JP2024037928A - Method, device, and anti-photoaging and/or dermal pigmentation inhibitor for preventing and/or improving photoaging and/or dermal pigmentation, screening method for anti-photoaging and/or dermal pigmentation inhibitors, evaluation method for anti-photoaging and/or dermal pigmentation-inhibiting cosmetic treatments, and evaluation method for photoaging and/or dermal pigmentation level - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide agents and methods for preventing and/or improving photoaging and/or dermal pigmentation.

SOLUTION: Provided is an anti-photoaging and/or dermal pigmentation inhibitor for preventing and/or improving photoaging and/or dermal pigmentation by increasing the ratio of M2 macrophages to M1 macrophages. Provided is a cosmetic method for preventing and/or improving photoaging and/or dermal pigmentation by increasing the ratio of M2 macrophages to M1 macrophages, the cosmetic method not comprising a treatment by a doctor or medical professional, in which increasing the ratio of M2 macrophages to M1 macrophages is achieved by applying to the skin a cosmetic containing anti-photoaging and/or dermal pigmentation inhibitors containing tranexamic acid methylamide, Hypericum erectum extract, Thymus vulgaris L. extract, Phellodendron amurense extract, and/or Eucalyptus extract.

SELECTED DRAWING: Figure 1d

COPYRIGHT: (C)2024,JPO&INPIT

Description

The present invention provides a method, a device, and an anti-photoaging and/or dermal pigmentation inhibitor for preventing or improving photoaging and/or dermal pigmentation by adjusting the M1/M2 balance, as well as an anti-photoaging and/or dermal pigmentation inhibitor. The present invention relates to a method for screening anti-photoaging and/or dermal pigmentation inhibitors as indicators, a method for evaluating cosmetic treatments for anti-photoaging and/or dermal pigmentation inhibition, and a method for evaluating the degree of photoaging and/or dermal pigmentation.

The aging phenomenon of human skin can be broadly divided into ``natural aging'' and ``photoaging.'' Photoaging is a phenomenon that is observed in areas that are exposed to light, and is skin-specific. Photoaging is thought to be the cause of the production of active oxygen and damage to cellular DNA caused by the effects of UV rays, which damages skin fibrous tissue and causes wrinkles and sagging skin to appear. For example, photoaging of the skin causes phenomena such as a decrease in collagen fibers and degeneration of elastin elastic fibers. In addition, melanocytes are also damaged and produce a large amount of melanin pigment, which causes age spots.

Pigmentation, including age spots and dullness, is caused by the accumulation of melanin produced by melanocytes in the basal layer of the epidermis. Melanin normally exists in the epidermis and basal layer, but because the epidermis turns over at a relatively rapid cycle, such melanin is easily excreted. On the other hand, melanin may exist in the dermis layer due to reasons such as melanin falling into the dermis through gaps in the basement membrane. The turnover cycle of dermal cells is much slower than that of the epidermis, so melanin often accumulates without being excreted. For these reasons, it is extremely difficult to improve dermal pigmentation.

As an anti-photoaging beauty method, for example, Patent Document 1 discloses an agent for preventing or suppressing skin photoaging by preventing inhibition of leukocyte elastase. Patent Document 2 discloses a photoaging inhibitor composition characterized by containing a plant extract of the genus Paffia of the family Amaranthaceae, which has a collagen synthesis promoting effect, and an animal-derived collagen peptide.

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

It has also been suggested that inflammation plays a role in 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 phagocytosis by macrophages can be used to improve dermal pigmentation, and Patent Document 15 suggests that dermal stains can be improved by attracting macrophages to fibroblasts that have taken up melanin that has fallen into the dermis and phagocytizing them. Disclose preventive/improving agents.

Patent No. 5657723 Japanese Patent Application Publication No. 2017-203004 Japanese Patent Application Publication No. 2018-177805 Special Publication No. 2014-504629 Japanese Patent Application Publication No. 2015-140334 Patent No. 6178088 Patent No. 6273304 Patent No. 5744409 Japanese Patent Application Publication No. 2018-140953 Japanese Patent Application Publication No. 2019-043855 Japanese Patent Application Publication No. 2013-053130 Japanese Patent Application Publication No. 09-187248 Japanese Patent Application Publication No. 2003-261455 Japanese Patent Application Publication No. 09-227367 Japanese Patent Application Publication No. 2018-072098 Patent No. 6535146 Japanese Patent Application Publication No. 2019-031482 Japanese Patent Application Publication No. 2007-063192

Journal of the American College of Cardiology, Vol. 62, No. 20, 2013, November 12, 2013:1890-901 Experimental & Molecular Medicine (2014) 46, e70; doi:10.1038/emm.2013.135 NATURE, VOL495, 28 MARCH 2013, pp524-530, doi:10.1038/nature11930 Journal of Investigative Dermatology, 2009 April; 129(4):1016-25. Epub 2008 Oct 9. Stem Cell Research & Therapy (2018) 9:88, https://doi.org/10.1186/s13287-018-0821-5 Journal of the European Academy of Dermatology and Venereology 2011 European Academy of Dermatology and Venereology, 2012, 26, 1577-1580 British Journal of Dermatology, 2005, 153, pp733-739 Pigment Cell Melanoma Research, Vol. 27, Issue 3, pp502-504 Ann Dermatol Vol. 28, No. 3, pp279-289, 2016 Endocrinology, 2011. 152(10): pp3779-90 British Journal of Dermatology, 2005. 153 Suppl 2: pp37-46 Experimental Dermatology, 2011. 20(11): pp953-5 Fujifilm Research & Development (No.55-2010):pp33-37

The object of the present invention is to provide a method, a device, an anti-photoaging and/or dermal pigmentation inhibitor, an anti-photoaging and/or dermal pigmentation inhibitor for preventing and/or improving photoaging and/or dermal pigmentation. The present invention provides a method for screening agents, a method for evaluating anti-photoaging and/or anti-dermal pigmentation cosmetic treatments, and a method for evaluating the degree of photoaging and/or dermal pigmentation.

（式中，定点AおよびBは，表皮または表皮が接着しているマトリックス上の任意の位置であり，ここで定点AとBを通る直線が伸展方向と平行である）
で算出される，（1）又は（2）に記載の方法。
（4）M1/M2バランスを調整することにより光老化及び／又は真皮色素沈着を予防及び／又は改善するための美容装置であって，
前記装置は，
物理刺激を生ずる刺激発生部と，
刺激発生部で発生した物理刺激を皮膚に付与する刺激付与部とを備え，
ここで，当該装置は，皮膚に対して微弱な物理刺激を適用する工程を行うための装置であり，ここで当該工程は，例えば，
（ａ）皮膚を0.1％以上50.0％以下の伸展率まで伸展すること；及び
（ｂ）伸展状態から回復すること；
並びに／又は，
（ａ－１）前記対象の皮膚を1μm～1000μm押圧すること；及び
（ｂ－１）前記対象の皮膚を押圧状態から回復させること；
を含むサイクルを60Ｈｚ以下の振動数で行うことであり，
ここで，伸展率は，上記式１により算出される，前記美容装置。
（5）抗光老化及び／又は真皮色素沈着抑制剤のスクリーニング方法であって，
生体試料に候補薬剤を施すこと；
候補薬剤を施した前後の生体試料におけるM1/M2バランスを測定すること；及び
前記候補薬剤を施した生体試料におけるM1/M2バランスが該薬剤を施す前と比較して改善する場合，前記薬剤に抗光老化及び／又は真皮色素沈着抑制作用があると評価すること；
を含む，前記方法。
（6）抗光老化及び／又は真皮色素沈着抑制美容処置の評価方法であって，
皮膚試料に美容処置を施すこと；
美容処置を施した前後の皮膚試料におけるM1/M2バランスを測定すること；及び
前記美容処置を施した皮膚試料におけるM1/M2バランスが該処置を施す前と比較して改善する場合，前記処置に抗光老化及び／又は真皮色素沈着抑制作用があると評価すること；
を含む，前記方法。
（7）1又は複数のコンピュータにより実行される光老化及び／又は真皮色素沈着度の評価方法であって，
あらかじめ設定した皮膚のM1/M2バランスの基準値に関するデータを取得する手順；
対象の皮膚のM1/M2バランスに関するデータを取得する手順；
前記基準値を参照し，前記対象の皮膚のM1/M2バランスに関するデータと比較して計算する手順；
前記計算手順による計算結果に基づいて前記皮膚の光老化及び／又は真皮色素沈着抑制度を評価する手順；及び
前記評価手順による評価結果を表示する手順；を有する，前記方法。
（8）光老化及び／又は真皮色素沈着度を評価するシステムであって，
あらかじめ設定した皮膚のM1/M2バランスの基準値に関するデータを記憶するデータベース部；
対象の皮膚のM1/M2バランスに関するデータを入力するデータ入力部；
前記データベース部で記憶されている基準値を参照し，前記データ入力部により入力された前記対象の皮膚のM1/M2バランスに関するデータと比較して計算する計算部；
前記計算部による計算結果に基づいて前記皮膚の光老化及び／又は真皮色素沈着度を評価する評価部；及び
前記評価部による評価結果を表示する表示部を有する，前記システム。
（9）M1/M2バランスを測定するための薬剤を含む，抗光老化及び／又は真皮色素沈着抑制剤のスクリーニングキット。
（10）M1/M2バランスを調整することにより光老化及び／又は真皮色素沈着を予防及び／又は改善するための抗光老化及び／又は真皮色素沈着抑制剤。
（11）以下の：

｛式中，Ｒ₁およびＲ₂は同一または異なり，水素原子，炭素数１～１８の直鎖状または分岐状アルキル基，炭素数５～８のシクロアルキル基，ベンジル基または下記式：

（但し，Ｘは低級アルキル基，低級アルコキシ基，ヒドロキシ基，アミノ基，ハロゲン原子を示し，ｎ＝０～３である）をそれぞれ示す｝
で表される化合物，又はその塩，
オトギリソウ抽出物，タイム抽出物，オウバク，及び／又はユーカリ抽出物を含む，(10)に記載の抗光老化及び／又は真皮色素沈着抑制剤。
（12）以下の：

