Method for evaluating degree of inhibition of melanin production

Abstract

To provide a means for evaluating the degree of suppression of melanin production using a specific miRNA as an index.SOLUTION: (a) isolating exosomes from a biological sample; (b) detecting miR - 6124, miR - 24 - 3p, miR - 12120, miR - 11181 - 3p, miR - 7108 - 3p, miR - 3144 - 5p, miR - 3610, miR - 6795 - 3p, miR - 6853 - 5p, miR - 4518, miR - 583, miR - 3661, miR - 671 - 3p, miR - 520f -, miR - 5p in the exosomes isolated in step (a); Also disclosed is a method comprising the steps of: quantifying the amount of at least one miRNA selected from the group consisting of miR-6850-5p; and evaluating the degree of suppression of melanin production on the basis of the result of the quantification of the miRNA obtained in step (b).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for assessing the degree of inhibition of melanin production. [Background technology]

[0002] The color of human skin and hair is determined by the amount of pigment melanin present in the skin and hair. Melanin is biosynthesized from tyrosine by the enzymes tyrosinase and dopa oxidase in pigment cells (melanocytes) present in the skin and hair bulb. Therefore, if melanin production is enhanced by activation of melanocytes or tyrosinase, the skin will turn brown and the hair will turn black.

[0003] One of the causes of skin blemishes and freckles is the activation of melanocytes due to stimulation of the skin from ultraviolet exposure, hormonal abnormalities, genetic factors, etc. Therefore, skin whitening agents have been developed that inhibit the activity of tyrosinase to suppress melanin production or reduce the amount of melanin produced.

[0004] On the other hand, there is also a high demand for tanning the skin by increasing melanin production, and people have traditionally tanned their skin by sunbathing or by irradiating it with ultraviolet rays indoors, or by using tanning agents.

[0005] Graying of hair is a physiological aging phenomenon in which melanin is reduced due to changes in hair matrix pigment cells, but the mechanism by which this occurs has not yet been fully elucidated, and hair dyeing is currently the main treatment. Therefore, there is a need to develop a method to turn gray hair into black hair.

[0006] Thus, the search for components that act directly or indirectly on melanocytes to change the amount of melanin is extremely useful for whitening skin, preventing or improving skin browning and graying of hair.

[0007] Various studies have been conducted on methods for controlling the amount of melanin in melanocytes. For example, Patent Document 1 discloses a simple method for searching for substances that control the amount of melanin in melanocytes, specifically, that keratinocyte-derived exosomes positively control the cell activity of melanocytes, including their proliferation and melanin production, and that melanin control agents can be evaluated or selected by using the secretion level of exosomes released from keratinocytes as an indicator.

[0008] Exosomes are membrane vesicles surrounded by a lipid bilayer membrane, and were previously thought to function to release unnecessary intracellular components. However, in recent years, it has become clear that many cells, including immune cells and tumor cells, release exosomes, and that these act as important messengers for the exchange of proteins and lipids between secretory cells (exosome-releasing cells) and their target cells (exosome-receiving cells).

[0009] However, there are still many unknowns regarding the effects of exosomes on melanocytes. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Japanese Patent Application Laid-Open No. 2016-103997 Summary of the Invention

[0011] The present inventors have diligently worked to elucidate the effects of exosomes on melanocytes and have found that specific miRNAs in exosomes contribute to the suppression of melanin production. The present invention is based on these findings.

[0012] Therefore, the present invention provides a means for evaluating the degree of suppression of melanin production using a specific miRNA as an indicator.

[0013] According to the present invention, the following inventions are provided. (1) An in vitro method for assessing the degree of inhibition of melanin production, comprising: (a) isolating exosomes from a biological sample; (b) quantifying the amount of at least one miRNA selected from the group consisting of miR-6124, miR-24-3p, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p in the exosomes isolated in step (a); and (c) evaluating the degree of suppression of melanin production based on the miRNA quantification results obtained in step (b); A method comprising: (2) The method according to (1), wherein the biological sample is derived from a human. (3) The method according to (2), wherein the human-derived biological sample is at least one selected from the group consisting of blood, stratum corneum, urine, and saliva. (4) A marker for evaluating the degree of suppression of melanin production, comprising at least one selected from the group consisting of miR-6124, miR-24-3p, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p. (5) A marker for evaluating the degree of inhibition of melanin production, comprising at least one marker selected from the group consisting of miR-6124, miR-24-3p, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p. (6) A method for screening a substance that inhibits melanin production, comprising: (i) contacting a candidate substance with a cell; (ii) isolating exosomes secreted from the cells contacted with the candidate substance; (iii) quantifying the expression level of at least one miRNA selected from the group consisting of miR-6124, miR-24-3p, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p in the exosomes isolated in step (ii); and (iv) determining whether the candidate substance is a substance that suppresses melanin production based on the miRNA quantification results obtained in step (iii); A method comprising: (7) The method according to (6), wherein the cells are Caco-2 cells derived from human intestinal epithelial cells. (8) A composition for promoting miRNA expression, comprising narcissus lily, The composition, wherein the miRNA is at least one selected from the group consisting of miR-6124, miR-24-3p, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p. (9) The composition described in (8), wherein the miRNA is contained in a cell-derived exosome. (10) The composition according to (9), wherein the cells are Caco-2 cells derived from human intestinal epithelial cells. (11) The composition according to any one of (8) to (10), which is a food composition. (12) A cosmetic preparation comprising exosomes containing at least one miRNA selected from the group consisting of miR-6124, miR-24-3p, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p.

[0014] The present invention provides a means for evaluating the degree of inhibition of melanin production using a specific miRNA as an indicator. The present invention also provides a method for screening for substances that inhibit melanin production using a specific miRNA as an indicator. Furthermore, the present invention also provides products obtained by screening. Additionally, the present invention also provides cosmetics containing a specific miRNA. [Brief explanation of the drawings]

[0015] [Figure 1] Figure 1 shows the results of comparing the effects of equol, oxyresveratrol, and narcoyuri on the expression level of miR-24-3p contained in exosomes secreted by Caco-2 cells. [Figure 2] Figure 2 shows the results of comparing the effects of equol, oxyresveratrol, and narcoyuri on the expression level of miR-6850-5p contained in exosomes secreted by Caco-2 cells. [Figure 3] Figure 3 shows the results of comparing the effects of equol, oxyresveratrol, and narcoyuri on the expression level of miR-12120 contained in exosomes secreted by Caco-2 cells. [Figure 4] Figure 4 shows the results of comparing the effects of each inhibitor on the melanin production-inhibiting effect of exosomes secreted by Caco-2. [Figure 5]Figure 5 shows the heat map used to identify seven miRNAs, miR-4518, miR-7108-3p, miR-3144-5p, miR-3610, miR-583, miR-6124, and miR-11181-3p, as having the effect of suppressing melanin production. [Figure 6] Figure 6 shows the heat map used to identify five miRNAs, miR-3661, miR-6795-3p, miR-6853-5p, miR-671-3p, and miR-520f-5p, as having the effect of suppressing melanin production. [Figure 7] Figure 7 shows the results of evaluating the predicted regulatory relationships between miR-6124, miR-11181-3p, miR-24-3p, and miR-12120 and melanin synthesis-related pathways using miRWalk. [Figure 8] FIG. 8 shows the results of evaluating the predicted regulatory relationships between miR-6124, miR-11181-3p, miR-24-3p, and miR-12120 and signaling pathways using the DAVID database. [Figure 9] Figure 9 shows the results of using miRWalk to evaluate the predicted regulatory relationships between miR-7108-3p, miR-3144-5p, miR-4518, miR-3610, and miR-583 and melanin synthesis-related pathways. [Figure 10] FIG. 10 shows the results of evaluating the predicted regulatory relationships between miR-7108-3p, miR-3144-5p, miR-4518, miR-3610, and miR-583 and signaling pathways using the DAVID database. [Figure 11] Figure 11 shows the results of evaluating the predicted regulatory relationships between miR-3661, miR-6795-3p, miR-6853-5p, miR-671-3p, and miR-520f-5p and signal transduction pathways, as well as the oxidative and inflammatory response pathways, using the DAVID database. [Figure 12]Figure 12 shows the results of comparing the effects of narcissus, oxyresveratrol, equol, and astaxanthin on the expression level of miR-6124 contained in exosomes secreted by Caco-2 cells. Specific Description of the Invention

[0016] One embodiment of the present invention provides an in vitro method for assessing the degree of suppression of melanin production, comprising: (a) isolating exosomes from a biological sample; (b) quantifying the amount of at least one miRNA selected from the group consisting of miR-24-3p, miR-6124, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p in the exosomes isolated in step (a); and (c) assessing the degree of suppression of melanin production based on the miRNA quantification results obtained in step (b).

