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 ART

[0002] Exosomes are lipid bilayer membrane-covered extracellular vesicles secreted by most cell types, and are generally defined by a particle size of 50 to 150 nm and several membrane-expressing marker proteins (such as CD9, CD63, and CD81). These vesicles contain cell membrane proteins, intracellular proteins, and further nucleic acids such as DNA, mRNA, and miRNA, and are said to be involved in intercellular interactions. In such intercellular interactions, physiological and pathological functions are drawing attention, and novel diagnostic methods for diseases using exosomes as indicators, and attempts to use exosomes themselves for disease treatment are also being investigated.

[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 and the like related to cell proliferation, differentiation and regeneration, and cosmetic compositions for skin whitening, wrinkle improvement or skin regeneration containing these as active ingredients have been developed (see Patent Document 1).

[0004] In recent years, people's demands for skin beauty and health are increasing day by day, and new types of compositions and compositions with more excellent cosmetic effects are required. PRIOR ART DOCUMENTS PATENT DOCUMENTS

[0005] PATENT DOCUMENT 1 Japanese Unexamined Patent Application Publication No. 2018-184446 SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0006] This invention has been made in view of the above circumstances, and aims to provide a new type of cosmetic composition that is excellent in cosmetic effects such as anti-aging effects. Specifically, the aim is to provide a composition that can promote the secretion of exosomes 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, etc., it is thought that a composition that can promote the secretion of such exosomes will consequently produce excellent cosmetic effects. [Means for solving the problem]

[0007] The present inventors, through diligent research to solve the above problems, have 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 adipose tissue-derived stem cells. Furthermore, it has been revealed that the exosome secretion-promoting effect of the above composition can be further enhanced by including a specific tetrapeptide in addition to these components. Moreover, it has been found that the exosomes secreted through the action of the above composition, which has excellent exosome secretion-promoting effect, are taken up by skin tissue, etc., resulting in excellent effects that lead to beauty, such as suppression of skin cell aging, promotion of cell proliferation, promotion of collagen production, and promotion of elastin production. Furthermore, it has been found that it also has an effect of M1 / M2 polarity conversion of cells, that is, the effect of changing the phenotype of inflammatory M1 macrophages into anti-inflammatory M2 macrophages. In other words, 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 of SEQ ID NO: 1 (Leu-Glu-His-Ala) 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 exosome uptake promoter into cells, 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. [6] The exosome uptake promoter into cells according to [5], further comprising a tetrapeptide. [7] The exosome uptake promoter into cells according to [6], wherein the tetrapeptide is a peptide represented by the amino acid sequence of SEQ ID NO: 1 (Leu-Glu-His-Ala) or a salt thereof. [8] An exosome uptake promoter into cells according to any one of [5] to [7], wherein the cells are dermal fibroblasts. [9] An exosome secretion promoter described in any of [1] to [4], used to promote cell proliferation.

[10] Exosome secretion promoters described in any of [1] to [4], used for M1 / M2 polarity reversal of cells.

[11] An exosome secretion promoter according to any one of [1] to [4], used for the suppression of cellular senescence.

[12] An exosome secretion promoter according to any one of [1] to [4], used to promote collagen and / or elastin production.

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

[14] An exosome uptake promoter into cells, as described in any of [5] to [8], used to promote cell proliferation.

[15] Exosome uptake promoters into cells, as described in any of [5] to [8], used for M1 / M2 polarity reversal of cells.

[16] An exosome uptake promoter into cells, as described in any of [5] to [8], used to inhibit cellular senescence.

[17] An exosome uptake promoter into cells, as described in any of [5] to [8], used to promote collagen and / or elastin production.

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

[19] A method for producing an exosome secretion promoter, comprising 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] A method for producing the exosome secretion promoter described in

[19] , further comprising the addition of 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 of SEQ ID NO: 1 (Leu-Glu-His-Ala) or a salt thereof.

[22] A method for producing an exosome uptake promoter into cells, comprising one or more 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 according to

[22] , further comprising the addition of a tetrapeptide.

