exosome uptake promoter" in the explanation of the section "exosome secretion promoter". Also, the explanation of the method for producing the exosome uptake promoter of the present invention can be applied as they are by replacing "exosome secretion promoter" with "exosome uptake promoter" in the explanation of the section "method for producing exosome secretion promoter".

[0047] The cells that will be promoted by the exosome uptake promoter of the present invention to uptake exosomes, i.e., that will take up a large number of exosomes by the exosome uptake promoter of the present invention, are preferably cells that constitute skin tissue, more preferably cells that constitute the epidermis or dermis of the skin, and particularly preferably cells that constitute epidermal keratinocytes or skin fibroblasts, in that the effects of the present invention can be easily obtained.

[0048] <Cosmetic composition for promoting exosome uptake> The present invention also includes a cosmetic composition for promoting exosome uptake, which contains the exosome uptake promoter of the present invention. The cosmetic composition for promoting exosome uptake of the present invention can be said to be characterized in that it contains at least one selected from the group consisting of glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract, and preferably further contains the above-mentioned specific tetrapeptide or a salt thereof. The explanation of the exosome uptake promoter, glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, soy protein extract, and tetrapeptide can be directly applied to the explanation of the exosome uptake promoter.

[0049] The cosmetic composition for promoting exosome uptake of the present invention may contain, in addition to the essential components and optional components described in the above section on exosome secretion promoters, other optional components that are preferably used in cosmetic compositions, as long as the effects of the present invention are not impaired. For specific explanations of each component and production method, the explanation in the section on cosmetic composition for promoting exosome secretion can be applied as is, with "cosmetic composition for promoting exosome secretion" read as "cosmetic composition for promoting exosome uptake".

[0050] <Method for promoting exosome uptake> The present invention also includes a method for promoting exosome uptake, characterized by using one or more selected from the group consisting of glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract. In the method for promoting exosome uptake of the present invention, it is preferable to further use the specific tetrapeptide in combination. Here, the above-mentioned use may be any method such as external use or internal use. According to the present invention, excellent cosmetic effects such as anti-aging effects can be obtained. Specifically, according to the method of the present invention, exosome secretion from cells, particularly adipose tissue-derived mesenchymal stem cells is promoted, and the secreted exosomes are taken into skin tissue, etc., resulting in cosmetic effects such as inhibition of cell aging in the skin, promotion of cell proliferation, promotion of collagen production, and promotion of elastin production. Furthermore, it can also have an effect of converting the M1 / M2 polarity of cells, that is, the effect of transforming inflammatory M1 macrophages into anti-inflammatory M2 macrophages. Therefore, the method of the present invention can be used for the purpose of anti-aging of the skin, regeneration of the skin, improving moisturizing properties, etc. For specific explanations of the various components used in the method for promoting exosome uptake, the explanations in the section on exosome secretion promoters can be applied as is. EXAMPLES

[0051] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0052] [Test 1: Extraction of exosomes] Human adipose tissue-derived mesenchymal stem cells (Human Adipose Derived Stem Cells, passage number P5, Kurabo) were cultured in a T75 cell culture flask (Corning Cell Bind) at 5,000 cells / cm. 2The cells were seeded so that the cells were 100% pure and cultured at 37°C in a 5% carbon dioxide environment for a total of 5 days, with the medium being replaced once every 2 days. After washing with phosphate buffer (Kohjin Bio Co., Ltd.), the medium was replaced with a medium containing the components shown in Table 2 below (Comparative Example 1, Examples 1 to 9). Each component was added so that the final concentration (mass%) was as shown in Table 2 below. After 48 hours, the culture supernatant was collected in a tube (Merck Amicon (registered trademark) Ultra) and centrifugal filtration was performed (5,000 x g, 20 minutes, followed by 4,000 x g, 10 minutes). The total volume was adjusted to 1.5 mL by adding phosphate buffer, and exosomes were extracted using an exosome purification column (GL Sciences EVSecond L70). The obtained exosomes were evaluated as described below.

[0053] [Evaluation 1: Exosome secretion] The particle concentration and particle size distribution of each exosome extracted in the above Test 1 were measured and analyzed using a nanoparticle analysis system (NanoSight, manufactured by Quantum Design Japan), and the particle concentration with a particle diameter of 50-150 nm was defined as the amount of exosome secretion. The ratio of the amount of exosome secretion in each Example to the amount of exosome secretion in Comparative Example 1, in which no component was added, was calculated, and this value was defined as the exosome secretion promoting ability of each Example, and is shown in Table 2 below.