｛式中，Ｒ₁およびＲ₂は同一または異なり，水素原子，炭素数１～１８の直鎖状または分岐状アルキル基，炭素数５～８のシクロアルキル基，ベンジル基または下記式：

（但し，Ｘは低級アルキル基，低級アルコキシ基，ヒドロキシ基，アミノ基，ハロゲン原子を示し，ｎ＝０～３である）をそれぞれ示す｝
で表される化合物，又はその塩，
オトギリソウ抽出物，タイム抽出物，オウバク，及び／又はユーカリ抽出物を含む，M1/M2バランス調整／改善剤。
（13）(10)～(12)のいずれか1項に記載の薬剤を含む組成物。
（14）M1/M2バランスを調整することは，(10)～(12)のいずれか1項に記載の薬剤を投与することにより達成される，（1）又は（2）に記載の方法。 As a result of intensive research into the inhibitory effects on photoaging and dermal pigmentation, the present inventors found that an imbalance in the M1/M2 balance of macrophages is associated with photoaging and dermal pigmentation. Based on such findings, anti-photoaging and/or dermal pigmentation inhibitors and methods for evaluating anti-photoaging and/or dermal pigmentation-inhibiting cosmetic treatments for preventing and/or ameliorating photoaging and/or dermal pigmentation. established. Using such a method, we have also developed a new cosmetic method, device, and anti-photoaging and/or dermal pigmentation inhibitor that have anti-photoaging and/or dermal pigmentation-inhibiting effects.
The following inventions were completed:
(1) A beauty method for preventing and/or improving photoaging and/or dermal pigmentation by adjusting the M1/M2 balance.
(2) The method according to (1), wherein adjusting the M1/M2 balance means increasing the ratio of M2 to M1.
(3) Adjusting the M1/M2 balance includes a step of applying a weak physical stimulus to the skin, where the step includes, for example, (a) stretching the target skin by 0.1% or more and 50.0% or less; (b) recovering from the extended state;
and/or
(a-1) Pressing the subject's skin by 1 μm to 1000 μm; and (b-1) recovering the subject's skin from the pressed state;
This is achieved by applying physical stimulation to the subject's skin that includes cycles at a frequency of 60Hz or less.
Here, the extension rate is

(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 straight line passing through fixed points A and B is parallel to the stretching direction.)
Calculated by the method described in (1) or (2).
(4) A beauty device for preventing and/or improving photoaging and/or dermal pigmentation by adjusting the M1/M2 balance,
The device includes:
a stimulus generating part that generates a physical stimulus;
and a stimulation applying part that applies physical stimulation generated in the stimulation generating part 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 here includes, for example,
(a) stretching the skin to a stretching rate of 0.1% or more and 50.0% or less; and (b) recovering from the stretched state;
and/or
(a-1) Pressing the subject's skin by 1 μm to 1000 μm; and (b-1) recovering the subject's skin from the pressed state;
It is a cycle including
Here, the extension rate is calculated by the above formula 1 in the beauty device.
(5) A screening method for anti-photoaging and/or dermal pigmentation inhibitors, comprising:
applying a candidate drug to a biological sample;
measuring the M1/M2 balance in the biological sample before and after administering the candidate drug; and if the M1/M2 balance in the biological sample treated with the candidate drug improves compared to before administering the drug; To be evaluated as having anti-photoaging and/or dermal pigmentation suppressing effects;
The method described above.
(6) An evaluation method for anti-photoaging and/or dermal pigmentation suppression cosmetic treatment, comprising:
performing cosmetic treatments on skin samples;
Measuring the M1/M2 balance in the skin sample before and after the cosmetic treatment; and if the M1/M2 balance in the skin sample after the cosmetic treatment improves compared to before the treatment, the treatment To be evaluated as having anti-photoaging and/or dermal pigmentation suppressing effects;
The method described above.
(7) A method for evaluating photoaging and/or dermal pigmentation degree performed by one or more computers,
Procedure for acquiring data regarding the preset reference value of skin M1/M2 balance;
Procedures for obtaining data regarding the M1/M2 balance of the subject's skin;
A step of calculating by referring to the reference value and comparing it with data regarding the M1/M2 balance of the subject's skin;
The method described above, comprising: a step of evaluating the degree of inhibition of photoaging and/or dermal pigmentation of the skin based on the calculation result of the calculation step; and a step of displaying the evaluation result of the evaluation step.
(8) A system for evaluating photoaging and/or dermal pigmentation,
A database unit that stores data regarding preset reference values of skin M1/M2 balance;
A data input section for inputting data regarding the M1/M2 balance of the target skin;
a calculation unit that refers to a reference value stored in the database unit and calculates by comparing it with data regarding the M1/M2 balance of the subject's skin input by the data input unit;
The system further comprises: an evaluation section that evaluates the degree of photoaging and/or dermal pigmentation of the skin based on calculation results by the calculation section; and a display section that displays the evaluation results by the evaluation section.
(9) A screening kit for anti-photoaging and/or dermal pigmentation inhibitors, including a drug for measuring M1/M2 balance.
(10) An anti-photoaging and/or dermal pigmentation inhibitor for preventing and/or improving photoaging and/or dermal pigmentation by adjusting the M1/M2 balance.
(11) The following:

{In the formula, R ₁ and R ₂ are the same or different and are a hydrogen atom, a linear or branched alkyl group having 1 to 18 carbon atoms, a cycloalkyl group having 5 to 8 carbon atoms, a benzyl group, or the following formula:

(However, X represents a lower alkyl group, a lower alkoxy group, a hydroxy group, an amino group, or a halogen atom, and n = 0 to 3)}
A compound represented by or a salt thereof,
The anti-photoageing and/or dermal pigmentation inhibitor according to (10), which contains Hypericum perforatum extract, thyme extract, Aspericula extract, and/or Eucalyptus extract.
(12) The following:

{In the formula, R ₁ and R ₂ are the same or different and are a hydrogen atom, a linear or branched alkyl group having 1 to 18 carbon atoms, a cycloalkyl group having 5 to 8 carbon atoms, a benzyl group, or the following formula:

(However, X represents a lower alkyl group, a lower alkoxy group, a hydroxy group, an amino group, or a halogen atom, and n = 0 to 3)}
A compound represented by or a salt thereof,
M1/M2 balance adjusting/improving agent containing Hypericum perforatum extract, thyme extract, Aspergillus perforatum extract, and/or Eucalyptus extract.
(13) A composition containing the drug according to any one of (10) to (12).
(14) The method according to (1) or (2), wherein adjusting the M1/M2 balance is achieved by administering the drug according to any one of (10) to (12).

With the method, device, and drug of the present invention, photoaging and/or dermal pigmentation can be prevented and/or improved by adjusting the M1/M2 balance. Furthermore, by using the M1/M2 balance as an index, it becomes possible to objectively evaluate the degree of photoaging and/or dermal pigmentation, and based on this method, it is possible to evaluate photoaging and/or dermal pigmentation. Drugs and cosmetic treatments for prevention and/or improvement can be explored.

Figure 1a shows the types of macrophage markers used in Experiment 1, as well as the age, skin type, and gender of the subjects. Figure 1b is a micrograph showing macrophages (CD11b: red) and M1 macrophages (CD86: green) in skin tissue of young and elderly subjects. Figure 1c is a micrograph showing macrophages (CD11b: red) and M2 macrophages (CD206: green) in skin tissue of young and elderly subjects. Figure 1d is a graph showing the number of M1, M2 macrophages and the total number of all macrophages in skin tissue of young and elderly subjects (cells/mm ² ). The left panel in Figure 1e is a micrograph showing co-staining of M1 macrophages (CD86: red) or M2 macrophages (CD206: red) and procollagen (green) in skin tissues of young and elderly subjects. The rectangles in each of the four panels shown on the left are enlargements of characteristic parts. The right figure in Figure 1e is a schematic diagram of the relationship between M1, M2, fibroblasts, and collagen production/destruction. Figure 2a shows an outline of the method for Experiment 2. Figure 2b is a micrograph of M0, M1 and M2 macrophages induced in Experiment 2. The upper panel of FIG. 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 panel of FIG. 2c is a graph showing the expression levels of mRNA of cell surface markers (M1: CD86, M2: CD206) of M1 and M2 macrophages. It is corrected by the GAPDH mRNA expression level and shown as a relative value (%) with M0 as 100%. Figure 3a shows an outline of the method for Experiment 3. Figure 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 is a micrograph showing collagen (red) and hyaluronic acid (green) in fibroblasts supplemented with M1 and M2 macrophage supernatants according to Experiment 3. Figure 3d is a photomicrograph showing β-galactosidase (β-Gal) in fibroblasts supplemented with M1 and M2 macrophage supernatants according to Experiment 3. Figure 3e shows the results of the senescence-associated β-galactosidase (SA β-Gal) assay in fibroblasts supplemented with M1 and M2 macrophage supernatants according to Experiment 3 (left panel: SA β-gal relative to the total number of DAPI-positive cells). It is a graph showing the percentage of positive cells (%)) and the total number of DAPI-positive cells per well (right figure). Figure 3f shows the melanogenesis-enhancing factors (HGF, ET1, bFGF, IL-1alpha, SCF) and melanogenesis-inhibiting factors (clusterin, DKK1) in fibroblasts to which M1 and M2 macrophage supernatants were added according to Experiment 3. Fig. 2 is a graph showing the mRNA expression level of . Figure 4a is a photomicrograph showing β-Gal in young and old fibroblasts supplemented with M1 and M2 macrophage supernatants according to experiment 4. Figure 4b shows the results of the SA β-Gal assay (SA β-gal relative to the total number of DAPI-positive cells) in young (top) and old fibroblasts (bottom) supplemented with M1 and M2 macrophage supernatants according to experiment 4. FIG. 3 is a graph showing the percentage of positive cells (%) and the total number of DAPI-positive cells per well. Figure 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 is a micrograph showing collagen (red) and DAPI-stained nuclei (blue) in young and old fibroblasts supplemented with M1 and M2 macrophage supernatants according to Experiment 3. 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 according to Experiment 5. The upper left photo shows fibroblasts cultured without the addition of macrophage supernatant. FIG. 6 is a micrograph showing macrophages (CD68: red) and M1 macrophages (CD86: green) or M2 macrophages (CD206: green) in the 3D model created according to Experiment 6. The triangle area indicates the area where CD68 and CD86 or CD68 and CD206 are double stained. Figure 7 is a micrograph showing the nucleus (blue) stained with p21 (red) and DAPI in the 3D model created according to Experiment 6. Figure 8 is a graph showing the ratio (%) of the number of p21-positive cells 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 according to Experiment 6. . Figure 9 is a graph showing the number of total macrophages, 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 as 100% (bar on the left: irradiation (-)), and the relative value (%) of the cell number of each macrophage with irradiation is expressed as the bar on the right (irradiation (-)). +)) Shown. Figure 10 shows the stretch stimulation settings used in Experiment 8. Figure 11 shows the equipment and skin samples used in Experiment 8. Figure 12 shows a comparison of the numbers of M1 and M2 macrophages and the total number of all macrophages when stretch stimulation was applied to the ex vivo skin model (stretch) and when it was not applied (control) according to Experiment 8. The figure on the left is a graph showing the number of macrophages per 1 mm ² of skin sample (cells/1 mm ² ). The figure on the right is a graph showing the ratio (%) of each macrophage (M1, M2) to the total number of macrophages. Figure 13 shows the results of the screening performed in Experiment 9. The bars indicate the CD86 expression level (CD86/GAPDH value) when each concentration of Hypericum perforatum extract (0.1%), thyme extract (0.1%), and tranexamic acid methylamide (0.03%, 0.06%) was added, and the control (CD86/GAPDH value). This is a graph showing relative values (%) when (cont) is set to 100%. FIG. 14 shows an example of the device of the present invention. Figure 15 shows the results of the screening performed in Experiment 10. The bars represent the CD86 expression level (CD86/GAPDH value) when adding each concentration of Aspergillus extract (0.01%) and Eucalyptus extract (0.1%), and the relative value (CD86/GAPDH value) when the control (cont) is taken as 100%. %). Figure 16a shows 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 according to Experiment 11. This is a graph showing relative values (%) when the results in 100% are taken as 100%. Figure 16b shows the mRNA expression levels of each collagen production/maturation factor (COL1A1, COL1A2, HSP47, and ADAMTS-2) in fibroblasts to which M1 and M2 macrophage supernatants were added according to Experiment 11. This is a graph showing relative values (%) when 100%. Figure 17a shows the age of each subject from whom samples were obtained in Experiment 12, by young and old groups. FIG. 17b is a graph showing the number of M1 and M2 macrophages (cells/mm ² ) in the skin tissue (50 μm from just below the basement membrane) of young and elderly subjects in Experiment 12. FIG. 17c is a graph showing the number of M1 and M2 macrophages (cells/mm ² ) in the skin tissue (200 μm from just below the basement membrane) of young and elderly subjects in Experiment 12. FIG. 17d is a graph showing the number of CD68-negative cells, M1, and M2 macrophages showing 3/4 collagen positivity in the skin tissues of young and elderly subjects in Experiment 12 (cells/mm ² ). Figure 18a shows the appearance of fibroblasts, M1 macrophages, and M2 macrophages 24 hours after melanin addition in Experiment 13. Cells that phagocytose melanin have a circular shape as shown in the figure on the right. Figure 18b shows the amount of melanin per cell (melanin/almar blue) taken up by fibroblasts, M1 macrophages, and M2 macrophages 24 hours after melanin addition in Experiment 13. Figure 18c shows the appearance of M1 macrophages and M2 macrophages 5 days after melanin addition in Experiment 13. The upper part of FIG. 19 is a graph showing the number of melanophagocytic M1 macrophages and M2 macrophages in the dermal layer counted according to Experiment 14 at each age. The lower part of Figure 19 shows the number of melanophagocytic M1 macrophages and M2 macrophages as an average for all subjects.

