JP7439059B2 - Methods and devices for preventing and/or improving photoaging and/or dermal pigmentation, anti-photoaging and/or dermal pigmentation inhibitors, screening methods for anti-photoaging and/or dermal pigmentation inhibitors, and anti-photoaging and/or dermal pigmentation inhibitors. Evaluation method of cosmetic treatment for suppressing photoaging and/or dermal pigmentation, and evaluation method of photoaging and/or dermal pigmentation degree - Google Patents

Methods and devices for preventing and/or improving photoaging and/or dermal pigmentation, anti-photoaging and/or dermal pigmentation inhibitors, screening methods for anti-photoaging and/or dermal pigmentation inhibitors, and anti-photoaging and/or dermal pigmentation inhibitors. Evaluation method of cosmetic treatment for suppressing photoaging and/or dermal pigmentation, and evaluation method of photoaging and/or dermal pigmentation degree Download PDF

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JP7439059B2
JP7439059B2 JP2021514255A JP2021514255A JP7439059B2 JP 7439059 B2 JP7439059 B2 JP 7439059B2 JP 2021514255 A JP2021514255 A JP 2021514255A JP 2021514255 A JP2021514255 A JP 2021514255A JP 7439059 B2 JP7439059 B2 JP 7439059B2
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photoaging
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聡 堀場
純一 細井
雅哉 高木
有宇子 松浦
健太朗 加治屋
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Description

本発明は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又は改善するための方法,装置,および抗光老化及び/又は真皮色素沈着抑制剤,並びにM1/M2バランスを指標とする抗光老化及び/又は真皮色素沈着抑制剤のスクリーニング方法,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,及び光老化及び/又は真皮色素沈着度の評価方法等に関する。 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.

ヒト皮膚の老化現象は大きく「自然老化」と「光老化」に分けられる。光老化は露光する部位特異的に認められる現象で,皮膚特異的である。光老化では,UVなどの影響で活性酸素の産生や,細胞のDNAの損傷などが誘発され,皮膚線維組織が損傷し,しわやたるみといった表現型が現れる原因と考えられている。例えば,皮膚光老化によりコラーゲンからなる膠原線維の減少やエラスチンからなる弾性線維の変性といった現象がみられる。また,メラノサイトもダメージを受け,メラニン色素が多く作られてしまい,しみ等を引き起こす原因となる。 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.

また,しみやくすみをはじめとする色素沈着は,表皮の基底層にあるメラノサイトで作り出されるメラニンが蓄積することにより起こる。メラニンは通常表皮や基底層に存在するが,表皮は比較的早いサイクルでターンオーバーするため,このようなメラニンは排出されやすい。一方,メラニンが基底膜の隙間から真皮に落ちこんでしまう等の理由によりメラニンが真皮層に存在する場合がある。真皮細胞のターンオーバーのサイクルは表皮に比べて非常に遅いため,このようなメラニンは排出されずに蓄積されてしまうことが多い。このような理由により,真皮の色素沈着の改善は非常に困難である。 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.

抗光老化美容法として,例えば,特許文献1では,白血球エラスターゼの阻害を予防することによる皮膚光老化の予防又は抑制剤を開示する。特許文献2では,コラーゲン合成促進作用を有するヒユ科パフィア属の植物抽出物と動物由来コラーゲンペプチドとを含有することを特徴とする光老化抑制剤組成物を開示する。 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.

真皮における色素沈着を改善するための美容法として,非特許文献13では,基底膜を強化することによりメラニンの真皮への落ちこみを防止することを提案する。 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.

また,皮膚の光老化現象において炎症が一因となることも示唆されており,抗炎症剤も多く開発されている。特許文献3では,アディポネクチン発現増加とともにオートファジー活性化を誘導する化合物を含む抗老化用化粧料組成物を開示する。真皮の色素沈着を改善するにはマクロファージによる貪食作用を利用することも示唆されており,特許文献15では,真皮へ落ち込んだメラニンを取り込んだ線維芽細胞にマクロファージを誘引し貪食させることによる真皮シミ予防・改善剤を開示する。 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.

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

Journal of the American College of Cardiology, Vol. 62, No. 20, 2013, November 12, 2013:1890-901Journal 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.2013.135Experimental & Molecular Medicine (2014) 46, e70; doi:10.1038/emm.2013.135 NATURE, VOL495, 28 MARCH 2013, pp524-530, doi:10.1038/nature11930NATURE, VOL495, 28 MARCH 2013, pp524-530, doi:10.1038/nature11930 Journal of Investigative Dermatology, 2009 April; 129(4):1016-25. Epub 2008 Oct 9.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/s13287-018-0821-5Stem 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, 1577-1580Journal 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, pp733-739British Journal of Dermatology, 2005, 153, pp733-739 Pigment Cell Melanoma Research, Vol. 27, Issue 3, pp502-504Pigment Cell Melanoma Research, Vol. 27, Issue 3, pp502-504 Ann Dermatol Vol. 28, No. 3, pp279-289, 2016Ann Dermatol Vol. 28, No. 3, pp279-289, 2016 Endocrinology, 2011. 152(10): pp3779-90Endocrinology, 2011. 152(10): pp3779-90 British Journal of Dermatology, 2005. 153 Suppl 2: pp37-46British Journal of Dermatology, 2005. 153 Suppl 2: pp37-46 Experimental Dermatology, 2011. 20(11): pp953-5Experimental Dermatology, 2011. 20(11): pp953-5 Fujifilm Research & Development (No.55-2010):pp33-37Fujifilm Research & Development (No.55-2010):pp33-37

本発明の課題は,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方法,装置,および抗光老化及び/又は真皮色素沈着抑制剤,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,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,並びに光老化及び/又は真皮色素沈着度の評価方法の提供にある。 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.

本発明者らは光老化と真皮色素沈着の抑制効果について鋭意研究の結果,マクロファージのM1/M2バランスの不均衡が光老化及び真皮色素沈着に関連することを見出した。かかる発見に基づき,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,並びに抗光老化及び/又は真皮色素沈着抑制美容処置の評価方法を確立した。このような方法により,抗光老化及び/又は真皮色素沈着抑制作用を奏する新規な美容方法,装置,および抗光老化及び/又は真皮色素沈着抑制剤も開発した。
以下の発明を完成するに至った:
(1)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方法。
(2)M1/M2バランスを調整することは,M1に対するM2の比率を上昇させることである,(1)に記載の方法。
(3)M1/M2バランスを調整することは,皮膚に対して微弱な物理刺激を適用する工程を含み,ここで当該工程は,例えば
(a)対象の皮膚を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物理刺激を対象の皮膚に与えることにより達成される,
ここで,伸展率は,

Figure 0007439059000001
(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:
Figure 0007439059000002
{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:
Figure 0007439059000003
(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:
Figure 0007439059000004
{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:
Figure 0007439059000005
(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
Figure 0007439059000001
(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:
Figure 0007439059000002
{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:
Figure 0007439059000003
(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:
Figure 0007439059000004
{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:
Figure 0007439059000005
(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).

本発明の方法,装置,および薬剤により,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/又は改善することができる。また,M1/M2バランスを指標とすることで,光老化及び/又は真皮色素沈着度を客観的に評価することが可能になり,このような方法に基づいて光老化及び/又は真皮色素沈着を予防及び/又は改善するための薬剤や美容処置が探索可能になる。 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.

