Agent for preventing and / or improving photoaging and / or dermal pigmentation, cosmetic method using the same, and cosmetic device for applying the method

Abstract

The present invention provides an agent for preventing and / or improving photoaging and / or dermal pigmentation comprising Thujae Semen extract as an active ingredient. The present invention also provides a cosmetic method which includes a step for applying the agent for preventing and / or improving photoaging and / or dermal pigmentation to skin to be treated, and a cosmetic device to be applied in said method.

Description

[Technical Field]

[0001] The present invention relates to an agent for preventing and / or improving photoaging and / or dermal pigmentation, a cosmetic method using the same, and a cosmetic device for applying the method. [Background technology]

[0002] The aging phenomenon of human skin can be broadly divided into "natural aging" and "photoaging." Photoaging is a skin-specific phenomenon that occurs in specific areas exposed to light. In photoaging, the effects of UV rays and other factors induce the production of reactive oxygen species and damage to cellular DNA, which is thought to damage skin fibrous tissue and cause phenotypes such as wrinkles and sagging. For example, skin photoaging can result in a decrease in collagen fibers made of collagen and degeneration of elastic fibers made of elastin. Melanocytes are also damaged, resulting in the overproduction of melanin pigment, which can cause age spots and other skin conditions.

[0003] Furthermore, pigmentation, including age spots and dullness, occurs due to the accumulation of melanin produced by melanocytes in the basal layer of the epidermis. Melanin is normally present in the epidermis and basal layer, but because the epidermis has a relatively fast turnover cycle, this melanin is easily excreted. However, melanin can sometimes be present in the dermis layer due to reasons such as it falling into the dermis through gaps in the basement membrane. Because the turnover cycle of dermal cells is much slower than that of the epidermis, this melanin often accumulates without being excreted. For these reasons, improving dermal pigmentation is extremely difficult.

[0004] As anti-photoaging cosmetic methods, for example, Patent Document 1 discloses an agent for preventing or inhibiting skin photoaging by preventing the inhibition of leukocyte elastase. Patent Document 2 discloses a photoaging inhibitor composition characterized by containing a plant extract of the genus Pfaffia of the Amaranthaceae family, which has the effect of promoting collagen synthesis, and animal-derived collagen peptides.

[0005] As a cosmetic method for improving pigmentation in the dermis, Non-Patent Document 13 proposes to prevent melanin from sinking into the dermis by strengthening the basement membrane.

[0006] It has also been suggested that inflammation is a contributing factor to the photoaging phenomenon of the skin, and many anti-inflammatory agents have been developed. Patent Document 3 discloses an anti-aging cosmetic composition containing a compound that increases adiponectin expression and induces autophagy activation. It has also been suggested that the phagocytic action of macrophages can be utilized to improve dermal pigmentation, and Patent Document 8 discloses an agent for preventing and improving dermal spots by attracting macrophages to fibroblasts that have taken up melanin that has fallen into the dermis and causing them to phagocytose. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 5657723 [Patent Document 2] Japanese Patent Application Publication No. 2017-203004 [Patent Document 3] Japanese Patent Application Publication No. 2018-177805 [Patent Document 4] Special Publication No. 2014-504629 [Patent Document 5] Japanese Patent Application Laid-Open No. 2015-140334 [Patent Document 6] Patent No. 6178088 [Patent Document 7] Patent No. 6273304 [Patent Document 8] Japanese Patent Application Publication No. 2018-072098 [Patent Document 9] Patent No. 4781842 [Patent Document 10] International Publication No. 2012 / 057123 [Non-patent literature]

[0008] [Non-licensed document 1] Journal of the American College of Cardiology, Vol. 62, No. 20, 2013, November 12, 2013:1890-901 [Non-licensed document 2] Experimental & Molecular Medicine (2014) 46, e70; doi:10.1038 / emm.2013.135 [Non-licensed document 3] NATURE, VOL495, 28 MARCH 2013, pp524-530, doi:10.1038 / nature11930

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

[0009] An object of the present invention is to provide an agent for preventing and / or improving photoaging and / or dermal pigmentation, a cosmetic method using the same, and a cosmetic device for applying the method. [Means for solving the problem]

[0010] As a result of an intensive search for an agent for preventing and / or improving photoaging and / or dermal pigmentation, the present inventors discovered that agave extract regulates the M1 / M2 balance of macrophages, thereby exerting the effect of preventing and / or improving photoaging and / or dermal pigmentation. Based on this discovery, the present invention was developed. Specifically, the present invention includes the following aspects.

[0011] [1] An agent containing limonene extract as an active ingredient, for preventing and / or improving photoaging and / or dermal pigmentation. [2] The agent according to item 1, which prevents and / or improves photoaging and / or dermal pigmentation by adjusting the M1 / M2 balance. [3] The agent according to item 2, wherein adjusting the M1 / M2 balance means increasing the ratio of M2 to M1.

[0012] [4] A cosmetic method for preventing and / or improving photoaging and / or dermal pigmentation in a subject, comprising: (a) applying the agent according to any one of items 1 to 3 to the skin of a subject; Including beauty methods. [5] The cosmetic method further includes a step of applying a weak physical stimulus to the skin, wherein the step of applying the physical stimulus includes, for example, (b-1) stretching the skin of the subject to a stretch rate of 0.1% or more and 50.0% or less, wherein the stretch rate is

number

[0013] [6] A beauty device for use in the beauty method described in item 5, The device comprises: a stimulus generator that generates physical stimuli; a stimulus applying unit that applies the physical stimulus generated by the stimulus generating unit to the skin, Here, the device is a device for performing a step of applying a weak physical stimulus to the skin, and the step includes, for example, (i-1) stretching the skin to a stretch rate of 0.1% or more and 50.0% or less; and (ii-1) recovering the skin from a stretched state; and / or, (i-2) pressing the skin to a depth of 1 μm to 1000 μm; and (ii-2) recovering the skin from the pressure state; and performing a cycle including the cycle at a frequency of 60 Hz or less, Here, the extension rate is calculated using the above formula 1. The cosmetic device. [Effects of the Invention]

[0014] By applying the present invention, it is possible to regulate the M1 / M2 balance of macrophages, thereby preventing and / or ameliorating photoaging and / or dermal pigmentation. [Brief explanation of the drawings]