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

The present inventors discovered that the balance between these M1 macrophages and M2 macrophages (M1/M2 balance) is disrupted specifically in areas exposed to light and in areas where pigmentation occurs, and we have found that the balance between M1 and M2 macrophages (M1/M2 balance) is disrupted, and we aim to adjust the M1/M2 balance. We discovered that this is particularly important in preventing and improving photoaging and pigmentation. Furthermore, the present inventors have discovered that using the M1/M2 balance as an index of skin photoaging and pigmentation, favorable effects can be produced when specific physical stimuli are applied to the target skin. More specifically, even on photoaged skin or skin with pigmentation, when physical stimulation is applied at a specific stretching rate and at a frequency of 60Hz or less, such as 1Hz or less or 10Hz or less, M1 /M2 balance was found to be adjusted/improved. In addition, the present inventors used the M1/M2 balance as an index of skin photoaging and pigmentation, and as a result of screening various substances, the compounds of the present invention including Hypericum perforatum extract, tranexamic acid methylamide, thyme extract, It was also discovered that extracts of Aspergillus orientalis and eucalyptus have the effect of adjusting/improving the M1/M2 balance and function as anti-photoaging agents and anti-pigmentation agents. Hypericum perforatum extract has been reported to have an effect of promoting type I collagen production, a hair growth effect, etc. (Patent Documents 9 and 10). The compounds of the present invention, including tranexamic acid methylamide, have been reported to have an effect on improving rough skin, a whitening effect, an antiallergic effect, etc. (Patent Documents 11, 14, etc.). Thyme extract has been reported to have antiallergic and antibacterial effects (Patent Documents 12 and 13). Oubaku is used as an antidiarrheal drug, a stomach medicine, an oral drug for stomach weakness and loss of appetite, and a topical drug for bruises and sprains. It has also been reported to have antibacterial, antioxidant, skin barrier function improvement effects, and pigment cell activation effects ( Patent Documents 16, 17). Eucalyptus extract has been reported to have antibacterial and antioxidant effects (Patent Document 18). However, it was discovered for the first time by the present inventors that these substances have the effect of adjusting/improving the M1/M2 balance.

Therefore, the present invention provides methods, devices, and anti-photoaging and/or anti-pigmentation agents for preventing and/or ameliorating photoaging and/or pigmentation by adjusting the M1/M2 balance. The method of the present invention is a method for cosmetic purposes, and may not be a treatment by a doctor or medical professional.

As used herein, pigmentation refers to the deposition of pigments such as melanin in the dermis and epidermis. Although the present invention is effective for both the dermis and epidermis, it is particularly promising as a countermeasure against pigmentation in the dermis, since there are limited methods for improving it other than phagocytosis by melanophages. By applying the agent of the present invention, spots, dullness, dark circles, etc. caused by pigmentation are improved. In addition, since the pigment is injected into the dermal layer, it is effective in erasing tattoos and other tattoos that are difficult to remove once the pigment is injected.

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, the measurement may be performed by quantifying M1-specific cytokines such as IL-1beta and TNF-alpha, or M2-specific cytokines such as IL-10. However, the markers are not limited to the above markers as long as they can measure the M1/M2 balance.

As used herein, M1/M2 balance may refer to the ratio of the number of M1 macrophages to the number of M2 macrophages, and the amount of mRNA of M1 macrophage markers (e.g., CD86, CD80, iNOS, etc.) It may also refer to the ratio of mRNA amounts of M2 macrophage markers (eg, 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 melanophagocytic activity, so adjustment/improvement of the M1/M2 balance can be achieved by changing the ratio of M2 to M1 (M2 ratio). number/number of M1, and/or amount of M2 marker mRNA/amount of M1 marker mRNA). The increase may be, for example, an increase that has a statistically significant difference (e.g., Student's t test) with a significance level of 5%, and/or, for example, 1% or more, 5% or more, 10% or more, It may be an increase of 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 can be achieved by keeping the ratio of M2 to M1 (number of M2/number of M1 and/or amount of M2 marker mRNA/amount of M1 marker mRNA) within a certain range, for example, approximately It may be from 4/6 to about 9/1, from about 5/5 to about 8/2, from about 5/5 to about 7/3, etc., or it may be close to the above range. It may be to maintain within a range, or it may be to maintain homeostasis around the above range.

Adjustment/improvement of M1/M2 balance may be achieved by applying physical stimulation to the skin such as stretching stimulation, pressing stimulation, massage, etc. Adjustment/improvement of M1/M2 balance includes a step of applying a weak physical stimulus to the skin, and the device of the present invention is a device for performing the step of applying a weak physical stimulus to the skin. In this step, for example, a cycle including (a) stretching the skin to a stretching rate of 0.1% or more and 50.0% or less; and (b) recovering from the stretched state is carried out at a frequency of 60 Hz or less. There may be.

The extension rate is calculated using Equation 1 above. Physical stimulation may be performed at an extension rate of 0.001% or more and 80.0% or less, 0.01% or more and 60.0% or less, 0.1% or more and 50.0% or less, preferably 0.1% or more and 50.0% or less. For example, 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%, 10.0% to 30.0%, etc. Any range of elongation ratio can be adopted.

Extension speed refers to the speed (%/s) until the maximum extension rate (%) is reached in one cycle. Recovery speed refers to the speed (%/s) of returning from the maximum extension rate to the non-extension state. Extension speed/recovery speed: 0.010%/s to 40%/s, 0.05%/s to 30%/s, 0.10%/s to 20%/s, 0.2%/s to 15%/s, 0.3%/s It may be performed at any speed such as s to 10%/s. The extension rate and recovery rate may be the same or different.

Adjustment/improvement of M1/M2 balance includes a step of applying a weak physical stimulus to the skin, and the device of the present invention is a device for performing the step of applying a weak physical stimulus to the skin. , where the step includes, for example, (a-1) pressing the subject's skin by 1 μm to 1000 μm; and (b-1) recovering the subject's skin from the pressed state; at a frequency of 60 Hz or less. It may be performed at a frequency of .

"Pressing the skin by 1 μm to 1000 μm" refers to pressing the skin to a depth of 1 μm to 1000 μm from the outermost surface of the skin. The depth of the pressure can be arbitrarily set from the outermost surface of the skin, such as 1 μm to 1000 μm, 10 μm to 1000 μm, 10 μm to 300 μm, 10 μm to 100 μm, etc.

The frequency of vibration refers to the number of cycles per second, where one cycle is defined as the cycle from the start of extension or pressure to recovery to a non-extension or non-pressure state. A cycle may include maintaining an extended or pressed state and/or resting in a non-extended or non-pressed state for a period of time. For example, during one cycle, (a') after (a) and before (b), or after (a-1) and before (b-1), 1 second for 0 seconds to 30 minutes. holding the extension or pressure for ~20 minutes, between 5 seconds and 10 minutes, or between 10 seconds and 5 minutes; and/or (b') after (b) and before (a) of the next cycle; or After (a-1) and before (b-1), 0 seconds to 30 seconds, 0 seconds to 20 seconds, 0 seconds to 10 seconds, 1 second to 10 seconds, 1 second to 20 seconds, 1 second to It may further include resting in a non-stretched or non-pressed state for 10 seconds. The frequency may be, for example, 0.0000001 Hz or more and 10 kHz or less, 0.000001 Hz or more and 1 kHz or less, 0.00001 Hz or more and 100 Hz or less, preferably 0.0001 Hz or more and 60 Hz or less, and 0.0001 Hz or more and 10 Hz or less. For example, 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, 0.5H z or more and 50Hz or less, 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, 0.1H z or more and 1Hz or less, Any frequency range can be adopted, such as 1Hz to 60Hz, 1Hz to 10Hz, 1Hz to 5Hz, etc.