図1aは,実験1で使用したマクロファージのマーカーの種類,並びに被験者の年齢,スキンタイプ,及び性別を示す。Figure 1a shows the types of macrophage markers used in Experiment 1, as well as the age, skin type, and gender of the subjects. 図1bは,若齢者及び高齢者の皮膚組織における,マクロファージ(CD11b:赤)及びM1マクロファージ(CD86:緑)を示す顕微鏡写真である。Figure 1b is a micrograph showing macrophages (CD11b: red) and M1 macrophages (CD86: green) in skin tissue of young and elderly subjects. 図1cは,若齢者及び高齢者の皮膚組織における,マクロファージ(CD11b:赤)及びM2マクロファージ(CD206:緑)を示す顕微鏡写真である。Figure 1c is a micrograph showing macrophages (CD11b: red) and M2 macrophages (CD206: green) in skin tissue of young and elderly subjects. 図1dは,若齢者及び高齢者の皮膚組織における,M1,M2マクロファージ数及び全マクロファージの総数を示すグラフである(cells/mm2)。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 ). 図1e左図は,若齢者及び高齢者の皮膚組織における,M1マクロファージ(CD86:赤)又はM2マクロファージ(CD206:赤)及びプロコラーゲン(緑)の共染色を示す顕微鏡写真である。左図の4つの各パネル内に存在する長方形は,特徴的な部位を拡大したものである。図1e右図は,M1,M2,線維芽細胞,コラーゲン産生/破壊の関係を模式化した図である。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. 図2aは,実験2の方法の概略を示す。Figure 2a shows an outline of the method for Experiment 2. 図2bは,実験2により誘導されたM0,M1及びM2マクロファージの顕微鏡写真である。Figure 2b is a micrograph of M0, M1 and M2 macrophages induced in Experiment 2. 図2cの上図は,実験2により誘導されたM0,M1及びM2マクロファージが産出するサイトカイン(IL-1beta,TNF-alpha,IL-10)の遺伝子発現量を示すグラフである。図2cの下図は,M1及びM2マクロファージの細胞表面マーカー(M1:CD86,M2:CD206)のmRNAの発現量を示すグラフである。GAPDHのmRNA発現量で補正し,M0の場合を100%とした相対値(%)として示す。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%. 図3aは,実験3の方法の概略を示す。Figure 3a shows an outline of the method for Experiment 3. 図3bは,実験3に従いM1及びM2マクロファージの上清を添加した線維芽細胞におけるプロコラーゲン量(μg/well)を示す。Figure 3b shows the amount of procollagen (μg/well) in fibroblasts to which M1 and M2 macrophage supernatants were added according to Experiment 3. 図3cは,実験3に従いM1及びM2マクロファージの上清を添加した線維芽細胞におけるコラーゲン(赤)及びヒアルロン酸(緑)を示す顕微鏡写真である。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. 図3dは,実験3に従いM1及びM2マクロファージの上清を添加した線維芽細胞におけるβ-ガラクトシダーゼ(β-Gal)を示す顕微鏡写真である。Figure 3d is a photomicrograph showing β-galactosidase (β-Gal) in fibroblasts supplemented with M1 and M2 macrophage supernatants according to Experiment 3. 図3eは,実験3に従いM1及びM2マクロファージの上清を添加した線維芽細胞における,老化関連β-ガラクトシダーゼ(SA β-Gal)アッセイの結果(左図:DAPI陽性細胞の総数に対するSA β-gal陽性細胞の割合(%)),及びウェルあたりのDAPI陽性細胞の総数(右図)を示すグラフである。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). 図3fは,実験3に従いM1及びM2マクロファージの上清を添加した線維芽細胞における,各メラノジェネシス亢進因子(HGF,ET1,bFGF,IL-1alpha,SCF)並びにメラノジェネシス抑制因子(clusterin,DKK1)のmRNA発現量を示すグラフである。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 . 図4aは,実験4に従いM1及びM2マクロファージの上清を添加した若齢及び老齢線維芽細胞におけるβ-Galを示す顕微鏡写真である。Figure 4a is a photomicrograph showing β-Gal in young and old fibroblasts supplemented with M1 and M2 macrophage supernatants according to experiment 4. 図4bは,実験4に従いM1及びM2マクロファージの上清を添加した若齢(上)及び老齢線維芽細胞(下)における,SA β-Galアッセイの結果(DAPI陽性細胞の総数に対するSA β-gal陽性細胞の割合(%))及びウェルあたりのDAPI陽性細胞の総数を示すグラフである。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. 図4cは,実験4に従いM1及びM2マクロファージの上清を添加した若齢及び老齢線維芽細胞におけるプロコラーゲン量(μg/well)を示す。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. 図4dは,実験3に従いM1及びM2マクロファージの上清を添加した若齢及び老齢線維芽細胞におけるコラーゲン(赤)及びDAPIにより染色した核(青)を示す顕微鏡写真である。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. 図5は,実験5に従いM1及びM2マクロファージの上清を添加した新生児包皮由来線維芽細胞における,I型コラーゲン(赤)及びマクロファージ(CD68:緑)を示す顕微鏡写真である。左上の写真はマクロファージの上清を添加せず培養した線維芽細胞を示す。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. 図6は,実験6に従い作成した3Dモデルにおけるマクロファージ(CD68:赤)およびM1マクロファージ(CD86:緑)又はM2マクロファージ(CD206:緑)を示す顕微鏡写真である。三角で示す部分は,CD68およびCD86,又はCD68およびCD206が二重染色されている部分を示す。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. Triangular areas indicate areas where CD68 and CD86 or CD68 and CD206 are double stained. 図7は,実験6に従い作成した3Dモデルにおけるp21(赤)及びDAPIにより染色した核(青)を示す顕微鏡写真である。Figure 7 is a micrograph showing the nucleus (blue) stained with p21 (red) and DAPI in the 3D model created according to Experiment 6. 図8は,実験6に従い作成した3Dモデルの各層(左:上層=表皮細胞層,右:下層=線維芽細胞層)における全細胞数に対するp21陽性細胞数の割合(%)を示すグラフである。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. . 図9は,実験7に従いソーラーシュミレーターを照射したex vivo皮膚モデルにおけるマクロファージ全体,M1及びM2マクロファージの数を示すグラフである。照射を行わない場合の各マクロファージの細胞数を100%とし(左側のバー:照射(-)),それに対する照射を行った各マクロファージの細胞数の相対値(%)を右側のバー(照射(+))に示す。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. 図10は,実験8で行った伸展刺激の設定を示す。Figure 10 shows the stretch stimulation settings used in Experiment 8. 図11は,実験8で使用した装置および皮膚試料を示す。Figure 11 shows the equipment and skin samples used in Experiment 8. 図12は,実験8に従いex vivo皮膚モデルに伸展刺激を与えた場合(stretch)と与えない場合(control)における,M1,M2マクロファージ数及び全マクロファージの総数の比較を示す。左図は,皮膚試料1mm2あたりのマクロファージ数(cells/1mm2)を示すグラフである。右図は,全マクロファージの総数に対する各マクロファージ(M1,M2)の割合(%)を示すグラフである。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. 図13は,実験9で行ったスクリーニングの結果を示す。バーはそれぞれ各濃度のオトギリソウ抽出物(0.1%),タイム抽出物(0.1%),トラネキサム酸メチルアミド(0.03%,0.06%)を添加した場合のCD86発現量(CD86/GAPDH値)を,対照(cont)を100%としたとき相対値(%)で示すグラフである。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%. 図14は,本発明の装置の一例を示す。FIG. 14 shows an example of the device of the present invention. 図15は,実験10で行ったスクリーニングの結果を示す。バーはそれぞれ各濃度のオウバク抽出物(0.01%),ユーカリ抽出物(0.1%)を添加した場合のCD86発現量(CD86/GAPDH値)を,対照(cont)を100%としたとき相対値(%)で示すグラフである。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%. %). 図16aは,実験11に従いM1及びM2マクロファージの上清を添加した線維芽細胞における各コラーゲン分解酵素(MMP-1,MMP-2)および炎症性サイトカイン(IL-1β)のmRNA発現量を,M1での結果を100%としたとき相対値(%)で示すグラフである。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%. 図16bは,実験11に従いM1及びM2マクロファージの上清を添加した線維芽細胞における各コラーゲン産生・成熟化因子(COL1A1,COL1A2,HSP47,およびADAMTS-2)のmRNA発現量を,M1での結果を100%としたとき相対値(%)で示すグラフである。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%. 図17aは,実験12におけるサンプルを取得した各被験者の年齢を若齢者及び高齢者群ごとに示す。Figure 17a shows the age of each subject from whom samples were obtained in Experiment 12, by young and old groups. 図17bは,実験12における若齢者及び高齢者の皮膚組織(基底膜直下から50μm)における,M1,M2マクロファージ数を示すグラフである(cells/mm2)。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. 図17cは,実験12における若齢者及び高齢者の皮膚組織(基底膜直下から200μm)における,M1,M2マクロファージ数を示すグラフである(cells/mm2)。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. 図17dは,実験12における若齢者及び高齢者の皮膚組織における3/4コラーゲン陽性を示すCD68陰性細胞,M1,M2マクロファージ数を示すグラフである(cells/mm2)。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 ). 図18aは,実験13において,メラニン添加から24時間後の線維芽細胞,M1マクロファージ,およびM2マクロファージの様子を示す。メラニンを貪食している細胞は右図で示すような円状の形態をとる。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. 図18bは,実験13において,メラニン添加から24時間後の線維芽細胞,M1マクロファージ,M2マクロファージが取り込んだ細胞あたりのメラニン量(melanin/almar blue)を示す。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. 図18cは,実験13において,メラニン添加から5日後のM1マクロファージとM2マクロファージの様子を示す。Figure 18c shows the appearance of M1 macrophages and M2 macrophages 5 days after melanin addition in Experiment 13. 図19上は,実験14に従いカウントした真皮層におけるメラニン貪食M1マクロファージとM2マクロファージの数を各年齢ごとに示すグラフである。図19下は,メラニン貪食M1マクロファージとM2マクロファージの数を全被験者における平均として示す。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.

マクロファージは,体内の様々な組織に局在し,異物や病原体に対する免疫応答を惹起する細胞であり,炎症にも関与することが知られている。マクロファージは,未分化状態のM0マクロファージ(以下M0とする略記することがある)からM1タイプとM2タイプに分化する。M1マクロファージ(以下M1と略記することがある)は炎症型として,M2マクロファージ(以下M2とする略記することがある)はリペア型(抗炎症型)として知られている。M1マクロファージとM2マクロファージのバランスの不均衡は,肥満,2型糖尿病,動脈硬化といった疾患と関連することが報告されている(特許文献4~6,非特許文献1~5)。しかしながら,皮膚光老化や色素沈着とM1/M2バランスとの関係については不明であった。 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.

本発明者らは,光のあたった皮膚や色素沈着が起こる部位では特異的にこれらM1マクロファージとM2マクロファージのバランス(M1/M2バランス)が乱れることを発見し,M1/M2バランスを調整することが,光老化や色素沈着の予防改善においてとりわけ重要であることを発見した。さらに,本発明者らは,M1/M2バランスを皮膚光老化や色素沈着の指標とし,特定の物理刺激を対象の皮膚に与えると好ましい効果が奏されることも発見した。より具体的には,光老化した皮膚や色素沈着が起こっている皮膚であっても,特定の伸展率で,例えば1Hz以下,10Hz以下といった60Hz以下の振動数で行う物理刺激を与えると,M1/M2バランスが調整/改善されることがわかった。また,本発明者らは,M1/M2バランスを皮膚光老化や色素沈着の指標とし,各種物質をスクリーニングした結果,オトギリソウ抽出物,トラネキサム酸メチルアミドをはじめとする本発明の化合物,タイム抽出物,オウバク,及びユーカリ抽出物にM1/M2バランスを調整/改善する効果があり,抗光老化剤や抗色素沈着剤として機能することも発見した。オトギリソウ抽出物には,I型コラーゲン産生促進作用や育毛作用等が報告されている(特許文献9,10)。トラネキサム酸メチルアミドをはじめとする本発明の化合物には,肌荒れ改善作用,美白作用,抗アレルギー作用等が報告されている(特許文献11,14等)。タイム抽出物には,抗アレルギー作用や抗菌作用等が報告されている(特許文献12,13)。オウバクは,止瀉薬,胃薬,胃弱や食欲不振に対する内服薬,あるいは打ち身,捻挫に対する外用薬としても用いられ,抗菌,抗酸化,皮膚バリア機能改善効果,色素細胞活性化効果等も報告されている(特許文献16,17)。ユーカリ抽出物は,抗菌,抗酸化作用などが報告されている(特許文献18)。しかしながら,これらの物質が,M1/M2バランスを調整/改善作用を有することは,本発明者らによって今回初めて見出された。 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.

よって,本発明は,M1/M2バランスを調整することにより光老化及び/又は色素沈着を予防及び/又は改善するための方法,装置,および抗光老化及び/又は色素沈着抑制剤を提供する。本発明の方法は,美容を目的とする方法であり,医師や医療従事者による治療ではない場合がある。 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.