[0015] [Figure 1a] FIG. 1a shows the types of macrophage markers used in Experiment 1, as well as the age, skin type, and sex of the subjects. [Figure 1b] Figure 1b is a photomicrograph showing macrophages (CD11b: red) and M1 macrophages (CD86: green) in skin tissue from young and elderly individuals. [Figure 1c] Figure 1c is a photomicrograph showing macrophages (CD11b: red) and M2 macrophages (CD206: green) in skin tissue from young and elderly individuals. [Figure 1d] Figure 1d is a graph showing the number of M1, M2 macrophages, and the total number of macrophages in skin tissue from young and elderly subjects (cells / mm2). [Figure 1e] The left panel of Figure 1e is a photomicrograph showing co-staining of M1 macrophages (CD86: red) or M2 macrophages (CD206: red) and procollagen (green) in skin tissue from young and elderly individuals. The rectangles in each of the four panels on the left represent enlarged views of characteristic areas. The right panel of Figure 1e is a schematic diagram of the relationship between M1, M2, fibroblasts, and collagen production / destruction. [Figure 2a] Figure 2a shows an outline of the method for Experiment 2. [Figure 2b] Figure 2b shows micrographs of M0, M1, and M2 macrophages induced in Experiment 2. [Figure 2c] The upper graph in Figure 2c is a graph showing the gene expression levels of cytokines (IL-1beta, TNF-alpha, IL-10) produced by M0, M1, and M2 macrophages induced in Experiment 2. The lower graph in Figure 2c is a graph showing the mRNA expression levels of cell surface markers (M1: CD86, M2: CD206) in M1 and M2 macrophages. The values ​​are corrected for GAPDH mRNA expression levels and shown as relative values ​​(%) with M0 being set at 100%. [Figure 3a] Figure 3a shows an outline of the method for Experiment 3. [Figure 3b] FIG. 3b shows the amount of procollagen (μg / well) in fibroblasts to which M1 and M2 macrophage supernatants were added according to Experiment 3. [Figure 3c] FIG. 3c is a photomicrograph showing collagen (red) and hyaluronic acid (green) in fibroblasts supplemented with supernatants from M1 and M2 macrophages according to experiment 3. [Figure 3d] FIG. 3d is a photomicrograph showing β-galactosidase (β-Gal) in fibroblasts to which M1 and M2 macrophage supernatants were added according to Experiment 3. [Figure 3e]Figure 3e is a graph showing the results of a senescence-associated β-galactosidase (SA β-Gal) assay in fibroblasts to which M1 and M2 macrophage supernatants were added according to Experiment 3 (left panel: percentage of SA β-gal positive cells relative to the total number of DAPI positive cells), and the total number of DAPI positive cells per well (right panel). [Figure 3f] Figure 3f is a graph showing the mRNA expression levels of each melanogenesis-enhancing factor (HGF, ET1, bFGF, IL-1alpha, SCF) and melanogenesis-inhibiting factor (clusterin, DKK1) in fibroblasts to which M1 and M2 macrophage supernatants were added according to Experiment 3. [Figure 4a] FIG. 4a is a photomicrograph showing β-Gal in young and old fibroblasts to which M1 and M2 macrophage supernatants were added according to Experiment 4. [Figure 4b] Figure 4b is a graph showing the results of the SA β-Gal assay (percentage of SA β-gal positive cells relative to the total number of DAPI-positive cells) and the total number of DAPI-positive cells per well in young (top) and old (bottom) fibroblasts to which M1 and M2 macrophage supernatants were added according to Experiment 4. [Figure 4c] FIG. 4c shows the amount of procollagen (μg / well) in young and old fibroblasts to which M1 and M2 macrophage supernatants were added according to Experiment 4. [Figure 4d] FIG. 4d is a photomicrograph showing collagen (red) and DAPI-stained nuclei (blue) in young and old fibroblasts to which M1 and M2 macrophage supernatants were added according to Experiment 3. [Figure 5] Figure 5 is a micrograph showing type I collagen (red) and macrophages (CD68: green) in neonatal foreskin-derived fibroblasts to which M1 and M2 macrophage supernatants were added in Experiment 5. The upper left photograph shows fibroblasts cultured without the addition of macrophage supernatants. [Figure 6]Figure 6 is a micrograph showing macrophages (CD68: red) and M1 macrophages (CD86: green) or M2 macrophages (CD206: green) in the 3D model created in Experiment 6. The triangles indicate areas double-stained for CD68 and CD86, or CD68 and CD206. [Figure 7] FIG. 7 is a photomicrograph showing p21 (red) and DAPI-stained nuclei (blue) in a 3D model prepared according to Experiment 6. [Figure 8] Figure 8 is a graph showing the percentage (%) of p21-positive cells relative to the total number of cells in each layer (left: upper layer = epidermal cell layer, right: lower layer = fibroblast layer) of the 3D model created in accordance with Experiment 6. [Figure 9] Figure 9 is a graph showing the number of total macrophages and M1 and M2 macrophages in an ex vivo skin model irradiated with a solar simulator according to Experiment 7. The cell number of each macrophage without irradiation is set to 100% (bar on the left: irradiation (-)), and the relative value (%) of the cell number of each macrophage after irradiation is shown in the bar on the right (irradiation (+)). [Figure 10] Figure 10 shows the settings for the stretch stimulation performed in Experiment 8. [Figure 11] FIG. 11 shows the apparatus and skin samples used in Experiment 8. [Figure 12] Figure 12 shows a comparison of the numbers of M1 and M2 macrophages and the total number of all macrophages when a stretch stimulus was applied to the ex vivo skin model (stretch) and when it was not applied (control) in Experiment 8. The graph on the left shows the number of macrophages per mm2 of skin sample (cells / mm2). The graph on the right shows the percentage (%) of each macrophage type (M1, M2) relative to the total number of all macrophages. [Figure 13] FIG. 13 shows an example of the device of the present invention. [Figure 14a]Figure 14a is a graph showing the mRNA expression levels of each collagen-degrading enzyme (MMP-1, MMP-2) and inflammatory cytokine (IL-1β) in fibroblasts to which M1 and M2 macrophage supernatants were added in Experiment 9, expressed as relative values ​​(%) when the results for M1 were set at 100%. [Figure 14b] Figure 14b is a graph showing the mRNA expression levels of each collagen production and maturation factor (COL1A1, COL1A2, HSP47, and ADAMTS-2) in fibroblasts to which M1 and M2 macrophage supernatants were added in Experiment 9, expressed as relative values ​​(%) when the results for M1 were set at 100%. [Figure 15a] Figure 15a shows the ages of the subjects from whom samples were taken in Experiment 10, divided into the young and elderly groups. [Figure 15b] Figure 15b is a graph showing the number of M1 and M2 macrophages (cells / mm2) in skin tissue (50 μm from just below the basement membrane) of young and elderly subjects in Experiment 10. [Figure 15c] Figure 15c is a graph showing the number of M1 and M2 macrophages (cells / mm2) in skin tissue (200 μm from just below the basement membrane) of young and elderly subjects in Experiment 10. [Figure 15d] FIG. 15d is a graph showing the number of CD68-negative cells, M1, and M2 macrophages that were 3 / 4 collagen-positive in the skin tissues of young and elderly subjects in Experiment 10 (cells / mm2). [Figure 16a] Figure 16a shows the appearance of fibroblasts, M1 macrophages, and M2 macrophages 24 hours after melanin addition in Experiment 11. Cells phagocytosing melanin take on a circular morphology as shown in the right image. [Figure 16b] FIG. 16b shows the amount of melanin (melanin / almar blue) taken up per cell by fibroblasts, M1 macrophages, and M2 macrophages 24 hours after the addition of melanin in Experiment 11. [Figure 16c] Figure 16c shows the appearance of M1 and M2 macrophages in Experiment 11, 5 days after melanin addition. [Figure 17] The top of Figure 17 is a graph showing the number of melanin-engulfing M1 macrophages and M2 macrophages in the dermis layer for each age group, as counted according to Experiment 12. The bottom of Figure 17 shows the average number of melanin-engulfing M1 macrophages and M2 macrophages across all subjects. [Figure 18] Figure 18 shows the results of the screening conducted in Experiment 13. The bars are graphs showing the expression levels (marker / GAPDH value) of each M1 and M2 marker when various concentrations of liquorice extract (0.3 ppm, 1.0 ppm, 3.0 ppm) were added, expressed as relative values ​​(%) when the control (no liquorice hydrolysis: control) was set at 100%. [Figure 19a] Figure 19a shows the state of immature (M0) THP-1 cells 30 minutes after addition of melanin to which 3 ppm of unhydrolyzed limonene (control) or 1.0 ppm or 3.0 ppm of limonene hydrolysate was added in Experiment 14. [Figure 19b] Figure 19b is an enlarged view of Figure 19a. The black arrow indicates a macrophage ingesting melanin. [Figure 20] Figure 20 shows the results of analysis of the intracellular activity of M0 macrophages, M1 macrophages, and M2 macrophages in Experiment 15 using a flux analyzer: (A) oxygen consumption rate (OCR), (B) extracellular acidification rate (ECAR), and (C) OCR / ECAR. [Figure 21] Figure 21 shows the results of analysis using a flux analyzer to determine the effect of the addition of lixivivir ginseng extract on intracellular activity of M0 macrophages 48 hours after the addition in Experiment 16. (A) Normalized OCR, (B) Basal OCR (OCR value at the third point of measurement A). [Figure 22] Figure 22 shows the results of a flux analyzer analysis of the effect of the addition of lixivivir ginseng extract on intracellular activity in M0 macrophages 48 hours after the addition in Experiment 16. (A) Normalized OCR, (B) OCR of mitochondrial respiration (the difference between the 3rd and 12th measurement points in A). [Figure 23] Figure 23 shows the results of analysis using a flux analyzer of the effects of representative unsaturated fatty acids contained in the citrus extract alone on intracellular activity 48 hours after addition of M0 macrophages in Experiment 17. (A) Basal OCR, (B) OCR of mitochondrial respiration. [Figure 24] Figure 24 shows the results of analysis using a flux analyzer to determine the effects of each component contained in the citrus extract on intracellular activity 24 hours after addition of M0 macrophages in Experiment 18. (A) Basal OCR, (B) OCR of mitochondrial respiration. [Figure 25] Figure 25 shows an outline of the method for Experiment 19. [Figure 26] Figure 26 shows the results of analysis using a flux analyzer to determine the effects of the citrus extract or each component contained in the citrus extract on intracellular activity during M1 macrophage differentiation in Experiment 19. (A) Basal OCR, (B) OCR of mitochondrial respiration, (C) OCR / ECAR. [Figure 27] 27 shows the expression levels of SDF-1α in the supernatants of M1 and M2 macrophages. (A) Outline of Experiment 20, (B) Amount of SDF-1α in the culture supernatants of M1 and M2 macrophages. [Figure 28] Figure 28 shows the effect on SDF-1α expression in skin fibroblasts cultured with M1 or M2 macrophage supernatant in Experiment 21. (A) Outline of Experiment 21, (B) Amount of SDF-1α in fibroblasts 3 days after culture with M1 or M2 macrophage culture supernatant. DETAILED DESCRIPTION OF THE INVENTION

[0016] Macrophages are cells that are localized in various tissues within the body and initiate immune responses against foreign substances and pathogens, and are known to be involved in inflammation. Macrophages differentiate from undifferentiated M0 macrophages (hereinafter sometimes abbreviated as M0) into M1 and M2 types. M1 macrophages (hereinafter sometimes abbreviated as M1) are known as inflammatory types, while M2 macrophages (hereinafter sometimes abbreviated as M2) are known as repair types (anti-inflammatory types). An imbalance between M1 and M2 macrophages has been reported to be associated with diseases such as obesity, type 2 diabetes, and arteriosclerosis (Patent Documents 4-6, Non-Patent Documents 1-5). However, the relationship between skin photoaging, pigmentation, and the M1 / M2 balance was unknown.

[0017] The present inventors discovered that the balance between M1 and M2 macrophages (M1 / M2 balance) is disrupted specifically in skin exposed to light or in areas where pigmentation occurs. They also found that adjusting the M1 / M2 balance is particularly important for preventing and improving photoaging and pigmentation in the dermis. Using the M1 / M2 balance as an indicator, the present inventors searched for substances that can prevent and improve photoaging and / or dermal pigmentation. They discovered that haxinin regulates the M1 / M2 balance, leading to the development of the present invention, which can prevent and improve photoaging and / or pigmentation in the skin, particularly the dermis. Haxinin has been reported to act on fibroblasts to promote their proliferation, promote the production of collagen and hyaluronic acid, and inhibit melanin production (Patent Documents 9 and 10). However, the present inventors are the first to have discovered that haxinin has the ability to regulate and improve the M1 / M2 balance.

[0018] The inventors have also found that the M1 / M2 balance can be adjusted by applying specific physical stimuli to the subject's skin. More specifically, even in photoaged or pigmented skin, the M1 / M2 balance can be adjusted / improved by applying weak physical stimuli at a specific stretch rate and a frequency of 60 Hz or less, such as 1 Hz or less, or 10 Hz or less.

[0019] Therefore, the present invention provides an agent for preventing and / or improving photoaging and / or dermal pigmentation by adjusting the M1 / M2 balance, a cosmetic method using the same, and a cosmetic device for applying the method. The method of the present invention is a method for cosmetic purposes and may not be treatment by a doctor or medical professional.

[0020] In this specification, pigmentation refers to the deposition of pigments in the dermis and epidermis, and includes, for example, pigmentation of melanin and the like caused by photoaging, as well as artificially injected pigments (such as tattoos). The present invention is effective for both the dermis and epidermis, but is particularly expected as a measure against dermal pigmentation, in the sense that there are limited methods of improvement other than phagocytosis by melanophages. Application of the present invention improves spots, dullness, dark circles, and other pigmentation-related appearances. In addition, because the pigment is injected into the dermis layer, it is also effective in erasing tattoos and other pigments, which are difficult to remove once the pigment is injected.