[0017] Generally, melanin is produced by the oxidation of tyrosine by copper ion-dependent tyrosinase in the melanosomes of melanocytes to produce dihydroxyphenylalanine (DOPA), which is then converted to dopaquinone by DOPA-oxidase, which then polymerizes non-enzymatically in melanocytes to dopachrome and indolequinone. The melanin produced in this way is called eumelanin (true melanin or black melanin) and is dark brown in color. In addition to true melanin, other melanins include orange-red pheomelanin (sub-melanin or flesh-colored melanin), which is biosynthesized from dopaquinone and cysteine ​​via dihydrobenzothiazine, and trichochrome.

[0018] In the present invention, the suppression of melanin production is not particularly limited, and may be caused by, for example, a decrease in the cell proliferation activity of melanocytes, a decrease in the number of melanocytes, a decrease in melanin production, a decrease in the expression level of factors involved in melanin production (master regulator of melanin production: Microphthalmia-associated transcription factor (MITF), enzymes involved in melanin production: TYR, TRP1, TRP2, etc.), and their genes, etc.

[0019] The melanin in the present invention is not particularly limited, but is preferably true melanin.

[0020] The miRNAs used in the present invention are not particularly limited, but are preferably human (hsa-miRNA). The sequences of hsa-miR-6124, hsa-miR-24-3p, hsa-miR-6850-5p, hsa-miR-12120, etc. are registered in publicly known databases (e.g., the miRBase database) in association with accession numbers, and those skilled in the art can unambiguously determine the sequences.

[0021] The in vitro method of the present invention for evaluating the degree of inhibition of melanin production comprises the step (a): isolating exosomes from a biological sample.

[0022] In the in vitro method for evaluating the degree of inhibition of melanin production of the present invention, a biological sample is used as a measurement sample. The animal from which this biological sample is derived may be any animal, and is not particularly limited, but is preferably a mammal, for example, a primate such as a human or chimpanzee, a pet animal such as a dog or cat, a livestock animal such as a cow, a horse, a sheep, or a goat, or a rodent such as a mouse or a rat, and more preferably a human. The biological sample derived from a human is not particularly limited, and examples that can be used include blood, stratum corneum, urine, and saliva.

[0023] Therefore, according to one preferred embodiment of the present invention, the measurement sample in the in vitro method for evaluating the degree of inhibition of melanin production is a biological sample derived from a human, and more preferably at least one selected from the group consisting of blood, stratum corneum, urine, and saliva.

[0024] The exosomes (also called exosomes) used in step (a) of the present invention are granular substances with a diameter of approximately 50 to 150 nm secreted from cells, and the cells are not particularly limited. At least a portion of the exosomes used in step (a) of the present invention contains at least one member selected from the group consisting of miR-24-3p, miR-6124, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p, preferably miR-24-3p, miR-6124, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p, and preferably miR-24-3p, miR-6124, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5 At least one miRNA selected from the group consisting of miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, and miR-4518 is included, more preferably at least one miRNA selected from the group consisting of miR-24-3p, miR-6124, miR-12120, and miR-11181-3p, more preferably at least one miRNA selected from the group consisting of miR-24-3p and miR-6124.

[0025] According to one embodiment of the present invention, at least a portion of the exosomes in step (a) of the present invention contains miR-24-3p.

[0026] According to another embodiment of the present invention, at least a portion of the exosomes in step (a) of the present invention contains miR-6124.

[0027] According to another embodiment of the present invention, at least a portion of the exosomes in step (a) of the present invention contains miR-12120.

[0028] According to another embodiment of the present invention, at least a portion of the exosomes in step (a) of the present invention contains miR-11181-3p.

[0029] According to another embodiment of the present invention, at least a portion of the exosomes in step (a) of the present invention contains miR-7108-3p.

[0030] According to another embodiment of the present invention, at least a portion of the exosomes in step (a) of the present invention contains miR-3144-5p.

[0031] According to another embodiment of the present invention, at least a portion of the exosomes in step (a) of the present invention contains miR-3610.

[0032] According to another embodiment of the present invention, at least a portion of the exosomes in step (a) of the present invention contains miR-6795-3p.

[0033] According to another embodiment of the present invention, at least a portion of the exosomes in step (a) of the present invention contains miR-6853-5p.

[0034] According to another embodiment of the present invention, at least a portion of the exosomes in step (a) of the present invention contains miR-4518.

[0035] According to another embodiment of the present invention, at least a portion of the exosomes in step (a) of the present invention contains miR-583.

[0036] According to another embodiment of the present invention, at least a portion of the exosomes in step (a) of the present invention contains miR-3661.

[0037] According to another embodiment of the present invention, at least a portion of the exosomes in step (a) of the present invention contains miR-671-3p.

[0038] According to another embodiment of the present invention, at least a portion of the exosomes in step (a) of the present invention contains miR-520f-5p.

[0039] According to another embodiment of the present invention, at least a portion of the exosomes in step (a) of the present invention contains miR-6850-5p.

[0040] The separation of exosomes in step (a) of the present invention may be carried out using a known method, and is not particularly limited to, for example, ultracentrifugation, polymer precipitation, immunoprecipitation, size exclusion chromatography, tangential flow filtration, ion exchange, etc.

[0041] The in vitro method of the present invention for evaluating the degree of inhibition of melanin production comprises step (b): quantifying the amount of at least one miRNA selected from the group consisting of miR-24-3p, miR-6124, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p in the exosomes isolated in step (a).

[0042] In step (b) of the present invention, the amount of at least one miRNA selected from the group consisting of miR-24-3p, miR-6124, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p can be quantified by, but is not limited to, reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction (Competitive RT-PCR), real-time reverse transcription polymerase chain reaction (Real-time RT-PCR), RNase protection assay (RPA), or the like. This can be done by the Northern blotting assay, Northern blotting, etc.

[0043] The in vitro method for evaluating the degree of inhibition of melanin production of the present invention comprises step (c): evaluating the degree of inhibition of melanin production based on the quantification results of miRNA obtained in step (b).

[0044] As shown in the Examples below, exosomes with a high melanin production inhibitory effect have a high expression level of at least one miRNA selected from the group consisting of miR-24-3p, miR-6124, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p. Therefore, the evaluation in step (c) of the present invention can be performed by, for example, evaluating sample X as having a higher suppression of melanin production when the quantitative value of the miRNA obtained in step (b) for sample X is higher than the quantitative value of the miRNA obtained in step (b) for sample Y.

[0045] According to another embodiment of the present invention, there is provided a marker for evaluating the degree of inhibition of melanin production. This marker refers to a biomarker, and is at least one selected from the group consisting of miR-24-3p, miR-6124, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p, preferably miR-24-3p, miR-6124, miR-12120, miR- It may comprise or consist of at least one selected from the group consisting of miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, and miR-4518, more preferably at least one selected from the group consisting of miR-24-3p, miR-6124, miR-12120, and miR-11181-3p, more preferably at least one selected from the group consisting of miR-24-3p and miR-6124.

[0046] According to one embodiment of the present invention, there is provided a marker for assessing the degree of suppression of melanin production, which marker comprises or consists of miR-24-3p.

[0047] According to another embodiment of the present invention, there is provided a marker for assessing the degree of suppression of melanin production, which comprises or consists of miR-6124.

[0048] According to another embodiment of the present invention, there is provided a marker for evaluating the degree of suppression of melanin production, which marker comprises or consists of miR-12120.

[0049] According to another embodiment of the present invention, there is provided a marker for evaluating the degree of suppression of melanin production, which marker comprises or consists of miR-11181-3p.

[0050] According to another embodiment of the present invention, there is provided a marker for assessing the degree of suppression of melanin production, which marker comprises or consists of miR-7108-3p.

[0051] According to another embodiment of the present invention, there is provided a marker for assessing the degree of suppression of melanin production, which marker comprises or consists of miR-3144-5p.

[0052] According to another embodiment of the present invention, there is provided a marker for evaluating the degree of suppression of melanin production, which comprises or consists of miR-3610.

[0053] According to another embodiment of the present invention, there is provided a marker for assessing the degree of suppression of melanin production, which comprises or consists of miR-6795-3p.