本明細書において,色素沈着は,真皮および表皮におけるメラニン等の色素の沈着を指す。本発明は,真皮および表皮のいずれにも有効であるが,とりわけ,メラノファージによる貪食以外に改善方法が限られているという意味で真皮の色素沈着に対する対策として大きく期待される。本発明の剤の適用により,色素沈着によるしみ,くすみ,くま等が改善され。また,真皮層に色素を注入するため一旦色素を入れてしまうと消すのが困難である入れ墨やタトゥーなどの色消しにも有効である。 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マクロファージは,CD86,CD80,iNOS等のマーカーで測定できる。M2マクロファージは,CD206,CD163,Agr1,等のマーカーで測定できる。M1とM2を含むマクロファージ全体のマーカーとしては,CD11b,CD68などが挙げられる。追加的又は代替的に,IL-1beta,TNF-alphaといったM1特異的なサイトカインやIL-10といったM2特異的なサイトカインを定量することによって測定してもよい。しかしながら,M1/M2バランスを測定可能なマーカーであれば,上記マーカーに限定されない。 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.

本明細書において,M1/M2バランスとは,M1マクロファージの数とM2マクロファージの数の比率を指すものであってもよく,M1マクロファージのマーカー(例えば,CD86,CD80,iNOS等)のmRNA量とM2マクロファージのマーカー(例えば,CD206,CD163,Agr1等)のmRNA量の比率を指すものであってもよい。光老化状態の皮膚ではM1の比率が高くM2の比率が低くなり,またメラニン貪食作用が高いのはM2であることから,M1/M2バランスの調整/改善は,M1に対するM2の比率(M2の数/M1の数,及び/又はM2マーカーのmRNA量/M1マーカーのmRNA量)を上昇させることであってもよい。上昇は,例えば,有意水準を5%とした統計学的有意差(例えばスチューデントのt検定)を有する上昇であってもよく,及び/又は,例えば1%以上,5%以上,10%以上,20%以上,30%以上,40%以上,50%以上,60%以上,70%以上,80%以上,90%以上,100%以上の上昇であってもよい。あるいは,M1/M2バランスの調整/改善は,M1に対するM2の比率(M2の数/M1の数,及び/又はM2マーカーのmRNA量/M1マーカーのmRNA量)を一定の範囲内,例えば,約4/6~約9/1,約5/5~約8/2,約5/5~約7/3等にすることであってもよく,上記範囲に近づけることであってもよく,上記範囲内に維持することであってもよく,上記範囲を中心に恒常性を保つことであってもよい。 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.

M1/M2バランスの調整/改善は,伸展刺激,押下刺激,マッサージ等の物理刺激を皮膚に与えることによって達成されてもよい。M1/M2バランスの調整/改善は皮膚に対して微弱な物理刺激を適用する工程を含み,本発明の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;を含むサイクルを60Hz以下の振動数で行うことであってもよい。 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.

伸展率は,上記式1により算出される。物理刺激は,0.001%以上80.0以下,0.01%以上60.0%以下,0.1%以上50.0%以下,好ましくは,0.1%以上50.0%以下の伸展率で行ってもよい。例えば,0.1%以上1.0%以下,0.1%以上5.0%以下,0.1%以上10.0%以下,0.1%以上20.0%以下,0.1%以上30.0%以下,1.0%以上5.0%以下,1.0%以上10.0%以下,1.0%以上20.0%以下,1.0%以上30.0%以下,1.0%以上50.0%以下,10.0%以上20.0%以下,10.0%以上30.0%以下,等任意の範囲の伸展率が採用できる。 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.

伸展速度は,1サイクル中で最大伸展率(%)に達するまでの速度(%/s)を指す。回復速度は,最大伸展率から非伸展状態に戻る速度(%/s)を指す。伸展速度・回復速度は,0.010%/s~40%/s,0.05%/s~30%/s,0.10%/s~20%/s,0.2%/s~15%/s,0.3%/s~10%/sなど任意の速度で行ってもよい。伸展速度と回復速度は同じでも異なっていてもよい。 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.

M1/M2バランスの調整/改善は,皮膚に対して微弱な物理刺激を適用する工程を含み,,本発明の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-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ってもよい。 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 .

「皮膚を1μm~1000μm押圧する」とは,皮膚の最表面から1μm~1000μmの深度まで皮膚を押圧することを指す。押圧の深度は,皮膚の最表面から1μm~1000μm,10μm~1000μm,10μm~300μm,10μm~100μm等任意に設定可能である。 "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.

振動数は,伸展又は押圧開始から非伸展又は非押圧状態まで回復するサイクルを1サイクルとした場合の1秒あたりのサイクル数を指す。1サイクルには,一定の時間,伸展又は押圧状態を維持すること及び/又は非伸展又は非押圧状態で休止することを含めてもよい。例えば,1サイクル中に,(a’)(a)の後かつ(b)の前に,あるいは(a-1)の後かつ(b-1)の前に,0秒~30分間,1秒~20分間,5秒~10分間,又は10秒~5分間伸展又は押圧状態を保持すること;及び/又は(b’)(b)の後かつ次のサイクルの(a)の前に,あるいは(a-1)の後かつ(b-1)の前に,0秒~30秒間,0秒~20秒間,0秒~10秒間,1秒~10秒間,1秒~20秒間,1秒~10秒間非伸展又は非押圧状態で休止すること,を更に含んでもよい。振動数は,例えば,0.0000001Hz以上10kHz以下,0.000001Hz以上1kHz以下,0.00001Hz以上100Hz以下,好ましくは,0.0001Hz以上60Hz以下,0.0001Hz以上10Hz以下であってもよい。例えば,0.001Hz以上60Hz以下,0.01Hz以上60Hz以下,0.001Hz以上10Hz以下,0.01Hz以上10Hz以下,0.1Hz以上60Hz以下,0.1Hz以上10Hz以下,0.5Hz以上60Hz以下,0.5Hz以上50Hz以下,0.5Hz以上10Hz以下,0.5Hz以上5Hz以下,0.5Hz以上1Hz以下,0.001Hz以上0.01Hz以下,0.001Hz以上0.1Hz以下,0.001Hz以上1Hz以下,0.01Hz以上1Hz以下,0.1Hz以上1Hz以下,1Hz以上60Hz以下,1Hz以上10Hz以下,1Hz以上5Hz以下,等任意の範囲の振動数が採用できる。 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.

市販の美顔器,マッサージ機器等には,RF波といった0.3~300MHz程度の周波数を有する電磁波を用いるものや,1MHzから7MHz程度の周波数を有する超音波を用いるものなどがある。これらの周波数/振動数と比べ,本発明の方法/装置が採用する振動数は極めて低い。従来の美顔器等のように皮膚に強い振動数を与えると皮膚に赤み,圧力痕,傷,痛み,炎症といった悪影響を与えるリスクがあるが,本発明のような振動数を採用すると上述のリスクが低く非侵襲的な物理刺激が可能になる。本発明者らにより,伸展率および振動数が高すぎると刺激が強すぎるため,これらの値を適正値に調節することにより皮膚を優しく刺激するほうが好ましいことが発見されたためである。 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.

また,従来,当該分野において一般的に利用されているモーター等の機構を利用する美容機器類ではそのモーターの機械的機構から60Hzを超える振動数しか選択できないことが当業者の技術常識であった。従来技術における美容機器類の限界である「60Hz」以下の振動数,例えば,60Hz以下,10Hz以下,1Hz以下といった本発明のような振動数を採用するには,特別な機械を作成する必要性が求められていた。更には,このような低い振動数では,本発明の効果を奏するには「弱すぎる」という固定概念もあり,これまで検討がほとんどされていなかった。しかしながら,本発明者らは,従来の技術常識から考えると非常に低い振動数を用いて実際に皮膚に物理刺激を与えてみたところ,驚くべきことに,このような低い振動数の優しい刺激でも良好な効果が奏された。 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.

EMS機器等では低中周波の装置も市販されているものの,これらは特に筋肉や皮下脂肪等の深い層で作用させるように設計されており,本願発明のような皮膚表層に対する影響は不明である。また,このような装置は周波数が低くても電流を流す際にビリビリとした刺激を伴うこともあり,皮膚に優しい刺激を与える本願発明とは異なる。一方,本発明は,超音波や電流,磁場といったエネルギーを加えず,直接皮膚に伸展刺激を与えるという簡便な方法による美容法が可能である。更に,このような振動数を有する伸展刺激は,優しい刺激でありながら,実施例に記載のようにM1/M2バランスの調整/改善作用を奏する。よって,本発明の方法/装置を使用すると,皮膚に悪影響を与えず,光老化及び/又は色素沈着の予防及び/又は改善効果が期待される。 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.

物理刺激は,美顔器などの器具,実験的な装置,ヒトの手や器具を用いたマッサージ,顔面のエクササイズによるものであってもよく,接触又は非接触により達成されるものであってもよい。一態様では,物理刺激を生ずる刺激発生部と,物理刺激を接触又は非接触により付与する刺激付与部を備えた機器を用いて,皮膚に対して機械的に発生された物理刺激を付与することができる。物理刺激は,例えば,皮膚を引張,押圧,叩く,揉む,吸引するといった接触によって達成されてもよく,及び/又は,例えば,超音波や空気圧または水圧のようなもので皮膚に衝撃波をあたえることで変位させるといった非接触により達成されてもよい。顔面のエクササイズとしては,頬を膨らませることや,目を見開くことなどが行うことができる。マッサージとしては,施術を受ける対象自身又は美容部員などの施術者による手やローラー等の器具を用いたマッサージが挙げられる。しかしながら,本発明の物理刺激の範囲内であれば限定されない。 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. I can do it. 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.

本発明の装置として,例えば,使用者の皮膚に接触し本発明の物理刺激を付与する皮膚接触部を備える美容装置が挙げられる。例えば,把持部及び皮膚伸展部又は皮膚押圧部から構成されるものであってもよい。例えば,図14左に記載の装置は,皮膚接触部が皮膚に触れることにより,皮膚を特定の振動数および伸展率にて伸展するように設計されている。 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.

また,例えば,本発明の装置は,電源と,刺激発生部と,皮膚刺激部を備え,電源は電気信号を発生し,刺激発生部は,電源からの電気信号を物理的な刺激に変換して物理刺激を発生し,皮膚刺激部は,刺激発生部で発生された物理刺激を受け取り使用者の皮膚に物理刺激を与えるものであってもよい。 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.