Commercially available facial beauty devices, massage devices, etc. include those that use electromagnetic waves such as RF waves with a frequency of about 0.3 to 300 MHz, and those that use ultrasonic waves with a frequency of about 1 MHz to 7 MHz. Compared to these frequencies/frequencies, the frequencies employed by the method/apparatus of the present invention are extremely low. Applying strong vibrations to the skin as with conventional facial beauty devices has the risk of causing negative effects such as redness, pressure marks, scars, pain, and inflammation on the skin, but using the frequency of the present invention eliminates the above-mentioned risks. This makes non-invasive physical stimulation possible. This is because the present inventors have discovered that stimulation is too strong if the extension rate and vibration frequency are too high, so it is preferable to gently stimulate the skin by adjusting these values to appropriate values.

Furthermore, it has been common knowledge among those skilled in the art that in beauty equipment that uses mechanisms such as motors that are commonly used in the field, only frequencies exceeding 60 Hz can be selected due to the mechanical mechanism of the motor. . In order to adopt a frequency of vibration below 60Hz, which is the limit of conventional beauty equipment, for example, below 60Hz, below 10Hz, below 1Hz, as in the present invention, it is necessary to create a special machine. was required. Furthermore, there is a fixed concept that such a low vibration frequency is "too weak" for the present invention to have the effect, and so far little research has been done. However, when the present inventors actually applied physical stimulation to the skin using a vibration frequency that is extremely low considering conventional technical common sense, they were surprised to find that even gentle stimulation with such a low vibration frequency did not affect the skin. A good effect was achieved.

Although low and medium frequency devices such as EMS devices are commercially available, these are specifically designed to act on deep layers such as muscles and subcutaneous fat, and it is unclear whether they will have an effect on the surface layer of the skin as in the present invention. . Furthermore, even if the frequency of such a device is low, it may cause a tingling stimulus when the current is applied, which is different from the present invention, which provides a gentle stimulus to the skin. On the other hand, the present invention enables a simple beauty treatment method in which stretching stimulation is applied directly to the skin without applying energy such as ultrasound, electric current, or magnetic field. Furthermore, the stretching stimulus having such a frequency is a gentle stimulus, but 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 pigmentation without adversely affecting the skin.

Physical stimulation may be through 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, a mechanically generated physical stimulus is applied to the skin using a device that includes a stimulus generating section that generates a physical stimulus and a stimulus applying section that applies the physical stimulus by contact or non-contact. Can be done. Physical stimulation may be achieved by contact, e.g. by pulling, pressing, tapping, kneading, suctioning the skin, and/or by applying shock waves to the skin, e.g. by ultrasound, air pressure or water pressure. This may also be accomplished non-contact, such as by displacement. Facial exercises include puffing out the cheeks and opening the eyes. Examples of massage include massage using instruments such as hands or rollers by the subject himself or a practitioner such as a beauty salon staff member. However, it is not limited as long as it is within the scope of the physical stimulation of the present invention.

An example of the device of the present invention is a beauty device that includes a skin contact portion that contacts the user's skin and applies the 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 in FIG. 14 is designed to stretch the skin at a specific frequency and extension rate by touching the skin with the skin-contacting part.

Further, for example, the device of the present invention includes a power source, a stimulation generating section, and a skin stimulating section, where the power source generates an electrical signal, and the stimulation generating section converts the electrical signal from the power source into a physical stimulation. The skin stimulation section may receive the physical stimulation generated by the stimulation generating section and apply the physical stimulation to the user's skin.

For example, the device shown on the left in FIG. 14 includes a grip section, a power source, a control section that controls physical stimulation, a stimulation generation section, and a skin contact section that includes a skin stimulation section and a skin fixation section. The user holds the grip part and places the skin contact part on the skin, fixes the skin with the skin fixing part, and operates the control part to send an electrical signal from the power source to the stimulation generating part to cause physical stimulation. It is designed so that the physical stimulus is transmitted to the skin stimulation part, and the skin is stretched at a specific frequency and extension rate by the skin stimulation part while being fixed to the skin fixation part. For example, the stimulation generator may be driven by a motor or the like and convert electrical signals into physical stimulation. Further, the skin stimulation part shown on the left side of FIG. 14 is one that applies stretching stimulation to the skin, but it may also be one that gives pressure stimulation to the skin, for example.

Alternatively, the device of the present invention may be a beauty device that includes a power source, a control section that controls physical stimulation, a stimulation generation section, and a skin contact section that includes a skin contact surface made of a sheet-like material. good. As an example, the skin-contact part of such a beauty device is shown on the right side of Figure 14. The sheet-like material may be capable of passing an electric current and convert an electrical signal from a power source into a physical stimulus. Such sheet materials include dielectric elastomer actuators (DEAs), conductive polymers, IPMCs, PVC gels, and Mckinnen types.

The power source of the device of the present invention may be an internal power source or an external power source, and may be rechargeable. Further, the device of the present invention may be one that uses data stored in a mobile phone or the cloud, or may be remotely operated wirelessly.

The physical stimulation may be one that achieves skin stretching by applying physical stimulation by contact or non-contact, such as in a surgical technique. The physical stimulus may be applied in a direction parallel to the skin surface, that is, in the lateral direction, or in a direction perpendicular to the skin surface, that is, in the longitudinal direction, or in any direction such as an oblique direction or a torsional direction. can.

The number of cycles of physical stimulation is not limited. For example, any number of cycles such as 10 to 500 cycles, 20 to 400 cycles, 30 to 300 cycles, 40 to 200 cycles, and 50 to 100 cycles may be performed. For example, as described in the Examples, 27 cycles may be sufficient.

Further, any number of these cycles may be considered as one set, and this set may be performed in any number of sets, for example, 1 to 100 sets, 2 to 50 sets, or 3 to 10 sets.

The time for performing physical stimulation is also not limited. For example, the cycle may be repeated for a certain period of time, such as 5 minutes to 3 hours, 10 minutes to 2 hours, or 30 minutes to 1 hour, with or without a rest period.

The time interval between cycles or sets is also not limited. For example, a stretch or pressure stimulus may be performed alone in one or more sets, and one or more sets may be applied daily, on the 2nd, 3rd, 4th, 5th, 6th, and 7th. It may be performed regularly or irregularly, continuously or intermittently, such as once, once every 1, 2, 3, or 4 weeks.

However, the vibration frequency, extension rate, number of cycles, and frequency are not limited to the above, as long as sufficient stimulation is achieved to exhibit skin anti-photoaging and/or pigmentation suppressing effects. The waveform for physical stimulation can also be arbitrarily set to a rectangular wave, sine wave, triangular wave, sawtooth wave, etc.

Alternatively, adjusting/improving the M1/M2 balance may be by administering an M1/M2 balance adjusting/improving agent or a composition containing the same. The present inventors have discovered that the compounds of the present invention, Hypericum perforatum extract, thyme extract, Laminaria perforatum extract, and/or Eucalyptus extract function as such an M1/M2 balance adjusting/improving agent. Accordingly, the present invention comprises a compound of the present invention, a Hypericum perforatum extract, a thyme extract, an extract of Hypericum perforatum, an extract of Hypericum perforatum, and/or an extract of Eucalyptus; Also provided are M1/M2 balance adjusting/improving agents, anti-photoaging agents, pigmentation inhibitors, and compositions containing them, which contain the extract as an active ingredient.

｛式中，Ｒ₁およびＲ₂は同一または異なり，水素原子，炭素数１～１８の直鎖状または分岐状アルキル基，炭素数５～８のシクロアルキル基，ベンジル基または下記式：

（但し，Ｘは低級アルキル基，低級アルコキシ基，ヒドロキシ基，アミノ基，ハロゲン原子を示し，ｎ＝０～３である）をそれぞれ示す｝
で表される化合物，又はその塩を指す。特に好ましくは，本発明の化合物は，Ｎ－メチル－トランス－４－（アミノメチル）シクロヘキサンカルボキサミド（トラネキサム酸メチルアミド（C₉H₁₉C₁N₂O））又はその塩である。上述の化合物は，例えば特許文献１４に記載のような方法で合成してもよく，市販品を用いてもよい。 As used herein, the "compound of the present invention" refers to the following formula:

{In the formula, R ₁ and R ₂ are the same or different and are a hydrogen atom, a linear or branched alkyl group having 1 to 18 carbon atoms, a cycloalkyl group having 5 to 8 carbon atoms, a benzyl group, or the following formula:

(However, X represents a lower alkyl group, a lower alkoxy group, a hydroxy group, an amino group, or a halogen atom, and n = 0 to 3)}
Refers to the compound represented by or its salt. Particularly preferably, the compound of the invention is N-methyl-trans-4-(aminomethyl)cyclohexanecarboxamide (tranexamic acid methylamide (C ₉ H ₁₉ C ₁ N ₂ O)) or a salt thereof. The above-mentioned compounds may be synthesized, for example, by the method described in Patent Document 14, or commercially available products may be used.

Hypericum erectum is a perennial plant of the genus Hypericum in the family Hypericumaceae. The extract of Hypericum perforatum used in the present invention is preferably an extract of the aerial parts of Hypericum perforatum, but since seeds, roots, etc. also contain active ingredients, extracts of one or more of these may be used. You can also use A commercially available extract of Hypericum perforatum can also be used.

Thymus is a perennial plant belonging to the Lamiaceae family and the genus Thymus. In the present invention, various types of thyme such as common thyme (T. vulgaris), citrus thyme (T. x citriodorus), and wild thyme (T. serpyllum) can be used. The thyme extract used in the present invention is preferably an extract of the whole thyme plant, but since seeds, flowers, roots, etc. also contain active ingredients, extracts of one or more of these may be used. You can also use objects. Commercially available thyme extracts can also be used.

Phellodendron amurense is a crude drug made from the dried bark of Phellodendron amurense or Phellodendron chinense. Auricularis or its extract may be produced by a conventional method, or a commercially available product may be used.

Eucalyptus is a tree of the genus Eucalyptus in the family Myrtaceae. The eucalyptus extract used in the present invention is preferably an extract of eucalyptus leaves, but since seeds, flowers, bark, roots, etc. also contain active ingredients, one or more of these may be used. Extracts can also be used. The eucalyptus extract may be produced by a conventional method, or a commercially available product may be used.