[0054] [Table 2]

[0055] Glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract promoted exosome secretion from adipose tissue-derived mesenchymal stem cells (Examples 1, 3, 5, 7, and 8). Furthermore, the inclusion of a tetrapeptide significantly improved the exosome secretion-promoting ability (Examples 2, 4, and 6). The inclusion of licorice leaf extract and soy protein extract significantly improved the exosome secretion-promoting ability (Example 9).

[0056] [Evaluation 2: Intracellular uptake of exosomes] Each exosome extracted in the above test 1 was membrane stained using ExoSparkler Exosome Membrane Labeling Kit (DOJINDO). The stained exosomes were seeded at 5,000 cells / well in a 96-well plate (Corning Cell Bind), and administered at 100 μL / well to normal human dermal fibroblasts (Kurabo Normal Human Dermal Fibroblasts, passage number P33) cultured for 24 hours at 37 ° C. in a 5% carbon dioxide environment. After 6 hours of culture, images were taken with ImageXpress (Molecular Devices) (16 fields / well), and intracellular exosomes were measured using an exosome counting program. The ratio of the number of exosomes in each example to the number of exosomes in Comparative Example 1 to which no components were added was calculated, and the value was taken as the exosome cellular uptake ability of each example, as shown in Table 3 below. The image of Example 2 is also shown in Figure 1.

[0057] [Evaluation 3: Cell proliferation by exosomes] The exosomes extracted in the above test 1 were seeded at 3,500 cells / well in a 96-well plate (Corning Cell Bind), and 100 μL / well was administered to normal human dermal fibroblasts (Kurabo Normal Human Dermal Fibroblasts, passage number P33) cultured for 24 hours at 37 ° C. in a 5% carbon dioxide environment. After 72 hours of culture, the cells stained with Hoechst33342 (DOJINDO) were imaged with ImageXpress (Molecular Devices) (16 fields / well), and the number of cells was counted using a nucleus counting program. The ratio of the number of cells in each example to the number of cells in Comparative Example 1 to which no components were added was calculated, and the value was taken as the cell proliferation ability by the exosomes in each example, as shown in Table 3 below.

[0058] [Evaluation 4: M1 / M2 polarity conversion by exosomes] Human monocyte-derived cell line THP-1 (ATCC, passage number P12) suspended in a medium containing 100 nM Phorbol 12-Myristate 13-Acetate (Fujifilm Wako Pure Chemical Industries, Ltd.) was seeded at 300,000 cells / well in a 12-well plate (Corning Cell Bind) and cultured for 24 hours at 37°C under 5% carbon dioxide. After 24 hours, it was confirmed that the cells had changed into adherent M0 macrophages, and then (i) 20 ng / mL IFNγ and 10 pg / mL LPS, or (ii) 20 ng / mL IL-4 and 20 ng / mL IL-13 were added and cultured for 72 hours to induce (i) M1 macrophages and (ii) M2 macrophages, respectively. After 72 hours, the cells induced to M1 macrophages were administered 1 mL / well of a sample in which the exosomes extracted in Test 1 were mixed with each Example. Comparative Example 1 was administered 1 mL / well of medium. After 72 hours, the expression levels of TNFα, IL-6, IL-1ra, and IL-10 in the culture supernatant were measured using Quantikine ELISA (R&D). The ratio of the expression level of each Example to the expression level of Comparative Example 1 in which no components were added was calculated, and the value was taken as the change in expression level due to exosomes in each Example, and is shown in Table 3 below.

[0059] [Table 3]

[0060] Exosomes secreted from adipose tissue-derived mesenchymal stem cells cultured in the presence of glycogen were taken up in large amounts by dermal fibroblasts. Exosomes secreted from adipose tissue-derived mesenchymal stem cells cultured in the presence of glycogen and tetrapeptide were taken up in even larger amounts by dermal fibroblasts. When observing the dermal fibroblasts of Example 2, exosomes stained in white were observed (Figure 1), but exosomes stained in white were not observed in the dermal fibroblasts of Comparative Example 1. Exosomes secreted from adipose tissue-derived mesenchymal stem cells cultured in the presence of glycogen improved the proliferation ability of skin fibroblasts. Exosomes secreted from adipose tissue-derived mesenchymal stem cells cultured in the presence of glycogen and a tetrapeptide further improved the proliferation ability of skin fibroblasts. Exosomes secreted from adipose tissue-derived mesenchymal stem cells cultured in the presence of glycogen, and exosomes secreted from adipose tissue-derived mesenchymal stem cells cultured in the presence of glycogen and the tetrapeptide, suppressed the expression of TNFα and IL-6 in inflammatory M1 macrophages and promoted the expression of IL-1ra and IL-10 in anti-inflammatory M2 macrophages, i.e., transformed inflammatory M1 macrophages into anti-inflammatory M2 macrophages.