[0021] M1 macrophages can be measured using markers such as CD86, CD80, and iNOS. M2 macrophages can be measured using markers such as CD206, CD163, and Agr1. Markers for macrophages as a whole, including M1 and M2, include CD11b and CD68. Additionally or alternatively, measurements may be made by quantifying M1-specific cytokines such as IL-1beta and TNF-alpha, or M2-specific cytokines such as IL-10. However, markers that can measure the M1 / M2 balance are not limited to the above markers.

[0022] As used herein, the M1 / M2 balance may refer to the ratio of the number of M1 macrophages to the number of M2 macrophages, or may refer to the ratio of the mRNA amount of an M1 macrophage marker (e.g., CD86, CD80, iNOS, etc.) to the mRNA amount of an M2 macrophage marker (e.g., CD206, CD163, Agr1, etc.). In photoaged skin, the ratio of M1 is high and the ratio of M2 is low, and it is M2 that has high melanin phagocytosis, so adjusting / improving the M1 / M2 balance may mean increasing the ratio of M2 to M1 (the number of M2 / the number of M1, and / or the amount of mRNA of an M2 marker / the amount of mRNA of an M1 marker). The increase may be, for example, an increase that is statistically significant at a 5% significance level (e.g., Student's t-test) and / or an increase of, for example, 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more. Alternatively, adjustment / improvement of the M1 / M2 balance may involve bringing the ratio of M2 to M1 (number of M2 / number of M1, and / or amount of mRNA of M2 marker / mRNA amount of M1 marker) within a certain range, for example, about 4 / 6 to about 9 / 1, about 5 / 5 to about 8 / 2, about 5 / 5 to about 7 / 3, etc., or may involve approaching or maintaining within the above range, or maintaining homeostasis around the above range.

[0023] As used herein, the M1 / M2 balance may refer to the balance between the intracellular activity of M1 macrophages and that of M2 macrophages, and may be expressed, for example, using an index of intracellular activity primarily used by M1 macrophages (e.g., the state of a thawing system) and / or an index of intracellular activity primarily used by M2 macrophages (e.g., the state of aerobic respiration (mitochondrial respiration)). Intracellular activity can be evaluated, for example, by measuring the cellular oxygen consumption rate (OCR value), extracellular acidification rate (ECAR value), or OCR / ECAR value. As used herein, "increasing the ratio of M2 to M1" may refer to an increase in intracellular mitochondrial activity, which is primarily used by M2 macrophages; for example, it may refer to an increase in aerobic respiration (mitochondrial respiration) in a population containing M1 macrophages and M2 macrophages (e.g., aerobic respiration (mitochondrial respiration) becoming predominant in a population containing M1 macrophages and M2 macrophages), and / or a decrease in the activity of glycolysis, which is anaerobic respiration, in a population containing M1 macrophages and M2 macrophages; for example, it may refer to an increase in the OCR value in a population containing M1 macrophages and M2 macrophages, and / or a decrease in the ECAR value in a population containing M1 macrophages and M2 macrophages, and / or an increase in the OCR / ECAR value in a population containing M1 macrophages and M2 macrophages. Intracellular activity can be measured non-invasively and with high sensitivity over time by using, for example, the extracellular flux analyzer XFe24 (for 24 wells) manufactured by Agilent Technologies (formerly Seahorse Bioscience), to measure the state of glycolysis, which is the main energy metabolic pathway of cells, and aerobic respiration by mitochondria. However, this device is merely an example, and intracellular activity can be measured and evaluated using any device or method, and therefore the use of this device is not limited.

[0024] In photoaged skin, the ratio of M1 is high and the ratio of M2 is low, and it is M2 that has high melanin phagocytosis. Therefore, adjusting / improving the M1 / M2 balance may mean increasing the ratio of M2 intracellular activity to M1, for example, increasing the OCR value in a population containing M1 macrophages and M2 macrophages, and / or decreasing the ECAR value in a population containing M1 macrophages and M2 macrophages. As used herein, "increase or decrease" may mean an increase or decrease that is statistically significant at a significance level of 5% (e.g., Student's t-test), and / or an increase or decrease of, for example, 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more.

[0025] Hakushinin (Hakushijin) is a herbal medicine made from the dried seeds of Platycladus orientalis Franco (Thuja orientalis L., Biota orientalis Endl.) of the Cupressaceae family. It is preferable to use the seed kernel (endosperm) as the hakushinin used in the present invention, but the entire seed can also be used since the active ingredient is contained in the entire seed. Hakushinin can be produced by conventional methods, or a commercially available product can be used.

[0026] The method for preparing the lily extract that can be used in the present invention can be based on known methods (e.g., Patent Documents 9 and 10), but is not limited to these. For example, methods for preparing the lily extract that can be used in the present invention include a method in which plant seeds are extracted and the resulting plant extract is treated with alkali (hereinafter referred to as the first method), and a method in which plant seeds are crushed and aged at a temperature of 10 to 35°C and a relative humidity of 20 to 90% for one week or more, followed by extraction (hereinafter referred to as the second method).

[0027] The extraction solvent used in the first and second methods described above can be any volatile solvent commonly used in extraction, and in particular, polar and non-polar organic solvents such as alcohols (e.g., methanol and ethanol), hydrous alcohols, acetone, ethyl acetate, hexane, and ether can be used alone or in combination. However, acetone, ethyl acetate, and hexane are particularly preferred because they are inexpensive and can be easily concentrated under reduced pressure. Supercritical carbon dioxide extraction can also be used. In contrast, extraction solvents containing large amounts of water are not preferred because they inhibit the extraction of active ingredients.

[0028] Extraction is carried out for a certain period of time using a solvent in an amount 1 to 100 times, preferably 1 to 30 times, the mass of the seeds. The seeds used can be crushed appropriately if necessary, but they can also be used uncrushed if clogging during filtration is a problem. Repeated extraction with small amounts of solvent is also possible, and a reflux device such as a Soxhlet filter can also be used. When using supercritical carbon dioxide, extraction can be carried out under typical conditions of around 40°C and 20 to 40 MPa.

[0029] In the first method, the extract is hydrolyzed by alkaline treatment after removing the extraction solvent from these extracts. The alkaline treatment involves adding 0.2 to 2 times the volume of concentrated alkaline aqueous solution to the extract after solvent removal, thoroughly stirring and mixing, and then aging for 30 minutes to several days. The temperature during mixing is preferably in the range of room temperature to 70°C, more preferably in the range of 30 to 60°C. During aging, it is desirable to stir appropriately so that the hydrophilic and lipophilic components do not separate. Examples of concentrated alkaline aqueous solutions include aqueous solutions consisting of NaOH, KOH, etc. at a concentration of 1 to 10N.

[0030] When the extract hydrolyzed by alkaline treatment as described above is further neutralized with acid to near neutrality, an oily substance separates out. This oily substance exhibits extremely high anti-dermal pigmentation activity while also being highly safe for the skin. To promote the recovery of the oily substance, alcohol, acetone, etc. may be added as appropriate after alkaline treatment, and further operations such as bleaching, deodorization, desalination, and distillation may be carried out as necessary.

[0031] According to the second method for preparing lily extract, the seeds are crushed or compressed appropriately prior to extraction and then left to mature for a certain period of time at around room temperature. The aging period is not particularly limited as long as the desired effect is achieved. However, if the aging period is short (less than one week), it is difficult to achieve a sufficient whitening effect, and conversely, if it extends to several years, problems such as decay and oxidative odor may occur, which is undesirable. Furthermore, if necessary, odor can be suppressed during aging by adding antioxidants or by replacing the air with nitrogen.

[0032] The seeds are then aged in this way and extracted using the extraction solvent and method described above. After extraction, if necessary, procedures such as solvent removal, bleaching, deodorization, and distillation are performed. The extract obtained by crushing and aging these specific seeds and then extracting them exhibits the same anti-dermal pigmentation effect as the alkali-treated extract described above, while also being highly safe for the skin.

[0033] In the present invention, it is possible to provide a safe, plant-based agent with excellent anti-dermal pigmentation effects, which prevents and / or improves photoaging and / or dermal pigmentation, consisting of an extract prepared from the seeds of the plants shown above by the first or second method, and further to provide an external skin preparation containing this agent.

[0034] The agent of the present invention may be used in combination with any known substance known to regulate / improve the M1 / M2 balance, such as those described in Patent Documents 4 to 7. The route of administration can be selected arbitrarily, for example, transdermal administration, oral administration, subcutaneous administration, transmucosal administration, intramuscular administration, etc., but transdermal administration, which allows administration to specific locations on the skin, may be preferred in some cases to prevent and / or improve dermal pigmentation. Furthermore, for pigmentation occurring in the epidermis or dermis, transdermal administration may be preferred in some cases to allow delivery from the skin to the epidermis or dermis. Furthermore, the regulation / improvement of the M1 / M2 balance may be achieved, for example, by inducing differentiation into M2 macrophages, or by any other method for regulating / improving the M1 / M2 balance.

[0035] For example, the agent or composition that can be used in the present invention adjusts / improves the M1 / M2 balance, thereby suppressing photoaging and / or dermal pigmentation. The M1 / M2 balance adjusting / improving agent, anti-photoaging agent, pigmentation inhibitor, and anti-dermal pigmentation agent of the present invention (hereinafter, these may be collectively referred to as "the agent of the present invention") may contain any one of the above active ingredients alone, or may contain two or more of them in any combination and ratio.

[0036] The agent of the present invention can also be a composition in which the above-mentioned active ingredient is combined with one or more other ingredients, such as excipients, carriers, and / or diluents. The composition may have any composition or form, and may be appropriately selected depending on the active ingredient, application, and other conditions. The composition can be manufactured using conventional methods in a formulation in which excipients, carriers, and / or diluents, etc., and other ingredients are appropriately combined depending on the dosage form.