例えば,図14左に示す装置は,把持部と,電源と,物理的な刺激を制御する制御部と,刺激発生部と,皮膚刺激部と皮膚固定部を含む皮膚接触部とを備える。使用者が把持部を持って皮膚接触部を皮膚に当て,皮膚固定部により皮膚を固定するようにし,制御部を操作することにより電源からの電気的な信号が刺激発生部により物理的な刺激に変換され,物理刺激が皮膚刺激部に伝わり,皮膚が皮膚固定部に固定されつつ皮膚刺激部により特定の振動数および伸展率にて伸展されるように設計されている。例えば,刺激発生部はモーターなどにより駆動され電気信号を物理刺激に変換するものであってもよい。また,図14左に記載の皮膚刺激部は,皮膚に伸展刺激を与えるものであるが,例えば,皮膚に押圧刺激を与えるものであってもよい。 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.

あるいは,本発明の装置は,電源と,物理的な刺激を制御する制御部と,刺激発生部と,シート状の材料からなる皮膚接触面を含む皮膚接触部とを備える美容装置であってもよい。例として,図14右にこのような美容装置の皮膚接触部を示す。シート状の材料は,電流を流すことが可能で,電源からの電気的な信号を物理的な刺激に変換するものであってもよい。そのようなシート状の材料としては,誘電エラストマーアクチュエータ(DEA),導電ポリマー,IPMC,PVCゲル,Mckinnen型などが挙げられる。 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.

本発明の装置の電源は,内部電源又は外部電源であってもよく,充電式であってもよい。また,本発明の装置は,例えば,携帯電話やクラウドなどに保存されているデータを用いるものであっても,ワイヤレスでリモート操作されるものであってもよい。 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.

物理刺激は,上術のような接触又は非接触による物理的な刺激を与えることにより皮膚の伸展を達成するものであってもよい。物理刺激は,皮膚表面に対し平行方向,すなわち横方向に加えてもよいし,皮膚表面に対し垂直方向,すなわち縦方向に加えてもよいし,あるいは斜め方向,ねじれ方向等任意の方向が採用できる。 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.

物理刺激を行うサイクル数は限定されない。例えば,10~500サイクル,20~400サイクル,30~300サイクル,40~200サイクル,50~100サイクルといった任意の数のサイクル行なってもよい。例えば,実施例に記載のように,27サイクル行えば十分な場合もある。 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.

さらに,これらの任意の数のサイクルを1セットとし,このセットを任意の数のセット,例えば,1~100セット,2~50セット,又は3~10セット行ってもよい。 Further, an arbitrary number of these cycles may be considered as one set, and this set may be performed in any arbitrary number of sets, for example, 1 to 100 sets, 2 to 50 sets, or 3 to 10 sets.

物理刺激を行う時間も限定されない。例えば,休止時間を設けて又は設けずにサイクルを繰り返し5分~3時間,10分~2時間,30分~1時間といった一定時間行ってもよい。 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.

サイクル間又はセット間の時間間隔も限定されない。例えば,伸展又は押圧刺激は,1セット又は複数セットを単独で行うものであってもよく,1又は複数のセットを毎日,2日,3日,4日,5日,6日,7日に1回,1,2,3,4週間に1回等,連続的又は断続的に定期的又は不定期的に行うものであってもよい。 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.

しかしながら,皮膚抗光老化及び/又は色素沈着抑制作用を発揮するのに十分な刺激が達成されれば上記のような振動数,伸展率,サイクル数,頻度に限定されない。物理刺激を行う波形も,矩形波,正弦波,三角波,のこぎり波等任意に設定できる。 However, the vibration frequency, extension rate, number of cycles, and frequency are not limited to those mentioned 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.

あるいは,M1/M2バランスの調整/改善は,M1/M2バランス調整/改善剤又はそれを含有する組成物を投与することであってもよい。本発明者らにより,本発明の化合物,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がこのようなM1/M2バランス調整/改善剤として機能することが発見された。よって,本発明は,本発明の化合物,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からなる,あるいは本発明の化合物,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有効成分として含む,M1/M2バランス調整/改善剤,抗光老化剤,色素沈着抑制剤,およびそれらを含有する組成物も提供する。 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.

本願明細書において「本発明の化合物」は,下記の式:

Figure 0007439059000006
{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:
Figure 0007439059000007
(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:
Figure 0007439059000006
{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:
Figure 0007439059000007
(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.

オトギリソウ(Hypericum erectum)は,オトギリソウ科オトギリソウ属の多年生植物である。本発明に用いられるオトギリソウの抽出物としては,オトギリソウの地上部の抽出物が好ましいが,種,根等にも有効成分が含まれているので,これらのうちいずれか1又は2以上の抽出物を使用することもできる。オトギリソウの抽出物は,市販品を用いることもできる。 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.

タイム(Thymus)は,シソ科イブキジャコウソウ属の多年生植物である。本発明では,コモンタイム(T. vulgaris),シトラスタイム(T. x citriodorus),ワイルドタイム(T. serpyllum)等の各種タイムを用いることができる。本発明に用いられるタイム抽出物としては,タイムの全草の抽出物が好ましいが,種,花,根等にも有効成分が含まれているので,これらのうちいずれか1又は2以上の抽出物を使用することもできる。タイムの抽出物は,市販品を用いることもできる。 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.

オウバクは,ミカン科キハダ(Phellodendron amurense)又はシナキハダ(Phellodendron chinense)の樹皮を乾燥させた生薬である。オウバク又はその抽出物は,常法により製造してもよく市販品を用いることもできる。 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.

ユーカリは,フトモモ科ユーカリ属(Eucalyptus)の樹木である。本発明に用いられるユーカリ抽出物としては,ユーカリの葉の抽出物が好ましいが,種,花,樹皮,根等にも有効成分が含まれているので,これらのうちいずれか1又は2以上の抽出物を使用することもできる。ユーカリ抽出物は,常法により製造してもよく市販品を用いることもできる。 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.

抽出物を用いる場合,抽出方法は特に限定されるものではないが,溶媒を用いた抽出法が好ましい。抽出を行う際には,植物体をそのまま使用することもできるが,顆粒状や粉末状に粉砕して抽出に供した方が,穏和な条件で短時間に高い抽出効率で有効成分の抽出を行うことができる。抽出温度は特に限定されるものではなく,粉砕物の粒径や溶媒の種類等に応じて適宜設定すればよい。通常は,室温から溶媒の沸点までの範囲内で設定される。また,抽出時間も特に限定されるものではなく,粉砕物の粒径,溶媒の種類,抽出温度等に応じて適宜設定すればよい。さらに,抽出時には,撹拌を行ってもよいし,撹拌せず静置してもよいし,超音波を加えてもよい。 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, as it 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.

溶媒の種類は特に限定されるものではないが,水,含水エタノール,エタノール等の低級アルコール,ヘキサン等の有機溶媒,又はヘキサン/エタノールといったこれらの混合溶媒が好ましい。抽出は常温で行ってもよいが,加熱下で(例えば温水や熱水等の加熱した溶媒を用いて)行ってもよい。また,溶媒に酵素を加えて抽出処理を行ってもよい。酵素を加えることによって,植物の細胞組織を崩壊させることができ,これにより抽出効率をより高めることができる。酵素としては,細胞組織崩壊酵素を用いることが好ましい。このような酵素としては,例えば,ペクチナーゼ,セルラーゼ,ヘミセルラーゼ,α-アミラーゼ,フィターゼが挙げられる。これらの酵素は1種類を単独で用いてもよいし,2種以上を混合して用いてもよい。 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.

このような抽出操作により,有効成分が抽出され,溶媒に溶け込む。抽出物を含む溶媒は,そのまま使用してもよいが,滅菌,洗浄,濾過,脱色,脱臭等の慣用の精製処理を加えてから使用してもよい。また,必要により濃縮又は希釈してから使用してもよい。さらに,溶媒を全て揮発させて固体状(乾燥物)としてから使用してもよいし,該乾燥物を任意の溶媒に再溶解してから使用してもよい。 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.

また,原料の植物を圧搾することにより得られる圧搾液にも抽出物と同様の有効成分が含まれているので,抽出物の代わりに圧搾液を使用することもできる。 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.

しかしながら,M1/M2バランス調整/改善剤や組成物は上記物質に限定されず,任意の物質,例えば,特許文献4~7等に記載の物質といったM1/M2バランス調整/改善することが知られている公知の物質を使用してもよい。その投与経路も例えば,経皮投与,経口投与,皮下投与,経粘膜投与,筋肉内投与等任意に選択できるが,光老化は,光が当たった部分の皮膚に特異的にみられる現象であるため,皮膚の特定箇所に投与できる経皮投与が好ましい場合もある。また,表皮や真皮に起こる色素沈着は,皮膚から表皮や真皮に到達できるよう経皮投与が好ましい場合もある。また,M1/M2バランスの調整/改善は,例えば,M2マクロファージに分化誘導することであってもよく,その他任意のM1/M2バランス調整/改善法によるものであってもよい。 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.

例えば,本発明の薬剤又は組成物は,M1/M2バランスを調整/改善し,その結果皮膚の光老化及び/又は色素沈着を抑制する。本発明のM1/M2バランス調整/改善剤,抗光老化剤,色素沈着抑制剤(以降これらを総称して「本発明の剤」という場合がある。)は,上記の有効成分の何れか1種を単独で含有してもよく,2種類以上を任意の組み合わせ及び比率で含有してもよい。 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-mentioned active ingredients. The seeds may be contained alone, or two or more types may be contained in any combination and ratio.

本発明の剤は,上記の有効成分を,1種又は2種以上の他の成分,例えば賦形剤,担体及び/又は希釈剤等と組み合わせた組成物とすることもできる。組成物の組成や形態は任意であり,有効成分や用途等の条件に応じて適切に選択すればよい。当該組成物は,その剤形に応じ,賦形剤,担体及び/又は希釈剤等及び他の成分と適宜組み合わせた処方で,常法を用いて製造することができる。 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.

本発明の剤は,化粧品等に配合してヒト及び動物に使用し,或いは医薬製剤としてヒト及び動物に投与することができる。また,各種の飲食品,飼料に配合してヒト及び動物に摂取させてもよい。 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.