[24] The method for producing an exosome uptake promoter into cells according to

[23] , wherein the tetrapeptide is a peptide represented by the amino acid sequence of SEQ ID NO: 1 (Leu-Glu-His-Ala) or a salt thereof. A cosmetic composition for promoting exosome secretion, comprising any of the exosome secretion promoters from

[25] [1] to [4] or [9] to

[13] . A method for producing a cosmetic composition for promoting exosome secretion, comprising any of the exosome secretion promoters from

[26] [1] to [4] or [9] to

[13] . A cosmetic composition for promoting exosome uptake into cells, comprising any of the exosome uptake promoters into cells from

[27] , [5] to [8], or

[14] to

[18] . A method for producing a cosmetic composition for promoting exosome uptake into cells, comprising any of the exosome uptake promoters into cells from

[28] , [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 the use of tetrapeptides.

[31] The method for promoting exosome secretion according to

[30] , wherein the tetrapeptide is a peptide represented by the amino acid sequence of SEQ ID NO: 1 (Leu-Glu-His-Ala) 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] The method for promoting exosome uptake into cells, as described in

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

[34] The method for promoting exosome uptake into cells according to

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

[0009] According to the present invention, it is possible to provide a new type of cosmetic composition that exhibits excellent cosmetic effects such as an anti-aging effect. Specifically, according to the exosome secretion promoter of the present invention, exosome secretion from cells, particularly adipose tissue-derived stem cells, is promoted, and the exosomes with promoted secretion are taken up by skin tissue and the like, and as a result, effects linked to beauty such as inhibition of skin cell senescence, promotion of cell proliferation, promotion of collagen production, and promotion of elastin production can be achieved. Furthermore, it can also exert an M1 / M2 polarization switching effect on cells, that is, the effect of phenotypically changing inflammatory M1 macrophages into anti-inflammatory M2 macrophages. Therefore, the exosome secretion promoter of the present invention is suitably used for products intended for skin anti-aging, skin regeneration, improvement of moisturizing properties, and the like. More specifically, improvement of skin dullness, recovery of skin firmness, enhancement of skin firmness, and improvement of noticeable pores can be expected, and it is also effective in improving aging symptoms such as dryness, improving and enhancing skin tone, and whitening the skin. [Brief Description of Drawings]

[0010] [Figure 1] This is a diagram showing the state in which exosomes secreted from adipose tissue-derived mesenchymal stem cells cultured in the composition of Example 2 are taken up by human fibroblasts. [Figure 2] This is a diagram showing the cell senescence inhibitory effect of exosomes secreted from adipose tissue-derived mesenchymal stem cells cultured in the composition of Example 2 on skin fibroblasts, in comparison with the case of using the composition of Comparative Example (β-Galactosidase staining). [Modes for Carrying Out the Invention]

[0011] Hereinafter, the present invention will be described in detail. The terms used in the present specification are to be construed as having the meanings commonly used in the relevant technical field unless otherwise specifically noted.

[0012] <Exosome Secretion Promoter> The exosome secretion-promoting agent of the present invention is characterized by containing, as an active ingredient, one or more selected from the group consisting of glycogen, Centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, and soybean protein extract. The exosome secretion-promoting agent of the present invention can further enhance the exosome secretion-promoting effect by containing a specific tetrapeptide. By comprising the above specific component as an active ingredient, the exosome secretion-promoting agent of the present invention promotes exosome secretion from cells, particularly adipose tissue-derived stem cells, and the exosomes with promoted secretion are taken up by skin tissue and the like, as a result, effects linked to beauty such as suppression of skin cell senescence, promotion of cell proliferation, promotion of collagen production, and promotion of elastin production can be achieved. Furthermore, it can also exert an M1 / M2 polarization switching effect on cells, that is, the effect of phenotypically changing pro-inflammatory M1 macrophages into anti-inflammatory M2 macrophages. Hereinafter, each essential component, optional component, and the like contained in the exosome secretion-promoting agent of the present invention will be specifically described.