[0037] The agent of the present invention can be used by humans and animals by being incorporated into cosmetics, etc., or can be administered to humans and animals as a pharmaceutical preparation. It may also be incorporated into various foods, drinks, and feeds for consumption by humans and animals.

[0038] When the present invention is applied to external skin preparations such as cosmetics, pharmaceuticals, and quasi-drugs, the amount (dry mass) of the plant body or its extract can be appropriately determined depending on the type, purpose, form, method of use, etc. For example, 0.00001% to 50% of liquorice root extract (in the case of extracts or herbal medicines, converted to dry mass) can be blended in the total amount of the cosmetic.

[0039] In addition to the above ingredients, if necessary, ingredients typically used in skin topicals such as cosmetics, pharmaceuticals, and quasi-drugs, such as antioxidants, oils, UV protection agents, surfactants, thickeners, alcohols, powder ingredients, colorants, aqueous ingredients, water, various skin nutrients, etc., can be appropriately blended as needed within the scope that does not impair the effects of the present invention.

[0040] The topical skin preparation of the present invention can be applied to the outer skin as a cosmetic, quasi-drug, etc., and is particularly suitable as a cosmetic, and its formulation is not limited as long as it can be applied to the skin, and any formulation can be used, such as a solution system, solubilized system, emulsion system, powder dispersion system, water-oil two-layer system, water-oil-powder three-layer system, ointment, lotion, gel, aerosol, etc.

[0041] When the agent of the present invention is used as a cosmetic, it may be used in the form of lotion, emulsion, foundation, lipstick, lip balm, cleansing cream, massage cream, pack, hand cream, hand powder, body shampoo, body lotion, body cream, bath cosmetics, etc.

[0042] However, the forms that the agent and composition of the present invention can take are not limited to the dosage forms and forms described above. In addition, the agent or composition of the present invention may be used in combination with the device or method of the present invention or other devices or methods.

[0043] The subjects to which the methods, devices, agents, and compositions of the present invention are applied may be subjects in whom skin photoaging and / or pigmentation (e.g., dermal pigmentation) is objectively or subjectively recognized, or subjects wishing to prevent pigmentation. For example, the subjects may be subjects determined to have an imbalance in M1 / M2 balance. In one embodiment, the subjects may be subjects determined to have a high level of pigmentation (e.g., dermal pigmentation) using the M1 / M2 balance in the skin as an indicator. Alternatively, the subjects may be subjects concerned with phenotypes specific to skin photoaging, such as age spots, wrinkles, and sagging, or subjects concerned with epidermal pigmentation, such as age spots, dullness, birthmarks, and tattoo marks. Age spots, wrinkles, sagging, dullness, birthmarks, tattoo marks, and the like can be determined by visual assessment or using known indicators.

[0044] In one embodiment, the present invention provides a cosmetic method for preventing and / or ameliorating photoaging and / or dermal pigmentation in a subject, comprising: (a) A cosmetic method is provided, which includes the step of applying to the skin of a subject an agent for preventing and / or improving photoaging and / or dermal pigmentation, which agent contains a lily of the valley extract as an active ingredient.

[0045] In one embodiment, the cosmetic method of the present invention may further include, in addition to the above step (a), a step of applying a weak physical stimulus to the skin, for example, applying a weak physical stimulus to the skin such as a stretching stimulus, a pressing stimulus, or a massage. For example, the step of applying a weak physical stimulus to the skin may be performed at a frequency of 60 Hz or less, in a cycle including: (b-1) stretching the skin to a stretch rate of 0.1% or more and 50.0% or less; and (c-1) recovering from the stretched state.

[0046] The stretching rate is calculated using the above formula 1. Physical stimulation may be performed at a stretching rate of 0.001% to 80.0%, 0.01% to 60.0%, or 0.1% to 50.0%, preferably 0.1% to 50.0%. For example, any range of stretching rates can be used, such as 0.1% to 1.0%, 0.1% to 5.0%, 0.1% to 10.0%, 0.1% to 20.0%, 0.1% to 30.0%, 1.0% to 5.0%, 1.0% to 10.0%, 1.0% to 20.0%, 1.0% to 30.0%, 1.0% to 50.0%, 10.0% to 20.0%, or 10.0% to 30.0%.

[0047] The stretch rate refers to the speed (% / s) at which the maximum stretch rate (%) is reached during one cycle. The recovery rate refers to the speed (% / s) at which the muscle returns to a non-stretched state from the maximum stretch rate. The stretch rate and recovery rate may be set at any speed, such as 0.010% / s to 40% / s, 0.05% / s to 30% / s, 0.10% / s to 20% / s, 0.2% / s to 15% / s, or 0.3% / s to 10% / s. The stretch rate and recovery rate may be the same or different.

[0048] For example, the step of applying a weak physical stimulus to the skin may be performed at a frequency of 60 Hz or less, in a cycle including: (b-2) pressing the subject's skin by 1 μm to 1000 μm; and (c-2) allowing the subject's skin to recover from the pressed state.

[0049] "Pressing the skin 1 μm to 1000 μm" means pressing the skin to a depth of 1 μm to 1000 μm from the outermost surface of the skin. The depth of pressure can be set arbitrarily, such as 1 μm to 1000 μm, 10 μm to 1000 μm, 10 μm to 300 μm, or 10 μm to 100 μm from the outermost surface of the skin.

[0050] The vibration frequency refers to the number of cycles per second, where one cycle is defined as the period from the start of extension or compression to the return to a non-extension or non-compression state. One cycle may include maintaining the extension or compression state and / or resting in a non-extension or non-compression state for a certain period of time. For example, one cycle may further include maintaining the extension or compression state for 0 to 30 minutes, 1 to 20 minutes, 5 to 10 minutes, or 10 to 5 minutes after (b-1) and before (c-1), or after (b-2) and before (c-2); and / or resting in a non-extension or non-compression state for 0 to 30 seconds, 0 to 20 seconds, 0 to 10 seconds, 1 to 10 seconds, 1 to 20 seconds, or 1 to 10 seconds after (c-1) and before (b-1) of the next cycle, or after (b-2) and before (c-2). The vibration frequency may be, for example, 0.0000001 Hz to 10 kHz, 0.000001 Hz to 1 kHz, 0.00001 Hz to 100 Hz, preferably 0.0001 Hz to 60 Hz, or 0.0001 Hz to 10 Hz. For example, any range of vibration frequencies can be used, such as 0.001Hz to 60Hz, 0.01Hz to 60Hz, 0.001Hz to 10Hz, 0.01Hz to 10Hz, 0.1Hz to 60Hz, 0.1Hz to 10Hz, 0.5Hz to 60Hz, 0.5Hz to 50Hz, 0.5Hz to 10Hz, 0.5Hz to 5Hz, 0.5Hz to 1Hz, 0.001Hz to 0.01Hz, 0.001Hz to 0.1Hz, 0.001Hz to 1Hz, 0.01Hz to 1Hz, 0.1Hz to 1Hz, 1Hz to 60Hz, 1Hz to 10Hz, 1Hz to 5Hz, etc.

[0051] Commercially available facial beauty devices, massagers, and the like use electromagnetic waves, such as RF waves, with frequencies of approximately 0.3 to 300 MHz, or ultrasonic waves with frequencies of approximately 1 to 7 MHz. Compared to these frequencies / vibrations, the vibration frequency employed by the method / device of the present invention is extremely low. Applying high vibration frequencies to the skin, as with conventional facial beauty devices, carries the risk of adverse effects such as redness, pressure marks, scars, pain, and inflammation. However, the use of vibration frequencies such as those employed in the present invention reduces these risks and enables non-invasive physical stimulation. The inventors discovered that, because too high a stretch rate and vibration frequency result in too strong a stimulation, it is preferable to gently stimulate the skin by adjusting these values ​​to appropriate levels.

[0052] Furthermore, it has been common technical knowledge among those skilled in the art that beauty devices utilizing mechanisms such as motors commonly used in this field can only select vibration frequencies above 60 Hz due to the mechanical mechanism of the motor. To adopt frequencies below 60 Hz, which is the limit of beauty devices in the prior art, such as frequencies below 60 Hz, 10 Hz, or 1 Hz, as in the present invention, required the creation of special machinery. Furthermore, there was a preconceived notion that such low vibration frequencies were "too weak" to achieve the effects of the present invention, and little research had been conducted on them to date. However, the inventors actually applied physical stimulation to the skin using frequencies that were extremely low compared to conventional technical knowledge, and surprisingly, good effects were achieved even with gentle stimulation at such low frequencies.

[0053] Although low- to medium-frequency EMS devices are commercially available, these are designed to act on deep layers, such as muscles and subcutaneous fat, and their effects on the surface layer of the skin, as in the present invention, are unknown. Furthermore, even with low frequencies, these devices can produce a tingling sensation when passing current, which differs from the present invention, which provides gentle stimulation to the skin. In contrast, the present invention enables a simple cosmetic treatment that directly applies stretching stimulation to the skin without applying energy such as ultrasound, current, or magnetic fields. Furthermore, stretching stimulation with such frequencies is gentle, yet has the effect of adjusting / improving the M1 / M2 balance, as described in the Examples. Therefore, the use of the method / device of the present invention is expected to prevent and / or improve photoaging and / or dermal pigmentation without adversely affecting the skin.

[0054] Physical stimuli may be achieved by instruments such as facial massagers, experimental devices, massage using human hands or instruments, or facial exercises, and may be achieved by contact or non-contact. In one embodiment, mechanically generated physical stimuli can be applied to the skin using a device equipped with a stimulus generator that generates physical stimuli and a stimulus imparting unit that imparts physical stimuli by contact or non-contact. Physical stimuli may be achieved by contact, for example, by pulling, pressing, tapping, kneading, or suctioning the skin, and / or by non-contact, for example, by displacing the skin by applying shock waves using ultrasound, air pressure, or water pressure. Examples of facial exercises include puffing out the cheeks and widening the eyes. Examples of massages include massages using hands, rollers, or other instruments by the subject receiving the treatment or by a practitioner such as a beauty consultant. However, the scope of the physical stimuli of the present invention is not limited.