[0054] According to another embodiment of the present invention, there is provided a marker for assessing the degree of suppression of melanin production, which comprises or consists of miR-6853-5p.

[0055] According to another embodiment of the present invention, there is provided a marker for assessing the degree of suppression of melanin production, which comprises or consists of miR-4518.

[0056] According to another embodiment of the present invention, there is provided a marker for evaluating the degree of suppression of melanin production, which comprises or consists of miR-583.

[0057] According to another embodiment of the present invention, there is provided a marker for evaluating the degree of suppression of melanin production, which comprises or consists of miR-3661.

[0058] According to another embodiment of the present invention, there is provided a marker for assessing the degree of suppression of melanin production, which comprises or consists of miR-671-3p.

[0059] According to another embodiment of the present invention, there is provided a marker for assessing the degree of suppression of melanin production, which comprises or consists of miR-520f-5p.

[0060] According to another embodiment of the present invention, there is provided a marker for assessing the degree of suppression of melanin production, which marker comprises or consists of miR-6850-5p.

[0061] According to another embodiment of the present invention, there is provided a method for screening for a substance that inhibits melanin production. The method includes the steps of (i) contacting cells with a candidate substance, (ii) isolating exosomes secreted from the cells contacted with the candidate substance, (iii) quantifying the expression level of at least one miRNA selected from the group consisting of miR-24-3p, miR-6124, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p in the exosomes isolated in step (ii), and (iv) determining whether the candidate substance is a substance that suppresses melanin production based on the miRNA quantification results obtained in step (iii).

[0062] The term "substance" as used herein refers to a substance that can exert a desired physiological effect on a subject on its own, and is included in a composition in the hope that the composition will also exert the desired physiological effect. Furthermore, although a substance may exert a desired physiological effect on its own, its desired physiological effect may be additively or synergistically enhanced when used in combination with other substances. Substances of the present invention include, but are not limited to, small molecules, low-molecular-weight compounds, polymeric compounds, nucleic acid molecules, proteins, peptides (cyclic peptides), and the like.

[0063] The method of the present invention for screening for a substance that inhibits melanin production comprises step (i): contacting a candidate substance with a cell.

[0064] The cells used in step (i) of the present invention are not particularly limited, but are preferably Caco-2 cells derived from human intestinal epithelial cells.

[0065] The method of the present invention for screening for a substance that inhibits melanin production comprises step (ii): isolating exosomes secreted from cells that have been contacted with a candidate substance.

[0066] At least a portion of the exosomes in step (ii) of the present invention contains at least one selected from the group consisting of miR-24-3p, miR-6124, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p, preferably miR-24-3p, miR-6124, At least one miRNA selected from the group consisting of miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, and miR-4518 is included, more preferably at least one miRNA selected from the group consisting of miR-24-3p, miR-6124, miR-12120, and miR-11181-3p, more preferably at least one miRNA selected from the group consisting of miR-24-3p and miR-6124.

[0067] According to one embodiment of the present invention, the exosomes in step (ii) of the present invention contain miR-24-3p at least in part.

[0068] According to another embodiment of the present invention, at least a portion of the exosomes in step (ii) of the present invention contains miR-6124.

[0069] According to another embodiment of the present invention, at least a portion of the exosomes in step (ii) of the present invention contains miR-12120.

[0070] According to another embodiment of the present invention, the exosomes in step (ii) of the present invention contain miR-11181-3p at least in part.

[0071] According to another embodiment of the present invention, at least a portion of the exosomes in step (ii) of the present invention contains miR-7108-3p.

[0072] According to another embodiment of the present invention, at least a portion of the exosomes in step (ii) of the present invention contains miR-3144-5p.

[0073] According to another embodiment of the present invention, at least a portion of the exosomes in step (ii) of the present invention contains miR-3610.

[0074] According to another embodiment of the present invention, at least a portion of the exosomes in step (ii) of the present invention contains miR-6795-3p.

[0075] According to another embodiment of the present invention, the exosomes in step (ii) of the present invention contain miR-6853-5p at least in part.

[0076] According to another embodiment of the present invention, at least a portion of the exosomes in step (ii) of the present invention contains miR-4518.

[0077] According to another embodiment of the present invention, at least a portion of the exosomes in step (ii) of the present invention contains miR-583.

[0078] According to another embodiment of the present invention, at least a portion of the exosomes in step (ii) of the present invention contains miR-3661.

[0079] According to another embodiment of the present invention, the exosomes in step (ii) of the present invention contain miR-671-3p at least in part.

[0080] According to another embodiment of the present invention, at least a portion of the exosomes in step (ii) of the present invention contains miR-520f-5p.

[0081] According to another embodiment of the present invention, at least a portion of the exosomes in step (ii) of the present invention contains miR-6850-5p.

[0082] The separation of exosomes in step (ii) of the present invention may be carried out using a known method, and is not particularly limited to, for example, ultracentrifugation, polymer precipitation, immunoprecipitation, size exclusion chromatography, tangential flow filtration, ion exchange, etc.

[0083] The method of the present invention for screening for substances that inhibit melanin production comprises step (iii): quantifying the expression level of at least one miRNA selected from the group consisting of miR-24-3p, miR-6124, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p within the exosomes isolated in step (ii).

[0084] The quantification of the amount of at least one miRNA selected from the group consisting of miR-24-3p, miR-6124, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p in step (iii) of the present invention is not particularly limited, and may be performed by, for example, reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction (Competitive RT-PCR), real-time reverse transcription polymerase chain reaction (Real-time RT-PCR), RNase protection assay (RPA), or the like. This can be done by the Northern blotting assay, Northern blotting, etc.

[0085] The method of the present invention for screening for substances that suppress melanin production comprises step (iv): determining whether the candidate substance is a substance that suppresses melanin production based on the miRNA quantification results obtained in step (iii).

[0086] The determination in step (iv) of the present invention can be made, for example, by determining that the candidate substance is a substance that suppresses melanin production when the quantitative value of miRNA obtained in step (iii) for candidate substance x exceeds a predetermined threshold (e.g., when the quantitative value of miRNA obtained in step (iii) for candidate substance x is significant compared to that of the control group), and by not determining that the candidate substance is a substance that suppresses melanin production when the quantitative value is lower than the predetermined threshold.

[0087] Another embodiment of the present invention provides an agent for promoting miRNA expression comprising Narcoyle japonica, wherein the miRNA is at least one selected from the group consisting of miR-24-3p, miR-6124, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p, preferably miR-24-3p, miR-6124, or miR-12120. , miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, and miR-4518, more preferably at least one selected from the group consisting of miR-24-3p, miR-6124, miR-12120, and miR-11181-3p, more preferably at least one selected from the group consisting of miR-24-3p and miR-6124. Furthermore, the miRNA is preferably miRNA contained in cell-derived exosomes, more preferably miRNA contained in exosomes derived from Caco-2 cells derived from human intestinal epithelial cells. Such a miRNA expression promoter comprising Narcoyuri may be a composition for promoting miRNA expression, comprising Narcoyuri.

[0088] Another embodiment of the present invention provides an agent for promoting miRNA expression, comprising Narcoylium nigricans, wherein the miRNA is miR-24-3p.

[0089] Another embodiment of the present invention provides an agent for promoting miRNA expression, comprising Narcoylium nigricans, wherein the miRNA is miR-6124.

[0090] Another embodiment of the present invention provides an agent for promoting miRNA expression, comprising Narcoylium nigricans. The miRNA is miR-12120.

[0091] Another embodiment of the present invention provides an agent for promoting miRNA expression comprising Narcolius nigra, wherein the miRNA is miR-11181-3p.

[0092] Another embodiment of the present invention provides an agent for promoting miRNA expression, comprising Narcoylium narcissiflora, wherein the miRNA is miR-7108-3p.

[0093] Another embodiment of the present invention provides an agent for promoting miRNA expression, comprising Narcoylium nigricans, wherein the miRNA is miR-3144-5p.

[0094] Another embodiment of the present invention provides an agent for promoting miRNA expression comprising Narcolius nigra, wherein the miRNA is miR-3610.

[0095] Another embodiment of the present invention provides an agent for promoting miRNA expression, comprising Narcoylium nigricans, wherein the miRNA is miR-6795-3p.

[0096] Another embodiment of the present invention provides an agent for promoting miRNA expression, comprising Narcoylium nigricans, wherein the miRNA is miR-6853-5p.