[0013] In this invention, glycogen refers to a polymer whose constituent unit is α-D-glucose (grape sugar), linked only by α-1,4-glucosidic bonds and α-1,6-glucosidic bonds. Examples of such glycogen include plant glycogen derived from corn seeds, barley, rice, potatoes, etc., and animal glycogen derived from the liver and muscles of animals such as cattle and pigs, and from shellfish such as scallops, abalone, oysters, mussels, pearl oysters, and clams. In this invention, glycogen may be obtained by using natural products prepared by conventional methods, or it may be synthesized using enzymes. Examples of synthesis methods using the above enzymes include reacting sucrose and primer molecules (such as maltooligosaccharides or dextrin) with sucrose phosphorylase (EC2.4.1.7), α-glucan phosphorylase (EC2.4.1.1), and blanching enzyme (EC2.4.1.18), and reacting short-chain amylose with blanching enzyme (EC2.4.1.18). Glycogen synthesized by these methods is known to have the same chemical and physical structure as natural glycogen, and these enzyme-synthesized glycogens can also be used. Furthermore, the glycogen of the present invention may be not only the natural or synthetic glycogens described above, but also their derivatives or equivalents.

[0014] In the present invention, glycogen may be used that has been separated into fractions with appropriate molecular weights by methods well known to those skilled in the art. Examples of such separation methods include chromatographic separation methods such as gel filtration chromatography and HPLC, and membrane separation methods. Precipitation methods using solvents such as methanol and ethanol, either alone or in combination, are also possible. Furthermore, methods for derivatization (etherification, esterification, crosslinking, and grafting) of natural or synthetic glycogen include methods commonly used for starch modification, which are 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. From the viewpoint of promoting exosome secretion, the average molecular weight of glycogen in the present invention is preferably 300,000 to 1,500,000, more preferably 400,000 to 1,200,000, and more preferably 600,000 to 900,000.

[0015] In this invention, commercially available glycogen can also be used. Examples of such commercially available products include BIOSACCHARIDES LS / HG (manufactured by Laboratories Serobiologiques), derived from mussels, and Bioglycogen (Glico Nutritional Foods Co., Ltd.).

[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] In the present invention, the Centella asiatica leaf extract, bilberry leaf extract, and licorice leaf extract can be extracts obtained from each plant using techniques conventionally known to those skilled in the art. The extraction method and extraction solvent for each plant extract are not particularly limited. Furthermore, each plant extract may be decomposed, and the decomposition method is not particularly limited; known techniques can be used. Commercially available Centella asiatica leaf extract, bilberry leaf extract, and licorice leaf extract can also be used in the present invention. The content of 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 has an exosome secretion promoting effect on cells, and can be appropriately set depending on the type and content of other ingredients, the formulation form of the exosome secretion promoter, etc. From the viewpoint of more significantly exhibiting the exosome secretion-promoting effect of the present invention, for example, the amount 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 for 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 this invention is not particularly limited as long as it is derived from soybeans of the legume family, and one or more species of soybean plants may be selected and used. Any plant part may be used for the soy protein extract, and one or more parts may be selected and used. Furthermore, 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 this invention may be a commercially available product. The content of the soy protein extract in the exosome secretion promoter of this invention is not particularly limited as long as it is within the range that has an exosome secretion promoting effect, and is appropriately set depending on the type and content of other ingredients, the formulation form of the exosome secretion promoter, etc. From the viewpoint of more significantly exhibiting the exosome secretion promoting effect of this 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 for 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 preferably contains a specific tetrapeptide.

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

[0021] [Table 1]

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

[0023] In the present invention, a peptide salt refers to any pharmacologically acceptable salt of a peptide (including inorganic and organic salts), such as sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, hydrochloride salt, sulfate salt, nitrate salt, and 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.). Preferably, the salt is an ammonium salt, hydrochloride salt, sulfate salt, or acetate salt, and more preferably an ammonium salt or acetate salt.

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

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

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

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

[0028] In addition to the essential components described above, the exosome secretion promoter of the present invention may contain other optional components as long as they do not interfere with the effects of the present invention. Such optional components may include excipients, thickeners, binders, disintegrants, dispersants, surfactants, suspending agents, emulsifiers, stabilizers, gelling agents, sweeteners, acidulants, flavoring agents, salting agents, bittering agents, coloring agents, UV inhibitors, antioxidants, preservatives, antibacterial agents, pH adjusters, buffers, chelating agents, additives such as vitamins, flavorings, and minerals, amino acids or their salts, sugars, humectants, 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. Known components can be appropriately selected and used, and the product can be manufactured using conventional methods.