[0055] An example of a device that can be used in the present invention is a cosmetic device equipped with a skin contact part that comes into contact with the user's skin and applies the weak physical stimulation of the present invention. For example, it may be composed of a gripping part and a skin stretching part or a skin pressing part. For example, the device shown on the left side of Figure 13 is designed so that the skin stretches at a specific frequency and stretch rate when the skin contact part comes into contact with the skin.

[0056] Furthermore, for example, the device of the present invention may include a power source, a stimulus generating unit, and a skin stimulation unit, wherein the power source generates an electrical signal, the stimulus generating unit converts the electrical signal from the power source into a physical stimulus to generate the physical stimulus, and the skin stimulation unit receives the physical stimulus generated by the stimulus generating unit and applies the physical stimulus to the user's skin.

[0057] For example, the device shown on the left side of Figure 13 comprises a gripping unit, a power source, a control unit that controls physical stimulation, a stimulation generating unit, and a skin contact unit that includes a skin stimulation unit and a skin fixation unit. The user holds the gripping unit and places the skin contact unit against the skin, fixes the skin with the skin fixation unit, and operates the control unit so that the electrical signal from the power source is converted into a physical stimulation by the stimulation generating unit, and the physical stimulation is transmitted to the skin stimulation unit, and the skin is stretched at a specific vibration frequency and stretch rate by the skin stimulation unit while being fixed to the skin fixation unit. For example, the stimulation generating unit may be driven by a motor or the like and convert the electrical signal into a physical stimulation. Furthermore, the skin stimulation unit shown on the left side of Figure 13 applies a stretching stimulation to the skin, but it may also apply a pressure stimulation to the skin, for example.

[0058] Alternatively, the device of the present invention may be a cosmetic device comprising a power source, a control unit for controlling physical stimulation, a stimulation generating unit, and a skin contact portion including a skin contact surface made of a sheet-like material. For example, the skin contact portion of such a cosmetic device is shown on the right side of Figure 13. The sheet-like material may be capable of passing an electric current and convert an electrical signal from the power source into a physical stimulation. Examples of such sheet-like materials include Dielectric Elastomer Actuator (DEA), conductive polymer, IPMC, PVC gel, and Mckinnen type.

[0059] The power source of the device of the present invention may be an internal or external power source, or may be rechargeable. Furthermore, the device of the present invention may use data stored on a mobile phone or in the cloud, or may be remotely operated wirelessly.

[0060] The physical stimulation may be achieved by applying physical stimulation by contact or non-contact as in the above method to achieve skin stretching. The physical stimulation may be applied in a direction parallel to the skin surface, i.e., horizontally, or in a direction perpendicular to the skin surface, i.e., vertically, or in any direction, such as diagonally or twisting.

[0061] The number of cycles for which physical stimulation is performed is not limited. For example, any number of cycles may be performed, such as 10 to 500 cycles, 20 to 400 cycles, 30 to 300 cycles, 40 to 200 cycles, or 50 to 100 cycles. For example, as described in the examples, 27 cycles may be sufficient.

[0062] Furthermore, any number of these cycles may be considered as one set, and any number of these sets may be performed, for example, 1 to 100 sets, 2 to 50 sets, or 3 to 10 sets.

[0063] The duration of physical stimulation is not limited either. For example, the stimulation may be repeated in cycles with or without rest periods for a fixed period of time such as 5 minutes to 3 hours, 10 minutes to 2 hours, or 30 minutes to 1 hour.

[0064] The time interval between cycles or sets is also not limited. For example, the stretching or pressure stimulation may be performed as one or more sets alone, or as one or more sets may be performed continuously or intermittently, regularly or irregularly, such as every day, every 2, 3, 4, 5, 6, or 7 days, or once every 1, 2, 3, or 4 weeks.

[0065] However, as long as a sufficient stimulation is achieved to exert the skin anti-photoaging and / or pigmentation inhibitory effect, the vibration frequency, stretching rate, number of cycles, and frequency are not limited to those described above. The waveform of the physical stimulation can also be set arbitrarily, such as a square wave, sine wave, triangular wave, sawtooth wave, etc.

[0066] Cosmetic treatments include, but are not limited to, any treatment believed to be effective in inhibiting photoaging and / or pigmentation, such as the application of cosmetics containing the agent of the present invention or other ingredients. Cosmetics refer to cosmetics applied to the skin, such as lotions, emulsions, serums, creams, and foundations, but are not limited to these. The term "cosmetics" is intended to include all products applied to the skin that are not directly intended to improve skin condition, including sunscreens. Alternatively, cosmetic treatments may involve physical stimulation of the skin, such as stretching, pressing, or massaging. Cosmetic treatments may be a one-time treatment or continuous treatments over several days or weeks. Cosmetic treatments may be performed privately or at beauty salons, cosmetic stores, esthetic salons, etc. [Example]

[0067] The present invention will now be described in more detail with reference to examples, although the present invention is not limited to these examples.

[0068] Experiment 1: Tissue staining Skin from the corners of the eyes of young people (20s-30s) and elderly people (60s-70s) experiencing photoaging (as shown in Figure 1a) from Caucasians was frozen and thinly sliced, and then stained with the macrophage markers shown below. (1) M1 macrophage staining: Double staining was performed with goat-derived anti-human CD86 antibody (R&D) and rabbit-derived anti-human CD11b antibody (Abcam) (Figure 1b). (2) Staining of M2 macrophages: Double staining was performed with mouse anti-human CD206 antibody (BD) or mouse anti-human CD163 antibody (Leica) and rabbit anti-human CD11b antibody (Abcam) (Figure 1c). (3) Staining of total macrophages: Double staining was performed using mouse-derived anti-human CD68 antibody (Abcam) and rabbit-derived anti-human CD11b antibody (Abcam).

[0069] Cells doubly positive for antibodies (1) to (3) were counted as macrophages within 200 μm of the epidermis (Fig. 1d). To investigate the relationship between M1 and M2 macrophages and collagen production, cells were double-stained with goat anti-human CD86 antibody (R&D) and rat anti-procollagen antibody (Millipore), or mouse anti-human CD206 antibody (BD) and rat anti-procollagen antibody (Millipore) (Fig. 1e, left).

[0070] Histological staining of (1)–(3) is shown in Figure 1b and c, a graph of the counting results is shown in Figure 1d, and the results of double staining with M1 and M2 macrophage antibodies and anti-procollagen antibodies are shown in Figure 1e (left). As shown in Figure 1b, c, and d, elderly subjects with photoaging exhibited a high concentration of M1 macrophages, while M2 macrophages were reduced. More specifically, as shown in Figure 1b, M1 macrophages were present only around blood vessels in young subjects, whereas they were scattered throughout the tissue in elderly subjects. Furthermore, as shown in Figure 1c, M2 macrophages were present throughout the tissue in young subjects, whereas their number was reduced in elderly subjects. Furthermore, as shown in Figure 1d, the total number of macrophages (M1 number + M2 number) did not change between young and elderly subjects; only the M1 / M2 balance changed. In young individuals, the ratio of M2 to M1 (number of M2 / number of M1) was within the range of approximately 5 / 5 to approximately 7 / 3, whereas in elderly individuals, the ratio of M2 to M1 decreased dramatically, resulting in a significant disruption of the M1 / M2 balance, which was significantly different from that in young individuals.

[0071] Furthermore, as shown in the two upper left photographs of Figure 1e, the positions of M1 macrophages and procollagen were misaligned in both young and elderly subjects, whereas the positions of M2 macrophages and procollagen were consistent in the two lower left photographs. This suggests that M1 may act on fibroblasts to promote collagen breakdown, while M2 may act to promote collagen production, as shown in the schematic diagram on the right of Figure 1e.

[0072] Experiment 2: M1 / M2 differentiation stimulation of THP-1 cells Following the method described in Non-Patent Document 1, the human-derived cell line THP-1 was induced to differentiate into M1 and M2 macrophages. Specifically, as shown in Figure 2a, THP-1 cells were cultured in RPMI1640 (Nakalai) supplemented with 1 mM Na Pyruvate (Nakalai), 2 mM L-Glutamine (Nakalai), and 10% FBS. Subsequently, 100 nM PMA (Abcam) was added and stimulated for 24 hours to differentiate into macrophages. For M1 differentiation, 100 ng / mL LPS (Sigma) and 20 ng / mL IFNγ (R&D) were added for 24 hours, and for M2 differentiation, 20 ng / mL IL-4 (R&D) and 20 ng / mL IL-13 (R&D) were added for 24 hours. Microscopic observation confirmed the morphological changes shown in Figure 2b.

[0073] In addition, mRNA was extracted from differentiated or undifferentiated cells, and real-time PCR was performed using TaqMan Gene expression assay with probes for IL-1beta, TNF-alpha, and IL-10 (Applied Biosystems) to quantify expression levels (Figure 2c, top). Furthermore, PCR was performed in the same way using the same CD86 antibody (R&D) and CD206 antibody (BD) as in Experiment 1 to quantify expression levels (Figure 2c, bottom). Each value was normalized by the expression level of GAPDH mRNA.

[0074] As shown in Figure 2c, macrophages differentiated by the method described in Experiment 2 were shown to produce inflammatory cytokines characteristic of M1 (IL-1beta, TNF-alpha) and anti-inflammatory cytokines characteristic of M2 (IL-10). Furthermore, these differentiated macrophages showed increased expression of CD86 and CD206, surface markers of M1 and M2 macrophages, respectively. These results confirmed that differentiation induction was successful. Therefore, M1 and M2 macrophages differentiated by the method of Experiment 2 and undifferentiated M0 macrophages were used in the following Experiments 3 and 4.