[0097] Another embodiment of the present invention provides an agent for promoting miRNA expression, comprising Narcoylium nigricans, wherein the miRNA is miR-4518.

[0098] Another embodiment of the present invention provides an agent for promoting miRNA expression, comprising Narcoylium nigricans, wherein the miRNA is miR-583.

[0099] Another embodiment of the present invention provides an agent for promoting miRNA expression comprising Narcolius narcissiflora, wherein the miRNA is miR-3661.

[0100] Another embodiment of the present invention provides an agent for promoting miRNA expression, comprising Narcoylium nigricans, wherein the miRNA is miR-671-3p.

[0101] Another embodiment of the present invention provides an agent for promoting miRNA expression, comprising Narcoylium nigricans, wherein the miRNA is miR-520f-5p.

[0102] Another embodiment of the present invention provides an agent for promoting miRNA expression, comprising Narcoylium nigricans, wherein the miRNA is miR-6850-5p.

[0103] In the present invention, "narcoyle" refers to "Polygonatum falcatum," a perennial plant belonging to the genus Polygonatum of the family Liliaceae. The "narcoyle" used in the present invention may be either the whole plant or a part of it, but the rhizome is preferred as the part used, and it can be used as is or dried (e.g., dried powder) as needed. Furthermore, the "narcoyle" used in the present invention may also be a narcoyle extract produced using the whole narcoyle or a part of it.

[0104] The method for producing the narcoyle extract of the present invention is not particularly limited and can be produced according to a method commonly used in the art, for example, by extracting the raw material of the extract. The extraction method is not limited to, but includes, for example, hot water extraction, ultrasonic extraction, filtration, reflux extraction, solvent (e.g., ethanol) extraction, etc. These methods may be carried out alone or in combination of two or more. In addition, to obtain a highly pure extract, the extract may be further extracted one or more times using the same method.

[0105] The type of solvent used to produce the Narcoyle extract of the present invention is not particularly limited, and any solvent known in the art may be used as long as it can produce an extract having the desired effects of the present invention. Examples of such solvents include, but are not limited to, water, alcohols having 1 to 4 carbon atoms, ethyl acetate, acetone, chloroform, etc., and two or more of these may be used in combination.

[0106] If narcissus (including dried powder, extract, etc.) is commercially available, it may be used as long as it has the desired effect of the present invention.

[0107] The composition for promoting miRNA expression comprising Narcoyle of the present invention is preferably, but not limited to, a food composition. When used as a food composition, known ingredients commonly used in food compositions can be further incorporated. Examples of such ingredients include, but are not limited to, foods such as sugars, fruits, fruit juices, vegetables, and meats, as well as food additives such as sweeteners, high-sugar sweeteners (aspartame, sucralose, glycyrrhizin, saccharin, stevia, dulcin, trichlorosucrose, thaumatin, acesulfame potassium, etc.), flavorings, colorings, acidulants, antioxidants, emulsifiers, preservatives, and stabilizers. Furthermore, when used as a food composition, it may be formulated as a health-functioning food, such as a food with nutrient claims, a food for specified health use, or a food with functional claims. It may also be manufactured as a therapeutic diet (i.e., a diet intended to achieve a therapeutic purpose, or a diet prepared based on a doctor's dietary prescription and a nutritionist's menu), a dietary therapy diet, or a care diet.

[0108] The amount of narcoyle used in the composition of the present invention for promoting miRNA expression, which comprises narcoyle, can be determined appropriately depending on the application. For example, when the composition is made into a pharmaceutical composition or food composition to be ingested by humans, the amount used relative to the total amount of the composition is preferably 10 mg to 1 g, and more preferably 100 mg to 500 mg, in dry weight. Furthermore, when the composition is used in the in vitro methods or screening methods of the present invention, the composition is used so that the final concentration of narcoyle in the medium containing exosome-secreting cells is preferably 1 μg / mL to 5000 μg / mL, and more preferably 10 μg / mL to 500 μg / mL, in dry weight.

[0109] According to another embodiment of the present invention, there is provided a cosmetic preparation containing exosomes. The exosomes contain at least one exosome selected from the group consisting of miR-24-3p, miR-6124, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p, preferably miR-24-3p, miR-6124, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p. It comprises at least one miRNA selected from the group consisting of miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, and miR-4518, more preferably at least one miRNA selected from the group consisting of miR-24-3p, miR-6124, miR-12120, and miR-11181-3p, more preferably at least one miRNA selected from the group consisting of miR-24-3p and miR-6124.

[0110] According to another embodiment of the present invention, there is provided a cosmetic preparation containing exosomes, the exosomes containing miR-24-3p.

[0111] According to another embodiment of the present invention, there is provided a cosmetic preparation containing exosomes, the exosomes containing miR-6124.

[0112] According to another embodiment of the present invention, there is provided a cosmetic preparation containing exosomes, the exosomes containing miR-12120.

[0113] According to another embodiment of the present invention, there is provided a cosmetic preparation containing exosomes, the exosomes containing miR-11181-3p.

[0114] According to another embodiment of the present invention, there is provided a cosmetic preparation containing exosomes, the exosomes containing miR-7108-3p.

[0115] According to another embodiment of the present invention, there is provided a cosmetic preparation containing exosomes, the exosomes containing miR-3144-5p.

[0116] According to another embodiment of the present invention, there is provided a cosmetic preparation containing exosomes, the exosomes containing miR-3610.

[0117] According to another embodiment of the present invention, there is provided a cosmetic preparation containing exosomes, the exosomes containing miR-6795-3p.

[0118] According to another embodiment of the present invention, there is provided a cosmetic preparation containing exosomes, the exosomes containing miR-6853-5p.

[0119] According to another embodiment of the present invention, there is provided a cosmetic preparation containing exosomes, the exosomes containing miR-4518.

[0120] According to another embodiment of the present invention, there is provided a cosmetic preparation containing exosomes, the exosomes containing miR-583.

[0121] According to another embodiment of the present invention, there is provided a cosmetic preparation containing exosomes, the exosomes containing miR-3661.

[0122] According to another embodiment of the present invention, there is provided a cosmetic preparation containing exosomes, the exosomes containing miR-671-3p.

[0123] According to another embodiment of the present invention, there is provided a cosmetic preparation containing exosomes, the exosomes containing miR-520f-5p.

[0124] According to another embodiment of the present invention, there is provided a cosmetic preparation containing exosomes, the exosomes containing miR-6850-5p. [Example]

[0125] The present invention will be described in detail based on the following examples, but the present invention is not limited to these examples. Contents are expressed in mass % unless otherwise specified.

[0126] Cultivation of human colon cancer-derived cells (Caco-2 cells) The human intestinal epithelium model used was Caco-2 cells derived from human colon cancer. Caco-2 cells were inactivated (heated in a 56°C water bath for 30 minutes) and subcultured in DMEM medium containing 10% Fetal Bovine Serum (FBS) in a cell culture dish (Corning) at 37°C in the presence of 5% CO2. DMEM medium was prepared by dissolving 4.75 g of Dulbecco's Modified Eagle's Medium "Nissui" (Nissui Pharmaceutical) in 470 mL of Milli-Q water and sterilizing the solution under autoclaving. This was supplemented with 10 mL of 0.2 M (+)-glutamine (Fujifilm Wako Pure Chemical Industries, Ltd.), 10 mL of 10% NaHCO3 (Fujifilm Wako), 1 mL of 50,000 U / mL penicillin (Meiji Seika Pharma), and 1 mL of 0.05 mg / mL streptomycin (Meiji Seika Pharma), and then filled to 500 mL with sterile water. The 0.2 M (+)-glutamine was filter-sterilized, and the 10% NaHCO3 was autoclaved.

[0127] Culture of hTERT-immortalized dermal melanocytes The human melanocyte model used was hTERT-immortalized dermal melanocytes (human (Homo sapiens), obtained from ATCC). hTERT-immortalized dermal melanocytes were cultured in Dermal Cell Basal Medium (obtained from ATCC) as the basal medium, supplemented with a Melanocyte Growth Kit (obtained from ATCC), 1 mL of 50,000 U / mL penicillin (Meiji Seika Pharma), and 1 mL of 0.05 mg / mL streptomycin (Meiji Seika Pharma). FBS was added to a concentration of 20%, and the cells were subcultured in tissue culture dishes (Corning) at 37°C and 5% CO2.