When using an extract, the extraction method is not particularly limited, but an extraction method using a solvent is preferred. When performing extraction, it is possible to use the plant as it is, but it is better to grind it into granules or powder and use it for extraction, which allows the extraction of the active ingredients in a short time and with high extraction efficiency under mild conditions. It can be carried out. The extraction temperature is not particularly limited, and may be appropriately set depending on the particle size of the pulverized material, the type of solvent, etc. It is usually set within the range from room temperature to the boiling point of the solvent. Further, the extraction time is not particularly limited, and may be appropriately set depending on the particle size of the pulverized material, the type of solvent, the extraction temperature, etc. Furthermore, during extraction, stirring may be performed, the mixture may be allowed to stand without stirring, or ultrasonic waves may be applied.

The type of solvent is not particularly limited, but water, aqueous ethanol, lower alcohols such as ethanol, organic solvents such as hexane, or mixed solvents thereof such as hexane/ethanol are preferred. Extraction may be performed at room temperature, or may be performed under heating (for example, using a heated solvent such as hot water or hot water). Alternatively, the extraction process may be performed by adding an enzyme to the solvent. By adding enzymes, it is possible to disrupt the plant cell tissue, thereby further increasing the extraction efficiency. As the enzyme, it is preferable to use a cell tissue disintegrating enzyme. Examples of such enzymes include pectinase, cellulase, hemicellulase, α-amylase, and phytase. These enzymes may be used alone or in combination of two or more.

Through such extraction operations, the active ingredients are extracted and dissolved in the solvent. The solvent containing the extract may be used as is, or may be used after being subjected to conventional purification treatments such as sterilization, washing, filtration, decolorization, and deodorization. It may also be used after being concentrated or diluted if necessary. Further, it may be used after all the solvent has been evaporated to form a solid (dried product), or the dried product may be used after being redissolved in an arbitrary solvent.

Moreover, since the pressed liquid obtained by pressing the raw material plant also contains the same active ingredients as the extract, the pressed liquid can be used instead of the extract.

However, the M1/M2 balance adjusting/improving agent or composition is not limited to the above-mentioned substances, and may be any substance known to adjust/improve the M1/M2 balance, such as the substances described in Patent Documents 4 to 7. Any known substance may be used. The administration route can be arbitrarily selected, such as transdermal administration, oral administration, subcutaneous administration, transmucosal administration, and intramuscular administration, but photoaging is a phenomenon that is specifically observed in areas of the skin that are exposed to light. Therefore, transdermal administration, which can be administered to specific areas of the skin, may be preferable. In addition, for pigmentation that occurs in the epidermis or dermis, transdermal administration may be preferable so that it can reach the epidermis or dermis through the skin. Further, the adjustment/improvement of the M1/M2 balance may be, for example, by inducing differentiation into M2 macrophages, or by any other method for adjusting/improving the M1/M2 balance.

For example, the agent or composition of the invention adjusts/improves the M1/M2 balance, thereby inhibiting photoaging and/or pigmentation of the skin. The M1/M2 balance adjusting/improving agent, anti-photoaging agent, and pigmentation inhibitor of the present invention (hereinafter, these may be collectively referred to as "the agent of the present invention") may be any one of the above active ingredients. It may contain a single species or two or more species in any combination and ratio.

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 and form of the composition are arbitrary and may be appropriately selected depending on the conditions such as the active ingredient and the intended use. The composition can be manufactured using conventional methods in a formulation that is appropriately combined with excipients, carriers and/or diluents, and other components depending on the dosage form.

The agent of the present invention can be used for 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 mixed with various foods, beverages, and feeds and ingested by humans and animals.

When applying the present invention to external skin preparations such as cosmetics, pharmaceuticals, and quasi-drugs, the amount (dry mass) of the plant or its extract will vary depending on the type, purpose, form, usage method, etc. , can be determined as appropriate. For example, 0.00001% to 50% (in the case of extracts or herbal medicines, calculated on dry mass basis) of the compound of the present invention, Hypericum perforatum extract, thyme extract, Aspergillus extract, and/or Eucalyptus extract are added to the total amount of the cosmetic. Can be mixed.

In addition to the above ingredients, if necessary, ingredients normally used in external skin preparations such as cosmetics, pharmaceuticals, and quasi-drugs, such as antioxidants, oils, and ultraviolet protection agents, may be added to the extent that the effects of the present invention are not impaired. , a surfactant, a thickener, an alcohol, a powder component, a coloring material, an aqueous component, water, various skin nutrients, etc. can be appropriately blended as necessary.

The skin external preparation of the present invention can be particularly suitably applied as a cosmetic, such as a cosmetic applied to the outer skin, a quasi-drug, etc., and its dosage form is not limited as long as it can be applied to the skin. Any dosage form can be applied, 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.

When the agent of the present invention is used as a cosmetic product, it can be used in lotions, milky lotions, foundations, lipsticks, lip balms, cleansing creams, massage creams, packs, hand creams, hand powders, body shampoos, body lotions, body creams, bath cosmetics, etc. It may also be used as a form.

However, the possible forms of the agents and compositions of the present invention are not limited to the above-mentioned dosage forms and forms. Furthermore, 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.

The subjects to which the methods, devices, agents, and compositions of the present invention are applied may be those in which skin photoaging and/or pigmentation (e.g., dermal pigmentation) is observed objectively or subjectively. and/or a subject wishing to prevent pigmentation. For example, it may be a target that is determined to be out of M1/M2 balance. In one embodiment, the target may be a subject determined to have a high degree of skin photoaging and/or pigmentation (for example, degree of dermal pigmentation) based on the M1/M2 balance in the skin. Alternatively, the target may be a phenotype specific to photoaging of the skin, such as age spots, wrinkles, sagging, etc., or epidermis or pigmentation, such as spots, dullness, bruises, tattoo marks, etc. It may be a subject of concern. Spots, wrinkles, sagging, dullness, bruises, tattoo marks, etc. can be determined by visual perception or using known indicators.

Further, according to the present invention, the steps include: applying a candidate drug to a biological sample; measuring the M1/M2 balance in the biological sample before and after applying the candidate drug; and determining the M1/M2 balance in the biological sample treated with the candidate drug. Also provided is a method for screening an anti-photoaging agent, comprising: evaluating the agent as having an anti-photoaging and/or pigmentation-inhibiting effect if the agent improves compared to before application. Applying a cosmetic treatment to a skin sample; Measuring the M1/M2 balance in the skin sample before and after the cosmetic treatment; Comparing the M1/M2 balance in the skin sample to which the cosmetic treatment was applied with that before the treatment. A method for evaluating an anti-photoaging and/or anti-pigmentation cosmetic treatment is also provided, comprising: evaluating the treatment as having an anti-photoaging and/or anti-pigmentation effect if the treatment improves the skin condition. The method of the present invention makes it possible to screen candidate drugs or cosmetic treatments to see whether they have anti-photoaging and/or pigmentation-inhibiting effects, thereby making it possible to develop products and propose new skin care. That is, according to the present invention, an anti-photoaging agent and/or a pigmentation inhibitor and an anti-photoaging and/or pigmentation agent for preventing and/or improving photoaging and/or pigmentation by adjusting the M1/M2 balance are provided. An anti-deposition cosmetic treatment is provided. The pigmentation may be dermal pigmentation.

Any biological sample can be used, such as a skin sample or an immune cell sample. The skin sample may be a skin sample after collection, for example, a skin sample in an ex vivo state after being collected from an animal such as a human, or a skin sample in cultured skin cells, such as monolayer or multilayer cultured cells, cultured skin samples, etc. It may be in an in vitro state such as keratinocytes or cultured fibroblasts. Alternatively, it may be an artificial skin sample such as a 3D skin model. The immune cell sample may be immune cells present in the skin after collection, immune cells in blood after collection of blood infiltrating the skin, cultured immune cells, etc. Although the 3D skin model is not limited, it may be created, for example, by the methods described in Patent Document 8 and Non-Patent Documents 10 to 12, or by modifying such methods to make it easier to measure the M1/M2 balance. . The biological sample is not limited as long as the M1/M2 balance can be measured.

For example, in the screening method for anti-photoaging and/or pigmentation inhibitors, immune cells such as THP-1 are differentiated into M0, the drug is added and the culture supernatant is collected after culturing for a certain period of time. This may be achieved by quantifying the amount of M2-specific cytokines such as IL-10 using a measurement method such as the above. In such screening methods, cells differentiated into M2 by adding IL-4, IL-13, etc. may be used as a positive control, and cells in which candidate drugs are added to IL-4, IL-13 may also be used. You can also compare. In other words, M2 has a high ability to produce IL-10, so if IL-10 in the supernatant increases after M0 differentiation compared to the control cultured only in medium, it can be said that it has differentiated into M2. Such a drug may be considered an "improving agent", and here, it is also possible to evaluate whether there is a difference in the amount of IL-10 by comparing the presence or absence of the drug in the presence of IL-4, IL-13, etc. good.

In this specification, candidate drugs refer to drugs whose anti-photoaging and/or pigmentation suppressing effects are being investigated, and include drugs that are already on the market as products, as well as drugs that are in the development stage. It may also be a drug selected for use in cosmetics in the development of cosmetics.

Cosmetic treatments include, but are not particularly limited to, any treatments considered to be effective in inhibiting photoaging and/or pigmentation, such as application of cosmetics containing the agent of the present invention or other ingredients. Cosmetics refer to cosmetics applied to the skin, including but not limited to lotions, milky lotions, serums, creams, foundations, etc., and do not have the direct purpose of improving skin conditions. However, it is intended to include anything that is applied to the skin, including, for example, sunscreen. Alternatively, the cosmetic treatment may be, for example, applying physical stimulation such as stretching stimulation, pressing stimulation, massage, etc. to the skin. Cosmetic treatments may be one-time treatments or continuous treatments performed over a period of days to weeks. Cosmetic treatments may be performed privately, or at beauty salons, cosmetics stores, beauty salons, and the like.

In addition, the present invention can measure the M1/M2 balance by using antibodies that detect arbitrary markers such as CD86, CD206, CD163, etc. that can measure the number of M1 and M2, and drugs that measure the amount of mRNA of these markers. Also provided is a screening kit for anti-photoaging and/or anti-pigmentation agents, including agents for the purpose of: Furthermore, the present invention also provides the following methods, systems, and devices.