[0075] Experiment 3: Addition of M1 and M2 macrophage supernatant to fibroblasts (1) As shown in Figure 3a, THP-1 cells were differentiated into M1 or M2 cells or left undifferentiated in M0 cells using the same method as in Experiment 2. The supernatant was removed, the cells were washed once with PBS, and then cultured for 48 hours in medium. These supernatants, which contained secreted substances from M1 and M2 cells, such as pro- and anti-inflammatory cytokines, were added to neonatal fibroblasts. Cells supplemented with RPMI 1640 (Nakalai) were used as a control. After the addition of the supernatant, the fibroblasts were cultured for 72 hours, and the amount of procollagen in the supernatant was quantified using a PIP ELISA kit (TAKARA) (Figure 3b). Cell fractions were stained with a rabbit anti-human collagen antibody (CEDERLANE) and biotinylated hyaluronan-binding protein (HOKUDO) (Figure 3c). Furthermore, β-gal was used as a senescence indicator. After staining the cell nuclei with DAPI, intracellular β-gal was stained using a Senescence Detection Kit (Abcam) (Fig. 3d), and the numbers of β-gal-positive cells and DAPI-positive cells were counted (Fig. 3e).

[0076] Furthermore, to examine the contribution of M1 and M2 macrophages to melanin production, we added the supernatants of each macrophage to fibroblasts and cultured them in the same manner as above. Afterwards, we harvested the fibroblasts and isolated mRNAs. Real-time PCR was performed using probes for HGF, ET1, bFGF, IL-1alpha, SCF, and clusterin to quantify the mRNA expression levels (Fig. 3f).

[0077] The results are shown in Figures 3b–3f. Figure 3b shows the amount of procollagen after 72 hours of incubation in fibroblasts with the supernatants of each macrophage (M1, M2). Figure 3c shows the localization of collagen and hyaluronic acid in fibroblasts after 72 hours of incubation in the supernatants of each macrophage (M1, M2). Figures 3b and 3c demonstrate that M1 significantly suppresses collagen production. Figure 3d shows the intracellular β-gal levels in fibroblasts after 72 hours of incubation in the supernatants of each macrophage (M1, M2). Figure 3d suggests that M1 promotes senescence by increasing the number of β-gal-positive cells, while M2 suppresses senescence. Figure 3e shows a graph of the number of β-gal-positive and DAPI-positive cells. The right panel shows the total number of DAPI-positive cells per well. The right panel of Figure 3e suggests that M2 not only suppresses cellular senescence but also promotes cell proliferation. The left panel of Figure 3e shows the percentage (%) of β-gal-positive cells per total number of DAPI-positive cells, suggesting that M1 promotes senescence and cell death, while M2 inhibits senescence and promotes cell proliferation. Furthermore, as shown in Figure 3f, we found that mRNA expression of melanogenesis-related factors increased melanin production through M1, while suppressed melanin production through M2. As reported in Non-Patent Document 6, fibroblasts are known to secrete factors such as SCF and HGF upon light stimulation, such as UV light, leading to cell death. Furthermore, as reported in Non-Patent Documents 7-9, fibroblasts secrete factors such as HGF, ET1, bFGF, SCF, and clusterin upon light stimulation, which act directly or indirectly on melanocytes to produce melanin. Therefore, it is suggested that light-induced cell death and melanin production are due to an imbalance between M1 and M2.

[0078] Experiment 4: Addition of M1 and M2 macrophage supernatant to young and aged fibroblasts Next, to confirm whether the anti-aging effect of M2 macrophages is also useful for aged fibroblasts, i.e., whether they have a rejuvenating effect, M1 and M2 macrophage supernatants were added to young and old cells, and the differences in the effects of macrophage supernatants depending on the age of the fibroblasts were examined.

[0079] Specifically, each macrophage supernatant, collected in the same manner as in Experiment 3, was added to fibroblasts derived from newborn human foreskin (young-derived fibroblasts) and fibroblasts derived from 68-year-old humans (old-derived fibroblasts) and cultured for 72 hours. Subsequently, β-gal and DAPI staining was performed in the same manner as in Experiment 3, and the numbers of β-gal-positive and DAPI-positive cells were counted (Fig. 4a, 4b). Furthermore, M1 and M2 supernatants were added to young- and old-derived fibroblasts as in Experiment 3, and the fibroblasts were cultured for 72 hours. The amount of procollagen in the supernatants was quantified using a PIP ELISA kit (TAKARA) (Fig. 4c), and the fibroblasts were stained with a rabbit anti-human collagen antibody (CEDERLANE) and DAPI (Fig. 4d).

[0080] Figure 4a shows a β-gal staining diagram. Figure 4b shows a graph of the number of β-gal-positive cells and the total number of cells per well in young and old fibroblasts. The bottom panel of Figure 4a shows that the addition of M2 supernatant significantly reduced the number of β-gal-positive cells, suppressing senescence, even in old cells. Figure 4b also shows that M1 promotes senescence and M2 suppresses senescence, regardless of age. This is supported by the results shown in Figures 4c and 4d, where cells added with M1 supernatant produced significantly less collagen than cells added with M2 supernatant, regardless of age.

[0081] Experiment 5: Co-culture experiment of M1 macrophages, M2 macrophages and neonatal foreskin-derived fibroblasts The amount of collagen produced when neonatal foreskin-derived fibroblasts and the same number of M1 or M2 macrophages (prepared as in Experiment 2) were cultured in RPMI 1640 (Nakalai) was compared with that when neonatal foreskin-derived fibroblasts and half the number of M1 or M2 macrophages were cultured in RPMI. The amounts were stained with rabbit anti-human collagen antibody (CEDERLANE) and compared. Macrophages were also visualized by staining with mouse anti-human CD68 antibody (Abcam).

[0082] The results are shown in Figure 5. In neonatal foreskin-derived blastocytes, the addition of M1 supernatant reduced collagen, while the addition of M2 increased collagen. This indicates that even in young cells, an imbalance in the M1 / M2 balance affects collagen.

[0083] Experiment 6: Effects of M1 / M2 imbalance using a 3D skin model To investigate the effects of M1 / M2 macrophages on epidermal cells and fibroblasts, three types of 3D skin models with three-layer structures were created as shown in the table below.

[0084] [Table 1] The 3D skin model was created as follows: human dermal fibroblasts (0.2 × 10 cells) were placed in a cell culture insert (φ12 mm, average pore size of porous membrane: 0.4 μm). 6 The cells were seeded with 200 μM ascorbic acid 2-magnesium phosphate (APM) and 10% FBS-DMEM, and cultured for one week with a medium change every two days. A 0.5% type I collagen-10% FBS-DMEM solution containing human dermal fibroblasts was added to the control model, while 30,000 M1 and M2 macrophages differentiated as described in Experiment 2 were added to the M1 and M2 models, respectively. A collagen gel was then created on top of the human dermal fibroblasts and cultured for one to five days.

[0085] Furthermore, 5 × 10 epidermal keratinocytes were dispersed in Humedia-KG2 (Kurabo) medium. 5 The cells were seeded on top of the collagen gel at 100 cells / well, and a medium made by mixing Humedia-KG2 and 10% FBS-DMEM in a 1:1 ratio and adding 200 μM APM was added to the outside of the insert to the same liquid level as inside, and the cells were cultured for 3 days.

[0086] The medium inside the insert or glass ring was then removed, and the skin model medium (10% FBS-DMEM and Humedia-KG2 EGF(-) mixed 1:1 to prepare a 1.8 mM Ca solution) containing 200 μM APM, 10 μM N-hydroxy-2-[[(4-methoxyphenyl)sulfonyl]3-picolyl)amino]-3-methylbutanamide hydrochloride (CGS27023A (an MMP inhibitor)), and 10 μM BIPBIPU (a heparanase inhibitor) was added to the bottom of the insert. The insert was then cultured at an air-liquid boundary, with the inside of the insert exposed to air. The medium was changed every 2–3 days and the cells were cultured for 2 weeks.

[0087] After completing the culture, the skin model was fixed with 4% PFA / PBS (Nakarai) and stained with anti-CD206 antibody (Abcam), anti-CD68 antibody (Abcam), and anti-CD86 antibody (Abcam) in the same manner as in Experiment 1 to confirm the presence of M1 and M2 macrophages (Figure 6). The model was then stained with anti-p21 antibody (Abcam) and DAPI (VECTOR). The number of cells stained with DAPI in the epidermal cell layer and fibroblast layer was counted as the total cell count, and the number of cells that also stained with anti-p21 antibody was counted as the number of p21-positive cells. The ratio of p21-positive cells to the total cell count was calculated using the following formula.

number

[0088] The results are shown in Figures 7 and 8. As shown in these figures, in both the upper epidermal cell layer and the lower fibroblast layer adjacent to the intermediate layer, the number of p21-positive cells increased in the M1 model (M1) compared to the control model (cont), while it decreased in the M2 model (M2). This trend was consistent with Experiment 3, which used monolayer cultures. Therefore, it is suggested that even in a 3D skin model that is closer to human skin, M1 macrophages have the ability to promote senescence and cell death, while M2 macrophages have the ability to suppress senescence and cell death.

[0089] Experiment 7: Sunlight irradiation experiment on an ex vivo model using human skin Genoskin NativeSkin (skin taken from a 38-year-old woman) was cultured for one day in the provided culture medium after arrival. The next day, an optical filter was inserted into an Oriel 1000W solar simulator, and only UVA and UVB were irradiated at 11.5 J / cm. 2 The cells were irradiated and continued to be cultured in the accompanying culture medium (irradiation (+)). A sample that was not irradiated was used as a control (irradiation (-)). Five days after irradiation, the samples were collected, and M1 macrophages, M2 macrophages, and total macrophages were stained as in Experiment 1, and the numbers were counted and graphed.

[0090] The results are shown in Figure 9. When exposed to the solar simulator, the number of M1 cells increased the most. On the other hand, the increase in the number of M2 cells was much smaller than that of M1 cells. In other words, it was found that light stimulation disrupted the M1 / M2 balance, resulting in an increase in the proportion of M1 cells.