[0029] The dosage form of the exosome secretion promoter of the present invention is not limited to specific products or properties, and can be prepared as appropriate by known methods in any dosage form, such as liquid, gel, cream, paste, solid, aerosol, or patch. Examples of liquid forms include lotions, emulsions, serums, oral solutions, and syrups. Examples of solid forms include sticks, tablets, chewable tablets, granules, powders, capsules, and ointments. In terms of administration form, it can be either a topical or oral preparation, but from the viewpoint of achieving the effects of the present invention, a topical preparation is preferred. Topical preparations are not limited to pharmaceuticals or cosmetics. These can be manufactured using conventional methods.

[0030] The cells that can obtain an excellent exosome secretion-promoting effect with the exosome secretion-promoting agent of the present invention are not particularly limited as long as they can obtain the above effect, but examples include adipose-derived stem cells, bone marrow-derived stem cells, umbilical cord-derived stem cells, etc., and it is preferable that the cells are adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, or umbilical cord-derived mesenchymal stem cells, more preferably adipose-derived mesenchymal stem cells or umbilical cord-derived mesenchymal stem cells, and even more preferably adipose-derived mesenchymal stem cells.

[0031] The exosome secretion promoter of the present invention promotes exosome secretion from cells, particularly adipose tissue-derived stem cells, through 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, as well as a tetrapeptide or its salt used in combination. The secreted exosomes are then taken up by skin tissue, resulting in cosmetic effects such as suppression 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 on cells, i.e., it can change the phenotype of inflammatory M1 macrophages into anti-inflammatory M2 macrophages. Therefore, the exosome secretion promoter of the present invention is used for promoting cell proliferation, for M1 / M2 polarity conversion of cells, for suppressing cell aging, and for promoting collagen and / or elastin production.

[0032] Therefore, the exosome secretion promoter of the present invention may be used in pharmaceuticals, quasi-drugs, cosmetics, and foods for the above-mentioned effects, and can be suitably used in beauty compositions for the purpose of anti-aging, skin regeneration, and improved moisturizing properties of the skin, as described later.

[0033] [Method for manufacturing an 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 incorporating 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 to further incorporate the above-mentioned tetrapeptide or a salt thereof. The above-mentioned exosome secretion promoter can be produced by a conventional method by further incorporating 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 and incorporating other optional components (base or carrier, additive, etc.) as needed.

[0034] <Beauty composition for promoting exosome secretion> The present invention also includes a cosmetic composition for promoting exosome secretion, which contains the exosome secretion-promoting agent of the present invention described above. The cosmetic composition for promoting exosome secretion of the present invention may also be described as 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, and preferably further containing the above-mentioned specific tetrapeptide or a salt thereof. The descriptions of the exosome secretion-promoting agent, glycogen, centella asiatica leaf extract, bilberry leaf extract, licorice leaf extract, soy protein extract, and tetrapeptide can be directly applied to the descriptions in the section on exosome secretion-promoting agents.

[0035] The exosome secretion-promoting cosmetic composition of the present invention may contain, in addition to the essential and optional components described in the section on exosome secretion promoters above, other optional components that are suitably used in cosmetic compositions, as long as they do not impair the effects of the present invention. Other optional components are described below.

[0036] Optional components included in the exosome secretion-promoting cosmetic composition of the present invention include, for example, whitening ingredients, turnover promoters, anti-glycation ingredients, antioxidant ingredients, anti-aging ingredients, anti-inflammatory agents, cooling agents, bactericidal agents, vitamins, organic acids, moisturizing ingredients, polyhydric alcohols, emulsifiers, scrubbing agents, UV absorbing ingredients, UV scattering ingredients, astringent ingredients, peptides or their derivatives (excluding the tetrapeptide of the present invention), amino acids or their derivatives, cleansing ingredients, keratin softening ingredients, cell activating ingredients, blood circulation promoting ingredients, and penetration agents, in addition to the essential ingredients and optional components described in the section on exosome secretion promoters above.