Procedure for acquiring data regarding the reference value of the M1/M2 balance of the skin set in advance; Procedure for acquiring data regarding the M1/M2 balance of the target skin; Referring to the reference value, the M1/M2 balance of the target skin a step of calculating by comparing with data regarding; a step of evaluating the degree of photoaging and/or pigmentation of the skin based on the results calculated by the calculation step; and displaying the results evaluated by the evaluation step. method for evaluating the degree of photoaging and/or pigmentation of a subject's skin, performed by one or more computers, comprising:

The analysis section of the server calculates the M1/M2 balance of the skin through machine learning using a neural network method using training data based on the data regarding the M1/M2 balance of the skin and the degree of photoaging and/or pigmentation stored in the storage section. /A procedure for analyzing the relationship between M2 balance and photoaging and/or pigmentation degree; A procedure for the reception unit of the server to receive data on the M1/M2 balance of the target skin; , a step of evaluating and outputting the degree of photoaging and/or pigmentation of the target skin using the received M1/M2 balance of the target skin as input based on the analyzed relationship; A method for evaluating the degree of photoaging and/or pigmentation.

A database section that stores data regarding the reference value of M1/M2 balance of the skin set in advance; A data input section that inputs data regarding the M1/M2 balance of the target skin; Refers to the reference value stored in the database section; , a calculation unit that calculates the degree of photoaging and/or pigmentation of the skin based on the calculation result by the calculation unit by comparing it with data regarding the M1/M2 balance of the subject's skin input by the data input unit; A system for evaluating the degree of photoaging and/or pigmentation of a subject's skin, the system comprising: an evaluation section for evaluating; and a display section for displaying the results of the evaluation by the evaluation section.

A photoaging and/or pigmentation degree calculation device that calculates the degree of photoaging and/or pigmentation of the skin of a target using data regarding the M1/M2 balance of the skin of the target. The calculation unit includes a calculation unit that calculates the degree of photoaging and/or pigmentation of the subject when data regarding the M1/M2 balance of the skin is input; A photoaging and/or pigmentation degree calculation device having a trained neural network that has been subjected to machine learning processing using teaching data to calculate the estimated photoaging and/or pigmentation degree.

The methods, systems, and devices of the present invention enable objective photoaging and/or pigmentation measurements based on M/M2 balance.

Next, the present invention will be explained in more detail with reference to Examples. Note that the present invention is not limited to this.

Experiment 1: Histological staining The skin from the outer corners of the eyes of Caucasians from young people (20s to 30s) and elderly people (60s to 70s) who have undergone photoaging as shown in Figure 1a was frozen and sectioned. After cutting, the cells were stained with the following macrophage markers.
(1) Staining of M1 macrophages: Double staining was performed with a goat-derived anti-human CD86 antibody (R&D) and a rabbit-derived anti-human CD11b antibody (abcam) (Figure 1b).
(2) Staining of M2 macrophages: Double staining was performed with mouse-derived anti-human CD206 antibody (BD) or mouse-derived anti-human CD163 antibody (Leica) and rabbit-derived anti-human CD11b antibody (abcam) (Figure 1c) .
(3) Staining of total macrophages: Double staining was performed with mouse-derived anti-human CD68 antibody (abcam) and rabbit-derived anti-human CD11b antibody (abcam).

Cells that were double positive for antibodies (1) to (3) up to 200 μm just below the epidermis were counted as each macrophage (Figure 1d). In addition, in order to investigate the relationship between M1 and M2 macrophages and collagen production, we used a goat-derived anti-human CD86 antibody (R&D), a rat-derived anti-procollagen antibody (Millipore), or a mouse-derived anti-human CD206 antibody (BD). and a rat-derived anti-procollagen antibody (Millipore) (Fig. 1e, left).

The tissue staining of (1) to (3) is shown in Figures 1b and c, the 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 Figures 1b, c, and d, in elderly people undergoing photoaging, M1 macrophages were abundant, while M2 macrophages were decreased. More specifically, as shown in Figure 1b, in young subjects M1 macrophages were present only around blood vessels, whereas in older subjects they were scattered throughout the tissue. Additionally, as shown in Figure 1c, while M2 macrophages were present throughout the tissues in young subjects, their number decreased in older subjects. Furthermore, as shown in Figure 1d, the total number of macrophages (number of M1 + number of M2) did not change between young and elderly subjects, and only the M1/M2 balance changed. In young people, the ratio of M2 to M1 (number of M2/number of M1) is within the range of about 5/5 to about 7/3, while in the elderly, the ratio of M2 to M1 decreases sharply, and M1/ The M2 balance was greatly disrupted and was markedly different from that of young subjects.

Furthermore, as shown in the two upper left photographs in Figure 1e, M1 macrophages and procollagen are misaligned in both young and old subjects, while M2 macrophages and procollagen are misaligned, as shown in the lower left two photographs. For procollagen, positional agreement was observed. This suggests that M1 may act on fibroblasts to promote collagen destruction and M2 to promote collagen production, as shown in the schematic diagram on the right side of Figure 1e.

Experiment 2: M1 and M2 differentiation stimulation experiment of THP-1 According to the method described in Non-Patent Document 1, THP-1, a human-derived cell line, was induced to differentiate into M1 and M2 macrophages. Specifically, THP-1 was cultured in RPMI1640 (Nakalai) supplemented with 1mM Na Pyruvate (Nakalai), 2mM L Glutamine (Nakalai), and 10% FBS using the method shown in Figure 2a. Then, 100nM PMA (abcam) was further added and stimulated for 24 hours to differentiate into macrophages. To differentiate into M1, stimulate for 24 hours by adding 100ng/mL LPS (sigma) and 20ng/mL IFNγ (R&D), and to differentiate into M2, add 20ng/ml IL-4 (R&D) and 20ng/mL. IL13 (R&D) was added and stimulated for 24 hours. When observed under a microscope, it was confirmed that the morphology had changed as shown in Figure 2b.

In addition, we extracted mRNA from each differentiated or undifferentiated cell, and performed real-time PCR using TaqMan Gene expression assay using probes for IL-1beta, TNF-alpha, and IL-10 (Applied Biosystems). , the expression level was quantified (Fig. 2c, upper panel). Furthermore, using the same CD86 antibody (R&D) and CD206 antibody (BD) as in Experiment 1, PCR was performed in the same manner to quantify the expression level (Figure 2c, lower panel). Each value was corrected by the GAPDH mRNA expression level.

As shown in Figure 2c, the macrophages differentiated by the method described in Experiment 2 showed inflammatory cytokines (IL-1beta, TNF-alpha) characteristic of M1 and anti-inflammatory cytokines (IL-1beta, TNF-alpha) characteristic of M2, respectively. -10). Furthermore, these differentiation-induced macrophages showed increased expression of CD86 and CD206, which are 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 Experiments 3 and 4 below.

Experiment 3: Addition of M1 and M2 macrophage supernatant to fibroblasts As shown in Figure 3a, THP-1 was differentiated into M1 and M2 in the same manner as in Experiment 2, or left in the undifferentiated state of M0. After removing the supernatant and washing once with PBS, culture medium was added and cultured for 48 hours. The supernatants containing M1 and M2 secretions such as inflammatory and anti-inflammatory cytokines were added to neonatal fibroblasts. Cells supplemented with RPMI1640 (Nakalai) were used as a control. After adding the supernatant, fibroblasts were cultured for 72 hours, and the amount of procollagen in the supernatant was quantified using a PIP ELISA kit (TAKARA) (Figure 3b). The cell fraction was stained with rabbit-derived anti-human collagen antibody (CEDERLANE) and biotinylated hyaluronic acid binding protein (HOKUDO) (Figure 3c). Furthermore, using β-gal as an aging indicator, we stained the nucleus of the cell with DAPI, and then stained the intracellular β-gal using the Senescence Detection Kit (abcam) (Figure 3d) to detect β-gal positive cells and The number of DAPI-positive cells was counted (Fig. 3e).

Furthermore, in order to examine the contribution of M1 macrophages and M2 macrophages to melanin production, the supernatant of each macrophage was added to fibroblasts and cultured in the same manner as above. Thereafter, fibroblasts were collected, mRNA was collected, and real-time PCR was performed using probes for HGF, ET1, bFGF, IL-1alpha, SCF, and clusterin to quantify the mRNA expression levels of each (Figure 3f).

The results are shown in Figures 3b-3f. Figure 3b shows the amount of procollagen after adding the supernatant of each macrophage (M1, M2) to fibroblasts and culturing them for 72 hours. Figure 3c shows the localization of collagen and hyaluronic acid in fibroblasts after adding the supernatant of each macrophage (M1, M2) and culturing for 72 hours. From Figures 3b and 3c, it can be seen that M1 significantly suppresses collagen production. Figure 3d shows intracellular β-gal in fibroblasts after adding the supernatant of each macrophage (M1, M2) and culturing for 72 hours. Figure 3d suggests that M1 increases the number of β-gal positive cells and promotes aging, while M2 suppresses aging. Figure 3e shows a graph of counting the number of β-gal positive cells and DAPI positive cells. The figure on the right shows the total number of DAPI-positive cells per well. The right panel in Figure 3e suggests that M2 not only suppresses cell aging but also promotes cell proliferation. The left panel in Figure 3e shows the ratio (%) of the number of β-gal positive cells to the total number of DAPI positive cells, indicating that M1 has senescence and cell death promoting effects, while M2 has senescence suppressing and cell proliferation effects. is suggested. Furthermore, as shown in Figure 3f, we found that mRNA expression of melanogenesis-related factors acted to increase melanin production by M1, while suppressing melanin production by M2. As reported in Non-Patent Document 6, it is known that fibroblasts secrete factors such as SCF and HGF when stimulated with light such as UV, causing cell death. Furthermore, as reported in Non-Patent Documents 7 to 9, fibroblasts act directly or indirectly on melanocytes by secreting factors such as HGF, ET1, bFGF, SCF, and clusterin upon light stimulation. and produce melanin. Therefore, it is suggested that cell death and melanin production due to light stimulation are due to an imbalance of M1/M2.

Experiment 4: Addition experiment of M1 and M2 macrophage supernatants to young and aged fibroblasts Next, we investigated whether the anti-aging effect of M2 macrophages is also useful for aged fibroblasts, that is, whether it has a rejuvenating effect. For the purpose of confirmation, M1 and M2 macrophage supernatants were added to young and aged cells, and the effect of macrophage supernatants was examined depending on the age of the fibroblasts.

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

Figure 4a shows a staining diagram of β-gal. FIG. 4b shows a graph of the number of β-gal positive cells and the total number of cells per well of young-derived fibroblasts and aged-derived fibroblasts. The lower panel of Figure 4a shows that even in aged cells, addition of M2 supernatant significantly reduced β-gal positive cells and suppressed aging. Figure 4b also shows that M1 accelerates aging and M2 suppresses aging, regardless of age. This is also supported by the result that, as shown in Figures 4c and d, collagen production is significantly lower in cells treated with M1 supernatant than in M2 supernatant, regardless of age.