[0128] Samples and their preparation Three types of polyphenols (equol and oxyresveratrol (both manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and dried powder of Narcoyle (manufactured by Nippon Powder Pharmaceuticals Co., Ltd.)) were used as samples. These polyphenols were prepared to 10 mM using dimethyl sulfoxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereafter referred to as DMSO). 10 mg of dried powder of Narcoyle was dissolved in 1 mL of DMSO.

[0129] Purification of Caco-2-derived exosomes 1.4 × 10 Caco-2 cells in a 10 mL dish 6 Cells were seeded at the desired cell number per dish. After seeding, the cells were cultured in DMEM medium containing 10% Exosome-depleted FBS Media Supplement Heat Inactivated (System Biosciences). After 24 hours of culture, the prepared equol, oxyresveratrol, or narcoyuri dried powder was added to a final concentration of 10 μM or 10 μg / mL. 24 hours later, the culture supernatant was collected, and exosomes secreted by Caco-2 cells were purified using the following method.

[0130] The collected culture supernatant was centrifuged at 300 × g for 5 minutes to remove cells. The supernatant was then transferred to another tube and centrifuged at 1,200 × g for 20 minutes to remove cell debris. The supernatant was then transferred to another tube and centrifuged at 10,000 × g for 3 minutes to remove extracellular vesicles larger than exosomes. The culture supernatant, from which cells and large extracellular vesicles had been removed, was concentrated approximately 40-fold using a centrifugal ultrafiltration unit (AmIcon U1tra-15100K, Merck Millipore) with a molecular weight cutoff of 100,000. Exosomes were purified from the concentrated culture supernatant using the Mag Capture Exosome Isolation Kit PS Ver. 2 (Fujifilm Wako).

[0131] First, the buffers to be used in each step were prepared. 0.55 mL of Exosome Immobilizing / Washing Buffer (10x) and 495 mL of purified water were added to a 5 mL centrifuge tube, and 11 μL of Exosome Binding Enhancer (500x) was further added to prepare Exosome Immobilizing / Washing Buffer (1x). 15 μL of Exosome Elution Buffer (10x) and 135 μL of purified water were added to a 1.5 mL microtube not included in the kit to prepare Exosome Elution Buffer (1x). The prepared solutions were mixed thoroughly.

[0132] Next, Exosome Capture immobilized beads were prepared. 60 μL of Biotin Capture Magnetic Beads were transferred to the provided 1.5 mL Reaction Tube, and 500 μL of Exosome Capture Immobilizing Buffer (1x) was added. The beads were then suspended using a vortex mixer. The tube was spun down and placed on a magnetic stand for 1 minute. Once the magnetic beads had completely adhered to the tube wall, the supernatant was removed with a pipette.

[0133] Next, 500 μL of Exosome Capture Immobilizing Buffer (1×) and 10 μL of Biotin-labeled Exosome Capture were added to the tube, which was then removed from the magnetic stand and suspended using a vortex mixer. The tube was then incubated at room temperature for 10 minutes, with end-over-end mixing using a rotary centrifuge. The tube was then spun down and placed back on the magnetic stand for 1 minute. Once the magnetic beads had completely adhered to the tube wall, the supernatant was removed with a pipette.

[0134] Repeat operation 1 500 μL of Exosome Capture Immobilizing Buffer (1x) was added to the tube, which was then removed from the magnetic stand and suspended using a vortex mixer. The tube was then spun down and placed back on the magnetic stand for 1 minute. Once the magnetic beads had completely adhered to the tube wall, the supernatant was removed with a pipette. This procedure was repeated once more.

[0135] The above procedure completed the preparation of Exosome Capture-immobilized beads. Next, the Exosome Capture-immobilized beads were reacted with the culture supernatant concentrated as described above. The 40-fold concentrated culture supernatant was transferred to a tube, and 1 / 500th the volume of Exosome Binding Enhancer (500x) was added to the concentrated culture supernatant and suspended using a vortex mixer. The tube was spun down, and the sample was transferred to a tube containing Exosome Capture-immobilized beads (1.5 mL Reaction Tube) and mixed using a vortex mixer. The reaction was allowed to proceed for over 1 hour at room temperature while mixing by inversion using a rotary vortex mixer. The 1.5 mL Reaction Tube was then spun down and placed on a magnetic stand and left to stand for approximately 1 minute. After the magnetic beads had completely adhered to the tube wall, the supernatant was removed with a pipette, leaving the exosome-bound beads.

[0136] Next, the exosome-bound beads were washed. 1 mL of Exosome Immobilizing / Washing Buffer (1x) was added to the 1.5 mL Reaction Tube containing the exosome-bound beads and mixed using a vortex mixer. The 1.5 mL Reaction Tube was spun down, then placed on a magnetic stand and left to stand for approximately 1 minute. After the magnetic beads had completely adhered to the tube wall, the supernatant was removed with a pipette.

[0137] Repeat operation 2 1 mL of Exosome Immobilizing / Washing Buffer (1x) was added and suspended using a vortex mixer. The 1.5 mL Reaction Tube was spun down and placed on a magnetic stand. After leaving it for approximately 1 minute for the magnetic beads to completely adhere to the tube wall, the supernatant was removed. This step 2 was repeated once more.

[0138] These steps yielded washed exosome-bound beads. Exosome elution was performed as follows: 50 μL of Exosome Elution Buffer (1X) was added to the 1.5 mL Reaction Tube containing the washed exosome-bound beads, removed from the magnetic stand, and suspended using a vortex mixer. The tube was spun down, placed on the magnetic stand, and left to stand for 1 minute until the magnetic beads completely adhered to the tube wall. The supernatant was then transferred to a new, sterile 15 mL tube. An additional 50 μL of Exosome Elution Buffer (1X) was added to the magnetic beads remaining in the 1.5 mL Reaction Tube, removed from the magnetic stand, suspended using a vortex mixer, spun down, placed on the magnetic stand, and left to stand for 1 minute. After the magnetic beads completely adhered to the tube wall, the supernatant was transferred to a new, sterile 15 mL tube, yielding a total of 100 μL of exosome solution. [Table 1]

[0139] Next, the exosome content of the prepared exosome solution was quantified by measuring the protein concentration using the BCA method. Protein concentration was measured using the Micro BCA Protein Assay Kit (Thermo Fisher Scientific). 32 μL of the exosome solution was diluted 10-fold and used for measurement. Standard curve standards were prepared by serially diluting BSA with PBS from 0 to 2000 μg / mL. Two wells of a 96-well plate (Thermo Fisher Scientific) were used as one assay, and 150 μL of the standard and sample were added to each well in this order. Next, 150 μL of a mixture of Micro Reagent A, Micro Reagent B, and Micro Reagent C (included in the Micro BCA Protein Assay Kit) in a 25:24:1 ratio was added to each well. The reaction was incubated at 37°C for 2 hours in the dark, and the absorbance at 562 nm was measured using a spectrophotometer (Sunrise™, TECAN). Quantitation was performed based on a standard curve obtained by measuring the albumin standard ampules, and the protein concentration was determined.

[0140] Observation of melanin production by adding Caco-2-derived exosomes hTERT-immortalized dermal melanocytes were added to a final concentration of 8.0 × 10 5 Cells were seeded onto a 96-well plate at 100 cells / mL and cultured for 24 hours. Purified Caco-2-derived exosomes were added at 90 ng / well. α-MSH was also added at 100 nM. After 96 hours of culture, intracellular melanin production was observed using an EVOS microscope (EVOSM5000, Thermo Fisher Scientific). The results confirmed that melanin production induced by α-MSH treatment was suppressed by the addition of Caco-2-derived exosomes treated with various samples.