本発明を化粧品,医薬品,医薬部外品等の皮膚外用剤に適用する場合,植物体又はその抽出物の配合量(乾燥質量)は,それらの種類,目的,形態,利用方法などに応じて,適宜決めることができる。例えば,化粧料全量中に,本発明の化合物,オトギリソウ抽出物,タイム抽出物,オウバク抽出物,及び/又はユーカリ抽出物それぞれ0.00001%~50%(抽出物又は生薬の場合,乾燥質量換算)を配合できる。 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.

上記成分に加えて,さらに必要により,本発明の効果を損なわない範囲内で,通常化粧品,医薬品,医薬部外品等の皮膚外用剤に用いられる成分,例えば酸化防止剤,油分,紫外線防御剤,界面活性剤,増粘剤,アルコール類,粉末成分,色材,水性成分,水,各種皮膚栄養剤等を必要に応じて適宜配合することができる。 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.

本発明の皮膚外用剤は,外皮に適用される化粧料,医薬部外品等,特に好適には化粧料として適用可能であり,その剤型も皮膚に適用できるものであれば限定されず,溶液系,可溶化系,乳化系,粉末分散系,水-油二層系,水-油-粉末三層系,軟膏,化粧水,ゲル,エアゾール等,任意の剤型が適用される。 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.

本発明の剤を化粧品として用いる場合は,化粧水,乳液,ファンデーション,口紅,リップクリーム,クレンジングクリーム,マッサージクリーム,パック,ハンドクリーム,ハンドパウダー,ボディシャンプー,ボディローション,ボディクリーム,浴用化粧品等の形態として用いてもよい。 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.

しかしながら,本発明の剤および組成物の採り得る形態は,上述の剤型や形態に限定されるものではない。また,本発明の剤又は組成物は,本発明の装置又は方法あるいはその他の装置又は方法等と併用してもよい。 However, the forms that the agents and compositions of the present invention can take 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.

本発明の方法,装置,剤及び組成物を適用する対象は,皮膚光老化及び/又は色素沈着(例えば,真皮色素沈着)が客観的又は主観的に認められる対象であっても,皮膚光老化及び/又は色素沈着を予防したいと希望する対象であってもよい。例えば,M1/M2バランスが崩れていると判断された対象であってもよい。1実施形態では,皮膚におけるM1/M2バランスを指標として皮膚光老化度及び/又は色素沈着度(例えば,真皮色素沈着度)が高いと判断された対象であってもよい。あるいは,皮膚の光老化に特異的な表現型,例えば,しみ,しわ,たるみ等が気になる対象であってもよいし,表皮や色素沈着,例えば,しみ,くすみ,アザ,入れ墨の跡等が気になる対象であってもよい。しみ,しわ,たるみ,くすみ,アザ,入れ墨の跡等は,視感判定や公知の指標を用いることにより決定することができる。 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. Stains, wrinkles, sagging, dullness, bruises, tattoo marks, etc. can be determined by visual judgment or using known indicators.

また,本発明により,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;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/又は色素沈着抑制作用があると評価すること;を含む,抗光老化剤のスクリーニング方法も提供される。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;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/又は色素沈着抑制作用があると評価すること;を含む,抗光老化及び/又は色素沈着抑制美容処置の評価方法も提供される。本発明の方法により,候補薬剤又は美容処置が抗光老化及び/又は色素沈着抑制効果を有するか否かについてのスクリーニングが可能となり,製品開発や新たな肌ケアの提案が可能になる。つまり,本発明により,M1/M2バランスを調整することにより光老化及び/又は色素沈着を予防及び/又は改善するための抗光老化剤及び/又は色素沈着抑制剤および抗光老化及び/又は色素沈着抑制美容処置が提供される。色素沈着は,真皮色素沈着であってもよい。 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.

生体試料は,皮膚試料,免疫細胞試料といった任意の試料を使用できる。皮膚試料は,採取後の皮膚試料,例えば,ヒト等の動物から採取された後のex vivoの状態の皮膚試料であってもよいし,培養皮膚細胞,例えば,単層又は重層培養細胞,培養角化細胞又は培養線維芽細胞といったin vitroの状態であってもよい。あるいは,3D皮膚モデルなどの人工皮膚試料であってもよい。免疫細胞試料は,採取後の皮膚に存在する免疫細胞,皮膚に浸潤する血液を採取した後の血液中の免疫細胞,培養免疫細胞等であってもよい。3D皮膚モデルは限定されないものの,例えば特許文献8,非特許文献10~12に記載の方法により作成しても,M1/M2バランスを測定しやすいようにかかる方法を改変して作成してもよい。生体試料は,M1/M2バランスを測定することができれば限定されない。 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.

例えば,抗光老化及び/又は色素沈着抑制剤のスクリーニング法は,THP-1等の免疫細胞をM0に分化させたのち,薬剤を添加し一定時間培養した後の培養上清を回収し,ELISA等の測定法にてIL-10といったM2特異的なサイトカイン等の量を定量することによって達成されてもよい。このようなスクリーニング法において,陽性対照としてIL-4,IL-13等を添加してM2に分化させた細胞を用いてもよく,更に,IL-4,IL-13に候補薬剤を加えたものを比較してもよい。つまり,M2はIL-10産生能が高いため,上清中のIL-10がM0分化後,培地のみで培養した対照(control)と比較して増加していたら,M2へ分化したといえ,このような薬剤を「改善剤」としてもよく,ここで,IL-4,IL-13等の存在下で薬剤の有無を比較してIL-10の量に差が生じるかを評価してもよい。 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 a screening method, cells differentiated into M2 by adding IL-4, IL-13, etc. may be used as a positive control, and cells in which a candidate drug is 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 the M2 has differentiated. 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.

本明細書において候補薬剤とは,抗光老化及び/又は色素沈着抑制効果について調査が行われる薬剤のことをいい,製品として既に販売されている薬剤の他,開発段階の薬剤も含み,さらに化粧料の開発において化粧料用に選択された薬剤であってもよい。 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.

美容処置としては,特に限定されないが,例えば本発明の剤又はその他の成分を含む化粧料の塗布などの光老化及び/又は色素沈着抑制に有効と考えられている任意の処置が含まれる。化粧料とは,皮膚に適用される化粧料をいい,例えば化粧水,乳液,美容液,クリーム,ファンデーションなどをいうが,これらに限定されず,皮膚状態の改善を直接の目的とするものではないが,皮膚に塗布されるもの全てを含むことを意図し,例えば,日焼け止め剤なども包含するものとする。あるいは,美容処置は,例えば,伸展刺激,押下刺激,マッサージ等の物理刺激を皮膚に与えることであってもよい。美容処置は,一回の処置であってもよいし,数日から数週間にわたって行われる継続的な処置であってもよい。美容処置は個人的に行われてもよいし,美容室や,化粧品の販売店,エステサロンなどで行われてもよい。 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.

また,本発明は,例えば,M1やM2の数を測定可能なCD86,CD206,CD163,等の任意のマーカーを検出する抗体やそれらのマーカーのmRNA量を測定する薬剤といった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/又は色素沈着抑制剤のスクリーニングキットも提供する。更に本発明は,以下の方法,システム,装置も提供する。 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.

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;前記計算手順により計算された結果に基づいて前記皮膚の光老化及び/又は色素沈着度を評価する手順;並びに,前記評価手順により評価された結果を表示する手順;を有する,1又は複数のコンピュータにより実行される対象の皮膚の光老化及び/又は色素沈着度の評価方法。 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:

サーバの分析部が,記憶部に記憶された皮膚のM1/M2バランスと光老化及び/又は色素沈着度に関するデータに基づく教師データを用いたニューラルネットワークによる手法を用いた機械学習により,皮膚のM1/M2バランスと光老化及び/又は色素沈着度との関係を分析する手順;前記サーバの受信部が,対象の皮膚のM1/M2バランスのデータを受信する手順;並びに,前記サーバの評価部が,前記分析した関係に基づいて,前記受信した対象の皮膚のM1/M2バランスを入力として,対象の皮膚の光老化及び/又は色素沈着度を評価して出力する手順;を含む,対象の皮膚の光老化及び/又は色素沈着度の評価方法。 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.

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/又は色素沈着度を評価する評価部;並びに,前記評価部により評価した結果を表示する表示部を有する,対象の皮膚の光老化及び/又は色素沈着度を評価するシステム。 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.

対象の皮膚のM1/M2バランスに関するデータを用いて,対象の皮膚の光老化及び/又は色素沈着度を算出する光老化及び/又は色素沈着度算出装置であって,前記装置は,対象の皮膚のM1/M2バランスに関するデータが入力されると,前記対象の光老化及び/又は色素沈着度を算出する算出部を備え,前記算出部は,皮膚のM1/M2バランスに関するデータが入力された際に,推定される光老化及び/又は色素沈着度を算出するように,教師データを用いた機械学習処理が施された学習済みニューラルネットワークを有する,光老化及び/又は色素沈着度算出装置。 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 device comprising: 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 that has a trained neural network that has been subjected to machine learning processing using teaching data to calculate the estimated photoaging and/or pigmentation degree.

本発明の方法,システム,装置により,M/M2バランスに基づいた客観的な光老化及び/又は色素沈着度の測定が可能になる。 The methods, systems, and devices of the present invention enable objective photoaging and/or pigmentation measurements based on M/M2 balance.

次に実施例によって本発明をさらに詳細に説明する。なお,本発明はこれにより限定されるものではない。 Next, the present invention will be explained in more detail with reference to Examples. Note that the present invention is not limited to this.

実験1:組織染色
図1aに示す年齢の若齢者(20代~30代)及び光老化が起こっている高齢者(60代~70代)の白人由来の目尻の皮膚を凍結切片にし,薄切後,以下に示すマクロファージのマーカーで染色した。
(1)M1マクロファージの染色:ヤギ由来の抗ヒトCD86抗体(R&D)とウサギ由来の抗ヒトCD11b抗体(abcam)で二重染色を行った(図1b)。
(2)M2マクロファージの染色:マウス由来の抗ヒトCD206抗体(BD)又はマウス由来抗ヒトCD163抗体(Leica)とウサギ由来の抗ヒトCD11b抗体(abcam)で二重染色を行った(図1c)。
(3)総マクロファージの染色:マウス由来の抗ヒトCD68抗体(abcam)とウサギ由来の抗ヒトCD11b抗体(abcam)で二重染色を行った。
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).