[0091] Experiment 8: Stretch stimulation experiment on ex vivo model using human skin Next, we investigated how to adjust or improve the M1 / M2 balance. Sample: Native Skin (skin taken from a 38-year-old woman) (6-well size, approximately 2-2.5 cm in diameter) manufactured by Genoskin was used. Stretching conditions: A stretching device was created, as shown in Figure 11, with gripping parts that grip both ends of the skin in the well, and the skin was stretched by pulling the gripping parts. The well containing the tissue piece was placed horizontally, and the gripping parts were operated to stretch the skin from both ends, stretching it to a 10% stretch rate at a speed of 10% / s as shown in Figure 10, and the sample was returned to its original, unstretched state at a recovery rate of 10% / s. This constituted one cycle, and a total of 90 cycles were performed over 30 minutes. These 90 cycles constituted one set, and a total of three sets (270 cycles in total) were performed over three hours, with rest periods of 30 minutes to one hour between sets. A sample without stretching stimulation was used as a control (control).

[0092] Observation method: For skin samples with or without stretching, M1 macrophages, M2 macrophages, and total macrophages were stained and counted in the same manner as in Experiment 1, except that total macrophages were stained only with rabbit anti-human CD11b antibody (Abcam). The percentage (%) of each macrophage (M1, M2) relative to the total number of all macrophages was calculated and graphed.

[0093] The results are shown in Figure 12. Compared to the control, the total number of macrophages and the number of M1 macrophages in the stretched samples did not change significantly, but the number of M2 macrophages increased significantly. This indicates that stretch improves the M1 / M2 balance.

[0094] Experiment 9: Effect of M1 and M2 macrophage supernatant on collagen In addition to Experiment 3, to examine the contribution of M1 and M2 macrophages to collagen degradation, the supernatants of each macrophage were added to fibroblasts and cultured in the same manner as in Experiment 3, and mRNA was collected after 72 hours. Real-time PCR was performed using probes for MMP-1, MMP-2, and IL-1β (Taqman probe, Applied Biosystems) as indicators of collagen degradation to quantify the mRNA expression levels of each (Fig. 14a).

[0095] Furthermore, to examine the contribution of M1 and M2 macrophages to collagen production and maturation, the supernatants of each macrophage were added to fibroblasts and cultured in the same manner as in Experiment 3, and mRNA was collected after 72 hours. As indicators of collagen production and maturation, real-time PCR was performed using probes for COL1A1, COL1A2, HSP47, and ADAMTS-2 (TaqMan probe, Applied Biosystems) to quantify the mRNA expression levels of each (Fig. 14b).

[0096] Figures 14a and 14b confirm that M1 contributes to collagen degradation, while M2 contributes to collagen production and maturation.

[0097] Experiment 10: Collagen degradation and production in an ex vivo model using young and aged human skin As in Experiment 1, temple skin from young (20s-30s) and elderly (60s-80s) Caucasians of the ages shown in Figure 15a was frozen and sliced ​​into thin sections. After thin sectioning, positive cells were counted in the same manner as in Experiment 1 using anti-procollagen antibody (millipore) and anti-fragmented collagen (AdipoGen) to stain collagen cleavage fragments on the 3 / 4 side, as well as the same CD68, CD11b, CD206, and CD86 antibodies as in Experiment 1.

[0098] The results are shown in Figure 15b, c, and d. Compared to the young group, the elderly group had a higher proportion of M1 macrophages and a lower proportion of M2 macrophages. However, regardless of age, there were more 3 / 4 collagen-positive macrophages than 3 / 4 collagen-positive fibroblasts, and there was a tendency for the number of 3 / 4 collagen-positive M2 macrophages to be greater than the number of 3 / 4 collagen-positive M1 macrophages.

[0099] Experiment 11: In vitro experiment demonstrating the ability of melanin to be taken up by the dermis THP-1 cells were differentiated into M1 and M2 macrophages using the same method as in Experiment 3. A melanin solution (Sigma, Melanin-BioReagent, Synthetic, suitable for cell culture) was dissolved in PBS and adjusted to 0.02% W / V and added to differentiated M1 and M2 macrophages, as well as fibroblasts (Kurabo). After 24 hours, the cells were washed with PBS, photographed under a microscope, and then harvested. The number of harvested cells was measured and quantified using Alamar Blue (Life Technologies) to determine the amount of melanin, as described below.

[0100] Melanin quantification: Alamar Blue, adjusted to a 1:10 ratio with the respective cell culture media (macrophages: RPMI 1640, fibroblasts: 1DMEM), was added and the cells were cultured at 37°C for 30 minutes. Then, 100 µl of the supernatant was collected per well, and fluorescence was measured using an Ascent (Thermo) at excitation / emission: 544 nm / 590 nm. After measurement, the cells were washed with PBS, and 1 M NaOH was added. The cells were then incubated at room temperature for 3 hours until completely thawed. The cell solution was measured at OD475 using a POWERSCAN HT (DS PHARMA BIOMEDICAL).

[0101] The results are shown in Figures 16a-c. The unit "melanin / almar blue" in the figures is the relative value ((2) / (1)) of the fluorescence intensity ((1)) of the supernatant obtained by staining cultured cells with alamar blue and measuring the fluorescence intensity at 544 nm / 590 nm, and the intensity ((2)) of the absorbance (measured at 475 nm) of the supernatant obtained by subsequently lysing the cells and dissolving the melanin. This indicates the melanin content per cell. These figures show that M2 phagocytosed a very large amount of melanin. This tendency was already observed after 24 hours, but the difference became even clearer when the cells were cultured and observed for up to 5 days (Figure 16c).

[0102] Experiment 12: Ex vivo experiment demonstrating the ability of melanin to be incorporated into the dermis Because Experiment 11 showed that M2 macrophages phagocytose more melanin than fibroblasts or M1 macrophages, the dermal layer of human skin counted in Experiment 11 was observed ex vivo using an LSM880 (Carl Zeiss). When observed under bright field, M2 macrophages in both young and old subjects were stained black with melanin, whereas no melanin was present near M1 macrophages. This demonstrated ex vivo that M2 macrophages ingest more melanin than fibroblasts or M1 macrophages in the dermis, and their numbers were counted and graphed (Figure 17).

[0103] As shown in Figure 17, the number of M2 macrophages that take up melanin is greater than the number of M1 macrophages in both old and young people, and there is little difference in this trend between age groups. These results suggest that because it is M2 macrophages that take up melanin in the dermis, increasing the proportion of M2 macrophages and adjusting the M1 / M2 balance can prevent and / or improve photoaging and / or dermal pigmentation.

[0104] Experiment 13: Search for M1 suppressing / M2 inducing agents Using the various markers listed in Figure 18, we searched for agents that can prevent and / or improve photoaging and / or dermal pigmentation by adjusting or improving the M1 / M2 balance, and found that lixivivir ginseng has a strong M2-inducing effect. The lixivivir ginseng extract (with or without hydrolysis) used here was prepared with reference to Japanese Patent No. 4781842 (Patent Document 9) and International Publication No. WO 2012 / 0571243 (Patent Document 10), and was prepared according to the following procedure. It should be noted that the procedure for preparing the lixivivir ginseng extract that can be used in the present invention is not limited to the procedure described below.

[0105] <Preparation of Peach Blossom Extract> Unground holly seeds (with the outer shell removed, if necessary) were immersed in 5 volumes (v / w) of acetone, ethyl acetate, or hexane and extracted for 10 days at room temperature. Residue was removed first through a nylon mesh (100 mesh), followed by filtration through filter paper. After removing the solvent from the filtrate using a rotary evaporator, 0.5–15 N NaOH was added to the mixture at a concentration of 100–300 g / L (50°C) while stirring with a stirring blade. The mixture was treated for 5 hours with stirring at 200 rpm. Subsequently, 5 N H2SO4 was gradually added to lower the pH to approximately 1 while stirring, and the separated oily substance was collected. The collected oily substance was washed with an equal volume of water to remove impurities, salts, and excess acid. The oily substance was dried under reduced pressure and named the alkali-treated product (hydrolyzed hollyl holly extract). The extract before alkali treatment (unhydrolyzed coriander extract) was used as a control.

[0106] THP-1 cells were cultured overnight at 37°C in an undifferentiated M0 state. Subsequently, 3 ppm of unhydrolyzed (unalkali-treated) lixiviene (control) dissolved in DMSO or lixiviene hydrolysate (0.3 ppm, 1 ppm, or 3 ppm) was added, and the cells were cultured for two nights at 37°C. Cells were harvested, RNA was extracted, and the expression levels of CD86, CCR7, TNF-alpha, CD206, CD163, IL-10, and GAPDH were quantified by real-time PCR. The expression level of each gene was divided by that of GAPDH. Lixiviene hydrolysate concentration-dependently reduced the expression levels of CD86 and TNF-alpha genes and increased the expression levels of CD206 and IL-10 genes. These results demonstrate that lixiviene hydrolysate inhibits M1 induction and promotes M2 induction (Figure 18).

[0107] Experiment 14: Increased melanin phagocytosis by citrus Immature (M0) THP-1 cells were treated with 3 ppm of unhydrolyzed (untreated with alkali) lixivivir (control) in DMSO, or with lixivivir hydrolysate at 1.0 ppm or 3.0 ppm. After culturing at 37°C for 48 hours, melanin solution (Sigma) was added and the cells were observed under a microscope (Nikon Bio Station CT).

[0108] The results are shown in Figures 19a-b. Thirty minutes after the addition of melanin, the lixivivir saccharin-added macrophages had begun to take up melanin (black arrow in Figure 19b). After a further three hours, there were still many cells in the control that had not taken up melanin, but in the lixivivir saccharin hydrolysate-added sample, all cells had taken up melanin.

[0109] It has been shown that the proportion of M1 increases due to photoaging (Experiment 7), and a higher proportion of M2 tends to result in a higher amount of skin collagen (Experiments 9 and 10) and higher pigment phagocytosis (Experiments 11 and 12). This suggests that haikushinine, which was screened as an M2 inducer, and the weak physical stimulation of the present invention, which has the effect of improving the M1 / M2 balance, have a strong effect of suppressing pigmentation in the dermis.