Experiment 5: Co-culture experiment of M1 macrophages, M2 macrophages, and neonatal foreskin-derived fibroblasts When the same number of M1 or M2 macrophages (created by the method of Experiment 2) as neonatal foreskin-derived blast cells were cultured in RPMI1640 (Nakalai). The amount of collagen produced when neonatal foreskin-derived fibroblasts and half the number of M1 or M2 macrophages were cultured in RPMI was stained with a rabbit-derived anti-human collagen antibody (CEDERLANE) and compared. At the same time, macrophages were visualized by staining with mouse-derived anti-human CD68 antibody (abcam).

The results are shown in Figure 5. Even in neonatal foreskin-derived blast cells, collagen was reduced when M1 supernatant was added, whereas collagen was increased when M2 was added. In other words, it was found that even in young cells, when the M1/M2 balance is disrupted, collagen is affected.

Experiment 6: Effects of M1/M2 imbalance using 3D skin model
In order to examine the effects of M1/M2 macrophages on epidermal cells and fibroblasts, we created three types of 3D skin models with a three-layer structure as shown in the table below.

The 3D skin model was created as follows. Human dermal fibroblasts (0.2 × 10 ⁶ cells) were seeded in cell culture inserts (φ12 mm, average pore diameter of porous membrane: 0.4 μm), and 200 μM magnesium ascorbic acid-2-phosphate (APM) and 10% FBS-DMEM were added. The cells were cultured for one week with medium exchange once every two days. In addition, the control model is a 0.5% type I collagen-10% FBS-DMEM solution containing human dermal fibroblasts, the M1 model, and the M2 model are M1 macrophages and M2 macrophages differentiated according to the method of Experiment 2. A collagen gel containing 30,000 cells of each was prepared on top of human dermal fibroblasts and cultured for 1 to 5 days.

Furthermore, epidermal keratinocytes dispersed in Humedia-KG2 (Kurabo Industries) medium were seeded onto the collagen gel at 5 × ¹⁰ cells/well, and Humedia-KG2 and 10% FBS-DMEM were added to the outside of the insert. A medium mixed at 1:1 and supplemented with 200 μM APM was added to the same liquid level as the inside, and cultured for 3 days.

Then, remove the medium inside the insert or glass ring, and add 200 μM APM to the outside of the skin model medium (1:1 mixture of 10% FBS-DMEM and Humedia-KG2 EGF(-) to make Ca 1.8 mM). 10μM N-hydroxy-2-[[(4-methoxyphenyl)sulfonyl]3-picolyl)amino]-3-methylbutanamide hydrochloride (CGS27023A (MMP inhibitor)), 10μM BIPBIPU (heparanase inhibitor) in the insert. Air-liquid boundary culture was performed with the inserts added up to the height of the bottom and the inside of the insert exposed to air. Culture was carried out for 2 weeks with medium exchange once every 2 to 3 days.

After fixing the cultured skin model with 4% PFA/PBS (Nakarai), it was stained with anti-CD206 antibody (abcam), anti-CD68 antibody (abcam), and anti-CD86 antibody (abcam) in the same manner as in Experiment 1. The presence of M1 and M2 macrophages was confirmed (Figure 6). Afterwards, the cells were stained with anti-p21 antibody (abcam) and DAPI (VECTOR), and the number of cells stained with DAPI in each of the epidermal cell layer and fibroblast cell layer was counted as the total cell count, and among them, the number of cells stained with anti-p21 antibody was also stained. The number of cells was counted as the number of p21 positive cells. The ratio of the number of p21 positive cells to the total number of cells was determined using the following formula.

The results are shown in Figures 7 and 8. As shown in these figures, the number of p21-positive cells increased in the M1 model (M1) compared to the control model (cont) in both the upper epidermal cell layer and the lower fibroblast layer, which are in contact with the intermediate layer. On the other hand, it decreased in the M2 model (M2). This trend was consistent with Experiment 3 using monolayer cultures. Therefore, even in a 3D skin model that is closer to human skin, it is suggested that M1 macrophages have the effect of promoting aging and cell death, while M2 macrophages have the effect of suppressing aging and cell death.

Experiment 7: Solar irradiation experiment on ex vivo model using human skin
Genoskin's NativeSkin (skin collected from a 38-year-old woman) was cultured for one day in the provided culture solution upon arrival. The next day, an optical filter was placed in a 1000W solar simulator manufactured by Oriel, and the cells were irradiated with only UVA and UVB at 11.5 J/ ^cm2 , and culture was continued using the supplied culture medium (irradiation (+)). A sample that was not irradiated was used as a control (irradiation (-)). Samples were collected 5 days after irradiation, and as in Experiment 1, M1 macrophages, M2 macrophages, and total macrophages were stained, and their numbers were counted and graphed.

The results are shown in Figure 9. The M1 number increased the most when solar simulator irradiation was applied. On the other hand, the increase in the number of M2 was very small compared to the case of M1. In other words, it was found that optical stimulation disrupts the M1/M2 balance and increases the M1 ratio.

Experiment 8: Stretch stimulation experiment on an ex vivo model using human skin Next, we investigated ways to adjust or improve the M1/M2 balance.
Sample: Genoskin's NativeSkin (skin collected from a 38-year-old woman) (6 well size, approximately 2 to 2.5 cm in diameter) was used.
Stretching conditions: As shown in Figure 11, we created a stretching device that had grips that grip both ends of the skin in the well and stretched the skin by pulling the grips. The well containing the above tissue piece was placed horizontally, and the skin was stretched from both ends by operating the gripping part, and as shown in Figure 10, the skin was stretched at a speed of 10%/s to give a stretching rate of 10%. The sample was returned to its original unstretched state at a recovery rate of %/s. This was considered one cycle, and a total of 90 cycles were performed over 30 minutes. These 90 cycles were considered one set, and a total of 3 sets (270 cycles in total) were performed over 3 hours with a rest period of 30 minutes to 1 hour between sets. A sample to which no extension stimulation was applied was used as a control (control).

Observation method: Using the same method as in Experiment 1, except that total macrophages were stained with rabbit-derived anti-human CD11b antibody (abcam) for skin samples with or without stretching stimulation, M1 macrophages and M2 macrophages were stained. , total macrophages were stained and their numbers were counted. The ratio (%) of each macrophage (M1, M2) to the total number of all macrophages was determined and graphed.

The results are shown in Figure 12. Compared to the control, in samples subjected to stretch stimulation, the total number of macrophages and the number of M1 did not change much, but the number of M2 increased significantly. In other words, it was found that stretch stimulation improved the M1/M2 balance.

Experiment 9: Screening for anti-photoaging and/or pigmentation agents that prevent and/or improve photoaging and/or pigmentation by adjusting or improving M1/M2 balance
We screened for drugs that adjust or improve M1/M2 balance.
A total of 19 ingredients were examined as screening target drugs, including Hypericum perforatum extract, thyme extract, and tranexamic acid methylamide (N-methyl-trans-4-(aminomethyl)cyclohexanecarboxamide). Hypericum perforatum extract was an extract of the aerial parts of Hypericum perforatum purchased from Ichimaru Falcos. Thyme extract is a whole plant extract of wild thyme purchased from Koei Kogyo. Tranexamic acid methylamide was synthesized by the method described in Patent Document 14. Hypericum perforatum extract was dissolved in 50% ethanol, thyme extract in butylene glycol, and tranexamic acid methylamide in PBS.

The same THP-1 cells as in Experiment 2 were used in an undifferentiated M0 state and cultured overnight at 37°C. Each of the above screening target drugs was added to the medium at a concentration of 0.1% Hypericum perforatum extract, 0.1% thyme extract, and 0.06% and 0.03% tranexamic acid methylamide, and the same amount of these solvents was added as a control. and cultured at 37°C for two nights. Cells were collected, RNA was extracted, the expression levels of CD86 and GAPDH were quantified by real time PCR, and CD86/GAPDH was measured. We searched for a drug that reproducibly lowers CD86/GAPDH levels compared to the control (cont) as an M1 differentiation inhibitor.

The results are shown in Figure 13. Figure 13 shows that the addition of Hypericum perforatum extract, thyme extract, and tranexamic acid methylamide significantly decreased the CD86/GAPDH value, indicating that they have an inhibitory effect on M1 differentiation, and can be used as M1/M2 balance adjusting/improving agents. is suggested.

Experiment 10: Screening for anti-photoaging agents that prevent and/or improve photoaging and/or pigmentation by adjusting or improving M1/M2 balance. A total of eight different ingredients, including eucalyptus extract, were investigated in the same manner as in Experiment 9. The extract used was the dried bark of Amberjack of the family Rutaceae, and the extract of the eucalyptus leaf was used as the eucalyptus extract. The extract of Aspergillus japonica was dissolved in butylene glycol to a concentration of 0.01%, and the eucalyptus extract was dissolved in 50% ethanol to a concentration of 0.1%. These solvents were used as controls.

Addition of drugs, collection of cells, extraction of RNA, and measurement of CD86/GAPDH were performed in the same manner as in Experiment 9, and drugs that reproducibly lowered CD86/GAPDH values than the control (cont) were used as M1 differentiation inhibitors. I explored. The results are shown in Figure 15. Figure 15 suggests that, in addition to the drugs in Experiment 9, extracts of Aspergillus elegans and eucalyptus can also be used as M1/M2 balance adjusting/improving agents.

Experiment 11: Effect of M1 and M2 macrophage supernatants on collagen In addition to Experiment 3, in order to examine the contribution of M1 and M2 macrophages to collagen degradation, supernatants of M1 and M2 macrophages were added using the same method as Experiment 3. was added to fibroblasts and cultured, and mRNA was collected 72 hours later. Real time PCR was performed using probes for MMP-1, MMP-2, and IL-1β (Taqman probe manufactured by Applied Biosystems) as indicators of collagen degradation, and the expression levels of each mRNA were quantified (FIG. 16a).

Furthermore, in order to examine the contribution of M1 macrophages and M2 macrophages to collagen production and maturation, the supernatant of each macrophage was added to fibroblasts and cultured in the same manner as in Experiment 3, and after 72 hours. mRNA was collected. Real time PCR was performed using COL1A1, COL1A2, HSP47, and ADAMTS-2 probes (Taqman probe manufactured by Applied Biosystems) as indicators of collagen production and maturation, and the mRNA expression levels of each were quantified (FIG. 16b).