表皮直下200μmまでの(1)~(3)の抗体二重に陽性の細胞を各マクロファージとして計数した(図1d)。また,M1,M2マクロファージとコラーゲン産生との関わりを検討する目的で,ヤギ由来の抗ヒトCD86抗体(R&D)とラット由来の抗プロコラーゲン抗体(Millipore),又はマウス由来抗ヒトCD206抗体(BD)とラット由来の抗プロコラーゲン抗体(Millipore)で二重染色した(図1e左)。 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).

(1)~(3)の組織染色を図1b,cに,計数結果のグラフを図1dに,M1,M2マクロファージ抗体と抗プロコラーゲン抗体で二重染色した結果を図1e左に示す。図1b,c,dに示すように,光老化が起こっている高齢者では,M1マクロファージが多く見られる一方,M2マクロファージは減少していた。より具体的には,図1bに示すように,若齢者では血管周辺のみにM1マクロファージが存在する一方,高齢者では組織全体的に散在していた。また,図1cに示すように,若齢者ではM2マクロファージが組織全体に存在する一方,高齢者ではその数が減少していた。更に,図1dに示すように,若齢者と高齢者間で,マクロファージの総数(M1の数+M2の数)は変化せず,M1/M2バランスのみが変化していた。若齢者では,M1に対するM2の比率(M2の数/M1の数)が約5/5~約7/3の範囲内にある一方,高齢者ではM1に対するM2の比率が激しく減少しM1/M2バランスが大きく崩れ,若齢者のものとは著しく異なっていた。 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.

更には,図1e左上の2枚の写真のように,若齢者・高齢者にかかわらず,M1マクロファージとプロコラーゲンとの位置はずれている一方,左下の2枚の写真のようにM2マクロファージとプロコラーゲンでは位置の一致が見られた。このことから,図1e右図の模式図で示すような,M1がコラーゲンの破壊を促進し,M2がコラーゲン生成を促進するように線維芽細胞に作用している可能性が示唆される。 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.

実験2:THP-1のM1,M2分化刺激実験
非特許文献1に記載の方法に従い,ヒト由来の株化細胞であるTHP-1を用いて,M1,M2マクロファージへ分化誘導した。具体的には,図2aに示す方法で,THP-1をRPMI1640(Nakalai)に1mM Na Pyruvate(Nakalai),2mM L Glutamine(Nakalai),10% FBSを添加して培養した。その後,100nM PMA(abcam)をさらに加えて24時間刺激しマクロファージに分化させた。M1に分化させる際はさらに100ng/mL LPS(sigma)と20ng/mL IFNγ(R&D)を加えて24時間刺激し,M2に分化させる際はさらに20ng/ml IL-4(R&D)と20ng/mL IL13(R&D)を加えて24時間刺激した。顕微鏡で観察し,図2bに示すように形態が変化したことを確認した。
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.

また,それぞれの分化後又は未分化の状態のままの細胞のmRNAを抽出し,IL-1beta,TNF-alpha,IL-10のプローブ(Applied Biosystems)を用いてTaqMan Gene expression assayによるrealtime PCRを行い,発現量を定量した(図2c上図)。更に,実験1と同じCD86抗体(R&D),CD206抗体(BD)を用い,同様にPCRを行い発現量を定量した(図2c下図)。それぞれの値はGAPDHのmRNA発現量で補正した。 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.

図2cに示すように,実験2に記載の方法により分化させたマクロファージがそれぞれ,M1に特徴的な炎症性サイトカイン(IL-1beta,TNF-alpha)及びM2に特徴的な抗炎症性サイトカイン(IL-10)を産生することが示された。更に,これらの分化誘導マクロファージがそれぞれM1,M2マクロファージの表面マーカーであるCD86,CD206発現の増加を示した。これらの結果より,分化誘導が成功したことが確認された。よって,実験2の方法で分化させたM1,M2マクロファージ及び未分化のM0マクロファージを以下の実験3,4で用いた。 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.

実験3:M1,M2マクロファージ上清の線維芽細胞への添加実験
図3aに示すように,実験2と同様の方法でTHP-1をM1,M2に分化後又はM0の未分化の状態のまま,上清を除き,PBSで1度洗浄した後,培地を加えて48時間培養した。炎症性・抗炎症性サイトカインといったM1,M2の分泌物を含むそれらの上清を新生児由来線維芽細胞に添加した。対照(control)としてRPMI1640(Nakalai)を添加した細胞を用いた。上清の添加後,線維芽細胞を72時間培養し,上清中のプロコラーゲン量をPIP ELISA kit(TAKARA)で定量した(図3b)。細胞画分はウサギ由来の抗ヒトコラーゲン抗体(CEDERLANE)とビオチン化ヒアルロン酸結合タンパク(HOKUDO)で染色した(図3c)。さらに,β-galを老化指標として用い,細胞の核をDAPIで染めた後,細胞内のβ-galをSenescence Detection Kit(abcam)を用いて染色し(図3d),β-gal陽性細胞及びDAPI陽性細胞の数を計数した(図3e)。
Experiment 3: Addition of M1 and M2 macrophage supernatants 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).

更に,M1マクロファージ,M2マクロファージのメラニン産生への寄与度を検討するために,上記と同様にそれぞれのマクロファージの上清を線維芽細胞に添加して培養した。その後,線維芽細胞を回収してmRNAを回収し,HGF,ET1,bFGF,IL-1alpha,SCF,clusterinのプローブを用いてreal time PCRを行いそれぞれのmRNA発現量を定量した(図3f)。 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).

結果を図3b~3fに示す。図3bは,各マクロファージ(M1,M2)の上清を線維芽細胞に添加して72時間培養した後のプロコラーゲン量を示す。図3cは,各マクロファージ(M1,M2)の上清を添加して72時間培養した後の線維芽細胞におけるコラーゲン及びヒアルロン酸の局在を示す。図3b,cより,M1は顕著にコラーゲン産生を抑制することがわかる。図3dは,各マクロファージ(M1,M2)の上清を添加して72時間培養した後の線維芽細胞における細胞内β-galを示す。図3dより,M1ではβ-gal陽性細胞が増え老化を促進する一方,M2は老化を抑制することが示唆される。図3eは,β-gal陽性細胞及びDAPI陽性細胞の数を計数したグラフを示す。右図はウェルあたりのDAPI陽性細胞の総数を示す。図3e右図より,M2は細胞の老化を抑制するのみならず,細胞の増殖を促進することが示唆される。図3e左図は,DAPI陽性細胞総数あたりのβ-gal陽性細胞の数の割合(%)を示し,M1は老化及び細胞死促進作用がある一方,M2は老化抑制及び細胞増殖作用があることが示唆される。更に,図3fに示すように,メラノジェネシス関連因子のmRNA発現はM1によりメラニン産生を増加する方向に作用する一方,M2によりメラニン産生を抑制する方向に作用したこともわかった。非特許文献6に報告されるように,線維芽細胞は,UV等の光刺激によりSCF,HGF等の因子を分泌し細胞死を引き起こすことが知られている。また,非特許文献7~9に報告されるように,線維芽細胞は,光刺激によりHGF,ET1,bFGF,SCF,clusterin等の因子を分泌することによりメラノサイトに直接的又は間接的に作用してメラニンを産生する。よって,光刺激による細胞死やメラニン産生は,M1/M2のバランスの崩れによるものであることが示唆される。 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 right panel 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, it was also found that the mRNA expression of melanogenesis-related factors acted in the direction of increasing melanin production by M1, while it acted in the direction of 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.

実験4:M1,M2マクロファージ上清の若齢及び老齢線維芽細胞への添加実験
次に,M2マクロファージの抗老化効果が老化した線維芽細胞にも有用か,つまり,若返り効果があるか,を確認する目的で,M1,M2マクロファージ上清を若齢及び老齢細胞に添加し,線維芽細胞の年齢の違いによるマクロファージ上清の効果の違いを検討した。
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.

具体的には,実験3と同じ方法で収集したそれぞれのマクロファージ上清を,新生児ヒト包皮由来線維芽細胞(若齢由来線維芽細胞)と68歳ヒト由来線維芽細胞(老齢由来線維芽細胞)に添加し,72時間培養した。その後,実験3と同様の方法でβ-gal及びDAPI染色を行い,β-gal陽性細胞及びDAPI陽性細胞の数を計数した(図4a,4b)。更に,実験3と同様にM1,M2の上清を若齢および老齢由来線維芽細胞に添加し,線維芽細胞を72時間培養し,上清中のプロコラーゲン量をPIP ELISA kit(TAKARA)で定量し(図4c),ウサギ由来抗ヒトコラーゲン抗体(CEDERLANE)とDAPIで染色した(図4d)。 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).

図4aは,β-galの染色図を示す。図4bは若齢由来線維芽細胞および老齢由来線維芽細胞のβ-gal陽性細胞数及びウェルあたりの細胞総数の結果をグラフ化したものを示す。図4a下図より,老齢細胞であっても,M2上清を添加することによりβ-gal陽性細胞が著しく減少し老化が抑制されたことがわかった。また,図4bより,年齢にかかわらず,M1により老化が促進され,M2により老化が抑制されることもわかった。このことは,図4c,dに示されるように,年齢にかかわらず,M1上清を添加した細胞では,M2上清に比べて顕著にコラーゲン産生量が低いという結果によっても支持される。 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.

実験5:M1マクロファージ,M2マクロファージと新生児包皮由来線維芽細胞との共培養実験
新生児包皮由来芽細胞と同数のM1又はM2マクロファージ(実験2の方法で作成)をRPMI1640(Nakalai)で培養した時と新生児包皮由来線維芽細胞とその半分の数のM1又はM2マクロファージをRPMIで培養した時に産生されるコラーゲン量をウサギ由来抗ヒトコラーゲン抗体(CEDERLANE)で染色し比較した。同時にマウス由来抗ヒトCD68抗体(abcam)でも染色し,マクロファージを可視化した。
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).

結果を図5に示す。新生児包皮由来芽細胞でも,M1上清を添加するとコラーゲンが減少される一方,M2を添加するとコラーゲンが多く見られた。つまり,若齢細胞であっても,M1/M2バランスが崩れるとコラーゲンに影響を及ぼすことがわかった。 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.