[0110] Experiment 15: Intracellular activity of macrophages M0, M1, and M2 macrophages were differentiated from THP-1 cells using the same method as in Experiment 2. The intracellular activity of these macrophages was measured using an extracellular flux analyzer XFe24 (for 24 wells) (hereafter referred to as the "flux analyzer") manufactured by Agilent Technologies (formerly Seahorse Bioscience) with reference to Nat Immunol, 2014. 15(9): pp. 846-855 (Non-Patent Document 14). The flux analyzer is a device that enables non-invasive, highly sensitive measurement of the state of glycolysis, the main cellular energy metabolic pathway, and mitochondrial aerobic respiration over time.

[0111] Oligomycin (ATP synthase inhibitor) ("Oligo"), FCCP (uncoupler), and rotenone (mitochondrial complex I inhibitor) plus antimycin A (mitochondrial complex III inhibitor) ("Rot+Ant") were added at the indicated times, and the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a flux analyzer. The basal OCR and ECAR values ​​were measured 18.3 minutes after measurement. Each measurement was corrected for the cell number obtained by counting cell nuclei stained with Hoechst.

[0112] The results showed that mitochondrial respiratory activity increased in the order of M2 macrophages, M0 macrophages, and M1 macrophages (Figure 20).

[0113] Experiment 16: Effect of Puccinia chinensis extract on intracellular activity of macrophages

[0114] M0, M1, and M2 cells were prepared by differentiation induction from THP-1 cells in the same manner as in Experiment 2. To M0 cells, 0.3 ppm, 1 ppm, or 3 ppm of a limonene extract (limonene hydrolysate) prepared in the same manner as in Experiment 13 was added. After 48 hours of incubation, the oxygen consumption rate (OCR) was measured using a flux analyzer. Each measurement value was corrected for the cell number obtained by counting cell nuclei stained with Hoechst (Figure 21).

[0115] As a result, it was revealed that the addition of the lily extract significantly increased the basal OCR value 18.3 minutes after the start of measurement (the third measurement value) (Figure 21) and the mitochondrial respiration value (the basal OCR value 18.3 minutes after the start of measurement minus the non-mitochondrial respiration (oxygen consumption not related to mitochondria) 95.1 minutes after the start of measurement) (Figure 22).

[0116] Experiment 17: Effects of each component of the lily of the valley extract on the intracellular activity of macrophages (1)

[0117] It is known that liquorice extract contains unsaturated fatty acids, such as ω-3 fatty acids linolenic acid and juniperonic acid, and ω-6 fatty acid linoleic acid. It has been reported that unsaturated fatty acids (EPA, DHA) act on inflammation-related genes in M1 cells and suppress inflammation (Non-Patent Document 15 (Cell Metab. 2017 Feb 7;25(2):412-427)). Therefore, we investigated the effect of unsaturated fatty acids contained in liquorice extract on the intracellular activity of macrophages.

[0118] M0 and M2 cells were prepared by differentiation induction from THP-1 cells in the same manner as in Experiment 2. Linoleic acid, linolenic acid, or juniperonic acid (3 ppm) was added to M0 cells. After 48 hours, the oxygen consumption rate (OCR) was measured using a flux analyzer in the same manner as in Experiment 15. Each measurement value was corrected for the number of cells obtained by counting cell nuclei stained with Hoechst.

[0119] As a result, the basal OCR and mitochondrial respiration values ​​of M0 macrophages treated with linoleic acid, linolenic acid, and juniperonic acid contained in the lily of the valley extract were not significantly different from the control treated with only solvent (DMSO) (Figure 23).

[0120] Experiment 18: Effects of each component of the lily of the valley extract on the intracellular activity of macrophages (2)

[0121] The basal OCR and mitochondrial respiration values ​​of M0 were examined when the incubation time after the addition of the citrus extract or unsaturated fatty acids was changed to 24 hours. The experimental method was the same as that described in Experiment 17, except that the incubation time after the addition of the citrus extract or unsaturated fatty acids was changed to 24 hours.

[0122] As a result, no increase in basal OCR or mitochondrial respiration was observed 24 hours after the addition of the agave extract or each unsaturated fatty acid (FIG. 24).

[0123] Experiment 19: Effects of components contained in limonene extract on intracellular activity of macrophages during M1 differentiation

[0124] M0 cells were induced to differentiate from THP-1 cells in the same manner as in Experiment 2, and the changes in intracellular activity when liquor extract or unsaturated fatty acids (linoleic acid, linolenic acid, or juniperonic acid) were added during the process of further inducing differentiation into M1 cells were analyzed using a flux analyzer (Figure 25).

[0125] When M0 cells, which were differentiated from THP-1 cells, were further induced to differentiate into M1 cells, the oxygen consumption rate (OCR value) and extracellular acidification rate (ECAR value) were measured using a flux analyzer 24 hours after the addition of limonene extract or unsaturated fatty acids, in the same manner as in Figure 15.

[0126] As a result, significant increases in basal OCR and mitochondrial respiration were observed only when the liquor extract was added (Figures 26(A) and (B)). Only linolenic acid, which has been reported to have strong anti-inflammatory effects, was observed to have a strong inhibitory effect on ECAR values, resulting in an increase in the OCR / ECAR value.

[0127] Mitochondrial respiration in macrophages undergoing M1 differentiation was significantly increased by the citrus extract, but no significant increase was observed with the unsaturated fatty acids contained in the citrus extract alone, suggesting that the effect of increasing mitochondrial respiration may be a specific effect due to the mixture of various components contained in the citrus extract.

[0128] Experiment 20: Confirmation of SDF-1α in M1 and M2 macrophage supernatants

[0129] It has been reported that the expression level of SDF-1α (CXCL12) is reduced in fibroblasts present beneath skin pigmentation sites, and that a decrease in SDF-1 levels may be related to the formation of pigmentation on the skin (Non-Patent Document 16 (Theranostics. 2018 Sep 9;8(17):4620-4632)). Therefore, we investigated the expression level of SDF-1α (CXCL12) in M1 / M2 macrophages themselves.

[0130] THP-1 cells were differentiated into M1 and M2 cells using the same method as in Experiment 2. The supernatant was then removed, the cells were washed once with PBS, and RPMI (0.5% FBS) was added and the cells were cultured for 24 hours. The amount of SDF-1α (CXCL12) in the supernatant was examined using a cytokine array (RayBiotech) (Figure 27(A)).

[0131] As a result, it was revealed that the M2 supernatant contained more SDF-1α (CXCL12) than the M1 supernatant (FIG. 27(B)).

[0132] Experiment 21: Addition of M1 and M2 macrophage supernatant to fibroblasts (2)

[0133] We investigated the effects of factors produced by M1 and M2 macrophages on the expression of SDF-1α (CXCL12) in fibroblasts (Figure 28(A)).

[0134] THP-1 cells were differentiated into M1 and M2 cells using the same method as in Experiment 2. After removing the supernatant and washing once with PBS, the cells were cultured for 48 hours in RPMI (containing 10% FBS and 500 μM ascorbic acid). The supernatants, which contained secreted substances from M1 and M2 cells, such as inflammatory and anti-inflammatory cytokines, were added to skin fibroblasts. As a control, cells supplemented with RPMI (containing 10% FBS and 500 μM ascorbic acid) were used. After the addition of the supernatant, the fibroblasts were cultured for 72 hours. The fibroblasts were then harvested, mRNA was extracted, and the expression level of SDF-1α (CXCL12) was analyzed by RNA-seq (Figure 28(A)).

[0135] RNA-seq reads were mapped to the human genome (hg19) using STAR. The resulting count data were subjected to size correction and log2 transformation between each sample after filtering out low-expression genes. The log2(CPM) calculated for each sample using the above method was compared between two groups using a quasi-likelihood F-test using the glmQLFit and glmQLFTest functions of the R package edgeR. After correcting for multiple comparisons using the Benjamini-Hochberg method, q-values ​​were calculated.

[0136] As a result, it was revealed that the amount of SDF-1a expression in fibroblasts to which M1 supernatant had been added was significantly reduced (FIG. 28(B)).

[0137] These results suggest that photoaging and / or dermal pigmentation can be prevented and / or improved by adjusting or improving the M1 / M2 balance. Adjusting or improving the M1 / M2 balance can be achieved by applying stretching or pressure stimulation or by applying citrus extract, etc., and is expected to ultimately prevent and / or improve photoaging and / or dermal pigmentation.

Claims

1. An agent for preventing and / or improving photoaging and / or dermal pigmentation by increasing the ratio of M2 macrophages to M1 macrophages, comprising a citrus extract as an active ingredient.

2. 1. A cosmetic method for preventing and / or ameliorating photoaging and / or dermal pigmentation in a subject by increasing the ratio of M2 macrophages to M1 macrophages, comprising: (a) applying the agent according to claim 1 to the skin of a subject; Cosmetic procedures, excluding medical procedures, including:

3. The cosmetic method further includes a step of applying a weak physical stimulus to the skin, wherein the step of applying the physical stimulus includes: (b-1) stretching the skin of the subject to a stretch rate of 0.1% or more and 50.0% or less, wherein the stretch rate is: [Equation 1] (In the formula, fixed points A and B are arbitrary positions on the epidermis or the matrix to which the epidermis is attached, and the line passing through fixed points A and B is parallel to the stretching direction.) is calculated as: (c-1) restoring the subject's skin from a stretched state; and / or (b-2) pressing the skin of the subject by 1 μm to 1000 μm; and (c-2) restoring the skin of the subject from the pressure state; Including, wherein the cycles of (b-1) and (c-1) and / or (b-2) and (c-2) are carried out at a frequency of 60 Hz or less; The cosmetic method according to claim 2, comprising:

4. A beauty device for use in the beauty method according to claim 3, The device comprises: a stimulus generating unit that generates a physical stimulus; a stimulus applying unit that applies the physical stimulus generated by the stimulus generating unit to the skin, Here, the device is a device for performing a step of applying a weak physical stimulus to the skin, and the step includes: (i-1) stretching the skin to a stretch rate of 0.1% or more and 50.0% or less; and (ii-1) restoring the skin from a stretched state; and / or (i-2) pressing the skin by 1 μm to 1000 μm; and (ii-2) recovering the skin from the pressure state; and performing a cycle including the cycle at a frequency of 60 Hz or less, Here, the extension rate is calculated by the above formula 1. The cosmetic device.

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