[0141] Evaluation of miRNA expression levels involved in melanin production in Caco-2-derived exosomes Next, we evaluated the expression levels of miRNAs involved in melanin production in Caco-2-derived exosomes. First, RNA was purified according to the miRNeasy Serum / P1asma Kit (QIAGEN) product protocol. 30 μL of the sample Caco-2-derived exosome solution was mixed with 170 μL of Exosome Elution Buffer (1x) in a 1.5 mL sample tube to prepare a total volume of 200 μL of exosome mixture. Next, 1 mL of QIAzol Lysis Reagent was added, vortexed, and spun down. After allowing to stand at room temperature for 5 minutes, 200 μL of chloroform was added and vortexed for 15 seconds. After allowing to stand at room temperature for 3 minutes, the mixture was centrifuged at 12,000 × g for 15 minutes at room temperature. After centrifugation, 590 μL of the aqueous layer alone was transferred to a new 1.5 mL tube (not provided) and 900 μL of 100% ethanol was added. A 2 mL collection tube (included) and spin column were assembled, and 700 μL of the solution was added. The column was centrifuged at 8,000 × g for 15 seconds at room temperature. After centrifugation, the liquid in the collection tube was discarded, the remaining 800 μL was added, and the column was centrifuged again at 8,000 × g for 15 seconds at room temperature. The liquid in the collection tube was discarded, 700 μL of Buffer RWT was added, and the column was centrifuged at 8,000 × g for 15 seconds at room temperature. The liquid in the collection tube was discarded, 500 μL of Buffer RPE was added, and the column was centrifuged at 8,000 × g for 15 seconds at room temperature. The liquid in the collection tube was discarded, and 500 μL of 80% ethanol was added, and the column was centrifuged at 8,000 × g for 2 minutes at room temperature. The liquid in the collection tube was discarded, and the column was reassembled in a new 2 mL collection tube (included). The column's lid was opened, and the column was centrifuged at 20,000 × g for 5 minutes at room temperature to completely dry out the ethanol. The liquid in the collection tube was discarded, and the column was reassembled in a new 1.5 mL tube (included). 14 μL of RNase-free water was added to the center of the column membrane, and the column was centrifuged at 20,000 × g for 1 minute at room temperature. The resulting eluate (approximately 12 μL) was used as the RNA solution.

[0142] Additionally, cDNA synthesis was performed according to the MirX miRNA quantification kit (Mir-X™ miRNA First-Strand Synthesis Kit, Takara Bio Inc.) product protocol. 5 μL of mRQ Buffer (2x), 3.75 μL of RNA sample, and 1.25 μL of mRQ Enzyme were mixed in a 0.2 mL sample tube to prepare a total volume of 10 μL. The mixture was then heat-treated at 37°C for 1 hour and 85°C for 5 minutes. The mixture was diluted 6-fold with ddH2O to a volume suitable for subsequent experiments and used as a template for subsequent PCR.

[0143] Furthermore, quantitative real-time PCR was performed using the prepared cDNA as a template according to the MirX miRNA quantification kit (Mir-X™ miRNA First-Strand Synthesis Kit, manufactured by Takara Bio Inc.) product protocol. 34.2 μL of ddH2O, 1.8 μL each of primers (forward and reverse) diluted to 10 μM, 2.0 μL of template cDNA, and 45 μL of TB Green Advantage Premix (2x) were mixed into a 0.2 mL sample tube and suspended thoroughly.

[0144] The primers used were U6 (forward / reverse) primers for the housekeeping gene, 10 μM miRNA-specific primers (forward primers specific to each miR) for the target gene, and 10 μM mRQ3' primer (a reverse primer common to each miR included in the Mir-X™ miRNA qRT-PCR TB Green Kit, manufactured by Clontech) included in the kit. The mRQ3' primer was designed based on the full-length miRNA sequence and can be used as a miRNA-specific primer for qPCR against the entire mature miRNA sequence (typically 21–25 bases). Subsequently, 25 μL of each solution was added to three wells of a 96-well plate, and quantitative RT-PCR was performed using a Thermal Cycle Dicer Real Time system (Takara Bio). The reaction conditions were 95°C for 10 seconds (1 cycle), 95°C for 5 seconds (40 cycles), 60°C for 20 seconds (40 cycles), and 95°C for 60 seconds (40 cycles), 60°C for 30 seconds (40 cycles), and 95°C for 30 seconds (40 cycles). Detection was performed by FAM. U6 primers were used for the calibration curve. The relative gene expression levels were calculated by dividing the measured values ​​by the U6 expression levels. The sequences of the miRNA-specific primers for the target genes are shown in Table 2 below, and the sequences of the U6 primers for the housekeeping genes are shown in Table 3 below.

[0145] [Table 2]

[0146] [Table 3]

[0147] The results for miR-24-3p are shown in Figure 1. In Figure 1, Test Example 1 shows the relative expression level of the miR-24-3p gene in exosomes derived from Caco-2 cells without polyphenols. Test Example 2 shows the relative expression level of the miR-24-3p gene in exosomes derived from Caco-2 cells with oxyresveratrol. Test Example 3 shows the relative expression level of the miR-24-3p gene in exosomes derived from Caco-2 cells with equol. Test Example 4 shows the relative expression level of the miR-24-3p gene in exosomes derived from Caco-2 cells with narcoli (a kind of lily of the valley) added. Multiple comparisons were performed using one-way analysis of variance (ANOVA) to compare each sample-treated group with the control group using the Dunnett method. A p value of less than 0.05 was considered a significant increase in expression. In Figure 1, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. It was shown that the expression of miR-24-3p was significantly increased in exosomes when oxyresveratrol, equol, and narcoyle were added to Caco-2 cells.

[0148] The results for miR-6850-5p are shown in Figure 2. In Figure 2, Test Example 1 shows the relative expression level of the miR-6850-5p gene in exosomes derived from Caco-2 cells without polyphenols. Test Example 2 shows the relative expression level of the miR-6850-5p gene in exosomes derived from Caco-2 cells with oxyresveratrol. Test Example 3 shows the relative expression level of the miR-6850-5p gene in exosomes derived from Caco-2 cells with equol. Test Example 4 shows the relative expression level of the miR-6850-5p gene in exosomes derived from Caco-2 cells with narcoli (a kind of lily of the valley) added. Multiple comparisons were performed using one-way analysis of variance (ANOVA) to compare each sample-treated group with the control group using the Dunnett method. A p value of less than 0.05 was considered a significant increase in expression. In Figure 2, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. It was shown that the expression of miR-6850-5p was significantly increased in exosomes when oxyresveratrol, equol, and narcoyuri were added to Caco-2 cells.

[0149] The results for miR-12120 are shown in Figure 3. In Figure 3, Test Example 1 shows the relative expression level of the miR-12120 gene in exosomes derived from Caco-2 cells without polyphenols. Test Example 2 shows the relative expression level of the miR-12120 gene in exosomes derived from Caco-2 cells with oxyresveratrol. Test Example 3 shows the relative expression level of the miR-12120 gene in exosomes derived from Caco-2 cells with equol. Test Example 4 shows the relative expression level of the miR-12120 gene in exosomes derived from Caco-2 cells with narcoyle. Multiple comparisons were performed using one-way analysis of variance. Multiple comparisons of each sample-treated group against the control group were performed using the Dunnett method, and a p value of less than 0.05 was considered a significant increase in expression. In Figure 3, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. It was shown that when oxyresveratrol, equol, and narcoyle were added to Caco-2, the expression of miR-12120 in exosomes was significantly increased or tended to be increased.

[0150] Effects of miRNA inhibitors on melanin production in a melanin-containing skin model Next, we evaluated the effect of miRNA inhibitors on melanin production in a melanin-containing skin model. First, a melanocyte-containing three-dimensional skin model (MEL-300, Kurabo Industries, Ltd.) was purchased and cultured in a 37°C, 5% CO2 incubator according to standard methods. Four wells (n = 4) were designed for each condition. EPI-100NMMI13 culture medium was used. This medium contained appropriate amounts of melanin-stimulating factors bFGF, α-MSH, and KGF. The culture medium was changed every other day, and miRNA inhibitors (miR-24-3p inhibitor, miR-6850-5p inhibitor, and negative control inhibitor) were dissolved to the specified concentrations each time the culture medium was changed. Each agent was applied to the bottom side of the skin model, and the culture was carried out every other day according to the specified method. The culture was continued for 16 days after the start of culture.

[0151] Data analysis was performed assuming that the melanin level in the control group was the reference (melanin suppression rate (%) = 0%). As a negative control, L-(-) ascorbic acid (Kanto Chemical) was prepared at a concentration of 100 μg / mL in the culture medium. Caco-2-derived exosomes were prepared to evaluate the effects of adding miRNA inhibitors. Conventional Caco-2 culture before exosome collection was performed as described above. Cells were subcultured in DMEM medium containing 10% Fetal Bovine Serum (FBS) that had been inactivated (heated in a 56°C water bath for 30 minutes) at 37°C and 5% CO2 in a cell culture dish (Corning). However, to remove FBS-derived exosomes, the medium was replaced with serum-free DMEM medium before culture medium collection, and the cells were maintained in a confluent state.