[0037] [Method for producing a cosmetic composition that promotes 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 incorporating the exosome secretion promoting agent of the present invention, which is an essential component (a formulation characterized by incorporating 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 characterized by incorporating the above-mentioned tetrapeptide or a salt thereof). The above-mentioned exosome secretion promoting agent (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 comprising the above-mentioned tetrapeptide or a salt thereof), and other optional components (base or carrier, additive, etc.) as needed, can be appropriately selected, incorporated, and mixed by a conventional method.

[0038] The exosome secretion-promoting cosmetic composition of the present invention is a new type of cosmetic composition that excels in cosmetic effects such as anti-aging effects. Specifically, the exosome secretion-promoting cosmetic composition of the present invention promotes exosome secretion from cells, particularly adipose tissue-derived stem cells, by the exosome secretion-promoting agent of the present invention. The secreted exosomes are then taken up by skin tissue, etc., resulting in cosmetic effects such as suppression 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 on cells, that is, an effect of changing the phenotype of inflammatory M1 macrophages into anti-inflammatory M2 macrophages. Therefore, the exosome secretion-promoting cosmetic composition of the present invention is suitably used in products aimed at anti-aging of the skin, skin regeneration, and improvement of moisturizing properties. More specifically, it can be expected to improve skin dullness, restore skin firmness, improve skin elasticity, and reduce the appearance of pores. It also has effects on improving aging symptoms such as dryness, improving skin tone, and whitening the skin. Thus, the exosome secretion-promoting beauty composition 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, cleanser, or washing agent, and more preferably as a skincare product such as a beauty serum, lotion, emulsion, cream, or gel cream.

[0039] Furthermore, the exosome secretion-promoting beauty composition of the present invention can be used as a food composition. This food composition can be suitably used as a health food or nutritional supplement (including balanced nutrition foods, supplements, etc.). It is also suitable for use as a functional food (including foods for specified health uses (including those with disease risk reduction claims and standardized types)).

[0040] [Formulations (Topical Compositions)] If the exosome secretion-promoting cosmetic composition of the present invention is an external composition, its essential components and other optional components described above can be mixed with a base or carrier commonly used in cosmetics, pharmaceuticals, and quasi-drugs, and, if necessary, known additives added to cosmetics, pharmaceuticals, and quasi-drugs, such as surfactants, antioxidants, colorants, pearlescent agents, chelating agents, pH adjusters, preservatives, and thickeners, in accordance with conventional methods, and emulsified or solubilized as necessary to produce cosmetic compositions in various formulation forms.

[0041] The formulation form of the exosome secretion-promoting cosmetic composition of the present invention is not particularly limited when it is an external composition. Examples include liquid formulations (lotions, serums, etc.), cream formulations (emulsions, creams, gel creams, balms, etc.), gel formulations, jelly formulations, sherbet formulations, aerosol formulations, foam formulations, spray formulations, patches, stick formulations, and other ointments, solid formulations, etc. These formulations can be manufactured by conventional methods, for example, the method described in the General Provisions for Preparations of the 17th Revised Japanese Pharmacopoeia. Furthermore, depending on the desired stability, feel, etc., the dosage form such as emulsion or solubilization can be selected as appropriate. In addition, for example, a patch may be made by impregnating a nonwoven fabric sheet, gel sheet, etc., with the cosmetic composition, lotion composition, etc.

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

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

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

[0045] <Methods to promote 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 exosome secretion promoting method of the present invention, it is preferable to further use the above-mentioned specific tetrapeptide in combination. Here, the above use may be by any method, such as topical application or oral administration. 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, especially adipose tissue-derived stem cells, is promoted, and the secreted exosomes are taken up by skin tissue, etc., resulting in cosmetic effects such as suppression of skin cell aging, promotion of cell proliferation, promotion of collagen production, and promotion of elastin production. Furthermore, it can also produce an M1 / M2 polarity conversion effect on cells, that is, an effect of changing the phenotype of 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, skin regeneration, improvement of moisturizing properties, etc. Furthermore, the specific details of the various components used in the exosome secretion promotion method can be directly applied to the explanation in the section on exosome secretion promoters.