実験6:3D皮膚モデルを用いたM1/M2バランスの崩れによる影響
M1/M2マクロファージが表皮細胞や線維芽細胞へ及ぼす影響を検討するために,以下の表に記載のような3層構造からなる三種類の3D皮膚モデルを作成した。

Figure 0007439059000008
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.
Figure 0007439059000008

3D皮膚モデルの作成は次のように行った。セルカルチャーインサート(φ12mm,多孔膜の平均孔径:0.4μm)にヒト真皮線維芽細胞(0.2×106個)を播種し,200μM アスコルビン酸―2-リン酸マグネシウム(APM),10%FBS-DMEMを用いて2日に1回培地交換して1週間培養した。その上に,対照モデルにはヒト真皮線維芽細胞を含んだ0.5%I型コラーゲン-10%FBS-DMEM溶液,M1モデル,M2モデルにはさらに実験2の方法で分化させたM1マクロファージ,M2マクロファージをそれぞれ30,000細胞添加したものをそれぞれ分注して,ヒト真皮線維芽細胞の上にコラーゲンゲルを作製し,1~5日間培養した。 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 using the method of Experiment 2. A collagen gel was prepared on human dermal fibroblasts by dispensing 30,000 cells each and culturing them for 1 to 5 days.

さらにHumedia-KG2(クラボウ)培地中に分散した表皮角化細胞を5×105個/ウェルとなるようにコラーゲンゲルの上に播種し,インサート外にHumedia-KG2と10%FBS-DMEMを1:1で混合し200μM APMを添加した培地を内側と同じ液面高さまで添加して3日間培養した。 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.

その後,インサートまたはガラスリング内の培地を取り除き,外側に皮膚モデル用培地(10%FBS-DMEMとHumedia-KG2 EGF(-)を1:1で混合してCa 1.8mMに調製)に200μM APM,10μM N-ヒドロキシ-2-[[(4-メトキシフェニル)スルホニル]3-ピコリル)アミノ]-3-メチルブタンアミド塩酸塩(CGS27023A(MMP阻害剤)),10μM BIPBIPU(ヘパラナーゼ阻害剤)をインサートの底面の高さまで添加して,インサート内部を空気に曝した状態で気液境界培養を行った。2~3日に1回培地交換して2週間培養した。 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). 10 μM N-hydroxy-2-[[(4-methoxyphenyl)sulfonyl]3-picolyl)amino]-3-methylbutanamide hydrochloride (CGS27023A (MMP inhibitor)), 10 μM 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 continued for 2 weeks with medium exchange once every 2 to 3 days.

培養を終えた皮膚モデルを4% PFA/PBS(Nakarai)で固定後,実験1と同様の方法で抗CD206抗体(abcam),抗CD68抗体(abcam),抗CD86抗体(abcam)で染色し,M1,M2マクロファージの存在を確認した(図6)。その後,抗p21抗体(abcam),DAPI(VECTOR)で染色し,表皮細胞層および線維芽細胞層それぞれにおいてDAPIで染色された細胞の数を全細胞数とし,そのうち抗p21抗体にも染色された細胞の数をp21陽性細胞数として計数した。全細胞数に対するp21陽性細胞数の割合を以下の式により求めた。

Figure 0007439059000009
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.
Figure 0007439059000009

結果を図7,8に示す。これらの図に示すように,中間層に接している上層の表皮細胞層および下層の線維芽細胞層のいずれでも対照モデル(cont)に対し,M1モデル(M1)ではp21陽性細胞が増加し,一方M2モデル(M2)では減少していた。この傾向は,単層培養物を用いた実験3と一致していた。従って,ヒト皮膚により近い3D皮膚モデルにおいても,M1マクロファージは老化及び細胞死促進作用がある一方,M2マクロファージは老化及び細胞死抑制作用があることが示唆される。 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.

実験7:ヒト皮膚を用いたex vivoモデルへの太陽光照射実験
Genoskin社製NativeSkin(38歳女性から採取後の皮膚)を到着後付属の培養液で1日培養した。翌日にOriel社製1000Wソーラーシミュレーターに光学フィルターを入れて,UVAとUVBのみを11.5 J/cm2照射し,付属の培養液で培養を続けた(照射(+))。対照として照射を行わない試料を用いた(照射(-))。照射5日後サンプルを回収し,実験1と同様にM1マクロファージ,M2マクロファージ,総マクロファージを染色し,その数を数えてグラフとした。
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.

結果を図9に示す。ソーラーシミュレーターを照射すると,M1数は最も増加した。一方,M2数の増加はM1の場合に比べて非常に小さかった。つまり,光刺激により,M1/M2のバランスが崩れM1の比率が増加することがわかった。 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.

実験8:ヒト皮膚を用いたex vivoモデルへの伸展刺激実験
次に,M1/M2バランスを調整又は改善する方法について検討した。
試料:Genoskin社製NativeSkin(38歳女性から採取後の皮膚)(6well size,直径約2~2.5cm)を使用した。
伸展条件:図11に示すようなウェル内の皮膚の両端を把持する把持部を有し,把持部を引っ張ることにより皮膚を伸展させる伸展器具を作成した。上記組織片が入ったウェルを水平に設置し,把持部を操作して両端から皮膚を伸展させ図10に示すように10%の伸展率となる伸展を10%/sの速度で行い,10%/sの回復速度にて試料を元の非伸展状態に戻した。これを1サイクルとし,合計90サイクルを30分間かけて行った。この90サイクルを1セットとし,セット間に30分~1時間の休止時間を設けて合計3セット(合計270サイクル)を3時間かけて行った。対照として伸展刺激を行わない試料を用いた(control)。
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).

観察方法:伸展刺激を与えた又は与えない皮膚試料について,総マクロファージの染色をウサギ由来の抗ヒトCD11b抗体(abcam)のみで染色した以外は実験1と同様の方法で,にM1マクロファージ,M2マクロファージ,総マクロファージを染色し,その数を計数した。全マクロファージの総数に対する各マクロファージ(M1,M2)の割合(%)を求めてグラフとした。 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.

結果を図12に示す。対照に比べ,伸展刺激を与えた試料では,マクロファージの総数とM1の数はあまり変化がなかったが,M2の数は非常に増加した。つまり,伸展刺激により,M1/M2のバランスが改善することがわかった。 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.

実験9:M1/M2バランスを調整又は改善することにより光老化及び/又は色素沈着を予防及び/又は改善する抗光老化及び/又は色素沈着剤のスクリーニング
M1/M2バランスを調整又は改善する薬剤のスクリーニングを行った。
スクリーニング対象薬剤として,オトギリソウ抽出物,タイム抽出物,トラネキサム酸メチルアミド(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)を含む合計19種の成分について検討した。オトギリソウ抽出物は一丸ファルコスより購入したオトギリソウ地上部の抽出物である。タイム抽出物は香栄興業より購入したワイルドタイムの全草の抽出物である。トラネキサム酸メチルアミドは特許文献14に記載の方法で合成した。オトギリソウ抽出物は50%エタノール,タイム抽出物はブチレングリコール,トラネキサム酸メチルアミドはPBSに溶解した。
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.

実験2と同じTHP-1細胞をM0の未分化の状態のまま用い,37℃で一晩培養した。上記各スクリーニング対象薬剤を培地に対しオトギリソウ抽出物は0.1%,タイム抽出物は0.1%,トラネキサム酸メチルアミドは0.06%,0.03%となるようにそれぞれ添加し,対照にはこれらの溶媒を同量添加し,37℃で二晩培養した。細胞を回収してRNAを抽出し,CD86とGAPDHの発現量をreal time PCRで定量し,CD86/GAPDHを測定した。対照(cont)より再現性をもってCD86/GAPDH値が低下する薬剤を,M1分化抑制剤として探索した。 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. The cells were then 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.

結果を図13に示す。図13より,オトギリソウ抽出物,タイム抽出物,トラネキサム酸メチルアミドを添加するとCD86/GAPDH値が有意に減少し,M1分化抑制作用があることが示され,M1/M2バランス調整/改善剤として使用できることが示唆される。 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.

実験10:M1/M2バランスを調整又は改善することにより光老化及び/又は色素沈着を予防及び/又は改善する抗光老化剤のスクリーニング
更に多くの数の薬剤についてスクリーニングを行うために,オウバク抽出物,ユーカリ抽出物を含む合計8種の各種成分について実験9と同様の方法で検討した。オウバク抽出物はミカン科キハダ樹皮の乾燥物,ユーカリ抽出物はユーカリ葉の抽出物を使用した。オウバク抽出物はブチレングリコールに0.01%となるように溶解し,ユーカリ抽出物は50%エタノールに0.1%となるように溶解した。対照にはこれらの溶媒を用いた。
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.

実験9と同じ方法で,薬剤の添加,細胞の回収,RNAの抽出,CD86/GAPDHの測定を行い,対照(cont)より再現性をもってCD86/GAPDH値が低下する薬剤を,M1分化抑制剤として探索した。結果を図15に示す。図15より,実験9の薬剤に加え,オウバク,ユーカリ抽出物もM1/M2バランス調整/改善剤として使用できることが示唆される。 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.

実験11:M1,M2マクロファージ上清のコラーゲンに対する影響
実験3に加えて,M1マクロファージ,M2マクロファージのコラーゲン分解への寄与度を検討するために,実験3と同様の方法でそれぞれのマクロファージの上清を線維芽細胞に添加して培養し,72時間後mRNAを回収した。コラーゲン分解の指標としてMMP-1,MMP-2,およびIL-1βのプローブ(Applied Bioosystems社製Taqman probe)を用いてreal time PCRを行いそれぞれのmRNA発現量を定量した(図16a)。
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).

更に,M1マクロファージ,M2マクロファージのコラーゲン産生・成熟化への寄与度を検討するために,実験3と同様の方法でそれぞれのマクロファージの上清を線維芽細胞に添加して培養し,72時間後mRNAを回収した。コラーゲン産生・成熟化の指標としてCOL1A1,COL1A2,HSP47,およびADAMTS-2のプローブ(Applied Bioosystems社製Taqman probe)を用いてreal time PCRを行いそれぞれのmRNA発現量を定量した(図16b)。 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).