[0061] [Evaluation 5: Suppression of cell aging by exosomes] The exosomes extracted in the above test 1 were seeded at 3,500 cells / well in a 96-well plate (Corning Cell Bind), and 100 μL / well was administered to normal human dermal fibroblasts (Kurabo Normal Human Dermal Fibroblasts, passage number P33) cultured for 24 hours at 37 ° C. in a 5% carbon dioxide environment. After 72 hours of culture, the degree of cellular senescence was measured using β-galactosidase as an index using a Cellular Senescence Plate Assay Kit (DOJINDO). The ratio of β-galactosidase activity of Example 2 to the β-galactosidase activity of Comparative Example 1 to which no component was added was calculated, and the value was taken as a value indicating the cellular senescence inhibitory effect of Example 2, and is shown in Table 4 below. The smaller this value, the higher the cellular senescence inhibitory effect. The staining of β-galactosidase was also observed under a microscope. (Figure 2)

[0062] [Evaluation 6: Promotion of collagen production by exosomes] The exosomes extracted in the above test 1 were seeded at 50,000 cells / well in a 12-well plate (Corning Cell Bind) and administered at 1 mL / well to normal human dermal fibroblasts (Kurabo Normal Human Dermal Fibroblasts, passage number P33) cultured for 24 hours at 37 ° C. in a 5% carbon dioxide environment. After 72 hours of culture, the expression level of type 1 collagen in the culture supernatant was measured using Human Procollagen I alpha 1 DuoSet ELISA (R & D). The ratio of the collagen expression level of Example 2 to the collagen expression level of Comparative Example 1 to which no component was added was calculated, and the value was taken as the collagen production promoting ability of Example 2, and is shown in Table 4 below.

[0063] [Evaluation 7: Exosomes promote elastin production] The exosomes extracted in the above test 1 were seeded in a T25 cell culture flask (Corning Cell Bind) at 2,000,000 cells / well, and administered at 5 mL / well to normal human dermal fibroblasts (Kurabo Normal Human Dermal Fibroblasts, passage number P33) cultured for 24 hours at 37 ° C. in a 5% carbon dioxide environment. After 72 hours of culture, elastin in the cells was extracted using Fastin Elastin Assay (Biocolor), and the amount of intracellular α-elastin was measured by colorimetry. The ratio of the amount of elastin in Example 2 to the amount of elastin in Comparative Example 1 to which no component was added was calculated, and the value was taken as the elastin production promoting ability of Example 2, and is shown in Table 4 below.

[0064] [Table 4]

[0065] Exosomes secreted from adipose tissue-derived mesenchymal stem cells cultured in the presence of glycogen and tetrapeptide suppressed β-galactosidase activity, an indicator of cell aging, in skin fibroblasts, and promoted collagen and elastin production. It was observed that the skin fibroblasts of Comparative Example 1 had high β-galactosidase staining, while the skin fibroblasts of Example 2 had low β-galactosidase staining (Figure 2). [Industrial Applicability]

[0066] According to the present invention, a new type of cosmetic composition having excellent cosmetic effects such as anti-aging effects can be provided. Specifically, the exosome secretion promoter of the present invention promotes exosome secretion from cells, particularly adipose tissue-derived mesenchymal stem cells, and the secretion-promoted exosomes are taken up into skin tissues, etc., resulting in cosmetic effects such as inhibition of skin cell aging, promotion of cell proliferation, promotion of collagen production, and promotion of elastin production. Furthermore, it can also exert an M1 / M2 polarity conversion effect of cells, that is, an effect of transforming inflammatory M1 macrophages into anti-inflammatory M2 macrophages. Therefore, the exosome secretion promoter of the present invention is suitable for use in products aimed at anti-aging, skin regeneration, and improvement of moisturizing properties of the skin. More specifically, it is expected to improve dullness of the skin, restore skin firmness, improve skin firmness, and improve the visibility of pores, and is also effective in improving aging symptoms such as dryness, improving and enhancing skin tone, and whitening the skin.

Claims

1. Contains glycogen An exosome secretion promoter used to transform inflammatory M1 macrophages into anti-inflammatory M2 macrophages.

2. Further containing a tetrapeptide, The exosome secretion promoter according to claim 1, wherein the total number of amino acids in the tetrapeptide is 4.

3. The exosome secretion promoter according to claim 2, wherein the tetrapeptide is a peptide represented by the amino acid sequence (Leu-Glu-His-Ala) of SEQ ID NO: 1 or a salt thereof.

4. The exosome secretion promoter according to any one of claims 1 to 3, which promotes exosome secretion in adipose-derived mesenchymal stem cells.

5. The exosome secretion promoter according to any one of claims 1 to 4, further comprising one or more selected from the group consisting of Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soy protein extract.

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