[0152] The culture medium was then collected and used as an exosome-extracted and purified sample. Exosome extraction and purification were performed using the PureExo® Exosome Isolation Kit for Cell Culture Media (Cosmo Bio). Specifically, 4 mL of the collected culture supernatant (for one reaction) was centrifuged at 3000 × g for 15 minutes at 4°C to remove cells and debris. Approximately 4 mL of the clear supernatant (cell-free medium) was transferred to a new glass tube 1 and stored on ice. Solutions A, B, and C were added to glass tube 2 in the following order to prepare a mixture (15 mL total, 0.25 mL of solution A, 0.25 mL of solution B, and 1 mL of solution C, prepared as needed). Glass tube 2 was vortexed for 10 seconds, and 1.5 mL of the mixture from glass tube 2 was added to glass tube 1 (4 mL of cell-free medium). Glass tube 1 was tightly capped and gently inverted at least 10 times to mix well. The mixture was then incubated at 4°C for 30 minutes. After removing only the upper, colored layer of the three-layer mixture, the mixture was spun at 5,000 × g for 3 minutes to separate into three new layers, the middle layer of which was used as the exosome fraction.

[0153] The top layer of this newly separated solution was removed with a pipette. The pipette tip was inserted to the bottom of the tube, completely removing the bottom colorless layer. Only the remaining middle layer was left in the tube. This middle layer was transferred to a new 0.5 mL microcentrifuge tube and spun at 5000 × g for 3 minutes. The remaining liquid in the upper layer was removed. The tube was left to air dry at room temperature with the cap open for 5 minutes. 100 μL of 1x PBS, four times the volume of the remaining exosome fraction pellet, was added to the 0.5 mL tube. The fluff pellet was resuspended by vigorously pipetting up and down 40 times. The 0.5 mL tube was shaken at high speed on a horizontal shaker for 3 minutes, after which the tube was vigorously pipetted up and down 10 times.

[0154] The supernatant was transferred to a PureExo® column (included), and the column was spun at 1000 × g for 5 minutes to collect the flow-through (exosomes suspended in PBS). The presence of exosomes in the isolated exosome fraction was qualitatively confirmed using a NanoSight NS3000 (Quantum Design Japan), and the amount of exosomes was quantified using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific).

[0155] The medium of the melanin-containing skin model was supplemented every other day with Caco-2-derived exosome fraction at a concentration equivalent to 100 ng / mL. The Caco-2-derived exosome fraction contained a certain amount of miRNAs (miR-24-3p, miR-6850-5p, etc.) that contribute to melanin suppression. Inhibitors of these miRNAs were also added every other day at a concentration of 10 nM each. [Table 4]

[0156] Measuring melanin levels After 16 days of culture, the skin models were washed with Dulbecco's PBS(-) and then observed under a microscope. The three-dimensional skin models were then excised from the culture cups and placed in 15 mL tubes, one sample at a time. 200 μL of 1 M NaOH was added to each tube and incubated at 60°C for 16 hours. Then, 100 μL of each sample was transferred to a 96-well plate and measured at 405 nm using an absorbance meter. The melanin inhibition rate (%) was calculated using the following formula (1):

number

[0157] The melanin suppression rate for each sample was calculated using skin models (n=4 per group), and the mean ± standard deviation was calculated. A one-way analysis of variance was used for multiple comparisons between groups. A multiple comparison test for each test example against Test Example 9 was performed using the Dunnett method, with a p value of less than 0.05 being considered a significant increase in melanin or melanin suppression (* indicates p<0.05), and a p value of 0.05 or greater but less than 0.1 being considered a significant tendency toward melanin increase or melanin suppression. The results were as follows:

[0158] First, bird's-eye images of the skin model confirmed that melanin production was progressing in the control group (Test Example 5), while melanin formation was suppressed by the action of melanin reduction in the L-(-) ascorbic acid-containing condition (Test Example 6), confirming the appropriate operation of the test system. Next, compared to the control group, the group receiving the Caco-2-derived exosome fraction and a negative control miRNA inhibitor (Test Example 9) showed approximately 5% melanin suppression. On the other hand, the group receiving the Caco-2-derived exosome fraction and a miR-24-3p inhibitor (Test Example 7) or the group receiving the Caco-2-derived exosome fraction and a miR-6850-5p inhibitor (Test Example 8) showed increased melanin. Statistical analysis showed that melanin levels were significantly higher in Test Example 7 than in Test Example 9, and that melanin levels in Test Example 8 tended to be significantly higher than in Test Example 9 (Figure 4). This suggests that Caco-2-derived exosome fractions, particularly specific miRNAs in exosomes such as miR-24-3p and miR-6850-5p, contribute to melanin suppression in melanogenesis, and that increased expression of miR-24-3p and miR-6850-5p increases their abundance around melanocytes, thereby exerting their melanin-suppressing function.

[0159] These results demonstrate that the degree of melanin production inhibition can be predicted by comprehensively evaluating the expression levels of miR-24-3p, miR-6850-5p, and miR-12120 in exosomes derived from Caco-2 cells treated with Narcoyle and other herbs.

[0160] Screening of miRNAs expected to have melanin production inhibitory effects using microarray analysis Caco-2 were cultured for 24 hours using procedures similar to those described above in the sections on samples and their preparation methods, and purification of Caco-2-derived exosomes. After 24 hours, equol, oxyresveratrol, wolfberry hydroethanol extract, black wolfberry aqueous extract, astaxanthin, pineapple-derived ethanol extract (containing components such as phytol and glucosylceramide), Satsuma mandarin-derived ethanol / hexane extract (containing components such as β-cryptoxanthin), or nobiletin (negative control) were added to a final dry weight concentration of 30 μM or 30 μg / mL. 24 hours later, the culture supernatant was collected and the exosomes secreted by Caco-2 were purified. Furthermore, when the inhibitory effect of melanin synthesis in Caco-2 cells supplemented with equol, oxyresveratrol, wolfberry hydroethanol extract, black wolfberry aqueous extract, astaxanthin, pineapple-derived ethanol extract (containing components such as Phytol and glucosylceramide), Satsuma mandarin-derived ethanol / hexane extract (containing components such as β-cryptoxanthin), or nobiletin was examined, melanin was significantly suppressed in the groups supplemented with equol, oxyresveratrol, wolfberry hydroethanol extract, black wolfberry aqueous extract, astaxanthin, pineapple-derived ethanol extract, or Satsuma mandarin-derived ethanol / hexane extract.On the other hand, no melanin suppression was observed in the group supplemented with nobiletin. This was thought to be because in the groups to which equol, oxyresveratrol, wolfberry hydroethanol extract, black wolfberry aqueous extract, astaxanthin, pineapple-derived ethanol extract, and Satsuma mandarin-derived ethanol / hexane extract had been added, the components contained in the Caco-2 culture medium, particularly exosomes, changed and the changed exosomes affected melanin suppression, while in the group to which nobiletin had been added, the components contained in the Caco-2 culture medium did not change and did not affect melanin suppression.

[0161] miRNA expression levels in purified exosomes from each group were analyzed using miR microarrays (Human microRNA Oligo chips, manufactured by Toray Industries, Inc.). miR microarray analysis was performed according to the instructions provided with the product. Data on the expression changes of 2,622 miRNAs in each group were extracted, and common expression patterns were tracked between groups. Of the seven groups treated with equol, oxyresveratrol, wolfberry hydroethanol extract, black wolfberry aqueous extract, astaxanthin, pineapple ethanol extract, and Satsuma mandarin ethanol / hexane extract, four or more groups were selected for which the ratio of miRNA expression levels to that in the DMSO-treated group (negative control) was 1.5-fold or greater. Heat maps were then created based on the log2 ratios for each group. The heat maps were created using RStudio. The pheatmap package in R was used to create the heat maps, and the distance between miRNAs in the clustering was calculated using correlation distances.

[0162] Seven miRNAs - miR-4518, miR-7108-3p, miR-3144-5p, miR-3610, miR-583, miR-6124, and miR-11181-3p - were identified as having the effect of suppressing melanin production, with their expression levels increasing in all groups supplemented with equol, oxyresveratrol, wolfberry hydroethanol extract, black wolfberry aqueous extract, astaxanthin, pineapple-derived ethanol extract, and Satsuma mandarin-derived ethanol / hexane extract, but with almost no increase in expression observed in the nobiletin-supplemented group (Figure 5).