図16a,bより,M1はコラーゲン分解に寄与する一方,M2はコラーゲン産生・成熟化に寄与することが確認された。 From Figures 16a and b, it was confirmed that M1 contributes to collagen decomposition, while M2 contributes to collagen production and maturation.

実験12:若年及び高齢者のヒト皮膚を用いたex vivoモデルにおけるコラーゲンの分解及び産生
図17aに示す年齢の若齢者(20代~30代)と高齢者(60代~80代)の白人由来のこめかみの皮膚を実験1と同様に凍結切片にし,薄切後,3/4側のコラーゲン切断断片を染色する抗プロコラーゲン抗体(millipore),抗断片化コラーゲン(AdipoGen),並びに実験1と同じCD68,CD11b,CD206,CD86抗体を用い,実験1と同様の方法で陽性細胞をカウントした。
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, an anti-procollagen antibody (millipore), an anti-fragmented collagen (AdipoGen), and an anti-fragmented collagen (AdipoGen) were used to stain the collagen cut fragments on the 3/4 side. Positive cells were counted in the same manner as in Experiment 1 using the same CD68, CD11b, CD206, and CD86 antibodies.

結果を図17b,c,dに示す。若年群に比べて高齢群ではM1マクロファージの割合が高くM2マクロファージの割合が低くなっていた。しかしながら,年齢にかかわらず,3/4コラーゲン陽性を示す線維芽細胞に比べて3/4コラーゲン陽性を示すマクロファージの数が多く,さらに3/4コラーゲン陽性を示すM1マクロファージよりも3/4コラーゲン陽性を示すM2マクロファージ数が多い傾向にあった。 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 .

実験13:真皮におけるメラニンの取り込み能力を示すin vitro実験
実験3と同様の方法でTHP-1細胞を分化させて得たM1マクロファージとM2マクロファージに分化させた。分化したそれぞれのM1マクロファージ,M2マクロファージ,および線維芽細胞(クラボウ社製)にメラニン溶液(Sigma社,Melanin-BioReagent, Synthetic, suitable for cell culture)をPBSに溶解し0.02% W/Vに調整し添加した。24時間後に細胞をPBSで洗浄後,顕微鏡で撮影し,細胞を回収した。回収した細胞の細胞数をアラマーブルー(Life Technologies)で,メラニン量を下記のように計測し,定量した。
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.

メラニンの定量:
それぞれの細胞の培地(マクロファージ:RPMI1640, 線維芽細胞:1DMEM)で1:10に調整したアラマーブルーを添加後,37℃で30分間培養した。その後,その上清を100ul/well回収し,excitement/emission:544nm/590nmで蛍光をAscent(Thermo社)で計測した。計測後,PBSで洗浄し,1M NaOHを添加後室温で3時間インキュベートし,完全に融解した。この細胞溶液をOD475にてPOWERSCAN HT(DS PHARMA BIOMEDICAL)を用いて計測した。
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).

結果を図18a~cに示す。図中の単位「melanin/almar blue」は,培養細胞をアラマーブルーで染色し上清を蛍光を544nm/590nmで計測した強度((1))と,その後細胞を溶解しメラニンも溶解させたものを吸光度475nmで計測した強度((2))の相対値((2)/(1))であり,細胞あたりのメラニン含有量を示す。これらの図より,M2が非常に多くのメラニンを貪食していることがわかった。この傾向は24時間後でも既に見られたが,5日後まで細胞の培養を続けて観察したところ,その差はより明確となった(図18c)。 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).

実験14:真皮におけるメラニンの取り込み能力を示すEx vivoの実験
実験13によってM2マクロファージが線維芽細胞やM1マクロファージよりも多くのメラニンを貪食することがわかったため,実験13でカウントしたヒト皮膚の真皮層をLSM880(カールツァイス社製)を用いてex vivoで観察した。高齢でも若齢でも,明視野で観察した時にM2マクロファージはメラニンが黒く一緒に染まるのに対し,M1マクロファージの近くにはメラニンが存在しないことがわかった。これにより,真皮においてM2マクロファージが線維芽細胞やM1マクロファージより多くのメラニンをとりこんでいることがex vivoでわかり,その数をカウントしグラフ化した(図19)。
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).

図19より,老齢でも若年でもメラニンを取り込んでいるM2マクロファージの数がM1マクロファージの数より多く,この傾向には年齢差があまりみられなかった。この結果より,真皮のメラニンを取り込むのはM2マクロファージであるため,M2マクロファージの割合を増やしてM1/M2バランスを整えれば真皮における色素沈着を予防及び/又は改善できることが示唆される。 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.

光老化によりM1の割合が増加することが示され(実験7),M2の割合が高いほうが皮膚コラーゲン量が多くなり(実験11,12)色素貪食作用も高くなる(実験13,14)傾向が示されたため,M1分化抑制剤としてスクリーニングされたトラネキサム酸メチルアミド等の本発明の化合物,タイム抽出物,オウバク,及びユーカリ抽出物,並びにM1/M2バランス改善作用のある本発明の物理刺激は抗光老化及び/又は色素沈着抑制効果が高いことが示唆される。 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.

以上の結果により,光刺激により,M1/M2のバランスの不均衡が生ずることにより,光のあたった皮膚で光老化が引き起こされたり,真皮では色素が沈着するが,M1/M2のバランスを調整又は改善することにより,光老化及び/又は真皮色素沈着を予防及び/又は改善できることが示唆される。M1/M2のバランスの調整又は改善は,伸展刺激や押圧刺激を与えることや,トラネキサム酸メチルアミド等の本発明の化合物,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等のM1分化抑制作用がある成分をM1/M2バランス調整/改善剤として適用することにより達成され,ひいては光老化及び/又は真皮色素沈着の予防及び/又は改善が期待される。 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.

本発明により,光老化及び/又は真皮色素沈着の予防及び/又は改善,光老化及び/又は真皮色素沈着度の評価,抗光老化及び/又は真皮色素沈着抑制剤及び抗光老化及び/又は真皮色素沈着抑制美容処置の探索が可能になる。 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 (6)

M1マクロファージに対するM2マクロファージの比率を上昇させ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であって、
トラネキサム酸メチルアミド,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,抗光老化及び/又は真皮色素沈着抑制剤。
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 ,
An anti-photoaging and/or dermal pigmentation inhibitor comprising tranexamic acid methylamide, Hypericum perforatum extract, thyme extract, Aspergillus spp., and/or eucalyptus extract.
トラネキサム酸メチルアミド,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マクロファージに対するM2マクロファージの比率を上昇させるための薬剤。 An agent for increasing the ratio of M2 macrophages to M1 macrophages, comprising tranexamic acid methylamide, Hypericum perforatum extract, thyme extract, Aspergillus perforatum extract, and/or Eucalyptus extract. M1マクロファージに対するM2マクロファージの比率は,M2マクロファージの数/M1マクロファージの数又はM2マクロファージマーカーの量/M1マクロファージマーカーの量である,請求項1又は2に記載の薬剤。 The drug according to claim 1 or 2 , wherein the ratio of M2 macrophages to M1 macrophages is the number of M2 macrophages/the number of M1 macrophages or the amount of M2 macrophage marker/the amount of M1 macrophage marker. M1マクロファージに対するM2マクロファージの比率を上昇させ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
(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,
(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)
により算出される,前記美容装置。
A cosmetic device for preventing and/or improving photoaging and/or dermal pigmentation by increasing the ratio of M2 macrophages to M1 macrophages,
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 is :
(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;
A cycle including
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 a straight line passing through fixed points A and B is parallel to the stretching direction.)
The beauty device calculated by.
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マクロファージに対するM2マクロファージの比率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マクロファージに対するM2マクロファージの比率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マクロファージに対するM2マクロファージの比率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;を有し,
M1マクロファージに対するM2マクロファージの比率は,M2マクロファージの数/M1マクロファージの数又はM2マクロファージマーカーの量/M1マクロファージマーカーの量である,前記方法。
A method for evaluating photoaging and/or dermal pigmentation degree performed by one or more computers, the method comprising:
A procedure for obtaining data regarding a preset standard value of the ratio of M2 macrophages to M1 macrophages in the skin;
a procedure for obtaining data regarding the ratio of M2 macrophages to M1 macrophages in the skin of a subject;
a step of calculating by referring to the reference value and comparing it with data regarding the ratio of M2 macrophages to M1 macrophages in the skin of the subject;
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;
The method as described above, wherein the ratio of M2 macrophages to M1 macrophages is the number of M2 macrophages/the number of M1 macrophages or the amount of M2 macrophage marker/the amount of M1 macrophage marker.
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マクロファージに対するM2マクロファージの比率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マクロファージに対するM2マクロファージの比率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マクロファージに対するM2マクロファージの比率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し,
M1マクロファージに対するM2マクロファージの比率は,M2マクロファージの数/M1マクロファージの数又はM2マクロファージマーカーの量/M1マクロファージマーカーの量である,前記システム。
A system for evaluating photoaging and/or dermal pigmentation, the system comprising:
a database unit that stores data regarding a preset standard value of the ratio of M2 macrophages to M1 macrophages in the skin;
a data input section for inputting data regarding the ratio of M2 macrophages to M1 macrophages in the subject's skin;
a calculation unit that refers to a reference value stored in the database unit and calculates by comparing it with data regarding the ratio of M2 macrophages to M1 macrophages in the skin of the subject input by the data input unit;
an evaluation section that evaluates the degree of photoaging and/or dermal pigmentation of the skin based on the calculation results of the calculation section; and a display section that displays the evaluation results of the evaluation section;
The system, wherein the ratio of M2 macrophages to M1 macrophages is the number of M2 macrophages/the number of M1 macrophages or the amount of M2 macrophage marker/the amount of M1 macrophage marker.
JP2021514255A 2019-04-19 2020-04-20 Methods and devices for preventing and/or improving photoaging and/or dermal pigmentation, anti-photoaging and/or dermal pigmentation inhibitors, screening methods for anti-photoaging and/or dermal pigmentation inhibitors, and anti-photoaging and/or dermal pigmentation inhibitors. Evaluation method of cosmetic treatment for suppressing photoaging and/or dermal pigmentation, and evaluation method of photoaging and/or dermal pigmentation degree Active JP7439059B2 (en)

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