[0046] <Exosome uptake enhancer> 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. Furthermore, the exosome uptake promoter of the present invention can further promote the uptake of exosomes into cells by containing a specific tetrapeptide. By containing the above specific component as an active ingredient, the exosome uptake promoter of the present invention promotes the secretion of exosomes from cells, especially adipose tissue-derived stem cells, and the secreted exosomes are taken up in greater quantities by cells such as skin tissue, resulting in effects that contribute to beauty, such as suppression 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 on cells, that is, an effect that changes the phenotype of inflammatory M1 macrophages into anti-inflammatory M2 macrophages. Furthermore, the descriptions of each essential component, optional component, dosage form, and use of the exosome uptake promoter of the present invention can be applied as is, by replacing "exosome secretion promoter" with "exosome uptake promoter" in the description of "exosome secretion promoter." Similarly, the method for producing the exosome uptake promoter of the invention can be applied as is, by replacing "exosome secretion promoter" with "exosome uptake promoter" in the description of "method for producing an exosome secretion promoter."

[0047] The cells on which the exosome uptake promoter of the present invention promotes the uptake of exosomes, that is, cells that take up a large amount of exosomes with the exosome uptake promoter of the present invention, are preferably cells that make up skin tissue, more preferably cells that make up the epidermis or dermis of the skin, and among these, cells that make up epidermal keratinocytes or dermal fibroblasts are even more preferably.

[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 described above. The cosmetic composition for promoting exosome uptake of the present invention may also be described as 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, and preferably further containing the above-mentioned specific tetrapeptide or a salt thereof. The descriptions 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 descriptions in the section on the exosome uptake promoter.

[0049] The exosome uptake-promoting cosmetic composition of the present invention may contain, in addition to the essential and optional components described in the section on exosome secretion promoters, other optional components that are suitably used in cosmetic compositions, as long as they do not impair the effects of the present invention. For specific descriptions of each component and manufacturing methods, the description in the section on exosome secretion-promoting cosmetic composition can be applied as is, by replacing "exosome secretion-promoting cosmetic composition" with "exosome uptake-promoting cosmetic composition."

[0050] <Methods to promote 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 exosome uptake promoting method of the present invention, it is preferable to further use the above-mentioned specific tetrapeptide in combination. Here, the above use may be by any method, such as topical application or oral administration. 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, especially adipose tissue-derived mesenchymal stem cells, is promoted, and the secreted exosomes are taken up by skin tissue, etc., resulting in cosmetic effects such as suppression of skin cell aging, promotion of cell proliferation, promotion of collagen production, and promotion of elastin production. Furthermore, it can also produce an M1 / M2 polarity conversion effect of cells, that is, an effect of changing the phenotype of 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, skin regeneration, improvement of moisturizing properties, etc. Furthermore, the specific details of the various components used in the exosome uptake promotion method can be directly applied to the explanation in the section on exosome secretion promoters. [Examples]

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

[0052] [Experiment 1: Exosome Extraction] Human adipose tissue-derived mesenchymal stem cells (Human Adipose Derived Stem Cells, Kurabo Industries Ltd., passage number P5) are cultured in a T75 cell culture flask (Corning Cell Bind) at a rate of 5,000 cells / cm³. 2Seeds were seeded as shown, and the culture was performed for a total of 5 days at 37°C in a 5% carbon dioxide environment, with the culture medium being changed every two days. After washing with phosphate buffer (Kojin Bio), the culture medium was replaced with one containing the components shown in Table 2 below (Comparative Example 1, Examples 1-9). 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® Ultra) and centrifuged (5,000 × g, 20 minutes, then 4,000 × 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 Test 1 above were measured and analyzed using a nanoparticle analysis system (NanoSight, manufactured by Quantum Design Japan). The particle concentration with a particle diameter of 50-150 nm was defined as the exosome secretion amount. The ratio of the exosome secretion amount of each example to the exosome secretion amount of Comparative Example 1 (without added components) was calculated, and this value was defined as the exosome secretion promoting ability of each example, as shown in Table 2 below.