[0163] Furthermore, five miRNAs - miR-3661, miR-6795-3p, miR-6853-5p, miR-671-3p, and miR-520f-5p - were identified as having the effect of suppressing melanin production. These five miRNAs were found to have elevated expression in all groups to which astaxanthin, pineapple-derived ethanol extract, and Satsuma mandarin-derived ethanol / hexane extract had been added, and showed no significant increase in expression in the groups to which equol, oxyresveratrol, wolfberry hydroethanol extract, black wolfberry aqueous extract, and nobiletin had been added (Figure 6).

[0164] KEGG pathway analysis of identified miRNAs For the miRNAs identified using the above method, target genes of the identified miRNAs were predicted using miRWalk (URL: http: / / mirwalk.umm.uni-heidelberg.de / ), and the associated pathways of each were searched for using DAVID (URL: https: / / david.ncifcrf.gov / summary.jsp).

[0165] First, we predicted the target genes of miR-24-3p and miR-12120 using miRWalk and evaluated the predicted regulatory relationships between human genes and miRNAs. The results showed that miR-6124 and miR-11181-3p were highly correlated with melanogenesis-related pathways, particularly melanogenesis inhibition, which are associated with miR-24-3p and miR-12120 (Figure 7). Furthermore, a search for related pathways using the DAVID database revealed that miR-6124, miR-11181-3p, miR-24-3p, and miR-12120 influence signaling pathways, particularly the MAPK and PI3K-Akt signaling pathways, among metabolic pathways related to melanogenesis (Figure 8). The relationship between each miRNA and each pathway was evaluated based on the calculated P value obtained using the formula: -log10(P value). The larger the value obtained using the formula: -log10(P value), the stronger the relationship between each miRNA and each pathway. In Figures 7 and 8, each miRNA and each pathway are connected by an arrow whose thickness corresponds to the number obtained using the formula: -log10(P value). In other words, the larger the number obtained using the formula: -log10(P value), the thicker the arrow connecting each miRNA and each pathway, and the thickness of the arrow directly indicates the strength of the relationship between each miRNA and each pathway.

[0166] Next, similar evaluations were performed on miR-7108-3p, miR-3144-5p, miR-4518, miR-3610, and miR-583. These miRNAs were found to significantly affect melanin synthesis-related pathways, particularly melanogenesis and melanoma formation (Figure 9). These miRNAs were also thought to affect signal transduction pathways, particularly the MAPK signaling pathway, the PI3K-Akt signaling pathway, the Wnt signaling pathway, and the cAMP signaling pathway (Figure 10). As with Figures 7 and 8, the relationship between each miRNA and each pathway was evaluated based on the calculated P value obtained using the formula: -log10(P value). In Figures 9 and 10, each miRNA is connected to each pathway with an arrow of a line thickness corresponding to the value obtained using the formula: -log10(P value). In other words, the larger the number obtained by the formula: -log10(P value), the thicker the arrow connecting each miRNA and each pathway, and the thickness of the arrow directly indicates the strength of the relationship between each miRNA and each pathway.

[0167] Next, we performed a similar evaluation on miR-3661, miR-6795-3p, miR-6853-5p, miR-671-3p, and miR-520f-5p. These miRNAs were suggested to be involved in the regulation of both melanin synthesis-related pathways and signal transduction pathways, as well as the oxidative and inflammatory response pathways, particularly the AGE-RAGE signaling pathway and inflammatory mediators (TRPs) (Figure 11). As in Figures 7 and 8, the relationship between each miRNA and each pathway was evaluated based on the calculated P value obtained using the formula: -log10(P value). In Figure 11, arrows with line thicknesses corresponding to the values ​​obtained using the formula: -log10(P value) are used to connect each miRNA and each pathway. In other words, the larger the number obtained by the formula: -log10(P value), the thicker the arrow connecting each miRNA and each pathway, and the thickness of the arrow directly indicates the strength of the relationship between each miRNA and each pathway.

[0168] Evaluation of the expression levels of identified miRNAs involved in melanin production in Caco-2-derived exosomes Using the same procedures as described above in the section on evaluation of miRNA expression levels involved in melanin production in Caco-2-derived exosomes, we added Narcoyuri (Nippon Funa Yakuhin Co., Ltd.), oxyresveratrol, equol, or astaxanthin to Caco-2 cells to suppress melanin production, and then evaluated the expression levels of miR-6124 and miR-12120 in the prepared and purified Caco-2-derived exosomes.

[0169] Similar to the procedures described above for assessing the expression levels of miRNAs involved in melanin production in Caco-2-derived exosomes, the primers used were U6 (forward / reverse) primers for housekeeping genes, 10 μM miRNA-specific primers (forward primers specific to each miR) for target genes, and 10 μM mRQ3' primer (a reverse primer common to each miR in the Mir-X™ miRNA qRT-PCR TB Green Kit, manufactured by Clontech) included in the kit. The sequences of the miRNA-specific primers for the target genes are shown in Table 5 below, and the sequences of the U6 primers for the housekeeping genes are shown in Table 6 below.

[0170] [Table 5]

[0171] [Table 6]

[0172] The results for miR-6124 are shown in Figure 12. In Figure 12, Test Example 10 shows the relative expression level of the miR-6124 gene in exosomes derived from Caco-2 cells without polyphenols. Test Example 11 shows the relative expression level of the miR-6124 gene in exosomes derived from Caco-2 cells supplemented with narcoli (Dalmatian lily). Test Example 12 shows the relative expression level of the miR-6124 gene in exosomes derived from Caco-2 cells supplemented with oxyresveratrol. Test Example 13 shows the relative expression level of the miR-6124 gene in exosomes derived from Caco-2 cells supplemented with equol. Test Example 14 shows the relative expression level of the miR-6124 gene in exosomes derived from Caco-2 cells supplemented with astaxanthin. Multiple comparisons were performed using one-way analysis of variance. Multiple comparisons of the sample-supplemented groups against the control group were performed using the Dunnett method, and a p value of less than 0.05 was considered a significant increase in expression. In Figure 12, ** indicates p<0.01. The addition of narcoyle, oxyresveratrol, equol, and astaxanthin to Caco-2 cells significantly increased the expression of miR-6124 in exosomes. A similar trend was also observed for miR-12120.

Claims

1. 1. An in vitro method for assessing the degree of inhibition of melanin production, comprising: (a) isolating exosomes from a biological sample; (b) quantifying the amount of at least one miRNA selected from the group consisting of miR-6124, miR-24-3p, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p in the exosomes separated in step (a); and (c) evaluating the degree of suppression of melanin production based on the miRNA quantification results obtained in step (b). A method comprising:

2. The method of claim 1 , wherein the biological sample is derived from a human.

3. The method according to claim 2, wherein the human-derived biological sample is at least one selected from the group consisting of blood, stratum corneum, urine, and saliva.

4. A marker for evaluating the degree of inhibition of melanin production, comprising at least one selected from the group consisting of miR-6124, miR-24-3p, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p.

5. A marker for evaluating the degree of inhibition of melanin production, comprising at least one marker selected from the group consisting of miR-6124, miR-24-3p, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p.

6. A method for screening a substance that inhibits melanin production, comprising: (i) contacting a candidate substance with a cell; (ii) isolating exosomes secreted from the cells contacted with the candidate substance; (iii) quantifying the expression level of at least one miRNA selected from the group consisting of miR-6124, miR-24-3p, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p in the exosomes separated in step (ii); and (iv) determining whether the candidate substance is a substance that suppresses melanin production based on the miRNA quantification results obtained in step (iii); A method comprising:

7. The method according to claim 6, wherein the cells are Caco-2 cells derived from human intestinal epithelial cells.

8. A composition for promoting expression of miRNA, comprising Narcoylium nigricans, The composition, wherein the miRNA is at least one selected from the group consisting of miR-6124, miR-24-3p, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p.

9. The composition according to claim 8, wherein the miRNA is a miRNA contained in a cell-derived exosome.

10. The composition according to claim 9, wherein the cells are Caco-2 cells derived from human intestinal epithelial cells.

11. The composition according to any one of claims 8 to 10, which is a food composition.

12. A cosmetic preparation comprising exosomes containing at least one miRNA selected from the group consisting of miR-6124, miR-24-3p, miR-12120, miR-11181-3p, miR-7108-3p, miR-3144-5p, miR-3610, miR-6795-3p, miR-6853-5p, miR-4518, miR-583, miR-3661, miR-671-3p, miR-520f-5p, and miR-6850-5p.

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