From Figures 16a and b, it was confirmed that M1 contributes to collagen decomposition, while M2 contributes to collagen production and maturation.

Experiment 12: Collagen degradation and production in an ex vivo model using young and elderly human skin. Young (20s to 30s) and elderly (60s to 80s) Caucasians with the ages shown in Figure 17a. The skin of the derived temple was frozen sectioned in the same manner as in Experiment 1, and after thin sectioning, anti-procollagen antibody (millipore), anti-fragmented collagen (AdipoGen), which stains the collagen cut fragment on the 3/4 side, and experiment 1 and Positive cells were counted in the same manner as in Experiment 1 using the same CD68, CD11b, CD206, and CD86 antibodies.

The results are shown in Figure 17b, c, 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, the number of macrophages that are positive for 3/4 collagen is greater than the fibroblasts that are positive for 3/4 collagen, and the number of macrophages that are positive for 3/4 collagen is higher than that of M1 macrophages that are positive for 3/4 collagen. There was a tendency for the number of M2 macrophages showing .

Experiment 13: In vitro experiment showing melanin uptake ability in the dermis THP-1 cells were differentiated into M1 macrophages and M2 macrophages in the same manner as in Experiment 3. Melanin solution (Sigma, Melanin-BioReagent, Synthetic, suitable for cell culture) was dissolved in PBS and adjusted to 0.02% W/V for each differentiated M1 macrophage, M2 macrophage, and fibroblast (manufactured by Kurabo Industries). Added. After 24 hours, the cells were washed with PBS, photographed with a microscope, and collected. The cell number of the collected cells was measured and quantified by measuring the amount of melanin using Alamar Blue (Life Technologies) as described below.

Quantification of melanin:
After adding Alamar blue adjusted to 1:10 in the culture medium of each cell (macrophage: RPMI1640, fibroblast: 1DMEM), the cells were cultured at 37°C for 30 minutes. Thereafter, 100 ul/well of the supernatant was collected, and fluorescence was measured using Ascent (Thermo) at excitation/emission: 544 nm/590 nm. After measurement, it was washed with PBS, and 1M NaOH was added, followed by incubation at room temperature for 3 hours to completely melt it. This cell solution was measured at OD475 using POWERSCAN HT (DS PHARMA BIOMEDICAL).

The results are shown in Figures 18a-c. The unit "melanin/almar blue" in the figure is the intensity ((1)) obtained by staining cultured cells with Alamar blue and measuring the fluorescence of the supernatant at 544 nm/590 nm ((1)), and the intensity after which the cells were lysed and melanin was also dissolved. It is the relative value ((2)/(1)) of the intensity ((2)) measured at absorbance of 475 nm, and indicates the melanin content per cell. These figures show that M2 phagocytizes a large amount of melanin. This tendency was already observed after 24 hours, but when the cells were continued to be cultured and observed after 5 days, the difference became clearer (Figure 18c).

Experiment 14: Ex vivo experiment showing melanin uptake ability in the dermis Experiment 13 showed that M2 macrophages phagocytose more melanin than fibroblasts and M1 macrophages, so the dermal layer of human skin counted in Experiment 13 was observed ex vivo using LSM880 (Carl Zeiss). When observed in bright field, whether old or young, M2 macrophages were stained black with melanin, whereas melanin was not present near M1 macrophages. As a result, we found ex vivo that M2 macrophages take up more melanin than fibroblasts and M1 macrophages in the dermis, and we counted and graphed their numbers (Figure 19).

From Figure 19, the number of M2 macrophages taking up melanin was greater than the number of M1 macrophages in both old and young subjects, and there was not much difference in age in this trend. These results suggest that because M2 macrophages take up melanin from the dermis, increasing the proportion of M2 macrophages and adjusting the M1/M2 balance can prevent and/or improve pigmentation in the dermis.

It has been shown that the proportion of M1 increases with photoaging (Experiment 7), and the higher the proportion of M2, the higher the amount of skin collagen (Experiments 11 and 12) and the higher the chromophagocytic activity (Experiments 13 and 14). Therefore, the compounds of the present invention, such as tranexamic acid methylamide, which were screened as M1 differentiation inhibitors, thyme extract, Laminaria japonica, and eucalyptus extract, as well as the physical stimulus of the present invention, which has the effect of improving M1/M2 balance, are antiphotogenic. It is suggested that the effect of suppressing aging and/or pigmentation is high.

The above results show that light stimulation causes an imbalance of M1/M2, which causes photoaging in the exposed skin and pigmentation in the dermis, but the balance of M1/M2 is adjusted. It is suggested that photoaging and/or dermal pigmentation can be prevented and/or improved by or improving photoaging. Adjustment or improvement of the M1/M2 balance can be achieved by applying a stretching stimulus or a pressure stimulus, or by applying a compound of the present invention such as tranexamic acid methylamide, an extract of Hypericum perforatum, an extract of thyme, an extract of Aspericula, and/or an extract of Eucalyptus. This is achieved by applying an ingredient that has an anti-differentiation effect as an M1/M2 balance adjusting/improving agent, and is expected to prevent and/or improve photoaging and/or dermal pigmentation.

According to the present invention, prevention and/or improvement of photoaging and/or dermal pigmentation, evaluation of photoaging and/or dermal pigmentation degree, anti-photoaging and/or dermal pigmentation inhibitor, and anti-photoaging and/or dermal pigmentation It becomes possible to explore cosmetic treatments to suppress pigmentation.

Claims (14)

A beauty method for preventing and/or improving photoaging and/or dermal pigmentation by adjusting the M1/M2 balance. 2. The method of claim 1, wherein adjusting the M1/M2 balance is increasing the ratio of M2 to M1. Adjusting the M1/M2 balance includes a step of applying a weak physical stimulus to the skin, where the step includes, for example, (a) stretching the target skin to a stretching rate of 0.1% to 50.0%; and (b) recovering from the extended state;
and/or
(a-1) Pressing the subject's skin by 1 μm to 1000 μm; and (b-1) recovering the subject's skin from the pressed state;
This is achieved by applying physical stimulation to the subject's skin that includes cycles at a frequency of 60Hz or less.
Here, the extension rate is

(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 straight line passing through fixed points A and B is parallel to the stretching direction.)
The method according to claim 1 or 2, wherein the method is calculated by: A beauty device for preventing and/or improving photoaging and/or dermal pigmentation by adjusting M1/M2 balance,
The device includes:
a stimulus generating part that generates a physical stimulus;
and a stimulation applying part that applies physical stimulation generated in the stimulation generating part 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 here includes, for example,
(a) stretching the skin to a stretching rate of 0.1% or more and 50.0% or less; and (b) recovering from the stretched state;
and/or
(a-1) Pressing the subject's skin by 1 μm to 1000 μm; and (b-1) recovering the subject's skin from the pressed state;
It is a cycle including
Here, the extension rate is calculated by the above formula 1 in the beauty device. A method for screening anti-photoaging and/or dermal pigmentation inhibitors, comprising:
applying a candidate drug to a biological sample;
measuring the M1/M2 balance in the biological sample before and after administering the candidate drug; and if the M1/M2 balance in the biological sample treated with the candidate drug improves compared to before administering the drug; To be evaluated as having anti-photoaging and/or dermal pigmentation suppressing effects;
The method described above. An evaluation method for anti-photoaging and/or dermal pigmentation suppression cosmetic treatment, comprising:
performing cosmetic treatments on skin samples;
Measuring the M1/M2 balance in the skin sample before and after the cosmetic treatment; and if the M1/M2 balance in the skin sample after the cosmetic treatment improves compared to before the treatment, the treatment To be evaluated as having anti-photoaging and/or dermal pigmentation suppressing effects;
The method described above. A method for evaluating photoaging and/or dermal pigmentation degree performed by one or more computers, the method comprising:
Procedure for acquiring data regarding the preset reference value of skin M1/M2 balance;
Procedures for obtaining data regarding the M1/M2 balance of the subject's skin;
A step of calculating by referring to the reference value and comparing it with data regarding the M1/M2 balance of the subject's skin;
The method described above, comprising: a step of evaluating the degree of inhibition of photoaging and/or dermal pigmentation of the skin based on the calculation result of the calculation step; and a step of displaying the evaluation result of the evaluation step. A system for evaluating photoaging and/or dermal pigmentation, the system comprising:
A database unit that stores data regarding preset reference values of skin M1/M2 balance;
A data input section for inputting data regarding the M1/M2 balance of the target skin;
a calculation unit that refers to a reference value stored in the database unit and calculates by comparing it with data regarding the M1/M2 balance of the subject's skin input by the data input unit;
The system further comprises: an evaluation section that evaluates the degree of photoaging and/or dermal pigmentation of the skin based on calculation results by the calculation section; and a display section that displays the evaluation results by the evaluation section. A screening kit for anti-photoaging and/or dermal pigmentation inhibitors, including a drug for measuring M1/M2 balance. ) An anti-photoaging and/or dermal pigmentation inhibitor for preventing and/or improving photoaging and/or dermal pigmentation by adjusting the M1/M2 balance. below:

{In the formula, R ₁ and R ₂ are the same or different and are a hydrogen atom, a linear or branched alkyl group having 1 to 18 carbon atoms, a cycloalkyl group having 5 to 8 carbon atoms, a benzyl group, or the following formula:

(However, X represents a lower alkyl group, a lower alkoxy group, a hydroxy group, an amino group, or a halogen atom, and n = 0 to 3)}
A compound represented by or a salt thereof,
The anti-photoaging and/or dermal pigmentation suppressing agent according to claim 10, comprising Hypericum perforatum extract, thyme extract, Hypericum perforatum extract, and/or Eucalyptus extract. below:

{In the formula, R ₁ and R ₂ are the same or different and are a hydrogen atom, a linear or branched alkyl group having 1 to 18 carbon atoms, a cycloalkyl group having 5 to 8 carbon atoms, a benzyl group, or the following formula:

(However, X represents a lower alkyl group, a lower alkoxy group, a hydroxy group, an amino group, or a halogen atom, and n = 0 to 3)}
A compound represented by or a salt thereof,
M1/M2 balance adjusting/improving agent containing Hypericum perforatum extract, thyme extract, Aspergillus perforatum extract, and/or Eucalyptus extract. A composition comprising a drug according to any one of claims 10 to 12. 3. The method according to claim 1 or 2, wherein adjusting the M1/M2 balance is achieved by administering a drug according to any one of claims 10 to 12.

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