[0054] [Table 2]

[0055] Glycogen, Centella asiatica leaf extract, Vaccinium bilberry leaf extract, Licorice leaf extract, and Soy protein extract promoted the secretion of exosomes from adipose tissue-derived mesenchymal stem cells (Examples 1, 3, 5, 7, 8). Furthermore, the inclusion of tetrapeptides significantly improved the ability to promote exosome secretion (Examples 2, 4, 6). The inclusion of Licorice leaf extract and Soy protein extract significantly improved the ability to promote exosome secretion (Example 9).

[0056] [Evaluation 2: Intracellular uptake of exosomes] Each exosome extracted in Test 1 above was membrane-stained using the 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 P33) cultured for 24 hours at 37°C in a 5% carbon dioxide environment. After 6 hours of culture, images were acquired using ImageXpress (Molecular Devices) (16 fields / well), and intracellular exosomes were counted using an exosome counting program. The ratio of the number of exosomes in each example to the number of exosomes in Comparative Example 1 (without added components) was calculated, and this value was defined as the intracellular exosome uptake capacity of each example, as shown in Table 3 below. Images from Example 2 are shown in Figure 1.

[0057] [Rating 3: Cell proliferation mediated by exosomes] Exosomes extracted in Test 1 above were seeded at 3,500 cells / well in a 96-well plate (Corning Cell Bind) and cultured for 24 hours at 37°C in a 5% carbon dioxide environment. Normal human dermal fibroblasts (Kurabo Industries Ltd., passage number P33) were then administered 100 μL / well. After 72 hours of culture, cells stained with Hoechst 33342 (DOJINDO) were imaged using ImageXpress (Molecular Devices) (16 fields of view / well), and the number of cells was measured using a nuclear counting program. The ratio of the number of cells in each example to the number of cells in Comparative Example 1 (without added components) was calculated, and this value was defined as the cell proliferation capacity induced by exosomes in each example, as shown in Table 3 below.

[0058] [Evaluation 4: Exosome-mediated M1 / M2 polarity reversal] 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, phenotypic transformation to adherent M0 macrophages was confirmed. 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 the cells were cultured for 72 hours to induce (i) M1 macrophages and (ii) M2 macrophages, respectively. After 72 hours, cells induced to become M1 macrophages were administered 1 mL / well of a sample containing the exosomes extracted in Test 1 mixed with the ingredients from each example. Comparative Example 1 was administered 1 mL / well of culture 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 levels of each example to the expression level of Comparative Example 1 (without added components) was calculated, and this value represents the change in expression levels due to exosomes in each example, as 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 quantities by dermal fibroblasts. Exosomes secreted from adipose tissue-derived mesenchymal stem cells cultured in the presence of glycogen and tetrapeptide were taken up even more in large quantities by dermal fibroblasts. When dermal fibroblasts from Example 2 were observed, stained exosomes, shown in white, were observed (Figure 1), whereas no stained exosomes, shown in white, were observed in dermal fibroblasts from Comparative Example 1. Exosomes secreted from adipose tissue-derived mesenchymal stem cells cultured in the presence of glycogen improved the proliferative capacity of dermal fibroblasts. Exosomes secreted from adipose tissue-derived mesenchymal stem cells cultured in the presence of glycogen and tetrapeptide further improved the proliferative capacity of dermal 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 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. In other words, they transformed inflammatory M1 macrophages into anti-inflammatory M2 macrophages.

[0061] [Rating 5: Suppression of cellular senescence by exosomes] Exosomes extracted in Test 1 were seeded at 3,500 cells / well in a 96-well plate (Corning Cell Bind) and cultured for 24 hours at 37°C under 5% carbon dioxide. Normal human dermal fibroblasts (Kurabo Industries Ltd., passage P33) were then administered 100 μL / well. After 72 hours of culture, the degree of cellular senescence was measured using β-Galactosidase as an indicator with the Cellular Senescence Plate Assay Kit (DOJINDO). The ratio of β-Galactosidase activity in Example 2 to that of Comparative Example 1 (without added components) was calculated, and this value was used as the value indicating the cellular senescence inhibitory effect of Example 2, as shown in Table 4 below. A smaller value indicates a higher cellular senescence inhibitory effect. The staining properties of β-Galactosidase were also observed under a microscope (Figure 2).

[0062] [Rating 6: Collagen production is promoted by exosomes] Exosomes extracted in Test 1 above were seeded at 50,000 cells / well in a 12-well plate (Corning Cell Bind) and cultured for 24 hours at 37°C under 5% carbon dioxide. Normal human dermal fibroblasts (Kurabo Industries Ltd., passage number P33) were then administered 1 mL / well. After 72 hours of culture, the amount of type I collagen expressed in the culture supernatant was measured using Human Procollagen I alpha 1 DuoSet ELISA (R&D Corporation). The ratio of the collagen expression amount in Example 2 to the collagen expression amount in Comparative Example 1 (without added components) was calculated, and this value was defined as the collagen production promoting ability of Example 2, as shown in Table 4 below.

[0063] [Evaluation 7: Exosome-mediated promotion of elastin production] Exosomes extracted in Test 1 above were seeded at 2,000,000 cells / well in a T25 cell culture flask (Corning Cell Bind) and cultured for 24 hours at 37°C under 5% carbon dioxide. Normal human dermal fibroblasts (Kurabo Industries Ltd., passage number P33) were then administered 5 mL / well. After 72 hours of culture, elastin was extracted from the cells using Fastin Elastin Assay (Biocolor), and the amount of intracellular α-elastin was measured by colorimetric method. The ratio of the amount of elastin in Example 2 to the amount of elastin in Comparative Example 1 (without added components) was calculated, and this value was defined as the elastin production promoting ability of Example 2, as 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 cellular senescence, and promoted collagen and elastin production in dermal fibroblasts. Dermal fibroblasts from Comparative Example 1 showed high staining for β-Galactosidase, while dermal fibroblasts from Example 2 showed low staining for β-Galactosidase (Figure 2). [Industrial applicability]

[0066] The present invention provides a new type of cosmetic composition that excels in cosmetic effects such as anti-aging effects. Specifically, the exosome secretion promoter of the present invention promotes the secretion of exosomes from cells, particularly adipose tissue-derived mesenchymal stem cells. The secreted exosomes are then taken up by skin tissue, resulting in cosmetic effects such as suppression 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 on cells, that is, it can change the phenotype of inflammatory M1 macrophages into anti-inflammatory M2 macrophages. Therefore, the exosome secretion promoter of the present invention is suitably used in products aimed at anti-aging of the skin, skin regeneration, and improvement of moisturizing properties. More specifically, it can be expected to improve skin dullness, restore skin firmness, improve skin elasticity, and reduce the appearance of pores. It also has effects on improving aging symptoms such as dryness, improving skin tone, and whitening the skin.

Claims

1. It contains glycogen and tetrapeptide, The total number of amino acids in the above tetrapeptide is 4. An exosome secretion promoter wherein the above-mentioned tetrapeptide is a peptide represented by the amino acid sequence (Leu-Glu-His-Ala) of SEQ ID NO: 1 or a salt thereof.

2. An exosome secretion promoter according to claim 1, which promotes exosome secretion in adipose-derived mesenchymal stem cells.

3. The exosome secretion promoter according to claim 1 or 2, further comprising one or more selected from the group consisting of Centella asiatica leaf extract, Vaccinium bilberry leaf extract, Glycyrrhiza glabra leaf extract, and Soy protein extract.

4. An exosome secretion promoter according to any one of claims 1 to 3, which has an effect of suppressing cellular senescence by exosomes.

5. It contains glycogen and tetrapeptide, The total number of amino acids in the above tetrapeptide is 4. The above tetrapeptide is a peptide represented by the amino acid sequence (Leu-Glu-His-Ala) of SEQ ID NO: 1 or a salt thereof, which is an exosome uptake promoter into cells.

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